THE CHEMISTRY AND METABOLISM OF THE COMPOUNDS OF PHOSPHORUS BY IX M. KALCKAR LItvision of Nutrition and Physiology, The Public Health Research Institute of The City of New York, Inc., New York During the past year considerable new knowledge has been added to that previously available concerning the function of phosphate com- pounds in metabolism. The well-known relationship between oxidation and phosphorylation has been further consolidated and extended, and some completely new reactions involving phosphate transfer have been discovered, such as the cleavage of thio-ether linkages by adenosine- triphosphate, and the ability of the latter to phosphorylate pyridoxal. Inasmuch as other chapters in this volume are devoted to nucleic acids and phospholipoids, only brief mention will be given in this chap- . ter to studies dealing with these compounds. It is hardly necessary to add that European literature during the year is covered only incom- pletely due to the difficulty of obtaining European journals. For the same reason it has been necessary to include European publications which are one or two years old. My thanks are due to Professor J. RunnstrBm, Wennergren Institute of Experimental Biology, Stock- holm, Sweden, for valuable information regarding research in Sweden during the past few years. DETERMINATION OF PHOSPHATE A modification of the usual method for determination of phosphate has been described by Borei ( 1). Norberg (2) describes an ultramicro I determination of total phosphorus using a photoelectric microscope described by Caspersson (3). Lowry & Bessey (4) have devised an- other ultramicro phosphorus determination using the Beckman spec- trophotometer and narrow cells ( 1 or 2 mm. wide and 10 mm. deep). The method for phosphoglycerol determination has been found to lack specificity (5). Haas (6) has described a spectrophotometric micro method for the determination of glucosed-phosphate using dichlorophenolindophenol as hydrogen acceptor and the specific dehy- drogenase as catalyst. The method is also useful for the study of de- hydrogenase. 283 284 KALCKAR PHOSPHORUS COMPOUNDS 285 increase in acid phosphatase iri the region of the cytoplasm of nerve cells in which chromatolysis had been produced by axon section. Adrenalectomy caused a decrease in kidney and liver phosphatase (15). The phosphatase content returned to normal following injection of adrenal cortex extract. Inclusion bodies of vaccinia, herpes sim- plex, fowl pox, etc. were also investigated by histochemical techniques and found to contain no phosphatase (16). Changes in serum phosphatase under various conditions have been studied by a large number of investigators. Drill et d. (17) found that sodium cyanide in concentrations of 0.0001 to 0.1 molar has only slight inhibitory effect on serum phosphatase activity of normal dogs. However, when the serum phosphatase values were increased as a result of liver damage, `the addition of sodium cyanide inhibited the extra phosphatase activity markedly. Gould (18) found that fat feed- ing causes an increase in serum phosphatase. Herbert (19) found that prostate phosphatase can be distinguished from other acid serum phosphatases by its great lability. Thus one hour incubation of serum at 37" at pH 7.4 inactivates the prostate phosphatases, whereas the other acid phosphatases are not affected. Addition of 0.4 vol. of ethanol to prostate phosphatase in buffers from pH 4.8 to 7.4 renders it completely inactive after half hour incubation at room temperature, whereas the other acid phosphatases remain un- altered. Delory & King (20) have studied the kinetics of phosphatase action at different pH's and with different substrates. They found that the rate of hydrolysis of phosphate esters having low ionization constants is higher than that of esters having high constants. Moreover, the pH optimum is more alkaline for the former than for the latter. These ob- servations are consistent with the hypothesis of Martland & Robison (21) that the enzyme is a weak base whose undissociated molecule combines with a substrate to form a compound which breaks down into the products of hydrolysis. Alkaline phosphatase is apparently only active if amino groups as well as phenol groups are intact (18). Its behavior towards ketene treatment is similar to that of the lacto- genie hormone. Phosphatase from rat sarcoma has a pH optimum of 5.6 in the absence of magnesium or manganese. In the presence of those two metals the pH optimum is around 4.8 (22). The more specific phosphatases will be dealt with in another chapter. PHOSPHATASES Determination.- The King-Armstrong (7) method for the deter- mination of phosphatase has been modified by Binkley, Shank & Hoagland (8). Phosphotyrosine is used as substrate and the liberated tyrosine is determined calorimetrically. By this method phosphatase can be determined in 1 ml. of plasma. Determination of phosphatase in very small quantities of blood has been made possible by the ingen- ious method of Lowry & Bessey (4) in which a nitrophenyl phosphate reagent is used. Three c.mm. of plasma at pH 10 are incubated with 50 c.mm. of the reagent, and after the addition of sodium hydroxide the intensity of the color (400 mp.) produced by the liberation of the free sodium salt of nitrophenol is measured directly in a photoelectric calorimeter. The method is simple and direct, and particularly useful when large numbers of determinations are required. Menten et al. (9) have modified the histochemical phosphatase test. They used a mono-aryl phosphate which is hydrolyzed by phosphatase. The liberated aryl group reacts with a diazotized amine, forming a highly colored insoluble dye. Barium-P-naphthol phosphate was used as the phosphoric ester and the diazotized a-naphthol amine as the coupling substance. Histochemical techniques have been used by a number of investiga- tors to study phosphatases in normal and pathological tissues. Wilmer (10) using a histochemical technique found that the renal tubules in the cortex of aglomerular kidneys do not contain alkaline phosphatase, whereas the connective tissue contains considerable amounts of the enzyme. This finding is of interest in connection with the mechanism of sugar reabsorption proposed by Lundsgaard (11) which involves a phosphorylation-dephosphorylation cycle. If this theory is correct, it is only logical that kidneys in which no filtration takes place, and consequently no reabsorption, are devoid of phosphatase. The occur- rence of an acid phosphatase in these kidneys still remains a possibility. Wilmer (10) also found a decrease in phosphatase in the tubular cells when the tubular function was destroyed, as for instance in hydro- nephrosis or spontaneous interstitial nephritis. Wachstein (12) studied renal phosphatases in hemorrhagic kidney due to choline deficiency. He found a marked decrease in alkaline phosphatase in the damaged atrophic tubules and some increase in phosphatase in the vessels and in the glomeruli. Alkaline phosphatase was also found in ovarian fol- licles and corpora lutea (13). Bodian & Mellors (14) found a marked 286 KALCKAR FORMATION OF PHOSPHATE ANHYDRIDES Mann (23) has conducted some interesting studies on the phos- phorus metabolism of the mold Aspergillus niger. A specific metaphos- phatase was isolated from extracts of the mold. Inorganic pyrophos- phate and polymetaphosphates were isolated as ammonium and barium salts. The metaphosphates are readily converted into inorganic pyro- phosphate during the isolation procedure. The metaphosphate could be separated from the inorganic pyrophosphate by chemical procedures as well as by enzymatic techniques. Thus pyrophosphatase splits only inorganic pyrophosphate, whereas the metaphosphatase from mold hy- drolyzes metaphosphate as well as pyrophosphate. The metaphosphate isolated acted as a precipitating agent for proteins. Inorganic pyro- phosphate has been isolated from yeast (24), and recently Cori & Ochoa (25) have isolated considerable amounts of inorganic pyrophos- phate from liver extracts which oxidized dicarboxylic acids. It is not definitely established yet whether the pyrophosphate, the polymeta- phosphate or perhaps an organic complex represent the compound originally present in the cell. PHOSPHORUS COMPOUNDS 287 (27) are of interest. They find that the priming effect of polysac- charides is markedly increased by partial hydrolysis. The staining reaction with iodine disappears completely about the time the poly- saccharide begins to decline in activating power. All these observa- tions are in agreement with the formulation proposed by Cori et al. (28) as a result of their experiments with crystalline muscle phos- phorylase incubated with varying amounts of glycogen. Cori et al. for- mulated the polysaccharide formation catalyzed by phosphorylase as follows : glucose-l-phosphate plus terminal glucose units e maltosidic chain units plus inorganic phosphate. The terminal glucose units are the end groups of the highly branched glycogen molecule which serves as the primer of the polysaccharide formation. Sumner & Somers (29) have described a simplified method for the preparation of glucose-l-phosphate employing purified potato phos- phorylase. Doudoroff, Hassid & Barker (30) described the synthesis of two new sugars which appear to be analogs of sucrose. A phosphory- lase prepared from Pseudomonas saccharophila catalyzes the reversible splitting of sucrose into Cori ester. If 1-sorbose or d-ketoxylose is in- cubated with the enzyme in the presence of Cori ester, a reaction simi- lar to that observed in the presence of fructose and Cori ester takes place, i.e., inorganic phosphate is liberated and the amount of sugar PHOSPHATE COMPOUNDS AND ENZYMES INVOLVED IN CARBOHYDRATE METABOLISM Formation of phosphohexoses.- Phosphorylase has been studied by Sumner, Somers & Sisler (26). They found that the nature of the products synthesized from Cori ester by plant phosphorylase depends upon the kind and amount of polysaccharide added to prime the re- action. Thus a small quantity of achroodextrin will lead to the pro- duction of a substance giving a blue color with iodine. A larger quantity of achroodextrin will cause a product to be formed giving a red color with iodine, while addition of a very large amount of the dextrin will cause the production of a substance which gives no color at all with iodine. In each case the quantity of inorganic phosphate liberated is practically the same. The authors interpret their findings as follows : the enzyme adds anhydro-d-glucose molecules to whatever foundation it finds present. If a few dextrin molecules'are present it forms a chain sufficiently long to give a blue color with iodine. If many dextrin molecules are present, phosphorylase forms many poly- saccharide chains of intermediate length and the product resembles erythrodextrin. In this connection the experiments of Hidy & Day decreases. The synthetic compound appears to be the disaccharide glucoside-sorboside. Shapiro & Wertheimer (3 1) investigated phosphorylase activity in various animal tissues. They found a highly active phosphorylase in subcutaneous tissue. Phosphorylase was not found in the muscle of rats of less than ten days of age. In fourteen-day-old rats the enzvme ._. is already active. No decrease-in the glycogen phosphorylase of m&cle could bk demonstrated in adrenalectomized or in thyroidectomized rats. The inhibitory effect of glucose on muscle phosphorylase was diminished by adenylic acid. No glucose inhibition was found with potato phosphorylase. Transphosphorylation of hexoses.-The transphosphorylation of glucose to hexose monophosphate and of the latter to hexose diphos- phate by adenosinetriphosphate has been given much attention during recent years. Youngburg (32) studied aerobic phosphorylation of sugars in kidney cortex extract. He found that whereas hexoses were readily phosphorylated, no phosphorylation of pentoses took place. KALCKAR PHOSPHORUS COMPOUNDS 289 Klein (33), studying the metabolism of brain tissue, observed that the oxidation of fructose is accompanied by a phosphorylation. Huszak (34) found that the white and gray matter of the brain show a differ- ent carbohydrate metabolism. The white matter used' preferentially glycogen as metabolite, whereas free glucose was not utilized. The main metabolite in the gray matter was glucose, which was phosphory- lated by adenosinetriphosphate and subsequently oxidized. Phospho- pyruvic acid is also able to phosphorylate glucose, but only through the adenylic acid system. Lindberg (35), working in Runnstrom's Insti- tute, studied the carbohydrate metabolism of sea-urchin eggs during fertilization. He found that the dehydrogenases from ground sea- urchin eggs are strongly stimulated by the addition of hexose phos- phates, phosphoribose, and phosphogluconic acid. Lindberg further- more described a phosphoric ester, occurring in sea-urchin eggs, which has an activating effect upon the carbohydrate metabolism. This phos- phoric ester was also found in and isolated from beef brain. The phos- phoric ester was crystallized both as a brucine and an acridine salt, and the molecular weight was reported to be about 150. The sub- stance, which is acid stable, behaves in many ways like glycerophos- phate. In experimenting with egg pulp, it was found that the ester had a strongly enhancing effect on respiration. Furthermore, it caused a temporary accumulation of pentoses and of an unidentified acid. The amount of acid formed was of the same order of magnitude as that of the carbohydrates broken down. The author compared the phenome- non with that which occurs after fertilization. In both processes forma- tion of acids takes place presumably by oxidative decarboxylation of hexose. Greville & Lehmann (36) studied phosphate and carbohy- drate metabolism in extract of human muscle. They observed the well-known transphosphorylations and dephosphorylations. The enzymes catalyzing the phosphorylation of hexoses to hexose mono- or diphosphates are of considerable interest for an understand- ing of the regulation of carbohydrate metabolism. Until recently the phosphorylation of glucose to hexose monophosphate (catalyzed by the enzyme hexokinase) had never been clearly demonstrated in muscle extracts. The phosphorylation of hexose monophosphate to diphos- phate by adenosinetriphosphate was demonstrated in muscle extracts several years ago by Ostern (37) and his co-workers. Now, `Colowick & Price (38) have succeeded in demonstrating hexokinase in extracts from rat muscle. They furthermore report that the transphosphoryla- tion of glucose and hexose monophosphate requires the presence of reduced cozymase, oxidized coiymase being without activating effect. The coenzyme of the transphosphatase seems to be destroyed rapidly by an enzyme in the muscle extract which has not yet been identified. However, it is known that animal tissues contain a specific nucleosidase which splits off the pyridine base in pyridine nucleotides (39). The finding that the reduced cozymase is the activator of these transphos- phorylations may be of importance for the understanding of the so- called Pasteur effect, i.e., the suppression of fermentation by oxygen. In this connection some recent experiments of Engelhardt & Sakov (40) are of interest. They found that the addition of phenol, phenol oxidase, and the cytochrome system completely inactivates the trans- phosphorylation of hexose monophosphate. Kubowitz (41) has shown that the phenol oxidase system can reoxidize reduced cozymase. The nature of the coenzyme of the hexose monophosphate fermen- tation (42) has not been further clarified. cidin on bacterial metabolism (43). He- found that gramicidin in- creases the oxygen uptake of intact bacteria, provided glucose is the substrate, and that the uptake of phosphate is completely inhibited. In kidney extract, Hotchkiss (44) was able to show that the aerobic phos- phorylation of glucose is also completely inhibited in the presence or small amounts of gramicidin (30 to 40 pg. per ml.). Meyerhof 8z Beck (45) have purified the phosphotriose isomerase by ammonium sulfate fractionation and adsorption on cupric oxide. The preparation obtained was free of phosphohexose isomerase and of aldolase. The activity of the preparation was high but the stability low. Coupling between oxidation-reduction and uptake or liberation of phosphate.-The enzymatic formation of 1,3-diphosphoglyceric acid (phosphoglycerylphosphate) from phosphotriose discovered by War- burg & Christian (46) and Negelein & Brijmel (47) has been re- viewed in previous volumes. Biicher, continuing these studies, has purified and crystallized the enzyme catalyzing the equilibrium between diphosphoglyceric acid and the adenylic acid system (48). The enzyme was precipitated as a nucleoprotein from acidified alcoholic solution and subsequently crystallized in alkaline ammonium sulfate (0.6 satu- rated) containing inorganic pyrophosphate. This enzyme is the most active fermentation enzyme thus far isolated. An amount as small as 0.01 mg. per ml. can be detected readily in the optical test at 334 mp. (the test for reduced cozymase). The equilibrium catalyzed by the enzyme can be expressed as follows: 1,3-diphosphoglyceric acid plus 290 KALCKAR adenosinediphosphate s 3-phosphoglycerate plus adenosinetriphos- phate. Lipmann has continued his studies of the formation and the prop- erties of acetyl phosphate and has been able to throw light on many interesting problems concerning bacterial metabolism. Acetyl phos- phate was synthesized by Lipmann & Tuttle (49) according to a greatly simplified method in which monosilver dihydrogen phosphate reacts with acetyl chloride yielding monoacetyl phosphate. Acetyl phosphate is readily hydrolyzed both in the acid and in the alkaline range. At pH 5 the compound showed maximum stability. The addi- tion of substances which combine with phosphate greatly increases the hydrolysis of acetyl phosphate. Thus molybdate increases the hydroly- sis of acetyl phosphate in acid solution, whereas calcium ions which precipitate phosphate at alkaline reaction correspondingly increase the hydrolysis of the compound in the alkaline range. The formation of acetyl phosphate by oxidation of pyruvate in the presence of dry bacteria (Bacillus acidificans longissimus) has also been described by Lipmann (50). Acetyl phosphate was isolated as a silver salt and identified as disilver monoacetyl phosphate. In analogy with the findings of Biicher (48), it was found that acetyl phosphate is also able to transfer its phosphate group to the adenylic acid system. Hitherto the enzymatic formation of acetyl phosphate had been demonstrated only in the special system just mentioned. However, during the last year acetyl phosphate formation has been observed in preparations from other bacteria. Koepsell, Johnson & Meek (51) have succeeded in demonstrating the formation of acetyl phosphate in the oxidation of pyruvate by a dry preparation of Clostridiurn butyli- cum. They found that in the absence of glucose, inorganic phosphate is taken up and appears as labile phosphate. After fractionation with silver they found that the purified labile phosphate fraction contained both acetic and butyric acid, this fact indicating the formation of both acetyl and butyryl phosphates. If butyric acid was incubated with acetyl phosphate in the presence of the enzyme extract, considerable amounts of butyric acid were found in the silver precipitate. This was interpreted as indicating the presence of butyryl phosphate since ab- sorption of free butyric acid by the silver precipitate had been ex- cluded. They suggested the following reaction : acetyl phosphate plus butyrate --P acetate plus butyryl phosphate. Acetyl phosphate has also been shown to play a role in the phos- phoroclastic splitting of pyruvate into acetate and formate catalyzed PHOSPHORUS COMPOUNDS by an enzyme from Escherichia coli. Utter & Werkman (52) found that the splitting of pyruvate proceeds according to the equation: pyruvate plus phosphate = acetyl phosphate plus formate. Utter, Werkman Sr Lipmann (53) have been able to show that this phos- phoroclastic splitting is reversible. An enzyme preparation from E. coli was incubated with formic acid containing an excess of G3, and pyruvic acid containing ordinary carbon. After one hour the Cl3 con- centration in the formate had decreased considerably and was ac- counted for in the carboxylic group of the pyruvate. The carbon dioxide did not contain any excess CIJ, indicating the absence of Woods' equilibrium enzyme (54). Although the equilibrium of the . phosphoroclastic reaction is far toward the formation of acetyl phos- phate, it has nevertheless been possible to demonstrate chemically the formation of small amounts of pyruvate by incubating acetyl phosphate and formate with the enzyme preparation (55). The equilibrium con- stant is roughly lo-= for the reversed phosphoroclastic reaction. Acetyl phosphate may also play a role in animal tissue. Lipmann (56) has most recently described an enzyme occurring in skeletal muscle which rapidly and specifically dephosphorylates acetyl phos- phate. The stability of the enzyme toward acid as well as its high speci- ficity are features not ordinarily found among phosphatases. Its pres- ence in tissue might very well interfere seriously with any demonstra- tion of acetyl phosphate formation in animal tissue. The occurrence of such an enzyme in animal tissue, on the other hand, may also sug- gest that this compound actually is an intermediate in the carbohydrate metabolism of higher animals. The role of acetyl phosphate in the formation of acetylcholine is not known. However, it has been found that adenosinetriphosphate under anaerobic conditions greatly stimulates the formation of acetyl- choline in brain extracts (57, 58). Ochoa (59) studied a-ketoglutarate dehydrogenase from cell free suspensions of washed heart muscle. The a-ketoglutarate was oxidized only one step, i.e., to succinate and carbon dioxide, provided that the succinic dehydrogenase was inhibited by malonate. In the presence of glucose, three mols of phosphate were transferred to the sugar (form- ing hexose diphosphate) for each mol ketoglutarate oxidized to suc- cinate and carbon dioxide. Synthetic succinyl phosphate did not give rise to any phosphorylation of sugar but was rapidly dephosphorylated. Inorganic phosphate, magnesium ions, and muscle adenylic acid, or adenylpyrophosphate, were required for the activity of a-ketoglutarate KALCKAR PHOSPHORUS COMPOUNDS 293 by magnesium contains one atom of the metal. Biicher likewise found that the crystalline mercury salt of enolase contains one atom of mer- cury per molecule of enolase. The molecular weight of enolase was determined by a specially constructed apparatus applying the Tyndall effect as a measure of molecular size. Edestin, the molecular weight of which was determined by diffusion and sedimentation as well as by the Tyndall method, served as a standard. Biicher found that the molecular weight of the enolase decreased after dialysis at pH 5 and the activity disappeared ; if salt were added the molecular weight in- creased to the original value and activity was fully restored. dehydrogenase. Adenosinetriphosphate was five times as efficient an activator as adenylic acid. This difference was attributed to destruc- tion of adenylic acid by deaminase action. Long (60) studied the oxidation of a-ketobutyrate on minced pigeon brain. He likewise found inorganic phosphate to be an essential component in the oxidation of pyruvate as well as of a-ketobutyrate. Adenine nucleotides markedly increase the oxidation of pyruvate, pro- vided inorganic phosphate is present. The oxygen-pyruvate ratio is 1: 2 for the part of the pyruvate oxidation catalyzed by adenine nucleotides. Leloir & Mufioz (61) have studied the oxidation of butyric acid in liver extract. They found that the oxidation of this fatty acid is stimu- lated by the presence of a number of dicarboxylic acids. All the active dicarboxylic acids when added alone to liver extract are readily oxi- dized and give rise to the formation of phospho-enol pyruvic acid. No phosphopyruvate is formed in the absence of adenylic acid, cyto- chome-c, or inorganic phosphate. Malonate inhibits phosphopyruvate formation from succinate, fumarate, or citrate. Phosphopyruvate can replace dicarboxylic acids in increasing the rate of butyrate oxidation, provided that carbon dioxide is present, indicating that phosphopy- ruvate may be carboxylated. Lehninger (62) has studied fatty acid oxidation in homogenized liver preparations. He found that the oxidation of saturated fatty acids having four to eight carbon atoms by homogenized rat liver requires the presence of adenosinetri- or adenosinediphosphate. Adenylic acid is inactive. This finding is in agreement with the observations of Lang (63) and of Shapiro & Wertheimer (64) who demonstrated that palmitic acid dehydrogenase from liver extract requires the presence of adenylpyrophosphate. Lardy, Hansen & Phillips (65) found a phosphate uptake in sperm cells which is coupled to oxidations other than those of carbohydrate metabolism. The ultilization of phospholipids was suggested as a pos- sibility. Enolase.-The work of Warburg & Christian (66) on crystalline enolase was reviewed last year (67). It will be recalled that the amount of magnesium present in purified enolase was analyzed and found to be one gram atom magnesium per 52,000 grams enolase. Biicher (68), working in Warburg's laboratory, has recently deter- mined the molecular weight of crystalline enolase and found it to be 62,000, which means that one molecule of enolase when fully activated NUCLEOTIDES AND NUCLEIC ACIDS Analysis of nucleotid,es and nucleic acid in tissues.-Analyses of tissues like muscle, liver, and kidneys have been made both by ordi- nary chemical methods, by enzymatic methods, and by optical methods. Caspersson & Thorell (69) using the photoelectric quartz microscope studied the ultraviolet absorption at various wavelengths of muscle fibers from Drosophila funebris which possesses large segments, mak- ing it possible to investigate the isotropic and anisotropic sections separately. He found that the ratio between the absorption at 260 mu. to that at 280 mu. was much higher in the isotropic than in the aniso- tropic part, indicating that the adenine nucleotides (which according to Parnas (70) constitute more than 90 per cent of the muscle purines) may be confined exclusively to the isotropic part of the rest- ing muscle.. There is reason to believe, as pointed out by Bernal (71), that myosin is present in both parts. Hoagland, Lavin & Shank (72) have reached the same conclusions as Caspersson, using a direct tech- nique by which one part of the muscle fiber is photogranhed in nolar- - v- I ized light and the rest of the fiber is simultaneously photographed in ultraviolet light (73). With this method, it was shown in human muscle fibers that the dark isotropic sections continue into the ultra- violet field as dark absorbing bands. It is hardly necessary to add that without the knowledge gathered by chemical analysis of muscle tissue, we would not be in a position to interpret the optical analyses. A number of investigators have studied acid labile phosphorus in various tissues under various conditions. Wagtendonk (74) found a particularly marked lowering of the labile phosphate in liver and kid- ney of guinea pigs on a diet deficient in the so-called "antistiffness" factor. Whether the decrease in labile phosphate in this case is spe- 294 KALCKAR cific or nonspecific, it is difficult to decide. It is known that starvation of animals gives rise to the same phenomenon (75, 76). However, Wagtendonk's observations are of interest because the changes are much more marked than those observed in starved animals. There is reason to believe that the decrease in the labile phosphate is mainly due to a decrease of adenylpyrophosphate since the "adenylic acid frac- tion" was correspondingly increased. However, it would be of interest to know whether it is adenylic acid or inosinic acid which constitutes the main component of the so-called "adenylic acid fraction." Rapoport et al. (77) found that red cells obtained during phenyl- hydrazine reticulocytosis showed a substantial increase in the concen- tration of adenylpyrophosphate as related to the hemoglobin. Kabat (78) found an increase of acid labile phosphate in the brain of animals infected with poliomyelitis. Whether this labile phosphate can be identified with adenylpyrophosphate remains to be seen. More precise information about the content of nucleotides in small tissue samples can be obtained by using enzymatic methods, provided the enzymes employed are sufficiently purified. Thus Schmidt & Engel (79) in 1933 initiated new methods for purine analyses in tissue samples by using purified deaminases and measuring the ammonia liberated from various purines. It has recently been possible to meas- ure minute amounts of purine derivatives using Schmidt's enzymatic technique combined with ultraviolet spectroscopy. Thus a highly sensitive and specific method for the determination of muscle adenylic acid was developed by observing the change in the ultraviolet spectrum (the decrease in absorption at 265 mu) which takes place when the nucleotide is deaminated by adenylic acid deaminase (80). If the deaminase has been freed from impurities of myokinase, no decrease of absorption takes place when adenosinetri- or diphosphate is added. However, if a few micrograms of a specific adenylpyrophosphate from potato are added to the system, adenylic acid is formed and subse- quently deaminated, thus causing a fall in the absorption. By this method less than 0.5 mg. of muscle tissue can be analyzed for adenylic acid and adenylpyrophosphate (81). A similar sensitive method for hypoxanthine compounds has been developed (81) using the rise in absorption at 290 rnp which takes place when hypoxanthine is oxi- dized to uric acid by xanthine oxidase. Inosine requires the presence of nucleosidase, and inosinic acid requires both nucleosidase and phos- phatase in addition to xanthine oxidase before any rise in absorption will take place. This combination of optical and enzymatic methods PHOSPHORUS COMPOUNDS 295 might be a valuable tool for studying changes in the composition of nucleotides or nucleic acids, as well as their enzymes in animal tissue under pathological conditions. Anfinson (82) studied the distribution of diphosphopyridine nu- cleotide (DPN) in retina using the Cartesian diver technique ; triose- phosphate dehydrogenase was used as catalyst. The higher concentra- tions of DPN (4 pg. per mg. fat free solid) were found in the two synaptic regions. The rods and the outer nuclear layer contained less and the nerve fibers were very low in DPN. Davidson & Waymouth (83) studied the content of nucleotides and nucleic acids in various tissues by means of ordinary chemical methods. The concentration of nucleotides seems to be lower in tumor tissue than in the corresponding normal tissues. En.zymatic reactions involving adenosinetriphosphate.-Adenosine- triphosphate can participate in reversible transphosphorylations and in irreversible transphosphorylations, and it can undergo simple hy- drolysis. The first type of reaction includes the phosphorylation of amidines (creatine, arginine) and of carboxylic groups (phosphogly- ceric acid, acetic acid), and the phosphorylation of adenosinediphos- phate (phosphate dismutation). The irreversible transphosphoryla- tions include the phosphorylation of hydroxy groups such as the l- or 6-hydroxy groups of hexoses and that of pyridoxal. The phosphoryla- tion of hexoses has already been discussed. The phosphorylation of pyridbxal is a very recent observation (84) and of great interest be- cause the phosphorylated product is active as a coenzyme of the enzyme which brings about decarboxylation of tyrosine. A completely new type of transphosphorylation was discovered by Binkley (85), who found that the terminal group of adenosinetriphosphates splits the thio-ether linkage of cystathionine with the formation of cysteine and phosphohomoserine. The reaction is a strictly stoichiometric one, with one mol of phosphate being transferred per mol of cysteine liberated. Adenylpyrophosphatases.-A highly active adenylpyrophosphatase was isolated from potatoes (86). The enzyme splits both labile phos- phate groups from adenosinetriphosphate. It is activated by calcium ions. Inosinetriphosphate is hydrolyzed to inosinic acid. The rate is somewhat slower than for adenylpyrophosphate. The enzyme is readily adsorbed on myosin, a phenomenon of interest in the discus- sion of the possible identity of myosin with muscle adenosinetriphos- phatase (ATP-tase) (87). Myosin might be identical with ATP-tase PHOSPHORUS COMPOUNDS 297 of anomalous viscosity and flow birefringence. When a myosin solu- tion was incubated with a small amount of ATP its birefringence was decreased about half, and the relative viscosity was slightly decreased. The full effect of ATP was obtained at a 0.004 molar concentration. Although other substances can cause a decrease in flow birefringence, to do so they must be present at a much higher concentration than this. The changes of physical and chemical properties of myosin brought about by ATP-tase are spontaneous and reversible, and seem to be connected with the enzymatic action of the protein as an ATP- tase. Effects similar to those of ATP have been obtained so far only with inosinetriphosphate, whereas inorganic triphosphate, although hydrolyzed by myosin phosphatase, has no effects on the physico- chemical properties of myosin. Important contributions to our knowledge in this field have also been made from the Institute of Medical Chemistry in Szged by Szent- Gyiirgyi and his group (95). They observed a marked difference in the physico-chemical as well as the enzymatic properties of myosin, depending on the method of extraction. The myosin obtained by ex- tracting skeletal muscle ten minutes with potassium chloride shows a low viscosity ; this preparation is called myosin "A" by the Hungarian group. If the muscle, on the other hand, is extracted for several hours with alkaline potassium chloride, a myosin preparation is obtained which is highly viscous and which readily forms fibers when injected into distilled water. The second type of myosin is called myosin "B" (96). If a myosin B fiber is placed in a freshly prepared water extract of muscle it contracts and becomes opaque. A myosin A fiber shows no change under such circumstances. Three components are necessary for the effect on the myosin B fiber : potassium, magnesium, and adeno- sinetriphosphate (ATP). If a contracted myosin B fiber is subse- quently suspended in 0.2 M potassium chloride (containing magne- sium ions) and ATP is added, the fiber relaxes. However, if the re- laxed fiber is suspended in 0.1 M potassium chloride (containing magnesium ions) addition of ATP now produces contraction. Thus ATP addition can give rise to either contraction or relaxation, depend- ing upon the potassium chloride concentration. In order to bring a contracted fiber into a state of relaxation in the absence of ATP, po- tassium chloride concentrations as high as 0.6 M are required, and it is necessary to adjust to a quite alkaline pH range. Straub (97) has isolated a protein called "actin" from muscle which is soluble in alkalies. An actin solution remains liquid in the absence 296 KALCKAR but the possibility that the muscle ATP-tase is adsorbed on myosin has certainly not been excluded. The muscle deaminase has also been found in myosin even after three reprecipitations (88). A large number of investigators have continued the study of the myosin adenylpyrophosphatases. Ziff & Moore (89) studied myosine ATP-tase by means of electrophoresis and ultracentrifugation. Myosin was found to consist electrophoretically of one component to which 90 per cent of the triphosphatase activity is associated. The effect of oxidation and reduction on myosin ATP-tase has been studied extensively. Singer & Barron (90) found that mercap- tide-forming compounds as well as mild oxidizing agents inhibit ATP- tase. This inhibition was attributed to an oxidation of sulfhydryl groups since the inhibited enzyme could be reactivated by adding re- duced glutathione. They found a close parallelism between the num- ber of sulfhydryl groups attacked by mercuric p-chlorobenzoate and the degree of inhibition of enzyme activity, and interpret these findings as evidence in favor of Engelhardt's hypothesis that.muscle ATP-tase is identical with myosin. Ziff (91) found that stored myosin loses its ATP-tase activity but can be partly reactivated by cystine or gluta- thione, which also reactivates oxidized myosin. Mehl (92) observed a greater decrease in ATP-tase activity in stored rat muscle myosin when the activity was estimated at a pH of 9 than when determined at pH 6 or 7. He likewise found that oxidation and reduction have a much greater effect on the activity measured in the alkaline range than when measured in the acid range. Binkley, Ward 8z Hoagland (93) studied myosin from persons afflicted with hereditary muscle dys- trophy and found that the preparation contains an active ATP-tase. It was found that traces of copper completely inhibited the enzyme activity. The effect of copper was nullified by the addition of cyanide. Cyanide also increased the activity of fresh myosin preparations as well as preparations inactivated by oxidation with hydrogen peroxide. They devised a method of purification in which the myosin was pre- cipitated with copper and redissolved in cyanide buffer. In this way they succeeded in obtaining ATP-tase preparations of more constant activity. As might be expected, myokinase added to myosin ATP-tase re- sults in the dephosphorylation of adenosinediphosphate ( ADP) (86). This is merely due to the enzymatic conversion of ADP into adenylic acid and ATP, which is then subsequently hydrolyzed by ATP-tase. Dainty et al. (94) studied the particle shape of myosin by means 298 KALCKAR of salts. Upon addition of salt, the viscosity as well as the birefrin- gence is greatly increased. Actin is able to combine with myosin, form- ing more or less viscous complexes, depending on the viscosity of the original "actin." For a given actin preparation the maximal viscosity is reached by mixing one part actin to three parts of myosin, a ratio which according to the authors is very nearly the same as that found in skeletal muscle. If ATP is added to a viscous solution of an actin- myosin complex it causes a marked decrease in viscosity, approaching the viscosity found for myosin A. Szent-GyCrgyi concludes from this observation that ATP separates myosin B into actin and myosin A. After ATP has been hydrolyzed by phosphatase action, the viscosity is found to increase again, indicating that after the disappearance of ATP, actin and myosin A are again able to form a complex (myosin B). One mol ATP is able to effect a decrease in viscosity of 100,000 grams of myosin, which indicates that if the molecular weight of myo- sin is around 100,000, one mol of ATP reacts with one mol myosin (98). Szent-Gyijrgyi emphasizes that the ATP reacts with the myosin component and not with the actin component. This is further indicated by the fact that ATP is also able to decrease the viscosity of free myosin (myosin A) in salt solution. Szent-GyCrgyi interprets the difference in myosin obtained by various methods of extraction as follows. Extraction of fresh muscle, containing a large amount of ATP, with saline yields myosin A, leav- ing the actin in the insoluble residue. If, on the other hand, the muscle is extracted with alkaline potassium chloride overnight, the ATP is hydrolyzed, and a myosin-actin complex, myosin B, is obtained. Sus- pensions of myosin A or B in potassium chloride solutions of a strength between 0.1 and 0.2 M are flocculated by the addition of ATP. Sus- pensions of myosin in potassium chloride solutions stronger than 0.2 M go into solution upon addition of ATP, an effect which is other- wise obtained only by raising the potassium chloride concentration to 0.6 M and making the reaction alkaline. These observations are simi- lar to those just described for the myosin fiber. The effect of ATP on isolated muscle fibers has also been studied (99). It was found that minute amounts of ATP applied directly to the isolated muscle fiber cause a rapid contraction. Intra-arterial in- jection of ATP likewise gives rise to muscle contractions accompanied by electrical activity (100). The curarized muscle is just as sensitive to ATP as the non-curarized. ADP has the same effect as ATP, whereas adenylic acid, though just as effective in the non-curarized PHOSPHORUS COMPOUNDS 299 muscle, is much less effective in the curarized. Inorganic phosphate is inactive, whereas inorganic triphosphate and pyrophosphate release contractions. In smooth muscle, only ATP is active. Pharmacological eflect.-Green & Stoner (101) report that the toxic effect of ATP on rats is potentiated by magnesium salts. The studies of Bollmann & Flock (102) are of interest in connection with the problem whether ATP plays a major role in tourniquet shock and traumatic shock. They found that ATP is almost completely hydro- lyzed in muscles deprived of their blood supply. If the occlusion lasts more than three hours, little if any rephosphorylation takes place after the blood supply has been re-established. The decomposition products are relatively nontoxic and, presumably, consist mainly of inosinic acid. Tourniquet shock is, therefore, not caused by release of adenylic acid derivatives into the blood stream. Moreover, it is unlikely that adenylic acid compounds play a major role in traumatic shock, even though it has been found by means of the spectroscopic deaminase method just described (80) that the adenosine derivative concentration of the blood coming from a traumatized extremity is increased (103). That this influx of adenosine compounds is insufficient to exert a de- pressor effect is shown by the failure of injections of adenosine de- aminase plus phosphatase to effect the low blood pressure accompany- ing traumatic shock (103). And yet, this enzyme combination exerts a marked antagonistic effect on the fall in arterial blood pressure caused by infusion of ATP. Release of adenylic acid or its derivatives into the blood stream seems, therefore, to be at the most a secondary factor in the traumatic shock. Prosthetic groups.-Phosphorylated cdmpounds have been identi- fied as prosthetic groups in two important enzymes. Ratner et al. (104) found that the flavine adenine dinucleotide is the prosthetic group of glycine oxidase. Gunsalus et al. (84) found that phosphoryl- ated pyridoxal can activate the decarboxylation of tyrosine. They were able to obtain an active coenzyme, both by chemical phosphorylation as well as by enzymatic phosphorylation, using ATP as a phosphate donor. The experiments of Westenbrink & Veldman (105) indicate that phosphothiamine synthesized in the yeast cell is not all bound to car- boxylase, although it is present in a form in which it is attacked much more slowly by yeast phosphatase than is free phosphothiamine. Thus the phosphothiamine content of yeast may be increased 1700 per cent without increase in carboxylase activity. One must, however, bear in 300 KALCKAR mind that phosphothiamine seems to be the prosthetic group of a num- ber of dehydrogenases as well as carboxylases. Nucleic acids.-Davidson & Waymouth (106) studied a factor in pancreatin which increased nucleoprotein phosphorus of fibroblasts in vitro. The active material seems to be a mixture of polypeptide and nucleotide derivatives. Gulland et al. (107) reviewed critically pre- vious claims concerning the structure of nucleic acids in the dividing cell. Claude (108) f ound nucleic acid associated with the formed elements of the cell. Woodward (109) observed ribonuclease in the plague bacillus. Bain Sr Rusch (110) described a manometric deter- mination of ribonuclease. Desoxyribonucleic acid.-Avery, MacLeod & McCarty (111) isolated in a highly purified form a factor from type III pneumococci which is able to transform the unencapsulated R variant of Pneu- mococcus type II into the fully encapsulated cells of type III. The active factor was shown to be a highly polymerized specific desoxy- ribonucleic acid which is destroyed by phosphatase and by minute amounts of purified desoxyribonuclease. The wide significance of this work will be discussed elsewhere. PHOSPHORUS COMPOUNDS 301 PHOSPHOLIPIDS AND THEIR CONSTITUENTS Baer & McArthur (112) have synthesized phosphorylcholine by phosphorylation of choline halide with diphenylphosphoryl chloride in pyridine, with subsequent isolation of the diphenylphosphorylcholine as the chloroaurate. The latter compound is decomposed with metallic silver, yielding the free diphenylphosphorylcholine which in turn may be readily catalytically hydrogenated to the free phosphoryl- choline. Riley (113) studied the metabolism of phosphorylcholine. The betaine is readily dephosphorylated in vivo and the inorganic phosphate is excreted in the urine. Phosphorylcholine exerts an inhibition of the turnover of phospholipid in the liver. The inhibition appears to be limited to the noncholine phosphatide fraction. Phosphorylcholine, as a unit, is probably not utilized in the synthesis of phospholipids. Wagner- Jauregg & Lennartz (114) have synthesized dicholesterol pyrophosphate. Elliott & Lebet (115) have studied oxidation of phosphatides in brain extract. Ascorbic acid and iron salts or iron protein complexes greatly stimulate the oxygen intake. .STUDIES OFTHE METABOLISM OF PHOSPHATE COMPOUNDS IN VIVO Studies of carbohydrate metabolism in Z&JO by means of radioactive phosphate.-A number of publications dealing with the study of carbo- hydrate metabolism and with the rejuvenation in vivo of phosphate compounds such as adenosine polyphosphates and phosphocreatine have appeared this year. It will be recalled that Sacks in 1940 (116) expressed the belief that, contrary to what had been found in vitro, the formation of lactic acid in the working muscle in viva is inde- pendent of phosphorylations. The main evidence brought forward against the occurrence of a phosphate cycle in the working muscle was the finding that phosphocreatine and pyrophosphate from the muscle of animals injected with radioactive phosphate contained the same concentration of P,, whether the muscle was working or resting, that is, whether much or little lactic acid was being produced. The isotope concentrations of the two fractions were much lower than that found in the inorganic phosphate. Bollmann & Flock (117) obtained essen- tially the same results and drew the same conclusions. It was, how- ever, soon realized that the conditions under which these experiments were conducted did not permit valid conclusions about the rate of phosphate turnover in the phosphate compounds of the working muscle. The main difficulty encountered in these studies has always been, and still is, to obtain reliable figures for the isotopic concentra- tion of the inorganic phosphate in the muscle fiber. The reason for this difficulty must first of all be attributed to the very slow penetra- tion of phosphate into the muscle fiber (Hevesy, 118). As a conse- quence of this, the isotopic concentration of the extracellular inorganic phosphate is manifoldly higher than that of the inorganic phosphate in- side the muscle fiber. The very low isotope concentration of pyro- phosphate and phosphocreatine as compared to inorganic phosphate, observed by Sacks, and Bollmann & Flock must, therefore, be ascribed to a contamination of the cellular inorganic phosphate with the highly radioactive extracellular phosphate. In a later paper Sacks & Alt- schuler (119) have taken the extracellular phosphate into account but, nevertheless, still maintain that. there is an essential difference in metabolism between the resting and the working muscle with respect to the phosphate cycle. It seems justifiable to raise the question whether there actually is any conflict between in vitro and in z&o studies in this special case. 302 KALCKAR So far, we do not know of any experimental data from in vivo studies of phosphate metabolism which cannot be accounted for by the so- called Embden-Meyerhof scheme. One way of studying the rate of rejuvenation of phosphate com- pounds in muscle is to remove the extracellular phosphate by perfus- ing the muscle with saline. If, under these circumstances, the P,, con- centrations of phosphocreatine and adenylpyrophosphate are com- pared with that of the true intracellular inorganic phosphate, one finds a very rapid rejuvenation of the phosphate compounds of the muscle (120). Thus only twenty minutes after an intravenous injection of P,, the phosphocreatine phosphorus has an isotope concentration about 60 per cent that of the inorganic phosphate ; the same results are obtained for adenylpyrophosphate. In other words, the phosphorus turnover of this labile phosphate compound is already so high in rest- ing muscle that with present techniques it would be quite difficult to discover any substantial increase in the turnover during or after mus- cular work. This technical failure does not, however, justify us in claiming that a further increase of phosphorus turnover does not ac- tually take place during muscular contraction. The rate of rejuvenation of adenylpyrophosphate phosphorus and of phosphocreatine phos- phorus in resting rabbit muscle amounts to 20 to 30 pg. phosphorus per minute per g. of muscle, and there is every reason to expect that the rate will be manifoldly higher in working muscle. In the liver the rate of rejuvenation of adenylpyrophosphate phosphorus is about the same orcler of magnitude as that of resting muscle. The rate of rejuvenation of the two labile phosphates in adenosine- triphosphate has been studied using hexokinase as an instrument to differentiate between the terminal and the second phosphate group (121). In the resting rabbit muscle the P,, concentration was always found to be the same in both of the labile phosphate groups even when investigated shortly after the injection of radioactive phosphate. How- ever, Flock & Bollmann (122), using myosin ATP-tase as a tool to differentiate between the two labile groups of ATP, have found a dis- tinctly higher P 32 concentration in the terminal phosphate group as compared with that of the second group. In the studies of Kalckar et al. (120) the relative isotope concentra- tion of hexosemonophosphate phosphorus in resting muscle varied greatly but was usually found to be considerably lower than that of the pyrophosphate. However, in certain cases the isotope concentration of this ester (which was purified as the calcium salt and "washed" with PHOSPHORUS COMPOUNDS inert inorganic phosphate) was found to be as much as three times that of the pyrophosphate and more than twice that of the inorganic phos- phate (120). The same observation has been made by Sacks (119), who has been able to throw further light on this finding (123, 124). The interpretation of Sacks' figures are, however, somewhat difficult because no values for the P,, concentrations of the inorganic phos- phate were presented. Undoubtedly, the most important observation is that the high P,, concentrations of the "glucose monophosphate" phosphorus are never found in fed animals (in the post-absorptive phase) but only in fasted animals. The results might be interpreted as indicating that, before entering the cell, some form of hexose combines with extracellular phosphate, which is, of course, very rich in isotopic phosphate. The possibility suggested by Hotchkiss (43) that phos- phate from the environment reacts with a polysaccharide complex in the cell wall of microorganisms forming a hexosephosphate might very well be considered in the case of muscle. Sacks discusses a simi- lar hypothesis. The observations made by MyrbLk & Vasseur (125) that certain enzymes in the yeast cell seem to be located inside and cer- tain other enzymes outside a barrier may also be of interest in this con- nection. Kaplan & Greenberg (126,127) have published a number of papers dealing with the determination of phosphoric esters in liver by barium and mercury fractionation, and by hydrolysis curves. The justifica- tion for identifying the "seven minutes acid-hydrolyzable phosphorus" in the fraction of water-insoluble barium salt with the labile phos- phorus of pyrophosphate is open to criticism. The identification of these two fractions is justified when applied to acid filtrates from skeletal muscle, but the picture in the liver is much more complex. Inorganic pyrophosphate, for instance, has been found in liver (25), and other yet unknown acid labile phosphoric esters having insoluble barium salts may belong to this fraction. The authors report that in- sulin (in the presence of glucose) increases the amount, as well as the P,, content, of the labile groups of ATP. The changes observed are small and may'be secondary or nonspecific. It is known that reduction of food intake causes a decrease of the acid-labile phosphorus in the liver (75), an observation actually confirmed by Kaplan & Greenberg (76). It is, therefore, conceivable that the effect of insulin may be the result of its known effect in increasing the food intake. Rejuvenation of phosphorus ilz nucleic acids.-Brues et al. (128) investigated the turnover of phosphorus in the desoxyribonucleic acid 304 KALCKAR (nuclear nucleic acid) from liver and found that the rate of rejuvena- tion in non-growing liver is very slow (10 to 11 per cent rejuvenation after three days). As shown by Marshak (129) the rate of rejuvena- tion of ribonucleic acid (cytoplasmic nucleic acid) phosphorus is very rapid. Euler & Hevesy (130, 131, 132) studied the rate of rejuvena- tion of nucleic acid phosphorus in tumors. The radioactivity per mg. nucleic acid phosphorus was compared with that of the free inorganic phosphate. In two hours 2 to 3 per cent of the total nucleic acid phos- phorus of a Jensen sarcoma had been rejuvenated. The phosphorus of desoxyribonucleic acid of growing sarcoma was also turned over at a considerable rate (1.5 per cent per hour). Irradiation with 1,000 In- ternational Roentgen units decreased the rate of rejuvenation of nucleic acid phosphorus to half or one-third of that of the untreated sarcoma. This effect of x-rays appears before the fall in the number of mitoses occurs; Phosphate turnover of phospholipids.-Two independent groups have confirmed and extended previous reports (133) that choline deficiency causes a decrease in the synthesis of phospholipids in the liver and kidney of young rats in which damage of the two organs has been produced by the dietary regime. Patterson et al. (134) found the rate of rejuvenation of phospholipid phosphorus decreases in choline deficient rats. Boxer & Stetten (135) found correspondingly that the daily replacement of choline in phospholipids, which in normal rats amounts to 3.9 mg., is decreased to one-third in choline deficient rats. The effect consists in a retardation of the incorporation of new choline into the phosphatide without altering the quantity of choline present in the phosphatides. The reader is referred to the chapter on lipid metabolism for further information about this interesting topic. 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