Original Repository: Stanford University Libraries. Department of Special Collections and University Archives. Paul Berg Papers
Reproduced with permission of Paul Berg.
Protein Synthesis, Tumor Viruses, and Recombinant DNA, 1959-1975
April 20, 1971
I ordinarily don't respond so quickly, particularly when the distractions are as many as they are now. Nevertheless, I
did want to say a bit about the conA binding studies.
You may recall when we talked in Paris or in London, I can't remember which, I told you of our inability to confirm a
difference in the binding of conA to normal and transformed cells. We never like Sachs' experiments with the nickel labeled
conA' because we were worried about whether he was measuring nickel binding rather than conA binding to cells (the radioactive
nickel was not covalently bound to the conA and, in fact, is readily dissociable by dialysis). His data could easily have
been explained if transformed cells were a more efficient ion exchanger than normal cells. We used 125 I-labeled conA prepared
enzymatically and could detect no difference in binding even though the labeled conA preparation caused agglutination of transformed
cells but not of normal cells. I told this to Joe Sambrook in Paris, but he had repeated Sachs' experiment with nickel
labeled conA and obtained results similar to those of Sachs. Subsequently, he also prepared 125I-labeled conA and wheat germ
agglutinin and obtained the same results as we have. At first I was somewhat reluctant to believe Donna Arndt's data because
it was at variance with our expectations; nevertheless, she has tried in a variety of ways to find a difference but without
success; and Joe's confirmation of her findings makes me convinced that we're not wrong and that there has to be another
explanation for the differential agglutinability.
It's not a technical problem, we're not using either glass filters or Millipore filters. Our first set of experiments
were done with cell suspensions made with EDTA which were mixed with labeled conA, incubated under various conditions, centrifuged,
washed several times, and then the cells were dissolved and counted. Burger raised the objection that perhaps our EDTA treatment
was liberating some protease perhaps from 1% of the cells and this obliterated the difference. We accepted his suggestion
for doing the experiment in the following way: monolayers are washed and then exposed to varying concentrations of labeled
conA directly on the plate. After an appropriate time the layer is washed several times and then dissolved in dilute alkali
and aliquots of the alkaline solution are counted and the protein content determined. Again, there is no appreciable difference
based on protein content or cell number in conA binding.
Joe wrote me recently saying that some people in your lab have found a similar result and I assume that's what you refer
to in your letter.
I can imagine several ways to reconcile our results with the differential agglutinability. It seems to me one could imagine
that there are steric barriers in normal cells which prevent agglutination even though conA is bound; perhaps a divalent conA
molecule cannot cross link normal cells but can do so with transformed or trypsinized cells. Conceivably, the cell surface
of transformed cells undergoes an allosteric change following conA binding and this allosteric change makes the cells "sticky".
If normal cells don't undergo the allosteric change on conA binding, they might not become "sticky". A third possibility
is that the charge on the surface of normal cells prevents the agglutination even though conA is bound. If the charge on transformed
cells is altered (and there is evidence that this is so), there might be no charge repulsion to prevent agglutination. I am
sure there are other possible explanations, but at the moment I am inclined to believe that there is no such thing as cryptic
conA binding sites in normal cells.
All the best,
PS.: If we can make it to the Cancer Gordon Conference, we'll look forward to seeing you.