High-resolution two-dimensional polyacrylamide gel electrophoresis shows that at-least halfof 370 denatured polypeptides from hamster cells and human cells are indistinguishable in terms of isoelectric points and molecular weights. Molecular evolution may have been more conservative for this set of proteins than sequence studies on soluble proteins have implied. This may be a consequence of complexities of intracellular organization and the numerous macromolecular interactions in which most polypeptides, participate. It is suggested that the term "quinary structure" be used to refer to macromolecular interactions that are transient in vivou Such interactions will not be evident from the composition ofpurified proteins, but they may constitute an important source of constraints on changes in primary structure. (11)(12)(13). Surprisingly, all agree that the apparent frequency ofpolymorphisms is much less than older surveys based on enzymic activity had implied (14, 15).Obviously, it is also possible to use O'Farrell gels to compare the polypeptides made by different organisms. I shall show below that this yields results consistent with the studies on intrapopulation variation by the same technique-i.e., the similarities between distantly related mammals are remarkably large. I propose that this evolutionary conservatism can be understood as a consequence of intracellular organization, which involves the majority ofcellular polypeptides in extensive macromolecular interactions. RESULTS AND DISCUSSIONA convenient way to make pair-wise comparisons of complex protein mixtures is to combine a 3H-labeled preparation from cultured cells of one type with a "GC-labeled preparation from cells of a different type, examine the mixture of polypeptides by two-dimensional electrophoresis, and compare the pattern of 3H-labeled polypeptides with the "GC-labeled polypeptide pattern, by using double-label autoradiography (16). This process involves the preparation of two autoradiograms from the same gel; one is a fluorogram-which records both isotopes-and the other is a directautoradiogram-made on film that records only the "'C beta particles. A contact negative ofthe fluorogram is made and, when the "'C autoradiogram is placed on top of the negative, polypeptides that are labeled exclusively with 3H appear as white spots, whereas polypeptides that are labeled with 3H and 14C, or 14C alone, appear as black spots or spots with white halos. The method permits rapid visual identification of differences in complex patterns. Only the more abundant polypeptides have been surveyed in the examples presented here. The possibility that a study of rarer polypeptides would give quantitatively different results cannot be excluded. Fig. 1 shows such a comparison of Chinese hamster ovary (CHO) cell proteins with HeLa (human cervical carcinoma) cell proteins. Polypeptides that occur only in the human cells appear as white spots in A; polypeptides that occur only in the hamster cells appear as white spots in B; polypeptides that are common...

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The effects of fresh medium and serum on protein synthesis in suspension-cultured HeLa cells after growth to high cell density (> 5 x 10(5) cells/ml) were studied. Cells which were resuspended in fresh medium plus serum and grown for 24 hours (control) were compared with cells grown for 2 hours after resuspension (stimulated). The spectrum of proteins being synthesized by control and stimulated cells does not appear to be grossly different; that is, the weight and number average molecular weights of newly synthesized whole-cell protein are about the same in both cultures. Also, no significant differences were observed in the number of ribosomes per polysome or in the fraction of total ribosomes in polysomes. However, the transit times (combined elongation and termination times) were found to differ significantly; the average transit time for control cells was 2.24 minutes, while the average transit time for stimulated cells was 1.26 minutes. (An appendex evaluating the methodology involved in measuring the transit time is included.) In aggreement with the difference in transit time, the absolute rate of protein synthesis in stimulated cells was approximately 1.8 times the rate measured in control cells. These data are taken as evidence that under certain conditions, the rate of elongation and/or termination of polypeptide chains limits the overall rate of translation, and that cells can respond to growth conditions by changing the elongation and/or termination rate of protein synthesis.

The numbering systems for mammalian ribosomal proteins used in several laboratories have been correlated and a proposal for a standard system is presented.

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