Cytosolic NADP'-dependent malic enzyme (ME) from human tumor cells was characterized in detail and compared to ME from normal human tissues produced in recombinant E. coli. Kinetic properties, size as seen in SDS gels, and HPLC elution profiles of tryptic digests of human 'normal cell' ME and NADP'-ME from tumor cells were identical. Thus, NADP'-ME found in tumor cells does not constitute a tumor-specific isoform as suggested by other studies but is identical to the 'housekeeping protein' predominantly expressed in human liver and white adipose tissue.

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Frog sartorius muscles were isolated, treated with 1-fluoro-2, 4-dini-trobenzene at 0 degrees C, then stimulated tetanically at the length in situ and stretched with a Levin-Wyman ergometer during stimulation. The normal adenosine triphosphate breakdown during the tetanus was reduced by about half during the forced stretch. The tension was increased by about 70 percent, but resynthesis of adenosine triphosphate did not occur. Thus, on the basis of A. V. Hill's results, adenosine triphosphate is probably not the direct final energy source for muscular contraction, although it intimately participates in the process. The use of adenosine triphosphate during negative work was less than one-tenth that needed for positive work.

Anomalous sedimentation patterns arise when free ribosomes from sea urchin eggs are centrifuged at high speeds. Pressure-induced dissociation of the ribosomes during sedimentation can explain the peculiar behavior; the assumption of such dissociation also yields estimates of the equilibrium constant (as a function of KCI concentration) and the change in molecular volume (500 i 100 ml/mol) in the reaction: subunits :± ribosome. Such dissociation during centrifugation may explain many experiments in which apparent reduced sedimentation coefficients for ribosomes, and increased coefficients for the subunits, have been ascribed to conformational changes.Anomalies in the sedimentation characteristics of ribosomes and subunits under various ionic conditions have been reported in many studies. These results have been generally interpreted in terms of conformational changes in the ribosomes and ribosomal subunits (1-7). However, a recent study (8) showed that the sedimentation pattern of free ribosomes from sea urchins is strongly dependent not only on the ionic conditions but also on the gravitational field and the concentration of ribosomes. When these factors are appropriately varied, the ribosomes display an apparently reduced sedimentation velocity or dissociate into subunits of apparently increased velocities. The results were interpreted in terms of (a) the dissociation of ribosomes at a rather distinct point during ultracentrifugation, and (b) the notion that the dissociation is due to a shift in the equilibrium between subunits and ribosomes in the sucrose gradient.The cause of this dissociation is suggested by the results of studies of protein dissociation, which have shown that the high pressure experienced by particles in the analytical ultracentrifuge can induce dissociation of multimers into subunits if thejnolecular volume decreases upon dissociation (for a general treatment of this subject, see refs. 9 and 10). Some of these ideas were formulated by Josephs and Harrington (11,12) arudwere used to explain the anomalous sedimentation of myosin. In this report, we examine the effect of hydrostatic pressure on the dissociation of ribosomes during zonal centrifugation in sucrose gradients. We conclude that pressure does induce the 75S free ribosomes to dissociate into their 56S and 35S subunits, and that this behavior can explain various peculiar sedimentation patterns of both eukaryotic and prokaryotic ribosomes that had previously been ascribed to conformational changes. MATERIALS AND METHODSUnfertilized eggs of the sea urchin, Strongylocentrotus purpuratus, were used in these studies, since the bulk (>95%) of the ribosomes from this source are not complexed with mRNA and peptidyl-tRNA (8). The procedures used to obtain cell-free homogenates are described in detail elsewhere (13,8). The eggs were homogenized in TM buffer containing 0.25 M sucrose and the indicated concentrations of KCl. (TM buffer contains 5 mM MgCl2-50 mM triethanolamine, pH 7.8.) A 15,000 X g supernatant (S15) was prepared and ana...