Purification and kinetic properties of phosphofructokinase from Rana ridibunda erythrocytes.

Phosphofructokinase (PFK) from Rana ridibunda erythrocytes was purified about 570-fold by column chromatography on Cibacron Blue Sepharose. The resulting enzyme preparation had a specific activity of 1.94 U/mg protein and a pH maximum of 7.6. The molecular weight as determined by HPLC chromatography was 330,000 Da. The S0.5 value for fructose-6-phosphate (F6P) was 5.6 mM and the Km for ATP 0.87 mM. The enzyme was sensitive to inhibition by ATP which was increased with lower F6P concentrations. At physiological levels of 2,3-diphosphoglycerate (0.35 mumol/ml RBC), 20% of PFK activity was inhibited. Significant activations under cellular conditions were exercised by AMP and, to a lesser extent, by Pi. Micromolar concentrations of fructose-2,6-bisphosphate and glucose-1,6-bisphosphate were also potent activators of the erythrocyte enzyme. Fructose-1,6-bisphosphate (10-50) microM activated the enzyme to a limited extent. With respect to these effects, it is suggested that PFK is a significant enzyme in regulating the glycolytic flux of Rana ridibunda red blood cells. The existence of a regulatory mechanism controlled by the energy status of the red cell, as well as the state of oxygenation of haemoglobin, is discussed, in which PFK occupies a central role.

Mechanisms responsible for the limited lifespan and immortal phenotypes in cultured mammalian cells.

Normal mammalian cells have a limited lifespan in culture and hypotheses explaining cellular senescence usually fall into one of two categories. One of these postulates that random errors or damage accumulate in essential macromolecules and eventually outstrip the cell's capacity for resynthesis and repair. The second considers the changes when immortal clones are produced from normal cells and in particular the lifespans of hybrids when cells of differing growth potentials are fused. These data can be explained by postulating that the mortal phenotype is dominant and that trans-acting growth inhibitors are involved in limiting lifespan. But the results do not indicate if the inhibitors are the primary cause of senescence or a secondary effect induced by quite different initial events. We suggest that normal cells possess proof-reading mechanisms which monitor the accuracy of chromosome segregation and replication and which can induce the synthesis of growth inhibitors when they detect major errors in chromosome metabolism. It is further postulated that random damage accumulates during the growth of normal cells and eventually leads to detectable chromosome changes and the synthesis of inhibitors. Our hypothesis predicts that the emergence of immortal clones will be linked to the absence of active inhibitors and therefore to a loss in the fidelity of chromosome metabolism. Data are quoted which show that in contrast to normal cells, immortal clones have highly irregular karyotypes, amplify segments of their chromosomes, integrate exogenous DNA efficiently, maintain a constant level of 5-methylcytosine residues and have high frequencies of chromosomal aberrations. The mechanism of the proof-reading is unknown, but it may monitor changes in the patterns by which chromosome domains are attached to the nuclear matrix.

Biochemical and physiological responses of Tetrahymena pyriformis during adaptation to high glycerol concentrations.

Tetrahymena pyriformis grown in high glycerol concentrations (0.6 and 0.8 M) have lower a growth rate than controls. There is no detectable difference in the electrophoretic pattern of unlabeled lysates of cells adapted to high glycerol concentrations in comparison with controls. On the contrary, the electrophoretic pattern of lysates of labeled Tetrahymena pyriformis revealed a set of proteins which are synthesized more vigorously in the glycerol adapted cells. Evidence is presented that some of these peptides are distributed in the mitochondrial-lysosomal fraction of glycerol adapted cells.

Physiological response of Tetrahymena pyriformis during adaptation to elevated external salinity.

Tetrahymena pyriformis exhibited a complex physiological and biochemical response during sudden exposure to high (200 mM) NaCl concentrations. The growth rate was reduced significantly. Uptake of precursor molecules and synthesis of macromolecules was reduced to 30% of control. Sudden exposure of Tetrahymena pyriformis to 200 mM NaCl resulted in increased proteolysis of already existing proteins and affected the electrophoretic pattern of the total proteins.