Mechanistic studies on carboxypeptidase a from goat pancreas — part II: Evidence for carboxyl group

Studies with substrate analogues and the pH optimum indicated the involvement of carboxyl group in the active site of goat carboxypeptidase A. Chemical modification of the enzyme with 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide methoI -p-toluene sulphonate, a carboxyl specific reagent, led to loss of both esterase and peptidase activities. Protection studies showed that this carboxyl group was in the active site and was protected by Βp-phenylpropionic acid and glycyl-L-tyrosine. Kinetic studies also confirmed the involvement of carboxylic group because the enzyme modification with water soluble carbodiimide was a two step reaction which excluded the possibility of tyrosine or lysine which are known to give a one step reaction with this reagent     

Interaction of protein kinase C with phosphatidylserine. 1. Cooperativity in lipid binding.

The basis for the apparent cooperativity in the activation of protein kinase C by phosphatidylserine has been addressed using proteolytic sensitivity, resonance energy transfer, and enzymatic activity. We show that binding of protein kinase C to detergent-lipid mixed micelles and model membranes is cooperatively regulated by phosphatidylserine. The sigmoidal dependence on phosphatidylserine for binding is indistinguishable from that observed for the activation of the kinase by this lipid [Newton & Koshland (1989) J. Biol. Chem. 264, 14909-14915]. Thus, protein kinase C activity is linearly related to the amount of phosphatidylserine bound. Furthermore, under conditions where protein kinase C is bound to micelles at all lipid concentrations, activation of the enzyme continues to display a sigmoidal dependence on the phosphatidylserine content of the micelle. This indicates that the apparent cooperativity in binding does not arise because protein kinase C senses a higher concentration of phosphatidylserine once recruited to the micelle. Our results reveal that the affinity of protein kinase C for phosphatidylserine increases as more of this lipid binds, supporting the hypothesis that a domain of phosphatidylserine is cooperatively sequestered around the enzyme.     

Chromosomal assignment of amplified genes in hydroxyurea-resistant hamster cells

We have shown previously that cDNAs for the M1 and M2 subunits of ribonucleotide reductase, ornithine decarboxylase (ODC), and p5-8, a 55,000-Dalton protein, hybridize to amplified genomic sequences in a highly hydroxyurea-resistant hamster cell line. We have extended these observations to include two additional, independently isolated, hydroxyurea-resistant cell lines: SC8, a single-step hamster ovary cell line, and KH450, a multistep human myeloid leukemic cell line, have also undergone genomic amplification for sequences homologous to ODC and p5-8 cDNAs. However, neither SC8 nor KH450 contains amplified genomic sequences homologous to an M1 cDNA probe. A panel of mouse-hamster somatic cell hybrids was used to map sequences homologous to M1, M2, ODC, and 5-8 cDNAs in the hamster genome. The M2, ODC, and p5-8 cDNAs hybridized to DNA fragments that segregated with hamster chromosome 7. In contrast, M1 cDNA hybridized to DNA fragments that segregated with hamster chromosome 3. These data suggest that the genes RRM2, (M2), ODC, and p5-8, but not RRMI (M1), are linked and may have been co-amplified in the selection of the hydroxyurea-resistant hamster and human cell lines.     

The enzymes of phospholipid synthesis in Clostridium butyricum

We have examined extracts of Clostridium butyricum for several enzymes of phospholipid synthesis. Membrane particles were shown to catalyze the formation of CDP-diglyceride from [3H]CTP and phosphatidic acid. The reaction was dependent on Mg2+ and stimulated by monovalent cations. CDP-diglyceride formed in vitro was found to be a substrate for both phosphatidylglycerophosphate synthetase and phosphatidylserine synthetase. The formation of phosphatidylglycerophosphate from added CDP-diglyceride and [U-14C]sn-glycerol-3-phosphate was dependent on Mg2+ and Triton X-100. The dephosphorylation of endogenously-generated phosphatidylglycerophosphate to yield phosphatidylglycerol was observed to be pH-dependent. The formation of phosphatidylserine from CDP-diglyceride and L-[3-14C]serine was stimulated by Mg2+ and Triton X-100. dCDP-diglyceride was a suitable substrate for both phosphatidylglycerophosphate synthetase and phosphatidylserine synthetase. Phosphatidylserine decarboxylase activity was barely detectable in membrane particles from C. butyricum. The addition of E. coli membrane particles provided efficient phosphatidylserine decarboxylase activity in this system. Although plasmalogens are the principal lipids of C. butyricum, none of the products of phospholipid synthesis formed in vitro contained measurable amounts of plasmalogens. The subcellular distribution of both phosphatidylglycerophosphate synthetase and phosphatidylserine synthetase in C. butyricum was also studied. Both were found to be membrane-associated.     

Changes in the vulnerable period of the rat myocardium during hypoxia, hyperventilation and heart failure

Changes in the duration and size of the vulnerable period of the myocardium in the presence of respiratory changes were studied in acute experiments on rats. The limits of the vulnerable period were determined by directly stimulating the heart during ventilation via the enlarged respiratory dead space, during hyperventilation and during heart failure. In the control group (normal ventilation without enlargement of the dead space), the vulnerable period lasted 5.7 +/- 0.76 ms. During ventilation via the enlarged dead space, hypercapnic hypoxaemia developed and the vulnerable period was markedly prolonged (18.55 +/- 5.29 ms) by a shift of its inner limit to the left. Hyperventilation caused normoxic to hyperoxic hypocapnia and markedly reduced the duration of the vulnerable period (8.17 +/- 2.21 and 9.31 +/- 2.38 ms respectively). The vulnerable period lengthened the most in heart failure (25.46 +/- 3.93), mainly as a result of a shift of its outer limit. In all the experimental groups there was a shift of the vulnerable period to the right, which was fastest in hypercapnic hypoxaemia and slowest in hyperoxic hypocapnia. The administration of Inderal (3 mg/kg i.p.) or Arfonad (50 mg/kg i.p.) markedly shortened the vulnerable period during hypercapnic hypoxaemia (9.87 +/- 2.78 and 9.32 +/- 2.16 ms respectively), but did not block the shift. Lengthening of the vulnerable period during hypercapnic hypoxaemia was probably due to activation of sympathetic nerves via beta-adrenergic receptors.