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.     

Effect of etmozin on thrombocyte aggregation capacity

It was shown in in vitro experiments that etmozin at a concentration of 100 micrograms/ml significantly suppressed (by 21%) platelet aggregation induced by ADP, but it had no effect on platelet aggregation induced by arachidonic acid. In in vivo experiments etmozin was found to cause a marked suppression of tendon collagen-induced platelet aggregation in the doses 2-5 mg/kg having antiarrhythmic activity. Under suppressed platelet aggregation induced by indomethacin, the prostaglandin biosynthesis blocker etmozin displayed no antiaggregation effect. It is suggested that etmozin effects on ADP release from platelets play the main role in the mechanism of its antiaggregation action.     

Inhibition of cell wall synthesis and acylation of the penicillin binding proteins during prolonged exposure of growing Streptococcus pneumoniae to benzylpenicillin

Growing cultures of an autolysis-defective pneumococcal mutant were exposed to [3H]benzylpenicillin at various multiples of the minimal inhibitory concentration and incubated until the growth of the cultures was halted. During the process of growth inhibition, we determined the rates and degree of acylation of the five penicillin-binding proteins (PBPs) and the rates of peptidoglycan incorporation, protein synthesis, and turbidity increase. The time required for the onset of the inhibitory effects of benzylpenicillin was inversely related to the concentration of the antibiotic, and inhibition of peptidoglycan incorporation always preceded inhibition of protein synthesis and growth. When cultures first started to show the onset of growth inhibition, the same characteristic fraction of each PBP was in the acylated form in all cases, irrespective of the antibiotic concentration. Apparently, saturation of one or more PBPs with the antibiotic beyond these threshold levels is needed to bring about interference with normal peptidoglycan production and cellular growth. Although it was not possible to correlate the inhibition of cell wall synthesis or cell growth with the degree of acylation (percentage saturation) of any single PBP, there was a correlation between the amount of peptidoglycan synthesized and the actual amount of PBP 2b that was not acylated. In cultures exposed to benzylpenicillin concentrations greater than eight times the minimal inhibitory concentration, the rates of peptidoglycan incorporation underwent a rapid decline when bacterial growth stopped. However, in cultures exposed to lower concentrations of benzylpenicillin (one to six times the minimal inhibitory concentration) peptidoglycan synthesis continued at constant rate for prolonged periods, after the turbidity had ceased to increase. We conclude that inhibition of bacterial growth does not require a complete inhibition or even a major decline in the rate of peptidoglycan incorporation. Rather, inhibition of growth must be caused by an as yet undefined process that stops cell division when the rate of incorporation of peptidoglycan (or synthesis of protein) falls below a critical value.     

Two-dimensional gel analysis of swine histocompatibility antigens

Miniature swine MHC antigens from three inbred herds were examined by two-dimensional gel electrophoresis. These antigens were found to constitute a series of complex glycoproteins displaying haplotype-specific patterns that allowed the distinction of both class I and class II molecules among the three haplotypes. Selected outbred pig antisera reacted with a subset of class I antigens, suggesting the presence of at least two distinct molecular species among these antigens. Similarly, alloantisera reacting with mouse Ia antigens and a monoclonal anti-human DR were shown to immunoprecipitate a subset of class II molecules. Examination of the cells from two recombinant haplotypes demonstrated that both independent recombinational events took place between the class I and class II genes.     

Genetic and morphological characterization of ftsB and nrdB mutants of Escherichia coli.

The ftsB gene of Escherichia coli is believed to be involved in cell division. In this report, we show that plasmids containing the nrdB gene could complement the ftsB mutation, suggesting that ftsB is an allele of nrdB. We compared changes in the cell shape of isogenic nrdA, nrdB, ftsB, and pbpB strains at permissive and restrictive temperatures. Although in rich medium all strains produced filaments at the restrictive temperature, in minimal medium only a 50 to 100% increase in mean cell mass occurred in the nrdA, nrdB, and ftsB strains. The typical pbpB cell division mutant also formed long filaments at low growth rates. Visualization of nucleoid structure by fluorescence microscopy demonstrated that nucleoid segregation was affected by nrdA, nrdB, and ftsB mutations at the restrictive temperature. Measurements of beta-galactosidase activity in lambda p(sfiA::lac) lysogenic nrdA, nrdB, and ftsB mutants in rich medium at the restrictive temperature showed that filamentation in the nrdA mutant was caused by sfiA (sulA) induction, while filamentation in nrdB and ftsB mutants was sfiA independent, suggesting an SOS-independent inhibition of cell division.