Regulation of ATP synthesis catalyzed by the calcium pump of sarcoplasmic reticulum

The role of pH, KCl, ATP, water activity, and temperature in ATP synthesis from ADP and Pi was investigated in sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle. In totally aqueous medium, the synthesis of ATP was inhibited by ATP, KCl, and pH values above 6.5. When the water activity of the medium was decreased by the addition of 30% (v/v) dimethyl sulfoxide, the synthesis of ATP was no longer inhibited by ATP; it was activated by KCl and the optimum pH changed from 6.5 to 7.5. In totally aqueous medium, the concentration of MgCl2 needed for half-maximal synthesis of ATP was found to vary with the temperature of the assay medium; at 35 degrees C it was 1 mM and increased to a value higher than 10 mM when the temperature was decreased to 15 degrees C. In the presence of 30% dimethyl sulfoxide, maximal synthesis of ATP was attained in presence of 0.05 mM MgCl2 at both 15 and 35 degrees C. The hypothesis is raised that in the living cell water structure may play a role in regulating the synthesis of ATP observed during the reversal of the Ca2+ pump of the sarcoplasmic reticulum.     

Studies on functional domains of the regulatory subunit of bovine heart adenosine 3':5'-monophosphate-dependent protein kinase

The functional domains of the regulatory subunit of isozyme II of cAMP-dependent protein kinase were studied. It was shown using Edman degradation that the regulatory subunit contained a phosphorylated residue which was very close in primary sequence to the site most sensitive to hydrolysis by low trypsin concentrations as postulated previously (Corbin, J.D., Sugden, P.H., West, L., Flockhart, D.A., Lincoln, T.M., and McCarthy, D. (1978) J. Biol. Chem. 253, 3997-4003). Catalytic subunit incorporated 0.9 mol of 32P from [gamma-32P]ATP into a preparation of regulatory subunit that contained 1.1 mol of endogenous phosphate. After phosphorylation by the catalytic subunit, the regulatory subunit contained 2.2 mol of chemical phosphate. The effects of heat denaturation upon the rate and extent of phosphorylation of the regulatory subunit were compared with the effects of these treatments upon the cAMP binding and inhibitory domains. These data suggested that the regulatory subunit required factors in addition to an intact phosphorylatable primary sequence in order for inhibitory activity to be expressed. Such factors might be part of the secondary or tertiary structure of the protein. These studies are discussed with respect to the mechanism of inhibition of catalytic activity, and a model of the regulatory subunit structure is proposed.     

The effects of insulin on choline kinase activity in cultured rat liver cells

The effect of insulin on phosphatidylcholine biosynthesis in cultured rat liver cells was assessed by measuring changes in the activity of the first enzyme in the choline pathway of phosphatidylcholine biosynthesis, choline kinase (ATP: cholinephosphortransferase, EC 2.7.1.32), in the presence or absence of the hormone. Choline kinase specific activity in liver cells incubated for 18 hours in the presence of 10^?7M insulin increased two-fold from 3.4 ± 0.3 nmoles phosphorylcholine formed/min/mg protein to 7.5 ± 0.6 nmoles/min/mg protein. This effect was dose dependent and reversed by the addition of actinomycin D and cycloheximide. It is concluded that the increase in specific activity is due to synthesis of new enzyme rather than activation of existing enzyme.     

Identification of the subunits of F1F0-ATPase from bovine heart mitochondria.

An oligomycin-sensitive F1F0-ATPase isolated from bovine heart mitochondria has been reconstituted into phospholipid vesicles and pumps protons. this preparation of F1F0-ATPase contains 14 different polypeptides that are resolved by polyacrylamide gel electrophoresis under denaturing conditions, and so it is more complex than bacterial and chloroplast enzymes, which have eight or nine different subunits. The 14 bovine subunits have been characterized by protein sequence analysis. They have been fractionated on polyacrylamide gels and transferred to poly(vinylidene difluoride) membranes, and N-terminal sequences have been determined in nine of them. By comparison with known sequences, eight of these have been identified as subunits beta, gamma, delta, and epsilon, which together with the alpha subunit form the F1 domain, as the b and c (or DCCD-reactive) subunits, both components of the membrane sector of the enzyme, and as the oligomycin sensitivity conferral protein (OSCP) and factor 6 (F6), both of which are required for attachment of F1 to the membrane sector. The sequence of the ninth, named subunit e, has been determined and is not related to any reported protein sequence. The N-terminal sequence of a tenth subunit, the membrane component A6L, could be determined after a mild acid treatment to remove an alpha-N-formyl group. Similar experiments with another membrane component, the a or ATPase-6 subunit, caused the protein to degrade, but the protein has been isolated from the enzyme complex and its position on gels has been unambiguously assigned. No N-terminal sequence could be derived from three other proteins. The largest of these is the alpha subunit, which previously has been shown to have pyrrolidonecarboxylic acid at the N terminus of the majority of its chains. The other two have been isolated from the enzyme complex; one of them is the membrane-associated protein, subunit d, which has an alpha-N-acetyl group, and the second, surprisingly, is the ATPase inhibitor protein. When it is isolated directly from mitochondrial membranes, the inhibitor protein has a frayed N terminus, with chains starting at residues 1, 2, and 3, but when it is isolated from the purified enzyme complex, its chains are not frayed and the N terminus is modified. Previously, the sequences at the N terminals of the alpha, beta, and delta subunits isolated from F1-ATPase had been shown to be frayed also, but in the F1F0 complex they each have unique N-terminal sequences.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

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.     

Basal and HCG-stimulated testosterone production by interstitial cells of the Mongolian gerbil (Meriones unguiculatus)--I. Influence of preincubation length, medium composition and temperature

In the absence of HCG, production of testosterone by whole testes superfused in vitro was quite constant during the 5-hr superfusion period. Addition of 23-184 mIU/ml HCG caused a significant increase of testosterone production which was apparent from 30 min after start of superfusion. Basal and HCG-stimulated testosterone production by whole testes was significantly higher (400, 1950 ng/testis/5 hr, without and with 100 mIU HCG) than by isolated cells (200, 1350 ng/testis/5 hr). Incubation of isolated interstitial cells in medium 199 supplemented with fetal calf serum (FCS), (N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid, HEPES) and 3-isobutyl-methylxanthine (MIX), and in medium 199 without FCS, HEPES or MIX, gave similar testosterone responses. While centrifugation at 8000 g for 2 min drastically diminished testosterone formation by isolated interstitial cells, production was similar by cells incubated in either 0.5, 1.0 or 1.5 ml medium. A significant decrease of testosterone synthesis by isolated interstitial cells was found when cells were stored at 4 degrees C for 2 days and then were incubated at 35 degrees C for 6 hr without or with 1-1000 microIU HCG. While isolated interstitial cells incubated at 5 degrees C did not produce testosterone at all, testosterone production increased to 49.5 +/- 3.9 ng/10(5) cells (30 degrees C) and 24.1 +/- 1.1 ng/10(5) cells (40 degrees C), respectively. HCG-stimulated testosterone production was maximal when interstitial cells were incubated at 34 degrees C.