CTP regulates membrane-binding activity of the nucleoid occlusion protein Noc.

ATP- and GTP-dependent molecular switches are extensively used to control functions of proteins in a wide range of biological processes. However, CTP switches are rarely reported. Here, we report that a nucleoid occlusion protein Noc is a CTPase enzyme whose membrane-binding activity is directly regulated by a CTP switch. In Bacillus subtilis, Noc nucleates on 16 bp NBS sites before associating with neighboring non-specific DNA to form large membrane-associated nucleoprotein complexes to physically occlude assembly of the cell division machinery. By in vitro reconstitution, we show that (1) CTP is required for Noc to form the NBS-dependent nucleoprotein complex, and (2) CTP binding, but not hydrolysis, switches Noc to a membrane-active state. Overall, we suggest that CTP couples membrane-binding activity of Noc to nucleoprotein complex formation to ensure productive recruitment of DNA to the bacterial cell membrane for nucleoid occlusion activity.

CTP synthetase and its role in phospholipid synthesis in the yeast Saccharomyces cerevisiae.

CTP synthetase is a cytosolic-associated glutamine amidotransferase enzyme that catalyzes the ATP-dependent transfer of the amide nitrogen from glutamine to the C-4 position of UTP to form CTP. In the yeast Saccharomyces cerevisiae, the reaction product CTP is an essential precursor of all membrane phospholipids that are synthesized via the Kennedy (CDP-choline and CDP-ethanolamine branches) and CDP-diacylglycerol pathways. The URA7 and URA8 genes encode CTP synthetase in S. cerevisiae, and the URA7 gene is responsible for the majority of CTP synthesized in vivo. The CTP synthetase enzymes are allosterically regulated by CTP product inhibition. Mutations that alleviate this regulation result in an elevated cellular level of CTP and an increase in phospholipid synthesis via the Kennedy pathway. The URA7-encoded enzyme is phosphorylated by protein kinases A and C, and these phosphorylations stimulate CTP synthetase activity and increase cellular CTP levels and the utilization of the Kennedy pathway. The CTPS1 and CTPS2 genes that encode human CTP synthetase enzymes are functionally expressed in S. cerevisiae, and rescue the lethal phenotype of the ura7Deltaura8Delta double mutant that lacks CTP synthetase activity. The expression in yeast has revealed that the human CTPS1-encoded enzyme is also phosphorylated and regulated by protein kinases A and C.

Peak power output is maintained in rabbit psoas and rat soleus single muscle fibers when CTP replaces ATP.

The chemomechanical coupling mechanism in striated muscle contraction was examined by changing the nucleotide substrate from ATP to CTP. Maximum shortening velocity [extrapolation to zero force from force-velocity relation (Vmax) and slope of slack test plots (V0)], maximum isometric force (Po), power, and the curvature of the force-velocity curve [a/Po (dimensionless parameter inversely related to the curvature)] were determined during maximum Ca2+-activated isotonic contractions of fibers from fast rabbit psoas and slow rat soleus muscles by using 0.2 mM MgATP, 4 mM MgATP, 4 mM MgCTP, or 10 mM MgCTP as the nucleotide substrate. In addition to a decrease in the maximum Ca2+-activated force in both fiber types, a change from 4 mM ATP to 10 mM CTP resulted in a decrease in Vmax in psoas fibers from 3.26 to 1.87 muscle length/s. In soleus fibers, Vmax was reduced from 1.94 to 0.90 muscle length/s by this change in nucleotide. Surprisingly, peak power was unaffected in either fiber type by the change in nucleotide as the result of a three- to fourfold decrease in the curvature of the force-velocity relationship. The results are interpreted in terms of the Huxley model of muscle contraction as an increase in f1 and g1 coupled to a decrease in g2 (where f1 is the rate of cross-bridge attachment and g1 and g2 are rates of detachment) when CTP replaces ATP. This adequately accounts for the observed changes in Po, a/Po, and Vmax. However, the two-state Huxley model does not explicitly reveal the cross-bridge transitions that determine curvature of the force-velocity relationship. We hypothesize that a nucleotide-sensitive transition among strong-binding cross-bridge states following Pi release, but before the release of the nucleotide diphosphate, underlies the alterations in a/Po reported here.

Isolation and characterization of a DNA helicase from cytosolic extracts of calf thymus.

A DNA helicase has been isolated from calf thymus tissue. The enzyme was enriched from crude cytosolic extracts by batchwise chromatography on phosphocellulose, followed by 35% ammonium sulfate precipitation, and subsequent chromatography on phenyl-Sepharose, single-stranded DNA cellulose, and AcA 44 gel filtration. The DNA helicase had a Stokes' radius of about 45 A and a sedimentation coefficient of 4.3 S. The most purified fractions contained three polypeptides with apparent molecular weights of 110, 65, and 34 kDa. UV crosslinking with radioactive dATP stained all three major polypeptides. The helicase catalyzed the unwinding of a DNA primer from a single-stranded DNA template in an ATP- or dATP-dependent manner. DNA unwinding was also observed with CTP or dCTP, but with reduced efficiency. The helicase translocated from 3' to 5' on the single-stranded template it was bound to. Relationships between this DNA helicase and other calf thymus helicases will be discussed.

ATP-liganded form of aspartate transcarbamoylase, the logical regulatory target for allosteric control in divergent bacterial systems.

In Escherichia coli, the mechanism for regulatory control of aspartate transcarbamoylase is clear; CTP allosterically inhibits catalysis in direct competition with ATP. However, both CTP and ATP may be activators or may have no effect on aspartate transcarbamoylases from other enteric bacteria. A common regulatory logic observed was that the ATP-activated enzymes were rendered less active as the result of competition with CTP, regardless of the independent effects.

Multistep regulation of Leydig cell function.

LH controls Leydig cell steroidogenesis by interaction with specific membrane receptors initiating membrane coupling events. Stimulation of the androgen pathways occurs mainly through cAMP mediated mechanism including LH induced guanyl nucleotide binding, membrane phosphorylation and adenylate cyclase activation. cAMP dependent kinase activation presumably causes phosphorylation of key proteins of the steroidogenic pathway and consequent increase in testosterone production. The hormone also appears to facilitate the androgen stimulus by a cyclic AMP independent mechanism located at the plasma membrane or intracellular sites. The stimulatory event can be negatively influenced by the action of certain peptide hormones (i.e. angiotensin II) through the guanyl nucleotide inhibitory subunit of adenylate cyclase (Gi). In recent studies we have presented evidence for a Ca2+ sensitive kinase system present in purified cell membranes. Gpp(NH)p, GTP, and phospholipid in presence of nanomolar Ca2+ induce phosphate incorporation into Mr 44,500 substrate with marked inhibition at microM Ca2+. Similarly a biphasic pattern of activation was observed with adenylate cyclase activity. Membrane phosphorylation may be a modifier of LH-stimulated adenylate cyclase activity and possibly other LH induced actions in the activated Leydig cell membrane. Furthermore we have defined the stimulatory effects of forskolin on all Leydig cell cyclic AMP pools and have provided additional evidence of functional compartmentalization and/or cAMP independent facilitory stimulus of steroidogenesis by the trophic hormone. The demonstration of a novel high affinity inhibitory action of forskolin upon adenylate cyclase activity and cyclic AMP generation mediated by the Gi subunit of adenylate cyclase has provided a new approach for direct evaluation of functional inhibitory influence of Gi subunit in the Leydig cell. The cultured fetal Leydig cell system has provided a useful model to elucidate mechanisms involved in the development of gonadotropin induced estradiol mediated desensitization of steroidogenesis. We have isolated from the fetal testis a small population (2-5% of total) of transitional cells with morphological characteristics of cells found in 15 day postnatal testis but functional capabilities of the adult cell. We have also demonstrated after appropriate treatment (i.e. estrogen, and frequent or a high gonadotropin dose) the emergence of a functional adult-like cell type from the fetal Leydig cell population.

Inhibition of dolichyl phosphate biosynthesis by compactin in cultured rat hepatocytes.

Isolated rat hepatocytes were cultured in monolayer for about 24 h. During this period the cells exhibited constant protein and lipid synthesis. When the culture medium contained compactin, a competitive inhibitor of the 3-hydroxyl-3-methylglutary-coenzyme-A reductase, dolichyl-P synthesis was inhibited by 91% at the end of the incubation, as estimated by the incorporation of [3H]acetate and by 77% as estimated by the incorporation of 32Pi. These results indicate that in primary cultures of rat hepatocytes dolichyl monophosphate is mainly synthesized through a de novo process, while phosphorylation through the CTP-mediated kinase is of limited functional importance.

[3H]Ouabain binding and Na+, K+-ATPase in resealed human red cell ghosts.

The interaction of the cardiac glycoside [3H]ouabain with the Na+, K+ pump of resealed human erythrocyte ghosts was investigated. Binding of [3H]ouabain to high intracellular Na+ ghosts was studied in high extracellular Na+ media, a condition determined to produce maximal ouabain binding rates. Simultaneous examination of both the number of ouabain molecules bound per ghost and the corresponding inhibition of the Na+, K+-ATPase revealed that one molecule of [3H]ouabain inhibited one Na+, K+-ATPase complex. Intracellular magnesium or magnesium plus inorganic phosphate produced the lowest ouabain binding rate. Support of ouabain binding by adenosine diphosphate (ADP) was negligible, provided synthesis of adenosine triphosphate (ATP) through the residual adenylate kinase activity was prevented by the adenylate kinase inhibitor Ap5A. Uridine 5'-triphosphate (UTP) alone did not support ouabain binding after inhibition of the endogenous nucleoside diphosphokinase by trypan blue and depletion of residual ATP by the incorporation of hexokinase and glucose. ATP acting solely at the high-affinity binding site of the Na+, K+ pump (Km approximately 1 microM) promoted maximal [3H]ouabain binding rates. Failure of 5'-adenylyl-beta-gamma-imidophosphate (AMP-PNP) to stimulate significantly the rate of ouabain binding suggests that phosphorylation of the pump was required to expose the ouabain receptor.

The influence of divalent cations and substrate concentration on the incorporation of myo-inositol into phospholipids of isolated bovine oligodendrocytes.

The incorporation of myo-inositol into phosphatidylinositol by two routes (CTP-independent and CTP-independent) has been investigated in homogenates prepared from isolated bovine oligodendrocyte perikarya. The CTP-dependent route has the higher maximum velocity of inositol incorporation and can utilise either Mn2+ or Mg2+ as a divalent ion cofactor. This route of inositol incorporation is also strongly inhibited by Ca2+ ions at concentrations less than 1 mM. The primary site of the inhibitory action appears to be the enzyme CDP-diglyceride inositol phosphatidyl transferase (EC 2.7.8.11) though synthesis of CDP-diacylglycerol is also inhibited by endogenous Ca2+ present in the oligodendrocyte homogenate. In contrast, CTP-independent inositol incorporation into phosphatidylinositol is only stimulated by Mn2+ (Zn2+,Cu2+, Mg2+, Ca2+ and Co2+ are ineffective) and is not inhibited by Ca2+, at least up to a concentration of 1 mM.

Effects of nucleoside triphosphates on choleragen-activated brain adenylate cyclase.

To investigate the effects of nucleoside triphosphates on the activation of adenylate cyclase by choleragen and on the stability and catalytic function of the choleragen-activated enzyme, we treated samples of particulate preparation from bovine brain successively in three separate incubations with extensive washing between each step. In incubation I, choleragen and NAD were pesent to activte the adenylate cyclase. In incubation II, conditions were varied to assess enzyme stability. Finally, adenylate cyclase activity was assayed with ATP or adenylyl imidodiphosphate [App-(NH)p] as the substrate. Even when assays contained an optimal concentration of GTP, nucleoside triphosphate (plus a regenerating system) was required in incubation I for maximal choleragen activation; in order of effectiveness, GTP > ITP > ATP greater than or equal to CTP = UTP. During incubation II (at 30 degrees C), activity of the choleragen-treated fractions was essentially completely stable when 100 microM GTP (plus a regenerating system) was present. ITP and ATP were less effective. Activation produced by guanylyl imidodiphosphate was more stable than that resulting from choleragen, GTP, and NAD. After activation of membranes with choleragen, NAD, and GTP, nucleoside triphosphate plus a regenerating system (but not NAD or additional choleragen) was essential for expression of maximal activity. In order of effectiveness, GTP > ITP > ATP greater than or equal to CTP = UTP. It appears that GTP, which was effective in micromolar concentrations, plays an important role not only in the activation of adenylate cyclase by choleragen but also in the stabilization and expression of the catalytic function of the activated enzyme.