Inhibition of cholesterol biosynthesis by fluorinated mevalonate analogues

The conversion of mevalonate to cholesterol in rat liver homogenates (IC50 = 0.01-1.0 mM) is inhibited by 6- (I), 6,6-di- (II), and 6,6,6-trifluoromevalonate (III), as well as 4,4-difluoromevalonate (IV). Addition of compound I, III, or IV to rat liver homogenates results in the accumulation of 5-phospho- and 5-pyrophosphomevalonate. The conversion of isopentenyl pyrophosphate to cholesterol is not inhibited by the fluorinated analogues. It thus appears likely that the decarboxylation of mevalonate 5-pyrophosphate is inhibited. Rat liver homogenates catalyze the phosphorylation of I and III. The inhibition of the decarboxylation of mevalonate 5-pyrophosphate by I and III was demonstrated directly with partially purified decarboxylase. Compound I is a remarkably effective inhibitor of the decarboxylation (Ki = 10 nM). Similar results were reported by Nave et al. [Nave, J. F., d'Orchymont, H., Ducep, J. B., Piriou F., & Jung, M. J. (1985) Biochem. J. 227, 247]. It is likely that the phosphorylated or pyrophosphorylated forms of all inhibitors tested are responsible for inhibition. We also describe a chemical method for the synthesis of mevalonate 5-pyrophosphate.     

Hormonal Regulation, and Intracellular Localization of a 33-kD Cationic Peroxidase in Excised Cucumber Cotyledons

Ethylene enhanced chlorosis and levels of 33-kilodalton cationic peroxidase (33-CPO) in excised cucumber (Cucumis sativus L. cv `Poinsett 76') cotyledons. Compared to other hormones, such as kinetin, indoleacetic acid, gibberellic acid, and abscisic acid, ethylene was the only effective promoter of 33-CPO synthesis. The hypothesis that peroxidase plays a role in chlorophyll degradation was tested by comparing levels of 33-CPO in cotyledons treated with compounds thought to either retard (kinetin, indoleacetic acid and gibberellic acid), or promote (abscisic acid and methyl jasmonate [MJ]) senescence. It was concluded that 33-CPO did not play a role in senescence since no direct correlation between chlorophyll content and 33-CPO was observed. MJ was as effective as ethylene in inducing senescence. However, ethylene did not appear to be involved in the action of MJ. Using immunocytochemistry, 33-CPO was found to be located primarily around starch grains and near the plasmalemma. High levels of 33-CPO were also found in cells destined to be vascular tissue.     

Substrate specificity of nicotinamide methyltransferase isolated from porcine liver

Nicotinamide methyltransferase (EC 2.1.1.1) has been purified over 1300-fold from porcine liver. The enzyme is electrophoretically homogeneous, exhibiting a relative molecular mass of 27,000. In addition to acting on nicotinamide and close structural analogs such as thionicotinamide and 3-acetylpyridine, the enzyme actively accommodates poor analogs such as quinoline, isoquinoline, and 1,2,3,4-tetrahydroisoquinoline as methyl group acceptors. The enzyme may thus have the function of detoxicating numerous alkaloids in vivo. In some cases, the action of the enzyme might paradoxically increase the toxicities of substrates, but the hepatotoxic antibiotic pyrazinamide, which we considered as potentially such an enzyme-activated electrophile, did not function detectably as a substrate for the isolated enzyme.     

Neplanocin A. Actions on S-adenosylhomocysteine hydrolase and on hormone synthesis by GH4C1 cells

We have investigated the biochemical actions of Neplanocin A (Nepl A), a carbocyclic adenosine analog, on purified calf liver S-adenosylhomocysteine hydrolase and in the GH4C1 strain of functional rat pituitary cells. Addition of 1 mol of Nepl A/2 mol of S-adenosylhomocysteine hydrolase subunit led to rapid and complete inactivation. Concomitant with inactivation, half of the enzyme-bound NAD was reduced and adenine was released stoichiometrically from Nepl A. In GH4C1 cells Nepl A caused a dose-dependent rapid (within 5 min) and irreversible inactivation of S-adenosylhomocysteine hydrolase and concomitant increase in intracellular S-adenosylhomocysteine. In cells treated with Nepl A for 4-5 days, methylation of DNA cytosine was depressed approximately 50%, and the level of cytoplasmic prolactin mRNA was elevated 2-fold. While acute (30 min) release of prolactin from intracellular stores was unaffected, Nepl A acted in a dose- and time-dependent manner to increase the production of both prolactin and growth hormone, the two hormones synthesized and secreted by GH4C1 cells. The lowest effective dose was 0.12 microM, the concentration required to decrease S-adenosylhomocysteine hydrolase activity by 50%. By 4-7 days the production of both hormones in Nepl A-treated cells was increased 2-3 times above control. The action on hormone production persisted for at least 7 days after removal of Nepl A from the culture medium. We conclude that Nepl A inhibits S-adenosylhomocysteine hydrolase, raises cellular S-adenosylhomocysteine, decreases bulk DNA methylation, and increases hormone synthesis in GH4C1 cells.     

Incorporation of beta-fluoroasparagine into peptides prevents N-linked glycosylation. In vitro studies with synthetic fluoropeptides

Previously, we reported that incorporation of threo-beta-fluoroasparagine into cellular protein inhibits N-linked glycosylation. We now show that short synthetic peptides which contain N-acetyl-threo-beta-fluoroasparagine fail to undergo glycosylation in a cell-free system except at extremely high substrate concentrations. An N-benzoyl-threo-beta-fluoroasparagine-containing peptide has a 100-fold lower Vmax/Km than the analogous N-benzoyl-asparagine-containing peptide. Substitution of a fluorine for a hydrogen on the beta-carbon of asparagine weakens the ability of the peptide to bind the oligosaccharyltransferase. A 100-fold excess of acetyl-threo-beta-fluoroasparaginyl-leucyl-threonine methylamide over acetyl-asparaginyl-leucyl-threonine methylamide inhibited glycosylation of the latter peptide by less than 10%. Both threo-beta-fluoroasparagine and erythro-beta-fluoroasparagine-containing peptides are glycosylated at the same rate. Glycofluoropeptides generated from beta-fluoroasparagine-containing peptides were N-glycosylated. These cell-free studies with synthetic fluoropeptides suggest that incorporation of beta-fluoroasparagine into cellular protein inhibits N-linked glycosylation by rendering protein substrates ineffective for glycosylation. In the course of this work, we also demonstrate that the N-linked glycosylating enzyme acts only on L-asparagine-containing peptides and not on D-asparagine peptides.     

Mode of interaction of beta-hydroxy-beta-methylglutaryl coenzyme A reductase with strong binding inhibitors: compactin and related compounds

The sodium salts of compactin (1) and trans-6-[2-(2,4- dichloro-6-hydroxyphenyl)ethyl]-3,4,5,6-tetrahydro-4-hydroxy-2H-pyran- 2-one (3) are inhibitors of yeast beta-hydroxy-beta-methylglutaryl coenzyme A (HMG-CoA) reductase. The dissociation constants are 0.24 X 10(-9) and 0.28 X 10(-9) M, respectively. Similar values have been reported for HMG-CoA reductase from mammalian sources [Endo, A., Kuroda, M., & Tanzawa, K. (1976) FEBS Lett. 72, 323; Alberts, A. W., et al. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 3957]. The structures of these compounds marginally resemble that of any substrates of HMG-CoA reductase. We, therefore, investigated the basis for the strong interaction between HMG-CoA reductase and these inhibitors. HMG-CoA and coenzyme A (CoASH), but not reduced nicotinamide adenine dinucleotide phosphate (NADPH), prevent binding of compactin to the enzyme. HMG-CoA, but not CoASH or NADPH, prevents binding of 3 to the enzyme. We also investigated the inhibitory activity of molecules that resemble structural components of compactin. Compactin consists of a moiety resembling 3,5-dihydroxyvaleric acid that is attached to a decalin structure. The sodium salt of DL-3,5-dihydroxyvaleric acid inhibits HMG-CoA reductase competitively with respect to HMG-CoA and noncompetitively with respect to NADPH. The dissociation constant for DL-3,5-dihydroxyvaleric acid, derived from protection against inactivation of enzyme by iodoacetic acid, is (2.1 +/- 0.9) X 10(-2) M. Two decalin derivatives (structurally identical with or closely related to the decalin moiety of compactin) showed no detectable inhibition. If the lack of inhibition is due to their limited solubility, the dissociation constant of these decalin derivatives may be conservatively estimated to be greater than or equal to 0.5 mM. Simultaneous addition of decalin derivatives and DL-3,5-dihydroxyvaleric acid does not lead to enhanced inhibition. The sodium salt of (E)-6-[2-(2-methoxy-1-naphthalenyl)ethenyl]-3,4,5,6- tetrahydro-4-hydroxy-2H-pyran-2-one (6) inhibits HMG-CoA reductase competitively with respect to HMG-CoA and noncompetitively with respect to NADPH. The inhibition constant (vs. HMG-CoA) is 0.8 microM. CoASH does not prevent binding of 6 to enzyme. Compound 6, therefore, behaves analogously to compound 3. We propose that these inhibitors occupy two sites on the enzyme: one site is the hydroxymethylglutaryl binding domain of the enzyme active site and the other site is a hydrophobic pocket located adjacent to the active site.(ABSTRACT TRUNCATED AT 400 WORDS)     

Partition of unit-copy miniplasmids to daughter cells. I. P1 and F miniplasmids contain discrete, interchangeable sequences sufficient to promote equipartition

Hybrids formed by insertion of the plasmid maintenance regions of P1 or F into a lambda delta att vector form stable unit-copy plasmids in their Escherichia coli host. They must therefore both be substrates for an accurate cellular partition apparatus that ensures that all daughter cells inherit a plasmid copy. Analysis of deletion mutants of both types of hybrid showed that, although the P1 and F plasmid maintenance regions differ in sequence and specificity, they are similar in general organization. Each contains an approximately 3 X 10(3) base-pair region that is essential for replication (rep) and an adjacent but separable 3 X 10(3) base-pair region that is essential for the stability of plasmid maintenance (par). Each par region is thought to specify the recognition of the plasmid as a substrate for equipartition. The deletion mutants provide sources of isolated rep and par sequences from both P1 and F DNA. These elements were then used to construct composite plasmids with novel combinations and arrangements of rep and par sequences. Heterologous constructions containing P1 rep and F par or F rep and P1 par sequences were maintained faithfully. We conclude that par regions are both necessary and sufficient to promote equipartition of replicating plasmid DNA. This activity is exerted only in cis but otherwise seems to be independent of the position or orientation of the par sequences within the DNA. Both P1 and F par regions include DNA sequences (incB of P1, incD of F) that we propose are analogues of the centromeres of eukaryotic chromosomes. The remaining portions of the par regions are known to encode protein products that, we believe, act at the inc sites. Extra copies of these inc sites appear to exert incompatibility by competition for the cellular partition apparatus.     

Stimulation of lettuce seed germination by ethylene

Ethylene increased the germination of freshly imbibed lettuce (Lactuca sativa L. var. Grand Rapids) seeds. Seeds receiving either red or far-red light or darkness all showed a positive response to the gas. However, ethylene was apparently without effect on dormant seeds, those which failed to germinate after an initial red or far-red treatment. Carbon dioxide, which often acts as a competitive inhibitor of ethylene, failed to clearly reverse ethylene-enhanced seed germination. While light doubled ethylene production from the lettuce seeds, its effect was not mediated by the phytochrome system since both red and far-red light had a similar effect.     

Mechanism of Action of Abscission Accelerators

Abscission zone explants of Gossypium hirsutum L., Cassia fistula L., and Coleus blumei Benth. were used to investigate correlations between endogenous rates of ethylene evolution and time of abscission. Additions of 0.1 nl/ml ethylene to the explants markedly accelerated abscission; continuous aeration of the explants, to prevent accumulation of small amounts of endogenously produced ethylene, inhibited abscission compared with that of sealed controls. Substances that stimulated abscission simultaneously accelerated ethylene evolution on all three species and at any position of application. The positional effects of auxin are explained as being due to differences in transport in the explant. Thus, distally applied auxin inhibits abscission, regardless of the accelerated rate of ethylene evolution, by being rapidly transported to the abscission zone. Auxin applied proximally stimulates abscission because it is unable to move as rapidly to the abscission zone and the ethylene effect becomes dominant. Ethylene was found to be most effective on aged tissues, and it is concluded that abscission rates are determined by an increase in sensitivity of the tissue to the ethylene that is already being produced.     

A protein that binds to the P1 origin core and the oriC 13mer region in a methylation-specific fashion is the product of the host seqA gene.

The P1 plasmid replication origin P1oriR is controlled by methylation of four GATC adenine methylation sites within heptamer repeats. A comparable (13mer) region is present in the host origin, oriC. The two origins show comparable responses to methylation; negative control by recognition of hemimethylated DNA (sequestration) and a positive requirement for methylation for efficient function. We have isolated a host protein that recognizes the P1 origin region only when it is isolated from a strain proficient for adenine methylation. The substantially purified 22 kDa protein also binds to the 13mer region of oriC in a methylation-specific fashion. It proved to be the product of the seqA gene that acts in the negative control of oriC by sequestration. We conclude that the role of the SeqA protein in sequestration is to recognize the methylation state of P1oriR and oriC by direct DNA binding. Using synthetic substrates we show that SeqA binds exclusively to the hemimethylated forms of these origins forms that are the immediate products of replication in a methylation-proficient strain. We also show that the protein can recognize sequences with multiple GATC sites, irrespective of the surrounding sequence. The basis for origin specificity is primarily the persistence of hemimethylated forms that are over-represented in the natural. DNA preparations relative to controls.