Alternative reactions of ouabain with a Na+ and K+ ATPase

Alternative reactions of ouabain with Na⁺ + K⁺ ATPase are described which may be interpreted by assuming that a conformational change takes place. Each conformational form appears to be dependent upon the cationic environment. The reaction of ouabain with one form inhibits the dephosphorylation step and inhibits the binding of ATP when it reacts with another conformational form.     

Inhibition by Thiopeptin of Bacterial Protein Synthesis

Thiopeptin, a sulfur-containing antibiotic, was found to inhibit protein synthesis in a bacterial ribosomal system. The pretreatment of ribosomal subunits with the antibiotic revealed that thiopeptin may act on the 50 S ribosomal subunit. The elongation of peptide chain on the ribosome is more profoundly blocked by the antibiotic than the initiation of protein synthesis. It was demonstrated that thiopeptin inhibits elongation factor (EF)-Tu-dependent GTP hydrolysis and binding of aminoacyl-tRNA to the ribosome. The peptidyl transferase-catalyzed puromycin reaction is not significantly affected by the antibiotic. Thiopeptin inhibits EF-G-associated GTPase reaction, and translocation of peptidyl-tRNA and mRNA from the acceptor site to the donor site. Protein synthesis in ribosomal systems, obtained from rat liver and rabbit reticulocytes are insensitive to the antibiotic.     

Effect of Inhibition of Macromolecule Synthesis on the Association of Bacteriophage T4 DNA with Membrane

The "Mg(2+)-Sarkosyl crystals" (M band) technique distinguishes between membrane-bound and free intracellular DNA. This procedure was employed to investigate the nature of the reactions necessary to convert input T4 DNA to a rapidly sedimenting form. Energy poisoning inhibits this attachment reaction. Neither protein nor DNA synthesis appears to be required, but experiments with rifampin and extensively irradiated T4 suggest that RNA synthesis is involved. These results were confirmed by a second procedure for the determination of rapidly sedimenting DNA.     

The in vitro simulation of IAA-induced modification of Avena cell wall polysaccharides by an exo-glucanase

Treatment of Avena coloeptile segments with auxin promotes adecrease in the noncellulosic glucose component of the cellwalls. The decrease in glucan may be simulated in these cellwalls in vitro by an exo-glucanase which produces glucose directlyas the sole reaction product. These results also reveal thatglucans of Avena cell wall polysaccharides are readily susceptibleto exo-enzyme attack. Nojirimycin, a potent inhibitor of ß-glucosidasesand exo-glucanases, inhibits the enzymatic release of glucosefrom Avena cell walls in vitro. Since nojirimycin inhibits IAA-inducedgrowth of Avena coleoptiles, the evidence presented supportsthe hypothesis that IAA-induced growth may be mediated by anexo-glucanase. (Received February 14, 1975; )     

Real-time measurements of acetylcholine-induced release of ATP from bovine medullary chromaffin cells

Long-term treatment with hydralazine is sometimes associated with deposition of immune complexes and development of systemic lupus erythematosus (SLE) as an adverse side-effect. Hydralazine inhibits the covalent binding reaction of the complement protein C4. We show that when hydralazine inhibits C4, it becomes covalently bound to the polypeptide chain containing the active site thiol ester. C4 is encoded at 2 adjacent polymorphic loci, C4A and C4B, within the major histocompatibility complex. We show that hydralazine binds more efficiently to the C4A than to the C4B gene product and suggest that C4 type may predispose patients to hydralazine-induced SLE.     

Regulation of UDPG pyrophosphorylase in Acetabularia mediterranea

The kinetic properties of UDPG pyrophosphorylase (glucosyl-1-phosphate uridyl transferase, EC 2.7.7.9) suggest that it may play a key role in the regulation of metabolism in Acetabularia mediterranea. The enzyme-catalyzed reaction is readily reversible in vitro, and has been assayed in both directions. The enzyme shows substrate inhibition by UDPG and UTP at substrate concentrations in excess of 2 mM. The kinetic behavior of the enzyme is consistent with the hypothesis that it catalyzes an ordered bisubstrate biproduct reaction in which G-1-P is the leading substrate, and UTP is the leading product. A plot of initial velocity vs. PPi concentration is sigmoid, indicating a cooperative homotropic effect. PGAL inhibits the reaction in the direction: UTP + G-1-P leads to UDPG + PPi It has no effect on the reverse reaction. The responses of the enzyme may serve to regulate the allocation of G-1-P between anabolic and catabolic pathways.     

The inhibition of parathyroid-hormone actions on gluconeogenesis and phosphate transport by 3-mercaptopicolinate in rabbit renal proximal tubules.

By using a glucose microassay and the technique for isolated renal-tubule perfusion in vitro, the addition of 3-mercaptopicolinate, a gluconeogenesis inhibitor which inhibits phosphoenolpyruvate carboxykinase specifically, was found to abolish the effects of parathyroid hormone on gluconeogenesis and phosphate-transport rate in isolated rabbit renal proximal straight tubules, suggesting that these parathyroid-hormone actions may share some unknown, yet 3-mercaptopicolinate-inhibitable, intracellular processes.     

Covalent binding of 3-pyridinealdehyde nicotinamide adenine dinucleotide and substrate to glyceraldehyde 3-phosphate dehydrogenase.

Glyceraldehyde 3-phosphate dehydrogenase (D-glyceraldehyde-3-phoshate:nicotinamide adenine dinucleotide oxidoreductase (phosphorylating), EC 1.2.1.12) forms a complex with 3-pyridinealdehyde-NAD which survives precipitation with 7% perchloric acid. The molar ratio bound 3-pyridinealdehyde-NAD to the enzyme is 2.5 to 2.9. Lactate, malate, and alcohol dehydrogenases do not form acid-precipitable complexes with 3-pyridinealdehyde-NAD. 3-Pyridinealdehyde-deamino-NAD or glyceraldehyde 3-phosphate also forms an acid-stable complex with glyceraldehyde 3-phosphate dehydrogenase; however, NAD, 3-acetylpyridine-NAD, or thionicotinamide-NAD does not produce an acid-stable complex. Incubation of the glyceraldehyde 3-phosphate dehydrogenase with glyceraldehyde 3-phosphate, acetyl phosphate, iodoacetic acid, or iodosobenzoate inhibits the formation of the acid-stable complex with 3-pyridinealdehyde-NAD. Glyceraldehyde 3-phosphate or 3-pyridinealdehyde-NAD also prevents carboxymethylation of the active site cysteine-149 by[14-C]iodoacetic acid. These studies indicate that the aldehyde group of 3-pyridinealdehyde-NAD forms a thiohemiacetal linkage with cysteine-149 which is the substrate binding site for the dehydrogenase reaction. These findings may account for the fact that 3-pyridinealdehyde-NAD strongly inhibits the dehydrogenase and esterase activities of 3-pyridinealdehyde-NAD forms a thiohemiacetal linkage with cysteine-149 which is the substrate binding site for the dehydrogenase reaction. These findings may account for the fact that 3-pyridinealdehyde-NAD strongly inhibits the dehydrogenase and esterase activities of glyceraldehyde 3-phosphate dehydrogenase which require reduced cysteine-149. However, the analogue does not inhibit the acetyl phosphates activity of the enzyme for which the active site sulfhydryl residues must be oxidized.     

Inhibition of pancreatic islet monoamine oxidase by adrenergic antagonists and ethanol.

Although the alpha-adrenergic antagonist phentolamine potentiates glucose-stimulated insulin secretion of intact animals, it either does not alter, or it inhibits in vitro insulin secretion. This may be because in the higher concentration used in in vitro studies, phentolamine exerts a second pharmacological effect that counterbalances its primary effect of blocking monoamine action. We recently demonstrated that pancreatic islets contain substantial amounts of monoamine oxidase (MAO), and that MAO inhibitors such as iproniazid and tranylcypromine can alter insulin secretion. In the present study, we determined if other drugs that affect insulin secretion, alter the MAO activity of homogenates of rabbit pancreatic islets (collagenase technique) or liver. Phentolamine, phenoxybenzamine and propranolol (10 muM and 100 muM) inhibit islet and hepatic MAO. Haloperidol (10muM) inhibits hepatic but not islet MAO, while haloperidol (10muM) does not inhibit MAO in either tissue. Ethanol (270 to 2.7mM) inhibits islet MAO. Hepatic MAO is inhibited by high (270 to 180mM) but not by low (27 to 2.7mM) concentrations of ethanol. Collagenase digestion does not increase the sensitivity of islet and liver MAO to inhibition by phentolamine or ethanol. In the absence of added monoamines, phentolamine and phenoxybenzamine do not alter basal or glucose-stimulated insulin secretion from rabbit pancreas. Preincubation of rabbit pancreas with the serotonin precursor 5-hydroxytryptophan (5-HTP) increases the beta cell serotonin content and inhibits glucose-stimulated insulin secretion. Alpha adrenergic antagonists not only fail to block, but actually potentiate the serotonin inhibition of insulin secretion. We conclude that inhibition of islet MAO may cause an increase in islet monoamine content and these monoamines may alter in vitro insulin secretion. One mechanism through which adrenergic antagonists and ethanol modify in vitro insulin secretion may be by inhibiting pancreatic islet MAO.     

Albumin competitively inhibits glycation of less abundant proteins

Glycation, a non-enzymatic reaction between glucose and protein is the primary cause of diabetic complications. Albumin, the most abundant plasma protein undergoes glycation both in vivo and in vitro. The influence of albumin on glycation of less abundant proteins has not been addressed. For the first time, we show that albumin competitively inhibits the glycation of less abundant proteins. This study suggests that at least in the initial stages of diabetes, albumin may protect other proteins from glycation.     

The retroactive action of paradoxical sleep deprivation and immobilization stress on the mechanisms of memory and passive avoidance in rats

Influence was studied of 48-hours deprivation of paradoxical sleep (DPS) and immobilization stress on conditioned reaction of fear and security--components of passive avoidance conditioned reflex (CRPA), with simultaneous studying of vertical, horizontal activity, grooming reactions and defecations in an illuminated chamber. The obtained results allow to conclude that DPS inhibits reproduction of memory traces of fear reaction but intensifies the process of learning and activity of memory traces of security reaction. Depending on condition of CRPA elaboration, DPS may disturb or, on the contrary, prolong preservation of passive avoidance. It has been found that the immobilization stress does not influence CRPA preservation and does not weaken the reaction of fear but increases the horizontal activity. It is suggested that the observed changes in behaviour and trace processes are connected with disturbance of sensory informational homeostasis of the nervous system at DPS.     

Regulation of glutaminase by exogenous glutamate,ammonia and 2-oxoglutarate in synaptosomal enriched preparation from rat brain

Phosphate activated glutaminase in synaptosomal enriched preparation from rat brain is very sensitive to inhibition by low concentration of glutamate, ammonia and 2-oxoglutarate when added to the incubation medium at pH 7.6. By increasing the concentration of either of these compounds up to 0.5 mM a pronounced initial inhibition is followed by little or no further effect when the concentration is increased beyond this level. By lowering the pH of the reaction mixture to 7.0, the inhibition by glutamate is almost abolished and that of ammonia reduced. Glutamate inhibits mainly the N-ethylmaleimide-sensitive fraction of glutaminase which previously is suggested to be localized to the outer phase of the mitochondrial inner membrane, whereas ammonia inhibits both the N-ethylmaleimidesensitive and-insensitive fraction. Evidence has been produced to show that the inhibition by 2-oxoglutarate is caused by glutamate formation by aminotransferase reactions. Since 2-oxoglutarate is produced by the tricarboxylic acid cycle, the operation of this cycle may regulate the glutaminase reaction by controlling glutamate formation via the aminotransferase reactions.Abbreviations used NEM N-ethylmaleimide - PAG phosphate activated glutaminase - AOA aminooxyacetic acid     

Product inhibition of potato tuber pyrophosphate:fructose-6-phosphate phosphotransferase by phosphate and pyrophosphate

The product inhibition of potato (Solanum tuberosum) tuber pyrophosphate:fructose-6-phosphate phosphotransferase by inorganic pyrophosphate and inorganic phosphate has been studied. The binding of substrates for the forward (glycolytic) and the reverse (gluconeogenic) reaction is random order, and occurs with only weak competition between the substrate pair fructose-6-phosphate and pyrophosphate, and between the substrate pair fructose-1,6-bisphosphate and phosphate. Pyrophosphate is a powerful inhibitor of the reverse reaction, acting competitively to fructose-1,6-biphosphate and noncompetitively to phosphate. At the concentrations needed for catalysis of the reverse reaction, phosphate inhibits the forward reaction in a largely noncompetitive mode with respect to both fructose-6-phosphate and pyrophosphate. At higher concentrations, phosphate inhibits both the forward and the reverse reaction by decreasing the affinity for fructose-2,6-bisphosphate and thus, for the other three substrates. These results allow a model to be proposed, which describes the interactions between the substrates at the catalytic site. They also suggest the enzyme may be regulated in vivo by changes of the relation between metabolites and phosphate and could act as a means of controlling the cytosolic pyrophosphate concentration.     

The bioflavonoid quercetin inhibits neutrophil degranulation, superoxide production, and the phosphorylation of specific neutrophil proteins

Quercetin, a C-kinase antagonist, inhibits neutrophil degranulation and superoxide production induced by f-met-leu-phe, solid phase IgG, zymosan treated serum and a phorbol ester (PMA). Quercetin is more effective in inhibiting degranulation (IC50 = 20 uM) than superoxide production (IC50 = 80 microM). Neutrophil activation by PMA is accompanied by the phosphorylation of neutrophil proteins of 205, 170, 130, 91, 77, 67, 56, 47, 39, 34, 27, and 20 kilodaltons; quercetin also inhibits the phosphorylation of these proteins. Dose-response studies indicated that phosphorylation of the 67 kilodalton protein was particularly sensitive to inhibition by quercetin at concentrations that also inhibit neutrophil degranulation and superoxide production. These results suggest that phosphorylation of the 67 kilodalton protein may be an important intracellular reaction associated with neutrophil activation.     

Effects of quaternary ligands on the inhibition of acetylcholinesterase by arsenite.

Arsenite inhibits acetylcholinesterase in a second-order reaction. The rate and equilibrium constants depend upon pH and have values on the order of 10(2) M-1 min-1 and 10(5) M (dissociation), respectively. Some quaternary ammonium ligands completely block the arsenite inhibition of the enzyme, others decrease the rate of the reaction and some, notably pyridine-2 aldoxime methiodide, greatly accelerate the rate of the reaction, up to 220-fold. Accelerators may bind at a separate enzyme site distinct form the anionic site involved in substrate binding. Although the kinetic data are consistent with a covalent reaction between arsenite and acetylcholinesterase, chemical evidence excludes the involvement of sulfhydryl groups which are usually implicated in arsenite inhibition.     

Inhibition of cdc2 activation by INH/PP2A.

INH, a type 2A protein phosphatase (PP2A), negatively regulates entry into M phase and the cyclin B-dependent activation of cdc2 in Xenopus extracts. INH appears to be central to the mechanism of the trigger for mitotic initiation, as it prevents the premature activation of cdc2. We first show that INH is a conventional form of PP2A with a B alpha regulatory subunit. We next explore the mechanism by which it inhibits cdc2 activation by examining the effect of purified PP2A on the reaction pathways controlling cdc2 activity. Our results suggest that although PP2A inhibits the switch in tyrosine kinase and tyrosine phosphatase activities accompanying mitosis, this switch is a consequence of the inhibition of some other rate-limiting event. In the preactivation phase, PP2A inhibits the pathway leading to T161 phosphorylation, suggesting that this activity may be one of the rate-limiting events for transition. However, our results also suggest that the accumulation of active cdc2/cyclin complexes during the lag is only one of the events required for triggering entry into mitosis.     

Ultraviolet light modification of the prostaglandin receptor

Prostaglandin E₁ elicits minimal inhibition of ultraviolet lightinduced platelet aggregation. Exposure to ultraviolet light progressively inhibits prostaglandin-induced responses in both the ADP-aggregating platelet and in uterine smooth muscle. This effect, which is cumulative and lasting, may be prevented or fully reversed by a sulfhydryl-oxidizing agent. Ultraviolet light may, therefore, initiate reduction of critical disulfide groups in the vicinity of the prostaglandin receptor. The technique of ultraviolet irradiation may serve as a useful tool in the specific determination of prostaglandins in biological systems.     

Interacting effects of dibutylchloromethyltin chloride, 2,3-dimercaptopropanol,and other reagents on mitochondrial respiration and K+ flux

The oxidative phosphorylation inhibitor DBCT (dibutylchloromethyltin chloride) inhibits state 3 respiration at a concentration less than that which stimulates K+ flux into respiring rat liver mitochondria. Inhibition of ADP-stimulated respiration by DBCT can be reversed or blocked by the dithiol 2,3-dimercaptopropanol. The data are consistent with previous suggestions that DBCT may interact with the ATP synthase via reaction with a dithiol group. The stimulation of K+ influx by DBCT is partially reversed by concentrations of 2-mercaptoethanol which fail to affect the inhibition of state 3 respiration by DBCT. The combination of DBCT plus 2,3-dimercaptopropanol inhibits mitochondrial K+ influx. The inhibitory effect of dicyclohexylcarbodiimide on K+ influx is not expressed in the presence of DBCT. Atractyloside has little effect on K+ influx in the presence or absence of DBCT. The combination of DBCT plus uncoupler induces a net loss of endogenous K+. Consideration is given to the alternative hypotheses that the acceleration of K+ influx by DBCT may involve either a direct link to the energy transduction apparatus, or may occur via separate activation of a passive transport mechanism.     

A novel reaction of reticulocyte peptide-chain elongation factor, EF2, with guanosine nucleotides

The formation of phenylalanyl puromycin from phenylalanyl-tRNA, bound nonenzymically or enzymically to reticulocyte ribosomes, requires the peptide-chain elongation factor, EF2², and GTP. However the GTP analogue, GDPCP, may replace GTP to a significant extent in this reaction. Other purine or pyrimidine nucleotides have little or no activity. Multistep experiments with either GTP or GDPCP indicate that binding of EF2 to the ribosome for subsequent peptide formation may be a portion of the activity of the EF2 (independent of the translocation reaction) during the elongation process. Neomycin inhibits the formation of phenylalanyl puromycin using either GTP or GDPCP in this system.     

Effect of vanadate on the vacuolar ATPase of Acer pseudoplatanus

The ATPase of vacuoles isolated from Acer pseudoplatanus cells is strongly inhibited by vanadate, a specific inhibitor of plasma membrane ATPase. The degree of inhibition depends upon the ionic composition, ATP and magnesium concentrations of the reaction medium and the inhibition is reversed by EDTA which complexes vanadate. In absence of factor which may interfere with the effectiveness of inhibitor, vanadate inhibits the ATPase non-competitively.