Unfolding and inactivation of Penaeus penicillatus acid phosphatase during denaturation by guanidine hydrochloride

Neuromuscular terminals of a single motoneuron to four muscles (CPV7a, GM5a, CV2, and CV3) in the stomach of the blue crab Callinectes sapidus showed structural evidence for the exocytotic release of dense-core vesicles exclusively at synapses. The primary evidence was the appearance of dense cores in the synaptic cleft, accompanied by indentations of the presynaptic or postsynaptic membrane. In their simplest form, these consisted of an omega-shaped figure of the presynaptic membrane enclosing one dense core, denoting release of a single dense-core vesicle. A larger indentation of the presynaptic membrane enclosing several dense cores denoted multiple release. A more complex form of multiple release was where the presynaptic membrane was normal, but the postsynaptic membrane elaborated into a sac projecting into the granular sarcoplasm and filled with dense cores. The postsynaptic sac in some instances was compressed into a thin, fingerlike extension, which lacked dense cores and, at its distal end, separated into small cisternae, suggesting a mechanism for membrane recycling. Profiles depicting single and multiple releases of dense-core vesicles were found more frequently at neuromuscular terminals that release relatively large amounts of transmitter with a single stimulus, such as CV2 and CV3, compared to those releasing smaller amounts, such as CPV7a and GM5a. The disparity in release sites among the four muscles of this single motor unit and the fact that many of the multiple-release figures were closely adjacent to the active zones for transmitter release suggest a possible modulatory role for dense-core vesicles in synaptic transmission. Such modulation may be long lasting, as implied by the postsynaptic sacs, which may permit prolonged release of the contents of their dense cores into the synaptic cleft. This is in keeping with the functional role of these stomach muscles, which is to be continuously active for long periods of time.     

Kinetics of inactivation of green crab (Scylla Serrata) alkaline phosphatase during removal of zinc ions by ethylenediaminetetraacetic acid disodium

Green crab (Scylla Serrata) alkaline phosphatase (EC 3.1.3.1) is a metalloenzyme, the each active site in which contains a tight cluster of two zinc ions and one magnesium ion. The kinetic theory of the substrate reaction during irreversible inhibition of enzyme activity previously described by Tsou has been applied to a study on the kinetics of the course of inactivation of the enzyme by ethylenediaminetetraacetic acid disodium (EDTA). The kinetics of the substrate reaction with different concentrations of the substrate p-nitrophenyl phosphate (PNPP) and inactivator EDTA suggested a complexing mechanism for inactivation by, and substrate competition with, EDTA at the active site. The inactivation kinetics are single phasic, showing the initial formation of an enzyme-EDTA complex is a relatively rapid reaction, followed a slow inactivation step that probably involves a conformational change of the enzyme. Zinc ions are finally removed from the enzyme. The presence of metal ions apparently stabilizes an active-site conformation required for enzyme activity.     

Apoptosis in barley aleurone during germination and its inhibition by abscisic acid

During germination of barley grains, DNA fragmentation was observed in the aleurone. The appearance of DNA fragmentation in the aleurone layer, observed by TUNEL staining in aleurone sections, started near the embryo and extended to the aleurone cells far from the embryo in a time dependent manner. The same spatial temporal activities of hydrolytic enzymes such as alpha-amylase were observed in aleurone. DNA fragmentation could also be seen in vitro under osmotic stress, in isolated aleurone. During aleurone protoplast isolation, a very enhanced and strong DNA fragmentation occurred which was not seen in protoplast preparations of tobacco leaves. ABA was found to inhibit DNA fragmentation occurring in barley aleurone under osmotic stress condition and during protoplast isolation, while the plant growth regulator gibberellic acid counteracted the effect of ABA. Addition of auxin or cytokinin had no significant effect on DNA fragmentation in these cells. To study the role of phosphorylation in ABA signal transduction leading to control of DNA fragmentation (apoptosis), the effects of the phosphatase inhibitor okadaic acid and of phenylarisine oxide on apoptosis were studied. We hypothesize that the regulation of DNA fragmentation in aleurone plays a very important role in spatial and temporal control of aleurone activities during germination. The possible signal transduction pathway of ABA leading to the regulation of DNA fragmentation is discussed.     

Identification of human seminal acid phosphatase by electrophoresis

Recent advances in forensic science in the identification of human seminal acid phosphatase are presented, with particular attention to the acrylamide gel electrophoretic method. In that method a difficulty in distinguishing seminal acid phosphatase from certain fecal phosphatases has been observed and an attempt is made here to distinguish the phosphatases from one another experimentally by differential substrate specificity. 4-Methylumbelliferyl phosphate and alpha-naphthyl acid phosphate are used as reaction substrates. Although seminal and vaginal acid phosphatases are differentiated by the modified method, fecal phosphatase is not clearly differentiated from seminal acid phosphatase.     

Mechanism-based inactivation of gamma-aminobutyric acid aminotransferase by 3-amino-4-fluorobutanoic acid

The mechanism of inactivation of the pyridoxal 5'-phosphate (PLP)-dependent enzyme gamma-aminobutyric acid (GABA) aminotransferase by 3-amino-4-fluorobutanoic acid (2) has been investigated. As in the case of the homologue, 4-amino-5-fluoropentanoic acid (1), 2 equiv of radiolabeled inactivator become covalently attached to the enzyme, and no transamination, as determined by the lack of conversion of [1-14C] alpha-ketoglutarate into [1-14C] glutamate during inactivation, was observed. In the case of 1, the conclusion was that inactivation was completely the result of modification of the coenzyme and that there was no metabolic turnover; every enzyme molecule catalysed the conversion of one molecule of inactivator to the activated species, which inactivated the enzyme by an enamine mechanism. With 2, however, 6.7 +/- 0.7 equiv of fluoride ions were released during inactivation, and it took 7.6 +/- 0.7 inactivator molecules to inactivate each enzyme dimer. Since no transamination was occurring, another metabolic event besides inactivation must result from the PLP form of the enzyme. Inactivation of GABA amino-transferase with [1,2-14C]-2 produced [14C] acetoacetic acid (about 5.5 equiv) as the metabolite. The 1.93 +/- 0.25 equiv of radioactivity covalently bound to the enzyme after inactivation with [1,2-14C]-2 and gel filtration were completely released by base treatment. HPLC analysis showed that three radioactive compounds, identified as 2, the product of reaction of PLP with acetone (3), and the product of reaction of PLP with acetoacetate (4), were detected. The release of 3 and 4 and the prevention of release of radioactivity by treatment with sodium borohydride are consistent with the formation of covalent intermediates that have beta-carbonyl-like character, such as 6 and/or 7 (Scheme 2). Inactivation of [3H] PLP-reconstituted GABA aminotransferase with 2 followed by gel filtration then base denaturation released all of the radioactivity as a mixture of PLP, 3, and 4. Inactivation with [1,2-14C]-2 resulted in the release of 1.37 equiv of 14CO2, which was shown to be the result of decarboxylation of the acetoacetate/4 after release from the enzyme. These results are not consistent with a Michael addition mechanism (Scheme 3), but are consistent with inactivation by an enamine mechanism; release of the enamine five out of seven turnovers accounts for the formation of acetoacetate as the metabolite. To account for the detection of PLP and 2 after denaturation, it is suggested that a nonproductive formation of the Schiff base of PLP with 2 occurs in the second subunit of the enzyme; this complex is released and hydrolysed to PLP and 2 upon base denaturation.     

乳酸直接缩聚制备聚乳酸的动力学

研究了乳酸直接缩聚制备聚乳酸的反应动力学,结果表明:在常压和无催化剂条件下,当温度达到124℃时,乳酸单体开始缩聚,在反应程度为14.15%～62.75%的范围内缩聚反应符合3级反应,其动力学方程式为-d[COOH]/dt=1.057×10-5[COOH]3.还计算了在不同温度下的反应速率常数k,并根据ARRHENNIS公式求得反应活化能E=4.34 kJ·mol-1.最后,比较了理论计算与实际测量的数均分子量与时间的关系.     

Kinetics of heat inactivation of Venezuelan equine encephalomyelitis virus

Thermal inactivation of Venezuelan Equine Encephalomyelitis Virus (VEEV) was studied at temperatures from 26 degrees to 55 degrees C. Inactivation of infectivity took place by two thermodynamically different reactions, one of which predominated at temperatures below 44 degrees C and the other at higher temperatures. The presence of 1 or 2 M NaCl stabilized the VEE virus at low temperatures but enhanced the inactivation at high temperatures. This latter effect at temperatures higher than 50 degrees C, is associated with the occurrence of two-component survival curves. The different effects of hypertonic NaCl concentrations at the two ranges of temperature, are related to different mechanisms of inactivation operating at each range (protein denaturation and nucleic acid-RNA breakdown). Different kinetics of thermal inactivation at 55 degrees C were observed between virus strains with different virulence. However, no significant correlations was found between the virulence of the eleven VEE virus strains studied and their thermostability at 37 degres and 55 degrees C.     

Kinetics of inhibition of rabbit reticulocyte peptidyltransferase by anisomycin and sparsomycin

A detailed kinetic study was carried out on the inhibitory mechanisms of two eukaryotic peptidyltransferase drugs (I), anisomycin and sparsomycin. In an in vitro system from rabbit reticulocytes, AcPhe-puromycin is produced in a pseudo-first-order reaction from the preformed AcPhe-tRNA/poly(U)/80S ribosome complex (complex C) and excess puromycin (S). This reaction is inhibited by anisomycin and sparsomycin through different mechanisms. Anisomycin acts as a mixed noncompetitive inhibitor. The product, AcPhe-puromycin, is derived only from C according to the puromycin reaction. On the other hand, sparsomycin reacts with complex C in a two-step reaction, [REACTION; SEE TEXT] An initial rapid binding of the drug produces the encounter complex CI. During this step and before conversion of CI to C*I, sparsomycin behaves as a competitive inhibitor. The rapidly produced CI is isomerized slowly to a conformationally altered species C*I in which I is bound more tightly. The rate constants of this step are k6 = 2.1 min-1 and k7 = 0.095 min-1. Moreover, the low value of the association rate constant k7/Ki' (2 x 10(5) M-1 sec-1), provides insight into the rates of possible conformational changes occurring during protein synthesis and supports the proposal that sparsomycin is the first example of a slow-binding inhibitor of eukaryotic peptidyltransferase. When complex C is preincubated with concentrations of sparsomycin of >8 Ki and then reacts with a mixture of puromycin and sparsomycin, the inhibition becomes linear mixed noncompetitive and involves C*I instead of CI. During this phase, AcPhe-puromycin is produced from a new, modified ribosomal complex with a lower catalytic rate constant. Thus, sparsomycin also acts as a modifier of eukaryotic peptidyltransferase activity.     

Kinetics of leaching of copper concentrates by nitric acid

Based on the study of the kinetics of leaching of copper concentrates by nitric acid, the limiting stages, reaction rate constants, activation energies, and general and partial orders of reactions of the components involved have been determined. An effect of films of reaction products on the limiting stage of the leaching of sulfides by nitric acid has been revealed. It has been shown that in the case of high oxidation potentials no films of elementary sulfur and iron oxide are formed, and the rate of the process is controlled by external diffusion.     

Oxidative inactivation of brain alkaline phosphatase responsible for hydrolysis of phosphocholine

Alkaline phosphatase, one of the enzymes responsible for the conversion of phosphocholine into choline, was purified from bovine brain membrane, where the phosphatase is bound as glycosylphosphatidylinositol-linked protein, and subjected to oxidative inactivation. The phosphatase activity, based on the hydrolysis of p-nitrophenyl phosphate and phosphocholine, decreased slightly after the exposure to H2O2. Inclusion of Cu2+ in the incubation with 1 mM H2O2 led to a rapid decrease of activity in a time- and concentration-dependent manner. In comparison, the H2O2/Cu2+ system was much more effective than the H2O2/Fe2+ system in inactivating brain phosphatase. In a further study, it was observed that the hydroxy radical scavengers mannitol, ethanol, or benzoate failed to prevent against H2O2/Cu2+-induced inactivation of the phosphatase, excluding the involvement of extraneous hydroxy radicals in metal-catalyzed oxidation. In addition, it was found that both substrates, p-nitrophenyl phosphate and phosphocholine, and an inhibitor, phosphate ion, at their saturating concentrations exhibited a remarkable, although incomplete, protection against the inactivating action of H2O2/Cu2+. A similar protection was also expressed by divalent metal ions such as Mg2+ or Mn2+. Separately, it was found that H2O2/Fe2+-induced inactivation was prevented by p-nitrophenyl phosphate or Mg2+ but not phosphate ions. Thus, it is implied that phosphocholine-hydrolyzing alkaline phosphatase in brain membrane might be one of enzymes susceptible to metal-catalyzed oxidation.     

Biological inactivation of 5-oxo-6,8,11,14-eicosatetraenoic acid by human platelets

Neutrophil-derived 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is a potent activator of neutrophils and eosinophils. In the present study we examined the biosynthesis and metabolism of this substance by platelets. Although platelets contain an abundant amount of 5-hydroxyeicosanoid dehydrogenase, the enzyme responsible for the formation of 5-oxo-ETE, they synthesize only very small amounts of this substance from exogenous 5-hydroxyeicosatetraenoic acid (5-HETE) unless endogenous NADPH is converted to NADP+ by addition of phenazine methosulfate. Similarly, relatively small amounts of 5-oxo-ETE were formed by A23187-stimulated mixtures of platelets and neutrophils, which instead formed substantial amounts of two 12-hydroxy metabolites of this substance, 5-oxo-12-HETE and 8-trans-5-oxo-12-HETE, which were identified by comparison with authentic chemically synthesized compounds. These metabolites were also formed from 5-oxo-ETE by platelets stimulated with thrombin or A23187. In contrast, unstimulated platelets converted 5-oxo-ETE principally to 5-HETE. Neither 5-oxo-12-HETE nor 8-trans-5-oxo-12-HETE had appreciable effects on neutrophil calcium levels or platelet aggregation at concentrations as high as 10 micromol/L, but both blocked 5-oxo-ETE-induced calcium mobilization in neutrophils with IC50 values of 0.5 and 2.5 micromol/L, respectively. We conclude that platelets can biologically inactivate 5-oxo-ETE. Unstimulated platelets convert 5-oxo-ETE to 5-HETE, with a 99% loss of biological potency, whereas stimulated platelets convert this substance to 12-hydroxy metabolites, which possess antagonist properties.     

ATP-dependent inactivation of Escherichia coli gamma-glutamylcysteine synthetase by L-glutamic acid gamma-monohydroxamate

Incubation of Escherichia coli gamma-glutamylcysteine synthetase with L-glutamic acid gamma-monohydroxamate and ATP caused slow but irreversible inhibition of the enzyme, and more than 90% activity was lost in three days. The enzyme was not inactivated when ATP was absent or L-aspartic acid beta-monohydroxamate was substituted for L-glutamic acid gamma-monohydroxamate, suggesting that the inactivation process reflected a mechanism-based reaction of L-glutamic acid gamma-monohydroxamate and ATP.     

Changes of acid phosphatase activity of fast and slow rat muscles during ontogenetic development

Total IgG, IgG subclass and IgE antibodies specific for grass pollen allergens were measured by the red cell linked antigen-antiglobulin reaction (RCLAAR) in a serum samples from nineteen patients who had undergone a course of hyposensitization. Increases in both specific IgG and IgE antibodies were seen after treatment in most patients. In the IgG subclasses the predominant response was for IgG1 and IgG4 antibodies. Attempts were made to correlate the antibody responses with the clinical response and the results are discussed with reference to the possible mechanisms of hyposensitization.     

Kinetics of slow reversible inhibition of human muscle creatine kinase by planar anions

The toxicity of NO3- and NO2- to mammals has been widely publicized. However, the kinetic mechanism of inhibition of human muscle creatine kinase by NO3- and NO2- has not been explored. The kinetic theory of the substrate reaction during the modification of enzyme activity previously described by Tsou (Adv. Enzymol. Related Areas Mol. Biol. 1988, 61, 381-436) has been applied to a study of the kinetics of slow reversible inhibition of human muscle creatine kinase by planar anions (NO3- and NO2-). The kinetic equation of the substrate reaction was derived from theoretical analysis and experimental data, then simplified. The microscopic rate constants for the reaction of the inhibitors with the enzyme were obtained from the simplified equation for the substrate reaction in the presence of the inhibitors. The results show that the apparent forward rate constant A is dependent on ATP concentration, indicating competition between the inhibitor (NO3- or NO2-) and ATP. The results also suggest that binding of creatine-MgADP and the anion with the enzyme is very tight, since their binding constants are much higher than those for normal substrates.     

Kinetics of iridium reduction by hydrogen in hydrochloric acid solution

The effects of stirring speed, initial iridium ion concentration, hydrogen pressure, hydrochloric acid, chloride concentrations, seeding, and temperature on the reduction rate of iridium ion by hydrogen have been investigated. The whole process of reduction occurs in a complicated heterogeneous stage. The reduced metallic Ir serves itself as a catalyst for the reduction. The rate in the earlier stage of the reduction (including the nucleation period) is controlled by chemical reaction; the apparent activation energy obtained in the temperature range of 363 to 423 K was 76.1 kJ/mol. The order of the reaction with respect to the iridium ion concentration and the hydrogen partial pressure was found to be one, and the reaction rate increased with the increase of HC1 and Cl concentrations. The rate in the later stage was controlled by diffusion, and the apparent activation energy was reduced to 25.5 kJ/mol. The reaction mechanism has been given by the analysis of experimental results. Formerly Graduate Student, Department of Extractive Metallurgy, Institute of Precious Metals     

A protein phosphatase is involved in the inhibition of histone deacetylation by sodium butyrate

Treatment of cells with sodium butyrate is known to increase histone acetylation by inhibiting deacetylases. Here we have observed, in cultured hepatoma cells, that the potent serine-threonine phosphatase inhibitors, okadaic acid or calyculin A, inhibited phosphatase activity and concomitantly decreased the histone acetylation classically maintained by sodium butyrate. These results suggest that a protein phosphatase may mediate the sodium butyrate effect on deacetylases. Since we have previously found that such a protein would also mediate the sodium butyrate effect on gene expression, we propose that a phosphatase activity constitutes an early and essential step in the sodium butyrate-triggered signalling pathway.     

The activation of glycogen synthase by insulin switches from kinase inhibition to phosphatase activation during adipogenesis in 3T3-L1 cells

The effects of insulin and platelet-derived growth factor (PDGF) on glycogen synthase activation were compared in 3T3-L1 fibroblasts and adipocytes. In the fibroblasts, PDGF elicited a stronger phosphorylation of mitogen-activated protein kinase (MAPK) and AKT than did insulin. Both agents caused a comparable stimulation of receptor autophosphorylation, MAPK, and phosphatidylinositol 3-kinase (PI3-K) activation in the adipocytes. However, adipogenesis resulted in the uncoupling of PI3-K activation by PDGF from subsequent AKT phosphorylation. The relative contributions of glycogen synthase kinase-3 (GSK-3) inactivation and protein phosphatase-1 (PP1) activation in the regulation of glycogen synthase in both cell types were evaluated. Insulin and PDGF caused a small increase in glycogen synthase a activity in the fibroblasts. Additionally, both agents caused a similar inhibition of GSK-3, while having no effect on PP1 activity. Following differentiation, insulin treatment resulted in a 5-fold stimulation of glycogen synthase, whereas PDGF was without effect. Both agents caused a comparable inhibition of GSK-3 activity in the adipocytes, whereas only insulin activated PP1. Finally, wortmannin completely blocked the stimulation of PP1 by insulin in 3T3-L1 adipocytes, indicating that PI3-K inhibition can impinge on PP1 activation. Cumulatively these results suggest that the weak activation of glycogen synthase in 3T3-L1 fibroblasts is mediated by GSK-3 inactivation, whereas in the more metabolically active adipocytes, the insulin-specific activation of glycogen synthase is mediated by PP1 activation.