Ping Pong Bi Bi机制的非稳态酶动力学分布函数图论研究

以非稳态酶动力学的布尔函数图形方法,来研究一类PingPongBiBi机制的非记酶动力学问题,推导出此类反应的非稳态酶动力学方程,并对此动力学方程进行了讨论,分析了此类PingPongBiBi机制酶反应体系的非稳态酶动力学方程。     

Random Bi Bi机制的非稳态酶动力学布尔函数图论研究

本文以非稳态酶动力学的布尔函数图形方法^[1],来研究一类Random Bi Bi机制的非稳态酶动力学问题,推导同此类反应的非稳态酶动力学方程,并对此动力学方程进行了讨论,分析了此类Random Bi Bi机制酶反应体系的非稳态酶动力学方程。     

非稳态酶活化动力学的布尔函数图论分析

以非稳态酶动力学的布尔函数图形方法研究非稳态酶活化动力学问题,推导出此类反应的非稳态酶动力学方程,并对此动力学方程进行了讨论,分析了酶活化反应体系的非稳态酶动力学过程．     

竞争性抑制的非稳态酶动力学布尔函数图论研究

以非稳戊酶动力学的布尔函数图形方法,来研究一类竞争性抑制的非稳态酶动力学问题,推导出此类反应的百稳态酶动力学方程,并对此动力学方程进行了讨论,分析了此类竞争性抑制酶反应体系的非稳态酶动力学问题。     

反竞争性抑制的非稳态酶动力学布尔函数图解研究

以非稳态酶动力学的布尔函数图形方法,来研究一类反竞争性抑制的非稳态酶动力学问题,推导出此类反应的非稳态酶动力学方程,并对此动力学方程进行了讨论,分析了此类反竞争性制酶反应体系的非稳态酶动力学问题。     

一类非竞争性抑制的非稳态酶动力学布尔函数图解研究

本文以非稳态酶动力学的布尔函数图形方法^[1],来研究一类非竞争性抑制的非稳态酶动力学问题,推导出此类反应的非稳态酶动力学方程,并对此动力学方程进行了讨论,分析了此类非竞争性抑制的非稳态酶动力学的动力学过程。     

黑曲霉过氧化氢酶发酵过程的数学模型

研究了黑曲霉发酵生产过程氧化氢酶的分批发酵动力学,并建立了发酵过程菌体生长,基质消耗及酶合成的随时间变化的数学模型。Logistic方程,Luedekin-Piret方程及与Luedeking-Piret方程相似的基质消耗方程能够很好地分别描述黑曲霉细胞的生长,发酵产酶过程及葡萄糖的消耗,过氧化氢酶的发酵合成是生长耦联的,研究中还将3个动力学模型的预测值和实验值进行了比较。     

从莫诺方程谈起——生物类本科专业教学中3个相似方程的探讨

介绍并比较探讨了生物类相关本科专业课程教学中涉及的3个相似方程：微生物细胞生长动力学中的莫诺方程、酶促反应动力学中的米氏方程、吸附分离过程中的等温吸附方程,以供相关专业教师的教学和学生的学习参考。     

内切酶切割环状DNA切割速度的计算

研究内切酶动力学,至今还没有很好的酶活测定方法.1976年Forsblom,S.等人用内切酶切割线状DNA分子时,研究了DNA片段的浓度对时间的依赖性,阐明了DNA上不同内切酶切点的切割速度与相应片段浓度的相关性,推导出一系列计算酶活力的方程.作者曾多次使用这套方程进行内切酶动力学研究,确实较其他方法方便可行.我们依据同     

酶促水解大豆分离蛋白动力学模型的研究

本文对AS1.398中性蛋白酶在pH6.9和温度49℃条件下水解大豆分离蛋白的动力学机制进行了研究．结果表明：酶水解速率随水解反呈指数递减．为了解释实验结果,我们提出了如下假设：对底物而言水解反应终为零级反应,水解过程中由于游离酶攻击酶-底物中间络合物而造成的不可逆酶变性是一个二级动力学过程．在此基础上,由实验数据推导得到了描述AS1.398中性蛋白酶催化水解大豆分离蛋白的动力学方程,该方程可用于指导和优化酶解反应实验．     

3-硝基-N-甲基水杨酰胺对琥珀酸细胞色素c还原酶的抑制作用

 我们发现3-硝基-N-甲基水杨酰胺对猪心线粒体呼吸链的琥珀酸细胞色素c还原酶活力有抑制作用。抑制部位在呼吸链中PMS和DCPIP的电子给体之间。抑制性质表现为可逆非竞争性。     

Regulatory aspects of mitochondrial phospholipase A2 from rat liver: effects of proteins, phospholipids and calcium ions

This paper deals with the search for specific inhibitors or activators of the mitochondrial phospholipase A2. Convincing evidence for the existence of proteins in the mitochondrial or cytosolic fraction that function as specific regulators of this enzyme was not obtained. The enzymatic activity appeared to be inhibited at low substrate concentrations by lipocortin isolated from human monocytes. However, at higher substrate concentrations, the inhibition disappeared, suggesting either that lipocortin sequestered the phospholipid substrate or that the putative inactive complex of enzyme and lipocortin dissociated in the presence of excess phospholipids. The hydrolysis of the neutral phospholipid phosphatidylethanolamine was stimulated by the presence of cardiolipin and phosphatidylglycerol. It is unlikely that this is caused merely by the negative charge of these phospholipids, since other negatively charged phospholipids did not show this effect. Using a phospholipid extract from mitochondria as substrate, the enzymatic activity as a function of the Ca2+ concentration was determined. Only one enzyme activity plateau was observed. The calculated KCa2+ value of 0.05 mM suggests that the mitochondrial phospholipase A2 could be regulated strictly by the modulation of the free Ca2+ concentration in vivo. The two activity plateaus observed previously upon variation of the Ca2+ concentration using phosphatidylethanolamine as substrate could be explained by a Ca2+-induced transition of the phospholipid structure.     

Phospholipid modulation of low-Km cyclic AMP phosphodiesterase activity on rat adipocyte microsomes

The ability of nine phospholipids to alter the activity of low-Km cyclic AMP phosphodiesterase was examined in microsomal fractions of rat adipocytes. The enzyme was activated by phosphatidylserine (21% at 300 microM) and phosphatidylglycerol (36% at 300 microM). The activation was concentration dependent over the range 1-1000 microM. Six other phospholipids were without effect. Phosphatidylinositol 4-phosphate inhibited the activity of the enzyme over the same range of concentrations (26% at 300 microM). Phosphatidylserine also activated a partially purified preparation of the enzyme, whereas phosphatidylinositol 4-phosphate was ineffective. The mechanism of the activation of the enzyme by phosphatidylserine and phosphatidylglycerol involved an increase in the apparent Vmax of the enzyme, while the inhibition by phosphatidylinositol 4-phosphate was associated with an increase in the Km of the enzyme for substrate. The phospholipid modulators of low-Km cyclic AMP phosphodiesterase activity did not alter the activity of high-Km cyclic AMP phosphodiesterase. The ability of phospholipids to alter the activity of low-Km cyclic AMP phosphodiesterase in native membranes suggests a possible role for phospholipids in metabolic regulation.     

Identification of inhibitors of the E. coli cyclopropane fatty acid synthase from the screening of a chemical library: In vitro and in vivo studies

Using an automated coupled colorimetric assay for the Escherichia coli cyclopropane fatty acid synthase (CFAS), we have screened an academic chemical library of 3040 compounds, to identify new inhibitors of this enzyme. We identified 8 compounds as potent inhibitors of this enzyme, with IC(50) ranging from 1 to 10 microM, in the presence of 750 microM S-adenosyl-l-methionine and 1 mg/mL phospholipids. We conducted kinetic analyses of the inhibition of the CFAS using dioctylamine and three inhibitors identified in this report: sinefungin, 1, a synthetic S-adenosyl-l-homocysteine analog, 2, and an indoloquinolizine derivative, 3. The inhibition patterns observed were interpreted assuming that the E. coli CFAS operated via an ordered Bi Bi mechanism with binding of S-adenosyl-l-methionine first. Dioctylamine was the most potent inhibitor with a competitive inhibition constant of 130 nM with respect to the phospholipids. Compound 2 bound to the two substrate-binding sites of the enzyme suggesting that it acted as a bisubstrate analog (apparent inhibition constant, K(I)=6 microM). Compound 2 was also found to completely inhibit cyclopropanation of the phospholipids in growing E. coli cells, at 150 microM. This molecule is thus the first inhibitor of a cyclopropane synthase that is active in vivo, contrary to sinefungin and other analogs that are only active on the isolated enzyme.     

Biosynthesis in Escherichia coli of sn-glycerol 3-phosphate, a precursor of phospholipid. Palmitoyl-CoA inhibition of the biosynthetic sn-glycerol-3-phosphate dehydrogenase.

Homogeneous biosynthetic sn-glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) of Escherichia coli was potently inhibited by palmitoyl-CoA and other long chain acyl-CoA thioesters. The concentration dependence of this inhibition was not cooperative. Enzyme activity was inhibited 50% at 1 microM palmitoyl-CoA; thus, this inhibition occurred at concentrations below the critical micellar concentration of palmitoyl-CoA. Palmitoyl-CoA was a reversible, noncompetitive inhibitor with respect to both NADPH and dihydroxyacetone phosphate. Palmitoyl-CoA did not affect the quaternary structure of the enzyme. This inhibition could be prevented or reversed by the addition of phospholipid vesicles prepared from E. coli phospholipids. Palmitoyl-CoA did not alter the kinetics of inhibition by sn-glycerol 3-phosphate, which is a proven physiological regulator of this enzyme. Decanoyl-CoA, dodecanoyl-CoA, myristoyl-CoA, palmitoyl-(1,N6-etheno)CoA, stearoyl-CoA, and oleoyl-CoA inhibited sn-glycerol-3-phosphate dehydrogenase at concentrations below their critical micellar concentrations. Palmitate inhibited sn-glycerol-3-phosphate dehydrogenase activity 50% at 200 microM. Palmitoyl-carnitine, deoxycholate, taurocholate, and dodecyl sulfate were more potent inhibitors than Triton X-100, Tween-20, or Tween-80. Palmitoyl-acyl carrier protein at concentrations up to 50 microM had no effect on sn-glycerol-3-phosphate dehydrogenase activity. The possible physiological role of long chain fatty acyl-CoA thioesters in the regulation of sn-glycerol 3-phosphate and phospholipid biosynthesis in E. coli is discussed.     

Phospholipid-enzyme interactions of chitin synthase of Coprinus cinereus

Abstract The effect of phospholipids on chitin synthase activity has been studied with digitonin-solubilized and partially purified preparations from Coprinus cinereus . When cholate was used as detergent, it inhibited enzyme activity, but this inhibition was reversed by increasing concentrations of phospholipids. Preincubation with cholate and phospholipid caused irreversible loss of activity. When sonicated with solubilized enzyme preparation, dimyristoyl phosphatidyl choline strongly stimulated activity, while dioleoyl phosphatidyl choline was inhibitory. The Arrhenius plot of the effect of temperature on enzyme activity contained breaks, characteristic of a membrane-bound enzyme. It is suggested that chitin synthase requires an annulus of phospholipids for activity.     

The side-chain cleavage of cholesterol sulfate--III. The effect of adrenodoxin, membrane phospholipids and Tween 80 on the kinetics of oxidation of the sterol sulfate by a reconstituted cholesterol desmolase system

This paper reports the Km values of a reconstituted cholesterol side-chain cleavage system for cholesterol sulfate, cholesterol, and adrenodoxin, determined under several experimental conditions. The Km values for adrenodoxin change depending on whether cholesterol or its sulfate is used as the substrate. Moreover, the Km values for both of the substrates and for adrenodoxin are greatly modulated by both membrane phospholipids, isolated from adrenal mitochondria, and Tween 80, 0.002%. In the absence of detergents or phospholipids, the enzyme system shows a high affinity for cholesterol sulfate, but is inhibited when high concentrations of the sterol sulfate are added to the incubation mixture. Raising the concentration of adrenodoxin in the assay mixture prevents the substrate inhibition. When cholesterol sulfate is incorporated into micelles containing the phospholipids, the enzyme system does not display substrate inhibition, and the kinetics of cleavage of the sterol sulfate are relatively independent of the concentration of adrenodoxin in the assay mixture. In the absence of phospholipids, the apparent kinetics of cleavage of cholesterol and its sulfate are quite different from each other, but when incorporated into micelles containing phospholipids, the kinetics of cleavage of the two substrates are similar to each other.     

Correlation between the effect of the anti-neoplastic ether lipid 1-O-octadecyl-2-O-methyl-glycero-3-phosphocholine on the membrane and the activity of protein kinase Calpha.

The antineoplastic ether phospholipid 1-O-octadecyl-2-O-methyl-sn-glycero-3-phophocholine (ET-18-OCH3) was incorporated into dimyristoylglycerophosphocholine (Myr2Gro-PCho)/dimyristoylglycerophosphoserine (Myr2Gro-PSer) (4 : 1 molar ratio) mixtures. Electron microscopy showed that the addition of ET-18-OCH3 reduced the size of the vesicles. Small vesicles could be detected even at 60 mol% ET-18-OCH3. Sedimentation studies showed the increasing presence of phospholipids in the supernatant, while turbidity measurements indicated a decrease in absorbance as the ET-18-OCH3 concentration was increased. These findings may be explained by the formation of small vesicles and/or mixed micelles. Infrared spectroscopy showed that at 60 mol% the fluidity of the membrane was considerably increased at temperatures below the phase transition, with only a small increase in the proportion of gauche isomers after the gel-to-fluid phase transition of this sample. On the other hand, protein kinase Calpha (PKCalpha) activity progressively decreased when ET-18-OCH3 was incorporated into multilamellar vesicles, reaching a minimum value at 20 mol%, this inhibition being attributed to the modification of the membrane produced by a cone-shaped molecule. At higher concentrations, however, ET-18-OCH3 activated the enzyme with a maximum being attained at 50 mol%. This activation being attributed to the formation of small vesicles and/or micelles. At still higher concentrations of ET-18-OCH3 the enzyme was once again inhibited, inhibition being almost complete at 80 mol%. When PKC was assayed using large unilamellar vesicles a slight activation was observed at very low ET-18-OCH3 concentrations.     

Studies on the purified, lipid-reconstituted (Na+ + Mg2+)-ATPase from Acholeplasma laidlawii B membranes. Dependence of enzyme activity on lipid headgroup and hydrocarbon chain structure

The purified (Na+ + Mg2+)-ATPase from Acholeplasma laidlawii B membranes was successfully reconstituted with a number of different phospho- and glycolipids, and the ability of these lipids to support the function of this enzyme was evaluated by their ability to increase the specific activity of the purified enzyme and by their ability to restore its lipid-phase state-dependent properties which were lost during purification. The incorporation of this ATPase into liposomes composed of the endogenous membrane lipids of the organism, or of zwitterionic phospholipids such as phosphatidylcholine or phosphatidylethanolamine, results in a full reconstitution of its activity and its lipid-phase state-dependent properties. In contrast, anionic phospholipids alone, or in combination with zwitterionic phospholipids at concentrations higher than 10 mol % of the anionic phospholipid, cause an irreversible inhibition of this ATPase. However, when combined with neutral glycolipids, larger amounts of anionic phospholipid can be tolerated without enzyme inhibition. Phosphatidylcholines with acyl chains of 14-24 linear carbon atoms and varying degrees of branching and unsaturation successfully reconstitute the enzyme, in marked contrast to the shorter chain homologues, which were ineffective. Our results indicate that the full expression of the activity of the A. laidlawii B ATPase requires a host lipid bilayer membrane of low to moderate negative surface charge which is predominantly liquid-crystalline and of a minimal bilayer thickness. Once such requirements are met, the enzyme exhibits considerable flexibility regarding the nature of the lipids which can effectively support its function. In particular, the activity of the A. laidlawii B ATPase is not very sensitive to lipid "fluidity" in the liquid-crystalline state.     

Effect of acidic phospholipids on the structural properties of recombinant cytosolic human glyoxalase II

A peculiar characteristic of highly concentrated cytosolic recombinant human glyoxalase II (GII) solutions is to undergo partial precipitation. Previous work indicated that anionic phospholipids (PLs) exert a noncompetitive inhibition on the enzymatic activity of the soluble enzyme. In this study, FTIR spectroscopy was used to analyze the structural properties and the thermal stability of the soluble protein in the absence and in the presence of liposomes made of different phospholipids (PLs). The structural analysis was performed on the precipitate as well. The interaction of acidic PLs with GII lowered the thermal stability of the enzyme and inhibited protein intermolecular interactions (aggregation) brought about by thermal denaturation. Infrared data indicated that ionic and hydrophobic interactions occur between GII and acidic PLs causing small changes in the secondary structure of the enzyme. No interactions of the protein with egg phosphatidylcholine liposomes were detected. The results are consistent with the destabilization of the protein tertiary structure, and indicate that GII possesses hydrophobic part(s) that interact with the acyl chains of PLs. Data on precipitated GII did not show remarkable modification of secondary structure, suggesting that hydrophobic stretches of the enzyme may also be involved in the protein-protein association (precipitation) at high GII concentration. The alterations in the GII structure and the noncompetitive inhibition exerted by acidic PLs are strictly related.