Preparation and partial characterization of a soluble site-to-site directed enzyme complex composed of alcohol dehydrogenase and lactate dehydrogenase

A soluble, bifunctional enzyme complex has been prepared by crosslinking lactate dehydrogenase and alcohol dehydrogenase with glutaraldehyde. The crosslinking was performed on a solid phase while the active sites of alcohol dehydrogenase and lactate dehydrogenase were held adjacent to one another with the aid of a bis-NAD analog. Subsequently, the enzyme complex was released from the solid phase. The soluble enzyme complex was then purified by using NAD-Sepharose as an affinity adsorbent. Based on gel filtration experiments, the complex was estimated to consist of one of each dehydrogenase. By using a third enzyme, lipoamide dehydrogenase, which competes with lactate dehydrogenase for NADH produced by alcohol dehydrogenase, the effect of site-to-site orientation was studied. It was found that about 83% of the NADH produced by alcohol dehydrogenase was oxidized by site-to-site oriented lactate dehydrogenase compared to a figure of only about 61% obtained in an identical system of separate enzymes. This indicates that given two alternative routes, the preference for the one to lactate dehydrogenase over the one to lipoamide dehydrogenase is enhanced when lactate dehydrogenase and alcohol dehydrogenase are site-to-site oriented.     

Purification of Alcohol Dehydrogenase from Saccharomyces cerevisiae Using Magnetic Dye-Ligand Affinity Nanostructures

Reactive Green 19 was covalently immobilized onto magnetic nanostructures for purification of alcohol dehydrogenase from Saccharomyces cerevisiae. The Reactive Green 19 immobilized magnetic nanostructures were characterized by Fourier transform infrared spectroscopy, electron spin resonance, atomic force microscope, and energy dispersive X-ray analysis. Particle size of nanostructures was found to be roughly 70 nm. Alcohol dehydrogenase adsorption experiments were investigated under different conditions in batch system (i.e., medium pH, alcohol dehydrogenase concentration, temperature, and ionic strength). Maximum alcohol dehydrogenase adsorption capacity was found to be 176.09 mg/g polymer while nonspecific alcohol dehydrogenase adsorption onto plain magnetic nanostructures was negligible (19.4 mg/g polymer). Alcohol dehydrogenase molecules were desorbed by using 1.0 M NaCl with 98.4 % recovery. Alcohol dehydrogenase from S. cerevisiae was purified 45.63-fold in single step with dye-immobilized magnetic nanostructures, and purity of alcohol dehydrogenase was shown by silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

A chemiluminometric method for NADPH and NADH using a two-enzyme bioreactor and its application to the determination of magnesium in serum

Chemiluminometric methods are described for the automated flow injection analysis of NADPH and NADH using an immobilized enzyme column reactor and serum magnesium. This application is for the clinical analysis of NADPH and NADH. The reactor for NADPH and NADH contains immobilized L-glutamate dehydrogenase and L-glutamate oxidase, and that for serum magnesium immobilized hexokinase, glucose-6-phosphate dehydrogenase, L-glutamate dehydrogenase and L-glutamate oxidase. When the sample is introduced into the four-enzyme bioreactor, hydrogen peroxide is produced in proportion to the concentration of serum magnesium by the successive reactions. A co-immobilized hexokinase/glucose-6-phosphate dehydrogenase/glutamate dehydrogenase column reactor gave better efficiency compared with an enzyme column which was prepared by packing co-immobilized hexokinase/glucose-6-phosphate dehydrogenase and immobilized glutamate dehydrogenase to make two layers. Magnesium in serum was determined with 1 microL of the sample without carry-over and for an assay time of approximately 15 s. The present method is sensitive (detection limit 0.1 nmol) because Mg2+ is recycled in a column, and gives perfect linearity of the data up to 3.0 mmol/L with satisfactory precision, reproducibility, and accurate reaction recoveries.     

Application of high-performance liquid chromatography to the purification, disintegration and molecular mass determination of pyruvate dehydrogenase multi-enzyme complexes from different sources

The pyruvate dehydrogenase complex is associated with the inner mitochondrial membrane. A gentle and rapid purification procedure, especially for the very unstable pyruvate dehydrogenase complex from the extremely thermophilic organism Thermus aquaticus, is described. This procedure is based essentially on a combination of hydrophobic interaction and of adsorption chromatography by the rapid fast protein liquid chromatographic technique. Applying the same method, a relative molecular mass of 9.1 . 10(6) daltons was obtained by gel filtration on Superose 6 HR 10/30 for the pyruvate dehydrogenase complex from T. aquaticus. The same column served to resolve the pyruvate dehydrogenase complex into its enzyme components.     

Determination of lipoyllysine derived from enzymes by liquid chromatography

Lipoyllysine was liberated from the commercial enzymes bovine heart alpha-ketoglutarate dehydrogenase and pyruvate dehydrogenase. After incubation of the enzymes with pronase for 4 h, the lipoyllysine liberated was determined by high-performance liquid chromatography with ultraviolet detection at 340 nm. Standard lipoyllysine was synthesized in our laboratory. The specific determination of lipoyllysine with ultraviolet detection only at 340 nm could be utilized for the enzyme hydrolysate samples. Recoveries of lipoyllysine added (5.0 micrograms) to a reaction mixture containing protease and bovine serum albumin or ovalbumin model proteins (1.0 mg) were 116.8 and 119.5%, respectively. The lipoyllysine content in beef heart alpha-ketoglutarate dehydrogenase was 0.55 microgram/mg of enzyme and 0.83 microgram/mg of enzyme protein in beef heart pyruvate dehydrogenase.     

Ethanol elimination in regenerating rat liver: the roles of alcohol dehydrogenase and acetaldehyde.

The rate of ethanol elimination in vivo was studied with rats in which the energy consumption of the liver was increased by partial hepatectomy. Immediately after partial hepatectomy the activity of alcohol dehydrogenase in the liver remnant was not changed from that of the livers of sham-operated controls, but the rate of ethanol removal was significantly faster. Twenty-four h after the partial hepatectomy the activity of alcohol dehydrogenase was only 48 % of the activity measured in unoperated control rats. Therefore it is concluded that in normal liver the activity of ADH is in excess. In partially hepatectomized rats the rate of ethanol elimination was linearly correlated with the activity of alcohol dehydrogenase, which suggests that when the rate of NADH reoxidation is markedly increased, as in regenerating rat liver, the rate of ethanol elimination may be limited by the activity of alcohol dehydrogenase. The activity of aldehyde dehydrogenase and the concentration of acetaldehyde in the tail blood were not significantly changed from the level of unoperated rats during oxidation of ethanol.     

基于可见吸收信号的乳酸脱氢酶光纤传感器

报道一种测定乳酸脱氢酶活力的基于可见吸收信号的光纤生物传感器，在该传感体系中，通过辅酶Ｉ的氧化还原对（ＮＡＤ＾＋／ＮＡＤＨ）将乳酸脱氢酶和心肌黄酶催化的两个脱氢过阳以耦合，第一个脱氢过程对分析对象进行了化学识别，第二个脱氢过程引起可见吸收信号的变化，该传感器对０～４００Ｕ／Ｌ的乳酸脱氢酶有线性响应关系，检测下限为４８ＵＬ，该传感器已用于人体血清中乳酸脱氢酶活力的测定。     

Strategy for the isolation of native dehydrogenases with potential for biosensor development from the organism Hyphomicrobium zavarzinii ZV580

Dehydrogenases are interesting candidates for the development of electrochemical biosensors. Most dehydrogenases are characterised by a comparatively broad substrate spectrum, yet highly specific enzymes exist as well. A specific formaldehyde dehydrogenase has, e.g., been described for the organism Hyphomicrobium zavarzinii ZV580. Isolation of enzymes from their natural source instead of a recombinant expression renders the isolation more challenging, as common tools such as affinity tags are no longer available. In this contribution, we develop chromatographic procedures for such isolation tasks. The previously described formaldehyde dehydrogenase was isolated by two procedures, one based on affinity chromatography, the other on hydroxyapatite. Neither procedure yielded an active enzyme. In addition two dehydrogenases, a formaldehyde and a methylamine dehydrogenase, were found in the cell free extract, which had not been described previously. Both enzymes could be isolated to near purity by a sequence of hydroxyapatite and anion exchange chromatography. The new formaldehyde dehydrogenase requires reconstitution with calcium and pyrroloquinoline quinone in order to become active. The enzyme shows no cross-reactivity with methylamine or methanol. The methylamine dehydrogenase catalyses the conversion of methylamine into formaldehyde, hence it could become a technical catalyst for the inverse reaction. This enzyme consists of two types of subunit and may be one of the rare alpha,beta-methylamine dehydrogenases.     

The Pyruvate Dehydrogenase Multienzyme Complex

The three enzymes pyruvate dehydrogenase, dihydrolipoamide transacetylase, and dihydrolipoamide dehydrogenase constitute the pyruvate dehydrogenase multienzyme complex of E. coli; in mammals the complex also contains a kinase and a phosphatase. Multienzyme complexes are structural, functional, and regulatory units enabling the organism to operate more economically than with single enzymes. The pyruvate dehydrogenase multienzyme complex may stand at the switch-point between energy metabolism and gluconeogenesis.     

THE PHOTOMORPHOGENETIC CONTROL OF THE DEVELOPMENT OF PLASTID GLYCERALDEHYDE-PHOSPHATE DEHYDROGENASE AND PHOSPHORIBULOKINASE ACTIVITIES IN COTYLEDONS OF MUSTARD SEEDLINGS

The plastid glyceraldehyde-phosphate dehydrogenase and phosphoribulokinase of mustard cotyledon extracts were activated by preincubation with ATP and with ATP and dithiothreitol respectively. By in vitro activation prior to assay, it was possible to determine the potential activities, which appear to have been directly proportional to the amount of each enzyme protein present. In this way it was possible to deduce the net synthesis of these two enzymes. The induction of synthesis of glyceraldehyde-phosphate dehydrogenase and phosphoribulokinase, by continuous far red or white light were similar hut net synthesis in continuous far red continued longer for glyceraldehyde-phosphate dehydrogenase than for phosphoribulokinase. The kinetics of the development of the glyceraldehyde-phosphate dehydrogenase potential activity were very close to those reported by Bruning et al. (1975) for this enzyme. The data do not permit elimination of either the single switch or multiple switches hypotheses for the action of phytochrome. Continuous white illumination gave results similar to those for continous far red for the net synthesis of the two enzymes but it was more effective than far-red in bringing about enzyme activation in vivo.     

Determination of dehydrogenase substrates by Clark-type oxygen electrodes and photosensitized coenzyme oxidation

The photosensitized oxidation of NADPH by oxygen can be used for the determination of the reduced coenzymes by means of a Clark oxygen electrode. This method is suitable for coupling to enzyme-catalysed dehydrogenation reactions and thus for the determination of glucose-6-phosphate with glucose-6-phosphate dehydrogenase and of glucose with the combined ATP/hexokinase/glucose-6-phosphate dehydrogenase system, even with the use of immobilized mediators.     

Determination of molecular mass, Stokes' radius, frictional coefficient and isomer-type of non-denatured proteins by time-dependent pore gradient gel electrophoresis

Molecular mass, Stoke's radius, frictional coefficient and isomer-type of non-denatured proteins can be obtained by time-dependent gradient gel electrophoresis by evaluating the resulting data using a two-step mathematical procedure. Provided a histochemical staining procedure is available to locate the position of an enzyme in the gel, crude cell extracts can be used for estimating their molecular size properties. The computation of molecular properties of non-denatured proteins is demonstrated for isozymes of aspartate aminotransferase (EC 2.6.1.1), peroxidase (EC 1.11.1.42) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) from current-year needles of spruce. The resulting data as well as those which were calculated for esterase (EC 3.1.1.1), glutamate dehydrogenase (EC 1.4.1.4), isocitrate dehydrogenase (EC 1.4.1.42), and shikimate dehydrogenase (EC 1.1.1.25) are in accordance with those reported in the literature. The method described may be applied to various scientific areas such as genetics or environmental pollution. It could be shown here that current-year needles of injured spruce (damage class 3) contained two more peroxidase isozymes and one more glucose-6-phosphate dehydrogenase isozyme than those from non-injured trees. These differences may mark two genotypes of spruce of different susceptibilities towards present-day air and soil pollutants.     

Enzymatic synthesis of (R) and (S) 1-deuterohexanol

This paper describes practical enzymatic procedures for the synthesis of (R) and (S) 1-deuterohexanol, a useful building block for chiral poly isocyanated liquid crystals. Alcohol dehydrogenases from horse liver and Pseudomonas catalyzed the reduction of hexanal with deuterated NAD (NADD) resulting in 50% and 89% yields of (R) and (S) 1-deuterohexanol, respectively. The deuterated cofactor was regenerated in situ by alcohol dehydrogenase catalyzed oxidation of ethanol-d6 or 2-propanol-d8. The (S) alcohol was also synthesized by the horse liver alcohol dehydrogenase reduction of 1-deuterohexanal, which was prepared chemically from hexanal. The yields of the reaction were greatly increased by the use of a biphasic system or with the immobilized enzyme in anhydrous organic solvents. Horse liver alcohol dehydrogenase was stabilized by immobilization on PAN or noncovalent entrapment on XAD resin.     

Principles of multienzyme purifications by affinity chromatography

Recently in our laboratory, up to 20 different enzymes and their genetic variants have been purified from mouse andDrosophila by affinity chromatography. By virtue of the specific coenzyme requirements, up to ten different enzymes could be copurified from a single tissue extract either by biospecific elutions with different coenzymes or inhibitors, or by sequential passages of the extract through several cofactor-related affinity columns. Important principles were developed to purify enzymes exhibiting low affinity to the affinity columns. By “affinity filtration” of the extract through the affinity column, enzymes of low affinity can be retarded and separated effectively from strongly bound and nonadsorbed proteins. By the “saturation readsorption” procedure, enzymes of low affinity could be effectively separated from those of high affinity by overloading of the extracts on the affinity columns. Readsorption of the leaked low affinity enzymes to a second affinity column often results in better enzyme purification because of the elimination of competitive high affinity enzymes. With the application of these principles, the following enzymes and their genetic variants were highly purified via a single- or two-step affinity column procedure: lactate dehydrogenase-A, lactate dehydrogenase-B, lactate dehydrogenase-X, phosphoglycerate kinase-A, phosphoglycerate kinase-B, cytoplasmic and mitochondrial isocitrate dehydrogenase, malate dehydrogenase, malic enzyme, glucose-6-phosphate dehydrogenase, glutathione reductase, phosphoglucose isomerase and pyruvate kinase from mouse tissues; alcohol dehydrogenase, malate dehydrogenase, α-glycerol-phosphate dehydrogenase, malic enzyme, and glucose-6-phosphate dehydrogenase fromDrosophila.     

Purification and characterization of three forms of class III alcohol dehydrogenase

We have resolved and characterized three forms of human and rat hepatic class III alcohol dehydrogenase. Separations were carried out in narrow immobilized pH gradients. Both in humans and rats the three forms were visualized by enzyme staining with cinnamol, but not with ethanol. They were insensitive to the inhibitory effect of pyrazole. The isoelectric points were approximately from 6.3-6.4, from 5.9-6.0 and 5.6. Each electroeluted enzyme extract, purified further by analytical isoelectric focusing over the pH range from 5-6 or 6-7, revealed a single band by enzyme and silver staining and by Western blotting followed by avidin-biotin staining. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate of each extract revealed a single molecular mass species corresponding to class III alcohol dehydrogenase (ADH). All forms of class III alcohol dehydrogenase were recognized by antisera raised against total class III ADH.     

镍离子与酵母乙醇脱氢酶的相互作用

不同体系中, 金属离子与蛋白以不同的结合方式相互作用. 酵母乙醇脱氢酶是一个含锌金属酶, 它可催化乙醇脱氢为乙醛的反应. 本文应用紫外-可见光谱、荧光光谱、差示扫描量热法等技术研究了二价镍离子与酵母乙醇脱氢酶的相互作用. 镍离子与酶结合后在320 nm出现了紫外吸收带, 同时荧光光谱反映了酶的构象变化, 紫外与荧光光谱均展现了结合过程的双相动力学. 镍离子与酶的相互作用导致了酶由四聚体向二聚体的解离; 在酶热变性过程中, 镍离子增加了乙醇脱氢酶的变性温度和变性焓. 研究工作揭示了镍离子与酶相互作用复杂和深层的作用机制.     

Interaction of NAD-dependent dehydrogenases with human erythrocyte membranes

Interaction of D-glyceraldehyde-3-phosphate dehydrogenase (GPDH) and ladate dehydrogenase with human erythrocyte membranes was studied. Under the conditions of low ionic strength, both enzymes bound to the membranes with similar affinities (Kd ≈ 1 μM). The binding was accompanied by complete inhibition of GPDH and by a 65–75% inhibition of ladate dehydrogenase (LDH). Increasing the ionic strength to physiologically meaningful values (0.15M) completely abolished the inactivation of both dehydrogenases in the presence of erythrocyte membranes, but did not preclude their binding. These results suggest that different modes of enzyme-membrane interaction can be realized under the conditions of low and high ionic strength. They also indicate that GPDH and LDH are capable of functioning in a membrane-bound state.     

Central Carbon Metabolism in Marine Bacteria Examined with a Simplified Assay for Dehydrogenases

A simplified assay platform was developed to measure the activities of the key oxidoreductases in central carbon metabolism of various marine bacteria. Based on microplate assay, the platform was low-cost and simplified by unifying the reaction conditions of enzymes including temperature, buffers, and ionic strength. The central carbon metabolism of 16 marine bacteria, involving Pseudomonas, Exiguobacterium, Marinobacter, Citreicella, and Novosphingobium were studied. Six key oxidoreductases of central carbon metabolism, glucose-6-phosphate dehydrogenase, pyruvate dehydrogenase, 2-ketoglutarate dehydrogenase, malate dehydrogenase, malic enzyme, and isocitrate dehydrogenase were investigated by testing their activities in the pathway. High activity of malate dehydrogenase was found in Citreicella marina, and the specific activity achieved 22 U/mg in cell crude extract. The results also suggested that there was a considerable variability on key enzymes’ activities of central carbon metabolism in some strains which have close evolutionary relationship while they adapted to the requirements of the niche they (try to) occupy.     

辅酶NADH与色氨酸共振能量转移的荧光动力学研究

采用时间相关单光子计数技术, 结合紫外-可见吸收光谱和稳态荧光光谱, 对不同环境下的色氨酸和辅酶还原型烟酰胺腺嘌呤二核苷酸(NADH)之间的共振能量转移荧光动力学进行了研究. 单体色氨酸、 牛血清白蛋白以及乳酸脱氢酶蛋白与NADH之间相互作用的光谱数据表明, 只有存在NADH结合位点的乳酸脱氢酶和NADH之间发生了荧光共振能量转移. 进一步通过加入丙酮酸来阻断乳酸脱氢酶和NADH之间的荧光共振能量转移通道, 时间分辨荧光光谱和衰减相关光谱(DAS)证实, 蛋白结合位点的存在是NADH和色氨酸之间发生荧光共振能量转移的前提条件. DAS揭示了乳酸脱氢酶平均荧光寿命的减小主要是源于色氨酸中7.35 ns的荧光寿命成分与NADH之间的荧光共振能量转移, 同时给出了NADH和色氨酸之间的能量转移效率, 为研究NADH和蛋白之间的相互作用提供了新思路.     

EFFECT OF LIGHT ON OSCILLATIONS OF ENZYME ACTIVITY DURING PHOTOMORPHOGENESIS IN CHENOPODIUM RUBRUM L.

An approach to determine the photomorphogenic effect of light (white or continuous far-red) on the development of rhythmic enzyme activity in Chenopodium rubrum L. is described. Previous results, obtained from mature seedlings grown in white light, demonstrated stable oscillations with periods ranging between 12 and 15 h for all of the enzymes tested. The present results, obtained during deetiolation, were complicated by the presence of a higher frequency component with a period of about 6 h. When the various oscillating components were defined, the analysis showed: (1) the enzymes of the Krebs cycle (malate and isocitrate dehydrogenase), the closely associated glutamate dehydrogen-ase, and the glycolytic pathway ((NAD) glyceraldehyde-3-phosphate dehydrogenase) had a dominant period in the range of 12–15 h, (2) those of the oxidative pentose phosphate pathway were either weakly circadian (glucose-6-phosphate dehydrogenase) or apparently arhythmic (6-phosphogluconate dehydrogenase), (3) the (NADP) glyceraldehyde-3-phosphate dehydrogenase from the Calvin cycle was circadian when kept in continuous darkness but becomes 15 h when placed in light, and (4) only the Calvin cycle enzyme is affected by light in the level of its activity and in its oscillatory behavior.