Rabbit liver alcohol dehydrogenase: purification and properties

Alcohol dehydrogenase [EC 1.1.1.1] was purified to homogeneity from rabbit liver by water extraction, DEAE-cellulose treatment, affinity chromatography on 5'-AMP-Sepharose and gel filtration on Sephadex G-150 using dithiothreitol as a stabilizer. The purified enzyme has an estimated molecular weight of 72,000 and consists of two subunits with a molecular weight of about 36,000 each. The enzyme contains 4 g-atoms of zinc and 18 sulfhydryl groups per mol of protein and exhibits maximal activity at pH 10.8, with a second maximum at pH 7.5. The apparent Km values for ethanol and NAD+ are 0.45 mM and 53.19 microM, respectively, at pH 10.8 and 3.33 mM and 6.94 microM, respectively, at pH 7.5. The enzyme oxidizes ethanol most readily among the aliphatic alcohols studied and has very low substrate specificity for methanol. Among steroid alcohols, 5 beta-androstan-3 beta-ol-17-one serves as a substrate for the enzyme. Pyrazole and 4-methylpyrazole (which are well known alcohol dehydrogenase inhibitors), sulfhydryl reagents, heavy metal ions and metal-chelating agents inactivate the enzyme.     

Purification and some properties of cytoplasmic aspartate aminotransferase from sheep liver

1. A procedure for the purification of the cytoplasmic isoenzyme of aspartate aminotransferase from sheep liver is described. 2. The purified isoenzyme shows a single component in the ultracentrifuge at pH7.6 and forms a single protein band on agar-gel electrophoresis at pH6.3 or 8.6, as well as when stained for protein or activity after polyacrylamide-gel or cellulose acetate electrophoresis at pH8.8. 3. Immunoelectrophoresis on agar gel yields only one precipitin arc associated with the protein band, with rabbit antiserum to the purified isoenzyme. By immunodiffusion, cross-reaction was detected between the cytoplasmic isoenzymes from sheep liver and pig heart, but not between the cytoplasmic and mitochondrial sheep liver isoenzymes. 4. The s(20,w) of the enzyme is 5.69S and the molecular weight determined by sedimentation equilibrium is 88900; 19313 molecules of oxaloacetate were formed/min per molecule of enzyme at pH7.4 and 25 degrees C. 5. The amino acid composition of the isoenzyme is presented. It has about 790 residues per molecule. 6. The holoenzyme has a maximum of absorption at 362nm at pH7.6 and 25 degrees C. 7. A value of 2.1 was found for the coenzyme/enzyme molar ratio. 8. The purified enzyme revealed two bands of activity on polyacrylamide-gel electrophoresis at pH7.4 and an extra, faster, band in some circumstances. These bands occurred even when dithiothreitol was present throughout the isolation procedure. 9. Three main bands were obtained by electrofocusing on polyacrylamide plates with pI values 5.75, 5.56 and 5.35. 10. Structural similarities with cytoplasmic isoenzymes from other organs are discussed.     

Purification and characterization of a novel form of 20 alpha-hydroxysteroid dehydrogenase from Clostridium scindens.

We have purified a steroid-inducible 20 alpha-hydroxysteroid dehydrogenase from Clostridium scindens to apparent homogeneity. The final enzyme preparation was purified 252-fold, with a recovery of 14%. Denaturing and nondenaturing polyacrylamide gradient gel electrophoresis showed that the native enzyme (Mr, 162,000) was a tetramer composed of subunits with a molecular weight of 40,000. The isoelectric point was approximately pH 6.1. The purified enzyme was highly specific for adrenocorticosteroid substrates possessing 17 alpha, 21-dihydroxy groups. The purified enzyme had high specific activity for the reduction of cortisone (Vmax, 280 nmol/min per mg of protein; Km, 22 microM) but was less reactive with cortisol (Vmax, 120 nmol/min per mg of protein; Km, 32 microM) at pH 6.3. The apparent Km for NADH was 8.1 microM with cortisone (50 microM) as the cosubstrate. Substrate inhibition was observed with concentrations of NADH greater than 0.1 mM. The purified enzyme also catalyzed the oxidation of 20 alpha-dihydrocortisol (Vmax, 200 nmol/min per mg of protein; Km, 41 microM) at pH 7.9. The apparent Km for NAD+ was 526 microM. The initial reaction velocities with NADPH were less than 50% of those with NADH. The amino-terminal sequence was determined to be Ala-Val-Lys-Val-Ala-Ile-Asn-Gly-Phe-Gly-Arg. These results indicate that this enzyme is a novel form of 20 alpha-hydroxysteroid dehydrogenase.     

Studies on the Xylanase from Streptomyces

Xylanase from Streptomyces xylophagus nov. sp. has been purified by ammonium sulfate fractionation and chromatography on DEAE-cellulose column. The purification of the enzyme was 276-fold with a yield of 18.6% on the basis of the activity per weight of total nitrogen. The purified enzyme was homogeneous on moving-boundary electrophoresis. Optimum pH and temperature for the enzyme activity were 6.2 and 55~60°C, respectively. The enzyme was stable up to 40°C and in the range of pH from 5.3 to 7.3, but inactivated at higher than 50°C and at extreme pH values of 2.4 and 9.4. Hydrolyzed products of xylan by the enzyme were xylose and xylobiose.     

Studies on Pectolytic Enzymes of Molds

Exopolygalacturonase from Coniothyrium diplodiella has been purified by ammonium sulfate fractionation, chromatography on DEAE-cellulose and column zone electrophoresis. The enzyme was concentrated about 5-fold with a yield of 0.24% on the basis of polygalacturonase activity per weight of total nitrogen. The purified enzyme was homogenous On free-boundary electrophoresis. The enzyme was most active in the pH range 4.0~4.5. The enzyme was stable at 50°C and pH range of 3.5~6.0, but inactivated at higher than 55°C. Hydrolysis of pectic acid by the enzyme went to completion via galacturonic acid liberation from the end of the chain, but pectin was little affected by the enzyme.     

Glyceraldehyde-3-phosphate dehydrogenase of horseshoe crab (Tachypleus tridentatus).

Glyceraldehyde-3-phosphate dehydrogenase [ED 1.2.1.12] was purified from the horseshoe crab, a living fossil, and its properties were examined. 1 The purified enzyme was homogeneous as judged by various tests. The enzyme, like enzymes from other sources, was a tetramer with a subunit molecular weight of 36,000. The kinetic parameters and pH optimum were also similar to those of other enzymes, though the enzyme was more stable against heat and pH denaturations. 2 Analysis of SH groups showed that there were 4 SH groups per subunit, one of which was essential for the enzyme activity and was highly reactive. 3. CD spectra of the enzyme suggested that the enzyme had a very high content of beta-structure (ca. 45 per cent). 4. The horseshoe crab enzyme could form a hybrid in vitro with the rabbit muscle enzymes in concentrated salt solution at acidic pH. 5. There results indicate that the enzyme has overall structural similarity to other enzymes and that the enzyme is highly conserved during a long period of evolution. Some discussions on the structure and activity of the horseshoe crab enzyme are made in comparison with the enzymes from other sources.     

Purification and characterization of the cytochrome oxidase from alkalophilic Bacillus firmus RAB

A cytochrome oxidase was purified 52-fold from membranes of alkalophilic Bacillus firmus RAB by extraction with Triton X-100, ion-exchange and hydroxyapatite chromatography, and gel filtration. On denaturing gels, the purified enzyme dissociated into two subunits of 56,000 and 40,000 Mr as well as a cytochrome c with an Mr of approximately 14,000. Heme contents calculated for an enzyme with a molecular weight of 110,000 were found to be 2 mol of heme a and 1 mol of heme c per mol of cytochrome oxidase; approximately 2 mol of copper per mol of purified enzyme was also found. Enzyme activity was observed in assays using reduced yeast or horse heart cytochrome c. Activity of the purified enzyme was optimal at pH 6.0 and in the presence of added lipids. Impure, membrane-associated activity exhibited a broader pH range for optimal activity extending to alkaline values.     

Purification and characterization of an acid phosphatase that displays phosphotyrosyl-protein phosphatase activity from bovine cortical bone matrix

An acid phosphatase activity that displayed phosphotyrosyl-protein phosphatase has been purified from bovine cortical bone matrix to apparent homogeneity. The overall yield of the enzyme activity was greater than 25%, and overall purification was approximately 2000-fold with a specific activity of 8.15 mumol of p-nitrophenyl phosphate hydrolyzed per min/mg of protein at pH 5.5 and 37 degrees C. The purified enzyme was judged to be purified based on its appearance as a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (silver staining technique). The enzyme could be classified as a band 5-type tartrate-resistant acid phosphatase isoenzyme. The apparent molecular weight of this enzyme activity was determined to be 34,600 by gel filtration and 32,500 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of reducing agent, indicating that the active enzyme is a single polypeptide chain. Kinetic evaluations revealed that the acid phosphatase activity appeared to catalyze its reaction by a pseudo Uni Bi hydrolytic two-step transfer reaction mechanism and was competitively inhibited by transition state analogs of Pi. The enzyme activity was also sensitive to reducing agents and several divalent metal ions. Substrate specificity evaluation showed that this purified bovine skeletal acid phosphatase was capable of hydrolyzing nucleotide tri- and diphosphates, phosphotyrosine, and phosphotyrosyl histones, but not nucleotide monophosphates, phosphoserine, phosphothreonine, phosphoseryl histones, or low molecular weight phosphoryl esters. Further examination of the phosphotyrosyl-protein phosphatase activity indicated that the optimal pH at a fixed substrate concentration (50 nM phosphohistones) for this activity was 7.0. Kinetic analysis of the phosphotyrosyl-protein phosphatase activity indicated that the purified enzyme had an apparent Vmax of approximately 60 nmol of [32P]phosphate hydrolyzed from [32P]phosphotyrosyl histones per min/mg of protein at pH 7.0 and an apparent Km for phosphotyrosyl proteins of approximately 450 nM phosphate group. In summary, the results of these studies represent the first purification of a skeletal acid phosphatase to apparent homogeneity. Our observation that this purified bovine bone matrix acid phosphatase was able to dephosphorylate phosphotyrosyl proteins at neutral pH is consistent with our suggestion that this enzyme may function as a phosphotyrosyl-protein phosphatase in vivo.     

Purification and characterization of extracellular pectate lyase from Bacillus subtilis

Bacillus subtilis strain SO113 secretes a pectate lyase which is produced during the exponential death phase of growth, just before sporulation. This extracellular pectate lyase, which produces unsaturated products from polygalacturonate, was purified 35-fold from the culture supernatant of Bacillus subtilis by a CM Sephadex chromatography. It has an isoelectric point of about 9.6 and an Mr of 42,000. Optimum activity occurred at pH 8.4 and at 42 degrees C. Calcium has a stimulative effect on the enzyme activity while EDTA leads to enzyme inactivation. The pectate lyase has a specific activity of 131 mumol of aldehyde groups per min and per mg of protein. The Km of the purified enzyme for polygalacturonic acid was 0.862 g.l-1 and the Vmax for polygalacturonic acid hydrolysis was 1.475 mumol of unsaturated products per min and per mg of protein. By using monoclonal antibodies raised against Erwinia chrysanthemi 3937 pectate lyases, it was shown that pectate lyases b and c of this strain are immunologically closely related to the Bacillus subtilis pectate lyase.     

In vitro stability of nitrate reductase from wheat leaves: I. Stability of highly purified enzyme and its component activities

NADH-nitrate reductase has been highly purified from leaves of 8-day-old wheat (Triticum aestivum L. cv. Olympic) seedlings by affinity chromatography, using blue dextran-Sepharose 4B. Purification was assessed by polyacrylamide gel electrophoresis. The enzyme was isolated with a specific activity of 23 micromoles nitrite produced per minute per milligram protein at 25 C. At pH 7.5, the optimum pH for stability of NADH-nitrate reductase, this enzyme, and a component enzyme reduced flavin adenine mononucleotide (FMNH₂)-nitrate reductase has a similar stability at both 10 and 25 C. Two other component enzymes—methylviologen-nitrate reductase and NADH-ferricyanide reductase—also have a similar but higher stability. At this pH the Arrhenius plot for decay of NADH-nitrate reductase and methylviologen-nitrate reductase indicates a transition temperature at approximately 30 C above which the energy of activation for denaturation increases. FMNH₂-nitrate reductase and NADH-ferricyanide reductase do now show this transition. The energy of activation for denaturation (approximately 9 kcal per mole) of each enzyme is similar between 15 and 30 C. The optimum pH for stability of the component enzymes was: NADH-ferricyanide reductase, 6.6; FMNH₂-nitrate reductase and methylviologen-nitrate reductase, 8.9. All of our studies indicate that the NADH-ferricyanide reductase was the most stable component of the purified nitrate reductase (at pH 6.6, t_½ [25 C] = 704 minutes). Data are presented which suggest that methylviologen and FMNH₂ do not donate electrons to the same site of the nitrate reductase protein.     

Isolation and characterization of an alkaline phosphatase from pea thylakoids

Endogenous dephosphorylation of the light-harvesting chlorophyll-protein complex of photosystem II in pea (Pisum sativum, L. cv Progress 9) thylakoids drives the state 2 to state 1 transition; the responsible enzyme is a thylakoid-bound, fluoride-sensitive phosphatase with a pH optimum of 8.0 (Bennett J [1980] Eur J Biochem 104: 85-89). An enzyme with these characteristics was isolated from well-washed thylakoids. Its molecular mass was estimated at 51.5 kD, and this monomer was catalytically active, although the activity was labile. The active site could be labeled with orthophosphate at pH 5.0. High levels of alkaline phosphatase activity were obtained with the assay substrate, 4-methylumbelliferyl phosphate (350 micromoles per minute per milligram purified enzyme). The isolated enzyme functioned as a phosphoprotein phosphatase toward phosphorylated histone III-S and phosphorylated, photosystem II-enriched particles from pea, with typical activities in the range of 200 to 600 picomoles per minute per milligram enzyme. These activities all had a pH optimum of 8.0 and were fluoride sensitive. The enzyme required magnesium ion for maximal activity but was not dependent on this ion. Evidence supporting a putative function for this phosphatase in dephosphorylation of thylakoid proteins came from the inhibition of this process by a polyclonal antibody preparation raised against the partially purified enzyme.     

Studies on Lipase from Candida cylindracea

Lipase from Candida cylindracea has been purified by ammonium sulfate precipitation, sodium deoxycholate treatment, ethanol-ether precipitation and chromatography on SE-Sephadex and Sephadex G-100 columns. The purification of the enzyme was 33.4-fold with a yeild of 18.0% on the basis of activity per weight of protein. The purified enzyme was homogeneous on ultracentrifugation and electrophoresis. Optimum pH for the hydrolysis of olive oil was 7.2 by the assay method using a polyvinylalcohol-emulsified system and 5.2 by the assay method using a shaken system without a macromolecular emulsifier. Optimum temperature was 45°C. The enzyme was stable up to 15°G and in the range of pH from 2.0 to 8.5. Sodium taurocholate showed either an activating or an inhibiting effect at pH 7.0, depending on the sodium taurocholate concentration and on the assay system.     

极耐热性b-葡萄糖醛酸酶的高效表达和酶学性质及其 应用

从海栖热袍菌克隆出编码热稳定性b-葡萄糖醛酸酶基因, 以热激载体pHsh为表达质粒, 在大肠杆菌中得到高效表达。基因表达产物通过一步热处理后, 酶纯度达电泳均一。纯化重组酶酶学性质研究表明, b-葡萄糖醛酸酶的最适反应温度为80oC, 最适反应pH为5.0, pH 5.8~ 8.2之间酶的稳定性较好, 80oC的半衰期为2 h, SDS-PAGE结果显示分子量为65.9 kD, 与理论推算值相吻合。以对硝基苯-b-葡萄糖醛酸苷(pNPG)为底物时, 其动力学参数Km值0.18 mmol/L, Vmax值为312 u/mg。初步的应用分析表明, 该重组酶能催化甘草酸转化为甘草次酸。     

Studies on the Enzymes Acting on Araban

Arabanase was purified by various procedures of gel filtrations and column chromatographies from the submerged culture filtrate of Aspergillus niger grown on the medium containing beet-araban as a carbon source, and the properties of the enzyme was examined. The purified enzyme was homogeneous protein in ultracentrifugal analysis, the optimum pH for enzymatic action was 4.0. The enzyme was thermostable at pH 6.0, namely, even after heating at 98°C for 10 minutes, 20.8 per cent of the initial activity still remained. The enzyme hydrolyzed beet-araban with producing l-arabinose.     

D-glucose dehydrogenase from Bacillus megaterium M 1286: purification, properties and structure.

1) Glucose dehydrogenase from Bacillus megaterium has been purified to a specific activity of 550 U per mg protein. The homogeneity of the purified enzyme was demonstrated by gel electrophoresis and isoelectric focusing. 2) The amino acid composition has been determined. 3) The molecular weight of the native enzyme was found to be 116000 by gel permeation chromatography, in good agreement with the values of 120000 and 118000, which were ascertained electrophoretically according to the method of Hedrick and Smith and by density gradient centrifugation, respectively. 4) In the presence of 0.1% sodium dodecylsulfate and 8M urea, the enzyme dissociates into subunits with a molecular weight of 30000 as determined by dodecylsulfate gel electrophoresis. These values indicate that the native enzyme is composed of four polypeptide chains, each probably possessing one coenzyme binding site, which can be concluded from fluorescent titration of the NADH binding sites. 5) In polyacrylamide disc electrophoresis, samples of the purified enzyme exhibit three bands of activity, which present the native (tetrameric) form of glucose dehydrogenase and two monomeric forms (molecular weight 30000), arising under the conditions of pH and ionic strength of this method. 6) The enzyme shows a sharp pH optimum at pH 8.0 in Tris/HCl buffer, and a shift of the pH optimum to pH 9.0 in acetate/borate buffer. The limiting Michaelis constant at pH 9.0 for NAD is 4.5 mM and 47.5 mM for glucose. The dissociation constant for NAD is 0.69 mM. 7) D-Glucose dehydrogenase is highly specific for beta-D-glucose and is capable of using either NAD or NADP. The enzyme is insensitive to sulfhydryl group inhibitors, heavy metal ions and chelating agents.     

Purification and characterization of avian liver 3-hydroxy-3-methylglutaryl coenzyme A lyase

3-Hydroxy-3-methylglutaryl-CoA lyase has been purified to homogeneity from avian liver mitochondria. Affinity chromatography of a partially purified preparation on agarose hexane 3',5'-ADP produces enzyme of high specific activity (351 units/mg). A total purification of 1750-fold over the mitochondrial matrix fraction is achieved. The purified enzyme is stable when stored in 30% glycerol with millimolar levels of dithiothreitol. Divalent cations (e.g. Mg2+, Mn2+) and thiol-protecting agents stimulate enzyme activity under assay conditions. The enzyme binds hydroxymethylglutaryl-CoA with a Km = 8 microM. Optimal enzyme activity, measured at pH = 8.9, is 7-fold higher than activity at physiological pH. The apparent molecular weight of the native enzyme, estimated by gel filtration on Sephadex G-100, is approximately 49,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggests that the enzyme is a dimer, composed of 27,000-dalton subunits. Assuming one active site per subunit, a turnover number of 158 s-1 (pH 8.2; 30 degrees C) is calculated. Antibodies have been prepared against homogeneous hydroxymethylglutaryl-CoA lyase. Ouchterlony double diffusion patterns verify the homogeneity of the preparation. Incubation of enzyme with antiserum results in virtually complete inhibition of enzyme activity.     

Purification and characterization of cathepsin D from herring muscle (Clupea harengus)

Cathepsin D was purified and concentrated 469-fold from a homogenate of Clupea harengus muscle. The purified enzyme is a monomer with a molecular weight of 38000-39000. It is inhibited by pepstatin and has optimal activity at pH 2.5 with hemoglobin as the substrate. The isoelectric point is at pH 6.8. Glycosidase treatment and binding to Concanavalin A indicated that the enzyme contains one N-linked carbohydrate moiety of the high-mannose type per molecule. The first 21 amino acid residues of the N-terminal showed high similarity to cathepsin D from antarctic icefish liver (Chionodraco hamatus) and trout ovary (Oncorhynchus mykiss). Digestion of the beta-chain of oxidized insulin resulted in preferential cleavage at Leu(15)-Tyr(16), (47%), Tyr(16)-Leu(17) (34%) and Ala(14)-Leu(15) (18%). Incubation with myofibrils from herring muscle at pH 4.23 showed that the enzyme mainly degraded myosin, actin and tropomyosin.     

Purification and characterization of a lysine aminopeptidase from Kluyveromyces marxianus

A lysine aminopeptidase was purified from the yeast Kluyveromyces marxianus. This enzyme was purified 100-fold from a soluble extract obtained at 100,000g. The purification procedure consisted in fractionated precipitation with ammonium sulfate and five chromatography steps. The native enzyme had a molecular mass of 46 kDa assessed through gel filtration. This aminopeptidase depicted an optimal pH of 7.0 and was stable at a pH range of 4-8, its optimal temperature was 45 degrees C and the enzyme became unstable at temperatures above 55 degrees C. The isoelectric point of the purified enzyme was 4.4. Michaelis constant and Vmax for L-lysine-p-nitroanilide were 0.33 mM and 2.2 mM min(-1) per milligram of protein, respectively. The enzyme was strongly inhibited by bestatin, o-phenanthroline and, to a lesser extent, by EDTA, suggesting that this enzyme is a metalloprotease. Our results suggest that the lysine aminopeptidase from Kluyveromyces marxianus might be of biotechnological relevance.     

Chemical modification of a xylanase from a thermotolerant Streptomyces. Evidence for essential tryptophan and cysteine residues at the active site.

Extracellular xylanase produced in submerged culture by a thermotolerant Streptomyces T7 growing at 37-50 degrees C was purified to homogeneity by chromatography on DEAE-cellulose and gel filtration on Sephadex G-50. The purified enzyme has an Mr of 20,463 and a pI of 7.8. The pH and temperature optima for the activity were 4.5-5.5 and 60 degrees C respectively. The enzyme retained 100% of its original activity on incubation at pH 5.0 for 6 days at 50 degrees C and for 11 days at 37 degrees C. The Km and Vmax. values, as determined with soluble larch-wood xylan, were 10 mg/ml and 7.6 x 10(3) mumol/min per mg of enzyme respectively. The xylanase was devoid of cellulase activity. It was completely inhibited by Hg2+ (2 x 10(-6) M). The enzyme degraded xylan, producing xylobiose, xylo-oligosaccharides and a small amount of xylose as end products, indicating that it is an endoxylanase. Chemical modification of xylanase with N-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide and p-hydroxymercuribenzoate (PHMB) revealed that 1 mol each of tryptophan and cysteine per mol of enzyme were essential for the activity. Xylan completely protected the enzyme from inactivation by the above reagents, suggesting the presence of tryptophan and cysteine at the substrate-binding site. Inactivation of xylanase by PHMB could be restored by cysteine.     

Purification and some properties of tyrosinase from aspergillus flavipes 56003

A method for isolation and purification of tyrosinase from the fungus Aspergillus flavipes 56003 was developed. The method includes extraction with water, concentration on DEAE-cellulose, gel-filtration on Acrylex P-150, and ion-exchange chromatography on DEAE-Toyopearl 650M. The tyrosinase was purified to apparent homogeneity according polyacrylamide gel electrophoresis and ultracentrifugation. The tyrosinase is a 130-kD protein with pI 4.6. It contains two copper atoms. The Km and Vmax for tyrosine hydroxylation are 0.3 mM and 1300 &mgr;moles/min per mg at pH 6.8, and for dehydrogenation of 3,4-dihydroxyphenylalanine (DOPA) they are 5 mM and 16000 &mgr;moles/min per mg, respectively. Hydroxylation of monophenols has a characteristic lag period. The rate of tyrosine and DOPA oxidation is maximal at pH 6.0-6.8. The half-life of the enzyme at 50 degrees C is 40 min. The hydroxylase activity of the tyrosinase is more stable at neutral pH, whereas the dehydrogenase activity is more stable at acidic pH (4.0). The absorption spectrum of the enzyme has a maximum at 290 mn and a shoulder in the 320-400-nm region.