Purification and some properties of beta-N-acetyl-D-glucosaminidase from the cabbage butterfly (Pieris rapae)

A beta-N-acetyl-D-glucosaminidase (NAGase) from the cabbage butterfly (Pieris rapae) was purified. The purified enzyme was a single band on polyacrylamide gel electrophoresis and the specific activity was determined to be 8715 U/mg. The molecular weight of whole enzyme was determined to be 106 kDa by gel filtration, and the result of SDS-PAGE showed that the enzyme was a heterodimer, which contained two subunits with different mass of 59.5 and 57.2 kDa. The optimum pH and optimum temperature of the enzyme for the hydrolysis of p-nitrophenyl-N-acetyl-beta-D-glucosaminide (pNP-NAG) were investigated to be at pH 6.2 and at 42 degrees C, respectively, and the Michaelis-Menten constant (K(m)) was determined to be 0.285 mM at pH 6.2 and 37 degrees C. The stability of the enzyme was investigated and the results showed that the enzyme was stable at the pH range from 4.0 to 9.0 and at the temperature below 45 degrees C. The activation energy was 83.86 kJ/mol. The reaction of this enzyme with pNP-NAG was judged to be Ordered Bi-Bi mechanism according to the inhibitory behaviors of the products. The ionization constant, pK(e), of ionizing group at the active site of the enzyme was found to be 5.20 at 39.0 degrees C, and the standard dissociation enthalpy (DeltaH(o)) was determined to be 2.18 kcal/mol. These results showed that the ionizing group of the enzyme active center was the carboxyl group. The results of chemical modification also suggested that carboxyl group was essential to the enzyme activity. Moreover, Zn(2+), Hg(2+), Cu(2+) had strongly inhibitory effects on the enzyme activity.     

Molecular characterization of a novel type of prostamide/prostaglandin F synthase, belonging to the thioredoxin-like superfamily

Prostaglandin F (PGF) ethanolamide (prostamide F) synthase, which catalyzed the reduction of prostamide H(2) to prostamide F(2alpha), was found in mouse and swine brain. The enzyme was purified from swine brain, and its amino acid sequence was defined. The mouse enzyme consisted of a 603-bp open reading frame coding for a 201-amino acid polypeptide with a molecular weight of 21,669. The amino acid sequence placed the enzyme in the thioredoxin-like superfamily with Cys(44) being the active site. The enzyme expressed in Escherichia coli as well as the native enzyme catalyzed not only the reduction of prostamide H(2) to prostamide F(2alpha) but also that of PGH(2) to PGF(2alpha). The V(max) and K(m) values for prostamide H(2) were about 0.25 micromol/min.mg of protein and 7.6 microm, respectively, and those for PGH(2) were about 0.69 micromol/min.mg of protein and 6.9 microm, respectively. Neither PGE(2) nor PGD(2) served as a substrate for this synthase. Based on these data, we named the enzyme prostamide/PGF synthase. Although the enzyme showed a broad specificity for reductants, reduced thioredoxin preferentially served as a reducing equivalent donor for this enzyme. Moreover, Northern and Western blot analyses in addition to the prostamide F synthase activity showed that the enzyme was mainly distributed in the brain and spinal cord, and the immunohistochemical study in the spinal cord showed that the enzyme was found mainly in the cytosol. These results suggest that prostamide/PGF synthase may play an important functional role in the central nervous system.     

Invertase reversibly immobilized onto polyethylenimine-grafted poly(GMA–MMA) beads for sucrose hydrolysis

The epoxy group containing poly(glycidyl methacrylate-co-methylmethacrylate) poly(GMA–MMA) beads were prepared by suspension polymerisation and the beads surface were grafted with polyethylenimine (PEI). The PEI-grafted beads were then used for invertase immobilization via adsorption. The immobilization of enzyme onto the poly(GMA–MMA)–PEI beads from aqueous solutions containing different amounts of invertase at different pH was investigated in a batch system. The maximum invertase immobilization capacity of the poly(GMA–MMA)–PEI beads was about 52 mg/g. It was shown that the relative activity of immobilized invertase was higher then that of the free enzyme over broader pH and temperature ranges. The Michaelis constant (K_m) and the maximum rate of reaction (V_max) were calculated from the Lineweaver–Burk plot. The K_m and V_max values of the immobilized invertase were larger than those of the free enzyme. The immobilized enzyme had a long-storage stability (only 6% activity decrease in 2 months) when the immobilized enzyme preparation was dried and stored at 4 °C while under wet condition 43% activity decrease was observed in the same period. After inactivation of enzyme, the poly(GMA–MMA)–PEI beads can be easily regenerated and reloaded with the enzyme for repeated use.     

Purification and characterization of a novel glutathione S-transferase from Atactodea striata

A novel GST isoenzyme was purified from hepatopancreas cytosol of Atactodea striata with a combination of affinity chromatography and reverse-phase HPLC. The molecular weight of the enzyme was determined to be 24 kDa by SDS-PAGE electrophoresis and 48 kDa by gel chromatography, in combination with GST information from literature revealed that the native enzyme was homodimeric with a subunit of M(r) 24 kDa. The purified enzyme, exhibited high activity towards 1-chloro-2,4-dinitrobenzene (CDNB) and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl). Kinetic analysis with respect to CDNB as substrate revealed a K(m) of 0.43 mM and V(max) of 0.24 micromol/min/mg and a specific activity of 108.9 micromol/min/mg. The isoelectric point of the enzyme was 5.5 by isoelectric focusing and its optimum temperature was 38 degrees C and the enzyme had a maximum activity at approximately pH 8.0. The amino acid composition was also determined for the purified enzyme.     

豇豆初生叶多胺氧化酶的催化特性

从豇豆幼苗 (6d苗龄 )初生叶提纯得到的多胺氧化酶 (EC 1 .4.3 .6 )属于二胺氧化酶 ,最有效的底物是 1 ,4 二胺丁烷 (腐胺 )、1 ,5 二胺戊烷 (尸胺 )、1 ,6 二胺己烷、1 ,1 0 二胺癸烷等α 二胺 ,其催化活性随二胺类底物碳链的增长而相应减弱。豇豆多胺氧化酶对亚精胺和精胺也具有较高的催化活性。另外 ,底物腐胺和尸胺的浓度超过 2mmol/L或亚精胺和精胺浓度超过 3mmol/L时会对酶活性有抑制效应。以腐胺和尸胺为底物时 ,酶的最适 pH约为7.0 ,而以亚精胺和精胺为底物时其最适pH为 6 .5。该酶的催化活性还随反应介质的离子强度增加而降低。K ,Ca2 和Mg2 (皆为 1 0mmol/L)对酶活性无明显抑制作用 ,而同样浓度的Mn2 ,Zn2 ,Fe2 ,Co2 和Cd2 则对酶活性有不同程度的抑制作用。金属螯合剂EDTA(1 0mmol/L)和腺苷蛋氨酸脱羧酶抑制剂甲基乙二醛 双脒腙 (0 .1mmol/L)可抑制酶活性约 80 % ,而铜结合剂KCN(1 .0mmol/L)、羰基试剂羟胺 (0 .1mmol/L)和氨基胍 (0 .1mmol/L)可导致该酶完全失活     

Exploration of pressure-induced dissociation of pyruvate oxidase.

The dissociation of pyruvate oxidase (PO) caused by pressure up to 220 MPa at various conditions was explored by measuring the intrinsic fluorescence spectra and polarization. At 5 degrees C and pH 7.6 the standard volume change (deltaV0) and free energy upon dissociation of the enzyme is -220 ml/mol and 29.83 kCal/mol, respectively. It was found that FAD was irreversibly removed during the pressure-dissociation of the enzyme. A much smaller standard volume change (-153 ml/mol) and lower free energy (24.92 kCal/mol) of apo-pyruvate oxidase (apo-PO) compared with the native enzyme indicated that FAD played very important role in stabilizing the enzyme and significantly influenced the standard volume change. The substrate pyruvic acid can significantly stabilize the enzyme against pressure in spite the standard volume for the enzyme in this case has a big increase relative to the native enzyme. The comparison of the intrinsic fluorescence of the native and the activated enzyme obtained by limited proteolysis indicated that the physical separation of alpha-peptide from the enzyme only occurred when the subunits were dissociated from each other under pressure.     

游离及固定化果糖基转移酶部分酶学性质的比较研究

 从诱变、筛选的米曲霉GX0 0 10菌株所产生的果糖基转移酶 ,经过纯化和固定化操作分别制备游离酶和固定化酶 ,对两者的酶学性质进行了比较研究 .结果表明 ,两者在蔗糖转化为蔗果低聚糖的酶促反应中 ,最适pH为 5 5,在pH5 0～ 7 5之间酶活性相对稳定 .游离酶和固定化酶的适宜温度范围分别是 4 5～ 52℃和 4 0～ 55℃ .在 55℃保温 60min ,酶活性保存率分别是 61 6%和 87 5% .固定化酶的热稳定性提高 .0 1mmol LHg2 +和 1mmol LAg+能完全抑制游离酶的活性 ,但只能部分抑制固定化酶的活性 ,1mmol L的Ti2 +能完全抑制两者的活性 .以蔗糖为底物时 ,游离酶的米氏常数Km=2 15mmol L ,而固定化酶Km =386mmol L .游离酶只能使用一次 ,固定化酶反复使用 54次后 ,剩余活力为 55 2 % .用 55% (W V)蔗糖溶液与固定化酶在pH5 0 ,4 6℃下作用 12h ,可获得61 5% (总低聚糖 总糖 )产物 ,其中蔗果五糖含量达到 7 2 % .     

Molecular and catalytic properties of angiotensin converting enzyme-I from bovine seminal plasma

Angiotensin converting enzyme [EC 3.4.15.1] was shown to exist in two distinct forms in bovine seminal plasma. The higher molecular weight form of the enzyme (angiotensin convering enzyme I) was purified to homogeneity by Sephadex G-200 gel filtration, and DEAE-Sepharose, blue Sepharose, and concanavalin A-Sepharose column chromatography. Final recovery of the enzyme was 9.0. The molecular weight of the enzyme was estimated to be 8 x 10(5) by the gel filtration method. A value of 4.6 x 10(5) was obtained for the reduced and denatured enzyme by dodecylsulfate polyacrylamide gel electrophoresis. The Stokes' radius, diffusion coefficient, and intrinsic viscosity of the purified enzyme were determined to be 95 A, 2.3 x 10(-7) cm2/s, and 6.76 ml/g. The enzyme had a specific activity of 105.12 mumol/min/mg protein for hippurylhistidylleucine. The Km value for hippurylhistidylleucine was found to be 20 mM. Studies with EDTA suggest that metal ions which are tightly bound are required for its activity. The enzyme was inhibited by some heavy metal ions but did not required sulfhydryl groups for its activity. Trypsin treatment of the urea-denatured enzyme produced a catalytically active fragment with an Mr of 30,000. Chemical hydrolysis of the native enzyme did not produce any active fragment.     

Studies of the catalytic mechanism of microsomal UDP-glucuronyltransferase. Alpha-glucuronidase activity and its stimulation by phospholipids

The rate at which a specific, purified form of microsomal UDP-glucuronyltransferase (designated as the GT2P type of this enzyme) catalyzes the hydrolysis of UDP-glucuronic acid was measured with pure, delipidated enzyme and enzyme reconstituted with different lysophosphatidylcholines. This activity of the GT2P type of UDP-glucuronyltransferase is referred to as alpha-glucuronidase activity. For delipidated enzyme, the rate of hydrolysis of UDP-glucuronic acid catalyzed by GT2P extrapolated to infinite concentrations of UDP-glucuronic acid was 1 X 10(-9) mol/min/mg of protein. This compares with a rate of glucuronidation of p-nitrophenol of 96 X 10(-9) mol/min/mg of enzyme, for delipidated enzyme. Addition of oleoyl- or myristoyllysophosphatidylcholine to GT2P did not affect the alpha-glucuronidase activity significantly. This activity was stimulated, however, in the presence of compounds that bind at the aglycone site but that do not undergo glucuronidation. alpha-Glucuronidase activity extrapolated to infinite concentration of UDP-glucuronic acid was 4.0 X 10(-9) mol/min/mg for delipidated enzyme assayed in the presence of less than saturating concentrations of p-nitrophenyl phenyl ether. Moreover, when the aglycone site of GT2P was occupied by ethers, the alpha-glucuronidase activity of this enzyme was enhanced by addition of phospholipids to delipidated enzyme. The extent of activation of the alpha-glucuronidase activity of GT2P, when the aglycone site was occupied, depended on the acyl chain of the lipid added to delipidated enzyme. These data indicate that the GT2P form of UDP-glucuronyltransferase catalyzes the hydrolysis of UDP-glucuronic acid at a significant rate and that lysophosphatidylcholines can influence this rate.     

Enzyme-Catalyzed oxidation of cholesterol in physically characterized water-in-oil microemulsions

The enzymatic conversion of cholesterol to cholestenone by cholesterol oxidase (Brevibacterium sp.)in reversed micelles in a system composed of AOT/isooctane/water/cholesterol has been examined. The catalytic activity of the enzyme was correlated with the physicochemical properties of water in water-in-oil (w/o) microemulsion systems. In a system consisting of 3 wt % AOT in isooctane, reversed micelles started to form as the [H(2)O]/[AOT] (e.g., the w(0)) ratio increased above 4-5. The formation of reversed micelles with a core of neat (bulk) water was verified from determinations of both the partial molar volume of water and the scissors vibration of water [with Fourier transform infrared (FTIR) spectroscopy] in the w/o microemulsion systems. A plot of enzyme activity vs. w(0) indicated that the hydration of enzyme molecules per se was not sufficient to give rise to catalytic activity. Instead, it appeared that the formation of an aqueous micellar core was necessary for full activation of the enzyme. Based on micelle size distribution analysis, it was estimated that about one micelle per one thousand contained an enzyme molecule. Since the apparent reaction rate could be markedly enhanced by increasing the enzyme/water ratio, we conclude that the number of enzyme-containing micelles was an important rate-limiting factor in the system.     

Purification and properties of acyl/alkyl dihydroxyacetone-phosphate reductase from guinea pig liver peroxisomes

The peroxisomal acyl/alkyl dihydroxyacetone-phosphate reductase (EC 1.1.1.101) was solubilized and purified 5500-fold from guinea pig liver. The enzyme could be solubilized by detergents only at high ionic strengths in presence of the cosubstrate NADPH. Peroxisomes, isolated from liver by a Nycodenz step density gradient centrifugation, were first treated with 0.2% Triton X-100 to remove the soluble and a large fraction of the membrane-bound proteins. The enzyme was solubilized from the resulting residue by 0.05% Triton X-100, 1 M KCl, 0.3 mM NADPH, and 2 mM dithiothreitol in Tris-HCl buffer (10 mM) at pH 7.5. The enzyme was further purified after precipitating it by dialyzing out the KCl and then resolubilized with 0.8% octyl glucoside in 1 M KCl (plus NADPH and dithiothreitol). The second solubilized enzyme was purified to homogeneity (370-fold from peroxisomes) by gel filtration in a Sepharose CL-6B column followed by affinity chromatography on an NADPH-agarose gel matrix. NADPH-agarose was prepared by reacting periodate-oxidized NADP+ to adipic acid dihydrazide-agarose and then reducing the immobilized NADP+ with NaBH4. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified enzyme showed a single homogeneous band with an apparent molecular weight of 60,000. The molecular weight of the native enzyme was estimated to be 75,000 by size exclusion chromatography. Amino acid analysis of the purified protein showed that hydrophobic amino acid comprised 27% of the molecule. The Km value of the purified enzyme for hexadecyldihydroxyacetone phosphate (DHAP) was 21 microM, and the Vmax value in the presence of 0.07 mM NADPH was 67 mumol/min/mg. The turnover number (Kcat), after correcting for the isotope effect of the cosubstrate NADP3H, was calculated to be 6,000 mol/min/mol of enzyme, assuming the enzyme has a molecular weight of 60,000. The purified enzyme also used palmitoyldihydroxyactone phosphate as a substrate (Km = 15.4 microM, and Vmax = 75 mumol/min/mg). Palmitoyl-DHAP competitively inhibited the reduction of hexadecyl-DHAP, indicating that the same enzyme catalyzes the reduction of both acyl-DHAP and alkyl-DHAP. NADH can substitute for NADPH, but the Km of the enzyme for NADH (1.7 mM) is much higher than that for NADPH (20 microM). The purified enzyme is competitively (against NADPH) inhibited by NADP+ and palmitoyl-CoA. The enzyme is stable on storage at 4 degrees C in the presence of NADPH and dithiothreitol.     

Purification to homogeneity, characterization and monoclonal antibodies of phospholipid-sensitive Ca2+-dependent protein kinase from spleen.

A phospholipid-sensitive Ca2+-dependent protein kinase was purified to homogeneity, for the first time, from extracts of pig spleen, employing the steps of DEAE-cellulose, octyl-agarose, Sephacryl S-200 and phosphatidylserine-Affigel 10 affinity chromatographies. The purified enzyme appeared as a single protein band on both analytical (non-denaturing) and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, having a minimum mol.wt. of 68 000 +/- 200. The molecular weight of the enzyme was also determined to be 74 500 +/- 4600 by gel filtration and 80 000 based on its sedimentation coefficient (5.52 S) and Stokes radius (3.52 +/- 0.09 nm), indicating that the enzyme was a monomeric protein. The frictional ratio (f/f0) of the enzyme was 1.24, indicating it was non-globular in shape. The enzyme had a pI of 5.3, and a pH optimum of 6.5 for its reaction. Amino acid analysis indicated that the enzyme apparently was not similar to myosin light-chain kinase (a calmodulin-sensitive species of Ca2+-dependent protein kinase) or cyclic AMP-dependent and cyclic GMP-dependent protein kinases. The enzyme had an apparent Km for ATP of 7.5 microns. Histone H1 and myelin basic protein were effective substrates for the enzyme, with apparent Km values of 0.3 and 0.2 microns, and Vmax, values of 0.06 and 0.09 mumol/min per mg of enzyme respectively. The enzyme activity was dependent on both phosphatidylserine (apparent Ka = 6.25 micrograms/ml) and Ca2+ (apparent Ka = 160 microns). Calmodulin was unable to substitute for the phospholipid as a cofactor, nor was it a subunit of the enzyme. Sr2+ and Ba2+ could partially mimic Ca2+ to activate the enzyme in the presence of phosphatidylserine. An endogenous substrate protein (mol.wt. 41 000) for the enzyme was found in the total, solubilized fraction of pig spleen. Monoclonal antibodies against the enzyme interacted similarly with the homogeneous and impure enzyme; the antibodies, however, did not bind to cyclic nucleotide-dependent protein kinases.     

Alanine racemase of alfalfa seedlings (Medicago sativa L.): first evidence for the presence of an amino acid racemase in plants

We demonstrated several kinds of D-amino acids in plant seedlings, and moreover alanine racemase (E.C.5.1.1.1) in alfalfa (Medicago sativa L.) seedlings. This is the first evidence for the presence of amino acid racemase in plant. The enzyme was effectively induced by the addition of L- or D-alanine, and we highly purified the enzyme to show enzymological properties. The enzyme exclusively catalyzed racemization of L- and D-alanine. The K(m) and V(max) values of enzyme for L-alanine were 29.6 x 10(-3) M and 1.02 mol/s/kg, and those for D-alanine are 12.0 x 10(-3) M and 0.44 mol/s/kg, respectively. The K(eq) value was estimated to be about 1 and indicated that the enzyme catalyzes a typical racemization of both enantiomers of alanine. The enzyme was inactivated by hydroxylamine, phenylhydrazine and some other pyridoxal 5'-phosphate enzyme inhibitors. Accordingly, the enzyme required pyridoxal 5'-phosphate as a coenzyme, and enzymologically resembled bacterial alanine racemases studied so far.     

Phosphatidylcholine makes specific activity of the purified Ca(2+)-ATPase from plasma membranes independent of enzyme concentration.

Ca(2+)-ATPase of plasma membranes (PMCA) was isolated from either human or pig red cells by calmodulin-affinity chromatography and supplemented with phosphatidylcholine (PC). The specific activity of the purified PMCA diluted in media with detergent (C(12)E(10)) was very low, and increased with the concentration of the enzyme along a curve that reached the maximum at 8 microg/ml with K(0.5)=1.2-2.5 microg/ml. Such behavior has been described and attributed to self-association of the enzyme (D. Kosk-Kosicka and T. Bzdega, J. Biol. Chem. 263 (1988) 18184-18189). After heat-inactivation, the PMCA was as effective an activator as the intact enzyme, increasing, to the maximum, the specific activity of diluted enzyme with K(0. 5)=2.2 microg/ml. The inactivated PMCA failed to increase the activity of concentrated enzyme, suggesting that activation did not depend on interaction of intact with denatured enzyme molecules. When enough PC was added to the reaction medium to make its final concentration 16-33 microg/ml, the specific activity of the PMCA was maximum and independent of enzyme concentration. Under these conditions, activation by calmodulin lowered to 10%. As a function of the concentration of pure PC, maximum specific activity was reached along a curve with K(0.5)=4 microg/ml. This curve was identical to that of activation at increasing enzyme concentration, suggesting that, in the latter case, activation could have depended on PC contributed to the assay medium by the enzyme. The results show that PC made the purified PMCA solubilized in detergent reach maximum activity at any concentration of the enzyme.     

Defective enzyme causes lecithin-cholesterol acyltransferase deficiency in a Japanese kindred

Lecithin-cholesterol acyltransferase mass levels and activity and apolipoproteins A-I, A-II, B and D were measured in a Japanese family who have a familial lecithin-cholesterol acyltransferase deficiency. This analysis was performed to gain insight into the molecular basis of the enzyme deficiency and to compare findings in this family with other families with familial lecithin-cholesterol acyltransferase deficiency. The mass of the enzyme in plasma was determined by a sensitive double antibody radioimmunoassay, and enzyme activity was measured by using a common synthetic substrate comprised of phosphatidylcholine, cholesterol and apolipoprotein A-I liposomes prepared by a cholate dialysis procedure. The lecithin-cholesterol acyltransferase-deficient subject had an enzyme mass level that was 35% of normal (2.04 micrograms/ml, as compared with an average normal level of 5.76 +/- 0.95 micrograms/ml in 19 Japanese subjects) and an enzyme activity of less than 0.1% of normal (0.07 nmol/h per ml, as compared with normal levels of 100 nmol/h per ml). This subject also had lower levels of apolipoproteins: apolipoprotein A-I was 53 mg/dl (42% of normal), apolipoprotein A-II was 10.6 mg/dl (31% of normal), apolipoprotein B was 68 mg/dl (68% of normal), and apolipoprotein D was 3.6 mg/dl (60% of normal). The three obligate heterozygotes had enzyme mass levels ranging from 65% to 100% of normal and enzyme activity levels ranging from 23% to 65% of normal (23.4, 56.8, and 64.7 nmol/h per ml, respectively). The proband's sister had an enzyme mass level of 6.55 micrograms/ml (114% of normal) and an enzyme activity of only 64.8 nmol/h per ml (65% of normal), suggesting that she was also a heterozygote for lecithin-cholesterol acyltransferase deficiency. The obligate heterozygotes and the sister had normal apolipoprotein levels. We conclude that the lecithin-cholesterol acyltransferase deficiency in this family is due to the production of a defective enzyme that is expressed in the homozygote as well as in the heterozygotes, and, further, that this family's mutation differs from that reported earlier for other Japanese lecithin-cholesterol acyltransferase-deficient families.     

Cloning and sequencing of the leucine dehydrogenase gene from Bacillus sphaericus IFO 3525 and importance of the C-terminal region for the enzyme activity

The structural gene (leudh) coding for leucine dehydrogenase from Bacillus sphaericus IFO 3525 was cloned into Escherichia coli cells and sequenced. The open reading frame coded for a protein of 39.8 kDa. The deduced amino acid sequence of the leucine dehydrogenase from B. sphaericus showed 76–79% identity with those of leucine dehydrogenases from other sources. About 16% of the amino acid residues of the deduced amino acid sequence were different from the sequence obtained by X-ray analysis of the B. sphaericus enzyme. The recombinant enzyme was purified to homogeneity with a 79% yield. The enzyme was a homooctamer (340 kDa) and showed the activity of 71.7 μmol·min⁻¹·mg⁻¹) of protein. The mutant enzymes, in which more than six amino acid residues were deleted from the C-terminal of the enzyme, showed no activity. The mutant enzyme with deletion of four amino acid residues from the C-terminal of the enzyme was a dimer and showed 4.5% of the activity of the native enzyme. The dimeric enzyme was more unstable than the native enzyme, and the K_m values for -leucine and NAD⁺ increased. These results suggest that the Asn-Ile-Leu-Asn residues of the C-terminal region of the enzyme play an important role in the subunit interaction of the enzyme.     

Catabolism of indole-3-acetic acid in citrus leaves: identification and characterization of indole-3-aldehyde oxidase

A new enzyme, named indole-3-aldehyde oxidase (IAldO), was identified in citrus ( Citrus sinensis L. Osbeck cv. Shamouti) leaves. The enzyme was partially purified by (NH₄)₂SO₄ fractionation. Sephadex G-200 gel filtration and DEAE-cellulose ion exchange chromatography. IAldO catalyzes the oxidation of indole-3-aldehyde (IAld) to indole-3-carboxylic acid (ICA) with the production of H₂O₂. The enzyme is highly specific for IAld. The apparent K_M of the enzyme for IAld is 19 μ M . The optimum oxidation of IAld occurs at pH 7. 5. The molecular mass of the enzyme, as determined by Sepharose-6B gel filtration, is about 200 kDa. Based on inhibitor studies, it is concluded that IAldO is not a flavin-linked oxidase and there is no requirement for free sulfhydryl groups or divalent cations for maximum activity. The enzyme is strongly inhibited by benzaldehyde. Ethylene pretreatment, wounding and aging of leaf tissues did not affect enzyme activity, suggesting that the enzyme is constitutive in citrus tissues.     

Thermostable aspartase from a marine psychrophile,Cytophaga sp. KUC-1: molecular characterization and primary structure

We found that a psychrophilic bacterium isolated from Antarctic seawater, Cytophaga sp. KUC-1, abundantly produces aspartase [EC4.3.1.1], and the enzyme was purified to homogeneity. The molecular weight of the enzyme was estimated to be 192,000, and that of the subunit was determined to be 51,000: the enzyme is a homotetramer. L-Aspartate was the exclusive substrate. The optimum pH in the absence and presence of magnesium ions was determined to be pH 7.5 and 8.5, respectively. The enzyme was activated cooperatively by the presence of L-aspartate and by magnesium ions at neutral and alkaline pHs. In the deamination reaction, the K(m) value for L-aspartate was 1.09 mM at pH 7.0, and the S(1/2) value was 2.13 mM at pH 8.5. The V(max) value were 99.2 U/mg at pH 7.0 and 326 U/mg at pH 8.5. In the amination reaction, the K(m) values for fumarate and ammonium were 0.797 and 25.2 mM, respectively, and V(max) was 604 U/mg. The optimum temperature of the enzyme was 55 degrees C. The enzyme showed higher pH and thermal stabilities than that from mesophile: the enzyme was stable in the pH range of 4.5-10.5, and about 80% of its activity remained after incubation at 50 degrees C for 60 min. The gene encoding the enzyme was cloned into Escherichia coli, and its nucleotides were sequenced. The gene consisted of an open reading frame of 1,410-bp encoding a protein of 469 amino acid residues. The amino acid sequence of the enzyme showed a high degree of identity to those of other aspartases, although these enzymes show different thermostabilities.     

Purification and characterization of glutathione disulfide-stimulated Mg2+-ATPase from human erythrocytes

We have previously shown the presence of two different forms of glutathione disulfide (GSSG)-stimulated Mg2+-ATPases in human erythrocytes. We have now investigated a low-Km form of the enzyme from human erythrocytes. Purification of the enzyme was performed to apparent homogeneity involving procedures of affinity chromatography and gel filtration. The enzyme was composed of two non-identical subunits of Mr = 82K and 62K. The enzyme reconstituted into phospholipid vesicles showed both GSSG-stimulated Mg2+-ATPase activity (285 nmol Pi released/mg protein/min) and active GSSG transport activity (320 nmol GSSG/mg protein/min). The amino acid composition of the enzyme was similar to that of the enzyme purified from cytoplasmic membranes of human hepatocytes. These enzymes were immunologically cross reactive. These results indicate that this enzyme functions in the active transport of GSSG as it possibly does in hepatocytes.     

Synthesis and characterization of immobilized dopamine beta-hydroxylase in membrane-bound and solubilized formats

Dopamine beta-hydroxylase (DBH) catalyzes the beta-hydroxylation of dopamine to norepinephrine. The enzyme in chromaffin granules occurs in a soluble form and a form confined to the surrounding membrane. DBH was noncovalently immobilized in the hydrophobic interface of an immobilized artificial membrane (IAM) liquid chromatographic stationary phase and the resulting DBH-IAM stationary phase was enzymatically active and was shown to mimic the membrane-bound form of the enzyme. DBH was also covalently immobilized onto a silica-based support containing, glutaraldehyde-P (Glut-P). The resulting DBH-Glut-P interphase was also enzymatically active, reproducible and shown to display characteristics of the solubilized enzyme. The results demonstrate that the different immobilization methods utilized for the enzyme can be used to quantitatively and qualitatively determine the enzyme kinetic constants associated with enzyme/substrate and enzyme/inhibitor interactions for the two distinct forms of the enzyme. These new entities can be used in basic biochemical studies as well as in high throughput screening of substances for DBH substrate/inhibitor properties.