Studies of human kidney gamma-glutamyl transpeptidase. Purification and structural, kinetic and immunological properties.

gamma-Glutamyl transpeptidase, present in various mammalian tissues, transfers the gamma-glutamyl moiety of glutathione to a variety of acceptor amino acids and peptides. This enzyme has been purified from human kidney cortex about 740-fold to a specific activity of 200 units/mg of protein. The purification steps involved incubation of the homogenate at 37 degrees followed by centrifugation and extraction of the sediment with 0.1 M Tris-HCl buffer, pH 8.0, containing 1% sodium deoxycholate; batchwise absorption on DEAE-cellulose; DEAE-cellulose (DE52) column chromatography; Sephadex G-200 gel filtration; and affinity chromatography using concanavalin A insolubilized on beaded Agarose. Detergents were used throughout the purification of the enzyme. The purified enzyme separated into three protein bands, all of which had enzyme activity, on polyacrylamide disc electrophoresis in the presence of Triton X-100. The enzyme has an apparent molecular weight of about 90,000 as shown by Sephadex G-200 gel filtration, and appears to be a tetramer with subunits of molecular weights of about 21,000. The Km for gamma-glutamyl transpeptidase using the artificial substrate, gamma-glutamyl-p-nitroanilide, with glycylglycine as the acceptor amino acid was found to be about 0.8 mM. The optimum pH for the enzyme activity is 8.2 and the isoelectric point is 4.5. Both GSH and GSSG competitively inhibited the activity of gamma-glutamyl transpeptidase when gamma-glutamyl-p-nitroanilide was used as the substrate. Treatment of the purified enzyme with papain has no effect on the enzyme activity or mobility on polyacrylamide disc electrophoresis. The purified gamma-glutamyl transpeptidase had no phosphate-independent glutaminase activity. The ratio of gamma-glutamyl transpeptidase to phosphate-independent glutaminase changed significantly through the initial steps of gamma-glutamyl transpeptidase purification. These studies indicate that the transpeptidase and phosphate-independent glutaminase activities are not exhibited by the same protein in human kidney.     

Fractionation and characterization of cystine aminopeptidase (oxytocinase) and arylamidase of the human placenta.

Three activity peaks hydrolysing L-cystine-di-beta-naphthylamide (CysNA) and two activities hydrolysing L-leucine-beta-naphthylamide (LeuNA) were separated by gel filtration on Sepharose 6B from human placental tissue. The enzyme activities in the void volume and the solubilized enzyme activities with both substrates apparently are bound and free forms of the same enzymes (I) since detergent treatment caused a total disappearance of the activities in the void volume. The second distinct enzyme (II) was highly soluble and detected only with CysNA. The particle-bound enzyme(s) had a pH optimum at 6.5 with CysNA and at about 7.5 with LeuNA. They were highly sensitive to EDTA, could be reactivated by Co2+ and Zn2+ and were more sensitive to Ni2+ and L-methionine than the soluble enzyme II. The former enzyme(s) tolerated thermal treatment better than the soluble enzyme II. The solubilized free enzyme(s) I had a molecular weight of about 309,000. The soluble enzyme II was resistant to EDTA. Its optimum was at pH 6.0 and an estimate of 76,000 for the molecular weight was obtained.     

High and low molecular weight proteolytic activities in prawn (Metapenaeus brevicornis) muscle

Muscle extract of prawn (Metapenaeus brevicornis) expressed high azocoll lytic activity compared to extracts of many other prawn varieties; the activity was also inhibited to a small extent by dithiothreitol. Ammonium sulphate precipitation, subsequent extraction at pH 5.6 and chromatography revealed the occurrence of two types of azocoll lytic activities: one, high molecular weight (630 kDa) and the other low molecular weight (< 30 kDa) enzyme. The former was stimulated by dithiothreitol whereas the latter was inhibited. SDS PAGE of high molecular weight preparation did not show homogeneity but the profile was similar to that of the low molecular weight fraction. Gel filtration of high molecular weight enzyme following incubation at high pH revealed the formation of low molecular weight fractions having activity towards azocoll. Chymotrypsin-like activity associated with high molecular weight enzyme was also susceptible to dissociation by high pH. Azocoll lytic activity of both enzymes was strongly inhibited by 1,10-phenanthroline.     

Purification and Characterization of β-Fructofuranosidase from Aspergillus japonicus TIT-KJ1

A β-fructofuranosidase (EC 3.2.1.26) was purified to homogeneity from Aspergillus japonicus TIT-KJ1. The enyme had an optimum pH for activity of 5.4 and pH stability at 7.0–8.4. The optimum temperature at pH 5.4 was 60°C. The enzyme had a molecular weight of 236,000 with two subunits and an isoelectric point of pH 4.0. The enzyme was inactivated by 5 mM Hg^{2 +} and Ag⁺. The enzyme had a high transfructosylating activity. Treatment of 50% (w/v) sucrose with the enzyme under optimum conditions afforded more than 55% fructooligosaccharides.     

Activities and properties of cellulase and xylanase associated with Phragmites leaf litter in a seawater lake

The activities and properties of cellulases and xylanases associated with Phragmites communis leaves were followed during the course of their decomposition from autumn to summer in a seawater lake. Cellulases and xylanases of low optimum pH (4 or less) were detected on aerial dead leaves before submergence. These enzymes remained on the leaves immediately after submergence, but were replaced by enzymes with a higher optimum pH (about 5.5 to 6.5), the activity of which increased rapidly during the initial two weeks. The enzyme activities then declined with water temperature until about day 100, but gradually increased again thereafter. This successive change in enzyme activities closely correlated with that of the decomposition rates of cellulose and xylan. The molecular weight distribution of the cellulases and xylanases changed markedly in the latter half of the experimental period, which suggested a change in the flora of active decomposer microorganisms.     

Characterization of glucose-6-phosphate dehydrogenase variants in the Sudan--including GdKhartoum, a hyperactive slow variant.

Erythrocyte glucose-6-phosphate dehydrogenase (G6PD) was characterized in blood samples of 94 male subjects in Sudan having deficient and non-deficient electrophoretic variants. They comprised 44 GdB, 17 GdA, 19 GdB-, 11 GdA- and 3 nondeficient (GdKhartoum) variants. Biochemical characteristics including enzyme activity, electrophoretic mobility, Km for glucose-6-phosphate (G6P) and nicotinamide adenine dinucleotide phosphate (NADP), heat stability and pH optimum of all the common and deficient variants were consistent with the reported characteristics of these variants. The GdKhartoum variant had 90% mobility in TEB buffer and 100% in phosphate buffer, 120% activity, Km of 130 +/- 49 microns for G6P and 0.8 +/- 0.2 microns for NADP, lowered thermostability and an optimum pH of 7.6. This variant was not inhibited by 15 mM maleic acid, 10 mM iodoacetate and dehydro-iso-androsterone. All other variants were inhibited by dehydro-iso-androsterone but uninhibited by maleic acid and iodoacetate.     

Identity of maleate-stimulated glutaminase with gamma-glutamyl transpeptidase in rat kidney.

Gamma-Glutamyl transpeptidase was purified from rat kidney by a procedure involving Lubrol extraction, acetone precipitation, ammonium sulfate fractionation, treatment with bromelain, and column chromatography on DEAE-cellulose and Sephadex G-100. The final preparation (enzyme III), which exhibits a specific activity about 8-fold higher than that of the purified rat kidney transpeptidase previously obtained in this laboratory (enzyme I), was apparently homogeneous on polyacrylamide gel electrophoresis. Enzyme III is a glycoprotein containing 10% hexose, 7% aminohexose, and 1.5% sialic acid; a tentative molecular weight value of about 70,000 was obtained by gel filtration. Enzyme III has a much lower molecular weight and a different amino acid and carbohydrate content than the less active rat kidney transpeptidase preparation previously obtained, but obtained, but the catalytic properties of these preparations are virtually identical. It is suggested that bromelain treatment may liberate the transpeptidase from a brush border complex that contains other proteins. An improved method is described for the isolation of the higher molecular weight form of the enzyme (enzyme I) in which affinity chromatography on concanavalin A-Sephrose is employed. The purified transpeptidase (enzyme III) is similar to the phosphate-independent maleate-stimulated glutaminase preparation obtained from rat kidney by Katunuma and colleagues with respect to amino acid and carbohydrate content, apparent molecular weight, and relative transpeptidase and maleate-stimulated "glutaminase" activities. Both of these enzyme preparations are much more active in transpeptidation reactions with glutathione and related gamma-glutamyl compounds than with glutamine. In the absence of maleate, the enzyme catalyzes the utilization of glutamine (by conversion to gamma-glutamylglutamine, glutamate, and ammonia) at about 2% of the rate observed for catalysis of transpeptidation between glutathione and glycylglycine; the utilization of glutamine occurs about 8 times more rapidly in the presence of 0.1 M maleate. The transpeptidation and maleate-stimulated glutaminase reactions catalyzed by both enzyme preprations are inhibited by 5 mM L-serine in the presence of 5 mM sodium borate. Studies on gamma-glutamyl transpeptidase and maleate-stimulated glutaminase in the kidneys of fetal rats, newborn rats, and rats after weaning showed parallel development of these activities. The evidence reported here and earlier work in this laboratory strongly support the conclusion that maleate-stimulated glutaminase activity is a catalytic function of gamma-glutamyl transpeptidase. The studies on the ontogeny of gamma-glutamyl transpeptidase and other data are considered in relation to the proposal that this enzyme is involved in amino acid and peptide transport. Its possible role in renal formation of ammonia is also discussed.     

gamma-Glutamyl transpeptidase from WI-38 fibroblasts: purification and active site modification studies

γ-Glutamyl transpeptidase has been purified to homogeneity from WI-38 human fetal lung fibroblasts, following extraction with Triton X-100 in the absence of added proteases. The specific activity of the purified enzyme is 16 units/mg protein at the optimum of pH 8.0. Although this activity value is low, the WI-38 enzyme is very similar to previously described γ-glutamyl transpeptidases in its molecular properties. The native molecule (apparent molecular weight of 82,000) is composed of one light and one heavy subunit (apparent molecular weights of 20,000 and 62,000, respectively). Papain digestion reduces the native molecular weight to an apparent value of 73,000 by proteolysis of the heavy chain. The known active site modifying agent and glutamine analog 6-diazo-5-oxo-l-nor-leucine, completely inactivates the enzyme, coincident with its stoichiometric incorporation into the light subunit. This inactivation is accelerated by maleate and prevented by S-methylglutathione. The WI-38 γ-glutamyl transpeptidase is also inactivated by the fluorescent alkylating agent, 5-iodoacetamidofluorescein. Selective reaction of this reagent with an active site residue is suggested by prevention of the inactivation by S-methylglutathione, the stoichiometric incorporation of the fluorescein moiety, and the loss of one methionine residue per molecule of protein accompanying inactivation.     

Purification,characterization, and chemical modification of manganese peroxidase from Bjerkandera adusta UAMH 8258

Ten strains of Bjerkandera adusta from the University of Alberta Microfungus Collection and Herbarium (UAMH) were compared for manganese peroxidase production. The enzyme from B. adusta UAMH 8258 was chosen for further study. After purification the enzyme showed a molecular weight of 43 kDa on 15% SDS-PAGE, 36.6 kDa on matrix-assisted laser desorption ionization-time of flight mass spectrometry, and an isoelectric point of 3.55. The N-terminal amino acid sequence was determined to be VAXPDGVNTATNAAXXALFA, and the amino acid composition showed no tyrosine residues in the enzyme. Manganese peroxidase exhibited both Mn(II)-dependent (optimum pH 5) and Mn(II)-independent activity (optimum pH 3). The purified enzyme was chemically modified with cyanuric chloride-activated methoxypolyethylene glycol to enhance its surface hydrophobicity. The modified and native enzymes showed similar catalytic properties in the oxidation of Mn(II) and other substrates such as 2,6-dimethoxylphenol, veratryl alcohol, guaiacol, and 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonate). However, the modified enzyme showed greater resistance to denaturation by hydrogen peroxide and stability to organic solvents such as acetonitrile, N,N-dimethylformamide, tetrahydrofuran, methanol, and ethanol. The PEG-modified enzyme also showed greater stability to higher temperatures and lower pH than the native enzyme. Thus, chemical modification of manganese peroxidase from B. adusta increases its potential usefulness for applied studies. Received: 12 October 2001 / Accepted: 14 November 2001     

Studies on the molecular species of DNA polymerase extracted from rat ascites hepatoma cells.

DNA polymerase [EC 2.7.7.7] activities present in hypotonic extract from rat ascites hepatoma AH130 cells were eluted in three separable peaks on DEAE-cellulose column chromatography. Peak I activity had an alkaline pH optimum, and was relatively resistant to SH-blocking reagents and salt concentration. These properties of DEAE peak I are typical of low molecular weight DNA polymerase. DEAE peak II and peak III activities possessed properties corresponding to high molecular weight (6-8 S) polymerase; they showed maximal activity at neutral pH, and were sensitive to SH-blocking reagents and salt. No low molecular weight polymerase activity was released from DEAE peak II or peak III by salt treatment, though partial conversion from DEAE peak II to peak III was observed on the same treatment.     

Purification and characterization of an acid phosphatase from the commercial mushroom Agaricus bisporus

Acid phosphatase [AP; EC 3.1.3.2], a key enzyme involved in the synthesis of mannitol in Agaricus bisporus, was purified to homogeneity and characterized. The native enzyme appeared to be a high molecular weight type glycoprotein. It has a molecular weight of 145 kDa and consists of four identical 39-kDa subunits. The isoelectric point of the enzyme was found at 4.7. Maximum activity occurred at 65°C. The optimum pH range was between 3.5 and 5.5, with maximum activity at pH 4.75. The enzyme was unaffected by EDTA, and inhibited by tartrate and inorganic phosphate. The enzyme exhibits a K _m for p-nitrophenylphosphate and fructose-6-phosphate of 370 M and 3.1 mM, respectively. A broad substrate specificity was observed with significant activities for fructose-6-phosphate, glucose-6-phosphate, mannitol-1-phosphate, AMP and -glycerol phosphate. Only phosphomonoesters were dephosphorylated. Antibodies raised against the purified enzyme could precipitate AP activity from a cell-free extract in an anticatalytic immunoprecipitation test.     

Dihydrofolate Reductase in Plasmodium lophurae and Duckling Erythrocytes*

Dihydrofolate reductase activity in duckling erythrocytes was found to be low, while activity in erythrocytes heavily infected with small uninucleate trophozoites was like that of uninfected erythrocytes. Activity of the enzyme in erythrocytes infected with large multinucleate parasites, however, was greatly increased. This activity was 5 times higher in erythrocyte-free large trophozoites than in small ones. The dihydrofolate reductase of P. lophurae differed from the host enzyme in: greater molecular weight; higher sensitivity to pyrimethamine inhibition; pH optimum; substrate and cofactor specificity; and stimulation by salts. The parasite enzyme was partially purified by ammonium sulfate precipitation.     

Conversion of DNA polymerase extracted from rat ascites hepatoma cells

DNA polymerase extracted fresh from rat ascites hepatoma cells possesses high molecular weight, maximal activity at neutral pH, and high sensitivity to N-ethylmaleimide (NEM). After physical and chemical treatment of the enzyme fraction, the appearance of low molecular weight DNA polymerase was detected by means of Sephadex gel filtration or sucrose density gradient centrifugation. This low molecular weight DNA polymerase possesses alkaline pH optimum, preference of native DNA as template/primer, and relative resistance to NEM.     

Properties of L-glutamate decarboxylase from brains of adult and newborn mice.

Abstract— l -Glutamate 1-carboxy-lyase (EC 4.1.1.15) (GAD) and 4-aminobutyrate-2-oxo-glutarate aminotransferase (EC 2.6.1.19) (GABA-T) have been purified from mouse brain (Wu et al. 1973; Schousboe et al., 1973) and their properties have been extensively studied (Wu & Roberts , 1974; Schousboe et al., 1974). The above enzymes were prepared from a water lysate of crude mitochondrial fraction, which accounted for only 25–30% of total GAD or GABA-T activities in brain. A procedure has been developed which liberates approx 85% of total GAD and GABA-T activities into supernatant. Two distinct, well-separated peaks with GAD activity and a single peak with GABA-T activity were observed when a concentrated extract from brain of adult or newborn mice was chromatographed on Sephadex G-200 or Bio-Gel A–1.5 m. The first peak appeared in the void volume and is. therefore. an entity of high molecular weight. The second peak gave elution characteristics which were identical to those of the enzyme that had been purified previously (mol wt = 85,000). These two GAD peaks were also clearly separated on polyacrylamide gel electrophoresis. The GAD activities in the two peaks showed similar pH profiles (optimum, 6.5). K_m values (1–2 mM), immunodiffusion patterns and inhibitions by anti-GAD IgG prepared against GAD purified from synaptosome-containing crude mitochondrial fraction (60–80%). The physiological implications of high molecular weight and low molecular weight forms of GAD are discussed.     

Enzymic synthesis of levan and fructo-oligosaccharides by <Emphasis Type="Italic">Bacillus circulans</Emphasis> and improvement of levansucrase stability by carbohydrate coupling

Bacillus circulans was able to produce extracellular levansucrase using sucrose as carbon source optimally at 35°C. The enzymic synthesis of levan and fructo-oligosaccharides was studied using a 50% ethanol fraction of crude extract. The molecular weight of the synthesized levan was markedly affected by sucrose concentration, the molecular weight of levan decreased with increased sucrose concentration up to 32% whereby fructo-oligosaccharides were isolated. Temperature and the reaction time clearly affected the conversion of fructose to levan with molecular weight values ranging from 10 to 38 kDa. Identification of levan indicated that fructose was the building unit of the levan obtained. Thermal and pH stabilities of B. circulans levansucrase could be improved by enzyme glycosylation using sodium metaperiodate treatment. Chemical modification provides additional points of attachment of the enzyme to the support which offered the modified enzyme greater stabilization than did the free enzyme. The modified enzyme exhibited thermal tolerance up to 50°C, where it retained 88.25% of its activity, while the free enzyme only retained 64.55% of its original activity. The half-life significantly increased from 130 min for the free enzyme to 347 min for the modified enzyme at 50°C, however, it increased from 103 min for the free enzyme to 210 min for the modified enzyme at 60°C. Other properties i.e., the response to some metal ions as well as the ability to convert higher substrate levels and tolerance to an extension of the reaction periods were also improved upon modification. Obviously, the results obtained outlined the conditions leading to the formation of important high or low molecular weight or levan and fructo-oligosaccharides suitable for different industrial applications.     

Characterization of phosphatidylinositol-specific phospholipase C from cultured vascular smooth muscle cells.

Phosphoinositide-specific phospholipase C (PLC) activities have been partially purified from cultured vascular smooth muscle cells and analyzed for substrate specificity, calcium and pH requirements, and molecular weight. The purification procedure involved DEAE-cellulose and heparin-Sepharose chromatographies followed by Mono Q and size exclusion high performance liquid chromatography. This technique resolves multiple peaks of activity using phosphatidylinositol (PI) and PI 4,5-bisphosphate (PIP2) as substrates. The major peak was purified to near homogeneity as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. PLC activity in vascular smooth muscle cells can be divided into two types based on their calcium and pH requirements, substrate preferences, and molecular weights. The low molecular weight PLC hydrolyzes both PI and PIP2, has a molecular mass of 58 kDa, requires the most calcium for full activation, and has a PI-pH profile that shifts slightly with calcium concentration. Screening a cDNA library with oligonucleotides directed against several of the known PLCs identified a highly expressed PLC cDNA that is 99% homologous to PLC-alpha, suggesting that this low molecular weight peak in fact corresponds to PLC-alpha. The high molecular mass peak (157 kDa) shows much greater activity against PI than PIP2, is active at lower calcium concentrations, and has a PI-pH optimum of 5.0 regardless of calcium concentration. Each of the PIP2 PLC activities is strongly dependent on the relative levels of calcium and pH in the assay buffer. These observations suggest that vascular smooth muscle contains both a high and low molecular weight PLC whose activities are affected markedly by the changes in calcium and pH accompanying hormonal stimulation of the cell.     

Low molecular weight DNA ligase of chick embryo.

A nuclear DNA ligase from chick embryos was isolated by the non-aqueous method and partially purified. Its activity is several fold lower than that of the enzyme found in the cytoplasmic fraction of the chick embryos. The pH dependance curve shows a single optimum for the nuclear enzyme activity, over a very narrow pH range. The molecular weight of the nuclear enzyme is 82000 and the activity is inhibited with a low K_Iby d-ATP.     

Isolation of an extracellular polysaccharide (EPS) depolymerase produced byBradyrhizobium japonicum

Bradyrhizobium japonicum is capable of producing an acidic, high molecular weight, extracellular polysaccharide (EPS). An enzyme exhibiting EPS depolymerase activity was detected in cell lysates ofB. japonicum strain 2143. The depolymerase was active against the EPS produced by strain 2143 and the closely related EPS produced by strain 311b 110. Depolymerase activity was characterized by its ability to decrease the viscosity of EPS solutions, to decrease the molecular weight of EPS, and to catalyze the release of reducing groups from EPS. The depolymerase exhibited a sharp activity optimum at pH 6 and had a molecular weight of approximately 45 kD as determined by gel permeation chromatography. Analysis of depolymerase-treated EPS indicates that the enzyme acts as an endo-depolymerase, producing a relatively narrow size range of high molecular weight products.Contribution from the Missouri Agricultural Experiment Station, Journal Series Number 10:959.     

Properties of Invertases in Sugar Storage Tissues of Citrus Fruit and Changes in Their Activities during Maturation

Both acid and alkaline invertases were present in immature juice sacs of satsuma mandarin (Citrus‘Unshu Marcovitch”) fruit, in which sugar content was low. Maturing and mature juice sacs, in which sugar content increased steadily with time, were characterized by the presence of alkaline invertase and the absence of acid invertase. When the immature juice sacs were homogenized with 0.2 M sodium phosphate-citrate buffer (pH 8.0), almost all of the acid invertase activity was found in the solubilized fraction, whereas almost all of the alkaline invertase activity was present in the insoluble fraction. The distribution of alkaline invertase between the solubilized and insoluble fractions changed with the development of fruit. The acid invertase had a molecular weight of 69,000, optimum pH of 4.8–5.3, and K_m value for sucrose of 7.3 mM. The alkaline invertase had a molecular weight of 200,000, pH optimum of 7.2–7.7, and K_m value of 35.7 mM. The hydrolysing activities of both enzymes for raffinose were considerably less than those for sucrose. The alkaline invertase had lower activity for raffinose than the acid invertase.     

Characteristics of the endoglucanase encoded by a cel gene from Bacteroides succinogenes expressed in Escherichia coli

A cel gene from Bacteroides succinogenes inserted into the vector pUC8 coded for an enzyme which exhibited high hydrolytic activity on carboxymethylcellulose, p-nitrophenylcellobioside, and lichenan and low activity on laminarin and xylan. The enzyme was not synthesized by the Escherichia coli host when cells were cultured in complex medium containing added glucose. In the absence of added glucose, the endoglucanase and cellobiosidase activities synthesized were partitioned into the periplasmic space during growth, and practically all enzyme was located in the periplasm when the stationary phase of growth was reached. The enzyme exhibited 17- and sixfold higher Km values for the hydrolysis of carboxymethylcellulose and lichenan, respectively, than did the extracellular endoglucanase complex from B. succinogenes. The Cel endoglucanase had a pH optimum similar to that of the B. succinogenes enzyme except that the range was narrower, and the Cel endoglucanase was more readily inactivated on exposure to high temperature, detergents, and certain metals. Its activity was stimulated by calcium and magnesium. Nondenaturing polyacrylamide gel electrophoresis at different acrylamide concentrations revealed the presence of three endoglucanase components, two with molecular weights of 43,000 and one with a molecular weight of 55,000.