Further characterization of L-leucine-pyruvate transaminase from Acetobacter suboxydans.

L-Leucine-pyruvate transaminase obtained from Acetobacter suboxydans exhibited absorbance maxima to 280 and 332 nm. The 332 nm peak was derived from the coenzyme bound to the enzyme protein with the epsilon NH2 of a lysine residue. The transaminase showed reactivity against many L-amino acids. The relation between the reactivity and the structure of the amino donor is discussed. The Michaelis constants for L-leucine, pyruvate, L-alanine and alpha-ketoisocaproate were 6.7, 3.1, 7.1 and 0.9 mM, respectively. The equilibrium constant was 5.3. The activation energy at pH 5.0 was 8,800 cal/mol.

Nucleotide sequence of the yeast glutathione S-transferase cDNA.

The nucleotide sequence (658 bp) of the cDNA coding for glutathione S-transferase Y-2 of yeast Issatchenkia orientalis was obtained. The cDNA clone contains an open reading frame of 570 nucleotides encoding a polypeptide comprising 190 amino acids with a molecular weight of 21,520. The primary amino acid sequence of the enzyme exhibits only 25.0% and 21.1% identity with 177 and 151 amino acid residues of maize glutathione S-transferase I and rat glutathione S-transferase Yb2, respectively.

Presence of a single enzyme catalyzing the pyrophosphorolysis of UDP-glucose and UDP-galactose in Bifidobacterium bifidum.

The enzyme preparation catalyzing the pyrophosphorolyses of UDP-glucose and UDPgalactose almost at the same rate was purified about 900-fold from Bifidobacterium bifidum grown on glucose medium. The two activities were always associated with each other, and their activity ratio was always constant throughout the purification steps. The final preparations was revealed homogeneous by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. There was no significant difference in thermal stabilities of the two activities. From these results it was concluded that the UDPglucose and UDPgalactose pyrophosphorylase activities in B. bifidum are catalyzed by a single enzyme protein.

Purification and properties of guanylate kinase from baker's yeast.

Guanylate kinase (ATP:(d)GMP phosphotransferase, EC 2.7.4.8) was purified about 200-fold with 4% yield from baker's yeast. The enzyme preparation showed a single band on polyacrylamide gel electrophoresis and the molecular weight of the enzyme was calculated to be 25 000 by gel filtration. With ATP as a phosphate donor, the kinase used only GMP as a phosphate acceptor. Km values for ATP and GMP were 0.5 and 0.048 mM, respectively. The enzyme reacted optimally at pH 7.5. The enzyme was labile during storage at 4 degrees C and inactivation was prevented by 20% glycerol.

Inhibition of UDP-N-acetylglucosamine pyrophosphorylase by uridine.

UDP-N-acetylglucosamine pyrophosphorylases (UTP: 2-acetamido-2-deoxy-alpha-D-glucose-1-phosphate uridylyltransferase, EC 2.7.7.23) from baker's yeast and Neurospora crassa IFO 6178 were inhibited by uridine which is the nucleoside moiety of UDP-GlcNAc. The inhibition was shown in both directions of pyrophosphorolysis and of synthesis of UDP-GlcNAc. Kinetic analysis revealed that uridine demonstrated a noncompetitive type of inhibition with UDP-GlcNAc and competitive inhibition with PPi. The Ki values for the baker's yeast enzyme were 1.8 mM for UDP-GlcNAc and 0.16 mM for PPi, and the values for the Neurospora enzyme were 1.1 mM for UDP-GlcNAc and 0.15 mM for PPi, respectively. Uridine did not bind irreversibly to the enzyme, as the activity was restored with dialysis. No other nucleosides caused inhibition of the enzyme activity except uridine. Some uridine derivatives, such as 5-hydroxyuridine, 5,6-dihydrouridine and pseudouridine, also inhibited the enzyme activity. But doexyuridine showed only slight inhibition, and 5'-UMP and orotidine caused no inhibition of the enzyme activity.

Serological study using alpha-N-acetylgalactosaminidase from Acremonium sp.

alpha-N-Acetylgalactosaminidase, produced by Acremonium sp. destroyed blood group A active substance. This enzyme converted human erythrocytes from group A to O(H). The enzyme was ascertained to liberate N-acetylgalactosamine from group A erythrocytes membranes accompanying a decrease in hemagglutination inhibition activity of A erythrocyte-anti-A serum by the erythrocyte membranes. Glycoproteins and glycolipids extracted from A erythrocytes reduced their blood group A activity after the enzyme treatment. The enzyme also worked on Forssman hapten glycolipid.

Purification and some properties of L-fucose dehydrogenase from Agrobacterium radiobacter and its application to the assay of bound-fucose in glycoconjugates.

L-Fucose dehydrogenase was found in the cell extract of Agrobacterium radiobacter and purified to homogeneity about 480-fold with 16% recovery. The molecular weight of the enzyme was approx. 64,000. The enzyme was active in the neutral pH range, unlike other L-fucose or D-arabinose dehydrogenases which are active only in the alkaline pH range. Using this enzyme and alpha-L-fucosidase F-I of Bacillus circulans (Tsuji, Y., Yamamoto, K., Tochikura, T., Seno, T., Ohkubo, Y. and Yamaguchi, H. (1990) J. Biochem. 107, 324-330) simultaneously, we developed a new coupled enzymatic method in a single buffer system for determining bound-fucose in biological materials. The fucose released by alpha-L-fucosidase F-I was oxidized with L-fucose dehydrogenase in the presence of NAD+, and the NADH formed was measured by absorbance of ultraviolet or utilized to generate color in a reaction involving CuSO4 and neocuproine. Using these methods, bound-fucose in various oligosaccharides and proteins such as lacto-N-fucopentaoses and porcine gastric mucin were quantitated within 15 min.

Purification, properties and formation of arginine-alpha-ketoglutarate transaminase in Arthrobacter simplex.

Arginine-alpha-ketoglutarate transaminase was purified 460-fold with 1.4% yield from Arthrobacter simplex grown on arginine as a carbon source. The preparation was more than 90% pure on polyacrylamide gel electrophoresis, and the molecular weight of the enzyme was calculated to be 110 000. The enzyme exhibited absorption maxima at 280, 330 and 370 nm. The 370 nm peak decreased with increase in the 330 nm peak on addition of arginine Km values for arginine, alpha-ketoglutarate, glutamate and alpha-keto-delta-guanidinovalerate were 2.9, 8.1, 25 and 0.30 mM, respectively. Those for pyridoxal 5'-phosphate and pyridoxamine 5'-phosphate were 0.25 and 0.57 microM. The enzyme reacted optimally at pH 8.0--8.5. The synthesis of arginine-alpha-ketoglutarate transaminase was inducible by arginine and alpha-keto-delta-guanidinovalerate.

Resolution and complementation of the labile L-leucine-pyruvate transaminase. An intermediate during enzyme formation under nitrogen starvation in Gluconobacter suboxydans.

L-Leucine-pyruvate transaminase (mol. wt. 70 000) in Gluconobactersuboxydans synthesized during nitrogen starvation contained a labile form which changed to the stable one later. The labile enzyme (mol. wt. 70 000) dissocated to the two proteinaceous components: a cationic one (mol. wt. 10 000--20 000) and an anionic one (mol. wt. 50 000--60 000), during column chromatography on DEAE-cellulose. The enzyme activity was reconstructed when they were mixed. The reconstructed enzyme had almost the same molecular size and enzymatic properties as the labile and the native stable enzymes.

Primary structure of an N-linked sugar chain derived from glucoamylase of Rhizopus niveus.

The primary structure of the N-linked sugar chain of Rhizopus niveus glucoamylase (major component) was investigated. The carbohydrate moiety was released from the polypeptide backbone by Flavobacterium sp. endo-beta-N-acetylglucosaminidase digestion. Studies using the method of exoglycosidase digestion of the fluorescent pyridylamino derivative, gel-permeation chromatography on Bio-Gel P-4 and 400-MHz 1H-NMR spectroscopy revealed that the most abundant structure is (Man)8-GlcNac-ol.

Antiviral agents with activity against human retroviruses.

The ability of various known anti-HIV antivirals to inhibit four different strains of human immunodeficiency virus type 1 (HIV-1), a strain of type 2 (HIV-2), and a human T-cell lymphotropic virus type I (HTLV-I) was tested. The tested substances included two sulfated polysaccharides (lentinan sulfate and dextran sulfate) and a nonsulfated polysaccharide PSK, E-P-LEM, glycyrrhizin sulfate, and nucleoside analogues (AZT and DHT). The effects of the substances were measured quantitatively with two different assays: (i) inhibition of cell-free viral infection and (ii) inhibition of the fusion reaction induced by cell-to-cell infection. The results showed that cell-free infection of HIV-1 and HIV-2 was almost completely blocked in the presence of all of the substances tested. However, cell-to-cell infection by HIV-1, HIV-2, and HTLV-I was inhibited only by the polysaccharides, E-P-LEM, and glycyrrhizin sulfate but not by the two nucleoside analogues. Moreover, the extent of inhibition of the fusion reaction by the substances varied significantly from strain to strain in HIV-1.

Fusion activity dissociated from replication ability in feline immunodeficiency virus (FIV) in human cells.

Multinucleated-giant-cell formation followed by cell killing was observed after cocultivation of the feline immunodeficiency virus (FIV)-producing feline T-cell line 3201/FIV with various human cells, including T-cell lines carrying human T-cell lymphotropic virus type I (HTLV-I). The susceptibility to giant cell formation varied with the cell lines tested. Cocultivation of irradiated 3201/FIV cells with MT-2 cells resulted in giant cell formation as early as 2 h in culture, with striking resemblance to that induced by human immunodeficiency virus (HIV). MT-4 cells (HTLV-I positive) and H9 cells (HTLV-I negative) were less susceptible than MT-2 to the induction of syncytia. MOLT-4 cells (HTLV-I negative) had intermediate sensitivity to syncytia formation. No syncytia were observed in the monocytic cell line U-937 (HTLV-I negative). Syncytia formation between 3201/FIV and MT-2 cells was inhibited by polyclonal cat anti-FIV antisera but not polyclonal cat anti-feline leukemia virus (FeLV) antisera, goat anti-FeLV, uninfected specific-pathogen-free cat serum, human anti-HTLV-I antisera, or normal human and goat serum. Concentrated cell-free FIV supernatant from 3201/FIV also induced giant cells of MT-2 cells that were indistinguishable from those induced by cocultivation. Giant cells and extensive cell killing associated with giant cell formation declined and disappeared within 10 days. Surviving cells appeared to be of normal size and grew continuously without expressing FIV antigen or releasing infective virus. Although Southern blot analysis using probes specific for FIV could not detect proviral DNA in any of the five human cell lines cocultured with irradiated 3201/FIV cells, the polymerase chain reaction (PCR) technique detected FIV-specific DNA in MOLT-4 cells. DNA from the FIV-PCR positive MOLT-4 cells was PCR negative for endogenous FeLV-specific sequences, indicating that the MOLT-4 cell DNA was not contaminated with DNA from feline cells (i.e., 3201 cells). The FIV-MOLT-4 cells remained PCR positive for FIV after 40 passages, suggesting stable integration in the human cell line. These findings indicate that FIV is capable of forming proviral DNA in human T-lymphoid cells by cocultivation, although this FIV-carrying human cell line failed to produce replication-competent viruses.

Binding of adult T-cell leukemia virus to various hematopoietic cells.

A newly found human retrovirus, adult T-cell leukemia virus (ATLV) was shown by means of membrane immunofluorescence to bind to various hematopoietic cells including T-, B- and non-T, non-B-cell lines. Partially purified viral gp46 from culture fluids of ATL virus producer lines also bound efficiently to an ATLV-negative T-cell line, CCRF-CEM cells. When the viruses were pre-incubated with anti-ATLV-positive human sera, ATLV binding to the cells was clearly inhibited but not by pre-incubation with anti-ATLV-negative sera. These data suggest that: (1) ATLV binds not only to T-cells but also to multiple types of cells of hematopoietic origin; (2) anti-ATLV antibody-positive human sera have the blocking antibody for the binding of ATLV to lymphoid cells.

Anti-human immunodeficiency virus (HIV) agents are also potent and selective inhibitors of feline immunodeficiency virus (FIV)-induced cytopathic effect: development of a new method for screening of anti-FIV substances in vitro.

The ability of several known anti-HIV substances to inhibit feline immunodeficiency virus (FIV) was tested. The results showed that FIV infection of feline T-cells was almost completely blocked in the presence of all of the agents tested. However, FIV-induced syncytium formation between a human T-cell line (MT-2 cells) and a FIV-infected feline lymphocyte cell line (3201/FIV) was inhibited only by dextran sulfate and pradimicin A. The assay used to measure syncytium inhibition was rapid and did not use potentially hazardous human immunodeficiency virus (HIV)-infected cells. The efficacy results coincided with those of HIV studies.

Purification and properties of UDP-glucose (UDP-galactose) pyrophosphorylase from Bifidobacterium bifidum.

An enzyme having both UDP-glucose (UDP-Glc) and UDP-galactose (UDP-Gal) pyrophosphorylase activities was purified to homogeneity from Bifidobacterium bifidum. The molecular weight of the enzyme was about 200,000 and it appeared to be composed of four identical subunits. The purified enzyme showed almost the same reactivity towards UDP-Glc and UDP-Gal, and showed about 10% of this activity towards UDP-xylose at 8 mM. The enzyme required magnesium ions for maximum activity. The apparent equilibrium constants were about 2.5 for UDP-Glc pyrophosphorolysis and 1.1 for UDP-Gal pyrophosphorolysis. The enzyme activities were inhibited by various nucleotides (product or substrate analogs). Some sugar phosphates, such as fructose 6-P, erythrose 4-P, and 3-phosphoglycerate, stimulated the activities. These properties are discussed in relation to the significance of the enzyme in galactose metabolism of B. bifidum.

Purification and enzymatic properties of glyoxylate reductase II from baker's yeast.

Glyoxylate reductase II was purified about 2,400-fold from a cell extract of baker's yeast by protamine sulfate treatment, and column chromatographies on DEAE-cellulose, hydroxylapatite, Sephadex G-150, and phosphocellulose. The purified enzyme was electrophoretically homogeneous. The molecular weight was determined to be approximately 65,000 by gel filtration. The enzyme was greatly stabilized by the addition of 20% (v/v) glycerol. It catalyzed the reduction of glyoxylate and hydroxypyruvate and was specific for NADPH as an electron donor, but showed slight affinity towards NADH. The Michaelis constants for glyoxylate, hydroxypyruvate, NADPH, and NADH were found to be 16mM, 1.4 mM, 5.7 microM, and 0.43 mM, respectively. The enzyme was inhibited by p-chloromercuribenzoate (PCMB) and iodoacetate, but inhibition was prevented by dithiothreitol (DTT) or L-cysteine. The reduction of glyoxylate and hydroxypyruvate was not stimulated by anions.

Purification and properties of glyoxylate reductase I from baker's yeast.

The purification and properties of NADPH-linked glyoxylate reductase [EC 1. 1. 1. 79] from baker's yeast were studied. Two active fractions (peak I and peak II) were isolated by DEAE-cellulose column chromatography. The peak I fraction was purified to homogeneity by the criteria of disc gel electrophoresis and tentatively designated glyoxylate reductase I. Its molecular weight was calculated to be 31,000 from gel filtration measurements. The enzyme reduced glyoxylate 7 times faster than hydroxypyruvate and was specific for NADPH. The enzyme showed optimum activity between pH 5.5 and 7.2. The Michaelis constants for glyoxylate and NADPH were found to be 13 mM and 4 microM, respectively. The enzymic activity was not significantly affected by anions, except for nitrate and iodide, which were inhibitory.

Purification and some properties of a novel alpha-L-fucosidase capable of acting on alpha-(1----6)-L-fucosidic linkages from Bacillus circulans M28.

Two types of alpha-L-fucosidase (F-I and F-II), that differ in substrate specificity, were produced in the culture fluid by Bacillus circulans isolated from soil when the bacterium was cultivated on medium containing porcine gastric mucin. F-I was able to cleave the alpha-(1----2), alpha-(1----3), and alpha-(1----4)-L-fucosidic linkages in various oligosaccharides and glycoproteins, but not p-nitrophenyl alpha-L-fucoside, as previously reported [Y. Tsuji et al. (1990) J. Biochem. 107, 324-330]. F-II was purified from the culture fluid obtained with glucose medium by ammonium sulfate fractionation and various subsequent column chromatographies. The purified enzyme was found to be homogeneous on PAGE and its molecular weight was estimated to be approximately 250,000. The maximal activity was observed between pH 6.0 to 7.0, the stable pH range being 6.0 to 8.5. The enzyme specifically cleaved alpha-L-fucosidic bonds in low molecular weight substrates. The enzyme cleaved not only p-nitrophenyl alpha-L-fucoside, but also 2-fucosyllactose and 3-fucosyllactose. The enzyme was also able to act on the alpha-(1----6)-L-fucosidic linkages to N-acetylglucosamine in 6-O-alpha-L-fucopyranosyl-N-acetylglucosamine, and bi- and tetra-antennary oligosaccharides derived from porcine pancreatic lipase, which were not hydrolyzed by F-I.

Microbial endo-beta-N-acetylglucosaminidases acting on complex-type sugar chains of glycoproteins.

The activity and substrate specificity of endo-beta-N-acetylglucosaminidase [glycopeptide-D-mannosyl-N4-(N-acetyl-D-glucosaminyl)2-asparagine 1,4-N-acetyl-beta-glucosamino-hydrolase, EC 3.2.1.96] obtained from Mucor hiemalis (Endo-M) was compared with that of the enzyme obtained from Flavobacterium meningosepticum (Endo-F), which is the only enzyme available that acts on the complex oligosaccharides of asparagine-linked sugar chains in glycoproteins. They showed almost the same activities toward DNS-ovalbumin glycopeptide containing high-mannose and hybrid asparagine-linked oligosaccharides. However, Endo-M showed high activity towards DNS-asialotransferrin and DNS-transferrin glycopeptides, which contain complex biantennary oligosaccharides. Endo-M could weakly act even on DNS-asialofetuin glycopeptide containing complex triantennary oligosaccharides, while Endo-F could not. SDS-denatured asialotransferrin was deglycosylated by both enzymes in the presence of non-ionic detergent (NP-40) and EDTA, and the deglycosylated protein migrated to a lower molecular weight position than asialotransferrin on SDS-PAGE. However, even in the absence of detergent, Endo-M deglycosylated native asialotransferrin and transferrin. Deglycosylation of asialotransferrin was confirmed by means of Con A-Sepharose 4B column chromatography and SDS-PAGE.

Purification and characterization of alpha-L-fucosidase from Bacillus circulans grown on porcine gastric mucin.

Bacillus circulans isolated from soil was found to produce two types of alpha-L-fucosidase differing in substrate specificity. One was able to liberate L-fucose from porcine gastric mucin (PGM), but not from artificial substrates, including p-nitrophenyl and methyl alpha-L-fucosides, while the other acted not on PGM but on p-nitrophenyl alpha-L-fucoside. The production of the former enzyme was enhanced about 150 times as much by PGM added to the medium as by glucose. The alpha-L-fucosidase acting on PGM was purified from the culture fluid obtained with PGM medium by ammonium sulfate fractionation and subsequent column chromatography. The purified enzyme was found to be homogeneous by PAGE and its molecular weight was estimated to be approximately 285,000. The optimum pH was found to be 5.5 to 6.5 and the stable pH range was 4.5 to 9.0. The enzyme decomposed various blood group O(H) active substances such as PGM, human milk and human saliva, and moreover acted on A-, B-, and O-erythrocytes. The enzyme was shown to cleave alpha-(1----2)-, (1----3)-, and (1----4)-L-fucosidic linkages in various glycoproteins and oligosaccharides, but failed to hydrolyze alpha-(1----6)-L-fucosic linkages in 6-O-alpha-L-fucopyranosyl-N-acetylglucosamine and intact bovine thyroglobulin.