α-Mannosidase from Pineapple Fruit: Partial Purification and Action on Glycopeptides

α-Mannosidase [EC 3.2.1.24, α-D-mannoside mannohydrolase] from the acetone powder of pineapple fruit juice was purified 190-fold by column chromatographic procedures. The partially purified a-mannosidase was detected to be contaminated with little other glycosidases, using p-nitrophenyl derivatives of glycosides. The enzyme released mannose from both the carbohydrate moiety of stem bromelain and glycopeptide prepared from the parent protein. The enzyme split about 70% of the total mannose of ovalbumin glycopeptide.     

A new beta-1,2-N-acetylglucosaminyltransferase that may play a role in the biosynthesis of mammalian O-mannosyl glycans

Recent studies have shown that O-mannosyl glycans are present in several mammalian glycoproteins. Although knowledge on the functional roles of these glycans is accumulating, their biosynthetic pathways are poorly understood. Here we report the identification and initial characterization of a novel enzyme capable of forming GlcNAc beta 1-2Man linkage, namely UDP-N-acetylglucosamine: O-linked mannose beta-1,2-N-acetylglucosaminyltransferase in the microsome fraction of newborn rat brains. The enzyme transfers GlcNAc to beta-linked mannose residues, and the formed linkage was confirmed to be beta 1-2 on the basis of diplococcal beta-N-acetylhexosaminidase susceptibility and by high-pH anion-exchange chromatography. Its activity is linearly dependent on time, protein concentration, and substrate concentration and is enhanced in the presence of manganese ion. Its activity is not due to UDP-N-acetylglucosamine: alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I (GnT-I) or UDP-N-acetylglucosamine: alpha-6-D-mannoside beta-1,2-D-acetylglucosaminyltransferase II (GnT-II), which acts on the early steps of N-glycan biosynthesis, because GnT-I or GnT-II expressed in yeast cells did not show any GlcNAc transfer activity against a synthetic mannosyl peptide. Taken together, the results suggest that the GlcNAc transferase activity described here is relevant to the O-mannosyl glycan pathway in mammals.     

Amine Transaminase

An enzyme “amine transaminase”, which catalyzed transamination between amines and α-keto acids, was found to occur in certain fermentative bacteria, such as Escherichia coli and Aerobacter aerogenes. Using a partially purified enzyme preparation obtained from cell extract of E. coli, some properties of the enzyme were investigated. α-Ketoglutaric acid appeared to be the most efficient amino acceptor and substitution of α-ketoglutaric acid by other α-keto acid resulted in much lower activity. Putrescine, cadaverine and hexamethylenediamine were found to be active as amino donors, but the other monoamines, diamines and polyamines were inert. Treatment of the enzyme with acid ammonium sulfate resolved the enzyme into apo- and coenzyme. The apoenzyme was well reactivated by pyridoxal phosphate as well as pyridoxamine phosphate. Physiological role of the amine transaminase was suggested in relation to the metabolism of amines in bacterial cells.     

Synthesis of ascorbate and urate in the ovary of water buffalo

Blood flow interruption is associated with oxygen depletion and loss of factors for function and survival in downstream tissues or cells. Hypoxia and absence of gonadotropins trigger apoptosis and atresia in the ovary. We studied the antioxidant response of follicular cells to plasma deprivation in ovaries dissected from water buffalo. Aliquots of follicular fluid were aspirated from each antral follicle, before and during incubation of the ovaries at 39°C. Urate, ascorbate, retinol and α-tocopherol in the fluid were, titrated by High Performance Liquid Chromatography (HPLC) with spectrophotometric or spectrofluorimetric detection. The total antioxidant capacity of follicular fluid was determined as absorbance decrease, following addition of a source of radical chromophores. The more the incubation progressed, the higher levels of urate, ascorbate and total antioxidant capacity were found. Conversely, changes in concentration of the liposoluble antioxidants were not observed. Ascorbate synthesizing activity in the follicle was demonstrated by detecting the enzyme L-gulono-γ-lactone oxidase in microsomes prepared from granulosa cells. These cells were also analyzed for the expression of the enzyme CPP32. The enzyme level, measured as DEVD-p-nitroanilide cleaving activity, was found related with the immunoreactivity to anti-CPP32 antibodies. Negative correlation between the enzyme activity (which is known to be induced by peroxynitrite) and the follicular level of urate (which scavenges peroxynitrite) was also observed. The amount of nitrotyrosine, a product of peroxynitrite attack on proteins, was measured in follicular fluids by Enzyme Linked ImmunoSorbent Assay (ELISA). This amount was found positively correlated with the CPP32 activity, and negatively correlated with the urate level in follicular fluid. Alterations in concentrations of ascorbate or urate may be associated with oxidative stress during follicular atresia.     

Specific binding of sulfated proteoglycans to concanavalin A - Sepharose 4B.

More than 90 % of [³⁵S]proteoglycans isolated from the secretions of human skin fibroblasts bind to Concanavalin A-Sepharose 4B (Con A-Sepharose) in the presence of 1 M NaCl. Above pH 5.0 1 M concentrations of methyl-α-D-mannoside and other haptenic inhibitors for Con A-sugar interaction prevent binding of [³⁵S]proteoglycans, whereas equimolar concentrations of non-haptenic carbohydrates do not effect binding. Below pH 5.0 [³⁵S]proteoglycans bind to Con A-Sepharose in the presence of both methyl-α-D-mannoside and galactose. About 60 % of the proteoglycans bound at pH 4.0 are eluted at pH 7.5 in the presence of 1 M methyl-α-D-mannoside. [³⁵S] Glycosaminoglycans prepared from [³⁵S] proteoglycans do not bind to Con A-Sepharose in the presence of 1 M NaCl.These results indicate a [³⁵S]proteoglycan-Con A interaction via the protein core of the proteoglycan and the sugar binding sites of Con A.     

Selection of High Ubiquinone 10-Producing Strain of Tobacco Cultured Cells by Cell Cloning Technique

A novel enzyme, which was named N^α-benzyloxycarbonyl amino acid urethane hydrolase, was purified from a cell-free extract of Streptococcus faecalis R ATCC 8043, using N^α-benzyloxycarbonyl glycine as substrate. The enzyme was purified 1300-fold with an activity yield of 8%. The purified enzyme was homogeneous by disc electrophoresis. The molecular weight of the native enzyme is about 220,000 by gel filtration, and a molecular weight of 32,000 was determined for the reduced and denatured enzyme by gel electrophoresis in sodium dodecyl sulfate. The isoelectric point was 4.48. The enzyme was inhibited by p-chloromercuribenzoate. The presence of divalent cations (i.e., Co²⁺ or Zn²⁺) is essential for its activity.     

Expression, Purification, and Characterization of Recombinant α-N-Acetylgalactosaminidase Produced in the YeastPichia pastoris

α-N-Acetylgalactosaminidase (αNAGAL, EC 3.2.1.49) purified from chicken liver has been used in seroconversion of human erythrocytes. Blood group A, defined by the terminal α-linkedN-acetylgalactosamine, can be cleavedin vitroby αNAGAL, resulting in the underlying penultimate blood group H (O) epitope structure. In order to produce sufficient quantities of recombinant αNAGAL (rαNAGAL) for such studies, we expressed the cDNA encoding chicken liver αNAGAL inPichia pastoris,a methylotrophic yeast strain. The αNAGAL coding sequence was cloned into theEcoRI site of the vector pPIC 9 such that the protein was in the same reading frame as the secretion signal of yeast α-mating factor derived from the vector. AfterP. pastoristransformation, colonies were screened for high-level expression of rαNAGAL based on enzyme activity. As a result of methanol induction of high-density cell cultures in a fermentor, enzymatically active rαNAGAL was produced and secreted into the culture medium. The recombinant enzyme was purified over 150-fold by chromatography on a cation exchange column followed by an affinity column. Its homogeneity was confirmed by Coomassie blue-stained SDS–PAGE, Western blot, and N-terminal sequencing. The purified rαNAGAL has a molecular mass of approximately 50 kDa while its native counterpart has a molecular mass of 43 kDa. This discrepancy in size was eliminated by endoglycosidase treatment, suggesting that the recombinant protein was hyperglycosylated by the hostP. pastoriscells. rαNAGAL was further characterized in terms of specific activity, pH profile, kinetic parameters, and thermostability by comparing with αNAGAL purified from chicken liver. The data presented here suggest that by overexpressing rαNAGAL inP. pastorisand purifying with affinity chromatography one can readily obtain the quantity of enzyme needed for seroconversion studies.     

Studies on cell adhesion and recognition. III. The occurrence of α-mannosidase at the fibroblast cell surface, and its possible role in cell recognition

The occurrence of α-mannosidase activity at the surface of hamster embryo (NIL) fibroblasts is indicated by the following findings: (a) When NIL cells were incubated on the glass surfaces on which ovalbumin glycopeptides were covalently linked, a rapid release of free mannose from ovalbumin glycopeptides was observed as evidenced by analysis on gas chromatography/mass spectrometry. (b) Cell suspensions as well as intact cell monolayers hydrolyzed rapidly p-nitrophenyl-α-D-mannoside, and the time-course of the hydrolytic cleavage was linear from the moment of mixing of the substrate with the cells. The hydrolysis of the nitrophenyl glycosides of β-D-mannose, α-D-galactose, β-D-galactose, α-L-fucose, β-D-glucose, β-D-N-acetylgalactosamine and β-D-N-acetylglucosamine was negligible or more than ten times lower as compared with the hydolysis of α-D-mannoside. (c) No released or secreted activity of mannosidase could be detected under the conditions used. (d) Studies using known proportions of broken cells in the incubation mixture indicated that more than 90 percent of the mannosidase activity measured was attributable to intact cells and not to broken cells or cell fragments. (e) Hydrolysis of p-nitrophenyl-α-D-mannoside by cell monolayers was inhibited, in the order of decreasing inhibitory activity, by yeast mannan, ovalbumin, α-1,4-L-mannonolactone, α-methylmannoside, and mannose-6-phosphate. High inhibitory activity of the mannan polysaccharide and of ovalbumin favored the presence of the mannosidase activity at the cell surface, as these substrates may not penetrate rapidly into the cells. The following findings indicated that the cell surface mannosidase is mediating the cell adhesion based on the recognition of high-mannose-type glycopeptide: (a) Ovalbumin- coated plastic surfaces strongly promoted attachment and spreading of NIL fibroblasts, whereas the same ovalbumin coat did not promote attachment and spreading of some other cell types (BALB/c 3T3 fibroblasts and freshly prepared rat liver cells). (b) Digestion of ovalbumin with α-mannosidase greatly reduced the adhesion-mediating activity. (c) Cell adhesion to ovalbumin-coated surfaces was strongly inhibited by mannose tetrasaccharides, moderately by α-1,4-L-mannonolactone, and weakly by α- methylmannoside and mannose-6-phosphate. This order of the inhibitory activity for cell attachment is the same as that for the inhibition of mannosidic hydrolysis. The interpretation that the cell surface mannosidase is able to mediate cell adhesion is in agreement with previous studies suggesting that polyvalent glycosidase surfaces can promote cell adhesion to a degree similar to that caused by fibronectin and several lectins by interacting with their cell surface substrate site (the accompanying papers of this series).     

Synthesis and biological evaluation of esters of 16-formyl-17-methoxy-dehydroepiandrosterone derivatives as inhibitors of 5α-reductase type 2

In this study, we investigated the in vitro effect of 16-formyl-17-methoxy dehydroepiandrosterone derivatives on the activity of 5α-reductase type 2 (5α-R2) obtained from human prostate. The activity of different concentrations of these derivatives was determined for the conversion of labelled testosterone to dihydrotestosterone. The results indicated that an aliphatic ester moiety at the C-3 position of these derivatives increases their in vitro potency as inhibitors of 5α-R2 activity compared to finasteride®, which is considered to be a potent inhibitor of 5α-R2. In this case, the augmentation of the lipophilicity of these dehydroepiandrosterone derivatives increased their potency as inhibitors of 5α-R2. However, the presence of cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl rings as the cycloaliphatic ester moiety at C-3 of the formyl methoxy dehydroepiandrosterone scaffold did not inhibit the activity of this enzyme. This may be due to the presence of steric factors between the enzyme and the spatial structure of these derivatives.     

Properties of Crystalline α-Glucosidase from Mucor javanicus

Substrate and inhibitor specificities, and transglucosylation action of crystalline α-glucosidase from the mycelia of Mucor javanicus have been investigated. The enzyme hydrolyzed maltose, methyl-α-maltoside, and soluble starch liberating glucose, but little or not phenyl-α-glucoside, methyl-α-glucoside, sucrose, isomaltose, panose and dextran. The enzyme hydrolyzed phenyl-α-maltoside to glucose and phenyl-α-glucoside. The enzyme acted also as a glucosyltransferase when it was incubated with glucosyl donor such as maltose. Maltotriose was the principal transglucosylation product formed from maltose. The enzyme also catalyzed transglucosylation from maltose to riboflavin, pyridoxine, esculin and rutin. Tris and turanose inhibited the enzyme activity, but PCMB and EDTA did not. It is suggested that the enzyme activity is closely related to the histidine residue in the active center, from the inhibition experiments using diazonium-1-H-tetrazole and rose bengal.     

N-Glycosylation is requisite for the enzyme activity and Golgi retention of N-acetylglucosaminyltransferase III

UDP-N-acetylglucosamine:ß-D-mannoside ß-1,4N-acetylglucosaminyltransferaseIII (GnT-III, EC 2.4.1.144 [EC] ) is a glycoprotein involved in thebiosynthesis of N-linked oligosaccharides. Rat GnT-III containsthree potential Nglycosylation sites, which have been predictedto be Asn243, Asn261, and Asn399. To study the roles of Nglycosylationin the GnT-III function, rat GnT-III was expressed in COS-1cells under tunicamycin or castanospermine treatment. The tunicamycin-treatedGnT-III, which was not N-glycosylated, had almost no activity.The castanospermine-treated GnT-III was not localized in theGolgi, but glucosylation did not affect its activity. To clarifythe role of individual N-glycosylations, we obtained a seriesof mutant cDNAs in which some or all of the potential glycosylationsites were eliminated by site-directed mutagenesis, and expressedthem in COS-1 cells. All the mutants exhibited lower enzymeactivity than the wild-type, but deglycosylation at individualsites had different effects on the enzyme activity. The deglycosylationat Asn243 or Asn261 was more effective on the activity thanthat at Asn399. The enzyme activity decreased as the numberof glycosylation sites decreased. The null glycosylation mutanthad no activity, corresponding to the case of tunicainycin-treatedwild-type GnT-III. Kinetic analysis revealed that the deglycosylationat Asn243 or Asn.261 resulted in slightly lower affinity forthe donor substrate, but the other mutation did not significantlychange the K_m value for either the donor or acceptor. None ofthe mutant GnT-IIIs showed perinuclear localization or Golgiretention, that was observed for the wild-type protein. Thisis the first demonstration that the glycosyltransferase localizedin the Golgi apparatus requires N-glycosylation for its activityand retention. N-acetylglucosaminyltransferase III N-glycosylation Golgi apparatus glycoprotein protein folding     

Comparative Biochemical Studies of α-Glucosidases

The properties of brewer’s yeast α-glucosidase have been investigated. The enzyme was capable of hydrolyzing various α-glucosides and was active especially on aryl-α-glucosides in comparison with other α-glucosides and sugars. The rate of hydrolysis decreased in following order: phenyl-α-glucosides, sucrose, matlose and isomaltose.

The range of opt. temp, was 40~45°C and opt. pH, 6.5~7.0.

Cu⁺⁺ and Hg⁺⁺ inhibited strongly the enzyme activity and Zn⁺⁺, moderately. The enzyme was suggested to be a sulfhydryl enzyme from the inhibition experiments by SH-reagents and the effects of glutathione on the activity.

The enzyme synthesized some oligosaccharides from maltose. As the transglucosidation products, nigerose, isomaltose, kojibiose and maltotriose were detected by paperchromatography.

Pure nigerose was separated by splitting maltose with amyloglucosidase from the mixture of maltose and nigerose and by use of successive carbon column chromatography.     

Induction of glutathione peroxidase in response to inactivation by nitric oxide

To determine effect of nitric oxide (NO) on cellular glutathione peroxidase (GPX) level in living cells, we measured the activity, protein and mRNA of GPX in rat kidney (KNRK) cells under a high NO condition. Combined treatment of lipopolysaccharide (LPS, 1 μg/ml) and tumor necrosis factor-α (TNF-α, 50 ng/ml) synergistically enhanced (23-folds) nitrite production from KNRK cells. This was suppressed by an inducible NO synthase (iNOS) inhibitor (aminoguanidine, N-nitro-L-arginine methylester hydrochloride) and arginase. iNOS expression was detected by RT-PCR in the treated cells. GPX was inactivated irreversibly when the cells had been homogenized before exposure to a NO donor, S-nitroso-N-acetylpenicillamine (SNAP). In living KNRK cells, SNAP and LPS + TNF-α exerted a transient effect on the GPX activity. The treatment with SNAP (200 μM) or sodium nitroprusside (200 μM) enhanced GPX gene expression, which was blocked by a NO scavenger, 2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide. GPX mRNA was markedly increased by the treatment with LPS + TNF-α, and aminoguanidine blocked the effect. In cells metabolically labeled with ⁷⁵Se, LPS + TNF-α accelerated the incorporation of radioactivity into GPX molecule by 2.1-fold. These results suggest that inactivation of GPX by NO triggers a signal for inducing GPX gene expression in KNRK cells, thereby restoring the intracellular level of this indispensable enzyme.     

The M2-type isoenzyme of pyruvate kinase phosphorylates prothymosin α in proliferating lymphocytes

Prothymosin α (ProTα) is a multifunctional protein that, in mammalian cells, is involved in nuclear metabolism through its interaction with histones and that also has a cytosolic role as an apoptotic inhibitor. ProTα is phosphorylated by a protein kinase (ProTαK), the activity of which is dependent on phosphorylation. ProTα phosphorylation also correlates with cell proliferation. Mass spectrometric analysis of ProTαK purified from human tumor lymphocytes (NC37 cells) enabled us to identify this enzyme as the M2-type isoenzyme of pyruvate kinase. A study on the relationship between ProTαK activity and pyruvate kinase isoforms in NC37 cells and in other cell types confirmed that the M2 isoform is the enzyme responsible for ProTαK activity in proliferating cells. Yet, about 10% of the cellular pool of the M2 isoform shows specific affinity for ProTα and is responsible for ProTαK activity. This pool of M2 protein possesses no observable pyruvate kinase activity and changes its responses to various effectors of pyruvate kinase activity; however, these responses to PK effectors are maintained by the main cellular fraction containing the M2 isoform. Acquisition of ProTαK activity by M2 seems to be due to the phosphorylation of serine and threonine residues, which, besides being essential for its catalytic activity, induces a trimeric association of ProTαK. This association can be shifted to a tetrameric form by fructose 1, 6-bisphosphate, which results in a decrease in ProTαK activity.     

Acid-Stable α-Amylase of Black Aspergilli

The acid-stable α-amylase or the acid-unstable α-amylase from Aspergillus niger contained 24 moles or 7 moles mannose and 4 moles or 1 mole hexosamine per mole of protein, respectively.

The acid-stable α-amylase and the acid-unstable α-amylase contained calcium only, but not detectable amounts of other metals. Calcium contents of the both enzymes were converged to at least one gram atom per mole of enzyme by dialysis against acetate buffer. The last calcium could be removed under the suitable conditions by EDTA. Calcium removal by EDTA was accompanied by the loss of activity and by the little change of UV absorption spectra. The phenomenon caused by calcium removal were partially reversible. This last one atom of calcium seemed to be essential for the maintenance of active structure of α-amylase.     

Modification of α‐Amylase by Sodium Alginate

Sodium alginate, activated by periodate oxidation, was covalently linked to porcine pancreatic α‐amylase via reductive alkylation with NaBH₄. The enzyme‐polymer conjugate, purified by gel filtration on Fractogel EMD BioSEC (S), retained about 50% of the native specific amylolytic activity. The sugar content was estimated to be 712 mol of monosaccharides per mol of enzyme protein. An average of 11 amino groups out of 21 groups from α‐amylase were modified with the polysaccharide. The functional stability was improved for α‐amylase after conjugation with sodium alginate. In comparison with the native enzyme, the thermostability of α‐amylase was increased by this modification. In addition, the stability in the range of pH 5.0–11.0 was improved for the modified enzyme. The conjugate was also more resistant to denaturation by 0.3% sodium dodecylsulphate, retaining about 10% of its initial activity after 120 min of incubation. The formation of stabilizing salt bridges in the protein surface of the α‐amylase‐polysaccharide complex was confirmed by FT‐IR spectrometry. Attending to the results obtained, we conclude that the covalent attachment of the anionic polysac‐charide sodium alginate to the enzymes might be a useful and non‐expensive method for improving the stabilization of these biocatalysts under various denaturing conditions.     

Purification and enzymatic properties of α-galactosidase from Penicillium janthinellum

α-Galactosidase (E.C.3.2.1.22) from Penicillium janthinellum was purified by precipitation and fractionation with ammonium sulphate, cold acetone or ethanol, calcium phosphate gel, and column chromatographies on Sephadex G-100 and G-200. The enzyme was purified about 110.39-fold when Sephadex G-100 was used. α-Galactosidase exhibited the optimum pH and temperature at 4.5 and 60°C, respectively. The optimum enzyme stability was obtained at pH 3.5 for 24 h (at room temperature). The enzyme was found to be thermostable below 65°C up to 40 minutes and was gradually inactivated by increasing the temperature above this degree. The MICHAELIS constant was 0.55 mM for p-nitrophenyl-α-D-galactoside. The α-galactosidase activity was strongly inhibited by Hg⁺⁺ and slightly activated by Mn⁺⁺. The results show the possibility of producing a thermostable enzyme from a low-priced agricultural product, for instance, lupine.     

Synthesis of Analogs of Pestalotin

Starch-utilizing mutants of Escherichia coli which can grow well on starch or amylose as the sole carbon source were isolated. The maximal viable cell number of the starch-utilizing mutants on the polysaccharide media reached the same level (4 × 10⁹ cells/ml) as that with glucose medium after incubation for 24 hours at 37°C. The isolated mutants could produce more intracellular α-amylase than the wild-type strain, and the enzyme activity was detected in the extracellular fluid. Polyacrylamide gel electrophoresis showed that the intracellular and extracellular enzymes had similar electrophoretic mobilities. These observations suggested that the ability of growth on the polysaccharide media was due to the excreted α-amylase, which appeared to be identical with the intracellular enzyme.     

Propionate Enhances Synthesis and Secretion of Bile Acids in Primary Cultured Rat Hepatocytes via Succinyl CoA

To discover the role of propionate produced by colonic bacteria, this study examined the secretion of bile acids and cholesterol 7α-hydroxylase activity in the primary cultured hepatocytes. Addition of propionate (2 mM) to the medium for 48 h caused an increase in the bile acid secretion and enzyme activity, while acetate and butyrate had no significant influence. Bile acid secretion was increased by the addition of succinyl CoA and its precursor substances (α -ketoglutarate, valine, isoleucine, and methionine), but not malate and oxaloacetate, which are the metabolites of succinyl CoA. α -Ketoglutarate and valine also increased the activity of cholesterol 7α -hydroxylase. Since cholesterol 7α -hydroxylase is a microsomal cytochrome P-450 enzyme and the formation of δ-aminolevulinate from succinyl CoA in the mitochondria is the rate-controlling step for the subsequent synthesis of heme proteins, propionate may affect bile acid synthesis via elevation of mitochondrial succinyl CoA.