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PubMed1981

[beta-1,3-1,4-glucanase in spore-forming microorganisms. IV. Properties of some Bacillus-beta-glucan-hydrolases (author's transl)].

The beta-glucan-hydrolases produced by seven Bacillus species have been characterized with regard to some physicochemical properties. Ca-ions stabilize all tested glucanases. Optimum pH and optimum temperature were found to be different for the investigated enzymes. In the work presented here is given further characteristics of Bacillus-beta-glucan-hydrolases as pH-stability and temperature stability, sensibility on glucono-1,5-lactone, as well known inhibitor of carbohydrases, and electrophoretic mobility on cellulose acetate-sheets.

Bacillus↗
PubMed1994

Guanosine-inosine-preferring nucleoside N-glycohydrolase from Crithidia fasciculata.

Protozoan parasites are incapable of de novo purine biosynthesis and obtain purines by salvage pathways. A nucleoside hydrolase which prefers inosine and uridine as substrates (IU-nucleoside hydrolase) has been characterized and implicated in purine salvage in Crithidia fasciculata (Parkin, D. W., Horenstein, B. A., Abdulah, D. R., Estupiñán, B., and Schramm, V. L. (1991) J. Biol. Chem. 31, 20658-20665). Treatment of C. fasciculata with inhibitors of the IU-nucleoside hydrolase did not prevent cell growth, suggesting alternative enzymes. A guanosine-inosine-preferring enzyme (GI-nucleoside hydrolase) has been purified from extracts of C. fasciculata and characterized. The enzyme is an oligomer of M(r) 38,500 subunits. The Vmax/Km for guanosine, inosine, and adenosine are 3.2 x 10(6), 6.2 x 10(6), and 9.8 M-1 S-1, respectively. Deoxynucleosides, nucleotides, and pyrimidine nucleosides are poor substrates. The pH profile for Km is independent of pH, whereas both Vmax and Vmax/Km demonstrate that a single protonated base, pKa 7.7 is required for activity. The transition state inhibitors of IU-nucleoside hydrolase, 1,4-dideoxy-1,4-imino-1-(S)-phenyl-D-ribitol (Horenstein, B. A., and Schramm, V. L. (1993) Biochemistry 32, 9917-9925) and p-nitrophenylriboamidrazone (Boutellier, M., Horenstein, B. A., Semenyaka, A., Schramm, V.L., and Ganem, B. (1994) Biochemistry 33, 3994-4000), are unexceptional inhibitors of the GI-nucleoside hydrolase. The enzyme is inhibited by 3-deazaadenosine and 2-iodoadenosine with Km/Ki values of 145 and 61, respectively. The results demonstrate that this previously uncharacterized enzyme has distinct structure, kinetic, and chemical mechanisms relative to IU-nucleoside hydrolase. Metabolic studies with labeled inosine as the sole purine source indicated that the GI-enzyme is efficient for purine salvage in vivo.

Animals↗
PubMed2002

Polysaccharide analysis using carbohydrate gel electrophoresis: a method to study plant cell wall polysaccharides and polysaccharide hydrolases.

A method to characterize plant cell wall polysaccharides is presented. The complexity of the polymer structures and the large number of different charged and uncharged monosaccharides that make up plant polysaccharides have previously made analysis technically demanding and laborious. Polysaccharide analysis using carbohydrate gel electrophoresis (PACE) relies on derivatization of reducing ends of sugars and oligosaccharides with a fluorophore, followed by electrophoresis under optimized conditions in polyacrylamide gels. We show that PACE is a sensitive and simple tool for studying the monosaccharide composition of polysaccharides and of cell wall preparations. In combination with specific hydrolases, it can be used to analyze the structure of polysaccharides. Moreover, the specificity and kinetics of the plant polysaccharide hydrolases themselves can be quickly and effectively studied. PACE can detect as little as 500 fmol of monosaccharides and 100 fmol of oligosaccharides, and it is fast and quantitative.

Arabidopsis↗
PubMed1995

Purification, cloning, and functional expression of sucrose:fructan 6-fructosyltransferase, a key enzyme of fructan synthesis in barley.

Fructans play an important role in assimilate partitioning and possibly in stress tolerance in many plant families. Sucrose:fructan 6-fructosyltransferase (6-SFT), an enzyme catalyzing the formation and extension of beta-2,6-linked fructans typical of grasses, was purified from barley (Hordeum vulgare L.). It occurred in two closely similar isoforms with indistinguishable catalytic properties, both consisting of two subunits with apparent masses of 49 and 23 kDa. Oligonucleotides, designed according to the sequences of tryptic peptides from the large subunit, were used to amplify corresponding sequences from barley cDNA. The main fragment generated was cloned and used to screen a barley cDNA expression library. The longest cDNA obtained was transiently expressed in Nicotiana plumbaginifolia protoplasts and shown to encode a functional 6-SFT. The deduced amino acid sequence of the cDNA comprises both subunits of 6-SFT. It has high similarity to plant invertases and other beta-fructosyl hydrolases but only little to bacterial fructosyltransferases catalyzing the same type of reaction as 6-SFT.

Amino Acid Sequence↗
PubMed1981

The mannose 6-phosphate receptor of Chinese hamster ovary cells. Compartmentalization of acid hydrolases in mutants with altered receptors.

The localization of acid hydrolases was examined in Chinese hamster ovary cells with defective mannose 6-phosphate receptors; these mutants had been shown to exhibit reduced uptake and altered binding of exogenously added acid hydrolase (Robbins, A. R., Myerowitz, R., Youle, R. J., Murray, G. J., and Neville, D. M., Jr. (1981) J. Biol. Chem. 256, 10618-10622). Cells were grown in the presence of [3H]mannose, alpha-L-iduronidase and beta-hexosaminidase were immunoprecipitated sequentially, electrophoresed on polyacrylamide gels containing sodium dodecyl sulfate, and detected by fluorography. About 55% of the alpha-L-iduronidase and beta-hexosaminidase synthesized by the mutants in 12 h was found in the growth medium; parental cells secreted only approximately 15%. The mutants also secreted 2 to 6 times more alpha-mannosidase, beta-glucuronidase, and alpha-L-fucosidase than the parent as determined by measurements of enzyme activity. Intracellular levels of these enzymes were reduced in the mutants. The mutants secreted acid hydrolases in the precursor forms, within the cells these enzymes resided in lysosomes and were processed normally; thus, the mutants appeared aberrant only with respect to distribution of hydrolases between intracellular and extracellular compartments. [35S]methionine-labeled beta-hexosaminidase and alpha-L-iduronidase secreted by the mutants were taken up normally by both human fibroblasts and wild type CHO cells, and this uptake was inhibited by mannose 6-phosphate. Thus, the elevated secretion of acid hydrolases was not due to alteration of the mannose 6-phosphate recognition marker on the enzymes, but appears to result from alterations in the mannose 6-phosphate receptor.

Animals↗
PubMed1976

Enzymic solubilization of the human intestinal brush border membrane enzymes.

The releases of proteins, maltase, lactase, sucrase, trehalase, alkaline phosphatase, gamma-glutamyltransferase and leucylnaphthylamide-hydrolyzing activity from human intestinal brush bborder membrane vesicles by various enzymes (especially pancreatic proteases) have been studied. The brush border membrane enzymes are not solubilized by digestion with trypsin and chymotrypsin but are largely released after treatment with papain or elastase. Most of the enzymes are fully active after the proteolytic treatment. All proteins released by papain and elastase have been identified by electrophoresis to already known intestinal hydrolases. Electron microscopy of brush border membrane vesicles demonstrates "knob-like" structures (particles) attached to the external side of the membrane. During papain treatment, enzyme removal runs parallel with the disappearance of the particles. During elastase treatment it is not possible to correlate the release of the enzymic activities with the removal of the particles. The results indicate that most of the intestinal hydrolases are surface components attached to the external side of the membrane. They are in accord with the concept that the brush border membrane enzymes are organized within the membrane in a mosaic-like pattern.

Cell Membrane↗
PubMed2006

Thioimidoyl furanosides as first inhibitors of the alpha-L-arabinofuranosidase AbfD3.

Two sets of five thioimidoyl alpha-L-arabino- and beta-D-galactofuranosides were designed, synthesized and subjected to docking studies to evaluate their ability to be recognized by the active site of the alpha-L-arabinofuranosidase AbfD3. Further in vitro assays showed that the targeted furanosides are the first potent inhibitors of this furanosyl hydrolase and that the most efficient one, the thiazolyl alpha-L-arabinofuranoside 1, is a competitive inhibitor having a KI of 1.4 microM.

Bacteria↗
PubMed1981

Uptake and metabolism of sucrose by Streptococcus lactis.

Transport and metabolism of sucrose in Streptococcus lactis K1 have been examined. Starved cells of S. lactis K1 grown previously on sucrose accumulated [14C]sucrose by a phosphoenolpyruvate-dependent phosphotransferase system (PTS) (sucrose-PTS; Km, 22 microM; Vmax, 191 mumol transported min-1 g of dry weight of cells-1). The product of group translocation was sucrose 6-phosphate (6-O-phosphoryl-D-glucopyranosyl-1-alpha-beta-2-D-fructofuranoside). A specific sucrose 6-phosphate hydrolase was identified which cleaved the disaccharide phosphate (Km, 0.10 mM) to glucose 6-phosphate and fructose. The enzyme did not cleave sucrose 6'-phosphate(D-glucopyranosyl-1-alpha-beta-2-D-fructofuranoside-6'-phosphate). Extracts prepared from sucrose-grown cells also contained an ATP-dependent mannofructokinase which catalyzed the conversion of fructose to fructose 6-phosphate (Km, 0.33 mM). The sucrose-PTS and sucrose 6-phosphate hydrolase activities were coordinately induced during growth on sucrose. Mannofructokinase appeared to be regulated independently of the sucrose-PTS and sucrose 6-phosphate hydrolase, since expression also occurred when S. lactis K1 was grown on non-PTS sugars. Expression of the mannofructokinase may be negatively regulated by a component (or a derivative) of the PTS.

Biological Transport↗
PubMed2004

Cloning and expression of a novel Trichoderma viride laminarinase AI gene (lamAI).

The gene lamAI, which encodes a novel laminarinase AI of Trichoderma viride U-1, was cloned using RT-PCR in conjunction with the rapid amplification of cDNA ends (RACE) technique. The open reading frame consisted of 2,277 bp encoding a protein of 759 amino acid residues, including a 32-residue signal prepropeptide. The protein showed 91% sequence similarity to the putative Trichoderma virens beta-1,3-glucanase BGN1, but no significant similarity to fungal beta-1,6-glucanases or beta-1,3-glucanases from other organisms. On 40 h incubation with a solo carbon source, northern analysis revealed that the gene was induced by 0.5% laminaran from Eisenia bicyclis but was not by the same concentration of glucose. The lamAI cDNA was functionally expressed in the methylotrophic yeast Pichia pastoris, resulting in a recombinant enzyme with as high activity against laminaran as native LAMAI. Based on these data, the probable existence of endo-beta-1,3:1,6-glucan hydrolases as a subclass of endo-beta-1,3-glucanases in some mycoparasitic fungi is suggested.

Amino Acid Sequence↗
PubMed1992

Molecular and immunological characterization of ADP-ribosylarginine hydrolases.

Mono-ADP-ribosylation is a reversible modification of proteins with NAD:arginine ADP-ribosyltransferases and ADP-ribosylarginine hydrolases catalyzing the forward and reverse reactions, respectively. Hydrolase activities were present in a variety of animal species, with the highest specific activities found in rat and mouse brain, spleen, and testis. Rat and mouse hydrolases were dithiothreitol- and Mg(2+)-dependent, whereas the bovine and guinea pig enzymes were dithiothreitol-independent. A rat brain hydrolase was purified approximately 20,000-fold and represented the major approximately 39-kDa protein on denaturing gels. Immunoaffinity-purified rabbit polyclonal antibodies reacted with 39-kDa proteins from turkey erythrocytes and rat, mouse, and calf brains. A rat brain cDNA library was screened using oligonucleotide and polymerase chain reaction-generated cDNA probes. Inserts from two overlapping clones yielded a composite sequence that included a 1086-base pair open reading frame, which contained amino acid sequences found in the purified hydrolase. A hydrolase fusion protein, synthesized in Escherichia coli, reacted with anti-39-kDa polyclonal antibodies and exhibited Mg(2+)- and dithiothreitol-dependent hydrolase activity. A coding region cDNA hybridized readily to a 1.7-kilobase band in rat and mouse poly(A)+ RNA, but poorly to bovine, chicken, rabbit, and human poly(A)+ RNA. The immunological and molecular biological data are consistent with partial conservation of hydrolase structure across animal species.

Amino Acid Sequence↗
PubMed1997

Mono(ADP-ribosyl)transferases and related enzymes in animal tissues. Emerging gene families.

Mono-ADP-ribosylation, like phosphorylation, is an enzyme-catalyzed, reversible post-translational modification that modulates protein function. It was originally discovered as the pathogenic principle of diphtheria-, cholera-, and other potent bacterial toxins. By analogy, corresponding enyzmes were postulated to exist in animal tissues, and mounting biochemical evidence indicates that such enzymes, indeed, play important regulatory roles in cellular functions. The molecular cloning of the first mammalian mono(ADP-ribosyl)transferase from rabbit skeletal muscle, the finding of its homology to a well-studied T-cell marker, RT6, and the molecular cloning of additional gene family members from mammals and birds is providing fresh impetus to research in this field. Intriguingly, these vertebrate enzymes are predicted to be secretory or membrane proteins. They are expressed in lymphatic tissues, muscle, testis, bone marrow, and erythroblasts. Here we review the relationship between this novel family of eucaryotic mono(ADP-ribosyl)transferases (mADPRTs), ADP-ribosylating bacterial toxins, the poly(ADP-ribose)polymerase (PARP), the ADP-ribosyl cyclases, and the ADP-ribosylprotein hydrolase (ARH) in terms of their structure, enzymatic properties and possible biological functions.

ADP Ribose Transferases↗
PubMed1988

Synergism of rumen microbial hydrolases during degradation of plant polymers.

In isolated mixture of exocellular enzymes of rumen bacteria Ruminococcus flavefaciens, Butyrivibrio fibrisolvens and rumen fungus Neocallimastix frontalis, specific activities of cellulases, hemicellulases and glycosidases were determined. The highest specific activities were shown mostly for proteins of N. frontalis.

Animals↗
PubMed1989

Intracellular degradation of bleomycin hydrolase in two Chinese hamster cell lines in relation to their peplomycin susceptibility.

The Chinese hamster lung (V79) cell was intrinsically 10-times more resistant to peplomycin, a bleomycin-related antitumor antibiotic, than the Chinese hamster ovary (CHO) cell. This may be associated with the 3-times higher levels of recovery of bleomycin hydrolase activity of the V79 cell. The degradation of bleomycin hydrolase molecules in both V79 and CHO cells was examined using a monoclonal antibody specific for the enzyme. Labelling experiments showed that the bleomycin hydrolase in CHO cells was less stable than the comparable enzyme in V79 cells, and that 48 kDa subunits comprising bleomycin hydrolase (a homohexameric enzyme) molecules were degraded into 31 kDa forms in both cell lines. The 105,000 X g pellet (microsomes) fraction obtained after subcellular fractionation of CHO cells contained both 48 kDa subunit and 31 kDa forms of bleomycin hydrolase, while the 105,000 X g supernatant cytosol fraction yielded only 48 kDa subunit forms of the enzyme. Moreover, bleomycin hydrolase activity of both V79 and CHO cells was almost entirely recovered from the cytosol fraction. These results suggest that degradation of the 48 kDa subunit form of bleomycin hydrolase in these two lines of cultured cells into the 31 kDa form occurs on the plasma membrane or the endoplasmic reticulum, with which the resulting large number of bleomycin hydrolase molecules or degraded forms of the enzyme that have lost enzymatic activity are associated.

Animals↗
PubMed1992

Identification of glutamic acid 105 at the active site of Bacillus amyloliquefaciens 1,3-1,4-beta-D-glucan 4-glucanohydrolase using epoxide-based inhibitors.

Bacillus amyloliquefaciens 1,3-1,4-beta-D-glucan 4-glucanohydrolase (EC 3.2.1.73) was modified by the mechanism-based, affinity-labeling reagent [14C](3,4)-epoxybutyl beta-D-cellobioside. Following partial inactivation a completely inactivated enzyme preparation containing 1.1 mol of covalently bound inhibitor/mol of protein was obtained by chromatography on a cellulosic matrix. The inactivated enzyme was digested with endoproteinase Glu-C and radioactive peptides purified by reversed-phase high performance liquid chromatography (HPLC). The affinity label was esterified exclusively to the gamma-carboxylate of Glu105 in the sequence Gly-Thr-Pro-Trp-Asp-Glu-Ile-Asp-Ile-Glu109. The sequence motif Glu-(Ile/Leu)-Asp-Ile is found in many glucanases and xylanases and may therefore serve to identify the catalytic nucleophile in beta-glycanases, which otherwise exhibit a low degree of sequence identity. The esterification of Glu105 by the affinity label abolished endoproteinase Glu-C-mediated hydrolysis of the Glu-Ile106 peptide bond. Identification of phenylthiohydantoin-Glu105 during automated sequence analysis was not possible unless the affinity label was liberated by prior base hydrolysis. These observations formed the basis for the development of a highly sensitive approach for the identification of catalytic carboxylates in polysaccharide hydrolases employing non-radioactive inhibitors, comparative HPLC mapping, electrospray mass spectrometry, and Edman degradation.

Amino Acid Sequence↗
PubMed1984

Study of some lysosomal glycohydrolases in tumors of the nervous system.

The specific activities of five glycohydrolases of lysosomal origin (beta-N-acetylglucosaminidase, beta-glucuronidase, beta-galactosidase, alpha-mannosidase and alpha-fucosidase) were measured in different types of primary and metastatic tumors of the human nervous system. The activities of these hydrolytic enzymes in samples of tumor tissue were compared with those in the white matter of 'control' tissue. The specific activities of beta-glucuronidase and beta-N-acetylglucosaminidase were significantly higher (P less than 0.001) in each group of tumors than in normal cerebral matter. The activities of the other hydrolases were sometimes significantly increased in primary tumors, but not always. In metastatic tumors, they were also significantly higher (P less than 0.01).

Acetylglucosaminidase↗
PubMed1992

Establishment of human squamous carcinoma cell lines highly and minimally sensitive to bleomycin and analysis of factors involved in the sensitivity.

Human squamous carcinoma cell lines that were highly and minimally sensitive to bleomycin were established from clinical specimens and designated as SCCKN and SCCTF, respectively. Although these cell lines showed a similar growth doubling time in vitro, SCCTF was approximately ten times less sensitive to bleomycin than SCCKN. The bleomycin high and low sensitivities were stable even at the 70-cell passage level in vitro. In addition, nude mouse tumors produced by SCCTF were less sensitive to bleomycin that those produced by SCCKN, and the ratio of the mean tumor weight in bleomycin-treated mice to that in control mice was 89.2% in SCCTF and 18.8% in SCCKN. As compared with SCCKN, SCCTF also was less sensitive to peplomycin (5-fold), mitomycin C (2.3-fold), cis-diamine dichloroplatinum (2.5-fold), and vincristine (6.5-fold). Analyses of low bleomycin sensitivity showed that SCCTF had an approximately 20% decreased cellular accumulation and retention of bleomycin, 1.2-fold increase of bleomycin hydrolase activity, elevated DNA repair activity, and increased poly(adenosine diphosphate-ribose) polymerase activity as compared with SCCKN.

Animals↗
PubMed1999

Unsaturated glucuronyl hydrolase of Bacillus sp. GL1: novel enzyme prerequisite for metabolism of unsaturated oligosaccharides produced by polysaccharide lyases.

The bacterium Bacillus sp. GL1 assimilates two kinds of heteropolysaccharides, gellan and xanthan, by using extracellular gellan and xanthan lyases, respectively, and produces unsaturated saccharides as the first degradation products. A novel unsaturated glucuronyl hydrolase (glycuronidase), which was induced in the bacterial cells grown on either gellan or xanthan, was found to act on the tetrasaccharide of unsaturated glucuronyl-glucosyl-rhamnosyl-glucose produced from gellan by gellan lyase, and the enzyme and its gene were isolated from gellan-grown cells. The nucleotide sequence showed that the gene contained an ORF consisting of 1131 base pairs coding a polypeptide with a molecular weight of 42,859. The purified enzyme was a monomer with a molecular mass of 42 kDa and was most active at pH 6.0 and 45 degrees C. Because the enzyme can act not only on the gellan-degrading product by gellan lyase, but also on unsaturated chondroitin and hyaluronate disaccharides produced by chondroitin and hyaluronate lyases, respectively, it is considered that the unsaturated glucuronyl hydrolase plays specific and ubiquitous roles in the degradation of oligosaccharides with unsaturated uronic acid at the nonreducing terminal produced by polysaccharide lyases.

Amino Acid Sequence↗
PubMed1984

Reversed-phase paired-ion high-performance liquid chromatographic method for the separation and quantification of multiple bleomycin congeners.

A rapid, linear gradient chromatographic technique for separating and quantifying copper(II)-chelated bleomycin congeners is described. This method is also capable of separating divalent from trivalent metal chelates; determining the purity of many chemically modified bleomycins; and assaying bleomycin hydrolase activity on complex mixtures. Quantification at 280 nm is sensitive to 50 pmol and is linear at least up to 10 nmol per injection.

Animals↗