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PubMed2001

xln23 from Streptomyces chattanoogensis UAH23 encodes a putative enzyme with separate xylanase and arabinofuranosidase catalytic domains.

The xylanase gene xysA of Streptomyces halstedii JM8 was used to isolate a DNA fragment from a gene library of Pstl-digested chromosomal DNA of the lignocellulolytic actinomycete Streptomyces chattanoogensis CECT-3336. Nucleotide sequence analysis revealed a gene (xln23) encoding a bifunctional multimodular enzyme bearing two independent xylanase and alpha-L-arabinofuranosidase domains separated by a Ser/Gly-rich linker. The N terminus of the predicted protein showed high homology to family F xylanases. The C terminus was homologous to amino acid sequences found in enzymes included in the glycosyl hydrolase family 62 and, in particular, to those of alpha-L-arabinofuranosidase AbsB from Streptomyces lividans. PCR and RT-PCR experiments showed that the nucleotide sequences corresponding to each domain are arranged as expected on the chromosomal DNA and that they are cotranscribed. To our knowledge, this is the first description of xylanase and arabinofuranosidase domains in a same open reading frame.

Amino Acid Sequence↗

PubMed2001

Molecular cloning of an alpha-glucosidase-like gene from Penicillium minioluteum and structure prediction of its gene product.

The dexC cDNA, which is expressed in dextran-containing medium by the filamentous fungus Penicillium minioluteum, was cloned and sequence characterized. The cDNA sequence comprises 1859 bp plus a poly (A) tail, coding for a predicted protein of 597 amino acids. The genomic counterpart was isolated by PCR, finding three introns in its sequence. The dexC gene was located by Southern blot in the same 9-kb fragment that the previously isolated dextranase-encoding gene (dexA). Sequence analysis revealed that the deduced DexC protein belongs to glycosyl hydrolase family 13, showing a high sequence identity (58%) with Aspergillus parasiticus alpha-1,6-glucosidase. In addition, the high sequence identity (51%) between DexC protein and oligo-1,6-glucosidase of Bacillus cereus, with three-dimensional (3D) structure determined, leads us to proposed a 3D model for the structural core of DexC protein.

Amino Acid Sequence↗

PubMed1979

Isolation of lysosomal alpha-mannosidase mutants of Chinese hamster ovary cells.

A method is presented for detection of lysosomal hydrolase activity in situ in colonies of Chinese hamster ovary cells. This method was used to screen for mutants deficient in lysosomal alpha-mannosidase. Mutagenized cells were replicated onto filter papers [Esko, J. D. & Raetz, C. R. H. (1978) Proc. Natl. Acad. Sci. USA 75, 1190-1193] and allowed to divide for 11-14 days; cells on the filter replicas were lysed, and the filters were incubated with 4-methylumbelliferyl-alpha-D-mannopyranoside at pH 4.4 and then photographed under UV light. Colonies for which the replicas exhibited little or no fluorescence were picked from the master plates and purified; mutants with decreased alpha-mannosidase activity were obtained at a frequency of 1 per 1500 mutagenized cells. Analysis of one of these mutants showed that lysosomal alpha-mannosidase activity was 18% of that from wild-type cells, whereas the activities of alpha-mannosidases not of lysosomal origin were similar in the wild type and mutant (these included both Golgi-associated and cytosolic enzymes as well as a novel acid alpha-mannosidase seen only in cells from confluent culture). The mutant contained normal levels of other lysosomal hydrolases. Both wild-type and mutant cells secreted alpha-mannosidase into the medium at levels proportional to those found inside the cells. Incubation of mutant cells with secretions from the wild type resulted in partial restoration of enzyme activity. Added enzyme was localized in the lysosomal fractions; uptake of added enzyme was inhibited by mannose 6-phosphate and fructose 1-phosphate, which are known to inhibit uptake of lysosomal enzymes into human diploid fibroblasts.

Animals↗

PubMed1998

Glycosyl hydrolases from hyperthermophilic microorganisms.

Glycosyl hydrolases from hyperthermophiles are, thus far, the most widely studied enzyme class from these organisms. Not only are there many biotechnological opportunities for these enzymes, but the rapidly increasing amount of information about their genetic, biochemical and biophysical characteristics (recently genomic sequencing data for both P. furiosus and P. horikoshi have been published on the Internet) make them ideal candidates for the study of biocatalysis and protein thermostability at extremely high temperatures.

Archaea↗

PubMedDate not supplied

In vitro action of gastric mucosal lysosomal enzymes on intracellular gastric glycoproteins.

The results show that incubation of gastric mucosal cells from rat at pH approximately 4.5 or in the presence of aspirin is associated with a specific increase in the activity of some acid-hydrolases. Intracellular glycoproteins, isolated by non-degrative techniques from rat or dog fundic mucosal cells, were found to be potential bio-substrates for these acid-hydrolyses. This may suggest that cleavage of the carbohydrate moieties of the intracellular and mucosal cell wall glycoproteins is a fundamental step in the development of gastric ulceration. A model for gastric lesions is proposed and discussed in the light of the results obtained.

Animals↗

PubMed1996

Polysaccharide hydrolase production by the rumen fungus Caecomyces communis.

The anaerobic fungus Caecomyces communis was grown in a fermentor in either a discontinuous cultivation system or in a culture system with daily withdrawal and addition of fresh medium. Lowe and Orpin media were tested. The Lowe medium was best for the stimulation of enzyme production, the Orpin medium, for the stimulation of fungal growth and enzyme release. Xylanase activity was predominant among the polysaccharide hydrolases. Most of the enzymes studied were associated with cells except when the culture medium contained glucose or Ray grass hay. Enzymatic activities were constitutive, but their level was regulated by a carbon source. Cellulase production in both the cellular and extracellular fractions and the extracellular xylanase activity were stimulated by the presence of glucose. Cell-associated xylanase activity, however, was stimulated by glucose plus cellobiose. The presence of glucose enhanced enzyme release.

Animals↗

PubMed2001

Metabolism of sucrose and its five linkage-isomeric alpha-D-glucosyl-D-fructoses by Klebsiella pneumoniae. Participation and properties of sucrose-6-phosphate hydrolase and phospho-alpha-glucosidase.

Klebsiella pneumoniae is presently unique among bacterial species in its ability to metabolize not only sucrose but also its five linkage-isomeric alpha-d-glucosyl-d-fructoses: trehalulose, turanose, maltulose, leucrose, and palatinose. Growth on the isomeric compounds induced a protein of molecular mass approximately 50 kDa that was not present in sucrose-grown cells and which we have identified as an NAD(+) and metal ion-dependent 6-phospho-alpha-glucosidase (AglB). The aglB gene has been cloned and sequenced, and AglB (M(r) = 49,256) has been purified from a high expression system using the chromogenic p-nitrophenyl alpha-glucopyranoside 6-phosphate as substrate. Phospho-alpha-glucosidase catalyzed the hydrolysis of a wide variety of 6-phospho-alpha-glucosides including maltose-6'-phosphate, maltitol-6-phosphate, isomaltose-6'-phosphate, and all five 6'-phosphorylated isomers of sucrose (K(m) approximately 1-5 mm) yet did not hydrolyze sucrose-6-phosphate. By contrast, purified sucrose-6-phosphate hydrolase (M(r) approximately 53,000) hydrolyzed only sucrose-6-phosphate (K(m) approximately 80 microm). Differences in molecular shape and lipophilicity potential between sucrose and its isomers may be important determinants for substrate discrimination by the two phosphoglucosyl hydrolases. Phospho-alpha-glucosidase and sucrose-6-phosphate hydrolase exhibit no significant homology, and by sequence-based alignment, the two enzymes are assigned to Families 4 and 32, respectively, of the glycosyl hydrolase superfamily. The phospho-alpha-glucosidase gene (aglB) lies adjacent to a second gene (aglA), which encodes an EII(CB) component of the phosphoenolpyruvate-dependent sugar:phosphotransferase system. We suggest that the products of the two genes facilitate the phosphorylative translocation and subsequent hydrolysis of the five alpha-d-glucosyl-d-fructoses by K. pneumoniae.

Amino Acid Sequence↗

PubMed1972

Lysosomes of the arterial wall. I. Isolation and subcellular fractionation of cells from normal rabbit aorta.

Smooth muscle cells were dissociated from normal rabbit aorta by incubating the tissue in Hanks' solution containing elastase, collagenase, and hyaluronidase. The isolated cells contained significant amounts of the following acid hydrolases: N-acetyl-beta-glucosaminidase, N-acetyl-beta-galactosaminidase, beta-galactosidase, beta-glucuronidase, alpha-mannosidase, beta-glucosidase, acid phosphatase, and cathepsins C and D. The cells were disrupted and fractionated by isopycnic centrifugation on sucrose density gradients in the Beaufay automatic zonal rotor. Lysosomes with a modal density of 1.16 were identified by the distribution of these acid hydrolases and by the latency of N-acetyl-beta-glucosaminidase and beta-galactosidase. Other particulate enzymes studied in these sucrose gradients included cytochrome oxidase and monoamine oxidase (mitochondria), 5'-nucleotidase and leucyl-beta-naphthylamidase (plasma membrane), and catalase (? peroxisome). This microanalytical subcellular fractionation technique is applicable to the study of milligram quantities of many other tissues, both normal and pathological.

Acetyltransferases↗

PubMed1982

PEP4 gene function is required for expression of several vacuolar hydrolases in Saccharomyces cerevisiae.

The pep4-3 mutation results in a 90-95% reduction in the levels of five vacuolar hydrolases in yeast, including proteinases A and B, carboxypeptidase Y, RNase(s) and the repressible alkaline phosphatase. The mutation is without effect on two secreted glycoproteins, on an enzyme of the vacuolar membrane, and on a proteinase located outside of the vacuole. Mutations at the PEP4 locus exhibit a dosage effect on the levels of some, but not all, of the enzymes whose expression requires the function of the gene.

Alkaline Phosphatase↗

PubMed1979

Coordinate secretion of acid hydrolases in rat bile.

Three lysosomal glycosidases, beta-glucuronidase (EC 3.2.1.31), beta-galactosidase (EC 3.2.1.23), and N-acetyl-beta-glucosaminidase (EC 3.2.1.30) have been investigated in bile that was freshly collected from rats through a complete bile fistula. Assay conditions have been established on the basis of appropriate kinetic studies. The biliary excretion patterns for these enzymes were found to vary considerably from rat to rat during the 24-h collection period. In a given animal, however, the three hydrolases were excreted in parallel and showed a gradual increase in activity with time, most marked after 10- 12 h of collection. 24-h biliary outputs of the three hydrolases averaged congruent with3% of their respective contents in total liver, and bile diversion had no effect on hepatic glycosidase activity or total protein content. Other enzymes known to be associated primarily with mitochondria, endoplasmic reticulum, and cell sap were also detected in bile, generally in smaller amounts. The biliary excretion of the plasma membrane markers, alkaline phosphodiesterase I and 5'-nucleotidase, however, was comparable to that of the lysosomal hydrolases. Biliary excretion of total protein was relatively constant and corresponded to 3.0% of the total hepatic protein content per day, whereas biliary bile acid secretion decreased during the first 12 h and then remained constant. Exocytic bulk discharge of hepatocyte lysosomes is proposed as the most likely mechanism for the biliary excretion of lysosomal enzymes. These results call attention to the possible pathophysiologic significance of biliary excretion of hepatic lysosomal contents as a means of residue disposal.

Animals↗

PubMed1982

Purification and properties of extracellular xylan hydrolases of Streptomyces exfoliatus.

Fractionation of extracellular xylan hydrolases of a strain of Streptomyces exfoliatus MC1 (by salting out, molecular sieving and ion exchange chromatography) revealed the presence of five species of the enzyme. Three major fractions could be purified to homogeneity; two were apparently endohydrolases and the third an exo-xylan hydrolase. The three fractions showed different degrees of affinity to the substrate and differed considerably in their substrate specificities. One of the endo-enzymes was specific to xylan while the other could also attack cellulose, inulin and pectin. The exo-enzyme showed xylanolytic and cellulolytic functions only. The three fractions further differed in their response to the presence of metal ions, mercapto reagents and compounds. Although the pH and temperature optima were different, the three fractions functioned synergistically in the hydrolysis of xylan.

Glycoside Hydrolases↗

PubMed1999

Purification and properties of a basic endo-beta-1,6-glucanase (BGN16.1) from the antagonistic fungus Trichoderma harzianum.

The antagonistic fungus Trichoderma harzianum CECT 2413 produces at least two extracellular beta-1,6-glucanases, among other hydrolases acting on polysaccharides from fungal cell walls, when grown in chitin as the sole carbon source. We have previously reported on the purification and biochemical characterization of the major activity, which corresponds to an acidic enzyme named BGN16.2 [de la Cruz, J., Pintor-Toro, J.A., Benítez, T. & Llobell, A. (1995) J. Bacteriol. 177, 1864-1871]. In this paper, we report on the purification to electrophoretical homogeneity of BGN16.1, the second beta-1, 6-glucanase enzyme. BGN16.1 was purified by ammonium sulfate precipitation followed by adsorption and digestion of pustulan (a beta-1,6-glucan), chromatofocusing and gel-filtration chromatography. BGN16.1 is a non-glycosylated protein with an apparent molecular mass of 51 kDa and a basic isoelectric point (pI 7.4-7.7). The enzyme was active toward substrates containing beta-1,6-glycosidic linkages, including yeast cell walls. The Km was 0.8 mg x mL-1 with pustulan as the substrate. Reaction product analysis by HPLC clearly indicated that BGN16.1 has an endo-hydrolytic mode of action. The probable role of this enzyme in the antagonistic action of T. harzianum is also discussed.

Antibiosis↗

PubMed1983

Dietary-induced rapid decrease of microvillar carbohydrase activity in rat jejunoileum.

Activities of several carbohydrases and peptidases were determined in proximal, middle, and distal thirds of the jejunoileum of female 16-wk-old rats that were fed a high-starch (70 cal%), low-fat (7 cal%) diet for 2 wk and also in rats that (after this introductory period) were fed an isocaloric low-starch (5 cal%), high-fat (73 cal%) diet for 1, 2, and 3 days. The body weight changes, food intake, amount of protein per intestinal segment, and rate of enterocyte migration were practically the same in all groups during these experimental periods. The decreased intake of starch was followed by a rapid decrease (40-80%) of carbohydrases (lactase, sucrase, maltase, and glucoamylase) within the first 24 h in total intestinal homogenates--and as studied in cryostat serial sections--in all regions of the jejunal villus-crypt columns, and mainly in proximal and middle segments. In contrast, the activities of leucylnaphthylamidase and L-phenylalanylglycine hydrolase exhibited little change except for a slight temporary decrease of activity on the 1st day in the proximal segment only (25-30%). Thus these data show that a decrease of starch content in an isocaloric diet evokes a rapid decrease in the activity of microvillar carbohydrases and that activity of these enzymes both in mature and immature enterocytes is capable of reacting to a change (decrease) of dietary carbohydrate content.

Animals↗

PubMed1979

Partial purification and characterization of a bacteriolytic enzyme secreted by Tetrahymena.

Tetrahymena pyriformis strain HSM secretes large quantities of acid hydrolases into the culture medium. An enzyme secreted by the ciliate and capable of degrading walls of streptococci was identified and purified to a considerable degree. The pH optimum of this enzyme was 3--4, and it was eluted after cytochrome c from Sephadex G-75 columns. Unlike lysozyme, the enzyme was thermolabile at pH 2.9, but relatively thermostable at pH 8.1. It degraded 14C-labeled cell walls of streptococci releasing reducing groups. Cell walls prepared from different strains of streptococci differed in susceptibility to this enzyme, the most sensitive strain tested being of group A, type T12. It was shown in immunologic studies that this hydrolase released the group-specific carbohydrate from the walls. Secretions of Tetrahymena from early stationary-phase cultures had more bacteriolytic activity than those from cells from late stationary-phase cultures. Further, cells from cultures grown in glucose-supplemented medium secreted less of the enzyme than ciliates of comparable age grown in unsupplemented proteose-peptone. The newly isolated bacteriolytic enzyme, presumably of lysosomal origin, may be helpful in characterizing streptococcal cell walls.

Animals↗

PubMed2004

Content of saccharides and activity of alpha-glycosidases in Galleria mellonella larvae infected with entomopathogenic nematodes Heterorhabditis zealandica.

Concentration of glycogen, maltose, and trehalose as well as the activity of hydrolases catabolising them: alpha-amylases, glucoamylase, maltase, and trehalase were studied during a 48 h infection of Galleria mellonella (Lepidoptera) instar larvae infected with infective Heterorhabditis zealandica (20 nematodes/insect). The content of trehalose in the infected insects during the first day of infection was higher (p < 0.05) than in the controls. The concentration of maltose and glycogen were similar in both groups despite the fact that the activity of all examined alpha-glycosidases was generally much higher in the infected insects than in the controls.

Animals↗

PubMed1982

A shuttle mechanism for DNA-protein interactions. The regulation of poly(ADP-ribose) polymerase.

Previously it had been shown that poly(ADP-ribose) polymerase requires DNA for its activity and that this enzyme is auto-poly(ADP-ribosyl)ated. The studies reported here indicate that this self-modification inhibits the enzyme and decreases its affinity for DNA, as shown by sucrose gradient density centrifugation. The coupling of poly(ADP-ribose) polymerase with poly(ADP-ribose) glycohydrolase reactivates the polymerase by degrading poly(ADP-ribose) and restoring the polymerase-DNA complex. The assay of polymerase in the presence of glyco-hydrolase was made possible by use of a double-label assay involving release of 14C-labelled nicotinamide and the incorporation of 3H-labelled ADP-ribose from NAD+. These results provide the basis for a shuttle mechanism in which the polymerase can be moved on and off DNA by the action of these two enzymes. Mg2+ and histone H1 appear to activate the polymerase by increasing the affinity of the polymerase for DNA.

Animals↗

PubMed1989

Bleomycin hydrolase is a unique thiol aminopeptidase.

Bleomycin hydrolase, which hydrolyzes the carboxamide bond in the pyrimidoblamic acid moiety of the bleomycin molecule, also cleaved several p-nitroanilide substrates with a neutral or basic amino acid residue and dipeptide substrates such as L-leucyl-glycine. The activity of bleomycin hydrolase was inhibited by two thiol protease inhibitors, E-64 and leupeptin, as well as by N-ethylmaleimide. These results suggest that bleomycin hydrolase is a thiol aminopeptidase. Magnesium ion, sodium chloride, ethylenediaminetetraacetic acid and 1,2-dihydroxybenzene-3,5-disulfonic acid specifically activated the enzymatic hydrolysis of L-arginine-p-nitroanilide, but did not that of L-leucine-p-nitroanilide. Lineweaver-Burk plots showed that Km values of the enzymatic activity for L-arginine-p-nitroanilide were altered by these reagents, although Vmax values were almost unaltered.

1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium ↗

PubMed1990

Hydrolases from bovine seminal vesicle, prostate and Cowper's gland.

The bovine seminal plasma is formed mainly by secretions of epididymis and the glandular epithelia in ampulla, seminal vesicles, prostate and Cowper's glands. The contribution of each organ to the hydrolytic enzyme activities (glycosidases, exopeptidases, phospholipases) of the bull seminal plasma has been analyzed and is reviewed in this paper with special emphasis on the role of the accessory glands. Seminal vesicles seem to have a major role in the secretion of seminal plasma acid alpha-glucosidase, acid alpha-mannosidase and beta-N-acetylhexosaminidase, aminopeptidase A, dipeptidyl peptidase II and IV and gamma-glutamyl transpeptidase as well as Ca(2+)-dependent and Ca(2+)-independent phospholipases A2 with distinct substrate specificities, a choline-specific phospholipase C and a Co2+ (Mn2+)-activated sphingomyelinase. The enzyme pattern in the ampulla closely resembled that of the seminal vesicles and obviously contributes to the seminal plasma level of these hydrolases. The bull prostate and Cowper's glands contained a strong Ca(2+)-dependent phospholipase A2 activity. However, these glands may not contribute to the seminal plasma PLA2 activity. At ejaculation the epididymal spermatozoa are exposed to these enzymes. They may have a specific affinity to sugar, peptide or phospholipid residues at distinct sites of the sperm surface. These enzymes may also participate in the digestion of various other semen components to create a suitable milieu for the emitted spermatozoa.

Animals↗