Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.26 (invertase)
4,927 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Leucine beta-naphthylamidase associated with the microvilli membranes of rabbit small intestine was solubilized with papain [EC 3.4.22.2] and purified by Sephadex G-200 gel filtration, DEAE-cellulose column chromatography, passage through a column of Sepharose 4B coupled with anti-sucrase antibodies and preparative disc electrophoresis in polyacrylamide gel. The purified enzyme was homogeneous on ultracentrifugation and disc electrophoresis, but a double immunodiffusion test showed the presence of a minor component which was probably denatured enzyme. The molecular weight of the purified enzyme was estimated to be 225,000 by Sephadex G-200 gel filtration and the sedimentation coefficient (S-0-20, w) was found to be 6.90S. Purified enzyme required bovine serum albumin for maximal activity, perhaps for its protection from autodigestion. It hydrolyzed, in addition to L-leucine beta-naphthylamide, various L-amino acid beta-naphthylamides and dipeptides with a free alpha-amino group, but did not hydrolyze benzoyl-L-arginine beta-naphthylamide. Therefore, the purified enzyme is an aminopeptidase. Hg-2+ and Cu-2+ ions strongly inhibited the enzyme activity, but other metal ions and EDTA showed no or only slight effect. N-Ethylmaleimide exhibited a weak inhibition. Purified enzyme had an optimal pH and Km value for leucine beta-naphthylamide similar to those of enzymes from other sources. Antibodies against the purified enzyme were raised in guinea pigs. The antibodies obtained were found by double immunodiffusion to be specific for the enzyme. They precipitated the enzyme quantitatively and partially inhibited the enzyme activity.
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PMID:Purification and properties of leucine beta-naphthylamidase from rabbit small-intestinal mucosal cells. 23 93

Cleavage of yeast invertase by alpha-chymotrypsin produced a number of small glycopeptides that were highly active as elicitors of ethylene biosynthesis and phenylalanine ammonia-lyase in suspension-cultured tomato cells. Five of these elicitors were purified and their amino acid sequence determined. They all had sequences corresponding to known sequences of yeast invertase, and all contained an asparagine known to carry a N-linked small high mannose glycan. The most active glycopeptide elicitor induced ethylene biosynthesis and phenylalanine ammonia-lyase half-maximally at a concentration of 5-10 nM. Structure-activity relationships of the peptide part were analyzed by further cleavage of a defined glycopeptide elicitor with various proteolytic enzymes. Removal of the C-terminal phenylalanine enhanced the elicitor activity, whereas removal of N-terminal arginine impaired it. A glycopeptide with the peptide part trimmed to the dipeptide arginine-asparagine was still fully active as elicitor. Glycopeptides with identical amino acid sequences were further separated into fractions differing in the oligosaccharide side chain. A given peptide had high elicitor activity when carrying a glycan with 10-12 mannosyl residues (Man10-12GlcNAc2), a 3-fold lower activity when carrying Man9GlcNAc2 and a 100-fold lower activity when carrying Man8GlcNAc2. The oligosaccharides, released by endo-beta-N-acetylglucosaminidase H from the pure glycopeptide elicitors, acted as suppressors of elicitor-induced ethylene biosynthesis and phenylalanine ammonia-lyase activity. A series of such oligosaccharides in the size range of Man8-13GlcNAc was purified. The structure and composition of the purified oligosaccharides corresponded to the known small high mannose glycans of yeast invertase as verified by 1H NMR spectroscopy at 600 MHz. The highest suppressor activities were obtained with the oligosaccharides containing 10-12 mannosyl residues (Man10-12GlcNAc). The oligosaccharide Man8 GlcNAc was ineffective as a suppressor. Thus, the structural requirements for the free oligosaccharides to act as efficient suppressors were the same as for the oligosaccharide side chains of the glycopeptides for high elicitor activity. We propose that the glycan suppressors bind to the same recognition site as the glycopeptide elicitors without inducing a response.
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PMID:Elicitors and suppressors of the defense response in tomato cells. Purification and characterization of glycopeptide elicitors and glycan suppressors generated by enzymatic cleavage of yeast invertase. 158 15

We have investigated different leader sequences for their ability to direct the efficient secretion of human epidermal growth factor (hEGF) from Saccharomyces cerevisiae. We designed a consensus signal sequence which directs secretion of hEGF from yeast as efficiently as the yeast invertase signal sequence. However, secretion is increased over fivefold by the introduction, after the signal sequence, of a synthetic 19-amino acid (aa) pro-sequence containing a cleavage recognition site for the KEX2 protease. Even in the absence of an Asn-linked glycosylation site, secretion of hEGF using the synthetic prepro-leader was as efficient as that directed by the alpha-factor leader. The role of the KEX2 protease cleavage site was investigated by mutation of the yeast alpha-factor KEX2 site (cleavage after Lys-Arg). Cleavage was obtained with the following order of efficiency, Lys-Arg greater than Pro-Arg greater than Asp-Arg, although the sequence context was also found to affect efficiency.
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PMID:Secretion of human epidermal growth factor from Saccharomyces cerevisiae using synthetic leader sequences. 193 55

The SNF1 gene of Saccharomyces cerevisiae encodes a protein-serine/threonine kinase that is required for derepression of gene expression in response to glucose limitation. We present evidence that the protein kinase activity is essential for SNF1 function: substitution of Arg for Lys in the putative ATP-binding site results in a mutant phenotype. A polyhistidine tract near the N terminus was found to be dispensable. Deletion of the large region C terminal to the kinase domain only partially impaired SNF1 function, causing expression of invertase to be somewhat reduced but still glucose repressible. The function of the SNF4 gene, another component of the regulatory system, was required for maximal in vitro activity of the SNF1 protein kinase. Increased SNF1 gene dosage partially alleviated the requirement for SNF4. C-terminal deletions of SNF1 also reduced dependence on SNF4. Our findings suggest that SNF4 acts as a positive effector of the kinase but does not serve a regulatory function in signaling glucose availability.
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PMID:Mutational analysis of the Saccharomyces cerevisiae SNF1 protein kinase and evidence for functional interaction with the SNF4 protein. 255 46

Various gene fusions between the arginine permease and invertase have been constructed in order to obtain information about whether parts of the CAN1 gene product can induce secretion of biologically active invertase missing its own signal sequence. A construction containing 30 N-terminal amino acid residues of the CAN1 gene product fused to invertase was not secreted. When the CAN1 portion was elongated to 477 or 560 amino acid residues, secretion of the fusion proteins was observed. A fusion lacking 59 amino acids at the amino-terminal end of the arginine permease was also secreted. These results indicate that the amino-terminal end of the arginine permease is neither sufficient nor essential for membrane insertion; instead this enzyme should contain an internal targeting sequence facilitating secretion. Some general implications on the biosynthesis and topology of membrane proteins are also discussed as well as the homology with histidine permease.
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PMID:CAN1-SUC2 gene fusion studies in Saccharomyces cerevisiae. 332 76

In adult sparse-fur mutant mice, ornithine transcarbamylase (OTC) activity represents only 14% of the normal values. We studied the development of this activity from birth to adult period and demonstrated that the enzyme deficiency is already fully expressed at birth, in both the liver and the small intestine of mutants. Since OTC catalyzes the conversion of ornithine to citrulline, in the presence of carbamoyl-phosphate, the effect of a disturbed ornithine metabolism on the postnatal development of the small intestine has been evaluated. The normal appearance of sucrase as well as the normal increase of glucoamylase, trehalase, and alkaline phosphatase activities are delayed in sparse-fur mice compared with controls. Moreover, normal adult values are never attained. In contrast, the normal decline of lactase activity is impaired while leucylnaphthylamidase activity is unaffected. Cell proliferation, as evaluated by [3H]thymidine incorporation into DNA and mitotic index, is less active during the 3rd wk of life in mutants. These phenomena are closely associated with a transient weak arginase and ornithine decarboxylase activity in the small intestine. Since arginase catalyzes the conversion of arginine to orthithine, thus ensuring the availability of this substrate for ornithine decarboxylase activity, these results indicate a disturbance of polyamine metabolism in mutant enterocytes with a consequent delay in postnatal differentiation and proliferation. Sparse-fur mutant mouse may therefore represent a useful animal model for evaluating the role of ornithine metabolism in the maturation process of the small intestine.
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PMID:Postnatal maturation of enterocytes in sparse-fur mutant mice. 395 97

1. Growth of a biotin-requiring strain of Saccharomyces cerevisiae in a medium containing a suboptimum concentration of biotin for growth caused a decreased synthesis of ornithine carbamoyltransferase as compared with yeast grown in a medium containing an optimum concentration of biotin. Inclusion of the biotin homologues norbiotin or homobiotin, but not bishomobiotin, in the biotin-deficient medium caused an appreciable increase in ornithine carbamoyltransferase synthesis without affecting growth or synthesis of total RNA and protein. The addition of norbiotin to biotin-deficient medium had no effect on the respiratory activity of the yeast or on the synthesis of aspartate carbamoyltransferase, acid phosphatase, beta-fructofuranosidase or malate dehydrogenase. 2. Synthesis of acetylornithine deacetylase and acetylornithine acetyltransferase was slightly diminished by the imposition of biotin deficiency, but the effect was not as great as on ornithine carbamoyltransferase synthesis. Incorporation of norbiotin in the biotin-deficient medium had no marked effect on the synthesis of any other arginine-pathway enzyme except ornithine carbamoyltransferase. 3. l-Ornithine induced synthesis of ornithine carbamoyltransferase in yeast grown in biotin-deficient medium, but in yeast grown in this medium supplemented with norbiotin it repressed synthesis of the enzyme. l-Arginine had no detectable effect on ornithine carbamoyltransferase synthesis by the yeast grown in biotin-deficient medium with or without norbiotin. l-Aspartate repressed synthesis of ornithine carbamoyltransferase in biotin-deficient yeast and completely nullified the stimulatory effect of norbiotin on synthesis of the enzyme in this yeast. 4. There was no increase in ornithine carbamoyltransferase synthesis in biotin-deficient yeast incubated in phosphate buffer, pH4.5, containing glucose and biotin or norbiotin. In biotin-deficient yeast suspended in complete medium containing an optimum concentration of biotin, there was an increase in ornithine carbamoyltransferase synthesis only after the onset of growth.
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PMID:A specific requirement for biotin in the synthesis of ornithine carbamoyltransferase by yeast. 596 54

To evaluate the role of dietary polyamines in maturation of the rat small intestine, spermine was given orally twice daily to suckling pups from day 10 to day 14 postpartum at different doses: 0, 0.2, 0.5, 1, 2.5, and 5 mumol/dose. Compared to saline treated controls, spermine (5 mumol) produced significant increases in mucosal mass parameters (+12 to +57%, P < 0.05), induced prematurely an adult pattern of microvillous enzymes, and enhanced, respectively, by 19- and 3.5-fold (P < 0.01 vs controls) the concentration of the secretory component of p-immunoglobulins in villous and crypt cells. The response of microvillous enzymes (lactase, sucrase, maltase, and aminopeptidase) to spermine was dose-dependent and -specific since oral administration of arginine (5 mumol) or ornithine (5 mumol) was without effect. Intestinal changes were found to be significant (P < 0.05) for doses of spermine exceeding 1 mumol/day, which is in the range of the amount of polyamines provided by solid pellets at weaning (0.4 mumol/g). However, intestinal changes were undetectable at the physiological amounts of polyamines consumed by pups from rat milk during the suckling period (less than 0.3 mumol/day). Consistent with a direct effect of spermine on the intestinal cell, the cytosolic activity of ornithine decarboxylase was depressed by 27-fold (P < 0.005 vs controls) in the jejunum, while inhibition of ornithine decarboxylase by alpha-difluoromethylornithine did markedly decrease but did not suppress the cell response to spermine. Alternately, plasma corticosteronemia, which was virtually absent by day 14 in controls, ranged between 1.4 and 4.6 micrograms/dl in 60% (N = 9) of the spermine-treated rats.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Maturation of villus and crypt cell functions in rat small intestine. Role of dietary polyamines. 850 5

The efficacy of ornithine alpha-ketoglutarate (OKG) in preventing bacterial translocation and dissemination, metabolic disorders and changes in mucosal enzyme activities was assessed in a model of bacterial translocation in rats. Antibiotic decontamination was performed 4 d before intragastric inoculation with an Escherichia coli strain (10(10) bacteria/kg body). Two days later, the rats were given either a lipopolysaccharide (LPS) 0127:B8 or a saline injection and were deprived of food for 24 h. Enteral nutrition, [Osmolite, 880 kJ/(kg. d)] supplemented with either OKG (LPS + OKG) or glycine (Saline + Gly or LPS + Gly), was then given for 2 d. Urinary total nitrogen losses and 3-methylhistidine excretion were determined daily. On killing at d 3, bacterial translocation to the mesenteric lymph nodes (MLN) and dissemination to the spleen and liver were evaluated, jejunal mucosa enzyme activities were assayed and tissue free amino acids in muscles were measured. Endotoxin induced translocation from the gut lumen to the MLN in all groups, whereas dissemination occurred only in LPS-treated rats. OKG significantly reduced dissemination of the bacteria in the spleen. 3-Methylhistidine excretion was greater in the LPS + Gly group (+25%, P: < 0.05) than in either the LPS + OKG or Saline + Gly group. The group fed the OKG-enriched diet had higher muscular glutamine, ornithine and arginine concentrations than did the Gly-supplemented groups (P: < 0.05). Intestinal sucrase and aminopeptidase activities were higher in the LPS + OKG group than in the LPS + Gly group (-30%, P: < 0.05). OKG supplementation limits bacterial dissemination and metabolic changes after injury in rats and thus may be useful in the prevention of gut-derived sepsis in critically ill patients.
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PMID:Bacterial dissemination and metabolic changes in rats induced by endotoxemia following intestinal E. coli overgrowth are reduced by ornithine alpha-ketoglutarate administration. 1111 Aug 43

Purification and characterization of an extracellular invertase produced by Aspergillus ochraceus TS are reported. The enzyme was purified (42-fold) from culture filtrate by salt precipitation, ion-exchange and gel filtration. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of the purified enzyme showed a single band of molecular mass 66 kDa. The molecular mass of the native enzyme was found to be 130 kDa by gel filtration. The purity of the protein was also checked against its antiserum raised in rabbits by two-dimensional immunodiffusion in agarose gel and Western blot that showed a single band. It is a glycoprotein with mannose as its carbohydrate residue. The enzyme showed high affinity for sucrose with a Km of 3.5 mM. The amino acid analysis revealed a high proportion of acidic residues but it had a low content of cysteine, histidine and arginine comparable to other fungal invertases.
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PMID:Purification and characterization of an invertase produced by Aspergillus ochraceus TS. 1169 82


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