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)

Arthrobacter sp. K-1, isolated from soil, produces beta-fructofuranosidase. This enzyme preparation was separated into three fractions (I, II, and III) by butyl-Toyopearl 650 M column chromatography. The beta-fructofuranosidase I was purified to homogeneity by disc-electrophoresis after consecutive column chromatographies. The enzyme had a molecular weight of 52,000 by SDS-polyacrylamide gel electrophoresis and 51,000 by gel filtration with Ultrogel AcA 44, and an isoelectric point of 4.3. The enzyme was most active at pH 6.5-6.8 and at 55 degrees C and stable up to 45 degrees C at pH 6.5 for 30 min of incubation, and from pH 5.5 to 10.0 at 40 degrees C in 2 hr of incubation. This enzyme hydrolyzed sucrose, erlose, raffinose, fructosylxyloside, neokestose, and stachyose at the relative velocities of 100, 75, 61, 59, 54, and 11, but hardly hydrolyzed 1-kestose and nystose (less than 0.02).
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PMID:Purification and some properties of beta-fructofuranosidase I from Arthrobacter sp. K-1. 136 50

The extracellular domain of human tissue factor (TF, amino acids 1-217) was expressed in Saccharomyces cerevisiae, using the inducible yeast acid phosphatase promoter and the yeast invertase signal sequence to direct its secretion into the culture broth. Two active soluble forms sTF alpha (high molecular weight form) and sTF beta (low molecular weight form) were purified, the yield being approximately 10 and 1 mg/liter of culture supernatant, respectively. sTF alpha had an apparent molecular mass of 150 kDa on SDS-polyacrylamide gel electrophoresis and contained more than 200 residues of mannose/mol of protein. sTF beta had an apparent molecular mass of 37 kDa and contained 22 residues of mannose/mol of protein. N-Glycosidase F treatments of both rTFs reduced the apparent molecular mass to 35 kDa. The amino-terminal sequences and amino acid compositions of sTF alpha and sTF beta were consistent with those deduced from the cDNA sequence, thereby indicating that the difference in molecular mass is caused by heterogeneity of oligosaccharide structures. Of these recombinant TFs, sTF beta enhanced factor VIIa-amidolytic activity 40-fold toward the chromogenic substrate and 147-fold toward the fluorogenic substrate, affecting mainly the kcat value. The enhancement was comparable with that of TF purified from human placenta. The TF-mediated enhancement of factor VIIa-amidolytic activity was inhibited by heparin-activated antithrombin III, forming a high molecular weight complex. As treatment of sTF beta with denaturants such as guanidine hydrochloride or urea led to a biphasic loss of the activity, the extracellular domain of TF probably consists of two discrete domains. This expression system provides a significant amount of the extracellular domain of TF so that studies of interactions with factor VII are feasible.
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PMID:Expression of human soluble tissue factor in yeast and enzymatic properties of its complex with factor VIIa. 140 Apr 45

Soluble beta-fructofuranosidase with an intracellular location and an isoelectric point of 3.8 (isoenzyme I) was purified and characterized from dry seeds and seedlings of carrot (Daucus carota). The enzyme hydrolyzed sucrose with a Km of 5 mM and a broad pH optimum around 5.0. The purified protein, which was N-glycosylated with high-mannose-containing and high-xylose-containing complex glycans, eluted as a monomeric polypeptide with a molecular mass of 68,000 from a gel-filtration column. On SDS/PAGE, the protein separated in the presence of SDS and 2-mercaptoethanol into three polypeptides with molecular masses of 68, 43 and 25 kDa. The amount of the 68-kDa polypeptide was highest in dry seeds and decreased with increasing age of carrot seedlings. Amino acid sequence analysis and immunological studies showed that the 43-kDa and 25-kDa polypeptides were N-terminal and C-terminal proteolytic fragments of the 68-kDa polypeptide. A comparison of partial amino acid sequences of the soluble beta-fructofuranosidase with the complete sequence of carrot cell-wall beta-fructofuranosidase showed that their N-terminal sequences were different, whereas some of the internal tryptic peptide sequences were up to 70% identical.
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PMID:Purification and characterization of a soluble beta-fructofuranosidase from Daucus carota. 154 2

The common hookworm (Ancylostoma ceylanicum) infection of humans was studied in golden hamsters model system. Significant biochemical modulations were observed in hamster jejunal brush border membrane (BBM), the primary site of infection. Analysis of BBM at the peak of infection (3-weeks) revealed a marked decrease in the activities of sucrase, lactase and maltase, while activities of alkaline phosphatase, (Ca2+ + Mg2+)-ATPase and gamma-glutamyl transpeptidase were increased. Kinetic studies conducted with maltase, a superficially localised enzyme of jejunal BBM, revealed loss of enzyme active site during the infection. Among other constituents, the levels of cholesterol and triglycerides were significantly decreased with slight increase in phospholipid content in the infected animals. The hookworm infection also caused a decline in total hexose content indicating an altered membrane glycocalyx. Conversely, there was significant enhancement of hydroxyproline and sialic acid contents. SDS-PAGE analysis showed an enhancement in both low and high molecular weight proteins in jejunal BBM preparations of the infected group. Gel electrophoresis of glycoproteins further revealed the appearance of two additional peaks in the low molecular weight region and concomitant disappearance of a peak in the high molecular weight region. These results strongly support the view that the hookworm infection causes severe damage not to the site of attachment alone but also to the entire cell lining of the jejunum and therefore could influence overall digestion and absorption.
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PMID:Biochemical analysis of jejunal brush border membrane of golden hamster: pathogenic modulations due to ancylostomiasis. 159 19

To investigate the biosynthetic basis for the mosaic expression of brush border enzymes in confluent Caco-2 cells, a human colon carcinoma cell line exhibiting characteristics of adult small intestinal enterocytes, we have obtained a series of clones differing markedly in their growth rates, amounts of transforming growth factor-alpha/epidermal growth factor-like activity released into the culture medium, and sucrase-isomaltase (SI) activity. Other intestinal markers (aminopeptidase N, dipeptidylpeptidase IV, lactase, alkaline phosphatase and 'crypt cell antigen') displayed a much more limited variability in expression, suggesting that the Caco-2 cell clones we have obtained did not differ in their overall ability to differentiate. Immunofluorescence staining, metabolic labelling with radioactive methionine and hybridization analysis of SI mRNA abundance were used to investigate SI synthesis and its regulation in clones endowed with low, intermediate or high sucrase activity. The results obtained have demonstrated heterogeneous SI expression, even in clonal cell lines, and a negative correlation between SI expression and growth factor concentrations in the culture medium, suggesting an autocrine regulation of cell proliferation and differentiation in confluent Caco-2 cells. Pulse-chase experiments using the two clones endowed with the lowest and highest levels of SI activity, followed by immunoprecipitation of labelled SI with epitope-specific antibodies and SDS/PAGE analysis, suggested that both transcriptional and post-translational mechanisms play a role in the regulation of SI expression in intestinal cells.
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PMID:Clonal analysis of sucrase-isomaltase expression in the human colon adenocarcinoma Caco-2 cells. 176 23

The structure and catalytic function of rat intestinal sucrase-alpha-dextrinase (sucrase-isomaltase) were characterized in intact brush border membranes by differential denaturation in 1% SDS at 4, 37, 45, 55, and 100 degrees C, analysis by acrylamide electrophoresis, and subsequent renaturation by transfer to nitrocellulose and in situ analyses of immunoactivity and catalytic activity (immunoblotting and catalytic blotting). Both the sucrase and alpha-dextrinase activities were associated with two mature oligomers, with sucrase predominantly in a 250-260-kDa unit and dextrinase in a 330-350-kDa unit. While sucrase activity declined progressively in response to increasing temperature to 45 degrees C due to loss of active sites, alpha-dextrinase activity increased reciprocally (Vmax +176%). Three principal monomeric products of postinsertional processing comprise the oligomers: alpha, 140 kDa, which carries the sucrase active site; beta, 125 kDa, harboring the dextrinase active site; and gamma, 110 kDa, produced by removal of 185 amino acid residues from the N-terminus of the alpha. Rather than being a simple hybrid dimer, membrane-associated sucrase-alpha-dextrinase appears to consist of two major oligomeric forms having complex structural associations that dramatically affect the availability of the active catalytic sites at the brush border membrane surface.
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PMID:Structural and functional correlates of sucrase-alpha-dextrinase in intact brush border membranes. 193 64

A detergent solubilised sucrase from monkey small intestine has been purified 388-fold to gel electrophoretic homogeneity with an overall recovery of 36%. The molecular weight of the enzyme was 263 kDa by gel filtration. Electrophoresis in the presence of SDS indicates that the enzyme is a hetero-dimer. Mixed substrate inhibition studies and inhibition by PCMB and Tris suggest the presence of two catalytically active sites in the form of maltase and sucrase with isomaltase activity being common to both sites. Polyclonal antiserum against the purified enzyme showed a single continuous precipitin line with the purified antigen.
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PMID:Some properties of monkey intestinal sucrase. 211 33

Invertase (EC 3.2.1.26) was purified to homogeneity from exponentially growing cells of Schizosaccharomyces pombe fully de-repressed for synthesis of the enzyme, and was shown to be a high-molecular-mass glycoprotein that can be dissociated in the presence of 8 M-urea/1% SDS into identical subunits with an apparent molecular mass of 205 kDa. The carbohydrate moiety, accounting for 67% of the total mass, is composed of equimolar amounts of mannose and galactose. There is a small amount of glucosamine, which is probably involved in the linkage to the protein moiety, since the enzyme is sensitive to treatment with endoglycosidase H. The composition of the carbohydrate moiety resembles that found in higher-eukaryotic glycoproteins and differs from glycoproteins found in Saccharomyces cerevisiae. The protein portion of each subunit is a polypeptide of molecular mass 60 kDa, very similar to the invertase of Sacch. cerevisiae. Both proteins cross-react with antibodies raised against the protein fractions of the other, indicating that the two enzymes are similar.
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PMID:Purification and characterization of the invertase from Schizosaccharomyces pombe. A comparative analysis with the invertase from Saccharomyces cerevisiae. 218 35

The invertase (beta-fructofuranosidase, EC 3.2.1.26) of the rumen holotrich ciliate Isotricha prostoma has been purified. This is the first report of an enzyme purification from a known species of rumen protozoon. Cells were disrupted by ultrasonic treatment and the enzyme was purified from the cell-free extract by three successive liquid column chromatographies (Sepharose CL4B/octyl-Sepharose CL4B, DE52 DEAE-cellulose and concanavalin A-Sepharose 4B). This resulted in a 160-fold purification and a 15% yield. The major form of the purified enzyme was a tetramer with Mr about 350,000 that was readily dissociated by electrophoresis. The invertase was heterogeneous, as five types of monomers were shown by SDS/polyacrylamide-gel electrophoresis after denaturation. Part of this heterogeneity was due to different glycosylated forms of one of the polypeptides present in the purified enzyme. Isotricha prostoma invertase exhibited maximum activity at pH 5.5-6.0 and 50 degrees C. The kinetic properties of the purified enzyme were very similar to those of invertases from other sources such as yeast or plants (substrate and product inhibition, transferase activity).
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PMID:Purification and characterization of a heterogeneous glycosylated invertase from the rumen holotrich ciliate Isotricha prostoma. 251 52

A genetic library consisting of over 5000 clones with an average insert size of 6.9 kilobasepairs (kbp) of Streptococcus mutans GS-5 has been constructed in a bivalent plasmid vector pMK3, which is capable of replicating in Escherichia coli and Bacillus subtilis. The recombinant plasmid pSUCRI, containing a 6.0 kbp fragment of S. mutans GS-5 DNA, was the focus of this study. Using Southern hybridization, in vitro and in vivo gene expression techniques, and biochemical analysis, this clone was shown to encode the 55 kiloDalton (kDal) GS-5 gtfA gene product, as well as a 38 and a 66 kDal polypeptide. In addition to the gtfA gene, pSUCRI encodes a dextranase activity with specificity for alpha(1----6)-linked glucans, and with no detectable activity on mutan. The dextranase enzyme had an apparent molecular weight of 66 kDal as demonstrated by SDS-PAGE analysis of the proteins produced by a dextranase-negative deletion derivative. The pH optimum of the enzyme was approximately 6.0, and there was no detectable activity below pH 5.0. By subcloning various combinations of DNA fragments from pSUCRI, it was demonstrated that the dextranase gene (designated dexB) can be separated from the gtfA gene and still be efficiently expressed in both E. coli and B. subtilis. The dexB gene contained its own promoter and ribosome-binding site. The genetic linkage of the gtfA and dexB genes in the S. mutans GS-5 chromosome was confirmed by Southern hybridization and by the independent isolation of four distinct clones containing the gtfA gene and common flanking sequences. In addition to a glucosyltransferase and dextranase, an invertase-like activity is also encoded on pSUCRI, indicating that there is a cluster of genes on the S. mutans GS-5 chromosome which is devoted to the dissimilation of sucrose and concomitant synthesis or modification of glucans into a water-insoluble form, perhaps constituting an operon for glucan modification which can be coordinately regulated in response to environmental alterations.
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PMID:Tight genetic linkage of a glucosyltransferase and dextranase of Streptococcus mutans GS-5. 294 34


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