Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.2.1.108 (lactase)
 2,133 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The adult guinea-pig small intestinal microvillus membrane was purified approximately 25-fold by both cation-precipitation and differential centrifugation methods. Comparison by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed no substantial differences in polypeptide composition between the two preparations. One-dimensional SDS-PAGE and two-dimensional isoelectric focussing (IEF)/SDS-PAGE, together with Coomassie-blue, silver and lectin-staining, showed three major high molecular weight polypeptides, Mr 108 000, 116 000 and 127 000, as well as a 47 kDa protein (actin), as major constituents of the membrane. The proteins of Mr 108 000 and 116 000 were strongly concanavalin A reactive. A detailed two-dimensional IEF/SDS-PAGE map of the membrane was constructed. Sodium carbonate treatment showed the two concanavalin A-reactive glycoproteins, Mr 108 000 and 116 000, comprising the sucrase-isomaltase complex, to be loosely-associated 'extrinsic' microvillus membrane proteins. Two proteins, Mr 127 000 and 135 000, were tightly-associated 'intrinsic' microvillus proteins. Despite regional differences in specific activity of some small intestinal microvillar enzymes, most noticeably enterokinase (EC 3.4.21.9) and dipeptidyl peptidase IV (EC 3.4.14.x), no substantial regional differences were seen in microvillus membrane polypeptide composition. In contrast, a substantial increase in the major high molecular weight proteins of Mr 108 000 and 116 000 accompanied a 10-fold rise in sucrase-isomaltase activity, and loss of a major protein of Mr 131 000 accompanied the complete loss of lactase activity from the membrane during postnatal development.
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PMID:Two-dimensional isoelectric focussing/sodium dodecyl sulphate polyacrylamide gel electrophoretic mapping and some molecular characteristics of the proteins of the adult guinea-pig small intestinal microvillus membrane. 399 21

These studies examined the potential for reorganization and differentiation of dissociated 18-day fetal rat intestine. Cultures of trypsin-dissociated fetal intestine were maintained in vitro for 1 week on a three-dimensional matrix, then transplanted into syngeneic hosts. When harvested after 4 weeks, these transplants consistently demonstrated organotypic differentiation. Spherical structures containing crypts with frequent mitotic figures and villi lined with columnar epithelium had formed. PAS staining demonstrated positive epithelial cell brush borders, goblet cells, and luminal contents. Significant levels of the microvillus membrane enzymes lactase, sucrase, maltase, and alkaline phosphatase were present in the luminal contents. Sucrase-isomaltase, an enzyme characteristic of postweaning small intestine, was demonstrated by immunoprecipitation and SDS-PAGE. Thus, both morphological and biochemical maturation occurred in the transplants.
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PMID:Organotypic differentiation of trypsin-dissociated fetal rat intestine. 661 90

We tested the effect of dietary fat on the lipid composition and hydrolase activity of jejunal brush border membranes in piglets. Eighteen 5-wk-old piglets were divided into three groups and for 4 wk fed either an unsaturated low fat diet (3.2% corn oil), an unsaturated high fat diet (17.2% corn oil) or a saturated high fat diet (2.2% corn oil + 15% tallow). Brush border membranes were prepared from the jejunal mucosa and analyzed for cholesterol, phospholipid and fatty acids. The activities of sucrase-isomaltase, lactase-phlorizin hydrolase, maltase-glucoamylase, aminopeptidase and alkaline phosphatase were measured. Lactase-phlorizin hydrolase isoforms were immunopurified and separated by SDS-PAGE, and their relative proportions were measured by densitometry. The activities of the disaccharidases and alkaline phosphatase, but not aminopeptidase, were greater in animals fed the saturated high fat diet than in animals fed the unsaturated high fat diet. The fatty acid composition of the membranes generally reflected the composition of the diet. Correlation analysis demonstrated that the phospholipid, fatty acid and cholesterol compositions of the membranes were associated with the differences in brush border hydrolase activity.
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PMID:Jejunal brush border hydrolase activity is higher in tallow-fed pigs than in corn oil-fed pigs. 793 9

We estimated in vivo turnover rates of sucrase-isomaltase and lactase-phlorizin hydrolase in adult rats. Fed animals received a primed continuous infusion of phenylalanine (300 microCi, 150 mumol Phe/100 g of body weight for 30 s, then 7.5 microCi, 3.75 mumol Phe/min for 10 to 140 min). Sucrase-isomaltase and lactase-phlorizin hydrolase were immunoprecipitated from jejunal mucosal membranes; isoforms were separated by SDS-polyacrylamide gel electrophoresis. Endoglycosidase H digestions and (for lactase-phlorizin hydrolase) N-terminal amino acid sequencing were performed on all isoforms. Specific radioactivity of prosucrase-isomaltase and prolactase-phlorizin hydrolase isoforms reached isotopic equilibrium by 60 and 90 min, respectively. Specific radioactivity of brush border sucrase and lactase did not reach steady state. The isotope kinetic, N-terminal amino acid sequencing, and endoglycosidase H digestion data suggested that one of the high molecular weight lactase isoforms is a dimer of mature lactase. Compartmental modeling of specific radioactivity demonstrated that mean intracellular residence time is 59 min for prosucrase-isomaltase isoforms and 68 min for prolactase-phlorizin hydrolase isoforms. Mean residence time in the brush border was 5.8 h for sucrase and 7.8 h for lactase. Fractional synthesis rates were 414%/day for sucrase and 307%/day for lactase. Thus, in vivo brush border sucrase and lactase turn over at similar rates in the adult rat.
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PMID:In vivo sucrase-isomaltase and lactase-phlorizin hydrolase turnover in the fed adult rat. 851 93

We have estimated the synthesis rates in vivo of precursor and brush-border (BB) polypeptides of lactase phlorhizin hydrolase (LPH) in newborn pigs fed with water or colostrum for 24h post partum. At the end of the feeding period, piglets were anaesthetized and infused intravenously for 3h with L-[4-3H]- phenylalanine. Blood and jejunal samples were collected at timed intervals. The precursor and BB forms of LPH were isolated from jejunal mucosa by immunoprecipitation followed by SDS/PAGE, and their specific radioactivity in Phe determined. The kinetics of precursor and BB LPH labelling were analysed by using a linear compartmental model. Immunoisolated LPH protein consisted of five polypeptides [high-mannose LPH precursor (proLPHh), complex glycosylated LPH precursor (proLPHe), intermediate complex glycosylated LPH precursor (proLPH1i) and two forms of BB LPH]. The fractional synthesis rate (Ks) of proLPHh and proLPHc (approx. 5%/min) were the same in the two groups but the absolute synthesis rate (in arbitrary units, min-1) of proLPHh in the colostrum-fed animals was twice that of the water-fed animals. The Ks values of proLPHi polypeptides were significantly different (water-fed, 3.89%/min; colostrum-fed, 1.6%/min), but the absolute synthesis rates did not differ. The Ks of BB LPH was not different between experimental treatment groups (on average 0.037%/min). However, the proportion of newly synthesized proLPHh processed to BB LPH was 48% lower in colostrum-fed than in water-fed animals. We conclude that in neonatal pigs, the ingestion of colostrum stimulates the synthesis of proLPHh but, at least temporarily, disrupts the processing of proLPH polypeptides to the BB enzyme.
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PMID:Lactase phlorhizin hydrolase turnover in vivo in water-fed and colostrum-fed newborn pigs. 900 57

Intestinal glycohydrolases are enzymes involved in assimilating carbohydrate for nutrition. The avian forms of these enzymes, in particular the maltase-glucoamylase complex (MG), are not well characterised. This study encompassed characterisation of these enzymes from ostrich intestines, and the first kinetic analysis of an avian MG. Proteolytically solubilised MG from ileal brush border membrane vesicles was purified by Sephadex G-200 gel filtration and Tris-affinity-chromatography, while jejunal sucrase-isomaltase (SI) and MG were purified by Toyopearl-Q650 and phenyl-Sepharose chromatography. Amino acid sequences and compositions of enzyme subunits, resulting from SDS-PAGE, were determined. Kinetics of hydrolysis of linear oligosaccharides was studied. Ostrich MG and SI showed the highest activity in the jejunum, followed by the ileum and duodenum. No lactase or trehalase activity could be detected. The jejunal MG and SI, resulting from brush-border membrane vesicles, could not be separated during purification. However, a minor form of ileal MG was purified using Sephadex G-200 chromatography. Ileal MG contained three subunits of M(r) 145,000, 125,000 and 115,000. Although the N-terminal amino acid sequences bear no homology to SI, the M(r) 115,000 subunit shows homology to porcine MG in both sequence and amino acid composition. The pH optimum of maltose-, starch- and isomaltose-hydrolysing activity was 6.5 and that of sucrose-hydrolysing activity 5.5. The glycohydrolases were most active at 58 degrees C, but were quickly denatured above 60 degrees C. Sucrose- and starch-hydrolysing activities were more thermostable than maltose- and isomaltose-hydrolysing activities. Kinetic parameters (K(m), kcat and kcat/K(m)) for the hydrolysis of maltooligosaccharides, starch and glycogen are reported for ileal MG. Maltotriose and maltotetraose displayed partial inhibition of ileal MG. The study revealed large similarities between ostrich SI and MG in charge, size, shape and hydrophobicity, based on their inseparability by several methods. Measurement of the specificity constants for maltooligosaccharide hydrolysis by ileal MG revealed less efficient hydrolysis of longer substrates as compared to maltose and maltotriose.
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PMID:Ostrich intestinal glycohydrolases: distribution, purification and partial characterisation. 961 76

We have previously identified and purified a novel beta-glucosidase, designated PNGH (pyridoxine-5'-beta-D-glucoside hydrolase), from the cytosolic fraction of pig intestinal mucosal. PNGH catalyses the hydrolysis of PNG (pyridoxine-5'-beta-D-glucoside), a plant derivative of vitamin B6 that exhibits partial nutritional bioavailability in humans and animals. Preliminary amino acid sequence analysis indicated regions of close similarity of PNGH to the precursor form of LPH (lactase-phlorizin hydrolase), the beta-glucosidase localized to the brush-border membrane. We report in the present study amino acid sequence data for PNGH and results of Northern blot analyses, upon which we propose a common genomic origin of PNGH and LPH. Internal Edman sequencing of the PNGH band isolated by SDS/PAGE yielded data for 16 peptides, averaging 10.8 amino acids in length. These peptides from PNGH (approx. 140 kDa) were highly similar to sequences existing over most of the length of the >200 kDa precursor of rabbit LPH; however, we found no PNGH sequences that corresponded to approx. 350 amino acids between positions 463 and 812 of the LPH precursor, a region encoded by exon 7 of the LPH precursor gene (amino acids 568-784), and no sequences that corresponded to regions near the N-terminus. MS analysis of tryptic peptides yielded 25 peptides, averaging 15 amino acids, with masses that matched segments of the rabbit LPH precursor. Northern blot analysis of pig and human small intestinal polyadenylated mRNA using a non-specific LPH cDNA probe showed an expected approx. 6 kb transcript of the LPH precursor, but also an approx. 4 kb transcript that was consistent with the size predicted from the PNGH protein mass. Using a probe specific to the region encoded by exon 7, hybridization occurred only with the 6 kb transcript. Based on these observations, we propose that both PNGH and LPH enzymes have the same genomic origin, but differ in transcriptional and, possibly, post-translational processing.
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PMID:Partial amino acid sequence and mRNA analysis of cytosolic pyridoxine-beta-D-glucoside hydrolase from porcine intestinal mucosa: proposed derivation from the lactase-phlorizin hydrolase gene. 1497 28

The present study analyzed the existence of carbohydrases in camel pancreas compared to some other ruminants. Disaccharidases (maltase, cellobiase, lactase, trehalase and sucrase), glucoamylase and alpha-amylase were detected in pancreas of camel, sheep, cow and buffalo. Enzyme levels in sheep were lower than in the other ruminants. The highest level was detected for alpha-amylase (EC 3.2.1.2). Moderate activity levels were detected for glucoamylase (EC 3.2.1.3) and maltase (EC 3.2.1.20), while other disaccharidases showed very low activity. The results suggested that, in addition to alpha-amylase, glucoamylase and maltase may be synthesized and secreted from pancreas to the small intestine in ruminants. Camel pancreatic glucoamylase was purified and characterized. The purification procedure included glycogen precipitation and chromatography on DEAE-Sepharose and Sepharose 6B. The molecular mass was 58 kDa for native and denatured enzyme using gel filtration and SDS-PAGE, respectively. The enzyme had a pH optimum at 5.5 and a Km of 10 mg starch/mL with more affinity toward potato soluble starch than the other carbohydrates. Glucoamylase had a temperature optimum at 50 degrees C with heat stability up to 30 degrees C. The effect of different cations and inhibitors was examined. The camel pancreatic glucoamylase may possess an essential thiol.
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PMID:Carbohydrases in camel (Camelus dromedarius) pancreas. Purification and characterization of glucoamylase. 1562 12

The lactase gene lacb' from Aspergillus candidus was fused behind alpha-factor signal sequence in the Pichia pastoris expression vector pPIC9, then integrated into the genome of P. pastoris by recombination events. The P. pastoris recombinants for lactase overexpression were screened by enzyme activity analysis and SDS-PAGE. The lactase expressed in P. pastoris was glycosylated protein with an apparent molecular weight of 130 kD, while the deglycosylated lactase treated with Endo H had an apparent molecular weight of about 110 kD. The expression level of secreted lactase protein in recombinant P. pastoris was 6 mg/mL with enzymatic activity of 3600 U/mL in the 5 L fermenter, which was the highest among that of all kinds of recombinant strains reported now. The optimal pH and optimal temperature of the lactase are 5.2 and 60 degrees C. The Vmax, Km, and specific activity of the lactase are 3.3 micromol/min, 1.7 mmol/L and 706.5 +/- 2.6 U/mg, respectively. Compare to the lactase from Aspergillus oryzae ATCC 20423, the expressed lactase from A. candidus have better enzymatic properties including the high thermostability, high specific activity and wide pH range for enzyme reaction.
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PMID:[Overexpression of Aspergillus candidus lactase and analysis of enzymatic properties]. 1598 70

Disaccharidases (maltase, cellobiase, lactase, and sucrase), alpha-amylase, and glucoamylase in the camel small intestine were investigated to integrate the enzymatic digestion profile in camel. High activities were detected for maltase and glucoamylase, followed by moderate levels of sucrase and alpha-amylase. Very low activity levels were detected for lactase and cellobiase. Camel intestinal maltase-glucoamylase (MG) was purified by DEAE-Sepharose and Sephacryl S-200 columns. The molecular weight of camel small intestinal MG4 and MG6 were estimated to be 140,000 and 180,000 using Sephacryl S-200. These values were confirmed by SDS-PAGE, where the two enzymes migrated as single subunits. This study encompassed characterization of MGs from camel intestine. The Km values of MG4 and MG6 were estimated to be 13.3 mM and 20 mM maltose, respectively. Substrate specificity for MG4 and MG6 indicated that the two enzymes are maltase-glucoamylases because they catalysed the hydrolysis of maltose and starch with alpha-1,4 and alpha-1,6 glycosidic bonds, but not sucrose with alpha-1,2 glycosidic bond which was hydrolyzed by sucrase-isomaltase. Camel intestinal MG4 and MG6 had the same optimum pH at 7.0 and temperature optimum at 50 degrees C and 40 degrees C, respectively. The two enzymes were stable up to 50 degrees C and 40 degrees C, followed by strong decrease in activity at 60 degrees C and 50 degrees C, respectively. The effect of divalent cations on the activity of camel intestinal MG4 and MG6 was studied. All the examined divalent cations Ca(2+), Mn(2+), Mg(2+), Co(2+) and Fe(3+) had slight effects on the two enzymes except Hg(2+) which had a strong inhibitory effect. The effect of different inhibitors on MG4 and MG6 indicated that the two enzymes had a cysteine residue.
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PMID:Disaccharidase activities in camel small intestine: biochemical investigations of maltase-glucoamylase activity. 1709 55


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