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:3.1.3.5 (5'-nucleotidase)
3,167 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The subcellular distribution of adenyl cyclase was investigated in small intestinal epithelial cells. Enterocytes were isolated, disrupted and the resulting membranes fractionated by differential and sucrose gradient centrifugation. Separation of luminal (brush border) and contra-luminal (basolateral) plasma membrane was achieved on a discontinuous sucrose gradient. The activity of adenyl cyclase was followed during fractionation in relation to other enzymes, notably those considered as markers for luminal and contraluminal plasma membrane. The luminal membrane was identified by the membrane-bound enzymes sucrase and alkaline phosphatase and the basolateral region by (Na+ + K+)-ATPase. Enrichment of the former two enzymes in purified luminal plasma membrane was 8-fold over cells and that of (Na+ + K+)-ATPase in purified bisolateral plasma membranes was 13-fold. F--activated adenyl cyclase co-purified with (Na+ + K+)-ATPase, suggesting a common localization on the plasma membrane. The distribution of K+-stimulated phosphatase and 5'-nucleotidase also followed (Na+ + K+)-ATPase during fractionation.
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PMID:The surface membrane of the small intestinal epithelial cell. I. Localization of adenyl cyclase. 17 91

A subcellular fractionation method to isolate simultaneously apical and basolateral plasma membrane fractions from the human adenocarcinoma cell line Caco-2, grown on filter supports, is described. The method employs sucrose-density-gradient centrifugation and differential precipitation. The apical membrane fraction was enriched 14-fold in sucrase-isomaltase and 21-fold in 5'-nucleotidase compared with the homogenate. The basolateral membrane fraction was enriched 20-fold relative to the homogenate in K(+)-stimulated p-nitrophenylphosphatase. Alkaline phosphatase was enriched 15-fold in the apical membrane fraction and 3-fold in the basolateral membrane fraction. Analytical density-gradient centrifugation showed that this enzyme was a true constituent of both fractions, and experiments measuring alkaline phosphatase release following treatment with phosphatidylinositol-specific phospholipase C showed that in both membrane fractions the enzyme was glycosyl-phosphatidylinositol-linked. There was very little contamination of either membrane fraction by marker enzymes of the Golgi complex, mitochondria or lysosomes. Both membrane fractions were greater than 10-fold purified with respect to the endoplasmic reticulum marker enzyme alpha-glucosidase. Protein composition analysis of purified plasma membrane fractions together with domain-specific cell surface biotinylation experiments revealed the presence of both common and unique integral membrane proteins in each plasma membrane domain. The post-synthetic transport of endogenous integral plasma membrane proteins was examined using the devised subcellular fractionation procedure in conjunction with pulse-chase labelling experiments and immunoprecipitation. Five common integral membrane proteins immunoprecipitated by an antiserum raised against a detergent extract of the apical plasma membrane fraction were delivered with the same time course to each cell-surface domain.
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PMID:The post-synthetic sorting of endogenous membrane proteins examined by the simultaneous purification of apical and basolateral plasma membrane fractions from Caco-2 cells. 131 18

Biopsy specimens from 29 adenomas, 17 adenocarcinomas, and 6 synchronous adenomas in cancer patients and from uninvolved mucosa of all main segments of the large bowel were examined histologically and assayed for a series of organelle marker enzymes. Six enzymes--lactase, sucrase, alkaline phosphatase, 5'-nucleotidase, acid phosphatase, and N-acetyl-beta-D-glucosaminidase--showed less activity in adenomas than in adjacent uninvolved mucosa and in specimens from controls. Cancer tissue had higher gamma-glutamyltransferase and lower lactase, alkaline and acid phosphatases, and N-acetyl-beta-D-glucosaminidase activities than specimens from uninvolved mucosa in cancer patients and control patients. Enhanced alkaline phosphatase and N-acetyl-beta-D-glucosaminidase activities were seen in uninvolved mucosa of cancer patients as compared with those of adenoma and control patients. Evidence has been found for multienzyme analysis to identify adenomas with signs of malignant transformation and carcinomas with poor prognosis.
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PMID:Enzyme activities in biopsy specimens from large-bowel mucosa in colorectal adenomas and carcinomas. 362 77

The distribution of a series of mucosal enzymes along the large bowel was studied by analysis of homogenized biopsy specimens from five different segments, obtained from 20 control patients. The activities varied significantly between the segments for the membrane enzymes lactase (p less than 0.005), alkaline phosphatase (p less than 0.0005), leucyl-beta-naphthylamidase (p less than 0.0001), and 5'-nucleotidase (p less than 0.001) and the mitochondrial enzyme monoamine oxidase (p less than 0.0005) when tested by analysis of variance modified for repeated measurements. When paired comparisons between segments were evaluated, the enzyme activities of the proximal large bowel were significantly higher than those of distal segments. The levels of sucrase, neutral-alpha-glucosidase, gamma-glutamyltransferase, and lysosomal enzymes remained unchanged throughout the large intestine, as did the protein to DNA ratio. The results are compatible with the theory that different segments of the large bowel have different functions.
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PMID:Longitudinal distribution of mucosal enzymes in the human large bowel. 377 57

A technique is described for the isolation of a plasma-membrane fraction from the rat intestinal epithelial cell which is distinct from the microvillus membrane of that cell. The isolated fraction contains only about 0.2% of the sucrase activity in the original homogenate and negligible quantities of nuclear and mitochondrial membrane markers. It contains 12% of the total Na(+),K(+)-dependent adenosine triphosphatase and 7% of the alkaline phosphatase, with significant increments in specific activity of these enzymes. Multiple membrane preparations were highly reproducible with respect to the specific activities of the markers studied. The small intestine of one rat yields material containing about 1.3mg of protein. In addition an assay is described suitable for determining 5'-nucleotidase in the small intestine.
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PMID:Preparation and characterization of the lateral and basal plasma membranes of the rat intestinal epithelial cell. 456 93

Highly sensitive techniques have been used for the assay of a range of marker enzymes including lactase, sucrase, alkaline phosphatase, leucyl-beta-naphthylamidase (brush border), and 5'-nucleotidase (basolateral membrane) in jejunal biopsy homogenates from patients with adult coeliac disease with and without steatorrhoea and from a control group. The absorption of D-xylose and vitamin B12 was compared in the two groups with coeliac disease. All enzymes assayed were equally depressed in both groups of coeliac disease as compared with the controls. The absorption of D-xylose and vitamin B12 were reduced in the patients with steatorrhoea compared with those without steatorrhoea. The findings suggest that lack of steatorrhoea in some patients with coeliac disease is due to better preservation of the ileal function rather than to a less severe jejunal mucosal injury.
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PMID:Enzyme activities in jejunal biopsy samples from patients with adult coeliac disease with and without steatorrhoea. 632 31

To define reproducible conditions for the homogenization of small-intestinal biopsy samples, tissue homogenization has been studied by the use of three different homogenizers. Tissue samples of increasing wet weights (0.5-10.8 mg) were homogenized in a fixed volume (1 ml) before DNA and protein were determined. The DNA to protein ratio was calculated for all wet weights and used as a measure for reproducible homogenization. The minimum tissue wet weight needed for analysis (2 mg) was determined from the values obtained for the DNA to protein ratio. Highly sensitive techniques are described in detail for the assay of brush border (maltase, lactase, sucrase, neutral alpha-glucosidase, alkaline phosphatase, gamma-glutamyl transferase, leucyl-beta-naphthylamidase), basolateral membrane (5'-nucleotidase), and mitochondrial (succinate dehydrogenase) marker enzymes and for four acid hydrolases (acid phosphatase, acid beta-D-galactosidase, N-acetyl-beta-D-glucosaminidase, acid diesterase) in human and rat jejunal mucosa. Linear kinetics have been established for all enzyme assays. The optimal dilution of tissue homogenate for the assay of the various enzymes has been determined to enable the determination of a maximum number of enzymes in each homogenate. The range of enzyme activities in samples of human and rat jejunal mucosa has been determined.
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PMID:Enzyme activities in human and rat jejunal mucosa. 667 54

A series of marker enzymes for brush borders, basolateral membrane, and lysosomes were assayed in mucosal biopsy specimens from patients with untreated and treated coeliac disease and from controls. The brush border enzymes lactase, sucrase, neutral alpha-glucosidase, alkaline phosphatase, and leucyl-beta-naphthylamidase showed reduced activities in the untreated state and complete or partial normalization during treatment. The lysosomal marker enzyme acid phosphatase increased in activity in untreated coeliac disease and was normalized by treatment. The brush border enzyme gamma-glutamyl transferase was nearly normal in untreated patients and slightly increased in treated patients. The basolateral membrane marker, 5'-nucleotidase, was reduced both in untreated and treated patients, whereas the lysosomal marker N-acetyl-beta-glucosaminidase was normal in the untreated state and decreased during treatment. The possible pathogenetic role of the three latter enzymes in coeliac disease is discussed. The patterns of the other enzymes are suggested to be attributable to the morphologic changes in the mucosa.
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PMID:Jejunal mucosal enzymes in untreated and treated coeliac disease. 667 55

Microvillus membrane vesicles from pig small intestine, isolated by hypotonic lysis, Mg2+ aggregation of contaminants and differential centrifugation, have been further purified by immunoadsorbent chromatography. The vesicles adhere to an immunoadsorbent prepared by coupling antibodies raised against three of the principal proteins of the brush border membrane (aminopeptidase, sucrase-isomaltase and lactase) to Sepharose 4B. After the contaminants are removed by washing, the adherent vesicles are released from the immunoabsorbent by applying shear forces. The purity of the immunoadsorbed vesicles has been established by electron microscopy and by measuring the activity of marker enzymes. The enrichment factor is 1.17 +/- 0.02 for aminopeptidase and 0.70 +/- 0.05 for 5'-nucleotidase. The contamination of the preparation before immunoadsorption constitutes 10% of the membrane protein and consists mainly of basolateral membrane fragments as judged from marker enzyme determinations and the lipid composition.
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PMID:Purification of microvillus membrane vesicles from pig small intestine by immunoadsorbent chromatography. 710 46

Intestinal alkaline phosphatase (IAP) is a brush-border membrane ectoenzyme (BBM-IAP) that is released into the lumen (L-IAP) after a high-fat diet. We examined the effects of oil feeding and the addition of mixed-lipid micelles on the formation of L-IAP in oil-fed rat intestine, Caco-2 cell monolayers, and mouse intestinal loops. We localized IAP in the duodenum of rats fed corn oil using fluorescence microscopy with enzyme-labeled fluorescence-97 as substrate. Four hours after oil feeding, L-IAP increased approximately 10-fold accompanied by the loss of BBM-IAP, consistent with BBM-IAP release. Rat IAP isozyme mRNAs progressively increased 4-6 h after oil feeding, followed by the increase of IAP activity in the subapical location at 6 h, consistent with the restoration of IAP protein. Postprandial lipid-micelle components, sodium taurocholate with or without oleic acid, mono-oleylglycerol, cholesterol, or lysophosphatidylcholine (lysoPC) were applied singly or as mixed-lipid micelles to the apical surface of polarized Caco-2 cell monolayers. LysoPC increased L-IAP >10-fold over basal release. LysoPC released IAP into the apical medium more than other intestinal brush-border enzymes, 5'-nucleotidase, sucrase, aminopeptidase N, and lactase, without comparable lactate dehydrogenase release or cell injury. LysoPC increased human IAP mRNA levels by 1.5-fold in Caco-2 cells. Luminally applied lysoPC also increased release of IAP preferentially in mouse intestinal loops. These data show that lysoPC accelerates the formation of L-IAP from BBM-IAP, followed by enhanced IAP synthesis, suggesting the role that lysoPC might play in the turnover of brush-border proteins.
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PMID:Role of lysophosphatidylcholine in brush-border intestinal alkaline phosphatase release and restoration. 1940 15


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