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Hydrolysis of small peptides, like disaccharide hydrolysis, is an important function of the intestinal brush border, but little is known of the individual human peptidases. The purposes of this study were to detect all human brush border enzymes hydrolyzing dipeptides and tripeptides, identify the most discriminating substrate for each enzyme in order to permit assays in crude mixtures, and begin biochemical characterization of each enzyme. Four brush border peptidases were identified. Enzymes I (aspartate aminopeptidase, E.C. 3.4.11.7) and III (amino-oligopeptidase, E.C. 3.4.11.2) are known brush border enzymes. Enzymes II (membrane Gly-Leu peptidase) and IV (zinc stable Asp-Lys peptidase) have not been identified in human brush border previously. They are distinct from dipeptidyl aminopeptidase IV, carboxypeptidase, and gamma-glutamyl transferase. The substrate most discriminating for each enzyme is alpha-Glu-beta-naphthylamide for I (100% of the brush border activity for this substrate is due to enzyme I), glycylleucine for II (80%), leucyl-beta-naphthylamide for III (91%), and aspartyl-lysine in 5 mM Zn2+ for IV (63%). The enzymes are immunologically distinct and antibodies to each one localize to the brush border on immunohistochemical staining. Purification of 142-, 79-, 158-, and 46-fold was achieved for enzymes I through IV, respectively. Biochemical characteristics include slightly alkaline pH optima, molecular weights of 91,000-190,000, and evidence of metal ion involvement in activity. These studies provide necessary information for determining the role of brush border peptidase deficiencies in human disease. 相似文献
3.
Cellular and subcellular localization of the Nramp2 iron transporter in the intestinal brush border and regulation by dietary iron. 总被引:41,自引:15,他引:26
Genetic studies in animal models of microcytic anemia and biochemical studies of transport have implicated the Nramp2 gene in iron transport. Nramp2 generates two alternatively spliced mRNAs that differ at their 3' untranslated region by the presence or absence of an iron-response element (IRE) and that encode two proteins with distinct carboxy termini. Antisera raised against Nramp2 fusion proteins containing either the carboxy or amino termini of Nramp2 and that can help distinguish between the two Nramp2 protein isoforms (IRE: isoform I; non-IRE: isoform II) were generated. These antibodies were used to identify the cellular and subcellular localization of Nramp2 in normal tissues and to study possible regulation by dietary iron deprivation. Immunoblotting experiments with membrane fractions from intact organs show that Nramp2 is expressed at low levels throughout the small intestine and to a higher extent in kidney. Dietary iron starvation results in a dramatic upregulation of the Nramp2 isoform I in the proximal portion of the duodenum only, whereas expression in the rest of the small intestine and in kidney remains largely unchanged in response to the lack of dietary iron. In proximal duodenum, immunostaining studies of tissue sections show that Nramp2 protein expression is abundant under iron deplete condition and limited to the villi and is absent in the crypts. In the villi, staining is limited to the columnar absorptive epithelium of the mucosa (enterocytes), with no expression in mucus-secreting goblet cells or in the lamina propria. Nramp2 expression is strongest in the apical two thirds of the villi and is very intense at the brush border of the apical pole of the enterocytes, whereas the basolateral membrane of these cells is negative for Nramp2. These results strongly suggest that Nramp2 is indeed responsible for transferrin-independent iron uptake in the duodenum. These findings are discussed in the context of overall mechanisms of iron acquisition by the body. 相似文献
4.
S Van Biervliet E Eggermont H Carchon G Veereman K Deboeck 《Acta gastro-enterologica Belgica》1999,62(3):267-271
The study concerns the maltase, saccharase, lactase and alkaline phosphatase activity in small intestinal biopsy specimens from 61 consecutively admitted, untreated, Caucasian cystic fibrosis patients. A group of 319 age matched controls admitted during the same time period for undefined gastrointestinal or nutritional disorders acted as the controls. In order to eliminate morphological damage as a confounding factor, the enzyme activities were studied in small intestinal biopsy specimens having both normal stereomicroscopic and histological features. It was shown that neither maltase nor saccharase activity was different in the two groups, in contrast to lactase and alkaline phophatase activity, that was significantly lower in cystic fibrosis patients. The differences could not be explained by the nutritional status as judged by the body mass index. Lactase activity is known to be easily affected by numerous enteropathies. As the information on alkaline phosphatase activity is limited, the low activity is discussed in more detail. Taking into account the literature data, the low alkaline phosphatase activity is tentatively attributed either to enhanced release from the brush border or to the faulty handling of alkaline phophatase protein in the post-golgi compartments secondary to the accumulation of incorrectly glycosylated CFTR in the same cell structures. 相似文献
5.
The mechanism of acetate absorption in the small intestine is not yet established. One possible mechanism is by carrier mediated Na(+)-acetate cotransport since acetate, like glucose, stimulates intestinal Na+ and water absorption in vivo. Uptake of radioactive carbon acetate by small intestinal brush border membrane vesicles was not saturable or Na+ dependent and did not respond to osmotic shrinkage of the vesicles. This suggests that acetate binds to the membranes but is not transported into the intravesicular space and argues against carrier mediated Na+ acetate cotransport. These results are consistent with acetate absorption by a non-mediated diffusion and suggest that the stimulation of water and Na+ absorption by acetate in vivo is largely due to osmotic forces and solvent drag. 相似文献
6.
A myosinlike 105-110-kilodalton calmodulin-binding protein, brush border myosin I, found in the intestinal brush border has been linked to two seemingly disparate but possibly interacting functions of the brush border, namely, microvillar motility and vitamin D regulated calcium transport. If brush border myosin I were to function primarily as a myosinlike molecule powering cellular or microvillar motility, one might expect it to be found in a variety of tissues with microvilli such as the renal brush border and bile canaliculus. On the other hand, a more specialized function such as participation in vitamin D regulated calcium transport might dictate a more restricted tissue distribution for brush border myosin I. To determine the tissue distribution of brush border myosin I, we purified this protein to apparent homogeneity, generated antisera to it, and used the antisera to localize the protein within the intestinal epithelial cell by immunocytochemistry. We then screened a variety of other tissues (brain, lung, heart, liver, spleen, pancreas, kidney, and skeletal muscle) both for calmodulin-binding proteins as well as for brush border myosin I using Western blots and immunofluorescence. Our results indicate that the intestinal brush border myosin I is limited in its distribution to the intestinal brush border. 相似文献
7.
V M Rajendran S A Ansari J M Harig M B Adams A H Khan K Ramaswamy 《Gastroenterology》1985,89(6):1298-1304
This study characterizes the transport of [1-14C]glycyl-L-proline into purified brush border membrane vesicles prepared from human small intestine. Time-course uptake curves of glycyl-L-proline were similar under sodium thiocyanate or potassium thiocyanate gradient conditions (extravesicular greater than intravesicular) and did not show any overshoot phenomena. The transport of glycine and proline, however, was stimulated by the presence of sodium gradient. Measurement of peptide uptake with increasing medium osmolarity showed that glycyl-L-proline was transported into an osmotically reactive intravesicular space with insignificant binding to the surface of the vesicles. Only 2% of the glycyl-L-proline in the incubation media was hydrolyzed after 10 min of incubation. Also, there was no hydrolysis of peptide transported into the intravesicular space. The effects of increasing concentrations of glycyl-L-proline on uptake showed that uptake of the peptide was saturable and conformed to Michaelis-Menten kinetics with a Km of 4.1 +/- 0.5 mM and a Vmax of 1.53 +/- 0.07 nmol/mg protein X 0.5 min. Free amino acids did not inhibit the transport of glycyl-L-proline while dipeptides and tripeptides exerted appreciable inhibition (up to 60%). Our results show that human small intestinal brush border membrane vesicles transport glycyl-L-proline as an intact peptide by a carrier-mediated, Na+-independent process. 相似文献
8.
The transport of riboflavin across the brush border membrane of human intestine was examined using the established brush border membrane vesicle technique. Both osmolarity and temperature studies have concluded that the uptake of riboflavin by these vesicles is mostly the result of transport of riboflavin into an active intravesicular space with less binding to membrane surfaces. When an inwardly directed Na+ gradient was imposed, transport of riboflavin was linear with time for approximately 20 seconds of incubation and was significantly higher than in the presence of an identical K+ gradient. Initial rate of transport of riboflavin as a function of concentration was found to include a saturable component in the presence of an inwardly directed Na+ gradient but was linear in the presence of an identical K+ gradient. The apparent Km and Vmax of the Na+ stimulated transport process were found to be 7.26 mumols/L and 0.97 pmol/mg protein per 10 seconds, respectively. The addition of high concentrations of unlabeled riboflavin and its structural analogue lumiflavin to the incubation medium caused significant inhibition in the transport of 3H-riboflavin in the brush border membrane vesicle incubated in the presence of an inwardly directed Na+ gradient but not in vesicles incubated in the presence of an identical K+ gradient. Inducing a relatively positive intravesicular space with the use of valinomycin and an inwardly directed K+ gradient caused significant inhibition in the transport of riboflavin. On the other hand, inducing a relatively negative intravesicular space with the use of anions of different lipid permeabilities caused significant stimulation in the transport of riboflavin. These results demonstrate that riboflavin transport in human intestinal brush border membrane vesicle is through a carrier-mediated system. This system functions in the presence of a Na(+)-gradient and seems to transport the substrate by an electrogenic process. 相似文献
9.
This study presents a method of brush border membrane (BBM) preparation from the human small intestine using polyethylene glycol (PEG) precipitation and also looks at the effect of in vitro oxidant exposure on structural and functional alterations in the membrane. Isolated BBM were relatively pure as judged by 10- to 14-fold enrichment of marker enzymes with less than 1% contamination by other subcellular organelles. These membranes showed uphill transport of glucose and lipid analysis showed a cholesterol–phospholipid (C/P) ratio of 1.19. Isolated BBM were found to be susceptible to superoxide generated by xanthine oxidase (XO), resulting in lipid and protein oxidation along with altered glucose uptake. Superoxide exposure also resulted in phospholipid alterations, especially generation of lyso phospholipids. These changes were prevented by inhibiting XO by allopurinol or scavenging superoxide by superoxide dismutase (SOD). Other oxidants studied did not have significant affect on these membranes. These studies suggest that PEG can be used for preparation of BBM from the human small intestine and these membranes undergo structural and functional alterations on exposure to superoxide. 相似文献
10.
Summary Experimental diabetes mellitus in rats was induced by streptozotocin. Five days after administration of streptozotocin intestinal brush border hydrolases (maltase, sucrase, trehalase, lactase) and alkaline phosphatase were markedly elevated at all levels of the small intestine as measured in the total homogenate and in the isolated brush border preparation. Insulin treatment beginning 15 h after administration of streptozotocin was able to decrease the increased disaccharidase activity due to streptozotocin diabetes. In experimental diabetes mellitus of rats trransport as well as digestive functions of the intestinal mucosa are stimulated.This work has been presented at the meeting of the European Society for Clinical Investigation, Scheveningen, The Netherlands, April 1972 and has appeared in abstract form (1a) 相似文献
11.
Zymogram studies of peptide hydrolases from the human intestinal brush border and cytoplasmic fractions produced multiple bands--that is, up to seven--while the brush border membrane produced only a single band of enzyme activity. With all of the substrates tested except L-leucyl-L-leucyl-L-leucine, a band having anodic mobility identical with that produced by the brush border enzymes was produced by the cytoplasmic enzymes. With L-trileucine as a substrate, no overlapping band was produced. This band in the cytoplasmic fraction was heat sensitive, while that in the brush border fraction was not. Thus it would appear that there is a single human intestinal brush border peptide hydrolase capable of hydrolysing a variety of di- and tri-peptides. This peptide hydrolases of the brush border and the cytoplasmic fraction of human intestine are distinct. 相似文献
12.
Evolutionary matches of enzyme and transporter capacities to dietary substrate loads in the intestinal brush border 总被引:2,自引:0,他引:2
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Stacey L. Weiss Eric A. Lee Jared Diamond 《Proceedings of the National Academy of Sciences of the United States of America》1998,95(5):2117-2121
Safety factors of enzymes and transporters are defined as the ratio of Vmax (maximal reaction rates at high substrate concentrations) to the reaction rate under actual physiological conditions. Although corresponding safety factors have been measured for macroscopic biological structures and for human-engineered structures, safety factors have been little studied at the molecular level. Some evolutionary considerations suggest that safety factors should be modestly in excess of 1.0 (“enough but not too much”) and should tend to be similar for the various steps of a pathway consisting of two or more elements arranged in series. Hence we used a preparation of intact mouse small intestine to measure Vmax values (capacities) of brush-border sucrase (yielding glucose plus fructose) and of the brush-border glucose transporter, for comparison with each other and with dietary sucrose loads. Load was manipulated by varying dietary sucrose level or by studying lactating mice with increased energy requirements. Capacities both of sucrase and the glucose transporter increased with sucrose load (i.e., both proteins are inducible) and remained approximately matched to each other except on a carbohydrate-free diet. Their safety factors decreased from ca. 2.7 at low load to 1.0 at high load. Thus, neither sucrase nor the glucose transporter is the rate-limiting step for sucrose digestion; both steps are equally limiting. The modest safety factors and matched capacities must be genetically programmed through natural selection, with benefits of excess capacities being balanced against costs of biosynthetic energy and limited membrane space. 相似文献
13.
Immunoelectrophoretic studies on human small intestinal brush border proteins: cellular alterations in the levels of brush border enzymes after jejunoileal bypass operation.
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The amounts of lactase (EC 3.2.1.23), sucrase (EC 3.2.1.48), maltase (EC 3.2.1.20), microvillus aminopeptidase (microsomal EC3.4.11.2), and dipeptidyl peptidase IV (EC 3.4.14.X) in biopsies from proximal jejunum and distal ileum were studied by quantitative crossed immunoelectrophoresis and enzymatic assays in obese patients one and six months after jejunoileal bypass operation and compared with peroperative levels. They were related to DNA and protein content. The protein/DNA ratio fell 28-43% postoperatively. Except for ileal lactase and sucrase all enzymes showed decreased levels when expressed per mg protein and an even more pronounced decrease when related to DNA. Lactase and sucrase levels in ileum were increased or unchanged. A constant correlation between the amount of immunoreactive enzyme protein and enzymatic activity was shown for all enzymes except maltase. The results suggest that the bypass operation is followed by an increased amount of enterocytes devoid of or low in enzymatic activity and protein content. The amounts of lactase and sucrase in ileum are increased in relation to the other enzymes. No immunoreactive enzymes with zero or depressed activity were detected. 相似文献
14.
Prof. Dr. M. Stern MD M. Knauss A. Stallmach MD 《Digestive diseases and sciences》1995,40(11):2438-2445
To compare differentiation along the crypt-villus axis in adult rats with changes observed in postnatal maturation with respect to binding capacities for lectins and food proteins, crypts and villi were isolated byin vivo perfusion andin vitro incubation. Brush border membranes were prepared from adults and newborns, and binding of125I-labeled lectins and food proteins was assessed by airfuge ultracentrifugation. Crypt and villus membrane protein patterns looked almost identical, unlike newborn membranes. Considerable shifts in lectin binding to membranes were observed during postnatal maturation, but not in crypt-villus differentiation. For instance, fucose-specific lectin binding patterns in both preparations resembled the general adult mode. Contrary to differences in food protein binding between newborn and adult membranes, food protein binding did not show a consistent significant difference between membranes of crypt and villus origin in adult animals. In conclusion, membrane differentiation along the crypt-villus axis was found to follow a pattern dissimilar from neonatal maturation as far as protein and carbohydrate composition and food protein binding were concerned.Supported by Deutsche Forschungsgemeinschaft DFG Ste 305. 相似文献
15.
Hiroshi Shiozaki MD Masahiro Yoshioka MD Dr. Soichiro Miura MD Hiroyuki Imaeda MD Akira Morita MD Hitoshi Asakura MD Masaharu Tsuchiya MD Hiromasa Ishii MD 《Digestive diseases and sciences》1995,40(6):1193-1198
The mechanisms whereby the conjugated bile salts regulate the activities of the brush border membrane hydrolases and its physiological significance were investigated in rat small intestine, and comparisons were made with the action of pancreatic protease. Rat brush border membrane proteins were metabolically labeled with [35S]methionine, and isolated brush border membrane was incubated with taurocholate or pancreatic elastase. The activity of solubilized hydrolases was assayed and the molecular forms of the hydrolases were examined by SDS-PAGE. The activity and protein bands of alkaline phosphatase and sucrase-isomaltase were solubilized by taurocholate, while alkaline phosphatase was not solubilized by elastase. Solubilized sucrase-isomaltase molecules were proteolytically degraded by elastase, whereas the intact molecule of sucrase-isomaltase was solubilized by taurocholate. Next the physiological role of bile salts in brush border membrane hydrolase turnover were investigated using metabolic labeling of brush border membrane hydrolase and immunoprecipitation in biliary diversion rats. After three days of biliary diversion, a significant increase in alkaline phosphatase activity was observed. Although synthesis of alkaline phosphatase in biliary diversion rats was similar to that observed in control rats, biliary diversion rats showed 1.5-fold slower turnover of alkaline phosphatase when compared with control rats. These results suggest that conjugated bile salts in the intestinal lumen may cause a rapid turnover of brush border membrane hydrolases, which may be increased by the enhanced enzyme degradation. The mechanisms for the enhanced degradation appeared to be solubilization of hydrolases caused by the detergent activity of bile salts. Therefore, conjugated bile salts may play an important physiological role in the regulation of expression of the protease-resistant enzymes such as alkaline phosphatase. 相似文献
16.
Thyroid hormone differentially regulates rat intestinal brush border enzyme gene expression. 总被引:3,自引:0,他引:3
Thyroid hormone [triiodothyronine (T3)] has been shown to play a critical role in the growth and maturation of the mammalian small intestine, but its mechanism of action has not been well studied. In the current study, an animal model of hypothyroidism and hyperthyroidism was used to study the effects of T3 on the small intestine. Adult rats were treated with propylthiouracil for a 6-week period and then given injections of either saline (hypothyroid) or 30 micrograms/100 g body wt of T3 (hyperthyroid). Northern blot analyses showed marked differential regulation of brush border enzyme gene expression. Lactase messenger RNA (mRNA) levels decreased approximately 75% along the length of the small intestine, whereas sucrase levels were unchanged. The intestinal alkaline phosphatase mRNA species were upregulated by T3, especially the 3-kilobase band, which increased most dramatically in jejunum. Further experiments showed significant levels of both the alpha-1 and beta-1 T3 receptor mRNAs within the small intestinal mucosa. Histological examination showed that T3 treatment causes marked villus hyperplasia throughout the length of the small intestine. These results provide insight into the mechanism by which T3 exerts its influence on the growth and differentiation of the intestinal epithelium. 相似文献
17.
Substantial protection against the economically important nematode Haemonchus contortus has been achieved by immunizing sheep with a glycoprotein fraction isolated from the intestinal membranes of this parasite. This fraction has been termed Haemonchus galactose-containing glycoprotein complex (H-gal-GP) since it was originally isolated through its selective binding to lectins with a specificity for N-acetylgalactosamine. A major component of this highly protective antigen complex is a family of four zinc metalloendopeptidases, designated MEPs 1-4. Various combinations of these MEPs were evaluated in immunization-challenge trials in sheep. In two experiments a combination of all four MEPs, separated from the rest of the complex by gel filtration in 8 m urea, significantly reduced H. contortus egg counts by 45 and 50%, an effect not significantly different from that conferred by 8 m urea treatment of H-gal-GP itself. Similarly, MEP3 alone or MEPs 1, 2 and 4 in combination, electroeluted from the complex following SDS gel electrophoresis, each reduced egg counts by some 33%. The MEPs are therefore protective components of H-gal-GP and from previously published findings, it appears that MEP3 is the most effective member of this metalloendopeptidase family. However, there was no significant protection when sheep were immunized with fully reduced and denatured H-gal-GP or with bacterially expressed recombinant forms of MEP 1 or the principal domains of MEP3, suggesting that conformational epitopes on the MEPs are required for immunity. 相似文献
18.
Conformational changes in the intestinal brush border sodium-glucose cotransporter labeled with fluorescein isothiocyanate. 总被引:3,自引:2,他引:1
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B E Peerce E M Wright 《Proceedings of the National Academy of Sciences of the United States of America》1984,81(7):2223-2226
Fluorescein isothiocyanate (FITC) was used to label the rabbit intestinal brush border Na+-glucose carrier, identify the carrier protein on sodium dodecyl sulfate/polyacrylamide gel electrophoresis, and monitor the effect of ions and substrates on fluorescence quenching. Enriched brush border preparations were employed to study both glucose transport and FITC binding. FITC and a nonfluorescent analog (phenyl isothiocyanate, PITC) both inhibited Na+-dependent D-glucose transport irreversibly. Inhibition was blocked completely by the presence of Na+ and D-glucose during labeling. PITC was used to label nonspecific amino groups in the presence of glucose and Na+, and then the glucose carrier was labeled with FITC in the absence of substrates. Fluorescence of FITC bound to the carrier was quenched specifically with Na+ in a saturable fashion, and this indicates a Na+-dependent conformational change in the carrier. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of FITC-labeled membranes revealed specific labeling of a 71,000-dalton peptide. We conclude that Na+ induces a conformational shift in the 71,000-dalton glucose carrier, and this is quite consistent with the kinetics of Na+-dependent glucose transport in these membranes. 相似文献
19.
Disaccharidase activities in intestinal metaplasia - contribution of lysosomal brush border enzymes.
Maltase, sucrase, and lactase were measured at pH 4 and pH 6 in normal and intestinalized gastric mucosa. In the normal mucosa the low activities of maltase and lactase seemed to be entirely due to lysosomal enzymes with acid pH-optimum. In intestinal metaplasia, brush border maltase and sucrase, but not lactase, appeared. On the other hand, there was a significant increase in lysosomal lactase (beta-galactosidase) activity. 相似文献