Effect of Na+ ions on pH-dependent conformational changes in brush border sucrase-isomaltase in mice intestine.

Intestinal brush border sucrase-isomaltase (sucrose D-glucosidase E.C. 3.2.1.48, E.C. 3.2.1.10) exhibits pH-dependent stimulatory or inhibitory effects in response to Na+ ions. However, whether the enzyme undergoes conformational modulations as a function of pH and in the presence of alkali metal ions is not known. In this paper, we investigated the structural and functional relationship of purified murine sucrase in response to pH and Na+ ions using UV-CD fluorescence and spectroscopic studies. Kinetic studies revealed that at pH 5.0, the enzyme activation by Na+ ions was V-type, which changed to K-type at pH 7.2, whereas at alkaline pH (8.5), Na+ ions inhibited the enzyme activity and inhibition was uncompetitive in nature, affecting both the Km and Vmax components. Far UV-CD spectra of protein at pH 7.2 in the absence and presence of Na+ were almost overlapping, suggesting that secondary structure of protein was not affected upon addition of the salt. However, near UV-CD spectra indicated marked alterations in the tertiary structure of protein in presence of 50 mM Na+ ions. Increase in pH from 7.2 to 8.5 resulted in a marked rise in fluorescence intensity and red shift in lambda max due to tryptophan residues in the enzyme molecule. These findings suggested that alterations in enzyme activity as a function of pH and Na+ ions was associated with ionization of key amino acid residues together with structural modifications in the enzyme conformation around neutral or alkaline pH.

Anticuerpos monoclonales en la evaluación de enzimas del ribete estriado yeyunal de lactantes con diarrea persistente / Monoclonal antibodies in the assessment of the jejunal striatum border enzymes in infants with longstanding diarrhea

En las biopsias de mucosa yeyunal de 10 pacientes con diarrea persistente se estudió expresión de las enzimas lactasa, sacarasa-isomaltasa, maltasa y aminopeptidasa, del ribete estriado, mediante anticuerpos monoclonales y los resultados se contrastaron con los síntomas y signos clínicos, morfológicos (microscopía de luz), actividad disacaridásica (Dahlqvist) y la expresión de lactasa por un método histoquímico. Se obtuvo expresión de aminopeptidasa en criptas y vellosidades, mediante los anticuerpos correspondientes. La expresión por anticuerpos histoquímica y actividad enzimática (Dahlqvist) fueron concordantes para la expresión de lactasa en las vellosidades, mientras en las criptas se registró positividad sólo en 2 casos. En las vellosidades los anticuerpos monoclonales tendieron a producir más reacciones positivas para sacarasa-isomaltasa en los casos con menos daño morfológico, excepto en uno de deficiencia primaria; en las criptas el resultado fue positivo en todos, menos dos pacientes, en los que tampoco hubo positividad en las vellosidades. Los anticuerpos monoclonales pueden aportar información útil para entender mejor los mecanismos de daño y reparación de las enzimas del ribete estriado y estimar el pronóstico de la lesión

Expression of brush border enzymes in ethanol fed rat intestine.

The effect of feeding ethanol daily for 40 days was studied on various brush border enzymes in rat intestine. Brush border alkaline phosphatase (AP), lactase, gamma-glutamyltranspeptidase (gamma-GTP), p-nitrophenyl (PNP)-beta-D-galactosidase (P < 0.01) and sucrase (P < 0.001) were significantly enhanced while leucine aminopeptidase and PNP-beta-D-glucosidase activities were unaltered in ethanol fed rats compared to the controls. Kinetic studies revealed that an increase in Vmax together with a decrease in affinity in case of gamma-GTP and an increase in Vmax for AP and sucrase were responsible for the observed stimulation of enzyme activities in ethanol administered rats. Significant changes in enzyme activities were observed in different populations of enterocytes along the crypt-villus unit in the ethanol fed animals. These observations suggest that ethanol feeding modifies the brush border enzymes in rat intestine but the underlying mechanisms seem to be distinct in differentiating enterocytes.

Inhibition of rabbit intestinal brush border sucrase by indolin 2,3-dione (isatin) at pH 5.0.

Isatin (10 mM) inhibited the activity of rabbit brush border sucrase by 60% at pH 5.0 but it had no effect on enzyme activity around neutral pH. Isatin inhibition of sucrase was unaffected by Na+ ions but K+ and Cs+ ions reduced enzyme inhibition, partially. Kinetic analysis revealed that sucrase inhibition by isatin at acidic pH was non-competitive with Ki of the order 6.5-7.8 mM. Isatin together with 4 mM harmaline or iodoacetate (3 mM) or dithionitrobenzene (2 mM) yielded 80-85% inhibition of the enzyme. These observations suggest that inhibitory sites for isatin, harmaline and -SH group reacting agents are distinct in rabbit brush border sucrase.

Effect of feeding high fat, high fiber diet on brush border enzymes in mice intestine.

The effect of dietary fat content on brush border enzymes has been studied in mice intestine. The results obtained from 26 per cent fat (high fat; HF)-fed mice were compared with those fed 10 per cent fat (pair-fed; PF and ad libitum-fed). Brush border alkaline phosphatase (AP), leucineaminopeptidase (LAP) and gamma-glutamyltranspeptidase (gamma-GTP) activities were significantly enhanced while sucrase activity was reduced (P < 0.001) in HF group compared to the controls. Activities of lactase, p-nitrophenyl (PNP)-beta-D-glucosidase and PNP-beta-D-galactosidase were unaltered under these conditions. Kinetic studies with AP, sucrase and LAP revealed that changes in enzyme levels in response to HF diet were due to change in Vmax. Significant changes in enzyme activities as a consequence of HF intake were observed in enterocytes all along the crypt-villus unit as compared to the control group. These results indicated that feeding a fat-rich diet produced selective changes in brush border enzyme activities in mice intestine.

Alterations in rat intestinal sucrase and alkaline phosphatase activities in alloxan induced experimental diabetes.

Rat intestines revealed a significant loss of proteins after seven days of alloxan induced diabetes. The data suggested the presence of two forms of alkaline phosphatase (ALP) in normal rat intestines. Along with the loss of proteins from the intestines during diabetes, a form of ALP which appears to be loosely bound to the intestine is also flushed out. Total brush border membrane (BBM) proteins are relatively preserved from such leaching effect of alloxan induced diabetes. Thus, sucrase and another form of ALP which appears to be strongly bound to the BBM are flushed out at a slower rate as compared to the other intestinal proteins and loosely bound soluble ALP. BBM preparations from diabetic rat intestines showed lower ratios for BBM/intestinal homogenate sucrase or ALP activity/mg proteins as compared to the normal control rats. Such ratios, therefore, misdepict the purity as low for the BBM from diabetic rats which is merely because of the decreased contents of proteins in the intestinal homogenate during alloxan-induced acute experimental diabetes.

Effect of divalent metal ions on soluble and membrane-bound alkaline phosphatase activity in suckling rat intestine.

EDTA treatment of intestinal brush border membranes (BBM) and epithelial cell supernatant completely inhibited alkaline phosphatase (AP) activity in suckling rat intestine. AP activity was fully regained upon dialysis of the preparations against Zn2+ and to a lesser extent against Co2+, Ca2+ and Mn2+ ions. Other metal ions (Cd2+ and Mg2+) tested were essentially ineffective in restoring the enzyme activity. Considerable differences were observed in kinetic characteristics of the membrane-bound and soluble AP activities in response to various metal ions. There were apparent differences in Km, Vmax, energy of activation (Ea) and thermal stability of the soluble and membrane-bound AP activities, after metal ion substitutions. Nearly 35% AP activity was solubilized on sodium dodecyl sulphate treatment of brush borders (membrane protein: detergent ratio 1:3; w/w). Dialysis of detergent solubilized BBM against different metal ions reconstituted AP activity in the particulate fraction: the order of effectiveness was Zn greater than Ca greater than Mn greater than Co. The kinetic properties of the reconstituted AP were essentially similar to the non-integrated enzyme activity in response to various divalent metal ions examined. But there were apparent differences in Km, Vmax, Ea and thermal stability of the reconstituted AP activity compared to native brush border enzyme. The results suggest the unique requirement of Zn ions for stability and catalytic activity of the soluble and membrane-bound AP activity in suckling rat intestine.

Biochemical analysis of jejunal brush border membrane of golden hamster: pathogenic modulations due to ancylostomiasis.

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.

Maternal nutrition and development of intestinal functions: II--Effect of feeding high protein and high fat diets to lactating rats.

Effects of feeding high-protein (HP) and high-fat (HF) diets to lactating rats have been studied on the development of microvillus membrane enzymes and glycosylation in suckling rats. The activities of sucrase and lactase were significantly (P less than 0.01) decreased in the pups reared on HP fed dams. Alkaline phosphatase (AP), leucine aminopeptidase (LAP) and gamma-glutamyl-transpeptidase (gamma-GTP) activities were essentially similar in HP and pair-fed groups. Pups reared on dams fed HF-diet, revealed nearly a 20% increase in disaccharidase levels and a significant (P less than 0.05) decrease in AP activity compared to the pair-fed controls. The activities of LAP and GTP were unaffected under these conditions. Sialic acid content was unaltered, however, fucose level of the membranes was significantly reduced in pups nursed by mothers fed HP-(P less than 0.05) or HF-(P less than 0.01) diet. The binding of 125I-labelled wheat germ agglutinin and Ulex europeus agglutinin was in agreement to the data on sialic acid and fucose contents of the membranes. The binding of peanut agglutinin to microvillus membranes was enhanced by 31% and 21% in HP and HF groups, respectively. These findings suggest that the quality of maternal nutrition affects the enzymes and glycosylation of brush-borders in developing rat intestine.

Intestinal glucoamylase & other disaccharidases in children with protracted diarrhoea.

Brush border lactase, sucrase and glucoamylase activities were assessed in jejunal mucosal biopsy specimens from 34 children (median age 11 months; range 1.5-38) having protracted diarrhoea with failure to thrive and 8 well nourished children with normal jejunal mucosal histology (median age 10.2 months; range 2-37). All enzymes showed progressive decrease in activity which was directly in relation to increasing degree of mucosal injury (P less than 0.002). Lactase was significantly reduced even in patients with protracted diarrhoea and normal mucosa (P less than 0.05). Glucoamylase and sucrase were significantly reduced only in the presence of mucosal injury (P less than 0.01). Our data suggest that most children with protracted diarrhoea may not tolerate lactose containing feeds and may need lactose-free diets preferably based on starch. A small number of children with protracted diarrhoea, who have severe mucosal injury may not be able to handle even starch and may require diets based on short chain glucose polymers. The findings of this study, need to be corroborated with well-controlled metabolic balance studies.

Effect of methylglyoxal on protein thiol and amino groups in isolated rat enterocytes and colonocytes and activity of various brush border enzymes.

The effect of methylglyoxal on protein -SH and -NH2 groups in cytosolic and membranous fractions of epithelial cells lining the gastrointestinal tract of rat was investigated, using isolated villus and crypt cells (enterocytes) and colonocytes. It was found that 11-12% cytosolic protein -SH and 14-17% membrane protein -SH groups were lost when villus and crypt cells were treated with 2 mM methylglyoxal. In colonocytes, the corresponding loss in protein -SH groups was 46 and 30% under the same treatment. Similarly, 27-37% protein -NH2 group in the cytosolic fraction and 18-19% protein -NH2 group in membranous fractions of the enterocytes were lost by 2 mM methylglyoxal treatment. In colonocytes, the loss of protein -NH2 group was 30 and 15% in cytosolic and membranous fractions, respectively, under the same treatment. Effect of methylglyoxal on activity of various brush border enzymes such as alkaline phosphatase, gamma-glutamyl transpeptidase, leucine aminopeptidase, Mg2(+)-ATPase, sucrase and lactase was also studied. Alkaline phosphatase and gamma-glutamyl transpeptidase activities were inhibited to the extent of 30 and 15% respectively. There was no significant change in the activities of other enzymes after treating the brush border vesicles with 2 mM methylglyoxal. These findings show that methylglyoxal can cause loss of protein thiol and amino groups and enzyme activity in mucosal cells of rat gastrointestinal tract and the effect is more pronounced in colonocytes, which are in constant contact with bacterial metabolites.