Amphibian intestinal brush border enzymes during thyroxine-induced metamorphosis

Summary The development of intestinal brush border hydrolytic activities has been studied during thyroxine-induced metamorphosis of Rana catesbeiana. Alkaline phosphatase activity peaks at 3 and 10 days after the beginning of the thyroxine treatment. The cytochemical observations concerning alkaline phosphatase activity are in agreement with the biochemical data. At the ultrastructural level, alkaline phosphatase activity is particularly evident on the microvilli membranes of the enterocytes in the primary epithelium after 3 days and in the secondary epithelium after 10 days. -glutamyltranspeptidase exhibits an increase of activity between 7 and 10 days. On the other hand, glucoamylase, maltase, trehalase and leucylnapthylamidase activities decrease during thyroxine treatment, these enzymatic activities being lower than that normally observed after natural metamorphosis. The present study indicates that even though thyroxine is able to induce the morphological differentiation of the intestinal epithelium this hormone is unable to complete the enzymatic load of the new mucosa.This work has been supported by grants from France-Québec (M.D., J.H.) and from the Medical Research Council of Canada (D.M., J.S.H.)     

Amphibian intestinal brush border membranes-I. Isolation from Rana catesbeiana tadpole.

1. Intestinal brush border membrane vesicles have been isolated form Rana catesbeiana tadpole. 2. Electron microscopy of brush border membrane vesicles demonstrates a fairly homogenous preparation of vesicles, some of them still containing electron dense material. 3. The dense vesicles probably comprise both microvillus core and membrane. 4. Negative staining of vesicles reveals the presence of knob-like structures (particles) covering the outer surface of the membrane. 5. The membranous fraction is characterized by a high specific activity of alkaline phosphatase, trehalase, glucoamylase, maltase and gamma-glutamyltranspeptidase.     

Influence of triiodothyronine on the polypeptide composition of the intestinal brush border membrane during amphibian metamorphosis

Brush border fragments (BBF) were isolated from homogenates of intestinal epithelium prepared from four groups of tadpoles: premetamorphic larvae, thyrostatic larvae, spontaneously metamorphosed larvae, and triiodothyronine (T3)-induced froglets. Isolation was accomplished by a combination of both Ca2+ precipitation and differential centrifugation methods. These preparations were routinely enriched seven- to-eleven-fold for the two amphibian brush border marker enzymes, gamma-glutamyltransferase and maltase. Comparison by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with silver staining revealed the presence of a polypeptide of Mr 27,000 only after spontaneous and T3-induced metamorphosis. One-dimensional SDS-PAGE together with lectin staining showed six strongly concanavalin A reactive polypeptides (Mr 52,000, 57,000, 65,000, 80,000, 130,000 and 150,000) in both preparations examined. Immunoblot analyses allowed us to detect in both preparations the presence of villin (Mr 105,000), a cytoskeletal component of microvilli. Two-dimensional isoelectric focusing IEF/SDS-PAGE together with silver staining showed the polypeptides of Mr 41,500, 43,000, 60,500 and 101,000 to be specific components of the primary intestinal epithelium brush border. In contrast six polypeptides of Mr 27,000, 52,000, 58,000, 59,000 and 95,000 were only detected in intestinal BBF after spontaneous and T3-induced metamorphosis. Their presence is under the control of the thyroid hormone. The results provide new insight regarding the subcellular localization of polypeptides whose synthesis changes during spontaneous (Figiel et al., 1987) and T3-induced metamorphosis (Figiel et al., 1989).     

Enzymic solubilization of the human intestinal brush border membrane enzymes

The releases of proteins, maltase, lactase, sucrase, trehalase, alkaline phosphatase, γ-glutamyltransferase and leucylnaphthylamide-hydrolyzing activity from human intestinal brush border membrane vesicles by various enzymes (especially pancreatic proteases) have been studied.The brush border membrane enzymes are not solubilized by digestion with trypsin and chymotrypsin but are largely released after treatment with papain or elastase. Most of the enzymes are fully active after the proteolytic treatment. All proteins released by papain and elastase have been identified by electrophoresis to already known intestinal hydrolases.Electron microscopy of brush border membrane vesicles demonstrates “knob-like” structures (particles) attached to the external side of the membrane. During papain treatment, enzyme removal runs parallel with the disappearance of the particles. During elastase treatment it is not possible to correlate the release of th enzymic activities with the removal of the particles.The results indicate that most of the intestinal hydrolases are surface components attached to the external side of the membrane. They are in accord with the concept that the brush border membrane enzymes are organized within the membrane in a mosaic-like pattern.     

Enzymic solubilization of the human intestinal brush border membrane enzymes.

The releases of proteins, maltase, lactase, sucrase, trehalase, alkaline phosphatase, gamma-glutamyltransferase and leucylnaphthylamide-hydrolyzing activity from human intestinal brush bborder membrane vesicles by various enzymes (especially pancreatic proteases) have been studied. The brush border membrane enzymes are not solubilized by digestion with trypsin and chymotrypsin but are largely released after treatment with papain or elastase. Most of the enzymes are fully active after the proteolytic treatment. All proteins released by papain and elastase have been identified by electrophoresis to already known intestinal hydrolases. Electron microscopy of brush border membrane vesicles demonstrates "knob-like" structures (particles) attached to the external side of the membrane. During papain treatment, enzyme removal runs parallel with the disappearance of the particles. During elastase treatment it is not possible to correlate the release of the enzymic activities with the removal of the particles. The results indicate that most of the intestinal hydrolases are surface components attached to the external side of the membrane. They are in accord with the concept that the brush border membrane enzymes are organized within the membrane in a mosaic-like pattern.     

Comparative study of some intestinal brush border membrane enzymes during perinatal development of the mouse

Intestinal brush border membrane (bbm) fractions have been isolated from fetal and neonatal mice. The existence of discordant developmental patterns of intestinal enzymatic activity derived from total homogenate and bbm fraction was confirmed. It originates chiefly from two phenomena: (a) variations in the state of purity of brush border fractions, and (b) loss of brush border membrane enzyme activities in supernatant that increases with age. The phenomenon of solubility for glucoamylase and alkaline phosphatase is already present two days before birth.     

Hyaluronidase activity during thyroxine-induced tadpole metamorphosis.

Immersion of Rana catesbeiana tadpoles in 10^?7Ml-thyroxine gives rise to increases in brain and backskin hyaluronidase activity. After 10 days of immersion, there is a 1.9-fold increment in brain enzyme activity and a 2.5-fold increment in the backskin. The rise in activity occurs mainly between the seventh and tenth days of treatment. During the 10-day treatment, hyaluronate content in the backskin decreases to 22% of the control level while sulfated glycosaminoglycan increases markedly, but no significant change in brain glycosaminoglycan composition occurs. The onset of major metamorphic events was observed between the seventh and tenth days of immersion in thyroxine.     

Differential sensitivity of intestinal brush border enzymes to pancreatic and lysosomal proteases.

Isolated human intestinal brush border membranes were used as sources of enzyme to study their degradation by proteolytic enzymes. Human intestinal brush border hydrolases undergo degradation by two separate proteolytic systems. Sucrase and alkaline phosphatase are degraded by pancreatic proteases (e.g. chymotrypsin) at neutral pH, whereas trehalase is degraded by lysosomal extracts at acid pH. Both the membrane bound and membrane free isolated enzymes had similar sensitivity to proteolytic enzymes. Thus, initial removal from the membrane is not essential as a prerequisite to proteolysis. It is postulated that the brush border membrane of the intestine is subject to proteolysis by pancreatic enzymes from the external cell surface and by lysosomal proteases within the cell.     

Developmental changes in intestinal brush border enzymes of rats prenatally exposed to ethanol

The activities of lactase, sucrase, alkaline phosphatase (AP) and y-glutamyl transpeptidase (gamma-GTP) were studied in the intestinal brush border membranes of pups born to rat mothers exposed to ethanol (1 ml of 30% ethanol daily during gestation) at different days of postnatal development. The activities of lactase (at day 4-20) and sucrase (at day 20-30) were considerably reduced in response to prenatal exposure to ethanol, while AP (at day 4-30) and gamma-GTP activities were significantly enhanced (p < 0.05) at day 4, 8, 14 and 20, but there was no significant difference by day 30 of postnatal development. The observed changes in enzyme activities were corroborated by Western blot analysis of lactase, sucrase and AP. Kinetic studies revealed a change in Vmax without affecting apparent Km of enzymes under these conditions. The present findings suggest that in utero ethanol exposure to rats is embryotoxic and affects the postnatal development of various brush border enzymes, which persist long after the ethanol was withdrawn prior to birth.     

Comparison of two pig intestinal brush border peptidases with the corresponding renal enzymes.

Intestinal dipeptidyl peptidase IV and gamma-glutamyltransferase were compared to the corresponding kidney enzymes with respect to immunological and electrophoretic properties. The influences of selected effectors on the two enzymes were also studied. The two kidney peptidases exhibited the reaction of total identity with the corresponding intestinal enzymes in immunodiffusion. Furthermore, the intestinal dipeptidyl peptidase IV and gamma-glutamyl transferase showed the same inhibition patterns as the corresponding kidney enzymes and the acceptor specificity of the intestinal gamma-glutamyl-transferase was found to be identical to that of the kidney enzyme. The electrophoretic mobilities of dipeptidyl peptidase IV from the two organs differed greatly. The difference was almost abolished by treatment with neuraminidase, suggesting that the variation in mobility was due to different contents of sialic acid. It is suggested that the intestinal brush border peptidases, dipeptidyl peptidase IV and gamma-glutamyltransferase, are closely related to the corresponding enzymes obtained from the kidney.     

Molecular organization of the intestinal brush border

The brush border of enterocytes represents one of the more specialized apical poles of epithelial cells. It is formed by particularly well-developed apical plasma membrane microvilli, whose shape is ensured by a highly organized cytoskeleton. The molecular organization of the cytoskeleton is described. Whereas several cytoskeleton proteins are ubiquitous, villin is highly specific for intestinal cells and can be used as a differentiation marker of these cells. The major glycoproteins, in particular hydrolases, of the brush border membrane have been characterized. They have many common structural features, in particular their mode of integration into the membrane by their N-terminal hydrophobic sequences that also plays the role of the 'signal peptide' responsible for their co-translational insertions into the endoplasmic reticulum. Studies on the biosynthesis and intracellular pathway of aminopeptidase N strongly suggest that sorting of apical and basolateral glycoproteins could occur after their integration into the basolateral domain.     

Effect of glucose and insulin on small intestinal brush border enzymes in fasted rats

Fasting reduced small intestinal length. It also decreased mucosal weight, DNA and protein content, and concentrations of enterokinase, maltase, and sucrase in both duodenal and jejunal segments. In contrast, the concentrations of lactase and leucine aminopeptidase were not affected. Concomitantly, serum insulin levels dropped to one-fifth of the control levels while serum glucose concentrations showed a lesser degree of reduction. Glucose supplementation alone raised the serum insulin level, prevented the decrease in DNA content, and showed a protective effect on mucosal protein, mucosal weight, mucosal thickness, and villus height. Glucose also protected the sucrase and maltase concentrations; more significantly for maltase in the jejunal segment. Insulin alone, although it increased the serum insulin level to that found with glucose supplementation alone, had no protective effect on the loss in protein, DNA, and most enzymes except for maltase concentration in the jejunal segment. Addition of insulin to glucose did not modify the glucose effect on the contents of DNA, protein, and concentrations of sucrase and maltase. These results suggest that the glucose effect on the mucosa is not mediated by insulin. In addition, the retention of both maltase and sucrase activities through only glucose supplementation suggests the loss of maltase and sucrase in fasting is due to nutrient rather than specific substrate restriction.