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R K Montgomery

Publications and source records attributed to R K Montgomery.

At least 37 records · Page 2Linked to original sources
PubMed1990

Coordinate expression of lactase-phlorizin hydrolase mRNA and enzyme levels in rat intestine during development.

The development of rat intestinal lactase-specific activity displays a well-known post-weaning decline. In contrast, total lactase activity increases to reach maximal levels around weaning, and remains high subsequently. In order to elucidate the molecular basis for these patterns, a rat lactase cDNA was isolated and characterized, and used in the quantification of lactase mRNA during development. This lactase cDNA uniquely hybridized to a 6.8-kilobase mRNA in the small intestine. To assess the amount of lactase mRNA encoding for lactase enzyme activity in the small intestine, total intestinal RNA was isolated and analyzed by Northern and dot-blot hybridization. The pattern of total lactase mRNA during development followed that of total lactase activity, suggesting that over this time span the level of lactase activity is primarily controlled at the transcriptional level. However, the magnitude of increase of total lactase activity during lactation compared to that of total lactase mRNA suggests that additional mechanisms are involved in regulating lactase levels. Analysis of the regional distribution of lactase mRNA along the small intestine at 14 days revealed that mRNA was high in the proximal three regions, but was dramatically lower in the distal regions. Total lactase activity, in contrast, displayed maximum activity in the mid-intestine with decreased levels both proximally and distally. Thus, lactase activity in the intestine appears to be regulated during development predominantly by transcriptional mechanisms, while alterations during lactation, and along the proximal to distal gradient, are the result of other control mechanisms.

Aging↗
PubMed1990

Glycosylation of lactase-phlorizin hydrolase in rat small intestine during development.

Age-specific changes in glycosylation of rat intestinal lactase-phlorizin hydrolase were analyzed using enzyme immunoprecipitated from microvillus membranes of suckling, weaning, and adult rats, and carbohydrate moieties were examined by lectin affinity binding, metabolic labeling, and neuraminidase treatment. Lectin binding indicated the presence of N-linked and O-linked oligosaccharide chains containing mannose and galactose throughout development. An age-dependent shift in sialic acid and fucose was seen during the period of weaning; no fucose was detectable in lactase-phlorizin hydrolase until after the rats were 20 days of age, whereas sialic acid was reduced in adult lactase-phlorizin hydrolase. The presence of sialic acid in suckling intestines and fucose in adult was confirmed by metabolic labeling with appropriate radioactive precursors. Sodium dodecyl phosphate-polyacrylamide gel electrophoresis analysis of immunoprecipitated lactase-phlorizin hydrolase from the proximal and mid small intestine showed two bands of approximately 220 and 130 kilodaltons in all age groups. In the distal part of the adult small intestine, lactase-phlorizin hydrolase appeared as two bands of similar size to those found in the proximal and mid portions. In contrast, during the suckling and weaning periods, these distal bands were approximately 225 and 135 kilodaltons. [35S]-methionine labeling and fluorography of neonatal intestines confirmed these observations. The size difference between proximal and distal small intestines was virtually eliminated by neuraminidase treatment. These data indicate that the core structure of microvillus membrane lactase-phlorizin hydrolase, consisting of both N-linked and O-linked oligosaccharides, remains constant during development, although terminal sugars shift from predominantly sialic acid during the suckling period to fucose in adulthood. This alteration in glycosylation of the protein occurs in a different pattern from the postweaning decline in lactase specific activity. Consequently, age-dependent changes in glycosylation cannot account for the decrease in lactase-phlorizin hydrolase-specific activity observed during development.

Aging↗
PubMed1989

Further studies of glycosylation and intracellular transport of lactase-phlorizin hydrolase in rat small intestine.

Previous studies [Büller, Montgomery, Sasak & Grand (1987) J. Biol. Chem. 262, 17206-17211] have demonstrated that lactase-phlorizin hydrolase is inserted into the microvillus membrane (MVM) as a large precursor of approx. 220 kDa, which then undergoes two proteolytic cleavage steps to become the 130 kDa mature MVM protein. In order to assess the role of glycosylation in intracellular transport, the processing of this enzyme has been studied in the presence of castanospermine, an inhibitor of N-linked oligosaccharide modification and subsequent treatment with two endoglycosidases, endo-beta-N-acetyl-glucosaminidase (endo-H) and peptide:N-glycosidase-F (N-glycanase). We now show that the intracellular precursor (205 kDa) undergoes carbohydrate processing (220 kDa) and transport to the MVM where its further proteolytic cleavage is as described. Treatment of the intracellular 205 kDa precursor with either endo-H which cleaves only high-mannose N-linked oligosaccharides, or with N-glycanase, which cleaves both high-mannose and complex N-linked oligosaccharides, results in the conversion of the 205 kDa protein band to one of 195 kDa. These data suggest that the 205 kDa precursor contains only high-mannose N-linked carbohydrates, and that the unglycosylated nascent protein is 195 kDa. In the presence of castanospermine, an intracellular precursor of approx. 210 kDa is observed. When treated with endo-H or N-glycanase, this form also produces a protein of 195 kDa. The transport of the intracellular precursor to the MVM and further proteolytic processing is not blocked by the inhibitor. However, all MVM forms of lactase-phlorizin hydrolase show an increase of approx. 5 kDa. Treatment of these three MVM forms with endo-H indicates the increased presence of high mannose oligosaccharides in comparison with non-castanospermine-treated forms. The susceptibility to endo-H of the 130 kDa MVM band synthesized in the absence of castanospermine implies the presence of high-mannose N-linked oligosaccharides in the mature form of lactase-phlorizin hydrolase. Incubation of these MVM forms with N-glycanase further reduces their electrophoretic mobility, indicating the presence of complex N-linked oligosaccharides in the MVM forms, in contrast with the intracellular precursor. Altered glycosylation reduces but does not abolish intracellular transport of lactase-phlorizin hydrolase to the MVM.

Alkaloids↗
PubMed1989

New insights into lactase and glycosylceramidase activities of rat lactase-phlorizin hydrolase.

Lactase-phlorizin hydrolase, a small intestinal disaccharidase, has been considered mainly an enzyme important only for the hydrolysis of lactose. After weaning in most mammals lactase-specific activity falls markedly, and, functionally, adult mammals are considered to be lactase deficient. However, the persistence of low levels of lactase activity in adulthood has never been explained. In addition, it has been suggested that lactase-phlorizin hydrolase is associated with glycosylceramidase activity when the enzyme is prepared by column chromatography, but it is unclear whether this represents copurified activities or two catalytic sites on one peptide. The developmental patterns of lactase-phlorizin hydrolase and other disaccharidases were investigated in homogenates of total rat small intestine; lactase and several glycosylceramidases were measured in immunoprecipitates from these homogenates using a monoclonal antibody. The developmental pattern of total lactase activity showed a steady 2.3-fold increase to adult levels (specific activity decreased eightfold), whereas total phlorizin-hydrolase activity increased 10.7-fold (specific activity decreased threefold). As expected, levels of both total and specific sucrase and maltase activities increased during development. In lactating rats total lactase activity showed a significant increase compared with adult males. The developmental pattern of the enzyme activities for the glycolipid substrates was similar to that found for lactase, and the immunoprecipitated enzyme showed a 40- to 55-fold higher affinity for the glycolipids than for lactose. Galactosyl- and lactosylceramide inhibited lactose hydrolysis by 38%, without a competitive pattern, suggesting two different active sites for lactose and glycolipid hydrolysis, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed1989

Suckling rat colon synthesizes and processes active lactase-phlorizin hydrolase immunologically identical to that from jejunum.

To identify potential tissue-specific characteristics of intestinal glycoprotein synthesis and processing, rat intestinal lactase-phlorizin hydrolase (L-Ph) was studied after pulse-labeling of colonic explants from 5-d-old suckling rats in organ culture and the data compared to similar studies in rat jejunum. Histologic sections of 5-d-old proximal colon showed villus-like structures lined with columnar epithelial cells. Lactase and phlorizin hydrolase activities showed tissue-specific developmental patterns. Using a MAb to small intestinal L-Ph, we were able to immunoprecipitate from colon at different ages a protein that hydrolyzed lactose and phlorizin, and whose activity was not inhibited by p-chloromercuribenzoate. After pulse-labeling for 60 min and chase for 30 min, immunoprecipitated L-Ph from total homogenates of rat colonic explants appeared on fluorography of SDS-PAGE as one band of approximately 205 kD. With increasing time of chase, it took 240 min before the precursor form was converted to the intermediate form (equivalent to the 180-kD form in jejunum) and the mature form (equivalent to the 130-kD form in jejunum), although these conversions in the jejunum were observed within 60 min of chase, and only 30 min of pulse labeling. When compared on SDS-PAGE to immunoprecipitated jejunal L-Ph, the precursor form in the colon had a slightly higher apparent mol wt than the corresponding precursor form found in the endoplasmic reticulum-Golgi fraction of the jejunum. The intermediate as well as the mature L-Ph forms in the colon were also both somewhat higher in apparent molecular weight than the same bands in the microvillus membrane fraction from jejunal explants. Removal of N-linked oligosaccharides from jejunum and colonic forms of L-Ph produced bands on SDS-PAGE with identical mobility, suggesting that the proteins were the same. The data demonstrate that, in neonatal colon, enzymatically active L-Ph undergoes biosynthetic and processing events similar to those in the jejunum. During early life, colonic L-Ph may function in the salvage of lactose not absorbed in the small intestine.

Animals↗
PubMed1987

Biosynthesis, glycosylation, and intracellular transport of intestinal lactase-phlorizin hydrolase in rat.

The biosynthesis of rat intestinal lactase-phlorizin hydrolase was studied by pulse-labeling of jejunal explants from 5-day-old suckling rats in organ culture. Explants were either continuously labeled with [35S] methionine for 15, 30, and 60 min or pulse-labeled for 30 min and chased for various periods of time up to 6 h in the presence or absence of protease inhibitors (PI), leupeptin, phenylmethylsulfonyl fluoride, and soybean trypsin inhibitor. Lactase-phlorizin hydrolase was immunoprecipitated from microvillus membrane (MVM) and ER-Golgi fractions with monoclonal antibodies. After pulse-labeling, lactase-phlorizin hydrolase from the ER-Golgi fraction appeared on SDS-PAGE as one band of approximately 220 kDa, regardless of the presence or absence of PI in the culture media. The 220-kDa protein band could also be labeled after incubation with [2-3H]mannose. In the absence of PI, the 220-kDa band appeared in the MVM by 30 min chase, simultaneously with a 180-kDa band, and by 60 min of chase an additional band of 130 kDa was seen. With increasing time of chase, the relative intensity of the 130-kDa band increased, whereas that of the 220-kDa band decreased, suggesting a precursor-product relationship. When PI were added to the medium, the formation of the 180-kDa band was not affected, but the conversion of the 180-kDa protein to the 130-kDa protein was virtually blocked. These findings suggest that lactase-phlorizin hydrolase is initially synthesized as a glycosylated precursor of 220 kDa, which is transported to the MVM. There it undergoes the following two cleavages: first, to the 180-kDa form, which is not prevented by PI used in these experiments, and second, to the 130-kDa form inhibited by PI.

Animals↗
PubMed1987

Characterisation of humoral trophic factors involved in intestinal adaptation using a 3T3 cell growth factor assay.

Indirect evidence has suggested that circulating trophic factor(s) are involved in intestinal adaptation. A 3T3 fibroblast cell culture system was used to more directly delineate the presence of such factors. Rats were divided into four groups; C-unop, those undergoing no surgery, S, those in which a dorsal slit was made; C-op, those in which the peritoneum was incised; and R, those undergoing 80% intestinal resection. At the time of death at 24 hours, one week and two weeks postoperative, stimulation of DNA synthesis in the 3T3 cells was noted after incubation with platelet free plasma from the C-op and R groups, and simultaneously an increase in ileal DNA specific activity occurred. Characterisation of the plasma fraction with growth factor activity revealed it to have a MW of greater than 6,000 and less than 14,000 d. The factor(s) was resistant to reduction with DTT, and was partially inactivated by heating to 60 degrees C. The use of 3T3 cell growth factor assay system allows further characterisation of circulating factors involved in intestinal adaptation.

Adaptation, Physiological↗
PubMed1987

Dolichol content and isoprenolog distribution in developing rat small intestine.

We examined developmental changes in dolichol concentration and isoprenolog distribution during the postnatal development of rat small intestine. We isolated dolichols from rat small intestinal lipids and quantified the isoprenologs by high performance liquid chromatography. The concentration of intestinal dolichol was highest in the intestine of the newborn rat and lowest in intestine from 19-day-old and lactating female rats. The predominant isoprenolog at all ages examined was that with 18 isoprene units. There was a significant increase in isoprenolog 17 during the 3rd wk of postnatal development and an increase in isoprenolog 19 in the adult rat small intestine. The timing of the changes in dolichol content suggests that this might be an important control point in intestinal glycoprotein synthesis during development.

Animals↗
PubMed1986

Morphogenesis in vitro of dissociated fetal rat small intestinal cells upon an open surface and subsequent to collagen gel overlay.

The morphogenesis of cells dissociated from fetal rat intestine at 18 days of gestation was compared in vitro on two different substrates, tissue culture plastic and collagen gel. During the first 2 days in culture growth on both substrates was similar, resulting in the formation of layers of epithelial cells and of small lumina within them. On plastic, the cell layers contracted over time, resulting in the formation of small mounds of cells bearing on their surface small protrusions covered with epithelial cells which had densely packed microvilli on their apical surfaces. When the cultures on collagen gels were overlaid with more collagen gel, vesicles lined with epithelial cells developed. These cells were joined by junctional complexes and displayed an apical brush border which was periodic acid-Schiff and alkaline phosphatase positive. After 1 week in culture, when the vesicles reached their maximum extent, numerous epithelial cells were actively incorporating labeled thymidine and cells were being extruded into the lumen. These results demonstrate that the dissociated intestinal cells have the capacity to form intestine-like organoids in vitro and that surrounding the cells with the collagen gel allows expression of this potential. The collagen gel cultures thus undergo morphogenetic processes which can be modified by the surrounding environment and should provide a useful in vitro model system for the study of intestinal development.

Animals↗
PubMed1986

Use of a 3T3 cell growth factor assay for the delineation and characterization of humoral trophic factors involved in intestinal adaptation in the rat.

Indirect evidence has suggested that circulating trophic factors are involved in intestinal adaptation. A 3T3 fibroblast cell culture system was used to more directly delineate the presence of such factors. Rats were divided into four groups: C-unop, those undergoing no surgery; S, those in which a dorsal slit was made; C-op, those in which the peritoneum was incised; and R, those undergoing 80% intestinal resection. At the time of death at 24 h, 1 wk, and 2 wk postoperative, stimulation of DNA synthesis in the 3T3 cells was noted after incubation with platelet-free plasma from the C-op and R groups, and simultaneously an increase in ileal DNA specific activity occurred. Characterization of the plasma fraction with growth factor activity revealed it to have a molecular weight of greater than 6000 but less than 14,000. The factors were resistant to reduction with dithiothreitol, and were partially inactivated by heating to 60 degrees C. The use of a 3T3 cell growth factor assay system now makes it possible to further characterize circulating factors involved in intestinal adaptation.

Adaptation, Physiological↗
PubMed1986

Pathogenesis of Shigella diarrhea: rabbit intestinal cell microvillus membrane binding site for Shigella toxin.

This study examined the binding of purified 125I-labeled shigella toxin to rabbit jejunal microvillus membranes (MVMs). Toxin binding was concentration dependent, saturable, reversible, and specifically inhibited by unlabeled toxin. The calculated number of toxin molecules bound at 4 degrees C was 7.9 X 10(10) (3 X 10(10) to 2 X 10(11))/micrograms of MVM protein or 1.2 X 10(6) per enterocyte. Scatchard analysis showed the binding site to be of a single class with an equilibrium association constant, K, of 4.7 X 10(9) M-1 at 4 degrees C. Binding was inversely related to the temperature of incubation. A total of 80% of the labeled toxin binding at 4 degrees C dissociated from MVM when the temperature was raised to 37 degrees C, but reassociated when the temperature was again brought to 4 degrees C. There was no structural or functional change of MVM due to toxin as monitored by electron microscopy or assay of MVM sucrase activity. These studies demonstrate a specific binding site for shigella toxin on rabbit MVMs. The physiological relevance of this receptor remains to be determined.

Animals↗
PubMed1985

Synthesis and intracellular processing of sucrase-isomaltase in rat jejunum.

To examine directly the synthesis and intracellular processing of rat intestinal sucrase-isomaltase, [3H]leucine-labeled enzyme was solubilized from purified microsomal and microvillus fractions, immunoprecipitated, and analyzed. Immunologic identity between the microsomal and microvillus forms of the enzyme was demonstrated. Time-course studies using both intravenous and intraluminal labeling demonstrated an initial peak of microsomal sucrase-isomaltase labeling at 30 min followed by a sharp decline, and a subsequent rise in microvillus sucrase-isomaltase labeling, suggesting a precursor-product relationship. Fluorography and quantitative analysis of electrophoresed immunoprecipitates confirmed these results, and revealed the presence of two high molecular weight proteins in the microsomes. Susceptibility of the smaller high molecular weight precursor to cleavage with endo-beta-N-acetyl-glucosaminidase H indicated that this was the initially synthesized form of the glycoprotein. These data are consistent with the membrane flow hypothesis and the single polypeptide model suggested for sucrase-isomaltase synthesis.

Acetylglucosaminidase↗
PubMed1985

Intestinal lactase synthesis during postnatal development in the rat.

To elucidate the mechanism of the developmental decline in intestinal lactase activity at weaning, we examined lactase synthesis in suckling and adult rats. Lactase was purified to homogeneity from pooled intestines of newborn rats and used to raise a monospecific antibody. Using this antibody, we developed a quantitative immunoprecipitation assay for lactase. Intestinal microvillus membrane proteins were labeled in 15-day and adult rats by intraluminal pulse-chase with 3H-leucine, and newly synthesized lactase quantified by immunoprecipitation. When lactase synthesis was expressed as the quantity of microvillus membrane lactase synthesized relative to total microvillus membrane protein synthesized, a significantly greater proportion of 3H-leucine incorporation into lactase was demonstrated in the suckling animals. No structural differences between newly synthesized suckling and adult lactase were observed when they were compared by SDS-polyacrylamide gel electrophoresis and fluorography. These data suggest that a change in the rate of lactase synthesis plays a role in the postweaning decline in enzyme activity.

Animal Population Groups↗
PubMed1985

Development of carbohydrate absorption in the fetus and neonate.

Maturation of mechanisms for carbohydrate absorption occurs in a defined sequence during human fetal development. The intestinal enzymes, lactase, sucrase, maltase, isomaltase, and glucoamylase, are at mature levels in the term fetus. Mature levels of pancreatic amylase activity and glucose transport occur postnatally, and levels are low in both the term and preterm neonate. In the preterm infant, sucrase, maltase, and isomaltase are usually fully active, but lactase activity, which increases markedly from 24 to 40 weeks, may be low depending upon fetal age. Despite these developmental patterns, clinical lactose intolerance is uncommon. Postnatal adaptive responses to ingested carbohydrates lead to competent carbohydrate absorption. Inadequately absorbed carbohydrates are salvaged by colonic flora through fermentation of carbohydrates to hydrogen gas and short-chain fatty acids; the latter are readily absorbed by the colon. In this setting, carbohydrate tends to be absent from the stool. Noninvasive reflection of the status of carbohydrate absorption may be obtained from breath hydrogen testing, a technique of particular value in young infants.

Adaptation, Physiological↗
PubMed1984

Rat lingual lipase: partial purification, hydrolytic properties, and comparison with pancreatic lipase.

We have partially purified lingual lipase from the serous glands of rat tongue. With a combination of Triton X-100 extraction or Triton X-114 phase-separation techniques, Bio-Bead SM-2 treatment, dialysis, and gel filtration on Sephadex G-200 or Sephacryl S-300, we obtained a sparingly soluble lipid-free protein demonstrating hydrolytic activity against triglycerides and negligible phospholipase or cholesteryl esterase activities. Compared with homogenate, specific activities of the enzyme were enriched 3- to 5-fold prior to gel filtration and 10-fold after gel filtration. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration under denaturing conditions (6 M guanidine X HCl or 0.1% sodium dodecyl sulfate) revealed one major glycoprotein band with Mr approximately 50,000. Gel filtration of the active enzyme in 0.1% Triton X-100 gave an Mr approximately 270,000-300,000, suggesting extensive self-aggregation. With both tributyrin and triolein, the pH optimum of the purified enzyme was 4.0 and activity extended from pH 2.0 to 8.0. In contrast to purified human pancreatic lipase, lingual lipase hydrolyzed triglyceride emulsions and mixed micelles stabilized with both short-chain (dihexanoyl) and long-chain (egg) lecithin and were inhibited only slightly (18-25%) by micellar concentrations of two common bile salts, taurodeoxycholate and taurocholate. Our results suggest that the hydrolysis of dietary fat by lingual lipase may extend from the pharynx through the esophagus and stomach and into the upper small intestine.

Animals↗
PubMed1983

Organotypic differentiation of trypsin-dissociated fetal rat intestine.

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.

Animals↗
PubMed1981

Rat intestinal microvillus membrane sucrase-isomaltase is a single high molecular weight protein and fully active enzyme in the absence of luminal factors.

Sucrase-isomaltase immunoprecipitated from brush border of an intestinal transplant lacking pancreatic proteases was found to be a single, high molecular weight protein. Elastase digestion converted this protein into two subunits which co-migrated on electrophoresis with those normally found on the microvillus membrane. The high molecular weight form had full sucrase and isomaltase activities.

Animals↗