Biosynthesis of intestinal microvillar proteins. Pulse-chase labelling studies on maltase-glucoamylase, aminopeptidase A and dipeptidyl peptidase IV

The biogenesis of three intestinal microvillar enzymes, maltase-glucoamylase (EC 3.2.1.20), aminopeptidase A (aspartate aminopeptidase, EC 3.4.11.7) and dipeptidyl peptidase IV (EC 3.4.14.5), was studied by pulse-chase labelling of pig small-intestinal explants kept in organ culture. The earliest detectable forms of the enzymes were polypeptides of Mr 225000, 140000 and 115000 respectively. These were found to represent the enzymes in a 'high-mannose' state of glycosylation, as judged by their susceptibility to treatment with endo-beta-N-acetylglucosaminidase H (EC 3.2.1.96). After about 40-60 min of chase, maltase-glucoamylase, aminopeptidase A and dipeptidyl peptidase IV were further modified to yield the mature polypeptides of Mr 245000, 170000 and 137000 respectively, which were expressed at the microvillar membrane after 60-90 min of chase. The fact that the enzymes before reaching the microvillar membrane were found in a Ca2+-precipitated membrane fraction (intracellular and basolateral membranes), but not in soluble form, indicates that during biogenesis maltase-glucoamylase, aminopeptidase A and dipeptidyl peptidase IV are transported and assembled in a membrane-bound state.     

Kinetic characteristics of soluble and brush border alkaline phosphatase and sucrase activities in developing rat intestine: effect of hormones

Suckling rat intestine contains 35 and 65% of the cytosolic and membrane-bound alkaline phosphatase (AP) activities. The corresponding values for sucrase were 20 and 80% respectively. The amount of the soluble enzymes was reduced to 7-11% in adult rat intestine. Administration of cortisone, thyroxine or insulin to suckling animals induced adult type distribution of the enzymes. There were apparent differences in kinetic characteristics of soluble and brush border enzymes, but the kinetic properties of the normally developed and hormone-induced AP and sucrase were essentially similar. This suggested identical nature of these enzymes under these conditions. A biphasic Arrhenius plot was obtained for AP in weaned and hormone injected pups with a break point around 18 degrees C, while the soluble enzyme yielded a monophasic curve (Ea = 8-11 kcal/mole). Arrhenius plot for sucrase was monophasic in the suckling, hormone-injected and adult rat intestine (Ea = 8.3-15.1 kcal/mole). Membrane-bound enzymes were generally labile, while soluble enzyme activities were stable to heat treatment (sucrase at 50 degrees C and AP at 60 degrees C) in various experimental groups.     

Comparison of sialylation of maltase-glucoamylase in brush-border and soluble fractions of the small intestine of immature rats.

Two fractions of high-molar-mass soluble neutral maltase-glucoamylase (G1 and G2) of distal small intestine of 18-day-old rats separated on Sepharose 4B differ in sialylation which is reflected in their pI values obtained by chromatofocusing. The major soluble G1 fraction shows eight sialylated peaks converted by neuraminidase into a single fraction eluted at pH 4.21. Fraction G2 is less sialylated and neuraminidase causes its pI shift to 4.36. The chromatofocusing pattern suggests that G1 contains more acidic and G2 more basic glycoforms than their membrane-bound counterpart. Presence of less acidic pI values in the soluble G1 fraction of 18-day-old rats than in that of 13-day-old rats indicates that developmental decrease of sialylation concerns not only membrane-bound but also the soluble membrane-type of maltase-glucoamylase.     

Partial purification and properties of a rat brain phospholipase.

At least 90% of a membrane-bound phospholipase D was solubilized by extraction of freeze-dried rat brain with 0.8% Miranol H2M and 0.5% cholate. The bulk of base exchange reaction enzymes remained firmly bound to the particulate fraction under these conditions. The phospholipase D specific activity was enriched 240-fold by a purification protocol employing ammonium sulfate precipitation, and both Sepharose 4B and DEAE-cellulose column chromatography. The approximate molecular weight of the partially purified enzyme was calculated to be 200,000 based upon the elution profile from Sepharose 4B and Sephadex G-200 columns. The optimum pH was 6.0, and Km values for phosphatidylcholine and phosphatidylethanolamine were 0.75 mM and 0.91 mM, respectively. The enzyme activity was not dependent on the presence of divalent cation although Ca2+ and Fe2+ showed stimulatory effects.     

Succinyl trialanine p-nitroanilide-hydrolytic enzymes in human serum. Partial purification and characterization

The levels of two kinds of elastase-like enzymes, which are able to hydrolyze an artificial elastase substrate, suc-(Ala)3-pNA, but unable to hydrolyse a naturally occurring substrate, elastin, were found to be elevated in the sera of patients suffering from hepatobiliary disorders and other diseases accompanied by tissue damage. One of the enzymes was characterized as being sensitive to a chelating reagent, EDTA, and partially inactivated enzyme activity was recovered by the addition of calcium ion. The apparent molecular weight estimated by Sepharose 4B column chromatography showed a wide distribution from 200,000 to approximately 10,000,000, but all components were converted to a molecular weight of about 200,000 by treatment with 2% Triton X-100. The activity of this enzyme was partially reduced by the addition of anti-beta-lipoprotein antibody, showing that a part of the enzyme was affiliated with low and very low density lipoproteins in the serum. The level of the other enzyme was rarely increased in the sera of patients suffering from severe hepatic disorders. This enzyme was resistant to EDTA, and the apparent molecular weight was 150,000-200,000. It appeared not to be associated with lipid component. Both enzymes were assumed to be tissue-derived enzymes, because their activities were very low in the sera of healthy persons.     

Extracellular depolymerization of hyaluronic acid in cultured human skin fibroblasts

The chain length of [3H]hyaluronic acid synthesized by cultivating human skin fibroblasts in the presence of [3H]glucosamine was investigated. [3H]Hyaluronic acid obtained from the matrix fraction was excluded from a Sepharose CL-2B column irrespective of the incubation period, whereas that from the medium was depolymerized into a constant chain length (Mr = 40,000). The reducing and non-reducing terminals of the depolymerized hyaluronic acid were N-acetylglucosamine and glucuronic acid, respectively. Prolonged incubation produced no oligosaccharides as shown by examination of hyaluronidase digests, suggesting the presence of a novel endo-beta-N-acetylglucosaminidase in cultured human skin fibroblasts.     

Soluble neutral and acid maltases in the suckling-rat intestine. The effect of cortisol and development

The 100000g supernatants from 13-day-old suckling-rat intestinal homogenates contained 43.5% of the total intestinal maltase activity, compared with 7.1% in weaned adult rats aged 40 days. The soluble maltase activity was separated on Sepharose 4B into two quantitatively equal fractions at pH6.0, one containing a maltase with a neutral pH optimum and the other a maltase with an acid pH optimum. The neutral maltase was shown to be a maltase-glucoamylase identical with membrane-bound maltase-glucoamylase in molecular weight, heat-sensitivity, substrate specificity, K(m) for maltose and K(i) for Tris. The soluble enzyme was induced by cortisol, but the ratio of the soluble to bound enzyme fell during induction. Solubility of the neutral maltase was not accounted for by the action of endogenous proteinases under the preparative conditions used. It is postulated that the soluble neutral maltase is a membrane-dissociated form of the bound enzyme and that the relationship between these two forms is modulated by cortisol. The acid maltase generally resembled acid maltase of liver, muscle and kidney. It was shown to be a maltase-glucoamylase with optimal activity at pH3.0, and molecular weight of 136000 by density-gradient centrifugation. At pH3.0 its K(m) for maltose was 1.5mm. It was inhibited by turanose (K(i)=7.5mm) and Tris (K(i)=5.5mm) but not by p-chloromercuribenzoate or EDTA. Some 55% of its activity was destroyed by heating at 50 degrees C for 10min. The acid maltase closely resembled beta-glucuronidase and acid beta-galactosidase in its distribution in the intestine, response to tissue homogenization in various media, and decrease in activity with cortisol treatment and weaning, indicating that it was a typical lysosomal enzyme concentrated in the ileum.     

Disaccharidase activities in camel small intestine: biochemical investigations of maltase-glucoamylase activity

Disaccharidases (maltase, cellobiase, lactase, and sucrase), alpha-amylase, and glucoamylase in the camel small intestine were investigated to integrate the enzymatic digestion profile in camel. High activities were detected for maltase and glucoamylase, followed by moderate levels of sucrase and alpha-amylase. Very low activity levels were detected for lactase and cellobiase. Camel intestinal maltase-glucoamylase (MG) was purified by DEAE-Sepharose and Sephacryl S-200 columns. The molecular weight of camel small intestinal MG4 and MG6 were estimated to be 140,000 and 180,000 using Sephacryl S-200. These values were confirmed by SDS-PAGE, where the two enzymes migrated as single subunits. This study encompassed characterization of MGs from camel intestine. The Km values of MG4 and MG6 were estimated to be 13.3 mM and 20 mM maltose, respectively. Substrate specificity for MG4 and MG6 indicated that the two enzymes are maltase-glucoamylases because they catalysed the hydrolysis of maltose and starch with alpha-1,4 and alpha-1,6 glycosidic bonds, but not sucrose with alpha-1,2 glycosidic bond which was hydrolyzed by sucrase-isomaltase. Camel intestinal MG4 and MG6 had the same optimum pH at 7.0 and temperature optimum at 50 degrees C and 40 degrees C, respectively. The two enzymes were stable up to 50 degrees C and 40 degrees C, followed by strong decrease in activity at 60 degrees C and 50 degrees C, respectively. The effect of divalent cations on the activity of camel intestinal MG4 and MG6 was studied. All the examined divalent cations Ca(2+), Mn(2+), Mg(2+), Co(2+) and Fe(3+) had slight effects on the two enzymes except Hg(2+) which had a strong inhibitory effect. The effect of different inhibitors on MG4 and MG6 indicated that the two enzymes had a cysteine residue.     

Purification and properties of 4-hydroxybiphenyl UDP-glucuronyltransferase from bovine liver microsomes.

A UDP-glucuronyltransferase isoform glucuronizes phenolic xenobiotics such as 4-nitrophenol, and an isoform glucuronizing 4-hydroxybiphenyl has also been found in rat liver. We purified a UDP-glucuronyltransferase isoform glucuronizing 4-hydroxybiphenyl from bovine liver microsomes by solubilization with 0.7% sodium cholate followed by three column chromatographic separations using DEAE-Toyopearl 650S, UDP-hexanolamine Sepharose 4B, and hydroxyapatite. The purified bovine liver 4-hydroxybiphenyl UDP-glucuronyltransferase (named Bovine 4HBGT) had glucuronidation activities toward 4-hydroxybiphenyl and 4-methylumbelliferone but had little activity toward 4-nitrophenol and 1-naphthol. The apparent molecular mass of Bovine 4HBGT was 54,000 Da on SDS-PAGE, and this was decreased to 50,000 Da by digestion with endo-beta-N-acetylglucosaminidase H. These data suggest that Bovine 4HBGT consists of a 50,000 Da polypeptide and a high mannose type oligosaccharide chain(s) of about 4,000 Da. The NH2-terminal sequence of GT-3 was GKVLVWPVDFSXWINI. These properties of Bovine 4HBGT were very similar to those of rat UDP-glucuronyltransferase glucuronizing xenobiotics. However, the NH2-terminal sequence of Bovine 4HBGT had higher homology with that of rat liver 4-hydroxybiphenyl UDP-glucuronyltransferase than with that of rat liver 4-nitrophenol UDP-glucuronyltransferase.     

Oligosaccharides in lysosomal enzymes. Distribution of high-mannose and complex oligosaccharides in cathepsin D and beta-hexosaminidase

The distribution of the different types of oligosaccharides in cathepsin D and in beta-hexosaminidase synthesized in cultured human fibroblasts was studied by using endo-beta-N-acetylglucosaminidase H as a probe for high-mannose oligosaccharides. The enzymes were specifically labelled in the protein or the carbohydrate moiety. In both enzymes, resistant and cleavable oligosaccharides were found. The resistant oligosaccharides prevailed in the secreted enzymes. Precursor molecules of cathepsin D contained two oligosaccharide side chains. Multiple forms of the precursor are synthesized with both, one or none of two oligosaccharides sensitive to the action of the endo-beta-N-acetylglucosaminidase H. In fibroblasts unable to phosphorylate lysosomal enzymes (mucolipidosis II) the excessively secreted lysosomal enzymes contained predominantly oligosaccharides resistant to endo-beta-N-acetylglucosaminidase H.     

Partial characterization of the carbohydrate units of rat intestinal sucrase--isomaltase.

Sucrase--isomaltase was purified from rat intestinal microvillus membranes after injection of D-[2-3H]mannose and L-[6-3H]fucose, using a column of monoclonal antibody-protein A-Sepharose. After Pronase digestion and gel filtration of the glycopeptides labelled from both precursors, a major part of the radioactivity was recovered in asparagine-linked complex oligosaccharides, and a smaller amount in partially alkali-labile high-molecular-weight glycopeptides. Only a small amount of [3H]mannose was found in endo-beta-N-acetylglucosaminidase H-sensitive high-mannose oligosaccharides.     

Maltase-glucoamylase and trehalase in the rabbit small intestine and kidney brush border membranes during postnatal development, the effects of hydrocortisone

Kidney and intestinal brush border membranes were isolated from 14-day-old rabbits and papa?n solubilized maltase-glucoamylase was purified to almost homogeneity from both membranes. Maltase-glucoamylase from kidney and intestine have the same molecular weight (669,000 daltons by AcA 22 gel filtration) and the same Km (4 mM, for maltose). Tris (Ki = 12.5 mM, for maltose) is a non-competitive inhibitor for both enzymes. In intestine, maltase and glucoamylase have low activity during the first two postnatal weeks and then undergo a sharp increase during the next 2 weeks. In contrast, for trehalase, adult levels are reached about 6 days after birth. Hydrocortisone injection to 10 days rabbits causes precocious increases in the specific activities of trehalase (3.6 x), maltase (5.2 x) and glucoamylase (7.4 x). Conversely, kidney maltase, glucoamylase and trehalase activities rise gradually from birth, reaching adult levels by the end of the third week. Administration of hydrocortisone to suckling rabbit does not affect either trehalase or maltase and glucoamylase in kidney brush border membrane.     

Postnatal changes in N-linked oligosaccharides of glycoproteins in rat liver.

[3H]Mannose-labelled glycopeptides in the slices of livers from neonatal and 1-, 2-, 3- and 5-week-old rats were characterized by column chromatographies on Sephadex G-50 and concanavalin A-Sepharose and by endo-beta-N-acetylglucosaminidase H digestion. The proportion of complex-type glycopeptides was increased with time until 2 weeks post partum and then returned to the neonatal level. This was mainly due to the increased proportion of concanavalin A-bound (biantennary) species. These changes were accompanied by consistent changes in the activities of processing enzymes in liver microsomal fraction, especially of N-acetylglucosaminyltransferase I. Complex-type glycopeptides from neonatal and 2- and 5-week-old rat livers were further characterized by column chromatographies on Bio-Gel P-6 and DE 52 DEAE-cellulose in combination with neuraminidase digestion. No significant difference was found between concanavalin A-bound species from neonatal liver and those from liver 5 weeks post partum, most of which were sialylated. Concanavalin A-bound species 2 weeks post partum were comparatively smaller in size and less sialylated. On the other hand, there was no significant difference among concanavalin A-unbound species from the three different sources, most of which were sialylated. Since glycoproteins from regenerating rat liver also contain a higher proportion of complex-type oligosaccharides, as previously reported, such changes in N-linked oligosaccharides of glycoproteins may be related to control of the growth of liver cells.     

Structural and compositional differences between intracellular and secreted mucin of rat small intestine.

An investigation was undertaken to discover whether mucin purified from secretions in the lumen of rat small intestine differed in structure or composition from intracellular mucin purified from rat intestinal tissue. To do this, ligated loops were constructed in situ from previously washed intestinal segments and mucin purified separately from tissue homogenates or loop fluid. Secreted mucin (SM) differed from intracellular mucin (IM) by having a higher proportion of 'minor' mucin amino acids (aspartic acid, glutamic acid, glycine and alanine) and a lower proportion of 'major' amino acids (serine, proline and threonine). SM also contained less N-acetylgalactosamine and a small, but measureable, amount of mannose. Gel electrophoresis showed that SM penetrated the gel more readily and, unlike IM, gave a rather prominent, but diffuse, band having a midpoint position of Mr 200,000. After reduction both IM and SM gave rise to the putative 'link' component of Mr 118,000 and the 200,000-Mr band of SM disappeared. SM was included to a greater extent than IM on Sepharose CL-2B chromatography, suggesting a smaller size. With the use of CsCl-density-gradient ultracentrifugation of SM, a lighter species [buoyant density (rho) = 1.38 g/ml] enriched in the 200,000-Mr component, was separated from a heavier, more glycosylated, species (rho = 1.50 g/ml). Purified 200,000-Mr component had a composition identical with that of the 118,000-Mr 'link' component of IM, reacted in Western blots with an antibody specific for the 118,000-Mr 'link' component, and after reduction gave rise to a 118,000-Mr component on gel electrophoresis. Thus secreted mucin contains a 200,000-Mr component which appears to represent a disulphide-linked dimer of the previously described 118,000-Mr 'link' component of intracellular mucin.     

Subcellular localization of enterokinase (Enteropeptidase EC 3.4.21.9) in rat small intestine

The subcellular localization of enterokinase is controversial. In this study, enterokinase was extracted from a soluble fraction and a brush border fraction of rat small intestine by differential centrifugation. The soluble fraction contained 41% of the initial enterokinase activity while the brush border fraction contained only 4.6% of the initial activity. In contrast, alkaline phosphatase monitored as a brush border marker, yielded 26.3 in the brush border fraction and only 6% in the soluble fraction. Further separation of the soluble fraction on a Sepharose 4B column revealed three peaks of enterokinase activity. One small peak (3%) of a bound enzyme (M_r, 2·10^?6) and two larger peaks of free enzyme (M_r, 3·10⁵ and 9·10⁵). In contrast, alkaline phosphatase major fraction was in a high molecular weight peak of bound enzyme. When the brush border fraction was chromatographed only a single peak of bound enterokinase and alkaline phosphatase were found. In the lower part of the small intestine, no brush border-bound enterokinase was found, while the peak of alkaline phosphatase was the same as in the upper intestine. These data suggest that enterokinase activity in the rat intestine is mainly in a free form localized in the mucin and soluble fraction and to a negligible extent in the brush border.     

Acid phosphatase in rat liver lysosomal membranes: purification and characterization

Acid phosphatase associated with rat liver lysosomal membranes (M-APase) was purified about 4,200-fold over the homogenate with 10% recovery to apparent homogeneity, as determined from the pattern on polyacrylamide gel electrophoresis in the presence of SDS. The purification procedure included; preparation of lysosomal membranes, solubilization of the membranes with 1% Triton X-100, immunoaffinity chromatography, and gel filtration with FPLC equipped with a Sephacryl S-300HR column. The molecular weight, estimated by gel filtration through TSK SW 3000G, was approximately 320K and SDS gel electrophoresis showed that the enzyme is composed of four identical subunits with an apparent molecular weight of 67K. The enzyme contains about 24.3% carbohydrate consisting of mannose, galactose, fucose, N-acetylglucosamine, N-acetylgalactosamine, and N-acetylneuraminic acid in a molar ratio of 38:20:5:36:4:11, respectively. In addition, three soluble forms of acid phosphatase (C-APase I, II, and III) in lysosomal contents were separated from rat liver lysosomal contents with DEAE-Sephacel. These three enzymes were also purified using immunoaffinity chromatography followed by gel filtration. C-APase I, II, III, and M-APase have isoelectric points of 7.7-8.2, 6.6-7.0, 5.7-6.7, and 3.4-3.8, respectively. All four APases are sensitive to endo-beta-N-acetylglucosaminidase H. However, only C-APase III and M-APase are digestible with neuraminidase. Susceptibility of M-APase to neuraminidase in intact tritosomes was examined to study the topography of M-APase in tritosomal membranes. Neuraminidase susceptibility of M-APase was not observed in the intact tritosomes until the tritosomes had been disrupted by osmotic shock.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic properties of gamma-glutamyltranspeptidase and leucine-amino-peptidase in developing rat intestine: effect of hormones

Administration of cortisone and thyroxine produced adult-type increase in the activities of soluble and membrane-bound gamma-glutamyltranspeptidase (gamma-GTP) in suckling rat intestine. Membrane-bound enzyme activity remained unaltered while the soluble enzyme activity was reduced (27%) in insulin-injected pups. Kinetic analysis revealed that the observed changes in the enzyme levels were a consequence of altered Vmax with no change in apparent Km. A 2-fold increase in the Km value was observed in adult gamma-GTP activity compared to that of suckling animals. Membrane-bound and soluble gamma-GTP yielded similar values of the Ea (9.7-13.1 kcal/mole) but exhibited apparent differences in heat stability in the control and hormone-injected groups. Leucine-amino peptidase(LAP) activity was reduced to adult levels in insulin-treated suckling animals. Thyroxine- and cortisone-treatment did not affect soluble activity but significantly (P less than 0.001) augmented the membrane-bound LAP levels. This increase was due to enhanced (54-82%) Vmax with no change in Km. The observed decrease in LAP activity in response to insulin was due to reduced Vmax. There was no change in Ea (8-11.6 kcal/mole) except the value was raised to 19.1 kcal/mole in cortisone-injected pups. Both the soluble and membrane-bound LAP activities were quite resistant to heat inactivation upto 30 min at 60 degrees C except in weanling rats. Thus, the kinetic behaviour of normally developed and precociously induced gamma-GTP and LAP is essentially similar but there are apparent differences in the mode of action of insulin, cortisone and thyroxine in affecting the development of these enzymes.     

Purification and properties of UDP-glucuronyltransferase from kidney microsomes of beta-naphthoflavone-treated rat

Rat kidney microsomal UDP-glucuronyltransferase activities toward phenoic xenobiotics were enhanced about 4-5-fold by treatment of the animal with beta-naphthoflavone. The transferase activity toward serotonin, an endogenous substrate, was also enhanced about 7.5-fold. A form of UDP-glucuronyltransferase was purified from kidney microsomes of beta-naphthoflavone-treated rat by solubilization with sodium cholate and two steps of column chromatography, the first with DEAE-Toyopearl (fast flow rate liquid chromatography:FFLC) and the second with UDP-hexanolamine Sepharose 4B (affinity chromatography). These procedures gave about 39-fold purification and 11.5% yield of the transferase activity toward 1-naphthol. The preparation, tentatively termed "GT-2," was highly purified as judged from the single protein band (Mr 54,000) on sodium dodecylsulfate (SDS)-polyacrylamide slab gel electrophoresis. It catalyzed the glucuronidation of not only phenolic xenobiotics such as 1-naphthol, 4-nitrophenol, and 4-methylumbelliferone but also serotonin. From the result that apparent molecular weight of GT-2 was reduced to 50,000 by endo-beta-N-acetylglucosaminidase H (Endo H)-treatment, GT-2 was found to be a 50,000 Da polypeptide carrying "high mannose" type oligosaccharide chain(s). The NH2-terminal sequence of 20 residues of GT-2 was determined to be Asp-Lys-Leu-Leu-Val-Val-Pro-Gln-Asp-Gly-Ser-His-Trp-Leu-Ser-Met-Lys-Glu- Ile-Val . It was observed that there are two amino acids substitutions in the seven NH2-terminal residues in comparison with GT-1, which was purified from liver microsomes of 3-methylcholanthrene-treated rat. The NH2-terminal sequence of GT-2 was found to be homologous with the NH2-terminal sequence from the 26th to 46th amino acid residue of various UDP-glucuronyltransferase cloned by other investigators.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solubilization of adrenal medullary opioid receptors

The opioid-binding activity of digitonin extract of bovine adrenal medullary membranes was studied. [3H]Diprenorphine binding to the solubilized material was rapid and saturable. The dissociation constant of [3H]diprenorphine binding was 0.76 nM. Several opioids displaced the [3H]diprenorphine binding. The complex of [3H]diprenorphine and the solubilized binding sites was eluted as a single peak on a Sepharose 6B column and showed an apparent molecular weight of 200,000. These results indicate that active opioid receptors are solubilized with digitonin from bovine adrenal medullary membranes.