Homogenates Of Intestinal Mucosa Research Articles

Peptide hydrolases of the human small intestinal mucosa: Distribution of activities between brush border membranes and cytosol

Brush border membranes from frozen human small intestine have been purified using a method which did not involve the use of EDTA-containing buffers or the disruption of brush border fragments with high concentrations of Tris. On average a 24-fold increase in specific activity of α-glucosidase (brush border marker) was obtained in the final preparation which contained insignificant traces of enzyme marker activities from cytosol and lysosomes. The homogenates of human small intestinal mucosa were shown to contain enzymes capable of hydrolysing di-, tri-, and tetrapeptides as well as amino acid- and peptide-2-nephthylamides. Assuming a 100% location of α-glucosidase in the brush border membrane, distribution studies indicated that activities against tetrapeptides and leucyl-2-naphthylamide were located exclusively in the brush border membrane. A large proportion of activity against α-glutamyl-2-naphthylamide, γ-glutamyl-2-naphthylamide and glycyl-prolyl-2-naphthylamide were also recovered in the brush border membrane fraction. Depending on the substrate utilized, 33–87% of tripeptidase activity was located in the brush border membrane. However, 58–87% of dipeptidase activity was recovered in the soluble fraction.

Subcellular localization of monoglyceride acyltransferase, xanthine oxidation, NADP: Isocitrate dehydrogenase and alkaline phosphatase in the mucosa of the guinea-pig small intestine

1. 1. Rate dependent and isopycnic banding in a zonal rotor were used to analyse the subcellular sites of enzymes in homogenates of guinea-pig small intestinal mucosa. 2. 2. The results demonstrate the following localizations: monoglyceride acyltransferase—microsomal; xanthine oxidase and dehydrogenase—soluble phase, and NADP: isocitrate dehydrogenase—soluble phase and mitochondrial. 3. 3. Alkaline phosphatase is confined to brush borders and is absent from the basolateral plasma membrane. A variable proportion of the activity, up to 40% is on brush borders which during homogenization break up into particles of reduced density and slow sedimentation rate.

9] Solubilization of the receptor for intrinsic factor—B12 complex from guinea pig intestinal mucosa

Publisher Summary This chapter discusses an overview of solubilization of the receptor for intrinsic factor-B 12 complex from guinea pig intestinal mucosa. The absorption of dietary vitamin B 12 from the intestine in many animal species requires the presence of intrinsic factor (IF), a glycoprotein secreted by the gastric mucosa. Normally, this protein is produced in large excess over actual requirements and is rarely a limiting factor in absorption. In vitro studies employing everted intestinal sacs, mucosal homogenates, and microvillous membranes prepared from the distal, but not the proximal, small bowel show uptake of IF-B 12 complex in the presence of a physiologic pH and calcium ions. The implication of this is that a receptor for IF-B 12 complex exists at the surface of the ileal cell. This chapter describes solubilization of guinea pig ileal mucosal homogenates employing the detergent Triton X-100. A particle-free solution was obtained that contained all the IF-B 12 receptors estimated to be present on the starting intestinal membranes. It is expected that better quantitation of IF- B12 receptor will help investigators to elucidate the mechanisms of B 12 absorption and malabsorption. The method employed for assaying and quantitating this solubilized receptor is also described in the chapter.

Kinetic characterization of unspecific alkaline phosphatase at different villus sites of rat jejunum. A quantitative histochemical study.

A quantitative histochemical method to determine the apparent Km and Vmax values of rat intestinal unspecific alkaline phosphatase at different sites of the villi is described. Naphthol-As-Bi-phosphate (0.025-1.5 mM) is employed as substrate and Fast Blue B as coupling reagent, and the resulting azo-dye in the brush border membrane has an absorbance maximum at lambda 550 nm. The ratio between the absorbance at lambda 550 and lambda 500 nm is constant as calculated from automatically recorded spectra at different intense dye deposits. Its absorbance is a linear function of incubation time up to 3 min and thickness of the slices up to 10 micrometers both with medium (0.5 mM) and high (1.5 mM) substrate concentrations. Using the histochemical assay under comparable conditions in test tube experiments with homogenates of intestinal mucosa an app. Km of 0.26 +/- 0.081 mM (weighted regression analysis) and 0.28-0.084 mM (direct linear plotting) is determined, demonstrating an affinity to the histochemical substrate, which is about 10 times higher than for p-Nitro-phenyl-phosphate with the purified enzyme. The results obtained by scanning the total dye deposits along jejunal villi show considerable differences in enzymatic activity between single villi and an increase from the villus base up to the transition between medium and apical villus third. As well in the apical region as at the villus base saturation curves are obtained by determining the relationship between the absorbance and the substrate concentration under standard conditions (slice thickness 10 micrometers, incubation time 3 min, 37 degrees C, pH 8.3). Calculated by weighted regression analysis and direct linear plotting from the absorbance data of six female rats the medium app. kinetic data +/- SD from the jejunal villi read as follows. Apical: Km = 0.81 +/- 0.43 mM, Vmax = 3.99 +/- 1.217 absorbance units (A) and Km = 0.87 +/- 0.428 mM, Vmax = 4.02 +/- 1.191 A, respectively. Basal: Km = 0.82 +/- 0.261 mM, Vmax = 3.26 +/- 0.719 A and Km = 0.77 +/- 0.184 mM, Vmax = 3.04 +/- 0.518 AU, respectively. As demonstrated by factorial analysis of variance only Vmax is influenced by the villus position.

Mutagenicity and metabolism of dimethylnitrosamine and benzo[α]pyrene in tissue homogenates from inbred syrian hamsters treated with phenobarbital, 3-methylcholanthrene or polychlorinated biphenyls

There are significant differences between mice and hamsters in polycyclic hydrocarbon and nitrosamine metabolism. Homogenates of liver, lung and intestinal mucosa from 6 strains of Syrian golden hamster were compared for their ability to metabolize benzo[α]pyrene (BP) and dimethylnitrosamine (DMN) to mutagens. Females of strains MHA/SsLak, LSH/SsLak, CB/SsLak, PD4/Lak LHC/Lak and Lak:LVG (SYR) were either untreated or received phenobarbital (PB), 3-methylcholanthrene (MC) or polychlorinated biphenyls (AR) to induce drug-metabolizing enzymes. Salmonella typhimurium TA92 and TA98 were used as indicators of the formation of mutagens. Dimethyl-nitrosamine demethylase (DMND) was assayed using 1 mM DMN as substrate. Aryl hydrocarbon hydroxylase (AHH) was measured using benzo[α]pyrene as substrate. MC does not induce AHH activity in hamster liver, but is an excellent inducer of enzymes converting BP to mutagens. This lack of correlation between increased AHH activity and increased metabolism of BP to mutagen in liver is in marked contrast to correlations seen in mice. MC induces AHH in hamster lung and intestinal mucosa. AR induces AHH in liver, lung and intestinal mucosa. Activity of DMND in liver is not affected by treatment of hamsters with BP or AR, but is repressed approx. 30% by treatment with MC.

Immobilized Soybean Trypsin Inhibitor in the Stabilization, Resolution and Purification of Adenosine‐3′: 5′‐monophosphate‐Dependent Protein Kinases

The protein kinase dependent on adenosine 3′: 5′‐monophosphate (cAMP) in homogenates of rat intestinal mucosa was found to be remarkably unstable during its purification due (at least in part) to proteolysis. Since soybean trypsin inhibitor was found to protect the kinase from inactivation, an attempt was made to use this inhibitor, coupled to Sepharose, in order to extract and remove the proteolytic enzyme(s) responsible for the instability at an early stage of the purification. Indeed, this column excluded the undissociated form of cAMP‐dependent protein kinase (R2C2) in a considerably more stable form, illustrating the use of immobilized proteolytic inhibitors for stabilizing proteins in crude extracts containing potent proteases. But unexpectedly the same column retarded a protein kinase which was not activated by cAMP. The latter enzyme was identified as the free catalytic subunit (C) of cAMP‐dependent protein kinase since it was found to be inhibited by the Walsh‐Krebs specific protein inhibitor. On the basis of these observations, a method was developed for the purification of C from rabbit muscle which is based on (a) passage of the crude extract on the column and (b) biospecific dissociation of R2C2 by cAMP and rechromatography of the extract on the same column. This procedure yields > 300‐fold purification of C in the last step (with a recovery of 34%) and a rapid resolution between R2C2 and C. The same column can also be used for the resolution of type I and type II cAMP‐dependent protein kinases, and for > 40 fold purification of the type II form of the enzyme, in one step. The theoretical and practical implications of these results with regard to the use and abuse of biorecognition in chromatography are discussed.

Studies on the subcellular localization of the membrane-bound fraction of intestinal calcium-binding protein

Sorbitol density gradient centrifugation applied to intestinal mucosa homogenates resulted in a complete separation of soluble calcium-binding protein from the bound fraction of calcium-binding protein, providing further documentation of the bound pool of calcium-binding protein. The peak of the bound calcium-binding protein was not associated with the major peaks of any of the markers used, but was associated with minor peaks of alkaline phosphatase, RNA, and glucose-6-phosphatase. Lack of association of bound calcium-binding protein with (Na + + K +)-ATPase indicated that the bound calcium-binding protein is not on the basolateral membrane. Differential centrifugation fractionation indicated that the bound calcium-binding protein is not associated with nuclei or mitochondria. The bound calcium-binding protein also could not be detected in partially pyrified brush borders. Exclusion of the brush border and basolateral membranes as the location of the bound calcium-binding protein suggests an intracellular locate.

Mucosal coenzyme A-dependent cholesterol esterification after intestinal perfusion of lipids in rats.

Coenzyme A-dependent esterification of cholesterol was determined in intestinal mucosal homogenates prepared after duodenal perfusion of cholesterol-free lipid emulsions for 5 h in unanesthetized rats. Cholesterol esterification rates were lowest and the mucosal cholesterol pool was greatly reduced after the same lipid infusions that, in lymph fistula rats, had produced chylomicrons deficient in cholesterol esters. CoA-dependent esterification rates were sufficient to account for all the cholesterol esters secreted in mesenteric lymph chylomicrons. During triglyceride secretion by the gut, unesterified cholesterol for chylomicron membranes may be maintained both by suppressing mucosal CoA-dependent cholesterol ester formation and from a mobilizable unesterified cholesterol pool within the mucosa.

Unmasking of Actual and Potential Receptor Sites for Cholera Toxin in Intestinal Mucosal Homogenates

Endongenous and exogenous sialidases appear to unmask sialidase-stable and sialidase-labile gangliosides in intestinal mucosal homogenates by attacking glycoproteins. Exogenous (but not endogenous) sialidase then converts sialidase-labile gangliosides into the cholera toxin-binding, sialidase-stable ganglioside GM1 (galactosyl-N-acetylgalactosaminyl [sialosyl] lactosyl ceramide). Since Vibrio cholerae produces sialidase, these observations may be relevant to the course of cholera.

The influences of the route of administration on the metabolism and excretion of bitolterol, a new bronchodilator, in the rat.

1. [3H]Bitolterol, an ester prodrug to colterol (N-t-butyl-arterenol), when administered orally, was excreted mostly in the urine; approx. equal amounts of 3H were found in urine and faeces after intraperitoneal or intravenous injection. 2. Half the dose was excreted in the bile following parenteral administration, while only a small amount of radioactivity was found in bile after oral dosage. The biliary-excreted material consisted mainly of glucuronides, for all routes of administration. 3. The glucuronides of colterol and 3-O-methyl-colterol were excreted in urine after oral administration of bitolterol. In addition to the glucuronides, free colterol and 3-O-methyl-colterol were excreted in urine following parenteral administration. 4. A part of bitolterol was hydrolysed to colterol in rat stomach, and bitolterol was more rapidly hydrolysed to colteral with homogenates of intestinal mucosa than with stomach homogenates in vitro.

Intestinal peroxisomes of goldfish ( Carassius auratus)—examination for hydrolase, dehydrogenase and carnitine acetyltransferase activities

1. 1. Rate sedimentation and isopycnic centrifugation were used to analyse the subcellular sites of enzymes in homogenates of goldfish intestinal mucosa. 2. 2. The results allowed the following allocations to be made: carnitine acetyl transferase-mitochondrial and peroxisomal, xanthine dehydrogenase and NAD: α-glycerophosphate dehydrogenase soluble phase, NADP: isocitrate dehydrogenase soluble phase and mitochondrial, and 2-naphthyl laurate hydrolase microsomal and/or brush border. 3. 3. Histochemistry confirmed the use of alkaline phosphatase and 1-naphthyl acetate esterase as brush border and microsome markers respectively. 4. 4. Urate oxidase, allantomase, allantoicase, xanthine oxidase and glycollate/lactate oxidase, activities were undetectable, and 1-naphthyl palmitate hydrolase was present only as a contaminant from pancreas.

Ligation or external fistulation of the common bile duct in the rat. II. Intestinal disaccharidase activities.

72 h after ligation or external fistulation of the common duct the activities of maltase, sucrase and lactase in the homogenate of the small intestinal mucosa of the rat were determined. The experiments were performed in connexion with intestinal perfusion studies, and the disaccharidase activities were measured in unperfused intestinal segments as well as in intestinal loops which had previously been perfused with a sucrose-containing solution. After bile duct ligation, the sucrase and maltase activities in a previously perfused intestinal loop were not different from those in sham-operated animals, the lactase activity was diminished. In a nonperfused segment, the sucrase activity was greater, the maltase activity was unchanged, and the lactase activity was lower than in control animals. After bile duct fistulation, the sucrase, maltase and lactase activities in a perfused segment were lower than in sham-operated rats. In a nonperfused loop, the sucrase activity was greater, the maltase activity was unchanged, and the lactase activity was lower then in the corresponding control group. These data suggest that bile is a factor which influences the total mucosal disaccharidase activities, and, probably, the intracellular enzyme distribution.

Ornithine synthesis from glutamate in rat intestinal mucosa homogenates: Evidence for the reduction of glutamate to γ-glutamyl semialdehyde

For 25 years, glutamate has been considered to be the source of the carbon and nitrogen atoms of ornithine and proline synthesized in mammalian tissues. However, the first enzyme reaction for these syntheses, namely the reduction of glutamate to γ-glutamyl semialdehyde, has not been observed in homogenates of mammalian tissues. These studies present evidence for this reaction in rat intestinal mucosa homogenates incubated with [U-14C]glutamate. The 14C-labeled ornithine was separated from other radioactive chemicals by ion-exchange chromatography, and was specifically identified by its capacity to serve as a substrate for ornithine transcarbamylase. The synthesis of ornithine required ATP, Mg2+ and NADPH in addition to glutamate.

Developmental changes in the sucrase-isomaltase complex in rat intestinal mucosa.

Developmental changes in sucaras-isomaltase complex formation were investigated in intestinal mucosal homogenates and brush border membranes of 15-day-old, 18-day-old and adult rats using Sephadex G-200 column chromatography and polyacrylamide disc gel electrophoresis. Disaccharidases were solubilized by papain treatment. The molecular weight of the complex did not change during development, however, the activity ratio of sucrase to isomaltase increased during development. Furthermore, a significant amount of free isomaltase, which was probably not to be derived from intestinal brush border membrane, was detected before the weanling.

Investigation into the species-specific deacylation of penta-acetyl-gitoxin.

Penta-acetyl-gitoxin (PAG) shows species-specific deacylation to 16-acetyl-gitoxin (16 AG; I and III) or gitoxin (II and IV) by homogenates of liver and intestinal mucosa of man (I), rabbit (II), guinea-pig (III) and rat (IV), whereas it is degraded into tri- and tetra-acetates by homogenates of guinea-pig myocardium as well as by human blood and serum. The identity of the principal and chloroform-extractable metabolities in human urine after PAG administration with 16-AG has been demonstrated by mass spectrometry.

N-Substituted lysines as sources of lysine in nutrition.

Twenty seven alpha-N- and epsilon-N-substituted derivatives of lysine belonging to eight different classes: (1) natural dipeptides, (2) alpha-N-acyl-, (3)epsilon-N-acyl-, (4)epsilon-N-(alpha-amino acyl)-, (5)epsilon-N-(omega-amino acyl)-, (6)alpha-N-epsilon-N-di-amino acyl-, (7)epsilon-N-acylglycyl- and (8)Schiff's bases were synthesized. The "in vitro" utilization of some of them was tested by a rat growth assay. Only the derivatives which provided biologically available lysine were hydrolysed by one or more of the intestinal mucosa, liver or kidney homogenates. It is argued that derivatives which can be split by any of the above homogenates are potential sources of lysine. The derivatives of classes (1), (4), (6) and (8) are nearly as efficient as free lysine while lysine in classes (2) and (7) is not utilized at all. From the classes (3) and (5) only some are utilized: epsilon- formyl- and epsilon-acetyl- partially and epsilon-(gamma-glutamyl)-totally. The biologically available derivatives were 4 to 7 times less reactive than free lysine in the Maillard reaction and could therefore be used to fortify foods which have to be submitted to severe heat-treatments. A cheap method of synthesis of epsilon-(gamma-glutamyl)-lysine is proposed and its metabolic transit described.

Radioimmunoassay for the antigenic determinants of cholera toxin and its components.

A radioimmunoassay procedure is described for the detection of cholera toxin and its component polypeptide chains. Cholera toxin, A subunit, B subunit, alpha chain, and gamma chain were iodinated by the chloramine T procedure. Radiolabeling did not significantly alter the polyacrylamide electrophoretic migration patterns of the toxin or its components. Moreover, radiolabeled toxin, B subunit, and alpha chain preparations retained substantial ability to bind to intestinal mucosal homogenates. The minimal amount of antitoxin detectable with radiolabeled toxin was 0.04 antitoxin units/ml. Substitution of radiolabeled B subunit, A subunit, and alpha chain for radiolabeled toxin decreased the sensitivity of the test. Radiolabeled gamma chain did not bind to the antitoxin preparation. Competitive inhibition studies, with titrated anti-choleragen serum and radiolabeled toxin or components, indicated that the minimum concentration of toxin detectable was 7.0 x 10(-8) mumol/ml at a 90% inhibition level. The A subunit and alpha chain preparations inhibited the binding of the radiolabeled B subunit to antitoxin sites. Conversely, B subunit inhibited the binding of radiolabeled A subunit and alpha chain to antitoxin. The gamma chain did not show any reaction with antitoxin or cross-reaction with either whole toxin or its components. These results strongly suggest that the A subunit and the alpha chain contain antigenic determinant(s) that are common to the B subunit. The B subunit (beta chain) and the alpha chain of cholera toxin may therefore contain region(s) of chemical similarity.

Stimulation by immune complexes of mucus release from goblet cells of the rat small intestine.

Immune complexes (bovine serum albumin with rat antibodies to bovine serum albumin) formed in twofold antibody excess were injected into the duodenum of normal rats. In comparison to controls injected with antigen only, there was a marked increase in the percentage of disrupted goblet cells (an index of mucus release) in segments from the intestine of rats exposed for 3 hours to immune complexes in vivo. Similarly, there was a significant increase in 35S-labeled mucus recovered by filtration of intestinal wash, rinse, and mucosal homogenate fluids from rats exposed to immune complexes compared to those from rats exposed to bovine serum albumin or purified rat antiserum to bovine serum albumin alone. These findings suggest that certain immune complexes can stimulate mucus release from intact rat small intestine; enhanced mucus release may have a role in clearing the surface of complexes.

Phospholipases de l'intestin de rat : mode d'action

Phospholipase activities of rat intestinal mucosa homogenate have been determined from lysophosphatidylcholines [14C] and phosphatidylcholines [-3H-14C]. In the presence of phosphatidylcholines, at pH 6.5, the homogenate has a phospholipase B activity. At pH 8.5, a phospholipase A2 activity was shown. In the presence of lysophospatidylcholines, at pH 6.5, we notice a lysophospholipase A1 activity. A kinetic study of the reactions allows us to separate the activity B into a phospholipase A2 activity and a lysophospholipase A1 activity. Thus, it appears that the total phospholipase activity of rat intestinal mucosa would results from a phospholipase A2 activity and a lysophospholipase A1 activity.

THE EFFECT OF IMMUNE COMPLEXES ON GOBLET CELL MUCUS RELEASE: A possible new mechanism of gastrointestinal host defense

In previous in vitro studies, we noted that immune complexes prepared in antibody excess and exposed to everted gut sacs consistently stimulated excessive mucus release. In the present study, the effect of immune complexes on mucus release was investigated in intact rats. Complexes (bovine serum albumin (BSA)-rat anti-BSA antibody) formed in two-fold antibody excess were injected at laparotomy into the duodenum of normal rats. Rats injected with BSA or purified anti-BSA antibody served as controls. In comparison to the controls, there was a marked increase (P < 0.01) in percentage of disrupted goblet cells (an index of mucus release) in segments from the intestine of rats exposed to immune complexes in vivo. Similarly, there was a significant increase in radiolabelled (Na235SO4) mucus released into intestinal contents, rinse and mucosal homogenate fluids from rats exposed to immune complexes compared to those from rats exposed to BSA (p < 0.001) or purified rat anti-BSA antibody (p < 0.001) alone. Cholera toxin, used as a positive control, also produced a significant increase in percentage of disrupted goblet cells and in radiolabelled mucus release into each of the fluids tested. These findings strongly suggest that immune complexes can stimulate mucus release in intact rat small intestine; enhanced release of mucus may have an important role in clearing the intestine of immune complexes formed on the surface. (Supported in part by USPHS grants).