Localization of a new serine protease, ingobsin, in goblet cells in rat, pig and man

Summary A serine protease, ingobsin, that cleaves Lys-x and Arg-x, has been purified from rat duodenal tissue. By immunohistochemical methods, the enzyme was localized in goblet cells in the small intestine of rat, pig, and man. The immunoreactive cells were most numerous in the proximal part of the intestine. In the electron microscope, the immunoreaction was localized mainly to the rough endoplasmic reticulum of the goblet cells and to the secretion being extruded from the cells.     

Studies on mammalian intestinal peroxidase.

A peroxidase, purified from rat small intestine to apparent homogeneity as judged by polyacrylamide-gel electrophoresis, exhibited an absorbance ratio (A412/A280) of 0.783. Its Mr (44000 +/- 1000) and spectral properties were similar to those of the pig intestinal enzyme. The velocity constant for the reaction between rat intestinal peroxidase and hydrogen peroxide was found to be 1.8 x 10(7) M-1 . s-1. Benzhydroxamic acid inhibited the peroxidative oxidation of guaiacol by intestinal peroxidase from both species but the concentration required to cause half-inhibition of the enzyme from the rat was higher by one order of magnitude than for the pig enzyme. The amino acid composition of highly-purified pig intestinal peroxidase showed a relative abundance of basic amino acids (lysine and arginine) and was similar to that of lactoperoxidase, but not that of myeloperoxidase. The initial ten amino acid residues of this enzyme (the first reported partial sequence for a mammalian peroxidase) were also determined.     

Studies on cytosolic superoxide dismutase from intestinal mucosa

CuZn superoxide dismutase from monkey (Macaca radiata) intestinal mucosa was purified to homogenity. The enzyme showed a subunit molecular weight of 16000. The enzyme preparation from intestinal mucosa of rat, rabbit, guinea-pig and monkey was distinctly different in electrophoretic mobility and in elution profile on ion-exchange chromatography, possibly due to their difference in charge. The difference may not be due to glycosylation, since the enzyme was not stained for glycoprotein. Polyclonal antibody against purified monkey enzyme inhibited the activity of intestinal CuZn superoxide dismutase from rat, rabbit and guinea-pig. Thus it appears that intestinal CuZn superoxide dismutases from different sources, despite being similar in immunological and other properties, differ in certain amino acids and hence in charge.     

Concomitant detection of mucosal mast cells and eosinophils in the intestines of normal and nippostrongylus-immune rats

Summary The granules of mucosal mast cells (MMC) in the rat and man apparently poorly fixed with formaldehyde and special fixation techniques are normally used to demonstrate MMC glycosaminoglycans (GAG) in these two species. However such techniques do not permit the study of MMC granule enzyme cytochemistry or the demonstration of eosinophils. We have, therefore, examined some histochemical and immunocytochemical properties of MMC and eosinophils in normal and parasitised rats following various fixation procedures. Immersion fixation of rat intestine in 4% paraformaldehyde for 6 h not only facilitated the demonstration of MMC glycosaminoglycans with basic dyes but also permitted the concomitant staining of cosinophils with acidophilic dyes. A MMC granule-associated serine esterase was also demonstrated by enzyme cytochemistry and rat mast cell protease II was detected within MMC granules by immunocytochemistry. This new methodology obviates the requirement for separate fixation procedures in the identification and characterisation of MMC/eosinophil interactions in normal and parasitised rat intestinal mucosa.     

Species-related variations in antioxidant components of gastric and duodenal mucosa

Enzymatic and non-enzymatic antioxidant profiles of the gastric and duodenal mucosa of rat, rabbit, cat and pig were investigated and found to exhibit significant variations. Rat gastric and duodenal mucosa exhibited the highest levels of basal glutathione of the various tissues examined. The highest activity of glutathione reductase was found in the gastric and duodenal mucosa of rat as compared with that in these tissues from the other species. The gastric mucosa of cat and pig showed similar activities of glutathione peroxidase, which was significantly lower than those in rat or rabbit gastric mucosa. The activity of this antioxidant enzyme was similar in rat, rabbit and pig duodenal mucosa and lower than that in cat duodenal mucosa. Strong correlations were found between activities of the functionally coupled antioxidant enzymes glutathione peroxidase and glutathione reductase in gastric but not in duodenal mucosa. The activity of superoxide dismutase showed negligible regional or species-related variations in activity.     

Extrahepatic synthesis of apolipoprotein E

Apolipoprotein E (apoE) synthesis has been examined in rat and guinea pig tissues using in vitro translation and [35S]methionine labeling of tissue slices. A number of tissues not involved in lipoprotein synthesis synthesize a protein very similar to apoE, including the spleen, adrenal, kidney, testis, ovary, heart, and lung. Although the intestine is involved in lipoprotein synthesis, apoE synthesis could not be detected in intestinal mucosa. The protein synthesized by the extrahepatic tissues was identified as apoE by its electrophoretic mobility, its immunologic reactivity with a monospecific antibody and by limited proteolysis mapping with Staphylococcus aureus V8 protease. ApoE represented between 0.02 and 0.7% of the total protein synthesized in the extrahepatic tissues, indicating that apoE mRNA is a fairly abundant mRNA in these tissues. ApoE mRNA was also detected by hybridization with a rat apoE cDNA clone, which hybridized to a single mRNA 1250 nucleotides in length in rat liver and in extrahepatic tissues. Hybridization of the apoE clone to rat genomic DNA demonstrated that the apoE gene was more heavily methylated in intestinal mucosa, which did not synthesize apoE, than in liver, testis, or kidney. 35S labeling of peritoneal macrophages revealed that both rat and guinea pig macrophages synthesized and secreted apoE in vitro. Rhesus aortic smooth muscle cells also synthesized and secreted apoE. The possible functions of apoE synthesized in the peripheral tissues are considered.     

Properties of monoglycerol acyltransferase in rat adipocytes.

Previous studies by Coleman and Haynes (1986, J. Biol. Chem. 261, 224-228) indicated the presence of two different tissue specific monoacylglycerol acyltransferases (MGAT) in intestinal mucosa and suckling rat liver. The evidence was based upon the differential responses of these two isoenzymes to various treatments including, the effects of temperature, proteolysis, protein modification reagents, detergents, and divalent cations. In the present investigation, we have used some of these criteria to determine the identity of adipose enzyme with the MGAT present in liver and intestinal microsomes. The properties of adipose and intestinal enzymes were similar in several respects, but differed from the liver enzyme. This suggests the possibility that MGAT activities from adipose and intestine may be mediated by the same enzyme protein, whereas the liver MGAT may be a separate isoenzyme as proposed earlier. The most distinguishing feature of the liver enzyme was its ability to sustain both high temperature (52 degrees C) and resist proteolysis. However, when disrupted microsomal preparations were used, the liver enzyme was both thermolabile and protease sensitive, as observed for the intestinal and adipose MGAT. Possibly, the location of liver MGAT within the membranes may be responsible for such unique properties of liver MGAT.     

Esterolytic activities of rat intestinal mucosa. 1. Characterization, cellular distribution and subcellular localization of a glycerol-ester hydrolase.

The preferential cellular distribution in the villus tip and the subcellular localization in the endoplasmic reticulum of an intestinal glycerol-ester hydrolase from rat mucosa are described. The enzyme is shown not to be from either pancreatic or bacterial origin; it catalyzes the hydrolysis of short- and medium chain triglycerides and of p-nitrophenylacetate. Contrarily to the specificity found for the pig intestinal lipase (Serrero, Négrel and Ailhaud, 1975), no activity is detectable against acylCoA; a thiolester hydrolase different from the glycerol-ester hydrolase was demonstrated after differential solubilization and chromatographic separation. A high proportion of glycerol-ester hydrolase is present in the intestinal lumen; its possible complementary role in lipid degradation is discussed.     

Cloning, sequencing and further characterization of acylpeptide hydrolase from porcine intestinal mucosa.

Acylpeptide hydrolase was purified to homogeneity from porcine intestinal mucosa using a seven-step procedure including ammonium sulfate precipitation, gel filtration as well as anion exchange and affinity chromatography. The specific activity of the enzyme reached 105000 nmol/mg protein per min and the purification was as high as 5500-fold. This tetrameric enzyme is composed of four apparently identical subunits, the molecular mass of which was estimated to be 75 kDa, based on the results of amino acid analysis and gel electrophoresis performed under denaturing conditions. It is likely that the NH(2)-terminal residue may be acetylated, while serine was found to be the COOH-terminal residue. The hydrolytic activity of the enzyme toward N-acetyl-L-alanine p-nitroanilide at the optimum pH value was increased twofold in the presence of the chloride anion. The K(m) value calculated from the kinetics of the hydrolysis of acetylalanyl peptides was found to be 0.7+/-0.1 mM, whereas the V(max) values decreased from 200 to 50 nmol/min per microgram of enzyme, depending on the peptidic chain lengths. The V(max) value of the synthetic substrate (250 nmol/min per microgram of enzyme) was 25-500% higher than those of the acetylalanyl peptides, depending on the peptide chain length, although the enzyme affinity was slightly lower (1.8 mM as compared with 0.7 mM). In line with data on other animal species and on various tissues, the enzyme seemed likely to be a serine protease, since it was readily inhibited by diisopropyl fluorophosphate and diethyl pyrocarbonate. A 2377-nucleotide long cDNA coding for the enzyme was isolated from pig small intestine. The deduced amino acid sequence consisted of 731 residues and showed a single different amino acid with that of the porcine liver APH, except the N-terminal amino acid which is still probably lacking.     

Structural and immunological evidence for the identity of prolyl aminopeptidase with leucyl aminopeptidase

Prolyl aminopeptidase (EC 3.4.11.5) has been assumed to be a unique enzyme catalyzing specifically the removal of unsubstituted NH2-terminal L-prolyl residues from various peptides and to be distinct from leucyl aminopeptidase (EC 3.4.11.1). In the present study, prolyl aminopeptidases were purified to apparent homogeneity from pig small intestine mucosa and human liver and their NH2-terminal amino acid sequences were determined together with that of pig kidney leucyl aminopeptidase. The NH2-terminal 24-residue sequence of pig intestinal prolyl aminopeptidase was shown to be identical with that of pig kidney leucyl aminopeptidase. The NH2-terminal sequence of human liver prolyl aminopeptidase was also shown to be very similar to that of pig kidney leucyl aminopeptidase. Further, pig intestinal prolyl aminopeptidase and pig kidney leucyl aminopeptidase were immunologically indistinguishable. These lines of evidence strongly suggest that prolyl aminopeptidase is identical with leucyl aminopeptidase.     

Mechanism of activation of adenylate cyclase by Vibrio cholerae enterotoxin. Relations to the mode of activation by hormones.

The influence of Vibrio cholerae enterotoxin (choleragen) on the response of adenylate cyclase to hormones and GTP, and on the binding of 125I-labeled glucagon to membranes, has been examined primarily in rat adipocytes, but also in guinea pig ileal mucosa and rat liver. Incubation of fat cells with choleragen converts adenylate cyclase to a GTP-responsive state; (-)-isoproterenol has a similar effect when added directly to membranes. Choleragen also increases by two- to fivefold the apparent affinity of (-)-isoproterenol, ACTH, glucagon, and vasoactive intestinal polypeptide for the activation of adenylate cyclase. This effect on vasoactive intestinal polypeptide action is also seen with the enzyme of guinea pig ileal mucosa; the toxin-induced sensitivity to VIP may be relevant in the pathogenesis of cholera diarrhea. The apparent affinity of binding of 125I-labeled glucagon is increased about 1.5- to twofold in choleragen-treated liver and fat cell membranes. The effects of choleragen on the response of adenylate cyclase to hormones are independent of protein synthesis, and they are not simply a consequence to protracted stimulation of the enzyme in vivo or during preparation of the membranes. Activation of cyclase in rat erythrocytes by choleragen is not impaired by agents which disrupt microtubules or microfilaments, and it is still observed in cultured fibroblasts after completely suppressing protein synthesis with diphtheria toxin. Choleragen does not interact directly with hormone receptor sites. Simple occupation of the choleragen binding sites with the analog, choleragenoid, does not lead to any of the biological effects of the toxin.     

Effect of an extract of nereis virens on mammalian spermatozoa

Epididymal sperm of the mouse, rat, and guinea pig and ejaculated sperm of rabbits are cleaved at the head-tail junction by an extract of Nereis virens. Annelids are extracted with water and the extract is purified by ion exchange chromatography. Electron microscopy shows that the extract acts on the filaments connecting the capitulum of the tail with the basal plate lining the nuclear envelope. Following detachment, the basal plate remains with the head. The extract contains proteases as indicated by hydrolysis of tosyl arginine methyl ester (TAME), benzoyl arginine ethyl ester (BAEE), and Azocoll, a general protease substrate. The hydrolysis of TAME is inhibited by tosyl lysine chloromethyl ketone (TLCK), a trypsin inhibitor, but TLCK does not prevent head-tail separation by the Nereis extract. Similarly tosyl phenylalanine chloromethyl ketone (TPCK), a chymotrypsin inhibitor, and phosphoramidon and leucyltryptophan, both thermolysin and acrolysin inhibitors — singly or in combination — do not prevent hydrolysis of Azocoll. Head-tail separation activity of the extract was inhibited by dithiothreitol, which reduces disulfide bonds, and phenylmethyl sulfonyl fluoride, an inhibitor of serine proteases. These results indicate that the extract is a mixture of proteases, one being a serine protease similar to trypsin. Digestion of the connecting filaments with the pure proteases, trypsin and Staphylococcus aureus V8 protease, has yielded the following information on the proteins of the filaments. The accessibility of arginine and/or lysine peptide bonds to enzyme action is highest in rat sperm filaments, whereas those in the filaments of mouse, rabbit, and guinea pig sperm are less accessible than in the rat. Another possibility is that the total content of arginine and/or lysine varies between the species. The most dramatic difference is the enzymatic action on glutamyl peptide bonds of the filaments, the order being: mouse 〉 rat 〉 rabbit, with guinea pig sperm filaments completely resistant over the time course of the experiment.     

Isolation, characterization, and developmental expression of pig intestinal fatty acid-binding proteins

The goal of this study was to characterize and quantify intestinal fatty acid-binding proteins of the pig. Small intestinal mucosa from 13-19 kg pigs was homogenized and centrifuged to obtain cytosol. Isolation of fatty acid-binding proteins from delipidated cytosol was achieved using molecular sieve, oleic acid affinity, and ion exchange chromatography. Fatty acid-binding protein isolation was monitored using a fatty-acid binding assay in conjunction with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. Antisera to rat liver-fatty acid-binding protein cross reacted with an isolated intestinal fatty acid-binding protein of Mr = 13,000, whereas antisera to rat intestine-fatty acid-binding protein was not cross reactive with isolated pig intestinal proteins. These experiments identify a pig intestinal fatty acid-binding protein that exhibits strong immunochemical similarity to rat liver-fatty acid-binding protein. Cytosol prepared from intestinal mucosa of pigs at -4, 2, 4, 7, 15, 22, 28, and 35 d of age was assayed for fatty acid-binding protein activity. Preweaning fatty acid-binding protein activity in cytosol was maximal at 7 days of age when expressed as total jejunal fatty acid binding per kilogram bodyweight, intestinal or mucosal weight or milligram total protein. After weaning (21 d), fatty acid-binding protein activities declined to 28 days, but increased again by 35 days. Total soluble fatty acid-binding protein activity in pig intestine is regulated during postnatal development and this may account in part for the altered intestinal absorption of lipids observed in young pigs at weaning.     

Molecular cloning and expression of rat antisecretory factor and its intracellular localization.

Antisecretory factor (AF) was identified as a pituitary protein that inhibits the intestinal fluid secretion induced by cholera toxin. One aim of this study was to elucidate whether AF is also synthesized in the intestine or if AF produced in the pituitary is transported to the intestinal tract for its function there. cDNA clones encoding a protein proposed to be AF were isolated from rat pituitary gland and intestinal mucosa cDNA libraries. The nucleotide sequences of clones isolated from the rat pituitary gland and intestinal mucosa were identical. The deduced amino acid sequence was highly homologous to the sequence for subunit 5a of the human 26S protease that exists abundantly in the cytosol and nucleus. The production of AF in the intestine was confirmed by Northern blot and immunoblot analyses. Immunocytochemical observations of cells transfected with the rat AF cDNA showed that the AF protein was localized in the cytoplasm. These findings suggest that the protein proposed to be AF may be a cytoplasmic protein, it exists in the intestine rather than being transported from the pituitary gland, and it may function in intestinal cells.     

Cleavage of the rat intestinal 1,25-dihydroxyvitamin D3 receptor by an endogenous protease to a form with defective DNA binding

In this report we describe a form of the 1,25(OH)2D3 receptor which no longer binds to DNA. The defective form of the receptor was produced by the action of an endogenous protease. Rat intestinal receptors, obtained by a two-step procedure of a low salt homogenization followed by extraction of the chromatin pellet with high salt, fail to bind to DNA-cellulose. Inclusion of various serine protease inhibitors during the preparation protects against the loss of DNA binding. Sedimentation analysis in sucrose gradients indicates that the defective receptor is measurably smaller than the native receptor and is unable to aggregate normally under low salt conditions. The size difference, as determined by gel chromatography, is approximately 9,000 Da (56,000 for the protected receptor, 47,000 for the cleaved form). The elution from DEAE-cellulose indicates that the overall charge of both intact and cleaved receptor forms is very similar. Cell fractionation and mixing experiments suggest the enzyme may be located in the lysosomal compartment, organelles which are susceptible to breakage during the extraction procedure. The results demonstrate that an endogenous enzyme preferentially cleaves the 1,25(OH)2D3 DNA binding site resulting in a receptor with altered characteristics. Such an enzymatic activity has not been previously described for the 1,25(OH)2D3 receptor from other tissues or species. Since rat intestine is a classically studied target organ, these findings have additional relevance in receptor purification or other studies to characterize the receptor.     

The serine protease from rat liver and hepatoma 8999. Location and role in mitochondrial protein degradation.

1. Hepatoma 8999 showed extremely high activity of serine protease, but similar activities of other lysosomal proteases to those of normal rat liver. 2. Serine protease from rat liver formed a single immunoprecipitation band against antiserum to purified protease from hepatoma 8999. 3. The serine proteases in rat liver and hepatoma 8999 were restricted to the inner membranes of the mitochondrial fraction. 4. Polyacrylamide gel electrophoresis with sodium dodecylsulfate showed that hepatoma 8999 mitochondria contained less of the slowest moving protein component than rat liver mitochondrial protein. This component was found to be the best substrate for mitochondrial serine protease in both liver and hepatoma 8999. 5. The role of serine protease in mitochondrial protein degradation is discussed on the basis of these results.     

Characterisation of two serine protease inhibitors expressed in the pituitary gland

Serine protease inhibitors (serpins) are a family of structurally related proteins that play key roles in the regulation of proteolytic homeostasis. We have isolated a novel intracellular serpin, termed raPIT5a, from the rat pituitary gland. Northern blot analysis indicated raPIT5a mRNA expression in a range of tissues, including the adrenal gland and the brain. In situ hybridisation histochemistry revealed raPIT5a mRNA expression in specific cell populations in the rat pituitary gland, adrenal gland, and pancreas. Based on sequence similarities to other intracellular serpins, we predicted raPIT5a may inhibit the pro-apoptotic serine protease granzyme B. We confirmed this experimentally by identification of a stable inhibitory complex between granzyme B and raPIT5a. To determine whether granzyme B or granzyme B-related enzymes were expressed in the rat pituitary gland, we performed PCR using primers predicted to amplify granzyme B and two other published granzyme sequences. We identified rat natural killer protease-1 (RNKP-1), the rat homologue of granzyme B, and a novel putative serine protease highly similar to granzyme-like protein III (GLP III), which we termed GLP IIIa. These data suggest raPIT5a may regulate apoptosis in the pituitary by inhibition of granzyme B or GLP IIIa, or members of the caspase enzyme family which have similar substrate specificity. We have also identified expression of a second serpin, called neuroserpin, in pituitary tissue and found that it alters the morphology of the AtT20 corticotrope cell line, presumably through changes in cell adhesion. These results identify new roles for serpins in pituitary cell function.     

Purification and characterization of prolyl endopeptidase from pig brain

The prolyl endopeptidase from pig brain was purified to homogeneity according to SDS-gel electrophoresis and visualization with the silver staining procedure. The molecular weight of prolyl endopeptidase was estimated as 70 kDa, and the isoelectric point as 4.9. The molecular properties of prolyl endopeptidase from pig brain are therefore similar to those of prolyl endopeptidases from other mammalian tissues. Diisopropylfluorophosphate, diethylpyrocarbonate and p-chloromercuribenzoic acid are strong irreversible inhibitors of prolyl endopeptidase from pig brain. We showed that diisopropylfluorophosphate und diethylpyrocarbonate act as competitive inhibitors with respect to substrate. Therefore it is assumed that at least one serine and one histidine residue are located at the active site of this enzyme. This result supports the assumption that the prolyl endopeptidase from pig brain is a typical serine protease. Substance P, thyreoliberin, beta-casomorphin-5 and morphiceptin are hydrolysed by prolyl endopeptidase in vitro.     

Outer membrane-associated serine protease of intestinal spirochetes

Pathogenic intestinal spirochetes cause damage to the intestinal mucosa of humans and animals by an unknown mechanism. The purpose of this study was to assess the pathogenic intestinal spirochetes Serpulina hyodysenteriae, Serpulina pilosicoli, and Brachyspira aalborgi and the non-pathogenic commensal intestinal spirochetes Serpulina innocens and Treponema succinifaciens for protease activity. A partially heat stable, subtilisin-like, serine protease was identified in the outer membrane of all spirochetes and thus may be essential for survival in the intestinal environment. The outer membrane protease may indirectly contribute to intestinal damage caused by pathogenic spirochetes during association with the mucosal surface of the host.     

Glucose metabolism in the mucosa of the small intestine: Enzymes of the pentose phosphate pathway

1. The occurrence of five enzymes of the pentose phosphate pathway in cell-free preparations of the mucosa of rat small intestine is described. These enzymes were found to be localized mainly in the supernatant fraction (6240000g-min.). 2. The properties of glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were studied with respect to K(m) values for substrates and NADP(+), pH optima and the effects of p-chloromercuribenzoate and palmitoyl-CoA. Higher total and specific activities of these two dehydrogenases were noted in the proximal half of the small intestine of the rat than in the distal half. 3. The specific activities of glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in the mucosa of the small intestine of the rat, cat, rabbit and guinea pig were compared. 4. In the rat the specific activities of ribose 5-phosphate isomerase, transketolase and transaldolase were higher in the supernatant fractions from the intestinal mucosa than in those from the liver. 5. The role of the pentose phosphate pathway is discussed in relation to the metabolism of hexose phosphates in the intestinal mucosa.