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PubMed1989

Lysosomal hydrolases of human vascular cells: response to agonists of endothelial function.

Endothelial injury has been proposed as a feature of a wide variety of vascular diseases, and release of endothelial lysosomal hydrolases could contribute to the pathological changes seen. We have determined the relative activities of 14 glycosidases, two esterases and four peptide hydrolases in human umbilical vein endothelial cells and investigated whether known agonists of endothelial function, or materials known to modulate hydrolase secretion in other phagocytic cells, influenced the activity or secretion of these enzymes by human umbilical vein endothelial cells. Hexosaminidase, beta-galactosidase, beta-glucuronidase and alpha-iduronidase accounted for most of the measured glycosidase activity. Acid phosphatase activity greatly exceeded arylsulphatase activity, and most of the measured peptidase activity was due to acid peptidases. Optimum pH and apparent Km values were determined for the most abundant hydrolases. Exposure of human umbilical vein endothelial cells to bradykinin, thrombin or interleukin-1 resulted in negligible release of either hexosaminidase or lactate dehydrogenase (LDH), in contrast to phorbol myristate acetate, which caused a parallel, dose-dependent release of both enzymes. Treatment of these cells with calcium ionophore A23187, trypsin or platelet-activating factor, caused less than 10% release of either hexosaminidase or LDH. Agents known to modulate lysosomal enzyme secretion by other phagocytic cells failed to induce selective secretion of lysosomal enzymes by human umbilical vein endothelial cells.

Bradykinin↗
PubMed1988

N-benzoyl-L-tyrosyl-p-aminobenzoic acid hydrolase: a metalloendopeptidase of the human intestinal microvillus membrane which degrades biologically active peptides.

Particulate fractions of human small intestinal mucosa contain an enzyme capable of hydrolyzing N-benzoyl-L-tyrosyl-p-aminobenzoic acid (PABA-peptide), a substrate used for clinical purposes to assess exocrine function of the pancreas (PABA test, pancreas function test). In this paper we describe the purification of PABA-peptide hydrolase (PPH) by immunoaffinity chromatography using a monoclonal antibody (Mab), HBB 3/716/36, bound to protein A-Sepharose, and the characterization of the purified enzyme. The final preparation of the enzyme was in the immobilized form, i.e., bound to Mab-protein A-Sepharose, and showed a 765-fold enrichment over the mucosal homogenate. The enrichment factor in purified microvillus membranes was comparable to that of sucrase-isomaltase, a microvillar marker enzyme. This, together with immunoelectron microscopy using protein A-gold, indicated that PPH is located in the apical membrane of intestinal epithelial cells. The enzyme was found to be present throughout the small intestine with the activity in distal ileum being 4.5-fold higher than that in the proximal duodenum. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the immunoaffinity-purified PPH under reducing conditions revealed a polypeptide band with a relative molecular weight (Mr) of 100,000; under nonreducing conditions a major band with Mr 200,000 was observed. This indicates that PPH consists of two subunits with Mr 100,000 each, which are held together by one or more disulfide bonds. Two-dimensional polyacrylamide gel electrophoresis of the enzyme showed marked microheterogeneity, with pI's ranging from 6.0 to 6.85, probably due to glycosylation. The Km for PABA-peptide was 16.7 mM, and the pH optimum was 7.5-8.0 PPH activity was not inhibited by phenylmethylsulfonyl fluoride; pepstatin, leupeptin, amastatin, bestatin, puromycin, iodoacetate, or phosphoramidon. Activity was affected by captopril and Zinkov inhibitor, and in particular by thiol and chelating reagents. Chelator-inhibited PPH could be reactivated by bivalent metal ions, Zn2+ being the most effective. The enzyme catalyzed the hydrolysis of peptides including insulin B-chain, angiotensins I and II, bradykinin and bradykinin derivatives, oxytocin, and substance P, in each case yielding reproducible peptide fragments. On the basis of amino acid analysis of the products it could be concluded that peptides are hydrolyzed preferentially after an aromatic residue.

4-Aminobenzoic Acid↗
PubMed1985

Synaptosomal degradation of substance P and some other neuropeptides.

Synaptosomes purified from spinal cord and from different rat brain areas exhibit peptide hydrolase activity, cleaving substance P (SP), bradykinin, THRH, LHRH, and neurotensin. The lowest activity for all the peptides tested was found in spinal cord, while the region with the highest degrading activity depended on the substrate: for substance P, it was striatum and cortex; for bradykinin, hypothalamus, and medulla oblongata; for THRH, striatum; for LHRH, midbrain; and for neurotensin, hippocampus. Degradation of substance P takes place at the plasma membrane of synaptosomes. Synaptosome ghosts cleave substance P (pH optimum 7-9, Km-2.5 X 10(-5) M, Vmax-130 nmol . hr-1 . mg protein-1) and also a number of its C-terminal fragments. Effects of the inhibitors show that several different classes of peptidases and proteases are involved in the degradation process. Peptide cleavage represents the probable pathway of synaptosomal inactivation of substance P.

Animals↗
PubMed1985

Expression and intracellular transport of microvillus membrane hydrolases in human intestinal epithelial cells.

A panel of monoclonal antibodies was produced against purified microvillus membranes of human small intestinal enterocytes. By means of these probes three disaccharidases (sucrase-isomaltase, lactase-phlorizin hydrolase, and maltase-glucoamylase) and four peptidases (aminopeptidase N, dipeptidylpeptidase IV, angiotension I-converting enzyme, and p-aminobenzoic acid peptide hydrolase) were successfully identified as individual entities by SDS PAGE and localized in the microvillus border of the enterocytes by immunofluorescence microscopy. The antibodies were used to study the expression of small intestinal hydrolases in the colonic adenocarcinoma cell line Caco 2. This cell line was found to express sucrase-isomaltase, lactase-phlorizin hydrolase, aminopeptidase N, and dipeptidylpeptidase IV, but not the other three enzymes. Pulse-chase studies with [35S]methionine and analysis by subunit-specific monoclonal antibodies revealed that sucrase-isomaltase was synthesized and persisted as a single-chain protein comprising both subunits. Similarly, lactase-phlorizin hydrolase was synthesized as a large precursor about twice the size of the lactase subunits found in the human intestine. Aminopeptidase N and dipeptidylpeptidase IV, known to be dimeric enzymes in most mammals, were synthesized as monomers. Transport from the rough endoplasmic reticulum to the trans-Golgi apparatus was considerably faster for the peptidases than for the disaccharidases, as probed by endoglycosidase H sensitivity. These results suggest that the major disaccharidases share a common biosynthetic mechanism that differs from that for peptidases. Furthermore, the data indicate that the transport of microvillus membrane proteins to and through the Golgi apparatus is a selective process that may be mediated by transport receptors.

Antibodies, Monoclonal↗
PubMed1998

Peptidase activities of the multicatalytic protease in rat liver after voluntary and intragastric ethanol administration.

Ethanol consumption slows down the rate of hepatic protein catabolism. The present study was conducted to determine whether ethanol consumption, given by voluntary (pair) feeding or by intragastric administration, affected the peptidase activities of the proteasome in rat liver. Rats were pair-fed liquid diets containing either ethanol or isocaloric maltose-dextrin. A separate group of animals was intragastrically infused continuously with similar liquid diets containing either ethanol or isocaloric dextrose. Crude liver homogenates and their cytosolic fractions were assayed for their chymotrypsin-like (Cht-L), trypsin-like (T-L), and peptidyl-glutamyl-peptide hydrolase (PGPH) activities, using specific fluorogenic peptides as substrates. Voluntary ethanol feeding did not affect the three peptidase activities of the proteasome. However, intragastric ethanol administration caused a 35% to 40% decline in the Cht-L and the T-L activities, but did not significantly change the PGPH activity. The lower peptidase activities in cytosol samples from intragastrically ethanol-fed rats were not restored to control levels by overnight dialysis, nor by the inclusion of low levels of sodium dodecyl sulfate (SDS) or of 0.5 mmol/L adenosine triphosphate (ATP) in the proteasome assay mixture. Immunoblot analyses using anti-rat liver proteaseome exhibited equal levels of immunoreactive proteasome subunits in livers of control and ethanol-fed rats. Similar results were obtained when blots were probed with antibody made specifically against the proteasome subunit, LMP-7. The results indicate that intragastric, but not voluntary, ethanol consumption differentially affects the separate catalytic activities of the proteasome without affecting its steady-state levels. Such changes may be related to the degree of ethanol-induced oxidative stress.

Animals↗