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1.
We studied the effect of parenteral morphine and naloxone administration on intestinal mucosal Prostaglandin E2 (PGE2) and 3',5' cyclic adenosine monophosphate (cAMP) levels and on indomethacin-induced intestinal ulceration in the rat. Compared to the control group, morphine significantly decreased whereas naloxone markedly increased both PGE2 and cAMP mucosal levels, respectively. Morphine or naloxone alone did not cause mucosal injury. However, when given with indomethacin, morphine significantly potentiated the ulcerogenic effect of indomethacin while naloxone exerted a protective effect. These results suggest that opioid peptides may play a role in modulation of intestinal mucosal PGE2 and cAMP levels. In addition, enhancement of indomethacin-induced ulcer formation by morphine and amelioration by naloxone might be in part mediated through their effect on mucosal PGE2 and cAMP levels. 相似文献
2.
Relations between plasma membrane and lysosomal membrane. 2. Quantitative evaluation of plasma membrane marker enzymes in the lysosomes 总被引:4,自引:0,他引:4
We have quantified, in cultured rat fibroblasts, the association to the lysosomal membrane of two classical plasma membrane markers, 5'-nucleotidase and alkaline phosphodiesterase I. To isolate highly purified lysosomal preparations, lysosomes were loaded with horseradish peroxidase (2-h cell uptake, 16-h chase) and isolated by isopycnic centrifugation in linear Percoll gradients, followed by a 3,3'-diaminobenzidine-induced density shift in sucrose gradients. Purified lysosomal preparations contained up to 50% of N-acetyl-beta-glucosaminidase of the homogenate. This lysosomal enzyme was enriched 33-fold in the most purified preparations. In the electron microscope, these preparations appeared to be highly purified and only contained organelles filled with diaminobenzidine reaction products. Analysis of purified preparations indicates that 0.5-0.8% of 5'-nucleotidase, but as much as 10.9-14.3% of alkaline phosphodiesterase I activities of the homogenate, are associated with lysosomes. After freezing-thawing, these activities remained essentially membrane-associated. The larger value obtained for alkaline phosphodiesterase I could not be ascribed to other lysosomal enzymes, as no such activity was detected at acidic pH. These two plasma membrane markers are thus unevenly distributed in the lysosomal compartment. 相似文献
3.
The effect of indomethacin, a non-steroidal anti-inflammatory drug upon purified calpain has been studied. Also, its effects upon Ca2+-mediated degradation of cytoskeletal proteins (neurofilament) in spinal cord homogenate has been investigated. A dose-dependent inhibition of purified calpain activity was observed. A 50% inhibition of 14C-caseinolytic activity was obtained with less than 1.1 mM of indomethacin while the activity was completely inhibited at 3.3 mM concentration. The inhibitory effect of ketorlac, another non-steroidal anti-inflammatory drug, upon calpain was weaker than that of indomethacin. The degradation of myelin basic protein (MBP) by cathepsin B, a lysosomal cysteine protease, was significantly inhibited by indomethacin. It also inhibited the Ca2+-mediated degradation of neurofilament protein (NFP) in spinal cord homogenate. The extent of NFP degradation was analyzed by SDS-PAGE and the inhibition shown by indomethacin was weaker than that observed with leupeptin and the calpain inhibitor E64-d. The inhibitory effect of indomethacin on the activity of multicatalytic proteinase complex was negligible. These results suggest that indomethacin, a non-steroidal anti-inflammatory drug and cyclooxygenase inhibitor also inhibits proteinases, including cathepsin B and calpain. 相似文献
4.
The isolated myenteric plexus-longitudinal muscle preparation from guinea pigs which had previously been administered morphine chronically 100 mg/kg/day, 7 days) shows a pronounced contraction when exposed to naloxone. Exposure of the preparation to indomethacin (6.7 micrograms/ml) for 10 minutes prior to naloxone inhibits this withdrawal response. A single dose of indomethacin, 1 mg/kg, given on the 6th day on morphine, also inhibits the naloxone contraction. These results suggest the involvement of a prostaglandin in the opiate withdrawal response in the ileum. 相似文献
5.
Normal rat liver lysosomal membranes in the form of membrane vesicles have been purified using Percoll density gradient centrifugation. Lysosomes (density = 1.111) were purified approximately 63 +/- 12-fold (mean +/- standard deviation, n = 5) using a gradient of Percoll made isotonic with sucrose and buffered to pH 7.0. These lysosomes were then exposed to 10 mM methionine methyl ester, pH 7.0, the uptake of which resulted in swelling and breakage of the lysosomes with subsequent vesicle formation. These vesicles (density = 1.056) were further separated from residual mitochondrial and plasma membrane enzyme activities using a second Percoll density gradient. Marker enzyme analysis and electron microscopy indicated that the lysosomal membranes were essentially free of both beta-hexosaminidase, a soluble lysosomal enzyme, and contaminating organelles. The specific activity of lysosomal ATPase in the lysosomal membranes was fourfold greater than in the intact lysosomes. 相似文献
6.
《Journal of Physiology》1997,91(3-5):189-197
There exists a considerable controversy in the literature with regard to the effect of either opiate receptor blockade or that of morphine in different gastric and intestinal ulcer models in the rat. We performed experiments to evaluate the effects of naloxone and morphine on gastric acid secretion and gastric mucosal damage in different experimental models of gastric mucosal injury, namely in indomethacin-, HCl (0.6N)- and ethanol (96%)-models. We found that: 1) 10 mg/kg naloxone ip given twice, effectively protected gastric mucosa against indomethacin (30 mg/kg ip) and against the acid-dependent injury caused by 0.6 N HCl (1 mL ig), but not against the non acid-dependent injury caused by 96% ethanol (1 mL ig); 2) morphine (10 + 10 mg/kg ip) increased ulcers in the HCl-model, but had no effect in the two other models; 3) this ulcer-aggravating effect of morphine in the HCl-model was blocked by pretreatment of 2 mg/kg ip naloxone; and 4) both naloxone (5 + 5 and 10 + 10 mg/kg ip) significantly decreased gastric acid secretion in 1-h pylorus ligated rats. We conclude that: 1) naloxone dose-dependently protects against the indomethacin- and HCl-, but not against the ethanol-induced gastric mucosal damage; 2) morphine aggravates the HCl-induced ulcerogenesis; and 3) both opiod receptor agonist and antagonist decrease gastric acid secretion. 相似文献
7.
An enzyme hydrolyzing flavin-adenine dinucleotide (FAD) to flavin mononucleotide and AMP was identified and purified from rat liver lysosomal (Tritosomal) membranes. The purified enzyme showed a single band on silver-stained denaturing gels with an apparent Mr 70,000. Periodate-Schiff staining after denaturing gel electrophoresis of whole membrane preparations revealed that this enzyme is one of the major glycoproteins in lysosomal membranes. FAD appeared to be the preferred substrate for the purified enzyme; equivalent concentrations of NAD or CoA were hydrolyzed at about one-half of the FAD rate. Negligible activity (less than or equal to 16%) was noted with ATP, TTP, ADP, AMP, FMN, pyrophosphate, or p-nitrophenylphosphate. The enzyme was inhibited by EDTA or dithiothreitol. It was stimulated by Zn, and was not affected by Ca or Mg ions, nor by p-chloromercuribenzoate. The pH optimum for FAD hydrolysis was 8.5-9 with an apparent Km of 0.125 mM. Antibodies prepared against the purified enzyme partially (50%) inhibited FAD phosphohydrolase activity in lysosomal membrane preparations but had no effect on the soluble lysosomal acid pyrophosphatase known to hydrolyze FAD. This enzyme could not be detected immunochemically in preparations of microsomes, Golgi, plasma membranes, mitochondrial membranes, or the soluble lysosomal fraction, suggesting that the enzyme is different from either soluble lysosomal acid pyrophosphatase or other FAD hydrolyzing activities in the liver cell. 相似文献
8.
Intracellular localization and enzymatic activities of lysosomal enzymes (cathepsin B, N-acetyl-beta-glucosaminidase, and beta-glucuronidase) were studied in control rats and after induction of caerulein pancreatitis. In control rats high enzymatic activities were found in the postnuclear 1000 g fraction (purified zymogen granules). The corresponding subcellular fraction in pancreatitis animals additionally contained larger secretory vacuoles and autophagosomes and revealed a marked increase in lysosomal enzyme activities. Immunolabelling studies at the ultrastructural level for trypsinogen and cathepsin B demonstrated a colocalization of lysosomal and digestive enzymes in zymogen granules in healthy controls. After induction of pancreatitis immunolabelling still demonstrated a colocalisation of cathepsin B and trypsinogen in secretory granules and newly formed Golgi-derived secretory vacuoles. Concomitantly appearing autophagosomes were, however, only labelled for cathepsin B. It is concluded that segregation of lysosomal and digestive enzymes is incomplete in normal acinar cells resulting in a colocalization in zymogen granules. In pancreatitis colocalization in secretory granules is maintained, whereas only lysosomal enzymes were sufficiently transferred into autophagic vacuoles. No indication for impaired mechanisms of molecular sorting of lysosomal and digestive enzymes in caerulein-induced pancreatitis was found. 相似文献
9.
Purification and immunological quantification of rat liver lysosomal glycosidases. 总被引:1,自引:0,他引:1
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Although lysosomal enzyme activities are known to vary in response to numerous physiological and pharmacological stimuli, the relationship between lysosomal enzyme activity and enzyme concentration has not been systematically studied. Therefore we developed radioimmunoassays for two lysosomal glycosidases in order to determine lysosomal enzyme concentration. beta-Galactosidase and beta-glucuronidase were purified from rat liver 2780-fold and 1280-fold respectively, by using differential centrifugation, affinity chromatography, ion-exchange chromatography and molecular-sieve chromatography. Polyclonal antibodies to these enzymes were raised in rabbits, and two radioimmunoassays were established. Antibody specificity was shown by: (i) selective immunoprecipitation of enzyme activity; (ii) identical bands of purified enzyme on SDS/polyacrylamide-gel electrophoresis and immunoelectrophoresis; (iii) single immunoreactive peaks in molecular-sieve chromatography experiments. Sensitivities of the assays were such that 15 ng of beta-galactosidase and 45 ng of beta-glucuronidase decreased the ratio of bound to free radiolabel by 50%; minimal detectable amounts of immunoreactive enzymes were 2 ng and 10 ng respectively. The assays were initially used to assess the effects of physiological perturbations (i.e. fasting and age) on enzyme concentrations in rat liver; these experiments showed that changes in enzyme concentrations do not always correlate with changes in enzyme activities. This represents the first report of radioimmunoassays for lysosomal glycosidases. The results suggest that these radioimmunoassays provide useful technology for the study of regulatory control mechanisms of the concentrations of lysosomal glycosidases in mammalian tissues. 相似文献
10.
Summary Intracellular localization and enzymatic activities of lysosomal enzymes (cathepsin B,N-acetyl-β-glucosaminidase, and β-glucuronidase) were studied in control rats and after induction of caerulein pancreatitis.
In control rats high enzymatic activities were found in the postnuclear 1000g fraction (purified zymogen granules). The corresponding subcellular fraction in pancreatitis animals additionally contained
larger secretory vacuoles and autophagosomes and revealed a marked increase in lysosomal enzyme activities. Immunolabelling
studies at the ultrastructural level for trypsinogen and cathepsin B demonstrated a colocalization of lysosomal and digestive
enzymes in zymogen granules in healthy controls. After induction of pancreatitis immunolabelling still demonstrated a colocalisation
of cathepsin B and trypsinogen in secretory granules and newly formed Golgi-derived secretory vacuoles. Concomitantly appearing
autophagosomes were, however, only labelled for cathepsin B. It is concluded that segregation of lysosomal and digestive enzymes
is incomplete in normal acinar cells resulting in a colocalization in zymogen granules. In pancreatitis colocalization in
secretory granules is maintained, whereas only lysosomal enzymes were sufficiently transferred into autophagic vacuoles. No
indication for impaired mechanisms of molecular sorting of lysosomal and digestive enzymes in caerulein-induced pancreatitis
was found. 相似文献
11.
Lysosomal enzymes possess a common antigenic determinant in the cellular slime mold, Dictyostelium discoideum 总被引:6,自引:0,他引:6
Antisera have been prepared against two lysosomal enzymes of the cellular slime mold, Dictyostelium discoideum. The two purified enzyme preparations used for immunization, N-acetylglucosaminidase and beta-glucosidase-1, show no cross-contamination with each other and no significant contamination by other lysosomal enzymes. However, antisera raised against either enzyme bind equally well to seven different lysosomal enzymes and show no preference for the enzyme against which they were raised. A total of 10 different antisera have been examined and all show similar results. Preadsorption of antisera with either purified enzyme removes all antibody activity against the other enzyme. Evidence is presented which indicates that the same species of antibodies are responsible for the precipitation of seven lysosomal enzymes. These data are discussed in terms of the proposal that the antigen that is shared by the lysosomal enzymes is a post-translational modification of the enzyme proteins. We have sought to further characterize the distribution of this common antigen among cellular proteins. We show that N-acetylglucosaminidase and beta-glucosidase-1 represent less than 5% of the total common antigen containing proteins in the cell. Precipitation of 35S-labeled cellular proteins from vegetative cells indicates that as much as 15-30% of the total cell protein may possess the common antigen. Preadsorption experiments confirm that all of the proteins immunoprecipitated in these experiments are recognized by the same antibodies that precipitate the lysosomal enzyme activities. Most of the labeled proteins are secreted into the medium along with the lysosomal enzyme activities during axenic growth. During the developmental phase of the life cycle of Dictyostelium, the total amount of the common antigen decreases about 2-fold relative to total cell protein. However, the synthesis of antigenic proteins continues throughout most of development. 相似文献
12.
Mitochondrial and Peroxisomal β-Oxidation of Stearic and Lignoceric Acids by Rat Brain 总被引:1,自引:1,他引:0
Crude subcellular fractions were prepared from adult rat brains by differential centrifugation of brain homogenates. Greater than 98% of the cellular mitochondrial marker enzyme activity sedimented in the heavy and light mitochondrial pellets, and less than 1% of the activity sedimented in microsomal pellets. Lysosomal marker enzyme activities mainly (71-78% of cellular activity) sedimented in the heavy and light mitochondrial pellets. Significant amounts of the lysosomal marker enzyme activity also sedimented in the crude microsomal pellets (9-13% of total) and high-speed supernatants (14-16% of total). The specific activities of microsomal and peroxisomal marker enzyme activities were highest in the crude microsomal pellets. Fractionation of the crude microsomal pellets on Nycodenz gradients resulted in the separation of the bulk of the remaining mitochondrial, lysosomal, and microsomal enzyme activities from peroxisomes. Fatty acyl-CoA synthetase activities separated on Nycodenz gradients as two distinct peaks, and the minor peak of the activities was in the peroxisomal enriched fraction. Fatty acid beta-oxidation activities also separated as two distinct peaks, and the activities were highest in the peroxisomal enriched fractions. Mitochondria were purified from the heavy mitochondrial pellets by Percoll density gradients. Fatty acyl-CoA synthetase and fatty acid beta-oxidation activities were present in both the purified mitochondrial and peroxisomal enriched fractions. Stearoyl-CoA synthetase activities were severalfold greater compared to lignoceroyl-CoA synthetase, and stearic acid beta-oxidation was severalfold greater compared to lignoceric acid beta-oxidation in purified mitochondrial and peroxisomal enriched fractions.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
13.
Purification and characterization of human lysosomes from EB-virus transformed lymphoblasts 总被引:8,自引:0,他引:8
Erik Harms Jürgen Kartenbeck Gholamreza Darai Jerry Schneider 《Experimental cell research》1981,131(2):251-266
A method was developed for the isolation of unmodified lysosomes of human origin using cultured EB-virus transformed lymphoblasts. The cells were lysed carefully by repeated resuspension in buffered isotonic sucrose. A crude granular fraction derived from this lysate was further purified by isopyknic centrifugation in an isotonic colloidal silica gel gradient and by free-flow electrophoresis. The following relative specific activities (mean ± S.D.) of lysosomal marker enzymes were measured in a pooled lysosomal fraction obtained from the final electrophoresis step (representing less than 0.1% of the initial protein): β-N-acetylglucosaminidase 85.6 ± 15.5; β-galactosidase 87.6 ± 13.4; acid β-glycerophosphatase 41.7 ± 3.5; β-glucuronidase 36.6 ± 6.1. With respect to the final two enzymes the recovery within this pooled fraction was 5–6% of the initial lysate. The great differences in relative specific activities achievable may be due mainly to different extralysosomal portions of the lysosomal marker enzymes, as was found for acid β-glycerophosphatase which was largely distributed within non-lysosomal structures in lymphoblasts when studied by histochemical staining. The final fraction consisted almost exclusively of lysosomes when examined by electron microscopy. Most lysosomes appeared club-shaped immediately after cell lysis and throughout the preparation procedure. Examination by electron microscopy and measurement of the latency of lysosomal enzyme activity revealed an exceptional integrity of the lysosomal membrane. This method provides the opportunity to study highly purified lysosomes from patients with lysosomal disorders. 相似文献
14.
1. Intracellular distribution of three types of beta-galactosidases (beta-D-galactoside galactohydrolase EC 3.2.1.12) i.e. hetero beta-galactosidase, lactase and acid beta-galactosidase, was studied by examining the properties of subcellular fractions isolated by a systematic fractionation of mucosal cells of the small intestine of the hog. Localization of hetero beta-galactosidase in cytosol could be shown. 2. Localization of lactase in the brush borders was shown by analyzing the purified brush borders prepared separately. 3. To domonstrate the lysosomal localization of acid belta-galactosidase, lysosomes were purified separately and their extract was chromatographed on a hydroxylapatite column. The activities of various enzymes in the purified lysosomes as well as in the intermediary fractions obtained during lysosome purification and the pattern of the hydroxylapatite chromatography led us to conclude that acid beta-galactosidase is a lysosomal enzyme. 相似文献
15.
Purification and properties of major alpha-D-mannosidase in the luminal fluid of porcine epididymis.
Y Z Jin F Dacheux J L Dacheux S Bannai Y Sugita N Okamura 《Biochimica et biophysica acta》1999,1432(2):382-392
A lysosomal type alpha-D-mannosidase was successfully purified by DEAE-Sephacel, Red-Amicon and Superdex 200 column chromatographies from porcine cauda epididymal fluid. The purified enzyme consisted of 63 and 51 kDa subunits at equimolar amounts. It cleaved alpha1-2 linked mannosyl residues and less but significantly cleaved alpha1-3 and alpha1-6 linked mannosyl residues in the high-mannose oligosaccharides. The optimal pH to hydrolyze oligosaccharide was in the acidic pH range (pH 3.5 approximately 4.0).Total alpha-D-mannosidase activities in the porcine epididymal fluid increased from proximal to distal caput epididymis, which maintained to cauda epididymis. At least two kinds of alpha-D-mannosidase (lysosomal type enzyme and 135 kDa alpha-D-mannosidase (MAN2B2)) were contained in the porcine epididymal fluid. The activity of the lysosomal type enzyme is much higher than MAN2B2 at the physiological pH.These results suggest that the lysosomal type alpha-D-mannosidase is the predominantly active enzyme in the luminal fluid of porcine epididymis and that it participates in the glycoprotein modification on the sperm surface during epididymal transit. 相似文献
16.
《Comp. Biochem. Physiol. C, Comp. Pharmacol. Toxicol.》1994,107(3):423-428
Copper deficiency in rats resulted in a decrease of liver cytosolic and lysosomal CuZnSOD activity (by 71% and by 55%, respectively) and in an increase of mitochondrial MnSOD (by 185%). The content of copper and zinc decreased by 64% and 38%, respectively, and that of manganese increased by 47%. Cytosolic CuZnSOD activity, both in control and copper-deficient rats, increased (by 71.5 units/mg protein and by 83.0 units/mg protein, respectively) after indomethacin treatment. Rat liver contained four CuZnSOD (cytosolic and lysosomal) and one MnSOD isoenzyme. Neither copper deficiency nor indomethacin treatment changed the position and the number of bands. The CuZnSOD-band with pI ∼ 5.7 was greatly decreased by copper deficiency and was not restored to normal after indomethacin treatment. 相似文献
17.
In vertebrates, mannose 6-phosphate receptors [MPR300 (Mr 300 kDa) and MPR46 (Mr 46 kDa)] are highly conserved transmembrane glycoproteins that mediate transport of lysosomal enzymes to lysosomes. Our studies have revealed the appearance of these putative receptors in invertebrates such as the molluscs and deuterostomes. Starfish tissue extracts contain several lysosomal enzyme activities and here we describe the affinity purification of α-fucosidase. The purified enzyme is a glycoprotein that exhibited a molecular mass of ∼56 kDa in SDS-PAGE under reducing conditions. It has also cross-reacted with an antiserum to the mollusc enzyme suggesting antigenic similarities among the two invertebrate enzymes. LC–MS/MS analysis of the proteolytic peptides of the purified enzyme in combination with de novo sequencing allowed us to do partial amino acid sequence determination of the enzyme. These data suggest that this invertebrate enzyme is homologous to the known mammalian enzyme. The purified enzyme exhibited a mannose 6-phosphate dependent interaction with the immobilized starfish MPR300 protein. Our results demonstrate that the lysosomal enzyme targeting pathway is conserved even among the invertebrates. 相似文献
18.
Heterogeneity of lysosomes originating from rat liver parenchymal cells. Metabolic relationship of subpopulations separated by density-gradient centrifugation. 总被引:6,自引:1,他引:5
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1. A crude lysosomal fraction obtained by differential centrifugation of a rat liver homogenate was subjected to zonal centrifugation in iso-osmotic self-generating gradients composed of modified colloidal silica (Percoll). Analysis of relevant marker-enzyme activities shows a continuous band of considerably purified lysosomal particles in the density range 1.04--1.11 g/ml. 2. A relationship between age and buoyant density of the parenchymal lysosomal subpopulations is indicated by the distribution of 125I-labelled asialoglycoproteins in the heterogeneous lysosomes during the catabolism of the glycoprotein. The labelled asialoglycoprotein first appeared in lysosomal particles of low density, which with time progressively acquired a higher density. Furthermore, 30 min after administration the 125I-labelled asialocaeruloplasmin recovered in the light lysosomes was less degraded than the material recovered in the heavy lysosomes. 3. A lysosomal enzyme (arylsulphatase) was found to possess considerably higher isoelectric points in the heavy lysosomes than in the light lysosomes, which is consistent with a relationship between age and density of the lysosomes. 相似文献
19.
Identification of latent procathepsins B and L in microsomal lumen: characterization of enzymatic activation and proteolytic processing in vitro 总被引:7,自引:0,他引:7
Procathepsins B and L in the hepatic endoplasmic lumen were identified as having a molecular weight of 39,000 by immunoblot analysis. The proenzymes were then purified to remove the mature enzymes by concanavalin A-Sepharose chromatography. The concanavalin A-adsorbed fractions containing the proenzymes showed no appreciable activities of cathepsins B and L. When those fractions were incubated at pH 3.0, the enzymatic activities markedly increased: the activities of cathepsins B and L after 36 h incubation were 60 and 210 times those of the controls, respectively. Immunoblot analysis showed that after 36 h incubation the proenzymes disappeared and the mature enzymes increased. Thus the proenzymes were processed to the mature enzymes under acidic conditions of pH 3.0. The marked increases of enzymatic activities and the conversion of the proenzymes to the mature forms were completely blocked with pepstatin, which is a potent inhibitor of aspartic proteases. The results strongly suggested that a processing protease for procathepsins B and L might be cathepsin D, a major lysosomal aspartic protease. Indeed, lysosomal cathepsin D could convert microsomal procathepsin B to the mature enzyme in vitro. Therefore, procathepsins B and L seem first to be synthesized as enzymatically inactive forms in endoplasmic reticulum and successively may be converted into active forms by cathepsin D in lysosomal compartments. 相似文献