Lysosomal hydrolases in calf thyroid.
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ABSTRACT:
SUBMITTER: Herveg JP
PROVIDER: S-EPMC1265169 | biostudies-other | 1966 Aug
REPOSITORIES: biostudies-other
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Project description:1. The following fractions were prepared from rat kidney and characterized ultrastructurally, biochemically and enzymically: (a) an ordinary rough microsomal (RM(1)) fraction; (b) a special rough microsomal (RM(2)) fraction enriched seven- to nine-fold in acid hydrolases over the homogenate; (c) a smooth microsomal (SM) fraction; (d) a Golgi (GM) fraction enriched 2.5-fold in acid hydrolases and 10-, 15- and 20-fold in sialyltransferase, N-acetyl-lactosamine synthetase and galactosyltransferase respectively; (e) a lysosomal (L) fraction enriched 15- to 23-fold in acid hydrolases. The frequency of Golgi sacs and tubules seen in the electron microscope and the specific activity of the three glycosyltransferases in these fractions increased in the order: RM(2)<RM(1)<SM<GM. 2. Five lysosomal hydrolases, acid phosphatase, beta-N-acetyl-hexosaminidase, beta-galactosidase, beta-glucuronidase and arylsulphatase, were characterized in these fractions with respect to (a) solubility on freeze-thawing and (b) electrophoretic mobility in polyacrylamide gels. 3. In the RM(2) fraction each of these hydrolases occurred largely or exclusively as a single bound basic form coincident with cationic glycoprotein bands in gels (Goldstone et al., 1973). 4. In the L fraction these hydrolases were present largely as soluble, acidic (anionic) forms. 5. The solubility, electrophoretic heterogeneity and anodic mobility of these hydrolases increased progressively in subcellular fractions in the order: RM(2)<RM(1)<SM<GM<L. 6. These findings, together with evidence cited in the text showing that N-acetylneuraminic acid residues are responsible for the solubility and electronegative charge of these acidic forms and incorporation of these residues into the Golgi apparatus, support the following scheme for the biosynthesis of lysosomal enzymes. Each hydrolase is synthesized as a bound basic glycoprotein enzyme in a restricted portion of the rough endoplasmic reticulum. The soluble, acidic forms are generated as the nascent glycoprotein enzymes migrate through the Golgi apparatus through the attachment of sugar sequences containing N-acetylneuraminic acid.
| S-EPMC1177585 | biostudies-other
Project description:Investigation of the binding characteristics of acid beta-D-galactosidase, N-acetyl-beta-D-glucosaminidase, alpha-D-galactosidase and alpha-L-fucosidase from patients with mucolipidosis II and mucolipidosis III to concanavalin A--Sepharose 4B revealed a 2--10-fold decrease in the proportion of enzyme activities from patients with mucolipidoses II and III that adsorbed on the lectin. Neuraminidase treatment of the unadsorbed enzyme fraction did not significantly increased the proportion of enzyme activities that bound to the concanavalin A--Sepharose 4B. Characterization of acid beta-D-galactosidase from the adsorbed and unadsorbed enzyme fractions of mucolipidosis II and mucolipidosis III patients demonstrated identical apparent Km values of 0.22 mM with respect to 4-methylumbelliferyl beta-D-galactopyranoside, altered pH--activity profiles and heterogeneous isoelectric-focusing patterns. The results of this study support the suggestion of an alteration of a post-translational modification (possibly glycosylation) occurring in mucolipidosis II and mucolipidosis III common to the lysosomal hydrolases that affects the mannoserelated properties of these enzymes.
| S-EPMC1186389 | biostudies-other
Project description:Cathepsin B from calf liver was obtained by a method involving preparation of a lysosomal-mitochondrial pellet and treatment of this pellet with acetone. The material was extracted with an acid buffer, pH4.0, and then precipitated from the extract with acetone. The precipitate was dissolved in phosphate buffer, pH7.4, and subjected to gel filtration on Sephadex G-200 and G-100. The cathepsin B emerged in a range of molecular weight much lower than 50000 as a well-defined component. The purity of this material was checked by electrophoresis. To obtain maximum activity the enzyme had to be activated with a chelating agent and a reducing agent (i.e. EDTA and cysteine). A number of different substrates were used. The enzyme was active for the hydrolysis of both peptide bonds and ester bonds and had approximately equal reactivity in the two cases. The pH-dependence of the hydrolysis was the same with both substrates. The binding of the substrates was half-maximal at pH4.5 and at pH6.8. A thiol group occurred in the active centre but this group ought to have a much higher pK than that found in this enzyme.
| S-EPMC1184951 | biostudies-other
Project description:1. A partially purified lysosomal preparation was obtained from mouse liver sucrose homogenates by differential and discontinuous gradient centrifugation. 2. Triton X-100 or repeated freezing and thawing of the lysosomal suspension (subfraction B) allowed comparison of free and activated values for acid phosphohydrolase, beta-glucuronidase and N-acetylglucosaminidase in the presence and absence of ascorbate. 3. The distribution of hydrolase activities between supernatant and pellet after high-speed centrifugation was measured and the percentages of total enzyme found in the supernatant were: acid phosphohydrolase, 40.7; beta-glucuronidase, 51; N-acetylglucosaminidase, 39.4. 4. Differential rates of elution of the three hydrolases from the membrane fraction occurred with increasing Na(+) and K(+) concentrations, whereas complex biphasic elution curves were obtained as a function of bivalent cation concentration with Ca(2+) and Mg(2+). 5. Sucrose-density-gradient centrifugation of frozen-and-thawed subfraction B demonstrated highly significant changes in the protein gradient profile in the presence of a low concentration of bivalent cation, indicating membrane aggregation and enzyme-membrane association. 6. The data provide further evidence for the nature of lysosomal enzyme binding and indicate the presence of different enzyme-membrane bonds conferring structure-linked latency upon individual lysosomal enzymes.
| S-EPMC1186764 | biostudies-other
Project description:1. A partially purified lysosomal preparation was obtained from adult mouse livers by sucrose-density-gradient centrifugation of a large-granule fraction. 2. This lysosome-enriched subfraction was contaminated approx. 10% by mitochondrial cytochrome c oxidase and malate dehydrogenase. 3. Free acid phosphohydrolase and beta-glucuronidase contributed less than 10% of the total (Triton X-100-solubilized) activity in contrast with approx. 30% free N-acetyl-beta-d-glucosaminidase when assayed in an iso-osmotic incubation system. 4. Exposure of the lysosomal preparation to inorganic Hg(2+) ions and organic mercurials (p-chloromercuribenzoate, phenylmercuric acetate) induced an irreversible loss of structure-linked latency with resulting enzyme activation. 5. Maximal activation was related to log [Hg(2+)] and pH. The response was all-or-none for individual particles; the dose-response curve portrayed the variation in particle resistance within the lysosomal population. 6. l-Cysteine and GSH totally prevented Hg(2+) ion-induced hydrolase activation. Ascorbate provided approx. 50% protection. 7. The three lysosomal hydrolases were differentially activated at constant [Hg(2+)], suggesting a different pattern of binding, unique for each enzyme studied.
| S-EPMC1198364 | biostudies-other
Project description:1. Cytochalasin B (10mug/ml) enhances the release of rabbit polymorphonuclear leucocyte lysosomal acid hydrolases induced by retinol (vitamin A alcohol). 2. This effect is seen at doses of the vitamin that cause selective release of acid hydrolases and those causing more general enzyme release indicated by the loss of lactate dehydrogenase. 3. Cytochalasin B (2-50mug/ml) has no effect on the release of sedimentable acid hydrolases of intact granules obtained from disrupted polymorphonuclear leucocytes. 4. Cytochalasin B (2-10mug/ml) causes a time- and dose-dependent release of mouse peritoneal macrophage acid hydrolases. 5. This effect is selective at all doses of cytochalasin B used, since no release of lactate dehydrogenase, malate dehydrogenase and leucine 2-naphthylamidase was detected. 6. Treatment with cytochalasin B at doses of up to 10mug/ml for as long as 72h did not significantly change the total activities of any of the enzymes measured. 7. The lack of toxicity of cytochalasin B was shown by dye-exclusion tests and its failure to release radioactive colloidal gold stored in secondary lysosomes.
| S-EPMC1177785 | biostudies-other
Project description:In addition to increased differentiation of vascular smooth muscle cells into osteoblast-like phenotypes, the limited accumulation of osteoclasts in atherosclerotic plaques or their dysfunction may participate in potential mechanisms for vascular calcification. N-acetylglucosamine-1-phosphate transferase containing alpha and beta subunits (GNPTAB) is a transmembrane enzyme complex that mediates the vesicular transport of lysosomal hydrolases. GNPTAB may also regulate the biogenesis of lysosomal hydrolases from bone-marrow derived osteoclasts. In this study, the areas surrounding calcification in human atherosclerotic plaques contained high levels of GNPTAB and low levels of lysosomal hydrolases such as cathepsin K (CTSK) and tartrate-resistant acid phosphatase (TRAP), as demonstrated by immunohistochemistry and laser-capture microdissection-assisted mRNA expression analysis. We therefore hypothesized that GNPTAB secretion may suppress the release of CTSK and TRAP by vascular osteoclast-like cells, thus causing their dysfunction and reducing the resorption of calcification. We used human primary macrophages derived from peripheral blood mononuclear cells, an established osteoclast differentiation model. GNPTAB siRNA silencing accelerated the formation of functional osteoclasts as detected by increased secretion of CTSK and TRAP and increased their bone resorption activity as gauged by resorption pits assay. We concluded that high levels of GNPTAB inhibit secretion of lysosomal hydrolases in dysfunctional osteoclasts, thereby affecting their resorption potential in cardiovascular calcification.
| S-EPMC5753093 | biostudies-literature
Project description:The gastrocnemius, a fast-twitch white muscle, and the soleus, a slow-twitch red muscle, were studied in A/J mice. The specific activities of the lysosomal hydrolases, beta-D-glucuronidase, hexosaminidase, beta-D-galactosidase and arylsulphatase, the inner-mitochondrial-membrane enzyme cytochrome c oxidase, and the outer-mitochondrial-membrane enzyme monoamine oxidase, were greater in the soleus than in the gastrocnemius. The specific activities of the lysosomal hydrolases and cytochrome c oxidase in the gastrocnemius and soleus were substantially higher in male mice than in female mice. Orchiectomy abolished this sex difference. Testosterone increased the activities of the lysosomal hydrolases and cytochrome c oxidase and coincidentally induced muscle hypertrophy and an accretion of protein and RNA, but total DNA remained constant. Monoamine oxidase was unaffected by sex, orchiectomy and testosterone. These findings indicate that endogenous androgens regulate the activity of enzymes associated with lysosomes and the inner mitochondrial membrane, as well as muscle fibre growth in mouse skeletal muscle.
| S-EPMC1162340 | biostudies-other
Project description:ObjectivesLysosomal acid lipase (LAL) is the key enzyme, which degrades neutral lipids at an acidic pH in lysosomes. The role of LAL in various cellular processes has mostly been studied in LAL-knockout mice, which share phenotypical characteristics with humans suffering from LAL deficiency. In vitro, the cell-specific functions of LAL have been commonly investigated by using the LAL inhibitors Lalistat-1 and Lalistat-2.MethodsWe performed lipid hydrolase activity assays and serine hydrolase-specific activity-based labeling combined with quantitative proteomics to investigate potential off-target effects of Lalistat-1 and -2.ResultsPharmacological LAL inhibition but not genetic loss of LAL impairs isoproterenol-stimulated lipolysis as well as neutral triglyceride and cholesteryl ester hydrolase activities. Apart from LAL, Lalistat-1 and -2 also inhibit major cytosolic lipid hydrolases responsible for lipid degradation in primary cells at neutral pH through off-target effects. Their binding to the active center of the enzymes leads to a decrease in neutral lipid hydrolase activities in cells overexpressing the respective enzymes.ConclusionsOur findings are critically important since they demonstrate that commonly used concentrations of these inhibitors are not suitable to investigate the role of LAL-specific lipolysis in lysosomal function, signaling pathways, and autophagy. The interpretation of their effects on lipid metabolism should be taken with caution and the applied inhibitor concentrations in cell culture studies should not exceed 1 μM.
| S-EPMC9118473 | biostudies-literature
Project description:The abundant proteoglycan, aggrecan, is resorbed from growth plate cartilage during endochondral bone ossification, yet mice with genetically-ablated aggrecan-degrading activity have no defects in bone formation. To account for this apparent anomaly, we propose that lysosomal hydrolases degrade extracellular, hyaluronan-bound aggrecan aggregates in growth plate cartilage, and that lysosomal hydrolases are released from hypertrophic chondrocytes into growth plate cartilage via Ca(2+)-dependent lysosomal exocytosis. In this study we confirm that hypertrophic chondrocytes release hydrolases via lysosomal exocytosis in vitro and we show in vivo evidence for lysosomal exocytosis in hypertrophic chondrocytes during skeletal development. We show that lysosome-associated membrane protein 1 (LAMP1) is detected at the cell surface following in vitro treatment of epiphyseal chondrocytes with the calcium ionophore, ionomycin. Furthermore, we show that in addition to the lysosomal exocytosis markers, cathepsin D and β-hexosaminidase, ionomycin induces release of aggrecan- and hyaluronan-degrading activity from cultured epiphyseal chondrocytes. We identify VAMP-8 and VAMP7 as v-SNARE proteins with potential roles in lysosomal exocytosis in hypertrophic chondrocytes, based on their colocalisation with LAMP1 at the cell surface in secondary ossification centers in mouse tibiae. We propose that resorbing growth plate cartilage involves release of destructive hydrolases from hypertrophic chondrocytes, via lysosomal exocytosis.
| S-EPMC3509456 | biostudies-literature