Purification and characterization of microsomal and lysosomal beta-glucuronidase from rat liver by use of immunoaffinity chromatography.

Rat liver beta-glucuronidase (EC 3.2.1.31), both from microsomal and lysosomal fractions, were purified about 9500-fold over the homogenate with high yield using affinity chromatography prepared by coupling purified specific immunoglobulin G against rat preputial gland beta-glucuronidase to Sepharose 2B and isoelectric focusing. The purified enzymes appeared homogeneous on electrophoresis in polyacrylamide gel and had a molecular weight of approximately 310000. In dodecylsulfate polyacrylamide gel electrophoresis, the microsomal beta-glucuronidase showed a single band corresponding to a molecular weight of 79000, while the lysosomal beta-glucuronidase had three distinct bands which consisted of one major and two minor bands corresponding to molecular weight of 79000, 74000, and 70000, respectively. A broad pH activity curve with a single optimum at pH 4.4 was observed in both the microsomal and the lysosomal beta-glucuronidases. Immunological gel diffusion technique with rabbit antiserum against rat liver lysosomal beta-glucuronidase revealed that both enzymes had the same or quite similar antigenic determinants.     

Novel carbohydrate-binding activity of bovine liver beta-glucuronidase toward lactose/N-acetyllactosamine sequences

Beta-glucuronidase is a lysosomal enzyme that plays an essential role in normal turnover of glycosaminoglycans and remodeling of the extracellular matrix components in both physiological and inflammatory states. The regulation mechanisms of enzyme activity and protein targeting of beta-glucuronidase have implications for the development of a variety of therapeutics. In this study, the effectiveness of various carbohydrate-immobilized adsorbents for the isolation of bovine liver beta-glucuronidase (BLG) from other glycosidases was tested. Beta-glucuronidase and contaminating glycosidases in commercial BLG preparations bound to and were coeluted from adsorbents immobilized with the substrate or an inhibitor of beta-glucuronidase, whereas beta-glucuronidase was found to bind exclusively with lactamyl-Sepharose among the adsorbents tested and to be effectively separated from other enzymes. Binding and elution studies demonstrated that the interaction of beta-glucuronidase with lactamyl-Sepharose is pH dependent and carbohydrate specific. BLG was purified to homogeneity by lactamyl affinity chromatography and subsequent anion-exchange high-performance liquid chromatography (HPLC). Lactose was found to activate beta-glucuronidase noncompetitively, indicating that the lactose-binding site is different from the substrate-binding site. Binding studies with biotinyl glycoproteins, lipids, and synthetic sugar probes revealed that beta-glucuronidase binds to N-acetyllactosamine/lactose-containing glycoconjugates at neutral pH. The results indicated the presence of N-acetyllactosamine/lactose-binding activity in BLG and provided an effective purification method utilizing the novel carbohydrate binding activity. The biological significance of the carbohydrate-specific interaction of beta-glucuronidase, which is different from the substrate recognition, is discussed.     

Purification and characterization of rat liver microsomal beta-glucuronidase.

Beta-Glucuronidase (EC 3.2.1.31) has been isolated from rat-liver microsomes by a novel chromatographic method employing antibody to rat preputial gland beta-glucuronidase coupled to Sepharose. The purified enzyme, homogeneous by several methods, was purified some 1700-fold. The microsomal beta-glucuronidase has been characterized with respect to catalysis, stability, and molecular weight. The purified enzyme is a tetramer of 290 000 daltons. Comparative studies with lysosomal beta-glucuronidase indicate that while these two enzymes are electrophoretically distinct, they are catalytically and immunologically identical and have indistinguishable molecular dimensions. The results suggest that microsomal and lysosomal beta-glucuronidase are charge isomers.     

Clearance of lysosomal hydrolases following intravenous infusion. The role of liver in the clearance of beta-glucuronidase and N-acetyl-beta-D-glucosaminidase.

Certain highly purified forms of rat lysosomal glycosidases, β-glucuronidase and N-acetyl-β-d-glucosaminidase, are rapidly cleared from the circulation following intravenous infusion. Several lines of evidence are presented which indicate that the primary site of enzyme uptake is the liver. Clearance of the two enzymes was unaffected by nephrectomy, whereas it was abolished by evisceration. Tissue distribution experiments with native and [¹²⁵I]β-glucuronidase indicate the liver as the major, if not exclusive, site of enzyme uptake. Experiments with the isolated perfused liver showed clearance of certain enzyme preparations but not others. Those enzymes cleared by the isolated perfused liver were likewise cleared in vivo. Liver fractionation studies following infusion of large doses of β-glucuronidase revealed a rapid, short-lived increase in microsomal β-glucuronidase and a slower but larger increase in lysosomal β-glucuronidase. The results indicate that β-glucuronidase, N-acetyl-β-d-glucosaminidase, and probably other glycosidases are rapidly incorporated into the lysosomal compartment of liver.     

Influence of insulin on lysosomal activity and urea production in isolated parenchymal cells from rat liver.

Non-latent (free) activities of two lysosomal enzymes (acid phosphatase and beta-glucuronidase) and urea production were measured in purified rat liver parenchymal cells incubated in the presence and absence of insulin. Non-latent enzyme activity was measured by including 0.25M sucrose in the assay mixtures to provide osmotic protection to the lysosomes. Total enzyme activity was estimated with Triton X-100 in the homogenates. Insulin was found to inhibit ureogenesis and to reduce non-latent lysosomal enzyme activity in the hepatocytes in vitro. Our data support the idea that insulin inhibits autophagy in rat liver parenchymal cells. Such an effect of insulin may also explain the inhibitory action of insulin on urea production in the rat liver.     

Acetoacetyl-CoA synthetase specific activity and concentration in rat tissues

An antibody against acetoacetyl-CoA synthetase purified from rat liver was raised in rabbits. Utilizing the binding of antibody-antigen complexes to a nitrocellulose membrane, a sensitive enzyme-linked immunosorbent assay was developed to estimate the enzyme concentration in rat tissues. The enzyme concentration (microgram immunoreactive protein/mg protein) in rat liver cytosol was increased about 3-, 1.8- and 7-fold by feeding rats diets containing 5% cholestyramine, 0.2% ML-236B (compactin), and 5% cholestyramine plus 0.2% ML-236B for 4 days, respectively, and decreased about 1.8-fold by fasting the animals or 1.3-fold by feeding them a diet containing 5% cholesterol. Changes in the enzyme activity were almost parallel to those in the enzyme concentration, suggesting the physiological role of this enzyme in cholesterol biosynthesis. Immunoblotting of the hepatic cytosol also confirmed that the increase in enzyme concentration on cholestyramine and/or ML-236B feeding was due to an increase in an enzyme protein the same as the purified enzyme and not the isozymic protein. Among various rat tissues examined, the concentrations of immunologically crossreactive enzyme were higher in lipogenic tissues, such as brain, adipose tissue and liver, than in other tissues. The enzymes in these three tissues were identical in molecular weight determined by gel filtration and immunoblotting.     

Clearance of lysosomal hydrolases following intravenous infusion. Kinetic and competition experiments with beta-glucuronidase and N-acetyl-beta-D-glucosaminidase.

Clearance experiments with highly purified lysosomal glycosidases, β-glucuronidase and N-acetyl-β-d-glucosaminidase, following intravenous infusion revealed widely varying clearance profiles which depended on the tissue source of the enzyme. Normal rat serum β-glucuronidase and epididymal N-acetyl-β-d-glucosaminidase were cleared slowly from the circulation when compared with rat preputial gland β-glucuronidase, liver lysosomal β-glucuronidase, and liver lysosomal N-acetyl-β-d-glucosaminidase, respectively, which were cleared rapidly. Experiments comparing the catalytic properties and molecular dimensions of the enzymes revealed no differences between rapid and slow clearance forms. Kinetic analysis using the rapid clearance forms of β-glucuronidase has allowed the resolution of at least two components, rapid and slow. Clearance of the rapid component is saturable and appears to reflect binding or uptake by a limited number of sites. By contrast, the clearance rate of the slow component increased linearly with respect to dose and may be due to nonspecific or low-affinity binding. Competition experiments with β-glucuronidase-free lysosomal extract and highly purified lysosomal enzymes, but not serum glycoproteins or colloidal silver, suggest that one lysosomal enzyme inhibits clearance of others and that a common mechanism may be involved in their binding.     

Lysosomal Dysfunctions Associated with Mutations at Mouse Pigment Genes

Melanosomes and lysosomes share several structural and biosynthetic properties. Therefore, a large number of mouse pigment mutants were tested to determine whether genes affecting melanosome structure of function might also affect the lysosome. Among 31 mouse pigment mutants, six had 1.5- to 2.5-fold increased concentrations of kidney beta-glucuronidase. Three mutants, pale ear, pearl and pallid, had a generalized effect on lysosomal enzymes since there were coordinate increases in kidney beta-galactosidase and alpha-mannosidase. The effects of these three mutations are lysosome specific since rates of kidney protein synthesis and activities of three nonlysosomal kidney enzymes were normal. Also, the mutants are relatively tissue specific in that all had normal liver lysosomal enzyme concentrations.--A common dysfunction in all three mutants was a lowered rate of lysosomal enzyme secretion from kidney into urine. While normal C57BL/6J mice daily secreted 27 to 30% of total kidney beta-glucuronidase and beta-galactosidase, secretion of these two enzymes was coordinately depressed to 1 to 2%, 8 to 9% and 4 to 5% of total kidney enzyme in the pale-ear, pearl and pallid mutants, respectively. Although depressed lysosomal enzyme secretion is the major pigment mutant alteration, the higher lysomal enzyme concentrations in pearl and pallid may be partly due to an increase in lysosomal enzyme synthesis. In these mutants kidney glucuronidase synthetic rate was increased 1.4- to 1.5-fold.--These results suggest that there are several critical genes in mammals that control the biogenesis, processing and/or function of related classes of subcellular organelles. The mechanism of action of these genes is amenable to further analysis since they have been incorporated into congenic inbred strains of mice.     

Lysosomal alpha-D-mannosidase of rat liver. Purification and comparison with the golgi and cytosolic alpha-D-mannosidases

Rat liver contains alpha-D-mannosidases in lysosomes, Golgi membranes, and cytosol. The lysosomal enzyme has now been purified approximately 30,000-fold over the crude extract and is free of at least 13 other lysosomal hydrolases. The enzyme has an apparent molecular weight of 335,000 by molecular sieve chromatography and 200,000 by sucrose density centrifugation. It is a glycoprotein, as evidenced by its binding to a concanavalin A affinity column and by a positive periodic acid-Schiff stain. The enzyme has a pH optimum near 4.6. Although it is generally insensitive to a large variety of inorganic salts, chelating agents, and sulfhydryl reagents, prolonged exposure to ethylenediaminetetraacetic acid caused loss of activity, which could be restored by the addition of ZnSO4. Substrate specificity studies were performed on the purified lysosomal alpha-D-mannosidase, as well as on the purified Golgi and cytosolic alpha-D-mannosidases. The three enzymes exhibited only very limited activity on native glycoproteins, but were found to be active on glycopeptides and oligosaccharides, hydrolyzing 1 yields 2 and 1 yields 3 linkages, except that the Golgi enzyme had negligible activity towards the latter linkage. Immunological comparisons by antibody precipitation tests and double-diffusion plates indicated that the three enzymes are not immunologically related. The alpha-D-mannosidase isolated from rat epididymis was found to be immunologically very similar, if not identical, to the lysosomal enzyme isolated from rat liver.     

Iodinated fibroblast beta-glucuronidase as a ligand for receptor-mediated endocytosis.

Antibodies raised to human placental beta-glucuronidase were shown to cross-react with the beta-glucuronidase secreted by mouse 3T3 fibroblasts, but did not react with other lysosomal enzymes. The beta-glucuronidase secreted by 3T3 cells was purified 15000-fold by chromatography on an affinity column made from this antibody and resolved into a single component, of Mr 68000, by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Iodinated samples of purified enzyme were taken up into mouse peritoneal macrophages by receptor-mediated endocytosis at a rate similar to that calculated previously for unlabelled enzyme, and uptake was competitively inhibited by yeast mannan. Binding of beta-glucuronidase to macrophages was saturable, with a Kd of 7 X 10(-9)l/mol, an affinity comparable with that calculated for the binding of mannosylated bovine serum albumin (Kd 1.3 X 10(-9)l/mol), a ligand specific for mannose receptors. Four times as many molecules of mannosylated albumin (12000) as of beta-glucuronidase (3000), however, bound to each cell. This purification and iodination procedure did not therefore have any adverse effect on the uptake properties of secreted beta-glucuronidase, and provides a ligand with which to investigate binding and specific endocytosis into a range of different types of cell.     

Partial purification, properties, and subcellulsr distribution of rat liver phosphatidate phosphatase.

Phosphatidate phosphatase (EC 3.1.3.4Y was purified 15- to 20-fold from the soluble fraction of rat liver. The purification procedure involved calcium phosphate gel adsorption and elution, ammonium sulfact precipitation, and molecular-sieve chromatography. For the enzyme assay, and aqueous dispersion of phosphatidate, rather than "membrane-bound" phosphatidate, was used as substrate. The partially purified enzyme depends almost entirely on the presence of Mg2+ for its activity. Morover, the activity of the enzyme is stimulated by phosphatidylcholine. The enzyme exhibits a high substrate specificity for phosphatidate. The apparent Km for phosphatidate is approximately 0.05 mM. The optimum pH is between 7.4 and 7.6. The enzyme is inhibited by fluoride and by p-chloromercuribenzoate. The subcellular distribution of phosphatidate phosphatase in rat liver was studied by assaying the activity of the enzyme in the presence of Mg2+ and phosphatidylcholine. In contrast ot the results of previous studies, most of the enzyme activity was found in the soluble fraction.     

Rat superoxide dismutases. Purification, labeling, immunoassay, and tissue concentration

This study determines the validity of utilizing radioimmunoassay of CuZn and Mn superoxide dismutase in the rat for defining mechanism of control over mammalian tissue superoxide dismutase concentrations. To accomplish this, rat Mn and CuZn superoxide dismutase were purified. The CuZn superoxide dismutase dimer had a specific activity of 3600 units/mg of protein and a subunit Mr of 17,000. The Mn superoxide dismutase tetramer had a specific activity of 3700 units/mg of protein and a subunit Mr of 22,000. Both enzymes provided a single discrete protein band on disc gel electrophoresis. The purified enzymes were utilized to develop sensitive (less than 2.5 ng/ml Mn superoxide dismutase and less than 3.12 ng/ml CuZn superoxide dismutase) reproducible immunoassays the specificity of which was confirmed by tissue homogenate dilution and column chromatography. Immunoassay of these enzymes in rat tissues permitted clarification of existing data based on activity assays and demonstrated a trend for higher Mn superoxide dismutase concentrations in tissues of high mitochondrial content (with relative tissue concentrations comparable to man) and low superoxide dismutase concentrations in islets (providing an explanation for their sensitivity to free radical damage). This represents the first report of a radioimmunoassay for rat Mn superoxide dismutase, and the second report of successful purification of rat Mn superoxide dismutase (with higher specific activity and apparent purity and stability). The data support the proposition that these radioimmunoassays in rats will provide a useful system for investigation of mechanisms of control over tissue superoxide dismutase concentrations in mammalian tissues.     

Purification and immunochemical characterization of monoamine oxidase from rat and human liver.

1. Monoamine oxidase from rat and human liver was purified to homogeneity by the criterion of polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. 2. The enzyme activity was extracted from mitochondrial preparations by Triton X-100. The enzyme was purified by (NH4)2SO4 fractionation followed by chromatography on DEAE-cellulose, Sepharose 6B, spheroidal hydroxyapatite, and finally chromatography on diazo-coupled tyramine-Sepharose. 3. Distinct differences occur in the chromatographic behaviour of the two enzymes on both DEAE-cellulose and spheroidal hydroxyapatite. 4. It is unlikely that the purification of the enzymes on tyramine-Sepharose is due to affinity chromatography and reasons for this are discussed. 5. The purified enzymes did not oxidize-5-hydroxytryptamine and the relative activities of the enzymes with benzylamine were increased approx. 1.25-fold compared with the enzyme activities of mitochondrial preparations. 6. Immunotitration of enzyme activity in extracts of mitochondrial preparations from rat liver was carried out with 5-hydroxytryptamine, tyramine and benzylamine. The enzyme activities were completely immunoprecipitated by the same volume of antiserum. Similar results were obtained with the antiserum to the enzyme from human liver.     

Acid phosphatase in rat liver lysosomal membranes: purification and characterization

Acid phosphatase associated with rat liver lysosomal membranes (M-APase) was purified about 4,200-fold over the homogenate with 10% recovery to apparent homogeneity, as determined from the pattern on polyacrylamide gel electrophoresis in the presence of SDS. The purification procedure included; preparation of lysosomal membranes, solubilization of the membranes with 1% Triton X-100, immunoaffinity chromatography, and gel filtration with FPLC equipped with a Sephacryl S-300HR column. The molecular weight, estimated by gel filtration through TSK SW 3000G, was approximately 320K and SDS gel electrophoresis showed that the enzyme is composed of four identical subunits with an apparent molecular weight of 67K. The enzyme contains about 24.3% carbohydrate consisting of mannose, galactose, fucose, N-acetylglucosamine, N-acetylgalactosamine, and N-acetylneuraminic acid in a molar ratio of 38:20:5:36:4:11, respectively. In addition, three soluble forms of acid phosphatase (C-APase I, II, and III) in lysosomal contents were separated from rat liver lysosomal contents with DEAE-Sephacel. These three enzymes were also purified using immunoaffinity chromatography followed by gel filtration. C-APase I, II, III, and M-APase have isoelectric points of 7.7-8.2, 6.6-7.0, 5.7-6.7, and 3.4-3.8, respectively. All four APases are sensitive to endo-beta-N-acetylglucosaminidase H. However, only C-APase III and M-APase are digestible with neuraminidase. Susceptibility of M-APase to neuraminidase in intact tritosomes was examined to study the topography of M-APase in tritosomal membranes. Neuraminidase susceptibility of M-APase was not observed in the intact tritosomes until the tritosomes had been disrupted by osmotic shock.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proteolysis in mitochondrial preparations and in lysosomal preparations derived from rat liver

A method of preparing rat liver mitochondria with low residual contamination by lysosomal proteases is described. Preparations of mitochondria are divided into two equal portions, one of which is supplemented with a lysosomal fraction. The addition of the lysosomal fraction causes an increase in proteolysis of between 26- and 56-fold at pH 5.0 in four similar experiments. This increase matches the increase in the lysosomal marker beta-glucuronidase and indicates that all proteolysis at pH 5.0 is due to enzymes of the lysosomal fraction. Above pH 7.0, the addition of a lysosomal supplement increases proteolysis by 1.5- to 5-fold only, suggesting that in the absence of a lysosomal supplement very little of the observed proteolysis is due to enzymes of lysosomal origin. A method of calculating the contribution to total proteolysis of enzymes of the lysosomal fraction or of the mitochondrial fraction is described. The calculations show that at pH 7.0 and above, more than 93% of the observed proteolysis is due to enzymes originating in the mitochondrial fraction. The results support the view of other workers that rat liver mitochondria contain an endogenous neutral proteolytic system capable of degrading mitochondrial proteins to acid-soluble products.     

Effect of cations on structure-linked sedimentability of lysosomal hydrolases

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.     

Intracellular enzymes of collagen biosynthesis in rat liver as a function of age and in hepatic injury induced by dimethylnitrosamine. Purification of rat prolyl hydroxylase and comparison of changes in prolyl hydroxylase activity with changes in immunoreactive prolyl hydroxylase.

Prolyl hydroxylase was purified from newborn rats by affinity chromatography using poly(L-proline), and antiserum to the enzyme was prepared in rabbits. The rat prolyl hydroxylase was similar to the chick and human enzymes with respect to specific activity, molecular weight and molecular weights of the polypeptide chains. The activity of prolyl hydroxylase and the content of immunoreactive enzyme were measured in rat liver as a function of age in experimental hepatic injury. Active prolyl hydroxylase comprised about 13.2% of the total immunoreactive protein in the liver of newborn rats and the value decreased to about 3.6% at the age of 420 days. This decrease was due to a decrease in the enzyme activity, whereas only minor changes were found in the content of the immunoreactive protein. In hepatic injury, a significant increase was found in the ratio of active enzyme to total immunoreactive protein, owing to an increase in the enzyme activity. The data indicate that prolyl hydroxylase activity in rat liver is controlled in part by a mechanism which does not involve changes in the content of the total immunoreactive protein.     

Acid hydrolases of the rat uterus in relation to pregnancy, post-partum involution and collagen breakdown

1. The rat uterus contains acid cathepsin, beta-glucuronidase, beta-galactosidase, acid phosphatase and deoxyribonuclease II at concentrations comparable with those found in liver. Two non-hydrolytic uterine enzymes, cytochrome c oxidase and aspartate aminotransferase, display only 2-6% of the activity found in liver. 2. The concentrations of acid cathepsin and beta-glucuronidase are significantly decreased in pregnancy and increase 3-4-fold during post-partum involution. 3. The concentrations of beta-galactosidase and acid phosphatase are not decreased in pregnancy and increase only 2-3-fold during involution. 4. The concentrations of these four acid hydrolases increase linearly during the first 4 days post partum and reach their peak values at the same time that wet weight and collagen content fall to their lowest point. 5. The concentration of deoxyribonuclease is depressed in pregnancy but does not rise above normal in the post-partum period. 6. Only a small proportion of each hydrolytic activity can be isolated in the mitochondrial-lysosomal fraction of sucrose homogenates of the rat uterus. This proportion increases during involution. However, the extensive mitochondrial rupture occurring during homogenization indicates that the technique is probably too harsh to obtain a true measure of the proportion of lysosomes present in the intact tissue. 7. There are no significant changes in either the concentration or subcellular distribution of the five acid hydrolases in the livers of the experimental rats during pregnancy or involution. In each case the largest proportion of the activity is found in the mitochondrial-lysosomal fraction of liver homogenates. 8. The results are interpreted in terms of the lysosomal theory of intracellular digestion.     

Processing of the phosphorylated recognition marker in lysosomal enzymes. Characterization and partial purification of a microsomal alpha-N-acetylglucosaminyl phosphodiesterase

N-Acetylglucosamine(1)phospho(6)mannose groups recently identified in lysosomal enzymes were proposed to be precursors of the recognition markers terminating with mannose 6-phosphate (Tabas, I., and Kornfeld, S. (1980) J. Biol. Chem. 225, 6633-6639; Hasilik, A., Klein, U., Waheed, A., Strecker, G., and von Figura, K. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 7074-7078). To study the presumptive enzyme removing N-acetylglucosamine from the diester, an assay was developed using a radioactive oligosaccharide containing diester groups of the above structure. An alpha-N-acetylglucosaminyl phosphodiesterase cleaving this substrate in vitro was found in human placenta and in rat liver. The enzyme was solubilized from the microsomal fraction of human placenta and more than 800-fold purified with 75% yield. It is distinct from the lysosomal alpha-N-acetylglucosaminidase by the criteria of immunological cross-reactivity, substrate specificity, and heat stability. The partially purified enzyme cleaves alpha-N-acetylglucosamine phosphodiester bonds in oligosaccharides from lysosomal enzymes, in lysosomal enzymes, and in UDP-N-acetylglucosamine. We propose that the microsomal alpha-N-acetylglucosaminyl phosphodiesterase is involved in the processing of the phosphorylated recognition marker in lysosomal enzymes.     

Purification and characterization of acid phosphatase in rat liver lysosomal contents

Acid phosphatase in rat liver lysosomal contents, C-APase I, was purified about 5,700-fold over the homogenate with 8.0% recovery, to apparent homogeneity as determined from the pattern on polyacrylamide gel electrophoresis in the presence and in the absence of SDS. The purification procedures included; preparation of crude lysosomal contents, DEAE-Sephacel ion exchange chromatography, hydroxylapatite chromatography, and gel filtration with Sephacryl S-300. The enzyme is composed of three identical subunits with an apparent molecular weight of 48K. The enzyme contains about 11% carbohydrate and the carbohydrate moiety was composed of mannose, fucose, N-acetylglucosamine, and N-acetylgalactosamine in a molar ratio of 20:3:11:1. Sialic acid was not detected in the enzyme. Antisera against the purified C-APase I were raised in goat and the C-APase I was rapidly purified with high yield (10%) by using the specific antibodies coupled to Sepharose 6B.