Sephacryl S-200 Column Chromatography Research Articles

Levels of Ah-Receptor in Excessive Ascorbic Acid-Fed Guinea Pigs Treated with N-Nitrosodiethylamine. A Comparative Estimation by Hydroxylapatite Assay and Sephacryl Chromatography.

As the nutritional status of an individual is important in the regulation of xenobiotic metabolism brought about by the activation and detoxification enzymes of mixed function oxidase systems, the balance of these reactions ultimately determines the fate of xenobiotic agents and thereby modulates their toxic, mutagenic, and carcinogenic effects. Among the dietary factors vitamin C has attracted special attention because of its non-toxic nature and tumor prophylactic effects on a number of experimentally induced tumors. In this study we evaluated the effect of N-nitrosodiethylamine (NDEA), a potent environmental carcinogen, on the status of the Ah-receptor (involved in the induction of arylhydrocarbon hydroxylase) in guinea pigs fed excessive doses of ascorbic acid. The Ah-receptor was estimated by two methods, hydroxylapatite assay and Sephacryl S-300 column chromatography. We found that in both the assays the level of Ah-receptors was significantly decreased in excessive ascorbic acid-fed guinea pigs treated with NDEA. The hydroxylapatite method, which was more convenient, fast and economical is to be preferred for the assay of the Ah-receptor.

Amino acid sequence and some properties of lectin-D from the roots of pokeweed (Phytolacca americana).

Two pokeweed lectins, designated PL-D1 and PL-D2, have been isolated from the roots of pokeweed (Phytolacca americana) using chitin affinity column chromatography followed by gel filtration on a Sephacryl S-200 column and fast protein liquid chromatography on a Mono-Q column, and their amino acid sequences have been analyzed. PL-D1 consists of 84 amino acid residues and has a molecular mass of 9317, while PL-D2 has an identical sequence with PL-D1 except lack of the C-terminal Leu-Thr. PL-D is composed of two chitin-binding domains, A and B, with 50% homology with each other. Both PL-Ds did not agglutinate native rabbit erythrocytes, but showed about 0.1% of the agglutinating activity of wheat germ agglutinin toward trypsin-treated erythrocytes. In the presence of beta (1-->4) linked oligomers of N-acetyl-D-glucosamine, which inhibit the hemagglutination, PL-D1 had an ultraviolet-difference spectrum with maxima at 292-294 nm and 284-285 nm, attributed to the red shift of the tryptophan residue, suggesting the location of tryptophan residue(s) at or near saccharide-binding site of PL-D1.

Isolation and Characterization of a New Vitamin C Producing Enzyme (L-Gulono-γ-lactone Dehydrogenase) of Bacterial Origin

For the first time we found that the bacterial strain Gluconobacter oxydans DSM 4025 produced L-ascorbic acid from L-gulono-γ-lactone and therefore we isolated and purified the enzyme L-gulono-γ-lactone dehydrogenase catalyzing the oxidation reaction. G. oxydans DSM 4025 produced 8.57 and 13.9 mg of L-ascorbic acid per ml from 70.3 and 89.3 mg of L-gulono-γ-lactone per ml in the growing and resting cell systems, respectively. The enzyme was isolated from the soluble fraction of the cells of G. oxydans DSM 4025 by DEAE-cellulose, Q-Sepharose, hydroxylapatite, and Sephacryl S-300 column chromatographies. The molecular weight of the enzyme was 110, 000 and it consisted of three kinds of subunits of 61, 000, 32, 500, and 16, 5000. L-Gulono-γ-lactone was oxidized to L-ascorbic acid very rapidly in the presence of dyes, such as 2, 6-dichlorophenolindophenol or phenazine methosulfate, but oxygen was not available as an electron acceptor. The absorption spectrum of the reduced form of the native enzyme showed maxima at 416, 521, and 552 nm in the visible region, indicating the presence of cytochrome c. The enzyme isolated from G. oxydans DSM 4025 was entirely different from isofunctional enzymes of eucaryotic organisms.

Purification and characterization of a geniposide-hydrolyzing beta-glucosidase from Eubacterium sp. A-44, a strict anaerobe from human feces.

A geniposide-hydrolyzing beta-glucosidase was discovered in Eubacterium sp. A-44, a human intestinal anaerobe. The enzyme was intracellularly distributed in the bacterium, and purified to homogeneity from the extract using Butyl-Toyopearl 650M, Sephacryl S-300, hydroxyapatite and chromatofocusing column chromatography. The enzyme was a single polypeptide chain with the molecular weight of 90 kDa and the N-terminal amino acid sequence initiated from methionine up to the 29th residue did not show more than 50% homology against known protein sequences. A broad substrate specificity was shown for the beta-glucosidase to hydrolyze aryl beta-D-glucosides (p-nitrophenyl beta-D-glucopyranoside-pNPG, esculin and salicin), alkyl beta-D-glucosides (geniposide and amygdalin) and cellobiose. The Km values (mM) for various beta-D-glucosides were 0.068 for geniposide, 0.10 for pNPG, 0.21 for esculin, 0.22 for salicin, 2.9 for amygdalin, and 0.91 for cellobiose. The pH optimum with pNPG and geniposide as the substrates was 6.0. The enzyme was inhibited by sulfhydryl reagents, Cu2+, and nojirimycin bisulfite.

Internalization of human extracellular-superoxide dismutase by bovine aortic endothelial cells

The high heparin-affinity subtype C of the secretory exzyme extracellular-superoxide dismutase (EC-SOD) mainly exists on the outside of endothelial cell surface in the vasculature. Radioodinated recombinant EC-SOD C (r-EC-SOD C) bound to cultured bovine aortic endothelial cells (BAE cell) at 4°C with an association constant of 9.35 × 10 6 M −1 and maximum binding of 600 ng/dish (3109 ng/mg cellular protein). When incubated at 37°C for 1 h, some 125I-r-EC SOD C was no longer releasable by heparin treatment, suggesting that 125I-r-EC-SOD C was internalized by BAE cells. Since the internalization was inhibited in the presence of heparin in medium, this step was mediated by the binding to cell surface heparan sulfate proteoglycans. When cells containing internalized 125I-r-EC-SOD C were incubated in newly added medium at 37°C for up to 1 h, 54% of radioactivity was recovered in new medium. However, 71% of the radioactive materials released to the medium, presumably 125I-r-EC-SOD C and its metabolic products, had lost heparin binding activity. Much of internalized 125I-r-EC-SOD C was degraded to low molecular weight peptides, because 54% of the radioactive products released to the medium were trichloroacetic acid-soluble and 59% of them were below 10 kDa. About one-fourth of radioactive materials were recycled 125I-r-EC-SOD judged from heparin-HPLC and Sephacryl S-200 column chromatography. In the presence of chloroquine, lysosomal protease inhibitor from heparin-HPLC and Sephacryl S-200 column chromatography. In the presence of chloroquine, lysosomal protease inhibitor, the release of internalized 125I-r-EC-SOD C decreased to 59% compared with the control culture. The degradation was also inhibited by chloroquine because the release radioactive materials largely kept heparin affinity and were trichloroacetic acid insoluble. These findings suggest that expressed EC-SOD is internalized, followed by being metabolized or recycled by endothelial cells. This process may contribute to the regulation of the EC-SOD concentration in vascular system and heparin-binding ability of this enzyme.

Activation of p72syk by Thrombin in a Cell-Free System

Thrombin dramatically activated p72syk in a time- and dose-dependent fashion in extracts of resting porcine platelets in the presence of EDTA. Separation analysis using Sephacryl S-300 column chromatography has demonstrated that p72syk may exist as large (complex) and small (monomer) forms in resting platelets, and activation of p72syk was only observed in the fraction of large form. Pretreatment with ATP scavenger, GDPβS and protein phosphatase inhibitors had no effect on this activation. Furthermore, washed immuno-precipitates of large form p72syk were also activated by thrombin or fibrinogen. These results suggest that p72syk may associate with thrombin receptor or other agonist receptors and there may be a novel activation mechanism of non-receptor type protein-tyrosine kinase, which does not require the modification by other protein kinases, protein phosphatases and GTP binding proteins.

Purification and characterization of beta-fructofuranosidase from Bifidobacterium infantis.

beta-Fructofuranosidase activities of eight strains of Bifidobacteria, intestinal bacteria, were assayed and Bifidobacterium infantis was selected for purification of the enzyme. beta-Fructofuranosidase activity was recovered in the supernatant fraction after disruption of B. infantis cells with sonication and was purified to homogeneity by ammonium sulfate fractionation, and DEAE-cellulose, butyl-Toyopearl and Sephacryl S-300 column chromatographies. The enzyme (molecular weight (M.W.) 232000) was composed of three identical subunits (M.W. 75000) whose NH2-terminal amino acids were threonine. The enzyme was stable at pH 6-8, having the optimum activity at pH 6.0-6.2. The enzyme activity was stable under 40 degrees C and the optimal temperature was 55 degrees C. This enzyme catalyzed the hydrolysis of sucrose, 1-kestose, nystose, inulin and raffinose at the relative velocities of 100, 297, 365, 140 and 3.8, respectively, but did not catalyze the hydrolysis of maltose or cellobiose. These results indicated that this fructooligosaccharide hydrolyzing enzyme is a novel type of beta-fructofuranosidase.

Crystal Matrix Protein Is Related to Human Prothrombin

Crystal matrix protein (CMP) is the principal protein found in calcium oxalate (CaOx) crystals precipitated from whole human urine. It is a potent inhibitor of crystal aggregation and may therefore be important in the aetiology of kidney stone disease. CMP was isolated from CaOx crystals by EDTA dissolution and purified by Sephacryl S-200 column chromatography and reversed-phase high pressure liquid chromatography. Edman degradation revealed 81.8% sequence identity of the 11 N-terminal amino acids of CMP with the N-terminus of human prothrombin, which contains 10 γ-carboxyglutamic acid residues in the first 32 amino acids. The apparent relationship between CMP and prothrombin was confirmed when an antibody to human prothrombin reacted with CMP upon Western blotting of sodium dodecyl sulphate polyacrylamide gels of the protein.

Differential Solubilization of Membrane Lipids by Detergents: Coenrichment of the Sheep Brain Serotonin 5-HT 1A Receptor with Phospholipids Containing Predominantly Saturated Fatty Acids

Most membrane receptors lose binding activity during purification and we studied the correlation between this event and differential solubilization of membrane lipids by detergents. Both 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate [Chaps; high critical micellar concentration (cmc) ∼0.5%] and n-dodecyl β-D-maltoside (DDM; low cmc ∼0.009%) solubilized the 8-[ 3H]hydroxy-2-(di- n-propylamino)tetralin ([ 3H]8-OH-DPAT)-binding serotonin 5-HT 1A receptor (5-HT 1A-R) optimally at 2% (w/v) detergent concentration despite the widely differing cmc of the two detergents. In contrast, n-octyl-β-D-glucopyranoside (octyl glucoside; high cmc ∼0.7%), Thesit (low cmc ∼0.005%), and Triton X-100 (low cmc ∼0.013%) solubilized virtually no [ 3H]8-OH-DPAT-binding activity. The total mass of solubilized lipids was always low at 0.5% detergent concentration and attained a plateau between 1 and 2.5% for all detergents except octyl glucoside. The mass of octyl glucoside-solubilized lipids showed an increasing trend even at 3.0% detergent concentration. The total amount of solubilized lipid is unrelated to the amount of 5-HT 1A-R solubilized but the species of lipid is important. Thus Chaps and DDM, with diverse structures and cmc, both preferentially solubilized phospholipids enriched in saturated fatty acids (67 and 72%, respectively). In contrast, octyl glucoside, Triton X-100, and Thesit showed no preference in solubilizing phospholipids. Octyl glucoside, which solubilized significantly higher proportions of saturated fatty-acid-containing phosphatidylethanolamine (slightly higher saturated fatty acids in total phospholipids), also produced more (twofold higher) solubilized 5-HT 1A sites than Triton X-100 and Thesit. This suggests an optimum involvement of saturated fatty acid side chains in forming tightly packed vesicles which stabilize the 5-HT 1A-R more than the vesicles of higher fluidity formed by phospholipids containing higher proportions of cis-double-bonded unsaturated fatty acids. Indeed, delipidation of the 1.5% Chaps-solubilized receptor preparations by Sephacryl S-200 column chromatography eliminated essentially all [ 3H]8-OH-DPAT-binding activity. Therefore, for efficient recovery during solubilization and reconstitution of a prototypic heptahelical receptor (5-HT 1A), it is essential to stabilize the receptor protein through association with saturated phospholipids.

Induction of alpha‐galactosidase in Penicillium ochrochloron by guar (Cyamopsis tetragonobola) gum

High yields of extracellular alpha-galactosidase from fungal cultures were obtained by inducing enzyme production with guar gum (a galactomannan obtained from the seeds of Cyamopsis tetragonobola) as the sole carbon source. An alpha-galactosidase was isolated from the culture medium of Penicillium ochrochloron culture and purified 867-fold by CM-cellulose and Sephacryl S-200 column chromatography to apparent homogeneity. Gel-filtration data revealed an M(r) of 57,500, which was in close agreement with SDS/PAGE M(r) estimation, for a single band, of 60,200. The alpha-galactosidase activity is strictly dependent upon the pH and temperature of the incubation medium, being maximal at pH 4.5 and 55 degrees C respectively. This enzyme from P. ochrochloron was isolated and purified, devoid of beta-mannanase activity, which cleaves the main beta-mannan backbone of galactomannans and greatly diminishes its gel-promoting capacity. The properties of purified guar-gum-induced alpha-galactosidase activity in P. ochrochloron culture were evaluated in order to ascribe a possible application for alpha-galactosidase in the controlled generation of an improved guar-gum-based gel promoter.

Dye-linked L-malate dehydrogenase from thermophilic Bacillus species DSM 465. Purification and characterization.

The distribution of dye-linked L-malate dehydrogenase (L-malate: acceptor oxidoreductase, EC 1.1.99.16) was investigated in many thermophilic bacteria. The enzyme occurred widely in thermophilic spore-forming bacteria like bacilli and thermoactinomycetes. The enzyme was purified to homogeneity from a thermophile, Bacillus sp. DSM 465, with a 2.7% overall recovery by DEAE-Toyopearl column chromatography, Sephacryl S-400 column chromatography and preparative slab PAGE. The enzyme had a molecular mass of about 660 kDa and consisted of about ten subunits all with a molecular mass of 66 kDa. The enzyme retained its full activity upon heating at 55 degrees C for at least 60 min and with incubation at pH 5.0-10.0, 55 degrees C, for 10 min. The enzyme exclusively catalyzed L-malate dehydrogenation in the presence of an electron acceptor such as 2,6-dichloroindophenol. The Michaelis constants for L-malate and 2,6-dichloroindophenol were determined to be 1.67 mM and 0.050 mM, respectively. FAD was identified as a prosthetic group of the enzyme by HPLC.

Biochemical and immunological studies on soluble antigens ofEntamoeba histolytica

The soluble antigens of Entamoeba histolytica trophozoites were analysed in detail by biochemical and immunochemical methods. The antigen was highly complex and heterogeneous as revealed by Sephacryl S-300 column chromatography, which showed four distinct fractions. The molecular mass of fractions FI, FII, FIII and FIV was 660, 170, 65 and 13 kDa, respectively. Protein was the major constituent in crude soluble antigen (CSA) and fractions FI and FII (67%, 80% and 90%, respectively). Polysaccharide was predominant in the FIII fraction (59%). Antigenic activity observed after different physico-chemical treatments revealed that CSA and FI antigens were predominantly glycoprotein in nature. However, the antigenicity of FIII antigen was greatly reduced after sodium meta-periodate treatment, whereas no alteration in reactivity was discerned after trypsin treatment. Sodium dodecyl sulphate-polyacryl-amide gel electrophoresis (SDS-PAGE) analysis demonstrated nearly 28 Coomassie blue bands for CSA and 20, 16, 15 and 3 polypeptide bands for the FI, FII, FIII and FIV fractions, respectively. The molecular mass of the polypeptides of these bands ranged from 210 to 20 kDa. Antigenic activity was observed in CSA and in the first three fractions, both in counter immunoelectrophoresis (CIEP) and in enzyme-linked immunosorbent assay (ELISA). However, the highest antigenic activity was noted in fraction FI. Major immunoreactive polypeptides of CSA and FI antigens against whole trophozoite antibody were observed in the 10- to 170-kDa regions. However, major differences in the immunoreactivity of the two antigens were noted at 116 and 14 kDa for FI antigen and at 84, 30 and 20 kDa for CSA.(ABSTRACT TRUNCATED AT 250 WORDS)

The purification and characterization of a cysteine protease of Fasciola gigantica adult worms

A 26–28 kDa protease was isolated from Fasciola gigantica adult worms by a two-stage purification process of column chromatography in a Sephacryl S-200 column and affinity chromatography in an l-phenylalanine-agarose column. This protease is a cysteine (thiol) proteinase with an optimum pH of 4.5 and is not inhibited by anti F. gigantica immunoglobulin G. The enzyme was inhibited by protease inhibitors known to inhibit cysteine proteases but not by metallo-, aspartate or serine protease inhibitors. The effect of several protease inhibitors and anti- F. gigantica IgG was also assessed on the total proteolytic activity of F. gigantica. There appears to be a preponderance of cysteine protease activity in F. gigantica and there was a significant inhibition of total proteolytic activity by anti- F. gigantica IgG.

Biosynthesis of NAD-sorbitol dehydrogenase is induced by acclimation at 5°C in diapause eggs of the silkworm, Bombyx mori

1. 1. In diapausing eggs of the silkworm, Bombyx mori, activity of NAD-sorbitol dehydrogenase (EC 1.1.1.14, SDH) is almost negligible, but is increased by acclimation at 5°C (Yaginuma et al., 1990, J. comp. Physiol. B160, 277–285). To elucidate the mechanism regulating SDH activity, the following experiments were conducted. Anti-SDH serum was made in a mouse using purified sheep liver SDH. 2. 2. This antiserum reacted with Bombyx egg SDH purified partially by Blue Sepharose CL-6B and Sephacryl S-300 column chromatographies. 3. 3. SDS-PAGE and immunoblotting analyses using the antiserum showed that SDH activity was correlated with the amount of the enzyme protein. 4. 4. These results indicate that biosynthesis of SDH is induced by acclimation at 5°C in diapause eggs of B. mori.

Production of active and inactive renin by adrenal explant cultures and the existence of a prorenin activating enzyme in the adrenal gland.

We previously demonstrated high levels of inactive renin (IR) in nephrectomized rat plasma which provided evidence for extra-renal sources of IR. However, the sources of IR were unknown. This study examined whether the adrenal gland is capable of producing active renin (AR) and IR, using explants of adrenal capsules (glomerulosa portions) from control and nephrectomized (Nepex) rats. Explants from both control and Nepex rats produced large quantities of IR and small quantities of AR. The productions of both IR and AR in Nepex rats were significantly greater than in control rats. High potassium culture medium markedly increased IR and slightly, but significantly, enhanced AR in both control and Nepex rats. On the other hand, sodium had little effect on either IR or AR. A renin-angiotensin system has been reported in the adrenal gland. Recently, a prorenin activating enzyme was demonstrated in the kidney and the aortic wall. The next study was performed to demonstrate the existence of a prorenin activating enzyme in the adrenal gland, using inactive renin from culture medium from Nepex rat explants as the substrate. A 26 KD component in adrenal zona glomerulosa tissues isolated by Sephacryl S-200 column chromatography, converted IR to AR in IR rich medium. The pH optimum for prorenin activation by the enzyme was 6.5. Inhibitor studies indicate that this enzyme is a serine protease. These data suggest that (1) the adrenal gland is an extra-renal source of inactive renin and (2) a prorenin activating enzyme exists in the adrenal gland.

Recognition of a 56 kDa protein in partially purified rat hepatic nuclear thyroid hormone receptor by anti-human c-erb A beta antibody.

Human beta thyroid hormone receptor (c-erb A beta protein) produced by an Escherichia coli expression system was purified by sequential column chromatography followed by electroelution from an electrophoresis gel and an antibody was prepared. The antibody recognized a 56 kDa protein band in a partially purified rat hepatic nuclear thyroid hormone receptor fraction on Western blotting. Although multiple bands appeared on Western blotting of crude rat hepatic receptor preparations, a 56 kDa band was the most prominent and preadsorption of the antibody by purified c-erb A protein resulted in almost complete disappearance of the 56 kDa band, indicating that the 56 kDa band was formed by a specific antigen-antibody interaction. Furthermore, the 56 kDa protein appeared to co-elute with 3, 5, 3'-triiodo-L-thyronine binding activity in hydroxylapatite, Sephacryl S-200, and DNA-cellulose column chromatography of rat hepatic nuclear receptor, and sequential column purification resulted in selective enrichment of the 56 kDa band. These results suggest that the 56 kDa protein may be the major component of the rat hepatic thyroid hormone receptor.

Immunosuppressive Factor in Cow Follicular Fluid

The activity of the immunosuppressive fraction and proteinase inhibitor isolated from cow follicular fluid was investigated. The immunosuppressive factor was separated from the accompanying proteinase inhibitor by Sepharose 6B and Sephacryl S-200 column chromatography. In vitro, the fraction significantly reduced mitogen-induced lymphocyte proliferation. In vivo, the fraction inhibited mouse plaque formation. However, the factor had no effect on the development of intact or zona free embryos. The active components have a molecular mass of about 110,000.

Common antigens ofFasciola gigantica, Dicrocoelium hospesandSchistosoma bovisand their relevance to serology

The possession of common antigens by three trematode parasites which commonly occur together in ruminants in the tropics, Fasciola gigantica, Dicrocoelium hospes and Schistosoma bovis was studied in relation to the reliability of serodiagnosis of infection with these helminths. The crude antigenic extracts of the three trematodes were subjected to Sephacryl S-300 column chromatography and F. gigantica was fractionated into six peaks, S. bovis into nine peaks and D. hospes into seven peaks. Common antigens were found in these three trematodes in both the crude whole worm antigenic extracts and in the semi-purified fractions obtained by Sephacryl S-300 column chromatography. The implications of this finding and the limitation it imposes on the usefulness of serodiagnostic tests in routine use as regards their specificity are discussed in relation to previous studies.

Demonstration of putative membrane and cytosol steroid receptors for 17α, 20β-dihydroxy-4-pregnen-3-one in brook troutSalvelinus fontinalis oocytes by photoaffinity labelling using synthetic progestin 17,21-dimethyl-19-nor-pregn-4,9-diene-3,20-dione (R5020)

Cytosol from brook trout ovarian follicles (stages 1-3) was photoaffinity (PA) labelled using synthetic progestin 17,21-dimethyl-19-nor-pregn-4,9-diene-3,20-dione ([(3)H]R5020). The covalently bound cytosol protein had a relative mass of 501,000 Mr following Sephacryl S-300 column chromatography. The zona radiata membrane fraction from brook trout oocytes which had gone through the first phase of meiotic maturation (stages 6-7) was isolated by ultracentrifugation of the whole oocytes. The zona radiata solubilized protein presumably from the oocyte membrane was also PA labelled and found to give a peak at 355,000 Mr. The SDS PAGE of the cytosol and zona radiata PA labelled protein gave very similar subunits indicating that the membrane protein and the cytosol protein, both of which bind the maturation inducing steroid (MIS) 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-DHP), have similar subunit structures. The isolated zona radiata protein showed cooperativity of binding to [(3)H]17α,20β-DHP and PA labelling to [(3)H]R5020. The association constant (Ka) was 2.0×10(7)M(-1) and maximum binding capacity (Nmax) 427 fmoles/mg protein with MIS [(3)H]17α,20β-DHP.No evidence for nuclear binding of MIS [(3)H]17α,20β-DHP or PA labelling of [(3)H]R5020 to nuclei was observed. The nuclei were isolated from stages 1 and 3 fresh ovarian follicles of brook trout. The experimental evidence presented demonstrates the presence of MIS 17α,20β-DHP receptor-like protein from the zona radiata membranes by PA labelling in brook trout oocytes during final stages of maturation.

Hydrolysis of glycyrrhizin to 18β-glycyrrhetyl monoglucuronide by lysosomal β- D-glucuronidase of animal livers

Glycyrrhizin (GL), a main constituent of liquorice, was hydrolysed to 18β-glycyrrhetic acid mono-β- d-glucuronide (GAMG, glycyrrhetyl monoglucuronide) by rat liver homogenate, and the hydrolytic activity was localized in the lysosomes among the same subcellular fractions as acid β- d-glucuronidase activity ( p-nitrophenyl β- d-glucuronide (pNPG)-hydrolysing activity). Rat liver lysosomeshydrolysed GAMG to 18β-glycyrrhetic acid (GA) at only 30% rate compared with the rate of GL to GAMG. GA was also produced slowly from GL after time lag by the lysosomes. Thus, GL seems to be first hydrolysed to GAMG, which was successively hydrolysed slowly to GA. GL-hydrolysing activity was released together with acid β- d-glucuronidase activity from the lysosomes by sonication. Both activities from the sonicated lysosomes were eluted coincidentally on Sephacryl S-300 and butyl-Toyopearl 650M column chromatography, indicating that both activities are exhibited by the same enzyme. Moreover, GL-hydrolysing activity was inhibited strongly with d-saccharic acid 1,4-lactone, a specific inhibitor of β- d-glucuronidases of various origins. pH optimum of GL-hydrolysing activity was found to be 5.6, different from that (less than 4.0) of pNPG-hydrolysing activity. K m for GL was found to be 2 × 10 −5 M. Although hepatic lysosomes from mouse and cattle hydrolysed GAMG to GA similarly to those from rat, the hydrolysis of GAMG was not detected in lysosomes of human and porcine livers. Accordingly, lysosomal β- d-glucuronidases from human and porcine livers converted GL to GAMG only.