Characterization of different proteolytic activities in Trypanosoma brucei brucei.

The variant surface glycoprotein of African trypanosomes is released after overnight incubation of parasites at 4 degrees C in pH 5.5 phosphate glucose buffer and may be purified by Concanavalin A Sepharose affinity chromatography. The addition of proteinase inhibitors during the parasite incubation is necessary to prevent the proteolysis of the variant surface glycoprotein by the trypanosomal released proteinases. Using this procedure without the addition of proteinase inhibitors, the proteolytic activities, released from the bloodstream forms Trypanosoma brucei brucei variant AnTat 1.1, were separated by Concanavalin-A Sepharose affinity chromatography. The unretained material (F1) shows hydrolytic activity against the two synthetic substrates Z-Phe-Arg-AMC and Z-Arg-Arg-AMC, which is stimulated by dithiothreitol, but not inhibited by E-64, and characterized by an alkaline pH optimum and an estimated molecular mass of 80-100 kDa. The Michaelis constant for the substrates Z-Arg-Arg-AMC and Z-Phe-Arg-AMC was, respectively, 2.8 and 6.7 microM. The retained material eluted by addition of 1% methyl-alpha-D-mannopyranoside (F2) shows hydrolytic activity against the synthetic substrate Z-Phe-Arg-AMC, which is stimulated by dithiothreitol, inhibited by E-64, active between pH 6.0 and 8.0, and could be separated into two peaks of activity by HPLC, one peak of high molecular mass (greater than 70 kDa) and the other peak of lower molecular mass (30-70 kDa). By electrophoresis in gels containing gelatin as substrate, this fraction contains several proteins with gelatinolytic activity, whereas the unretained fraction F1 did not have any gelatinolytic activity.     

Cathepsin L--a latent proteinase in guinea pig sperm

Guinea pig spermatozoa were found to contain a fully-latent cysteine proteinase that could be unmasked by incubating epididymal sperm for 2 hr at pH 3.5 and 37 degrees C. The proteinase was identified as cathepsin L (EC 3.4.22.15) on the basis of its optimal hydrolysis of benzyloxycarbonyl-Phe-Arg-7-(4-methyl)coumarylamide (Z-Phe-Arg-NMec) at pH 5.5; lack of action on Z-Arg-Arg-NMec and Arg-NMec; urea-enhanced digestion of azocasein; marked sensitivity to thiol reagents, leupeptin, Z-Phe-Phe-CHN2, and L-trans-epoxy-succinylleucylamido(3-methyl)butane (Ep-475 or E-64-c); and insensitivity to pepstatin and serine proteinase inhibitors. Gossypol, a male antifertility agent, was inhibitory. The unmasking phenomenon was reversibly inhibited by HgCl2 and mersalyl acid, and prevented by leupeptin and Ep-475, but not by pepstatin.     

The purification and properties of cathepsin L from rabbit liver.

Cathepsin L was purified from rabbit liver by a method involving whole-tissue homogenization, pH precipitation, ammonium sulphate fractionation and chromatography on CM-Sephadex C-50, phenyl-Sepharose and Sephadex G-75. Pure enzyme was obtained without the necessity of laborious subcellular fractionation techniques. The Mr of the enzyme was determined to be 29 000 by gel filtration, and affinity for concanavalin A-Sepharose indicated that it was a glycoprotein. A novel technique for detection of enzyme activity in agarose isoelectrofocusing gels showed that the enzyme existed in multiple isoenzymic forms with pI values ranging from 5.0 to 5.9. The enzyme catalysed the hydrolysis of azocasein, collagen and Z-Phe-Arg-NMec (where Z and NMec indicate benzyloxycarbonyl and N-methylcoumarin derivative respectively) optimally at pH 5.2, 3.3 and 6.0 respectively. In addition, cathepsin L was found to degrade benzoyl-Phe-Val-Arg-NMec and 3-carboxypropionyl-Ala-Phe-Lys-NMec. However, cathepsin B also cleaved all of these substrates. One major difference between these two enzymes was in their Michaelis constants for Z-Phe-Arg-NMec; cathepsin B had Km 75 microM whereas that of cathepsin L was 0.7 microM. Cathepsin L was inhibited by all of the usual chemical inhibitors of thiol proteinases as well as the more specific inhibitors Z-Phe-Phe-CHN2, Z-Phe-Ala-CHN2, compound E-64 and compound Ep-475. Active-site titration with compound E-64 showed that the purified sample contained 80% active protein, which had kcat. 20s-1 for the substrate Z-Phe-Arg-NMec. Antibodies were raised to active cathepsin L, and these did not cross-react with cathepsin B, thus demonstrating that these two enzymes are immunologically distinct.     

Characterization of different proteolytic activities in Trypanosoma brucei brucei

The variant surface glycoprotein of African trypanosomes is released after overnight incubation of parasites at 4°C in pH 5.5 phosphate glucose buffer and may be purified by Concanavalin A Sepharose affinity chromatography [1]. The addition of proteinase inhibitors during the parasite incubation is necessary to prevent the proteolysis of the variant surface glycoproteins by the trypanosomal released proteinases. Using this procedure without the addition of proteinase inhibitors, the proteolytic activities, released from the bloodstream forms Trypanosoma brucei brucei variant AnTat 1.1, were separated by Concanavalin-A Sepharose affinity chromatography. The unretained material (F1) shows hydrolytic activity against the two synthetic substrates Z-Phe-Arg-AMC and Z-Arg-Arg-AMC, which is stimulated by dithiothreitol, but not inhibited by E-64, and characterized by and alkaline pH optimum and an estimated molecular mass of 80–100 kDa. The Michaelis constant for the substrates Z-Arg-Arg-AMC and Z-Phe-Arg-AMC was, respectively, 2.8 and 6.7 μM. The retained material eluted by addition of 1% methyl-α-D-mannopyranoside (F2) shows hydrolytic activity against the synthetic substrate Z-Phe-Arg-AMC, which is stimulated by dithiothreitol, inhibited by E-64, active between pH 6.0 and 8.0, and could be separated into two peaks of activity by HPLC, one peak of high molecular mass (> 70 kDa) and the other peak of lower molecular mass (30–70 kDa). By electrophoresis in gels containing gelatin as substrate, this fraction contains several proteins with gelatinolytic activity, whereas the unretained fraction F1 did not have any gelatinolytic activity.     

Purification and properties of different isoforms of bovine cathepsin B

A rapid purification procedure is described for cathepsin B from bovine liver. After preparation of crude lysosomal extracts, the method only involves DEAE Zeta-Prep-Disk chromatography, gel filtration, and fast protein liquid chromatography on Mono-S column. Two active peaks (P1 and P2) of cathepsin B were distinguished. Both presented uncleaved (relative mass (Mr) 30,000) and cleaved (Mr 25,000 + Mr 5000) chains, but different isoforms as revealed by isoelectrofocusing. These two different populations of cathepsin B isoforms nevertheless exhibited similar enzymatic properties. Km and kcat were 114 microM and 52 s-1, and 125 microM and 75 s-1, for hydrolysis of Z-Arg-Arg-NMec by P1 and P2, respectively. Both were rapidly inhibited by low concentrations of E-64 or leupeptin, but were unaffected by cathepsin-L-specific inhibitor Z-Phe-Phe-CHN2.     

Variant surface glycoprotein of Trypanosoma brucei brucei AnTat 1.1: influence of the isolation conditions upon the disulfide linked dimer/monomer ratio

1. Using the variant surface glycoprotein (VSG) isolation procedure described by Baltz et al. ([1976] Ann. Immunol. (Inst. Pasteur) 127 C, 761-774) which involves suspension of the trypanosomes in a pH 5.5 buffer, the Antwerpen trypanozoon antigenic type (AnTat) 1.1 VSG is mainly obtained as a disulfide linked dimeric form with a trace amount of a monomeric form. 2. The use of a parasite suspension buffer at pH 7.0 results in a slight decrease of the VSG dimer/monomer ratio. 3. pH 5.5 and 7.0 supernatants of centrifuged parasite suspensions were submitted to kinetic incubations at different temperatures and pH, and we found conditions involving transformation of the AnTat 1.1 VSG dimer into the AnTat 1.1 VSG monomer (shifting the pH 5.5 supernatant to pH 7.0 and incubation at room temperature). 4. This transformation of the AnTat 1.1 VSG dimer into the AnTat 1.1 VSG monomer is activated by the addition of 1 mM reduced glutathione, and is inhibited by the addition of 1 mM oxidized glutathione or 0.1 mM N-ethylmaleimide or cadmium acetate.     

Human kidney cathepsins B and L. Characterization and potential role in degradation of glomerular basement membrane.

Cathepsins B and L were purified from human kidney. SDS/polyacrylamide-gel electrophoresis demonstrated that cathepsins B and L, Mr 27000-30000, consist of disulphide-linked dimers, subunit Mr values 22000-25000 and 5000-7000. The pH optimum for the hydrolysis of methylcoumarylamide (-NHMec) substrates (see below) is approx. 6.0 for each enzyme. Km and kcat. are 252 microM and 364s-1 and 2.2 microM and 25.8 s-1 for the hydrolysis of Z-Phe-Arg-NHMec (where Z- represents benzyloxycarbonyl-) by cathepsins B and L respectively, and 184 microM and 158 s-1 for the hydrolysis of Z-Arg-Arg-NHMec by cathepsin B. A 10 min preincubation of cathepsin B (40 degrees C) or cathepsin L (30 degrees C) with E-64 (2.5 microM) results in complete inhibition. Under identical conditions Z-Phe-Phe-CHN2 (0.56 microM) completely inhibits cathepsin L but has little effect on cathepsin B. Incubation of glomerular basement membrane (GBM) with purified human kidney cathepsin L resulted in dose-dependent (10-40 nM) GBM degradation. In contrast, little degradation of GBM (less than 4.0%) was observed with cathepsin B. The pH optimum for GBM degradation by cathepsin L was 3.5. Cathepsin L was significantly more active in degrading GBM than was pancreatic elastase, trypsin or bacterial collagenase. These data suggest that cathepsin L may participate in the lysosomal degradation of GBM associated with normal GBM turnover in vivo.     

Phospholipase A2 from Trypanosoma brucei gambiense and Trypanosoma brucei brucei: inhibition by organotins

Activity and kinetics of phospholipase A2 (PLA2) from Trypanosoma brucei gambiense (Wellcome strain) and Trypanosoma brucei brucei (GUTat 3.1) were examined using two different fluorescent substrates. The activity in the supernatants of sonicated parasites was Ca2+-independent, strongly stimulated by Triton X-100 with optimum activity at 37 degrees C and pH 6.5-8.5. To encourage a possible interaction between the parasite enzyme and organotin compounds, fatty acid derivatives of dibutyltin dichloride were synthesized and evaluated as potential inhibitors of PLA2. The enzyme from the two-trypanosome species differ with respect to kinetic parameters and are noncompetitively inhibited by the organotin compounds. The Michaelis constant (KM) for PLA2 from T. b. brucei is 63.87 and 30.90 microM while for T. b. gambiense it is 119.64 and 32.91 microM for the substrates 1,2-bis-(1-pyrenebutanoyl)-sn-glycero-3-phosphocholine (PBGPC) and 2-(12-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine (NBDC12-HPC), respectively.     

Differences in midgut serine proteinases from larvae of the bruchid beetles Callosobruchus maculatus and Zabrotes subfasciatus

Proteinase activities in the larval midguts of the bruchids Callosobruchus maculatus and Zabrotes subfasciatus were investigated. Both midgut homogenates showed a slightly acidic to neutral pH optima for the hydrolysis of fluorogenic substrates. Proteolysis of epsilon-aminocaproil-Leu-Cys(SBzl)-MCA was totally inhibited by the cysteine proteinase inhibitors E-64 and leupeptin, and was activated by 1.5 mM DTT in both insects, while hydrolysis of the substrate Z-ArgArg-MCA was inhibited by aprotinin and E-64, which suggests that it is being hydrolysed by serine and cysteine proteinases. Gel assays showed that the proteolytic activity in larval midgut of C. maculatus was due to five major cysteine proteinases. However, based on the pattern of E-64 and aprotinin inhibition, proteolytic activity in larval midgut of Z. subfasciatus was not due only to cysteine proteinases. Fractionation of the larval midgut homogenates of both bruchids through ion-exchange chromatography (DEAE-Sepharose) revealed two peaks of activity against Z-ArgArg-MCA for both bruchid species. The fractions from C. maculatus have characteristics of cysteine proteinases, while Z. subfasciatus has one non-retained peak of activity containing cysteine proteinases and another eluted in a gradient of 250-350 mM NaCl. The proteolytic activity of the retained peak is higher at pH 8.8 than at pH 6.0 and corresponds with a single peak that is active against N-p-tosyl-GlyGlyArg-MCA, and sensitive to 250 microM aprotinin (90% inhibition). The peak contains a serine proteinase which hydrolyzes alpha-amylase inhibitor 1 from the common bean (Phaseolus vulgaris). Arch.     

Lysosomal and non-lysosomal peptidyl hydrolases of the bloodstream forms of Trypanosoma brucei brucei

African trypanosomes have thiol-dependent proteolytic activity that resembles some of the cathepsin-like activity found in mammalian lysosomes [Lonsdale-Eccles, J. D. & Mpimbaza, G. W. N. (1986) Eur. J. Biochem. 155, 469-473]. Here we show that this activity is found in lysosome-like organelles which we have isolated (density = 1.082 g/cm3 in Percoll) from bloodstream forms of Trypanosoma brucei brucei. They are approximately 250 nm in diameter, are bounded by a single limiting membrane, and contain acid phosphatase. The predominant proteolytic and peptidolytic activity of these organelles has a pH optimum about 6.0, exhibits latency, and has the characteristics of mammalian cathepsin L (and possibly cathepsin H) with respect to its hydrolysis of small fluorogenic peptidyl substrates such as benzyloxycarbonyl-phenylalanyl-arginyl-7-amido-4-methylcoumarin. This substrate appears to be a good marker for trypanosomal lysosomes. The cathepsin-L-like activity is inhibited by the thiol-protease inhibitors, E-64, cystatin, leupeptin and mercurial compounds. The proteolytic activity of the lysosome-like fraction is observed as a single band of activity with an approximate molecular mass of 27 kDa when measured after electrophoresis in the fibrinogen-containing sodium dodecyl sulphate/polyacrylamide gels. The addition of mammalian serum to this purified fraction, or to whole trypanosome homogenates, results in the appearance of additional bands of activity, with a concomitant increase in the total observed proteolytic activity. The serum of some species of animal (e.g. goat and guinea pig) appear to lack the ability to generate this new and increased activity, while rat, rabbit, human and bovine sera exhibit varying capacities to generate the new activity, the cow being the most effective. The apparent molecular masses of the new bands of activity are different for each mammalian species, suggesting that the activator is a species-specific molecule or class of molecules. We also show that Trypanosoma brucei contains soluble peptidolytic activity with an alkaline pH optimum. It is inhibited by the serine-protease inhibitor diisopropylfluorophosphate, but not by inhibitors such as phenylmethylsulphonyl fluoride, alpha 1-antitrypsin, or aprotinin. Nor is it inhibited by the thiol-protease-specific inhibitors E-64 or cystatin, although it is susceptible to inhibition by tosyllysylchloromethane, leupeptin, HgCl2 and p-chloromercuribenzoate. This enzymic activity has a preference for arginyl residues in the primary binding site (the P1 position), as also does the activity from the lysosomes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Activity of lysosomal cysteine proteinase during differentiation of rat skeletal muscle.

Cysteine-proteinase activities were measured in extracts of pre- and post-fusion populations of rat myogenic line L6 cells and in extracts of whole rat muscle. Activities of cathepsins B, L and H were compared. The substrates used included Z-Phe-Arg-NMec (cathepsins B and L), Z-Arg-Arg-NMec (cathepsin B), and Arg-NMec (cathepsin H) (where Z = benzyloxycarbonyl, and NMec = 4-methyl-7-coumarylamide); the enzyme activities were more specifically differentiated by appropriate concentrations of the inhibitors Z-Phe-Phe-CHN2 (CHN2 = diazomethane), bestatin and E-64 [L-trans-epoxysuccinyl-leucylamido(4-guanidino)butane]. These experiments have demonstrated the feasibility of determining the cysteine-proteinase activities of myoblasts from a single (60 mm-diameter) Petri dish, with enzyme concentrations in the range of 5-20 ng/ml. Specific activities of the enzymes in L6 cells increased 2-20-fold after fusion. Concentrations of cysteine proteinases in extracts from cultured myoblasts were two orders of magnitude greater than those in muscle-tissue extracts. Cultured-cell extracts contained endogenous inhibitor(s) to purified rat cathepsins B, L and H.     

Cysteine proteinase content of rat muscle lysosomes. Evidence for an unusual proteinase activity

Lysosomal cysteine proteinases were fractionated from partially purified rat muscle lysosomes. By gel filtration on Sephadex G75, cathepsin D was separated from two thiol-requiring proteolytic fractions of Mr 25 000 and 55 000, respectively. By chromatofocusing, the first fraction (Mr = 25 000) was resolved into three isoenzymic forms of cathepsin H, eluted at pH 5.8, 6.0 and 7.2, respectively, and two isoenzymic forms of cathepsin B, eluted at pH 5.5 and 5.25. Cathepsin H isoenzymes hydrolyzed Arg-NNap and BANA, were totally inhibited by 1 mM p-CMB and only to 60% by 5.10(-5) M leupeptin. The two forms of cathepsin B which degraded Z-Phe-Arg-NMec, Z-Arg-Arg-NNap and BANA were very sensitive to p-CMB and leupeptin. In addition to cathepsins B and H, a typical cathepsin-L- like activity was found in this fraction but only as a very minor component. The high Mr fraction (Mr = 55 000) contained a cysteine proteinase hydrolyzing, at pH 6.0, Z-Phe-Arg-NMec, and to a lesser extent Z-Arg-Arg-NNap and BANA. Unlike cathepsins B and H, it was very sensitive to p-CMB and HgCl2 and was fully activated only in the presence of 10 mM DTT, and inhibited to 93% by 2.10(-8) M leupeptin. By chromatofocusing, it was resolved into several isoenzymatic forms, eluted between pH 5.8 and 4.0.     

Identification and partial purification of a unique phenolic steroid sulphotransferase in rat liver cytosol.

Phenolic steroid sulphotransferase activity for both oestradiol and oestrone was identified in male rat liver cytosol in the 30 000-40 000 Mr fractions on gel filtration when activity was assayed at pH 5.5 (pH optimum 5.5-6.0). Activity for oestradiol but not oestrone was found in the 60 000-70 000-Mr range when assayed at pH 8.0 (pH optimum biphasic, 5.5-6.0 and 7.0-8.0). Km for oestradiol (1.3 microM) was lower than published values for hydroxysteroid sulphotransferases (15-35 microM) and previously reported oestradiol sulphotransferases (71-85 microM). At above 2 microM-oestradiol phenolic sulphotransferase activity exhibited substrate inhibition. The phenolic steroid sulphotransferase activity was found to be distinct in chromatofocusing from organic-anion-binding and bile acid-binding proteins previously identified in this Mr range. Further purification on hydroxyapatite yielded a 44-fold enriched fraction that contained two monomeric bands, Mr 32 500 and 29 500.     

Purification to homogeneity of human placental acid sphingomyelinase

Acid sphingomyelinase was purified to homogeneity from human placenta in the presence of a dialyzable detergent, n-octyl-beta-D-glucopyranoside. The major steps in the procedure included column chromatographies with Con A-Sepharose, sphingosylphosphorylcholine-Sepharose 4B, hexyl-agarose, and Mono P. The purified enzyme with pI 7.4 had a specific activity of approx 170,000 units/mg protein with a yield of 3.6%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single protein band of Mr 62,000. Gel filtration with a Superose 12 column gave a single peak, and the enzyme in the presence 50 mM n-octyl-beta-D-glucopyranoside was of Mr 123,000, indicating that the native enzyme occurs in a dimeric form. The optimal pH was 5.5 with both sphingomyelin and an artificial substrate, 2-N-hexadecanoylamino-4-nitrophenylphosphorylcholine. The Km values were 55 microM with sphingomyelin and 340 microM with the artificial substrate. The enzyme activity was not affected by Mg2+ (1-5 mM), confirming that the enzyme is acid sphingomyelinase. The enzyme was stable at -80 degrees C for more than 4 months. In addition to the enzyme with pI 7.4, the Mono P chromatofocusing gave two peaks (pI 7.0 and 6.7) possessing the enzymatic activity.     

Purification and properties of inositol-1,4-bisphosphatase from bovine brain.

Inositol-1,4-bisphosphatase has been purified 13,000-fold from bovine brain supernatant. The enzyme is monomeric, with an apparent subunit Mr of 40,000. Maximal hydrolytic rates were observed in Tris buffer, pH 7.8, in the presence of 9 mM-Mg2+. The enzyme acted as a 1-phosphatase, hydrolysing both inositol 1,4-bisphosphate [Ins(1,4)P2] (Km 0.04 mM) and inositol 1,3,4-trisphosphate [Ins(1,3,4)P3] (Km 0.5 mM) to inositol 4-phosphate and inositol 3,4-bisphosphate respectively. Li+ inhibited the hydrolysis of both substrates in an uncompetitive manner, with apparent Ki values of 9.63 mM and 0.46 mM for Ins(1,4)P2 and Ins(1,3,4)P3 respectively.     

Presence of a thiol protease in regenerating rat-liver nuclei. Partial purification and some properties

1. Nuclei of regenerating rat liver washed with Triton X-100 were found to contain a new protease. Since the enzymatic activity for degrading ribosomal proteins was inhibited in vivo by administration of E-64, a thiol protease inhibitor, the enzyme may participate in the degradation of newly synthesized ribosomal proteins and histones in regenerating rat liver nuclei as reported previously by us [Biochem. Biophys. Res. Commun. 75, 525-531 (1077)]. The optimum pH was 5.5. 2. The enzyme was extracted from washed nuclei and partially purified by gel filtration through Sepharose 6B. Its molecular weight was about 40 000. A maximal activity of partially purified enzyme was observed in the presence of 1 mM EDTA and 2 mM dithiothreitol at pH 5.5 It was inhibited by thio reagents, E-64, leupeptin and hevy metal ions. The enzyme degraded ribosomal proteins endoproteolytically and degraded most proteins tested as substrates, although liver cell sap proteins and serum albumin were less degraded than ribosomal proteins and histones, alpha-N-Benzoylarginine-beta-naphthylamide and benzoylarginine amide were not hydrolyzed.     

On the substrate specificity of cathepsin L

A view is given on the maximal hydrolysis of proteins by cathepsin L (EC 3.4.22.15) in dependence on the pH. The overall degradation of several proteins at pH values lower than pH 6.0 implies a very broad specificity, whereas at pH 7.0 and 7.5 cathepsin L seems to act on proteins cleaving only restricted specific peptide bonds. Some kinetic constants are given for the three synthetic substrates of cathepsin L which are known so far: Bz-Arg-NH2, Z-Lys-OPhNO2 and Z-Phe-Arg-NMec. They cannot be used as completely specific substrates of cathepsin L, because all of them are hydrolysed by cathepsin B and also other proteinases.     

Chick embryo liver beta-glucuronidase. Comparison of activity on natural and artificial substrates.

The beta-glucuronidase in homogenates of 12-day chick embryo livers catalyzed the release of glucuronic acid from 4-methylumbelliferyl-beta-D-glucuronide and from the nonreducing terminals of the hexasaccharides of chondroitin-6-SO4 and chondroitin-4-SO4 at rates of 143, 114, and 108 nmol of glucuronic acid/h/mg of protein, respectively, when assayed at pH 3.5 in 0.05 M sodium acetate buffer. During a 60-fold purification of the enzyme, the ratios of the activities on these substrates did not change. When 4-methylumbelliferyl-beta-D-glucuronide was used as substrate the enzyme was active at pH values from 3.0 to 5.5, with maximal activity between pH values 4.0 and 4.5. Concentrations of NaCl from 0.15 to 0.3 M inhibited the activity at low pH values but activated the enzyme between pH 4.0 and 5.5. The enzyme was active on the chondroitin-6-SO4 hexasaccharide from pH 3.0 to 5.5, with a broad optimum between 3.0 and 4.5. NaCl inhibited the activity on the oligosaccharide substrate at all pH values. Eadie-Scatchard plots of rates of 4-methylumbelliferyl-beta-D-glucuronide hydrolysis at substrate concentrations ranging from 2 to 1000 microM showed multiple kinetic forms of the enzyme, a form with a Km of approximately 11 microM, and a second form with a Km of approximately 225 microM. The pH optimum of the low Km form was 3.5 to 4.0; that of the high Km form was pH 4.5. NaCl inhibited the activity of the low Km form, but activated the high Km form of the enzyme. Chondroitin SO4 oligosaccharides competed with 4-methylumbelliferyl-beta-D-glucuronide for the low Km form of the enzyme but had little effect on the hydrolysis of 4-methylumbelliferyl-beta-D-glucuronide by the high Km form of the enzyme. The activities of the beta-glucuronidase on tetra-, hexa-, octa-, and decasaccharides of chondroitin-6-SO4 and chondroitin-4-SO4, measured using a new assay procedure which can detect the formation of 1 nmol of product, were similar, although rates were somewhat lower for the higher oligosaccharides. With the exception of the chondroitin-4-SO4 tetrasaccharide, all of the oligosaccharide substrates saturated the enzyme at concentrations of 20 to 30 microM, indicating Km values of less than 10 to 15 microM for the oligosaccharides. Highly purified beta-glcuronidases from human placenta and from rat preputial gland also showed multiple kinetic forms when assayed using 4-methylumbelliferyl-beta-D-glucuronide as substrate.     

Properties of a highly purified mitochondrial deoxyguanosine kinase

Deoxyguanosine kinase, purified over 6000-fold from beef liver mitochondria by means of deoxyguanosine-3'-(4-aminophenyl phosphate)-Sepharose affinity chromatography, was nearly homogeneous. It phosphorylates only deoxyguanosine and deoxyinosine among the natural nucleosides, with apparent Km values of 4.7 and 21 microM, respectively. Among nucleoside analogs tested, only arabinosylguanine (Ki = 125 microM) and 8-aza-deoxyguanosine (Ki = 450 microM) competed with deoxyguanosine. The relative molecular mass of the enzyme is 56,000, as determined by equilibrium sedimentation, and sodium dodecyl sulfate-gel electrophoresis suggests two subunits of Mr 28,000. The pH optimum for enzyme activity is 5.5, but optimum enzyme stability is seen at pH 7.0. Triton X-100 increased the stability of the enzyme markedly. ATP is the best phosphate donor at pH 5.5, but pyrimidine triphosphates such as dTTP and UTP are more efficient donors at pH 7.4. The activation energy, at pH 5.5, was estimated to be 10.9 kcal/mol. Amino acid modification experiments suggest the involvement of arginine, cysteine, and probably histidine. The inactivation of the enzyme by modification of these amino acid residues was time and pH dependent. Both substrates protected the enzyme from inactivation in every case but that of photooxidation by Rose Bengal, where only deoxyguanosine prevented inactivation.     

Human N-acetylgalactosamine-4-sulphate sulphatase. Purification, monoclonal antibody production and native and subunit Mr values.

Initial purification of N-acetylgalactosamine-4-sulphate sulphatase from human liver homogenates containing approx. 1 mg of enzyme in 26 g of soluble proteins was achieved by a six-column chromatography procedure and yielded approx. 40 micrograms of a single major protein species. Enzyme thus prepared was used to produce N-acetylgalactosamine-4-sulphate sulphatase-specific monoclonal antibodies. The use of a monoclonal antibody linked to a solid support facilitated the purification of approx. 0.5 mg of N-acetylgalactosamine-4-sulphate sulphatase from a similar liver homogenate. Moreover the enzyme isolated contained a single protein species, shown by SDS/polyacrylamide-gel electrophoresis to have an Mr of 57,000, which dissociated into subunits of Mr 43,000 and 13,000 in the presence of reducing agents. Essentially identical enzyme preparations were isolated from homogenates of human kidney and lung and from concentrated human urine. The native protein Mr of enzyme from human liver and kidney was assessed by gel-permeation chromatography to be 43,000 on Ultrogel AcA and Bio-Gel P-150. The liver N-acetylgalactosamine-4-sulphate sulphatase was shown to have pH optima of approx. 4 and 5.5 with the oligosaccharide substrate (GalNAc4S-GlcA-GalitolNAc4S) and fluorogenic substrate (methylumbelliferyl sulphate) respectively. Km values of 60 microM and 4 mM and Vmax. values of 2 and 20 mumol/min per mg were determined with the oligosaccharide and fluorogenic substrates respectively.