Purification and characterization of rat liver glutaminase

Phosphate-dependent glutaminase (EC 3.5.1.2) from livers of starved rats was purified about 400-fold to near homogeneity. The specific activity of the final pool was more than 30 U/mg protein. For the rapid quantification of the enzyme activity a simple and sensitive assay, based on the determination of the produced ammonia with an o-phthalaldehyde reagent, was developed which avoids massive dilution of the samples. The enzyme preparation involved extraction of the enzyme from sonified isolated mitochondria after treatment with a brief hypotonic shock followed by ammonium sulphate precipitation, ion-exchange and hydroxyapatite chromatography. A major improvement was the stabilization of the enzyme by chymostatin protecting it from degradation by a protease of presumably lysosomal origin. In the presence of chymostatin or leupeptin the half-life of glutaminase in a crude mitochondrial preparation subsequent to mild treatment with digitonin could be increased to more than 200 h. The relative molecular mass of the protein (Mr 170,500) was estimated by sucrose gradient ultracentrifugation. The molecular mass of the subunits (Mr 57,000) was determined by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. These results suggest a protein composed of three subunits of identical molecular mass. The molecular data clearly differentiate liver glutaminase from the phosphate-dependent glutaminase present in kidney.     

Purification and characterization of rat liver glycosylasparaginase.

1. Rat liver glycosylasparaginase [N4-(beta-N-acetylglucosaminyl)-L-asparaginase, EC 3.5.1.26] was purified to homogeneity by using salt fractionation, CM-cellulose and DEAE-cellulose chromatography, gel filtration on Ultrogel AcA-54, concanavalin A-Sepharose affinity chromatography, heat treatment at 70 degrees C and preparative SDS/polyacrylamide-gel electrophoresis. The purified enzyme had a specific activity of 3.8 mumol of N-acetylglucosamine/min per mg with N4-(beta-N-acetylglucosaminyl)-L-asparagine as substrate. 2. The native enzyme had a molecular mass of 49 kDa and was composed of two non-identical subunits joined by strong non-covalent forces and having molecular masses of 24 and 20 kDa as determined by SDS/polyacrylamide-gel electrophoresis. 3. The 20 kDa subunit contained one high-mannose-type oligosaccharide chain, and the 24 kDa subunit had one high-mannose-type and one complex-type oligosaccharide chain. 4. N-Terminal sequence analysis of each subunit revealed a frayed N-terminus of the 24 kDa subunit and an apparent N-glycosylation of Asn-15 in the same subunit. 5. The enzyme exhibited a broad pH maximum above 7. Two major isoelectric forms were found at pH 6.4 and 6.6. 6. Glycosylasparaginase was stable at 75 degrees C and in 5% (w/v) SDS at pH 7.0.     

Purification and characterization of rat liver alpha-N-acetylglucosaminyl phosphodiesterase

In an earlier report we described the identification of an alpha-N-acetylglucosaminyl phosphodiesterase that is capable of cleaving the outer phosphodiester-linked alpha-N-acetylglucosamine residues present on the high mannose oligosaccharides of newly synthesized lysosomal enzymes (Varki, A., and Kornfeld, S. (1980) J. Biol. Chem. 255, 8398-8401). We have now purified this enzyme 1800-fold with a 24% yield from rat liver, using subcellular fractionation, differential extraction with Triton X-10, DEAE-cellulose chromatography, heparin-Sepharose chromatography, concanavalin A-Sepharose affinity chromatography, and gel filtration on Sephacryl S-300. The purified preparation is free of lysosomal alpha-N-acetylglucosaminidase. The enzyme exhibited a single form on both the ion exchange and gel filtration steps. It has a broad pH optimum between 6.0-8.0 and is unaffected by divalent cations or reducing agents. The enzyme cleaves alpha-N-acetylglucosamine residues from five different locations on the high mannose oligosaccharide. In the case of molecules with one phosphodiester, the rate of cleavage is not affected by the size of the underlying oligosaccharide or the presence or absence of an asparagine-linked peptide. Molecules with two phosphodiesters are cleaved in a nonrandom manner. The enzyme has no activity toward p-nitrophenyl-alpha-N-acetylglucosamine but is capable of cleaving phosphodiester-linked N-acetylglucosamine in molecules such as UDP-N-acetylglucosamine, indicating that it can only hydrolyze N-acetylglucosamine residues that are alpha-linked to a phosphate group.     

Purification and characterization of S-phenacylglutathione reductase from rat liver

An enzyme catalyzing the reduction of S-(2,4-dichlorophenacyl)glutathione to 2',4'-dichloroacetophenone was purified to apparent homogeneity by ion exchange, gel filtration, and hydroxylapatite chromatography from rat hepatic cytosol. The molecular weight was 30,000-37,000. The enzyme is distinct from the glutathione S-transferases, mercaptopyruvate sulfurtransferase, and glyoxalase I. Substrate specificity studies showed that S-phenacylglutathiones are the preferred first substrates and that several thiols (glutathione, mercaptoethanol, L-cysteine, or cysteamine) serve as reducing substrates. The enzyme serves to convert toxic alpha-haloketones, which react rapidly and nonenzymatically with glutathione, to nontoxic alkyl ketones.     

Purification and characterization of rat liver minoxidil sulphotransferase.

Minoxidil (Mx), a pyrimidine N-oxide, is used therapeutically as an antihypertensive agent and to induce hair growth in patients with male pattern baldness. Mx NO-sulphate has been implicated as the agent active in producing these effects. This paper describes the purification of a unique sulphotransferase (ST) from rat liver cytosol that is capable of catalysing the sulphation of Mx. By using DEAE-Sepharose CL-6B chromatography, hydroxyapatite chromatography and ATP-agarose affinity chromatography, Mx-ST activity was purified 240-fold compared with the activity in cytosol. The purified enzyme was also capable of sulphating p-nitrophenol (PNP) at low concentrations (less than 10 microM). Mx-ST was purified to homogeneity, as evaluated by SDS/PAGE and reverse-phase h.p.l.c. The active form of the enzyme had a molecular mass of 66,000-68,000 Da as estimated by gel exclusion chromatography and a subunit molecular mass of 35,000 Da. The apparent Km values for Mx, 3'-phosphoadenosine 5'-phosphosulphate and PNP were 625 microM, 5.0 microM and 0.5 microM respectively. However, PNP displayed potent substrate inhibition at concentrations above 1.2 microM. Antibodies raised in rabbits to the pure enzyme detected a single band in rat liver cytosol with a subunit molecular mass of 35,000 Da, as determined by immunoblotting. The anti-(rat Mx-ST) antibodies also reacted with the phenol-sulphating form of human liver phenol sulphotransferase, suggesting some structural similarity between these proteins.     

Purification and partial characterization of rat liver soluble catechol-O-methyltransferase

The rat liver soluble catechol-O-methyltransferase (EC 2.1.1.6.) has been purified utilizing a combination of conventional chromatography and HPLC. The purified enzyme has a molecular mass of 25 kDa, a pI of 5.1, and exists in two forms which differ in the nature of their intramolecular disulfide bonds. This difference causes these two protein forms to behave differently in reversed phase chromatography.     

Purification and characterization of cytosolic sialidase from rat liver

Sialidase has been purified from rat liver cytosol 83,000-fold by sequential chromatography on DEAE-cellulose, CM-cellulose, Blue-Sepharose, Sephadex G-200, and heparin-Sepharose. When subjected to sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis, the purified cytosolic sialidase moved as a single protein band with Mr = 43,000, a value similar to that obtained by sucrose density gradient centrifugation. The purified enzyme was active toward all of the sialooligosaccharides, sialoglycoproteins, and gangliosides tested except for submaxillary mucins and GM1 and GM2 gangliosides. Those substrates possessing alpha 2----3 sialyl linkage were hydrolyzed much faster than those with alpha 2----6 or alpha 2----8 linkage. The optimum pH was 6.5 for sialyllactose and 6.0 for orosomucoid and mixed brain gangliosides. The activity toward sialyllactose was lost progressively with the progress of purification but restored by addition of proteins such as bovine serum albumin. In contrast, neither reduction by purification nor restoration by albumin was observed for the activity toward orosomucoid. When mixed gangliosides were the substrate, bile acids were required for activity and this requirement became almost absolute after the enzyme had been purified extensively. Intracellular distribution study showed that about 15% of the neutral sialidase activity was in the microsomes. The enzyme could be released by 0.5 M NaCl; the released enzyme was indistinguishable from the cytosolic sialidase in properties.     

Purification and characterization of calmodulin from rat liver mitochondria

Mitochondrial calmodulin of rat liver was purified and classified. It co-migrated with bovine brain calmodulin in non-denaturing polyacrylamide gel electrophoresis, SDS-polyacrylamide gel electrophoresis and isoelectric focusing. The mitochondrial calmodulin activated Ca²⁺-dependent phosphodiesterase of bovine brain in the presence of Ca²⁺. About 80% of the mitochondrial calmodulin was proved to be of cytosol origin. It was easily detached by washing with buffer containing EGTA. The other 20% was intramitochondrial calmodulin; half of it was in the matrix space, and half in the membrane.     

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.     

Purification and characterization of the rat liver gamma-butyrobetaine hydroxylase

The biosynthesis of carnitine from lysine and methionine involves five enzymatic reactions. -butyrobetaine hydroxylase (BBH; EC 1.14.11.1) is the last enzyme of this pathway. It catalyzes the reaction of hydroxylation of -butyrobetaine to carnitine. This enzyme had never been purified to homogeneity from rat tissue. This paper describes the purification and characterization of the rat liver BBH. This protein has been purified some 413 fold by ion exchange, affinity and gel-filtration chromatographies and appears as a dimere of 43,000 Daltons subunits by PAGE. The affinity chromatography column used in the purification process utilizes 3-(2,2,2-trimethylhydrazinium)propionate (THP), a BBH inhibitor, as the ligand. Polyclonal antibodies were raised against the liver enzyme. They were able to precipitate BBH activity in either a crude liver extract or a purified fraction of the enzyme. Furthermore, it crossreacts with a 43 kDa protein in the liver. No evidence for extra hepatic enzyme was found.     

Purification and characterization of an alpha-ketoisocaproate oxygenase of rat liver

Rat liver contains a cytosolic alpha-ketoisocaproate oxygenase which oxidatively decarboxylates and hydroxylates alpha-ketoisocaproate to form beta-hydroxyisovalerate. This oxygenase was purified to near homogeneity. The oxygenase is unstable during purification, unless 5% monothioglycerol is added. The purified enzyme is stable in the presence of 5% monothioglycerol for 3 weeks at 4 degrees C and at least 10 weeks at -80 degrees C. The molecular weight of the alpha-ketoisocaproate oxygenase as determined to be 46,000 and 51,000 using denaturing and nondenaturing conditions, respectively, indicating a monomer. The alpha-ketoisocaproate oxygenase requires Fe2+; other metal ions did not replace Fe2+. Ascorbate activates the enzyme at subsaturating levels of Fe2+, by regenerating Fe2+. The activity is markedly affected by the type of buffer used. For example, the oxygenase activity increased 2- to 3-fold when 0.1 M maleate was used. Iron chelators, such as ADP and EDTA, are inhibitory. The ratio of decarboxylation of 1 mM alpha-[1-14C] ketoisocaproate (as measured by 14CO2 release) to decarboxylation of 1 mM alpha-[1-14C]keto-gamma-methiolbutyrate is 1.0 for all purification fractions, indicating that a single enzyme catalyzes the decarboxylation of both substrates. The apparent Km and Vmax values of the alpha-ketoisocaproate oxygenase using optimized assay conditions are 0.32 mM and 130 nmol/min/mg of protein for alpha-ketoisocaproate and 1.9 mM and 247 nmol/min/mg of protein for alpha-keto-gamma-methiolbutyrate. The principal product of the purified alpha-ketoisocaproate oxygenase, using alpha-ketoisocaproate as a substrate, is beta-hydroxyisovalerate, although small amounts of a compound, which has the chromatographic properties of isovalerate, are also produced.     

Purification and characterization of a calcium-dependent protease from rat liver

A calcium-dependent protease, previously identified in rat liver and designated peak II [DeMartino, G. N. (1981) Arch. Biochem. Biophys. 211, 253-257], was purified and characterized. The calcium-dependent proteolytic activity was accounted for by an 80 000-dalton protein. Depending on the method of purification, we found that this protease could be associated with a 28 000-dalton subunit, which was devoid of protease activity. The catalytic characteristics of the two different forms of the protease were indistinguishable. Each was half-maximally activated by approximately 250 microM calcium.     

Purification and characterization of thiol proteinase inhibitor from rat liver

A thiol proteinase inhibitor was purified from rat liver by essentially the same procedure as reported previously (Kominami, E., Wakamatsu, N., and Katunuma, N. (1981) Biochem. Biophys. Res. Commun. 99, 568-575), but without heat treatment. The purified inhibitor appears homogeneous on polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate and displayed no multiple forms. The inhibitor has Mr = 12,500 and contains 50.5% of polar amino acid residues, 9.3% aromatic amino acids, and no tryptophan. The presence of 2 half-cystines/molecule and the absence of free thiol groups indicate that the inhibitor possesses one disulfide bridges. The inhibitor inhibits cathepsin H by forming an enzyme-inhibitor complex in a molar ratio of 1:1. It inhibits most thiol proteinases such as cathepsin H, L, B, and C, papain, and ficin, but not calcium-activated neutral proteinase or serine proteinases or carboxyl proteinases. The inhibitor was found in various rat tissues. Immunological diffusion analysis with anti-liver thiol proteinase inhibitor serum indicated that the rat liver inhibitor is immunologically identical with the inhibitors from other rat tissues. On subcellular fractionation of rat liver, the thiol proteinase inhibitor was recovered in the cytosol fraction.     

Purification and characterization of cathepsin J from rat liver.

Cathepsin J has been partially purified [Liao, J. C. R. & Lenney, J. F. (1984) Biochem. Biophys. Res. Commun. 124, 909-916], but its detailed properties are still unknown. In this study, we have purified cathepsin J completely and characterized it. It was purified to homogeneity from the mitochondrial-lysosomal fraction of rat liver by acid treatment, followed by ammonium sulfate precipitation (20-65%), and chromatographies on S-Sepharose, ConA-Sepharose, Affi-gel 501, HPLC DEAE-5PW and HPLC TSK G3000SW. Cathepsin J was found to be a lysosomal high-molecular-mass cysteine protease of about 160 kDa consisted of two different subunits. One subunit (alpha subunit) was a glycoprotein with a molecular mass of 19-24 kDa which was reduced to 19 kDa by treatment with endoglycosidase F. It has the amino acid sequence LPESWDWRNVR at its N-terminus, which was very similar to those at the N-termini of rat cathepsins B, H and L. The other subunit (beta subunit) was a glycoprotein with a molecular mass of 17 kDa, which was reduced to 14 kDa by treatment with endoglycosidase F. It had DTPANETYPDLLG at its N-terminus, which had no similarity with the N-terminal sequences of other cathepsins. Cathepsin J showed strong affinity for synthetic substrates such as N-benzyloxycarbonyl-phenylalanyl-arginine 4-methyl-coumaryl-7-amide and glycyl-arginine beta-naphthylamide. It was activated by thiol reagents and chloride ion and was inhibited by cysteine protease inhibitors. However, its initial inhibition constant Ki(initial) by N-(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucine-3- methylbutylamide (E-64-c) was 1800 nM, which was 100-500 times those of cathepsins B and L. Many properties of cathepsin J were similar to those of cathepsin C (dipeptidylaminopeptidase I) reported as a lysosomal cysteine protease with dipeptidyl-aminopeptidase activity [McDonald, J. K., Reilly, T. J. & Ellis, S. (1964) Biochem. Biophys. Res. Commun. 16, 135-140]. Furthermore, antiserum against rat liver cathepsin C reacted with rat liver cathepsin J. These findings suggested that cathepsin J is identical with cathepsin C.     

Purification and characterization of aldehyde dehydrogenase from rat liver mitochondria

Nicotinamide adenine dinucleotide- and nicotinamide adenine dinucleotide phosphate-dependent dehydrogenase activities from rat liver mitochondria have been copurified to homogeneity using combined DEAE, Sepharose, and affinity chromatographic procedures. The enzyme has a native molecular weight of 240,000 and subunit molecular weight of 60,000. The enzyme is tetrameric consisting of four identical subunits as revealed by electrophoresis and terminal analyses. A partial summary of physical properties is provided. The amino acid composition by acid hydrolysis is reported. Specific activities for various NAD(P)+ analogs and alkanal substrates were compared. The action of the effectors chloral hydrate, disulfiram, diethylstilbestrol, and Mg2+ and K+ ions were also investigated.     

Purification and characterization of phosphoinositide 3-kinase from rat liver

Phosphoinositide 3-kinase was purified 27,000-fold from rat liver. The enzyme was purified by acid precipitation of the cytosol followed by chromatography on DEAE-Sepharose, S-Sepharose, hydroxylapatite, Mono-Q, and Mono-S columns. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified phosphoinositide 3-kinase preparation contained an 85-kDa protein and a protein doublet of approximately 110 kDa. The 85- and 110-kDa proteins focus together on native isoelectric focusing gels and are cross-linked by dithiobis(succinylamide propionate), showing that the 110- and 85-kDa proteins are a complex. The apparent size of the native enzyme, as determined by gel filtration, is 190 kDa. The 85-kDa subunit is the same protein previously shown to associate with polyoma virus middle T antigen and the platelet-derived growth factor receptor (Kaplan, D. R., Whitman, M., Schaffhausen, B., Pallas, D. C., White, M., Cantley, L., and Roberts, T. M. (1987) Cell 50, 1021-1029). The two proteins co-migrate on two-dimensional gels; and, using a Western blotting procedure, 32P-labeled middle T antigen specifically blots the 85-kDa protein. The purified enzyme phosphorylates phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate. The apparent Km values for ATP were found to be 60 microM with phosphatidylinositol 4-phosphate or phosphatidylinositol 4,5-bisphosphate as the substrate. The apparent Km for phosphatidyinositol is 60 microM, for phosphatidylinositol 4-phosphate is 9 microM, and for phosphatidylinositol 4,5-bisphosphate is 4 microM. The maximum specific activity using phosphatidylinositol as the substrate is 0.8 mumol/mg/min. The enzyme requires Mg2+ with an optimum of 5 mM. Substitution of Mn2+ for Mg2+ results in only approximately 10% of the Mg2(+)-dependent activity. Physiological calcium concentrations have no effect on the enzyme activity. Phosphoinositide 3-kinase has a broad pH optimum around 7.