Purification and characterization of a binding protein related to the Z class of cytosolic proteins in guinea-pig liver cytosol (guinea-pig Z protein).

The purification and characterization of a low-molecular-mass binding protein from female guinea-pig liver cytosol is reported. Its molecular mass (14.4 kDa), amino acid composition, abundance and biological properties identify it as belonging to the Z class of liver cytosolic proteins [Levi, A.J., Gatmaitan, Z. & Arias, I.M. (1969) J. Clin. Invest. 48, 2956-2167]. Among the most important members of this class of proteins are the fatty-acid-binding proteins (FABPs) and the sterol carrier protein2 (SCP2). The guinea-pig Z protein (G-ZP) has some similarities in its amino acid composition and NH2-terminal sequence with those of the rat liver FABP, but its isoelectric point is basic (pI 8.85), like that of SCP2. We also examined its binding affinities for a number of ligands bound by these two proteins. The results show that the purified G-ZP binds dehydroepiandrosterone sulfate, estrone sulfate, oleic acid and cholesterol, but shows no affinity for free steroids such as estrone and DHEA. Thus it can be said that G-ZP has some characteristics of FABPs and some of SCP2 but seems, however, to be different from both these proteins. The purified G-ZP inhibits microsomal DHEA sulfate sulfatase activity in a mixed noncompetitive way. This protein could be involved in the transport and/or metabolism of sulfated steroids.     

Purification of N-hydroxy-2-acetylaminofluorene reductase from rabbit liver cytosol

N-Hydroxy-2-acetylaminofluorene reductase was purified from rabbit liver cytosol by fractionation with ammonium sulfate, and chromatography with DEAE-cellulose, Sephadex G-200 and hydroxylapatite. The purified enzyme was homogeneous by the criterion of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 34,000 by the electrophoresis and by gel filtration on Sephadex G-200. The enzyme required cysteine, glutathione, dithiothreitol, 2-mercaptoethanol, NADPH or NADH as an electron donor. The enzyme activity was inhibited by p-chloromercuribenzoic acid, N-ethylmaleimide, cupric sulfate or disulfiram, but little by oxygen.     

Z protein: Isolation and characterization of multiple forms in rat liver cytosol

The Z protein fraction of rat liver cytosol contains one or more proteins which have been associated with organic anion transport, fatty acid metabolism, and aminoazodye binding. To study the possible identity of these proteins and investigate their function, Z was purified using ammonium sulfate fractionation, gel filtration, and preparative isoelectric focusing. Three protein fractions were obtained (pI 5.2, 6.0, 7.3) which reacted specifically with anti-Z IgG. These three fractions were homogenous as determined by several electrophoretic systems. Monospecific antibody prepared against two of the proteins cross-reacted specifically with all three. Each fraction bound BSP with different affinity; acidic Z bound the least BSP. The molecular weight of each fraction was 12,500 as determined by SDS-gel electrophoresis. Amino acid analyses of the three Z protein bands were virtually identical. Heterogeneity in Z probably results from interaction of the protein with ampholytes or exogenous ligands.     

Purification of oxysterol binding protein from hamster liver cytosol

We have purified to apparent homogeneity an oxysterol binding protein from cytosol of hamster livers. This protein, which corresponds to the protein described by Taylor and Kandutsch (Taylor, F. R., and Kandutsch, A. (1985) Chem. Phys. Lipids 38, 187-194), binds oxysterols such as 25-hydroxycholesterol but does not bind cholesterol or steroid hormones in vitro. It may participate in the feedback repression of enzymes of cholesterol biosynthesis and the low density lipoprotein receptor. The protein was purified more than 40,000-fold with a series of ion exchange chromatography steps. The final preparation contained a doublet of peptides with molecular weights (Mr) of 101,000 and 96,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These components formed a complex that migrated on gel filtration with an apparent Mr of 280,000 in the absence or presence of 25-hydroxycholesterol. The amino acid sequence of a tryptic peptide from this protein complex was obtained, and a monoclonal antipeptide antibody was prepared. The antibody stained both the 101,000- and 96,000-Da proteins on immunoblots, suggesting that these two components are closely related and that one may be a modified or proteolyzed form of the other. With the purified protein now available, it should become possible to determine the role, if any, that this protein plays in the regulation of intracellular cholesterol metabolism.     

Purification and some properties of rabbit liver cytosol thioltransferase

Thioltransferase was purified 650-fold from rabbit liver by procedures including acid treatment, heat treatment, gel filtration on Sephadex G-50, column chromatography on DEAE-cellulose, isoelectric focusing (pH 3.5-10) and gel filtration on Sephadex G-75. The final enzyme preparation was almost homogeneous in polyacrylamide gel electrophoretic analysis. Only one active peak with an apparent molecular weight (Mr) of 13,000 was detected by gel filtration on Sephadex G-50 and only a single protein band with a molecular weight of 12,400 was detected by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Isoelectric focusing revealed only one enzyme species, having an isoelectric point (pI) of 5.3. The enzyme has an optimum pH about 3.0 with S-sulfocysteine and GSH as substrates. The purified enzyme utilized some disulfides including S-sulfocysteine, alpha-chymotrypsin, trypsin, bovine serum albumin, and insulin as substrates in the presence of GSH. The enzyme does not act as a protein : disulfide isomerase (the activity of which can be measured in terms of reactivation of randomly reoxidized soybean Kunitz trypsin inhibitor). The enzyme activity was inhibited by chloramphenicol, but not by bacitracin. The inhibition by chloramphenicol was non-competitive (apparent K1 of 0.5 mM). Thioltransferase activity was found in the cytosol of various rabbit tissues.     

Purification and characterization of formaldehyde dehydrogenase from rat liver cytosol.

Formaldehyde dehydrogenase was purified to electrophoretic and column chromatographic homogeneity from rat liver cytosolic fraction by a procedure which includes ammonium sulfate precipitation, DEAE-cellulose-, hydroxyapatite-, Mono Q-chromatography, and gel filtration. Its molecular mass was estimated to be 41 kDa by gel filtration and SDS-PAGE, suggesting that it is a monomer. It utilized neither methylglyoxal nor aldehydes except formaldehyde as a substrate. It has been reported that liver class III alcohol dehydrogenase and formaldehyde dehydrogenase are the same enzyme and oxidize formaldehyde and long chain primary alcohols. However, the enzyme examined here did not use n-octanoi as a substrate. The Km values for formaldehyde and NAD+ were 5.09 and 2.34 microM at 25 degrees C, respectively. The amino acid sequences of 10 peptides obtained from the purified enzyme after digestion with either V8 protease or lysyl endopeptidase were determined. From these results, the enzyme was proved to be different from the previously described mammalian formaldehyde dehydrogenase and is the first true formaldehyde dehydrogenase to be isolated from a mammalian source.     

Purification and characterization of a dimer form of the cAMP-dependent protein kinase from mouse liver cytosol

A protein kinase that phosphorylates histones and polysomal proteins was partially purified from mouse liver cytosol. The active enzyme has a molecular mass of 100 kDa and a phosphorylatable subunit of 54 kDa. Biochemical as well as immunological data suggest that the enzyme is a heterodimer composed of the catalytic subunit of cyclic AMP-dependent protein kinase and the R_II regulatory subunit. This RC form does not seem to dissociate upon activation with 3, 5 cyclic AMP and exhibits identical specificity as the classical cAMP-dependent protein kinase (2.7.1.37). The enzyme is affected by the 3, 5 cyclic phosphates of adenosine mainly, but also of guanosine, uridine and cytidine in a substrate-dependent manner. Cyclic nucleotides slightly stimulate phosphate incorporation into histones, while phosphorylation of polysomal proteins in intact polysomes is dramatically increased. The substrate- specific stimulatory effects of 3, 5 cyclic nucleotides are due to repression of the inhibition exerted upon the reaction, by negatively charged macromolecules such as RNA, DNA and to a lesser extent heparin.     

Purification and characterization of 3-hydroxyisobutyrate dehydrogenase from rabbit liver

3-Hydroxyisobutyrate dehydrogenase (3-hydroxy-2-methyl propanoate: NAD+ oxidoreductase, EC 1.1.1.31) was purified 1800-fold from rabbit liver by detergent extraction, differential solubility in polyethylene glycol and (NH4)2SO4, and column chromatography on DEAE-Sephacel, phenyl-Sepharose, CM(carboxymethyl)-Sepharose, Affi-Gel Blue, and Ultrogel AcA-34. The enzyme had a native Mr of 74,000 and appeared to be a homodimer with subunit Mr = 34,000. The enzyme was specific for NAD+. It oxidized both S-3-hydroxyisobutyrate and R-3-hydroxyisobutyrate, but the kcat/Km was approximately 350-fold higher for the S-isomer. Steady state kinetic analysis indicates an ordered Bi Bi reaction mechanism with NAD+ binding before 3-hydroxyisobutyrate. The enzyme catalyzed oxidation of S-3-hydroxyisobutyrate between pH 7.0 and 11.5 with optimal activity between pH 9.0 and 11.0. The enzyme apparently does not have a metal ion requirement. Essential sulfhydryl groups may be present at both the 3-hydroxyisobutyrate and NAD+ binding sites since inhibition by sulfhydryl-binding agents was differentially blocked by each substrate. The enzyme is highly sensitive to product inhibition by NADH which may play an important physiological role in regulating the complete oxidation of valine beyond the formation of 3-hydroxyisobutyrate.     

Purification and characterization of two phosphorylase phosphatases from rabbit liver

Two phosphorylase phosphatase activities (I and III) have been purified from rabbit liver, with respective molecular weights of 117,000 and 230,000. Phosphatase III contained three different subunits of molecular weights 35,000, 67,000 and 80,000. Phosphatase I although majoritary in the preparation, was not homogeneous. Both phosphatases were dissociated by 2-mercaptoethanol treatment, releasing a catalytic subunit with a molecular weight of about 35,000. Phosphatases I and III activities responded very differently to incubation with trypsin and to ethanol precipitation. Phosphatase III was much more sensitive to inactivation by several ions and ATP than phosphatase I. On the basis of the obtained data, phosphatase I can be classified as a type-1 phosphatase and phosphatase III as a type-1 phosphatase.     

Purification and characterization of a carboxylesterase from rabbit liver lysosomes

Carboxylesterase [EC 3.1.1.1] was purified from rabbit liver lysosomes by means of detergent solubilization, and by hydroxyapatite, phenyl-Sepharose and chromatofocusing column chromatographies. The purified enzyme appeared to be homogeneous on SDS-polyacrylamide gel electrophoresis and its molecular weight was estimated to be 58,000. This enzyme was eluted at an isoelectric point of approximately 5.8 by chromatofocusing, and exhibited a broad pH optimum of between 6.0 and 9.0. The enzyme hydrolyzed 4-methylumbelliferyl esters of saturated fatty acids (C2-C12), and it also hydrolyzed p-nitrophenylacetate, methyl butyrate, and tributyrin, but not acetanilide. Its activity was completely inhibited by diisopropyl-fluorophosphate (DFP) and phenylmethylsulfonyl fluoride (PMSF) at 10(-4) M, but was not affected by eserine, or by alpha- or beta-naphthyl acetate at 10(-3) M. Various metal ions (Mg2+, Mn2+, Ca2+, Co2+, Cu2+, Zn2+, Ni2+) at 10(-3) M also had no effect on the enzyme activity.     

Purification and characterization of a multifunctional calmodulin-dependent protein kinase from canine myocardial cytosol

A calmodulin-dependent protein kinase from canine myocardial cytosol was purified 1150-fold to apparent homogeneity with a 1.5% yield. The purified enzyme had a M_r of 550,000 with a sedimentation coefficient of 16.6 S, and showed a single protein band with a M_r of 55,000 (55K protein), determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme had a specific activity of 1.6 μmol/mg protein/min, and K_a values of 67 nM and 1.1 μM for calmodulin and Ca²⁺, respectively, using chicken gizzard myosin light chain as substrate. Calmodulin bound to the 55K protein. The purified enzyme had a broad substrate specificity. Endogenous proteins including glycogen synthase, phospholamban, and troponin I from the canine heart were phosphorylated by the enzyme. These results suggest that the purified enzyme works as a multifunctional protein kinase in the Ca²⁺, calmodulin-dependent cellular functions of the canine myocardium, and that the enzyme resembles enzymes detected in the brain, liver, and skeletal muscle.     

Purification and characterization of NADP+-dependent 3 alpha-hydroxysteroid dehydrogenase from mouse liver cytosol

A monomeric 3 alpha-hydroxysteroid dehydrogenase with a molecular weight of 34,000 was purified to apparent homogeneity from mouse liver cytosol. The enzyme catalyzed the reversible oxidation of the 3 alpha-hydroxy group of C19-, C21-, and C24-steroids, reduced a variety of carbonyl compounds, and was inhibited by SH-reagents, synthetic estrogens, anti-inflammatory drugs, prostaglandins, and delta 4-3-ketosteroids. Although these properties are similar to those of the enzyme from rat liver cytosol, the mouse enzyme exhibited low dehydrogenase activity toward benzene dihydrodiol and some alicyclic alcohols, it showed a strict cofactor specificity for NADP(H), and high substrate inhibition was observed in the reverse reaction. In addition, dexamethasone, deoxycorticosterone, and medroxyprogesterone acetate inhibited the mouse enzyme competitively at low concentrations and noncompetitively at high concentrations, whereas hexestrol, indomethacin, and prostaglandin A1 were competitive inhibitors. Steady-state kinetic measurements in both directions indicated that the reaction proceeds through an ordered bi bi mechanism with the cofactors binding to the free enzyme. The 3-ketosteroid substrates inhibited the enzyme uncompetitively at elevated concentrations, suggesting that the substrates bind to the enzyme.NADPH complex and to the enzyme NADP+ complex.     

Purification and characterization of the alpha-D-mannosidase of rat liver cytosol.

Three forms of alpha-D-mannosidase have previously been identified in rat liver, and each is localized in a different subcellular fraction: lysosomes, Golgi membranes, and cytosol. This communication reports the purification and characterization the cytosolic form. The enzyme was purified 12,000-fold in good yield to approximately 90% purity with the aid of the competitive inhibitor mannosylamine and dithioerythritol as stabilizers. The molecular weight of the enzyme is in the range of 372,000 to 490,000 depending on the method used. Since the subunit molecular weight is 110,000 by sodium dodecyl sulfate polyacrylamide electrophoresis, the enzyme is probably a tetramer. The pH optimum was shown to be between 5.5 and 5.9 (in the presence of 1 mM CoCl2) with the substrate p-nitrophenyl-alpha-D-mannoside. Normal Michaelis-Menten kinetics were observed with a Km of 0.14 mM. Mannosylamine was a competitive inhibitor with a Ki of 0.007 mM. The purified enzyme, stabilized by Co2+, Mn2+, and Fe2+ under some conditions, was unstable at low protein concentrations. Since an electrophoresed sample showed a positive periodic acid-Schiff stain, the enzyme may contain carbohydrate. The availability of purified cytosolic alpha-D-mannosidase should now make it possible to carry out substrate specificity, immunological, and structural studies which may shed light on the biological role of this enzyme.     

Purification and characterization of 7beta-hydroxysteroid dehydrogenase from rabbit liver microsomes

7beta-Hydroxysteroid dehydrogenase (7beta-HSD), a specific enzyme active in the metabolization of 7beta-hydroxycholesterol, was purified about 300-fold from male rabbit liver microsomes using ion exchange, hydroxylapatite, 2'5'ADP Sepharose 4B, and high-performance liquid chromatography on the basis of its catalytic activity. The specific activity of the purified enzyme was 276 nmol/min/mg protein. The molecular weight of the purified enzyme was 34,000. The preferred coenzyme was beta-NADP+. The optimum pH for oxidation was around 7.7 in potassium phosphate buffer, and 11.0 in glycine-NaOH buffer. The purified enzyme catalyzed the synthesis of not only 7beta-hydroxycholesterol but also corticosterone and hydrocortisone. Enzyme activities toward these three substrates accompanied all purification steps of 7beta-HSD. The amino acid sequence of the N-terminal of the purified enzyme showed that 7beta-HSD had sequence similarity to rabbit type I 11beta-hydroxysteroid dehydrogenase (11beta-HSD), indicating that 7beta-HSD may belong to the rabbit type I 11beta-HSD family and may play the same role in the metabolism of 11-hydroxysteroids and 7-hydroxysterols.     

Purification and properties of an angiotensin-binding protein from rabbit liver particles

An angiotensin II-binding protein was purified more than 8000-fold after solubilization from rabbit liver particles with digitonin. The procedure comprised fractionation with ammonium sulfate, chromatography on DEAE-cellulose and Affi-Gel 501, gel filtration through Sephacryl S-200, and chromatography with hydroxylapatite. The purified preparation exhibited Kd and Bmax values of 6.7 nM and 8.4 nmol of angiotensin II bound/mg protein. The latter figure represents more than 60% of the theoretical value calculated for a protein of Mr 75,000 as estimated for the major protein component by gel electrophoresis. The purified preparation displayed comparable or slightly higher affinities for various angiotensin antagonists and angiotensin III than that for angiotensin II, whereas angiotensin I as well as the hexapeptide and smaller carboxy-terminal fragments were less tightly bound. Binding of angiotensin II by the isolated protein was highly dependent upon the presence of p-chloromercuriphenylsulfonic acid and also required ethylene diaminetetraacetic acid which could be almost completely replaced by ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid but not by o-phenanthroline.     

Purification and characterization of cytosol phosphoenolpyruvate carboxykinase from bullfrog (Rana catesbeiana) liver.

Cytosol PEP carboxykinase has been purified to electrophoretic homogeneity from bullfrog liver homogenate. The enzyme is a single polypeptide chain with a molecular weight of approximately 72,000-75,000. The purified enzyme catalyzed oxaloacetate decarboxylation (nucleoside triphosphate-supported), phosphoenolpyruvate carboxylation, and an exchange reaction between oxaloacetate and [14C]HCO3-in the presence of ITP or CTP. Manganese is absolutely required for the enzyme-catalyzed phosphoenolpyruvate carboxylation, whereas it can be replaced by Mg2+ for the oxaloacetate decarboxylation and the exchange reaction. The optimal pH of each reaction is dependent on the divalent metal ion used. The dependence of the enzyme activity on Mn2+ is markedly different in the phosphoenolpyuvate carboxylation and the oxaloacetate decarboxylation reactions.     

Purification and characterization of the isozymes of phosphoenolpyruvate carboxykinase from rabbit liver

Procedures are described for the purification of the mitochondrial and cytosolic isozymes of phosphoenolpyruvate carboxykinase from rabbit liver. Examination of the purified isozymes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated apparent homogeneity and identical molecular weights of approximately 65,000. Gel filtration chromatography of the native isozymes, however, yielded apparent molecular weights of 68,000 and 56,000 for the cytosolic and mitochondrial isozymes, respectively. The isoelectric points as determined by chromatofocusing were 5.8 for the mitochondrial isozyme and 5.0 for the cytosolic isozyme. The purified isozymes were readily separable on ion-exchange columns, with the cytosolic isozyme showing the greater affinity. A minor amount of cross-reactivity was apparent when each isozyme was immunotitrated with polyclonal antibodies raised in goat against the opposite isozyme. Peptide maps obtained by high pressure liquid chromatography of both tryptic digests and cyanogen bromide digests of the isozymes showed that many of the peaks were not coincident, suggesting that differences in the sequences are found throughout the primary structures of the isozymes.     

Purification and partial characterization of glycogen synthase kinase-3 from rabbit liver

Summary Glycogen synthase kinase-3 (GSK-3) was purified from rabbit liver to homogeneity by ultracentrifugation, ion-exchange chromatography on DEAE-cellulose, Cellulose phosphate, CM-Sephadex and Fast Protein Liquid Chromatography (FPLC) on Mono-S column. The enzyme was purified approximately 20,000 fold with an approximate 2% recovery. The purified enzyme showed a single band on SDS-polyacrylamide gel electrophoresis. GSK-3 is a monomeric enzyme with a molecular weight of 50,000–52,000 as derived from SDS-polyacrylamide gel electrophoresis and gel filtration. The purified enzyme was indeed a GSK-3 since it phosphorylated three sites, i.e., 3a, 3b, and 3c on liver glycogen synthase. GSK-3 incorporated up to 2.6 mol Pi/mol glycogen synthase subunit with a concomitant inactivation of glycogen synthase activity.