Hypoxanthine phosphoribosyltransferase from human brain: purification and partial characterization

A facile and rapid purification procedure, based upon the heat denaturation of extraneous proteins and GMP-Sepharose affinity chromatography, has been used to purify hypoxanthine phosphoribosyltransferase from human brain. A homogeneous enzyme preparation, as judged by sodium dodecyl sulfate and gradient polyacrylamide gel electrophoresis, was obtained. The subunit molecular weight of the enzyme was estimated as 24,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The native molecular weight, determined by gradient gel electrophoresis, was approximately 100,000. These results suggest human brain hypoxanthine phosphoribosyltransferase is a tetramer, consistent with recent results reported for the human erythrocyte enzyme. At least three charge variant forms of the human brain enzyme were distinguished by nondenaturing polyacrylamide gel electrophoresis, electrofocusing, and chromatofocusing. Acidic pI values of approximately 5.7, 5.5, and 5.0 were estimated for the three major species.     

Purification and Properties of Succinyl-CoA:3-Oxo-Acid CoA-Transferase from Rat Brain

Abstract: Rat brain succinyl-CoA:3-oxo-acid CoA-transferase (3-Oxo-acid CoA-transferase, EC 2.8.3.5), the first committed enzyme in the oxidation of ketone bodies in mitochondria, was purified to apparent homogeneity as judged by polyacrylamide gel electrophoresis. The enzyme has an apparent molecular weight of 90,000 as determined by (3-150 Sephadex chromatography, and an apparent subunit molecular weight of 53,000 as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The specific activity of the purified enzyme was approximately 161 μmol/min/mg of protein. Initial velocity studies of the forward reaction (acetoacetate → acetoacetyl-CoA) are consistent with a "ping pong" mechanism. Substrate inhibition appears above approximately 1 m M acetoacetate. Apparent K_m, values were 70 μM for acetoacetate and 156 μ M for succinyl-CoA (the forward reaction), and 59 μ M for acetoacetyl-CoA and 25 m M for succinate (the reverse reaction). These values are markedly different from those reported for this enzyme from pig heart.     

Purification de l'alcool deshydrogénase de tomate par chromatographie d'affinité

Tomato alcohol dehydrogenase has been purified 99-fold by affinity chromatography on Blue Sepharose CL-6B with 37% yield. The enzyme so obtained is homogenous in polyacrylamide gel electrophoresis. By adding 20% glycerol to the extraction and purification buffers, an enzyme is obtained which is stable for several months at 4°. The molecular weight values determined by gel filtration (Sephadex G 200) and polyacrylamide gradient gel electrophoresis on one hand and by polyacrylamide gel electrophoresis in sodium dodecyl sulfate on the other, show that the enzyme exists in dimeric form.     

Purification and properties of an NADP-specific 6-phosphogluconate dehydrogenase from Streptococcus faecalis.

A procedure is described for the purification of 6-phosphogluconate dehydrogenase (6-phospho-D-gluconate:NADP oxidoreductase (decarboxylating) EC 1.1.1.44) from cell extracts of Streptococcus gaecalis. A 180-fold purification was achieved with an over-all yield of about 12% and an average specific activity of 14. The enzyme was homogeneous as determined by polyacrylamide gel electrophoresis, immunoelectrophoresis, and sedimentation equilibrium, studies. Its weight average molecular weight, as measured by sedimentation equilibrium, was 108,000 +/- 3,600. Other methods employed for molecular weight determinations gave values that ranged between 106,000 and 115,000. An analysis of the enzyme by sodium dodecyl sulfate polyacrylamide gel electrophoresis showed it to be a dimer composed of subunits having equal molecular weight. The amino acid composition of the streptococcal enzyme is reported. The apparent Km values for NADP and 6-phosphogluconate were calculated from kinetic data and found to be 0.015 mM and 0.024 mM, respectively. Kinetic studies also indicated that the binding of one substrate did not affect the apparent affinity of the enzyme for the other substrate.     

Purification and properties of ornithine decarboxylase from rat liver

Ornithine decarboxylase was purified to homogeneity, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and polyacrylamide gel electrofocusing, about 710,000-fold with a 35% yield from the liver cytosol of thioacetamide-treated rats. The final specific activity was approximately 24,400 nmol/min/mg of protein. The apparent molecular weight of the enzyme determined by gel filtration analyses on Sephacryl S-200 was 55,000 in the presence of 0.25 M NaCl and 145,000 in its absence. The minimum molecular weight of the enzyme was determined to be 54,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point of the enzyme was estimated as 5.7 in the presence of 8 M urea. Some catalytic properties of the enzyme were also studied.     

3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase. Purification and molecular characterization of the phenylalanine-sensitive isoenzyme from Escherichia coli.

The phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (7-phospho-2-keto-3-deoxy-D-arabino-heptonate D-erythrose-4-phosphate lyase (pyruvate phosphorylating), EC 4.2.1.15) was purified to apparent homogeneity from extracts of Escherichia coli K12. The enzyme has a molecular weight of 140,000 as judged by gel filtration and sedimentation equilibrium analysis. The enzyme has a subunit molecular weight of 35,000 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, suggesting that the native form of the enzyme is a tetramer. This was confirmed by cross-linking the enzyme with dimethylsuberimidate and by analyzing the cross-linked material by gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme shows a narrow pH optimum between pH 6.5 and 7.0. The enzyme is stable for several months when stored at -20 degrees C in buffers containing phosphoenolpyruvate. Removal of phosphoenolpyruvate causes an irreversible inactivation of the enzyme. The enzyme is strongly inhibited by L-phenylalanine and to a lesser degree by dihydrophenylalanine. Molecular parameters of the previously isolated tyrosine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase from E. coli (Schoner, R., and Herrmann, K.M. (1976) J. Biol. Chem. 251, 5440-5447) are compared with those of the phenylalanine-sensitive isoenzyme from the same organism.     

delta-Aminolevulinic acid synthetase from rat liver mitochondria. Purification and properties.

delta-Aminolevulinic acid synthetase has been purified from liver mitochondria of young, uninduced rats. After nonionic detergent solubilization of mitochondrial inner membrane-matrix fractions, the enzyme was purified to a specific activity of approximately 2,000 nmol of delta-aminolevulinic acid formed/h/mg of protein at 30 degrees C, by means of ammonium sulfate precipitation, diethylaminoethyl cellulose chromatography, Sephacryl chromatography, and preparative gel electrophoresis. The purified enzyme preparation thus obtained was apparently homogeneous as judged by its migration as a single band with a molecular weight of 58,000 +/- 6,000 upon electrophoresis in sodium dodecyl sulfate polyacrylamide gels. The native enzyme probably exists as a dimer with a molecular weight of approximately 120,000. A pH optimum of 7.5 and an isoelectric point of 4.5 were also determined. Both monovalent cations and hemin strongly inhibited the activity of the purified enzyme.     

Prenyltransferase from Saccharomyces cerevisiae. Purification to homogeneity and molecular properties.

Prenyltransferase (EC 2.5.1.1) has been purified to homogeneity from the supernatant fraction of yeast by ammonium sulfate fractionation, diethylaminoethyl-cellulose and hydroxylapatite chromatography, and column isoelectric focusing techniques. The active enzyme from isoelectric focusing columns emerged as a single symmetrical peak with specific activities 15- to 35-fold higher than previously reported preparations. The enzyme was found to be homogeneous by continuous polyacrylamide gel electrophoresis at pH 8.4 and discontinuous polyacrylamide gel electrophoresis at pH 6.9 as well as sodium dodecyl sulfate polyacrylamide electrophoresis at pH 7.0. By means of gel chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis, the protein was shown to be a dimer with a molecular weight of 84,000 plus or minus 10%. The isoelectric point of the enzyme was determined to be 5.3. The enzyme synthesizes farnesyl and geranylgeranyl pyrophosphates from dimethylallyl, geranyl, and farnesyl pyrophosphates. Michaelis constants for the enzyme were 4, 8, and 14 mu M for isopentenyl, dimethylallyl, and geranyl pyrophosphates, respectively.     

Sulfation and molecular weight of fibronectin shed by human melamona cells

Intrinsic radiolabeling, affinity chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis of fibronectin shed by cultured human melanoma cells unexpectedly showed that it is sulfated and has a higher apparent molecular weight than plasma fibronectin. The implications of these findings are discussed.     

Purification of nitric oxide synthase from rat macrophages.

Nitric oxide (NO) synthase (EC 1.14.23) has been purified to apparent homogeneity from rat macrophages. The purification procedure involves affinity chromatography with adenosine 2',5'-diphosphate-agarose and gel filtration chromatography on a Superose 12 HR 10/30 column. The apparent molecular weight is 300,000 by gel filtration. On polyacrylamide gel electrophoresis in sodium dodecyl sulfate, the enzyme migrates as a single protein band with Mr = 150,000. The purified enzyme is colorless, and an absorption maximum is observed at 280 nm. The half-life of the enzyme activity is 6 h at pH 7.4 and 4 degrees C. The enzyme activity required the presence of NADPH, (6R)-5,6,7,8-tetrahydro-L-biopterin, and dithiothreitol. Although the cerebellar and endothelial enzyme require Ca2+ and calmodulin, these are not required by the macrophage enzyme. The macrophage nitric oxide synthase (an inducible enzyme) seems to be different from the cerebellar and endothelial enzyme (a constitutive enzyme).     

Purification and characterization of cholesterol esterase from porcine pancreas.

A protein catalyzing the hydrolysis of cholesterol esters and p-nitrophenyl acetate has been purified 200-fold from porcine pancreas. The enzyme is homogenous as judged by polyacrylamide gel electrophoresis and exhibits a molecular weight of 80 000 as determined by sodium dodecyl sulfate electrophoresis and gel filtration. Activity toward p-nitrophenylacetate exhibits a broad pH optimum and is influenced by a group with a pKa of 5.5--6.0. The enzyme is completely inhibited by diisopropylfluorophosphate at concentrations as low as 10(-5) M, suggesting that it is a serine esterase. Partial inhibition was observed with p-chloromercuribenzoate.     

Oxidation of fatty alcohol in the cotyledons of jojoba seedlings.

An externally accessible polypeptide has been purified from hepatoma tissue culture cells. The purification involves four steps: deoxycholate extraction of whole cells, isoelectric focusing of deoxycholate-insoluble material in the presence of 8 m urea and Triton X-100, hydroxylapatite chromatography in the presence of sodium dodecyl sulfate, and preparative acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The final preparation is homogeneous, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by isoelectric focusing in polyacrylamide. The polypeptide has an apparent molecular weight of 55,000 and is labeled following in situ lactoperoxidase-catalyzed iodination of the hepatoma tissue culture cells. The polypeptide can also be labeled by growing cells in the presence of labeled amino acids, but is not labeled by growth in labeled sugars. The purified protein does not react with the periodate-Schiff reagent. Hence, it does not appear to be a glycoprotein that contains mannose, fucose, glucosamine, or sialic acids.     

Isolation of collagen glucosyltransferase as a homogeneous protein from chick embryos

Collagen glucosyltransferase was isolated as a homogeneous protein from chick embryos by a procedure consisting of ammonium sulphate fractionation, two affinity chromatographies and two gel filtrations. The specific activity of the purified enzyme was 32,000 times that of the 15,000 x g supernatant of the embryo homogenate, and the enzyme was pure when examined by sodium dodecyl sulphate polyacrylamide gel electrophoresis using three different gel compositions. The molecular weight of the enzyme was about 72,000-78,000 by sodium dodecyl sulphate polyacrylamide gel electrophoresis, the value being dependent on the gel composition. The apparent molecular weight by gel filtration was dependent on the purity and protein concentration. The sedimentation coefficient S20,w was 4.7. The data suggest that the enzyme molecule consists of one polypeptide chain.     

Purification and characterization of a plasminogen activator secreted by cultured human pancreatic carcinoma cells.

A plasminogen activator secreted by cultured human pancreatic carcinoma (Mia PaCa-2) cells has been purified to apparent homogeneity by procedures including Sepharose-L-arginine methyl ester affinity chromatography, Sephadex G-200 gel filtration, isoelectric focusing, and sodium dodecyl sulfate gel electrophoresis. The plasminogen activator shares many properties with urokinase including: molecular weight (55 000), isoelectric point (8.7), heat stability (60 degrees C, 30 min), PH stability (1.5-10), and its mode of activation of plasminogen. The intracellular enzyme is membrane bound and can be solubilized by detergent. Solubilized activator has a molecular weight similar to that of the secreted enzyme as determined by sodium dodecyl sulfate gel electrophoresis. The production of plasminogen activator by Mia PaCa-2 cells is totally inhibited by actinomycin D and cycloheximide.     

Liver microsomal expoxide hydrase. Solubilization, purification, and characterization.

Epoxide hydrase was solubilized from liver microsomes of phenobarbital-treated rats by treatment with cholate and purified to apparent homogeneity by ammonium sulfate fractionation and column chromatography in the presence of the nonionic detergent Emulgen 911 on DEAE-cellulose and hydroxylapatite. The purified enzyme preparation had a single major band with a molecular weight of 53,000 to 54,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Other studies indicated that in the absence of sodium dodecyl sulfate, purified epoxide hydrase exists as high molecular weight aggregates. The preparation was essentially free of heme and flavin, but still contained small amounts of lipids and Emulgen 911.     

Purification and properties of beta-N-acetylglucosaminidase from Escherichia coli.

beta-N-acetylglucosaminidase (EC 3.2.1.30) has been purified from Escherichia coli K-12 to near homogeneity based on polyacrylamide gel electrophoresis in both 0.5% sodium dodecyl sulfate and in 6 M urea at pH 8.5. The purified enzyme shows a pH optimum of 7.7 and the Km for p-nitrophenyl-beta-D-2-acetamido-2-deoxyglucopyranoside is 0.43 mM. The molecular weight of this enzyme, determined by both Sephadex gel filtration and by sodium dodecyl sulfate gel electrophoresis, is equivalent to 36,000. It is shown to be a soluble cytoplasmic enzyme. Studies on the substrate specificites of the purified enzyme indicate that this enzyme is an exo-beta-N-acetylglucosaminidase.     

Isolation of phospholipase A2 from sheep erythrocyte membranes in the presence of detergents

Isolation of phospholipase A₂ (EC 3.1.1.4) from sheep erythrocyte membranes was carried out by a combination of (1) extraction of membranes at low ionic strength, (2) solubilization of extracted membranes with sodium dodecyl sulfate, (3) replacement of dodecyl sulfate with cholate by means of gel exclusion chromatography and (4) affinity chromatography on dialkyl-phosphatidylcholine-Sepharose in the presence of cholate. The phospholipase was prepared with good yield and purified to near homogeneity, as judged by sodium dodecyl sulfate gel electrophoresis. The protein is a minor component of the sheep erythrocyte membrane and has an apparent molecular weight of 18 500.     

Isolation and characterization of yeast nicotinamide adenine dinucleotide kinase.

NAD+ kinase (ATP:NAD+ 2'-phosphotransferase, EC 2.7.1.23) from yeast has been purified utilizing ion-exchange and NAD+-agarose affinity chromatography to give a 2100-fold purification. The apparent homogeneity of the enzyme preparation was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and analytical ultracentrifugation. The enzyme has a subunit molecular weight of 31,000, and a native molecular weight of 124,000, and is, thus, probably a tetramer. The single form of the enzyme has an apparent isoelectric point of 5.85. Initial velocity studies in the forward direction with both substrates gave intersecting Lineweaver-Burk plots, and this suggests a sequential mechanism in which both substrates are bound before products are released. Replots of these data were linear and gave Km values for NAD+ and ATP of 0.68 mM and 2.3 mM, respectively.     

Purification and characterization of a long-chain acyl-CoA hydrolase from rat liver microsomes.

A long-chain acyl-CoA hydrolase from rat liver microsomes has been purified by solvent extraction and gel chromatography to homogeneity as judged by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. The enzyme was a monomer of molecular weight 59 000. In a sucrose gradient it sedimented at 4.3 S. The isoelectric point, pI was 6.9, and the Stokes radius was approx. 31 A. The enzyme hydrolyzed long-chain fatty acyl-CoA (C7--C18) with maximum activity for palmitoyl-CoA. Bovine serum albumin activation of the enzyme was related to the ratio acyl-CoA/bovine serum albumin, and at high ratios, acyl-CoA inhibited the enzyme activity. Disregarding the substrate inhibition, an apparent Km of 65 nmol/mg protein or 1-10(-7) M and a V of 750 nmol/mg protein per min were calculated. The enzyme was inhibited by p-hydroxymercuribenzoate and N-ethylmaleimide. Reactivation by means of dithiothreitol was not complete.     

Subunit structure of Mycobacterium smegmatis fatty acid synthetase. Evidence for identical multifunctional polypeptide chains

Fatty acid synthetase from Mycobacterium smegmatis has been purified to near homogeneity as judged by a variety of electrophoretic criteria under both native and dissociating conditions. A single protein band was obtained on gel electrophoresis in sodium dodecyl sulfate or 8 M urea at various pH values and on isoelectric focusing in 8 M urea. A subunit molecular weight of about 290,000 was found by polyacrylamide gel electrophoresis in sodium dodecyl sulfate or by sedimentation equilibrium ultracentrifugation in 6 M guanidine HCl. Quantitative Quantitative determination of pantetheine, of flavin, and of the number of fatty acids synthesized during a single enzyme turnover all yield values corresponding to a stoichiometry of about 1 mol per mol of subunit, providing strong evidence that M. smegmatis fatty acid synthetase is an oligomer of identical, multifunctional polypeptide chains.