Purification and characterization of myosin from bovine retina

Myosin has been isolated from bovine retinae and characterised by its ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity, its mobility in sodium dodecyl sulphate polyacrylamide gels and by electron microscopy. The purified myosin shows high ATPase activity in the presence of EDTA or Ca²⁺ and a low activity in the presence of Mg²⁺. The Mg²⁺-dependent ATPase activity is stimulated by rabbit skeletal muscle actin. The presumptive retinal myosin possesses a major component which has a mobility in sodium dodecyl sulphate polyacrylamide gel electrophoresis similar to that of the heavy chain of bovine skeletal mucle myosin. Electron microscopy showed retinal myosin to form bipolar filaments in 0.1 M KCl. It is concluded that the retina possesses a protein with enzymic and structural properties similar to those of muscle myosin.     

Purification and characterization of selenium-glutathione peroxidase from hamster liver

Hamster liver glutathione peroxidase was purified to homogeneity in three chromatographic steps and with 30% yield. The purified enzyme had a specific activity of approximately 500 μmol cumene hydroperoxide reduced/min/mg of protein at 37 °C, pH 7.6, and 0.25 mm GSH. The enzyme was shown to be a tetramer of indistinguishable subunits, the molecular weight of which was approximately 23,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A single isoelectric point of 5.0 was attributed to the active enzyme. Amino acid analysis determined that selenocysteine, identified as its carboxymethyl derivative, was the only form of selenium. One residue of cysteine was found to be present in each glutathione peroxidase subunit. The presence of tryptophan was colorimetrically determined. Pseudo-first-order kinetics of inactivation of the enzyme by iodoacetate was observed at neutral pH with GSH as the only reducing agent. An optimal pH of 8.0 at 37 °C and an activation energy of 3 kcal/mol at pH 7.6 were found. A ter-uni-ping-pong mechanism was shown by the use of an integrated-rate equation. At pH 7.6, the apparent second-order rate constants for reaction of glutathione peroxidase with hydroperoxides were as follows: k₁ (t-butyl hydroperoxide), 7.06 × 10⁵ mm min^?1; k₁ (cumene hydroperoxide), 1.04 × 10⁶ mm^?1 min^?1; k₁ (p-menthane hydroperoxide), 1.2 × 10⁶ mm^?1 min^?1; k₁ (diisopropylbenzene hydroperoxide), 1.7 × 10⁶ mm^?1 min^?1; k₁ (linoleic acid hydroperoxide), 2.36 × 10⁶ mm^?1 min^?1; k₁ (ethyl hydroperoxide), 2.5 × 10⁶ mm^?1 min^?1; and k₁ (hydrogen peroxide), 2.98 × 10⁶ mm^?1 min^?1. It is concluded that for bulky hydroperoxides, the more hydrophobic the substrate, the faster its reduction by glutathione peroxidase.     

Purification and characterization of a protein inhibitor of calcium-dependent proteases from rat liver

Soluble extracts of rat liver contain a protein inhibitor of calcium-dependent proteases. The inhibitor has an apparent M_r = 250,000 and is separated from the calcium-dependent proteases by gel-filtration chromatography in the presence of EGTA. The inhibitor has been purified by affinity chromatography using a calcium-dependent protease covalently linked to Affi-Gel 15. The inhibitor specifically binds to this affinity resin in a calcium-dependent manner and elutes in the presence of EDTA or EGTA. The purified inhibitor appears as a single protein with M_r = 125,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Presumably it is a dimer under nondenaturing conditions. The inhibitor inhibits each of two calcium-dependent proteases from rat liver and from other tissues and species. However, it has no effect on any other protease tested.     

Purification and immunochemical characterization of a low-pI form of UDP glucuronosyltransferase from mouse liver

A liver UDP glucuronosyltransferase (GT) enzyme from either phenobarbital- or 3-methylcholanthrene-treated C57BL/6N mice was isolated by phenyl-Sepharose, DEAE-ion exchange, and UDP hexanolamine chromatographic steps. This enzyme had a broad substrate specificity and was mainly responsible for the microsomal capacity to glucuronidate testosterone, 1-naphthol, and morphine. This UDP glucuronosyltransferase ( GTM1 ) appeared to be at least 95% homogeneous and had a subunit molecular weight of 51,000 using sodium dodecyl sulfate-polyacrylamide gel and two-dimensional gel electrophoreses. Antibodies prepared against the purified protein developed a single immunoprecipitin line by double-diffusion analysis with purified antigen and with solubilized microsomes from both control and drug-induced C57BL/6N and DBA/2N mice. A precipitin line was also observed with microsomal proteins which isoelectrofocused at approximately pH 6.7, but not with those which isoelectrofocused at approximately pH 8.5. GTM1 was, therefore, designated at low-pI form. Immunopurified antibody preferentially inhibited and immunoprecipitated GT activities toward testosterone, 1-naphthol, and morphine. To a lesser extent, activities toward phenolphthalein, 3-hydroxybenzo[a]pyrene, and estrone were inhibited while activities toward 4-nitrophenol and 4-methylumbelliferone were not affected. All activities, however, were immunoadsorbed in the presence of protein A-Sepharose. This observation can be explained by the following results. Immunoprecipitates from labeled microsomes contained primarily a 51,000-Da protein. When the immune complexes were adsorbed with protein A-Sepharose, a 54,000-Da protein as well as the expected 51,000-Da GTM1 was detected. This 54,000-Da protein was associated with the glucuronidation of 3-hydroxybenzo[a]pyrene and 4-nitrophenol, and was designated GTM2 .     

Purification and characterization of glucose-6-phosphate dehydrogenase from Aspergillus parasiticus

Glucose-6-phosphate dehydrogenase (EC 1.1.1.49) was purified from mycelium of Aspergillus parasiticus (1-11-105 Whl). The enzyme had a molecular weight of 1.8 × 10⁵ and was composed of four subunits of apparently equal size. The substrate specificity was very strict, only glucose 6-phosphate and glucose being oxidized by NADP or thio-NADP. Zinc ion was a powerful inhibitor of the enzyme, inhibition being competitive with respect to glucose 6-phosphate, with K_i about 2.5 μm. Other divalent metal ions which also serve as inhibitors are nickel, cadmium, and cobalt. It is proposed that the stimulation of polyketide synthesis by zinc ion may be mediated in part by inhibition of glucose-6-phosphate dehydrogenase.     

Purification and characterization of the soluble form of mitochondrial adenosine triphosphatase from sweet potato

The soluble form of mitochondrial adenosine triphosphatase was purified in an electrophoretically and immunologically pure form from sweet potato root tissue. The enzyme consisted of six kinds of subunits with different molecular weights (52,500, 51,500, 35,500, 26,000, 23,000, and 12,000), and its molecular weight was about 370,000. Adenosine triphosphatase associated with the submitochondrial particles was oligomycin-sensitive and heat-labile, whereas the soluble form of the enzyme was oligomycin-insensitive and cold-labile. The enzyme in either the membrane-bound or the soluble form showed negative cooperativity. Both experiments with polyacrylamide gel electrophoresis and immunological methods suggest that some of the subunits, probably those with molecular weights of 52,500 and 51,500, are dissociated from the enzyme protein during storage of the enzyme preparations.     

Purification and molecular characterization of human fibroblast interferon

Human fibroblast interferon was purified from serum-containing culture medium by a combination of concanavalin A or Blue Dextran Sepharose affinity chromatography with high-performance liquid chromatography to material exhibiting a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The interferon could be chromatographed and purified at acidic pH in volatile buffers on RP-8, RP-18, cyclohexyl, phenylalkyl, diphenyl, cyanopropyl, and diol supports. A specific activity averaging around 4 × 10⁸ units/mg was found for the pure material with a molecular weight of 20,000–21,000 after 20,000- to 50,000-fold purifications. In some preparations, low activity levels were also found at positions corresponding to 10,000, 17,000–18,000, 35,000, and 40,000 daltons. Amino acid and amino sugar analysis, partial NH₂- and COOH-terminal sequences, and tryptic peptide patterns determined at the picomole level are reported for the purified interferon.     

Purification and characterization of a xylobiose- and xylose-producing endo-xylanase from Aspergillus niger

A xylanase from a commercial Aspergillus niger pentoglycanase was purified to homogeneity by column chromatography on Ultrogel AcA 54, SP-Sephadex, Sephadex G-50, and SP-Sephadex. The enzyme hydrolyzed xylotriose slowly to xylose and xylobiose, and xylotetraose and higher xylo-oligosaccharides rapidly to mixtures of smaller xylo-oligosaccharides, with xylobiose and xylose being the preponderant final products. The anomeric configuration of the products was inverted, in contrast to the behavior of most other carbohydrases that initially produce mixtures of oligosaccharides. This enzyme is a glycoprotein having an amino acid composition high in acidic residues. Its molecular weight is 20,800 and its isoelectric point is at pH 6.7. Optimal pH values for activity and stability are between 4 and 6 and, in a 20-min assay, maximal activity is attained at 55°.     

Purification and characterization of the individual glutathione S-transferases from sheep liver

The glutathione S-transferases (EC 2.5.1.18) have been purified to electrophoretic homogeneity from 105,000g supernatant of sheep liver homogenate by employing a combination of gel filtration on Sephadex G-150 and affinity chromatography on S-hexylglutathione-linked Sepharose-6B columns. Approximately 70% of the original glutathione S-transferase activity toward 1-chloro-2,4-dinitrobenzene and glutathione peroxidase activity toward cumene hydroperoxide could be recovered by this purification method. Of particular importance in developing this procedure was the fact that the enzyme preparation obtained after affinity column chromatography represented all the isozymes of sheep liver glutathione S-transferases. Further purification by CM-cellulose and DEAE-cellulose column chromatography resolved the glutathione S-transferases into seven distinct cationic isozymes designated C-1, C-2, C-3, C-4, C-5, C-6, and C-7 and five overlapping anionic transferases designated A-1, A-2, A-3, A-4, and A-5, respectively, in the order of their elution from the ion-exchange columns. The sodium dodecyl sulfate SDS-gel electrophoretic data on subunit composition revealed that cationic enzymes are composed of two subunits with an identical Mr of 24,000 whereas a predominant subunit with Mr of 26,000 was observed in all anionic isozyme peaks except A-1. Cationic isozymes accounted for approximately 98% of the total peroxidase activity associated with the glutathione S-transferase whereas only A-1 of the anionic isozymes displayed some peroxidase activity. Isozyme C-4 was found to be the most abundant glutathione S-transferase in the sheep liver. Characterization of the individual transferases by their specificity toward a number of selected substrates, subunit composition, and isoelectric points showed some similarities to those patterns for human liver glutathione S-transferases.     

Purification of rat liver monoamine oxidase by octyl glucoside extraction and reconstitution

Catalytically active isoenzymes of rat liver monoamine oxidase have been copurified from the outer mitochondrial membrane by a novel method involving repetitive solubilization with octyl-β-d-glucopyranoside followed by reconstitution into lipid vesicles. As analyzed using sodium dodecyl sulfate-gel electrophoresis, the purified enzyme migrates as a single band of protein of molecular weight 60,000. The preparation is capable of metabolizing 576 nmol serotonin and 777 nmol β-phenylethylamine/min/mg protein. Apparent K_m values and sensitivity to the inhibitor clorgyline are very similar for the purified and outer mitochondrial membrane-bound enzyme when determined with the substrates β-phenylethylamine, serotonin, and tyramine.     

Purification and characterization of a vanadate-sensitive nucleotide tri- and diphosphatase with acid pH optimum from rat bone

Rat bone was extracted with KCl and Triton X-100, and a tartrate-resistant acid phosphatase activity was purified by protamine sulfate precipitation, ion-exchange chromatography (CM-cellulose), and gel filtration on Sephadex G-200 according to previously described procedures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining demonstrated a major band with an apparent monomer molecular size of approximately 14,000 Da. The enzyme is active with p-nitrophenylphosphate (p-NPP) but exhibits a 5- to 10-fold higher affinity towards several nucleotides of which ATP and ADP are the most readily hydrolyzed substrates based on kinetic studies. Based on sensitivity towards proteolytic treatment and detergent removal, as well as pH-optimum studies, a single enzyme was found to be responsible for activity towards nucleotide phosphates as well as p-NPP. This nucleotide tri- and diphosphatase constitutes around 15% of the total acid phosphatase activity in rat bone. The activity with ATP as substrate in contrast to that with p-NPP was inhibited in a noncompetitive fashion by MgCl2, sodium metavanadate, and p-chloromercuribenzoate. Enzyme activity with p-NPP and ATP is dependent on the presence of KCl and detergent and is activated by Fe3+ and ascorbate. The reported characteristics of the enzyme suggest that it functions as a unique membrane acid ATPase.     

Purification and chemical characterization of polysaccharides obtained from Lampteromyces japonicus by concanavalin A-sepharose affinity chromatography

Two classes of neutral polysaccharide which could not be separated from each other by conventional methods were isolated from the fungus, Lampteromyces japonicus, by affinity chromatography using concanavalin A-Sepharose. The polysaccharide retained on the concanavalin A-Sepharose column was eluted with 0.05 M methyl α-d-mannopyranoside and appeared to be α-mannan, while that which passed through the column was virtually all β-glucan.Both polysaccharides were subjected to Smith-type degradation, methylation, acetolysis and glucosidase treatment. The results indicated that the α-mannan contained predominantly α-(1 → 2)-linked side chains branching from an α-(1 → 6)-linked backbone at the (1 → 2,6)-linked mannopyranosyl residues. Galactose was attached to approximately one-quarter of the non-reducing mannose terminals. The β-glucan seemed to contain mainly (1 → 6)-linked side chains branching from a (1 → 3)-linked backbone at the (1 → 3,6)-linked glucopyranosyl residues.     

Purification, stabilization, and characterization of rat hepatic triglyceride lipase

Hepatic triglyceride lipase (H-TGL) was purified to near homogeneity from heparin-containing rat liver perfusates with the following column chromatography steps: heparin-Sepharose affinity chromatography, anion-exchange chromatography on DEAE-Sephacel, and gel filtration on Ultrogel AcA 34. A final specific activity of 45,000 μmol fatty acid/mg/h was obtained with an overall 31% recovery of catalytic activity. The heparin-Sepharose step resulted in a 20-fold purification, while the DEAE and gel filtration steps led to further purification with complete recovery of activity. An extensive survey of various detergents as potential stabilizers of H-TGL activity led to the selection of Triton N-101 for use in the column buffers of the DEAE and gel filtration steps. Relative to initial H-TGL activity upon dilution in buffer without detergent, recoveries between 90 and 100% were consistently obtained with Triton N-101-containing buffers following a 24-h incubation at 20°C. In contrast after a 24-h incubation at 20°C those control samples lacking detergent were at least 95% inactivated. The highly purified H-TGL exhibited a single major band by sodium dodecyl sulfate-electrophoresis. The use of DEAE chromatography and stabilization of H-TGL with Triton N-101 are the improvements in purification that resulted in an 8-fold enhancement in specific activity relative to the highest previous report of purification from rat liver perfusates.     

Purification and properties of phosphorylase from baker's yeast

A rapid, reliable method for purification of phosphorylase, yielding 200-400 mg pure phosphorylase from 8 kg of pressed baker's yeast, is described. The enzyme is free of phosphorylase kinase activity but contains traces of phosphorylase phosphatase activity. Phosphorylase constitutes 0.5-0.8% of soluble protein in various strains of yeast assayed immunochemically. The subunit molecular weight (Mr) of yeast phosphorylase is around 100,000. The enzyme is composed of two subunits in various ratios, differing slightly in molecular weight and N-terminal sequence. Both are active. Only the enzyme species containing the larger subunit can form tetramers and higher oligomers. The activated enzyme is dimeric. Correlated with specific activity (1 to 110 U/mg), phosphorylase contained between less than 0.1 to 0.74 covalently bound phosphate per subunit. Inactive forms of phosphorylase could be activated by phosphorylase kinase and [gamma-32P]ATP with concomitant phosphorylation of a single threonine residue in the aminoterminal region of the large subunit. The small subunit was not labeled. The incorporated phosphate could be removed by yeast phosphorylase phosphatase, resulting in loss of activity of phosphorylase, which could be restored by ATP and phosphorylase kinase.     

Purification and characterization of an extracellular H2O2-requiring diarylpropane oxygenase from the white rot basidiomycete, Phanerochaete chrysosporium

An H2O2-requiring oxygenase found in the extracellular medium of ligninolytic cultures of the white rot fungus Phanerochaete chrysosporium was purified by DEAE-Sepharose ion-exchange chromatography and gel filtration on Sephadex G-100. Sodium dodecyl sulfate (SDS)-disc gel electrophoresis indicated that the purified protein was homogeneous. The Mr of the enzyme as determined by gel filtration and SDS-polyacrylamide gel electrophoresis was 41,000. The absorption spectrum of the enzyme indicated the presence of a heme prosthetic group. The absorption maximum of the native enzyme (407 nm) shifted to 435 nm in the reduced enzyme and to 420 nm in the reduced-CO complex. The pyridine hemochrome absorption spectrum indicated that the enzyme contained one molecule of heme as iron protoporphyrin IX. Both CN- and N-3 bound readily to the native enzyme, indicating an available coordination site and that the heme iron was high spin. The purified enzyme generated ethylene from 2-keto-4-thiomethyl butyric acid, and oxidized a variety of lignin model compounds, including the diarylpropane, 1-(3'4'-diethoxyphenyl)1,3-dihydroxy-2-(4"-methoxyphenyl)propane (I); a beta-ether dimer, 1-(4'-ethoxy-3'-methoxyphenyl)glycerol-beta-guaiacyl ether (V); an olefin, 1-(4'-ethoxy-3'-methoxyphenyl)-1,2 propene (III); and a diol, 1-(4'-ethoxy-3'-methoxyphenyl)-1,2-propane diol (IV). The products found were equivalent to the metabolic products previously isolated from intact ligninolytic cultures.     

Chicken fumarase. I. Purification and characterization.

Fumarase from chicken heart is purified 400 times from the crude muscle extract. The isolation procedure includes ammonium sulfate fractionations, Bio-Gel P-300 column chromatography and electrofocusings on pH-gradients from pH 3 to 10 and from pH 7 to 9. Chicken fumarase behaves as an homogeneous protein in sedimentation, diffusion and electrofocusing studies; the protein possesses a single amino-terminal residue: lysine. The analysis of the CD and ORD spectra suggests the presence of 60-65 p. cent of alpha-helix, 0 - 5 p. cent of beta-structure with the remaining portions of the protein in an unordered conformation. Chicken fumarase is found to be composed of 4 subunits of identical molecular weight (51.000) and devoid of disulfide bridges. Finally, the physicochemical properties of chicken fumarase are compared with those of the porcine enzyme.     

Studies on Turbatrix aceti beta-N-acetylglucosaminidase. 1. Purification and physicochemical characterization

N-Acetyl-beta-D-glucosaminidase was purified, from the culture medium of the nematode Turbatrix aceti, to homogeneity, as judged by electrophoresis in polyacrylamide gel and ultracentrifugation. The purification scheme involved the following steps: (i) concentration of the culture medium by ultra-filtration by an Amicon PM-30 membrane; (ii) ammonium sulfate precipitation; (iii) DEAE-Sephadex and (iv) Sephadex G-200 chromatography; and (v) affinity chromatography on succinyldiaminopropyl amino-Sepharose bearing the ligand p-aminophenyl 2-acetamido-2-deoxy-1-thio-beta-D-glucopyranoside. The molecular weight of the enzyme was 112,000 +/- 4800 and 124,000 as determined by polyacrylamide gel electrophoresis and by gel filtration through Sephacryl S-200, respectively. The enzyme showed a pH optimum of 4.8 for N-acetylglucosaminidase and 5.4 for N-acetylgalactosaminidase. The detailed substrate specificity studies were carried out on both synthetic and natural oligosaccharides and glycopeptides. The chitin oligosaccharides and asialo-agalacto complex type as well as high mannose-type glycoproteins such as fetuin and ovalbumin, respectively, were good substrates for the enzyme. Substrate analogs in which the oxygen atom of the acetamido group was replaced by sulfur atom proved to be poor substrates.     

Bovine adrenocortical self-phosphorylating protein kinase,SPK 380: Purification and characterization

Recently, we described the partial purification and characterization of a novel adrenocortical cyclic nucleotide-independent protein kinase, PK 380, that catalyzes the phosphorylation of an endogenous peptide (120,000 daltons) and a serine residue(s) of the α subunit (38,000 daltons) of the eucaryotic initiation factor eIF-2 (Y. Kuroda, W. C. Merrick, and R. K. Sharma, 1982, Arch. Biochem. Biophys.213, 271–275). In the present communication we describe the purification to apparent homogeneity and characterization of this protein kinase (SPK 380). As shown by sucrose density sedimentation, the native enzyme has a molecular weight of 356,000. The protein is composed of three identical subunits of M_r 120,000. Polyacrylamide-gel isoelectric focusing electrophoresis revealed a single peak with pI 4.5. SPK 380 self-phosphorylated a histidine residue(s) of its 120,000-dalton peptide. This reaction utilized the terminal phosphate of ATP; GTP was inactive. Divalent cations (5 mm Mn²⁺ or 10 mm Mg²⁺) were essential for optimum activity. Thiol reagents (N-ethylmaleimide, p-chloromercuriphenylsulfonic acid) inhibited the kinase, indicating a sulfhydryl-group requirement for enzyme activity.     

Immunological similarity of milk sulfhydryl oxidase and kidney glutathione oxidase

A radioimmunoassay for sulfhydryl oxidase, a membrane enzyme, was developed using antibodies raised to the bovine milk enzyme which had been purified by transient covalent affinity chromatography on a cysteinylsuccinamidopropyl-glass matrix. Bovine milk sulfhydryl oxidase and bovine kidney sulfhydryl oxidase (“glutathione oxidase”) appear to be immunologically identical as evidenced by parallel responses in radioimmunoassays. Antibodies raised to the purified milk sulfhydryl oxidase can immunoprecipitate glutathione oxidase activity, but not γ-glutamyltransferase (“transpeptidase”) activity, from bovine kidney preparations.     

Fatty acid synthetase from chloroplasts of soybean cotyledons: ACP activation and CoA inhibition

Fatty acid synthetase from chloroplasts of soybean cotyledons was activated by preincubation with acyl carrier protein and dithiothreitol. The synthetase reaction had a 3–10 min lag which was not eliminated by the preincubation. Acetyl-CoA and malonyl-CoA had no effect on the activation. Fatty acid synthetase from spinach chloroplasts was neither activated by preincubation nor had a lag. The variability of the activity of the soybean enzyme with preincubation suggested that the fatty acid synthetase was present in two forms and that the acyl carrier protein caused conversion to the active form. This fatty acid synthetase and the same synthetase from spinach chloroplasts were inhibited by CoA. The type of inhibition by CoA in soybean was competitive with respect to malonyl-CoA and the Ki was 80μM.