Partial purification and charcterization of phosphatidylinositol kinase from bovine brain

Purification of Phosphatidylinositol (PI) kinase was attempted from bovine brain. A seven step purification protocol increased the specific activity 100×but attempts at further purification were unsuccessful. Labeling of the partially purified PI kinase with the ATP analog fluorosulfonylbenzoyl adenosine reproducibly identified three bands on polyacrylamide gel electrophoresis of 76 K, 45 K, and 29 K, one of which likely represents PI kinase. Kinetic studies showed aK _m of 17 M for ATP, 0.02 mg/ml for PI and aV _m of 1830 pmol/min/mg protein for ATP and 820 pmol/min/mg protein for PI.     

Purification and properties of pyridoxal kinase from bovine brain

A 27,000-fold purification of pyridoxal kinase from bovine brain tissue has been achieved by a combination of ammonium sulfate fractionation, DEAE-cellulose chromatography, hydroxyapatite chromatography, Sephadex G-150 gel filtration, Blue Sepharose CL-6B chromatography, and Phenyl-Superose chromatography. The final chromatography step yields a homogeneous preparation of high specific activity (2105 nmol/min/mg protein). The molecular mass of the native enzyme was estimated to be approximately 80,000 on gel filtration. The subunit molecular mass was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis to be approximately 39,500. This indicates that pyridoxal kinase is a dimeric enzyme.     

Rapid purification and properties of protein kinase C from bovine adrenal medulla

Protein kinase C was purified from bovine adrenal medulla using a rapid procedure which resulted in an approximate 1500-fold increase in specific activity. The characteristics of the enzyme are reported and for the first time it is possible to compare the effects of TPA on secretion from intact and permeabilized cells with the effect of TPA on protein kinase C purified from the same secretory tissue.     

An improved purification procedure and properties of casein kinase II from brain

A simple and short purification procedure applicable to casein kinase II has been developed, for fully characterizing the enzyme from calf cerebral cortex cytosol. The procedure consists of four chromatographic steps: DEAE-cellulose, phosphocellulose, phosvitin-Sepharose and ATP-agarose which yields 87% pure casein kinase II. The purified enzyme shows three major bands with apparent molecular masses of 42, 38, and 27 kDa by polyacrylamide gel electrophoresis in sodium dodecyl sulfate and is self-autophosphorylated on its 27 kDa polypeptide. The enzyme shows all the characteristics described for casein kinase II from other sources: it is independent of cyclic nucleotides, calcium/phospholipids, and double-stranded poly(I).poly(C); it can utilize both ATP and GTP as phosphoryl donors and can phosphorylate both casein and phosvitin but not histone. The kinetic studies establish that theK _m for ATP is 12.5 M and 25.1 M when using phosvitin and casein respectively as phosphoryl acceptors. TheK _m for phosvitin is 0.91 mg/ml and for casein 1.43 mg/ml, while theV _max is 315 nmol/min/per mg protein and 479 nmol/min/per mg protein for phosvitin and casein respectively. The activity of the kinase is highly stimulated by KCl or NaCl, and almost completely inhibited by heparin concentrations of 1 g/ml (92%). This inhibition is reduced to only 33% in the presence of optimal KCl concentrations (150 mM). Spermine stimulates enzyme activity, whilst hemin produces a slight inhibition.     

Purification and properties of pyruvate kinase type M1 from bovine brain

1. Pyruvate kinase type M1 was purified from bovine brain about 241-fold with 38% yield. 2. Specific activity of the enzyme was above 217 U/mg of protein (25 degrees C), relative mol. wt of the subunit--57,000 (+/- 2000) and pH optimum--6.8-7.2. 3. The enzyme shoved hyperbolic kinetics with Km value for PEP of 0.04 mM and for ADP of 0.3 mM. 4. Inorganic phosphate and ATP at concentrations below 4 mM showed activating effect, 1-phenylalanine and ATP above 6 mM--an inhibiting effect on the enzyme. 5. Inhibition by 1-phenylalanine was prevented by fructose-1,6-bisphosphate.     

Partial purification and some properties of a bovine brain trypsin inhibitor

Using hemoglobin modified by pyridoxal 5'-phosphate as substrate, a trypsin inhibitor from bovine brain was purified by extraction at pH 4.5, ion-exchange chromatography on DEAE-Sephadex A-50, gel filtration on Sephadex G-100 and isoelectric focusing. On a column of Sephadex G-100 the inhibitor exhibited a molecular mass of 78 kDa. The iso-electric point of the inhibitor was 4.3-4.4. The dissociation constant (Ki) for the complex of bovine trypsin and brain inhibitor was estimated to be 3.7 X 10(-10)M as tested with a protein substrate, and 2.4 X 10(-10)M when tested with a synthetic substrate. During purification two other brain trypsin inhibitors were detected.     

Isolation, purification and study of the properties of pyruvate kinase from the bovine adrenal cortex

Pyruvate kinase (ATP: pyruvate 2-0-phosphotransferase, EC 2. 7. 1. 40) from bovine adrenal cortex was purified 243 fold. The whole purification procedure included ammonium sulphate fractionation, heat treatment, Sephadex HW-55 chromatography and phosphocellulose chromatography. The specific activity of the preparation is 15.6 U/mg at 30 degrees C, the yield--36%. Pyruvate kinase showed only one protein band as judged by sodium dodecyl sulphate acrylamide gel electrophoresis. The enzyme displayed a hyperbolic saturation curve with respect to P-enolpyruvate. The apparent Km for this substrate was 0.55 X 10(-4) M, pH optimum--6.8-7.0. K+ concentrations above 0.1 M inhibit the enzyme.     

Partial purification and characterization of sialate O-acetylesterase from bovine brain

From bovine brain an esterase was purified 2,600-fold in an overall yield of 5.6%. For the isolation ion-exchange chromatographies, gel filtration, and preparative isoelectric focusing were used. The molecular mass is 56 kDa after gel chromatography on Sephacryl S-200 and 51 kDa after HPLC, the pH-optimum at 7.4, and the isoelectric point in the range of pH 5.8-6.1, as estimated from preparative isoelectric focusing. The substrate specificity of this enzyme was tested with various naturally occurring O-acylated sialic acids, synthetic carbohydrate acetates, and other esters. Besides aromatic acetyl esters such as e.g. alpha-naphthyl acetate, the highest preference was for N-acetyl-9-O-acetylneuraminic acid, followed by N-acetyl-4-O-acetylneuraminic acid. Other primary acetyl esters such as 6-O-acetylated D-glucose and 2-acetamido-2-deoxy-D-mannose were not hydrolyzed. The 9-O-acetyl derivative of the naturally occurring unsaturated sialic acid 2-deoxy-2,3-didehydro-N-acetylneuraminic acid, however, is a substrate for this esterase. Whereas N-acetyl-9-O-acetylneuraminic acid as a component of sialyllactose is nearly as well hydrolyzed as the corresponding free sialic acid, O-acetylated sialoglycoconjugates with high molecular weights (mucins, serum glycoproteins, gangliosides) are not hydrolyzed by this esterase. N-Acetylated sialic acids are better substrates than the analogous N-glycoloyl derivatives. Esterification of the carboxyl function of sialic acids prevents the action of the esterase on the O-acetyl groups. The enzyme has no carboxyl esterase or amidase activity, and does not act on acetylcholine. It hydrolyzes almost exclusively acetyl esters. Inhibition studies suggest that it has a catalytically active serine residue.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solubilization and partial purification of GABAB receptor from bovine brain

gamma-Aminobutyric acid (GABA)B receptor has been solubilized and partially purified by an affinity column chromatography. GABAB receptor was solubilized by 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) in the presence of asolectin. The solubilized GABAB receptor was adsorbed on baclofen-coupled epoxy-activated Sepharose 6B. The affinity matrix adsorbed 80% of the solubilized [3H]GABA binding activity to GABAB receptor, and approximately 75% of the adsorbed activity could be eluted with 1 M KC1. GABAB receptor binding in the fraction eluted from affinity column was displaced by GABA, baclofen and 2-hydroxy saclofen in a dose-dependent manner. Furthermore, the purified GABAB receptor showed approximately 2800-fold purification as compared with the original solubilized fraction and possessed the specific binding activity of 17.68 p mol/mg of protein. This binding consisted of a single binding site with a dissociation constant of 64.4 nM. The present results indicate that affinity column chromatographic procedures using baclofen-coupled epoxy-activated Sepharose 6B are suitable for the partial purification of GABAB receptor from cerebral tissues.     

The purification and properties of an aryl beta-hexosidase from bovine liver.

An aryl β-hexosidase was purified 800-fold from bovine liver. The purified enzyme hydrolyzed p-nitrophenyl glycosylpyranoside derivatives of β-d-galactose, β-d-glucose, β-d-xylose, β-d-mannose, and α-l-arabinose, but did not hydrolyze several other p-nitrophenyl glycosides. The enzyme also catalyzed hydrolysis of a variety of plant arylglucosides. Disaccharides, polysaccharides, glycolipids, glycoproteins, and glycosaminoglycans containing terminal nonreducing β-d-galactopyranosyl or β-d-glucopyranosyl residues were not hydrolyzed. The pH optima for the several substrates tested ranged from 7.0 to 9.5. The purified enzyme was homogeneous by disc gel electrophoresis and had a molecular weight of 41,000 by Sephadex gel filtration and 46,000 by disc gel electrophoresis performed in the presence of sodium dodecyl sulfate. The enzyme readily transferred glycosyl residues from susceptible β-galactosides or β-glucosides to other sugars; the resulting products were not hydrolyzed by the enzyme. Methyl α-d-glucopyranoside was the most efficient carbohydrate acceptor compound tested. The enzyme exhibited a K_m for p-nitrophenyl β-d-galactopyranoside of 1.78 × 10^?3m and for p-nitrophenyl β-d-glucopyranoside, 2.50 × 10^?3m when incubations were conducted in the presence of 0.15 m methyl α-d-glucopyranoside. Aryl β-hexosidase was found in the cytosol of all mammalian livers tested, but could not be detected in liver of birds, reptiles, or fish; low levels were detected in frog liver. Analysis of bovine extracts indicated that the enzyme occurred in liver, kidney, and intestinal mucosa; it was not detected in testis, spleen, serum, or muscle.     

Microsomal NADH-cytochrome b5 reductase of bovine brain: purification and properties

Bovine brain microsomal NADH-cytochrome b5 (cyt. b5) reductase [EC 1.6.2.2] was solubilized by digestion with lysosomes, and purified 8,500-fold with a 20% recovery by procedures including affinity chromatography on 5'-AMP-Sepharose 4B. The purified enzyme showed one band of a molecular weight of 31,000 on polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS). Polyacrylamide gel electrophoresis of the purified enzyme without SDS revealed a major band with a faint minor band, both of which exhibited NADH-cyt. b5 reductase activity. The isoelectric points of these components were 6.0 (major) and 6.3 (minor). The apparent Km values of the purified enzyme for NADH and ferricyanide were 1.1 and 4.2 microM, respectively. The apparent Km value for cyt. b5 was 14.3 microM in 10 mM potassium phosphate buffer (pH 7.5). The apparent Vmax value was 1,190 mumol cyt. b5 reduced/min/mg of protein. The NADH-cyt. b5 reductase activity of the purified enzyme was inhibited by sulfhydryl inhibitors and flavin analogues. Inhibition by phosphate buffer or other inorganic salts of the enzyme activity of the purified enzyme was proved to be of the competitive type. These properties were similar to those of NADH-cyt. b5 reductase from bovine liver microsomes or rabbit erythrocytes, although the estimated enzyme content in brain was about one-twentieth of that in liver (per g wet tissue). An immunochemical study using an antibody to purified NADH-cyt. b5 reductase bovine liver microsomes indicated that NADH-cyt. b5 reductase from brain microsomes is immunologically identical to the liver microsomal enzyme.     

Complete purification and immunochemical analysis of S-adenosylmethionine synthetase from bovine brain

We have purified S-adenosylmethionine (AdoMet) synthetase about 3000-fold from bovine brain extract. The Km values of the enzyme for L-methionine and ATP were 10 and 50 microM, respectively. An apparent molecular mass of the enzyme was estimated to be 160 kDa by gel filtration on a Sephacryl S-200 column. Sucrose density gradient centrifugation gave a sedimentation coefficient of 8 S. Polyacrylamide gel electrophoresis of the purified enzyme in native system revealed a single protein band, whereas two polypeptide bands with molecular masses of 48 kDa (p48) and 38 kDa (p38) were observed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme. Antibody against bovine brain AdoMet synthetase was prepared by injecting the purified enzyme into a rabbit. Immunoblot analysis revealed that the antibody recognized both p48 and p38 in the impure enzyme preparations from bovine brain as well as in the purified enzyme. Specific antibodies against p48 and p38 were separated from the immunoglobulin fraction by an affinity purification, both of which inhibited the enzyme activity. These results indicate that AdoMet synthetase from bovine brain consists of two different polypeptides, p48 and p38.