Purification and properties of low-Km aldehyde reductase from ox brain

A low-Km aldehyde reductase (alcohol:NADP+ oxidoreductase, EC 1.1.1.2), which may be identical with aldose reductase (alditol:NADP+ 1-oxidoreductase, EC 1.1.1.21), has been purified from ox brain to homogeneity. It was shown to be a monomer with Mr values of 31 000 and 35 100 being obtained by gel filtration and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, respectively. The enzyme catalyses the NADPH-dependent reduction of a number of aromatic and sugar aldehydes. The activity of the enzyme with 133 microM NADH was about one-third of that with 120 microM NADPH. Activity with both these coenzymes was optimum at pH 6.2 and was inhibited by increasing the ionic strength with KCl, NaCl or NaNO3. In contrast, the activity was stimulated by sodium phosphate. The activity with NADH as the coenzyme was more sensitive to stimulation by phosphate and to inhibition by increasing ionic strength than that determined with NADPH.     

Purification of the high-Km aldehyde reductase from rat brain and liver and from ox brain.

A procedure is described that yields an apparently homogeneous preparation of the high-Km aldehyde reductase from rat brain. This procedure is also applicable to the purification of this enzyme from rat liver and ox brain. In the latter case, however, the purified preparation could be resolved into two protein bands, both of which had enzyme activity, by polyacrylamide-gel electrophoresis. Since a sample of the ox brain enzyme from an earlier step in the purification procedure only showed the presence of a single band of activity after electrophoresis, this apparent multiplicity probably results from modification of the enzyme, possibly by oxidation, during the final step of the purification. A number of properties of the rat brain enzyme were determined and these were compared with those of the enzyme from rat liver. The two preparations were similar in their stabilities, behaviour during purification, kinetic properties, electrophoretic mobilities and amino acid compositions. Antibodies to the rat liver enzyme cross-reacted with that from brain and the inhibition of both these preparations by the antiserum was similar, further supporting the view that the enzymes from these two sources were closely similar if not identical.     

Purification and kinetic properties of ox brain histamine N-methyltransferase.

Histamine N-methyltransferase (EC 2.1.1.8) was purified 1100-fold from ox brain. The native enzyme has an Mr of 34800 +/- 2400 as measured by gel filtration on Sephadex G-100. The enzyme is highly specific for histamine. It does not methylate noradrenaline, adrenaline, DL-3,4-dihydroxymandelic acid, 3,4-dihydroxyphenylacetic acid, 3-hydroxytyramine or imidazole-4-acetic acid. Unlike the enzyme from rat and mouse brain, ox brain histamine N-methyltransferase did not exhibit substrate inhibition by histamine. Initial rate and product inhibition studies were consistent with an ordered steady-state mechanism with S-adenosylmethionine being the first substrate to bind to the enzyme and N-methylhistamine being the first product to dissociate.     

Purification of an alkaline ribonuclease from soluble fraction of beef brain.

A ribonuclease (ribonucleate 3-pyrimidine-oligonucleotidohydrolase, EC 3.1.4.22) was purified 8300-fold from soluble fraction of beef brain and its properties were investigated. The enzyme is an endonuclease capable of hydrolyzing tRNA, rRNA, poly(C), but shows no activity towards poly(U), poly(A), and poly(G). The preparation is free of deoxyribonuclease, non-specific phosphodiesterase and phosphomonoesterase activity. The enzyme has a pH optimum of 7.6, is not heat stable, has a molecular weight of 25 000, and has a K-m of 134 mu rRNA and K-m of 1600 mug poly(C) per ml.     

Purification of glutamate dehydrogenase from ox brain and liver. Evidence that commercially available preparations of the enzyme from ox liver have suffered proteolytic cleavage.

1. A rapid procedure, involving ion-exchange chromatography on DEAE-cellulose and affinity chromatography on GTP-Sepharose, was used to purify glutamate dehydrogenase from ox brain and liver. 2. Preparations purified in this way differed from those of the ox liver enzyme that were obtained from commercial suppliers in their mobilities on polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. This difference appears to result from the occurrence of limited proteolysis during the preparation of the latter enzyme samples. 3. N-Terminal sequence analysis showed the presence of four amino acid residues in the enzyme prepared in this study that were not present in those obtained from the commercial sources and which have not been reported in previous studies on the sequence of the ox liver enzyme. 4. A preliminary examination of the enzyme prepared in this way indicated that the Michaelis constants for the substrates are similar to those obtained from the commercial preparation, but that the response to allosteric effectors was modified.     

Purification of soluble acetylcholinesterase from Japanese quail brain by affinity chromatography.

The purification of a soluble acetylcholinesterase from Japanese quail brain using affinity chromatography on concanavalin A-Sepharose and edrophonium-Sepharose is described. The affinity matrix was synthesized by coupling an inhibitor edrophonium to epoxy-activated Sepharose. Acetylcholinesterase was purified 10,416-fold with a specific activity of 2500 U/mg protein. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and mercaptoethanol gave only one band with a molecular weight of 62.5 kDa. The molecular weight of the purified acetylcholinesterase was estimated to be 245.5 kDa by gel chromatography on Sephacryl S-200 under nondenaturing conditions. Based on the molecular weight obtained by both SDS-PAGE and gel filtration the purified acetylcholinesterase was assumed to be a tetrameric form.     

Preparative isolation of polyphosphoinositide fractions from ox brain

A simple preparative method for chromatographic isolation of pure fractions of di- and triphosphoinositides (1-phosphatidylinositol 4-phosphate and 1-phosphatidylinositol 4,5-bisphosphate) from ox brain is described. Polyphosphoinositide fractions have been obtained by ion-exchange chromatography of the lipid extract using gradient elution with 0-0.6 M ammonium acetate in chloroform/methanol/water (20:9:1) from a DEAE-cellulose column. Before chromatography, divalent metal ions were removed from the lipid extract by passing through a Dowex-50 (H+) column and lipids were converted to the sodium salt by neutralisation with sodium hydroxide in methanol solution. After chromatography, fractions of di- and triphosphoinositides were precipitated in methanol/water mixture (1:1) by evaporation in a vacuum to a final concentration of about 4 M ammonium acetate. Necessary salts of di- and triphosphoinositides were obtained by passing the ammonium salts of the lipids through Dowex-50 (H+) and neutralising with corresponding base in methanol solution. About 0.35 mmol of diphosphoinositide and 0.63 mmol of triphosphoinositide were obtained from 1 kg of wet ox brain tissue.     

Arylsulphatases in human brain: separation, purification, and certain properties of the two soluble arylsulphatases

Abstract— Arylsulphatases (aryl-sulphate sulphohydrolases; E.C. 3.1.6.1) in the soluble subcellular fraction (105000g, 2 h) of human brain were partially purified by ammonium sulphate fractionation, ion exchange chromatography, and Sephadex gel filtration. Potassium-4-methylumbelliferone-sulphatase (MUS-sulphatase) adsorbed on DEAE-cellulose was purified approximately 700-fold over activity in the soluble fraction and the unadsorbed MUS-sulphatase was similarly purified approximately 600-fold. The arylsulphatase adsorbed to DEAE-cellulose exhibited a K_m value for MUS of 12.5 mM and a pH optimum of 5.7, whereas the unadsorbed arylsulphatase exhibited a K_m value for MUS of 8.3 mM and a pH optimum of 5.4. The molecular weights of the two enzymes were approximately 109,600 and 51,300, respectively. Sulphate (0.5 mM) showed pronounced mixed inhibition only of the unadsorbed arylsulphatase. Ag⁺ ions (0.25 mM) showed 96 per cent inhibition of the adsorbed arylsulphatase, whereas an activation of the unadsorbed arylsulphatase was observed.     

Purification of soluble tetrodotoxin-sensitive protein from the cytoplasmic fraction of bovine brain tissue

Highly purified protein preparations promoting TTX-dependent sodium permeability in the liposomal membrane were separated from the cytoplasmic fraction of bovine brain tissue. The protein was purified using anion-exchange and separation chromatography and gel filtration. The separated protein was found to be a high-molecular acidic glycoprotein forming 55 kD subunits during denaturation under reducing conditions. It was thought to contribute to the formation of voltage-dependent sodium channels in the cell membrane.A. A. Bogomolets Institute of Physiology and A. V. Palladin Institute of Biochemistry, Academy of Sciences of the USSR, Kiev. Translated from Neirofiziologiya, Vol. 19, No. 2, pp. 202–209, March–April, 1987.     

Purification and characterization of a beta-galactoside-binding soluble lectin from rat and bovine brain

A beta-galactoside-binding activity has been detected in mammalian brain extracts using a hemagglutination test and a nerve cell aggregation assay. Inhibition studies suggested the involvement of lectin-carbohydrate interactions in these processes. In an attempt to explore further the biological role of brain lectins, the beta-galactoside-binding activity has been purified to apparent homogeneity from bovine and rat brain by salt extraction of the brain tissue and affinity chromatography on asialofetuin-agarose. The molecular weights determined by gel filtration, under native conditions on Ultrogel AcA-34, were 30,000 for the bovine brain lectin and 32,000 for the rat brain lectin; polyacrylamide gel electrophoresis in SDS gave molecular weights of 15,000 and 16,000, respectively, suggesting that the two brain lectins are dimers. Both lectins have an isoelectric point of 3.9. Amino acid composition data indicate that both lectins contain high proportions of glycine and acidic amino acids. The lectins are specific for beta-D-galactosides and related sugars and the configuration of carbon atoms 1, 2 and 4 seems of primary importance. Moreover, the nerve cell aggregation-promoting activity of the purified lectin is 300-fold that of the crude extracts.