Purification and properties of aromatic L-amino acid decarboxylase (4.1.1.28) of rat brain]

L-aromatic aminoacid decarboxylase has been purified more than thousand times from homogenates of rat brain, in several steps : centrifugation, DEAE-cellulose, CM cellulose, hydroxylapatite, DEAE sephadex. Its properties have been studied, most of them on an intermediate fraction of the purification, because of the instability of the purified enzyme in spite of the addition of different stabilizing agents : the enzyme decarboxylates 5-hydroxytryptophan (5 HTP) and DOPA in a ratio constant throughout the purification but does not decarboxylate tryptophan, tyrosine, histidine at a measurable rate. Optimum pH, Km, Vm, have been measured with 5 HTP and DOPA as substrates. The enzyme has a molecular weight of 115.000, an apparent isoelectric point of 6,4-6,5. It is inhibited by serotonin, dopamine, some cations : Cu++, Fe++, Ni++ by N-ethylmaleimide, sodium dodecylsulfate. Some pyridoxal-5 phosphate (PLP) remains strongly bound to the enzyme. For relatively weak concentrations of substrate, the enzyme is inhibited by an excess of PLP ; for weak concentrations of PLP, the enzyme in inhibited by an excess of substrate, particularly of DOPA. We also observe a spontaneous decarboxylation of the substrates that reaches a plateau and is enhanced by high concentrations of PLP, by serotonin, dopamine, Cu++ and reduced by mercaptoethanol and the presence of crude or boiled homogenates. Several possible explanations of the spontaneous decarboxylation and of the enzymic inhibitions by an excess of PLP and by the substrates are given.     

Purification and properties of arginase of rat kidney

l-Arginase from rat kidney was partially purified and some properties were compared with those of l-arginase of rat liver. The kidney enzyme was firmly bound to the mitochondrial fraction and after solubilization required arginine or an unknown factor in tissue extracts for stabilization after dialysis. The two enzymes differed also in stability with respect to acetone treatment, heating or freezing. In further contrast with liver arginase, arginase from kidney was not adsorbed to CM-cellulose at pH7.5 and its activity was not increased by incubation with Mn(2+). Other differences were seen in relative specificities for substrates, ratio of hydrolysis rates with high and low concentrations of arginine and effects of certain inhibitors. Antisera prepared to pure liver arginase did not cross-react with partially purified kidney arginase.     

Purification and properties of acid phosphatases isolated from Owenia fusiformis.

Acid phosphatase (EC. 3.1.3.2) has been separated by molecular sieving into two fractions and these fractions were purified by Sephadex ion-exchange chromatography. One of the purified enzymes (fraction II) was purified 830 fold and had a specific activity of 34 international units per mg protein at 37 degrees C and at a pH of 4.9. The Km value with p-nitrophenylphosphate as substrate was 9.10(-4) M and the kinetic studies showed no possibilities of control by allosteric transitions, and no effect of metabolites (amino acids) on the reaction velocity.     

Purification and partial characterization of rat liver soluble catechol-O-methyltransferase

The rat liver soluble catechol-O-methyltransferase (EC 2.1.1.6.) has been purified utilizing a combination of conventional chromatography and HPLC. The purified enzyme has a molecular mass of 25 kDa, a pI of 5.1, and exists in two forms which differ in the nature of their intramolecular disulfide bonds. This difference causes these two protein forms to behave differently in reversed phase chromatography.     

Purification and properties of methyl sulfoxide reductases from rat kidney

Two kinds of enzymes (tentatively designated methyl sulfoxide reductases I and II) responsible for the reduction of the methyl sulfoxide group on various xenobiotics have been purified about 223- and 155-fold, respectively, from rat kidney cytosol. The molecular weight was determined to be 12,000 +/- 1000 for methyl sulfoxide reductase I and 24,000 +/- 1000 for methyl sulfoxide reductase II. Thioredoxin or dithiothreitol is essential in order for the reducing activity to occur. The respective Km values of p-bromophenylmethyl sulfoxide were 2.75 and 1.30 mM for methyl sulfoxide reductases I and II. Replacement of the methyl group on the sulfur atom with a longer alkyl group or phenyl group caused a markedly low or negligible substrate activity.     

Purification and properties of alpha-aminoadipate aminotransferase from rat kidney.

Previous studies with rat kidney preparations indicated that alpha-aminoadipate aminotransferase (AadAT) and kynurenine aminotransferase (KAT) activities are associated with a single protein. However, recent studies from our laboratory demonstrated that AadAT and KAT activities belong to two different proteins. AadAT from rat kidney supernatant fraction was purified by affinity chromatography to electrophoretic homogeneity. This rapid and efficient procedure improved the yield and the degree of purification over previously published methods and separated AadAT from KAT. The molecular weight of the enzyme was estimated to be 89,000 by Sephadex G-200 gel filtration chromatography. SDS-PAGE indicated that the enzyme is composed of two apparently identical subunits. Absorption spectra and the kinetic properties of AadAT are reported.     

Purification and properties of UDP-glucuronyltransferase from kidney microsomes of beta-naphthoflavone-treated rat

Rat kidney microsomal UDP-glucuronyltransferase activities toward phenoic xenobiotics were enhanced about 4-5-fold by treatment of the animal with beta-naphthoflavone. The transferase activity toward serotonin, an endogenous substrate, was also enhanced about 7.5-fold. A form of UDP-glucuronyltransferase was purified from kidney microsomes of beta-naphthoflavone-treated rat by solubilization with sodium cholate and two steps of column chromatography, the first with DEAE-Toyopearl (fast flow rate liquid chromatography:FFLC) and the second with UDP-hexanolamine Sepharose 4B (affinity chromatography). These procedures gave about 39-fold purification and 11.5% yield of the transferase activity toward 1-naphthol. The preparation, tentatively termed "GT-2," was highly purified as judged from the single protein band (Mr 54,000) on sodium dodecylsulfate (SDS)-polyacrylamide slab gel electrophoresis. It catalyzed the glucuronidation of not only phenolic xenobiotics such as 1-naphthol, 4-nitrophenol, and 4-methylumbelliferone but also serotonin. From the result that apparent molecular weight of GT-2 was reduced to 50,000 by endo-beta-N-acetylglucosaminidase H (Endo H)-treatment, GT-2 was found to be a 50,000 Da polypeptide carrying "high mannose" type oligosaccharide chain(s). The NH2-terminal sequence of 20 residues of GT-2 was determined to be Asp-Lys-Leu-Leu-Val-Val-Pro-Gln-Asp-Gly-Ser-His-Trp-Leu-Ser-Met-Lys-Glu- Ile-Val . It was observed that there are two amino acids substitutions in the seven NH2-terminal residues in comparison with GT-1, which was purified from liver microsomes of 3-methylcholanthrene-treated rat. The NH2-terminal sequence of GT-2 was found to be homologous with the NH2-terminal sequence from the 26th to 46th amino acid residue of various UDP-glucuronyltransferase cloned by other investigators.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and properties of an NADPH-linked aldehyde reductase from rat kidney

Rat kidney was shown to contain two NADPH-linked aldehyde reductases (alcohol:NADP+) oxidoreductase, EC 1.1.1.2) with different substrate affinities. The high-Km aldehyde reductase, which was purified to apparent homogeneity, had a molecular weight of 32 000 as determined by Sephadex G-100 gel filtration, and of 37 000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The purified enzyme reduced various aliphatic aldehydes of different carbon-chain lengths besides many chemicals containing aldehyde groups. The Km values for n-hexadecanal and n-octadecanal were 8 microM and 4 microM, respectively. Bovine serum albumin (1.8 mM) stimulated the reduction of n-hexadecanal and n-octadecanal, and increased the Vmax values by about 15-fold without changing the Km values. The kidney enzyme was not distinguishable from the brain and liver high-Km aldehyde reductases in mobility on polyacrylamide gel electrophoresis, immunological properties, peptide maps or substrate specificity.     

Purification and properties of alpha-aminoadipate aminotransferase from rat liver and kidney mitochondria.

Recently we reported an affinity chromatography method to purify alpha-aminoadipate aminotransferase (AadAT) activity from rat kidney supernatant fraction. Using the same affinity column, we purified AadAT activities from rat kidney and liver mitochondria. The physical and kinetic properties such as pH optima, Km for substrates, molecular weight, subunit structure, isoelectric pH, electrophoretic mobility and inhibition by dicarboxylic acids of mitochondrial AadAT were similar to those of the AadAT from rat kidney supernatant fraction. These results indicate that AadAT from different subcellular fractions is structurally and immunologically identical.     

Purification, properties and regulation of urocaninase from rat liver]

Urocaninase (EC 4.2.1.4.9) from rat liver homogenate has been purified, using protein precipitation at pH 4,8, ammonium sulfate fractionation, gel-filtration through Sephadex G-200 and chromatography on DEAE-cellulose. Upon DEAE-cellulose chromatography urocaninase is separated from the proteins possessing the activity of 3',5'-AMP-dependent protein kinase. The purified enzyme becomes activated after addition of ATP and exogenous protein kinase or one of the fractions resulting from DEAE-cellulose chromatography. Using [gamma-32P]ATP, it has been shown that such activation is accompanied by incorporation of at least one phosphate residue into the enzyme molecule. The mol. weight of urocaninase as determined by gel-filtration is about 110 000. The Km value for urocanate is 15 . 10(-6) M, the isoelectric point lies at 5,6. The mechanism of regulation of the urocaninase activity in rat liver is discussed.     

Polymorphism of the catechol-O-methyltransferase gene in Han Chinese patients with psoriasis vulgaris

PSORIASIS VULGARIS IS DEFINED BY A SERIES OF LINKED CELLULAR CHANGES IN THE SKIN: hyperplasia of epidermal keratinocytes, vascular hyperplasia and ectasia, and infiltration of T lymphocytes, neutrophils and other types of leukocytes in the affected skin. Catechol-O-methyltransferase (COMT) 158 polymorphism can reduce the activity of the COMT enzyme that may trigger defective differentiation of keratinocytes in psoriasis. Immunocytes can degrade and inactivate catecholamines via monamine oxidase (MAO) and COMT in the cells. We hypothesized that the COMT-158G > A polymorphism was associated with the risk of psoriasis vulgaris in Han Chinese people. In a hospital-based case-control study, 524 patients with psoriasis vulgaris and 549 psoriasis-free controls were studied. COMT-158 G > A polymorphism was genotyped using the PCR sequence-specific primer (PCR-SSP) technique. We found no statistically significant association between the COMT-158 allele A and the risk of psoriasis vulgaris (p = 0.739 adjusted OR = 1.03; 95% CI = 0.81-1.31). This suggests that the COMT-158 G > A polymorphism may not contribute to the etiology of psoriasis vulgaris in the Han Chinese population.     

Affinity labeling of catechol-O-methyltransferase with N-iodoacetyl-3,5-dimethoxy-4-hydroxyphenylethylamine

Catechol-O-methyltransferase is inactivated rapidly by incubation with N-iodoacetyl-3,5-dimethoxy-4-hydroxyphenylethylamine; not by the N-acetyl analogue. Iodoacetate or iodoacetamide produce slight inactivation. Inactivation is first order with respect to enzyme activity. A kinetic analysis suggests the formation of a dissociable enzyme-inhibitor complex prior to inactivation. Substrate, 3,4-dihydroxybenzoate, protects the enzyme from alkylation and loss of activity.     

Zein of maize grain. Preparation and characterization]

A laboratory procedure for isolation and purification of zein from grains of 4 varieties of Maize was described. The preparations were characterized by their physicochemical properties. Upon polyacrylamide gel electrophoresis in sodium dodecyl sulphate (SDS), native zein (from INRA 260 hybrid) was resolved into 2 major classes with average molecular weights of 45,000 and 22,000. After reduction with mercaptoethanol zein contained only two subunits of 22,000 and 24,000 daltons. Upon starch gel electrophoresis in 6 M urea at pH 3.5, native zein exhibited five major or medium intensity bands and several minor ones. The latter, under reducing conditions, disappeared to reinforce the major bands or to yield some new minor bands. Amino acid analysis revealed a very low content of lysine. The NH2-terminal amino acids were determined to be threonine and phenylalanine with a preponderance of the former. Zeins isolated from the varieties studied appeared tohave the same NH2-terminal residues and similar amino acid compositions with an arginine/histidine ratio ranging from 1.1 to 1.2. They differed in relative importance of components, detected by electrophoresis in the presence of SDS or urea. Changes in zein characteristics with the grain genotype allow one to conclude that the components of molecular weights of 22,000 and 24,000 consist of several subunits differing in charge and amino acid content.     

Purification and properties of bovine kidney ribonucleases

Two RNases (RNases K1 and K2) were purified from bovine kidney by means of column chromatography on phospho-cellulose, Sephadex G-50, CM-cellulose, heparin-Sepharose, nd agarose-APUP. They were named RNase K1 and RNase K2 in order of elution from the heparin-Sepharose column. The purity of RNase K1 thus obtained was about 90% by SDS-disc electrophoresis. RNase K2 was purified to homogeneity by SDS- and pH 4.3 disc electrophoresis. The yield of RNase K2 was 3.4 mg from 11 kg of kidneys. The antigenic properties of the two bovine renal RNases were studied by Ouchterlony's double diffusion analysis. RNase K1 and RNase A were serologically indistinguishable. RNase K2 did not cross-react immunologically with RNase K1 or RNase A. The molecular weights of these RNases determined by gel-filtration on Sephadex G-50 were 13,400 and 14,600 for RNase K1 and RNase K2, respectively. The pH optima for RNase K1 and RNase K2 were 8.5 and 6.5, respectively. Both RNase K1 and RNase K2 were as acid stable as RNase A. RNase K2 was less heat-stable than RNase K1 and RNase A. Although both renal RNases were pyrimidine nucleotide-specific enzymes, RNase K1 and RNase A were more preferential or cytidylic acid than RNase K2. The chemical composition of RNase K2 was determined. RNase K2, like human urinary RNase Us, contained one tryptophan residue. The N-terminal sequences of RNase K2 and RNase Us were determined by Edman degradation. Rnase K2 had a homologous sequence of about 10 amino acid residues with the sequence of RNase Us, a typical non-secretory RNase, within the N-terminal 30 residues.