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C Nakai

Publications and source records attributed to C Nakai.

At least 37 records · Page 2Linked to original sources
PubMed1988

Purification and properties of catechol 1,2-dioxygenase (pyrocatechase) from Pseudomonas putida mt-2 in comparison with that from Pseudomonas arvilla C-1.

Catechol 1,2-dioxygenase (pyrocatechase) has been purified to homogeneity from Pseudomonas putida mt-2. Most properties of this enzyme, such as the absorption spectrum, iron content, pH stability, pH optimum, substrate specificity, Km values, and amino acid composition, were similar to those of catechol 1,2-dioxygenase obtained from Pseudomonas arvilla C-1 [Y. Kojima et al. (1967) J. Biol. Chem. 242, 3270-3278]. These two catechol 1,2-dioxygenases were also found, from the results of Ouchterlony double diffusion, to share several antigenic determinants. The molecular weight of the putida enzyme was estimated to be 66,000 and 64,000 by sedimentation equilibrium analysis and Sephadex G-200 gel filtration, respectively. The enzyme gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, corresponding to Mr 32,000. The NH2-terminal sequence, which started with threonine, was determined up to 30 residues by Edman degradation. During the degradation, a single amino acid was released at each step. The NH2-terminal sequence up to 20 residues was identical to that of the beta subunit of the arvilla enzyme, with one exception at step 16, at which arginine was observed instead of glutamine. The COOH-terminal residue was deduced to be arginine on carboxypeptidase A and B digestions and on hydrazinolysis. These results indicate that the putida enzyme consists of two identical subunits, in contrast to the arvilla enzyme which consists of two nonidentical subunits, alpha and beta [C. Nakai et al. (1979) Arch. Biochem. Biophys. 195, 12-22], although these two enzymes have very similar properties.

Amino Acid Sequence↗
PubMed1984

Purification of a major serum protein of rainbow trout (Salmo gairdneri) homologous to the third component of mammalian complement.

The present paper describes the purification and characterization of a major serum protein of the rainbow trout (Salmo gairdneri) required for complement-related functions (target cell lysis and opsonization). This protein, termed C3-1, was identified as the third component (C3) of rainbow trout complement. It is homologous to component C3 of human and other mammalian complements based on its structural and functional characteristics. Rainbow trout C3-1 is a beta-globulin of Mr = 190,000 composed of two polypeptide chains (Mr = 128,000 alpha-chain and Mr = 74,000 beta-chain) linked by disulfide bonds. The chain structure of C3-1 is, therefore, very similar to those of the third and fifth components of mammalian complement. C3-1 retains, on its alpha-chain, a unique hidden thiol site that can be exposed upon attachment of methylamine in the same way as human C3 and C4. The amino acid composition and NH2-terminal sequence of C3-1 show significant homology to mammalian C3 and to cobra venom factor (cobra C3).

Amino Acid Sequence↗
PubMed1983

Complete nucleotide sequence of the metapyrocatechase gene on the TOI plasmid of Pseudomonas putida mt-2.

Metapyrocatechase which catalyzes the oxygenative ring cleavage of catechol to form alpha-hydroxymuconic epsilon-semialdehyde is encoded by the xylE gene on the TOL plasmid of Pseudomonas putida mt-2. We have cloned the xylE region in Escherichia coli and determined the nucleotide sequence of the DNA fragment of 985 base pairs around the gene. The fragment included only one open translational frame of sufficient length to accommodate the enzyme. The predicted amino acid sequence consisted of 307 residues, and its NH2- and COOH-terminal sequences were in perfect agreement with those of the enzyme recently determined (Nakai, C., Hori, K., Kagamiyama, H., Nakazawa, T., and Nozaki, M. (1983) J. Biol. Chem. 258, 2916-2922). A mutant plasmid was isolated which did not direct the synthesis of the active enzyme. This plasmid had a DNA region corresponding to the NH2-terminal two-thirds of the polypeptide. From the deduced amino acid sequence, the secondary structure was predicted. Around 10 base pairs upstream from the initiator codon for metapyrocatechase, there was a base sequence which was complementary to the 3'-end of 16 S rRNAs from both E.coli and Pseudomonas aeruginosa. A preferential usage of C- and G-terminated codons was found in the coding region xylE, which contributed to the relatively high G + C content (57%) of this gene.

Amino Acid Sequence↗
PubMed1982

Simple, direct measurement of lipoprotein X in serum.

This relatively simple method for quantitative estimation of lipoprotein X is based on assay of phospholipids in precipitates of lipoprotein X. We first remove other lipoproteins by precipitation with phosphotungstic acid solution. Lipoprotein X is then precipitated from the supernatant fluid with an alkaline solution of magnesium ion, and phospholipid is determined in this precipitate. Results are linearly related to concentration of lipoprotein X. The CV is less than 3%. Results correlated well with those for agar gel electrophoresis.

Cholestasis↗
PubMed1977

Protein inhibitors of phosphorylase phosphatase and cyclic AMP-dependent protein kinase from rabbit skeleta muscle.

A heat-and acid-stable protein inhibitor of phosphorylase phosphatase is present in a highly purified preparation of protein inhibitor of cyclic AMP-dependent protein kinase from rabbit skeletal muscle. Although these two inhibitors have strikingly similar properties to each other, such as sensitivity to trypsin and behavior on gel permeation chromatography, they can be separated by polyacrylamide disc gel electrophoresis. This indicates that the phosphatase-inhibitory and kinase-inhibitory activities reside with different protein species. The inhibition of both the enzymes is not altered by incubating the inhibitor preparation with a general phosphoprotein phosphatase, with phosvitin kinase, or with cyclic AMP-dependent protein kinase. Inhibition of phosphorylase phosphatase is of a non-competitive type supporting the idea that the phosphatase inhibitor is not an alternative substrate for the enzyme. Inhibition of phosphatase activity is selective in that it does no occur when phosphorylated histone or phosphorylated protamine are used as substrates.

Animals↗
PubMed1977

Inhibition of rabbit skeletal muscle phosphorylase phosphatase by spermine.

The effect of three naturally occurring polyamines (putrescine, spermidine, and spermine) on the activity of rabbit skeletal muscle phosphorylase phosphatase was investigated. Only spermine significantly inhibited the enzyme. The mode of inhibition (ki value of 0.3 mM) of the phosphatase by spermine appears to be different from that caused by divalent metal ions or by other organic cations, such as arginine and lysine esters, since it is noncompetitive with respect to the substrate, phosphorylase a.

Animals↗
PubMed1975

Effects of magnesium on the kinetic properties of bovine heart glycogen synthase D.

Highly purified glycogen synthase D, free of synthase kinase and phosphatase activities, was prepared from bovine heart. The enzyme had no activity without glucose 6-phosphate. Kinetic studies of this enzyme at various concentrations of UDP-glucose demonstrated that there was no cooperativity with respect to the substrate at any concentration of glucose-6-P with or without Mg2+. Glucose 6-phosphate increased the maximum velocity (Vmax) of the enzyme, but had very little or no effect on the Michaelis constant for UDP-glucose (Km equals 0.33 mM). Free Mg2+ gave a high Vmax at all glucose 6-phosphate concentrations without affecting the Km for the substrate. The double reciprocal plots of reaction rates versus glucose 6-phosphate concentration were biphasic and were interpreted as evidence for two kinetic forms, each with a glucose 6-phosphate binding site of different affinity (A1/2 values equals 0.31 and 1.1 mM). High Mg2+ concentrations nearly abolished the biphasic kinetic behavior of glucose 6-phosphate, suggesting that the Vmax of both enzyme forms was the same at saturating concentration of Mg2+ and glucose 6-phosphate and that magnesium ion might have no effect on the binding of glucose 6-phosphate or on the state of the equilibrium between two forms. Plots of reaction velocity versus Mg2+ concentration showed no cooperativity of Mg2+ activation in the presence or absence of glucose 6-phosphate. Both kinetic forms of glycogen synthase D had the same affinity for Mg2+ (A1/2 is approximately equal to 4 mM). Studies on the inhibition of the enzyme by Pi, ATP, and UTP were carried out with assays specific for the form of synthase with A1/2 for glucose 6-phosphate equals 0.31 mM (high affinity form) and the form with A1/2 for glucose 6-phosphate equals 1.1 mM (LOW AFFINITY FORM) BY ASSAYING WITH AND WITHOUT 5.0 MM free Mg2+, respectively. Both forms of synthase exhibited positive cooperativity with respect to UDP-glucose when inhibited by UTP, but not with Pi or ATP. Thus, each form of the enzyme had more than one UDP-glucose site, and these sites showed cooperativity only in the presence of a uridine nucleotide inhibitor. In the absence of Mg2+ (low affinity form), the inhibitors, Pi, ATP, and UTP, all induced positive cooperativity with respect to glucose 6-phosphate binding to this enzyme. The positive cooperativity induced by ATP was obliterated by adding free Mg2+ (high affinity form), but that induced by other inhibitors was affected slightly or not at all by the cation. These results indicate that each of the enzyme forms (high or low affinity forms) has more than one glucose 6-phosphate site and that these may function in a cooperative manner. The preceding findings are interpreted in relation to the importance of Mg2+ in the regulation of glycogen synthase D activity as well as the regulation of glycogen synthase phosphatase activity in heart.

Adenosine Triphosphate↗
PubMed1975

Assay for adenylate cyclase and cyclic nucleotide phosphodiesterases and the preparation of high specific activity 32-P-labeled substrates.

Simple one step assay methods for adenylate cyclase (ATP pyrophosphate-lyase (cyclizing) EC 4.6.1.1) and cyclic nucleotide phosphodiesterases (3',5'-cyclic nucleotide 5'-nucleotidohydrolase EC 3.1.4.17) have been developed. [alpha-32-P] ATP is used as the substrate for adenylate cyclase. Acid-heat destruction of [32-P] ATP remaining after the cyclase reaction followed by Zn-Ba treatment quantitatively leaves cyclic [32-P] AMP in the supernatant essentially free from other 32-P-containing compounds. This assay method requires no corrections for recovery and routinely yields blank values less than 0.03 per cent. If higher sensitivity is desired, a simple 5 min alumina column step can be introduced into the procedure which quantitatively elutes cyclic [32-P] AMP directly into a liquid scintillation vial and lowers the blank values to less than 0.002 per cent. This method is rapid and easily performed, without sacrificing high reliability, specificity, or sensitivity. One step phosphodiesterase assays are easily accomplished using 32-P-labeled cyclic nucleotides as substrates. Descending paper chromatography of the reaction mixture on individual 2 cm wide paper strips gives a complete and quantitative separation of all possible products including [5'-32-P] AMP and [5'-32-P] GMP from their respective 32-P-labeled 3',5'-cyclic nucleotides in 1-2 h. The paper strips are cut, inserted in scintillation vials without scintillant and the 32-P-products determined by Cerenkov counting. Low blank values of less than 0.5 per cent and the use of high specific activity 32-P-labeled cyclic nucleotide substrates make this method the most reliable and most sensitive phosphodiesterase assay described to date. Because of the simplicity, specificity, and high sensitivity obtainable with these assay methods using 32-P-labeled substrates, we have also devised simple conditions for the preparation and purification of [alpha-32-P] ATP, cyclic [32-P] AMP and cyclic [32-P] GMP with specific activities in excess of 100 Ci/mmol. These high specific activity 32-Plabeled cyclic nucleotides are important for these new assay methods and are also useful to follow purification recovery of endogenous cyclic AMP and cyclic GMP from biological materials before protein binding or radioimmunological isotope displacement assays when performed in the femtomole range.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗