Takamitsu Yorifuji

Crystalline arginine racemase.

Abstract Arginine racemase has been purified from the extract of Pseudomonas graveolens approximately 2,300-fold by a procedure including column chromatography on DEAE-cellulose, DEAE-Sephadex and Sephadex G-150. The purified enzyme was crystallized. The crystalline enzyme is homogeneous upon analysis by ultracentrifugation and its molecular weight is 167,000. The enzyme exhibits absorption maxima at 280 mμ and 420 mμ, and contains firmly bound pyridoxal 5′-phosphate.

A simple approximation of weight-based protein measurement with HPLC apparatus

In this report, we describe a simple refractometric method which uses an HPLC apparatus equipped with a differential refractometric detector and a signal integrator. This method requires very small amounts of sample proteins and gives protein concentrations approximate to those obtained on a dry weight basis

Guanidinobutyrase for L-Arginine Degradation in Brevibacterium helvolum

Guanidinobutyrase (guanidinobutyrate amidinohydrolase, EC. 3.5.3.7) catalyzing the third step of the arginine oxygenase pathway in Brevibacterium helvolum IFO 12073 (ATCC 11822) was purified to homogeneity and characterized. The enzyme had a molecular weight of 190,000 and was composed of four apparently identical subunits with a molecular weight of 45,000. The E(1%) value at 280 nm of the enzyme protein was 2.4. The enzyme contained 0.5 mol of firmly bound Zn(2+) per mol of subunit. The enzyme was highly specific for 4-guanidinobutyrate, but had a weak activity toward L- and D-arginine. The Michaelis constant (Km) for 4-guanidinobutyrate was 2.9 mM. The optimum pH was 9.0. Strong mixed type inhibition was observed with thioglycolate and several other thiol compounds. These properties were compared with those of the enzyme of fluorescent Pseudomonas and discussed.

Metabolisms of Nucleosides in Bacteria: Part IV. Pyrimidine Nucleoside Phosphorylase in Erwinia carotovora and Corynebacterium sepedonicumPart V. Purine Nucleoside Phosphorylases in Erwina carotovoia and Corynebactcrium sepedonicum

Pyrimidine nucleoside phosphorylases obtained from Er. carotovora and Cor. sepedonicum were purified by means of ammonium sulfate fractionation and DEAE-cellulose column chromatography. Some properties of these enzyme were also studied. The enzyme from Cor. sepedonicum catalyzed the formation and the degradation of uridine only, although the enzyme from Er. carotovora catalyzed the formation of thymine riboside as well as uridine. Optimum pH of the enzyme from Cor. sepedonicum was 9.0 and that of Er. carotovora was 7.0.Purine nucleoside phosphorylases obtained from Er. carotovora and Cor. sepedonicum were partially purified and some properties of these enzymes were studied.Purine nucleoside phosphorylases obtained from Er. carotovora and Cor. sepedonicum catalyzed the formation of inosine, guanosine, adenosine and xanthosine, though the reaction rate was different with each enzyme.

[41] Arginine racemase (Pseudomonas graveolens)☆

Publisher Summary This chapter describes the assay, purification, and properties of arginine racemase. This enzyme catalyzes the conversion of either D- or L-arginine to the racemate. The assay method is based on the measurement of L-arginine formed from D-arginine. L-Arginine is determined by measuring the formation of urea released by arginase, by a slight modification of the method of Archibald. The crystalline enzyme can be stored at 3–7° as a suspension in 0.01 M potassium phosphate buffer, pH 7.3, containing 10 -4 M pyridoxal 5′-phosphate and 60% saturated ammonium sulfate without loss of activity for periods of over six months. The enzyme, when examined in the presence of 0.04 M glycine buffer, has a maximum activity in the pH range 10.0–10.6. The enzyme exhibits absorption maxima at 280 m μ and 420 m μ with an absorbance ratio of 5:1. The arginine racemase is inhibited by hydroxylamine and L-ornithine. L-Ornithine inhibits the enzyme noncompetitively for both D-arginine and pyridoxal 5′-phosphate. The enzyme also catalyzes the racemization of several amino acids other than arginine.