Taneaki Higashi

High purification and properties of Pseudomonas cytochrome oxidase

Abstract Pseudomonas cytochrome oxidase was purified further by acetone fractionation. Its specific activity was increased 4–8 fold by this procedure. The resulting enzyme solution was green. In oxidized form, its absorption peaks were at 635, 410 and 356 mμ, and there was a shoulder around 450 mμ. After reduction with hydrosulfite there were absorption bands at 625, 551, 548, 522 and 418 mμ, and a shoulder around 470 mμ. The activity of the purified oxidase was inhibited in the presence of a low concentration of cyanide or carbon monoxide. The absorption spectrum of the oxidase in the presence of cyanide was unaffected except for a slight increase in absorption at 410 mμ in the oxidized form. Carbon monoxide scarcely influenced the spectrum of the oxidized form. However, in the reduced form, the absorption peak at 625 mμ was flattened and the shoulder around 470 mμ disappeared, indicating that these absorption bands corresponded to the a - and γ-absorption peak of cytochrome a 2 , respectively. Purification increased the likelihood that P -cytochrome oxidase is a protein complex consisting of a cytochrome a 2 -moiety and at least two other cytochrome moieties (the cytochrome 551 -moiety and cytochrome 548 -moiety). Full activity was observed in the presence of a minimum of 20% oxygen. A reaction mechanism for P -cytochrome oxidase is discussed on the basis of these results.

Organ-specific difference in the sugar chains of γ-glutamyltranspeptidase

Sugar chains of gamma-glutamyltranspeptidase purified from neonatal mouse liver and adult mouse kidney were quantitatively released as oligosaccharides from the polypeptide backbone by hydrazinolysis. A comparative study of the structures of the oligosaccharides has revealed that the GlcNAc beta 1 leads to 4Man beta 1 leads to group is found in the sugar chains of kidney enzyme but not in those of liver enzyme. This is considered as an organ-specific difference common to mammals because the same phenomenon was found in bovine and rat enzymes.

Decrease of glutathione and induction of γ-glutamyltransferase by dibutyryl-3′,5′-cyclic AMP in rat liver

Abstract Administration of dibutyryl-3′,5′-cyclic AMP to rats caused marked, but temporary, decrease of liver glutathione. This decrease appeared to be catalyzed by γ-glutamyltransferase, because it occured concomitantly with induction of the enzyme and increase of cysteine in the liver. The biological half-life of hepatic γ-glutamyltransferase was estimated to be about 3 hours. It is proposed that the physiological levels of glutathione and γ-glutamyltransferase in the liver are controlled by 3′,5′-cyclic AMP, and that liver glutathione may serve as a reservoir of cysteine, which can be mobilized by the transferase.

The structures of the carbohydrate moieties of mouse kidney γ-glutamyltranspeptidase: Occurrence of X-antigenic determinants and bisecting N-acetylglucosamine residues☆

Paper electrophoresis and Bio-Gel P-4 column chromatography of the oligosaccharides released from mouse kidney gamma-glutamyltranspeptidase by hydrazinolysis gave fractionation patterns quite distinct from those of the bovine and rat kidney enzymes. Structural studies of the fractionated oligosaccharides by sequential exoglycosidase digestion in combination with methylation analysis showed that mouse kidney gamma-glutamyltranspeptidase contains a series of bisected complex-type asparagine-linked sugar chains with the following oligosaccharides as their outer chain moieties: GlcNAc beta 1----, Sia alpha 2----Gal beta 1----4GlcNAc beta 1----, Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----, and sialylated N-acetyllactosamine repeating sugar chains. Some of these sugar chains were found for the first time in glycoproteins.