G. Rotilio

THE COMPLETE AMINO ACID SEQUENCE OF HUMAN Cu/Zn SUPEROXIDE DISMUTASE

D. BARRA, F. MARTINI, J. V. BANNISTER*, M. E. SCHININP;, G. ROTILIO, W. I-I. BANNISTER+ and F. BOSSA Istituto di Chimica Biologica e Centro di Biologia Molecolare de1 CNR, University of Rome, Rome, Italy, *Inorganic Chemistry Department, University of Oxford, Oxford OXI 3QR, England and +Nuffield Department of Clinical Biochemistry, Radcliffe Infirmary, University of Oxford, Oxford OX2 6HE, England

Erythrocuprein and singlet oxygen

McCord and Fridovich [ 1,2] have shown that erythrocuprein, the copper and zinc containing protein extracted from red blood cells, can enzymatically disproportionate superoxide radicals (Oh), an intermediate of the reduction of oxygen, the generation of which in some Ha 0s -producing biological oxidations has been demonstrated [3]. Khan has recently pointed out [4] that dismutation of Og gives rise to the production of the highly reactive singlet state of oxygen, which can be revealed by its own luminescence; indeed Arneson [5] observed that oxidation of xan’thine by xanthine oxidase produced luminescence, which was abolished by the presence of erythrocuprein. On the basis of these data, the main purpose of the following paper is to show that erythrocuprein has a quenching effect on luminescence phenomena which are reasonably ascribed to singlet oxygen produced even in the absence of superoxide intermediates. Thus the physiological role of erythrocuprein could be to catalyze the singlet-triplet conversion of the oxygen states: in other words to afford an enzyme-regulated dismutation liberating in solution as a product the inert ground state triplet oxygen, rather than simply accelerating the dismutation reaction.

Pyridoxal phosphate as a prosthetic group of pig kidney diamine oxidase

Abstract Pig kidney diamine oxidase was obtained in homogeneous form by the criteria of gel electrophoresis and ultracentrifugation. A new modification of the purification procedure is described. The presence of pyridoxal phosphate in the enzyme was demonstrated by the following studies: ( 1 ) spectrophotometric observations on the enzyme treated with carbonyl reagents; ( 2 ) chromatography of pyridoxal phosphate semicarbazone obtained from the enzyme; and ( 3 ) reactivation of apo-aspartate aminotransferase by diamine oxidase derivatives.

Effect of ionic strength on the activity of bovine superoxide dismutase.

In studies of activity of superoxide dismutase (SOD), see [l] , two kinds of methods have been used. For routine detection of activity, the inhibition by the enzyme of some 0; dependent reactions has been utilized [2-41. For studies of the catalytic mechanism and when a precise evaluation of rate constants is needed, pulse radiolytic methods have been the only successful ones so far [S--8]. Recently we have applied the polarographic method of the kinetic currents to kinetic studies of the dismutation of O;, which allows the determination of the rate constants by a very simple procedure [9]. Moreover it presents some advantages with respect to pulse radiolysis in so far that the composition of the solution under study can be varied in a way which would disturb straightforward pulse radiolysis experiments. For example, variation of ionic strength and type of ions of the solution, can be somewhat limited in pulse radiolysis, because of the possible production of secondary radicals. In this report we present results on the effect of ionic strength and different species of anions and cations on the activity of SOD as a part a program.

Kinetic study of O2− dismutation by bovine superoxide dismutase. Evidence for saturation of the catalytic sites by O2−

Dismutation of O2− by bovine copper-zinc superoxide di smutase has been studied at different O2− concentrations with a polarographic method. Saturation of the enzyme by the substrate was observed and Km and Vmax values were calculated. Inhibition by OH− and CN− was shown to be of the competitive type. The data support on inner sphere mechanism for the reaction between O2− and copper.

Superoxide dismutase, glutathione peroxidase and catalase in oxidative hemolysis. A study of Fanconi's anemia erythrocytes

Abstract Superoxide dismutase, glutathione peroxidase and catalase were assayed in the erythrocytes of three patients of Fanconis anemia. Superoxide dismutase was found to be significantly decreased, as previously reported. The enzymes metabolizing H 2 O 2 are normal (glutathione peroxidase in the higher limits of the normal value). The abnormal erythrocytes were found to be as resistant (perhaps more resistant) as normal red blood cells to oxidative hemolysis induced by drugs. Malonyl dialdehyde production was found to be comparable to that of normal erythrocytes. It is concluded that a significant (30–40%) deficiency of superoxide dismutase, when associated to normal values of H 2 O 2 -removing enzymes, does not affect the antioxidative defense capability of erythrocytes, even in conditions of augmented oxidative injury.

19F relaxation as a probe of the oxidation state of Cu, Zn superoxide dismutase. Studies of the enzyme in steady-state turnover

Summary 19F nmr relaxation proved to be a proper method to evaluate the Cu2+/Cu+ ratio at the active site of Cu, Zn superoxide dismutase in either equilibrium or turnover conditions. In the steady-state under fluxes of O2−, the enzyme was found to contain 50% Cu2+, in accord with the equal rates of copper catalytic reduction and oxidation. Previous results giving 75% Cu at the steady-state without change of the overall catalytic constant were confirmed for samples subjected to freeze-drying or freezing-thawing.

Correlation between superoxide dismutase, glutathione peroxidase and catalase in isolated rat hepatocytes during fetal development

Abstract Superoxide dismutase, glutathione peroxidase and catalase activities were determined in isolated fetal rat hepatocytes of various ages and compared with the values of neonatal and adult cells. The developmental pattern of superoxide dismutase and glutathione peroxidase were very similar with a low constant activity in the fetal cells and a postnatal burst. On the contrary catalase begins to increase already since the 18th day of the fetal life. The results suggest a functional correlation of superoxide dismutase and glutathione peroxidase in the antioxidative enzyme defense of liver cells.

The Cu,Zn superoxide dismutase isoenzymes of Xenopus laevis : purification, identification of a heterodimer and differential heat sensitivity

The three Cu,Zn superoxide dismutase electromorphs of the amphibian Xenopus laevis were purified by an original procedure. N-terminal sequence analysis demonstrated that they are two different homodimers (AA and BB) and a hybrid heterodimer (AB), arising from the co-expression of duplicated genes. The three forms have the same pI, same enzyme activity and EPR spectra, but different heat-sensitivity, form BB being more resistant than form AA, with form AB showing intermediate sensitivity. Thermostability of BB and the control bovine enzyme was enhanced by a tenfold increase in protein concentration. It is suggested that the higher heat sensitivity of the AA isoenzyme is related to the presence of an extra Cys residue and to an easier dissociation of the protein dimer into monomers.

pH dependence of redox properties of the type 2 Cu-depleted tree laccase

The laccases are the best chara~te~zed copper oxidases having three types of copper centres [I]. They contain one blue paramagnetic copper (Type l), one non-blue paramagnetic copper (Type 2), and one copper pair undetectable by EPR (Type 3) [I]. Fu~he~ore they can be selectively depleted of Type 2 Cu, resulting in an inactive enzyme which can be reactivated by reconstitution with copper ions [2,3]. Some discrepancies exist in the literature on the properties of the Japanese Rhus vewicifera lactase selectively depleted of Type 2 Cu [4,5]. They concern the spectroscopic properties of the remaining Type 1 and Type 3 Cu and the redox behaviour of the enzyme. We have now reexamined such properties, with particular attention to their dependence on pH, since different pH conditions had apparently been used in previous reports [4,5]. A definite pH dependence was found of the redox behaviour and the stability of Type 2 Cudepleted lactase.