Noriyuki Shiraishi

IDENTIFICATION OF S100B PROTEIN AS COPPER-BINDING PROTEIN AND ITS SUPPRESSION OF COPPER-INDUCED CELL DAMAGE

We have isolated from bovine brain a protein with a high capacity to inhibit the copper ion-catalyzed oxidation ofl-ascorbate and identified it as S100b protein, an EF-hand calcium-binding protein, by sequencing its proteolytic peptides. Copper binding studies showed that this protein has four copper-binding sites per dimeric protein molecule with a dissociation constant of 0.46 μm and that in the presence ofl-ascorbate, copper ions bind to a total of six binding sites with a great increase in affinity. Furthermore, we examined whether S100b protein can prevent copper-induced cell damage. Bovine S100b protein was found to suppress dose-dependently the hemolysis of mouse erythrocytes induced by CuCl2. We transformed Escherichia coli cells with pGEX-5X-3 vector containing a cDNA for rat S100b protein, so that this protein could be expressed as a fusion protein with glutathioneS-transferase. The transformed cells were demonstrated to be markedly resistant to a treatment with CuCl2 plus H2O2 as compared with the control cells expressing glutathione S-transferase alone. These results indicate that S100b protein does suppress oxidative cell damage by sequestering copper ions.

Increased Metallothionein Content in Rat Liver Induced by X Irradiation and Exposure to High Oxygen Tension

X irradiation and exposure to high oxygen tension are known to induce lipid peroxidation. The effects of these stresses on hepatic content of metallothionein, which may be involved in the regulation of zinc and copper metabolism, have been studied. The amount of metallothionein in rat liver was increased 11-fold by a high dose of X irradiation (1000 R). Increased metallothionein content (about 15 times) was also observed in liver of rats exposed to high oxygen tension for 3 days.

Copper-catalyzed autoxidations of GSH and L-ascorbic acid: mutual inhibition of the respective oxidations by their coexistence.

Glutathione (GSH) is known to inhibit copper-catalyzed autoxidation of L-ascorbic acid (AA); in this study, AA was found to conversely inhibit copper-catalyzed autoxidation of GSH. To elucidate the mechanism of the mutual inhibition of the autoxidations of these two reducing substances in their coexistence, we have kinetically investigated these phenomena. The study of the former phenomenon revealed that GSH forms a 1:1 chelate with Cu(+) and thereby prevents the autoxidation of AA. By the analysis of the latter phenomenon, it was postulated that the inhibition of GSH oxidation by AA is due to rapid reduction of thiyl radical of GSH by AA rather than competition of AA with GSH in the reduction of Cu(2+). The effect of GSH on the formation of hydroxyl radical by the copper-catalyzed autoxidation of AA was also studied and it was found that the hydroxyl radical formation was delayed dose-dependently by GSH with time lags comparable to those of the oxidation of AA. Because there are several lines of evidence that redox-active copper ions are released from tissues under pathological conditions, it is possible that such copper ions coexist with AA and GSH in vivo, and in such a situation, GSH may exert an inhibitory effect on the hydroxyl radical formation caused by the autoxidation of AA.

Fragmentation and dimerization of copper-loaded prion protein by copper-catalysed oxidation

Prion protein consists of an N-terminal domain containing a series of octapeptide repeats with the consensus sequence PHGGGWGQ and a C-terminal domain composed of three alpha-helices and two short beta-strands. Several studies have shown that the N-terminal domain binds five Cu2+ ions. In the present study, we have investigated copper-catalysed oxidation of a recombinant mouse prion protein, PrP23-231. The copper-loaded PrP23-231 was found to be carbonylated by incubation with dopamine. Besides the formation of carbonyls, a cross-linked species with the dimeric size and C-terminally truncated species were generated. These reactions were retarded in the presence of Cu+- and Cu2+-specific copper chelators, catalase, and SOD (superoxide dismutase), but not in the presence of various bivalent metal ions. Together, these results indicate that the copper bound to prion protein undergoes catalytic cycling in the presence of catecholamines and causes the oxidation of the protein.

Mitochondrial damage in galactosamine-induced liver intoxication in rats

Mitochondria isolated from livers of rats which received D-galactosamine (375 mg/kg body wt., four times) demonstrated a marked decrease in respiratory control ratios, the ADP/O ratios, and state 3 respiration rates and an increase in state 4 respiration rates. The aberration was profound with site I being altered prior to sites II and III. Quantitation of phospholipids revealed a reduction of total phospholipids per mg protein with decreases in phosphatidylcholine and phosphatidylethanolamine contents. Caldiolipin was the only phospholipid which remained unaltered. Fatty acid composition was altered in these phospholipids; caldiolipin was altered most severely, showing reductions in linoleic and arachidonic acids, and an elevation in saturated fatty acids and in some other small components of fatty acids. In phosphatidylethanolamine, palmitic acid decreased, whereas stearic and docosahexenoic acids increased. These changes were smaller in phosphatidylcholine fatty acids. These mitochondria were also characterized by an altered composition in high molecular weight polypeptide components. By experiments with normal mitochondria in vitro, galactosamine, but not other aminohexoses, was proved to be an uncoupling agent of the oxidative phosphorylation system. Electron microscopic observation demonstrated that both in vivo and in vitro treatments with galactosamine induced marked disorganization of mitochondrial structures. These results suggest that mitochondrial damage is also included in galactosamine-induced hepatic lesion.

Carbonyl formation on a copper-bound prion protein fragment, PrP23-98, associated with its dopamine oxidase activity

The amino‐terminal part of prion protein (PrP), containing a series of octapeptide repeats with the consensus sequence PHGGGWGQ, has been implicated in the binding of copper ion. This region possesses amino acid residues susceptible to oxidation, such as histidine, lysine, arginine and proline. In this study, we have investigated copper‐catalyzed oxidation of an N‐terminal part of human PrP, PrP23–98, that was prepared by the recombinant DNA technique. Carbonyl formations on copper‐bound PrP23–98 induced by dopamine and L‐ascorbate were analyzed kinetically, and it was found that the redox cycling of PrP23–98‐bound copper, especially induced by dopamine, was coupled to the formation of carbonyls on the protein.

Permeability changes of phospholipid liposomes caused by pancreatic phospholipase A2: analysis by means of phase transition release

Many biological messages are recognized by the binding of the ligands to specific receptors of the outer surface of the cell membrane [l-4]. These bindings then initiate certain chemical and physical changes in the membrane. One of the early changes in membrane state is the activation of phospholipase A2 (PLA2) [5]. This reaction has many important functions such as in biosynthesis of prostaglandins, in physicochemical changes of biomembranes and in regulation of the activities of other membrane bound enzymes. Therefore, the activation of membrane bound PLA2 has a key role in the mechanisms of cell activation. One of the important features of the regulation of PLA2 is the dependency of activation on membrane structure. Many investigations were carried out on the analogies with much better defined systems using soluble phospholipase, though this system is essentially different from those occurring in natural membranes. In those studies, it was proposed that a particular region of PLA2, so called interface recognition site, is involved in interaction with phospholipid structure [6]. Moreover, it was considered that phosphatidylcholine can be hydrolysed only near the transition temperature where lipid in liquid crystalline phase and in the gel phase coexist ]7,81. We have therefore applied the phase transition release technique to the study of the activation mechanism as related to the changes in physiological characteristics of the membrane and bring evidence that the maximal carboxyfluorescein release Phospholipase A2 (Phospholipid liposome) Dipalmitoylphosphatidylcholine

Combination of NADPH and copper ions generates proteinase K-resistant aggregates from recombinant prion protein.

Recent studies have demonstrated that the octapeptide repeats of the N-terminal region of prion protein may be responsible for de novo generation of infectious prions in the absence of template. Here we demonstrate that PrP-(23–98), an N-terminal portion of PrP, is converted to aggregates upon incubation with NADPH and copper ions. Other pyridine nucleotides possessing a phosphate group on the adenine-linked ribose moiety (the reduced form of nicotinamide adenine dinucleotide 3′-phosphate, nicotinic acid adenine dinucleotide phosphate, and NADP) were also effective in promoting aggregation, but NADH and NAD had no effect. The aggregation was attenuated by the metal chelator EDTA or by modification of histidyl residues with diethyl pyrocarbonate. The aggregates are amyloid-like as judged by the binding of thioflavin T, a fluorescent probe for amyloid, but do not exhibit fibrillar structures according to electron micrography. Interestingly the aggregates were resistant to proteinase K digestion. Likewise NADPH and zinc ions caused aggregation of PrP-(23–98), but the resulting aggregates were susceptible to degradation by proteinase K. Upon incubation with NADPH and copper ions, the full-length molecule PrP-(23–231) also formed proteinase K-resistant amyloid-like aggregates. Because it is possible that PrP, NADPH, and copper ions could associate in certain tissues, the aggregation observed in this study may be involved in prion initiation especially in the nonfamilial types of prion diseases.

Occurrence of two missense mutations in Cu‐ATPase of the macular mouse, a menkes disease model

We have investigated the genetic defect of the Cu‐ATPase gene (Atp7a) in the macular mouse, a genetic model of classical Menkes disease. Northern blot analysis showed that its placenta and kidney possess a normal amount of the Cu‐ATPase mRNA of the normal size; sequencing analysis revealed two missense mutations, His674Arg and Ser1381Pro, in a PCR‐amplified cDNA for mutant Cu‐ATPase. The latter mutation was suspected to affect the function of the ATPase, because it lies in the transmembrane segment that is thought to form a channel for the transportation of copper ions.

Distribution of protein-bound zinc in serum of analbuminemic rat.

The distribution of protein-bound zinc in serum of rat with analbuminemia was analyzed with gel filtration and affinity chromatography. From the profiles of chromatography, the zinc present in analbuminemic rat serum is composed of two principal species in similar to that of Sprague-Dawley rat: one fraction is firmly bound to alpha 2-macroglobulin, and a second fraction is more loosely bound to various proteins.