〔研究ノート〕ヒト血清アルブミンの糖化反応による カルボキシメチルリジンの生成箇所の同定

　　Human serum albumin（HSA）is an abundant plasma protein and is modified by glucose in plasma. The incubation of HSA with glucose at 37℃ for four weeks resulted in the formation of carboxymethyllysine in 11 sites of lysine residues of HSA（65, 190 . . .Ⅰ, 199, 233, 313, 378 . . .Ⅱ, 402, 432, 519, 525, 573 . . . Ⅲ）. The same experiments without glucose showed no carboxymethyllysine formation.　　Although early studies established that four lysine residues（Lys199, Lys281, Lys439 and Lys525）had been modified by nonenzymatic glycosylation, carboxymethylations of Lys281 and Lys439 were not detected in this experiment. Most carboxymethylation of lysine residues in HSA was located in domain Ⅲ in our study

種々の生体物質のペルオキシナイトライト消去能

Peroxynitrite, which is formed from the reaction of superoxide radical and nitric oxide, can cause specific structural modifications in proteins such as the addition of a nitro group onto aromatic residues. These modifications can cause perturbations in the properties of the proteins, such as conformation, catalytic activity, susceptibility to proteolysis, and immunogenicity, resulting in several human diseases. Finding a specific peroxynitrite scavenger is, therefore, of considerable importance. Accordingly, we assessed the scavenging ability of several biomolecules purchased from commercial sources on nitration of a protein by peroxynitrite at physiologic CO2 condition in vitro. In sixteen molecules tested, reduced redox-cofactors, and polyphenol-compounds were effective scavengers against peroxynitrite-mediated protein nitration in vitro and one of these, epigallocatechine was the most effective. It was also noticed that tryptophan and serotonin creatinine sulfate possessed relatively high scavenging abilities against peroxynitrite-mediated protein nitration in vitro

米ぬか水抽出物のペルオキシナイトライト消去能

Oxidative stress is known as one of the major causes of diseases and is known to be initiated by reactive oxygen species(ROS).Peroxynitrite(PON)is a highly reactive ROS, which can nitrate and oxidize biomolecules, and is produced by the rapid reaction between superoxide and nitric oxide. In tissues of patients with a number of diseases, 3-nitrotyrosine has been detected. These results strongly suggest that there is formation of PON in the tissues in vivo. Therefore, from the standpoint of preventive medicine, it is important to clarify the PON scavenging activities of dietary antioxidants. In this study, we used three methods to evaluate the efficacy of rice bran to protect proteins from peroxynitrite-mediated modification. By coexistence of the rice bran extract in the reaction mixture, inhibition of pig heart lactate dehydrogenase by 1mM PON treatment was suppressed about 70%, and the formation of 3-nitrotyrosine and yellow pigment production by 1mM PON in bovine serum albumin were inhibited about 75% and 60%, respectively. These results suggest that there is strong scavenging activity of PON in the extract of rice bran

Porphyromonas gingivalis スーパーオキシドジスムターゼの構造における72位Leu をTrp に置換した影響

Porphyromonas gingivalis contains a single constitutive superoxide dismutase (SOD) that is active with either iron or manganese at the active site. The aim of this work was to evaluate the effect of the Leu ₇2 to Trp mutation on the structure of P. gingivalis SOD (Pg SOD) using lectrophoretic characterization. Leu ₇2, which is located near the active site metal, is substituted with Trp in aligned amino acid sequences of iron–containing SOD. The results of electrophoretic characterization and the expressed activity of mutant SOD suggest that mutant SOD have the same gross structure as wild–type SOD. We herein conclude that the integrity of Leu ₇2 is a necessary requisite for the metal–tolerant activity of Pg SOD

Porphyromonas gingivalis SOD における活性中心近傍に局在するアミノ酸残基の金属特異的活性における関与：Leu ₇2 Trp およびLeu ₇6 Phe の2残基変異による検討

The role of superoxide dismutase (SOD) as a radical scavenger in Porphyromonas gingivalis is well documented. P. gingivalis SOD (Pg SOD), which is characterized by a metal–tolerant activity, can use either iron or manganese as a cofactor. Leu ₇2 and Leu ₇6, located near the active–site metal, are characteristic amino acid sequences of Pg SOD proteins, although they are substituted to Trp in the ₇2 position and Phe in the ₇6 position in most iron–containing SOD (Fe–SOD) proteins. In the present study, we constructed a mutant of the enzyme with changes from Leu ₇2 to Trp and Leu ₇6 to Phe. This mutant SOD was examined with respect to its metal–dependent activity and structural characterization. We herein conclude the integrity of Leu ₇2 and Leu ₇6 is a necessary requisite for the metal–tolerant activity of Pg SOD

Porphyromonas gingivalis SOD の活性中心金属の近位には，トレオニンがセリンよりも優先的に選択される:155位Gly をSer に変異させた影響

Summary In this study, we analyzed the cambialistic superoxide dismutases (SODs) of Porphyromonas gingivalis (Pg–SODs) with a mutation directed at glycine position 155 to introduce serine. Glycine 155 is a highly conserved outer sphere in manganese–containing SODs (Mn–SODs), even though threonine is substituted at this position in most iron–containing SODs (Fe–SODs). Conversion of glycine 155 may affect the metal–specific activity of SODs, including that of cambialistic Pg–SODs.Previously, we reported that a Pg–SOD Gly155Thr mutant exhibited a substantially changed metalspecific activity from that of a cambialistic type to an Fe–specific type. Although serine and threonine equally contribute to protein function, serine has never been observed at position 155 in SODs. In order to elucidate this phenomenon, we created a Pg–SOD mutant Gly155Ser. The specific Fedependent activity of this mutant was almost identical to the wild–type SOD, whereas the Mndependent activity exhibited a 60% reduction. The ultraviolet–visible absorption of Fe– and Mnreconstituted mutant SODs did not exhibit characteristic absorption spectra. Similar to the wild–typeSODs, the mutant SODs exhibited a single band with identical mobilities after separation by polyacrylamide gel electrophoresis. However, their behavior after anion–exchange chromatography differed from that of the wild–type SODs. Thus, Gly155 is considered to be an essential residue for maintaining the hydrogen–bond network for Mn–specific and Fe/Mn–tolerant activity. Gly155 was possibly retained instead of Ser during the evolution of SODs owing to its increased efficiency in maintaining a dimeric structure

〔報 文〕シイタケ子実体のスーパーオキシドジスムターゼの精製と諸性質

Crude extract of Lentinus edodes(L. edodes)contained more than three kinds of superoxide dismutases(SODs)which were distinguished electrophoretically. All of these were found to be insensitive to H2O2 and cyanide. A centrifugal study of the crude extract in 0.25M sucrose solution revealed that most of the SOD activities are located in the cytoplasmic fraction. We purified one of these superoxide dismutases from pilei of L. edodes to homogeneity by ammonium sulfate fractionation, DE-32 ion-exchange, Sephadex G-100 gel filtration and Butyl-toyopearl hydrophobic chromatographies. The purified enzyme showed that a single protein band coincided with the single SOD activity band in native polyacrylamide gel electrophoresis(PAGE). The purified SOD showed a single band in PAGE in the presence of sodium dodecyl sulfate(SDS-PAGE)and its subunit molecular weight was estimated to 23±0.5kDa. The subunit molecular weight of SOD was also estimated by LC-MS analysis to be 22,184Da. Using the sedimentation equilibrium centrifugation method the molecular mass of native SOD was estimated to be 84,240Da. These observations suggest that the enzyme is a tetramer composed of subunits of equal size. Metal analysis of the native enzymes revealed 0.64g-atoms Mn per mole subunit in the preparations whose specific activity were 3500U/mg. Direct analysis of N-terminal amino acid of this enzyme by Edman degradation using a protein sequencer cannot detect any amino acid residue, but amino acid residue of N-terminal appeared as serine residue after the treatment of the enzyme with tetrafluoroacetic acid in a vapor phase at 60℃ for 10min. Therefore, the N-terminal amino acid was modified with acetyl group. The modification of N-terminal amino acid is the first example of Mn-SOD. Some of other physiological and biochemical properties of the enzyme were also investigated

ペルオキシナイトライトによるトリプトファンの反応生成物

トリプトファンとペルオキシナイトライトを反応させてからすぐに逆相HPLCで分離させ,ニトロソ化物,ニトロシル化物等不安定な物質を同定することや,前報でトリプトファンの方がチロシンよりも未確認の生成物が多く検出されたということから,これらの未確認生成物を同定することを目的として,実験を進めた。その結果,キヌレニン,7-ニトロトリプトファン,6-ニトロトリプトファン,4-ニトロトリプトファン,5-ニトロトリプトファンが確認された。また,反応直後と数日後の比較から,報告にあるニトロソ化物,ニトロシル化物以外の,不安定な生成物が確認された。It is well known that tyrosine residues in proteins are the target of peroxynitrite to form nitrotyrosine residues. On the other hand, it became clear that tryptophan residues in proteins are the target of peroxynitrite to form tryptophan derivatives. However, the reaction products of tryptophan with peroxynitrite are not so clear. In our previous investigation, we reported that tryptophan was easier to react with peroxynitrite than tyrosine, and that a lot of unknown reaction products had been detected in the reaction. In the reaction of tryptophan with peroxynitrite, it was reported that the reaction is complicated and formed some unstable products, N^1-nitro- and N^1-nitroso derivatives. In this study, we experimented on the reaction of the tryptophan by peroxynitrite. As a result, kynurenine, 7-nitrotryptophan, 6-nitrotryptophan, 4-nitrotryptophan and 5-nitrotryptophan were identified. And two other unstable products which were not N^1-nitro- and N^1-nitroso derivatives were found