Comparative properties of envelope-associated arginine-gingipains and lysine-gingipain of Porphyromonas gingivalis.
Two arginine specific proteinases (Arg-gingipain [RGP-A, RGP-B]) and a lysine specific proteinase (Lys-gingipain [KGP]) were purified from materials of the envelope of Porphyromonas gingivalis solubilized by a detergent by the sequential procedures of ion-exchange chromatography, affinity chromatography, and isoelectric focusing. Each purified enzyme showed a single stained band on SDS-PAGE. The three enzymes were commonly activated by reducing reagents such as mercaptoethanol, dithiothreitol and cysteine. RGP-B was activated markedly by glycyl-glycine and KGP was activated significantly by EDTA. Both RGP-A and RGP-B were inhibited by p-hydroxymercuribenzoate, tosyl-L-lysine chloromethyl ketone (TLCK), N-ethylmaleimide, EDTA, leupeptin, L-trans-epoxy-succinylleucylamido-(4-guanidino)butane and antipain. KGP was inhibited by p-hydroxymercuribenzoate, N-ethylmaleimide and TLCK. The molecular masses of RGP-A and RGP-B were the same (43 kDa), and that of KGP was 48 kDa. The hydrolytic activities of RGPs and KGP to chromogenic synthetic substrates were limited to the compounds with arginine and lysine in the P-1 positions, respectively. When IgG was treated with the three enzymes separately, it was demonstrated that two new fragments of 34 kDa and 15 kDa were generated in each reaction product. Hemoglobin was almost exhaustively digested by RGP-B, but substantial degradation could not be induced by RGP-A or KGP treatment.
Department of Oral Microbiology, Matsumoto Dental University School of Dentistry, Shiojiri-Shi, Japan. fujimura@po.mdu.ac.jp
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Fujimura
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Hirai
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Shibata
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Nakayama
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Nakamura
D018829:Adhesins, Bacterial; D001120:Arginine / Q000378:metabolism; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D000080867:Gingipain Cysteine Endopeptidases; D006388:Hemagglutinins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006454:Hemoglobins / Q000378:metabolism; D007074:Immunoglobulin G / Q000378:metabolism; D007525:Isoelectric Focusing; D008239:Lysine / Q000378:metabolism; D016966:Porphyromonas gingivalis / Q000201:enzymology*; D011480:Protease Inhibitors; D013379:Substrate Specificity
D018829:Adhesins, Bacterial; D000080867:Gingipain Cysteine Endopeptidases; D006388:Hemagglutinins; D006454:Hemoglobins; D007074:Immunoglobulin G; D011480:Protease Inhibitors; D001120:Arginine; D003546:Cysteine Endopeptidases; D008239:Lysine