Cellular locations of proteinases and association with vesicles in Porphyromonas gingivalis.
We found that locations of arginine-specific gingipain (RGP) in the cellular fractions in the crude extract, envelope, vesicles, and culture supernatants were 48%, 16%, 17%, and 31%, respectively, and the corresponding values of lysine-specific gingipain (KGP) were 47%, 10%, 7%, and 36%, respectively. Although the molecular mass of RGP in the culture supernatant had been determined as 43 kDa, and that of KGP had been as 48 kDa, molecular masses of both proteinases solubilized from the vesicles were estimated to be over 1,500 kDa, since they eluted in the void volume of the column in the gel filtration on Sephacryl S-300. There was no reduction of molecular size by the following treatment with SDS, high-concentration NaCl, or urea. Interestingly, the occurrence of the macromolecular forms could not observed in other enzymes tested such as monopeptidyl, dipeptidyl, and tripeptidyl peptidases, as well as alkaline phosphatase. Therefore, occurrence of the macromolecular forms may be restricted to the proteinases. When the vesicle and culture supernatants containing free RGP and KGP were mixed and incubated, neither RGP nor KGP seemed to bind to vesicles. RGP bound to the vesicle was found to be more stable to heat treatment than the free form, suggesting that association of RGP with the vesicle caused heat stability of this enzyme.
Department of Oral Health, Graduate School of Oral Medicine, Shiojiri-Nagano, Japan.
S
S
Oishi
M
M
Miyashita
A
A
Kiso
Y
Y
Kikuchi
O
O
Ueda
K
K
Hirai
Y
Y
Shibata
Setsuo
S
Fujimura
D018829:Adhesins, Bacterial / Q000302:isolation & purification / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology*; D003546:Cysteine Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005374:Filtration / Q000379:methods; D000080867:Gingipain Cysteine Endopeptidases; D006358:Hot Temperature; D006801:Humans; D008828:Microbiological Techniques; D010518:Periodontitis / Q000382:microbiology*; D016966:Porphyromonas gingivalis / Q000201:enzymology* / Q000648:ultrastructure
D018829:Adhesins, Bacterial; D004791:Enzyme Inhibitors; D000080867:Gingipain Cysteine Endopeptidases; D003546:Cysteine Endopeptidases