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Cellular locations of proteinases and association with vesicles in Porphyromonas gingivalis.

Oishi S, Miyashita M, Kiso A, Kikuchi Y, Ueda O, Hirai K, Shibata Y, Fujimura S - Eur. J. Med. Res. (2010)

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Oral Health, Graduate School of Oral Medicine, Shiojiri-Nagano, Japan.

ABSTRACT
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.

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Gel filtration on Sephacryl S-300 of RGP purified from culture supernatant. The column (2.6 by 94 cm) was eluted with 50 mM Tris-HCl, pH 8.0 containing 200 mM NaCl.
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Figure 1: Gel filtration on Sephacryl S-300 of RGP purified from culture supernatant. The column (2.6 by 94 cm) was eluted with 50 mM Tris-HCl, pH 8.0 containing 200 mM NaCl.

Mentions: The purified RGP of culture supernatant (43 kDa) eluted at elution volume of 335 ml in the gel filtration on Sephacryl S-300 (Figure 1); however, the RGP solubilized from vesicles eluted in the void volume of the column (205 ml) with a negligible peak of the activity at the corresponding position of the purified culture supernatant RGP (Figure 2). These findings indicate that the molecular mass of RGP originated from the vesicles may be over 1,500 kDa (by the manual of Sephacryl S series). RGP eluted at the void volume was temporarily referred to as macromolecular form RGP. Gel filtration profiles of KGP were substantially the same as those of RGP; however, significant amounts of activity in vesicle sample were detected in the valid volume, and the approximate ratio of the activities in the void volume and to the valid volume was 2 to 1. To examine whether 43 kDa RGP was able to convert to the macromolecular form simply by incubation with vesicles, mixtures of vesicle and the purified 43 kDa or raw RGP (concentrated culture supernatant) were subjected to gel filtration, but no shift of the elution volume of 43 kDa RGP to the void volume was seen.


Cellular locations of proteinases and association with vesicles in Porphyromonas gingivalis.

Oishi S, Miyashita M, Kiso A, Kikuchi Y, Ueda O, Hirai K, Shibata Y, Fujimura S - Eur. J. Med. Res. (2010)

Gel filtration on Sephacryl S-300 of RGP purified from culture supernatant. The column (2.6 by 94 cm) was eluted with 50 mM Tris-HCl, pH 8.0 containing 200 mM NaCl.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3351907&req=5

Figure 1: Gel filtration on Sephacryl S-300 of RGP purified from culture supernatant. The column (2.6 by 94 cm) was eluted with 50 mM Tris-HCl, pH 8.0 containing 200 mM NaCl.
Mentions: The purified RGP of culture supernatant (43 kDa) eluted at elution volume of 335 ml in the gel filtration on Sephacryl S-300 (Figure 1); however, the RGP solubilized from vesicles eluted in the void volume of the column (205 ml) with a negligible peak of the activity at the corresponding position of the purified culture supernatant RGP (Figure 2). These findings indicate that the molecular mass of RGP originated from the vesicles may be over 1,500 kDa (by the manual of Sephacryl S series). RGP eluted at the void volume was temporarily referred to as macromolecular form RGP. Gel filtration profiles of KGP were substantially the same as those of RGP; however, significant amounts of activity in vesicle sample were detected in the valid volume, and the approximate ratio of the activities in the void volume and to the valid volume was 2 to 1. To examine whether 43 kDa RGP was able to convert to the macromolecular form simply by incubation with vesicles, mixtures of vesicle and the purified 43 kDa or raw RGP (concentrated culture supernatant) were subjected to gel filtration, but no shift of the elution volume of 43 kDa RGP to the void volume was seen.

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Oral Health, Graduate School of Oral Medicine, Shiojiri-Nagano, Japan.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus