Purification and characterization of a protease from Porphyromonas gingivalis capable of degrading salt-solubilized collagen.
An enzyme capable of hydrolyzing the substrate 4-phenylazobenzyloxycarbonyl-L-prolyl-leucyl-glycyl-prolyl-D-ar gin ine (pZ-peptide), pZ-peptidase, was purified from the oral bacterium Porphyromonas gingivalis. pZ-peptidase hydrolyzed salt-solubilized type I collagen from rat skin, rat plasma low-molecular-weight kininogen, and transferrin at room temperature in the presence of calcium and dithiothreitol. pZ-peptidase did not cleave acid-soluble type I calf skin collagen, type V placental collagen, lysozyme, albumin, or human plasma fibrinogen. Furthermore, the purified enzyme did not hydrolyze N-alpha-benzoyl-DL-Arg-p-nitroanilide, Gly-Pro-p-nitroanilide, N-p-tosyl-Gly-Pro-Arg-p-nitroanilide, N-p-tosyl-Gly-Pro-Lys-p-nitroanilide, azoalbumin, or azocasein. Under reducing conditions, the native enzyme migrated as a single band at 120 kDa on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. However, when heated to 100 degrees C for 10 min in SDS under reducing conditions, the enzyme migrated as a major band at 50 kDa and a minor band at 60 kDa on SDS-polyacrylamide gel electrophoresis. Zymography using calf skin gelatin revealed the gelatin-cleaving activity of the enzyme as evidenced by a diffuse band in the range of 120 to 300 kDa under reducing conditions at room temperature, suggesting that this is the native form of the enzyme. However, incubation at 50 degrees C for 10 min under reducing conditions showed gelatin-cleaving activity at a distinct band of 60 kDa. A minimum temperature of 50 degrees C was required to dissociate the 60-kDa chain from the native complex in active form on gelatin zymography. The ability of the enzyme to cleave other proteins, including kininogen and transferrin, suggests that it has specificity for the Pro-X-Gly sequence found in several proteins, including collagen.
Sojar
H T
HT
Department of Oral Biology, State University of New York, Buffalo 14214.
Lee
J Y
JY
Bedi
G S
GS
Genco
R J
RJ
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002850:Chromatography, Gel; D003094:Collagen / Q000378:metabolism*; D010450:Endopeptidases / Q000737:chemistry / Q000302:isolation & purification*; D005780:Gelatin / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016966:Porphyromonas gingivalis / Q000201:enzymology*; D051381:Rats; D014168:Transferrin / Q000378:metabolism
D000596:Amino Acids; D014168:Transferrin; D005780:Gelatin; D003094:Collagen; D010450:Endopeptidases