Massetti AP, Mengoni F, Contini C, Sebastiani G, Folgori F, Vullo V, Sorice F; International Conference on AIDS.

Int Conf AIDS. 1992 Jul 19-24; 8: B138 (abstract no. PoB 3310).

Institute of Infectious Disease, University La Sapienza, Rome, Italy.

OBJECTIVES: To characterize and purify proteases of P. carinii in order to study their role in host-parasite relationship. METHODS: P. carinii proteases were identified by a technique of copolimerization of substrate in polyacrylamide gels. P. carinii cysts purified from a human lung and sonicated were applied on a 7.5% polyacrylamide gel containing 0.1% gelatin and subjected to SDS-PAGE under non reducing conditions. Gels were incubated overnight at 37 degrees C either in phosphate buffered saline, pH 7.5 (PBS) or in PBS containing protease inhibitors and then Coomassie stained; proteases were identified as clear bands on a blue background. In order to purify P. carinii proteases, sonicated cysts were absorbed on a hydroxylapatite (HPT) column and eluted with phosphate buffer of increasing ionic strength (10-400 mM) or with MgCl2 or CaCl2 solutions. Eluted fractions were concentrated and analyzed by copolimerization of substrate in gel. RESULTS: Six protease bands (MW 100-50 kDa) were identified in P. carinii. The activity of the band at approximately 90 kDa was specifically inhibited by 10 mM EDTA. By purification of the different P. carinii components by HPT chromatography, it was possible to reveal the presence of a band of 90 kDa with proteolytic activity in the fractions eluted with 100 mM phosphate buffer or with 3 mM MgCl2 or CaCl2. In phosphate buffer also a 60 kDa protease was present, while with MgCl2 or CaCl2 the band at 90 kDa was the only band which was shown by copolimerization of substrate in gel. CONCLUSIONS: The technique of copolimerization of substrate in gel allowed us to identify in P. carinii 6 bands with proteolytic activity; the band at 90 kDa was inhibited by 10 mM EDTA, a chelating agent of bivalent ions which selectively inhibits metalloproteases, and might therefore belong to this class of proteases. The 90 kDa protease was eluted by HPT column by 100 mM phosphate buffer and by 3 mM MgCl2 or CaCl2. This elution pattern suggests that the protease is a highly basic protein, since bivalent cations at low concentrations selectively elute from HPT proteins with an isoelectric point greater than pH 8.

Publication Types:

• Meeting Abstracts

Keywords:

• Edetic Acid
• Electrophoresis, Polyacrylamide Gel
• Endopeptidases
• Gelatin
• Humans
• Hydrogen-Ion Concentration
• Isoelectric Point
• Kinetics
• Metalloproteases
• Peptide Hydrolases
• Protease Inhibitors
• isolation & purification

Other ID:

• 92401037

UI: 102198750

From Meeting Abstracts