An organic solvent-stable protease (PST-01 protease) in a culture broth of organic solvent-tolerant Pseudomonas aeruginosa PST-01 was purified by successive hydrophobic interaction chromatography using Butyl-Toyopearl gels. The purified enzyme was homogeneous as determined by SDS-polyacrylamide gel electrophoresis. PST-01 protease had a molecular mass of 38 kDa. The optimum temperature and pH for casein hydrolysis were 55 degrees C and 8.5, respectively. PST-01 protease was stable at pH 8-12 and below 50 degrees C and was determined to be a metalloprotease which was inhibited by EDTA, 1,10-phenanthroline, and phosphoramidon. PST-01 protease inhibited by EDTA was reactivated completely by the addition of zinc or cobalt ions. The stability of PST-01 protease in solutions containing water-soluble organic solvents or alcohols was higher than that in the absence of organic solvent. Furthermore, in general, PST-01 protease was more stable than commercially available proteases, namely, subtilisin Carlsberg, thermolysin, and alpha-chymotrypsin, in the presence of water-soluble organic solvents or alcohols.

Changes of chicken breast myosin during storage at 2 ЊC and 37 ЊC were monitored immunochemically. Anti-myosin subfragment-1 (S-1) monoclonal antibody, which recognized epitopes within the 27 kDa fragment of S-1, and the anti-myosin rod polyclonal antiserum, were prepared. Myosin degradation products were not detected in muscle extracts stored for 3 weeks at 2 ЊC. In contrast, storage at 37 ЊC brought about the degradation of myosin heavy chain to immunologically detectable small fragments. While, myosin rod produced during the conditioning period was not decomposed into any small filaments. Namely, storage of muscle at 37 ЊC resulted in minor amounts of myosin heavy chain degradation, with initial conversion to rod and S-1 fragments, and subsequent breakdown occurred in the S-1 region only. Immunoblot assay also suggested that the pattern of changes in myosin heavy chain in muscle incubated at 37 ЊC was similar to that produced by in vitro digestion with cathepsin D.

Recently, two new kits, HITAZYME (Hitachi Chemical Co., Ltd.) and SERO IPALISA (Savyon Diagnostics, Ltd.), for the assay of anti-C. trachomatis antibodies by the enzyme immunoassay (EIA) method have been developed and put into clinical application. In the study reported here, the authors investigated the clinical usefulness of these assay kits, together with the IPAzyme and micro-IF test, in the diagnosis of cases of urogenital tract C. trachomatis infections. 1. The positive rates for IgA antibodies, which are considered to be an indicator of active infection, obtained with the HITAZYME and SERO IPALISA kits in the 82 antigen-positive cases were significantly (p < 0.005) higher than the rates obtained with the IPAzyme and micro-IF test. These results showed the usefulness of the HITAZYME and SERO IPALISA kits for detecting C. trachomatis infections. 2. A comparison was made of the assay results obtained with the HITAZYME and SERO IPALISA kits, and it was found that there was a large number of cases (142) that tested negative for IgA antibodies with the HITAZYME but positive with the SERO IPALISA kit. We carried out a confirmatory test on the specimens of cases for which the results obtained with the HITAZYME and SERO IPALISA kits were not in agreement. This test employed the Western blotting method using COMC (the antigen extracted from EB of C. trachomatis strain L2 and used in the HITAZYME kit) and whole EB of C. trachomatis strain L2 (the antigen used in the SERO IPALISA kit). The results showed a significantly higher degree of agreement between the HITAZYME kit data and the Western blotting data than between the SERO IPALISA kit data and the Western blotting data. 3. In addition, with the objective of investigating the existence of cross reactivity with anti-C. pneumoniae antibodies, we performed Western blotting using as the antigen crude whole EB of C. pneumoniae strain TW-183. The results showed that anti-C. pneumoniae antibodies were detected in 25 of 35 (71.4%) cases that were negative with C. trachomatis antigen and the HITAZYME kit and positive with the SERO IPALISA kit. These findings indicate a strong possibility that these cases positive with the SERO IPALISA kit are due to a cross reaction with anti-C. pneumoniae antibodies.(ABSTRACT TRUNCATED AT 400 WORDS)

No abstract