1990 Fiscal Year Final Research Report Summary
Partial Purification and Some Characteristics of Proteinase Induced by Staphylococcal Exfoliative Toxin.
| Project/Area Number |
63570481
|
|---|
| Research Category |
Grant-in-Aid for General Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Research Field |
Dermatology
|
|---|
| Research Institution | Showa University |
|---|
Principal Investigator |
TAKIUCHI Iwao Showa University, Medicine prof., 医学部, 教授 (90102342)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
TERAMOTO Teruyo Showa University, Medicine Assistant, 医学部, 助手 (10192204)
WATANABE Hideyoshi Showa University, Medicine Assistant, 医学部, 助手 (20221690)
|
|---|
| Project Period (FY) |
1988 – 1990
|
| Keywords | Staphylococcus aureus / Exfoliative toxin / Epidermis / Protease / SSSS |
|---|
| Research Abstract |
When staphylococcal exfoliative toxin (ET) was incubated with the epidermis that had been separated from newborn mice dermis by incubating it in 0.24 M NH_4Cl (pH 9.5), a substantial increase in caseinolytic activity was observed. A production of the activity reached a maximum at 12hrs. Whereas, no activity was detected when it was incubated without ET. Our findings suggest that the exfoliation in SSSS is induced by proteinase caused by ET. An attempt to purify the casein-hydrolyzing enzyme was made after 12hrs incubation at 37^ﾟC. The enzyme was partially purified by several chromatographies using Sephadex G-50 and G-75, TSK-60 and TSK-DEAE-5-PW column. However, on SDS-PAGE the partially purified fractions exhibited four protein bands. When the partially purified enzyme was preincubated with EDTA or EGTA, substantial inhibition of the activity was observed ; however, no recovery of the activity was detected after the addition of CaCl_2. Treatment of the enzyme with PMSF and NEM caused little inactivation of the activity. Enzyme activity retained almost 100 % of the initial activity following 2min incubation at 60^ﾟC, but was completely inactivated after 4min. When rabbits or mice were immunized using the partially purified fractions, two precipitation lines were demonstrated by Ouchterlony double diffusion experiments. However, enzyme activity was not inhibited by the rabbit and mouse IgG which was obtained from the anti-sera using protein A affinity chromatography. At present, we were unable to demonstrate the localization of the enzyme with an immunoperoxidase staining.
Finally, we found the result when epidermis alone which were obtained by heat separation was incubated at 37^ﾟC for 12hrs, same enzyme activity was detected. This problem is also under investigation on our laboratory.
|
