| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1986: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1985: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Research Abstract |
A serine proteinase isolated from Aspergillus sojae seemed to be homogeneous by free boundary electrophoresis at pH 4.0 and 7.8, and the sedimentation pattern showed it to be monodisperse.
However, the presence of a major band and four minor bands was noted on disc gel electrophoresis at pH 9.4. These were termed in order of their decreasing mobility towards the cathode, Sep <I> , Sep <II> , Sep <III> , Sep <IV> and Sep <V> .
When angiotensin was digested with the multiple forms of serine proteinase at pH 7.5 and 30゜C, each form hydrolyzed the <Tyr^4> - <Ile^5> bond. Increasing the mobility toward the cathod from Sep <IV> to Sep <II> affects the <K_m> values only slightly, but caused a remarkable increase in the catalytic rate <k_(cat)> . When the value of <k_(cat)> / <K_m> are plotted against electrophoretic mobility of the serine proteinase toward the cathod, the curve shows age-related change like a growth curve. The maximum <k_(cat)> / <K_m> value is shown with Sep <II> .
When Sep <V> was incubated in Tris-HCl buffer at pH 8.0 and 30゜C for one night, Sep <IV> was observed on SDS gel electrophoresis. Similar molecular conversion was observed with the incubation of the molecular species Sep <IV> and Sep <III> , respectively.
Similar molecular weight values of these multiple forms were found on SDS gel electrophoresis. It was, however, important to learn that the SDS gel electrophoresis of Sep <I> shows a major band with molecular weight of 36 K and two lower molecular weight fraction of 19 K and 17 K when incubated at 30゜C in a pH 8.0 solution. These multiple forms have the same N- and C- terminal amino acid residues. And no significant differences and only minor conformational changes were observed.
This is the first report that the catalytic activities of electrophoretic species of the serine proteinases shows age-related change.
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