| Budget Amount *help |
¥4,100,000 (Direct Cost: ¥4,100,000)
Fiscal Year 1990: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 1989: ¥2,800,000 (Direct Cost: ¥2,800,000)
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| Research Abstract |
A proteolytic measurement of various isoenzymes activities and the optimal conditions for selective proteolytic inactivation of cytosolic aspartate aminotransferase to determine mitochondrial aspartate aminotransferase in serum were investigated by use of purified bytosolic aspartate aminotransferase inactivating proteases. Some of the proteases, such as, alpha-chymotrypsin, subtilisin, protease 401 and proteinase K, also specifically inactivated cytosolic aspartate aminotransferase, but not mitochondrial aspartate aminotransferase. The protease 401 and proteinase K were the heat stable for storage. Protease 401 was found to be effective over a pH range 6.0-10.0. A pH of 9.5 with 0.5% albumin in the reagent mixture was determined to be optimal for inactivation of cytosolic aspartate aminotransferase and preservation of mitochondrial aspartate aminotransferase, lactic dehydrogenase, and malic dehydrogenase in the assay procedure. The presence of serum endogeneous protein inhibitors such
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as alpha_1-antitrypsin and alpha_2-macroglobin did not inhibit protease 401 and proteinase K.
Total mitochondrial aspartate aminotransferase, the sum of apo- and holo-mitochondrial aspartate aminotransferase activity in human serum, was measured by using proposed method. Cytosolic aspartate aminotransferase is completely inactivated, and apo-mitochondrial aspartate aminotransferase is completely activated by pyridoxal 5' -phosphate within 5 min. Results by the proposed method correlated well with those by an immunochemical method (r=0.994,n=145) and the regression curve was Y=0.95X + 1.60 (X ; proteolytic method) and showed excellent inhibitory activity of the protease for holo-and apo-cytoslic aspartate aminotransferase up to 5000 U/L more than 200-fold the upper limit and activation of mitochondrial apo-aspartate aminotransferase up to 2000 U/L in the presence of 100 mumol of pyridoxal 5' -phosphate per liter. Within-run CVs were good (1.37-7.49%). Mean values for total mitochondrial aspartate aminotransferase activities in serum of the healthy subjects were 4.8 (SD 0.9) and 1.8 (SD 0.8) U/L, respectively (n=154). Various common interferents tested not affects this assay. Less
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