Alkalophile NADH dehydrogenase consisting of two 65-kDa subunits was changed by subtilisin into an enzyme species consisting of two 38-kDa subunits. The amino acid composition and enzyme activity per molecule of the subtilisin-treated enzyme were almost the same as those of the native enzyme, respectively. On mixing with phospholipid liposome, the conformation of the native enzyme was changed, as suggested by the changes in the type of Arrhenius plot and of CD spectrum and enzyme activity. These conformational properties of the subtilisin-treated enzyme, on the other hand, were not affected by liposome. Gel filtration of the subtilisin-treated enzyme mixed with the liposome showed no binding of the protein to liposome.
We have developed a sensitive and simple staining method for use in electrophoretic analysis of serum alpha-amylase (EC 3.2.1.1) isoenzymes. The principle of this method is as follows. alpha-Amylase hydrolyzes 4-nitrophenylmaltoheptaoside to generate oligosaccharide, which is then converted to gluconolactone in the presence of oligosaccharide dehydrogenase (no EC no. assigned), with concomitant reduction of 1-methoxy-5-methylphenazinium methylsulfate (1-m-PMS) to 1-m-PMSH. The hydrogen in 1-m-PMSH is then transferred to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to yield formazan. Each of the isoenzymes can then be measured densitometrically. The mean (and SD) values for total amylase, P1, S1, and S2 as determined by this method with sera from 25 healthy adults in fasting were 251 (64), 104 (35), 126 (40), and 22 (11) U/L, respectively. Between-assay CVs (n = 10) for determinations of P1, S1, and S2 were 3.54%, 4.03%, and 7.01%, respectively.
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