Kurganov B I, Schors E I, Livanova N B, Chebotareva N A, Eronina T B, Andreeva I E, Makeeva V P, Pekel N D
AN Bach Institute of Biochemistry, Russian Academy of Sciences, Moscow.
Biochimie. 1993;75(6):481-5. doi: 10.1016/0300-9084(93)90114-8.
The kinetics of tryptic proteolysis of rabbit skeletal muscle phosphorylase b has been registered by the diminishing of protein fluorescence intensity at lambda = 335 nm (excitation at 290 nm) or by the disappearance of the enzyme activity (0.02 M Hepes buffer, pH 6.8, 37 degrees C). The first procedure showed that flavins (riboflavin, FMN, FAD) protected the enzyme against tryptic digestion. Microscopic dissociation constants for the complexes of phosphorylase b with riboflavin, FMN and FAD were calculated from dependences of the initial digestion rate on the flavin concentration. They where equal to 30 +/- 1, 15.8 +/- 0.2 and 36 +/- 1 microM, respectively. No influence of FMN on the rate of the tryptic hydrolysis of phosphorylase b was observed when using the second procedure (enzyme activity test). FMN completely prevents the formation of 69-, 81- and 85-kDa fragments during 20 min incubation of phosphorylase b with trypsin.
通过在λ = 335 nm处蛋白质荧光强度的减弱（激发波长为290 nm）或酶活性的消失（0.02 M Hepes缓冲液，pH 6.8，37℃）来记录兔骨骼肌磷酸化酶b的胰蛋白酶解动力学。第一种方法表明，黄素（核黄素、FMN、FAD）可保护该酶免受胰蛋白酶消化。根据初始消化速率对黄素浓度的依赖性，计算了磷酸化酶b与核黄素、FMN和FAD复合物的微观解离常数。它们分别等于30±1、15.8±0.2和36±1 μM。当使用第二种方法（酶活性测试）时，未观察到FMN对磷酸化酶b胰蛋白酶水解速率的影响。在磷酸化酶b与胰蛋白酶孵育20分钟期间，FMN完全阻止了69 kDa、81 kDa和85 kDa片段的形成。
