J. Biochem, 1983, Vol. 94, No. 1 181-188
© 1983 Japanese Biochemical Society
research-article |
Mechanism of the Activation of 
-AminoIevulinate Synthetase in Rhodopseudomonas spheroides by Rat Liver Mitochondrial Fraction1
Department of Biochemistry, Yamagata University School of Medicine Yamagata, Yamagata 990-23
2 To whom correspondence should be addressed.
When rat liver mitochondria were incubated with L-[35S]cystine or L-[14C]cystine in the presence of 
-cystathionase [EC 4.4.1.1
[EC]
], the mitochondrial proteins incorporated 35S, but not 14C, showing that only the S-atom was incorporated from L-cystine into the mitochondrial proteins by the action of -cystathionase.
The incorporation of 35S into the mitochondrial proteins was not effectively inhibited by dilution with the cold sulfide ion which was added to the reaction mixture at a concentration 5 times higher than that of L-[35S]cystine used as a substrate. Therefore, the S-atom of L-cystine may not be incorporated into mitochondrial proteins through the sulfide ion produced by -cystathionase.
The S-atom incorporated into mitochondrial proteins was fairly stable under acidic conditions, but was released as sulfide by incubating the proteins with thiols such as dithiothreitol. The MS-labeled mitochondrial proteins were digested by pronase (nonspecific proteinase), and a 35S-labeled compound obtained in the hydrolysate was identified as cystine trisulfide by Dowex 50 column chromatog-raphy and high-voltage paper electrophoresis. It was assumed, therefore, that the 35S-atom of [35S]cystine is incorporated to form a cystine trisulfide structure with two cysteine residues in the mitochondrial protein.
The subfractionation of the 35S-labeled mitochondria was performed by using digitonin and lubrol, revealing that the outer membranous, intermembranous space and inner membranous proteins were highly labeled.
The mitochondrial protein incorporating the S-atom from L-cystine was capable of converting the inactive form of 
-aminolevulinate synthetase of Rhodopseudomonas spheroides to the active form. Therefore, the mechanism of this activation reaction seems to be the same as that of the "regulator" protein found in R. spheroides cells.
1 This work was supported in part by Scientific Research Grant 57570120 from the Ministry of Education, Science and Culture of Japan.