Identification of a Membrane Protein Involved in Mitochondrial Phosphate Transport

Abstract

A specific labeling by radioactive N-ethylmaleimide of a protein involved in phosphate transport was obtained by protecting 1 of the 2 SH groups of the transport system with low concentrations of mersalyl. Subsequently, the other free SH groups were blocked with excess N-ethylmaleimide. Removal of mersalyl by cysteine and subsequent incubation with labeled N-ethylmaleimide results in a "specific" binding of N-ethylmaleimide to 1 SH group functionally involved in phosphate transport. The isolated inner membrane fraction of the labeled [rat liver] mitochondria was subjected to dodecylsulfate gel electrophoresis. The difference of the radioactivity pattern on the dodecylsulfate-polyacrylamide gel of inner membrane proteins, labeled with N-[14C]ethylmaleimide in the absence and with N-[3H]-ethylmaleimide in the presence of mersalyl during preincubation of mitochondria, shows only 1 main labeled peak. The same labeled peak is obtained from the difference of labeling after preincubation with a constant low concentration of mersalyl at 32.degree. C and at 0.degree. C. The position of the labeled peak on the dodecylsulfate-polyacrylamide gel corresponds to a protein of MW of 26,500 .+-. 800. The amount of 1 of the 2 SH groups, involved in phosphate transport, was estimated to be 30 nmol/g of mitochondrial protein.