PURIFICATION OF A GTP-BINDING PROTEIN LOCALIZED IN MITOCHONDRIA

A particulate fraction consisting of heavy organelles such as nuclei a nd mitochondria was prepared from Ehrlich ascites tumor cells. From th is fraction we have purified a GTP-binding protein with a molecular ma ss of 33 kDa (MTG33) by guanidine hydrochloride extraction followed by four steps of column chromatography. The K-d value of MTG33 for GTP w as 17 nM. [alpha-P-32]GTP-billding to MTG33 was inhibited by GTP and G DP, but not appreciably by ATP, CTP, UTP, or GMP. MTG33 hydrolyzed GTP to GDP at a rate of 4.5 mmol/min/mol protein. Subcellular fractionati on analysis of mouse liver revealed that the heavy mitochondrial fract ion contained the highest level of MTG33. Furthermore, dual immunofluo rescence examination indicated that the staining of NIH 3T3 cells with anti-MTG33 antibody is coincident with the distribution of mitochondr ial succinate dehydrogenase. Of the mouse organs examined, the heart c ontained the highest level of MTG33. These results strongly suggest th at MTG33 is a GTP-binding protein located in mitochondria.