PART I: METHIONINE REQUIREMENT OF LIVER EPITHELIAL CELLS IN CULTURE. PART II: ETHIONINE IN PROTEIN SYNTHESIS

The lipotropes, methionine, choline, folic acid and vitamin B(,12), influence the metabolism and carcinogenic activity of several known hepatocarcinogens. Lipotrope metabolism is itself altered by carcinogen administration and is abnormal in tumors. Normal and transformed rat liver epithelial cells were grown in medium in which methionine was replaced by homocysteine. Growth constants for the two normal cell lines grown on homocysteine were only slightly lower than those observed with the same lines in complete medium. However, the growth constants of three transformed lines grown on homocysteine were significantly lower than the corresponding values on complete medium. Supplementation of homocysteine medium with thymidine, adenosine and guanosine or thymidine alone did not stimulate cell growth. Normal and transformed cell lines were grown in medium containing varying concentrations of leucine and methionine. The transformed cells showed no disproportionately increased requirement for methionine over leucine. The growth constants of the cells grown on homocysteine paralleled their cellular levels of N('5)-methyltetrahydrofolate: homocysteine methyltransferase. Ethionine, the ethyl analog of methionine, is carcinogenic. In procaryotes it has been demonstrated in other laboratories that ethionine can be aminoacylated to the isoacceptors of methionine transfer ribonucleic acid. Furthermore, protein containing ethionine has been isolated in crystalline form from the culture medium of bacteria. Similar studies have not been reported for eucaryotic systems. Transfer ribonucleic acid and aminoacyl synthetases were isolated from rat liver. Transfer ribonucleic acid was aminoacylated with radioactive methionine and ethionine and fractionated by reverse phase column chromatography. The elution profiles of the transfer ribonucleic acid aminoacylated with ethionine and methionine were the same except that ethionyl-transfer ribonucleic acid was more hydrophilic. Conditions of optimum aminoacylation of purified initiator and internal reading methionine isoacceptors were determined. Ethionine may, therefore, be able to initiate protein synthesis as well as replace methionine at internal sites of protein. Preliminary experiments in this laboratory using a purified internal reading isoacceptor indicate the latter to be true. Incorporation into protein, particularly regulatory protein, may be the mechanism of ethionine carcinogenesis.