The long term objectives of this program are to study the chemistry, enzymology and biological significance of protein methylation. In particular, cytochrome c methylation in fungi and plants will be used as a model system. This will be pursued with the following aims: (1) Exploration of the possible effects of methylation on various aspects of the in vitro translation of mRNA of yeast iso-l cytochrome c, employing the wheat germ extract in vitro translation system. In particular, the effect of methylation on (a) the completion of the polypeptide chain, (b) the rate of synthesis, (c) import into the mitochondria, and (d) the stability of the newly synthesized cytochrome c will be examined. (2) Studies on methylation-induced changes in cytochrome c conformation. Changes in tertiary structure by methyl and other chemical substituents will be monitored by pI value shift, optical rotatory dispersion and circular dichroism, thermal and acid-denaturation. (3) Comparative enzymological studies on methylated and unmethylated cytochrome c. This includes an assessment of the binding and catalytic activity towards cytochrome c reductase and oxidase, and cytochrome c peroxidase. (4) Identification, purification, and subsequent characterization of three enzymes implicated to exist from observations during the past grant period. (a) An enzyme which dealkylates methylated cytochrome c in vivo. (b) An enzyme which methylates the Res-86 lysine of cytochrome c. (c) Histone-specific protein methylase III from fungal or plant sources. (5) Identification of a mutant strain that is deficient in the cytochrome c methylation reaction in order to evaluate the reaction's physiological function in the organism, i.e., growth rate, the rate of respiration.
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