Cytochrome c-specific protein-lysine methyltransferase from Neurospora crassa. Purification, characterization, and substrate requirements.

Identification ofNG-Methylarginine Residues in Human Heterogeneous RNP Protein A1: Phe/Gly-Gly-Gly-Arg-Gly-Gly-Gly/Phe Is a Preferred Recognition Motif

“…The methylated A1 thus obtained contained 1.45 mol of [methyl-3 H] groups per mol of protein and gave a single band of 34 kDa on SDS-PAGE [see Figure 8, lane 1 of Rajpurohit et al (1994)]. This level of methylation is significantly above that observed for other proteins that have been methylated in Vitro previously [i.e., the maximal level of in Vitro methylation of lysine 72 in cytochrome c was only 0.2% (Durban et al, 1978)].…”

Cytochrome c-specific methylase from wheat germ

“…Enzymatic Assay. The routine assay of the enzymatic methylation of horse heart cytochrome c was carried out by the procedure previously described (Durban et al, 1978) in a final volume of 0.25 mL. The final assay mixture contained 0.1 M glycine-NaOH (pH 9.0), 5.7 mM mercaptoethanol, was performed as described by Paik & Kim (1967).…”

“…Apocytochrome c was prepared similar to the method described by Fisher et al (1973). The residue 1-65 CNBr peptide was prepared from apocytochrome c by the previously described procedure (Durban et al, 1978).…”

“…Much work has been done in seeking to understand the nature and function of cytochrome c methylation, particularly in fungal organisms. The methylases that catalyze the reaction were purified from two fungal sources, Neurospora crassa and baker's yeast, and were found to be highly specific in regard to their exclusive recognition of cytochrome c as a substrate (Durban et al, 1978;DiMaria et al, 1979). In addition, they exhibit a further specificity in that they recognize only the residue 72 lysine as a site of methylation when horse heart cytochrome c is used as a substrate (DiMaria et al, 1979). This observation correlates well with the in vivo occurrence of trimethyllysine at exclusively the residue 72 in the cytochromes c of N. crassa and baker's yeast (DeLange et al, 1969(DeLange et al, , 1970.…”

Protein methylation in mitochondria

“…In yeast, cytochrome c (Cyc1p) is trimethylated at Lys-78 (precursor numbering), whereas the corresponding residue in mammals, referred to as Lys-72 (mature numbering), is unmethylated ( 98 ). The responsible enzymatic activity, cytochrome c-KMT, was partially purified from various sources and shown to mediate methylation of cytochrome c of fungal and mammalian origin ( 99 , 100 ), but it took nearly two decades until the corresponding gene, CTM1 , was identified in yeast ( 101 , 102 ). It was demonstrated that cytochrome c from the ΔCtm1 strain was completely unmethylated, and recombinant Ctm1p was shown to specifically target unmethylated cytochrome c (from mammals or ΔCtm1 yeast) in vitro ( 102 ).…”