Identification of N-acetylmethionine as the product released during the NH2-terminal processing of a pseudo-class I actin.
Genes for the various isoactins define two classes of actin. Class I actin genes code for Met-Asp(Glu)-actin, and class II actin genes code for Met-X-Asp(Glu)-actin where X is usually cysteine. Amino termini of both are removed in an acetylation-dependent processing reaction yielding acetyl-Asp(Glu)-actin. Both classes are processed at approximately equal rate (t1/2 = 15 min) in vivo. In vitro, class II actins are 90% processed by endogenous enzymes after 60 min in a rabbit reticulocyte lysate system, whereas class I actins are only minimally processed during this period. Using site-directed mutagenesis of a human skeletal muscle isoactin coupled with in vitro transcription and translation methods, we have synthesized a pseudo-class I actin in which the penultimate cysteine has been changed to an aspartic acid, thus placing a class I amino terminus on an otherwise class II actin molecule. The pseudo-class I actin was less than 20% processed during the translation period as determined by peptide mapping. It was further processed by exogenous processing enzyme at a rate compatible with a class I actin. These results indicate that the major actin determinant controlling differential actin-processing rates is the amino-terminal residue being cleaved, not the remaining structure of the actin molecule. We have also demonstrated for the first time that N-acetylmethionine is the immediately released product from the amino terminus of a pseudo-class I actin during processing.
Sheff
D R
DR
Department of Biochemistry, University of Iowa College of Medicine, Iowa City 52242.
Rubenstein
P A
PA
D000107:Acetylation; D000199:Actins / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002845:Chromatography; D006801:Humans; D008715:Methionine / Q000031:analogs & derivatives* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism; D014176:Protein Biosynthesis; D051381:Rats; D012697:Serine Endopeptidases; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic; D014357:Trypsin
D000199:Actins; D010446:Peptide Fragments; C006379:N-acetylmethionine; D008715:Methionine; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin