The ribonuclease activity of the cytotoxin alpha-sarcin. The characteristics of the enzymatic activity of alpha-sarcin with ribosomes and ribonucleic acids as substrates.
The ribonuclease activity of the cytotoxic protein alpha-sarcin has been characterized. When rat liver ribosomes or 60 S ribosomal subunits were the substrates, alpha-sarcin cleaved a single oligonucleotide of about 488 residues, the alpha-fragment, from the 3' end of 28 S rRNA. In contrast, 40 S ribosomal subunits were not affected by alpha-sarcin. The alpha-fragment was cleaved from 28 S rRNA in 80 S ribosomes when the concentration of alpha-sarcin was 3 x 10(-8) M and the toxin retained its specificity even when the concentration was 3 x 10(-5) M. The turnover number (kcat) for the reaction of alpha-sarcin with ribosomes was 55 min-1, establishing that the toxin acts catalytically. When total rRNA or 28 S rRNA was the substrate, alpha-sarcin caused extensive progressive digestion of the nucleic acids; however, no formation of the alpha-fragment occurred. The extent of the digestion of 28 S rRNA was related to the concentration of alpha-sarcin, but the amount of the toxin required was somewhat greater than that needed with ribosomes. Digestion of homopolynucleotides with alpha-sarcin indicated that the protein is specific for purines. When [32P]5 S rRNA was the substrate, alpha-sarcin cleaved on the 3' side of purines in both single- and double-stranded regions of the molecule. The results suggest that the unusual specificity of alpha-sarcin, in that it cleaves only one of more than 7000 phosphodiester bonds in the ribosome, is a property both of the cytotoxin and of the ribosome.
D000818:Animals; D004722:Endoribonucleases; D005656:Fungal Proteins / Q000378:metabolism*; D006868:Hydrolysis; D012313:RNA / Q000378:metabolism*; D012335:RNA, Ribosomal / Q000378:metabolism; D051381:Rats; D012260:Ribonucleases / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism
D005656:Fungal Proteins; D012335:RNA, Ribosomal; C014801:alpha-sarcin; D012313:RNA; D004722:Endoribonucleases; D012260:Ribonucleases