Abolition of ATPase activities of skeletal myosin subfragment 1 by a new selective proteolytic cleavage within the 50-kilodalton heavy chain segment.
We have isolated and chemically characterized several 5-thio-2-nitrobenzoate-subfragment 1 derivatives (TNB-S-1) generated by the reaction of 5,5'-dithiobis(2-nitrobenzoic acid) (DNTB, up to 10-fold molar excess) with native S-1, N-acetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine-S-1 (AEDANS-S-1), and N,N'-p-phenylenedimaleimide-S-1 (pPDM-S-1) at 4 degrees C, pH 8.0. The reaction of the reagent with AEDANS-S-1, which has a blocked -SH1 group, induced the formation of an intramolecular cystine disulfide between two vicinal -SH groups in S-1; in contrast, the treatment of pPDM-S-1 with DTNB resulted in the formation of TNB mixed disulfides only. The incorporation of the TNB groups (up to 3 mol/mol of S-1) into the native or premodified S-1 led to a local conformational change in the 50K heavy chain region that was fully reversed upon disulfide reduction. Exploiting this peculiarity of the DTNB-modified S-1's, we have realized a highly selective proteolysis of the S-1 heavy chain by thrombin and chymotrypsin, which do not act at all on the normal S-1. The 95K heavy chain was cut by thrombin into two fragments with apparent masses of 68K and 30K, whereas the "connector segments" and the light chains were unaffected. The two new fragments were issued from a primary peptide-bound cleavage between Lys-560 and Ser-561 within the amino acid sequence of the 50K region (M. Elzinga, personal communication).(ABSTRACT TRUNCATED AT 250 WORDS)
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors*; D000818:Animals; D002417:Cattle; D002918:Chymotrypsin / Q000378:metabolism; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D007700:Kinetics; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology*; D015879:Myosin Subfragments; D009218:Myosins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010446:Peptide Fragments / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011817:Rabbits; D013917:Thrombin / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D015879:Myosin Subfragments; D010446:Peptide Fragments; D004228:Dithionitrobenzoic Acid; D002918:Chymotrypsin; D014357:Trypsin; D013917:Thrombin; D000251:Adenosine Triphosphatases; D009218:Myosins