Proteolytic fragmentation of Dictyostelium myosin and localization of the in vivo heavy chain phosphorylation site.
Dictyostelium myosin was associated into dimers and small oligomers at very low ionic strength, filamentous at intermediate ionic strength, and monomeric in solution conditions of high ionic strength. These different associations were probed by fragmenting myosin with chymotrypsin, trypsin, or V-8 protease. All three proteases digested monomeric myosin giving rise to multiple fragments with a wide range of molecular weights. Filamentous myosin was not digested by the V-8 protease, was preferentially cleaved at a single site in the middle of the heavy chain by chymotrypsin, and was cleaved at several sites by trypsin. If the reaction was carried out in very low ionic strength, however, two of these proteases generated stable fragments of high molecular weight. Electron microscopic analysis of these stable fragments showed that tails were shorter than in intact myosin, indicating that the cleavage sites were in the rod portion of the molecule. Under the same conditions of enzymatic digestion, myosin that had been radio labeled in vivo with 32P was analyzed by SDS-PAGE and autoradiography. By comparing the state of phosphorylation and the size of the stable fragments, it was determined that the heavy chain phosphorylation site was located between 55 and 70 kD from the tip of the myosin tail, near a region where the tail displayed sharp bends.
Kuczmarski
E R
ER
Department of Cell Biology and Anatomy, Northwestern University Medical School, Chicago, Illinois 60611.
Routsolias
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L
Parysek
L M
LM
D000818:Animals; D002918:Chymotrypsin / Q000494:pharmacology; D004023:Dictyostelium / Q000032:analysis*; D008854:Microscopy, Electron / Q000379:methods; D009218:Myosins / Q000032:analysis* / Q000378:metabolism*; D010766:Phosphorylation; D012697:Serine Endopeptidases / Q000494:pharmacology; D014357:Trypsin / Q000494:pharmacology
D012697:Serine Endopeptidases; D002918:Chymotrypsin; C028344:glutamyl endopeptidase; D014357:Trypsin; D009218:Myosins