title: Effect of limited trypsin digestion on the biochemical kinetics of skeletal myosin subfragment-1.
abstract: We have investigated the effect of limited trypsin digestion of chymotryptic myosin Subfragment-1 (S-1) on its kinetic properties. We find that Vmax (i.e., the extrapolated maximal ATPase activity at infinite actin) remains approximately constant, independent of the period of digestion. We also find that the apparent actin activation constant, KATPase, and the apparent dissociation constant, Kbinding, are both significantly weakened by trypsin digestion of S-1, and that these kinetic parameters change in concert. In addition, we investigated the effect of limited trypsin digestion on the initial phosphate burst. We find that trypsin digestion has no effect on the rate of the tryptophan fluorescence enhancement that occurs after ATP binds to digested S-1, but that the magnitude of the fluorescence enhancement falls approximately 40% with digestion. Digested S-1 also showed anomalous behavior in that the fluorescence magnitude increased and the fluorescence rate dropped in the presence of actin. Trypsin digestion also decreased the magnitude of the chemically measured Pi burst approximately 35%, but this magnitude was essentially unaffected by actin. A possible explanation for this behavior is discussed.
authors.affiliation: Department of Medicine, State University of New York, Stony Brook 11790.
authors.forename: V A
authors.initials: VA
authors.lastname: Harwalkar
authors.forename: M P
authors.initials: MP
authors.lastname: White
authors.forename: D T
authors.initials: DT
authors.lastname: Annis
authors.forename: F
authors.initials: F
authors.lastname: Zervou
authors.forename: L A
authors.initials: LA
authors.lastname: Stein
mesh_terms: D000199:Actins / Q000378:metabolism; D002918:Chymotrypsin / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D015879:Myosin Subfragments / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification; D011485:Protein Binding; D014357:Trypsin / Q000378:metabolism / Q000494:pharmacology*
chemical_list: D000199:Actins; D015879:Myosin Subfragments; D010446:Peptide Fragments; D002918:Chymotrypsin; D014357:Trypsin