Abstract
Hydrolysis of highly enriched [gamma-18O]ATP in unlabeled water by acto-heavy meromyosin at low actin concentration was found to be heterogeneous in that significant amounts of both the species containing 0 or 3 18O-labeled oxygens/phosphate were formed. Detailed quantitative comparison with theoretical distributions over a wide range of actin concentrations, however, indicated that the pathway which catalyzed ATP hydrolysis with a low extent of exchange only made a significant contribution at a low actin concentration and did not represent a major fraction of the total hydrolysis seen at higher actin concentrations. This low exchange component was also detected in the dependence on actin of the steady state ATPase. At low actin the steady state ATPase rate increased more rapidly as a function of actin concentration than predicted by the Km and Vmax for actin activation observed at moderate to high actin levels. This extra ATP hydrolysis at low actin correlates with that predicted for the low exchange pathway both with respect to the fraction of the ATP hydrolyzed and to its dependence on the actin concentration.
Highlights
Hydrolysis of highly enriched [y-'"OIATP in unlabeled water by acto-heavy meromyosin at low actin concentration was found to be heterogeneous in that significant amounts of both the species containing 0 or 3 "0-labeled oxygens/phosphate were formedD. etailed quantitative comparison with theoretical distributions over a wide range of actin concentrations,indicated that the pathway which catalyzed ATP hydrolysis with a low extent of exchange only made a significant contribution at a low actin concentration anddid not represent amajor fraction of the total hydrolysis seen athigher actin concentrations
At moderate actin concentrations the predominant flux of ATP hydrolysis occurs via steps 4, 8, 2, 9, and 6 sequentially as fist demonstrated by Lymn and Taylor [5]
Thedistribution shifted from mainlyP'"O0 at low actin tomainly P1'03at high actin in a smooth and continuous manner with hydrolysis at intermediate actin concentrations resulting in Pi which contained substantial amountosf P"O1 and P"02 in quantitative agreement with the theoretical treatmentfor a homogeneous model
Summary
" Purified by ion exchange chromatol gra.Dhl v onDEAE-cellulose in 50 mM imidazole-HCI, pH7.0, with a 0-0.25 M NaCl gradient (400 X 400 ml) on a column (2.5 X 45 &). Analysis of the exchange during hydrolysiswith the above is largely due to the extremseensitivity of the theoret- crude HMM before ultracentrifugation showed little increase ical distribution in this region on the exact valuoef the P"O1/ in thelow exchange component as shown in Tabl1e11.Further. Comparison with the0.65 PCtheoretical distribu- purification of the HMMby ion exchangechromatography on tion indicates that the discrepancy maybe viewed as a slight DEAE-cellulose or gel filtration on SepharoseCL-2B resulted over production of P'*Oo rather than P"03 and this point is in the virtually completeloss of unfragmented myosin heavy developed further in the "Discussion.") Theseresultsare chain, but did not decrease the contribution of the low exconsistent with a model in which the absolute rate of ATP change component asalso seen in Table 111.If anything, the hydrolysis in the low exchange pathway increases rapidly at contribution increased slightly. The absolute rate of ATP hydrolysis by the highexchange The results strengthen theconclusion that the low exchange component continues to increase and dominaattehsigh actin. component is producedby a species of HMM
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