Analysis of the bound nucleotide in the acto-heavy meromyosin in vitro motility assay.
Muscle contraction and various forms of cell motility are driven by the interaction of actin and myosin with the simultaneous binding and hydrolysis of ATP. The process can be reconstituted in in vitro motility assays, where actin filaments slide over myosin in an ATP-dependent fashion. We have recently shown that in vitro actin motility persists at unexpectedly low, nanomolar free ATP concentrations if the actomyosin is pretreated with millimolar levels of the nucleotide (10). In these experiments, however, the amount of bound ATP--which could potentially support motility--was not exactly known. In the present work, the amount of nucleotide bound in the in vitro motility assay is directly measured by using radiolabeled ATP analogs in a novel capillary binding assay. The results indicate that although a low quantity of nucleotide remains bound, it is stable and does not seem to be available to support motility.
Kellermayer
M S
MS
Central Laboratory, University Medical School of Pécs, Hungary.
D000199:Actins / Q000737:chemistry / Q000302:isolation & purification / Q000502:physiology; D000255:Adenosine Triphosphate / Q000032:analysis* / Q000378:metabolism; D000818:Animals; D002465:Cell Movement; D009119:Muscle Contraction; D018482:Muscle, Skeletal / Q000502:physiology; D015879:Myosin Subfragments / Q000737:chemistry* / Q000302:isolation & purification / Q000502:physiology; D011817:Rabbits
D000199:Actins; D015879:Myosin Subfragments; D000255:Adenosine Triphosphate