Life and Medical Sciences
Cell & Developmental Biology
Wiley InterScience Backfile Collection 1832-2000
In current purification strategies, affinity for microtubules or calmodulin is used to identify and purify cytoplasmic dynein-like ATPase from cell-free extracts of unfertilized sea urchin eggs. However, affinity purification procedures, though they define dynein-like ATPase activity, have not yet proven to be quantitative. An alternative purification strategy capable of producing a high yield of enzyme would require a specific assay in order to monitor cytoplasmic dynein purity at each step.In this study, we make a detailed comparison of the effects of EHNA on 22 different ATP-metabolizing enzyme activities, including 13 Mg++-ATPases. We isolate cytoplasmic dynein-like ATPase activity from three species of sea urchin eggs and sperm and show by means of dose-response curves that their sensitivities to inhibition by EHNA are very similar to one another. We demonstrate further that the EHNA dose-response characteristics of fourteen other ATP-metabolizing enzyme activities, including seven nondynein Mg++-ATPases, differ quantitatively from those of dynein-like ATPases.In studies of three other agents (vanadate, Ca++/calmodulin, and Triton X-100), we find that dynein-like ATPases vary by two orders of magnitude in their sensitivities to inhibition by vanadate, and little or no stimulation by either Ca++/calmodulin or Triton X-100 is seen. Our results suggest that inhibition by EHNA is a universal and specific property of dynein-like ATPases, which ultimately should prove useful in the quantitative purification and characterization of cytoplasmic dynein-like ATPase (s).
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