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Evidence for functional differences between two flagellar dynein ATPases (1986)

Penningroth, Stephen M. ; Peterson, Dolores D.

New York, NY : Wiley-Blackwell

Cell Motility and the Cytoskeleton 6 (1986), S. 586-594

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ISSN: 0886-1544

Keywords: sea urchin sperm ; motilily ; two dynein ATPases ; force generation ; power output ; Life and Medical Sciences ; Cell & Developmental Biology

Source: Wiley InterScience Backfile Collection 1832-2000

Topics: Biology , Medicine

Notes: Energy coupling in flagellar motility was investigated using demembranated, reactivated sea urchin spermatozoa (Arbacia punctulata). The ATP-dependence of ATPase activity was investigated for ATP concentrations ranging from 4 μM to 600 μM ATP. Using Eadie-Scatchard plot analysis, we identified two axonemal dynein ATPase activities. Their apparent Michaelis constants were calculated to be equal to 4 μM and 161 μM ATP, and they were referred to, respectively, as the high-affinity dynein ATPase (HADA) and the low-affinity dynein ATPase (LADA). Investigation of movement-coupled ATPase activity (difference between the ATPase activities of reactivated and broken, immotile spermatozoa) indicated that HADA and LADA were both 65% movement-coupled. The apparent Michaelis constants of movement-coupled HADA and LADA, 12 μM and 271 μM ATP, respectively, were two- to four-fold greater than the apparent Michaelis constants of movement-uncoupled HADA and LADA. The apparent Michaelis constants for force generation and beat frequency of reactivated spermatozoa were determined to be 24 μM and 290 μM ATP, respectively. These results raise the possibility that flagellar force generation is controlled primarily by movement-coupled HADA, and that flagellar beat frequency is controlled primarily by movement-coupled LADA. Thus, mechanochemical activity in flagellar motility may be divided between two enzymatically and functionally distinct classes of flagellar dyneins.

Additional Material: 4 Ill.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1002/cm.970060607

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An EHNA-senstive ATPase in unfertilized sea urchin eggs (1985)

Penningroth, Stephen M. ; Rose, Patricia ; Cheung, Anne ; [et al.]

New York, NY : Wiley-Blackwell

Cell Motility and the Cytoskeleton 5 (1985), S. 61-75

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ISSN: 0886-1544

Keywords: dynein ; erythro-9-[3-2-(hydroxynonyl)]adenine (EHNA) ; ATPase ; inhibition ; axoneme ; cytoplasm ; Life and Medical Sciences ; Cell & Developmental Biology

Source: Wiley InterScience Backfile Collection 1832-2000

Topics: Biology , Medicine

Notes: 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).

Additional Material: 6 Ill.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1002/cm.970050106

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