A marine natural product inhibitor of kinesin motors

Science

Volumn 280, Issue 5361, 1998, Pages 292-295

  Sakowicz, Roman ^a   Berdelis, Michael S ^a   Ray, Krishanu ^a   Blackburn, Christine L ^b   Hopmann, Cordula ^b   Faulkner, D John ^b   Goldstein, Lawrence S B ^a  

^a UNIVERSITY OF CALIFORNIA   (United States)

^b UNIVERSITY OF CALIFORNIA   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

ADENOSINE TRIPHOSPHATASE; ADOCIASULFATE 2; ENZYME INHIBITOR; KINESIN; NATURAL PRODUCT; UNCLASSIFIED DRUG;

ARTICLE; BINDING KINETICS; CELL DIVISION; CHEMICAL STRUCTURE; DROSOPHILA MELANOGASTER; EMBRYO DEVELOPMENT; ENZYME INHIBITION; MARINE ENVIRONMENT; MICHAELIS CONSTANT; MICROTUBULE; NONHUMAN; PRIORITY JOURNAL; SPONGE (PORIFERA);

ADENOSINE DIPHOSPHATE; ADENOSINE TRIPHOSPHATASES; ADENOSINE TRIPHOSPHATE; ANIMALS; BINDING SITES; CELL DIVISION; DROSOPHILA; ENZYME INHIBITORS; HELA CELLS; HUMANS; KINESIN; KINETICS; MICROTUBULES; MITOSIS; PORIFERA; SULFURIC ACID ESTERS;

ADOCIA; DROSOPHILA MELANOGASTER; HALICLONA; MELANOGASTER; PORIFERA;

EID: 0032502817     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.280.5361.292     Document Type: Article

References (25)

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23. 32P]ATP at room temperature for 15 min and stored on ice. One-microliter aliquots were diluted into 100 μl of "chase mix" containing pyruvate kinase (0.5 mg/ml), 2 mM phosphoenolpyruvate, and AS-2. At different times, 5-μl aliquots of the chase mix were quenched in 100 μl of 1 M Hcl, 1 mM ATP, and 1 mM ADP. The amount of ADP accessible to pyruvate kinase and converted to ATP was determined by thin-layer chromatography on PEI-cellulose and phospholmager (Molecular Dynamics) quantitation.
24. Embryos were collected every 20 min and dechorionated. They were desiccated for 7 min and pressure-injected with either the AS-2 solution in injection buffer [5 mM KCl and 100 mM sodium phosphate (pH 7.5)] or with buffer alone as a control. Batches of 20 embryos were injected, and at least three batches were injected for each concentration of AS-2 and the control. After injection, the embryos were developed for 20 to 30 min at room temperature inside a moist chamber and were fixed, devitelinized, immunostained for tubulin (Fig. 3, C, D, and F), and counterstained with 4′,6′-diamidino-2-phenylindole (DAPI) (0.01 mg/ml) (Fig. 3, A through D and F). Images were recorded on a Bio-Rad MRC-1024 confocal laser scanning microscope using LaserSharp software.
25. We thank the Republic of Palau and the State of Koror for marine research permits, M. K. Harper for identification of the sponge, E. Komives and B. Yang for advice on enzyme kinetics, S. Farlow for kinesin constructs, and K. Wood for Xenopus CENP-E constructs. This work was supported by the Cancer Research Fund of the Damon Runyon-Walter Winchell Foundation Fellowship (R.S.), by NIH (L.S.B.G., C.L.B., and D.J.F.), and by the California Sea Grant College Program (C.L.B., C.H., and D.J.F.). L.S.B.G. is an investigator at the Howard Hughes Medical Institute.