Release of granule contents from sea urchin egg cortices. New assay procedures and inhibition by sulfhydryl-modifying reagents.
We have developed a rapid turbidimetric assay for the release of cortical granule contents from cortices prepared from eggs of the sea urchin, Strongylocentrotus purpuratus. The decrease in turbidity of cortex suspensions which occurs when the free calcium ion concentration is increased to 0.38-0.62 microM can be followed spectrophotometrically. Kinetic experiments demonstrate that this calcium-triggered turbidity change occurs rapidly and with no detectable lag period. Evidence indicating that the observed decrease in turbidity results from the release of cortical granule contents was obtained by correlating the free calcium ion concentration required to initiate the turbidity change with the free calcium ion concentration required in microscopic and enzymatic assays. All three assays exhibited similar calcium dependence. In the microscopic assay, morphological changes are used to assess the extent of cortical granule exocytosis. The enzymatic assay is based upon the latency of ovoperoxidase, a cortical granule enzyme. Ovoperoxidase catalyzed a 30-125-fold increase in the incorporation of [125I]iodine into trichloroacetic acid-precipitable cortex protein at and above threshold calcium ion concentrations. We have utilized the turbidimetric assay to screen several potential inhibitors of the cortical reaction. In confirmation of previous reports, we find that the phenothiazine drugs, chlorpromazine and trifluoperazine, are inhibitory. Sulfhydryl-modifying reagents, N-ethylmaleimide and sodium tetrathionate, are also inhibitory. Inhibition of cortical granule enzyme release by N-ethylmaleimide was confirmed with the ovoperoxidase latency assay.
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002746:Chlorpromazine / Q000494:pharmacology; D003594:Cytoplasmic Granules / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D005089:Exocytosis / Q000187:drug effects; D005260:Female; D007700:Kinetics; D009391:Nephelometry and Turbidimetry; D010063:Ovum / Q000187:drug effects / Q000378:metabolism*; D010544:Peroxidases / Q000378:metabolism; D011398:Promethazine / Q000494:pharmacology; D012617:Sea Urchins; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014268:Trifluoperazine / Q000494:pharmacology
D013439:Sulfhydryl Reagents; D014268:Trifluoperazine; D010544:Peroxidases; D011398:Promethazine; D002118:Calcium; D002746:Chlorpromazine