Isolation and localization of a spectrin-like protein from echinoderm sperm.
Thyone sperm undergo an explosive acrosome reaction resulting in the extension of a 90 microns long acrosomal process. In unreacted sperm, profilamentous actin is sequestered within the profilactin cup (Tilney: Journal of Cell Biology 69:73-89 1976), which consists of four major polypeptides: actin, profilin, and a 250/235 kDa equimolar doublet (TS 250/235). Dialysis of profilactin preparations into an actin assembly buffer resulted in the formation of acrosomal-like macromolecular aggregates containing actin, TS 250/235, and several other polypeptides as detected by SDS-PAGE. TS 250/235 was purified by subjecting extracts of pH solubilized profilactin cups to DEAE and phosphocellulose ion exchange chromatography. TS 250/235 demonstrated immunocrossreactivity with affinity purified polyclonal antibodies raised against S. purpuratus egg spectrin. As determined by biotinylated-calmodulin overlays, both subunits of TS 250/235 bound calmodulin in a Ca(++)-sensitive manner. Electron microscopy of low angle, rotary shadowed replicas of TS 250/235 revealed an elongate rod-shaped molecule with an average contour length of 203 nm. By indirect immunofluorescence, TS 250/235 was found to be uniformly distributed throughout the profilactin cup of the unreacted sperm. This distribution of TS 250/235 correlated with the location of monomeric actin as determined by localization studies utilizing fluorescent-DNase-1. Upon sperm activation, the cellular distribution of TS 250/235 dramatically changed and was observed both along the length and at the base of the extended acrosomal process.
D'Andrea
L
L
Department of Biological Sciences, Rutgers University, Newark, New Jersey 07102.
Fishkind
D J
DJ
Begg
D A
DA
Bonder
E M
EM
D000177:Acrosome; D000199:Actins / Q000737:chemistry; D000818:Animals; D001710:Biotin; D002147:Calmodulin; D003850:Deoxyribonuclease I; D004447:Echinodermata / Q000032:analysis* / Q000166:cytology; D019793:Fluorescein; D005452:Fluoresceins; D015151:Immunoblotting; D008297:Male; D008856:Microscopy, Fluorescence; D010590:Phalloidine; D051304:Profilins; D011506:Proteins / Q000737:chemistry; D012235:Rhodamines; D013049:Spectrin / Q000276:immunology / Q000302:isolation & purification*; D013094:Spermatozoa / Q000737:chemistry*
D000199:Actins; D002147:Calmodulin; D005452:Fluoresceins; D051304:Profilins; D011506:Proteins; D012235:Rhodamines; C024782:profilactin; D013049:Spectrin; D010590:Phalloidine; D001710:Biotin; D003850:Deoxyribonuclease I; D019793:Fluorescein