We have purified SP-22, a substrate protein for mitochondrial ATP-dependent protease in bovine adrenal cortex. Native SP-22 showed an M(r) of 350,000 +/- 20,000, and was composed of more than 10 molecules of an M(r) 21,600 subunit. Subcellular and submitochondrial fractionation of adrenocortical tissues revealed that SP-22 was localized in the mitochondrial matrix, suggesting that SP-22 is a natural substrate for ATP-dependent protease, a matrix enzyme. The concentration of SP-22 in adrenocortical mitochondrial fractions was 16 +/- 3 micrograms/mg proteins (mean +/- SD, n = 6) as determined by radioimmunoassay using specific anti-SP-22 antibody. Adrenal cortex showed the highest concentration among the 15 bovine tissues tested, followed by liver, renal cortex, adrenal medulla, heart, and renal medulla. We determined the amino acid sequence of SP-22, which is composed of 195 amino acids. Amino acid 47 was not identified by the sequencer. FAB-mass spectrometry of AA47-AA55 fragment revealed that AA47 was cysteine-sulfinic acid (Cys-SO2H). By a homology search in the NBRF-PIR data base, SP-22 was found to be 91% homologous to murine erythroleukemia cell MER-5 protein, which may have an important role in the induction of differentiation. SP-22 was also homologous to the C22 component of alkyl hydroperoxide reductase in Salmonella typhimurium, thiol-specific antioxidant in Saccharomyces cerevisiae, and some other proteins. Since a segment around AA47 was highly conserved, this residue may be important for the biochemical functions of SP-22.

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Calyculin A, a protein phosphatase inhibitor, induced cleavage-like morphological change in unfertilized sea urchin eggs. A contractile ring-like apparatus containing both filamentous actin and myosin was formed in the cleavage furrow. Wheat germ agglutinin receptors were also found in the same region. The eggs did not develop further after constriction of the ring. No aster-like microtubular structure was found in the calyculin A-treated eggs. The cleavage was not inhibited by the antimicrotubule drug griseofulvin. Calyculin A also increased histone Hi kinase activity and induced chromosome condensation. These changes also occurred in the presence of emetine (an inhibitor of protein synthesis) and aphidicolin (an inhibitor of DNA synthesis). It is suggested that calyculin A induced these changes in the sea urchin eggs by inhibiting the activity of protein phosphatase 1.The motive force of cleavage in animal cells is generated by the interaction between actin filaments and myosin in the contractile ring (CR) or the contractile arc (1, 2). It has been proposed (3) that induction of the cleavage furrow is mediated by an as-yet-uncharacterized substance that is transported from the mitotic apparatus to the cortical layer via astral microtubules at a specific stage ofcell division. The CR forms rapidly but reduces its volume during contraction and disappears soon after cytokinesis (4). The mechanisms that induce this sequence of events and the nature of the signal that stimulates the furrow formation have not yet been elucidated. One interesting approach to this problem is to identify substances that induce cleavage or a specific stage in the cleavage process.Calyculin A (CL-A), a tumor-promoting substance isolated from the marine sponge Discodermia calyx (5), is a specific inhibitor of protein phosphatase 1 (PP1) (IC50 = 0.5-1 nM) and protein phosphatase 2A (PP2A) (IC50 = 2 nM) in rabbit skeletal muscle (6). It has been shown to induce oocyte maturation in starfish (7) and stimulate contraction of smooth muscle (6), effects also induced by the phosphatase inhibitor okadaic acid (OA). We report here that CL-A induces a CR-like apparatus in unfertilized sea urchin eggs. The simultaneous condensation of chromosomes was also observed. MATERIALS AND METHODSMaterials. The species of sea urchins used in this study were Anthocidaris crassispina, Clypeaster japonicus, Diadema savignyi, Diadema setosum, Echinometra mathaei, Echinostrephus Wheat germ agglutinin (WGA) receptors were visualized by staining with fluorescein-conjugated WGA (1 ,ug/ml; Vector Laboratories). To visualize DNA, the eggs were stained with 4',6-diamidino-2-phenylindole (0.25 ,Ag/ml). The chemicals were dissolved in buffer A. Isolation of egg cortices on protamine-coated glass surface was carried out as described (9). The sample was examined with a Nikon Optiphot microscope equipped with ordinary epifluorescence optics or confocal optics (MRC-00; Bio-Rad). Triton X-100 for 10 min on ice. The, egg suspension was passed through a nylon mesh (50 u...

Flux of K+ and changes in intracellular Ca2+ in the sperm of salmonid fishes were measured with spectrophotometry, ion electrode, microscopic fluorometry, and radioisotope accumulation. Release of K+ occurred at the initiation of sperm motility which is induced by decrease in external K+ and the K+ efflux and sperm motility were inhibited by K+ channel blockers. Intracellular Ca2+ increased within a short period in K(+)-free condition, and the accumulation of 45Ca in sperm cells was higher in motile sperm than that in immotile sperm. The efflux of K+ and the increase in intracellular Ca2+ were suppressed when external K+ concentration increased, i.e., sperm remained immotile. These results suggest that efflux of K+ through K+ channel and subsequent increase in intracellular Ca2+ are prerequisite for the initiation of sperm motility.

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