Partial activation of sea-urchin eggs by bonellin

We here describe further studies on the action of bonellin on sea-urchin eggs. Bonellin brings about Some of the changes that are known to occur in the egg upon fertilization. In particular, it appears to cause the increased rate of incorporation of amino acids into proteins, the increase of the voltage noise, and the exocytosis of some of the cortical granules. A comparison with the effect of ammonia is discussed.     

Purification and characterization of arginine kinase from sea-urchin eggs

In most invertebrates, creatine kinase is replaced by arginine kinase, which catalyzes reversibly the transfer of a phosphate group between adenosine triphosphate and arginine. In sea-urchin larvae, arginine kinase only is expressed whereas in adult sea-urchins both arginine kinase and creatine kinase can be found in the same tissue. In order to study their developmental regulation and properties, we have purified arginine kinase to homogeneity from the eggs of the sea-urchin Paracentrotus lividus. The purification involves ethanol and ammonium sulfate precipitations, followed by an anion-exchange chromatography, an affinity chromatography and a gel filtration. A 500-fold increase in specific activity leads to a specific activity of 360 IU/mg protein at 25 degrees C. Arginine kinase (pI = 5.7) is rapidly and irreversibly inactivated at 45 degrees C. Amino acid composition and Km values (2.08 mM for phospho-L-arginine and 1.25 mM for ADP) are also given. Determination of molecular mass by gel filtration and separation by SDS/polyacrylamide gel electrophoresis indicate that the enzyme is an 81-kDa dimer of two subunits of 42 kDa.     

Unusual enthalpy changes which accompany the titration of dimyristoylphosphatidylcholine vesicles with Triton X-100

The enthalpy changes which accompany the titration of 0.1% and 0.25% small unilamellar and multiameller vesicle samples of dimyristoylphosphatidylcholine with 2% Triton X-100 in 0.067 M phosphate buffer (pH 7.4) containing 0.15 M NaCl have been determined by titration calorimetry at 21 degrees C and 28 degrees C, the enthalpy change for both type of vesicles was zero within the limits of experimental error. At 21 degrees C, the multilamellar vesicle samples exhibited an enthalpy change of 1.35 +/- 0.48 and 2.47 +/- 0.98 kcal/mol dimyristoylphosphatidylcholine which was complete at a molar ratio of dimyristoylphosphatidylcholine to Triton of 3.21 +/- 0.84 and 5.77 +/- 1.05 for 0.1% and 0.25% dimyristoylphosphatidylcholine solutions, respectively. An exothermic transition of -2.39 +/- 0.30 and -2.05 +/- 0.69 kcal/mol phospholipid followed by an endothermic transition of 1.37 +/- 0.12 and 1.94 +/- 0.20 kcal/mol dimyristoylphosphatidylcholine was observed at 21 degrees C for 0.1% and 0.25% small unilamellar vesicle samples, respectively. In addition the nearly athermal association of the small unilemellar vesicle samples at 21 degrees C was observed, which may be an appropriate model for biological membrane fusion.     

Histochemical studies of jelly coat of sea-urchin eggs during oogenesis

Summary Jelly coat of sea-urchin eggs consists of polysaccharides and glycoproteins. Some properties of jelly coat have already been investigated, but not histochemically. The oogenesis in Paracentrotus lividus was studied histologically and the oocytes were classified into six different stages. The extracellular jelly appeared first around the growing oocytes II which remained attached to the germinal epithelium. The jelly became thicker when the oocyte approached maturation. Histochemical analysis revealed that the jelly consists of mucopolysaccharide-protein-complexes. The polysaccharide component is composed of both neutral and acid mucopolysaccharides. The former are amylase-resistant. The acid mucopolysaccharides contain both carboxyl and sulfate groups, which are in close proximity to vicinal hydroxyl groups. Sulfated mucopolysaccharide is hyaluronidase-resistant. Sialic acid could not be clearly demonstrated, because it seems to be resistant to neuraminidase. Pepsin digestion indicated the masking of acidic groups by proteins which compete with basic dyes (Alcian blue, Azure A, coriphosphine etc.). Proteolytic digestion enhanced dye-binding ability of jelly, but removed also some of the periodate-reactive mucosubstances. Also a protein component could be demonstrated histochemically. No histochemical difference between jelly coat of oocytes and that of eggs has been found. The possible molecular structure of jelly coat is discussed.     

Slow calcium waves accompany cytokinesis in medaka fish eggs

Animal cells are cleaved by the formation and contraction of an extremely thin actomyosin band. In most cases this contractile band seems to form synchronously around the whole equator of the cleaving cell; however in giant cells it first forms near the mitotic apparatus and then slowly grows outwards over the cell. We studied the relationship of calcium to such contractile band growth using aequorin injected medaka fish eggs: we see two successive waves of faint luminescence moving along each of the first three cleavage furrows at approximately 0.5 micron/s. The first, narrower waves accompany furrow extension, while the second, broader ones, accompany the subsequent apposition or slow zipping together of the separating cells. If the first waves travel within the assembling contractile band, they would indicate local increases of free calcium to concentrations of about five to eight micromolar. This is the first report to visualize high free calcium within cleavage furrows. Moreover, this is also the first report to visualize slow (0.3-1.0 micron/s) as opposed to fast (10-100 microns/s) calcium waves. We suggest that these first waves are needed for furrow growth; that in part they further furrow growth by speeding actomyosin filament shortening, while such shortening in turn acts to mechanically release calcium and thus propagates these waves as well as furrow growth. We also suggest that the second waves act to induce the exocytosis which provides new furrow membrane.     

On the water-soluble protein of sea urchin eggs and its changes during the early development

Summary In the egg of sea urchin,Hemicentrotus pulcherrimus, changes in the amount of water-soluble protein were observed chromatographically. There were three kinds of group (A, B, and C) in the protein pattern by thin-layer chromatography with Sephadex gel (G-200) and each group contained several components. The molecular weight was 30,000–90,000 in group A, 90,000–480,000 in group B, and more than 480,000 in group C, respectively. The area which belonged to group C reduced remarkably upon fertilization and the opposite aspect was observed in groups A and B. This suggests the possibility of the attack of proteolytic enzyme which induces subsequently an increase of small molecular substances. In later stages, the area of group C might recovered neary to that of the unfertilized egg at a sacrifice of group A. The rearrangement of proteins possibly may take place after the fertilization.The relationship between the water-soluble protein and fertilization phenomena is discussed with the presence of the experimental results.     

Respiratory behaviour of sea-urchin spermatozoa. II. Sperm-activating substance obtained from jelly coat of sea-urchin eggs.

A sperm-activating substance (SAS) was obtained from the jelly coat of sea-urchin ova and its chemical properties were investigated in three sea-urchin species. The SAS was partially purified from the jelly coat of Pseudocentrotus eggs through several steps of purification by procedures consisting of charcoal adsorption, ion-exchange chromatography on DEAE-Sephadex A-25 column, and gel-filtration on Sephadex G-15 columns. The partially purified SAS was found to contain a ninhydrin-positive material and is inactivated by pronase digestion. The molecular weight of SAS was estimated as about 630 by gel-filtration through Sephadex G-25 and the isoelectric-point of SAS is located at about pH 5.3 by isoelectrofocusing method. The SAS is non-volatile, alcohol-soluble, and labile in a diluted alkaline or acid solution. The origin of SAS is discussed.