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.     

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.     

Induction of calcium-dependent, localized cortical granule breakdown in sea-urchin eggs by voltage pulsation

A technique that employs a high-voltage pulses to produce pores in cell membranes (Kinosita and Tsong (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 1923) has been used to investigate the role of Ca2+ in the early events of activation of sea-urchin eggs. Exposure of eggs to a voltage pulse of 1 kV/cm for 100 microseconds resulted in localized exocytosis of the contents of cortical granules and development of a partial fertilization envelope. This effect was triggered by entrance of Ca2+ through the voltage-induced pores. In a medium containing 100 microM Ca2+ and 45Ca2+ tracer, the voltage-treated eggs admitted 3.6 +/- 0.3 fmol Ca2+/egg within a few seconds. Untreated eggs took up only 1.0 +/- 0.2 fmol/egg after minutes of incubation. Furthermore, depletion of Ca2+ or the presence of EGTA in the external medium prevented elevation of the fertilization envelope by the voltage pulsation. Delay in Ca2+ addition after the voltage pulsation reduced the fraction of eggs that developed partial fertilization envelope. Loss of essential cytoplasmic components during the delay period is judged unlikely, since these eggs were viable, could form partial fertilization envelopes if re-pulsed in the presence of Ca2+, and could develop to normal blastula stage embryos upon fertilization with sperm. Thus, we interpret this effect as due to a resealing of pores; the half-life of pores being 20 s. The elevation of partial fertilization envelopes occurred only at the loci facing the anode, and multiple pulses with mixing resulted in the formation of multiple fertilization envelopes. These envelopes were stable for up to several hours; further propagation (wave spreading) was not observed. The above results indicate that a primary reaction in the sequence of steps in fertilization envelope formation involves Ca2+ to trigger cortical granule breakdown and formation of the fertilization envelope.     

Induction of calcium-dependent, localized cortical granule breakdown in sea-urchin eggs by voltage pulsation

A technique that employs a high-voltage pulses to produce pores in cell membranes (Kinosita and Tsong (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 1923) has been used to investigate the role of Ca²⁺ in the early events of activation of sea-urchin eggs. Exposure of eggs to a voltage pulse of 1 kV/cm for 100 μs resulted in localized exocytosis of the contents of cortical granules and development of a partial fertilization envelope. This effect was triggered by entrance of Ca²⁺ through the voltage-induced pores. In a medium containing 100 μM Ca²⁺ and ⁴⁵Ca²⁺ tracer, the voltage-treated eggs admitted 3.6±0.3 fmol Ca²⁺/egg within a few seconds. Untreated eggs took up only 1.0±0.2 fmol/egg after minutes of incubation. Furthermore, depletion of Ca²⁺ or the presence of EGTA in the external medium prevented elevation of the fertilization envelope by the voltage pulsation. Delay in Ca²⁺ addition after the voltage pulsation reduced the fraction of eggs that developed partial fertilization envelope. Loss of essential cytoplasmic components during the delay period is judged unlikely, since these eggs were viable, could form partial fertilization envelopes if re-pulsed in the presence of Ca²⁺, and could develop to normal blastula stage embryos upon fertilization with sperm. Thus, we interpret this effect as due to a resealing of pores; the half-life of pores being 20 s. The elevation of partial fertilization envelopes occured only at the loci facing the anode, and multiple pulses with mixing resulted in the formation of multiple fertilization envelopes. These envelopes were stable for up to several hours; further propagation (wave spreading) was not observed. The above results indicate that a primary reaction in the sequence of steps in fertilization envelope formation involves Ca²⁺ to trigger cortical granule breakdown and formation of the fertilization envelope.     

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.     

The periodic change in adenosine 3',5'-monophosphate concentration in sea-urchin eggs

The level of adenosine 3',5'-monophosphate (cyclic AMP) in the eggs of the sea urchin, Anthocidaris crassispina, was found to change periodically after fertilization. The minimum and maximum levels of cyclic AMP were 1.0 X 10(-7)M and 1.5 X 10(-6)M, respectively. The activity of adenylate cyclase in a 105 000 X g precipitate reached a plateau at 20 min after fertilization and stayed constant for at least 2 h. It was also found that 1.0 mM CaCl2 increased the activity of adenylate cyclase in the same precipitate from unfertilized eggs. In contrast, phosphodiesterase activity changed periodically and correlated with cyclic AMP levels in the eggs. Up to a concentration of 1.5 X 10(-6)M cyclic AMP, phosphodiesterase activity was low, but it became activated when the level of cyclic AMP rose beyond this level. These results indicate that the change in the intracellular level of cyclic AMP is regulated mainly by the change in phosphodiesterase activity.     

The relationship between hydrophobicity and interfacial tension of proteins

Charge-free hydrophobic gels of Hjerten et al. (Hjerten, S., Rosengren, J. and Pahlman, S. (1974) J. Chromatogr. 101, 281–288) were used for hydrophobic affinity chromatography. The effective hydrophobicity of proteins was expressed as their retention volumes from columns of butylepoxy- and hexylepoxy-Sepharose 4B. The effective hydrophobicity was also estimated by a partition method of Shanbhag and Axelsson ((1975) Eur. J. Biochem. 60, 17–22) from the partition coefficients of proteins between two phases, poly (ethylene glycol) and dextran. The former contained a hydrophobic ligand, palmitate.A close correlation was observed between the hydrophobicities determined by the two methods. However, no significant relationship was observed between these effective hydrophobicities and the average hydrophobicity of Bigelow ((1967) J. Theoret. Biol. 16, 187–211) that was calculated from the total amino acid composition of each protein.The interfacial tensions at the 0.2% protein/corn oil interface revealed negative correlations with the effective hydrophobicities determined by both methods indicating lower interfacial tensions with more hydrophobic proteins.     

The relationship between hydrophobicity and interfacial tension of proteins.

Charge-free hydrophobic gels of Hjerten et al. (Hjerten, S., Rosengren, J. and Pahlman, S. (1974) J. Chromatogr. 101, 281--288) were used for hydrophobic affinity chromatography. The effective hydrophobicity of proteins was expressed as their retention volumes from columns of butylepoxy- and hexylepoxy-Sepharose 4B. The effective hydrophobicity was also estimated by a partition method of Shanbhag and Axelsson ((1975) Eur. J. Biochem. 60, 17--22) from the partition coefficients of proteins between two phases, poly (ethylene glycol) and dextran. The former contained a hydrophobic ligand, palmitate. A close correlation was observed between the hydrophobicities determined by the two methods. However, no significant relationship was observed between these effective hydrophobicities and the average hydrophobicity of Bigelow ((1967) J. Theoret. Biol. 16, 187--211) that was calculated from the total amino acid composition of each protein. The interfacial tensions at the 0.2% protein/corn oil interface revealed negative correlations with the effective hydrophobicities determined by both methods indicating lower interfacial tensions with more hydrophobic proteins.