A novel actin filament-capping protein from sea urchin eggs: a 20,000-molecular-weight protein-actin complex

A novel protein factor which reduced the low-shear viscosity of rabbit skeletal muscle actin was purified from a 0.6 M KCl-extract of an insoluble fraction of sea urchin eggs by ammonium sulfate fractionation, gel filtration column chromatography, DNase I column chromatography, and hydroxylapatite column chromatography. This protein factor was shown to be a one-to-one complex of a 20,000-molecular-weight protein and egg actin. This protein complex accelerated the initial rate of actin polymerization, but reduced the steady-state viscosity of F-actin. It inhibited at substoichiometric amounts the elongation of actin filaments on sonicated F-actin fragments and depolymerization of F-actin induced by dilution. In addition, it increased the critical concentration of actin for polymerization. All these effects of this protein complex on actin could be explained by the "capping the barbed end" of the actin filament by the complex. The 20,000-molecular-weight protein which was separated from actin also possessed the barbed end-capping activities, but differed from the complex in that it did not accelerate the polymerization of actin.     

Identification of a calsequestrin-like protein from sea urchin eggs

Following studies on calcium transport by isolated smooth endoplasmic reticulum from unfertilized sea urchin eggs (Oberdorf, J. A., Head, J. F., and Kaminer, B. (1986) J. Cell Biol. 102, 2205-2210) we have purified and partially characterized a calsequestrin-like protein from this organelle isolated from eggs from Strongylocentrotus droebachiensis and Arbacia punctulata. Muscle calsequestrin from sarcoplasmic reticulum is well characterized as a calcium storage protein. The egg protein resembles calsequestrin in its behavior in purification steps, electrophoretic mobility, blue staining with Stains-all on polyacrylamide gels, and its calcium binding and amino acid composition. Purification was attained with DEAE-cellulose and hydroxyapatite chromatography. The egg protein Mr of 58,000 in the Laemmli gel system is reduced to 54,000 under Weber-Osborn (neutral) conditions, thus showing a pH dependence in its mobility, although less than occurs with muscle calsequestrins. 25% of its amino acids are acidic and 10% basic. It binds 309 nmol of Ca2+/mg of protein, within the range reported for cardiac calsequestrin. Antigenically, the sea urchin egg protein is related to cardiac calsequestrin capable of binding anti-cardiac calsequestrin antibody.     

A novel ceramide trihexoside from the eggs of the sea urchin, Hemicentrotus pulcherrimus.

Glucosylceramide (Glc beta 1-1Cer) and a novel ceramide trihexoside (Gal beta 1-6Gal beta 1-6Glc beta 1-1Cer) were purified from the eggs of the sea urchin, Hemicentrotus pulcherrimus. Their chemical structures were determined by gas-liquid chromatography, methylation analysis, chromic acid oxidation, enzymatic hydrolysis, enzyme-linked immunosorbent assay, fast atom bombardment mass spectrometry, and proton nuclear magnetic resonance spectroscopy. The ceramide trihexoside has a novel carbohydrate structure, and its core structure, Gal beta 1-6Glc, is also novel. The ceramide moieties of these glycolipids are almost identical. Two fatty acids, 22:1 and 22h:1, constitute more than 80% of the total acids. Long-chain bases are all phytosphingosine, approximately 90% of which is n-t18:0. The finding of melibiosylceramide (Gal alpha 1-6Glc beta 1-1Cer) from the eggs of another sea urchin species [Kubo, H. et al. (1988) J. Biochem. 104, 755-760] and the present finding of the novel ceramide trihexoside suggest that there are a variety of unique sugar structures in sea urchin glycosphingolipids.     

Protein kinase C from sea urchin eggs

1. Protein kinase C is considered to be ubiquitous in tissues and organs; however, its isolation and characterization have been principally with adult mammalian tissues. 2. There is increasing evidence for the importance of this enzyme during early development. 3. In this study, protein kinase C has been identified and partially characterized in cytosolic fraction from sea urchin eggs. 4. The enzyme was resolved from other protein kinase activities by ion exchange chromatography. 5. Phosphatidylserine and Ca2+ were required for protein kinase C to be active. 6. Diacylglycerol and phorbol ester enhanced the activation of the enzyme.     

Chloride permeability of sea urchin eggs.

We have examined the content and permeability of chloride in sea urchin eggs. After fertilization there is a large increase in the permeability to chloride. We discuss the mechanism underlying this permeability change and the generalized increase in ion permeability observed after fertilization.     

An 100-kDa Ca2+-sensitive actin-fragmenting protein from unfertilized sea urchin egg

An actin-modulating protein was purified from unfertilized eggs of sea urchin, Hemicentrotus pulcherrimus, by means of DNase I affinity and DEAE-cellulose column chromatographies. This protein was a globular protein with a Stokes radius of 41-42 nm and consisted of a single polypeptide chain having an apparent molecular mass of 100 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Gel filtration chromatography revealed that one 100-kDa protein molecule binds two or three actin monomers in the presence of Ca2+, but such binding was not observed in the absence of Ca2+. The effect of the 100-kDa protein on the polymerization of actin was studied by viscometry, spectrophotometry and electron microscopy. The initial rate of actin polymerization was decreased at a very low molar ratio of 100-kDa protein/actin. Acceleration of the initial rate of polymerization occurred at a relatively high, but still substoichiometric, molar ratio of 100-kDa protein/actin. The 100-kDa protein produced fragmentation of muscle actin filaments at Ca2+ concentrations greater than 0.3 microM as revealed by viscometry and electron microscopy. Evidence was also presented that the 100-kDa protein binds to the barbed end of the actin filament.     

The mitotic apparatus-associated 51-kDa protein from sea urchin eggs is a GTP-binding protein and is immunologically related to yeast polypeptide elongation factor 1 alpha

We investigated the biochemical characteristics of the 51-kDa protein that is a major mitotic apparatus-associated basic protein of sea urchin eggs (Toriyama, M., Ohta, K., Endo, S., and Sakai, H. (1988) Cell Motil. Cytoskeleton 9, 117-128). The amino acid composition of the 51-kDa protein was apparently different from those of tubulin, actin, histones, and myelin basic protein; yet it was similar to those of polypeptide elongation factors 1 alpha (EF-1 alpha). In addition, antibody to EF-1 alpha from yeast cross-reacted with the 51-kDa protein. [3H] GTP binding activity was detected in the phosphocellulose-purified fraction (PC fraction) which predominantly contained the 51-kDa protein and was shown to be specific to GTP, GDP, guanylyl imidodiphosphate, and ITP. Photo-affinity labeling using [alpha-32P]8-azidoguanosine triphosphate (8-azido-GTP) demonstrated that a 51-kDa polypeptide in the PC fraction specifically bound 8-azido-GTP. This GTP-binding polypeptide was bound to a GTP affinity column, could be eluted by the addition of GTP, and was immunoreactive with anti-51-kDa protein antibodies. When the PC fraction was applied to a gel filtration chromatography column, GTP binding activity was completely coeluted with the 51-kDa protein. Furthermore, the PC fraction and the gel filtration-purified fraction had EF-1 alpha activity: [14C]Phe-tRNA transferring activity to ribosomes in the presence of poly(U) and ribosome-dependent GTPase activity. The results indicate that the mitotic apparatus-associated 51-kDa protein is a GTP-binding protein and suggest that it is structurally and functionally related to yeast EF-1 alpha.     

Phosphorylation of a 225-kDa centrosomal component in mitotic CHO cells and sea urchin eggs

Components of centrosomes are those among cellular proteins that are phosphorylated at the transition from interphase to mitosis. Using an anti-phosphoprotein antibody (CHO3) directed against isolated mitotic CHO spindles, we identified a 225-kDa centrosomal phosphocomponent in mitotic CHO cells and in cleaving sea urchin eggs. The 225-kDa protein is tightly attached to the centrosome, which allowed us to separate it from other spindle-associated factors by high salt extraction. Phosphorylation of the 225-kDa protein occurred during mitosis. This was shown by isotope labeling on gels as well as by visualization of thiophosphorylated centrosomes with an anti-thiophosphoprotein antibody (M. Cyert, T. Scherson, and M. W. Kirschner, 1988, Dev. Biol. 129, 209) after preincubation with ATP-gamma-S in vivo and in vitro. Mitotic spindles isolated from CHO cells retained their ability to phosphorylate the centrosomal component, whereas sea urchin spindles did not, possibly due to loss or inactivation of protein kinase(s) during spindle isolation. The enzyme associated with isolated CHO spindles was extractable by high salt treatment and was capable of phosphorylating many spindle components, including the 225-kDa centrosomal protein of CHO cells and sea urchin embryos. Such high salt extracts contain protein kinases, including cell cycle control protein kinase p34cdc2, suggesting that the enzyme responsible for centrosomal phosphorylation could be p34cdc2 or other downstream mitotic kinases activated by the action of p34cdc2.     

Preparation of plasma membranes from fertilized sea urchin eggs

A new method is presented for preparation of highly purified plasma membranes from fertilized sea urchin eggs. The purified plasma membranes are in vesicle form and are highly enriched in ouabain inhibitable, Na+/K+ ATPase activity. Analysis of membrane proteins by sodium dodecyl sulfate-gel electrophoresis indicates that several high-molecular-weight proteins characteristic of plasma membranes from unfertilized eggs are absent in plasma membranes from fertilized eggs.     

Cytasters from sea urchin eggs parthenogenetically activated by procaine

A method is presented for the isolation of cytasters from unfertilized sea urchin eggs parthenogenetically activated by procaine. These cytasters do not appear to contain centrioles. The microtubules seem to grow out from the condensed chromosomes. The chromosomes have an unusual morphology.     

Ribosomal proteins of sea urchin eggs

Summary Ribosomal proteins from unfertilized eggs of three sea urchin species, Pseudocentrotus depressus, Hemicentrotus pulcherrimus, and Anthocidaris crassispina, were analyzed. Species-specific differences were observed in the profiles of large subunit proteins on two-dimensional slab gels, though the number of ribosomal proteins and the molecular weights of their counterparts were the same. The small subunit proteins revealed similarities in the electrophoretic profiles and in the phosphorylation patterns among these three species.     

Fertilization kinetics of sea urchin eggs

Eggs and sperm of the sea urchin Paracentrotus lividus of the Mediterranean are used for an in vitro study of fertilization kinetics. The results are analyzed in terms of two models. One of these models assumes that all sperm-egg encounters lead to permanent attachment; the other (less realistically) assumes that sperm continue their random search after an unsuccessful encounter. More than 100 spermatozoa per egg are needed to achieve a fertilization ratio of more than 95%. There are two explanations for this: only 1% of the egg surface is subject to fertilization, or only 1% of spermatozoa are intrinsically able to fertilize. In the same context, chemotactic attraction and the role of the jelly are discussed. Comparison with earlier work of Rothschild and Swann and of Hultin and Hagström clarifies some discrepancies between and within these papers.