Isolation of a protease from sea urchin eggs before and after fertilization

Fodor, E; Ako, Harry; Walsh, Kenneth A.

doi:10.1021/bi00693a022

Cited by 58 publications

(38 citation statements)

References 21 publications

(17 reference statements)

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“…However, other investigators reported that S purpuratus eggs contained and secreted a single serine protease at fertilization [Fodor et al, 19751. These conflicting results may reflect the fact that the cortical granule protease appears to be secreted from the eggs complexed to a nonsedimentable particle [Fodor et al, 1975;Grossman et al, 1973al . Preliminary observations suggest that the enzymatic and biologic functions of the cortical granulederived protease may be regulated by its association with this particle [Csernansky et al, 19761.…”

Section: Serine Proteasementioning

confidence: 99%

Secretory functions of egg cortical granules in fertilization and development: A critical review

Schuel

1978

Gamete Research

214

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Section: Serine Proteasementioning

confidence: 99%

Secretory functions of egg cortical granules in fertilization and development: A critical review

Schuel

1978

Gamete Research

214

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“…The cortical granule protease was purified by SBTI affinity chromatography (Fodor et al, 1975), and column fractions were concentrated and washed in 10 mM Tris, pH 4.0, buffer in Centricon-10 centrifugal filter devices (Millipore, Beverly, MA). Samples were then diluted in 10 mM Tris, pH 8.0, buffer and protease activity was measured by benzoyl-l-arginine ethylester (BAEE) as described previously (Schwert and Takenaka, 1955).…”

Section: Protease Activity Assaymentioning

confidence: 99%

Regulated Proteolysis by Cortical Granule Serine Protease 1 at Fertilization

Haley

Wessel

2004

MBoC

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Cortical granules are specialized organelles whose contents interact with the extracellular matrix of the fertilized egg to form the block to polyspermy. In sea urchins, the granule contents form a fertilization envelope (FE), and this construction is critically dependent upon protease activity. An autocatalytic serine protease, cortical granule serine protease 1 (CGSP1), has been identified in the cortical granules of Strongylocentrotus purpuratus eggs, and here we examined the regulation of the protease activity and tested potential target substrates of CGSP1. We found that CGSP1 is stored in its full-length, enzymatically quiescent form in the granule, and is inactive at pH 6.5 or below. We determined the pH of the cortical granule by fluorescent indicators and micro-pH probe measurements and found the granules to be pH 5.5, a condition inhibitory to CGSP1 activity. Exposure of the protease to the pH of seawater (pH 8.0) at exocytosis immediately activates the protease. Activation of eggs at pH 6.5 or lower blocks activation of the protease and the resultant FE phenotypes are indistinguishable from a protease-null phenotype. We find that native cortical granule targets of the protease are ␤-1,3 glucanase, ovoperoxidase, and the protease itself, but the structural proteins of the granule are not proteolyzed by CGSP1.Whole mount immunolocalization experiments demonstrate that inhibition of CGSP1 activity affects the localization of ovoperoxidase but does not alter targeting of structural proteins to the FE. The mistargeting of ovoperoxidase may lead to spurious peroxidative cross-linking activity and contribute to the lethality observed in protease-null cells. Thus, CGSP1 is proteolytically active only when secreted, due to the low pH of the cortical granules, and it has a small population of targets for cleavage within the cortical granules. INTRODUCTIONPolyspermy is lethal to the eggs of most species. If more than one sperm fertilizes the egg, then the newly formed zygote will cleave abnormally and die. One effective way to block polyspermy is for the egg to rapidly modify its extracellular surface after sperm activation so that subsequent sperm cannot reach the egg cell surface. A conserved mechanism for this modification is for the fertilizing sperm to induce the egg to exocytose contents from specialized organelles called cortical granules. The contents of the granule serve to modify the extracellular matrix of the egg, making it impenetrable to additional sperm fusions. The cortical granules have a specific and singular role during development: these organelles are present only in eggs and oocytes, and most of the known content proteins are expressed selectively in oocytes. Although the molecular mechanisms used to block polyspermy are different between animals, all mammals, most vertebrates, and many invertebrates, including the sea urchin share this fundamental process Wessel et al., 2001).Cortical granules of the sea urchin contain a diverse population of proteins, including structural molecules, enzym...

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“…Plasminogen activator has been shown to be involved in ovula-tion (40). Other enzymes have been isolated from sea urchin eggs (41) including a homogenous enzyme similar to bovine trypsin in its molecular weight and susceptibility to inhibitors diisopropylfluro-phosphate and soybean trypsin inhibitor (42). The presence of the a,-protease inhibitor in ovarian tissue might serve to prevent extensive autodigestion of the tissue by tryptic enzymes following ovulation.…”

Section: ~2mentioning

confidence: 99%