Evidence that hatching enzyme of the sea urchin Strongylocentrotus purpuratus is a chymotrypsin-like protease

1988 ◽  
Vol 66 (11) ◽  
pp. 1200-1209 ◽  
Author(s):  
Laura L. Post ◽  
Regina Schuel ◽  
Herbert Schuel
Keyword(s):  
Ethyl Ester ◽  
Proteolytic Activity ◽  
Sea Urchin ◽  
Trypsin Inhibitor ◽  
Strongylocentrotus Purpuratus ◽  
Soybean Trypsin Inhibitor ◽  
Chloromethyl Ketone ◽  
Hatching Enzyme ◽  
Sea Urchin Eggs ◽  
Chymotrypsin Inhibitors

The sea urchin blastula secretes a hatching enzyme (HE) that dissolves the fertilization envelope. HE was collected from the supernatant seawater of cultures of hatched Strongylocentrotus purpuratus blastulae, and concentrated 20 times by ultrafiltration. The proteolytic activity of HE using casein as substrate was inhibited by the chymotrypsin inhibitors, chymostatin and N-tosyl-L-phenylalanine chloromethyl ketone. The activity was not inhibited by inhibitors (antipain, elastatinal, pepstatin, phosphoramidon, soybean trypsin inhibitor, and Nα-p-tosyl-L-lysine chloromethyl ketone) of other types of proteases. HE did not hydrolyze the synthetic trypsin substrate, α-N-benzoyl-L-arginine ethyl ester, but did hydrolyze the synthetic substrate of chymotrypsin, N-benzoyl-L-tyrosine ethyl ester (BTEE). The BTEEase activity of HE was completely inhibited by the chymotrypsin inhibitors chymostatin and 2-nitro-4-carboxyphenyl N,N-diphenylcarbamate (NCDC). Chymostatin inhibited the natural hatching of sea urchin blastulae. Application of HE to freshly fertilized sea urchin eggs, 2 h after insemination, caused premature dispersal of the hardened fertilization envelope. Chymostatin and NCDC inhibited HE-induced lysis of the fertilization envelope, while inhibitors of other types of proteases were ineffective. These data suggest that sea urchin HE is a chymotrypsin-like protease we call "chymostrypsin."

Inositol-1,4,5-trisphosphate injection mimics fertilization potentials in sea urchin eggs

1986 ◽  
Vol 250 (2) ◽  
pp. C340-C344 ◽  
Author(s):  
B. E. Slack ◽  
J. E. Bell ◽  
D. J. Benos
Keyword(s):  
Membrane Potential ◽  
Partial Response ◽  
Sea Urchin ◽  
Time Course ◽  
Strongylocentrotus Purpuratus ◽  
Sea Urchin Eggs ◽  
Low Calcium ◽  
Inositol 1,4,5 Trisphosphate

The effects of inositol-1,4,5-trisphosphate (IP3) and of diacylglycerol (DAG) and its analogues on the membrane potential of eggs from the sea urchin Strongylocentrotus purpuratus were examined. Injection of IP3 into eggs resulted in a change in membrane potential that was similar in magnitude and time course to the fertilization potential elicited by sperm attachment. In low-calcium seawater, IP3 injection elicited a partial response. DAG and its analogues phorbol myristyl acetate and 1-oleoyl-2-acetylglycerol did not affect membrane potential either when applied by perfusion or when injected. The results indicate that IP3, but not DAG or its analogues, may be involved in the generation of the fertilization potential triggered by the interaction of sperm with sea urchin eggs.

The Respiratory and Glycolytic Enzymes of Sea-Urchin Eggs

1954 ◽  
Vol 31 (2) ◽  
pp. 208-217
Author(s):  
MARTYNAS YČAS
Keyword(s):  
Cytochrome C ◽  
Sea Urchin ◽  
Strongylocentrotus Purpuratus ◽  
Lactic Dehydrogenase ◽  
Glycolytic Pathway ◽  
Endogenous Respiration ◽  
Centrifugal Field ◽  
Lytechinus Pictus ◽  
Sea Urchin Eggs ◽  
Sea Urchin Egg

1. Activity corresponding to phosphoglucomutase, phosphohexoisomerase, aldolase, triosephosphate dehydrogenase, enolase and lactic dehydrogenase has been demonstrated in homogenates prepared from unfertilized sea-urchin eggs (Strongylocentrotus purpuratus and Lytechinus pictus). 2. The presence of cytochromes a and b1 has been confirmed. These cytochromes sediment in a relatively low centrifugal field. 3. No cytochrome c could be demonstrated, although cytochrome c is both reduced and oxidized by homogenates, and addition of cytochrome c increases the endogenous respiration and oxidation of succinate. 4. These results support the view that the usual glycolytic pathway operates in the sea-urchin egg and is the principal route of oxidation of carbohydrate.

Cathepsin B and aminopeptidase in the posterior midgut of Phymata wolffii Stål (Hemiptera: Phymatidae)

1985 ◽  
Vol 63 (6) ◽  
pp. 1288-1291 ◽  
Author(s):  
Jon G. Houseman ◽  
P. E. Morrison ◽  
A. E. R. Downe
Keyword(s):  
Molecular Weight ◽  
Trypsin Inhibitor ◽  
Cathepsin B ◽  
Ethylenediaminetetraacetic Acid ◽  
Soybean Trypsin Inhibitor ◽  
Aminopeptidase Activity ◽  
Chloromethyl Ketone ◽  
Posterior Midgut ◽  
Hydrolysis Of

The posterior midgut of the phymatid Phymata wolffii Stål contains cathepsin B and aminopeptidase activity. Identification of cathepsin B was based on maximal hydrolysis of benzoyl-DL-arginine-2-naphthylamide and benzoyl-DL-arginine-p-nitroanilide at pH 5.8 and 5.5, respectively. Cathepsin B hydrolysis of the tested substrates was activated by thiol chemicals and ethylenediaminetetraacetic acid (EDTA) and inhibited by tosyl-L-lysine chloromethyl ketone, iodoacetamide, and soybean trypsin inhibitor. Aminopeptidase hydrolyzed leucine-p-nitroanilide maximally at pH 7.8 and hydrolysis of the substrate was activated by magnesium and inhibited by EDTA, dithiothreitol, glutathione, and cysteine. The molecular weight of cathepsin B was 40 000 and was greater than 150 000 for aminopeptidase.

scholarly journals Transition from mitosis to interphase in sea urchin first division: immunofluorescence studies of tubulin distribution in methacrylate sections.

1987 ◽  
Vol 35 (3) ◽  
pp. 343-349 ◽  
Author(s):  
P J Harris ◽  
B P Rubin
Keyword(s):  
Low Temperature ◽  
Methyl Methacrylate ◽  
Sea Urchin ◽  
Strongylocentrotus Purpuratus ◽  
Immunofluorescence Staining ◽  
Cleavage Cycle ◽  
Sea Urchin Eggs ◽  
Immunofluorescence Studies ◽  
Flat Embedding ◽  
Cell Thickness

Previous immunofluorescence studies of microtubule distribution in fertilized sea urchin eggs have suffered from poor resolution caused by cell thickness, unavoidable artifacts resulting from excessive flattening, or extraction by detergents of membranes and other lipid-containing structures that may be of interest in relation to the microtubules. To avoid these difficulties, we have developed a fixation and embedding protocol based on buffered paraformaldehyde fixation and butyl-methyl methacrylate embedment, which allows immunofluorescence staining of 0.5-1 micron sections. Polymerization artifacts are reduced by polymerizing the methacrylate at a relatively low temperature (40-45 degrees C) and by flat embedding for more uniform polymerization. Using this method, we have examined mitotic stages in the first cleavage cycle of the sea urchin Strongylocentrotus purpuratus. We provide evidence that the interphase microtubules that appear after first division are not derived from the mitotic asters but are new structures growing from organizing centers within the degenerating mitotic asters. During the transition from mitosis to interphase, there is a temporary overlap of old and new microtubules to form a very large composite aster at telophase before the old structure finally disappears.

Human Plasma and Serum Trypsin-like Esterase Activity

1966 ◽  
Vol 12 (6) ◽  
pp. 350-359 ◽  
Author(s):  
Roman B Rutkowski
Keyword(s):  
Calcium Oxalate ◽  
Human Plasma ◽  
Ethyl Ester ◽  
Trypsin Inhibitor ◽  
Spectrophotometric Method ◽  
Esterase Activity ◽  
Soybean Trypsin Inhibitor ◽  
Serum Trypsin ◽  
Healthy Males

Abstract A spectrophotometric method using benzoyl arginine ethyl ester (BAEE) is described for the assay of serum and plasma trypsin-like esterase activity. The method was used to find activities in serum and plasma (anticoagulated with oxalate and heparin) from 12 healthy males at 3 different reaction pH's. The effects of calcium, oxalate, heparin, soybean trypsin inhibitor, ovomucoid, and heat on this activity were also measured. Values of esterase activity were significantly different, indicating a variable enzyme or inhibitor composition in the 3 sets of samples. Plasma from heparinized blood gave unique activity responses to pH and to the agents tested for effect.

Proteolytic activity of rabbit perivitelline spermatozoa

Zygote ◽  
1999 ◽  
Vol 7 (2) ◽  
pp. 143-149 ◽  
Author(s):  
M. Valdivia ◽  
T. Sillerico ◽  
A. De Ioannes ◽  
C. Barros
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Serine Protease ◽  
Proteolytic Activity ◽  
Trypsin Inhibitor ◽  
Zona Pellucida ◽  
Gelatin Film ◽  
Soybean Trypsin Inhibitor ◽  
Substrate Film

Acrosin, an acrosomal serine protease, has been associated with binding of spermatozoa and their penetration through the zona pellucida. This study was aimed at determining whether the remaining proacrosin/acrosin system on rabbit perivitelline spermatozoa still has proteolytic activity and whether this activity is involved in further penetration of unfertilised rabbit eggs. Eight hundred and sixty-five rabbit perivitelline spermatozoa were evaluated by the gelatin-substrate film technique for the detection of acrosin on individual spermatozoan. Fifteen per cent of the studied spermatozoa showed small digestion halos on the gelatin film. The proteolytic activity of rabbit perivitelline spermatozoa was inhibited in the presence of 1 mg/ml of soybean trypsin inhibitor (SBTI) or with 20 μg/ml of a mixture of the monoclonal anti-proacrosin/acrosin antibody. In vitro fertilisation occurred in 21.8% of rabbit oocytes co-incubated with perivitelline spermatozoa and was completely inhibited when oocytes were incubated with 600 μg/ml of a mixture of three anti-acrosin monoclonal antibodies (ACRO-A8C10, ACRO-C2B10 and ACRO-C5F10). Inseminations in the presence of anti-cholera monoclonal antibody (irrelevant to spermatozoa) resulted in 17.6% fertilisation. These results support the idea that the residual proacrosin/acrosin system in perivitelline spermatozoa might be involved in spermatozoal binding and/or second penetration through the zona pellucida.

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