scholarly journals Colchicine-binding activity distinguishes sea urchin egg and outer doublet tubulins.

1984 ◽  
Vol 99 (1) ◽  
pp. 37-41 ◽  
Author(s):  
L Wilson ◽  
H P Miller ◽  
T A Pfeffer ◽  
K F Sullivan ◽  
H W Detrich
Keyword(s):  
Sea Urchin ◽  
Strongylocentrotus Purpuratus ◽  
Binding Activity ◽  
Binding Affinities ◽  
Regulatory Site ◽  
Colchicine Binding Site ◽  
Sea Urchin Egg ◽  
Enthalpy And Entropy Changes ◽  
Enthalpy And Entropy ◽  
Significant Chemical

The colchicine-binding activity of tubulin has been utilized to distinguish the tubulins from two distinct microtubule systems of the same species, the sea urchin Strongylocentrotus purpuratus. We have analyzed the colchicine-binding affinities of highly purified tubulins from the unfertilized eggs and from the flagellar outer doublet microtubules by van't Hoff analysis, and have found significant differences in the free energy, enthalpy, and entropy changes characterizing the binding of colchicine to the two tubulins. The data indicate that significant chemical differences in the tubulins from the two functionally distinct microtubule systems exist, and that the differences are expressed in the native forms of the tubulins. Our findings are discussed in terms of the possibility that the colchicine-binding site may be an important regulatory site on the tubulin molecule.

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.

scholarly journals Hierarchies of protein cross-linking in the extracellular matrix: involvement of an egg surface transglutaminase in early stages of fertilization envelope assembly.

1988 ◽  
Vol 107 (6) ◽  
pp. 2447-2454 ◽  
Author(s):  
D E Battaglia ◽  
B M Shapiro
Keyword(s):  
Cell Surface ◽  
Sea Urchin ◽  
Physiological Function ◽  
Strongylocentrotus Purpuratus ◽  
Cross Linking ◽  
Early Step ◽  
Transglutaminase Activity ◽  
Sea Urchin Egg ◽  
A Cell ◽  
Secretory Products

The involvement of transglutaminase activity in fertilization envelope (FE) formation was investigated using eggs from the sea urchin, Strongylocentrotus purpuratus. Eggs fertilized in the presence of the transglutaminase inhibitors, putrescine and cadaverine, had disorganized and expanded FEs with inhibition of the characteristic I-T transition. The permeability of the FE was increased by these agents, as revealed by the loss of proteins from the perivitelline space and the appearance of ovoperoxidase activity in supernates from putrescine-treated eggs. [3H]putrescine was incorporated into the FE during fertilization in a reaction catalyzed by an egg surface transglutaminase that could also use dimethylcasein as a substrate in vitelline layer-denuded eggs. Egg secretory products alone had no transglutaminase activity. The cell surface transglutaminase activity was transient and maximal within 4 min of activation. The enzyme was Ca2+ dependent and was inhibited by Zn2+. We conclude that sea urchin egg surface transglutaminase catalyzes an early step in a hierarchy of cross-linking events during FE assembly, one that occurs before ovoperoxidase-mediated dityrosine formation (Foerder, C. A., and B. M. Shapiro. 1977. Proc. Natl. Acad. Sci. USA. 74:4214-4218). Thus it provides a graphic example of the physiological function of a cell surface transglutaminase.

scholarly journals Properties of tubulin in unfertilized sea urchin eggs. Quantitation and characterization by the colchicine-binding reaction.

1976 ◽  
Vol 69 (3) ◽  
pp. 599-607 ◽  
Author(s):  
T A Pfeffer ◽  
C F Asnes ◽  
L Wilson
Keyword(s):  
Sea Urchin ◽  
Competitive Inhibition ◽  
Binding Constants ◽  
Binding Activity ◽  
Decay Rates ◽  
Cell Protein ◽  
Soluble Cell ◽  
Sea Urchin Egg ◽  
Different Temperatures ◽  
Delta G

The colchicine-binding assay was used to quantitate the tubulin concentration in unfertilized Strongylocentrotus purpuratus eggs and to characterize pharmacological properties of this tubulin. Specificity of colchicine binding to tubulin was demonstrated by apparent first-order decay colchicine-binding activity with stabilization by vinblastine sulfate, time and temperature dependence of the reaction, competitive inhibition by podophyllotoxin, and lack of effect of lumicolchicine. The results demonstrate that the minimum tubulin concentration in the unfertilized egg is 2.71 mg per milliliter or 5.0% of the total soluble cell protein. Binding constants and decay rates were determined at six different temperatures between 8 degrees C and 37 degrees C, and the thermodynamic parameters of the reaction were calculated. delta H0=6.6 kcal/mol, delta S0=46.5 eu, and, at 13 degrees C, delta G=-6.7 kcal/mol. The association constants obtained were similar to those of isolated sea urchin egg vinblastine paracrystals (Bryan, J. 1972. Biochemistry. 11:2611-2616) but approximately 10 times lower than that obtained for purified chick embryo brain tubulin at 37 degrees C (Wilson, L.J.R. Bamburg, S.B. Mizel, L. Grisham, and K. Creswell. 1974. Fed Proc. 33:158-166). Therefore, the lower binding constants for colchicine in tubulin-vinblastine paracrystals are not due to the paracrystalline organization of the tubulin, but are properties of the sea urchin egg tubulin itself.

Phosphate-Transport Appearance in the Sea-Urchin Egg. II. Analysis of the Genome Function with Centrifuged Eggs and Ethidium Bromide

1974 ◽  
Vol 52 (6) ◽  
pp. 1186-1191
Author(s):  
Joël de la Noüe
Keyword(s):  
Ethidium Bromide ◽  
Sea Urchin ◽  
Phosphate Uptake ◽  
Strongylocentrotus Purpuratus ◽  
Nuclear Genome ◽  
Phosphate Transport ◽  
Carrier System ◽  
Purple Sea Urchin ◽  
Sea Urchin Egg ◽  
Phosphate Carrier

Ethidium bromide, an inhibitor of mitochondrial transcription, inhibits phosphate uptake, valine incorporation, and uridine incorporation in fertilized eggs of Strongylocentrotus purpuratus, the purple sea urchin. Phosphate uptake, valine incorporation, and valine influx are also inhibited by this drug in artificially activated nucleate and anucleate egg fragments. Ethidium bromide does not affect the egg respiration and it has no measurable effect on ATP level and labeling. It is concluded that the post-fertilization appearance of the phosphate-carrier system does not require the participation of the nuclear genome but that of the mitochondrial one. It is likely that the same proposal holds for the L-valine-transport system. Some effects of ethidium bromide on development are exposed.

The GTP-binding protein RhoA localizes to the cortical granules of Strongylocentrotus purpuratus sea urchin egg and is secreted during fertilization

2000 ◽  
Vol 79 (2) ◽  
pp. 81-91 ◽  
Author(s):  
Patricia Cuéllar-Mata ◽  
Guadalupe Martínez-Cadena ◽  
Juana López-Godínez ◽  
Armando Obregón ◽  
Jesús García-Soto
Keyword(s):  
Sea Urchin ◽  
Binding Protein ◽  
Strongylocentrotus Purpuratus ◽  
Cortical Granules ◽  
Gtp Binding Protein ◽  
Gtp Binding ◽  
Sea Urchin Egg

scholarly journals Monoclonal antibodies to dynein subunits reveal the existence of cytoplasmic antigens in sea urchin egg.

1984 ◽  
Vol 98 (5) ◽  
pp. 1842-1850 ◽  
Author(s):  
G Piperno
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibodies ◽  
Sea Urchin ◽  
Strongylocentrotus Purpuratus ◽  
Apparent Molecular Weight ◽  
Mitotic Apparatus ◽  
Cellular Fraction ◽  
Sea Urchin Egg ◽  
Monospecific Antibodies

Monoclonal antibodies directed against subunits of a sea urchin flagellar dynein were used to test for the presence of cytoplasmic antigens in preparations of fertilized eggs and mitotic apparati . A 9-10 S complex composed of 330,000-, 134,000-, and 126,000-mol-wt subunits was isolated from outer arms of Strongylocentrotus purpuratus sperm flagella and used to characterize the antibodies. Seven monospecific antibodies to the 330,000 subunit and two against the 134,000 subunit of the 9-10 S complex were identified by binding to nitrocellulose blots of electrophoretograms resolving polypeptides from different dynein preparations. The antibodies were applied also to blots of polypeptides from fertilized sea urchin egg at the first metaphase and a cellular fraction of mitotic apparati . Three of the antibodies to the 330,000 subunit bound to a cytoplasmic polypeptide of approximately the same molecular weight and the two antibodies to the smaller subunits recognized a polypeptide of 124,000 apparent molecular weight. Both antigens appeared to be enriched in the fraction containing mitotic apparati . These results indicate that polypeptides similar to two subunits of the 9-10 S complex are present in eggs at metaphase, and they are apparently associated with the mitotic apparatus.

scholarly journals The purification of a 50 kDa protein-actin complex from unfertilized sea-urchin (Strongylocentrotus purpuratus) eggs

1989 ◽  
Vol 257 (3) ◽  
pp. 809-815 ◽  
Author(s):  
R M Golsteyn ◽  
D M Waisman
Keyword(s):  
Sea Urchin ◽  
Binding Protein ◽  
Strongylocentrotus Purpuratus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Binding Activity ◽  
Stable Complex ◽  
Chelex 100 ◽  
Activity Peak ◽  
Sds Page

The 100,000 g supernatant from the unfertilized egg of the sea urchin Strongylocentrotus purpuratus has been fractionated on DEAE-cellulose and analysed for Ca2+-binding activity by the Chelex-100 competitive Ca2+-binding activity assay. The major peak of Ca2+-binding activity was subjected to further purification and the Ca2+-binding protein responsible for this Ca2+-binding-activity peak has been isolated and characterized. Non-denaturing polyacrylamide-gel electrophoresis (PAGE) followed by 45Ca2+ autoradiography suggested a molecular mass of 80 kDa for the Ca2+-binding protein. SDS/PAGE revealed that the 80 kDa protein consisted of a 1:1 molar complex of proteins of 50 and 42 kDa. The 42 kDa protein was identified as actin. The complex was not dissociated by extensive dialysis against an EGTA-containing buffer. The EGTA-stable complex was named ‘50K-A’.

Evidence that a secretory component of the sea urchin egg is routed to yolk platelets after fertilization

1989 ◽  
Vol 47 ◽  
pp. 838-839
Author(s):  
M. C. Valdizan ◽  
G. L. Decker
Keyword(s):  
Sea Urchin ◽  
Colloidal Gold ◽  
Sea Water ◽  
Strongylocentrotus Purpuratus ◽  
Secretory Component ◽  
Time Points ◽  
Sea Urchin Egg ◽  
Cortical Vesicles ◽  
The Individual ◽  
Lowicryl K4m

During fertilization the sea urchin egg undergoes a global wave of secretion (the cortical reaction) followed by a period of endocytotic activity involved in membrane retrieval. Using a monoclonal antibody (MAb 1223) that recognizes a 130-kDa glycoprotein found in both the egg and embryo of Strongylocentrotus purpuratus, we recently suggested that glycoproteins bearing the 1223 epitope are stored in the cortical vesicles of the egg, secreted during fertilization, and subsequently endocytosed and routed to yolk platelets, a possible site of degradation.In the current study, to determine whether fertilized eggs could internalize an antibody probe directed at the cell surface, we coupled MAb 1223 to colloidal gold and presented it to eggs that had been stripped of fertilization envelopes and hyaline layers. Gold-conjugated mouse IgG (preimmune), bovine serum albumin (BSA), and goat-antimouse IgG served as controls. Colloidal gold (8 and 15 nm) was prepared and conjugated to BSA or antibody as previously described. For localization of the 1223 antigen in unfertilized eggs MAb 1223-gold was diluted 1:20 in buffer and applied to sections of eggs embedded in Lowicryl K4M. To remove the envelopes and hyaline layers, 20-30 seconds after addition of sperm the eggs were suspended in ice-cold artificial sea water containing 25 mM EGTA and passed through a nylon screen (64 μm mesh). Subsequently, 1 ml aliquots of the denuded eggs [10% suspension (vol/vol)] were gravity settled, removed from the Ca2+-chelator and resuspended in complete sea water. After a second wash, 20 μl aliquots of the eggs were transferred to fresh 1.0 ml volumes of sea water equilibrated at 14°C in the presence of the individual gold probes. The final dilution of the gold probes in sea water was 1:100. After gentle mixing of the eggs and gold probes, at time points 5,15,30 and 60 minutes specimens were fixed and embedded in Spurr resin as previously described.

scholarly journals THE EFFECTS OF CARBON MONOXIDE INHIBITION ON ATP LEVEL AND THE RATE OF MITOSIS IN THE SEA URCHIN EGG

1963 ◽  
Vol 17 (2) ◽  
pp. 315-319 ◽  
Author(s):  
David Epel
Keyword(s):  
Carbon Monoxide ◽  
Sea Urchin ◽  
Strongylocentrotus Purpuratus ◽  
Specific Inhibitor ◽  
Atp Synthesis ◽  
Luciferase Assay ◽  
Basic Premise ◽  
Atp Level ◽  
Sea Urchin Egg ◽  
Kinetics Of

The requirements for ATP synthesis during the various phases of mitosis were investigated in the oxygen-requiring eggs of the sea urchin, Strongylocentrotus purpuratus. CO in the dark, a specific inhibitor of respiration, was used to inhibit ATP synthesis. The kinetics of respiratory inhibition were determined by analyzing ATP levels with the luciferin-luciferase assay. The kinetics of mitotic inhibition were determined by analysis of the rate of mitosis. It was found that CO inhibition resulted in a decrease in the normal ATP level. Coincident with this decrease was a decrease in the rate of mitosis which stops completely when the ATP drops below 50 per cent of the normal level. With the use of various degrees of CO inhibition, the rate of mitosis is shown to be related to the resultant ATP level. These results contradict the basic premise of the energy reservoir hypothesis, and also disagree with other reports that cells in mitosis are insensitive to inhibitors of energy metabolism. Data are presented which demonstrate that these conflicting reports result from insufficient inhibition of ATP synthesis. The above findings all indicate that mitosis depends on the continuous synthesis and utilization of ATP.

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