scholarly journals The in Vivo Rate of Glucose-6-Phosphate Dehydrogenase Activity in Sea Urchin Eggs Determined with a Photolabile Caged Substrate

1995 ◽  
Vol 169 (2) ◽  
pp. 733-744 ◽  
Author(s):  
Robert R. Swezey ◽  
David Epel
Keyword(s):  
Dehydrogenase Activity ◽  
Sea Urchin ◽  
Sea Urchin Eggs ◽  
Glucose 6 Phosphate Dehydrogenase

Effects of redox agents on glucose-6-phosphate dehydrogenase isozymes and enzyme activity in sea urchin eggs and zygotes

1985 ◽  
Vol 234 (3) ◽  
pp. 459-470
Author(s):  
Mary Lee Sparling (Barber) ◽  
Timothy Killeen ◽  
Yolanda Sinisgallo ◽  
Craig Yabuta
Keyword(s):  
Enzyme Activity ◽  
Sea Urchin ◽  
Sea Urchin Eggs ◽  
Redox Agents ◽  
Glucose 6 Phosphate Dehydrogenase

scholarly journals Effects of caffeine and other methylxanthines on the development and metabolism of sea urchin eggs. Involvement of NADP and glutathione.

1976 ◽  
Vol 68 (3) ◽  
pp. 440-450 ◽  
Author(s):  
J Nath ◽  
J I Rebhun
Keyword(s):  
Cell Division ◽  
Sea Urchin ◽  
Reductase Activity ◽  
Inhibitory Effect ◽  
Pentose Phosphate ◽  
Nad Kinase ◽  
Mitotic Apparatus ◽  
Sea Urchin Eggs ◽  
Glutathione Reductase Activity

Methylxanthines (MX) inhibit cell division in sea urchin and clam eggs. This inhibitory effect is not mediated via cAMP. MX also inhibit respiration in marine eggs, at concentrations which inhibit cleavage. Studies showed that no changes occurred in ATP and ADP levels in the presence of inhibitory concentrations of MX, indicating an extra-mitochondrial site of action for the drug. Subsequent studies revealed decreased levels of NADP+ and NADPH, when eggs were incubated with inhibitory concentrations of MX, but no change in levels of NAD+ and NADH. MX did not affect the pentose phosphate shunt pathway and did not have any effect on the enzyme NAD+ -kinase. Further studies showed a marked inhibitory effect on the glutathione reductase activity of MX-treated eggs. Reduced glutathione (GSH) could reverse the cleavage inhibitory effect of MX. Moreover, diamide, a thiol-oxidizing agent specific for GSH in living cells, caused inhibition of cell division in sea urchin eggs. Diamide added to eggs containing mitotic apparatus (MA) could prevent cleavage by causing a dissolution of the formed MA. Both MX and diamide inhibit a Ca2+-activated ATPase in whole eggs. The enzyme can be reactivated by sulfhydryl reducing agents added in the assay mixture. In addition, diamide causes an inhibition of microtubule polymerization, reversible with dithioerythritol. All experimental evidence so far suggests that inhibition of mitosis in sea urchin eggs by MX is mediated by perturbations of the in vivo thiol-disulfide status of target systems, with a primary effect on glutathione levels.

scholarly journals Cell cycle-dependent phosphorylation of the 77 kDa echinoderm microtubule-associated protein (EMAP) in vivo and association with the p34cdc2 kinase

1996 ◽  
Vol 109 (12) ◽  
pp. 2885-2893 ◽  
Author(s):  
E. Brisch ◽  
M.A. Daggett ◽  
K.A. Suprenant
Keyword(s):  
Cell Cycle ◽  
Sea Urchin ◽  
Time Course ◽  
Mitotic Apparatus ◽  
Sea Urchin Eggs ◽  
Spindle Poles ◽  
A Cell ◽  
Cycle Dependent ◽  
Phosphopeptide Mapping

The most abundant microtubule-associated protein in sea urchin eggs and embryos is the 77 kDa echinoderm microtubule-associated protein (EMAP). EMAP localizes to the mitotic spindle as well as the interphase microtubule array and is a likely target for a cell cycle-activated kinase. To determine if EMAP is phosphorylated in vivo, sea urchin eggs and embryos were metabolically labeled with 32PO4 and a monospecific antiserum was used to immunoprecipitate EMAP from 32P-labeled eggs and embryos. In this study, we demonstrate that the 77 kDa EMAP is phosphorylated in vivo by two distinct mechanisms. In the unfertilized egg, EMAP is constitutively phosphorylated on at least five serine residues. During the first cleavage division following fertilization, EMAP is phosphorylated with a cell cycle-dependent time course. As the embryo enters mitosis, EMAP phosphorylation increases, and as the embryo exits mitosis, phosphorylation decreases. During mitosis, EMAP is phosphorylated on 10 serine residues and two-dimensional phosphopeptide mapping reveals a mitosis-specific site of phosphorylation. At all stages of the cell cycle, a 33 kDa polypeptide copurifies with the 77 kDa EMAP, regardless of phosphorylation state. Antibodies against the cdc2 kinase were used to demonstrate that the 33 kDa polypeptide is the p34cdc2 kinase. The p34cdc2 kinase copurifies with the mitotic apparatus and immunostaining indicates that the p34cdc2 kinase is concentrated at the spindle poles. Models for the interaction of the p34cdc2 kinase and the 77 kDa EMAP are presented.

scholarly journals GLUCOSE-6-PHOSPHATE AND 6-PHOSPHOGLUCONATE DEHYDROGENASES FROM EGGS OF THE SEA URCHIN, ARBACIA PUNCTULATA

1955 ◽  
Vol 38 (4) ◽  
pp. 431-439 ◽  
Author(s):  
M. E. Krahl ◽  
A. K. Keltch ◽  
C. P. Walters ◽  
G. H. A. Clowes
Keyword(s):  
Oxygen Consumption ◽  
Dehydrogenase Activity ◽  
Sea Urchin ◽  
Maximal Activity ◽  
Glycolytic Pathway ◽  
Supernatant Fraction ◽  
Arbacia Punctulata ◽  
Rate Of Oxygen Consumption ◽  
Wet Weight ◽  
Glucose 6 Phosphate Dehydrogenase

1. Glucose-6-phosphate and 6-phosphogluconate dehydrogenases have been found in homogenates of Arbacia eggs; 95 per cent of the activity toward each substrate is recovered in the supernatant fraction after centrifuging at 20,000 g for 30 minutes. 2. With glucose-6-phosphate as substrate) the rate of TPN reduction by the supernatant fraction from 1 gm. wet weight unfertilized or fertilized eggs was 1.8 to 3.0 micromoles per minute; this rate is sufficient to support a rate of oxygen consumption 24 times that observed for unfertilized, and 6 times that for fertilized, eggs. Pentose was formed from glucose-6-phosphate at a rate 0.3 to 0.5 that of TPN reduction, when both rates were expressed as micromoles per minute. 3. The concentrations of glucose-6-phosphate and 6-phosphogluconate for half maximal activity were each approximately 0.00004 M for the respective enzymes in the supernatant fraction. Maximal activity toward 6-phosphogluconate was 50 to 60 per cent of that toward glucose-6-phosphate. Glucose-6-phosphate dehydrogenase activity was 50 per cent inhibited in presence of 0.00006 M 2,4,5-trichlorophenol. 4. Reduction of DPN by the supernatant fraction in presence of fructose-1,6-diphosphate and ADP was 0.1 to 0.2 micromoles per minute per gm. wet eggs, indicating that the glycolytic pathway can metabolize glucose-6-phosphate at about 5 per cent the rate at which it can be oxidized by the TPN system from unfertilized or fertilized Arbacia eggs. 5. Phosphoglucomutase, hexose isomerase, and a phosphatase for fructose-1,6-diphosphate also appear to be present in Arbacia eggs.

scholarly journals Stable, resealable pores formed in sea urchin eggs by electric discharge (electroporation) permit substrate loading for assay of enzymes in vivo.

1989 ◽  
Vol 1 (1) ◽  
pp. 65-74 ◽  
Author(s):  
R R Swezey ◽  
D Epel
Keyword(s):  
Small Molecules ◽  
Sea Urchin ◽  
Cell Types ◽  
Atp Content ◽  
Fluorescent Indicators ◽  
Sea Urchin Eggs ◽  
Minimal Loss ◽  
Endogenous Metabolites ◽  
Intracellular Milieu

We describe a simple electroporation procedure for loading suspensions of unfertilized sea urchin eggs with impermeant small molecules under conditions that allow close to 90% successful fertilization and development. Poration is carried out in a low-Ca2+ medium that mimicks the intracellular milieu. The induced pores remain open for several minutes in this medium, allowing loading of the cells; resealing is achieved by adding back millimolar calcium ions to the medium. While the pores are open, an influx of exogenous molecules and efflux of endogenous metabolites takes place, and the eggs can lose up to 40% of their ATP content and still survive. Introduced metabolites are utilized by the cells, e.g., introduced 3H-thymidine is incorporated into DNA. This procedure will be useful for loading impermeant substrates into eggs, permitting in vivo assessment of metabolism, and also for introducing other interesting impermeant molecules, such as inhibitors, fluorescent indicators, etc. Though the details may differ, the principle of electroporation in an intracellular-like medium may prove to be useful for loading other cell types with minimal loss of viability.

The effects of mitotic inhibitors on the structure of vinblastine-induced tubulin paracrystals from sea-urchin eggs

1976 ◽  
Vol 20 (1) ◽  
pp. 91-100
Author(s):  
D. Starling
Keyword(s):  
Sea Urchin ◽  
Ultrastructural Level ◽  
Tubulin Polymerization ◽  
Sea Urchin Eggs ◽  
Mitotic Inhibitors ◽  
Vinblastine Sulphate ◽  
Tubulin Paracrystals ◽  
Careful Standardization

Vinblastine sulphate (VLB) is known to induce in vivo formation of tubulin paracrystals in sea-urchin eggs. Corresponding paracrystals have been prepared in the presence of both vinblastine sulphate and other mitoclasic agents. Careful standardization of conditions was required to restrict the formation of alternative forms of the paracrystals induced by vinblastine alone. Comparisons were made between preparations in terms of paracrystal shape, size, proportion of eggs containing paracrystals, number per egg and their relative times of first appearance. A correlation between such properties were established. Comparison of paracrystals at the ultrastructural level showed them all to be similar regardless of the drugs present during their formation. The implications of tubulin polymerization in the presence of mitoclasic agents are discussed and mechanisms for paracrystal enhancement by combinations of such drugs are suggested. Some similarities of paracrystal and microtubule seeding are discussed together with the activation of tubulin in the pool.

scholarly journals Fertilization triggers unmasking of maternal mRNAs in sea urchin eggs.

1987 ◽  
Vol 7 (11) ◽  
pp. 3947-3954 ◽  
Author(s):  
J L Grainger ◽  
M M Winkler
Keyword(s):  
Sea Urchin ◽  
Gradient Centrifugation ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
High Salt ◽  
Translation System ◽  
Sea Urchin Eggs ◽  
Translational Activity

Fertilization of sea urchin eggs results in a large increase in the rate of protein synthesis which is mediated by the translation of stored maternal mRNA. The masked message hypothesis suggests that messenger ribonucleoprotein particles (mRNPs) from unfertilized eggs are translationally inactive and that fertilization results in alterations of the mRNPs such that they become translationally active. Previous workers have isolated egg mRNPs by sucrose gradient centrifugation and have assayed their translational activity in heterologous cell-free systems. The conflicting results they obtained are probably due to the sensitivity of mRNPs to artifactual activation and inactivation. Previously, we demonstrated that unfractionated mRNPs in a sea urchin cell-free translation system were translationally inactive. Now, using large-pore gel filtration chromatography, we partially purified egg mRNPs while retaining their translationally repressed state. Polysomal mRNPs from fertilized eggs isolated under the same conditions were translationally active. The changes in the pattern of proteins synthesized by fractionated unfertilized and fertilized mRNPs in vitro were similar to those changes observed in vivo. Treatment of egg mRNPs with buffers containing high salt and EDTA, followed by rechromatography, resulted in the activation of the mRNPs and the release of an inhibitor of translation from the mRNPs. Analysis of the inhibitory fraction on one-dimensional sodium dodecyl sulfate gels indicated that this fraction contains a complex set of proteins, several of which were released from high-salt-EDTA-activated mRNPs and not from inactive low-salt control mRNPs. One of the released proteins may be responsible for the repression of egg mRNPs in vitro and be involved in the unmasking of mRNPs at fertilization.

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