scholarly journals ADP-ribosyl cyclase: an enzyme that cyclizes NAD+ into a calcium-mobilizing metabolite.

1991 ◽  
Vol 2 (3) ◽  
pp. 203-209 ◽  
Author(s):  
H C Lee ◽  
R Aarhus
Keyword(s):  
Sea Urchin ◽  
Exchange Chromatography ◽  
Reaction Products ◽  
Sea Urchin Eggs ◽  
Tissue Extracts ◽  
Adp Ribosyl Cyclase ◽  
Sea Urchin Egg ◽  
Sds Page ◽  
Cyclic Adp Ribose ◽  
Charge Analysis

Cyclic ADP-ribose (cADPR) is a metabolite of NAD+ that is as active as inositol trisphosphate (IP3) in mobilizing intracellular Ca2+ in sea urchin eggs. The activity of the enzyme responsible for synthesizing cADPR is found not only in sea urchin eggs but also in various mammalian tissue extracts, suggesting that cADPR may be a general messenger for Ca2+ mobilization in cells. An aqueous soluble enzyme, thought to be an NADase, has been purified recently from the ovotestis of Aplysia californica (Hellmich and Strumwasser, 1991). This paper shows that the Aplysia enzyme catalyzes the conversion of NAD+ to cADPR and nicotinamide. The Aplysia enzyme was purified by fractionating the soluble extract of Aplysia ovotestis on a Spectra/gel CM column. The purified enzyme appeared as a single band of approximately 29,000 Da on SDS-PAGE but could be further separated into multiple peaks by high-resolution, cation-exchange chromatography. All of the protein peaks had enzymatic activity, indicating that the enzyme had multiple forms differing by charge. Analysis of the reaction products of the enzyme by anion-exchange high-pressure liquid chromatography (HPLC) indicated no ADP-ribose was produced; instead, each mole of NAD+ was converted to equimolar of cADPR and nicotinamide. The identification of the product as cADPR was further substantiated by proton NMR and also by its Ca(2+)-mobilizing activity. Addition of the product to sea urchin egg homogenates induced Ca2+ release and desensitized the homogenate to authentic cADPR but not to IP3. Microinjection of the product into sea urchin eggs elicited Ca2+ transients as well as the cortical exocytosis reaction. Therefore, by the criteria of HPLC, NMR, and calcium-mobilizing activity, the product was identical to cADPR. To distinguish the Aplysia enzyme from the conventional NADases that produce ADP-ribose, we propose to name it ADP-ribosyl cyclase.

scholarly journals Ca2+ release triggered by nicotinate adenine dinucleotide phosphate in intact sea urchin eggs

1995 ◽  
Vol 312 (3) ◽  
pp. 955-959 ◽  
Author(s):  
C M Perez-Terzic ◽  
E N Chini ◽  
S S Shen ◽  
T P Dousa ◽  
D E Clapham
Keyword(s):  
Sea Urchin ◽  
Specific Inhibitor ◽  
Intact Cells ◽  
Sea Urchin Eggs ◽  
Sea Urchin Egg ◽  
Cyclic Adp Ribose ◽  
Inositol 1,4,5 Trisphosphate ◽  
Dose Dependent Increase ◽  
Dose Dependent

Nicotinate adenine dinucleotide phosphate (NAADP) was recently identified [Lee and Aarhus (1995) J. Biol. Chem. 270, 2152-2157; Chini, Beers and Dousa (1995) J. Biol. Chem. 270, 3116-3223] as a potent Ca(2+)-releasing agent in sea urchin egg homogenates. NAADP triggered Ca2+ release by a mechanism that was distinct from inositol 1,4,5-trisphosphate (InsP3)- and cyclic ADP-ribose (cADPR)-induced Ca2+ release. When NAADP was microinjected into intact sea urchin eggs it induced a dose-dependent increase in cytoplasmic free Ca2+ which was independent of the extracellular [Ca2+]. The Ca2+ waves elicited by microinjections of NAADP originated at the site of injection and swept across the cytosol. As previously found in sea urchin egg homogenates, NAADP-induced Ca2+ release in intact eggs was not blocked by heparin or by prior desensitization to InsP3 or cADPR. Thio-NADP, a specific inhibitor of the NAADP-induced Ca2+ release in sea urchin homogenates [Chini, Beers and Dousa (1995) J. Biol. Chem. 270, 3116-3223] blocked NAADP (but not InsP3 or cADPR) injection-induced Ca2+ release in intact sea urchin eggs. Finally, fertilization of sea urchin eggs abrogated subsequent NAADP-induced Ca2+ release, suggesting that the NAADP-sensitive Ca2+ pool may participate in the fertilization response. This study demonstrates that NAADP acts as a selective Ca(2+)-releasing agonist in intact cells.

Cyclic ADP-ribose activates caffeine-sensitive calcium channels from sea urchin egg microsomes

1998 ◽  
Vol 274 (2) ◽  
pp. C430-C439 ◽  
Author(s):  
Claudio F. Pérez ◽  
Juan José Marengo ◽  
Ricardo Bull ◽  
Cecilia Hidalgo
Keyword(s):  
Sea Urchin ◽  
Microsomal Fraction ◽  
Ruthenium Red ◽  
Single Channels ◽  
Channel Activity ◽  
Sea Urchin Eggs ◽  
Sea Urchin Egg ◽  
Cyclic Adp Ribose ◽  
Inositol 1,4,5 Trisphosphate ◽  
Intracellular Ca

Adenosine 5′-cyclic diphosphoribose [cyclic ADP-ribose (cADPR)], a metabolite of NAD+ that promotes Ca2+ release from sea urchin egg homogenates and microsomal fractions, has been proposed to act as an endogenous agonist of Ca2+ release in sea urchin eggs. We describe experiments showing that a microsomal fraction isolated from Tetrapigus nyger sea urchin eggs displayed Ca2+-selective single channels with conductances of 155.0 ± 8.0 pS in asymmetric Cs+ solutions and 47.5 ± 1.1 pS in asymmetric Ca2+ solutions. These channels were sensitive to stimulation by Ca2+, ATP, and caffeine, but not inositol 1,4,5-trisphosphate, and were inhibited by ruthenium red. The channels were also activated by cADP-ribose in a Ca2+-dependent fashion. Calmodulin and Mg2+, but not heparin, modulated channel activity in the presence of cADP-ribose. We propose that these Ca2+ channels constitute the intracellular Ca2+-induced Ca2+ release pathway that is activated by cADP-ribose in sea urchin eggs.

scholarly journals Nicotinamide inhibits cyclic ADP-ribose-mediated calcium signalling in sea urchin eggs

1996 ◽  
Vol 319 (2) ◽  
pp. 613-617 ◽  
Author(s):  
Jaswinder K SETHI ◽  
Ruth M EMPSON ◽  
Antony GALIONE
Keyword(s):  
Sea Urchin ◽  
Calcium Release ◽  
Calcium Signalling ◽  
Hydrolytic Activity ◽  
Calcium Release Channels ◽  
Adp Ribosyl Cyclase ◽  
Sea Urchin Egg ◽  
Cyclic Adp Ribose ◽  
Single Polypeptide ◽  
Hydrolysis Of

Cyclic ADP ribose (cADPR) is a potent Ca2+-releasing agent, and putative second messenger, the endogenous levels of which are tightly regulated by synthetic (ADP-ribosyl cyclases) and degradative (cADPR hydrolase) enzymes. These enzymes have been characterized in a number of mammalian and invertebrate tissues and their activities are often found on a single polypeptide. β-NAD+, cGMP and nitric oxide (NO) have been reported to mobilize Ca2+ in the sea urchin egg via the cADPR-mediated pathway. We now report that in sea urchin egg homogenates, nicotinamide inhibits the Ca2+-mobilizing action of β-NAD+, cGMP and NO, but has no effect on cADPR-induced Ca2+ release. Moreover, nicotinamide inhibits cGMP-induced regenerative Ca2+ waves in the intact sea urchin egg. By successfully separating the cADPR-metabolizing machinery from that which releases Ca2+, we have shown that nicotinamide inhibits cADPR-mediated Ca2+ signalling at the level of cADPR generation. Importantly, nicotinamide had no effect upon the hydrolysis of cADPR, and its selective action on cyclase activity was supported by its inhibition of purified Aplysia ADP-ribosyl cyclase, which does not exhibit detectable hydrolytic activity. The action of nicotinamide in blocking Ca2+ release by β-NAD+, cGMP and NO strongly suggests that these agents act as modulators of cADPR synthesis rather than to sensitize calcium release channels to cADPR.

scholarly journals Comparison of Ca2+ mobilizing activities of cyclic ADP-ribose and inositol trisphosphate.

1990 ◽  
Vol 1 (3) ◽  
pp. 279-290 ◽  
Author(s):  
P J Dargie ◽  
M C Agre ◽  
H C Lee
Keyword(s):  
Sea Urchin ◽  
Inositol Trisphosphate ◽  
Specific Receptor ◽  
Receptor System ◽  
Sea Urchin Eggs ◽  
Sea Urchin Egg ◽  
Mammalian Tissues ◽  
Cyclic Adp Ribose ◽  
Half Maximal Effective Concentration

We have previously shown that a metabolite of NAD+ generated by an enzyme present in sea urchin eggs and mammalian tissues can mobilize intracellular Ca2+ in the eggs. Structural determination established it to be a cyclized ADP-ribose, and the name cyclic ADP-ribose (cADPR) has been proposed. In this study, Ca2+ mobilizations induced by cADPR and inositol trisphosphate (IP3) in sea urchin egg homogenates were monitored with Ca2+ indicators and Ca2(+)-specific electrodes. Both methods showed that cADPR can release Ca2+ from egg homogenates. Evidence indicated that it did not act as a nonspecific Ca2(+)-ionophore or as a blocker of the microsomal Ca2(+)-transport; instead, it was likely to be operating through a specific receptor system. This was supported by its half-maximal effective concentration of 18 nM, which was 7 times lower than that of IP3. The receptor for cADPR appeared to be different from that of IP3 because heparin, an inhibitor of IP3 binding, had no effect on the cADPR action. The Ca2+ releases induced by cADPR and IP3 were not additive and had an inverse relationship, indicating overlapping stores were mobilized. Microinjection of cADPR into intact eggs induced transient intracellular Ca2+ changes and activated the cortical reaction. The in vivo effectiveness of cADPR was directly comparable with IP3 and neither required external Ca2+. In addition, both were effective in activating the eggs to undergo multiple nuclear cycles and DNA synthesis. These results suggest that cADPR could function as a second messenger in sea urchin eggs.

scholarly journals Different Ca2+-releasing abilities of sperm extracts compared with tissue extracts and phospholipase C isoforms in sea urchin egg homogenate and mouse eggs

2000 ◽  
Vol 346 (3) ◽  
pp. 743-749 ◽  
Author(s):  
Keith T. JONES ◽  
Miho MATSUDA ◽  
John PARRINGTON ◽  
Matilda KATAN ◽  
Karl SWANN
Keyword(s):  
Phospholipase C ◽  
Sea Urchin ◽  
Tissue Extracts ◽  
Sea Urchin Egg ◽  
Boar Sperm ◽  
Specific Activities ◽  
Mammalian Eggs ◽  
Mouse Eggs

A soluble phospholipase C (PLC) from boar sperm generates InsP3 and hence causes Ca2+ release when added to sea urchin egg homogenate. This PLC activity is associated with the ability of sperm extracts to cause Ca2+ oscillations in mammalian eggs following fractionation. A sperm PLC may, therefore, be responsible for causing the observed Ca2+ oscillations at fertilization. In the present study we have further characterized this boar sperm PLC activity using sea urchin egg homogenate. Consistent with a sperm PLC acting on egg PtdIns(4,5)P2, the ability of sperm extracts to release Ca2+ was blocked by preincubation with the PLC inhibitor U73122 or by the addition of neomycin to the homogenate. The Ca2+-releasing activity was also detectable in sperm from other species and in whole testis extracts. However, activity was not observed in extracts from other tissues. Moreover recombinant PLCβ1, -γ1, -γ2, -∆1, all of which had higher specific activities than boar sperm extracts, were not able to release Ca2+ in the sea urchin egg homogenate. In addition these PLCs were not able to cause Ca2+ oscillations following microinjection into mouse eggs. These results imply that the sperm PLC possesses distinct properties that allow it to hydrolyse PtdIns(4,5)P2 in eggs.

scholarly journals Nicotinic acid-adenine dinucleotide phosphate mobilizes Ca2+ from a thapsigargin-insensitive pool

1996 ◽  
Vol 315 (3) ◽  
pp. 721-725 ◽  
Author(s):  
Armando A. GENAZZANI ◽  
Antony GALIONE
Keyword(s):  
Nicotinic Acid ◽  
Sea Urchin ◽  
Cross Talk ◽  
Inositol Trisphosphate ◽  
Sea Urchin Eggs ◽  
Bivalent Cations ◽  
Sea Urchin Egg ◽  
Different Characteristics ◽  
Degree Of Overlap

Nicotinic acid–adenine dinucleotide phosphate (NAADP) is a novel intracellular Ca2+ releasing agent recently described in sea-urchin eggs and egg homogenates. Ca2+ release by NAADP is independent of that induced by either inositol trisphosphate (InsP3) or cyclic adenosine dinucleotide phosphate (cADPR). We now report that in sea urchin egg homogenates, NAADP releases Ca2+ from a Ca2+ pool that is distinct from those that are sensitive to InsP3 and cADPR. This organelle has distinct Ca2+ uptake characteristics: it is insensitive to thapsigargin and cyclopiazoic acid, but maintenance of the pool shows some requirement for ATP. Although the different Ca2+ pools have different characteristics, there appears to be some degree of overlap or cross-talk between the NAADP- and cADPR/InsP3-sensitive Ca2+ pools. Ca2+-induced Ca2+ release is unlikely to account for the apparent overlap between stores, since NAADP-induced Ca2+ release, in contrast with that stimulated by cADPR, is not potentiated by bivalent cations.

Ryanodine Activates Sea Urchin Eggs. (sea urchin egg/activation/ryanodine/inositol phosphate/heparin)

1992 ◽  
Vol 34 (1) ◽  
pp. 37-42 ◽  
Author(s):  
C. Sardet ◽  
I. Gillot ◽  
A. Ruscher ◽  
P. Payan ◽  
J.-P. Girard ◽  
...  
Keyword(s):  
Sea Urchin ◽  
Inositol Phosphate ◽  
Egg Activation ◽  
Sea Urchin Eggs ◽  
Sea Urchin Egg

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.

Sign in / Sign up

Export Citation Format

Share Document