Ectoenzymatic breakdown of diadenosine polyphosphates by Xenopus laevis oocytes.
Xenopus laevis oocytes exhibit ectoenzymatic activity able to hydrolytically cleave extracellular diadenosine polyphosphates (Ap(n)A). The basic properties of this ectoenzyme were investigated using as substrates di-(1,N(6)-ethenoadenosine) 5',5"'-P(1),P(4)-tetraphospate [epsilon-(Ap(4)A)] and di-(1,N(6)-ethenoadenosine) 5',5"'-P(1),P(5)-pentaphospate [epsilon-(Ap(5)A)], fluorogenic derivatives of Ap(4)A and Ap(5)A, respectively. epsilon-(Ap(4)A) and epsilon-(Ap(5)A) are hydrolysed by folliculated oocytes according to hyperbolic kinetics with K(m) values of 13.4 and 12.0 microM and Vmax values of 4.8 and 5.5 pmol per oocyte per min, respectively. The ectoenzyme is activated by Ca(2+) and Mg(2+), reaches maximal activity at pH 8--9 and is inhibited by suramin. Defolliculated oocytes also hydrolyse both substrates with similar K(m) values but V(max) values are approximately doubled with respect to folliculated controls. Chromatographic analysis indicates that extracellular epsilon-(Ap(4)A) and epsilon-(Ap(5)A) are first cleaved into 1,N(6)-ethenoAMP (epsilon-AMP) + 1,N(6)-ethenoATP (epsilon-ATP) and epsilon-AMP + 1,N(6)-ethenoadenosine tetraphosphate (epsilon-Ap(4)), respectively, which are catabolized to 1,N(6)-ethenoadenosine (epsilon-Ado) as the end product by folliculated oocytes. Denuded oocytes, however, show a drastically reduced rate of epsilon-Ado production, epsilon-AMP being the main end-product of extracellular epsilon-(Ap(n)A) catabolism. Results indicate that, whereas the Ap(n)A-cleaving ectoenzyme appears to be located mainly in the oocyte, ectoenzymes involved in the dephosphorylation of mononucleotide moieties are located mainly in the follicular cell layer.
Department of Neurobiology and Behavior, University of California, Irvine, USA.
J S
JS
Aguilar
R
R
Reyes
A C
AC
Asensio
S
S
Oaknin
P
P
Rotllán
R
R
Miledi
D000241:Adenosine / Q000031:analogs & derivatives / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002413:Cations, Divalent / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D015226:Dinucleoside Phosphates / Q000378:metabolism*; D005453:Fluorescence; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis / Q000187:drug effects; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009865:Oocytes / Q000166:cytology / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D013498:Suramin / Q000494:pharmacology; D014982:Xenopus laevis / Q000378:metabolism*
D002413:Cations, Divalent; D015226:Dinucleoside Phosphates; D013498:Suramin; D008274:Magnesium; D000241:Adenosine; D002118:Calcium