Regulation of agonist-receptor binding by G proteins and divalent cations in spermatozoa solubilized A1 adenosine receptors.
Solubilized A1 adenosine receptor (A1AR) was used to investigate the effect of several cations on agonist-binding characteristics and GTP hydrolysis. It was shown by Western blot with G beta-M14 that this preparation contains both G proteins and receptor. The role of the receptor molecule is to facilitate the activation of G proteins as alpha-GTP complex, and GTP hydrolysis has important consequences for the basic deactivation mechanism. Divalent cations, such as Mn2+, Ca2+, and Mg2+, potentiated the agonist-specific binding: Mn2+ had the highest apparent affinity with half-maximal effect at 50 microM. Binding assays, performed in the presence of 100 microM Mn2+, showed an increase in the apparent affinity of the binding sites, whereas, in the presence of 1 mM Mg2+, significant alteration of the apparent affinity, but not of the number of sites, was detected. Concentrations of 1 mM Mg2+ and 100 microM Mn2+ enhanced GTPase activity, whereas 5 mM Ca2+ resulted in the increase of Vmax values without significant alterations of K(m). In the presence of A1-specific agonists, Mn2+ and Mg2+ caused a decrease of Vmax values and an increase of GTP affinity. Other cations, such as Co2+, Cd2+, Cu2+, and Zn2+, inhibited the binding capacity but caused almost no changes in GTP hydrolysis kinetics.
Minelli
A
A
Dipartimento di Biologia Cellulare e Molecolare, Università di Perugia, Italia.
Allegrucci
C
C
Rosati
R
R
Mezzasoma
I
I
D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002413:Cations, Divalent / Q000378:metabolism* / Q000494:pharmacology; D002499:Centrifugation, Density Gradient; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism / Q000494:pharmacology; D008297:Male; D008345:Manganese / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding; D058906:Purinergic P1 Receptor Agonists; D018047:Receptors, Purinergic P1 / Q000378:metabolism*; D013094:Spermatozoa / Q000378:metabolism*
D002413:Cations, Divalent; D058906:Purinergic P1 Receptor Agonists; D018047:Receptors, Purinergic P1; D008345:Manganese; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D008274:Magnesium; D002118:Calcium