Occurrence of an Inhibitory Guanine Nucleotide-Binding Regulatory Component of the Adenylate Cyclase System in cyc Variants of S49 Lymphoma Cells

Authors

  • Karl H. JAKOBS,

    1. Pharmakologisches Institut der Ruprecht-Karl-Universität Heidelberg, Im Neuenheimer Feld 366, D-6900 Heidelberg 1, Federal Republic of Germany
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  • Ulrich GEHRING,

    1. Institut für Biologische Chemie der Ruprecht-Karl-Universität Heidelberg, Im Neuenheimer Feld 501, D-6900 Heidelberg 1, Federal Republic of Germany
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  • Bernhard GAUGLER,

    1. Physiologisch-Chemisches Institut der Julius-Maximilians-Universität Würzburg, Koellikerstraße 2, D-8700 Würzburg, Federal Republic of Germany
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  • Thomas PFEUFFER,

    1. Physiologisch-Chemisches Institut der Julius-Maximilians-Universität Würzburg, Koellikerstraße 2, D-8700 Würzburg, Federal Republic of Germany
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  • Günter SCHULTZ

    1. Pharmakologisches Institut der Ruprecht-Karl-Universität Heidelberg, Im Neuenheimer Feld 366, D-6900 Heidelberg 1, Federal Republic of Germany
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Abstract

In membranes of S49 lymphoma cyc variants, which lack a functional guanine nucleotide-binding component (Ns protein) mediating adenylate cyclase stimulation by hormones, guanine nucleotides decreased the forskolin-stimulated adenylate cyclase activity by maximally 40–60%. The potency order of the guanine nucleotides studied was guanosine 5′-[γ-thio]triphosphate(GTP[S])> guanosine 5′[β,γ-imido]triphosphate ≫ GTP > guanosine 5′-[β-thio]diphosphate (GDP[S]). GTP and GDP[S] acted as partial inhibitors; they competitively antagonized the GTP[S]-induced inhibition, which was half-maximal and maximal at about 3 nM and 100 nM GTP[S], respectively. Cholera toxin did not enhance the inhibitory action of GTP. The cyc adenylate cyclase inhibition by GTP[S] occurred after a short lag period and persisted after washing the membranes. The inhibition was not affected by the pH (6.5–9.0) of the incubation medium and was largely independent of the concentrations of MgATP (up to 200 μM) and Mg2+ (up to 10 mM). In contrast, Mn2+ potently reduced the GTP[S]-induced cyc adenylate cyclase inhibition. Similarly as observed with forskolin, GTP[S] decreased the cyc adenylate cyclase activity stimulated by purified, preactivated Ns protein. Apart from Mn2+, inhibition of the cyc adenylate cyclase by GTP[S] was prevented or reversed by various treatments, which have been shown to obliterate hormone-induced adenylate cyclase inhibition in other cell types, such as by N-ethylmaleimide, by limited proteolysis with trypsin and by a factor extracted from bovine sperm particles. The data indicate that in membranes of cyc variants, which lack a functional Ns protein, a guanine nucleotide-binding component (Ni) is present, which mediates adenylate cyclase inhibition by guanine nucleotides. Such a component apparently also mediates adenylate cyclase inhibition by hormones in other cell types. Comparison of adenylate cyclase stimulation and inhibition by guanine nucleotides suggests that Ni is activated and inactivated by similar but not identical mechanisms as the Ns protein and that the relative activity states of these coupling components determine the activity of the adenylate cyclase.

Abbreviations
cyc

variants of S49 lymphoma cells, which lack a functional stimulatory guanine nucleotide-binding regulatory component of the adenylate cyclase system

Ns or Ns protein

guanine nucleotide-binding regulatory protein mediating adenylate cyclase stimulation by hormones and guanine nucleotides

Ni

guanine nucleotide-binding regulatory component mediating adenylate cyclase inhibition by hormones and guanine nucleotides

cAMP or cyclic AMP

adenosine 3′,5′-monophosphate

GTP[S]

guanosine 5′-[γ-thio]triphosphate

GuoPP[NH]P

guanosine 5′-[β,γ-imido]triphosphate

GDP[S]

guanosine 5′-[β-thio]diphosphate

Hepes

4-(2-hydroxyethyl)-1-piperazineethanesulfonate

Enzymes
 

Adenylate cyclase (EC 4.6.1.1)

 

creatine kinase (EC 2.7.3.2)

 

trypsin (EC 3.4.21.4)

 

GTPase (EC 3.6.1.-)

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