Abstract

Both the catalytic protein (C) and the guanine nucleotide-binding regulatory protein (G/F) of rabbit hepatic adenylate cyclase were solubilized in active form in sodium cholate at high ionic strength. Upon precipitation by (NH4)SO4, C was selectively aggregated such that it was largely excluded from Ultrogel AcA 34 when chromatographed in cholate solution at high ionic strength. G/F eluted with KD congruent to 0.5. Thus C can be completely resolved from G/F by this procedure. Rabbit hepatic C prepared in this way resembles the C activity previously described by us in the cyc- mutant of S49 lymphoma cells (Ross, E. M., Howlett, A. C., Ferguson, K. M., and Gilman, A. G. (1978) J. Biol. Chem. 253, 6401-6412). Similarities include 7- to 10-fold stimulation by Mn2+ relative to Mg2+ and a complete lack of sensitivity to fluoride or guanine nucleotides. Reconstitution of hepatic C with hepatic G/F causes restoration of stimulation up to 25-fold by either of these activators in the presence of Mg2+. While C is relatively unstable in cholate under these conditions, especially at temperatures over 4 degrees C, it is stabilized by removal of cholate and by addition of phospholipid. These procedures allow the study of the catalytic protein of adenylate cyclase from a hormone-responsive tissue in the absence of endogenous regulatory protein or membrane lipid and also provide a starting point for the purification of the catalyst.

Highlights

  • RESULTS(NHI)IS01/15mM Na cholate (pH8) and stirred for 1 h

  • C so far has been only partially resolved from G/F in detergent extracts of pigeon erythrocyte membranes (4), and the only preparation of C which has beenmoderately well described is the plasma membrane of the S49 lymphoma cell eyc- mutant, which is genetically deficient in G/F activity (5,6)

  • Volume of Buffer A (10 mM Na Hepes/0.lCt (v/v) 2-mercap- stimulated adenylate cyclase activity in plasma membranes of cyc toethanol), and homogenized with four strokes a t low speed with a S49 lymphoma cells, which are deficient in G/F (5.6)

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Summary

RESULTS

(NHI)IS01/15mM Na cholate (pH8) and stirred for 1 h. The resuspended material cholate and 0.6 mM (NH,),SO,, about 90% of the Mn"-stimwas applied to a column of Ultrogel AcA34 equilibrated with 0.1 M ulated adenylatecyclase is solubilized. A comparable fraction of the Mg'+ plus GTP-y-S-stimulated activity is solubilized, butthis activity is nota good indication of theextent of solubilization of C This value can be increased up to 2-fold by addition of excess G/F anddoes not always dilute linearly. Protein was frequently diluted into buffer containing DMPC This essary to dilute the supernatanta total of at least 12-fold into thedetergent-free assaymixturein ordertomeasurethe correct activityr,eflecting the instability of cholate-solubilized.

Resolution of CataPlyrotitcein of Adenylate Cyclase
Assay conditions
Findings
DISCUSSION

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