Na(+)-K(+)-ATPase-G protein coupling in myocardial sarcolemma: separation and reconstitution.
The cholinergic agonist carbachol produces a concentration-dependent (half-maximum inhibitory concentration = 0.9 microM) decrease in the Na(+)-K(+)-adenosine triphosphatase (ATPase) activity of rabbit cardiac sarcolemma that occurred only in the presence of guanosine 5'-[gamma-thio]triphosphate (0.1 microM GTP gamma S) and reached 40% inhibition. The inhibition is blocked by the muscarinic receptor antagonist atropine (10 microM) and is abolished in sarcolemma treated with pertussis toxin (20 micrograms/ml) in the presence of 100 microM NAD. GTP gamma S alone reduces Na(+)-K(+)-ATPase activity by 45% (half-maximum inhibitory = 1 microM). The apparent affinity of the enzyme for GTP gamma S is increased approximately 10-fold in the presence of 1 microM carbachol. In sarcolemma solubilized with the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS, 10 mM), the GTP gamma S-dependent inhibition of the Na(+)-K(+)-ATPase is also observed. Gel filtration of a CHAPS extract of sarcolemma on a Sepharose CL-6B column resulted in a separation of Na(+)-K(+)-ATPase and pertussis toxin-sensitive Gi activities. Na(+)-K(+)-ATPase activity that was separated on the column lost its sensitivity to the inhibitory action of guanine nucleotides. Inhibitory effects (20-30%) of guanosine 5'-triphosphate analogues [Gpp(NH)p, GTP gamma S, or Gpp(CH2)p] at micromolar concentrations were restored when the Na(+)-K(+)-ATPase activity was recombined with fractions that contained the pertussis toxin-sensitive Gi protein(s). Similar concentrations of guanosine 5'-triphosphate, guanosine 5'-diphosphate, guanosine-5'-[beta-thio]diphosphate, or App(NH)p were unable to induce the Gi protein-mediated attenuation of Na(+)-K(+)-ATPase activity in the reconstitution system.(ABSTRACT TRUNCATED AT 250 WORDS)
Danilenko
M P
MP
Laboratory of Molecular Endocrinology, USSR Cardiology Research Center, Moscow.
Turmukhambetova
V C
VC
Yesirev
O V
OV
Tkachuk
V A
VA
Panchenko
M P
MP
D000818:Animals; D002217:Carbachol / Q000494:pharmacology; D002793:Cholic Acids / Q000494:pharmacology; D002850:Chromatography, Gel; D019204:GTP-Binding Proteins / Q000032:analysis / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000494:pharmacology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D009206:Myocardium / Q000378:metabolism*; D009994:Osmolar Concentration; D010042:Ouabain / Q000494:pharmacology; D011817:Rabbits; D012508:Sarcolemma / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012995:Solubility
D002793:Cholic Acids; D006150:Guanine Nucleotides; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D010042:Ouabain; D002217:Carbachol; D019204:GTP-Binding Proteins; D000254:Sodium-Potassium-Exchanging ATPase; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate