Effects of guanine nucleotides and Mg on human erythrocyte Ni and Ns, the regulatory components of adenylyl cyclase.
The effect of GTP analogs and Mg on the structure of Ns and Ni, the stimulatory and inhibitory regulatory components of adenylyl cyclase, were studied in a comparative manner. Both N proteins, which are alpha beta gamma heterotrimers that differ in their alpha subunits, when exposed to GTP analogs underwent a Mg-dependent conformational change that was not dependent on subunit dissociation. This was seen both as change in sedimentation behavior at 4 degrees C from 4 S to about 3 S and by a property of the new conformation to retain guanine nucleotide tightly bound to it. Warming to 32 degrees C promoted subunit dissociation, each protein giving a mixture of 2 S alpha G and 2 S beta gamma complexes. For both Ns and Ni, these reactions were reversible: 2 S complexes of Ns and Ni associated to 3 S forms on cooling to 4 degrees C, provided the Mg concentration was at or below 10 mM and detergent concentration was below 1%, and the 3 S complexes of these proteins reverted to 4 S forms and released the nucleotide in the cold on chelation of free Mg with EDTA. Reconstitution assays with Ns-deficient membranes from cyc- S49 lymphoma cells revealed that the 3 S form of Ns is a "pre-active" form of the protein. The scheme below summarizes these findings, where G represents a guanine nucleotide. (Formula: see text) Ns and Ni differ in that more Mg is necessary to promote the 4 S to 3 S conversion of Ns than of Ni, and in that both the 2 S to 3 S to 4 S conversions proceeded more readily with Ni than with of Ns. Mg could not be shown to promote subunit dissociation. The above scheme is suggested as a plausible description of the reaction sequence leading from an unactivated to an activated N protein.
D000262:Adenylyl Cyclases / Q000097:blood*; D002499:Centrifugation, Density Gradient; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D006150:Guanine Nucleotides / Q000494:pharmacology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D006801:Humans; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology*; D013696:Temperature; D013873:Thionucleotides / Q000494:pharmacology; D013997:Time Factors
D006150:Guanine Nucleotides; D046911:Macromolecular Substances; D013873:Thionucleotides; D006165:Guanylyl Imidodiphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D000262:Adenylyl Cyclases; D008274:Magnesium