Michael E. Maguire

Adenylate cyclase assay with adenylyl imidodiphosphate and product detection by competitive protein binding

Abstract An assay for adenylate cyclase (EC 4.6.1.1) is described using adenylyl imidodiphosphate (AMP-PNP) as substrate. Methods are described for introducing the cyclase reaction mixture directly into a protein binding assay for the cyclic nucleotide without purification of cyclic AMP. Significant substrate depletion and regenerating systems are avoided by this method, and blank values are negligible. The assay is capable of reproducibly detecting adenylate cyclase activity with less than 5 μg of protein from rat cerebral cortex.

Hormone receptors and the control of cyclic AMP metabolism in parental and hybrid somatic cells.

Publisher Summary The study of cyclic AMP (cAMP) metabolism and its regulation by hormones in clonal cultured cell systems offers advantages over investigations in more heterogeneous preparations. Cultured cell lines that display differentiated functions pertinent to the cAMP regulation are readily available. The conventional biochemical and pharmacological experimentation is facilitated because these clones are genetically homogenous. Furthermore, the opportunity exists to manipulate the genetic composition of such replicating cell lines. Thus, attempts are being made in several laboratories to dissect the cAMP system using hybrid somatic cells and the selection of mutant or variant cell clones with specific alterations in the response pathway. This chapter reviews the study of effects of β -adrenergic agents and prostaglandins on cAMP synthesis in parental cell clones and hybrid cells formed by fusion of appropriate parental strains. It defines the relationship between receptors for these agents and adenylate cyclase. The chapter also analyzes responses of hybrid cells to catecholamines and PGE 1 , the β -adrenergic receptor, effects of guanine nucleotides on binding to β -adrenergic receptors, PGE 1 binding activity, and receptor binding in cell lines lacking adenylate cyclase activity.

Loss of cytosolic Mg2+ binding sites in the Thermotoga maritima CorA Mg2+ channel is not sufficient for channel opening

The CorA Mg2+ channel is a homopentamer with five-fold symmetry. Each monomer consists of a large cytoplasmic domain and two transmembrane helices connected via a short periplasmic loop. In the Thermotoga maritima CorA crystal structure, a Mg2+ is bound between D89 of one monomer and D253 of the adjacent monomer (M1 binding site). Release of Mg2+ from these sites has been hypothesized to cause opening of the channel. We generated mutants to disrupt Mg2+ interaction with the M1 site. Crystal structures of the D89K/D253K and D89R/D253R mutants, determined to 3.05 and 3.3 Å, respectively, showed no significant structural differences with the wild type structure despite absence of Mg2+ at the M1 sites. Both mutants still appear to be in the closed state. All three mutant CorA proteins exhibited transport of 63Ni2+, indicating functionality. Thus, absence of Mg2+ from the M1 sites neither causes channel opening nor prevents function. We also provide evidence that the T. maritima CorA is a Mg2+ channel and not a Co2+ channel.