Advances in knowledge of magnesium physiology and biochemistry indicate that Mg2+ may be an intracellular regulatory ligand. Mg2+ activates or inhibits numerous important enzyme systems at specific Mg2+ binding sites. Mg2+ transport systems are regulated by a variety of hormones in a manner that will cause changes in intracellular Mg2+ content and probably free concentration within specific compartments. In addition, Mg2+ deficiency results alters the regulation of at least one Mg2+- regulated system, adenylate cyclase. To further study the role of intracellular Mg2+, this proposal will focus three aspects of Mg2+ biochemistry in eukaryotic cells. First, the cellular regulation of Mg2+ influx will be studied. Protein kinase C, cyclic AMP-dependent protein kinase and a third pathway via the beta-adrenergic receptor but independent of cyclic AMP are known to modulate the rate of Mg2+ influx and possibly its intracellular compartmentation. The interaction of these three pathways and the mechanism by which they alter Mg2+ influx will be studied in our model system, the murine S49 lymphoma cell These studies will allow insight into the potential role(s) of hormonal regulation of Mg2+ transport and intracellular Mg2+. Second, to gain further insight into Mg2+'s potential intracellular role(s), the effect(s) of Mg2+ deficiency on cell response to hormone will be studied. Clonal lines of the S49 lymphoma cell able to grow in 30 muM Mg2+ versus a normal Mg2+ concentration of 800 muM have been isolated; they exhibit decreased cell Mg2+ content and compromised cyclic AMP synthesis in response several agonists. We will investigate which component(s) of the receptor-cyclase-kinase pathway may be defective in Mg2+ deficiency and whether other abnormal cell responses are present. Finally, the only currently available tool for study of Mg2+ transport is the very short-lived isotope, 28Mg, thus placing severe limitations on the experimental approaches available for study of the actual mechanism of Mg2+ transport. Our development of a strain of the bacterium Salmonella typhimurium in which all genes for Mg2+ transport have been deleted provides an opportunity to develop an important additional tool to study the mechanism of Mg2+ transport, namely the gene(s) for a Mg2+ transport protein. We will screen a yeast genomic library in the S. typhimurium strain to identify a eukaryotic gene(s) for Mg2+ transport by complementation and thus allowing an eventual approach to study of the mechanism of Mg2+ transport.
Showing the most recent 10 out of 11 publications
