18O-Labeling of guanosine monophosphate upon hydrolysis of cyclic guanosine 3':5'-monophosphate by phosphodiesterase

The hydrolysis of cGMP by phosphodiesterase was conducted in [18O]water to determine the site of bond cleavage and the stoichiometry of 18O incorporation into 5'-GMP. Three different forms of phosphodiesterase including a calmodulin-calcium-dependent enzyme in its basal and activated states were examined. The hydrolysis of cGMP catalyzed by each of the forms of phosphodiesterase proceeded with incorporation of 1 18O atom recoverable in the phosphate moiety of each molecule of 5'-GMP generated. No molecular species of phosphate deriving from the 5'-GMP generated containing two or three 18O were detectable. These results indicate that the phosphodiesterase-catalyzed hydrolysis of cGMP proceeds by nucleophilic substitution at phosphorus resulting in P-O bond cleavage. The stoichiometry of 18O incorporation indicates that the reaction proceeds without phosphate-water oxygen exchange when the hydrolytic reaction is catalyzed by diverse forms of phosphodiesterase in the basal or activated state. These considerations of the phosphodiesterase reaction help to establish the validity of monitoring the rate of enzyme-catalyzed hydrolysis of cGMP as a function of the rate of 18O-labeling of the phosphate of 5'-GMP when the reaction proceeds in a medium of predetermined 18O enrichment.     

Magnesium effects on activation of skinned fibers from striated muscle

The intracellular Ca movements that control contraction and relaxation of striated muscle are regulated by the membrane potential and influenced by Mg2+. In skinned fibers, the internal composition can be manipulated directly by Ca movements estimated from isometric force transients, net changes in sarcoplasmic reticulum (SR) Ca, and 45Ca flux between fiber and bath. Stimulated Ca release, unlike unstimulated 45Ca efflux at low external [Ca2+], is highly [Mg2+]-sensitive at 20 C. Force and tracer measurements indicate three major sites of Mg2+-Ca2+ interaction in situ: Mg2+ can stimulate the SR active Ca transport system, inhibit a Ca2+-dependent Ca efflux pathway of SR, and shift the force-[Ca2+] relation, presumably by reducing Ca2+ binding to myofilament regulatory sites. These mechanisms constrain the resting Ca flux and are adaptive during relaxation. However, analysis of CI-stimulated 45Ca release and reaccumulation suggests that the depolarization process may inhibit Mg2+-dependent Ca influx, the membrane potential controlling both efflux and influx; recent studies on voltage-clamped cut fibers support this hypothesis. The Ca2+ and Mg2+ dependence of caffeine-stimulated 45Ca efflux suggests that Mg2+ inhibition of the Ca2+-dependent efflux pathway is small during rapid Ca2+ efflux. Therefore, both Mg2+ mechanisms, which minimize net release, may be reversed during normal activation.     

Separate Intramolecular Targets for Protein Kinase A Control N-Methyl-d-aspartate Receptor Gating and Ca2+ Permeability

Protein kinase A (PKA) enhances synaptic plasticity in the central nervous system by increasing NMDA receptor current amplitude and Ca²⁺ flux in an isoform-dependent yet poorly understood manner. PKA phosphorylates multiple residues on GluN1, GluN2A, and GluN2B subunits in vivo, but the functional significance of this multiplicity is unknown. We examined gating and permeation properties of recombinant NMDA receptor isoforms and of receptors with altered C-terminal domain (CTDs) prior to and after pharmacological inhibition of PKA. We found that PKA inhibition decreased GluN1/GluN2B but not GluN1/GluN2A gating; this effect was due to slower rates for receptor activation and resensitization and was mediated exclusively by the GluN2B CTD. In contrast, PKA inhibition reduced NMDA receptor-relative Ca²⁺ permeability (P_Ca/P_Na) regardless of the GluN2 isoform and required the GluN1 CTD; this effect was due primarily to decreased unitary Ca²⁺ conductance, because neither Na⁺ conductance nor Ca²⁺-dependent block was altered substantially. Finally, we show that both the gating and permeation effects can be reproduced by changing the phosphorylation state of a single residue: GluN2B Ser-1166 and GluN1 Ser-897, respectively. We conclude that PKA effects on NMDA receptor gating and Ca²⁺ permeability rely on distinct phosphorylation sites located on the CTD of GluN2B and GluN1 subunits. This separate control of NMDA receptor properties by PKA may account for the specific effects of PKA on plasticity during synaptic development and may lead to drugs targeted to alter NMDA receptor gating or Ca²⁺ permeability.     

mit-Mutations in the structural gene of subunit III of cytochrome oxidase in Saccharomyces cerevisiae

Two-dimensional electrophoretic analysis of the mitochondrial translation products of four mit-mutants indicate that subunit III of cytochrome oxidase is the only mitochondrial translation product affected by mutations in the oxi2 region of the mtDNA. Mitochondria of two of these mutants synthesize new products which coprecipitate with an anticytochrome oxidase antiserum and produce proteolytic digests similar to those of subunit III of the enzyme complex. These data strongly support the suggestion that the oxi2 region of the yeast mtDNA contains the structural gene of subunit III of cytochrome oxidase.