Marvin I. Siegel

Phospholipase C and phospholipase D are activated independently of each other in chemotactic peptide-stimulated human neutrophils.

When cytochalasin B‐treated neutrophils were stimulated with fMet‐Leu‐Phe (fMLP) in the presence of Ca2+, phospholipase C (PLC) activity, as measured by inositol‐1,4,5‐trisphosphate (IP3) formation, preceded phospholipase D (PLD)‐catalyzed breakdown of choline‐ containing phosphoglycerides to form choline and dirady 1‐sn ‐glycero‐3‐phosphate (phosphatidic acid), suggesting a possible link between PLC and PLD. However, in the absence of cytochalasin B or extracellular Ca2+, PLC was fully activated by fMLP with minimal activation of PLD, indicating that PLC activation alone is not sufficient for PLD activation. Full activation of PLD by fMLP required the simultaneous presence of both Ca2+ and cytochalasin B, a condition that caused no further enhancement of PLC. This result suggests that PLD products are not involved in the regulation of PLC activation. Furthermore, under conditions of complete inhibition of PLC by phorbol 12‐myristate 13‐acetate (PMA), there was no inhibition of PLD, showing that fMLP can activate PLD in the absence of PLC. Treatment of intact neutrophils with pertussis toxin inhibited both PLC and PLD, with PLC inhibition occurring at lower concentrations than PLD inhibition. These differential effects of pertussis toxin and the observed lack of inhibition of fMLP‐stimulated PLD by PMA, which is believed to inactivate G‐proteins involved in PLC activation, imply that PLC and PLD are linked to fMLP receptors through distinct G‐proteins. Taken together, these observations suggests that, in fMLP‐stimulated neutrophils, PLC and PLD are activated through independent mechanisms.

Interaction of ribulose diphosphate carboxylase with 2-carboxyribitol diphosphate, an analogue of the proposed carboxylated intermediate in the CO2 fixation reaction

Abstract Support for the participation of a six-carbon carboxylated intermediate (or transition state) in the D-ribulose-1,5 diphosphate carboxylase-catalyzed reaction was derived from kinetic and binding studies with 2-carboxy-D-ribitol-1,5-diphosphate (CRDP), an analog of the proposed intermediate and potent inhibitor of the reaction. Evidence is presented that following the formation of a carboxylase·Mg·CRDP complex, a slow temperature-dependent conformational change occurs locking the enzyme in an inhibited state from which release of the inhibitor is extremely slow.

Epinephrine stimulation of fat cell adenylate cyclase: Regulation by guanosine-5′-triphosphate and magnesium ion

Abstract Guanosine-5′-triphosphate (GTP) affects the activity and responsiveness of the fat cell adenylate cyclase to epinephrine. In the absence of free magnesium ion, the presence of GTP is an absolute requirement for epinephrine stimulation. Half-maximal activation of both basal and stimulated adenylate cyclase activity occurs at a GTP concentration of 0.6 mM. In the presence of 5 mM MgCl 2 , GTP is no longer required but enhances epinephrine stimulation. The half-maximal concentration for this effect occurs at approximately 10 μM GTP. At high (5 mM) magnesium ion concentrations GTP inhibits basal adenylate cyclase activity. GTP lowers the apparent affinity of adenylate cyclase for ATP while simultaneously increasing the velocity of the catalytic reaction, possibly by competing with ATP at the active site as well as by binding at a regulatory site. This effect is observed on both basal and epinephrine-stimulated activities. Epinephrine itself raises the apparent K M for ATP and the V max of adenylate cyclase. The interdependence of these effects suggests that transient changes in the levels of GTP, ATP, and magnesium ions in the fat cell may directly regulate the responsiveness of adenylate cyclase to epinephrine.

Guanylate cyclase. Existence of different forms and their regulation by nucleotides in calf uterus

The activity of calf uterus guanylate cyclase (EC 4.6.1.2) exists in at least two and most probably three distinct forms. The cytosolic enzyme exhibits hyperbolic substrate curves with respect to GTP and Mn2+, while the particulate cyclases (nuclear and microsomal)display sigmoidal (GTP) and hyperbolic (Mn2+) relationships. The Hill coefficient for the GTP dependence is 0.9 for the cytosolic, 1.5 for the nuclear, and 1.4 for the microsomal enzyme. The cytosolic enzyme has a Km for GTP of 70 muM while half maximal velocity occurs at 90 and 100 muM GTP for the nuclear and microsomal enzymes, respectively. The Ka for Mn2+ is 0.57, 0.71 or 0.75 mM for the cytosolic, nuclear, or microsomal enzyme, respectively.

Characterization of a digitonin-solubilized bovine brain H3 histamine receptor coupled to a guanine nucleotide-binding protein.

Abstract: The H3 receptor is a high‐affinity histamine receptor that inhibits release of several neurotransmitters, including histamine. We have characterized H3 receptor binding in bovine brain and developed conditions for its solubilization. Particulate [3H]histamine binding showed an apparently single class of sites (KD= 4.6 nM; Bmax= 78 fmol/mg of protein). Of the detergents tested, digitonin at a detergent/protein ratio of 1:1 (wt/wt) yielded the greatest amount of solubilized receptors, typically 15–30% of particulate binding. Neither equilibrium binding of [3H]histamine to receptors (KD= 6.1 nM; Bmax= 92 fmol/mg of protein) nor the inhibitor profile was substantially altered by digitonin solubilization. However, solubilization did increase the rate of [3H]histamine association with and dissociation from the receptor. Size‐exclusion chromatography indicated an apparent molecular weight of 220,000 for the solubilized receptor, and peak binding from this column retained its guanine nucleotide sensitivity. These last two observations are consistent with the solubilized receptor occurring in complex with a guanine nucleotide‐binding protein.

5 Ribulose-1,5-Diphosphate Carboxylase

Publisher Summary This chapter discusses the molecular properties and catalytic process of ribulose-1,5-diphosphate carboxylase. The identification by Calvin and his colleagues of phosphoglyceric acid as the first stable radioactive product formed during a brief exposure of algae to 14 CO 2 led ultimately to the discovery that the carboxylation of ribulose diphosphate is the initial step in the photosynthetic carbon cycle. It was observed that the ribulose diphosphate became labeled early and that illumination caused a decrease in its steady state concentration and a concomitant rise in the concentration of 3-phosphoglycerate. When the supply of CO 2 was cut off, the ribulose diphosphate level increased and the level of 3-phosphoglycerate decreased correspondingly. Ribulose diphosphate carboxylases from higher plants have complex quaternary structures as evidenced by their appearance in the electron microscope, high molecular weights, and numbers of substrate, cofactor, inhibitor, and antibody binding sites. The amino acid compositions of several ribulose diphosphate carboxylases have been reported. The molecular activity of the homogeneous spinach ribulose diphosphate carboxylase, which is typical of most of the plant carboxylases, is 1340 moles of ribulose diphosphate carboxylated per minute per mole of enzyme at pH 7.9. Ribulose diphosphate carboxylase from spinach appears to be absolutely specific for D -ribulose 1,5-diphosphate.