Presence Of Guanosine Triphosphate Research Articles

Divalent cations regulate glucagon binding. Evidence for actions on receptor-Ns complexes and on receptors uncoupled from Ns.

The effects of Mg2+ or ethylenediaminetetraacetic acid (EDTA) on 125I-glucagon binding to rat liver plasma membranes have been characterized. In the absence of guanosine 5'-triphosphate (GTP), maximal binding of 125I-glucagon occurs in the absence of added Mg2+. Addition of EDTA or Mg2+ diminishes binding in a dose-dependent manner. In the presence of GTP, maximal binding occurs in the presence of 2.5 mM Mg2+ (EC50 = 0.3 mM) while EDTA or higher concentrations of Mg2+ diminish binding. Response to exogenous Mg2+ or EDTA depends on the concentration of Mg2+ in the membranes and may vary with the method used for membrane isolation. Solubilized 125I-glucagon-receptor complexes fractionate on gel filtration columns as high molecular weight, GTP-sensitive complexes in which receptors are coupled to regulatory proteins and lower molecular weight, GTP-insensitive complexes in which receptors are not coupled to other components of the adenylyl cyclase system. In the absence of GTP, 40 mM Mg2+ or 5 mM EDTA diminishes receptor affinity for hormone (from KD = 1.2 +/- 0.1 nM to KD = 2.6 +/- 0.3 nM) and the fraction of 125I-glucagon in high molecular weight receptor-Ns complexes without affecting site number (Bmax = 1.8 +/- 0.1 pmol/mg of protein). Thus, while GTP promotes disaggregation of receptor-Ns complexes, Mg2+ or EDTA diminishes the affinity with which these species bind hormone. In the presence of GTP, hormone binds to lower affinity (KD = 9.0 +/- 3.0 nM), low molecular weight receptors uncoupled from Ns.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylate cyclase stimulation by VIP in rat and human parotid membranes

Adenylate cyclase activity was stimulated by vasoactive intestinal peptide (VIP) in rat parotid membranes, in the presence of 100 μM guanosine triphosphate (GTP). The threshold concentration of VIP was 300 nM and the activity doubled at the maximal VIP concentration tested (30 μM). The relative potency of peptides of the VIP family was: VIP > peptide histidine isoleucinamide (PHI) > secretin. The β-adrenergic agent isoproterenol was a more efficient activator of rat parotid adenylate cyclase and its stimulatory effect, like that of VIP, depended on the presence of GTP. The effects of VIP and isoproterenol were both potentiated by 10 μM forskolin. By comparison with rat parotid preparations, membranes from a human parotid gland responded similarly to the VIP family of peptides (VIP > PHI > secretin). In both rat and human parotid membranes, two proteins (M r 44 kDa and 53 kDa) of the α-subunit of Ns (the guanyl nucleotide-binding stimulatory protein) were labelled by ADP-ribosylation, in the presence of cholera toxin. Taken together, these results indicate that VIP receptors, when coupled to Ns, were able to activate the adenylate cyclase system in rat and human parotid membranes.

Identification of a guanosine triphosphate-binding site on guinea pig liver transglutaminase. Role of GTP and calcium ions in modulating activity.

Guanosine 5'-triphosphate (GTP) was found to inhibit guinea pig liver transglutaminase activity as measured by [3H]putrescine incorporation into casein. GDP and GTP-gamma-S also inhibited enzyme activity (GTP-gamma-S greater than GTP greater than GDP). Kinetic studies showed that GTP acted as a reversible, noncompetitive inhibitor and that CaCl2 partially reversed GTP inhibition. GTP also inhibited rat liver and adult bovine aortic endothelial cell transglutaminase, but did not inhibit Factor XIIIa activity. Guanosine monophosphate (GMP), cyclic GMP, and polyguanylic acid did not inhibit enzyme activity. Guinea pig liver transglutaminase adsorbed well to GTP-agarose affinity columns, but not to CTP-agarose columns, and the binding was inhibited by the presence of calcium ions. Specific binding of GTP to transglutaminase was demonstrated by photoaffinity labeling with 8-azidoguanosine 5'-[gamma-32P] triphosphate, which was inhibited by the presence of GTP or CaCl2. GTP inhibited trypsin proteolysis of guinea pig liver transglutaminase without affecting the trypsin proteolysis of chromogenic substrates. Proteolytic protection was reversed by the addition of calcium. This study demonstrates that GTP binds to transglutaminase and that both GTP and calcium ions function in concert to regulate transglutaminase structure and function.

Effect of sonication on nucleotide-dependent light scattering changes in retinal rod outer segment suspensions

Near-infrared light scattering from suspensions of rod outer segment fragments is a useful probe of visible-light-activated changes in peripheral membrane proteins in photoreceptor cells. Limited sonication of suspensions has been shown to increase the amplitude of light induced turbidity changes in the presence of guanosine triphosphate by a factor of 2. Further sonication led to a decrease in the signal amplitude by an order of magnitude. This reduction has been puzzling, since the activity of the GTP-binding protein (as measured by GTP hydrolysis turnover number) was unaffected by the range of sonication used. This effect of sonication is investigated here using a novel, Reticon-based apparatus that measures the angular distribution of scattered light from samples as small as 1 microliter. The results show that even at high rhodopsin concentrations (125 microM) with millimeter path lengths, significant amounts of unscattered light are transmitted by the samples. A simple phenomenological theory that assumes a constant fractional change in scattering power (15%), independent of amount of sonication, explains the effect of sonication on the angle dependence data as well as the original turbidity data. The results have general relevance for optimization of light-scattering studies of membrane systems.

Cholera toxin action on rabbit corpus luteum membranes: effects on adenylyl cyclase activity and adenosine diphospho-ribosylation of the stimulatory guanine nucleotide-binding regulatory component.

Cholera toxin elicited 5- to 7-fold stimulation of adenylyl cyclase activity. Half-maximal activation was at 4.42 micrograms/ml cholera toxin. Cholera toxin-mediated activation was time dependent. At 0.1 mM ATP, both guanosine triphosphate (GTP) and nicotinamide adenine dinucleotide (NAD+) were required for cholera toxin activation of luteal adenylyl cyclase. The concentrations of GTP and NAD+ required for half-maximal activation were 1 and 200 microM, respectively. The GTP requirement could be eliminated by increasing the ATP concentration to 1.0 mM. Guanosine-5'-O-(2-thiodiphosphate) [GDP beta S] did not support cholera toxin activation of the luteal enzyme. Cholera toxin treatment increased GTP-stimulated activity, did not significantly alter guanyl-5'-yl imidodiphosphate [GMP-P(NH)P]-stimulated activity, and depressed NaF-stimulated activity. Furthermore, toxin treatment resulted in a 3.4-fold reduction in the Kact values for ovine luteinizing hormone (oLH) to activate adenylyl cyclase. A similar reduction in Kact values for oLH was obtained when concentration-effect curves performed in the presence of GMP-P(NH)P were compared to those performed in the presence of GTP. In addition, luteal membranes treated with cholera toxin and [32P]NAD+ were subjected to autoradiographic analysis following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This treatment resulted in the [32P] adenosine diphospho (ADP)-ribosylation of a 45,000-dalton protein doublet, corresponding to the alpha subunit of the stimulatory guanine nucleotide-binding regulatory component (Ns). As with activation of adenylyl cyclase activity, cholera toxin-specific [32P] ADP-ribosylation was time dependent and increased with increasing concentrations of cholera toxin. GTP, GMP-P(NH)P, and NaF, but not GDP beta S, were capable of supporting [32P] ADP-ribosylation of the protein doublet. oLH did not alter the ability of cholera toxin to ADP-ribosylate the protein activation of luteal adenylyl cyclase activity is due to the ADP-ribosylation of the alpha subunit of Ns and the concomitant inhibition of a GTPase associated with adenylyl cyclase.

Increase in Mg2+ affinity as a possible mechanism of chymotryptic activation of adenylate cyclase in rat heart membranes.

Chymotryptic activation of adenylate cyclase from rat heart was examined with respect to guanosine triphosphate (GTP), adenosine triphosphate (ATP) and Mg2+ concentrations. The activation was inhibited by aprotinin. In the absence or presence of GTP, chymotrypsin stimulated the cyclase at the same ratio. GTP exerted stimulatory (up to 0.1 mM) and inhibitory (at 1 mM) effects on the cyclase. Both effects were not affected by chymotrypsin. ATP at low concentrations increased the cyclase activity dose dependently and at high concentrations decreased the cyclase activity. Chymotrypsin retarded the appearance of the decreasing phase. This effect of chymotrypsin was similar to that of increasing the concentration of Mg2+ in the reaction mixture. According to double-reciprocal plots of Mg2+ concentrations and the cyclase activity, chymotrypsin diminished Km for Mg2+ with little effect on the Vmax. Hill plots of the same data showed that the Hill coefficient was about 1 and was not altered by chymotrypsin. These results suggest that chymotryptic activation of adenylate cyclase is mainly due to increasing the affinity for Mg2+ of the system.

Adenylate Cyclase Activity in Fetal Rabbit Myocardium

Adenylate cyclase activity [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] was determined in vitro in fetal rabbit myocardial membranes from individual fetal pups at 21 to 31 days gestation (term, 31 days). Basal and NaF-stimulated adenylate cyclase activities did not change during 21-31 days gestation. Significant stimulation of the enzyme by l-isoproterenol was observed only in the presence of guanosine triphosphate (100 microM). Under these conditions, maximal adenylate cyclase stimulation by l-isoproterenol (100 microM) was significantly higher at 25-31 than at 21 days gestation. Moreover, EC50 (Kact) for l-isoproterenol at 25-31 days was significantly lower than at 21 days gestation. We conclude that, in fetal rabbit myocardial membranes, there is an increase in the sensitivity of adenylate cyclase stimulation by l-isoproterenol from 21 to 25-31 days gestation.

Distribution of parathyroid hormone-sensitive adenylate cyclase in isolated rabbit renal cortex microvessels and glomeruli

The effect of the synthetic amino-terminal fragment of bovine parathyroid hormone, bPTH-(1–34), on the adenylate cyclase of microvessels and glomeruli isolated from rabbit kidney cortex was studied in the presence and absence of guanosine triphosphate (GTP). bPTH-(1–34) stimulated the vascular and glomerular adenylate cyclase in a dose-dependent manner with apparent ED 50 values of 11.5 nM and 64 nM respectively, in the absence of GTP. 10 −4M GTP greatly amplified the vascular response to bPTH-(1–34) while, in the glomeruli, both GTP, and bPTH-(1–34) had only additive effects. In the presence of GTP, vascular and glomerular apparent ED 50 were 190 nM and 64 nM respectively. [Nle 8, Nle 18, Tyr 34] -bPTH-(3–34) amide, described as a PTH antagonist, inhibited the action of bPTH-(1–34) in the microvessels and to a lesser extent in the glomeruli. PTH is therefore a potent stimulator of adenylate cyclase in rabbit renal microvessels and glomeruli, and may play a role in the regulation of renal blood flow and glomerulo-tubular feedback control.

Calmodulin stimulates adenylate cyclase activity and increases dopamine activation in bovine retina

Adenylate cyclase activity in bovine retina is highly responsive to Ca2+ and the endogenous Ca2+-binding protein, calmodulin (CaM). CaM stimulated adenylate cyclase activity in washed particulate fractions of bovine retina by 6.6-fold in a Ca2+-dependent manner. Activation of adenylate cyclase activity by CaM was maximal at 0.12 microM free Ca2+. The apparent Ka for calmodulin stimulation of adenylate cyclase was 67 nM and the apparent Vmax was 116 pmol/min/mg of protein above basal activity. Adenylate cyclase activity in bovine retina was stimulated approximately 50% by guanosine 5'-triphosphate (GTP), but the nonhydrolyzable GTP analogue, guanosine-5'-(beta, gamma-imido)triphosphate (Gpp(NH)p), was able to activate the enzyme nearly 5-fold. CaM and Gpp(NH)p appeared to be partially competitive activators of adenylate cyclase in the retina particulate fraction. Dopamine stimulated adenylate cyclase activity in the presence of GTP with an apparent Ka of 1.0 microM and an apparent Vmax of 66 pmol/min/mg of protein. Ca2+ and CaM increased the apparent Vmax of the dopamine-stimulated adenylate cyclase activity more than 2-fold to a level of 146 pmol/min/mg of protein but did not alter the apparent Ka. This suggests that CaM is an endogenous modulator of dopamine-stimulated adenylate cyclase activity in the retina. CaM-stimulated adenylate cyclase activity may be a common component to retina since we found this activity in retinas from rabbit, rat, and goldfish as well as cow.

Calcium inhibition of parathyroid adenylate cyclase.

Parathyroid adenylate cyclase activity has previously been reported to be inhibited by calcium. However, the concentrations of calcium required to inhibit this enzyme were, in most instances, several orders of magnitude higher than cytoplasmic calcium concentrations. We undertook this study to determine the effects of calcium at submicromolar, as well as higher concentrations on parathyroid adenylate cyclase activity. A partially purified membrane fraction was obtained from normal porcine parathyroid glands using buffers free of divalent cation chelators. Relatively high concentrations of EGTA were then used in the incubation mixture to achieve the desired low ionic calcium concentrations. Addition of 0.5 mM EGTA to the reaction mixture resulted in an approximate 30% increase in basal adenylate cyclase activity and a similar percentage increase in the activity measured in the presence of guanosine triphosphate or NaF, known activators of parathyroid adenylate cyclase. EGTA also stimulated the activity measured in the presence of forskolin, which itself markedly stimulated this enzyme in a concentration-dependent manner. The effects of calcium added in amounts calculated to achieve ionic concentrations of 0.5 X 10(-7) M to 1 X 10(-3) M on the forskolin-activated enzyme activity was investigated. The resultant calcium inhibition curve was found to be stepwise in appearance. Analysis of the data using a mathematical model which assumes multiple, independent calcium-binding sites indicated the presence of two inhibitory sites. One had a high affinity for calcium, Kdiss 0.4-1.5 X 10(-6) M. This site accounted for 50-70% of the calcium-inhibitable activity. The second had a considerably lower affinity, Kdiss = 1.0-5.0 X 10(-4) M. Similar affinities were obtained from experiments in which guanosine triphosphate was used to activate the enzyme instead of forskolin. It is suggested that the high affinity calcium-binding site may be involved in the physiological regulation of parathyroid adenylate cyclase activity and that the previously reported estimates of the calcium sensitivity of parathyroid adenylate cyclase represent composites of the high and low affinity calcium-inhibitable processes.

Coupling of the canine renal parathyroid hormone receptor to adenylate cyclase: modulation by guanyl nucleotides and N-ethylmaleimide.

Guanyl nucleotides greatly enhance PTH stimulation of renal adenylate cyclase activity. As part of an investigation of the mechanism of this potentiation, we evaluated the effect of guanosine triphosphate (GTP) and a GTP analog, guanylimidodiphosphate [Gpp(NH)p] on the specific binding of l25I-labeled bovine (b) PTH-(l–34) to canine renal cortical plasma membranes. Dissociation of [l25I]bPTH-(l–34) from receptors was slow (t½ 200 min) in the absence of added guanyl nucleotide, whereas in the presence of GTP or Gpp(NH)p (100 μM), dissociation was rapid (t½ 1 min). The increase in dissociation rate prompted by Gpp(NH)p was reflected in a 3- to 4-fold decrease in PTH receptor affinity, from 5 nM to 17 nM. In contrast to the slow dissociation rate of the l25I-labeled agonists [Nle8,Nle18,Tyr34]bPTH-(l–34)amide and bPTH(l–34), the dissociation rate of the l25I-labeled antagonist analog of PTH [Nle8,Nle18,Tyr34]bPTH-(3–34)amide was rapid (t½ 2 min). Moreover, binding of the labeled PTH antagonist analog was not...

Structure-function relationships in Escherichia coli translational elongation factor G: modification of lysine residues by the site-specific reagent pyridoxal phosphate.

Translational elongation factor G (EF-G) of Escherichia coli was modified with the selective, site-specific lysine reagent pyridoxal phosphate (PLP). The reaction results in the modification of a maximum of 12 lysine residues, one of which is essential for guanosine 5'-triphosphate (GTP) binding and whose modification is inhibited by the presence of GTP. Formation of a reversible adduct between 2,3-butanedione and an essential arginine similarly located in the GTP binding site [Rohrbach, M.S., & Bodley, J. W. (1977) Biochemistry 16, 1360-1363] also protects EF-G from PLP inactivation, suggesting that these two residues are spatially close to each other in the native factor. The essential lysine residue was found in the trypsin-resistant fragment T4 (Mr 41 000). In addition to the lysine essential for GTP binding, at least one further lysine was found to be important for EF-G function, since GTP-protected, PLP-modified EF-G molecules fully competent in binding to 50S ribosomal subunits showed decreased activity in 50S- and 70S-dependent GTP hydrolysis. It is likely that a PLP-modified lysine impairs the interaction of the factor with 30S ribosomal subunits and/or a conformational change of the factor required for the hydrolysis of GTP.

Alterations of catecholamine-sensitive adenylate cyclase in gerbil cerebral cortex after bilateral ischemia

Alterations in central concentrations of cyclic nucleotides occur in mammalian brain during ischemic episodes. Our aim was to evaluate the catecholamine sensitivity of adenylate cyclase using homogenates of the gerbil cerebral cortex during periods of bilateral ischemia and recirculation. After 15-min ischemia without recirculation, the sensitivity of adenylate cyclase was usually enhanced to norepinephrine (NE) and dopamine (DA) alone or in the presence of guanosine triphosphate (GTP) but not 5′-guanylyl imidodiphosphate [Gpp(NH)p]. After 60-min ischemia (no recirculation), the sensitivity of the enzyme to NE and DA showed either no change from sham controls or a slight decrease. Responses, however, in the presence of GTP remained elevated. When the animals were subjected to 15-min ischemia followed by 15-min recirculation, enzyme activation by NE and DA with or without GTP and Gpp(NH)p was usually greater than controls. After 60-min ischemia plus 15-min recirculation, however, enzyme responsiveness to catecholamines and NaF was attenuated. No changes in either high and low K m cyclic AMP phosphodiesterase or in guanylate cyclase were observed during ischemic episodes. The results indicate that the catecholamine-elicited activity of adenylate cyclase became elevated as a consequence of a short duration of ischemia. Longer periods of ischemia thought to be irreversible showed deficits in adenylate-cyclase sensitivity to catecholamines unless GTP was present. In the presence of GTP, enzyme sensitivity was restored as long as recirculation was prevented. Thus adverse consequences of recirculation on neuronal membranes or receptors could be evaluated by their state of GTP sensitivity.

73] Isolation and assay of a phosphodiesterase inhibitor from retinal rod outer segments

Publisher Summary This chapter discusses the isolation and assay of a phosphodiesterase inhibitor from retinal rod outer segments. Photolyzed rhodopsin activates a cyclic guanosine monophosphate (cGMP) phosphodiesterase in rod outer segments (ROS) in the presence of guanosine triphosphate (GTP). It has been proposed that photolyzed rhodopsin, GTP, and light-activated GTPase counteract the effects of this inhibitor on the phosphodiesterase. The most effective method for isolating this inhibitor takes advantage of its tight binding to the phosphodiesterase. The inhibitor copurifies with the phosphodiesterase and can then be isolated from the phosphodiesterase by heat treatment. Heat treatment was used to isolate the inhibitor from the phosphodiesterase. The highest yields of inhibitor were obtained by treating 0.6 mg/ml purified phosphodiesterase at pH 12.0 for 5 min at 100°. The pH was then lowered to 5.9 to precipitate the denatured phosphodiesterase.

Adenylate cyclase from human decidua parietalis

Adenylate cyclase (EC 4.6.1.1) activity was demonstrated in membrane fractions of human decidua parietalis. Adenylate cyclase activity was associated with membrane fractions of decidua but not chorion or amnion. Prostaglandins PGE1 and PGE2 but neither PGD2 nor PGF2α stimulated this activity at gestational ages 18 to 40 weeks. Fluoride and guanosine triphosphate stimulated decidual adenylate cyclase; however, epinephrine-stimulated adenylate cyclase activity was demonstrable only in the presence of guanosine triphosphate. The possible roles of adenylate cyclase activity in the decidua are discussed.

Stimulation of adenylate cyclase in isolated rat glomeruli by prostaglandins.

The effect of prostaglandins and various agents on cyclic adenosine 3',5'-monophosphate generation was studied in isolated rat glomeruli. Specific activity of adenylate cyclase in the glomeruli showed a 12-fold increase over the crude homogenate and a fivefold increase over a tubular preparation. Prostacyclin (PGI2) preferentially stimulated adenylate cyclase of isolated glomeruli at a concentration as low as 10(-9) M. Prostaglandins (PGE1, PGE2, and PGA2) and parathyroid hormone (1-34 synthetic PTH fragment) increased adenylate cyclase in glomeruli with maximal stimulation at 2 X 10(-5) M and 2-4 microgram/ml, respectively. No inhibition of PGE2 on PTH stimulation was observed. Isoproterenol (2 X 10(-4) M) caused a small stimulation, while PGF2alpha, arginine vasopressin, and angiotensin II were ineffective. The presence of guanosine triphosphate in the adenylate cyclase assay enhanced basal and PGE2- and PTH-stimulated activity, but had no effect on NaF stimulation. These findings show an effect of prostaglandins and PTH on the glomerular cAMP system and raise the possibility of a physiological action of these agents on the glomerulus.

Guanosine 5′-triphosphate inhibits growth and stimulates differentiated functions in B16 melanoma cells

Exogenous guanosine 5′-triphosphate (GTP) at a concentration of 0.5 mM causes in vitro growth inhibition and induces morphological and biochemical differentiation of B16 melanoma cells. After two days in the presence of GTP, cell proliferation is markedly reduced. Cessation of cell proliferation is followed by the extension of numerous dendrite-like processes and marked increase in melanin production. Other nucleotides such as guanosine 5′-diphosphate (GDP), guanosine 5′-monophosphate (GMP), guanosine 3′:5′-cyclic-monophosphoric acid (cGMP) or adenosine 5′-triphosphate (ATP) have little or no effect on cell morphology or melanin production in B16 melanoma cells, although these compounds retard cell proliferation similar to GTP. These findings are discussed in light of a possible relationship between cell proliferation and differentiation.

Binding of aminoacyl-tRNA to ribosomes promoted by elongation factor Tu. Studies on the role of GTP hydrolysis.

The role of guanosine triphosphate (GTP) in the elongation factor Tu(EF-Tu)promoted binding of aminoacyl-tRNA to ribosomes has been investigated. In the presence of EF-Tu and GTP, phenylalanyl-tRNA (Phe-tRNA) was bound to the A site of the poly(U).ribosome complex having N-acetylphenylalanyl-tRNA on its P site. EF-Tu could be utilized repeatedly in this binding reaction in the presence of GTP but was used only once and remained bound to ribosomes when GTP was replaced by 5'guanylyl methylenediphosphonate (Gpp(CH2)p), a nonhydrolyzable analog of GTP. The binylalanyl-tRNA, while little dipeptide was formed in the latter case. However, when EF-Tu and Gpp(CH2)p bound to the ribosomal complex were released by centrifugation through 10% sucrose containing 0.2 mM GDP, the yield of the dipeptide was correspondingly increased. It was concluded that the role of GTP in this reaction is to facilitate the transfer of Phe-tRNA to ribosomes by virtue of the high affinity of EF-Tu.GTP for ribosomes. The subsequent conversion of EF-Tu.GTP to EF-Tu.GDP, a form of EF-Tu with low affinity for ribosomes as well as for Phe-tRNA, resulted in the detachment of EF-Tu. Thus, the hydrolysis of GTP seems to be required for the release of EF-Tu from ribosomes, which is necessary for peptidyl transfer and the reutilization of EF-Tu. A freely reversible interaction between ribosome and Phe-tRNA.EF-Tu.Gpp(CH2)p complex was also demonstrated.

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

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.

Demonstration of a guanosine triphosphate-dependent enzymatic binding of aminoacyl-ribonucleic acid to Escherichia coli ribosomes.

Multiple enzyme fractionS< have been shown to be required for the synthesis of polypeptides from aminoacyl-sRNA in both mammalian and microbial systems.1'6 In the reticulocyte system two enzyme fractions have been founid to be required for the polyuridylic acid-(poly U)-directed synthesis of polyphenylalanine.7 One of these fractions was found to enihance the binding of phenylalanyl-sRNA to reticulocyte ribosomes in the presence of guanosine 5'-triphosphate (GTP) at concentrationis of Mlg++ that are close to the optimum for the over-all polymerization reaction.8' Three complemenitary polymerization factors isolated from extracts of Escherichia coli and Pseudomonas fluorescens10 have been shown to be required for polyphenylalanine synthesis with washed E. coli ribosomes. Several investigators"1-"3 have demonstrated that poly U-directed binding of phenylalanylsRNA to ribosomes occurs at high concentrations of i\Ig++ in the absence of GTP and soluble enzyme fractions (nonenzymatic binding); however, no enhancement of the biniding of phenylalanyl-sRNA to ribosomes in the presence of GTP and purified enzyme fractionis (enizymatic biniditng) has beeni observed previously in microbial systems.12 13 In the present study, ani extract of L'. coli W has been separated into two fractionls, both of which are required for the transfer of aminoacyl-sRNA to polypeptide with washed E. coli W ribosomes. One of these fractionis was founid to be required for the GTP-depeiidenit, enzymatic binding of aminoacyl-sRNA to E. coli ribosomes. These findings appear to be analogous to those previously reported in the reticulocyte system.8'