Roger A. Johnson

The enzymatic preparation of [α-32P]nucleoside triphosphates, cyclic [32P]AMP, and cyclic [32P]GMP

Abstract A method has been developed for the enzymatic preparation of α-32P-labeled ribo- and deoxyribonucleoside triphosphates, cyclic [32P]AMP, and cyclic [32P]GMP of high specific radioactivity and in high yield from 32Pi. The method also enables the preparation of [γ-32P]ATP, [γ-32P]GTP, [γ-32P]ITP, and [γ-32P]-dATP of very high specific activity and in high yield. The preparation of the various [α-32P]nucleoside triphosphates relies on the phosphorylation of the respective 3′-nucleoside monophosphates with [γ-32P]ATP by polynucleotide kinase and a subsequent nuclease reaction to form [5′-32P]nucleoside monophosphates. The [5′-32P]nucleoside monophosphates are then converted enzymatically to the respective triphosphates. All of the reactions leading to the formation of [α-32P]nucleoside triphosphates are carried out in the same reaction vessel, without intermediate purification steps, by the use of sequential reactions with the respective enzymes. Cyclic [32P]AMP and cyclic [32P]GMP are also prepared enzymatically from [α-32P]ATP or [α-32P]GTP by partially purified preparations of adenylate or guanylate cyclase. With the exception of the cyclases, all enzymes used are commercially available. The specific activity of 32P-labeled ATP made by this method ranged from 200 to 1000 Ci/mmol for [α-32P]ATP and from 5800 to 6500 Ci/mmol for [γ-32P]ATP. Minor modifications of the method should permit higher specific activities, especially for the [α-32P]nucleoside triphosphates. Methods for the use of the [α-32P]nucleoside phosphates are described for the study of adenylate and guanylate cyclases, cyclic AMP- and cyclic GMP phosphodiesterase, cyclic nucleotide binding proteins, and as precursors for the synthesis of other 32P-labeled compounds of biological interest. Moreover, the [α-32P]nucleoside triphosphates prepared by this method should be very useful in studies on nucleic acid structure and metabolism and the [γ-32P]nucleoside triphosphates should be useful in the study of phosphate transfer systems.

Effects of glucagon on cyclic AMP and carbohydrate metabolism in livers from diabetic rats

Abstract Cyclic AMP metabolism in normal and diabetic livers was studied using the isolated perfused rat liver and plasma membrane and supernatat fractions from rat liver. Liver from alloxan- or streptozotocin-diabetic rats had increased tissue levels of cyclic AMP and showed increased release of cyclic AMP during perfusion. Addition of glucagon or cyclic AMP to the medium produced little or no increase in the high rates of glucose production and lactate gluconeogenesis in diabetic livers. Low concentrations of glucagon (2·10 −10 M or less) did not increase tissue accumulation or release of cyclic AMP in livers from diabetic rats but were effective in normal livers. Higher concentrations of the hormone produced normal responses in diabetic livers. Basal or fluoride-stimulated adenylate cyclase activity in plasma membranes isolated from diabetic livers was not altered, but the enzyme was subnormally normally responsive to concentrations of glucagon within the range 10 −10 −10 −8 M. Insulin (10 −11 −10 −6 M) added in vitro was without effect on basal or glucagon-stimulated plasma membrane adenylate cyclase. Neither modulation of the Mg 2+ concentration, nor addition of Ca 2+ , GTP, theophylline or ouabain caused the emergence of an insulin effect. Rat liver plasma membranes contained phosphodiesterase activity with two apparent K m values of about 0.5 and 70 μM. The activity of the low K m enzyme was decreased in plasma membranes from diabetic rats and was increased by insulin treatment of the rats in vivo. Both low and high K m activities were also decreased in supernatant fractions from livers of diabetic rats. Insulin at 10 −9 or 10 −7 M concentration had no effect in vitro on plasma membrane phosphodiesterase activities.

Analysis of adenosine 3′,5′-monophosphate with luciferase luminescence☆

Abstract A sensitive, relatively simple, assay with wide range linearity has been developed for adenosine 3′,5′-monophosphate (cyclic AMP). It is based on the conversion of cyclic AMP to adenosine triphosphate (ATP), which is then measured by its luminescent reaction with luciferase. A linear standard curve for cyclic AMP was demonstrated using samples of 100 μl containing from 7.2 × 10−9 to 7.2 × 10−6M cyclic AMP. The assay was used to measure the rise in human urine cyclic AMP levels produced by glucagon infusion. Urine samples needed only to be filtered and buffered prior to assay and values agreed with those obtained using another assay method.

Characteristics of the enzymatic hydrolysis of 5′-adenylylimidodiphosphate: Implications for the study of adenylate cyclase☆

Abstract The characteristics of the hydrolysis of 5′-adenylylimidodiphosphate [AMP-P(NH)P] by partially purified plasma membranes from rat liver are described. Hydrolysis was less with membranes from fat cells and was poor with a detergent-dispersed preparation from rat cerebellum. The Chromatographic behavior of the principal degradation products suggests that AMP-P(NH)P is first hydrolyzed to 5−AMP, which is then hydrolyzed further to adenosine. The adenosine is shown to inhibit adenylate cyclase noncompetitively with respect to substrate and in a cation-dependent manner. Sensitivity to inhibition by adenosine was markedly enhanced by agents that stimulated adenylate cyclase. The characteristics of the initial hydrolysis of AMP-P(NH)P fit best those of nucleotide pyrophosphatase and support the conclusion that several of the various phosphatase activities present in membranes may be due to the same enzyme. Under conditions shown to be linear with respect to time and membrane protein concentration, hydrolysis of AMP-P(NH)P exhibited a pH optimum between 9.5 and 10. At pH 9.5, hydrolysis occurred with a K m of about 20 μ m and a V of about 220 nmol (min) 1 (mg of protein) −1 . The initial hydrolysis of AMP-P(NH)P was inhibited in a linear-competitive manner by ATP, ADP, 5′-AMP, GTP, 5′-guanylylimidodiphosphate, NAD + , and p -nitrophenyl-dTMP and in a noncompetitive manner by UDP-glucose. Adenosine 3′:5−cyclic phosphate and guanosine 3′:5′-cyclic phosphate were not inhibitory at concentrations up to 1 m m . ATP, GTP, and 5′-guanylylimidodiphosphate were also hydrolyzed in a manner comparable to that for AMP-P(NH)P. Hydrolysis of AMP-P(NH)P did not require the presence of added metal, and some metals were inhibitory. Activity was inhibited by dithiothreitol (50% at m ) and by EDTA (50% at about 10 m m ). Following pretreatment with EDTA or dithiothreitol, the readdition of certain metals, especially Zn or Co, caused some restoration of hydrolytic activity. The evidence suggests that hydrolytic activity involves the participation of bound metal and that the enzyme is a metallo-protein.

Regulation of adenosine-sensitive adenylate cyclase from rat brain striatum.

An adenosine‐sensitive adenylate cyclase has been characterized from rat brain striatum. In whole homogenates as well as in particulate fractions, N6‐phenylisopropyl adenosine (PIA), 2‐chloroadenosine, and adenosine N′‐oxide were equipotent in stimulating adenylate cyclase. Although GTP inhibited basal as well as PIA‐stimulated activity of whole homogenates, the enzyme showed an absolute dependency on GTP for stimulation by PIA, dopamine, epinephrine, and norepinephrine in a particulate fraction derived from discontinuous sucrose gradient centrifugation. Adenosine exerts two effects on this adenylate cyclase, stimulation at low concentrations and inhibition at high concentrations, suggesting the presence of two adenosine binding sites. The stimulation of adenylate cyclase by PIA was dependent on the concentration of Mg2‐. The degree of stimulation by PIA was greater at a low concentration of Mg2+, which suggests that stimulation by PIA was accompanied by increasing the apparent affinity for Mg2+. Activation of adenylate cyclase by PIA was blocked by theophylline or 3‐isobutyl‐ 1‐methylxanthine (IBMX). The pH optimum for basal or (PIA + GTP)‐stimulated activities was broad, with a peak between 8.5 and 9.5. In the presence of GTP, stimulation by an optimal concentration of PIA was additive, with maximal stimulation by the catecholamines. Phospholipase A2 treatment at a concentration of 1 U/ml for 5 min completely abolished the stimulatory effect of dopamine, whereas PIA‐stimulated activity remained unaltered. These data suggest that rat brain striatum either has a single adenylate cyclase, which is stimulated by catecholamines and adenosine by distinct mechanisms, or has different cyclase populations, stimulated by either adenosine or catecholamines.

Role of Phospholipids in Coupling of Adenosine and Dopamine Receptors to Striatal Adenylate Cyclase

Abstract: Treatment of striatal washed particles with phospholipase A2 or C abolished the activation of adenylate cyclase by dopamine but not by N6‐phenylisopropyl adenosine (PIA). The inhibition of dopamine‐sensitive cyclase was dependent on Ca2+ and increased with time and phospholipase concentration. F‐‐sensitive cyclase was not affected by phospholipase A2 treatment, but was enhanced by phospholipase C treatment. Phospholipase D did not affect basal, PIA, dopamine, or F‐‐sensitive cyclase activities. The observed effects of phospholipase A2 were not due to either the detergent effect of lysophospholipids or to contaminating proteases. Ropamine‐sensitive cyclase, inactivated by pretreatment with phospholipase A2, was restored by asolectin (a soybean mixed phospholipid), phosphatidylcholine, phosphatidylethanol‐amine, or phosphatidylserine, but not by phosphatidylinositol. Phosphatidylserine and phosphatidylcholine were equipotent in restoring dopamine‐sensitive activity. Lubrol‐PX, a nonionic detergent, abolished completely the dopamine‐sensitive cyclase activity, whereas PIA‐sensitive activity was slightly inhibited. In contrast, digitonin inhibited dopamine‐ and PIA‐sensitive cyclase activity in a parallel fashion. Lubrol‐PX released some adenylate cyclase into a 16,000 ×g supernatant fraction that was stimulated by PIA but not by dopamine. Removal of most of the free detergent by Bio‐bead SM 2 enhanced stimulation by PIA but did not restore sensitivity to dopamine. Asolectin also did not restore the activity of dopamine‐sensitive cyclase. The data suggest that the requirement for phospholipids for the coupling of dopamine and adenosine receptors to the striatal adenylate cyclase may be different and that the adenosine receptors may be more tightly coupled to the enzyme than are dopamine receptors.

Detergent dispersion of adenylate cyclase from partially purified rat liver plasma membranes.

Abstract Adenylate cyclase (EC 4.6.1.1) of partially purified rat liver plasma membranes was dispersed by a number of nonionic detergents. The presence of F− stabilized adenylate cyclase activity during dispersion by detergent. Both dispersed and intact membrane adenylate cyclase preparations were more active in the presence of Mn2+ than Mg2+. The specific activity of the dispersed enzyme was higher than either the basal or F−-stimulated cyclase activity of intact membranes, but the dispersed enzyme did not respond to glucagon or to further addition of F−.

Evidence for a new activator of rat liver phosphofructokinase

Abstract A low molecular weight compound that activates purified rat liver phosphofructokinase has been isolated and partially purified from rat hepatocyte extracts. It can be separated from both fructose bisphosphate and AMP on DEAE-Sephadex. Incubation of rat hepatocytes with glucagon lowers the level of this activator, and this accounts for the inhibition of phosphofructokinase that was observed in hepatocyte extracts (S. Pilkis, et al. (1979) Biochem. Biophys. Res. Commun. 88 , 960–967). Other characteristics of this activator are described which suggest that it is not any of the known effectors of rat liver phosphofructokinase.

Regulation of platelet adenylate cyclase by adenosine.

The stimulatory and inhibitory effects of adenosine on the adenylate cyclases of human and pig platelets were studied. Stimulation occurred at lower concentrations than did inhibition, and the stimulatory effect was prevented by methylxanthines. Stimulation by adenosine was immediate in onset and was reversible, under conditions when cyclic AMP formation was linear with respect to time and protein concentration. The stimulatory and inhibitory effects could be distinguished further by the use of various analogues of adenosine and could be prevented by adenosine deaminase. The data suggest that both stimulation and inhibition were due to adenosine itself and not one of its degradation products and that in the platelet preparation, neither formation nor degradation of adenosine during the adenylate cyclase incubation appreciably influenced measured activity. Stimulation by adenosine was additive with the effects of GMP-P(NH)P, and alpha- or beta-adrenergic stimulation, but was abolished by prostaglandin E1 or by NaF. Prostaglandin E1 and NaF increased the sensitivity of adenylate cyclase to inhibition by adenosine. The data suggest that guanyl-5-yl-(beta-gamma-imino)diphosphate and/or adrenergic stimulation and adenosine exert their effects on adenylate cyclase by distinct mechanisms, but that prostaglandin E1 or F- and adenosine increase enzyme activity by mechanisms which may involve common intermediates in the coupling to adenylate cyclase.

Calcium regulation of guanine nucleotide activation of hepatic adenylate cyclase

Pretreatment of isolated rat liver plasma membranes by washing with NaHCO3 buffer or by exposure to the chelator ethyleneglycol bis(beta-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA) with or without the ionophore A23187, produced a decrease in the sensitivity of adenylate cyclase (ATP pyrophosphate-lyase (cyclizing) EC 4.6.1.1) to subsequent stimulation by NaF or guanosine 5-(beta-gamma-imino)triphosphate (GPP(NH)P). Sensitivity to activation by the nucleotide could be restored by addition of the lyophilized and ashed wash or by addition of Ca2+, Mg2+ or Mn2+. The factor extracted from the membranes by these various treatments which was responsible for loss of stimulation was identified as Ca2+. Determination of the metal ion content of isolated membranes by atomic absorption spectrometry indicated that Ca2+ was the only divalent cation present in sufficient concentration to support persistent activation by either NaF or GPP(NH)P. Pretreatment of liver plasma membranes with trifluoperazine, which inhibits the action of Ca2+-dependent regulator protein in other enzyme systems, reduced GPP(NH)P activation of adenylate cyclase and caused marked depletion of membrane Ca2+. The effects of low concentrations (less than 100 microM) of the phenothiazine could be reversed totally by Ca2+ and partly by regulator protein. At higher concentrations of trifluoperazine, slight restoration of enzyme activation was seen with either agent. The hypothesis is presented that Ca2+ interacts with the nucleotide (GTP or GDP) regulatory site(s) of the adenylate cyclase. This interaction may be regulator-protein-dependent and may be important in determining the sensitivity of the enzyme to nucleotide activation in vivo.