A new method for removing nonionic detergent that allows reconstitution of dopamine-sensitive adenylate cyclase.

The macromolecular components of dopamine-sensitive adenylate cyclase are soluble in solutions of the nonionic detergents Brij 56 or digitonin, however response of the adenylate cyclase to dopamine is lost. Removal of the nonionic detergent by replacement with cholate and phospholipid, followed by removal of the cholate, restored the dopamine sensitivity of adenylate cyclase. By this method, the functional complex was reassembled from two separate solutions of components, one deficient in adenylate cyclase activity, and the other unresponsive to dopamine.     

Transient complexes. A structural model for the activation of adenylate cyclase by hormone receptors (guanine nucleotides/irradiation inactivation)

1. The irradiation-inactivation procedure was used to study changes in the state of association of the protein components of adenylate cyclase in intact rat liver plasma membranes by measurement of alterations in the target size determined from the catalytic activity of the enzyme. 2. A decrease in target size at 30 degrees C in response to p[NH]ppG (guanosine 5'-[betagamma-imido]triphosphate) or GTP was demonstrated, which we take to reflect the dissociation of a regulatory subunit. The effect of GTP is potentiated by glucagon. This effect is not observed at 0 degrees C. 3. An increase in target size was observed in response to glucagon in the absence of guanine nucleotides, which we take to reflect the association of glucagon receptor with adenylate cyclase. 4. We propose a model for the activation of adenylate cyclase by glucagon in which the binding of the hormone to its receptor causes an initial association of the receptor with the catalytic unit of the enzyme and a regulatory subunit to form a ternary complex. The subsequent activation of the adenylate cyclase results from the dissociation of the ternary complex to leave a free catalytic unit in the activated state. This dissociation requires the binding of a guanine nucleotide to the regulatory subunit. 5. The effects of variation of temperature on the activation of adenylate cyclase by glucagon and guanine nucleotides were examined and are discussed in relation to the irradiation-activation data. 6. The effectiveness of hormones, guanine nucleotides and combinations of hormone and guanine nucleotides as activators of adenylate cyclase in both rat liver and rat fat-cell plasma membranes was studied and the results are discussed in relation to the model proposed, which is also considered in relation to the observations published by other workers.     

Solubilization of the [8-lysine]vasopressin receptor and adenylate cyclase from pig kidney plasma membranes.

Adenylate cyclase and the [8-lysine]vasopressin receptor were solubilized from pig kidney medulla membranes using the nonionic detergent Triton X-100. Optimal conditions for solubilization were under continuous stirring in a medium containing 0.5% (/v) Triton X-100, 100 mM Tris-HCl, pH 8, and 10 mM MgCl2. Both adenylate cyclase activity and [3H][8-lysine]vasopressin binding activity were recovered in a -26,000 X g supernatant of detergent-treated membranes. The yield of solubilized adenylate cyclase was nearly 100%. The soluble enzyme was no longer sensitive to antidiuretic hormone but was slightly activated by sodium fluoride. The affinity of the soluble receptor for [8-lysine]vasopresin was les than that of the membrane-bound receptor (mean apparent Km values, respectively 10(-7) M and 2 X 10(-8) M), however binding cooperativity was preserved. Hill coefficients were 1.42 for the soluble receptor and 1.50 for the membrane receptor. The soluble receptor discriminated as efficiently as did the membrane receptor between [8-lysine-a1vasopressin and oxytocin. The yield of spolubilized receptor was only 30% despite the fact that all binding activity had disappeared from the residual pellet of detergent-treated membranes. When the membranous receptors were occupied before solubilization and the latter was performed under conditions in which dissociation of the hormone-receptor comples is slow, i.e. at low temperature, 65% to 100% of the hormone-receptor complex was recovered in the soluble fraction. The soluble hormone-receptor complex partially dissociated on rewarming whereas the free hormone concentration was kept unchanged in the medium. The residual binding capacity, which was 30% of the initial value, was identical with that determined when the receptor was solubilized in free form before incubation with labeled hormone. It was concluded that (a) solubilization of the receptor molecules was complete, (b) during solubilization two forms of the receptor appear, of which only one is accessible to the hormone, (c) occupancy of the receptor by the hormone prevented the formation of the nonaccessible form, and (d) some component or components of the soluble fraction might be responsible for the loss in apparent affinity.     

Solubilization and separation of the glucagon receptor and adenylate cyclase in guanine nucleotide-sensitive states.

Adenylate cyclase in liver membranes was solubilized with Lubrol PX and partially purified by gel filtration. The partially purified enzyme was susceptible to activation by guanyl-5'-yl imidodiphosphate (Gpp(NH)p). Studies on the binding of [3H]Gpp(NH)p to various fractions eluted from the gels revealed that an upper limit of 1% of the Gpp(NH)p binding sites is associated with adenylate cyclase activity stimulated by the nucleotide. The glucagon receptor, pretagged with 125I-glucagon in the membranes, solubilized with Lubrol PX, and fractionated on the same gel columns, eluted in a peak fraction that overlaps with, but is separate from, adenylate cyclase in its Gpp(NH)p-stimulated form. Addition of GTP to the solubilized glucagon-receptor complex caused complete dissociation of the complex, as has been shown with the membrane-bound form of the complex. Since the GTP-sensitive form of the glucagon receptor complex separates from the Gpp(NH)p-sensitive form of adenylate cyclase, it is concluded that the receptor and the enzyme are separate molecules, each associated with a distinct nucleotide regulatory site or component. These findings are discussed in terms of the possible structure of the hormone-sensitive state of adenylate cyclase.     

Activation of adenylate cyclase by forskolin in rat brain and testis

Detergent-dispersed adenylate cyclase from rat cerebrum was detected in two components, one sensitive to Ca2+ and calmodulin and another sensitive to fluoride or guanyl-5'-yl imidodiphosphate (Gpp(NH)p). The enzyme activity of both components was markedly augmented by forskolin assayed in the presence or absence of other enzyme activators (e.g., NaF, Gpp(NH)p, calmodulin). The catalytic subunit fraction in which G/F protein was totally lacking was also activated by forskolin. During 1-35 days of postnatal development, the basal adenylate cyclase activities in either cerebrum and cerebellum particulate preparations progressively increased. While the fluoride sensitivity of the cerebrum and cerebellum enzyme increased during postnatal development, the responsiveness to forskolin remained unaltered. There was no enhancement of soluble adenylate cyclase (from rat testis) by forskolin under the assay conditions in which there was a marked stimulatory action on the particulate enzyme. The results seen with the solubilized enzyme, with either Lubrol PX or cholate, indicate that the effects of forskolin on the cyclase do not require either G/F protein or calmodulin and the results of our study of brain enzymes support this view. Data on soluble testis cyclase (a poor or absent response to forskolin by this enzyme) imply that it lacks a protein (other than the catalytic unit) which could confer greater stimulation. The present results do not rule out an alternative explanation that forskolin stimulates adenylate cyclase by a direct interaction with the catalytic subunit, if the catalytic proteins do differ widely in various species of cells and their response to this diterpene.     

Separation of soluble adenylate and guanylate cyclases from the mature rat testis.

The mature rat testis contains both a soluble guanylate cyclase and a soluble adenylate cyclase. Both these soluble enzymes prefer manganous ion for activity. It is known that guanylate cyclase can, when activated by a variety of agents, catalyze the formation of cyclic AMP. The following experiments were performed to determine whether the testicular soluble adenylate and guanylate cyclase activities were carried on the same molecule. Analysis of supernatants from homogenized rat testis by gel filtration and sucrose density gradient centrifugation showed that the two activities were clearly separable. The molecular weight of guanylate cyclase is 143 000, while that of adenylate cyclase is 58 000. Treatment of the column fractions with 0.1 mM sodium nitroprusside allowed guanylate cyclase activity to be expressed with Mg(2+) as well as with Mn(2+). Sodium nitroprusside did not affect the metal ion or substrate specificity of adenylate cyclase. These experiments show that adenylate and guanylate cyclase activities are physically separable.     

Adenylate cyclase activity and motor behavior following cerebral ischemia in the unanesthetized gerbil

Five minutes of bilateral carotid occlusion in unanesthetized gerbils produced substantial changes in spontaneous locomotor activity. Behavior was decreased after 1 hr of reperfusion and was increased at 24 hrs post-ischemia. Adenylate cyclase activity was measured in homogenates of frontal cortex and hippocampus at 90 min and 24 hrs following 5 min of cerebral ischemia. Enzyme activity was determined in the absence and presence of the activators guanosine-5'-triphosphate (GTP), guanylyl-5'-imidodiphosphate (GppNHp), isoproterenol (Iso) plus GTP, and forskolin (Fors) plus GTP. Homogenates responded with expected increases over basal adenylate cyclase activity with addition of all activators. An additional small increase in isoproterenol-stimulated activity was observed in frontal cortex homogenates at 90 min post-ischemia. No other significant changes in adenylate cyclase activity were observed after either 90 min or 24 hrs of reperfusion. The substantial increases in locomotor activity evident at 24 hrs after transient ischemia are not associated with measurable changes in adenylate cyclase activity in homogenates of frontal cortex or hippocampus.     

Separation of soluble adenylate and guanylate cyclases from the mature rat testis

The mature rat testis contains both a soluble guanylate cyclase and a soluble adenylate cyclase. Both these soluble enzymes prefer manganous ion for activity. It is known that guanylate cyclase can, when activated by a variety of agents, catalyze the formation of cyclic AMP. The following experiments were performed to determine whether the testicular soluble adenylate and guanylate cyclase activities were carried on the same molecule. Analysis of supernatants from homogenized rat testis by gel filtration and sucrose density gradient centrifugation showed that the two activities were clearly separable. The molecular weight of guanylate cyclase is 143 000, while that of adenylate cyclase is 58 000.Treatment of the column fractions with 0.1 mM sodium nitroprusside allowed guanylate cyclase activity to be expressed with Mg²⁺ as well as with Mn²⁺. Sodium nitroprusside did not affect the metal ion or substrate specificity of adenylate cyclase.These experiments show that adenylate and guanylate cyclase activities are physically separable.     

Human platelet adenylate cyclase: persistent binding of guanine nucleotide is central to the regulation of both hormone-stimulated and basal activities

Prostaglandin E1 stimulation of human platelet adenylate cyclase, in purified plasma membranes, occurs without the addition of exogenous GTP. Possible contamination of the adenylate cyclase assay mixture by GTP either from nonspecifically bound nucleotide in the plasma membrane or from the substrate ATP was ruled out as follows: (a) variation of the membrane concentration, repeated washing, inclusion of EDTA, GDP beta S, or GMP in the wash step, or UDP in the assay, are all without effect, and (b) analysis of the substrate by high-performance liquid chromatography revealed no contaminating GTP. Other prostaglandins (I2, E2, D2) also activate cyclase without the addition of GTP. In sharp contrast, stimulation of adenylate cyclase in the human neutrophil plasma membrane by prostaglandin E1 shows an obligatory requirement for GTP, under identical assay conditions. GDP beta S pretreatment amplifies the fold cyclase stimulation by GTP in the presence and absence of prostaglandin E1, by lowering the basal activity. This alteration occurs without lowering the GTP-independent prostaglandin E1 activation, and is specific for inhibitory guanine nucleotides (GDP beta S, GMP, GDP) in the pretreatment. Extensive washing with buffer or incubation with other nucleotides, epinephrine, or prostaglandin E1 prior to the assay, is without effect. GTP gamma S treatment of the membrane induces a high-activity state and abolishes the GDP beta S effect on basal activity as well as prostaglandin E1 activation of cyclase. The results suggest distinct patterns of prostaglandin stimulation in platelet and neutrophil cyclase systems, and further imply that guanine nucleotide, prebound to specific sites within the GTP-regulatory proteins, may modify the kinetic characteristics of platelet adenylate cyclase.     

Age-related parallel decline in beta-adrenergic receptors, adenylate cyclase and phosphodiesterase activity in rat erythrocyte membranes.

The activities of three components of the cyclic AMP system were compared in erythrocyte ghost membranes prepared from the blood of rats at various ages from 1.5 to 15 months. The apparent number of β-adrenergic receptor sites, adenylate cyclase activity and cyclic AMP phosphodiesterase activity all declined about 50% in the membranes from the older animals (>5 months) as compared to the 1.5 month ones. The soluble erythrocyte phosphodiesterase also declined with age, but the decline did not parallel that of the membrane-associated activity. In contrast, there was no age-related change in the number of β-adrenergic receptors in membranes from the brains of the same animals. In erythrocyte ghosts, both the ratio of isoproterenol-stimulated adenylate cyclase activity to basal activity and the ratio of sodium fluoride-stimulated activity to basal were constant with age. Neither the dissociation constant for the β-adrenergic receptor nor the Michaelis constant for the phosphodiesterase changed as a function of age. Together with other data in the literature, these results suggest a close functional association of the components of the cyclic AMP system in the mature erythrocyte membrane, and support a physiological role for the cyclic AMP mediated β-adrenergic effects in the red blood cell.     

Changes in rat testicular adenylate cyclase activities and gonadotrophin binding during unilateral experimental cryptorchidism

Unilaterally cryptorchid rats were examined at 3, 8, 15, 22 and 28 days after operation. There was a selective decrease in the adenylate cyclase (ATP pyrophosphate--lyase (cyclizing), EC 4.6.1.1) responses to gonadotrophin stimulation in the abdominal testis. This was associated with a parallel decrease in specific FSH and LH binding. There was no reduction in the response of testicular adenylate cyclases to prostaglandin (PG) E-1 or fluoride stimulation, indicating that both the GTP binding protein (N-component) and the catalytic subunit of the adenylate cyclase complexes were intact. The reduction in FSH-responsive adenylate cyclase activity in the abdominal testis was not due to a change in the Km for adenylate cyclase activation, but was due to a reduction in maximal velocities. Unilateral cryptorchidism was also associated with a rapid decline in soluble Mn2+-dependent adenylate cyclase activity in germ cells (spermatids). By 3 days after operation there was an 82% decrease in germ cell adenylate cyclase activity. The loss of soluble Mn2+-dependent adenylate cyclase activity was associated with a parallel decrease in Sertoli cell secretion of androgen binding protein, indicating that Sertoli cell factors may be important for the maintenance of germ cell adenylate cyclase activity. The desensitization of the gonadotrophin--responsive adenylate cyclases and the loss of gonadotrophin receptors in Leydig and Sertoli cells were not due to changes in plasma gonadotrophin values because LH concentrations were within normal limits and plasma FSH was only marginally elevated in the cryptorchid rats. No significant alterations of any of these parameters were seen in the scrotal testis of unilaterally cryptorchid rats when compared to values for intact controls.     

Localization of particulate guanylate cyclase in plasma membranes and microsomes of rat liver.

The subcellular localization of guanylate cyclase was examined in rat liver. About 80% of the enzyme activity of homogenates was found in the soluble fraction. Particulate guanylate cyclase was localized in plasma membranes and microsomes. Crude nuclear and microsomal fractions were applied to discontinuous sucrose gradients, and the resulting fractions were examined for guanylate cyclase, various enzyme markers of cell components, and electron microscopy. Purified plasma membrane fractions obtained from either preparation had the highest specific activity of guanylate cyclase, 30 to 80 pmol/min/mg of protein, and the recovery and relative specific activity of guanylate cyclase paralleled that of 5'-nucleotidase and adenylate cyclase in these fractions. Significant amounts of guanylate cyclase, adenylate cyclase, 5'-nucleotidase, and glucose-6-phosphatase were recovered in purified preparation of microsomes. We cannot exclude the presence of guanylate cyclase in other cell components such as Golgi. The electron microscopic studies of fractions supported the biochemical studies with enzyme markers. Soluble guanylate cyclase had typical Michaelis-Menten kinetics with respect to GTP and had an apparent Km for GTP of 35 muM. Ca-2+ stimulated the soluble activity in the presence of low concentrations of Mn-2+. The properties of guanylate cyclase in plasma membranes and microsomes were similar except that Ca-2+ inhibited the activity associated with plasma membranes and had no effect on that of microsomes. Both particulate enzymes were allosteric in nature; double reciprocal plots of velocity versus GTP were not linear, and Hill coefficients for preparations of plasma membranes and microsomes were calculated to be 1.60 and 1.58, respectively. The soluble and particulate enzymes were inhibited by ATP, and inhibition of the soluble enzyme was slightly greater. While Mg-2+ was less effective than Mn-2+ as a sole cation, all enzyme fractions were markedly stimulated with Mg-2+ in the presence of a low concentration of Mn-2+. Triton X-100 increased the activity of particulate fractions about 3- to 10-fold and increased the soluble activity 50 to 100%.     

Enzymatic formation of inosine 3',5'-monophosphate and of 2'-deoxyguanosine 3',5'-monophosphate. Inosinate and deoxyguanylate cyclase activity.

Enzymes in particulate fractions from sea urchin sperm and in soluble fractions from rat lung were shown to catalyze the formation of inosine 3',5'-monophosphate (cyclic IMP) and of 2'-deoxyguanosine 3',5'-monophosphate (cyclic dGMP) from ITP and dGTP, respectively. With sea urchin sperm particulate fractions, Mn2+ was an essential metal cofactor for inosinate, deoxyguanylate, guanylate and adenylate cyclase activities. Heat-inactivation studies differentiated inosinate and deoxyguanylate cyclase activities from adenylate cyclase, but indicated an association of these activities with guanylate cyclase. Preincubation of sea urchin sperm particulate fractions with trypsin altered in a very similar manner guanylate, inosinate, and deoxyguanylate cyclase activities, and various metals and metal-nucleotide combinations protected the three cyclase activities to comparable degrees against trypsin. The relative guanylate, deoxyguanylate and inosinate cyclase activities at 0.1 mM nucleoside triphosphate were 1.0, 0.5 and 0.08, respectively. With these three cyclase activities, plots of reciprocal velocities against reciprocal Mn2+-nucleoside triphosphate concentrations were concave upward, suggesting positive homotropic effects. With rat lung soluble preparations, relative guanylate, deoxyguanylate, inosinate and adenylate cyclase activities at 0.09 mM nucleoside triphosphate were 1.0, 1.7, 0.1 and 0, respectively. MnGTP was a competitive inhibitor of deoxyguanylate cyclase activity (Ki equals 12.2 muM) and MndGTP was a competitive inhibitor of guanylate cyclase activity (Ki equals 16.2 muM). Inhibition studies using ITP were not conducted. When soluble fractions from rat lung were applied to Bio-Gel A 1.5 m columns, elution profiles of guanylate, deoxyguanylate and inosinate cyclase activities were similar. These results suggest that deoxyguanylate, guanylate and inosinate cyclase activities reside within the same protein molecule.     

The Escherichia coli adenylate cyclase complex: Activation by phosphoenolpyruvate

A model for the regulation of the activity of Escherichia coli adenylate cyclase is presented. It is proposed that Enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) interacts in a regulatory sense with the catalytic unit of adenylate cyclase. The phosphoenolpyruvate (PEP)-dependent phosphorylation of Enzyme I is assumed to be associated with a high activity state of adenylate cyclase. The pyruvate or sugar-dependent dephosphorylation of Enzyme I is correlated with a low activity state of adenylate cyclase. Evidence in support of the proposed model involves the observation that Enzyme I mutants have low cAMP levels and that PEP increases cellular cAMP levels and, under certain conditions, activates adenylate cyclase, Kinetic studies indicate that various ligands have opposing effects on adenylate cyclase. While PEP activates the enzyme, either glucose or pyruvate inhibit it. The unique relationships of PEP and Enzyme I to adenylate cyclase activity are discussed.     

Manganese and manganese-ATP interactions with bovine sperm adenylate cyclase

The adenylate cyclase of mammalian spermatozoa shares some of the properties of the isolated catalytic component from somatic cell adenylate cyclases. One of these properties is the large apparent stimulation by Mn2+. We have used the direct linear plot according to Eisenthal and Cornish-Bowden to explore whether this apparent stimulation is due to direct stimulation by Mn2+ or due to complexation of free ATP, a postulated inhibitor of cyclase activity. We have observed the activity of the particulate adenylate cyclase from bovine caudal epididymal spermatozoa as a function of calculated equilibrium values for the concentrations of Mn2+, free ATP, and the enzyme's substrate, the manganese-ATP complex. Direct linear plots for activity and substrate concentration over the apparent inhibitory concentration range of free ATP give the pattern expected for a hyperbolic substrate response. By contrast, direct linear plots in which Mn2+ concentration varies over its apparent stimulatory range show that as Mn2+ concentration increases, activities are higher than would be predicted for a hyperbolic substrate response. We conclude that for particulate bovine sperm adenylate cyclase, free ATP is not strongly inhibitory, and Mn2+ is a positive effector, reaching half-maximal stimulation at 0.2 mM. The unique nature of the sperm adenylate cyclase and its possible regulation by Mn2+ under physiological conditions is discussed.     

"Spare" beta-adrenergic receptors of rat white adipocyte membranes

The apparent equilibrium dissociation constants for the interaction of isoproterenol with beta-receptors and adenylate cyclase were determined under the same conditions in rat adipocyte membranes and were compared with the apparent dissociation constant for the interaction of isoproterenol with cyclic AMP accumulation in the adipocyte. From these determinations, it was calculated that the occupancy of less than 4% of the receptor population is required for half-maximal stimulation of adenylate cyclase in membranes and cyclic AMP accumulation in intact cells, provided that receptor-binding and adenylate cyclase assays are performed in the presence of guanine nucleotides. Since guanine nucleotides are also required for adenylate cyclase activation in intact cells, it is concluded that the beta-receptors of rat adipocytes are "spare" receptors.     

Inhibitory effects of pentacaine and some related local anaesthetics on rat hepatic adenylate cyclase

In the present study effects of a new local anaesthetics, pentacaine (trans-2-pyrolidinocyclohexylester of 3-pentyloxyphenylcarbamic acid), and of some chemically related compounds on rat hepatic adenylate cyclase activity were studied under various experimental conditions. As compared with tetracaine, the local anaesthetics tested showed stronger inhibitory effects, regardless of the type of stimulating agents used to activate adenylate cyclase. The most potent effect was observed with pentacaine. Its inhibitory effects on glucagon, guanylylimidodiphosphate (Gpp/NH/p), sodium fluoride or forskolin stimulated activity suggest that it may directly act on the catalytic unit of adenylate cyclase. The same conclusion can be drawn based on its inhibitory effects on adenylate cyclase, regardless ATP concentrations used as the enzyme substrate, and on octylpyranoside solubilized enzyme activated by preincubation of the enzyme preparation with Gpp/NH/p. Structure-activity studies have suggested that the pentacaine molecule as a whole and none of its parts alone or its analogs are responsible for the inhibitory effect. However, the inhibitory effects of these compounds on the rat adenylate cyclase activity do not correlate with their local anaesthetic properties. The possibility of using adenylate cyclase inhibitors to decrease cyclic AMP production under pathological conditions, like in cholera, known to be due to a high adenylate cyclase activity, is discussed.     

Incorporation of rat brain adenylate cyclase into artificial phospholipid vesicles.

Adenylate cyclase was solubilized from rat brain particulate fraction with the nonionic detergent, Nonidet P-40. Incubation of detergent-solubilized adenylate cyclase with liposomes prepared from egg yolk phosphatidylcholine results in virtually quantitative incorporation of the enzyme activity into phospholipid vesicles. Incorporation of adenylate cyclase into liposomes results in an approximately 10- to 20-fold purification relative to the solubilized preparation giving a final specific activity of about 50 nmol of cAMP min-1 mg-1. The detergent-solubilized adenylate cyclase migrates as a broad band between 14 and 33% sucrose on density gradient centrifugation, separated from the endogenous phospholipid. Following overnight incubation of the solubilized enzyme with exogenous phospholipid, all enzyme activity is found in a narrow band between 7 and 9% sucrose, co-migrating with the phospholipid. The adenylate cyclase could not be released from the liposomes by extraction with high ionic strength, low ionic strength-EDTA, or sonication. Treatment of liposomal adenylates cyclase with soluble proteases or immobilized trypsin destroys enzyme activity. Thus, it is likely that a functionally important part of the enzyme molecule is exposed on the outer surface of the liposome. Optimal conditions for the incorporation of adenylate cyclase into liposomes, and some effects of manipulating the phospholipid composition on enzyme activity are reported.     

Mechanism of catabolite repression in Escherichia coli bacteria: interaction between transport proteins and adenylate cyclase

The mechanism of catabolite repression caused by sugar transported via the phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS) and stipulated by the decrease of the adenylate cyclase activity was studied. It was demonstrated that the sensitivity of the adenylate cyclase and beta-galactosidase synthesis to methyl-L-D-glucoside (MeGlc) or sorbitol is correlated with the content and activity of glucose (EIIGlc) or mannitol enzyme II of the PTS, correspondingly. Under anaerobic conditions the cells become insensitive to catabolic repression caused by MeGlc and the adenylate cyclase activity does not decrease in the presence of the sugar despite the increased rate of MeGlc transport. The adenylate cyclase activity of the mutant with the Tn5 transposone inserted into the ptsG gene does not change in the presence of MeGlc, while the activity of adenylate cyclase and the differential rate of beta-galactosidase synthesis increase in these bacteria. The data obtained confirm the hypothesis on the "catabolite signal" which is generated when the substrate binds to its transporter, i. e. adenylate cyclase reacts to the conformational changes in the transporter being complexed with it. The strength of this complex depends on the affinity of adenylate cyclase for the transporter and on the value of the membrane potential, delta mu H+ A model is proposed, which explains the necessity of factor IIIGlc for EIIGlc binding to adenylate cyclase.     

Interaction of the insecticide 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane with the beta-receptor adenylate cyclase system in Chang liver cell membranes

Interaction of the insecticide 1,1,1-trichloro-2,2-bis-(p-chlorophenyl)-ethane (DDT) with beta-receptor binding and adenylate cyclase activity of biological membranes has been studied. Following exposure of cultured Chang liver cells to DDT, maximal binding of the catecholamine antagonist [125I]-iodohydroxybenzylpindolol (HYP) to isolated cell membranes was decreased by 30% whereas the dissociation constant remained unchanged. Both basal activity and maximal isoproterenol-stimulated activity of adenylate cyclase were not altered. The isoproterenol concentration required for half-maximal stimulation of the enzyme was increased about 2-fold as was the agonist concentration required for half-maximal displacement of the antagonist HYP at the beta-receptor binding site. Thus, coupling efficiency of hormone-stimulated adenylate cyclase activity was not influenced by the presence of DDT in these membranes. The data show that interaction of DDT with the beta-receptor adenylate cyclase complex is restricted to the receptor component. Enzyme activity is directly linked to changes of agonist binding at the beta-receptor. Interference of DDT with signal transduction via 'fluidization' of membrane lipids has not been detected.