Adenylate cyclase system of bovine adrenal plasma membranes.

The adenylate cyclase system present in a preparation enriched in plasma membranes derived from bovine adrenal cortex was investigated in considerable detail. This system is stimulated by adrenocorticotropic hormone (ACTH), by biologically active analogs of this hormone, and by fluoride ion. The preparation contains sodium-potassium- and magnesium-dependent ATPases that are markedly inhibited by 50 mM sodium fluoride. Incorporation of a pyruvate phosphokinase ATP generating system into the adenylate cyclase assay medium provided constant substrate levels. In the presence of the ATP generating system, the rate of cyclic AMP formation (basal, fluoride, and ACTH-activated) was proportional to enzyme concentration and was linear with time. Proportionality with respect to enzyme concentration as concerned the hormone-activated adenylate cyclase was achieved only when the ratio of hormone to enzyme protein was kept constant. The temperature optimum of the adenylate cyclase, basal or activated, was approximately 30 degrees. Michaelis-Menten kinetics were observed when the ratio of Mg2+ to ATP was approximately 6:1. Both calcium and ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid completely inhibited the adenylate cyclase system at concentrations of 5 and 0.5 mM, respectively. GTP was inhibitory at concentrations of 10-2 M but had little effect at lower concentrations. Freezing in liquid nitrogen and storage at -60 degrees exerted little effect on the fluoride-stimulated enzyme but lowered hormone stimulated activity. Preincubation in the presence of ACTH afforded a high degree of stabilization of the enzyme system while preincubation with a biologically inactive analog afforded no protection.     

The effect of temperature on the activity of the adenylate cyclase system of liver plasma membranes

The rat liver adenylate cyclase system shows a discontinuity in the Arrhenius plots at 20°C in the nonstimulated activity (basal) with activation energies of 16 and 28 Kcal/mole. The discontinuity disappears when the enzyme is stimulated either by glucagon, sodium fluoride, 5′ guanylyl-imidodiphosphate or glucagon plus 5′ guanylyl-imidodiphosphate and the energy of activation was the same with all the compounds tested. If the activator was initially in contact with the membranes at 0°C the energy of activation was similar to that observed below the break (26 Kcal/mole) but it changed to that above the break if the compound contacted the membranes at temperatures above the break (22–24°C). We discuss the possibility of two different conformations of the enzyme; both conformations can be “frozen” by any of the compounds tested, “isolating” the enzyme from any subsequent physical change of the membrane due to temperature.     

Purification of the calmodulin-sensitive adenylate cyclase from bovine cerebral cortex

A calmodulin-sensitive adenylate cyclase was purified 3000-fold from bovine cerebral cortex using DEAE-Sephacel, calmodulin-Sepharose, and two heptanediamine-Sepharose column steps. The purified enzyme activity was stimulated by calmodulin, forskolin, 5'-guanylyl imidodiphosphate, and NaF. The molecular weight of the protein component was estimated as 328 000 with a smaller form of Mr 153 000 obtained in the presence of Mn2+. The most highly purified preparations contained major polypeptides of 150 000, 47 000, and 35 000 daltons on sodium dodecyl sulfate (SDS) gels. Photoaffinity labeling of the preparation with azido[125I]iodocalmodulin gave one product of 170 000 daltons on SDS gels. It is proposed that the catalytic subunit of the calmodulin-sensitive enzyme is 150 000 +/- 10 000 daltons and that the enzyme exists as a complex of one catalytic subunit and the stimulatory guanyl nucleotide regulatory complex. These data are consistent with the previous report that the catalytic subunit of this enzyme has a molecular weight of 150 000 +/- 10 000 [Andreasen, T.J., Heideman, W., Rosenberg, G.B., & Storm, D.R. (1983) Biochemistry 22,2757].     

The activity of dopamine-stimulated adenylate cyclase from rat brain striatum is modulated by temperature and the bilayer-fluidizing agent, benzyl alcohol

Hepatocytes were isolated by collagenase perfusion of livers from rats that had been allowed access to a carbohydrate-rich diet or laboratory chow or had been deprived of food 48h before use. By incubation with l-[4,5-(3)H]leucine and precipitation with anti-(L-type pyruvate kinase) sera the rates of synthesis and degradation of L-type pyruvate kinase were measured in freshly prepared cells and hepatocytes maintained in monolayer culture for up to 5 days. Hepatocytes from carbohydrate-rich-diet-fed rats synthesized more L-type pyruvate kinase than did cells from chow-fed animals, which in turn synthesized more than cells from 48h-starved rats. Hepatocytes maintained in culture for up to 5 days synthesized L-type pyruvate kinase at similar rates to freshly prepared cells. The degradation of [(3)H]leucine-labelled L-type pyruvate kinase was shown to be biphasic. A phase with t((1/2)) (half-time) 4.9h and a duration of 8-10h was followed by a phase with t((1/2)) 79.2h. Cells from chow-fed and carbohydrate-rich-diet-fed rats showed similar patterns of degradation of L-type pyruvate kinase. The addition of 2mm-fructose and 0.1mum-insulin to the culture medium increased the t((1/2)) of the rapid phase to 12h in cells isolated from carbohydrate-rich-diet-fed rats, but not in cells from chow-fed rats. The secondary, slower, phase of degradation remained unaffected. The degradation of fructose 1,6-bisphosphatase and total cell protein followed first-order kinetics. The half-life of fructose 1,6-bisphosphatase was 41.0h in cells from chow-fed animals and 48.5h in cells from carbohydrate-rich-diet-fed donors. Fructose and insulin did not affect the rate of enzyme degradation. We propose that there is a role for protein catabolism in the short-term and long-term control of L-type pyruvate kinase concentration.     

Calmodulin activates adenylate cyclase from rabbit heart plasma membranes

It was shown that calmodulin (CM) activates the adenylate cyclase (AC) of rabbit heart light sarcolemma in the presence of micromolar free Ca2+ concentrations and this effect is blocked by trifluoroperazine and troponin I. GTP (in the presence of isoproterenol) and Gpp(NH)p are able to increase the CM-dependent activity of enzyme. It was concluded that there is no special CM-dependent "form' of AC in the heart and the common catalytic component of AC can be regulated both by CM and guanine nucleotide-binding regulatory component (N-protein). In the presence of Ca2+ and guanine nucleotide heart AC exists as a complex: CM-catalytic component-N-protein.     

Adenylate cyclase of bovine adrenal cortex plasma membranes. Divergence between corticotropin and fluoride combined effects with forskolin

The diterpene forskolin maximally stimulated bovine adrenal cortex adenylate cyclase activity 9-fold with a concentration producing half-maximum effect (ED50) of about 4 microM. The effects of forskolin and the fully active corticotropin fragment ACTH (I 24) were additive over nearly the whole range of concentration of both effectors, indicating separate and independent mechanisms of action. By contrast, 10 mM NaF blocked forskolin action in the nanomolar range of the diterpene concentration, while it allowed a partial stimulation by forskolin in the micromolar range. NaF thus reveals a heterogeneity of forskolin action in the adrenal cortex plasma membranes. Moreover, our data suggest that ACTH and NaF activation effects, both mediated by the stimulatory regulatory protein Gs, proceed through different mechanisms.     

Lysophosphatidylcholines can modulate the activity of the glucagon-stimulated adenylate cyclase from rat liver plasma membranes.

1. Synthetic lysophosphatidylcholines inhibit the glucagon-stimulated adenylate cyclase activity of rat liver plasma membranes at concentrations two to five times lower than those needed to inhibit the fluoride-stimulated activity. 2. Specific 125I-labelled glucagon binding to hormone receptors is inhibited at concentrations similar to those inhibiting the fluoride-stimulated activity. 3. At concentrations of lysophosphatidylcholines immediately below those causing inhibition, an activation of adenylate cyclase activity or hormone binding was observed. 4 These effects are essentially reversible. 5. We conclude that the increased sensitivity of glucagon-stimulated adenylate cyclase to inhibition may be due to the lysophosphatidylcholines interfering with the physical coupling between the hormone receptor and catalytic unit of adenylate cyclase. 6. We suggest that, in vivo, it is possible that lysophosphatidylcholines may modulate the activity of adenylate cyclase only when it is in the hormone-stimulated state.     

Thyrotropin binding to and adenylate cyclase activity of porcine thyroid plasma membranes.

Plasma membranes have been purified from porcine thyroid gland homogenate by discontinuous sucrose gradient centrifugation. The preparations contained specific binding sites for thyrotropin but not for luteinizing hormone or the beta subunits of thyrotropin and luteinizing hormone. Optimum conditions of 125I-labeled thyrotropin binding were pH 6.0-6.5 and 37 degrees C. Thyrotropin binding was reduced by divalent (Ca2+, Mg2+) and monovalent cations (Na+, K+, Li+), 50% inhibition being obtained at 10 mM and 50 mM respectively. Displacement curves of 125I-labeled bovine or porcine thyrotropin by the unlabeled hormone from three species was in the order of increasing concentrations (bovine greater than porcine greater than human) which is the order of decreasing biological activity of these hormone preparations in the assay in vivo in the mouse. The validity of the results was established by controlling that porcine membranes bound the native and the 125I-labeled hormones with equal affinity. A single type of high-affinity (Kd = 0.28 nM) binding sites was detected for bovine and porcine thyrotropins. In contrast, porcine plasma membranes bound human thyrotropin with a lower affinity (Kd = 70 nM). A good correlation was found at equilibrium and in the conditions of the cyclase assay, between receptor occupancy and adenylate cyclase activation for the three hormones.     

Inhibition of adenylate cyclase activity by polyamines in human erythrocyte plasma membranes

Polyamines (spermidine, spermine and putrescine) inhibited the adenylate cyclase activity in a concentration dependent manner in human erythrocyte plasma membranes. Spermidine (Spd) exhibited more inhibitory effect than spermine (Spm) and putrescine (Put). On the contrary, the addition of amino acids (arginine, glutamine and lysine) did not influence the basal enzyme activity. Other cations (polylysine, polyarginine and polyglutamine) also did not affect the enzyme activity. Addition of all the three polyamines (Spd, Spm and Put) in the reaction mixture exhibited moderate inhibitory effect on the adenylate cyclase activity whether it was basal or activated with sodium fluoride or with forskolin. Since the three polyamines exhibited maximum inhibitory effect at 10 microM concentration which is within physiological limit for mammalian tissues, we suggest that there may be a regulatory function of these molecules on adenylate cyclase activity in human erythrocytes.     

Effect of serum lipoproteins on the adenylate cyclase activity of rat liver plasma membranes.

Four rat lipoprotein classes [lymph chylomicrons, VLD (very-low-density), LD (low-density) and HD (high-density) lipoproteins] were tested for their ability to affect basal adenylate cyclase (EC 4.6.1.1) activity of rat liver plasma membranes. All the lipoproteins, with the exception of lymph chylomicrons, effectively increase the enzyme activity. VLD lipoproteins are the most active class (67% maximal increase), followed by HD lipoproteins (33%) and LD lipoproteins (23%). The effect of VLD lipoproteins is additive to that elicited by GTP or GTP plus glucagon (at least within a certain concentration range). VLD lipoproteins affect only the Vmax. of the enzyme, not the Km.     

ANF stimulation of detergent-dispersed particulate guanylate cyclase from bovine adrenal cortex

Particulate guanylate cyclase from bovine adrenal cortex can be stimulated by ANF. A 2-fold stimulation of the enzyme was obtained with 100 nM ANF and a half-maximal stimulation, with a 5 nM dose. The stimulation by ANF persisted for at least 30 min. Various detergents, such as Triton X-100, Lubrol PX, cholate, CHAPS, digitonin and zwittergent, stimulated several-fold the activity of particulate guanylate cyclase. However, only Triton X-100 dispersed particulate guanylate cyclase without affecting its response to ANF. The dose-response curve of ANF stimulation of the particulate and the Triton X-100 dispersed enzyme was similar. The dispersion of a fully responsive guanylate cyclase to ANF will help us to uncover the type of interactions between guanylate cyclase and ANF. It will also be used as a first step for the purification of an ANF-sensitive particulate guanylate cyclase.     

Regulation of secretion from the adrenal medulla. Evidence for adenylate cyclase activity in secretory vesicle membranes.

Adenylate cyclase activity has been found in purified secretory vesicle membranes from the adrenal medulla. Activity was detected both by formation of radioactive cAMP from [alpha-32P]ATP and by the competitive protein binding assay for cAMP. Activity was highest at pH 8.0 to 8.5, and was stimulated by sodium fluoride and GppNHp, a GTP analogue known to stimulate adenylate cyclase activity in plasma membrane preparations. The reaction rate was strongly dependent on the molar ratio of Mg2+:ATP in the system. This is the first demonstration of adenylate cyclase in a secretory vesicle membrane.     

Distribution of parathyroid hormone-stimulated adenylate cyclase in plasma membranes of cells of the kidney cortex.

Free flow electrophoresis was employed to separate renal cortical plasma membranes into luminal (brush border microvilli) and contraluminal (basal-lateral membrane) fractions. During the separation adenylate cyclase activity was found to parallel the activity of Na+-K+-activated ATPase, an enzyme which is present in contraluminal but not in luminal membranes. In the basal-lateral membrane fraction the specific activities of adenylate cyclase and Na+-K+-activated ATPase were 4.4 and 4.6 times greater, respectively, than in the brush border fraction. The adenylate cyclase of the basal-lateral membrane fraction was specifically stimulated by parathyroid hormone which maximally increased enzyme activity eightfold. The biologically active (1-34) peptide fragment of paratyhroid hormone produced a 350% increase in adenylate cyclase activity. In contrast, calcitonin, epinephrine and vasopressin maximally stimulated the enzyme by only 55, 35 and 30%, respectively. These results indicate that adenylate cyclase, specifically stimulated by parathyroid hormone, is distributed preferentially in the contraluminal region of the plasma membrane of renal cortical epithelial cells.