Inhibition of adenylate cyclase by polyadenylate

The effects of ribo- and deoxyribonucleic acids on the activity of detergent-dispersed adenylate cyclases from rat and bovine brain were examined. Mn2+ (10 mM)-activated adenylate cyclase was inhibited by micromolar concentrations of poly(A) (IC50 congruent to 0.45 microM). This inhibition was directly due to poly(A) and was not mediated by: (a) protein contamination of the poly(A) preparation, (b) metal chelation, (c) formation of an acid-soluble inhibitor of adenylate cyclase, (d) effects on the specific activity of [alpha-32P]ATP, (e) competition with MnATP for binding to adenylate cyclase, or (f) diversion of substrate to an alternate polymerase reaction. Inhibition of adenylate cyclase by poly(A) was on the enzyme's catalytic unit, as purified preparations of the enzyme from bovine brain were inhibited by poly(A). This inhibition by poly(A) was not likely mediated via the enzyme's "P"-site, through which activated forms of the enzyme are selectively inhibited by specific adenosine phosphates. In contrast with inhibition by the "P"-site agonist 3' AMP, inhibition of adenylate cyclase by poly(A) was slow in onset and was not reversible by dilution and showed a different metal-dependence. Inhibition of adenylate cyclase was relatively specific for poly(A) as poly(U) caused less than 50% inhibition and deoxyribonucleic acids had no effect. The potency and specificity of the inhibition of adenylate cyclase by poly(A) imply a biochemically interesting interaction that is possibly also of physiological significance.     

Inhibition of the catalytic subunit of ram sperm adenylate cyclase by adenosine

Adenosine inhibits ram sperm adenylate cyclase activity which is membrane-bound and comprises only the catalytic subunit. The inhibition parameters of adenylate cyclase by adenosine were not modified when the enzyme was purified 3 to 5,000 fold. Optimal inhibition by adenosine was found to require a high concentration of manganese, and exhibited a noncompetitive pattern up to a concentration of 1 mM adenosine. Adenosine was the most potent inhibitor among various analogs tested with the following rank order of potencies: adenosine greater than 2'O-methyladenosine greater than 2'deoxyadenosine much greater than 2 chloroadenosine. Studies with agonists and antagonists of the "R"-type adenosine receptor led us to conclude that adenosine inhibits ram sperm adenylate cyclase via a "P"-site carried by the catalytic subunit itself.     

Guanine nucleotide activation and inhibition of adenylate cyclase as modified by islet-activating protein, pertussis toxin, in mouse 3T3 fibroblasts

Guanine nucleotide regulation of membrane adenylate cyclase activity was uniquely modified after exposure of 3T3 mouse fibroblasts to low concentrations of islet-activating protein (IAP), pertussis toxin. The action of IAP, which occurred after a lag time, was durable and irreversible, and was associated with ADP-ribosylation of a membrane Mr = 41,000 protein. GTP, but not Gpp(NH)p, was more efficient and persistent in activating adenylate cyclase in membranes from IAP-treated cells than membranes from control cells. GTP and Gpp(NH)p caused marked inhibition of adenylate cyclase when the enzyme system was converted to its highly activated state by cholera toxin treatment or fluoride addition, presumably as a result of their interaction with the specific binding protein which is responsible for inhibition of adenylate cyclase. This inhibition was totally abolished by IAP treatment of cells, making it very likely that IAP preferentially modulates GTP inhibitory responses, thereby increasing GTP-dependent activation and negating GTP-mediated inhibition of adenylate cyclase.     

Adenosine Inhibition of Calmodulin-Sensitive Adenylate Cyclase from Bovine Cerebral Cortex

Calmodulin (CaM)-sensitive adenylate cyclase has recently been purified extensively from bovine brain. In this study, the sensitivity of the CaM-sensitive adenylate cyclase to adenosine and adenosine analogs was examined. The highly purified enzyme preparation retained sensitivity to inhibition by adenosine and adenosine analogs with ribose ring modifications, but not to those with purine ring modifications. Adenosine inhibition of this enzyme was not dependent on GTP and was noncompetitive with respect to ATP. Enzyme that had been dissociated from functional guanine nucleotide binding protein interactions by gel filtration in the presence of the zwitterionic detergent 3-[3-(cholamidopropyl)-dimethylammonio]-propanesulfonate and Mn2+ retained sensitivity to adenosine inhibition. The Ki for adenosine inhibition of the CaM-sensitive adenylate cyclase was approximately 2.6 X 10(-4) M. 5'-Guanylylimidodiphosphate and CaM did not affect the Ki of 3'-deoxyadenosine for the enzyme, but the presence of Ca2+ in the millimolar range raised the Ki by a factor of 5. These results show that the CaM-sensitive form of adenylate cyclase from bovine brain is subject to adenosine inhibition, and strongly suggest that this inhibition is due to interaction of ligands with a purine-specific ("P") site located on the catalytic subunit of the enzyme.     

Potent and cooperative feedback inhibition of adenylate cyclase activity by calcium in pituitary-derived GH3 cells

Calcium (Ca2+) ion concentrations that are achieved intracellularly upon membrane depolarization or activation of phospholipase C stimulate adenylate cyclase via calmodulin (CaM) in brain tissue. In the present study, this range of Ca2+ concentrations produced unanticipated inhibitory effects on the plasma membrane adenylate cyclase activity of GH3 cells. Ca2+ concentrations ranging from 0.1 to 0.8 microM exerted an increasing inhibition on enzyme activity, which reached a plateau (35-45% inhibition) at around 1 microM. This inhibitory effect was highly cooperative for Ca2+ ions, but was neither enhanced nor dependent upon the addition of CaM (1 microM) to EGTA-washed membranes. The inhibition was greatly enhanced upon stimulation of the enzyme by vasoactive intestinal peptide (VIP) and/or GTP. Prior exposure of cultured cells to pertussis toxin did not affect the inhibition of plasma membrane adenylate cyclase activity by Ca2+, although in these membranes, hormonal (somatostatin) inhibition was significantly attenuated. Maximally effective concentrations of Ca2+ and somatostatin produced additive inhibitory effects on adenylate cyclase. The addition of phosphodiesterase inhibitors demonstrated that inhibitory effects of Ca2+ were not mediated by Ca2(+)-dependent stimulation of a phosphodiesterase activity. These observations provide a mechanism for the feedback inhibition by elevated intracellular Ca2+ levels on cAMP-facilitated Ca2+ entry into GH3 cells, as well as inhibitory crosstalk between Ca2(+)-mobilizing signals and adenylate cyclase activity.     

Presence of a functional inhibitory GTP-binding regulatory component, Gi, linked to adenylate cyclase in adipocytes of ob/ob mice

It has been reported recently (Begin-Heick, N. (1985) J. Biol. Chem. 260, 6187-6193) that adipocytes from the obese mouse strain (ob/ob), unlike normal mice (+/+), lack functional Gi, a GTP-regulated protein complex that mediates inhibition of adenylate cyclase. In contrast, we have found functional Gi linked to inhibition of adenylate cyclase in adipocyte membranes from both ob/ob and +/+ mice. This conclusion is based on observation of: 1) GTP-dependent inhibition of adenylate cyclase by antilipolytic agents, such as prostaglandin E2, nicotinic acid, and the adenosine receptor agonist, phenylisopropyladenosine (PIA); 2) classical biphasic GTP kinetics, with stimulation by low and inhibition by high concentrations of GTP; and 3) elimination of cyclase inhibition by antilipolytic agents upon treatment of ob/ob adipocytes with pertussis toxin. Upon treatment with pertussis toxin and [32P] NAD, purified adipocyte membranes from ob/ob mice incorporated twice as much radioactivity per unit membrane protein than those from +/+ mice in the 40,000-42,000 region. The inhibitory actions of PIA on adenylate cyclase were blocked by the adenosine receptor antagonists, theophylline and isobutylmethylxanthine. However, in contrast to other known inhibitory adenosine receptors, relatively high (100 nM) PIA concentrations were required for half-maximal inhibition of adenylate cyclases from both +/+ and ob/ob adipocytes. The adipocyte adenylate cyclase from both mouse strains were approximately equally susceptible to inhibition by nicotinic acid and prostaglandin E2. However, the ob/ob cyclase was inhibited by 47% with PIA, whereas the enzyme from the +/+ mouse was inhibited by only 27% (p less than 0.01). This greater inhibition by adenosine may contribute to abnormal fat metabolism in adipocytes from ob/ob mice.     

Evidence for the existence of a sulfhydryl group in the adenylate cyclase active site

6-Cloro-9-beta-d-ribofuranosylpurine 5'-triphosphate (CIRTP) and 6-mercapto-9-beta-d-ribofuranosylpurine 5'-triphosphate (SRTP) irreversibly inhibit adenylate cyclase from rat brain. Adenosine 5'-[beta, gamma -imido] triphosphate protects the enzyme against inactivation by CIRTP and SRTP and acts as a competitive inhibitor with respect to ATP with the Ki value 2 X 10(-4) M. Study of the pH-dependence of the rate of the enzyme inactivation by CIRTP showed that pK for the group modified by this compound is equal to 7.45. Inactivation is first order with respect to the enzyme; the saturation effect is observed at the increased concentration of CIRTP. The k2 and KI values for irreversible inhibition of brain adenylate cyclase by CIRTP were 0.25 min-1 and 1.9 X 10(-4) M, respectively. Adenylate cyclase inhibition by SRTP is also time-dependent. Partial protection against the enzyme inactivation was observed. Dithiothreitol restores the activity of SRTP-inactivated adenylate cyclase. The results obtained indicate the presence of an -SH group in the purine amino group binding area of the enzyme active site.     

Comparison of the Effects of Ions and GTP on Substance P Binding to Membrane-Bound and Solubilized Specific Sites

Mg2+ increased but Na+ and GTP decrease [3H]substance P (SP) binding to rat cerebral cortical membranes and to 10 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS)-solubilized membrane fraction. To determine the binding parameters that are modified by the cations and GTP, inhibition experiments of [3H]SP binding by unlabeled SP were performed in both of the preparations. Nonlinear least-squares regression analysis of data in the membrane fraction indicated that optimal fitting of the inhibition curves in the presence of 10 mM MgCl2 was attained with a two-site model, corresponding to a "high-affinity (H)" and a "low-affinity (L)" state. By omitting MgCl2, or by addition of NaCl and GTP, the [3H]SP specific binding was decreased, the H state disappeared, and the L state and a new "super-low affinity (SL)" state observed. The SP/[3H]SP inhibition curves in the cerebral cortical membranes by in vivo treatment with pertussis toxin (islet-activating protein) were similar to that in the presence of GTP in control membranes. The effects of MgCl2, NaCl, and GTP were greater in the CHAPS-solubilized fraction than in the membrane fraction. In contrast to the membrane fraction, the inhibition curves of [3H]SP binding by unlabeled SP in the presence of MgCl2 in the CHAPS-solubilized fraction were best fitted to a one-site model. The KD value was relatively close to that of the low-affinity state in the membrane fraction. Even with the addition of NaCl or GTP, or by reducing MgCl2 concentration to 1 mM, although the inhibition curves consistently fit the one-site model, the KD values changed only slightly.     

Acidic phospholipid species inhibit adenylate cyclase activity in rat liver plasma membranes.

Incubation of rat liver plasma membranes with liposomes of dioleoyl phosphatidic acid (dioleoyl-PA) led to an inhibition of adenylate cyclase activity which was more pronounced when fluoride-stimulated activity was followed than when glucagon-stimulated activity was followed. If Mn2+ (5 mM) replaced low (5 mM) [Mg2+] in adenylate cyclase assays, or if high (20 mM) [Mg2+] were employed, then the perceived inhibitory effect of phosphatidic acid was markedly reduced when the fluoride-stimulated activity was followed but was enhanced for the glucagon-stimulated activity. The inhibition of adenylate cyclase activity observed correlated with the association of dioleoyl-PA with the plasma membranes. Adenylate cyclase activity in dioleoyl-PA-treated membranes, however, responded differently to changes in [Mg2+] than did the enzyme in native liver plasma membranes. Benzyl alcohol, which increases membrane fluidity, had similar stimulatory effects on the fluoride- and glucagon-stimulated adenylate cyclase activities in both native and dioleoyl-PA-treated membranes. Incubation of the plasma membranes with phosphatidylserine also led to similar inhibitory effects on adenylate cyclase and responses to Mg2+. Arrhenius plots of both glucagon- and fluoride-stimulated adenylate cyclase activity were different in dioleoyl-PA-treated plasma membranes, compared with native membranes, with a new 'break' occurring at around 16 degrees C, indicating that dioleoyl-PA had become incorporated into the bilayer. E.s.r. analysis of dioleoyl-PA-treated plasma membranes with a nitroxide-labelled fatty acid spin probe identified a new lipid phase separation occurring at around 16 degrees C with also a lipid phase separation occurring at around 28 degrees C as in native liver plasma membranes. It is suggested that acidic phospholipids inhibit adenylate cyclase by virtue of a direct headgroup specific interaction and that this perturbation may be centred at the level of regulation of this enzyme by the stimulatory guanine nucleotide regulatory protein NS.     

Improvement of reconstitution of the Cl(-)-translocating ATPase isolated from Acetabularia acetabulum into liposomes and several anion pump characteristics.

The improved reconstitution of the Mono Q-III fraction, a Cl(-)-translocating ATPase, isolated from Acetabularia acetabulum (Ikeda et al. (1990) Biochemistry 29, 2057-2065) into liposomes rendered transport properties of this enzyme clear. The liposomes were prepared by the reversed-phase method using egg lecithin and cholesterol in a molar ratio of 2:1 and the purified ATPase was incorporated into the liposomes by a dialysis for 3 h. About 80% of the ATPase was incorporated into the liposomes. The weight ratio of the enzyme to lipid was 1:400-600. A sigmoid curve was obtained when the Cl(-)-transport activity of the enzyme was plotted against Cl- concentration. Hill's plot afforded a half-substrate concentration [S]0.5 of 45 mM and a Hill's coefficient n of 2.33. Effects of Br- and F- on the Cl(-)-transport were also examined in the reconstituted system, both halide ions decreased the 36Cl- efflux significantly. These kinetic data are in good agreement with the electrophysiological data presented by Tittor et al. ((1983) J. Membr. Biol. 75, 129-139).     

Heterologous desensitization of the inhibitory A1 adenosine receptor-adenylate cyclase system in rat adipocytes. Regulation of both Ns and Ni

Adenosine, acting via A1 adenosine receptors, can inhibit adenylate cyclase activity in adipocytes. To assess the effects of chronic adenosine agonist exposure on the A1 adenosine receptor system of adipocytes, rats were infused with (-)-phenylisopropyladenosine or vehicle for 6 days and membranes were prepared. Basal as well as isoproterenol-, sodium fluoride-, and forskolin-stimulated adenylate cyclase activities were significantly increased (approximately 2-fold) in membranes from treated animals. (-)-Phenylisopropyladenosine-mediated inhibition of forskolin-stimulated adenylate cyclase activity was significantly (p = 0.0001) attenuated in membranes from treated rats (20.1 +/- 2.1% inhibition) versus controls (31.6 +/- 2.3% inhibition). Prostaglandin E1-induced inhibition of forskolin-stimulated adenylate cyclase activity was also attenuated: 11.7 +/- 3.6 versus 23.2 +/- 4.6% (p = 0.001). Using the A1 adenosine receptor agonist radioligand (-)-N6-(3-[125I]iodo-4-hydroxyphenylisopropyl)adenosine, 32% fewer high affinity binding sites were detected in membranes from treated animals (p less than 0.04). Photoaffinity labeling with N6-2-(3-[125I]iodo-4-azidophenyl)ethyladenosine revealed no gross difference in receptor structure. The number of beta-adrenergic receptors as well as the percentage of receptors in the high affinity state as assessed by (-)-3-[125I]iodocyanopindolol binding were the same in both groups. In membranes from treated rats, the amount of [alpha-32P]NAD incorporated by pertussis toxin into the alpha subunit of the inhibitory guanine nucleotide regulatory protein (Ni) was decreased by 37 +/- 11%. Concurrently, the quantity of label incorporated by cholera toxin into the alpha subunit of the stimulatory guanine nucleotide regulatory protein (Ns) was increased by 44 +/- 14% in treated membranes. Finally, the capacity of Ns solubilized from treated membranes to stimulate adenylate cyclase activity when reconstituted into cyc- S49 lymphoma cell membranes was enhanced by approximately 50% compared to control. Thus, heterologous desensitization, manifested by a diminished capacity to inhibit adenylate cyclase and an enhanced responsiveness to stimulatory effectors, can be induced in the A1 adenosine receptor-adenylate cyclase system of adipocytes. A decrease in Ni alpha subunit concomitant with an increase in Ns alpha subunit quantity and activity may represent the biochemical mechanism of desensitization in this system.     

Galanin inhibits adenylate cyclase of rat brain membranes.

The ubiquitous neuropeptide, galanin, strongly inhibits adenylate cyclase in rat brain membranes. While basal enzyme activity was not altered, galanin from 10(-11) M to 5 x 10(-7) M decreased forskolin- and VIP-stimulated adenylate cyclase with a half-maximal effect being elicited by 0.7 nM neuropeptide and a maximal 80% inhibition of the enzyme activity. The galanin fragments (2-29) and (1-15) dose-dependently inhibited the forskolin-stimulated adenylate cyclase, while the fragments (3-29) and (10-29) were found inactive. These results indicate that the regulatory action of galanin in the central nervous system involves the coupling of galanin receptors to the inhibition of the adenylate cyclase system.     

Inhibition of adenylate cyclase by the 2',3'-dialdehyde of adenosine triphosphate

The periodate-oxidized analog of ATP, 2',3'-dialATP, competitively inhibited bovine brain and rat liver adenylate cyclase. The apparent Ki for inhibition of brain adenylate cyclase by 2',3'-dialATP was 196 microM in the presence of Mg2+ and 37 microM in the presence of Mn2+. The Ki values for inhibition of rat liver adenylate cyclase by 2',3'-dialATP were 48 and 30 microM in the presence of Mg2+; and Mn2+, respectively. Adenylate cyclase activity was irreversibly inactivated by 2'3'-dialATP in the presence of NaCNBH3 and the kinetics for loss in enzyme activity were pseudo-first order. Both ATP and Tris protected adenylate cyclase from irreversible inhibition by 2',3'-dialATP and NaCNBH3. It is proposed that 2',3'-dialATP forms a Schiff's base with an amino group at the active site of the enzyme and that Na-CNBH3 reduction of this Schiff's base causes irreversible modification of the catalytic subunit. The Km for 2',3'-dialATP inactivation, the maximal rate constant of inactivation, and protection of the enzyme by ATP were not affected by the presence or absence of free Mg2+. These data indicate that a divalent cation is not required for binding of 2',3'-dialATP to the active site of adenylate cyclase.     

Modulation of adenylate cyclase activity by sulfated glycosaminoglycans. I. Inhibition by heparin of gonadotrophin-stimulated ovarian adenylate cyclase.

Heparin inhibits (I50 = 2 microgram/ml) the activity of luteinizing hormone and human chorionic gonadotropin-stimulated adenylate cyclase in purified rat ovarian plasma membranes. Unstimulated enzyme activity and activity stimulated by NaF, GTP or guanosine 5'-(beta,gamma-imido)triphosphate were inhibited to a lesser extent. Human chorionic gonadotropin binding to this membrane preparation was inhibited by heparin (I50 = 6 microgram/ml). The inhibition with respect to hormone concentration was of a mixed type for hormone binding and adenylate cyclase stimulation. Inhibition by heparin was not eliminated at saturating hormone concentration. The degree of inhibition was unaffected by the order in which enzyme, hormone and heparin were introduced into the assay system. Heparin (3 microgram/ml) did not affect the pH activity relationship of basal and hormone-stimulated adenylate cyclase activity and did not change the dependence of enzyme activity on magnesium ion concentration. The inhibitory action of heparin cannot be solely attributed to interference with either catalysis or hormone binding. The possibility is considered that the highly charged heparin molecule interferes with enzyme receptor coupling, by restricting the mobility of these components or by effecting their conformation.     

Forskolin-resistant Y1 mutants harbor defects associated with the guanyl nucleotide-binding regulatory protein, Gs

The properties of the adenylate cyclase from forskolin-resistant mutants of Y1 adrenocortical tumor cells was compared with the properties of the enzyme from parental Y1 cells in order to localize the site of mutation. In parental Y1 cells, forskolin stimulated adenylate cyclase activity with kinetics suggestive of an interaction at two sites; in mutant cells, forskolin resistance was characterized by a decrease in enzymatic activity at both sites. Forskolin potentiated the enzyme's responses to NaF and guanyl-5'-yl imidodiphosphate (Gpp(NH)p) in parent and mutant clones, and the mutant enzyme showed the same requirements for Mg2+ and Mn2+ as did the parent enzyme. The adenylate cyclase associated with forskolin-resistant mutants was insensitive to ACTH and was less responsive to Gpp(NH)p than was the parent enzyme. In parental Y1 cells and in the forskolin-resistant mutants, cholera toxin catalyzed the transfer of [32P]ADP-ribose from [32P]NAD+ into three membrane proteins associated with the alpha subunit of Gs; however, the amount of labeled ADP-ribose incorporated into mutant membranes was reduced by as much as 70%. Both parent and mutant membranes were labeled by pertussis toxin to the same extent. The insensitivity of the mutant adenylate cyclase to ACTH and Gpp(NH)p and the selective resistance of the mutant membranes to cholera toxin-catalyzed ADP-ribosylation suggest that a specific defect associated with Gs is involved in the mutation to forskolin resistance in Y1 cells.     

Enzymes of adenylate metabolism and their role in hibernation of the white-tailed prairie dog, Cynomys leucurus

AMP deaminase (AMPD) and adenylate kinase (AK) were purified from skeletal muscle of the white-tailed prairie dog, Cynomus leucurus, and enzyme properties were assayed at temperatures characteristic of euthermia (37 degrees C) and hibernation (5 degrees C) to analyze their role in adenylate metabolism during hibernation. Total adenylates decreased in muscle of torpid individuals from 6.97 +/- 0. 31 to 4.66 +/- 0.58 micromol/g of wet weight due to a significant drop in ATP but ADP, AMP, IMP, and energy charge were unchanged. The affinity of prairie dog AMPD for AMP was not affected by temperature and did not differ from that of rabbit muscle AMPD, used for comparison. However, both prairie dog and rabbit AMPD showed much stronger inhibition by ions and GTP at 5 degrees C, versus 37 degrees C, and inhibition by inorganic phosphate, NH(4)Cl, and (NH(4))(2)SO(4) was much stronger at 5 degrees C for the prairie dog enzyme. Furthermore, ATP and ADP, which activated AMPD at 37 degrees C, were strong inhibitors of prairie dog AMPD at 5 degrees C, with I(50) values of 1 and 14 microM, respectively. ATP also inhibited rabbit AMPD at 5 degrees C (I(50) = 103 microM). Strong inhibition of AMPD at 5 degrees C by several effectors suggests that enzyme function is specifically suppressed in muscle of hibernating animals. By contrast, AK showed properties that would maintain or even enhance its function at low temperature. K(m) values for substrates (ATP, ADP, AMP) decreased with decreasing temperature, the change in K(m) ATP paralleling the decrease in muscle ATP concentration. AK inhibition by ions was also reduced at 5 degrees C. The data suggest that adenylate degradation via AMPD is blocked during hibernation but that AK maintains its function in stabilizing energy charge.     

Calcitonin-sensitive adenylate cyclase in rat renal tubular membranes.

1. Renal tubular membranes from rat kidneys were prepared, and adenylate cyclase activity was measured under basal conditions, after stimulation by NaF or salmon calcitonin. Apparent Km value of the enzyme for hormone-linked receptor was close to 1 x 10(-8) M. 2. The system was sensitive to temperature and pH. pH was found to act both on affinity for salmon calcitonin-linked receptor and maximum stimulation, suggesting an effect of pH on hormone-receptor binding and on a subsequent step. 3. KCl was without effect areas whereas CoCl and CaCl2 above 100 muM and MnCl2 above 1 muM inhibited F- -and salmon calcitonin-sensitive adenylate cyclase activities. The Ca2+ inhibition of the response reflected a fall in maximum stimulation and not a loss of affinity of salmon calcitonin-linked receptor for the enzyme. 4. The measurement of salmon calcitonin-sensitive adenylate cyclase activity as a function of ATP concentration showed that the hormone increases the maximum velocity of the adenylate cyclase. GTP, ITP and XTP at 200 muM did not modify basal, salmon calcitonin- and parathyroid hormone-sensitive adenylate cyclase activities. 5. Basal, salmon calcitonin- and F- -sensitive adenylate cyclase activities decreased at Mg2+ concentrations below 10 mM. High concentrations of Mg2+ (100 mM) led to an inhibition of the F- -stimulated enzyme. 6. Salmon calcitonin-linked receptor had a greater affinity for adenylate cyclase than human or porcine calcitonin-linked receptors. There was no additive effect of these three calcitonin peptides whereas parathyroid hormone added to salmon calcitonin increased adenylate cyclase activity, thus showing that both hormones bound to different membrane receptors. Human calcitonin fragments had no effect on adenylate cyclase activity. 7. Salmon calcitonin-stimulated adenylate cyclase activity decreased with the preincubation time. This was due to progressive degradation of the hormone and not to the rate of binding to membrane receptors.     

Interactions of platinum complexes with the essential and nonessential sulfhydryl groups of thymidylate synthetate.

Thymidylate synthetase (methylenetetrahydrofolate:deoxyuridylate C-methyltransferase) from Lactobacillus casei was progressively inactivated when incubated at 25 degrees C, pH 6.8, in the presence of trans-Pt(NH3)2Cl2. The inhibition appeared to be irreversible, and the rate ofa ctivity loss was dependent on the inhibitor concentration. The corresponding cis isomer was incapable of inhibiting the enzyme under the same conditions. The presence of 2-mercaptoethanol protected the enzyme from inhibition, but did not reactivate enzyme preparations which had been inhibited prior to the addition of the thiol. The interactions of cis- and trans-Pt(NH3)2Cl2 with the enzyme's sulfhydryl (-SH) groups were inferred from the results of spectrophotometric titrations of the enzyme with 5,5'-dithiobis(2-nitrobenzoic acid) and p-hydroxymercuribenzoate. The results suggested that the cis isomer reacted with an average of 1.3 of the enzyme's 4-SH groups and that these were not essential for catalysis. The trans isomer reacted with a total of approximately 2.5 -SH groups, 1.2 of which are essential for catalysis. Neither the trans isomer nor a combination of both isomers was able to react with 1.2 of the 4 -SH groups. Further evidence that the Pt complexes are interacting with enzyme's -SH groups was obtained by reversibly blocking the -SH groups of thymidylate synthetase, and demonstrating the resistance of these preparations to inhibition by the trans Pt complex. Possible explanations for the preferential inhibition of thymidylate synthetase by only one of the two geometric isomers of Pt(NH3)2Cl2 are considered.     

Purification and properties of squid mantle adenylate kinase. Role of NADH in control of the enzyme.

Adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3) from the mantle muscle of the squid, Loligo pealeii, was purified over 170-fold to homogeneity as judged by polyacrylamide and starch gel electrophoresis. The tissue contains a single isozyme of adenylate kinase, the enzyme from cytoplasmic and mitochondrial compartments (90 and 10% of total activity, respectively) being identical in physical and kinetic properties. Molecular weight was found to be 27,000 +/- 400. The enzyme shows a pH optimum of 8.2 in the forward (APD utilizing) and 7.4 in the reverse direction. Michaelis constants for ADP, ATP, and AMP are 0.70, 0.13, and 0.15 mM, respectively, with optimal Mg2+:adenylate ratios being 1:2 for ADP and 1:1 for ATP. A comparison of mass action ratios with the equilibrium constant indicated that squid adenylate kinase is held out of equilibrium in resting, but not active, muscle. A search for metabolic modulators of adenylate kinase revealed that NADH (Ki of 0.1 mM) was the only modulator which exerted a significant effect within its in vivo concentration range. The data presented indicate that NADH inhibition is the factor maintaining adenylate kinase in a nonequilibrium state in resting muscle and that release of this inhibition can serve to integrate adenylate kinase into the known scheme of intermediary metabolism in this tissue. A sharp drop in NADH levels at the onset on muscular work co-ordinates that activation of aerobic metabolism in this tissue and allows adenylate kinase to return to equilibrium function. At equilibrium, the enzyme can function to ampligy the concentration of AMP, a potent activator and deinhibitor of key glycolytic and Krebs cycle enzymes. The effect of modulators of adenylate kinase in preventing denaturation by heat or proteolysis revealed that NADH and substrates induced conformational changes in the enzyme which rendered it less susceptible to denaturation. The conformation state induced by NADH differed from that induced by substrate.