The HLB dependency for detergent solubilization of hormonally sensitive adenylate cyclase.

The HLB dependency for the solubilization of membrane proteins and adenylate cyclase activity from a plasma membrane-enriched fraction from rat liver has been determined. The HLB (hydrophilic/lipophilic/balance) number of a detergent is an empirical measure of its relative hydrophobicity. Detergent HLB numbers vary systematically with the length of the ethylene oxide chain for a homologous series of detergents such as the Triton X series. These detergents have a constant hydrophobic moiety, octylphenyl, and a variable polar portion, polyethoxyethanol. Basal-NaF-epinephrine-, and glucagon-stimulated adenylate cyclase activities were solubilized in the HLB range of 16.8-17.4. Solubilization was most effective in 0.01 M Tris buffers at pH 7.5 containing 1-5 mM mercaptoethanol, 1 mM MgCl2, and 0.1% Triton X-305. The detergent to membrane protein ratio used in these studies was 3:1. Criteria for solubilization included lack of sedimentation at 100,000 X g, the absence of particulate material in the supernatant when examined by electron microscopy, and inclusion of hormonally sensitive adenylate cyclase activity in Sephadex G-200 gels. The apparent molecular weight of the solubilized enzyme was approximately 200,000 in the presence of Triton X-305. The solubilized enzyme was stimulated 5-fold by NaF, 7-fold by glucagon, and 20-fold by epinephrine compared to the particulate enzyme used in this study which was stimulated 10-fold, 3.4-fold, and 4-fold by NaF, epinephrine, and glucagon, respectively. The solubilized enzyme is stable for several weeks when stored at -60 degrees C.     

Absorption and fluorescence characteristics of photo-activated adenylate cyclase nano-clusters from the amoeboflagellate Naegleria gruberi NEG-M strain

The spectroscopic characteristics of BLUF (BLUF = sensor of blue light using flavin) domain containing soluble adenylate cyclase (nPAC = Naegleria photo-activated cyclase) samples from the amoeboflagellate Naegleria gruberi NEG-M strain is studied at room temperature. The absorption and fluorescence spectroscopic development in the dark was investigated over two weeks. Attenuation coefficient spectra, fluorescence quantum distributions, fluorescence quantum yields, and fluorescence excitation distributions were measured. Thawing of frozen nPAC samples gave solutions with varying protein nano-cluster size and varying flavin, tyrosine, tryptophan, and protein color-center emission. Protein color-center emission was observed in the wavelength range of 360-900 nm with narrow emission bands of small Stokes shift and broad emission bands of large Stokes shift. The emission spectra evolved in time with protein nano-cluster aging.     

Current Modulation of Guanylate Cyclase Pathway Activity—Mechanism and Clinical Implications

For years, guanylate cyclase seemed to be homogenic and tissue nonspecific enzyme; however, in the last few years, in light of preclinical and clinical trials, it became an interesting target for pharmacological intervention. There are several possible options leading to an increase in cyclic guanosine monophosphate concentrations. The first one is related to the uses of analogues of natriuretic peptides. The second is related to increasing levels of natriuretic peptides by the inhibition of degradation. The third leads to an increase in cyclic guanosine monophosphate concentration by the inhibition of its degradation by the inhibition of phosphodiesterase type 5. The last option involves increasing the concentration of cyclic guanosine monophosphate by the additional direct activation of soluble guanylate cyclase. Treatment based on the modulation of guanylate cyclase function is one of the most promising technologies in pharmacology. Pharmacological intervention is stable, effective and safe. Especially interesting is the role of stimulators and activators of soluble guanylate cyclase, which are able to increase the enzymatic activity to generate cyclic guanosine monophosphate independently of nitric oxide. Moreover, most of these agents are effective in chronic treatment in heart failure patients and pulmonary hypertension, and have potential to be a first line option.     

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.     

Vasopressin-sensitive kidney adenylate cyclase: modulation of the hormonal response.

Vasopressin-sensitive pig kidney adenylate cyclase is sensitive to several effectors, such as Mg2+, other divalent cations, and guanyl nucleotides. The purpose of the present study was to compare the main characteristics of adenylate cyclase activation by vasopressin, Mg2+, and GMPPNP, respectively. Mg2+ ions were shown to exert at least three different effects on adenylate cyclase. The substrate of the adenylate cyclase reaction is the Mg-ATP complex. Mg2+ interacts with an enzyme regulatory site. Finally, Mg2+ can modulate the hormonal response, with Mg2+ ions affecting the coupling function--that is, the quantitative relationship between receptor occupancy and adenylate cyclase activation. At all the magnesium concentrations tested, from 0.25 mM to 16 mM, adenylate cyclase activation was not a direct function of receptor occupancy. At low Mg2+ concentrations, adenylate cyclase activation dose-response curve to the hormone tended to be superimposable to the hormone dose-binding curve. These results suggest a role of magnesium at the coupling step between the hormone-receptor complex and adenylate cyclase response. Cobalt, but not calcium, ions could exert the same effects as Mg2+ ions on this coupling step. GMPPNP induced considerable adenylate cyclase activation (15 to 35 times the basal value). Activation by GMPPNP was highly time and temperature dependent. At 30 degrees C, a 20 to 60 min preincubation period in the presence of GMPPNP was needed to obtain maximal activation. The higher the dose of GMPPNP in the medium, the longer it took to reach equilibrium. At 15 degrees C, activation was still increasing with time after 3 hr preincubation in the presence of the nucleotide. GMPPNP was active in a 10(-8)M to 10(-5)M concentration range. Unlike the results obtained with lysine vasopressin, the kinetic characteristics of dose-dependent adenylate cyclase activation curves by GMPPNP were unaffected by varying Mg2+ concentrations except for the increase in velocity when raising Mg2+ concentration. It was not clear whether or not the activation processes by the hormone and by GMPPNP had common mechanisms.     

Some kinetic and chromatographic properties of detergent-dispersed adenylate cyclase.

Studies on the reaction kinetics and chromatographic properties of detergent-dispersed adenylate cyclase are described. Detergent-dispersed enzyme was prepared from whole rat cerebellum and from partially purified plasma membranes from rat liver. Data were simulated to fit kinetic models for which an inhibitor is added in constant proportion to the variable substrate. Models were chosen to distinguish whether the adenylate cyclase reaction may be controlled by an inhibitory action of free ATP--4 (or HATP--3) or by a stimulatory action of free divalent cations. The various kinetic models were then tested with the dispersed brain adenylate cyclase with both Mg++ and Mn++ and in two different buffer systems. The experimental data indicate that this enzyme has a distinct cation binding site, but exhibits no significant inhibition by HATP--3 or ATP--4. The detergent-dispersed adenylate cyclase both from liver plasma membranes and from brain have been chromatographed on anion exchange material and have been subjected to gel filtration. The presence of detergent was required for elution of cyclase activity from DEAE-Sephadex but was not required when DEAE-agarose was used. Dispersed brain cyclase was also chromatographed on agarose-NH(CH2)3NH(CH2)3-NH2 which exhibits both ionic and hydrophobic properties. Fifty percent of the applied activity was recovered with a fivefold increase in specific activity. The data suggest that the relative effectiveness of a given chromatographic procedure for detergent-dispersed adenylate cyclase may reflect the influence of both hydrophobic and ionic factors.     

The cytochemical localization of adenylate cyclase activity in mucous and serous cells of the salivary gland.

The present study was undertaken to localize adenylate cyclase activity in salivary glands by cytochemical means. For the study, serous parotid glands and mixed sublingual glands of the rat were used. Pieces of the fixed glands were incubated with adenosine triphosphate (ATP) or adenylyl-imidodiphosphate (AMP-PNP) as substrate: inorganic pyrophosphate or PNP liberated upon the action of adenylate cyclase on the substrates is precipitated by lead ions at their sites of production. In both glands, the reaction product was detected along the myoepithelial cell membranes in contact with secretory cells, indicating that a high level of adenylate cyclase activity occurs in association with these cell membranes. The association with a high level of the enzyme activity might be related to the contractile nature of myoepithelial cells which are supposed to aid secretory cells in discharging secretion products. A high level of adenylate cyclase activity was also detected associated with serous secretory cells (acinar cells of the parotid gland and demilune cells of the sublingual gland), but not with mucous secretory cells. In serous cells, deposits of reaction product were localized along the extracellular space of the apical cell membrane bordering the lumen. This is the portion of the cell membrane which fuses with the granule membranes during secretion. Since the granule membranes are not associated with a detectable level of adenylate cyclase activity, it appears that the enzyme activity becomes activated or associated with the granule membranes as they become part of the cell membrane by fusion. The association with a high level of adenylate cyclase activity appears to be related to the ability of the membrane to fuse with other membranes. It is likely, since the luminal membrane of mucous cells which does not fuse with mucous granule membranes during secretion is not associated with a detectable enzyme activity.     

Purification and some properties of squalene-2,3-epoxide: lanosterol cyclase from rat liver

Squalene-2,3-epoxide: lanosterol cyclase was purified from rat liver in five steps as a soluble and homogeneous protein. The purified enzyme showed a single band on SDS-polyacrylamide gel electrophoresis with a molecular weight of 75 kD. In its native state it behaved as a homo-dimer. The isoelectric point of 5.5 and the apparent Km value for (3S)-squalene-epoxide of 55 microM were estimated for the cyclase.     

The regulatory control of beta-receptor dependent adenylate cyclase.

The characteristics of the beta-receptor in turkey erythrocyte adenylate cyclase were studied using both kinetics of enzyme activation and direct binding measurement of the beta-agonists and antagonists to the beta-receptor. The regulatory ligands Gpp(NH)p and Ca2+ do not have any direct effect on the beta-receptor, but modulate the enzyme activity through the interaction with specific regulatory sites.     

Effect of transmembrane Ca2+ gradient on conformation and enzyme activity of reconstituted adenylate cyclase.

Adenylate cyclase from bovine brain cortex was reconstituted into asolectin liposomes with (500-fold) or without transmembrane Ca2+ gradient. The enzyme activity of four types of proteoliposomes (the active center of enzyme exposing outside) was compared. The highest adenylate cyclase activity was observed in the vesicles with outside lower Ca2+ concentration (approximately 10(-6) mol/L, similar to the physiological condition). If the transmembrane Ca2+ gradient was in the inverse direction (i.e. outside higher Ca2+ concentration, 0.5 mmol/L), a lowest enzymatic activity would appear. The difference in enzymatic activity between the two types of proteoliposomes could be diminished following the addition of Ca2+ ionophore A23187. Proteoliposomes without transmembrane Ca2+ gradient exhibited intermediate activities. The conformation difference of adenylate cyclases in the above-mentioned proteoliposomes was also detected by measuring intrinsic fluorescence and fluorescence quenching with KI.     

Mass spectrometric analysis of recombinant adenylate cyclase toxin from Bordetella pertussis strain 18323/pHSP9

The adenylate cyclase toxin-hemolysin (ACT) is a key virulence factor of the whooping cough agent Bordetella pertussis (Bp). The major cytotoxic activity of this 1706-residue protein consists of its capacity to invade a variety of eukaryotic cells directly across their cytoplasmic membrane and to deliver into cells a catalytic adenylate cyclase domain. This causes impairment of immune effector cells and apoptosis of lung macrophages by uncontrolled conversion of ATP to cAMP. The adenylate cyclase toxin-hemolysin acquires biological activity upon post-translational amide-linked palmitoylation of the epsilon-amino group of lysine 983 (K983) by the accessory fatty acyltransferase, CyaC. However, an additional conserved acylation site can be identified in ACT at lysine 860 (K860) and this residue is palmitoylated when recombinant ACT is produced in Escherichia coli (r-Ec-ACT). In this paper we report the double acylation of r-Bp-ACT secreted by a recombinant Bp strain 18323/pHSP9. This strain overproduces ACT from an oligocopy plasmid carrying the entire cya locus of Bordetella pertussis 18323. Palmitoylation of both conserved lysines (K860 and K983) of r-Bp-ACT expressed by this Bp strain was found. In addition, an error in the deduced protein sequence was identified, with Leu being the real residue at position 1001 and not the Val residue given in the published gene sequence. We also discuss these results in comparison with those from recombinant ACT expressed in E. coli strain K12 XL1-Blue. The analytical approach for characterization of the fatty acylation of ACT from strain 18323/pHSP9 consisted of multiple proteolytic digestion procedures (trypsin, Asp-N), microcapillary liquid chromatography/tandem mass spectrometry and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.     

The roles of cysteines in the heme domain of human soluble guanylate cyclase

Soluble guanylate cyclase(sGC) is a critical heme-containing enzyme involved in NO signaling.The dimerization of sGC subunits is necessary for its bioactivity and its mechanism is a striking and an indistinct issue.The roles of heme domain cysteines of the sGC on the dimerization and heme binding were investigated herein.The site-directed mutations of three conserved cysteines(C78A,C122A and C174S) were studied systematically and the three mutants were characterized by gel filtration analysis,UV-vis spectroscopy and heme transfer examination.Cys78 was involved in heme binding but not referred to the dimerization,while Cys174 was demonstrated to be involved in the homodimerization.These results provide new insights into the cysteine-related dimerization regulation of sGC.     

Effective inhibition by pentobarbital of forskolin-stimulated adenylate cyclase activity in rat brain

The effect of pentobarbital on the adenylate cyclase system was examined in synaptosomal membranes from rat brain. Pentobarbital inhibited forskolin-stimulated enzyme activity more effectively than the basal and Mn2(+)-stimulated enzyme activities. The degree of inhibition of the enzyme activity by pentobarbital was increased by the presence of forskolin in a concentration-dependent manner. No significant difference is observed in the degree of the inhibition by pentobarbital between the basal and forskolin-stimulated activities in the membranes prepared from the peripheral tissues.     

Boric acid inhibits adenosine diphosphate-ribosyl cyclase non-competitively

Adenosine diphosphate-ribosyl cyclase (ADP-ribosyl cyclase) is a ubiquitous enzyme in eukaryotes that converts NAD+ to cyclic-ADP-ribose (cADPR) and nicotinamide. A quantitative assay for cADPR was developed using capillary electrophoresis to separate NAD+, cADPR, ADP-ribose, and ADP with UV detection (254 nm). Using this assay, the apparent Km and Vmax for Aplysia ADP-ribosyl cyclase were determined to be 1.24+/-0.05 mM and 131.8+/-2.0 microM/min, respectively. Boric acid inhibited ADP-ribosyl cyclase non-competitively with a Ki of 40.5+/-0.5 mM. Boric acid binding to cADPR, determined by electrospray ionization mass spectrometry, was characterized by an apparent binding constant, KA, of 655+/-99 L/mol at pH 10.3.     

Requirement for guanosine triphosphate in the activation of adenylate cyclase by cholera toxin.

The activation of adenylate cyclase in lysed pigeon erythrocytes requires, among several cofactors, a nucleotide which may be ATP, GTP, or many other triphosphates. However, after removal of endogenous nucleotides by gel filtration or by adsorption onto charcoal the requirement can be met only by GTP, or an analog of GTP. The GTP is required during the activation of the cyclase by toxin even if GTP is also included during the subsequent adenylate cyclase assay, conducted without toxin. In the presence of GTP it is possible to assay for the cytosolic protein that is also required for the action of cholera toxin. By gel filtration, its apparent molecular weight is 15,000--20,000.     

Product identification and adenylyl cyclase activity in chloroplasts of Nicotiana tabacum

In view of the ongoing debate on plant cyclic nucleotide metabolism, especially the functional presence of adenylyl cyclase, a novel detection method has been worked out to quantify the reaction product. Using uniformly labelled (15)N-ATP as a substrate for adenylyl cyclase, a qualitative and quantitative liquid chromatography/electrospray ionisation tandem mass spectrometry (LC/ESI-MS/MS) method was developed to measure de novo formed (15)N-adenosine 3',5'-cyclic monophosphate. Adenylyl cyclase activity was observed in chloroplasts obtained from Nicotiana tabacum cv. Petit Havana and the kinetic parameters and influence of various metabolic effectors are discussed in their context.     

Interfacial composition and structural parameters of water/C12-s-C12 x 2Br/n-hexanol/n-heptane microemulsions studied by the dilution method

The interfacial composition of the stable water/C12-s-C12 x 2Br/n-hexanol/n-heptane microemulsions has been studied in detail by dilution method. The results showed a marked maximum amount of the n-hexanol populating on the surfaces of droplets (represented as a = n(a)i/n(s), where n(a)i and n(s) are respectively the moles of n-hexanol and gemini surfactant on the surface of droplets) with increasing water content. At a constant level of water addition (the molar ratio of water to surfactant W0 = 20), a decreased with increasing the spacer length in the C12-s-C12 x 2Br molecule. The structural parameters of a w/o microemulsion were also estimated by analyzing the data of dilution experiments, and we found that the radius of the water pool was very sensitive to the increment of water content. The radius of the water pool varied from 0.74 to 5.35 nm with increasing W0 from 10 to 50. The variation extent reached 4.61 nm. In the cases of water/CPC/n-butanol/isopropyl myristate and water/CTAB/n-butanol/isopropyl myristate, however, the corresponding variation extents were only 1.22 and 1.68 nm, respectively, when increasing comparable water content. The ratio of N(a)/N(2C), where N(a) and N(2C) are respectively the average numbers of n-hexanol and the total average numbers of alkyl chains of gemini surfactant populating on per droplet surface, decreased obviously with increasing water content at W0 > 15. This indicated that C12-2-C12 x 2Br favored to form large droplets that were suitable to solubilize more water.     

Effect of light on soluble guanylyl cyclase activity in Pharbitis nil seedlings

Cyclic GMP acts as a chemical switch in plant cells to modulate cellular reactions. However, its metabolism has not been extensively explored and is still poorly understood. Previous experiments suggest that an endogenous cGMP system could participate in the mechanism of phytochrome controlled photoperiodic flower induction in Pharbitis nil. In order to gain further information on the role of cGMP, we have begun to study the enzyme of cGMP synthesis. In this article, the presence of the enzyme with guanylyl cyclase (GC) activity in soluble protein fractions of P. nil is reported. A large portion of the enzymatic activity is present in the cotyledons, where enzyme activity amounted to 0.45 pmol cGMP/min/mg protein. The enzyme exhibited a K(m) 0.5mM for GTP. A plot of 1/v versus 1/[GTP] was linear and V(max) was 0.74 pmol cGMP/min/mg protein. It was shown that the anti-sGC antibody recognise a 40 kDa protein. Moreover, the NO-donor, sodium nitroprusside (SNP) and YC-1, as a NO-independent stimulator, enhanced enzyme activity. The NS 2028 (a potent GC inhibitor) treatments provoked a 3-fold reduction of the enzyme activity in comparison to the untreated fractions. Furthermore, the influence of light on GC activity was analysed. It was noted that cGMP level increased in cool white light, and darkness inhibited enzyme activity. Exposure to blue light acts to stimulate cGMP formation, whereas in red light a rapid decrease in GC activity was observed that returned to the high level when far-red light was applied after the red light treatment. The results presented in this work strongly argue that an enzyme with guanylyl cyclase activity is present in P. nil organs and its activity is controlled by light via the photoreceptors-dependent pathways.     

PARTIAL CHARACTERIZATION and LIGHT-INDUCED REGULATION OF GTP-BINDING PROTEINS IN Lemna paucicostata

Binding of GTP-binding proteins with [³⁵S]GTP7S in the extract containing membrane components of Lemna paucicostata 441 was inhibited by red or far red light by 20 to 25%, but blue light showed no or little effect. The plant used for the preparation of the extract was subjected to single darkness for 8 h, as both red and far red light inhibit flowering. The extract treated with 1% Lubrol was fractionated by gel filtration. Four species of GTP-binding proteins, GL1, GL2, GL3 and GL4 were detected with Km values 3, 7, 80 and 4 n M , respectively. GL1, GL2 and GL3 were ADP-ribosylated by pertussis toxin. The extract activated by [³⁵S]GTP-γS in darkness, under red light or under far red light was treated with 1% Lubrol and subsequent gel filtration of the extracts made it possible to detect GTP-binding protein with a small molecular weight only in an extract labeled in darkness. The reduction in the molecular weight of GTP-binding protein from the larger molecule associated with the binding of [³⁵S]GTPγS was confirmed by rechromatography of the larger molecule activated by [³⁵S]GTPγS in darkness. The binding of GL2 and/or GL3 with [³⁵S]GTPγS was suggested to be inhibited by red or far red light.