Melittin interactions with adenylate cyclase

Melittin, a basic polypeptide from bee venom, inhibits basal and thyrotropin-stimulated adenylate cyclase of beef thyroid membranes with a K_i ≈ 10 μM. Although this property resides in the basicC-terminal and not the N-terminal portion of the molecule, inhibition is due primarily to its detergent-like nature rather than the charge effects. There is also a small enhancing effect of both basal and thyrotropin-stimulated adenylate cyclase of 0.3–3 μM melittin.     

Activation of human platelet adenylate cyclase by a bovine sperm component

The latent cysteine proteinase present in ascitic fluid of patients with neoplasia and released from ascites cells in culture has been partially purified and the enzyme after pepsin activation was shown to be immunologically related to the lysosomal proteinase, cathepsin B. The latent form was characterized as a single chain of Mr 40 000 as determined by SDS-polyacrylamide gel electrophoresis under reducing conditions followed by Western blotting and immune staining with an antiserum to human cathepsin B. Using the same techniques the enzyme after pepsin activation gave a single band of Mr 33 000. Analysis by isoelectric focusing showed that the latent enzyme before and after pepsin treatment is composed of several acidic isoenzymes. These findings suggest that this latent proteinase represents a precursor form of cathepsin B which is released extracellularly rather than being processed and directed to the lysosome.     

Adenosine interactions with thyroid adenylate cyclase

Adenosine and certain adenosine analogues inhibit beef thyroid membrane adenylate cyclase. The inhibition has a rapid onset, is not directly on the catalytic or nucleotide regulatory sites, occurs with all activators tested (ITP, Gpp(NH)p, TSH, and F^?), and is seen also in mouse and human thyroid membranes. Addition of manganous ion, which activates adenylate cyclase, markedly enhances the inhibition by adenosine analogues. The order of potencies is: 2′,5′-dideoxyadenosine > 5′-deoxyadenosine > 2′-deoxy-3′-phosphoadenosine > 2′-deoxyadenosine > adenosine > adeninexyloside > adenine arabinoside. Purinemodified analogues are either inactive or stimulate slightly at high concentrations. This chemical specificity, the Mn²⁺ requirement, and the lack of reversal by theophylline, suggest that these membranes have little “R” site activity (stringent for the ribose moiety) and primarily contain a “P” site that has stringent purine requirement but permits changes in the ribose moiety. This site appears to be associated with the catalytic unit since it persists in solubilized adenylate cyclase.     

Calmodulin-mediated adenylate cyclase from mammalian sperm

Calmodulin (CaM), the calcium binding protein that modulates the activity of a number of key regulatory enzymes, is present at high levels in sperm. To determine whether CaM regulates adenylate cyclase in mammalian sperm, the actions of EGTA and selected CaM antagonists on a solubilized adenylate cyclase from mature equine sperm were examined. The activity of equine sperm adenylate cyclase was inhibited by EGTA in a concentration-dependent manner with a half-maximal inhibitory concentration (IC50) of 2 mM. Equine sperm adenylate cyclase was also inhibited in a concentration-dependent manner by the CaM antagonists chlorpromazine and calmidazolium (IC50 = 400 and 50 microM, respectively). The inhibition of enzyme activity by these agents correlated with their known potency and specificity as anti-CaM agents. The activity of the enzyme in the presence of 200 microM calmidazolium was restored by the addition of authentic CaM (EC50 = 15 microM); full activity was restored by the addition of 50 microM CaM. La3+, an ion that dissociates CaM from tightly bound CaM-enzyme systems, inhibited equine sperm adenylate cyclase (IC50 = 1 mM). Incubation of equine sperm adenylate cyclase with La3+ dissociated endogenous CaM from the enzyme so that most of the enzyme bound to a CaM-Sepharose column equilibrated with Ca2+. Specific elution of CaM-binding proteins from the CaM-Sepharose column with EGTA yielded a CaM-depleted adenylate cyclase fraction that was stimulated 2-fold by the addition of exogenous CaM.     

Identification of sea urchin sperm adenylate cyclase

Calmodulin (CaM) affinity chromatography of a detergent extract of sea urchin sperm yielded approximately 20 major proteins. One of these proteins, of Mr 190,000, was purified and used to immunize rabbits. After absorption with living sperm, the serum reacted monospecifically on one- and two-dimensional Western immunoblots with the Mr 190,000 protein. The anti-190-kD serum inhibited 94% of the adenylate cyclase (AC) activity of the CaM eluate. An immunoaffinity column removed 95% of the AC activity, and the purified (but inactive) Mr 190,000 protein was eluted from the column. The antiserum also inhibited 23% of the activity of bovine brain CaM-sensitive AC and 90% of the activity of horse sperm CaM-sensitive AC. These data support the hypothesis that the Mr 190,000 protein is sea urchin sperm AC. Although this AC bound to CaM, it was not possible to demonstrate directly a Ca2+ or CaM sensitivity. However, two CaM antagonists, calmidazolium and chlorpromazine, both inhibited AC activity, and the inhibition was released by added CaM, suggesting the possibility of regulation of this AC by CaM. Indirect immunofluorescence showed the Mr 190,000 protein to be highly concentrated on only the proximal half of the sea urchin sperm flagellum. This asymmetric localization of AC may be important to its function in flagellar motility. This is the first report of the identification of an AC from animal spermatozoa.     

Mechanism of action of forskolin on adenylate cyclase: effect on bovine sperm complemented with erythrocyte membranes

The mechanism of action of forskolin stimulation of adenylate cyclase was investigated by examining its effects on the enzyme's Mg2+ activated catalytic unit (C) from bovine sperm, both preceding and following complementation with human erythrocyte membranes as a source of guanine nucleotide regulatory protein (N). Prior to complementation, sperm C was not activated by either NaF (10 mM) or 5'-guanylyl-beta-gamma-imidodiphosphate (Gpp(NH)p, 10 microM), suggesting that functional N was not present in this preparation. Forskolin (100 microM) was also without effect on C. Following complementation of the sperm membranes with those of erythrocytes, Mg2+-dependent sensitivity to forskolin, NaF, and Gpp(NH)p was imparted to C. Our findings are incompatible with the current hypothesis that forskolin stimulates adenylate cyclase by direct activation of C. Rather, the data suggest that the activation process occurs through an effect on N or by augmentation of the interaction between the components of the adenylate cyclase complex.     

Calcium and adenosine affect human sperm adenylate cyclase activity

The adenylate cyclase activity of human ejaculated spermatozoa in broken-cell preparations was investigated. In the presence of 5 mM metal cations and 0.1 mM ATP, the relative enzyme activity with Mn²⁺, Ca²⁺, Mg²⁺, Ba²⁺ was 1.00, 0.28, 0.22, and 0.03, respectively. Added Ca²⁺ appeared to activate the enzyme in the presence of Mn²⁺ or Mg²⁺. The human sperm adenylate cyclase was stimulated by ～ 2-fold by free Ca²⁺ (lmM) in the presence of Mg²⁺ (5 mM). If the GTP analogue, 5′-guanylyl imidophosphate (Gpp(NH)p) was added to the sperm homogenate in the presence of 200 μM ethylene-glycol-bis (β-aminoethylether) N,N′-tetraacetic acid (EGTA), the adenylate cyclase activity was increased by approximately 25%, but with the addition of 280 μM Ca²⁺ there was a decrease in enzyme activity. A similar response to low concentrations of Ca²⁺ was obtained after complementation of the sperm enzyme with the guanine nucleotide regulatory component from human erythrocytes, where the addition of 40 μM Gpp(NH)p, 200 μM EGTA, and Ca²⁺ (≤ 160 μM) stimulated the sperm enzyme ～ 3–4-fold, but the further addition of Ca²⁺ (280 μM, final) neutralized the stimulatory effect. The addition of adenosine, and the nucleotides 5′-AMP and 5′-ADP inhibited the enzyme, whereas guanine and 5′-GMP had no appreciable effect. Human follicular fluid and serum also had little direct effect on the sperm adenylate cyclase. These resuls suggest that Ca²⁺ might be an important physiological modulator of the human sperm adenylate cyclase.     

Bull sperm adenylate cyclase: localization and partial characterization.

A purine-nucleoside phosphorylase (purine-nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1) from bovine thyroid tissue has been purified 670-fold utilizing the techniques of ammonium sulfate precipitation, ion-exchange and molecular-exclusion chromatography, and polyacrylamide-gel electrophoresis. The protein has an apparent molecular weight of 90,000, a single isoelectric point at 5.6, and a Michaelis constant of 0.028 mm for inosine. Double-reciprocal plots of the reaction rate for the phosphorylase-catalyzed reaction versus phosphate or arsenate concentration display a downward trend at high substrate concentrations. Two apparent Michaelis constants of 0.38 and 1.49 mm were determined for phosphate.     

Receptor-related interactions of opiates with PGE-induced adenylate cyclase in brain

The inhibition by opiates of the PGE2-induced formation of cAMP in slices from rat brain striatum was investigated. A maximal, 3.5-fold increase over the basal level of cAMP was obtained with an EC50 for PGE2 of 3 microM. Opiate agonists of both mu and kappa type were inhibitory. The IC50 values for morphine, levorphanol and ethylketocyclazocine (EKC) were 110 nM, 80 nM and 25 nM, respectively. These values were similar to the potencies of the compounds in displacing stereospecifically bound 3H-etorphine in rat brain membranes. As evidenced by the inactivity of dextrorphan, the inhibition of PGE2-dependent cAMP formation was stereospecific. Also ineffective were the opiate antagonists naloxone, naltrexone and MR 2266. These compounds did, however, reverse the inhibition by agonists, displaying thereby selectivity toward the putative mu and kappa opiates. Thus, the inhibition by morphine was antagonized to a greater degree by naloxone than by MR 2266, and the action of EKC was blocked more effectively by MR 2266 relative to naloxone.     

Cytochemical localization of adenylate cyclase in bovine cumulus-oocyte complexes

The ultrastructural localization of adenylate cyclase was studied in bovine cumulus-oocyte complexes. Adenylate cyclase was observed on the plasma membrane of the oocyte and occasionally on the plasma membrane of cumulus cells. The cytochemical observations presented demonstrate that there is more adenylate cyclase in cumulus-oocyte complexes after in vitro stimulation with forskolin. The presence of adenylate cyclase upon the oocyte was more pronounced. In addition adenylate cyclase appeared to be localized on the cumulus cells, especially between junctional complexes of cumulus cells and on cumulus cell processes contacting the oocyte. The cumulus cells never showed the presence of adenylate cyclase in the absence of forskolin. No changes in the presence of adenylate cyclase were observed during in vitro meiotic maturation.     

Different structural requirements for adenylate cyclase toxin interactions with erythrocyte and liposome membranes

The bifunctional Bordetella adenylate cyclase toxin-hemolysin (ACT) penetrates target cell membranes, forms cation-selective channels and subverts cellular signaling by catalyzing uncontrolled conversion of ATP to cAMP. While primarily targeting phagocytes expressing the alphaMbeta2 integrin (CD11b/CD18), the toxin can also penetrate mammalian erythrocytes lacking the receptor and membrane endocytosis. We sought here to analyze the membrane interactions of ACT in a liposome model. Insertion of ACT into liposome membranes required calcium and caused leakage of entrapped fluorescent probes due to liposome disruption, as indicated by similar release kinetics for the approximately 398 Da FITC probe and its approximately 4400 Da dextran conjugate. However, the non-acylated proACT, which does not penetrate cellular membranes, exhibited higher capacity to bind and lyze liposomes than the mature toxin, showing that the fatty-acyl modification was not required for penetration of ACT into the lipid bilayer. Individual deletions within the channel-forming, acylation and repeat domains of ACT abolished its capacity to disrupt both liposomes and erythrocytes. In contrast to erythrocyte binding, however, the liposome binding was only lost upon a simultaneous deletion of both the channel-forming and acylation domains, suggesting that the acylation domain was also involved in liposome penetration of ACT. Moreover, substitutions of glutamates 509 and 516 by lysines, which strongly enhanced the channel-forming and hemolytic activity of ACT, did not affect its capacity to disrupt liposomes. This shows that the mechanism of ACT action in cellular membranes is not fully reproduced in liposome membranes.     

Stimulation of partially purified adenylate cyclase from bull sperm by bicarbonate

Solubilized and partially purified adenylate cyclase from bull sperm was found to be specifically activated (up to 6-fold) by sodium bicarbonate (NaHCO3) and to a lesser extent by NaNO3. Other sodium salts were either ineffective (e.g. NaCOOH) or inhibitory (e.g. NaHSO3, NaHSO4 and Na2B4O7). Stimulation by NaHCO3 was dose-dependent in the range of 0-40 mM and was greater when enzyme activity was assayed in the presence of magnesium as compared with manganese ions. Bicarbonate seems to affect maximal enzyme velocity (Vmax) and has no effect on the Km of adenylate cyclase for Mn-ATP. Stimulation of adenylate cyclase by NaHCO3 coincided with the elution pattern of the enzyme as recorded following chromatography on DEAE-cellulose or gel filtration on BioGel P-100. These results suggest that in the course of stimulation of sperm adenylate cyclase, bicarbonate is likely to interact directly with the enzyme. Furthermore, this intrinsic and unique property of sperm adenylate cyclase may explain results reported by others on the stimulation of cAMP production by bicarbonate in intact and broken sperm preparations and suggest a biochemical basis for enhanced sperm motility associated with high bicarbonate concentrations.     

Adenosine and gpp(NH)p modulate mouse sperm adenylate cyclase

The possible roles of adenosine and the GTP analogue Gpp(NH)p in regulating mouse sperm adenylate cyclase activity were investigated during incubation in vitro under conditions in which after 30 min the spermatozoa are essentially uncapacitated and poorly fertile, whereas after 120 min they are capacitated and highly fertile. Adenylate cyclase activity, assayed in the presence of 1 mM ATP and 2 mM Mn²⁺, was determined by monitoring cAMP production. When adenosine deaminase (1 U/ml) was included in the assay to deplete endogenous adenosine, enzyme activity was decreased in the 30-min suspensions but increased in the 120-min samples (P < 0.02). This suggests that endogenous adenosine has a stimulatory effect on adenylate cyclase in uncapacitated spermatozoa but is inhibitory in capacitated cells. Since the expression of adenosine effects at low nucleoside concentrations usually requires guanine nucleotides, the effect of adding adenosine in the presence of 5 x 10^–5 M Gpp(NH)p was examined. While either endogenous adenosine or adenosine deaminase may have masked low concentration (10^?9?10^?7 M) effects of exogenous adenosine, a marked inhibition (P < 0.001) of adenylate cyclase activity in both uncapacitated and capacitated suspensions was observed with higher concentrations (>10^?5 M) of adenosine. Similar inhibition was also observed in the absence of Gpp(NH)p, suggesting the presence of an inhibitory P site on the enzyme. In further experiments, the effects of Gpp(NH)p in the presence and absence of adenosine deaminase were examined. Activity in 30-min suspensions was stimulated by the guanine nucleotide and in the presence of adenosine deaminase this stimulation was marked, reversing the inhibition seen with adenosine deaminase alone. In capacitated suspensions the opposite profile was observed, with Gpp(NH)p plus adenosine deaminase being inhibitory; again, this was a reversal of the effects obtained in the presence of adenosine deaminase alone, which had stimulated enzyme activity. These results suggest the existence of a stimulatory adenosine receptor site (R_a) on mouse sperm adenylate cyclase that is expressed in uncapacitated spermatozoa and an inhibitory receptor site (R_i) that is expressed in capacitated cells, with guanine nucleotides modifying the final response to adenosine. It is concluded that adenosine and guanine nucleotides may regulate mouse sperm adenylate cyclase activity during capacitation.     

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].