Adenylate cyclase activity in lymphocyte subcellular fractions. Characterization of non-nuclear adenylate cyclase.

Human peripheral lymphocytes were broken in a Dounce homogenizer and subcellular fractions enriched in plasma membranes or microsomal particles and mitochondria were isolated by centrifugation through a discontinuous sucrose gradient. Various agents that promote cyclic AMP accumulation in intact lymphocytes were compared in their ability to stimulate adenylate cyclase activity in the individual fractions. Plasma-membrane-rich fractions that were essentially free of other subcellular particles as judged by electron microscopy and marker enzyme measurements responded to fluoride, but weakly or not at all to prostaglandin E1 and other prostaglandins. Microsomal and mitochondrial-rich fractions responded markedly to both prostaglandin E1 and fluoride. In some, but not all, experiments phytohaemagglutinin produced a modest increase in enzyme activity in plasma-membrane-rich fractions. Catecholamines, histamine, parathyrin, glucagon and corticotropin produced little or no response. In the absence of theophylline, adenosine (1-10 micronM) stimulated basal enzyme activity, although at higher concentrations the responses to prostaglandin E1 and fluoride were inhibited. GTP (1-100 micronM) and GMP(5-1000 micronM) respectively inhibited or stimulated the response to fluoride, whereas the converse was true with prostaglandin E1.     

Adenylate cyclase activity of membrane fractions isolated from sea urchin sperm

The adenylate cyclase activity of sperm membrane fragments isolated from Lytechinus pictus sperm according to Cross [20] has been studied. Two distinct fractions preferentially coming from the flagellar plasma membrane are obtained. Surface I¹²⁵-labeling experiments performed by Cross [20] indicate that these membranes are representative of the entire sperm plasma membrane. Both fractions are enriched in their adenylate cyclase activity: the specific activity of the top membranes is eightfold higher than in whole sperm, whereas that of the middle membranes is 15-fold higher. The cyclase seems to be associated with the membranes. Lytechinus pictus egg jelly has no effect or slightly inhibits the adenylate cyclase activity of the isolated sperm plasma membrane fragments. Mg⁺⁺ and Na⁺ stimulated their cyclase activity about sevenfold at 2.5 mM Mn⁺⁺ and 3.2 mM ATP. At this ATP to Mn⁺⁺ ratio, high concentrations of Ca⁺⁺ have a small stimulatory effect.     

Characterization of Neurospora crassa mutant stratins deficient in adenylate cyclase activity.

Mycelial extracts from Neurospora crassa strains having any one of five different mutations of the cr-1 allele (“crisp”) exhibited an adenylate cyclase specific activity 2 to 3% of that found in wild-type strains. The enzyme deficiency seemed to be specific for the cr-1 mutation but not for the “crisp” morphology and recessive in heterocaryons carrying mutated and wild-type cr-1 alleles. The reduced adenylate cyclase activity detected in extracts from cr-1 mutants was not due to an impairment in the extraction of membranes, to a preferential inactivation of the enzyme after extraction, or to the presence of inhibitors.     

Adenylate cyclase activity in cultured epithelial cells.

The cyclic AMP metabolism of cultured epithelial cells was investigated. Epinephrine or 1-methyl,3-isobutylxanthine (MIX) alone had no effect on cyclic AMP levels in intact cells, whereas the combination of the two agents yielded a 6- to 10-fold increase in cyclic AMP levels. Both basal and stimulated cyclic AMP levels decreased with increasing cell density. Cell-free adenylate cyclase preparations were stimulated markedly by epinephrine or isoproterenol in the absence of MIX. Since the epithelial cells were found to have a relatively small amount of cyclic nucleotide phosphodiesterase (PDE) activity, the requirement for MIX to visualize intact cell responsiveness to epinephrine could be explained only partially by its PDE inhibitory properties.     

Evidence for reversability of age-related decrease in human lymphocyte adenylate cyclase activity

Age-related loss of adenylate cyclase responsiveness to guanyl nucleotide was demonstrated in lymphocytes freshly isolated from human subjects. Enzyme activity of cells from young (<40 years) and elderly (>65 years) subjects were markedly sensitive to inhibition by non-ionic detergents. When enzyme activity in the presence of guanyl nucleotide and low concentrations of Triton X-100 was determined in a mixture of cells from the young and aged donors, the activity was 40±17 percent (mean ± S.D.) greater than anticipated from the activity of the cells of the two age groups assayed separately. The detergent range which facilitated the enhanced enzyme activity was too low to extract the catalytic subunit of adenylate cyclase from the cells. These results further suggest that in man, changes distal to receptors contribute to diminished responsiveness of lymphocyte adenylate cyclase as a function of age. In addition, these age-related changes may be partially reversible by reconstitution with factors from cells from younger subjects.     

The participation of adenylate cyclase in lymphocyte capping

In this study, we have observed that cells increase their intracellular cAMP to relatively high levels during receptor capping induced by either ligand-dependent (anti-Thy-1 antibody) or ligand-independent (colchicine) treatment. In addition, we have found that under capping conditions, membrane-bound adenylate cyclase is induced to co-cap with independent membrane molecules such as Thy-1 antigens. These findings suggest that the binding of anti-Thy-1 to its receptors or treatment with colchicine induces the molecular reorganization of membrane-bound adenylate cyclase which may be responsible for activating the contractile machinery required for the collection of surface receptors into a cap structure.     

Adenylate cyclase activity in cultured epithelial cells

Summary The cyclic AMP metabolism of cultured epithelial cells was investigated. Epinephrine or 1-methyl, 3-isobutylxanthine (MIX) alone had no effect on cyclic AMP levels in intact cells, whereas the combination of the two agents yielded a 6- to 10-fold increase in cyclic AMP levels. Both basal and stimulated cyclic AMP levels decreased with increasing cell density. Cell-free adenylate cyclase preparations were stimulated markedly by epinephrine or isoproterenol in the absence of MIX. Since the epithelial cells were found to have a relatively small amount of cyclic nucleotide phosphodiesterase (PDE) activity, the requirement for MIX to visualize intact cell responsiveness to epinephrine could be explained only partially by its PDE inhibitory properties. This study was supported in part by Grant PDT-16B, American Cancer Society.     

Soluble adenylate cyclase activity in Neurospora crassa.

A soluble form of adenylate cyclase was extracted from mycelia of Neurospora crassa wild-type strains. This enzyme activity was purified by chromatography on hexyl-amino-Sepharose, agarose and Blue Sepharose and preparative polyacrylamide-gel electrophoresis. On sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, peak fractions from the later purification steps showed a main polypeptide band with an apparent molecular weight of about 66 000. The following hydrodynamic and molecular parameters were established for the Neurospora soluble adenylate cyclase activity: sedimentation coefficient, 6.25 S; Stokes radius, 7.3 nm; partial specific volume, 0.74 ml/g; molecular weight, 202 000; frictional ratio, 1.65. The isoelectric point of this enzyme activity was 4.65. The enzyme was not activated by GTP, [beta gamma-imido]GTP, fluoride or cholera toxin.     

Muscarinic cholinergic receptor mediated inhibitory transduction of adenylate cyclase activity in subcellular fractions from rat heart: improved detection in sodium phosphate buffer

Summary Cholinergic inhibition of myocardial adenylate cyclase activity in cell-free fractions has been known for many years, although the reported degrees of inhibition have been rather modest (20–30%), notably in rat heart fractions. The present study conducted with rat heart subcellular fractions document following major findings: (1) Myocardial adenylate cyclase activity and notably its cholinergic inhibition in cell-free fractions are notoriously labile to storage at 4°C whereas its stimulation by beta adrenergic receptor agonists or forskolin are reasonably well preserved during storage. (2) Among four buffers (Tris, glycylglycine, imidazole and sodium phosphate) examined, sodium phosphate buffer afforded the best preservation of cholinergic inhibitory response of adenylate cyclase. (3) The commonly used biochemical buffers, notably imidazole, exerted deleterious effect on the cholinergic inhibition of myocardial adenylate cyclase such that it was considerably attenuated or barely detectable; this explains, in part, the reported poor inhibition of myocardial enzyme by others. (4) Imidazole buffer, on the other hand, augmented beta adrenergic and forskolin stimulated adenylate cyclase activity. The likely significance of these findings is discussed from consideration that the observed differential influence of buffers results from differential actions on the interactions between the components (receptor/coupling G proteins/catalyst) comprising autonomic receptor coupled adenylate cyclase system in rat heart.     

Adenylate cyclase activity in zona-free mouse oocytes

Zona-free mouse oocytes, prepared by either chemical or enzymatic treatment, possess adenylate cyclase activity, since forskolin elevated cAMP to similar levels in either these oocytes or in oocytes with intact zonae. In addition, it was shown that oocyte isolation conditions can affect 'basal' cAMP levels.     

Distribution of adenylate cyclase among membrane fractions of rat liver.

Adenylate cyclase activity was detected in plasma membranes, Golgi apparatus, and endoplasmic reticulum from rat liver. Adenylate cyclase activities of purified membranes were determined biochemically by two methods. In one, the synthesis of radioactive cyclic AMP from ATalpha32P was monitored. In the other, the synthesis of cyclic AMP was quantitiated using a protein which specifically binds cyclic AMP. The enzyme activity was responsive to activation by both glucagon and sodium fluoride although differences in degree of activation were noted comparing plasma membrane, Golgi apparatus, and endoplasmic reticulum. Cytochemical studies, using both whole tissue and purified cell fractions and conducted in parallel, confirmed the biochemical results. Deposition of lead phosphate, enhanced by glucagon and NaF with samples incubated with appropriate substrates, was not restricted to plasma membranes of hepatocytes but was present in intracellular membranes as well. Adenylate cyclase of rat hepatocytes appears more widely distributed among internal membranes than previously recognized.     

Presence of adenylate cyclase activity in Golgi and other fractions from rat liver. I. Biochemical determination

The distribution of adenylate cyclase (AC) in Golgi and other cell fractions from rat liver was studied using the Golgi isolation procedure of Ehrenreich et al. In liver homogenate the AC activity was found to decay with time, but addition of 1 mM EGTA reduced the rate of enzyme loss. The incorporation of 1 mM EGTA into the sucrose medium used in the initial two centrifugal steps of the Golgi isolation method stabilized the enzyme activity throughout the entire procedure and resulted in good enzyme recovery. In such preparations, AC activity was demonstrated to be associated not only with plasma membranes but also with Golgi membranes and smooth microsomal membranes as well. Furthermore, under the conditions used, enzyme activity was also associated with the 105,000 g x 90 min supernatant fraction. The specific activity of the liver homogenate was found to be 2.9 pmol-mg protein-1-min-1, the nonsedimentabel and microsomal activity was of the same order of magnitude, but the Golgi and plasma membrane activities were much higher. The specific activity of plasma membrane AC was 29 pmol-mg proten-1-min-1. The Golgi activity varied in the three fractions, with the highest activity (14 pmol) in GF1 lowest activity (1.8) in GF2, and intermediate activity (5.5) in GF3, when the Golgi activity was corrected for the presence of content protein, the activity in GF1 became much higher (9 x) than that of the plasma membrane while the activities in GF2 and GF3 were comparable to that of plasma membrane. In all locations studied, the AC was sensitive to NaF stimulation, especially the enzyme associated with Golgi membranes. The activities in plasma and microsomal membranes were stimulated by glucagon, whereas the Golgi and nonsedimentable AC were not.     

Characterization of adenylate cyclase fromEscherichia coli

Adenylate cyclase activity was detected and characterized in cell-free preparations of different strains ofEscherichia coli; it was localized not only in the membrane fraction but also in the cytoplasm, the localization differing from strain to strain. The adenylate cyclase activity is highly dependent on the method used for disintegration of cells. The best results were obtained when using vortexing of the cell suspension with ballotini beads. The pH optimum of adenylate cyclase in cell-free preparations was found to be 9.0 –9.5. The enzyme has an absolute requirement for Mg²⁺ and is inhibited by sodium fluoride and inorganic diphosphate. Release of adenylate cyclase from the membrane leads to an immediate loss of the activity; it was found that adenylate cyclase is quite labile and hence it could not yet been purified. The method used to determine adenylate cyclase activity and cyclic AMP is described.     

Guanylate cyclase activity in Escherichia coli mutants defective in adenylate cyclase.

Guanylate cyclase, which catalyzes the synthesis of guanosine 3',5'-monophosphate, has been assayed in several strains of Escherichia coli. They include wild-type cells and mutants defective in adenylate cyclase, which is responsible for the synthesis of adenosine 3',5'-phosphate. Our results demonstrate that adenylate cyclase and guanylate cyclase are two different enzymes in E. coli and suggest that the gene that encodes adenylate cyclase also plays a regulatory role in the synthesis of guanylate cyclase.