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

Nuclei from purified human peripheral lymphocytes were prepared by incubations with Triton X-100 to disrupt the cells, followed by sucrose-density gradient centrifugation. The nuclei were pure as judged by phase-contrast microscopy and had low contents of non-nuclear marker enzymes. In addition, nuclei prepared from lymphocytes surface-labelled with 125I had only 2-7% of the radioactivity bound to intact lymphocytes. At 3.3 mM-Ca2+ and 100 micronM-ATP a fluoride-sensitive adenylate cyclase was demonstrated in nuclei prepared in 0.2% Triton X-100 or 0.33% Triton X-100. There was linear accumulation of cyclic AMP for 10 min in both preparations. The apparent Km for ATP was 90 micronM. Adenylate cyclase activity was augmented by 1.0 mM-Mn2+ and inhibited at higher concentrations. Ca2+ showed two peaks of stimulation, at 1.0-2.5 mM- and above 10 mM-Ca2+. Mg2+ was inhibitory at all concentrations. EDTA OR EGTA only slightly decreased adenylate cyclase activity, suggesting that another metal ion may be necessary for activity. Adenylate cyclase activity was stimulated by 10mM-isoproterenol and 10 micronM-adrenaline in the presence of a phosphodiesterase inhibitor. Phytohaemagglutinin and prostaglandin E1 alone or in combination with isoproterenol had no effect on nuclear adenylate cyclase activity in either nuclei preparation. These results indicate that human lymphocyte nuclei contain one or several adenylate cyclases which differ from adenylate cyclases found in other subcellular fractions of these cells with regard to their bivalentcation requirements and responsiveness to pharmacological agents.     

The purification and characterization of plasma membranes and the subcellular distribution of adenylate cyclase in mouse parotid gland.

1. Plasma membranes have been purified 17-fold from mouse parotid gland homogenates prepared in hypertonic sucrose media using differential centrifugation. The method is fast and simple. The membranes were characterised by electron microscopy, enzyme composition and chemical composition. Further purification was achieved by isopycnic centrifugation in discontinuous sucrose gradients. 2. The purified membranes contain an adenylate cyclase activity which is stimulated by isoproterenol and fluoride. Only 50% of the total adenylate cyclase activity sedimented in the plasma membrane fraction. The rest of the activity resided in the crude nuclear and mitochondrial pellets. However, this adenylate cyclase activity was not associated with these organelles but with membrane fragments in the pellets. Purified nuclei did not contain adenylate cyclase activity. 3. Adenylate cyclase activity was also localised by electron microscopic cytochemistry. Besides being found at the plasma membrane, large amounts of adenylate cyclase were found in a small proportion of the vesicles within the acinar cells, which appeared to be secondary lysosomes. 4. Adenylate cyclase activities, under standard assay conditions, are proportional to the time of incubation and the concentration of enzyme. The enzyme requires both Mg-2+ and CA-2+ for activity. Isoproterenol increased activity 2-fold and this increase is abolished by beta-adrenergic blocking agents.     

Subcellular Localization of Adenylate Cyclase in the Shoot Apices of Bryum argenteum Hedw

Adenylate cyclase activity has been detected in the shoot apicesof Bryum argenteum at the ultrastructural level, using adenylylimidodiphosphate as substrate. Heavy deposits of the reactionproduct were observed along the plasma membrane, tonoplast andthe membranous system of developing plastids. No clear stainingwas associated with other subcellular membranes. Mosses, adenylate cyclase, Bryum, cyclic AMP     

Distribution of Protein I, a Synapse-Specific Phosphoprotein, and Adenylate Cyclase in the Rat Spinal Cord

The longitudinal and transverse distributions of the synapse-specific phosphoprotein Protein I and adenylate cyclase in the rat spinal cord were studied. Protein I was found to be enriched in all cervical and midlumbar (L3-L5) segments, and sparse in midthoracic and sacral segments. Adenylate cyclase activity was high in all cervical and lumbosacral segments, and low in mid-thoracic segments. Cross sectionally, both Protein I and adenylate cyclase were more enriched in the dorsal half than in the ventral half in the various segments studied. The similar topographical distributions of Protein I and adenylate cyclase in the spinal cord support the idea that adenylate cyclase may be intimately associated with Protein I in the nervous system, and could thereby regulate the state of in vivo phosphorylation of Protein I through formation of cyclic AMP.     

Adenylate cyclase and cyclic AMP phosphodiesterase in Bradyrhizobium japonicum bacteroids.

Adenylate cyclase and cyclic AMP (cAMP) phosphodiesterase have been identified and partially characterized in bacteroids of Bradyrhizobium japonicum 3I1b-143. Adenylate cyclase activity was found in the bacteroid membrane fraction, whereas cAMP phosphodiesterase activity was located in both the membrane and the cytosol. In contrast to other microorganisms, B. japonicum adenylate cyclase remained firmly bound to the membrane during treatment with detergents. Adenylate cyclase was activated four- to fivefold by 0.01% sodium dodecyl sulfate (SDS), whereas other detergents gave only slight activation. SDS had no effect on the membrane-bound cAMP phosphodiesterase but strongly inhibited the soluble enzyme, indicating that the two enzymes are different. All three enzymes were characterized by their kinetic constants, pH optima, and divalent metal ion requirements. With increasing nodule age, adenylate cyclase activity increased, the membrane-bound cAMP phosphodiesterase decreased, and the soluble cAMP phosphodiesterase remained largely unchanged. These results suggest that cAMP plays a role in symbiosis.     

Cytochemical demonstration of adenylate cyclase in cardiac muscle: effect of dimethyl sulfoxide.

Adenylate cyclase (AC) activity was evaluated after perfusion fixation of rat and dog myocardium with 4% paraformaldehyde (PFA), 2% glutaraldehyde (GA) or a combination of both, in cacodylate buffer. Dimethyl sulfoxide (DMSO) was added to the fixatives and its effect on the preservation of cell organelles and enzyme activity was determined. Adenylate cyclase activity was preserved best after fixation with 4% paraformaldehyde but this fixative did not provide for optimal maintenance of structure. Prefixation with 2% glutaraldehyde and 5% dimethyl sulfoxide provided the most effective preservation of both structural and enzymatic integrity. Precipitation of lead diphosphoimide was the morphologic indicator of sites of adenylate cyclase activity. The most intense precipitate was in the lumen of junctional sarcoplasmic reticulum in close contact with T-tubules and in subsarcolemmal cisternae. Evidence of activity was also seen on the intracellular aspect of the sarcolemmal membrane and in the nexus segment of the intercalated discs. Alloxan was effective as an inhibitor of adenylate cyclase activity only if the concentration of the activating substance sodium fluoride (NaF) was 20 mM or lower.     

A Rapid Anterograde Axonal Transport of Carboxypeptidase H in Rat Sciatic Nerves

Abstract: Using the highly sensitive HPLC-fluorophotometry technique, anterograde and retrograde axonal transport of carboxypeptidase H (CPH), a putative pro-hormone processing enzyme that removes a basic amino acid from the C-terminus of a precursor peptide, was measured 12–72 h after double ligations of rat sciatic nerves. CPH-like activity in rat sciatic nerves was 60-fold lower than that in the pituitary gland. CPH-like enzyme activity was rapidly accumulated in the proximal segment and peaked 48 h after ligation. The axonal flow was 100 mm/day, indicating that CPH in rat sciatic nerves is rapidly transported to the nerve terminals as an active form. The properties of the enzyme were similar to those of CPH in the brain: The pH optimum is at 5.5, and the molecular mass is ∼50 kDa. These results suggest that active CPH in the PNS is transported by a rapid anterograde axonal flow and may play a role in converting proneuropeptides to active neuropeptides under the axonal transport.     

Calpain-mediated degradation of rapidly and slowly axonally transported proteins in retinal ganglion cells

Labelled axonally transported proteins belonging to four different phases of transport in the retinal ganglion cells of the rabbit were used as substrates in order to study proteolytic degradation in axons and nerve terminals.Proteins of both rapidly and slowly transported phases of axonal transport were easily degraded in small intact pieces of the superior colliculus.Addition of the Ca-dependent neutral protease, calpain, to isolated soluble and membrane fractions from the superior colliculus resulted in an increased rate of degradation of axonally transported components. The effects of calpain was most marked toward components in phases II and V of axonal transport in this system (Karlsson and Sjöstrand, 1971; Willard and Hulebak, 1977). The latter phase contains slowly transported neurofilament and microtubular protein while the former one contains rapidly transported membrane proteins.     

Subcellular location of adenylate cyclase in rat cardiac muscle

Crude homogenates of rat cardiac muscle were fractionated in order to examine the subcellular location of adenylate cyclase in this tissue. The fractionation procedure employed differential centrifugation of homonized material, followed by collagenase treatment, centrifugation on a discontinuous sucrose density gradient and extraction with 1 M KCl. The particulate fraction obtained by this procedure contained a high specific activity and yield of adenylate cyclase, moderate levels of mitochondria and low levels of sarcoplasmic reticulum and contractile protein as judged by marker enzyme activities. Adenylate cyclase was purified 20-fold with a 33% yield from the crude homogenate, while mitochondrial, sarcoplasmic reticulum and contractile protein yields were 5, 0.4 and 0.7% respectively. The membrane fractions prepared in this manner were examined by sodium dodecyl sulfate · gel electrophoresis.Adenylate cyclase copurified with ouabain-sensitive (Na⁺ + K⁺)-ATPase, a plasma membrane marker enzyme, and not with Ca²⁺-accumulating activity, which is associated with the sarcoplasmic reticulum. The distribution of marker enzyme activities indicates that heart adenylate cyclase is not located in the sarcoplasmic reticulum but is localized predominantly, if not exclusively, in the plasma membrane.     

Physiological desensitization of carbohydrate permeases and adenylate cyclase to regulation by the phosphoenolpyruvate:sugar phosphotransferase system in Escherichia coli and Salmonella typhimurium. Involvement of adenosine cyclic 3',5'-phosphate and inducer

Adenylate cyclase and a number of carbohydrate transport systems are subject to regulation by the phosphoenolpyruvate:sugar phosphotransferase system. These sensitive carbohydrate transport systems are desensitized to regulation by the phosphotransferase system, and adenylate cyclase is deactivated when cells are grown in medium containing cyclic AMP. These effects are specific for cyclic AMP and are potentiated by the genetic loss of cyclic AMP phosphodiesterase. Inclusion in the growth medium of an inducer of a sensitive transport system also promotes desensitization of that particular transport system. Inducer-promoted desensitization is specific for the particular target transport system, while cyclic AMP-promoted desensitization is general and affects several systems. Desensitization of the permeases to regulation, and inactivation of adenylate cyclase, are slow processes which are blocked by chloramphenicol and are therefore presumably dependent on protein synthesis. Several sugar substrates of the phosphotransferase system are capable of regulating the sensitive carbohydrate transport systems. The evidence suggests that desensitization to this regulation does not result from a direct effect on the functioning of Enzyme I, a small heat-stable protein of the phosphotransferase system, HPr, or an Enzyme II of the phosphotransferase system, but specifically uncouples the permease systems from regulation.     

Adenylate cyclase responsiveness of human insulinomas

Adenylate cyclase activity was assayed in a crude particulate fraction of one benign and one malignant human insulinoma. Adenylate cyclase of both tumours responded to 5'-guanylyl-imidodiphosphate, sodium fluoride, glucagon and prostaglandin E2, and in addition the adenylate cyclase of the benign tumour responded to isoprenaline. Glucose and prostaglandin I2 (prostacyclin) did not stimulate the adenylate cyclase in either tumour, although prostaglandin I2 stimulated insulin secretion in cultures of the benign tumour. The in vitro responsiveness of the adenylate cyclase to glucagon did not correlate closely with the effect of glucagon on insulin secretion in vivo.     

Acrylamide-Induced Increases in Deposition of Axonally Transported Glycoproteins in Rat Sciatic Nerve

The axonal transport of proteins, glycoproteins, and gangliosides in sensory neurons of the sciatic nerve was examined in adult rats exposed to acrylamide via intraperitoneal injection (40 mg/kg of body weight/day for nine consecutive days). The L5 dorsal root ganglion was injected with either [35S]methionine to label proteins or [3H]glucosamine to label, more specifically, glycoproteins and gangliosides. At times ranging from 2 to 6 h later, the sciatic nerve and injected ganglion were excised and radioactivity in consecutive 5-mm segments determined. In both control and acrylamide-treated animals, outflow profiles of [35S]methionine-labeled proteins showed a well defined crest which moved down the nerve at a rate of approximately 340 mm/day. Similar outflow profiles and transport rates were seen for [3H]glucosamine-labeled glycoproteins in control animals. However, in animals treated with acrylamide, the crest of transported labeled glycoprotein was severely attenuated as it moved down the nerve. This finding suggests that in acrylamide-treated animals, axonally transported glycoproteins were preferentially transferred (unloaded or exchanged against unlabeled molecules) from the transport vector to stationary axonal structures. We also examined the clearance of axonally transported glycoproteins distal to a ligature on the nerve. The observed impairment of clearance in acrylamide-treated animals relative to controls is supportive of the above hypothesis. Acrylamide may directly affect the mechanism by which axonally transported material is unloaded from the transport vector. Alternatively, the increased rate of unloading might reflect an acrylamide-induced increase in the demand for axonally transported material.     

Anterograde axonal transport of endopeptidase 24.15 in rat sciatic nerves

Axonal transport of endopeptidase 24.15 (EP24.15), a putative neuropeptide degrading-enzyme, was examined in the proximal, middle, and distal segments of rat sciatic nerves using a double ligation technique. At 48h after ligation, a significant amount of the axonal transport of EP24.15 activity was found in the proximal segment, while axonal transport of deamidase activity, a lysosomal enzyme, increased in both proximal and distal segments. Western blot analysis of EP24.15 showed that EP24.15 immunoreactivity in the proximal segment was 1.8-fold higher than that in the middle segment. The immunohistochemical analysis of the segments also showed an increase in the immunoreactive EP24.15 in the proximal segment in comparison with that in the middle segment. In the distal segment, no axonal transport of EP24.15 was found in all methods examined, indicating that EP24.15 is mainly transported by an anterograde axonal flow. These observations suggest that EP24.15 may be involved in the metabolism of neuropeptides in nerve terminals or synaptic clefts.     

Rapid Migration of Inositol Phospholipids with Axonally Transported Substances in the Rabbit Optic Pathway

Following an intraocular injection of myo-[2-3H]inositol, the axonal transport of labelled water-soluble substances and inositol phospholipids was investigated. Evidence was obtained for a rapid axonal transport of a relatively small amount of labelled inositol phospholipids. In contrast to other axonally transported phospholipids, there was no significant accumulation of labelled, rapidly transported inositol phospholipids in the nerve terminal region at later time intervals following the isotope administration.     

Cellular Origin and Biosynthesis of Rat Optic Nerve Proteins: A Two-Dimensional Gel Analysis

High resolution 2DGE (two-dimensional gel electrophoresis) was used to characterize neuronal and glial proteins of the rat optic nerve, to examine the phases of intraaxonal transport with which the neuronal proteins are associated, and to identify the ribosomal populations on which these proteins are synthesized. Neuronal proteins synthesized in the retinal ganglion cells were identified by injecting the eye with L-[35S]methionine, followed by 2DGE analysis of fast and slow axonally transported proteins in particulate and soluble fractions. Proteins synthesized by the glial cells were labeled by incubating isolated optic nerves in the presence of L-[35S]methionine and then analyzed by 2DGE. A number of differences were seen between filamentous proteins of neurons and glia. Most strikingly, proteins in the alpha- and beta-tubulin region of the 2D gels of glial proteins were distinctly different than was observed for axonal proteins. As expected, neurons but not glia expressed neurofilament proteins, which appeared among the slow axonally transported proteins in the particulate fraction; significant amounts of the glial filamentous protein, GFA, were also labeled under these conditions, which may have been due to transfer of amino acids from the axon to the glial compartment. The fast axonally transported proteins contained relatively large amounts of high-molecular-weight acidic proteins, two of which were shown to comigrate (on 2DGE) with proteins synthesized by rat CNS rough microsomes; this finding suggests that rough endoplasmic reticulum may be a major site of synthesis for fast transported proteins. In contrast, the free polysome population was shown to synthesize the principal components of slow axonal transport, including tubulin subunits, actin, and neurofilament proteins.     

Histochemical localization of adenylate cyclase and phosphodiesterase activities in the folliate papillae of the rabbit I. Light microscopic observations

Adenylate cyclase and cyclic AMP phosphodiesterase activitieson the foliate papillae of rabbit were studied by means of histochemistry.In unfixed papillae the reaction product for adenylate cyclaseactivity was localized in the apex of taste buds, lamina proprioand connective tissue core of the papillae, but in fixed papillaeit was limited to the apex of taste buds. The reaction productfor cyclic AMP phosphodiesterase activity was limited to theapex of taste buds in unfixed and fixed papillae. Neither anacceleratory nor an inhibitory effect of sweet and bitter substanceson the adenylate cyclase and cyclic AMP phosphodiesterase activitieswas demonstrated, but NaCl prevented the formation of reactionproduct for the adenylate cyclase activity at the apex of tastebuds.     

Axonal transport of synaptic vesicle proteins in the rat optic nerve

The optic nerve, as a part of the central nervous system (CNS), has been used to study axonal transport for decades. The present study has concentrated on the axonal transport of synaptic vesicle proteins in the optic nerve, using the “stop-flow/nerve crush” method. After blocking fast axonal transport, distinct accumulations of synaptic vesicle proteins developed during the first hour after crush-operation and marked increases were observed up to 8 h postoperative. Semiquantitative analysis, using cytofluorimetric scanning (CFS) of immunoincubated sections, revealed that the ratio between distal accumulations (organelles in retrograde transport) and proximal accumulations (organelles in anterograde transport) was much higher (up to 80–90%) for the transmembrane proteins than that for surface adsorbed proteins (only 10–20%). The pattern of axonal transport in the optic nerve was comparable to that in the sciatic nerve. However, clathrin and Rab3a immunoreactivities were accumulated in much lower amounts than that in the sciatic nerve. Most synaptic vesicle proteins were colocalized in the axons proximal to the crush. A differential distribution of synaptobrevin I and II, however, was observed in the optic nerve axons; synaptobrevin I was present in large-sized axons, while synaptobrevin II immunoreactivity was present in most axons, including the large ones. The two isoforms were, thus, partially colocalized. The results demonstrate that (1) cytofluorimetric scanning techniques could be successfully used to study axonal transport not only in peripheral nerves, but also in the CNS; (2) synaptic vesicles are transported with fast axonal transport in this nerve; and (3) some differences were noted compared with the sciatic nerve, especially for Rab3a and clathrin. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 237–250, 1997.     

Adenylate Cyclases in the Vertebrate Retina: Distribution and Characteristics in Rabbit and Ground Squirrel

Adenylate cyclase activity and the effects of EGTA, 5'-guanylylimidodiphosphate (GPP(NH)P), and dopamine were measured in microdissected layers of rod-dominant (rabbit) and cone-dominant (ground squirrel) retinas, The distribution of basal enzyme activity was similar in both species, with the highest levels found in the inner plexiform and photoreceptor cell inner segment layers, EGTA inhibited adenylate cyclase in the inner retina of both species and stimulated activity in rabbit outer and inner segment layers, but had no effect in these layers from ground squirrel. Enzyme activity was stimulated in all regions by GPP(NH)P, except in the outer segments of the photoreceptors. Dopamine stimulated the enzyme in the outer and inner plexiform and inner nuclear layers in rabbit, but only in the inner plexiform layer in ground squirrel. These data demonstrate that the enzymatic characteristics of adenylate cyclase vary extensively from region to region in vertebrate retina and suggest that cyclic AMP may have multiple roles in this tissue. A model for the distribution of the different forms of adenylate cyclase in retina is proposed.     

Axonal transport blockade and denervation have qualitatively different effects upon skeletal muscle metabolism

The activity and isoenzyme pattern of muscle lactic dehydrogenase (LDH) was measured at different times after axonal transport blockade by colchicine or after denervation. After denervation, total LDH activity decreased and the isoenzyme pattern was altered, LDH-1 being the most affected form. In contrast, after axonal transport blockade there was a decrease in LDH activity but the isoenzyme pattern was not modified. Denervation abolishes both nerve-evoked muscle activity and the release of neuro trophic substances from the nerve whereas colchicine blocks axonal transport without affecting the nerve capacity to conduct action potentials or neuromuscular transmission. It is then concluded that nerve-evoked muscle activity is the most important factor in the regulation of muscle LDH isoenzyme distribution. On the other hand, muscle metabolism can also be regulated by axonally transported molecules. The results presented here show that there is a qualitative difference between the effects of denervation and those of axonal transport blockade upon the muscle, since only denervation altered the isoenzyme pattern of muscle LDH.