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.     

Modulation of adenylate cyclase activity by sulfated glycosaminoglycans. II. Effects of mucopolysaccharides and dextran sulfate on the activity of adenylate cyclase derived from various tissues.

Heparin was found to be the most potent inhibitor of rat ovarian luteinizing hormone-sensitive adenylate cyclase (I50 = 2 microgram/ml) when compared to other naturally occurring glycosamin oglycans. This inhibition was also apparent when this enzyme was stimulated by follicle-stimulating hormone or prostaglandin E2. Heparin was also found to inhibit glucagon-sensitive rat hepatic adenylate cyclase, and the prostaglandin E1-sensitive enzyme from rat ileum and human platelets. In contrast, heparin stimulated the dopamine sensitive adenylate cyclase from rat caudate nucleus. The sulfated polysugar dextran sulfate exerts similar effects on adenylate cyclase activity of the rat ovary and was shown to inhibit hormone binding to rat ovarian plasma membrane in a manner similar to that exerted by heparin. In contrast to heparin, dextran sulfate inhibited dopamine-sensitive adenylate cyclase from rat caudate nucleus.     

Isolation of plasma membranes from rat lungs. Effect of age on the subcellular distribution of adenylate cyclase activity

A simple and rapid method of isolating plasma membranes from rat lungs is described. The method involves homogenization of tissue in isotonic sucrose-buffered medium followed by differential and sucrose density gradient centrifugation. Plasma membranes obtained by this procedure were essentially free from other subcellular contamination. Plasma membranes isolated from 2-day-old rat lungs showed 6 to 7-fold purification of adenylate cyclase and 5′-nucleotidase activities compared to the original homogenate In contrast, plasma membranes from 35-day-old rat lungs showed no purification of adenylate cyclase activity although 5′-nucleotidase activity showed similar enrichment. These results suggest that adenylate cyclase activity is not a reliable marker for plasma membranes from adult rat lungs.     

Isolation of plasma membranes from rat lungs: effect of age on the subcellular distribution of adenylate cyclase activity

A simple and rapid method of isolating plasma membranes from rat lungs is described. The method involves homogenization of tissue in isotonic sucrose-buffered medium followed by differential and sucrose density gradient centrifugation. Plasma membranes obtained by this procedure were essentially free from other subcellular contamination. Plasma membranes isolated from 2-day-old rat lungs showed 6 to 7-fold purification of adenylate cyclase and 5'-nucleotidase activities compared to the original homogenate. In contrast, plasma membranes from 35-day-old rat lungs showed no purification of adenylate cyclase activity although 5'-nucleotidase activity showed similar enrichment. These results suggest that adenylate cyclase activity is not a reliable marker for plasma membranes from adult rat lungs.     

Modulation of adenylate cyclase activity by sulfated glycosaminoglycans II. Effects of mucopolysaccharides and dextran sulfate on the activity of adenylate cyclase derived from various tissues

Heparin was found to be the most potent inhibitor of rat ovarian luteinizing hormone-sensitive adenylate cyclase (I₅₀ = 2 μg/ml) when compared to other naturally occurring glycosaminoglycans. This inhinibition was also appparent when this enzyme was stimulated by follicle-stimulating hormone or prostaglandin E ₂. Heparin was also found to inhibit glucagon-sensitive rat hepatice adenylate cyclase, and the prostaglandin E₁-sensitive enzyme from rat ileum and human platelets. In contrast, heparin stimulated the dopamine sensitive adenylate cyclase from rat caudate nucleus. The sulfade polysugar dextran sulfate exerts similar effects on adenylate cyclase activity of the rat ovary was shown to inhibit hormone binding to rat ovarian plasma membrane in a manner similar to that exerted by heparin. In contrast to heparin, dextran sulfate inhibited dopamine-sensitive adenylate cyclase from rat caudate nucleus.     

Properties of rat liver plasma membrane adenylate cyclase after chromatography on O-diethylaminoethyl-cellulose and agarose-hexane-GTP: Sensitivity of partially purified and purified enzyme to Lubrol-PX, 5′-guanylylimidodiphosphate,and glucagon

Partially purified rat liver plasma membranes were enriched to yield a more glucagon-sensitive membrane fraction which was solubilized with Lubrol-PX. The supernate obtained after centrifugation at 165,000g was subjected to O-diethylaminoethyl anion exchange chromatography. An adenylate cyclase fraction was eluted and purified further by chromatography on agarose-hexane-GTP. The enzyme adsorbed to the affinity resin and was eluted with 0.5 m Tris-HCl. The protein isolated by chromatography on the affinity resin was homogenous by conventional acrylamide gel electrophoresis; one band was observed in sodium dodecyl sulfate. The purified enzyme was free of nucleotide phosphohydrolases found in the parent solubilized membrane preparation. The anion exchange product was not sensitive to glucagon; Lubrol-PX and 5′-guanylylimidodiphosphate [Gpp(NH)p] decreased the activity of this fraction. In the presence of detergent or guanyl nucleotide, glucagon, at 10^?6m, increased enzyme activity by 30 and 21%, respectively, to a statistically significant degree, but not above basal levels. Adenylate cyclase was also purified by subjecting the 165,000g supernate directly to agarose-hexane-GTP; agarose-hexane-ATP or agarose-hexane was not effective. The affinity-derived material was associated with 85 nmol of Lubrol-PX/mg of protein. When calculated on the basis of a molecular weight of 150,000 for detergent-free protein after gel filtration on Bio-Gel A-0.5 m, there was 13 mol of detergent/mol of the enzyme obtained by chromatography on the affinity resin. The direct affinity product was insensitive to glucagon and Gpp(NH)p; enzyme activity varied as a function of Lubrol concentration.     

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.     

Structural and functional polarity of canalicular and basolateral plasma membrane vesicles isolated in high yield from rat liver

A method has been developed for routine high yield separation of canalicular (cLPM) from basolateral (blLPM) liver plasma membrane vesicles of rat liver. Using a combination of rate zonal floatation (TZ- 28 zonal rotor, Sorvall) and high speed centrifugation through discontinuous sucrose gradients, 9-16 mg of cLPM and 15-28 mg of blLPM protein can be isolated in 1 d. cLPM are free of the basolateral markers Na+/K+-ATPase and glucagon-stimulatable adenylate cyclase activities, but are highly enriched with respect to homogenate in the "canalicular marker" enzyme activities leucylnaphthylamidase (48-fold), gamma-glutamyl-transpeptidase (60-fold), 5'-nucleotidase (64-fold), alkaline phosphatase (71-fold), Mg++-ATPase (83-fold), and alkaline phosphodiesterase I (116-fold). In contrast, blLPM are 34-fold enriched in Na+/K+-ATPase activity, exhibit considerable glucagon-stimulatable adenylate cyclase activity, and demonstrate a 4- to 15-fold increase over homogenate in the various "canalicular markers." cLPM have a twofold higher content of sialic acids, cholesterol; and sphingomyelin compared with blLPM. At least three canalicular-(130,000, 100,000, and 58,000 mol wt) and several basolateral-specific protein bands have been detected after SDS PAGE of the two LPM subfractions. Specifically, the immunoglobin A-binding secretory component is restricted to blLPM as demonstrated by immunochemical techniques. These data indicate virtually complete separation of basolateral from canalicular LPM and demonstrate multiple functional and compositional polarity between the two surface domains of hepatocytes.     

Complement-mediated production of plasma-membrane vesicles from rat fat-cells.

1. Rat isolated fat-cells were coated with rabbit anti-(rat erythrocyte) antibody and incubated with fresh guinea-pig serum for 25 min at 37 degrees C, which resulted in a more than 95% release of the cytosolic enzyme lactate dehydrogenase. 2. Under these conditions fragmentation of the plasma membrane was examined by following the plasma-membrane markers 5'-nucleotidase, adrenaline-sensitive adenylate cyclase and membrane-bound rabbit immunoglobulin G through a differential-centrifugation fractionation procedure. 3. Approx. 50% of the plasma-membrane markers remained associated with triacylglycerol. Of the remainder more than half was pelleted by centrifugation at 10 000 g for 30 min. 4. The 10 000 g supernatant was fractionated by centrifugation on a sucrose density gradient (15-50%, w/w). This procedure resulted in the production of two visible white bands on the density gradient. The bands consisted of vesicles derived from the plasma membrane, since they coincided with peaks of 5'-nucleotidase activity, contained membrane-bound immunoglobulin G and the denser one had adenylate cyclase activity. The phospholipid and protein contents of the vesicles were determined and compared with those in purified plasma membrane. 5. It is suggested that complement-mediated lysis of rat fat-cells caused the production of plasma-membrane vesicles that differ in composition from the whole plasma membrane.     

Purification of bovine adrenal-cortex plasma-membrane vesicles containing a highly corticotropin-sensitive adenylate-cyclase system and angiotensin-II-binding sites.

The purpose of this experimental investigation was to provide a purified plasma membrane fraction containing a highly hormone-responsive adenylate cyclase system. Bovine adrenal cortex was homogenised and a washed pellet (450 000 X g - min) was fractionated by zonal centrifugation in a sucrose and dextran gradient. Adenylate cyclase activity was purified up to 60-fold to a specific activity of 55, 340 and 210 pmol of adenosine 3':5'-monophosphate (cyclic AMP) produced/minute per mg of protein at 38 degrees C for the basal, adrenocorticotrophin and fluoride-activated states, respectively. The time course of the adenylate cyclase activity is linear. The concentration necessary for half-maximal stimulation by adrenocorticotrophin-(1-24)-tetracosipeptide is 0.5 muM. The high hormone-responsiveness of the membrane preparation allows one to demonstrate activation of adenylate cyclase by very weakly agonistic adrenocorticotrophin fragments. The F- activated state can be detergent-dispersed by Lubrol and shows a Km (ATP) different from that of either the basal or adrenocorticotrophin-stimulated state. Other marked enzymes such as 5'-nucleotidase, glucose-6-phosphatase and cytochrome oxidase were followed during purification. The plasma membrane fraction shows rather homogeneous, relatively large vesicles (mean diameter 0.5 mum). It contains high-affinity binding sites for angiotensin II (about 2 pmol per mg protein) with an apparent association constant of 2 X 10(7) (1/mol) at 12 degrees C. The yield, 20 mg of membrane protein per preparation, may make it a tool in either affinity-labelling studies with the peptide hormones mentioned or the starting point for solubilisation and purification of adenylate cyclase.     

Dimethylnitrosamine inhibits the glucagon-stimulated adenylate cyclase activity of rat liver plasma membranes and decreases plasma membrane fluidity

The effect of the hepatocarcinogen dimethylnitrosamine on rat liver plasma membrane adenylate cyclase activity and lipid fluidity was assessed. Glucagon-stimulated adenylate cyclase activity exhibited a complex response to increasing concentrations of dimethylnitrosamine, whereas fluoride-stimulated adenylate cyclase activity was progressively inhibited. Maximal inhibitory effects were observed at a concentration of 15 mM in both cases. The activity of detergent-solubilized adenylate cyclase was unaffected by dimethylnitrosamine. ESR analysis using a fatty acid spin probe showed that dimethylnitrosamine produced a marked, dose-dependent reduction in the fluidity of the plasma membrane with a maximal effect occurring at 20 mM. Dimethylnitrosamine also elevated the temperature at which the lipid phase separation occurred in rat liver plasma membranes, from 28 degrees C to 31 degrees C. The non-carcinogenic but structurally similar compound, dimethylamine hydrochloride neither inhibited adenylate cyclase nor decreased plasma membrane fluidity. It is suggested that the decrease in membrane fluidity, induced by dimethylnitrosamine, via its effects on membrane fluidity, could influence plasma membrane function and cellular regulation.     

Changes in plasma membrane enzyme activities during liver regeneration in the rat.

Changes in activities of plasma membrane enzymes during liver regeneration may be related to the maintenance of hepatic function or to the regulation of cell proliferation. Plasma membranes were isolated from rat livers at various times after partial hepatectomy, and the specific activities of alkaline phosphatase, (Na+ + K+)-ATPase, leucine aminopeptidase, 5'-nucleotidase, and adenylate cyclase (basal and with glucagon or epinephrine) were measured. Alkaline phosphatase and (Na+ + K+)-ATPase activity increased 3.6-fold and 2-fold respectively, during the first 48 h after partial hepatectomy. The time of onset and duration of change suggest that these increases in activity are involved in the maintenance of bile secretion. Decreases in leucine aminopeptidase activity at 48--108 h and in 5'-nucleotidase activity at 12--24 h were observed, which may be involved in the restoration of protein and accumulation of RNA. The basal activity of adenylate cyclase increased after partial hepatectomy. The response of adenylate cyclase to epinephrine showed a transitory increase between 36 and 108 h after surgery, while the response to glucagon was decreased by approximately 50% at all time points through 324 h after surgery. These changes in the hormone responsiveness of adenylate cyclase are similar to those previously observed in fetal and preneoplastic liver.     

Endocytosis of beta-adrenergic ligands by rat liver. Comparison of beta-adrenergic receptor and adenylate cyclase distribution in endosome and plasma-membrane fractions.

The internalization of beta-adrenergic receptors was investigated in rat livers perfused with an agonist ([3H]isoprenaline) or an antagonist ([125I]iodocyanopindolol). Analytical centrifugation of liver homogenates indicated that the ligands were transferred rapidly to endosomal and lysosomal positions in sucrose gradients. Endosome fractions contained beta-adrenergic binding sites, but adenylate cyclase activity was low and poorly activated by isoprenaline. The results indicate that the receptor-regulatory-protein-adenylate cyclase complex was disassembled during uptake of beta-adrenergic ligands, with the adenylate cyclase being retained at the plasma membrane.     

Studies on isolated subcellular components of cat pancreas. I. Isolation and enzymatic characterization.

Pancreas of the cat was fractionated into its subcellular components by centrifugation through an exponential ficoll-sucrose density gradient in a zonal rotor. This enables a preparation of four fractions enriched in plasma membranes, endoplasmic reticulum, mitochondria and zymogen granules, respectively. The first fraction, enriched by 9- to 15-fold in the plasma membrane marker enzymes, hormone-stimulated adenylate cyclase, (Na+K+)-ATPase, and 5'-nucleotidase, is contaminated by membranes derived from endoplasmic reticulum but is virtually free from mitochondrial and zymogen-granule contamination. The second fraction from the zonal gradient shows only moderate enrichment of the above marker enzymes but contains a considerable quantity of plasma membrane marker enzymes and represents mostly rough endoplasmic reticulum. The third fraction contains the bulk of mitochondria and the fourth mainly zymogen granules as assessed by electron microscopy and marker enzymes for both mitochondria and zymogen granules, namely succinic dehydrogenase, trypsin and amylase. Further purification of the plasma membrane fractions by differential and sucrose step-gradient centrifugation yields plasma membranes enriched 40-fold in basal and hormone-stimulated adenylate cyclase and (Na+K+)-ATPase.     

Partial purification from rat and pig liver of cytosolic stimulators of hormone-sensitive adenylate cyclase: quantitation and resolution of two components

Procedures were carried out to isolate from liver cytosol the protein activators of hormone-sensitive adenylate cyclase. A method for quantifying amounts of activator protein was used to monitor recovery after each isolation step. The activator proteins were precipitable by ammonium sulfate (30-60% saturation) and partially recoverable from the precipitate. On gel filtration of cytosol, stimulatory activity for glucagon-sensitive adenylate cyclase was recovered in two peaks representing proteins with molecular weights of 49,000 and 25,500. Exposure to GTP-Sepharose reduced liver cytosol's content of stimulatory factors for glucagon-sensitive adenylate cyclase by up to 70%. However, soluble protein adenylate cyclase activators distinct from GTP could not be subsequently eluted from the affinity matrix. Purification efforts were thwarted by factor instability and large losses during simple and conventional steps despite the use of a variety of protein stabilizers and protease inhibitors. If the problem of stimulator instability can be overcome, large-scale purification should be possible using pig liver as a starting material.     

Studies on isolated subcellular components of cat pancreas

Summary Pancreas of the cat was fractionated into its subcellular components by centrifugation through an exponential ficoll-sucrose density gradient in a zonal rotor. This enables a preparation of four fractions enriched in plasma membranes, endoplasmic reticulum, mitochondria and zymogen granules, respectively. The first fraction, enriched by 9- to 15-fold in the plasma membrane marker enzymes, hormone-stimulated adenylate cyclase, (Na⁺K⁺)-ATPase, and 5-nucleotidase, is contaminated by membranes derived from endoplasmic reticulum but is virtually free from mitochondrial and zymogen-granule contamination. The second fraction from the zonal gradient shows only moderate enrichment of the above marker enzymes but contains a considerable quantity of plasma membrane marker enzymes and represents mostly rough endoplasmic reticulum. The third fraction contains the bulk of mitochondria and the fourth mainly zymogen granules as assessed by electron microscopy and marker enzymes for both mitochondria and zymogen granules, namely succinic dehydrogenase, trypsin and amylase. Further purification of the plasma membrane fractions by differential and sucrose step-gradient centrifugation yields plasma membrane enriched 40-fold in basal and hormone-stimulated adenylate cyclase and (Na⁺K⁺)-ATPase.     

Comparison of bone and osteosarcoma adenylate cyclase. Partial purification of membranes and kinetic properties of enzyme.

The purpose of this study was to compare the adenylate cyclase of a tumour (rat osteosarcoma) growing in vivo with that of fast-growing embryonic bone. In the tumour the enzyme activity per total protein or DNA (under the same assay conditions) was 6--10-fold lower than in embryonic bone. To characterize this difference, we examined the kinetic properties of the enzyme in partially purified plasma membranes from the two tissues. A purification procedure based on differential centrifugation and discontinuous-sucrose-gradient centrifugation yielded a 10-fold increase in the specific activities of adenylate cyclase and 5'-nucleotidase in bone. The same procedure yielded an enriched membrane preparation from the tumour, but, relative to 5'-nucleotidase, a loss of 30% in adenylate cyclase occurred, which could not be recovered from another fraction. Kinetic analysis revealed that the lower adenylate cyclase activity in the tumour was due to a decrease in Vmax.. There was no significant difference in Ks (approx. 0.15 mM), and in the Km for GTP and p[NH]ppG. There were marked differences, however, in the extent of stimulation by p[NH]ppG, GTP and hormone, which was greater in tumour, and in the K1 for adenosine inhibition, which was 140 microM in bone and 500 microM in tumour. Under maximum stimulatory conditions, the enzyme activity in the tumour approached that in bone. The kinetic differences between bone and tumour enzyme were decreased by detergent solubilization, suggesting that the membrane environment plays a role in the generation of the observed differences.     

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.     

Changes in plasma membrane enzyme activities during liver regeneration in the rat

Changes in activities of plasma membrane enzymes during liver regeneration may be related to the maintenance of hepatic function or to the regulation of cell proliferation. Plasma membranes were isolated from rat livers at various times after partial hepatectomy, and the specific activities of alkaline phosphatase, (Na⁺ + K⁺)-ATPase, leucine aminopeptidase, 5′-nucleotidase, and adenylate cyclase (basal and with glucagon or epinephrine) were measured. Alkaline phosphatase and (Na⁺ + K⁺)-ATPase activity increased 3.6-fold and 2-fold respectively, during the first 48 h after partial hepatectomy. The time of onset and duration of change suggest that these increases in activity are involved in the maintenance of bile secretion. Decreases in leucine aminopeptidase activity at 48–108 h and in 5′-nucleotidase activity at 12–24 h were observed, which may be involved in the restoration of protein and accumulation of RNA. The basal activity of adenylate cyclase increased after partial hepatectomy. The response of adenylate cyclase to epinephrine showed a transitory increase between 36 and 108 h after surgery, while the response to glucagon was decreased by approximately 50% at all time points through 324 h after surgery. These changes in the hormone responsiveness of adenylate cyclase are similar to those previously observed in fetal and preneoplastic liver.