The size of adenylate cyclase and guanylate cyclase from the rat renal medulla

The size distribution of adenylate cyclase from the rate renal medulla solubilized with the nonionic detergents Triton X-100 and Lubrol PX was determined by gel filtration and by centrifugation in sucrose density gradients made up in H₂O or D₂O. The physical parameters of the predominant from in Triton X-100 are ²20,w, 5.9 S; Stokes radius, 62 A; partial specific volume (v ), 0.74 ml/g; mass, 159,000 daltons; f/f₀, 1.6; axial ratio (prolate ellipsoid), 11. For the minor form the values are : ²20,w, 3.0; Stokes radius, 28 A; mass, 38,000 daltons; f/f₀, 1.2. The corresponding values determined in Lubrol PX are similar. The value of v for the enzyme indicates that it binds less than 0.2 mg detergent/mg protein. Since interactions with detergents probably substitute for interactions with lipids and hydrophobic amino acid side chains, these findings suggest that no more than 5% of the surface of adenylate cyclase is involved in hydrophobic interactions with other membrance components. Thus, most of the mass of the enzyme is not deeply embedded in the lipid bilayer of the plasma membrance. Similar studies have been performed on the soluble guanylate cyclase of the rate renal medulla. In the absence of detergent, the molecular properties of this enzyme are: ^s20,w, 6.3 S; Stokes radius, 54 A, v , 0.75 ml/g; mass, 154,000 daltons f/f₀, 1.4; axial ratio, 7. The addition of 0.1% Lubrol PX to this soluble enzyme increases its activity two- to fourfold and changes the physical properties to : ^s20,w, 5.5 S; Stokes radius, 62 A; v , 0.74 ml/g; mass, 148,000 daltons; f/f₀, 1.6; axial ratio, 11. These results show that Lubrol PX activates the enzyme by causing a conformational change with unfolding on the polypeptide chain. Guanylate cyclase from the particulate cell fraction can be solubilized with Lubrol PX but has properties quite different from those of the enzyme in the soluble cell fraction. It is a heterogeneous aggregrate with ^s20,w, 10 S; Stokes radius, 65 A; mass about 300,000 daltons. The conditions which solubilize guanylate cyclase also solubilize adenylate cyclase and the two activities can be separated on the same sucrose gradient.     

Subcellular distribution and activation by non-ionic detergents of guanylate cyclase in cerebral cortex of rat

Non-ionic detergents stimulated particulate guanylate cyclase activity in cerebral cortex of rat 8- to 12-fold while stimulation of soluble enzyme was 1.3- to 2.5-fold. Among various detergents, Lubrol PX was the most effective one. The subcellular distribution of guanylate cyclase activity was examined with or without 0.5% Lubrol PX. Without Lubrol PX two-thirds of the enzyme activity was detected in the soluble fraction. In the presence of Lubrol PX, however, two-thirds of guanylate cyclase activity was recovered in the crude mitochondrial fraction. Further fractionation revealed that most of the particulate guanylate cyclase activity was associated with synaptosomes. The sedimentation characteristic of the particulate guanylate cyclase activity was very close to those of choline acetyltransferase and acetylcholine esterase activities, two synaptosomal enzymes. When the crude mitochondrial fraction was subfractionated after osmotic shock, most of guanylate cyclase activity as assayed in the absence of Lubrol PX was released into the soluble fraction while the rest of the enzyme activity was tightly bound to synaptic membrane fractions. The total guanylate cyclase activity recovered in the synaptosomal soluble fraction was 6 to 7 times higher than that of the starting material. The specific enzyme activity reached more than 1000 pmol per min per mg protein, which was 35-fold higher than that of the starting material. The membrane bound guanylate cyclase activity was markedly stimulated by Lubrol PX. Guanylate cyclase activity in the synaptosomal soluble fraction, in contrast, was suppressed by the addition of Lubrol PX. The observation that most of guanylate cyclase activity was detected in synaptosomes, some of which was tightly bound to the synaptic membrane fraction upon hypoosmotic treatment, is consistent with the concept that cyclic GMP is involved in neural transmission.     

Highly purified particulate guanylate cyclase from rat lung: characterization and comparison with soluble guanylate cyclase

Guanylate cyclase was purified 1000-fold from washed rat lung particulate fractions to a final specific activity of 500 nmoles cyclic GMP produced/min/mg protein by a combination of detergent extraction and chromatography on concanavalin A-Sepharose, GTP-agarose, and blue agarose. Particulate guanylate cyclase has a molecular weight of 200 000 daltons, a Stokes radius of 48 A and a sedimentation coefficient of 9.4 while the soluble form has a molecular weight of 150 000 daltons, a Stokes radius of 44 A, and a sedimentation coefficient of 7.0. Whereas the particulate enzyme is a glycoprotein with a specific affinity for concanavalin A and wheat germ agglutinin, the soluble form of guanylate cyclase did not bind to these lectins. Purified particulate guanylate cyclase did not cross-react with a number of monoclonal antibodies generated to the soluble enzyme. While both forms of the enzyme could be regulated by the formation of mixed disulfides, the particulate enzyme was relatively insensitive to inhibition by cystine. With GTP as substrate both forms of the enzyme demonstrated typical kinetics, and with GTP analogues negative cooperativity was observed with both enzyme forms. These data support the suggestion that the two forms of guanylate cyclase possess similar catalytic sites, although their remaining structure is divergent, resulting in differences in subcellular distribution, physical characteristics, and antigenicity.     

Guanylate cyclase and cyclic guanosine 3':5'-monophosphate phosphodiesterase activities and cyclic guanosine 3':5'-monophosphate levels in normal and transformed fibroblasts in culture.

To investigate the role of guanosine 3':5'-monophosphate (cyclic GMP) in cultured cells we have measured guanylate cyclase and cyclic GMP phosphodiesterase activities and cyclic GMP levels in normal and transformed fibroblastic cells. Guanylate cyclase activity is found almost exclusively in the particulate fraction of normal rat kidney (NRK) and BALB 3T3 cells. Enzyme activity is stimulated 3- to 10-fold by treatment with the detergent Lubrol PX. However, enhancement of guanylate cyclase by fibroblast growth factor could not be demonstrated under a variety of assay conditions. In both NRK and BALB 3T3 cells guanylate cyclase activity is low during logarithmic growth and increases as the cells crowd together and growth slows. Guanylate cyclase activity is undetectable in homogenates of NRK cells transformed by the Kirsten sarcoma virus (KNRK cells) either in the presence or absence of Lubrol PX. Guanylate cyclase activity is also greatly decreased in NRK cells transformed by Moloney, Schmidt-Ruppin, or Harvey viruses. BALB 3T3 cells transformed by RNA viruses (Kirsten, Harvey, or Moloney), by a DNA virus (SV40), by methylcholanthrene, or spontaneously, all have diminished but readily detectable guanylate cyclase activity. Cyclic GMP phosphodiesterase activity is found predominately in the soluble fraction of NRK cells. This activity increases slightly as NRK cells enter the stationary growth phase. Cyclic GMP phosphodiesterase activity is undetectable in two clones of KNRK cells under a variety of assay conditions, and is decreased relative to the level present in NRK cells in a third KNRK clone. However, both Moloney- and Schmidt-Ruppin-transformed NRK cells have a phosphodiesterase activity similar to that found in NRK cells. Boiled supernatant from both NRK and KNRK cells is observed to appreciably enhance the activity of activator-deficient phosphodiesterase from bovine heart. This result indicates that the absence of cyclic GMP phosphodiesterase activity in KNRK cells is not due to a loss of the phosphodiesterase activator. The intracellular concentration of cyclic GMP is found to be very low in transformed NRK cells when compared to levels measured in confluent NRK cells. The low levels of cyclic GMP in transformed NRK cells reflect the greatly decreased guanylate cyclase activity observed in these cells. These results do not appear to support the suggestion that cyclic GMP promotes the growth of fibroblastic cells.     

Rat testis mitochondrial adenylate cyclase. Partial purification and characterization.

1. Adenylate cyclase (EC 4.6.1.1) from rat testis mitochondria has been solubilized by treatment with the non-ionic detergent Lubrol PX. The soluble enzyme was further purified by DEAE-cellulose chromatography. 2. The specific activity of the adenylate cyclase eluted from the DEAE-cellulose column was found to be four times higher than that of an intact mitochondrial preparation. At this step the enzyme shows a sedimentation coefficient of 4.2 S and a diffusion coefficient (D) of 3.12 - 10- minus 7 cm-2/sec. 3. Solubilization of the adenylate cyclase resulted in loss of responsiveness to gonadotrophic hormones. Addition of phosphatidylserine to the soluble preparation partially restored the activation of adenylate cyclase by human chorionic gonadotrophin. 4. The results of this study suggest that the activity of the adenylate cyclase may be dependent on the membrane-bound phospholipids and that the enzyme attached to the mitochondrial membranes has some properties which are similar to the adenylate cyclase found to be associated with other membrane systems of the cell.?     

Proof of guanylate cyclase activity in the coronary artery of cattle]

Guanylate cyclase activities were identified in a soluble fraction and a particular fraction obtained from the Arteria coronaria of cattle. The Km-value was 1.0 +/- 0.7 - 10(-4) M for the enzyme substrate complex of the guanylate cyclase of the soluble fraction and 9.2 +/- 1.5 - 10(-4) M for the particular fraction. For the enzyme activity of the soluble fraction Mn++ cannot be replaced by Ca++ or Mg++, whereas for the enzyme activity of the particulate fraction Mn++ can be replaced by Mg++ but not by Ca++. The guanylate cyclase of the particulate fraction can be activated by acetylcholine. This activation can be cancelled by atropine. Acetylcholine exerts no influence on the guanylate cyclase activity of the soluble fraction. ATP inhibits the enzyme activities of both fractions whereas cAMP shows no influence on the guanylate cyclase activity.     

Guanylate cyclase activity of human lymphocytes from peripheral blood, thymus, and tonsils. A comparative study

The subcellular repartition and the distinctive properties of guanylate cyclase (EC 4.6.1.2) vary according to the lymphocyte population studied and according to the presence of detergent. Guanylate cyclase of non-adherent peripheral lymphocytes and of thymus lymphocytes is recovered by more than 90% in the soluble fraction of the homogenate. Kinetics according to the substrate (5'-GTP-Mn2+) is Michaelian, the Ca2+ ion acts as an activator, especially in the case of blood lymphocytes, and the detergent has no effect on the enzyme activity. On the other hand, the guanylate cyclase of tonsil lymphocytes reside in the particulate fraction. It has non-Michaelian kinetics for the substrate, a strong stimulating effect of detergent, and an inhibitory effect of Ca2+. A comparison of the enzymatic activities of unseparated and of non-adherent tonsil lymphocytes obtained from the same donor points to a correlation between their T and B properties: predominant soluble activity in the T population and particulate guanyl cyclase activity in the B subset.     

Activation of guanylate cyclase in cerebral cortex of rat by hydroxylamine.

Hydroxylamine actived guanylate cyclase in particulate fraction of cerebral cortex of rat. Activation was most remarkable in crude mitochondrial fraction. When the crude mitochondrial fraction was subjected to osmotic shock and fractionated, guanylate cyclase activity recovered in the subfractions as assayed with hydroxylamine was only one-third of the starting material. Recombination of the soluble and the particulate fractions, however, restored guanylate cyclase activity to the same level as that of the starting material. When varying quantities of the particulate and soluble fractions were combined, enzyme activity was proportional to the quantity of the soluble fraction. Heating of the soluble or particulate fraction at 55 degrees for 5 min inactivated guanylate cyclase. The heated particulate fraction markedly activated guanylate cyclase activity in the native soluble fraction, while the heated soluble fraction did not stimulate enzyme activity in the particulate. The particulate fraction preincubated with hydroxylamine at 37 degrees for 5 min followed by washing activated guanylate cyclase activity in the soluble fraction in the absence of hydroxylamine. Further fractionation of the crude mitochondrial fraction revealed that the factor(s) needed for the activation by hydroxylamine is associated with the mitochondria. The mitochondrial fraction of cerebral cortex activated guanylate cyclase in supernatant of brain, liver, or kidney in the presence of hydroxylamine. The mitochondrial fraction prepared from liver or kidney, in turn, activated soluble guanylate cyclase in brain. Activation of guanylate cyclase by hydroxylamine was compared with that of sodium azide. Azide activated guanylate cyclase in the synaptosomal soluble fraction, while hydroxylamine inhibited it. The particulate fraction preincubated with azide followed by washing did not stimulate guanylate cyclase activity in the absence of azide. The activation of guanylate cyclase by hydroxylamine is not due to a change in the concentration of the substrate GTP, Addition of hydroxylamine did not alter the apparent Km value of guanylate cyclase for GTP. Guanylate cyclase became less dependent on manganese in the presence of hydroxylamine. Thus the activation of guanylate cyclase by hydroxylamine is due to the change in the Vmax of the reaction.     

Trypanosoma cruzi adenylate cyclase activity. Purification and characterization.

Adenylate cyclase activity associated with Trypanosoma cruzi sedimentable fractions was solubilized by treatment with the non-ionic detergent Lubrol PX and 0.5 M-(NH4)2SO4. The following hydrodynamic and molecular parameters were established for a partially purified enzyme-detergent complex: sedimentation coefficient 6.2 S; Stokes radius 5.65 nm; partial specific volume 0.83 ml/g; Mr 244 000; frictional ratio 1.33. A Mr of about 124 000 was calculated for the detergent-free protein from these parameters. The pI of this enzyme activity was 6.2. A monoclonal antibody to T. cruzi adenylate cyclase was obtained, which inhibited cyclase activities from several lower eukaryotic organisms. The T. cruzi adenylate cyclase was further purified by using this antibody in immunoaffinity chromatographic columns. Fractions obtained after this chromatography showed, on SDS/polyacrylamide-gel electrophoresis, a main polypeptide band with an apparent Mr of about 56 000, which specifically reacted with the monoclonal antibody.     

Effect of alamethicin, gramicidin S and melittin upon the particulate guanylate cyclase from rat lung.

The channel-forming antibiotic alamethicin activated rat lung particulate guanylate cyclase (GTP pyrophosphate-lyase (cyclizing) EC 4.6.1.2), and the activated enzyme was further stimulated by sodium nitroprusside when a thiol such as 2-mercaptoethanol was present. Similar effects were seen with the antibiotic gramicidin S and with melittin, a polypeptide purified from bee venom. All of these agents are amphiphilic polypeptides. Nitroprusside was not able to stimulate both particulate and soluble enzyme treated with the nonionic amphiphile, Lubrol PX, suggesting that the membrane-active polypeptides had a different mechanism of action. These polypeptides are known to alter the membrane matrix by binding to phospholipid, and we suggest that this alteration allowed greater access of substrate and of nitroprusside to the enzyme. Lubrol PX, however, may interact preferentially with the enzyme, and thus block nitroprusside activation. The most potent of these agents was melittin, which stimulated nitroprusside activation at a concentration which had little effect by itself (7 microns), and at which others have demonstrated lytic effects on cells.     

Purification and properties of guanylate cyclase from the synaptosomal soluble fraction of rat brain.

Guanylate cyclase (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2) was purified 2250-fold from the synaptosomal soluble fraction of rat brain. The specific activity of the purified enzyme reached 41 nmol cyclic GMP formed per min per mg protein at 37 degrees C. In the purified preparation, GTPase activity was not detected and cyclic GMP phosphodiesterase activity was less than 4% of guanylate cyclase activity. The molecular weight was approx. 480 000. Lubrol PX, hydroxylamine, or NaN3 activated the guanylate cyclase in crude preparations, but had no effect on the purified enzyme. In contrast, NaN3 plus catalase, N-methyl-N'-nitro-N-nitrosoguanidine or sodium nitroprusside activated the purified enzyme. The purified enzyme required Mn2+ for its activity; the maximum activity was observed at 3-5 mM. Cyclic GMP activated guanylate cyclase activity 1.4-fold at 2 mM, whereas inorganic pyrophosphate inhibited it by about 50% at 0.2 mM. Guanylyl-(beta,gamma-methylene)-diphosphonate and guanylyl-imidodiphosphate, analogues of GTP, served as substrates of guanylate cyclase in the purified enzyme preparation. NaN3 plus catalase or N-methyl-N'-nitro-N-nitrosoguanidine also remarkably activated guanylate cyclase activity when the analogues of GTP were used as substrates.     

Stimulation of guanylate cyclase of fibroblasts by free fatty acids.

The membranous guanylate cyclase of Balb 3T3 fibroblasts was stimulated by a fraction of calf serum extracted by ether. Stimulation was observed with Mg2+ as the only bivalent cation in the presence of Lubrol PX. The activator co-chromatographed with free fatty acids, and several of these were found to stimulate guanylate cyclase. Among the saturated fatty acids, myristic acid had the highest activity. Stimulating activity diminished as the hydrocarbon chain of the fatty acid was lengthened or shortened. Introduction of an unsaturated bond enhanced the activation by the longer fatty acids. This pattern of specificity is similar to that observed for the effect of fatty acids on many other membranous functions. Under appropriate conditions fatty acids were found to stimulate guanylate cyclase activity in the absence of Lubrol PX. The relationship among the effects of Mg2+, Mn2+, Lubrol PX, and fatty acids on enzyme activity was examined. On the basis of these studies, it appears that fatty acids stimulate the enzyme by a mechanism different from nonionic detergents or Mn2+.     

Factors affecting the activity of guanylate cyclase in lysates of human blood platelets.

1. Under optimal ionic conditions (4 mM-MnCl2) the specific activity of guanylate cyclase in fresh platelet lysates was about 10nmol of cyclic GMP formed/20 min per mg of protein at 30 degrees C. Activity was 15% of optimum with 10mM-MgCl2 and negligible with 4mM-CaCl2. Synergism between MnCl2 and MgCl2 or CaCl2 was observed when [MnCl2] less than or equal to [GPT]. 2. Lower than optimal specific activities were obtained in assays containing large volumes of platelet lysate, owing to the presence of inhibitory factors that could be removed by ultrafiltration. Adenine nucleotides accounted for less than 50% of the inhibitory activity. 3. Preincubation of lysate for 1 h at 30 degrees C increased the specific activity of platelet guanylate cyclase by about 2-fold. 4. Lubrol PX (1%, w/v) stimulated guanylate cyclase activity by 3--5-fold before preincubation and by about 2-fold after preincubation. Triton X-100 was much less effective. 5. Dithiothreitol inhibited the guanylate cyclase activity of untreated, preincubated and Lubrol PX-treated lysates and prevented activation by preincubation provided that it was added beforehand. 6. Oleate stimulated guanylate cyclase activity 3--4-fold and arachidonate 2--3-fold, whereas palmitate was almost inactive. Pretreatment of lysate with indomethacin did not inhibit this effect of arachidonate. Oleate and arachidonate caused marked stimulation of guanylate cyclase in preincubated lysate, but inhibited the enzyme in Lubrol PX-treated lysate. 7. NaN3 (10mM) increased guanylate cyclase activity by up to 7-fold; this effect was both time- and temperature-dependent. NaN3 did not further activate the enzyme in Lubrol PX-treated lysate. 8. The results indicated that preincubation, Lubrol PX, fatty acids and NaN3 activated platelet guanylate cyclase by different mechanisms. 9. Platelet particulate fractions contained no guanylate cyclase activity detectable in the presence or absence of Lubrol PX that could not be accounted for by contaminating soluble enzyme, suggesting that physiological aggregating agents may increase cyclic GMP in intact platelets through the effects of intermediary factors. The activated and inhibited states of the enzyme described in the present paper may be relevant to the actions of these factors.     

The metabolism of cyclic nucleotides in the guinea-pig pancreas. Adenylate cyclase and guanylate cyclase.

Adenylate cyclase from the guinea-pig pancreas was activated in a dose-dependent manner by both secretin and cholecystokinin-pancreozymin, but in contrast with results in other species the hormones were approximately equipotent. All other hormones and transmitter substances tested were without any effect on adenylate cyclase activity. Guanylate cyclase activity was shown to have both particulate and supernatant components in the guinea-pig pancreas. The particulate enzyme, but not the supernatant enzyme, was markedly activated by Triton X-100, and most of the induced activity was released into the supernatant. The supernatant enzyme was specifically Mn2+-dependent, but, even though Mn2+ was maximally effective at a concentration of 3 mM, activity could be raised further by increasing Ca2+ concentration. The particulate enzyme, by contrast, was relatively Mn2+-independent. Activity of the particulate guanylate cyclase was enhanced by phosphatidylserine. The supernatant enzyme displayed classical Michaelis-Menten kinetics, but the particulate enzyme deviated markedly from such kinetics. Under none of the conditions used was any significant activation of guanylate cyclase observed with any of the secretogen hormones or transmitter substances.     

Apparent activation of rabbit lung membrane adenylate cyclase by cytosolic proteins possessing adenylate kinase activity.

Lung cytosolic fraction (23500 x g supernatant) activates cAMP synthesis by lung membrane adenylate cyclase (AC). 23 kDa and 29 kDa proteins were isolated from rabbit lung cytosolic fraction in a homogeneous state, as 'activators' of lung membrane AC. Both of these proteins possess high adenylate kinase (AK) activity and are able to mimic the 'activating' effect of lung cytosol on the lung membrane AC in the standard incubation mixture devoid of adenylate kinase. The activating effect is abolished in the presence of adenylate kinase inhibitor DAPP and after heat- or trypsin-treatment of the cytosolic fraction. Commercial adenylate kinase or nonionic detergent Lubrol PX activate cAMP synthesis by lung membrane AC in a similar manner to that of cytosolic fraction. In the presence of commercial adenylate kinase or Lubrol PX no activating effect of the cytosolic fraction on lung membrane AC is revealed. The ability of cytosolic fraction, commercial adenylate kinase, Lubrol PX or purified 23 kDa and 29 kDa proteins to activate cAMP synthesis by lung membrane AC correlates with their ability to support the constant ATP (AC substrate) concentration in the AC assay mixture. Our data indicate that 'activation' of lung membrane AC in the presence of cytosolic fraction may be produced by cytosolic adenylate kinase activity which regenerates ATP from AMP in the presence of creatine kinase and creatine phosphate providing the substrate for cAMP synthesis by AC.     

Sea urchin sperm guanylate cyclase antibody. Cross-reactivity various rat tissue guanylate cyclases.

After the repeated injection of sea urchin sperm guanylate cyclase into rabbits, antibodies to the enzyme were formed. These antibodies inhibited the particulate or the Triton-dispersed forms of the sperm enzyme by greater than 97%. The sperm adenylate cyclase, cyclic GMP phosphodiesterase, adenosine triphosphatase, guanosine triphosphatase, and 5'-nucleotidase enzymes were not affected by the antiserum. The antiserum inhibited the Triton-dispersed guanylate cyclase from rat heart, liver, lung, spleen, and kidney but did not inhibit the soluble form of the enzyme from any of these tissues. The inhibition of the Triton-dispersed enzyme in these tissues was partial, however, ranging from 30% (liver) to 70% (heart). These results provide evidence that adenylate cyclase is antigenically different from guanylate cyclase, and that the soluble form of guanylate cyclase is antigenically different from a particulate form of the enzyme in various rat tissues.     

Disaggregation of adenylate cyclase during polyacrylamide-gel electrophoresis in mixtures of ionic and non-ionic detergents

1. Adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] solubilized from the rat liver plasma membrane with 1% Lubrol PX and partially purified by gel filtration in buffer containing 0.01% Lubrol PX was physically characterized by polyacrylamide-gel electrophoresis. 2. The molecular radius determined for the partially purified enzyme was 4.9nm, compared with the value of 3.9nm obtained for the enzyme before gel filtration. 3. This difference, representing an approximate doubling of the molecular volume of the enzyme, implied that aggregation with itself or other proteins had occurred during partial purification. 4. Aggregation was not reversed by electrophoresis in the presence of high Lubrol concentrations. 5. Substitution of deoxycholate or N-dodecylsarcosinate for Lubrol PX either for solubilization or during electrophoresis led to poorer resolution of membrane proteins at concentrations giving greater than 70% loss of enzyme activity. 6. Partially purified adenylate cyclase was electrophoresed in the presence of mixed micelles of Lubrol PX and deoxycholate or Lubrol PX and N-dodecylsarcosinate. Different mixtures were examined simultaneously in a suitable apparatus. 7. Electrophoresis in the presence of 0.1% Lubrol plus 0.03% deoxycholate decreased the molecular radius of the cyclase to 4.0nm, with greater than 90% recovery of enzymic activity. The net charge of the enzyme was also increased, indicating ionic detergent binding. 8. With 0.1% Lubrol plus 0.03% N-dodecylsarcosinate the molecular radius was 4.3nm, recovery approx. 50% and net charge similar to that seen in Lubrol plus deoxycholate. 9. The resolution of cyclase from bulk protein, on an analytical scale, was improved in the presence of detergent mixtures, as compared with resolution in Lubrol alone. 10. The results demonstrate the usefulness of polyacrylamide-gel electrophoresis to detect and overcome aggregation problems with membrane proteins and suggest that detergent mixtures in specific ratios may be useful in the purification of adenylate cyclase and other intrinsic membrane proteins.     

Differential effects of detergents upon the soluble and particulate guanylate cyclases from rat lung.

The enzyme guanylate cyclase is present in both particulate and soluble form in rat lung homogenates. As previously reported, the soluble enzyme can be activated by preincubation in the presence of O₂. The inactive (nonactivated) soluble enzyme is also stimulated by nonionic detergents, in the order Tween 20 > Lubrol PX > Triton X-67 > Triton X-100. The activated enzyme, however, was inhibited by these detergents in the reverse order. Sodium deoxycholate and lysolecithin were potent inhibitors of both inactive and activated enzyme. The activity of the particulate enzyme was stimulated by Lubrol PX > Triton X-100 > Triton X-67 > Tween 20. At a low concentration of lysolecithin or deoxycholate the particulate activity was increased; however, when detergent/protein > 1, inhibition was seen. In the case of deoxycholate, the inhibition could be reversed if excess deoxycholate was removed either by chromatography or by forming mixed micelles with Lubrol PX; however, deoxycholate inhibition of the soluble enzyme was irreversible. The stimulation by detergents of the particulate enzyme was apparently the result of solubilization. The effects upon the activity of the soluble enzyme were interpreted in terms of a model which assumes two hydrophobic regions on the enzyme surface. The two regions differ in hydrophobicity with the more hydrophobic region only being exposed as a result of activation. Interaction of a nonionic detergent with the less hydrophobic region stimulates activity, while interaction with the more hydrophobic region results in inhibition.     

Endogenous activating factor for guanylate cyclase in synaptosomal-soluble fraction of rat brain.

When the crude mitochondrial fraction of rat brain was homogenized with distilled water and centrifuged, most of guanylate cyclase activity was detected in the soluble fraction. The total guanylate cyclase activity recovered in the soluble fraction was 5- to 8-fold higher than that of the crude mitochondrial fraction. The greater recovery of guanylate cyclase activity was found to be due to a release of an endogenous activating factor for guanylate cyclase. The activating factor was partially purified by acid extraction followed by a gel filtration and ion exchange resin columns. The factor was a dialyzable small molecule. The molecular weight was estimated to be between 300 and 600 by a Sephadex G-15 column and Diaflo ultrafilter membranes. It was stable in dilute acids, but labile in alkaline solution. It was readily soluble in water, but insoluble in organic solvents. Treatment with various enzymes, so far as tested, failed to abolish the activity. The activating factor stimulated the initial velocity of the reaction. It altered neither the Km value for GTP nor the dependency of the enzyme on divalent metals. The activation by the factor was due to an increase in the Vmax of the reaction. The activation was prevented by lysolecithin, Lubrol PX, hydroxylamine, methylhydroxylamine, or hemoglobin.     

Guanylate cyclase. Subcellular distribution in cardiac muscle, skeletal muscle, cerebral cortex and liver.

1. Guanylate cyclase of every fraction studied showed an absolute requirement for Mn2+ ions for optimal activity; with Mg2+ or Ca2+ reaction was barely detectable. Triton X-100 stimulated the particulate enzyme much more than the supernatant enzyme and solubilized the particulate-enzyme activity. 2. Substantial amounts of guanylate cyclase were recovered with the washed particulate fractions of cardiac muscle (63-98%), skeletal muscle (77-93%), cerebral cortex (62-88%) and liver (60-75%) of various species. The supernatants of these tissues contained 7-38% of total activities. In frog heart, the bulk of guanylate cyclase was present in the supernatant fluid. 3. Plasma-membrane fractions contained 26, 21, 22 and 40% respectively of the total homogenate guanylate cyclase activities present in skeletal muscle (rabbit), cardiac muscle (guinea pig), liver (rat) and cerebral cortex (rat). In each case, the specific activity of this enzyme in plasma membranes showed a five- to ten-fold enrichment when compared with homogenate specific activity. 4. These results suggest that guanylate cyclase, like adenylate cyclase, and ouabain-sensitive Na+ + K+-dependent ATPase (adenosine triphosphatase), is associated with the surface membranes of cardiac muscle, skeletal muscle, liver and cerebral cortex; however, considerable activities are also present in the supernatant fractions of these tissues which contain very little adenylate cyclase or ouabain-sensitive Na+ + K+-dependent ATPase activities.