Guanylate cyclase from the rat renal medulla. Physical properties and comparison with adenylate cyclase.

Guanylate cyclase from the rat renal medulla is found in both the soluble and particulate fractions of the cell. Sucrose density gradient centrifugation and gel filtration in H2O and D2O indicate that the enzyme from the soluble cell fraction has the following properties: S20w, 6.3 S; Stokes radius, 54 A; partial specific volume, 0.75 ml/g; mass, 154,000 daltons; f/fo, 1.4; axial ratio (prolate ellipsoid), 7. The addition of 0.1% Lubrol PX to this fraction activates the enzyme and changes thartial specific volume, 0.74 ml/g; mass, 148,000 daltons; f/fo, 1.6; axial ratio (prolate ellipsoid), 11. These findings show that detergent activates the enzyme by changing its conformation and not simply by dispersing nonsedimentable membrane fragments. The dimensions of this guanylate cyclase in detergent are very similar to those of detergent-solubilized adenylate cyclase from the same tissue (Neer, E.J. (1974) J. Biol. Chem. 249, 6527-6531). Guanylate cyclase can be solubilized from the particulate cell fraction with 1% Lubrol PX but has properties quite different from those of the guanylate cyclase in the soluble cell fraction. It is a large aggregate with a value of S20,w of about 10 S, Stokes radius of 65 A, and a mass of approximately 300,000 daltons. However, the peaks of guanylate cyclase activity in column effluents and sucrose density gradients are very broad indicating a mixture of different size proteins. The conditions used to solubilize guanylate cyclase from the particulate fraction also solubilize adenylate cyclase, and the two activities can be separated on the same sucrose gradient. Studies of this sort require a rapid, accurate guanylate cyclase assay. We have developed an assay for guanylate cyclase activity which meets these criteria by adapting the competitive protein binding assay for guanosine cyclic 3':5' monophosphate originally described by Murad et al. (Murad, F., Manganiello, V., and Vaughn, M. (1971) Proc. Natl. Acad. Sci. U.S.A. 68, 736-739).     

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.     

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.     

Hydrodynamic properties of the regulatory component of adenylate cyclase

Hydrodynamic parameters of the regulatory component of adenylate cyclase, G/F, have been estimated by gel filtration and sucrose density gradient centrifugation. In solutions containing Lubrol 12A9, the protein has an apparent molecular weight of 130,000. G/F from various sources and resolved from the catalytic moiety of the enzyme by different techniques behaves similarly. Consistent with our previous proposal that this protein is the site of action of both guanine nucleotides and fluoride, treatment with these activating ligands causes a reduction in both the sedimentation coefficient and the Stokes radius of G/F. These changes suggest a loss of mass of approximately 40,000 daltons. Nevertheless, this alteration is fully reversible when ligands are removed, even if the liganded protein is first fractionated by gel filtration or sucrose density gradient centrifugation.     

Protein activator of cyclic 3':5'-nucleotide phosphodiesterase of bovine or rat brain also activates its adenylate cyclase.

Bovine or rat brain adenylate cyclase (EC 4.6.1.1) solubilized by Lubrol PX contained an activator which was separated from the enzyme by an anionic exchange resin column. Dissociation of the activator from adenylate cyclase rendered the enzyme less active, and reconstituting with an exogenous activator restored full enzyme activity. A pure protein activator of cyclic 3′:5′-nucleotide phosphodiesterase (EC 3.1.4.17) isolated from bovine brain also stimulated this adenylate cyclase. Stimulation of adenylate cyclase by the activator required Ca⁺⁺, the effect being immediate and reversible. Although the activator was specific, it lacked tissue specificity; an activator isolated from bovine brain cross-activated effectively adenylate cyclase from rat, and vice versa. These findings indicate that brain adenylate cyclase required an activator for activity and that this activator is functionally identical to the protein activator of phosphodiesterase (J.B.C. 249: 4943–4954, 1974).     

The effect of gonadotropins on the mitochondrial adenylate cyclase of rat testis

The formation of adenosine 3′:5′-cyclic monophosphate from ATP by testicular mitochondria of immature and mature rats was increased to the same extent by addition of either human chorionic gonadotropin or luteinizing hormone. Follicle stimulating hormone was found to be more active in stimulating adenylate cyclase activity in testicular mitochondria of immature rats. The stimulatory effect of gonadotropins were not suppressed by Ca⁺⁺ complexing agent ethylene-glycol-bis-(β-amino-ethyl ether) N,N′-tetra-acetic acid. The detergent Lubrol PX, solubilized 75–80% of the mitochondrial adenylate cyclase. The solubilized enzyme was activated by sodium fluoride but not by gonadotropins. The present results indicate a direct effect of gonadotropins on the adenylate cyclase attached to mitochondrial membranes.     

Solubilization and conversion of hepatic adenylate cyclase to a form requiring MnATP as substrate

A general feature of membrane-bound adenylate cyclase systems is the “lability” of the basal enzyme to dispersion by detergents. A stable form of the detergentsolubilized enzyme is obtained only if the membrane-bound enzyme is first pretreated with fluoride or Gpp(NH)p. However, we have found with the basal hepatic enzyme that the lability is evident primarily when MgATP is used as substrate; substitution of MnATP for MgATP reveals that substantial basal activity survives detergent treatment. This effect is independent of the detergent; it is seen with either Lubrol PX or with deoxycholate. In addition to the altered substrate requirement, the membrane-bound and solubilized forms of the basal enzyme exhibit other differences. In contrast to the membrane-bound form, the solubilized enzyme shows (1) weak stimulation by Gpp(NH)p; (2) little inhibition by adenosine, (3) strong inhibition by P_i or PP_i, and (4) and apparent loss of the Me²⁺-reactive regulatory site. Such dissimilarities between membranebound and solubilized cyclase are not seen if the membranes are pretreated with Gpp(NH)p prior to exposure to detergents. The characteristics of the solubilized basal hepatic enzyme are similar to those of the naturally occurring soluble adenylate cyclase found in mature rat testes. It would appear that separation of adenylate cyclase from components that confer regulation by divalent cation and guanine nucleotides produces a form of the enzyme that will turnover only MnATP; this may represent the free catalytic moiety. Such preparations could be useful in reconstructing some of the regulatory functions of adenylate cyclase seen in its membrane-bound form.     

Isoelectric focusing of brain adenylate cyclase

Mouse brain adenylate cyclase has been solubilized with Lubrol PX and separated by isoelectric focusing on polyacrylamide gels. The enzyme activity has been measured with a sensitive assay isolating cyclic AMP from Dowex and alumina columns. The technique allows a one-step analysis of this membrane enzyme from a heterogeneous sample within 6 hr.     

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.     

Cholera-toxin-dependent ADP-ribosylation of the adenylate cyclase regulatory protein in turkey erythrocyte membranes

Incubation of turkey erythrocyte membranes with cholera toxin and [³²P]NAD caused toxin-dependent incorporation of ³²P into a 42,000 M_r peptide which could be distinguished from toxin-independent ³²P incorporation into other membrane proteins. The radiolabeled 42,000 M_r peptide could be extracted from the membranes using Lubrol PX. When toxin-treated membranes were incubated with isoproterenol and GMP before detergent solubilization, the 42,000 M_r labeled peptide was adsorbed by GTP-γ-agarose which, with the same conditions, adsorbed the adenylate cyclase guanine nucleotide regulatory protein. The labeled peptide and guanine nucleotide regulatory protein activity were coeluted from the affinity matrix by guanylyl-β,γ-imidodiphosphate, GDP, and GMP. Guanosine 5′-O-(2-thiodiphosphate), an analog of GDP which blocks guanine nucleotide- and fluoride-stimulated adenylate cyclase activity, caused elution of labeled peptide which exhibited no regulatory protein activity. Our data support the view that the 42,000 M_r peptide is part of the adenylate cyclase guanine nucleotide regulatory protein. The labeled peptide allows identification of both active and inactive regulatory protein and should be useful in monitoring the purification of the regulatory protein from turkey erythrocytes.     

Solubilization and separation of the glucagon receptor and adenylate cyclase in guanine nucleotide-sensitive states.

Adenylate cyclase in liver membranes was solubilized with Lubrol PX and partially purified by gel filtration. The partially purified enzyme was susceptible to activation by guanyl-5'-yl imidodiphosphate (Gpp(NH)p). Studies on the binding of [3H]Gpp(NH)p to various fractions eluted from the gels revealed that an upper limit of 1% of the Gpp(NH)p binding sites is associated with adenylate cyclase activity stimulated by the nucleotide. The glucagon receptor, pretagged with 125I-glucagon in the membranes, solubilized with Lubrol PX, and fractionated on the same gel columns, eluted in a peak fraction that overlaps with, but is separate from, adenylate cyclase in its Gpp(NH)p-stimulated form. Addition of GTP to the solubilized glucagon-receptor complex caused complete dissociation of the complex, as has been shown with the membrane-bound form of the complex. Since the GTP-sensitive form of the glucagon receptor complex separates from the Gpp(NH)p-sensitive form of adenylate cyclase, it is concluded that the receptor and the enzyme are separate molecules, each associated with a distinct nucleotide regulatory site or component. These findings are discussed in terms of the possible structure of the hormone-sensitive state of adenylate cyclase.     

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.?     

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.     

Properties of detergent-dispersed myocardial adenylate cyclase

Adenylate cyclase from rabbit ventricle was solubilized in 30 to 50% yield by the nonionic detergent Lubrol PX. The detergent, when present in the assay at concentrations above 0.05%, rapidly inactivated the enzyme in assays conducted above 26 °C; assays were valid only when conducted below this temperature. The solubilized enzyme was eluted from diethylaminoethyl (DEAE)-Bio-Gel A (DEAE-agarose) with 100 mm NaCl in a yield of 25% and was free of detergent. Several properties of the solubilized detergent-free enzyme were similar to properties of the native membrane-bound species. The K_m for substrate was 0.1 mm, the K_a for Mg²⁺ was 2.5 mm, and ATP in excess of Mg²⁺ was inhibitory. The enzyme was activated by F^? and guanyl-5′-yl imidodiphosphate [Gpp(NH)p] in a time- and temperature-dependent manner, and activation by the latter was persistent. Activation by F^? and Gpp(NH)p reduced the K_a for Mg²⁺. Activation by Gpp(NH)p was increased by Mg²⁺; the apparent K_a for activation was 0.1 μm. Multiple binding sites for Gpp(NH)p were present: one class with a K_d value of 0.11 μm was probably associated with activation of the enzyme. The soluble enzyme was insensitive to catecholamines, in both the presence and the absence of Gpp(NH)p. Sensitivity to catecholamines was not restored by the addition of phospholipids, particularly phosphatidyl inositol, in either the presence or the absence of Gpp(NH)p, and this phospholipid did not increase the sensitivity of the membrane-bound enzyme to epinephrine. Catecholamine binding sites were present, and their association with adenylate cyclase was seemingly not affected by phospholipids.     

Variations in the levels of cyclic adenosine 3':5'-monophosphate and in the activities of adenylate cyclase and cyclic adenosine 3':5'-monophosphate phosphodiesterase during aerobic morphogenesis of Mucor rouxii

Particulate cell fractions of mycelium of Mucor rouxii contain adenylate cyclase activity which can be partially solubilized by 2% Lubrol PX. The enzyme requires Mn²⁺ and its activity is not modified by NaF or guanosine nucleotides. Mycelial extracts also contain cyclic adenosine 3′:5′-monophosphate phosphodiesterase activity, 60% of which is soluble. This activity shows characteristic low K_m (1 μm) for cyclic AMP and does not hydrolyze cyclic guanosine 3′:5′-monophosphate. It requires Mn²⁺ ions for maximal activity and is not inhibited by methylxanthines or activated by imidazole. Both enzymatic activities vary during the aerobic life cycle of the fungus. The spores have the highest levels of adenylate cyclase and cAMP phosphodiesterase, which decrease during the aerobic development. At the round cell stage, phosphodiesterase activity reaches 40% of the activity of the spores and varies only slightly thereafter. At this stage the specific activity of adenylate cyclase is 25% of the activity of ungerminated spores, and from this stage on, the activity increases up to the end of the logarithmic phase. Intracellular levels of cyclic AMP have been measured during aerobic germination. The variations of the intracellular level are tentatively explained by unequal variations in the activities of adenylate cyclase and cyclic AMP phosphodiesterase. A continuous increase of the extracellular cyclic AMP level during aerobic development has also been found, which cannot be accounted for solely by variations in the cyclase and diesterase activities.     

Dopamine receptors in canine caudate nucleus

Summary Physiological, pharmacological, histochemical and biochemical studies indicate that dopamine receptors are heterogenous in the, central nervous system with each individual functions. This review describes pharmacological and biochemical characteristics of dopamine receptors, particularly in canine caudate nucleus, which have been studied in our laboratory with a brief comparison to the current studies by other workers in similar research fields.Two distinct dopamine receptors have been characterized by means of [³H]dopamine binding to the synaptic membranes from canine caudate nucleus. One of the receptors with a Kd of about 3 M for dopamine may be associated with adenylate cyclase and referred to as D, receptor. The other receptor with a Kd of about 10 nM for dopamine is independent of adenylate cyclase and referred to as D₂. A photochemical irreversible association of [³H]dopamine with the membraneous receptors makes it possible to separate D₁ and D₂ receptors from one another by gel filtration on a Sephadex G-200 column after solubilization with Lubrol PX. On the basis of selective inhibition of [³H]dopamine binding to D₁ and D₂ receptors, dopamine antagonists can be classified into three classes: D₁-selective (YM-09151-2), D₂-selective (sulpiride) and nonselective (haloperidol, chlorpromazine). Effects of these typical antagonists on the metabolism of rat brain dopamine suggest that D₁ receptor is more closely associated with the neuroleptic-induced increase in dopamine turnover. Studies with 28 benzamide derivatives and some classical neuroleptics reveal that apomorphine-induced stereotypy displays a greater association with D₁ than with D₂ receptors.Dopamine-sensitive adenylate cyclase in canine caudate nucleus can be solubilized with Lubrol PX in a sensitive form to either dopamine, Gpp(NH)p or fluoride. Sephadex G-200 gel filtration separates adenylate cyclase from D₁ receptors with a concomitant loss of dopamine sensitivity. Addition of the D₁ receptor fraction to the adenylate cyclase restores the responsiveness to dopamine. The solubilized dopamine-unresponsive adenylate cyclase can be further separated into two distinct fractions by a batch-wise treatment with GTP-sepharose: a catalytic unit which does not respond to fluoride, and a guanine nucleotide regulatory protein. The regulatory protein confers distinct responsiveness to Gpp(NH)p and fluoride upon adenylate cyclase. These results indicate that dopamine-sensitive adenylate cyclase is composed of at least three distinct units; D₁ receptor, guanine nucleotide regulatory protein and adenylate cyclase.     

The role of phospholipids in TSH stimulation of adenylate cyclase in thyroid plasma membranes

Treatment of bovine thyroid plasma membranes with phospholipase A or C inhibited the stimulation of adenylate cyclase activity by thyroid-stimulating hormone (TSH). In general, basal and NaF-stimulated adenylate cyclase activity was not influenced by such treatment. When plasma membranes were incubated with 1–2 units/ml phospholipase A, subsequent addition of phosphatidylcholine or phosphatidylserine but not phosphatidylethanolamine partially restored TSH stimulation. Phosphatidylcholine was more effective than phosphatidylserine in that it caused greater restoration of the TSH response and smaller amounts of phosphatidylcholine were active. However, when the TSH effect was obliterated by treatment of plasma membranes with 10 units/ml phospholipase A, phospholipids were unable to restore any response to TSH. Lubrol PX, a nonionic detergent, inhibited basal, TSH- and NaF-stimulated adenylate cyclase activities in thyroid plasma membranes. Although phosphatidylcholine partially restored TSH stimulation of adenylate cyclase activity in the presence of Lubrol PX, it did not have a similar effect on the stimulation induced by NaF. These results indicate that phospholipids are probably essential components in the system by which TSH stimulates adenylate cyclase activity in thyroid plasma membranes. The effects do not seem to involve the catalytic activity of adenylate cyclase but the data do not permit a distinction between decreased binding of TSH to its receptor or impairment of the signal from the bound hormone to the enzyme activity.     

Preparative isoelectric focusing and some properties of solubilized adenylate cyclase from rat brain

A new procedure for the purification of rat brain adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) is presented. The enzyme solubilized in Lubrol PX was purified either by molecular sieving or by hydrophobic chromatography, followed by a preparative isoelectric focusing step. For this purpose, a new isoelectric focusing technique was developed which allows a good resolution of adenylate cyclase in a short period of time. When resolved by this procedure, the enzyme migrated as a single molecular species with a pI of 6.3. When isoelectric focusing was performed in the presence of EGTA, two distinct peaks of activity could be detected at pI 6.1 and 7.3. This suggests that adenylate cyclase consists of two subunits held together by divalent ions. It is shown that the purified adenylate cyclase has a smaller sedimentation coefficient and is less hydrophobic than the native one. We conclude that the adenylate cyclase containing complex was at least partially disaggregated by this procedure.     

Solubilization of hormone-responsive adenylate cyclase from human renal cortex

Adenylate cyclase activity from human renal cortical plasma membranes remained in the 100,000 xg supernatant (2 hrs) following treatment with 0.25% Lubrol PX in 10mM Tris buffer (pH 7.45), 1 mM EDTA, 0.25 M sucrose, and 5 mM NaF. Solubilization decreased total adenylate cyclase activity by at least one-half; responsiveness to calcitonin, glucagon and guanyl nucleotides, but not to parathyroid hormone, was preserved. Glucagon and calcitonin-stimulated adenylate cyclase eluted near the void volume on Sephadex G200 columns; two other peaks of non-hormone stimulated activity eluted later.     

Increase of proton electrochemical potential and ATP synthesis in rat liver mitochondria irradiated in vitro by helium-neon laser

Particulate adenylate cyclase (AC) and guanylate cyclase (GC) activities localized in the ciliary membrane from Paramecium were solubilized by a two-step procedure using the detergents Brij 56 and Lubrol PX. The enzymes remained in the supernatant after a 100 000 × g centrifugation. Upon gel chromatography, AC and GC were almost completely separated proving that each enzyme is a distinct molecular entity. Solubilization of GC was achieved with the calmodulin subunit remaining firmly attached to the catalytic part. Antibodies against calmodulin inhibited the enzyme as did La³⁺ and EGTA. AC activity appeared to be regulated specifically by K⁺, enzyme activity being enhanced up to 100% by 15 mM K⁺. Na⁺ and Li⁺ were inactive.