Solubilization and characterization of hormone- responsive phosphodiesterase activity of rat fat cells.

Fat cells particulate phosphodiesterase activity can be solubilized in high yield (80--100%) in a buffer system (30 mM Tris - HCl, pH 8.0) containing non-ionic detergents (0.1% Brij 30, 1.0% Triton X-100), salt (3.0 mM MgSO4, 5.0 mM NaBr) and dithiothreitol (5.0 mM). Polyacrylamide gel electrophoresis of the solubilized enzyme activity indicated the presence of two bands of activities of different electrophoretic mobilities, both of which hydrolyzed cyclic AMP and cyclic GMP. The solubilized activity eluted from DEAE Bio-Gel columns as a somewhat broad profile with at least two peaks of activity. Activity against both cyclic AMP and cyclic GMP eluted in similar but not identical patterns. The solubilized enzyme and DEAE column eluates wxhibited low (less than 1 micronM) Michaelis constants for cyclic AMP and cyclic GMP. In addition, the increases in phosphodiesterase activity induced by incubation of intact fat cells with insulin or adrenocorticotropic hormone are maintained in the solubilized state.     

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Fat cell particulate phosphodiesterase activity can be solubilized in high yield (80–100%) in a buffer system (30 mM Tris · HCl, pH 8.0) containing non-ionic detergents (0.1% Brij 30, 1.0% Triton X-100), salt (3.0 mM MgSO₄, 5.0 mM NaBr) and dithiothreitol (5.0 mM). Polycrylamide gel electrophoresis of the solubilized enzyme activity indicated the presence of two bands of activities of different electrophoretic mobilities, both of which hydrolyzed cylic AMP and cyclic GMP. The solubilized activity eluted from DEAE Bio-Gel columns as a somewhat broad profile with at least two peaks of activity. Activity against both cyclic AMP and cyclic GMP eluted in similar but not identical patterns. The solubilized enzyme and DEAE column eluates exhibited low (<1 μM) Michaelis constants for cyclic AMP and cyclic GMP. In addition, the increase in phosphodiesterase activity induced by incubation of intact fat cells with insulin or adrenocorticotropic hormone are maintained in the solubilized state.     

Solubilization of adenylate cyclase of brain membranes by lipid peroxidation

Adenylate cyclase in the membrane fractions of bovine and rat brains, but not in rat liver plasma membranes, was solubilized by treatment with Fe²⁺ (10 μM) plus dithiothreitol (5 mM). Solubilization of the enzyme by these agents was completely prevented by simultaneous addition of N,N′-diphenyl-p-phenylenediamine (DPPD), an inhibitor of lipid peroxidation. Ascorbic acid also solubilized the enzyme from the brain membranes. Lipid peroxidation of the brain membranes was characterized by a selective loss of phosphatidylethanolamine. Solubilization of membrane-bound enzymes by Fe²⁺ plus dithiothreitol was not specific for adenylate cyclase, because phosphodiesterase, thiaminediphosphatase and many other proteins were also solubilized. Solubilized adenylate cyclase had a high specific activity and was not activated by either NaF, 5′-guanylyl imidodiphosphate (Gpp[NH]p) or calmodulin. These results suggested that lipid peroxidation of the brain membranes significantly solubilized adenylate cyclase of high specific activity.     

Purification, characterization and production of rabbit antibodies to rat liver particulate, high-affinity, cyclic AMP phosphodiesterase

The cyclic nucleotide phosphodiesterase (EC 3.4.16) activities of a rat liver particulate fraction were analyzed after solubilization by detergent or by freeze-thawing. Analysis of the two extracts by DEAE-cellulose chromatography revealed that they contain different complements of phosphodiesterase activities. The detergent-solubilized extract contained a cyclic GMP phosphodiesterase, a low affinity cyclic nucleotide phosphodiesterase whose hydrolysis of cyclic AMP was activated by cyclic GMP and a high affinity cyclic AMP phosphodiesterase. The freeze-thaw extract contained a cyclic GMP phosphodiesterase and two high affinity cyclic AMP phosphodiesterase, but no low affinity cyclic nucleotide phosphodiesterase. The cyclic AMP phosphodiesterase activities from the freeze-thaw extract and from the detergent extract all had negatively cooperative kinetics. One of the cyclic AMP phosphodiesterases from the freeze-thaw extract (form A) was insensitive to inhibition by cyclic GMP; the other freeze-thaw solubilized cyclic AMP phosphodiesterase (form B) and the detergent-solubilized cyclic AMP phosphodiesterase were strongly inhibited by cyclic GMP. The B enzyme appeared to be converted into the A enzyme when the particulate fraction was stored for prolonged periods at -20 degrees C. The B form was purified extensively, using DEAE-cellulose, a guanine-Sepharose column and gel filtration. The enzyme retained its negatively cooperative kinetics and high affinity for both cyclic AMP and cyclic GMP throughout the purification, although catalytic activity was always much greater for cyclic AMP. Rabbit antiserum was raised against the purified B enzyme and tested via a precipitin reaction against other forms of phosphodiesterase. The antiserum cross-reacted with the A enzyme and the detergent-solubilized cyclic AMP phosphodiesterase from rat liver. It did not react with the calmodulin-activated cyclic GMP phosphodiesterase of rat brain, the soluble low affinity cyclic nucleotide phosphodiesterase of rat liver or a commercial phosphodiesterase preparation from bovine heart. These results suggest a possible interrelationship between the high affinity cyclic nucleotide phosphodiesterase of rat liver.     

Effects of dithiothreitol on insulin-sensitive phosphodiesterase in rat fat cells

Dithiothreitol activates the low-Km membrane-bound cyclic AMP phosphodiesterase when incubated with the enzyme in a cell-free system. To investigate the mechanism of its activation, we studied the effect of protease inhibitors. Isolated fat cells obtained from Sprague-Dawley rats were incubated in Krebs-Henseleit Hepes buffer, pH 7.4, at 37 degrees C with and without insulin (2 nM, 10 min). A crude microsomal fraction prepared by differential centrifugation was suspended in 0.25 M sucrose containing 10 mM Tes buffer, pH 7.5, with and without 2 mM dithiothreitol and protease inhibitors at 4 degrees C for 48 h. Dithiothreitol stimulated the phosphodiesterase, in a time-dependent manner. As little as 0.02 mM dithiothreitol activated the enzyme, and the maximally effective dose was 2-10 mM. Among the various protease inhibitors tested, antipain, leupeptin, chymostatin and E-64 were the most effective in preventing activation of the enzyme by dithiothreitol. Antipain also inhibited release of the enzyme from the bound fraction. These results suggest that activation of the low-Km phosphodiesterase by dithiothreitol may be provoked by stimulation of an endogenous thiol protease.     

Stimulation of hamster adipocyte cyclic 3':5'-nucleotide phosphodiesterase activity by ionophore A23187 and calcium.

Incubation of hamster isolated fat cells with the ionophore A23187 and calcium for 20 minutes caused 30-40% increases in the cyclic 3':5'-nucleotide phosphodiesterase (EC 3.1.4.17) activity of adipocyte homogenates when either 0.6 micron cyclic AMP or 0.6 micron cyclic GMP was the enzyme substrate. The stimulation of adipocyte cyclic AMP phosphodiesterase activity by A23187 and calcium was not antagonized by the adrenergic receptor blocking agents phentolamine and propranolol. The changes in enzyme activity produced by the ionophore and calcium were not associated with elevated intracellular cyclic AMP levels. Furthermore, A23187 and calcium acted to enhance adipocyte phosphodiesterase activity before, but not after, homogenization of the fat cells. These data suggest that the phosphodiesterase activity of hamster isolated fat cells may, at least in part, be regulated by fluctuations in intracellular calcium concentrations.     

Solubilization of tryptophan-5-monooxygenase from the pineal glands and existance of an activating substance in the tissue extract

A particle bound tryptophan-5-monooxygenase in bovine pineal glands was solubilized by a combination of high pH (PH 8.5), high salt concentration (0.5 M-KCl or NaCl), and sonication (20 kHz, 200 W, 30min), We recovered 75% or more of the monooxygenase activity of the tissue homogenate in the 105,000 g supernatant thus confirming our previous study (Hori et al., 1976). The solubilized enzyme preparation contained an activating substance which was also particle bound and which activated the monooxygenase when preincubated together with the enzyme and dithiothreitol. The solubilized tryptophan-5-monooxygenase was purified about 6-fold over the tissue homogenate using ammonium sulphate fractionation and column chromatography on hydroxylapatite and Sephacryl S-200. The activating substance was separated from the monooxygenase during hydroxylapatite column chromatography. The activating substance was shown to be different from phospholipids, such as L-α-lysophosphatidyl choline and L-α-phosphatidyl-L-serine, and from heparin or bovine serum albumin, although bovine serum albumin significantly activated the monooxygenase. Further experiments have suggested that both the monooxygenase and the activating substance are modified by dithiothreitol and that these modified materials interact to give the active form of the monooxygenase.     

Insulin-sensitive phosphodiesterase. Its localization, hormonal stimulation, and oxidative stabilization.

As it was shown previoulsy by others, the membrane-bound phosphodiesterase (cyclic adenosine 3':5'-monophosphate phosphodiesterase) of rat epididymal fat cells was stimulated when intact cells were exposed to insulin. The levels of stimulation observed in the present study in the cell homogenate and microsomal fraction were approximately 2.0- to 2.5-fold and 2.5- to 3.0-fold, respectively, when the initial substrate level was 100 nM and insulin concentration was 1 to 3 nM. When the microsomal fraction was subjected to a sucrose density gradient centrifugation, most of the insulin-sensitive phosphodiesterase activity was fractionated into the "light" microsomal fraction which was rich in NADH2:potassium ferricyanide:oxidoreductase) and low in 5'-AMPase, adenylate cyclase, and insulin-binding activities. The latter three activities were mostly fractionated into the "heavy" microsomal fraction. Both basal and insulin-stimulated phosphodiesterase activities were low when cells were homogenized in the presence of N-ethylmaleimide or p-chloromercuribenzoate. The insulin-stimulated enzyme activity was also low when cells were homogenized in the presence of --SH compounds (e.g. dithiothreitol) or certain metal-chelating agents (e.g. ethylene glycol bis(beta-aminoethyl ehter)-N,N'-tetraacetate (EGTA)), or in a nitrogen atmosphere. The effect of EGTA was prevented by the addition of certain heavy metal ions but not by the addition of Ca2+ or Ca2+ plus Mg2+ ions. When cells were homogenized in the presence of certain oxidants (e.g. diamide, sodium tetrathionate, or air), a high plus-insulin activity was observed; this activity was not lowered by subsequent treatment of the enzyme with N-ethylmaleimede, EGTA, or fresh cell homogenate that was prepared in the presence of EGTA. However, the activity of an apparently oxidized enzyme could still be lowered by treatment woth dithiothreitol. A partially purified enzyme in the enzyme in the microsomal fraction was fairly stable both in basal and insulin-stimulated states (fully active after 35 days when kept at -20degrees). EGTA added to the homogenization buffer lowered the basal phosphodiesterase activity, but this effect was reversed by the addition of Ca2+ ions. EGTA also decreased the enzyme activity that was stimulated by norepinephrine. However, neither EGTA nor dithiothreitol had any effect on the activities of 5'-AMPase, NADH-dehydrogenase, and malate dehydrogenase of fat cells. The above data indicate that most of the insulin-sensitive phosphodiesterase and the so-called "cell membrane markers" are associated with different subcellular particles in the cell homogenate. In addition, the data seem to indicate that the insulin-stimulated phosphodiesterase has certain --SH groups and that the activity of the enzyme is stabilized when the --SH groups are oxidized by certain oxidants including molecular oxygen. It is suggested that the air oxidation of the enzyme is catalyzed by a trace of certain heavy metal ions and, therefore, can be blocked by a metal-chelating agent.     

Properties and distribution of cyclic AMP phosphodiesterase from rat liver.

1. Phosphodiesterase activity in rat liver supernatant and solubilized rat liver particulate fractions was chromatographed on Q Sepharose and several characteristics of each peak determined. 2. Rat liver supernatant contained four peaks of activity. The first two of these corresponded to type I and II phosphodiesterases. The fourth peaks was similar to a type V activity and the third peak could not be definitely classified. 3. Particulate activity solubilized by mild protease treatment also contained four peaks of activity. The first two corresponded to the first two from the supernatant, the fourth was a type IV enzyme which is the insulin activated phosphodiesterase. The third peak could not be definitely characterized. 4. Particulate activity solubilised by Triton X-100 contained three peaks. Two had the properties of a type IV enzyme but only one of these was immunologically identified as the insulin sensitive enzyme. The remaining activity was similar to the chymotrypsin peak 3 activity. 5. Most of the particulate phosphodiesterase of rat liver is found in a microsomal fraction, and most is the insulin sensitive type IV enzyme.     

Involvement of arginine residues in the activation of calmodulin-dependent 3',5'-cyclic-nucleotide phosphodiesterase

Pretreatment of an affinity-purified, brain calmodulin (CaM)-dependent phosphodiesterase (EC 3.1.4.17) with p-hydroxyphenylglyoxal (pHPG), a specific arginine-modifying reagent, resulted in a time-dependent loss in CaM-stimulated hydrolysis of cyclic AMP and cyclic GMP with no change in basal, CaM-independent activity. The loss in CaM-stimulated activity was preceded by a transient increase in CaM-dependent activity. Phenylglyoxal was 10-fold more effective than pHPG in promoting the loss of CaM-stimulated activity with a second-order rate constant of 13.3 M-1 min-1. Other arginine-modifying reagents, 1,2-cyclohexanedione and 2,3-butanedione, were not effective. The pHPG-modified enzyme was activated by 100 microM lysophosphatidylcholine to levels comparable to CaM-stimulated activity. The arginyl-modified enzyme was also activated by chymotrypsin and trypsin but not to the extent of the untreated enzyme stimulated with CaM. The presence of CaM during chemical modification with pHPG protected the enzyme from inactivation. Both the extent of activation and the amount of CaM necessary for 50% maximal activation were affected by pHPG treatment of the enzyme. The approximate number of modified arginines estimated by [7-14C]phenylglyoxal incorporation and amino acid analysis after complete inactivation of CaM stimulation was seven residues per catalytic subunit assuming enzyme homogeneity. The Stokes radius and sedimentation coefficient of the enzyme were unchanged by the modification. These results suggest that arginine residues are critical for functional interaction between phosphodiesterase and CaM and that controlled modification can selectively alter CaM-stimulated enzyme activity.     

Decreased expression and function of adipocyte hormone-sensitive lipase in subcutaneous fat cells of obese subjects.

Decreased lipolytic effect of catecholamines in adipose tissue has repeatedly been demonstrated in obesity and may be a cause of excess accumulation of body fat. However, the mechanisms behind this lipolysis defect are unclear. The role of hormone-sensitive lipase was examined using abdominal subcutaneous adipocytes from 34 obese drug-free and otherwise healthy males or females and 14 non-obese control subjects. The enzyme catalyzes the rate-limiting step of the lipolysis pathway. The maximum lipolytic capacity of fat cells was significantly decreased in obesity when measured using either a non-selective beta-adrenergic receptor agonist (isoprenaline) or a phosphodiesterase resistant cyclic AMP analogue (dibutyryl cyclic AMP). Likewise, enzyme activity, protein expression, and mRNA of hormone-sensitive lipase were significantly decreased in adipocytes of obese subjects. The findings were not influenced by age or gender. The data suggest that a decreased expression of hormone-sensitive lipase in subcutaneous fat cells, which in turn causes decreased enzyme function and impaired lipolytic capacity of adipocytes, is present in obesity. Impaired expression of the hormone-sensitive lipase gene might at least in part explain the enzyme defect.     

Enzyme-inhibitor interactions as a basis for heterogeneity of extracellular cyclic AMP phosphodiesterases in Dictyostelium discoideum

We have previously characterized three forms of cyclic-AMP phosphodiesterase obtained after dithiothreitol activation of the enzyme from the extracellular medium during late vegetative growth of Dictyostelium discoideum (Toorchen, D. and Henderson, E.J. (1979) Biochem. Biophys. Res. Commun. 87, 1168–1175). This communication presents evidence supporting the earlier hypothesis that the observed heterogeneity of enzyme species is due to formation of complexes between an endogenous inhibitor protein and a common catalytic polypeptide. Dithiothreitol inactivates the inhibitor, but does not cause its release from the catalytic unit. Additional evidence is presented for the presence of a similar catalytic polypeptide in the extracellular phosphodiesterase produced during the first 8 h of developmetn, except that this species is a phosphoprotein.     

Proteolytic activation of calmodulin-dependent cyclic nucleotide phosphodiesterase

Purified calmodulin-stimulated cyclic nucleotide phosphodiesterase from brain, a homodimer of 59-kDa subunits, was activated by limited proteolysis with trypsin, alpha-chymotrypsin, Pronase, or papain and could not be further stimulated by addition of Ca2+ and calmodulin. Proteolysis increased Vmax and had little effect on the Km for cGMP. Treatment with alpha-chymotrypsin in the presence of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) produced, sequentially, 57- and 45-kDa peptides from the bovine and 55-, 53-, and 38-kDa peptides from the ovine enzyme. This protease-treated phosphodiesterase exhibited a Stokes radius of 3.9 nm and an S20,w value of 4.55; comparison with the hydrodynamic properties observed for native enzyme (4.3 nm, 5.95 S) strongly suggests a dimeric protein of Mr approximately 80,000-90,000. The proteolyzed species does not interact significantly with calmodulin immobilized on agarose, nor does it show complex formation with 2-dimethylaminonaphthalene-1-sulfonyl-calmodulin even at micromolar concentrations of protein. Proteolysis, in the presence of calmodulin plus Ca2+, fully activated phosphodiesterase, producing the same intermediate peptides; however, final peptides from the bovine and ovine enzymes were 47 and 42 kDa, respectively, indicating a new, specific conformation of the enzyme. When EGTA was added to such incubations, these peptides were cleaved to those of the size seen when proteolysis was carried out entirely in the presence of EGTA. The initial rate of activation was increased by the presence of Ca2+ and calmodulin, suggesting that, in complex, phosphodiesterase exhibits a site with increased susceptibility to proteolysis. Since calmodulin can still interact with a fully activated form of the enzyme, it appears that retention of calmodulin binding can occur concomitantly with damage to that portion of the phosphodiesterase molecule responsible for suppression of its basal catalytic activity.     

Mapping of the hormone-sensitive lipase binding site on the adipocyte fatty acid-binding protein (AFABP). Identification of the charge quartet on the AFABP/aP2 helix-turn-helix domain

The hormone-sensitive lipase (HSL) and adipocyte fatty acid-binding protein (AFABP/aP2) form a physical complex that affects basal and hormone-stimulated adipocyte fatty acid efflux. Previous work has established that AFABP/aP2-HSL complex formation requires that HSL be in its activated, phosphorylated form and AFABP/aP2 have a bound fatty acid. To identify the HSL binding site of AFABP/aP2 a combination of alanine-scanning mutagenesis and fluorescence resonance energy transfer was used. Mutation of Asp17, Asp18, Lys21, or Arg30 (but not other amino acids in the helix-turn-helix region) to alanine inhibited interaction with HSL without affecting fatty acid binding. The cluster of residues on the helical domain of AFABP/aP2 form two ion pairs (Asp17-Arg30 and Asp18-Lys21) and identifies the region we have termed the charge quartet as the HSL interaction site. To demonstrate direct association, the non-interacting AFABP/aP2-D18K mutant was rescued by complementary mutation of HSL (K196E). The charge quartet is conserved on other FABPs that interact with HSL such as the heart and epithelial FABPs but not on non-interacting proteins from the liver or intestine and may be a general protein interaction domain utilized by fatty acid-binding proteins in regulatory control of lipid metabolism.     

Comparison of phospholipid effects on insulin-sensitive low Km cyclic AMP phosphodiesterase in adipocyte plasma membranes and microsomes

Both adipocyte plasma membranes and microsomes possess insulin-sensitive low K_m cyclic AMP phosphodiesterase activity. The activity of the enzyme from both sources was susceptible to activation by several anionic phospholipids. Activators of the plasma membrane enzyme were lysophosphatidylglycerol > lysophosphatidylcholine > lysophosphatidylserine > phosphatidylserine > phosphatidylglycerol. These same phospholipids activated the microsomal enzyme but the extent of activation by each phospholipid was reversed. Neutral phospholipids and other anionic phospholipids were without effect. The phospholipids had no effect on high K_m cAMP phosphodiesterase in either membrane. The results suggest that the phospholipid headgroup was an important determinant for enzyme activation by phospholipid. The increased susceptibility of the plasma membrane enzyme to lysophospholipid may be attributed to a difference in the plasma membrane enzyme compared to the microsomal membrane enzyme or to differences in plasma membrane and microsomal membrane phospholipid composition and their ability to regulate low K_m cAMP phosphodiesterase activity.     

Characterization of phosphodiesterase catalytic sites by means of cyclic nucleotide derivatives

Cyclic nucleotide derivatives have been used as a tool to characterize distinct catalytic sites on phosphodiesterase enzyme forms: the cGMP-stimulated enzyme from rat liver and the calmodulin-sensitive enzyme from rat or bovine brain. Under appropriate assay conditions, the analogues showed linear competitive inhibition with respect to cAMP (adenosine 3',5'-monophosphate) as substrate. The inhibition sequence of the fully activated cGMP-stimulated phosphodiesterase was identical to the inhibition sequence of the desensitized enzyme, i.e. the enzyme which has lost its ability to be stimulated by cGMP. The inhibition pattern could, therefore, not be attributed to competition with cGMP at an allosteric-activating site. Also, the inhibition sequence of the calmodulin-sensitive phosphodiesterase was maintained whether activity was basal or fully stimulated by calmodulin. When cAMP and cGMP, with identical chemical ligands substituted at the same position, were compared as inhibitors of the calmodulin-sensitive phosphodiesterase, the cGMP analogues were always the more potent suggesting that, for that enzyme, the catalytic site was sensitive to a guanine-type cyclic nucleotide structure. Comparing the two phosphodiesterases, it was possible to establish both similar and specific inhibitor potencies of cyclic nucleotide derivatives. In particular, the two enzymes exhibited large differences in analogue specificity modified at C-6, 6-chloropurine 3',5'-monophosphate or purine 3',5'-monophosphate.     

Role of phosphoprotein phosphatases in reversible deactivation of chicken adipose tissue hormone-sensitive lipase.

The reversible deactivation of chicken adipose tissue hormone-sensitive lipase alpha(previously activated with Mg2+ ATP and adenosine 3':5'-monophosphate) required Mg2+ and was inhibited by phosphate. These results are consistent with the assumption that deactivation of the protein kinase-activated enzyme is catalyzed by a lipase phosphatase. Cholesterol ester is catalyzed by a lipase phosphatase. Cholesterol ester hydrolase similarly was activated and reversibly deactivated. The activity of endogenous lipase phosphatase in pH 5.2 precipitate fractions was reduced, and in some cases eliminated, by incubation at 50 degrees for 20 min in buffer containing 20% glycerol. Heating at 50 degrees greatly increased the apparent percentage activation of triglyceride and cholesterol ester hydrolases but this was due to a selective decrease in basal (nonactivated) hydrolase activities. Essentially all endogenous lipase phosphatase could be removed by treatment of the pH 5.2 precipitate fraction with ATP-Sepharose affinity gel. The addition of a partially purified preparation of rat liver phosphorylase phosphatase deactivated triglyceride and cholesterol ester hydrolases. The deactivation process was concentration, 5 mM) and was inhibited by 5 mM phosphate and by phosphorylase alpha. Reversible deactivation of hormone-sensitive lipase alpha was also observed with crude prepa- and by phosphorylase alpha. Reversible deactivation of hormone-sensitive lipas alpha was also observed with crude preparations of phosphoprotein phosphatases from rat and turkey hearts, and from rat epididymal fat pads. Thus, hormone-sensitive lipase is deactivated by a variety of phosphoprotein phosphatases from different tissues and different species, implying a low degree of specificity for the deactivating system.     

Forskolin-induced change of the size of adenylate cyclase

Forskolin, a potent activator of cyclic AMP generating systems, has been proposed to act directly on the catalytic unit of adenylate cyclase. Nevertheless, some arguments indicate a possible role of the guanosine triphosphate-binding regulatory protein in forskolin action on adenylate cyclase. In this study, we have observed an increase in the apparent sedimentation coefficient of solubilized adenylate cyclase, elicited by forskolin, both in rat liver (from 6.4 +/- 0.1 to 7.2 +/- 0.1 S) and rat striatum (from 6.7 +/- 0.1 to 7.6 +/- 0.1 S). On both systems, a similar increase in the sedimentation coefficient was observed after preactivation of the enzyme with guanosine 5'-(beta, gamma-imido)triphosphate (Gpp(NH)p). In contrast to the Gpp(NH)p effect, the forskolin action was found to be reversible. Simultaneous pretreatments of adenylate cyclase with forskolin and Gpp(NH)p did not induce additive increases of the apparent sedimentation coefficient of adenylate cyclase. The modification of the size of solubilized adenylate cyclase was corroborated by gel filtration studies. In rat liver membranes, the Stokes radius of the solubilized enzyme increased from 59 +/- 1 A for basal state to 65 +/- 1 A for forskolin preactivated state. A possible explanation of our findings is that forskolin may stabilize the complex between the GTP-binding regulatory protein and the catalytic unit of adenylate cyclase in a reversible manner.     

Purification of the putative hormone-sensitive cyclic AMP phosphodiesterase from rat adipose tissue using a derivative of cilostamide as a novel affinity ligand

A "low Km" cAMP phosphodiesterase with properties of a peripheral membrane protein accounts for approximately 90% of total cAMP phosphodiesterase activity in particulate (100,000 X g) fractions from rat fat cells. Incubation of fat cells with insulin for 10 min increased particulate (but not soluble) cAMP phosphodiesterase activity, with a maximum increase (approximately 100%) at 1 nM insulin. Most of the increase in activity was retained after solubilization (with non-ionic detergent and NaBr) and partial purification (approximately 20-fold) on DEAE-Sephacel. The solubilized enzyme from adipose tissue was purified approximately 65,000-fold to apparent homogeneity (yield approximately 20%) by chromatography on DEAE-Sephacel and Sephadex G-200 and affinity chromatography on aminoethyl agarose conjugated with the N-(2-isothiocyanato)ethyl derivative of the phosphodiesterase inhibitor cilostamide (OPC 3689). A 63,800 +/- 200-Da polypeptide (accounting for greater than 90% of the protein eluted from the affinity column) was identified by polyacrylamide gel electrophoresis in sodium dodecyl sulfate (with or without reduction). Enzyme activity was associated with the single protein band after electrophoresis under nondenaturing conditions. On gel permeation, Mr(app) was 100,000-110,000, suggesting that the holoenzyme is a dimer. A pI of 4.9-5.0 was estimated by isoelectric focusing. At 30 degrees C, the purified enzyme hydrolyzed both cAMP and cGMP with normal Michaelis-Menten kinetics; the pH optimum was 7.5. The Km(app) for cAMP was 0.38 microM and Vmax, 8.5 mumol/min/mg; for cGMP, Km(app) was 0.28 microM and Vmax, 2.0 mumol/min/mg. cGMP competitively inhibited cAMP hydrolysis with a Ki of approximately 0.15 microM. The enzyme was also inhibited by several OPC derivatives and "cardiotonic" drugs, but not by RO 20-1724. It was very sensitive to inhibition by agents which covalently modify protein sulfhydryls, but not by diisopropyl fluorophosphate. The activation by insulin and other findings indicate that the purified enzyme, which seems to belong to a subtype of low Km cAMP phosphodiesterases that is specifically and potently inhibited by cGMP, cilostamide, other OPC derivatives, and certain cardiotonic drugs, is likely to account for the hormone-sensitive particulate low Km cAMP phosphodiesterase activity of rat adipocytes.     

Effects of sulfonylureas on membrane-bound low Km cyclic AMP phosphodiesterase in rat fat cells

The effects of sulfonylureas and a biguanide on membrane-bound low K_m cyclic AMP phosphodiesterase and lipolysis were examined in rat fat cells. Pharmacologically active sulfonylureas, such as tolbutamide (10 mM), acetohexamide (10 mM) and glibenclamide (200 μM) activated the phosphodiesterase when incubated with fat cells and suppressed lipolysis induced by isoproterenol. However, neither of these actions was observed in the presence of a pharmacologically inactive sulfonylurea, carboxytolbutamide (10 mM) and a biguanide, buformin (500 μM). Tolbutamide (0.5–10 mM) activated the enzyme, concentration dependently, and this manner of activation appears to coincide with that of the suppressive effect on the lipolysis. The time course of the enzyme activation was similar to that seen with insulin. K_m, optimal pH and sensitivity to temperature of the enzyme from tolbutamide-treated cells were the same as those of the enzyme from control and insulin-treated cells. Direct incubation of the enzyme from control cells with tolbutamide did not affect the activity, while as little as 10 μM 3-isobutyl-1-methylxanthine markedly inhibited the enzyme. Tolbutamide continued to activate the enzyme in cells in which insulin receptor had been destroyed by trypsin-pretreatment. These results are compatible with the idea that the enzyme activated by sulfonylurea and that activated by insulin may be the same species of phosphodiesterase and that the antilipolytic action of sulfonylurea may be mediated by the activation of the enzyme which does not occur through the insulin receptor.