Characterization of particulate cyclic nucleotide phosphodiesterases from bovine brain: purification of a distinct cGMP-stimulated isoenzyme

In the absence of detergent, approximately 80-85% of the total cGMP-stimulated phosphodiesterase (PDE) activity in bovine brain was associated with washed particulate fractions; approximately 85-90% of the calmodulin-sensitive PDE was soluble. Particulate cGMP-stimulated PDE was higher in cerebral cortical gray matter than in other regions. Homogenization of the brain particulate fraction in 1% Lubrol increased cGMP-stimulated activity approximately 100% and calmodulin-stimulated approximately 400-500%. Although 1% Lubrol readily solubilized these PDE activities, approximately 75% of the cAMP PDE activity (0.5 microM [3H]cAMP) that was not affected by cGMP was not solubilized. This cAMP PDE activity was very sensitive to inhibition by Rolipram but not cilostamide. Thus, three different PDE types, i.e., cGMP stimulated, calmodulin sensitive, and Rolipram inhibited, are associated in different ways with crude bovine brain particulate fractions. After solubilization and purification by chromatography on cGMP-agarose, heparin-agarose, and Superose 6, the brain particulate cGMP-stimulated PDE cross-reacted with antibody raised against a cGMP-stimulated PDE purified from calf liver supernatant. The brain enzyme exhibited a slightly greater subunit Mr than did soluble forms from calf liver or bovine brain, as evidenced by protein staining or immunoblotting after polyacrylamide gel electrophoresis under denaturing conditions. Incubation of brain particulate and liver soluble cGMP-stimulated PDEs with V8 protease produced several peptides of similar size, as well as at least two distinct fragments of approximately 27 kDa from the brain and approximately 23 kDa from the liver enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

The identification of a new cyclic nucleotide phosphodiesterase activity in human and guinea-pig cardiac ventricle. Implications for the mechanism of action of selective phosphodiesterase inhibitors.

Four cyclic nucleotide phosphodiesterase (PDE) activities were separated from low-speed supernatants of homogenates of human cardiac ventricle by DEAE-Sepharose chromatography, and designated PDE I-PDE IV in order of elution with an increasing salt gradient. PDE I was a Ca2+/calmodulin-stimulated activity, and PDE II was an activity with a high Km for cyclic AMP which was stimulated by low concentrations of cyclic GMP. Human ventricle PDE III had Km values of 0.14 microM (cyclic AMP) and 4 microM (cyclic GMP), and showed simple Michaelis-Menten kinetics with both substrates. PDE IV is a previously unrecognized activity in cardiac muscle, the human enzyme having Km values of 2 microM (cyclic AMP) and 50 microM (cyclic GMP). PDE III and PDE IV were not activated by cyclic nucleotides or calmodulin. Four PDE activities were also isolated from guinea-pig ventricle, and had very similar kinetic properties. By gel filtration, the Mr of PDE III was 60,000, and that of PDE IV 45,000. The drug SK&F 94120 selectively and competitively inhibited PDE III with a Ki value of 0.8 microM (human), showing simple hyperbolic inhibition kinetics. Rolipram (Schering ZK 62711) and Ro 20-1724 (Roche), which have previously been reported to inhibit PDE III-like activities strongly, were shown to be weak inhibitors of human and guinea-pig PDE III enzymes (Ki values greater than 25 microM), but potent inhibitors of PDE IV [Ki values 2.4 microM (Rolipram) and 3.1 microM (Ro 20-1724) with human PDE IV]. The inhibition in all cases demonstrated simple hyperbolic competition. These observations suggest that the previously reported complex inhibition of PDE III-type activities from cardiac muscle was caused by incomplete separation of the PDE III from other enzymes, particularly PDE IV.     

Preliminary identification and role of phosphodiesterase isozymes in human basophils.

We attempted to identify and establish the role of cyclic nucleotide phosphodiesterase (PDE) isozymes in human basophils by using standard biochemical techniques as well as describing the effects of isozyme-selective and nonselective inhibitors of PDE. The nonselective PDE inhibitors, theophylline and 3-isobutyl-1-methylxanthine, inhibited anti-IgE-induced release of histamine and leukotriene C4 (LTC4) from basophils. This inhibition was accompanied by elevations in cAMP levels. Rolipram, an inhibitor of the low Km cAMP-specific PDE (PDE IV), inhibited the release of both histamine and LTC4 from activated basophils and increased cAMP levels in these cells. In contrast, mediator release from basophils was not inhibited by either siguazodan or SK&F 95654, inhibitors of the cGMP-inhibited PDE (PDE III) or zaprinast, an inhibitor of the cGMP-specific PDE (PDE V). SK&F 95654 failed to elevate basophil cAMP in these experiments whereas zaprinast induced significant increases in cAMP content. The inhibitory effect of rolipram on mediator release was potentiated by siguazodan or SK&F 95654, but not by zaprinast. SK&F 95654 also enhanced the ability of rolipram to increase cAMP content. Forskolin, a direct activator of adenylate cyclase, inhibited IgE-dependent release of mediators from basophils and increased cAMP levels in these cells. These effects were enhanced by rolipram, but not by SK&F 95654 or zaprinast. The cell permeant analog of cAMP, dibutyryl cAMP, inhibited mediator release from these cells, a property not shared by either dibutyryl-cGMP or sodium nitroprusside, an activator of soluble guanylate cyclase. The presence of both PDE III and PDE IV was confirmed by partially purifying and characterizing PDE activity in broken cell preparations. Overall, these data lend support to the hypothesis that cAMP inhibits mediator release from basophils and suggest that the major PDE isozyme responsible for regulating cyclic AMP content in these cells is PDE IV, with a minor contribution from PDE III. However, the finding that zaprinast caused increases in cAMP without inhibiting mediator release indicates that cAMP accumulation is not invariably linked to an inhibition of basophil activation.     

Triton solubilization of proteins from pig liver mitochondrial membranes

Various aspects of membrane solubilization by the Triton X-series of nonionic detergents were examined in pig liver mitochondrial membranes. Binding of Triton X-100 to nonsolubilized membranes was saturable with increased concentrations of the detergent. Maximum binding occurred at concentrations exceeding 0.5% Triton X-100 (w/v). Solubilization of both protein and phospholipid increased with increasing Triton X-100 to a plateau which was dependent on the initial membrane protein concentration used. At low detergent concentrations (less than 0.087% Triton X-100, w/v), proteins were preferentially solubilized over phospholipids. At higher Triton X-100 concentrations the opposite was true. Using the well-defined Triton X-series of detergents, the optimal hydrophile-lipophile balance number (HLB) for solubilization of phosphatidylglycerophosphate synthase (EC 2.7.8.5) was 13.5, corresponding to Triton X-100. Activity was solubilized optimally at detergent concentrations between 0.1 and 0.2% (w/v). The optimal protein-to-detergent ratio for solubilization was 3 mg protein/mg Triton X-100. Solubilization of phosphatidylglycerophosphate synthase was generally better at low ionic strength, though total protein solubilization increased at high ionic strength. Solubilization was also dependent on pH. Significantly higher protein solubilization was observed at high pH (i.e., 8.5), as was phosphatidylglycerophosphate synthase solubilization. The manipulation of these variables in improving the recovery and specificity of membrane protein solubilization by detergents was examined.     

Solubilization in high yield of opioid receptors retaining high-affinity delta, mu and kappa binding sites

The binding sites for opiates (agonist and antagonist) and opioid peptides can be solubilized from rat brain membranes with digitonin in the presence of Mg2+ (10 mM). High affinity and high capacity binding to the soluble delta, mu, and kappa receptors is obtainable when the membranes are treated in Mg2+ (30 degrees C, 60 min) prior to solubilization. The yields of solubilized binding sites extracted with digitonin, 40-90%, are higher than those obtained from Mg2+-pretreated membranes with other detergents commonly used for receptor solubilization. The stability of the digitonin-soluble opioid receptor at room temperature makes it useful for purification and characterization.     

Coexpression of human cAMP-specific phosphodiesterase activity and high affinity rolipram binding in yeast.

Studies by various investigators have demonstrated that the low Km, cAMP-specific phosphodiesterase (PDE IV) is selectively inhibited by a group of compounds typified by rolipram and Ro 20-1724. In addition to inhibiting the catalytic activity of PDE IV, rolipram binds to a high affinity binding site present in brain homogenates. Although it has been assumed that the high affinity rolipram-binding site is PDE IV, no direct evidence has been produced to support this assumption. The present studies were undertaken to determine whether the rolipram-binding site is coexpressed with PDE IV catalytic activity in Saccharomyces cerevisiae genetically engineered to express human recombinant monocytic PDE IV (hPDE IV). Expressing hPDE IV cDNA in yeast resulted in a 20-fold increase in PDE activity that was evident within 1 h of induction and reached a maximum by 3-6 h. The recombinant protein represented hPDE IV as judged by its immunoreactivity, molecular mass (approximately 88 kDa), kinetic characteristics (cAMP Km = 3.1 microM; cGMP Km greater than 100 microM), sensitivity to rolipram (Ki = 0.06 microM), and insensitivity to siguazodan (PDE III inhibitor) and zaprinast (PDE V inhibitor). Saturable, high affinity [3H] (R)-rolipram-binding sites (Kd = 1.0 nM) were coexpressed with PDE activity, indicating that both binding activity and catalytic activity are properties of the same protein. A limited number of compounds were tested for their ability to inhibit hPDE IV catalytic activity and compete for [3H](R)-rolipram binding. Analysis of the data revealed little correlation (r2 = 0.35) in the structure-activity relationships for hPDE IV inhibition versus competition for [3H] (R)-rolipram binding. In fact, certain compounds (e.g. (R)-rolipram Ro 20-1724) possessed a 10-100-fold selectivity for inhibition of [3H] (R)-rolipram binding over hPDE IV inhibition, whereas others (e.g. dipyridamole, trequinsin) possessed a 10-fold selectivity for PDE inhibition. Thus, although the results of these studies demonstrate that hPDE IV activity and high affinity [3H](R)-rolipram binding are properties of the same protein, they do not provide clear cut evidence linking the binding site with the PDE inhibitory activity of rolipram and related compounds.     

Activation of rabbit liver high affinity cAMP (type IV) phosphodiesterase by a vanadyl-glutathione complex. Characterization of the role of the sulfhydryl.

Activation of rabbit liver microsomal high affinity cAMP phosphodiesterase (Type IV PDE) by vanadyl-glutathione complexes was studied as a possible model of insulin stimulation of the enzyme in a cell-free system. The effect of VO.2GSH activation of PDE was a 21-fold decrease in the IC50 value for cGMP inhibition and a 2.6-fold increase in the Vmax of the higher affinity cAMP catalytic site. Cyclic AMP and cGMP substrate affinities and cGMP hydrolysis were unaffected by VO.2GSH activation. Selective Type IV PDE inhibitors and cGMP analogs indicated that VO.2GSH complexes activated the cGMP-inhibitable form of the Type IV PDE activities which co-localized in hepatic microsomes. The Type IV PDE activating complex appears to consist minimally of vanadyl ion and 2 oxidized electron donor compounds. The components of the electron donor required to achieve an enzyme activation complex are: 1) a free -SH group as the electron donor for vanadate reduction and 2) a minimum structure of cysteamine (NH2-CH2-CH2-SH). Maximal activation of the enzyme required near 2:1 molar ratios of either glutathione or cysteamine mixed with sodium orthovanadate. Active vanadyl-cysteamine complexes were isolated by reverse- phase high performance liquid chromatography. Tungsten, niobium, and tantalum, but not manganese, chromium, or molybdenum, substituted for vanadium to form enzyme-activating complexes with glutathione. VO.RSH complex activation occurred rapidly upon addition to microsomes and was reversible. We conclude from these studies that VO.RSH complexes and insulin activate the same form of Type IV PDE in rabbit liver microsomes; our findings are discussed with respect to the involvement of a possible electron transfer enzyme oxidation in the activation mechanism.     

Triton X-100 solubilization of mitochondrial inner and outer membranes

Rat liver mitochondrial inner and outer membranes were subjected to the solubilizing effect of the nonionic detergent Triton X-100 under various conditions. After centrifugation, the supernatants (containing the solubilized fraction) and pellets were characterized chemically and/or ultrastructurally. The detergent seems to act by inducing a phase transition from membrane lamellae to mixed protein-lipid-detergent micelles. Different electron-micro-scopy patterns are shown by the inner membranes after treatment with different amounts of surfactant, whereas the corresponding images from outer membranes vary but slightly. Selective solubilization of various components is observed, especially in the case of the inner membrane. Some membrane lipids (e.g., cardiolipin) are totally solubilized at detergent concentrations when others, such as sphyngomyelin, remain in the membrane. Other inner-membrane components (flavins, cytochromes, coenzymeQ) show different solubilization patterns. This allows the selection of conditions for optimal solubilization of a given membrane component with some degree of selectivity. The influence of Triton X-100 on various mitochondrial inner-membrane enzyme activities was studied. The detergent seems to act especially through disruption of the topology of the functional complexes, although the activity of the individual enzymes appears to be preserved. Relatively simple enzyme activities, such as ATPase, are more or less solubilized according to the detergent concentration, whereas the more complex succinate-cytochromec reductase activity practically disappears even at low Triton X-100 concentrations.     

Adenosine-3':5'-monophosphate-dependent and plasma-membrane-associated protein kinase from bovine corpus luteum. Solubilization and properties of solubilized enzyme.

The solubilization of plasma membrane fractions FI and FII associated protein kinases has been attempted using monovalent salts of high ionic strength and various detergent treatments. Extraction of FI and FII plasma membranes with high ionic strength salt solutions did not release more than 20% of the protein kinase activity. Similarly, monovalent salts released little adenosine 3':5'-monophosphate (cyclic AMP) binding activity, but after extraction binding capacity of cyclic [3H]AMP to plasma membranes was increased about 150-200%. Triton X-100 was a better solubilizing agent that Lubrol WX or deoxycholate. In addition to solubilization, 0.1% Triton X-100 also stimulated the protein kinase activity 150-200%. The properties of Triton X-100 solubilized FI and FII and purified cytosol KII were characterized with respect to protein substrate specificity, effect of cyclic AMP, cyclic nucleotide specificity, effects of divalent metal ion and gonadotropins. Upon sucrose density gradient centrifugation, FI solubilized protein kinase and cyclic AMP binding activities co-sedimented with a sedimentation coefficient of 6.3 S. The FII solubilized protein kinase sedimented as two components with sedimentation coefficients of 7.7 S and 5.5 S. The cyclic AMP binding activity also sedimented as two components with sedimentation coefficient 6.7 S and 5.5 S. Cyclic AMP caused dissociation of solubilized protein kinase from FI into a single catalytic (4.8 S) and two cyclic AMP binding subunits (8.1 S and 6.7 S). FII solubilized enzyme was dissociated into one catalytic (4.8 S) and one cyclic AMP binding subunit (6.3 S). Fractionation of FI and FII solubilized enzymes on DEAE-cellulose column chromatography resolved them each into two peaks Ia, Ib and IIa, IIb, respectively. Peaks Ib and IIb were more sensitive to cyclic AMP STIMULATION THAN Ia and IIa peaks. From these studies it is concluded that the plasma-membrane associated and cytosol protein kinases have similar catalytic properties but differ in some of their physical properties.     

Comparison of the phosphodiesterase inhibitory subunit interactions of frog and bovine rod outer segments.

The rod outer segments of the bovine and frog retina possess a cyclic GMP phosphodiesterase (PDE) that is composed of two larger subunits, alpha and beta (P alpha beta), which contain the catalytic activity and a smaller gamma (P gamma) subunit which inhibits the catalytic activity. We studied the binding of P gamma to P alpha beta in both the bovine and frog rod outer segment membranes. Analysis of these data indicates that there are two classes of P gamma binding sites per P alpha beta in both species. The activation of PDE by the guanosine 5'-[gamma-thio]triphosphate form of the alpha subunit of transducin, T alpha.GTP gamma S, was also studied. These data indicate that the two classes of P gamma binding sites contribute to the formation of two classes of binding sites for T alpha.GTP gamma S. We demonstrate solubilization of a portion of the P gamma by T alpha.GTP gamma S in both species. There is also present, in both species, a second class of P gamma which is not solubilized even when it is dissociated from its inhibitory site on P alpha beta by T alpha.GTP gamma S. The amount of full PDE activity which results from release of the solubilizable P gamma is about 50% in the frog PDE but only approx. 17% in the bovine PDE. We also show that activation of frog rod outer segment PDE by trypsin treatment releases the PDE from the membranes. This type of release by trypsin has already been demonstrated in bovine rod outer segments [Wensel & Stryer (1986) Proteins: Struct. Funct. Genet. 1, 90-99].     

Inhibition, by a protease inhibitor, of the solubilization of the F1-portion of the Mg2+-stimulated adenosine triphosphatase of Escherichia coli.

The effects of two protease inhibitors on the solubilization of the membrane-bound Mg2+-adenosine triphosphatase (Mg-ATPase) of Escherichia coli were investigated. p-Aminobenzamidine prevented the solubilization of the Mg-ATPase during treatment of membranes with low-ionic-strength buffers containing ethylenediaminetetraacetic acid. p-Aminobenzamidine did not prevent subsequent solubilization of the Mg-ATPase by treatment of the membranes with chloroform. This method of solubilization yielded a preparation of similar apparent molecular weight but with a 10-fold-increased specific activity as compared with the Mg-ATPase solubilized by washing with low-ionic-strength buffer. However, in contrast to the latter preparation, the chloroform-solubilized Mg-ATPase did not reconstitute ATP-dependent energization of stripped membranes, which were prepared by low-ionic-strength washing in the absence of p-aminobenzamidine. Another protease inhibitor, epsilon-amino-n-caproic acid, did not effect the solubilization of the Mg-ATPase, but did inhibit the loss of activity occurring during concentration, by ultrafiltration, of the Mg-ATPase solublized by the low-ionic-strength treatment.     

Effect of Isozyme-Selective Inhibitors of Phosphodiesterase on Histamine-Stimulated Cyclic AMP Accumulation in Guinea-Pig Hippocampus

Addition of histamine (0.1 mM) to guinea-pig hippocampal slices causes a 20- to 30-fold increase in the accumulation of cyclic AMP compared with basal levels. This accumulation represents a balance between cyclic AMP production by adenylate cyclase and cyclic AMP breakdown mediated by phosphodiesterase (PDE). However, brain tissues are known to contain several different PDE isozymes. To determine which are involved in this response to histamine, the effect of isozyme-specific PDE inhibitors on cyclic AMP accumulation was examined in the hippocampus. MB 22948 (0.1 mM), an inhibitor of PDEs I and II, had no significant effect on the response to either 1 microM or 0.1 mM histamine. SKF 94120 (0.1 mM), a PDE III inhibitor, was also without effect in the presence of 1 microM histamine, although with 0.1 mM histamine, it caused a weak (1.25-fold compared with control), but statistically significant, enhancement of cyclic AMP accumulation. However, both rolipram (0.1 mM), a PDE IV inhibitor, and 3-isobutyl-1-methylxanthine (0.1 or 1 mM), an inhibitor of all forms of PDE, significantly increased cyclic AMP accumulation (2.8- to 6.5-fold compared with controls), and the relative size of this effect decreased with increasing histamine concentration. It is concluded that PDE IV is the main PDE isozyme involved in cyclic AMP turnover in guinea-pig hippocampal slices responding to histamine.     

Solubilization of the [8-lysine]vasopressin receptor and adenylate cyclase from pig kidney plasma membranes.

Adenylate cyclase and the [8-lysine]vasopressin receptor were solubilized from pig kidney medulla membranes using the nonionic detergent Triton X-100. Optimal conditions for solubilization were under continuous stirring in a medium containing 0.5% (/v) Triton X-100, 100 mM Tris-HCl, pH 8, and 10 mM MgCl2. Both adenylate cyclase activity and [3H][8-lysine]vasopressin binding activity were recovered in a -26,000 X g supernatant of detergent-treated membranes. The yield of solubilized adenylate cyclase was nearly 100%. The soluble enzyme was no longer sensitive to antidiuretic hormone but was slightly activated by sodium fluoride. The affinity of the soluble receptor for [8-lysine]vasopresin was les than that of the membrane-bound receptor (mean apparent Km values, respectively 10(-7) M and 2 X 10(-8) M), however binding cooperativity was preserved. Hill coefficients were 1.42 for the soluble receptor and 1.50 for the membrane receptor. The soluble receptor discriminated as efficiently as did the membrane receptor between [8-lysine-a1vasopressin and oxytocin. The yield of spolubilized receptor was only 30% despite the fact that all binding activity had disappeared from the residual pellet of detergent-treated membranes. When the membranous receptors were occupied before solubilization and the latter was performed under conditions in which dissociation of the hormone-receptor comples is slow, i.e. at low temperature, 65% to 100% of the hormone-receptor complex was recovered in the soluble fraction. The soluble hormone-receptor complex partially dissociated on rewarming whereas the free hormone concentration was kept unchanged in the medium. The residual binding capacity, which was 30% of the initial value, was identical with that determined when the receptor was solubilized in free form before incubation with labeled hormone. It was concluded that (a) solubilization of the receptor molecules was complete, (b) during solubilization two forms of the receptor appear, of which only one is accessible to the hormone, (c) occupancy of the receptor by the hormone prevented the formation of the nonaccessible form, and (d) some component or components of the soluble fraction might be responsible for the loss in apparent affinity.     

Cyclic nucleotide phosphodiesterase from a particulate fraction of rat heart. Solubilization and characterization of a single enzymatic form

Approximatively 2–8% of the cyclic nucleotide phosphodiesterase activity of a crude 1000 g supernatant from rat heart was associated with the washed 105,000 g pellet fraction. This activity exhibited biphasic Lineweaver-Burk plots over a large range of cyclic nucleotides concentrations. Concave-Bownward plots were obtained with cyclic AMP as the assay substrate, while cyclic GMP gave rise to concave-upward plots. Treatment of this particulate fraction by freezing and thawing and then with 2% Lubrol PX released the major part of phosphodiesterase activity into the supernatant (70 and 90% for cyclic AMP and cyclic GMP phosphodiesterase activities respectively). Isoelectric focusing of the solubilized enzyme revealed a single peak of phosphodiesterase activity. While the Lineweaver-Burk plots of cyclic AMP phosphodiesterase activity were not markedly modified by detergent treatment kinetic plots of cyclic GMP phosphodiesterase activity underwent a drastic transformation during the overall solubilization procedure. The substantial increase in the cyclic GMP rate of hydrolysis observed at low substrate level might explain the difference in the apparent yield of solubilization between cyclic AMP and cyclic GMP phosphodiesterase activities.     

The isolation and characterization of cyclic nucleotide phosphodiesterases from Morris hepatoma 5123tc(h) and rat liver

Four main phosphodiesterase (PDE) forms were resolved and partially purified from rat liver and Morris hepatoma 5123tc(h). The activities of the high Km cyclic nucleotide PDE (form II) in hepatoma were markedly reduced compared to liver, while the activities of the low Km cAMP PDE (form III) and low Km cyclic nucleotide PDE (form IV) in hepatoma were markedly higher than those of liver. The partially purified low Km cAMP PDE's (forms III and IV) from liver showed non-linear Lineweaver-Burk plots, whereas the same enzyme forms in hepatoma displayed linear kinetics. Activation of low Km cGMP PDE activity by calmodulin was found with form I in liver whereas in hepatoma form II was responsive to calmodulin.     

Stabilization of soluble active rat liver glucagon receptor

Active glucagon receptor was solubilized with 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonate (Chaps) from rat liver plasma membranes but rapidly (less than 8 h) lost activity. Either inclusion of 1X Hanks' balanced salt solution in the 3 mM Chaps solubilization buffer or its addition after solubilization increased the percentage of total binding attributable to specific glucagon binding from approximately 10 to greater than 80%; of great importance, it increased the stability from near zero binding at 8 h to 50% binding at 48 h (4 degrees C). Of the Hanks' solution components, either NaCl (137 mM) or CaCl2 (1.26 mM) was effective in increasing specific binding to approximately 70 and 60% respectively: Mg salts were ineffective. Soluble receptor binding activity was assayed by dextran-coated charcoal adsorption of free hormone. The assay is rapid, simple, and reproducible. It is suitable for monitoring receptor activity during purification and molecular characterization. Competition binding studies gave an IC50 value of 10-20 nM (slope factor approximately 1), with or without GTP. Dissociation assays revealed GTP sensitivity when receptors were solubilized either as glucagon-receptor complexes or free receptor. Active glucagon-receptor complexes could be eluted from wheat germ lectin-agarose: neither concanavalin A-agarose nor soybean agglutinin-agarose bind receptor. A glucagon degrading activity which co-solubilized with the receptor but did not require detergent for extraction was distinguishable from the soluble receptor not only by solubility but also by its heat stability (30 degrees C), its inhibition by bacitracin, its affinity for glucagon, its retention of activity for at least 1 week at 4 degrees C, and its size.     

Solubilization and reconstitution of cholinephosphotransferase from sarcoplasmic reticulum: stabilization of solubilized enzyme by diacylglycerol and glycerol

Cholinephosphotransferase (CDPcholine: 1,2-diacylglycerol cholinephosphotransferase, EC 2.7.8.2), which catalyzes the terminal step in phosphatidylcholine synthesis via the CDPcholine pathway, is present in sarcoplasmic reticulum from rabbit skeletal muscle (Cornell, R. and MacLennan, D.H. (1985) Biochim. Biophys. Acta 835, 567-576). The conditions for solubilization and reconstitution of this enzyme were investigated as a preliminary step towards its eventual purification. The activity was not released by treatment of membranes with 1 M KCl, but was solubilized after dissolution of membranes with detergents. Cholinephosphotransferase was inactivated by cholate, deoxycholate, Triton X-100, octylglucoside, Tween-20 or SDS at concentrations which solubilize the membrane. However, the activity could be fully recovered after reconstituting the membrane by adding excess lipid (soybean) and removing detergent by gel filtration, dialysis or by absorption to Bio-Beads. When the membrane was solubilized with octylglucoside or cholate at weight ratios of detergent: membrane protein of at least 10, the activity was irreversibly lost unless stabilizers were added with detergent. The substrate diacylglycerol and glycerol were effective stabilizers.     

Insulin-induced formation of macromolecular complexes involved in activation of cyclic nucleotide phosphodiesterase 3B (PDE3B) and its interaction with PKB

Fractionation of 3T3-L1 adipocyte membranes revealed that PDE3B (phosphodiesterase 3B) was associated with PM (plasma membrane) and ER (endoplasmic reticulum)/Golgi fractions, that insulin-induced phosphorylation/activation of PDE3B was greater in internal membranes than PM fractions, and that there was no significant translocation of PDE3B between membrane fractions. Insulin also induced formation of large macromolecular complexes, separated during gel filtration (Superose 6 columns) of solubilized membranes, which apparently contain phosphorylated/activated PDE3B and signalling molecules potentially involved in its activation by insulin, e.g. IRS-1 (insulin receptor substrate-1), IRS-2, PI3K p85 [p85-subunit of PI3K (phosphoinositide 3-kinase)], PKB (protein kinase B), HSP-90 (heat-shock protein 90) and 14-3-3. Expression of full-length recombinant FLAG-tagged murine (M) PDE3B and M3BDelta604 (MPDE3B lacking N-terminal 604 amino acids) indicated that the N-terminal region of MPDE3B was necessary for insulin-induced activation and recruitment of PDE3B. siRNA (small interfering RNA) knock-down of PDE3B indicated that PDE3B was not required for formation of insulin-induced complexes. Wortmannin inhibited insulin-induced assembly of macromolecular complexes, as well as phosphorylation/activation of PKB and PDE3B, and their co-immunoprecipitation. Another PI3K inhibitor, LY294002, and the tyrosine kinase inhibitor, Genistein, also inhibited insulin-induced activation of PDE3B and its co-immunoprecipitation with PKB. Confocal microscopy indicated co-localization of PDE3B and PKB. Recombinant MPDE3B co-immunoprecipitated, and co-eluted during Superose 12 chromatography, to a greater extent with recombinant pPKB (phosphorylated/activated PKB) than dephospho-PKB or p-DeltaPKB [pPKB lacking its PH domain (pleckstrin homology domain)]. Truncated recombinant MPDE3B proteins and pPKB did not efficiently co-immunoprecipitate, suggesting that structural determinants for their interaction reside in, or are regulated by, the N-terminal portion of MPDE3B. Recruitment of PDE3B in macromolecular complexes may be critical for regulation of specific cAMP pools and signalling pathways by insulin, e.g. lipolysis.     

Modified cell ELISA to determine the solubilization of cell surface proteins: Applications in GPI-anchored protein purification

A major step in purifying membrane bound proteins involves the solubilization of the protein of interest from the cell membranes. Glycosylphosphatidyl inositol (GPI)-anchored proteins pose a singular problem in this solubilization step since they are found in detergent-resistant membrane complexes and accordingly are insoluble in cold Triton X-100. In this study we have developed a modified cell ELISA that determines the solubility of these cell surface proteins under various solubilization conditions. Using this non-radioactive method we show that the combination of saponin/Triton X-100 at 4 degrees C solubilized GPI-anchored proteins more efficiently than Triton X-100 at 4 degrees C. The combination of saponin/Triton X-100 at 4 degrees C avoids the potential of activating proteases that occurs when using Triton X-100 at 37 degrees C. Furthermore, our method also shows the saponin/Triton X-100 solubilized GPI-anchored proteins equivalent to the more expensive octyl beta-glucoside. This is a particularly important consideration in large-scale protein purification. This method obviates the need to use radioactivity, gel electrophoresis and immunoblotting procedures. The solubilization conditions determined by this modified ELISA are readily translated to the practical application of large-scale protein purification as demonstrated in the purification of two different recombinant GPI-anchored proteins, GPI-hB7-1 (CD80) and GPI-mICAM-1 (CD54).     

Characterization of a bovine cone photoreceptor phosphodiesterase purified by cyclic GMP-sepharose chromatography

The biochemical bases for the differences in cone and rod photoreceptor physiology have not been thoroughly examined because of the difficulty in obtaining cone photoreceptor components. We report here the purification and preliminary characterization of a bovine cyclic GMP phosphodiesterase (PDE) which is enriched in cone photoreceptors. The cone PDE was purified at least 15,000-fold to apparent homogeneity from bovine retinas by DEAE-cellulose and cGMP-Sepharose affinity chromatography. The trypsin-activated cone PDE hydrolyzed cGMP with efficiency similar to that of the rod PDE. However, a number of characteristics distinguished the cone PDE from the rod isozyme including the subunit structure. As previously reported, the apparent molecular weight of the cone PDE large subunit (alpha') was slightly larger than either of the large subunits of the rod PDE (93,500 versus 88,000 and 84,000). Three other smaller polypeptides were associated with the alpha' subunit (Mr = 11,000, 13,000, and 15,000), one of which (11,000) may be identical to the rod PDE gamma subunit. Cone phosphodiesterase binds at least 10-fold more cyclic GMP/mol of PDE than the rod photoreceptor isozyme. Cyclic GMP binds to this noncatalytic site with high affinity (Kd = 11 nM) and dissociates very slowly (t1/2 = 10-20 min at 37 degrees C). Purified rod transducin activated the cone PDE in solution to at least 90% of the trypsin-activated level. The concentration of rod transducin required for half-maximal activation of cone PDE (15 nM) was 50-fold lower than that necessary for half-maximal activation of rod PDE. Thus several properties of the cone phosphodiesterase clearly distinguish it from the rod isozyme and could account for some differences in cone and rod physiology.