Discriminative insulin antagonism of stimulatory effects of various cAMP analogs on adipocyte lipolysis and hepatocyte glycogenolysis

Although insulin effectively blocked hormone-stimulated glycerol output in adipocytes or phosphorylase activation in hepatocytes, the inhibitory effect of insulin on cAMP analog-stimulated cells depended on the cAMP analog used. Of the 20 analogs tested in adipocytes and 13 tested in hepatocytes, the effects of about half of them were effectively blocked by insulin, whereas the effects of many of them were not inhibited at all. In order to approach the explanation for this discriminative insulin action, the inhibitory effects of insulin on the responses to the analogs in the intact cells were correlated with the in vitro cAMP analog specificity for the hepatocyte cAMP-dependent protein kinase isozymes and the low Km, hormone-sensitive phosphodiesterases from both cell types. No correlation was found between insulin resistance of analog-stimulated hepatocyte phosphorylase and the concentration of analog required in vitro for half-maximal activation of either type I or type II cAMP-dependent protein kinase from hepatocytes. However, a good correlation was found between insulin resistance of cAMP analog-stimulated responses and the analog I50 values for the phosphodiesterase from both cell types. Using a new method capable of measuring hydrolysis at low analog concentrations, several of those analogs which had relatively low, but not high, phosphodiesterase I50 values were shown to be directly hydrolyzed by the low Km adipocyte phosphodiesterase. The insulin inhibition of cell responses when stimulated by hydrolyzable analogs, but not by poorly hydrolyzable analogs, is best explained by insulin stimulation of the low Km phosphodiesterases from both cell types.     

The identification and characterization of two cyclic nucleotide phosphodiesterases from bovine adrenal medulla

Two soluble cyclic nucleotide phosphodiesterase activities, designated Peak I (Mr = 216,000) and Peak II (Mr = 230,000), have been isolated from bovine adrenal medulla by DEAE-cellulose chromatography. Peak I has Ca2+-independent, cGMP-specific phosphodiesterase activity and Peak II has cGMP-stimulated cyclic nucleotide phosphodiesterase activity. Peak I hydrolyzes cGMP with hyperbolic kinetics and demonstrates a Km of 23 microM. Peak II hydrolyzes cGMP with hyperbolic kinetics but hydrolyzes cAMP with slightly sigmoidal kinetics and demonstrates Km values of 54 +/- 0.7 microM cGMP and 38 +/- 6 microM cAMP. Cyclic AMP and cGMP are competitive inhibitors of each other's hydrolysis, suggesting that these nucleotides may be hydrolyzed at the same catalytic site. Micromolar concentrations of cGMP cause a 5-fold stimulation of the hydrolysis of subsaturating concentrations of cAMP by the Peak II phosphodiesterase. Half-maximal activation occurs at 0.5 microM cGMP and the result of activation is a decrease in the apparent Km for cAMP. Stimulation of the hydrolysis of subsaturating concentrations of cGMP by cAMP was also detected; however, cAMP is a less potent activator of the enzyme than cGMP. Cyclic AMP causes a 1.5-fold stimulation of cGMP hydrolysis and half-maximal activation occurs at 2.5 microM cAMP.     

Activation of rat parotid low Km cyclic AMP phosphodiesterase by isoproterenol

Approximately 94% of rat parotid cyclic AMP phosphodiesterase activity measured at a substrate concentration of 0.1 microM cyclic AMP was found in the 100,000 X g supernatant while the remaining enzyme activity was in the particulate fraction. Incubation of parotid slices with 10 microM isoproterenol resulted in approximately 40% activation of the cyclic AMP phosphodiesterase activity of the 100,000 X g supernatant. The enzyme activity in the particulate fraction was unaffected. The activation resulted from an increase in the value of the Vmax while the apparent Km (0.51 microM) was unaffected. The concentration of isoproterenol required to give half-maximal activation was 0.34 microM. The activation was rapid, became significant after 2 min and reached maximum after 30 min incubation of the parotid slices with isoproterenol. The activation of the enzyme activity by isoproterenol could be blocked by propanolol but was unaffected by cycloheximide. Dibutyryl-cyclic AMP was also effective while phenylephrine and carbamylcholine were ineffective in increasing the activity of the enzyme.     

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.     

Cyclic GMP-dependent stimulation of the membrane-bound insulin-sensitive cAMP phosphodiesterase from rat adipocytes

The insulin-sensitive cAMP phosphodiesterase (phosphodiesterase) in rat adipocytes is a membrane-bound low Km enzyme that can be recovered in a crude microsomal fraction (Fraction P-2). The action of this enzyme to hydrolyze cAMP is known to be inhibited by cGMP; nevertheless, it was found in our present study that under selected conditions, the enzyme can also be stimulated by cGMP as well as some other nucleotide derivatives. The maximum cGMP-dependent stimulation was observed when the enzyme in Fraction P-2 was incubated with 10 microM cGMP for 5-20 min at 37 degrees C in the presence of Mg2+, washed, and then assayed in the absence of added cGMP. The level of this stimulation was close to, but less than, that achieved by insulin in intact cells. The actions of the cGMP- and insulin-stimulated enzymes to hydrolyze labeled cAMP were inhibited in an identical manner by cilostamide (Ki = 0.10 microM), griseolic acid (Ki = 0.19 microM), unlabeled cAMP (Km = 0.20 microM), and cGMP (Ki = 0.16 microM), all added to the assay system. Also, the basal, insulin-stimulated, and cGMP-activated enzymes were identically inhibited by a polyclonal antibody raised against a purified membrane-bound low Km phosphodiesterase from bovine adipose tissue. When the same antibody was used for the Western blot analysis of Fraction P-2, it immunoreacted with a single band of protein (165 kDa). These observations indicate that the insulin-sensitive phosphodiesterase in rat adipocytes can be stimulated with 10 microM cGMP and that this stimulation is detectable only after the nucleotide has been eliminated since the enzyme would be strongly inhibited by the nucleotide if the latter exists in the assay system. It is proposed that the insulin-sensitive phosphodiesterase, which is often referred to as a Type IV enzyme, is functionally similar to the Type II enzymes that are known to be stimulated by a low concentration of cGMP and inhibited by higher concentrations of the same nucleotide.     

Activation of dolichol-phosphate mannosyltransferase by dibutryl cyclic AMP in rat liver

Radiolabeled mannose incorporation into secretory glycoproteins and immunoprecipitable fibronectin in the incubation media significantly increased (105 and 32 percent respectively) with a corresponding increase in the levels of dolichol-phosphate mannose, dolichol-diphosphate oligosaccharides and dolichol-phosphate mannosyltransferase activity in the rat liver slices when incubated with dibutryl cAMP and ATP. Dibutryl cAMP activated maximally this enzyme in the presence of ATP in the incubation medium. The activation of the enzyme resulted in a two fold increase in Vmax with no apparent change in the Km for GDP mannose. Phosphorylation the rat liver microsomes with catalytic subunit of cAMP dependent protein kinase, resulted in the activation of dolichol-phosphate mannosyltransferase. These results suggest that cAMP modulates protein glycosylation by activating dolicholphosphate mannosyltransferase activity. The activation of this enzyme could be through phosphorylation/dephosphorylation mechanism involving a cAMP dependent protein kinase.     

Regulation of Ca2+/calmodulin-dependent cyclic nucleotide phosphodiesterase by the autophosphorylated form of Ca2+/calmodulin-dependent protein kinase II

The 63-kDa subunit, but not the 60-kDa subunit, of brain calmodulin-dependent cyclic nucleotide phosphodiesterase was phosphorylated in vitro by the autophosphorylated form of Ca2+/calmodulin-dependent protein kinase II. When calmodulin was bound to the phosphodiesterase, 1.33 +/- 0.20 mol of phosphate was incorporated per mol of the 63-kDa subunit within 5 min with no significant effect on enzyme activity. Phosphorylation in the presence of low concentrations of calmodulin resulted in a phosphorylation stoichiometry of 2.11 +/- 0.21 and increased about 6-fold the concentration of calmodulin necessary for half-maximal activation of the phosphodiesterase. Peptide mapping analyses of complete tryptic digests of the 63-kDa subunit revealed two major (P1, P4) and two minor (P2, P3) 32P-peptides. Calmodulin-binding to the phosphodiesterase almost completely inhibited phosphorylation of P1 and P2 with reduced phosphorylation rates of P3 and P4, suggesting the affinity change of the enzyme for calmodulin may be caused by phosphorylation of P1 and/or P2. When Ca2+/calmodulin-dependent protein kinase II was added without prior autophosphorylation, there was no phosphorylation of the 63-kDa phosphodiesterase subunit or of the kinase itself in the presence of a low concentration of calmodulin, and with excess calmodulin the phosphodiesterase subunit was phosphorylated only at P3 and P4. Thus the 63-kDa subunit of phosphodiesterase has a regulatory phosphorylation site(s) that is phosphorylated by the autophosphorylated form of Ca2+/calmodulin-dependent protein kinase II and blocked by Ca2+/calmodulin binding to the subunit.     

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 Km 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 greater than lysophosphatidylcholine greater than lysophosphatidylserine greater than phosphatidylserine greater than 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 Km 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 Km cAMP phosphodiesterase activity.     

Kinetic characterization of the Ca2+-pumping ATPase of cardia sarcolemma in four states of activation

The Ca2+ dependence of the Ca2+-pumping ATPase of bovine cardiac sarcolemma was studied for four states of activation: (a) unactivated, (b) cAMP-dependent protein kinase (cAMP protein kinase C-subunit)-activated, (c) calmodulin (CAM)-activated, and (d) CAM plus cAMP protein kinase C-subunit-activated. Analysis of the Ca2+ dependence of active transport gave the following Vmax (nanomoles Ca2+/(mg x min], Km (nM) for Ca2+, and Hill coefficient values for the four states at pH 7.4, 37 degrees C: (a) 1.7 +/- 0.3, 1800 +/- 100, 1.6 +/- 0.1; (b) 3.1 +/- 0.5, 1100 +/- 100, 1.7 +/- 0.1; (c) 15.0 +/- 2.5, 64 +/- 1.4, 3.7 +/- 0.2; and (d) 36.0 +/- 6.5, 63 +/- 1.7, 3.7 +/- 0.1. CAM has the most dramatic effect, increasing the apparent Ca2+ affinity by a factor of 28, increasing the Hill coefficient 2.0 units to a value approaching 4 and increasing the Vmax by a factor of 9 or 12. The effective Ca2+ concentration (EC50) for the Ca2+-induced activation of the enzyme in the presence of 5 microM calmodulin is close to the Km for Ca2+ for the CAM-activated state (64 nM). Activation by cAMP protein kinase C-subunit had only minor effects on the Km and Hill coefficient, but increased the Vmax of both the unactivated and the CAM-activated forms of the pump by factor of 1.8 and 2.4, respectively. Analysis suggests that CAM activation is the result of direct binding of Ca2-CAM or high complexes, conferring higher Ca2+ affinity to the enzyme. Analysis suggests that regulatory phosphorylation (cAMP protein kinase C-subunit) increases the rates of processes subsequent to or distinct from Ca2+ binding. The CAM-activated form of the pump was further characterized. Unexpectedly, this form of the enzyme is stimulated a factor of 1.9 by ADP, with half-maximal stimulation between 0.4 and 0.7 mM. Analysis of the progress curves for uptake show that the CAM-activated enzyme is highly resistant to inhibition by transported Ca2+, with an IC50 of 32 mM. The implications of these findings for the pump mechanism and for its role in the regulation of cardiac contractility are discussed.     

Phospholipid modulation of low-Km cyclic AMP phosphodiesterase activity on rat adipocyte microsomes

The ability of nine phospholipids to alter the activity of low-Km cyclic AMP phosphodiesterase was examined in microsomal fractions of rat adipocytes. The enzyme was activated by phosphatidylserine (21% at 300 microM) and phosphatidylglycerol (36% at 300 microM). The activation was concentration dependent over the range 1-1000 microM. Six other phospholipids were without effect. Phosphatidylinositol 4-phosphate inhibited the activity of the enzyme over the same range of concentrations (26% at 300 microM). Phosphatidylserine also activated a partially purified preparation of the enzyme, whereas phosphatidylinositol 4-phosphate was ineffective. The mechanism of the activation of the enzyme by phosphatidylserine and phosphatidylglycerol involved an increase in the apparent Vmax of the enzyme, while the inhibition by phosphatidylinositol 4-phosphate was associated with an increase in the Km of the enzyme for substrate. The phospholipid modulators of low-Km cyclic AMP phosphodiesterase activity did not alter the activity of high-Km cyclic AMP phosphodiesterase. The ability of phospholipids to alter the activity of low-Km cyclic AMP phosphodiesterase in native membranes suggests a possible role for phospholipids in metabolic regulation.     

Phosphorylation and activation of calmodulin-sensitive cyclic nucleotide phosphodiesterase by a brain Ca2+, calmodulin-dependent protein kinase

A Ca2+, calmodulin-dependent protein kinase from rat brain with a MW of 640,000 phosphorylated calmodulin-sensitive phosphodiesterase from the brain cytosol. The Km of the enzyme for the phosphodiesterase was 5.0 microM and the Vmax was 212 nmol/mg/min. The amount of phosphate incorporated into the phosphodiesterase was 0.7 mol/mol subunit. Phosphorylation of the phosphodiesterase enhanced the enzyme activity by about 20% for hydrolysis of a higher concentration of cyclic AMP.     

Ca2+-independent cyclic GMP phosphodiesterases from rat liver and HTC hepatoma cells.

We have separated and characterized a Ca2+- and calmodulin-insensitive cyclic nucleotide phosphodiesterase from rat liver supernatant as well as an analogous enzyme from HTC hepatoma cells. Chromatography of rat liver supernatant on DEAE-cellulose in the presence and subsequently in the absence of 0.1 mM-CaCl2 resulted in the separation of two distinct phosphodiesterase activities, both of which preferentially hydrolysed cyclic GMP rather than cyclic AMP. One enzyme, E-Ib, was activated in the presence of Ca2+ and calmodulin, and the other, E-Ia, was not. The E-Ia enzyme, which did not bind to calmodulin-Sepharose, had Mr 325 000 and displayed anomalous kinetic behaviour [Km (cyclic GMP) 1.2 microM; Km (cyclic AMP) 15.4 microM]. The E-Ib enzyme, which bound to calmodulin-Sepharose in the presence of Ca2+, had Mr 150 000 and exhibited Michaelis-Menten kinetics for hydrolysis of cyclic GMP [Km (basal) 6.5 microM; Km (activated) 12.0 microM]. E-Ia activity was diminished by incubation with alpha-chymotrypsin and was unaffected by the action of a rat kidney lysosomal proteinase. Partial hydrolysis of E-Ib enzyme by alpha-chymotrypsin or the kidney proteinase resulted in irreversible activation of the enzyme. The E-I enzyme isolated from HTC hepatoma cells was similar to the rat liver E-Ia enzyme in many respects. Its apparent Mr was 325 000. Its activity was unaffected by calmodulin in the presence of Ca2+ or by incubation with the kidney proteinase, and was decreased by digestion with alpha-chymotrypsin. Unlike the liver E-Ia enzyme, however, the hepatoma enzyme exhibited normal kinetic behaviour, with Km (cyclic GMP) 3.2 microM. Although HTC cells contain two other phosphodiesterases analogous to those in rat liver and a calmodulin-like activator of phosphodiesterase, no calmodulin-sensitive phosphodiesterase was detected.     

Tyrosine kinase-independent inhibition of cyclic-AMP phosphodiesterase by genistein and tyrphostin 51.

The phosphodiesterase activity in the HT4.7 neural cell line was pharmacologically characterized, and phosphodiesterase isozyme 4 (PDE4) was found to be the predominant isozyme. The Km for cAMP was 1-2 microM, indicative of a "low Km" phosphodiesterase, and the activity was inhibited by PDE4-selective inhibitors rolipram and Ro20-1724, but not PDE3- or PDE2-selective inhibitors. Calcium, calmodulin, and cGMP, regulators of PDE1, PDE2, and PDE3, had no effect on cAMP hydrolysis. The protein tyrosine kinase inhibitor, genistein, inhibited HT4.7 cAMP phosphodiesterase activity by 85-95% with an IC50 of 4 microM; whereas daidzein, an inactive structural analog of genistein, had little effect on phosphodiesterase activity. This is a common pharmacological criterion used to implicate the regulation by a tyrosine kinase. However, genistein still inhibited phosphodiesterase activity with a mixed pattern of inhibition even when ion-exchange chromatography was used to partially purify phosphodiesterase away from the tyrosine kinase activity. Moreover, tyrphostin 51, another tyrosine kinase inhibitor, was found to also inhibit partially purified phosphodiesterase activity noncompetitively. These data suggest that HT4.7 phosphodiesterase activity is dominated by PDE4 and can be regulated by genistein and tyrphostin 51 by a tyrosine kinase-independent mechanism.     

Properties of multiple kinetic forms of soluble cyclic nucleotide phosphodiesterase activity of rat colonic mucosa

Soluble phosphodiesterase (EC 3.1.4.1) activity is 3-5-fold lower in superficial colonic epithelial cells compared to that in cells isolated from the lower colonic crypt. Higher phosphodiesterase activity in lower crypt cells is correlated with a 5-fold higher rate of incorporation of [3H]thymidine into DNA in these cells. DEAE-cellulose chromatography of the soluble fraction of superficial and proliferative colonic epithelial cells resulted in separation of three enzyme forms: (1) fraction I, an enzyme which hydrolyzes both cAMP and cGMP with high affinity (apparent Km cAMP = 5 +/- 1 microM, Km cGMP = 2.5 +/- 0.5 microM) and is stimulated 3-6-fold by Ca2+ plus calmodulin; (2) fraction II, a form which hydrolyzes both cAMP and cGMP with low affinity (S0.5 cAMP = 52 +/- 7 microM, S0.5 cGMP = 17 +/- 4 microM), exhibits positive copperativity with respect to substrate and shows cGMP stimulation of cAMP hydrolysis and (3) fraction III, a cAMP-specific form which exhibits biphasic kinetics, a low Km for cAMP (Km cAMP = 5 +/- 1 microM) and does not hydrolyze cGMP. The pattern of distribution of phosphodiesterase activities on DEAE-cellulose was similar in superficial and proliferative colonic epithelial cells. The higher specific activity in proliferative cells was reflected in higher activities of each of the three chromatographically distinct forms of the enzyme. In contrast to epithelial cells, the soluble fraction of homogenates of the submucosa and supporting cells exhibited phosphodiesterase forms I and II and was lacking in the form corresponding to fraction III of epithelial cells.     

Hormone-sensitive particulate cAMP phosphodiesterase activity in 3T3-L1 adipocytes. Regulation of responsiveness by dexamethasone

Confluent 3T3-L1 fibroblasts incubated for 72 h with methylisobutylxanthine, dexamethasone, and insulin differentiate and acquire phenotypic characteristics of mature adipocytes, including hormone-sensitive cAMP phosphodiesterase activity located in a particulate fraction of homogenates. About 10 days after initiating differentiation, a maximally effective concentration of insulin (100 pM) increased particulate cAMP phosphodiesterase activity 40 to 60% in 8 min; activation persisted for at least 30 min in the presence of insulin. Incubation of adipocytes for 6-8 min with agents that increased cAMP, e.g. 1 microM epinephrine, 0.1 microM isoproterenol, corticotropin (2 mu units/ml), or thyroid-stimulating hormone (15 ng/ml), also increased particulate phosphodiesterase activity 40-60%. Changes in phosphodiesterase activity produced by epinephrine tended to lag behind changes in cAMP. Insulin, epinephrine, and corticotropin increased Vmax, not Km (0.5 microM), for cAMP. Particulate phosphodiesterase activity, solubilized with detergent, eluted in a single peak from DEAE-Bio-Gel. Insulin and epinephrine increased the activity eluted in this peak. Neither insulin nor lipolytic hormones increased activity in soluble fractions from differentiated cells or particulate or soluble fractions from undifferentiated cells. Incubation of adipocytes for 48 h with 1 microM dexamethasone prevented insulin-induced activation of the particulate phosphodiesterase and did not alter basal activity. After incubation for 72 h with 0.1 microM dexamethasone, insulin and epinephrine activation were abolished. These effects of dexamethasone on hormonal regulation of particulate phosphodiesterase activity could account for some of the so-called permissive effects of glucocorticoids on cAMP-mediated processes as well as the "anti-insulin" effects of glucocorticoids.     

Characterization of calmodulin-activated protein kinase activity of rat adipocyte endoplasmic reticulum fraction

Calmodulin-activated protein kinase activity in the endoplasmic reticulum fraction of rat adipocytes was identified and characterized. The major endogenous protein substrate of the calmodulin-activated kinase activity has an apparent molecular weight of 54,000 as determined by sodium dodecyl sulfate gel electrophoresis. The calmodulin-activated component of the activity was saturated at 10 microM ATP. Calcium or calmodulin alone did not increase the activity, but the simultaneous presence of calcium and calmodulin increased activity three to four-fold. Half-maximal activation of this activity occurred at 8 microM Ca2+. The addition of increasing amounts of calmodulin caused a concentration-dependent activation in the presence of calcium, which was saturable at high calmodulin concentrations. Magnesium was required for activity, with half-maximal activity occurring at 230 microM. The antipsychotic drug trifluoperazine inhibited the activation of the protein kinase activity by calmodulin, but had a negligible effect on the basal activity. Half-maximal inhibition occurred at 63 microM. Phosphorylation of the 54,000 mol. wt band was independent of cAMP, cGMP and the combination of cAMP and cAMP-dependent protein kinase. Calmodulin-activated protein kinase phosphorylated both phosphoserine and phosphothreonine residues in the 54,000 mol. wt substrate. These experiments have partially characterized a calmodulin-activated protein kinase activity from adipocytes, which appears to be a unique activity of unknown function.     

Tyrosine Hydroxylase Activation and Inactivation by Protein Phosphorylation Conditions

Tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, catalyzes the conversion of tyrosine to DOPA, Cyclic AMP-dependent protein phosphorylation conditions alter tyrosine hydroxylase activity in rat striatal homogenates. In agreement with other laboratories, we find that short-term pre-incubation (3 min) of extracts under phosphorylating conditions (Mg . ATP, cAMP) increases enzyme activity two- to tenfold over control as measured during a subsequent 15-min assay. We now report that preincubation under phosphorylating conditions for longer periods (30 min) results in a loss of activity to levels equal to or below that of the control enzyme. Addition of purified bovine brain protein kinase catalytic subunit and Mg . ATP enhances activation and increases the rate of inactivation. To demonstrate that inactivation is not associated with proteolytic degradation or irreversible denaturation, the inactivated form of the enzyme can be reactivated. The protein kinase inhibitor protein decreases the activation process and prevents inactivation of the enzyme to below control values. The sedimentation coefficient is not changed by phosphorylation conditions (S = 8.8 +/- 0.1). Although the apparent Km of the enzyme for the 6-methyltetrahydropterine (6-MPH4) cofactor is reduced (0.86 mM, control; 0.32 mM, activated), it is also reduced in the inactivated form (0.38 mM). The Ki for dopamine is increased from 4.5 microM for the control to 28 microM for the activated enzyme, whereas the inactivated form of the enzyme exhibits a Ki of 10 microM. Removal of catecholamines by gel filtration fails to alter activity and the apparent cofactor Km. Moreover, both the activated and the inactivated states persist following gel filtration. It therefore appears that the activation-inactivation process is not mediated solely by the modulation of enzyme feedback inhibition or changes in the Km for 6-MPH4. We also describe a coupled decarboxylase assay in which labeled dopamine is resolved from the precursors tyrosine and DOPA by low-voltage paper electrophoresis.     

Characterization of a Ca2+-calmodulin-stimulated cyclic GMP phosphodiesterase from bovine brain

A calmodulin-stimulated form of cyclic nucleotide phosphodiesterase from bovine brain has been extensively purified (1000-fold). Its specific activity is approximately 4 mumol min-1 (mg of protein)-1 when 1 microM cGMP is used as the substrate. This form of calmodulin-sensitive phosphodiesterase activity differs from those purified previously by showing a very low maximum hydrolytic rate for cAMP vs. cGMP. The purification procedure utilizing ammonium sulfate precipitation, ion-exchange chromatography on DEAE-cellulose, gel filtration on Sephacryl S-300, isoelectric focusing, and affinity chromatography on calmodulin-Sepharose and Cibacron blue-agarose results in a protein with greater than 80% purity with 1% yield. Kinetics of cGMP and cAMP hydrolysis are linear with Km values of 5 and 15 microM, respectively. Addition of calcium and calmodulin reduces the apparent Km for cGMP to 2-3 microM and increases the Vmax by 10-fold. cAMP hydrolysis shows a similar increase in Vmax with an apparent doubling of Km. Both substrates show competitive inhibition with Ki's close to their relative Km values. Highly purified preparations of the enzyme contain a major protein band of Mr 74 000 that best correlates with enzyme activity. Proteins of Mr 59 000 and Mr 46 000 contaminate some preparations to varying degrees. An apparent molecular weight of 150 000 by gel filtration suggests that the enzyme exists as a dimer of Mr 74 000 subunits. Phosphorylation of the enzyme preparation by cAMP-dependent protein kinase did not alter the kinetic or calmodulin binding properties of the enzyme. Western immunoblot analysis indicated no cross-reactivity between the bovine brain calmodulin-stimulated gGMP phosphodiesterase and the Mr 60 000 high-affinity cAMP phosphodiesterase present in most mammalian tissues.     

The role of protein kinases in anoxia tolerance in facultative anaerobes: purification and characterization of a protein kinase that phosphorylates pyruvate kinase

A protein kinase which phosphorylates pyruvate kinase (PK) in vitro was purified and characterized from the foot muscle of the anoxia-tolerant gastropod mollusc Busycon canaliculatum. Purification involved four steps: poly(ethylene glycol) fractionation, affinity chromatography on Blue agarose, ion-exchange chromatography on phosphocellulose and preparative isoelectric focusing (pI = 5.5). The activity was monitored by following changes in pyruvate kinase I50 values for L-alanine which have previously been linked to changes in the degree of enzyme phosphorylation. The correlation between enzyme phosphorylation and changes in the L-alanine inhibition constant was also directly demonstrated in the present paper by radioactively labelling PK with [tau-32P]ATP. The final purified protein kinase solution gave a single band on SDS-gel electrophoresis with a molecular weight of 37,000 +/- 2000. Kinetic analysis of the purified protein kinase (PK-kinase) showed a pH optimum of 7.0, an absolute requirement for magnesium ions (Km = 1.29 mM), a relatively high affinity for MgATP (Km = 57 microM), and inhibition by increasing salt concentrations (I50 = 55 mM KCl). The protein kinase activity was not affected by either spermine, heparin, cAMP, cGMP or concentrations of CaCl2 less than 10 mM. The enzyme did not phosphorylate either phosphofructokinase or glycogen phosphorylase, two enzymes that are also phosphorylated during anoxia in whelks. The purified enzyme is different from the catalytic subunit of cAMP-dependent protein kinase as shown by the inability of cAMP to stimulate the protein kinase at all stages of the preparation; cAMP did not activate either crude enzyme, the 7% poly(ethylene glycol) supernatant, or any of the column eluant peak fractions when measured by changes in pyruvate kinase kinetic parameters.     

Regulatory properties of phosphorylase from chicken skeletal muscle

The main kinetic parameters for purified phosphorylase kinase from chicken skeletal muscle were determined at pH 8.2: Vm = 18 micromol/min/mg; apparent Km values for ATP and phosphorylase b from rabbit muscle were 0.20 and 0.02 mM, respectively. The activity ratio at pH 6.8/8.2 was 0.1-0.4 for different preparations of phosphorylase kinase. Similar to the rabbit enzyme, chicken phosphorylase kinase had an absolute requirement for Ca2+ as demonstrated by complete inhibition in the presence of EGTA. Half-maximal activation occurred at [Ca2+] = 0.4 microM at pH 7.0. In the presence of Ca2+, the chicken enzyme from white and red muscles was activated 2-4-fold by saturating concentrations of calmodulin and troponin C. The C0.5 value for calmodulin and troponin C at pH 6.8 was 2 and 100 nM, respectively. Similar to rabbit phosphorylase kinase, the chicken enzyme was stimulated about 3-6-fold by glycogen at pH 6.8 and 8.2 with half-maximal stimulation occurring at about 0.15% glycogen. Protamine caused 60% inhibition of chicken phosphorylase kinase at 0.8 mg/ml. ADP (3 mM) at 0.05 mM ATP caused 85% inhibition with Ki = 0.2 mM. Unlike rabbit phosphorylase kinase, no phosphorylation of the chicken enzyme occurred in the presence of the catalytic subunit of cAMP-dependent protein kinase. Incubation with trypsin caused 2-fold activation of the chicken enzyme.