Calcium-dependent cyclic nucleotide phosphodiesterase from brain identification of phospholipids as calcium-independent activators.

Phosphatidyl inositol and lysophosphatidyl choline have been identified as activators of a partially purified brain cyclic nucleotide phosphodiesterase previously shown to be regulated in vitro by Ca²⁺ and a Ca²⁺-binding protein. Microgram quantities of either phospholipid produced a linear, immediate and reversible activation of the enzyme in the absence of Ca²⁺ and the Ca²⁺-dependent regulator (CDR). Fatty acids were also found to activate the phosphodiesterase to varying degrees, with oleic acid being the most effective. Phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine and lysophosphatidyl ethanolamine were not effective as activators. Only sodium dodecyl sulfate, of a variety of nonionic, cationic, and anionic detergents tested, activated the phosphodiesterase. Sodium dodecyl sulfate produced a modest degree of activation over a narrow concentration range, followed by enzyme denaturation at higher concentrations.The interaction of the phosphodiesterase with the phospholipid activators has been compared to its interaction with the Ca²⁺·CDR complex. Both Ca²⁺·CDR and lysophosphatidyl choline decreased the thermal stability of the enzyme to a similar extent. The apparent K_m of the lysophosphatidyl choline-dependent phosphodiesterase activity was approximately 30 μm with guanosine-3′,5′-monophosphate (cGMP) as substrate and 1 mm with adenosine-3′,5′-monophosphate (cAMP) as substrate. With increasing lysophosphatidyl choline concentration, the apparent K_m for each nucleotide remained unchanged while the V increased. The apparent K_d for Mg²⁺ of the lysophosphatidyl choline-dependent phosphodiesterase activity was approximately 3 μm and was unaffected by lysophosphatidyl choline concentration. Activation of the phosphodiesterase by lysophosphatidyl choline was characterized by a high degree of positive cooperativity, exhibiting a Hill coefficient of 3.8. Fluphenazine was a competitive inhibitor of both Ca²⁺·CDR and lysophosphatidyl choline activation of the enzyme.     

Calcium-dependent cyclic nucleotide phosphodiesterase from brain: comparison of adenosine 3',5'-monophosphate and guanosine 3',5'-monophosphate as substrates.

A Ca²⁺-dependent cyclic nucleotide phosphodiesterase has been partially purified from extracts of porcine brain by column chromatography on Sepharose 6 B containing covalently linked protamine residues, ammonium sulfate salt fractionation, and ECTEOLA-cellulose column chromatography. The resultant preparation contained a single form of cyclic nucleotide phosphodiesterase activity by the criteria of isoelectric focusing, gel filtration chromatography on Sephadex G-200, and electrophoretic migration on polyacrylamide gels. When fully activated by the addition of Ca²⁺ and microgram quantities of a purified Ca²⁺-binding protein (CDR), the phosphodiesterase hydrolyzed both adenosine 3′,5′-monophosphate (cyclic AMP) and guanosine 3′,5′-monophosphate (cyclic GMP), with apparent K_m values of 180 and 8 μm, respectively. Approximately 15% of the total enzymic activity was present in the absence of added CDR and Ca²⁺. This activity exhibited apparent K_m values for the two nucleotides identical to those observed for the maximally activated enzyme. Competitive substrate kinetics and heat destabilization studies demonstrated that both cyclic nucleotides were hydrolyzed by the same phosphodiesterase. The purified enzyme was identical to a Ca²⁺-dependent phosphodiesterase present in crude extract by the criteria of gel filtration chromatography, polyacrylamide-gel electrophoresis, and kinetic behavior.Apparent K_m values of the Ca²⁺-dependent phosphodiesterase for cyclic AMP and cyclic GMP were lowered more than 20-fold as CDR quantities in the assay were increased to microgram amounts, whereas the respective maximal velocities remained constant. The apparent K_m for Mg²⁺ was lowered more than 50-fold as CDR was increased to microgram amounts. Half-maximal activation of the phosphodiesterase occurred with lower amounts of CDR as a function of either increasing degrees of substrate saturation or increasing concentrations of Mg²⁺. At low cyclic nucleotide substrate concentrations i.e., 2.5 μm, cyclic GMP was hydrolyzed at a fourfold greater velocity than cyclic AMP. At high substrate concentrations (millimolar range) cyclic AMP was hydrolyzed at a threefold greater rate than cyclic GMP.     

Calcium-dependent adenylate cyclase from rat cerebral cortex: Activation by guanine nucleotides

The adenylate cyclase activity of a participate preparation of rat cerebral cortex is composed of at least two contributing components, one of which requires a Ca²⁺-dependent regulator protein (CDR) for activity (Brostrom, C. O., Brostrom, M. A., and Wolff, D. J. (1977) J. Biol. Chem.252, 5677–5685). Each of these components of the activity was activated by GTP and its synthetic analog, 5-guanylylimidodiphosphate (Gpp(NH)p). The component of the adenylate cyclase activity which did not respond to CDR (CDR-independent activity) was stimulated approximately 60% by 100 μm GTP and 3.5-fold by 100 μm Gpp(NH)p. Concentrations of GTP required for maximal activation of the CDR-dependent adenylate cyclase component decreased as CDR concentrations in the assay were increased. Similarly, GTP pr Gpp(NH)p lowered the concentration of CDR required to produce half-maximal activation of this enzyme form. At saturating CDR concentrations, however, increases in activity were not observed with the addition of these nucleotides. The CDR-dependent component responded biphasically (activation followed by inhibition) to increasing free Ca²⁺ concentrations; both phases of this response occurred at lower free Ca²⁺ concentrations with GTP present in the assay. The concentration of chlorpromazine which inhibited activation of adenylate cyclase by CDR was elevated when GTP was present. The CDR-dependent form of activity, which is stabilized by CDR to thermal inactivation, was also stabilized by Gpp(NH)p. The increase in stability produced by Gpp(NH)p did not require the presence of CDR, and stabilization with both Gpp(NH)p and CDR was greater than that obtained with either Gpp(NH)p or CDR alone.     

Calmodulin-stimulated cyclic nucleotide phosphodiesterase from Neurosproa crassa

Cyclic nucleotide phosphodiesterase has been partially purified by calmodulin-Sepharose affinity chromatography from a soluble extract of Neurospora crassa. The phosphodiesterase activity remained bound to the affinity column even in the presence of 6 M urea and could only be eluted by calcium chelation. The enzyme exhibits cAMP and cGMP phosphodiesterase activities. Both activities can be enhanced by calmodulin in a Ca²⁺-dependent manner. Stimulation of cyclic nucleotide phosphodiesterase by calmodulin can be inhibited by calmodulin antagonists such as pimozide, trifluoperazine and chlorpromazine.     

Effects of CuCl2 on the hydrolysis of cyclic GMP and cyclic AMP by the activator-dependent cyclic nucleotide phosphodiesterase from bovine heart

CuCl₂ non-comepetitively inhibited the hydrolysis of cyclic GMP and cyclic AMP by the activator-dependent phosphodiesterase from bovine heart in the presence of 5 mM Mg²⁺, 10 μM Ca²⁺ and phosphodiesterase activator with K_i values of approximately 2 μM for both substrates. CuCl₂ inhibition was also non-competitive with Mg²⁺, Ca²⁺ and phosphodiesterase activator. Dialysis demonstrated that CuCl₂ inhibition in reversible. Treatment of the enzyme with p-hydroxymercuribenzoate resulted in the loss of enzyme activity, suggesting the presence of sulfhydryl groups essential for enzyme activity. The inhibitory activity of CuCl₂ was not additive with that p-hydroxymercuribenzoate, therefore CuCl₂ may inhibit enzyme activity by binding to one or more essential sulfhydryl groups. CuCl₂ also inhibited the hydrolysis of cyclic AMP by the cyclic AMP-specific phosphodiesterase from bovine heart with an I₅₀ value of 18 μM. Several effects of Cu²⁺ are discussed which have been noted in other studies and might be due, in part, to changes in cyclic nucleotide levels following alterations in phosphodiesterase activity.     

Multiple forms of cyclic nucleotide phosphodiesterase from bovine carotid artery smooth muscle.

DEAE-cellulose chromatography, in the presence and absence of Ca²⁺, of the 16,000g supernatant from bovine carotid artery smooth muscle has been used to separate four different types of cyclic nucleotide phosphodiesterase (3′:5′-cyclic-nucleotide 5′-nucleotidohydrolase, EC 3.1.4.17) activity, designated types A, B, C, and D. Type A is a high affinity, cyclic AMP-specific form of phosphodiesterase (K_m = 1.6 μM) and elutes at relatively high ionic strength. Type B is a high affinity (K_m = 2 μM), cyclic GMP-specific form which elutes at low ionic strength. Type C is a mixed substrate form, displaying anomalous kinetics for the hydrolysis of both cyclic AMP and cyclic GMP. It elutes from DEAE-cellulose at an ionic strength intermediate to that of types A and B. Type D is also a mixed substrate form of phosphodiesterase. However, its elution pattern from DEAE-cellulose differs, depending on whether Ca²⁺ is present or not, suggesting a Ca²⁺-dependent interaction between this enzyme form and the acidic Ca²⁺-dependent regulator protein (CDR). The hydrolytic activity of type D is stimulated by CDR, and activation requires the simultaneous presence of Ca²⁺ and CDR. Kinetic analysis of cyclic AMP hydrolysis by type D gives a linear double reciprocal plot; activation has no effect on the K_m but increases the velocity approximately sixfold. Activation of cyclic GMP hydrolysis apparently affects both the K_m and V. At all concentrations tested, the degree of activation is higher with cyclic AMP than with cyclic GMP. It is suggested that while the activable form of phosphodiesterase may play a relatively minor role in the overall hydrolysis of cyclic nucleotides, Ca²⁺-dependent activation may have a more important role in regulating the level of cyclic AMP than that of cyclic GMP in vascular smooth muscle.     

REGULATION OF CYCLIC NUCLEOTIDE PHOSPHODIESTERASES OF CEREBRAL CORTEX BY Ca2+ AND CYCLIC GMP

Cyclic nucleotide phosphodiesterase activity of porcine cerebral cortical extracts was measured with 0.1–100 μM-cyclic AMP and cyclic GMP and found to be dependent on both Ca²⁺ and added cyclic nucleotides. With decreasing substrate concentration activity with cyclic GMP became more dependent on Ca²⁺ whereas hydrolysis of cyclic AMP became less dependent. Cyclic GMP at 3 μM stimulated the hydrolysis of 0.1–10μM-cyclic AMP in the absence of Ca²⁺ (< 10^-10M) but inhibited activity with 200 μM-Ca²⁺ present. This differential, substrate- and Ca²⁺-dependent regulation was attributed to the presence of at least two types of phosphodiesterase distinguishable by DEAE-column chromatography. In the absence of Ca²⁺, activity with 1 μM-cyclic GMP eluted in one minor peak followed by two major peaks, D-I and D-II. Activity with 1 μM-cyclic AMP eluted almost entirely in D-II. Hydrolysis of cyclic AMP in D-II was activated by cyclic GMP. With added Ca²⁺ plus a Ca²⁺-dependent regulator (CDR), activity with 1 μM-cyclic GMP was markedly increased and eluted entirely at D-I. Total activity with 1 μM-cyclic AMP was only moderately increased and eluted as D-I with a shoulder at D-II. Elution profiles with 100 μM-substrate were relatively independent of substrate, with D-I predominant with Ca²⁺·CDR present and D-II predominant in its absence. Kinetic analysis of rechromatographed D-I showed a 20- to 40-fold activation by Ca²⁺·CDR that was largely due to an increase in V_max, with only 50% decreases in K_m Both substrates competitively inhibited hydrolysis of the other with K_i values equal to their respective K_m values (1.7 μM for cyclic GMP and 48 μM for cyclic AMP with Ca²⁺-CDR present). Studies with theophylline and trifluoperazine indicate differential, substrate-dependent inhibitions of both enzymes. These findings demonstrate that phosphodiesterase activity in neural tissue is subject to regulation by Ca²⁺, cyclic GMP, and inhibitors in a complex, substrate-specific and concentration-dependent manner.     

Cyclic nucleotide phosphodiesterases of rabbit renal cortex. Characterization of brush border membrane activities.

Cyclic nucleotide phosphodiesterase activity in brush border membranes, isolated from proximal tubule cells of the rabbit renal cortex, was investigated. Brush border cAMP phosphodiesterase activity was tightly bound to the membrane and was distinguished from the soluble phosphodiesterase activity of the renal cortex cytosol. Multiple forms of the brush border membrane cAMP phosphodiesterase activity, dependent on the concentration of substrate, were found. When assayed with 1 μm or 1 mm cAMP, activities differed in pH optimum, effects of various divalent cations, inhibition by metal ion chelators and reactivation by metals, thermolability, sensitivity to inhibitors and specificity.Renal brush border membranes also possessed cGMP phosphodiesterase activity. cAMP was a relatively poor inhibitor of the hydrolysis of 1 μm cGMP and the hydrolysis of 1 μm cAMP was virtually insensitive to cGMP. These findings suggest that the low substrate concentration-dependent cAMP phosphodiesterase was distinct from the low substrate concentration-dependent cGMP phosphodiesterase.Heat-stable effectors of phosphodiesterase activity were found in the renal cortex. One effector activated soluble cAMP phosphodiesterase. Activation was decreased by EGTA, enhanced by Ca²⁺ and diminished by preincubating the effector with proteolytic enzymes. The other heat-stable effector inhibited brush border membrane phosphodiesterase activity. Inhibition was unaffected by metal ions, unaffected by preincubating the effector with proteolytic enzymes, but diminished by preincubation with phospholipase C and neuraminidase.It is suggested that changes in the activity of the enzyme (or enzymes), which in turn controls, in part, the effective concentration of cAMP at its site (or sites) of action in the renal cell, may be significant in regulating hormonal-dependent transport in the proximal tubule.     

Properties of a cyclic nucleotide phosphodiesterase of amphibian oocytes that is activated by calmodulin and calcium,by tryptic proteolysis,and by phospholipids

A calmodulin-Ca²⁺-stimulated cyclic nucleotide phosphodiesterase (EC 3.1.4.17) which hydrolyzed both cGMP and cAMP has been purified about 2000-fold from ovaries of the amphibian Xenopus laevis. Gel filtration through Sephadex G-200 indicated a molecular weight of 140,000. A single, major protein band of molecular weight 66,000 was observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In addition to the stimulation by calmodulin-Ca²⁺, the enzyme was activated 5- to 10-fold by proteolysis and by certain phospholipids. Trypsin activation of the enzyme caused a reduction in the native molecular weight to 90,000 and a loss of the capacity to be stimulated by calmodulin-Ca²⁺ or by phospholipids. The phosphodiesterase was stimulated by low concentrations (0.1 μg/ml) of lysophosphatidylcholine and lysophosphatidylethanolamine. This response did not require calcium ions. Phosphatidylinositol, fatty acids, progesterone, and phospholipase C had little or no effect on activity. Simultaneous addition of 1 mm 2-chloro-10-(3-aminopropyl)phenothiazine and lysophosphatidylcholine to the enzyme did not diminish the stimulatory effect of the phospholipid. The activation of the enzyme by all three agents resulted in an increase in the maximum velocity of the reaction without significant modification of the apparent K_m values for cGMP (5 μm) or cAMP (30 μm). It was suggested that trypsin removed an inhibitory domain from the enzyme and that calmodulin and phospholipids interact with this same domain, eliminating its capacity to inhibit the active center of the enzyme.     

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.     

Calcium-dependent cyclic nucleotide phosphodiesterase inhibition of basal activity by heat-stable factors from rat cerebrum

The boiled supernatant fraction from rat cerebrum contained factors which inhibited the basal activity of a Ca²⁺-dependent phosphodiesterase from rat cerebrum. Two inhibitory fractions were isolated by DEAE-cellulose or Sephadex chromatography and were deemed proteins, based on their sensitivity to trypsin digestion. The inhibitory fractions eluted from DEAE-cellulose columns prior to the Ca²⁺-dependent activator protein. The inhibitory factors, unlike the activator protein, were stable to heat treatment under alkaline conditions. The inhibitory factors caused both an increase in K_m for cyclic GMP and a decrease in $\text{V}$. In the presence of calcium ions and purified activator protein, the Ca²⁺-dependent phosphodiesterase was not inhibited by the factors, but instead was slightly stimulated. The inhibitory factors caused a slight apparent stimulation of a Ca²⁺-independent phosphodiesterase from rat cerebrum but this proved instead to be a nonspecific stabilizing effect which was mimicked by bovine serum albumin. After prolonged alkaline treatment, the purified activator protein caused a modest Ca²⁺-independent activation of Ca²⁺-dependent phosphodiesterase. The inhibitory factors antagonized the activation of Ca²⁺-dependent phosphodiesterase by alkaline treated activator protein or by lysophosphatidylcholine. The inhibitory factors had no effect on activity of trypsinized Ca²⁺-dependent phosphodiesterase. Of various other proteins, only casein mimicked the effects of the inhibitory factors on phosphodiesterase activity.     

cAMP controls rod photoreceptor sensitivity via multiple targets in the phototransduction cascade

In early studies, both cyclic AMP (cAMP) and cGMP were considered as potential secondary messengers regulating the conductivity of the vertebrate photoreceptor plasma membrane. Later discovery of the cGMP specificity of cyclic nucleotide–gated channels has shifted attention to cGMP as the only secondary messenger in the phototransduction cascade, and cAMP is not considered in modern schemes of phototransduction. Here, we report evidence that cAMP may also be involved in regulation of the phototransduction cascade. Using a suction pipette technique, we recorded light responses of isolated solitary rods from the frog retina in normal solution and in the medium containing 2 µM of adenylate cyclase activator forskolin. Under forskolin action, flash sensitivity rose more than twofold because of a retarded photoresponse turn-off. The same concentration of forskolin lead to a 2.5-fold increase in the rod outer segment cAMP, which is close to earlier reported natural day/night cAMP variations. Detailed analysis of cAMP action on the phototransduction cascade suggests that several targets are affected by cAMP increase: (a) basal dark phosphodiesterase (PDE) activity decreases; (b) at the same intensity of light background, steady background-induced PDE activity increases; (c) at light backgrounds, guanylate cyclase activity at a given fraction of open channels is reduced; and (d) the magnitude of the Ca²⁺ exchanger current rises 1.6-fold, which would correspond to a 1.6-fold elevation of [Ca²⁺]_in. Analysis by a complete model of rod phototransduction suggests that an increase of [Ca²⁺]_in might also explain effects (b) and (c). The mechanism(s) by which cAMP could regulate [Ca²⁺]_in and PDE basal activity is unclear. We suggest that these regulations may have adaptive significance and improve the performance of the visual system when it switches between day and night light conditions.     

Studies On Cyclic Nucleotide Metabolism In Tetrahymena Pyriformis: Partial Characterization of Cyclic Amp- and Cyclic Gmp-Dependent Phosphodiesterases*

SYNOPSIS. Cyclic nucleotide phosphodiesterase [EC 3.1.4.17] was examined in Tetrahymena pyriformis strain NT-1. Enzymic activity was associated with the soluble and the particulate fractions, whereas most of the cyclic GMP phosphodiesterase activity was localized in the soluble fraction: the activities were optimal at pH 8.0–9.0. Although very low activities were detected in the absence of divalent cations, they were significantly increased by the addition of either Mg²⁺ or Mn^2-. A kinetic analysis of the properties of the enzymes yielded 2 apparent K_III values ranging in concentration from 0.5 to 50 μM and from 0.1 to 62 μ M for cyclic AMP and GMP. respectively. A Ca²⁺-dependent activating factor for cyclic nucleotide phosphodiesterase was extracted from Tetrahymena cells, but this factor did not stimulate guanylate cyclase [EC 4.6.1.2] activity in this organism. On the other hand, Tetrahymena also contained a protein activator which stimulated guanylate cyclase in the presence of Ca²⁺, although this activator did not stimulate the phosphodiesterase. the results suggested that Tetrahymena might contain 2 types of Ca²⁺-dependent activators, one specific for phosphodiesterase and the other for guanylate cyclase.     

Calmodulin-like activity in the soluble fraction of Escherichia coli

A heat-stable factor with properties similar to those of calmodulin was found in the fraction containing Ca²⁺-dependent cyclic AMP phosphodiesterase of $\text{Escherichia}\text{coli}$. The factor activated such enzymes as cyclic nucleotide phosphodiesterase of bovine brain, (Ca²⁺,Mg²⁺)ATPase of human erythrocyte menbrane and myosin light chain kinase of rabbit myometrium in a Ca²⁺-dependent fashion with an apparent K_a of 5 × 10^?5$\text{M}$. The factor and brain calmodulin had no effect on the phosphodiesterase of $\text{E.}\text{coli}$. It may be concluded that calmodulin or a calmodulin-like protein occurs in prokaryotes.     

Purification and characterization of human lung calmodulin-independent cyclic AMP phosphodiesterase

A high-affinity calmodulin-independent cyclic AMP phosphodiesterase was purified to homogeneity from human lung tissue. This enzyme has a molecular weight of 60,000, a sedimentation coefficient of 3.2–3.4 S, and an isoelectric pH of 4.6–4.8. Neither Ca²⁺ nor calmodulin (in the presence or absence of added Ca²⁺) stimulates the enzymatic activity. This enzyme appears to be very similar to that described previously from dog kidney (W. J. Thompson, P. M. Epstein, and S. J. Strada, (1979) Biochemistry18, 5228–5237). Hydrolysis of cyclic AMP is greatly enhanced by Mg²⁺ (25–30× at 10 mm Mg²⁺) and Mn²⁺ (20× at 10 mm Mn²⁺). Zn²⁺, Cu²⁺, and Co²⁺ are ineffective at these concentrations. Cyclic AMP is the exclusive substrate with a K_m of 0.7–0.8 μm. The I₅₀ of cyclic GMP is 1 mm using 1 μm cyclic AMP as substrate. In contrast, aminophylline, MIX, and SQ 20009 have I₅₀s of 0.28, 0.021, and 0.001 mm, respectively). The purified enzyme is susceptible to temperature inactivation and protease degradation. Significant (10%) inhibition is seen at 37 °C for 20 min. Trypsin, at 0.1 μg/ml, destroys 50% of the activity in 30 min at 25 °C. Our observations concerning its lability to temperature and proteases coupled with its lack of response to calmodulin suggest this enzyme is a basic catalytic subunit of other cyclic AMP phosphodiesterases present within human lung tissue.     

Partial purification of adenosine 3',5'-cyclic monophosphate phosphodiesterases from rat pancreas in the presence of excess protease inhibitors.

(i) Three forms of cyclic AMP phosphodiesterases (3′,5′-cyclic AMP 5′-nucleotidohydrolase, EC 3.1.4.17), F1, F2-I and F2-II, were partially purified from the soluble fraction of rat pancreas in the presence of excess protease inhibitors by DEAE-cellulose column chromatography and gel filtration and were characterized. (ii) F2-II, which was purified 31-fold, exhibited a single peak of activity on both polyacrylamide-gel electrophoresis and isoelectric focusing. The enzyme had a molecular weight of about 70,000, an isoelectric point of 3.9, and an optimal pH around 8.5 and required Mg²⁺ or Mn²⁺ but not Ca²⁺ for activity. The K_m values of this enzyme for cyclic AMP and cyclic GMP were 1 and 50 μm, respectively, while V values of this enzyme for cyclic AMP and cyclic GMP were 36.1 and 12.6 nmol min^?1 (mg of protein)^?1, respectively. Cyclic GMP competitively inhibited hydrolysis of cyclic AMP by this enzyme. Ro20-1724 [4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone] also inhibited hydrolysis of cyclic AMP competitively, with a K_i value of 1 μm. (iii) Fraction F1, which was purified 10-fold, had a molecular weight of more than 500,000 and required Mg²⁺ for activity. Its K_m values for cyclic AMP were 1 and 5 μm. Its K_m value for cyclic GMP was 45 μm. Fraction F2-I, which was purified 26-fold, had a molecular weight of about 70,000. The ratio of the initial velocity of hydrolysis of cyclic GMP to that of cyclic AMP was 0.5 at a substrate concentration of 1 μm.     

Ca2+/calmodulin-dependent protein kinase in Saccharomyces cerevisiae

Ca²⁺-dependent chromatography of soluble cytosolic proteins on calmodulin-Sepharose gave a fraction that exhibited Ca²⁺- and calmodulin-dependent phosphorylation of several polypeptides, including 60, 56 and 45 kDa species. At 0.2 μM beef calmodulin the phosphorylation was optimal at 3 μM free Ca²⁺, and at 80 μM free Ca²⁺ it was half-maximal at about 0.1 μM beef calmodulin. It is concluded that the fraction contains calmodulin-dependent protein kinase(s) which is (are) autophosphorylated or associated with substrates.     

Isolation of calcium pump system and purification of calcium ion-dependent ATPase from heart muscle

The procedure for the isolation of the highly active fraction of sarcoplasmic reticulum from pigeon and dog hearts is described. The method is based on the partial loading of heart microsomes with calcium and oxalate ions and the precipitation of loaded vesicles in sucrose and potassium chloride concentration gradients. Preparations obtained possess high activity of Ca²⁺-dependent ATPase and are also able to accumulate up to 10 μmol Ca²⁺ per mg protein. Purification of sarcoplasmic reticulum membranes is accompanied by a decrease in concentration of cytochrome a+a₃ and an increase in the content of [³²P]phosphoenzyme. The basic components in “calcium-oxalate preparation” from hearts are proteins with molecular weights of about 100 000 (Ca²⁺-dependent ATPase) and 55 000 Calcium-oxalate preparation from pigeon hearts was used for subsequent purification of Ca²⁺-dependent ATPase. Specific activity of purified enzyme from pigeon hearts is 12–16 μmol P_i/min per mg protein. Enzyme activity of purified Ca²⁺-dependent ATPase is inhibited by EGTA and is not sensitive to azide, 2,4-dinitrophenol and ouabain. The data obtained demonstrate the similarity of calcium pump systems and Ca²⁺-dependent ATPases isolated from heart and skeletal muscles.     

High tyrosine-specific protein kinase activity in normal human peripheral blood lymphocytes

Tyrosine-specific protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) activity was measured in normal human nonadherent peripheral blood lymphocytes using synthetic peptide substrates having sequence homologies with either pp60^src or c-myc. A high level of tyrosine-specific protein kinase activity was found associated with the cell particulate fraction (100 000 × g pellet). High-pressure liquid chromatography and phosphoamino acid analysis of the synthetic peptide substrates substantiated the phosphorylation of tyrosine residues by the particulate fraction enzyme. The human enzyme was also capable of phosphorylating a synthetic random polymer of 80% glutamic acid and 20% tyrosine. Enzyme activity was half-maximal with 22 μM Mg·ATP and had apparent K_m values for the synthetic peptides from 1.9 to 7.1 mM. The enzyme preferred Mg²⁺ to Mn²⁺ for optimal activity and was stimulated 2–5-fold by low levels (0.05%) of some ionic as well as non-ionic detergents including deoxycholate, Nonidet P-40 and Triton X-100. The enzyme activity was not stimulated by N⁶;O^2′-dibutyryl cyclic AMP (100 μM), N⁶;O^2′-dibutyryl cyclic GMP (100 μM), Ca²⁺ (200 μM), insulin (1 μg/ml) or homogeneous human T-cell growth factor (3 μg/ml) under the conditions used. Alkaline-resistant phosphorylation of particulate proteins in vitro revealed protein bands with M_r 59 000 and 54 000 suggesting that there are endogenous substrates for the human lymphocyte tyrosine protein kinase.     

Regulation by a β-adrenergic receptor of a Ca2+-independent adenosine 3′,5′-(cyclic)monophosphate phosphodiesterase in C6 glioma cells

The hormonal control of cyclic nucleotide phosphodiesterase (EC 3.1.4.17) activity has been studied by using as a model the isoproterenol stimulation of cyclic AMP phosphodiesterase activity in C6 glioma cells. A 2-fold increase in cyclic AMP phosphodiesterase specific activity was observed in homogenates of isoproterenol-treated cells relative to control. This increase reached a maximum 3 h after addition of isoproterenol, was selective for cyclic AMP hydrolysis, was reproduced by incubation with 8-Br cyclic AMP but not with 8-Br cyclic GMP and was limited to the soluble enzyme activity. The presence of 0.1 mM EGTA did not alter the magnitude of the increase in phosphodiesterase activity. Moreover, the calmodulin content in the cell extracts was not changed after isoproterernol. DEASE-Sephacel chromatography of the 100 000×g supernatant resolved two peaks of phosphodiesterase activity. The first peak hydrolyzed both cyclic nucleotides and was activated by Ca²⁺ and purified calmodulin. The second peak was specific for cyclic AMP but it was Ca²⁺- and calmodulin-insensitive. Isoproterenol selectively increased the specific activity of the second peak. Kinetic analysis of the cyclic AMP hydrolysis by the induced enzyme reveled a non-linear Hofstee plot with apparent K_m values of 2–5 μM. Cyclic GMP was not hydrolyzed by this enzyme in the absence or presence of calmodulin and failed to affect the kinetics of the hydrolysis of cyclic AMP. Gel filtration chromatography of the induced DEASE-Sephacel peak resolved a single peak of enzyme activity with an apparent molecular weight of 54 000.