A calcium-sensitive fluorescent analog of calmodulin based on a novel calmodulin-binding fluorophore

Structure-activity studies of tetramethinemerocyanine fluorophores enabled the synthesis of novel dyes which showed spectral changes during reversible, calcium-dependent association with calmodulin. These spectral changes were greatly enhanced in dyes with a quaternary nitrogen and specifically placed hydrophobic chains. One such dye was covalently attached to calmodulin, producing a calmodulin analog with calcium-sensitive fluorescence. The analog, MeroCaM, showed a calcium-induced 3.4-fold increase in excitation ratio (608/532 nm excitation, 623 nm emission), which was fully reversed by lowering free calcium levels. MeroCaM's excitation ratio showed a half-maximal change at 300-400 nM calcium, below calcium concentrations reported to produce half-maximal saturation of calcium-calmodulin binding. However, the calcium dependence of MeroCaM's phosphodiesterase activation paralleled that of calmodulin. MeroCaM's fluorescence changes therefore appear to reflect primarily calcium binding to high affinity sites. MeroCaM's maximal phosphodiesterase activation was 30-40% that of calmodulin. In myosin light chain kinase activation, MeroCaM and calmodulin displayed indistinguishable maximal activation levels and concentration dependence of activation. Changes in MeroCaM's calcium affinity induced by magnesium, phosphodiesterase, and melittin were similar to those reported for calmodulin. Experiments with melittin revealed that target protein interaction could alter the fluorescence changes produced by calcium binding. MeroCaM showed promising brightness and photostability when imaged in individual living fibroblasts. The long excitation and emission wavelengths of MeroCaM, and the strong dependence of its excitation ratio on calcium concentrations, suit it well for use as a probe of calmodulin-dependent calcium signaling in living cells, as well as for experiments in vitro.     

Identification of beta-endorphin residues 14-25 as a region involved in the inhibition of calmodulin-stimulated phosphodiesterase activity

The inhibition of the calmodulin-mediated stimulation of bovine brain cyclic nucleotide phosphodiesterase activity (3':5'-cyclic adenosine monophosphate 5'-nucleotidohydrolase, EC 3.1.4.17) by the 31-residue opiate peptide beta-endorphin has been investigated. Using conditions in which porcine brain calmodulin (6 nM) is limiting (i.e., to give a 3-fold, Ca2+-dependent stimulation of enzymic activity toward cyclic guanosine monophosphate), the domain of beta-endorphin responsible for the inhibition was mapped by using a series of deletion peptides. beta-Endorphin exhibited an ED50 of several micromolar under the conditions employed, and several amino-terminal deletion peptides were essentially as inhibitory as the parent peptide. Methionine enkephalin and various carboxy-terminal deletion peptides had no demonstrable effect at concentrations of 100-200 microM. Peptides 1-25 and 1-27 (C' fragment) inhibited the calmodulin-dependent activity of phosphodiesterase, but higher concentrations were required than of beta-endorphin. Studies using combined amino- and carboxy-terminal deletion peptides demonstrate that peptide 14-25 was the shortest peptide examined that was capable of inhibiting calmodulin stimulation of phosphodiesterase activity under the conditions used. There was no evidence to indicate that the amino-terminal region comprising residues 1-13 of beta-endorphin contributes to the measured inhibition of calmodulin-stimulated enzymic activity. The circular dichroic spectra of calmodulin, beta-endorphin, and mixtures of the two were obtained, and the ellipticity of the peptide-protein mixtures at 221 nm exceeded that expected by assuming simple additivity. This finding is consistent with a direct interaction of beta-endorphin with calmodulin which seems to lead to enhanced helicity of one or both components.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium- and calmodulin-dependent phosphorylase kinase activity in porcine uterine smooth muscle

Porcine uterine smooth muscle phosphorylase kinase has been partially purified. The enzyme was activated about 1.5-2.0-fold by exogenous calmodulin. Half maximal stimulation was observed at about 100 nM calmodulin. The activation was dependent on calcium and was maximum at pH 7.5 in the range of pH from 6 to 9. This activation was completely abolished by 100 microM trifluoperazine. The result suggested that unlike slow and cardiac muscles, phosphorylase kinase of uterine smooth muscle showed similar response to calmodulin with that of fast muscle. The physiological role of the calcium and calmodulin-dependent activation of myometrium phosphorylase kinase is briefly discussed.     

Inhibition of a Low Km GTPase Activity in Rat Striatum by Calmodulin

In rat striatum, the activation of adenylate cyclase by the endogenous Ca2+-binding protein, calmodulin, is additive with that of GTP but is not additive with that of the nonhydrolyzable GTP analog, guanosine-5'-(beta, gamma-imido)triphosphate (GppNHp). One possible mechanism for this difference could be an effect of calmodulin on GTPase activity which has been demonstrated to "turn-off" adenylate cyclase activity. We examined the effects of Ca2+ and calmodulin on GTPase activity in EGTA-washed rat striatal particulate fractions depleted of Ca2+ and calmodulin. Calmodulin inhibited GTP hydrolysis at concentrations of 10(-9)-10(-6) M but had no effect on the hydrolysis of 10(-5) and 10(-6) M GTP, suggesting that calmodulin inhibited a low Km GTPase activity. The inhibition of GTPase activity by calmodulin was Ca2+-dependent and was maximal at 0.12 microM free Ca2+. Maximal inhibition by calmodulin was 40% in the presence of 10(-7) M GTP. The IC50 for calmodulin was 100 nM. In five tissues tested, calmodulin inhibited GTP hydrolysis only in those tissues where it could also activate adenylate cyclase. Calmodulin could affect the activation of adenylate cyclase by GTP in the presence of 3,4-dihydroxyphenylethylamine (DA, dopamine). Calmodulin decreased by nearly 10-fold the concentration of GTP required to provide maximal stimulation of adenylate cyclase activity by DA in the striatal membranes. The characteristics of the effect of calmodulin on GTPase activity with respect to Ca2+ and calmodulin dependence and tissue specificity parallel those of the activation of adenylate cyclase by calmodulin, suggesting that the two activities are closely related.(ABSTRACT TRUNCATED AT 250 WORDS)     

Peptide-activator of cyclic nucleotide phosphodiesterases in the rat and human uterus

The presence of the cyclic nucleotide phosphodiesterase peptide-activator previously isolated from the calf myometrium was studied in uterine tissue of rats at different functional states (in puberty, estrus and diestrus stages, pregnancy) and in human myometrium and myoma. The peptide isolated from calf myometrium, immature rat myometrium and uterine tissue in myoma caused a 3-4-fold stimulation of the phosphodiesterase activity.     

Preparation of an enzymatically active cross-linked complex between brain cyclic nucleotide phosphodiesterase and 3-(2-pyridyldithio)propionyl-substituted calmodulin

Cyclic nucleotide phosphodiesterase (0.07 nM) was activated by near stoichiometric concentrations of [3-(2-pyridyldithio)propionyl]calmodulin (PDP-CaM) after initial incubation of these proteins at 200-fold higher concentrations; activity in assays with EGTA was 80% of that in the presence of Ca2+. The enzyme incubated with native calmodulin under identical conditions required approximately 1 nM for half-maximal activation, and no activation was observed in the absence of calcium. These data suggested formation of a covalent complex between phosphodiesterase and PDP-CaM. On high-performance gel-permeation chromatography in the presence of metal chelators, the complex appeared considerably larger than the native enzyme. Incubation of phosphodiesterase with the thiolated (inactivated) form of PDP-CaM did not change its chromatographic behavior, indicating that reactive sulfhydryl groups were involved in complex formation. Although the total activities recovered from chromatography were not significantly different, maximal activation of PDP-CaM-phosphodiesterase complex was only approximately 20%, whereas the control enzyme was activated 6-8-fold by Ca2+ plus calmodulin. Kinetics of cGMP hydrolysis in the presence of EGTA by the isolated complex differed from those of control enzyme but were indistinguishable from those of control enzyme assayed with saturating Ca2+ and CaM. The calmodulin antagonists W-7 and trifluoperazine had relatively little effect on activity of the PDP-CaM-phosphodiesterase complex. Incubation of the complex with dithiothreitol dramatically increased its Ca2+ and calmodulin responsiveness, suggesting that reduction of the disulfide cross-link released phosphodiesterase from the complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential Regulation by Calmodulin of Basal, GTP-, and Dopamine-Stimulated Adenylate Cyclase Activities in Bovine Striatum

The concentration requirements of calmodulin in altering basal, GTP-, and dopamine-stimulated adenylate cyclase activities in an EGTA-washed particulate fraction from bovine striatum were examined. In the bovine striatal particulate fraction, calmodulin activated basal adenylate cyclase activity 3.5-fold, with an EC50 of 110 nM. Calmodulin also potentiated the activation of adenylate cyclase by GTP by decreasing the EC50 for GTP from 303 +/- 56 nM to 60 +/- 10 nM. Calmodulin did not alter the maximal response to GTP. The EC50 for calmodulin in potentiating the GTP response was only 11 nM as compared to 110 nM for activation of basal activity. Similarly, calmodulin increased the maximal stimulation of adenylate cyclase by dopamine by 50-60%. The EC50 for calmodulin in eliciting this response was 35 nM. These data demonstrate that calmodulin can both activate basal adenylate cyclase and potentiate adenylate cyclase activities that involve the activating GTP-binding protein, Ns. Mechanisms that involve potentiation of Ns-mediated effects are much more sensitive to calmodulin than is the activation of basal adenylate cyclase activity. Potentiation of GTP-stimulated adenylate cyclase activity by calmodulin was apparent at 3 and 5 mM MgCl2, but not at 1 or 10 mM MgCl2. These data further support a role for calmodulin in hormonal signalling and suggest that calmodulin can regulate cyclic AMP formation by more than one mechanism.     

Calmodulin activates bovine-cardiac myosin light-chain kinase by increasing the affinity for myosin light-chain 2

The basic mechanism by which calmodulin activates bovine-cardiac muscle myosin light-chain kinase was investigated using highly purified preparations of mixed bovine-cardiac myosin light chains or isolated myosin light chain 2. The apparent contamination of these substrate proteins by calmodulin, as detected by activation of calmodulin-sensitive phosphodiesterase, was less than 4 parts/million and was undetectable by antibodies against calmodulin. The apparent KA for calmodulin was 2 nM and 20 nM in the presence of isolated myosin light-chain 2 and mixed myosin light chains, respectively. Purified bovine cardiac troponin C activated myosin light-chain kinase by about 10% at a concentration of 2 microM. Mixed myosin light chains were phosphorylated in the absence and presence of calmodulin and in the presence of calcium with a V of 11.1 and 11.0 mumol phosphate transferred min-1 (mg enzyme)-1, respectively. The apparent Km values for mixed myosin light chains were 8.0 and 0.35 mg/ml in the absence and presence of calmodulin, respectively. Similarly calmodulin lowered the Km value for isolated myosin light-chain 2 over 20-fold and increased the V value only about 1.5-fold. Activity observed in the absence of calmodulin was dependent on the presence of calcium and was suppressed by chelating free calcium either before or during a phosphorylation reaction. The apparent KA for calcium was 1.2 microM and 0.4 microM in the absence and presence of calmodulin. Activity in the absence of calmodulin was inhibited at very high concentrations of the 'specific' calmodulin antagonists W-7, trifluoperazine and R24571 with apparent IC50 values of 0.3 mM, 0.2 mM and 0.02 mM. Antibiotics raised against calmodulin suppressed completely the kinase activity in the presence of calmodulin but had no effect on the activity measured in its absence. These results suggest that calmodulin stimulates the activity of bovine-cardiac myosin light-chain kinase by increasing over 20-fold the affinity for its substrate myosin light-chain 2.     

Biochemical events associated with activation of smooth muscle contraction

Biochemical events associated with activation of smooth muscle contraction were studied in neurally stimulated bovine tracheal smooth muscle. A latency period of 500 ms preceded increases in isometric force and myosin light chain phosphorylation. However, stimulation resulted in the rapid hydrolysis of inositol phospholipids as demonstrated by increases in inositol phosphates by 500 ms. Inositol trisphosphate increased 2-fold with no significant change in inositol tetrakisphosphate. The apparent activation state of myosin light chain kinase was assessed indirectly through measurements of the fractional activation of a second calmodulin-dependent enzyme, cyclic nucleotide phosphodiesterase. The fractional activation of cyclic nucleotide phosphodiesterase increased after neural stimulation to a maximal extent by 500 ms and remained at this level for at least 4 s. The monophosphorylation of myosin light chain increased after 500 ms and reached a maximum value by 2 s. Diphosphorylation also occurred but to a much lesser extent. Fractional activation of cyclic nucleotide phosphodiesterase and myosin light chain phosphorylation both decreased after 10 min continuous stimulation, although the force response remained at a maximal level. These observations demonstrate that inositol trisphosphate formation and activation of cyclic nucleotide phosphodiesterase (and hence most likely myosin light chain kinase) by calmodulin precede myosin light chain phosphorylation and that these events are sufficiently rapid to mediate the contractile response of neurally stimulated tracheal smooth muscle.     

Ca2+-calmodulin-activated cyclic nucleotide phosphodiesterase from the rabbit myometrium

Two forms of soluble phosphodiesterase of cyclic nucleotides separating by DEAE-cellulose ion-exchange chromatography and not only differing in physicochemical and catalytic parameters but also differently regulated by calmodulin are found in the doe myometrium. Calmodulin with 10(-7)-10(-5) M concentrations of Ca2+ promotes the two-fold activation of the 3':5'-AMP (but not of 3':5'-GMP) hydrolysis by the first form of phosphodiesterase. Trifluoperazine (10 microM) lowers the activating action of calmodulin. The second form of soluble phosphodiesterase is not sensitive to the action of both calmodulin and Ca2+. 3':5'-GMP (10 microM) inhibits the 3':5'-AMP hydrolysis by the first form of phosphodiesterase; calmodulin exerts no effect on this process. The data obtained testify to the possible participation of Ca2+ and calmodulin in Ca2+-calmodulin-dependent phosphodiesterase regulation of the content of cyclic nucleotides (3':5'-AMP, in particular) in the doe myometrium.     

Plasma membrane Ca(2+) pump isoform 3f is weakly stimulated by calmodulin

Isoform 3f of the plasma membrane Ca(2+) pump is a major isoform of this pump in rat skeletal muscle. It has an unusual structure, with a short carboxyl-terminal regulatory region of only 33 residues when compared with the 77 to 124 residues found in the other isoforms. Also, whereas the regulatory regions of the other isoforms, downstream of the alternative splice, consist of two homologous groups, the sequence of 3f is not related to either group. A synthetic peptide representing the calmodulin binding domain of isoform 3f had a much lower calmodulin affinity (with a K(d) of 15 nM) than the corresponding peptide of isoform 2b (K(d) value was 0.2 nM). The characteristics of this domain were further studied by making chimeras of the 3f regulatory region with the catalytic core of isoform 4 and by making the full-length isoform 3f. Both constructs bound to calmodulin-Sepharose. The chimera was fully active without calmodulin, showing no stimulation of activity when calmodulin was added. The full-length isoform 3f was slightly activated by calmodulin. These data show that the regulatory region of isoform 3f is only a weak autoinhibitor of the enzyme, in contrast to the properties of all the other isoforms studied so far. Rather, this isoform is a special-purpose, constitutively active form of the enzyme, expressed primarily in skeletal muscle and as a minor isoform in brain.     

Substrate selectivity and sensitivity to inhibition by FK506 and cyclosporin A of calcineurin heterodimers composed of the alpha or beta catalytic subunit.

The calcineurin (CaN) alpha and beta catalytic subunit isoforms are coexpressed within almost all cell types. The enzymatic properties of CaN heterodimers comprised of the regulatory B subunit (CnB) with either the alpha or beta catalytic subunit were compared using in vitro phosphatase assays. CaN containing the alpha isoform (CnA alpha) has lower K(m) and higher V(max) values than CaN containing the beta isoform (CnA beta) toward the PO4-RII, PO4-DARPP-32(20-38) peptides, and p-nitrophenylphosphate (pNPP). CaN heterodimers containing the alpha or beta catalytic subunit isoform displayed identical calmodulin dissociation rates. Similar inhibition curves for each CaN heterodimer were obtained with the CaN autoinhibitory peptide (CaP) and cyclophilin A/cyclosporin A (CyPA/CsA) using each peptide substrate at K(m) concentrations, except for a five- to ninefold higher IC50 value measured for CaN containing the beta isoform with p-nitrophenylphosphate as substrate. No difference in stimulation of phosphatase activity toward p-nitrophenylphosphate by FKBP12/FK506 was observed. At low concentrations of FKBP12/FK506, CaN containing the alpha isoform is more sensitive to inhibition than CaN containing the beta isoform using the phosphopeptide substrates. Higher concentrations of FKBP12/FK506 are required for maximal inhibition of beta CaN using PO4-DARPP-32(20-38) as substrate. The functional differences conferred upon CaN by the alpha or beta catalytic subunit isoforms suggest that the alpha:beta and CaN:substrate ratios may determine the levels of CaN phosphatase activity toward specific substrates within tissues and specific cell types. These findings also indicate that the alpha and beta catalytic subunit isoforms give rise to substrate-dependent differences in sensitivity toward FKBP12/FK506.     

Characterization of the phosphatase activity of a baculovirus-expressed calcineurin A isoform.

Calcineurin A was purified by calmodulin-Sepharose affinity chromatography from Sf9 cells infected with recombinant baculovirus containing the cDNA of a rat calcineurin A isoform. The Sf9-expressed calcineurin A has a low basal phosphatase activity in the presence of EDTA (0.9 nmol/min/mg) which is stimulated 3-5-fold by Mn2+. Calmodulin increased the Mn2+ stimulated activity 3-5-fold. Bovine brain calcineurin B increased the A subunit activity 10-15-fold, and calmodulin further stimulated the activity of reconstituted A and B subunits 10-15-fold (644 nmol/min/mg). The Km of calcineurin A for 32P-RII pep (a peptide substrate (DLDVPIPGRFDRRVSVAAE) for CaN), was 111 microM with or without calmodulin, and calmodulin increased the Vmax about 4-fold. The Km of reconstituted calcineurin A plus B for 32P-RII pep was 20 microM, and calmodulin increased the Vmax 18-fold without affecting the Km. CaN A467-492, a synthetic autoinhibitory peptide (ITSFEEAKGLDRINERMPPRRDAMP) from calcineurin, inhibited the Mn2+/calmodulin-stimulated activities of the reconstituted enzyme and the A subunit with IC50's of 25 microM and 90 microM, respectively. The reconstitution of the phosphatase activity of an expressed isoform of calcineurin A by purified B subunit and calmodulin may facilitate comparative studies of the regulation of calcineurin A activity by the B subunit and calmodulin.     

Characterization of A-kinase-anchoring disruptors using a solution-based assay

Subcellular localization of PKA (cAMP-dependent protein kinase or protein kinase A) is determined by protein-protein interactions between its R (regulatory) subunits and AKAPs (A-kinase-anchoring proteins). In the present paper, we report the development of the Amplified Luminescent Proximity Homogeneous Assay (AlphaScreen) as a means to characterize AKAP-based peptide competitors of PKA anchoring. In this assay, the prototypic anchoring disruptor Ht31 efficiently competed in RIIalpha isoform binding with RII-specific and dual-specificity AKAPs (IC50 values of 1.4+/-0.2 nM and 6+/-1 nM respectively). In contrast, RIalpha isoform binding to a dual-specific AKAP was less efficiently competed (IC50 of 156+/-10 nM). Characterization of two RI-selective anchoring disruptors, RIAD (RI-anchoring disruptor) and PV-38 revealed that RIAD (IC50 of 13+/-1 nM) was 20-fold more potent than PV-38 (IC50 of 304+/-17 nM) and did not compete in the RIIalpha-AKAP interaction. We also observed that the kinetics of RII displacement from pre-formed PKA-AKAP complexes and competition of RII-AKAP complex formation by Ht31 differed by an order of magnitude when the component parts were mixed in vitro. No such difference in potency was seen for RIalpha-AKAP complexes. Thus the AlphaScreen assay may prove to be a valuable tool for detailed characterization of a variety of PKA-AKAP complexes.     

Inhibition of Calmodulin-Stimulated Phosphodiesterase Activity by Vasoactive Intestinal Peptide

The effects of certain peptides of the glucagon family on calmodulin activity were determined from their capacity to inhibit a calmodulin-dependent form of phosphodiesterase. Vasoactive intestinal peptide and secretin were potent inhibitors of calmodulin activity, having IC50 values of 0.5 microM and 2 microM, respectively. By contrast, glucagon failed to inhibit calmodulin activity even at concentrations of 100 microM. None of these compounds significantly inhibited the basal activity of phosphodiesterase at concentrations up to 100 microM. These findings support the suggestion that important structural features of peptides for anticalmodulin activity include a net positive charge and a hydrophobic surface.     

Stimulatory effect of vasoactive intestinal peptide (VIP) on cyclic AMP production in rat peritoneal macrophages.

Vasoactive intestinal peptide (VIP) stimulated cyclic AMP production in rat peritoneal macrophages. The stimulatory effect of VIP was dependent on time, temperature and cell concentration, and was potentiated by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). At 15 degrees C, the response occurred in the 0.1-1000 nM range of VIP concentrations. Half maximal stimulation of cellular cyclic AMP (ED50) was obtained at 1.2 +/- 0.5 nM VIP, and maximal stimulation (about 3-fold basal level) was obtained between 100-1000 nM. The cyclic AMP system of rat peritoneal macrophages showed a high specificity for VIP. The order of potency observed in inducing cyclic AMP production was VIP greater than rGRF greater than hGRF greater than PHI greater than secretin. Glucagon, insulin, pancreastatin and octapeptide of cholecystokinin did not modify cyclic AMP levels at concentrations as high as 1 microM. The beta-adrenergic agonist isoproterenol increased the cyclic AMP production and show additive effect with VIP. Somatostatin inhibits the accumulation of cyclic AMP in the presence of both vasoactive intestinal peptide and isoproterenol. The finding of a VIP-stimulated cyclic AMP system in rat peritoneal macrophages, together with the previous characterization of high-affinity receptors for VIP in the same cell preparation, strongly suggest that VIP may be involved in the regulation of macrophage function.     

Identification and characterization of a Ca2+-calmodulin-sensitive cyclic nucleotide phosphodiesterase in a human lymphoblastoid cell line.

This study examines the pattern and regulatory properties of cyclic nucleotide phosphodiesterases in a human lymphoblastoid B-cell line (RPMI 8392) established from a patient with acute lymphocytic leukaemia. In this cell line, phosphodiesterase activity measured at 0.25 microM-cyclic AMP is approx. 7-fold greater than that in isolated human peripheral-blood lymphocytes, and 16% of the phosphodiesterase activity in RPMI 8392 cells is associated with particulate fractions. Phosphodiesterase activity in crude fractions of this cell line is reproducibly stimulated by about 60-80% by Ca2+-calmodulin. In the presence of 20 nM-calmodulin, half-maximal stimulation occurs at 0.7 microM-Ca2+. The cytosolic phosphodiesterase activity of RPMI 8392 cells is separated into two forms by DEAE-Sephacel chromatography. The first form is eluted at approx. 0.2 M-sodium acetate, catalyses the hydrolysis of both cyclic AMP and cyclic GMP, and is stimulated 3-fold by Ca2+-calmodulin. This form exhibits non-linear kinetics for cyclic AMP in the absence of calmodulin, with extrapolated Km values of 0.8 and 4 microM, and non-linear kinetics in the presence of calmodulin, with extrapolated Km values of 0.5 and 1 microM. The Vmax. values are increased approx. 3-fold by calmodulin. The second form is eluted at approx. 0.6 M-sodium acetate, is specific for cyclic AMP, and insensitive to stimulation by Ca2+-calmodulin. The Ca2+-calmodulin-sensitive phosphodiesterase from the DEAE-Sephacel column can be adsorbed to a calmodulin-Sepharose affinity column and eluted with EGTA. This enzymic activity can also be immunoprecipitated by a monoclonal antibody directed against a calmodulin-bovine heart phosphodiesterase complex. This study documents the existence of Ca2+-calmodulin-sensitive phosphodiesterase in a cultured lymphoblastoid cell line derived from a leukaemic patient.     

Calcium- and calmodulin-independent modulation of calmodulin-sensitive hypothalamic cyclic nucleotide phosphodiesterase activity by the (11–19) fragment of thymosin β4

A fragment (11–19) of thymosin 4 was found to stimulate phosphodiesterase activity even in the absence of calcium and calmodulin. Half-maximal enzyme activation occurred with 10 nM peptide, and was further increased by phospholipids such as phosphatidylserine. The mechanism of stimulation is an increase in the V_max of cAMP degradation without a substantial change in the K_m for the substrate. In the presence of calcium ions and calmodulin the peptide was also stimulatory.     

Allosteric regulation of Ca2+-calmodulin-dependent phosphodiesterase activity from the bovine brain

The Ca2+-calmodulin-dependent interaction of phosphodiesterase with phenyl-Sepharose was demonstrated. BSA caused incomplete competitive inhibition of phosphodiesterase activation by calmodulin. The 17-fold increase of the constant for phosphodiesterase activation by calmodulin was accompanied by an insignificant rise in the maximum rate of cAMP hydrolysis; in this case the value of the inhibition constant amounted to Ki approximately 6 microM. In the absence of calmodulin saturating concentrations of BSA reduced the enzyme activity nearly 3-4-fold. The effect of BSA on phosphodiesterase was incompetitive with respect to cAMP (Ki approximately 1.4 microM). Both phenomena are characteristic of incompetitive binding of BSA to the enzyme with respect to cAMP and calmodulin. Gel filtration data reflect the changes in the enzyme molecular weight during its interaction with BSA. All the above reactions of the enzyme are reversible.     

Purification and characterization of myosin light-chain kinase from porcine myometrium and its phosphorylation and modulation by cyclic AMP-dependent protein kinase

Myosin light-chain kinase was purified from porcine myometrium to apparent homogeneity at about 262-fold with an Mr of 130 000 as determined by SDS-polyacrylamide gel electrophoresis and a sedimentation coefficient of 4.5 S. The approximate content of the soluble myosin light-chain kinase was estimated to be about 0.85 microM. The purified enzyme exhibited strict substrate specificity only for 20-kDa myosin light chain and Ka values of 0.6 nM and 0.3 microM for calmodulin and Ca2+, respectively. The enzyme was phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase, which resulted in a decrease in the affinity for calmodulin of 4-7-fold without effect on the Vmax. The maximal amount of phosphate incorporated into the enzyme was 0.5-0.8 and 1.0-1.4 mol per mol of the enzyme in the presence and absence of Ca2+ and calmodulin, respectively. In the presence of a subsaturating concentration of calmodulin, the enzyme showed a lower sensitivity for Ca2+ by phosphorylation.