Modulation of an Intracellular Calmodulin-Stimulated Ca2+-Pumping ATPase in Cauliflower by Trypsin (The Use of Calcium Green-5N to Measure Ca2+ Transport in Membrane Vesicles)

The effect of controlled trypsin digestion of a calmodulin-stimulated Ca2+-ATPase in low-density intracellular membranes from cauliflower (Brassica oleracea L.) inflorescences was investigated. Ca2+ uptake into vesicles was measured either continuously with the fluorescent Ca2+ indicator Calcium Green-5N or with a radio-active filter technique. Trypsin treatment of vesicles resulted in a 3-fold activation of Ca2+ uptake and loss of calmodulin sensitivity. Immunoblotting experiments with an antiserum raised against the Ca2+-ATPase showed that the trypsin activation was accompanied by a decrease in the amount of intact Ca2+-ATPase (111 kD) and by successive appearances of polypeptides of 102 and 99 to 84 kD. 125I-Calmodulin overlays showed that only the intact Ca2+-ATPase bound calmodulin. Removal of the calmodulin-binding domain (about 9 kD) was not enough to obtain full activation. Trypsin proteolysis resulted in a Ca2+ concentration necessary for half-maximal activity of 0.5 [mu]M, whereas a value of about 2 [mu]M was obtained with untreated membranes in the presence of calmodulin. Without trypsin treatment or calmodulin the activity was not saturated even at 57 [mu]M free Ca2+. The data suggest that trypsin digestion and calmodulin activate the cauliflower Ca2+-ATPase by at least partly different mechanisms.     

Activation of the Ca2+-ATPase of human erythrocyte membrane by an endogenous Ca2+-dependent neutral protease

Limited proteolysis of the plasma membrane calcium transport ATPase (Ca2+-ATPase) from human erythrocytes by trypsin produces a calmodulin-like activation of its ATP hydrolytic activity and abolishes its calmodulin sensitivity. We now demonstrate a similar kind of activation of the human erythrocyte membrane Ca2+-ATPase by calpain (calcium-dependent neutral protease) isolated from the human red cell cytosol. Upon incubation of red blood cell membranes with purified calpain in the presence of Ca2+ the membrane-bound Ca2+-ATPase activity was increased and its sensitivity to calmodulin was lost. In contrast to the action of other proteases tested, proteolysis by calpain favors activation over inactivation of the Ca2+-ATPase activity, except at calpain concentrations more than 2 orders of magnitude higher. Exogenous calmodulin protects the Ca2+-ATPase against calpain-mediated activation at concentrations which also activate the Ca2+-ATPase activity. Calcium-dependent proteolytic modification of the Ca2+-ATPase could provide a mechanism for the irreversible activation of the membrane-bound enzyme.     

Heat-stable calmodulin-binding protein in rat testis. Inhibition of calmodulin-stimulated cyclic nucleotide phosphodiesterase activity

An inhibitor of procine brain calmodulin-dependent cyclic nucleotide phosphodiesterase was purified about 940-fold from rat testis. This inhibitor inhibited the calmodulin-induced activation of the enzyme without affecting its basal activity. The inhibitor activity was counteracted by a high concentration of calmodulin, but was not by a high concentration of Ca2+. The analysis on polyacrylamide disc gel electrophoresis demonstrated that the inhibitor and calmodulin form a complex in the presence of Ca2+ but not in the presence of excess amount of EGTA. This inhibitor also inhibited the calmodulin-induced activation of Ca2+, Mg2+ -ATPase of human erythrocytes. The inhibitor appeared to be a heat-stable protein, since the inhibitor activity was not attenuated by boiling up to 9 min but was completely abolished by tryptic or chymotryptic digestion. The molecular weights of the inhibitor determined by linear polyacrylamide gradient gel electrophoresis under nondenaturing conditions and sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 40,000 and 32,000, respectively. Thus, the inhibitor is suggested to be a calmodulin-binding protein composed of a monomer which has unique properties different from those of other tissues.     

Calmodulin. An activator of human erythrocyte (Ca2+ + Mg2+)ATPase phosphorylation

The effect of purified calmodulin on the calcium-dependent phosphorylation of human erythrocyte membranes was studied. Under the conditions employed, only one major peak of phosphorylation was observed when solubilized membrane proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of this phosphorylated protein band was estimated to be 130000 and in the presence of purified red blood cell calmodulin, the rate of phosphorylation of this band was increased. These data suggest that calmodulin activation of (Ca2+ + Mg2+)-ATPase could be a partial reflection of an increased rate of phosphorylation of the (Ca2+ + Mg2+)-ATPase of human erythrocyte membranes.     

Characterization of a (Ca2+ + Mg2+)-ATPase activator bound to human erythrocyte membranes

Incubation of human erythrocyte ghosts with an equal volume of 0.2 mM EDTA in isotonic KCl decreased both the activity and Ca2+ sensitivity of the (Ca2+ + Mg2+)-ATPase remaining associated with the membrane. Readdition of the EDTA-extract activated the (Ca2+ + Mg2+)-ATPase activity. The activator activity was trypsin sensitive, heat stable and retained by a phenothiazine affinity column, consistent with properties expected of calmodulin. However, unlike calmodulin, the activity was not retained by DEAE Sephadex A-50 and it eluted from Sephacryl S-200 as heterogeneous peaks of activator activity of apparent molecular weight between 107,000 and 178,000. Nevertheless, the activator in the EDTA extract both before and after gel filtration contained calmodulin, as determined by radioimmunoassay and by its activation of calmodulin - deficient phosphodiesterase. SDS-gel electrophoresis of the activator isolated by gel filtration showed a protein of Mr 56,000 in addition to a low molecular weight protein corresponding to calmodulin. It is suggested that the red cell membrane contains a calmodulin binding protein which tightly binds calmodulin as a polymeric complex in a Ca2+-independent manner.     

Purification and Properties of ent-Kaurene Synthase B from Immature Seeds of Pumpkin

ent-Kaurene synthase B (KSB) was purified 291-fold from a crude enzyme preparation from endosperm of pumpkin (Cucurbita maxima L.). Separation of ent-kaurene synthase A and KSB was achieved by hydrophobic interaction chromatography. The fractions containing KSB activity were further purified by diethylaminoethyl, phenyl, and hydroxyapatite column chromatography. Using sodium dodecyl phosphate-polyacrylamide gel electrophoresis, the purest enzyme preparation showed a major band at an apparent molecular mass of 81 kD. The amount of protein in this band was correlated with KSB activity after diethylaminoethyl and hydroxyapatite chromatography. The N terminus of the 81-kD protein was blocked. Therefore, the protein was partially digested with protease and the amino acid sequences of the resulting major peptide fragments were analyzed. A polyclonal antibody was raised against a synthetic peptide based on the longest peptide fragment combined with a keyhole limpet hemocyanin. The antibody recognized only the 81-kD denatured protein and not the native KSB. The properties of KSB were examined using the phenyl-purified enzyme preparation. The Km value for copalyl pyrophosphate was 0.35 [mu]M, and the optimal pH was 6.8 to 7.5. The KSB activity required divalent cations such as Mg2+, Mn2+, and Co2+, whereas Cu2+, Ca2+, and Ba2+ inhibited the activity.     

The effect of calmodulin on the interaction of carbodiimides with the purified human erythrocyte (Ca2+ + Mg2+)-ATPase

The activity of the solubilized and purified (Ca2+ + Mg2+)-ATPase from human erythrocyte membranes was inhibited by N,N'-dicyclohexylcarbodiimide in a concentration-dependent manner. The carbodiimide prevented formation of the phosphorylated intermediate during the catalytic cycle of the enzyme. Treatment of the enzyme with N,N'-dicyclohexyl[14C]carbodiimide resulted in the formation of a 14C-labelled polypeptide corresponding to the enzyme monomer (molecular weight 136,000). The tryptic fragmentation of this 14C-labelled enzyme resulted in the formation of three major 14C-labelled fragments with molecular weights of 58,000, 36,500 and 23,000, the latter two probably representing transmembrane and calmodulin-binding domains of the enzyme, respectively. In the absence of calmodulin, 6.7 molecules of N,N'-dicyclohexyl[14C]carbodiimide covalently bound to each molecule of Ca2+-ATPase; in the presence of calmodulin, the number of molecules of carbodiimide bound was 13.1. The binding of N,N'-dicyclohexylcarbodiimide to the (Ca2+ + Mg2+)-ATPase greatly reduced its ability to bind to a calmodulin-agarose gel.     

Purification and characterization of a myosin heavy chain kinase from Dictyostelium discoideum

A Dictyostelium discoideum myosin heavy chain kinase has been purified 14,000-fold to near homogeneity. The enzyme has a Mr = 130,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and greater than 700,000 as determined by gel filtration on Bio-Gel A-1.5m. The enzyme has a specific activity of 1 mumol/min X mg when assayed at a Dictyostelium myosin concentration of 0.3 mg/ml. A maximum of 2 mol of phosphate/mol of myosin is incorporated by the kinase, and the phosphorylated amino acid is threonine. Phosphate is incorporated only into the myosin heavy chains, not into the light chains. The actin-activated Mg2+-ATPase of Dictyostelium myosin is inhibited 70-80% following maximal phosphorylation with the kinase. The myosin heavy chain kinase requires 1-2 mM Mg2+ for activity and is most active at pH 7.0-7.5. The activity of the enzyme is not significantly altered by the presence of Ca2+, Ca2+ and calmodulin, EGTA, cAMP, or cGMP. When incubated with Mg2+ and ATP, phosphate is incorporated into the myosin heavy chain kinase, perhaps by autophosphorylation.     

Purification and characterization of phosphatidylinositol-specific phospholipase C from bovine platelets

Phosphatidylinositol-specific phospholipase C was purified to homogeneity from soluble fraction of bovine platelets by ammonium sulfate fractionation, hydrophobic chromatography, DEAE ion exchange chromatography and gel filtration. The purified enzyme has a narrow pH optimum ranging from 6.5 to 7.5 and the molecular weight of the enzyme was estimated to be 143,000 by sodium dodecyl sulfate slab gel electrophoresis. The purified enzyme requires Ca2+ strictly for activity, which was markedly enhanced in the presence of arachidonate. No enhancement of the activity was observed in the presence of purified calmodulin. The activity was markedly inhibited in the presence of quinacrine but no inhibition by indomethacin was observed.     

Calmodulin-mediated regulation of calcium transport and (Ca2+ + Mg2+)-activated ATPase activity in isolated cardiac sarcoplasmic reticulum

A severalfold activation of calcium transport and (Ca2+ + Mg2+)-activated ATPase activity by micromolar concentrations of calmodulin was observed in sarcoplasmic reticulum vesicles obtained from canine ventricles. This activation was seen in the presence of 120 mM KCl. The ratio of moles of calcium transported per mol of ATP hydrolyzed remained at about 0.75 when calcium transport and (Ca2+ + Mg2+)-activated ATPase activity were measured in the presence and absence of calmodulin. Thus, the efficiency of the calcium transport process did not change. Stimulation of calcium transport by calmodulin involves the phosphorylation of one or more proteins. The major 32P-labeled protein, as determined by sodium dodecyl sulfate slab gel electrophoresis, was the 22,000-dalton protein called phospholamban. The Ca2+ concentration dependency of calmodulin-stimulated microsomal phosphorylation corresponded to that of calmodulin-stimulated (Ca2+ + Mg2+)-activated ATPase activity. Proteins of 11,000 and 6,000 daltons and other proteins were labeled to a lesser extent. A similar phosphorylation pattern was obtained when microsomes were incubated with cAMP-dependent protein kinase and ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. Phosphorylation produced by added cAMP-dependent protein kinase and calmodulin was additive. These studies provided further evidence for Ca2+-dependent regulation of calcium transport by calmodulin in sarcoplasmic reticulum that could play a role in the beat-to-beat regulation of cardiac relaxation in the intact heart.     

Effect of acylphosphatase on human erythrocyte membrane Ca2(+)-ATPase

We studied the effect of human acylphosphatase on the activity of human erythrocyte membrane Ca2(+)-ATPase. Both the acylphosphatase that is contained in hemolysate and the purified enzyme isolated from red blood cells were able to stimulate Ca2(+)-ATPase activity in erythrocyte membranes. Given the same acylphosphatase activity, however, the hemolysate showed higher stimulatory effect than the purified enzyme. Acylphosphatase stimulation was additive to that induced by calmodulin, thus indicating that acylphosphatase acts in a calmodulin-independent manner. Trifluoperazine, a calmodulin antagonist, did not inhibit acylphosphatase-induced stimulation of Ca2(+)-ATPase activity. Acylphosphatase significantly decreased the rate of Ca2+ influx into inside-out erythrocyte membrane vescicles, thus acting as Ca2+ pump inhibitor. Taken together these findings indicate that acylphosphatase is a soluble, non-calmodulin activator of erythrocyte membrane Ca2(+)-ATPase and might be involved in the control of calcium transport across the plasma membrane.     

Calmodulin and Ca2+- and Calmodulin-Dependent Protein Kinase in Rat Anterior Pituitary Gland

Calmodulin and Ca2+- and calmodulin-dependent protein kinase were identified in the rat anterior pituitary gland. The concentration of calmodulin was 1.18 +/- 0.11 microgram/mg protein (n = 7) in the cytosol fraction. The calmodulin of the anterior pituitary gland co-migrated with brain calmodulin on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The Ka value of the partially purified enzyme for Ca2+ was 3.3 microM in the presence of 0.30 microM calmodulin. Trifluoperazine and chlorpromazine, calmodulin-interacting agents, inhibited enzyme activity, with Ki values of 1.3 and 2.6 X 10(-5) M, respectively. The enzyme was resolved into two peaks of activity, with sedimentation coefficients of 5.5 S and 16.5 S, by sucrose density gradient centrifugation. At least nine proteins were phosphorylated by the enzyme in a Ca2+- and calmodulin-dependent manner. In light of these results, the possibility that calmodulin and the calmodulin-activatable protein kinase system are involved in the mediation of the Ca2+ effect on hormone release from the anterior pituitary gland must be given consideration.     

Purification and partial characterization of a calmodulin-stimulated nucleoside triphosphatase from pea nuclei

A nucleoside triphosphatase/deoxynucleoside triphosphatase associated with the chromatin fraction from a highly purified preparation of pea nuclei has been isolated and characterized. The purified enzyme has a molecular weight of 47,000 as checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and it has an isoelectric point of 6.6. In the presence of divalent cations (Mg2+ = Mn2+ greater than Ca2+), this enzyme hydrolyzes nucleoside triphosphates or deoxynucleoside triphosphates. Hydrolysis is optimal at pH 7.5 and is significantly inhibited by relatively low concentrations of quercetin, but is not sensitive to vanadate, nitrate, or oligomycin. The enzyme has a rather broad nucleotide substrate specificity and has a Km for MgATP2- of 0.6 mM. The enzyme activity is stimulated over 3-fold by Ca2+ and calmodulin, and the stimulation is blocked by the Ca2+ chelator EGTA and by the calmodulin antagonists compound 48/80 and chlorpromazine.     

Activation of the erythrocyte Ca2+-ATPase by either self-association or interaction with calmodulin

The octaethyleneglycol mono-n-dodecyl ether solubilized Ca2+-ATPase purified from human erythrocytes has been studied to determine the physical mechanism of its activation by calmodulin. The dependence of Ca2+-ATPase activity on the enzyme concentration shows a transformation from a calmodulin-dependent to a fully active calmodulin-independent form. The transformation is cooperative with a half-maximal activation at 10-20 nM enzyme. This suggests that at higher enzyme concentrations interactions between Ca2+-ATPase polypeptide chains substitute for calmodulin-enzyme interactions, resulting in activation. In support of this interpretation, the inclusion of higher octaethyleneglycol mono-n-dodecyl ether concentrations shifts the half-maximal transformation to higher enzyme concentrations. Regardless of the detergent concentration, calmodulin decreases by about 2-fold the enzyme concentration required to observe half-maximal Ca2+-ATPase activation, without affecting the maximal velocity or cooperativity. This indicates that calmodulin facilitates interactions between enzyme molecules. The fluorescein-5'-isothiocyanate-modified Ca2+-ATPase shows an increase in fluorescence polarization which occurs over the same narrow concentration range that is seen with the Ca2+-ATPase activity, confirming association of enzyme molecules. Stimulation of the Ca2+-ATPase activity by calmodulin has revealed a stoichiometry of 0.73, with a dissociation constant of 1.6 nM calmodulin. We have demonstrated by use of calmodulin-Sepharose chromatography that both the calmodulin-dependent and independent Ca2+-ATPase forms bind calmodulin, even though stimulation of activity is seen only with the former one. Our data suggest the following two mechanisms for the Ca2+-ATPase activation: self-association of enzyme molecules or interaction with calmodulin.     

Partial Purification of a Na+ -ATPase from the Plasma Membrane of the Marine Alga Heterosigma akashiwo

A Na+ -ATPase was partially purified from plasma membranes of the marine alga Heterosigma akashiwo. The plasma membranes of H. akashiwo cells were collected by differential centrifugation with subsequent discontinuous gradient centrifugation. Na+ -ATPase activity was associated with the resultant plasma membrane fraction and was stimulated to the greatest extent in the presence of 100 to 200 mM Na+, 10 mM K+, and 5 mM Mg2+ ions, pH 8.0. The Km value for Na+ ions was 12.2 mM. An apparent Km value for ATP was 880 [mu]M. A 140-kD phosphorylated intermediate was also detected in the same fraction in the presence of both Mg2+ and Na+ ions, and this protein was dephosphorylated upon the addition of K+ ions. We could partially purify the 140-kD protein after solubilization by Suc monolaurate and fractionation by sequential column chromatography on Sephacryl S-300, DEAE-Sepharose CL-6B, and Mono-Q columns. The purified 140-kD polypeptide could also be phosphorylated and be detected after acid sodium dodecyl sulfate-polyacryl-amide gel electrophoresis in the presence of Na+ and Mg2+ ions.     

Ca2+-stimulated, Mg2+-dependent ATPase in bovine thyroid plasma membranes

An isolated plasma membrane fraction from bovine thyroid glands contained a Ca2+-stimulated, Mg2+-dependent adenosine triphosphatase ((Ca2+ + Mg2+)-ATPase) activity which was purified in parallel to (Na+ + K+)-ATPase and adenylate cyclase. The (Ca2+ + Mg2+)-ATPase activity was maximally stimulated by approx. 200 microM added calcium in the presence of approx. 200 microM EGTA (69.7 +/- 5.2 nmol/mg protein per min). In EGTA-washed membranes, the enzyme was stimulated by calmodulin and inhibited by trifluoperazine.     

Purification of the Plasma Membrane Ca2+-ATPase from Radish Seedlings by Calmodulin-Agarose Affinity Chromatography

The Ca²⁺-ATPase of the plasma membrane (PM) of germinating radish (Raphanus sativus L.) seeds was purified by calmodulin (CaM)-affinity chromatography using a batch procedure. PM purified by aqueous two-phase partitioning was solubilized with n-dodecyl β-d-maltoside and applied to a CaM-agarose matrix. After various washings with decreasing Ca²⁺ concentrations, the Ca²⁺-ATPase was eluted with 5 mm ethylenediaminetetraacetate (EDTA). The EDTA-eluted fraction contained about 25% of the loaded Ca²⁺-ATPase activity, with a specific activity 70-fold higher than that of the starting PM fraction. The EDTA-eluted fraction was highly enriched in a 133-kD polypeptide, which was identified as the PM Ca²⁺-ATPase by ¹²⁵I-CaM overlay and fluorescein-isothiocyanate labeling. The PM Ca²⁺-ATPase cross-reacted with an antiserum against a putative Ca²⁺-ATPase of the Arabidopsis thaliana chloroplast envelope.     

Controlled Proteolysis Activates the Plasma Membrane Ca2+ Pump of Higher Plants (A Comparison with the Effect of Calmodulin in Plasma Membrane from Radish Seedlings)

The effects of calmodulin and of controlled trypsin treatments on the activity of the Ca2+ pump were investigated in plasma membrane purified from radish (Raphanus sativus L.) seedlings. Treatment of the plasma membrane with ethylenediaminetetra-acetate (EDTA), which removed about two-thirds of the plasma membrane-associated calmodulin, markedly increased the stimulation of the Ca2+ pump by calmodulin. In EDTA-treated plasma membrane, stimulation by calmodulin of the Ca2+ pump activity was maximal at low free Ca2+ (2-5 [mu]M) and decreased with the increase of free Ca2+ concentration. The Ca2+ pump activity was stimulated also by a controlled treatment of the plasma membrane with trypsin: the effect of trypsin treatment depended on the concentration of both trypsin and plasma membrane proteins and on the duration of incubation. Stimulation of the Ca2+ pump activity by trypsin treatment of the plasma membrane was similar to that induced by calmodulin both in extent and in dependence on the free Ca2+ concentration in the assay medium. Moreover, the Ca2+ pump of trypsin-treated plasma membrane was insensitive to further stimulation by calmodulin, suggesting that limited proteolysis preferentially cleaves a regulatory domain of the enzyme that is involved in its activation by calmodulin.     

Bepridil and cetiedil reversibly inhibit thyroid hormone stimulation in vitro of human red cell Ca2+-ATPase activity

Thyroid hormone (10(-11) to 10(-10) M) stimulates plasma membrane Ca2+-ATPase activity in vitro in various tissues, including the human red cell (RBC), by a calmodulin-requiring mechanism. Bepridil and cetiedil are Ca2+ antagonists with an intracellular (calmodulin-antagonist) site of action, as well as an effect on the calcium channel in excitable tissues. We have studied the actions of bepridil and cetiedil on Ca2+-ATPase in a channel-free membrane (RBC) to determine effectiveness of these agents as inhibitors of thyroid hormone action on the enzyme. Dose-response studies showed that thyroid hormone stimulation of Ca2+-ATPase activity in vitro was significantly inhibited by as little as 2 x 10(-5) M bepridil and cetiedil. IC50 values of bepridil and cetiedil for thyroid hormone response of the enzyme were 5 x 10(-5) and 2 x 10(-5) M, respectively, whereas IC50s of these agents for enzyme activity in the absence of thyroid hormone were both 10(-4) M. Progressive addition of purified rat testis calmodulin in vitro (10-150 ng calmodulin/mg membrane protein) restored hormone responsiveness in the presence of bepridil and cetiedil. Binding of labeled thyroid hormone by RBC membranes was unaffected by bepridil and cetiedil (up to 2 x 10(-4) M). Thus, bepridil and cetiedil are Ca2+ antagonists that reversibly inhibit thyroid hormone action on human RBC Ca2+-ATPase by a calmodulin-dependent mechanism. Thyroid hormone effect on Ca2+-ATPase is more susceptible to bepridil and cetiedil inhibition than is basal enzyme activity.     

Purification of D-myo-inositol 1,4,5-trisphosphate 3-kinase from rat brain

The ATP-dependent, calmodulin-sensitive 3-kinase responsible for the conversion of D-myo-inositol 1,4,5-trisphosphate to D-myo-inositol 1,3,4,5-tetrakisphosphate has been purified 2,700-fold from rat brain to a specific activity of 2.3 mumol/min/mg protein. A method of purification is described involving chromatography on phosphocellulose, Orange A dye ligand, calmodulin agarose, and hydroxylapatite columns. Neither the highly purified enzyme nor enzyme eluting from the phosphocellulose column were activated by Ca2+. However, enzyme in the 100,000 x g supernatant from rat brain was activated by Ca2+ over the range from 10(-7) to 10(-6) M and Ca2+ sensitivity of the purified enzyme was restored by the addition of calmodulin. The enzyme has a catalytic subunit Mr of 53,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Size exclusion chromatography of the purified enzyme on a Superose 12 column gave a Mr value of 70,000, indicating that the purified enzyme was present as a monomer. In contrast, the 100,000 x g supernatant and the purified enzyme after addition of calmodulin and 10(-6) M Ca2+ chromatographed on size exclusion chromatography with a Mr of 150,000-160,000. These results imply that the native enzyme is a dimeric structure of two catalytic subunits plus calmodulin. The purified enzyme showed a Km of 0.21 +/- 0.08 microM for D-myo-inositol 1,4,5-trisphosphate and had a pH optimum of 8.5. Addition of calmodulin increased both the Km and the Vmax of the purified enzyme about 2-fold. The high affinity of the 3-kinase for D-myo-inositol 1,4,5-trisphosphate together with its activation by Ca2+/calmodulin suggests that this enzyme may exert an important regulatory role in inositol phosphate signaling by promoting the formation of additional inositol polyphosphate isomers.