Calcium-dependent regulation of NAD kinase.

An activator protein of NAD kinase from the pea, $\text{Pisum}\text{satavum}$ L., has been shown to be Ca²⁺-dependent. This plant activator protein also stimulates the activity of modulator protein dependent-cyclic nucleotide phosphodiesterase from porcine brain. This stimulation is similar to that observed with modulator protein isolated from animal sources. Furthermore, Ca²⁺-dependent modulator proteins isolated from porcine brain, bovine brain, and the coelenterate, $\text{Renilla}$, will regulate the NAD kinase activity of peas. Other common properties of the plant activator protein and animal modulator proteins are their acidic nature, heat stabilities, similar Stokes' radii, and their interactions with troponin I.     

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.     

Structure–function relationships of molluscan troponin T revealed by limited proteolysis

Molluscan troponin regulates muscle contraction through a novel Ca²⁺-dependent activating mechanism associated with Ca²⁺-binding to the C-terminal domain of troponin C. To elucidate the further details of this regulation, we performed limited chymotryptic digestion of the troponin complex from akazara scallop striated muscle. The results indicated that troponin T is very susceptible to the protease, compared to troponin C or troponin I. The cleavage occurred at the C-terminal extension, producing an N-terminal 33-kDa fragment and a C-terminal 6-kDa fragment. This extension is conserved in various invertebrate troponin T proteins, but not in vertebrate troponin T. A ternary complex composed of the 33-kDa fragment of troponin T, troponin I, and troponin C could be separated from the 6-kDa troponin T fragment by gel filtration. This complex did not show any Ca²⁺-dependent activation of the Mg-ATPase activity of rabbit-actomyosin–scallop-tropomyosin. In addition, the actin–tropomyosin-binding affinity of this complex was significantly decreased with increasing Ca²⁺ concentration. These results indicate that the C-terminal extension of molluscan troponin T plays a role in anchoring the troponin complex to actin–tropomyosin filaments and is essential for regulation.     

Structural evidence for co-evolution of the regulation of contraction and energy production in skeletal muscle

Skeletal muscle phosphorylase kinase (PhK) is a Ca²⁺-dependent enzyme complex, (αβγδ)₄, with the δ subunit being tightly bound endogenous calmodulin (CaM). The Ca²⁺-dependent activation of glycogen phosphorylase by PhK couples muscle contraction with glycogen breakdown in the “excitation-contraction-energy production triad.” Although the Ca²⁺-dependent protein-protein interactions among the relevant contractile components of muscle are well characterized, such interactions have not been previously examined in the intact PhK complex. Here we show that zero-length cross-linking of the PhK complex produces a covalent dimer of its catalytic γ and CaM subunits. Utilizing mass spectrometry, we determined the residues cross-linked to be in an EF hand of CaM and in a region of the γ subunit sharing high sequence similarity with the Ca²⁺-sensitive molecular switch of troponin I that is known to bind actin and troponin C, a homolog of CaM. Our findings represent an unusual binding of CaM to a target protein and supply an explanation for the low Ca²⁺ stoichiometry of PhK that has been reported. They also provide direct structural evidence supporting co-evolution of the coordinate regulation by Ca²⁺ of contraction and energy production in muscle through the sharing of a common structural motif in troponin I and the catalytic subunit of PhK for their respective interactions with the homologous Ca²⁺-binding proteins troponin C and CaM.     

Purification and characterization of myosin from bovine retina

Myosin has been isolated from bovine retinae and characterised by its ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity, its mobility in sodium dodecyl sulphate polyacrylamide gels and by electron microscopy. The purified myosin shows high ATPase activity in the presence of EDTA or Ca²⁺ and a low activity in the presence of Mg²⁺. The Mg²⁺-dependent ATPase activity is stimulated by rabbit skeletal muscle actin. The presumptive retinal myosin possesses a major component which has a mobility in sodium dodecyl sulphate polyacrylamide gel electrophoresis similar to that of the heavy chain of bovine skeletal mucle myosin. Electron microscopy showed retinal myosin to form bipolar filaments in 0.1 M KCl. It is concluded that the retina possesses a protein with enzymic and structural properties similar to those of muscle myosin.     

Comparison of calcium-binding proteins. Bovine heart and brain protein activators of cyclic nucleotide phosphodiesterase and rabbit skeletal muscle troponin C.

In previous studies we have shown that the activation of bovine heart cyclic nucleotide phosphodiesterase by purified protein activator is completely dependent on the presence of Ca2+ and that the protein activator Ca2+ complex is probably the true activator for the enzyme (Teo, T.S. and Wang, J.H. (1973) J. Biol. Chem. 248, 5930-5955). More recent studies have led us to believe that the mechanism of the Ca2+ activation of phosphodiesterase resembles that of the Ca2+ activation of muscle contraction and that the protein activator may play a role similar to troponin. In the present study we show that the protein activator resembles rabbit muscle troponin C in amino acid composition, molecular weight, isoelectric point, and ultraviolet absorption spectrum. Preliminary structural studies also indicate that these two proteins may have evolved from a common ancestral protein through gene duplication. This argument is strengthened by the finding that the tryptic peptide map of the bovine heart protein activator is indistinguishable from that of the bovine brain phosphodiesterase activator protein for which preliminary sequence information also suggests homology to troponin C (Watterson, D.M., Harrelson, W.G., Jr., Keller, P.M., Sharief, F., and Vanaman, T.C. (1976) J. Biol. Chem. 251, 4501-4513).     

Modulator binding protein antagonizes activation of (Ca2+ + Mg2+)-ATPase and Ca2+ transport of red blood cell membranes

Red blood cells contain a protein that activates membrane-bound (Ca²⁺ + Mg²⁺)-ATPase and Ca²⁺ transport. The red blood cell activator protein is similar to a modulator protein that stimulates cyclic AMP phosphodiesterase. Wang and Desai [Journal of Biological Chemistry 252:4175–4184, 1977] described a modulator-binding protein that antagonizes the activation of cyclic AMP phosphodiesterase by modulator protein. In the present work, modulator-binding protein was shown to antagonize the activation of (Ca²⁺ + Mg²⁺)-ATPase and Ca²⁺ transport by red blood cell activator protein. The results further demonstrate the similarity between the activator protein from human red blood cells and the modulator protein from bovine brain.     

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.     

Mechanism of stimulation of Ca2+ plus Mg2+ -dependent phospodiesterase from rat cerebral cortex by the modulator protein and Ca2+

The activity of phosphodiesterase (“Ca²⁺ plus Mg²⁺-dependent” phosphodiesterase) of a preparation from brain was found to depend on the presence of both Ca²⁺ and a protein factor called modulator. It was shown by gel filtration that the active enzyme-modulator complex (MW, about 200,000) was formed from the modulator (MW, 28,000) and an inactive enzyme (MW, about 150,000) in the presence of Ca²⁺. When EGTA was added, this active enzyme-modulator complex dissociated into inactive enzyme and modulator. These results, together with the finding of Teo and Wang that Ca²⁺ binds to the modulator, could explain the stimulatory effect of Ca²⁺ on this enzyme as follows: The “Ca²⁺ plus Mg²⁺-dependent” phosphodiesterase may exist as the inactive free form in equilibrium with the active enzymemodulator (Ca²⁺) complex, and Ca²⁺, through binding to the modulator, may shift the equilibrium towards formation of the active enzyme-modulator (Ca²⁺) complex, thereby increasing the activity of the mixture. On decreasing the concentration of Ca²⁺, the process is reversible.     

Purification of calmodulin from bean rust uredospores

Calmodulin has been isolated from uredospores of the bean rust fungus and purified to apparent homogeneity as judged using sodium dodecyl sulfatepolyacrylamide gels. The protein substituted for bovine calmodulin as an activator of Ca²⁺-dependent cyclic nucleotide phosphodiesterase. Its molecular mass was 15.6 kDa in the presence of Ca²⁺ and 17.0 kDa in its absence. ThepI was 4.4. The amino acid composition was generally similar to those of calmodulin from other fungi.     

Troponin C-like protein in chicken gizzard muscle.

1. A troponin C-like protein was prepared from frozen chicken gizzard by preparative polyacrylamide gel electrophoresis and its apparent molecular weight was estimated to be about 15,500 daltons. 2. In urea gel electrophoresis, the mobility of the troponin C-like protein increased slightly in the presence of Ca2+, like that of skeletal muscle troponin C. On the other hand, the mobility of the the troponin C-like protein in glycerol gel electrophoresis, unlike that of skeletal muscle troponin C, was significantly decreased by Ca2+. 3. In alkaline gel electrophoresis, the troponin C-like protein formed a Ca2+-dependent complex with troponin I or troponin T from skeletal muscle. 4. The troponin C-like protein could neutralize the inhibitory effect of skeletal muscle troponin I on the Mg2+-activated ATPase of actomyosin from rabbit skeletal muscle, but could not confer Ca2+-sensitivity on the actomyosin in the presence of troponin I and troponin T from skeletal muscle.     

Signal tranduction: regulation of cAMP concentration in cardiac muscle by calmodulin-dependent cyclic nucleotide phosphodiesterase

The bovine heart calmodulin-dependent phosphodiesterase can be phosphorylated by cAMP-dependent protein kinase, resulting in a decrease in the enzyme's affinity for calmodulin. The phosphorylation of calmodulin-dependent phosphodiesterase is blocked by Ca²⁺ and calmodulin and reversed by the calmodulin-dependent phosphatase. The dephosphorylation is accompanied by an increase in the affinity of the phosphodiesterase for calmodulin. The CaM-dependent phosphodiesterase isozymes of heart and brain are regulated by calmodulin, but the affinity for calmodulin are different. Furthermore, the bovine heart CaM-dependent phosphodiesterase isozyme in stimulated at much lower Ca²⁺ concentration than the bovine brain isozymes. Results from this study suggest that the activity of this phosphodiesterase is precisely regulated by cross-talk between Ca²⁺ and cAMP signalling pathways.     

An endogenous inhibitor protein of brain adenylate cyclase and cyclic nucleotide phosphodiesterase.

Brain adenylate cyclase (EC 4.6.1.1) and cyclic nucleotide phosphodiesterase (EC 3.1.4.17) require an endogenous Ca²⁺-dependent activator protein for full activity (Cheung et al. (1975) Biochem. Biophys. Res. Commun. 66, 1055–1062). We now describe another brain factor which inhibited both brain adenylate cyclase and phosphodiesterase in vitro. The factor appeared to be a protein; it was inactivated by incubation with trypsin, but not with ribonuclease or deoxyribonuclease. Gel filtration with a calibrated column indicated a molecular weight of 80,000 and a Stokes' radius of 3.85 nm. In the presence of Ca²⁺, the inhibitor interacted with the activator protein to form an inhibitor activator complex. This makes the activator unavailable to adenylate cyclase or phosphodiesterase, resulting in a decrease of enzyme 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.     

Chemical modification studies on the Ca2+-dependent protein modulator: the role of methionine residues in the activation of cyclic nucleotide phosphodiesterase.

Methionine residues have been implicated in the activation of cyclic nucleotide phosphodiesterase by the Ca2+-dependent protein modulator [Walsh, M., & Stevens, F.C. (1977) Biochemistry 16,2742-2749]. Treatment of the modulator with N-chlorosuccinimide in the presence of Ca2+ resulted in selective oxidation of methionine residues at positions 71,72, 76, and, possibly, 109 in the modulator sequence. These residues lie on the surface of the molecule exposed to solvent. This modification has several effects on the modulator protein: (1) the Ca2+-binding properties of the oxidized modulator are changed with apparent loss of high-affinity binding sites, (2) the oxidized protein no longer interacts with phosphodiesterase, and (3) troponin C like activities, viz., Ca2+-dependent change in mobility on urea-polyacrylamide gel electrophoresis and formation of a urea-stable complex with troponin I, are lost upon oxidation of the modulator. The phosphodiesterase binding domain of the modulator protein appears to be located between the second and third Ca2+-binding loops, a region of the molecule known from previous partial proteolysis studies [Walsh, M., Stevens, F.C., Kuznicki, J., & Drabikowski, W.(1977), J. Biol. Chem. 252, 7440-7443] to be exposed in the presence of Ca2+.     

Interaction of cardiac troponin with cardiotonic drugs: a structural perspective

Over the 40 years since its discovery, many studies have focused on understanding the role of troponin as a myofilament based molecular switch in regulating the Ca²⁺-dependent activation of striated muscle contraction. Recently, studies have explored the role of cardiac troponin as a target for cardiotonic agents. These drugs are clinically useful for treating heart failure, a condition in which the heart is no longer able to pump enough blood to other organs. These agents act via a mechanism that modulates the Ca²⁺-sensitivity of troponin; such a mode of action is therapeutically desirable because intracellular Ca²⁺ concentration is not perturbed, preserving the regulation of other Ca²⁺-based signaling pathways. This review describes molecular details of the interaction of cardiac troponin with a variety of cardiotonic drugs. We present recent structural work that has identified the docking sites of several cardiotonic drugs in the troponin C-troponin I interface and discuss their relevance in the design of troponin based drugs for the treatment of heart disease.     

Detection of calcium-dependent regulatory protein binding components using 125I-labeled calcium-dependent regulatory protein.

The calcium-dependent regulatory protein (CDR) purified from bovine brain was iodinated with Na[125I]I using the lactoperoxidase-glucose oxidase system. The iodinated protein retained its ability to stimulate the Ca2+-sensitive CDR-depleted cyclic nucleotide phosphodiesterase from bovine heart. Stimulation of the phosphodiesterase by 125I-CDR was Ca2+-dependent and the labeled protein had a Ka for activation of cyclic nucleotide phosphodiesterase that was 4 times greater than unmodified CDR. 125I-CDR formed a Ca2+-dependent complex with the partially purified cyclic nucleotide phosphodiesterase which was detectable by autorradiography following electrophoresis of the complex on nondenaturing gels. This technique was used to detect CDR binding components in crude homogenates prepared from bovine heart and brain.     

Purification and characterization of calmodulin from rat liver mitochondria

Mitochondrial calmodulin of rat liver was purified and classified. It co-migrated with bovine brain calmodulin in non-denaturing polyacrylamide gel electrophoresis, SDS-polyacrylamide gel electrophoresis and isoelectric focusing. The mitochondrial calmodulin activated Ca²⁺-dependent phosphodiesterase of bovine brain in the presence of Ca²⁺. About 80% of the mitochondrial calmodulin was proved to be of cytosol origin. It was easily detached by washing with buffer containing EGTA. The other 20% was intramitochondrial calmodulin; half of it was in the matrix space, and half in the membrane.     

Resolution and characterization of two soluble calcium-dependent protein kinases from silver beet leaves

Two soluble Ca²⁺-dependent protein kinases (enzymes I and II) have been extensively purified from silver beet leaf tissue by means of a protocol involving batch-wise elution from DEAE-cellulose, Ca²⁺-dependent binding to phenyl-Sepharose, gradient elution from DEAE-Sephacel, gel filtration and binding to Cibacron F3GA-Sepharose CL-6B. Protein kinases I and II are resolved on gradient elution from DEAE-Sephacel and are further distinguished by their different K_m values for ATP and large differences in relative rates of phosphorylation of histone H1, casein and bovine serum albumin (the latter two proteins are relatively poor substrates for enzyme II but not enzyme I). Both enzymes have similar molecular weights as determined from gel filtration (56000 ± 2000 and 57000 ± 3000 for enzymes I and II, respectively). Both enzymes are absolutely dependent on free Ca²⁺ for activity with maximal histone H1 kinase activity being obtained at 0.5 μM free Ca²⁺. A millimolar concentration of Mg²⁺ is required in addition to a micromolar concentration Ca²⁺ for maximal activity. Both enzymes specifically phosphorylate serine residues of histone H1, are thiol activated and are inhibited by lanthanides and a range of calmodulin antagonists and inhibitors of protein kinase C.     

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.