Regulation of calmodulin binding to P-57. A neurospecific calmodulin binding protein

P-57 is a neural-specific calmodulin binding protein with novel calmodulin binding properties. P-57 exhibits higher affinity for calmodulin-Sepharose in the absence of free Ca2+ than in the presence of Ca2+ (Andreasen, T.J., Luetje, C.W., Heideman, W. & Storm, D.R. (1983) Biochemistry 22, 4615-4618; Cimler, B. M., Andreasen, T.J., Andreasen, K.I. & Storm, D.R. (1985) J. Biol. Chem. 260, 10784-10788). In this study, the dissociation constants for P-57 and immunopurified 5-[[(iodoacetylamino)ethyl]-amino]-1-naphthalenesulfonic acid-labeled calmodulin (AEDANS-CaM) were determined under low and high ionic strength conditions. In the absence of added KCl, the dissociation constants for the P-57 X AEDANS-CaM complex were 2.3 X 10(-7) +/- 6 X 10(-8) M and 1.0 X 10(-6) +/- 3 X 10(-7) M in the presence and absence of excess Ca2+ chelator. The addition of KCl to 150 mM increased the Ca2+-independent and -dependent dissociation constants to 3.4 X 10(-6) +/- 9 X 10(-7) M and 3.0 X 10(-6) +/- 9 X 10(-7) M, respectively. The association of P-57 with AEDANS-CaM under low Ca2+ conditions was determined as a function of KCl concentrations. By taking into account the amount of P-57 found in brain and its affinity for calmodulin, it is concluded that most or all of the CaM would be complexed to P-57 in unstimulated cells. P-57 was phosphorylated by the Ca2+-phospholipid-dependent protein kinase (protein kinase C) with a phosphate:protein molar ratio of 1.3. Phosphoamino acid analysis demonstrated phosphorylation at a serine residue. CaM decreased the rate of phosphorylation of P-57 by protein kinase C, and phosphorylation prevented P-57 binding to calmodulin-Sepharose. P-57 was not phosphorylated by the catalytic subunit of the cAMP-dependent protein kinase. It is proposed that P-57 binds and localizes calmodulin at specific sites within the cell and that free calmodulin is released locally in response to phosphorylation of P-57 by protein kinase C and/or to increases in intracellular free Ca2+. This regulatory mechanism, which appears to be specific to brain, would serve to decrease the response time for Ca2+-calmodulin-regulated processes.     

Purification of a novel calmodulin binding protein from bovine cerebral cortex membranes

A new calmodulin (CaM) binding protein, designated P-57, has been purified to apparent homogeneity from bovine cerebral cortex membranes. In contrast to other calmodulin binding proteins, P-57 has higher affinity for calmodulin in the absence of bound Ca2+ than in its presence. The protein was purified by DEAE-Sephacel chromatography and two CaM-Sepharose affinity column steps. The first CaM-Sepharose column was run in the presence of Ca2+; the second was run in the presence of chelator in excess of Ca2+. P-57 was adsorbed by CaM-Sepharose only in the absence of bound Ca2+ and was eluted from the second column by buffers containing Ca2+. Sodium dodecyl sulfate (SDS)-polyacrylamide gels of the purified protein showed only one band at Mr 57 000. The major form of the protein on Bio-Gel A-1.5m and native polyacrylamide gradient gel electrophoresis ran with an apparent Stokes radius of 41 A. Photoaffinity labeling of P-57 with azido[125I]calmodulin yielded one cross-linked product on SDS gels with an Mr of 70 000. This interaction occurred only when excess ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid was present and was inhibited by the presence of Ca2+ in excess of chelator. It appears that P-57 has novel binding properties for calmodulin distinct from all other calmodulin binding proteins described thus far.     

The calmodulin binding domain of the plasma membrane Ca2+ pump interacts both with calmodulin and with another part of the pump

Synthetic peptides corresponding to the calmodulin-binding domain of the human erythrocyte Ca2+ pump were prepared representing residues 2-29 (C28W), 2-21 (C20W), 2-16 (C15W), and 16-29 (C14) of the sequence (James, P., Maeda, M., Fisher, R., Verma, A. K., Krebs, J., Penniston, J. T., and Carafoli, E. (1988) J. Biol. Chem. 263, 2905-2910). Peptides C28W, C20W, and C15W bound to calmodulin with an apparent 1:1 stoichiometry in the presence of Ca2+ and inhibited the activation of the Ca2+ pump by calmodulin, while C14 was ineffective. Substituting tyrosine (C28Y) or alanine (C28A) for the tryptophan residue lowered the affinity for calmodulin. The estimated Kd values for the calmodulin-peptide complexes were 0.1 nM for C28W, 5-15 nM for C20W, C28Y, and C28A, and 700-1700 nM for C15W. The Ca2+ pump in inside-out erythrocyte membrane vesicles was activated by proteolytic removal of the endogenous calmodulin-binding domain. Addition of C20W or C28W then inhibited calmodulin-independent Ca2+ transport, while a calmodulin-binding peptide from another enzyme had no effect. The inhibition of the pump by C20W was purely competitive with Ca2+, while C28W decreased the Vmax and increased the K1/2 for Ca2+, restoring the pump activity nearly to its low basal level. The results suggest that a calmodulin-binding peptide from any enzyme has two kinds of specificity: it shares with peptides from other enzymes the ability to bind to calmodulin, but only it has the specificity to interact with its own (proteolytically activated) enzyme.     

Identification and characterization of the calmodulin-binding domain of neuromodulin, a neurospecific calmodulin-binding protein

Neuromodulin (formerly designated P-57) is an abundant, neural specific, calmodulin-binding protein which exhibits higher affinity for calmodulin in the absence of free Ca2+ than in the presence of free Ca2+. In this study a series of proteolytic fragments of neuromodulin were systematically screened for calmodulin-Sepharose binding activity. A 9-amino acid fragment, designated M1-C1 and having the sequence RGHITRKKL, was identified as the putative CaM-binding domain of neuromodulin. Two heptadecapeptides, designated FP57-Phe and FP57-Trp, were synthesized, each containing the M1-C1 sequence and the four flanking amino acids from each site. The FP57-Trp peptide contained a tryptophan residue in place of the native phenylalanine. Anti-FP57-Phe antibody binding to neuromodulin was inhibited by preincubation of antibodies with excess FP57-Phe. 125I-CaM gel overlay of neuromodulin was inhibited by anti-FP57-Phe antibodies. Addition of CaM to FP57-Trp increased peptide tryptophanyl fluorescence. In the presence of Ca2+, the stoichiometry of the FP57-Trp.CaM complex was 1:1, FP57-Trp binding to CaM was competitive with neuromodulin. The Ca2+-independent dissociation constant of the FP57-Phe.CaM complex was 0.41 microM. The Ca2+-dependent affinity of the complex could not be measured directly but appeared to be significantly greater than the Ca2+-independent affinity.     

Physicochemical and hydrodynamic characterization of P-57, a neurospecific calmodulin binding protein

P-57 is a neurospecific calmodulin binding protein that was discovered by virtue of its unusual interactions with calmodulin-Sepharose [Andreasen, T. J., Luetje, C. W., Heideman, W., & Storm, D. R. (1983) Biochemistry 22, 4615-4618; Cimler, B. M., Andreasen, T. J., Andreasen, K. I., & Storm, D. R. (1985) J. Biol. Chem. 260, 10784-10788]. In contrast to other calmodulin binding proteins, P-57 has higher affinity for calmodulin-Sepharose in the absence of calcium compared to that in the presence of calcium. In this study, we report the chemical and physical properties of P-57 purified from detergent-solubilized bovine brain membranes. The amino acid composition of P-57 is distinctive in that it contains a single phenylalanine residue with no other aromatic amino acids and a relatively high percentage of proline and alanine. In the presence of 0.05% Lubrol PX, its predicted secondary structure from circular dichroism spectroscopy is 1% alpha-helix, 21% beta-sheet, and 78% random coil. The hydrodynamic characteristics of the protein-detergent complex and the molecular weight of the protein were determined by gel filtration and sucrose density gradient sedimentation in the presence and absence of calmodulin. The P-57-detergent complex has an apparent Stokes radius (Rs) of 4.58 nm and a sedimentation coefficient (S20,w) of 1.44 S while the Stokes radius and S20,w for the P-57-calmodulin-detergent complex are 5.33 nm and 2.32 S, respectively. Perrin analysis of a 5-[[[(iodoacetyl)amino]ethyl]amino]-1-naphthalenesulfonic acid (AEDANS) derivative of P-57 confirmed the Stokes radius determined by gel filtration.(ABSTRACT TRUNCATED AT 250 WORDS)     

The calmodulin-binding domain as an endogenous inhibitor of the p-nitrophenylphosphatase activity of the Ca2+ pump from human red cells.

Digestion of red cell membranes with chymotrypsin elicited p-nitrophenylphosphatase activity. During digestion, the p-nitrophenylphosphatase appeared in parallel with the activation of the Ca(2+)-ATPase (in the absence of calmodulin). The chymotrypsin-activated p-nitrophenylphosphatase was inhibited by C20W, a 20 amino acid peptide modelled after the sequence of the calmodulin-binding site of the red cell Ca2+ pump (Vorherr et al. (1990) Biochemistry 29, 355-365). On the contrary, the (ATP + Ca(2+)-dependent p-nitrophenylphosphatase activity of intact red cell membranes was not affected by C20W. Ca2+ inhibited the chymotrypsin-induced p-nitrophenylphosphatase (Ki for Ca2+ = 2 microM). In the absence of ATP, C20W and Ca2+ did not interact in apparent affinity as inhibitors of this activity. On the other hand, in the presence of 2 mM ATP, Ca2+ antagonized the inhibition produced by C20W. The results are consistent with the idea that the calmodulin-binding site is an 'autoinhibitory domain' of the Ca2+ pump, and that removal of this domain by proteolysis, or its modification by calmodulin binding is the reason for the activation of both the ATPase and the p-nitrophenylphosphatase activity of the pump. The results presented in this paper give new information about the mechanism of the two kinds of p-nitrophenylphosphatase and about the nature of the apparent competition between C20W and Ca2+.     

Calmodulin-binding proteins and calmodulin-regulated enzymes in dog pancreas.

Calmodulin was isolated and purified to homogeneity from dog pancreas. Highly purified subcellular fractions were prepared from dog pancreas by zonal sucrose-density ultracentrifugation and assayed for their ability to bind 125I-calmodulin in vitro. Proteins contained in these fractions were also examined for binding of 125I-calmodulin after their separation by polyacrylamide-gel electrophoresis in SDS. Calmodulin-binding proteins were detected in all subcellular fractions except the zymogen granule and zymogen-granule membrane fractions. One calmodulin-binding protein (Mr 240,000), observed in a washed smooth-microsomal fraction, has properties similar to those of alpha-fodrin. The postribosomal-supernatant fraction contained three prominent calmodulin-binding proteins, with apparent Mr values of 62,000, 50,000 and 40,000. Calmodulin-binding proteins, prepared from a postmicrosomal-supernatant fraction by Ca2+-dependent affinity chromatography on immobilized calmodulin, exhibited calmodulin-dependent phosphodiesterase, protein phosphatase and protein kinase activities. In the presence of Ca2+ and calmodulin, phosphorylation of smooth-muscle myosin light chain and brain synapsin and autophosphorylation of a Mr-50,000 protein were observed. Analysis of the protein composition of the preparation by SDS/polyacrylamide-gel electrophoresis revealed a major protein of Mr 50,000 which bound 125I-calmodulin. This protein shares characteristics with the calmodulin-dependent multifunctional protein kinase (kinase II) recently observed to have a widespread distribution. The possible role of calmodulin-binding proteins and calmodulin-regulated enzymes in the regulation of exocrine pancreatic protein synthesis and secretion is discussed.     

Free radical production from the aerobic oxidation of reduced pyridine nucleotides catalysed by phenazine derivatives

Calcium-accumulating vesicles were isolated by differential centrifugation of sonicated platelets. Such vesicles exhibit a (Ca2+ + Mg2+)-ATPase activity of about 10 nmol (min . mg)-1 and an ATP-dependent Ca2+ uptake of about 10 nmol (min . mg)-1. When incubated in the presence of Mg[gamma-32P]ATP, the pump is phosphorylated and the acyl phosphate bond is sensitive to hydroxylamine. The [32P]phosphate-labeled Ca2+ pump exhibits a subunit molecular weight of 120 000 when analyzed by lithium dodecyl sulfate-polyacrylamide gel electrophoresis. Platelet calcium-accumulating vesicles contain a 23 kDa membrane protein that is phosphorylatable by the catalytic subunit of cAMP-dependent protein kinase but not by protein kinase C. This phosphate acceptor is not phosphorylated when the vesicles are incubated in the presence of either Ca2+ or Ca2+ plus calmodulin. The latter protein is bound to the vesicles and represents 0.5% of the proteins present in the membrane fraction. Binding of 125I-labeled calmodulin to this membrane fraction was of high affinity (16 nM), and the use of an overlay technique revealed four major calmodulin-binding proteins in the platelet cytosol (Mr = 94 000, 87 000, 60 000 and 43 000). Some minor calmodulin-binding proteins were enriched in the membrane fractions (Mr = 69 000, 57 000, 39 000 and 37 000). When the vesicles are phosphorylated in the presence of MgATP and of the catalytic subunit of cAMP-dependent protein kinase, the rate of Ca2+ uptake is essentially unaltered, while the Ca2+ capacity is diminished as a consequence of a doubling in the rate of Ca2+ efflux. Therefore, the inhibitory effect of cAMP on platelet function cannot be explained in such simple terms as an increased rate of Ca2+ removal from the cytosol. Calmodulin, on the other hand, was observed to have no effect on the initial rate of calcium efflux when added either in the absence or in the presence of the catalytic subunit of the cyclic AMP-dependent protein kinase, nor did the addition of 0.5 microM calmodulin result in increased levels of vesicle phosphorylation.     

Calmodulin-binding proteins from brain and other tissues.

The calmodulin contents of rabbit brain, lung, kidney and liver, of bovine aorta and uterus, and of chicken gizzard have been determined. 2. The calmodulin in all of these tissues has been shown to be present in the form of very stable complexes with several other proteins. 3. A calmodulin-binding protein of mol.wt. 22 000 has been purified in high yield from bovine brain. It has been shown to interact with calmodulin and rabbit skeletal-muscle troponin C in a Ca2+-dependent manner. 4. The 22 000-mol.wt. protein inhibits the activation of bovine brain phosphodiesterase by calmodulin, but has very little affect on the activation of myosin light-chain kinase. 5. Calmodulin-binding proteins of mol.wts. 140000, 77000 and 61000 have also been partially purified from rabbit brain by affinity chromatography and have been shown to interact in a Ca2+-dependent manner with calmodulin. 6. The apparent molecular weights of the calmodulin-calmodulin-binding protein complexes, determined by gel filtration in the presence of 6M-urea, have been shown to be similar for most of the mammalian tissues examined. 7. By using 125I-labelled calmodulin, similar complexes have been demonstrated in rabbit skeletal muscle, although they are present at much lower concentrations.     

140 mouse brain proteins identified by Ca2+-calmodulin affinity chromatography and tandem mass spectrometry

Calmodulin is an essential Ca2+-binding protein that binds to a variety of targets that carry out critical signaling functions. We describe the proteomic characterization of mouse brain Ca2+-calmodulin-binding proteins that were purified using calmodulin affinity chromatography. Proteins in the eluates from four different affinity chromatography experiments were identified by 1-DE and in-gel digestion followed by LC-MS/MS. Parallel experiments were performed using two related control-proteins belonging to the EF-hand family. After comparing the results from the different experiments, we were able to exclude a significant number of proteins suspected to bind in a nonspecific manner. A total of 140 putative Ca2+-calmodulin-binding proteins were identified of which 87 proteins contained calmodulin-binding motifs. Among the 87 proteins that contained calmodulin-binding motifs, 48 proteins have not previously been shown to interact with calmodulin and 39 proteins were known calmodulin-binding proteins. Many proteins with ill-defined functions were identified as well as a number of proteins that at the time of the analysis were described only as ORFs. This study provides a functional framework for studies on these previously uncharacterized proteins.     

Functional domain structure of calcineurin A: mapping by limited proteolysis

Limited proteolysis of calcineurin, the Ca2+/calmodulin-stimulated protein phosphatase, with clostripain is sequential and defines four functional domains in calcineurin A (61 kDa). In the presence of calmodulin, an inhibitory domain located at the carboxyl terminus is rapidly degraded, yielding an Mr 57,000 fragment which retains the ability to bind calmodulin but whose p-nitrophenylphosphatase is fully active in the absence of Ca2+ and no longer stimulated by calmodulin. Subsequent cleavage(s), near the amino terminus, yield(s) an Mr 55,000 fragment which has lost more than 80% of the enzymatic activity. A third, slower, proteolytic cleavage in the carboxyl-terminal half of the protein converts the Mr 55,000 fragment to an Mr 42,000 polypeptide which contains the calcineurin B binding domain and an Mr 14,000 fragment which binds calmodulin in a Ca2+-dependent manner with high affinity. In the absence of calmodulin, clostripain rapidly severs both the calmodulin-binding and the inhibitory domains. The catalytic domain is preserved, and the activity of the proteolyzed 43-kDa enzyme is increased 10-fold in the absence of Ca2+ and 40-fold in its presence. The calcineurin B binding domain and calcineurin B appear unaffected by proteolysis both in the presence and in the absence of calmodulin. Thus, calcineurin A is organized into functionally distinct domains connected by proteolytically sensitive hinge regions. The catalytic, inhibitory, and calmodulin-binding domains are readily removed from the protease-resistant core, which contains the calcineurin B binding domain. Calmodulin stimulation of calcineurin is dependent on intact inhibitory and calmodulin-binding domains, but the degraded enzyme lacking these domains is still regulated by Ca2+.     

Calmodulin and Ca2+-dependent phosphorylation and dephosphorylation of 63-kDa subunit-containing bovine brain calmodulin-stimulated cyclic nucleotide phosphodiesterase isozyme

Bovine brain contains calmodulin-dependent cyclic nucleotide phosphodiesterase isozymes which are composed of two distinct subunits: Mr 60,000 and 63,000. The 60-kDa but not the 63-kDa subunit-containing isozyme can be phosphorylated by cAMP-dependent protein kinase resulting in decreased affinity of this subunit toward calmodulin (Sharma, R. K., and Wang, J. H. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 2603-2607). In contrast, purified 63-kDa subunit-containing isozyme has been found to be phosphorylated by a preparation of bovine brain calmodulin-binding proteins in the presence of Ca2+ and calmodulin. The phosphorylation resulted in the maximal incorporation of 2 mol of phosphate/mol of the phosphodiesterase subunit with a 50% decrease in the enzyme affinity toward calmodulin. At a constant calmodulin concentration of 6 nM, the phosphorylated isozyme required a higher concentration of Ca2+ for activation than the nonphosphorylated phosphodiesterase. The Ca2+ concentrations at 50% activation by calmodulin of the nonphosphorylated and phosphorylated isozymes were 1.1 and 1.9 microM, respectively. Phosphorylation can be reversed by the calmodulin-dependent phosphatase, calcineurin, but not by phosphoprotein phosphatase 1. The results suggest that the Ca2+ sensitivities of brain calmodulin-dependent cyclic nucleotide phosphodiesterase isozymes can be modulated by protein phosphorylation and dephosphorylation mechanisms in response to different second messengers.     

Interaction of a fluorescent N-dansylaziridine derivative of troponin I with calmodulin in the absence and presence of calcium

Rabbit skeletal muscle troponin I was covalently labeled with N-dansylaziridine, resulting in a fluorescent labeled protein. This derivative (DANZTnI) and native troponin I (TnI) inhibited calmodulin (CaM) stimulation of bovine heart Ca2+-sensitive cyclic nucleodite phosphodiesterase with identical inhibition constants. Association of DANZTnI with calmodulin was monitored directly by changes in flourescence intensity in the presence of Ca2+ and by changes in fluorescence anisotropy in the absence of Ca2+. Quantitation of the affinity of calmodulin for calmodulin-binding proteins in both the presence and absence of Ca2+ is necessary for prediction of the extent of interaction of both Ca2+ and calmodulin-binding proteins with calmodulin in vivo. The dissociation constants for the DANZTnI-calmodulin-l4Ca2+ and DANZTnI-calmodulin complexes were 20 nM and 70 micrometers, respectively. These dissociation constants define a free energy coupling of-4.84 kcal/mol of troponin I for binding of Ca2+ and troponin I to calmodulin. The Ca2+ dependence for troponin I-calmodulin complex formation predicted from these experimentally determined parameters was closely approximated by the Ca2+ dependence for complex formation between troponin I and fluorescent 5-[[[(iodoacetyl)amino]ethyl]-amino]-1-napthalenesulfonic acid derivatized calmodulin as determined by fluorescence anisotropy. Complex formation occurred over a relatively narrow range of Ca2+ concentration, indicative of positive heterotropic cooperativity for Ca2+ and troponin I binding to calmodulin.     

Tryptophan 1093 is largely responsible for the slow off rate of calmodulin from plasma membrane Ca2+ pump 4b

Tryptophan 1093 resides in the 28-residue calmodulin-binding/autoinhibitory domain of the plasma membrane Ca(2+) pump (PMCA). Previous studies with the isolated calmodulin-binding/autoinhibitory peptide from PMCA have shown that mutations of the tryptophan residue decrease the affinity of the peptide for calmodulin and its affinity as an inhibitor of proteolytically activated pump. In this study, the PMCA mutation in which tryptophan 1093 is converted to alanine (W1093A) was constructed in the full-length PMCA isoform 4b. The mutant pump was expressed in COS cells, and its steady state and pre-steady state kinetic properties were examined. The W1093A pump exhibited an increased basal activity in the absence of calmodulin, so the activation was approximately 2-fold (it is 10-fold in the wild type). The W1093A mutation also lowered the steady state affinity for calmodulin from K(0.5) of 9 nm for wild type to 144 nm (assayed at 700 nm free Ca(2+)). Pre-steady state measurements of the rate of activation by Ca(2+)-calmodulin revealed that the W1093A mutant responded 2.5-fold faster to calmodulin. In contrast to these relatively modest effects, the half-time of inactivation of the mutant was reduced by more than 2 orders of magnitude from 41 min to 7 s. We conclude that tryptophan 1093 does not play a substantial role in Ca(2+)-calmodulin recognition; rather it functions primarily to slow the inactivation of the calmodulin-activated pump.     

Isolation of S-100 binding proteins from brain by affinity chromatography

S-100-binding proteins, and calmodulin-binding proteins were isolated from S-100- and calmodulin-depleted bovine brain extract by Ca2+-dependent affinity chromatography using S-100- and calmodulin-coupled Sepharose columns respectively. The majority of the protein (80 to 90%) including calcineurin that bound to S-100 also bound to calmodulin and vice versa, suggesting both proteins may regulate common targets. However these two regulatory proteins also bind few other proteins specific for each. These include cyclic nucleotide phosphodiesterase, 55k, and 220k proteins for calmodulin and 24k, 42k, and 90k proteins for S-100. Certain proteins also specifically bound to S-100 both in Ca2+-dependent and independent ways. In glial cells S-100 protein may replace calmodulin in regulating Ca2+-influenced functions.     

Activation of a calmodulin-dependent phosphatase by a Ca2+-dependent protease

A calmodulin-dependent protein phosphatase (calcineurin) was converted to an active, calmodulin-independent form by a Ca2+-dependent protease (calpain I). Proteolysis could be blocked by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, leupeptin, or N-ethylmaleimide, but other protease inhibitors such as phenylmethanesulfonyl fluoride, aprotinin, benzamidine, diisopropyl fluorophosphate, and trypsin inhibitor were ineffective. Phosphatase proteolyzed in the absence of calmodulin was insensitive to Ca2+ or Ca2+/calmodulin; the activity of the proteolyzed enzyme was greater than the Ca2+/calmodulin-stimulated activity of the unproteolyzed enzyme. Proteolysis of the phosphatase in the presence of calmodulin proceeded at a more rapid rate than in its absence, and the proteolyzed enzyme retained a small degree of sensitivity to Ca2+/calmodulin, being further stimulated some 15-20%. Proteolytic stimulation of phosphatase activity was accompanied by degradation of the 60-kilodalton (kDa) subunit; the 19-kDa subunit was not degraded. In the absence of calmodulin, the 60-kDa subunit was sequentially degraded to 58- and 45-kDa fragments; the 45-kDa fragment was incapable of binding 125I-calmodulin. In the presence of calmodulin, the 60-kDa subunit was proteolyzed to fragments of 58, 55 (2), and 48 kDa, all of which retained some ability to bind calmodulin. These data, coupled with our previous report that the human platelet calmodulin-binding proteins undergo Ca2+-dependent proteolysis upon platelet activation [Wallace, R. W., Tallant, E. A., & McManus, M. C. (1987) Biochemistry 26, 2766-2773], suggest that the Ca2+-dependent protease may have a role in the platelet as an irreversible activator of certain Ca2+/calmodulin-dependent reactions.     

Phosphorylation and characterization of bovine heart calmodulin-dependent phosphodiesterase.

Calmodulin-dependent phosphodiesterase was purified to apparent homogeneity from the total calmodulin-binding fraction of bovine heart in a single step by immunoaffinity chromatography. The isolated enzyme had significantly higher affinity for calmodulin than the bovine brain 60-kDa phosphodiesterase isozyme. The cAMP-dependent protein kinase was found to catalyze the phosphorylation of the purified cardiac calmodulin-dependent phosphodiesterase with the incorporation of 1 mol of phosphate/mol of subunit. The phosphodiesterase phosphorylation rate was increased severalfold by histidine without affecting phosphate incorporation into the enzyme. Phosphorylation of phosphodiesterase lowered its affinity for calmodulin and Ca2+. At constant saturating concentrations of calmodulin (650 nM), the phosphorylated calmodulin-dependent phosphodiesterase required a higher concentration of Ca2+ (20 microM) than the nonphosphorylated phosphodiesterase (0.8 microM) for 50% activity. Phosphorylation could be reversed by the calmodulin-dependent phosphatase (calcineurin), and dephosphorylation was accompanied by an increase in the affinity of phosphodiesterase for calmodulin.     

Interaction between chicken gizzard caldesmon and tropomyosin

Chicken gizzard muscle caldesmon has been examined for ability to interact with tropomyosin from chicken gizzard muscle by using fluorescence enhancement of tropomyosin labeled with dansyl chloride (DNS) and affinity chromatography. The binding of caldesmon to tropomyosin was regulated by Ca2+ and calmodulin, i.e., at low ionic strength most of the caldesmon bound to tropomyosin-Sepharose 4B was co-eluted by adding calmodulin only in the presence of Ca2+, but not in its absence. This regulation by Ca2+ and calmodulin was also suggested by fluorescence measurements. Actin- and calmodulin-binding sites on the caldesmon molecule were located in the 38K fragment (Fujii, T., Imai, M., Rosenfeld, G.C., & Bryan, J. (1987) J. Biol. Chem. 262, 2757-2763). When 38K-enriched fraction was applied to the tropomyosin-Sepharose, the 38K fragment was retained by the column and could be eluted by adding Ca2+ and calmodulin.     

Differential recognition of calmodulin-enzyme complexes by a conformation-specific anti-calmodulin monoclonal antibody

An anti-calmodulin monoclonal antibody having an absolute requirement for Ca2+ has been produced from mice immunized with a mixture of calmodulin and calmodulin-binding proteins. Radioimmune assays were developed for the determination of its specificity. the epitope for this antibody resides on the COOH-terminal half of the mammalian protein. Plant calmodulin or troponin C had little reactivity. The apparent affinity of the antibody for calmodulin was increased approximately 60-fold in the presence of heart calmodulin-dependent phosphodiesterase. The presence of heart phosphodiesterase in the radioimmune assay greatly enhanced the sensitivity for calmodulin. The intrinsic calmodulin subunit of phosphorylase kinase and calmodulin which was bound to brain phosphodiesterases was also recognized with high affinity by the antibody. The antibody reacted poorly with calmodulin which was bound to heart or brain calcineurin, skeletal muscle myosin light chain kinase, or other calmodulin-binding proteins. In direct binding experiments, most of the calmodulin-binding proteins studied were unreactive with the antibody. This selectivity allowed purification of heart and two brain calmodulin-dependent cyclic nucleotide phosphodiesterase isozymes on immobilized antibody affinity columns. Phosphodiesterase activity was adsorbed directly from crude samples and specifically eluted with EGTA. Isozyme separation was accomplished using a previously described anti-heart phosphodiesterase monoclonal antibody affinity support. The brain isozymes differed not only in reactivity with the anti-phosphodiesterase antibody, but also in apparent subunit molecular weight, and relative specificity for cAMP and cGMP as substrates. The calmodulin activation constants for the brain enzymes were 10-20-fold greater than for the heart enzyme. The data suggest that the binding of ligands to Ca2+/calmodulin induce conformation changes in calmodulin which alter reactivity with the anti-calmodulin monoclonal antibody. The differential antibody reactivity toward calmodulin-enzyme complexes indicates that target proteins either induce very different conformations in calmodulin and/or interact with different geometries relative to the antibody binding site. The anti-calmodulin monoclonal antibody should be useful for the purification of other calmodulin-dependent phosphodiesterases as well as isozymes of phosphorylase kinase.     

Calmodulin binding proteins in human erythrocyte membranes

Human erythrocyte membranes reveal different calmodulin-binding proteins determined by a 125I-calmodulin gel overlay procedure. Beside the well-established Ca2+-transport ATPase, other proteins (205, 91, 72 and 42 kDa) bind calmodulin in a Ca2+-dependent manner. Two proteins of the human erythrocyte membrane are able to bind calmodulin only in the absence of Ca2+. One of them (76 kDa) is probably an integral, the other (240 kDa) a peripheral protein.