Identification of the Ca(2+)-dependent calmodulin-binding region of chromogranin A.

Chromogranin A (CGA), the most abundant protein in bovine adrenal chromaffin granules, is a high-capacity, low-affinity Ca(2+)-binding protein found in most neuroendocrine cells, and binds calmodulin (CaM) in a Ca(2+)-dependent manner. The binding of chromogranin A to calmodulin was determined by measuring the intrinsic tryptophan fluorescence of chromogranin A in the presence and absence of Ca2+. Binding was specifically Ca(2+)-dependent; neither Mg2+ nor Mn2+ could substitute for Ca2+. Chelation of Ca2+ by EGTA completely eliminated the chromogranin A-calmodulin interaction. CaM binding was demonstrated by a synthetic CGA peptide representing residues 40-65. When the CGA peptide and CaM were mixed in the presence of 15 mM CaCl2, the intrinsic tryptophan fluorescence emission underwent a substantial blue-shift, shifting from 350 to 330 nm. Like the intact CGA, the peptide-CaM binding was specifically Ca(2+)-dependent, and neither Mg2+ nor Mn2+ could induce the binding. Calmodulin bound both to CGA and to the synthetic CGA peptide with a stoichiometry of one to one. The dissociation constants (Kd) determined by fluorometric titration were 13 nM for the peptide-CaM binding and 17 nM for intact CGA-CaM binding. The Kd values are comparable to those (approximately 10(-9) M) of other CaM-binding proteins and peptides, demonstrating a tight binding of CaM by CGA. The CaM-binding CGA residues 40-65 are 100% conserved among all the sequenced CGAs in contrast to 50-60% conservation found in the entire sequence, implying essential roles of this region.     

The effects of chemical modification of calmodulin on Ca2+-induced exposure of a hydrophobic region. Separation of active and inactive forms of calmodulin

Native calmodulin binds four calcium ions per molecule and exhibits strong Ca2+-dependent binding to phenyl-Sepharose. In contrast, calmodulin inactivated by oxidation of methionine residues or by deamidation binds fewer calcium ions (two per molecule) and shows relatively weak interaction with phenyl-Sepharose. Calmodulin inactivated by modification of lysine residues still is able to bind four calcium ions per molecule and shows strong binding to phenyl-Sepharose similar to native calmodulin. The results suggest that complete exposure of calmodulin's hydrophobic region occurs only after the binding of four ions of calcium to the calmodulin molecule. Thus, phenyl-Sepharose hydrophobic interaction chromatography might be used to separate active calmodulin from inactive forms of calmodulin obtained by oxidation or heat treatment for prolonged periods. As an example, phenyl-Sepharose chromatography can be used to separate free iodide and inactivated species of calmodulin readily from the active, iodinated form of calmodulin following iodination.     

Characterization of Calelectrin, a Ca2+-Binding Protein Isolated from the Electric Organ of Torpedo marmorata

We report a fast (less than 1 day) and efficient (2-3 mg protein/100 g tissue) isolation method for calelectrin, a protein of Mr 34,000 in the electric organ of Torpedo marmorata that binds to membranes in the presence of Ca2+. Purified protein was used to investigate the nature of its interaction with membranes and with Ca2+. Calelectrin binds to liposomes composed of total extractable lipids from the electric organ in a Ca2+-dependent and -specific manner with half-maximal binding between 3 and 7 microM free Ca2+. This binding is totally inhibited by 1 mM mercaptoethanol. It is also shown that calelectrin directly binds Ca2+ in solution by two techniques: at 1 and 10 microM Ca2+ it binds 45Ca2+ as measured by gel permeation chromatography, and it contains saturable Tb3+-binding sites that are Ca2+-displaceable. An investigation of the protein's endogenous fluorescence shows that although it contains both tryptophan and tyrosine, there is no change in the apparent quantum yield as a function of Ca2+. Ca2+-dependent hydrophobic affinity chromatography of the total soluble proteins from Torpedo electric organ shows that Torpedo calelectrin, like calmodulin and mammalian calelectrins, is specifically retained in the presence of Ca2+ and eluted by EGTA. Calelectrin also contains high-affinity sites for hydrophobic fluorescence probes such as N-phenyl-1-naphthylamine, 2-CP-toluidinylnaphthalene-6-sulfonic acid, and 1-anilinonaphthalene-8-sulfonic acid, which again unlike calmodulin, show no changes as a function of Ca2+. We conclude that calelectrin is a Ca2+-binding protein whose binding to the lipid moieties of membranes is regulated by physiological change in the Ca2+ concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of the yeast calmodulin gene: calmodulin is an essential protein

Calmodulin was purified from Saccharomyces cerevisiae based on its characteristic properties. Like other calmodulins, the yeast protein is small, heat-stable, acidic, retained by hydrophobic matrices in a Ca2+-dependent manner, exhibits a pronounced Ca2+-induced shift in electrophoretic mobility, and binds 45Ca2+. Using synthetic oligonucleotide probes designed from the sequences of two tryptic peptides derived from the purified protein, the gene encoding yeast calmodulin was isolated. The gene (designated CMD1) is a unique, single-copy locus, contains no introns, and resides on chromosome II. The amino acid sequence of yeast calmodulin shares 60% identity with other calmodulins. Disruption or deletion of the yeast calmodulin gene results in a recessive-lethal mutation; thus, calmodulin is essential for the growth of yeast cells.     

Calmodulin modulates initiation but not termination of spontaneous Ca2+ sparks in frog skeletal muscle

Calmodulin is a ubiquitous Ca(2+) sensing protein that binds to and modulates the sarcoplasmic reticulum Ca(2+) release channel, ryanodine receptor (RYR). Here we assessed the effects of calmodulin on the local Ca(2+) release properties of RYR in permeabilized frog skeletal muscle fibers. Fluorescently labeled recombinant calmodulin in the internal solution localized at the Z-line/triad region. Calmodulin (0.05-5.0 micro M) in the internal solution (free [Ca(2+)](i) approximately 50-100 nM) initiated a highly cooperative dose-dependent increase in Ca(2+) spark frequency, with a half-maximal activation (K) of 1.1 micro M, a Hill coefficient (n) of 4.2 and a fractional maximal increase in frequency (R) of 17-fold. A non-Ca(2+) binding mutant of calmodulin elicited a similar highly cooperative dose-dependent increase in spark frequency (K = 1.0 micro M; n = 3.7; R = 12-fold). Spatiotemporal properties of Ca(2+) sparks were essentially unaffected by either wild-type or mutant calmodulin. An N-terminal extension of calmodulin, (N+3)calmodulin, that binds to but does not activate RYR at nM [Ca(2+)] in sarcoplasmic reticulum vesicles, prevented the calmodulin-induced increase in spark frequency. These data suggest that exogenous Ca(2+)-free calmodulin cooperatively sensitizes the Ca(2+) release channel to open, but that Ca(2+) binding to the added calmodulin does not play a significant role in the termination of Ca(2+) sparks.     

Calmodulin in adult mammalian skeletal muscle: localization and effect on sarcoplasmic reticulum Ca2+ release

Calmodulin is a ubiquitous Ca2+ binding protein that binds to ryanodine rectors (RyR) and is thought to modulate its activity. Here we evaluated the effects of recombinant calmodulin on the rate of occurrence and spatial properties of Ca2+ sparks as an assay of activation in saponin-permeabilized mouse myofibers. Control myofibers exhibited a time-dependent increase and subsequent decrease in spark frequency. Recombinant wild-type calmodulin prevented the time-dependent appearance of Ca2+ sparks and decreased the derived Ca2+ flux from the sarcoplasmic reticulum during a spark by approximately 37%. A recombinant Ca2+-insensitive form of calmodulin resulted in an instantaneous increase in spark frequency as well as an increase in the derived Ca2+ flux by approximately 24%. Endogenous calmodulin was found to primarily localize to the Z-line. Surprisingly, removal of endogenous calmodulin did not alter the time dependence of Ca2+ spark appearance. These results indicate that calmodulin may not be essential for RyR1-dependent Ca2+ release in adult mammalian skeletal muscle.     

Calmodulin-dependent regulation of a lipid binding domain in the v-SNARE synaptobrevin and its role in vesicular fusion

Trans SNARE complex assembly is an essential step in Ca2+-dependent membrane fusion, although the SNARE proteins do not bind Ca2+ ions. Studies to evaluate how the Ca2+sensor protein calmodulin might regulate this process led to the identification of a consensus calmodulin binding motif in the v-SNARE VAMP2. This sequence (residues 77-90) is situated precisely C-terminal to the tetanus toxin (TeNT) and botulinum B toxin cleavage site (76Q-F77) close to the transmembrane anchor. The same domain also binds acidic phospholipids and Ca2+/calmodulin or lipid binding are mutually exclusive. Directed mutagenesis of basic or hydrophobic residues within this motif reduced interactions with both Ca2+/calmodulin and phospholipids to a similar extent. The effects of these mutations on Ca2+-dependent exocytosis was explored using an hGH release assay in permeabilized pheochromocytoma PC12 cells. Treatment of cells with tetanus toxin (TeNT), which cleaves endogenous VAMP, abolished secretion. Secretion could be re-established by transfecting TeNT-resistant VAMP with mutations (Q76V,F77W) in the cleavage site. However rescue of exocytosis was abolished when additional mutations (K83A,K87V or W89A,W90A) were introduced that inhibited calmodulin and phospholipid binding to VAMP. Thus calmodulin and/or phospholipid binding to the membrane proximal region of VAMP is required for Ca2+-dependent exocytosis. We speculate that interactions between cis phospholipids at the vesicle surface and the membrane proximal region of VAMP inhibits SNARE complex assembly. Displacement of these interactions by Ca2+/calmodulin may promote SNARE complex assembly and lead to trans interactions between the membrane proximal region of VAMP and phospholipids in the plasma membrane.     

Fluorescence investigations of calmodulin hydrophobic sites

Calmodulin activation of target enzymes depends on the interaction between calmodulin hydrophobic regions and some enzyme areas. The Ca2+ induced exposure of calmodulin hydrophobic sites was studied by means of 2-p-toluidinylnaphthalene-6-sulfonate, a fluorescent probe. Scatchard and Job plots showed that the calmodulin-Ca42+ complex bound two molecules of this hydrophobic probe, with KD congruent to 1.4 X 10(-4) M. These sites are not totally exposed until calmodulin has bound four Ca2+ per molecule, so the conformational change is not over before the four specific Ca2+ - binding sites are saturated with Ca2+.     

Structural basis for calmodulin as a dynamic calcium sensor

Calmodulin is a prototypical and versatile Ca(2+) sensor with EF hands as its high-affinity Ca(2+) binding domains. Calmodulin is present in all eukaryotic cells, mediating Ca(2+)-dependent signaling. Upon binding Ca(2+), calmodulin changes its conformation to form complexes with a diverse array of target proteins. Despite a wealth of knowledge on calmodulin, little is known on how target proteins regulate calmodulin's ability to bind Ca(2+). Here, we take advantage of two splice variants of SK2 channels, which are activated by Ca(2+)-bound calmodulin but show different sensitivity to Ca(2+) for their activation. Protein crystal structures and other experiments show that, depending on which SK2 splice variant it binds to, calmodulin adopts drastically different conformations with different affinities for Ca(2+) at its C-lobe. Such target protein-induced conformational changes make calmodulin a dynamic Ca(2+) sensor capable of responding to different Ca(2+) concentrations in cellular Ca(2+) signaling.     

Identification of a peptide inhibitor of the cardiac sarcolemmal Na(+)-Ca2+ exchanger

The deduced amino acid sequence of the cardiac sarcolemmal Na(+)-Ca2+ exchanger has a region which could represent a calmodulin binding site. As calmodulin binding regions of proteins often have an autoinhibitory role, a synthetic peptide with this sequence was tested for functional effects on Na(+)-Ca2+ exchange activity. The peptide inhibits the Na(+)-dependent Ca2+ uptake (KI approximately 1.5 microM) and the Nao(+)-dependent Ca2+ efflux of sarcolemmal vesicles in a noncompetitive manner with respect to both Na+ and Ca2+. The peptide is also a potent inhibitor (KI approximately 0.1 microM) of the Na(+)-Ca2+ exchange current of excised sarcolemmal patches. The binding site for the peptide on the exchanger is on the cytoplasmic surface of the membrane. The exchanger inhibitory peptide binds calmodulin with a moderately high affinity. From the characteristics of the inhibition of the exchange of sarcolemmal vesicles, we deduce that only inside-out sarcolemmal vesicles participate in the usual Na(+)-Ca2+ exchange assay. This contrasts with the common assumption that both inside-out and right-side-out vesicles exhibit exchange activity.     

Site-specific derivatives of wheat germ calmodulin. Interactions with troponin and sarcoplasmic reticulum

Wheat germ calmodulin (CaM) was derivatized at its single cysteine (Cys27) with either the fluorescent reagent, N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonic acid (I-EDANS) or the photoactivable cross-linker benzophenone-4-maleimide. Comparison of the native and derivatized wheat germ CaMs with native bovine testis CaM indicates that the concentrations of these proteins required for half-maximal stimulation of either erythrocyte membrane Ca2+-ATPase activity or cardiac sarcoplasmic reticulum phosphorylation are very similar. Affinity labeling of troponin subunits with 125I- and benzophenone-4-maleimide-labeled CaM demonstrates CaM binding to troponin I (TnI) and troponin T (TnT) in binary complexes, as well as to both subunits in the CaM.TnI.TnT ternary complex. This suggests that both subunits are within 10 A of Cys27 of calmodulin. Affinity labeling of cardiac sarcoplasmic reticulum vesicles with 125I- and benzophenone-4-maleimide-labeled CaM exhibits a Ca2+- and Mg2+-dependent labeling of phospholamban, as shown previously with bovine calmodulin (Louis, C.F., and Jarvis, B. (1982) J. Biol. Chem. 257, 15187-15191). Thus, it appears that Ca2+-binding site I of calmodulin is at or near binding sites of calmodulin for TnI, TnT, and phospholamban. Analysis of the time-resolved fluorescence decay curves of I-EDANS-labeled calmodulin indicates a major component with a lifetime of 11.9 ns (+Ca2+), which accounts for 81% of the total fluorescence. The lifetime decreases slightly to 11.3 ns in the absence of Ca2+. Fluorescence anisotropy experiments indicate that I-EDANS-labeled CaM binds TnI with Kd = 6 x 10(-8) M in the presence of Ca2+. This study suggests that these single-site derivatives will be useful for characterizing a variety of calmodulin-receptor interactions because they lack ambiguities inherent in less specific labeling methods.     

Calelectrins are a ubiquitous family of Ca2+-binding proteins purified by Ca2+-dependent hydrophobic affinity chromatography by a mechanism distinct from that of calmodulin

The calelectrins, a heterogeneous group of three new Ca2+-binding proteins of M 67 000, 35 000 and 32 500, copurify with calmodulin during Ca2+-dependent hydrophobic affinity chromatography (Südhof et al., Biochemistry, in press, 1984). This property is exploited for the rapid purification of all three calelectrins including for the first time the Mr 35 000, from commercially available acetone powders from several bovine tissues (heart, liver, brain, pancreas and testis). The nature of the Ca2+-dependent interaction of the calelectrins with hydrophobic affinity matrices has been investigated. As with calmodulin, the Ca2+-binding sites of all three purified calelectrins can be probed with Tb3+ which binds to them in a stoichiometric, saturable and Ca2+-displaceable manner. However, using several hydrophobic fluorescence probes which bind to the proteins, contrary to calmodulin no Ca2+-dependent exposure of hydrophobic sites could be detected in any of the three purified proteins. Therefore the Ca2+-dependent purification of the calelectrins on hydrophobic affinity columns seems not to involve the surface exposure of hydrophobic sites and the calelectrins have in this respect little similarity to calmodulin.     

Calmodulin activation and inhibition of skeletal muscle Ca2+ release channel (ryanodine receptor).

The calmodulin-binding properties of the rabbit skeletal muscle Ca2+ release channel (ryanodine receptor) and the channel's regulation by calmodulin were determined at < or = 0.1 microM and micromolar to millimolar Ca2+ concentrations. [125I]Calmodulin and [3H]ryanodine binding to sarcoplasmic reticulum (SR) vesicles and purified Ca2+ release channel preparations indicated that the large (2200 kDa) Ca2+ release channel complex binds with high affinity (KD = 5-25 nM) 16 calmodulins at < or = 0.1 microM Ca2+ and 4 calmodulins at 100 microM Ca2+. Calmodulin-binding affinity to the channel showed a broad maximum at pH 6.8 and was highest at 0.15 M KCl at both < or = 0.1 MicroM and 100 microM Ca2+. Under condition closely related to those during muscle contraction and relaxation, the half-times of calmodulin dissociation and binding were 50 +/- 20 s and 30 +/- 10 min, respectively. SR vesicle-45Ca2+ flux, single-channel, and [3H]ryanodine bind measurements showed that, at < or = 0.2 microM Ca2+, calmodulin activated the Ca2+ release channel severalfold. Ar micromolar to millimolar Ca2+ concentrations, calmodulin inhibited the Ca(2+)-activated channel severalfold. Hill coefficients of approximately 1.3 suggested no or only weak cooperative activation and inhibition of Ca2+ release channel activity by calmodulin. These results suggest a role for calmodulin in modulating SR Ca2+ release in skeletal muscle at both resting and elevated Ca2+ concentrations.     

Ca2+- and Mg2+-dependent association of phosphorylase kinase with human erythrocyte membranes

The interaction of rabbit muscle phosphorylase kinase (EC 2.7.1.38) with human erythrocyte membranes was investigated. It was found that at pH 7.0 the kinase binds to the inner face of the erythrocyte membrane (inside-out vesicles) and that this binding is Ca2+- and Mg2+-dependent. The sharpest increase in the binding reaction occurs at concentrations between 70 and 550 nM free Ca2+. Erythrocyte ghost or right-side out erythrocyte vesicles showed a significantly lower capacity to interact with phosphorylase kinase. Autophosphorylated phosphorylase kinase shows a similar Ca2+-dependent binding profile, while trypsin activation of the kinase and calmodulin decrease the original binding capacity by about 50%. Heparin (200 micrograms/ml) and high ionic strength (50 mM NaCl) almost completely blocks enzyme-membrane interaction; glycogen does not affect the interaction.     

Localization of a felodipine (dihydropyridine) binding site on calmodulin

The fluorescent dihydropyridine calcium antagonist drug felodipine binds to calmodulin (CaM) in a Ca2+-dependent manner. Its binding can be regulated by the interaction of CaM antagonist drugs through allosteric mechanisms [Mills, J.S., & Johnson, J.D. (1985) Biochemistry 24, 4897]. Here, we have examined the binding of a nonspecific hydrophobic fluorescent probe molecule TNS (toluidinylnaphthalenesulfonate) and of felodipine to CAM and several of its proteolytic fragments. While TNS interacts with sites on both the amino-terminal half of the protein [proteolytic fragment TR1C (1-77)] and carboxy-terminal half [proteolytic fragment TR2C (78-148)], felodipine binding shows more selectivity. It binds in a Ca2+-dependent manner to the proteolytic fragments TM1 (1-106) and TR2E (1-90) but exhibits only weak affinity for TR1C (1-77) and TR2C (78-148). Furthermore, felodipine exhibits selectivity over TNS and trifluoperazine (TFP) in blocking the tryptic cleavage between residues 77 and 78. These studies indicate a selective binding of felodipine to a hydrophobic site existing in residues 1-90 and suggest that productive binding requires amino acids in the region 78-90. Although the felodipine binding site is preserved in fragment 1-106, the allosteric interactions between the prenylamine and the felodipine binding sites that are observed with intact CaM are not observed in this fragment. Rather, prenylamine simply displaces felodipine from its binding site on this fragment. Our results are consistent with calmodulin containing not less than two allosterically related hydrophobic drug binding sites. One of these sites (felodipine) appears to be localized in region 1-90 and the other one in region 78-148.     

The effect of calmodulin on the phosphoprotein intermediate of Mg2+-dependent Ca2+-stimulated adenosine triphosphatase in human erythrocyte membranes.

The effect of calmodulin on the formation and decomposition of the Ca2+-dependent phosphoprotein intermediate of the (Mg2+ + Ca2+)-dependent ATPase in erythrocyte membranes was investigated. In the presence of 60 microM-Ca2+ and 25 microM-MgCl2, calmodulin (0.5-1.5 microgram) did not alter the steady-state concentration of the phosphoprotein, but increased its rate of decomposition. Higher calmodulin concentrations significantly decreased the steady-state concentration of phosphoprotein. Calmodulin (0.5-1.7 microgram) increased Ca2+-transport ATPase activity by increasing the turnover rate of its phosphoprotein intermediate. Increasing the MgCl2 concentration from 25 microM to 250 microM increased the (Mg2+ + Ca2+)-dependent ATPase activity, but decreased the concentration of the phosphoprotein intermediate. Similarly to calmodulin, MgCl2 increased the turnover rate of the Ca2+-transport ATPase complex (about 3-fold). At the higher MgCl2 concentration calmodulin did not further affect the decomposition of the phosphoprotein intermediate. It was concluded that both calmodulin and MgCl2 increase the turnover of the Ca2+-pump by enhancing the decomposition of the Ca2+-dependent phosphoprotein intermediate.     

Isolation, purification and cell-free synthesis of calmodulin from the pig anterior pituitary gland.

Calmodulin was extracted and purified from pig anterior pituitary gland. The protein was characterized by its migration on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis in the presence of Ca2+ or EGTA, its U.V. spectrum between 240 and 290 nm and the activation of calmodulin-deficient cyclic AMP phosphodiesterase. The yield was 370 mg/kg wet wt. mRNA was also extracted from the same tissue and translated in a wheat-germ cell-free translation system. Translated calmodulin was identified by its heat-stability, its co-migration with authentic anterior-pituitary calmodulin on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, its acidic isoelectric point (4.15) on flat-bed isoelectric focusing, its Ca2+-dependent binding to fluphenazine-Sepharose 6B, and its co-elution from this gel with authentic unlabelled calmodulin with EGTA buffer. Calmodulin was not translated as a precursor form. In this tissue it was calculated that calmodulin accounted for 0.5-1% of the total translated protein.     

Calmodulin Affinity for Brain Coated Vesicle Proteins

A systematic characterization of the affinity of calmodulin for brain coated vesicles was undertaken. Binding of 125I-labeled calmodulin to coated vesicles was saturable and competed with unlabeled calmodulin, but not with troponin-C. Scatchard analysis revealed one high-affinity, low-capacity binding site, KD = 3.9 +/- 0.6 nM, Bmax = 16.3 +/- 2.4 pmol/mg, and one low-affinity, high-capacity binding site, KD = 102 +/- 15.0 nM, Bmax = 151 +/- 23.0 pmol/mg. Radioimmunoassay revealed that coated vesicles contain 1.05 microgram calmodulin/mg protein. Because this value remained constant even after removal of clathrin, the major coat protein, from the coated vesicle, it is apparent that calmodulin is associated with the vesicle per se rather than with its clathrin lattice. When a Triton X-100-treated extract of coated vesicles was passed through a Sepharose 4B-calmodulin affinity column, polypeptides with Mrs (molecular weights) of 100,000, 55,000, and 30,000 bound in a Ca2+-dependent manner. A 30,000 Mr protein doublet purified from coated vesicles was completely eluted by EGTA from the calmodulin affinity column, confirming that this protein doublet represents one of the coated vesicle calmodulin binding sites. Because calmodulin stimulated [Ca2+-Mg2+]-ATPase activity as well as Ca2+ uptake in coated vesicles, it is postulated that the 100,000 and 55,000 Mr calmodulin binding proteins represent the [Ca2+-Mg2+]-ATPase complex, the other coated vesicle calmodulin binding site.     

Preparation and properties of calcium-dependent resins with increased selectivity for calmodulin

Calmodulin from both animal and plant sources is known to bind a number of hydrophobic compounds with resultant inhibition of calmodulin function. Some of these compounds, including certain phenothiazine and naphthalene sulfonamide derivatives, have been previously shown to be useful in the chromatographic isolation of calmodulin, when covalently linked to a solid support. With the exception of fluphenazine linked to epoxide-activated Sepharose, these resins have the undesirable characteristics of requiring high salt concentrations in the elution buffer for efficient elution of calmodulin, thus decreasing the selectivity for this protein. The synthesis of nine Sepharose-ligand affinity resins is reported. Some of the ligands are newly synthesized naphthalene sulfonamide and phenothiazine derivatives. The synthetic ligands have been coupled to three types of Sepharose: epoxide-activated, CNBr-activated, and carbodiimide-activated. The properties of these resins are reported and their relative abilities to act selectively in the isolation of calmodulin are compared. 2-Trifluoromethyl-10-aminopropyl phenothiazine (TAPP), when linked to epoxide-activated Sepharose, was found to be the most useful for calmodulin isolation in terms of its combined stability, capacity, and ability to select for calmodulin. This resin was found to behave as a true affinity resin. A quantitative evaluation of its affinity behavior was consistent with the presence of two high-affinity Ca2+-dependent phenothiazine binding sites on calmodulin, in apparent agreement with previous reports which involved the use of different methods.     

Calmodulin-regulated binding of the 90-kDa heat shock protein to actin filaments

We have found that the 90-kDa heat shock protein (HSP90) prepared from a mouse lymphoma exists in homodimeric form under physiological conditions and has the ability to bind to F-actin (Koyasu, S., Nishida, E., Kadowaki, T., Matsuzaki, F., Iida, K., Harada, F., Kasuga, M., Sakai, H., and Yahara, I. (1986) Proc. Natl. Acad. Sci. U.S.A., in press). Here we show that calmodulin regulates the binding of HSP90 to F-actin in a Ca2+-dependent manner. The binding of HSP90 to F-actin occurred optimally under physiological solution conditions, i.e. in 2 mM MgCl2 + 100 mM KCl. The binding was saturable in a molar ratio of about 1 HSP90 (dimer) to 10 actins. HSP90 was dissociated from F-actin by the binding of tropomyosin to F-actin. Calmodulin was found to inhibit the binding of HSP90 to F-actin in a Ca2+-dependent manner. Moreover, the equilibrium gel filtration demonstrated that calmodulin binds to HSP90 in the presence of Ca2+, but not in the absence of Ca2+. These data indicate that HSP90 complexed with Ca2+-calmodulin is unable to bind to F-actin. Ca2+-dependent interaction of HSP90 with calmodulin as well as calmodulin-regulated binding of HSP90 to F-actin revealed here may provide new insight into the function of HSP90 and the regulation of actin structure in cells.