©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
A New Function for Adducin
CALCIUM/CALMODULIN-REGULATED CAPPING OF THE BARBED ENDS OF ACTIN FILAMENTS (*)

(Received for publication, November 21, 1995; and in revised form, January 24, 1996)

Philip A. Kuhlman (1) Christine A. Hughes (2) Vann Bennett (2) Velia M. Fowler (1)(§)

From the  (1)Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037 and the (2)Howard Hughes Medical Institute and the Departments of Biochemistry and Cell Biology, Duke University Medical Center, Durham, North Carolina 27710

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Adducin is a membrane skeleton protein originally described in human erythrocytes that promotes the binding of spectrin to actin and also binds directly to actin and bundles actin filaments. Adducin is associated with regions of cell-cell contact in nonerythroid cells, where it is believed to play a role in regulating the assembly of the spectrin-actin membrane skeleton. In this study we demonstrate a novel function for adducin; it completely blocks elongation and depolymerization at the barbed (fast growing) ends of actin filaments, thus functioning as a barbed end capping protein (K 100 nM). This barbed end capping activity requires the intact adducin molecule and is not provided by the NH(2)-terminal globular head domains alone nor by the COOH-terminal extended tail domains, which were previously shown to contain the spectrin-actin binding, calmodulin binding, and phosphorylation sites. A novel difference between adducin and other previously described capping proteins is that it is down-regulated by calmodulin in the presence of calcium. The association of stoichiometric amounts of adducin with the short erythrocyte actin filaments in the membrane skeleton indicates that adducin could be the functional barbed end capper in erythrocytes and play a role in restricting actin filament length. Our experiments also suggest novel possibilities for calcium regulation of actin filament assembly by adducin in erythrocytes and at cell-cell contact sites in nonerythroid cells.


INTRODUCTION

Precise control of actin filament length is an important functional consideration in a number of tissues including striated muscle (1) and the erythrocyte membrane skeleton(2) . These two examples are similar with respect to the uniform lengths of the filament population, although the actual lengths themselves are different, 1 µm in skeletal muscle (3) and 33-37 nm in erythrocytes (4, 5, 6) . These uniform length distributions suggest strict regulation of actin filament growth because pure actin filaments polymerize to an exponential length distribution at steady state in vitro(7) . In muscle, control of actin filament growth is achieved by capping the fast growing (barbed) ends with capZ (8, 9) and by capping the slow growing (pointed) ends with tropomodulin(10, 11, 12) . In erythrocytes, tropomodulin caps the pointed ends of the short actin filaments(11, 13, 14) , but a barbed end capping protein has not yet been identified.

A conserved mechanism of actin filament length regulation between striated muscle and erythrocytes would require a barbed end capping protein to be identified in erythrocytes. There are five known actin binding proteins that are associated with the short erythrocyte actin filaments: spectrin, band 4.1, tropomyosin, tropomodulin, band 4.9, and adducin. Most of these proteins can be ruled out because they bind along the sides of actin filaments and have been shown directly not to block actin polymerization from the barbed filament ends (for reviews see (15, 16, 17) ). However, several observations raised the possibility that adducin, which was initially characterized as a calmodulin-binding protein(18) , might be a candidate for a novel erythrocyte barbed end capping protein. Adducin is associated in stoichiometric amounts with the short erythrocyte actin filaments (one alpha,beta heterodimer per filament) (18) and has been shown to bind to spectrin-actin complexes and promote spectrin binding to actin (K = 80 nM)(19, 20) , as well as to bind directly to F-actin (K = 280 nM) and to bundle actin filaments(20, 21) . Curiously, although the 39-kDa NH(2)-terminal globular ``head'' domains of both alpha and beta adducin contain a region of sequence homology to the actin-binding domain of the alpha-actinin family of actin binding proteins(22) , the isolated head domains of either alpha or beta adducin do not appear to bind F-actin or spectrin-actin complexes in cosedimentation assays (23) . Indeed, the 33-kDa COOH-terminal extended tail domains of both the alpha and beta adducin subunits were recently shown to be sufficient for binding to spectrin-actin complexes and for recruitment of additional spectrin molecules(24) . Thus, the functional significance of the actin binding sequence homology in the head domain of adducin remained an open question and suggested that adducin might have additional actin-binding properties.

In this study, we show that whole adducin blocks elongation and depolymerization from the fast growing (barbed) ends of actin filaments. In contrast, neither the isolated head or tail domains have barbed end capping activity on their own. We further demonstrate that unlike other barbed end capping proteins, the capping activity of adducin is down-regulated by calmodulin in the presence of calcium. Sequencing of adducin cDNAs from erythrocytes and other tissues has demonstrated that adducins are a unique family of proteins associated with the spectrin-based membrane skeleton in erythroid and nonerythroid cells (for a review see (16) ). The combination of adducin's calcium/calmodulin-regulated barbed end capping, actin bundling, and enhancement of spectrin-actin binding activities indicates that adducins represent a new type of regulated actin filament binding and barbed end capping protein.


EXPERIMENTAL PROCEDURES

Purification of Proteins

Actin was purified from rabbit skeletal muscle acetone powder (25) and subsequently gel filtered on Superose 6 to remove actin nuclei and minor contaminants. Pyrene-labeled actin was prepared by the method of Kouyama and Mihashi (26) with the modifications of Weber et al.(27) ; labeled and unlabeled actin was stored on ice in buffer A (see below) and used within 1 week. Human erythrocyte adducin was prepared from the low salt extract of human erythrocyte ghosts by the method of Bennett (28) as modified by Hughes and Bennett (24) and stored at -80 °C in storage buffer (10% sucrose, 10 mM sodium phosphate, 0.05% Tween 20, 1 mM sodium azide, 1 mM EDTA, and 1 mM dithiothreitol). Calmodulin was purified from bovine brain as described(29) . Nonmuscle capping protein (30) was purified from erythrocyte cytosol. (^1)The adducin tail domains were expressed using the bacterial expression vector pGEMEX and purified as described in Hughes and Bennett(24) . The adducin head domain was purified after digestion of human erythrocyte adducin with trypsin(23) .

Actin Polymerization Assays

Buffer A (2 mM Tris-HCl, 0.2 mM Na(2)ATP, 0.5 mM 2-mercaptoethanol, 0.2 mM CaCl(2), 0.005% sodium azide, final pH = 8.0 at 25 °C) was used in all assays. In all pyrene fluorescence experiments the amount of labeled actin was 5% to prevent internal quenching of the probe. The assay used to quantitate inhibition of actin polymerization at the barbed ends utilized the method of Casella et al. (8) in which polymerization was seeded using a crude low ionic strength extract of erythrocyte membranes containing spectrin-actin oligomeric complexes(8, 31) . Briefly, 5 µM G-actin was primed for rapid polymerization by addition of Mg to 0.2 mM and incubated for 5 min. Next, all other components of the assays were added (e.g. adducin at the concentrations indicated in the figures, 50 µg/ml spectrin-actin complexes), and actin polymerization was initiated by increasing the KCl concentration to 1 mM and the MgCl(2) concentration to 0.4 mM (final). Actin polymerization was followed by monitoring the increased pyrene fluorescence of labeled F-actin (excitation at 365 nm and emission of 407 nm) using a SLM8000 spectrofluorimeter. The fluorescence data were saved to a computer disc for subsequent analysis. Actin depolymerization was initiated by dilution of a 20 µM stock of F-actin (polymerized overnight at 4 °C) to a final concentration of 1 µM in buffer A alone or buffer A containing adducin or capping protein; actin depolymerization was indicated by a decrease in pyrene fluorescence. The sample temperature was maintained at a constant 25 °C for all actin polymerization experiments and at 37 °C for actin depolymerization experiments using a circulating water bath. Initial rates of polymerization and depolymerization were determined using the linear regression analysis feature of the graphics program Cricket Graph running on a Macintosh.

Actin Critical Concentration Assay

5 µM G-actin in buffer A was mixed with increasing amounts of adducin or capping protein and polymerized by the addition of KCl and Mg to 100 and 2 mM, respectively. After 24 h of incubation at 4 °C, F-actin was collected by sedimentation for 1 h at 435,000 times g in a Beckman TL100 rotor at 4 °C. (The sensitivity of adducin to proteolytic degradation during prolonged incubations at room temperature made it necessary to do this experiment at 4 °C.) The supernatant was carefully removed, and the G-actin remaining in the supernatant was precipitated by the addition of 10% trichloroacetic acid using tRNA as a carrier(32) . The precipitated G-actin and the F-actin were solubilized in equivalent volumes of SDS sample buffer and electrophoresed on 12% SDS-polyacrylamide gels(33) . The amount of actin in each sample was determined by pyridine elution of Coomassie Blue from the stained gel slices (34) and quantitated spectroscopically by reading the absorbance at 605 nm with reference to an actin standard curve.


RESULTS

Adducin Inhibits the Polymerization and Depolymerization of Actin from the Barbed Filament End

We used spectrin-actin complexes (8, 31) to seed actin polymerization in a low salt buffer (1 mM KCl, 0.4 mM Mg) where actin alone would otherwise not polymerize(8) . Under these conditions actin polymerization is expected to be limited to the barbed end due to the low salt concentration as well as the presence of the pointed end actin capping protein tropomodulin in the spectrin-actin complexes (11) . (^2)Fig. 1A shows that adducin dramatically suppressed the initial rate of polymerization from the spectrin-actin seeds. We quantitated the level of inhibition (capping) as a percentage of the initial polymerization rate in the absence of any adducin (100% capping indicates the complete cessation of actin polymerization). Increasing concentrations of adducin correlated with increasing inhibition of actin polymerization; plotting adducin concentration against the percentage of inhibition demonstrated that saturating concentrations of adducin can completely inhibit actin elongation from the barbed filament end (Fig. 1B). The adducin concentration that produced 50% capping was taken to be the K; under these conditions the K for adducin was approximately 100 nM. The inhibitory effect of adducin on actin elongation is specific for the barbed filament end because adducin does not inhibit the rate of elongation from the pointed ends of gelsolin-capped actin filaments (data not shown).


Figure 1: Effect of adducin on the rate of actin polymerization from the barbed ends of spectrin-actin seeds. A, raw data. Elongation was initiated by the addition of spectrin-actin seeds and salts to a 5 µM G-actin solution (5% pyrenyl-actin) containing increasing concentrations of adducin as indicated. B, percentage of inhibition of elongation (percent capping) plotted against increasing adducin concentrations. (The polymerization rate is calculated as a percentage of the initial rate of polymerization in the absence of adducin and is inversely proportional to capping.) The concentration of adducin required to produce 50% inhibition of polymerization (100 nM) was taken as a measure of the K.



Not only can adducin completely inhibit elongation from the barbed ends of actin filaments, but saturating amounts of adducin (700 nM) also greatly reduce the depolymerization rate of F-actin that has been diluted below its critical concentration (Fig. 2). This effect is primarily due to inhibition of depolymerization from the barbed end because under the conditions of our assays the higher off rate of the barbed ends dominates the depolymerization kinetics; for example, even complete inhibition of depolymerization at the pointed end in these assays would have been expected to lead to only about a 10% reduction in the rate of depolymerization(35) . Furthermore, we observe a similar reduction in the initial rate of actin depolymerization in the presence of saturating amounts (30 nM) of nonmuscle capping protein isolated from erythrocytes (Fig. 2), a known barbed end capping protein (30) .^1 The initial rates of depolymerization for actin in the presence of adducin or capping protein were 53 and 56 arbitrary units, respectively, as compared with a rate of 754 arbitrary units for actin alone. Interestingly, after the initial period, the rate of actin depolymerization in the presence of adducin was slower than in the presence of capping protein. We attribute this difference to an effect of adducin on the pointed filament end because adducin reduced (but did not block) the rate of actin depolymerization from the pointed ends of gelsolin capped actin filaments (data not shown). The inhibitory effect of adducin on the rate of actin depolymerization from the barbed filament ends also indicates that adducin does not function by exclusively sequestering actin monomers; monomer sequestering would have been expected to lead to an increase in the rate of actin disassembly, which was also not observed (see below). Finally, these data also rule out possible artifacts in the nucleated actin polymerization assay in Fig. 1from fluorescence signal quenching due to light scattering and demonstrate that protein aggregation does not occur in these assays. Taken together, the ability of adducin to inhibit both actin polymerization and depolymerization indicates that adducin is a barbed end capping protein with a capping affinity of 100 nM.


Figure 2: Effect of adducin on the rate of actin depolymerization. Depolymerization of pyrene-labeled F-actin was initiated by dilution of a 20 µM stock to a final concentration of 1 µM into buffer A with or without adducin or nonmuscle capping protein as indicated. All measurements utilized a single actin stock to maintain a constant filament number in the assay. Final concentrations in the assay of intact adducin were 700 nM, and that of nonmuscle capping protein was 30 nM. Under the low salt conditions used in this assay, the final actin concentration after dilution (1 µM) is less than the actin critical concentration under these conditions (approx120 µM). This leads to the complete disassembly of the actin filaments into monomers, as indicated by the decrease in the pyrene fluorescence signal for the pure actin curve almost to the baseline. CP, nonmuscle capping protein.



Complete capping of barbed filament ends by previously described barbed end capping proteins results in an increase of the actin monomer concentration at steady state with the filaments, reflecting the higher critical concentration for the pointed ends(35, 36) . Fig. 3A shows that saturating concentrations of nonmuscle capping protein isolated from erythrocytes resulted in about a 3-fold increase in the amount of actin monomer remaining in the supernatant at steady state from 0.4 to 1.3 µM. In contrast, increasing concentrations of adducin (from 100 to 700 nM) led to a considerably smaller increase in the amount of actin monomer in the supernatant from 0.3 to 0.6 µM (Fig. 3B). This result suggests that adducin may reduce the critical concentration at the pointed end of the filament in addition to blocking monomer addition and loss at the barbed filament end. This interpretation is consistent with the observation that adducin reduces the rate of depolymerization while increasing the rate of elongation at the pointed filament end (data not shown). This may be a consequence of adducin reducing the actin monomer off rate for the pointed filament end by its binding along the sides of actin filaments. Finally, the observation that increasing amounts of adducin do not lead to continuously increasing amounts of nonpolymerizable actin also demonstrates that the effects of adducin on polymerization kinetics at the barbed filament end are not solely due to monomer sequestration.


Figure 3: The effect of increasing concentrations of nonmuscle capping protein (A) or adducin (B) on actin critical concentration. The extent of actin polymerization was determined after a 24-h incubation by centrifugation followed by SDS-polyacrylamide gel electrophoresis of supernatants and pellets as described under ``Experimental Procedures.'' The amount of actin remaining in the supernatant was taken to be the actin monomer concentration (actin critical concentration) and plotted versus the concentration of nonmuscle capping protein and adducin using the same y axis to facilitate comparison. The actin critical concentration in the absence of adducin or capping protein varied from about 0.3-0.4 µM (this experiment) down to about 0.05 µM in other experiments; however, the relative increases in the actin critical concentration in the presence of adducin or nonmuscle capping protein were similar in all experiments.



In an effort to determine whether the actin filament capping activity of adducin could be localized to either the NH(2)-terminal globular head or to the COOH-terminal extended tail domains, we tested bacterially expressed tails (24) and a head-neck domain prepared by trypsin digestion of human erythrocyte adducin(23) . Neither the head nor the tail domains (tested up to final concentrations of 1 and 5 µM, respectively) had any significant effects on actin polymerization or depolymerization in our assays (data not shown). Therefore, the barbed end capping activity of adducin appears to be an attribute of the entire molecule requiring both the heads and tails.

The Effect of Calmodulin on the Barbed End Capping Activity of Adducin

Calmodulin has previously been shown to bind to adducin with a K(d) of 0.2-0.5 µM in the presence of calcium(18) . Calcium-dependent binding of calmodulin to adducin prevents adducin from binding to F-actin as well as reducing its ability to bind to spectrin-actin complexes and recruit additional spectrin molecules to the spectrin-actin complexes(19, 20) . Fig. 4A shows that calmodulin in conjunction with calcium was also able to reverse the inhibitory effect of adducin on actin polymerization from the barbed filament end. A plot of the percentage of adducin capping activity against calmodulin concentration produced a curve with half-maximal reversal of capping at about 2 µM calmodulin (Fig. 4A). The ability of calmodulin to eliminate the barbed end capping activity of adducin was also seen in actin depolymerization experiments as evidenced by the return of the depolymerization rate in the presence of adducin plus calmodulin to that of pure actin (Fig. 4B).


Figure 4: Effect of calmodulin on the ability of adducin to inhibit actin polymerization (A) and depolymerization (B) in the presence of calcium. A, percentage of capping calculated as described in the legend to Fig. 1B plotted against increasing concentrations of calmodulin. The ability of 200 nM adducin to inhibit polymerization from spectrin-actin seeds was tested as described above except with the inclusion of increasing concentrations of calmodulin as indicated. The standard assay conditions contained 0.2 mM CaCl(2) (see ``Experimental Procedures''). B, effect of calmodulin on the ability of adducin to inhibit the rate of actin depolymerization in the presence of calcium. This assay was performed as described above in Fig. 2but with diluting the F-actin into a solution containing adducin and calmodulin to give final concentrations of 700 nM and 10 µM, respectively. Calmodulin had no effect on actin polymerization in the absence of adducin (not shown).




DISCUSSION

Using both a nucleated actin polymerization assay (8) and an F-actin depolymerization assay, we have shown that adducin caps the barbed ends of actin filaments with a K approx 100 nM. The association of stoichiometric amounts of adducin with the short actin filaments in the erythrocyte membrane skeleton (18) implies that adducin may indeed be the functional barbed end cap in erythrocytes and supports our hypothesis that the mechanism of restriction of actin filament length in erythrocytes is based on capping actin filaments at both ends to prevent growth or shrinking, similar to striated muscle. Although it has been assumed that the barbed ends of the actin filaments in the erythrocyte membrane skeleton are uncapped, this idea stems mainly from the use of purified membranes or isolated spectrin-actin oligomeric complexes to nucleate actin polymerization(32, 37, 38, 39) . Our results raise the possibility that the capping activity of adducin might have been inactivated during hemolysis and/or the extraction procedures used to prepare membranes and purify the spectrin-actin complexes used in these experiments.

Calmodulin has been demonstrated to regulate the mechanical properties of the erythrocyte membrane in the presence of calcium(40) . The inhibitory effect of calcium/calmodulin on the ability of adducin to cap the barbed ends of actin filaments suggests that the effect of calmodulin on membrane mechanics may be partly mediated via changes in actin filament polymerization. Calcium/calmodulin also inhibits adducin binding to F-actin and to spectrin-actin complexes(19, 20) , as well as inhibiting spectrin-protein 4.1 cross-linking of actin filaments(41, 42) . These observations emphasize that calmodulin is likely to regulate membrane mechanical properties via multiple effects on membrane skeleton organization.

The capping affinity of adducin is considerably lower than has been described for other barbed end capping proteins (e.g. gelsolin = 1 pM; capping protein = 1 nM; for reviews see (9) and (36) ) and is similar to the affinity of adducin for binding to the sides of actin filaments and bundling them (K(d) = 280 nM)(20) . It is also striking that the inhibitory effect of calmodulin on the barbed end capping activity of adducin has a similar dependence on calmodulin concentration as that of calmodulin's inhibition of adducin side binding to F-actin and to spectrin-actin complexes(19, 20) . Furthermore, the calmodulin binding site on adducin has been proposed to be located on the midregion of the COOH-terminal tail domains(43) ; yet dissection of adducin into NH(2)-terminal head and COOH-terminal tail domains shows that the entire molecule is required for barbed end capping activity. Therefore, the barbed end capping activity of adducin may be functionally linked to its ability to bind along the sides of actin filaments.

The actin-binding sequence homology in the NH(2)-terminal head domain of the adducin alpha and beta subunits diverges significantly from the consensus actin-binding domain found in the functional members of the alpha-actinin protein family (similarity: alpha-adducin 47.9%, beta-adducin 40.7%; identity: alpha-adducin 21.6%, beta-adducin 12.8%). (^3)In addition, both the alpha and beta subunits of adducin deviate from the sequence found in alpha-actinin by the presence of an extra portion of sequence; in the case of the alpha subunit, this is in one of the regions identified as the actin-binding site(44) . The divergence from the consensus actin-binding sequence has been suggested to explain the lack of binding to actin filaments by the head domain of adducin(22) . However, we would propose that this actin-binding site in the NH(2)-terminal head domain of adducin may have, through divergent evolution, developed the capability to bind to actin so as to cap the barbed end of the filament. Actin monomers in the filament are oriented such that subdomains 1 and 3 are exposed at the barbed end of the actin filament(45, 46) . It may be significant that a portion of the alpha-actinin actin binding site has been demonstrated to be on subdomain 1 of actin(47, 48, 49) .

Based on these considerations, we propose that the barbed end capping activity of the NH(2)-terminal head domain is reliant on the side binding of the adducin COOH-terminal tails to actin filaments. It is likely that the COOH-terminal tail domains of the alpha and beta subunits possess an F-actin binding site based on their ability to bind to spectrin-actin complexes and to promote spectrin binding to actin (24) . Binding of the tails along each strand of the actin filament in a polarized fashion (21) could position the head domains to bind to a site in subdomain 1 of the terminal actin monomer at the barbed filament end, hence interfering with association of new actin monomers as well as stabilizing the filament and preventing monomer dissociation. A model depicting binding of adducin tails to actin and capping the barbed end of the actin filament by adducin heads is shown in Fig. 5. In addition to promoting spectrin binding to actin, binding of adducin tails along the sides of the erythrocyte actin filaments may also play a role in stabilizing them at their pointed filament ends, as suggested by the actin critical concentration experiment (Fig. 3). The multifunctional actin binding properties of adducin may not be unique; tensin is another protein that has been shown to cap actin filament barbed ends as well as to bind along the sides of actin filaments and bundle them(50, 51) .


Figure 5: A schematic model for actin capping by adducin. The barbed end of an erythrocyte actin filament is shown with six associated spectrin molecules(4, 6) . For clarity only the NH(2)-terminal actin-binding region of beta-spectrin is depicted, and alpha-spectrin, protein 4.9, protein 4.1, and the tropomodulin and tropomyosin at the pointed end of the actin filament have been omitted. The NH(2)-terminal adducin head domains form a tetramer (24) at the barbed end of the filament blocking the exchange of actin monomers. The extended COOH-terminal adducin tails lie along each strand of the actin filament forming contacts between the actin and spectrin molecules, enhancing their interaction. We propose that this lateral actin binding by the adducin tails is responsible for maintenance of the cap at the end of the filament. Recruitment of extra spectrin molecules is either due to increased affinity of spectrin for the complex or the reorganization of the bound spectrin to accommodate more molecules. Hence adducin plays a crucial role in the organization of the molecular interactions at the barbed end of the actin filaments in erythrocytes.



The actin capping properties of adducin suggest that adducin could provide a link between membranes and the barbed ends of spectrin-associated actin filaments. In erythrocytes, adducin has been shown to bind directly to stomatin(52) , a membrane protein that is associated with ion channels and has been genetically linked to hereditary stomatocytosis(2) . In nonerythroid cells, adducin is associated with the spectrin-based membrane skeleton at cell-cell contact sites on the lateral borders of the plasma membrane of epithelial cells(53) . Protein kinase C phosphorylation of adducin in cultured epithelial cells induces redistribution of adducin away from cell-cell contact sites(53) . Functional linkage of adducin's barbed end capping activity to the filament side binding activity of the tails may imply that phosphorylation of tails by protein kinase C (23) could also regulate adducin's capping function, as we have demonstrated here for calcium/calmodulin. It is tempting to speculate that adducin could be a regulatory target for signal transduction pathways that lead to remodeling of the actin cytoskeleton at cell-cell contact sites.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants GM34225 (to V. M. F.) and DK29808 (to V. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Cell Biology, MB24, The Scripps Research Inst., 10666 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 619-554-8277; Fax: 619-554-8753; Velia{at}riscsm.scripps.edu.

(^1)
P. A. Kuhlman and V. M. Fowler, manuscript in preparation.

(^2)
V. M. Fowler, unpublished observations.

(^3)
P. A. Kuhlman, unpublished observations.


ACKNOWLEDGEMENTS

We are grateful to Dr. Charles Cochrane for allowing us to use the spectrofluorimeter and to Lydia Davis (Duke Medical Center) for the generous gift of calmodulin. We acknowledge the Scripps General Clinical Research Center for the provision of normal human blood (supported by National Institutes of Health Grant M01 RR00833).


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