Characterization of acidic Ca(2+)-independent phospholipase A2 of bovine lung

The Gram-positive bacterium Staphylococcus aureus infects diverse tissues and causes a wide spectrum of diseases, suggesting that it possesses a repertoire of distinct molecular mechanisms promoting bacterial survival in disparate in vivo environments. Signature-tag transposon mutagenesis screening of a 1520-member library identified numerous S. aureus genetic loci affecting growth and survival in four complementary animal infection models including mouse abscess, bacteraemia and wound and rabbit endocarditis. Of a total of 237 in vivo attenuated mutants identified by the murine models, less than 10% showed attenuation in all three models, emphasizing the advantage of screening in diverse disease environments. The largest gene class identified by these analyses encoded peptide and amino acid transporters, some of which were important for S. aureus survival in all animal infection models tested. The identification of staphylococcal loci affecting growth, persistence and virulence in multiple tissue environments provides insight into the complexities of human infection and on the molecular mechanisms that could be targeted by new antibacterial therapies.     

Reduction of myocardial cross-bridge turnover rate in presence of EMD 53998, a novel Ca(2+)-sensitizing agent

We have studied the effect of EMD 53998 (5-(1-(3,4-dimethoxybenzoyl)-1,2,3,4-tetrahydrochinolin-6-yl)-6-me thyl-3,6-dihydro-2H-1,3,4-thiadiazin-2-one) on cross-bridge turnover rate at varying Ca2+ concentrations. Cross-bridge cycling rate was estimated both by adenosine triphosphatase measurements and determination of mechanical characteristics of constantly activated fibres, which is assumed to reflect cross-bridge kinetics. The results indicate that the turnover rate of myocardial cross-bridges was reduced in the presence of EMD 53998 at low Ca2+ concentrations (pCa greater than or equal to 6.25), but not at higher Ca2+ concentrations (pCa less than or equal to 5.85).     

Mechanisms of isoflurane-increased submaximum Ca2+-activated force in rabbit skinned femoral arterial strips

The role of ftsK in the growth of Escherichia coli was examined by turning off its expression. This resulted in smooth filaments without constrictions, indicating that FtsK was required at an early step in septation. Consistent with this, FtsK was found to localize to the septum in 70% of the cells, indicating that it was recruited relatively early in this process. FtsK localization required the function of FtsZ and FtsA but not FtsI and FtsQ. Consistent with this, Z rings were present in FtsK-depleted filaments. Subcellular localization of FtsK confirmed that it was a membrane protein. Only the first 202 amino acids of FtsK were essential for its role in membrane localization, cell division and viability. The expression of ftsK increased as part of the SOS response, and increased expression of ftsK conferred increased resistance to DNA damage.     

Effects of cyclic adenosine monophosphate (cAMP) on sarcoplasmic reticulum Ca(2+)-loading in failing rabbit and human cardiac trabeculae

The response of cardiac SR Ca(2+)-loading to cAMP in failing rabbit and human myocardium was examined. Right ventricular (RV) trabeculae were isolated and mounted for isometric tension measurement. They were treated with saponin to permeabilise the sarcolemma but retain SR function, and bathed in a mock intracellular solution including adenosine triphosphate (ATP) and buffered calcium. Caffeine (10 mM) was used to release calcium from the SR. The amplitude of the caffeine-induced contracture was used as a quantitative gauge of the calcium content of the SR. Trabeculae were isolated from rabbits with coronary ligation-induced heart failure (LIG, n = 11), sham operated controls (SH, n = 10), isoprenaline-infused rabbits (ISO, 7 days mini-osmotic pump 100 micrograms/kg.h; n = 7) and saline-infused controls (SAL, n = 7). Failing human RV trabeculae were obtained at the time of cardiac transplantation. Failing rabbit trabeculae demonstrated increased baseline caffeine-induced contractures compared with controls, the response to cAMP was similar in the two groups (LIG 9.3 +/- 2.8 vs SH 10.6 +/- 3.2% Fmax; P = 0.55), There was no difference in the baseline SR Ca(2+)-loading in ISO trabeculae compared with SAL controls but there was a marked difference in the response to cAMP (11.1 +/- 5.4 vs 4.2 +/- 2.1% Fmax, P = 0.02). SR Ca(2+)-loading in failing human RV trabeculae was related to the severity of LV dysfunction (r = 0.59, P = 0.04) and demonstrated a marked cAMP-induced enhancement of caffeine-contracture (20.2 +/- 4.7% increase of Fmax) which was greater in patients with low compared with high ejection fraction. While beta-receptors are known to be down regulated in heart failure these results suggest that the scope for cAMP-mediated enhancement of SR Ca(2+)-loading is maintained.     

Activation of protein kinase C induces cortical granule exocytosis in a Ca(2+)-independent manner, but not the resumption of cell cycle in porcine eggs

N-Acetylneuraminic acid (1) is a common sugar in many biological recognition processes. Neuraminidase enzymes recognize and cleave terminal sialic acids from cell surfaces. Viral entry into host cells requires neuraminidase activity, thus inhibition of neuraminidase is a useful strategy for development of drugs for viral infections. A recent crystal structure for influenza viral neuraminidase with sialic acid bound shows that the sialic acid is in a boat conformation [Prot Struct Funct Genet 14: 327 (1992)]. Our studies seek to determine if structural pre-organization can be achieved through the use of sialyllactones. Determination of whether siallylactones are pre-organized in a binding conformation requires conformational analysis. Our inability to find a systematic study comparing the results obtained by various computational methods for carbohydrate modeling led us to compare two different conformational analysis techniques, four different force fields, and three different solvent models. The computational models were compared based on their ability to reproduce experimental coupling constants for sialic acid, sialyl-1,4-lactone, and sialyl-1,7-lactone derivatives. This study has shown that the MM3 forcefield using the implicit solvent model for water implemented in Macromodel best reproduces the experimental coupling constants. The low-energy conformations generated by this combination of computational methods are pre-organized toward conformations which fit well into the active site of neuraminidase.     

The oxidative inactivation of sarcoplasmic reticulum Ca(2+)-ATPase by peroxynitrite

The oxidative inactivation of rabbit skeletal muscle Ca(2+)-ATPase in sarcoplasmic reticulum (SR) vesicles by peroxynitrite (ONOO-) was investigated. The exposure of SR vesicles (10 mg/ml protein) to low peroxynitrite concentrations ( < or = 0.2 mM) resulted in a decrease of Ca(2+)-ATPase activity primarily through oxidation of sulfhydryl groups. Most of this deactivation (ca.70%) could be chemically reversed by subsequent reduction of the enzyme with either dithiothreitol (DTT) or sodium borohydride (NaBH4), indicating that free cysteine groups were oxidized to disulfides. The initial presence of 5 mM glutathione failed to protect the SR Ca(2+)-ATPase activity. However, as long as peroxynitrite concentrations were kept < or = 0.45 mM, the efficacy of DTT to reverse Ca(2+)-ATPase inactivation was enhanced for reaction mixtures which initially contained 5 mM glutathione. At least part of the disulfides were formed intermolecularly since gel electrophoresis revealed protein aggregation which could be reduced under reducing conditions. The application of higher peroxynitrite concentrations ( > or = 0.45 mM) resulted in Ca(2+)-ATPase inactivation which could not be restored by exposure of the modified protein to reducing agents. On the other hand, treatment of modified protein with NaBH4 recovered all SR protein thiols. This result indicates that possibly the oxidation of other amino acids contributes to enzyme inactivation, corroborated by amino acid analysis which revealed some additional targets for peroxynitrite or peroxynitrite-induced processes such as Met, Lys, Phe, Thr, Ser, Leu and Tyr. Tyr oxidation was confirmed by a significant lower sensitivity of oxidized SR proteins to the Lowry assay. However, neither bityrosine nor nitrotyrosine were formed in significant yields, as monitored by fluorescence spectroscopy and immunodetection, respectively. The Ca(2+)-ATPase of SR is involved in cellular Ca(2+)-homeostasis. Thus, peroxynitrite mediated oxidation of the Ca(2+)-ATPase might significantly contribute to the loss of Ca(2+)-homeostasis observed under biological conditions of oxidative stress.     

Characterization of Ca(2+) flows essential in ornithine decarboxylase induction by L-asparagine in rat hepatoma cells using Ca(2+) flow inhibitors

L-Asparagine stimulates bi-directional Ca(2+) flows and induces ornithine decarboxylase in Reuber H-35 hepatoma cells. Previously it has been shown that these effects are completely, but reversibly inhibited by lanthanum chloride. In this study we examined the role(s) of Ca(2+) flows using more specific Ca(2+) flow inhibitors. It was shown that ornithine decarboxylase induction was inhibited by CdCl(2) and verapamil at concentrations above 1 mu M and 100 mu M respectively, but was unaffected by as much as 300 mu M NiCl(2), 1 mM nifedipine, or 10 mu M omega-conotoxin. Enzyme induction was blocked by the Ca(2+)-ATPase pump antagonists vanadate and Compound 48/80 in a dose-dependent manner. These results, taken together with the observations that extracellular Ca(2+) is essential for enzyme induction but a substantial elevation of cytoplasmic [Ca(2+)] is not, suggest that Ca(2+) inflow independent of the receptor-activated Ca(2+) channels, and the Ca(2+)-ATPase mediated Ca(2+) out-flow, are both important factors in the action of L-asparagine.     

Ca(2+)-dependent and Ca(2+)-independent isozymes of protein kinase C mediate exocytosis in antigen-stimulated rat basophilic RBL-2H3 cells. Reconstitution of secretory responses with Ca2+ and purified isozymes in washed permeabilized cells

Rat basophilic RBL-2H3 cells, which exhibit Ca(2+)-dependent secretion of granules when stimulated with antigen, contained the Ca(2+)-dependent alpha and beta and the Ca(2+)-independent delta, epsilon, and zeta isoforms of protein kinase C. These isoforms associated, to variable extents (i.e. delta the most and zeta the least), with the membrane fraction upon antigen stimulation but without external Ca2+; only the Ca(2+)-independent isoforms did so. Both types of isozymes were probably necessary for optimal responses to antigen as indicated by the following observations. All Ca(2+)-dependent isozymes were degraded in cells treated with 20 nM phorbol 12-myristate 13-acetate for 6 h, whereas the Ca(2+)-independent isozymes were not degraded and were retained when the cells were subsequently permeabilized and washed. Cells so treated still exhibited antigen-induced secretion (25-33% of normal) which was suppressed by selective inhibitors of protein kinase C (Ro31-7549 and calphostin C) thereby indicating a possible contribution of the Ca(2+)-independent isozymes in secretion. Normally, washed permeabilized cells lost all isozymes of protein kinase C and failed to secrete in response to antigen. A full secretory response to antigen could be reconstituted by the subsequent addition of nanomolar concentrations of either beta or delta isozymes of protein kinase C (other isozymes were much less effective) but only in the presence of 1 microM free Ca2+ to indicate distinct roles for Ca2+ and protein kinase C in exocytosis.     

The structure of calcyclin reveals a novel homodimeric fold for S100 Ca(2+)-binding proteins

The S100 calcium-binding proteins are implicated as effectors in calcium-mediated signal transduction pathways. The three-dimensional structure of the S100 protein calcyclin has been determined in solution in the apo state by NMR spectroscopy and a computational strategy that incorporates a systematic docking protocol. This structure reveals a symmetric homodimeric fold that is unique among calcium-binding proteins. Dimerization is mediated by hydrophobic contacts from several highly conserved residues, which suggests that the dimer fold identified for calcyclin will serve as a structural paradigm for the S100 subfamily of calcium-binding proteins.     

Force regulation by Ca2+ in skinned single cardiac myocytes of frog

Atrial and ventricular myocytes 200 to 300 microm long containing one to five myofibrils are isolated from frog hearts. After a cell is caught and held between two suction micropipettes the surface membrane is destroyed by briefly jetting relaxing solution containing 0.05% Triton X-100 on it from a third micropipette. Jetting buffered Ca2+ from other pipettes produces sustained contractions that relax completely on cessation. The pCa/force relationship is determined at 20 degrees C by perfusing a closely spaced sequence of pCa concentrations (pCa = -log[Ca2+]) past the skinned myocyte. At each step in the pCa series quick release of the myocyte length defines the tension baseline and quick restretch allows the kinetics of the return to steady tension to be observed. The pCa/force data fit to the Hill equation for atrial and ventricular myocytes yield, respectively, a pK (curve midpoint) of 5.86 +/- 0.03 (mean +/- SE.; n = 7) and 5.87 +/- 0.02 (n = 18) and an nH (slope) of 4.3 +/- 0.34 and 5.1 +/- 0.35. These slopes are about double those reported previously, suggesting that the cooperativity of Ca2+ activation in frog cardiac myofibrils is as strong as in fast skeletal muscle. The shape of the pCa/force relationship differs from that usually reported for skeletal muscle in that it closely follows the ideal fitted Hill plot with a single slope while that of skeletal muscle appears steeper in the lower than in the upper half. The rate of tension redevelopment following release restretch protocol increases with Ca2+ >10-fold and continues to rise after Ca2+ activated tension saturates. This finding provides support for a strong kinetic mechanism of force regulation by Ca2+ in frog cardiac muscle, at variance with previous reports on mammalian heart muscle. The maximum rate of tension redevelopment following restretch is approximately twofold faster for atrial than for ventricular myocytes, in accord with the idea that the intrinsic speed of the contractile proteins is faster in atrial than in ventricular myocardium.     

Evidence for mitochondrial Ca(2+)-induced Ca2+ release in permeabilised endothelial cells

Generally most intracellular Ca2+ is stored in the endoplasmic reticulum (ER) and mitochondria. Recently a mitochondrial Ca(2+)-induced Ca2+ release (mCICR) mechanism, unconnected with ryanodine receptors (RyR's), has been shown in tumour cells. The existence of a mitochondrial Ca2+ release mechanism in BAE cells was investigated using saponin-permeabilised BAE cells. When buffered intracellular solution were 'stepped' from 10 nM to 10 microM free Ca2+, the mitochondrial inhibitors CN (2 mM), FCCP (1 microM), and RR (20 microM) significantly reduced total CICR by approximately 25%. The ER Ca(2+)-ATPase inhibitor thapsigargin (100 nM) had no effect. Furthermore, cyclosporin A (200 nM), an inhibitor of the mitochondrial permeability transition pore (PTP), abolished total CICR. Therefore, the novel ryanodine-caffeine insensitive CICR mechanism previously reported in BAE cells involves mitochondrial Ca2 release. It is proposed that in BAE cells, mCICR occurs via the mitochondrial PTP and may be physiologically important in endothelial cell Ca2+ signalling.     

Taurine regulation of Ca2+ uptake and (Ca(2+)+Mg2+)-ATPase in developing chick B-cells

1. The objective of the present study was to determine the effect of age and taurine on chick B cell calcium uptake and membrane (Ca(2+)+Mg2+)-ATPase activity in 1-4-week-old chicks. 2. The calcium uptake rate decreased with age (P < 0.05) and was further decreased by taurine (P < 0.05). 3. (Ca(2+)+Mg2+)-ATPase activity increased with age (P < 0.05) and was stimulated by taurine (P < 0.05). 4. The data demonstrate that the flux of calcium across the B-cell membrane changes during early post-hatch development, and that taurine regulates both the influx and efflux of calcium in chick B-cells.     

The C2 domain of the Ca(2+)-independent protein kinase C Apl II inhibits phorbol ester binding to the C1 domain in a phosphatidic acid-sensitive manner

There are two protein kinase Cs (PKCs) in the Aplysia nervous system, PKC Apl I, which is homologous to the Ca(2+)-activated PKC family, and PKC Apl II, which is homologous to the Ca(2+)-independent PKCs epsilon and eta. Purified PKC Apl I requires much less phosphatidylserine for activation than does purified PKC Apl II, and this may explain why the neurotransmitter serotonin activates PKC Apl I but not PKC Apl II in the intact nervous system [Sossin, W. S., Fan, X., and Baseri, F. (1996) J. Neurosci. 16, 10-18]. PKC Apl II's requirement for high levels of phosphatidylserine may be mediated by its C2 domain, since removal of this domain allows PKC Apl II to be activated at lower concentrations of phosphatidylserine. To begin to understand how this inhibition is mediated, we generated fusion proteins containing the C1 and C2 domains from PKC Apl II and determined their lipid dependence for phorbol ester binding. Our results indicate that the presence of the C2 domain lowers the affinity of protein kinase C activators for the C1 domains and this inhibition can be removed by phosphatidylserine. Phosphatidic acid, however, is much more potent than phosphatidylserine in reducing C2 domain-mediated inhibition, suggesting that phosphatidic acid may be a required cofactor for the activation of PKC Apl II.     

Mastoparan stimulates exocytosis at a Ca(2+)-independent late site in stimulus-secretion coupling. Studies with the RINm5F beta-cell line

Mastoparan, a tetradecapeptide from wasp venom, stimulated exocytosis in a concentration-dependent manner, which was enhanced by pertussis toxin pre-treatment, in the insulin secreting beta-cell line RINm5F. Mastoparan (3-20 microM) also elevated cytosolic free calcium concentration ([Ca2+]i), a rise that was not attenuated by nitrendipine. Divalent cation-free Krebs-Ringer bicarbonate (KRB) medium with 0.1 mM EGTA nullified the mastoparan-induced increase in [Ca2+]i, suggesting that the peptide increased Ca2+ influx but not through the L-type voltage-dependent Ca2+ channel. Depletion of the intracellular Ca2+ pool did not affect the mastoparan-induced elevation of [Ca2+]i. Remarkably, in divalent cation-free KRB medium with 0.1 mM EGTA and 2 microM thapsigargin in which mastoparan reduced [Ca2+]i, the mastoparan-stimulated insulin release was similar to that in normal Ca(2+)-containing KRB medium. Inhibitors of protein kinase C, such as bisindolylmaleimide, staurosporine, and 1-O-hexadecyl-2-O-methyl-rac-glycerol did not suppress the mastoparan-stimulated insulin release. Mastoparan at 10-20 microM did not increase cellular cAMP levels, nor did mastoparan at 5-10 microM affect [3H]arachidonic acid release. In conclusion, although mastoparan increased [Ca2+]i, this increase was not involved in the stimulation of insulin release. Rather, the data suggest that mastoparan directly stimulates exocytosis in a Ca(2+)-independent manner. As GTP-binding proteins (G proteins) are thought to be involved in the process of exocytosis and as mastoparan is known to exert at least some of its effects by activation of G proteins, an action of mastoparan to activate the putative stimulatory Ge (exocytosis) protein is likely.     

Signal transduction versus buffering activity in Ca(2+)-binding proteins

The large raspberry aphid, Amphorophora idaei, has several biotypes described by their abilities to overcome plant resistance genes. Bioassays of field populations showed a strong shift towards A1 resistance-breaking biotypes since the 1960s. RFLP analysis of the rDNA cistron was used to study variation found within and between standard clones of three A. idaei biotypes and twenty-nine field populations collected over 3 years. Probing genomic DNA with the ribosomal DNA probe pBG 35 produced consistent differences in RFLPs between standard clones of biotypes. However, analysis of field populations gave more complex RFLP patterns that were not biotype-specific, unlike characteristic intergenic spacer (IGS) patterns reported for Schizaphis graminum biotypes. All but one sample collected from separate fields showed considerable genetic diversity within populations, attributed to alate migrations of parthenogenetic females in summer and males in autumn.     

Inhibition by mibefradil, a novel calcium channel antagonist, of Ca(2+)- and volume-activated Cl- channels in macrovascular endothelial cells

1. We have studied the effects of mibefradil, a novel calcium antagonist, on the resting potential and ion channel activity of macrovascular endothelial cells (calf pulmonary artery endothelial cells, CPAE). The patch clamp technique was used to measure ionic currents and the Fura-II microfluorescence technique to monitor changes in the intracellular Ca2+ concentration, [Ca2+]i. 2. Mibefradil (10 microM) hyperpolarized the membrane potential of CPAE cells from its mean control value of -26.6 +/- 0.6 mV (n = 7) to -59.8 +/- 1.7 mV (n = 6). A depolarizing effect was observed at higher concentrations (-13.7 +/- 0.6 mV, n = 4, 30 microM mibefradil). 3. Mibefradil inhibited Ca(2+)-activated Cl- currents, ICl,Ca, activated by loading CPAE cells via the patch pipette with 500 nM free Ca2+ (Ki = 4.7 +/- 0.18 microM, n = 8). 4. Mibefradil also inhibited volume-sensitive Cl- currents, ICl,vol, activated by challenging CPAE cells with a 27% hypotonic solution (Ki = 5.4 +/- 0.22 microM, n = 6). 5. The inwardly rectifying K+ channel, IRK, was not affected by mibefradil at concentrations up to 30 microM. 6. Ca2+ entry activated by store depletion, as assessed by the rate of [Ca2+]i-increase upon reapplication of 10 mM extracellular Ca2+ to store-depleted cells, was inhibited by 17.6 +/- 6.5% (n = 8) in the presence of 10 microM mibefradil. 7. Mibefradil inhibited proliferation of CPAE cells. Half-maximal inhibition was found at 1.7 +/- 0.12 microM (n = 3), which is similar to the concentration for half-maximal block of Cl- channels. 8. These actions of mibefradil on Cl- channels and the concomitant changes in resting potential might, in addition to its effect on T-type Ca2+ channels, be an important target for modulation of cardiovascular function under normal and pathological conditions.     

Different effects of endothelin-3 on the Ca2+ discharge induced by agonists and Ca(2+)-ATPase inhibitors in human platelets

1. The present study demonstrates that endothelin-3 (ET-3), previously shown to attenuate thrombin-evoked aggregation of human platelets, delayed the dose-dependent aggregatory response to thapsigargin (Tg). As this Ca(2+)-ATPase inhibitor induces platelet activation in part through the depletion of internal Ca(2+)-stores, we examined the influence of ET-3 on Ca2+ discharge from internal pools. 2. Cytosolic Ca2+ concentration was evaluated with Fura-2 in the absence of Ca2+ influx. Platelet preincubation for 15 min with 5 x 10(-7) M ET-3 decreased the Ca2+ release evoked by thrombin and U46619, a thromboxane-mimetic. However, ET-3 did not affect Ca2+ movements induced by 1 microM ADP. Addition of Tg (0.5 to 5 microM) to resting platelets induced a cytosolic [Ca2+] rise with concentration-dependent increase of the initial rate and decrease of the time to reach the peak. ET-3 slowed down these dose-dependent effects with a more marked influence on the responses induced by low concentrations of Tg. 3. ET-3 did not modify the Ca2+ response to another Ca(2+)-ATPase inhibitor, 2,5-di-(tert-butyl)-1,4-benzohydroquinone(tBuBHQ). The thromboxane A2 receptor antagonist, SQ 29548, reduced by 53% the calcium signal evoked by 1 microM Tg, which became similar to that induced by 15 microM tBuBHQ. Under these conditions, the ET-3 effects were suppressed. A subsequent addition of thrombin induced a substantial further Ca2+ increase which was again sensitive to ET-3. 4. ET-3 attenuates Ca2+ mobilization from an internal pool dependent on the stimulation of thrombin and thromboxane A2 receptors and insensitive to the direct effect of Ca2+-ATPase inhibitors. The small but significant inhibitory effect of ET-3 leads us to propose that endothelin-3 acts as a modulator of platelet activation.     

Control of heat produced during ATP hydrolysis by the sarcoplasmic reticulum Ca(2+)-ATPase in the absence of a Ca2+ gradient

The amount of heat produced during the hydrolysis of ATP by the sarcoplasmic reticulum Ca(2+)-ATPase was found to vary depending on the Ca2+ concentration in the medium. When the CaCl2 concentration is raised from 0.1 to 2.0 mM a part of the energy derived from ATP hydrolysis is not dissipated as heat but it is used by the enzyme to resenthesize a small fraction of the ATP previously cleaved. Thus, Ca2+ seems to regulate the ATPase in such a way as to vary the fraction of energy derived from ATP hydrolysis which is converted into heat and that which is conserved as chemical energy.     

Interaction of Arabidopsis kinesin-like calmodulin-binding protein with tubulin subunits: modulation by Ca(2+)-calmodulin

Kinesin-like calmodulin-binding protein (KCBP) is a recently identified novel kinesin-like protein that appears to be unique to and ubiquitous in plants. KCBP is distinct from all other known KLPs in having a calmodulin-binding domain adjacent to its motor domain. We have used different regions of KCBP to study its interaction with tubulin subunits and the regulation of this interaction by Ca(2+)-calmodulin. The results show that the carboxy-terminal part of the KCBP, with or without calmodulin-binding domain, binds to tubulin subunits and this binding is sensitive to nucleotides. In the presence of Ca(2+)-calmodulin the motor with calmodulin-binding domain does not bind to tubulin. This Ca(2+)-calmodulin modulation is abolished in the presence of antibodies specific to the calmodulin-binding domain of KCBP. Similar binding studies with the carboxy-terminal part of KCBP lacking the calmodulin-binding domain show no effect of Ca(2+)-calmodulin. These results indicate that Ca(2+)-calmodulin modulates the interaction of KCBP with tubulin subunits and this modulation is due to the calmodulin-binding domain in the KCBP. Calcium-dependent calmodulin modulation of KCBP interaction with tubulin suggests regulation of KCBP function by calcium, the first such regulation of a kinesin heavy chain among all the known kinesin-like proteins.