Egg-shell calcium and the hatching of Globodera rostochiensis

The Ca²⁺ content of Globodera rostochiensis egg-shells was investigated by X-ray microanalysis. When intact eggs were treated with 5 mM sodium 1, 2-di (2-aminoethoxy) ethane-N,N,N',N',-tetra-acetate (EGTA) only part of the egg-shell Ca²⁺ was removed. Similarly treated opened egg-shells lost almost all their Ca²⁺. We think that Ca²⁺ of intact egg-shells which is accessible to EGTA is in the outer part of the shell and that the inaccessible Ca²⁺ is in the inner lipoprotein layer. Much Ca²⁺ was removed from opened egg-shells by the hatching agents Zn²⁺, La³⁺ and decationised potato-root exudate, but none by dilute acetic acid or Mg²⁺. Hatching agents, by binding to or replacing Ca²⁺, may change lipoprotein membrane structure. Eggshells treated with potato-root exudate contained about 3 times as much Ca²⁺ as untreated shells, because the treatment makes additional binding sites available. Our results suggest that three types of Ca²⁺ -binding sites occur in the egg-shell.     

The role of Ca2+ in the activity of Mycobacterium tuberculosis DNA gyrase

DNA gyrase is the only type II topoisomerase in Mycobacterium tuberculosis and needs to catalyse DNA supercoiling, relaxation and decatenation reactions in order to fulfil the functions normally carried out by gyrase and DNA topoisomerase IV in other bacteria. We have obtained evidence for the existence of a Ca²⁺-binding site in the GyrA subunit of M. tuberculosis gyrase. Ca²⁺ cannot support topoisomerase reactions in the absence of Mg²⁺, but partial removal of Ca²⁺ from GyrA by dialysis against EGTA leads to a modest loss in relaxation activity that can be restored by adding back Ca²⁺. More extensive removal of Ca²⁺ by denaturation of GyrA and dialysis against EGTA results in an enzyme with greatly reduced enzyme activities. Mutation of the proposed Ca²⁺-binding residues also leads to loss of activity. We propose that Ca²⁺ has a regulatory role in M. tuberculosis gyrase and suggest a model for the modulation of gyrase activity by Ca²⁺ binding.     

Identification and characterisation of Ca-pectate binding peroxidases inArabidopsis thaliana

The Arabidopsis genome encodes many secretory guaiacol peroxidases (class III plant peroxidases, EC 1.11.1.7). These higher plant enzymes are found either in the vacuole or in the apoplast, where several functions have been attributed to them. Their localisation within the cell wall matrix is most likely important for their activity. In the present work, a gel consisting of polygalacturonate chains cross-linked by Ca²⁺ and embedded in polyacrylamide was used to separate proteins from Arabidopsis leaves having an affinity for the Ca²⁺-mediated conformation of pectin. This chromatographic technique selected a small number of cationic isoperoxidases able to bind to Ca²⁺-pectate but not to Ca²⁺-alginate, a polyuronate gel similar to Ca²⁺-pectate. This result suggested that some of the Arabidopsis peroxidases have an affinity for pectin in vivo. Such a property could allow them to be properly distributed within the cell wall network. In addition, eleven cDNAs encoding an Arabidopsis peroxidase were expressed in the baculovirus-insect cell system. The capacity of the resulting recombinant peroxidases to bind Ca²⁺-pectate and Ca²⁺-alginate was also assessed. It appeared that 3 of them exhibited a Ca²⁺-pectate binding activity that was resistant to the action of NaCl. The binding of these recombinant peroxidases to Ca²⁺-alginate was much weaker than to Ca²⁺-pectate, confirming the specificity of the interaction with the pectic structure.     

Protein phosphorylation and its regulation by calcium and calmodulin in membrane fractions from zucchini hypocotyls

Protein-kinase activity has been found to be associated with a membrane fraction obtained from dark-grown zucchini (Cucurbita pepo L., cv. Senator) hypocotyl hooks. Proteins of this membrane fraction were used as protein substrates. The effects of Mg²⁺, Na⁺ and K⁺ on phosphorylation, measured as ³²P incorporation, was investigated. The kinetics of phosphorylation of the individual protein peptides indicate the presence of specific phosphatase activity. Phosphorylation activity is strongly influenced by Ca²⁺. One peptide (relative molecular weight: 180,000) exhibits strong inhibition of ³²P incorporation at physiological Ca²⁺ concentrations between 0.1 and 1 μM. Phosphorylation of about 10 other proteins was enhanced by Ca²⁺, being maximal in most cases at a concentration of about 3 μM free Ca²⁺. Five out of these 10 peptides show increased phosphorylation in the presence of 1 μM calmodulin. This calmodulin-dependent enhancement of phosphorylation could be completely inhibited by the calmodulin antagonist fluphenazine. Cyclic AMP was found to have no stimulating effect on protein phosphorylation.     

Properties of enzyme activities involved in protein phosphorylatino-dephosphorylation of the thyroid plasma membranes

Bovine thyroid tissue exhibited cAMP-dependent and Ca²⁺-dependent protien kinase activities as well as a basal (cAMP- and Ca²⁺-independent) one, and phosphoprotein phosphatase activity. Although the former two protein kiniase activities were not clearly demonstrated using endogenous protein as substrate, they were clearly shown in soluble, particulate and plasma membrane fractions using exogenous histones as substrate. The highest specific activities were in the plasma membrane. The apparent K_m values of cAMP and Ca²⁺ for the membrane-bound protein kinase were 5·10^?8 M and 8.3·10^?4M (in the presence of 1 mM EGTA), respectively. The apparent K_m values of Mg²⁺ were 7·10^?4 M (without cAMP and Ca²⁺, 5·10^?4 M (with cAMP) and 1.3·10^?3 M (with Ca²⁺), and those ATP were 3.5·10^?5 M (with or without cAMP) and 8.5·10^?5 M (with Ca²⁺). The Ca²⁺-dependent protein kinase could be dissociated from the membrane by EGTA-washing. The enzyme activity so released was further activated by added phospholipid (phosphatidylserine/1,3-diolein), but not by calmodulin. Phosphoprotein phosphatase activity was also clearly demonstrated in all of the fractions using ³²P-labeled mixed histones as substrate. The activity was not modified by either cAMP or Ca²⁺, but was sitmulated by a rather broad range (5–25 mM) of Mg²⁺ and Mn²⁺. NaCl and substrate concentrations also influenced the activity. Pyrophosphate, ATP, inorganic phosphate and NaF inhibited the activity in a dose-dependent manner. Trifluoperazine, chlorpromazine, dibucaine and Triton X-100 (above 0.05%, w/v) specifically inhibited the Ca²⁺-dependent protein kinase in plasma membranes. Repetitive phosphorylation of intrinsic and extrinsic proteins by the membrane-bound enzyme activities clearly showed an important co-ordination of them at the step of protein phosphorylation. These findings suggest that these enzyme activities in plasma membranes may contribute to regulation of thyroid function in response to external stimuli.     

Modulation of lyso-platelet-activating factor:acetyl-CoA acetyltransferase from rat splenic microsomes. The role of calcium ions

The enzyme lyso-platelet-activating factor:acetyl-CoA acetyltransferase (EC 2.3.1.67) was assayed in microsomal fractions from rat spleens. The addition of micromolar Ca²⁺ rapidly enhanced acetyltransferase activity and this activation was reversed by the addition of EGTA in excess of Ca²⁺. The effect of Ca²⁺ was on the apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of the enzyme for the substrate acetyl-CoA without showing any significant effect on the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ of the acetylation reaction. When microsomes were isolated in the presence of 5 mM EGTA, to remove endogenous calmodulin, the same enhancing effect of Ca²⁺ on the acetylation reaction was observed. The addition of exogenous calmodulin to this preparation had no effect on the enzyme activity. Preincubation of spleen microsomes with the calmodulin inhibitor trifluoperazine decreased acetyltransferase in both the presence and the absence of Ca²⁺, indicating an effect of this drug independently of calmodulin. The addition of Mg-ATP to the assay mixture also had no effect on the acetylation reaction. These data suggest that Ca²⁺ modulates acetyltransferase activity from rat spleen microsomes by a mechanism that seems to be independent of calmodulin or protein phosphorylation.     

Adenylate Cyclase in Silkworm: Regulation by Calcium and a Calcium-binding Protein

Homogenates of silkworm pupal fat body were separated into particulate and supernatant fractions by centrifugation. The particulate fraction was further washed with EGTA. Adenylate cyclase activity of the washed particulate fraction was stimulated 2-fold by the addition of supernatant fraction in the presence of low concentrations of Ca²⁺. The activating factor in supernatant was heat-stable, non-dialyzable and trypsin-sensitive, and shown to be a Ca²⁺-dependent regulator protein. For the activation of adenylate cyclase by the regulator protein, the optimum concentrations of free Ca²⁺ were in a range of 2 µm, and higher concentrations of Ca²⁺ were inhibitory.     

Calcium-dependent binding of mouse epididymal spermatozoa to the zona pellucida.

The minimal requirements and characteristics of epididymal sperm binding to the zona pellucida of the mouse egg were investigated using a new stop-fix centrifugation technique. This assay provided a precise physical definition of the association between the spermatozoon and the zona and permitted quantitation of the binding reaction at short time intervals. The results demonstrated that Ca²⁺ is an essential physiological component required for binding to occur. Sperm preincubated for 60 min in a simplified medium lacking Ca²⁺ did not acquire the ability to bind to eggs. In contrast, if sperm preincubation occurred in this medium supplemented with 1.7 mM Ca²⁺, binding was identical to that observed following sperm preincubation in the complete culture medium which supports both capacitation and fertilization in vitro. The Ca²⁺-dependent binding reaction was rapid, reversed by EGTA, specific for Ca²⁺, and did not require the transport of Ca²⁺ into the cell. Sperm bound to the zona surface following preincubation with Ca²⁺ were capable of fertilization in vitro when the eggs were subsequently transferred to the culture medium. It is proposed that this binding reaction represents a part of capacitation and not the acrosome reaction.     

Effects of calcium on the activities of cytosolic antioxidative enzymes and IAA oxidase during in vitro adventitious rooting of mung bean seedlings

The effects of Ca²⁺ on antioxidative enzymes and indole-3-acetic acid (IAA) oxidase during adventitious rooting were investigated in mung bean (Vigna radiata). CaCl₂ significantly promoted the formation and growth of adventitious roots. EGTA (a Ca²⁺ chelator) or ruthenium red (a Ca²⁺-channel blocker) significantly inhibited root formation and growth, but these inhibitory effects could be partially reversed by CaCl₂. Furthermore, inclusion of 5 mM CaCl₂ significantly increased superoxide dismutase (SOD) activity by 10% at 3 h and catalase (CAT) activity by an average of 29.6% at each time point. CaCl₂ decreased peroxidase (POD) activity by 9.4% and 21% at 12 and 24 h, respectively, and ascorbate peroxidase (APX) activity by an average of 13.9% at each time point. These CaCl₂-induced changes in enzymatic activities were similar to changes caused by indole-3-butyric acid (IBA). Treatment with EGTA or ruthenium red decreased SOD activity by an average of 18.4% and 15.2%, respectively; POD activity by 27.4% and 57.6%, respectively; APX activity by 10.3% and 15.6%, respectively; and CAT activity by 19.3% and 5.2%, respectively, when compared with CaCl₂. In addition, CaCl₂ increased IAA oxidase activity by an average of 5.5% beginning at 6 h, whereas EGTA significantly decreased IAA oxidase activity by 29.2%, 22.9%, and 13.5% at 6, 9, and 12 h, respectively. The inhibitory effects of EGTA could be partially suppressed by addition of CaCl₂. These results imply that the stimulative effect of Ca²⁺ on adventitious rooting is partially related to Ca²⁺-induced changes in the activities of antioxidative enzymes and IAA oxidase.     

Calcium Transport and Local Pool Regulate Polycystin-2 (TRPP2) Function in Human Syncytiotrophoblast

Polycystin-2 (PC2, TRPP2) is a Ca²⁺-permeable, nonselective cation channel implicated in Ca²⁺ transport and epithelial cell signaling. Although PC2 may contribute to Ca²⁺ transport in human term placenta, the regulatory mechanisms associated with Ca²⁺ handling in this tissue are largely unknown. In this work we assessed the regulation by Ca²⁺ of PC2 channel function from a preparation of apical membranes of human syncytiotrophoblast (PC2_hst) reconstituted in a lipid bilayer system. Addition of either EGTA or BAPTA to the cis hemi-chamber, representing the cytoplasmic domain of the channel, and lowering Ca²⁺ to ∼0.6–0.8 nM, inhibited spontaneous PC2_hst channel activity, with a time response dependent on the chelator tested. EGTA reduced PC2_hst channel currents by 86%, with a t_1/2 = 3.6 min, whereas BAPTA rapidly and completely (100%) eliminated channel activity with a t_1/2 = 0.8 min. Subsequent titration with Ca²⁺ reversed the inhibition, which followed a Hill-type function with apparent dissociation constants of 1–5 nM, and 4 Ca²⁺ binding sites. The degree of inhibition by the cis Ca²⁺ chelator largely depended on increasing trans Ca²⁺. This was consistent with measurable Ca²⁺ transport through the channel, feeding the regulatory sites in the cytoplasmic domain. Interestingly, the reconstituted in vitro translated PC2 (PC2_iv) was completely insensitive to Ca²⁺ regulation, suggesting that the regulatory sites are not intrinsic to the channel protein. Our findings demonstrate the presence of a Ca²⁺ microdomain largely accessible through the channel that controls PC2 function in human syncytiotrophoblast of term placenta.     

Purification and biochemical characterization of two extracellular peroxidases from Phanerochaete chrysosporium responsible for lignin biodegradation

Two extracellular peroxidases from Phanerochaete chrysosporium, namely a lignin peroxidase (LiP) and manganese peroxidase (MnP), were purified simultaneously by applying successively, ultrafiltration, ion-exchange and gel filtration chromatography. LiP and MnP have a molecular mass of 36 and 45 kDa, respectively. The optimal pHs for LiP and MnP activities were 3.0 and 4.5, respectively. Both peroxidases showed maximal activity at 30 °C and moderate thermostability. MnP activity was strongly inhibited by Fe²⁺, Zn²⁺, Mg²⁺ and Hg²⁺, and enhanced by Mn²⁺, Ca²⁺ and Cu²⁺. LiP activity was enhanced by Ca²⁺, Na⁺ and Co²⁺ and it was inhibited in the presence of K⁺, Hg⁺, Fe²⁺, Mg²⁺ and high concentrations of Cu²⁺ and Zn²⁺. The K_m and V_max for LiP toward veratryl alcohol as a substrate were 0.10 mM and 15.2 U mg⁻¹, respectively and for MnP toward Mn²⁺, they were respectively 0.03 mM and 25.5 U mg⁻¹. The two peroxidases were also able to break down rice lignin in a small-scale solid state treatment system. Data suggest these two peroxidases may be considered as potential candidates for the development of enzyme-based technologies for lignin degradation.     

Choline incorporation into phospholipids by microsomal fractions from L5178Y lymphoma

Microsomal fractions prepared from mouse lymphoma L5178Y grown in culture incorporated choline into cellular phospholipids when either choline or CDP-choline was used as the labeled precursor. Incorporation of label from CDP-choline was stimulated by Mg²⁺ and inhibited by Ca²⁺. In contrast, incorporation of label from choline required Ca²⁺ and was inhibited by EGTA. The characteristics of incorporation indicated that L5178Y cells have the capacity to utilize choline for phospholipid synthesis through both the Kennedy pathway and Ca²⁺-stimulated choline exchange.     

cAMP,not Ca2+/calmodulin,regulates the phosphorylation of acetylcholine receptor in Torpedo californica electroplax

The regulation of the phosphorylation of the acetylcholine receptor in electroplax membranes from Torpedo californica and of purified acetylcholine receptor was investigated. The phosphorylation of the membrane-bound acetylcholine receptor was not stimulated by Ca²⁺/calmodulin, nor was it inhibited by EGTA, but it was stimulated by the catalytic subunit of cAMP-dependent protein kinase, and was blocked by the protein inhibitor of cAMP-dependent protein kinase. Purified acetylcholine receptor was not phosphorylated by Ca²⁺/calmodulin-dependent protein kinase activity in electroplax membranes, nor by partially purified Ca²⁺/calmodulin-dependent protein kinases from soluble or particulate fractions from the electroplax. Of the four acetylcholine receptor subunits, termed α, β, γ and δ, only the γ- and δ-subunits were phosphorylated by the cAMP-dependent protein kinase (+cAMP), or by its purified catalytic subunits.     

Heat shock-triggered Ca2+ mobilization accompanied by pectin methylesterase activity and cytosolic Ca2+ oscillation are crucial for plant thermotolerance

Apoplastic Ca²⁺ concentration controls membrane permeability, cell wall stabilization and cell integrity; however, little is known about its role in thermotolerance in plants. Here, we report that the acquired thermotolerance of etiolated rice seedlings (Oryza sativa) was abolished by an exogenously supplied Ca²⁺ chelator, EGTA, related to increased cellular content leakage during heat shock (HS) treatment. Thermotolerance was restored by the addition of Ca²⁺ during EGTA incubation. Pectin methylesterase (EC 3.1.1.11), a cell-wall remodeling enzyme, was activated in response to HS and its elevated activity was related to the recovery of the HS-released Ca²⁺ concentration. EGTA interfered with the capability of HS to increase oscillation of [Ca²⁺]_cyt content. We assume that heat-activated PME activity is involved in cell-wall localized Ca²⁺. The removal of apoplastic Ca²⁺ might participate in HS signaling to induce HS protein expression and cell-wall remodeling to retain plasma membrane integrity, prevent cellular content leakage and confer thermoprotection.Key words: Ca²⁺, cell wall, EGTA, HSP, HSR, pectin methylesterase, thermotoleranceBiological organisms have developed a remarkable number of strategies to adapt environmental changes. Induction of heat shock protein (HSP) expression is one of the best characterized responses to elevated temperature and plays an important role in the acquisition of thermotolerance.¹ Besides HSPs, Ca²⁺ has a role in the plant adaptation to stress,² but little evidence of apoplastic Ca²⁺ homeostasis contributes to the adaptation to heat stress, which is crucial to elucidate its mechanisms. Our previous study showed that the recovery of HS-released Ca²⁺ in Ca²⁺-pectate reconstitution through pectin methylesterase (PME) activity is required for cell-wall remodeling during HS in soybean, which retains plasma membrane integrity and co-ordinates with HSPs to confer thermotolerance.³ Here, we demonstrated that administering the extracellular Ca²⁺ chelator EGTA to rice plants inhibited the development of thermotolerance, which could be overcome by the addition of Ca²⁺. We also investigated HS inducing apoplastic Ca²⁺ mobilization to increase cytosolic Ca²⁺ level and that, combined with Ca²⁺-pectate networks, may be a universal response for the acquisition of thermotolerance in planta.     

H-mediated coupling of transmembrane Ca fluxes in vegetative Trichoderma viride mycelia suggested by the study of ageing and adaptation to extreme Ca concentrations

The adaptation to extreme concentrations of Ca²⁺ and its consequence on the properties of the ⁴⁵Ca²⁺ transport were studied in submerged mycelia of Trichoderma viride. The adaptation to low [Ca²⁺]_o did not cause changes in kinetic parameters of the ⁴⁵Ca²⁺ influx but the adaptation to high [Ca²⁺]_o increased the K_M(Ca2+). The V_max of the ⁴⁵Ca²⁺ influx decreased with the age of (non-adapted) mycelia with concomitant decrease of the K_M(Ca2+) these changes were prevented in mycelia adapted to high Ca²⁺. High [Ca²⁺]_o decreased the stimulation by the uncoupler, 3, 3′, 4′, 5-tetrachloro salicylanilide (TCS) (30 μM), as compared to the control, whereas the Ca²⁺ chelator, EGTA, stimulated it. In the aged mycelia, the stimulation by TCS of the ⁴⁵Ca²⁺ influx faded away, in parallel with the activity of the H⁺-ATPase. The ⁴⁵Ca²⁺ efflux from mycelia was affected by TCS in a similar way as the ⁴⁵Ca²⁺ influx. The results demonstrate the adaptive responses of transport processes participating in the mycelial Ca²⁺ homeostasis and ageing are in agreement with a notion that both Ca²⁺-influx and-efflux are coupled by the H⁺-homeostasis at the plasma membrane.     

Calcium mediated cytokinin action on chlorophyll synthesis in isolated embryo of Scots pine

Chlorophyll (Chl) synthesis in isolated Scots pine embryos depended on exogenous application of cytokinin (CK) and Ca²⁺. At a constant benzyladenine (BA) level (4.4×10^?5 M) 10^?4 to 10^?2 M Ca²⁺ concentrations in mineral medium were optimum for Chl biosynthesis under both light and dark. At a zero or very low (10^?6 M) concentration of external Ca²⁺, Chl synthesis was relatively more Ca²⁺-dependent in embryos cultured in darkness than in the light, which suggested that the light: (a) stimulated the transport of Ca²⁺ from external sources to cytosol, and/or (b) interacted with Ca²⁺ directly in the pathway of Chl biosynthesis. The need of external Ca²⁺ was evidenced in experiments with modulators of Ca²⁺-transport systems. The reduction of the inward current of Ca²⁺ from readily accessible external sites by chelating agent (ethylene glycol-bis (beta-aminoethyl ether-N,N,N′N′-tetraacetic acid, EGTA) and Ca²⁺-channel blockers canceled the formation of Chl. The effect of EGTA depended on the level of external Ca²⁺. Inhibitory action of Ca²⁺-channel blockers depended on their kind and concentration: at the 10^?5 M concentration La³⁺>verapamil>nifedipine inhibited Chl formation. In the presence of Ca²⁺, the Ca²⁺-agonist A 23187 mimicked the BA effect and about 92% of Chl was synthesized as compared with the BA variant. Low concentrations of calmodulin antagonists reduced the amounts of Chl. Calmodulin was included in a second messenger system for BA action in promoting Chl biosynthesis in isolated Scots pine embryos.     

The effect of pH on the rate of relaxation of isolated barnacle myofibrillar bundles

CO₂-induced acidosis in barnacle muscle fibres prolongs the relaxation phase of the electrically stimulated contraction (Ashley, C.C., Franciolini, F., Lea, T.J. and Lignon, J. (1979) J. Physiol. 296, 71P). In order to test if this effect is due to a direct action of H⁺ on the relaxation kinetics of the myofilaments, isolated myofibrillar bundles were contracted and relaxed in Ca²⁺ buffer solutions at pH 6.0 and 7.1, in the presence of 20 mM caffeine to inactivate the sarcoplasmic reticulum. At pH 7.1, the relaxation half-time was reduced from 1.5 to 0.3 s as the EGTA concentration in the relaxing solution was progressively increased from 0.3 to 50 mM. The resulting curve was shifted in the direction of increasing EGTA concentration by lowering the pH to 6.0. This effect could be explained by the reduction in affinity of Ca²⁺ for EGTA at pH 6.0, since relaxation half-times for a given relaxing pCa (calculated from the contaminating Ca²⁺ concentrations in the relaxing solutions) were shorter (by about 40%) at pH 6.0 compared with 7.1. However, similar experiments using the new Ca²⁺-chelating agent 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), which is much less pH sensitive than EGTA, indicated that there was no significant difference between relaxation half-times at pH 6.0 and 7.1 for a given relaxing pCa. It is concluded that because no prolongation of relaxation of the myofibrils was observed on lowering the pH from 7.1 to 6.0, the effect of CO₂ on the relaxation of intact muscle fibres is probably due to a modification of sarcoplasmic reticulum activity.     

Polar calcium flux in sunflower hypocotyl segments : I. The effect of auxin

The flux of Ca²⁺ at the apical or basal ends of short sunflower (Helianthus annuus L.) hypocotyl segments was monitored using a Ca²⁺-specific electrode. A higher Ca²⁺ efflux was observed at the apical end relative to the basal end, indicating a net polar flux of Ca²⁺. The extreme low mobility of Ca²⁺ in the isolated segment makes it likely that the observed Ca²⁺ fluxes are of localized origin, that is, from the parenchyma cells close to the exposed cut ends and may represent acropetal transport of Ca²⁺ at the cellular level. The rate of Ca²⁺ efflux depended on the concentration of Ca in the seedling medium. Incubation of hypocotyl segments in 10 mm CaCl₂ for 24 h did not eliminate the net acropetal flux of Ca²⁺ at the apical end.     

Extracellular calmodulin-binding proteins in plants: purification of a 21-kDa calmodulin-binding protein

A 21-kDa calmodulin (CaM)-binding protein and a 19-kDa calmodulin-binding protein were detected in 0.1 M CaCl₂ extracts of Angelica dahurica L. suspension-cultured cells and carrot (Daucus carota L.) suspension-cultured cells, respectively, using a biotinylated cauliflower CaM gel-overlay technique in the presence of 1 mM Ca²⁺. No bands, or very weak bands, were shown on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels overlayed with biotinylated cauliflower CaM when 1 mM Ca²⁺ was replaced by 5 mM EGTA, indicating that the binding of these two CaM-binding proteins to CaM was dependent on Ca²⁺. Less 21-kDa CaM-binding protein was found in culture medium of Angelica dahurica suspension cells; however, a 21-kDa protein was abundant in the cell wall. We believe that the 21-kDa CaM-binding protein is mainly in the cell wall of Angelica dahurica. Based on its reaction with periodic acid-Schiff (PAS) reagent, this 21-kDa protein would appear to be a glycoprotein. The 21-kDa CaM-binding protein was purified by a procedure including Sephadex G-100 gel filtration and CM-Sepharose cation-exchange column chromatography. The purity reached 91% according to gel scanning. The purified 21-kDa CaM-binding protein inhibited the activity of CaM-dependent NAD kinase and the degree of inhibition increased with augmentation of the 21-kDa protein, which appeared to be the typical characteristic of CaM-binding protein.