Simultaneous phosphorylation of three human calpactins by kinase C.

We evaluated the concurrent phosphorylation of reconstituted mixtures of three purified human placental calpactins (or lipocortins) by purified bovine brain protein kinase C (PKC). Calpactin-I (p36 or lipocortin-II), calpactin-II (p38 or lipocortin-I), and a 70-kilodalton calpactin-related protein, calpactin-p70, when present together as substrates for PKC, all demonstrated comparable kinetic parameters (Vmax values = 0.3-0.5 nmol phosphate incorporated/min), with calpactin-II and calpactin-p70 exhibiting lower apparent Km values (40 and 30 nM, respectively) than did calpactin-I (Km, 200 nM). Because of the higher Vmax/Km ratios for calpactin-II and calpactin-70 (12.5 and 10.0, respectively) compared with the ratio for calpactin-I (2.0), our data suggest that, intracellularly, where all three calpactins might be co-localized, the higher molecular mass calpactins could be preferred substrates for PKC. Nonetheless, the requirement for relatively high calcium concentrations (greater than or equal to 0.5 mM) suggests that PKC-mediated phosphorylation of the calpactins may take place only in restricted intracellular compartments, wherein calcium concentrations might transiently reach levels much higher than those that are normally found intracellularly (less than or equal to 0.25 mM).     

Isolation of a new member of the S100 protein family: amino acid sequence, tissue, and subcellular distribution

A low molecular mass protein which we term S100L was isolated from bovine lung. S100L possesses many of the properties of brain S100 such as self association, Ca++-binding (2 sites per subunit) with moderate affinity, and exposure of a hydrophobic site upon Ca++-saturation. Antibodies to brain S100 proteins, however, do not cross react with S100L. Tryptic peptides derived from S100L were sequenced revealing similarity to other members of the S100 family. Oligonucleotide probes based on these sequences were used to screen a cDNA library derived from a bovine kidney cell line (MDBK). A 562-nucleotide cDNA was sequenced and found to contain the complete coding region of S100L. The predicted amino acid sequence displays striking similarity, yet is clearly distinct from other members of the S100 protein family. Polyclonal and monoclonal antibodies were raised against S100L and used to determine the tissue and subcellular distribution of this molecule. The S100L protein is expressed at high levels in bovine kidney and lung tissue, low levels in brain and intestine, with intermediate levels in muscle. The MDBK cell line was found to contain both S100L and the calpactin light chain, another member of this protein family. S100L was not found associated with a higher molecular mass subunit in MDBK cells while the calpactin light chain was tightly bound to the calpactin heavy chain. Double label immunofluorescence microscopy confirmed the observation that the calpactin light chain and S100L have a different distribution in these cells.     

The calpactin light chain is tightly linked to the cytoskeletal form of calpactin I: studies using monoclonal antibodies to calpactin subunits

Calpactins are a family of related Ca++-regulated cytoskeletal proteins. To analyze the expression and cytoskeletal association of calpactins we raised monoclonal antibodies with specificity for the heavy or light chains of calpactin I or to calpactin II. Comparison of the tissue distribution of calpactin I heavy and light chains by Western blots revealed that these subunits are coordinately expressed. Both soluble and cytoskeletal forms of the heavy chain of calpactin I were detected in human fibroblasts whereas only a soluble pool of calpactin II was found. These two forms of the calpactin I heavy chain differed both in their state of association with the light chain and in their rate of turnover. Both the soluble pool of the calpactin I heavy chain and calpactin II turned over three to four times faster than the cytoskeletal pool of heavy and light chains. Immunofluorescence microscopy revealed that the calpactin I light chain was present exclusively in the cytoskeleton whereas the calpactin I heavy chain distribution was more diffuse. No difference in the amount of light chain or the cytoskeletal attachment of phosphorylated calpactin I heavy chain was found in Rous sarcoma virus-transformed chick embryo fibroblasts compared with their normal counterpart. The antibody to the light chain of calpactin I was microinjected into cultured fibroblasts and kidney epithelial cells. In many cases antibody clustering was observed with the concomitant aggregation of the associated calpactin I heavy chain. The distribution of fodrin and calpactin II in injected cells remained unchanged. These results are consistent with the existence of two functionally distinct pools of calpactin I which differ in their association with the cytoskeleton.     

Identification of the 32 kDa components of bovine lens EDTA-extractable protein as endonexins I and II.

EDTA-extractable protein (EEP) is a mixture of major lens membrane proteins with molecular masses ranging from 32 kDa to 40 kDa. These bind to the lens membrane in a Ca2(+)-dependent manner. In the present study we have identified and purified two distinct 32 kDa components of EEP (designated as EEP 32-1 and EEP 32-2) from bovine lens that inhibit phospholipase A2 activity. Both EEP 32-1 and EEP 32-2 bind to phospholipid-containing liposomes and actin filaments in a Ca2(+)-dependent fashion. Immunochemical studies and two-dimensional electrophoreses demonstrate that the two proteins are distinct from one another. Both EEP 32-1 and EEP 32-2 are clearly different from calpactin (lipocortin) or its proteolytic fragments because they did not react with anti-[human placenta calpactin (lipocortin)] antibody. Our results also indicate that EEP 32-1 is very similar to endonexin I and that EEP 32-2 corresponds to endonexin II.     

Regulation of calpactin I phospholipid binding by calpactin I light-chain binding and phosphorylation by p60v-src.

Calpactins I and II are proteins that bind Ca2+, phospholipids, actin and spectrin; they are also major substrates of oncogene and growth-factor-receptor tyrosine kinases. Since calpactins have been proposed to provide a link between membrane lipids and the cytoskeleton, we examined in detail the interactions between purified calpactin I and phospholipid liposomes. We focused on the Ca2+-dependence, the effects of phosphorylation of calpactin I by p60v-src (the protein kinase coded for by the Rous-sarcoma-virus oncogene), and the effects of the binding of calpactin I light chain to calpactin I heavy chain. Binding of the light chain to the heavy chain increased the affinity of calpactin I for phosphatidylserine (PS) liposomes. The opposite effect was observed for phosphorylation by p60v-src; phosphorylation decreased the affinity of calpactin I for PS liposomes. These two opposite effects appeared to be independent, since phosphorylation did not prevent light-chain binding to the heavy chain. Calpactin I was found, by the use of three different techniques, to bind to phospholipid liposomes at less than 10(-8) M free Ca2+. This result is in contrast with those of previous studies, which indicated that greater than 10(-6) M free Ca2+ was required. Our findings suggest that calpactin I may be bound to phospholipids in vivo at Ca2+ concentrations of about 1.5 x 10(-7) M, typical of resting unstimulated cells, and that this interaction may be modulated by light-chain binding and phosphorylation by p60v-src.     

Mammary gland Ca2+-binding (-dependent) proteins: Identification as calelectrins and calpactin I/p36

Calcium-binding (-dependent) proteins (CBPs) associated with the spreading of mammary epithelial cell cultures have been identified as various calelectrins and calpactins (p36). In immunoblot analysis, the CBPs of 30–36 kD and 68–70 KD variously react with different calelectrin and calpactin I monomer/p36 antisera. The same immunoreactive proteins were shown to be present in virgin mammary glands and collagen gel mouse mammary epithelial cell cultures. The mammary CBPs show extensive immunochemical relatedness; however, they fail to show cross-reaction with antiserum to calpactin II (lipocortin) antiserum. These immunoreactive CBPs comigrate in electrophoresis with ³⁵S-methionine-labeled CBPs isolated from mammary epithelial cell cultures. Unlike calmodulin, the mammary CBPs that correspond to calelectrins and calpactin I monomer/p36 are not stable to thermal denaturation. The mammary CBPs bind to epithelial cell membranes in a Ca²⁺-dependent manner and are differentially released from ruptured cells, compared with calmodulin, suggesting subcellular localization. Phenothiazineagarose and phenylagarose are equivalent in their ability to bind the mammary CBPs. Thus, mammary gland CBPs of 30–36 kD and 68–70 kD have been shown to be related or equivalent to the calelectrins and to calpactin I monomer/p36. Since these proteins are known to bind Ca²⁺, we conclude that the mammary gland CBPs are also Ca²⁺-binding proteins. The mammary gland CBPs are immunologically related and probably represent members of a larger family of related proteins.     

Characterization of monoclonal antibodies to Acanthamoeba myosin-I that cross-react with both myosin-II and low molecular mass nuclear proteins

We characterized nine monoclonal antibodies that bind to the heavy chain of Acanthamoeba myosin-IA. Eight of these antibodies bind to myosin-IB and eight cross-react with Acanthamoeba myosin-II. All but one of the antibodies bind to a 30-kD chymotryptic peptide of myosin-IA that derives from the COOH terminus of the molecule, and to tryptic peptides as small as 17 kD, hence these epitopes are clustered closely together on the heavy chain. None of the antibodies prevent heavy chain phosphorylation by myosin-I heavy chain kinase. One antibody inhibits the K+-EDTA ATPase activity and three antibodies inhibit the actin- activated Mg++-ATPase activity of myosin-I under the set of conditions that we tested. When fluorescent antibody staining of both whole cells and isolated nuclei is done, several of these monoclonal antibodies react strongly with nuclei. These antibodies also stain the cytoplasmic matrix, especially the cortex near the plasma membrane. All nine of the monoclonal antibodies bind to polypeptides of 30-34 kD that are highly enriched in nuclei isolated from Acanthamoeba. There is no myosin-I in the isolated nuclei, so the 30-34-kD polypeptides, not myosin-I, are responsible for the nuclear staining.     

Calcium-dependent regulation of actin filament bundling by lipocortin-85

Lipocortin-85 (L-85, calpactin-I/lipocortin-II heterotetramer) binds to F-actin in the presence of calcium with high affinity and in a cooperative manner. Quantitative analysis of binding curves indicate an apparent Kd (L-85) of 0.226 microM +/- 0.153 (2 S.D., n = 3), a stoichiometry of L-85/actin of 1:1.9 and a Hill coefficient of 1.37 +/- 0.14 (2 S.D., n = 3). Large anisotropic bundles were visualized by electron microscopy under these conditions, and quantitation of bundling by both low speed sedimentation and light scattering yielded apparent Kd values between 0.12 and 0.27 microM L-85. Filament bundling was dependent upon calcium, and the calcium sensitivity was increased by raising the molar ratio of lipocortin-85/F-actin. At saturating levels of L-85, apparent K0.5 values of 0.1-2 microM Ca2+f were obtained. The monomeric heavy chain, lipocortin-II, bundled F-actin to a much lesser extent and at much higher concentrations than for lipocortin-85. Bundling of F-actin by lipocortin-I was not detected at molar ratios of lipocortin-I to actin as high as 2.5 mol/mol (lipocortin-I/actin). At 5-10 microM Ca2+f and saturating levels of L-85, F-actin bundling progressed very rapidly with a t0.5 of 6 s. The process was quickly reversed by the addition of excess EGTA, and bundles could be reformed by the addition of a second burst of 5-10 microM Ca2+f. Thus, our data suggest that lipocortin-85 can rapidly regulate F-actin bundling in a calcium-dependent manner at physiologically relevant calcium levels.     

Calpactin-like proteins in human spermatozoa

Polyclonal antibodies directed against human calpactin I (p36) and calpactin II (p35) have been employed to investigate the distribution of calpactin-like proteins in human spermatozoa. Calpactins are a family of Ca2+-regulated cytoskeletal proteins that are major substrates of oncogene and growth factor receptor protein tyrosine kinases. The existence of a Triton-soluble 37-kDa protein antigenically related to calpactin II from somatic cells was revealed by Western blot analysis of human sperm extracts. The 37-kDa protein was not released from spermatozoa after experimental induction of the acrosome reaction by A23187 and Ca2+. Treatment of sperm homogenates with an EGTA-containing buffer partially solubilized the 37-kDa protein from the corpuscolate matter. Indirect immunofluorescence microscopy showed that anticalpactin II binds specifically to the sperm tail and to a band-like structure encircling the sperm head at the equatorial segment. In contrast, antibodies to calpactin I were found to bind to the tail midpiece, but failed to bind to Western blots of sperm proteins. This is the first immunological and biochemical report on the presence of calpactin proteins in a germ cell, the human spermatozoon.     

Cloning and expression of cDNA for human endonexin II, a Ca2+ and phospholipid binding protein

Endonexin II is a member of the family of Ca2+-dependent phospholipid binding proteins known as annexins. We cloned human endonexin II cDNA and expressed it in Escherichia coli. The apparent size and Ca2+-dependent phospholipid binding properties of purified recombinant endonexin II were indistinguishable from those of the placental protein. A single mRNA of approximately 1.6 kilobase pairs was found to be expressed in human cell lines and placenta and was in close agreement with the length of the cDNA clone (1.59 kilobase pairs). The cDNA predicted a 320-amino acid protein with a sequence that was in agreement with the previously determined partial amino acid sequence of endonexin II isolated from placenta. Endonexin II contained 58, 46, and 43% sequence identity to protein II, calpactin I (p36, protein I), and lipocortin I (p35), respectively. The partial sequence of bovine endonexin I was aligned with the sequence of endonexin II to give 63% sequence identity. Like these other proteins, endonexin II had a 4-fold internal repeat of approximately 70 residues preceded by an amino-terminal domain lacking similarity to the repeated region. It also had significant sequence identity with 67-kDa calelectrin (p68), a protein with an 8-fold internal repeat. Comparing the amino-terminal domains of these four proteins of known sequence revealed that, in general, only endonexin II and protein II had significant sequence identity (29%). Endonexin II was not phosphorylated by Ca2+/phospholipid-dependent enzyme (protein kinase C) even though it contained a threonine at a position analogous to the protein kinase C phosphorylation sites of lipocortin I, calpactin I, and protein II.     

Evidence for Ca(2+)-mediated F-actin/phospholipid binding of human sperm calpactin II.

We have previously reported the presence in human spermatozoa of calpactin II, a calcium-binding component of the membrane skeleton (Berruti, Exper. Cell Res. 179, 374, 1988). Reported here are studies which show the ability of sperm calpactin II to interact with filamentous actin and acidic phospholipids in a Ca(2+)-dependent fashion. At high Ca2+ concentrations (greater than 1 mM) sperm calpactin II binds to actin filament and it is resolubilized by EGTA. Liposome binding experiments reveal that sperm calpactin II does associate with phospholipidic vesicles at micromolar free Ca2+ levels. These interaction properties together with the cellular distribution of the protein revealed by immunofluorescence analysis in Ca2+ and ionophore A23187-treated human spermatozoa allow to hypothesize a physiological role for calpactin II in sperm Ca(2+)-mediated events.     

Phospholipid-dependent Ca2+ binding by the 36-kDa tyrosine kinase substrate (calpactin) and its 33-kDa core

A 36-kDa protein, which is a component of the membrane skeleton, has been shown to co-localize with spectrin in addition to serving as a major substrate for tyrosine-protein kinases. This protein, which will be referred to as calpactin (for calcium-dependent phospholipid and actin binding protein), was isolated from bovine intestine as the complex with a 10-kilodalton light chain and the Ca2+ binding was analyzed by equilibrium dialysis with 45Ca2+ in the presence or absence of phospholipid. Although Ca2+ binding by calpactin alone was negligible at micromolar free Ca2+, it was greatly enhanced by liposomes containing phosphatidylserine or phosphatidylinositol. A proteolytic derivative of calpactin, termed the "core," which has lost the site of association with the light chain in addition to the site of tyrosine phosphorylation by pp60src, was also found to contain this high affinity phospholipid enhanced Ca2+-binding activity. Scatchard plots reveal that each calpactin monomer or core polypeptide bound 2 Ca2+ ions with a Kd of 4.5 X 10(-6) M at 200 micrograms of phosphatidylserine/ml. Liposome binding experiments confirmed that calpactin as a complex with light chain as well as calpactin monomer or the 33-kDa core interact with phosphatidylserine liposomes in a Ca2+-dependent manner.     

A 32-kDa protein associated with phospholipase A2-inhibitory activity from human placenta

Two monomeric 32-kDa proteins, termed 32K-I (pI 5.8) and 32K-II (pI 5.1), were isolated from human placenta, which was solubilized by a Ca2+-chelator. Only 32K-I was associated with PLA2-inhibitory activity. CNBr peptide mapping indicated that 32K-I was distinct from 32K-II and two 36-kDa proteins, called calpactin I and II or lipocortin II and I, which have been shown to possess PLA2-inhibitory activity. 32K-I bound to PS in a Ca2+-dependent manner. 32K-I was detected in many tissues except brain, cardiac and skeletal muscle.     

The protein-tyrosine kinase substrate, calpactin I heavy chain (p36), is part of the primer recognition protein complex that interacts with DNA polymerase alpha

Primer recognition proteins (PRP) stimulate the activity of DNA polymerase alpha on DNA substrates with long single-stranded template containing few primers. Purified PRP from HeLa cells and human placenta are composed of two subunits of 36,000 (PRP 1) and 41,000 (PRP 2) daltons. By amino acid sequence homology, we have identified PRP 2 as the glycolytic enzyme 3-phosphoglycerate kinase. Here we present data that establishes PRP 1 to be the protein-tyrosine kinase substrate, calpactin I heavy chain. Amino acid sequence analysis of six tryptic peptides of PRP 1 followed by homology search in a protein sequence data base revealed 100% identity of all six peptides with the deduced amino acid sequence of human calpactin I heavy chain. The activities of PRP and calpactin I coelute on gel filtration columns, and a high correlation of PRP and calpactin I activities was seen at different stages of purification. A rabbit polyclonal anti-chicken calpactin I antibody was shown to cross-react with PRP 1 polypeptide at various stages of PRP purification, and the homogeneous preparation of PRP exhibits 3-phosphoglycerate kinase (PRP 2) and calpactin I (PRP 1) activities. PRP activity is neutralized by a mouse monoclonal anti-calpactin II antibody although having no effect on the polymerase alpha activity itself. Calpactin II has a 50% amino acid sequence homology with calpactin I. However, PRP 1 is not calpactin II as shown by lack of cross-reaction to a monoclonal anti-calpactin II antibody on Western blots. Calpactin I and 3-phosphoglycerate kinase, purified independently, cannot be efficiently reconstituted into the PRP complex, indicating that their association in the PRP complex involves specific protein-protein interactions that remain to be elucidated. The biochemical and immunological data presented here revealing the identity of PRP 1 as calpactin I provide evidence for one physiological role of calpactin I in the cell.     

Epidermal-growth-factor-stimulated phosphorylation of calpactin II in membrane vesicles shed from cultured A-431 cells.

Membrane vesicles shed from intact A-431 epidermoid carcinoma cells and harvested in the presence of Ca2+ contained epidermal-growth-factor (EGF) receptor/kinase substrates of apparent molecular masses 185, 85, 70, 55, 38 and 27 kDa. The 38 kDa substrate (p38) was recognized by an antibody that had been raised against the human placental EGF receptor/kinase substrate calpactin II (lipocortin I). The A-431 and placental substrates, isolated by immunoprecipitation after phosphorylation in situ, yielded identical phosphopeptide maps upon limited proteolytic digestion with each of five different enzymes. The A-431-cell vesicular p38 is therefore calpactin II. EGF treatment of the intact A-431 cells before inducing vesiculation was not necessary for the substrate to be present within the vesicles. Our data thus indicate that receptor internalization is not a prerequisite for receptor-mediated phosphorylation of calpactin II. The ability of the protein to function as a substrate for the receptor/kinase depended upon the continued presence of Ca2+ during the vesicle-isolation procedure. EGF-stimulated phosphorylation of calpactin II was much less pronounced in vesicles prepared from A-431 cells in the absence of Ca2+, although comparable amounts of the protein were detectable by immunoblotting. Calpactin II therefore appears to be sequestered in a Ca2+-modulated manner within shed vesicles, along with at least four other major targets for the EGF receptor/kinase. The vesicle preparation may be a useful model system in which to study the phosphorylation and function of potentially important membrane-associated substrates for the receptor.     

Strongylocentrotus purpuratus spindle tubulin. II. Characteristics of its sensitivity to Ca++ and the effects of calmodulin isolated from bovine brain and S. purpuratus eggs

Tubulin was extracted from spindles isolated from embryos of the sea urchin Strongylocentrotus purpuratus and purified through cycles of temperature-dependent assembly and disassembly. At 37 degrees C, the majority of the cycle-purified spindle tubulin polymer is insensitive to free Ca++ at concentrations below 0.4 mM, requiring free Ca++ concentrations greater than 1 mM for complete depolymerization. However, free Ca++ at concentrations above 1 microM inhibits initiation of polymer formation without significantly inhibiting the rate of elongation onto existing polymer. At 15 degrees C and 18 degrees C, temperatures that are physiological for S. purpuratus embryos, spindle tubulin polymer is sensitive to free Ca++ at micromolar concentrations such that 3-20 microM free Ca++ causes complete depolymerization. Calmodulin purified from either bovine brain or S. purpuratus eggs does not affect the Ca++ sensitivity of the spindle tubulin at 37 degrees C, although both increase the Ca++ sensitivity of cycle-purified bovine brain tubulin. These results indicate that cycle-purified spindle tubulin and cycle-purified bovine brain tubulin differ significantly in their responses to calmodulin and in their Ca++ sensitivities at their physiological temperatures. They also suggest that, in vivo, spindle tubulin may be regulated by physiological levels of intracellular Ca++ in the absence of Ca++-sensitizing factors.     

cDNA structure and expression of calpactin, a peptide involved in Ca2(+)-dependent cell aggregation in sponges.

Aggregation of cells of the marine sponge Geodia cydonium is mediated by an aggregation factor (AF) particle of Mr 1.3 X 10(8). It is now reported that the AF particle is associated with calpactin, which was ascribed a role in the cell-adhesion process. In order to identify the sequence similarity to other members of the lipocortin family, the cDNA of sponge calpactin was cloned and found to display an 80% sequence similarity to vertebrate calpactin II but only a 47% similarity to calpactin I. The calpactin gene, which contains the consensus sequence coding for the amino acids G-T-D-E, was expressed in Escherichia coli and subsequently purified to a 37000-Mr polypeptide. Both the p32 and the p37 are provided with approximately two Ca2+ ions/molecule and the property to bind to phospholipids. The dissociation constant (calpactin-Ca2+) was in the absence of phospholipids in the range 500-700 microM-Ca2+ but in their presence about 20-30 microM-Ca2+. On the basis of (i) inhibition studies with antibodies to calelectrin and (ii) competition experiments with soluble phospholipids (both chemically defined as well as total homologous membrane lipids) we conclude that the AF-associated calpactin and plasma-membrane-bound phospholipid(s) are involved in cell-cell aggregation in sponges.     

Inhibitory effects of cordycepin (3'-deoxyadenosine), a component of Cordyceps militaris, on human platelet aggregation induced by thapsigargin

Cordycepin (3'-deoxyadenosine) is an adenosine analog, isolated from Cordyceps militaris, and it has been used as an anticancer and anti-inflammation ingredient in traditional Chinese medicine. We investigated the effects of cordycepin (3'-deoxyadenosine) on human platelet aggregation, which was induced by thapsigargin, a tumor promoter, and determined the cytosolic free Ca2+ levels ([Ca2+]i) (an aggregation-stimulating molecule) and cyclic-guanosine monophosphate (cGMP) (an aggregation-inhibiting molecule). Cordycepin inhibited thapsigargin-induced platelet aggregation in a dose-dependent manner, and it clearly reduced the levels of [Ca+]i, which was increased by thapsigargin (1 microM) or U46619 (3 microM). Cordycepin also increased the thapsigargin-reduced cGMP levels. Accordingly, our data demonstrated that cordycepin may have a beneficial effect on platelet aggregation-mediated thrombotic diseases through the [Ca2+]i-regulating system such as cGMP.     

Endothelin and Ca++ agonist Bay K 8644: different vasoconstrictive properties

The mechanism of vasoconstriction induced by endothelin was investigated in rat isolated aorta in comparison with the Ca++ agonist, Bay K 8644. Endothelin (EC50 = 4 nM) induced a slow and sustained contraction in control medium whereas the one elicited by Bay K 8644 (EC50 = 14 nM) necessitating a partly K+ depolarized medium was fast with superimposed rhythmic contraction. By opposition with Bay K 8644, endothelin contraction was not inhibited by the calcium antagonists (1 microM), nifedipine, diltiazem and D 600, and substantially persisted in Ca++ free medium or after depletion of intracellular Ca++ by phenylephrine (1 microM). These data show that endothelin does not act as an activator of potential dependent Ca++ channels but probably through specific receptor(s) as suggested by its mode of vasoconstriction.     

Characterization of the interaction between calpactin I and fodrin (non-erythroid spectrin)

Calpactin I, a calcium-binding protein associated with the membrane cytoskeleton, has been reported to bind to a calcium-dependent manner to fodrin, to certain phospholipids, and to F-actin. We have investigated the interaction between calpactin I and fodrin. Using a gel filtration assay, we observed one or more calpactin I molecules were bound calcium-dependently only at high concentrations of calpactin (greater than 1 microM), indicating that the interaction is of only moderate affinity. At higher concentrations of calpactin I, the calpactin coprecipitated with fodrin in a calcium-dependent manner. The molar ratio of calpactin to fodrin tetramer in the precipitate was greater than 25:1, indicating that the calpactin binds to a large number of sites. Moreover, the monomeric form of calpactin I (p36), which did not induce precipitation of fodrin, showed no evidence of saturation in its binding to fodrin even when more than 30 mol of p36 were bound per mole of fodrin tetramer. Several proteins other than fodrin, including clathrin, alpha-actinin, and neurofilament-H, also interacted calcium-dependently with calpactin I in the gel filtration assay. These results demonstrate that the interaction between calpactin and fodrin is not of high affinity, is not readily saturated, and is not specific for fodrin. Our results suggest that calpactin's interaction with fodrin is a particular example of a calcium-dependent, but promiscuous, binding of calpactin to proteins.