Cyclic nucleotide regulation of teleost rod photoreceptor inner segment length

Retinal rod photoreceptors of teleost fish elongate in the light and shorten in the dark. Rod cell elongation and shortening are both mediated by actin-dependent mechanisms that occur in the inner segment myoid and ellipsoid. The intracellular signaling pathways by which light and dark regulate the actin cytoskeleton in the inner segment are unknown. To investigate the intracellular signals that regulate teleost rod motility, we have been using mechanically isolated rod inner/outer segments (RIS-ROS) obtained from the retinas of green sunfish, Lepomis cyanellus. In culture, RIS-ROS retain the ability to elongate in response to light; myoids elongate 15-20 microns in length during 45 min of light culture. A pharmacological approach was taken to investigate the role of cyclic nucleotides, cyclic nucleotide-dependent kinases, and protein phosphatases in the regulation of RIS-ROS motility. Millimolar concentrations of cAMP and cGMP analogues were both found to inhibit light-induced myoid elongation and two cyclic nucleotide analogues, SpCAMPS and 8BrcGMP, promoted myoid shortening after RIS-ROS had elongated in response to light. The cyclic nucleotide- dependent kinase inhibitor, H8, mimicked light by promoting myoid elongation in the dark. The effects of H8 were dose dependent, with maximal elongation occurring at concentrations of approximately 100 microM. Similar to the effects of cyclic nucleotide analogues, the phosphatase inhibitor, okadaic acid (0.1-10 microM), inhibited light- induced elongation and promoted shortening. The results presented here suggest that RIS-ROS motility is regulated by protein phosphorylation: phosphorylation in the dark by cyclic nucleotide-dependent protein kinases promotes rod shortening, while dephosphorylation in the light promotes rod elongation.     

Actin-dependent myoid elongation in teleost rod inner/outer segments occurs in the absence of net actin polymerization.

In the retinas of teleost fish, rod photoreceptors elongate in response to light. Light-activated elongation is mediated by the myoid of the rod inner segment and is actin-dependent. Inner segment F-actin filaments form bundles running parallel to the cell's long axis. We examined the mechanism of rod elongation using mechanically-detached rod fragments, consisting of the motile inner segment and sensory outer segment (RIS-ROS). When RIS-ROS are isolated from dark-adapted green sunfish and cultured in the light, they elongate 15 microns at 0.3-0.6 microns/min. Elongation was inhibited 65% by 0.1 microM Cytochalasin D, suggesting a requirement for actin assembly. To determine the extent of assembly during elongation, we used three approaches to measure the F-actin content in RIS-ROS: detection of pelletable actin by SDS-PAGE after detergent-extraction of RIS-ROS; quantification of fluorescein-phalloidin binding by fluorimetry, fluorescence-activated cell sorting and image analysis; estimation of total F-actin filament length by electron microscopy. All three assays indicated that no net assembly of RIS-ROS F-actin accompanied myoid elongation. An increase in F-actin content within the elongated myoid was counterbalanced by a decrease in F-actin content within the 13 microvillus-like calycal processes located at the end of the inner segment opposite to the growing myoid. O'Connor and Burnside (Journal of Cell Biology 89:517-524, 1981) showed that minus-ends of rod F-actin filaments are oriented towards the elongating myoid while plus-ends are oriented towards the shortening calycal processes. Our observations suggest that RIS-ROS elongation entails actin polymerization at the minus-ends of filaments coupled with depolymerization at the filament plus-ends.     

Calmodulin-binding proteins are developmentally regulated in gametes and embryos of fucoid algae

Calcium-binding proteins and calmodulin-binding proteins were identified in gametes and zygotes of the marine brown algae Fucus vesiculosus, Fucus distichus, and Pelvetia fastigiata using gel (SDS-PAGE) overlay techniques. A calcium current appears to be important during cell polarization in fucoid zygotes (K.R. Robinson and L.F. Jaffe, 1975, Science 187, 70-72; K.R. Robinson and R. Cone, 1980, Science 207, 77-78), but there are no biochemical data on calcium-binding proteins in these algae. By using a sensitive 45Ca2+ overlay method designed to detect high-affinity calcium-binding proteins, at least 9-11 polypeptides were detected in extracts of fucoid gametes and zygotes. All samples had calcium-binding proteins with apparent molecular weights of about 17 and 30 kDa. A 17-kDa calcium-binding protein was purified by calcium-dependent hydrophobic chromatography and was identified as calmodulin by immunological and enzyme activator criteria. A 125I-calmodulin overlay assay was used to identify potential targets of calmodulin action. Sperm contained one major calmodulin-binding protein of about 45 kDa. Eggs lacked major calmodulin-binding activity. A 72-kDa calmodulin-binding protein was prominent in zygotes from 1-65 hr postfertilization. Both calmodulin-binding proteins showed calcium-dependent binding activity. Overall, the data suggest that the appearance and distribution of certain calcium-binding and calmodulin-binding proteins are under developmental regulation, and may reflect the different roles of calcium during fertilization and early embryogenesis.     

The cytoskeletal system of nucleated erythrocytes. II. presence of a high molecular weight calmodulin-binding protein

Calmodulin was detected in dogfish erythrocyte lysates by means of phosphodiesterase activation. Anucleate dogfish erythrocyte cytoskeletons bound calmodulin. Binding of calmodulin was calcium- dependent, concentration-dependent, and saturable. Cytoskeletons consisted of a marginal band of microtubules containing primarily tubulin, and trans-marginal band material containing actin and spectrinlike proteins. Dogfish erythrocyte ghosts and cytoskeletons were found to contain a calcium-dependent calmodulin-binding protein, CBP, by two independent techniques: (a) 125I-calmodulin binding to cytoskeletal proteins separated by SDS PAGE, and (b) in situ azidocalmodulin binding in whole anucleate ghosts and cytoskeletons. CBP, with an apparent molecular weight of 245,000, co-migrated with the upper band of human and dogfish erythrocyte spectrin. CBP was present in anucleate ghosts devoid of marginal bands and absent from isolated marginal bands. CBP therefore appears to be localized in the trans- marginal band material and not in the marginal band. Similarities between CBP and high molecular weight calmodulin-binding proteins from mammalian species are discussed.     

A Similar Calmodulin-Binding Protein Expressed in Chromaffin, Synaptic, and Neurohypophyseal Secretory Vesicles

The presence of calmodulin-binding proteins in three neurosecretory vesicles (bovine adrenal chromaffin granules, bovine posterior pituitary secretory granules, and rat brain synaptic vesicles) was investigated. When detergent-solubilized membrane proteins from each type of secretory organelle were applied to calmodulin-affinity columns in the presence of calcium, several calmodulin-binding proteins were retained and these were eluted by EGTA from the columns. In all three membranes, a 65-kilodalton (63 kilodaltons in rat brain synaptic vesicles) and a 53-kilodalton protein were found consistently in the EGTA eluate. 125I-Calmodulin overlay tests on nitrocellulose sheets containing transferred chromaffin and posterior pituitary secretory granule membrane proteins showed a similarity in the protein bands labeled with radioactive calmodulin. In the presence of 10(-4) M calcium, eight major protein bands (240, 180, 145, 125, 65, 60, 53, and 49 kilodaltons) were labeled with 125I-calmodulin. The presence of 10 microM trifluoperazine (a calmodulin antagonist) significantly reduced this labeling, while no labeling was seen in the presence of 1 mM EGTA. Two monoclonal antibodies (mAb 30, mAb 48), previously shown to react with a cholinergic synaptic vesicle membrane protein of approximate molecular mass of 65 kilodaltons, were tested on total membrane proteins from the three different secretory vesicles and on calmodulin-binding proteins isolated from these membranes using calmodulin-affinity chromatography. Both monoclonal antibodies reacted with a 65-kilodalton protein present in membranes from chromaffin and posterior pituitary secretory granules and with a 63-kilodalton protein present in rat brain synaptic vesicle membranes. When the immunoblotting was repeated on secretory vesicle membrane calmodulin-binding proteins isolated by calmodulin-affinity chromatography, an identical staining pattern was obtained. These results clearly indicate that an immunologically identical calmodulin-binding protein is expressed in at least three different neurosecretory vesicle types, thus suggesting a common role for this protein in secretory vesicle function.     

Calcium-induced phosphorylations and [125I]calmodulin binding in renal membrane preparations

Calcium-induced phosphorylated intermediates and calmodulin-binding proteins in membrane preparations from th renal cortex were analyzed by SDS-polyacrylamide gel electrophoresis at low pH, protein electroblotting and [¹²⁵I]calmodulin overlay. Two calcium-induced phosphoproteins were found, with a molecular mass of 135 and 115 kDa, respectively. By comparing different preparations characterized by marker enzymes, it was shown that the 135 kDa phosphoprotein is localized in the basal-lateral fragment of the plasma membrane, whereas the 115 kDa phosphoprotein is more pronounced in preparations containing a high proportion of endoplasmic reticulum. A prominent calmodulin-binding protein comigrated with the 135 kDa phosphoprotein; there was no calmodulin binding to polypeptides in the molecular mass range of the 115 kDa phosphoprotein. Partial proteolysis by trypsin and the effect of $\text{20 μ}\text{M La}{}^{\mathrm{2+}}$ on the formation of phosphoproteins before and after trypsinization support the conclusion that the 135 kDa protein can be identified with the plasma membrane calcium pump, whereas the 115 kDa phosphoprotein is the phosphorylated intermediate of a different type of calcium pump probably originating from the endoplasmic reticulum. Calmodulin binding in renal membrane preparations analyzed on Laemmli-type slab gels revealed that there are many calmodulin-binding proteins in our preparations. We have identified one band with the renal calcium pump localized in the basal-lateral membrane. Another calmodulin-binding protein migrating at 108 kDa, is not localized in the basal-lateral membrane and could be one of the calmodulin-binding proteins originating from the cytoskeleton.     

Calmodulin-Binding Proteins in Chromaffin Cell Plasma Membranes

Abstract: Calmodulin-binding proteins present in chromaffin cell plasma membranes were isolated and directly compared with calmodulin-binding proteins present in chromaffin granule membranes. Chromaffin cell plasma membranes were prepared using Cytodex 1 microcarriers. Marker enzyme studies on this preparation showed a nine- to 10–fold plasma membrane enrichment over cell homogenates and a low contamination of these plasma membranes by subcellular organelles. Plasma membranes prepared in this manner were solubilized with Triton X-100 and applied to a calmodulin-affinity column in the presence of calcium. Several major calmodulin-binding proteins ( 240, 105 , and 65 kilodaltons) were eluted by an EGTA-containing buffer. ¹²⁵I-Calmodulin overlay experiments on nitrocellulose sheets containing both chromaffin plasma and granule membranes showed that these two membranes have several calmodulin-binding proteins in common ( 65, 60, 53 , and 50 kilodaltons), as well as unique calmodulin-binding proteins (34 kilodaltons in granule membranes and 240 and 160 kilodaltons in plasma membranes). The 65–kilodalton calmodulin-binding protein present in both membrane types was shown to consist of two isoforms (pI 6.0 and 6.2) by two-dimensional gel electrophoresis. Previous experiments from our laboratory, using two monoclonal antibodies (mAb 30 and mAb 48) specific for a rat brain synaptic vesicle membrane protein (p65), showed that the monoclonal antibodies reacted with a 65–kilodalton calmodulin-binding protein present in at least three neurosecretory vesicles (chromaffin granules, neurohypophyseal granules, and rat brain synaptic vesicles). When these monoclonal antibodies were tested on chromaffin cell plasma membranes and calmodulin-binding proteins isolated from these membranes, they recognized a 65–kilodalton protein. These results indicate that an immunologically identical calmodulin-binding protein is expressed in both chromaffin granule membranes (as well as other secretory vesicle membranes) and chromaffin cell plasma membranes, thus suggesting a possible role for this protein in granule/plasma membrane interaction.     

Stimulation of Cell Elongation in Teleost Rod Photoreceptors by Distinct Protein Kinase Inhibitors

Abstract: The rod photoreceptors of teleost retinas elongate in the light. To characterize the role of protein kinases in elongation, pharmacological studies were carried out with rod fragments consisting of the motile inner segment and photosensory outer segment (RIS-ROS). Isolated RIS-ROS were cultured in the presence of membrane-permeant inhibitors that exhibit selective activity toward specific serine/threonine protein kinases. We report that three distinct classes of protein kinase inhibitors stimulated elongation in darkness: (1) cyclic AMP-dependent protein kinase (PKA)-selective inhibitors (H-89 and KT5720), (2) a protein kinase C (PKC)-selective inhibitor (GF 109203X) that affects most PKC isoforms, and (3) a kinase inhibitor (H-85) that does not affect PKC and PKA in vitro. Other kinase inhibitors tested neither stimulated elongation in darkness nor inhibited light-induced elongation; these include the myosin light chain kinase inhibitors ML-7 and ML-9, the calcium-calmodulin kinase II inhibitor KN-62, and inhibitors or activators of diacylglycerol-dependent PKCs (sphingosine, calphostin C, chelerythrine, and phorbol esters). The myosin light chain kinase inhibitors as well as the PKA and PKC inhibitors H-89 and GF 109203X all enhanced light-induced elongation. These observations suggest that light-induced RIS-ROS elongation is inhibited by both PKA and an unidentified kinase or kinases, possibly a diacylglycerol-independent form of PKC.     

Phosphorylation reactions in bovine rod outer segments studied by 32P-labelling of intact retina

The protein phosphorylation pattern in the intact bovine retina has been investigated by labelling with 32P-phosphate under incubation conditions that preserve the electrical photoresponse of the photoreceptor cells. The phosphorylation of rod outer segment proteins was analysed after isolation of outer segments from the labelled retina. The global influence of light, Ca2+ and the phosphodiesterase inhibitor, isobutylmethylxanthine, on protein phosphorylation in rod outer segments was analysed. A 12 kDa protein is the most prominent phosphorylated species in the intact bovine retina. Its phosphorylation is increased by light and/or Ca2+. Evidence is presented that this strongly phosphorylated protein is not located in the outer segment, and we suggest that it may be a synaptic protein. Retinal rod outer segment membrane proteins with apparent molecular weights of 245, 226, 125, 110, 50, 46, 38 and 20 all show light-stimulated phosphorylation. Lowering the extracellular Ca2+ levels results in a decrease of the phosphorylation level of some of these proteins, viz. at 125, 50, 38 and probably at 20 kDa. Such proteins, whose phosphorylation level is influenced both by light and by elevated Ca2+, are candidates for mediators of phototransduction. The phosphorylated species at 245, 226, 110, 50 and 20 kDa are enriched in rod outer segment plasma membrane preparations. These protein species could participate in the light-regulated modulation of the Na+-conductance of the plasma membrane.     

Calmodulin-binding protein detection using a non-radiolabeled calmodulin fusion protein

Calmodulin-binding proteins are involved in numerous cellular signaling pathways. The biotinylated-calmodulin overlay is a nonradioactive method widely used to detect calmodulin-binding proteins in tissue and cell samples. This method has several limitations; therefore, we developed a nonradioactive calmodulin-binding protein detection overlay using an S-tag-labeled calmodulin fusion protein. An expression system was used to generate a calmodulin fusion protein with an S-tag label, a 15 amino acid sequence that binds to a 105 amino acid S-protein. The S-protein is conjugated to horseradish peroxidase for final detection with a chemiluminescent substrate. The S-tag calmodulin was compared to purified calmodulin and biotinylated calmodulin in a calmodulin-dependent phosphodiesterase assay. The results of the calmodulin-dependent phosphodiesterase assay indicate that S-tag calmodulin induces higher phosphodiesterase activity than biotinylated calmodulin and lower activity than purified calmodulin. A comparison of the biotinylated and S-tag calmodulin overlay assays indicate that S-tag calmodulin is more sensitive than biotinylated calmodulin in the detection of calcineurin, a known calmodulin-binding protein. The overlay assay results also indicate that the S-tag calmodulin and biotinylated calmodulin detect similar calmodulin-binding proteins in colon epithelial cells. In conclusion, the S-tag calmodulin overlay assay is a consistent, sensitive, and rapid nonradioactive method to detect calmodulin-binding proteins.     

Actin-depolymerizing factor, ADF/cofilin, is essentially required in assembly of Leishmania flagellum

ADF/cofilins are ubiquitous actin dynamics-regulating proteins that have been mainly implicated in actin-based cell motility. Trypanosomatids, e.g. Leishmania and Trypanosoma, which mediate their motility through flagellum, also contain a putative ADF/cofilin homologue, but its role in flagellar motility remains largely unexplored. We have investigated the role of this protein in assembly and motility of the Leishmania flagellum after knocking out the ADF/cofilin gene by targeted gene replacement. The resultant mutants were completely immotile, short and stumpy, and had reduced flagellar length and severely impaired beat. In addition, the assembly of the paraflagellar rod was lost, vesicle-like structures were seen throughout the length of the flagellum and the state and distribution of actin were altered. However, episomal complementation of the gene restored normal morphology and flagellar function. These results for the first time indicate that the actin dynamics-regulating protein ADF/cofilin plays a critical role in assembly and motility of the eukaryotic flagellum.     

Calmodulin-binding proteins in brain

It is now widely accepted that actions of intracellular Ca²⁺ are mediated by a four-domain Ca²⁺-binding protein, calmodulin. Brain is especially rich in calmodulin, containing about 400 mg (24 μmol) of EGTA-extractable calmodulin per kg of brain. However, only a fraction of the above amount is required for the calmodulin-activated enzymes and most of the rest may be assigned to calmodulin-binding proteins, proteins which are apparently devoid of enzyme activities but undergo Ca²⁺-dependent associations with calmodulin. Several of such proteins have been recently discovered in brain. These include a heat-labile 80 K phosphodiesterase inhibitor protein (calcineurin), a heat-stable 70 K phosphodiesterase inhibitor protein, a 50 K protein, myelin basic protein, tubulin, microtubule τ (tau) factor, a spectrin-like doublet protein (240 plus 235 K) (calspectin; fodrin) and a particle-associated 155 K protein.Functions of these calmodulin-binding proteins have not been fully elucidated yet. Some proteins may be calmodulin-regulated enzymes catalyzing yet unknown biochemical reactions, e.g. a protein phosphatase activity was found for calcineurin. Some proteins may interact with contractile elements or cytoskeleton of the cell, e.g. τ factor and calspectin interacted with tubulin and F-actin, respectively and tubulin itself is a calmodulin-binding protein. So, interesting possibilities are the regulation of the functions of cytoskeleton by calmodulin through these calmodulin-binding proteins. Regulation of microtubule assembly by Ca²⁺-dependent binding of calmodulin to tubulin and/or τ factor and possible involvement of calspectin in the mechanism regulating axonal transport of neuronal proteins have been suggested. Thus, the exploration of the regulating functions of Ca²⁺/calmodulin in brain depends largely upon the further study of the properties of these calmodulin-binding proteins.     

Calmodulin binding proteins in human erythrocyte membranes

Human erythrocyte membranes reveal different calmodulin-binding proteins determined by a 125I-calmodulin gel overlay procedure. Beside the well-established Ca2+-transport ATPase, other proteins (205, 91, 72 and 42 kDa) bind calmodulin in a Ca2+-dependent manner. Two proteins of the human erythrocyte membrane are able to bind calmodulin only in the absence of Ca2+. One of them (76 kDa) is probably an integral, the other (240 kDa) a peripheral protein.     

A Dictyostelium mutant lacking an F-actin cross-linking protein, the 120-kD gelation factor

Actin-binding proteins are known to regulate in vitro the assembly of actin into supramolecular structures, but evidence for their activities in living nonmuscle cells is scarce. Amebae of Dictyostelium discoideum are nonmuscle cells in which mutants defective in several actin-binding proteins have been described. Here we characterize a mutant deficient in the 120-kD gelation factor, one of the most abundant F-actin cross-linking proteins of D. discoideum cells. No F-actin cross-linking activity attributable to the 120-kD protein was detected in mutant cell extracts, and antibodies recognizing different epitopes on the polypeptide showed the entire protein was lacking. Under the conditions used, elimination of the gelation factor did not substantially alter growth, shape, motility, or chemotactic orientation of the cells towards a cAMP source. Aggregates of the mutant developed into fruiting bodies consisting of normally differentiated spores and stalk cells. In cytoskeleton preparations a dense network of actin filaments as typical of the cell cortex, and bundles as they extend along the axis of filopods, were recognized. A significant alteration found was an enhanced accumulation of actin in cytoskeletons of the mutant when cells were stimulated with cyclic AMP. Our results indicate that control of cell shape and motility does not require the fine-tuned interactions of all proteins that have been identified as actin-binding proteins by in vitro assays.     

Integral membrane protein interaction with Triton cytoskeletons of erythrocytes

The organization of erythrocyte membrane lipids and proteins has been studied following the release of cytoplasmic components with the non-ionic detergent Triton X-100. After detergent extraction, a detergent-resistant complex called the erythrocyte cytoskeleton is separated from detergent, solubilized lipid and protein by sucrose buoyant density sedimentation. In cytoskeletons prepared under isotonic conditions all of the major erythrocyte membrane proteins are retained except for the integral protein, glycophorin, which is quantitatively solubilized and another integral glycoprotein, band 3, which is only 60% removed. When cytoskeletons are prepared in hypertonic KCl solutions, band 3 is fully solubilized along with bands 2.1 and 4.2 and several minor components. The resulting cytoskeletons have the same morphology as those prepared in isotonic buffer but they are composed of only three major peripheral proteins, spectrin, actin and band 4.1. We have designated this peripheral protein complex the 'shell' of the erythrocyte membrane, and have shown that the attachment of band 3 to the shell satisfies the criteria for a specific interaction. Although Triton did affect erythrocyte shape, cytoskeleton lipid content and the activity of membrane proteases, there was no indication that Triton altered the attachment of band 3 to the shell. We suggest that band 3 attaches to the shell as part of a ternary complex of bands 2.1, 3 and 4.2.     

Proteins of rod outer segments of toad retina: binding with calmodulin and with GTP

Proteins of purified rod outer segments from toad retina were analysed by electrophoresis on polyacrylamide gel containing sodium dodecyl sulfate. The binding of proteins with calmodulin and with guanosine triphosphate was studied by electroblotting the proteins resolved by electrophoresis onto nitrocellulose sheets and by incubating the blots with labelled ligands. The results indicate that rod outer segments from toad retina contain nine proteins which bind to calmodulin and one protein, different from transducin, that binds to guanosine triphosphate.     

Modulation of calmodulin levels, calmodulin methylation, and calmodulin binding proteins during carrot cell growth and embryogenesis.

Carrot cell cultures were used to study the dynamics of calmodulin protein levels, calmodulin methylation, and calmodulin-binding proteins during plant growth and development. Comparisons of proliferating and nonproliferating wild carrot cells show that, while calmodulin protein levels does not vary significantly, substantial variation in post-translational methylation of calmodulin on lysine-115 is observed. Calmodulin methylation is low during the lag and early exponential stages, but increases substantially as exponential growth proceeds and becomes maximal in the postexponential phase. Unmethylated calmodulin quickly reappears within 12 h of reinoculation of cells into fresh media, suggesting that the process is regulated according to the cell growth state. Calmodulin and calmodulin-binding proteins were also analyzed during the formation and germination of domestic carrot embryos in culture. Neither calmodulin methylation nor calmodulin protein levels varied significantly during somatic embryogenesis. However, upon germination of embryos, the level of calmodulin protein doubled. By calmodulin overlay analysis, we have detected a major 54,000 M(r) calmodulin-binding protein that also increased during embryo germination. This protein was purified from carrot embryo extracts by calmodulin-Sepharose chromatography. Overall, the data suggest that calmodulin methylation is regulated depending upon the state of cell growth and that calmodulin and its target proteins are modulated during early plant development.     

Crystal structure of the alpha-actinin rod reveals an extensive torsional twist

BACKGROUND: Alpha-actinin is a ubiquitously expressed protein found in numerous actin structures. It consists of an N-terminal actin binding domain, a central rod domain, and a C-terminal domain and functions as a homodimer to cross-link actin filaments. The rod domain determines the distance between cross-linked actin filaments and also serves as an interaction site for several cytoskeletal and signaling proteins. RESULTS: We report here the crystal structure of the alpha-actinin rod. The structure is a twisted antiparallel dimer that contains a conserved acidic surface. CONCLUSIONS: The novel features revealed by the structure allow prediction of the orientation of parallel and antiparallel cross-linked actin filaments in relation to alpha-actinin. The conserved acidic surface is a possible interaction site for several cytoplasmic tails of transmembrane proteins involved in the recruitment of alpha-actinin to the plasma membrane.     

Calmodulin and calmodulin-binding proteins in the renal brush border

The calmodulin content of renal brush-border membrane vesicles, prepared by Mg2+-precipitation in EGTA-containing solutions, amounts to 1.8 micrograms per mg protein. The amount and the distribution of this EGTA-insensitive calmodulin was determined in membrane and cytoskeletal fractions prepared from the brush-border membrane vesicles by extraction with Triton X-100. The Triton X-100 insoluble pellet contains 21.2% of the protein and 52.2% of the EGTA-insensitive calmodulin, which amounts in this fraction to 4.4 micrograms per mg protein. Treatment of the Triton X-100 insoluble pellet, consisting of the microvillar core residue, with ATP and Mg2+ results in the solubilization of a relatively small number of proteins among which are actin, myosin, calmodulin and several calmodulin-binding proteins. The solubilization is partially reversible and a fraction of the proteins can be precipitated by centrifugation after the enzymatic hydrolysis of ATP. Readdition of ATP to the pellet results in the resolubilization of myosin, part of the actin, an 115-kDa calmodulin-binding protein and calmodulin. The calmodulin content of the final extract was 61.8 micrograms per mg protein. We have found roughly the same distribution pattern of calmodulin and ATP-solubilized, calmodulin-binding proteins in renal and intestinal brush-border preparations. The calmodulin content, however, as well as the relative amount of the calmodulin-binding proteins versus actin are about 4 to 5-times higher in intestinal than in renal microvillar core residues.