Molecular structure of the sarcomeric Z-disk: two types of titin interactions lead to an asymmetrical sorting of alpha-actinin.

The sarcomeric Z-disk, the anchoring plane of thin (actin) filaments, links titin (also called connectin) and actin filaments from opposing sarcomere halves in a lattice connected by alpha-actinin. We demonstrate by protein interaction analysis that two types of titin interactions are involved in the assembly of alpha-actinin into the Z-disk. Titin interacts via a single binding site with the two central spectrin-like repeats of the outermost pair of alpha-actinin molecules. In the central Z-disk, titin can interact with multiple alpha-actinin molecules via their C-terminal domains. These interactions allow the assembly of a ternary complex of titin, actin and alpha-actinin in vitro, and are expected to constrain the path of titin in the Z-disk. In thick skeletal muscle Z-disks, titin filaments cross over the Z-disk centre by approximately 30 nm, suggesting that their alpha-actinin-binding sites overlap in an antiparallel fashion. The combination of our biochemical and ultrastructural data now allows a molecular model of the sarcomeric Z-disk, where overlapping titin filaments and their interactions with the alpha-actinin rod and C-terminal domain can account for the essential ultrastructural features.     

A Ca2+-activated protease possibly involved in myofibrillar protein turnover. Partial characterization of the purified enzyme.

The purified Ca2+-activated protease (CAF) isolated from porcine skeletal muscle and capable of removing Z-disks from intact myofibrils is optimally active on either myofibril or casein substrates at pH 7.5 and in the presence of 1 mM Ca2+ and at least 2 mM 2-mercaptoethanol. No CAF activity is detected when 1 mM Mg2+, Mn2+, Ba2+, Co2+, Ni2+, and Fe2+ are added singly. When added with 1 mM Ca2+, Co2+, Cu2+, Ni2+, and Fe2+ inhibit, whereas Mg2+, Mn2+, and Ba2+ have no effect on CAF activity. CAF is irreversibly inhibited by iodoacetate but is unaffected by soybean trypsin inhibitor. S0/20,W=5.90 S, and sedimentation equilibrium molecular weight - 112 000 for purified CAF. Because purified CAF migrates as two polypeptide chains with molecular weights of 80 000 and 30 000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the CAF molecule must consist of one each of these two polypeptide chains. Approximate molecular dimensions of 38 X 220 A can be calculated for CAF from calibrated gel permeation column data or from S0/20,W and the molecular weight. Amino acid composition and physical properties of purified CAF distinguish it from the known catheptic enzymes and from other proteases found in blood or in granulocytes. Purified CAF removes Z-disks the 400-A periodicity associated with troponin in the I band and partly degrades M lines but causes no other ultrastructurally detectable effects when incubated with myofibrils. These results agree with the earlier finding that purified CAF degrades troponin, tropomyosin, and C-protein but has no effect on myosin, actin, or alpha-actinin, and suggest that CAF may have a physiological role in disassembly of intact myofibrils during metabolic turnover of myofibrillar proteins.     

Inhibition of CapZ during myofibrillogenesis alters assembly of actin filaments

The actin filaments of myofibrils are highly organized; they are of a uniform length and polarity and are situated in the sarcomere in an aligned array. We hypothesized that the barbed-end actin-binding protein, CapZ, directs the process of actin filament assembly during myofibrillogenesis. We tested this hypothesis by inhibiting the actin- binding activity of CapZ in developing myotubes in culture using two different methods. First, injection of a monoclonal antibody that prevents the interaction of CapZ and actin disrupts the non-striated bundles of actin filaments formed during the early stages of myofibril formation in skeletal myotubes in culture. The antibody, when injected at concentrations lower than that required for disrupting the actin filaments, binds at nascent Z-disks. Since the interaction of CapZ and the monoclonal antibody are mutually exclusive, this result indicates that CapZ binds nascent Z-disks independent of an interaction with actin filaments. In a second approach, expression in myotubes of a mutant form of CapZ that does not bind actin results in a delay in the appearance of actin in a striated pattern in myofibrils. The organization of alpha-actinin at Z-disks also is delayed, but the organization of titin and myosin in sarcomeres is not significantly altered. We conclude that the interaction of CapZ and actin is important for the organization of actin filaments of the sarcomere.     

Proteins Released from Myofibrils by CASF (Ca2+-activated sarcoplasmic factor) and Trypsin

The nature of the soluble proteins and peptides released from myofibrils by treatment with CASF (Ca²⁺-activated sarcoplasmic factor) was investigated by using Polyacrylamide gel electrophoresis in both a nondenaturing and a denaturing (sodium dodecyl sulfate=SDS) solvent and by using gel permeation chromatography on Sepharose 6B. Both CASF and trypsin treatment cause removal of Z-disks before causing other ultrastructurally detectable degradation of myofibrils. CASF treatment of myofibrils releases a protein that is identical to α-actinin, one of the known components of the Z-disk, on the basis of mobility in Polyacrylamide gel electrophoresis in a nondenaturing solvent and in SDS and on the basis of elution from gel permeation columns. Trypsin treatment of myofibrils releases a number of smaller molecular weight products that cannot be identified with any of the known myofibrillar proteins. Hence, CASF seems to remove Z-disks from myofibrils by means of a very specific proteolytic activity that releases α-actinin without extensively degrading it. Trypsin, on the other hand, probably seems to remove Z-disks by degrading α-actinin to peptides.     

Tetanic contractions impair sarcomeric Z-disk of atrophic soleus muscle via calpain pathway

The aim of this study was to determine whether or not over-activation of calpains during running exercise or tetanic contractions was a major factor to induce sarcomere lesions in atrophic soleus muscle. Relationship between the degrees of desmin degradation and sarcomere lesions was also elucidated. We observed ultrastructural changes in soleus muscle fibers after 4-week unloading with or without running exercise. Calpain activity and desmin degradation were measured in atrophic soleus muscles before or after repeated tetani in vitro. Calpain-1 activity was progressively increased and desmin degradation was correspondingly elevated in 1-, 2-, and 4-week of unloaded soleus muscles. Calpain-1 activity and desmin degradation had an additional increase in unloaded soleus muscles after repeated tetani in vitro. PD150606, an inhibitor of calpains, reduced calpain activity and desmin degradation during tetanic contractions in unloaded soleus muscles. The 4-week unloading decreased the width of myofibrils and Z-disk in soleus fibers. After running exercise in unloaded group, Z-disks of adjacent myofibrils were not well in register but instead were longitudinally displaced. Calpain inhibition compromised exercise-induced misalignment of the Z-disks in atrophic soleus muscle. These results suggest that tetanic contractions induce an over-activation of calpains which lead to higher degrees of desmin degradation in unloaded soleus muscle. Desmin degradation may loose connections between adjacent myofibrils, whereas running exercise results in sarcomere injury in unloaded soleus muscle.     

An 100-kDa Ca2+-sensitive actin-fragmenting protein from unfertilized sea urchin egg

An actin-modulating protein was purified from unfertilized eggs of sea urchin, Hemicentrotus pulcherrimus, by means of DNase I affinity and DEAE-cellulose column chromatographies. This protein was a globular protein with a Stokes radius of 41-42 nm and consisted of a single polypeptide chain having an apparent molecular mass of 100 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Gel filtration chromatography revealed that one 100-kDa protein molecule binds two or three actin monomers in the presence of Ca2+, but such binding was not observed in the absence of Ca2+. The effect of the 100-kDa protein on the polymerization of actin was studied by viscometry, spectrophotometry and electron microscopy. The initial rate of actin polymerization was decreased at a very low molar ratio of 100-kDa protein/actin. Acceleration of the initial rate of polymerization occurred at a relatively high, but still substoichiometric, molar ratio of 100-kDa protein/actin. The 100-kDa protein produced fragmentation of muscle actin filaments at Ca2+ concentrations greater than 0.3 microM as revealed by viscometry and electron microscopy. Evidence was also presented that the 100-kDa protein binds to the barbed end of the actin filament.     

Stimulation of the interaction between actin and myosin by Physarum caldesmon-like protein and smooth muscle caldesmon.

We have purified an actin-binding protein from the plasmodia of a lower eukaryote, Physarum polycephalum, with an apparent molecular mass of 210,000 daltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This protein bound to actin filaments with a stoichiometry of 1:7-8 in a Ca(2+)-calmodulin-dependent manner. Antibody raised against caldesmon from smooth muscle cross-reacted with the 210-kDa protein. In vitro motility assay revealed that the 210-kDa protein increased the sliding velocity of actin filaments on Physarum myosin. The 210-kDa protein more than doubled the actin-activated ATPase activity of Physarum myosin under comparative conditions of in vitro motility assay. Further increases in the concentration of the 210-kDa protein decreased its stimulatory effects. Ca(2+)-calmodulin prevented the stimulatory effects of the 210-kDa protein. Unexpectedly, smooth muscle caldesmon also increased the sliding velocity of actin filaments on smooth muscle myosin at lower concentrations. The well-known inhibitory effect of smooth muscle caldesmon on the actin-myosin interaction was observed with this motility assay when the concentration of the caldesmon was increased further. The stimulatory and inhibitory effects were confirmed by measurements of actin-activated ATPase activity of smooth muscle myosin. From estimations of the intracellular concentrations of the 210-kDa protein and smooth muscle caldesmon in vivo, it appears that effects of the former and the latter on actin-myosin interactions in vivo are stimulatory and inhibitory, respectively.     

Cytoplasmic gels from macrophages. Evidence for the involvement of four proteins in the calcium-dependent solation of actin networks

A method has been devised to study the influence of Ca2+ on the in vitro formation of actin gel networks. Under appropriate conditions low-Ca2+ cytosolic extracts (less than 1 nM) from macrophages rapidly formed a macromolecular complex composed of actin, filamin, alpha-actinin and two new proteins of 70 kDa and 55 kDa. [Pacaud, M. (1986) Eur. J. Biochem. 156, 521-530]. Increasing concentrations of free Ca2+ to 1-2 microM resulted in complete inhibition of the association of 70-kDa protein, a protein which associates actin filaments into parallel arrays. Concentrations of Ca2+ greater than 3 microM caused incorporation of two additional proteins, gelsolin and a 18-kDa polypeptide, with no change in either the actin or alpha-actinin content of the cytoskeletal structures. Use of a polyacrylamide gel overlay technique with 125I-calmodulin revealed that a high-Mr calmodulin-binding protein analogous to spectrin was also associated with these structures when micromolar Ca2+ was present. Similar assays with 45CaCl2 indicated that the 70-kDa protein binds Ca2+ with high affinity. It is thus suggested that Ca2+ might regulate the dynamic assembly of microfilaments through several target proteins, gelsolin, the 70-kDa protein and calmodulin.     

Calcium-induced weakening of skeletal muscle Z-disks

Structural changes in the Z disk were sensitively detected by measuring fragmentation indexes of myofibrils. The Ca2+-induced weakening of Z disks and the Z-disk removal by muscle calpain could be clearly distinguished by using muscle calpastatin, an endogenous inhibitor of muscle calpain. The Ca2+-induced weakening of Z disks occurred without concomitant release of alpha-actinin and had maxima at 10(-4) M Ca2+ and 45 degrees C and a minimum at pH 6.5, while the Z-disk removal by calpain had similar optima to the caseinolytic activity of calpain, at 10(-3) M Ca2+, 20 degrees C and pH 7.0. The Ca2+-induced weakening of Z disks is therefore not due to the proteolytic action of calpain. In postmortem muscle, moreover, the Ca2+-induced weakening of Z disks was inferred to be predominate over calpain proteolysis, and therefore to be the major factor in the characteristic weakening of Z disks.     

Calvasculin, an encoded protein from mRNA termed pEL-98, 18A2, 42A, or p9Ka, is secreted by smooth muscle cells in culture and exhibits Ca(2+)-dependent binding to 36-kDa microfibril-associated glycoprotein.

Calvasculin, an EF-hand protein with a molecular mass of 11 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is present abundantly in bovine aorta (Watanabe, Y., Kobayashi, R., Ishikawa, T., and Hidaka, H. (1992) Arch. Biochem. Biophys. 292, 563-569). This protein is synthesized constitutively by bovine aortic smooth muscle (BASM) cells and rat embryo fibroblast 3Y1 cells in culture. We discovered that calvasculin was secreted by BASM cells and 3Y1 cells. Immunofluorescence staining of BASM cells showed a granular distribution for calvasculin that was typical of a secreted protein. This protein bound with an extracellular matrix protein, 36-kDa microfibril-associated glycoprotein (36-kDa MAP), in a Ca(2+)-dependent manner in vitro. A stoichiometry analysis showed that the 36-kDa MAP bound 2.2 calvasculin eq/mol of protein. Solid-phase binding assays indicated a preferential affinity of native calvasculin for 36-kDa MAP among the extracellular matrices in a Ca(2+)-dependent manner. These results suggest that calvasculin, intracellular Ca(2+)-binding protein, is released to the extracellular space and binds with 36-kDa MAP.     

Isolation of F-actin filaments. Comparison of F-actin filament preparations from normal and dystrophic mouse muscle.

To investigate the dystrophic influence on the characteristics of actin, a method for the isolation of F-actin filaments from the skeletal muscle of small sizes, i.e., less than 0.5 g, was devised. In this method, minced muscle was treated with collagenase and hyaluronidase, and the isolated filaments were washed with adenosine triphosphate (ATP). Upon examination in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the ATP-washed filaments showed a protein component identical in mobility to actin in untreated myofibrils or to that prepared by the conventional method. Electron microscopic appearances of the filaments were similar to those of F-actin filaments described in the literature. The dimensions of the filaments were 0.5--2.5 micrometer in length and 60--70 A in diameter. The ability to activate the Mg-adenosine triphosphatase or myosin was found to be Ca2+ independent. In all aspects of the above characteristics, the filaments from leg muscles of 129/Re dydy dystrophic mice and their litter mates were observed to be identical.     

The stoichiometry and location of troponin I- and troponin C-like proteins in the myofibril of the bay scallop, Aequipecten irradians.

Localization and quantification studies were carried out on bay-scallop (Aequipecten irradians) striated-muscle troponin C- and troponin I-like proteins. Indirect immunofluorescence microscopy of scallop myofibrils stained with either rabbit anti-(scallop troponin I) or anti-(scallop troponin C) antibodies shows staining of all I-bands observed. The results of quantification studies using sodium dodecyl sulfate poly-acrylamide-gel electrophoresis of untreated scallop myofibrils, washed scallop myofibrils, and isolated scallop thin filaments indicate an actin/tropomyosin/troponin-C molar rationn of 7:1:1. The molar ratio for troponin I could not be determined in untreated myofibrils because of interfering bands; in washed myofibrils a value of 0.6 mol of troponin I/mol of tropomyosin was found. Purified scallop troponin C binds Ca2+ and interacts with scallop troponin I to relieve troponin I-induced inhibition of actomyosin ATPase. Although scallop troponin C is an acidic protein, it appears to be less acidic than troponin C from higher organisms. A calmodulin-like protein has been isolated from scallop striated muscle that activates bovine brain phosphodiesterase to the same extent as does brain calmodulin. Its amino acid composition and its electrophoretic mobility on alkaline 6 M-urea/polyacrylamide gels differs from that of scallop troponin C, and it appears not to be associated with thin filaments.     

Analysis of myofibrillar structure and assembly using fluorescently labeled contractile proteins

To study how contractile proteins become organized into sarcomeric units in striated muscle, we have exposed glycerinated myofibrils to fluorescently labeled actin, alpha-actinin, and tropomyosin. In this in vitro system, alpha-actinin bound to the Z-bands and the binding could not be saturated by prior addition of excess unlabeled alpha-actinin. Conditions known to prevent self-association of alpha-actinin, however, blocked the binding of fluorescently labeled alpha-actinin to Z-bands. When tropomyosin was removed from the myofibrils, alpha-actinin then added to the thin filaments as well as the Z-bands. Actin bound in a doublet pattern to the regions of the myosin filaments where there were free cross-bridges i.e., in that part of the A-band free of interdigitating native thin filaments but not in the center of the A- band which lacks cross-bridges. In the presence of 0.1-0.2 mM ATP, no actin binding occurred. When unlabeled alpha-actinin was added first to myofibrils and then labeled actin was added fluorescence occurred not in a doublet pattern but along the entire length of the myofibril. Tropomyosin did not bind to myofibrils unless the existing tropomyosin was first removed, in which case it added to the thin filaments in the l-band. Tropomyosin did bind, however, to the exogenously added tropomyosin-free actin that localizes as a doublet in the A-band. These results indicate that the alpha-actinin present in Z-bands of myofibrils is fully complexed with actin, but can bind exogenous alpha- actinin and, if actin is added subsequently, the exogenous alpha- actinin in the Z-band will bind the newly formed fluorescent actin filaments. Myofibrillar actin filaments did not increase in length when G-actin was present under polymerizing conditions, nor did they bind any added tropomyosin. These observations are discussed in terms of the structure and in vivo assembly of myofibrils.     

Alpha-actinin is a component of the Z-filament, a structural backbone of skeletal muscle Z-disks

The relationship between the ultrastructure and protein components of Z-disks was studied using psoas muscles of rabbit and breast muscles of chicken. Glycerinated fiber bundles of these muscles were treated with a solution containing 0.1 mM Ca2+ to induce a structural weakening of Z-disks non-enzymatically. During the treatment, clear geometrical configurations of Z-filaments could be observed along with removal of amorphous matrix under an electron microscope. Even after a prolonged treatment for 14 d in which all Z-disks were entirely weakened, entangled Z-filaments were left in the original region of the disks. Immunoelectron microscopic observations showed that antibodies against alpha-actinin bound to the entangled Z-filaments, forming dense lines at the position of the original Z-disks. On SDS-PAGE of Z-disk substances, alpha-actinin remained unchanged and Mr 75,000 and 55,000 proteins were removed during the Ca2+-treatment. We therefore conclude that alpha-actinin is a component of Z-filaments, and that the amorphous matrix is composed at least of these Mr 75,000 and 55,000 proteins.     

Localization of Z-protein in isolated Z-disk sheets of chicken leg muscle

Immunoblotting studies with antisera against Z-protein, desmin, and alpha-actinin showed that Z-protein is clearly distinguishable from desmin and alpha-actinin. Z-protein is not a proteolytic product of another protein but is an intrinsic component of chicken breast muscle myofibrils. In these experiments, an SDS extract of intact muscle was first electrophoresed in a polyacrylamide gel, and then proteins were transferred to a nitrocellulose paper sheet. Detection of each protein on the sheet was made possible by the application of the indirect immunofluorescence technique with the respective antiserum. Immunofluorescence microscope studies using these antisera revealed that Z-protein has the same distribution as alpha-actinin in isolated Z- disk sheets. Anti-Z-protein antiserum and anti-alpha-actinin antiserum stained the interior of Z-disks. On the other hand, antiserum against desmin stained the periphery of Z-disks in isolated Z-disk sheets.     

Stringent requirement for Ca2+ in the removal of Z-lines and alpha-actinin from isolated myofibrils by Ca2+-activated neutral proteinase

Treatment of isolated myofibrils with Ca2+-activated neutral proteinase (CANP) results in specific removal of Z-line and of alpha-actinin. To investigate the ionic requirement for these processes, we measured Z-line removal by phase-contrast and interference microscopy and alpha-actinin removal by sodium dodecyl sulphate/polyacrylamide-gel electrophoretic analysis of myofibrillar proteins. The proteolytic digestion of native purified proteins was measured directly on polyacrylamide gels and by the fluorescamine technique. We found that the removal of Z-line and alpha-actinin as well as the release of proteolytic degradation products from isolated myofibrils by CANP occur only in the presence of Ca2+; Sr2+, Ba2+, Mn2+, Mg2+, Co2+ and Zn2+ are all ineffective. In contrast with this stringent requirement for Ca2+, the proteolytic activity of CANP measured with denatured casein, native and denatured haemoglobin, native actin and tropomyosin also occurs in the presence of other bivalent cations, in the following order: Ca2+ greater than Sr2+ greater than Ba2+. These data suggest that only Ca2+ can produce the conformational change in myofibrils that renders them susceptible to the action of CANP, whereas its proteolytic activity is stimulated by several bivalent ions.     

Elevated levels of a calcium-activated muscle protease in rapidly atrophying muscles from vitamin E-deficient rabbits.

A Ca2+-activated proteolytic enzyme that partially degrades myofibrils was isolated from hind limb muscles of normal rabbits and rabbits undergoing rapid muscle atrophy as a result of vitamin E deficiency. Extractable Ca2+-activated protease activity was 3.6 times higher in muscle tissue from vitamin E-deficient rabbits than from muscle tissue of control rabbits. Ultrastructural studies of muscle from vitamin E-deficient rabbits showed that the Z disk was the first myofibrillar structure to show degradative changes in atrophying muscle. Myofibrils prepared from muscles from vitamin E-deficient rabbits showed partial or complete loss of Z-disk density. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that the amount of troponin-T (37 000 daltons) and alpha-actinin (96 000 daltons) was reduced in myofibrils from atrophying muscle as compared to myofibrils prepared from control muscle. In vitro treatment of purified myofibrils with purified Ca2+-activated proteolytic enzyme produced alterations in myofibrillar ultrastructure that were identical to the initial alterations occurring in myofibrils from atrophying muscle (i.e. weakening and subsequent removal of Z disks). Additonally the electrophoretic banding pattern of Ca2+-activated proteolytic enzyme-treated myofibrils is very similar to that of myofibrils prepared from muscles atrophying as a result of nutritional vitamin E deficiency. The possible role of Ca2+-activated proteolytic enzyme in disassembly and degradation of the myofibril is discussed.     

The rho-guanine nucleotide exchange factor domain of obscurin regulates assembly of titin at the Z-disk through interactions with Ran binding protein 9

Obscurin is an approximately 800-kDa protein composed of structural and signaling domains that organizes contractile structures in striated muscle. We have studied the Rho-GEF domain of obscurin to understand its roles in morphogenesis and signaling. We used adenoviral overexpression of this domain, together with ultrastructural and immunofluorescence methods, to examine its effect on maturing myofibrils. We report that overexpression of the Rho-GEF domain specifically inhibits the incorporation of titin into developing Z-disks and disrupts the structure of the Z-disk and Z/I junction, and alters features of the A/I junction. The organization of other sarcomeric markers, including alpha-actinin, was not affected. We identified Ran binding protein 9 (RanBP9) as a novel ligand of the Rho-GEF domain and showed that binding is specific, with an apparent binding affinity of 1.9 muM. Overexpression of the binding region of RanBP9 also disrupted the incorporation of titin into developing Z-disks. Immunofluorescence localization during myofibrillogenesis indicated that the Rho-GEF domain assembles into sarcomeres before RanBP9, which first occurs in myonuclei and later in development translocates to the myoplasm, where it colocalizes with obscurin. Both the Rho-GEF domain and its binding region on RanBP9 bind directly to the N-terminal Ig domains of titin, which flank the Z-disk. Our results suggest that the Rho-GEF domain interacts with RanBP9 and that both can interact with the N-terminal region of titin to influence the formation of the Z-disk and A/I junction.     

Characterization of components of Z-bands in the fibrillar flight muscle of Drosophila melanogaster

Twelve monoclonal antibodies have been raised against proteins in preparations of Z-disks isolated from Drosophila melanogaster flight muscle. The monoclonal antibodies that recognized Z-band components were identified by immunofluorescence microscopy of flight muscle myofibrils. These antibodies have identified three Z-disk antigens on immunoblots of myofibrillar proteins. Monoclonal antibodies alpha:1-4 recognize a 90-100-kD protein which we identify as alpha-actinin on the basis of cross-reactivity with antibodies raised against honeybee and vertebrate alpha-actinins. Monoclonal antibodies P:1-4 bind to the high molecular mass protein, projectin, a component of connecting filaments that link the ends of thick filaments to the Z-band in insect asynchronous flight muscles. The anti-projectin antibodies also stain synchronous muscle, but, surprisingly, the epitopes here are within the A-bands, not between the A- and Z-bands, as in flight muscle. Monoclonal antibodies Z(210):1-4 recognize a 210-kD protein that has not been previously shown to be a Z-band structural component. A fourth antigen, resolved as a doublet (approximately 400/600 kD) on immunoblots of Drosophila fibrillar proteins, is detected by a cross reacting antibody, Z(400):2, raised against a protein in isolated honeybee Z-disks. On Lowicryl sections of asynchronous flight muscle, indirect immunogold staining has localized alpha-actinin and the 210-kD protein throughout the matrix of the Z-band, projectin between the Z- and A-bands, and the 400/600-kD components at the I-band/Z-band junction. Drosophila alpha-actinin, projectin, and the 400/600-kD components share some antigenic determinants with corresponding honeybee proteins, but no honeybee protein interacts with any of the Z(210) antibodies.     

Calcium-activated neutral proteases (calpains) are carbohydrate binding proteins

Calcium-activated neutral proteases (calpain, EC 3.4.22.17) bind to agarose matrices (Bio-Gel A-150m, Sepharose 4B, and Ultrogel AcA 34) with high affinity in the presence of calcium. 6-O-beta-Galactopyranosyl-D-galactose, a disaccharide which closely resembles the repeating unit of the agarose matrices, completely blocks the binding of calpains and can release agarose-bound enzymes in the presence of calcium. At least 1 microM level of free calcium is required for binding. Other calcium binding proteins, including calmodulin, calpastatin, casein, and neurofilament proteins, fail to bind under the same conditions. Both calpain I and calpain II can be readily purified from crude enzyme preparations by agarose chromatography in the presence of calcium and leupeptin. Agarose-bound enzymes are eluted with calcium-free solutions or can be released in the presence of calcium by 1% Triton X-100, but not by 1 M urea or 20% ethylene glycol. Enzymes eluted from agarose are activated, as evidenced by the appearance of faster migrating forms (76 and 78 kDa) of the 80-kDa catalytic subunit of calpain I upon electrophoresis and by the increased sensitivity of calpain II to activation by micromolar levels of calcium. The electrophoretic migration of the 30-kDa regulatory subunit is, however, unaltered in enzyme fractions eluted from an agarose column. When the enzyme subunits are dissociated in 1 M NaSCN, only the 30-kDa subunit binds to the agarose matrix. Furthermore, neither calpain I nor calpain II binds to agarose when their 30-kDa subunit is autocatalyzed to an 18-kDa fragment, indicating that the NH2-terminal of the 30-kDa subunit is important for the binding of calpains to an agarose matrix.