Tissue distribution of high molecular weight calmodulin-binding protein

Polyclonal antibodies raised against bovine heart high molecular weight calmodulin-binding protein were used to study the distribution of this protein in diverse bovine tissues. The high molecular weight calmodulin-binding protein, in addition to bovine heart, is also present in lung and brain at much lower levels, but not in skeletal muscle, spleen, kidney or uterus.     

Desmocalmin: a calmodulin-binding high molecular weight protein isolated from desmosomes

A unique high molecular weight protein (240,000 mol wt) has been purified from isolated desmosomes of bovine muzzle epidermis, using low-salt extraction at pH 9.5-10.5 and gel-filtration followed by calmodulin-affinity column chromatography. This protein was shown to bind to calmodulin in a Ca2+-dependent manner, so we called it desmocalmin here. Desmocalmin also bound to the reconstituted keratin filaments in vitro in the presence of Mg2+, but not to actin filaments. By use of the antibody raised against the purified desmocalmin, desmocalmin was shown by both immunoelectron and immunofluorescence microscopy to be localized at the desmosomal plaque just beneath the plasma membrane. Judging from its isoelectric point and antigenicity, desmocalmin was clearly distinct from desmoplakins I and II, which were identified in the desmosomal plaque by Mueller and Franke (1983, J. Mol. Biol., 163:647-671). In the low-angle, rotary-shadowing electron microscope, the desmocalmin molecules looked like flexible rods approximately 100-nm long consisting of two polypeptide chains lying side by side. The similar rodlike structures were clearly identified in the freeze-etch replica images of desmosomes. Taken together, these findings indicate that desmocalmin could function as a key protein responsible for the formation of desmosomes in a calmodulin-dependent manner (Trinkaus-Randall, V., and I.K. Gipson, 1984, J. Cell Biol., 98:1565-1571).     

Novel bovine heart calmodulin-dependent protein kinase which phosphorylates a high molecular weight calmodulin-binding protein.

A novel calmodulin-dependent protein kinase has been isolated from bovine cardiac muscle by successive chromatography on DEAE-Sepharose 6B, Calmodulin-Sepharose 4B affinity and Sepharose 6B chromatography columns. The protein kinase was shown by gel filtration chromatography to have a molecular mass of 36,000 daltons. The highly purified protein kinase stoichiometrically phosphorylated the high molecular weight calmodulin-binding protein from cardiac muscle [Sharma RK (1990) J Biol Chem 265, 1152-1157] in a Ca2+/calmodulin-dependent manner. The phosphorylation resulted in the maximal incorporation of 1 mol of phosphate/mol of the high molecular weight calmodulin-binding protein. Other Ca2+/calmodulin-dependent protein kinases failed to phosphorylate the high molecular weight calmodulin-binding protein. The distinct substrate specificity of this protein kinase indicates that it is not related to the known calmodulin-dependent protein kinases and therefore constitutes a novel protein kinase.     

Cardiac high molecular weight calmodulin-binding protein is homologous to calpastatin I and calpastatin II

Calpastatin is an endogenous inhibitor of calpain, which has been implicated in various physiological and pathological processes. In the present study we determined the molecular and inhibitory properties of HMWCaMBP, calpastatin I, and calpastatin II. Western blot analysis with antibodies raised against either full length HMWCaMBP or internal peptides that are common to all isoforms showed that all three homologs have common antigenic epitopes. However, additional Western blot analysis with N-terminal specific antibodies showed that all three proteins are different at the N-terminal end. HMWCaMBP is clearly different from two other homologues, calpastatin I and II, at the N-terminal end. In addition, HMWCaMBP also showed the same affinities for m-calpain as calpastatin I and calpastatin II. Our findings suggest that HMWCaMBP is the homolog of calpastatin and may be a CaM-binding form of calpastatin.     

The cytoskeletal system of nucleated erythrocytes. I. Composition and function of major elements

We have studied the dogfish erythrocyte cytoskeletal system, which consists of a marginal band of microtubules (MB) and trans-marginal band material (TBM). The TBM appeared in whole mounts as a rough irregular network and in thin sections as a surface-delimiting layer completely enclosing nucleus and MB. In cells incubated at 0 degrees C for 30 min or more, the MB disappeared but the TBM remained. MB reassembly occurred with rewarming, and was inhibited by colchicine. Flattened elliptical erythrocyte morphology was retained even when MBs were absent. Total solubilization of MB and TBM at low pH, or dissolution of whole anucleate cytoskeletons, yielded components comigrating with actin, spectrin, and tubulin standards during gel electrophoresis. Mass-isolated MBs, exhibiting ribbonlike construction apparently maintained by cross-bridges, contained four polypeptides in the tubulin region of the gel. Only these four bands were noticeably increased in the soluble phase obtained from cells with 0 degrees C- disassembled MBs. The best isolated MB preparations contained tubulin but no components comigrating with high molecular weight microtubule- associated proteins, spectrin, or actin. Actin and spectrin therefore appear to be major TBM constituents, with tubulin localized in the MB. The results are interpreted in terms of an actin- and spectrin- containing subsurface cytoskeletal layer (TBM), related to that of mammalian erythrocytes, which maintains cell shape in the absence of MBs. Observations on abnormal pointed erythrocytes containing similarly pointed MBs indicate further that the MB can deform the TBM from within so as to alter cell shape. MBs may function in this manner during normal cellular morphogenesis and during blood flow in vivo.     

The cytoskeletal system of nucleated erythrocytes. III. Marginal band function in mature cells

Marginal bands (MBs) of microtubules are believed to function during morphogenesis of nonmammalian vertebrate erythrocytes, but there has been little evidence favoring a continuing role in mature cells. To test MB function, we prepared dogfish erythrocytes with and without MBs at the same temperature by (a) stabilization of the normally cold- labile MB at 0 degree C by taxol, and (b) inhibition of MB reassembly at room temperature by nocodazole or colchicine. We then compared the responses of these cells to mechanical stress by fluxing them through capillary tubes. Before fluxing , cells with or without MBs had normal flattened elliptical shape. After fluxing , deformation was consistently observed in a much greater percentage of cells lacking MBs. The difference in percent deformation between the two cell types was highly significant. That the MB is an effector of cell shape was further documented in studies of the formation of singly or doubly pointed dogfish erythrocytes that appear during long-term incubation of normal cells at room temperature. On-slide perfusion experiments revealed that the pointed cells contain MBs of corresponding pointed morphology. Incubation of cells with and without MBs showed that they become pointed only when they contain MBs, indicating that the MB acts as a flexible frame which can deform and support the cell surface from within. To test this idea further, cells with and without MBs were exposed to hyperosmotic conditions. Many of the cells without MBs collapsed and shriveled , whereas those with MBs did not. The results support the view that the MB has a continuing function in mature erythrocytes, resisting deformation and/or rapidly returning deformed cells to an efficient equilibrium shape in the circulation.     

A high molecular weight peptide hydrolase in erythrocytes

1. A peptide hydrolase has been partially purified from the soluble fraction of erythrocyte lysates. 2. The enzyme has a molecular weight of approximately 600,000 and hydrolyses the chymotrypsin substrate glutaryl-Gly-Gly-Phe-7-amido-4-methylcoumarin (pH optimum 7.0) and the trypsin substrate CBZ-Gly-Gly-Arg-2-naphthylamide. The two activities could not be separated by the purification procedure used. 3. The activity towards glutaryl-Gly-Gly-Phe-7-amido-4-methylcoumarin in rat reticulocytes was four times that in mature erythrocytes. 4. Activity was abolished by 10 microM p-hydroxymercuriphenylsulphonic acid.     

Immunolocalization of the 110,000 molecular weight cytoskeletal protein of intestinal microvilli

The core structures of microvilli from absorptive cells of the intestinal epithelium are primarily composed of calmodulin (M_r 16,000), actin (M_r 43,000), villin (M_r 95,000) and a protein of M_r 110,000. We have isolated this protein and raised antibodies against it. The antibodies interact specifically with villin and M_r 110,000 polypeptides present in isolated microvilli or brush borders. However, after absorption on an immobilized villin preparation, these antibodies still immunoprecipitate the M_r 110,000 protein but not villin. Thus, these two proteins appear to share some antigenic determinants but also contain other determinants specific for each protein. Immunolocalization studies have been performed using specific antibodies against the M_r 110,000 protein. Immunofluorescent studies on thin frozen sections of intestinal cells show that this protein is located in the brush border and at the basolateral faces of these polarized cells. Immunoferritin studies on rat brush borders demembranated with the detergent Triton X-100 show the association of the M_r 110,000 protein with core filaments of microvilli, as well as with some filaments localized in the terminal web network.Using sealed, right-side-out vesicles prepared from pig intestinal mucosa in the presence of Ca²⁺ and Mg²⁺, a polypeptide of M_r 140,000 was found to be a major component of the Triton X-100 insoluble pellet. This protein is a minor component of an equivalent pellet obtained from isolated microvilli prepared in the presence of EDTA. The significance of this M_r 140,000 polypeptide associated with the core residue of intestinal microvilli is discussed.     

Structure and composition of the cytoskeleton of nucleated erythrocytes I. The presence of microtubule-associated protein 2 in the marginal band

The marginal band (MB) of nucleated erythrocytes is composed of a bundle of microtubules that encircles the cell immediately beneath the plasma membrane. When cells are lysed in buffer containing Triton X-100 the MB microtubules remain intact, and the nucleus remains suspended at the cell center by a filamentous network called the trans-MB material that connects the nucleus to the peripheral MB. When these lysed cells are prepared for indirect immunofluorescence by use of an antibody to chick brain microtubule-associated protein 2 (MAP 2), intense staining of the MB results; no staining is evident in the areas occupied by the nucleus or the trans-MB material. Controls demonstrate that the staining is specific, because no staining occurs with fluorescent goat antirabbit serum alone or when nonimmune serum is used as the first antibody. Furthermore, the fluorescence of the MB is not affected by pretreatment of the immune serum with purified tubulin, but staining is prevented by pretreatment of the immune serum with purified MAP 2. To determine which protein component of the MB was responsible for the positive immunofluorescence results, 125I-protein A staining was used after the protein components of the isolated cytoskeleton had been resolved by SDS-polyacrylamide gels. Controls showed that the antiserum could react on SDS gels with MAP 2 from purified chick brain microtubules. The results with the cytoskeletal proteins demonstrated that the antiserum reacted only with a high molecular weight protein having a molecular weight similar, but not identical, to that of chick brain MAP 2. Thus, it is concluded that a protein with antigenic characteristics similar to those of chick brain MAP 2 is a component of the MB. The results are discussed in terms of the possible function of MAP 2 in the MB.     

Identification of a high molecular weight actin-binding protein in skeletal muscle.

A large polypeptide having a molecular weight of 240,000 as determined by electrophoresis in the presence of sodium dodecyl sulfate has been identified in whole cell homogenates from chick skeletal muscle myoblasts and the rat myoblast L6 cell line. A similar polypeptide was identified in both thigh and breast chicken skeletal muscle, but the latter contained less of this protein per g of tissue. Antibodies made to gizzard filamin (an actin-binding protein having a molecular weight of 240,000) cross-reacted with the partially purified Mr = 240,000 protein from chicken skeletal muscle. With use of the indirect immunofluorescence technique, the filamin antibody localized in the Z-line region of chicken skeletal muscle myofibrils. These results indicate that skeletal muscle contains a filamin-like protein that may form an integral part of the myofibril structure.     

High molecular weight DNA from nucleated erythrocytes for use in pulsed-field gel electrophoresis (PFGE)

Nucleated erythrocytes of lower vertebrates provide a source of genomic DNA that can be used in pulsed-field gel electrophoresis (PFGE) studies. Difficulties reported in the preparation of chicken erythrocyte DNA for analysis by PFGE suggest that the presence of hemoglobin iron may result in iron-mediated DNA degradation. We report here modifications to the procedures established for isolation of high molecular weight DNA from mammalian cells. By increasing the volume of buffers and extending the incubation periods to allow for the removal of hemoglobin iron, we have successfully prepared channel catfish erythrocyte DNA suitable for analysis of PFGE.     

A high molecular weight centrosomal protein of mammalian cells is antigenically related to myosin II.

Available data on the molecular composition of the centrosome, the typical microtubule-organizing center of animal cells, are still fragmentary. To address this important issue we have taken advantage of centrosome isolation from a human lymphoblastic cell line (KE37) to generate a monoclonal antibody (mAb) library. Here we present the characterization of one of these mAbs (CTR56). On the basis of both its immunofluorescence staining pattern and its reactivity with a major 200 kD antigen on immunoblots, CTR56 has been tentatively classified as an anticellular myosin heavy chain. In light of cytological and biochemical data obtained in parallel with two other well-characterized myosin antibodies, it appears that myosin cannot be considered as a genuine centrosomal protein. We have resolved the paradoxical results with CTR56 by showing that in addition to the cellular myosin heavy chain, this antibody also recognizes a high molecular weight protein specifically enriched in centrosomal fractions. The possible biological significance of this finding is discussed in structural and functional terms.     

High molecular weight calmodulin-binding protein is phosphorylated by calmodulin-dependent protein kinase VI from bovine cardiac muscle

A high molecular weight calmodulin-binding protein (HMW CaMBP) from bovine heart cytosolic fraction was purified to apparent homogeneity. A novel CaM-dependent protein kinase was originally discovered when the total CaM-binding protein fraction from cardiac muscle was loaded on a gel filtration column. The CaM-dependent protein kinase was shown by gel filtration chromatography to have an apparent molecular mass of 36,000 daltons. The CaM-dependent protein kinase has been highly purified by sequential chromatography on DEAE-Sepharose C1 6B (to remove calmodulin), CaM-Sepharose 4B, phosphocellulose, Sepharose 6B gel filtration and Mono S column chromatographies. The highly purified protein kinase stoichiometrically phosphorylated the HMW CaMBP in a Ca²⁺/CaM-dependent manner. The phosphorylation resulted in the maximal incorporation of 1 mol of phosphate/mol of the HMW CaMBP. The distinct substrate specificity of this protein kinase indicates that it is not related to the known protein kinases (I, II, III, IV and V) that have been already characterized, therefore we would like to designate this novel kinase as a CaM-dependent protein kinase V1.     

Naturally occurring anti-band 3 autoantibodies recognize a high molecular weight protein on the surface of Plasmodium falciparum infected erythrocytes

Naturally occurring anti-band 3 autoantibodies bind to erythrocytes infected with a knobby variant of the human malaria Plasmodium falciparum (FCR-3 strain). The autoantibodies recognized a greater than 240 kDa protein in SDS extracts made from surface iodinated infected erythrocytes. The antigen was associated only with erythrocytes infected with a knobby variant, and was removed by trypsin treatment of intact infected cells. By two-dimensional peptide map analysis the antigen was shown to be structurally related to the human erythrocyte anion transporter, band 3.     

The cytoskeletal proteins of erythrocytes

The erythrocyte membrane skeleton is composed of the number of proteins isolated and characterized. One of the major proteins of cytoskeleton is actin presented in erythrocytes in the form of short protofilaments. This review will focus on the manner of attachment of actin protofilaments to the red cell membrane, and on the relationships between skeleton membrane proteins. Membrane skeleton proteins in erythrocytes are not unique. Recently a lot of proteins similar to the red cell membrane skeleton proteins were found in a wide variety of non-erythroid cells. This fact gives the opportunity to suppose the existence of a unique protein system in erythroid and non-erythroid cells which provides the attachment of actin filaments to cell membranes and which might be the centre for the assembling of actin structures in the cortical cytoplasm.     

Observations of the marginal band system of nucleated erythrocytes

The marginal band (MB) of nucleated erythrocytes (thos of nonmammalian vertebrates) is a continuous peripheral bundle of microtubules normally obscured by hemoglobin. Treatment of these elliptical cells with modified microtubule polymerization media containing Triton X-100 yields a semilysed system in which MB, nucleus, and trans-MB material (TBM) are visible under phase contrast. The TBM apparently interconnects structural components, passing around opposite sides of the nucleus and suspending it in native position. In uranyl acetatestained whole whole mounts (goldfish) examined by transmission electron microscopy, the TBM appears as a network. MBs of semilysed cells are relatively planar initially, but twist subsequently into a range of "figure-8" shapes with one of the two possible mirror-image configurations predominant. Nuclei and MBs can be released using proteolytic enzymes, to which the TBM seems most rapidly vulnerable. MBs thus freed are birefringent, generally untwisted, and much more circular than they are in situ. As a working hypothesis, it is prosposed that the flattened, elliptical shape of nucleated erythrocytes is a result of TBM tension applied asymmetrically across an otherwise more circular MB, and that the firure-8 configuration occurs when there is extreme TBM shrinkage or contraction.     

Cytoplasmic network arrays demonstrated by immunolocalization using antibodies to a high molecular weight protein present in cytoskeletal preparations from cultured cells

Antisera were raised in rabbits against the two major components of intermediate filament preparations from glia-derived C6 cells, polypeptides of M_r around 300 000 and 58 000 (vimentin). These, and a third antiserum raised against microtubule proteins from hog brain, were shown to be specific for their respective immunogens. The assay employed involved the separation of components of crude cell extracts or filament preparations by SDS-polyacrylamide gel electrophoresis, and their subsequent transfer to and immobilization on nitrocellulose sheets. Cross-reacting counterparts of the immunogens were found in various cell lines, including C6, BALB/c 3T3, SV101, CHO, HeLa and PtK2 cells. In indirect immunofluorescence studies, antibodies to the high-M_r polypeptide component stained dense cytoplasmic network arrays of seemingly short, irregularly oriented fibres and lines of dots, in fibroblasts and in HeLa cells, but not in PtK2 cells. In well spread cells these networks were clearly distinguishable in morphology from the fibres decorated by antibodies to either microtubule protein or vimentin. The network arrays were resistant towards treatments with Triton X-100 and colcemid. By double immunofluorescence microscopy of single cells, using an additional antibody preparation to vimentin raised in guinea pigs, it was shown that after prolonged colcemid treatment of BALB/c 3T3 cells both; vimentin filaments and the structures stained by antibodies to the high-M_r component, accumulated in corresponding areas of the cytoplasm. The possibilities are discussed that this novel network-like structure is of the intermediate filament type and that it might function as a cross-linker of cytoplasmic—in particular cytoskeletal—elements. To signify its fluorescent localization and its possible linking role it is proposed to call the high-M_r component of intermediate filament preparations from cultured cells ‘plectin’.     

The anchoring filament protein kalinin is synthesized and secreted as a high molecular weight precursor.

Kalinin, a recently characterized novel protein component of anchoring filaments, has been shown to be involved in keratinocyte attachment to culture substrates and to dermis in vivo, and to exist in keratinocyte-conditioned culture medium in two heterotrimeric forms of 440 and 400 kDa (Rousselle, P., Lunstrum, G.P., Keene, D.R., and Burgeson, R.E. (1991) J. Cell Biol. 114, 567-576). This study demonstrates that kalinin is initially synthesized in a cell-associated form estimated to be 460 kDa. By second dimension reduced electrophoresis, V8 protease digestion, and immunoblot analysis, we demonstrate that the cell form contains nonidentical subunits of 200, 155, and 140 kDa. The 440-kDa medium form is derived from the cell form by extracellular processing of the 200-kDa subunit to 165 kDa, a step which also occurs in skin organ culture. The 400-kDa form is derived from the 440-kDa form by extracellular processing of the 155 kDa-subunit to 105 kDa. The cell form is secreted by keratinocytes, deposited onto culture substratum, and is the form which facilitates attachment and adhesion of growing and spreading keratinocytes. It is also the form initially synthesized in skin organ culture. Kalinin purified from tissue, which appears to facilitate epithelial-mesenchymal cohesion in vivo, is closely related to the 400-kDa medium form purified from culture.