Studies on human porin. VI. Production and characterization of eight monoclonal mouse antibodies against the human VDAC "Porin 31HL" and their application for histotopological studies in human skeletal muscle.

We report on the production and characterization of eight monoclonal mouse antibodies against the complete human VDAC "Porin 31HL". The antigen used was purified from a total membrane preparation of the transformed human B-lymphocyte cell line H2LCL. In Western blots all eight mAbs react with a single 31-kDa band in solubilized H2LCL membrane preparations thus demonstrating their specificity for the human VDAC "Porin 31HL". Concerning the epitope specificity we show that all eight mAbs equally react with the N-terminal part of human porin. Moreover, we demonstrate the expression of VDAC in the sarcolemma by indirect immunoenzyme labelling of cryosections of human skeletal muscle applying four of our mAbs. These data support our recent observations on the expression of porin channels in the plasmalemma of different normal and transformed human cell lines. VDAC in the plasmalemma is discussed as the molecular basis of the Blatz and Magleby channel.     

Lipopolysaccharide tightly bound to porin monomers and trimers from Escherichia coli K-12.

Lipopolysaccharide (LPS) bound to isolated porin was detected on polyacrylamide gels by using a carbohydrate-specific silver stain and on Western blots by using anti-lipid A monoclonal antibodies. Porin was isolated from Escherichia coli JF733 (Ra chemotype) and D21f2 (Re chemotype). Isolated porin was separated from loosely associated LPS by polyacrylamide gel electrophoresis (PAGE) in sodium dodecyl sulfate (SDS). Unheated porin traveled on gels as aggregates, presumably trimers, with an apparent molecular weight of 78,000 to 83,000. After heating to 100 degrees C for 2 min in SDS, the porin traveled as a monomer with a molecular weight of 36,000. The unheated, high-molecular-weight trimer band reacted in the gel with the carbohydrate-specific silver stain, while the heated monomer band showed no staining. In contrast, lipid A-specific monoclonal antibodies showed reactivity on Western blots to the 36,000-molecular-weight band but not to the trimer. Finally, both monomer and trimer bands were isolated from gels and rerun by SDS-PAGE. LPS was released from the trimer preparation when the sample was heated, but the monomer band that was formed by heating the trimer isolate still reacted with anti-lipid A antibodies. Quantitative Limulus amebocyte lysate analysis revealed an approximately equal molar ratio of LPS to protein in the electroeluted porin monomer. Thus, some but not all of the LPS could be released from trimer complexes by boiling in SDS. The isolated monomer did not release more LPS on boiling in SDS a second time but still had LPS tightly bound, as detected by lipid A-specific monoclonal antibodies.     

Positive reactions on Western blots do not necessarily indicate the epitopes on antigens are continuous

Epitope mapping (identification of an antigenic site recognized by an antibody) is an important component of vaccine development and immunological assays. It is widely accepted that in Western blots, antibodies react exclusively with continuous epitopes: discontinuous epitopes are assumed to be irreversibly destroyed by electrophoresis under the denaturing conditions used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Here, we demonstrate that the epitopes recognized by four different monoclonal antibodies were identified as discontinuous epitopes when characterized by radioimmunoprecipitation assays and enzyme-linked immunosorbent assays, yet each of these antibodies reacted with the corresponding antigen on Western blots. Reaction on Western blots may be due to epitope renaturation during or after the transfer of the protein to a membrane. Therefore, positive reactions on Western blots do not necessarily indicate that epitopes are continuous and this caveat should be kept in mind while characterizing them.     

Preparation and characterization of monoclonal antibodies to several frog rod outer segment proteins

Monoclonal antibodies to proteins important in phototransduction in the frog rod outer segment have been obtained. These include 6 different antibodies to rhodopsin, 50 to a guanine nucleotide binding protein (G-protein; 40,000 daltons), and 2 to cytoplasmic proteins. The antigens used were Percoll-purified rod outer segments, a rod outer segment soluble protein fraction, or a soluble plus peripheral membrane protein fraction. Antibodies were assayed by solid phase assay using a fluorogenic detection system. Proteins to which antibodies bound were assayed on Western blots, and the sensitivities of three different detection systems were compared. Most antibodies bound to only one rod outer segment protein band on Western blots. Immunofluorescence microscopy demonstrated binding of both anti-rhodopsin and anti-G-protein to isolated frog rod outer segments. Antibodies were purified from either culture supernatants or ascites fluid on protein A affinity columns. Two purified anti-G-protein antibodies have binding affinities to 125I-labeled G-protein of less than 10(-6) M-1. Of 11 antibodies to frog or bovine G-protein tested in solid phase and Western blot assays, all bind to the alpha rather than the beta or gamma subunits. Procedures developed here are being used in preparing other antibodies that affect reactions in the phototransduction pathway.     

Modulation of the activity of the transverse tubule Mg2+ATPase from frog skeletal muscle by a monoclonal antibody in vitro

We have established several hybridoma lines that produce monoclonal antibodies against transverse tubule (t-tubule) proteins from frog skeletal muscle. The specificity of these antibodies was characterized by ELISA and Western immunoblotting with purified t-tubule, sarcoplasmic reticulum and partially purified sarcolemmal membranes. One of the monoclonal antibodies (2/34.4) recognizes a band of 109 000 Da in immunoblots. When purified frog t-tubule vesicles were preincubated with this antibody we observed an increase in the rate of the Mg²⁺-ATPase enzyme (up to six fold) which was dependent on antibody concentration. Immunocytological experiments done on cryostat sections of frog muscle indicate that the antigen recognized by this antibody is localized mainly at the level of the t-tubules (I band) and to a lesser extent at the sarcolemma. These results indicate that monoclonal antibody 2/34.4 recognizes the t-tubule Mg²⁺-ATPase and modulates its activity. This antibody should be useful as a probe on studies designed to study the physiological function of the enzyme.Abbreviations t-tubules transverse-tubules - mAb monoclonal antibody - SR sarcoplasmic reticulum - SL sarcolemma     

Immunological characterization of phosphoprotein phosphatases

Phosphoprotein phosphatases regulate the biological activities of proteins through their involvement in cyclic phosphorylation/dephosphorylation cascades. A variety of multimeric phosphatases have been isolated and grouped into several classes, termed type 1 and types 2A, 2B, and 2C. To elucidate the relationship between the different phosphoprotein phosphatases, highly purified enzymes from soil amoebae, turkey gizzards, bovine heart and brain, and rabbit skeletal muscle and reticulocytes were tested for immunological antigenic relatedness. Two heterologous antibody preparations were employed for this purpose. One was made against an Acanthamoeba type 2A phosphatase and the other was made to bovine brain phosphatase type 2B (calcineurin, holoenzyme). Specific subunit cross-reactivity was examined by protein blot ("Western") analysis. The antibody to the type 2A phosphatase reacted with the catalytic subunits of every type 2 enzyme tested, including both the catalytic and Ca2+-binding subunits of the Ca2+/calmodulin-dependent type 2B phosphatase (calcineurin), bovine cardiac type 2A phosphatase, and turkey gizzard smooth muscle phosphatase-1 (type 2A1). It did not react with any type 1 phosphatase (catalytic subunit or ATP-Mg-dependent). The antigenic relatedness of calcineurin and the bovine cardiac type 2A phosphatase (Mr 38,000) was demonstrated further by protein blot analysis showing that the anti-calcineurin antibody cross-reacted with both enzymes. The mutual cross-reactivity poses an intriguing problem because these enzymes are so different in their molecular structures and modes of regulation. The degree of evolutionary conservation exhibited by the antigenic cross-reactivity of the type 2 enzymes from a broad range of species and tissues suggests a strong selective pressure on maintaining one or more features of these important regulatory enzymes.     

Monoclonal antibodies directed against skeletal muscle actin

The development of a monoclonal antibody directed against rabbit skeletal muscle monomeric actin is described. The production of the monoclonal antibody followed a standard hybridoma technique, the antibody being purified by affinity chromatography. It was found to be of the IgM class. Antibody specificity for rabbit skeletal actin was demonstrated by radioimmunoassay. The antibody failed to bind to actin in Western Blot experiments, presumably due to modification of the antigenic determinant on actin during the Western Blot procedure. The antibody was also shown to bind to two other isotypes of actin, i.e. actin from squid mantle muscle and bovine myocardium.     

Biochemical and immunohistochemical evidence for a non-muscle myosin at the neuromuscular junction in bovine skeletal muscle.

We identified 220-kD protein in bovine skeletal muscle homogenate by affinity chromatography on an agarose column and subsequent SDS-PAGE. Peptide mass fingerprinting (MALDI mass spectrometry) and internal sequence analysis revealed that this protein has homology with several members of the myosin superfamily, particularly with human cardiac beta-myosin heavy chain (beta-MHC). A rabbit polyclonal antibody against the 220-kD protein specifically stained a 220-kD band in Western blots of skeletal muscle homogenate. Immunohistochemical experiments on cryostat sections demonstrated that in skeletal muscle this protein is exclusively localized at the neuromuscular junctions, no immunoreactivity being present at the myofibril level. Because of its relative homology with cardiac beta-MHC, we also investigated the distribution of the 220-kD protein in bovine heart. In cardiac fibers, 220-kD protein-related immunoreactivity was restricted to the intercalated disks, whereas myofibrils were completely devoid of specific immunoreactivity. This distribution pattern was completely different from that of cardiac beta-MHC, which involved myofibrils. Because of the above biochemical and immunohistochemical features, the 220-kD protein we have identified is suggested to be a novel member of the non-muscle (non-sarcomeric) myosin family.     

STUDIES ON MUSCLE DIFFERENTIATION. V. ANTIGENICITIES OF MYOSIN AND OF ACTIN FROM FROG SKELETAL MUSCLES1

Both intact and denatured preparations of myosin and actin from frog skeletal muscles produced in rabbits antisera containing antibodies against authentic myosin and actin, respectively, though being contaminated with antibodies against other proteins. Antigenicity of our frog myosin as revealed in agar diffusion tests was indistinguishable from that of cardiac muscle myosin from the same species. Similarly, skeletal muscle myosins from other amphibians shared to a certain extent immunological characteristics with our frog myosin, but those from avian and mammalian materials did not. Similarity in antigenicity was also demonstrated among our skeletal muscle actin, cardiac muscle actin from the same species and skeletal muscle actin from the other anurans studied. However, skeletal muscle actin from an urodele could not clearly be correlated in its immunological properties with our frog actin, and those from avian and mammalian materials were antigenically different from our frog actin. Thus, the degree of antigenic similarity of these muscle proteins seemed to be correlated with the phylogenic relationship of the animals so far studied. The results also indicated that our antisera could only be applied to immuno-cytological and immuno-embryological studies of myosin and actin when the antisera absorbed with the corresponding antigen preparations were used as negative controls.     

Myotonic dystrophy protein kinase monoclonal antibody generation from a coiled-coil template

Myotonic dystrophy protein kinase (DMPK) was the initial representative of a ubiquitous protein kinase family that regulates cell size and shape. DMPK is highly expressed in heart and skeletal muscle and transgenic over-expression induces cardiac hypertrophy. The characterization of DMPK has been limited by the paucity of immunological reagents with high affinity and well-defined specificity. Amino acid sequence data was used to predict the surface exposure of the coil-coiled domain of DMPK. These exposed amino acids were substituted into an extremely stable coiled-coil template to produce a peptide antigen. Sera from mice immunized with the peptide conjugated to keyhole limpet hemocyanin were screened against recombinant DMPK using Western blots. Murine spleens expressing DMPK antibodies were used to produce hybridoma cell lines. Hybridoma supernatants were further screened against recombinant DMPK and four clonal hybridoma cell lines expressing DMPK antibodies were generated. These four monoclonal antibodies recognized recombinant DMPK in Western blots of COS-1 cell lysates expressing high levels of recombinant DMPK and immunoprecipitated recombinant DMPK from COS-1 cell lysates. The identity of the immunoprecipitated DMPK was confirmed by MALDI-TOF mass spectrometry and peptide mass fingerprinting. DMPK was the only protein detected in the immunoprecipitates, indicating the high specificity of the antibodies. Western blots immunostained with two of the monoclonal antibodies specifically recognized the two isoforms of endogenous DMPK, DMPK-1 and DMPK-2, that are expressed at low levels in the human heart. The recognition of low amounts of DMPK-1 and DMPK-2 indicates the high affinity of these antibodies. A human heart lysate was subjected to ammonium sulfate precipitation and column chromatography to produce a fraction that was enriched in DMPK. One of the monoclonal antibodies immunoprecipitated endogenous DMPK from this fraction. This antibody was used for immuno-localization studies of an adenoviral DMPK construct, expressed in adult mouse cardiac myocytes. This construct was localized to the intercalated disc, the site of endogenous DMPK, indicating that this antibody is applicable to immuno-localization studies. This study demonstrates the utility of the described procedure for generation of specific monoclonal antibodies with high affinity for epitopes in coiled-coiled domains of mammalian proteins expressed at low levels.     

Preparation and characterization of the major isotype of parvalbumin from skeletal muscle of the toad (Bufo bufo japonicus)

The major isotype of parvalbumin has been isolated from the skeletal muscle of the toad, Bufo bufo japonicus. Unlike the skeletal muscle of every frog so far examined (Rana esculenta, Rana temporaria, and Rana catesbeiana), which contains two major isotypes of parvalbumins, toad skeletal muscle has been shown to contain only one isotype, but the content of parvalbumin in toad skeletal muscle was similar to the sum of those of the two isotypes in skeletal muscles of frogs. This feature of toad skeletal muscle is advantageous to clarify the physiological role of parvalbumin. The relative molecular mass of toad parvalbumin was estimated to be 12,200 by SDS-polyacrylamide gel electrophoresis. The isoelectric point was determined to be 4.81 by polyacrylamide gel isoelectric focusing. The amino acid composition indicated that toad parvalbumin corresponds to bullfrog (R. catesbeiana) pI 4.97 parvalbumin, showing that toad parvalbumin is genetically an alpha-parvalbumin. It was also revealed by the amino acid composition that toad parvalbumin is distinctly different from any of the parvalbumins from frogs. The ultraviolet spectrum of toad parvalbumin is consistent with its amino acid composition. The ultraviolet difference spectrum of the Ca2+-loaded form vs. the metal-free form indicates that some Phe residues in the toad parvalbumin molecule are affected by a conformational change associated with Ca2+ binding. On electrophoresis in polyacrylamide gel in 14 mM Tris and 90 mM glycine, the metal-free and Mg2+-loaded forms of toad parvalbumin migrated twice as fast as the Ca2+-loaded form.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of pregnant sheep myometrium myosin light chain kinase

Myosin light chain kinase (MLCK) has been purified from the myometrium of pregnant sheep. The Mr of the enzyme was determined from SDS-polyacrylamide gels to be 160,000. It requires Ca2+ and calmodulin for activation, and phosphorylates the 20,000-Da light chains of myosin at a rapid rate. The specific activity for the myosin light chains from turkey gizzards and rabbit uterine muscle are 7.7 and 5.4 mumol/min/mg, respectively. The Km for the former substrate is 40 microM and the Vmax of the reaction is 19 mumol/min/mg. Polyclonal antibodies raised against the enzyme cross-reacted with pregnant sheep myometrium (psm), turkey gizzard (tg), and chicken gizzard MLCK. Affinity purification of the antibodies on tg-MLCK Sepharose resulted in the preparation of two fractions of antibodies with different reactivity toward these proteins. Fraction A antibodies which did not bind to the affinity column cross-reacted only with psm-MLCK while Fraction B antibodies which bound to the column cross-reacted with all three proteins. Western blots of extracts of turkey gizzards, human myometrium, and various tissues from sheep showed cross-reactivity of both fractions of antibodies with a 160,000-Da protein in the extracts of sheep smooth muscles. Only Fraction B antibodies cross-reacted with a protein (130,000 Da) in turkey gizzards and human myometrium extracts. Prolonged tryptic digestion of psm-MLCK produced large fragments Mr approximately 60,000 which appears to be similar to that formed from tg-MLCK, and some smaller peptides. Fraction A antibodies cross-reacted with the small peptides while Fraction B antibodies cross-reacted with the large fragments but not vice versa. Further analysis of the tryptic peptides suggests that the epitopes of Fraction A antibodies are localized in a peptide which appears to be in the NH2-terminal region of the molecule.     

Interspecific differences in molecular weights of skeletal myosin,actin, troponin C and tropomyosin in the frogs <Emphasis Type="Italic">Hyla japonica</Emphasis> and <Emphasis Type="Italic">Xenopus tropicalis</Emphasis>

Molecular weights of the skeletal muscle myosin, actin, troponin C and tropomyosin were compared in two frog species, Hyla japonica and Xenopus tropicalis, by SDS-PAGE and Western blot. Polyclonal antibody was produced using H. japonica skeletal muscle as the antigen. Polyclonal antibodies to nematode (Caenorhabditis elegans), mold slime (Physarum polycephalum), crab (Pagurus japonicus) and chicken skeletal muscle were also used. In H. japonica, the molecular weights of skeletal myosin, actin, troponin C and tropomyosin were 230, 42, 19 and 38 kDa, respectively, by using anti-C. elegans paramyosin, anti-P. polycephalum actin, anti-crab troponin C and anti-chicken gizzard tropomyosin antibodies. Molecular weights of the same proteins in X. tropicalis detected by the same antibodies were 230, 43, 20 and 40 kDa, respectively. In total, 29 protein bands were detected in H. japonica skeletal muscle and 24 bands in X. tropicalis by SDS-PAGE. The results revealed interspecific differences in molecular weights of selected skeletal muscle proteins and in the total skeletal muscle protein profiles between the two frog species.     

Human red blood cell insulin-degrading enzyme and rat skeletal muscle insulin protease share antigenic sites and generate identical products from insulin.

The mechanisms of cellular insulin degradation remain uncertain. Considerable evidence now exists that the primary cellular insulin-degrading activity is a metallothiol proteinase. Two similar degrading activities have been purified and characterized. Insulin protease has been purified from rat skeletal muscle and insulin-degrading enzyme from human red blood cells. Whereas the two degrading activities share a number of similar properties, significant differences have also been reported; and it is not at all established that they are the same enzyme. To examine this, we have compared antigenic and catalytic properties of the two enzymatic activities. Monoclonal antibodies against the red blood cell enzyme adsorb the skeletal muscle enzyme; and on Western blots, the antibodies react with an identical 110-kDa protein. Immunoaffinity-purified enzymes from both red blood cells and skeletal muscle degrade [125I]iodo(B26)insulin to the same products as seen with purified insulin protease and with intact liver and kidney. Chelator-treated muscle and red blood cell enzymes can be reactivated with either Mn2+ or Ca2+. Thus, insulin-degrading enzyme and insulin protease have similar properties. These results support the hypothesis that these activities reside in the same enzyme.     

Subcellular sorting of isoactins: selective association of gamma actin with skeletal muscle mitochondria

We describe two subpopulations of actin antibodies isolated by affinity chromatography from a polyclonal antibody to chicken gizzard actin. One subpopulation recognizes gamma actins from smooth muscle and nonmuscle cells, but does not recognize alpha actin from skeletal muscle. The other subpopulation recognizes determinants that are common to alpha actin from skeletal muscle and the two gamma actin isotypes. Neither antibody recognizes cytoplasmic beta actin. Both antibodies recognize only actins or molecules with determinants that are also present in actins. By immunofluorescence we found that the anti-gamma actin colocalizes with mitochondria in fibers of mouse diaphragm, and that it does not bind detectably to the 1 bands of sarcomeres. The antibody that recognizes both alpha and gamma actins stains 1 bands intensely, as expected. We interpret these observations as preliminary evidence for selective association of gamma actin with skeletal muscle mitochondria and, more broadly, as evidence for subcellular sorting of isoactins.     

Binding of myosin cross-bridges to thin filaments of rabbit skeletal muscle.

Cross-linking of myosin subfragment 1 (S1) with a molar excess of actin in vitro reveals the presence of an actin-S1-actin complex. It is absolutely essential that actin be present in molar excess over S1 so that the decoration of F-actin with S1 be incomplete. However, the excess of actin may not be available in the overlap zone of sarcomeres of skeletal muscle. We therefore found it necessary to test for the presence of the actin-S1-actin complex in vivo. Myofibrils from rabbit skeletal muscle were reacted with zero-length cross-linker, the products were resolved by polyacrylamide gel electrophoresis and analyzed by Western blots using antibodies against actin and against heavy and light chains of myosin. The cross-linking produced the evidence of formation of actin-S1-actin complex.     

Generation of subunit-specific antibody probes for Torpedo acetylcholinesterase: cross-species reactivity and use in cell-free translations

The assembly of the collagen tailed A12 form of acetylcholinesterase (AChE) is regulated by muscle contraction. To begin to study this regulation, we derived antibody probes for the three subunits (100 kd, catalytic, and collagen tail) of AChE purified from Torpedo californica electric tissue. These included a polyclonal antiserum that recognizes all 3 subunits and 19 monoclonal antibodies; 16 of the monoclonals recognized the catalytic subunit, 2 recognized the tail subunit, and 1 recognized the 100 kd subunit on Western blots. We used immunohistochemical procedures to show that several of the anticatalytic and one of the antitail monoclonals cross-reacted with frog muscle AChE and Western blotting to show that several of the anticatalytic monoclonals cross-react with rat brain AChE. These antibodies were then used to immunoprecipitate AChE precursors from a cell-free translation system. There were generally three primary translation products, corresponding to the three enzyme subunits. Therefore, each subunit is probably derived from a separate mRNA. Occasionally there were two translation products corresponding to the catalytic subunit alone. The catalytic subunit was glycosylated following addition of canine microsomal membranes to the translation mix. The mRNA coding for this subunit appeared to be present in the poly(A)- RNA pool.     

UCP2 and UCP3 rise in starved rat skeletal muscle but mitochondrial proton conductance is unchanged

The relationship between UCP2 and UCP3 expression and mitochondrial proton conductance of rat skeletal muscle was examined. Rats were starved for 24 h and the levels of UCP2 and UCP3 mRNA and UCP3 protein were determined by Northern and Western blots. Proton conductance was measured by titrating mitochondrial respiration rate and membrane potential with malonate. Starvation increased UCP2 and UCP3 mRNA levels more than 5-fold and 4-fold, respectively, and UCP3 protein levels by 2-fold. However, proton conductance remained unchanged. These results suggest either that Northern and Western blots do not reflect the levels of active protein or that these UCPs do not catalyse the basal proton conductance in skeletal muscle mitochondria.     

Phosphoprotein phosphatase inhibitor-2. Identification as a species of molecular weight 31,000 in rabbit muscle, liver, and other tissues

Specific antibodies, raised to purified rabbit skeletal muscle inhibitor-2, were used to analyze for the presence of inhibitor-2 in extracts of rabbit skeletal, cardiac, and diaphragm muscles, liver, kidney, brain, and lung. Direct analyses of the extracts by "Western blotting" revealed several immunoreactive species, apparent molecular weights in the range 26,000-136,000, as well as species with the electrophoretic mobility of inhibitor-2, apparent molecular weight 31,000. When supernatants from boiled extracts were similarly analyzed, most of the immunoreactive material was lost and the species corresponding to inhibitor-2 became prominent. Liver and muscle were studied in more detail; immunoprecipitates from either boiled or unboiled extracts were analyzed by Western blotting. The dominant polypeptide now was the species of apparent molecular weight 31,000, corresponding to inhibitor-2. Higher molecular weight species (115,000 in muscle and 136,000 in liver) were also detectable. The amount of inhibitor-2 detected in immunoprecipitates was not greatly different whether unboiled or boiled tissue extracts were used. In addition, extraction of the precipitates by boiling released material that inhibited purified type 1 protein phosphatase. The results suggest that inhibitor-2 is widely distributed in rabbit tissues and is found predominantly as a form of apparent molecular weight 31,000. In particular, the study provides direct demonstration of a species in rabbit liver with similar properties to rabbit muscle inhibitor-2.