Muscle Membrane Protein Research Articles

Significance of antibodies reactive with a 64 kDa eye muscle membrane antigen in patients with thyroid autoimmunity.

SDS-polyacrylamide gel electrophoresis and Western blotting for antibodies reactive with a 64 kDa protein in pig eye muscle membrane was carried out in patients with lid lag and retraction, but no other signs of ophthalmopathy, associated with thyroid disease or nonimmunologic goiter and in patients with Graves' hyperthyroidism without ophthalmopathy who were studied prospectively to determine the relationship of eye muscle antibodies to clinical features of the ophthalmopathy as they appeared in this group of predisposed patients. Seventy-one percent of euthyroid patients with lid lag and retraction but no established ophthalmopathy had detectable serum antibodies to a 64 kDa eye muscle membrane protein. Much smaller proportions had antibodies to proteins of other MW. In normal subjects with previously detectable antibodies to a 64 kDa protein, serum titers, determined by carrying out immunoblotting at serum dilutions of 1:25-1:6400, were low (< or = 1:100) in all cases tested. On the other hand, titers were higher (1:200-1:6400) in 16 of 22 patients with established ophthalmopathy and in 5 of 7 patients with lid lag and retraction tested. Titers tended to be lower in patients with ophthalmopathy of > or = 3 years duration than in those of < or = 1 year duration. Antibody titers were low (1:25) in 6 of 7 patients with Graves' hyperthyroidism without evident eye disease tested. Antibodies to a 64 kDa eye muscle membrane protein were predictive of the development of ophthalmopathy in patients with Graves' hyperthyroidism studied prospectively for periods of 8-42 months.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of excitation-contraction-coupling components in chronically stimulated canine skeletal muscle.

The chronic stimulation of predominantly fast-twitch mammalian skeletal muscle causes a transformation to physiological characteristics of slow-twitch skeletal muscle. Here, we report the effects of chronic stimulation on the protein components of the sarcoplasmic reticulum and transverse tubular membranes which are directly involved in excitation-contraction coupling. Comparison of protein composition of microsomal fractions from control and chronically stimulated muscle was performed by immunoblot analysis and also by staining with Coomassie blue or the cationic carbocyanine dye Stains-all. Consistent with previous experiments, a greatly reduced density was observed for the fast-twitch isozyme of Ca(2+)-ATPase, while the expression of the slow-twitch Ca(2+)-ATPase was found to be greatly enhanced. Components of the sarcolemma (Na+/K(+)-ATPase, dystrophin-glycoprotein complex) and the free sarcoplasmic reticulum (Ca(2+)-binding protein sarcalumenin and a 53-kDa glycoprotein) were not affected by chronic stimulation. The relative abundance of calsequestrin was slightly reduced in transformed skeletal muscle. However, the expression of the ryanodine receptor/Ca(Ca2+)-release channel from junctional sarcoplasmic reticulum and the transverse tubular dihydropyridine-sensitive Ca2+ channel, as well as two junctional sarcoplasmic reticulum proteins of 90 kDa and 94 kDa, was greatly suppressed in transformed muscle. Thus, the expression of the major protein components of the triad junction involved in excitation-contraction coupling is suppressed, while the expression of other muscle membrane proteins is not affected in chronically stimulated muscle.

Stimulation of Calmodulin Binding to Skeletal Muscle Membrane Proteins by 1,25-Dihydroxy-Vitamin D 3

Previous work has shown that 1,25-dihydroxy-vitamin D3 rapidly increases calmodulin levels of skeletal muscle membranes without altering the muscle cell calmodulin content. Therefore, the effects of the sterol on the binding of calmodulin to specific muscle membrane proteins were investigated. Soleus muscles from vitamin D-deficient chicks were treated in vitro for short intervals (5-15 min) with physiological concentrations of 1,25-dihydroxy-vitamin D3. Proteins of mitochondria and microsomes isolated by differential centrifugation were separated on sodium dodecyl sulfate polyacrylamide gels. Calmodulin-binding proteins were identified by a [125I]calmodulin gel overlay procedure followed by autoradiography. 1,25-Dihydroxy-vitamin D3 increased the binding of labelled calmodulin to a major, calcium-independent, calmodulin-binding protein of 28 Kda localized in microsomes, and to minor calmodulin-binding proteins of 78 and 130 Kda proteins localized in mitochondria. The binding of [125I]calmodulin to these proteins was abolished by flufenazine or excess non-radioactive calmodulin. 1,25-Dihydroxy-vitamin D3 rapidly increased muscle tissue Ca uptake and cyclic AMP levels and stimulated the phosphorylation of several membrane proteins including those whose calmodulin-binding capacity potentiates. Analogously to the sterol, forskolin increased membrane calmodulin content, calmodulin binding to the 28 Kda microsomal protein and 45Ca uptake by soleus muscle preparations. Forskolin also induced a similar profile of changes in muscle membrane protein phosphorylation as the hormone. These results suggest that 1,25-dihydroxy-vitamin D3 affects calmodulin distribution in muscle cells through cyclic AMP-dependent phosphorylation of membrane calmodulin-binding proteins. These changes may play a role in the stimulation of muscle Ca uptake by the sterol.

The cyclic nucleotide-dependent phosphorylation of aortic smooth muscle membrane proteins

Membrane proteins of M r 240,000, 130,000, and 85,000 (GS-proteins) were rapidly and selectively phosphorylated in particulate fractions of rabbit aortic smooth muscle in the presence of [Mg- 32P]ATP and low concentrations of cGMP ( K a = 0.01 μM) or cAMP ( K a = 0.2 μM). The effects of both cyclic nucleotides in this preparation were mediated entirely by an endogenous, membrane-bound form of cGMP-dependent protein kinase (G-kinase). The GS-proteins were also phosphorylated by the soluble form of G-kinase purified from bovine lung; this effect was most evident following removal of endogenous G-kinase from the membranes using Na 2CO 3 and high salt washes. The membranebound and cytosolic forms of G-kinase phosphorylated the M r 130,000 GS-protein with the same specificity as determined by two-dimensional peptide mapping. Despite this functional homology between the two forms of G-kinase, only the particulate enzyme appears to play a role in phosphorylating the GS-proteins. Although little endogenous cAMP-dependent protein kinase (A-kinase) activity was detected in washed aortic smooth muscle membranes, the GS-proteins could be phosphorylated when purified A-kinase catalytic subunit was added to this preparation. Peptide mapping of the M r 130,000 GS-protein indicated that A-kinase phosphorylated a subset of the same peptides labeled by the two forms of G-kinase. The endogenous A-kinase of rabbit aortic smooth muscle homogenates was also found to phosphorylate the GS-proteins. Since the intracellular concentrations of cGMP or cAMP can be selectively elevated by different stimuli, these results suggest several possible mechanisms by which the phosphorylation state of the GS-proteins may be regulated by cyclic nucleotides: (i) activation of the membrane-bound G-kinase by cGMP or cAMP; and (ii) activation of cytosolic A-kinase by cAMP.

Na channels in skeletal muscle concentrated near the neuromuscular junction.

Neuronal function depends crucially on the spatial segregation of specific membrane proteins, particularly the segregation associated with sites of synaptic contact. Understanding the factors governing this localization of proteins is a major goal of cellular neurobiology. A conspicuous example of synaptic specialization is the almost exclusive localization of vertebrate skeletal muscle acetylcholine (ACh) receptors to the subsynaptic membrane of the neuromuscular junction (for example, refs 1,2). The localization of other membrane proteins in skeletal muscle has been much less studied, but a knowledge of their distribution is crucial for understanding the factors governing regional specialization. We have explored the distribution in muscle of the voltage-gated Na channel responsible for the action potential using the loose patch-clamp technique, and have measured Na currents in 5-10 micron-diameter membrane patches as a function of distance from the end plate region of snake and rat muscle fibres. Here we report that the Na current density immediately adjacent to the endplate is 5-10-fold higher than at regions away from the endplate. The increased Na current density falls off rapidly with distance, reaching the background level 100-200 micron from the endplate. Although one might expect ACh receptors to be concentrated near the region of ACh release, such a concentration for Na channels, which propagate the impulse throughout the length of the cell, is surprising and suggests that factors similar to those responsible for concentrating ACh receptors at the endplate also operate to concentrate Na channels.

Concerted phosphorylation of endogenous tracheal smooth muscle membrane proteins by Ca 2+ · calmodulin-, cyclic GMP- and cyclic AMP-dependent protein kinases

FEBS LettersVolume 139, Issue 2 p. 309-312 Full-length articleFree Access Concerted phosphorylation of endogenous tracheal smooth muscle membrane proteins by Ca2+ · calmodulin-, cyclic GMP- and cyclic AMP-dependent protein kinases G.Kurt Hogaboom, G.Kurt Hogaboom Department of Pharmacology and Toxicology, West Virginia University Medical Center, USASearch for more papers by this authorCarol A. Emler, Carol A. Emler Department of Biochemistry, West Virginia University Medical Center, USASearch for more papers by this authorFred R. Butcher, Fred R. Butcher Department of Biochemistry, West Virginia University Medical Center, USASearch for more papers by this authorJeffrey S. Fedan, Corresponding Author Jeffrey S. Fedan Department of Pharmacology and Toxicology, West Virginia University Medical Center, USA Physiology Section, Appalachian Laboratory for Occupational Safety and Health, NIOSH, CDC, DHHS, Morgantown, WV 26505, USAAddress correspondence to J. S. F. at: Appalachian Laboratory for Occupational Safety and Health, WV 26505, USASearch for more papers by this author G.Kurt Hogaboom, G.Kurt Hogaboom Department of Pharmacology and Toxicology, West Virginia University Medical Center, USASearch for more papers by this authorCarol A. Emler, Carol A. Emler Department of Biochemistry, West Virginia University Medical Center, USASearch for more papers by this authorFred R. Butcher, Fred R. Butcher Department of Biochemistry, West Virginia University Medical Center, USASearch for more papers by this authorJeffrey S. Fedan, Corresponding Author Jeffrey S. Fedan Department of Pharmacology and Toxicology, West Virginia University Medical Center, USA Physiology Section, Appalachian Laboratory for Occupational Safety and Health, NIOSH, CDC, DHHS, Morgantown, WV 26505, USAAddress correspondence to J. S. F. at: Appalachian Laboratory for Occupational Safety and Health, WV 26505, USASearch for more papers by this author First published: March 22, 1982 https://doi.org/10.1016/0014-5793(82)80877-6Citations: 22AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL References 1 R.H. Kretsinger, Adv. Cyclic Nucl. Res., 11, (1979), 1– 26. 2 F. Wuytack, R. Casteels, Biochim. Biophys. Acta, 595, (1980), 257– 263. 3 G.K. Hogaboom, J.S. Fedan, Biochem. Biophys. Res. Commun., 99, (1981), 737– 744. 4 F. Wuytack, G. DeSchutter, R. Casteels, FEBS Lett., 129, (1981), 297– 300. 5 R.C. Bhalla, R.C. Webb, D. Singh, T. Brock, Am. J. Physiol., 234, (1978), H508– H514. 6 J.G. Sarmiento, R.A. Janis, T.M. Lincoln, Fed. Proc. FASEB, 40, (1981), 1831a– 7 M. Tada, F. Ohmori, M. Yamada, H. Abe, J. Biol. Chem., 254, (1979), 319– 326. 8 U.K. Laemmli, Nature, 227, (1970), 680– 685. 9 M.M. Bradford, Anal. Biochem., 72, (1976), 248– 254. 10 R.M. Levin, B. Weiss, J. Pharmacol. Exp. Ther., 208, (1979), 454– 459. 11 J.E. Casnellie, H.E. Ives, J.D. Jamieson, P. Greengard, J. Biol. Chem., 255, (1980), 3770– 3776. 12 R.J.A. Grand, S.V. Perry, R.A. Weeks, Biochem. J., 177, (1979), 521– 529. 13 J.C. Khoo, G.N. Gill, Biochim. Biophys. Acta, 584, (1979), 21– 32. 14 H.G. Nimmo, P. Cohen, Adv. Cyclic Nucl. Res., 8, (1977), 145– 266. 15 T.A. Langan, Adv. Cyclic Nucl. Res., 3, (1973), 99– 153. 16 W.B. Huttner, L.J. DeGennaro, P. Greengard, J. Biol. Chem., 256, (1981), 1482– 1488. 17 C.J. LePeuch, J. Haiech, J.G. Demaille, Biochemistry, 18, (1979), 5150– 5157. 18 D.B. Rylatt, N. Embi, P. Cohen, FEBS Lett., 98, (1979), 76– 80. Citing Literature Volume139, Issue2March 22, 1982Pages 309-312 ReferencesRelatedInformation

Allogeneic cytolysis of reconstituted membrane vesicles.

The successful use of lipid bilayer model membranes as targets for cytotoxic lymphocytes is described. Lipid vesicles were made from a mixture of dipalmitoyl lecithin, dimyristoyl lecithin, and cholesterol. Membrane proteins of LSTRA or EL4 tumor cells (as source of H-2 antigens), human eye muscle membrane proteins (as supporting proteins), and 51Cr marker were inserted into the lipid vesicles. Incubation of the reconstituted vesicles with lymphocytes sensitized in mixed lymphocyte cultures against allogeneic cells resulted in the specific release of intravesicular 51Cr. Vesicle damage was mediated by thymus-derived lymphocytes. H-2 antigens could be incorporated into vesicles without eye muscle proteins. However, immune damage of the vesicles could not be demonstrated when vesicles inserted with H-2 antigens in the absence of eye muscle proteins were used as targets.

Cell-mediated lysis of lipid vesicles containing eye muscle protein: implications regarding pathogenesis of Graves ophthalmopathy.

We prepared artificial vesicles that are lysed upon cell-mediated immunological attack by human lymphocytes. These vesicles are made from a mixture of dimyristoyl lecithin, dipalmitoyl lecithin, and cholesterol, have eye muscle membrane protein (EMP) inserted into the bilayer wall, and contain intravesicular (99m)Tc marker. Injury to the vesicular membrane was assessed by measurement of (99m)Tc release. Thyroglobulin (Tg) and Tg-anti-Tg complex (TgA) bind to EMP-vesicles to an extent equal to or greater than to native eye muscle membranes in vitro; this binding requires the presence of normal human IgG. The role of Tg, TgA, IgG, and peripheral blood lymphocytes in altering membrane permeability was analyzed. Incubation of vesicles for up to 3 hr alone, with added IgG alone, or with further addition of Tg or TgA did not result in (99m)Tc release. Addition of lymphocytes from normal donors to the above four preparations showed release in the presence of TgA. Lymphocytes from each of eight patients with Graves ophthalmopathy caused release not only in the presence of TgA, but also in the presence of Tg. Separation of a patient's lymphocytes into high- and low-affinity rosette-formers (T and K cells, respectively) showed that cell-mediated vesicle lysis in the presence of TgA was greater with K cells than with T cells, while vesicle lysis in the presence of Tg was greater with T cells than with K cells. Vesicles made with inserted Tg but lacking EMP were not lysed by such T cells. Lymphocytes failed to induce permeability changes in vesicles containing other inserted proteins obtained from human nonextraocular muscle, liver, spleen, or adrenal, even if Tg or TgA were present. The results support the concept that muscle cell damage in Graves ophthalmopathy is immunological, cell-mediated, and of two types: (i) K lymphocytes reacting to immune complex, TgA, on the eye muscle cell surface (i.e., antibody-dependent cytotoxicity) and (ii) sensitized T lymphocytes reacting to Tg on the eye muscle cell surface. An antigenic role for EMP is possible, but has not been unequivocally proven.

Macromolecular characterization of muscle membranes. Endogenous membrane kinase and phosphorylated protein substrate from normal and denervated muscle.

Light density membranes derived from the "microsomal" fraction of rat skeletal muscle contained an endogenous protein kinase which catalyzed the phosphorylation of an endogenous membrane substrate. No other membrane fraction contained any significant protein kinase activity. The optimal specific activity of the enzyme in these membranes was 350 pmol/mg/min. The endogenous muscle membrane protein kinase required magnesium, was stimulated by micromolar concentrations of calcium, had a pH optimum between 7.0 and 7.5, and demonstrated a K-m for ATP of 2.6 times 10 minus 5 M. The enzyme was markedly heat labile and demonstrated a linear Arrhenius plot with an apparent energy of activation of 12,100 cal/mol. There was no stimulation by cyclic nucleotides; and neither monovalent cations nor various neurotransmitters exerted any effect. It is presently unclear where the membranes exhibiting protein phosphorylation are localized within the muscle fiber. Enzyme markers suggest that these membranes are not derived from sarcolemma or sarcoplasmic reticulum but may originate in transverse tubules. The membrane phosphorylation was largely confined to a polypeptide with an apparent molecular weight of 28,000. Phosphorylation could also be detected in a lower molecular weight substrate as well as two polypeptides with apparent molecular weights of 95,000 and 56,000. The M-r-28,000 endogenous protein kinase substrate was isolated by preparative gel electrophoresis in sodium dodecyl sulfate. High voltage electrophoresis of a partial acid hydrolysate of the phosphorylated M-r-28,000 substrate identified the phosphate bond to be that of phosphoserine. The amino acid composition of the substrate was neither strongly acidic nor basic. It had a high content of glycine, glutamic acid, serine, and lysine. Hydrophobic residues constituted only 45% of the total composition. Following muscle denervation for 10 days, there was a significant decrease in the amount of the M-r-28,000 polypeptide as well as the extent of phosphorylation.

Muscle membrane protein kinase in myotonic muscular dystrophy.

MYOTONIC muscular dystrophy is an inherited disorder of man with manifestations in many organ systems including skeletal muscle myotonia and dystrophy, testicular atrophy, cataract formation, hypercatabolism of IgG, cranial bone malformations, and glucose intolerance with enhanced insulin levels (see ref. 1 for clinical references). Our previous experiments demonstrated a significant diminution in the phosphorylation of endogenous membrane proteins of frozen erythrocyte ghosts from myotonic patients1. In subsequent studies the technique of electron magnetic resonance was employed to compare the erythrocyte membrane milieu of normal and myotonic paients2. Following incorporation of 5-nitroxide methyl stearate into erythrocyte membranes, the spin label was located in a less polar and somewhat more fluid region in myotonic membranes than in normal membranes. Although such experiments do not specify the molecular membrane abnormality or its possible relationship to protein phosphorylation, they do support the suggestion that myotonic muscular dystrophy is a disease resulting from a basic membrane abnormality.