High Molecular Weight Polypeptides Research Articles

The outer arm dynein α‐heavy chains of sea urchin sperm flagella and embryonic cilia are different

AbstractSea urchin sperm outer arm dynein is a multi‐subunit protein composed of heavy chains, intermediate chains, and light chains. We prepared monoclonal and affinity‐purified polyclonal antibodies to heavy and intermediate chain subunits and examined whether the embryonic ciliary axonemes ofthe same species contain the polypeptides sharing epitopes with them. Ciliary axonemes contained a high molecular weight polypeptide with the exact same mobility as flagellar β‐heavy chain. This polypeptide also shared epitopes with it. In contrast, no polypeptide had the exact same molecular weight as flagellar α‐heavy chain and shared epitopes with it. Western blots showed that ciliary axonemes also contain three polypeptides sharing epitopes with the respective flagellar intermediate chain. The present results revealed that the α‐heavy chains of flagellar and ciliary outer arm dyneins are different.

Antioxidative Effect of Bovine Serum Albumin Hydrolyzates and Their Synergistic Effect with Antioxidants

Bovine serum albumin hydrolyzates (BSA-H) were prepared by the hydrolysis of BSA with pepsin, trypsin, chymotrypsin, or the combinations of these enzymes. In the presence of BSA-H, linoleic acid was oxidized in an ethanol-phosphate buffer (pH 7.0) system at 60°C in the dark, and the extent of lipid oxidation was measured by the ferric thiocyanate method.The antioxidative activity of BSA-H depended on the degree of hydrolysis but not on the particular enzyme or enzyme combination used. The antioxidative activity of BSA-H, however, was not as strong as ordinary antioxidants such as butylhydroxyanisol, butylhydroxytoluene, α-tocopherol or sodium ascorbate.BSA-H used with each antioxidant considerably enhanced in each case antioxidative activity, thus indicating BSA-H to be a useful synergists.BSA-H prepared with pepsin was separated into several peaks by Sephadex G-25 chromatography, and a particular group, comprised of relatively high molecular weight polypeptides, showed strong synergistic effect with each antioxidant.

Human DNA polymerase α catalytic polypeptide binds ConA and RCA and contains a specific labile site in the N-terminus

The catalytic polypeptide of DNA polymerase alpha is often observed in vitro as a family of phosphopolypeptides predominantly of 180 and 165 kDa derived from a single primary structure. The estimated Mr of this polypeptide deduced from the full-length cDNA is 165 kDa. Immunoblot analysis with polyclonal antibodies against peptides of the N- and C-termini of the deduced primary sequence indicates that the observed family of polypeptides from 180 kDa to lower molecular weight results from proteolytic cleavage from the N-terminus. Antibodies against the N-terminal peptide detect only the 180 kDa species suggesting that this higher molecular weight polypeptide may be the result of posttranslational modification of the 165 kDa primary translation product. The catalytic polypeptide is not only phosphorylated but is also found to react with lectins ConA and RCA. N-terminal sequencing of the isolated catalytic polypeptide from human cells and of the recombinant fusion proteins indicates that the often observed 165 kDa polypeptide is the in vitro proteolytic cleavage product of the modified 180 kDa protein at the specific site between lys123 and lys124 within the sequence -RNVKKLAVTKPNN-.

Molecular analysis of the tight junction

The tight junction (zonula occludens) constitutes a selectively permeable barrier in the paracellular pathway of most epithelia. It is also thought to play a role in the maintenance of the cell surface compositional asymmetry characteristic of epithelial cells. The identification of ZO-1 and cingulin, the first two proteins found to be exclusively associated with the tight junction, permits novel investigations of this important epithelial cell structure at the biochemical level.ZO-1 is a high molecular weight polypeptide (>200 kD) found at the tight junctions of a variety of epithelia as well as endothelia, and ultrastructural localization studies on isolated liver plasma membranes indicate that this molecule is clustered at the points of membrane contact on the cytoplasmic surface of the junction. Physical analysis demonstrates ZO-1 to be an elongated, monomeric, phosphorylated protein, peripherally associated with the junctional membrane. Cingulin was originally isolated from chicken intestine, and, like ZO-1, is a peripheral membrane component of the junction which exhibits an elongated shape. Antibodies directed against this molecule show two primary bands at 140 kD and 108 kD on immunoblots of several epithelial tissues, although the relationship between these two elements remains undefined. Little information currently exists regarding the relationship of ZO-1 and cingulin to each other or to tight junction structure or function. We report here a comparison of the immunocytochemical properties of these junctional components as well as an examination of the phosphorylation state of ZO-1.

Epithelial cell heterogeneity in mammalian thymus: Monoclonal antibody to high molecular weight keratins exclusively binds to Hassall's corpuscles

Hassall's corpuscles represent a subset of medullary thymic epithelial cells whose origin and function within the thymus still remain largely unknown. The present study shows that Hassall's corpuscles can be defined by their intracellular content in specific keratin subunits. Two monoclonal anti-keratin antibodies were used: KL1, directed to high molecular weight keratins, and KL4, specific for high and medium molecular weight polypeptides. In vivo, KL1 exclusively binds to Hassall's corpuscles of five mammalian species including mouse, rat, guinea-pig, rabbit and pig. Thus KL1 appears as an exclusive marker of Hassall's corpuscles in a large number of mammals. In vitro, thymic epithelial cells gave rise in certain species to Hassall's corpuscles. In contrast to its in vivo reactivity, KL1 never labelled Hassall's corpuscles developed in vitro. These data strongly support the following conclusions: (1) Hassall's corpuscles derive from medullary epithelial cells; (2) they represent advanced stages of thymic epithelial maturation; (3) thymic epithelial cell differentiation is impaired in vitro. Furthermore, this study provides additional evidence that thymic epithelium heterogeneity reflects different stages in epithelial maturation.

Transdermal iontophoretic drug delivery: mechanistic analysis and application to polypeptide delivery.

Three factors are of primary importance in determining the iontophoretic flux of a charged solute: the electrochemical potential gradient across the skin, an increase in skin permeability to passive transport due to iontophoresis (loosely defined as skin damage), and a current-induced water flux. The latter two factors can also affect the transport of uncharged solutes during iontophoresis. A method of correcting for the skin damage effect is introduced. The contributions of the water transport effect relative to that of the applied voltage drop for charged solutes is estimated. It is shown that the water transport contribution is generally lower than the contribution due to the applied voltage drop. The observed iontophonetic flux of the enhancement factors due to the applied voltage drop alone are compared with the theoretical predictions based on the constant field assumption. It is shown that the theoretical predictions are higher than the experimental observations. This work also examines, for the first time, a synergism of iontophoresis and pretreatment with a chemical penetration enhancer as a means for delivering high molecular weight polypeptides. It is shown that a 2-h pretreatment with absolute ethanol followed by iontophoresis dramatically increases the permeability coefficient of insulin through human skin.

AMINO ACID ANALYSIS OF BEER POLYPEPTIDES

The principles of amino acid analysis of proteins and polypeptides are reviewed. Analysis of the amino acid composition of dialysed beer material prepared from a wide variety of commercial and pilot brewery beers showed that the principal amino acids comprised glutamic acid/glutamine, proline, glycine and aspartic acid/asparagine. The results from the analysis of a series of pilot brewery beers brewed under standardised conditions showed that the composition of the grist may influence the amino acid composition of beer polypeptide fractions. Dialysed beer material prepared from beer brewed from grists containing torrified wheat, wheat flour and malted wheat contained greater proportions of glutamic acid/glutamine compared to material prepared from all malt beers. Further fractionation and analysis of dialysed beer material prepared from pilot brewery beers suggested that fractions MW>60000 contained polypeptide material derived from yeast mannan-protein. In addition fractions MW>60000 prepared from beer brewed from grists containing torrified wheat, wheat flour or all malted wheat may contain high molecular weight polypeptide material derived from wheat proteins. The results from the analysis of fraction MW 40,000–60000 prepared from beers brewed from grists containing all malt, 80% malt and 20% torrified wheat and 50% malt and 50% malted wheat are consistent with the presence of polypeptide material derived from cereal albumins and globulins whereas fractions MW 40,000–60000 prepared from beers brewed from 80% malt and 20% wheat flour and 100% malted wheat may contain polypeptide material derived from wheat prolamins and glutelins. The amino acid composition of fraction MW 20,000–40,000 from all pilot brewery beers investigated is consistent with the presence of polypeptide material derived from cereal prolamins and glutelins. The amino acid composition of beer polypeptide fractions may be used to detect the use of wheat adjuncts in beer brewing.

Comparison of the calcium release channel of cardiac and skeletal muscle sarcoplasmic reticulum by target inactivation analysis

The calcium release channel of sarcoplasmic reticulum which triggers muscle contraction in excitation-contraction coupling has recently been isolated. The channel has been found to be morphologically identical with the feet structures of the junctional face membrane of terminal cisternae and consists of an oligomer of a unique high molecular weight polypeptide. In this study, we compare the target size of the calcium release channel from heart and skeletal muscle using target inactivation analysis. The target molecular weights of the calcium release channel estimated by measuring ryanodine binding after irradiation are similar for heart (139,000) and skeletal muscle (143,000) and are smaller than the monomeric unit (estimated to be about 360,000). The target size, estimated by measuring polypeptide remaining after irradiation, was essentially the same for heart and skeletal muscle, 1,061,000 and 1,070,000, respectively, indicating an oligomeric association of protomers. Thus, the calcium release channel of both cardiac and skeletal muscle reacts uniquely with regard to target inactivation analysis in that (1) the size by ryanodine binding is smaller than the monomeric unit and (2) a single hit leads to destruction of more than one polypeptide, by measuring polypeptide remaining. Our target inactivation analysis studies indicate that heart and skeletal muscle receptors are structurally very similar.

The formation and translational activity of polysomes from developing lupin seeds

The formation and in vitro translational activity of total, free and membrane‐bound polysomes from various stages of developing cotyledons of yellow lupin seeds (Lupinus luteus L. cv. Iryd) has been investigated. The early stages of seed formation were characterized by a low level of polysomes that progressively increased. The main features of the cotyledons at the middle phase of development were full expansion growth and the highest amount of polysomes observed in all three poly so me fractions. In The final stages of emhryogenesis. the seed dehydration was accompanied by‐gradual loss of all types of polysomes, at which the membrane‐attached formations were degraded earlier than the free ones. By means of a wheat germ‐derived cell‐free system for protein synthesis, a correlation was demonstrated between cotyledon growth, polysome formation and their capacity for protein synthesis in vitro. As compared to the free polysomes, both the total and membrane‐bound formations were more active in protein synthesis in vitro. Analysis of the translational products by means of immunoprecipitation and gel electrophoresis followed by fluorography showed that only membrane‐bound polysomes produced polypeptides of higher molecular weight, including subunits of a legumin‐like protein.

Ovarian protein synthesis in the South American white shrimp,Penaeus vannamei, during the reproductive cycle

Summary Ovarian development was induced by eyestalk ablation in the South American white shrimp, Penaeus vannamei. During the gonadotrophic cycle, ovarian protein increased from virtually undetectable levels in undeveloped ovaries to >400 mg in mature ovaries. Total protein was separated by SDS-PAGE and high molecular weight polypeptides of ~175–200 kDa were abundant in vitellogenic ovaries but absent in previtellogenic ovaries. These polypeptides are probably the yolk polypeptides. We determined in vitro protein synthesis rates for the ovary and hepatopancreas during ovarian development. Tissues from animals at the onset of vitellogenesis incorporated radiolabelled amino acids at the most active rate. Using autoradiography, we demonstrated that the ovary synthesized many polypeptides, including the major, putative yolk polypeptides. We did not detect synthesis of these polypeptides by the hepatopancreas.

Anti‐200 kDa neurofilament antibody cross‐reacts with microtubule‐associated protein‐2 (MAP‐2)

An antibody prepared against purified 200-kDa neurofilament (NF) subunit was found to cross-react with a high molecular weight polypeptide present in rat brain cytoskeletal extracts starting from early postnatal development. This polypeptide was found to be highly concentrated in microtubule preparations and was specifically recognized as the 280-kDa microtubule-associated protein-2 (MAP-2). Our results, besides suggesting the existence of common epitopes between the heavy NF subunit and MAP-2, suggest the possibility of an interaction between two of the most important neuronal cytoskeletal organelles: NFs and microtubules.

An autoradiographic analysis of vitellogenin synthesis and secretion in the fat body of the stick insect Bacillus rossius

The fat body of the stick insect Bacillus rossius was studied with a view to clarifying the metabolic pathway leading to secretion of vitellogenin (VG). Electrophoretic analysis of ovarian follicles and hemolymph from egg-laying females showed that the two tissues shared a common polypeptide composition consisting of five major polypeptides with molecular weights ranging from 60–180 Kd. Following in vivo exposure to [ 35S]-methionine for up to 24 h, these polypeptides were labeled in a stage- and time-dependent manner, suggesting that they were transferred from the hemolymph to the oocyte during vitellogenesis. Fat body pulse-labeled with [ 35S]-methionine for up to 240 min and immunoprecipitated with an anti-yolk serum was labeled only in a fraction containing high molecular weight polypeptides. We presume these polypeptides to be VG precursors bearing a precursor-product relationship with the five major polypeptides of the hemolymph and developing ovarian follicles. Fat body exposed in vivo to [ 3H]-leucine for time intervals ranging from 20–240 min were processed for EM autoradiography. The results of this analysis showed that incorporated radioactivity was progressively transferred from the endoplasmic reticulum to the Golgi apparatus and from there to the composite granules. The data provided in this study are consonant with previous findings by which composite granules were shown to contain two compartments differing both in content and origin. In addition, the autoradiographical data of in vivo labeled fat body demonstrate that only the material partitioned into the electron-dense compartment of these granules is exocytosed.

Quantitation of the dynein pool in unfertilized sea urchin eggs

A dynein-like ATPase activity has been isolated previously from soluble extracts of unfertilized sea urchin eggs. However, the use of non-quantitative isolation techniques, in particular affinity for microtubules or Ca2+/calmodulin, has precluded accurate estimates of dynein pool size. We have taken the unique approach of using dynein-like ATPase activity to quantitate the egg dynein pool. This approach is based on the isolation by anion-exchange chromatography on DEAE-Sephacel of a peak of dynein-like ATPase activity comprising 65% of soluble ATPase activity in the cytosolic extract. Identification of cytoplasmic dynein was based on dose-dependent inhibition by erythro-9-[3-(2-hydroxynonyl)]adenine and orthovanadate, low GTPase activity and a sedimentation coefficient of 12 S. Two high molecular weight polypeptides corresponding to the A- and D-bands of axonemal dynein were shown to copurify with dynein-like ATPase activity and to undergo specific photocrosslinking with [alpha-32P]ATP, suggesting that they were egg dynein catalytic polypeptides. The specific ATPase activity of these putative catalytic polypeptides was determined to be 1.2 mumol.min-1.mg-1. The specific dynein-like ATPase activity of the crude soluble extract of unfertilized sea urchin eggs was determined to be 0.004 mumol.min-1.mg-1. The concentration of putative dynein catalytic polypeptides was therefore determined from the ratio of the specific activities of crude to pure cytoplasmic dynein catalytic polypeptide to be 0.33% of soluble protein, or 99 pg per egg. This is approximately 3-fold greater than the mass of dynein catalytic polypeptides estimated to be present in cilia at the blastula stage of sea urchin embryonic development. The large amount of cytoplasmic dynein in unfertilized eggs suggests that it could act as a precursor of embryonic ciliary dynein. Three minor peaks of ATPase activity were also resolved from cytosolic extracts and shown to be dynein-like. However, their GTPase activities were 2-4-fold higher than that of cytoplasmic dynein, raising the possibility that egg cytoplasm may contain several isoforms of dynein.

Genomic locus for a 140 kDa structural protein (pp150) of human cytomegalovirus in strains Towne and AD169

An HCMV specific clone was isolated from a genomic library of human cytomegalovirus (HCMV) DNA cloned into the expression vector λgt11. This clone (λ111-1) expressed an HCMV/β-galactosidase fusion protein which was reactive with rabbit antibody prepared against purified HCMV virions and dense bodies as well as human HCMV immune serum. By probing Western blots of HCMV virion proteins or HCMV-infected cells with antibody prepared against the fusion protein, the authentic gene product of clone λ111-1 was identified as a high molecular weight polypeptide of 140. Probing the restriction digests of HCMV DNA with insert DNA from the immunoreactive λgt11 clone permitted us to localize the coding sequence for the 140 kDa polypeptide to the long unique region (map coordinates of 0.16–0.18) on HCMV Towne and AD169 genomes.

Partial extraction of inner dynein arms from an outer arm-less mutant of Chlamydomonas reinhardtii.

Flagellar axonemes that lack the outer dynein arms and a part of the inner dynein arms were prepared by extracting demembranated axonemes of an outer-arm less mutant of Chlamydomonas with a solution containing 0.3 M KC1 and 10 μM ATP. When reactivated with 1 mM ATP, the extracted axonemes, whose beat frequency before the extraction had been 26.9±2.6 Hz (26°C, n=10), beat with a reduced frequency of 14.9±3.7 Hz (26°C, n=10). The decrease in beat frequency depended on the KC1 concentration and on the presence of ATP in the extracting solution. The bend angle and the curvature of bend were not affected by the extraction. Electron microscopic observation showed that 28.5% of the inner arms were weak in appearance or lost in the extracted axoneme. Electrophoresis of the axonemal proteins indicated that one of the high molecular weight polypeptides of the inner dynein arm was reduced in quantity. These results suggest that some part of the inner arms has the function of increasing the flagellar beat frequency but is not essential for flagellar motility.

Monoclonal antibodies to a proenkephalin A fusion peptide synthesized in Escherichia coli recognize novel proenkephalin A precursor forms.

Monoclonal antibodies have been generated to a chimeric peptide comprised of Escherichia coli beta-galactosidase fused to the amino acid sequence 69-207 of human preproenkephalin A. Two monoclonal antibodies, PE-1 and PE-2, were identified by their ability to recognize the same segment of proenkephalin A fused to the cII gene product of the E. coli bacteriophage lambda. The binding domains of PE-1 and PE-2 have been broadly located, with respect to the primary translation product, within the amino acid sequences 152-207 and 84-131, respectively. Immunoblot analysis of total bovine adrenomedullary chromaffin granule lysate reveals PE-1 and PE-2 immunoreactive forms of observed molecular mass 35, 33, 29, 24, 22, and 15 kDa, and an 18-kDa PE-1 immunoreactive form. Separation of granule membranes from their contents reveals differential membrane association of these high molecular weight polypeptides. There is preliminary evidence that PE-1 may be detecting a subset of polypeptides where shortening from the NH2 terminus has occurred. We postulate that the 35-kDa form represents the intact bovine enkephalin precursor of predicted molecular mass 27.3 kDa. This experimental approach should be generally applicable to the generation of antibodies which will recognize intact peptide precursors together with their post-translational cleavage products.

Enhanced synthesis and secretion of type IV collagen and entactin during adipose conversion of 3T3-L1 cells and production of unorthodox laminin complex.

Synthesis and secretion of basement membrane proteins by 3T3-L1 adipocytes were studied by metabolic labeling of the cells with [35S]methionine. Enhanced synthesis and secretion of many polypeptides of high molecular weight were observed by stimulating the adipose conversion of 3T3-L1 fibroblasts with dexamethasone, 1-methyl-3-isobutylxanthine, and insulin. Among these polypeptides, alpha 1(IV) and alpha 2(IV) chains of collagen were identified based on specific immunoprecipitation and digestion with bacterial collagenase. Synthesis and secretion of alpha 1(IV) and alpha 2(IV) chains were negligible in the fibroblasts, but remarkably enhanced in adipocytes. Based on specific immunoprecipitation of a sulfated polypeptide of 150 kDa, enhanced (6-fold) synthesis and secretion of entactin were also demonstrated. Immunoprecipitation with anti-laminin antiserum showed synthesis of three polypeptides with sizes corresponding to B subunits but failed to demonstrate synthesis of the A subunit. Synthesis of these laminin-related polypeptides remained constant during the conversion. Nonreducing sodium dodecyl sulfate electrophoresis showed intracellular assembly of three laminin-related polypeptides into binary and ternary complexes in a similar sequence of AB1B2 formation via B1B2 in embryonal carcinoma F9 (Morita, A., Sugimoto, E., and Kitagawa, Y. (1985) Biochem. J. 229, 259-264). The ternary complex of laminin in 3T3-L1 cells had a size significantly smaller than AB1B2 complex in F9 cells. In this complex, a novel subunit appears to take the place of the A subunit. Thus, a novel laminin complex is produced by 3T3-L1 cells.

Identification and purification of a transverse tubule coupling protein which binds to the ryanodine receptor of terminal cisternae at the triad junction in skeletal muscle.

In fast twitch skeletal muscle, the signal for excitation-contraction coupling is transferred from transverse tubule across the triad junction; calcium is thereby released from the terminal cisternae of sarcoplasmic reticulum triggering muscle contraction. Recently, the feet structures of terminal cisternae, which bridge the gap at the triad junction, have been identified as the ryanodine receptor and in turn with the calcium release channels of sarcoplasmic reticulum. The latter consists of an oligomer of a single high molecular weight polypeptide (Mr 360,000). This study attempts to identify the component in the transverse tubule which ligands with the foot structure to form the triad junction. The purified ryanodine receptor, derivatized with sulfosuccinimidyl-2-(p-azidosalicylimido)-1,3'-dithiopropionate (SASD), a thiol-cleavable, 125I-iodinatable, and photoactive probe, was shown to selectively cross-link to a protein with Mr of 71,000 in isolated transverse tubules. This coupling protein was purified from transverse tubule by solubilization with the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) and then purified by sequential column chromatography. In the absence of sulfhydryl agents, the purified polypeptide has an Mr of 61,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A complementary approach using SASD was employed to confirm association of the coupling protein with the ryanodine receptor of terminal cisternae. We conclude that the transverse tubule coupling protein together with the ryanodine receptor (foot structure) is involved in the liganding between transverse tubule and terminal cisternae of sacroplasmic reticulum.

Regulation of axonemal Mg 2+-ATPase from Paramecium cilia: effects of Ca 2+ and cyclic nucleotides

Ciliary activity is regulated by Ca 2+ and cyclic nucleotides, but the molecular mechanisms of the regulation are unknown. We have tested the ability of Ca 2+ and cyclic nucleotides to alter ciliary Mg 2+-ATPase or to stimulate phosphorylation of axonemal dynein. Mg 2+-ATPase activity in cilia and axonemes from Paramecium was stimulated 2-fold by micromolar Ca +, but this Ca 2+ sensitivity was lost upon solubilization of the dyneins from the axoneme. The Ca 2+-sensitive component of ciliary Mg 2+-ATPase activity was inhibited by the dynein inhibitors vanadate and Zn 2+, but was insensitive to the calmodulin antagonists calmidazolium and melittin. Dynein activity in the high-salt extract from axonemes was also insenstive to calmidazolium. Calmodulin did not sediment with 22 S or 12 S dyneins on sucrose gradients containing Ca 2+, but it did sediment in the region from 19 S to 14 S. Mg 2+-ATPase activity in ciliary fractions was unaltered in the presence of cAMP or cGMP. However, polypeptides associated with the 22 S and 12 S dyneins, as well as proteins of 19 S, 15 S, and 8 S, were substrates for endogenous ciliary kinases. High molecular weight polypeptides that sedimented at 22 S and 19 S were phosphorylated in a cyclic nucleo-stimulated manner.

Ultrastructure of the calcium release channel of sarcoplasmic reticulum.

This study is concerned with the characterization of the morphology of the calcium release channel of sarcoplasmic reticulum (SR) from fast-twitch skeletal muscle, which is involved in excitation-contraction coupling. We have previously purified the ryanodine receptor and found it to be equivalent to the feet structures, which are involved, in situ, in the junctional association of transverse tubules with terminal cisternae of SR. The receptor is an oligomer of a single high molecular weight polypeptide and when incorporated into phospholipid bilayers, has channel conductance which is characteristic of calcium release in terminal cisternae of SR. The purified channel can be observed by electron microscopy using different methods of sample preparation, with complementary views being observed by negative staining, double staining, thin section and rotary shadowing electron microscopy. Three views can be observed and interpreted: (a) a square face which, in situ, is junctionally associated with the transverse tubule or junctional face membrane; (b) a rectangle equivalent to the side view; and (c) a diamond shape equivalent to the side view, of which the base portion appears to be equivalent to the transmembrane segment. Negative staining reveals detailed substructure of the channel. A computer averaged view of the receptor displays fourfold symmetry and ultrastructural detail. The dense central mass is divided into four domains with a 2-nm hole in the center, and is enclosed within an outer frame which has a pinwheel appearance. Double staining shows substructure of the square face in the form of parallel linear arrays (six/face). The features of the isolated receptor can be correlated with the structure observed in terminal cisternae vesicles. Sections tangential to the junctional face membrane reveal that the feet structures (23-nm squares) overlap so as to enclose smaller square spaces of approximately 14 nm/side. We suggest that this is equivalent to the transverse tubule face and that the terminal cisternae face is smaller (approximately 17 nm/face) and has larger alternating spaces as a consequence of the tapered sides of the foot structures. Image reconstruction analysis appears to be feasible and should provide the three-dimensional structure of the channel.