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PubMed2002

Effect of adenosine 5'-[beta,gamma-imido]triphosphate on myosin head domain movements.

Conventional and saturation transfer electron paramagnetic resonance spectroscopy (EPR and ST EPR) was used to study the orientation of probe molecules in muscle fibers in different intermediate states of the ATP hydrolysis cycle. A separate procedure was used to obtain ST EPR spectra with precise phase settings even in the case of samples with low spectral intensity. Fibers prepared from rabbit psoas muscle were labeled with isothiocyanate spin labels at the reactive thiol sites of the catalytic domain of myosin. In comparison with rigor, a significant difference was detected in the orientation-dependence of spin labels in the ADP and adenosine 5'-[beta,gamma-imido]triphosphate (AdoPP[CH2]P) states, indicating changes in the internal dynamics and domain orientation of myosin. In the AdoPP[CH2]P state, approximately half of the myosin heads reflected the motional state of ADP-myosin, and the other half showed a different dynamic state with greater mobility.

Adenylyl Imidodiphosphate↗

PubMed1983

Differentiation of posterior pararenal space infection from psoas abscess by gallium imaging.

Three of four patients whose cases fit the clinical description of psoas abscess proved on gallium imaging to have infection in the posterior pararenal space sparing the psoas muscle. This space provides a route for spread of infection connecting the spine, the anterior abdominal wall, the scrotum, the anterior thigh, and the gluteal region as demonstrated by the cases presented. Clinical differentiation between posterior pararenal space infection and psoas abscesses is difficult and CT studies may not demonstrate the process when the psoas space is not involved.

Abdomen↗

PubMed2000

Primary pyogenic psoas abscess in children.

Primary pyogenic psoas abscess, although quite a common condition, particularly in the tropics, is often overlooked as a clinical entity, probably because a psoas abscess has been traditionally associated with tuberculous spondylitis. The abscess is easily diagnosed by ultrasonography (US). Treatment by open drainage and antibiotics effective against Staphylococcus aureus results in complete reversal of symptoms and signs. In our series of 55 cases in the pediatric age group (0-12 years), pain and flexion at the hip were the most frequent clinical features at presentation. US was diagnostic in all cases in which it was performed. All except 1 patient showed complete resolution with extraperitoneal drainage, antibiotics, and skin traction. Although 4% of the cases were associated with suppurative external-iliac lymphadenitis, the remaining ones arose de novo in the psoas sheath, suggesting a primary pyomyositis of the psoas muscle.

Anti-Bacterial Agents↗

PubMed1996

Orientation of intermediate nucleotide states of indane dione spin-labeled myosin heads in muscle fibers.

We have used electron paramagnetic resonance to study the orientation of myosin heads in the presence of nucleotides and nucleotide analogs, to induce equilibrium states that mimic intermediates in the actomyosin ATPase cycle. We obtained electron paramagnetic resonance spectra of an indane dione spin label (InVSL) bound to Cys 707 (SH1) of the myosin head, in skinned rabbit psoas muscle fibers. This probe is rigidly immobilized on the catalytic domain of the head, and the principal axis of the probe is aligned nearly parallel to the fiber axis in rigor (no nucleotide), making it directly sensitive to axial rotation of the head. On ADP addition, all of the heads remained strongly bound to actin, but the spectral hyperfine splitting increased by 0.55 +/- 0.02 G, corresponding to a small but significant axial rotation of 7 degrees. Adenosine 5'-(adenylylim-idodiphosphate) (AMPPNP) or pyrophosphate reduced the actomyosin affinity and introduced a highly disordered population of heads similar to that observed in relaxation. For the remaining oriented population, pyrophosphate induced no significant change relative to rigor, but AMPPNP induced a slight but probably significant rotation (2.2 degrees +/- 1.6 degrees), in the direction opposite that induced by ADP. Adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) relaxed the muscle fiber, completely dissociated the heads from actin, and produced disorder similar to that in relaxation by ATP. ATP gamma S plus Ca induced a weak-binding state with most of the actin-bound heads disordered. Vanadate had negligible effect in the presence of ADP, but in isometric contraction vanadate substantially reduced both force and the fraction of oriented heads. These results are consistent with a model in which myosin heads are disordered early in the power stroke (weak-binding states) and rigidly oriented later in the power stroke (strong-binding states), whereas transitions among the strong-binding states induce only slight changes in the axial orientation of the catalytic domain.

Adenine Nucleotides↗

PubMed1997

Sarcomere length dependence of the rate of tension redevelopment and submaximal tension in rat and rabbit skinned skeletal muscle fibres.

1. We examined the hypothesis that in skeletal muscle the steep relationship between twitch tension and sarcomere length (SL) within the range 2.30 to 1.85 microns involves SL-dependent alterations in the rate of tension development. 2. In skinned preparations of both rat slow-twitch and rabbit fast-twitch skeletal muscle fibres the rate of tension redevelopment (ktr) at 15 degrees C was reduced at short SL (approximately 2.00 microns) compared with a longer SL (approximately 2.30 microns). In submaximally activated fibres, the decrease in ktr over this range of lengths was greater in fast-twitch fibres (38% reduction) than in slow-twitch fibres (14% reduction). 3. Ca2+ sensitivity of tension, as assessed as the pCa (-log[Ca2+]) for half-maximal activation, or pCa50, decreased to a greater extent in rabbit fast-twitch skeletal muscle fibres than in slow-twitch fibres from both rabbit and rat when SL was reduced from approximately 2.30 to approximately 1.85 microns. The delta pCa50 over this SL range was 0.24 +/- 0.07 pCa units in fast-twitch fibres from rabbit psoas muscle. The delta pCa50 for slow-twitch fibres from rabbit and rat soleus muscle was 0.08 +/- 0.02 and 0.10 +/- 0.04 pCa units, respectively. 4. Osmotic compression of both slow-twitch and fast-twitch fibres at a SL of 2.00 microns increased ktr to values similar to those obtained at a SL of 2.30 microns in the absence of dextran. This result indicates that the slower rate of tension redevelopment at short SL is due in large part to the increase in interfilament lattice spacing associated with shorter SL. 5. Taken together, these results suggest that length dependence of twitch tension is, in part, due to length dependence of isometric cross-bridge interaction kinetics, an effect that is mediated by length-dependent changes in interfilament lattice spacing.

Animals↗

PubMed1998

Fluorescence polarization transients from rhodamine isomers on the myosin regulatory light chain in skeletal muscle fibers.

Fluorescence polarization was used to examine orientation changes of two rhodamine probes bound to myosin heads in skeletal muscle fibers. Chicken gizzard myosin regulatory light chain (RLC) was labeled at Cys108 with either the 5- or the 6-isomer of iodoacetamidotetramethylrhodamine (IATR). Labeled RLC (termed Cys108-5 or Cys108-6) was exchanged for the endogenous RLC in single, skinned fibers from rabbit psoas muscle. Three independent fluorescence polarization ratios were used to determine the static angular distribution of the probe dipoles with respect to the fiber axis and the extent of probe motions on the nanosecond time scale of the fluorescence lifetime. We used step changes in fiber length to partially synchronize the transitions between biochemical, structural, and mechanical states of the myosin cross-bridges. Releases during active contraction tilted the Cys108-6 dipoles away from the fiber axis. This response saturated for releases beyond 3 nm/half-sarcomere (h.s.). Stretches in active contraction caused the dipoles to tilt toward the fiber axis, with no evidence of saturation for stretches up to 7 nm/h.s. These nonlinearities of the response to length changes are consistent with a partition of approximately 90% of the probes that did not tilt when length changes were applied and 10% of the probes that tilted. The responding fraction tilted approximately 30 degrees for a 7.5 nm/h.s. release and traversed the plane perpendicular to the fiber axis for larger releases. Stretches in rigor tilted Cys108-6 dipoles away from the fiber axis, which was the opposite of the response in active contraction. The transition from the rigor-type to the active-type response to stretch preceded the main force development when fibers were activated from rigor by photolysis of caged ATP in the presence of Ca2+. Polarization ratios for Cys108-6 in low ionic strength (20 mM) relaxing solution were compatible with a combination of the relaxed (200 mM ionic strength) and rigor intensities, but the response to length changes was of the active type. The nanosecond motions of the Cys108-6 dipole were restricted to a cone of approximately 20 degrees half-angle, and those of Cys108-5 dipole to a cone of approximately 25 degrees half-angle. These values changed little between relaxation, active contraction, and rigor. Cys108-5 showed very small-amplitude tilting toward the fiber axis for both stretches and releases in active contraction, but much larger amplitude tilting in rigor. The marked differences in these responses to length steps between the two probe isomers and between active contraction and rigor suggest that the RLC undergoes a large angle change (approximately 60 degrees) between these two states. This motion is likely to be a combination of tilting of the RLC relative to the fiber axis and twisting of the RLC about its own axis.

Animals↗

PubMed1993

Identification of the regions conferring calmodulin-like properties to troponin C.

The structural and functional correlations between troponin C (TnC) and calmodulin (CaM) were investigated by mutagenizing a synthetic cDNA coding rabbit skeletal muscle TnC. Compared with TnC, calmodulin lacks the N-terminal alpha-helical arm (N-helix), and its central helix is shorter due to the absence of 88KGK90 residues. Deleting both regions concomitantly (delta Nt delta KGK) elicited CaM-like regulation as tested (i) by smooth muscle contractility (maximal tension = 80 +/- 5% Po of control) and (ii) by the activation of phosphodiesterase (Vmax = 75 +/- 2% of control). The Ca(2+)-binding capacity of the mutant and the effect of the mutant on maximally Ca(2+)-activated tension of skinned rabbit psoas muscle fibers were both conserved. Furthermore, in the linker region of the central helix, replacing the TnC-characteristic 85EDAKGK90 successive residues with CaM-specific DTD residues generated a highly effective CaM mimic (Vmax = 96 +/- 2%) whether or not the N-helix was also retained. Apparent KD values (i.e. concentrations for half-maximal response) for the successful mutants were similar to each other but about 200-fold higher than that for CaM. A part of the alpha-helical linker region in CaM may unfold and bend to promote multiplicity of target interaction using all four hands (Ikura, M., Clore, G. M., Gronenborn, A. M., Zhu, G., Klee, C. B., and Bax, A. (1992) Science 256, 632-638; Meador, W. E., Means, A. R., and Quiocho, F. A. (1992) Science 257, 1251-1255). In contrast, our results suggest that the TnC central helix evolved to be less pliable by the combined influences of 85EDAKGK90 residues and the alpha-helical extension in N terminus, thereby keeping the N-terminal hands well separated from their C-terminal counterparts.

Amino Acid Sequence↗

PubMed1998

Potent stimulation of myofilament force and ATPase activity of skeletal muscle by eudistomin M, a novel Ca(++)-sensitizing agent from a Caribbean tunicate.

In the course of our survey of biologically active compounds from natural sources, eudistomins were isolated from a Caribbean tunicate Eudistoma olivaceum. In the present experiments, eudistomin M (Eud-M, > 10(-5) M) caused a concentration-dependent increase in the contractile response of skinned fibers from guinea pig skeletal psoas muscles to Ca++. The superprecipitation and ATPase activity of myosin B from fast skeletal muscles of rabbit back and leg were potentiated by this compound (> 10(-5) M) in a concentration-dependent manner. In skinned fibers, superprecipitation and the ATPase activity of myosin B, Eud-M shifted the concentration-response curve for Ca++ to the upper direction. Ca(++)-, K(+)-EDTA- or Mg(++)-ATPase was not affected by Eud-M. This compound had no effect on the ATPase activity of actomyosin reconstituted from actin and myosin in the presence or absence of troponin. However, the ATPase activity of actin-myosin-troponin-tropomyosin reconstituted system was increased significantly by Eud-M. These results suggest that Eud-M increases the Ca++ sensitivity of the contractile apparatus in skeletal muscles at least partially mediated through troponin-tropomyosin system and thus enhances the ATPase activity of myosin B and the contractile force of myofilament.

Actin Cytoskeleton↗

PubMed1980

The structure of segments of the anisotropic band of muscle. II. Preparation and properties of A segments from vertebrate skeletal muscle.

The anisotropic band of skeletal muscle is a complex structural assembly of the protein myosin and associated nonmyosin components. To study the relationships among these proteins, aggregates of thick myofilaments held together at the M line (A segments) have been prepared from fresh and glycerol extracted chicken pectoralis and rabbit psoas muscles and from fresh frog sartorius muscle. The structure of the A segments included several thick filaments, an M line, and a bare zone or pseudo-H zone, lateral to the M line. Most of the A segments exhibited a pattern of eleven periodic stripes in each half lateral to the bare zone. The A segments from fresh muscle displayed these stripes more consistently than did the A segments from glycerinated muscle. Some of the major stripes appeared to be double, and there were two subdivisions between the stripes nearest the bare zone. The more lateral of the major A band stripes, however, had one subdivision between them. The M line consisted of three prominent medial stripes and two fainter lateral stripes. In the M lines of rabbit A segments the lateral stripes were located well into the bare zone whereas the lateral stripes of M lines in chicken A segments were closer to the three medial M line stripes. Our results on the preparation and properties of A segments are compared with those of of the investigators.

Animals↗

PubMed1993

Filament overlap affects TnC extraction from skinned muscle fibres.

Recent studies on calcium regulation of muscle contraction selectively extract troponin C (TnC) from skinned skeletal muscle fibres with a low ionic strength rigor solution containing a Ca2+/Mg2+ chelator. As previous results from this laboratory and others demonstrate a crossbridge effect, especially rigor, on many of the properties of TnC, the effects of filament overlap on TnC extraction from skinned rabbit psoas muscle fibres were investigated. Tension-pCa relationships at a sarcomere length of 2.7 microns were determined before and after a 5 min TnC extraction at sarcomere lengths of 2.3, 2.5, 2.7, 3.1, 3.3 or 3.5 microns with 20 mM Tris, pH 7.8, 5 mM EDTA. The decrease in the post-extraction maximum Ca2+ activated tension, an indicator of the amount of TnC extracted, was linearly related to the overlap of the thick and thin filaments with decreases in tension being associated with a decrease in filament overlap. The smaller fibre diameter at the longer sarcomere length could facilitate diffusion of TnC from fibre segments. However, the wide range of measured diameters, 40-120 microns, accounted for only 14% of the observed tension decrement and shrinking the fibre with polyvinylpyrrolidone did not increase the tension decrement. Increasing the sarcomere length before extraction was also found to decrease the TnC content of fibre segments along with the post-extraction maximum tension. Thus, TnC appears to be preferentially extracted from non-overlap than overlap regions of the sarcomere. These results further indicate that rigor crossbridges affect TnC other than through increased Ca2+ binding and that under the conditions used here, they retard its extraction.

Animals↗

PubMed2001

Unusual presentation of psoas abscess in a child.

Primary abscess of the psoas muscle is relatively rare in infants and young children. The clinical presentation of the disease, with limping, fever, and abdominal pain, may be confused with conditions such as septic arthritis of the hip, osteomyelitis, or appendicitis. The authors present an unusual case of a ruptured left psoas abscess presenting as generalized peritonitis in a child. J Pediatr Surg 36:1859-1860.

Diagnosis, Differential↗

PubMed1978

Protein turnover in rat skeletal muscle: effects of hypophysectomy and growth hormone.

The role of growth hormone in regulating protein turnover was examined in a perfused preparation of rat skeletal muscle. The perfused muscle maintained in vivo levels of ATP and creatine phosphate and exhibited constant rates of oxygen consumption and protein synthesis. Hypophysectomy reduced the rate of protein synthesis, the concentration of RNA, and the efficiency of protein synthesis in gastrocnemius muscle to 30, 46, and 66 percent of normal, respectively. In vivo treatment of hypophysectomized (hypox) rats with bovine growth hormone (250 microgram/day for 5 days) resulted in small increases in protein synthesis and RNA, whereas synthesis/RNA was returned to near normal. Elevation of ribosomal subunits in psoas muscle indicated an inhibition of peptide-chain initiation in hypox rats that was reversed by in vivo growth hormone treatment. Thus, hypox rats exhibited both a decreased capacity and a decreased efficiency of protein synthesis. Growth hormone replacement primarily increased efficiency of protein synthesis. The rate of protein degradation and the activity of cathepsin D in gastrocnemius muscle were decreased by hypophysectomy. Growth hormone treatment had no significant effect on degradation.

Animals↗

PubMed1998

The cat vertebral column: stance configuration and range of motion.

This study examined the configuration of the vertebral column of the cat during independent stance and in various flexed positions. The range of motion in the sagittal plane is similar across most thoracic and lumbar joints, with the exception of a lesser range at the transition region from thoracic-type to lumbar-type vertebrae. The upper thoracic column exhibits most of its range in dorsiflexion and the lower thoracic and lumbar in ventroflexion. Lateral flexion is limited to less than 5 degrees at all segments. The range in torsion is almost 180 degrees and occurs primarily in the midthoracic region, T4-T11. Contrary to the depiction in most atlases, the standing cat exhibits several curvatures, including a mild dorsiflexion in the lower lumbar segments, a marked ventroflexion in the lower thoracic and upper lumbar segments, and a profound dorsiflexion in the upper thoracic (above T9) and cervical segments. The curvatures are not significantly changed by altering stance distance but are affected by head posture. During stance, the top of the scapula lies well above the spines of the thoracic vertebrae, and the glenohumeral joint is just below the bodies of vertebrae T3-T5. Using a simple static model of the vertebral column in the sagittal plane, it was estimated that the bending moment due to gravity is bimodal with a dorsiflexion moment in the lower thoracic and lumbar region and a ventroflexion moment in the upper thoracic and cervical region. Given the bending moments and the position of the scapula during stance, it is proposed that two groups of scapular muscles provide the major antigravity support for the head and anterior trunk. Levator scapulae and serratus ventralis form the lateral group, inserting on the lateral processes of cervical vertebrae and on the ribs. The major and minor rhomboids form the medial group, inserting on the spinous tips of vertebrae from C4 to T4. It is also proposed that the hypaxial muscles, psoas major, minor, and quadratus lumborum could support the lumbar trunk during stance.

Animals↗

PubMed1981

A proton gradient controls a calcium-release channel in sarcoplasmic reticulum.

Sarcoplasmic reticulum vesicles from mammalian skeletal muscle have previously been shown to develop a proton gradient (alkaline inside) of 0.15-0.5 pH units during active Ca2+ uptake. We found that dissipation of this gradient by the proton ionophores gramicidin, nigericin, and carbonyl cyanide p-trichloromethoxyphenylhydrazone caused a rapid transient tension in skinned rabbit psoas muscle fibers. Increases, but not decreases, in medium pH of approximately 0.2 units over the range from pH 6.5 to pH 7.5 also elicited transient tensions. In isolated vesicles, physiological levels of Ca2+ (3.3 microM), inhibited pH-induced Ca2+ release. Dicyclohexylcarbodiimide blocked pH- and ionophore-induced Ca2+ release under conditions in which it could bind to sarcoplasmic reticulum proteins but did not inhibit Ca2+ uptake. We propose that a proton gradient generated across sarcoplasmic reticulum membranes during Ca2+ uptake maintains a Ca2+ release channel in a closed conformation and that dissipation of this gradient permits the Ca2+ release channel to open. We further propose that elevated myoplasmic Ca2+ also causes the Ca2+ channel to close, permitting Ca2+ uptake through Ca2+/Mg2+-ATPase to function effectively. As the proteolipids of sarcoplasmic reticulum bind dicyclohexylcarbodiimide under conditions in which Ca2+ release is blocked and as they have previously been shown to have Ca2+ ionophoric activity, we propose that the Ca2+-release channel either resides in the proteolipids or is controlled by H+ fluxes through the proteolipids.

Animals↗

PubMedDate not supplied

Peripheral primitive neuroectodermal tumor (PPNET) of pelvic origin: report of a case arising from an unusual location.

We report a rare case of a peripheral primitive neuroectodermal tumor (PPNET) originating from the left ileopsoas muscle in an adult patient with neoplastic thrombosis of the left external iliac vein, the common femoral vein and the left popliteal vein. We performed a median laparotomy with an oblique left inguinal incision to remove the neoplasm, which consisted of a large mass surrounding the iliac-psoas muscles, extending from the transverse apophysis of the spinal column to Scarpa's triangle, and passing through the lacuna musculorum. Histopathological examination revealed a primitive neuroectodermal tumor (PNET) with focal areas of necrosis, hemorrhage and vascular invasion. Immunophenotyping was positive for CD99, NSE, and focally for CK. Ultrastructural examination of the neoplastic cells showed often multiple nuclei with dense chromatin and very large nucleoli. The patient was discharged ten days after the operation. Adjuvant treatment consisted of radiotherapy at a dose of 2000 cGy in five fractions followed by six cycles of chemotherapy. The venous thrombosis was treated by anticoagulant therapy and recanalization of the occluded veins was obtained after two months of therapy. An MRI scan, carried out 12 months later, showed a local relapse, which was treated with chemotherapy and arterial chemoembolization.

Aged↗

PubMed1975

Effect of dantrolene sodium on excitation-contraction coupling in frog skeletal muscle.

The effect of dantrolene sodium, 1-(5-(p-nitrophenyl)furfuryli-deneamino)hydantoin sodium hydrate, on electrical and mechanical response in frog skeltal muscles (whole muscles or single fibers) and on the biochemical properties of contractile proteins and fragmented sarcoplasmic reticulum isolated from frog or rabbit skeletal muscle was investigated. The peak tensions of twitch, tetanus and potassium contracture were significantly inhibited by dantrolene, without affecting the magnitude of resting potential, the amplitude and duration of action potential and the negative afterpotential. On the other hand, ATP-INDUCED SHORTENING OF GLYCEROL-EXTRACTED RABBIT PSOAS MUSCLE FIBERS, ATPase activity of frog myofibrils and Ca release induced by caffeine, Ca uptake and ATPase activity of fragmented sarcoplasmic reticulum of frog or rabbit muscle were not affected by dantrolene. Caffeine contracture was partially inhibited by dantrolene and was almost unchanged by it in potassium-depolarized muscele fiber. Nitrate ions and low concentration of caffeine rapidly recovered the twitch inhibition induced by dantrolene. These results suggested that dantrolene acts on the membrane of transverse tubules and possibly the triadic junction and that it inhibits the inward movement of Ca and subsequently decreases the release of activator Ca from sarcoplasmic reticulum.

Action Potentials↗

PubMed1991

Muscle injury following experimental intraoperative irradiation.

The paraaortic region of beagle dogs was irradiated to 15 to 55 Gy intraoperative irradiation, 10 to 47.5 Gy intraoperative irradiation following 50 Gy external beam irradiation in 25 fractions, or 50 to 80 Gy external beam irradiation in 30 fractions. Six MeV electrons were used for intraoperative irradiation, and external beam irradiation was done using photons from a 6 MV linear accelerator. The psoas muscle in the irradiation field was examined histomorphometrically 2 or 5 years after irradiation. The percentage of muscle fibers and capillaries decreased, whereas the percentage of connective tissue increased with increased dose for both intraoperative irradiation only and intraoperative irradiation plus external beam irradiation. The dose causing a 50% decrease in the percentage of muscle fibers was 21.2 Gy and 33.8 Gy at 2 and 5 years, respectively, after intraoperative irradiation alone, and 22.9 Gy and 25.2 Gy at 2 and 5 years, respectively, after intraoperative irradiation combined with 50 Gy external beam irradiation. The ED50 for severe vessel lesions was 19.2 Gy and 25.8 Gy at 2 and 5 years, respectively, after intraoperative irradiation alone and 16.0 Gy and 18.0 Gy at 2 and 5 years, respectively, after intraoperative irradiation combined with 50 Gy external beam irradiation. External beam irradiation alone caused a slight decrease in percentage of muscle fibers with increased dose, and vessel lesions were infrequent or mild. Radiation-induced muscle injury was characterized by loss of muscle fibers, decreased fiber size, severe vessel lesions, hemorrhage, inflammation, coagulation necrosis, and fibrosis. These histopathologic characteristics distinguish this muscle injury from that caused by neurogenic atrophy. These data indicate that radiation-induced muscle injury most likely was caused by injury of the supporting vasculature. The lesions produced were largely a function of the single intraoperative dose rather than the external beam fractionated doses. Furthermore, it appears that 20 to 25 Gy intraoperative irradiation combined with 50 Gy external beam irradiation may be near the maximum tolerated dose by sublumbar musculature and its supporting vasculature.

Animals↗

PubMed1993

Detection and localization of triadin in rat ventricular muscle.

Dyads (transverse tubule--junctional sarcoplasmic reticulum complexes) were enriched from rat ventricle microsomes by continuous sucrose gradients. The major vesicle peak at 36% sucrose contained up to 90% of those membranes which possessed dihydropyridine (DHP) binding sites (markers for transverse tubules) and all membranes which possessed ryanodine receptors and the putative junctional foot protein (markers for junctional sarcoplasmic reticulum). In addition, the 36% sucrose peak contained half of the vesicles with muscarine receptors. Vesicles derived from the nonjunctional plasma membrane as defined by a low content of dihydropyridine binding sites per muscarine receptor and from the free sarcoplasmic reticulum as defined by the M(r) 102K Ca2+ ATPase were associated with a diffuse protein band (22-30% sucrose) in the lighter region of the gradient. These organelles were recovered in low yield. Putative dyads were not broken by French press treatment at 8,000 psi and only partially disrupted at 14,000 psi. The monoclonal antibody GE4.90 against skeletal muscle triadin, a protein which links the DHP receptor to the junctional foot protein in skeletal muscle triad junctions, cross-reacted with a protein in rat dyads of the same M(r) as triadin. Western blots of muscle microsomes from preparations which had been treated with 100 mM iodoacetamide throughout the isolation procedure showed that cardiac triadin consisted predominantly of a band of M(r) 95 kD. Higher molecular weight polymers were detectable but low in content, in contrast with the ladder of oligomeric forms in rat psoas muscle microsomes. Cardiac triadin was not dissolved from the microsomes by hypertonic salt or Triton X-100, indicating that it, as well as skeletal muscle triadin, was an integral protein of the junctional SR. The cardiac epitope was localized to the junctional SR by comparison of its distribution with that of organelle markers in both total microsome and in French press disrupted dyad preparations. Immunofluorescence localization of triadin using mAb GE4.90 revealed that intact rat ventricular muscle tissue was stained following a well-defined pattern of bands every sarcomere. This spacing of bands was consistent with the interpretation that triadin was present in the dyadic junctional regions.

Animals↗