Dimeric structure of rat skeletal muscle AMP-deaminase subunit

AMP-deaminase from rat skeletal muscle was purified by affinity chromatography on phosphocellulose and gel-filtration on Sephadex G-200. It was established that disulfide bridges and hydrogen bonds were not essential for stability of enzyme oligomeric structure. The dimeric structure of enzyme subunit with Mr 76 kDa (S1) was detected by means of PAGE in the presence of SDS: besides the S1 there were also exhibited two additional bands with Mr 42 (S2) and 33 (S3) kDa. Repeated SDS-PAGE of S1 has revealed the same three protein bands. These results indicate the possibility of dissociation of S1-subunit into two subunits with close Mr values.     

Phosphorylation of the skeletal muscle AMP-deaminase by protein kinase C

Protein kinase C catalyzes phosphorylation of the rat skeletal muscle AMP-deaminase in the presence of calcium ions and phosphatidylserine. At the same time, the catalytic subunit of cAMP-dependent protein kinase fails to phosphorylate AMP-deaminase. Ca2+, phosphatidylserine-dependent phosphorylation decreases three-fold (from 0.6 to 0.2 mM) the Km value and does not affect Vmax. Protein kinase C-induced phosphorylation of AMP-deaminase, besides ADP-ribosylation, is suggested to be involved in regulating the AMP-deaminase activity in vivo.     

Inhibition by alanine of AMP-deaminase from rabbit skeletal muscle.

1. Alanine inhibits rabbit muscle AMP-deaminase while aspartate, histidine and glutamate are ineffective. 2. The degree and type of inhibition of AMP-deaminase by alanine depend on pH; at pH 6.5 alanine behaves like an allosteric effector exerting a negative heterotropic effect. At pH 7.0 the inhibition is non-competitive, Ki being as high as 19 mM. 3. The probable significance of the effect of alanine on AMP-deaminase in muscle metabolism is discussed.     

Lysosomes in skeletal muscle following denervation

Summary The in-vivo uptake of exogenously applied horseradish peroxidase and the activities of the lysosomal enzymes acid phosphatase and cathepsin D were studied histochemically and/or biochemically in innervated and 2–14 day-denervated tibialis anterior muscles of the mouse. The biochemically determined uptake of horseradish peroxidase showed a large increase already 4 days after denervation. The activities of the lysosomal enzymes increased in a more gradual fashion, and only cathepsin D showed an increase in activity when expressed as total activity per muscle. Histochemically horseradish peroxidase was found to be localized in muscle fibres in characteristic spindle-shaped segments after denervation. The main increase in the number of such segments per transverse section of the muscle occurred between 3 and 6 days after denervation. In serial sections these segments frequently showed positive staining also for acid phosphatase.It is concluded that exogenously applied horseradish peroxidase is taken up into the lysosomal system, which after denervation becomes organized into characteristic spindle-shaped segments in the muscle fibres. The endocytic activity of muscle fibres increases early after denervation. This is followed by a more gradual increase in activity of lysosomal enzymes and finally by an organization of the lysosomal system into characteristic spindle-shaped segments. The results are compatible with the working hypothesis that increased endocytosis may initiate lysosomal activation in denervated skeletal muscle.     

Reduction of skeletal muscle atrophy by a proteasome inhibitor in a rat model of denervation

The ubiquitin-proteasome system is the primary proteolytic pathway implicated in skeletal muscle atrophy under catabolic conditions. Although several studies showed that proteasome inhibitors reduced proteolysis under catabolic conditions, few studies have demonstrated the ability of these inhibitors to preserve skeletal muscle mass and architecture in vivo. To explore this, we studied the effect of the proteasome inhibitor Velcade (also known as PS-341 and bortezomib) in denervated skeletal muscle in rats. Rats were given vehicle or Velcade (3 mg/kg po) daily for 7 days beginning immediately after induction of muscle atrophy by crushing the sciatic nerve. At the end of the study, the rats were euthanized and the soleus and extensor digitorum longus (EDL) muscles were harvested. In vehicle-treated rats, denervation caused a 33.5 +/- 2.8% and 16.2 +/- 2.7% decrease in the soleus and EDL muscle wet weights (% atrophy), respectively, compared to muscles from the contralateral (innervated) limb. Velcade significantly reduced denervation-induced atrophy to 17.1 +/- 3.3% in the soleus (P < 0.01), a 51.6% reduction in atrophy associated with denervation, with little effect on the EDL (9.8 +/- 3.2% atrophy). Histology showed a preservation of muscle mass and preservation of normal cellular architecture after Velcade treatment. Ubiquitin mRNA levels in denervated soleus muscle at the end of the study were significantly elevated 120 +/- 25% above sham control levels and were reduced to control levels by Velcade. In contrast, testosterone proprionate (3 mg/kg sc) did not alleviate denervation-induced skeletal muscle atrophy but did prevent castration-induced levator ani atrophy, while Velcade was without effect. These results show that proteasome inhibition attenuates denervation-induced muscle atrophy in vivo in soleus muscles. However, this mechanism may not be operative in all types of atrophy.     

Subunit structure of AMP-deaminase from chicken and rabbit skeletal muscle.

The AMP-deaminases from chicken and rabbit muscle have been investigated by techniques which include sedimentation equilibrium, sodium dodecyl sulfate gel electrophoresis, amino acid analysis, NH2- and COOH-terminal analyses, and tryptic peptide mapping. The molecular weights of the native chicken (276,000) and rabbit (271,000) enzymes obtained by sedimentation equilibrium studies are in good agreement with values of 276,000 (chicken) and 275,000 (rabbit) calculated from amino acid analyses. The enzymes were reduced, carboxymethylated, and treated with either maleic or succinic anhydride in the presence of 6 M guanidine hydrochloride. Sodium dodecyl sulfate gel electrophoresis of the chemically modified enzymes resulted in a single electrophoretic species having an apparent molecular weight of 85,000. This observation is consistent with previous studies on the nonacylated enzymes and suggests that the muscle AMP-deaminases from chicken and rabbit do not contain noncovalent linkages which are readily disrupted by a large increase in negative charge. NH2-terminal analyses by the method of Stark and Amyth as well as the dansyl technique, indicate that the NH2-terminal positions of these enzymes are blocked. The enzymes are also resistant to digestion with carboxypeptidases A or B (or both) in the presence of sodium dodecyl sulfate. The most distinctive feature of the amino acid compositions of both the chicken and rabbit AMP-deaminases is the presende of eight half-cystine residues per 69,000 g of protein. Tryptic digests of the S-14C-carboxymethylated proteins were fractionated by ion exchange chromatography and high voltage electrophoresis. Six and five radioactiviely labeled peptides were detected in the electrophoretograms of the chicken and rabbit enzymes, respectively. This observation and the number of ninhydrinposition spots, together with the physical data on the molecular weights of the native enzymes and their subunits, suggest that the AMP-deaminases from chidken and rabbit muscle consist of four identical or very similar polypeptide chains.     

Myosin polymorphism in urodelan amphibian dorsal skeletal muscle

• 1.1. Myosin isoforms were analyzed in the dorsal skeletal muscle of four urodelan amphibian species using a modified pyrophosphate gel electrophoresis which allowed better discrimination than classical methods.
• 2.2. The three fast and the intermediate isomyosins were characterized by a specific heavy chain, respectively HCf and HCi, associated with different combinations of the fast light chains LC1f, LC2f and LC3f.
• 3.3. Slow myosin was characterized by one (P. waltlii, T. palmatus, S. maculata) or two (T. alpestris) isoforms, combining a specific slow myosin heavy chain (HCs) with slow light chains only in the case of P. waltlii, or with slow and fast light chains in the other species.

     

Inositol 1,4,5-triphosphate phosphatase activity in membranes isolated from amphibian skeletal muscle [corrected

The hydrolysis of [3H]inositol 1,4,5-trisphosphate by a soluble fraction and by isolated transverse tubule and sarcoplasmic reticulum membranes from frog skeletal muscle was studied. Transverse tubule membranes displayed rates of hydrolysis several-fold higher than those of sacroplasmic reticulum and soluble fraction; Km and Vmax were 25.2 microM and 44.1 nmol/mg/min, respectively. Transverse tubule membranes sequentially hydrolyzed inositol trisphosphate to inositol bisphosphate, inositol 1-phosphate and inositol, indicating that these membranes have inositol bis- and monophosphatases in addition to inositol trisphosphatase.     

Effect of denervation on pigeon slow skeletal muscle

Summary The slow anterior latissimus dorsi muscle (ALD) of the pigeon was denervated surgically and examined after varying post-operative intervals. Muscles were studied with respect to changes in weight, histological and ultrastructural alterations, and changes in size and number of fibers. The weights of the denervated muscles increased over the contralateral control, reaching a maximum hypertrophy in the first 18 days, but the hypertrophy persisted for several months. The fibers of the denervated muscle did not hypertrophy. They showed a gradation in size from the posterior to the anterior border, with the fibers in the anterior third of the muscle being the smallest. After measuring cross-sectional sizes from the anterior, middle, and posterior thirds of the muscle, the overall fiber change was one of atrophy.Morphologically, the fibers showed various signs of pathological changes, including nuclear proliferation, swelling and migration away from the sarcolemmal position, vacuolation, myofibril degeneration, connective-tissue infiltration and replacement of the fibers, and regenerative activities in the form of budding and myoblast formation. A condition termed a peripheral rim of degeneration is described. Although many abnormal conditions were found in these denervated muscles, much of the muscle appeared normal; the neurotrophic relationship of slow muscle is discussed.This investigation was supported in part by a Public Health Service Fellowship, 2 F 2 NB 35, 582, from the National Institute of Neurological Diseases and Stroke, and by an Ohio University Research Grant to R. Hikida; and a grant 5 RO 1 AN 10856 from the National Institute of Arthritis and Metabolic Diseases to W. Bock.The authors wish to acknowledge gratefully the skillful technical assistance of Mr. Lawrence Mezza and Miss Sally Mitchell.