Isolation and Characterization of Human Skeletal Muscle Creatine Kinase

Human skeletal muscle creatine kinase was purified by isoelectric focusing with a yield of greater than 60%. Two enzymic proteins, differing in specific activity, were obtained, and each final product produced only a single protein band when examined by electrophoretic methods. The proteins were composed of two subunits of about 41, 000 daltons each, and the amino acid compositions were similar.     

Acetylcholine Synthesizing Enzymes in Frog Skeletal Muscle

Acetylcholine synthesis in homogenates of frog sartorius muscle was measured by a radiometric method with a low blank. Choline acetyltransferase activity was very low (V_max, 2 nmol g¹ h^?1, K_mfor choline, approx. 50 μ, m ). The enzyme was found only in the endplate area and disappeared after denervation; it was inactivated by 4-(1-naphthylvinyl)pyridine. At high substrate concentrations its activity was overshadowed by the acetylcholine-synthesizing activity of a different enzyme not saturated by 10 mm -choline. The non-specific enzyme was present at and away from the endplate area, and it was not affected by denervation.     

Multiple Molecular Forms of Acetylcholine Receptors in Cultured Skeletal Muscle Cells: Subcellular Localization and Characterization

Abstract: Skeletal muscle cells of newborn rats, cultured in the absence of neuronal influence, were found to contain two types of cell surface acetylcholine receptors as demonstrated by isoelectric focusing. The isoelectric points of the two types of receptors were indistinguishable from those of junctional and extrajunctional types of receptors in mature animals. The cultured cells had two classes of intracellular α-bungarotoxin (αBT) binding components; one had the same sedimentation coefficient as that of surface receptors (9S), and the other had much smaller apparent molecular weights. Only a single major component was detected by isoelectric focusing analysis of the 9s intracellular aBT binding component, with a PI value close to that of the extra junctional receptor. These results suggest that the junctional and extrajunctional types of receptors may be synthesized through a common precursor.     

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.Download video file.^{(90M, mov)}     

Isolation and Characterization of an Enriched Golgi Fraction from Neurons of Developing Rat Brains

We report a method for the isolation of enriched fractions of intact Golgi apparatus from neurons of 10- to 12-day-old rat brains. Neurons were prepared according to a modified method of Farooq and Norton [J. Neurochem. 31, 887-894 (1978)]. Golgi-enriched fractions were obtained after centrifugation of postmitochondrial supernatants in a discontinuous sucrose gradient. Golgi fractions 1 and 2, recovered at the interfaces of 28-34% and 34-36% sucrose densities, respectively, were examined with morphometric and enzymatic methods. Morphometric analyses showed that 21-34% of fraction 1 and 11-29% of fraction 2 consisted of intact Golgi apparatus. Lysosomes, mitochondria, ribosomes, and rough endoplasmic reticulum contaminated fraction 1 (6-10%) and fraction 2 (14-26%). Golgi fraction 1 showed a 25- to 65-fold enrichment over neurons of UDP Gal:GlcNAc galactosyltransferase, CMP-sialic acid:lactosylceramide sialyltransferase, and PAPS:cerebroside sulfotransferase activities. Golgi fraction 2 showed a 8- to 23-fold enrichment over neurons of the activities of the above glycolipid- and glycoprotein-synthesizing enzymes. The activities of the possible marker enzymes rotenone-insensitive NADH-cytochrome c reductase, succinate-cytochrome c reductase, and arylsulfatase were low or minimally elevated in the Golgi fractions. A sevenfold enrichment of Na+, K+-ATPase activities was found in the Golgi fractions. This is consistent either with significant plasma membrane contamination or with the presence of this enzyme in the neuronal Golgi apparatus.     

Isolation of two Proteolipids from Rabbit Skeletal Muscle Sarcoplasmic Reticulum

We have isolated two proteolipids from rabbit skeletal muscle sarcoplasmic reticulum by chromatography on columns of Sepharose CL-6B and Sephadex LH-60. One, PL-II, is identical to the proteo-lipid previously obtained by others using organic solvent extraction. The other, PL-I, has an amino acid composition very similar to those of proteolipids we previously isolated from canine cardiac SR and lamb kidney (Na, K)-ATPase.     

Solubilization and Partial Purification of a Presynaptic Membrane Protein Ensuring Calcium-Dependent Acetylcholine Release from Proteoliposomes

In previous work, it was shown that cytoplasmic acetylcholine decreased on stimulation of Torpedo electric organ or synaptosomes in a strictly calcium-dependent manner. This led to the hypothesis that the presynaptic membrane contained an element translocating acetylcholine when activated by calcium. To test this hypothesis, the presynaptic membrane constituents were incorporated into the membranes of liposomes filled with acetylcholine. The proteoliposomes thus obtained released the transmitter in response to a calcium influx. The kinetics and calcium dependency of acetylcholine release were comparable for proteoliposomes and synaptosomes. The presynaptic membrane element ensuring calcium-dependent acetylcholine release is most probably a protein, since it was susceptible to Pronase, but only when the protease had access to the intracellular face of the presynaptic membrane. Postsynaptic membrane fractions contained very low amounts of this protein. It was extracted from the presynaptic membrane under alkaline conditions in the form of a protein-lipid complex of large size and low density which was partially purified. The specificity of the calcium-dependent release for acetylcholine was tested with proteoliposomes filled with equal amounts of acetylcholine and choline or acetylcholine and ATP. In both cases, acetylcholine was released preferentially. After cholate solubilization and gel filtration, the protein ensuring the calcium-dependent acetylcholine release was recovered at a high apparent molecular weight (between 600,000 and 200,000 daltons), its apparent sedimentation coefficient being 17S after cholate elimination. This protein is probably an essential coin of the transmitter release mechanism. We propose to name it mediatophore.     

Purification and Properties of an Aminopeptidase from Rabbit Skeletal Muscle

An aminopeptidase active on l-Val-l-Val-l-Val-l-Ala was purified from rabbit skeletal muscle by the method including ammonium sulfate precipitation, DEAE-cellulose chromatography, gel-filtration on Sephadex G–200, rechromatography on DEAE-cellulose, hydroxylapatite chromatography and rechromatography on Sephadex G–200. Polyacrylamide gel disc electrophoresis showed that the enzyme thus obtained was homogeneous. The specific activity of the purified enzyme was 1500 times that of the original muscle extract. The enzyme had an optimal pH in a range of 6.0~7.6 and was stable in pH 6.1~8.1. Molecular weight of the enzyme was estimated to be 160,000 from the result of gel-filtration on Sephadex G–200. The enzyme showed specificity for tri-, tetra-, penta-, and hexapeptides. The analytical data of liberated amino acids showed that the enzyme was an aminopeptidase active on these oligopeptides. The enzyme was strongly inhibited by N-ethyl-maleimide and EDTA.     

Acetylcholine and Calcium on Membrane Permeability and Contraction of Intestinal Smooth Muscle

Acetylcholine elicited a sustained contraction and an increase in potassium efflux in longitudinal muscle isolated from the guinea pig ileum. Stepwise increases in the calcium concentration of the bathing medium, from 0.06 to 36 mM generally reduced the increase in potassium efflux, but had a complex effect on the mechanical response. Contractions produced by high levels of acetylcholine became progressively larger or remained at a high magnitude as the calcium concentration was increased. Contractions produced by low levels of acetylcholine also improved initially, but were depressed again by the highest concentration of calcium introduced. Ethanol, in the appropriate concentration, inhibited completely the acetylcholine-induced contraction without reducing the increase in potassium efflux. Calcium reversed this effect. Both extracellular calcium and ethanol depressed the large, transient increase in muscle tone developed by fibers that were preincubated in a high calcium medium and then exposed to a calcium-free medium. These findings suggested that extracellular calcium ions react with two different sites in the membrane, a stabilizing site and a storage site. A muscle contraction is activated by calcium ions which diffuse from the storage site to the myoplasm. Calcium ions reacting with the stabilizing site impede this diffusion process. Part of the stimulatory effect of acetylcholine is derived from its capacity to counteract the action of calcium at the stabilizing site.     

Redistribution of Terbium Ions Across Acetylcholine Receptor-Enriched Membranes Induced by Agonist Desensitization

Using small-angle x-ray diffraction from centrifugally oriented acetylcholine receptor (AChR) enriched membranes coupled with anomalous scattering from terbium ions (Tb³⁺) titrated into presumed Ca²⁺ binding sites, we have mapped the distribution of Tb³⁺ perpendicular to the membrane plane using a heavy atom refinement algorithm. We have compared the distribution of Tb³⁺ in the closed resting state with that in the carbamylcholine-desensitized state. In the closed resting state we find 45 Tb³⁺ ions distributed in 10 narrow peaks perpendicular to the membrane plane. Applying the same refinement procedure to the data from carbamylcholine desensitized AChR we find 18 fewer Tb³⁺ ions in eight peaks, and slight rearrangements of Tb³⁺ density in the peaks near the ends of the AChR ion channel pore. These agonist dependent changes in the Tb³⁺ stoichiometry and distribution suggest a likely role for multivalent cations in stabilizing the different functional states of the AChR, and the changes in the Tb³⁺ distribution at the two ends of the pore suggest a potential role for multivalent cations in the gating of the ion channel.     

Nerve Stump Length and Membrane Changes in Denervated Skeletal Muscle

THE effect of nerve stump length on the rate of failure of neuromuscular transmission and on the degeneration of the neuromuscular apparatus has been studied in some detail^1–4 and there is general agreement that neuromuscular transmission fails and the nerve terminals degenerate more rapidly if the nerve is cut close to the muscle than if it is cut far away.     

Further Characterization of Aminopeptidase of Rabbit Skeletal Muscle

Further investigation on characterization was conducted on purified neutral aminopeptidase of 160,000 daltons from rabbit skeletal muscle. The enzyme possesses arylamidase activity. The greater part of leucine-β-naphthylamide hydrolyzing activity of the muscle extract was attributed to the enzyme. The Km value for Ala-Gly-Phe-Ala, the most cleavable substrate tested, was 0.25 mm. Substrate inhibition was observed for Val-Val-Val-Ala and Val-Val-Val. The enzyme was inhibited by puromycin in a non-competitive manner, Ki being 4 × 10^?6 m. The enzyme was also inhibited by insulin and the oxidized B-chain of insulin. The tetrapeptide with N-terminal residue of d configuration, tRNA, pyruvate and α-ketoglutarate had no effect on the enzyme. On the basis of all properties determined so far, this muscle aminopeptidase is concluded to be identical to none of the known aminopeptidases from other tissues.     

Isolation and Partial Characterization of Transfer RNAs from Astragalus bisulcatus

A procedure has been developed for the isolation of transfer RNA from the selenium accumulator plant Astragalus bisulcatus. This material appears free of interfering phenolic compounds, has a high guanosine to cytidine ratio, shows a major and modified nucleoside composition characteristic of plant transfer RNAs, and exhibits chromatographic and electrophoretic properties similar to transfer RNAs from other well studied bacterial and plant systems. RNAs isolated from A. bisulcatus seedlings incubated in the presence of ⁷⁵Se indicate some incorporation of radioactivity into the transfer RNAs, but at extremely low levels. The transfer RNAs were active in accepting amino acids, although their over-all levels of activity appeared low when compared with those from a homologous Escherichia coli aminoacylation reaction system.     

Isolation and Partial Characterization of a Mycotoxin from Penicillium roqueforti

Extracts of pure cultures of Penicillium roqueforti isolated from toxic feed samples and of P. roqueforti NRRL 849 were lethal to rats by either intraperitoneal or oral administration. Purification studies guided by this test led to the isolation of a major toxin which showed intraperitoneal and oral median lethal dose values in weanling rats of 11 and 115 mg/kg, respectively. Partial characterization of the crystalline compound, C(17)H(20)O(6), by infrared, ultra violet, PMR, and mass spectroscopy, and by several chemical transformations indicated the presence of three C-methyl substituents plus one acetoxy, one aldehyde, and one alpha,beta-unsaturated ketone group. Two oxygen atoms are present either in epoxide or ether form.     

Purification and Characterization of a Cl−-Activated Aminopeptidase from Bovine Skeletal Muscle

To elucidate the mechanisms involved in the increase in free amino acids during postmortem storage of meat, a novel aminopeptidase was purified from bovine skeletal muscle by ammonium sulfate fractionation and successive chromatographies such as DEAE-cellulose, Sephacryl S-200, Hydroxyapatite, Phenyl-Sepharose, and Hi-Trap affinity column chromatography. The molecular mass of the enzyme was found to be 58 kDa on SDS–PAGE. This enzyme had optimum pH at around 7.5, and preferably hydrolyzed Ala-β-naphthylamide (-NA) in amino acid-NAs. The activity was strongly inhibited by phenylmethansulfonyl fluoride (PMSF) and bestatin, suggesting that it is to be classified as a serine protease. Moreover, the activity was enhanced by chloride and nitrate ions, which is the most remarkable property of this enzyme. The enzyme appeared to be involved in the increase in free amino acids during postmortem storage of meat.     

Isolation and Characterization of an Outer Membrane Protein of Chlorobium tepidum

A protein was isolated from membranes of the green sulfur bacterium Chlorobium tepidum. This protein was characterized by gel electrophoresis, gel filtration, analytical ultracentrifugation and amino acid sequencing. The molecular weight of the purified protein was shown to be 26 kDa by SDS-PAGE. HPLC gelfiltration, SDS-PAGE and analytical ultracentrifugation are consistent with the presence of a homogenous protein in the preparations. Amino acid analysis was obtained from the isolated protein after fragmentation with Lys-C, trypsin and cyanogen bromide. The cleavage pattern resulting from these treatments combined with Edman sequencing yield a sequence allowing the identification of an integral membrane agglutinin in Chl. tepidum.     

Characterization of the Calcium-release Channel/Ryanodine Receptor from Zebrafish Skeletal Muscle

Calcium (Ca²⁺)-mediated signaling is fueled by two sources for Ca²⁺: Ca²⁺ can enter through Ca²⁺ channels located in the plasma membrane and can also be released from intracellular stores. In the present study the intracellular Ca²⁺ release channel/ryanodine receptor (RyR) from zebrafish skeletal muscle was characterized. Two RyR isoforms could be identified using immunoblotting and single-channel recordings. Biophysical properties as well as the regulation by modulators of RyR, ryanodine, ruthenium red and caffeine, were measured. Comparison with other RyRs showed that the zebrafish RyRs have features observed with all RyRs described to date and thus, can serve as a model system in future genetic and physiological studies. However, some differences in the biophysical properties were observed. The slope conductance for both isoforms was higher than that of the mammalian RyR type 1 (RyR1) measured with divalent ions. Also, inhibition by millimolar Ca²⁺ concentrations of the RyR isoform that is inhibited by high Ca²⁺ concentrations (teleost α RyR isoform) was attenuated when compared to mammalian RyRs. Due to the widespread expression of RyR these findings have important implications for the interpretation of the role of the RyR in Ca²⁺ signaling when comparing zebrafish with mammalian physiology, especially when analyzing mutations underlying physiological changes in zebrafish. Received: 15 February 2001/Revised: 1 June 2001     

Host-Pathogen Interactions: XIV. Isolation and Partial Characterization of an Elicitor from Yeast Extract

An elicitor of glyceollin accumulation in soybeans (Glycine max L.) has been isolated from a commercially available extract of brewers' yeast. Yeast is not a known pathogen of plants. The elicitor was isolated by precipitation in 80% (v/v) ethanol followed by column chromatography on DEAE-cellulose, sulfopropyl-Sephadex, and concanavalin A-Sepharose. Compositional and structural analysis showed the elicitor to be a glucan containing terminal, 3-, 6-, and 3,6-linked glucosyl residues. The yeast elicitor stimulates the accumulation of glyceollin in the cotyledons and hypocotyls of soybeans when as little as 15 nanograms or 100 nanograms of the elicitor is applied to the respective tissues. The yeast elicitor is very similar in both structure and absolute elicitor activity to an elicitor isolated from the mycelial walls of Phytophthora megasperma var. sojae, a pathogen of soybeans. These and other results of this laboratory suggest that plants are able to respond to the presence of a wide range of fungi by recognizing, as foreign to the plant, structural polysaccharides of the mycelial walls of the fungi.