scholarly journals The identification and properties of phosphatases in skeletal muscle with activity towards the inhibitory subunit of troponin, and their relationship to other phosphoprotein phosphatases

1976 ◽  
Vol 157 (2) ◽  
pp. 369-380 ◽  
Author(s):  
K P Ray ◽  
P J England
Keyword(s):  
Skeletal Muscle ◽  
Troponin I ◽  
Polyacrylamide Gel Electrophoresis ◽  
Rat Skeletal Muscle ◽  
Histone Fraction ◽  
Bivalent Metal ◽  
Phosphoprotein Phosphatases ◽  
Bivalent Metal Ions ◽  
Inhibitory Subunit Of Troponin ◽  
Minor Activity

1. Phosphoprotein phosphatases with activity towards the inhibitory subunit of troponin (troponin I), phosphorylase a and lysine-rich histone (fraction F1) have been fractionated from rat skeletal muscle by chromatography on Sephadex G-200 and polylysine-Sepharose. Six separate fractions were identified on the basis of substrate specificity and behaviour during chromatography. 2. All fractions showed similar Km values for any given protein substrate. The Km for troponin I (5 muM) was significantly lower than that previously reported. 3. Phosphatase activities towards troponin I and hosphorylase a did not show a requirement for bivalent-metal ions. Two of the fractions with only minor activity towards histone were activated by Mn2+. 4. Discontinuous polyacrylamide-gel-electrophoresis studies indicated that several of the fractions contained more than one phosphatase activity, and additionally showed that several of the activities could exist in different aggregation states. On the basis of these studies at least two phosphatases with activity only towards troponin I were identified. In addition, phosphorylase phosphatase (which has considerable activity towards troponin I) and a general phosphatase with activity towards all three substrates were found. 5. A fraction with mol.wt. of 150000 could be activated by freezing with 2-mercaptoethanol or by heating to 55 degrees C. This activation was accompanied by a decrease in mol.wt. to 25000. 6. The total amount of phosphatase with activity towards troponin I which was extracted would be sufficient to dephosphorylate all the troponin I present in skeletal muscle in approximately 10s.

Effect of denervation on the content of cardiac troponin-T and cardiac troponin-I in rat skeletal muscle

2007 ◽  
Vol 40 (5-6) ◽  
pp. 423-426 ◽  
Author(s):  
Salim Fredericks ◽  
Hans Degens ◽  
Godfrina McKoy ◽  
Katie Bainbridge ◽  
Paul O. Collinson ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Cardiac Troponin ◽  
Troponin I ◽  
Troponin T ◽  
Cardiac Troponin I ◽  
Cardiac Troponin T ◽  
Rat Skeletal Muscle

scholarly journals Purification and some physico-chemical and enzymic properties of a calcium ion-activated neutral proteinase from rabbit skeletal muscle

1979 ◽  
Vol 183 (2) ◽  
pp. 339-347 ◽  
Author(s):  
Jean-Louis Azanza ◽  
Jacques Raymond ◽  
Jean-Michel Robin ◽  
Patrick Cottin ◽  
André Ducastaing
Keyword(s):  
Skeletal Muscle ◽  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Troponin I ◽  
Proteinase Inhibitors ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Rabbit Skeletal Muscle ◽  
Neutral Proteinase

Ca2+-activated neutral proteinase was purified from rabbit skeletal muscle by a method involving DEAE-Sephacel chromatography, affinity chromatography on organomercurial–Sepharose and gel filtration on Sephacryl S-200 and Sephadex G-150. The SDS (sodium dodecyl sulphate)/polyacrylamide-gel-electrophoresis data show that the purified enzyme contains only one polypeptide chain of mol.wt. 73000. The purification procedure used allowed us to eliminate a contaminant containing two components of mol.wt. about 30000 each. Whole casein or α1-casein were hydrolysed with a maximum rate at 30°C, pH7.5, and with 5mm-CaCl2, but myofibrils were found to be a very susceptible substrate for this proteinase. This activity is associated with the destruction of the Z-discs, which is caused by the solubilization of the Z-line proteins. The activity of the proteinase in vitro is not limited to the removal of Z-line. SDS/polyacrylamide-gel electrophoresis on larger plates showed the ability of the proteinase to degrade myofibrils more extensively than previously supposed. This proteolysis resulted in the production of a 30000-dalton component as well as in various other higher- and lower-molecular-weight peptide fragments. Troponin T, troponin I, α-tropomyosin, some high-molecular-weight proteins (M protein, heavy chain of myosin) and three unidentified proteins are degraded. Thus the number of proteinase-sensitive regions in the myofibrils is greater than as previously reported by Dayton, Goll, Zeece, Robson & Reville [(1976) Biochemistry15, 2150–2158]. The Ca2+-activated neutral proteinase is not a chymotrypsin- or trypsin-like enzyme, but it reacted with all the classic thiol-proteinase inhibitors for cathepsin B, papain, bromelain and ficin. Thus the proteinase was proved to have an essential thiol group. Antipain and leupeptin are also inhibitors of the Ca2+-activated neutral proteinase.

Rat skeletal muscle phosphorylase kinase: turnover and control of isozyme levels in culture

1986 ◽  
Vol 250 (3) ◽  
pp. C365-C373 ◽  
Author(s):  
W. J. Salsgiver ◽  
J. C. Lawrence
Keyword(s):  
Skeletal Muscle ◽  
Kinase Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Proteolytic Processing ◽  
Alpha Subunit ◽  
Phosphorylase Kinase ◽  
Ionophore A23187 ◽  
Rat Skeletal Muscle ◽  
Alpha Subunits

The expression of phosphorylase kinase was investigated in rat skeletal muscle cells developing in vitro. The enzyme was immunoprecipitated from cells cultured in the presence of [35S]methionine, and the 35S-labeled alpha-, alpha'-, and beta-subunits of the kinase were resolved by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Fusion of myoblasts into myotubes was associated with marked increases in the amounts of kinase activity and the three 35S-labeled subunits. In 2-wk-old myotubes, the net amount of alpha'-subunit represented less than 20% of the total alpha-subunits (alpha + alpha'); however, alpha'-subunits appeared to be synthesized at least as rapidly as alpha-subunits. That alpha'-subunits were degraded more rapidly was confirmed by pulse-chase experiments, which also indicated that alpha'-subunits were not formed by proteolytic processing of the larger alpha-subunit. Inhibition of the spontaneous contractile activity of the myotubes with lidocaine markedly increased both phosphorylase kinase activity and the amounts of the 35S-labeled subunits. The divalent cation ionophore, A23187, decreased the alpha-subunits by 60%, but did not change levels of the alpha'-subunits. Taken together, the present results indicate that rat myotubes synthesize the two isozymes of phosphorylase kinase, and that levels of both are controlled by differentiation and muscle activity.

Calmodulin Binding Proteins In Platelet Actomypsin

1981 ◽  
Author(s):  
L Muszbek ◽  
J Harsfalvi
Keyword(s):  
Skeletal Muscle ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Binding Proteins ◽  
Troponin I ◽  
Polyacrylamide Gel Electrophoresis ◽  
Regulatory System ◽  
Inhibitory Protein ◽  
Calmodulin Binding ◽  
Actomyosin Complex

Platelet actomyosin (thrombosthenin) possesses a myosin-linked Ca2+ regulation and Ca2+ sensitivity is conferred to it by calmodulin through myosin light chain kinase. Calmodulin binding proteins if they are present in the actomyosin complex may have an important regulatory role in the contractile mechanism of platelet activation. To test this possibility an aceton powder was made from platelet actomyosin and extracted with an 8 M urea containing buffer. The extract was examined for the presence of calmodulin binding proteins by alkaline urea polyacrylamide gel electrophoresis. It was shown by this technique that some proteins in the actomyosin complex can form a Ca2+ dependent complex with both calmodulin and skeletal muscle troponin C (TNC is closely related to calmodulin) even in the presence of 8 M urea. Calmodulin binding proteins could be isolated from the extract by affinity chromatography in 8 M urea on TNC-Agarose column. 3 major proteins of 270 K, 6l K and 23 K molecular weight were eluted by EGTA and each of them was able to bind to calmodulin or TNC if Ca2+was present. At least one of these calmodulin binding proteins exerted a troponin I like effect when tested on reconstituted skeletal muscle actomyosin and the 23 K protein showed a close similarity to troponin I. the inhibitory protein of the actin linked Ca2+ regulatory system in skeletal muscle. It is presumed that calmodulin binding proteins may have a dual role in the regulation of platelet actomyosin. They can inhibit the Ca2+ dependent phosphorylation of myosin light chain and one or more of them may also exert an actin linked inhibitory effect.

scholarly journals Characterization of jack-bean α-D-mannosidase as a zinc metalloenzyme

1975 ◽  
Vol 147 (1) ◽  
pp. 83-90 ◽  
Author(s):  
S M Snaith
Keyword(s):  
Gel Electrophoresis ◽  
Metal Ion ◽  
Chelating Agents ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Chromatography ◽  
Enzyme Molecule ◽  
Bivalent Metal ◽  
Jack Bean ◽  
Bivalent Metal Ions

1. Two methods were used to obtain α-mannosidase free from unbound Zn2+, (a) by removal of excess of metal ion from preparations purified in the presence of Zn2+ and (b) by purification under conditions that eliminate the need to add Zn2+. 2. The purified enzyme is homogeneous on ultracentrifugation, polyacrylamide-gel electrophoresis and gel chromatography. 3. The molecular weight is estimated to be 230 000. 4. The enzyme contains between 470 and 565 mug of zinc/g of protein, corresponding to between 1.7 and 2 atoms of zinc/enzyme molecule. The contents of other metals are much lower. 5. The enzyme is inactivated by chelating agents and activity is restored by Zn2+. 6. No other metal ion was found to replace Zn2+ with retention of activity. Some bivalent metal ions, e.g. Cu2+, rapidly inactivate the enzyme. 7. The results indicate that jack-bean α-mannosidase exists naturally as a zinc-protein complex and may be considered as a metalloenzyme.

scholarly journals Fast and Slow Skeletal Troponin I in Serum from Patients with Various Skeletal Muscle Disorders: A Pilot Study

2005 ◽  
Vol 51 (6) ◽  
pp. 966-972 ◽  
Author(s):  
Jeremy A Simpson ◽  
Ralf Labugger ◽  
Christine Collier ◽  
Robert J Brison ◽  
Steve Iscoe ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Troponin I ◽  
Serum Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Fiber Types ◽  
Muscle Injuries ◽  
Serum Samples ◽  
Muscle Disorders ◽  
Skeletal Muscle Disorders

Abstract Background: Detection of skeletal muscle injury is hampered by a lack of commercially available assays for serum markers specific for skeletal muscle; serum concentrations of skeletal troponin I (sTnI) could meet this need. Moreover, because sTnI exists in 2 isoforms, slow (ssTnI) and fast (fsTnI), corresponding to slow- and fast-twitch muscles, respectively, it could provide insight into differential injury/recovery of specific fiber types. The purpose of this study was to investigate whether the 2 isoforms of sTnI and their modified forms are present in the blood of patients with various skeletal muscle disorders. Methods: Serial serum samples were obtained from 25 patients with various skeletal muscle injuries. Serum proteins were separated by a modified sodium dodecyl sulfate–polyacrylamide gel electrophoresis protocol followed by Western blotting for sTnI with monoclonal antibodies specific to ssTnI and fsTnI. Results: We observed (a) intact and, in some cases, degraded sTnI products; (b) evidence of posttranslational modifications in addition to proteolysis; and (c) differential detectability of both skeletal isoforms in the same patient. Conclusions: It is possible to monitor both sTnI isoforms; this could lead to the development of new diagnostic assays for skeletal muscle damage.

scholarly journals Purification of adenylosuccinate lyase from rat skeletal muscle by a novel affinity column. Stabilization of the enzyme, and effects of anions and fluoro analogues of the substrate

1987 ◽  
Vol 246 (2) ◽  
pp. 263-269 ◽  
Author(s):  
P J Casey ◽  
J M Lowenstein
Keyword(s):  
Skeletal Muscle ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Affinity Column ◽  
Rat Skeletal Muscle ◽  
Adenylosuccinate Lyase ◽  
Apparent Homogeneity ◽  
High Concentrations

Adenylosuccinate lyase from rat skeletal muscle was purified to apparent homogeneity by a combination of ion-exchange chromatography and affinity chromatography on agarose containing covalently bound adenylophosphonopropionate. The purified enzyme is stable when stored in 20% glycerol at −70 degrees C, and can be thawed and re-frozen with minimal loss of activity. Adenylosuccinate lyase has a specific activity of 11 mumol/min per mg of protein at 25 degrees C. Its subunit Mr is 52,000, by SDS/polyacrylamide-gel electrophoresis, and its apparent native Mr is approx. 200,000, by gel filtration. The purified enzyme has Km values for adenylosuccinate and 4-(N-succino)-5-aminoimidazole-4-carboxamide ribonucleotide (SAICAR) of 1.5 microM and approximately 1 microM respectively, in Hepes/KOH buffer, pH 7.4. Several monoanions and dianions activate the enzyme at low concentration; several of these inhibit the enzyme at high concentrations. Fluoro analogues of adenylosuccinate and SAICAR were synthesized by using highly purified adenylosuccinate synthase and SAICAR synthase respectively, and erythro-beta-fluoroaspartate in place of aspartate. Both analogues are competitive inhibitors of adenylosuccinate lyase in both of the reactions catalysed by the enzyme, with Ki values well below the Km values for the two substrates.

scholarly journals Ca2+ Regulation of Bovine Thrombosthenin

1977 ◽  
Author(s):  
L. Muszbek ◽  
J. Kuźnicki ◽  
W. Drabikowski
Keyword(s):  
Skeletal Muscle ◽  
Gel Electrophoresis ◽  
Troponin I ◽  
Polyacrylamide Gel Electrophoresis ◽  
Regulatory Proteins ◽  
Skeletal Muscle Myosin ◽  
Myosin Binding ◽  
Moving Band ◽  
Low Ionic Strength ◽  
Muscle Myosin

In order to reveal the type of its Ca2+-regulation bovine thrombosthenin - natural platelet actomyosin - was investigated by competitive actin and myosin binding assay and by urea gel electrophoresis. The Ca2+-sensitivity of low ionic strength Mg-ATPase activity of platelet actomyosin was not influenced by the addition of excess skeletal muscle actin free of regulatory proteins. In contrast, the replacement of platelet myosin by skeletal muscle myosin resulted in a hybrid actomyosin insensitive to Ca2+. If actomyosin was reconstructed from crude platelet actin and skeletal muscle myosin again no regulatory effect of Ca2+ could be observed. In the presence of EGTA a fast moving band with the mobility corresponding to muscle troponin C (TN-C) was detected by alkaline urea Polyacrylamide gel electrophoresis. However, if Ca2+ was added this protein, unlike TN-C, neither changed its mobility nor formed a complex even if muscle troponin I was included into the system. The experimental results indicate that our Ca2+ sensitive bovine thrombosthenin preparation does not contain TN-C like component and is not regulated by an actin -, but rather by a myosin-connected system.

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