Autolysis of the millimolar Ca2+-requiring form of the Ca2+-dependent proteinase from chicken skeletal muscle

1988 ◽  
Vol 66 (10) ◽  
pp. 1023-1031 ◽  
Author(s):  
Peter A. Nagainis ◽  
Frederick H. Wolfe ◽  
Shridhar K. Sathe ◽  
Darrel E. Goll
Keyword(s):  
Skeletal Muscle ◽  
Specific Activity ◽  
Maximal Activity ◽  
Chicken Breast ◽  
Ph Optimum ◽  
Tissue Extracts ◽  
Chicken Skeletal Muscle

The millimolar Ca2+-requiring form of the Ca2+-dependent proteinase from chicken breast skeletal muscle contains two subunit polypeptides of 80 and 28 kDa, just as the analogous forms of this proteinase from other tissues do. Incubation with Ca2+ at pH 7.5 causes rapid autolysis of the 80-kDa polypeptide to 77 kDa and of the 28-kDa polypeptide to 18 kDa. Autolysis of the 28-kDa polypeptide is slightly faster than autolysis of the 80-kDa polypeptide and is 90–95% complete after 10 s at 0 °C. Autolysis for 15 s at 0 °C converts the proteinase from a form requiring 250–300 μM Ca2+ to one requiring 9–10 μM Ca2+ for half-maximal activity, without changing its specific activity. The autolyzed proteinase has a slightly lower pH optimum (7.7 vs. 8.1) than the unautolyzed proteinase. The autolyzed proteinase is not detected in tissue extracts made immedately after death; therefore, the millimolar Ca2+-requiring proteinase is largely, if not entirely, in the unautolyzed form in situ.

scholarly journals Auto-ubiquitination of ubiquitin-activating enzymes from chicken breast muscle

1990 ◽  
Vol 267 (3) ◽  
pp. 751-757 ◽  
Author(s):  
J E Arnold ◽  
W Gevers
Keyword(s):  
Skeletal Muscle ◽  
Protease Inhibitor ◽  
Alkylating Agent ◽  
Alkali Treatment ◽  
Breast Muscle ◽  
Chicken Breast ◽  
Site Specificity ◽  
Neutral Ph ◽  
Chicken Skeletal Muscle

A soluble ubiquitin-depleted fraction from chicken skeletal muscle (fraction II), when incubated at neutral pH for several hours with 125I-ubiquitin and ATP, formed small amounts of a ubiquitin derivative (Mr 115,000) of the ubiquitin-activating enzyme E1 as well as certain similarly modified E2 species (Mr 37,000, 34,000 and 24,000). Treatment of such mixtures with NaOH during the incubations, even at early times, greatly enhanced the appearance of these entities; up to two-thirds of the thiolesters of ubiquitin bound to these proteins before alkali treatment were thus converted. The bonds involved had properties compatible with their being peptidic in nature, suggesting that auto-ubiquitination had occurred in each case. The protease inhibitor and alkylating agent tosyl-lysylchloromethane (‘TLCK’), when preincubated at 50 microM with fraction II for 2 h at 37 degrees C before the addition of 125I-ubiquitin and ATP, promoted the subsequent auto-ubiquitination of E1 and inhibited its adenylate-forming and thiolester-transferring activities. The findings have a bearing on the physiological substrate- and site-specificity of ubiquitin-conjugating reactions.

Multiple molecular forms of avian aldolases. IV. Purification and properties of chicken (Gallus domestkus) brain aldolase

1970 ◽  
Vol 48 (3) ◽  
pp. 322-333 ◽  
Author(s):  
Ronald R. Marquardt
Keyword(s):  
Specific Activity ◽  
Diffusion Constant ◽  
Sedimentation Coefficient ◽  
Breast Muscle ◽  
Molecular Forms ◽  
Chicken Breast ◽  
Rabbit Brain ◽  
Muscle Enzymes ◽  
Ph Optimum ◽  
Chicken Brain

Aldolase (fructose-1,6-diphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13) was purified from chicken (Gallus domesticus) brain tissue. The enzyme was shown to be homogeneous according to the following criteria: purification to a constant specific activity following sequential chromatography on DEAE and Sephadex, sedimentation velocity analysis, and electrophoresis on cellulose acetate strips.Several properties of the enzyme were determined including the Stokes radius (47 Å), diffusion constant (D020 w = 4.6 × 10−7 cm2/s), sedimentation coefficient (s020 w = 8.0), and molecular weight (155 000). The enzyme has a broad pH optimum centered around 7.2. The apparent Michaelis constants for fructose 1,6-diphosphate and fructose 1-phosphate were 7 × 10−5 M and 3 × 10−2 M, respectively. The activity ratio with the above two substrates was 30.Many of the molecular properties of this enzyme are similar to those of the rabbit brain enzyme and the muscle enzymes from both chickens and rabbits. The enzymic properties of chicken brain aldolase correspond more closely to those of the rabbit brain enzyme than they do to chicken breast muscle aldolase. The amino acid composition of chicken brain aldolase was found to be quite different from chicken breast muscle aldolase with respect to certain amino acids (methionine, cysteine, tryptophan, histidine, proline, aspartate, valine, and phenylalanine).

TISSUE AND SERUM CREATINE KINASE ACTIVITY IN HYPOTHYROID RATS

1968 ◽  
Vol 42 (4) ◽  
pp. 495-499 ◽  
Author(s):  
F. Q. NUTTALL
Keyword(s):  
Skeletal Muscle ◽  
Creatine Kinase ◽  
Protein Concentration ◽  
Kinase Activity ◽  
Specific Activity ◽  
Creatine Kinase Activity ◽  
Serum Creatine Kinase ◽  
Heart Activity ◽  
Total Protein Concentration ◽  
Tissue Extracts

SUMMARY In rats made hypothyroid by thyroidectomy, serum creatine kinase (CK) activity appears to be unchanged or slightly reduced. This is in contrast to the increased values reported in myxoedematous man. Brain CK activity was unchanged and heart activity was only slightly reduced in these animals. Skeletal muscle CK activity, however, was more definitely reduced and the reduction was highly significant. The lower tissue enzyme activity in heart and skeletal muscle could be explained by a reduction in total protein concentration of the tissue extracts from the thyroidectomized animals, since the specific activity of the enzyme from these tissues was essentially unchanged as compared with controls.

scholarly journals CYTOCHEMISTRY OF PHOSPHATASES OF THE SARCOPLASMIC RETICULUM

1966 ◽  
Vol 31 (3) ◽  
pp. 473-487 ◽  
Author(s):  
A. G. Engel ◽  
Lois W. Tice
Keyword(s):  
Microsomal Fraction ◽  
Specific Activity ◽  
Psoas Muscle ◽  
Thiol Group ◽  
Ph Optimum ◽  
Dependent Atpase ◽  
Synergistic Activation ◽  
Low Concentrations ◽  
Ouabain Inhibition

A microsomal fraction was isolated from rabbit psoas muscle by a modification of Muscatello's method. The fraction contained a Mg-dependent ATPase which had a pH optimum of 7.5. Activity was further stimulated by addition of Na or K or other monovalent cations to the reaction mixture, but synergistic activation by Na and K, and ouabain inhibition, could not be demonstrated. The enzyme hydrolyzed only ATP (adenosine triphosphate) and ITP (inosine triphosphate) at appreciable rates, but Na or K stimulated activity only when ATP was used as substrate. Activity was inhibited by Ca and by low concentrations of Na deoxycholate, and was sensitive to inhibition by thiol group reagents. The enzyme could be distinguished from another enzyme, also present in the fraction, which was Ca-activated, and which exhibited a wider substrate specificity, different pH activation characteristics, lower specific activity, lack of stimulation by Na or K, and less sensitivity to inhibition by deoxycholate and by thiol group reagents. These findings formed the basis for demonstration of the Mg-dependent ATPase in situ.

scholarly journals Monoclonal antibodies distinguish titins from heart and skeletal muscle.

1986 ◽  
Vol 102 (3) ◽  
pp. 1099-1108 ◽  
Author(s):  
C Hill ◽  
K Weber
Keyword(s):  
Skeletal Muscle ◽  
Monoclonal Antibodies ◽  
Immunofluorescence Microscopy ◽  
Primary Cultures ◽  
Immunoblot Analysis ◽  
Chicken Breast ◽  
Chicken Heart ◽  
Permanent Cell ◽  
Chicken Skeletal Muscle ◽  
Titin Antibodies

Murine monoclonal antibodies specific for titin have been elicited using a chicken heart muscle residue as antigen. The three antibodies T1, T3, and T4 recognize both bands of the titin doublet in immunoblot analysis on polypeptides from chicken breast muscle. In contrast, on chicken cardiac myofibrils two of the antibodies (T1, T4) react only with the upper band of the doublet indicating immunological differences between heart and skeletal muscle titin. This difference is even more pronounced for rat and mouse. Although all three antibodies react with skeletal muscle titin, T1 and T4 did not detect heart titin, whereas T3 reacts with this titin both in immunofluorescence microscopy and in immunoblots. Immunofluorescence microscopy of myofibrils and frozen tissues from a variety of vertebrates extends these results and shows that the three antibodies recognize different epitopes. All three titin antibodies decorate at the A-I junction of the myofibrils freshly prepared from chicken skeletal muscle and immunoelectron microscopy using native myosin filaments demonstrates that titin is present at the ends of the thick filaments. In chicken heart, however, antibodies T1 and T4 stain within the I-band rather than at the A-I junction. The three antibodies did not react with any of the nonmuscle tissues or permanent cell lines tested and do not decorate smooth muscle. In primary cultures of embryonic chicken skeletal muscle cells titin first appears as longitudinal striations in mononucleated myoblasts and later at the myofibrillar A-I junction of the myotubes.

Electron Probe Analysis of Muscle

1982 ◽  
Vol 40 ◽  
pp. 398-401
Author(s):  
A. V. Somlyo ◽  
H. Shuman ◽  
A. P. Somlyo
Keyword(s):  
Skeletal Muscle ◽  
Smooth Muscle ◽  
Sarcoplasmic Reticulum ◽  
Resting State ◽  
Binding Sites ◽  
Electron Probe ◽  
Frog Skeletal Muscle ◽  
Electron Probe Analysis ◽  
Probe Analysis

Electron probe analysis of frozen dried cryosections of frog skeletal muscle, rabbit vascular smooth muscle and of isolated, hyperpermeab1 e rabbit cardiac myocytes has been used to determine the composition of the cytoplasm and organelles in the resting state as well as during contraction. The concentration of elements within the organelles reflects the permeabilities of the organelle membranes to the cytoplasmic ions as well as binding sites. The measurements of [Ca] in the sarcoplasmic reticulum (SR) and mitochondria at rest and during contraction, have direct bearing on their role as release and/or storage sites for Ca in situ.

Glycogen phosphorylase in fish muscle: demonstration of three interconvertible forms

1990 ◽  
Vol 258 (2) ◽  
pp. C344-C351 ◽  
Author(s):  
H. Schmidt ◽  
G. Wegener
Keyword(s):  
Glycogen Phosphorylase ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Muscular Activity ◽  
Fish Muscle ◽  
Kinetic Properties ◽  
Phosphorylase Activity ◽  
Tissue Extracts

White skeletal muscle of crucian carp contains a single isoenzyme of glycogen phosphorylase, which was purified approximately 300-fold to a specific activity of approximately 13 mumol.min-1.mg protein-1 (assayed in the direction of glycogen breakdown at 25 degrees C). Tissue extracts of crucian muscle produced three distinct peaks of phosphorylase activity when separated on DEAE-Sephacel. Peaks 1 and 3 were identified, in terms of kinetic properties and by interconversion experiments, as phosphorylase b and a, respectively. Peak 2 was shown to be a phospho-dephospho hybrid. The three interconvertible forms of phosphorylase were purified and shown to be dimeric molecules at 20 degrees C. At 5 degrees C, a and the hybrid tended to form tetramers. The Mr of the subunit was estimated to be 96,400 from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The hybrid is kinetically homogeneous, and its kinetic properties are intermediate between those of b and a forms. The b, hybrid, and a forms of phosphorylase can be isolated from rapidly frozen muscle of crucian but in different proportions, depending on whether fish were anesthetized or forced to muscular activity for 20 s. Muscle of anesthetized crucian had 36, 36, and 28% of phosphorylase b, hybrid, and a forms, respectively, whereas the corresponding values for exercised fish were 12, 37, and 51%. Results suggest that three interconvertible forms of phosphorylase exist simultaneously in crucian muscle and that hybrid phosphorylase is active in contracting muscle in vivo.

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