Early over-expression of low-affinity [3H]ryanodine receptor sites in heavy sarcoplasmic reticulum fraction from dystrophic chicken pectoralis major

Isaac N Pessah, Mary J. Schiedt

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Abstract

Heavy sacroplasmic reticulum (SR membranes enriched in [3H]ryanodine receptor have been isolated from pectoralis major (PM) of normal line 412 and dystrophic line 413 chickens paired at various stages during post-hatch development. Normal PM 2 days ex ovo yields 17% lower protein recovery in the heavy SR subfractions compared to preparations from paired dystophic PM (0.80 vs 0.96% mg/g PM, respectively). By 2 weeks ex ovo, protein recovery in normal SR subfractions decreases over 3-fold to 0.24 mg/g PM, whereas yields from dystrophic PM increase to 1.67 mg/g PM. Dystrophic preparations consistently give 7-9-fold higher recoveries of protein in heavy SR subfractions compared to normal PM when examined at 2, 4 and 5.5 weeks ex ovo. [3H]ryanodine binding to normal SR from PM 2 days ex novo exhibits nonlinear Scatchard plots which resolve into high-(Kd,app = 18 nM; Bmax - 17 pmol /mg protein) and low-(Kd,app = 532 nM; Bmax = 2.6 pmol/mg protein) affinity binding sites, whereas dystrophic preparations exhibit only high-affinity [3H]ryanodine binding sites (Kd,app = 31 nM; Bmax = 2.1 pmol/mg protein). Both normal and dystrophic PM have similar capacities to bind [3H]ryanodine (2.6 vs. 2.0 pmol/g PM, respectively) at 2 days ex ovo. However, at 2, 4, and 5.5 weeks ex ovo the density of high-affinity [3H]ryanodine binding sites in normal SR drops dramatically to 3.5, 1.2, and 0.4 pmol/mg protein, respetively, and corresponds with disappearance of the low-affinity binding sites. In marked contrast, heavy SR membranes from distrophic PM 2, 4, and 5.5 weeks ex ovo, maintain their high-affinity binding sites for [3H]ryanodine and exhibit high densities of low-affinity binding sites (Kd,app = 725-4500 nM; Bmax = 15.4-25.1 pmol/mg protein). Early developmental over-expression of [3H]ryanodine binding capacity in dystrophic PM ranges from 34-fold to 388-fold that of normal PM at 2 weeks and 5.5 weeks, respectively, and correspond to the intensity with which high molecular weight doublets of Mr 340 000 and 320 000 stain on SDS-PAGE. Low-affinity [3H]ryanodine binding sites of dystrophic SR exhibit 4-6-fold higher sensitivity to activation by Ca2+ and altered sensitivity to activation by caffein and adenine nucleotides. These results demonstrate that over-expression of junctional SR and [3H]ryanodine receptors having altered radioligand binding properties is a very early event in the post-hatch development of dystrophic PM. Since the [3H]ryanodine receptor is a specific marker for the SR Ca2+ release channel at the muscle triad and a key component of excitation-contraction coupling, abnormal expression of [3H]ryanodine receptors may be of a fundamental importance to the etiology of muscular dystrophy in the chicken.

Original languageEnglish (US)
Pages (from-to)98-106
Number of pages9
JournalBBA - Biomembranes
Volume1023
Issue number1
DOIs
StatePublished - Mar 30 1990

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Ryanodine
Ryanodine Receptor Calcium Release Channel
Sarcoplasmic Reticulum
Chickens
Binding Sites
Application programs
Proteins
Hatches
Recovery
Chemical activation
Excitation Contraction Coupling
Reticulum
Membranes
Adenine Nucleotides
Muscular Dystrophies
Caffeine
Protein Binding
Polyacrylamide Gel Electrophoresis
Coloring Agents
Muscle

Keywords

  • Muscle development
  • Muscular dystrophy
  • Ryanodine receptor
  • Sarcoplasmic reticuluml
  • Skeletal muscle

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Cell Biology

Cite this

@article{882a1746cfef4b459f9c3af91d540138,
title = "Early over-expression of low-affinity [3H]ryanodine receptor sites in heavy sarcoplasmic reticulum fraction from dystrophic chicken pectoralis major",
abstract = "Heavy sacroplasmic reticulum (SR membranes enriched in [3H]ryanodine receptor have been isolated from pectoralis major (PM) of normal line 412 and dystrophic line 413 chickens paired at various stages during post-hatch development. Normal PM 2 days ex ovo yields 17{\%} lower protein recovery in the heavy SR subfractions compared to preparations from paired dystophic PM (0.80 vs 0.96{\%} mg/g PM, respectively). By 2 weeks ex ovo, protein recovery in normal SR subfractions decreases over 3-fold to 0.24 mg/g PM, whereas yields from dystrophic PM increase to 1.67 mg/g PM. Dystrophic preparations consistently give 7-9-fold higher recoveries of protein in heavy SR subfractions compared to normal PM when examined at 2, 4 and 5.5 weeks ex ovo. [3H]ryanodine binding to normal SR from PM 2 days ex novo exhibits nonlinear Scatchard plots which resolve into high-(Kd,app = 18 nM; Bmax - 17 pmol /mg protein) and low-(Kd,app = 532 nM; Bmax = 2.6 pmol/mg protein) affinity binding sites, whereas dystrophic preparations exhibit only high-affinity [3H]ryanodine binding sites (Kd,app = 31 nM; Bmax = 2.1 pmol/mg protein). Both normal and dystrophic PM have similar capacities to bind [3H]ryanodine (2.6 vs. 2.0 pmol/g PM, respectively) at 2 days ex ovo. However, at 2, 4, and 5.5 weeks ex ovo the density of high-affinity [3H]ryanodine binding sites in normal SR drops dramatically to 3.5, 1.2, and 0.4 pmol/mg protein, respetively, and corresponds with disappearance of the low-affinity binding sites. In marked contrast, heavy SR membranes from distrophic PM 2, 4, and 5.5 weeks ex ovo, maintain their high-affinity binding sites for [3H]ryanodine and exhibit high densities of low-affinity binding sites (Kd,app = 725-4500 nM; Bmax = 15.4-25.1 pmol/mg protein). Early developmental over-expression of [3H]ryanodine binding capacity in dystrophic PM ranges from 34-fold to 388-fold that of normal PM at 2 weeks and 5.5 weeks, respectively, and correspond to the intensity with which high molecular weight doublets of Mr 340 000 and 320 000 stain on SDS-PAGE. Low-affinity [3H]ryanodine binding sites of dystrophic SR exhibit 4-6-fold higher sensitivity to activation by Ca2+ and altered sensitivity to activation by caffein and adenine nucleotides. These results demonstrate that over-expression of junctional SR and [3H]ryanodine receptors having altered radioligand binding properties is a very early event in the post-hatch development of dystrophic PM. Since the [3H]ryanodine receptor is a specific marker for the SR Ca2+ release channel at the muscle triad and a key component of excitation-contraction coupling, abnormal expression of [3H]ryanodine receptors may be of a fundamental importance to the etiology of muscular dystrophy in the chicken.",
keywords = "Muscle development, Muscular dystrophy, Ryanodine receptor, Sarcoplasmic reticuluml, Skeletal muscle",
author = "Pessah, {Isaac N} and Schiedt, {Mary J.}",
year = "1990",
month = "3",
day = "30",
doi = "10.1016/0005-2736(90)90014-F",
language = "English (US)",
volume = "1023",
pages = "98--106",
journal = "Biochimica et Biophysica Acta - Biomembranes",
issn = "0005-2736",
publisher = "Elsevier",
number = "1",

}

TY - JOUR

T1 - Early over-expression of low-affinity [3H]ryanodine receptor sites in heavy sarcoplasmic reticulum fraction from dystrophic chicken pectoralis major

AU - Pessah, Isaac N

AU - Schiedt, Mary J.

PY - 1990/3/30

Y1 - 1990/3/30

N2 - Heavy sacroplasmic reticulum (SR membranes enriched in [3H]ryanodine receptor have been isolated from pectoralis major (PM) of normal line 412 and dystrophic line 413 chickens paired at various stages during post-hatch development. Normal PM 2 days ex ovo yields 17% lower protein recovery in the heavy SR subfractions compared to preparations from paired dystophic PM (0.80 vs 0.96% mg/g PM, respectively). By 2 weeks ex ovo, protein recovery in normal SR subfractions decreases over 3-fold to 0.24 mg/g PM, whereas yields from dystrophic PM increase to 1.67 mg/g PM. Dystrophic preparations consistently give 7-9-fold higher recoveries of protein in heavy SR subfractions compared to normal PM when examined at 2, 4 and 5.5 weeks ex ovo. [3H]ryanodine binding to normal SR from PM 2 days ex novo exhibits nonlinear Scatchard plots which resolve into high-(Kd,app = 18 nM; Bmax - 17 pmol /mg protein) and low-(Kd,app = 532 nM; Bmax = 2.6 pmol/mg protein) affinity binding sites, whereas dystrophic preparations exhibit only high-affinity [3H]ryanodine binding sites (Kd,app = 31 nM; Bmax = 2.1 pmol/mg protein). Both normal and dystrophic PM have similar capacities to bind [3H]ryanodine (2.6 vs. 2.0 pmol/g PM, respectively) at 2 days ex ovo. However, at 2, 4, and 5.5 weeks ex ovo the density of high-affinity [3H]ryanodine binding sites in normal SR drops dramatically to 3.5, 1.2, and 0.4 pmol/mg protein, respetively, and corresponds with disappearance of the low-affinity binding sites. In marked contrast, heavy SR membranes from distrophic PM 2, 4, and 5.5 weeks ex ovo, maintain their high-affinity binding sites for [3H]ryanodine and exhibit high densities of low-affinity binding sites (Kd,app = 725-4500 nM; Bmax = 15.4-25.1 pmol/mg protein). Early developmental over-expression of [3H]ryanodine binding capacity in dystrophic PM ranges from 34-fold to 388-fold that of normal PM at 2 weeks and 5.5 weeks, respectively, and correspond to the intensity with which high molecular weight doublets of Mr 340 000 and 320 000 stain on SDS-PAGE. Low-affinity [3H]ryanodine binding sites of dystrophic SR exhibit 4-6-fold higher sensitivity to activation by Ca2+ and altered sensitivity to activation by caffein and adenine nucleotides. These results demonstrate that over-expression of junctional SR and [3H]ryanodine receptors having altered radioligand binding properties is a very early event in the post-hatch development of dystrophic PM. Since the [3H]ryanodine receptor is a specific marker for the SR Ca2+ release channel at the muscle triad and a key component of excitation-contraction coupling, abnormal expression of [3H]ryanodine receptors may be of a fundamental importance to the etiology of muscular dystrophy in the chicken.

AB - Heavy sacroplasmic reticulum (SR membranes enriched in [3H]ryanodine receptor have been isolated from pectoralis major (PM) of normal line 412 and dystrophic line 413 chickens paired at various stages during post-hatch development. Normal PM 2 days ex ovo yields 17% lower protein recovery in the heavy SR subfractions compared to preparations from paired dystophic PM (0.80 vs 0.96% mg/g PM, respectively). By 2 weeks ex ovo, protein recovery in normal SR subfractions decreases over 3-fold to 0.24 mg/g PM, whereas yields from dystrophic PM increase to 1.67 mg/g PM. Dystrophic preparations consistently give 7-9-fold higher recoveries of protein in heavy SR subfractions compared to normal PM when examined at 2, 4 and 5.5 weeks ex ovo. [3H]ryanodine binding to normal SR from PM 2 days ex novo exhibits nonlinear Scatchard plots which resolve into high-(Kd,app = 18 nM; Bmax - 17 pmol /mg protein) and low-(Kd,app = 532 nM; Bmax = 2.6 pmol/mg protein) affinity binding sites, whereas dystrophic preparations exhibit only high-affinity [3H]ryanodine binding sites (Kd,app = 31 nM; Bmax = 2.1 pmol/mg protein). Both normal and dystrophic PM have similar capacities to bind [3H]ryanodine (2.6 vs. 2.0 pmol/g PM, respectively) at 2 days ex ovo. However, at 2, 4, and 5.5 weeks ex ovo the density of high-affinity [3H]ryanodine binding sites in normal SR drops dramatically to 3.5, 1.2, and 0.4 pmol/mg protein, respetively, and corresponds with disappearance of the low-affinity binding sites. In marked contrast, heavy SR membranes from distrophic PM 2, 4, and 5.5 weeks ex ovo, maintain their high-affinity binding sites for [3H]ryanodine and exhibit high densities of low-affinity binding sites (Kd,app = 725-4500 nM; Bmax = 15.4-25.1 pmol/mg protein). Early developmental over-expression of [3H]ryanodine binding capacity in dystrophic PM ranges from 34-fold to 388-fold that of normal PM at 2 weeks and 5.5 weeks, respectively, and correspond to the intensity with which high molecular weight doublets of Mr 340 000 and 320 000 stain on SDS-PAGE. Low-affinity [3H]ryanodine binding sites of dystrophic SR exhibit 4-6-fold higher sensitivity to activation by Ca2+ and altered sensitivity to activation by caffein and adenine nucleotides. These results demonstrate that over-expression of junctional SR and [3H]ryanodine receptors having altered radioligand binding properties is a very early event in the post-hatch development of dystrophic PM. Since the [3H]ryanodine receptor is a specific marker for the SR Ca2+ release channel at the muscle triad and a key component of excitation-contraction coupling, abnormal expression of [3H]ryanodine receptors may be of a fundamental importance to the etiology of muscular dystrophy in the chicken.

KW - Muscle development

KW - Muscular dystrophy

KW - Ryanodine receptor

KW - Sarcoplasmic reticuluml

KW - Skeletal muscle

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