Purification of sarcoplasmic reticulum vesicles from horse gluteal muscle.
Anal Biochem
; 610: 113965, 2020 12 01.
Article
em En
| MEDLINE
| ID: mdl-32956693
ABSTRACT
We have analyzed protein expression and enzyme activity of the sarcoplasmic reticulum Ca2+-transporting ATPase (SERCA) in horse gluteal muscle. Horses exhibit a high incidence of recurrent exertional rhabdomyolysis, with myosolic Ca2+ proposed, but yet to be established, as the underlying cause. To better assess Ca2+ regulatory mechanisms, we developed an improved protocol for isolating sarcoplasmic reticulum (SR) vesicles from horse skeletal muscle, based on mechanical homogenization and optimized parameters for differential centrifugation. Immunoblotting identified the peak subcellular fraction containing the SERCA1 protein (fast-twitch isoform). Gel analysis using the Stains-all dye demonstrated that calsequestrin (CASQ) and phospholipids are highly enriched in the SERCA-containing subcellular fraction isolated from horse gluteus. Immunoblotting also demonstrated that these horse SR vesicles show low content of glycogen phosphorylase (GP), which is likely an abundant contaminating protein of traditional horse SR preps. The maximal Ca2+-activated ATPase activity (Vmax) of SERCA in horse SR vesicles isolated using this protocol is 5â25-fold greater than previously-reported SERCA activity in SR preps from horse skeletal muscle. We propose that this new protocol for isolating SR vesicles will be useful for determining enzymatic parameters of horse SERCA with high fidelity, plus assessing regulatory effect of SERCA peptide subunit(s) expressed in horse muscle.
Palavras-chave
Centrifugation; Equidae [B01.050.150.900.649.313.984.235]; Gel electrophoresis; Intracellular membranes [A11.284.835.514]; Membrane-bound enzyme; Microsome; Muscle, Skeletal [A02.633.567]; Phospholipid; Phosphoprotein; Rhabdomyolysis [C05.651.807]; Sarcoplasmic reticulum calcium-transporting ATPases [D08.811.277.040.025.314.250.500]; Western blotting [E05.601.470.320.200]
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Músculo Esquelético
/
Vesículas Extracelulares
Tipo de estudo:
Prognostic_studies
Limite:
Animals
Idioma:
En
Revista:
Anal Biochem
Ano de publicação:
2020
Tipo de documento:
Article