Purification of sarcoplasmic reticulum vesicles from horse gluteal muscle

Autry, Joseph M.; Karim, Christine B.; Cocco, Mariana; Carlson, Samuel F.; Thomas, David D.; Valberg, Stephanie J.

doi:10.1016/j.ab.2020.113965

Cited by 4 publications

(35 citation statements)

References 70 publications

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“…In comparison to synthetic horse SLN standard, immunoblot analysis of four horse SR preps using pAb GS3379 determined that native SLN is expressed at 0.16 ± 0.015 nmol SLN/mg SR protein ( Figure 2 ). The expression level of SERCA in horse SR is estimated at ~2.1 nmol SERCA/mg SR protein, per content assessed by Coomassie densitometry and immunoblotting [ 58 ], using rabbit SR vesicles as a relative standard with 5.0‒6.4 nmol SERCA/mg SR protein [ 78 , 79 , 80 ]. Thus, immunoblotting using the anti-horse-SLN pAb 3379, with synthetic horse SLN peptide as quantitative standard, determined that SR vesicles from horse gluteal muscle express a minor stoichiometric ratio of ~0.06 SLN/SERCA (mol/mol), i.e., there are ~16 SERCA molecules per available SLN subunit.…”

Section: Resultsmentioning

confidence: 99%

“…Protein immunoblotting required 200 g of muscle in order to isolate highly-purified SR membranes [ 55 , 56 , 57 , 58 ]. This necessitated euthanasia, and therefore horses donated to the University of Minnesota due to severe orthopedic lameness, were used for biochemical studies of muscle SR proteins [ 58 ]. Muscle samples were obtained from four horses aged 10–18 years: three castrated males (two Quarter Horses and one Thoroughbred) and one female (Quarter Horse).…”

Section: Methodsmentioning

confidence: 99%

“…For SR isolation from rabbit skeletal muscle, New Zealand white rabbits (junior does less than six months of age) were provided by the Research Animal Resources Facility at the University of Minnesota. Euthanasia of rabbits was performed with appropriate palliative care consistent with American Veterinary Medical Association guideline, with IACUC protocol # 1611-34327A [ 58 ].…”

Section: Methodsmentioning

confidence: 99%

“…Following humane euthanasia of rabbits, the back and leg muscles per rabbit were harvested, and mechanical disruption and differential centrifugation were used to isolate SR vesicles [ 58 , 64 ]. Rabbit SR vesicles are defined as the centrifugal pellet isolated at 23,000× g max for 60 min (the “23KP” cellular fraction), which was preceded by three clarification spins: 4000× g max for 20 min, 4000× g max for 20 min, and 12,000× g max for 20 min (see the rabbit SR protocol flow-chart reported as Figure S1 in [ 58 ]). The protein concentration of rabbit SR vesicles was determined by the BCA assay (Pierce Biotechnology).…”

Section: Methodsmentioning

confidence: 99%

“…Protein immunoblotting required 200 g of muscle in order to isolate highly-purified SR membranes [55][56][57][58]. This necessitated euthanasia, and therefore horses donated to the University of Minnesota due to severe orthopedic lameness, were used for biochemical studies of muscle SR proteins [58].…”

Section: Animals and Samplesmentioning

confidence: 99%

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Sarcolipin Exhibits Abundant RNA Transcription and Minimal Protein Expression in Horse Gluteal Muscle

Autry

Karim

Perumbakkam

et al. 2020

Veterinary Sciences

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Ca2+ regulation in equine muscle is important for horse performance, yet little is known about this species-specific regulation. We reported recently that horse encode unique gene and protein sequences for the sarcoplasmic reticulum (SR) Ca2+-transporting ATPase (SERCA) and the regulatory subunit sarcolipin (SLN). Here we quantified gene transcription and protein expression of SERCA and its inhibitory peptides in horse gluteus, as compared to commonly-studied rabbit skeletal muscle. RNA sequencing and protein immunoblotting determined that horse gluteus expresses the ATP2A1 gene (SERCA1) as the predominant SR Ca2+-ATPase isoform and the SLN gene as the most-abundant SERCA inhibitory peptide, as also found in rabbit skeletal muscle. Equine muscle expresses an insignificant level of phospholamban (PLN), another key SERCA inhibitory peptide expressed commonly in a variety of mammalian striated muscles. Surprisingly in horse, the RNA transcript ratio of SLN-to-ATP2A1 is an order of magnitude higher than in rabbit, while the corresponding protein expression ratio is an order of magnitude lower than in rabbit. Thus, SLN is not efficiently translated or maintained as a stable protein in horse muscle, suggesting a non-coding role for supra-abundant SLN mRNA. We propose that the lack of SLN and PLN inhibition of SERCA activity in equine muscle is an evolutionary adaptation that potentiates Ca2+ cycling and muscle contractility in a prey species domestically selected for speed.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Animals and Samplesmentioning

confidence: 99%

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Sarcolipin Exhibits Abundant RNA Transcription and Minimal Protein Expression in Horse Gluteal Muscle

Autry

Karim

Perumbakkam

et al. 2020

Veterinary Sciences

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FBXL4 protects against HFpEF through Drp1-Mediated regulation of mitochondrial dynamics and the downstream SERCA2a

Abudureyimu,

Luo,

Jiang

et al. 2024

Redox Biology

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Isolation of the Sarcoplasmic Reticulum Ca2+-ATPase from Rabbit Fast-Twitch Muscle

Rivera-Morán,

Sampedro

2023

MPs

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The sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) is a membrane protein that is destabilized during purification in the absence of calcium ions. The disaccharide trehalose is a protein stabilizer that accumulates in the yeast cytoplasm when under stress. In the present work, SERCA was purified by including trehalose in the purification protocol. The purified SERCA showed high protein purity (~95%) and ATPase activity. ATP hydrolysis was dependent on the presence of Ca2+ and the enzyme kinetics showed a hyperbolic dependence on ATP (Km = 12.16 ± 2.25 μM ATP). FITC labeling showed the integrity of the ATP-binding site and the identity of the isolated enzyme as a P-type ATPase. Circular dichroism (CD) spectral changes at a wavelength of 225 nm were observed upon titration with ATP, indicating α-helical rearrangements in the nucleotide-binding domain (N-domain), which correlated with ATP affinity (Km). The presence of Ca2+ did not affect FITC labeling or the ATP-mediated structural changes at the N-domain. The use of trehalose in the SERCA purification protocol stabilized the enzyme. The isolated SERCA appears to be suitable for structural and ligand binding studies, e.g., for testing newly designed or natural inhibitors. The use of trehalose is recommended for the isolation of unstable enzymes.

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Purification of sarcoplasmic reticulum vesicles from horse gluteal muscle

Cited by 4 publications

References 70 publications

Sarcolipin Exhibits Abundant RNA Transcription and Minimal Protein Expression in Horse Gluteal Muscle

Sarcolipin Exhibits Abundant RNA Transcription and Minimal Protein Expression in Horse Gluteal Muscle

FBXL4 protects against HFpEF through Drp1-Mediated regulation of mitochondrial dynamics and the downstream SERCA2a

Isolation of the Sarcoplasmic Reticulum Ca2+-ATPase from Rabbit Fast-Twitch Muscle

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