Tetramyristoylcardiolipin

Manufactured by Avanti Polar Lipids

Sourced in United States

Tetramyristoylcardiolipin is a lipid compound used in various laboratory applications. It is a tetraester of myristic acid and cardiolipin, a phospholipid found in the inner mitochondrial membrane. Tetramyristoylcardiolipin is commonly used as a model system for studying the biophysical properties and interactions of cardiolipin-containing membranes in research settings.

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Lab products found in correlation

8 protocols using tetramyristoylcardiolipin

Nanodisc Formulation of Cardiolipins

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Dimyristoylphosphatidylcholine (PC), tetramyristoyl cardiolipin (CL) and tetralinoleoyl cardiolipin were purchased from Avanti Polar Lipids. Unless otherwise indicated, studies reported herein employed tetramyristoyl CL. Five mg aliquots were dissolved in 200 μL CHCl3:CH3OH (3:1 v/v), and dried under a stream of N₂ gas, creating a thin film on the walls of the vessel. Samples were lyophilized overnight to remove residual solvent. To formulate ND, 750 μL 20 mM HEPES buffer, pH 7.2, was added to a 5 mg aliquot of dried phospholipid. The sample was vortexed to disperse the phospholipid which ultimately appeared as an opaque suspension. Subsequently, 2 mg recombinant human apoA-I [22 (link)] in 500 μL HEPES buffer was added to the lipid suspension. The final volume of the mixture was 1.25 ml. PC ND were formulated by bath sonication of the PC/apoA-I mixture at 25 °C until the solution cleared (< 10 min). Tetramyristoyl CL ND (hereafter referred to as CL ND) were formulated in a similar manner, with the exception that bath sonication was performed at 48 °C. Tetralinoleoyl CL ND were formulated by bath sonication at 25 °C under an N₂ atmosphere.

Fox C.A., Ellison P., Ikon N, & Ryan R.O. (2019). Calcium-induced transformation of cardiolipin nanodisks. Biochimica et biophysica acta. Biomembranes, 1861(5), 1030-1036.

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Phospholipid Internal Standards Protocol

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Phospholipid internal standards 1,2-dimyristoyl-sn-glycero-3-phosphocholine (dMPC), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (dMPE), 1,2-dimyristoyl-sn-glycero-3-phospho-(10-rac-)glycerol (dMPG), 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine (dMPS), tetramyristoylcardiolipin (TMCL), 1,2-dipalmitoyl-sn-glycero-3-phosphatidylinositol (dPPI), N-palmitoyl-D-erythro-sphingosylphosphorylcholine (NPSM), 1-nonadecanoyl-2-hydroxy-sn-glycero-3-phosphocholine (LPC) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). Chloroform, methanol and acetonitrile were purchased from Fisher scientific (Leicestershire, UK); all the solvents were of high performance liquid chromatography (HPLC) grade and were used without further purification. All the other reagents and chemicals used were of the highest grade of purity commercially available. The water was of Milli-Q purity (Synergy1, Millipore Corporation, Billerica, MA).

Colombo S., Melo T., Martínez-López M., Carrasco M.J., Domingues M.R., Pérez-Sala D, & Domingues P. (2018). Phospholipidome of endothelial cells shows a different adaptation response upon oxidative, glycative and lipoxidative stress. Scientific Reports, 8, 12365.

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Lipid and Peptide Preparation for Antimicrobial Assays

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1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and tetramyristoylcardiolipin (TMCL) were purchased from Avanti Polar Lipids, Inc. (USA), and used without further purification.
The amidated peptides LF11-322 (PFWRIRIRR-NH₂, M = 1298.6 g/mole), its N-6-methyloctanoyl derivative 6-MO-LF11-322 (CH₃CH₂-CH₂(CH₃)-(CH₂)₄-CO-NH-PFWRIRIRR-NH₂, M = 1438.9 g/mole) and LF11-215 (FWRIRIRR-NH₂, M = 1201,5 g/mole) and its N-octanoyl derivative O-LF11-215 (CH₃-(CH₂)₆-CO-NH-FWRIRIRR-NH₂, M = 1327,7 g/mole) were purchased from PolyPeptide Laboratories (San Diego, CA, USA) (see Table 1).Peptides were dissolved in phosphate buffered saline (PBS, 20 mM NaPi, 130 mM NaCl, pH 7.4), if not otherwise indicated at a concentration of 3 mg/ml before each experiment.

Zweytick D., Japelj B., Mileykovskaya E., Zorko M., Dowhan W., Blondelle S.E., Riedl S., Jerala R, & Lohner K. (2014). N-acylated Peptides Derived from Human Lactoferricin Perturb Organization of Cardiolipin and Phosphatidylethanolamine in Cell Membranes and Induce Defects in Escherichia coli Cell Division. PLoS ONE, 9(3), e90228.

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Lipid Characterization and Modification

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See also the Key Resource Table below. Common chemicals were purchased from Fisher Scientific (Pittsburgh, PA) or Sigma-Aldrich (St. Louis, MO). Horse heart cytochrome c (without isotopic labeling) was purchased from Sigma-Aldrich (catalog number C7752). Dioleoyl phosphatidylcholine (1,2-dioleoyl-sn-glycero-3-phosphocholine, DOPC; C18:1), 1-Palmitoyl(D31)-2-oleoyl-sn-glycero-3-phosphocholine, (16:0D31-18:1 PC), tetramyristoyl-cardiolipin (1',3'-bis[1,2-dimyristoyl-sn-glycero-3-phospho]-sn-glycerol (sodium salt), TMCL), monounsaturated tetraoleoyl cardiolipin (1',3'-bis[1,2-dioleoyl-sn-glycero-3-phospho]-sn-glycerol (sodium salt), TOCL; C18:1), and bovine heart cardiolipin (BHCL; natural mix with mostly C18:2) were obtained from Avanti Polar Lipids (Alabaster, Alabama). Tetralinoleoyl-cardiolipin (1',3'-bis[1,2-dilinoleoyl-sn-glycero-3-phospho]-sn-glycerol (sodium salt) TLCL (C18:2) was obtained as a custom synthesis from Avanti Polar Lipids. Control samples of oxygenated TLCLs (containing 1-4 oxygen) were biosynthesized from TLCL in the reaction catalyzed by soybean lipoxidase (LOX, Sigma-Aldrich) in 50 mM HEPES buffer containing 100 μM DTPA (for transition metals chelation) and saturated with oxygen (before addition of LOX) at pH 7.4. Oxygenated TLCL molecular species were purified by preparative reverse phase HPLC.

Li M., Mandal A., Tyurin V.A., DeLucia M., Ahn J., Kagan V.E, & van der Wel P.C. (2019). Surface-binding to Cardiolipin Nanodomains Triggers Cytochrome C Pro-apoptotic Peroxidase Activity via Localized Dynamics. Structure (London, England : 1993), 27(5), 806-815.e4.

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Cardiolipin Synthesis and Characterization

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1’,3’‐bis[1,2‐dimyristoyl‐sn‐glycero‐3‐phospho]‐sn‐glycerol (tetramyristoyl cardiolipin, TMCL, 14:0) and 1’,3’‐bis[1,2‐dioleoyl‐sn‐glycero‐3‐phospho]‐sn‐glycerol (tetraoleoyl cardiolipin, TOCL, 18:1) were purchased from Avanti Polar Lipids (Alabaster, Alabama, USA). Bovine heart cardiolipin (BHCL; ≥80% tetralinoleoyl cardiolipin, TLCL, 18:2n6), L‐α‐phosphatidylcholine (PC), and cytochrome c (equine heart) were purchased from Sigma‐Aldrich Canada. 10‐acetyl‐3,7‐dihydroxyphenoxazine (ADHP) was purchased from Cayman Chemicals.

Wilkinson J.A., Silvera S, & LeBlanc P.J. (2021). The effect of cardiolipin side chain composition on cytochrome c protein conformation and peroxidase activity. Physiological Reports, 9(5), e14772.

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Lipid Extraction from N. crassa Mycelia

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Freeze dried mycelia of the N. crassa strains were crashed for 1 min at 20 Hz using a Mixermill (MM400, Retsch). The total protein content of each sample was determined by Bradford protein assay (Bio-Rad), after dissolving the sample in dH₂O.
Subsequently lipids were extracted from biomass corresponding to 500 μg total protein by adding 500 μL chloroform:methanol (2:1) containing 0.5 μM of the internal standards (tetramyristoylcardiolipin and N-heptadecanoyl-D-erythro-sphingosine [(C17 ceramide), Avanti Polar Lipids]. Mycelia were further homogenized 4 × 30 sec at 20 Hz and then incubated for 5 min in an ultrasonic bath. For phase separation, samples were centrifuged for 10 min at 14,000 rpm (4°C) and the organic phases were transferred to glass tubes. This chloroform:methanol extraction step was repeated once more. Combined organic phases were dried overnight and stored at -20°C until analysis.

Huber A., Oemer G., Malanovic N., Lohner K., Kovács L., Salvenmoser W., Zschocke J., Keller M.A, & Marx F. (2019). Membrane Sphingolipids Regulate the Fitness and Antifungal Protein Susceptibility of Neurospora crassa. Frontiers in Microbiology, 10, 605.

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Quantification of Cardiolipin in Heart Tissue

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Cardiolipin was quantified in a subset of the total cohort by using previously published methods with liquid chromatography coupled to electrospray ionization mass spectrometry in an API 4000 Mass Spectrometer (SCIEX, Framingham, Massachusetts) (20) (link). After BIOPS or BIOPS Plus 100 μM elamipretide treatment for 4 h, heart tissue was frozen without buffer at –80°C. Tissue pieces were homogenized by using a glass-on-glass homogenizer in phosphate-buffered saline and lipids extracted according to previously published methods with 1 mmol tetramyristoyl-cardiolipin as an internal standard (Avanti Polar Lipids, Alabaster, Alabama) 20 (link), 27 (link). Cardiolipin species were quantified per milligram of protein.

Chatfield K.C., Sparagna G.C., Chau S., Phillips E.K., Ambardekar A.V., Aftab M., Mitchell M.B., Sucharov C.C., Miyamoto S.D, & Stauffer B.L. (2019). Elamipretide Improves Mitochondrial Function in the Failing Human Heart. JACC: Basic to Translational Science, 4(2), 147-157.

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Preparation of Cardiolipin Nanodisc

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Tetralinoleoylcardiolipin [(18:2/18:2)₂-cardiolipin] and tetra-myristoylcardiolipin [(14:0/14:0)₂-cardiolipin] were purchased from Avanti Polar Lipids. Five mg of a given CL (stock solution in chloroform) was transferred to a glass tube and the solvent evaporated under a stream of N₂ gas. Residual solvent was removed under vacuum. The prepared lipid was dispersed in phosphate buffered saline (PBS; 20 mM sodium phosphate, 150 mM sodium chloride, pH 7.0) followed by the addition of 2 mg recombinant human apoA-I [21 (link)] in a final volume of 1mL. The sample was subjected to bath sonication under a N₂ atmosphere, with the temperature maintained between 22°C and 25 °C. During sonication, the turbid lipid dispersion became clear indicating apolipoprotein/phospholipid complexes (i.e. CL-ND) had formed. No pellet formed upon centrifugation. Control ND, containing dimyristoyl-phosphatidylcholine (Avanti Polar Lipids), were prepared in a similar manner. Where indicated, CL-ND were formulated in the presence (1 % w/w) of a flourescent CL (TopFlour-Cardiolipin; Avanti Polar Lipids).

Ikon N., Su B., Hsu F.F., Forteand T.M, & Ryan R.O. (2015). Exogenous cardiolipin localizes to mitochondria and prevents TAZ knockdown-induced apoptosis in myeloid progenitor cells. Biochemical and biophysical research communications, 464(2), 580-585.

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