MUT activity was determined by methods described previously [36 (link)]. Briefly, lysates were incubated with 5′-deoxyadenosylcobalamin (200 μM, C0884, Sigma-Aldrich Inc., St. Louis, MO) and a racemic mix of methylmalonyl-CoA (1 mM; M1762, Sigma-Aldrich Inc., St. Louis, MO) at 37 °C for 15 min. MUT enzyme reactions were terminated by the addition of 100 g/L trichloroacetic acid (2.5%; T6399 Sigma-Aldrich Inc., St. Louis, MO) with vortexing. Samples were centrifuged at 13,000g for 5 min. Chromatographic separation and quantification were accomplished with HPLC. Supernatants (20 μL) were injected and separated on a Poroshell EC-C18 120 HPLC column (695975–302, Agilent Technologies, Santa Clara, CA) equilibrated with100 mM acetic acid (A6283, Sigma Aldrich Inc., St. Louis, MO) in 100 mM sodium phosphate (AC343815000, Fisher Scientific, Waltham, MA) buffer, pH 7.0 (Solvent A). Solvent B was prepared by addition of 18% v/v methanol in Solvent A. Elution was performed with a linear methanol gradient: Solvent B increased from 0 to 95% from 0 to 15 min (95% Solvent B) and then stayed at 95% from 15 to 25 min (95%) with a flow rate of 0.5 mL/min.
M1762
Manufactured by Merck Group
The M1762 is a laboratory equipment product manufactured by Merck Group. It is designed for general laboratory use. The core function of the M1762 is to perform specific tasks required in a laboratory setting. No further details on the intended use of this product can be provided in an unbiased and factual manner.
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2 protocols using m1762
1
Methylmalonyl-CoA Mutase Activity Assay
2
Fibroblast PI Incorporation and MMUT Activity
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PI into acid-precipitable material of primary fibroblasts was assessed according to a protocol described previously42 (link) with modifications as described20 . MMUT enzyme activity assay was performed in fibroblast crude cell lysates as originally described43 ,44 (link) using recent modifications8 (link). MMUT enzyme activity in HEK cells was measured using the same protocol but without radiolabeled substrate (instead only 1 mM of methylmalonyl-CoA was used, Sigma M1762) and final succinate determination was performed by HPLC separation and electrospray ionization (ESI) tandem mass spectrometry (MS/MS) detection (SCIEX TripleQuad 5500 LC–MS/MS System).
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