Action in vivo and in vitro of 2-tetradecylglycidic acid, 2-tetradecylglycidyl-CoA and 2-tetradecylglycidylcarnitine on hepatic carnitine palmitoyltransferase

P. S. Brady, L. J. Brady

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Abstract

The effects of 2-tetradecylglycidic acid (TDGA), TDGA-CoA and TDGA-carnitine were examined in purified hepatic CPT (carnitine palmitoyltransferase) and in hepatic mitochondria and inverted submitochondrial vesicles derived from Sprague-Dawley rats. Since TDGA has been reported as a specific inhibitor of carnitine palmitoyltransferase-A (CPT-A), the focus was on kinetics and inhibition of CPT-A, and the relationship of this key enzyme to β-oxidation. After administration of TDGA in vivo to overnight-starved rats, the V(max.) of CPT in intact mitochondria and in inverted vesicles (CPT-B) was depressed by 66%. The s0,5 for palmitoyl-CoA and K(m) for carnitine were unchanged. The I50 (concn. giving 50% inhibition) for malonyl-CoA was significantly increased from 20 to 141 μM in intact mitochondria, but unchanged (199 versus 268 μM) in inverted vesicles. The addition in vitro of TDGA-CoA (0-1.0 μM) gave I50 values of 0.29 and 0.27 μM (S.E.M. = 0.19) in intact mitochondria from fed and 48 h-starved rats, and 0.81 and 1.57 μM (S.E.M. = 0.29) for inverted vesicles derived from fed and starved rats. Addition in vitro of TDGA-carnitine to mitochondria from starved rats yielded an I50 value of 27.7 mM (S.E.M. = 12.2) for L-[methyl-14C]carnitine release from palmitoyl-L-[methyl-14C]carnitine and 0.64 mM (S.E.M. = 0.07) for palmitoyl-L-[methyl-14C]carnitine formation from L-[methyl-14C]carnitine in intact mitochondria. Inverted vesicles were not measurably sensitive to TDGA-carnitine up to 500 μM for the assay of L-[methyl-14C]carnitine release, but were as sensitive as intact mitochondria when inhibition was determined in the direction of palmitoyl-L-[methyl-14C]carnitine formation (I50 = 0.54 ± 0.07 μM). When TDGA-CoA was added to intact mitochondria, then incubated for 5 min at room temperature and subsequently washed out, V(max.) of CPT decreased from 5.8 to 3.5 (S.E.M. = 0.6) in intact mitochondria, and from 17.2 to 6.3 (S.E.M. = 4.8) in inverted vesicles. The K(m) for L-carnitine and the s0.5 for palmitoyl-CoA increased 2-fold with TDGA-CoA pretreatment in both intact mitochondria and inverted vesicles. Detergent solubilization (0.05% Triton X-100) resulted in a complete loss of TDGA-CoA sensitivity (up to 1.0 μM measured). Sonicated mitochondria exhibited an I50 of 0.72 ± 0.03 μM. When sonicated mitochondria were partitioned into the sonicated supernatant and pellet after centrifugation at 100,000 g for 1 h, the supernatant had an I50 of 8.8 ± 3.2 μM and the pellet had an I50 of 0.84 ± 0.05 μM. Purified rat liver CPT had an I50 of 60-80 μM, which declined to 9.5 μM in the presence of 20% dimethyl sulphoxide and to 15 μM in phosphatidylcholine or cardiolipin liposomes. The data suggest that the membrane environment may be important to TDGA sensitivity.

Original languageEnglish (US)
Pages (from-to)801-809
Number of pages9
JournalBiochemical Journal
Volume238
Issue number3
DOIs
StatePublished - 1986

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