Regulatory properties of a mutant carnitine palmitoyltransferase in human skeletal muscle

Abstract

Carnitine palmitoyltransferase (EC 2.3.1.21) was studied in sonicated muscle homogenates of seven patients who had recurrent attacks of myoglobinuria and marked deficiency of carnitine palmitoyltransferase in the isotope exchange assay, and in control subjects.1. When L-palmitoylcarnitine was reduced from 0.5 mM to 0.05 mM in the isotope exchange assay, enzyme activity returned to normal in the patients but was not significantly altered in the controls.2. When the forward assay was performed in the presence of 80 pM palmitoyl-CoA and 0.1 % albumin, all patients showed normal carnitine palmitoyltransferase activity. The apparent K,,, values for DL-carnitine and palmitoyl-CoA were also normal in the patients.3. When albumin was omitted from the forward assay, 72-105% of the initial activity was observed in the controls, but only 31 -55% in the patients.4. When the palmitoyl-CoA concentration in the forward assay exceeded 0.08 mM the enzyme activity was inhibited in both patients and controls, but the inhibition was significantly greater in the patients.5. The addition of either L-palmitoylcarnitine or DL-palmitoylcarnitine to the forward assay progressively inhibited enzyme activity in both patients and controls, but the inhibition was significantly greater in the patients. In the controls but not the patients D-palmitoylcarnitine was less inhibitory than the L-isomer or the m-racemate.6. When the forward assay was performed with muscle homogenates preincubated with 0.4% Triton X-100 only 7 -21 % of the original enzyme activity remained in the patients, but 86 -110% was found in the controls.7. Increasing concentrations of malonyl-CoA inhibited both the forward and the isotope exchange assays. When the inhibition was maximal, only 14-18% of the CPT activity remained in homogenates of patients but 32-47% in homogenates of controls. The Zs0 (median inhibitory concentration) and Ki values for malonyl-CoA determined in the forward assay were not significantly different in the patients and controls.The data imply that CPT deficiency is caused by altered regulatory properties of a mutant enzyme and/or by altered interaction between the enzyme and its membranous environment rather than lack of catalytically active CPT I, I1 or both. The mutant CPT would be most vulnerable to inhibition by its substrate and/or product when lipid metabolism is stressed. This could also explain why the symptoms differ from muscle carnitine deficiency, and why so little lipid accumulates in muscle in CPT deficiency.Exercise-induced episodes of muscle weakness, pain, rhabdomyolysis and myoglobinuria with or without renal failure have been reported in patients with skeletal muscle CPT deficiency.Since the original description of the disease by DiMauro and DiMauro in 1973 [l] 25 additional cases have been reported [2-211. Depending on the assay system used, CPT activities in the patients ranged from 'not detectable' [l, 2, 8, 101 to 20 -30% of normal [4,11,15, 201. 'Partial deficiencies' in which 45 -60% of normal CPT activity was present also ...

Hepatic and Muscular Presentations of Carnitine Palmitoyl Transferase Deficiency: Two Distinct Entities

“…They demonstrated an impaired CPT activity in muscle mitochondria from muscular presentation patients in an assay similar to assay A performed with 0.1 % albumin and 200 pM palmitoyl CoA. This discrepancy between our data and those of Zierg and Engel (25) might be due to differences in the cellular material used (intact fibroblast mitochondria versus disrupted muscle mitochondria).…”

“…Conversely, in these cells, CPT activity was not impaired when measured in detergent conditions, suggesting an intact CPT2 activity. In patients' cell lines with muscular presentation, assay A failed to evidence a decreased activity, irrespective of the concentration of albumin (0.1 to I %) and palmitoyl CoA (10 to 300 pM) used in the assay (data not shown), contrasting with Zierg and Engel's results (25). They demonstrated an impaired CPT activity in muscle mitochondria from muscular presentation patients in an assay similar to assay A performed with 0.1 % albumin and 200 pM palmitoyl CoA.…”

Molecular characterization of inherited carnitine palmitoyltransferase II deficiency.

“…Thus, the kinetic properties of this mutant CPTase II are different from those described in a previous study (29), which provided evidence that in some patients with muscular CPTase II deficiency the mutant enzyme exhibited normal activity under optimal assay conditions but was abnormally inhibited by increasing concentrations of either palmitoyl-CoA or palmitoyl-L-carnitine. Metabolic labeling of patient's fibroblasts in a pulse-chase protocol demonstrated normal biosynthesis of a normal-sized CPTase II monomer that appeared less stable than the normal counterpart (Fig.…”

A case of carnitine palmitoyltransferase II deficiency in human skeletal muscle

“…This assay depends on the rate of incorporation of [3H]carnitine into a pool of palmitoylcarnitine in the presence of CoA [15]. The assay medium (final volume 1.0 ml at 30°C) was the same as that used for the forward assay except that the substrates were 2.5 mM L-[3H]carnitine (18.5 kBq), 100/~M or 0.5 mM palmitoylcarnitine and 0.2 mM CoA.…”