Zierz S, Engel A G
Eur J Biochem. 1985 May 15;149(1):207-14. doi: 10.1111/j.1432-1033.1985.tb08913.x.
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. 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. When the forward assay was performed in the presence of 80 microM palmitoyl-CoA and 0.1% albumin, all patients showed normal carnitine palmitoyltransferase activity. The apparent Km values for DL-carnitine and palmitoyl-CoA were also normal in the patients. 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. 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. 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 DL-racemate. 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. 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 I50 (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, II 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.
对7名患有复发性肌红蛋白尿且在同位素交换试验中肉碱棕榈酰转移酶明显缺乏的患者以及对照者的超声处理后的肌肉匀浆中的肉碱棕榈酰转移酶（EC 2.3.1.21）进行了研究。在同位素交换试验中，当L-棕榈酰肉碱从0.5 mM降至0.05 mM时，患者的酶活性恢复正常，而对照者的酶活性无明显变化。当在80 microM棕榈酰辅酶A和0.1%白蛋白存在的情况下进行正向试验时，所有患者的肉碱棕榈酰转移酶活性均正常。患者中DL-肉碱和棕榈酰辅酶A的表观Km值也正常。当正向试验中省略白蛋白时，对照者观察到初始活性的72 - 105%，而患者仅为31 - 55%。当正向试验中棕榈酰辅酶A的浓度超过0.08 mM时，患者和对照者的酶活性均受到抑制，但患者的抑制作用明显更大。在正向试验中添加L-棕榈酰肉碱或DL-棕榈酰肉碱均会逐渐抑制患者和对照者的酶活性，但患者的抑制作用明显更大。在对照者而非患者中，D-棕榈酰肉碱的抑制作用小于L-异构体或DL-外消旋体。当用预先与0.4% Triton X-100孵育的肌肉匀浆进行正向试验时，患者中仅保留了7 - 21%的原始酶活性，而对照者中为86 - 110%。丙二酰辅酶A浓度的增加会抑制正向试验和同位素交换试验。当抑制作用最大时，患者匀浆中仅保留了14 - 18%的CPT活性，而对照者匀浆中为32 - 47%。在正向试验中测定的丙二酰辅酶A的I50（半数抑制浓度）和Ki值在患者和对照者中无显著差异。数据表明，CPT缺乏是由突变酶的调节特性改变和/或酶与其膜环境之间的相互作用改变引起的，而不是缺乏具有催化活性的CPT I、II或两者。当脂质代谢受到压力时，突变的CPT最容易受到其底物和/或产物的抑制。这也可以解释为什么症状与肌肉肉碱缺乏不同，以及为什么在CPT缺乏时肌肉中积累的脂质如此之少。
