BOTH INSULIN AND EPIDERMAL GROWTH-FACTOR STIMULATE LIPOGENESIS AND ACETYL-COA CARBOXYLASE ACTIVITY IN ISOLATED ADIPOCYTES - IMPORTANCE OF HOMOGENIZATION PROCEDURE IN AVOIDING ARTIFACTS IN ACETYL-COA CARBOXYLASE ASSAY
BOTH INSULIN AND EPIDERMAL GROWTH-FACTOR STIMULATE LIPOGENESIS AND ACETYL-COA CARBOXYLASE ACTIVITY IN ISOLATED ADIPOCYTES - IMPORTANCE OF HOMOGENIZATION PROCEDURE IN AVOIDING ARTIFACTS IN ACETYL-COA CARBOXYLASE ASSAY
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DOI：
10.1042/bj2340279
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发表时间：
1986-03-01
影响因子：
4.1
通讯作者：
HARDIE, DG
中科院分区：
文献类型：
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作者：
HAYSTEAD, TAJ;HARDIE, DG
表皮生长因子（EGF）在分离的脂肪细胞中刺激脂肪生成3-4倍，在10 nM-EGF时达到一半最大效果。在同一批细胞中，胰岛素刺激脂肪生成15倍。2.脂肪细胞的冷冻和长时间的均质化导致大量丙酮酸羧化酶从破碎的线粒体中释放，并且足够的丙酮酸可以被带入该酶的测定中，从而对粗脂肪细胞提取物中乙酰辅酶A羧化酶的测定造成显著干扰。这可能解释了在一些先前的出版物中报道的脂肪细胞提取物中存在大量的柠檬酸盐非依赖性乙酰辅酶A羧化酶活性。这个问题可以通过非常短暂的冷冻而不冷冻来消除。3.通过使用修改的匀浆程序，EGF治疗的脂肪细胞显示出产生的乙酰辅酶A羧化酶活性的效果几乎与胰岛素相同。两种信使均增加Vmax。对别构激活剂柠檬酸盐的Ka没有显著影响。
Epidermal growth factor (EGF) stimulates lipogenesis by 3-4-fold in isolated adipocytes, with a half-maximal effect at 10 nM-EGF. In the same batches of cells insulin stimulated lipogenesis by 15-fold. 2. Freezing and prolonged homogenization of adipocytes results in release of large quantities of pyruvate carboxylase from broken mitochondria, and sufficient pyruvate can be carried through into assays for this enzyme to cause significant interference with assays of acetyl-CoA carboxylase in crude adipocyte extracts. This may account for the high amount of citrate-independent acetyl-CoA carboxylase activity reported to be present in adipocyte extracts in some previous publications. This problem may be eliminated by homogenizing very briefly without freezing. 3. By using the modified homogenization procedure, EGF treatment of adipocytes was shown to produce an effect of acetyl-CoA caroboxylase activity almost identical with that of insulin. Both messengers increase Vmax. without significant effect on the Ka for the allosteric activator, citrate.