Lyles G A, Holt A, Marshall C M
Department of Pharmacology and Clinical Pharmacology, Ninewells Hospital, Dundee, UK.
J Pharm Pharmacol. 1990 May;42(5):332-8. doi: 10.1111/j.2042-7158.1990.tb05421.x.
An ion exchange radiochemical assay has been developed to study the deamination of [14C]methylamine (MA) in homogenates of rat aorta and human umbilical artery, as well as in samples of human plasma. MA metabolism was found to be inhibited almost completely by 1 mM semicarbazide, but virtually unaffected by 0.1 mM clorgyline, suggesting that MA is a substrate for the semicarbazide-sensitive amino oxidase (SSAO) activities which also metabolize benzylamine (BZ) in these sources. Mean Km values for MA metabolism by aorta, umbilical artery and plasma were 182, 832 and 516 microM, respectively, with corresponding Vmax values in aorta and umbilical artery of 100 and 590 nmol (mg prot.)-1 h-1, and in plasma of 48 nmol (mL serum)-1 h-1. Kinetic constants determined for [14C]BZ metabolism in plasma (by an organic solvent extraction assay) and in umbilical artery (by the ion exchange assay) yielded mean Km values of 225 microM (plasma), 222 microM (umbilical artery), and Vmax values of 28 nmol (mL serum)-1 h-1 (plasma) and 377 nmol (mg prot.)-1 h-1 (umbilical artery). The deamination of [14C]MA was inhibited competitively by unlabelled BZ, with Ki values in umbilical artery and plasma of 220 and 172 microM, respectively. Also, metabolite formation from mixtures of [14C]BZ (200 microM) and [14C]MA (800 microM) was extremely close to that predicted for a single enzyme capable of metabolizing two alternative substrates in a competitive fashion.(ABSTRACT TRUNCATED AT 250 WORDS)
已开发出一种离子交换放射化学分析法，用于研究大鼠主动脉和人脐动脉匀浆以及人血浆样本中[¹⁴C]甲胺（MA）的脱氨作用。发现1 mM氨基脲几乎完全抑制MA代谢，但0.1 mM氯吉兰对其几乎无影响，这表明MA是氨基脲敏感型氨基氧化酶（SSAO）活性的底物，该酶在这些来源中也能代谢苄胺（BZ）。主动脉、脐动脉和血浆中MA代谢的平均Km值分别为182、832和516 μM，主动脉和脐动脉中相应的Vmax值分别为100和590 nmol（mg蛋白）⁻¹ h⁻¹，血浆中的Vmax值为48 nmol（mL血清）⁻¹ h⁻¹。通过有机溶剂萃取法测定血浆中[¹⁴C]BZ代谢以及通过离子交换法测定脐动脉中[¹⁴C]BZ代谢的动力学常数，得出平均Km值为225 μM（血浆）、222 μM（脐动脉），Vmax值为28 nmol（mL血清）⁻¹ h⁻¹（血浆）和377 nmol（mg蛋白）⁻¹ h⁻¹（脐动脉）。未标记的BZ竞争性抑制[¹⁴C]MA的脱氨作用，脐动脉和血浆中的Ki值分别为220和172 μM。此外，[¹⁴C]BZ（200 μM）和[¹⁴C]MA（800 μM）混合物的代谢物形成与预测的能够以竞争性方式代谢两种替代底物的单一酶的情况极为接近。（摘要截短于250字）
