Levin M, Weisz J, Bui Q D, Santen R J
Department of Medicine, Milton S. Hershey Medical Center, Pennsylvania State University, Hershey 17033.
J Steroid Biochem. 1987 Nov;28(5):513-20. doi: 10.1016/0022-4731(87)90510-3.
The ability of breast cancer tissues from postmenopausal women to form catechol estrogens was examined by using a product isolation assay. Initial assays were carried out in the presence of either: (a) NADPH, the co-factor for monooxygenase mediated catecholestrogen (CE) formation or; (b) light-activated Tween 80 (LAT-80), a putative organic hydroperoxide co-factor for peroxidatic activity. Under monooxygenase conditions, CE formation by homogenates of 10 tumors did not exceed that obtained with heat denatured tissue. In contrast, 17 of 20 tumors incubated with LAT-80 synthesized significant amounts of CE (8.5 +/- 1.17 2-hydroxyestradiol [2-OH-E2] and 12.8 +/- 2.4 nmol/g protein/10 min 4-hydroxyestradiol [4-OH-E2]). Substitution of cumene hydroperoxide, an organic hydroperoxide, for LAT-80 enhanced estrogen 2/4 hydroxylase (E-2/4-H) activity over 200-fold, making it possible to characterize systematically the peroxidatic activity. The properties of peroxidatic E-2/4-H activity were similar to those of soluble peroxidases isolated from brain, including an acidic pH optimum, localization in the soluble fraction, an apparent Km in the range of 80 microM and an apparent Vmax in the range of 4000 nmol/g/protein/10 min for both 2- and 4-OH-E2. Under optimal assay conditions, peroxidatic E-2/4-H activity was identified in 10 of 13 tumors (2480 +/- 580 nmol/g protein/10 min for 2-OH-E2 and 2790 +/- 600 for 4-OH-E2). The level of activity detected suggests a biological relevance for CE formation by breast cancer tissue.
采用产物分离分析法检测绝经后女性乳腺癌组织形成儿茶酚雌激素的能力。最初的实验在以下条件下进行：(a) 烟酰胺腺嘌呤二核苷酸磷酸（NADPH），单加氧酶介导儿茶酚雌激素（CE）形成的辅助因子；或(b) 光活化吐温80（LAT-80），一种假定的过氧化物酶活性有机氢过氧化物辅助因子。在单加氧酶条件下，10个肿瘤匀浆形成的CE不超过热变性组织。相反，20个肿瘤中有17个与LAT-80一起孵育时合成了大量CE（8.5±1.17纳摩尔/克蛋白质/10分钟2-羟基雌二醇[2-OH-E2]和12.8±2.4纳摩尔/克蛋白质/10分钟4-羟基雌二醇[4-OH-E2]）。用有机氢过氧化物异丙苯过氧化氢替代LAT-80可使雌激素2/4羟化酶（E-2/4-H）活性提高200倍以上，从而能够系统地表征过氧化物酶活性。过氧化物酶E-2/4-H活性的特性与从大脑中分离的可溶性过氧化物酶相似，包括最适酸性pH、定位于可溶性部分、2-OH-E2和4-OH-E2的表观米氏常数（Km）在80微摩尔范围内，表观最大反应速度（Vmax）在4000纳摩尔/克/蛋白质/10分钟范围内。在最佳检测条件下，13个肿瘤中有10个检测到过氧化物酶E-2/4-H活性（2-OH-E2为2480±580纳摩尔/克蛋白质/10分钟，4-OH-E2为2790±600纳摩尔/克蛋白质/10分钟）。检测到的活性水平表明乳腺癌组织形成CE具有生物学相关性。
