Sebti S M, DeLeon J C, Ma L T, Hecht S M, Lazo J S
Department of Pharmacology, Yale University, School of Medicine, New Haven, CT 06510.
Biochem Pharmacol. 1989 Jan 1;38(1):141-7. doi: 10.1016/0006-2952(89)90160-3.
Bleomycin (BLM) hydrolase is believed to protect both malignant and normal tissue from the toxicity of the antitumor drug BLM. Little is known about the substrate specificity of BLM hydrolase. Thus, we developed ion-paired reverse phase high speed liquid chromatography systems to assay for the metabolism of several BLM analogs. We found that BLM A2, BLM B2, tallysomycin S10b (TLM S10b), peplomycin (PEP), butylamino-3-propylamino-3-propylamine bleomycin (BAPP), deglyco bleomycin A2 (dgBLM A2) and bleomycinic acid were each metabolized by rabbit lung BLM hydrolase to a single metabolite. When compared to their corresponding parent compounds, these metabolites were 6- to 35-fold less potent in their ability to inhibit the proliferation of A-253 human head and neck squamous carcinoma cells in culture. Furthermore, we found that substitutions in various regions of the BLM molecule greatly affected the kinetic parameters of BLM hydrolase. For example, the Km with BLM B2 (0.056 +/- 0.005 mM) was 15-fold lower than that seen with BLM A2 (0.83 +/- 0.11 mM). In contrast, the Vmax was not affected markedly by these terminal amine substitutions but was influenced greatly by deletion of the carbohydrate groups of BLM. For example, a 4-fold higher Vmax was observed with dgBLM A2 compared to BLM A2. Thus, these results demonstrate that BLM hydrolase can recognize and metabolize a broad spectrum of BLM analogs regardless of their structural features. This enzymatic conversion resulted in the inactivation of the BLMs as demonstrated by a substantial decrease in their cytotoxicity. Furthermore, the terminal amine and carbohydrate regions, respectively, dictate the apparent affinity and the rate of metabolism of BLM hydrolase substrates.
博来霉素（BLM）水解酶被认为可保护恶性组织和正常组织免受抗肿瘤药物博来霉素的毒性影响。目前对BLM水解酶的底物特异性了解甚少。因此，我们开发了离子对反相高速液相色谱系统来检测几种博来霉素类似物的代谢情况。我们发现，BLM A2、BLM B2、 tallysomycin S10b（TLM S10b）、培洛霉素（PEP）、丁胺基-3-丙胺基-3-丙胺博来霉素（BAPP）、去糖基博来霉素A2（dgBLM A2）和博来霉素酸均可被兔肺BLM水解酶代谢为单一代谢产物。与相应的母体化合物相比，这些代谢产物在抑制培养的A-253人头颈鳞状癌细胞增殖方面的能力低6至35倍。此外，我们发现博来霉素分子不同区域的取代对BLM水解酶的动力学参数有很大影响。例如，BLM B2的米氏常数（Km）（0.056±0.005 mM）比BLM A2（0.83±0.11 mM）低15倍。相反，最大反应速度（Vmax）并未受到这些末端胺基取代的明显影响，但受博来霉素碳水化合物基团缺失的影响很大。例如，与BLM A2相比，dgBLM A2的Vmax高4倍。因此，这些结果表明，无论其结构特征如何，BLM水解酶都能识别并代谢多种博来霉素类似物。这种酶促转化导致博来霉素失活，其细胞毒性显著降低即证明了这一点。此外，末端胺基区域和碳水化合物区域分别决定了BLM水解酶底物的表观亲和力和代谢速率。
