Carboxypeptidase-mediated release of methotrexate from methotrexate alpha-peptides.
Carboxypeptidase-mediated release of methotrexate from methotrexate alpha-peptides.
复制标题
羧肽酶介导的甲氨蝶呤从甲氨蝶呤 α 肽的释放。
DOI：
10.1021/bi00431a047
复制
发表时间：
1989
期刊：
影响因子：
2.9
通讯作者：
Huennekens,FM
中科院分区：
文献类型：
--
作者：
Kuefner,U;Lohrmann,U;Montejano,YD;Vitols,KS;Huennekens,FM
1988年10月25日收到修订稿件摘要：甲氨蝶呤(MTX)-含有代表中性(丙氨酸)、酸性(天冬氨酸)和碱性(精氨酸)氨基酸的多肽是通过区域特异性路线合成的。通过薄层层析、高效液相色谱、元素分析、核磁共振和吸收光谱确定了MTX-Ala、MTX-Asp和MTX-Arg的纯度和真实性。这些多肽在羧基肽酶的作用下产生MTX和氨基酸。反应的监测方法为：用吲哚三酮法检测氨基酸，用高效液相色谱和分光光度法检测甲氨蝶呤。胰腺羧肽酶A(CP-A)对MTX-Ala、MTX-Asp和MTX-Arg的降解速度要慢得多。MTX-ALA也是胰腺羧肽酶B(CP-B)的底物；该酶和MTX-Arg的边缘活性被观察到。人血清仅对MTX-Arg有水解性；2-(硫代甲基)-3-(胍乙基)硫代丙酸酯对该活性的两相抑制与已知存在两种内源性羧基肽酶(CP-N)相一致。在适当的羧基肽酶存在下，MTX多肽对体外培养的LI210细胞的细胞毒作用显著增强。MTX-Ala的毒性远低于MTX(ID_(50)分别为2.0×6 M和2.4×8 M)，但在CP-A存在下，其ID_(50)提高到8.5×1CT8 M，与MTX-Asp/CP-A和MTX-Ala/CP-B的结果相似，MTX-Arg表现出良好的细胞毒性(ID_(50)为5.0×10~8M)，这是由于在培养液中的胎牛血清中CP-N的活性所致；
Revised Manuscript Received October 25, 1988 abstract: Methotrexate (MTX)-peptides containing representative neutral (alanine), acidic (aspartic acid), and basic (arginine) amino acids were synthesized by a regiospecific route. Purity and authenticity of MTX-Ala, MTX-Asp, and MTX-Arg were established by TLC, HPLC, elemental analysis, and NMR and absorbance spectra. These peptides were hydrolyzed by carboxypeptidases to yield MTX and the amino acids. Reactions were monitored by using a ninhydrin assay for the amino acids and HPLC and spec-trophotometric assays for MTX. Pancreatic carboxypeptidase A (CP-A) hydrolyzed MTX-Ala and, at a much slower rate, MTX-Asp and MTX-Arg. MTX-Ala was also a substrate for pancreatic carboxypeptidase B (CP-B); marginal activity was observed with thisenzyme and MTX-Arg. Human serum hydrolyzed only MTX-Arg; biphasic inhibition of this activity by 2-(mercaptomethyl)-3-(guanidinoethyl) thiopropionate was consistent with the known presence of two types of endogenous carboxypeptidase (CP-N). Cytotoxicity of the MTX peptides toward LI210 cells in culture was enhanced considerably in the presence of the appropriate carboxypeptidases. MTX-Ala was much less toxic than MTX (ID50 values of 2.0 X" 6 M and 2.4 X 8 M, respectively), but in the presence of CP-A the ID50 of the peptide improved to 8.5 X 1CT8 M. Similar results were obtained with MTX-Asp/CP-A and MTX-Ala/CP-B combinations. MTX-Arg showed good cytotoxicity (ID50 of 5.0 X 10~ 8 M), due to CP-N activity inthe fetal bovine serum of the culture medium; inclusion of CP-B lowered the ID50 to that of MTX. Possible clinical uses of MTX peptides are discussed.