Spontaneous deamidation of a protein antibiotic, neocarzinostatin, at weakly acidic pH. Conversion to a homologous inactive preneocarzinostatin due to change of asparagine 83 to aspartic acid 83 accompanied by conformational and biological alterations.
Spontaneous deamidation of a protein antibiotic, neocarzinostatin, at weakly acidic pH. Conversion to a homologous inactive preneocarzinostatin due to change of asparagine 83 to aspartic acid 83 accompanied by conformational and biological alterations.
复制标题
蛋白质抗生素新制癌菌素在弱酸性 pH 条件下自发脱酰胺。
DOI：
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发表时间：
1977
期刊：
影响因子：
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通讯作者：
K. Kuromizu
中科院分区：
文献类型：
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作者：
H. Maeda;K. Kuromizu
用天冬酰胺和谷氨酰胺分析了四-S-羧甲基-新卡西汀(NCS)和经羧基修饰的NCS(用水溶性碳二亚胺和[14C]甘氨酸甲酯修饰)的氨基转移酶M酶中天冬酰胺和谷氨酰胺的含量。用CM-纤维素柱层析从粗制NCS制剂中分离纯化了前胡萝卜素(Pre)。Pre中天冬酰胺的摩尔含量比NCS少1摩尔，在Pre中天冬酰胺被脱胺为天冬氨酸。用两种方法研究了在4℃pH 3.2或26℃0.1M醋酸中NCS向Pre的时间依赖转化：一是用CM-纤维素柱层析定量测定NCS和Pre；二是在密闭容器中透析时释放游离NH3。在实验误差范围内，无论是酸解后的氨基酸含量，还是用10%丙烯酰胺的十二烷基硫酸钠圆盘凝胶电泳法测定的表观分子量，以及N-端和C-端的氨基酸残基，Pre和NCS都没有区别。用Ouchterlony琼脂扩散法检测，NCS和Pre具有共同的抗原性。在醋酸纤维素膜上的电泳和胰酶消化的肽图上，两者只有很小的区别。然而，Pre完全没有生物活性。除了色谱图上的差异外，CD光谱还观察到构象差异，即240-265 nm区域的波谷较浅，表明Pre具有明显的疏松结构。这一解释得到了以下发现的支持，即Pronase对Pre的消化比NCS的更广泛。这些结果表明，NCS的单一天冬酰胺残基(ASN 83)在生物活性中起着重要作用，这种作用明显受构象的控制。
The amide content of neocarzinostatin (NCS), an antitumor protein, has been determined by analysing asparagine and glutamine in the Pronase-aminopeptidase M digests of tetra-S-carboxymethyl-NCS and carboxyl-modified NCS (modified with a water-soluble carbodiimide and [14C]glycine methyl ester). Preneocarzinostatin (PRE) was separated and purified from a crude NCS preparation by CM-cellulose column chromatography. PRE was found to contain one mole less asparagine than NCS, and asparagine was deamidated to aspartic acid in PRE. A time-dependent conversion of NCS to PRE at pH 3.2 at 4 degrees or in 0.1 M acetic acid at 26 degrees was studied in two ways; first, by quantitative determination of NCS and PRE by CM-cellulose column chromatography and second, by following the release of free NH3 during dialysis in an air-tight container. Within experimental error, PRE was indistinguishable from NCS in amino acid content after acid hydrolysis, as well as in apparent molecular weight as determined by SDS-disc gel electrophoresis (10% acrylamide), and N- and C-terminal amino acid residues. Both NCS and PRE shared a common antigenicity as determined by Ouchterlony's agar diffusion method. Only a slight difference between the two in electrophoresis on a cellulose acetate membrane and on a peptide map of the tryptic digest was demonstrated. PRE, however, was completely devoid of biological activity. In addition to the chromatographic difference, a conformational difference was observed by CD spectroscopy, namely, an apparently looser structure of PRE was indicated by the shallowness of the trough in the 240-265 nm region. This interpretation was supported by the finding that digestions by Pronase were more extensive with PRE than with NCS. These results indicate an important role of the single asparagine residue (Asn 83) of NCS in the biological activity, which is evidently governed by the conformation.