Chikuma T, Kocha T, Hanaoka K, Kato T, Ishii Y, Tanaka A
Department of Pharmaceutical Analytical Chemistry, Showa College of Pharmaceutical Sciences, Tokyo, Japan.
Neurochem Int. 1994 Oct;25(4):349-54. doi: 10.1016/0197-0186(94)90142-2.
In many peptide hormones and neuropeptides, the carboxyl-terminal alpha-amide structure is essential in eliciting their biological activity. In the present study, an enzymatic activity capable of converting 4-dimethylaminoazobenzene-4'-sulfonyl-Gly-L-Phe-Gly(Dabsyl-Gly-Phe -Gly) to 4-dimethylaminoazo-benzene-4'-sulfonyl-Gly-L-Phe-NH2(Dabsyl- Gly-Phe-NH2) was investigated in bovine hypothalamus. The concentrations of copper ion and ascorbic acid required for maximal enzyme activity were 16 microM and 2 mM, respectively. Amidating activity showed a pH profile with two pH optima at acidic pH (around 6.0) and neutral pH (around 7.5). Kinetic studies with the enzyme obtained from bovine hypothalamus demonstrated two distinct Km and Vmax values. The first Km and Vmax values were 142.9 microM and 22.2 pmol/microgram/h and the second Km and Vmax values were 22.7 microM and 4.44 pmol/microgram/h, respectively. Two molecular forms of amidating activity were identified by size-exclusion chromatography and the molecular weight of the two enzymes were estimated to be 49 kDa and 69 kDa.
在许多肽类激素和神经肽中，羧基末端的α-酰胺结构对于引发其生物活性至关重要。在本研究中，对牛下丘脑内一种能够将4-二甲基氨基偶氮苯-4'-磺酰基-Gly-L-Phe-Gly（Dabsyl-Gly-Phe-Gly）转化为4-二甲基氨基偶氮苯-4'-磺酰基-Gly-L-Phe-NH2（Dabsyl-Gly-Phe-NH2）的酶活性进行了研究。最大酶活性所需的铜离子和抗坏血酸浓度分别为16 microM和2 mM。酰胺化活性呈现出一种pH曲线，在酸性pH（约6.0）和中性pH（约7.5）处有两个最适pH值。对从牛下丘脑获得的该酶进行的动力学研究表明有两个不同的Km和Vmax值。第一个Km和Vmax值分别为142.9 microM和22.2 pmol/微克/小时，第二个Km和Vmax值分别为22.7 microM和4.44 pmol/微克/小时。通过尺寸排阻色谱法鉴定出两种酰胺化活性的分子形式，两种酶的分子量估计分别为49 kDa和69 kDa。
