Chikuma T, Hanaoka K, Loh Y P, Kato T, Ishii Y
Department of Pharmaceutical Analytical Chemistry, Showa College of Pharmaceutical Sciences, Tokyo, Japan.
Anal Biochem. 1991 Nov 1;198(2):263-7. doi: 10.1016/0003-2697(91)90423-q.
In many peptide hormones and neuropeptides, the carboxy-terminal alpha-amide structure is essential in eliciting biological activity. In the present study, a rapid and sensitive assay method for the determination of peptidylglycine alpha-amidating monooxygenase (PAM) activity has been reported. This method is based on the monitoring of the absorption at 460 nm of 4-dimethylaminoazobenzene-4'-sulfonyl-Gly-L-Phe-NH2 (Dabsyl-Gly-Phe-NH2), enzymatically formed from the substrate 4-dimethylaminoazobenzene-4'-sulfonyl-Gly-L-Phe-Gly, after separation by high-performance liquid chromatography (HPLC) using a C-18 reversed-phase column by isocratic elution. This method is sensitive enough to measure Dabsyl-Gly-Phe-NH2 at concentrations as low as 1 pmol and yield highly reproducible results and requires less than 5 min per sample for separation and quantitation. The concentrations of copper and ascorbic acid required for maximal enzyme activity were 1 microM and 2 mM, respectively. The pH optimum for PAM activity was 5.0 to 5.5. The Km and Vmax values were respectively 3.5 microM and 100 pmol/micrograms/h with the use of enzyme extract obtained from bovine pituitary. By using this method, PAM activity could be readily detected in a single rat saliva. The sensitivity of this assay method will also aid in the effort to examine the regulation of in vivo PAM activity.
在许多肽类激素和神经肽中，羧基末端α-酰胺结构对于引发生物活性至关重要。在本研究中，已报道了一种用于测定肽基甘氨酸α-酰胺化单加氧酶（PAM）活性的快速灵敏检测方法。该方法基于监测经高效液相色谱（HPLC）使用C-18反相柱等度洗脱后，由底物4-二甲基氨基偶氮苯-4'-磺酰基-Gly-L-Phe-Gly酶促形成的4-二甲基氨基偶氮苯-4'-磺酰基-Gly-L-Phe-NH2（Dabsyl-Gly-Phe-NH2）在460nm处的吸光度。该方法灵敏度足以检测低至1pmol浓度的Dabsyl-Gly-Phe-NH2，并产生高度可重复的结果，每个样品分离和定量所需时间不到5分钟。最大酶活性所需的铜和抗坏血酸浓度分别为1μM和2mM。PAM活性的最适pH为5.0至5.5。使用从牛垂体获得的酶提取物时，Km和Vmax值分别为3.5μM和100pmol/μg/h。通过使用该方法，可以很容易地在单个大鼠唾液中检测到PAM活性。这种检测方法的灵敏度也将有助于研究体内PAM活性的调节。
