A RADIOIMMUNOASSAY FOR MEASURING ALPHA-AMIDATING ENZYME-ACTIVITY

被引：13

作者：

JENG, AY ^{[1
]}

WONG, M ^{[1
]}

LOVATO, SJ ^{[1
]}

ERION, MD ^{[1
]}

GILLIGAN, JP ^{[1
]}

机构：

[1] UNIGENE LABS INC, FAIRFIELD, NJ 07006 USA

来源：

ANALYTICAL BIOCHEMISTRY | 1990年 / 185卷 / 02期

关键词：

D O I：

10.1016/0003-2697(90)90282-E

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

A sensitive α-amidating enzyme (αAE) assay using C-terminal glycine-extended substance P (SP-Gly) as a substrate was developed. The product, substance P (SP), was measured by a radioimmunoassay with specific polyclonal antibodies which recognize SP with an affinity 10,000-fold higher than that of SP-Gly. The sensitivity of the radioimmunoassay was 5 fmol. Enzyme activity could be readily detected with 25 ng αAE partially purified from the conditioned medium of rat medullary thyroid carcinoma CA-77 cells. The Km and Vmax values were 2.0 ± 0.2 μm and 1.7 ± 0.1 nmol/mg/min (mean ± SE, n = 3), respectively. The assay enabled the kinetic characterization of αAE from a single rat pituitary homogenate. Optimal Cu2+ required was 30 μm and greater than 3 mm of ascorbate was needed for maximal enzyme activity. The sensitivity of this assay will aid efforts to examine the regulation of in vivo αAE activity. © 1990.

引用

页码：213 / 219

页数：7

共 28 条

[1] MECHANISM OF C-TERMINAL AMIDE FORMATION BY PITUITARY ENZYMES [J].