Moran R G, Colman P D
Anal Biochem. 1984 Aug 1;140(2):326-42. doi: 10.1016/0003-2697(84)90174-x.
Previous methods for the measurement of folylpolyglutamate synthetase have been modified and combined to facilitate assay of this enzyme at the levels found in mammalian tissues. Batch adsorption of product onto charcoal allowed the rapid analysis of multiple samples of partially purified enzyme, e.g., column fractions. This technique, however, was unsuitable for the assay of folylpolyglutamate synthetase in crude cytosols due to the presence of interfering enzyme activities. On the other hand, the sequential use of charcoal adsorption and batch elution from DEAE-cellulose permitted isolation of the folate product from assay mixtures containing crude enzyme fractions. Under these conditions, interference from other enzyme activities and background values were low enough for the quantitation of 10 pmol of oligoglutamyl folate product. Folylpolyglutamate synthetase was measured in a series of mouse tissues and tumors. Enzyme activity was quite low in all cases. Mouse liver and kidney and some of the tumors studied had the highest levels (50-100 pmol product/h/mg protein); other tumors and spleen had lower levels. Enzyme activity was at the limit of detection in intestine and lung and was below detection in brain, heart, and skeletal muscle.
先前用于测定叶酰聚谷氨酸合成酶的方法已经过改进和整合，以便于在哺乳动物组织中发现的水平下对该酶进行检测。将产物分批吸附到活性炭上，可以对部分纯化的酶的多个样品（如柱层析馏分）进行快速分析。然而，由于存在干扰酶活性，该技术不适用于粗细胞溶质中叶酰聚谷氨酸合成酶的检测。另一方面，依次使用活性炭吸附和从DEAE-纤维素上分批洗脱，可以从含有粗酶馏分的检测混合物中分离叶酸产物。在这些条件下，其他酶活性的干扰和背景值足够低，足以定量10 pmol的寡聚谷氨酰叶酸产物。对一系列小鼠组织和肿瘤中的叶酰聚谷氨酸合成酶进行了检测。在所有情况下，酶活性都相当低。所研究的小鼠肝脏、肾脏和一些肿瘤的酶活性水平最高（50 - 100 pmol产物/小时/毫克蛋白质）；其他肿瘤和脾脏的酶活性水平较低。在肠道和肺中，酶活性处于检测限，而在脑、心脏和骨骼肌中则低于检测限。
