Assay of proteolytic enzymes by the fluorescence polarization technique. |
| |
Authors: | H Maeda |
| |
Affiliation: | National Institute of Arthritis, Metabolism and Digestive Diseases, National Institutes of Health, Bethesda, Maryland 20014 USA |
| |
Abstract: | Neuraminidase substrates of high specific activity (>300 μCi/μmol) were prepared by reduction of sialyllactose with NaB3H4, followed by separation of the 2 → 3 and 2 → 6 isomers of [3H]sialyllactitol by paper chromatography. Hydrolysis of sialyllactitol by neuraminidase was monitored by measuring the radioactivity in the neutral reaction product, which was separated from the charged substrate by passage over a small anion exchange column. The assay was applied to the neuraminidase activity of cultured human skin fibroblasts. The Km was found to be 1.1 mm for both substrates; the pH optimum, 4.0; the 2 → 3 isomer was hydrolyzed twice as fast as the 2 → 6. In several genetic disorders associated with neuraminidase deficiency, the activity toward both isomers was reduced almost completely (mucolipidoses I and II; Goldberg syndrome), or only partially (mucolipidosis III; adult myoclonus syndrome); however, the relative activity towards the two isomers remained approximately the same in all cases. |
| |
Keywords: | Address correspondence to Dr. Elizabeth F. Neufeld Bullding 10 Room 9N-238 National Institutes of Health 9000 Rockville Pike Bethesda Md. 20014. |
本文献已被 ScienceDirect 等数据库收录! |
|