Rabbit liver phosphorylase: Improvement of the purification procedure and assay of the inactive b form

✍ Scribed by Mikihiko Kobayashi; Donald J. Graves

• Publisher: Elsevier Science
• Year: 1982
• Tongue: English
• Weight: 510 KB
• Volume: 122
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(82)90256-1

✦ Synopsis

A new assay system for total phosphorylase activity (a + b form) from rabbit liver was developed by the use of 20% (v/v) 1,2-dimethoxyethane and 20 mM AMP, which provided high activation of the b form. In the absence of 1,Zdimetboxyethane and AMP, phosphorylase a had 100% of the activity (50 units/mg) obtained with this condition, whereas phosphorylase b had only 3%. The assay system can be used to determine total phosphorylase activity and the level of a and 6 forms in purified systems or crude liver extracts. A simple and convenient purification of rabbit liver phosphorylase b was attained by ethanol precipitation, ammonium sulfate precipitation, DE-52 chromatography, and AMP-Sepharose affinity column chromatography. The purified and homogeneous enzyme bad a specific activity of 12 units/mg at 16 mM glucose l-phosphate. Twenty-eight milligrams of enzyme was obtained from 400 g of liver tissue, representing a 461-fold purification. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate gave a single protein band, which corresponded closely to the mobility of rabbit muscle phosphorylase b.