Isolation of neuronal plasma membranes from the crayfish Procamburus clarkii, with an aqueous two phase polymer system followed by sucrose density gradient centrifugation

Abstract

An aqueous two phase polymer system (Dextran-polyethyleneglycol system was developed for isolation of plasma membrane fraction from nerves of the crayfish, Procamburus clarkii. The polymer system effectively reduced both mitochondrial and endoplasmic reticulum marker enzyme activity from a crude membrane fraction. The similar enrichment of (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) was shown by the polymer system as well as by the sucrose density gradient centrifugation. The purified plasma membrane fraction (PM) was obtained using the polymer system followed by sucrose density gradient centrifugation. The PM fraction had a high specific activity of (Na + K+)-ATPase of up to 17 times that in the homogenate, with smaller contamination by mitochondria and endoplasmic reticulum enzyme activities than any other membrane fraction. Electron micrographs of the PM fraction also supported the above evidences. The protein recovered from the PM fraction amounted to 1.1% of the total protein in the homogenate. The specific activity of acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) in the membrane fractions was less increased than that of (Na+ + K+)-ATPase. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis suggested that polypeptide chains of estimated molecular weight 115,000 and 31,000 were enriched in the plasma membranes of the crayfish nerves.