Preparation of membrane-bound and of solubilized [formula omitted] from rectal glands of Squalus acanthias. The effect of preparative procedures on purity, specific and molar activity

Abstract

A simple method is described for the routine preparation of larger quantities of purified (Na + + K +)-ATPase from the rectal glands from Squalus acanthias and for solubilization of the purified enzyme in a highly active form. Microsomes are prepared by homogenization of the glands in a Waring Blendor followed by differential centrifugation. They keep their activity for years when stored at −70°C. Based on the earlier method (Jørgensen, P.L. and Skou, J.C. (1971) Biochim. Biophys. Acta 233, 366–380), enzyme with a specific activity of 1500 μmol P i · mg −1 protein · h −1 was prepared by treating the microsomes with low concentrations of deoxycholate followed by differential centrifugation, and with a yield of 70% of the activity in the deoxycholate-treated microsomes. The purified enzyme can be dissolved in deoxycholate in the presence of cholesterol, and after a single centrifugation to remove undissolved enzyme, the specific activity of the solubilized enzyme is increased to 2400–2600 μmol P i · mg −1 protein · h −1. Precipitation of the solubilized enzyme leads to a decrease in specific activity to 1500 μmol P i · mg −1 protein · h −1 and to a decrease in molar activity.