Immunological and biochemical study on tissue and subcellular distributions of protein kinase FA (an activating factor of ATP.Mg-dependent protein phosphatase): A simplified and efficient procedure for high quantity purification from brain

Abstract

Although protein kinase F_A/GSK-3α (an activating factor of ATP.Mg-dependent protein phosphatase) has been established as a cytosolic enzyme in mammalian nonnervous tissues involved in the metabolic regulation, immunological and biochemical studies on tissue and subcellular distributions demonstrate that kinase F_A/GSK-3α is in fact a membrane-associated enzyme and most abundantly exists in brain particulate membrane fractions depending on the tissue homogenization conditions. For instance, when brain was homogenized in Polytron without 0.32M sucrose, approximately 40% of the total F_A/GSK-3α was found in the cytosol. However, when brain was homogenized in buffer containing 0.32M sucrose and in a glass homogenizer with Teflon pestle, more than 80% of the total F_A/GSK-3α was found associated with the particulate membrane fractions. By manipulating these findings, we have developed a simplified procedure for purification of homogeneous kinase F_A/GSK-3α in high recovery and in a substantial amount from brain tissue. The data explain why kinase F_A/GSK-3α cannot be isolated in a reasonable amount from most mammalian tissues for the past years. The specific pure antibody that can specifically recognize kinase F_A/GSK-3α from crude tissue extracts together with the high quantity purification of the enzyme as presented in this report provides an initial key step for studies on the role of kinase F_A/GSK-3α in the regulation of brain functions especially in the brain particulate membrane fractions.