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.
Although protein kinase FA/GSK-3 alpha (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 FA/GSK-3 alpha 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 0.32 M sucrose, approximately 40% of the total FA/GSK-3 alpha was found in the cytosol. However, when brain was homogenized in buffer containing 0.32 M sucrose and in a glass homogenizer with Teflon pestle, more than 80% of the total FA/GSK-3 alpha was found associated with the particulate membrane fractions. By manipulating these findings, we have developed a simplified procedure for purification of homogeneous kinase FA/GSK-3 alpha in high recovery and in a substantial amount from brain tissue. The data explain why kinase FA/GSK-3 alpha cannot be isolated in a reasonable amount from most mammalian tissues for the past years. The specific pure antibody that can specifically recognize kinase FA/GSK-3 alpha 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 FA/GSK-3 alpha in the regulation of brain functions especially in the brain particulate membrane fractions.
Yu
J S
JS
Institute of Biomedical Science, National Tsing Hua University, Hsinchu, Taiwan, Republic of China.
Yang
S D
SD
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001921:Brain / Q000201:enzymology*; D002458:Cell Fractionation; D002845:Chromatography; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000201:enzymology; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D038362:Glycogen Synthase Kinase 3; D015151:Immunoblotting / Q000379:methods; D007425:Intracellular Membranes / Q000201:enzymology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009842:Oligopeptides / Q000138:chemical synthesis / Q000276:immunology; D009928:Organ Specificity; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D011494:Protein Kinases / Q000032:analysis* / Q000302:isolation & purification* / Q000378:metabolism; D011817:Rabbits; D013347:Subcellular Fractions / Q000201:enzymology; D013552:Swine
D000906:Antibodies; D009842:Oligopeptides; D017886:Durapatite; D011494:Protein Kinases; D038362:Glycogen Synthase Kinase 3; D010749:Phosphoprotein Phosphatases