Purification and tissue-specific expression of casein kinase from the lactating guinea-pig mammary gland.
A serine-specific casein kinase, an integral membrane protein of the lactating guinea-pig mammary gland, has been purified from a Golgi-enriched membrane fraction, using a combination of sucrose gradient centrifugation and chromatography on ATP-agarose. The enzyme comprises a polypeptide of estimated Mr 74 000 as judged by sodium dodecyl sulphate/polyacrylamide gel electrophoresis, compared with a monomer Mr of 50 000 as determined by sucrose gradient centrifugation in the presence of 500 mM NaCl and 0.1% Triton X-100. Kinetic studies show that the purified enzyme exhibits kinetic constants distinctly different from the rabbit reticulocyte casein kinases I and II, whilst polyclonal antisera raised against the mammary gland enzyme did not cross-react with soluble liver or reticulocyte protein kinase activities. Immunoblotting and immunocytochemical analyses demonstrate the mammary gland enzyme's apparently unique location in lactating mammary gland tissue. Comparative studies with polyclonal antisera raised against bovine galactosyltransferase, show that casein kinase and galactosyltransferase have a similar intracellular localisation in the lactating mammary gland as judged by immunocytochemistry at the light level, but that casein kinase was unique to mammary gland whereas galactosyltransferase could be found in other tissues. The results extended our earlier observations which suggest a Golgi location for casein kinase, and demonstrate that future studies using this enzyme may well prove advantageous for the study of intracellular mechanisms involved in the biogenesis of organelles, in this instance the Golgi apparatus.
D000818:Animals; D047388:Casein Kinases; D011232:Chemical Precipitation; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006056:Golgi Apparatus / Q000201:enzymology; D006168:Guinea Pigs; D007120:Immunochemistry; D007700:Kinetics; D007774:Lactation; D008321:Mammary Glands, Animal / Q000201:enzymology*; D009928:Organ Specificity; D011247:Pregnancy; D011494:Protein Kinases / Q000096:biosynthesis / Q000302:isolation & purification*; D012156:Reticulocytes / Q000201:enzymology
D011494:Protein Kinases; D047388:Casein Kinases