A serinc-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 M , 74000 as judged by sodium dodecyl sulphate/polyacrylamidc gel electrophoresis, compared with a monomer M , of 50 000 as determined by sucrose gradient centrifugation in the presence of500 mM NaCl and 0.1 %, Triton X-1 00. Kinetic studics show that the purified enzyme exhibits kinetic constants distinctly different from the rabbit reticulocyte casein kinases I and 11, 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 tissuc. 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 Colgi 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, i n this instance the Golgi apparatus.We have previously dcscribed the presence in the lactating guinea-pig mainmary gland of a serine-specific casein kinasc, an integral membrane protein located in a Golgi-enriched membrane fraction [l]. Similar enzyme activities have been described in Golgi-enriched fractions isolated from mammary glands of othcr species [2-41. Other casein kinase activities isolated from diverse tissues and cells have also been described in the literature (see [S]). These are CAMP-independcnt and arc classified on the basis of their elution from DEAE-cellulose a s casein kinases 1 and 11; they differ from each other with respect to iiucleotide triphosphate substrate. Casein kinase 11, utilises both ATP and GTP [6] whikt casein kinase I has a preference for ATP. In additioi. cascin kinase I1 but not casein kiiiase I is inhibited by h e p r i n [7].Studies on the phosphorylation of guinea-pig caseins using lactating mammary gland explants in culture [8] or Xrnopus oocytes microinjected with milk protein mRNA [9] demonstrate that each contain partially processed unphosphorylatcd caseins sequestered within the secretory pathway, but that only those secreted by the guinea-pig mammary explants, not by the oocyte, are phosphorylated. These observations suggest that casein kinase is not an enzyme common to the secretory pathway of all cells, and may therefore be expressed in a tissue-specific manner.To determine whet...
Portions of a 125I-iodinated bovine serum albumin preparation were exposed to freezing, acetic acid (pH 3.5, 3.0 or 2.5), urea or formaldehyde, and the effect of these treatments on the rates of pinocytic uptake by yolk sacs from 17.5-day-pregnant rats cultured in vitro and of clearance from the rat bloodstream were studied. Uptake of albumin by the yolk sac was followed by rapid release of [125I]iodo-L-tyrosine into the culture medium. Similarly clearance of albumin in vivo was accompanied by the appearance of trichloroacetic acid-soluble radioactivity in the bloodstream. In both systems the rates of uptake of modified albumin preparations formed a series: formaldehyde or urea greater than acetic acid greater than freezing. The increased rates of uptake of modified albumin preparations could not be ascribed to the formation of aggregates nor, in the yolk-sac system, to an increase in the rate of pinosome formation. It is concluded that the various treatments to which the albumin was subjected increase to varying degrees the affinity of the albumin molecule for binding sites on that region of the plasma membrane from which pinocytic vesicles are formed. Some comparable experiments with native and desialylated human orosomucoid indicate that the rat yolk-sac epithelial cells do not possess the recognition system for uptake of asialoglycoproteins that exists on the surface of hepatic parenchymal cells.
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