Phosphorylation of human preprogastrin 93-101 by a Golgi membrane kinase from rat mammary gland.
The precursor of the acid-stimulating hormone gastrin contains a phosphorylation site which is immediately adjacent to a functionally important cleavage site, and which occurs in a sequence resembling the phosphorylation sites in casein. We have examined phosphorylation of human preprogastrin 93-101 with [gamma-32P]ATP by a Triton-solubilized Golgi membrane preparation from mammary glands of lactating rats. The activity of solubilized Golgi membranes was approx. an order of magnitude greater than that of intact vesicles suggesting a luminal orientation of the kinase. Incorporation of 32P was linear for up to 12 min at 30 degrees C, and the half-maximal rate of phosphorylation at 1 mM ATP was observed at peptide concentrations of 0.2 mM. The Km for ATP was 0.12 mM and the maximal velocity was 2.17 nmol of peptide per min per mg Golgi protein. Proteinase inhibitors (leupeptin, pepstatin, benzamidine) and p-nitrophenyl phosphate did not influence phosphorylation. The incorporation of 32P was inhibited by poly-L-lysine but not by heparin. We conclude that the phosphorylation site in progastrin is a substrate for a Golgi membrane kinase and that a similar enzyme might act on endogenous progastrin in vivo.
Vegh
M
M
Physiological Laboratory, University of Liverpool, UK.
Varro
A
A
D000595:Amino Acid Sequence; D000818:Animals; D005260:Female; D005755:Gastrins / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D007700:Kinetics; D007774:Lactation; D008321:Mammary Glands, Animal / Q000378:metabolism* / Q000648:ultrastructure; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism*; D010766:Phosphorylation; D011498:Protein Precursors / Q000378:metabolism*; D051381:Rats
D005755:Gastrins; D009842:Oligopeptides; D011498:Protein Precursors; C086562:preprogastrin (93-101); C042873:preprogastrin