Divalent cation activation of galactosyltransferase in native mammary Golgi vesicles.
Millimolar concentrations of manganese are required for maximal activation of purified galactosyltransferase (lactose synthase, EC 2.4.1.22), the enzyme that catalyzes addition of galactosyl groups to proteins and, in lactose synthesis, to glucose. To examine manganese activation of this enzyme under in vivo conditions, we studied intact, partially purified Golgi membranes from mouse mammary glands. In intact vesicles treated with the divalent cation ionophore, A23187, activation followed Michaelis-Menton kinetics with a Km of 3 microM; maximal activation was achieved below 10 microM manganese. In both detergent-solubilized and leaky vesicles the kinetics of manganese activation were consistent with the presence of two manganese-binding sites with dissociation constants about 40 microM and 20 mM. The difference is consistent with the presence in intact vesicles of an endogenous activator too large to traverse the membrane via A23187; this activator could bind to the low affinity manganese site allowing manganese or another divalent cation such as zinc to activate the enzyme at micromolar concentrations. The Km for UDP-galactose was found to be similar in the vesicular and solubilized preparations at micromolar and millimolar manganese concentrations, respectively, providing additional evidence for this hypothesis.
Witsell
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DL
Department of Physiology, University of Colorado School of Medicine, Denver 80262.
Casey
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CE
Neville
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MC
D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002413:Cations, Divalent / Q000494:pharmacology; D004789:Enzyme Activation; D005260:Female; D006056:Golgi Apparatus / Q000201:enzymology*; D007700:Kinetics; D007788:Lactose Synthase / Q000378:metabolism*; D008321:Mammary Glands, Animal / Q000201:enzymology*; D008345:Manganese / Q000494:pharmacology*; D051379:Mice; D008892:Milk / Q000201:enzymology; D008962:Models, Theoretical
D002413:Cations, Divalent; D000001:Calcimycin; D008345:Manganese; D007788:Lactose Synthase