Divalent cation activation of galactosyltransferase in native mammary Golgi vesicles

• Published in: The Journal of biological chemistry Vol. 265; no. 26; pp. 15731 - 15737
• Main Authors: WITSELL, D. L, CASEY, C. E, NEVILLE, M. C
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 15-09-1990
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Calcimycin - pharmacology; Cations, Divalent - pharmacology; Enzyme Activation; Enzymes and enzyme inhibitors; Female; Fundamental and applied biological sciences. Psychology; Golgi apparatus; Golgi Apparatus - enzymology; Kinetics; Lactose Synthase - metabolism; Mammary Glands, Animal - enzymology; manganese; Manganese - pharmacology; Mice; Milk - enzymology; Models, Theoretical; Transferases; Enzyme; Rodentia; Dissociation constant; Activation; Biomembrane; Binding site; Golgi apparatus; In vivo; Vertebrata; Mammalia; Enzymatic activity; Mouse; Lactalbumin; Cations; Scatchard plot; Kinetics; Mammary gland; Manganese Ions; Lactose synthase
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)55458-2

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.