Submicromolar manganese dependence of Golgi vesicular galactosyltransferase (lactose synthetase).
1. Manganese(II) buffers were set up with inorganic triphosphate, trimethylenediaminetetraacetate and tetramethylenediaminetetraacetate to study the Mn dependence of beta 1,4-galactosyltransferase (lactose synthetase) in preparations of rat mammary gland. 2. In intact particulate preparations, treated with the calcium ionophore A23187, lactose synthesis was abolished by chelators and restored by bivalent transition metal ions in a manner characteristic of activation site I of the pure enzyme. Ni(II) also activated, as did Mg at high concentration. 3. Only Mn(II) could restore endogenous rates, giving an apparent Km of 0.1-0.2 microM, and eliciting about 70% full activity without addition of a site II activator. 4. In purified Golgi membrane vesicles, Mn gave an apparent Km of 0.4 microM. This increased sharply to about 10 microM on permeabilization with filipin, lysis with detergents, solubilization with Triton X-100, or in the pure enzyme. Preparations of chemically undamaged Golgi vesicles, known to include a proportion of the enzyme on exposed membranes, exhibited both low-Km and high-Km components. 5. The response of particulate galactosyltransferase to apparently physiological concentrations of free Mn(II) ion is interpreted as either due to a sensitizing factor within the Golgi lumen, or to the accumulation of Mn at elevated concentrations. Alternatively, the high Km of the soluble enzyme may reflect proteolytic damage.
Kuhn
N J
NJ
School of Biochemistry, University of Birmingham, England.
Ward
S
S
Leong
W S
WS
D000818:Animals; D002413:Cations, Divalent; D005260:Female; D005372:Filipin / Q000494:pharmacology; D006056:Golgi Apparatus / Q000201:enzymology*; D007425:Intracellular Membranes / Q000201:enzymology; D007700:Kinetics; D007788:Lactose Synthase / Q000302:isolation & purification / Q000378:metabolism*; D008321:Mammary Glands, Animal / Q000201:enzymology*; D008345:Manganese / Q000494:pharmacology*; D008433:Mathematics; D051381:Rats; D011919:Rats, Inbred Strains
D002413:Cations, Divalent; D008345:Manganese; D005372:Filipin; D007788:Lactose Synthase