Purification, properties and cation activation of galactosyltransferase from lactating-rat mammary Golgi membranes.
Galactosyltransferase was purified from Golgi membranes of lactating-rat mammary gland and studied with respect to its physical and enzymic (lactose synthetase) properties. The enzyme occurred in both monomeric (43-46 kDa) and apparently dimeric (90 kDa) forms. It was very unstable except in the presence of phospholipid, detergent, or cations binding to site 2. The amino acid composition and the N-terminal sequence closely resembled that of the human and bovine milk enzymes, particularly in respect to a Pro-Pro-Pro-Pro sequence. Kinetic studies demonstrated a high-affinity Mn2+-binding site (1) essential for activity, and a low-affinity Mn2+-binding site (2) that could also bind spermidine or clupeine. Mn2+ binding at site 2 raised Vmax fivefold. Spermidine binding at site 2 enhanced Mn2+ binding at site 1, and influenced binding of glucose. At physiological glucose concentration, clupeine or spermidine activated nearly as well as 15 mM MnCl2 and are regarded as models of a natural cation activator that remains to be isolated. Evidence is given for an essential histidine residue in the galactosyltransferase. It is proposed that site 1 Mn2+ participates directly in the reaction mechanism, whereas site 2 is a regulator site allosterically activated by a basic protein.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000302:isolation & purification; D000818:Animals; D003026:Clupeine / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D005700:Galactosyltransferases / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D006056:Golgi Apparatus / Q000201:enzymology*; D007774:Lactation / Q000378:metabolism*; D008321:Mammary Glands, Animal / Q000201:enzymology*; D008345:Manganese / Q000494:pharmacology; D011247:Pregnancy; D051381:Rats; D011919:Rats, Inbred Strains; D013095:Spermidine / Q000494:pharmacology
D000596:Amino Acids; D008345:Manganese; D003026:Clupeine; D005700:Galactosyltransferases; D013095:Spermidine