Lysyl hydroxylase. Further purification and characterization of the enzyme from chick embryos and chick embryo cartilage.
A purification of up to 4000-fold is reported for lysyl hydroxylase (EC 1.14.11.4) from extract of chick-embryo homogenate and one of about 300-fold from extract of chick-embryo cartilage. Multiple forms of the enzyme were observed during purification from whole chick embryos. In gel filtration the elution positions of the two main forms corresponded to average molecular weights of about 580000 and 220000. These two forms could also be clearly separated in hydroxyapatite chromatography. In addition, some enzyme activity was always eluted between the two main peaks both in gel filtration and in hydroxyapatite chromatography. The presence of the two main forms was also observed when purifying enzyme from chick embryo cartilage. Both forms of the enzyme hydroxylated lysine in arginine-rich histone, which does not contain any -X-Lys-Gly- sequence. No difference was found between the enzyme from whole chick embryos and from chick embryo cartilage in this respect. Lysyl hydroxylase was found to have affinity for concanavalin A, indicating the presence of some carbohydrate residues in the enzyme molecule. Lysyl and prolyl hydroxylase activities increased when the chick embryo homogenate was assayed in the presence of lysolecithin. Preincubation of the homogenate either with lysolecithin or with Triton X-100 increased lysyl hydroxylase activity in homogenate, and in the 1500 x g and 150000 x g supernatants, suggesting that the increase in the enzyme activity was due to liberation of the enzyme from the membranes. Divalent cations were found to inhibit the activity of lysyl and prolyl hydroxylases in vitro. An inhibition of about 50% was achieved with 15 mM calcium 60 muM copper and 3 muM zinc concentrations. The mode of inhibition was tested with Cu2+, and was found to be competitive with Fe2+.
D000418:Albumins / Q000494:pharmacology; D000818:Animals; D002356:Cartilage / Q000201:enzymology*; D002413:Cations, Divalent / Q000494:pharmacology; D002642:Chick Embryo; D002846:Chromatography, Affinity; D003094:Collagen / Q000378:metabolism; D003208:Concanavalin A; D006657:Histones / Q000378:metabolism; D008244:Lysophosphatidylcholines / Q000494:pharmacology; D046911:Macromolecular Substances; D006899:Mixed Function Oxygenases / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D011092:Polyethylene Glycols / Q000494:pharmacology; D011188:Potassium / Q000494:pharmacology; D011393:Procollagen-Proline Dioxygenase / Q000378:metabolism; D012964:Sodium / Q000494:pharmacology
D000418:Albumins; D002413:Cations, Divalent; D006657:Histones; D008244:Lysophosphatidylcholines; D046911:Macromolecular Substances; D003208:Concanavalin A; D011092:Polyethylene Glycols; D003094:Collagen; D012964:Sodium; D006899:Mixed Function Oxygenases; D011393:Procollagen-Proline Dioxygenase; D011188:Potassium