Partial purification and characterization of protocollagen lysine hydroxylase from chick embryos.
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A procedure was developed for partial purification of protocollagen lysine hydroxylase, the enzyme which synthesizes the hydroxylysine in collagen. The procedure involves fractionation with (NH4)2SO4, fractionation with calcium phosphate gel, two successive chromatographies on DEAE-cellulose and gel filtration on 8% agarose. During the chromatography on DEAE-cellulose the protocollagen lysine hydroxylase was separated from protocollagen proline hydroxylase. The final preparation of the enzyme was up to 300-fold purified over the embryonic extract and up to 600-fold purified over the initial homogenate. At least three different forms of the enzyme were observed. In low ionic strength buffers, the enzyme formed large, insoluble aggregates which were not studied further. Two major forms were obtained in high salt buffers, and these eluted from gel filtration columns with apparent molecular weights of about 550 000 and 200 000 as estimated from the elution positions of globular proteins. Some enzyme activity eluted between the two major forms, and this may have represented an additional form of the enzyme or an interconversion of the two forms during the chromatography. The Km values were determined for -ketoglutarate, Fe2+ and ascorbate which are co-substrates or co-factors for the reaction and were found to be 50 M, 1 M and 50 M, respectively. Even in the presence of saturating concentrations of these co-substrates and co-factors, maximal enzymic activity was not observed unless serum albumin, catalase and dithiothreitol were added to the incubation mixture. p-Mercuribenzoate inhibited the enzyme and the inhibition was partially reversed by dithiothreitol, indicating that free sulfhydryl groups were required for enzymic activity. The results presented here demonstrate that protocollagen lysine hydroxylase is a separate enzyme from protocollagen proline hydroxylase but that it has several properties similar to those of the proline hydroxylase. 1972.
author list (cited authors)
Kivirikko, K. I., & Prockop, D. J.
complete list of authors
Kivirikko, KI||Prockop, DJ