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pubmed:abstractText |
Prolyl 4-hydroxylase was isolated in a highly purified form from a multi-cellular green alga, Volvox carteri, by a procedure consisting of ion-exchange chromatography and affinity chromatography on poly(L-hydroxyproline) coupled to Sepharose. Two other affinity-column procedures were also developed, one involving 3,4-dihydroxyphenylacetate and the other 3,4-dihydroxyphenylpropionate linked to Sepharose. The Km values of the Volvox enzyme for the co-substrates and the peptide substrate, as well as the inhibition constants for selected 2-oxoglutarate analogues, were similar to those of the enzyme from Chlamydomonas reinhardii, except that the Km for 2-oxoglutarate with the Volvox enzyme was 6-fold greater. The temperature optimum of the Volvox enzyme was also 10 degrees C higher. The apparent Mr of the Volvox enzyme by gel filtration was about 40,000, being similar to that reported for the Chlamydomonas enzyme but markedly lower than that of the vertebrate enzymes. A similar apparent Mr of about 40,000 was also found for prolyl 4-hydroxylase from the green alga Enteromorpha intestinalis, whereas the enzyme from various vascular plants gave an apparent Mr greater than 300,000. SDS/polyacrylamide-gel electrophoresis demonstrated in the highly purified Volvox enzyme the presence of a major protein band doublet with a Mr of about 65,000 and a minor doublet of Mr about 55,000-57,000. A polyclonal antiserum, prepared against the Mr-65,000 doublet, stained in immunoblotting the Mr-65,000 doublet as well as the alpha subunit, but not the beta subunit, of the vertebrate prolyl 4-hydroxylase. An antiserum against the beta subunit of the vertebrate enzyme stained in immunoblotting a Mr-50,000 polypeptide in a partially purified Volvox enzyme preparation, but did not stain either the Mr-65,000 or the Mr-55,000-57,000 doublet of the highly purified enzyme. The data thus suggest that the active Volvox carteri prolyl 4-hydroxylase is an enzyme monomer antigenically related to the alpha subunit of the vertebrate enzyme.
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