Kaska D D, Myllylä R, Günzler V, Gibor A, Kivirikko K I
Department of Medical Biochemistry, University of Oulu, Finland.
Biochem J. 1988 Nov 15;256(1):257-63. doi: 10.1042/bj2560257.
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.
通过离子交换色谱法和在与琼脂糖偶联的聚（L-羟脯氨酸）上进行亲和色谱法，从多细胞绿藻团藻（Volvox carteri）中以高度纯化的形式分离出脯氨酰4-羟化酶。还开发了另外两种亲和柱方法，一种涉及与琼脂糖连接的3,4-二羟基苯乙酸，另一种涉及与琼脂糖连接的3,4-二羟基苯丙酸。团藻酶对共底物和肽底物的Km值，以及对选定的2-氧代戊二酸类似物的抑制常数，与莱茵衣藻（Chlamydomonas reinhardii）的酶相似，只是团藻酶对2-氧代戊二酸的Km值大6倍。团藻酶的最适温度也高10℃。通过凝胶过滤法测得团藻酶的表观Mr约为40,000，与报道的衣藻酶相似，但明显低于脊椎动物的酶。从绿藻肠浒苔（Enteromorpha intestinalis）中分离的脯氨酰4-羟化酶也发现了约40,000的相似表观Mr，而来自各种维管植物的酶的表观Mr大于300,000。SDS/聚丙烯酰胺凝胶电泳显示，在高度纯化的团藻酶中存在一条主要的蛋白带双峰，Mr约为65,000，以及一条次要的双峰，Mr约为55,000 - 57,000。针对Mr-65,000双峰制备的多克隆抗血清，在免疫印迹中可使Mr-65,000双峰以及脊椎动物脯氨酰4-羟化酶的α亚基染色，但不能使β亚基染色。针对脊椎动物酶β亚基的抗血清，在免疫印迹中可使部分纯化的团藻酶制剂中的一条Mr-50,000多肽染色，但不能使高度纯化酶的Mr-65,000或Mr-55,000 - 57,000双峰染色。因此，数据表明活性团藻脯氨酰4-羟化酶是一种酶单体，在抗原性上与脊椎动物酶的α亚基相关。
