Pragobpol S, Gero A M, Lee C S, O'Sullivan W J
Arch Biochem Biophys. 1984 Apr;230(1):285-93. doi: 10.1016/0003-9861(84)90109-7.
Orotate phosphoribosyltransferase (OPRTase) and orotidylate decarboxylase (ODCase) have been found to be particulate in the kinetoplastid protozoan, Crithidia luciliae. Sucrose density centrifugation indicated that these two enzymes are associated with the glycosome, a microbody which appears to be unique to the Kinetoplastida and which contains many of the glycolytic enzymes. The particulate location of OPRTase and ODCase was considered to be favorable for channeling of orotidine-5'-monophosphate (OMP), the product of the first enzyme and substrate for the second. The degree of channeling was determined by double radioactively labeled experiments designed to determine the relative efficiency of endogenous and exogenous OMP as substrates of ODCase. The efficiency of channeling was high, with an approximate 50-fold preference for endogenous OMP. By comparison, the degree of channeling for the yeast enzymes, which are soluble and unassociated, was less than 2-fold. The OPRTase-ODCase enzyme complex was solubilized using Triton X-100 in the presence of dimethyl sulfoxide, glycerol, and phosphoribosyldiphosphate. The percentage recovery of the overall enzyme activity was approximately 20%. The degree of channeling was reduced by approximately 10-fold for the solubilized complex. The Km for OMP changed from 7.5 (+/- 1.8) to 1.6 (+/- 0.3) microM in the ODCase reaction. There was no alteration in the Km for orotate in the OPRTase reaction.
乳清酸磷酸核糖基转移酶（OPRTase）和乳清酸脱羧酶（ODCase）已被发现在动质体原生动物——路氏锥虫中是颗粒状的。蔗糖密度离心表明这两种酶与糖体相关，糖体是一种微体，似乎是动质体所特有的，并且含有许多糖酵解酶。OPRTase和ODCase的颗粒状定位被认为有利于乳清苷-5'-单磷酸（OMP）的通道化，OMP是第一种酶的产物和第二种酶的底物。通道化程度通过双放射性标记实验来确定，该实验旨在确定内源性和外源性OMP作为ODCase底物的相对效率。通道化效率很高，对内源性OMP的偏好约为50倍。相比之下，酵母中可溶且不相关的酶的通道化程度小于2倍。在二甲基亚砜、甘油和磷酸核糖二磷酸存在的情况下，使用 Triton X-100使OPRTase-ODCase酶复合物溶解。总酶活性的回收率约为20%。对于溶解的复合物，通道化程度降低了约10倍。在ODCase反应中，OMP的 Km从7.5（±(1.8)）变为1.6（±(0.3)）微摩尔。在OPRTase反应中，乳清酸的Km没有改变。
