Studies on a pyrimidine phosphoribosyltransferase from murine leukemia P1534J. Partial purification, substrate specificity, and evidence for its existence as a bifunctional complex with orotidine 5-phosphate decarboxylase.
Studies on a pyrimidine phosphoribosyltransferase from murine leukemia P1534J. Partial purification, substrate specificity, and evidence for its existence as a bifunctional complex with orotidine 5-phosphate decarboxylase.
复制标题
鼠白血病 P1534J 嘧啶磷酸核糖基转移酶的研究。
DOI：
10.1016/s0021-9258(19)41283-0
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发表时间：
1975
期刊：
影响因子：
--
通讯作者：
M. Guganig
中科院分区：
文献类型：
--
作者：
P. Reyes;M. Guganig
一种嘧啶磷酸核糖转移酶，以前被证明利用5-氟尿嘧啶，也可能利用尿嘧啶和甲酸(Reyes，P.2057-2062)，已经从小鼠白血病P1534J中纯化了大约100倍。约20%的原始活性被恢复以产生比活性为7.4摩尔的5-氟尿嘧啶利用/小时/毫克蛋白质的酶制剂。这种制剂的圆盘凝胶电泳法显示出一条主要的蛋白质条带，并伴随着几种微量污染物。重点研究了该酶的底物专一性。5-氟尿嘧啶、尿嘧啶和甲酸磷酸核糖转移酶活性在硫酸铵和鱼精蛋白分级过程中平行纯化，并一起从Sephadex TG-150和DEAE-纤维素柱上洗脱。三种磷酸核糖转移酶活性从Sephadex柱上洗脱出来，表观分子量在55,000到60,000之间。尽管存在配位分级，但在DEAE-纤维素层析过程中，优先损失了谷氨酸的活性。在其他条件下，ORotate活性继续以独特的方式表现，例如在蛋白质分解消化期间。然而，在后一种情况下，当消化在5 mM UMP存在的情况下进行时，所有三种活动都平行反应。以下结果提供了额外的证据支持这一观点，即所有三种磷酸核糖转移酶的活性可能是由同一种酶催化的：(A)当用5-氟尿嘧啶、尿嘧啶或Orotate测定酶活性时，5-磷酸核糖-P的表观Km没有显著变化；(B)5-氟尿嘧啶和尿嘧啶被发现是相互竞争的抑制剂；5-氟尿嘧啶对Orotate活性的影响同样是竞争性的；(C)在没有UMP的情况下，Orotate是5-FU和Uracil的非竞争性抑制剂，但当存在5 mM的UMP时，它成为这两种活性的竞争性抑制剂；(D)5-FU和Orotate的活性在5-20%的蔗糖梯度中共沉淀(尿嘧啶活性未检测到)；以及(E)各种正常小鼠组织显示出与P1534J酶制剂中几乎相同的5-FU和Uuracil与Orotate的活性比率。本研究中报道的表观Km和KI值表明，首选的嘧啶底物是旋转酸类。因此，这种酶似乎很可能在体内作为一种轮转型磷酸核糖转移酶发挥作用。在Sephadex G-150凝胶过滤过程中，(A)在Sephadex G-150凝胶过滤过程中一起洗脱，(B)在5~20%的蔗糖梯度中共沉淀，(C)在与硫酸铵和鱼精蛋白的分级过程中保持结合，以及(D)当先前受到弹性酶限制的酶制剂在蔗糖梯度中沉淀时，分离出磷酸核糖转移酶和脱羧酶组分。
A pyrimidine phosphoribosyltransferase, previously shown to utilize 5-fluorouracil and possibly also uracil and orotate (Reyes, P. (1969) Biochemistry 8, 2057-2062), has been purified about 100-fold from murine leukemia P1534J. Roughly 20% of the original activity was recovered to yield an enzyme preparation with a specific activity of 7.4 mumol of 5-fluorouracil utilized/hour/mg of protein. Disc gel electrophoresis of this preparation revealed the presence of a major band of protein accompanied by several trace contaminants. Emphasis was placed on a study of the substrate specificity of this enzyme. 5-Fluorouracil, uracil, and orotate phosphoribosyltransferase activities purified in parallel during fractionation with ammonium sulfate and protamine sulfate and eluted together from columns of Sephadex tG-150 and DEAE-cellulose. The three phosphoribosyltransferase activities eluted from the Sephadex columns with an apparent molecular weight of 55,000 to 60,000. In spite of this coordinate fractionation, preferential losses of orotate activity were experienced during DEAE-cellulose chromatography. Orotate activity continued to behave in a unique manner under other conditions, such as during proteolytic digestion. In the latter case, however, all three activities responded in parallel when digestion took place in the presence of 5mM UMP. The following results provided additional evidence to support the view that all three phosphoribosyltransferase activities may be catalyzed by the same enzyme: (a) the apparent Km for 5-phosphoribosyl 1-pyrophosphate (PP-ribose-P) did not change significantly when enzyme activity was measured with either 5-fluorouracil, uracil, or orotate; (b) 5-fluorouracil and uracil were found to be mutually competitive inhibitors; the effect of 5-fluorouracil on orotate activity was likewise competitive in nature; (c) in the absence of UMP, orotate was a noncompetitive inhibitor of 5-fluorouracil and uracil activities, but in the presence of 5mM UMP it became a competitive inhibitor of both of these activities; (d) 5-fluorouracil and orotate activities co-sedimented in 5 to 20% sucrose gradients (uracil activity was not examined); and (e) a wide variety of normal mouse tissues displayed virtually the same 5-fluorouracil to uracil to orotate activity ratio as found in P1534J enzyme preparations. The apparent Km and Ki values reported in this study indicate that the preferred pyrimidine substrate is orotate. It seems likely, therefore, that this enzyme functions in vivo as an orotate phosphoribosyltransferase. Orotate phosphoribosyltransferase and orotidine 5'-monophosphate (OMP) decarboxylase activities (a) eluted together during gel filtration on Sephadex G-150, (b) co-sedimented in 5 to 20% sucrose gradients, (c) remained associated during fractionation with ammonium sulfate and protamine sulfate, and (d) separated into a phosphoribosyltransferase and decarboxylase component when enzyme preparations previously subjected to limited proteolysis by elastase were sedimented in sucrose gradients...