Carrey E A, Hardie D G
Department of Biochemistry, The University, Dundee.
Eur J Biochem. 1988 Feb 1;171(3):583-8. doi: 10.1111/j.1432-1033.1988.tb13828.x.
We have examined the domain organization, and the locations of the sites phosphorylated by the cyclic-AMP-dependent protein kinase, in the multifunctional polypeptide of the pyrimidine-biosynthetic protein, CAD. Fragments produced after limited proteolysis by elastase or trypsin were separated by SDS/polyacrylamide gel electrophoresis and transferred onto nitrocellulose. The blots were probed with antibodies raised against the core aspartate carbamoyltransferase (ACTase) and dihydroorotase (DHOase) fragments to locate fragments containing these domains, and we also examined the locations of the phosphorylation sites by complete tryptic digestion of blotted, 32P-labelled fragments, followed by analytical isoelectric focussing. Our results are consistent with the domain order glutaminase(GLNase)-carbamoyl-phosphate synthetase-(CPSase)-DHOase-ACTase, as suggested by recently reported homologies between the predicted amino acid sequence for the Drosophila rudimentary gene product, and monofunctional CPSases/ACTases/DHOases. In particular, the finding of a 95-kDa elastase fragment which cross-reacted with both anti-DHOase and anti-ACTase antibodies rules out the previously suggested domain order: DHOase-GLNase-CPSase-ACTase. Phosphorylation by cyclic-AMP-dependent protein kinase accelerates cleavage of native CAD by both elastase and trypsin, and abolishes the protective effect of UTP. Site 1 is located close to the C-terminal end of the 160-kDa GLNase/CPSase region. Comparison with the predicted amino acid sequence of the Drosophila rudimentary gene revealed a strong homology between the tryptic peptide containing site 1 from hamster CAD, and a region at the extreme C-terminal end of the CPSase II domain of the Drosophila enzyme. Alignment of the Drosophila sequence and that of rat liver CPSase I, which is not phosphorylated by cyclic-AMP-dependent protein kinase, revealed that this putative site 1 region is missing in CPSase I. Site 2 could not be located with certainty, either from the limited proteolysis data, or from comparison of the sequence around this site and the sequence of the rudimentary gene. There were also one or more previously undetected minor phosphorylation site(s) located in the protease-sensitive hinge region between the DHOase and ACTase domains.
我们研究了嘧啶生物合成蛋白CAD的多功能多肽中的结构域组织，以及环磷酸腺苷依赖性蛋白激酶磷酸化位点的位置。通过弹性蛋白酶或胰蛋白酶进行有限蛋白酶解后产生的片段，经十二烷基硫酸钠/聚丙烯酰胺凝胶电泳分离，然后转移至硝酸纤维素膜上。用针对核心天冬氨酸氨甲酰基转移酶（ACTase）和二氢乳清酸酶（DHOase）片段制备的抗体对印迹进行检测，以定位包含这些结构域的片段，并且我们还通过对印迹的、经32P标记的片段进行完全胰蛋白酶消化，随后进行分析性等电聚焦，来研究磷酸化位点的位置。我们的结果与结构域顺序谷氨酰胺酶（GLNase）-氨甲酰磷酸合成酶（CPSase）-二氢乳清酸酶（DHOase）-天冬氨酸氨甲酰基转移酶（ACTase）一致，正如最近报道的果蝇rudimentary基因产物的预测氨基酸序列与单功能CPSases/ACTases/DHOases之间的同源性所表明的那样。特别是，发现一个95 kDa的弹性蛋白酶片段与抗DHOase和抗ACTase抗体均发生交叉反应，排除了先前提出的结构域顺序：DHOase-GLNase-CPSase-ACTase。环磷酸腺苷依赖性蛋白激酶的磷酸化加速了弹性蛋白酶和胰蛋白酶对天然CAD的切割，并消除了三磷酸尿苷（UTP）的保护作用。位点1位于160 kDa的GLNase/CPSase区域的C末端附近。与果蝇rudimentary基因的预测氨基酸序列进行比较发现，仓鼠CAD中包含位点1的胰蛋白酶肽段与果蝇酶的CPSase II结构域最末端C端的一个区域之间存在很强的同源性。果蝇序列与大鼠肝脏CPSase I（其不被环磷酸腺苷依赖性蛋白激酶磷酸化）的序列比对显示，CPSase I中缺少这个假定的位点1区域。无论是从有限蛋白酶解数据，还是从该位点周围的序列与rudimentary基因的序列比较中，都无法确定位点2的位置。在DHOase和ACTase结构域之间对蛋白酶敏感的铰链区中还存在一个或多个先前未检测到的较小的磷酸化位点。
