Polynucleotide phosphorylase [polyribonucleotide: orthophosphate nucleotidyltransferase, EC 2.7.7.8] was purified to near homogeneity from the photosynthetic bacterium, Rhodospirillum rubrum. The purified enzyme had a molecular weight of approximately 160,000, and consisted of two equivalent subunits of approximately 76,000 daltons. It catalyzed the three reactions described below. 2. In the exchange reaction of the beta-phosphate of nucleoside diphosphates with Pi by the purified enzyme in the presence of 3.3 mM Pi, 6.7 mMCl2, and 0.33 mM or 1.0 mM nucleotide at pH 8.0 and 20 degrees C, ADP, GDP, and CDP, and CDP were better substrates than UDP, while IDP and deoxyribonucleoside diphosphates hardly served as substrates. The ADP-Pi exchange activity was significantly inhibited by high concentrations of either ADP or Pi. 3. In the polymerization reaction of ribonucleoside diphosphates by the purified enzyme in the presence of 6.7 mM nucleotide and 6.7 mM MgCl2 at pH 8.0 and 20 degrees C, ADP was the best substrate; the activities relative to that with ADP were 55% with UD, 51% with CDP, and 48% with IDP, while GDP hardly served as a substrate, 4. In the phosphoryolysis reaction of polynucleoside diphosphates by the purified enzyme in the presence of 1.0 mM polynucleotide, 6.7 mM Pi, and 6.7 mM MgCl2 at pH 8.0 and 20 degrees C, poly[U] was the best substrate; the activities relative to that with poly[U] were 32% with poly[A], 28% with poly[I], 21% with poly[C], and 2% with yeast RNA, while poly[G] and yeast DNA hardly served as substrates. 5. The three kinds of activities of the purified enzyme described above were stimulated by divalent cations such as Mg2+, Mn2+, Cd2+, and Co2+.
摘要
多核苷酸磷酸化酶[聚核糖核苷酸：正磷酸核苷酸基转移酶，EC 2.7.7.8]从光合细菌红螺菌中纯化至近乎同质。纯化后的酶分子量约为160,000，由两个约76,000道尔顿的等同亚基组成。它催化下述三个反应。2. 在3.3 mM Pi、6.7 mM Cl2以及0.33 mM或1.0 mM核苷酸存在的情况下，于pH 8.0和20℃，纯化后的酶催化核苷二磷酸的β-磷酸与Pi的交换反应，其中ADP、GDP和CDP作为底物比UDP更好，而IDP和脱氧核糖核苷二磷酸几乎不作为底物。高浓度的ADP或Pi会显著抑制ADP-Pi交换活性。3. 在6.7 mM核苷酸和6.7 mM MgCl2存在的情况下，于pH 8.0和20℃，纯化后的酶催化核糖核苷二磷酸的聚合反应，其中ADP是最佳底物；相对于ADP的活性，UDP为55%，CDP为51%，IDP为48%，而GDP几乎不作为底物。4. 在1.0 mM多核苷酸、6.7 mM Pi和6.7 mM MgCl2存在的情况下，于pH 8.0和20℃，纯化后的酶催化多核苷二磷酸的磷酸解反应，其中聚[U]是最佳底物；相对于聚[U]的活性，聚[A]为32%，聚[I]为28%；聚[C]为21%，酵母RNA为2%，而聚[G]和酵母DNA几乎不作为底物。5. 上述纯化后的酶的三种活性受到Mg2+、Mn2+、Cd2+和Co2+等二价阳离子的刺激。
