Plagemann P G, Woffendin C
Department of Microbiology, University of Minnesota, Minneapolis 55455.
Biochim Biophys Acta. 1989 Jun 6;981(2):315-25. doi: 10.1016/0005-2736(89)90043-6.
Rapid kinetic techniques were used to study the transport and salvage of uridine and other nucleosides in mouse spleen cells. Spleen cells express two nucleoside transport systems: (1) the non-concentrative, symmetrical, Na+-independent transporter with broad substrate specificity, which has been found in all mammalian cells and is sensitive to inhibition by dipyridamole and nitrobenzylthioinosine; and (2) a Na+-dependent nucleoside transport, which is specific for uridine and purine nucleosides and resistant to inhibition by dipyridamole and nitrobenzylthioinosine. The kinetic properties of the two transporters were determined by measuring uridine influx in ATP-depleted cells and dipyridamole-treated cells, respectively. The Michaelis-Menten constants for Na+-independent and -dependent transport were about 40 and 200 microM, respectively, but the first-order rate constants were about the same for both transport systems. Nitrobenzylthioinosine-sensitivity of the facilitated nucleoside transporter correlated with the presence of about 10,000 high-affinity (Kd = 0.6 nM) nitrobenzylthioinosine-binding sites per cell. The turnover number of the nitrobenzylthioinosine-sensitive nucleoside transporter was comparable to that of mouse P388 leukemia cells. The activation energy of this transporter was 20 kcal/mol. Entry of uridine via either of the transport routes was rapidly followed by its phosphorylation and conversion to UTP. The Michaelis-Menten constant for the in situ phosphorylation of uridine was about 50 microM and the first-order rate constants for phosphorylation and transport were about the same. The spleen cells also efficiently salvaged adenosine, adenine, and hypoxanthine, but not thymidine.
采用快速动力学技术研究了小鼠脾细胞中尿苷及其他核苷的转运和补救过程。脾细胞表达两种核苷转运系统：（1）非浓缩性、对称的、不依赖钠离子的转运体，底物特异性广泛，在所有哺乳动物细胞中均有发现，对双嘧达莫和硝基苄硫肌苷的抑制敏感；（2）依赖钠离子的核苷转运体，对尿苷和嘌呤核苷具有特异性，对双嘧达莫和硝基苄硫肌苷的抑制有抗性。分别通过测量ATP耗竭细胞和双嘧达莫处理细胞中的尿苷流入量来确定这两种转运体的动力学特性。不依赖钠离子和依赖钠离子转运的米氏常数分别约为40和200微摩尔，但两种转运系统的一级速率常数大致相同。易化核苷转运体对硝基苄硫肌苷的敏感性与每个细胞中约10,000个高亲和力（解离常数Kd = 0.6纳摩尔）硝基苄硫肌苷结合位点的存在相关。硝基苄硫肌苷敏感的核苷转运体的周转数与小鼠P388白血病细胞的相当。该转运体的活化能为20千卡/摩尔。通过任何一种转运途径进入的尿苷随后迅速发生磷酸化并转化为UTP。尿苷原位磷酸化的米氏常数约为50微摩尔，磷酸化和转运的一级速率常数大致相同。脾细胞还能有效地补救腺苷、腺嘌呤和次黄嘌呤，但不能补救胸苷。
