Pisoni R L, Thoene J G
Department of Pediatrics and Communicable Diseases, University of Michigan Medical School, Ann Arbor 48109-2029.
J Biol Chem. 1989 Mar 25;264(9):4850-6.
Lysosomes contain enzymatic activities capable of degrading nucleic acids to their constituent nucleosides, but the manner by which these degradation products are released from the lysosome is unknown. To investigate this process, human fibroblast lysosomes, purified on Percoll density gradients, were incubated with [3H]adenosine at pH 7.0, and the amount of adenosine taken up by the lysosomes was measured. Adenosine uptake by fibroblast lysosomes attained a steady state by 12 min at 37 degrees C and was unaffected by the presence of 2 mM MgATP or changes in pH from 5.0 to 8.0. An Arrhenius plot was linear with an activation energy of 12.9 kcal/mol and a Q10 of 2.0. Lysosomal adenosine uptake is saturable, displaying a Km of 9 mM at pH 7.0 and 37 degrees C. Various nucleosides and the nucleobase, 6-dimethylaminopurine, strongly inhibit lysosomal adenosine uptake, whereas neither D-ribose or nucleotide monophosphates have any significant effect upon lysosomal adenosine uptake. On a molar basis, purines are recognized more strongly than pyrimidines. Changing the nature of the nucleoside sugar from ribose to arabinose or deoxyribose has little effect on reactivity with this transport system. The known plasma membrane nucleoside transport inhibitors, dipyridamole and nitrobenzylthioinosine, inhibit lysosomal nucleoside transport at relatively low concentrations (25 microM) relative to the Km of 9 mM for lysosomal adenosine uptake. The half-times of [3H]inosine and [3H]uridine efflux from fibroblast lysosomes ranged from 6 to 8 min at 37 degrees C. Trans effects were not observed to be associated with either inosine or uridine exodus. In contrast to adenosine uptake, adenine primarily enters fibroblast lysosomes by a route not saturable by high concentrations of various nucleosides. In conclusion, the saturability of lysosomal adenosine uptake and its specific, competitive inhibition by other nucleosides indicate the existence of a carrier-mediated transport system for nucleosides within fibroblast lysosomal membranes.
溶酶体含有能够将核酸降解为其组成核苷的酶活性，但这些降解产物从溶酶体中释放的方式尚不清楚。为了研究这一过程，在Percoll密度梯度上纯化的人成纤维细胞溶酶体在pH 7.0下与[3H]腺苷一起孵育，并测量溶酶体摄取的腺苷量。成纤维细胞溶酶体对腺苷的摄取在37℃下12分钟达到稳态，并且不受2 mM MgATP的存在或pH从5.0至8.0变化的影响。阿累尼乌斯图呈线性，活化能为12.9千卡/摩尔，Q10为2.0。溶酶体对腺苷的摄取是可饱和的，在pH 7.0和37℃下显示Km为9 mM。各种核苷和核碱基6-二甲基氨基嘌呤强烈抑制溶酶体对腺苷的摄取，而D-核糖或核苷酸单磷酸对溶酶体对腺苷的摄取没有任何显著影响。在摩尔基础上，嘌呤比嘧啶的识别更强。将核苷糖的性质从核糖改变为阿拉伯糖或脱氧核糖对与该转运系统的反应性影响很小。已知的质膜核苷转运抑制剂双嘧达莫和硝基苄硫肌苷在相对于溶酶体对腺苷摄取的9 mM Km的相对低浓度（25 microM）下抑制溶酶体核苷转运。在37℃下，[3H]肌苷和[3H]尿苷从成纤维细胞溶酶体流出的半衰期为6至8分钟。未观察到反式效应与肌苷或尿苷外流相关。与腺苷摄取相反，腺嘌呤主要通过高浓度各种核苷不可饱和的途径进入成纤维细胞溶酶体。总之，溶酶体对腺苷摄取的可饱和性及其被其他核苷的特异性竞争性抑制表明在成纤维细胞溶酶体膜内存在一种载体介导的核苷转运系统。
