Pisoni R L
Department of Pediatrics and Communicable Diseases, University of Michigan Medical School, Ann Arbor 48109-2029.
J Biol Chem. 1991 Jan 15;266(2):979-85.
The uptake of [32P]KH2PO4 by Percoll-purified human fibroblast lysosomes at pH 7.0 was investigated to determine if lysosomes contain a transport system recognizing phosphate. Lysosomal phosphate uptake was linear for the first 2 min, attained a steady state by 8-10 min at 37 degrees C, and was not Na+ or K+ dependent. Upon entering lysosomes, [32P]phosphate was rapidly metabolized to trichloroacetic acid-soluble and trichloroacetic acid-insoluble products. After 1-min incubations, 50% of the radioactivity recovered from lysosomes was in the form of inorganic phosphate; and after a 2.5-min incubation, 27% of the radioactivity was recovered as inorganic phosphate. When lysosomes are loaded with radioactivity by incubation with 0.03 mM [32P]KH2PO4 for 25 min and then washed at 4 degrees C, lysosomes fail to release the accumulated radioactivity during a subsequent incubation at 37 degrees C. Lysosomal phosphate uptake gave linear Arrhenius plots (Q10 = 1.8) and was inversely proportional to medium osmolarity. Phosphate uptake was maximal at pH 5-6, half-maximal at pH 7.1, with little transport activity at pH greater than 8, suggesting that the transport system recognizes the monobasic form of phosphate. Lysosomal phosphate uptake is saturable, displaying a Km of 5 microM at pH 7.0 and 37 degrees C. High specificity for phosphate is demonstrated since large concentrations of Na2SO4, NaHCO3, KCl, NaCl, 5'-AMP, or the anion transport inhibitor, 4,4'-diisothiocyanatostilbene-2,2'-disulfonate, have no effect on lysosomal phosphate transport. In contrast, the phosphate analog, arsenate, strongly inhibits lysosomal phosphate uptake in a competitive manner with a Ki of 7 microM. Pyridoxal phosphate, CTP, adenosine 5'-(beta,gamma-imino)triphosphate (AMP-PNP), and glucose 6-phosphate were found to be noncompetitive inhibitors of lysosomal phosphate uptake displaying Ki values of 80-250 microM. When lysosomes are incubated with [gamma-32P]ATP, the lysosomal membrane ATPase hydrolyzes the ATP to form inorganic phosphate which then enters lysosomes by this lysosomal phosphate transport route.
研究了在pH 7.0条件下，经Percoll纯化的人成纤维细胞溶酶体对[32P]KH2PO4的摄取情况，以确定溶酶体是否含有识别磷酸盐的转运系统。溶酶体对磷酸盐的摄取在最初2分钟呈线性，在37℃下8 - 10分钟达到稳态，且不依赖于Na+或K+。[32P]磷酸盐进入溶酶体后，迅速代谢为可溶于三氯乙酸和不溶于三氯乙酸的产物。孵育1分钟后，从溶酶体中回收的放射性中有50%是以无机磷酸盐的形式存在；孵育2.5分钟后，27%的放射性以无机磷酸盐的形式回收。当溶酶体通过与0.03 mM [32P]KH2PO4孵育25分钟加载放射性，然后在4℃下洗涤后，溶酶体在随后37℃的孵育过程中无法释放积累的放射性。溶酶体对磷酸盐的摄取给出线性的阿伦尼乌斯曲线（Q10 = 1.8），且与培养基渗透压成反比。磷酸盐摄取在pH 5 - 6时最大，在pH 7.1时为最大摄取量的一半，在pH大于8时几乎没有转运活性，这表明转运系统识别磷酸盐的一元形式。溶酶体对磷酸盐的摄取是可饱和的，在pH 7.0和37℃时Km为5 microM。由于高浓度的Na2SO4、NaHCO3、KCl、NaCl、5'-AMP或阴离子转运抑制剂4,4'-二异硫氰酸根合芪-2,2'-二磺酸盐对溶酶体磷酸盐转运没有影响，因此证明了对磷酸盐具有高特异性。相反，磷酸盐类似物砷酸盐以竞争方式强烈抑制溶酶体对磷酸盐的摄取，Ki为7 microM。发现磷酸吡哆醛、CTP、腺苷5'-(β,γ-亚氨基)三磷酸（AMP-PNP）和6-磷酸葡萄糖是非竞争性抑制溶酶体对磷酸盐的摄取，Ki值为80 - 250 microM。当溶酶体与[γ-32P]ATP孵育时，溶酶体膜ATP酶将ATP水解形成无机磷酸盐，然后通过这种溶酶体磷酸盐转运途径进入溶酶体。
