Andrews J C, Nolan J P, Hammerstedt R H, Bavister B D
Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park 16802-4504, USA.
Cytometry. 1995 Oct 1;21(2):153-9. doi: 10.1002/cyto.990210207.
Zinc stabilizes membranes and DNA and inhibits respiration in somatic cells. It is present in high concentrations in the male reproductive tract and may stabilize spermatozoa prior to fertilization. Herein, we evaluate N-(6-methoxy-8-quinolyl)-p-toluenesulfonamide (TSQ) for analysis of Zn2+ in phosphatidylcholine (PC) vesicles and living spermatozoa using spectrofluorometry and flow cytometry. TSQ:Zn fluorescence in decanol or PC vesicles was compared to that in aqueous buffer. Evaluation of cation specificity, kinetics of TSQ:Zn binding, quenching of TSQ by dithionite and Zn2+ chelation by D-penicillamine established that TSQ is more fluorescent in decanol or PC vesicles than in aqueous buffer, has a high affinity for lipid bilayers and is specific for Zn2+ compared to Mg2+ and Ca2+. Fluorescence measurement of vesicles with and without pretreatment with Zn2+ indicated that, in the absence of Zn2+, 90% of the residual TSQ fluorescence was destroyed by dithionite but > 50% was protected by the presence of Zn2+. When D-penicillamine was added the remaining fluorescence was quenched (T1/2 = 10 s) indicating that TSQ remains in/on the membrane. These results established that TSQ can be used to effectively evaluate Zn2+ in artificial membranes and sperm cells. Additional experiments will be necessary to explain the dynamics of TSQ:Zn:membrane interactions.
锌可稳定细胞膜和DNA，并抑制体细胞的呼吸作用。它在雄性生殖道中含量很高，可能在受精前使精子稳定。在此，我们使用荧光分光光度法和流式细胞术评估N-(6-甲氧基-8-喹啉基)-对甲苯磺酰胺(TSQ)用于分析磷脂酰胆碱(PC)囊泡和活精子中的Zn2+。将TSQ:Zn在癸醇或PC囊泡中的荧光与在水性缓冲液中的荧光进行比较。对阳离子特异性、TSQ:Zn结合动力学、连二亚硫酸盐对TSQ的淬灭以及D-青霉胺对Zn2+的螯合作用的评估表明，TSQ在癸醇或PC囊泡中比在水性缓冲液中更具荧光性，对脂质双层具有高亲和力，并且与Mg2+和Ca2+相比对Zn2+具有特异性。对经过和未经过Zn2+预处理的囊泡进行荧光测量表明，在没有Zn2+的情况下，90%的残余TSQ荧光被连二亚硫酸盐破坏，但>50%被Zn2+的存在所保护。当加入D-青霉胺时，剩余的荧光被淬灭(T1/2 = 10秒)，表明TSQ保留在膜内/膜上。这些结果表明，TSQ可用于有效评估人工膜和精子细胞中的Zn2+。需要进行更多实验来解释TSQ:Zn:膜相互作用的动力学。
