Dorsten F A, Wyss M, Wallimann T, Nicolay K
Department of in vivo NMR Spectroscopy, Bijvoet Center for Biomolecular Research, Utrecht University, Bolognalaan 50, NL-3584 CJ Utrecht, The Netherlands.
Biochem J. 1997 Jul 15;325 ( Pt 2)(Pt 2):411-6. doi: 10.1042/bj3250411.
The kinetics of the creatine kinase (CK) reaction were studied in suspensions of quiescent and active, intact sea-urchin spermatozoa in artificial seawater, using 31P-NMR magnetization transfer. In inactive sperm, no CK-mediated exchange flux was detected, whereas in activated motile sperm, the forward pseudo-first-order rate constant was 0.13+/-0.04 s-1 at 10 degrees C, corresponding to a steady-state CK flux of 3.1+/-0.5 mM.s-1. Intracellular pH shifted from 6.6+/-0.1 to 7.6+/-0.1 upon activation. The phosphocreatine (PCr)/ATP and PCr/Pi ratios were only marginally reduced in activated sperm, whereas the estimated cytosolic free ADP concentration increased remarkably from 9 microM in quiescent, to 114 microM in activated spermatozoa. The elevation of CK flux upon sperm activation is discussed in the light of the proposition that in sea-urchin spermatozoa, which are fuelled entirely by oxidative phosphorylation, high-energy phosphate transport is mediated by a 'CK/PCr shuttle'.
利用31P - NMR磁化转移技术,研究了静止和活跃的完整海胆精子悬浮于人工海水中时肌酸激酶(CK)反应的动力学。在未活化的精子中,未检测到CK介导的交换通量,而在活化的活动精子中,10℃时正向假一级速率常数为0.13±0.04 s-1,对应稳态CK通量为3.1±0.5 mM·s-1。活化后细胞内pH从6.6±0.1变为7.6±0.1。活化精子中磷酸肌酸(PCr)/ATP和PCr/Pi比值仅略有降低,而估计的胞质游离ADP浓度从静止精子中的9μM显著增加到活化精子中的114μM。根据海胆精子完全由氧化磷酸化供能,高能磷酸转运由“CK/PCr穿梭”介导这一观点,讨论了精子活化时CK通量的升高。