Reyes R, Carranco A, Huacuja L, Delgado N M
División de Biologia del Desarrollo, Unidad de Investigación Biomédica de Occidente, Guadalajara, Jalisco, Mexico.
Arch Androl. 1991 Mar-Apr;26(2):53-60. doi: 10.3109/01485019108987626.
The release and phosphorylation/dephosphorylation mechanisms of human spermatozoa histone during nuclei in vitro decondensation by heparin was studied. Washed sperm cells were incubated in the presence of 32P and in the absence or presence of heparin. The results showed an increase in the incorporation of 32P of 20 times greater in the presence of heparin than in the absence of heparin (the control sample). In some cases the incorporation of 32P into histones was confirmed by its isolation. To validate these results a phosphorylation kinetic of isolated sperm histone, used as a substrate, was performed. The amount of 32P was not a linear function of time, and maximal phosphorylation was reached in 60 min. A measurement of 32P incorporated as a function of the amount of histone, shows a linear relationship of up to 50 micrograms of protein, with a rapid saturation thereafter with the incorporation of 220 nm and with a KD = 442 x 10(-6) mol/L. 32P incorporation, independent of exogenous cAMP, was related to alkaline pH but was totally dependent on temperature--with a maximum of 37 degrees C. The only histone released was histone H-3. Phosphorylation/dephosphorylation is involved during male pronuclei formation.
研究了肝素诱导人精子细胞核体外解聚过程中组蛋白的释放及磷酸化/去磷酸化机制。将洗涤后的精子细胞在有或无肝素存在的情况下于32P环境中孵育。结果显示，与无肝素（对照样品）相比，有肝素存在时32P的掺入量增加了20倍。在某些情况下，通过对组蛋白进行分离证实了32P掺入其中。为验证这些结果，对用作底物的分离精子组蛋白进行了磷酸化动力学研究。32P的量不是时间的线性函数，60分钟时达到最大磷酸化。作为组蛋白量的函数对掺入的32P进行测量，结果显示在蛋白质含量高达50微克时呈线性关系，此后随着220纳米的掺入迅速饱和，KD = 442×10(-6)摩尔/升。32P的掺入与外源性环磷酸腺苷无关，与碱性pH有关，但完全依赖于温度——在37摄氏度时达到最大值。唯一释放的组蛋白是组蛋白H-3。在雄原核形成过程中涉及磷酸化/去磷酸化。
