摘要
利用染料亲和层析 (CibacornBlue柱 )和离子交换层析 (MacrosphereWCX柱 )对长角血蜱Haemaphysalislongicornis唾液腺的腺苷三磷酸双磷酸酶进行纯化 ,经SDS PAGE证实其分子量为 6 6kD。腺苷三磷酸双磷酸酶可以水解ATP和ADP ,但对AMP无水解作用 ,水解ATP和ADP的Km 值均为 0 2 μmol L ,Vmax值分别为 12 5和 15 6 μmol (min·mg)。腺苷三磷酸双磷酸酶水解ATP的中间产物是ADP ,最终产物是AMP和正磷酸。表明腺苷三磷酸双磷酸酶水解ATP的位点是 5′ 核苷酸的γ 磷酸键 ,水解ADP的位点是 5′ 核苷酸的 β 磷酸键。
Apyrase was isolated from soluble crude salivary glands extract of \%Haemaphysalis longicornis\% by a Cibacron Blue column and a Macrosphere weak cation-exchange column. Apyrase could hydrolyse ATP and ADP, but failed to hydrolyse AMP. The approach K m, obtained with ATP and ADP as substrates, were evaluated 0.2 μmol/L and the approach V max were 12.5 and 15.6 μmol/(min·mg) respectively. During the hydrolysis of ATP as monitored by HPLC, the UV-absorbing compounds observed were the ADP and AMP,and for ADP was the AMP only. Direct evidence showed that the hydrolysis site of ATP and ADP occurred by cleavage at the phosphate residues γ and β to the 5′ carbon atom of the ribose was obtained by enzymically hydrolyzing both [γ- 32 P] ATP and [β- 32 P] ADP and separating the products by ion-exchange HPLC. The mechanism of ATP and ADP hydrolysis is sequential removal of phosphate residues to produce, finally, AMP.
出处
《昆虫学报》
CAS
CSCD
北大核心
2002年第3期307-312,共6页
Acta Entomologica Sinica
基金
国家自然科学基金项目 ( 39170 5 91)
