长角血蜱唾液腺中腺苷三磷酸双磷酸酶的纯化及其酶切机制

利用染料亲和层析(Cibacorn Blue柱)和离子交换层析(Macrosphere WCX柱)对长角血蜱Haemaphysalis longicornis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66 kD。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K_m值均为0.2 μmol/L，V_max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。

Purification and mechanism of enzymic hydrolysis of apyrase from salivary glands of Haemaphysalis longicornis

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- ³²P］ATP and ［β-³²P］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.