›› 2002, Vol. 45 ›› Issue (3): 307-312.

• RESEARCH PAPERS • Previous Articles     Next Articles

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

CHENG Yuan-Guo1, WU Hou-Yong1,LI De-Chang2, LI Cheng-Wen1, ZHAO Tong-Yan1   

  • Online:2002-06-20 Published:2002-06-20

Abstract: 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 Km, obtained with ATP and ADP as substrates, were evaluated 0.2 μmol/L and the approach Vmax 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.

Key words: Haemaphysalis longicornis, salivary gland, apyrase, enzymic hydrolysis