Tsuda T, Kaya S, Funatsu H, Hayashi Y, Taniguchi K
Biological Chemistry, Graduate School of Science, Hokkaido University, Sapporo.
J Biochem. 1998 Jan;123(1):169-74. doi: 10.1093/oxfordjournals.jbchem.a021906.
The modification of Na+,K+-ATPase with increasing pyridoxal 5'-diphospho-5'-adenosine (AP2PL) concentrations resulted in saturation of the approximately 0.5 mol AP2PL probe incorporation into the Lys-480/mol catalytic alpha-chain and reduced the Na+,K+-ATPase activity to around half without affecting the phosphorylation by acetyl phosphate (AcP), and led to increases in the AP2PL fluorescence caused by ATP and AcP. Further modification with fluorescein 5'-isothiocyanate (FITC) resulted in approximately 0.9 mol FITC probe incorporation into the Lys-501/mol alpha-chain and reduced the activity to below 5% without affecting the phosphorylation by AcP and these fluorescence increases. The ATP binding capacity of the AP2PL-FITC enzyme was shown to be at least 50% of that of the control enzyme (approximately 0.8 mol/mol alpha-chain). This is the first direct demonstration that Na+-bound FITC-modified enzymes accept ATP with an affinity for ATP (K(1/2) > 150 microM) reduced by two orders of magnitude. The data also suggest half site reactivity of Lys-480 as to AP2PL and all site reactivity of Lys-501 as to FITC in the catalytic subunits.
随着5'-磷酸吡哆醛-5'-腺苷（AP2PL）浓度增加对钠钾ATP酶进行修饰，导致每摩尔催化性α链中约0.5摩尔AP2PL探针掺入达到饱和，钠钾ATP酶活性降低至约一半，而不影响乙酰磷酸（AcP）的磷酸化作用，并导致ATP和AcP引起的AP2PL荧光增加。用异硫氰酸荧光素（FITC）进一步修饰导致每摩尔α链中约0.9摩尔FITC探针掺入，活性降低至5%以下，而不影响AcP的磷酸化作用以及这些荧光增加。AP2PL - FITC酶的ATP结合能力显示为对照酶（约每摩尔α链0.8摩尔）的至少50%。这是首次直接证明与钠结合的FITC修饰酶接受ATP时，对ATP的亲和力（K(1/2) > 150 microM）降低了两个数量级。数据还表明，在催化亚基中，Lys - 480对AP2PL具有半位点反应性，而Lys - 501对FITC具有全位点反应性。
