Griffith C J, Rea P A, Blumwald E, Poole R J
Department of Biology, McGill University, 1205 Avenue Docteur Penfield, Montreal, Quebec, Canada H3A 1B1.
Plant Physiol. 1986 May;81(1):120-5. doi: 10.1104/pp.81.1.120.
The H(+)-ATPase of tonoplast vesicles isolated from red beet (Beta vulgaris L.) storage tissue was studied with respect to the kinetic effects of Cl(-) and NO(3) (-). N-Ethylmaleimide (NEM) was employed as a probe to investigate substrate binding and gross conformational changes of the enzyme. Chloride decreased the K(m) of the enzyme for ATP but caused relatively little alteration of the V(max). Nitrate increased K(m) only. Michaelis-Menten kinetics applied throughout with respect to ATP concentration. Nitrate yielded similar kinetics of inhibition in both the presence and absence of Cl(-). Other monovalent anions that specifically increased the K(m) of the ATPase for ATP were, in order of increasing K(i), SCN(-), ClO(4) (-), and ClO(3) (-). Sulfate, although inhibitory, manifested noncompetitive kinetics with respect to ATP concentration. ADP, like NO(3) (-), was a competitive inhibitor of the ATPase but ADP and NO(3) (-) did not interact cooperatively nor did either interfere with the inhibitory action of the other. It is concluded that NO(3) (-) does not show competitive kinetics because of its stereochemical similarity to the terminal phosphoryl group of ATP. NEM was an irreversible inhibitor of the tonoplast ATPase. Both Mg.ADP and Mg.ATP protected the enzyme from inactivation by NEM but Mg.ADP was the more potent of the two. Chloride and NO(3) (-) exerted little or no effect on the protective actions of Mg.ADP and Mg.ATP suggesting that neither Cl(-) nor NO(3) (-) are involved in substrate binding.
研究了从红甜菜（Beta vulgaris L.）贮藏组织中分离的液泡膜囊泡的H(+)-ATP酶对Cl(-)和NO(3) (-)的动力学效应。使用N-乙基马来酰亚胺（NEM）作为探针来研究该酶的底物结合和总体构象变化。氯离子降低了该酶对ATP的K(m)，但对V(max)的改变相对较小。硝酸盐仅增加K(m)。在整个ATP浓度范围内都符合米氏动力学。无论有无Cl(-)，硝酸盐都产生类似的抑制动力学。其他特异性增加ATP酶对ATP的K(m)的单价阴离子，按K(i)增加的顺序依次为SCN(-)、ClO(4) (-)和ClO(3) (-)。硫酸盐虽然具有抑制作用，但对ATP浓度表现出非竞争性动力学。ADP与NO(3) (-)一样，是ATP酶的竞争性抑制剂，但ADP和NO(3) (-)既不协同作用，也不相互干扰对方的抑制作用。得出的结论是，NO(3) (-)不表现出竞争性动力学是因为它与ATP的末端磷酰基在立体化学上相似。NEM是液泡膜ATP酶的不可逆抑制剂。Mg.ADP和Mg.ATP都能保护该酶不被NEM灭活，但Mg.ADP在两者中更有效。氯离子和NO(3) (-)对Mg.ADP和Mg.ATP的保护作用影响很小或没有影响，这表明Cl(-)和NO(3) (-)都不参与底物结合。
