Synthesis and removal of phenylalanine ammonia-lyase activity in illuminated discs of potato tuber parenchyme.
Synthesis and removal of phenylalanine ammonia-lyase activity in illuminated discs of potato tuber parenchyme.
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马铃薯块茎薄壁组织照明圆盘中苯丙氨酸解氨酶活性的合成和去除。
DOI：
10.1016/0304-4165(79)90384-2
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发表时间：
1979
期刊：
影响因子：
--
通讯作者：
V. S. Butt
中科院分区：
文献类型：
--
作者：
C. Lamb;T. Merritt;V. S. Butt
1.(1)用氧化氘（2H2O）中的氘（2H）密度标记，然后在CsCl密度梯度中离心至平衡，研究了辐照马铃薯（Solanum tuberosumcv King Edward）块茎组织中苯丙氨酸解氨酶（EC 4.3.1.5）的合成和去除。(2)酶水平的时间变化用公式(dE/dt) =ks−kde其中（dE/dt）是单位组织(E)的酶水平相对于时间(t)的变化率，k是酶的合成速率常数，k是活性酶的去除速率常数。(3)通过分析2h2o浓度、酶活性的发展和苯丙氨酸氨解酶浮力密度增加幅度之间的关系，确定了最佳的2h2o浓度。发现约40% （v/v）的2h2o浓度是最佳的，可以在不抑制酶活性发展的情况下实现酶的浮力密度的最大或接近最大的增加，从而避免了2h2o作为密度标签来源的主要缺点。(4)密度梯度离心后酶活性的重叠分布曲线通过最佳拟合的迭代方法解决，该方法允许在标记期结束时估计预先存在的未标记酶和新合成的标记酶的比例。这项技术的发展是为了获得酶合成的速率常数和在酶水平快速变化期间去除活性酶的速率常数。(5)光照圆盘中苯丙氨酸解氨酶活性的初始快速增加反映了在不激活已有酶和不去除活性酶的情况下酶合成速率常数的增加。突然过渡到酶活性下降的阶段是由(a)酶合成速率常数的降低和(b)去除活性酶的速率常数的急剧增加引起的。随后酶水平的稳定是由两个速率常数衰减到相对较低的水平引起的。(6)结果与假设一致，即组织分化过程中酶水平的快速调节是通过同时改变酶合成和活性酶去除的速率常数来实现的。
1.(1) The synthesis and removal of phenylalanine ammonia-lyase (EC 4.3.1.5) in illuminated discs of potato (Solanum tuberosumcv King Edward) tuber tissue has been investigated by density labelling with deuterium (2H) from deuterium oxide (2H2O) followed by centrifugation to equilibrium in a CsCl density gradient.2.(2) Temporal changes in enzyme level have been described in terms of the equation (dE/dt) =ks−kdEwhere (dE/dt) is the rate of change of enzyme level per unit of tissue (E) with respect to time (t),ksis the rate constant for synthesis of the enzyme andkdis the rate constant for the removal of active enzyme.3.(3) The optimal concentration of2H2O was determined by analysis of the relationship between2H2O concentration, development of enzyme activity and the magnitude of the increase in buoyant density of phenylalanine ammonialyase. A concentration of2H2O of about 40% (v/v) was found to be optimal, allowing achievement of maximal or near maximal increases in the buoyant density of the enzyme without inhibition of the development of enzyme activity, thereby circumventing the major drawback of2H2O as a source of density label.4.(4) The overlapping distribution profiles of enzyme activity after density gradient centrifugation were resolved by an iterative method of best fit which allows estimation of the proportions of pre-existing, unlabelled enzyme and newly synthesised, labelled enzyme at the end of the labelling period. This technique has been developed to obtain the rate constants for enzyme synthesis and for removal of active enzyme throughout the period of rapid change in enzyme level.5.(5) It is demonstrated that the initial rapid increase in phenylalanine ammonia-lyase activity in illuminated discs reflects an increase in the rate constant for enzyme synthesis in the absence of activation of pre-existing enzyme and in the absence of removal of active enzyme. The abrupt transition to a phase of decline in enzyme activity is caused by (a) a reduction in the rate constant for enzyme synthesis and (b) a dramatic increase in the rate constant for removal of active enzyme. The subsequent stabilisation of the enzyme level is caused by decay of both rate constants to relatively low levels.6.(6) The results are consistent with the hypothesis that rapid modulation of enzyme levels during tissue differentiation is achieved by simultaneous changes in the rate constants for both enzyme synthesis and for removal of active enzyme.