Croce A C, Bottiroli G, Prosperi E, Supino R, Stoward P J
Centro di Studio per l'Istochimica del CNR, Department of Animal Biology, University of Pavia, Italy.
Histochem J. 1988 Nov;20(11):595-602. doi: 10.1007/BF01324077.
The cytochemical quantification of catechol oxidase activity in fixed B16 melanoma cells was investigated using dopa as the substrate. Inhibitors showed that peroxidases do not significantly interfere. The kinetics of melanin formation were studied initially in solution with purified catechol oxidase. Two key parameters were identified: lag-time and the rate of melanin formation. The lag-time was taken as the time required by intermediates to reach a critical concentration at which the polymerization process starts and melanin production becomes measurable (at 640 nm). In solution, the lag-time decreases as the enzyme activity increases, particularly when the activity is very low. The rate at which melanin is formed by pure enzyme in solution is independent of dopa concentration when its activity is low but increases linearly with dopa concentration when the activity is comparatively high. In fixed melanoma cells, the lag-time decreases linearly with increases of dopa concentrations up to 20 mM; at concentrations higher than this, the lag decreases more slowly. In contrast, the rate of melanin production is unaffected by changes in dopa concentration. The lag-times of different cells lines incubated at the same substrate concentration decrease as the enzyme activity of the cells increases. The rate of melanin production seems to be affected by factors other than catechol oxidase activity, such as the intracellular organization and distribution of the enzyme.
以多巴为底物，对固定化的B16黑色素瘤细胞中儿茶酚氧化酶活性进行了细胞化学定量研究。抑制剂表明过氧化物酶不会产生显著干扰。最初在溶液中用纯化的儿茶酚氧化酶研究了黑色素形成的动力学。确定了两个关键参数：延迟时间和黑色素形成速率。延迟时间被定义为中间体达到临界浓度所需的时间，此时聚合过程开始，黑色素生成变得可测量（在640nm处）。在溶液中，随着酶活性增加，延迟时间缩短，尤其是当活性非常低时。当纯酶在溶液中的活性较低时，其形成黑色素的速率与多巴浓度无关，但当活性相对较高时，其与多巴浓度呈线性增加。在固定化的黑色素瘤细胞中，延迟时间随着多巴浓度增加至20 mM而线性缩短；高于此浓度时，延迟缩短得更慢。相反，黑色素生成速率不受多巴浓度变化的影响。在相同底物浓度下孵育的不同细胞系的延迟时间随着细胞酶活性增加而缩短。黑色素生成速率似乎受儿茶酚氧化酶活性以外的因素影响，如酶在细胞内的组织和分布。
