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>Comparison of the catalytic activities of ricin A-chain, maize rproRIP1, maizeRIP1, and two maize rproRIP1 deletion mutants interacting with an RNA 10-mer GAGA tetraloop.
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Comparison of the catalytic activities of ricin A-chain, maize rproRIP1, maizeRIP1, and two maize rproRIP1 deletion mutants interacting with an RNA 10-mer GAGA tetraloop.
Ribosome-inactivating proteins (RIPS) catalytically depurinate a conserved adenine in the ribosomal α-sarcin domain, and it is believed that the local structure surrounding the target adenine is (at least transiently) an RNA GAGA tetraloop with the first A in the GAGA sequence being the target adenine. Studies of the catalytic activity of ricin A-chain interacting with ribosomes and with RNA GAGA tetraloops, as well as the catalytic activities of a set of related maize RIPs interacting with ribosomes have been reported in the literature. The purpose of this research project was to extend our understanding of maize RIP1 catalytic activity by measuring and comparing the catalytic activities of ricin A-chain and a set of related maize RIPs interacting with an RNA 10-mer GAGA tetraloop (denoted by A-10) over a range of pH values from pH 3.0 to 6.0.; Timecourse experiments of A-10/enzyme reactions were used to measure catalytic activity by quantitative HPLC detection of the adenine freed during a succession of elapsed time intervals as the depurination reaction proceeded. The form of the timecourse data did not conform sufficiently to the integrated Michaelis-Menten equation to permit the use of nonlinear curve fitting to characterize the results in terms of the Michaelis-Menten parameters K M and kcat. Thus, the comparison of the activities of ricin A-chain and the various maize RIP variants both among themselves and as a function of pH, although definitive, is semiquantitative. For one of the enzymes, maizeRIP1, initial velocity experiments at high substrate concentrations yielded an approximate Vmax directly, which was then used to treat V max as a constant rather than a variable in fitting the data to the integrated Michaelis-Menten equation, and corresponding values for K M were obtained. However, these substrate concentrations were not sufficiently high to yield Vmax values for the other enzymes.; One unexpected result, with important ramifications concerning the conformation of the maize proRIP1, was evidence that maize rproRIP1, which is a zymogen with respect to ribosomes, may be catalytically active with respect to the small RNA tetraloop A-10.
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机译：核糖体失活蛋白（RIPS）在核糖体α-sarcin结构域中催化去嘌呤嘌呤的保守嘌呤，据信靶腺嘌呤周围的局部结构是（至少暂时地）GAGA序列中第一个A的RNA GAGA四环是目标腺嘌呤。在文献中已经报道了蓖麻蛋白A链与核糖体和RNA GAGA四环相互作用的催化活性，以及与玉米核糖体相互作用的一系列相关玉米RIP的催化活性的研究。该研究项目的目的是通过测量和比较蓖麻毒素A链和一组与RNA 10-mer GAGA四环相互作用的相关玉米RIP（由A-10表示）的催化活性来扩展我们对玉米RIP1催化活性的了解。 ）在pH值从3.0到6.0的范围内； A-10 /酶反应的时程实验用于通过定量HPLC检测随着去纯化反应的进行在一系列经过的时间间隔内释放的腺嘌呤来测量催化活性。时间过程数据的形式与整合的Michaelis-Menten方程不完全吻合，无法使用非线性曲线拟合来根据Michaelis-Menten参数K M 和k 猫。因此，尽管是确定的，但蓖麻蛋白A链和各种玉米RIP变体之间的相互作用以及作为pH的函数的比较虽然是确定的，但仍是半定量的。对于其中一种酶maizeRIP1，在高底物浓度下的初始速度实验直接产生了近似的V max ，然后将其用作常数而不是a的V max 。变量以使数据适合于集成的Michaelis-Menten方程，并获得K M 的对应值。但是，这些底物浓度不足以产生其他酶的V max 值。一个出乎意料的结果是，与玉米proRIP1的构型有关的重要方面，是玉米rproRIP1（相对于核糖体而言是酶原）相对于小RNA四环A-10可能具有催化活性的证据。
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