OPTIMAL CONDITIONS FOR THE USE OF PROTEIN L-ISOASPARTYL METHYLTRANSFERASE IN ASSESSING THE ISOASPARTATE CONTENT OF PEPTIDES AND PROTEINS
OPTIMAL CONDITIONS FOR THE USE OF PROTEIN L-ISOASPARTYL METHYLTRANSFERASE IN ASSESSING THE ISOASPARTATE CONTENT OF PEPTIDES AND PROTEINS
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DOI：
10.1016/0003-2697(91)90553-6
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发表时间：
1991-02-01
影响因子：
2.9
通讯作者：
ASWAD, DW
中科院分区：
文献类型：
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作者：
JOHNSON, BA;ASWAD, DW
蛋白质l-异天冬氨酸甲基转移酶为非典型异天冬氨酸键的酶促测定提供了基础，异天冬氨酸键对蛋白质微观异质性有重要贡献。甲基转移酶反应的低vmax和甲酯的不稳定性会影响准确的测定，不同的实验室在不同的条件下对相同蛋白质的异天冬氨酸含量得到了不同的值。为了研究这些条件的影响，并对实验进行优化，我们在不同条件下甲基化异天冬氨酸δ睡眠诱导肽。我们发现，在pH 6.2和30°C条件下，1 μm甲基转移酶在40分钟的反应中获得2 μm肽的化学计量修饰。利用从甲基化初始速率研究中获得的动力学常数的计算机模型预测了对酶浓度的相同要求。在酶浓度低于0.4 μm时，载体蛋白是保证甲基转移酶活性的必要条件。化学计量甲基化要求s -腺苷蛋氨酸的浓度远远超过肽的浓度；50 μ ms -腺苷蛋氨酸是完成10 μm肽修饰的最低要求。自发去甲基化在所有测试条件下都是显著的，因此甲酯本身从未达到总肽1 mol/mol的比例。这些结果表明，当反应在低肽浓度、高s -腺苷蛋氨酸浓度和高酶浓度下进行时，可以获得最准确的异天冬氨酸测量值。此外，当基于甲醇和甲酯的测量而不是仅基于完整甲酯的测量时，定量将明显更好。
Protein l-isoaspartyl methyltransferase provides a basis for enzymatic measurement of atypical, isoaspartyl linkages which make a major contribution to protein microheterogeneity. The low Vmaxof the methyltransferase reaction and the instability of the methyl ester can hinder accurate determinations, and different laboratories using different conditions have achieved discrepant values for the isoaspartate content of the same proteins. To investigate the effects of these conditions, and to optimize the assay, isoaspartyl δ sleep-inducing peptide was methylated under a variety of conditions. We found that 1 μm methyltransferase was required to obtain stoichiometric modification of 2 μm peptide in 40-min reactions at pH 6.2 and 30°C. A computer model utilizing kinetic constants obtained from studies on initial rates of methylation predicted the same requirement for enzyme concentration. Carrier protein was necessary for optimal methyltransferase activity at enzyme concentrations below 0.4 μm. Stoichiometric methylation required concentrations of S-adenosylmethionine to be in substantial excess over those of peptide; 50 μmS-adenosylmethionine is the minimum needed for complete modification of 10 μm peptide. Spontaneous demethylation was significant under all conditions tested, so that the methyl ester itself never reached a ratio of 1 mol/mol of total peptide. These results demonstrate that the most accurate measurements of isoaspartate are obtained when reactions are carried out at low peptide concentrations, high S-adenosylmethionine concentrations, and high enzyme concentrations. Moreover, quantitation will be significantly better when based on measurements of both methanol and methyl esters rather than on measurements of intact methyl esters alone.