Kagan H M, Lerch R M
Biochim Biophys Acta. 1976 May 20;434(1):223-32. doi: 10.1016/0005-2795(76)90054-4.
Dicarboxylic amino acids constitute the most numerous residues of insoluble elastin in which are potentially ionizable in the physiological range of pH. These residues are essential in facilitating productive electrostatic interaction between elastase and elastin. The present study has investigated the possibility that the glutamic and aspartic acid residues of elastin are amidated. Acid-labile amide-bound ammonia of elastin was quantitated after hydrolysis of the insoluble protein with 2 M HC1 by incubating aliquots of microdistilled hydrolysates with glutamate dehydrogenase, excess alpha-ketoglutarate, and reduced nicotinamide adenine dinucleotide and measuring the resultant decrease in A340 due to oxidation of the dinucleotide cofactor. It was found that ligament elastin purified by repeated autoclaving contains approximately 2.29 mumol of acid-labile amide nitrogen per 10 mg of protein, a value equivalent to approximately 70% of the total number of dicarboxylic amino acid residues. Independent analysis of the amide content was obtained by amino acid analysis of an esterified and reduced elastin sample in which the free dicarboxylic amino acid residues had been converted to the corresponding alcohol derivatives. This analysis indicated that autoclaved ligament elastin contains approximately 18 glutamine, 3 asparagine, 4 glutamic acid and 5 aspartic acid residues per 1000 residues, in good agreement with the analysis of total acid-labile ammonia. The esterified and reduced elastin derivative was nearly inert as an elastase substrate, consistent with a lack of free dicarboxylic amino acid residues. However, addition of sodium dodecyl sulfate to this elastin derivative restores enzyme-substrate charge complementarity, and the elastin-ligand complex was readily hydrolyzed by elastase at the fully stimulated rate, emphasizing the control such ligands can exert in elastolysis. The amide bonds of elastin were found to be significantly more resistant to hydrolysis by 0.1 M NaOH at 98 degrees C than were those of lysozyme or free amidated amino acids. The finding that most of dicarboxylic amino acid residues of elastin exist at neutral amides further emphasizes the apolar character of elastin and has bearing upon the metabolic susceptibility, ligand-binding ability and structural aspects of this connective tissue protein.
二羧酸氨基酸构成了不溶性弹性蛋白中数量最多的残基，这些残基在生理pH范围内具有潜在的可电离性。这些残基对于促进弹性蛋白酶与弹性蛋白之间有效的静电相互作用至关重要。本研究探讨了弹性蛋白的谷氨酸和天冬氨酸残基被酰胺化的可能性。在用2M盐酸水解不溶性蛋白质后，通过将微量蒸馏水解物的等分试样与谷氨酸脱氢酶、过量的α-酮戊二酸和还原型烟酰胺腺嘌呤二核苷酸一起孵育，并测量由于二核苷酸辅因子氧化导致的A340的下降，对弹性蛋白中酸不稳定的酰胺结合氨进行了定量。结果发现，通过反复高压灭菌纯化的韧带弹性蛋白每10mg蛋白质含有约2.29μmol酸不稳定的酰胺氮，该值约相当于二羧酸氨基酸残基总数的70%。通过对酯化和还原的弹性蛋白样品进行氨基酸分析获得了酰胺含量的独立分析结果，在该样品中游离的二羧酸氨基酸残基已转化为相应的醇衍生物。该分析表明，经高压灭菌的韧带弹性蛋白每1000个残基中约含有18个谷氨酰胺、3个天冬酰胺、4个谷氨酸和5个天冬氨酸残基，与总酸不稳定氨的分析结果吻合良好。酯化和还原的弹性蛋白衍生物作为弹性蛋白酶底物几乎没有活性，这与缺乏游离的二羧酸氨基酸残基一致。然而，向该弹性蛋白衍生物中加入十二烷基硫酸钠可恢复酶-底物电荷互补性，并且弹性蛋白-配体复合物很容易被弹性蛋白酶以完全刺激的速率水解，这强调了此类配体在弹性蛋白水解中可发挥的控制作用。发现弹性蛋白的酰胺键在98℃下比溶菌酶或游离酰胺化氨基酸的酰胺键对0.1M氢氧化钠水解的抗性明显更强。弹性蛋白的大多数二羧酸氨基酸残基以中性酰胺形式存在这一发现进一步强调了弹性蛋白的非极性特征，并与这种结缔组织蛋白的代谢敏感性、配体结合能力和结构方面有关。
