A novel post-translational modification found in rat liver fatty acid-binding ptotein. Its physiological significance.

脂肪酸结合蛋白是一类对长链脂肪酸具有高亲和力的低分子量胞质蛋白。以前我们发现，大鼠肝脏脂肪酸结合蛋白中最酸性的分子种类选择性地结合花生四烯酸，花生四烯酸是生理上重要的类花生酸前体。在本研究中，我们确定这种修饰的物种是异戊酰-105蛋白。这种修饰被认为是在老化蛋白质中发现的自发和非酶促过程。然而，我们的研究结果强烈表明异戊酰键形成的生理意义，因为大鼠肝脂肪酸结合蛋白是相当短的寿命的蛋白质和改性的物种优先结合花生四烯酸。我们推测某些细胞成分可能参与了这一过程，并试图鉴定它们。为此，首先发明了一种检测和定量修饰物种的方法。蛋白质用甲醇HCl甲基化，然后用硼氢化锂还原。通过这种方法，异丙酰基残基被转化为异高丝氨酸，而正常的丙酰基残基被鉴定为高丝氨酸。这些产物可以用普通的氨基酸分析仪进行定量，但灵敏度有待提高。还可以通过在碳酸氢钠缓冲液中孵育蛋白质来制备异戊酰-105物质。这允许获得大量的改性物质用于表征。还观察到天冬酰胺-2的自发但特异性脱酰胺化。在本研究过程中，发现了一种新的分子物种，其在半胱氨酸-69处具有与半胱氨酸混合的二硫化物，并对其功能进行了表征。

Fatty acid-binding proteins are low-molecular-mass cytosolic protein with high affinity for long chain fatty acids. Previously we found that the most acidic molecular species of rat liver fatty acidbinding protein selectively bound arachidonate, the physiologically important precursor of eicosanoids. In the present investigation, we identified this modified species to be isoaspartyl-105 protein. This modification has been thought to be a spontaneous and non-enzymatic process found in aged proteins. However, our finding strongly suggests the physiological significance of the isoaspartyhl-bond formation, since rat liver fatty acid-binding protein is rather short-lived protein and the modified species preferentially binds arachidonate. We supposed that some cellular component (s) may working on this process, and tried to identify them. For this purpose, a method to detect and quantify the modified species was first invented. The protein was methylated with mathanolic HCl and then reduced with lithium borohydride. By this method, isoaspartyl residues were converted to isohomoserine, while normal aspartyl residues were identify as homoserine. These products could be quantified by the ordinary amino acid analyzer, though some improvement for sensitivity was necessary. Isoaspartyl-105 species could also be prepared by incubating the protein in sodium bicarbonate buffer. This allowed to obtain large amounts of the modified species for characterization. Spontaneous but specific deamidation at asparagine-2 was also observed. During the course of this study a new molecular species having a mixed disulfide with cysteine at cysteine-69 was discovered and characterized for the function.