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

点击翻译按钮获取中文摘要

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.