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Free D-amino acid content in some archaea was investigated and D-forms of several amino acids were found in them. In the acidothermophilic archaeon, Thermoplasma acidophilum, the proportion of D-aspartate (D-Asp) to total Asp was as high as 39.7%. Crude extracts of Thermoplasma acidophilum had Asp-specific racemase activity that was pyridoxal 5'-phosphate (PLP)-dependent. The relative insensitivity to a SH-modifying reagent distinguished this activity from those of the PLP-independent Asp racemases found in other hyperthermophilic archaea (Matsumoto, M., et al., J. Bacteriol. 181, 6560-6563 1999). Thus, high levels of d-Asp should be produced by a new type(s) of Asp-specific racemase in Thermoplasma acidophilum, although the function of d-Asp in this archaeon remains unknown.

In our previous studies, we found a relationship between the fluorescence characteristics (the fluorescence intensity, the maximum excitation and emission wavelengths) and Hammett substituent constants (ςp) of the substituent groups at the 4- and 7-positions of benzofurazan compounds, which enabled us to predict the fluorescence characteristics of the benzofurazan compounds. This prompted us to design a new fluorogenic derivatization reagent having a benzofurazan skeleton for carboxylic acids along this line of thought. Accordingly, the fluorogenic reagent, 7-acetylamino-4-mercapto-2,1,3-benzoxadiazole (AABD-SH) was synthesized. The reagent itself fluoresced very weakly but fluoresced strongly after the reaction with carboxylic acids. AABD-SH reacted with carboxylic acids within 5 min at room temperature in the presence of triphenylphosphine and 2,2′-dipyridyl disulfide. The derivatives of five carboxylic acids were separated on a reversed phase column and detected fluorimetrically at 524 nm with excitation at 368 nm. The detection limits were 10–20 fmol (signal-to-noise ratio of 3).

d-Aspartate (d-Asp) is found in prolactin (PRL)-containing cells of the rat anterior pituitary gland [Lee et al., Brain Res. 838, 193-199, 1999]. In order to determine whether d-Asp is actually produced by the anterior pituitary gland and whether it plays a physiological role in PRL function, a PRL-secreting clonal strain of rat pituitary tumor cells (GH(3)) was employed in this study. HPLC analysis and immunocytochemical staining detected the presence and synthesis of d-Asp in the cytoplasm of these cells. In addition, thyrotropin-releasing hormone-stimulated PRL secretion was increased in a dose-dependent fashion by d-Asp from these cells. These results suggest that the anterior pituitary gland synthesizes d-Asp and that d-Asp acts as a messenger in this gland.