Analysis of aspartic acid and asparagine metabolism in Xenopus laevis oocytes using a simple and sensitive HPLC method.
The amino acids in methanol-soluble extracts of Xenopus oocytes were measured using a method involving precolumn derivatization with phenylisothiocyanate and reverse phase HPLC of the derivatized amino acids. This technique allows the estimation of asparagine and glutamine pools in oocytes, estimated as 70 and 283 pmoles per oocyte, respectively. The pool sizes of the other amino acids were similar to previously reported results obtained using conventional ion exchange chromatography and postcolumn derivatization with ninhydrin. The advantages of the method developed here include picomolar sensitivity and the enhanced resolution of asparagine and glutamine from other amino acids. The kinetics of aspartic acid and asparagine utilization were monitored following microinjection of oocytes with [3H]aspartic acid and [14C]asparagine. The aspartic acid pool turned over rapidly with a half-time of < 30 min. The asparagine pool was metabolized much more slowly and appeared to be utilized almost completely for protein synthesis. The absolute rate of protein synthesis in oocytes was calculated from the incorporation data and chemical pool measurements as approximately 25 ng/hr-oocyte. The methodology developed here may be useful in experimental situations involving limited amounts of biological material.
O'Connor
C M
CM
Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545.
D000596:Amino Acids / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D001216:Asparagine / Q000378:metabolism*; D001224:Aspartic Acid / Q000378:metabolism*; D002250:Carbon Radioisotopes; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D005260:Female; D007700:Kinetics; D009865:Oocytes / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013997:Time Factors; D014316:Tritium; D014982:Xenopus laevis
D000596:Amino Acids; D002250:Carbon Radioisotopes; D014316:Tritium; D001224:Aspartic Acid; D001216:Asparagine