Study of leucine-enkephalin in rat brain by a new high-performance liquid chromatographic method.
A specific method is presented for the assay of leucine-enkephalin (Leu-Enk) and its metabolites by reversed-phase high-performance liquid chromatography on a muBondapak C18 column with a mobile phase of methanol-water and 0.005 M tetrabutylammonium phosphate. The four substances are resolved by a linear program from 8 to 70% methanol in 25 min. Detection is achieved by monitoring the absorbance at 280 nm. Time of analysis can be reduced by means of a new high-speed liquid chromatography package. This method allows the study of the effect of Phe-Ala on the cerebral metabolism of Leu-Enk. For this purpose, membrane preparations from rat striatum were incubated in the presence of [3H]Leu-Enk with different concentrations of Phe-Ala from 10(-7) to 10(-3) M during 1 h at 37 degrees C. Collected eluates were counted by liquid scintillation. The results suggest the presence of three membrane enzymes which generate the three metabolites, Tyr, Tyr-Gly-Gly and Tyr-Gly, in order of abundance. Maximum inhibition of [3H]Leu-Enk degradation is obtained at a concentration of 10(-3) M Phe-Ala.
D000818:Animals; D001923:Brain Chemistry; D002851:Chromatography, High Pressure Liquid; D003342:Corpus Striatum / Q000032:analysis; D004743:Enkephalin, Leucine / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains
D004743:Enkephalin, Leucine