Degradation Kinetics Of Methionine(5)-Enkephalin By Cerebrospinal Fluid: In Vitro Studies

Changes in the levels or biochemistry of cerebrospinal fluid (CSF) neuropeptides with opioid-like properties have been suggested to reflect alterations in specific biological processes. We have determined various kinetic parameters for methionine(5)-enkephalin (MET) degradation by CSF samples from nonneurological patients. Study subjects included 9 males (51-67 years of age) and 5 females (47-61 years of age). Aliquots, removed from an incubation vessel containing buffer, CSF, and peptide [tyr-3',5'-H-3(N)MET], were analyzed for tyrosine and other degradation products. Essentially all of the labeled tyrosine from the added MET was recovered as free amino acid after 60 minutes of incubation (1:2 ratio, vol:vol; optimum pH 7.4; and temperature 37 degrees C); other possible peptide metabolites (>3%) were not detected. Irrespective of age or gender, the peptide's degradation half-life and initial velocity values were in a limited range; t(1/2) 26.2 +/- 5.5 and 20.8-33.8 minutes, and I-v 0.03 +/- 0.01 and 0.02-0.03 pg of peptide per milligram protein per minute. K-m and V-max values were 0.19 +/- 0.02 and 0.17-0.21 mM, and 9.8 +/- 2.2 and 7.6-12.0 mu mol.L-1.min(-1), respectively. Neither CSF sample storage time (up to a year) nor repeated freezing and thawing (up to 3 times over a year) altered the kinetics or products of this reaction. These preliminary findings might serve as reference values when conducting similar studies using CSF from patients diagnosed with specific neurological conditions; significant alterations in MET degradation profile in such a population could provide valuable biological markers for diagnostic and treatment purposes.