Degradation kinetics of methionine5-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-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(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°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; t1/2 26.2 ± 5.5 and 20.8-33.8 minutes, and Iv 0.03 ±0.01 and 0.02-0.03 pg of peptide per milligram protein per minute. Km and Vmax values were 0.19 ± 0.02 and 0.17-0.21 mM, and 9.8 ± 2.2 and 7.6-12.0 μmol·L·min, 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.
Mosnaim
Aron D
AD
Department of Cellular and Molecular Pharmacology, Rosalind Franklin University of Medicine and Science/Chicago Medical School, N. Chicago, IL 60064, USA. aron.mosnaim@rosalindfranklin.edu
Chuang
FuCheng
F
Wolf
Marion E
ME
D000368:Aged; D000596:Amino Acids / Q000032:analysis; D015415:Biomarkers; D004744:Enkephalin, Methionine / Q000134:cerebrospinal fluid*; D005260:Female; D006207:Half-Life; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D008875:Middle Aged; D013048:Specimen Handling; D014443:Tyrosine / Q000032:analysis
D000596:Amino Acids; D015415:Biomarkers; D014443:Tyrosine; D004744:Enkephalin, Methionine