Hydrolysis of [Leu]enkephalin by chick plasma in vitro.
The in vitro hydrolysis of [Leu]enkephalin added to plasma collected from 2-day-old chicks was studied with two different techniques: thin-layer chromatography separation of intact [3H]-[Leu]enkephalin from its [3H]-Tyr-containing metabolites and high-performance liquid chromatography-electrochemical detection assay of [Leu]enkephalin disappearance and Tyr-containing metabolite accumulation. The radiometric assay evaluated enkephalin hydrolysis at close to presumed physiological concentrations of this peptide, whereas the liquid chromatography assay necessitated 100-fold higher peptide concentrations to achieve adequate sensitivity. Similar results were obtained with both techniques. We found that the in vitro hydrolysis of [Leu]enkephalin is more rapid in chick plasma (half-life, 0.7-1 min) than in rat (half-life, 2-2.5 min) or mouse (half-life, 9-14 min) plasma. Comparison of the rate of enkephalin hydrolysis and pattern of metabolite accumulation in the absence vs. the presence of various peptidase inhibitors suggested that a bestatin-sensitive aminopeptidase, probably aminopeptidase M, is the primary enzyme responsible for the hydrolysis of enkephalin by chick plasma, and that less than 1% of the total hydrolysis of [Leu]-enkephalin by chick plasma is attributable to dipeptidyl carboxy-peptidase activity. This pattern of enzyme activities differs from that which we identified previously in rat and mouse plasma.
Shibanoki
S
S
Department of Pharmacology, Nihon University School of Medicine, Tokyo, Japan.
Weinberger
S B
SB
Beniston
D
D
Nudelman
K A
KA
Schulteis
G
G
Bennett
E L
EL
Rosenzweig
M R
MR
Ishikawa
K
K
Martinez
J L
JL
D000626:Aminopeptidases / Q000502:physiology; D000818:Animals; D002645:Chickens / Q000097:blood; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D004743:Enkephalin, Leucine / Q000378:metabolism*; D006868:Hydrolysis; D066298:In Vitro Techniques; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D008297:Male; D011691:Puromycin / Q000494:pharmacology
D011691:Puromycin; D004743:Enkephalin, Leucine; D000626:Aminopeptidases; D007930:Leucine; C012211:ubenimex