Release in vivo of Met-enkephalin and encrypted forms of Met-enkephalin from brain and spinal cord of the anesthetized cat.
Processing of the proenkephalin molecule will result in peptide fragments in which the pentapeptide YGGFM is included. We have employed a molecular sieve (2 kDa) separation, enzyme hydrolysis radioimmunoassay (RIA) treatment sequence which permits concurrent measurement of Met-enkephalin (Enk) and several enkephalin-encrypting (X-Enk) peptides in a single sample. Using this protocol, the release of Enk and X-Enk (total Enk-Enk) greater and less than 2 kDa from spinal cord and the mesencephalic aqueductal grey was assessed under resting conditions and during stimulation of the sciatic nerve in the chloralose-urethane anesthetized cat. Under resting conditions measurable levels of Enk (10.5 +/- 4.7; 9.1 +/- 2.1 pg/min) and X-Enk (47.8 +/- 19.7; 45.7 +/- 12.3 pg/min) are found in aqueductal and spinal superfusates, respectively. The X-Enk measured under resting and evoked conditions in aqueductal and spinal perfusates is associated almost exclusively (greater than 90-95%) with fragments greater than 2 kDa in size. There results, showing the relative absence of detectable levels of X-Enk forms greater than 2 kDa, were confirmed by reverse phase chromatography. During sciatic nerve stimulation, the levels of both Enk and X-Enk were mildly elevated in spinal and ventricular perfusates. With the addition of thiorphan (10(-5) M), though there was no effect on the resting release of either Enk or X-Enk, the levels of Enk measured under evoked conditions were significantly augmented in both ventricular and spinal perfusates.
Lucas
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Department of Gastroenterology, Mayo Clinic, Rochester, MN.
Yaksh
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D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism*; D043424:Carboxypeptidase B; D002268:Carboxypeptidases; D002415:Cats; D002850:Chromatography, Gel; D004558:Electric Stimulation; D004744:Enkephalin, Methionine / Q000031:analogs & derivatives* / Q000378:metabolism*; D005260:Female; D006221:Halothane; D006868:Hydrolysis; D008297:Male; D008969:Molecular Sequence Data; D011863:Radioimmunoassay; D013116:Spinal Cord / Q000378:metabolism*; D015244:Thiorphan / Q000494:pharmacology; D014357:Trypsin
D004744:Enkephalin, Methionine; D015244:Thiorphan; D002268:Carboxypeptidases; D043424:Carboxypeptidase B; D014357:Trypsin; D006221:Halothane