title: Assay of preproenkephalin A processing enzymes in human lumbar cerebrospinal fluid.
abstract: Vmax and Km measurements have been obtained for endogenous peptidases that are important for methionine enkephalin (ME) homeostasis in humans. Those peptidases in human lumbar cerebrospinal fluid (CSF) act upon several synthetic biologically significant peptides that are also contained within the preproenkephalin Ahuman,1-267 molecule. The amount of endogenous methionine enkephalin-like immunoreactivity (ME-li) in human lumbar CSF is 74.1 +/- 5.7 fmol ME-li/ml CSF (n = 56; mean +/- SE). The kinetic parameters of the various enzymes that inactivate exogenous, synthetic methionine enkephalin (ME, YGGFM) and that also produce ME from two different portions of the preproenkephalin Ahuman,1-267 precursor molecule were determined. The enzyme that inactivates synthetic ME to FM, and that correlates to the rate of decrease of ME, has a Vmax = 560 +/- 43.3 nmol/ml/min and a Km = 4514 +/- 373 microM (n = 56; mean +/- SE). Preproenkephalin Ahuman,186-193 (PA = YGGFMRGL) was added to CSF samples to characterize those processing and converting enzymes that produce the ME pentapeptide. Vmax, as measured by the rate of the decrease of PA to produce YGGFMR, was 0.192 +/- 0.038 nmol/ml/min and a Km of 513 +/- 121 microM (n = 10; mean +/- SE). Similarly, a bovine analog to preproenkephalin Ahuman,128-140 (PPEhuman, GSEILAKRYGGFM; PPEbovine,125-137, GGEVLGKRYGGFM) was used to characterize that enzyme system that produces ME from an N-terminally extended ME peptide. That endopeptidase had a Vmax of 0.120 +/- 0.048 nmol/ml/min with a Km of 734 +/- 296 microM (n = 10). Those endogenous enzymes in human CSF may relate to the proopiomelanocortin convertase enzymes that contain the subtilisin-like catalytic domain.
authors.affiliation: Charles B. Stout Neuroscience Mass Spectrometry Laboratory, Department of Neurology, University of Tennessee, Memphis 38163.
authors.forename: T
authors.initials: T
authors.lastname: Kaneko
authors.forename: G
authors.initials: G
authors.lastname: Wood
authors.forename: W L
authors.initials: WL
authors.lastname: Crouch
authors.forename: D M
authors.initials: DM
authors.lastname: Desiderio
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D010450:Endopeptidases / Q000134:cerebrospinal fluid*; D004744:Enkephalin, Methionine / Q000134:cerebrospinal fluid; D004745:Enkephalins / Q000134:cerebrospinal fluid*; D006801:Humans; D007700:Kinetics; D008161:Lumbosacral Region; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000134:cerebrospinal fluid; D011498:Protein Precursors / Q000134:cerebrospinal fluid*
chemical_list: D004745:Enkephalins; D010446:Peptide Fragments; D011498:Protein Precursors; D004744:Enkephalin, Methionine; C033660:preproenkephalin; D010450:Endopeptidases