Substance P inactivating enzymes in human cerebrospinal fluid.

The Km and Vmax values were determined for enzymes in human lumbar cerebrospinal fluid (CSF) that inactivate synthetic substance P (SP = RPKPQQFFGLM-NH2) and produce metabolic products. For the human lumbar CSF samples analyzed in this study, Km = 2.24 +/- 0.93 mM and Vmax = 0.113 +/- 0.035 nmol/ml/min (n = 10; mean +/- SEM) for the rate of decrease of SP. HPLC analysis of the incubated synthetic peptide fragments demonstrated that the primary enzymatically produced fragment is SP(3-11), with minor amounts in decreasing order of SP(1-4), SP(1-7), and SP(1-9). Electrospray ionization mass spectrometry (ESI-MS) confirmed the appropriate molecular weights for the four peptides, SP(3-11), SP(1-4), SP(1-7), and SP(1-9). These data demonstrate that the primary enzyme in human lumbar CSF that acts on synthetic SP is a post-proline cleaving enzyme (PPCE).

Assay of preproenkephalin A processing enzymes in human lumbar cerebrospinal fluid.

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.