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Neuropeptide‐Metabolizing Peptidases in Neuro‐2a Neuroblastoma and C 6 Glioma Cells
Author(s) -
Bel Elaine A.,
Gambarini Angelo G.,
Martins Antonio R.
Publication year - 1986
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1986.tb00701.x
Subject(s) - endopeptidase , bradykinin , neuropeptide , prolyl endopeptidase , chemistry , biochemistry , enzyme , neprilysin , peptide , peptide bond , microbiology and biotechnology , biology , receptor
Mouse Neuro‐2a neuroblastoma and rat C 6 glioma cloned cells were screened for neuropeptide‐metabolizing peptidases using a kininase bioassay combined with a time‐course bradykinin‐product analysis, and a fluorimetnc assay for prolyl endopeptidase. The complementary peptide products Arg 1 → Phe 5 /Ser 6 → Arg 9 and Arg 1 → Pro 7 /Phe 8 ‐Arg 9 were released during bradykinin (Arg 1 ‐Pro 2 ‐Pro 3 ‐Gly 4 ‐Phe 5 ‐Ser 6 ‐Pro 7 ‐Phe 8 ‐Arg 9 ) inactivation by homogenates of Neuro‐2a and C 6 cells. The 1:1 stoichiometry of the complementary fragments and their high yields, at 10% bradykinin inactivation, demonstrated the sites of hydrolysis. The initial rate of Phe 5 ‐Ser 6 bond cleavage was six‐fold higher than that of the Pro 7 ‐Phe 8 bond. These sites of cleavage can be attributed to enzymes similar to endqpeptidase A (Phe 5 ‐Ser 6 ) and prolyl endopeptidase (Pro 7 ‐Phe 8 ) on the basis of the specificity and sensitivity to inhibitors of the kininase activity in Neuro‐2a and C 6 cell homogenates. Kininase and prolyl endopeptidase specific activities (fmol/min/cell) were 10.5 and 12.4 for Neuro‐2a, and 1.5 and 2 for C 6 homogenate, respectively. The recovery of kininase activity was 2.2‐fold higher in the particulate than in the soluble (105,000 g for 1 h) neuronal fraction, whereas the amount of prolyl endopeptidase activity was about the same in both fractions. Kininase and prolyl endopeptidase activities in C 6 cells were recovered mostly in the soluble fraction. Prolyl endopeptidase specific activity decreased 10‐fold in serum‐starved Neuro‐2a cultured cells, with no change in activity in similarly treated C 6 cells. In contrast, kininase specific activity in both cell types was essentially unaffected on serum‐deprivationinduced differentiation.