Evidence for pyroglutamyl peptidase I and prolyl endopeptidase activities in the rat insulinoma cell line RINm 5F: lack of relationship with TRH metabolism.
Thyrotropin-Releasing hormone (TRH)-degrading pyroglutamyl peptidase I (PGP I) and prolylendopeptidase (PE) activities have been demonstrated in rat insulinoma RINm 5F cell line. These two enzymes catalyze the conversion of TRH to Histydyl-Proline-Diketopiperazine and to acid TRH respectively. After cell fractionation, we found all the PGP I and PE activities in the cytosolic fraction. The membrane-bound PGP II activity is not detectable in the RINm 5F cells. Further investigations on these two cytosolic enzymes show that pyroglutamyl- and proline-containing peptides are inhibitors of each TRH-degrading enzyme. Gel filtration chromatography on Sephadex G100 shows that PGP I and PE activity have an apparent molecular mass of about 18 kDa and 57 kDa, respectively. Kinetic analysis with TRH as substrate, gives a Km of 44 microM and 235 microM, and a Vmax of 1.49 and 8.80 pmol/min/micrograms protein for PGP I and PE, respectively. Immunoreactive TRH, His-Pro-Diketopiperazine and acid TRH levels in the cell line extracts are 2.2 +/- 0.9, 22.5 +/- 11.1 and 28.7 +/- 14.6 pg/1O6 cells, respectively. When cells have been incubated for 2 to 72 hours with a P.E. inhibitor (Z-Gly-Pro-CHN2) at 5 x 10(-7) M, both cell PGP I and PE activities are inhibited. No change in the cellular content of immunoreactive TRH, His-Pro-Diketopiperazine and acid TRH have been observed in treated cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Salers
P
P
Laboratoire de Neuroendocrinologie Experimentale, INSERM U 297, Faculté de Médecine Nord, Marseille, France.
Ouafik
L H
LH
Giraud
P
P
Dutour
A
A
Maltese
J Y
JY
Oliver
C
C
D007516:Adenoma, Islet Cell / Q000188:drug therapy / Q000201:enzymology*; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D003978:Diazomethane / Q000031:analogs & derivatives / Q000494:pharmacology; D010450:Endopeptidases / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D000085822:Prolyl Oligopeptidases; D011480:Protease Inhibitors / Q000494:pharmacology; D011751:Pyroglutamyl-Peptidase I / Q000378:metabolism*; D011863:Radioimmunoassay; D051381:Rats; D012697:Serine Endopeptidases; D013347:Subcellular Fractions / Q000201:enzymology; D013379:Substrate Specificity; D013973:Thyrotropin-Releasing Hormone / Q000378:metabolism*
D011480:Protease Inhibitors; C047930:N-benzyloxycarbonylglycyl-proline diazomethyl ketone; D013973:Thyrotropin-Releasing Hormone; D003978:Diazomethane; D010450:Endopeptidases; D011751:Pyroglutamyl-Peptidase I; D012697:Serine Endopeptidases; D000085822:Prolyl Oligopeptidases