Glucagon-(19-29), a Ca2+ pump inhibitory peptide, is processed from glucagon in the rat liver plasma membrane by a thiol endopeptidase.
Glucagon-(19-29) is 1000-fold more potent that glucagon as an inhibitor of the liver plasma membrane calcium pump, which suggests that this peptide fragment is naturally occurring. Since glucagon-(19-29) is undetectable in plasma, the processing of glucagon into its (19-29) fragment may occur upon interaction of glucagon with its target tissues. The use of a specific radioimmunoassay for glucagon-(19-29) in association with the separation and identification of peptides by high performance liquid chromatography revealed that, upon incubation at 37 degrees C with hepatic plasma membranes, glucagon is processed into its (19-29) C-terminal fragment. The identity of the fragment was confirmed by amino acid sequencing. The processing activity was inhibited by reagents of the thiol group and by 1,10-phenanthroline, suggesting that a thiol endopeptidase containing a catalytically active metal is involved in this processing. Following its production, glucagon-(19-29) was degraded with a half-life of less than 10 s. This degradation was inhibited by bacitracin and by the aminopeptidase inhibitors bestatin and amastatin. When glucagon was incubated with liver plasma membranes in the absence of inhibitors, the accumulation of glucagon-(19-29) reached a maximum at 2 min (1% of initial glucagon), followed by a slow decline. In the presence of bacitracin and bestatin, the amounts of glucagon-(19-29) obtained from glucagon increased continuously, 1 and 2% of glucagon being transformed after 10 and 30 min, respectively. The production of glucagon-(19-29) did not appear to be associated with the binding of glucagon to its receptors, since (i) guanosine 5'-(3-O-thio)triphosphate, a compound which decreases the glucagon-receptor interaction, could not decrease the conversion of glucagon into glucagon-(19-29); (ii) a glucagon analogue which displays a strongly decreased affinity for the hepatic glucagon receptors was processed similarly to glucagon. The conversion also occurs upon incubation with intact hepatoma cells in monolayer culture. These observations suggest that, under physiological conditions, glucagon is processed in liver by cleavage of the Arg17-Arg18 basic doublet, leading to the production of a fragment which is known to display an original biological specificity, namely the modulation of the hepatocyte plasma membrane calcium pump.
Blache
P
P
Centre National de la Recherche Scientifique-Institut National de la Santé et de la Recherche Médicale de Pharmacologie-Endocrinologie, Montpellier, France.
Kervran
A
A
Dufour
M
M
Martinez
J
J
Le-Nguyen
D
D
Lotersztajn
S
S
Pavoine
C
C
Pecker
F
F
Bataille
D
D
D000255:Adenosine Triphosphate / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D001414:Bacitracin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D005934:Glucagon / Q000276:immunology / Q000378:metabolism* / Q000494:pharmacology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D008099:Liver / Q000378:metabolism*; D008114:Liver Neoplasms, Experimental / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism; D011498:Protein Precursors / Q000276:immunology / Q000378:metabolism*; D051381:Rats; D014407:Tumor Cells, Cultured
D010446:Peptide Fragments; D011498:Protein Precursors; D001414:Bacitracin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000255:Adenosine Triphosphate; D005934:Glucagon; D003546:Cysteine Endopeptidases; D000252:Calcium-Transporting ATPases; D007930:Leucine; C012211:ubenimex; D002118:Calcium