Purification and preliminary characterization of new peptide inhibitor of insulin from pork pancreas.
A new active peptide was purified from the acid-alcohol extract of pork pancreas. It markedly suppressed the insulin activity detected by either in vivo mouse convulsion assay or in vitro free-fat cell assay. When the extract was subjected to chromatography on a carboxymethylcellulose column, the insulin fraction completely passed through the column, whereas the glucagon fraction was absorbed. The fact that the total apparent biological activity of insulin in the exclusive eluate was higher than in the original extract and the insulin radioimmunoactivity remained unchanged led to the discovery of a potent insulin inhibitor in the extract. The inhibitor was separated from glucagon and insulin in the extract by ion-exchange chromatography on a carboxymethylcellulose column followed by gel filtration on a Bio-Gel P-6 column and finally purified by reverse-phase high-performance liquid chromatography (HPLC) on a C-18 column. The antagonistic effect of this inhibitor on insulin was dose dependent with an ED50 of 2 x 10(-10) M, which was the same level used for insulin in vitro assay (1.7 x 10(-10) M). Amino acid analysis of the inhibitor showed that it was rich in arginine and glycine. It was estimated to be approximately 3000 Mr. The NH2-terminal of the peptide was proved to be blocked because it could not be degraded by Edman degradation. Based on the physicochemical and biochemical characteristics of the inhibitor and compared with other active peptides known to be in the pancreas, the inhibitor is probably a new active peptide that might play an important role in homeostasis of carbohydrate metabolism.
Ma
J N
JN
Shanghai Institute of Biochemistry, Academia Sinica, Shanghai, China.
Zhu
B L
BL
Chi
C W
CW
D000273:Adipose Tissue / Q000187:drug effects / Q000378:metabolism*; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004305:Dose-Response Relationship, Drug; D005947:Glucose / Q000378:metabolism; D007328:Insulin / Q000494:pharmacology*; D007329:Insulin Antagonists / Q000302:isolation & purification* / Q000494:pharmacology / Q000633:toxicity; D007700:Kinetics; D008055:Lipids / Q000096:biosynthesis; D008297:Male; D008970:Molecular Weight; D010179:Pancreas / Q000737:chemistry*; D010455:Peptides / Q000302:isolation & purification* / Q000494:pharmacology / Q000633:toxicity; D051381:Rats; D011919:Rats, Inbred Strains; D012640:Seizures / Q000139:chemically induced; D013552:Swine
D000596:Amino Acids; D007328:Insulin; D007329:Insulin Antagonists; D008055:Lipids; D010455:Peptides; D005947:Glucose