Conversion of biosynthetic human proinsulin to partially cleaved intermediates by collagenase proteinases adsorbed to isolated rat adipocytes.
Studies of the biological activity of proinsulin have resulted in widely varying conclusions. Relative to insulin, the biological activity of proinsulin has been reported from less than 1% to almost 20%. Many of the assays in vitro for the biological potency of proinsulin have utilized isolated rat adipocytes. To examine further the interaction of proinsulin with rat adipocytes, we prepared specifically-labelled proinsulin isomers that were iodinated on tyrosine residues corresponding to the A14, A19, B16 or B26 residue of insulin. These were incubated with rat adipocytes and their metabolism was examined by trichloroacetic acid precipitation, by Sephadex G-50 chromatography, and by h.p.l.c. chromatography. By trichloroacetic acid-precipitation assay, there was little or no proinsulin degradation. By G-50 chromatography and subsequent h.p.l.c. analysis, however, we found that the labelled proinsulin isomers were converted rapidly and almost completely to materials which eluted differently on h.p.l.c. from intact proinsulin. This conversion was due primarily to proteolytic activity which adsorbed to the fat cells from the crude collagenase used to isolate the cells. Two primary conversion intermediates were found: one with a cleavage at residues 23-24 of proinsulin (the B-chain region of insulin), and one at residues 55-56 in the connecting peptide region. These intermediates had receptor binding properties equivalent to or less than intact proinsulin. These findings show that isolated fat cells can degrade proinsulin to intermediates due to their contamination with proteolytic activity from the collagenase used in their preparation. Thus the previously reported range in biological activities of proinsulin in fat cells may have arisen from such protease contamination. Finally, the present findings demonstrate that a sensitive assay for degradation of hormones is required to examine biological activities in isolated cells.
Duckworth
W C
WC
Veterans Administration Medical Center, Omaha, NE.
Peavy
D E
DE
Hamel
F G
FG
Liepnieks
J
J
Brunner
M R
MR
Heiney
R E
RE
Frank
B H
BH
D000273:Adipose Tissue / Q000187:drug effects / Q000378:metabolism*; D000327:Adsorption; D000818:Animals; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D005033:Ethylmaleimide / Q000494:pharmacology; D066298:In Vitro Techniques; D007457:Iodine Radioisotopes; D008297:Male; D003012:Microbial Collagenase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010436:Pepstatins / Q000494:pharmacology; D010618:Phenanthrolines / Q000494:pharmacology; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D011384:Proinsulin / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains
D007457:Iodine Radioisotopes; D010436:Pepstatins; D010618:Phenanthrolines; C014195:Streptomyces pepsin inhibitor; D010664:Phenylmethylsulfonyl Fluoride; D011384:Proinsulin; D003012:Microbial Collagenase; D005033:Ethylmaleimide; C031375:pepstatin; C025205:1,10-phenanthroline