Liberation of immunoreactive somatomedin-C from its binding proteins by proteolytic enzymes and heparin.
Most of the somatomedin in serum exists as a high molecular weight complex (approximately equal to 140,000) composed of binding protein subunits and small molecular weight somatomedin. These binding proteins may interfere with measurements of the somatomedins by various radioligand assays. In a companion paper, we reported that when serum is incubated at neutral pH the detectability of the somatomedin-C (Sm-C) is increased. Using gel chromatography, however, it was not possible to demonstrate release of free Sm-C from the macromolecular complex. In the studies reported here, incubation of heparinized plasma at pH 7.4 followed by gel chromatography in a heparin-containing buffer caused 70-80% of the immunoreactive Sm-C to shift from the gamma-globulin region to a molecular weight which approximates that of free Sm-C. This conversion is a time-dependent process which is inhibited by the proteolytic enzyme inhibitor antipain. Similar changes in the elution profile of Sm-C were observed when heparinized plasma was acidified and chromatographed in heparin. These findings suggest that 1) at neutral pH, serum proteolytic enzymes reduce the affinity between small molecular weight Sm-C and its binding proteins. 2) At acid pH, a similar effect is observed. 3) In the presence of heparin, reassociation of these components does not occur. These results suggest a possible mechanism whereby the somatomedin macromolecular complex could be disrupted so that small molecular weight somatomedin is made available to tissues.
D000976:Antipain / Q000494:pharmacology; D002352:Carrier Proteins / Q000097:blood*; D002850:Chromatography, Gel; D006493:Heparin; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D018969:Insulin-Like Growth Factor Binding Proteins; D007334:Insulin-Like Growth Factor I; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000097:blood*; D013002:Somatomedins / Q000097:blood*
D002352:Carrier Proteins; D018969:Insulin-Like Growth Factor Binding Proteins; D013002:Somatomedins; D000976:Antipain; D007334:Insulin-Like Growth Factor I; D006493:Heparin; D010447:Peptide Hydrolases