N-terminal truncated insulin-like growth factor-I in human urine.
Urinary insulin-like growth factor-I (IGF-I) from healthy human subjects was examined using two antisera directed toward the whole molecule (WM) and the N-terminal of IGF-I. Pooled urine samples from normal adults were dialyzed, lyophilized, then subjected to Sephacryl S-200 chromatography. The gel filtration profile of immunoreactive IGF-I measured by RIA using WM antiserum showed two peaks. Of the total IGF-I, approximately 40% was free, and the rest was present as a 50-kilodalton complex. To characterize the IGF-I forms present in those two peaks, antibody capture enzyme-linked immunoassays (EIA) using the two antisera were established for detection of intact IGF-I and N-terminal-truncated IGF-I variants. The WM antibody recognizes intact IGF-I and des(1-3)-IGF-I, an N-terminal-truncated variant, equally well, whereas the N-terminal IGF-I antibody recognizes intact IGF-I, but not des(1-3)-IGF-I (< 1% cross-reactivity). As both antibodies show similar cross-reactions with IGF-II, the difference between IGF-I levels recognized by the two antisera was considered to indicate the presence of N-terminal-truncated IGF-I variants. Of the free immunoreactive IGF-I in the urine, 64% was not recognized by N-terminal IGF-I antiserum and was considered to represent N-terminal-truncated IGF-I. In contrast, only 6% of the IGF-I present in the 50-kilodalton fraction was truncated. Urine samples from normal human subjects were analyzed by RIA with WM antiserum and EIA with both WM and N-terminal IGF-I antisera after extraction of IGF-I from binding proteins. IGF-I values measured by EIA with the WM antiserum correlated well with those values obtained by RIA using WM antiserum (r = 0.98; P < 0.001). The total urinary IGF-I level measured by EIA with the WM antiserum was 216.0 +/- 41.1 ng/L (mean +/- SEM), and 35.2 +/- 6.1% of this was considered to represent N-terminal-truncated IGF-I. Using an immobilized biotinylated peptide corresponding to the N-terminal six amino acids of IGF-I, we detected proteolytic activity toward the N-terminal of IGF-I in all four human serum samples tested. In contrast, only two of seven urine samples had detectable protease activity, and in these samples, activity was very low compared to that in serum.(ABSTRACT TRUNCATED AT 400 WORDS)
Yamamoto
H
H
Department of Internal Medicine, University of Manitoba, Winnipeg, Canada.
Murphy
L J
LJ
D000328:Adult; D002352:Carrier Proteins / Q000652:urine; D002850:Chromatography, Gel; D005260:Female; D006801:Humans; D007124:Immunoenzyme Techniques; D018969:Insulin-Like Growth Factor Binding Proteins; D007334:Insulin-Like Growth Factor I / Q000652:urine*; D008297:Male; D010446:Peptide Fragments / Q000652:urine*; D010447:Peptide Hydrolases / Q000097:blood / Q000652:urine; D011863:Radioimmunoassay; D013002:Somatomedins / Q000378:metabolism
D002352:Carrier Proteins; D018969:Insulin-Like Growth Factor Binding Proteins; D010446:Peptide Fragments; D013002:Somatomedins; C058957:insulin-like growth factor 1, des-(1-3)-; D007334:Insulin-Like Growth Factor I; D010447:Peptide Hydrolases