Cancer-related urinary proteinase inhibitor, EDC1: a new method for its isolation and evidence for multiple forms.
During the past several years, numerous laboratories have reported isolation and purification of proteinase inhibitors from human urine. Many of these molecules were incompletely characterized and some of them may have been artifacts in part because of harsh procedures used for their isolation. Consequently, there is disagreement and confusion regarding the biochemical characteristics of these inhibitors. We previously reported the isolation of a proteinase inhibitor, EDC1, from the urine of a leukemic patient. This molecule, M(r) 30 kDa, was antigenically related to plasma inter-alpha-trypsin inhibitor (IATI) and inhibited the growth of a virally transformed B cell line. Immunoreactive EDC1 was also the major component of low molecular weight proteinuria observed in cancer patients. We now report a new method for the isolation of EDC1 from urine of patients with adenocarcinomas of colon and lung and melanoma and compare its partial amino acid sequence with that of HI 30, a proteinase inhibitor previously isolated from pooled normal urine by Hochstrasser et al. [Hoppe-Seyler's Z Physiol Chem 357:153-162, 1976]. Our method involves i) a batchwise cation exchange, ii) gel filtration chromatography, iii) anion exchange chromatography on FPLC, and iv) reverse phase C18 chromatography on HPLC. This method is mild and results in an overall yield of 0.4 to 1.2 mg of EDC1/liter urine. On the basis of the partial N-terminal amino acid sequence of its N terminal (38 residues) and middle regions (29 residues), EDC1 appears to be identical with HI30. Surprisingly, during this isolation procedure, another proteinase inhibitor, M(r) 22 kDa, which cross-reacted with antisera to EDC1 and IATI, was also isolated. The 22 kDa molecule was a major component of the IATI related urinary molecules and was identical with the 30 kDa EDC1 in which first the 15 N terminal residues were clipped. The lower M(r) inhibitor was not an artifact formed during storage or isolation procedure because the Western blot analysis of fresh cancer and normal urine revealed the 30 and 22 kDa molecules. Thus, both the 30 kDa EDC1 (or HI30) and its clipped variant, the 22 kDa molecule, are physiologic components of IATI related urinary proteinase inhibitors and excretion of both forms may be increased in patients with advanced cancer.
Chawla
R K
RK
Department of Medicine, Emory University School of Medicine, Decatur, Georgia.
Lawson
D H
DH
Ahmad
M
M
Travis
J
J
D000230:Adenocarcinoma / Q000652:urine*; D000595:Amino Acid Sequence; D015153:Blotting, Western; D006023:Glycoproteins / Q000737:chemistry / Q000276:immunology / Q000652:urine*; D006801:Humans; D008545:Melanoma / Q000652:urine*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry; D011480:Protease Inhibitors / Q000737:chemistry / Q000276:immunology / Q000652:urine*
D006023:Glycoproteins; D010446:Peptide Fragments; D011480:Protease Inhibitors; C029236:glycoprotein EDC1, human