Evidence for stable homodimers and heterodimers of gamma-glutamyltranspeptidase subunits under protein-denaturing conditions.
gamma-Glutamyltranspeptidase is synthesized as a core glycosylated propeptide (Mr 75,000) which is subsequently cleaved to yield a stable heterodimeric structure (subunit Mr 50,000 and 30,000). The propeptide represents an insignificant mass of the transpeptidase but higher molecular weight bands designated H1 (Mr 85,000) and H2 (Mr 100,000) are readily observed by protein staining or immunoblot analysis of the enzyme or crude membranes after SDS-polyacrylamide gel electrophoresis. Although H1 and H2 represent the predominant antigenic forms of transpeptidase in tissues which exhibit relatively low specific enzyme activity, neither their structure nor their physiological function is known. In order to determine the relationship between H1 and H2, and the large (L) and small (S) subunits of the transpeptidase, individual bands (H1, H2, L and S) of the purified renal enzyme were cut from a Coomassie-stained SDS gel, eluted and re-electrophoresed. Isolated S produced S and dimers of S (Mr 60,000), while isolated L produced L and dimers of L corresponding to H2. Equivalent mixtures of L and S also produced H1. Utilizing IgG affinity-purified against either L or S, immunoblot analysis confirmed that H2 is a dimer of L, and H1 is a heterodimer of L and S. However, monoclonal IgG which recognizes both transpeptidase propeptide and native heterodimer did not react with H1. Thus, it is clear that isolated L and S can form and maintain unique dimeric structures during SDS-polyacrylamide gel electrophoresis. With this information it should now be possible to ascertain the basis for the apparent predominance of H1 and H2 in non-renal tissues.
D000818:Animals; D000906:Antibodies; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004797:Enzyme-Linked Immunosorbent Assay; D007668:Kidney / Q000201:enzymology; D046911:Macromolecular Substances; D008871:Microvilli / Q000201:enzymology; D008970:Molecular Weight; D011489:Protein Denaturation; D051381:Rats; D005723:gamma-Glutamyltransferase / Q000302:isolation & purification / Q000378:metabolism*
D000906:Antibodies; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D046911:Macromolecular Substances; D005723:gamma-Glutamyltransferase