Structural studies and two-dimensional gel electrophoresis of gamma-glutamyl transpeptidase.
The heterodimeric enzyme gamma-glutamyl transpeptidase (EC 2.3.2.2) was isolated from adult rat kidney and purified to homogeneity for structural studies using papain solubilization and multiple chromatographies. Two-dimensional gel electrophoresis was found to resolve the active papain-purified enzyme into at least 18 components. Seven components with apparent molecular masses of 23,000-26,000 and isoelectric point range of 5.4-7.0 constitute the light subunit, and 11 components with apparent molecular mass of 51,000-53,000 and isoelectric point range of 5.8-7.1 constitute the heavy subunit. Immunoblot analysis of two-dimensional gels showed that all of these components are immunoreactive with a mixture of the two antibodies generated separately against the light and heavy subunits. Preparative subunit separation was achieved using reverse-phase HPLC under acidic but nonreducing conditions. N-Terminal amino acid sequencing of the separated subunits of the papain-purified enzyme yielded sequence information for the first 32 residues of the heavy chain with the N-terminal starting sequence Gly-Lys-Pro-Asp-His-Val-Tyr-Ser-Arg-Ala, and for the first 36 residues of the light subunit with the N-terminal starting sequence Thr-Ala-His-Leu-Ser-Val-Val-Ser-Glu-Asp.
Cone
J L
JL
Glowinski
I B
IB
Wirth
P J
PJ
Grantham
P H
PH
Roller
P P
PP
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003101:Collodion; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D007120:Immunochemistry; D007527:Isoenzymes / Q000302:isolation & purification; D007668:Kidney / Q000201:enzymology; D008297:Male; D010206:Papain; D010446:Peptide Fragments / Q000032:analysis; D051381:Rats; D005723:gamma-Glutamyltransferase / Q000302:isolation & purification*
D007527:Isoenzymes; D010446:Peptide Fragments; D003101:Collodion; D005723:gamma-Glutamyltransferase; D010206:Papain