Isolation of gamma-glutamyltransferase from human liver, and comparison with the enzyme from human kidney.

Abstract

We isolated gamma-glutamyltransferase [(gamma-glutamyl)-peptide:amino acid gamma-glutamyltransferase, EC 2.3.2.2] from human liver and compared some of its properties with the same enzyme prepared from human kidney. The enzymes from these two sources are very similar with respect to initial velocity kinetic constants, pH optima of the transpeptidation and autotransfer reactions, heat stability, competitive inhibition by glutathione of the colorimetric assay in which gamma-glutamyl-4-nitroanilide is substrate, stability of catalytic activity to trypsinization, and relative rates of transfer of the gamma-glutamyl moiety from gamma-glutamyl-4-nitroanilide and L-[glycine-2-3/]glutathione to some amino acids and small peptides. The kidney enzyme is inhibited more by the gamma-glutamyl acceptor substrate, glycylglycine, as reflected in a sevenfold lower value for the inhibition constant KiA. Major differences were observed in the lectin-binding properties of liver gamma-glutamyltransferase compared to the kidney enzyme. Lectin-binding property differences are retained for the trypsinized form of the liver and kidney enzymes, although the degree of precipitation was less for certain lectins as compared to the untreated enzyme. Lectin-binding properties were reversed by carbohydrates specific for each lectin. We adapted the histochemical staining technique of Rutenberg et al. [J. Histochem. Cytochem. 17, 517 (1969)] to the detection of gamma-glutamyltransferase activity in acrylamide gels; diffusion artifacts are minimized and the color produced is stable for several days. Untreated and trypsinized forms of the liver enzyme both migrated faster in acrylamide gels (as single bands) than did the corresponding forms of the kidney enzyme.

Enzyme profile of rat bile ductular epithelial cells in reference to the resistance phenotype in hepatocarcinogenesis†

“…Not surprisingly, the hepatic GGT associated with the bile ductular cell hyperplasia possessed catalytic properties which were very similar to those demonstrated for the GGTs of normal rat and human kidney (34)(35)(36)(37)(38).…”

“…The bile ductular epithelial cell isolate was prepared by first mincing the tissue fragments in 50 ml Leibowitz L-15 tissue culture medium, pH 7.4, supplemented with 1 gm per liter bovine serum albumin, 36 mM HEPES, 8.3 mM a-D(+)-ghcose, 0.1 FM insulin, 2 mM L-glutamine, 3% fetal calf serum, 100,000 units per liter penicillin G and 100 mg per liter streptomycin sulfate. To this was added 18,000 units collagenase, 35,000 units hyaluronidase, 6,500 units deoxyribonuclease I and 5 mg soybean trypsin inhibitor, and the mix was then vigorously shaken for 50 min at 37°C. The resulting cell suspension was cooled at 4°C and consecutively filtered through four Nitex Swiss nylon monofilament screens (TETKO, Elmford, NY) having mesh pore diameters of 253, 100, 60 and 20 pm, respectively.…”

Clinical application of an enzyme‐linked immunosorbent assay detecting hepatoma‐specific γ‐glutamyltransferase

“…27 Datura stramonium agglutinin (DSA) has strong affinity for the GGT with a complex structure of saccharide, and low affinity for the GGT with a simple sugar chain. 28 Our laboratory successfully separated the isoenzyme of GGT using DSA-sepharose affinity chromatography, and clinical evaluation of DSA-GGT showed good specificity and sensitivity for the diagnosis of HCC. 19 Nevertheless, these measurements of the isoenzyme of GGT were based on electrophoresis or column chromatography, which were complicated, time-consuming and unsuitable for handling large numbers of serum samples.…”

“…Some data showed the isoenzyme of GGT with characteristics of a more complex structure of saccharide 27 . Datura stramonium agglutinin (DSA) has strong affinity for the GGT with a complex structure of saccharide, and low affinity for the GGT with a simple sugar chain 28 . Our laboratory successfully separated the isoenzyme of GGT using DSA‐sepharose affinity chromatography, and clinical evaluation of DSA‐GGT showed good specificity and sensitivity for the diagnosis of HCC 19 …”

Gamma-Glutamyltransferase Fractions in Human Plasma and Bile: Characteristic and Biogenesis

“…In adult liver, GGT is localized in the biliary pole of hepatocytes and in cholangiocytes and hence secreted in bile [3] , [5] . Plasma GGT is supposed to origin from liver [6] , [7] and early studies demonstrated that it is associated with several carriers having different molecular weights, densities and charges [8] , [9] . Nevertheless, the mechanism of release, the structures and the clinical significance of such carriers have not been completely characterized.…”