Active deglycosylated mammalian gamma-glutamyl transpeptidase.
gamma-Glutamyl transpeptidase, a highly glycosylated heterodimeric enzyme that is usually attached to the external surface of cell membranes, is of major importance in the metabolism of glutathione. The enzyme, which has been isolated from many animal sources, contains a large amount of carbohydrate, which is linked to both protein subunits. Previous work has not shown whether such carbohydrate is needed for enzyme activity nor indicated its functional role. Notably, gamma-glutamyl transpeptidase isolated from Escherichia coli, which exhibits about 80% amino acid sequence homology with the rat enzyme, has only about 0.1% of its specific enzymatic activity and is not glycosylated. Here we treated the highly glycosylated gamma-glutamyl transpeptidases isolated from rat and pig kidneys with a mixture of glycosidases and then separated two completely active gamma-glutamyl transpeptidase fractions from each species. One fraction was completely devoid of carbohydrate and was fully active as compared with the respective isolated enzymes, but differed in solubility and stability. The other fraction, which contained 10-20% of the initially bound carbohydrate, exhibited a marked increase in susceptibility to proteases. The oligosaccharide chains of gamma-glutamyl transpeptidase may protect against protease action (including self-destruction by the inherent protease activity of the light subunit) during synthesis of the active enzyme from its single chain precursor, as well as after enzyme synthesis.
Smith
T K
TK
Department of Biochemistry, Cornell University Medical College, New York, New York 10021.
Meister
A
A
D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000201:enzymology; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D012995:Solubility; D013552:Swine; D005723:gamma-Glutamyltransferase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D002241:Carbohydrates; D010446:Peptide Fragments; D005723:gamma-Glutamyltransferase; D006026:Glycoside Hydrolases