Abstract Gamma-glutamyl compounds include antioxidants, inflammatory molecules, drug metabolites and neuroactive compounds. Two cell surface enzymes have been identified that metabolize gamma-glutamyl compounds, gamma-glutamyl transpeptidase GGT1 and gamma-glutamyl leukotrienase GGT5. There is controversy in the literature regarding the substrate specificity of these enzymes. To address this issue, we have developed a method for comprehensive kinetics analysis of compounds as substrates for GGT enzymes. Our assay is sensitive, quantitative and is conducted at physiologic pH. These data provide essential information regarding the target enzymes for developing treatments for inflammatory diseases such as asthma and cardiovascular disease in humans.
|Published (Last):||8 March 2019|
|PDF File Size:||20.84 Mb|
|ePub File Size:||3.61 Mb|
|Price:||Free* [*Free Regsitration Required]|
Abstract Gamma-glutamyl compounds include antioxidants, inflammatory molecules, drug metabolites and neuroactive compounds. Two cell surface enzymes have been identified that metabolize gamma-glutamyl compounds, gamma-glutamyl transpeptidase GGT1 and gamma-glutamyl leukotrienase GGT5.
There is controversy in the literature regarding the substrate specificity of these enzymes. To address this issue, we have developed a method for comprehensive kinetics analysis of compounds as substrates for GGT enzymes.
Our assay is sensitive, quantitative and is conducted at physiologic pH. These data provide essential information regarding the target enzymes for developing treatments for inflammatory diseases such as asthma and cardiovascular disease in humans. This assay is invaluable for studies of oxidative stress, drug metabolism and other pathways that involve gamma-glutamyl compounds. Keywords: Glutathione, gamma-glutamyl substrates, gamma-glutamyl transpeptidase, gamma-glutamyl leukotrienase, glutamate assay Introduction Many extracellular gamma-glutamyl compounds have been identified in humans and they serve a variety of functions.
Metabolites of both endogenous and exogenous compounds form GSH S-conjugates and are excreted from the cell. S-nitroso-GSH is a transport form of nitric oxide [ 3 ; 4 ].
For other compounds, such as aflatoxin B1, conjugation to GSH and transport out of the cell are steps in the detoxification and excretion of the compound [ 6 ]. Additional gamma-glutamyl compounds have also been identified including gamma-glutamyl taurine, an endogenous modulator of excitatory aminoacidergic neurotransmission in the brain [ 7 ].
Studies with GGT knockout mice indicate that there are other gamma-glutamyl compounds yet to be discovered that play a role in osteogenesis and other physiologic processes [ 8 ; 9 ; 10 ; 11 ; 12 ; 13 ; 14 ]. GSH cannot be taken up intact by most cells and there is no evidence that GSH S-conjugates can be transported into the cell [ 9 ; 15 ]. The initial step in their metabolism is cleavage of the gamma-glutamyl bond [ 16 ; 17 ; 18 ].
GGT1 and GGT5 are the only two extracellular enzymes that have been shown to cleave gamma-glutamyl bonds [ 19 ; 20 ; 21 ].
Both enzymes are expressed in mice and in humans and data regarding the substrates for these two enzymes is largely derived from GGT1 and GGT5 knockout mice [ 2 ; 19 ; 20 ; 21 ; 22 ; 23 ]. GGT1 knockout mice cannot breakdown GSH into its constituent amino acids as it passes through the proximal tubules of the kidney [ 9 ]. They excrete large amounts of GSH in their urine, which leads to a fatal cysteine deficiency [ 9 ].
Supplementing the mice with N-acetylcysteine restores cysteine levels, but does not correct deficiencies in bone resorption [ 12 ]. No gamma-glutamyl compound has been shown to modulate osteoclasts. These data suggest that the gamma-glutamyl compound that is essential in this process has yet to be identified.
Studies with GGT1 knockout mice have shown reduced activation of cisplatin to a nephrotoxin, implicating a cisplatin-S-GSH conjugate as a metabolite in its activation [ 8 ]. Gamma-glutamyl leukotrienase GGT5 is the only other member of the GGT family of enzymes that has been shown, to date, to have catalytic activity [ 2 ; 21 ].
To date, there has not been any method to evaluate the substrate specificity of gamma-glutamyl enzymes. We have developed a novel assay and done a comprehensive kinetic analysis of the metabolism of a series of gamma-glutamyl compounds by two human enzymes gamma-glutamyl transpeptidase GGT1 and gamma-glutamyl leukotrienease GGT5. The initial steps in the reaction catalyzed by GGT1 are the cleavage of the gamma-glutamyl bond of the substrate and the formation of a transient ES complex [ 16 ; 17 ; 18 ].
All but the gamma-glutamyl group of the substrate is released. In the ES complex, the gamma-carbon of the gamma-glutamyl substrate forms an acyl bond with the side chain of the Thr in the active site of the enzyme [ 16 ; 17 ; 18 ; 25 ; 26 ] Fig. Hydrolysis of the esterified enzyme can occur, releasing glutamate and regenerating free enzyme [ 25 ; 26 ]. In the presence of a high concentration of an acceptor amino acid or dipeptide, a transpeptidation reaction occurs in which the ES complex is resolved when the gamma-glutamyl group is transferred to the amine of the acceptor, thereby forming a new gamma-glutamyl compound [ 27 ].
The transfer of the gamma-glutamyl group to a dipeptide acceptor, occurs by a modified Ping-Pong mechanism [ 17 ; 28 ]. The degree to which GGT1 catalyzes the hydrolysis versus transpeptidation reaction is influenced by both pH and the presence of an acceptor amino acid or peptide [ 27 ; 29 ; 30 ].
Pourquoi votre taux de gamma-GT est-il élevé
A blood test is when a sample of blood is taken from the body to be tested in a lab. Doctors order blood tests to check things such as the levels of glucose , hemoglobin, or white blood cells. This can help them find problems like a disease or medical condition. Sometimes, blood tests can help them see how well an organ such as the liver or kidneys is working.
The active site of GGT is known to be located in the light subunit. Medical applications[ edit ] GGT is predominantly used as a diagnostic marker for liver disease. Latent elevations in GGT are typically seen in patients with chronic viral hepatitis infections often taking 12 months or more to present. Elevated serum GGT activity can be found in diseases of the liver, biliary system, and pancreas.
Gamma-GT : décryptage de sa prise de sang