Term
| Discovery of GSTs happened in the 1880's when dogs were given bromobenzene and excreted a ______-containing metabolite called ______. |
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Definition
Sulfur-containing
mercapturic acid |
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Term
| The mercapturic acid excreted in urine from dogs given bromobenzene was shown to be the ____________ derivative of bromobenzene. |
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Definition
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Term
| What is the name of the tripeptide that is the source of cysteine in the N-acety-cysteine derivative (mercapturic acid) of bromobenzene, etc.? |
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Definition
| gamma-glutamylcysteinylglycine named glutathione |
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Term
| __________ substances are common substrates for glutathione transferases (GSTs) |
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Definition
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Term
| Describe the GST (mercapturic) pathway of bromobenzene. |
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Definition
Bromobenzme + CYP = bromobenzene with an oxirane ring
bromobenzene(w/ oxirane ring) + GST + GSH = glutathione conjugate(bromobenzne with OH and gamma-glutamylcysteinylglycine).... -H2O to restore aromatic ring and loss of OH group
bromobenzene glutathione conjugate + gamma glutamyl transpeptidase = bromobenzene cysteinyl-glycine conjugate
bromobenzene cysteinyl-glycine conjugate + cysteinyl-glycinase = cysteine conjugate
cysteine conjugate + N-acetyltransferase = N-acetylcysteine conjugate (Mercapturic Acid!) |
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Term
| What is the cosubstrate for GST? |
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Definition
| GSH (gamma-glutamylcysteinylglycine) |
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Term
| Is GSH available in high concentrations in the body? At what concentration in most tissues? The eye? Liver? |
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Definition
Yes, in the liver 5mM.
The lens of the eye has the most (10mM)
Most tissues >0.2 mM |
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Term
| Why do cells generally contain ample amounts of GSH? |
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Definition
| Used as a cellular antioxidant |
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Term
| What affects the cellular concentration of GSH? |
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Definition
The protein nutritional status of the cell (source of amino acid components)
Exposure to xenobiotics |
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Term
| How is the concentration of GSH controlled in the cell? |
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Definition
| By a negative feedback loop with the [GSH] inhibiting the gamma-glutamylcysteine synthetase |
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Term
| What is the role of buthionine sulfoximine on the concentration of cellular GSH? |
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Definition
| Buthionine sulfoximine inhibits gamma-glutamylcusteine synthetase (reduces [GSH]) |
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Term
| What role does diethyl maleate have on cellular [GSH]? |
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Definition
| Diethyl maleate is a substrate for glutation conjugation and will lower the concentration of GSH in the cell by forming a GSH conjugate. |
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Term
| How do oxidizing agents such as glutathione disulfide affect cellular GSH? |
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Definition
| During futile cycling of thiocarbamides and 2-mercaptoimidazole by FMO, GSH can be converted to glutathione disulfide GSSG |
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Term
Give the equation for reduction of GSSG
How is it catalyzed? Does it happen readily? |
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Definition
GSSG + NADPH + H+ ---> 2GSH + NADP+
Catalyzed by glutathione reductase
Happens readily in cells (with NADPH present) |
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Term
| What are common substrates for the GST enzyme superfamily? |
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Definition
Electrophiles:
aryl halides, aklyl halides, epoxides, alpha-beta unsaturated ketones, aliphatic sulfate esters |
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Term
| How do GST's affect the toxicity of drugs? |
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Definition
| GSH conjugates are generally detoxication products. |
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Term
| Does GST act as an isomerase? |
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Definition
Yes, for some substrates
(also, GSH is used, but not consumed) |
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Term
| What is the reaction of GST (w GSH) with aklyl halides like dibromoethane? |
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Definition
Formation of an episulfonium ion which is highly reactive and fan form an ethyl-glutathione aduct of a DNA base
NOT a detoxification pathway of GST |
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Term
| What happens when alpha-beta unsaturated ketones react with GST? How is it facilitated? |
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Definition
A Michael-type 1,4-addition of GS
Facilitated by a Tyrosine in N-term of GST that H-bonds with GSH to make it more reactive
Tyrosine/Serine in Substrate binding site that H-bonds to oxygen in ketone to stabilize it. |
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Term
| What happens to sulfate esters that react with GST? |
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Definition
| H2SO4 leaves and GS anchors to the R group |
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Term
| What happen nitroglycerine reacts with GST? |
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Definition
| Nitroglylcerine becomes a nitro alcohol and the GS binds to the NO2 leaving group... this transition state then reacts with GSH to form GSSG and HNO2 |
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Term
| Describe the reaction of steroid type molecules with GST. |
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Definition
| GST acts as a steroid isomerase, where the location of a double-bond is changed. The [GSH] remains constant, as it is needed but not consumed in the reaction. |
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Term
| Describe the reaction of Maleylacetoacetate and GST? |
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Definition
GST acts as a Maleylacetoacetate Isomerase and converts 4-Maleylacetoacetate (CIS) to 4-Fumarylacetoacetate (TRANS)...
[GSH] remains constant, as it is needed for the reaction to occur, but is not consumed |
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Term
| What happens with dichloroacetate reacts with GST? |
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Definition
Dichloroacetate gets converted to glyoxylate (via GST(z)!) a reaction that does not consume GSH, but requires it in the RxN.
The Glyoxylate product and the chlroo-glutathione intermediate can form adducts to residues in the GST(zeta) protein |
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Term
| Describe the structure of GST |
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Definition
Exists as a dimer (generally homo), with each monomeric subunig weighting ~22,000-29000(ligandin).
Has one GSH binding site (G-site) and one substrate binding site(H-site). |
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Term
|
Definition
When the most abundant protein of GST was discovered, it was shown to bind a number of lipophilic organic molecules (azo-dye carcinogens, bilirubin, and polyciclic aromatic hydrocarbons)
GSTA1-1 (alpha class of GST) is the most abundant form of GST (about 5% of the liver cytosolic protein) |
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Term
| What is s-aryl-transferase? |
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Definition
Another name for GST (GSTA1-1)/ligandin
Dimer, monomeric weight 22000-29000, 5% cytoslic liver protein, most abundant GST
name no longer used |
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Term
| What is the most abundant GST in liver? |
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Definition
| GST A1-1, this protin is about 5% of the liver cytosolic protein |
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Term
| Which enzyme is responsible for making GSH conjugates? |
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Definition
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Term
| Which enzyme is responsible for the isomerization of double bonds (specifically in sterols) |
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Definition
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Term
| Describe the "ligandin" function of GSTs. Is it reversible? |
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Definition
The "ligandin function" is important for intracelular transport of organic molecules (including potentially toxic compounds)
Yes, this binding is reversible |
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Term
| In what case would GST bind irriversibly to an electrophile? |
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Definition
| If there's insufficient amounts of GSH in the cell, the electrophile will covalently bond to cysteine, serine, or threonine groups in the GST protein. |
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Term
| To what three amino acids in GST would an elctrophile bind in the case of insufficient GSH in the cell? |
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Definition
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Term
| In which fraction of the cell were GST's first discovered? |
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Definition
| Cytosolic fraction, (however, there is now evidence for microsomal and mitochondrial forms) |
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Term
| Describe the nomenclature of GST |
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Definition
Families/class - Roman capital letters (corresponding to Greek letters)
A alpha
M mu
P phi
T theta
Z zeta
S sigma
O omega
K kappa
Subfamilies - Arabic numbers (homodimers = same ; heterodimers = different) |
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Term
Historically, if two GST forms showed cross reactivity to the same antibody, they were considered to be in the same _________.
Today, with the advent of proteomics, a limit of _____% sequence identity. However, most GST's are >___% identical. (For humans, this puts GST's of the same class on the same chromosome. |
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Definition
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Term
| Most forms of GST (except ____ and _____) use CDNB as a ______. |
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Definition
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Term
| In which three families of GST's are there considerable overlap, making them less selective enzymes? |
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Definition
Alpha, Mu, Pi
(all have conserved tyrosine residue in GSH-binding site) |
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Term
| Describe the importance of the G-site in GST. |
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Definition
The G-site is the GSH binding site of GST. It binds and facilitates the formation of the GS- anion and ehances catalysis by lowering pKa.
GSH pKa 9.5
GST-bound GS- pKa 7.5 |
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Term
| What do alpha, mu, and pi GST's have in common? |
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Definition
A conserved tyrosine residue in the GSH-binding site
low selectivity |
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Term
| What to theta and zeta classes of GST's have in common? |
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Definition
| A conserved serine residue in the GSH binding site, activate thiol gp. |
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Term
| The alpha, mu, and pi classes of GST's are readily inducible by chemicals... name inducers of alpha and mu. |
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Definition
Alpha - induced by phenobarbital, 3-methylcholanthrene, and various antioxidants
Pi - induced by antioxidants |
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Term
| What is the response element involved with induction of GST A by 3MC? |
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Definition
| Dioxin Response Element (DRE) is the regulatory response element for 3MC induction of GST alpha |
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Term
| What is the response element involved with antioxidants and pro-oxidants for Alpha and Pi classes of GST's? |
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Definition
| ARE (antioxidant response element) |
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Term
| Explain the significance of thiol modification of Keap1 cytoskeletal binding protein. |
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Definition
Modification of the cysteine residues on Keap1 allos Nrf2 to accumulate and enter the nucleus.
The Nrf2 then forms a heterodimer with Maf. The dimer binds to ARE and enhances transcription of GSTA4-4 and glutamate cysteine modulatory subunites (and thus synthesis of GSH). |
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Term
| How does activation of the ARE lead to decrased amounts of 4-HNE in the cell? |
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Definition
Transactivation of the ARE-driven genes by the Nrf2-Maf dimer increases the production of GSTA4, glutamate cysteine ligase catalytic, and glutamate cysteine modulatory elements.
The elevation in the amounts of GSTA4-4 and GST allow increased metabolism of 4-HNE and its elimination from the cell via MRP. |
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Term
| How is 4-HNE eliminated from the cell? |
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Definition
| Metabolism using GSTA4-4/GSH and elimination via MRP |
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Term
| How does phenobarbital affect GST? |
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Definition
First of all, leads to an increase in liver size
Induces GST A1-1 specifically (ligandin)
Studies showed that maximal induction with PB lead to ~13% GST A1-1 liver cytosolic protein. |
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Term
| Are polymorphisms common in GSTs? |
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Definition
Yes
Numerous alleles have been identified for common GSTs |
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Term
| Why don't some people express an active GST M1-1 allele that detoxifies carcinogens in cigarette smoke? |
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Definition
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Term
| Why don't some people express activeGST T1-1 allele? |
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Definition
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Term
| Why are GST P's overexpressed in certain cancers? |
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Definition
Multi Drug Resistance phenomenon
Expression of GST P in blood is suggested as a tumor marker |
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Term
| Name two physiologically produced electrophiles that have been propsed as regulators of GST expression |
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Definition
| hydroxynonenal and maleylacetoacetate |
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Term
| Does age affect GST expression? |
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Definition
| Yes, fetus has none essentially, increases with age until ~20 yrs and then starts to slowly decrease (although varies greatly between individuals) |
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Term
Describe the tissue distribution of GSTs?
Where are alpha, mu and pi forms mostly found? |
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Definition
Found mainly in liver
Widespread in tissues - (especially intestine & kidney)
Alpha - lung
Mu - lung
Pi - placenta |
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Term
| How can calss A, M, and P GST's be isolated from liver cytosol? |
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Definition
Affinity chromatography:
Media: Glutathione-agarose or S-hexylthutathione coupled to epoxy-activated agarose
Add liver cytosol, GSTs will bind to medium, wash with buffer to remove all other proteins.
Wash with high concentration of GSH to remove GST's from medium.
Reverse phase HPLC onlarge pore columns, tetected by UV/fluorescence |
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Term
| In HPLC analysis of GST enzymes, wildtype male and female rats both express the ______ gene the most (similar to ligandin gene in humans)... However, when GST Z form is knocked out, the ______ gene is massively induced in both male and female rats. |
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Definition
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Term
In the mercapturic pathway:
Where does glutathione conugation occur? By what enzyme? |
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Definition
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Term
In the mercapturic pathway
Where does cleavage of gamma-glutamyl group occur? By what enzyme? |
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Definition
Bile/Kidney (not in liver)
gamma-glutamyl transpeptidase - high levels in the kidney on outside of cell membranes, also found in the cytosol bound to the membrane |
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Term
In the mercapturic pathway:
Where does cleavage of the glycine group occur? By what enzyme? |
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Definition
Hydrolysis of cysteinyl-glycine occurs in the kidney, intestine, and other sites (not liver)
Enzymes that catalyze this rxn:
Cysteinylglycinase
Aminopeptidase-M
(Both are membrane-associated zinc metalloproteinases) |
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Term
| Which enzymes are zinc metalloproteinases? |
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Definition
| Cysteinylglycinases and Aminopeptidase-M's (those that hydrolyze cysteinyl-glycine conjugates) |
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Term
In the mercapturic pathway:
Where does acetylation of cysteine occur? By what enzyme? |
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Definition
Kidney
Cystein S-conjugate N-aetyl transferase, requires Acetyl-coenzyme A as the acetyl donor
Product is a non-toxic, readily excreted mercapturic acid (N-acetylcysteine conjugate) |
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Term
| Where does most deacetylation occur? What are the reactants and products of deacetylation? |
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Definition
The kidney - it has high deacetylase activity
Mercapturic acid can be deacetylated to reform cysteine conjugates |
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Term
| Which enzyme is pyridoxal phosphate dependent? |
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Definition
| Beta-lyases that form thiol compounds, pyruvate, and ammonia from cysteine |
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Term
| How do GSH conjugates like mercapturic acid and cysteine conjugates transport in the body? |
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Definition
| Several transporter proteins assist in transport. |
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Term
| The liver is the main site of ________. But the _______ and ________ are important in further processing. |
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Definition
GSH conjugates
Kidney & Intestine |
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