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Role of Metabolism in Chemically Induced Nephrotoxicity
Published in Robin S. Goldstein, Mechanisms of Injury in Renal Disease and Toxicity, 2020
The requirement for metabolism of GSH S-conjugates to the corresponding cysteine S-conjugates was demonstrated by showing that irreversible inhibition of γ-glutamyltransferase with acivicin prevented the cytotoxicity of DCVG (Lash and Anders, 1986), of S-(2-chloro-1,1,2-trifluoroethyl)glutathione (Dohn et aL, 1985b), and of the GSH S-conjugate of hexachlorobutadiene (Jones et al., 1986), that stimulation of γ-glutamyltransferase activity by addition of glycylglycine as a cosubstrate potentiated the toxicity of DCVG (Lash and Anders, 1986), and that inhibition of dipeptidase activity prevented the cytotoxicity of both DCVG and the corresponding cysteinylglycine S-conjugate in isolated kidney cells (Lash and Anders, 1986).
The Modification of Cysteine
Published in Roger L. Lundblad, Chemical Reagents for Protein Modification, 2020
Cleavage at S-cyano residues has also been reported by Marshall and Cohen.68 In these studies the enzyme was first reacted with 5,5’-dithiobis (2-nitrobenzoic acid) in 0.020 M 4-morpholinepropanesulfonic acid-0.1 M KC1, pH 7.1 for 3 h at 25°C. Conversion to the S-cyano derivative was accomplished by reaction in 0.2 M KCN, pH 8.1. Cleavage was accomplished by incubation at pH 8.0 at 50°C for 24 h. The reaction of 2-nitro-5-thiocyanobenzoic acid with phosphofructokinase has been studied by Ogilvie.69 Approximately 1 mol of cysteine is available for modification in the native enzyme with an approximately stoichiometric excess of reagent (1.06 mol 2-nitro-5-thiocyanobenzoic acid per mole enzyme protomer) (Figure 30). The modification with 2-nitro-5-thiocyanobenzoic acid was performed in 0.025 M glycylglycine — 0.025 M sodium phosphate, pH 7.2 (containing 1 mM EDTA, 0.4 mM fructose-6-phosphate, and 0.1 mM ATP) at 24°C. Cleavage at the S-cyanocysteinyl residue is accomplished by incubation of the modified protein in 0.2 M Tris-acetate, pH 8.1 containing 2% sodium dodecyl sulfate at 37°C. Approximately 20% of the total phosphofructokinase was cleaved after 48 h of incubation at 37°C. This would correspond to approximately 40% cleavage of the S-cyanylated protein. Cleavage at S-cyano derivatives of cysteine is covered in detail in Chapter 5.
Methods of Protein Iodination
Published in Erwin Regoeczi, Iodine-Labeled Plasma Proteins, 2019
Hunter and Ludwig,133 who did extensive studies using methyl benzimidate and methyl acetimidate, found that amidine formation was strongly pH dependent; the optimal pH not only depended on the type of imidoester used but also on the reactive amino group. Thus, when using methyl benzimidate, optimal reaction with ϵ-aminocaproic acid was observed at pH 9.5 to 10, whereas with glycylglycine at pH 7.6. Insulin reacted with imidoesters much the same way as predicted from model compounds. Amidine formation was also dependent on the temperature: a decrease from 25 to 1°C reduced the kapp of the reaction approximately fivefold.
Intracellular trafficking and cytotoxicity of a gelatine–doxorubicin conjugate in two breast cancer cell lines
Published in Journal of Drug Targeting, 2020
Mohammed M. Alvi, Rachel E. Nicoletto, Bayan A. Eshmawi, Hyun (Kate) Kim, Christopher R. Cammarata, Clyde M. Ofner
This procedure was modified from a previous report based on aqueous conditions [35]. Briefly, 100 mg of gelatine was dissolved in 4 ml of formamide at pH 7. A solution of glycylglycine (GG, 30.5 mg) was prepared in formamide at pH 3. The pH was adjusted to 7, the GG solution was added to the gelatine solution, and EDC (24.6 mg) was added for a 3 h reaction. The product was precipitated with 45 ml ice cold absolute ethanol, centrifuged, followed by dissolution in 5 ml of 5 mM ammonium acetate and passed through a solid phase extraction column (PD-10 desalting column, GE Healthcare, NJ). The 3.5 ml collected solution was lyophilised. A 6 ml formamide solution with 132 µl of hydrazine hydrate was added to the gelatine-GG lyophilised powder, stirred for 1 h, then EDC (27.6 mg) was added and maintained at pH 6 for 3 h. The gelatine-GG-hydrazide (Gel-GG-Hz, precursor) was collected by ethanol precipitation and desalting described above, lyophilised and stored at −20 °C. A 10-fold molar excess of DOX (approximately 60 mg) to hydrazide groups was added to the precursor solution in formamide with 10 mM aniline and 200 mg of anhydrous sodium sulphate at pH 5.6 and stirred for 24 h in the dark. The product was purified with two ethanol precipitations and four desalting steps, lyophilised and stored. Prior to use, the GDOX powder was purified by one extra desalting step.
A novel αVβ3 ligand-modified HPMA copolymers for anticancer drug delivery
Published in Journal of Drug Targeting, 2018
Fengling Wang, Lian Li, Wei Sun, Lijia Li, Yuanyuan Liu, Yuan Huang, Zhou Zhou
N-Methacryloyl-glycylglycine (MA-GG-OH). MS (ESI): m/z 201.1 [M + H+]; purity > 99.0% (HPLC); 1H NMR 600 MHz [(CD3)2SO]: δ 1.88 s, 3H (CH3); 3.76 d, J = 6.0 Hz 4H (Gly-Gly); 5.37 m, 1H (CH2=); 5.74 s, 1H (CH2=); 8.12 t, J = 6.0 Hz 1H (NH); 8.17 t, J = 6.0 Hz 1H (NH); 12.57 s, 1H(CO-OH).
Porphyromonas gingivalis laboratory strains and clinical isolates exhibit different distribution of cell surface and secreted gingipains
Published in Journal of Oral Microbiology, 2021
Christine A. Seers, A. Sayeed M. Mahmud, N. Laila Huq, Keith J. Cross, Eric C. Reynolds
Maintaining a reduced environment is necessary for optimum gingipain activity and a reducing agent such as cysteine is an important component of a gingipain proteolytic assay to protect the catalytic cysteine from oxidation. Glycylglycine can stimulate gingipain proteolytic activities in the presence of substrates, including BApNA, azocasein, and azocoll [47]. The activity enhancement in the presence of glycylglycine was originally suggested by Potempa et al. [9] to be due to the elimination of non-productive, high-affinity binding of the substrate arginine side chain. However, Zhang et al. [24] subsequently showed that gingipains behave as transpeptidases, with it likely that the transfer reaction permits faster product release, thus enhancing the rate of substrate turnover. The stimulatory effect of cysteine beyond that required for active site cysteine reduction indicates it may also behave as a transpeptidation acceptor molecule. This was indicated by the higher whole-cell Arg-gingipain Vmax of strains ATCC 33277 and W50 in the presence of 20 mM cysteine relative to the whole-cell Arg-gingipain Vmax in the presence of 20 mM DTT (Figure 4). This result is consistent with the previous report where it was shown that among the thiol-containing compounds, cysteine (0–50 mM) produced a 1.4-fold higher stimulatory effect than that of either DTT or β-mercaptoethanol in the presence of 100 mM glycylglycine [47]. However, the addition of 200 mM cysteine to the assay had an adverse effect on the gingipain function. This could be due to the chelation of calcium (Ca2+), an ion that stabilizes gingipains [47]. Relative to the assay containing 200 mM cysteine, the addition of 200 mM glycylglycine was found to only have a positive effect on Arg-gingipain activities produced by 10 of the 14 strains of P. gingivalis (Table 2). This may suggest that differences exist between the enzymes of these strains that are sufficient to affect substrate-acceptor molecule interactions during catalysis.