A method for the detection of asparagine deamidation and aspartate isomerization of proteins by MALDI/TOF-mass spectrometry using endoproteinase Asp-N. (49/339)

A method was established for evaluating Asn deamidation and Asp isomerization/racemization. To detect the subtle changes in mass that accompany these chemical modifications, we used a combination of enzyme digestion by endoproteinase Asp-N, which selectively cleaves the N-terminus of L-alpha-Asp, and MALDI/TOF-mass spectrometry. To achieve better resolution, we employed digests of (15)N-labeled protein as an internal standard. To demonstrate the advantages of this method, we applied it to identify deamidated sites in mutant lysozymes in which the Asn residue is mutated to Asp. We also identified the deamidation or isomerization site of the lysozyme samples after incubating them under acidic or basic conditions.  (+info)

Human thymine DNA glycosylase (TDG) and methyl-CpG-binding protein 4 (MBD4) excise thymine glycol (Tg) from a Tg:G mispair. (50/339)

The repair enzymes thymine DNA glycosylase (TDG) and methyl-CpG-binding protein 4 (MBD4) remove thymines from T:G mismatches resulting from deamination of 5-methylcytosine. Thymine glycol, a common DNA lesion produced by oxidative stress, can arise from oxidation of thymine or from oxidative deamination of 5-methylcytosine, and is then present opposite adenine or opposite guanine, respectively. Here we have used oligonucleotides with thymine glycol incorporated into different sequence contexts and paired with adenine or guanine. We show that TDG and MBD4 can remove thymine glycol when present opposite guanine but not when paired with adenine. The efficiency of these enzymes for removal of thymine glycol is about half of that for removal of thymine in the same sequence context. The two proteins may have evolved to act specifically on DNA mismatches produced by deamination and by oxidation-coupled deamination of 5-methylcytosine. This repair pathway contributes to mutation avoidance at methylated CpG dinucleotides.  (+info)

Deamination of newly-formed dopamine in rat renal tissues. (51/339)

1. The present study has examined the formation of dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) in slices of the rat renal cortex and the renal medulla loaded with exogenous L-beta-3,4-dihydroxyphenylalanine (L-DOPA). The effects of pargyline and of two selective inhibitors of monoamine oxidase (MAO) types A and B, respectively Ro 41-1049 and Ro 19-6327, on the deamination of newly-synthesized dopamine in kidney slices incubated with exogenous L-DOPA were also tested. The assay of L-DOPA, dopamine, noradrenaline and DOPAC was performed by means of h.p.l.c. with electrochemical detection. 2. Incubation of renal slices with exogenous L-DOPA resulted in a concentration-dependent accumulation of dopamine and DOPAC; the tissue levels of newly-formed dopamine and DOPAC in slices of the renal medulla were 6-8% of those in cortical slices. 3. Pargyline (0.1 mM) produced a marked decrease (84% reduction) in the formation of DOPAC in kidney slices loaded with 1.0 mM L-DOPA; this effect was accompanied by a 17% increase in the accumulation of dopamine. Similar effects were obtained at higher concentrations of pargyline (0.5 and 1.0 mM). At 5.0 and 10.0 mM pargyline, a marked decrease (46 and 76% reduction) in the accumulation of newly-formed dopamine was observed. 4. The accumulation of dopamine and DOPAC was found to be time-dependent in experiments in which tissues were incubated with 5 and 10 microM L-DOPA for 5, 10, 20 and 30 min. Pargyline (0.1 mM) produced an increase in the accumulation of dopamine at all incubation periods and decreased the formation of DOPAC. 6. It is concluded that deamination of newly-formed dopamine in kidney slices loaded with L-DOPA constitutes an important mechanism of amine inactivation. The results presented also suggest that most of the MAO, located inside the compartment where the synthesis of dopamine occurs, is of the A type.  (+info)

Structural and functional properties of hen egg-white lysozyme deamidated by protein engineering. (52/339)

The structural and functional properties of lysozymes genetically deamidated at positions 103 (N103D) and 106 (N106D) were studied by a protein engineering technique. The wild-type and mutant lysozymes were expressed in Saccharomyces cerevisiae and purified from the cultivation medium in two steps by cation-exchange chromatography on CM-Toyopearl. The lytic activity of deamidated lysozymes was almost the same as that of wild lysozyme, although the optimal pH of activity was slightly shifted to lower pH by the deamidation. The Gibbs free energy changes of unfolding (delta G) at 20 degrees C for N103D and N106D were almost the same as that of wild-type. On the other hand, the structural flexibility of lysozymes, estimated by protease digestion, was significantly increased by the deamidation. The surface functional properties of deamidated lysozymes were considerably enhanced, compared to those of wild-type lysozyme. These results suggest that structural flexibility is an important governing factor in surface functional properties of proteins, regardless of their structural stability.  (+info)

An in vitro interethnic comparison of monoamine oxidase activities between Japanese and Caucasian livers using rizatriptan, a serotonin receptor 1B/1D agonist, as a model drug. (53/339)

AIMS: Monoamine oxidase (MAO) is located in human liver, and catalyses the oxidative deamination step of many xenobiotics. However, whether there exists an interethnic difference in MAO activities has, to our knowledge, not been clarified. We aimed to assess the MAO type A (MAO-A) involvement in the metabolic pathway of rizatriptan (RIZ), an antimigraine 5-hydroxytryptamine (5-HT)1B/1D agonist, and the interethnic difference in MAO activities between Caucasians and Japanese using RIZ as a model drug in in vitro experiments. METHODS: Oxidative deaminase activities were determined with the subcellular fractions of Japanese livers and the microsomal fraction of Caucasian livers using RIZ, 5-HT (MAO-A substrate) and 2-phenylethylamine (PEA) (MAO-B substrate) as substrates. RESULTS: The oxidative deaminase activities of RIZ vs. 5-HT were highly (r = 0.87 and 0.96, P < 0.001) correlated with each other in both the microsomal and mitochondrial fractions of Japanese livers. Subsequent results were obtained from in vitro experiments using liver microsomes based upon these findings. The oxidative deaminase activities of RIZ were inhibited completely by the nanomolar-order concentration of clorgyline and Ro 41-1049 (MAO-A selective inhibitors), but not by that of Ro 16-6491 (MAO-B selective inhibitor). The majority of the mean Michaelis-Menten values for three substrates toward MAO obtained from six Japanese and six Caucasian liver microsomes reached no significant differences between the two ethnic groups. The mean microsomal oxidative deaminase activities assessed in 18 Japanese and 20 Caucasian livers using the three substrates also showed no significant differences between the two ethnic groups. CONCLUSIONS: RIZ is mainly metabolized by MAO-A and the in vitro oxidative deaminase activities mediated via MAO-A and -B do not appear to differ between Japanese and Caucasians.  (+info)

Involvement of SSAO-mediated deamination in adipose glucose transport and weight gain in obese diabetic KKAy mice. (54/339)

Semicarbazide-sensitive amine oxidase (SSAO) is located on outer surfaces of adipocytes and endothelial and vascular smooth muscle cells. This enzyme catalyzes deamination of methylamine and aminoacetone, leading to production of toxic formaldehyde and methylglyoxal, respectively, as well as hydrogen peroxide and ammonium. Several lines of evidence suggest that increased SSAO activity is related to chronic inflammation and vascular disorders related to diabetic complications. We found that a highly potent and selective SSAO inhibitor, (E)-2-(4-fluorophenethyl)-3-fluoroallylamine (FPFA), was capable of reducing numbers of atherosclerotic lesions as well as weight gain in obese KKAy mice fed an atherogenic diet. SSAO inhibitors cause a moderate and long-lasting hyperglycemia. Such an increase in serum glucose is a result of reduction of glucose uptake by adipocytes. SSAO-mediated deamination of endogenous methylamine substrates induces adipocyte glucose uptake and lipogenesis. Highly selective SSAO inhibitors can effectively block induced glucose uptake. The results suggest that increased SSAO-mediated deamination may be concomitantly related to obesity and vascular disorders associated with type 2 diabetes.  (+info)

Effect of deamidation of asparagine 146 on functional and structural properties of human lens alphaB-crystallin. (55/339)

PURPOSE: To elucidate the effect of deamidation on the structural and functional properties of human alphaB-crystallin. METHODS: Site-directed mutagenesis was used to generate three deamidated mutants of alphaB-crystallin: N78D, N146D, and N78D/N146D. The mutations were confirmed by DNA sequencing and matrix-assisted desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Recombinant native alphaB-crystallin (wild type [WT]) and the three mutated alphaB species were expressed, and each species was purified to homogeneity by ion-exchange chromatography followed by hydrophobic interaction chromatography. The structural and functional properties compared with WT protein were investigated, respectively, by static light scattering (SLS), circular dichroism (CD), and fluorescence spectroscopy and by determining chaperone activity with the use of three substrates. RESULTS: Native WT and the N78D mutant showed relatively higher chaperone activity compared with the N146D and N78D/N146D mutants with all the substrates. Further, during binding experiments with 1-anilino-8-naphthalenesulfonate (ANS), the WT and N78D mutant showed relatively more solvent-exposed hydrophobic residues than the N146D and N78D/N146D mutants. On determining far-UV circular dichroism and tryptophan (Trp) fluorescence spectra, significant secondary and tertiary structural changes were observed in the N146D and N78D/N146D mutants compared with WT and the N78D mutant. The static light scattering data showed a high order of oligomerization in all the three mutants. N146D and N78D/N146D formed the largest oligomers of 750 and 770 kDa, respectively, compared with WT (580 kDa). CONCLUSIONS: The results show that the deamidation of N146 but not of N78 have profound effects on the structural and functional properties of alphaB-crystallin.  (+info)

The Bacillus subtilis counterpart of the mammalian 3-methyladenine DNA glycosylase has hypoxanthine and 1,N6-ethenoadenine as preferred substrates. (56/339)

The AAG family of 3-methyladenine DNA glycosylases was initially thought to be limited to mammalian cells, but genome sequencing efforts have revealed the presence of homologous proteins in certain prokaryotic species as well. Here, we report the first molecular characterization of a functional prokaryotic AAG homologue, i.e. YxlJ, termed bAag, from Bacillus subtilis. The B. subtilis aag gene was expressed in Escherichia coli, and the protein was purified to homogeneity. As expected, B. subtilis Aag was found to be a DNA glycosylase, which releases 3-alkylated purines and hypoxanthine, as well as the cyclic etheno adduct 1,N(6)-ethenoadenine from DNA. However, kinetic analysis showed that bAag removed hypoxanthine much faster than human AAG with a 10-fold higher value for k(cat), whereas the rate of excision of 1, N(6)-ethenoadenine was found to be similar. In contrast, it was found that bAag removes 3-methyladenine and 3-methylguanine approximately 10-20 times more slowly than human AAG, and there was hardly any detectable excision of 7-methylguanine. It thus appears that bAag has a minor role in the repair of DNA alkylation damage and an important role in preventing the mutagenic effects of deaminated purines and cyclic etheno adducts in Bacillus subtilis.  (+info)