Purification and partial characterization of glyoxalase I from bovine brain.

Glyoxalase I was purified to homogeneity from bovine brain using affinity chromatography on S-hexylglutathione-Sepharose 6B with a yield of 22%. The enzyme was a dimer (44,000 Daltons) composed of, apparently, identical subunits (22,000 Daltons), as shown by SDS electrophoresis, and contained one mole of Zn2+/monomer. The active site metal ion, Zn2+, was removed by dialysis against EDTA, but the activity of the apoenzyme obtained was not completely restored after addition of Co2+ and Zn2+ (<25%), while a recovery of 50% was obtained after addition of Mg2+. The enzyme was inhibited by S-bromobenzylglutathione and S-p-nitrobenzylglutathione with a Ki value of 21 microM and 32 microM, respectively. The highest dissociation constant observed for the brain enzyme with respect to that reported for human erythrocytes, or other mammalian forms of enzyme could be related to a tissue-specific dependence of the glyoxalase I activity.

Functional residues on the enzyme active site of glyoxalase I from bovine brain.

Bovine brain glyoxalase I was investigated in order to identify amino acid residues essential for its catalytic activity. This enzyme is a 44-kDa dimeric protein which exhibits a characteristic intrinsic fluorescence, with an emission peak centered at 342 nm. The total of eight tryptophan residues/molecule was estimated by using a fluorescence titration method. Low values of Stern Volmer quenching constants for the quenchers used indicated that the tryptophan residues are relatively buried in the native molecule. Similar results were obtained for glyoxalase I, purified from yeast and human erythrocytes. The activity of bovine brain glyoxalase I was found to be particularly sensitive to 2,3-butanedione and diethylpyrocarbonate, selective reagents for arginine and histidine residues, respectively. A minor effect was observed by treatment of the enzyme with other amino acid-specific reagents. A protective effect of the competitive inhibitor S-hexylglutathione was observed for all reagents used, indicating the presence of modified amino acids in or near the enzyme active site.

Effect of 17-beta estradiol and epidermal growth factor on DNA and RNA labeling in astroglial cells during development, maturation and differentiation in culture.

Growth factors stimulate astroglial and neuronal proliferation and differentiation in culture. Estrogens markedly influence astroglia, and are key factors participating in neurodegeneration. The aim of the present study was to investigate interactions between estradiol (E2) and epidermal growth factor (EGF) during astroglia development, maturation and differentiation in culture. DNA or RNA labeling in 16 or 40 or 60 days in vitro (DIV) astrocyte cultures treated for 24 or 48 h with EGF and/or E2 was evaluated. A significant increase in DNA labeling in 16 DIV astrocyte cultures treated for 24 h with EGF (5 ng/ml) and E2 (1 nM) was found. EGF (5 or 10 ng/ml) addition in the last 24 h in 48 h E2 (1 or 5 nM)-treated astrocyte cultures at 16 DIV caused a slight, but significant increase in DNA labeling. No differences in RNA labeling were observed in 16 DIV astrocyte cultures treated for 24 or 48 h with EGF (5 or 10 ng/ml) in the presence of E(2) (1 or 5 nM). A significant stimulation in DNA labeling was shown in 40 DIV astrocyte cultures treated for 48 h with E2 (1 or 5 nM) in the presence of EGF (5 or 10 ng/ml) added in the last 24 h. In well differentiated astroglial cell cultures (60 DIV), DNA labeling was remarkably increased after 24 h treatment with 1 nM E2 or 5 ng/ml EGF. Co-addition of 1 nM E2 and 5 ng/ml EGF for 24 h reduced [methyl-(3)H]thymidine incorporation, when data are compared to E2- or EGF-treated cultures. Addition of EGF in the presence of E2 for 48 h or only in the last 24 h caused a significant decrease of [methyl-(3)H]thymidine incorporation in comparison with EGF-treated cultures at 60 DIV or with untreated cultures. Treatment of cultures for 24 h with EGF (5 or 10 ng/ml) alone or in combination with E2 (1 or 5 nM) induced a strong increase of RNA labeling in 60 DIV astrocyte cultures. Addition for 48 h of E2 (1 or 5 nM) or EGF (5 or 10 ng/ml) alone or in association stimulated significantly RNA labeling in astrocyte cultures at 60 DIV. When 60 DIV astrocyte cultures were treated for 48 h with E2 (1 or 5 nM) in the presence of EGF (5 or 10 ng/ml) added only in the last 24 h, a potentiating effect of RNA labeling was observed. The above results suggest that interaction between growth factors and estrogens may contribute to regulate astroglia development, maturation and differentiation.

Functional residues at the active site of bovine brain adenosine deaminase.

Brain adenosine deaminase was investigated in order to identify amino acid residues essential for its catalytic activity. The pH dependence of log Vmax shows that the enzyme activity depends on two ionizing groups with pK values of 5.4, that must be unprotonated, and 8.4, that must be protonated, for the catalysis. These same groups are observed in the Vmax/Km profiles. The plausible role of histidine residues at the active site of brain adenosine deaminase was proved by chemical modification with (DEP). The histidine specific reagent inactivated the enzyme following a pseudo first-order kinetics with a second-order rate constant of 8.9 10(-3) (+/- 1.8 10(-3)) M-1 min-1. The inhibition of the enzyme with PCMBS was studied monitoring the enzyme activity after incubation with the inhibitor. Brain adenosine deaminase exhibited a characteristic intrinsic tryptophan fluorescence with an emission peak centered at 335 nm. Stern-Volmer quenching parameters in the presence of acrylamide and iodide indicated that tryptophan residues are buried in the native molecule. Tryptophan residues also showed a high heterogeneity that was increased after binding of ground- and transition-state analogs to the enzyme.

Reinvestigation of the role of thiol groups of glyoxalase I purified from yeast (Saccharomyces cerevisiae).

Glyoxalase I has been purified to homogeneity from Saccharomyces cerevisiae and tested with two different thiol reagents, i.e., 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) and 1-chloro-2,4-dinitrobenzene (CDNB). DTNB reacts with four thiol groups per molecule of enzyme and leads to a complete inhibition which is not reversed by addition of the disulfide-reducing agent dithiothreitol. On the other hand, CDNB slightly affects the glyoxalase-I activity and alkylates only one thiol residue/enzyme. In agreement, DTNB reacts with three thiol residues of the CDNB-reacted enzyme and no reactivation is observed after dithiothreitol treatment. The peptide containing the CDNB-reactive thiol group has been isolated and the sequence overlaps the segment 58-63 of the only known primary structure of glyoxalase I from Pseudomonas putida.

Adenosine deaminase from Saccharomyces cerevisiae: kinetics and interaction with transition and ground state inhibitors.

Several adenosine analogs, such as coformycin, 2'-deoxycoformycin and erythro-9-(3-nonyl-p-aminobenzyl)adenine (EHNA), which are strong inhibitors of mammalian adenosine deaminase, are much weaker inhibitors of the Saccharomyces cerevisiae enzyme. The specificity of the yeast enzyme is more restricted than that of mammalian adenosine deaminase, particularly towards the ribose moiety and around position 6 and 1 of the substrate. The sulphydryl group appears to be more masked in the yeast than in the mammalian enzyme. The kinetic effects of pH with adenosine substrate and with the inhibitor purine riboside are reported. The findings on specificity and pH kinetic effects can be interpreted in a model involving proton transfer from the -SH group of the enzyme to the N-1 atom of the substrate.

Adenosine deaminase from bovine brain: purification and partial characterization.

Bovine brain adenosine deaminase cytoplasmatic form was purified about 450 fold by salt fractionation, column chromatography on DEAE-cellulose, octyl-sepharose 4B and affinity chromatography on CH-sepharose 4B 9-(p-aminobenzyl)adenine. The purified enzyme was homogeneous on disc gel electrophoresis; the enzyme had a molecular mass of about 65 kDa with an isoelectric point at pH 4.87. The Km values for adenosine and 2'-deoxyadenosine were 4 x 10(-5) and 5.2 x 10(-5) M, respectively. The enzyme showed a great stability to temperature with a half life of 15 hours at 53 degrees C significantly different compared to that known for other mammalian forms of this enzyme. Aza and deaza analogs of adenosine and erythro-9-(2-hydroxy-3-nonyl) adenine were good inhibitors of the bovine brain enzyme with little difference with respect to those reported for the adenosine deaminases purified from other sources. Kinetic constants for the association and dissociation of coformycin and 2'-deoxycoformycin with the bovine brain adenosine deaminase are reported.

Adenosine deaminase from Saccharomyces cerevisiae: purification and characterization.

Adenosine deaminase from Saccharomyces Cerevisiae was purified about 1600 fold by salt fractionation, ion exchange and affinity chromatography. Some physico-chemical properties have been determined: the molecular weight of the enzyme by gel filtration is 85,000 daltons; one -SH is readily titrated by paramercuribenzoate per 78,000 mol. weight; optimum pH is 7; Km for adenosine is 40.7 microM; 2'-deoxyadenosine is not a substrate. Deazaadenosine analogues are good inhibitors, while erythro-9-(2-hydroxy-3-nonyl) adenine binds with low affinity. These properties are compared with those of other adenosine deaminases.

The effect of the presence of the metal prosthetic groups on the subunit structure of bovine superoxide dismutase in sodium dodecyl sulfate.

Dissociation into protomers of bovine superoxide dismutase by sodium dodecyl sulfate (SDS) depends on the metal prosthetic group and incubation time in the presence of detergent. The holoenzyme containing either copper and zinc or copper and cobalt is not dissociated. The fully metal-free apoenzyme is dissociated into protomers after short preincubation in SDS. The copper-free enzyme, still containing zinc or cobalt, is dissociated to a significant extent only after 24 hours preincubation in SDS. This effect is associated with a gradual alteration of the native zinc site, as followed by optical spectra of the homologous cobalt enzyme. Removal of SDS results in significant reassociation of protomers which is apparently independent of the presence of metals.