The kinetic mechanism of the reactions catalyzed by the glutamate synthase from Azospirillum brasilense

The reactions catalyzed by glutamate synthase from Azospirillum brasilense have been investigated by a combination of absorption spectroscopy, steady-state kinetic measurements and experiments with stereospecifically labelled substrate. The data show that both L-glutamine-dependent and ammonia-dependent reactions of the glutamate synthase from A. brasilense follow an identical two-site uni-uni bi-bi kinetic mechanism, in which the enzyme is alternately reduced by NADPH and oxidized by the iminoglutarate formed on addition of ammonia to the C2 of 2-oxoglutarate. The spectroscopic experiments support the involvement of the enzyme chromophores (flavins and iron-sulfur centers) in both reactions. Finally, using stereospecifically labelled NADPH, we showed that the enzyme from Azospirillum is specific for the transfer of the 4S hydrogen of NADPH. During the catalysis of both L-glutamine-dependent and ammonia-dependent reactions, this hydrogen atom equilibrates with the solvent. The data obtained with glutamate synthase from A. brasilense, a diazotroph, differ significantly from those regarding the ammonia-dependent reaction of other glutamate synthases. The ammonia-dependent activity of glutamate synthase from Azospirillum is not physiologically significant, representing only a segment of the overall physiological L-glutamine-dependent activity and requiring the enzyme flavins and iron-sulfur centers. Finally, the data are not consistent with the hypothesis [Geary, L. E. & Meister, A. (1977) J. Biol. Chem. 252, 3501-3508] that the small subunit of glutamate synthase is endowed with a glutamate-dehydrogenase-like activity.     

Effect of Zn(II) and Mg(II) on phosphohydrolytic activity of rat matrix-induced alkaline phosphatase

Rat matrix-induced alkaline phosphatase is an enzyme which requires magnesium and zinc ions for its maximal activity. Two Zn(II) ions and one Mg(II) ion are bound to each subunit of native dimeric enzyme. The presence of magnesium ion (10-100 microM) or zinc ion (7-20 nM) alone is sufficient to stimulate apoenzyme activity. However maximal activity (264 U/mg) requires the presence of both ions. Binding of Zn(II) ions to the Mg(II) binding site causes a strong inhibition of the apoenzyme while the binding of Mg(II) on Zn(II) binding site is not sufficient to stimulate PNPPase activity of the apoenzyme. Binding of both ions to the enzyme molecule did not change the apparent dissociation constant for PNPP hydrolysis.     

Immunoelectron microscopic localization of D-amino acid oxidase in rat kidney and liver

Summary The intracellular localization ofd-amino acid oxidase in rat kidney and liver has been investigated using the indirect immunogold postembedding technique. Different fixation and embedding conditions for optimal preservation of antigenicity and fine structure have been tested. Immunolabelling was possible only in tissues embedded in polar resins (glycol methacrylate and Lowicryl K4M). In kidney the enzyme was demonstrable only in the peroxisomes of the proximal tubule, where it was associated with the peroxisome core. The enzyme was present in all the peroxisomes of the proximal tubule and appeared to be codistributed with catalase. Control experiments and quantitative analysis confirmed the specificity of thed-amino acid oxidase immunolocalization. All the other cells in kidney failed to demonstrate any labelling. In liver, the immunolabelling was present in the matrix of the hepatocyte peroxisomes, whereas no traces of the enzyme were found in the nucleoid. The intensity of the immunolabelling in liver peroxisomes was lower than in kidney. No specific labelling was observed in cells other than hepatocytes.     

Purification and characterization of glutamate synthase from Azospirillum brasilense.

Growth conditions for Azospirillum brasilense Sp6 were devised for maximal expression of glutamate synthase. The enzyme levels were largely affected by the type and concentration of the nitrogen source. A 10-fold increase in the synthesis of the enzyme was observed at a limiting concentration of ammonia. The enzyme was purified to homogeneity by a procedure which was fairly rapid and allowed a good recovery of enzyme (30%). Azospirillum glutamate synthase is a complex iron-sulfur flavoprotein with a stoichiometry of 1 flavin adenine dinucleotide:1 flavin mononucleotide:8 Fe:8 S per protomer with a molecular weight of 185,000. The protomer is composed of two dissimilar subunits with molecular weights of 135,000 and 50,000. Kinetic parameters were determined. Km values for NADPH, 2-oxoglutarate, and L-glutamine were 6.25, 29, and 450 microM, respectively. The optimum pH was about 7.5. Complete reduction of the enzyme under anaerobic conditions was obtained either by NADPH (in the presence of a regenerating system) or dithionite or by photochemical reduction (in the presence of EDTA and 5-deazariboflavin). No stable long-wavelength intermediates were observed.     

Respiration and mitochondrial ATPase in energized mitochondria during isoproterenol-induced cell injury of myocardium.

1. Respiration of mitochondria, membrane potential and mitochondrial ATPase under energized conditions were studied in rat myocardium during cell injury induced by treatment with isoproterenol. 2. Increase in the state 4 rate of respiration and ADP:O ratio, as well as decrease in the state 3 rate and Respiratory Control Ratio (RCR) were found. 3. The optimum pH for RCR and for maximum ATPase activity was shifted to lower values. 4. The state 3 respiration was more sensitive to oligomycin inhibition. 5. The mitochondria showed lower ability to generate membrane potential. 6. An increase in the K0.5 values for catalytic sites II and III of mitochondrial ATPase at pH 7.4 and 5.5 was found. 7. These results are consistent with alterations on the integrity of mitochondrial membrane, and corroborate with the hypothesis of changes on the mitochondrial ATPase during isoproterenol-induced cell injury of myocardium.     

Accumulated SET protein up-regulates and interacts with hnRNPK,increasing its binding to nucleic acids,the Bcl-xS repression,and cellular proliferation

SET and hnRNPK are proteins involved in gene expression and regulation of cellular signaling. We previously demonstrated that SET accumulates in head and neck squamous cell carcinoma (HNSCC); hnRNPK is a prognostic marker in cancer. Here, we postulate that SET and hnRNPK proteins interact to promote tumorigenesis. We performed studies in HEK293 and HNSCC (HN6, HN12, and HN13) cell lines with SET/hnRNPK overexpression and knockdown, respectively. We found that SET and/or hnRNPK protein accumulation increased cellular proliferation. SET accumulation up-regulated hnRNPK mRNA and total/phosphorylated protein, promoted hnRNPK nuclear location, and reduced Bcl-x mRNA levels. SET protein directly interacted with hnRNPK, increasing both its binding to nucleic acids and Bcl-xS repression. We propose that hnRNPK should be a new target of SET and that SET–hnRNPK interaction, in turn, has potential implications in cell survival and malignant transformation.     

In vitro metabolism study of a new nitrosyl ruthenium complex [Ru(NH·NHq)(terpy)NO]3+ nitric oxide donor using rat microsomes

A new nitrosyl ruthenium complex [Ru(NH·NHq)(terpy)NO]³⁺ nitric oxide donor was recently developed and due to its excellent vasodilator activity, it has been considered as a potential drug candidate. Drug metabolism is one of the main parameters that should be evaluated in the early drug development, so the biotransformation of this complex by rat hepatic microsomes was investigated. In order to perform the biotransformation study, a simple, sensitive and selective HPLC method was developed and carefully validated. The parameters evaluated in the validation procedure were: linearity, recovery, precision, accuracy, selectivity and stability. Except for the stability study, all the parameters evaluated presented values below the recommended by FDA guidelines. The stability study showed a time-dependent degradation profile. After method validation, the biotransformation study was accomplished and the kinetic parameters were determined. The biotransformation study obeyed the Michaelis–Menten kinetics. The V_max and K_m were, respectively, 0.1625 ± 0.010 μmol/mg protein/min and 79.97 ± 11.52 μM. These results indicate that the nitrosyl complex is metabolized by CYP450.     

Impact of adenosine nucleotide translocase (ANT) proline isomerization on Ca<Superscript>2+</Superscript>-induced cysteine relative mobility/mitochondrial permeability transition pore

Mitochondrial membrane carriers containing proline and cysteine, such as adenine nucleotide translocase (ANT), are potential targets of cyclophilin D (CyP-D) and potential Ca²⁺-induced permeability transition pore (PTP) components or regulators; CyP-D, a mitochondrial peptidyl-prolyl cis-trans isomerase, is the probable target of the PTP inhibitor cyclosporine A (CsA). In the present study, the impact of proline isomerization (from trans to cis) on the mitochondrial membrane carriers containing proline and cysteine was addressed using ANT as model. For this purpose, two different approaches were used: (i) Molecular dynamic (MD) analysis of ANT-Cys⁵⁶ relative mobility and (ii) light scattering techniques employing rat liver isolated mitochondria to assess both Ca²⁺-induced ANT conformational change and mitochondrial swelling. ANT-Pro⁶¹ isomerization increased ANT-Cys⁵⁶ relative mobility and, moreover, desensitized ANT to the prevention of this effect by ADP. In addition, Ca²⁺ induced ANT “c” conformation and opened PTP; while the first effect was fully inhibited, the second was only attenuated by CsA or ADP. Atractyloside (ATR), in turn, stabilized Ca²⁺-induced ANT “c” conformation, rendering the ANT conformational change and PTP opening less sensitive to the inhibition by CsA or ADP. These results suggest that Ca²⁺ induces the ANT “c” conformation, apparently associated with PTP opening, but requires the CyP-D peptidyl-prolyl cis-trans isomerase activity for sustaining both effects.     

The anti-cancer agent nemorosone is a new potent protonophoric mitochondrial uncoupler

Nemorosone, a natural-occurring polycyclic polyprenylated acylphloroglucinol, has received increasing attention due to its strong in vitro anti-cancer action. Here, we have demonstrated the toxic effect of nemorosone (1-25 μM) on HepG2 cells by means of the MTT assay, as well as early mitochondrial membrane potential dissipation and ATP depletion in this cancer cell line. In mitochondria isolated from rat liver, nemorosone (50-500 nM) displayed a protonophoric uncoupling activity, showing potency comparable to the classic protonophore, carbonyl cyanide m-chlorophenyl hydrazone (CCCP). Nemorosone enhanced the succinate-supported state 4 respiration rate, dissipated mitochondrial membrane potential, released Ca(2+) from Ca(2+)-loaded mitochondria, decreased Ca(2+) uptake and depleted ATP. The protonophoric property of nemorosone was attested by the induction of mitochondrial swelling in hyposmotic K(+)-acetate medium in the presence of valinomycin. In addition, uncoupling concentrations of nemorosone in the presence of Ca(2+) plus ruthenium red induced the mitochondrial permeability transition process. Therefore, nemorosone is a new potent protonophoric mitochondrial uncoupler and this property is potentially involved in its toxicity on cancer cells.