João Meireles Ribeiro

Isoelectric points of proteins: theoretical determination

Three methods for calculating the isoelectric points (pI) of proteins, provided that their amino acid compositions are known, are described. The comprehensive and abridged procedures involve solutions of polynomial equations of different degrees depending on whether pK values of the specific acid-base residues or the means of some of those values, respectively, are adopted. In the simplified procedure, approximate pI values of proteins can be determined easily with the help of calculated values, included in this paper, related to the amino acid composition of proteins.

Specific ADP-ribose pyrophosphatase from Artemia cysts and rat liver: effects of nitroprusside, fluoride and ionic strength

One specific ADP-ribose pyrophosphatase (ADPRibase) has been identified in Artemia cysts, following a protocol that in rat liver allows the identification of three ADPRibases. Artemia ADPRibase resulted similar, but not identical, to rat liver ADPRibase-I with respect to known and novel properties disclosed in this work. In the presence of Mg2+, Artemia ADPRibase was highly specific for ADP-ribose and showed a low, 0.7 microM Km. Preincubation with the nitric oxide donor nitroprusside and dithiothreitol, elicited dose- and time-dependent, severalfold increase of Km and decrease of Vmax. At saturating ADP-ribose concentrations, fluoride was a strong inhibitor (IC50 approximately equal to 10-20 microM), whereas bringing ionic strength to 0.3-1.3 mol/l doubled the activity measured at lower or higher strengths. The novel fluoride and ionic strength effects were studied also with rat liver ADPRibase-I. Differences between the Artemia enzyme and ADPRibase-I concerned molecular weight (31,000 versus 38,500, respectively), Mn2+ ability to substitute for Mg2+ as the activating cation (better for the rat enzyme), and Vmax decrease by nitroprusside (not seen with the rat enzyme). The results are discussed in relation with the role of specific ADPRibases as protective factors limiting free ADP-ribose accumulation and protein glycation, and as targets for cytotoxic agents.

An algorithm for the computer calculation of the coefficients of a polynomial that allows determination of isoelectric points of proteins and other macromolecules

The isoelectric point (pI) of a macromolecule containing any number of acid-base residues can be expressed as a polynomial whose coefficients are related to both the number of acid-base residues present in the molecule and their K values. Polynomials of degree higher than 5 are too complicated to be solved manually. An algorithm is here presented which allows, with few sentences written in BASIC, the calculation of the polynomial coefficients. With this knowledge and using standard polynomial solving programs, the isoelectric point of the macromolecule can be calculated.

Glycine-enhanced inhibition of rat liver nucleotide pyrophosphatase/phosphodiesterase-I by EDTA: a full account of the reported inhibition by commercial preparations of acidic fibroblast growth factor (FGF-1).

The earlier reported inhibition of rat liver nucleotide pyrophosphatase/phosphodiesterase I (EC 3.1.6.9/EC 3.1.4.1; NPP/PDE) by culture‐grade acidic fibroblast growth factor (FGF‐1) correlates with a low‐M r contaminant. 1H‐NMR analyses revealed EDTA in the total‐volume fractions of a gel‐filtration experiment, where all the inhibitory activity of the FGF‐1 preparation was recovered. NPP/PDE inhibition by EDTA (and by unfractionated FGF‐1 or the EDTA‐containing fractions) was time‐dependent, blocked by the substrate p‐nitrophenyl‐dTMP, and strongly enhanced by glycine. The use of glycine buffers in earlier work was critical to the apparent inhibition by FGF‐1. The results point to a conformational change favored by glycine that may be relevant to the biological role of NPP/PDE.

Hydrolysis of the phosphoanhydride linkage of cyclic ADP-ribose by the Mn2+-dependent ADP-ribose/CDP-alcohol pyrophosphatase

Cyclic ADP‐ribose (cADPR) metabolism in mammals is catalyzed by NAD glycohydrolases (NADases) that, besides forming ADP‐ribose, form and hydrolyze the N 1‐glycosidic linkage of cADPR. Thus far, no cADPR phosphohydrolase was known. We tested rat ADP‐ribose/CDP‐alcohol pyrophosphatase (ADPRibase‐Mn) and found that cADPR is an ADPRibase‐Mn ligand and substrate. ADPRibase‐Mn activity on cADPR was 65‐fold less efficient than on ADP‐ribose, the best substrate. This is similar to the ADP‐ribose/cADPR formation ratio by NADases. The product of cADPR phosphohydrolysis by ADPRibase‐Mn was N 1‐(5‐phosphoribosyl)‐AMP, suggesting a novel route for cADPR turnover.

ADP‐ribose pyrophosphatase‐I partially purified from livers of rats overdosed with acetaminophen reveals enzyme inhibition in vivo reverted in vitro by dithiothreitol

Free ADP‐ribose reacts nonenzymatically with proteins and can lead to intracellular damage. The low‐Km ADP‐ribose pyrophosphatase‐I (ADPRibase‐I) is well suited to control free ADP‐ribose and nonenzymatic ADP‐ribosylation. In vitro, the acetaminophen metabolite N‐acetyl‐p‐benzoquinoneimine (NAPQI) decreases ADPRibase‐I Vmax and increases Km, effects not reverted by dithiothreitol (DTT) and attributed to enzyme arylation. The present study was conducted to test whether acetaminophen overdose affected ADPRibase‐I in vivo. Rats pretreated with 3‐methylcholanthrene and L‐buthionine‐[S,R]‐sulfoximine to potentiate acetaminophen toxicity received an intraperitoneal dose of either acetaminophen (800 mg/kg; n = 5) or vehicle (n = 3). ADPRibase‐I partially purified from acetaminophen‐overdosed rats showed a decreased Vmax (0.32 ± 0.09 versus 0.60 ± 0.03 mU/mg of liver protein; p < 0.01) not reverted by DTT and an increased Km for ADP‐ribose (1.39 ± 0.31 versus 0.67 ± 0.05 μM; p < 0.01) that, contrary to the in vitro NAPQI effect, was reverted by DTT. Incubation of partially purified ADPRibase‐I from normal rat liver with oxidized glutathione elicited a time‐ and dose‐dependent, DTT‐reverted increase of Km, without change of Vmax. The results indicate that the activity of ADPRibase‐I can be regulated by thiol exchange and that the increase of Km elicited by acetaminophen overdosage was related to the oxidative stress caused by the drug. It remains to be seen whether an increase of free ADP‐ribose concomitant to ADPRibase‐I inhibition could contribute to the hepatotoxicity of acetaminophen.

Isoelectric point, electric charge, and nomenclature of the acid–base residues of proteins

The main object of this work is to present the pedagogical usefulness of the theoretical methods, developed in this laboratory, for the determination of the isoelectric point (pI) and the net electric charge of proteins together with some comments on the naming of the acid‐base residues of proteins

Rat liver ADP-ribose pyrophosphatase-I as an in vitro target of the acetaminophen metabolite N-acetyl-p-benzoquinoneimine

N-acetyl-p-benzoquinoneimine (NAPQI) is the metabolite responsible for acetaminophen hepatotoxicity. ADP-ribose pyrophosphatase-I (ADPRibase-I; EC 3.6.1.13) hydrolyzes protein-glycating ADP-ribose. The results show NAPQI-dependent alterations of ADPRibase-I leading to strong inhibition: a fast Km increase produced by low concentrations, and a time-dependent Vmax decrease by higher NAPQI concentrations. Both effects were prevented by thiols, but not reverted by them, nor by gel filtration of NAPQI-treated enzyme. Liver ADPRibase-I can be a target of NAPQI-dependent arylation. The inhibition or inactivation of the enzyme would contribute to increasing the free ADP-ribose concentration and nonenzymatic ADP-ribosylation, which is coherent with results linking free ADP-ribose-producing pathways to acetaminophen toxicity.

The Characterization of Escherichia coli CpdB as a Recombinant Protein Reveals that, besides Having the Expected 3´-Nucleotidase and 2´,3´-Cyclic Mononucleotide Phosphodiesterase Activities, It Is Also Active as Cyclic Dinucleotide Phosphodiesterase.

Endogenous cyclic diadenylate phosphodiesterase activity was accidentally detected in lysates of Escherichia coli BL21. Since this kind of activity is uncommon in Gram-negative bacteria, its identification was undertaken. After partial purification and analysis by denaturing gel electrophoresis, renatured activity correlated with a protein identified by fingerprinting as CpdB (cpdB gene product), which is annotated as 3´-nucleotidase / 2´,3´-cyclic-mononucleotide phosphodiesterase, and it is synthesized as a precursor protein with a signal sequence removable upon export to the periplasm. It has never been studied as a recombinant protein. The coding sequence of mature CpdB was cloned and expressed as a GST fusion protein. The study of the purified recombinant protein, separated from GST, confirmed CpdB annotation. The assay of catalytic efficiencies (kcat/Km) for a large substrate set revealed novel CpdB features, including very high efficiencies for 3´-AMP and 2´,3´-cyclic mononucleotides, and previously unknown activities on cyclic and linear dinucleotides. The catalytic efficiencies of the latter activities, though low in relative terms when compared to the major ones, are far from negligible. Actually, they are perfectly comparable to those of the ‘average’ enzyme and the known, bona fide cyclic dinucleotide phosphodiesterases. On the other hand, CpdB differs from these enzymes in its extracytoplasmic location and in the absence of EAL, HD and DHH domains. Instead, it contains the domains of the 5´-nucleotidase family pertaining to the metallophosphoesterase superfamily, although CpdB lacks 5´-nucleotidase activity. The possibility that the extracytoplasmic activity of CpdB on cyclic dinucleotides could have physiological meaning is discussed.

Bifunctional homodimeric triokinase/FMN cyclase: contribution of protein domains to the activities of the human enzyme and molecular dynamics simulation of domain movements.

Background: Triokinase, which phosphorylates dihydroxyacetone and fructose-derived glyceraldehyde, remains molecularly unidentified. Results: Human DAK gene encodes homodimeric triokinase/FMN cyclase formed by two-domain subunits. Although kinase activity requires intact homodimers, cyclase requires only a truncated, single domain subunit. Conclusion: Triokinase/FMN cyclase identity and bifunctionality are established. Significance: This study molecularly dissects a bifunctional enzyme of unusual specificity and finishes the molecular identification of fructose pathway enzymes. Mammalian triokinase, which phosphorylates exogenous dihydroxyacetone and fructose-derived glyceraldehyde, is neither molecularly identified nor firmly associated to an encoding gene. Human FMN cyclase, which splits FAD and other ribonucleoside diphosphate-X compounds to ribonucleoside monophosphate and cyclic X-phosphodiester, is identical to a DAK-encoded dihydroxyacetone kinase. This bifunctional protein was identified as triokinase. It was modeled as a homodimer of two-domain (K and L) subunits. Active centers lie between K1 and L2 or K2 and L1: dihydroxyacetone binds K and ATP binds L in different subunits too distant (≈14 Å) for phosphoryl transfer. FAD docked to the ATP site with ribityl 4′-OH in a possible near-attack conformation for cyclase activity. Reciprocal inhibition between kinase and cyclase reactants confirmed substrate site locations. The differential roles of protein domains were supported by their individual expression: K was inactive, and L displayed cyclase but not kinase activity. The importance of domain mobility for the kinase activity of dimeric triokinase was highlighted by molecular dynamics simulations: ATP approached dihydroxyacetone at distances below 5 Å in near-attack conformation. Based upon structure, docking, and molecular dynamics simulations, relevant residues were mutated to alanine, and kcat and Km were assayed whenever kinase and/or cyclase activity was conserved. The results supported the roles of Thr112 (hydrogen bonding of ATP adenine to K in the closed active center), His221 (covalent anchoring of dihydroxyacetone to K), Asp401 and Asp403 (metal coordination to L), and Asp556 (hydrogen bonding of ATP or FAD ribose to L domain). Interestingly, the His221 point mutant acted specifically as a cyclase without kinase activity.