Mario Cappiello

THIOL DEPENDENT OXIDATION OF ENZYMES : THE LAST CHANCE AGAINST OXIDATIVE STRESS

1. A survey of known effects of oxidized thiols on enzyme activity reveals a potential concerted action on metabolic pathways determining an impairment of anabolic reduction processes and an activation of the oxidative arm of the hexose monophosphate shunt. Thus it appears that, following oxidative stress, the increase of disulphides may act in restoring a reduced state in the cell by specifically channelling the metabolic energy flux.

Distribution of UDP-glucuronosyltransferase and its endogenous substrate uridine 5′-diphosphoglucuronic acid in human tissues

SummaryThe activity of UDP-glucuronosyltransferase (UDPGT) and the concentration of its endogenous substrate, 5′-diphosphoglucuronic acid (UDPGA), have been measured in human liver, kidney, lung and intestinal mucosa.The activity of UDPGT was tissue- and substrate-dependent. The liver/kidney and liver/intestine ratios for UDPGT varied over one order of magnitude with three substrates. The highest activity of UDGPT in extrahepatic tissues was in the kidney, with 1-naphthol as substrate; it was about half of the hepatic activity.The concentration (μmol · kg−1) of UDPGA was 279 (liver), 17.4 (kidney), 19.3 (intestinal mucosa) and 17.2 (lung), it was at least 15-fold higher in liver than the other tissues, and the concentration in kidney, lung and intestinal mucosa was similar.The kinetics of UDPGT in a liver homogenate at varying concentrations of UDPGA and fixed concentration of 1-naphthol, ethinyloestradiol, and morphine was also measured. The apparent kM for UDPGT depended upon the chemical nature of the UDPGA-acceptor substrate; average values of kM were 63, 300, and 700 μmol · 1−1 for 1-naphthol, ethinyloestradiol and morphine respectively. These values are, respectively, lower, similar to and higher than the hepatic concentration of UDPGA.Under certain circumstances UDPGA may be the limiting factor in the in vivo glucuronidation of drugs by extrahepatic tissues.

Specifically Targeted Modification of Human Aldose Reductase by Physiological Disulfides

Aldose reductase is inactivated by physiological disulfides such as GSSG and cystine. To study the mechanism of disulfide-induced enzyme inactivation, we examined the rate and extent of enzyme inactivation using wild-type human aldose reductase and mutants containing cysteine-to-serine substitutions at positions 80 (C80S), 298 (C298S), and 303 (C303S). The wild-type, C80S, and C303S enzymes lost >80% activity following incubation with GSSG, whereas the C298S mutant was not affected. Loss of activity correlated with enzyme thiolation. The binary enzyme-NADP+ complex was less susceptible to enzyme thiolation than the apoenzyme. These results suggest that thiolation of human aldose reductase occurs predominantly at Cys-298. Energy minimization of a hypothetical enzyme complex modified by glutathione at Cys-298 revealed that the glycyl carboxylate of glutathione may participate in a charged interaction with His-110 in a manner strikingly similar to that involving the carboxylate group of the potent aldose reductase inhibitor Zopolrestat. In contrast to what was observed with GSSG and cystine, cystamine inactivated the wild-type enzyme as well as all three cysteine mutants. This suggests that cystamine-induced inactivation of aldose reductase does not involve modification of cysteines exclusively at position 80, 298, or 303.

New role for leucyl aminopeptidase in glutathione turnover.

A manganese-dependent cysteinyl-glycine hydrolysing activity has been purified to electrophoretic homogeneity from bovine lens. The characterization of the purified enzyme (molecular mass of the native protein, molecular mass of the subunit and extensive primary structure analysis) allowed the unequivocal attribution of the cysteinyl-glycine hydrolysing activity, which is usually associated with alanyl aminopeptidase (EC 3.4.11.2) or membrane-bound dipeptidase (EC 3.4.13.19), to LAP (leucyl aminopeptidase; EC 3.4.11.1). Analysis of the pH dependence of Cys-Gly hydrolysis catalysed by LAP, supported by a molecular modelling approach to the enzyme-substrate conformation, gave insights into the ability of the enzyme to recognize Cys-Gly as a substrate. Due to the effectiveness of LAP in hydrolysing Cys-Gly (K(m)=0.57 mM, kcat=6.0x10(3) min(-1) at pH 7.4 and 25 degrees C) with respect to other dipeptide substrates, a new role for this enzyme in glutathione turnover is proposed.

Oxidative modification of aldose reductase induced by copper ion. Definition of the metal-protein interaction mechanism.

Aldose reductase (ALR2) is susceptible to oxidative inactivation by copper ion. The mechanism underlying the reversible modification of ALR2 was studied by mass spectrometry, circular dichroism, and molecular modeling approaches on the enzyme purified from bovine lens and on wild type and mutant recombinant forms of the human placental and rat lens ALR2. Two equivalents of copper ion were required to inactivate ALR2: one remained weakly bound to the oxidized protein whereas the other was strongly retained by the inactive enzyme. Cys303 appeared to be the essential residue for enzyme inactivation, because the human C303S mutant was the only enzyme form tested that was not inactivated by copper treatment. The final products of human and bovine ALR2 oxidation contained the intramolecular disulfide bond Cys298-Cys303. However, a Cys80-Cys303 disulfide could also be formed. Evidence for an intramolecular rearrangement of the Cys80-Cys303 disulfide to the more stable product Cys298-Cys303 is provided. Molecular modeling of the holoenzyme supports the observed copper sequestration as well as the generation of the Cys80-Cys303disulfide. However, no evidence of conditions favoring the formation of the Cys298-Cys303 disulfide was observed. Our proposal is that the generation of the Cys298-Cys303 disulfide, either directly or by rearrangement of the Cys80-Cys303 disulfide, may be induced by the release of the cofactor from ALR2 undergoing oxidation. The occurrence of a less interactive site for the cofactor would also provide the rationale for the lack of activity of the disulfide enzyme forms.

Distribution of 2-naphthol sulphotransferase and its endogenous substrate adenosine 3′-phosphate 5′-phosphosulphate in human tissues

SummaryThe activity of sulphotransferase towards 2-naphthol and the concentration of its endogenous substrate, adenosine 3′-phosphate 5′-phosphosulphate (PAPS), have been measured in five specimens of human liver, lung, and kidney, and the mucosa from the ileum and the ascending, descending and sigmoid colon.The activity of 2-naphthol sulphotransferase (mean nmol·min−1·mg−1 protein) was 1.82 (liver); 0.034 (kidney); 0.19 (lung); 0.64 (ileum); 0.47 (ascending colon); 0.50 (descending colon); 0.40 (sigmoid colon).The concentration of PAPS (mean nmol·g−1 wet tissue) was 22.6 (liver); 4.8 (kidney); 4.3 (lung); 12.8 (ileum); 8.1 (ascending colon); 7.5 (descending colon); 6.2 (sigmoid colon).The concentration of PAPS and the activity of 2-naphthol sulphotransferase were higher in the liver than in the extrahepatic tissues. There was significant difference between ileum and ascending colon, both the activity of sulphotransferase and the concentration of PAPS being higher in the former.2-Naphthol sulphotransferase activity and the concentration of PAPS have consistent distribution patterns. Differences between the tissues studied were more marked for sulphotransferase than for its endogenous substrate.

Ribose 1-phosphate and inosine activate uracil salvage in rat brain

The purpose of this study was to determine the mechanism by which inosine activates pyrimidine salvage in CNS. The levels of cerebral inosine, hypoxanthine, uridine, uracil, ribose 1-phosphate and inorganic phosphate were determined, to evaluate the Gibbs free energy changes (deltaG) of the reactions catalyzed by purine nucleoside phosphorylase and uridine phosphorylase, respectively. A deltaG value of 0.59 kcal/mol for the combined reaction inosine+uracil <==> uridine+hypoxanthine was obtained, suggesting that at least in anoxic brain the system may readily respond to metabolite fluctuations. If purine nucleoside phosphorolysis and uridine phosphorolysis are coupled to uridine phosphorylation, catalyzed by uridine kinase, whose activity is relatively high in brain, the three enzyme activities will constitute a pyrimidine salvage pathway in which ribose 1-phosphate plays a pivotal role. CTP, presumably the last product of the pathway, and, to a lesser extent, UTP, exert inhibition on rat brain uridine nucleotides salvage synthesis, most likely at the level of the kinase reaction. On the contrary ATP and GTP are specific phosphate donors.

A New Approach to Control the Enigmatic Activity of Aldose Reductase

Aldose reductase (AR) is an NADPH-dependent reductase, which acts on a variety of hydrophilic as well as hydrophobic aldehydes. It is currently defined as the first enzyme in the so-called polyol pathway, in which glucose is transformed into sorbitol by AR and then to fructose by an NAD+-dependent dehydrogenase. An exaggerated flux of glucose through the polyol pathway (as can occur in diabetes) with the subsequent accumulation of sorbitol, was originally proposed as the basic event in the aethiology of secondary diabetic complications. For decades this has meant targeting the enzyme for a specific and strong inhibition. However, the ability of AR to reduce toxic alkenals and alkanals, which are products of oxidative stress, poses the question of whether AR might be better classified as a detoxifying enzyme, thus raising doubts as to the unequivocal advantages of inhibiting the enzyme. This paper provides evidence of the possibility for an effective intervention on AR activity through an intra-site differential inhibition. Examples of a new generation of aldose reductase “differential” inhibitors (ARDIs) are presented, which can preferentially inhibit the reduction of either hydrophilic or hydrophobic substrates. Some selected inhibitors are shown to preferentially inhibit enzyme activity on glucose or glyceraldehyde and 3-glutathionyl-4-hydroxy-nonanal, but are less effective in reducing 4-hydroxy-2-nonenal. We question the efficacy of D, L-glyceraldehyde, the substrate commonly used in in vitro inhibition AR studies, as an in vitro reference AR substrate when the aim of the investigation is to impair glucose reduction.

In vitro assessment of salvage pathways for pyrimidine bases in rat liver and brain

In this paper we extend our previous observation on the mobilization of the ribose moiety from guanosine to xanthine catalyzed by rat liver extracts (Giorgelli et al., Biochim. Biophys. Acta 1335 (1997) 16-22). The data show that in rat liver and brain extracts the activated ribose, stemming from inosine and guanosine phosphorolysis as ribose 1-phosphate, can be used to salvage uracil to uracil nucleotides. Uridine is an intermediate. The salvage process occurs even in the presence of excess inorganic phosphate suggesting that uridine phosphorylase may function in vivo as an anabolic enzyme. Ribose 5-phosphate cannot substitute for inosine, guanosine or ribose 1-phosphate as ribose donor. When inorganic phosphate was substituted with arsenate, hindering the formation of ribose 1-phosphate, no ribose transfer could be observed. A similar pathway occurs at the deoxy level. The deoxyribose moiety of deoxyinosine can be used to salvage thymine to thymine nucleotides, again in the presence of excess inorganic phosphate. Our results introduce a novel aspect of the salvage pathway, in which ribose 1-phosphate seems to play a pivotal role.

In vitro recycling of α-D-ribose 1-phosphate for the salvage of purine bases

Abstract In this paper, we extend our previous observation on the mobilization of the ribose moiety from a purine nucleoside to a pyrimidine base, with subsequent pyrimidine nucleotides formation (Cappiello et al., Biochim. Biophys. Acta 1425 (1998) 273–281). The data show that, at least in vitro, also the reverse process is possible. In rat brain extracts, the activated ribose, stemming from uridine as ribose 1-phosphate, can be used to salvage adenine and hypoxanthine to their respective nucleotides. Since the salvage of purine bases is a 5-phosphoribosyl 1-pyrophosphate-dependent process, catalyzed by adenine phosphoribosyltransferase and hypoxanthine guanine phosphoribosyltransferase, our results imply that Rib-1P must be transformed into 5-phosphoribosyl 1-pyrophosphate, via the successive action of phosphopentomutase and 5-phosphoribosyl 1-pyrophosphate synthetase; and ,in fact, no adenosine could be found as an intermediate when rat brain extracts were incubated with adenine, Rib-1P and ATP, showing that adenine salvage does not imply adenine ribosylation, followed by adenosine phosphorylation. Taken together with our previous results on the Rib-1P-dependent salvage of pyrimidine nucleotides, our results give a clear picture of the in vitro Rib-1P recycling, for both purine and pyrimidine salvage.