Biomedical subjects

G E Martorana

Publications and source records attributed to G E Martorana.

At least 19 recordsLinked to original sources

PubMed2006

Alterations of energy metabolism and glutathione levels of HL-60 cells induced by methacrylates present in composite resins.

OBJECTIVES: Methacrylic compounds such as 2-hydroxyethyl methacrylate (HEMA), triethylene glycol dimethacrylate (TEGDMA) and bisphenol A glycerolate (1 glycerol/phenol) dimethacrylate (Bis-GMA) are largely present in auto- or photopolymerizable composite resins. Since the polymerization reaction is never complete, these molecules are released into the oral cavity tissues and biological fluids where they could cause local adverse effects. The aim of this work was to verify the hypothesis that the biological effects of HEMA, TEGDMA and Bis-GMA - at a non-cytotoxic concentration - depend on the interaction with mitochondria and exert consequent alterations of energy metabolism, GSH levels and the related pathways in human promyelocytic cell line (HL-60). METHODS: The biological effects of methacrylic monomers were determined by analyzing the following parameters: GSH concentration, glucose-6-phosphate dehydrogenase (G6PDH) and glutathione reductase (GR) activity, oxygen and glucose consumption and lactate production along with cell differentiation and proliferation. RESULTS: All monomers induced both cellular differentiation and decrease in oxygen consumption. Cells treated with TEGDMA and Bis-GMA showed a significant enhancement of glucose consumption and lactate production. TEGDMA and HEMA induced GSH depletion stimulating G6PDH and GR activity. CONCLUSIONS: All the monomers under study affect the metabolism of HL-60 cells and show differentiating activity. Since alterations in cellular metabolism occurred at compound concentrations well below cytotoxic levels, the changes in energy metabolism and glutathione redox balance could be considered as potential mechanisms for inducing clinical and sub-clinical adverse effects and thus providing useful parameters when testing biocompatibility of dental materials.

Bisphenol A-Glycidyl Methacrylate↗

PubMed2004

Methionine 35 oxidation reduces toxic and pro-apoptotic effects of the amyloid beta-protein fragment (31-35) on isolated brain mitochondria.

Amyloid beta-peptide (AbetaP), the central constituent of senile plaques in Alzheimer's disease (AD) brain, has been shown to be a source of free radical oxidative stress that may lead to neurodegeneration. In particular, it is well known that oxidation of methionine 35, is strongly related to the pathogenesis of AD, since it represents the residue in AbetaP most susceptible to oxidation in vivo. In the present study, we used the fragment 31-35 of AbetaP, which has a single methionine at residue 35, in order to investigate the influence of the oxidation state of methionine-35 on the toxic and pro-apoptotic effects induced by Abeta(31-35) on isolated brain mitochondria. The obtained results show that exposure of isolated mitochondria from rat brain to AbetaP(31-35) determines (i) a large release of cytochrome c (ii) a significant reduction in mitochondrial respiration and (iii) a slight drop in the mitochondrial membrane potential (deltapsi). In contrast, the amplitude of these events resulted attenuated or completely abrogated in isolated brain mitochondria exposed to the AbetaP(31-35)Met35OX, in which methionine 35 was oxidized to methionine sulfoxide. We have further characterized the action of AbetaP(31-35) and Abeta(31-35)Met35OX peptide on PC12 cells. Although these two peptides, compromised mitochondrial function at a different extent as assessed by MTT reduction, neither one of them decreased cell viability as measured by Trypan Blue exclusion assay. The results obtained in this study support the hypothesis that the oxidative state of Met-35 may play a critical role in the mechanisms responsible of neurotoxicity exerted by this peptide.

Amyloid beta-Peptides↗

PubMed2003

Bezafibrate induces a mitochondrial derangement in human cell lines: a PPAR-independent mechanism for a peroxisome proliferator.

Bezafibrate is a hypolipidemic drug that belongs to the group of peroxisome proliferators because it binds to peroxisome proliferator-activated receptors type alpha (PPARs). Peroxisome proliferators produce a myriad of extraperoxisomal effects, which are not necessarily dependent on their interaction with PPARs. An investigation on the peculiar activities of bezafibrate could clarify some of the molecular events and the relationship with the biochemical and pharmacological properties of this class of compounds. In this view, the human acute promyelocytic leukemia HL-60 cell line and human rabdomiosarcoma TE-671 cell line were cultured in media containing bezafibrate and a number of observations such as spectrophotometric analysis of mitochondrial respiratory chain enzymes, NMR metabolite determinations, phosphofructokinase enzymatic analysis, and differentiation assays were carried on. Bezafibrate induced a derangement of NADH cytochrome c reductase activity accompanied by metabolic alterations, mainly a shift to anaerobic glycolysis and an increase of fatty acid oxidation, as shown by NMR analysis of culture supernatants where acetate, lactate, and alanine levels increased. On the whole, the present results suggest a biochemical profile and a therapeutic role of this class of PPARs ligands more complex than those previously proposed.

Acetates↗

PubMed2001

Is homocysteine a pro-oxidant?

High plasma homocysteine concentrations have been found to be associated with atherosclerosis and thrombosis of arteries and deep veins. The oxidative damage mediated by hydrogen peroxide production during the metal-catalyzed oxidation of homocysteine is to date considered to be one of the major pathophysiological mechanisms for this association. In this work, a very sensitive and accurate method was employed to measure the effective production of H2O2 during homocysteine oxidation. Furthermore, the interaction of homocysteine with powerful oxidizing species (hypochlorite, peroxynitrite, ferrylmyoglobin) was evaluated in order to ascertain the putative pro-oxidant role of homocysteine. Our findings indicate that homocysteine does not produce H2O2 in a significant amount (1/4000 mole/mole ratio of H2O2 to homocysteine). Moreover, homocysteine strongly inhibits the oxidation of luminol and dihydrorhodamine by hypochlorite or peroxynitrite and rapidly reduces back ferrylmyoglobin, the oxidizing species, to metmyoglobin. All these results should, in our opinion, lead to a rethinking of the commonly held view that homocysteine oxidation is one of the main causative mechanisms of cardiovascular damage.

Animals↗

PubMed2001

Human heart cytosolic reductases and anthracycline cardiotoxicity.

Anthracyclines are a class of antitumor drugs widely used for the treatment of a variety of malignancy, including leukemias, lymphomas, sarcomas, and carcinomas. Different mechanisms have been proposed for anthracycline antitumor effects including free-radical generation, DNA intercalation/binding, activation of signaling pathways, inhibition of topoisomerase II and apoptosis. A life-threatening form of cardiomyopathy hampers the clinical use of anthracyclines. According to the prevailing hypothesis, anthracyclines injure the heart by generating damaging free radicals through iron-catalyzed redox cycling. Although the "iron and free-radical hypothesis" can explain some aspects of anthracycline acute toxicity, it is nonetheless disappointing when referred to chronic cardiomyopathy. An alternative hypothesis implicates C-13 alcohol metabolites of anthracyclines as mediators of myocardial contractile dysfunction ("metabolite hypothesis"). Hydroxy metabolites are formed upon two-electron reduction of the C-13 carbonyl group in the side chain of anthracyclines by cytosolic NADPH-dependent reductases. Anthracycline alcohol metabolites can affect myocardial energy metabolism, ionic gradients, and Ca2+ movements, ultimately impairing cardiac contraction and relaxation. In addition, alcohol metabolites can impair cardiac intracellular iron handling and homeostasis, by delocalizing iron from the [4Fe-4S] cluster of cytoplasmic aconitase. Chronic cardiotoxicity induced by C-13 alcohol metabolite might be primed by oxidative stress generated by anthracycline redox cycling ("unifying hypothesis"). Putative cardioprotective strategies should be aimed at decreasing C-13 alcohol metabolite production by means of efficient inhibitors of anthracycline reductases, as short-chain coenzyme Q analogs and chalcones that compete with anthracyclines for the enzyme active site, or by developing novel anthracyclines less susceptible to reductive metabolism.

Anthracyclines↗

PubMed1998

Antioxidant properties of 2,3-dimethoxy-5-methyl-6-(10-hydroxydecyl)-1,4-benzoquinone (idebenone).

Idebenone [2,3-dimethoxy-5-methyl-6-(10-hydroxydecyl)-1,4-benzoquinone] is a synthetic analogue of coenzyme Q that is currently employed in the treatment of vascular and degenerative diseases of the central nervous system. There is some evidence to suggest that idebenone might function as an antioxidant; however, it has not been demonstrated whether this function pertains to the quinone or hydroquinone form of idebenone. Here we demonstrate that idebenone can scavenge a variety of free radical species, including organic radicals such as 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) and diphenylpicrylhydrazyl, peroxyl and tyrosyl radicals, and peroxynitrite. Idebenone can also redox couple with hypervalent species of Mb or Hb, thus preventing lipid peroxidation promoted by these species. Likewise, idebenone inhibits microsomal lipid peroxidation induced by ADP-iron complexes or organic hydroperoxides. In so doing, idebenone prevents the destruction of cytochrome P450, which otherwise would accompany lipid peroxidation. Irrespective of the experimental system under investigation, idebenone functions by virtue of the electron-donating properties of the hydroquinone form. Redox coupling of this hydroquinone with free radicals generates the quinone compound, which per se lacks antioxidant activity. In many experiments, the antioxidant effects of idebenone become appreciable at approximately 2 microM, which is well in the range of plasma levels attainable in patients given oral doses of this drug. Moreover, comparative experiments have shown that the antioxidant efficiency of idebenone varies from no less than 50% to slightly more than 100% of that of vitamin E or Trolox. We would therefore propose that the neuroprotective effects of idebenone can be attributed, at least in part, to its ability to function as an antioxidant, involving redox cycling between hydroquinone and quinone.

Antioxidants↗

PubMed1997

Defective plasma antioxidant defenses and enhanced susceptibility to lipid peroxidation in uncomplicated IDDM.

Oxidative stress is postulated to be increased in patients with IDDM. Accumulating evidence suggests that oxidative cell injury caused by free radicals contributes to the development of IDDM complications. On the other side, a decreased efficiency of antioxidant defenses (both enzymatic and nonenzymatic) seems to correlate with the severity of pathological tissue changes in IDDM. Thus, we determined plasma antioxidant defenses, measuring the total radical-trapping antioxidant capacity (TRAP) and the two markers of oxidative stress, lipid hydroperoxides (ROOHs) and conjugated dienes, in 72 patients with well-controlled IDDM and without evident complications, compared with 45 nondiabetic subjects. Compared with control subjects, IDDM patients showed significantly reduced plasma TRAP (669 +/- 131 vs. 955 +/- 104 micromol/l, P < 0.001) and significantly increased levels of ROOHs (7.13 +/- 2.11 vs. 2.10 +/- 0.71 micromol/l, P < 0.001) and conjugated dienes (0.0368 +/- 0.0027 vs. 0.0328 +/- 0.0023 arbitrary units [AU], P < 0.01), especially in the trans-trans conformation (0.0340 +/- 0.0028 vs. 0.0259 +/- 0.0022 AU, P < 0.001), with a concurrent reduction of conjugated dienes in the cis-trans conformation (0.0028 +/- 0.0011 vs. 0.0069 +/- 0.0012 AU, P < 0.001). The oxidative parameters studied did not appear to be correlated with metabolic control (HbA1c levels) and lipid profile (cholesterol or triglyceride levels). The reduced TRAP and the increased ROOH and conjugated diene plasma levels, together with the decreased ratio of cis-trans/trans-trans conjugated dienes, which reflects an altered redox status of plasma, indicate that in IDDM patients, oxidative stress is enhanced and antioxidant defenses are defective, regardless of diabetes duration, metabolic control, or presence of complications.

Adult↗

PubMed1996

NA+/K+ ATPase impairment and experimental glycation: the role of glucose autoxidation.

Non enzymatic glycation could be involved in the early impairment of Na+/K+ ATPase that occurs in sciatic nerve of diabetic rats. In fact, decrease of Na+/K+ ATPase activity is one of the first alterations showed in experimental diabetic neuropathy. In this respect, it is known that in the presence of transition metals under physiological conditions, glucose can autoxidize yielding hydrogen peroxide (H2O2) and free radical intermediates, which, in turn, inhibit the cation pump. Our experiments were designed to determine if glucose autoxidation has any relevance in the early steps of Na+/K+ ATPase experimental glycation. Compared experiments with and without the sodium borohydride (NaBH4) reduction step demonstrated that incubation of brain Na+/K+ ATPase with glucose 6-phosphate (G 6-P) and trace metals induced a significant decrease in enzyme activity dramatically enhanced by addition of copper (Cu2+). A concomitant production of H2O2 was noticed. The presence of diethylenetriaminepentaacetic acid (DTPA), a strong metal chelator, completely prevented Na+/K+ ATPase impairment and hydrogen-peroxide formation. No gross structural and conformational alterations of the enzyme can be demonstrated by intrinsic and extrinsic fluorescence measurements. Our results suggest that during the exposure of brain NA+/K+ ATPase to glucose 6-phosphate in vitro (experimental glycation), the decrease in activity can be correlated, at lease in the early phases, to metal-catalyzed production of oxidative species, such as H2O2, through the glucose autoxidation process, and not to glucose attachment to the enzyme. Since plasma hydroperoxides and copper appear to be elevated in diabetic patients with complications, our data suggest a critical role for oxidative reactions in the pathophysiology of the chronic complications of diabetes like neuropathy.

Animals↗

PubMed1995

Self-similarity properties of alpha-crystallin supramolecular aggregates.

The supramolecular aggregation of alpha-crystallin, the major protein of the eye lens, was investigated by means of static and dynamic light scattering. The aggregation was induced by generating heat-modified alpha-crystallin forms and by stabilizing the clusters with calcium ions. The kinetic pattern of the aggregation and the structural features of the clusters can be described according to the reaction limited cluster-cluster aggregation theory previously adopted for the study of colloidal particles aggregation systems. Accordingly, the average mass and the hydrodynamic radius of alpha-crystallin supramolecular aggregates grow exponentially in time. The structure factor of the clusters is typical of fractal aggregates. A fractal dimension df approximately 2.15 was determined, indicating a low probability of sticking together of the primitive aggregating particles. As a consequence, the slow-forming clusters assemble a rather compact structure. The basic units forming the fractal aggregates were found to have a radius about twice (approximately 17 nm) that of the native protein and 5.3 times its size, which is consistent with an intermediate molecular assembly corresponding to the already known high molecular weight forms of alpha-crystallin.

Animals↗

PubMed1994

The interaction of short chain coenzyme Q analogs with different redox states of myoglobin.

Two-equivalent oxidation of metmyoglobin (MbIII) by hydrogen peroxide (H2O2) yields an oxoferryl moiety (MbIV) plus a protein radical which presumably originates from the conversion of tyrosines to tyrosyl radicals (-MbIV). In the absence of electron donors, MbIII oxidation is followed by (i) heme degradation or (ii) tyrosyl radical-dependent reactions, such as irreversible dimerization or covalent binding of the heme group to the apoprotein. Moreover, the oxidizing equivalents of H2O2-activated MbIII promote the peroxidative decomposition of polyunsaturated fatty acids. In this study, water-soluble short chain coenzyme Q analogs (CoQ1H2 and CoQ2H2) were found to reduce the oxoferryl moiety, preventing heme degradation and regenerating MbIII and, more slowly, MbIIO2. CoQ1H2 and CoQ2H2 were also found to reduce tyrosyl radicals generated by UV irradiation of tyrosine solutions. Accordingly, CoQ1H2 and CoQ2H2 effectively prevented tyrosyl radical-dependent reactions such as the dimerization of sperm whale myoglobin and heme-apoprotein covalent binding in horse heart myoglobin. By competing for the oxidizing equivalents of hypervalent myoglobin, CoQ1H2 and CoQ2H2 also prevented the peroxidation of arachidonic acid. Collectively, these studies suggest that the proposed function of coenzyme Q as a naturally occurring antioxidant might well relate to its ability of reducing H2O2-activated myoglobin. Coenzyme Q should therefore mitigate cardiac or muscular dysfunctions that are caused by an abnormal generation of H2O2.

Acetylation↗

PubMed1994

Free radical production by activated haem proteins: protective effect of coenzyme Q.

The interaction of hydrogen peroxide with haem proteins leads readily to the formation of myoglobin and/or haemoglobin higher oxidation states (MbIV and/or HbIV), which are capable of promoting the oxidation of cellular costituents and are probably to blame for myocardic tissue damage in ischaemia/reperfusion. This study supports the evidence that the reduced form of Coenzyme Q, like other reducing agents, has an antioxidant activity exerted through the progressive reduction of ferryl forms (MbIV and/or HbIV) back to met and oxy forms (Mb and/or HbIIO2). Furthermore, the strong inactivation afforded by ferryl states of myoglobin on several enzymes, especially creatine kinase (CK), can be prevented by the addition of ubiquinol which protects the enzyme from the oxidative modifications. The ability of ubiquinol to recycle ferryl states of haem proteins provides a novel antioxidant mechanism for Coenzyme Q, besides its direct or indirect antiperoxidative activity, and may represent an important defense mechanism against oxidative tissue injury.

Animals↗

PubMed1993

Antioxidant effect of coenzyme Q on hydrogen peroxide-activated myoglobin.

In recent years increased attention has been focused on the reduced forms of coenzyme Q as antioxidant compounds inhibiting lipid peroxidation in model systems and in biological membranes, but in spite of extensive experimental evidences the molecular mechanisms responsible for the antioxidant activity of ubiquinones are still debated. Ferrylmyoglobin and/or its free radical form are regarded as powerful oxidizing agents capable of promoting oxidation of essential cellular constituents, particularly cell membranes. Therefore, we investigated the effects of ubiquinol on the formation and survival of ferryl species of myoglobin and on metmyoglobin itself. The addition of a threefold molar excess of hydrogen peroxide to a solution of metmyoglobin induces the rapid formation of a compound with the spectral characteristics of ferrylmyoglobin. The reaction is complete within 4 min, producing up to 76% of ferrylmyoglobin, which remains stable for at least 30 min. The addition of ubiquinol-1 to the same solution provokes a rapid and progressive reduction of ferrylmyoglobin to metmyoglobin and oxymyoglobin. Ubiquinol-1, furthermore, is also capable of protecting metmyoglobin against oxidation when added in the solution before hydrogen peroxide. Ubiquinol-1, indeed, is effective at both limiting the maximal ferrylmyoglobin level attained (59% inhibition) and accomplishing complete removal of the ferryl form (in about 15 min). The results demonstrate that ubiquinol is capable of reducing both ferrylmyoglobin and metmyoglobin to oxymyoglobin, providing a novel antioxidant mechanism for coenzyme Q.

Antioxidants↗

PubMed1992

Conformational stability of bovine alpha-crystallin. Evidence for a destabilizing effect of ascorbate.

Short-term incubation of bovine alpha-crystallin with ascorbate alters the protein conformational stability. The denaturation curves with urea and guanidinium-chloride show different patterns, suggesting a deviation from a two-state mechanism owing to the presence of one or more intermediates in the unfolding of ascorbate-modified alpha-crystallin. Furthermore, the latter protein profiles are shifted to lower denaturant concentrations indicating a destabilizing action of ascorbate, which is capable of facilitating protein dissociation into subunits as demonstrated by gel filtration with 1.5 M-urea. The decrease in conformational stability cannot be ascribed to any major structural alteration, but rather to localized changes in the protein molecule. In fact, no difference between native and ascorbate-treated alpha-crystallin can be detected by amino acid analysis but perturbation of the tryptophan and tyrosine environment is indicated by alterations in intrinsic fluorescence. Furthermore, turbidity and light-scattering measurements suggest an involvement of the lysine side chains, since aggregability patterns with acetylsalicylic acid are significantly altered. The ascorbate-destabilizing effect on the conformational stability of alpha-crystallin, probably exerted through oxidative modification of amino acid residues and/or the formation of covalent adducts, provokes unfavourable steric interactions between residues along the polypeptide chains, thus favouring aggregation and insolubilization of crystallins which can lead to cataract formation, as also demonstrated by proteolytic digestion patterns which show a lower rate of degradation of the ascorbate-modified alpha-crystallin.

Acrylamide↗

PubMed1992

Enzyme inactivation by metal-catalyzed oxidation of coenzyme Q1.

Ubiquinol-1 in aerated aqueous solution inactivates several enzymes--alanine aminotransferase, alkaline phosphatase, Na+/K(+)-ATPase, creatine kinase and glutamine synthetase--but not isocitrate dehydrogenase and malate dehydrogenase. Ubiquinone-1 and/or H2O2 do not affect the activity of alkaline phosphatase and glutamine synthetase chosen as model enzymes. Dioxygen and transition metal ions, even if in trace amounts, are essential for the enzyme inactivation, which indeed does not occur under argon atmosphere or in the presence of metal chelators. Supplementation with redox-active metal ions (Fe3+ or Cu2+), moreover, potentiates alkaline phosphatase inactivation. Since catalase and peroxidase protect while superoxide dismutase does not, hydrogen peroxide rather than superoxide anion seems to be involved in the inactivation mechanism through which oxygen active species (hydroxyl radical or any other equivalent species) are produced via a modified Haber-Weiss cycle, triggered by metal-catalyzed oxidation of ubiquinol-1. The lack of efficiency of radical scavengers and the almost complete protection afforded by enzyme substrates and metal cofactors indicate a 'site-specific' radical attack as responsible for the oxidative damage.

Alanine Transaminase↗

PubMed1991

Metal-catalyzed oxidation of human serum albumin: conformational and functional changes. Implications in protein aging.

Mild oxidative stress, as elicited by ascorbate, oxygen, and trace metals, affects the binding properties of human serum albumin via purely conformational changes. In fact, no gross alteration can be observed in the electrophoretic and chromatographic patterns of albumin, whereas localized modifications are indicated by the changes in absorption and fluorescence spectra and in polarization degree. The oxidized protein presents a small increase of bityrosine production and a time-dependent increase in the content of carbonyl groups, whereas proteolytic susceptibility is unchanged. A higher affinity for cis-parinaric acid and a slight loss of solubility in high salt indicate a greater surface hydrophobicity. Pinpoint denaturation of the albumin molecule is also suggested by a decreased "esterase" activity in the presence of p-nitrophenyl acetate. Conformational stability evaluated through thermal shock and addition of moderate amounts of guanidine indicate that the oxidized protein is more heat-resistant, less flexible, and more rigid than the native one. Although limited, structural damages afforded by the oxidative stress cause alterations of albumin binding properties as documented by experiments with probes and physiological ligands. The loss of biological activity of human serum albumin induced by ascorbate system appears of medical relevance, because it can affect drug metabolism and particularly drug tolerance in the elderly.

Ascorbic Acid↗

PubMed1990

Prooxidant action of desferrioxamine: enhancement of alkaline phosphatase inactivation by interaction with ascorbate system.

Desferrioxamine (DFO) nearly doubles alkaline phosphatase oxidative inactivation by the ascorbate system. The effect is dependent on ascorbate and desferrioxamine concentrations, exhibiting in both cases a saturation mechanism. Conversion of desferrioxamine to ferrioxamine abolishes the prooxidant action. Desferrioxamine also increases ascorbate-dependent oxygen consumption and nitroblue tetrazolium reduction. Superoxide dismutase, which blocks the desferrioxamine enhancing effect on enzyme inactivation, markedly slows down nitroblue tetrazolium reduction as well as oxygen consumption by ascorbate plus desferrioxamine, while it fails to protect against the ascorbate system alone. Therefore, in the presence of desferrioxamine, the metal-catalyzed ascorbate autooxidation becomes superoxide-dependent and thus inhibitable by superoxide dismutase. Catalase, peroxidase, and ascorbate oxidase protect alkaline phosphatase from inactivation by both ascorbate and ascorbate-desferrioxamine systems. Hemin shields the enzyme from ascorbate plus DFO attack but not from ascorbate alone. In air-saturated solution, desferrioxamine seems to mediate one electron transfer from ascorbate to oxygen, generating superoxide anions, which can either trigger a Fenton reaction or produce desferal nitroxide radicals. In the absence of oxygen, ascorbate alone is ineffective, but the ascorbate plus desferrioxamine system still inactivates the enzyme; catalase, peroxidase, and ascorbate oxidase, but not superoxide dismutase, afford protection.

Alkaline Phosphatase↗

PubMed1989

Time-resolved fractionation of bone and liver alkaline phosphatase activities with a 'peeling-off' method.

A procedure for the selective fractionation of the bone and liver alkaline phosphatase activity in tissue extracts and human sera is proposed. Optimized conditions of the assay are: urea 3.7 mol/l in 0.5 mol/l DEA buffer, pH 9.8; 0.5 mmol/l MgCl2; 10.0 mmol/l p-nitrophenyl phosphate. The sample is diluted 1:20 in the reagent solution and the activity is recorded for 10 min at 37 degrees C. By means of a computerized or manual graphic analysis, based on 'peeling-off' the exponentials, the two differently urea-sensitive subforms are identified and the slow-(liver) and the fast-decaying (bone) activities are easily discriminated and their respective values calculated. Interference due to the intestinal isoenzyme can be also accounted for. The analytical variability is very satisfactory (within run CV = 7.5 and 4.5% for osseous and hepatic form, respectively; day-to-day CV less than 10% for both). The lower limits of detection are about 10 U/l and the serum or plasma reference values together with the influence on the assay of hemoglobin and protein content are also investigated.

Adult↗

PubMed1988

Mixed function oxidation and enzymes: kinetic and structural properties of an oxidatively modified alkaline phosphatase.

No major structural alteration of alkaline phosphatase can be observed in the early stages of enzyme oxidative inactivation by the ascorbate model system. Fluorescence changes of protein-bound 8-anilino-1-naphthalenesulfonic acid suggest, however, that localized modifications take place. Oxidized alkaline phosphatase displays less catalytic efficiency (decrease of Vmax), while retaining the other kinetic properties, including the same affinity for substrates and inhibitors and the same activation energy of the native enzyme. Typical features of the modified protein are a decreased thermal stability and a biphasic heat inactivation profile, which make the oxidized form quite similar to aged enzymes. The lower response to Mg2+ activation indicates that the magnesium binding sites of alkaline phosphatase are probably the targets of the ascorbate system oxidative modifications.

Alkaline Phosphatase↗