Human erythrocyte δ-aminolevulinate dehydratase inhibition by monosaccharides is not mediated by oxidation of enzyme sulfhydryl groups

Description
Human erythrocyte δ-aminolevulinate dehydratase inhibition by monosaccharides is not mediated by oxidation of enzyme sulfhydryl groups

Please download to get full document.

View again

of 6
All materials on our website are shared by users. If you have any questions about copyright issues, please report us to resolve them. We are always happy to assist you.
Information
Category:

Communications

Publish on:

Views: 0 | Pages: 6

Extension: PDF | Download: 0

Share
Tags
Transcript
  Human erythrocyte  d -aminolevulinate dehydrataseinhibition by monosaccharides is not mediated by oxidationof enzyme sulfhydryl groups D. Gabriel, L. Pivetta, V. Folmer, J.C.M. Soares, G.R. Augusti,C.W. Nogueira, G. Zeni, J.B.T. Rocha * Departamento de Quı´ mica, Centro de Cie ˆncias Naturais e Exatas, Universidade Federal de Santa Maria,97105-900 Santa Maria, RS, Brazil  Received 8 June 2004; revised 1 December 2004; accepted 30 March 2005 Abstract The heme pathway enzyme  d -aminolevulinate dehydratase is a good marker for oxidative stress and metal intoxication. Thissulfhydryl enzyme is inhibited in such oxidative pathologies as lead, mercury and aluminum intoxication, exposure to seleniumorganic species and diabetes. Oxidative stress is a complicating factor in diabetes, inducing non-enzymatic glucose-mediatedreactions that change protein structures and impair enzyme functions. We have studied the effects of high glucose, fructose andribose concentrations on  d -ALA-D activity in vitro. These reducing sugars inhibited  d -ALA-D with efficacies in the orderfructose Z ribose O glucose. The possible mechanism of glucose inhibition was investigated using lysine, DTT, and  t -butylamine.Oxidation of the enzyme’s critical sulfhydryl groups was not involved because DTT had no effect. We concluded that highconcentrations of reducing sugars or their autoxidation products inhibit  d -ALA-D by a mechanism not related to thiol oxidation.Also, we are not able to demonstrate that the formation of a Schiff base with the critical lysine residue of the enzyme is involved inthe inhibition of   d -ALA-D by hexoses.   2005 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords:  Ebselen; Diabets; Porphobilinogen synthase; Oxidative stress 1. Introduction Diabetes is characterized by hyperglycemia and istreated with insulin or drugs to ameliorate this symptom(Strowig and Raskin, 1992; Pinero-Pilona et al., 2002). Thediseaseleadstodebilitatingsecondarycomplicationsthat shorten the patient’s life span. Recently, themolecular mechanisms underlying these secondary com-plications have been more thoroughly investigated.Evidence suggests that non-enzymatic glucose-mediatedreactions such as autoxidation (Hunt et al., 1988; Wolff andDean,1987a,b;Carubellietal.,1994;Parthibanetal.,1995),proteincross-linking(Namikietal.,1977;Lietal., 1996; Day et al., 1979; Beswick and Harding, 1985) and AGEs formation (Strowig and Raskin, 1992; Sensi et al.,1995; Soluis et al., 1999; Rahbar et al., 1999; Chevalieret al., 2002; Forbes et al., 2003) are involved. There havebeen numerous studies on the deleterious effects of hyperglycemia on the properties of physiologicallyabundant proteins such as hemoglobin (Schwartz,1995), albumin (Day et al., 1979), and collagen (Fu et al., 1992). However, published data on the effects of  * Corresponding author. Fax: C 55 552208031. E-mail address:  jbtrocha@yahoo.com.br (J.B.T. Rocha).1065-6995/$ - see front matter   2005 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.doi:10.1016/j.cellbi.2005.03.017 www.elsevier.com/locate/cellbi Cell Biology International 29 (2005) 669 e 674  hyperglycemia on less abundant proteins, such as  d -aminolevulinate dehydratase ( d -ALA-D), are rare (Ca-ballero et al., 1998). d -Aminolevulinate dehydratase ( d -ALA-D), an en-zyme in the heme biosynthesis pathway, is essential forall aerobic organisms. It is a marker for oxidative stressbecause its active sulfhydryl group renders it highlysensitive to pro-oxidant elements (Maciel et al., 2000;Folmer et al., 2002, 2003; Soares et al., 2002), which impair its function (Rodrigues et al., 1989; Rocha et al.,1993, 1995; 2004; Barbosa et al., 1998; Flora, 1999, 2000; Farina et al., 2001; Jacques-Silva et al., 2001). This enzyme catalyzes the asymmetric condensation of twomolecules of 5-aminolevulinic acid ( d -ALA) to form themonopyrrole porphobilinogen (PBG) (Jaffe et al., 1995;Sassa et al., 1989; Sassa, 1998) (Fig. 1). In subsequent steps, PBG is assembled into tetrapyrrole molecules,which constitute the prosthetic groups of physiologicallysignificant proteins such as hemoglobin, cytochromesand enzymes such as catalase.In the presumed mechanism of PBG assembly, a lysylamino group in  d -ALA-D reacts with the carbonylgroup of the first molecule of   d -ALA forming a Schiff base (Jaffe et al., 1995). Subsequently, the free aminogroup of the  d -ALA-D bound  d -ALA reacts with thecarbonyl group of the second  d -ALA molecule, forminga second Schiff base. This latter step requires Zn 2 C andreduced sulfhydryl groups (Jaffe et al., 1995) and isblocked by sulfhydryl reagents such as MMTS (methylmethanethiosulfonate). Inhibition of the enzyme leadsto disturbances of heme biosynthesis as well as in-termediate accumulation, which has been shown toinduce pro-oxidant events (Pereira et al., 1992; Becharaet al., 1993).Importantly,  d -ALA-D activity is impaired in di-abetic patients and in animal models of the disease(Folmer et al., 2002, 2003). Furthermore, high concen- tration of glucose and other reducing sugars inhibits  d -ALA-D in vitro (Caballero et al., 1998). This inhibitionis prevented by acetyl salicylic acid and is not associatedwith increased production of thiobarbituric acid species.The molecular mechanism underlying  d -ALA-D impair-ment after in vitro (or even in vivo) exposure to glucoseis still not completely understood, but may be causedeither by glycation of the active site lysine residueinvolved in Schiff’s base formation with the first  d -ALAmolecule, or by oxidation of the essential reducedcysteinyl residues of the enzyme (Caballero et al.,1998; Fernandez-Cuartero et al., 1999). In this study,we investigated the capacities of amino-containingsubstance, DTT (a sulfhydryl reducing agent) or  d -ALA itself to modulate the inhibitory action of reducingsugars. Furthermore, we showed that short-term pre-incubation of blood  d -ALA-D with high concentrationsof reducing sugar (glucose, fructose or ribose) hada similar inhibiting effect on the enzyme to long-termpre-incubation. 2. Materials and methods 2.1. Blood preparation Venous blood (10 ml) was collected from 20 healthyfasting male human normoglycemic (below 100 mg/dl)volunteers from our workgroup (University FederalSanta Maria, RS, Brazil) into heparinized tubes andcentrifuged. The concentrated erythrocytes were mixedwith Triton X 100 to effect hemolysis. 2.2.  d -ALA-D assay Blood  d -ALA-D activity was assayed by the methodof  Berlin and Schaller (1974) by measuring the rate of product formation (porphobilinogen), except that84 mM potassium phosphate buffer, pH 6.4, and2.4 mM  d -ALA were used. In some experiments( n Z 4), the  d -ALA concentration was varied from0.75 to 3.0 mM and glucose or fructose concentrationsof 200 and 400 mM were used. The time of pre-incubation with monosaccharides was 1 h. The reactionwas started by adding 200  m l hemolyzed blood to theabove medium and stopped after 90 min with 10% TCAcontaining 10 mM HgCl 2 . The reaction product wasdetermined using modified Ehrlich’s reagent at 555 nm,with a molar absorption coefficient of 6.1 ! 10 4 M  1 forthe Ehrlich-porphobilinogen salt. The reaction rates Fig. 1. Asymmetric condensation of two 5-aminolevulinic acid ( d -ALA) molecules catalyzed by  d -aminolevulinate dehydratase ( d -ALA-D).670  D. Gabriel et al. / Cell Biology International 29 (2005) 669 e 674  were linear with respect to incubation time and proteinconcentration under all experimental conditions. 2.3. Statistics Statistical analyses were performed by one, two orthree-way ANOVA followed by Duncan’s multiplerange test (when the univariate test revealed an  F   valueassociated with a  p ! 0.05). Results were consideredstatistically significant when  p ! 0.05. 3. Results Fructose, ribose and glucose caused significant in-hibition of   d -ALA-D activity (  p ! 0.01 for each sugarseparately; Fig. 2). However, glucose inhibited  d -ALA-D with lower potency than fructose and ribose. IC 50  toglucose, fructose and ribose were 367, 225 and 282 mM,respectively (one-way ANOVA followed by Duncan’smultiple range test revealed a significant differencebetween glucose and fructose or ribose potency asinhibitors;  p ! 0.01). The IC 50  value for  d -ALA-Dinhibition by glucose is very similar to those reportedpreviously by Caballero et al. (1998) after 20 h of pre-incubation with glucose. The inhibitory effect of glucoseand fructose (200 or 400 mM) was not modified whenthe enzymatic reaction was carried out in the presenceof 0.75 or 3 mM of   d -ALA (data not shown). Thisindicated that the inhibitory action of reducing sugars isnon-competitive.The inhibitory action of glucose was also investigatedin the presence of 2.5 and 10 mM of borohydride. Thiswas done with the intention of stabilizing the putativeSchiff base between the enzyme and glucose. However,these concentrations of borohydride did not modify theinhibitory effect of glucose (data not shown). Higherconcentrations were not used to avoid changes in the pHof the reaction mixture.DTT, which maintains  d -ALA-D activity in vitrowhen the enzyme is challenged with oxidizing agents,afforded no protection against inhibition by glucose,fructose or ribose (Fig. 3). Two-way ANOVA revealeda significant main effect of DTT (  p ! 0.01) and of sugar(  p ! 0.01). These results indicate that although DTTcaused an increase in enzyme activity, it was not efficientin counteracting the inhibitory effect of glucose, fructoseand ribose. This indicates that sulfhydryl groupoxidation is not involved in the inhibition. Moreover,ebselen (2  m mol/l), an organochalcogenide with antiox-idant activity (Mugesh et al., 2001; Nogueira et al.,2004), was similarly ineffective (Fig. 4), indicating that reactive oxygen species are not likely to be involved in  d -ALA-D inhibition by glucose after short-term pre-incubation periods. Two-way ANOVA revealed onlya significant main effect of glucose (  p ! 0.01). Low-molecular weight amines (lysine and  t -butylamine) alsofailed to protect  d -ALA-D from inhibition by glucose Fig. 2. Inhibition of human erythrocyte  d -aminolevulinate dehydratase( d -ALA-D) by reducing sugars. Values are means G SEM ( n Z 6).(Control activity without glucose, ribose or fructose, 256.72 nmol of PBG/ml of erythrocytes/h.)Fig. 3. Absence of antagonism by DTT of   d -aminolevulinatedehydratase ( d -ALA-D) activity from human erythrocytes inhibitedby glucose, fructose and ribose. Values are means G SE ( n Z 4).(Control Z basal activity, 203.06 nmol of PBG/ml of erythrocytes/h.)Fig. 4. Absence of antagonism by ebselen of   d -aminolevulinatedehydratase ( d -ALA-D) activity from human erythrocytes inhibitedby glucose ( n Z 4). Values are means G SE. (Basal activity (100%)was 214.65 nmol of PBG/ml of erythrocytes/h.)671 D. Gabriel et al. / Cell Biology International 29 (2005) 669 e 674  (Fig. 5a and b). Two-way ANOVA for each amineseparately revealed only a significant main effect of glucose (  p ! 0.01). The main effects of amines were notsignificant.Zn 2 C , an essential ion for mammalian  d -ALA-D,caused a small but significant activation of the bloodenzyme at 1 mmol/l but was inhibitory at higherconcentrations (Fig. 6). Zn 2 C had no effect on theinhibition of   d -ALA-D by high concentrations of glucose. The effects of glucose and Zn 2 C were notmodified by the inclusion of lysine in the reactionmedium (Fig. 6). Indeed, three-way ANOVA revealeda significant main effect of glucose (  p ! 0.01) and of Zn 2 C (  p ! 0.01), but the effect of lysine was notsignificant. 4. Discussion Glycosylation plays a significant role in protein aging(Day et al., 1979; Hunt et al., 1988; Carubelli et al., 1994). The reaction starts with the reversible formationof a Schiff base between glucose (or other reducingsugar) and protein amino groups. Subsequently, theunstable Schiff base is rearranged to form a stableAmadori product (Sensi et al., 1995). In vivo, Amadoriproducts are formed slowly and play an important rolein the development of diabetes complications (Chevalieret al., 2002). In vitro protein glycosylation can beachieved in short-term experiments with high non-physiological concentrations of reducing sugars. In thepresent study we showed that exposure to highconcentrations of glucose, fructose or ribose causedsignificant inhibition of blood  d -ALA-D. The concen-trations of monosaccharides are not physiological;however, short-term exposure to high concentrationsof reducing sugars can help to elucidate, at least in part,the mechanism through which sugars inhibit  d -ALA-D,which in turn may have some pathological significance.The possible mechanisms of inhibition were investi-gatedandwereachedthefollowingconclusions.First,theoxidationofessentialthiolgroupsisnotinvolvedbecausethe thiol protecting and reducing agent DTT wasineffective in counteracting inhibition by glucose andfructose. Consequently, in short-term in vitro models of glycosylation, the formation of free radicals that accom-panies the glycosylation of amino groups and sugarautoxidation (Namiki et al., 1977), which could poten-tiallyoxidizeessentialenzyme e SHgroups,seemstoplayno significant role in  d -ALA-D inhibition. This wasfurtherconfirmedbythefailureofebselen,anantioxidant(Mugesh et al., 2001; Nogueira et al., 2004), to modify inhibition by glucose or fructose. Second, high concen-trations of compounds containing amino groups such aslysine and  t -butylamine were similarly ineffective. Thissuggests that the amino groups of these low-molecularweightaminesaremuchlessreactivethanthelysylaminogroup at the active center of blood  d -ALA-D or thatglucose does not react with this residue in the activecenter of the enzyme. However, it must be emphasizedthat amino groups in free amino acids react rapidlywith reducing sugars only under extreme conditions of temperature and pH (Namiki et al., 1977), but in vitro Fig. 5. Glucose inhibition of   d -aminolevulinate dehydratase ( d -ALA-D) activity from human erythrocytes: absence of antagonism by lysine(A) or by  t -butylamine (B) of   d -ALA-D inhibition by glucose(400 mM). Values are means G SE ( n Z 5). (Control activity (100%)was 201.96 nmol of PBG/ml of erythrocytes/h to A and 187.10 nmol of PBG/ml of erythrocytes/h for B.)Fig. 6.  d -Aminolevulinate dehydratase ( d -ALA-D) activity in humanerythrocytes: influence of Zn 2 C and lysine on enzyme inhibition byglucose. Values are means G SE ( n Z 4). (Control activity was184.70 nmol of PBG/ml of erythrocytes/h.)672  D. Gabriel et al. / Cell Biology International 29 (2005) 669 e 674  glycosylationofhemoglobinoccurswithinafewhoursof exposuretohighglucoseconcentrations(Caballeroetal.,1998). In a previous study, we observed that in vivo,hemoglobin glycosylation correlates with  d -ALA-Dinhibition in mice (Folmer et al., 2003). Consequently, we proposed that the reducing sugarmay inhibit  d -ALA-D by binding to an enzyme e substrate intermediate. One potential candidate is theamino group of the first  d -ALA molecule bound to d -ALA-D. In fact, we observed that pre-incubation withglucose followed by its withdrawal from the medium,before the enzyme reaction was started with  d -ALA, didnot result in enzyme inhibition. If glucose inhibited  d -ALA-D by interacting with its active center lysil residue,one would expect that inhibition could not be com-pletely overcome by removing glucose from the reactionmedium after a previous pre-incubation with theenzyme. The relationship of these artificial conditions(high reducing sugar concentrations) to the inhibition of  d -ALA-D in vivo after dietary or pathological hyper-glycemia deserves further investigation (Gugliucci andAllard, 1996; Al-Zuhair and Mohamed, 1998; Mon-tenegro et al., 2002). However, the fact that  d -ALA-Dinhibition promoted by hyperglycemia in vivo (Folmeret al., 2002; 2003) is not reversed by DTT suggests thatthe mechanism underlying  d -ALA-D inhibition both invitro and in vivo does not involve e SH group oxidation. Acknowledgements Financial support by FAPERGS, CAPES and CNPqis gratefully acknowledged. References Al-Zuhair H, Mohamed HE. Vitamin C attenuation of the de-velopment of type I diabetes mellitus by interferon-alpha.Pharmacol Res 1998;38:59 e 64.Barbosa NBV, Rocha JBT, Zeni G, Emanuelli T, Beque MC,Braga AL. Effect of organic forms of selenium on  d -amino-levulinate dehydratase from liver, kidney, and brain of adult rats.Toxicol Appl Pharmacol 1998;149:243 e 53.Bechara EJH, Medeiros MHG, Monteiro HP, Hermes-Lima M,Pereira B, Demasi M, et al. A free radical hypothesis of leadpoisoning and inborn porphyrias associated with 5-aminolevulinicacid overload. Quim Nova 1993;16:385 e 92.Berlin A, Schaller KH. European standardized method for thedetermination of delta-aminolevulinic acid dehydratase activity inblood. Z Klin Chem Klin Biochem 1974;12:389 e 90.Beswick HT, Harding JJ. Aldehydes or dicarbonyls in non-enzymicglycosylation of proteins. Biochem J 1985;226:385 e 9.Caballero FA, Gerez EN, Polo CF, Vazquez ES, Batlle AMC.Reducing sugars trigger  d -aminolevulinate dehydratase inactiva-tion: evidence of in vitro aspirin prevention. Gen Pharmacol 1998;31:441 e 5.Carubelli R, Schineider JE, Pye QN, Floyd R. Cytotoxic effects of autoxidative glycation. Free Radic Biol Med 1994;18:265 e 9.Chevalier F, Chobert JM, Dalgalarrondo M, Choiset Y, Haertle T.Maillard glycation of beta-lactoglobulin induces conformationchanges. Nahrung 2002;46:58 e 63.Day JF, Thornburg SR, Baynes JW. Non-enzymatically glycosylatedalbumin. J Biol Chem 1979;254:595 e 7.Farina M, Folmer V, Bolzan RC, Andrade LH, Zeni G, Braga AL,et al. Selenoxides inhibit  d -aminolevulinic acid dehydratase.Toxicol Lett 2001;119:27 e 37.Fernandez-Cuartero B, Rebollar JL, Batlle A, Enriquez DE,Salamanca R. Delta aminolevulinate dehydratase (ALA-D) activityin human and experimental diabetes mellitus. Int J Biochem CellBiol 1999;31:479 e 88.Flora SJS. Arsenic-induced oxidative stress and its reversibilityfollowing combined administration of   N  -acetylcysteine and meso2,3-dimercaptosuccinic acid in rats. Clin Exp Pharmacol Physiol1999;26:865 e 9.FloraSJS.Possiblehealthhazardsassociatedwiththeuseoftoxicmetalsin semiconductor industries. J Occup Health 2000;42:105 e 10.Folmer V, Soares JCM, Rocha JBT. Oxidative stress in mice isdependent on the free glucose content in the diet. Int J BiochemCell Biol 2002;34:1279 e 85.Folmer V, Soares JCM, Gabriel D, Rocha JBT. A high fat diet inhibitsdelta-aminolevulinate dehydratase and increases lipid peroxidationin mice ( Mus musculus ). J Nutr 2003;133:2165 e 70.Forbes JM, Thallas V, Thomas MC, Founds HW, Burns WC,Jerums G, et al. The breakdown of preexisting advanced glycationend products is associated with reduced renal fibrosis inexperimental diabetes. FASEB J 2003;17:1762 e 4.Fu MX, Knecht KJ, Thorpe SR, Baynes JW. Role of oxygen incrosslinking and chemical modification of collagen by glucose.Diabetes 1992;41:42 e 8.Gugliucci A, Allard MF. Glycation of hepatocyte cytosolic proteinsin streptozotocin-induced diabetic rats. Biochem Biophys ResCommun 1996;229:952 e 8.Hunt JV, Dean RT, Wolff SP. Hydroxyl radical production andautoxidative glycosylation. Glucose oxidation as the cause of protein damage in the experimental glycation model of diabetesmellitus and aging. Biochem J 1988;256:205 e 12.Jacques-Silva MC, Nogueira CW, Broch LC, Flores EMM,Rocha JBT. Diphenyl diselenide and ascorbic acid changesdeposition of selenium and ascorbic acid in brain of mice.Pharmacol Toxicol 2001;44:119 e 25.Jaffe EK, Ali S, Mitchell LW, Taylor KM, Volin M, Markham GD.Characterization of the role of the stimulatory magnesium of  Escherichia coli   porphobilinogen synthase. Biochemistry 1995;34:244 e 51.Li D, Devaraj S, Fuller C, Bucala R, Jialal I. Effect of alpha-tocopherol on LDL oxidation and glycation: in vitro and in vivostudies. J Lipid Res 1996;37:1978 e 86.Maciel EM, Bolzan RC, Braga AL, Rocha JBT. Diphenyl diselenideand diphenyl ditelluride differentially affects  d -aminolevulinatedehydratase from liver, kidney and brain of mice. J Biochem MolToxicol 2000;14:310 e 9.Montenegro RMJ, Montenegro AP, Fernandes MI, De Moraes RR,Elias JJ, Gouveia LM, et al. Triglyceride-induced diabetes mellitusin congenital generalized lipodystrophy. J Pediatr EndocrinolMetab 2002;15:441 e 7.Mugesh G, du Mont WW, Sies H. Chemistry of biologically importantsynthetic organoselenium compounds. Chem Rev 2001;101:2125 e 79.Namiki M, Haiashi T, Ohta Y. Novel free radical formed by theamino e carbonyl reactions of sugars with amino acids, amine andproteins. Adv Exp Med Biol 1977;86B:471 e 501.Nogueira CW, Zeni G, Rocha JBT. Organoselenium and organo-tellurium compounds: toxicology and pharmacology. Chem Rev2004;104:6255 e 85.Parthiban A, Vijayalingam S, Shanmugasundaram KR, Mohan R.Oxidative stress and the development of diabetic complications e 673 D. Gabriel et al. / Cell Biology International 29 (2005) 669 e 674
Related Search
Similar documents
View more...
We Need Your Support
Thank you for visiting our website and your interest in our free products and services. We are nonprofit website to share and download documents. To the running of this website, we need your help to support us.

Thanks to everyone for your continued support.

No, Thanks