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Cell Biology International (2005) 29, 669–674 (Printed in Great Britain)
Human erythrocyte δ-aminolevulinate dehydratase inhibition by monosaccharides is not mediated by oxidation of enzyme sulfhydryl groups
D. Gabriel, L. Pivetta, V. Folmer, J.C.M. Soares, G.R. Augusti, C.W. Nogueira, G. Zeni and J.B.T. Rocha*
Departamento de Química, Centro de Ciências Naturais e Exatas, Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS, Brazil


Abstract

The heme pathway enzyme δ-aminolevulinate dehydratase is a good marker for oxidative stress and metal intoxication. This sulfhydryl enzyme is inhibited in such oxidative pathologies as lead, mercury and aluminum intoxication, exposure to selenium organic species and diabetes. Oxidative stress is a complicating factor in diabetes, inducing non-enzymatic glucose-mediated reactions that change protein structures and impair enzyme functions. We have studied the effects of high glucose, fructose and ribose concentrations on δ-ALA-D activity in vitro. These reducing sugars inhibited δ-ALA-D with efficacies in the order fructose=ribose>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 high concentrations of reducing sugars or their autoxidation products inhibit δ-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 in the inhibition of δ-ALA-D by hexoses.


Keywords: Ebselen, Diabetes, Porphobilinogen synthase, Oxidative stress.

*Corresponding author. Fax: +55 552208031.


1 Introduction

Diabetes is characterized by hyperglycemia and is treated with insulin or drugs to ameliorate this symptom (Strowig and Raskin, 1992; Pinero-Pilona et al., 2002). The disease leads to debilitating secondary complications that shorten the patient's life span. Recently, the molecular mechanisms underlying these secondary complications have been more thoroughly investigated. Evidence suggests that non-enzymatic glucose-mediated reactions such as autoxidation (Hunt et al., 1988; Wolff and Dean, 1987a,b; Carubelli et al., 1994; Parthiban et al., 1995), protein cross-linking (Namiki et al., 1977; Li et al., 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; Chevalier et al., 2002; Forbes et al., 2003) are involved. There have been numerous studies on the deleterious effects of hyperglycemia on the properties of physiologically abundant proteins such as hemoglobin (Schwartz, 1995), albumin (Day et al., 1979), and collagen (Fu et al., 1992). However, published data on the effects of hyperglycemia on less abundant proteins, such as δ-aminolevulinate dehydratase (δ-ALA-D), are rare (Caballero et al., 1998).

δ-Aminolevulinate dehydratase (δ-ALA-D), an enzyme in the heme biosynthesis pathway, is essential for all aerobic organisms. It is a marker for oxidative stress because its active sulfhydryl group renders it highly sensitive 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 two molecules of 5-aminolevulinic acid (δ-ALA) to form the monopyrrole 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 physiologically significant proteins such as hemoglobin, cytochromes and enzymes such as catalase.


Fig. 1

Asymmetric condensation of two 5-aminolevulinic acid (δ-ALA) molecules catalyzed by δ-aminolevulinate dehydratase (δ-ALA-D).


In the presumed mechanism of PBG assembly, a lysyl amino group in δ-ALA-D reacts with the carbonyl group of the first molecule of δ-ALA forming a Schiff base (Jaffe et al., 1995). Subsequently, the free amino group of the δ-ALA-D bound δ-ALA reacts with the carbonyl group of the second δ-ALA molecule, forming a second Schiff base. This latter step requires Zn2+ and reduced sulfhydryl groups (Jaffe et al., 1995) and is blocked by sulfhydryl reagents such as MMTS (methyl methanethiosulfonate). Inhibition of the enzyme leads to disturbances of heme biosynthesis as well as intermediate accumulation, which has been shown to induce pro-oxidant events (Pereira et al., 1992; Bechara et al., 1993).

Importantly, δ-ALA-D activity is impaired in diabetic patients and in animal models of the disease (Folmer et al., 2002, 2003). Furthermore, high concentration of glucose and other reducing sugars inhibits δ-ALA-D in vitro (Caballero et al., 1998). This inhibition is prevented by acetyl salicylic acid and is not associated with increased production of thiobarbituric acid species. The molecular mechanism underlying δ-ALA-D impairment after in vitro (or even in vivo) exposure to glucose is still not completely understood, but may be caused either by glycation of the active site lysine residue involved in Schiff's base formation with the first δ-ALA molecule, or by oxidation of the essential reduced cysteinyl residues of the enzyme (Caballero et al., 1998; Fernandez-Cuartero et al., 1999). In this study, we investigated the capacities of amino-containing substance, DTT (a sulfhydryl reducing agent) or δ-ALA itself to modulate the inhibitory action of reducing sugars. Furthermore, we showed that short-term pre-incubation of blood δ-ALA-D with high concentrations of reducing sugar (glucose, fructose or ribose) had a similar inhibiting effect on the enzyme to long-term pre-incubation.

2 Materials and methods

2.1 Blood preparation

Venous blood (10ml) was collected from 20 healthy fasting male human normoglycemic (below 100mg/dl) volunteers from our workgroup (University Federal Santa Maria, RS, Brazil) into heparinized tubes and centrifuged. The concentrated erythrocytes were mixed with Triton X 100 to effect hemolysis.

2.2 δ-ALA-D assay

Blood δ-ALA-D activity was assayed by the method of Berlin and Schaller (1974) by measuring the rate of product formation (porphobilinogen), except that 84mM potassium phosphate buffer, pH 6.4, and 2.4mM δ-ALA were used. In some experiments (n=4), the δ-ALA concentration was varied from 0.75 to 3.0mM and glucose or fructose concentrations of 200 and 400mM were used. The time of pre-incubation with monosaccharides was 1h. The reaction was started by adding 200μl hemolyzed blood to the above medium and stopped after 90min with 10% TCA containing 10mM HgCl2. The reaction product was determined using modified Ehrlich's reagent at 555nm, with a molar absorption coefficient of 6.1×104M−1 for the Ehrlich-porphobilinogen salt. The reaction rates were linear with respect to incubation time and protein concentration under all experimental conditions.

2.3 Statistics

Statistical analyses were performed by one, two or three-way ANOVA followed by Duncan's multiple range test (when the univariate test revealed an F value associated with a p<0.05). Results were considered statistically significant when p<0.05.

3 Results

Fructose, ribose and glucose caused significant inhibition of δ-ALA-D activity (p<0.01 for each sugar separately; Fig. 2). However, glucose inhibited δ-ALA-D with lower potency than fructose and ribose. IC50 to glucose, fructose and ribose were 367, 225 and 282mM, respectively (one-way ANOVA followed by Duncan's multiple range test revealed a significant difference between glucose and fructose or ribose potency as inhibitors; p<0.01). The IC50 value for δ-ALA-D inhibition by glucose is very similar to those reported previously by Caballero et al. (1998) after 20h of pre-incubation with glucose. The inhibitory effect of glucose and fructose (200 or 400mM) was not modified when the enzymatic reaction was carried out in the presence of 0.75 or 3mM of δ-ALA (data not shown). This indicated that the inhibitory action of reducing sugars is non-competitive.


Fig. 2

Inhibition of human erythrocyte δ-aminolevulinate dehydratase (δ-ALA-D) by reducing sugars. Values are means±SEM (n=6). (Control activity without glucose, ribose or fructose, 256.72nmol of PBG/ml of erythrocytes/h.)


The inhibitory action of glucose was also investigated in the presence of 2.5 and 10mM of borohydride. This was done with the intention of stabilizing the putative Schiff base between the enzyme and glucose. However, these concentrations of borohydride did not modify the inhibitory effect of glucose (data not shown). Higher concentrations were not used to avoid changes in the pH of the reaction mixture.

DTT, which maintains δ-ALA-D activity in vitro when the enzyme is challenged with oxidizing agents, afforded no protection against inhibition by glucose, fructose or ribose (Fig. 3). Two-way ANOVA revealed a significant main effect of DTT (p<0.01) and of sugar (p<0.01). These results indicate that although DTT caused an increase in enzyme activity, it was not efficient in counteracting the inhibitory effect of glucose, fructose and ribose. This indicates that sulfhydryl group oxidation is not involved in the inhibition. Moreover, ebselen (2μmol/l), an organochalcogenide with antioxidant 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 δ-ALA-D inhibition by glucose after short-term pre-incubation periods. Two-way ANOVA revealed only a significant main effect of glucose (p<0.01). Low-molecular weight amines (lysine and t-butylamine) also failed to protect δ-ALA-D from inhibition by glucose (Fig. 5a and b). Two-way ANOVA for each amine separately revealed only a significant main effect of glucose (p<0.01). The main effects of amines were not significant.


Fig. 3

Absence of antagonism by DTT of δ-aminolevulinate dehydratase (δ-ALA-D) activity from human erythrocytes inhibited by glucose, fructose and ribose. Values are means±SE (n=4). (Control=basal activity, 203.06nmol of PBG/ml of erythrocytes/h.)


Fig. 4

Absence of antagonism by ebselen of δ-aminolevulinate dehydratase (δ-ALA-D) activity from human erythrocytes inhibited by glucose (n=4). Values are means±SE. (Basal activity (100%) was 214.65nmol of PBG/ml of erythrocytes/h.)


Fig. 5

Glucose inhibition of δ-aminolevulinate dehydratase (δ-ALA-D) activity from human erythrocytes: absence of antagonism by lysine (A) or by t-butylamine (B) of δ-ALA-D inhibition by glucose (400mM). Values are means±SE (n=5). (Control activity (100%) was 201.96nmol of PBG/ml of erythrocytes/h to A and 187.10nmol of PBG/ml of erythrocytes/h for B.)




Zn2+, an essential ion for mammalian δ-ALA-D, caused a small but significant activation of the blood enzyme at 1mmol/l but was inhibitory at higher concentrations (Fig. 6). Zn2+ had no effect on the inhibition of δ-ALA-D by high concentrations of glucose. The effects of glucose and Zn2+ were not modified by the inclusion of lysine in the reaction medium (Fig. 6). Indeed, three-way ANOVA revealed a significant main effect of glucose (p<0.01) and of Zn2+ (p<0.01), but the effect of lysine was not significant.


Fig. 6

δ-Aminolevulinate dehydratase (δ-ALA-D) activity in human erythrocytes: influence of Zn2+ and lysine on enzyme inhibition by glucose. Values are means±SE (n=4). (Control activity was 184.70nmol of PBG/ml of erythrocytes/h.)


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 formation of a Schiff base between glucose (or other reducing sugar) and protein amino groups. Subsequently, the unstable Schiff base is rearranged to form a stable Amadori product (Sensi et al., 1995). In vivo, Amadori products are formed slowly and play an important role in the development of diabetes complications (Chevalier et al., 2002). In vitro protein glycosylation can be achieved in short-term experiments with high non-physiological concentrations of reducing sugars. In the present study we showed that exposure to high concentrations of glucose, fructose or ribose caused significant inhibition of blood δ-ALA-D. The concentrations of monosaccharides are not physiological; however, short-term exposure to high concentrations of reducing sugars can help to elucidate, at least in part, the mechanism through which sugars inhibit δ-ALA-D, which in turn may have some pathological significance.

The possible mechanisms of inhibition were investigated and we reached the following conclusions. First, the oxidation of essential thiol groups is not involved because the thiol protecting and reducing agent DTT was ineffective in counteracting inhibition by glucose and fructose. Consequently, in short-term in vitro models of glycosylation, the formation of free radicals that accompanies the glycosylation of amino groups and sugar autoxidation (Namiki et al., 1977), which could potentially oxidize essential enzyme –SH groups, seems to play no significant role in δ-ALA-D inhibition. This was further confirmed by the failure of ebselen, an antioxidant (Mugesh et al., 2001; Nogueira et al., 2004), to modify inhibition by glucose or fructose. Second, high concentrations of compounds containing amino groups such as lysine and t-butylamine were similarly ineffective. This suggests that the amino groups of these low-molecular weight amines are much less reactive than the lysyl amino group at the active center of blood δ-ALA-D or that glucose does not react with this residue in the active center of the enzyme. However, it must be emphasized that amino groups in free amino acids react rapidly with reducing sugars only under extreme conditions of temperature and pH (Namiki et al., 1977), but in vitro glycosylation of hemoglobin occurs within a few hours of exposure to high glucose concentrations (Caballero et al., 1998). In a previous study, we observed that in vivo, hemoglobin glycosylation correlates with δ-ALA-D inhibition in mice (Folmer et al., 2003).

Consequently, we proposed that the reducing sugar may inhibit δ-ALA-D by binding to an enzyme–substrate intermediate. One potential candidate is the amino group of the first δ-ALA molecule bound to δ-ALA-D. In fact, we observed that pre-incubation with glucose followed by its withdrawal from the medium, before the enzyme reaction was started with δ-ALA, did not result in enzyme inhibition. If glucose inhibited δ-ALA-D by interacting with its active center lysil residue, one would expect that inhibition could not be completely overcome by removing glucose from the reaction medium after a previous pre-incubation with the enzyme. The relationship of these artificial conditions (high reducing sugar concentrations) to the inhibition of δ-ALA-D in vivo after dietary or pathological hyperglycemia deserves further investigation (Gugliucci and Allard, 1996; Al-Zuhair and Mohamed, 1998; Montenegro et al., 2002). However, the fact that δ-ALA-D inhibition promoted by hyperglycemia in vivo (Folmer et al., 2002; 2003) is not reversed by DTT suggests that the mechanism underlying δ-ALA-D inhibition both in vitro and in vivo does not involve –SH group oxidation.

Acknowledgements

Financial support by FAPERGS, CAPES and CNPq is gratefully acknowledged.

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Received 8 June 2004/1 December 2004; accepted 30 March 2005

doi:10.1016/j.cellbi.2005.03.017


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
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