Biochimica et Biophysica Acta 924 (1987) 557-561 Elsevier
Irreversible inhibition of hexosaminidase C by medium-chain monocarboxylic acids and Triton X-100 Michel Hardy, Robert Salvayre, Arlette Maret and Louis Douste-Blazy Laboratoire de Biochimie et INSERM Unitd 101, Facultd de Mddecine, Toulouse (France)
(Received 15 December 1986)
Key words: Hexosaminidase C; Monocarboxylic acid; Triton X-100; Hydrophobic interaction
The neutral fl-N-acetylhexosaminidase (hexosaminidase C) from human brain was partially purified (separated from lysosomal ~-N-acetylhexosaminidases by chromatography on a Con A-Sepharose column). Hexosaminidase C was inhibited by medium-chain fatty acids (monocarboxylic acids with chain-length between C~ and C9) , whereas shorter-chain monocarboxylic acids showed no inhibitory effect. Studies on the inhibition mechanism showed an irreversible and pH-dependent inhibition which progresses with time and which is not reversed by the removal of fatty acids (by Bio-Beads SM-2). Similar inhibitory effects were also obtained using Triton X-100 (but not with homologous alkylamines). These results suggest that the hexosaminidase C inactivation is related to the hydrophobic properties of the inhibitor which acts as a denaturing agent mainly at acidic pH. The possibility has been discussed that this inactivation effect of monocarboxylic acid on hexosaminidase C could constitute a molecular model of the toxicity of mediumchain-length fatty acids.
Two major groups of hexosaminidase (fl-Nacetylhexosaminidase, EC 18.104.22.168) are present in mammalian tissues: the lysosomal acidic hexosaminidases and the neutral cytosolic hexosaminidase C. The ubiquitous lysosomal hexosaminidases A and B [1-4], I and P [5,6], M  and S  are involved in the pathogenesis of GM2-gangliosidoses [9,10]. Hexosaminidases A and B, the most extensively studied enzymes [9-13], showed hydrophobic propertie s of the fl subunit enzymatic Abbreviations: MU-GlcNAc, 4-methylumbelliferyl-2-acetamido-2-deoxy-fl-D-glucopyranoside; GlcNAcLone, 2acetamido-2-deoxy-o-gluconolactone; Cn, monocarboxylic acids with n carbons, number (1 ~ n ~<9); hexosaminidase C, the neutral /LN-acetylhexosaminidase; GA2, N-acetylgalactosaminyl-fll -~ 4-galactosyl-fll ~ 4-glucosyl-fll -~ 1-ceramide. Correspondence: Dr. Robert Salvayre, Laboratoire de Biochimie, Facult~ de M~decine, 37 all6es Jules Guesde, 31073 Toulouse Cedex, France.
site as demonstrated by the binding of glycolipids (GA2) [9-13] and the competitive inhibitory effect of medium-chain fatty acids (Hardy et al., unpublished data). The cytosolic hexosaminidase C is not involved in gangliosidosis pathogenesis [14,15]; its physiological function and its natural substrate remain unknown [16-19]. Hexosaminidase C exhibits hydrophobic properties as demonstrated by the binding to octyl- and phenyl-Sepharose and elution by Triton X-100 . In order to state more precisely these hydrophobic properties of hexosaminidase C and to compare them with those previously reported concerning the lysosomal enzymes, we have studied the interaction of monocarboxylic acids and Triton X-100 with hexosaminidase C. We report in this paper the irreversible inhibitory effect of these amphiphilic compounds on hexosaminidase C and we discuss the inactivation mechanism and its possible occurrence during physiopathological manifestations.
0304-4165/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
Enzyme preparation was performed by the following procedure: 20% homogenate of human neonate brain (frozen at - 7 0 ° C until use) was prepared by sonication in distilled water (three times for 15 s). After centrifugation at 220 000 X g for 1 h, hexosaminidase C was partially purified on a Con A-Sepharose column (7 x 1 cm) (Pharmacia, Uppsala, Sweden); hexosaminidase C was not bound by this lectin, in contrast to lysosomal hexosaminidases. This preparation contained a very small amount of lysosomal hexosaminidases, as previously demonstrated . /3-N-Acetylhexosaminidase activity was determined by the standard enzyme assay which contained 1.2 mM of the substrate, MU-GlcNAc (Koch-Light, Colnbrook, U.K.), and 0.4 M citrate/phosphate buffer (pH 6.0). The reaction mixture was incubated at 37 °C for 20 min (enzyme reaction was linear up to 30 min). The reaction was stopped and the liberated methylumbelliferone was fluorometrically determined as previously described . Inhibition experiments were performed by preincubating hexosaminidase C in 0.15 mM citrate-phosphatebuffered medium with monocarboxylic acid (Fluka, Buchs, Switzerland) or Triton X-100 at variable concentration. Other experiments were performed varying the preincubation time or pH of the medium for variable preincubation times. Immediately after the preincubation, the buffered substrate was added and residual enzyme activity was determined in standard conditions. Enzyme activity was expressed as percent of the initial activity. For fatty acid removal by Bio-Beads SM-2 (Bio-Rad, Richmond, CA, U.S.A.), the enzyme was preincubated with inhibitor for 15 min at 37 °C, then Bio-Beads were added (batch procedure) and the mixture was vigorously shaken for 15 min at 4 ° C [22,23]. After removing monocarboxylic acid, the residual enzyme activity was determined by standard enzyme assay. The removal of monocarboxylic acid by Bio-Beads was tested by using radiolabelled fatty acid, [1-~4C]octanoic acid (NEN, Paris, France). The inhibition of hexosaminidase C by monocarboxylic acids is reported in Figs. 1 and 2; as shown in Fig. 1A, hexosaminidase C was not inhibited by acetate, propionate or butyrate. In contrast, monocarboxylic acids with chain length higher than five carbons showed a clear inhibitory
Fig. 1. Inhibitory effect of monocarboxylic acids on hexosaminidase C: concentration, carbon chain length and pH dependence. (A) Hexosaminidase C solution (in 0.15 mM citrate/phosphate buffer pH 5.0) was preincubated at 37°C for 15 min with variable concentrations (between 0 and 20 mM) of monocarboxylic acids (from formic C 1 to nonanoic C9 acids). Immediately after the preincubation, the residual enzyme activity was determined using the standard enzyme assay. Inset: enzyme inhibition at fixed concentration of monocarboxylic acid (10 mM) vs. carbon chain length of monocarboxylic acid (C). Other conditions (temperature and time of preincubation) as described above. (B) Hexosaminidase C in 0.15 mM citrate/phosphate buffer pH 5.0 ( ) or pH 6.0 (. . . . . . ) was preincubated at 37°C for 15 min with variable concentrations (between 0 and 20 mM) of pentanoic acid, C 5 (zx), and hexanoic acid, C6 (l), or without monocarboxylic acid (O). After preincubation, the residual enzyme activity was determined using standard assay.
effect, which increased with monocarboxylic acid concentration. Moreover, a stronger inhibitory effect was observed with the longer-chain monocarboxylic acids (as demonstrated by the 50% inhibition which occurred under experimental conditions used in Fig. 1A, at about 30 mM for C 5, at 7.5 mM for C 7, and at 3.5 for C9). It is noteworthy that the inhibitory effect of mono-
lb 2'0 30 4b min. preincubation time
Fig. 2. Irreversible inactivation of hexosaminidase C by medium-chain monocarboxylic acids. Hexosaminidase C solution (in 0.15 mM citrate/phosphate buffer pH 5.0) was preincubated at 37 ° C for variable periods of time with 10 mM of pentanoic acid, C 5 (zx), or nonanoic acid, C 9 (11), or without monocarboxylic acid (O). Aliquots of the preincubation mixture were taken up at the indicated time and the residual enzyme activity was determined in the standard conditions immediately after the end of the preincubation period.
carboxylic acids occurred at concentrations lower (e.g., C 5, 10 mM in Fig. 2) than the critical micelle concentration, evaluated at around 250 mM for C5, 30 mM for C6, 20 mM for C7 and 10 mM for C 8 by the dye spectral shift method of Bonsen et al. . This suggested that micelles are not required for obtaining an inhibitory effect. Preincubation of hexosaminidase C with monocarboxylic acids (the residual activity being determined immediately after the preincubation by the standard assay) demonstrated that the inhibition is time-dependent (Fig. 2). This suggests an irreversible inhibitory effect (or inactivation) of monocarboxylic acids on hexosaminidase C. Moreover, the rate of inactivation was dependent on pH, as shown in Fig. lB. In order to demonstrate the irreversibility suggested by the above-reported experiments, we tested the effect of removing inhibitors by Bio-Beads: monocarboxylic acid (C8) was almost completely removed (97%) from the incubation medium by Bio-Beads, but we observed no reactivation of hexosaminidase C. We have shown that the low amount (3%, i.e., 0.3 mM) of the non-removed C 8 induced no appreciable inhibition when it was used in the inhibition standard conditions (see Fig. 1A). This experiment strongly supports the hypothesis of the irreversible inactivation of the enzyme by monocarboxylic acids. On the other hand, we have tested the effect of GlcNAcLone, a competitive inhibitor of
hexosaminidases : the time-dependent inactivation of hexosaminidase C by monocarboxylic acids was not affected by the presence of GlcNAcLone, thus this competitive inhibitor showed no protective effect against the inactivation (data not shown). We observed in preliminary experiments a non-negligible inhibitory effect of Triton X-100, although it has been used by several authors [19,24,25] to prepare hexosaminidase C. Similarly to monocarboxylic acids, Triton X-100 induced a time-dependent inactivation of hexosaminidase C (Fig. 3C). Moreover, this inactivation was also pH-dependent (Fig. 3A and B): Triton X-100 induced a shift of stability curves towards the most alkaline pH; at pH 5.0, the presence of a low concentration of Triton X-100 was very critical, since the enzyme showed a good stability in buffer but was rapidly inactivated when 0.1% Triton X-100 was added. The mechanism of interaction of monocarboxylic acids with hexosaminidase C probably does not involve an ionic interaction, since the neutral surfactant, Triton X-100, exhibited the same in-
0 10 20 3o 4brain preincubation time
Fig. 3. Inhibition of hexosaminidase C by Triton X-100. Each experiment was performed using preincubation of the hexosaminidase C solution (in 0.15 mM citrate-phosphate at indicated pH) with or without Triton X-100 (Tx 100) in variable conditions. (A) Preincubation at pH varying between 4.0 and 7.0 without (O) or with 0.2 g / l (ill) or 1 g/1 (A) of Triton X-100 for 15 rain at 37 o C. (B) Preincubation at pH 5.0 (~,) or pH 6.0 (©) for 15 min at 37 °C with variable concentrations of Triton X-100. (C) Preincubation at pH 5.0 without (O) or with 0.2 g / l Triton X-100 (11) for variable periods of time. In each case, the preincubation was followed by determination of residual enzyme activity using the standard enzyme assay.
activating effect as medium-chain fatty acids, and also because of the stronger inhibitory effect of these monocarboxylic acids at lower pH, i.e., at lower concentration of ionized monocarboxylic acids (see Fig. 1B). Moreover, alkylamines with 6, 8 or 10 carbons (which are ionized in our experimental conditions) showed no inhibitory effect at the tested concentrations (between 0 and 10 mM). A mechanism involving hydrophobic interaction during the inactivation of hexosaminidase C by medium-chain fatty acids is more probable because the maximal inactivating effect was observed with the longest-chain monocarboxylic acids tested. This is also consistent with the higher inactivating effect observed when pH decreased; at lower pH, the hydrophobic properties of monocarboxylic acids increased. Moreover, such a hydrophobic interaction is in agreement with the existence of the hydrophobic properties of hexosaminidase C reported by Overdijk et al. . Experiments of pH-inactivation without and with monocarboxylic acids or Triton X-100 (demonstrating a shift of stability curves towards less acidic pH in the presence of detergents) show that pH-denaturation and inactivation by hydrophobic interaction are two additive mechanisms which probably act by destabilizing the protein conformation; pH-inactivation probably by breaking ionic or other stabilizing bonds, and hydrophobic inactivation by laying open the core of hexosaminidase C. This probably leads to an irreversible change in hexosaminidase C conformation, as suggested by experiments in which fatty acids were removed by Bio-Beads. In conclusion, these results demonstrating the inactivating effect of medium-chain-length fatty acids and Triton X-100 could be of practical and physiopathological relevance. From a practical point of view, the stability of hexosaminidase C decreased at acidic pH and in the presence of detergent (e.g., Triton X-100). Therefore, in contrast to several authors [19,26,27], we recommend the use of a neutral pH medium for purifying or studying the enzyme and avoidance of the use of detergent. We do not know what the physiological role of hexosaminidase C is. However, various pathophysiological manifestations  seem to be due to the toxicity of high levels of short- and medium-chain
monocarboxylic acids. At the present time, no general mechanism explains their toxicity. Although our study was achieved in vitro, we may speculate that the irreversible inhibition of hexosaminidase C described here could constitute a molecular model of the in vivo toxicity of medium-chain monocarboxylic acids on enzymes. Thanks are due to Mrs Y. Jonquirre for correcting the English manuscript. This work was supported by grants from the University Paul Sabatier (Toulouse) and from INSERM (R~seau No. 850024).
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