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A radiometric assay for sialidase acting on ganglioside GD1a
ANALYTICAL
BIOCHEMISTRY
78, 333-339 (1977)
A Radiometric Assay for Sialidase Acting on Ganglioside GDla J. SCHRAVEN,C.&P,G.
NOWOCZEK,AND
K. SANDHOFF
Max-Planck-lnstitut fir Psychiatric. Neurochemische Abteilung, Kraepelinstra@ 2, 8000 Miinchen 40, West Germany Received April 1, 1976; accepted November 11, 1976 A sensitive radiometric assay has been designed for measuring the activity of sialidase extracted from calf brain using as substrate tritium-labelled ganghoside Gn,, (61.5 &i/~mol). The substrate was labelled by saturating the double bond of the sphingosine moiety with tritium gas in the presence of platinum catalyst. After incubation with sialidase, the substrate, [3H]ganglioside G,,,, and its degradation product, [3H]ganglioside GMl, are separated by thin-layer chromatography on silica gel 60 and their radioactivities are measured in a liquid scintillation counter. Using the tritiated substrate less than 0.04 nmol of product can be detected. The standard error of the method was found to be 26%. Under optimal reaction conditions the test is applicable for a concentration of protein up to 120 &200 ~1 of incubation mixture.
Most of the sialidase activity of the brain is membrane bound with the highest specific activity in neuronal plasma membranes (l-3). In these membranes sialidase occurs together with its endogenous substrates: gangliosides (l-4) and possibly glycoproteins (5,6). Sialidase of brain membranes is usually determined by measuring the amount of sialic acid produced in the enzymatic reaction by the thiobarbituric acid method (7). However, this method frequently does not give reliable results with crude enzymes, which contain a variety of interfering substances (7- 13). In these cases, isolation of the free sialic acid must be performed prior to its assay (8). But when applied to crude particulate enzyme preparations of low activity, sialic acid released often falls close to the lower limit of sensitivity of the calorimetric method (2-5 nmol; refs. 1,7,14). Therefore, a more sensitive radiometric assay has been developed: Less than 0.04 nmol of product formed can be quantified. The test uses a natural substrate, ganglioside GD1,,’ labelled in its sphingosine portion. This paper describes its application to the measurement of calf brain sialidase activity. ’ Abbreviations used: ganglioside G,,,, AcNeu-(a2 + 3) Gal-(/31 ---f 3)GalNAc(PI -+ 4) [AcNeu-(cY2 + 3)] Gal@1 -+ 4)Glc-(PI -1’) ceramide; gangtioside GM,, Gal(pl ---) 3)GalNAc-(PI + 4) [AcNeu-(a2 + 3)]Gal-(PI --) 4)Glc-(p I + 1’) ceramide; TCA, trichloroacetic acid. 333 Copyright 0 1977 by Academic Prerr, Inc. All rights of reproduction in any form reserved.
ISSN ooO3-2697
334
SCHRAVEN
MATERIALS
ET
AL.
AND METHODS
Materials Solvents, sucrose, and the other chemicals used were all reagent grade and obtained from Merck (Darmstadt). Silica gel for column chromatography and plastic-backed sheets of silica gel 60 for thin-layer chromatography from Merck Darmstadt were used. Gangliosides from bovine brain and Unisolve I were purchased from Koch-Light (U.K.). Tritium gas was obtained from the Radiochemical Centre, Amersham (U.K.), Triton X-100 from Serva, Heidelberg, and neuraminidase (C/. perfringens) from Boehringer , Mannheim . Preparation
of [3H]Ganglioside
G,,,
Ganglioside GDla was prepared from a mixture of bovine brain gangliosides according to the method described by Svennerholm (15). The purified ganglioside G,,, (300 mg) was dissolved in 3 ml of dimethyl sulfoxide; 8 ml of benzene and approximately 200 mg of platinum oxide were added. Platinum oxide was reduced by a brief exposure of the mixture to hydrogen gas at room temperature. The reaction mixture was shaken at 40°C for 24 hr at atmospheric pressure in the presence of hydrogen containing 10 Ci of tritium gas. (The labelling procedure can also be performed by the Radiochemical Centre, Amersham, England, if sufficient safety facilities are not otherwise available.) After incubation, the mixture was filtered and the solution was evaporated under a stream of nitrogen. In order to remove labile radioactivity, the residue was redissolved in 100 ml of chloroform-methanol (l:l, v/v) and evaporated to dryness. This exchange procedure was repeated six times until the radioactivity of the residue reached a constant value. The labelled product was purified by column chromatography on silica gel according to the method of Svennerholm (IS). The isolated [3H]ganglioside G,,, (88 mg) had a specific radioactivity of 192 &i/pmol and contained 33.1% (w/w) sialic acid (7). After hydrolysis, 98% of its total radioactivity appeared in the sphingosine fraction. The ganglioside appeared to be homogeneous as checked by several thin-layer systems using silica gel plates and the following solvent mixtures: (i) n-butanol-pyridine-0.55% KC1 (6:3:2, v/v/v; Fig. 1); (ii) chloroform-methanol-2.5 N ammonia solution (60:40:9, v/v/v); (iiia) chloroform-methanol-water (14:6: 1, v/v/v); after drying rechomatographed with (iiib) chloroform-methanol-2.5 N ammonia solution (60:38:8, v/v/v). Protein
Determination
Protein was measured by the method of Lowryet al. (16) with crystalline bovine serum albumin as standard. Protein samples containing Triton
A RADIOMETRIC
33s
ASSAY FOR SIALIDASE
--I t ;tart
Solvent
t
Front
FIG. 1. Thin-layer chromatogram of the incubation mixture. Substrate, 16.2 nmol of [3H]ganglioside GnIa; enzyme, (. . . .) Triton X-100 extracted enzyme, ( ) Triton X-100 extracted enzyme heated for 10 min at 100°C. For the purpose of illustrating the degree of separation of substrate and product, and the absence of a product in the blank incubations with denatured enzyme, a high concentration of enzyme (1.5 mg of protein) was used in this experiment. In this case the incubation was performed in the presence of 0.7% Triton X-100, which was necessary to maintain high enzyme reactivity at the protein concentration used. For other assay conditions, see Materials and Methods.
X-100 were assayed in the presence of sodium dodecyl sulfate (0.5 to 1%) which gave a linear relationship between protein concentration and optical density. Sialidase Preparations Fresh calf cerebra were obtained from the local slaughter house. Starting with a 10% homogenate of cortical grey matter in 0.32 M sucrose, a crude microsomal fraction was sedimented from a 13,OOOg (30 min) supernatant by centrifugation at 150,OOOg for 60 min. The microsomal pellet was washed twice with 10 mM Tris buffer, pH 7.2 in order to remove the sucrose. Aliquots were stored in 10 mM Tris buffer (26 mg of protein/ml) at -20°C. An enzyme extract was prepared by treating the microsomal fraction (10 mg of protein/ml) in 10 mM Tris buffer, pH 7.2 with 0.2% Triton X- 100 at 4°C. The mixture was sonicated twice for 10 set, stirred for 1 hr at 4°C and centrifuged for 1 hr at 150,OOOg. The supernatant, referred to as “Triton extract,” was used as the enzyme preparation. Assay of Sialidase
with [3H]Ganglioside
G,,,
Incubation conditions. [3HJGanglioside GD,, (16.2 nmol in chloroform/ methanol, 2: 1, v/v) which had been diluted with unlabelled ganglioside GoI,
336
SCHRAVEN
ET AL.
to give a specific activity of 61.5 @Zi/pmol was dried under a stream of nitrogen. The residue was resuspended by sonicating at 4°C in acetate buffer and, if required, in Triton X- 100. The final incubation volume of 200 ~1 contained, under standard conditions, Triton extract (up to 120 pg of protein) or the microsomal preparation (up to 150 pg of protein) in a final buffer concentration of 0.15 M, pH 4.2. The incubation mixture was shaken intensively using a Vortex mixer and incubated for 40 min at 37°C and the reaction was stopped by cooling down to 0°C. Blanks were run with enzyme preparations inactivated by heating for 10 min at 100°C or by the addition of TCA at room temperature to give a final concentration of 1.5%, which did not change the pH value of the incubation mixture. Analysis. Aliquots of the incubation mixtures (40 ~1) were applied to thin layers of silica gel 60 and cochromatographed with standards of ganglioside GD,, and ganglioside G,,. For the separation of substrate and product the solvent system n-butanol-pyridine-0.55% KC1 (6:3:2, v/v/v) was used. In some cases the thin layer sheet was dried by a stream of air for about 6 hr and rechromatographed with chloroform containing 0.38% (v/v) acetic acid in order to remove the pyridine and increase the counting efficiency of the radioscanner. The substrate and product spots, separated by at least 3-5 cm, were localized using a radio scanner (Fa. Berthold, Germany) (Fig. 1). Substrate and product spots were cut out with scissors and put into plastic vials containing 15 ml of Unisolve I. After adding 4.5 ml of water and shaking intensively, a stable gel formed. The radioactivity measured in a liquid scintillation counter (Mark II, Nuclear Chicago) increased linearly with the amount of gangliosides tested up to 200,000 cpm. Formation of product is calculated from its percentage of total radioactivity. Determination
of Sialic Acid Content of Enzyme Preparations
Microsomal and supernatant enzyme preparations were hydrolysed with 0.05 N sulfuric acid at 80°C for 90 min and centrifuged (56OOg, 5 min). The sialic acid content was determined in the supernatant according to the procedure of Warren (7). The amounts of the individual gangliosides were determined by densitometry after separation by thin-layer chromatography as described previously (17,18). RESULTS
AND DISCUSSION
Membrane-bound sialidase degrades a variety of sialic acid-containing glycosphingolipids (14,19,20). Among the natural substrates, ganglioside G Dla is a prominent and readily degradable one (14,19,20). In order to develop a sensitive assay of the enzyme activity. it was labelled by saturating the double bond in the sphingosine moiety with a mixture of tritium and hydrogen gas. The purified [3H]ganglioside Go,, (192 PCilpmol)
A RADIOMETRIC
TABLE END~GENOUS
SUBSTRATES
FOR SIALIDASE ENZYME
Total NeuAc
337
ASSAY FOR SIALIDASE 1 IN MICROSOMES
AND
SUPERNATANT
(nmoVmg of protein) Total ganglioside NeuAc
Microsomes
69
31
Supernatant enzyme (0.2% Triton X-100)
19
5
Ganglioside GD,, 7 0.5
Ganglioside-bound NeuAc susceptible for sialidase 16 2
appeared to be radiochemically homogenous (Fig. 1) and contained 98% of its radioactivity in the dihydrosphingosine moiety. As shown in Table 1, microsomes and, to a smaller extent, the Triton X- 100 extract also contain endogenous ganglioside substrates, which dilute the added [3H]ganglioside GD,, in the incubation mixture, but this dilution is negligible in the case of Triton X-100 extract. The exogenous substrate (16.2 nmol of [3H]ganglioside G,,,) is diluted only by 0.04 to 0.24 nmol of ganglioside-bound sialic acid accessible for sialidase (Table 1) when 20 to 120 pg of enzyme protein were used for the test. On the other hand, microsomes containing 100 pg of protein add 1.6 nmol of lipid-bound sialic acid accessible to sialidase, which dilute the added substrate by 10%. This dilution can also be avoided by preincubation of particulate enzyme preparation (14,21). Sialidase splits [3H]ganglioside G Dla into sialic acid and [3H]ganglioside GMI, the amount of which can be measured reproducibly and sensitively in a liquid scintillation counter after separation from the substrate by thin-layer chromatography. The solvent system employed allows a complete separation of both substrate and product without any overlapping (Fig. 1). The assay procedure employed yields essentially a linear increase of radioactivity counted with the amount of ganglioside applied to the thin-layer plate up to 5 nmol. Down to 0.04 nmol of [3H]ganglioside GM1 formed can be quantitatively detected under the conditions used. The standard deviation of the assay lies between 5 and 6% of the mean within the range of 0.5 to 5 nmol of [3H]ganglioside GM1 formed. The radioactive test showed that sialidase is completely inactivated by the addition of 1.5% of TCA at room temperature as well as by heating for 10 min at 100°C (Fig. 1). Therefore, TCA-inactivated microsomes and their Triton X-100 extracts have also been used successfully for blank incubations when applying the calorimetric thiobarbituric acid test (7) to the determination of sialidase activity in the microsomal preparation. Activation of particulate sialidase has been reported over a narrow range by Triton X-100 (14,20) and by Triton CF-54 (14). The addition of 0.1% Triton X-100 to the incubation mixture results in an optimal stimulation for
338
SCHRAVEN
ET AL.
FIG. 2. Effect of increasing concentrations of Triton X-100 on the activity of sialidase from calf brain microsomes toward rH]ganglioside GoI,. For assay conditions, see Materials and n , 259 pg of microsomal protein; 0 0,129 pg of microsomal protein. Methods. n -
a protein concentration up to 150 pg, whereas higher detergent concentrations exert an inhibitory effect (Fig. 2). Lower concentrations essentially result in a nonlinear protein dependence of the enzyme activity. On the other hand, 0.1% Triton X- 100 does not fully activate higher protein concentrations. In this case, higher detergent concentrations would be needed. A presupposition for a quantitative enzyme assay is a linear increase of activity with the amount of enzyme protein added. This seems to be valid for the particulate as well as for the extracted enzyme only under special conditions, i.e., in the presence of 0.1% Triton X-100 and up to 120 pg of crude enzyme protein added. For the microsomal preparation, a less than linear increase of activity is observed in the absence of detergent. Sialidase.was active in the range between pH 4 and 5 with an optimum at pH 4.2 to 4.5. At pH 4.2, product formation increases linearly with time up to 40 min and then levels off. This seems to be due to the instability of the enzyme preparation. Whereas the extracted enzyme is stable over several weeks at -20°C its half-life in the absence of exogenous substrate at 4°C is about 20 hr and at 37°C only about 12 min. The physical state of substrate and enzyme in the detergent-containing incubation mixture is unknown. Therefore, under standard conditions, i.e., in the presence of 0.1% Triton X-100, only an apparent MichaelisMenten constant of 84 PM can be determined and it is not surprising that the values vary for different Triton X-100 concentrations used in the incubation mixtures. In this test, substrate concentrations on the order of the apparent K, value give rise to a linear time and protein dependence.
A RADIOMETRIC
ASSAY FOR SIALIDASE
339
Concentrations higher than twice the K, result in an apparent substrate inhibition. Despite the complex kinetics of sialidase, the radioactive test can be used under the defined conditions as a sensitive enzyme assay. The assay has been found to be suitable for the determination of other sialidases such as neuraminidase from Cl. perfringens. Recently, a radiometric assay for sialidase has been described (22) using a water soluble substrate (a-o-N-acetylneuraminosyl-(2 + 3’) lactit [3H]ol) in the absence of detergents. This test has been applied to a variety of neuraminidases and, in comparison with the test using a natural, lipid (amphiphilic) substrate, might facilitate the elucidation of the properties of membrane-bound sialidases. REFERENCES 1. Schengnmd, C. L., and Rosenberg, A. (197O)J. Bib!. Chem. 245, 61%-6200. 2. Tettamanti, G., Morgan, J. G., Gombos, G., Vincendon, G., and Mandel, P. (1972) Brain Res. 47, 515-518. 3. Editorial Note (1974) in Brain Res. 66, 557. 4. Wiegandt, H. (1%7) .I. Neurochem. 14, 671-674. 5. Roukema, P. A., and Heijlman, J. (1970) J. Neurochem. 17, 773-780. 6. Heijlman, J., and Roukema, P. A. (1972) J. Xeurochem. 19,2567-2575. 7. Warren, L. (1959)J. Biol. Chem. 234, 1971-1975. 8. Horvat, A., and Touster, 0. (1968) J. Biol. Chem. 243, 4380-4390. 9. Weissbach, A., and Hurwitz, J. (1959) J. Biol. Chem. 234, 705-709. 10. Preiss, J., and Ashwell, G. (1%2) J. Biol. Chem. 237, 309-316. 11. Carubelli, R., Bhavanandan, V. P., and Gottschalk, A. (1%5) Biochim. Biophys. Acra 101, 67-82.
12. 13. 14. 15.
Eichberg, J., and Karnovsky, M. L. (1966)Biochim. Biophys. Acta 124, 118-124. Brown, C. R., Srivastava, P. N., and Hartee, E. F. (1970) Biochem. J. 118, 123-133. Ghman, R., Rosenberg, A., and Svennerholm, L. (1970) Biochemistry 9, 3774-3782. Svennerholm, L. (1970) in Methods of Carbohydrate Chemistry (Whistler, R. L., and Wolfrom, M. L., eds.), Vol. VI, pp. 464-474, Academic Press, New York. 16. Lowry, 0. H., Rosebrough. N. J.. Farr, A. L., and Randall, R. J. (1951)J. Biol. Chem. 193, 265-275.
17. Sandhoff, K., Harzer, K., and Jatzkewitz, H. (1968) Hoppe-Seyler’s 2. Physiol. Chem. 349, 283-287. 18.
Harzer, K., Wassle, W., Sandhoff, K., and Jatzkewitz,
H. (1968) Z. Anal. Chem. 243,
527-536. 19. Leibowitz, Z., and Gatt, S. (1968) Biochim. Biophys. Acta 152, 136-143. 20. Tettamanti, G., Cestaro, B., Lombardo, A., Preti, A., Venerando, B., and Zambotti. V. (1974) Biochim. Biophys. Acra 350, 415-424. 21. Preti, A., Lombardo, A., and Tettamanti, G. (1970) Ital. J. Biochem. 19, 371-385. 22. Bhavanandan, V. P., Yeh, A. K., and Carubelli, R. (1975)Anal. Biochem. 69,385-394.
BIOCHEMISTRY
78, 333-339 (1977)
A Radiometric Assay for Sialidase Acting on Ganglioside GDla J. SCHRAVEN,C.&P,G.
NOWOCZEK,AND
K. SANDHOFF
Max-Planck-lnstitut fir Psychiatric. Neurochemische Abteilung, Kraepelinstra@ 2, 8000 Miinchen 40, West Germany Received April 1, 1976; accepted November 11, 1976 A sensitive radiometric assay has been designed for measuring the activity of sialidase extracted from calf brain using as substrate tritium-labelled ganghoside Gn,, (61.5 &i/~mol). The substrate was labelled by saturating the double bond of the sphingosine moiety with tritium gas in the presence of platinum catalyst. After incubation with sialidase, the substrate, [3H]ganglioside G,,,, and its degradation product, [3H]ganglioside GMl, are separated by thin-layer chromatography on silica gel 60 and their radioactivities are measured in a liquid scintillation counter. Using the tritiated substrate less than 0.04 nmol of product can be detected. The standard error of the method was found to be 26%. Under optimal reaction conditions the test is applicable for a concentration of protein up to 120 &200 ~1 of incubation mixture.
Most of the sialidase activity of the brain is membrane bound with the highest specific activity in neuronal plasma membranes (l-3). In these membranes sialidase occurs together with its endogenous substrates: gangliosides (l-4) and possibly glycoproteins (5,6). Sialidase of brain membranes is usually determined by measuring the amount of sialic acid produced in the enzymatic reaction by the thiobarbituric acid method (7). However, this method frequently does not give reliable results with crude enzymes, which contain a variety of interfering substances (7- 13). In these cases, isolation of the free sialic acid must be performed prior to its assay (8). But when applied to crude particulate enzyme preparations of low activity, sialic acid released often falls close to the lower limit of sensitivity of the calorimetric method (2-5 nmol; refs. 1,7,14). Therefore, a more sensitive radiometric assay has been developed: Less than 0.04 nmol of product formed can be quantified. The test uses a natural substrate, ganglioside GD1,,’ labelled in its sphingosine portion. This paper describes its application to the measurement of calf brain sialidase activity. ’ Abbreviations used: ganglioside G,,,, AcNeu-(a2 + 3) Gal-(/31 ---f 3)GalNAc(PI -+ 4) [AcNeu-(cY2 + 3)] Gal@1 -+ 4)Glc-(PI -1’) ceramide; gangtioside GM,, Gal(pl ---) 3)GalNAc-(PI + 4) [AcNeu-(a2 + 3)]Gal-(PI --) 4)Glc-(p I + 1’) ceramide; TCA, trichloroacetic acid. 333 Copyright 0 1977 by Academic Prerr, Inc. All rights of reproduction in any form reserved.
ISSN ooO3-2697
334
SCHRAVEN
MATERIALS
ET
AL.
AND METHODS
Materials Solvents, sucrose, and the other chemicals used were all reagent grade and obtained from Merck (Darmstadt). Silica gel for column chromatography and plastic-backed sheets of silica gel 60 for thin-layer chromatography from Merck Darmstadt were used. Gangliosides from bovine brain and Unisolve I were purchased from Koch-Light (U.K.). Tritium gas was obtained from the Radiochemical Centre, Amersham (U.K.), Triton X-100 from Serva, Heidelberg, and neuraminidase (C/. perfringens) from Boehringer , Mannheim . Preparation
of [3H]Ganglioside
G,,,
Ganglioside GDla was prepared from a mixture of bovine brain gangliosides according to the method described by Svennerholm (15). The purified ganglioside G,,, (300 mg) was dissolved in 3 ml of dimethyl sulfoxide; 8 ml of benzene and approximately 200 mg of platinum oxide were added. Platinum oxide was reduced by a brief exposure of the mixture to hydrogen gas at room temperature. The reaction mixture was shaken at 40°C for 24 hr at atmospheric pressure in the presence of hydrogen containing 10 Ci of tritium gas. (The labelling procedure can also be performed by the Radiochemical Centre, Amersham, England, if sufficient safety facilities are not otherwise available.) After incubation, the mixture was filtered and the solution was evaporated under a stream of nitrogen. In order to remove labile radioactivity, the residue was redissolved in 100 ml of chloroform-methanol (l:l, v/v) and evaporated to dryness. This exchange procedure was repeated six times until the radioactivity of the residue reached a constant value. The labelled product was purified by column chromatography on silica gel according to the method of Svennerholm (IS). The isolated [3H]ganglioside G,,, (88 mg) had a specific radioactivity of 192 &i/pmol and contained 33.1% (w/w) sialic acid (7). After hydrolysis, 98% of its total radioactivity appeared in the sphingosine fraction. The ganglioside appeared to be homogeneous as checked by several thin-layer systems using silica gel plates and the following solvent mixtures: (i) n-butanol-pyridine-0.55% KC1 (6:3:2, v/v/v; Fig. 1); (ii) chloroform-methanol-2.5 N ammonia solution (60:40:9, v/v/v); (iiia) chloroform-methanol-water (14:6: 1, v/v/v); after drying rechomatographed with (iiib) chloroform-methanol-2.5 N ammonia solution (60:38:8, v/v/v). Protein
Determination
Protein was measured by the method of Lowryet al. (16) with crystalline bovine serum albumin as standard. Protein samples containing Triton
A RADIOMETRIC
33s
ASSAY FOR SIALIDASE
--I t ;tart
Solvent
t
Front
FIG. 1. Thin-layer chromatogram of the incubation mixture. Substrate, 16.2 nmol of [3H]ganglioside GnIa; enzyme, (. . . .) Triton X-100 extracted enzyme, ( ) Triton X-100 extracted enzyme heated for 10 min at 100°C. For the purpose of illustrating the degree of separation of substrate and product, and the absence of a product in the blank incubations with denatured enzyme, a high concentration of enzyme (1.5 mg of protein) was used in this experiment. In this case the incubation was performed in the presence of 0.7% Triton X-100, which was necessary to maintain high enzyme reactivity at the protein concentration used. For other assay conditions, see Materials and Methods.
X-100 were assayed in the presence of sodium dodecyl sulfate (0.5 to 1%) which gave a linear relationship between protein concentration and optical density. Sialidase Preparations Fresh calf cerebra were obtained from the local slaughter house. Starting with a 10% homogenate of cortical grey matter in 0.32 M sucrose, a crude microsomal fraction was sedimented from a 13,OOOg (30 min) supernatant by centrifugation at 150,OOOg for 60 min. The microsomal pellet was washed twice with 10 mM Tris buffer, pH 7.2 in order to remove the sucrose. Aliquots were stored in 10 mM Tris buffer (26 mg of protein/ml) at -20°C. An enzyme extract was prepared by treating the microsomal fraction (10 mg of protein/ml) in 10 mM Tris buffer, pH 7.2 with 0.2% Triton X- 100 at 4°C. The mixture was sonicated twice for 10 set, stirred for 1 hr at 4°C and centrifuged for 1 hr at 150,OOOg. The supernatant, referred to as “Triton extract,” was used as the enzyme preparation. Assay of Sialidase
with [3H]Ganglioside
G,,,
Incubation conditions. [3HJGanglioside GD,, (16.2 nmol in chloroform/ methanol, 2: 1, v/v) which had been diluted with unlabelled ganglioside GoI,
336
SCHRAVEN
ET AL.
to give a specific activity of 61.5 @Zi/pmol was dried under a stream of nitrogen. The residue was resuspended by sonicating at 4°C in acetate buffer and, if required, in Triton X- 100. The final incubation volume of 200 ~1 contained, under standard conditions, Triton extract (up to 120 pg of protein) or the microsomal preparation (up to 150 pg of protein) in a final buffer concentration of 0.15 M, pH 4.2. The incubation mixture was shaken intensively using a Vortex mixer and incubated for 40 min at 37°C and the reaction was stopped by cooling down to 0°C. Blanks were run with enzyme preparations inactivated by heating for 10 min at 100°C or by the addition of TCA at room temperature to give a final concentration of 1.5%, which did not change the pH value of the incubation mixture. Analysis. Aliquots of the incubation mixtures (40 ~1) were applied to thin layers of silica gel 60 and cochromatographed with standards of ganglioside GD,, and ganglioside G,,. For the separation of substrate and product the solvent system n-butanol-pyridine-0.55% KC1 (6:3:2, v/v/v) was used. In some cases the thin layer sheet was dried by a stream of air for about 6 hr and rechromatographed with chloroform containing 0.38% (v/v) acetic acid in order to remove the pyridine and increase the counting efficiency of the radioscanner. The substrate and product spots, separated by at least 3-5 cm, were localized using a radio scanner (Fa. Berthold, Germany) (Fig. 1). Substrate and product spots were cut out with scissors and put into plastic vials containing 15 ml of Unisolve I. After adding 4.5 ml of water and shaking intensively, a stable gel formed. The radioactivity measured in a liquid scintillation counter (Mark II, Nuclear Chicago) increased linearly with the amount of gangliosides tested up to 200,000 cpm. Formation of product is calculated from its percentage of total radioactivity. Determination
of Sialic Acid Content of Enzyme Preparations
Microsomal and supernatant enzyme preparations were hydrolysed with 0.05 N sulfuric acid at 80°C for 90 min and centrifuged (56OOg, 5 min). The sialic acid content was determined in the supernatant according to the procedure of Warren (7). The amounts of the individual gangliosides were determined by densitometry after separation by thin-layer chromatography as described previously (17,18). RESULTS
AND DISCUSSION
Membrane-bound sialidase degrades a variety of sialic acid-containing glycosphingolipids (14,19,20). Among the natural substrates, ganglioside G Dla is a prominent and readily degradable one (14,19,20). In order to develop a sensitive assay of the enzyme activity. it was labelled by saturating the double bond in the sphingosine moiety with a mixture of tritium and hydrogen gas. The purified [3H]ganglioside Go,, (192 PCilpmol)
A RADIOMETRIC
TABLE END~GENOUS
SUBSTRATES
FOR SIALIDASE ENZYME
Total NeuAc
337
ASSAY FOR SIALIDASE 1 IN MICROSOMES
AND
SUPERNATANT
(nmoVmg of protein) Total ganglioside NeuAc
Microsomes
69
31
Supernatant enzyme (0.2% Triton X-100)
19
5
Ganglioside GD,, 7 0.5
Ganglioside-bound NeuAc susceptible for sialidase 16 2
appeared to be radiochemically homogenous (Fig. 1) and contained 98% of its radioactivity in the dihydrosphingosine moiety. As shown in Table 1, microsomes and, to a smaller extent, the Triton X- 100 extract also contain endogenous ganglioside substrates, which dilute the added [3H]ganglioside GD,, in the incubation mixture, but this dilution is negligible in the case of Triton X-100 extract. The exogenous substrate (16.2 nmol of [3H]ganglioside G,,,) is diluted only by 0.04 to 0.24 nmol of ganglioside-bound sialic acid accessible for sialidase (Table 1) when 20 to 120 pg of enzyme protein were used for the test. On the other hand, microsomes containing 100 pg of protein add 1.6 nmol of lipid-bound sialic acid accessible to sialidase, which dilute the added substrate by 10%. This dilution can also be avoided by preincubation of particulate enzyme preparation (14,21). Sialidase splits [3H]ganglioside G Dla into sialic acid and [3H]ganglioside GMI, the amount of which can be measured reproducibly and sensitively in a liquid scintillation counter after separation from the substrate by thin-layer chromatography. The solvent system employed allows a complete separation of both substrate and product without any overlapping (Fig. 1). The assay procedure employed yields essentially a linear increase of radioactivity counted with the amount of ganglioside applied to the thin-layer plate up to 5 nmol. Down to 0.04 nmol of [3H]ganglioside GM1 formed can be quantitatively detected under the conditions used. The standard deviation of the assay lies between 5 and 6% of the mean within the range of 0.5 to 5 nmol of [3H]ganglioside GM1 formed. The radioactive test showed that sialidase is completely inactivated by the addition of 1.5% of TCA at room temperature as well as by heating for 10 min at 100°C (Fig. 1). Therefore, TCA-inactivated microsomes and their Triton X-100 extracts have also been used successfully for blank incubations when applying the calorimetric thiobarbituric acid test (7) to the determination of sialidase activity in the microsomal preparation. Activation of particulate sialidase has been reported over a narrow range by Triton X-100 (14,20) and by Triton CF-54 (14). The addition of 0.1% Triton X-100 to the incubation mixture results in an optimal stimulation for
338
SCHRAVEN
ET AL.
FIG. 2. Effect of increasing concentrations of Triton X-100 on the activity of sialidase from calf brain microsomes toward rH]ganglioside GoI,. For assay conditions, see Materials and n , 259 pg of microsomal protein; 0 0,129 pg of microsomal protein. Methods. n -
a protein concentration up to 150 pg, whereas higher detergent concentrations exert an inhibitory effect (Fig. 2). Lower concentrations essentially result in a nonlinear protein dependence of the enzyme activity. On the other hand, 0.1% Triton X- 100 does not fully activate higher protein concentrations. In this case, higher detergent concentrations would be needed. A presupposition for a quantitative enzyme assay is a linear increase of activity with the amount of enzyme protein added. This seems to be valid for the particulate as well as for the extracted enzyme only under special conditions, i.e., in the presence of 0.1% Triton X-100 and up to 120 pg of crude enzyme protein added. For the microsomal preparation, a less than linear increase of activity is observed in the absence of detergent. Sialidase.was active in the range between pH 4 and 5 with an optimum at pH 4.2 to 4.5. At pH 4.2, product formation increases linearly with time up to 40 min and then levels off. This seems to be due to the instability of the enzyme preparation. Whereas the extracted enzyme is stable over several weeks at -20°C its half-life in the absence of exogenous substrate at 4°C is about 20 hr and at 37°C only about 12 min. The physical state of substrate and enzyme in the detergent-containing incubation mixture is unknown. Therefore, under standard conditions, i.e., in the presence of 0.1% Triton X-100, only an apparent MichaelisMenten constant of 84 PM can be determined and it is not surprising that the values vary for different Triton X-100 concentrations used in the incubation mixtures. In this test, substrate concentrations on the order of the apparent K, value give rise to a linear time and protein dependence.
A RADIOMETRIC
ASSAY FOR SIALIDASE
339
Concentrations higher than twice the K, result in an apparent substrate inhibition. Despite the complex kinetics of sialidase, the radioactive test can be used under the defined conditions as a sensitive enzyme assay. The assay has been found to be suitable for the determination of other sialidases such as neuraminidase from Cl. perfringens. Recently, a radiometric assay for sialidase has been described (22) using a water soluble substrate (a-o-N-acetylneuraminosyl-(2 + 3’) lactit [3H]ol) in the absence of detergents. This test has been applied to a variety of neuraminidases and, in comparison with the test using a natural, lipid (amphiphilic) substrate, might facilitate the elucidation of the properties of membrane-bound sialidases. REFERENCES 1. Schengnmd, C. L., and Rosenberg, A. (197O)J. Bib!. Chem. 245, 61%-6200. 2. Tettamanti, G., Morgan, J. G., Gombos, G., Vincendon, G., and Mandel, P. (1972) Brain Res. 47, 515-518. 3. Editorial Note (1974) in Brain Res. 66, 557. 4. Wiegandt, H. (1%7) .I. Neurochem. 14, 671-674. 5. Roukema, P. A., and Heijlman, J. (1970) J. Neurochem. 17, 773-780. 6. Heijlman, J., and Roukema, P. A. (1972) J. Xeurochem. 19,2567-2575. 7. Warren, L. (1959)J. Biol. Chem. 234, 1971-1975. 8. Horvat, A., and Touster, 0. (1968) J. Biol. Chem. 243, 4380-4390. 9. Weissbach, A., and Hurwitz, J. (1959) J. Biol. Chem. 234, 705-709. 10. Preiss, J., and Ashwell, G. (1%2) J. Biol. Chem. 237, 309-316. 11. Carubelli, R., Bhavanandan, V. P., and Gottschalk, A. (1%5) Biochim. Biophys. Acra 101, 67-82.
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