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Continuous spectrophotometric assay of pantetheinase activity
ANALYTICAL
BIOCHEMISTRY
Continuous
142, 17% 18 1 ( 1984)
Spectrophotometric
Assay of Pantetheinase
Activity’
SILVESTRO DUPRI?,~ ROBERTA CHIARALUCE,* MIRELLA NARDINI,* CARLO~ANNELLA,$ GIORGIO FUCCI,* ANDDORIANO CAVALLINI* *Institute of Biological Chemistry. University “‘La Sapienza? Rome; TChair of Biological Chemistry, % Institute of General Biology, University of L’Aquila. L’Aquila; and $Institute of Biochemistry, University of Parma, Parma, Italy Received January 3, 1984 A continuous spectrophotometric assayfor pantetheinase determination using S-pantetheine3-pyruvate as substrate is described. The enzymatic hydrolysis of this new substrate leads to the formation of S-cysteamine-3-pyruvate, which cyclizes in a non-rate-limiting step to give 2H- 1+thiazin-5,6dihydro-3-carboxylic acid (aminoethylcysteine ketimine), a compound exhibiting a strong absorption at 296 nm. The assay is optimized with respect to pH, buffer, and substrate concentration. Prereduction of the enzyme and some properties of the reaction are also studied. The assayis simple, rapid, very sensitive, and specific. Q 1984 Academic mess, IX KEY WORDS: &enzymes; Cysteamine; Hydrolases; Pantetheinase activity; Pantetheine; Spectrophotometry.
Pantetheinase (EC 3.5.1.-; pantetheine hydrolase), the pantetheine-hydrolyzing enzyme which has been isolated from two sources and recently actively studied (l-5), is the key enzyme of pantetheine degradative pathways (3). It is also the crucial enzyme in the cysteamine pathway of taurine biosynthesis (6). Various reports of medical research on therapeutical applications of pantothenate derivatives, especially pantethine, in lipid metabolism dysfunctions have recently appeared. A renewed interest in pantetheinase and enzymes related to the intermediate metabolism of pantothenate derivatives has surfaced (for detailed references see (4)). Several procedures for the determination of pantetheinase activity have been described (1,3,5,7-9); however, most of them are tedious, time consuming, and require special equipment. In this paper we describe a simple, continuous spectrophotometric method for the assay of pantetheinase activity, which is ’ This work was supported by the Ministero della Pubblica Istruzione and is in partial full6llment of a Ph.D. in Biochemistry (University “La Sapienza,” Rome) for M. Nardini.
based on the ability of this enzyme to hydrolyze the pantetheine-bromopyruvate adduct (s-pantetheine-3-pyruvate), releasing a cyclic sulfur-nitrogen compound (aminoethylcysteine ketimine) which exhibits strong absorbance at about 300 nm (10). Preliminary studies (11) have already suggested that pantetheinase is active on 9pantetheine-3-pyruvate, and this property is definitively demonstrated in this paper. MATERIALS
AND METHODS
Chemicals. Aminocthylcysteine ketimine was prepared as previously described (10). Bromopyruvic acid, D,L-homocysteine, dithiothreitol, glutathione, and NADH were Sigma (USA) products. Mercaptoethanol and mercaptopropionic acid were from Pluka (Switzerland). All other chemicals were Merck products. Lactate dehydrogenase (850 units/ mg) was purchased from Boehringer (BRD). Hog kidney D-amino acid oxidase (17 units/ mg) was a Sigma product. Preparation of o-pantetheine, DPantethine was prepared starting from Dpantolactone 175
0003-2697184 $3.00 Copyright 0 1984 by Academic Press, Inc. All rights of reploduction in any form rcsuvcd.
176
DUPRe ET AL.
(Fluka, Switzerland), according to Viscontini et al. (12) with minor modifications. The preparation was purified by passage through a Dowex 2 column (2 X 6 cm) (OH- form, 200-400 mesh). Pantethine was reduced following the procedure previously described (11); pantethine (40 IIIM) was reacted with 1 M cysteine at pH 8.5 in 5 ml final volume. After 30 min at room temperature under a Nz stream, the solution was passed through a Dowex 50 column (2 X 6 cm) (H+ form, X8; 200-400 mesh). Pantetheine was eluted with water, brought to pH 4-5, concentrated to about 10 mM in vacua at 4O”C, and titrated with Ellman’s reagent (13). Partial hydrolysis of pantetheine has been observed during concentration at stronger acidic pH values. Pantetheine solutions must be used as soon as prepared. Preparation of S-pantetheine-3-pyruvate. 9 pantetheine-3-pyruvate was prepared essentially by following the procedure described for the preparation of Spyruvoyl glutathione (14,15). Bromopyruvic acid and pantetheine react rapidly at room temperature, and the reaction at pH 8 was complete within 5 min. Stoichiometry was demonstrated by adding pantetheine (100 mM), in portions of 20 rmol, to 100 rmol bromopyruvic acid dissolved in 10 ml 0.1 M phosphate buffer, pH 8.0. Five minutes after each addition, residual bromopyruvate was titrated by a lactate dehydrogenase-NADH coupled reaction ( 16). A linear relationship corresponding to a 1: 1 stoichiometry was obtained, and -SH groups tested with nitroprusside reagent (17) disap peared after the addition of stoichiometric amounts of bromopyruvate. Under these conditions oxidation of sulthydryl groups was negligible. This preparation cannot be used routinely, since a slight excess of free bromopyruvate strongly inhibits pantetheinase activity (unpublished data); on the other hand, the presence of small amounts of pantetheine also interferes with the enzyme assay. Therefore, we standardized two slightly different procedures, both equivalent and fully satisfactory; the first one allows to obtain the pure adduct in solution.
First, pantetheine ( 10 mM) was reacted with 20 mM bromopyruvate in 10 ml 0.1 M phosphate buffer, pH 8.0, at room temperature. After 10 min, homocysteine (20 mM) was added in order to scavenge excess bromopyruvate. After 10 min the mixture was passed through a Dowex 50 column (2 X 6 cm) (H+ form, X8; 200-400 mesh), and 9 pantetheine-3-pyruvate was eluted with 30 ml water. Under these conditions the bromopyruvate-homocysteine adduct was completely retained (18). The effluent was neutralized with 4 N KOH and stored at 4% Second, pantetheine and bromopyruvic acid were reacted in the conditions reported for the first preparation. After 10 min, 20 mM mercaptoethanol was added and the solution was used as such. Quantitation of 9pantetheine-3-pyruvate was obtained by hydrolysis, either enzymatic
0.6
-
0.4
-
! I I
b
“Ill
PIG. 1. Spectrophotometric analyses of S-pantetheine3-pyruvate, and synthetic and enzymatically produced aminoethylcysteine ketimine. (a) SPantetheine-3-pyruvate (0.1 mM) in 0.1 M phosphate buffer, pH 8.0. (b) (dotted line) S-Pantetheine-3-pyruvate (0.1 mM) in 0.1 M phosphate buffer, pH 8.0, after 10 min incubation at 30°C with 0.042 unit pantetheinase; this spectrum was recorded against blank without substrate. (b) (full line) Authentic aminoethylcysteine ketimine (0.1 mM) in 0.1 M phosphate buffer, pH 8.0.
CONTINUOUS
SPECTROPHOTOMETRIC
or acidic. Exhaustive enzymatic hydrolysis with pantetheinase (0.042 units, pH 8.0) of 100 nmol 9pantetheine-3-pyruvate released 95-100 nmol aminoethylcysteine ketimine, as calculated from A absorbance at 296 nm (10) (Fig. 1). Acidic hydrolysis with 4 N HCl (lOO°C, 20 min) yielded 93%, determined from A absorbance at 296 nm after neutralization at pH 7.6. Since the presence of the mercaptoethanol-bromopyruvate adduct does not interfere at all with the pantetheinase activity determination, we have routinely used the second preparation. Attempts to obtain S-pantetheine-3-pyruvate in solid form were unsuccessful because of its instability; on the contrary, 5 mM buffered solutions at pH 8.0 were stable for at least 3 days if stored at 4°C and for a week if stored below 0°C. Preparation of pantetheinase.Pantetheinase was prepared from horse kidney by the procedure described by Dupre et al. (1). Enzyme preparations with specific activities of 0.05 unit/mg were used throughout this work (one unit is the amount of enzyme which hydrolyzed 1 pmol substrate/min at pH 7.6 and 37“C, with the radiolabeled procedure described in (1)). Less purified fractions were occasionally used. Test conditions for pantetheinase activity determination. Pantetheinase (up to 0.05 unit) was preincubated with 30 pmol mercaptoethan01 in 0.2 ml (final volume) 0.5 M Kphosphate buffer, pH 8.0, for 10 min at 30°C. After prereduction, water was added to a final volume of 0.9 ml, and the reaction was primed with 0.1 ml substrate solution
ASSAY OF PANTETHEINASE
ACTIVITY
177
(500 nmol) in a 1-ml quartz cuvette ( 1 cm light path) thermostated at 30°C. Aminoethylcysteine ketimine production was followed at 296 nm (10). Spectrophotometric analyses were performed with a Beckman 5260 spectrophotometer. Polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis was performed according to Ornstein and Davis ( 19), with the exception that only one buffer (12 mM, Tris-glycine, pH 8.2) was used. Gels at 7.5% acrylamide were prerun at 5 mA/tube for 40 min to remove persulfate. About 200~pg ahquots of protein were run for 15 min at 1 mA/tube, and then for 30 min at 3 mA/ tube. Gels were removed from the glass tubes with water, and fractionated with water in a Gilson gel divider. Each fraction (2 mm smashed slices in 0.5 ml water) was divided in two parts, and enzymatic activities on pantetheine and on S-pantetheine-3-pyruvate were determined. Activity with pantetheine was assayed by incubation with 1 rmol pantetheine in 1 ml 0.1 M phosphate buffer, pH 8.0, at 37°C for 1 h. Cysteamine enzymatitally produced was determined according to the method recently described (20). Enzymatic activity with 9pantetheine-3-pyruvate was assayed with the spectrophotometric method reported above. RESULTS
The spectrophotometric procedure for the assay of pantetheinase activity with S-pantetheine-Zpyruvate as substrate may be summarized as follows: enzymatic
pantothenoyl-NH-CHz-CHZ-S-CHZ-CO-OH M (9pantetheine-3-pyruvate) NH2-CH2-CHz-S-CHz-CO-COOH (S-cysteamine-3-pyruvate) spontaneous )
COOH (aminoethylcysteine
ketimine)
+ Pantothenic
acid
178
DUPRti
As previously described, S-cysteamine-f pyruvate rapidly and quantitatively cyclizes into a ketimine ring which strongly absorbs at 296 nm (eM = 6200) (10). At this wavelength S-pantetheine-3-pyruvate has an absorption of about 4% (CM = 250) (Fig. la). Spectrophotometric analysis after exhaustive hydrolysis of 9pantetheine-3-pyruvate with purified pantetheinase (Fig. lb) confirmed the enzymatic production of aminoethylcysteine ketimine. This product exhibited spontaneous decarboxylation and autoxidation at neutral pH (lo), which was relevant at 10 mM concentration. At lower concentrations (x0.5 mM) under the assay conditions, less than 2% absorbance decrease at 296 nm in 1 h was observed. Experiments performed with polyacrylamide gel electrophoresis demonstrated that enzymatic activities with pantetheine and Spantetheine-3-pyruvate were electrophoretitally indistinguishable (Fig. 2). These results, together with those previously described (1 l), demonstrated that the same enzyme was responsible for both activities. Pantetheinase activity was therefore continuously monitored, with S-pantetheine-3-
ET AL.
pyruvate as substrate, by following absorbance changes at 296 nm. For quantitative purposes, an apparent cM = 5950 was used to account for negative contribution in absorbance during substrate consumption. The absorbance increased linearly with time, and linearity was retained for up to 80% of the reaction time. The reaction started without apparent lag phase, and this fact indicated that the ring closure of S-cysteamine-3-pyruvate was not rate limiting. A lag phase could be observed in the presence of small amounts of pantethine in the 9pantetheine-3-pyruvate solution, when the reduction of pantethine, performed in an incorrect way, was not complete. Absorbance of the blank without enzyme did not increase with time. Pantetheine hydrolytic activity from various sources was increased in the presence of several thiols, and preincubation steps with reducing agents have been described (1,3,4,7). The S-pantetheine-3-pyruvate hydrolytic activity was increased by various thiols. Preincubation of the enzyme for 10 min at pH 8.0 (0.5 M phosphate buffer) with 150 mM thiol enhances activity about 3.5 times with mercaptoethanol, dithiothreitol, and cysteine,
> =
z ; m .-o-
60 -
; 40P z (Yc 20 : ; ; 2 .---
--.-
0 -.-.-.-. 5
IO
15
20
25
I 30
Fractions
FIG. 2. Coincidence of S-pantetheine-3-pyruvate and pantetheine hydrolytic activities tier gel electrophoresis. Polyacrylamide gel electrophoresis of pantetheinase (0.01 unit, 200 ccgprotein), gel fractionation, and determination of enzymatic activities were performed as described under Materials and Methods. The amount of cysteamine produced (nmol/h) (left) and of aminoethylcysteine ketimine (nmol/h) (right) are reported on the ordinates.
CONTINUOUS
SPEff ROPHOTOMETRIC
ASSAY OF PANTETHEINASE
ACTIVITY
Materials and Methods provided maximal reactivation. Various buffers have been used to investigate pH dependence of the reaction, and optimization of the method has been achieved by carrying out the reaction in 0.1 M phosphate buffer, pH 8.0 (Fig. 3). Above pH 6 the extinction of the chromophore was pH independent and was not influenced by the buffer used. On the contrary, the rate of substrate hydrolysis was affected by the concentration of Tris ions. Determination of K, for 9pantetheine-3pyruvate by Lineweaver-Burk plots yielded a value of 2.8 X 10m5 M (Fig. 4). The rate of reaction was linearly dependent on the amount of protein incubated within a wide concentration range (Fig. 5). The lower limit was about 2 X 10m4 pmol/min unit pantetheinase. A comparison between former assay procedures and the assay described above is given in Table 1. The method was also tested on less purified pantetheinase fractions, and was found to be reliable even when the specific activity was as low as 1.5 X 10e4 units/mg protein.
.04
.E .03 E EC L% - .02 ; : .o 1
FIG. 3. The pH profile of enzymatic activity. Pantetheinase (0.007 unit) was prereduced with 30 pm01 mercap toethanol in 0.2 ml (final volume) 0.05 M phosphate buffer, pH 8.0, for 10 min at 30°C. Buffers were added to the final concentrations shown. The reaction (1 ml final volume) was started with 500 nmol substrate. A, 0.1 M acetate buffer; n , 0.1 M phosphate buffer, A, 0.1 M borate-NaOH buffer; 0, 0.1 M Tris-HCl buffer, 0, 0.5 M Tris-HCl buffer. Below pH 6 aminoethylcysteine ketimine extinction becomes pH dependent; the real concentration was evaluated by comparison with authentic aminoethylcysteine ketimine samples (right ordinate).
and about 3 times with glutathione and mercaptopropionic acid. The activation step has been optimized with mercaptoethanol, and standardized conditions as given under
DISCUSSION
Substrate specificity of pantetheinase has been studied in detail ( 1,4), and one thioether,
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so
/
B N ;i
l
:
>E
! 40
t
/
-20
.’
/
9’
7
I
-40
.’
20 1
/
.
179
I
40 6-Pyruvoyl
.
I
.
60 Pantetheine
I
60
I
100 ( m Me’)
FIG. 4. Double-reciprocal plot for S-pantetheine-3-pyruvate. Pantetheinase (0.0075 unit) was incubated in the test conditions given under Materials and Methods, in the presence of variable amounts of S-pantetheine-3-pyruvate (from 0.01 to 0.5 mM).
180
DUPRB ET AL.
l .lO
/
-
.
c
i ; c
.08 -
:w
.06-
.
/
/
. ; 4 9
.04
.
/
FIG. 5. Dependence of rate on protein concentration. Variable amounts of pantetheinase were tested, as described under Materials and Methods.
S-ethyl pantetheine, was reported to be hydrolyzed (4). The pantetheinase assay described in this paper takes advantage of the ability of the enzyme to hydrolyze a new substrate, Spantetheine-3-pyruvate, with high efficiency. This substrate specificity confirms the ability of pantetheinase to use thioethers of pantetheine as substrates. The method has been optimized in respect to pH, substrate concentration, and preactivation of the enzyme.
Between 7.5 and 8.5, the pH is not critical for activity, as a plateau in the pH dependence was observed. Phosphate buffer must be used because Tris buffer of similar pH and concentration slows down the reaction. The pHdependence curve reported in Fig. 3 is different from that reported for the same enzyme, using pantetheine as substrate (1). This may be due either to the different substrates used or to the fact that the continuous method at 296 nm allows initial rate measurements. The former pH curve, derived from activity measurements lasting 1 h, clearly reflects the pH dependence of the reaction itself and the stability of both enzyme and substrate at the pH used. These facts may also explain the different K,,, value for 9pantetheine-3-pyruvate (2.8 X IO-’ M), which is of the same order as that reported for the pig kidney enzyme with Dpantetheine (4), but quite different from that previously given for the horse kidney enzyme (2). The prereduction step of the substrate is not necessary with S-pantetheine-3-pyruvate. Aminothiols, mainly cysteamine, interfere with the method at the high concentrations used for prereduction, probably by reacting with the carbonylic function of S-pantetheine-
TABLE 1 COMPAFUS~N
Method I) SPantetheine-3pyruvate assay
AMONG
Substrate 9pantetheine-3pyruvate
VARIOUS
PANTETHEINASE
Temperature (“C) 30
37
Acnvrn
PROCEDURES
Enzyme (units)
A Absorbance in 10 min
0.0037
0.210
0.0075
0.420
35 71 Aminoethyl
0.0037 0.0075
0.300 0.600
50 101
1ii Cysteamine
2) Mercaptide assay
Pantetheine
37
0.0037 0.0075
0.080 0.175
3) Radiolabeled
[ “C]Pantetheine
37
0.0037 0.0075
-
asSaY
Enzymatic product (nmol/lO min)
-
cysteine ketimine
37 [ ‘*C]Pantothenic 75 acid
Note.S;Pantetheine3-pyruvate assay was carried out as described under h4aterials and Methods. A coefficient of variation of 3.5 (n = 7) was obtained. Mercaptide assaywas performed as reported by Wittwer e2 al. (7), in 1 ml final volume instead of 3 ml. Radiolabeled assay was as reported by Dupr6 et al. (I).
CONTINUOUS
SPECTROPHOTOMETRIC
3-pyruvate to give stable thiazolidine rings (2 I). For prereduction of the enzyme, dithiothreitol (or dithioerythritol) is less suitable, because its oxidized form absorbs at about 300 nm. The procedure reported in this paper has some advantages over the other continuous spectrophotometric method, which is based on the absorption of the mercaptide ion of cysteamine at 240 nm (7). For instance, our method shows an increase in sensitivity by about a factor of four, does not need deoxygenated conditions, and the absorbance of proteins at 296 nm is lower than at 240 nm. Perhaps the greatest advantages of the Spantetheine-3-pyruvate-based method are its simplicity and good sensitivity, which also make the procedure suitable for routine pantetheinase dosages. The sensitivity of the method proposed may be further increased at the nanomole level by monitoring the amount of aminoethylcysteine ketimine formed with the method recently described (20), based on D-amino acid oxidase activity inhibition. REFERENCES 1. Dupti, S., Graziani, M. T., Rosei, M. A., Fabi, A., and Del Groso, E. (1970) Eur. J. B&hem. 16, 571-578. 2. Dupre, S., and Cavallini, D. (1979) in Methods in Enzymology (McCormick, D. B., and Wright, L. D., eds.), Vol. 62, pp. 262-267, Academic Press, New York. 3. Abiko, Y. (1975) in Metabolic Pathways (Greenberg, D. M., ed.), 3rd. ed., Vol. 7, pp. l-25, Academic Press, New York. 4. Wittwer, C. T., Burkard, D., Ririe, K., Rasmussen, R., Brown, J., Wyse, B., and Hansen, R. G. (1983) .I. Biol. Chem. 258, 9733-9738.
ASSAY OF PANTETHEINASE
ACTIVITY
181
5. Orloff, S., Butler, J. D., Towne, D., Murkhejee, A. B., and Schulman, J. D. (1981) Pediatr. Rex 15, 1063-1067. 6. Cavallini, D., Scandurra, R., Dupe, S., Federici, G., Santoro, L., Ricci, G., and Barra, D. (1976) in Taurine (Huxtable, R., and Barbeau, A., eds.), pp. 59-65, Raven Press, New York. 7. Wittwer, C., Wyse, B., and Hansen, G. (1982) Anal. Biochem. 122,2 13-222. 8. Dupm, S., Antonucci, A., Piergrossi, P., and Aureli, M. (1976) Ital. J. B&hem. 25,229-235. 9. Cavallini, D., De Marco, C., and Crifo, C. (1964) Boll. Sot. Ital. Biol. Sper. 40, 1973-1977. 10. Cavallini, D., Ricci, G., Federici, G., Costa, M., Pensa, B., Matarese, R. M., and Achilli, M. (1982) in Structure and Function in Biochemical Systems (Bossa, F., Chiancone, E., Finazzi-Agro, A., and Strom, R., eds.), pp. 359-374, Plenum, New York. 11. Chiaraluce, R., Nardini, M., Dupre, S., Ricci, G., and Cavallini, D. (1983) IRC’S Med. Sci. 11, 1119-1120. 12. Viscontini, M., Adank, K., Merckling, N., Eberhardt, K., and Karrer, P. (1954) Helv. Chim. Acfa 37, 375-377. 13. Ellman, G. L. (1959) Arch. Biochem. Biophys. 82, 70-77. 14. Avi-Dor, Y. (1960) B&hem. J. 76, 370-374. 15. Tate, S. S., and Meister, A. (1974) J. Biol. Chem. 249,7593-7602. 16. Berghauser, J., Falderbaum, I., and Woenckhaus, C. (1971) Hoppe-Seyler’s Z. Physiol. Chem. 352, 52-58. 17. Toennies, G., and Levine, T. F. (1934) J. Biol. Chem. 105, 115-121. 18. Ricci, G., Santoro, L., Achilli, M., Matarese, R. M., Nardini, M., and Cavallini, D. (1983) J. Biol. Chem. 258, 1051 I-10517. 19. Omstein, L., and Davis, B. J. (1964) Ann. N. Y. Ad. Sci. 121, 32 l-330. 20. Ricci, G., Nardini, M., Chiaraluce, R., Dupre, S., and Cavallini, D. (1983) J. Appl. Biochem. 5, 320-329. 21. Schubert, M. P. (1936) J. Biol. Chem. 114, 341350.
BIOCHEMISTRY
Continuous
142, 17% 18 1 ( 1984)
Spectrophotometric
Assay of Pantetheinase
Activity’
SILVESTRO DUPRI?,~ ROBERTA CHIARALUCE,* MIRELLA NARDINI,* CARLO~ANNELLA,$ GIORGIO FUCCI,* ANDDORIANO CAVALLINI* *Institute of Biological Chemistry. University “‘La Sapienza? Rome; TChair of Biological Chemistry, % Institute of General Biology, University of L’Aquila. L’Aquila; and $Institute of Biochemistry, University of Parma, Parma, Italy Received January 3, 1984 A continuous spectrophotometric assayfor pantetheinase determination using S-pantetheine3-pyruvate as substrate is described. The enzymatic hydrolysis of this new substrate leads to the formation of S-cysteamine-3-pyruvate, which cyclizes in a non-rate-limiting step to give 2H- 1+thiazin-5,6dihydro-3-carboxylic acid (aminoethylcysteine ketimine), a compound exhibiting a strong absorption at 296 nm. The assay is optimized with respect to pH, buffer, and substrate concentration. Prereduction of the enzyme and some properties of the reaction are also studied. The assayis simple, rapid, very sensitive, and specific. Q 1984 Academic mess, IX KEY WORDS: &enzymes; Cysteamine; Hydrolases; Pantetheinase activity; Pantetheine; Spectrophotometry.
Pantetheinase (EC 3.5.1.-; pantetheine hydrolase), the pantetheine-hydrolyzing enzyme which has been isolated from two sources and recently actively studied (l-5), is the key enzyme of pantetheine degradative pathways (3). It is also the crucial enzyme in the cysteamine pathway of taurine biosynthesis (6). Various reports of medical research on therapeutical applications of pantothenate derivatives, especially pantethine, in lipid metabolism dysfunctions have recently appeared. A renewed interest in pantetheinase and enzymes related to the intermediate metabolism of pantothenate derivatives has surfaced (for detailed references see (4)). Several procedures for the determination of pantetheinase activity have been described (1,3,5,7-9); however, most of them are tedious, time consuming, and require special equipment. In this paper we describe a simple, continuous spectrophotometric method for the assay of pantetheinase activity, which is ’ This work was supported by the Ministero della Pubblica Istruzione and is in partial full6llment of a Ph.D. in Biochemistry (University “La Sapienza,” Rome) for M. Nardini.
based on the ability of this enzyme to hydrolyze the pantetheine-bromopyruvate adduct (s-pantetheine-3-pyruvate), releasing a cyclic sulfur-nitrogen compound (aminoethylcysteine ketimine) which exhibits strong absorbance at about 300 nm (10). Preliminary studies (11) have already suggested that pantetheinase is active on 9pantetheine-3-pyruvate, and this property is definitively demonstrated in this paper. MATERIALS
AND METHODS
Chemicals. Aminocthylcysteine ketimine was prepared as previously described (10). Bromopyruvic acid, D,L-homocysteine, dithiothreitol, glutathione, and NADH were Sigma (USA) products. Mercaptoethanol and mercaptopropionic acid were from Pluka (Switzerland). All other chemicals were Merck products. Lactate dehydrogenase (850 units/ mg) was purchased from Boehringer (BRD). Hog kidney D-amino acid oxidase (17 units/ mg) was a Sigma product. Preparation of o-pantetheine, DPantethine was prepared starting from Dpantolactone 175
0003-2697184 $3.00 Copyright 0 1984 by Academic Press, Inc. All rights of reploduction in any form rcsuvcd.
176
DUPRe ET AL.
(Fluka, Switzerland), according to Viscontini et al. (12) with minor modifications. The preparation was purified by passage through a Dowex 2 column (2 X 6 cm) (OH- form, 200-400 mesh). Pantethine was reduced following the procedure previously described (11); pantethine (40 IIIM) was reacted with 1 M cysteine at pH 8.5 in 5 ml final volume. After 30 min at room temperature under a Nz stream, the solution was passed through a Dowex 50 column (2 X 6 cm) (H+ form, X8; 200-400 mesh). Pantetheine was eluted with water, brought to pH 4-5, concentrated to about 10 mM in vacua at 4O”C, and titrated with Ellman’s reagent (13). Partial hydrolysis of pantetheine has been observed during concentration at stronger acidic pH values. Pantetheine solutions must be used as soon as prepared. Preparation of S-pantetheine-3-pyruvate. 9 pantetheine-3-pyruvate was prepared essentially by following the procedure described for the preparation of Spyruvoyl glutathione (14,15). Bromopyruvic acid and pantetheine react rapidly at room temperature, and the reaction at pH 8 was complete within 5 min. Stoichiometry was demonstrated by adding pantetheine (100 mM), in portions of 20 rmol, to 100 rmol bromopyruvic acid dissolved in 10 ml 0.1 M phosphate buffer, pH 8.0. Five minutes after each addition, residual bromopyruvate was titrated by a lactate dehydrogenase-NADH coupled reaction ( 16). A linear relationship corresponding to a 1: 1 stoichiometry was obtained, and -SH groups tested with nitroprusside reagent (17) disap peared after the addition of stoichiometric amounts of bromopyruvate. Under these conditions oxidation of sulthydryl groups was negligible. This preparation cannot be used routinely, since a slight excess of free bromopyruvate strongly inhibits pantetheinase activity (unpublished data); on the other hand, the presence of small amounts of pantetheine also interferes with the enzyme assay. Therefore, we standardized two slightly different procedures, both equivalent and fully satisfactory; the first one allows to obtain the pure adduct in solution.
First, pantetheine ( 10 mM) was reacted with 20 mM bromopyruvate in 10 ml 0.1 M phosphate buffer, pH 8.0, at room temperature. After 10 min, homocysteine (20 mM) was added in order to scavenge excess bromopyruvate. After 10 min the mixture was passed through a Dowex 50 column (2 X 6 cm) (H+ form, X8; 200-400 mesh), and 9 pantetheine-3-pyruvate was eluted with 30 ml water. Under these conditions the bromopyruvate-homocysteine adduct was completely retained (18). The effluent was neutralized with 4 N KOH and stored at 4% Second, pantetheine and bromopyruvic acid were reacted in the conditions reported for the first preparation. After 10 min, 20 mM mercaptoethanol was added and the solution was used as such. Quantitation of 9pantetheine-3-pyruvate was obtained by hydrolysis, either enzymatic
0.6
-
0.4
-
! I I
b
“Ill
PIG. 1. Spectrophotometric analyses of S-pantetheine3-pyruvate, and synthetic and enzymatically produced aminoethylcysteine ketimine. (a) SPantetheine-3-pyruvate (0.1 mM) in 0.1 M phosphate buffer, pH 8.0. (b) (dotted line) S-Pantetheine-3-pyruvate (0.1 mM) in 0.1 M phosphate buffer, pH 8.0, after 10 min incubation at 30°C with 0.042 unit pantetheinase; this spectrum was recorded against blank without substrate. (b) (full line) Authentic aminoethylcysteine ketimine (0.1 mM) in 0.1 M phosphate buffer, pH 8.0.
CONTINUOUS
SPECTROPHOTOMETRIC
or acidic. Exhaustive enzymatic hydrolysis with pantetheinase (0.042 units, pH 8.0) of 100 nmol 9pantetheine-3-pyruvate released 95-100 nmol aminoethylcysteine ketimine, as calculated from A absorbance at 296 nm (10) (Fig. 1). Acidic hydrolysis with 4 N HCl (lOO°C, 20 min) yielded 93%, determined from A absorbance at 296 nm after neutralization at pH 7.6. Since the presence of the mercaptoethanol-bromopyruvate adduct does not interfere at all with the pantetheinase activity determination, we have routinely used the second preparation. Attempts to obtain S-pantetheine-3-pyruvate in solid form were unsuccessful because of its instability; on the contrary, 5 mM buffered solutions at pH 8.0 were stable for at least 3 days if stored at 4°C and for a week if stored below 0°C. Preparation of pantetheinase.Pantetheinase was prepared from horse kidney by the procedure described by Dupre et al. (1). Enzyme preparations with specific activities of 0.05 unit/mg were used throughout this work (one unit is the amount of enzyme which hydrolyzed 1 pmol substrate/min at pH 7.6 and 37“C, with the radiolabeled procedure described in (1)). Less purified fractions were occasionally used. Test conditions for pantetheinase activity determination. Pantetheinase (up to 0.05 unit) was preincubated with 30 pmol mercaptoethan01 in 0.2 ml (final volume) 0.5 M Kphosphate buffer, pH 8.0, for 10 min at 30°C. After prereduction, water was added to a final volume of 0.9 ml, and the reaction was primed with 0.1 ml substrate solution
ASSAY OF PANTETHEINASE
ACTIVITY
177
(500 nmol) in a 1-ml quartz cuvette ( 1 cm light path) thermostated at 30°C. Aminoethylcysteine ketimine production was followed at 296 nm (10). Spectrophotometric analyses were performed with a Beckman 5260 spectrophotometer. Polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis was performed according to Ornstein and Davis ( 19), with the exception that only one buffer (12 mM, Tris-glycine, pH 8.2) was used. Gels at 7.5% acrylamide were prerun at 5 mA/tube for 40 min to remove persulfate. About 200~pg ahquots of protein were run for 15 min at 1 mA/tube, and then for 30 min at 3 mA/ tube. Gels were removed from the glass tubes with water, and fractionated with water in a Gilson gel divider. Each fraction (2 mm smashed slices in 0.5 ml water) was divided in two parts, and enzymatic activities on pantetheine and on S-pantetheine-3-pyruvate were determined. Activity with pantetheine was assayed by incubation with 1 rmol pantetheine in 1 ml 0.1 M phosphate buffer, pH 8.0, at 37°C for 1 h. Cysteamine enzymatitally produced was determined according to the method recently described (20). Enzymatic activity with 9pantetheine-3-pyruvate was assayed with the spectrophotometric method reported above. RESULTS
The spectrophotometric procedure for the assay of pantetheinase activity with S-pantetheine-Zpyruvate as substrate may be summarized as follows: enzymatic
pantothenoyl-NH-CHz-CHZ-S-CHZ-CO-OH M (9pantetheine-3-pyruvate) NH2-CH2-CHz-S-CHz-CO-COOH (S-cysteamine-3-pyruvate) spontaneous )
COOH (aminoethylcysteine
ketimine)
+ Pantothenic
acid
178
DUPRti
As previously described, S-cysteamine-f pyruvate rapidly and quantitatively cyclizes into a ketimine ring which strongly absorbs at 296 nm (eM = 6200) (10). At this wavelength S-pantetheine-3-pyruvate has an absorption of about 4% (CM = 250) (Fig. la). Spectrophotometric analysis after exhaustive hydrolysis of 9pantetheine-3-pyruvate with purified pantetheinase (Fig. lb) confirmed the enzymatic production of aminoethylcysteine ketimine. This product exhibited spontaneous decarboxylation and autoxidation at neutral pH (lo), which was relevant at 10 mM concentration. At lower concentrations (x0.5 mM) under the assay conditions, less than 2% absorbance decrease at 296 nm in 1 h was observed. Experiments performed with polyacrylamide gel electrophoresis demonstrated that enzymatic activities with pantetheine and Spantetheine-3-pyruvate were electrophoretitally indistinguishable (Fig. 2). These results, together with those previously described (1 l), demonstrated that the same enzyme was responsible for both activities. Pantetheinase activity was therefore continuously monitored, with S-pantetheine-3-
ET AL.
pyruvate as substrate, by following absorbance changes at 296 nm. For quantitative purposes, an apparent cM = 5950 was used to account for negative contribution in absorbance during substrate consumption. The absorbance increased linearly with time, and linearity was retained for up to 80% of the reaction time. The reaction started without apparent lag phase, and this fact indicated that the ring closure of S-cysteamine-3-pyruvate was not rate limiting. A lag phase could be observed in the presence of small amounts of pantethine in the 9pantetheine-3-pyruvate solution, when the reduction of pantethine, performed in an incorrect way, was not complete. Absorbance of the blank without enzyme did not increase with time. Pantetheine hydrolytic activity from various sources was increased in the presence of several thiols, and preincubation steps with reducing agents have been described (1,3,4,7). The S-pantetheine-3-pyruvate hydrolytic activity was increased by various thiols. Preincubation of the enzyme for 10 min at pH 8.0 (0.5 M phosphate buffer) with 150 mM thiol enhances activity about 3.5 times with mercaptoethanol, dithiothreitol, and cysteine,
> =
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; 40P z (Yc 20 : ; ; 2 .---
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0 -.-.-.-. 5
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15
20
25
I 30
Fractions
FIG. 2. Coincidence of S-pantetheine-3-pyruvate and pantetheine hydrolytic activities tier gel electrophoresis. Polyacrylamide gel electrophoresis of pantetheinase (0.01 unit, 200 ccgprotein), gel fractionation, and determination of enzymatic activities were performed as described under Materials and Methods. The amount of cysteamine produced (nmol/h) (left) and of aminoethylcysteine ketimine (nmol/h) (right) are reported on the ordinates.
CONTINUOUS
SPEff ROPHOTOMETRIC
ASSAY OF PANTETHEINASE
ACTIVITY
Materials and Methods provided maximal reactivation. Various buffers have been used to investigate pH dependence of the reaction, and optimization of the method has been achieved by carrying out the reaction in 0.1 M phosphate buffer, pH 8.0 (Fig. 3). Above pH 6 the extinction of the chromophore was pH independent and was not influenced by the buffer used. On the contrary, the rate of substrate hydrolysis was affected by the concentration of Tris ions. Determination of K, for 9pantetheine-3pyruvate by Lineweaver-Burk plots yielded a value of 2.8 X 10m5 M (Fig. 4). The rate of reaction was linearly dependent on the amount of protein incubated within a wide concentration range (Fig. 5). The lower limit was about 2 X 10m4 pmol/min unit pantetheinase. A comparison between former assay procedures and the assay described above is given in Table 1. The method was also tested on less purified pantetheinase fractions, and was found to be reliable even when the specific activity was as low as 1.5 X 10e4 units/mg protein.
.04
.E .03 E EC L% - .02 ; : .o 1
FIG. 3. The pH profile of enzymatic activity. Pantetheinase (0.007 unit) was prereduced with 30 pm01 mercap toethanol in 0.2 ml (final volume) 0.05 M phosphate buffer, pH 8.0, for 10 min at 30°C. Buffers were added to the final concentrations shown. The reaction (1 ml final volume) was started with 500 nmol substrate. A, 0.1 M acetate buffer; n , 0.1 M phosphate buffer, A, 0.1 M borate-NaOH buffer; 0, 0.1 M Tris-HCl buffer, 0, 0.5 M Tris-HCl buffer. Below pH 6 aminoethylcysteine ketimine extinction becomes pH dependent; the real concentration was evaluated by comparison with authentic aminoethylcysteine ketimine samples (right ordinate).
and about 3 times with glutathione and mercaptopropionic acid. The activation step has been optimized with mercaptoethanol, and standardized conditions as given under
DISCUSSION
Substrate specificity of pantetheinase has been studied in detail ( 1,4), and one thioether,
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-20
.’
/
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7
I
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/
.
179
I
40 6-Pyruvoyl
.
I
.
60 Pantetheine
I
60
I
100 ( m Me’)
FIG. 4. Double-reciprocal plot for S-pantetheine-3-pyruvate. Pantetheinase (0.0075 unit) was incubated in the test conditions given under Materials and Methods, in the presence of variable amounts of S-pantetheine-3-pyruvate (from 0.01 to 0.5 mM).
180
DUPRB ET AL.
l .lO
/
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.
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.
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FIG. 5. Dependence of rate on protein concentration. Variable amounts of pantetheinase were tested, as described under Materials and Methods.
S-ethyl pantetheine, was reported to be hydrolyzed (4). The pantetheinase assay described in this paper takes advantage of the ability of the enzyme to hydrolyze a new substrate, Spantetheine-3-pyruvate, with high efficiency. This substrate specificity confirms the ability of pantetheinase to use thioethers of pantetheine as substrates. The method has been optimized in respect to pH, substrate concentration, and preactivation of the enzyme.
Between 7.5 and 8.5, the pH is not critical for activity, as a plateau in the pH dependence was observed. Phosphate buffer must be used because Tris buffer of similar pH and concentration slows down the reaction. The pHdependence curve reported in Fig. 3 is different from that reported for the same enzyme, using pantetheine as substrate (1). This may be due either to the different substrates used or to the fact that the continuous method at 296 nm allows initial rate measurements. The former pH curve, derived from activity measurements lasting 1 h, clearly reflects the pH dependence of the reaction itself and the stability of both enzyme and substrate at the pH used. These facts may also explain the different K,,, value for 9pantetheine-3-pyruvate (2.8 X IO-’ M), which is of the same order as that reported for the pig kidney enzyme with Dpantetheine (4), but quite different from that previously given for the horse kidney enzyme (2). The prereduction step of the substrate is not necessary with S-pantetheine-3-pyruvate. Aminothiols, mainly cysteamine, interfere with the method at the high concentrations used for prereduction, probably by reacting with the carbonylic function of S-pantetheine-
TABLE 1 COMPAFUS~N
Method I) SPantetheine-3pyruvate assay
AMONG
Substrate 9pantetheine-3pyruvate
VARIOUS
PANTETHEINASE
Temperature (“C) 30
37
Acnvrn
PROCEDURES
Enzyme (units)
A Absorbance in 10 min
0.0037
0.210
0.0075
0.420
35 71 Aminoethyl
0.0037 0.0075
0.300 0.600
50 101
1ii Cysteamine
2) Mercaptide assay
Pantetheine
37
0.0037 0.0075
0.080 0.175
3) Radiolabeled
[ “C]Pantetheine
37
0.0037 0.0075
-
asSaY
Enzymatic product (nmol/lO min)
-
cysteine ketimine
37 [ ‘*C]Pantothenic 75 acid
Note.S;Pantetheine3-pyruvate assay was carried out as described under h4aterials and Methods. A coefficient of variation of 3.5 (n = 7) was obtained. Mercaptide assaywas performed as reported by Wittwer e2 al. (7), in 1 ml final volume instead of 3 ml. Radiolabeled assay was as reported by Dupr6 et al. (I).
CONTINUOUS
SPECTROPHOTOMETRIC
3-pyruvate to give stable thiazolidine rings (2 I). For prereduction of the enzyme, dithiothreitol (or dithioerythritol) is less suitable, because its oxidized form absorbs at about 300 nm. The procedure reported in this paper has some advantages over the other continuous spectrophotometric method, which is based on the absorption of the mercaptide ion of cysteamine at 240 nm (7). For instance, our method shows an increase in sensitivity by about a factor of four, does not need deoxygenated conditions, and the absorbance of proteins at 296 nm is lower than at 240 nm. Perhaps the greatest advantages of the Spantetheine-3-pyruvate-based method are its simplicity and good sensitivity, which also make the procedure suitable for routine pantetheinase dosages. The sensitivity of the method proposed may be further increased at the nanomole level by monitoring the amount of aminoethylcysteine ketimine formed with the method recently described (20), based on D-amino acid oxidase activity inhibition. REFERENCES 1. Dupti, S., Graziani, M. T., Rosei, M. A., Fabi, A., and Del Groso, E. (1970) Eur. J. B&hem. 16, 571-578. 2. Dupre, S., and Cavallini, D. (1979) in Methods in Enzymology (McCormick, D. B., and Wright, L. D., eds.), Vol. 62, pp. 262-267, Academic Press, New York. 3. Abiko, Y. (1975) in Metabolic Pathways (Greenberg, D. M., ed.), 3rd. ed., Vol. 7, pp. l-25, Academic Press, New York. 4. Wittwer, C. T., Burkard, D., Ririe, K., Rasmussen, R., Brown, J., Wyse, B., and Hansen, R. G. (1983) .I. Biol. Chem. 258, 9733-9738.
ASSAY OF PANTETHEINASE
ACTIVITY
181
5. Orloff, S., Butler, J. D., Towne, D., Murkhejee, A. B., and Schulman, J. D. (1981) Pediatr. Rex 15, 1063-1067. 6. Cavallini, D., Scandurra, R., Dupe, S., Federici, G., Santoro, L., Ricci, G., and Barra, D. (1976) in Taurine (Huxtable, R., and Barbeau, A., eds.), pp. 59-65, Raven Press, New York. 7. Wittwer, C., Wyse, B., and Hansen, G. (1982) Anal. Biochem. 122,2 13-222. 8. Dupm, S., Antonucci, A., Piergrossi, P., and Aureli, M. (1976) Ital. J. B&hem. 25,229-235. 9. Cavallini, D., De Marco, C., and Crifo, C. (1964) Boll. Sot. Ital. Biol. Sper. 40, 1973-1977. 10. Cavallini, D., Ricci, G., Federici, G., Costa, M., Pensa, B., Matarese, R. M., and Achilli, M. (1982) in Structure and Function in Biochemical Systems (Bossa, F., Chiancone, E., Finazzi-Agro, A., and Strom, R., eds.), pp. 359-374, Plenum, New York. 11. Chiaraluce, R., Nardini, M., Dupre, S., Ricci, G., and Cavallini, D. (1983) IRC’S Med. Sci. 11, 1119-1120. 12. Viscontini, M., Adank, K., Merckling, N., Eberhardt, K., and Karrer, P. (1954) Helv. Chim. Acfa 37, 375-377. 13. Ellman, G. L. (1959) Arch. Biochem. Biophys. 82, 70-77. 14. Avi-Dor, Y. (1960) B&hem. J. 76, 370-374. 15. Tate, S. S., and Meister, A. (1974) J. Biol. Chem. 249,7593-7602. 16. Berghauser, J., Falderbaum, I., and Woenckhaus, C. (1971) Hoppe-Seyler’s Z. Physiol. Chem. 352, 52-58. 17. Toennies, G., and Levine, T. F. (1934) J. Biol. Chem. 105, 115-121. 18. Ricci, G., Santoro, L., Achilli, M., Matarese, R. M., Nardini, M., and Cavallini, D. (1983) J. Biol. Chem. 258, 1051 I-10517. 19. Omstein, L., and Davis, B. J. (1964) Ann. N. Y. Ad. Sci. 121, 32 l-330. 20. Ricci, G., Nardini, M., Chiaraluce, R., Dupre, S., and Cavallini, D. (1983) J. Appl. Biochem. 5, 320-329. 21. Schubert, M. P. (1936) J. Biol. Chem. 114, 341350.