ANALYTICAL
BIOCHEMISTRY
82, 204-209 (1977)
A Spectrophotometric Assay for Adenine Phosphoribosyltransferase C. SALERNO* AND A. GIACOMELLO~ * Institute
of Biological Chemistry and Center t Institute of Rheumatology,
of Molecular University
Biology of Rome,
of C.N.R. Italy
Rome,
and
Received March 9, 1977; accepted May 9, 1977 The present paper describes a new spectrophotometric assay for adenine phosphoribosyltransferase activity which is highly reproducible, rapid, sensitive, and simpler than the isotopic assays for this enzyme. This assay is based on the quantitative measurement of the product AMP by a NADH-coupled enzyme method.
In recent years, a number of enzymatic abnormalities that may lead to an accelerated rate of de ~OVO purine biosynthesis and hyperuricemia have been described (1). The demonstration of the deficiency of hypoxanthine-guanine phosphoribosyltransferase (HGPRT)’ (EC 2.4.2.8) in the Lesch-Nyhan syndrome and in some patients with gout (2,3) led to a reevaluation of the importance of the “salvage pathway” in the regulation of purine metabolism. Adenine phosphoribosyltransferase (APRT) (EC 2.4.2.7), a closely related enzyme which also operates in the “salvage pathway,” catalyzes the transfer of the phosphoribosyl moiety of phosphoribosylpyrophosphate (PRPP) to the N-9 position of adenine, with production of adenine monophosphate (AMP) and pyrophosphate (PPi). Although at least four families with a genetically determined partial deficiency of APRT activity in circulating erythrocytes have been described (4-7), at present, no disease has been unequivocally associated with this enzyme defect. Elevated APRT activity has been observed in erythrocytes from patients with the Lesch-Nyhan syndrome and from some gouty subjects with partial HGPRT deficiency (2). Despite the theoretically important role of APRT in puke metabolism, an understanding of the clinical and biochemical significance of alterations in APRT activity requires further investigation. Existing methods for the quantitative assay of this enzyme are rather complex and expensive and I Abbreviations used; HGPRT, hypoxanthine-guanine phosphoribosyltransferase; APRT, adenine phosphoribosyltransferase; MK, myokinase; PK, pyruvate kinase; LDH, lactate dehydrogenase; PRPP, phosphoribosylpyrophosphate; AMP, adenosine 5’-monophosphate; ATP, adenosine 5’-triphosphate; PEP, phosphoenolpyruvate; PP,, pyrophosphate. 204 Copyright All rights
0 1977 by Academic Press. Inc. of reproduction in any form reserved.
ISSN 0003-2697
ADENINE
PHOSPHORIBOSYLTRANSFERASE
ASSAY
205
require scintillation equipment which is not usually available in the clinical laboratory. These methods are based upon separation of the isotopically labeled product from its substrate by high-voltage electrophoresis (3,8), paper (9) and thin-layer (10) chromatography, or precipitation of the nucleotide as the lanthanum salt (11). The present paper describes a rapid, sensitive, and simple spectrophotometric assay of APRT activity. MATERIALS
All enzymes were commercial samples from Boehringer A. G. ATP and ADP from Fluka were purified by ion-exchange chromatography according to Caldwell (12). Other reagents were high-purity commercial samples from Boehringer A. G., Sigma, and Merck A. G. PRPP was dissolved in water, stored frozen, and kept in a ice bath before use. METHODS
APRT was assayed in erythrocyte lysate from human heparinized venous blood. Hemolysates were prepared according to published procedures (3,lO). Plasma and leukocytes were removed by centrifugation. Erythrocytes were washed twice in isotonic saline and were lysed by freezing and thawing twice in a dry ice-acetone bath. The cell debris was removed by centrifugation, and the hemolysates were dialyzed against 0.01~ Tris-HCI buffer, pH 7.4, containing 5 mM MgS04, for 6 hr at 4°C. Proteins in hemolysates were measured by the method of Lowry et al. (13). APRT was assayed by a two-step procedure: First, the APRT-catalyzed reaction was conducted at 37°C and was terminated by addition of perchloric acid; second, the reaction product, AMP, was then measured spectrophotometrically by the NADH-coupled enzyme method of Adam (14). The incubation mixtures for the two-step assay were designed after published procedures (3,10,14). Step 1: APRT-Catalyzed
Reaction
The reaction mixture contained: 50 mM Tris-HCl, pH 7.4,5 mM MgS04, 0.96 mM adenine, 1.4 mM PRPP tetrasodium salt, and 1.7- 10.3 mg/ml of protein from dialyzed hemolysate. All incubations were conducted at 37”C, and PRPP was added last to initiate the reaction. One-milliliter aliquots of the reaction mixture were added at different periods of time to 1 ml of 1 M ice-cold perchloric acid. The precipitated protein was removed by centrifugation. A 1.5-ml volume of the supernatant was neutralized by adding 0.3 ml of a solution containing 0.5 M triethanolamine-HCl and 2.0 M K&O,. After incubation in an ice bath for 10 min, the potassium per-
206
SALERNO
AND
GIACOMELLO
chlorate precipitate was removed by centrifugation, and the supernatant was used for the quantitative determination of AMP. Step 2: AMP Assay
AMP was measured by the following
NADH-linked
AMP + ATP % 2 ADP + 2 PEP 2 pyruvate
2ADP
PK, Mg++, K+
+ 2 NADH
+ 2 H+ -
method:
> 2 ATP + 2 pyruvate
LDH
2 lactate + 2 NAD+.
The reaction mixture contained 1 ml of the supernatant obtained in Step 1, 1 pmol of PEP, 0.25 pmol of NADH, 0.12 pmol of ATP, 40 pmol of MgS04, 130 hmol of KCl, 3 IU of LDH, 3 IU of PK in a final volume of 1.27 ml. The first spectrophotometric reading was made at 340 nm to determine the initial absorbance. A lo-p1 volume of 500 III/ml of MK was then added to the mixture, and the reaction was followed to its completion at 340 nm. The reaction went to completion after 30 min at 25°C. AMP concentration was calculated from the optical density variation at 340 nm, assuming a molar extinction coefficient of 6220 for NADH and considering that 2 mol of NAD are formed from 1 mol of AMP (14). RESULTS
AND DISCUSSION
The calibration curve for the AMP assay obtained by applying the procedure described in Step 2 on AMP solutions of known concentration is reported in Fig. 1. The relationship between the AMP concentration and the optical density variation at 340 nm is linear in the range studied. Two
[AMP]
FIG. 1. Calibration
x 10~ M
curve for the enzymatic assay of AMP.
ADENINE
PHOSPHORIBOSYLTRANSFERASE
ASSAY
207
moles of NAD are formed from one mole of AMP, confirming the results of Adam (14). From the same experiment, a standard deviation of ? 1.3 nmol was obtained for the AMP assay. No difference in the relationship between AMP concentration and optical density variation at 340 nm was observed, when known aliquots of AMP (4 x IO+ to 10-4~) were added to the reaction mixture for APRT assay in the absence of PRPP, and protein precipitation and supernatant neutralization were carried out as described in Step 1. Other control experiments showed that PRPP at the concentrations employed and PPi (product of the APRT-catalyzed reaction) added in equimolar amounts to AMP (3 x lo+ to 5.2 x 10e5M) do not influence the AMP assay. Under the assay conditions described, the APRT-catalyzed reaction followed a linear course during the first hour (Fig. 2); the slope of the straight line describing the reaction progress was a linear function of the hemolysate protein concentration (Fig. 3). Control experiments without PRPP or adenine displayed no activity. Other control experiments showed that AMP does not break down significantly during incubation with erythrocyte hemolysate under the conditions of the APRT assay. Trivial effects and the presence of APRT activity in the enzyme system used for the AMP determination do not occur since no activity was observed with acid-denatured red cell lysate. The precision of the APRT activity determination was -t 1.4 nmol/hr/mg of protein hemolysate on a day when I5 measurements were carried out on same blood sample The mean hemolysate APRT activity in 21 healthy adult males (mean age, 35 years) with serum uric acid concentrations within the normal range (4.10-5.62 mg/lOO ml) was 23 2 7.7 (SD) nmol/hr/mg of protein hemolysate. This result is comparable with those obtained by other authors using
20
40
TIME FIG. 2. Progress curve of the APRT-catalyzed protein in the APRT assay was 6.6 mg/ml.
I
1
60
I
(min.)
reaction. The concentration of hemolysate
208
SALERNO
AND GIACOMELLO
I
V
4
2
I
I
6
I
8
I
IO
FIG. 3. APRT activity as a function of hemolysate concentration. Optical density variation at 340 nm in 20 min as a function of hemolysate protein concentration.
isotopic procedures, incubation temperature of 37-38”C, and the same standard conditions of pH, substrate and magnesium ion concentration (Table 1). In Table 1, the age and sex distributions of the subjects studied are not included since APRT activity in dialyzed erythrocyte hemolysates has been demonstrated to be independent of these factors (9). This new assay for red blood cell APRT activity is rapid and sensitive and eliminates the expense and equipment requirements of radioisotopic assays. This assay is suitable for routine laboratory use. Furthermore, even though the present investigators have confined their work to determining APRT activity in human red blood cells, the method should be TABLE APRT
ACTIVITY
IN DIALYZED
1
ERYTHROCYTE
HEMOLYSATE~
Mean values
Standard deviation
Number of subjects studied
Reference
Kelley et al. (3) (radiochemical)
31.2 15.36
6 2.44
32 8
3 15
Emerson et al. (8) (radiochemical)
17.2 20.1
2.9 2.5
24 22
8 16
Cartier and Hamet (9) (radiochemical)
34.8 21
3.3 4.55
29 30
9 17
Sperling et al. (10) (radiochemical)
10.44
2.4
30
10
This paper (spectrophotometric)
23
7.7
21
Assay method
This paper
a Activity expressed as nanomoles of AMP formed per milligram of protein per hour.
ADENINE
PHOSPHORIBOSYLTRANSFERASE
ASSAY
209
generally applicable to detection of enzyme activity in other tissues, provided that dialysis or other means have been employed to remove endogenous interfering substances and that such tissues contain no phosphatase converting AMP to adenosine. REFERENCES 1. Kelley, W. N., and Wyngaarden, J. B. (1974) in Advances in Enzymology (A. Maister, ed.), Vol. 41, pp. l-33, John Wiley, New York. 2. Seegmiller, J. E., Rosenbloon, F. M., and Kelley, W. N. (1967)Science 155, 1682- 1684. 3. Kelley, W. N., Rosenbloon, F. M., Henderson, J. F., and Seegmiller, J. E. (1967) Proc. Nat. Acad. Sci. USA 57, 1735-1739. 4. Kelley, W. N., Levy, R. I., Rosenbloon, F. M., Henderson, J. F., and Seegmiller, J. E. (l%S) J. Clin. Invest. 47,2281-2288. 5. FOX, I. H., Meade, J. C., and Kelley, W. N. (1973) Amer. J. Med. 55, 614-620. 6. Delbarre, F., Auscher, C., Amor, B., and De Gery, A. (1974) in Purine Metabolism in Man (Sperling, 0.. De Vries, A., and Wyngaarden, J. B., eds.), pp. 333-339, Plenum Press, New York. 7. Emmerson, B. T., Gordon, R. B., and Thompson L. (1974) in Purine Metabolism in Man (Sperling, O., De Vries, A., and Wyngaarden, J. B., eds.), pp. 327-331, Plenum Press, New York. 8. Emmerson, B. T., Thompson, C. J., and Wallace. D. C. (1972) Ann. Znt. Med. 76, 285-287. 9. Cartier, P., and Hamet, M. (1968) Clin. Chim. Acta 20, 205-214, 10. Sperling, O., Frank, M.. Ophir, R., Liberman, U. A., Adam. A., and De Vries. A. (1970) Eur. J. C/in. Biol. Res. 15, 942-947. 11. Bakay, B., Telfer, M. A.. and Nyhan. W. L. (1969) Biochem. Med. 3, 230-235. 12. Caldwell, I. C. (1969) J. Chromatogr. 44, 331-341. 13. Lowry, 0. H.. Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193,265-275. 14. Adam, H. (1961) Biochem. 2. 335, 25-36. 15. Mueller, M. M., and Stemberg, H. (1974) in Purine Metabolism in Man (Sperling, O., De Vries, A., and Wyngaarden, J. B., eds.), pp. 187-194, Plenum Press, New York. 16. Gordon, R. B., Thompson, L., and Emmerson, B. T. (1974) M&&o&m 23, 921-927. 17. Delbarre, F., Cattier, P., Auscher, C., De Gery, A., and Hamet, M. (1970) Presse M&d. 78, 729-734.
BIOCHEMISTRY
82, 204-209 (1977)
A Spectrophotometric Assay for Adenine Phosphoribosyltransferase C. SALERNO* AND A. GIACOMELLO~ * Institute
of Biological Chemistry and Center t Institute of Rheumatology,
of Molecular University
Biology of Rome,
of C.N.R. Italy
Rome,
and
Received March 9, 1977; accepted May 9, 1977 The present paper describes a new spectrophotometric assay for adenine phosphoribosyltransferase activity which is highly reproducible, rapid, sensitive, and simpler than the isotopic assays for this enzyme. This assay is based on the quantitative measurement of the product AMP by a NADH-coupled enzyme method.
In recent years, a number of enzymatic abnormalities that may lead to an accelerated rate of de ~OVO purine biosynthesis and hyperuricemia have been described (1). The demonstration of the deficiency of hypoxanthine-guanine phosphoribosyltransferase (HGPRT)’ (EC 2.4.2.8) in the Lesch-Nyhan syndrome and in some patients with gout (2,3) led to a reevaluation of the importance of the “salvage pathway” in the regulation of purine metabolism. Adenine phosphoribosyltransferase (APRT) (EC 2.4.2.7), a closely related enzyme which also operates in the “salvage pathway,” catalyzes the transfer of the phosphoribosyl moiety of phosphoribosylpyrophosphate (PRPP) to the N-9 position of adenine, with production of adenine monophosphate (AMP) and pyrophosphate (PPi). Although at least four families with a genetically determined partial deficiency of APRT activity in circulating erythrocytes have been described (4-7), at present, no disease has been unequivocally associated with this enzyme defect. Elevated APRT activity has been observed in erythrocytes from patients with the Lesch-Nyhan syndrome and from some gouty subjects with partial HGPRT deficiency (2). Despite the theoretically important role of APRT in puke metabolism, an understanding of the clinical and biochemical significance of alterations in APRT activity requires further investigation. Existing methods for the quantitative assay of this enzyme are rather complex and expensive and I Abbreviations used; HGPRT, hypoxanthine-guanine phosphoribosyltransferase; APRT, adenine phosphoribosyltransferase; MK, myokinase; PK, pyruvate kinase; LDH, lactate dehydrogenase; PRPP, phosphoribosylpyrophosphate; AMP, adenosine 5’-monophosphate; ATP, adenosine 5’-triphosphate; PEP, phosphoenolpyruvate; PP,, pyrophosphate. 204 Copyright All rights
0 1977 by Academic Press. Inc. of reproduction in any form reserved.
ISSN 0003-2697
ADENINE
PHOSPHORIBOSYLTRANSFERASE
ASSAY
205
require scintillation equipment which is not usually available in the clinical laboratory. These methods are based upon separation of the isotopically labeled product from its substrate by high-voltage electrophoresis (3,8), paper (9) and thin-layer (10) chromatography, or precipitation of the nucleotide as the lanthanum salt (11). The present paper describes a rapid, sensitive, and simple spectrophotometric assay of APRT activity. MATERIALS
All enzymes were commercial samples from Boehringer A. G. ATP and ADP from Fluka were purified by ion-exchange chromatography according to Caldwell (12). Other reagents were high-purity commercial samples from Boehringer A. G., Sigma, and Merck A. G. PRPP was dissolved in water, stored frozen, and kept in a ice bath before use. METHODS
APRT was assayed in erythrocyte lysate from human heparinized venous blood. Hemolysates were prepared according to published procedures (3,lO). Plasma and leukocytes were removed by centrifugation. Erythrocytes were washed twice in isotonic saline and were lysed by freezing and thawing twice in a dry ice-acetone bath. The cell debris was removed by centrifugation, and the hemolysates were dialyzed against 0.01~ Tris-HCI buffer, pH 7.4, containing 5 mM MgS04, for 6 hr at 4°C. Proteins in hemolysates were measured by the method of Lowry et al. (13). APRT was assayed by a two-step procedure: First, the APRT-catalyzed reaction was conducted at 37°C and was terminated by addition of perchloric acid; second, the reaction product, AMP, was then measured spectrophotometrically by the NADH-coupled enzyme method of Adam (14). The incubation mixtures for the two-step assay were designed after published procedures (3,10,14). Step 1: APRT-Catalyzed
Reaction
The reaction mixture contained: 50 mM Tris-HCl, pH 7.4,5 mM MgS04, 0.96 mM adenine, 1.4 mM PRPP tetrasodium salt, and 1.7- 10.3 mg/ml of protein from dialyzed hemolysate. All incubations were conducted at 37”C, and PRPP was added last to initiate the reaction. One-milliliter aliquots of the reaction mixture were added at different periods of time to 1 ml of 1 M ice-cold perchloric acid. The precipitated protein was removed by centrifugation. A 1.5-ml volume of the supernatant was neutralized by adding 0.3 ml of a solution containing 0.5 M triethanolamine-HCl and 2.0 M K&O,. After incubation in an ice bath for 10 min, the potassium per-
206
SALERNO
AND
GIACOMELLO
chlorate precipitate was removed by centrifugation, and the supernatant was used for the quantitative determination of AMP. Step 2: AMP Assay
AMP was measured by the following
NADH-linked
AMP + ATP % 2 ADP + 2 PEP 2 pyruvate
2ADP
PK, Mg++, K+
+ 2 NADH
+ 2 H+ -
method:
> 2 ATP + 2 pyruvate
LDH
2 lactate + 2 NAD+.
The reaction mixture contained 1 ml of the supernatant obtained in Step 1, 1 pmol of PEP, 0.25 pmol of NADH, 0.12 pmol of ATP, 40 pmol of MgS04, 130 hmol of KCl, 3 IU of LDH, 3 IU of PK in a final volume of 1.27 ml. The first spectrophotometric reading was made at 340 nm to determine the initial absorbance. A lo-p1 volume of 500 III/ml of MK was then added to the mixture, and the reaction was followed to its completion at 340 nm. The reaction went to completion after 30 min at 25°C. AMP concentration was calculated from the optical density variation at 340 nm, assuming a molar extinction coefficient of 6220 for NADH and considering that 2 mol of NAD are formed from 1 mol of AMP (14). RESULTS
AND DISCUSSION
The calibration curve for the AMP assay obtained by applying the procedure described in Step 2 on AMP solutions of known concentration is reported in Fig. 1. The relationship between the AMP concentration and the optical density variation at 340 nm is linear in the range studied. Two
[AMP]
FIG. 1. Calibration
x 10~ M
curve for the enzymatic assay of AMP.
ADENINE
PHOSPHORIBOSYLTRANSFERASE
ASSAY
207
moles of NAD are formed from one mole of AMP, confirming the results of Adam (14). From the same experiment, a standard deviation of ? 1.3 nmol was obtained for the AMP assay. No difference in the relationship between AMP concentration and optical density variation at 340 nm was observed, when known aliquots of AMP (4 x IO+ to 10-4~) were added to the reaction mixture for APRT assay in the absence of PRPP, and protein precipitation and supernatant neutralization were carried out as described in Step 1. Other control experiments showed that PRPP at the concentrations employed and PPi (product of the APRT-catalyzed reaction) added in equimolar amounts to AMP (3 x lo+ to 5.2 x 10e5M) do not influence the AMP assay. Under the assay conditions described, the APRT-catalyzed reaction followed a linear course during the first hour (Fig. 2); the slope of the straight line describing the reaction progress was a linear function of the hemolysate protein concentration (Fig. 3). Control experiments without PRPP or adenine displayed no activity. Other control experiments showed that AMP does not break down significantly during incubation with erythrocyte hemolysate under the conditions of the APRT assay. Trivial effects and the presence of APRT activity in the enzyme system used for the AMP determination do not occur since no activity was observed with acid-denatured red cell lysate. The precision of the APRT activity determination was -t 1.4 nmol/hr/mg of protein hemolysate on a day when I5 measurements were carried out on same blood sample The mean hemolysate APRT activity in 21 healthy adult males (mean age, 35 years) with serum uric acid concentrations within the normal range (4.10-5.62 mg/lOO ml) was 23 2 7.7 (SD) nmol/hr/mg of protein hemolysate. This result is comparable with those obtained by other authors using
20
40
TIME FIG. 2. Progress curve of the APRT-catalyzed protein in the APRT assay was 6.6 mg/ml.
I
1
60
I
(min.)
reaction. The concentration of hemolysate
208
SALERNO
AND GIACOMELLO
I
V
4
2
I
I
6
I
8
I
IO
FIG. 3. APRT activity as a function of hemolysate concentration. Optical density variation at 340 nm in 20 min as a function of hemolysate protein concentration.
isotopic procedures, incubation temperature of 37-38”C, and the same standard conditions of pH, substrate and magnesium ion concentration (Table 1). In Table 1, the age and sex distributions of the subjects studied are not included since APRT activity in dialyzed erythrocyte hemolysates has been demonstrated to be independent of these factors (9). This new assay for red blood cell APRT activity is rapid and sensitive and eliminates the expense and equipment requirements of radioisotopic assays. This assay is suitable for routine laboratory use. Furthermore, even though the present investigators have confined their work to determining APRT activity in human red blood cells, the method should be TABLE APRT
ACTIVITY
IN DIALYZED
1
ERYTHROCYTE
HEMOLYSATE~
Mean values
Standard deviation
Number of subjects studied
Reference
Kelley et al. (3) (radiochemical)
31.2 15.36
6 2.44
32 8
3 15
Emerson et al. (8) (radiochemical)
17.2 20.1
2.9 2.5
24 22
8 16
Cartier and Hamet (9) (radiochemical)
34.8 21
3.3 4.55
29 30
9 17
Sperling et al. (10) (radiochemical)
10.44
2.4
30
10
This paper (spectrophotometric)
23
7.7
21
Assay method
This paper
a Activity expressed as nanomoles of AMP formed per milligram of protein per hour.
ADENINE
PHOSPHORIBOSYLTRANSFERASE
ASSAY
209
generally applicable to detection of enzyme activity in other tissues, provided that dialysis or other means have been employed to remove endogenous interfering substances and that such tissues contain no phosphatase converting AMP to adenosine. REFERENCES 1. Kelley, W. N., and Wyngaarden, J. B. (1974) in Advances in Enzymology (A. Maister, ed.), Vol. 41, pp. l-33, John Wiley, New York. 2. Seegmiller, J. E., Rosenbloon, F. M., and Kelley, W. N. (1967)Science 155, 1682- 1684. 3. Kelley, W. N., Rosenbloon, F. M., Henderson, J. F., and Seegmiller, J. E. (1967) Proc. Nat. Acad. Sci. USA 57, 1735-1739. 4. Kelley, W. N., Levy, R. I., Rosenbloon, F. M., Henderson, J. F., and Seegmiller, J. E. (l%S) J. Clin. Invest. 47,2281-2288. 5. FOX, I. H., Meade, J. C., and Kelley, W. N. (1973) Amer. J. Med. 55, 614-620. 6. Delbarre, F., Auscher, C., Amor, B., and De Gery, A. (1974) in Purine Metabolism in Man (Sperling, 0.. De Vries, A., and Wyngaarden, J. B., eds.), pp. 333-339, Plenum Press, New York. 7. Emmerson, B. T., Gordon, R. B., and Thompson L. (1974) in Purine Metabolism in Man (Sperling, O., De Vries, A., and Wyngaarden, J. B., eds.), pp. 327-331, Plenum Press, New York. 8. Emmerson, B. T., Thompson, C. J., and Wallace. D. C. (1972) Ann. Znt. Med. 76, 285-287. 9. Cartier, P., and Hamet, M. (1968) Clin. Chim. Acta 20, 205-214, 10. Sperling, O., Frank, M.. Ophir, R., Liberman, U. A., Adam. A., and De Vries. A. (1970) Eur. J. C/in. Biol. Res. 15, 942-947. 11. Bakay, B., Telfer, M. A.. and Nyhan. W. L. (1969) Biochem. Med. 3, 230-235. 12. Caldwell, I. C. (1969) J. Chromatogr. 44, 331-341. 13. Lowry, 0. H.. Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193,265-275. 14. Adam, H. (1961) Biochem. 2. 335, 25-36. 15. Mueller, M. M., and Stemberg, H. (1974) in Purine Metabolism in Man (Sperling, O., De Vries, A., and Wyngaarden, J. B., eds.), pp. 187-194, Plenum Press, New York. 16. Gordon, R. B., Thompson, L., and Emmerson, B. T. (1974) M&&o&m 23, 921-927. 17. Delbarre, F., Cattier, P., Auscher, C., De Gery, A., and Hamet, M. (1970) Presse M&d. 78, 729-734.
