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Presence, preliminary properties and partial purification of 5-phosphoribosylpyrophosphate amidotransferase from Artemia sp.
114
Biochimica et Biophysica Acta, 1033 (1990) 114-1.17
Elsevier BBAGEN 20253
BBA Report
Presence, preliminary properties and partial purification of 5-phosphoribosylpyrophosphate amidotransferase from A rtemia sp. Antonio Liras, Luisa Argomaniz and Pilar Llorente Departamento de Bioqulmica and lnstituto de lnoestigaciones Biom~dicas del C. S.L C., Facultad de Medicina de la Universidad Aut6noma de Madrid, Madrid (Spain)
(Received 20 July 1989)
Key words: Phosphoribosylpyrophosphate amidotransferase; Purine; ( A rtemia)
5'-Phosphoribosylpyrophosphate amidotransferase, which catalyzes the synthesis of phosphoribosylamine in the de novo synthesis of purine nucleotides, has been detected and partially purified approx. 800-fold from A rtemia sp. nauplii. The apparent K m values for 5'-phosphoribosyi 1-pyrophosphate as substrate were 0.7 mM and 0.4 mM in the presence of glutamine and ammonia as nitrogenous sources, respectively, and the enzymatic activity was inhibited by purine 5'-ribonucleotide compounds and 5', 5 " .pl, p4.diguanosine tetraphosphate.
5 '-Phosphoribosylpyrophosphate amidotransferase (PRPP-AT) (EC 2.4.2.14) catalyzes the irreversible reaction from 5'-phosphoribosyl 1-pyrophosphate (PRPP) to form 5'-phosphoribosyl-l-amine (PRA), the initial specific intermediate of purine biosynthesis de novo, in the presence of glutamine and/or ammonia as nitrogenous source, and which is considered the primary site of feedback regulation of this pathway [1,2]. This enzyme has been purified from a number of eukaryotic cells, including avian liver [3], rat hepatocytes [4] and human tissues [5], with a high requirement for divalent cations [6] and the presence of nonheme iron in the molecule [7], which implies an extreme instability of the purified enzyme to oxidation reactions. PRPP-AT is competitively inhibited with respect to PRPP [8] by purine 5'-ribonucleotides, end products of the de novo purine biosynthetic pathway. Previous studies in our laboratory [9] have demonstrated that purine de novo biosynthesis is operative in early larval development of Artemia sp., as measured by in vivo incorporation of [u-t4C]glycine and [t4C]bicarbonate as purine precursors, into cellular purines, and that the rate of incorporation increases during larval development of Artemia.
Abbreviation: PRPP-AT, phosphoribosyl; GP4G, 5', 5 ,,,.pl p4_di_ guanosine tetraphosphate; PRA, 5'-phosphoribosyl-l-amine Correspondence: P. Liorente, Instituto Investigaciones Biom6dicas del C.S.I.C., Arzobispo Morcillo 4, 28029 Madrid, Spain.
In this paper, we show the presence and partial purification of PRPP-AT from Arternia nauplii, its characterization with respect to some kinetic properties and its inhibition by purine nucleotides and GP4G, the major nucleotide in encysted embryos of the brine shrimp Artemia [10,11]. Artemia cysts, from San Francisco Bay Brand Co., were treated as described [12], and growth of Artemia nauplii was initiated by incubation of freshly hydrated cysts in Dutrieu saline medium [13] diluted 5-fold. At the appropriate developmental stages, PRPP-AT was assayed by radiometric procedures which were slightly modified [9]. Assay A determines the PRPP-dependent conversion of [14C]glutamine to [14C]glutamate [6] in the presence of glutamine as a nitrogenous source and the purified enzyme preparation (approx. 0.2 mg protein). The reaction was initiated by addition of 1.0 #Ci [t4C]glutamine and stopped after 30 min of incubation at 37°C by spotting a 20 #l aliquot of the reaction mixture on Whatman No. 3 MM chromatography paper with 5 #l of L-glutamine and L-glutamate as a carrier (0.5 #mol each). Glutamate and glutamine were then separated by high-voltage electrophoresis and the spots were identified with ninhydrin, cut out and the radioactivity in each amino acid was determined. Assay B determines the formation of a stable adduct between the phosphoribosylamine and [35S]cysteine [14], to evaluate the role of ammonia as nitrogen donor for the enzyme. The reaction was started with the enzyme extract and was stopped by the addition of 10 #l of 200 mM disodium EDTA, after an incubation at 37°C for
0304-4165/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
115 TABLE I Purification of PRPP-A T from Artemia nauplii
/0
o ~
e~...~o
1.0
o
Step
Volume Protein Activity a Spec.act. Purifi(ml) (mg) (mU) (mU/mg) cation (fold)
Precipitation at pH 5.0 8.0 (NH4)2SO4 (0-55%) 2.2 ATP-agarose • 0.7
285.0
1.4
0.005
m w
i I0
1
/7 .'
m
33.0 0.2
3.3 0.8
0.100 3.950
20 790
The activity was assayed by determining the conversion of []4Clglutamine to [14C]glutamate.
g
/--
o tla
c
1.6 |
u
o
dul gg
I •
30 min. A 20/~1 aliquot of reaction mixture and 10 #1 of carrier, 0.1 M ribose 5-phosphate/L-cysteine, were spotted on W h a t m a n No. 3 M M c h r o m a t o g r a p h y paper. L-Cysteine and PRA-cysteine complex were separated b y h i g h - v o l t a g e e l e c t r o p h o r e s i s , identified with ninhydrin and their radioactivity was determined. Protein was determined by the m e t h o d of L o w r y et al. [15]. Partial purification of P R P P - A T was carried out at 4 ° C and buffers used were degassed under v a c u u m to remove dissolved oxygen. The purification steps from 40-h-old Artemia nauplii are shown in Table I. Animals, which were at different developmental stages after incubation of cysts in growth medium, were homogenized with 2 vol. of ice-cold 50 m M Tris-HC1, ( p H 7.5) containing 0.2 M sucrose, 20 m M 2-mercaptoethanol and 2 m M MgC12, and the h o m o g e n a t e was centrifuged at 20000 × g for 10 min, and the supernatant after centrifugation at 100 000 × g was slowly adjusted to p H 5.1 by addition of 5% acetic acid with stirring for exactly 2 min. The precipitate was separated by centrif-
0.|
<
0
i
i
o
, I
IU
I/l~ppJ,~'~ 0
i
0
I
!
1
l
I
4
S
(pApP) . u
Fig. 2. Michaelis-Menten plots for PRPP-AT. The enzyme activity was determined by assay A (e) and assay B (o) at various PRPP concentrations, as described in Materials and Methods. The inset shows the Lineweaver-Burk plot of initial velocity versus PRPP concentration for determination of the g m values.
ugation at 20000 × g for 10 min, adjusted to p H 7.0 with 1.0 M K O H and P R P P - A T was precipitated b y the addition of a m m o n i u m sulfate to 55% saturation for 2 h. T h e e n z y m e preparation was suspended in 50 m M Tris-HC1, p H 7.5 buffer and desalted through a Sephadex G-25 column. The fractions containing peak enz y m e activity were p o o l e d a n d applied to an A T P - a g a r o s e c o l u m n (1 × 8 cm) (Fig. 1) equilibrated with 20 m M Tris-HC1, p H 7.9 buffer, containing 5 m M MgC12 and 5 m M dithiothreitol. P R P P - A T activity was eluted at a flow rate of 5 m l / h by 5 m M P R P P in the
I0,000
~
I00
PR~ A
0
'~
v
0.4
!
H 0 Q g~d
6,000
>..
>. FF"0
I,-
w
50
I,-
0
z 0.2 no~5 m
< ill >N Z gl
10
20 30 FRACTIONS
40
60
Fig. 1. Purification of PRPP-AT activity from Artemia nauplii by affinity chromatography on an ATP-agarose column. PRPP-AT was assayed by determining the conversion of []4C]glutamine to [14C]glutamate.
L Q.
NONE AMP ADP GMP GDP~0.1
0.6
1.0
2.0
J
GP~4G[lllM)
Fig. 3. Inhibition of PRPP-AT activity by purine nucleotides. The enzyme activity was determined by assay A in the presence of 1 mM purine nucleotides, except for enzyme assay with GP4G at different concentrations: 0.1, 0.5, 1.0 and 2.0 mM. The control enzyme assay was performed in the absence of purine nucleotides.
116 same buffer. An 800-fold purification was obtained with this procedure with respect to the acid-precipitation step. In the absence of nucleotide inhibitors, the substrate-veloeity curves of the purified Artemia PRPPAT were hyperbolic, using PRPP as substrate. Apparent K m values for PRPP calculated from double-reciprocal plots (Fig. 2) in the presence of glutamine and ammonia as nitrogenous sources, were 0.7 mM and 0.4 mM, respectively. The V~x value in the presence of glutamine was 15-fold greater than that which was obtained with ammonia. Purified PRPP-AT activity was inhibited by purine nucleotides (Fig. 3). At a 1 mM concentration, the inhibition by diphosphates was consistently more potent than inhibition by monophosphates and the most potent inhibitor was ADP. AMP, GMP and GP4G (at 0.1 mM and 0.5 mM) produce 40-60% inhibition - intermediate inhibition - and GDP, ADP and GP4G (above 1.0 mM) produce 60% to 95% inhibition - high inhibition. PRPP-AT activity increases during early larval development of Artemia, approx. 7 fold in 2-day-old Artemia with respect to newly hatched nauplii. At the nauplii hatching, nearly undetectable PRPP-AT activity was determined (0.6 m U / m g protein) and from 10, 20, 30, 40 and 50 h posthatching the PRPP-AT activity was 1.9, 2.6, 3.4, 3.8 and 4.2 m U / m g protein, respectively. Because Artemia nauplii are able to synthesize purine nucleotides using the de novo pathway [9], it is important to study the PRPP-AT activity which catalyzes the first reaction, the primary site of control in this pathway. We have purified this enzyme from Artemia nauplii grown in optimal salt concentration (0.1 M) for de novo purine biosynthesis (unpublished results). The procedure described here for purification, including in particular an acid precipitation step that implies a lack of contaminating glutaminase activity which may obscure PRPP-AT activity, and ATP-agarose affinity chromatography. The specific activity of our preparations, although lower, are comparable to that obtained by other authors [16,17]. As shown by the present study and unlike the PRPPAT activity from pigeon liver [8] or S. pombe [18], the Artemia enzyme did not show PRPP sigmoidal kinetics with glutamine and/or ammonia. The K m values for PRPP are within the concentration range previously reported for this enzyme in other systems [6,18,19]. Although Artemia PRPP-AT can utilize both glutamine and ammonia as nitrogen donors, the higher enzyme activity (Vmax) observed in the presence of glutamine when compared to that with ammonia and the slightly different K m values, lead us to consider the possibility that two independent isoenzymatic forms may be present in Artemia nauplii as has been described in human lymphoblasts [20]. On the other hand, our studies indicate that the reaction catalyzed by Artemia PRPP-AT
can be inhibited by purine nucleotides as feedback control by end-products in the purine nucleotide de novo biosynthesis pathway. Artemia PRPP-AT was highly sensitive to purine nucleotides such ADP, GDP and GP4G , while AMP and GMP are less effective, with a similar inhibition to that reported from pigeon liver [21]. However, important differences in the inhibition effect by purine nucleotides are dependent upon the degree of enzyme purification [6,22]. A 50% inhibition of enzyme activity has been obtained by 0.4 mM GPaG, which is consistent with our previous results [9] in demostrating that early enzymatic steps from the de novo purine biosynthetic pathway are regulated by this nucleotide in Artemia. PRPP-AT increases its activity during development of Artemia nauplii, in a qualitatively similar form to that observed for [U-14C]glycine incorporation into formylglycinamide ribonucleotide (FGAR) [9]. This increase, its inhibition by purine nucleotides, the finding of large quantities of the unusual nucleotide GP4G in the encysted embryos of Artemia and its progressive disappearance throughout development [23] together with GDP, which decreases to a barely detectable level in 3-day-old Artemia nauplii, leads us to consider the role of guanyl compounds, especially GP4G and GDP, as feasible potential regulators and modulators of de novo purine biosynthesis in Artemia. Then inactivation of derepression of this enzyme activity could play an important role in de novo purine biosynthesis in A rtemia. References 1 Wyngaarden, J.B. (1972) in Current Topics in Cellator Regulation (Horecker, B.L. and Stadman, E.R., eds.), Vol. 5, pp. 135-176, Academic Press, New York. 2 Wyngaarden, J.B. (1973) in The Enzymes of Glutamine Metabolism. (Prusiner, S. and Stadtman, E.R., eds.), pp. 365-386, Academic Press, San Diego. 3 Rowe, P.B. and Wyngaarden, J.B. (1968) J. Biol. Chem. 243, 6373-6383. 4 Itakura, M., Tsuchiya, M., Yamashita, K. and Oda, T. (1986) J. Pharmacol. Exp. Therap. 237, 794-798. 5 Itakura, M. and Holmes, E.W. (1979) J. Biol. Chem. 254, 333-338. 6 Holmes, E.W., McDonald, J.A., McCord, J.M., Wyngaarden, J.B. and Kelley, W.N. (1973) J. Biol. Chem. 248, 144-150. 7 Averill, B.A., Dwivedi, A., Debrurmer, P., Vollmer, S.J., Wong, J.Y. and Switzer, R.L. (1980) J. Biol. Chem. 255, 6007-6010. 8 Rowe, P.B., Coleman, M.D. and Wyngaarden, J.B. (1970) Biochemistry 9, 1498-1505. 9 Llorente, P., Carratalh, M., Liras, A. and Rotllhn, P. (1987) in Artemia Research and its Applications. (Decleir, W., Moens, L., Slegers, H., Jaspers, E. and Sorgeloos, P., eds.), Vol. 2, pp. 243-252, Universa Press, Wetteren. 10 Finamore, F.J. and Warner, A.H. (1963) J. Biol. Chem. 238, 344-348. 11 Clog,g, J.S., Warner, A.H. and Finamore, F.J. (1967) J. Biol. Chem. 242, 1938-1943. 12 Miralles, J., Sebastian, J., and Heredia, C.F. (1978) Biochim. Biophys. Acta 518, 326-332.
117 13 Dutrien, J. (1960) Arch. Zool. Exp. Gen. 99, 1-133. 14 King, (3.L. and Holmes, E.W. (1976) Anal. Biochem. 75, 30-39. 15 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. 16 Tsuda, M., Katunuma, N. and Weber, G. (1979) J. Biochem. 85, 1347-1354. 17 Hahn, R., Oberrauch, W. and Mecke, D. (1979) Biochim. Biophys. Acta 566, 152-156. 18 Nagy, M. (1970) Biochim. Biophys. Acta 198, 471-481.
19 Bagnara, S.S., Brox, L.W. and Henderson, J.F. (1974) Biochim. Biophys. Acta 350, 171-182. 20 Reem, G.H. (1974) J. Biol. Chem. 249, 1696-1703. 21 Wyngaarden, J.B. and Ashton, D.M. (1959) J. Biol. Chem. 234, 1492-1496. 22 ReenL G.H. and Friend, C. (1967) Science 157, 1203-1204. 23 Warner, A.H. and Finamore, F.J. (1967) J. Biol. Chem. 242, 1933-1937.
Biochimica et Biophysica Acta, 1033 (1990) 114-1.17
Elsevier BBAGEN 20253
BBA Report
Presence, preliminary properties and partial purification of 5-phosphoribosylpyrophosphate amidotransferase from A rtemia sp. Antonio Liras, Luisa Argomaniz and Pilar Llorente Departamento de Bioqulmica and lnstituto de lnoestigaciones Biom~dicas del C. S.L C., Facultad de Medicina de la Universidad Aut6noma de Madrid, Madrid (Spain)
(Received 20 July 1989)
Key words: Phosphoribosylpyrophosphate amidotransferase; Purine; ( A rtemia)
5'-Phosphoribosylpyrophosphate amidotransferase, which catalyzes the synthesis of phosphoribosylamine in the de novo synthesis of purine nucleotides, has been detected and partially purified approx. 800-fold from A rtemia sp. nauplii. The apparent K m values for 5'-phosphoribosyi 1-pyrophosphate as substrate were 0.7 mM and 0.4 mM in the presence of glutamine and ammonia as nitrogenous sources, respectively, and the enzymatic activity was inhibited by purine 5'-ribonucleotide compounds and 5', 5 " .pl, p4.diguanosine tetraphosphate.
5 '-Phosphoribosylpyrophosphate amidotransferase (PRPP-AT) (EC 2.4.2.14) catalyzes the irreversible reaction from 5'-phosphoribosyl 1-pyrophosphate (PRPP) to form 5'-phosphoribosyl-l-amine (PRA), the initial specific intermediate of purine biosynthesis de novo, in the presence of glutamine and/or ammonia as nitrogenous source, and which is considered the primary site of feedback regulation of this pathway [1,2]. This enzyme has been purified from a number of eukaryotic cells, including avian liver [3], rat hepatocytes [4] and human tissues [5], with a high requirement for divalent cations [6] and the presence of nonheme iron in the molecule [7], which implies an extreme instability of the purified enzyme to oxidation reactions. PRPP-AT is competitively inhibited with respect to PRPP [8] by purine 5'-ribonucleotides, end products of the de novo purine biosynthetic pathway. Previous studies in our laboratory [9] have demonstrated that purine de novo biosynthesis is operative in early larval development of Artemia sp., as measured by in vivo incorporation of [u-t4C]glycine and [t4C]bicarbonate as purine precursors, into cellular purines, and that the rate of incorporation increases during larval development of Artemia.
Abbreviation: PRPP-AT, phosphoribosyl; GP4G, 5', 5 ,,,.pl p4_di_ guanosine tetraphosphate; PRA, 5'-phosphoribosyl-l-amine Correspondence: P. Liorente, Instituto Investigaciones Biom6dicas del C.S.I.C., Arzobispo Morcillo 4, 28029 Madrid, Spain.
In this paper, we show the presence and partial purification of PRPP-AT from Arternia nauplii, its characterization with respect to some kinetic properties and its inhibition by purine nucleotides and GP4G, the major nucleotide in encysted embryos of the brine shrimp Artemia [10,11]. Artemia cysts, from San Francisco Bay Brand Co., were treated as described [12], and growth of Artemia nauplii was initiated by incubation of freshly hydrated cysts in Dutrieu saline medium [13] diluted 5-fold. At the appropriate developmental stages, PRPP-AT was assayed by radiometric procedures which were slightly modified [9]. Assay A determines the PRPP-dependent conversion of [14C]glutamine to [14C]glutamate [6] in the presence of glutamine as a nitrogenous source and the purified enzyme preparation (approx. 0.2 mg protein). The reaction was initiated by addition of 1.0 #Ci [t4C]glutamine and stopped after 30 min of incubation at 37°C by spotting a 20 #l aliquot of the reaction mixture on Whatman No. 3 MM chromatography paper with 5 #l of L-glutamine and L-glutamate as a carrier (0.5 #mol each). Glutamate and glutamine were then separated by high-voltage electrophoresis and the spots were identified with ninhydrin, cut out and the radioactivity in each amino acid was determined. Assay B determines the formation of a stable adduct between the phosphoribosylamine and [35S]cysteine [14], to evaluate the role of ammonia as nitrogen donor for the enzyme. The reaction was started with the enzyme extract and was stopped by the addition of 10 #l of 200 mM disodium EDTA, after an incubation at 37°C for
0304-4165/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
115 TABLE I Purification of PRPP-A T from Artemia nauplii
/0
o ~
e~...~o
1.0
o
Step
Volume Protein Activity a Spec.act. Purifi(ml) (mg) (mU) (mU/mg) cation (fold)
Precipitation at pH 5.0 8.0 (NH4)2SO4 (0-55%) 2.2 ATP-agarose • 0.7
285.0
1.4
0.005
m w
i I0
1
/7 .'
m
33.0 0.2
3.3 0.8
0.100 3.950
20 790
The activity was assayed by determining the conversion of []4Clglutamine to [14C]glutamate.
g
/--
o tla
c
1.6 |
u
o
dul gg
I •
30 min. A 20/~1 aliquot of reaction mixture and 10 #1 of carrier, 0.1 M ribose 5-phosphate/L-cysteine, were spotted on W h a t m a n No. 3 M M c h r o m a t o g r a p h y paper. L-Cysteine and PRA-cysteine complex were separated b y h i g h - v o l t a g e e l e c t r o p h o r e s i s , identified with ninhydrin and their radioactivity was determined. Protein was determined by the m e t h o d of L o w r y et al. [15]. Partial purification of P R P P - A T was carried out at 4 ° C and buffers used were degassed under v a c u u m to remove dissolved oxygen. The purification steps from 40-h-old Artemia nauplii are shown in Table I. Animals, which were at different developmental stages after incubation of cysts in growth medium, were homogenized with 2 vol. of ice-cold 50 m M Tris-HC1, ( p H 7.5) containing 0.2 M sucrose, 20 m M 2-mercaptoethanol and 2 m M MgC12, and the h o m o g e n a t e was centrifuged at 20000 × g for 10 min, and the supernatant after centrifugation at 100 000 × g was slowly adjusted to p H 5.1 by addition of 5% acetic acid with stirring for exactly 2 min. The precipitate was separated by centrif-
0.|
<
0
i
i
o
, I
IU
I/l~ppJ,~'~ 0
i
0
I
!
1
l
I
4
S
(pApP) . u
Fig. 2. Michaelis-Menten plots for PRPP-AT. The enzyme activity was determined by assay A (e) and assay B (o) at various PRPP concentrations, as described in Materials and Methods. The inset shows the Lineweaver-Burk plot of initial velocity versus PRPP concentration for determination of the g m values.
ugation at 20000 × g for 10 min, adjusted to p H 7.0 with 1.0 M K O H and P R P P - A T was precipitated b y the addition of a m m o n i u m sulfate to 55% saturation for 2 h. T h e e n z y m e preparation was suspended in 50 m M Tris-HC1, p H 7.5 buffer and desalted through a Sephadex G-25 column. The fractions containing peak enz y m e activity were p o o l e d a n d applied to an A T P - a g a r o s e c o l u m n (1 × 8 cm) (Fig. 1) equilibrated with 20 m M Tris-HC1, p H 7.9 buffer, containing 5 m M MgC12 and 5 m M dithiothreitol. P R P P - A T activity was eluted at a flow rate of 5 m l / h by 5 m M P R P P in the
I0,000
~
I00
PR~ A
0
'~
v
0.4
!
H 0 Q g~d
6,000
>..
>. FF"0
I,-
w
50
I,-
0
z 0.2 no~5 m
< ill >N Z gl
10
20 30 FRACTIONS
40
60
Fig. 1. Purification of PRPP-AT activity from Artemia nauplii by affinity chromatography on an ATP-agarose column. PRPP-AT was assayed by determining the conversion of []4C]glutamine to [14C]glutamate.
L Q.
NONE AMP ADP GMP GDP~0.1
0.6
1.0
2.0
J
GP~4G[lllM)
Fig. 3. Inhibition of PRPP-AT activity by purine nucleotides. The enzyme activity was determined by assay A in the presence of 1 mM purine nucleotides, except for enzyme assay with GP4G at different concentrations: 0.1, 0.5, 1.0 and 2.0 mM. The control enzyme assay was performed in the absence of purine nucleotides.
116 same buffer. An 800-fold purification was obtained with this procedure with respect to the acid-precipitation step. In the absence of nucleotide inhibitors, the substrate-veloeity curves of the purified Artemia PRPPAT were hyperbolic, using PRPP as substrate. Apparent K m values for PRPP calculated from double-reciprocal plots (Fig. 2) in the presence of glutamine and ammonia as nitrogenous sources, were 0.7 mM and 0.4 mM, respectively. The V~x value in the presence of glutamine was 15-fold greater than that which was obtained with ammonia. Purified PRPP-AT activity was inhibited by purine nucleotides (Fig. 3). At a 1 mM concentration, the inhibition by diphosphates was consistently more potent than inhibition by monophosphates and the most potent inhibitor was ADP. AMP, GMP and GP4G (at 0.1 mM and 0.5 mM) produce 40-60% inhibition - intermediate inhibition - and GDP, ADP and GP4G (above 1.0 mM) produce 60% to 95% inhibition - high inhibition. PRPP-AT activity increases during early larval development of Artemia, approx. 7 fold in 2-day-old Artemia with respect to newly hatched nauplii. At the nauplii hatching, nearly undetectable PRPP-AT activity was determined (0.6 m U / m g protein) and from 10, 20, 30, 40 and 50 h posthatching the PRPP-AT activity was 1.9, 2.6, 3.4, 3.8 and 4.2 m U / m g protein, respectively. Because Artemia nauplii are able to synthesize purine nucleotides using the de novo pathway [9], it is important to study the PRPP-AT activity which catalyzes the first reaction, the primary site of control in this pathway. We have purified this enzyme from Artemia nauplii grown in optimal salt concentration (0.1 M) for de novo purine biosynthesis (unpublished results). The procedure described here for purification, including in particular an acid precipitation step that implies a lack of contaminating glutaminase activity which may obscure PRPP-AT activity, and ATP-agarose affinity chromatography. The specific activity of our preparations, although lower, are comparable to that obtained by other authors [16,17]. As shown by the present study and unlike the PRPPAT activity from pigeon liver [8] or S. pombe [18], the Artemia enzyme did not show PRPP sigmoidal kinetics with glutamine and/or ammonia. The K m values for PRPP are within the concentration range previously reported for this enzyme in other systems [6,18,19]. Although Artemia PRPP-AT can utilize both glutamine and ammonia as nitrogen donors, the higher enzyme activity (Vmax) observed in the presence of glutamine when compared to that with ammonia and the slightly different K m values, lead us to consider the possibility that two independent isoenzymatic forms may be present in Artemia nauplii as has been described in human lymphoblasts [20]. On the other hand, our studies indicate that the reaction catalyzed by Artemia PRPP-AT
can be inhibited by purine nucleotides as feedback control by end-products in the purine nucleotide de novo biosynthesis pathway. Artemia PRPP-AT was highly sensitive to purine nucleotides such ADP, GDP and GP4G , while AMP and GMP are less effective, with a similar inhibition to that reported from pigeon liver [21]. However, important differences in the inhibition effect by purine nucleotides are dependent upon the degree of enzyme purification [6,22]. A 50% inhibition of enzyme activity has been obtained by 0.4 mM GPaG, which is consistent with our previous results [9] in demostrating that early enzymatic steps from the de novo purine biosynthetic pathway are regulated by this nucleotide in Artemia. PRPP-AT increases its activity during development of Artemia nauplii, in a qualitatively similar form to that observed for [U-14C]glycine incorporation into formylglycinamide ribonucleotide (FGAR) [9]. This increase, its inhibition by purine nucleotides, the finding of large quantities of the unusual nucleotide GP4G in the encysted embryos of Artemia and its progressive disappearance throughout development [23] together with GDP, which decreases to a barely detectable level in 3-day-old Artemia nauplii, leads us to consider the role of guanyl compounds, especially GP4G and GDP, as feasible potential regulators and modulators of de novo purine biosynthesis in Artemia. Then inactivation of derepression of this enzyme activity could play an important role in de novo purine biosynthesis in A rtemia. References 1 Wyngaarden, J.B. (1972) in Current Topics in Cellator Regulation (Horecker, B.L. and Stadman, E.R., eds.), Vol. 5, pp. 135-176, Academic Press, New York. 2 Wyngaarden, J.B. (1973) in The Enzymes of Glutamine Metabolism. (Prusiner, S. and Stadtman, E.R., eds.), pp. 365-386, Academic Press, San Diego. 3 Rowe, P.B. and Wyngaarden, J.B. (1968) J. Biol. Chem. 243, 6373-6383. 4 Itakura, M., Tsuchiya, M., Yamashita, K. and Oda, T. (1986) J. Pharmacol. Exp. Therap. 237, 794-798. 5 Itakura, M. and Holmes, E.W. (1979) J. Biol. Chem. 254, 333-338. 6 Holmes, E.W., McDonald, J.A., McCord, J.M., Wyngaarden, J.B. and Kelley, W.N. (1973) J. Biol. Chem. 248, 144-150. 7 Averill, B.A., Dwivedi, A., Debrurmer, P., Vollmer, S.J., Wong, J.Y. and Switzer, R.L. (1980) J. Biol. Chem. 255, 6007-6010. 8 Rowe, P.B., Coleman, M.D. and Wyngaarden, J.B. (1970) Biochemistry 9, 1498-1505. 9 Llorente, P., Carratalh, M., Liras, A. and Rotllhn, P. (1987) in Artemia Research and its Applications. (Decleir, W., Moens, L., Slegers, H., Jaspers, E. and Sorgeloos, P., eds.), Vol. 2, pp. 243-252, Universa Press, Wetteren. 10 Finamore, F.J. and Warner, A.H. (1963) J. Biol. Chem. 238, 344-348. 11 Clog,g, J.S., Warner, A.H. and Finamore, F.J. (1967) J. Biol. Chem. 242, 1938-1943. 12 Miralles, J., Sebastian, J., and Heredia, C.F. (1978) Biochim. Biophys. Acta 518, 326-332.
117 13 Dutrien, J. (1960) Arch. Zool. Exp. Gen. 99, 1-133. 14 King, (3.L. and Holmes, E.W. (1976) Anal. Biochem. 75, 30-39. 15 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. 16 Tsuda, M., Katunuma, N. and Weber, G. (1979) J. Biochem. 85, 1347-1354. 17 Hahn, R., Oberrauch, W. and Mecke, D. (1979) Biochim. Biophys. Acta 566, 152-156. 18 Nagy, M. (1970) Biochim. Biophys. Acta 198, 471-481.
19 Bagnara, S.S., Brox, L.W. and Henderson, J.F. (1974) Biochim. Biophys. Acta 350, 171-182. 20 Reem, G.H. (1974) J. Biol. Chem. 249, 1696-1703. 21 Wyngaarden, J.B. and Ashton, D.M. (1959) J. Biol. Chem. 234, 1492-1496. 22 ReenL G.H. and Friend, C. (1967) Science 157, 1203-1204. 23 Warner, A.H. and Finamore, F.J. (1967) J. Biol. Chem. 242, 1933-1937.