BIOCHEMICAL
MEDICINE
AND
METABOLIC
BIOLOGY
47, 232-241 (1992)
Purification and Properties of AMP-Deaminase GRZEGORZ NOWAK Department
of Biochemistry,
Academic
AND KRYSTIAN
Medical
School,
from Human Kidney
KALETHA~
SO-211 Gdatisk,
ul. Dgbinki
1, Poland
Received January 24, 1992 AMP-deaminase from human kidney (cortex and medulla) was purified and the physicochemical properties were characterized. The enzyme from both portions of the kidney exhibited identical kinetics and regulatory properties. At optimal pH (6.6), the AMPdeaminase studied exhibited a distinctly sigmoidal substrate saturation kinetics, with the half-saturation parameter (.S,,..5)as high as 10 mM. ATP at 1 mM strongly activated the enzyme, decreasing S,,, nearly lo-fold. The activating effect of ADP was less strong. Orthophosphate inhibited the enzyme, but the inhibition observed was weak (K, = 16 mM) and had a pure competitive character. At pH 7.2, physiological for the-kidney cortex, orthophosphate inhibition became even weaker and became partially competitive. Variations in the adenylate energy charge had potent effects on the activity of AMP-deaminase, depending on the size of the total adenine nucleotide pool examined. The results of gel filtration and SDS-PAGE indicated that human kidney AMP-deaminase is an oligomeric enzyme composed of four, probably identical, subunits weighing about 37 kDa each. Q iw Academic Press. Inc.
AMP-deaminase (EC 3.5.4.6), the enzyme catalyzing irreversible hydrolytic deamination of adenylic acid, is widely distributed in vertebrate tissues (1). The physiological significance of AMP-deaminase in cellular metabolism is still unclear, but the existence of the tissue-specific enzyme variants (2) indicates that it must be connected with the metabolic specificity of the tissue. Among many roles postulated for this enzyme, stabilization of the adenylate energy charge (3), regulation of the purine nucleotide pool size (4), and replenishment of cellular citric acid cycle intermediates (5) are most frequently quoted. In the kidney cortex, the participation of AMP-deaminase (through the purine nucleotide cycle) in ammoniagenesis also has been proposed (6). Despite many experimental efforts (7-9), the role of the purine nucleotide cycle in kidney cortex ammonia production remained unclear until now. Arguments questioning such a role have included the fact that (skeletal muscle) AMP-deaminase is inhibited by orthophosphate (10). It was recently recognized (11,12) that a multigene family consisting of three different genes is responsible for production of AMP-deaminase in various rat ’ To whom correspondence should be addressed. 232 08854505/92 $5.00 Copyright 0 1992 by Academic Press, Inc. AI1 rights of reproduction in any form reserved.
PURIFICATION
AND PROPERTIES
OF AMP-DEAMINASE
233
and human tissues. The ampdl gene encodes isozyme M, which is specific to mature skeletal muscle. The ampd3 gene encodes isozyme El, which is specific to erythrocytes. The ampd2 gene, encoding isozyme L of AMP-deaminase, is expressed in most other tissues of the adult rat and human (11,12). putatively including those of uterus and kidney. Little attention has been paid to AMP-deaminase from the kidney thus far. In 1980 Prus et al. (13) partially purified AMP-deaminase from pig kidney and described some of its properties, mainly the influence of liposomes. In this paper some properties of AMP-deaminase purified from human kidney are described. MATERIALS Isolation
AND METHODS
of the Enzyme
Human kidneys (from the Department of Forensic Medicine, taken in the course of autopsies of young, healthy subjects, 12-24 h after death) were washed, weighed, and frozen at -20°C. After fat and membranes were removed, kidneys were dissected along their longitudinal axis, and the cortex was separated from the medulla by dissection through the corticomedullary junction. The obtained tissue was homogenized in 3 vol (v/w) of extraction buffer (0.089 M phosphate buffer, pH 6.5, containing 0.18 M KC1 and 1 mM thioethanol) using a Waring blender-type homogenizer and then centifuged twice (first 30 min at 3OOOg and then 30 min at 18,000g). AMP-deaminase was subsequently isolated by phosphocellulose chromatography, essentially according to the procedure of Smiley et al. (14). The column (2.6 x 20 cm) with the enzyme adsorbed on the phosphocellulose was washed first with 1 liter of the extraction buffer and then with 0.2 liter of 0.4 M KC1 solution. After this the enzyme was eluted stepwise with 0.75 M KC1 and then with a linear (0.75-2.0 M) KC1 gradient. The most active fractions of the main (second) activity peak (see Fig. 1) were pooled and readsorbed onto phosphocellulose. The adsorbed enzyme was washed twice (first with 100 ml of 0.4 M KC1 and then with 100 ml of 0.75 M KCl) and then eluted with a linear (0.75-2.0 M) KC1 gradient. The activity released formed one symmetrical peak correlated very well with the concomitant protein peak (not shown here). No AMP-deaminase activity was detected in the 0.75 M KC1 eluate during this chromatography. The enzyme preparation obtained was purified about 1800-fold, and its specific activity (measured at 10 mM substrate concentration) was about 15 ~mol/min/mg of protein. This preparation (free from adenosine deaminase and 5’-nucleotidase activity) was used for kinetic experiments. For electrophoretic analysis the rechromatographed enzyme preparation was concentrated on a Schleicher-Schuell collodion apparatus to about 1.5 ml, applied to a 60 x 1.5 cm Sepharose CL-6B column, and eluted with 0.1 M sodium cacodylate buffer, pH 7.0, with the addition of 0.5 M KCl. Enzyme Assay Activity of AMP-deaminase was estimated calorimetrically, according to the phenol-hypochlorite method of Chaney and Marbach (15). The incubation me-
234
NOWAK
AND KALETHA
dium, in a final volume of 0.5 ml, contained 0.1 M sodium cacodylate buffer, pH 6.6 or 7.2, 100 mM potassium chloride, the substrate (nine different concentrations ranging from 0.4 to 51.2 mM), and, where indicated, the effector (ATP, ADP, or orthophosphate). After equilibration of the incubation medium at 30°C 50 /..d of the enzyme solution (containing about 2 pg of protein) was added to start the reaction. All incubations were carried out for 20 min, and the velocity of the reaction was determined from the mean amount of ammonia liberated in three parallel incubations. No evidence of enzyme denaturation was observed, as judged from the proportionality of the ammonia liberation versus time for the period of reaction. The kinetic parameters of the reaction (maximum velocity of the reaction, V,,, ; half-saturation constant, So,5; and cooperativity coefficient, nn) were computed as described previously (16). Protein was measured by the method of Bradford (17). Adenylate energy charge was generated as described previously (18). The equilibrium concentrations of the nucleotides were calculated on the basis of the value of 0.8 for the equilibrium constant of the adenylate kinase reaction (3). The total concentration of adenine nucleotides was kept constant at either 2.5 or 10 mM. SDS-PAGE was performed as described previously (19). Reagents ATP and ADP (Na+ salts), 5’-AMP (free acid), and AMP analogues (listed in Table 2) were supplied by Sigma (St. Louis, MO); cellulose phosphate was from Whatman (Maidstone, Kent, UK). Electrophoretic reagents were supplied by BioRad (Richmond, CA) and LMW standards by Pharmacia (Uppsala, Sweden). All other chemicals were from Polskie Odczynniki Chemiczne (Gliwice, Poland). RESULTS measured in human kidney extract was about protein and was the same in both parts (cortex
The activity of AMP-deaminase 0.008 pmol/min/mg of extractable and medulla) of the organ. Figure 1 presents the results of chromatography of human kidney cortex AMPdeaminase on a phosphocellulose column. As may be seen from this figure, the enzyme eluted from the column in the form of two well-separated activities. The same elution profile was obtained when AMP-deaminase from kidney medulla was chromatographed. The activity eluted by 0.75 M KC1 always constituted less than 10% of the total activity released. The enzyme form present in this eluate manifested a hyperbolic type of substrate saturation kinetics (not shown here), with an So.5 of (evaluated at pH 6.6) about 3 mM. The greater part of the AMPdeaminase activity eluted from the column at a higher (1.0-1.1 M KCl) concentration. Figure 2 presents a set of kinetic curves produced by human kidney cortex AMP-deaminase (the rechromatographed enzyme form eluted within the second, main activity peak presented in Fig. l), while measured at optimal pH 6.6 in the absence (control conditions) and in the presence of several important regulatory ligands. As may be seen from this figure, in the absence of regulatory ligands,
PURIFICATION
AND PROPERTIES
OF AMP-DEAMINASE
235
2 s
1.4
E d 12 ‘=-
I2 i
Fraction
number
FIG. 1. Elution profile of human kidney AMP-deaminase tions of 4 ml volume were collected.
20
from a phosphocellulose column. Frac-
30
40mM
[AMPI
FIG. 2. Effect of substrate concentration on the velocity of the reaction catalyzed by human kidney AMP-deaminase. The reaction was measured in the absence (x) or in the presence of 1 mM ATP (A), 1 mM ADP (V), or 2.5 mr+r orthophosphate (H).
236
NOWAK
AND KALETHA
TABLE 1 The Effect of Regulatory Ligands on S,, and V,,,, Values of the Reaction Catalyzed by Human Kidney AMP-Deaminase Effector added None 1 mM ATP 1 mM ADP 2.5 rnM Pi 2.5 mM Pi + 1 mM ATP
Scl.5 (m4 9.9 1.1 3.2 12.7 2.8
V nmx (% of the control)
(1.3) (0.9) (1.1) (2.2) (1.1)
Note. Assay conditions are 0.1 M cacodylate buffer, pH 6.6, with 0.1 represent SD from three experiments.
100 106 105 101 102 M
2.0 1.1 1.4 1.9 1.2
KCl. The values in parentheses
the enzyme studied followed distinctly sigmoidal (nH = 2.0) substrate kinetics with the half-saturation constant value (,!&) as high as 10 mM (Table 1). The addition of 2.5 mM orthophosphate to the incubation medium further increased this value to about 13 mM, hardly influencing the shape of the kinetic curve. The observed inhibitory effect was weak (Ki = 16.1 ( + 1.8) mM) and had a pure competitive character (Fig. 3). In contrast to the above, ATP strongly activated human kidney cortex AMP-deaminasethe kinetic curve produced by 1 mM ATPactivated enzyme had a regular hyperbolic shape, and the value of S0.5 was as low as 1 mM, i.e., lo-fold lower than that calculated for control conditions (Table 1). In the presence of both ATP (1 mM) and orthophosphate (2.5 mM), the kinetic curve (not shown on Fig. 2) again had a regular hyperbolic shape, but the value of S,,, was almost 3-fold higher (Table 1). The activating effect of ADP was weaker than that of ATP, and the shape of the kinetic curve produced by 1 mM ADP-activated enzyme deviated slightly from a regular hyperbola (Table 1, Fig. 2). None of the effecters tested influenced the maximum velocity of the reaction (Table 1). At pH 7.2, physiological for the kidney cortex (20), the effecters investigated had similar modes of regulation, but the effects observed (especially that exerted by orthophosphate) were less accentuated. The value of &, 12.4 (-+ 1.8) mM under control conditions, was 13.7 (k2.2) mM after the addition of 2.5 mM orthophosphate to the incubation medium. Further increases in orthophosphate concentration to 5 and 10 mM raised the value of S,,, to 14.9 (22.6) and 16.1 (& 3.1) mM, respectively. Insertion of the experimental data into the Hill-type equation (21) allowed the generation of a set of nonlinear, hyperbolic relationships (Fig. 3) characteristic of a partially competitive type of inhibition (21,22). The curves presented in Fig. 4 demonstrate the response of kidney cortex AMPdeaminase to variation in adenylate energy charge value, measured at physiological pH (7.2) and at two different concentrations of the adenylate pool. It may be seen from this figure that the character of the above response depends very much on the concentration of the adenylate pool tested. At a nucleotide pool concentration of 2.5 mM, the response curve manifested a clearly bell-shaped profile, and an optimum at OS-O.6 energy charge value could be observed. With an
PURIFICATION
-0.5
AND PROPERTIES
0.0
k% II
237
OF AMP-DEAMINASE
0.5
1.0
FIG. 3. Inhibition of human kidney AMP-deaminase by orthophosphate (Loftfield-Eigner plot). The experimental kinetic data were inserted into a Hill-type equation (22) giving a set of straight line or hyperbolic relationships, characteristic of pure competitive or partially competitive inhibition, respectively. The influence of orthophosphate was measured at substrate concentrations of 6.4 mM (A) and 12.8 mM (0).
adenylate nucleotide pool concentration of 10 mM, a plateau between 0.3 and 0.7 energy charge value was evident. In the physiologically important range of adenylate energy charge (0.7 to 0.9), the activity of AMP-deaminase increased very sharply with the decrease of the energy charge value. The results presented in Table 2 indicate that human kidney cortex AMPdeaminase has high substrate specificity. Among several substrate analogues tested only dAMP was perceivably deaminated by enzyme from both parts of this organ. The gel filtration of the twice chromatographed AMP-deaminase on a Sepharose CLdB column (not shown here) revealed the presence of one symmetrical activity peak eluting just behind the aldolase (158 kDa) standard. This allowed us to estimate the molecular weight of the native enzyme at about 150 kDa. SDSpolyacrylamide gel electrophoresis (Fig. 5) disclosed the presence of one distinct
238
NOWAK
6.0
I
4.0
I *
2.0
l-9
AND KALETHA
MEDICINE
AND
METABOLIC
BIOLOGY
47, 232-241 (1992)
Purification and Properties of AMP-Deaminase GRZEGORZ NOWAK Department
of Biochemistry,
Academic
AND KRYSTIAN
Medical
School,
from Human Kidney
KALETHA~
SO-211 Gdatisk,
ul. Dgbinki
1, Poland
Received January 24, 1992 AMP-deaminase from human kidney (cortex and medulla) was purified and the physicochemical properties were characterized. The enzyme from both portions of the kidney exhibited identical kinetics and regulatory properties. At optimal pH (6.6), the AMPdeaminase studied exhibited a distinctly sigmoidal substrate saturation kinetics, with the half-saturation parameter (.S,,..5)as high as 10 mM. ATP at 1 mM strongly activated the enzyme, decreasing S,,, nearly lo-fold. The activating effect of ADP was less strong. Orthophosphate inhibited the enzyme, but the inhibition observed was weak (K, = 16 mM) and had a pure competitive character. At pH 7.2, physiological for the-kidney cortex, orthophosphate inhibition became even weaker and became partially competitive. Variations in the adenylate energy charge had potent effects on the activity of AMP-deaminase, depending on the size of the total adenine nucleotide pool examined. The results of gel filtration and SDS-PAGE indicated that human kidney AMP-deaminase is an oligomeric enzyme composed of four, probably identical, subunits weighing about 37 kDa each. Q iw Academic Press. Inc.
AMP-deaminase (EC 3.5.4.6), the enzyme catalyzing irreversible hydrolytic deamination of adenylic acid, is widely distributed in vertebrate tissues (1). The physiological significance of AMP-deaminase in cellular metabolism is still unclear, but the existence of the tissue-specific enzyme variants (2) indicates that it must be connected with the metabolic specificity of the tissue. Among many roles postulated for this enzyme, stabilization of the adenylate energy charge (3), regulation of the purine nucleotide pool size (4), and replenishment of cellular citric acid cycle intermediates (5) are most frequently quoted. In the kidney cortex, the participation of AMP-deaminase (through the purine nucleotide cycle) in ammoniagenesis also has been proposed (6). Despite many experimental efforts (7-9), the role of the purine nucleotide cycle in kidney cortex ammonia production remained unclear until now. Arguments questioning such a role have included the fact that (skeletal muscle) AMP-deaminase is inhibited by orthophosphate (10). It was recently recognized (11,12) that a multigene family consisting of three different genes is responsible for production of AMP-deaminase in various rat ’ To whom correspondence should be addressed. 232 08854505/92 $5.00 Copyright 0 1992 by Academic Press, Inc. AI1 rights of reproduction in any form reserved.
PURIFICATION
AND PROPERTIES
OF AMP-DEAMINASE
233
and human tissues. The ampdl gene encodes isozyme M, which is specific to mature skeletal muscle. The ampd3 gene encodes isozyme El, which is specific to erythrocytes. The ampd2 gene, encoding isozyme L of AMP-deaminase, is expressed in most other tissues of the adult rat and human (11,12). putatively including those of uterus and kidney. Little attention has been paid to AMP-deaminase from the kidney thus far. In 1980 Prus et al. (13) partially purified AMP-deaminase from pig kidney and described some of its properties, mainly the influence of liposomes. In this paper some properties of AMP-deaminase purified from human kidney are described. MATERIALS Isolation
AND METHODS
of the Enzyme
Human kidneys (from the Department of Forensic Medicine, taken in the course of autopsies of young, healthy subjects, 12-24 h after death) were washed, weighed, and frozen at -20°C. After fat and membranes were removed, kidneys were dissected along their longitudinal axis, and the cortex was separated from the medulla by dissection through the corticomedullary junction. The obtained tissue was homogenized in 3 vol (v/w) of extraction buffer (0.089 M phosphate buffer, pH 6.5, containing 0.18 M KC1 and 1 mM thioethanol) using a Waring blender-type homogenizer and then centifuged twice (first 30 min at 3OOOg and then 30 min at 18,000g). AMP-deaminase was subsequently isolated by phosphocellulose chromatography, essentially according to the procedure of Smiley et al. (14). The column (2.6 x 20 cm) with the enzyme adsorbed on the phosphocellulose was washed first with 1 liter of the extraction buffer and then with 0.2 liter of 0.4 M KC1 solution. After this the enzyme was eluted stepwise with 0.75 M KC1 and then with a linear (0.75-2.0 M) KC1 gradient. The most active fractions of the main (second) activity peak (see Fig. 1) were pooled and readsorbed onto phosphocellulose. The adsorbed enzyme was washed twice (first with 100 ml of 0.4 M KC1 and then with 100 ml of 0.75 M KCl) and then eluted with a linear (0.75-2.0 M) KC1 gradient. The activity released formed one symmetrical peak correlated very well with the concomitant protein peak (not shown here). No AMP-deaminase activity was detected in the 0.75 M KC1 eluate during this chromatography. The enzyme preparation obtained was purified about 1800-fold, and its specific activity (measured at 10 mM substrate concentration) was about 15 ~mol/min/mg of protein. This preparation (free from adenosine deaminase and 5’-nucleotidase activity) was used for kinetic experiments. For electrophoretic analysis the rechromatographed enzyme preparation was concentrated on a Schleicher-Schuell collodion apparatus to about 1.5 ml, applied to a 60 x 1.5 cm Sepharose CL-6B column, and eluted with 0.1 M sodium cacodylate buffer, pH 7.0, with the addition of 0.5 M KCl. Enzyme Assay Activity of AMP-deaminase was estimated calorimetrically, according to the phenol-hypochlorite method of Chaney and Marbach (15). The incubation me-
234
NOWAK
AND KALETHA
dium, in a final volume of 0.5 ml, contained 0.1 M sodium cacodylate buffer, pH 6.6 or 7.2, 100 mM potassium chloride, the substrate (nine different concentrations ranging from 0.4 to 51.2 mM), and, where indicated, the effector (ATP, ADP, or orthophosphate). After equilibration of the incubation medium at 30°C 50 /..d of the enzyme solution (containing about 2 pg of protein) was added to start the reaction. All incubations were carried out for 20 min, and the velocity of the reaction was determined from the mean amount of ammonia liberated in three parallel incubations. No evidence of enzyme denaturation was observed, as judged from the proportionality of the ammonia liberation versus time for the period of reaction. The kinetic parameters of the reaction (maximum velocity of the reaction, V,,, ; half-saturation constant, So,5; and cooperativity coefficient, nn) were computed as described previously (16). Protein was measured by the method of Bradford (17). Adenylate energy charge was generated as described previously (18). The equilibrium concentrations of the nucleotides were calculated on the basis of the value of 0.8 for the equilibrium constant of the adenylate kinase reaction (3). The total concentration of adenine nucleotides was kept constant at either 2.5 or 10 mM. SDS-PAGE was performed as described previously (19). Reagents ATP and ADP (Na+ salts), 5’-AMP (free acid), and AMP analogues (listed in Table 2) were supplied by Sigma (St. Louis, MO); cellulose phosphate was from Whatman (Maidstone, Kent, UK). Electrophoretic reagents were supplied by BioRad (Richmond, CA) and LMW standards by Pharmacia (Uppsala, Sweden). All other chemicals were from Polskie Odczynniki Chemiczne (Gliwice, Poland). RESULTS measured in human kidney extract was about protein and was the same in both parts (cortex
The activity of AMP-deaminase 0.008 pmol/min/mg of extractable and medulla) of the organ. Figure 1 presents the results of chromatography of human kidney cortex AMPdeaminase on a phosphocellulose column. As may be seen from this figure, the enzyme eluted from the column in the form of two well-separated activities. The same elution profile was obtained when AMP-deaminase from kidney medulla was chromatographed. The activity eluted by 0.75 M KC1 always constituted less than 10% of the total activity released. The enzyme form present in this eluate manifested a hyperbolic type of substrate saturation kinetics (not shown here), with an So.5 of (evaluated at pH 6.6) about 3 mM. The greater part of the AMPdeaminase activity eluted from the column at a higher (1.0-1.1 M KCl) concentration. Figure 2 presents a set of kinetic curves produced by human kidney cortex AMP-deaminase (the rechromatographed enzyme form eluted within the second, main activity peak presented in Fig. l), while measured at optimal pH 6.6 in the absence (control conditions) and in the presence of several important regulatory ligands. As may be seen from this figure, in the absence of regulatory ligands,
PURIFICATION
AND PROPERTIES
OF AMP-DEAMINASE
235
2 s
1.4
E d 12 ‘=-
I2 i
Fraction
number
FIG. 1. Elution profile of human kidney AMP-deaminase tions of 4 ml volume were collected.
20
from a phosphocellulose column. Frac-
30
40mM
[AMPI
FIG. 2. Effect of substrate concentration on the velocity of the reaction catalyzed by human kidney AMP-deaminase. The reaction was measured in the absence (x) or in the presence of 1 mM ATP (A), 1 mM ADP (V), or 2.5 mr+r orthophosphate (H).
236
NOWAK
AND KALETHA
TABLE 1 The Effect of Regulatory Ligands on S,, and V,,,, Values of the Reaction Catalyzed by Human Kidney AMP-Deaminase Effector added None 1 mM ATP 1 mM ADP 2.5 rnM Pi 2.5 mM Pi + 1 mM ATP
Scl.5 (m4 9.9 1.1 3.2 12.7 2.8
V nmx (% of the control)
(1.3) (0.9) (1.1) (2.2) (1.1)
Note. Assay conditions are 0.1 M cacodylate buffer, pH 6.6, with 0.1 represent SD from three experiments.
100 106 105 101 102 M
2.0 1.1 1.4 1.9 1.2
KCl. The values in parentheses
the enzyme studied followed distinctly sigmoidal (nH = 2.0) substrate kinetics with the half-saturation constant value (,!&) as high as 10 mM (Table 1). The addition of 2.5 mM orthophosphate to the incubation medium further increased this value to about 13 mM, hardly influencing the shape of the kinetic curve. The observed inhibitory effect was weak (Ki = 16.1 ( + 1.8) mM) and had a pure competitive character (Fig. 3). In contrast to the above, ATP strongly activated human kidney cortex AMP-deaminasethe kinetic curve produced by 1 mM ATPactivated enzyme had a regular hyperbolic shape, and the value of S0.5 was as low as 1 mM, i.e., lo-fold lower than that calculated for control conditions (Table 1). In the presence of both ATP (1 mM) and orthophosphate (2.5 mM), the kinetic curve (not shown on Fig. 2) again had a regular hyperbolic shape, but the value of S,,, was almost 3-fold higher (Table 1). The activating effect of ADP was weaker than that of ATP, and the shape of the kinetic curve produced by 1 mM ADP-activated enzyme deviated slightly from a regular hyperbola (Table 1, Fig. 2). None of the effecters tested influenced the maximum velocity of the reaction (Table 1). At pH 7.2, physiological for the kidney cortex (20), the effecters investigated had similar modes of regulation, but the effects observed (especially that exerted by orthophosphate) were less accentuated. The value of &, 12.4 (-+ 1.8) mM under control conditions, was 13.7 (k2.2) mM after the addition of 2.5 mM orthophosphate to the incubation medium. Further increases in orthophosphate concentration to 5 and 10 mM raised the value of S,,, to 14.9 (22.6) and 16.1 (& 3.1) mM, respectively. Insertion of the experimental data into the Hill-type equation (21) allowed the generation of a set of nonlinear, hyperbolic relationships (Fig. 3) characteristic of a partially competitive type of inhibition (21,22). The curves presented in Fig. 4 demonstrate the response of kidney cortex AMPdeaminase to variation in adenylate energy charge value, measured at physiological pH (7.2) and at two different concentrations of the adenylate pool. It may be seen from this figure that the character of the above response depends very much on the concentration of the adenylate pool tested. At a nucleotide pool concentration of 2.5 mM, the response curve manifested a clearly bell-shaped profile, and an optimum at OS-O.6 energy charge value could be observed. With an
PURIFICATION
-0.5
AND PROPERTIES
0.0
k% II
237
OF AMP-DEAMINASE
0.5
1.0
FIG. 3. Inhibition of human kidney AMP-deaminase by orthophosphate (Loftfield-Eigner plot). The experimental kinetic data were inserted into a Hill-type equation (22) giving a set of straight line or hyperbolic relationships, characteristic of pure competitive or partially competitive inhibition, respectively. The influence of orthophosphate was measured at substrate concentrations of 6.4 mM (A) and 12.8 mM (0).
adenylate nucleotide pool concentration of 10 mM, a plateau between 0.3 and 0.7 energy charge value was evident. In the physiologically important range of adenylate energy charge (0.7 to 0.9), the activity of AMP-deaminase increased very sharply with the decrease of the energy charge value. The results presented in Table 2 indicate that human kidney cortex AMPdeaminase has high substrate specificity. Among several substrate analogues tested only dAMP was perceivably deaminated by enzyme from both parts of this organ. The gel filtration of the twice chromatographed AMP-deaminase on a Sepharose CLdB column (not shown here) revealed the presence of one symmetrical activity peak eluting just behind the aldolase (158 kDa) standard. This allowed us to estimate the molecular weight of the native enzyme at about 150 kDa. SDSpolyacrylamide gel electrophoresis (Fig. 5) disclosed the presence of one distinct
238
NOWAK
6.0
I
4.0
I *
2.0
l-9
AND KALETHA
