125
Biochimica et Biophysics @ Elsevier/North-Holland
Acta, 531 (1978) Biomedical
125-130
Press
BBA Report ___~_ ~~~~ BBA 51230
ENZYME ACTIVITIES LIVERS
CLAUDIA
MOORE,
IN LIPID-MODIFIED
TEN-CHING
LEE, NELSON
MEMBRANES
STEPHENS
and FRED
OF RAT
SNYDER
Medical and Health Sciences Division, Oak Ridge Associated Universities, and University of Tennessee-Oa& Ridge Graduate School of Biomedical Sciences, Oak Ridge, Term. 37830 (U.S.A.) (Received
June 2nd, 1978)
Summary The unnatural amino alcohol, N-isopropylethanolamine, is incorporated into phospholipids of various membrane fractions of rat liver after it is injected intraperitoneally. When this base analog was incorporated into the phospholipids of plasma membranes, the activity of 5’-nucleotidase in cell-free homogenates and in purified plasma membranes decreased by approx. 20--50% of that in the control preparations. Arrhenius plots for 5’-nucleotidase in both the control and isopropylethanolamine-treated plasma membranes had break points at the same temperatures. The enzymes from both control and isopropylethanolamine-treated membranes had identical apparent Michaelis constants, but different maximum velocities. Removal of phospholipids from control and isopropylethanolamine-treated plasma membrane preparations by phospholipase C treatment did not affect their 5’nucleotidase activities. In addition, incubation of control membranes in the presence of free isopropylethanolamine (l----l0 mM) did not alter the 5’-nucleotidase activity. Collectively, these data indicate the lowered 5’-nucleotidase activity in the isopropylethanolamine-treated membranes is due to a decreased amount of the enzyme and cannot be explained by the modification of the phospholipids within the membrane where the enzyme resides. Three other membrane-bound enzyme activities (NADH:cytochrome c reductase (EC 1.6.99.3), NADPH:cytochrome c reductase (EC 1.6.2.4), and succinic acid dehydrogenase (EC 1.3.99.1)) were not affected by the isopropylethanolamine treatment.
Membrane modifications induced by the incorporation of unnatural analogs of amino alcohols into lipids have been used to assess how po1.a.r head groups in phospholipids can affect the structural and functional properties of cell membranes [l-13]. In our laboratory, we have demonstrated that the isomeric analog of choline, N-isopropylethanolamine, can be incorporated into
126
the phospholipid fraction of L-M cells grown as monolayers [l-2] and subcellular membranes of the rat liver [3]. Incorporation of the analog into phosphatidyl-isopropylethanolamine of mouse L-M cells caused alterations in the glycerolipid biosynthesis by inhibiting the transport of the choline into the cells [23] and the incorporation of choline and ethanolamine into phosphatidylcholine and phosphatidylethanolamine [ 11. Incorporation of isopropylethanolamine also caused a decrease in stearoyl-CoA desaturation by preferentially inhibiting the terminal oxidase activity of the desaturase complex [ 21. It could not be determined whether the decreased activity was due to the presence of a modified lipid or to some other effect of the analog on the synthesis or regulation of the enzyme. When the analog was incorporated into lipids in rat livers, the phosphatidyl-isopropylethanolamine constituted 9% of the total phospholipids and was distributed in the plasma membrane, mitochondria, and microsomal fractions. In addition, a second unnatural phospholipid analog, phosphatidyl-Nmethyl-isopropylethanolamine (3---4% of total phospholipid phosphorus), was formed in the liver by N-methylation of the phosphatidyl-isopropylethanolamine [ 141. Although the phosphatidyl-isopropylethanolamine was formed at the expense of phosphatidylcholine and phosphatidylethanolamine, its fatty acid composition did not resemble either of these phospholipids [ 31. Schroeder et al. [5] have shown that when two other base analogs, N-methylethanolamine and NJ’-dimethylethanolamine, were incorporated into membrane phospholipids of the L-M cells, the phase transition temperatures, as measured by two fluorescent probes, were not altered in the plasma membrane, mitochondria, or microsomal fractions. This indicated that L-M cells have a mechanism by which they may maintain their membrane fluidity. In the present study, we examined the effect of a polar head group modification on the activities of several membrane-bound enzymes in rat livers. Only 5’-nucleotidase (EC 3.1.3.5) was altered by isopropylethanolamine. Analysis of kinetic data, the Arrhenius plots, the effect of removal of phospholipids from plasma membranes by phospholipase C treatment, and the effect of free isopropylethanolamine base on enzyme activity demonstrated that the isopropylethanolamine reduced 5’-nucleotidase activity by a mechanism that does not involve the alteration in the polar head group of the phospholipids in the plasma membrane. The isopropylethanolamine, synthesized as previously described [ 11, was dissolved in saline and adjusted to pH 7.0 with HCl. Buffalo-strain rats (male, 200-300 g, Simonsin Laboratories, Gilroy, Calif.), maintained on a Purina Laboratory Chow diet, were fasted the day before isopropylethanolamine was injected and throughout the remaining experimental period. We injected rats intraperitoneally with either isopropylethanolamine (20 mg/lOO g body weight per day) or saline for 4 days and then killed them 1 h after the final injection. The livers were removed immediately and homogenized in 5 mM Tris-HCl buffer (pH 7.4) containing 0.5 mM CaCl, and 0.25 M sucrose. We isolated the plasma membrane, mitochondrial, and microsomal fractions from control (saline-injected) and treated (isopropylethanolamine-injected) rat livers as described previously 131. Cell-free homogenates were made by homogenizing the
127
livers in the same manner and centrifuging them twice at 180 X g for 10 min. The cell-free supernatant was then assayed for 5’-nucleotidase activity. Plasma membrane phospholipids were hydrolyzed with phospholipase C (EC 3.1.4.3) (Bacillis cereus, from Grand Island Biological Co.) by a modification of the method of Emmelot and Bos 1151. Tubes containing 2.0-2.5 mg plasma membrane protein, 5 mM Tris-HCl (pH 7.3), 0.1 mM CaCl, , and 2OOpg of phospholipase C were incubated at 37°C for 1 h. After the reaction was terminated by placing the tubes in ice, the incubation mixtures were centrifuged for 30 min at 100 000 X g. The pellet was suspended in water and assays for phospholipid phosphorus and 5’-nucleotidase activities were performed. 5’-Nucleotidase activity was assayed in a total volume of 1 ml by the method of Song and Bodansky [ 161. After terminating the reaction by adding 1 ml 10% trichloroacetic acid, we measured the phosphate released from AMP by the method of Bartlett [17]. We obtained the Arrhenius plots by incubating the samples for 30 min at temperatures from 25 to 60°C using 10 mM AMP as substrate. The incubations for the kinetic studies, were carried out for 10 and 20 min at 3’7°C with substrate concentrations between 0.25 and 5 mM AMP. Total lipids were extracted from the plasma membranes by the method of Bligh and Dyer except the methanol contained 2% glacial acetic acid [ 181. The phospholipids were resolved by two-dimensional thin-layer chromatography on silica gel HR layers: first in chloroform/methanol/acetic acid (50 : 25 : 8, v/v), and then, after drying the plate for 1 h in a vacuum desiccator, in chloroform/methanol/ammonium hydroxide (65 : 35 : 5, v/v). Phosphorus was determined [19] on an aliquot of total phospholipids and in each phospholipid class that could be visualized after spraying the layers with concentrated HzS04 and charring at 180-200°C. Activities of NADH- and NADPH-dependent cytochrome c reductase were assayed by the method of Dallner [ 201. Succinic acid dehydrogenase was assayed as described by Eibl et al. [Zl]. Of the four enzymes studied, two from the microsomal fraction (NADHand NADPH:cytochrome c reductase), one from mitochondria (succinate dehydrogenase), and one from plasma membranes (5’~nucleotidase), only the latter was significantly affected by the isopropylethanolamine incorporation (Table I). We chose to study this enzyme further to determine if the effect of TABLE
I
EFFECT OF N-ISOPROPYLETHANOLAMINE ON ENZYME SUBCELLULAR FRACTIONS OF RAT LIVER Subcellular
fraction
Enzyme
ACTIVITIES Specific
activities
MicrosomaJ(3) Microsomal Mitochondrkl
(3) (3)
(4)*
5’-Nucleotidase (pg Pi/min per mg protein) NADH:cytochrome c reductase &mol/min per mg protein) NADPH:cytochrome c reductase (nmol/min per mg protein) Succinic dehydrogenase (nmollmin per mg protein)
~~__ *The numbers in&zheses indicate the numberof%%&?liular standard error: each preparation was assayed in duplicate. **p = < 0.01.
f SE.
Control
Isopropylethanolaminetreated
20.8 f 0.64**
12.4 * 1.87**
-____ Plasma membrane
IN DIFFERENT
0.39 f 0.03
0.40
f. 0.04
71.7 f 0.54
71.2 f 0.22
10.0 f 0.17
10.6 f 0.03
preparations
used to calculate
the
128
isopropylethanolamine was due solely to its presence as phosphatidyl-isopropylethanolamine in the membrane or whether some other mechanism was involved. The alteration in the 5’-nucleotidase activity could result from the purification steps involved in the isolation of the plasma membranes. To eliminate this possibility, we assayed three separate cell-free homogenates from livers of isopropylethanolamine-injected and control rats for 5’-nucleotidase activity. The enzyme from the treated rats also demonstrated a significant decrease in its total activity (isopropylethanolamine-treated = 168.1 f 7.8 pg Pi/ min per g tissue; control = 227.9 f 7.5 pg Pi/min per g tissue; the P value is < 0.01); this eliminates the purification of the plasma membrane fraction as a factor affecting the enzyme’s activity. The Arrhenius plots (Fig. 1) of the 5’-nucleotidase activity in plasma membranes of livers from control and isopropylethanolamine-injected rats showed a similar relationship for the plasma membrane lipids and the activity of this enzyme. The graphs paralleled one another with the break points in the curves for the two preparations occurring at identical temperatures (35, 37,45 and 50°C). The major difference between the two plots was the decrease (20-50%) in the overall activity of the 5’-nucleotidase in the plasma membranes of the rats injected with isopropylethanolamine. The parallel nature of the two curves indicates that the 5’-nucleotidase activity is not directly affected by the presence of a modified polar head group in the plasma membrane phospholipids. Therefore, we examined the type of inhibition in 5’-nucleotidase activity caused by isopropylethanolamine treatment.
.
3.1
32
l.OOO/Temperature Fig.
1. Arrhenius
ethanolamine-injected
plot
of (0)
5’-nucleotidase rats.
activity
in plasma
membranes
from
control
(*)
and isopropyl-
129
A double reciprocal plot of the activity of the enzyme versus substrate concentration (Fig. 2) revealed that the affinity of the enzyme for its substrate was not affected by the analog injection. Both enzyme preparations had a K, of about 0.3 mM. However, the maximum velocity of the 5’-nucleotidase from the preparations containing the analog was about 50% lower than that for the control enzyme (9.1 versus 1.8.9 pg phosphorus/min per mg protein). This is consistent with a noncompetitive type of inhibition and indicates that the amount of active enzyme was lowered either from decreased synthesis or through an irreversible binding of the phosphatidyl-isopropylethanolamine to the enzyme in such a way as to render it inactive. We further investigated the possibility that the phosphatidyl-isopropylethanolamine molecules in the plasma membrane could be acting as a noncompetitive inhibitor of the 5’nucleotidase. This was done by removing the phosphatidyl-isopropylethanolamine from the plasma membrane; up to 80% of the membrane phospholipids were removed by phospholipase C hydrolysis. Two-dimensional thin-layer chromatography and phosphorus analysis revealed that all of the phosphatidyl-isopropylethanolamine was removed and that sphingomyelin made up the remaining 20% of the phospholipase C-resistant phospholipids in the membranes. Even after removal of the isopropylethanol-
Fig. 2. Lineweaver-Burk plot of 5’-nucleotidase injected (cl) rats.
activity in control (0) and isopropylethanolamine-
TABLE II EFFECT OF PHOSPHOLIPASE C TREATMENT ON 5’-NUCLEOTIDASE AND PHOSPHOLIPIDS FROM CONTROL AND ISOPROPYLETHANOLAMINE-TREATED RAT LIVER PLASMA MEMBRANES Values are the average of duplicates. Control Phospholipase C 5’-Nucleotidase 018 phosphorus/min per mg protein) Phospholipid phosphorus (%Jo)
Isopropylethanolamine-treated
(-)
(+)
(-)
(+)
20.4 100
20.0 20.5
16.4 100
16.8 20.4
-
130
amine base from the phospholipids by this treatment, the 5’-nucleotidase activity was still inhibited in the analog-injected membrane preparations as compared with that in control membranes (Table II). This was also in agreement with the data in which the 5’nucleotidase activity remained unchanged after removing phospholipids from normal membranes with phospholipase A2 [ll], i.e., the 5’-nucleotidase does not require phospholipids for expression of its activity [ 221. Finally, we examined the effect of free isopropylethanolamine base on 5’nucleotidase activity of control membranes. We saw no change in the specific activity of the enzyme with any of the base analog concentrations (1, 5, and 10 mM) used (data not shown). These data indicate that the lowered activity of the 5’-nucleotidase in the isopropylethanolamine-injected rat liver plasma membranes was due to a decreased amount of the enzyme present. Since other laboratories have shown that polar head group analogs can alter enzyme activities strictly by their presence in the modified membrane (see, e.g., Engelhard et al. [7]), we conclude that amino alcohols can alter enzyme activities by different means. Our data suggest that the isopropylethanolamine analog acted as an inhibitor of cellular processes involving the regulation of membrane enzyme activities, perhaps at the point of protein synthesis. This work was supported by the United States Department of Energy, American Cancer Society (Grant K-701), National Cancer Institute (Grant CA 11949-08), and National Cancer Institute (Training Grant CA 09104). References 1
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Biochimica et Biophysics @ Elsevier/North-Holland
Acta, 531 (1978) Biomedical
125-130
Press
BBA Report ___~_ ~~~~ BBA 51230
ENZYME ACTIVITIES LIVERS
CLAUDIA
MOORE,
IN LIPID-MODIFIED
TEN-CHING
LEE, NELSON
MEMBRANES
STEPHENS
and FRED
OF RAT
SNYDER
Medical and Health Sciences Division, Oak Ridge Associated Universities, and University of Tennessee-Oa& Ridge Graduate School of Biomedical Sciences, Oak Ridge, Term. 37830 (U.S.A.) (Received
June 2nd, 1978)
Summary The unnatural amino alcohol, N-isopropylethanolamine, is incorporated into phospholipids of various membrane fractions of rat liver after it is injected intraperitoneally. When this base analog was incorporated into the phospholipids of plasma membranes, the activity of 5’-nucleotidase in cell-free homogenates and in purified plasma membranes decreased by approx. 20--50% of that in the control preparations. Arrhenius plots for 5’-nucleotidase in both the control and isopropylethanolamine-treated plasma membranes had break points at the same temperatures. The enzymes from both control and isopropylethanolamine-treated membranes had identical apparent Michaelis constants, but different maximum velocities. Removal of phospholipids from control and isopropylethanolamine-treated plasma membrane preparations by phospholipase C treatment did not affect their 5’nucleotidase activities. In addition, incubation of control membranes in the presence of free isopropylethanolamine (l----l0 mM) did not alter the 5’-nucleotidase activity. Collectively, these data indicate the lowered 5’-nucleotidase activity in the isopropylethanolamine-treated membranes is due to a decreased amount of the enzyme and cannot be explained by the modification of the phospholipids within the membrane where the enzyme resides. Three other membrane-bound enzyme activities (NADH:cytochrome c reductase (EC 1.6.99.3), NADPH:cytochrome c reductase (EC 1.6.2.4), and succinic acid dehydrogenase (EC 1.3.99.1)) were not affected by the isopropylethanolamine treatment.
Membrane modifications induced by the incorporation of unnatural analogs of amino alcohols into lipids have been used to assess how po1.a.r head groups in phospholipids can affect the structural and functional properties of cell membranes [l-13]. In our laboratory, we have demonstrated that the isomeric analog of choline, N-isopropylethanolamine, can be incorporated into
126
the phospholipid fraction of L-M cells grown as monolayers [l-2] and subcellular membranes of the rat liver [3]. Incorporation of the analog into phosphatidyl-isopropylethanolamine of mouse L-M cells caused alterations in the glycerolipid biosynthesis by inhibiting the transport of the choline into the cells [23] and the incorporation of choline and ethanolamine into phosphatidylcholine and phosphatidylethanolamine [ 11. Incorporation of isopropylethanolamine also caused a decrease in stearoyl-CoA desaturation by preferentially inhibiting the terminal oxidase activity of the desaturase complex [ 21. It could not be determined whether the decreased activity was due to the presence of a modified lipid or to some other effect of the analog on the synthesis or regulation of the enzyme. When the analog was incorporated into lipids in rat livers, the phosphatidyl-isopropylethanolamine constituted 9% of the total phospholipids and was distributed in the plasma membrane, mitochondria, and microsomal fractions. In addition, a second unnatural phospholipid analog, phosphatidyl-Nmethyl-isopropylethanolamine (3---4% of total phospholipid phosphorus), was formed in the liver by N-methylation of the phosphatidyl-isopropylethanolamine [ 141. Although the phosphatidyl-isopropylethanolamine was formed at the expense of phosphatidylcholine and phosphatidylethanolamine, its fatty acid composition did not resemble either of these phospholipids [ 31. Schroeder et al. [5] have shown that when two other base analogs, N-methylethanolamine and NJ’-dimethylethanolamine, were incorporated into membrane phospholipids of the L-M cells, the phase transition temperatures, as measured by two fluorescent probes, were not altered in the plasma membrane, mitochondria, or microsomal fractions. This indicated that L-M cells have a mechanism by which they may maintain their membrane fluidity. In the present study, we examined the effect of a polar head group modification on the activities of several membrane-bound enzymes in rat livers. Only 5’-nucleotidase (EC 3.1.3.5) was altered by isopropylethanolamine. Analysis of kinetic data, the Arrhenius plots, the effect of removal of phospholipids from plasma membranes by phospholipase C treatment, and the effect of free isopropylethanolamine base on enzyme activity demonstrated that the isopropylethanolamine reduced 5’-nucleotidase activity by a mechanism that does not involve the alteration in the polar head group of the phospholipids in the plasma membrane. The isopropylethanolamine, synthesized as previously described [ 11, was dissolved in saline and adjusted to pH 7.0 with HCl. Buffalo-strain rats (male, 200-300 g, Simonsin Laboratories, Gilroy, Calif.), maintained on a Purina Laboratory Chow diet, were fasted the day before isopropylethanolamine was injected and throughout the remaining experimental period. We injected rats intraperitoneally with either isopropylethanolamine (20 mg/lOO g body weight per day) or saline for 4 days and then killed them 1 h after the final injection. The livers were removed immediately and homogenized in 5 mM Tris-HCl buffer (pH 7.4) containing 0.5 mM CaCl, and 0.25 M sucrose. We isolated the plasma membrane, mitochondrial, and microsomal fractions from control (saline-injected) and treated (isopropylethanolamine-injected) rat livers as described previously 131. Cell-free homogenates were made by homogenizing the
127
livers in the same manner and centrifuging them twice at 180 X g for 10 min. The cell-free supernatant was then assayed for 5’-nucleotidase activity. Plasma membrane phospholipids were hydrolyzed with phospholipase C (EC 3.1.4.3) (Bacillis cereus, from Grand Island Biological Co.) by a modification of the method of Emmelot and Bos 1151. Tubes containing 2.0-2.5 mg plasma membrane protein, 5 mM Tris-HCl (pH 7.3), 0.1 mM CaCl, , and 2OOpg of phospholipase C were incubated at 37°C for 1 h. After the reaction was terminated by placing the tubes in ice, the incubation mixtures were centrifuged for 30 min at 100 000 X g. The pellet was suspended in water and assays for phospholipid phosphorus and 5’-nucleotidase activities were performed. 5’-Nucleotidase activity was assayed in a total volume of 1 ml by the method of Song and Bodansky [ 161. After terminating the reaction by adding 1 ml 10% trichloroacetic acid, we measured the phosphate released from AMP by the method of Bartlett [17]. We obtained the Arrhenius plots by incubating the samples for 30 min at temperatures from 25 to 60°C using 10 mM AMP as substrate. The incubations for the kinetic studies, were carried out for 10 and 20 min at 3’7°C with substrate concentrations between 0.25 and 5 mM AMP. Total lipids were extracted from the plasma membranes by the method of Bligh and Dyer except the methanol contained 2% glacial acetic acid [ 181. The phospholipids were resolved by two-dimensional thin-layer chromatography on silica gel HR layers: first in chloroform/methanol/acetic acid (50 : 25 : 8, v/v), and then, after drying the plate for 1 h in a vacuum desiccator, in chloroform/methanol/ammonium hydroxide (65 : 35 : 5, v/v). Phosphorus was determined [19] on an aliquot of total phospholipids and in each phospholipid class that could be visualized after spraying the layers with concentrated HzS04 and charring at 180-200°C. Activities of NADH- and NADPH-dependent cytochrome c reductase were assayed by the method of Dallner [ 201. Succinic acid dehydrogenase was assayed as described by Eibl et al. [Zl]. Of the four enzymes studied, two from the microsomal fraction (NADHand NADPH:cytochrome c reductase), one from mitochondria (succinate dehydrogenase), and one from plasma membranes (5’~nucleotidase), only the latter was significantly affected by the isopropylethanolamine incorporation (Table I). We chose to study this enzyme further to determine if the effect of TABLE
I
EFFECT OF N-ISOPROPYLETHANOLAMINE ON ENZYME SUBCELLULAR FRACTIONS OF RAT LIVER Subcellular
fraction
Enzyme
ACTIVITIES Specific
activities
MicrosomaJ(3) Microsomal Mitochondrkl
(3) (3)
(4)*
5’-Nucleotidase (pg Pi/min per mg protein) NADH:cytochrome c reductase &mol/min per mg protein) NADPH:cytochrome c reductase (nmol/min per mg protein) Succinic dehydrogenase (nmollmin per mg protein)
~~__ *The numbers in&zheses indicate the numberof%%&?liular standard error: each preparation was assayed in duplicate. **p = < 0.01.
f SE.
Control
Isopropylethanolaminetreated
20.8 f 0.64**
12.4 * 1.87**
-____ Plasma membrane
IN DIFFERENT
0.39 f 0.03
0.40
f. 0.04
71.7 f 0.54
71.2 f 0.22
10.0 f 0.17
10.6 f 0.03
preparations
used to calculate
the
128
isopropylethanolamine was due solely to its presence as phosphatidyl-isopropylethanolamine in the membrane or whether some other mechanism was involved. The alteration in the 5’-nucleotidase activity could result from the purification steps involved in the isolation of the plasma membranes. To eliminate this possibility, we assayed three separate cell-free homogenates from livers of isopropylethanolamine-injected and control rats for 5’-nucleotidase activity. The enzyme from the treated rats also demonstrated a significant decrease in its total activity (isopropylethanolamine-treated = 168.1 f 7.8 pg Pi/ min per g tissue; control = 227.9 f 7.5 pg Pi/min per g tissue; the P value is < 0.01); this eliminates the purification of the plasma membrane fraction as a factor affecting the enzyme’s activity. The Arrhenius plots (Fig. 1) of the 5’-nucleotidase activity in plasma membranes of livers from control and isopropylethanolamine-injected rats showed a similar relationship for the plasma membrane lipids and the activity of this enzyme. The graphs paralleled one another with the break points in the curves for the two preparations occurring at identical temperatures (35, 37,45 and 50°C). The major difference between the two plots was the decrease (20-50%) in the overall activity of the 5’-nucleotidase in the plasma membranes of the rats injected with isopropylethanolamine. The parallel nature of the two curves indicates that the 5’-nucleotidase activity is not directly affected by the presence of a modified polar head group in the plasma membrane phospholipids. Therefore, we examined the type of inhibition in 5’-nucleotidase activity caused by isopropylethanolamine treatment.
.
3.1
32
l.OOO/Temperature Fig.
1. Arrhenius
ethanolamine-injected
plot
of (0)
5’-nucleotidase rats.
activity
in plasma
membranes
from
control
(*)
and isopropyl-
129
A double reciprocal plot of the activity of the enzyme versus substrate concentration (Fig. 2) revealed that the affinity of the enzyme for its substrate was not affected by the analog injection. Both enzyme preparations had a K, of about 0.3 mM. However, the maximum velocity of the 5’-nucleotidase from the preparations containing the analog was about 50% lower than that for the control enzyme (9.1 versus 1.8.9 pg phosphorus/min per mg protein). This is consistent with a noncompetitive type of inhibition and indicates that the amount of active enzyme was lowered either from decreased synthesis or through an irreversible binding of the phosphatidyl-isopropylethanolamine to the enzyme in such a way as to render it inactive. We further investigated the possibility that the phosphatidyl-isopropylethanolamine molecules in the plasma membrane could be acting as a noncompetitive inhibitor of the 5’nucleotidase. This was done by removing the phosphatidyl-isopropylethanolamine from the plasma membrane; up to 80% of the membrane phospholipids were removed by phospholipase C hydrolysis. Two-dimensional thin-layer chromatography and phosphorus analysis revealed that all of the phosphatidyl-isopropylethanolamine was removed and that sphingomyelin made up the remaining 20% of the phospholipase C-resistant phospholipids in the membranes. Even after removal of the isopropylethanol-
Fig. 2. Lineweaver-Burk plot of 5’-nucleotidase injected (cl) rats.
activity in control (0) and isopropylethanolamine-
TABLE II EFFECT OF PHOSPHOLIPASE C TREATMENT ON 5’-NUCLEOTIDASE AND PHOSPHOLIPIDS FROM CONTROL AND ISOPROPYLETHANOLAMINE-TREATED RAT LIVER PLASMA MEMBRANES Values are the average of duplicates. Control Phospholipase C 5’-Nucleotidase 018 phosphorus/min per mg protein) Phospholipid phosphorus (%Jo)
Isopropylethanolamine-treated
(-)
(+)
(-)
(+)
20.4 100
20.0 20.5
16.4 100
16.8 20.4
-
130
amine base from the phospholipids by this treatment, the 5’-nucleotidase activity was still inhibited in the analog-injected membrane preparations as compared with that in control membranes (Table II). This was also in agreement with the data in which the 5’nucleotidase activity remained unchanged after removing phospholipids from normal membranes with phospholipase A2 [ll], i.e., the 5’-nucleotidase does not require phospholipids for expression of its activity [ 221. Finally, we examined the effect of free isopropylethanolamine base on 5’nucleotidase activity of control membranes. We saw no change in the specific activity of the enzyme with any of the base analog concentrations (1, 5, and 10 mM) used (data not shown). These data indicate that the lowered activity of the 5’-nucleotidase in the isopropylethanolamine-injected rat liver plasma membranes was due to a decreased amount of the enzyme present. Since other laboratories have shown that polar head group analogs can alter enzyme activities strictly by their presence in the modified membrane (see, e.g., Engelhard et al. [7]), we conclude that amino alcohols can alter enzyme activities by different means. Our data suggest that the isopropylethanolamine analog acted as an inhibitor of cellular processes involving the regulation of membrane enzyme activities, perhaps at the point of protein synthesis. This work was supported by the United States Department of Energy, American Cancer Society (Grant K-701), National Cancer Institute (Grant CA 11949-08), and National Cancer Institute (Training Grant CA 09104). References 1
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