0013-7227/90/1273-1194$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society
Vol. 127, No. 3 Printed in U.S.A.
Calcium-Dependent Protein Kinase Activity Is Decreased in Diabetic Rat Sciatic Nerve ROBERT A. GABBAY, LINDA SICONOLFI-BAEZ, AND HAROLD E. LEBOVITZ Department of Medicine, Division of Endocrinology, State University of New York Health Science Center, Brooklyn, New York 11203
presence of Ca+2 revealed an additional activity in these homogenates which probably represents the Ca+2-phospholipid-dependent protein kinase (protein kinase-C). The diabetic state did not appear to alter that activity. The Ca+2-dependent protein kinase was sensitive to agents known to inhibit calmodulindependent protein kinase or protein kinase-C. The IC50 values of the inhibitors for the Ca+2-dependent protein kinase, however, differed from those reported for the other two kinases. {Endocrinology 127: 1194-1198, 1990)
ABSTRACT. The effects of 4 weeks of streptozotocin-induced diabetes in the rat on sciatic nerve homogenate protein kinase activities were studied. There was a 47 ± 7% inhibition of Ca+2dependent protein kinase activity in nerves from the diabetic rats compared to that in their paired normal controls. This Ca+2dependent activity did not require the addition of phospholipid and was only minimally affected by Sephadex G-50 gel filtration, suggesting that endogenous phospholipid activation was not responsible for this activity. The addition of phospholipid in the
D
kg streptozotocin (Zanosar, Upjohn Co., Kalamazoo, MI), and the other was maintained as nondiabetic controls. Rats were considered to be diabetic if their blood glucose was consistently above 22 mmol/liter. Rats made diabetic by this technique in our laboratory are insulinopenic, with mean plasma insulin values of 13 jtU/ml. Four weeks after the induction of diabetes, diabetic and control rats were anesthetized with sodium pentobarbital, and the sciatic nerves were removed (5). The extraneural tissues were dissected away, and the nerves were homogenized in a 20 mmol/liter Tris-HCl buffer, pH 7.5, containing 0.25 mol/liter sucrose, 10 mmol/liter EGTA, 2 mmol/liter EDTA, 1 mmol/liter phenylmethylsulfonylfluoride, and 20 ng/ ml leupeptin at 4 C in a Polytron PT 10/35 (Brinkmann Instruments, Westbury, NY) four times, 10 sec each. The nerves were homogenized at a concentration of 40 mg weight/ ml buffer. Leupeptin was included as a potent inhibitor of the Ca+2-activated neutral protease, which proteolytically activates protein kinase-C, eliminating its phospholipid and Ca+2 requirement (6). The homogenate was centrifuged for 1 min in a Beckman B microfuge (Palo Alto, CA), the supernatant was removed, and Triton X-100 was added to a final concentration of 0.2%. After 2 h at 4 C, protein kinase activity was determined. Triton treatment was necessary to demonstrate the observed protein kinase activity.
IABETIC neuropathy is characterized by a decrease in nerve conduction velocity associated with a diminished Na,K-ATPase activity. A hyperglycemia-mediated depletion of nerve myo-inositol has been implicated as the basis for these metabolic derangements (1). Greene and Lattimer (2) have hypothesized that the effects of reduced myo-inositol levels are mediated through alterations in phosphatidylinositol turnover, thus causing a reduction in the levels of diacylglycerol, the endogenous activator of protein kinase-C, and inositol-l,4,5-trisphosphate, a mediator of calcium release (3). They were able to restore depressed Na,K-ATPase activity in sciatic nerve preparations from diabetic rabbits with specific activators of protein kinase-C (4). Their studies suggested a central role for protein kinase-C and diminished protein phosphorylation in the pathogenesis of diabetic neuropathy. The aim of the present study was to determine whether streptozotocin-induced diabetes in the rat caused an alteration in measurable protein kinase-C activity in sciatic nerves. Materials and Methods
Nerve preparation Male Wistar rats, weighing 180-200 g, were obtained from Charles Rivers (Boston, MA). Rats of identical ages were randomly assigned to two groups. One was injected iv with 60 mg/ Received February 12,1990. Address all correspondence and requests for reprints to: Harold E. Lebovitz, M.D., State University of New York Health Science Center, 450 Clarkson Avenue, Box 1205, Brooklyn, New York 11203.
Determination of protein kinase activity Protein kinase activity was assayed using a modification of the method of Kitano et al. (7). Briefly, 32P incorporation from [T-32P]ATP (3 Ci/nmol; Amersham, Arlington Heights, IL) into lysine-rich histone III-S (Sigma Chemical Co., St. Louis, MO) was measured. The reaction was begun by the addition of 100 jxl of the nerve homogenate Triton-treated
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CALCIUM-DEPENDENT KINASE IN DIABETES supernatant into 150 n\ assay medium at 31 C. The resultant concentration of assay medium reagents was 20 mmol/liter pH 7.5 Tris-HCL, 10 mmol/liter magnesium acetate, 10 Mmol/liter ATP, 200 Mg/ml histone III-S, 0.5 ^Ci [ Y - 3 2 P ] A T P , and, where indicated, 100 Mg/ml L-a-phosphatidyl-L-serine (PS), 20 /ig/ml diolein (Sigma), 5 mmol/liter CaCl2. This calcium concentration was necessary to overcome the 4 mmol/liter EGTA present from the addition of nerve homogenate buffer. PS-diolein mixtures were prepared as previously described (7). Phospholipid additions, when made, were added directly to the nerve homogenate supernatant before addition to the assay mixture containing calcium. The reaction was terminated at 5 min by the addition of 1 ml 25% trichloroacetic acid. Phosphorylated histone was collected on nitrocellulose filters (HAWP, Millipore, Bedford, MA), washed three times with 5 ml 25% trichloroacetic acid, and counted by Cerenkov counting in a liquid scintillation spectrophotometer. Stimulated activity is defined as that found in the presence of activators (Ca2+ or Ca2+ with PS and diolein) minus the basal activity in their absence. Statistical analyses were performed using Student's t test. The results are presented as the mean ± SE. Removal of endogenous phospholipid from nerve homogenates A modification of a centrifuged column procedure was used to remove low mol wt compounds from the nerve homogenate supernatants (8). Gel filtration columns were prepared in 3-ml syringes with Sephadex G-50 (Pharmacia, Inc., Piscataway, NJ) with a sample to bed volume ratio of 0.2. A 400-jttl nerve homogenate supernatant was applied to the column. Under these conditions, greater than 93% of the protein was recovered, and less than 5% of a low mol wt marker (750 gm/ mol) passed through the column. The effluent was assayed for protein kinase activity as described above.
Results Identification of a calcium-dependent protein kinase in sciatic nerve homogenates During a series of experiments to develop an assay for protein kinase-C activity in rat sciatic nerve homogenate, we observed a calcium-stimulated protein kinase activity that did not require the addition of exogenous phospholipid. In the presence of a final assay concentration of 4 mmol/liter EGTA, the calcium activation of protein kinase was maximal at 5 mmol/liter and decreased with higher calcium concentrations. As shown in Fig. 1, this calcium-dependent protein kinase activity was linear with homogenate protein concentration. A further increase in the calcium-stimulated activity was observed after the addition of PS and diolein (Fig. 1). The difference between the two activities, i.e. that stimulated in the presence of 5 mmol/liter Ca+2 alone and that in the presence of 5 mmol/liter Ca+2 plus PS and diolein (Fig. 1), probably represents protein kinase-C activity. No activity was detected when PS and diolein were added to the assay system in the absence of Ca+2.
1195
40
30-
Stimulated Kinase Activity (pmolPO4/min)
20
10-
10
20 Total Protein (ng)
FIG. 1. Nerve homogenate protein kinase activity stimulated by Ca2+ or Ca2+ plus PS and diolein. Rat sciatic nerve homogenates were prepared and assayed as described in Materials and Methods. PSdiolein additions were made directly to the nerve homogenate before addition to assay buffer containing Ca2+. Stimulated activity is that found in the presence of activators (Ca2+ or Ca2+ plus PS-diolein) minus the basal activity in their absence. Assays contained 4 mM EGTA and, where indicated, 5 mM CaCl2, 100 Mg/ml PS, and 20 Mg/ml diolein. Results are expressed as the mean ± SE of triplicate determinations from a representative experiment.
To determine whether the nerve homogenates contained sufficient endogenous phospholipids to activate protein kinase-C in the presence of calcium alone, we performed a series of experiments in which nerve homogenate supernatants were reassayed after Sephadex G-50 chromatography. Under conditions in which greater than 95% of added [14C]phosphatidyl-L-serine-dipalmitoyl was retained on the column and greater than 95% of the protein was recovered, there was only an 8% decrease in the calcium-stimulated protein kinase activity of the nerve homogenate effluent. The ability of calcium and exogenous phospholipid to stimulate the protein kinase activity of the Sephadex G-50 effluent was unchanged. Effects of diabetes on nerve homogenate calciumdependent protein kinase The calcium-stimulated protein kinase activity was decreased by up to 70% in 4-week streptozotocin diabetic rats compared to that in paired normal controls (Fig. 2). Although the absolute calcium-dependent protein kinase activity varied among the individual sets of diabetic and normal rats (Table 1), a consistent and statistically significant decrease in activity was present in the diabetic rats compared to that in their paired normal controls (mean decrease, 47 ± 6.8%; P < 0.01). The calcium- and PS-diolein-stimulated protein kinase activity (protein kinase-C activity) did not show any consistent differ-
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CALCIUM-DEPENDENT KINASE IN DIABETES
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Endo • 1990 Vol 127 • No 3
nase) and protein kinase-C are given. The spectrum of inhibition of the calcium-dependent nerve homogenate kinase differs from that for both protein kinase-C and calmodulin-dependent kinases. Stimulated Kinase Activity (pniol PO4/min)
Discussion
20
40
60
80
100
Studies of sciatic nerves removed from diabetic rabbits and rats and compared to nerves from control animals have shown that chronic hyperglycemia causes 1) an accumulation of sorbitol and fructose, 2) a depletion of myo-inositol, and 3) a decrease in Na,K-ATPase activity (1). A major function of myo-inositol is as a precursor for inositol-l,4,5-triphosphate, which mobilizes intracellular Ca+2, and diacylglycerol, which activates protein kinase-C (3). Protein kinase-C activators have been shown to normalize Na,K-ATPase activity in peripheral nerves from alloxan-diabetic rabbits (4). Greene and Lattimer (13) have proposed that nerves from diabetic animals have decreased protein kinase-C activity, which is responsible for the decreased Na,K-ATPase activity. Greene and co-workers in a preliminary report (14) described a diabetes-induced decrease in nerve protein kinase activity which required both Ca+2 and phospholipid for activation. Simpson and Hawthorne (15), on the other hand, have reported that cytosolic protein kinaseC activity in rat sciatic nerve is unaffected by 6 weeks of streptozotocin-induced diabetes. Our data are at variance with both previous reports. We observed a calcium-dependent protein kinase activity in rat sciatic nerve homogenates that was markedly reduced in 4-week streptozotocin-diabetic rats compared to their paired controls. This activity was measurable only after Triton X-100 addition to the homogenate supernatant. Several lines of evidence support the conclusion that the calcium-dependent protein kinase activity measured in the present study is not protein kinaseC. In contrast to protein kinase-C, the activity we report did not require the addition of exogenous phospholipid
120
Total Protein (ng) FlG. 2. Effect of diabetes on Ca2+-dependent protein kinase activity. Sciatic nerve homogenates from rats that were diabetic for 4 weeks and their paired normal controls were prepared and assayed as described in Materials and Methods. Stimulated activity was determined in the presence of 5 mM Ca2+ and 4 DIM EGTA, as described in Fig. 1. Results are expressed as the mean ± SE for triplicate determinations from a representative experiment. • , Data from normal rats; O, data from diabetic rats.
ences between nerve homogenates from diabetic rats and their paired normal controls (data not shown). Characterization of the calcium-dependent protein kinase activity in nerve homogenates The nature of the calcium-dependent protein kinase activity in the sciatic nerve homogenate was examined using a spectrum of known inhibitors of protein kinaseC (H7 and polymyxin-B) or calmodulin-dependent protein kinases (trifluoperazine and W7). Table 2 lists our experimentally determined IC50 values of the nerve homogenate calcium-dependent protein kinase for polymyxin-B, H7, trifluoperazine, and W7. For comparison, the known IC50 values or Ki values of these agents for calmodulin-dependent kinase (myosin light chain kinase or rat cerebral cortex calmodulin-dependent protein ki-
TABLE 1. Effects of diabetes on calcium-dependent protein kinase activity at two protein concentrations pmol phosphate transferred/min Exp. no.
1 2 3 4 5
at 25 Mg protein (0.1 mg/min)
at 50 ng protein (0.2 mg/ml)
Normal
Diabetic
% Inhibition
Normal
Diabetic
% Inhibition
5.07 8.97 6.16 4.40 2.98
2.39 5.77 4.03 1.27 1.46
53% 36% 34%
8.93 17.91 9.06 7.25 7.70
4.79 9.77 6.76 2.34 4.63
46% 45%
Mean ± SEM (%)
71% 51%
49 ± 6.7°
25% 68% 40%
45 ± 6.9°
2+
Sciatic nerve preparations were assayed for Ca -dependent protein kinase activity as described in Fig. 2 and Materials and Methods. Each experiment represents a different pair of experiment animals. °P
Vol. 127, No. 3 Printed in U.S.A.
Calcium-Dependent Protein Kinase Activity Is Decreased in Diabetic Rat Sciatic Nerve ROBERT A. GABBAY, LINDA SICONOLFI-BAEZ, AND HAROLD E. LEBOVITZ Department of Medicine, Division of Endocrinology, State University of New York Health Science Center, Brooklyn, New York 11203
presence of Ca+2 revealed an additional activity in these homogenates which probably represents the Ca+2-phospholipid-dependent protein kinase (protein kinase-C). The diabetic state did not appear to alter that activity. The Ca+2-dependent protein kinase was sensitive to agents known to inhibit calmodulindependent protein kinase or protein kinase-C. The IC50 values of the inhibitors for the Ca+2-dependent protein kinase, however, differed from those reported for the other two kinases. {Endocrinology 127: 1194-1198, 1990)
ABSTRACT. The effects of 4 weeks of streptozotocin-induced diabetes in the rat on sciatic nerve homogenate protein kinase activities were studied. There was a 47 ± 7% inhibition of Ca+2dependent protein kinase activity in nerves from the diabetic rats compared to that in their paired normal controls. This Ca+2dependent activity did not require the addition of phospholipid and was only minimally affected by Sephadex G-50 gel filtration, suggesting that endogenous phospholipid activation was not responsible for this activity. The addition of phospholipid in the
D
kg streptozotocin (Zanosar, Upjohn Co., Kalamazoo, MI), and the other was maintained as nondiabetic controls. Rats were considered to be diabetic if their blood glucose was consistently above 22 mmol/liter. Rats made diabetic by this technique in our laboratory are insulinopenic, with mean plasma insulin values of 13 jtU/ml. Four weeks after the induction of diabetes, diabetic and control rats were anesthetized with sodium pentobarbital, and the sciatic nerves were removed (5). The extraneural tissues were dissected away, and the nerves were homogenized in a 20 mmol/liter Tris-HCl buffer, pH 7.5, containing 0.25 mol/liter sucrose, 10 mmol/liter EGTA, 2 mmol/liter EDTA, 1 mmol/liter phenylmethylsulfonylfluoride, and 20 ng/ ml leupeptin at 4 C in a Polytron PT 10/35 (Brinkmann Instruments, Westbury, NY) four times, 10 sec each. The nerves were homogenized at a concentration of 40 mg weight/ ml buffer. Leupeptin was included as a potent inhibitor of the Ca+2-activated neutral protease, which proteolytically activates protein kinase-C, eliminating its phospholipid and Ca+2 requirement (6). The homogenate was centrifuged for 1 min in a Beckman B microfuge (Palo Alto, CA), the supernatant was removed, and Triton X-100 was added to a final concentration of 0.2%. After 2 h at 4 C, protein kinase activity was determined. Triton treatment was necessary to demonstrate the observed protein kinase activity.
IABETIC neuropathy is characterized by a decrease in nerve conduction velocity associated with a diminished Na,K-ATPase activity. A hyperglycemia-mediated depletion of nerve myo-inositol has been implicated as the basis for these metabolic derangements (1). Greene and Lattimer (2) have hypothesized that the effects of reduced myo-inositol levels are mediated through alterations in phosphatidylinositol turnover, thus causing a reduction in the levels of diacylglycerol, the endogenous activator of protein kinase-C, and inositol-l,4,5-trisphosphate, a mediator of calcium release (3). They were able to restore depressed Na,K-ATPase activity in sciatic nerve preparations from diabetic rabbits with specific activators of protein kinase-C (4). Their studies suggested a central role for protein kinase-C and diminished protein phosphorylation in the pathogenesis of diabetic neuropathy. The aim of the present study was to determine whether streptozotocin-induced diabetes in the rat caused an alteration in measurable protein kinase-C activity in sciatic nerves. Materials and Methods
Nerve preparation Male Wistar rats, weighing 180-200 g, were obtained from Charles Rivers (Boston, MA). Rats of identical ages were randomly assigned to two groups. One was injected iv with 60 mg/ Received February 12,1990. Address all correspondence and requests for reprints to: Harold E. Lebovitz, M.D., State University of New York Health Science Center, 450 Clarkson Avenue, Box 1205, Brooklyn, New York 11203.
Determination of protein kinase activity Protein kinase activity was assayed using a modification of the method of Kitano et al. (7). Briefly, 32P incorporation from [T-32P]ATP (3 Ci/nmol; Amersham, Arlington Heights, IL) into lysine-rich histone III-S (Sigma Chemical Co., St. Louis, MO) was measured. The reaction was begun by the addition of 100 jxl of the nerve homogenate Triton-treated
1194
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CALCIUM-DEPENDENT KINASE IN DIABETES supernatant into 150 n\ assay medium at 31 C. The resultant concentration of assay medium reagents was 20 mmol/liter pH 7.5 Tris-HCL, 10 mmol/liter magnesium acetate, 10 Mmol/liter ATP, 200 Mg/ml histone III-S, 0.5 ^Ci [ Y - 3 2 P ] A T P , and, where indicated, 100 Mg/ml L-a-phosphatidyl-L-serine (PS), 20 /ig/ml diolein (Sigma), 5 mmol/liter CaCl2. This calcium concentration was necessary to overcome the 4 mmol/liter EGTA present from the addition of nerve homogenate buffer. PS-diolein mixtures were prepared as previously described (7). Phospholipid additions, when made, were added directly to the nerve homogenate supernatant before addition to the assay mixture containing calcium. The reaction was terminated at 5 min by the addition of 1 ml 25% trichloroacetic acid. Phosphorylated histone was collected on nitrocellulose filters (HAWP, Millipore, Bedford, MA), washed three times with 5 ml 25% trichloroacetic acid, and counted by Cerenkov counting in a liquid scintillation spectrophotometer. Stimulated activity is defined as that found in the presence of activators (Ca2+ or Ca2+ with PS and diolein) minus the basal activity in their absence. Statistical analyses were performed using Student's t test. The results are presented as the mean ± SE. Removal of endogenous phospholipid from nerve homogenates A modification of a centrifuged column procedure was used to remove low mol wt compounds from the nerve homogenate supernatants (8). Gel filtration columns were prepared in 3-ml syringes with Sephadex G-50 (Pharmacia, Inc., Piscataway, NJ) with a sample to bed volume ratio of 0.2. A 400-jttl nerve homogenate supernatant was applied to the column. Under these conditions, greater than 93% of the protein was recovered, and less than 5% of a low mol wt marker (750 gm/ mol) passed through the column. The effluent was assayed for protein kinase activity as described above.
Results Identification of a calcium-dependent protein kinase in sciatic nerve homogenates During a series of experiments to develop an assay for protein kinase-C activity in rat sciatic nerve homogenate, we observed a calcium-stimulated protein kinase activity that did not require the addition of exogenous phospholipid. In the presence of a final assay concentration of 4 mmol/liter EGTA, the calcium activation of protein kinase was maximal at 5 mmol/liter and decreased with higher calcium concentrations. As shown in Fig. 1, this calcium-dependent protein kinase activity was linear with homogenate protein concentration. A further increase in the calcium-stimulated activity was observed after the addition of PS and diolein (Fig. 1). The difference between the two activities, i.e. that stimulated in the presence of 5 mmol/liter Ca+2 alone and that in the presence of 5 mmol/liter Ca+2 plus PS and diolein (Fig. 1), probably represents protein kinase-C activity. No activity was detected when PS and diolein were added to the assay system in the absence of Ca+2.
1195
40
30-
Stimulated Kinase Activity (pmolPO4/min)
20
10-
10
20 Total Protein (ng)
FIG. 1. Nerve homogenate protein kinase activity stimulated by Ca2+ or Ca2+ plus PS and diolein. Rat sciatic nerve homogenates were prepared and assayed as described in Materials and Methods. PSdiolein additions were made directly to the nerve homogenate before addition to assay buffer containing Ca2+. Stimulated activity is that found in the presence of activators (Ca2+ or Ca2+ plus PS-diolein) minus the basal activity in their absence. Assays contained 4 mM EGTA and, where indicated, 5 mM CaCl2, 100 Mg/ml PS, and 20 Mg/ml diolein. Results are expressed as the mean ± SE of triplicate determinations from a representative experiment.
To determine whether the nerve homogenates contained sufficient endogenous phospholipids to activate protein kinase-C in the presence of calcium alone, we performed a series of experiments in which nerve homogenate supernatants were reassayed after Sephadex G-50 chromatography. Under conditions in which greater than 95% of added [14C]phosphatidyl-L-serine-dipalmitoyl was retained on the column and greater than 95% of the protein was recovered, there was only an 8% decrease in the calcium-stimulated protein kinase activity of the nerve homogenate effluent. The ability of calcium and exogenous phospholipid to stimulate the protein kinase activity of the Sephadex G-50 effluent was unchanged. Effects of diabetes on nerve homogenate calciumdependent protein kinase The calcium-stimulated protein kinase activity was decreased by up to 70% in 4-week streptozotocin diabetic rats compared to that in paired normal controls (Fig. 2). Although the absolute calcium-dependent protein kinase activity varied among the individual sets of diabetic and normal rats (Table 1), a consistent and statistically significant decrease in activity was present in the diabetic rats compared to that in their paired normal controls (mean decrease, 47 ± 6.8%; P < 0.01). The calcium- and PS-diolein-stimulated protein kinase activity (protein kinase-C activity) did not show any consistent differ-
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CALCIUM-DEPENDENT KINASE IN DIABETES
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Endo • 1990 Vol 127 • No 3
nase) and protein kinase-C are given. The spectrum of inhibition of the calcium-dependent nerve homogenate kinase differs from that for both protein kinase-C and calmodulin-dependent kinases. Stimulated Kinase Activity (pniol PO4/min)
Discussion
20
40
60
80
100
Studies of sciatic nerves removed from diabetic rabbits and rats and compared to nerves from control animals have shown that chronic hyperglycemia causes 1) an accumulation of sorbitol and fructose, 2) a depletion of myo-inositol, and 3) a decrease in Na,K-ATPase activity (1). A major function of myo-inositol is as a precursor for inositol-l,4,5-triphosphate, which mobilizes intracellular Ca+2, and diacylglycerol, which activates protein kinase-C (3). Protein kinase-C activators have been shown to normalize Na,K-ATPase activity in peripheral nerves from alloxan-diabetic rabbits (4). Greene and Lattimer (13) have proposed that nerves from diabetic animals have decreased protein kinase-C activity, which is responsible for the decreased Na,K-ATPase activity. Greene and co-workers in a preliminary report (14) described a diabetes-induced decrease in nerve protein kinase activity which required both Ca+2 and phospholipid for activation. Simpson and Hawthorne (15), on the other hand, have reported that cytosolic protein kinaseC activity in rat sciatic nerve is unaffected by 6 weeks of streptozotocin-induced diabetes. Our data are at variance with both previous reports. We observed a calcium-dependent protein kinase activity in rat sciatic nerve homogenates that was markedly reduced in 4-week streptozotocin-diabetic rats compared to their paired controls. This activity was measurable only after Triton X-100 addition to the homogenate supernatant. Several lines of evidence support the conclusion that the calcium-dependent protein kinase activity measured in the present study is not protein kinaseC. In contrast to protein kinase-C, the activity we report did not require the addition of exogenous phospholipid
120
Total Protein (ng) FlG. 2. Effect of diabetes on Ca2+-dependent protein kinase activity. Sciatic nerve homogenates from rats that were diabetic for 4 weeks and their paired normal controls were prepared and assayed as described in Materials and Methods. Stimulated activity was determined in the presence of 5 mM Ca2+ and 4 DIM EGTA, as described in Fig. 1. Results are expressed as the mean ± SE for triplicate determinations from a representative experiment. • , Data from normal rats; O, data from diabetic rats.
ences between nerve homogenates from diabetic rats and their paired normal controls (data not shown). Characterization of the calcium-dependent protein kinase activity in nerve homogenates The nature of the calcium-dependent protein kinase activity in the sciatic nerve homogenate was examined using a spectrum of known inhibitors of protein kinaseC (H7 and polymyxin-B) or calmodulin-dependent protein kinases (trifluoperazine and W7). Table 2 lists our experimentally determined IC50 values of the nerve homogenate calcium-dependent protein kinase for polymyxin-B, H7, trifluoperazine, and W7. For comparison, the known IC50 values or Ki values of these agents for calmodulin-dependent kinase (myosin light chain kinase or rat cerebral cortex calmodulin-dependent protein ki-
TABLE 1. Effects of diabetes on calcium-dependent protein kinase activity at two protein concentrations pmol phosphate transferred/min Exp. no.
1 2 3 4 5
at 25 Mg protein (0.1 mg/min)
at 50 ng protein (0.2 mg/ml)
Normal
Diabetic
% Inhibition
Normal
Diabetic
% Inhibition
5.07 8.97 6.16 4.40 2.98
2.39 5.77 4.03 1.27 1.46
53% 36% 34%
8.93 17.91 9.06 7.25 7.70
4.79 9.77 6.76 2.34 4.63
46% 45%
Mean ± SEM (%)
71% 51%
49 ± 6.7°
25% 68% 40%
45 ± 6.9°
2+
Sciatic nerve preparations were assayed for Ca -dependent protein kinase activity as described in Fig. 2 and Materials and Methods. Each experiment represents a different pair of experiment animals. °P
