750
BIOCHEMICAL SOCIETY TRANSACTIONS
(Magnesium plus Sodium plus Potassium Ion)-Stimulated Adenosine Triphosphatase in Rat Sciatic Nerve PRANAB K. DAS, GARTH M. BRAY, ALBERT J. AGUYAO and MICHAEL RAMINSKY
Division of Neurology, Montreal General Hospital Research Institute, Montreal, Que., Canada The presence of at least two types of ATPase* (EC 3.6.1.3, ATP phosphohydrolase) has been reported in almost all tissues (Bonting et al., 1961). The original finding by Skou (1957) on the presence of a specific ATPase in crab nerve (which requires the presence of critical concentrations of Na+, K+ and MgZ+for maximal activity) is of particular interest in the present investigation. This latter type of ATPase, which is intimately associated with the active transport of Na+ and K+ across the nerve axonal membrane, leading to impulse transmission (Pincus et al., 1970), is specifically inhibited by ouabain. Information about ATPases from mammalian nerves is sparse, except for some early reports by Libet (1948) and Abood &Gerard (1954). Also, Khan & Ochs (1974) reported that ATPase systems in cat sciatic nerve are largely composed of the Mgz+- or Cazfactivated type. The reason for the lack of information on ATPase of peripheral nerves from individual animals is probably the unavailability of adequate amounts of the tissues. Our present interest has grown out of a detailed study by Sharma & Thomas (1974) showing that the conduction velocity in sciatic nerves of streptozotocin-treated diabetic rats is significantly lower than normal without there being any demonstrable morphological abnormalities. Since a basic component of nerve conduction is the permeability changes in the membrane, involving the ouabain-sensitive (Na+ K+)-ATPase which controls the sodium pump (Dahl & Hokin, 1974), we decided to study the characteristics of this enzyme activity. It was, therefore, considered of primary importance to define conditions for its quantitative measurement and its normal distribution and then to compare the values with those from experimental diabetic animals under conditions in which the optimum activity could be measured. Here we present some studies on the properties of ATPase from rat sciatic-nerve homogenates.
+
Materials and methods For the evaluation of an ATPase-assay method and studies on some aspects of properties of the enzymes, male Sprague-Dawley rats, weighing 300-400 g, were used. Individual animals were anaesthetized with intraperitoneal injections of sodium pentobarbital. Both sciatic nerves were removed from the sciatic notch to the popliteal fossa, immersed initially in 200 mM-sucrose/Tris/HCI buffer, pH 7.4, and stripped of their epineurial tissues. By using a Polytron homogenizer, the desheathed nerves were homogenized for at least three periods (not exceeding 15 s each) in 2 ml of specified buffer solutions, at W ' C . The homogenates were centrifuged lightly (lo00 rev./min for 2 min) at 4"C, and the supernatants formed the starting material in all the experiments except those for subcellular frationations. Differential centrifugation of the crude homogenates was carried by a method similar to that of Das & Sayer (1975). Protein concentrations and ATPase activities were measured as a routine by the methods of Lowry et al. (1951) and Bonting et al. (1961) respectively, with appropriate control blanks and standards for each individual experiment. For the ATPase assay, portions of the homogenized nerves or suspensions of subcellular fractions were incubated at 37°C in 2 ml of three different solutions: solution A contained ~ ~ M - A T P , 2m~-MgC1,, 100nM-EDTA in 100 mM-Tris/HCl buffer, pH 7.4; solution B was the same as solution A, but contained in addition, 60m-NaCI and S ~ M - K C Isolution ; C contained 60m-NaCI and 100nwouabain. After 1h the reaction was stopped
* Abbreviation: ATPase, adenosine triphosphatase. 1976
75 1
563rd MEETING, LONDON
with 0 . 5 d of ice-cold trichloroacetic acid (15 %, w/v). After centrifugation, 1 . 5 d of the supernatant was added to 1.5ml of colour reagent for the determination of Pi. Activity measured in solution B represented total (Mg2++Na++K+)-ATPase.Ouabain-resistant activity was measured in solution C and the ouabain-sensitive component was calculated as the difference between the activities in solutions B and C. Activity in solution A represented MgZ+-ATPase, and, under standard conditions, this activity was similar to that in solution C. All the activities were expressed as pmol of Pi/h per mg of protein. Results and discussion
ATPase activities (both ouabain-resistant and ouabain-sensitive) were most stable in 50 mM-Tris/HCI buffer, pH 7.5, containing 100nM-dithiothreitol and 2 W 5 0 0 m ~ sucrose at 4°C. In the absence of dithiothreitol, the ouabain-sensitive enzyme was particularly unstable. Freezing at -20°C and subsequent thawing caused aninitial loss of 20 and 40 % of the ouabain-resistant and ouabain-sensitive enzymes respectively. In general, storage appeared to decrease the ouabain-sensitive/ouabain-resistant enzyme ratio. Homogenates prepared in the above buffer were used in subsequent investigations. Both the ATPase activities were linear for periods ranging from 10 to 60 min and were proportional to increasing amounts of tissue between 10 and 65mg of protein. Specific activities of about 7.8 and 3.0 ,umol/h per mg of protein were observed for ouabain-resistant and ouabain-sensitive ATPases respectively along the entire length of dissected nerve. Incubation of the homogenates above 40°C for 1-2 h caused inactivation of both the components. However, at 60°C, ouabain-resistant enzyme and 90 % of ouabainsensitive enzyme were lost. A linear increase in all ATPase activities was observed when the activities were determined at increasing temperature between 15" and 40°C. Similarly when various samples were incubated for 1h a t 4°C over a pH range 6.2-8.6 in Tris/HCl buffer (in one experiment 5Om~-imidazole/HCIbuffer was used for pH 6.2-6.7), i t was observed that ouabain-resistant enzyme activity remained more or less unaffected over the pH range 7.0-8.6, and a decrease of 20% was observed at acid pH values between 6.2 and 6.7; the maximum activity for oubain-sensitive enzyme was observed in the homogenates kept at pH values 7.3-7.6, and 50 and 30% of this activity were observed in the samples kept at pH 6.2 and 8.2 respectively. When the activity was measured in reaction media of different pH, with the application of appropriate corrections for any possible denaturation, a sharp optimum for ouabainsensitive ATPase was observed at p H 7.5, whereas ouabain-resistant ATPase activity increased to a flat optimum around pH 7.6-8.2. Determination of the velocity with respect to ATP concentration, in the presence and absence of 100 nwouabain, and in the presence of constant concentrations of 2.0 mMgCI,, 60m~-NaC1and 5rm-KCI at 37°C indicated that maximum activity for all ATPase activities was reached when the ATP concentration was in a 1 :1 ratio with MgCI2. This maximum activity was maintained even when ATP exceeded Mg2+concentration by 3: 1. Under this condition a K, value for (Mg2+ Na+ K+)-ATPase of 0 . 1 8 m ~was obtained. Both ouabain-sensitive and ouabain-resistant ATPase hydrolysed the following nucleotides in decreasing order of effectiveness: ATP > ITP > GTP > ADP > AMP. However, the ouabain-resistant enzyme had equal and the ouabain-sensitive enzyme 60% of ATP-hydrolytic activity when ITP was used as substrate. Total enzyme activity was almost zero when no cations were present in the reaction medium. Similarly, when the reaction mixture contained only 60 mM-NaC1 and ~ ~ M - Kwithout CI any MgCl, negligible activity could be measured. Increasing concentrations of Mgz+activated the ouabain-resistant enzyme to a maximum value at 2-3 m when ATP was kept constant at 2 m ~ Similarly, . maximum activity for the ouabainsensitive enzyme was also achieved at similar concentrations of MgZf. This latter activity appeared to be inhibited when the Mgz+concentration was raised to 6 m ~ . A similar type of increase in ouabain-resistant ATPase activity was found when increasing concentrations of Ca2+instead of Mgz+were used. Again maximum activity
+
voi. 4
+
BIOCHEMICAL SOCIETY TRANSACTIONS
752
was observed when the concentration of CaZ+was 3 m~ and remained constant even after the concentration was increased to 6 m ~Under . the same conditions, only a minor amount of ouabain-sensitive enzyme activity could be detected at lower concentrations of Caz+(O.O5m), and no activity was seen at higher Ca2+concentration. Our observation is compatible with an earlier observation that Ca2+is inhibitory to (Na+ K+)activated ATPase (Skou, 1957). This was also true even when Mgz+ was present. An additional activity, up to 40-50 %, of ouabain-resistant enzyme was measured when 2m-MgC12 was added to the reaction mixture, after the concentration of CaZ+for maximal activity was held at 3mM. The present observation appeared to be contradictory to those of Khan & Ochs (1974), who observed a slight inhibition of Mg2+-ATPase of a preparation from cat sciatic nerve. Our results, at this stage cannot be compared with those of Khan & Ochs (1974), since we were using crude homogenates instead of a purer fraction. However, it appears that the rat sciatic-nerve homogenates contain three different ATPases: (1) ouabain-resistant (Mg2+ Na+ K+)-stimulated; (2) ouabainsensitive (Mg2+ Na+ K+)-stimulated; (3) Ca2+-stimulated, (MgZ+ Na+ K+)stimulated. From preliminary investigations on the effects of Na+ and K+ by the approaches used bySkou(1957) and Albers & Koval(l962) it appeared that an optimal ratio of Na+: K+ is 12:1, when the total concentration of the univalent ions is 65 mM. Enzyme activities in different fractions of homogenates were measured, and percentage distribution of the ouabain-resistand and ouabain-sensitive enzymes in each fraction was calculated. The results indicate that the ‘m’ fraction, sedimented at 145000g after centrifugation for 60 min, contained major portions of Mgz+-dependent (Na+ K+)enhanced ouabain-sensitive ATPase. Electron-microscopic examination of this fraction revealed numesous closed membrane vesicles. It is concluded that rat sciatic nerves contain two major types of ATPase: (1) one depends on the presence of Mg2+and ATP in a ratio of 1 :1 ; (2) the other additionally requires the presence of Na+ and K+ and is inhibited by 100nM-ouabain. The latter enzyme accounts for one-fifth to one-third of the total enzyme activities.
+
+
+
+
+
+
+
+
P. K. D. is at present a Senior Humboldt Fellow at the Institute of Human Genetics, University of Hamburg, Hamburg, Federal Republic of Germany, and is indebted to the Humboldt Foundation for a travelling grant to attend the meeting.
Abood, L. G., & Gerard, R. W. (1954) J. Cell. Comp. Physiol. 43,379-392 Albers, R. W. & Koval, G. V. (1962) Life Sc. 5,219-222 Bonting, S. L., Simon, K. A. & Hawkins, N. M. (1961) Arch. Biochem. Biophys. 95,416-423 Dahl, J. L. & Hokin, L. E. (1974) Annu. Rev. Biochern. 44,327-356 Das, P . K. &Sayer, K. (1975) Enzyme 19,225-232 Khan, M . A. & Ochs, S. (1974) Bruin Rex. 81,413-426 Libet, B. (1948) Fed. Proc. Fed. Am. SOC.Exp. Biol. 7,95 Lowry, 0.H., Rosebrough,N. J., Farr,A. L. &Randall, R. J. (1951)J. Biol. Chem. 193,265-275 Pincus, J. H., Grove, I., Marino, B. B. & Glaser, G. E. (1970) Arch. Neurol. 22,566-571 Sharma, A. K. & Thomas, P. K. (1974) J. Neurol. Sci. 23,l-15 Skou. J. C. (1957) Biochim. Biophys. Acta 23,394-401
1976
BIOCHEMICAL SOCIETY TRANSACTIONS
(Magnesium plus Sodium plus Potassium Ion)-Stimulated Adenosine Triphosphatase in Rat Sciatic Nerve PRANAB K. DAS, GARTH M. BRAY, ALBERT J. AGUYAO and MICHAEL RAMINSKY
Division of Neurology, Montreal General Hospital Research Institute, Montreal, Que., Canada The presence of at least two types of ATPase* (EC 3.6.1.3, ATP phosphohydrolase) has been reported in almost all tissues (Bonting et al., 1961). The original finding by Skou (1957) on the presence of a specific ATPase in crab nerve (which requires the presence of critical concentrations of Na+, K+ and MgZ+for maximal activity) is of particular interest in the present investigation. This latter type of ATPase, which is intimately associated with the active transport of Na+ and K+ across the nerve axonal membrane, leading to impulse transmission (Pincus et al., 1970), is specifically inhibited by ouabain. Information about ATPases from mammalian nerves is sparse, except for some early reports by Libet (1948) and Abood &Gerard (1954). Also, Khan & Ochs (1974) reported that ATPase systems in cat sciatic nerve are largely composed of the Mgz+- or Cazfactivated type. The reason for the lack of information on ATPase of peripheral nerves from individual animals is probably the unavailability of adequate amounts of the tissues. Our present interest has grown out of a detailed study by Sharma & Thomas (1974) showing that the conduction velocity in sciatic nerves of streptozotocin-treated diabetic rats is significantly lower than normal without there being any demonstrable morphological abnormalities. Since a basic component of nerve conduction is the permeability changes in the membrane, involving the ouabain-sensitive (Na+ K+)-ATPase which controls the sodium pump (Dahl & Hokin, 1974), we decided to study the characteristics of this enzyme activity. It was, therefore, considered of primary importance to define conditions for its quantitative measurement and its normal distribution and then to compare the values with those from experimental diabetic animals under conditions in which the optimum activity could be measured. Here we present some studies on the properties of ATPase from rat sciatic-nerve homogenates.
+
Materials and methods For the evaluation of an ATPase-assay method and studies on some aspects of properties of the enzymes, male Sprague-Dawley rats, weighing 300-400 g, were used. Individual animals were anaesthetized with intraperitoneal injections of sodium pentobarbital. Both sciatic nerves were removed from the sciatic notch to the popliteal fossa, immersed initially in 200 mM-sucrose/Tris/HCI buffer, pH 7.4, and stripped of their epineurial tissues. By using a Polytron homogenizer, the desheathed nerves were homogenized for at least three periods (not exceeding 15 s each) in 2 ml of specified buffer solutions, at W ' C . The homogenates were centrifuged lightly (lo00 rev./min for 2 min) at 4"C, and the supernatants formed the starting material in all the experiments except those for subcellular frationations. Differential centrifugation of the crude homogenates was carried by a method similar to that of Das & Sayer (1975). Protein concentrations and ATPase activities were measured as a routine by the methods of Lowry et al. (1951) and Bonting et al. (1961) respectively, with appropriate control blanks and standards for each individual experiment. For the ATPase assay, portions of the homogenized nerves or suspensions of subcellular fractions were incubated at 37°C in 2 ml of three different solutions: solution A contained ~ ~ M - A T P , 2m~-MgC1,, 100nM-EDTA in 100 mM-Tris/HCl buffer, pH 7.4; solution B was the same as solution A, but contained in addition, 60m-NaCI and S ~ M - K C Isolution ; C contained 60m-NaCI and 100nwouabain. After 1h the reaction was stopped
* Abbreviation: ATPase, adenosine triphosphatase. 1976
75 1
563rd MEETING, LONDON
with 0 . 5 d of ice-cold trichloroacetic acid (15 %, w/v). After centrifugation, 1 . 5 d of the supernatant was added to 1.5ml of colour reagent for the determination of Pi. Activity measured in solution B represented total (Mg2++Na++K+)-ATPase.Ouabain-resistant activity was measured in solution C and the ouabain-sensitive component was calculated as the difference between the activities in solutions B and C. Activity in solution A represented MgZ+-ATPase, and, under standard conditions, this activity was similar to that in solution C. All the activities were expressed as pmol of Pi/h per mg of protein. Results and discussion
ATPase activities (both ouabain-resistant and ouabain-sensitive) were most stable in 50 mM-Tris/HCI buffer, pH 7.5, containing 100nM-dithiothreitol and 2 W 5 0 0 m ~ sucrose at 4°C. In the absence of dithiothreitol, the ouabain-sensitive enzyme was particularly unstable. Freezing at -20°C and subsequent thawing caused aninitial loss of 20 and 40 % of the ouabain-resistant and ouabain-sensitive enzymes respectively. In general, storage appeared to decrease the ouabain-sensitive/ouabain-resistant enzyme ratio. Homogenates prepared in the above buffer were used in subsequent investigations. Both the ATPase activities were linear for periods ranging from 10 to 60 min and were proportional to increasing amounts of tissue between 10 and 65mg of protein. Specific activities of about 7.8 and 3.0 ,umol/h per mg of protein were observed for ouabain-resistant and ouabain-sensitive ATPases respectively along the entire length of dissected nerve. Incubation of the homogenates above 40°C for 1-2 h caused inactivation of both the components. However, at 60°C, ouabain-resistant enzyme and 90 % of ouabainsensitive enzyme were lost. A linear increase in all ATPase activities was observed when the activities were determined at increasing temperature between 15" and 40°C. Similarly when various samples were incubated for 1h a t 4°C over a pH range 6.2-8.6 in Tris/HCl buffer (in one experiment 5Om~-imidazole/HCIbuffer was used for pH 6.2-6.7), i t was observed that ouabain-resistant enzyme activity remained more or less unaffected over the pH range 7.0-8.6, and a decrease of 20% was observed at acid pH values between 6.2 and 6.7; the maximum activity for oubain-sensitive enzyme was observed in the homogenates kept at pH values 7.3-7.6, and 50 and 30% of this activity were observed in the samples kept at pH 6.2 and 8.2 respectively. When the activity was measured in reaction media of different pH, with the application of appropriate corrections for any possible denaturation, a sharp optimum for ouabainsensitive ATPase was observed at p H 7.5, whereas ouabain-resistant ATPase activity increased to a flat optimum around pH 7.6-8.2. Determination of the velocity with respect to ATP concentration, in the presence and absence of 100 nwouabain, and in the presence of constant concentrations of 2.0 mMgCI,, 60m~-NaC1and 5rm-KCI at 37°C indicated that maximum activity for all ATPase activities was reached when the ATP concentration was in a 1 :1 ratio with MgCI2. This maximum activity was maintained even when ATP exceeded Mg2+concentration by 3: 1. Under this condition a K, value for (Mg2+ Na+ K+)-ATPase of 0 . 1 8 m ~was obtained. Both ouabain-sensitive and ouabain-resistant ATPase hydrolysed the following nucleotides in decreasing order of effectiveness: ATP > ITP > GTP > ADP > AMP. However, the ouabain-resistant enzyme had equal and the ouabain-sensitive enzyme 60% of ATP-hydrolytic activity when ITP was used as substrate. Total enzyme activity was almost zero when no cations were present in the reaction medium. Similarly, when the reaction mixture contained only 60 mM-NaC1 and ~ ~ M - Kwithout CI any MgCl, negligible activity could be measured. Increasing concentrations of Mgz+activated the ouabain-resistant enzyme to a maximum value at 2-3 m when ATP was kept constant at 2 m ~ Similarly, . maximum activity for the ouabainsensitive enzyme was also achieved at similar concentrations of MgZf. This latter activity appeared to be inhibited when the Mgz+concentration was raised to 6 m ~ . A similar type of increase in ouabain-resistant ATPase activity was found when increasing concentrations of Ca2+instead of Mgz+were used. Again maximum activity
+
voi. 4
+
BIOCHEMICAL SOCIETY TRANSACTIONS
752
was observed when the concentration of CaZ+was 3 m~ and remained constant even after the concentration was increased to 6 m ~Under . the same conditions, only a minor amount of ouabain-sensitive enzyme activity could be detected at lower concentrations of Caz+(O.O5m), and no activity was seen at higher Ca2+concentration. Our observation is compatible with an earlier observation that Ca2+is inhibitory to (Na+ K+)activated ATPase (Skou, 1957). This was also true even when Mgz+ was present. An additional activity, up to 40-50 %, of ouabain-resistant enzyme was measured when 2m-MgC12 was added to the reaction mixture, after the concentration of CaZ+for maximal activity was held at 3mM. The present observation appeared to be contradictory to those of Khan & Ochs (1974), who observed a slight inhibition of Mg2+-ATPase of a preparation from cat sciatic nerve. Our results, at this stage cannot be compared with those of Khan & Ochs (1974), since we were using crude homogenates instead of a purer fraction. However, it appears that the rat sciatic-nerve homogenates contain three different ATPases: (1) ouabain-resistant (Mg2+ Na+ K+)-stimulated; (2) ouabainsensitive (Mg2+ Na+ K+)-stimulated; (3) Ca2+-stimulated, (MgZ+ Na+ K+)stimulated. From preliminary investigations on the effects of Na+ and K+ by the approaches used bySkou(1957) and Albers & Koval(l962) it appeared that an optimal ratio of Na+: K+ is 12:1, when the total concentration of the univalent ions is 65 mM. Enzyme activities in different fractions of homogenates were measured, and percentage distribution of the ouabain-resistand and ouabain-sensitive enzymes in each fraction was calculated. The results indicate that the ‘m’ fraction, sedimented at 145000g after centrifugation for 60 min, contained major portions of Mgz+-dependent (Na+ K+)enhanced ouabain-sensitive ATPase. Electron-microscopic examination of this fraction revealed numesous closed membrane vesicles. It is concluded that rat sciatic nerves contain two major types of ATPase: (1) one depends on the presence of Mg2+and ATP in a ratio of 1 :1 ; (2) the other additionally requires the presence of Na+ and K+ and is inhibited by 100nM-ouabain. The latter enzyme accounts for one-fifth to one-third of the total enzyme activities.
+
+
+
+
+
+
+
+
P. K. D. is at present a Senior Humboldt Fellow at the Institute of Human Genetics, University of Hamburg, Hamburg, Federal Republic of Germany, and is indebted to the Humboldt Foundation for a travelling grant to attend the meeting.
Abood, L. G., & Gerard, R. W. (1954) J. Cell. Comp. Physiol. 43,379-392 Albers, R. W. & Koval, G. V. (1962) Life Sc. 5,219-222 Bonting, S. L., Simon, K. A. & Hawkins, N. M. (1961) Arch. Biochem. Biophys. 95,416-423 Dahl, J. L. & Hokin, L. E. (1974) Annu. Rev. Biochern. 44,327-356 Das, P . K. &Sayer, K. (1975) Enzyme 19,225-232 Khan, M . A. & Ochs, S. (1974) Bruin Rex. 81,413-426 Libet, B. (1948) Fed. Proc. Fed. Am. SOC.Exp. Biol. 7,95 Lowry, 0.H., Rosebrough,N. J., Farr,A. L. &Randall, R. J. (1951)J. Biol. Chem. 193,265-275 Pincus, J. H., Grove, I., Marino, B. B. & Glaser, G. E. (1970) Arch. Neurol. 22,566-571 Sharma, A. K. & Thomas, P. K. (1974) J. Neurol. Sci. 23,l-15 Skou. J. C. (1957) Biochim. Biophys. Acta 23,394-401
1976
