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A characterization of the ouabain sensitivity of heart microsomal ATPase
162
SHORT COMMUNICATIONS
BBA 43082
A characterization of the ouabain
sensitivity
of heart microsomal ATPase
An ATPase which is Mg2+ activated, (Na + + K+)-stimulated, and ouabain inhibited has been isolated from rabbit heart. The characteristics of our preparation are similar to those of an enzyme from sarcoplasmic reticulum of guinea-pig heart a, and to the (Na + +K+)-transport ATPase described in the classical paper of SKOU2. Ouabain inhibition is an essential criterion for the recognition 3 of this type of transport ATPase. We noted, however, that small deviations in the technique of preparation resulted in failure to reveal what CSXK¥4 has called the "hidden activity". Indeed, an apparently relatively minor modification of the method led to ouabain stimulation. It was therefore evident that the preparative technique contributed to the "visibility" of this transport ATPase and that delineation of the factors which influence the activity pattern might contribute to an understanding of the enzymatic action. Frozen rabbit hearts were macerated in IO vol. of cold o.I M Tris buffer (pH 7.2) containing Na +, K + and Mg2+ as chloride salts and 0.25 M sucrose. The resulting slurry was centrifuged at 600 >,~ g for 20 rain and the supernatant was dialyzed for 24 h against Tris-sucrose buffer with 5 mM EDTA (disodium salt). The preparation was then centrifuged at IOOOO (30 rain), 20000 (30 rain) and 80000 × g (30 min), and in each instance the pellet was rejected. The supernatant was then centrifuged at iooooo × g (7° min) and the final pellet was resuspended in 2 ml of buffer. This material constituted the enzyme. Protein content was determined. Reaction mixtures (o.i ml enzyme, 0.8 ml substrate, o.r ml water or inhibitor in aqueous solution) were incubated at 420 for I h. The substrate was Tris-sucrose buffer with disodium- or T r i s - A T P (usually 0.3 mg/ml of reaction mixture) together with Na +, K +, Mg2+ as chloride salts, and, sometimes, sodium deoxycholate. After incubation, o.I ml of 50 % trichloroacetic acid was added and the mixture centrifuged. The supernatant was assayed for inorganic phosphate. Substrate levels affect reaction rate when below 0.3 mg ATP per ml reaction mixture. The reaction mixture contained o.ooi M MgC12, 0.002 M KC1, o.ooi M NaC1. These amounts represent optimal exogenous metal levels. The enzyme preparation shows a single activity peak at pH 8.1. Inhibition by ouabain with respect to pH was constant over the range pH 6.8-9.2. The temperature optimum is 42° . The time course of phosphate hydrolysis at 37 ° is greatest in the first moments and then falls off sharply. Similar experiments in which much higher substrate levels were present yielded the same result. A number of the conditions of preparation of the enzyme contributed to the effect of ouabain upon enzyme activity. Ouabain inhibition was only occasionally seen in a fresh membrane preparation. Indeed, most fresh preparations were stimulated rather than inhibited by ouabain, Storage at 4 °, however, consistently yielded an enzyme which was inhibited by ouabain. Sodium deoxycholate, urea, and sonication accomplished the same end. Sonication time was critical : 8 sec, 2 % inhibition; IO see, 8 % ; 15 sec, 14 % ; 45 sec, 16 % ; 60 sec, 6 %. Increasing the sucrose concentration resulted uniformly in stimulation by ouabain (Table I). Inhibition of this ATPase system by sodium azide appears to be independent Biochim. Biophys. Acta, 12o (z966) I 6 2 - I 6 5
163
SHORT COMMUNICATIONS
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Biochim. Biophys. Acta, 12o (1966) 162-165
16 4
SHORT COMMUNICATIONS
of inhibition by ouabain. In a typical experiment, a preparation aged 2 days was inhibited 5.6 o, by 5" lO-5 M ouabain and q % by the same concentration of sodium azide; ouabain and azide together lowered activity 14 % (control, 39.4/*g Pl/h per mg protein; 5 " I o - 5 M ouabain, 37.2; 5 . i o - 5 M azide, 35.5; azide + o u a b a i n , 33.3). p-Hydroxymercuribenzoate (IO-4 M) inhibited preparations 4o-6o % and these were not further inhibited by ouabain. Inhibition by ouabain was affected by K +. Maxim u m ouabain inhibition occurred when o.oo2 M KC1 was present. When the K + level was increased, ouabain inhibition was much less than in the control. (In a representative experiment, where 0.002 M KC1 was present, IO a M ouabain inhibited 6 1 % ; where 0.02 M KC1 was present in the incubation mixture, ouabain inhibited 32 % Eeach represents the mean of 6 determinations]). A transport ATPase activity exists in particulate preparations from rabbit heart. The activity pattern is consonant with the criteria for this type of transport enzyme. The cardiac glycoside effect is, in vitro, a function, not only in degree but in kind, of the chemical and physical environment of the enzyme. The experiments described above were conducted with the substrate level sufficient to saturate the enzyme. Frequently in the transport literature, first-order rather than zero-order conditions have been chosen, upon the rationale, presumably, that physiologic ATP levels would be maintained. While the point is germaine, no practical scale would seem to be at hand bv which the amount of ATP actually proximal to the microsome, in the living cell, could be estimated. Also pertinent to the design of our experiments is the fact that reaction rate falls off rapidly after the first few moments. Comparability of activity with impure enzyme preparations is always an equivocal matter; a particular caution m a y be noted for this present system. The rapid deceleration of reaction rate (which does not result from substrate exhaustion) is not explicable with finality. However, the observation does bear comment as it draws attention to the difficulty of converting data describing one transport ATPase system to the form used in describing another. This results from the fact that investigators use arbitrary incubation times which are interpreted with correction factors in terms of activity/time. A variety of environmental factors has been related to the onabain sensitivity and activating-metal requirements of transport ATPase. Enzyme activity characteristics (i.e., the extent of ouabain inhibition, of metal activation, etc.) have been shown to be affected by incubation temperature, by potassium concentration 5 and by altered nuances of manipulation in enzyme preparation ~. SCHWARTZ 1, and SCHWARTZ AND LASETER 7, relate membrane characteristics which change with age and which change in the presence of altered chemical environments to the response of the enzyme to steroids and to metals. Erythrocyte ghost preparations have also been modified b y sonication 8 and by ageing 9. BRow.'," el al. 1° observed, under varying environmental conditions, changes in ouabain sensitivity in both degree and direction. Ouabain stimulation resulted as consistently under one given set of conditions as did inhibition under a second set. This same result has been obtained in rabbit-heart membrane ATPase. The percentage of stimulation effected by ouabain in experiments with this enzyme were, however, considerably smaller than in the earlier work. Stimulation of transport ATPase by cardiac steroids is known in a variety of systems. It has been described for preparations from guinea-pig hearO 1, chicken ,'0
~
Biochim. Biophys. Acta, ~2o (1966) 162-165
165
SHORT COMMUNICATIONS
kidney 12, rabbit brain 13, at low concentration, and at higher concentrations in individual experiments in embryo chicken heart 14. Several explanations for these phenomena exist. We have earlier put forward a hypothesis based upon supposition of a conformational effect of ouabain upon the enzyme itself relative to a metal-binding site z°. This would suppose that ouabain affects the enzyme conformation in a manner which the pre-reaction conformation of the protein disposes. In consequence, availability of the binding site for potassium might be made greater or less by a single action of ouabain. This thesis may be economically applied to the present observations. This work was supported in part by U.S. Public Health Service Research Grant H E 09 571; in part by a Grant-in-Aid from the American Heart Association.
Biochemistry Department, University of Texas Medical Branch, Galveston, Texas (U.S.A.) I 2 3 4 5 6 7 8 9 io ii 12 13 14
HARRY DARROW BROWN
A. SCHWARTZ, Biochem. Biophys. Res. Commun., 9 (1962) 3Ol. J. C. SKOU, Biochim. Biophys. Acta, 23 (1957) 394J. C. SKOU, Physiol. Rev., 45 (1965) 596. T. Z. CS~KY, Rev. Physiol,, 27 (1965) 415 . K. AH~aED AND J. D. JUDAH, Canad. J. Biochem., 43 (1965) 877. M. V. RILEY, Exptl. Eye Res., 3 (1964) 76. A. SCHWARTZ AND A. H. LASETER, Biochem. Pharmacol., 13 (1964) 337. A. ASKARI AND J. C. FRATANTONI, Biochim. Biophys. Acta, 71 (1963) 229. L. E. HOKIN AND D. REASA,. Biochim. Biophys. Acta, 9o (1964) 176. H. D. BROWN, ~h~. J. 2~-EUCERE, A. M. ALTSCHUL AND W. J. EVANS, Life Sci., (1965) 1439. K. S. LEE AND D. H. YU, Biochem. Pharmacol., 12 (1963) 1253. R. F. PALMER AND B. R. NECHAY,J. Pharmacol. Exptl. Therap., 146 (1964) 92. S. L. BUNTING, N. M. HAWKINS AND M. R. CANADY,Biochem. Pharmacol., 13 (1964) 13. R. L. KLEIN, Biochim. Biophys. Acta, 73 (1963) 488.
Received September 27th, 1965 Revised manuscript received November i2th, 1965 Biochim. Biophys. Acta, 12o (1966) 162-165
BBA 43 077
Nicotinamide-adenine dinucleotide dehydrogenase activity of human erythrocyte membranes Evidence accumulated in the last few years supports the concept that the cell membrane is a metabolically active structure, where a "vectorial" assembly of enzymes may be closely related with transport processes 1. A number of enzymic activities has been found in membranes of bacteria and liver cells2,3. In red blood cell "ghosts", (Na+-K+-MgZ+)-activated ATP phosphohydrolase (EC 3.6.1.3) (ref. 4), AMP aminohydrolase (EC 3.5.4.6) (ref. 5), D-glyceraldehyde-3-ph°sphate:NAD oxidoreducta~e (phosphorylating)-ATP:D-glyceraldehyde 3-phosphotransferase complex (EC 1.2.1.12 and 2.7.I.28), together with a triple enzyme sequence of pentose phosphate pathway*, have been described. Also, incorporation of fatty acids into phospholipids has been detected in erythrocyte ghosts 7. Other findings seem to indicate that the red-cell membrane has, as a feature in common with other biological membranes, Biochim. Biophys. Acta, 12o (1966) 165-169
SHORT COMMUNICATIONS
BBA 43082
A characterization of the ouabain
sensitivity
of heart microsomal ATPase
An ATPase which is Mg2+ activated, (Na + + K+)-stimulated, and ouabain inhibited has been isolated from rabbit heart. The characteristics of our preparation are similar to those of an enzyme from sarcoplasmic reticulum of guinea-pig heart a, and to the (Na + +K+)-transport ATPase described in the classical paper of SKOU2. Ouabain inhibition is an essential criterion for the recognition 3 of this type of transport ATPase. We noted, however, that small deviations in the technique of preparation resulted in failure to reveal what CSXK¥4 has called the "hidden activity". Indeed, an apparently relatively minor modification of the method led to ouabain stimulation. It was therefore evident that the preparative technique contributed to the "visibility" of this transport ATPase and that delineation of the factors which influence the activity pattern might contribute to an understanding of the enzymatic action. Frozen rabbit hearts were macerated in IO vol. of cold o.I M Tris buffer (pH 7.2) containing Na +, K + and Mg2+ as chloride salts and 0.25 M sucrose. The resulting slurry was centrifuged at 600 >,~ g for 20 rain and the supernatant was dialyzed for 24 h against Tris-sucrose buffer with 5 mM EDTA (disodium salt). The preparation was then centrifuged at IOOOO (30 rain), 20000 (30 rain) and 80000 × g (30 min), and in each instance the pellet was rejected. The supernatant was then centrifuged at iooooo × g (7° min) and the final pellet was resuspended in 2 ml of buffer. This material constituted the enzyme. Protein content was determined. Reaction mixtures (o.i ml enzyme, 0.8 ml substrate, o.r ml water or inhibitor in aqueous solution) were incubated at 420 for I h. The substrate was Tris-sucrose buffer with disodium- or T r i s - A T P (usually 0.3 mg/ml of reaction mixture) together with Na +, K +, Mg2+ as chloride salts, and, sometimes, sodium deoxycholate. After incubation, o.I ml of 50 % trichloroacetic acid was added and the mixture centrifuged. The supernatant was assayed for inorganic phosphate. Substrate levels affect reaction rate when below 0.3 mg ATP per ml reaction mixture. The reaction mixture contained o.ooi M MgC12, 0.002 M KC1, o.ooi M NaC1. These amounts represent optimal exogenous metal levels. The enzyme preparation shows a single activity peak at pH 8.1. Inhibition by ouabain with respect to pH was constant over the range pH 6.8-9.2. The temperature optimum is 42° . The time course of phosphate hydrolysis at 37 ° is greatest in the first moments and then falls off sharply. Similar experiments in which much higher substrate levels were present yielded the same result. A number of the conditions of preparation of the enzyme contributed to the effect of ouabain upon enzyme activity. Ouabain inhibition was only occasionally seen in a fresh membrane preparation. Indeed, most fresh preparations were stimulated rather than inhibited by ouabain, Storage at 4 °, however, consistently yielded an enzyme which was inhibited by ouabain. Sodium deoxycholate, urea, and sonication accomplished the same end. Sonication time was critical : 8 sec, 2 % inhibition; IO see, 8 % ; 15 sec, 14 % ; 45 sec, 16 % ; 60 sec, 6 %. Increasing the sucrose concentration resulted uniformly in stimulation by ouabain (Table I). Inhibition of this ATPase system by sodium azide appears to be independent Biochim. Biophys. Acta, 12o (z966) I 6 2 - I 6 5
163
SHORT COMMUNICATIONS
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Biochim. Biophys. Acta, 12o (1966) 162-165
16 4
SHORT COMMUNICATIONS
of inhibition by ouabain. In a typical experiment, a preparation aged 2 days was inhibited 5.6 o, by 5" lO-5 M ouabain and q % by the same concentration of sodium azide; ouabain and azide together lowered activity 14 % (control, 39.4/*g Pl/h per mg protein; 5 " I o - 5 M ouabain, 37.2; 5 . i o - 5 M azide, 35.5; azide + o u a b a i n , 33.3). p-Hydroxymercuribenzoate (IO-4 M) inhibited preparations 4o-6o % and these were not further inhibited by ouabain. Inhibition by ouabain was affected by K +. Maxim u m ouabain inhibition occurred when o.oo2 M KC1 was present. When the K + level was increased, ouabain inhibition was much less than in the control. (In a representative experiment, where 0.002 M KC1 was present, IO a M ouabain inhibited 6 1 % ; where 0.02 M KC1 was present in the incubation mixture, ouabain inhibited 32 % Eeach represents the mean of 6 determinations]). A transport ATPase activity exists in particulate preparations from rabbit heart. The activity pattern is consonant with the criteria for this type of transport enzyme. The cardiac glycoside effect is, in vitro, a function, not only in degree but in kind, of the chemical and physical environment of the enzyme. The experiments described above were conducted with the substrate level sufficient to saturate the enzyme. Frequently in the transport literature, first-order rather than zero-order conditions have been chosen, upon the rationale, presumably, that physiologic ATP levels would be maintained. While the point is germaine, no practical scale would seem to be at hand bv which the amount of ATP actually proximal to the microsome, in the living cell, could be estimated. Also pertinent to the design of our experiments is the fact that reaction rate falls off rapidly after the first few moments. Comparability of activity with impure enzyme preparations is always an equivocal matter; a particular caution m a y be noted for this present system. The rapid deceleration of reaction rate (which does not result from substrate exhaustion) is not explicable with finality. However, the observation does bear comment as it draws attention to the difficulty of converting data describing one transport ATPase system to the form used in describing another. This results from the fact that investigators use arbitrary incubation times which are interpreted with correction factors in terms of activity/time. A variety of environmental factors has been related to the onabain sensitivity and activating-metal requirements of transport ATPase. Enzyme activity characteristics (i.e., the extent of ouabain inhibition, of metal activation, etc.) have been shown to be affected by incubation temperature, by potassium concentration 5 and by altered nuances of manipulation in enzyme preparation ~. SCHWARTZ 1, and SCHWARTZ AND LASETER 7, relate membrane characteristics which change with age and which change in the presence of altered chemical environments to the response of the enzyme to steroids and to metals. Erythrocyte ghost preparations have also been modified b y sonication 8 and by ageing 9. BRow.'," el al. 1° observed, under varying environmental conditions, changes in ouabain sensitivity in both degree and direction. Ouabain stimulation resulted as consistently under one given set of conditions as did inhibition under a second set. This same result has been obtained in rabbit-heart membrane ATPase. The percentage of stimulation effected by ouabain in experiments with this enzyme were, however, considerably smaller than in the earlier work. Stimulation of transport ATPase by cardiac steroids is known in a variety of systems. It has been described for preparations from guinea-pig hearO 1, chicken ,'0
~
Biochim. Biophys. Acta, ~2o (1966) 162-165
165
SHORT COMMUNICATIONS
kidney 12, rabbit brain 13, at low concentration, and at higher concentrations in individual experiments in embryo chicken heart 14. Several explanations for these phenomena exist. We have earlier put forward a hypothesis based upon supposition of a conformational effect of ouabain upon the enzyme itself relative to a metal-binding site z°. This would suppose that ouabain affects the enzyme conformation in a manner which the pre-reaction conformation of the protein disposes. In consequence, availability of the binding site for potassium might be made greater or less by a single action of ouabain. This thesis may be economically applied to the present observations. This work was supported in part by U.S. Public Health Service Research Grant H E 09 571; in part by a Grant-in-Aid from the American Heart Association.
Biochemistry Department, University of Texas Medical Branch, Galveston, Texas (U.S.A.) I 2 3 4 5 6 7 8 9 io ii 12 13 14
HARRY DARROW BROWN
A. SCHWARTZ, Biochem. Biophys. Res. Commun., 9 (1962) 3Ol. J. C. SKOU, Biochim. Biophys. Acta, 23 (1957) 394J. C. SKOU, Physiol. Rev., 45 (1965) 596. T. Z. CS~KY, Rev. Physiol,, 27 (1965) 415 . K. AH~aED AND J. D. JUDAH, Canad. J. Biochem., 43 (1965) 877. M. V. RILEY, Exptl. Eye Res., 3 (1964) 76. A. SCHWARTZ AND A. H. LASETER, Biochem. Pharmacol., 13 (1964) 337. A. ASKARI AND J. C. FRATANTONI, Biochim. Biophys. Acta, 71 (1963) 229. L. E. HOKIN AND D. REASA,. Biochim. Biophys. Acta, 9o (1964) 176. H. D. BROWN, ~h~. J. 2~-EUCERE, A. M. ALTSCHUL AND W. J. EVANS, Life Sci., (1965) 1439. K. S. LEE AND D. H. YU, Biochem. Pharmacol., 12 (1963) 1253. R. F. PALMER AND B. R. NECHAY,J. Pharmacol. Exptl. Therap., 146 (1964) 92. S. L. BUNTING, N. M. HAWKINS AND M. R. CANADY,Biochem. Pharmacol., 13 (1964) 13. R. L. KLEIN, Biochim. Biophys. Acta, 73 (1963) 488.
Received September 27th, 1965 Revised manuscript received November i2th, 1965 Biochim. Biophys. Acta, 12o (1966) 162-165
BBA 43 077
Nicotinamide-adenine dinucleotide dehydrogenase activity of human erythrocyte membranes Evidence accumulated in the last few years supports the concept that the cell membrane is a metabolically active structure, where a "vectorial" assembly of enzymes may be closely related with transport processes 1. A number of enzymic activities has been found in membranes of bacteria and liver cells2,3. In red blood cell "ghosts", (Na+-K+-MgZ+)-activated ATP phosphohydrolase (EC 3.6.1.3) (ref. 4), AMP aminohydrolase (EC 3.5.4.6) (ref. 5), D-glyceraldehyde-3-ph°sphate:NAD oxidoreducta~e (phosphorylating)-ATP:D-glyceraldehyde 3-phosphotransferase complex (EC 1.2.1.12 and 2.7.I.28), together with a triple enzyme sequence of pentose phosphate pathway*, have been described. Also, incorporation of fatty acids into phospholipids has been detected in erythrocyte ghosts 7. Other findings seem to indicate that the red-cell membrane has, as a feature in common with other biological membranes, Biochim. Biophys. Acta, 12o (1966) 165-169