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A modified method for studying the interaction of isolated cardiac cell membrane with ions and drugs
ANALYTICAL
BIOCHEMISTRY
75, 345-355 (1976)
A Modified Method for Studying the interaction of isolated Cardiac Cell Membrane with Ions and Drugs Doo HEE KANG AND KWANG Soo LEE Department
of Pharmacology, Medical Center,
State University Brooklyn, New
of New York, York 11203
Downstate
Received October 6, 1975; accepted April 27. 1976 A small amount (0.5 mg) of isolated membrane fragments of rabbit cardiac muscle was dried on Corning cover glasses at 4°C under reduced pressure. The membrane fragments so dried did not come off the cover glasses during incubation in reaction mixtures and subsequent washing. The Mg3+- and Na+-K+-activated adenosine triphosphatase (ATPase) activities of dried membrane fragments were similar and comparable to those of original membrane fragments before drying. Furthermore, the specific binding of ouabain to phosphorylated intermediate forms of Na+-K+-activated ATPase and the ATPdependent “Na binding to the membrane were found to occur in dried membrane fragments. Retention of these vital characteristics of cell membrane, the requirement of small quantities of membrane material, and an advantage of instantaneous removal of membrane fragments from reaction mixtures make this preparation uniquely suited for certain kinds of investigations on the cellular membrane.
Plasma membrane preparations obtained from various cells have been found by many investigators to interact with ions and drugs in vitro (l-4). In most of these studies, membrane preparations were incubated in media containing isotopically labeled drugs or ions and precipitated by centrifugation. Subsequently, radioactivity in the membrane precipitate was counted for determination of material bound to membranes. Thus, in these methods, usually a relatively large amount of membrane preparations and a relatively long time for centrifugation are required. With few exceptions, such as the red blood cell (RBC) membrane which can be prepared easily on a large scale, the yield of purified membrane in large quantities is rather difficult in most of cellular membrane preparations including liver and muscle cells. Also, the centrifugation method of studying ion or drug interaction with membrane preparations presents the inconvenience of repeated centrifugations which require considerable time, thus making it difficult or even impossible to limit the time of exposure of membrane to reaction mixtures. In a search for a method to overcome these disadvantages, various methods of membrane preparation were tried, and it was found that an adaptation of the method of Koketsu et al. (5) was the most successful among all preparations investigated. Previously, Koketsu ef al. (5) de345 Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
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vised a simple method for the study of calcium binding to the skeletal sarcolemma isolated from bull frog. In their study, a small amount of the membrane preparation dried on a Lucite plate was incubated in medium containing 45Ca and subsequently was washed by dipping the Lucite plate in washing solutions. It was found that the dried membrane fragments had the same 45Ca-binding characteristics as those obtained with nondried, conventionally prepared membranes which were obtained by the centrifugation method. Koketsu er al. (5) investigated only the passive 45Ca binding to the dried membrane, and no study has been made on vital characteristics of the dried membrane preparation. Although the dried membrane method offers various advantages, such as rapid separation of membrane from reacting mixtures and a longer duration of preservation, the degree of retention of vital membrane characteristics is unknown. The present study is aimed at investigating this unknown aspect of dried membrane preparations obtained from cardiac tissues. The most important and well-established function of the cellular membrane is the Na+-K+-activated adenosine triphosphatase (membrane ATPase) activity, and one of specific characteristics of the membrane ATPase is the binding of ouabain, a specific inhibitor of the enzyme, to intermediary phosphorylated forms of the enzyme (6- 11). In order to ascertain the degree of preservation of vital characteristics in the membrane fragments prepared by the drying procedure, Na+-K+ ATPase activity and specific binding of ouabain to the enzyme were studied with the dried membrane fragments. Thus, a comparison of Na+-K+-activated ATPase activities of nondried and dried membrane preparations and an investigation of the specific binding of [3H]ouabain to dried membrane preparations under various experimental conditions were made in the present study. METHODS Preparation
of Cardiac
Cell Membrane
Fragments
Cardiac muscle membrane preparation was first isolated from rabbit heart according to the method employed by Rosenthal et al. (12) and Kono and Colowick (13) with some modifications. After the isolation, the drying method of Koketsu et al. (5) was applied to the membrane preparation as follows. Hearts were taken out immediately after rabbits were sacrificed by a blow on the base of the head, placed in an ice-cold 10 mM ethylenediaminetetraacetate (EDTA) solution at pH 7.0 (solution A), and immediately perfused with 30 to 50 ml of solution A which was forced by syringe through the coronary artery to wash out blood trapped in the vascular bed. Auricles, auriculo-ventricular valves, fat, and connective tissues were trimmed off. Ventricles were minced with
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scissors into small pieces, then suspended in a beaker containing solution A. In most experiments, minced tissues from five hearts were suspended in 100 ml of solution A. The beaker was then gently agitated in a Dubnoff water bath at 37°C for 30 min, after which the beaker was taken out and cooled in ice. Following two washings with ice-cold solution A, the cardiac tissue was suspended in 500 ml of solution A which was approximately 20 times the tissue volume. An aliquot of tissue suspension was transferred into a glass tissue grinder and homogenized with a Teflon pestle driven by motor (1200 t-pm). Five to six up-anddown strokes were usually sufficient to obtain the desired degree of homogenization. The homogenates were filtered through two layers of cheesecloth, and the filtrate was centrifuged at IOOOg in a clinical centrifuge for 3 min. Precipitates obtained by the above procedure were pooled and suspended in 450 ml of solution A. This suspension of precipitates was contaminated with a considerable amount of nuclei and RBC as checked by phase-contrast microscopy. This contamination was eliminated as follows: The suspension was transferred into long glass tubes of 150-ml capacity and was placed standing in ice for 60 min. This procedure allowed small tissue fragments and partially broken cells to settle at the bottom of tubes, membrane fragments forming loosely packed precipitates above the bottom. The supematant was suctioned off, and the loosely packed membrane precipitate was resuspended in solution A. This procedure was repeated four or more times, until contamination with nuclei and RBC was negligible as examined by phase-contrast microscopy. The final precipitate was suspended for 30 min in 500 ml of 10 mM EDTA solution containing 0.4 M LiBr, pH 7.0 (solution B), to extract actomyosin and was centrifuged at 110% in a clinical centrifuge for 3 min. After the supernatant was suctioned off, the precipitate was then dispersed evenly in 500 ml of solution B by one or two gentle strokes in a homogenizer. After 30 min of standing it was centrifuged, and the supernatant was decanted. The final precipitate was suspended in 600 ml of 10 mM EDTA solution containing 1.0 M LiBr. pH 7.0 (solution C), and stored overnight in a cold room at 4°C with mild stirring with a magnetic stirrer. The suspension was then centrifuged in a clinical centrifuge at 1 IOOg for 5 min, and the precipitate was resuspended in the same volume of solution C and stirred for another hour followed by centrifugation. The last procedure was repeated once more. The precipitate was washed with 200 ml of 10 mM Tris solution (pH 7.0) three times. Between each wash the suspension was stirred by a magnetic I stirrer for 30 min. The precipitate obtained from the last procedure was suspended in a small amount (less than 10 ml) of 10 mM Tris solution (pH 7.0) and ultrasonicated for 1 or 2 min at 60 W with a cell disruptor (Model W 185 D, Branson Sonic Power Co.). The treated material was allowed to
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stand in a lo-ml test tube in ice. The small amount of tissue which settled down was discarded, and a homogenous supernatant was obtained. The degree of mitochondrial contamination as estimated by measuring cytochrome oxidase and succinic dehydrogenase activities according to the method of Smith (21) was less than 1%. The degree of contamination by the sarcoplasmic reticulum was measured by comparing the active Ca2+ uptake of the present membrane preparation to that of cardiac sarcoplasmic reticulum, according to the method of Lee and Choi (22). It was found that there was no measurable activity of sarcoplasmic reticulum. Thus, the present preparation was found to be not contaminated to a measurable degree by mitochondria and sarcoplasmic reticulum, and it contained mostly membrane fragments from cardiac tissue as judged by Na+-K+ ATPase activity, as shown in the present study. About 0.2 ml of cardiac membrane fragments (about 0.5 mg of protein) was spread over a Corning cover glass (18 x 18 mm), which was then placed in a desiccator containing silica gel. The desiccator (with vacuum fittings) was stored in a cold room (4”C), and samples were dried overnight under reduced pressure of 10 mm Hg. Layering of the membranes onto the cover glasses was consistently even and firm. The thickness of spread membranes appeared to be even as observed under a microscope, and there was no measurable variation in protein content of cover glasses treated in the same manner. Measurement
of Ionic or Drug Binding
Four to eight dried preparations on cover glasses were placed in separated slots of a rack made of Plexiglass and having a vertical arrangement of slots similar to a tissue stain jar commonly used in histology laboratories. The rack had perforated walls so that an incubation mixture or a washing solution was freely accessible to the cover glasses. Before incubation, a rack containing samples was placed for 10 min in a beaker containing 10 mM Tris buffer of the same pH as the incubation medium in order to wet and equilibrate the membranes. The rack was taken out, drained, carefully wiped, transferred into a beaker with solutions containing isotopically labeled agents, and incubated for 10 min at 37°C in experimental solutions. Following incubation, the rack was taken out and washed by dipping it two or three times for 1 to 2 set into a beaker containing 800 ml of 20 mM Tris solution. This was repeated twice in two separate beakers. After the final washing the rack was drained, and cover glasses were taken out with forceps, gently placed on filter papers which absorbed most of the solution adhering to them, and dried completely at room temperature or under a heat lamp. Dried cover glasses were placed in vials containing 10 ml of Bray’s solution and radioactivity
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of vials was counted in Packard Tri-Carb liquid scintillation spectrometer. The presence of cover glasses in vials did not significantly interfere with counting. When 24Na was used, the cover glasses after drying were wrapped with a piece of Parafilm, crushed into many pieces, and placed into test tubes before the determination of radioactivity in a Nuclear Chicago well-type counter. Any deviation from the above standard procedure will be specified. Measurement
of ATPase
Activity
With nondried preparations, Mg2+-activated and Na+-K+-activated ATPase activities were measured by the methods employed in this laboratory (14). Reaction media for Na+-K+-activated ATPase contained 0.125 M Tris buffer (pH 7.4), 3 mM MgClz, 3 mM ATP, 100 mM NaCI, and 10 mM KCl. Reaction media for Mgz+ ATPase contained 10e4 M ouabain in addition to the above components. With dried preparations, cover glasses were incubated in a petri dish containing the same incubation mixture used with nondried preparations; the reaction was stopped by addition of trichloroacetic acid to the reaction mixtures, and the cover glasses were taken out. The inorganic phosphate content in the supernatant after centrifugation of reaction mixtures was determined by the method of Fiske and Subbarow (15), and the protein content was measured by the method of Lowry et al. (20). RESULTS ATPase
Activities
of Dried and Nondried
Preparations
Membrane fragments isolated from the same heart muscle were divided into two groups; one group was dried on cover glasses as described in Methods, and the other was used immediately or stored in a freezer without drying until the time of use. Conditions of incubation were the same for dried and nondried preparations except that incubation of nondried preparations was carried out in test tubes and that of cover glass preparations in a petri dish. The incubation time was 10 min. The results obtained in ATPase experiments are shown in Table 1. The results show that there is no significant difference between Na+-K+ ATPase activities of dried and nondried preparations, indicating that drying does not alter the enzymatic activity of membrane fragments. [3H]Ouabain
Binding
to Membrane
Fragments
Ouabain was found to bind specifically to phosphorylated intermediates of Na+-K+-activated ATPase (8,10.11). In the following experiments, experimental conditions were varied so as to increase or decrease phosphorylated intermediates of Na+-K+-activated ATPase, and the de-
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1
ATPASE ACTIVITIES IN DIUED AND NONDRIED MEMBRANE FRAGMENTS Activity ATPase Mg+-activated Na+-K+-activated
Nondried preparations
Dried, coverglass preparations
1.09 2 0.16 2.72 ” 0.21
0.88 2 0.1 2.63 2 0.12
a Average of 10 experiments. Activity is expressed as micromoles of Pi per milligram of protein per hour.
gree of [3H]ouabain binding to dried membrane fragments under these variable experimental conditions was investigated. First Ca2+ was shown to inhibit the Na+-K+ ATPase activity, possibly through competition between Ca-ATP and Mg-ATP for the enzyme (16). Thus it is possible that formation of phosphorylated intermediates may be inhibited in the presence of Ca2+, resulting in decreased binding of [3H]ouabain to dried membrane fragments. To investigate this possibility, membrane fragments were incubated with and without 1 mM Ca2+ in media containing various concentrations of Mg2+. Radioactivities bound to membranes after incubation were plotted by the
FIG. 1. Effect of ions on [!‘H]ouabain binding to dried membrane fragments (A) Incubation was carried out in a medium containing 50 mM Tris (pH 7.4), 100 mM NaCl, 4 mM ATP (Tris), 10-r M [3H]ouabain, and 0.5, 1.0, 2.0, or 4.0 mM MgCl,. (B) Incubation was carried out in the same mixture as (A) except that the concentration of MgCl, was fixed at 3 mM in all mixtures and the concentration of NaCl was varied. (C) Incubation was carried out in the same medium as (B) except that the NaCl concentration was fixed at 100 mM and the KC1 concentration was varied. Incubation time was 10 min. at 37°C. (- 0 -) with 1 mM CaCl,; (- x -) without CaCl,.
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Lineweaver-Burk method and are shown in Fig. 1A. In the absence of Ca2+, the amount of [3H]ouabain bound to membrane fragments increased as the Mg2+ concentration increased. Data in Fig. 1A show that the presence of 1 mM Ca2+ inhibits E3H]ouabain binding to membrane fragments when the Mg2+ concentration of the medium is less than 2 mM; however, this inhibitory effect of Ca2+ is overcome by increasing the Mg2+ concentration to above 2 mM. It has been shown that Na+ promotes phosphorylation of the membrane ATPase, whereas K+ dephosphorylates the enzyme (10, 11, and others). Thus, effects of Na+ and K+ on [3H]ouabain binding to the dried membrane preparation were investigated. As can be seen in Fig. 1, the binding of ouabain to membrane fragments increased as the Na+ concentration increased and decreased as the K+ concentration increased. The presence and absence of Ca2+ in media did not influence the above effects, of Na+ and K+ under these experimental conditions which employed 3 mM Mg2+ in the media. Effects of ATP on 24Na Binding
to Membrane
Fragments
Since the ATP-dependent 24Na binding was found to occur in nondried conventional membrane fragments (18), this phenomenon was studied in the present dried preparations. The dried membrane fragments were incubated in a medium containing 20 mM Tris (pH 7.4), 10 mM NaCl with 20 &i/ml of 24Na, and 3 mM MgCl, in the presence or absence of 3 mM ATP at 37°C and washed in a large volume of 2 mM Tris (pH 7.4) buffer solution by dipping cover glasses for a varying number of times. The duration of incubation was 10 min since, in preliminary experiments, the maximal binding was found to occur in 10 min. Also, it was found that the amounts of radioactivity bound to cover glasses were strictly proportional to amounts of homogenate spread over the cover glasses. Two sets, each set containing eight cover glasses with membrane fragments, were incubated with and without ATP in the medium, and radioactivities bound to the membrane after a number of washings are shown in Fig. 2. In three separate experiments, radioactivity values among cover glasses belonging to the same experimental group were so close that no statistical treatment was necessary. As can be seen in this figure, washout curves after the second washings were linear in experiments both with and without ATP in the incubation medium, and the 24Na binding to dried membrane fragments in the presence of ATP was always found to be higher than that in the absence of ATP after the same number of washings. The same type of experiment was carried out with dried RBC membrane preparations. Human RBC obtained from a blood bank were pooled and lysed in distilled water and subsequently washed twice with 20 mM
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CPM.lb
IO
6
I .o Q6. 0.6 . 05. 44. 0.3. 0.2 t
FIG. 2. ATP-dependent Na+ binding to dried membrane fragments. Incubation was carried out in a medium containing 20 mM Tris (pH 7.4), 10 mM NaCl with 20 @/ml of 24Na, 3 mM MgCl*, with and without 3 mM ATP at 37°C for 10 min. The cover glass with membrane fragments was washed in a large volume of 2 mM Tris (pH 7.4) solution by dipping for a varying number of times.
ethyleneglycol-bis-(p-aminoethyl ether)-N,N’-tetraacetic acid (EGTA) solution. After washing with 2 mM Tris solution, membrane precipitates were ultrasonified and dried on cover glasses as described in preparation of cardiac cell membrane fragments. When the RBC membrane was incubated with and without ATP in the same manner as the cardiac membrane fragments, it was found that the presence of ATP increased 24Na binding to the RBC membrane in a manner similar to cardiac membrane fragments. DISCUSSION
The cover glass method was originally introduced by Koketsu et al. (5) for the study of passive binding of Ca2+ to the skeletal muscle membrane of bull frog. They compared calcium binding to the dried muscle
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membrane to that of the conventional, nondried membrane and found no difference between the two membrane preparations with regard to the amount of Ca2+ bound to membranes. From this they concluded that the dried membrane behaved in the same way as the nondried membrane with respect to passive calcium binding. However, the drying procedure might still influence some other characteristics involving active processes of the membrane. Therefore, some of the biochemical and pharmacological properties involved with active processes of the cellular membrane were investigated in membranes prepared from the cardiac tissue by the drying method. As mentioned previously, it is known that isolated cellular membranes have Na+-K+-activated ATPase activity and bind cardiac glycosides and Na+ in a specific manner in the presence of ATP (3,6,7,10,1719). The sodium pump mechanism is one of the most important functions of the cell membrane and it is well known that the Na+-K+-activated ATPase activity is intimately associated with the active sodium transport mechanism (7, and others). Therefore, the Na+-K+-activated ATPase activity of dried membrane fragments was investigated to ascertain the degree of retention of vital characteristics in these preparations. As shown in Table 1, no significant difference was found between Na+-K+-activated ATPase activities of dried and nondried membrane fragments. It is significant that the Na+-K+-activated ATPase activity is not impaired by the drying procedure since this is the most important known enzymatic function of cell membranes. If the Na+-K+-activated ATPase activity is intact in the dried preparations, then some phenomena associated with enzymatic processes of the ATPase would be observed in the dried preparations as well as in the nondried preparations. It was previously reported that [3H]ouabain binding to Na+-K+-activated ATPase is increased in the presence of Na+, which promotes phosphorylation of the enzyme, and is decreased in the presence of K+, which promotes dephosphorylation of the enzyme (10,ll). The same characteristic effects of Na+ and K+ were observed to occur with binding of ouabain to the present dried preparations. Thus, it can be seen in this study that the amount of [3H]ouabain bound to dried membrane fragments progressively increases as the Na+ concentration increases (see Fig. 1). Also, the 3H binding to dried membrane fragments decreases as the K+ concentration increases. These findings suggest that both Na+-stimulated phosphorylation and K+-stimulated dephosphorylation of the enzyme also occur in these dried membrane fragments. It indicates that vital characteristics of membranes’ enzymatic processes are not impaired during the drying procedure and are retained in the final dried preparations. The inhibitory effect of Ca2+ on Na+-K+-activated ATPase activity was also utilized for the investigation of the enzymatic processes of
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the dried preparations. In this study, as mentioned previously, [3H]ouabain binding was considered to be an index of formation of phosphorylation intermediates. With regard to the effect of Ca2+, it appears (Fig. 1A) that Mg2+ increases the [3H]ouabain binding, and this effect of Mg2+ is antagonized by Ca2+ in a competitive manner. However, when the Mg2+ concentration is high, Ca 2+ has no influence on the effects of Na+ and K+ in causing the phosphorylation and dephosphorylation, respectively, of Na+-K+-activated ATPase. From these results, it is suggested that Ca2+ inhibits the Na+-K+-activated ATPase of cardiac muscle membrane by competing with Mg2+ in the process of the formation of a phosphorylated intermediate. This supports the finding of Epstein and Whittam (16) in kidney cortex and RBC membrane where they found that Ca2+ (or Ca2+-ATP) competed with Mg2+ (Mg2+-ATP) but not with Na+. However, this finding is at variance with the results of Portius and Repke (19), who found that Ca2+ competed with Na+ as well as Mg2+ for the activation of Na+-K+-activated ATPase. The reason for the difference in results obtained by these investigators is unknown. It may be due to the difference in tissues from which the enzymes were prepared, since Portius and Repke used guinea pigs, whereas the present investigation used rabbits. Lastly, the ATP-dependent 24Na binding was also demonstrated in these dried preparations. Jarnefelt and Stedingk (18) incubated a brain microsomal fraction obtained from rabbit in media containing 24Na with and without ATP. They found that 24Na binding to the brain microsomal fraction, which supposedly contains neuronal membrane fragments among other cellular components, was increased in the presence of ATP, and they attributed this increase to the active binding of Na+ to the membrane in the presence of ATP. The results obtained with the present dried preparations (Fig. 2) indicate that similar ATP-dependent Na+ binding to the membrane fragments also occurs after drying. This is another indication that the active process of the ion transport mechanism of the membrane may not be influenced by the drying procedure. The simplicity, retention of vital characteristics, requirements of a very small quantity of membrane material, and an easy limitation of time of membrane interaction with solutions (up to a few seconds), among other advantages, make this drying technique useful for many studies of membrane characteristics such as washout curves and ionic or drug interactions. ACKNOWLEDGMENTS This work was supported by Grants HE-2875 and HL 16706 from the National Heart and Lung Institute, National Institutes of Health, United States Public Health Service. The second author is a career scientist of the Health Research Council City of New York. This investigation is a part of Ph.D. thesis work by the first author.
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REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 1 I. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
Sanui, H., and Pace. N. (1959) J. Gen. Physiol. 42, 1325. Sanui, H., and Pace, N. (1962) J. Cell. Camp. Physiol. 59, 251. Schwartz, A., Matsui, H., and Laughter, A. H. (1968) Science 160, 323. Jamefelt, J. (1961) Biochem. Biophys. Res. Commun. 6, 285. Koketsu, K., Kitamura, R., and Tanaka, R. (1964) Amer. J. Physiol. 207, 509. Bonting. S. L.. Caravaggio. L. L., and Hawkins, N. M. (1962) Arch. Biochem. Biophys. 98, 413. Skou, J. C. (1%5) Physiol. Rev. 45, 596. Matsui, H., and Schwartz, A. (1967) Fed. Proc. 26, 398. Portius, H. J., and Repke, K. R. H. (1%7) Acrn Biol. Med. Ger. 19, 907. Albers. R. W., Koval, G. J., and Siegel, G. J. (1968) Mol. Pharmacol. 4, 324. Post, R. L., Kume, S., Tobin, T.. Orcutt. B., and Sen. A. K. (1%9) J. Gen. Physiol. 54, 306s. Rosenthal, S. L., Edelman, P. M., and Schwartz, I. L. (1%5) Biochim. Biophys. Acta 109. 512. Kono, T.. and Colowick, S. P. (l%I) Arch. Biochem. Biophys. 93, 520. Lee, K. S., and Yu, D. H. (1963) Biochem. Pharmacol. 12, 1253. Fiske, C. H., and Subbarow, Y. S. (1925) J. Biol. Chem. 66, 375. Epstein, F. H., and Whittam, R. (1966) Biochem. J. 99, 232. Cummins, J., and Hyden, H. (1962) Biochim. Biophys. Acta 60, 271. Jarnefelt, J., and Von Stedingk. L. V. (1%3) Acta Physiol. Stand. 57, 328. Portius. H. J., and Repke, K. R. H. (1967) Acta Biol. Med. Ger. 19, 879. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall. R. (1951) J. Biol. Chem. 193, 265. Smith, L. (1955) in Methods in Enzymology (Colowick, S. P. and Kaplan, N. O., eds.), Vol. 2. pp. 732, Academic Press, New York. Lee, K. S., and Choi. S. J. (1%6) J. Pharmacol. 153, 114.
BIOCHEMISTRY
75, 345-355 (1976)
A Modified Method for Studying the interaction of isolated Cardiac Cell Membrane with Ions and Drugs Doo HEE KANG AND KWANG Soo LEE Department
of Pharmacology, Medical Center,
State University Brooklyn, New
of New York, York 11203
Downstate
Received October 6, 1975; accepted April 27. 1976 A small amount (0.5 mg) of isolated membrane fragments of rabbit cardiac muscle was dried on Corning cover glasses at 4°C under reduced pressure. The membrane fragments so dried did not come off the cover glasses during incubation in reaction mixtures and subsequent washing. The Mg3+- and Na+-K+-activated adenosine triphosphatase (ATPase) activities of dried membrane fragments were similar and comparable to those of original membrane fragments before drying. Furthermore, the specific binding of ouabain to phosphorylated intermediate forms of Na+-K+-activated ATPase and the ATPdependent “Na binding to the membrane were found to occur in dried membrane fragments. Retention of these vital characteristics of cell membrane, the requirement of small quantities of membrane material, and an advantage of instantaneous removal of membrane fragments from reaction mixtures make this preparation uniquely suited for certain kinds of investigations on the cellular membrane.
Plasma membrane preparations obtained from various cells have been found by many investigators to interact with ions and drugs in vitro (l-4). In most of these studies, membrane preparations were incubated in media containing isotopically labeled drugs or ions and precipitated by centrifugation. Subsequently, radioactivity in the membrane precipitate was counted for determination of material bound to membranes. Thus, in these methods, usually a relatively large amount of membrane preparations and a relatively long time for centrifugation are required. With few exceptions, such as the red blood cell (RBC) membrane which can be prepared easily on a large scale, the yield of purified membrane in large quantities is rather difficult in most of cellular membrane preparations including liver and muscle cells. Also, the centrifugation method of studying ion or drug interaction with membrane preparations presents the inconvenience of repeated centrifugations which require considerable time, thus making it difficult or even impossible to limit the time of exposure of membrane to reaction mixtures. In a search for a method to overcome these disadvantages, various methods of membrane preparation were tried, and it was found that an adaptation of the method of Koketsu et al. (5) was the most successful among all preparations investigated. Previously, Koketsu ef al. (5) de345 Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
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vised a simple method for the study of calcium binding to the skeletal sarcolemma isolated from bull frog. In their study, a small amount of the membrane preparation dried on a Lucite plate was incubated in medium containing 45Ca and subsequently was washed by dipping the Lucite plate in washing solutions. It was found that the dried membrane fragments had the same 45Ca-binding characteristics as those obtained with nondried, conventionally prepared membranes which were obtained by the centrifugation method. Koketsu er al. (5) investigated only the passive 45Ca binding to the dried membrane, and no study has been made on vital characteristics of the dried membrane preparation. Although the dried membrane method offers various advantages, such as rapid separation of membrane from reacting mixtures and a longer duration of preservation, the degree of retention of vital membrane characteristics is unknown. The present study is aimed at investigating this unknown aspect of dried membrane preparations obtained from cardiac tissues. The most important and well-established function of the cellular membrane is the Na+-K+-activated adenosine triphosphatase (membrane ATPase) activity, and one of specific characteristics of the membrane ATPase is the binding of ouabain, a specific inhibitor of the enzyme, to intermediary phosphorylated forms of the enzyme (6- 11). In order to ascertain the degree of preservation of vital characteristics in the membrane fragments prepared by the drying procedure, Na+-K+ ATPase activity and specific binding of ouabain to the enzyme were studied with the dried membrane fragments. Thus, a comparison of Na+-K+-activated ATPase activities of nondried and dried membrane preparations and an investigation of the specific binding of [3H]ouabain to dried membrane preparations under various experimental conditions were made in the present study. METHODS Preparation
of Cardiac
Cell Membrane
Fragments
Cardiac muscle membrane preparation was first isolated from rabbit heart according to the method employed by Rosenthal et al. (12) and Kono and Colowick (13) with some modifications. After the isolation, the drying method of Koketsu et al. (5) was applied to the membrane preparation as follows. Hearts were taken out immediately after rabbits were sacrificed by a blow on the base of the head, placed in an ice-cold 10 mM ethylenediaminetetraacetate (EDTA) solution at pH 7.0 (solution A), and immediately perfused with 30 to 50 ml of solution A which was forced by syringe through the coronary artery to wash out blood trapped in the vascular bed. Auricles, auriculo-ventricular valves, fat, and connective tissues were trimmed off. Ventricles were minced with
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scissors into small pieces, then suspended in a beaker containing solution A. In most experiments, minced tissues from five hearts were suspended in 100 ml of solution A. The beaker was then gently agitated in a Dubnoff water bath at 37°C for 30 min, after which the beaker was taken out and cooled in ice. Following two washings with ice-cold solution A, the cardiac tissue was suspended in 500 ml of solution A which was approximately 20 times the tissue volume. An aliquot of tissue suspension was transferred into a glass tissue grinder and homogenized with a Teflon pestle driven by motor (1200 t-pm). Five to six up-anddown strokes were usually sufficient to obtain the desired degree of homogenization. The homogenates were filtered through two layers of cheesecloth, and the filtrate was centrifuged at IOOOg in a clinical centrifuge for 3 min. Precipitates obtained by the above procedure were pooled and suspended in 450 ml of solution A. This suspension of precipitates was contaminated with a considerable amount of nuclei and RBC as checked by phase-contrast microscopy. This contamination was eliminated as follows: The suspension was transferred into long glass tubes of 150-ml capacity and was placed standing in ice for 60 min. This procedure allowed small tissue fragments and partially broken cells to settle at the bottom of tubes, membrane fragments forming loosely packed precipitates above the bottom. The supematant was suctioned off, and the loosely packed membrane precipitate was resuspended in solution A. This procedure was repeated four or more times, until contamination with nuclei and RBC was negligible as examined by phase-contrast microscopy. The final precipitate was suspended for 30 min in 500 ml of 10 mM EDTA solution containing 0.4 M LiBr, pH 7.0 (solution B), to extract actomyosin and was centrifuged at 110% in a clinical centrifuge for 3 min. After the supernatant was suctioned off, the precipitate was then dispersed evenly in 500 ml of solution B by one or two gentle strokes in a homogenizer. After 30 min of standing it was centrifuged, and the supernatant was decanted. The final precipitate was suspended in 600 ml of 10 mM EDTA solution containing 1.0 M LiBr. pH 7.0 (solution C), and stored overnight in a cold room at 4°C with mild stirring with a magnetic stirrer. The suspension was then centrifuged in a clinical centrifuge at 1 IOOg for 5 min, and the precipitate was resuspended in the same volume of solution C and stirred for another hour followed by centrifugation. The last procedure was repeated once more. The precipitate was washed with 200 ml of 10 mM Tris solution (pH 7.0) three times. Between each wash the suspension was stirred by a magnetic I stirrer for 30 min. The precipitate obtained from the last procedure was suspended in a small amount (less than 10 ml) of 10 mM Tris solution (pH 7.0) and ultrasonicated for 1 or 2 min at 60 W with a cell disruptor (Model W 185 D, Branson Sonic Power Co.). The treated material was allowed to
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stand in a lo-ml test tube in ice. The small amount of tissue which settled down was discarded, and a homogenous supernatant was obtained. The degree of mitochondrial contamination as estimated by measuring cytochrome oxidase and succinic dehydrogenase activities according to the method of Smith (21) was less than 1%. The degree of contamination by the sarcoplasmic reticulum was measured by comparing the active Ca2+ uptake of the present membrane preparation to that of cardiac sarcoplasmic reticulum, according to the method of Lee and Choi (22). It was found that there was no measurable activity of sarcoplasmic reticulum. Thus, the present preparation was found to be not contaminated to a measurable degree by mitochondria and sarcoplasmic reticulum, and it contained mostly membrane fragments from cardiac tissue as judged by Na+-K+ ATPase activity, as shown in the present study. About 0.2 ml of cardiac membrane fragments (about 0.5 mg of protein) was spread over a Corning cover glass (18 x 18 mm), which was then placed in a desiccator containing silica gel. The desiccator (with vacuum fittings) was stored in a cold room (4”C), and samples were dried overnight under reduced pressure of 10 mm Hg. Layering of the membranes onto the cover glasses was consistently even and firm. The thickness of spread membranes appeared to be even as observed under a microscope, and there was no measurable variation in protein content of cover glasses treated in the same manner. Measurement
of Ionic or Drug Binding
Four to eight dried preparations on cover glasses were placed in separated slots of a rack made of Plexiglass and having a vertical arrangement of slots similar to a tissue stain jar commonly used in histology laboratories. The rack had perforated walls so that an incubation mixture or a washing solution was freely accessible to the cover glasses. Before incubation, a rack containing samples was placed for 10 min in a beaker containing 10 mM Tris buffer of the same pH as the incubation medium in order to wet and equilibrate the membranes. The rack was taken out, drained, carefully wiped, transferred into a beaker with solutions containing isotopically labeled agents, and incubated for 10 min at 37°C in experimental solutions. Following incubation, the rack was taken out and washed by dipping it two or three times for 1 to 2 set into a beaker containing 800 ml of 20 mM Tris solution. This was repeated twice in two separate beakers. After the final washing the rack was drained, and cover glasses were taken out with forceps, gently placed on filter papers which absorbed most of the solution adhering to them, and dried completely at room temperature or under a heat lamp. Dried cover glasses were placed in vials containing 10 ml of Bray’s solution and radioactivity
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of vials was counted in Packard Tri-Carb liquid scintillation spectrometer. The presence of cover glasses in vials did not significantly interfere with counting. When 24Na was used, the cover glasses after drying were wrapped with a piece of Parafilm, crushed into many pieces, and placed into test tubes before the determination of radioactivity in a Nuclear Chicago well-type counter. Any deviation from the above standard procedure will be specified. Measurement
of ATPase
Activity
With nondried preparations, Mg2+-activated and Na+-K+-activated ATPase activities were measured by the methods employed in this laboratory (14). Reaction media for Na+-K+-activated ATPase contained 0.125 M Tris buffer (pH 7.4), 3 mM MgClz, 3 mM ATP, 100 mM NaCI, and 10 mM KCl. Reaction media for Mgz+ ATPase contained 10e4 M ouabain in addition to the above components. With dried preparations, cover glasses were incubated in a petri dish containing the same incubation mixture used with nondried preparations; the reaction was stopped by addition of trichloroacetic acid to the reaction mixtures, and the cover glasses were taken out. The inorganic phosphate content in the supernatant after centrifugation of reaction mixtures was determined by the method of Fiske and Subbarow (15), and the protein content was measured by the method of Lowry et al. (20). RESULTS ATPase
Activities
of Dried and Nondried
Preparations
Membrane fragments isolated from the same heart muscle were divided into two groups; one group was dried on cover glasses as described in Methods, and the other was used immediately or stored in a freezer without drying until the time of use. Conditions of incubation were the same for dried and nondried preparations except that incubation of nondried preparations was carried out in test tubes and that of cover glass preparations in a petri dish. The incubation time was 10 min. The results obtained in ATPase experiments are shown in Table 1. The results show that there is no significant difference between Na+-K+ ATPase activities of dried and nondried preparations, indicating that drying does not alter the enzymatic activity of membrane fragments. [3H]Ouabain
Binding
to Membrane
Fragments
Ouabain was found to bind specifically to phosphorylated intermediates of Na+-K+-activated ATPase (8,10.11). In the following experiments, experimental conditions were varied so as to increase or decrease phosphorylated intermediates of Na+-K+-activated ATPase, and the de-
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ATPASE ACTIVITIES IN DIUED AND NONDRIED MEMBRANE FRAGMENTS Activity ATPase Mg+-activated Na+-K+-activated
Nondried preparations
Dried, coverglass preparations
1.09 2 0.16 2.72 ” 0.21
0.88 2 0.1 2.63 2 0.12
a Average of 10 experiments. Activity is expressed as micromoles of Pi per milligram of protein per hour.
gree of [3H]ouabain binding to dried membrane fragments under these variable experimental conditions was investigated. First Ca2+ was shown to inhibit the Na+-K+ ATPase activity, possibly through competition between Ca-ATP and Mg-ATP for the enzyme (16). Thus it is possible that formation of phosphorylated intermediates may be inhibited in the presence of Ca2+, resulting in decreased binding of [3H]ouabain to dried membrane fragments. To investigate this possibility, membrane fragments were incubated with and without 1 mM Ca2+ in media containing various concentrations of Mg2+. Radioactivities bound to membranes after incubation were plotted by the
FIG. 1. Effect of ions on [!‘H]ouabain binding to dried membrane fragments (A) Incubation was carried out in a medium containing 50 mM Tris (pH 7.4), 100 mM NaCl, 4 mM ATP (Tris), 10-r M [3H]ouabain, and 0.5, 1.0, 2.0, or 4.0 mM MgCl,. (B) Incubation was carried out in the same mixture as (A) except that the concentration of MgCl, was fixed at 3 mM in all mixtures and the concentration of NaCl was varied. (C) Incubation was carried out in the same medium as (B) except that the NaCl concentration was fixed at 100 mM and the KC1 concentration was varied. Incubation time was 10 min. at 37°C. (- 0 -) with 1 mM CaCl,; (- x -) without CaCl,.
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Lineweaver-Burk method and are shown in Fig. 1A. In the absence of Ca2+, the amount of [3H]ouabain bound to membrane fragments increased as the Mg2+ concentration increased. Data in Fig. 1A show that the presence of 1 mM Ca2+ inhibits E3H]ouabain binding to membrane fragments when the Mg2+ concentration of the medium is less than 2 mM; however, this inhibitory effect of Ca2+ is overcome by increasing the Mg2+ concentration to above 2 mM. It has been shown that Na+ promotes phosphorylation of the membrane ATPase, whereas K+ dephosphorylates the enzyme (10, 11, and others). Thus, effects of Na+ and K+ on [3H]ouabain binding to the dried membrane preparation were investigated. As can be seen in Fig. 1, the binding of ouabain to membrane fragments increased as the Na+ concentration increased and decreased as the K+ concentration increased. The presence and absence of Ca2+ in media did not influence the above effects, of Na+ and K+ under these experimental conditions which employed 3 mM Mg2+ in the media. Effects of ATP on 24Na Binding
to Membrane
Fragments
Since the ATP-dependent 24Na binding was found to occur in nondried conventional membrane fragments (18), this phenomenon was studied in the present dried preparations. The dried membrane fragments were incubated in a medium containing 20 mM Tris (pH 7.4), 10 mM NaCl with 20 &i/ml of 24Na, and 3 mM MgCl, in the presence or absence of 3 mM ATP at 37°C and washed in a large volume of 2 mM Tris (pH 7.4) buffer solution by dipping cover glasses for a varying number of times. The duration of incubation was 10 min since, in preliminary experiments, the maximal binding was found to occur in 10 min. Also, it was found that the amounts of radioactivity bound to cover glasses were strictly proportional to amounts of homogenate spread over the cover glasses. Two sets, each set containing eight cover glasses with membrane fragments, were incubated with and without ATP in the medium, and radioactivities bound to the membrane after a number of washings are shown in Fig. 2. In three separate experiments, radioactivity values among cover glasses belonging to the same experimental group were so close that no statistical treatment was necessary. As can be seen in this figure, washout curves after the second washings were linear in experiments both with and without ATP in the incubation medium, and the 24Na binding to dried membrane fragments in the presence of ATP was always found to be higher than that in the absence of ATP after the same number of washings. The same type of experiment was carried out with dried RBC membrane preparations. Human RBC obtained from a blood bank were pooled and lysed in distilled water and subsequently washed twice with 20 mM
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KANG
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CPM.lb
IO
6
I .o Q6. 0.6 . 05. 44. 0.3. 0.2 t
FIG. 2. ATP-dependent Na+ binding to dried membrane fragments. Incubation was carried out in a medium containing 20 mM Tris (pH 7.4), 10 mM NaCl with 20 @/ml of 24Na, 3 mM MgCl*, with and without 3 mM ATP at 37°C for 10 min. The cover glass with membrane fragments was washed in a large volume of 2 mM Tris (pH 7.4) solution by dipping for a varying number of times.
ethyleneglycol-bis-(p-aminoethyl ether)-N,N’-tetraacetic acid (EGTA) solution. After washing with 2 mM Tris solution, membrane precipitates were ultrasonified and dried on cover glasses as described in preparation of cardiac cell membrane fragments. When the RBC membrane was incubated with and without ATP in the same manner as the cardiac membrane fragments, it was found that the presence of ATP increased 24Na binding to the RBC membrane in a manner similar to cardiac membrane fragments. DISCUSSION
The cover glass method was originally introduced by Koketsu et al. (5) for the study of passive binding of Ca2+ to the skeletal muscle membrane of bull frog. They compared calcium binding to the dried muscle
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membrane to that of the conventional, nondried membrane and found no difference between the two membrane preparations with regard to the amount of Ca2+ bound to membranes. From this they concluded that the dried membrane behaved in the same way as the nondried membrane with respect to passive calcium binding. However, the drying procedure might still influence some other characteristics involving active processes of the membrane. Therefore, some of the biochemical and pharmacological properties involved with active processes of the cellular membrane were investigated in membranes prepared from the cardiac tissue by the drying method. As mentioned previously, it is known that isolated cellular membranes have Na+-K+-activated ATPase activity and bind cardiac glycosides and Na+ in a specific manner in the presence of ATP (3,6,7,10,1719). The sodium pump mechanism is one of the most important functions of the cell membrane and it is well known that the Na+-K+-activated ATPase activity is intimately associated with the active sodium transport mechanism (7, and others). Therefore, the Na+-K+-activated ATPase activity of dried membrane fragments was investigated to ascertain the degree of retention of vital characteristics in these preparations. As shown in Table 1, no significant difference was found between Na+-K+-activated ATPase activities of dried and nondried membrane fragments. It is significant that the Na+-K+-activated ATPase activity is not impaired by the drying procedure since this is the most important known enzymatic function of cell membranes. If the Na+-K+-activated ATPase activity is intact in the dried preparations, then some phenomena associated with enzymatic processes of the ATPase would be observed in the dried preparations as well as in the nondried preparations. It was previously reported that [3H]ouabain binding to Na+-K+-activated ATPase is increased in the presence of Na+, which promotes phosphorylation of the enzyme, and is decreased in the presence of K+, which promotes dephosphorylation of the enzyme (10,ll). The same characteristic effects of Na+ and K+ were observed to occur with binding of ouabain to the present dried preparations. Thus, it can be seen in this study that the amount of [3H]ouabain bound to dried membrane fragments progressively increases as the Na+ concentration increases (see Fig. 1). Also, the 3H binding to dried membrane fragments decreases as the K+ concentration increases. These findings suggest that both Na+-stimulated phosphorylation and K+-stimulated dephosphorylation of the enzyme also occur in these dried membrane fragments. It indicates that vital characteristics of membranes’ enzymatic processes are not impaired during the drying procedure and are retained in the final dried preparations. The inhibitory effect of Ca2+ on Na+-K+-activated ATPase activity was also utilized for the investigation of the enzymatic processes of
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the dried preparations. In this study, as mentioned previously, [3H]ouabain binding was considered to be an index of formation of phosphorylation intermediates. With regard to the effect of Ca2+, it appears (Fig. 1A) that Mg2+ increases the [3H]ouabain binding, and this effect of Mg2+ is antagonized by Ca2+ in a competitive manner. However, when the Mg2+ concentration is high, Ca 2+ has no influence on the effects of Na+ and K+ in causing the phosphorylation and dephosphorylation, respectively, of Na+-K+-activated ATPase. From these results, it is suggested that Ca2+ inhibits the Na+-K+-activated ATPase of cardiac muscle membrane by competing with Mg2+ in the process of the formation of a phosphorylated intermediate. This supports the finding of Epstein and Whittam (16) in kidney cortex and RBC membrane where they found that Ca2+ (or Ca2+-ATP) competed with Mg2+ (Mg2+-ATP) but not with Na+. However, this finding is at variance with the results of Portius and Repke (19), who found that Ca2+ competed with Na+ as well as Mg2+ for the activation of Na+-K+-activated ATPase. The reason for the difference in results obtained by these investigators is unknown. It may be due to the difference in tissues from which the enzymes were prepared, since Portius and Repke used guinea pigs, whereas the present investigation used rabbits. Lastly, the ATP-dependent 24Na binding was also demonstrated in these dried preparations. Jarnefelt and Stedingk (18) incubated a brain microsomal fraction obtained from rabbit in media containing 24Na with and without ATP. They found that 24Na binding to the brain microsomal fraction, which supposedly contains neuronal membrane fragments among other cellular components, was increased in the presence of ATP, and they attributed this increase to the active binding of Na+ to the membrane in the presence of ATP. The results obtained with the present dried preparations (Fig. 2) indicate that similar ATP-dependent Na+ binding to the membrane fragments also occurs after drying. This is another indication that the active process of the ion transport mechanism of the membrane may not be influenced by the drying procedure. The simplicity, retention of vital characteristics, requirements of a very small quantity of membrane material, and an easy limitation of time of membrane interaction with solutions (up to a few seconds), among other advantages, make this drying technique useful for many studies of membrane characteristics such as washout curves and ionic or drug interactions. ACKNOWLEDGMENTS This work was supported by Grants HE-2875 and HL 16706 from the National Heart and Lung Institute, National Institutes of Health, United States Public Health Service. The second author is a career scientist of the Health Research Council City of New York. This investigation is a part of Ph.D. thesis work by the first author.
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Sanui, H., and Pace. N. (1959) J. Gen. Physiol. 42, 1325. Sanui, H., and Pace, N. (1962) J. Cell. Camp. Physiol. 59, 251. Schwartz, A., Matsui, H., and Laughter, A. H. (1968) Science 160, 323. Jamefelt, J. (1961) Biochem. Biophys. Res. Commun. 6, 285. Koketsu, K., Kitamura, R., and Tanaka, R. (1964) Amer. J. Physiol. 207, 509. Bonting. S. L.. Caravaggio. L. L., and Hawkins, N. M. (1962) Arch. Biochem. Biophys. 98, 413. Skou, J. C. (1%5) Physiol. Rev. 45, 596. Matsui, H., and Schwartz, A. (1967) Fed. Proc. 26, 398. Portius, H. J., and Repke, K. R. H. (1%7) Acrn Biol. Med. Ger. 19, 907. Albers. R. W., Koval, G. J., and Siegel, G. J. (1968) Mol. Pharmacol. 4, 324. Post, R. L., Kume, S., Tobin, T.. Orcutt. B., and Sen. A. K. (1%9) J. Gen. Physiol. 54, 306s. Rosenthal, S. L., Edelman, P. M., and Schwartz, I. L. (1%5) Biochim. Biophys. Acta 109. 512. Kono, T.. and Colowick, S. P. (l%I) Arch. Biochem. Biophys. 93, 520. Lee, K. S., and Yu, D. H. (1963) Biochem. Pharmacol. 12, 1253. Fiske, C. H., and Subbarow, Y. S. (1925) J. Biol. Chem. 66, 375. Epstein, F. H., and Whittam, R. (1966) Biochem. J. 99, 232. Cummins, J., and Hyden, H. (1962) Biochim. Biophys. Acta 60, 271. Jarnefelt, J., and Von Stedingk. L. V. (1%3) Acta Physiol. Stand. 57, 328. Portius. H. J., and Repke, K. R. H. (1967) Acta Biol. Med. Ger. 19, 879. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall. R. (1951) J. Biol. Chem. 193, 265. Smith, L. (1955) in Methods in Enzymology (Colowick, S. P. and Kaplan, N. O., eds.), Vol. 2. pp. 732, Academic Press, New York. Lee, K. S., and Choi. S. J. (1%6) J. Pharmacol. 153, 114.