The effect of deoxycholate on rat brain microsomes and microsomal (Na+K+)-ATPase

Brain Research, 52 (1973) 345-358 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

345

THE EFFECT OF DEOXYCHOLATE ON RAT BRAIN MICROSOMES AND MICROSOMAL (Na÷-K+)-ATPase

BENT FORMBY

Zoophysiological Laboratory C, August Krogh Institute, University of Copenhagen, Copenhagen (Denmark) (Accepted August 22nd, 1972)

SUMMARY

Under carefully controlled conditions for the treatment of rat brain microsomal membranes with deoxycholate, a marked increase in the activities of (Na+-K÷) ATPase and total ATPase were observed. The relationship between the increase in enzymic activities and the amount of protein rendered soluble could not explain the observed activation, since an approximately 50 ~o decrease in protein content of the microsomal membranes after deoxycholate treatment yielded 5-fold activation of (Na+-K+)-ATPase. Polyacrylamide gel electrophoresis in a sodium dodecyl sulphate system revealed minor significant changes in the number of membrane proteins after treatment with deoxycholate, as only one slow and two fast moving bands had disappeared. Kinetic studies of the solubilization of membrane protein made it difficult to consider deoxycholate as an enzyme activator. Calculation of sedimentation coefficients emphasized that deoxycholate splits the microsomal membrane into smaller subunits" a homogeneous part having (Na+-K+)ATPase activity and a heterogeneous part carrying ouabain-insensitive (MgZ+)ATPase. It is concluded that a deoxycholate-induced decrease in particle size increases the number of sites for substrate and activators, and it is suggested that (Na+-K+)ATPase in vivo could be controlled by some membrane-bound factors that are removed by deoxycholate treatment.

INTRODUCTION

The specific activity of the membrane-bound sodium-potassium activated ATPase, (Na+-K+)-ATPase (ouabain-sensitive; ATP phosphohydrolase, EC 3.6.1.3)

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can be markedly increased after treatment of the membrane with detergents such as deoxycholate 5-7, sodium dodecyl sulphate a and lubrolS,lL The present paper describes an investigation of the mechanism of deoxycholate activation of ( N a + - K ) ATPase from rat brain. As an aqueous solution of active (Na+-K+)-ATPase cannot be obtained by using classical methods for dissolving enzymes, the activity of the enzyme is believed to depend on its close integration into the membrane structure. Jorgensen and Skou 6 analysed in detail the effect ofdeoxycholate on preparations from the outer medulla of rabbit kidney and reported that deoxycholate removes membrane protein whereby the access of substrate and activators is facilitated to their respective sites on the membrane-bound enzyme. On the other hand, Uesugi et al. ~2 found, in studies with bovine brain microsomes, that deoxycholate did not remove any membrane protein and that the increase in specific activity, therefore, could be due to some type of enzyme activation. The following aspects of the problem are here investigated: (i) the relationship between deoxycholate activation of (Na÷-K+)-ATPase and the amount of microsomal membrane protein rendered soluble, (ii) scanning of microsomal membrane protein removed by deoxycholate treatment, (iii) kinetics of solubilization of membrane protein, and (iv) change in microsomal size and density induced by treatment with the detergent. EXPERIMENTAL

Tissue preparation. Fresh rat brains were homogenized at 0 °C in a solution containing 0.25 M sucrose, 30 m M histidine-HC1 buffer (pH 6.54) and 1.0 m M EDTA. The homogenate (diluted to 10~, w/w) was centrifuged at 600 × g for 10 rain, and the resulting supernatant was centrifuged at 10,000 × g for 20 min to sediment a pellet of heavy microsomes and mitochondria. The supernatant from this was centrifuged at 40,000 × g for 100 min to sediment microsomal membranes, which were then washed and finally suspended in 30 m M histidine-HC1 buffer (pH 6.54) to give about 7 mg membrane protein per ml. The membranes were stored at --35 °C. Before use, thawed membranes were centrifuged at 150,000 × g for 30 min to remove non-membranous protein. Enzymic activity. Assays of ATPase activity were carried out using two standard incubation media: (i) ouabain-insensitive (Mg~+)-ATPase activity was assayed in a medium containing 0.1 ml diluted enzyme preparation (2-3 mg membrane protein per ml), 5.0 m M Tris-ATP, 5.0 m M MgClz, 0.1 m M ouabain and 30 m M histidineHC1 buffer (pH 7.28) in a total volume of 0.6 ml; (ii) total ATPase activity was assayed in a (Mg2+-Na+-K+)-medium containing 0.1 ml enzyme preparation (2-3 mg membrane protein per ml), 5.0 m M Tris-ATP, 100 m M NaC1, 20 m M KC1, 5.0 m M MgCI2 and 30 m M histidine-HC1 buffer (pH 7.28) in a total volume of 0.6 ml. The enzymic reactions were allowed to run for 20 min at 30 °C whereafter aliquots of 100 #1 were removed for phosphate determination by the procedure of Stanton 11. The difference between the total ATPase activity and the ouabain-insensitive (MgZ+)-ATPase activity gave the (Na+-K+)-ATPase activity.

EFFECT OF DEOXYCHOLATE ON RAT BRAIN MICROSOMES

347

Protein was measured by the procedure of Lowry et alp using bovine serum albumin as standard. Incubation of the microsomal membranes with deoxycholate. During initial studies the pH, temperature, length of incubation time and concentration of deoxycholate were varied as described by Jorgensen and Skou 6. In agreement with these authors the following procedure was found to give maximal activation: 2-3 mg membrane protein were incubated in 30 mM histidine-HCl buffer (pH 6.58) with 1.0 mM EDTA and sodium deoxycholate for 30 min at room temperature in a total volume of 1.0 ml. The incubation mixture was then centrifuged at 150,000 x g for 30 min. The pellet was washed with 30 mM histidine-HC1 buffer (pH 6.58) and finally resuspended in the histidine buffer with 2 mg membrane protein/ml and used within 2 h. Polyacrylamide gel electrophoresis. To remove lipids 2.0 ml samples of diluted microsomal membranes were shaken with 3.0 ml chloroform-ethanol (3:1, v/v). After centrifugation at 600 × g for 20 min, the lower phase was removed by gentle suction with a pasteur pipette. The upper phase was then centrifuged at 1500 x g for 20 min, and the resulting pellet was washed several times with 30 mM histidine-HCl buffer (pH 6.58). Finally the pellet was dried overnight at room temperature. The resulting dry proteins were solubilized (0.25 ml/mg) by incubation at 37 °C for 3 h in a solution containing 1.0 ~o sodium dodecyl sulphate (SDS), 1.0 ~ 2-mercaptoethanol and 0.01 M sodium phosphate buffer (pH 7.20). Each sample was diluted with glycerol and sodium phosphate buffer to give a final SDS concentration of 0.5 and a final glycerol concentration of 20 ~o. For electrophoresis, 75 tzl aliquots (150200/zg protein) were used. Polyacrylamide gels were made as follows. Small pore gel: 5.0~ acrylamide, 0.25~ N,N'-methylenebisacrylamide, 0.003~ N,N,N',N'-tetramethylenetylenediamine, 0.085~ (NH4)2S2Os, and 0.1 ~ SDS in 0.5 M Tris-HC1 buffer (pH 7.50), were added to glass tubes (5.0 mm x 7.5 cm) to give 6.5 cm gel columns; after being overlayered with water the gels were allowed to polymerize for 1 h. Large pore gel: 3.0~ acrylamide, 0.0007 ~ riboflavin, and 0.1 ~o SDS in 0.3 M Tris-HC1 buffer (pH 5.50); after being overlayered with water, the gels were

1.200 -

E

O.800-

a 0.~0,

o 0

0.40

0.80

1.20

> 1.60 Protein (mg/ml)

Fig. 1. Variation of the absorbance of microsomal membrane suspensions in 30 m M histidine--HCl buffer (pH 6.58) with particle concentration in terms of membrane protein. 4, 540 rim.

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allowed to photopolymerize for 1 h. In both electrode compartments 0.01 M sodium phosphate buffer (pH 7.20) in 0.1 ~ SDS was used. After electrophoresis for 15 min without samples at a constant current of 5 mA per tube, electrophoresis with samples was carried out for 2 h at room temperature at a constant current of 5 mA per tube. All the proteins migrated towards the anode. After electrophoresis the gels were removed from the glass tubes and stained for 90 min in 1 700amido black in 7 ~o acetic acid and destained with frequent changes of 7 ~ acetic acid. Densitometric scans of the gels were accomplished with a Joyce-Loebl densitometer. Kinetic experiments. Various concentrations of microsomal membranes were suspended in 30 m M histidine-HC1 buffer (pH 6.58) and turbidity was measured at 540 nm. As shown in Fig. l, a straight line relationship was observed between particle concentration in terms of membrane protein, and absorbance. The turbidity, z, of a suspension of spherical particles is defined by the relationship = In Io/I = a 2" N • K where Io and I are, respectively, the intensities of the incident and transmitted light, a is the radius of the scattering particles, N is the number of particles per unit volume, and K is the scattering coefficient (i.e. the fraction of light incident on the particle that is scattered by the particle). Assuming that K is constant in the interval studied, then any tendency to decrease 3, will give rise to decreased values for a and/or N. An initial velocity (Vo) for the change in particle concentration as induced by detergent was defined as mg membrane protein solubilized per ml solution per sec. It was measured as follows: 1.5 ml microsomal membrane suspension (2 mg membrane protein/ml) was rapidly mixed with 0.5 ml solution of deoxycholate in 30 m M histidine-HCl buffer (pH 6.58), and the turbidity was recorded at intervals of 5 sec. The initial slope of the curve for turbidity vs. time was determined and used as a measure of the initial velocity Vo. The turbidity from deoxycholate-solubilized membrane protein was insignificant.

Calculation of energy of activation for the solubilization of membrane protein. Using the integrated form of the Arrhenius equation, In k = (--Ea/RT) + C, the change in energy of activation, Ea, can be calculated, assuming that energies of activation are independent of temperature in the interval used and that the initial velocity (Vo) is directly proportional to the specific rate constant (k) at constant deoxycholate concentration and may be substituted for it. Analytical differential centrifugation. The sedimentation of microsomes and the various components present after treatment with detergent was performed as described by Anderson 1 and Cotman et al. 4. An MSE No. 59113 angle-head rotor was spun in an MSE superspeed-50 centrifuge equipped with temperature control and a ratemeter which accurately indicated the speed in rev./min. A 9.0 ml sample of particle suspensions was placed in each tube (total volume 10 ml). After centrifugation the tubes were rotated very slowly through 180° until the pellet was fiat in the bottom of the tube 1. Of the upper fluid, 7.0 ml was carefully removed and defined as supernatant, and the remaining 2.0 ml defined as sediment. With the geometry of rotor and tube used, these volumes resulted in a radius of the menisci before (Ro) and after (Rp)

EFFECT OF DEOXYCHOLATE ON RAT BRAIN MICROSOMES

60'

349

f'\'-,

50

40

30

20 E

10

l ' \ ° " ° " - e ~ o

0:4

o:e

I:=

.):4 ooc ,..gl,.,.,l

Ii

Fig. 2. Dependence of the activity of total ATPase (O O), (Na+-K+)-ATPase ( A - - ~ k ) and ouabain-insensitive (Mg2+)-ATPase ( [] []) on the concentration of deoxycholate. For experimental conditions, see text.

removal of the 7.0 ml supernatant with the following dimensions: Ro, 4.71 cm; and Rp, 6.21 cm. The time at speed was defined as the time between acceleration and deceleration. All experiments were conducted with the temperature control set at 5 °C. Calculations. The number o f g min was related to ro2t by the formula ~o~t = (g min • 60 • 980)/Ray where g is force times gravity at Rav; rain, minutes of centrifugation time; Ray, average radius in cm. The o~2t values of interest ranged from 5 • 107 to 5 • 1010 (corresponding to 6000 g min and 1200,000 g min). Graphically, co2t values were plotted along the abscissa with (Na+-K+)-ATPase or ouabain-insensitive (Mg2+)ATPase activity on the ordinate. The sedimentation coefficients (corrected to 20 °C) corresponding to the oj2t values used were calculated from the following equation with Ct/Co = 0 1 S --

2 co2t

In [ R°2 Ct [ Rp 2 ~- C ~ o

( •1

Ro2)J Rp 2

where Ro ----- radius of meniscus; Rp = radius of partition, or plane of the surface of the pellet volume;

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Co = concentration of particles, or enzymic activity at the start of the run ; Ct = concentration of particles, or enzymic activity at the conclusion of the run. An ideal particle curve for a lOaS particle was obtained by solving the same equation for a series of values of Ct/Co. RESULTS

Ef[ect of deoxycholate on enzymic activity As already demonstrated by Jorgensen and Skou 6, the results show that the activating effect of deoxycholate on (Na+-K÷)-ATPase is highly dependent on the pH of the incubation medium, the presence of EDTA, and the length of incubation. Fig. 2 shows the activation by deoxycholate of total ATPase, (Na+-K+)-ATPase and ouabaininsensitive (Mg2+)-ATPase. The activity of both total ATPase and (Na+-K÷)-ATPase increased with increasing concentrations of deoxycholate, the highest degree of activation being obtained with a deoxycholate concentration of 1.2 mg/ml, whereas the activity of ouabain-insensitive(Mg2+)-ATPase decreased with increasing concentrations of deoxycholate. This concentration of deoxycholate is larger than that described by J~rgensen and Skou 6 for obtaining maximal activation. However, the discrepancy is supposedly due to the larger concentration of membrane protein used here during the incubation with deoxycholate (e.g. 2 mg/ml vs. 0.25 mg/ml). Fig. 3 shows the effect of deoxycholate on the liberation of protein from the microsomal membranes. After incubation with various concentrations of deoxycholate, the incubation mixtures were centrifuged at 150,000 × g for 30 min, and the content of protein in sediment and supernatant was measured. From the straight line relationship in Fig. 3, it is seen that a deoxychotate concentration of 1.2 mg/ml incubation medium decreased the total protein in the sediment to about 40 %. Note that the same deoxycholate concentration increased total ATPase activity about 2-fold and (Na+-K+)-ATPase activity Protein in Supernetant Protein in Sediment

2,0 1.6. 1.2, 0.8. 0.4, 0

0

o:s

1:o

1~5 DOC ma/ml

Fig. 3. Distributionof membranous protein betweensupernatantand sedimentafter treatment with various concentrationsof deoxycholate.

EFFECT OF DEOXYCHOLAIE ON RAT BRAIN MICROSOMES

351

6O

g

e.

so, 4030-

a.

i

L

s'*

20s j

&

s

100

o

;o

|

2o

!

30

4'0

~'o

6'0

Percent Protein in Supernatant

Fig. 4. Dependence of the activity of total ATPase and (Na+-K+)-ATPase on the percentage of microsomal membrane protein rendered soluble by increasing concentrations of deoxycholate. (See also Fig. 3.) Enzymic activities were assayed in the sediment after centrifugation of incubation media at 150,000 x g for 30 min.

about 5-fold, as shown in Fig. 2. Ouabain-insensitive (Mg~+)-ATPase or (Na+-K+) ATPase activities were not detectable in the supernatant fraction after centrifugation of the incubation media at 150,000 x g for 30 min. Fig. 4 shows the linearity between the activities of total ATPase and (Na+-K+)-ATPase and the percentage of membrane protein rendered soluble by increasing concentrations of deoxycholate. The activation by deoxycholate of (Na+-K+)-ATPase appears to be irreversible since the activated level of enzymic activity was retained after resuspension of the microsomal sediment in a medium devoid of detergent.

Polyacrylamide gel electrophoresis Initial experiments showed that electrophoresis in SDS gave the best separation of the microsomal proteins. The use of a low pH (5.5) in the large pore gel gave a better separation than the system described by Ornstein 1°. Fig. 5 shows electrophoresis of untreated microsomal membranes revealing at least 16 bands. Fig. 5 also shows electrophoresis of sediment and supernatant obtained after centrifugation of microsomal membranes treated with deoxycholate. Only minor changes are seen in the number of bands in detergent-treated and untreated microsomal membranes, respectively. Band 6 together with bands 14 and 18 appear to have been removed from the precipitate almost entirely. Band 6 reappears in the supernatant with almost unchanged mobility. Band 15 in the supernatant appears to travel more rapidly than the correspondingly numbered band in the precipitate, which could be due to the presence of different proteins in a single spot. Figs. 6a-c show densitometric scans of the 3 gels in Fig. 5, and demonstrate the disappearance of 3 bands (6, 14 and 18). However, it is obvious from Fig. 6b that some membrane-bound proteins were partially solubilized and migrated among

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B. FORMBk

Fig. 5. Polyacrylamide gel electrophoresis of microsomal membranes (number 1). After treatment with 0.8 mg deoxycholate/ml incubation media, the sample was centrifuged at 150,000 x g for 30 rain. Sediment (number 2) and supernatant (numbers 3 and 4) were applied to gel electrophoresis. Per tube, 75 itl aliquots (150-200/~g protein) were used. The arrow indicates band 6 removed after treatment with detergent.

353

EFFECT OF DEOXYCHOLATE ON RAT BRAIN MICROSOMES

a

12

16

11 10

1817 ~

e

2 43

T 14

C

12

9

-

T

i

j/

~

"

,.-.%]

Fig. 6. Densitometer scans of the gels in Fig. 5. a, Number 1 ; b, number 3; c, number 2. The proteins migrated towards the anode (+). the faster bands. Membrane fractions with better defined morphology could make such a finding more conclusive. Kinetics

Fig. 7 shows the logarithm of initial solubilization velocity vs. the logarithm of the concentration of deoxycholate at 20 o and 38 °C. A break in these curves is observed at a detergent concentration of 0.7 mg/ml. This could be the critical micelle concentration (cmc), in good agreement with the value of 0.6 mg/ml found by Jorgensen e by measuring the surface tension of the incubation media. Also note from Fig. 7 that the curves continue to rise with a smaller, but still appreciable, concentration dependence. Since the concentration of dissociated deoxycholate anions remains constant above cmc, a direct interaction between micelles and microsomal membranes is indicated 2. The slopes of the curves below the cmc suggest a faster interaction and much greater concentration dependence than above the cmc. The plots at 20 ° and 38 °C remain almost parallel below the cmc, with the same vertical separation. An Arrhenius plot of these data yields an energy of activation of 1 kcal/

354

log

B. FORMBY

vo

1.2. 0.8' 0.4.

cmc

0

o'.1

0'.2

o73

d4

0"5 ,.g [oocl

Fig. 7. Logarithm of initial velocity of solubilization of membrane proteins (mg protein/ml/sec) vs. logarithm of deoxycholate concentration (DOC), in mM. The microsomal membrane concentration was 1 mg/ml and the temperatures were 20 °C ( A - - A ) and 38 °C ( • • ) . The arrow indicates the break at the critical micelle concentration (cmc). mole, for each type o f solubilization, showing that the r a t e - d e t e r m i n i n g step in the interaction o f m i c r o s o m a l m e m b r a n e s with either micelles or m o n o m e r i c deoxycholate anions could be the sameL Sedimentation properties

It was shown a b o v e that d e o x y c h o l a t e increases the activity of (Na+-K+) A T P a s e several-fold w i t h o u t n o t i c e a b l e changes in the activity o f ouabain-insensitive (Mg2+)-ATPase. A f t e r t r e a t m e n t o f m i c r o s o m a l m e m b r a n e s with deoxycholate, ouabain-insensitive (Mg2+)-ATPase m a y thus be located on particles different f r o m those possessing ( N a + - K + ) - A T P a s e activity. Figs. 8 a n d 9 show the s e d i m e n t a t i o n profiles o f m i c r o s o m a l m e m b r a n e s with a n d w i t h o u t t r e a t m e n t with detergent. ( N a ~K + ) - A T P a s e a n d ouabain-insensitive (MgZ+)-ATPase were used as m a r k e r enzymes.

100 80' 60' 40' m 20'

)

0 107 105

104

S20,W 103

102

101

Fig. 8. Sedimentation curves for microsomes. The points indicate the percentage (Na+-K+)-ATPase activity remaining in the supernatant after centrifugation of a suspension of rat brain microsomes (2 mg protein/ml) in 30 mM histidine-HC1 buffer (pH 6.58) for the integrals (w2t) shown on the abscissa. The to~t scale on the abscissa was aligned with the sedimentation scale as described in text. The dotted line shows an ideal curve for a 10aS particle. ~rrows indicate observed sedimentation coefficients in Svedberg units corrected to water at 20 °C. A - - - - A , Native particles; • • , particles treated with deoxycholate (0.8 mg/ml).

355

EFFECT OF DEOXYCHOLATE ON RAT BRAIN MICROSOMES

100

Z

806040eI

20-

e~e

0 10 r

109

10 s

10 s

10 4

4,

S,O,W 1~)s

loll

101~, 1()z

~)2t > 10'

Fig. 9. Sedimentation curves for microsomes. The points indicate the percentage ouabain-insensitive

(Mg~+)-ATPase activity remaining in the supernatant after centrifugation of a suspension of microsomes (2 mg protein/ml) in 30 mM histidine-HCl buffer (pH 6.58) for the integrals (c02t)shown on the abscissa. The dotted projection which intercepts at 5 • 10sS represents an average sedimentation coefficientfor approximately 55 ~ of the microsomai particles. Arrows indicate observed sedimentation coefficientsin Svedberg units corrected to water at 20 °C. A A, Native particles; • •, particles treated with deoxycholate (0.8 mg/ml). The dotted line in Fig. 8 shows how a 108S particle of uniform size and density would behave. When compared with the ideal particle curve it is obvious from Figs. 8 and 9 that microsomal membranes before treatment with detergent exhibit homogeneity. With (Na+-K+)-ATPase and ouabain-insensitive (Mg2+)-ATPase as marker enzymes these particles were completely removed from the supernatant after 3.8 • 109 co2t, corresponding to equal sedimentation coefficients of 3 • 103S. However, after treatment of the microsomal membranes with deoxycholate (0.8 mg/ml), a considerable change in the sedimentation properties was observed. With (Na+-K+)-ATPase as marker enzyme, Fig. 8 shows a drift from the ideal particle curve, and 2.7.1010 co2t was required to remove the particles from the supernatant completely. The sedimentation coefficient was 4.102S. With ouabain-insensitive (Mg2+)-ATPase as marker enzyme, the dotted projection in Fig. 9 intercepts the x axis at 5.10nS, which represents an average sedimentation coefficient for approximately 55 ~o of the microsomal particles. The remaining and smaller particles (approximately 45 ~ ) were not completely sedimented after 6.5 • 1010 os2t and therefore represent particles of another size and density class. This observation emphasizes that treatment with deoxycholate decreases the size of the particles exhibiting both (Na~-K+)-ATPase and ouabaininsensitive (Mg2+)-ATPase activities, but also that smaller particles only carrying ouabain-insensitive (Mg2+)-ATPase activity are being formed. DISCUSSION

Under carefully controlled conditions for the treatment of rat brain microsomal membranes with deoxycholate6, a marked increase in the specific activity of (Na+-K+)ATPase and of total ATPase was observed, whereas the specific activity of ouabain-

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insensitive (Mg~+)-ATPase decreased. The activation of total ATPase and (Na -K +)ATPase was maximal at 1.2 mg deoxycholate per ml incubation medium. This concentration is 2-fold higher than that found by Jorgensen and Skou 6. One explanation for this discrepancy must be the much higher membrane protein concentration used in the present experiments. As previously shown, the activating effect of a given concentration of deoxycholate decreases if the protein concentration is increased 6. Differences in the nature of the preparations may also be taken into account. A considerable amount of membrane protein was rendered water soluble when microsomal membranes were incubated with increasing concentrations of deoxycholate, and a linear relation was found with the increase in specific activity of total ATPase, whereas the rise in the specific activity of (Na+-K+)-ATPase was 4-5-fold. One simple interpretation would seem to be that some of the proteins adsorbed to native membrane particles actually inhibit the (Na+-K÷)-ATPase. However, solubilized membrane protein was not found to inhibit (Na÷-K+)-ATPase activated by deoxycholate treatment. Polyacrylamide gel electrophoresis in an SDS system revealed minor changes in the number of microsomal membrane proteins after treatment with deoxycholate. One slow and two fast moving bands had disappeared. Densitometric scanning of the gels clearly showed that other proteins were partially solubilized after treatment with deoxycholate. It has been shown 6 that deoxycholate does not bind to membranes having high specific (Na+-K+)-ATPase activity. This observation makes it unlikely that the activation is due to an enzyme-activator function of deoxycholate, as further supported by kinetic studies of the solubilization of membrane protein. The calculation of initial velocity of membrane protein solubilization as a function of deoxycholate concentration indicates a direct interaction between the microsomal membranes and deoxycholate micelles above the critical micelle concentration, although it is slower than the interaction with monomeric deoxycholate anions below the critical micelle concentration. The energy of activation remained the same in either case, about 1 kcal/ mole. The change in turbidity of microsomal membrane suspensions, which was recorded for the calculation of the initial velocity, is subject to some experimental uncertainty. For example the effect of increasing deoxycholate concentrations upon light scattering by the various particles, the role of aggregation initiated by higher deoxycholate concentrations and the change in refractive indices are difficult to evaluate. However, because the microsomal fraction is a population of membrane particles which are similar in composition, over-all organization and physicochemical properties, the errors might be considered constant over the range of deoxycholate concentrations studied. The method of differential centrifugation can be used for characterizing the sedimentation of various particles from brain homogenates 4. Microsomal membranes isolated from rat brain showed characteristics similar to those expected for a homogeneous particulate population, as demonstrated by sedimentation plots of (Na~-K +)ATPase and ouabain-insensitive (Mg2+)-ATPase activities. Since the sedimentation coefficients are the same for both enzymes, it appears that they are bound to mere-

EFFECT OF DEOXYCHOLATEON RAT BRAIN MICROSOMES

357

branes o f the same size and density. However, treatment o f the microsomal membranes with deoxycholate resulted in a change in size and/or density. Thus, the (Na+-K+) ATPase activity decreased in sedimentation coefficient from 3 • 108S to 4 . 102S, and the ouabaJn-insensitive (Mg2+)-ATPase appeared to become more heterogeneous. M o s t o f this activity has a sedimentation coefficient o f 5 • 102S, close to the 4 • 10sS value calculated for the (Na+-K+)-ATPase particles. The remaining activity sedimented m o r e slowly. The results presented suggest that deoxycholate removes a large a m o u n t o f protein from, and splits, the microsomal m e m b r a n e into smaller subunits, the more h o m o g e n e o u s part having (Na+-K+)-ATPase activity and some o f the ouabaininsensitive (Mg2+)-ATPase, and the smaller, more heterogeneous part carrying the remaining part of the ouabain-insensitive (Mg2+)-ATPase. The strong increase in (Na+-K+)-ATPase activity induced by deoxycholate cannot be explained by an enzyme-activator effect as suggested by Uesugi et al. 1~, but is evidently due to a change in the microsomal m e m b r a n e composition and structure. On the basis o f this conclusion it is possible that (Na+-K+)ATPase activity and/or ouabain-insensitive (Mg2+)-ATPase in vivo are controlled by m e m b r a n e - b o u n d factors that are partially and irreversibly (.9) released in soluble form by deoxycholate. ACKNOWLEDGEMENTS This "nvestigation was supported by the Danish Medical Research Council. The a u t h o r thanks Miss Nina Hoffding for skilful technical help.

REFERENCES 1 ANDERSON,N. G., Analytical techniques for cell fractions. VIII. Analytical differential centrifugation in angle-head rotors, Analyt. Biochem., 23 (1968) 72-83. 2 AUBORN,J. J., EYRING,E. M., ANDCHOULES,G. L., Kinetics of sodium dodecyl sulfate solubilization of Mycoplasma laidlawii plasma membranes, Proc. nat. Acad. Sci. (Wash.), 68 (1971) 1996-1998. 3 CHAN,P. C., Reversible effect of sodium dodecyl sulfate on human erythrocyte membrane adenosine triphosphatase, Biochim. biophys. Acta (Amst.), 135 (1967) 53-60. 4 COTMAN,C., BROWN,D. H., HARRELL,B. W., ANDANDERSON,N. G., Analytical differential centrifugation: An analysis of the sedimentation properties of synaptosomes, mitochondria and lysosomes from rat brain homogenates, Arch. Biochem., 136 (1970) 436-447. 5 JORGENSEN,P. L., Regulation of the (Na + + K+)-activated ATP hydrolyzing enzyme system in rat kidney. I. The effect of adrenalectomy and the supply of sodium on the enzyme system, Biochim. biophys. Acta (Amst.), 151 (1968) 212-224. 6 JORGENSEN,P. L., AND SKOU, J. C., Purification and characterization of (Na + ÷ K+)-ATPase. I. The influence of detergents on the activity of (Na + + K+)-ATPase in preparations from the outer medulla of rabbit kidney, Biochim. biophys. Acta (Amst.), 233 (1971) 366-380. 7 JORGENSEN,P. L., AND SKOU, J. C., Purification and characterization of (Na + + K+)-ATPase. II. Preparation by zonal centrifugation of highly active (Na + + K+)-ATPase from the outer medulla of rabbit kidneys, Biochim. biophys. Acta (Amst.), 233 (1971) 381-394. 8 KAHLENBERG,A., DULAK,N. C., DIXON,J. F., GALSWORTHY,P. R., ANDHOKIN,L. E., Studies on the characterization of the sodium-potassium transport adenosinetriphosphatase. V. Partial purification of the lubrol-solubilized beef brain enzyme, Arch. Biochem., 131 (1969) 253-262.

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9 LowRY, O. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 10 ORNSTEIN, L., Disc electrophoresis. I. Background and theory, Ann. N.Y. Acad. Sci., 121 (1964) 321-350. I I STANTON,M. G.~ Determination of inorganic phosphate in the presence of ATP, Analyt. Biochem., 22 (1968) 27-35. 12 UESUGI, S., DULAK, N. C., DIXON, J. F., HEXUM, T. D., DAHL, J. L., PERDUE, J. F., AND HOKIN, L. E., Studies on the characterization of the sodium-potassium transport adenosinetriphosphatase. VI. Large scale purification and properies of a lubrol-solubilized bovine brain enzyme, d. biol. Chem., 246 (1971) 531 543.