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Physiol. Plant. 35: 77-79.1975 ELECTROPHORETIC MOBILITY OF MEMBRANE PARTICLES 77 Effect of Na% K% Mg^^ and ATP on the Relative Electrophoretic Mobility of Sugar Beet Membrane Particles with NaK ATPase Activity By JAN KARLSSON Botanical Institute, University of Stockholm, Lilla Frescati, S-104 05 Stockholm, Sweden (Received 4 June, 1975) Abstract Electrophoretic measurements on membrane coated particles were performed with a Zytopherometer. Tris-HCl buffer 0.2 M pH 7.0 at 37°C with addition of different combinations of Na"*', K+, Mg^"^ and ATP was used as test medium. The membranes were of two types, an untreated preparation with low NaK ATPase activity and a deoxycholate treated preparation with high NaK ATPase activity. There was no marked difference in reaction between the two types of membranes. To both types of membranes Mg^"*" gave a strong positive and ATP a shght negative addition to the membrane charge. In the presence of ATP Na+ gave a higher charge contribution than did K"*" or a combination of Na"^ and K+. This implies that K+ gives a higher affinity for ATP than Na+ does and/or that ATP mediates a higher affinity for Na+ than for K+. Introduction In an earlier paper (Karlsson et al. 1971) we reported relative electrophoretic mobilites for sugar beet membrane fragments with different lipid patterns and different NaK ATPase activities. The NaK ATPase activity is influenced by the cations: Na+, K"^ and Mg^+ and also by its substrate ATP (Skou 1957). Activation of the NaK ATPase may in part be due to changes in membrane charges influenced by the presence of these ions, so that the changes in membrane charges would be particularly pronounced for membranes rich in NaK ATPase activity. The present work was initiated to test this hypothesis, even though it was realized that the presence of other membrane constituents than the ATPases, might make the result difficult to evaluate. Some of the data in this paper were also presented at the 8th FEBS congress (Karlsson et al. 1972). Material and Methods Membrane preparations were made as earlier described (Karlssone/fl/. 1971, KyWnetal. 1972). Untreated prepara- 6 tion (A) with low NaK ATPase activity and preparation (B) treated 1 h with 0.1 % deoxycholate and containing higher NaK ATPase activity, were used. The electrophoretic mobility was measured for silica par- ticles, which were coated with membrane fragments (or un- coated for controls). Suspensions were made by addingO. 15 g washed silica powder to 100 ml 0.2 M tris-HCl-buffer (pH 7.0 at 37°C), which contained cations as indicated in "Results". This high concentration of tris was needed to ensure good conductivity. For experiments, 2 ml of a mem- brane preparation (or for controls 2 ml sucrose-histidine- HCI-buff"er) were added to 20 ml of the silica suspension. The velocities of the particles were determined at 37 C in a cell electrophoresis apparatus, a Zytopherometer (Fuhr- mann and Ruhenstroth-Bauer 1965, Tribukait 1968). The velocity of a particle was first measured in one direction, after which the direction of the current was reversed and the same particle followed in the opposite direction. For each series 16 particles were followed this way. Controls were measured as described but with uncoated silica. Two controls were run for each media, one as the first series and one as the last. Silica particles have a negative charge. The direction of the coated particles was the same as that of the silica particles, which means that also coated particles are negative. Calculations and statistics: Only relative electrophoretic mobilities are given, as there was a drift in the Zytophero- meter. The relative electrophoretic mobilities for the mem- brane coated particles (R) were calculated as percentage of the mean electrophoretic mobility for the two uncoated control series for each medium according to the following equations: R±S = MxlOO MxlOO

Effect of Na+, K+, Mg2+ and ATP on the Relative Electrophoretic Mobility of Sugar Beet Membrane Particles with NaK ATPase Activity

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Page 1: Effect of Na+, K+, Mg2+ and ATP on the Relative Electrophoretic Mobility of Sugar Beet Membrane Particles with NaK ATPase Activity

Physiol. Plant. 35: 77-79.1975 ELECTROPHORETIC MOBILITY OF MEMBRANE PARTICLES 77

Effect of Na% K% Mg^^ and ATP on the Relative Electrophoretic Mobilityof Sugar Beet Membrane Particles with NaK ATPase Activity

By

JAN KARLSSON

Botanical Institute, University of Stockholm, Lilla Frescati,S-104 05 Stockholm, Sweden

(Received 4 June, 1975)

Abstract

Electrophoretic measurements on membrane coated particleswere performed with a Zytopherometer. Tris-HCl buffer 0.2 MpH 7.0 at 37°C with addition of different combinations of Na"*',K+, Mg " and ATP was used as test medium. The membraneswere of two types, an untreated preparation with low NaKATPase activity and a deoxycholate treated preparation withhigh NaK ATPase activity. There was no marked difference inreaction between the two types of membranes. To both types ofmembranes Mg "*" gave a strong positive and ATP a shght negativeaddition to the membrane charge. In the presence of ATP Na+gave a higher charge contribution than did K"*" or a combinationof Na" and K+. This implies that K+ gives a higher affinity forATP than Na+ does and/or that ATP mediates a higher affinityfor Na+ than for K+.

Introduction

In an earlier paper (Karlsson et al. 1971) we reportedrelative electrophoretic mobilites for sugar beet membranefragments with different lipid patterns and different NaKATPase activities. The NaK ATPase activity is influencedby the cations: Na+, K" and Mg + and also by its substrateATP (Skou 1957). Activation of the NaK ATPase may inpart be due to changes in membrane charges influenced bythe presence of these ions, so that the changes in membranecharges would be particularly pronounced for membranesrich in NaK ATPase activity. The present work was initiatedto test this hypothesis, even though it was realized that thepresence of other membrane constituents than the ATPases,might make the result difficult to evaluate.

Some of the data in this paper were also presented at the8th FEBS congress (Karlsson et al. 1972).

Material and Methods

Membrane preparations were made as earlier described(Karlssone/fl/. 1971, KyWnetal. 1972). Untreated prepara-

6

tion (A) with low NaK ATPase activity and preparation(B) treated 1 h with 0.1 % deoxycholate and containinghigher NaK ATPase activity, were used.

The electrophoretic mobility was measured for silica par-ticles, which were coated with membrane fragments (or un-coated for controls). Suspensions were made by addingO. 15 gwashed silica powder to 100 ml 0.2 M tris-HCl-buffer(pH 7.0 at 37°C), which contained cations as indicated in"Results". This high concentration of tris was needed toensure good conductivity. For experiments, 2 ml of a mem-brane preparation (or for controls 2 ml sucrose-histidine-HCI-buff"er) were added to 20 ml of the silica suspension.The velocities of the particles were determined at 37 C ina cell electrophoresis apparatus, a Zytopherometer (Fuhr-mann and Ruhenstroth-Bauer 1965, Tribukait 1968). Thevelocity of a particle was first measured in one direction,after which the direction of the current was reversed and thesame particle followed in the opposite direction. For eachseries 16 particles were followed this way. Controls weremeasured as described but with uncoated silica. Two controlswere run for each media, one as the first series and one asthe last.

Silica particles have a negative charge. The direction ofthe coated particles was the same as that of the silica particles,which means that also coated particles are negative.

Calculations and statistics: Only relative electrophoreticmobilities are given, as there was a drift in the Zytophero-meter. The relative electrophoretic mobilities for the mem-brane coated particles (R) were calculated as percentage ofthe mean electrophoretic mobility for the two uncoatedcontrol series for each medium according to the followingequations:

R±S =MxlOO MxlOO

Page 2: Effect of Na+, K+, Mg2+ and ATP on the Relative Electrophoretic Mobility of Sugar Beet Membrane Particles with NaK ATPase Activity

78 JAN KARLSSON Physiol. Plant. 35. 1975

Q = (2)

. + 54):2 (3)where:R = relative electrophoretic mobility for membrane coated

silica particles, in percentage of uncoated controls runin similar media.

Sx = standard deviations as indicated by the indices.M = mean electrophoretic mobility for membrane coated

particlesQ = mean electrophoretic mobility for uncoated particles.

In order to compare the relative electrophoretic mobilitiesobtained in different media, the following equation was used

Table 2. Significant (P < O.OOI) differences in relative electrophore-tic mobilities as influenced by ions.

t =

where n = particles observed, ni = «2 = 16.

Results and Discussion

There was no marked difference in reaction between thetwo types of membranes, on the contrary both types ofmembranes usually were influenced by ions in a similar way(Table 1). But the measurements confirmed that the deoxy-cholate treated membranes have a higher negative surfacecharge (Karlsson etal. 1971), new data not shown. From thedata in Table 1, the differences in relative electrophoreticmobility obtained indifferent media, were calculated. As forthe influence of specific iotis or ionic combinations, otilysuch differences are given that are common for both pre-parations and have a probability of less than 0.1 % (Table2). This precautions has been taken in order to avoid report-ing differences of minor significance.

Mg + gave a strong positive addition to the membranecharge, and ATP gave a slight negative charge contribution(Table 2). The Mg + -effect is dominant when both ions arepresent together. It has been shown that MgATP is thesubstrate for the NaK ATPase (Hexum etal. 1970, Lindberget al. 1974), but it has also been shown that the enzyme has

Table 1. Relative electrophoretic mobilities ± SE of untreated (A)and deoxycholate-treated (B) membrane fragments in differentionic media.

Na 25 mM NoIons Na 50 mM K 50 mM K 25 mM additions

ATPl mM A 71.1 ±2.6B 75.8 ±2.8

ATP 1 mM A 70.5 ± 2.0Mg3mM B 67.9 ±2.8Mg3mM A 62.5 ±1.9

B 64.5 ±2.1No A 77.6 ± 2.1

additions B 75.4 ± 3.2

87.6 ±2.7 94.4 ±3.0 79.8 ± 2.995.5 ±3.0 92.6 ±3.0 90.0 ±2.761.4 ±L8 72.8 ±2.2 61.7 ±2.262.3 ± 2.0 69.3 ± 2.45 68.5 ± 2.361.6 ±2.7 65.5 ±2.1 63.2 ±1.471.6 ±2.5 73.1 ±2.0 65.0 ± 2.073.6 ±2.0 76.2 ±2.0 71.4 ±2.581.9 ±2.2 91.9 ±3.2 76.8 ±2.7

Preparation

ABABABABABABABABAB

Differencebetween

I

ATPATPATPATPATPATPATPATPATPATPATPATP—

KK

Na + KNa + K

IP

ATP + MgATP + MgATP + MgATP + MgATP + MgATP + Mg

MgMgMgMgMgMg

ATP + MgATP + Mg

NaNaNaNa

Other ionsin the medium

KK

Na + KNa + K

——KK

Na + KNa + K

——KK

ATPATPATPATP

Differencein rel.

el. phor.mobilities

26.232.321.623.318.221.426.023.928.919.516.624.912.118.716.619.723.316.8

^ II is the ion or combination that gives the least electro-phoretic mobility, i.e. makes the membranes more positive thanI does.

a high affinity for Mg " and that the binding of Mg^+ tothe enzyme is a prerequisite for its activity (Fahn et al. 1966).The binding of the positive Mg^* ion to the membrane sur-face could facilitate the binding of ATP to the enzyme andin that way be one part of the Mg^^-dependence of thesugar beet NaK ATPase.

In the presence of ATP, Na"*" gave a higher positive con-tribution to the membrane charge (i.e. decreased the velocityof the particles) than did K" or combination of Na+ and K+.This indicates that Na" and K" influence the affinity of themembrane for ATP in different ways and/or that the influenceof ATP gives different affinities for Na+ and K+. K+ givesa higher affinity for ATP than Na+ does and/or ATPmediates a higher affinity for Na" than for K^.

It is likely that the effect of K'* is dominant, as there is ahighly significant difference between (Na" + ATP) and(Na" + K" + ATP) media, but no such difference betweenK+ + ATP) and (Na+ + K+ + ATP) media. That the affinityfor ATP of animal NaK ATPase is influenced by Na+ andK" has been found by flow dialysis work (Hegyvary and Post1971), in which case Na* enhanced the affinity for ATP andK""" decreased the affinity in the absence of

Acknowledgements are due to Professor A. Kylin for valuablediscussions and for reading the manuscript; Professor B.Tribukait for supplying me with his apparatus and his technicalstaff; the Laboratory engineers Agneta Osterdahl and GunBjorklund for the skilful measurements; and the Swedish NaturalScience Research Foundation and C. F. Lundstrom Foundationfor financial support.

Page 3: Effect of Na+, K+, Mg2+ and ATP on the Relative Electrophoretic Mobility of Sugar Beet Membrane Particles with NaK ATPase Activity

Physiol. Plant. 35. 1975 ELECTROPHORETIC MOBILITY OF MEMBRANE PARTICLES 79

References

Fahn, S., Koval, G. J. & Albers, R. W. 1966. Sodium-potassium-activated adenosine triphosphatase of Electrophorus electricorgan. I. An associated sodium-activated transphosphoryla-tion. — J. Biol. Chem. 241: 1882-1889.

Fuhrmann, G. F. & Ruhenstroth-Bauer, G. 1965. Cell electro-phoresis employing a rectangular measuring cuvette. — InCell Electrophoresis (E. J. Embrose, ed.), pp. 22-65. Churchill,London.

Hegyvary, C. SL Post, R. L. 1971. Binding of adenosine triphos-phate to sodium and potassium ion-stimulated adenosine tri-phosphatase. — J. Biol. Chem. 246: 5234-5240.

Hexum, T., Samson, F. E. & Hines, R. H. 1970. Kinetic studies ofmembrane(Na+ + K+ + Mg^+)-ATPase.—Biochim.Biophys.Acta 212: 322-331.

Karlsson, J., Tribukait, B. & Kylin, A. 1971. Electrical mobilitiesof fragments of sugar beet root membranes with properties as(Na+ + K+)-activated adenosine triphosphatases. — Proc. 1st

Europ. Biophys. Congr. Ill, Verlag der Wiener MedizinischenAkademie, Wien, pp. 75-79.

. 1972. Electrical mobilities of membrane fragments with(Na + K)-ATPase activity in relation to ATP and cations ofthe medium. —Abstr. Commun. Meet. Fed. Eur. Biochem.Soc. 8, North-Holland, Amsterdam, abstr. 33.

Kylin, A., Kuiper, P. J. C. & Hansson, G. 1972. Lipids from sugarbeet in relation to the preparation and properties of (sodium+ potassium)-activated adenosine triphosphatases. — Physiol.Plant. 26: 271-278.

Lindberg, S., Hansson, G. & Kylin, A. 1974. Kinetic studies ofa (Na+ + K+ + Mg^^) ATPase in sugar beet roots. I. Mg de-pendence. ~ Ibid. 32:103-107.

Skou, J. C. 1957. The influence of some cations on an adenosinetri-phosphatase from peripheral nerves. — Biochim. Biophys.Acta 23: 394^01.

Tribukait, B. 1968. Rhythmical changes of the electrophoreticmobility of erythrocytes after irridation with increasing X-raydoses. — Nature 219: 382-383.

Page 4: Effect of Na+, K+, Mg2+ and ATP on the Relative Electrophoretic Mobility of Sugar Beet Membrane Particles with NaK ATPase Activity