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Adenosine triphosphate-dependent transport of rubidum into erythrocyte ghost
BIOCHIMICA ET BIOPHYSICA ACTA
Preliminary
135
Notes
PN "r256
Adenosine triphosphate
SeRb B Y GHOST CELLS
T h e i n c u b a t i o n m i x t u r e contained 4 ° pmoles of t r i e t h a n o l a m i n e - H C l (pH 7.6), i p m o l e of ATP, I p m o l e of MgC12, 0.3 p m o l e of SeRb (25ooo counts/rain), ghost cells (prepared from 0.3 ml of erythrocytes) a n d other c o m p o n e n t s as indicated below in a final volume of 0.5 ml. Incubation was carried o u t a t 37 ° for I h. L i b e r a t i o n of Pz f r o m A T P was determined b y a slight modification of t h e m e t h o d of FISKE A~D SUBEAROW v. The binding of SeRb b y t h e ghosts was assayed as described in Fig. x. Additions (+) or omissions (--)
Nil + Na + (o. x M) q- K + (o.I M) --ATP + Ouabain (io -8 M) (5" 10--8 M )
- - Mg 2+ + N a + (o.x M), K + (o.i M)
.4 TPase #mole Pilh
0.95 o.96 0.99 -o.95
,aRb binding (founts/rain)
296o 35oo I76o 650 ioo
I.IO
~4 0
0.06 1.2 3
950 I25o
A study of the increase of ~Rb in the ghost cells in relation to time is i!lustrated in Fig. x. The rate of incorporation was increased in the presence of ATP. A saturation level was reached in about z or 3 h, when the ATP added was hydrolyzed almost completely. Contrary to this finding the sodium binding by the microsomes of brain tissue 4 or the calcium uptake by the skeletal muscle endoplasmic reticulum 5 reached Biochim. Biophys. ,4cta, 75 (x963) I 3 5 - t 3 6
X~6
PRELIMINARY NOTES
saturation in a very short time, even in the presence of sufficient amount of ATP. ATP-depende~'~ rubidium uptake by ghost cells was affected by various reagents (Table I). It was depressed in the presence of K + but not by Na +. This is in accordance with a usual conception that Rb + and K + are similar electrochemically and have si~ affinities for the c ~ e r of K + transport. It is well ~ o w n that an active cationic transport through the membrane of the cell is inhibited by ouabain with little effect on passive transpo~, In erythrocytes, ouabain slows the decline of ATP level, with tittle effect on the ATP-generative processes s. This is due possibly to the inhibitory action of ouabain on the membrane ATPase. Both rubidi_um uptak~ and Na+-, K +dependent ATPase of ghost cells were inhibited markedly by the addition of ouabain.
0
COUIT
fJ ~,cc~
g 8 ~o¢
/
(~0 - - - - ~
1 Time ( h )
2
3
Fig. x. The binding o f ~ R b by ghost cells of erythrocytes in relation to time. Ghosts were suspended in 0.08 M triethanolamine-HCl buffer (pH 7.6) containing SeRb (25ooo counts/min) and i pmole of MgCli in the presence ( Q - - I ) , or absence ( O - - O ) , of z/,mole of ATP in a total volume of o. 5 ml. Incubation was carried out at 37 °. Binding of SeRb was assayed, at appropriate intervals, by measuring t|~e decrease in radioactivity of the supernatant after removal of the ghosts by centrifugation.
The characteristics of the ATP-dependent binding of Rb + by ghost cells axe similar to the known properties of the active transport of K + and Mg2+-activated Na +-, K+-dependent ATPase, which is considered to be an intimate component of the active cation transportation pump.
Department of Physiological Chemistry and Nut.+ition, Faculty of Medicine, The University of Tokyo, Tokyo (japan)
TAKASHI HASHIMOTO HARUHISA YOSHIKAWA
1 M. TATIBANA, A. SHIMAMUNE, M. NAKAO, Y. ISHIt, T. HASHIMOTO AND H. YOSHIKAWA, Folia H~matol., 79 (x962) 470. = R. L. POST, C. R. MERRIT, C. R. KINSOLVINGAND C. D. ALBRIGHT,f . Biol. Chem., 235 (196o) I796. a D. C. TOSTESON, R. H. MOULTONAND M. BLAUNSTEIN,Federation Proc., I9 (I96o) I28. * J. J~RNEFELT, Bioohem. Biophys. Res. Commun., 6 (I96I) 28~. s W. HASSELnACtlA~V M. MAKtNOSS, Biochem. Biophys. Res. Commun., 7 (I962) I32. e ]. F. HOFFMAN,J. Gen. Physiol., 45 (x962) 837. * C. H. FmKE A~D Y. SuneARow, J. Biol. Chem., 66 (i925) 375. s E. T. DUNHAM, Federation Proc., x6 (x957) 33-
Received February 7th, x963 B;ochim. Biophys. ,qcta, 75 (I963) x35-136
Preliminary
135
Notes
PN "r256
Adenosine triphosphate
SeRb B Y GHOST CELLS
T h e i n c u b a t i o n m i x t u r e contained 4 ° pmoles of t r i e t h a n o l a m i n e - H C l (pH 7.6), i p m o l e of ATP, I p m o l e of MgC12, 0.3 p m o l e of SeRb (25ooo counts/rain), ghost cells (prepared from 0.3 ml of erythrocytes) a n d other c o m p o n e n t s as indicated below in a final volume of 0.5 ml. Incubation was carried o u t a t 37 ° for I h. L i b e r a t i o n of Pz f r o m A T P was determined b y a slight modification of t h e m e t h o d of FISKE A~D SUBEAROW v. The binding of SeRb b y t h e ghosts was assayed as described in Fig. x. Additions (+) or omissions (--)
Nil + Na + (o. x M) q- K + (o.I M) --ATP + Ouabain (io -8 M) (5" 10--8 M )
- - Mg 2+ + N a + (o.x M), K + (o.i M)
.4 TPase #mole Pilh
0.95 o.96 0.99 -o.95
,aRb binding (founts/rain)
296o 35oo I76o 650 ioo
I.IO
~4 0
0.06 1.2 3
950 I25o
A study of the increase of ~Rb in the ghost cells in relation to time is i!lustrated in Fig. x. The rate of incorporation was increased in the presence of ATP. A saturation level was reached in about z or 3 h, when the ATP added was hydrolyzed almost completely. Contrary to this finding the sodium binding by the microsomes of brain tissue 4 or the calcium uptake by the skeletal muscle endoplasmic reticulum 5 reached Biochim. Biophys. ,4cta, 75 (x963) I 3 5 - t 3 6
X~6
PRELIMINARY NOTES
saturation in a very short time, even in the presence of sufficient amount of ATP. ATP-depende~'~ rubidium uptake by ghost cells was affected by various reagents (Table I). It was depressed in the presence of K + but not by Na +. This is in accordance with a usual conception that Rb + and K + are similar electrochemically and have si~ affinities for the c ~ e r of K + transport. It is well ~ o w n that an active cationic transport through the membrane of the cell is inhibited by ouabain with little effect on passive transpo~, In erythrocytes, ouabain slows the decline of ATP level, with tittle effect on the ATP-generative processes s. This is due possibly to the inhibitory action of ouabain on the membrane ATPase. Both rubidi_um uptak~ and Na+-, K +dependent ATPase of ghost cells were inhibited markedly by the addition of ouabain.
0
COUIT
fJ ~,cc~
g 8 ~o¢
/
(~0 - - - - ~
1 Time ( h )
2
3
Fig. x. The binding o f ~ R b by ghost cells of erythrocytes in relation to time. Ghosts were suspended in 0.08 M triethanolamine-HCl buffer (pH 7.6) containing SeRb (25ooo counts/min) and i pmole of MgCli in the presence ( Q - - I ) , or absence ( O - - O ) , of z/,mole of ATP in a total volume of o. 5 ml. Incubation was carried out at 37 °. Binding of SeRb was assayed, at appropriate intervals, by measuring t|~e decrease in radioactivity of the supernatant after removal of the ghosts by centrifugation.
The characteristics of the ATP-dependent binding of Rb + by ghost cells axe similar to the known properties of the active transport of K + and Mg2+-activated Na +-, K+-dependent ATPase, which is considered to be an intimate component of the active cation transportation pump.
Department of Physiological Chemistry and Nut.+ition, Faculty of Medicine, The University of Tokyo, Tokyo (japan)
TAKASHI HASHIMOTO HARUHISA YOSHIKAWA
1 M. TATIBANA, A. SHIMAMUNE, M. NAKAO, Y. ISHIt, T. HASHIMOTO AND H. YOSHIKAWA, Folia H~matol., 79 (x962) 470. = R. L. POST, C. R. MERRIT, C. R. KINSOLVINGAND C. D. ALBRIGHT,f . Biol. Chem., 235 (196o) I796. a D. C. TOSTESON, R. H. MOULTONAND M. BLAUNSTEIN,Federation Proc., I9 (I96o) I28. * J. J~RNEFELT, Bioohem. Biophys. Res. Commun., 6 (I96I) 28~. s W. HASSELnACtlA~V M. MAKtNOSS, Biochem. Biophys. Res. Commun., 7 (I962) I32. e ]. F. HOFFMAN,J. Gen. Physiol., 45 (x962) 837. * C. H. FmKE A~D Y. SuneARow, J. Biol. Chem., 66 (i925) 375. s E. T. DUNHAM, Federation Proc., x6 (x957) 33-
Received February 7th, x963 B;ochim. Biophys. ,qcta, 75 (I963) x35-136