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The molecular size of the calcium-transport ATPase of sarcotubular vesicles estimated from radiation inactivation
260
SHORT COMMUNICATIONS
BBA 73O5O
The molecular size of the calcium-transport ATPase of sarcotubular vesicles estimated from radiation inactivation S a r c o t u b u l a r vesicles from muscle b e h a v e as a simple a c t i v e - t r a n s p o r t s y s t e m ; aside from a basic ATPase, tile presence of Ca 2+ induces an a c t i v a t e d A T P a s e coupled with a massive i n w a r d flow of Ca 2+ (e.g. HASSELBACH AND MAK~NOSE~, EBASHi AXe) LIP.~IAXX2). I n this work, we have i n v e s t i g a t e d w h e t h e r a molecular-size range could be established, with t h e m e t h o d of r a d i a t i o n i n a c t i v a t i o n , of the s t r u c t u r a l entities responsible for (i) the basic ATPase, (ii) the Ca2+-activated ATPase, a n d (iii) the t r a n s p o r t function. I n t h e first stage of the work (with Mr. C. CHAMBERLAIN, i 9 6 4 i965) , we s u r v e y e d the p r o b l e m a n d its m e t h o d o l o g y , with p r e l i m i n a r y results on all three properties, in the second stage (with Mr. P. SHEetER, I966 I967) , means were found to i m p r o v e the m e t h o d s for the e x p e r i m e n t s u n d e r (i) a n d (ii), and these results will be r e p o r t e d here. W i t h respect to (iii), the indications are t h a t this function is much more susceptible to i n a c t i v a t i o n , indicative of a larger t a r g e t volume. This does not, however, indicate a larger size of the t r a n s p o r t u n i t ; the results could also be e x p l a i n e d b y assuming t h a t , once a vesicle has received a few hits, it becomes n o n - r e t e n t i v e a n d so does not allow the d e t e c t i o n of its Ca 2+ u p t a k e . W e believe this to be the most likely e x p l a n a t i o n , b u t the radiological m e t h o d o l o g y is n o t sufficiently precise to c o n d u c t a c c u r a t e studies on this point, so t h a t we have n o t further p u r s u e d question (iii). Vesicles were p r e p a r e d from r a b b i t muscle in the p u r e s t form o b t a i n a b l e (SERAYDARIAN AND MOMMAERTSa). F r o m the final suspension in sucrose, t h e y were washed twice b y u l t r a c e n t r i f u g a t i o n with o.I M KC1. Aliquots of o.6 ml were p i p e t t e d into glass a m p o u l e s a n d d r i e d in vac.uo from the frozen state. E a c h a m p o u l e was then e v a c u a t e d , flushed twice for eo lnin with pure N2, e v a c u a t e d , a n d sealed. This p r o c e d u r e leads to little change in the Ca2+-ATPase per mg of protein, b u t reduces the basic A T P a s e to a b o u t o. 4 times the original a c t i v i t y . The closed vials were placed, at room t e m p e r a t u r e , in the electron b e a l n of the 6-MeV accelerator (Varian Associates, Palo Alto, Calif. ; cf. [-IAIMSON AND KARZMARK~) of the D e p a r t m e n t of R a d i o l o g y , U C L A Medical Center. The dose r a t e was a b o u t 2.5' IO a r a d s . rain ~, checked occasionally with a liquid d o s i m e t e r (FRICKE AND MORSE a, SHALEK AND SINCLAIR~). Doses, from I '4 Mrads, were v a r i e d b y changing the exposure times. The t u b e s were then k e p t at @2 ° until a s s a y e d within ~4 h; a f t e r opening, the original a m o u n t s of w a t e r were a d d e d back. F o r the assays (@ ref. 3), there was o. 4 m g vesicle p r o t e i n in I ml of m e d i u m , o.I M KC1, 5 mM potassiuln oxalate, 5 mini MgC1.,, xo mM imidazole (pH 7.o), 5 mM A T P a n d o.55 mM e t h y l e n e glycol-bis-(/~-anfinoethylether)-N,A:'-tetraacetic acid. F o r the basic ATPase, phosp h a t e analyses a were p e r f o r m e d after o, 4, 8 a n d r2 rain of i n c u b a t i o n at 25 °. F o r the Ca2+-ATPase, CaC12 was a d d e d to a final c o n c e n t r a t i o n of 0.5 raM, a n d this was the trigger for the a s s a y ; the r e a c t i o n times were (o), I5, 3o, 45, a n d 6o sec, a n d the basic r a t e was s u b t r a c t e d from the observed. The results were e v a l u a t e d according to simple t a r g e t theory, as evolved in the writings of LEA a n d co-workersT, 8 a n d HUTCHINSON a n d POLLARD a n d their coworkers.9 13. A s s u m i n g t h a t an average of 75 eV are required per i n a c t i v a t i n g event, Biochb~z. t~iophys. Acta, r63 (I968) -'6(~-2()~
SHORT COMMUNICATIONS
267
we originated the formula M = (D0.aT)-l'o.72" IO 12, where D0.a7 is the dosage in rads at which the control activity is reduced to a fraction e -1, and M is the molecular weight in Daltons. This t h e o r y has an extensive empirical foundation in the s t u d y of enzymes and viruses (@ refs. 7-13) and, while this work was in progress, has been applied to the target sizes of ATPase in erythrocyte ghosts b y KEPNER AND M A C E Y 14, and of an ( N a + + K + ) - a c t i v a t e d ATPase from brain microsomes b y NAKAO et a / . 1 5 ; values of lO 6 and o.5" lO 6, respectively, were obtained for these cases, which have similarities to the point investigated here. The experiments to be reported were done on four separate preparations from different rabbits. E a c h of these was made up into 2o samples, so t h a t each control or irradiated data point was run in quadruplicate. The ATP-splitting b y the basic ATPase was a rectilinear function of time; likewise for the Ca2+-ATPase b u t for a non-zero intercept at zero time. This indicates an initial burst, which m a y be related to the rapid initial Ca 2+binding process studies b y EBASHI AND YAMANOUCH116. This initial burst was also inactivated with increased dose but the accuracy was not satisfactory. The linear reaction rates were plotted semilogarithmically as a function of dose, and regression lines drawn with the least-squares procedure; the results are stated with the o.95 confidence intervals according to the S t u d e n t ' s t-test, Table I gives a breakdown of the calculated target sizes for each of the 4 series separately as well as for the aggregate. TABLE
l
RESULTS
OF
4
SERIES
OF
EXPERIMENTS
ON
RADIATION
INACTIVATION
T a r g e t sizes i n D a l t o n s , C o n f i d e n c e i n t e r v a l s , ± o.95.
Expt.
Basic A TPase
Ca2+-activated A TPase
85 ooo
21o oooi9
i
21o ooo
2
18o o o o ~
~
80 ooo
16o ooo ± 5 ° ooo
o ooo
3 4
IOO ooo ~ 4 ° ooo 1 4 o o o o E 7o o o o
17o o o o ± 45 ooo 2ooooo ± 34ooo
Combined:
16o ooo ± 45 ooo
I 9 o ooo ± 34 ooo
The target sizes for the basic and Ca2+-activated ATPases, respectively, are not significantly different, and it cannot be decided whether these activities are seated in the same or different units; the preferred removal of basic ATPase b y washing m a y be due to the selective elimination of a separate activity, and the remaining activities m a y reflect two manifestations, without and with Ca ~+, of the same active unit. This unit, or these units, of a mass of the order of 175ooo Daltons, thus appear to be the entities responsible for ATP-splitting, and m a y also be the units of the Ca 2+ transport. It is of interest to compare this size with the result of HASSELBACH AND S E R A Y D A R I A N 17, according to which there is one critical sulfhydryl group, essential for transport, per IOOOOO g of protein. If our target volume includes lipid as well (of which there is about o,35 g per g protein in KCl-washed preparations (ref. 3); cf. MARTONOSI18), these values would come closer, t h o u g h clearly there are m a n y Biochim. Biophys. Acla, 163 (1968) 266 268
268
SHORT COMMUNICATIONS
r e a s o n s w h y t h e y d o n o t h a v e t o b e i d e n t i c a l , in v i e w of tile h e t e r o g e n e o u s p r o t e i n c o m p o s i t i o n of t h e v e s i c l e s (MOMMAERTS 19, MARTONOSI2°). W h i l e t h e a b s o l u t e a c c u r a c y of t h e t a r g e t - m e t h o d is l i m i t e d , a n d is o f t e n s t a t e d t o a l l o w a n a m b i g u i t y b y a f a c t o r of 2 (@ refs. 6 I2), i t s p o w e r lies i n t h e f a c t of a l l o w i n g t h e s i z e - d e t e r m i n a t i o n of a f u n c t i o n a l l y d e f i n e d u n i t r e g a r d l e s s of i t s s t a t e of a s s o c i a t i o n . I t is of i n t e r e s t t h a t a n a c t i v i t y b a s i c t o a t r a n s p o r t p r o c e s s is f o u n d t o r e s i d e w i t h i n a l i p o - p r o t e i n u n i t of s u c h s m a l l size as is i n d i c a t e d b y o u r w o r k . W e a r e i n d e b t e d t o P r o f e s s o r A M o s NORMAN, D e p a r t m e n t of R a d i o l o g y , for h i s c o u n s e l i n t h e i n i t i a l s t a g e of tile w o r k , a n d for a r r a n g i n g t h e c o o p e r a t i o n of t h e t e c h n i c a l s t a f f of t h e a c c e l e r a t o r a n d of his g r a d u a t e s t u d e n t s Mr. C. CHAMBERLAIN ( p r e s e n t a d d r e s s : D e p a r t m e n t of R a d i o l o g y , S t a t e U n i v e r s i t y of N e w Y o r k , U p s t a t e M e d i c a l C e n t e r , S y r a c u s e , N. Y.) a n d Mr. P. SPIEGLER. T h i s w o r k w a s s u p p o r t e d in p a r t b y p r o g r a m - p r o j e c t g r a n t No. H E - I 1 3 5 1 of t h e N a t i o n a l H e a r t I n s t i t u t e of H e a l t h , B e t h e s d a , Md.
The Los Angeles County Heart Association Cardiovascular Research Laboratory and the D e p a r t m e n t of PlLvsiology, U . C . L . A . School of Medicine, Los Angeles, Calif. 9oo24 ( U . S . A . ) ! 2 3 4 5 6 7 8 9 IO II I2 13
14 15 16 17 I8 19 2o
KLARA VEGH PETER SPIEGLER CRAIG CHAMBERLAIN W . F. H . M. MOMMAERTS
\V. [IASSELt~,ACH AND 31. MAK1NOS1,;, Biochem. Z., 333 (I961) 518. S. l{BaSnI AXD F. LIP.MANN, J. (?ell Biol., 14 (1962) 389. K. SERAYDARIAN AND \V. F. H. M. MOMMAERTS, ./. Cell Biol., 26 (I965) 64t. J. HAIMSON ANI) C. J. [(ARZMARK, Brit. J. Radiol., 36 (1963) 65o. H. FRlCKE ANI) S. 1. MORSE, Am. J. l?oenlgenol. Radium Therapy, 18 (1927) 420. R. J. SHAL~;K AND W. K. SINCLAIR, Syrup. on Radiation Biology and Cancer, r958, University of Texas Press, Austin, 1958, p. 149. D. E. LEA, Actions of Radialions on Living Cells, Cambridge, 1946 , p. 316. D. E. LEA, I'L M. SMITH, B. H()LMI~;S ANII I)v. ]~'IARKHAM,Parasitology, 36 (I944) 11o. F. HUTCHINSON, Science, I34 (1901) 533. F. HUTCHINSON, Syrup. on Cellular Radiation Biology, University of Texas 3I. D. Anderson Hospital and T~**nor Institute, 1964, Williams and \Vilkins, Baltimore, 1965, p. 86. [". HUTCHINSON AND E. ('. POLLARD, Mechanisms in Radiobiology, Vol. t, Academic Press, New York, 196~, p. 71 . [r. (-. POLLARD, l~ev. Alod. l°hysics, 31 (1959) 273. l{. C. POLLARD, 19\r. 1{. (;UILD, F. HUTCtIINSON AND ]{. ]3. SETLO\V, Progr. l~iophys. Biophys. CheIn., 5 (1955) 72. G. R. KEPNER AND I~.. I. MaCEY, Biochem. Biophys. Res. Commun., 23 (1966) 2o2. 3I. NAKAO, 1(. NAGANO, T. NAKAO, N. MIzuxo, Y. TASHIMA, M. FUJITA, H. MA~'D:\ AND H. MATSUDAIRA, ~liochem. Biophys. Res. Commun., 29 (1967) 588. S. F.BASHI AND 1. YAMANOUCHI, .[. Biochem. Tokyo, 55 (1964) 5o4 . \V. HASSELBACH AND K. SERAYDARIAN, Biochem. Z., 345 (I966) I59. A. MARTONOSI, Federation Proc., 23 (i964) 913. \V. F. 1t. M. ~IOMMAERTS, Proe. Natl. Acad. Nci. U.S., 58 (1967) 2476. A. MARTONOSI, j . Biol. Chem., 243 (I968) 71 .
R e c e i v e d J u l y 8 t h , 1968
Biockim. Biophys. Acta, 163 (1968) 266-268
SHORT COMMUNICATIONS
BBA 73O5O
The molecular size of the calcium-transport ATPase of sarcotubular vesicles estimated from radiation inactivation S a r c o t u b u l a r vesicles from muscle b e h a v e as a simple a c t i v e - t r a n s p o r t s y s t e m ; aside from a basic ATPase, tile presence of Ca 2+ induces an a c t i v a t e d A T P a s e coupled with a massive i n w a r d flow of Ca 2+ (e.g. HASSELBACH AND MAK~NOSE~, EBASHi AXe) LIP.~IAXX2). I n this work, we have i n v e s t i g a t e d w h e t h e r a molecular-size range could be established, with t h e m e t h o d of r a d i a t i o n i n a c t i v a t i o n , of the s t r u c t u r a l entities responsible for (i) the basic ATPase, (ii) the Ca2+-activated ATPase, a n d (iii) the t r a n s p o r t function. I n t h e first stage of the work (with Mr. C. CHAMBERLAIN, i 9 6 4 i965) , we s u r v e y e d the p r o b l e m a n d its m e t h o d o l o g y , with p r e l i m i n a r y results on all three properties, in the second stage (with Mr. P. SHEetER, I966 I967) , means were found to i m p r o v e the m e t h o d s for the e x p e r i m e n t s u n d e r (i) a n d (ii), and these results will be r e p o r t e d here. W i t h respect to (iii), the indications are t h a t this function is much more susceptible to i n a c t i v a t i o n , indicative of a larger t a r g e t volume. This does not, however, indicate a larger size of the t r a n s p o r t u n i t ; the results could also be e x p l a i n e d b y assuming t h a t , once a vesicle has received a few hits, it becomes n o n - r e t e n t i v e a n d so does not allow the d e t e c t i o n of its Ca 2+ u p t a k e . W e believe this to be the most likely e x p l a n a t i o n , b u t the radiological m e t h o d o l o g y is n o t sufficiently precise to c o n d u c t a c c u r a t e studies on this point, so t h a t we have n o t further p u r s u e d question (iii). Vesicles were p r e p a r e d from r a b b i t muscle in the p u r e s t form o b t a i n a b l e (SERAYDARIAN AND MOMMAERTSa). F r o m the final suspension in sucrose, t h e y were washed twice b y u l t r a c e n t r i f u g a t i o n with o.I M KC1. Aliquots of o.6 ml were p i p e t t e d into glass a m p o u l e s a n d d r i e d in vac.uo from the frozen state. E a c h a m p o u l e was then e v a c u a t e d , flushed twice for eo lnin with pure N2, e v a c u a t e d , a n d sealed. This p r o c e d u r e leads to little change in the Ca2+-ATPase per mg of protein, b u t reduces the basic A T P a s e to a b o u t o. 4 times the original a c t i v i t y . The closed vials were placed, at room t e m p e r a t u r e , in the electron b e a l n of the 6-MeV accelerator (Varian Associates, Palo Alto, Calif. ; cf. [-IAIMSON AND KARZMARK~) of the D e p a r t m e n t of R a d i o l o g y , U C L A Medical Center. The dose r a t e was a b o u t 2.5' IO a r a d s . rain ~, checked occasionally with a liquid d o s i m e t e r (FRICKE AND MORSE a, SHALEK AND SINCLAIR~). Doses, from I '4 Mrads, were v a r i e d b y changing the exposure times. The t u b e s were then k e p t at @2 ° until a s s a y e d within ~4 h; a f t e r opening, the original a m o u n t s of w a t e r were a d d e d back. F o r the assays (@ ref. 3), there was o. 4 m g vesicle p r o t e i n in I ml of m e d i u m , o.I M KC1, 5 mM potassiuln oxalate, 5 mini MgC1.,, xo mM imidazole (pH 7.o), 5 mM A T P a n d o.55 mM e t h y l e n e glycol-bis-(/~-anfinoethylether)-N,A:'-tetraacetic acid. F o r the basic ATPase, phosp h a t e analyses a were p e r f o r m e d after o, 4, 8 a n d r2 rain of i n c u b a t i o n at 25 °. F o r the Ca2+-ATPase, CaC12 was a d d e d to a final c o n c e n t r a t i o n of 0.5 raM, a n d this was the trigger for the a s s a y ; the r e a c t i o n times were (o), I5, 3o, 45, a n d 6o sec, a n d the basic r a t e was s u b t r a c t e d from the observed. The results were e v a l u a t e d according to simple t a r g e t theory, as evolved in the writings of LEA a n d co-workersT, 8 a n d HUTCHINSON a n d POLLARD a n d their coworkers.9 13. A s s u m i n g t h a t an average of 75 eV are required per i n a c t i v a t i n g event, Biochb~z. t~iophys. Acta, r63 (I968) -'6(~-2()~
SHORT COMMUNICATIONS
267
we originated the formula M = (D0.aT)-l'o.72" IO 12, where D0.a7 is the dosage in rads at which the control activity is reduced to a fraction e -1, and M is the molecular weight in Daltons. This t h e o r y has an extensive empirical foundation in the s t u d y of enzymes and viruses (@ refs. 7-13) and, while this work was in progress, has been applied to the target sizes of ATPase in erythrocyte ghosts b y KEPNER AND M A C E Y 14, and of an ( N a + + K + ) - a c t i v a t e d ATPase from brain microsomes b y NAKAO et a / . 1 5 ; values of lO 6 and o.5" lO 6, respectively, were obtained for these cases, which have similarities to the point investigated here. The experiments to be reported were done on four separate preparations from different rabbits. E a c h of these was made up into 2o samples, so t h a t each control or irradiated data point was run in quadruplicate. The ATP-splitting b y the basic ATPase was a rectilinear function of time; likewise for the Ca2+-ATPase b u t for a non-zero intercept at zero time. This indicates an initial burst, which m a y be related to the rapid initial Ca 2+binding process studies b y EBASHI AND YAMANOUCH116. This initial burst was also inactivated with increased dose but the accuracy was not satisfactory. The linear reaction rates were plotted semilogarithmically as a function of dose, and regression lines drawn with the least-squares procedure; the results are stated with the o.95 confidence intervals according to the S t u d e n t ' s t-test, Table I gives a breakdown of the calculated target sizes for each of the 4 series separately as well as for the aggregate. TABLE
l
RESULTS
OF
4
SERIES
OF
EXPERIMENTS
ON
RADIATION
INACTIVATION
T a r g e t sizes i n D a l t o n s , C o n f i d e n c e i n t e r v a l s , ± o.95.
Expt.
Basic A TPase
Ca2+-activated A TPase
85 ooo
21o oooi9
i
21o ooo
2
18o o o o ~
~
80 ooo
16o ooo ± 5 ° ooo
o ooo
3 4
IOO ooo ~ 4 ° ooo 1 4 o o o o E 7o o o o
17o o o o ± 45 ooo 2ooooo ± 34ooo
Combined:
16o ooo ± 45 ooo
I 9 o ooo ± 34 ooo
The target sizes for the basic and Ca2+-activated ATPases, respectively, are not significantly different, and it cannot be decided whether these activities are seated in the same or different units; the preferred removal of basic ATPase b y washing m a y be due to the selective elimination of a separate activity, and the remaining activities m a y reflect two manifestations, without and with Ca ~+, of the same active unit. This unit, or these units, of a mass of the order of 175ooo Daltons, thus appear to be the entities responsible for ATP-splitting, and m a y also be the units of the Ca 2+ transport. It is of interest to compare this size with the result of HASSELBACH AND S E R A Y D A R I A N 17, according to which there is one critical sulfhydryl group, essential for transport, per IOOOOO g of protein. If our target volume includes lipid as well (of which there is about o,35 g per g protein in KCl-washed preparations (ref. 3); cf. MARTONOSI18), these values would come closer, t h o u g h clearly there are m a n y Biochim. Biophys. Acla, 163 (1968) 266 268
268
SHORT COMMUNICATIONS
r e a s o n s w h y t h e y d o n o t h a v e t o b e i d e n t i c a l , in v i e w of tile h e t e r o g e n e o u s p r o t e i n c o m p o s i t i o n of t h e v e s i c l e s (MOMMAERTS 19, MARTONOSI2°). W h i l e t h e a b s o l u t e a c c u r a c y of t h e t a r g e t - m e t h o d is l i m i t e d , a n d is o f t e n s t a t e d t o a l l o w a n a m b i g u i t y b y a f a c t o r of 2 (@ refs. 6 I2), i t s p o w e r lies i n t h e f a c t of a l l o w i n g t h e s i z e - d e t e r m i n a t i o n of a f u n c t i o n a l l y d e f i n e d u n i t r e g a r d l e s s of i t s s t a t e of a s s o c i a t i o n . I t is of i n t e r e s t t h a t a n a c t i v i t y b a s i c t o a t r a n s p o r t p r o c e s s is f o u n d t o r e s i d e w i t h i n a l i p o - p r o t e i n u n i t of s u c h s m a l l size as is i n d i c a t e d b y o u r w o r k . W e a r e i n d e b t e d t o P r o f e s s o r A M o s NORMAN, D e p a r t m e n t of R a d i o l o g y , for h i s c o u n s e l i n t h e i n i t i a l s t a g e of tile w o r k , a n d for a r r a n g i n g t h e c o o p e r a t i o n of t h e t e c h n i c a l s t a f f of t h e a c c e l e r a t o r a n d of his g r a d u a t e s t u d e n t s Mr. C. CHAMBERLAIN ( p r e s e n t a d d r e s s : D e p a r t m e n t of R a d i o l o g y , S t a t e U n i v e r s i t y of N e w Y o r k , U p s t a t e M e d i c a l C e n t e r , S y r a c u s e , N. Y.) a n d Mr. P. SPIEGLER. T h i s w o r k w a s s u p p o r t e d in p a r t b y p r o g r a m - p r o j e c t g r a n t No. H E - I 1 3 5 1 of t h e N a t i o n a l H e a r t I n s t i t u t e of H e a l t h , B e t h e s d a , Md.
The Los Angeles County Heart Association Cardiovascular Research Laboratory and the D e p a r t m e n t of PlLvsiology, U . C . L . A . School of Medicine, Los Angeles, Calif. 9oo24 ( U . S . A . ) ! 2 3 4 5 6 7 8 9 IO II I2 13
14 15 16 17 I8 19 2o
KLARA VEGH PETER SPIEGLER CRAIG CHAMBERLAIN W . F. H . M. MOMMAERTS
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R e c e i v e d J u l y 8 t h , 1968
Biockim. Biophys. Acta, 163 (1968) 266-268