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The 2,4-dinitrophenol-responsive adenosinetriphosphatase of rat-liver mitochondria
PRELIMINARY
395
NOTES
The 2,4-dinitrophenol-responsive adenosinetriphosphatase of rat-liver mitochondria Various i n v e s t i g a t o r s 1-a h a v e p o s t u l a t e d t h a t D N P a c t i v a t i o n of m i t o c h o n d r i a l A T P a s e is a m a n i f e s t a t i o n of the s y s t e m of o x i d a t i v e p h o s p h o r y l a t i o n . I t was of interest in this r e g a r d when LARDY ~ showed t h a t desiccation of r a t - l i v e r m i t o c h o n d r i a gave powders t h a t c o n t a i n e d A T P a s e a c t i v i t y . E x t r a c t s of such powders possessed A T P a s e a c t i v i t y u n s e d i m e n t e d at 18,ooo × g a n d responsive to b o t h D N P a n d Mg ++. This r e p o r t presents results of further s t u d y of the A T P a s e activities present in such powders. E a r l y in this w o r k it was found t h a t a significant percentage of t h e D N P - r e s p o n s i v e A T P a s e of the powders is n o t s e d i m e n t e d in 60 min a t lO5,OOO × g. Since such p r e p a r a t i o n s w o u l d present a d v a n t a g e s in purification a t t e m p t s , t h e y h a v e been e m p l o y e d routinely. E x t r a c t s are p r e p a r e d b y homogenizing the powders in 0.25 M sucrose. T h e suspension is clarified a t IO5,OOO × g, a n d the s u p e r n a t a n t is lyophilized for r e c o n s t i t u t i o n as needed. P h o s p h a t e f o r m a t i o n in the presence of A T P is d e t e r m i n e d b y a modified M a r t i n a n d D o t y procedure 4. T h e original a s s a y for D N P - r e s p o n s i v e A T P a s e * e m p l o y e d 0.09 21//KC1 a t p H 7.4. T h i s w o r k has shown t h a t p H 8.5 is o p t i m a l for t h e D N P response (Pl f o r m a t i o n in presence of D N P less PI f o r m a t i o n in absence of D N P ) . I n addition, t h e m a x i m u m response occurs in the absence of a d d e d salts such as KC1 or NaC1. I n Table I t h e TABLE I ION INTERFERENCE
IN RESPONSE
OF ATPAsE
TO 2,4-DINITROPHENOL
Conditions: o.o6M Tris buffer, pH 8.5; o.o12 M ATP; enzyme containing 1.3 mg protein; additions of KC1 and DNP as indicated; in volume of o. 5 ml. Incubation 2 min at 31 °. Pt formation was measured directly on the assay system by a modified Martin and Doty procedure4. (ltmoles P i forraed/mg proteinj × ~ro2 Added KCl (M)
--
5.IO~ M 5" lO-5 3I 5" IO~t M i.io~M
DNP DNP DNP DNP
--
4.2 lO.5 7 .8 9.4 11.2
0.04
0.08
o.r2
6.5 7.2 7.4 lO.3 14.2
5.1 o 1.6 2.5 6.9
4.3 o o 1.6 5.6
results show t h a t a d d e d KC1 diminishes the response to D N P . Similar results occur w i t h NaC1. P r e s u m a b l y , a n y m o n o v a l e n t - c a t i o n r e q u i r e m e n t the e n z y m e has is m e t b y the s y s t e m as constituted. The i t e m of g r e a t e s t interest in T a b l e I, however, concerns the first column of figures where no KC1 was added. U n d e r these conditions, two c o n c e n t r a t i o n s of D N P differing 2oo-fold possess a p p r o x i m a t e l y the same c a p a c i t y to s t i m u l a t e Pl release f r o m ATP. I n the original a s s a y s y s t e m s, in t h e presence of 0.0 9 M KC1, I . lO -4 to I . lO -3 M D N P h a d been found to yield the m a x i m a l response. These findings gave a h i n t of a m u l t i p h a s e aspect in t h e response to D N P a n d Abbreviations: DNP, 2,4-dinitrophenol; ATP, adenosine triphosphate; Tris, tris(hydroxymethyl) aminomethane; Pt, inorganic phosphate Biochim. Biophys. Acta, 44 (196o) 395-396
396
PRELIMINARY NOTES
therefore prompted reinvestigation of the nature of ATPase response over a wide range of DNP concentrations. Fig. I shows extracts that of mitochondrial acetone powders in 0.25 M sucrose exhibit characteristic multiphasic curves of ATPase reponse to DNP. Peaks of response occur at I. IO-8, I. lO -6 and I. lO -3 M DNP. Curves A and B were obtained with two different powders, the second of which had a lower specific activity at more dilute DNP concentrations. Similar results have been obtained with other acetone powders. 30
25
20
f.u moles Pi \ 2 ormed / mg/lO protein ] I 5 I0
5
0
9
7
6 - log [DN P]
5
4
3
2
Fig. I. Effects of variation of D N P concentration on ATPase activity. Conditions: 0.06 M Tris, p H 8.5; o.o12 M ATP; D N P in 0. 5 ml. Curve A: 1.o 4 mg protein incubated 2 min at 28 °. Curve B: 1.25 mg protein incubated I I min at 28 °.
These results m a y have other interpretations, but it is our feeling that they give direct evidence for the three DNP-responsive ATPases postulated by SLATER5. While SLATER'S results indicated the presence of three such enzymes with pH optima at 6.3, 7.4 and 8.5, these results were obtained at pH 8.5. Work is in progress to ascertain if the enzymes more sensitive to DNP have greater activity at lower pH values. This work was supported by grants from the National Science Foundation, North Carolina Heart Association, an American Cancer Society Institutional Grant.
Department of Biochemistry, University of North Carolina Chapel Hill, North Carolina (U.S.A.)
RALPH PENNIALL*
1 F. E. HUNTER, in W. D. MCELROY AND H. B. GLASS, Phosphorus Metabolism, Vol. I, J o h n s H o p k i n s Press, Baltimore, 1951, p. 297. 2 H. A. LARDY AND H. WELLMAN, J. Biol. Chem., 2Ol (1953) 357. 3 C. COOPER AND A. L. LEHNINGER, J. Biol. Chem., 224 (1957) 561. 4 1~.. PENNIALL,Analytical Biochem., in the press. 5 D. K. MYERS AND E. C. SLATER, Biochem. J., 67 (1957) 558. * Fellow of the American H e a r t Association.
Received September i2th, 196o Biochim. Biophys. Acta, 44 (196o) 395-396
395
NOTES
The 2,4-dinitrophenol-responsive adenosinetriphosphatase of rat-liver mitochondria Various i n v e s t i g a t o r s 1-a h a v e p o s t u l a t e d t h a t D N P a c t i v a t i o n of m i t o c h o n d r i a l A T P a s e is a m a n i f e s t a t i o n of the s y s t e m of o x i d a t i v e p h o s p h o r y l a t i o n . I t was of interest in this r e g a r d when LARDY ~ showed t h a t desiccation of r a t - l i v e r m i t o c h o n d r i a gave powders t h a t c o n t a i n e d A T P a s e a c t i v i t y . E x t r a c t s of such powders possessed A T P a s e a c t i v i t y u n s e d i m e n t e d at 18,ooo × g a n d responsive to b o t h D N P a n d Mg ++. This r e p o r t presents results of further s t u d y of the A T P a s e activities present in such powders. E a r l y in this w o r k it was found t h a t a significant percentage of t h e D N P - r e s p o n s i v e A T P a s e of the powders is n o t s e d i m e n t e d in 60 min a t lO5,OOO × g. Since such p r e p a r a t i o n s w o u l d present a d v a n t a g e s in purification a t t e m p t s , t h e y h a v e been e m p l o y e d routinely. E x t r a c t s are p r e p a r e d b y homogenizing the powders in 0.25 M sucrose. T h e suspension is clarified a t IO5,OOO × g, a n d the s u p e r n a t a n t is lyophilized for r e c o n s t i t u t i o n as needed. P h o s p h a t e f o r m a t i o n in the presence of A T P is d e t e r m i n e d b y a modified M a r t i n a n d D o t y procedure 4. T h e original a s s a y for D N P - r e s p o n s i v e A T P a s e * e m p l o y e d 0.09 21//KC1 a t p H 7.4. T h i s w o r k has shown t h a t p H 8.5 is o p t i m a l for t h e D N P response (Pl f o r m a t i o n in presence of D N P less PI f o r m a t i o n in absence of D N P ) . I n addition, t h e m a x i m u m response occurs in the absence of a d d e d salts such as KC1 or NaC1. I n Table I t h e TABLE I ION INTERFERENCE
IN RESPONSE
OF ATPAsE
TO 2,4-DINITROPHENOL
Conditions: o.o6M Tris buffer, pH 8.5; o.o12 M ATP; enzyme containing 1.3 mg protein; additions of KC1 and DNP as indicated; in volume of o. 5 ml. Incubation 2 min at 31 °. Pt formation was measured directly on the assay system by a modified Martin and Doty procedure4. (ltmoles P i forraed/mg proteinj × ~ro2 Added KCl (M)
--
5.IO~ M 5" lO-5 3I 5" IO~t M i.io~M
DNP DNP DNP DNP
--
4.2 lO.5 7 .8 9.4 11.2
0.04
0.08
o.r2
6.5 7.2 7.4 lO.3 14.2
5.1 o 1.6 2.5 6.9
4.3 o o 1.6 5.6
results show t h a t a d d e d KC1 diminishes the response to D N P . Similar results occur w i t h NaC1. P r e s u m a b l y , a n y m o n o v a l e n t - c a t i o n r e q u i r e m e n t the e n z y m e has is m e t b y the s y s t e m as constituted. The i t e m of g r e a t e s t interest in T a b l e I, however, concerns the first column of figures where no KC1 was added. U n d e r these conditions, two c o n c e n t r a t i o n s of D N P differing 2oo-fold possess a p p r o x i m a t e l y the same c a p a c i t y to s t i m u l a t e Pl release f r o m ATP. I n the original a s s a y s y s t e m s, in t h e presence of 0.0 9 M KC1, I . lO -4 to I . lO -3 M D N P h a d been found to yield the m a x i m a l response. These findings gave a h i n t of a m u l t i p h a s e aspect in t h e response to D N P a n d Abbreviations: DNP, 2,4-dinitrophenol; ATP, adenosine triphosphate; Tris, tris(hydroxymethyl) aminomethane; Pt, inorganic phosphate Biochim. Biophys. Acta, 44 (196o) 395-396
396
PRELIMINARY NOTES
therefore prompted reinvestigation of the nature of ATPase response over a wide range of DNP concentrations. Fig. I shows extracts that of mitochondrial acetone powders in 0.25 M sucrose exhibit characteristic multiphasic curves of ATPase reponse to DNP. Peaks of response occur at I. IO-8, I. lO -6 and I. lO -3 M DNP. Curves A and B were obtained with two different powders, the second of which had a lower specific activity at more dilute DNP concentrations. Similar results have been obtained with other acetone powders. 30
25
20
f.u moles Pi \ 2 ormed / mg/lO protein ] I 5 I0
5
0
9
7
6 - log [DN P]
5
4
3
2
Fig. I. Effects of variation of D N P concentration on ATPase activity. Conditions: 0.06 M Tris, p H 8.5; o.o12 M ATP; D N P in 0. 5 ml. Curve A: 1.o 4 mg protein incubated 2 min at 28 °. Curve B: 1.25 mg protein incubated I I min at 28 °.
These results m a y have other interpretations, but it is our feeling that they give direct evidence for the three DNP-responsive ATPases postulated by SLATER5. While SLATER'S results indicated the presence of three such enzymes with pH optima at 6.3, 7.4 and 8.5, these results were obtained at pH 8.5. Work is in progress to ascertain if the enzymes more sensitive to DNP have greater activity at lower pH values. This work was supported by grants from the National Science Foundation, North Carolina Heart Association, an American Cancer Society Institutional Grant.
Department of Biochemistry, University of North Carolina Chapel Hill, North Carolina (U.S.A.)
RALPH PENNIALL*
1 F. E. HUNTER, in W. D. MCELROY AND H. B. GLASS, Phosphorus Metabolism, Vol. I, J o h n s H o p k i n s Press, Baltimore, 1951, p. 297. 2 H. A. LARDY AND H. WELLMAN, J. Biol. Chem., 2Ol (1953) 357. 3 C. COOPER AND A. L. LEHNINGER, J. Biol. Chem., 224 (1957) 561. 4 1~.. PENNIALL,Analytical Biochem., in the press. 5 D. K. MYERS AND E. C. SLATER, Biochem. J., 67 (1957) 558. * Fellow of the American H e a r t Association.
Received September i2th, 196o Biochim. Biophys. Acta, 44 (196o) 395-396