The inhibition of succinate dehydrogenase by oxaloacetate

The inhibition of succinate dehydrogenase by oxaloacetate

210 PRELIMINARY NOTES BBA 61123 The inhibition of succinate dehydrogenaseby oxaloacetate It is generally believed that the inhibition of succinat...

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210

PRELIMINARY NOTES

BBA 61123

The inhibition of succinate

dehydrogenaseby oxaloacetate

It is generally believed that the inhibition of succinate dehydrogenase (EC i .3.99. i) by oxaloacetate z is competitive in nature ~. In this laboratory DERVARTANIAN AND VEEGER3 found that this is the case with the purified enzyme4 and reported an inhibition constant of 1.5 #M. WOJTCZAKand co-workersS, 6, however, have reported that a non-competitive inhibition is obtained if the initial reaction rate is measured, and a typically competitive inhibition only if the steady-state rate of oxidation is taken. Furthermore, they state that neither the inhibition by oxaloacetate nor its partial reversal by succinate is instantaneous. A K, of 0.2 ffM was reported for the constant rate of oxidation. We have re-examined the problem with the purified enzyme isolated by a modification 3 of the method of WANG,Tsou AND WANG4. Fig. 1 shows that, in disagreement with WOJTCZAKet al.S, 6, the inhibition is instantaneous. However, in agreement with W O J T C Z A K et al., after a steady rate of oxidation for about 1. 5 rain, the degree of inhibition increased, to reach a new level after about 30 sec to (as in the experiment illustrated in Fig. I) several minutes. The increased inhibition is not due to the formation of fumarate. The addition of I mM fumarate had no appreciable effect on the activity of the enzyme in the absence of oxaloacetate, or on the degree of inhibition by oxaloacetate, either that directly measured or after the secondary inhibitory state is reached. In disagreement with W O J T C Z A K et al.5, 8, the inhibition found immediately on the addition of oxaloacetate was competitive. Fig. 2 shows a typical experiment from which values of K, of 4. 5 and 4.3 ffM may be calculated. Similar results were obtained

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16C I 14C /12C



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8C 6C

2o

40

~0.~

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4

Time (min)

6

o_6 0 0

- 800

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o 1/[S] (M -1)

8&o

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Fig. I. I n h i b i t i o n of s u c c i n a t e d e h y d r o g e n a s e b y oxaloacetate. P h o s p h a t e buffer (pH 7.8), ioo m M ; K3Fe(CN)e, 5 mY[; succinate, io m M ; bovine s e r u m a l b u m i n , i m g / m ] ; e n z y m e , 0.06 m g / m l . At t h e arrow, o x a l o a c e t a t e was a d d e d to a c o n c e n t r a t i o n of 6. 3 ffM. Fig. 2. Double-reciprocal plot of p r i m a r y inhibition of s u c c i n a t e d e h y d r o g e n a s e b y o x a l o a c e t a t e . P h o s p h a t e buffer (pH 7.8), ioo m M ; K3Fe(CN)8, 6 m M ; bovine s e r u m a l b u m i n , I m g / m l ; E D T A , I m M ; e n z y m e (240 u n i t s * / m g protein), 0.08 m g p r o t e i n per ml. T h e velocity is in a r b i t r a r y units. 0 - - 0 , no o x a l o a c e t a t e ; A - - A , 5.4 ffM o x a l o a c e t a t e ; O - - O , lO.8 # M oxaloacetate. T h e o x a l o a c e t a t e c o n c e n t r a t i o n s were d e t e r m i n e d with m a l a t e d e h y d r o g e n a s e a n d N A D H . T h e oxaloa c e t a t e used c o n t a i n e d io % p y r u v a t e (as d e t e r m i n e d w i t h l a c t a t e d e h y d r o g e n a s e a n d N A D H ) .

Biochim. Biophys. Acta, 132 (1967) 21o-212

PRELIMINARY NOTES

211

over a large r a n g e of o x a l o a c e t a t e c o n c e n t r a t i o n s (o.6-135 #M) a n d succinate concent r a t i o n s (o.2-1oo mM). The K i v a r i e d b e t w e e n 1. 7 a n d 6.3 # M in 14 m e a s u r e m e n t s in this range. I n t h e e x p e r i m e n t shown in Fig. 2, t h e reaction was s t a r t e d b y a d d i n g e n z y m e to an otherwise c o m p l e t e r e a c t i o n m i x t u r e . I n h i b i t i o n was also i n s t a n t a n e o u s a n d c o m p e t i t i v e (Kt = 2.6-3.1 #M) when t h e order of a d d i t i o n was t h e same as in Fig. i. T h e s e c o n d a r y inhibition was also found to be p u r e l y c o m p e t i t i v e , w i t h a K i of 0.6-0.7 ffM. W h e n t h e e n z y m e was p r e - i n c u b a t e d for IO rain w i t h a high c o n c e n t r a t i o n of o x a l o a c e t a t e a n d t h e n d i l u t e d to t h e c o n c e n t r a t i o n s usual in t h e a s s a y of e n z y m e a c t i v i t y before a d d i n g t h e succinate or K3Fe(CN)6, t h e g r e a t e r inhibition was f o u n d i m m e d i a t e l y . I n this case, however, the inhibition was p a r t l y n o n - c o m p e t i t i v e a n d p a r t l y c o m p e t i t i v e . The K , of t h e c o m p e t i t i v e c o m p o n e n t was 0. 7 #M, a n d t h a t of t h e n o n - c o m p e t i t i v e c o m p o n e n t 0.6 ffM.

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%-

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290

Oxaloacetate concn. (IJM)

Fig. 3. Titration of the succinate dehydrogenase with oxaloacetate, followed spectrophotometrically at 5"/0 raft. To 1.8 ml of enzyme (24o units4/mg protein; 3 mg protein per ml) in a I-cm cell was added from a Hamilton microsyringe various amounts of i mM oxaloacetate. Finally an excess oxaloacetate was added in the form of a I o mM solution. The total volume added was o.3 ml. The absorbances were corrected for dilution. The initial absorbance was 0.20o.

The r e a c t i o n b e t w e e n t h e e n z y m e a n d o x a l o a c e t a t e , as m e a s u r e d b y t h e increase in a b s o r b a n c e a t 570 m # on a d d i n g t h e i n h i b i t o r to the enzyme~, 7, has also been f u r t h e r studied. E x p e r i m e n t s w i t h the stopped-flow a p p a r a t u s showed t h a t the r e a c t i o n was a l w a y s c o m p l e t e in 4 sec. I n close a g r e e m e n t w i t h t h e value p r e v i o u s l y reported~, 7, a dissociation c o n s t a n t of 3 #M m a y be c a l c u l a t e d from the d a t a given in Fig. 3. (In this t i t r a t i o n , the c o n c e n t r a t i o n s of e n z y m e a n d o x a l o a c e t a t e are of t h e same order of m a g n i t u d e , a n d in calculating t h e K o a correction for t h e a m o u n t of i n h i b i t o r b o u n d to t h e e n z y m e was made.) The r a p i d r e a c t i o n b e t w e e n e n z y m e a n d o x a l o a c e t a t e , as followed spectrop h o t o m e t r i c a l l y , a n d t h e a g r e e m e n t b e t w e e n t h e K i o b t a i n e d for t h e p r i m a r y inh i b i t i o n a n d t h e KD from t h e t i t r a t i o n s u p p o r t s t h e conclusion3, 7 t h a t t h e spectroscopically identified c o m p o u n d is t h e E I complex of t h e p r i m a r i l y i n h i b i t e d enzyme. The n a t u r e of the s e c o n d a r y inhibition is u n d e r s t u d y . Bio~him. Biophys. Acta, i32 (i967) 21o-212

212

PRELIMINARY NOTES

We wish to thank Professor C. VEEGER and Dr. D. V. DERVARTANIANfor useful suggestions and Mr. S. VAN DALSEM for technical assistance.

Laboratory of Biochemistry, B. C. P. Jansen Institute*, University of Amsterdam, (The Netherlands)

W . P . ZEYLEMAKER E . C . SLATER

13. DAS, Biochem. J., 31 (1937) 1116. 13. PARDEE AND V. R. POTTER, J. Biol. Chem, 176 (1948) lO85. V. DERVARTANIAN AND C. VEEGER, Biochim. Biophys. Acta, 92 (1964) 233. Y. WANG, C. L. T s o u AND Y. L. WANG, Sci. Sinica Peking, 5 (1956) 73. B. WOJTCZAK, in J. M. TAGER, S. PAPA, E. QUAGLIARIELLO AND E. C. SLATER, Regulation of Metabolic Processes in Mitochondria, BBA Library, Vol. 7, Elsevier, A m s t e r d a m , 1966, P. 539. 6 L. WOJTCZAK, A. B. WOJTCZAK AND L. ERNSTER, Abstracts Third Meeting of the Federation of European Biochemical Societies, Warsaw, z966, Academic Press, London, and P W N , Warsaw, 1966, p. 124. 7 D. V. DERVARTANIAN, W. P. ZEYL]~iMAKER AND C. VEEGER, in E. C. SLATER, Flavins and Flavoproteins, BtBA Library, Vol. 8, Elsevier, A m s t e r d a m , 1966, p. 183. I 2 3 4 5

N. A. D. T. A.

Received October I3th, 1966 * Postal address: Plantage Muidergracht 12, Amsterdam-C.

Biochim. Biophys. Acta, 132 (1967) 21o-212

BBA 6 1 1 2 1

~-D-Xylosidase in pig kidney It has recently been reported a that rat liver possesses fl-xylosidase activity. Little is known of the distribution of this enzyme in other mammalian tissues, although highly active preparations have been demonstrated in bacteria, plants and fungi 2,s. It is possible that the enzyme occurs in a number of organs where the cleavage of the link between xylose and serine in chondroitin sulphate-protein complexes m a y be an important step in the catabolism of the mucoid residues, but lack of a suitably sensitive method of assay has so far hampered investigation of this enzyme. By using the fluorigenic substrate, 4-methylumbelliferyl fl-I)-xylosidO, we have been able to detect and examihe a fl-xylosidase in pig kidney. The substrate (i ml of I mM solution) in 0.2 M phosphate-citrate buffer is incubated with I ml of a 0.040//0 (w/v) homogenate of the tissue in water for 30 rain before adding 3 ml ofo.5 M glycineNaOH buffer (pH io.4) to stop the reaction and develop the fluorescence of the liberated aglycone. Fluorescence was measured on a Locarte LF.5 fluorimeter at 44 ° m#, (activation at 340-380 m/z). Under these conditions the enzyme had a broad p H optimum of 5-6 (Fig. I), the rapid loss of activity in more acid conditions being caused by irreversible denaturation. The working substrate concentration used does not allow complete saturation of the enzyme (Fig. 2), but higher concentrations are precluded by its low solubility. It has been shown that the rat liver enzyme is sedimented in isotonic sucrose homogenates along with the acid phosphatase-rich particles, and the fl-xylosidase activity is latent until released by detergent or hypotonic conditions 1. I f thus appears Bioehim. Biophys. Acta, 132 (1967) 212-214