Plant Science Letters, 1 (1973) 11--14 © Elsevier Scientific Publishing Company, Amsterdam - - Printed in The Netherlands
ACTIVATION OF NADP--MALATE DEHYDROGENASE BY LIPOATE *
NEMATOLLAH AHMADI and IRWIN P. TING **
Department of Biology, University of California, Riverside, Calif. 92502 (U.S.A.) (Received August 28th, 1972)
The NADP-specific malate dehydrogenase of spinach leaf chloroplasts, a light-activated enzyme, is activated in vitro by the naturally-occurring disulfide compound, lipoate. Other compounds which tend to form cyclic disulfides on oxidation such as dithiothreitol (DTT) and trimethylene dithiol will also activate in vitro. Monosulfhydryl compounds such as glutathione, mercaptoethanol, and cysteine do not activate appreciably. These data suggest that lipoate or a similar disulfhydryl compound may play an in vitro role in the light activation of chloroplast NADP--malate dehydrogenase.
Recently, a protein with NADP--malate dehydrogenase activity (L-malate: NADP oxidoreductase) was isolated from plant tissue 1-4, and shown to be localized in chloroplasts 1,4. In vivo, the enzyme is light-activated and rapidly inactivated in the dark 2,3. For in vitro activity, a requirement for DTT was demonstrated ~ - 4 In this report, we present data indicating that lipoate will also activate NADP--malate dehydrogenase whereas other naturally-occurring sulfhydryl compounds such as glutathione, acetyl-CoA, and L-cysteine do not activate. MATERIALS AND METHODS
The enzyme was prepared from fresh spinach leaf tissue by extraction in 0.05 mM tris buffer, pH 7.5, containing 1 mM DTT, and I mM EDTA. The * Supported b y NSF Grant GB-25878. ** To whom all correspondence should be directed. Abbreviations: BAL, 2,3-dimercapto-l-propanol; DTE, dithioerythreitol; DTT, dithiothreitol.
latter preparation was precipitated between 20 and 65% ammonium sulfate saturation. The precipitate was redissolved in 5 mM, pH 7.0, sodium phosphate buffer, and further purified by anion-exchange column chromatography on DEAE-cellulose and by gel filtration through Sephadex G-200. Details of preparation and assay were described previously 4 The enzyme was partially or completely inactivated by exhaustive dialysis against tris buffer 4. Reactivation b y a variety of chemicals was tested by incubation at 25 or 0.° The following c o m p o u n d s were tested at 5 mM concentrations: DTT, reduced glutathione, BAL, and ~-mercaptoethanol all from Cal Biochem, San Diego, Calif.; 2-mercaptobenzothiazole, trimethylenedithiol, and dithiodiglycol from K & K Laboratories, Hollywood, Calif.; and coenzyme-A, DTE, reduced D,L-6,8-thioctic acid (lipoate), monothioglycerol 2,6
Addition of the following c o m p o u n d s did not result in appreciable activation: coenzyme-A, 2,6-dithiopurine, 2,4-dithiopyrimidine, NADPH, thiamine pyrophosphate, L-cysteine, glutathione, mercaptobenzothiazole, monothioglycerol, dithiodiglycol, and ~-mercaptoethanol. Of the c o m p o u n d s tested, only those which form cyclic disulfides or linear polymers were effective activators. When an inactive preparation was incubated with dithiols at 25 ° for 60 min, the following activation order was observed: DTT ~-- DTE > lipoate trimethylene dithiol > BAL (Table I). TABLE I A C T I V A T I O N O F N A D P - - M A L A T E D E H Y D R O G E N A S E BY D I T H I O L C O M P O U N D S a
D T T (%)
DTT DTE Lipoate Trimethylenedithiol BAL
100 100 70 63 21
a I n c u b a t i o n w a s at 25 ° for 60 rain in the presence o f 5 m M dithiol. A n inactivated preparation w a s i n c u b a t e d at 25 ° for 6 0 rain in the p r e s e n c e o f 5 m M dithiol. A f t e r i n c u b a t i o n , the preparation w a s assayed for N A D P - - m a l a t e d e h y d r o g e n a s e . 100% = 0.45 A/mini0.1 ml preparation.
When an activated preparation was incubated in the presence of 5 mM DTT or lipoate at 2 5 ° , activation was completed in about 75 min ( F i g . 1 ) . In this experiment, the DTT resulted in a b o u t a 19-fold activ~ttion whereas the lipoate resulted in a 15-fold activation. When incubated for 15 h at 0 ° , 2 mM
/ / o
t // 0'1 1" / // 0
Fig. 1. T i m e course o f N A D P - - m a l a t e dehydrogenase activation in the presence o f 5 mM D ~ F or 5 m M lipoate. 25 °.
DTT or lipoate was sufficient to activate maximally (Fig. 2). If 5 mM DTT was added to the lipoate-activated preparation, the activity was increased to the level of the DTT activation (see Figs. I and 2). The Michaelis constants with oxaloacetate as the variable substrate were: DTT-activated = 0.03 raM, lipoate-activated = 0.024 mM. The latter results are an average of two separate experiments and agree quite well with published results 2,4 The initial rate of heat inactivation at 40 ° was approximately 10 times as rapid with the lipoate-activated as with the DTT-activated preparation (DTT = 0.8% min -1 ; lipoate = 7.3% rain -1 ).
,o - ~ . ~ - - - ~
~ 6 7 Conc. (raM)
Fig. 2. Activation o f N A D P - - m a l a t e dehydrogenase as a function o f D T T or lipoate concentration. Preparations were incubated overnight at 0 ° prior to assay.
It should be noted that the lipoate activation of NADP--malate dehydrogenase does n o t seem to be comparable to the ~-ketodehydrogenase systems requiring ATP, thiamine, and lipoate s ; however, like NADP--malate dehydrogenase, a 30-min preincubation with lipoate was necessary to obtain full activity of b o t h pyruvate and ~-ketoglutarate dehydrogenases 6. Neither pyruvate nor ~-ketoglutarate were effective substrates, and ATP did not enhance the reaction when in the presence of DTT or lipoate. Of the c o m p o u n d s tested, only those disulfides which are n o t readily reversible on oxidation were effective activators. DTT and lipoate b o t h form cyclic disulfides and their redox potentials are similar ~,s. Monothiol compounds tested with high redox potentials were not effective. Earlier reports that sulfhydryls of green plants increase in the light 9, that lipoate is involved in chloroplast photometabolism 10, and that NADP-malate dehydrogenase is light-activated 2,3, tend to suggest an in vivo relationship between lipoate and NADP--malate dehydrogenase. Naturally-occurring cyclic disulfides have been suggested to function in enzymatic activation ~1 We propose, therefore, that lipoate or a similar disulfide c o m p o u n d could be the natural in vivo activator of NADP--malate dehydrogenase. Further research will be necessary to establish for certain the nature of the in vivo activation. REFERENCES
1 2 3 4 5 6 7 8 9 10 11
M.D. Hatch and C.R. Slack, Biochem. Biophys. Res. Commun., 34 (1969) 589. H.S. Johnson and M.D. Hatch, Biochem. J., 119 (1970) 273. H.S. Johnson, Biochem. Biophys. Res. Commun., 43 ( 1 9 7 1 ) 7 0 3 . I.P. Ting and V. Rocha, Arch. Biochem. Biophys., 147 (1971) 156. S.K. Mitra and D.P. Burma, Biol. Chem., 240 (1965) 4072. L.J. Reed, F.R. Leach and M. Koike, J. Biol. Chem., 232 (1958) 123. W.W. Cleland, Biochemistry, 3 (1964) 480. Handbook of Biochemistry, The Chemical Rubber Co., Cleveland, Ohio, 1968. W.M. Dugger and I.P. Ting, Recent Advan. Phytochem., 3 (1970) 31. L.J. Reed, Advan. Enzymol., 18 (1957) 319. M. Tokushige, O. Hayaishi and K. Morita, Arch. Biochem. Biophys., 122 (1967) 522.