Possible functions of the NADP-linked isocitrate dehydrogenase and ...

260s Biochemical Society Transactions ( 1 993) 21 Possible functions of the NADP-linked dehydrogenase and H + -transhydrogenase mitochondria

isocitrate in heart

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I,EONID A. SAZANOV AND J . BAZ JACKSON School of Biochemistry, University of Birmingham, Birmingham B15 211, U.K. The NAD- and NADP-linked isocitrate dehydrogenases (ICDH) of mitochondria catalyze an equivalent step in the citric acid cycle but it is widely held that NAD-ICDH carries most of the flux through the cycle 111. Although NADP-ICDH is at least 10 times more active in vitro than the NAD-linked enzyme [21, its physiological role, especially in heart, where there are no major consumers of NADPH has not been established. The role of H+-transhydrogenase (H +! -thase) in mitochondria is also not completely understood (for reviews see (3,41). One of the pmibilities is that the H -thase functions as a "redox buffer", preventing uncontrolled changes in the NAD(P) redox state or depletion of hp 131. It remains to be established under what physiological conditions this "buffering" effect is important. We propose that NADP-ICDH and H +-thase have a joint function in heart mitochondria. Two possible cases are worthy of consideration. I ) To allow dissipation of damagingly high levels of the transmembrane proton electrochemical gradient When the demand for ATP in heart is low (as in state 4 respiration), @ is high and the action of H i -thase (catalysinq the "forward", energy-linked, reaction: NADH NADP t HfoUt + NADPH t NADt t H in) leads to the dissipation of ip and causes the NADPH/NADP ratio to increase. In principle this could be sufficient to drive the NADP-ICDH in "reverse", from a-ketoglutarate (KG) to isocitrate (IC), while the thermodynamics of the NAD-linked enzyme still favour the "forward" direction. The net result of continued operation of H -thase and cycling around the ICDH is only dissipation of ("futile cycling"). It would operate only above a critical value fixed by the nucleotide mass action ratio and the value of hp. Because of the logarithmic dependence o n the the switch could act within a fairly narrow range, in contrast to other processes that limit the build-up of hp, such as "respiratory control". 2) Another Issibility is that the concerted abion of NADP. under certain conditions (pg. during ICDH and H -thase transitions between the equivalent of States 4 and 3, when a relatively "rapid" response is necessary) could account for a substantial part of respiratory activity through NADPt 3 NADPH -+ NADH pathway. Note that such a system of two sequential enzymes might he more effectively regulated in vivo by hp, than would NAD-ICDH by the thermodynamic "back pressure" of NADH ("respiratory control"). The high capacity of NADP-ICDH would allow for a more rapid increase in respiration in response to the increased demand for ATP. If the "futile cycling" mechanism does take place during conditions of low load, then it would further increase the sensitivity of iswitrate dehydrogenases to changes in ATP demand. If the above mechanisms are to play significant role in the control of mitwhondrial metabolism, then the capacity of the enzymes involved must be adequate relative to the turnover of the citric acid cycle and the rate of resFirdtion. Data on such activities are available in the literature. However, they were obtained under different conditions and by different research groups. Most where reported in IS] but with the im exception of the reverse NADP-ICDH and the forward H -thase reactions. Therefore it was necessary to record rates on the same mitochondria1 preparation and using plausible in vivo concentrations of substrates. We have measured the rates of respiration, the activities of NAD-ICDH,NADP-ICDHand H thase reactions (Tab.l) in intact beef heart mitochondria, submitochondria1 particles (SMP) and a soluble enzyme fraction obtained by sonication from the same mitochondria. Preparations were essentially according to Low and Vallin 161.

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Abbreviations used: NAD(NADP)-ICDH - NAD(NADP) linked isocitrate dehydrogenase, H -thase H transhydrogenase, IC - isocitrate, KG - a-ketoglutarate.

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TABLE 1. T e at s f itoc ondria reactions I nmol obtained in three different preparations. Rate Respiration State 4 State 3 NADP-ICDH forward reverse NAD-ICDH forward Hi-thase forward reverse

(IC

-P

KG)

IC) (IC + KG) (NADH + thio-NADP') (NADPH + AcPyAD')

(KG

+

15-30 70-130 250-500 20-40

5-10 25-40 65-110

Respiration was measured (in nmol 101) with an oxygen electrode in 0.25M sucrose, 33mM glucose, 5mM MgCl 12mM K2HP04, O.lmM EDTA medium @H 7.4) with 5 m h pyruvate and 5mM malate as substrate. To induce sdate 3, ADP (ImM) was added. ICDH reactions were assayed spectrophotometrically by addition of soluble mitochondrial protein (0.1 mglml) to 30 mM Triethanolamine-CI buffer (PH 7.4) with 2mM MnS04 or MgCI2. For the forward NADPICDH reaction O.lmM isocitrate and O.lmM NADPt were added, for the reverse reaction, 0.6 mM a-ketoglutarate, 0.1 mM NADPH and 20mM NaHC03 and for the forward NAD-ICDH reaction, 2mM isocitrate, 0.6mM NADt and ImM ADP. NADlCDH was inactive in the reverse direction. Activity of H +-thase was assayed spectrophotometrically by addition of SMP (0.3 mglml) to 0.25M sucrose, 0.1M K HPO4 medium (pH 7.4), containing in the range 0-10 mM hgC1 I-25pM rotenone, tpg/mg.prot. olygomycin, and 0.1-0.3md' of NADH t thioNADP + or NADPH t acetylpyridine adenine dinucteotide (AcPyAD'). lOmM succinate or 2mM ATP were used for energization of SMP. Rates per mg of mitochondria1 protein for soluble proteins and SMP were estimated on the basis that SMP protein comprises about 40%, and soluble protein about 50% of' total mitochondrial protein [7,8]. The values show the range of activities recorded in different preparations. The values presented in the 1ah.l tie within the ran ye of previously published data, the activity of NADP-ICDH bheing closer to higher limits, while that of NAD-ICDH - to lower limits. As evident from the Tab. I , the activity of NADP-ICDH in the reverse and H +--&ax in forward direction are more than adequate to support State 4 respiration according to our mechanism of "futile cycling" and are even higher than the activity of NAD-ICDH in forward direction. Very high activity of NADP-ICDH in forward direction is consistent with the "rapid response" mechanism. The activity of NADP-ICDH in both directions in the presence of possible endogenous concentrations of reaction products (0.05-0.ImM IC together with 0.4-2mM KG [5,91) might be only 15.30% less than the maximal values (initial rates obtained in absence of products) reported in Tab.1. The value for NAD-ICDH activity may be underestimated, since this enzyme is a subject for complex allosteric regulation in vivo. However, the remarkably high activities of NADP-ICDH and H t -thase support our view that these enzymes together do play significant role in metabolism in heart mitochondria. Validation of these hypotheses requires further investigation. We acknowledge financial support from the Wellcome Trust (fellowship to L. A. S.) . 1. McComack, J.G. & Denton, R.M. (1986) Trends Biochem. Sci. 11. 258-262. 2. Smiih, G.M. & Plaut, G.W.E. (1979) Eur.J.Biochem. 97, 283-295. 3. Rydstrom, J. & Hoek, J.B. (1988) Biochem.J. 254, 1-10. 4. Jackson. J.B. (1991) J.Bioenerg.Biomembr. 23, 715-741. 5. Hansford, R.G. &'Johnson, E.N. (1975) J.Biol.Chem. 250, 8361-8375. 6. Low, H. & Vallin, I. (1963) Biochim.Biophys.Acta 69,361374. 7. Wanders, R.J.A. Van Doom, H.E. & Tager, J.M. (1981) Eur.J.Biochem. 116, 609-614. 8. Nicholls, D.G. & Garland, P.B. (1969) Biochem.J. 114, 215225. 9. LaNoue, K.F., B la, J & Williamsom, J.R. (1972) J.Biol.Chem. 247, 667-779. .