PRELIMINARY NOTES

PN 6r054Energy-linked incorporation of diphosphopyridine nucleotide into rat-livermitochondria

PURVIS AND LOWENSTEINI have previously shown that DPN could be in corp o­rated into mitochondria in vitro, and ac hieve d repl acements of 40-50% of the mito­chond rial pool. This incorporat ion of DPN was proportion al to t he time of incu bationand to mitochondrial protein. The per cent replacement of mitochondrial DPN alsowas dependent on the concent ration of DPN incubated with t he mitochondria. Theincorporation, however, was much slower t han that of metal ions" or adenine nuc1eo­tides" but was specific for DPN in that nicotin amide, nicotinic acid, and TPN werenot incorporated to a significant extent. The pr esent communication shows that thisincorporat ion, like that of metal ions is energy lin ked ; t hat it requires substrate, isinhibited b y respiratory inhibitors like An timycin and KCN, and is uncoupled by2,4-d initrophenol, and illustrates that energy is required for the specific incorporationof a large molecule the size of a nucleotide as well as with small metal ions. The in­corporation of adenine nu cleotides, although much more rap id , has not been shownto be energy linked except in the case where Ca2+ ions are added". This lat te r pointis the sub ject of another communicat ions .

Since the mitochondria were cent rifuged an d washe d twice and zero ti meincorporations of DPN wer e routi nely done to monitor the washing, the percentagereplacement of the DPN pool refers t o the DPN bound to the crist ae or in the matrix

TA BLE I

INH IB I TION A ND STIMULATION OF THE INCO R PORATION OF [14C]DPN I N R AT-LIVE R MI TOCH ON D RIA

The incubation mixture cont a ined 10 mM Tris (pH 7.4). 80 m M NaC!, 10 mM MgCI•. 4.0 mMsodi um potassium phosph ate, 1 0 mM sodium succinate, 63.5 m rzmolcs of [uC]DPN having aspecific ac t ivity of 1.32' lOG counts/min!,umo le, 14.0 mg mitochondrial protein , and wh ere in­dicated An t imycin, I ,ug/mg p ro t ein , 0.050 mM :lA- d in itrophenol and 4,0 m M CaCl. , Fi n alvolume 1.2 rnl. Temperature : 25° . Incubation time: 30 min. Zero time incorporation of [14C]DPNis su btract ed from t he va lues in Colu mn I .

Additions

DPN 83 750 counts/minDPN, Ant im ycinDPN, 2.4-dinitrophe nolDPN, CaCI.

Cou nts jmi»incorporate d perI O mg protein in30 min'

% mitochondri al roplacement:Specific activ ity DPN isolated 100Specifi c activ i ty DPN ini tial X

2·5 50.32

0. 32

9. 30

, Corrected for pyridin e nucleot ide loss during incu bation and separa t ion of m it och on­dria and su pernatant.

of the mitochondria. This distinction is necessary in view of evi dence" which indicat esthat sma ll molecules un der the molecular weight of inulin pass through the mito­chondria l membra ne readily , but do not en ter t he crist ae or pass through this barrierto the rna trix,

The counts/min incorporated per I O mg of mitochondrial protein in 30 min is

Biocbim. Biophys. Acta, 99 (1965) 191-194

192

TABLE II

PRELIMINARY NOTES

EFFECT OF DIFFERENT RESPIRATORY INfiIBITORS ON [14C]DPN INCORPORATION INTO MITOCHON­

DRIA

The incubation mixture contained the same as Table I except that 160 m,umoles of [14C]DPNcontaining 220000 counts/min (specific activity of 1.37' 106 counts/minjzzmole) was incubatedwith 11.0 mg of mitochondrial protein, and either 10 mM sodium succinate or 10 mM sodiummalate plus 10 mM sodium glutamate. 2 mM KCN, Antimycin, I ltg/mg protein, 3.2 . 10-3 mMrotonone and o.oyo mM 2,4-dinitrophenol were added as indicated. Final volume 1.2 ml. Temper­ature 2jo. Incubation time: 30 min. Zero incorporation of ['4C]DPN is subtracted from the valuesin Column 2.

Additions Counts/min Couruslmin. %mitochondrial replacemeni:incorporated incorporated pel' Specific activity DPN isolated

X 100in 30 min IO mg protein Specific activity DPN initialin 30 min'

DPN, succinate" 149 0.17DPN, succinate 2420 1460 5. 18DPN, succinate, rotonone 2260 1225 4-42DPN, succinate, Antimycin,

rotonone 268 79 0.67DPN, succinate, KCN 350 130 0·92DPN, malate, glutamate 1710 1047 3·93DPN, glutamate, malate,

rotonone 248 74 0.81DPN, glutamate, malate,

2,4-dinitrophenol 180 20 0.30

, Corrected for pyridine nucleotide loss during incubation and separation of mitochondriaand supernatant and zero time incorporation.

• , Zero time incorporation.

also shown in Tables I and II. From these values the per cent incorporation of addedDPN into mitochondria may be calculated. During this incorporation of DPN thereis less than 10% loss of pyridine nucleotides from the mitochondria and virtually noprotein leakage during the incubation. Nevertheless the values are corrected for thissmall loss to prevent an underestimation of the per cent incorporation.

[Nicotinamide-J_14C]DPN was prepared and incorporated into mitochondriaaccording to the procedure of PURVIS AND LOWENSTEIN l except that the incubationconditions were those of CARAFOLI AND LEHNINCTER4 minus CaC12• DPN was isolatedand purified to constant specific activity in two solvent systems and assayed accordingto PURVIS', Protein was determined by the biuret method.

Table I shows that in the presence of 53,aM [I4C]DPN there is a 2.55% replace­ment of the mitochondrial DPN pool in 30 min. Since the percentage replacement ofthe mitochondrial pool is proportional to the concentration of DPN in the suspendingmedia and the time, this value agrees very well with the per cent replacement obtainedwith larger DPN concentrations'. This is equivalent to 49.7% replacement per hourat the physiological concentration of 495 ,aM. This incorporation is almost completelyinhibited by the respiratory inhibitor, Antimycin, and is completely uncoupled by2A-dinitrophenol. The last line in Table I shows for comparison purposes the in­corporation of DPN in the presence of 4-0 roM CaC12 • This 4-fold increased incorpora­tion- indicates that once permeability of the membrane is altered DPN enters andleaves the mitochondria rapidly.

Biochim, Biophys. Acta, 99 (196j) 191-194

PRELIMINARY NOTES 193

The effect of different respiratory inhibitors on DPN incorporation in the pres­ence of succinate and of malate plus glutamate is shown in Table II. Both KCN andAntimycin almost completely inhibit DPN incorporation with succinate as substrate;the inhibition with KCN, however, is not as complete as with Antimycin. Rotenone,an inhibitor which blocks DPNH2 but not succinate oxidation", only causes a slightinhibition of DPN incorporation with succinate in contrast to almost completelyinhibiting incorporation with malate and glutamate. The difference in DPN incorpo­ration with succinate in the presence and absence of rotonone probably reflects thecontribution of endogenous substrate passing through DPNH2 20 mM malonate (notshown) completely inhibits the effect of succinate but does not decrease the incorpora­tion in the absence of substrate.

The inhibition of DPN incorporation in the presence of respiratory inhibitorsshows that electron flow is necessary to support incorporation. The complete inhibi­tion by 2,4-dinitrophenol (Tables I and II) indicates that electron flow is necessaryto generate a high energy intermediate to support the incorporation. The completeoxidation of pyridine nucleotides in the presence of z.a-dinitrophenol" shows thatincorporation is not dependent on the percentage of DPN in the oxidized form. Con­comitant swelling experiments with and without 2,4-dinitrophenol indicate thatswelling and contraction are not involved in DPN incorporation. The inhibition ofDPN incorporation by Antimycin and 2,{-dinitrophenol is very similar to the energy­linked incorporation of metal ions recently reported by various laboratories9- 11, andstrongly suggests that DPN incorporation into mitochondria is energy linked. Theincorporation of DPN is not dependent on the nicotinamide-ribose linkage for N­methyl [7-14C]nicotinamide is incorporated ten times more slowly than DPN (ap­proximately the same as nicotinamide). Nor does 2,4-dinitraphenol which almostcompletely inhibits DPN incorporation alter the incorporation of N-methyl nicotin­amide.

This energy-linked incorporation is only partially dependent on the presenceof substrate. This partial dependency is probably due to endogenous substrate sincethe omission of added substrate lowers the incorporation of DPN by 50% and 20 mMmalonate plus 3 I-tM rotonone decreases this small level of incorporation further. Theper cent replacement of the mitochondrial pool in 30 min was: DPN, 1.65; DPN minussuccinate, 0.78; DPN minus succinate plus malonate (20 mM) and rotonone (3,uM),0.46; DPN minus phosphate, 1.98; and DPN plus 2,4-dinitrophenol (50 ftM), 0.30. Asexpected both the incorporation supported by endogenous and added substrate wascompletely abolished by 2,4-dinitrophenol. DPN incorporation into rat-liver mito­chondria was not dependent on the presence of phosphate in the media. The absenceof phosphate also has no effect on the energy-linked accumulation of Mn2+ (ref. 10)and low levels of Ca2+ (ref. 12) in rat-liver mitochondria, and Mg2+ (ref.13) in beef­heart mitochondria.

The technical assistance of Mr. M. WALDER is gratefully acknowledged. Thiswork was supported by a grant from the National Science Foundation (GB-224).

Department oj Chemistry,University oj Rhode Island,Kingston, R.I. (U.S.A.)

MICHAEL GREENSPAN

JOHN L. PURVIS

Biochim. Biopliys. Acta, 99 (1965) 191-194

194 PRELIMINARY NOTES

I ]. L. PURVIS AND]. M. LOWENSTEIN, J. Bioi. Chem., 236 (1961) 2794­2 C. S. ROSSI AND A. L. LEHNINGER, Biochem, Z., 338 (1963) 698.3 A. BRUNI, S. LUCIANI AND A. R. CONTESSA, Nature, 201 (1964) 1219.4 E. CAROFOLI AND A. L. LEHNINGER, Biochem, Biopbys. Res. Commun., 16 (1964) 66.5 M. D. GREENSPAN AND J. L. PURVIS, Biochim. Biophys. Acta, 99 (1965) 167.6 G. P. BRIERLEY, personal communication.7 ]. L. PURVIS, Biochim. Biophys. Acta, 38 (19 6::» 435.8 L. ERNSTER, G. DALLNER AND G. F. AZZONE, J. BiA. Chem., 238 (1963) 1124.9 G. P. BRIERLEY, E. MURER, E. BACHMANN AND D. E. GREEN, ]. Bioi. Chem., 238 (1963) 3482.

10 ]. B. CHAPPELL, M. COHN AND G. D. GREVILLE, in B. CHANCE, Energy-linked Functions ofMitochondria, Academic Press, New York, 1963, p. 219.

II F. D. VASINGTON AND ]. V. MURPHY, J. Bioi. Chem., 237 (1962) 2670.12 C. S. ROSSI AND A. L. LEHNINGER, J. Bioi. cu-«, 239 (1964) 397I.13 G. P. BRIERLEY, E. MURER AND R. L. O'BRIEN, Biochim, Biophys. Acta, 88 (1964) 645.

Received October roth, 1964

Biochim. Biopbys. Acta, 99 (1965) 191-194

PN 6ro59The possible role of ascorbic acid in the a.-hydroxyacid decarboxylase of brainmicrosomes

Microsomes obtained from rat brain have been shown1,2 to contain an oxidativedecarboxylase system which acts on a-hydroxystearic and a-hydroxytricosanoic acid,according to the scheme:

I IIRCH2CHOHCOOH ---7 RCH2COCOOH -;.. RCH2COOH

-C02

The system shows a requirement of the dialysate of the non-particulatefraction of rat brain homogenate for NAD+ and ATP. Omission ofthe latter cofactorresulted ina lower depression of the overall reaction than did omission of NAD+.The a-keto-fatty acid is a likely intermediate and is probably bound to the enzyme.It was then postulated that these findings, together with results from studies in vivoSlend support to an a-oxidation system of degradation for the 20 to 26 carbon fattyacids of the sphingolipids.

Recently, we have undertaken a series of studies in order to isolate the activeconstituent of the dialysate. During these studies the sucseptibility of this factor,or factors, to oxidizing agents became apparent. The possibility that the unknownfactor was a reducing compound or that a component of the system could be activatedby such a compound, was therefore tested. It can be seen from the results in Table Ithat the two sulfhydryl compounds may partially replace the dialysate in the de­carboxylation of a-hydroxystearate and that only ascorbate can replace it com­pletely. Reduced glutathione and (oASH, as we previously reported-, were in­active. The same was found for cystine and dehydroascorbic acid (prepared by theoxidation of ascorbic acid by bromine).

The possibility that an intermediate oxidation product of ascorbic acid4- 6 canactivate an NADH oxidase system, coupled with the dehydrogenation step (ReactionI), was then tested by incubating microsomes with NADH. Under the experimental

Biochim. Biophys. Acta, 99 (19 65) I94-I97