Communication

Reversibility of the Mitochondrial Isocitrate Dehydrogenase Reaction in the Perfused Rat Liver

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 269, No. 44, Issue of November 4, pp. 27179-27182, 1994 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

kinetic properties of the NAD"1CDH are consistent with its operation in vivo in the direction of aKG formation(1, 4). NAD+-ICDH is regulated by a variety of positive (Ca2+, ADP, citrate) and negative (ATP, NADH, NADPH) effectors (5). In contrast, NADP+-ICDH has no known allosteric effector a n d could operatein the directionof ICIT formation; its afflnity for NADPH is 100-fold greater than for NADP+(61, and its K,,, for CO, (1.6 m ~ i s)in the range of the physiological concentration EVIDENCE FROM ISOTOPOMER ANALYSIS OF (1.5 m, Ref. 7). CITRIC ACID CYCLE INTERMEDIATES* There is a consensus view that NAD+-ICDH catalyzes the (Received for publication, August 16, 1994, and in revised form, aKG (4,8,9).However, the function of the conversion of ICIT to September 12, 1994) mitochondrial NADP+-ICDH is uncertain (4, 8-11). Very reChristine Des Rosiersl, Charles A. FernandezS, cently, Sazanov and Jackson (12) proposed that, in the mitoFrance David% and Henri Brunengraberll(1 chondrial matrix,a substrate cycle operates betweenICIT and From the $Department of Nutrition, University of aKG, where NAD+-ICDH generates aKG and NADP+-ICDH Montreal, Montreal, Quebec H3C 357, Canada and the regenerates ICIT. The NADPH used in the reverse reaction Departments of Wutrition andBiomedical would be supplied by the H+h-anshydrogenase driven bythe Engineering, Case Western Reserve University, proton electrochemical gradient. The net balance of the ICIT z Cleveland, Ohio 44106 aKG cycle would be the dissipation of the gradient. The ICIT 2 The reversal of the mitochondrial isocitrate dehydro- aKG cycle provides a mechanism by which the flux throughthe genase reaction was investigated in rat livers perfused CAC is (i) more tightly controlled by the modifiers of NAD+with [U-lsC,]glutamate or [U-'sC,lglutamine. The mass ICDH and (ii) directly controlled by the energy state of the isotopomer distribution of citric acid cycle intermedi- inner mitochondrial membrane. This hypothesis is supported ates extracted from the livers was determined by gas by studies conducted in isolated liver mitochondria. However, chromatography-mass spectrometry. Citrate was enthe operation of an ICIT s aKG cycle has not been demon"C. The formariched in an isotopomer containing five strated in intact liver. tion of this isotopomer can only be explained by the reversal of the isocitrate dehydrogenase reaction. Cal- In the course of investigating (13) gluconeogenesis in isolated rat livers perfused with [U-13C,llactate a n d [U-13C,]pyruvate, culation of kineticparameters from themassisotomass isotopomer analysisof CAC intermediates suggestedthe pomer data reveals a rapid interconversion of isocitrate reversal of the mitochondrial ICDH reaction in the intact liver. and a-ketoglutarate. This interconversion results in an 6 of citrate and mito- Briefly, t h e data were compatible with an isotopic exchange isotopic exchange between carbon between C-6of citrate and CO,. If that is the case, we reasoned chondrial CO, that can affect the calculation of citric perfused with [U-13C,]glutamate or acid cycle kinetic parameters. Thus, the reversal of the that, in rat livers [U-'3C,]glutamine, reversal of the ICDH reaction would result isocitrate dehydrogenase reaction should be included in in the formationof M+5 citrate. The present study was underisotope labeling models of the citric acid cycle. taken to testthis hypothesis.

EXPERIMENTALPROCEDURES Isocitrate dehydrogenase (ICDH)' catalyzes the conversion of Materials-Chemicalswere obtained fromSigma-Aldrich and enthreo-q-isocitrate (ICIT) to a-ketoglutarate (aKG)as shown by zymesfrom Boehringer Mannheim. [U-13C,lGlutamateand [U-13C,]Reaction 1. glutamine (98%)were obtained from Cambridge Isotopes. The derivatwas ization agent N-methyl-N-(t-butyldimethylsilyl~trifluoroacetamide threo-n,-Isocitrate + NAD(P)+u aKG + HCO; + NAD(P)H +H+ obtained from Regis Chemical Co. (Morton Grove, IL). Perfusion Experiments-Livers from 24-h fasted male SpragueREACTION 1 Dawley rats (Charles River Laboratories) were perfused (14) with nonLiver contains three ICDH: one cytosolic and two mitochon- recirculating Krebs-Ringer bicarbonate buffer containing 4 m~ glucose, M lactate, 0.2 m~ pyruvate, 0.2 m M acetate, 0.2 m M octanoate, and drial. The cytosolic ICDH uses NADP+ and generates NADPH 1 m M glutamine. After a 10-min equilibraLiver mitochondria either 0.5 m~ glutamate or 0.5 m for fatty acidand cholesterol syntheses(1). contain a NAD+-ICDH a n d a NADP+-ICDH (2), which are part tion, glutamate or glutamine was replaced by [U-'3C,lglutamate or [U-'3C,lglutamine and the experiment continued for20 minbefore of the citric acid cycle (CAC). In rat liver mitochondria, the freeze-clampingof the liver. NAD"1CDH is associated with otherCAC enzymes: fumarase, Analytical Procedures-'ho-gram samples of frozen livers were exand aconitase (3). The tracted with 7 ml of 8% sulfosalicylic acid and 1 ml of 5 M hydroxylamalate dehydrogenase, citrate synthase, mine-HC1. After centrifugation, the extract was brought to pH 8 with * This work was supported by Grant MA-9575 (to C. D. R.) from the KOH and incubated for 60min at 65 "C to convert ketoacids to hydroxMedical Research Council of Canada, Grant DK35543 (to H. B.) from amates. The solution was acidified topH 1-2 with HCI, saturated with the National Institutes of Health, and a grant from the Nutrition De- NaC1, and extracted three times for 10 min with 12 ml of ethyl acetate. velopment Fund of the Cleveland Mt. Sinai Medical Center. The costs of The pooled extract was evaporated and theresidue reacted with 50 pl of at 60 "C for 1h, to publication of this article were defrayed in partby the payment of page N-methyl-N-~t-butyldimethylsilyl~trifluoroacetamide charges. This article must therefore be hereby marked "advertisement" convert analytes to tert-butyldimethylsilyl derivatives. One or two pl were injected into a Hewlett Packard MS engine (HP 5890 gas chroin accordance with 18 U.S.C. Section 1734 solely to indicate this fact. matograph and HP 5989 mass spectrometer) equipped with a HP-5 11 To whom correspondence should be addressed. The abbreviations used are: ICDH, isocitrate dehydrogenase; aKG, capillary column (25 or 50 m x 0.2 mm, inner diameter, 0.33 pm film a-ketoglutarate; CAC, citric acid cycle; ICIT, threo-ne-isocitrate;O M , thickness). Split ratio was 20/1 for lactate and succinate (splitless for all oxaloacetate. other compounds).Carrier gas was helium (1ml/min), and column head

27179

27180

Reversibility of Isocitrate Dehydrogenase in Liver

TABLE I Isotopomer distribution of metabolites labeled from W-I3C Jglutamate Livers from 24-h-starved rats were perfused with non-recirculating buffer containing 4 m~ glucose, 1 m~ lactate, 0.2 m~ pyruvate, 0.2 m~ acetate, 0.2 m M octanoate, and 0 . 5m M glutamate. After 10 min of equilibration, glutamate was replaced by [U-'3C]glutamate and the experiment continued for 20 min. The isotopomer distribution of metabolites isolated from the freeze-clampedlivers has been corrected for natural abundance of heavy isotopes (16, 17). The molar fraction of each isotopomeris expressed as a percentage (mean f S.D.). Influent glutamate was 94%M+5. Numbers printed in bold are used in the computations presented under"Discussion."
M
M+l

M+2

M+3

M+4

M+5

M+6

Citrate aKG Succinate Fumarate Malate Lactate Pyruvate

82.9 * 0.9 2.4 71.3 f 0.4 1.8 81.1 * 1.4 94.5 f 1.0 93.5 f 0.6 99.1 f 0.2 97.9 * 0.5

* 0.6
f 0.2 0.8 f 0.1 0.1 f 0.9 0.0 f 0.2 0.2 f 0.2

0.2 0.1 0.6 * 0.1

1.9 * 0.4 1.5 * 0.1 1.3 2 0.1 0.8 f 0.9 1.3 * 0.1 0.4 0.1

2 . 1*0 . 3 1 . 1
0.3 * 0.03 1 . 3 0.5

2.2 f 0.3 1.4 f 0.1 1 . 0 f 0.04 1.8 * 0.7

2 . 1 f 0.4 1 0 2 . 4 1 5 1 . 0 7 1 5 . 1 8 . 2 2 . 8 f 0.5 f 0 . 3

1 0 . 1 8 . 4

-0.8

0.4

pressurewas 200 kilopascals. Threecolumntemperatureprograms were used. For lactate, pyruvate, succinate, aKG, and malate, the program was: 50-m column,100 "Cfor 1 min, increase 5 Wmin until 205 "C, 35 "C/min until 275 "C, 20 min at 275 "C. For citrate, the programwas: 25-m column,100 "Cfor 4 min, increase 5 "C/min until 275 "C, 5 min at 275 "C. Forfumarate, the program was: 2 min at 80 "C, increase by 5 Wmin until 185 "C, 25min at 185 "C, increase 35 Wmin until 300 "C, 5 min at 300 "C. Very long chromatographic runs were necessary to avoid interferences at all m / z monitored. Ions monitored were m / z 261-264 (lactate), 274-277 (pyruvate), 289-293 (succinate), 287-291 (fumarate), 419423 (malate), 446-451 (aKG), and 459465 (citrate). The enrichments of influent [U-'3C5]glutamate and [U-'3C51glutamine were measured as described in Ref. 15. Areas under each fragmentogramweredetermined bycomputer integration and corrected for naturally occurring heavy isotopes (16, 17). RESULTS AND DISCUSSION

Table shows I data from four liver perfusions with [U-'3C,]glutamate. The mass isotopomer distributions of liver CAC intermediates, lactate, and pyruvate have been corrected for naturally occurring heavy isotopes (16, 17). Influent [U-13C,]glutamate was 94% enriched in M+5 isotopomer. The corresponding M+5 enrichment of aKG was 24%. This 4-fold dilution reflectsthe influx of unlabeled carbon in theCAC. The to isotovery low enrichments of aKG and citrate in M+l M+4 pomers show that unlabeled carbon enters the CAC both as acetyl-coA and oxaloacetate (OAA). In livers perfused under similar conditions with [3-'3C11actate + [3-'3C]pyruvate (14, E ) , we showed that very little acetyl-coA is formed from pyruvate. Most of the acetyl-coA derives from octanoate. The striking finding in this study is that citrate is 10.8% enriched in M+5 isotopomer. The M+5 enrichment of citrate amounts to 10.8/24, i.e. 45% of the M+5 enrichment of aKG. One perfusion was conducted with [U-'3C,lglutamine. The distribution of the heaviest isotopomers of the compounds assayed was: M+5 glutamine (94%), M+5 citrate (4.5%), M+5 aKG (13.6%), M+4 succinate (18.2%), M+4 fumarate (4.0%), M+4 malate (3.6%), M+3 lactate (0.3%), and M+3 pyruvate (0.7%). There are only two pathways by which M+5 glutamate or M+5 glutamine can yield M+5 citrate. The first involves M+5 glutamate + M+5 aKG M+4 succinate + M+4 OAA + M+3 pyruvate + M+2 acetyl-coA. Some of the M+4 OAA could become M+3 in the pyruvate cycle: M+4 OAA + M+3 phosphoenolpyruvate 4 M+3 pyruvate + M+3 OAA. Then, M+3 OAA could combine with M+2 acetyl-coA to form M+5 citrate. However, one would expect to find even more M+6 citrate resulting from the condensation of M+4 OAA with M+2 acetylCoA. No M+6 citrate wasfound. This firstmechanism can thus be excluded because of (i) absence of M+6 citrate, (ii)very low enrichments in M+4 malate and M+3 pyruvate, and (iii) very low enrichments in M+l t o M+4 isotopomers of citrate and aKG.
-j

The second and only mechanism that canexplain the formation of M+5 citratefrom M+5 aKG is the reversalof reactions catalyzed by aconitase and ICDH. Aconitase is fully reversible (18). However, the sequence of the ICDH and aKG dehydrogenase reactions is considered irreversible in intact cells, given the cyclical and continuous nature of the CAC. This would not be the case when the mitochondrial [NADH]/[NAD+l ratio is elevated, as is the case during ethanol oxidation in the liver. Then, the flux through the CAC can be decreased to almost zero (19). However, under our conditions, livers were perfused with medium containing a redox buffer made upof 1m M lactate and 0.2 m M pyruvate. This [lactate]/[pyruvate] ratio of 5, which is at the oxidized end of the physiological range, imposed a similarly oxidized [NADHl/[NAD+] ratio in the liver cytosol. In other perfusions with similar substrates, we measured a [R-phydroxybutyratel/[acetoacetatel ratio of 1.1 (14, 151, which is also fairly oxidized. Since the [R-@-hydroxybutyrate]/ [acetoacetate] ratio reflects the mitochondrial [NADH]/[NAD+l ratio, we can exclude a n inhibition of aKG dehydrogenase and NAD+-ICDHby a redox shift. In the metabolic sequence aKG + succinate + fumarate + malate, most of the dilution in the enrichment in fully labeled isotopomers (M+5 for citrate and aKG, M+4 for the others) occurs at the level of fumarate, about 5-fold. The strong dilution of fumarate is caused by the influx of unlabeled OAA derived from pyruvate. OAA interconverts with malate and fumarate through the very active and reversible malate dehydrogenase and fumarase reactions. The identical M+4 enrichments of fumarate and malate show that the reversible fumarase reaction is sufficiently rapid to achieve isotopic equilibrium. The enrichment of M+4 succinate (16%) is lower than that of M+5 aKG but higher than that of M+4 malate (2.8%). This confirms that the succinate dehydrogenase is reversible (20),although it does not reach isotopic equilibrium. The mass isotopomer distributions of the various metabolites in Table I allow calculation of certain relative input fluxes. Since M+5 aKG is the only source of M+5 citrate, the balance of M+5 mass isotopomers of citrate and adjacent metabolites yields the formula shownby Equation 1, where FC,,,, is the fractional contribution of aKG to citratevia the reversalof the ICDH reaction.
(FC,K~,,,)'(MF,+,.K~)

= (MFM+6CIT)

(Eq. 1)

MF,,, and MFM+5 CIT are themole fractions of M+5 aKG and M+5 citrate, respectively. Using the mole fractions of Table I, one calculates a 45% fractional contribution of aKG t o citrate, via the reversal of ICDH; the remaining 55% of citrate molecules come from OAA. These percentages can be introduced in the balance of M+4 mass isotopomers of citrate and adjacent metabolites, using similar notations (Equation2).

Reversibility of Isocitrate Dehydrogenase in Liver

0.55.(MFM+40,) + O . ~ ~ . ( M F M + ~ , , K G )
=
(MFM+~c,T)

27181

(Eq. 2)

PYR

Solving this equationyields the mole fraction of mitochondrial M+4 OAA, i.e. 2.6%. This enrichmentis comparable to whatwe measured for M+4 malate (3.1%, Table I) and fumarate(2.8%), suggesting that the latter enrichments are representative of mitochondrial metabolites. One can thus consider mitochondrial OAA, malate, and fumarate as a single pool whose average M+4 enrichment is 2.8%, for which M+4 succinate is the only source of M+4 isotopomers. The M+4 isotopomer balance around mitochondrial OAA, malate (MAL), and fumarate (FUM) yields Equation 3. (FC~UC-F""ALI~AA).(MFM+~SUC) =M (F M + F ,L J ~ ~ ~ O IA A )
=

7.5

0.028

(Eq. 3)

From this equation, the fractional contribution of mitochondrial succinate to the poolof fumarate, malate, and OAA is 18%. Consequently, the fractional contribution of pyruvate to this pool is 82%. The M+4 isotopomer balance around succinate (SUC) yields Equation 4.
(FCaKG+SUC)'(MFM+5 aKG) ~ ~ O

FIG.1. Relative rates of citric acid cycle reactions. Rates of various CAC reactions, including the reversal of the combined aconitase + ICDH reactions, were calculated from the data of Table I using the equations developed under "Results and Discussion." Rates are expressed relative to the net flux through the CAC, i.e. the rate of the aKG dehydrogenase reaction. Abbreviations: CIT, citrate; SUC, succinate; GLU, glutamate; PYR, pyruvate; OAA, oxaloacetate; MAL, malate; FUM, fumarate.

or [l-13C]pyruvate, the specific activity or enrichment of C-6 of citrate will be decreased. In contrast, labeled bicarbonate will be incorporated into C-6 of citrate and into other metabolites From thisequation,thefractional contribution of aKG to following [6-14C]- or [6-l3C]citrate cleavage to [1-l4C1- or succinate is 61%, Consequently the fractional contribution of [1-13C]OAA.Such a scheme was proposed by Heath and Rose mitochondrial fumarate t o succinate is 39%. The flux ratio (25) in their study of Hl4CO; fixation in liver metabolites, by (pyruvate (PYR) carboxylase)/(CAC) equals Equation 5, where DAdamo and Haft (26) t o explain the labeling of hepatic lipids the CAC flux is equal to the irreversible flux from aKG to and glucose from [2-14C]glutamateand [5-14C]glutamateand by succinate. Kellehe? to account for the incorporation of [5-14Clglutamine into lipids and CO, by rat hepatoma cells. PC/CAC = (FCpm-FwO,Y (Eq. 5) Kelleher (27) developed equations to determine rate con[FC,.KG+S,C)'(FCSUC-FU"AUOAA)I = 7.5 stants of various CAC reactions from the distributionof 14C on Such a high ratio has also reported previously by others (21-24) carbons of citrate labeled from [14C]acetate,-pyruvate, or -succinate. This model, which includes reversible reactions between and by us (15). and aKG, could be used with the corresponding Since the measured M+5 molar enrichment of influent glu- citrate tamate is 94%, a M+5 mass isotopomer balance around aKG ['3C]substrates. However, this would require a technique for measuring the 13C enrichment of each carbon of citrate. Beyields Equation 6. cause of the chemical symmetry of its molecule, citrate must (FCGLU-aKG)'(MFM+5 GLU) + (FCCIT-mKG)' 0%. 6) first be cleaved into its acetyl-coA and OAA moieties before measuring the labeling pattern of these moieties by NMR of gas (MFM+5 CIT) = (MFM+5 aKG) chromatography-mass spectrometry. An alternative approach Thus, the fractional contributions of citrate and glutamate to is t o use uniformly labeled 13C-substrates and to apply mass aKG are 84 and 16%, respectively. isotopomer analysis to the calculation of CAC and gluconeogenLiver pyruvate contains1.3% of M+3 isotopomer, which must esis parameters (28, 29). However, a reversible ICDH reaction arise from the action of pyruvate kinase on M+3 phosphoenol- would also affect the profiles of mass and positional isotopyruvate formed from [U-'3C,10AA and [1,2,3-'3C,10AA. As- pomers of citrate, OAA, phosphoenolpyruvate, and glucose. suming that the M+4 and M+3 enrichments of malate repre- Therefore, incompatible parameters could be calculated from sent that of the O M precursor of phosphoenolpyruvate, the the isotopomer profiles of these metabolites. To avoid such M+3 enrichment of phosphoenolpyruvate (PEP) is shown by problems, models of the CAC based on isotopomer analysis Equation 7. should include the reversal of the ICDH reaction. Models of the CAC that incorporate a reversible ICDH reac(MF,,, = (MF,,, -) + 0.5.(MFM+, -) = 4.2% (Eq. 7) tion would help to test some of the assumptionsof the Sazanov In this equation, the factor 0.5 reflects the assumptionof com- and Jacksonhypothesis (12)on the functions of the NAD'- and plete randomization of OAA. The calculated M+3enrichment of NADP+-ICDH in liver mitochondria. Our demonstration of a phosphoenolpyruvate is 3 times higher than the measured M+3 rapid interconversion of ICIT and a-ketoglutarateis consistent enrichment of tissue pyruvate. This is due to the continuous with thishypothesis. However, under ourconditions, mass isoinflux of unlabeled lactateandpyruvateinthe inflowing topomer distribution analysis does not differentiate substrate perfusate. cycling between ICIT and aKG from the reversibility of the The above calculations, summarized in Fig. 1,illustrate the ICDH reaction(s). power of mass isotopomer distribution analysis. Such computations could not have been achieved with radioactive tracers or REFERENCES with singly labeled 13C-substrates. 1. Dalziel, K. (1975) in The Enzvmes (Boyer, P. D., ed) Val. 13, .. pp. 140,Academic Press, New York One importantconsequence of the reversibility of the ICDH 2. Plaut, G . W. E., and Aogaichi, T.(1967) Biochem. Biophys. Res. Commun. 28, reaction in the intactliver is its impact on the labeling pattern 628434 3. Robinson. J. B., Jr, and Srere, P.A. (1985) J. Biol. Chem. 260, 10800-10805 of citrate. The reversal of the ICDH reaction causes anisotopic exchange between C-6 of citrate and mitochondrial CO,. Consequently, in experiments with tracers such [l-l4C1pyruvate as J. K. Kelleher, personal communication.
(FcFUh-SUC)' (MFM+4 F A

(Eq. 4)

= A ( )M F M + SUC) ~ = 0.16

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Reversibility of Isocitrate Dehydrogenase in Liver

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