Modulation of branched-chain α-keto acid decarboxylase activity in rat liver mitochondria by hypophysectomy

ARCHIVES

OF

BIOCHEMISTRY

Modulation STEPHEN Division

ofHuman

AND

BIOPHYSICS

176, 225-234 (1976)

of Branched-Chain a-Keto Acid Decarboxylase Rat Liver Mitochondria by Hypophysectomy’ GENE

SULLIVAN,

JOSEPH

DANCIS,

AND

Activity

in

RODY P. COX

Genetics, Departments of Medicine, Pharmacology, and Pediatrics, New York University Medical Center, 550 First Avenue, New York, New York 10016 Received

March

5, 1976

A reliable and reproducible assay was developed for measuring mitochondrial cr-keto acid decarboxylase activity using ferricyanide as the electron acceptor. This method permitted the functional isolation and investigation of the decarboxylase step of the branched-chain a-keto acid dehydrogenases in rat liver mitochondria. Pyruvate and (Yketoglutarate decarboxylases are known to be separate and distinct enzymes from the branched-chain cY-keto acid decarboxylases and were studied as controls. The relative specific activities of rat liver mitochondrial decarboxylases as measured by the ferricyanide assay showed that pyruvate and o-ketoglutarate were decarboxylated twice as rapidly as cY-ketoisovalerate and four to ten times as fast as cu-keto-P-methylvalerate and cu-ketoisocaproate. The three branched-chain cr-keto acids individually inhibit pyruvate and o-ketoglutarate decarboxylases. Inactivation of mitochondrial branched-chain cu-keto acid decarboxylase activity by freezing and thawing and by prolonged storage resulted in a proportional decrease in decarboxylase activity toward each of the three branched-chain cu-keto acids. However, hypophysectomy was found to increase decarboxylase activity with oc-keto-p-methylvalerate to four times normal and with o-ketoisovalerate to three times normal, but the activity with cY-ketoisocaproate was not changed. Hypophysectomy did not alter mitochondrial decarboxylase activity with pyruvate, (Yketoglutarate, or a-ketovalerate. The finding that hypophysectomy differentially alters the mitochondrial decarboxylase activity with the three branched-chain a-keto acids suggests either that there is more than one substrate-specific enzyme with branchedchain cu-keto acid decarboxylase activity or that there is a modification of one enzyme such that the catalytic activity is selectively altered toward the three substrates.

The three branched-chain cr-keto acids, a-ketoisovaleric acid (KIV),2 cY-keto-pmethylvaleric acid (KMV), and a-ketoisocaproic acid (KIC), are oxidatively decarboxvlated in mammals bv mitochondrial enzymes which catalyze” the following overall reaction:

0 11 R-C-COOH

’ Supported by research grants, AM 14528 and HD 04526, from the National Institutes of Health and from the Samuel A. Berger Foundation. Stephen Gene Sullivan is a Medical Scientist Trainee, Grant No. 5 TO5 GM01668. * The cu-keto acid substrates will be abbreviated as follows: PYR, pyruvate; KG, cr-ketoglutarate; KIV, a-ketoisovalerate; KMV, a-keto-P-methylvalerate; KV, cy-ketovalerate; KIC, cY-ketoisocaproic acid. Other abbreviations used: TPP, thiamine pyrophosphate; LIP, lipoic acid; FAD, flavin-adenine dinucleotide.

The enzyme is labile and attempts at isolation of submitochondrial preparations have been unsatisfactory. The structure of the branched-chain a-keto acid dehydrogenases is unknown but the mechanism of catalysis is assumed to be similar to the closely related enzymes, pyruvate (PYR) dehydrogenase (EC 1.2.4.1) and cr-ketoglutarate (KG) dehydrogenase (EC 1.2.4.2). The latter two enzymes have three different polypeptide moieties: a decarboxylase

? + R-A-S-CoA

225 Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved

+ NAD+ f CoA-SH

+ NADH

+ H+ + CO,.

226

SULLIVAN,

DANCIS

(ENZ A), a transacylase (ENZ B), and a dehydrogenase (ENZ C). The mechanism of PYR and KG dehydrogenases has been shown to consist of the following reactions (1, 2): 0

II

R-C-COOH

+ EN2 A-TPP-H

[ll

OH I -+ ENZ A-TPP-C-R

+ CO,;

I!I OH I ENZ A-TPP-C-R

S

+ ENZ B-LIP-

II

k

$

S-C-R; --f ENZ A-TPP-H

+ ENZ B-LIPSH

ENZ B-LIP-

S-i-,

+ CoA-SH

I

SH 0 II + CoA-S-C-R;

-SH --j ENZ B-LIPISH SH ENZ B-LIP-

+ ENZ C-FAD SH [41

-+ ENZ B-LIP-

ENZ C-FADH,

I

S 1 + ENZ C-FADH,, S

+ NAD+

151

+ ENZ C-FAD

+ NADH

+ H+.

The activity of the entire complex can be measured by collecting liberated CO, or following the reduction of NAD+. Alternatively, the decarboxylase activity can be functionally isolated from the transacylase and dehydrogenase activities by adding ferricyanide to the reaction mixture (3). The ferricyanide releases the enzyme bound product at the decarboxylase step and replaces the later steps by oxidizing the product to the simple acid (4): R-CHOH-TPP-ENZ 4 R-COOH

+ 2Fe(CN)t-

+ H,O + 2Fe(CN)3,+ 2H+ + H-TPP-ENZ.

Most studies on branched-chain

cy-keto

AND

COX

acid metabolism have measured the overall dehydrogenase reaction (5-7). The ferricyanide assay as described by Gubler has been used to study the branchedchain cu-keto acid decarboxylases3 (8) but the assay has not been carefully characterized. Using purified rat liver mitochondria we have studied the requirements and the kinetics of the reaction and have modified the assay appropriately. The branched-chain cr-keto acid decarboxylase activity with KIV, KMV, and KIC has been compared to the activity of the well-characterized PYR and KG decarboxylases. Inhibition of PYR and KG decarboxylases by the branched-chain cw-keto acids has been studied using the ferricyanide assay and the results compared to published data on the inhibition of the overall reaction (7, 9, 10). It is not known whether there are one, two, or three enzymes responsible for decarboxylase activity with KIV, KMV, and KIC. Since the enzymes cannot yet be isolated, a search was made for methods of uncovering functional independence of the three decarboxylase activities. Mixed substrate experiments yielded equivocal results. Simple inactivation experiments showed no significant differential change in relative activity with the three substrates. Unequivocal differential effects on the activity of branched-chain a-keto acid decarboxylases were provided by altering the hormonal milieu of rats by hypophysectomy. MATERIALS

AND

METHODS

Chemicals. The sodium salts of pyruvic acid (PYR), a-ketoglutaric acid (KG), cy-ketoisovaleric acid (KIV), m-a-keto-p-methylvaleric acid (DLKMV), cY-ketoisocaproic acid (KIC), and cy-ketovaleric acid (KV) were obtained from Sigma. Preparation of rat liver mitochondria. Mitochondria were prepared from livers of adult SpragueDawley rats by the method of Hogeboom (11). The final washed mitochondrial pellet was suspended in a small volume of 0.25 M sucrose to give a concentration of 14 to 36 mg of mitochondrial protein per ml. The protein concentration was determined by the ’ For clarity, the term rw-keto acid dehydrogenase is used to refer to the enzyme complex catalyzing the overall reaction. The term cY-keto acid decarboxylase is used to refer to the first step of the overall reaction isolated by ferricyanide.

MITOCHONDRIAL method of Lowry et al. (12) after digesting the mitochondria with 4 N NaOH. Mitochondrial suspensions were frozen and stored in aliquots at -70°C until used. These preparations remained stable on storage for 4 months at -70°C and exhibited kinetics similar to freshly prepared mitochondria assayed before freezing. Assay of wketo acid decarboxylases. cY-Keto acid decarboxylase activity was measured by a modification of the method of Gubler in which reduction of ferricyanide is followed spectrophotometrically. Mitochondrial suspensions scatter light, making the reading of spectroscopic absorbance inaccurate by the usual methods. This problem can be avoided by turning the cuvettes so that the light beam traverses the frosted glass side of the cuvettes, and a scattered light source is used for the measurements (8). Testing cuvettes with various concentrations of ferricyanide in the presence of mitochondria verified the validity of the method. The standard reaction mixture contained 20 mM sodium phosphate (pH 7.4), 2.7 mM K,Fe(CNjG, 5 mM MgC&, 0.54 mM Na,-EDTA, one or more cy-keto acids at saturating concentration (5 mM KIV, 10 mM DL-KMV, 5 mM KIC, 5 m&i PYR, 1.25 mM KG, or 5 mM KV), 0.30 to 0.50 mg of mitochondrial protein, and 0.25 M sucrose to make a final volume of 370 ~1. The reaction was started by the addition of substrate to the cuvette and studies were carried out at room temperature. Fresh solutions of a-keto acids and K,Fe(CN), were prepared daily. Optical density at 420 nm was measured sequentially in three or four cuvettes (2-mm light path) with a Beckman DU spectrophotometer with a Gilford 2000 attachment. Five separate readings on each cuvette were recorded every 2 min. The assays were routinely carried out for 5 min. However, the reaction was linear for at least 20 min or until ferricyanide became limiting. The first cuvette served as a control containing all the reactants except substrate. It therefore measured the endogenous mitochondrial reduction of Fe(CN):and controlled for mechanical or electronic variation in the recording device. The endogenous reaction was subtracted from the experimental results. Rates are expressed in units of AOD x 109minute where 1.0 AOD x lo3 (a change of 0.001 optical density unit) represents 2 nmol of Fe(CN):reduced. Hypophysectomy. Six white male Sprague-Dawley rats weighing 150 g were studied. Three were hypophysectomized and three were sham hypophysectomized. Postoperatively, rats were fed normal rat chow and water ad libitum. Two weeks after surgery four rats (two sham operated and two hypophysectomized) were sacrificed and liver mitochondria were prepared. One week later the two remaining animals were sacrificed and mitochondria were prepared. At the time of sacrifice the sham-hypophysectomized rats weighed 300 to 330 g,

227

DECARBOXYLASES

and the hypophysectomized rats weighed 150 to 160 g. The animals were sacrificed between 7:00 and 10:00 AM. Rats were obtained from and surgery was performed by Charles River Breeding Laboratories, Inc., North Wilmington, Mass. RESULTS

Characterization Assay

of the Decarboxylase

Gubler’s assay for mitochondrial decarboxylase activity contained phosphate buffer, mitochondria, a-keto acid, and ferricyanide. To this solution Gubler also added ATP, MgCl*, and EDTA. It appeared advisable to investigate the effects of these latter components singly and in combination on the kinetics of the rehction. When all three components (ATP, MgC&, and EDTA) were omitted from the assay there was no detectable decarboxylase activity. Either MgCl, or EDTA alone supported the decarboxylase activity of mitochondria whereas with ATP alone there was ho activity. Whenever ATP was included in the assay mixture with EDTA and MgC12, decarboxylase activity with PYR was essentially linear but KG had an initial lag phase lasting 3 to 4 min and then reached linearity, and KIC showed a markedly enhanced initial rate (burst) of activity followed by a linear rate with ATP also caused a high lower activity. endogenous rate of ferricyanide reduction in the absence of a-keto acid substrate (Table I, Experiments 1, 2, 3, and 5). Furthermore, ATP was found to be an inhibitor of the branched-chain a-keto acid decarboxylases as previously reported (6). For those reasons ATP was omitted from our assay. The presence of EDTA and MgCl, in the reaction mixture gave the lowest endogenous activity (Table I, Experiment 41,that is, ferricyanide reduction in the absence of the cu-ketoacid substrate, and the reaction with a-keto acid substrates was linear. Therefore, reaction mixtures used for all subsequent studies contained EDTA and MgCl, without added ATP. With this system the endogenous or background reaction was less than 20% of that observed with the a-keto acid substrates, except for KIC, where the endogenous reaction occasionally reached 50% of the total activity. In all cases the endoge-

228

SULLIVAN,

DANCIS

nous reaction was subtracted from that observed in the presence of substrate. The branched-chain cr-keto acid decarboxylase activity was compared to the activity of PYR and KG decarboxylases. Figure 1 shows representative experiments comparing the decarboxylase activity of rat mitochondria for each of five substrates (PYR, KG, KIV, KMV, and KIC) at saturating concentrations. The reaction rate was linear with time except for pyruvate decarboxylase which is labile (13) and showed a variable decrease in activity within 2 to 6 min. Therefore, the rate of reaction with PYR was determined as an initial velocity. The rates calculated from Fig. 1 and expressed as AOD x 103/minute are: PYR, 20.6; KG, 11.6; KIV, 7.9; DLKMV, 2.8; and KIC, 1.9. The endogenous reaction in each case was less than 0.6. The reaction was linear with respect to mitochondrial concentration for all five substrates (Fig. 2). Reciprocal plots of substrate concentration versus velocity were also linear (Fig. 3). The activity of KIC decarboxylase was too low with substrate concentrations below 5 mM to obtain reliable data. No substrate inhibition was present up to concentrations of 10 mM PYR, 2.5 mM KG, 10 mM KIV, 20 mM DL-KMV and 10 mM KIC. The K,‘s calculated from the plots in Fig. 3 are PYR, 0.036 mM; KG, 0.11 mM; KIV, 0.071 mM; DL-Kh!V, 0.060 mM. It was previously reported (5, 7) that TABLE

I

Additions 1.6 mM ATP + + + -

+ -

5rnM M&L + + +

+ -

COX

0

0.54 rnM EDTA + + +

+

ON BY RAT

Endogenous activity” 20.0 17.2 11.7 1.7 22.3 4.4 4.4

n Ferricyanide reduction was measured as described in Materials and Methods except that a-keto acid substrates were omitted from the reaction mixture. Activity is expressed as AOD x lo3 per minute per milligram of mitochondrial protein.

4 8 TIME (MINI

FIG. 1. a-Keto acid decarboxylase activity with substrate at saturating concentrations. Each curve represents a separate experiment. In each experiment 0.36 mg of mitochondrial protein was used. Conditions were described in Materials and Methods. 5 mM PYR (01; 1.25 mM KG (Al; 5 mM KIV (01; 10 mM DL-KMV (A); 5 mM KIC (01.

0

EFFECT OF ATP, MgCl, AND EDTA ENDOCENOUS FERRICYANIDE REDUCTION LIVER MITOCHONDRIA

Experiment

AND

.2

.4

.6

mg OF MITOCHONLXIALPROTEIN FIG. 2. Effect of concentration of mitochondria on a-keto acid decarboxylase activity. 5 mM PYR (0); 1.25 mM KG (A); 5 mM KIV (0); 10 mM DL-KMV (A,;5 mMKIC (0).

KIC at concentrations above 2 mM caused inhibition of the overall reaction. With the ferricyanide assay inhibition was not observed at 10 mM concentrations of KIC, suggesting that the site of KIC inhibition must be later than the decarboxylase step. The ferricyanide assay is based on the assumption that the overall reaction is intercepted after the decarboxylase step before acylation of lipoic acid. Therefore, as expected, the addition of the cofactors 5

MITOCHONDRIAL

mM NAD+ and 5 mM CoA to the reaction mixture singly and in combination had no effect on ferricyanide reduction by KIV. However, CoA increased the endogenous reaction 5-lo-fold making measurements of the specific reduction of ferricyanide with a-keto acid difficult. NAD+ caused no change in the endogenous reaction. Arsenite is thought to inhibit a-keto acid dehydrogenases by binding to dithiols generated by the transacylase and dehydrogenase steps of the reaction. The overall reaction with KG is nearly completely inhibited by 1O-4M arsenite, whereas the reaction with ferricyanide is only slightly inhibited (20-40%) at higher concentrations (14). In our assay ferricyanide reduction with KIV was inhibited by only 25% with 10e3 M arsenite, supporting the assumption that ferricyanide intercepts the overall reaction at the decarboxylase step. Inhibition of PYR and KG Decarboxylases by Branched-Chain a-Keto Acids

It is known that branched-chain a-keto acids are inhibitors of the overall reaction catalyzed by PYR and KG decarboxylases (7, 9, 10). Using the ferricyanide assay it was possible to test whether this inhibition occurred at the level of the decarboxylase subunit. Mixed substrate experiments at saturating concentrations for each substrate (Table II, Experiments 1-6) showed that all three branched-chain a-keto acids are inhibitors of mitochondrial decarboxylase activity with PYR and KG. In each experiment the rate with competing substrates is lower than the rate with PYR and KG alone.

050

0-

229

DECARBOXYLASES

Inhibition of Branched-Chain Decarboxylases by Mixtures strates

a-Keto Acid of Two Sub-

Mutual inhibition among branchedchain a-keto acid dehydrogenases using the overall reaction has previously been reported in liver mitochondria from rat (5) and beef (7). Our results using the ferricyanide assay, that isolates the first step of the reaction, shows a similar inhibition (Table II, Experiments 7-9). This inhibition might be the result of competition between the two substrates for one enzyme or cross-inhibition of two enzymes by the heterologous substrates. Inspection of these inhibition data cannot distinguish unequivocally between these possibilities. Inactivation of Branched-Chain a-Keto Acid Decarboxylases by Storage and Freeze-Thawing

Mitochondria slowly lost decarboxylase activity on prolonged storage at -70°C. After 4 months of storage at -70°C one preparation lost 50,30, and 43% of its original decarboxylase activity with KIV, KMV, and KIC, respectively. Freezethawing also lowers the activity of the mitochondrial branched-chain a-keto acid decarboxylases. In one experiment slow freeze-thawing caused losses of decarboxylase activity of 42,63, and 50% with KIV, KMV, and KIC respectively. Concordant losses of decarboxylase activity with all three substrates are consistent with the hypothesis of one decarboxylase. The possibility of three decarboxylases is neither supported nor negated by these results.

050100150

025

4 (rnrbl-0 FIG. 3 Lineweaver-Burk plots relating a-keto acid decarboxylase activity to concentration substrate. Graphs are reciprocal plots of reaction velocity versus substrate concentration. each experiment 0.36 mg of mitochondrial protein was used.

of In

230

SULLIVAN,

DANCIS

Effects of Hypophysectomy

Hypophysectomy has been reported to affect mitochondrial function, and there is evidence that the mitechondrion may be an important target for pituitary hormones (15). Alteration of the hormonal milieu of rats by hypophysectomy was investigated in an attempt to affect the relative decarboxylase activity differentially for the three branched-chain a-keto acids. Rats weighing 150 g were hypophysectomized and sacrificed 2 weeks later. Liver mitochondria from hypophysectomized animals showed a marked increase in KIV and KMV decarboxylase activity when compared to untreated controls, The KMV activity was increased more (four times normal) than KIV (twice normal). The decarboxylase activity with KIC, PYR, and KG as substrates was similar for both normal and hypophysectomized rats. To corroborate these results further, more extensive studies were carried out with sham-hypophysectomized and hypophysectomized rats. The branchedchain a-keto acid decarboxylase activities of mitochondrial preparations from six rats are shown in Table III. Although there are some variations between observations on one preparation and between different animals in the same treatment group, the differences between hypophysectomized and sham-operated animals are striking with respect to the decarboxTABLE

AND

COX

ylation of KIV and KMV substrates. Hypophysectomy increases the activity of KIV decarboxylase to approximately three times normal and that of KMV to about four times normal, whereas the KIC decarboxylase activity is unchanged. Table IV demonstrates that despite variations between experiments in the absolute levels of KIV and KMV decarboxylase activities the ratio remains constant. This ratio is decreased in hypophysectomized animals, emphasizing the finding that KMV decarboxylase activity is increased to a greater extent than KIV activity. The activities of pyruvate, a-ketoglutarate, and a-ketovalerate decarboxylases were unchanged in hypophysectomized animals (Table V), indicating the selectivity of the increase in the KIV and KMV decarboxylase activities. DISCUSSION

The ferricyanide assay permits functional isolation and investigation of the mitochondrial decarboxylation of the (Yketo acids without the problem of physically separating the decarboxylase from the macromolecular complex in which it is presumed to reside. The latter approach has been particularly difficult with the branched-chain a-keto acid decarboxylase because of the major losses in enzyme activity that accompany disruption of the mitochondria and because of the possible II

DECARBOXYLASE ACTIVITY WITH MIXTURES OF Two SUBSTRATES AT SATURATING CONDITIONS” ExperiSubstrate DecarboxylSubstrate DecarboxylMixtures of ment ase activityb ase activity the two substrates, decarboxylase activity 1 2 3 4 5 6 7 8 9

5 mM PYR 5 FM PYR 5 mM PYR 1.25 mM KG 1.25 mM KG 1.25 mM KG 5 mM KIV 5 mM KIV 10 mM DL-KMV

42.8 35.0 38.7 27.4 25.0 30.8 13.4 13.5 8.2

5 mM KIV 10 mh%DL-KMV 5 mM KIC 5 mM KIV 10 mM DL-KMV 5 mM KIC 10 mM DL-KMV 5 mM KIC 5 mM KIC

a Reaction rates were determined at V concentrations for each substrate substrates each of which is at the V concentration. b Decarboxylase activity is expressed as AOD x lo3 per minute per milligram

18.1 9.5 2.6 15.0 7.9 2.1 9.2 2.0 1.8 and with

37.6 22.9 10.3 16.1 10.0 5.0 10.5 2.8 3.4 a mixture

of mitochondrial

of two

protein.

MITOCHONDRIAL

TABLE DECARBOXYLASE

ACTIVITY

III

OF LIVER MITOCHONDRIA HYPOPHYSECTOMIZED

Animal

FROM SHAM-HYPOPHYSECTOMIZED RATS

Decarboxylase 5 mM KIV

10 mM DL-KMV

5 mM KIC

10.8 k 2.2 (7) 8.1 k 2.2 (7) 6.0 k 1.4 (5)

2.2 k 0.5 (4) 1.7 + 0.1 (3) 1.7 k 1.2 (3)

Hypox 1 Hypox 2 Hypox 3

26.4 k 3.1 (8) 51.6 ” 12.4 (6) 30.2 2 7.2 (6)

26.4 -+ 4.4 (6) 40.4 k 4.5 (8) 29.2 ? 7.6 (6)

1.6 + 0.3 (3) 2.4 k 0.7 (6) 1.9 + 0.4 (3)

TABLE

5lllM KIV

1 2 3 4 5 6

Sham Sham Sham Sham Sham Sham

7 8 9

Hypox Hypox Hypox Hypox Hypox Hypox

1 1 2 2 3 3 1 1 2 2 3 3

300 to 330 g; hypophysectomized (hypox) The specific activities of this preparation

TABLE IN INDIVIDUAL

Decarboxylase ty”

Animal’

iment

per milligram of mitochondrial protein. number of observations on each animal

IV

ACTIVITY

EXPERIMENTS WITH LIVER MITOCHONDRIA SHAM-HYPOPHYSECTOMIZED AND HYPOPHYSECTOMIZED RATS

11 12

activity”

16.6 + 2.5 (7) 13.0 k 2.5 (5) 8.8 f 1.3 (7)

DECARBOXYLASE

10

AND

Sham lb Sham 2 Sham 3

a Decarboxylase activity is expressed as AOD x lo3 per minute The values are the mean activity + standard deviation with the shown in parentheses. * At the time of sacrifice sham-operated (sham) animals weighed animals, 150 to 160 g. As a control a 150-g normal rat was assayed. were KIV, 9.1; KMV, 5.1; KIC, 1.5.

Exper-

231

DECARBOXYLASES

activi-

FROM

10 l,lM DLKMV

RATS

Ratio of

reaction rates

Animal”

Decarboxylase

(KIVI KMV)

15.6 13.6 13.8 12.2 7.1 8.7

9.3 8.2 8.2 7.3 4.4 5.1

1.7 1.7 1.7 1.7 1.6 1.7

21.1 26.7 36.2 52.9 25.3 26.7

19.8 23.6 31.6 42.9 18.0 22.2

1.1 1.1 1.1 1.2 1.4 1.2

V

PYRUVATE, WKETOGLUTARATE, AND (YKETOVALERATE DECARBOXYLASE ACTIVITIES OF LIVER MITOCHONDRIA FROM SHAMHYPOPHYSECTOMIZED AND HYPOPHYSECTOMIZED

activity

5 mM PYR

1.25 mM KG

Sham 1 Sham 2 Sham 3

37.8 35.6 27.8

31.1 22.2 24.4

51.1 47.8 40.0

Hypox Hypox Hypox

18.4 31.1 22.2

31.1 45.6 33.3

40.0 47.8 33.3

1 2 3

a See footnote b Decarboxylase

103/min/mg

5mMKV

b, Table III. activity

mitochondrial

is expressed

as AOD

x

protein.

was modified by the elimination of ATP, thereby achieving a low endogenous re(1See footnote b, Table III. duction of ferricyanide and eliminating irb Decarboxylase activity is expressed as AOD X regularities of the kinetics.. The reasons lo3 per minute per milligram of mitochondrial profor the ATP effects were not investigated tein. further but might be related to perturbapresence of a soluble branched-chain (Y- tions in pools of mitochondrial couples keto acid decarboxylase of relatively low (ATP/ADP, GTP/GDP, NADH/NAD+, activity (6, 16, 17). Gubler’s technique has acyl-CoA/CoA) which are known to affect been modified making the results more PYR and KG dehydrogenase activities by reproducible and kinetically valid. Only product inhibition or other mechanisms 0.3 mg of mitochondrial protein is required (18-20). The ferricyanide assay, as modified, is a per assay making possible a large series of studies with one preparation of rat liver valid approach for studying the a-keto mitochondria. Gubler’s reaction mixture acid decarboxylase activity of mitochon-

232

SULLIVAN,

DANCIS

dria. The assay exhibits simple enzyme kinetics (Figs. 1-3). Decarboxylase activity is not significantly inhibited by arsenite, supporting the hypothesis that ferricyanide intercepts the reaction prior to the transacylation step. The use of freezethaw preparations makes transport of substrate into the mitochondria an unlikely complication in interpreting results. Further confirmation of the validity of the assay was obtained by comparing the inhibition of ferricyanide reduction by mixtures of substrates with published reports on the inhibition of the overall reaction (5, 7, 9, 10). The question of whether there are one, two, or three separate branched-chain (Yketo acid dehydrogenases has not been resolved because of difficulty in separating or purifying the enzyme activities. In the present study alterations of the hormonal state were attempted in order to determine if the activity of the branched-chain cr-keto acid decarboxylases for the three substrates could be differentially altered. Hypophysectomy markedly increases the decarboxylase activity of rat liver mitochondria with KIV and KMV as substrates without altering the activity with KIC, PYR, KG, and KV. Furthermore, the increase in KIV and KMV decarboxylase activities are quantitatively different (Table IV). These findings demonstrate that the three branched-chain cr-keto acid decarboxylase activities can be functionally separated. The simplest explanation for our observations on differential alterations in branched-chain a-keto acid decarboxylase activities is that three decarboxylases exist, one specific for each branched-chain CYketo acid. Any hypothesis suggesting a decarboxylase specific for two or three of the branched-chain cY-keto acids must include changes in active or allosteric sites in order to account for the differential changes in activity observed after hypophysectomy. The question of the number of decarboxylases for the branched-chain a-keto acids is important relative to the understanding of the genetic defect in maple syrup urine disease. If there are three decarboxylases, then the genetic defect which affects all three branched-chain LY-

AND

COX

keto acids must be located in some other common polypeptide of the multienzyme complex. Our data provide no information concerning the transacylase and dehydrogenase subunits. Previous studies also have suggested that there may be more than one branched-chain a-keto acid dehydrogenase. A soluble branched-chain a-keto acid dehydrogenase for KMV and KIC has been partially purified from beef liver (16, 17). This enzyme has no measurable KIV dehydrogenase activity. It has been suggested that soluble KMV-KIC dehydrogenase is a cytoplasmic enzyme and differs from the mitochondrial dehydrogenases in that it does not require exogenous CoA for activity (6). The mitochondrial branched-chain cr-keto acid dehydrogenases require both CoA and NAD+ for full activity (6). Elsas et al. (21) reported that cultured human fibroblasts disrupted by freezing and thawing have a dehydrogenase activity for KMV that is apparently different from the branched-chain a-keto acid dehydrogenase active with all three branched-chain substrates. The KMV dehydrogenase requires TPP but not exogenous CoA and NAD+ and therefore might be related to the soluble KMV-KIC dehydrogenase reported in beef liver that also does not require exogenous CoA. In our investigations washed mitochondria were used, eliminating the possibility that differences in subcellular localization of branchedchain cY-keto acid decarboxylase activity could explain the observed results. In maple syrup urine disease all three branched-chain c-u-keto acid dehydrogenase activities are concordantly reduced by this mutation (22-24). However, the genetic defect in maple syrup urine disease has recently been shown to be heterogeneous at the level of the mutant enzyme by complementation studies in heterokaryons derived from fusing cells of patients with classical and/or variant forms of this disease (25). In complemented heterokaryons the restoration of branched-chain cY-keto acid decarboxylase activity for leucine as substrate was much greater than for valine. This finding supports the concept of separate decarboxylase activities (25).

MITOCHONDRIAL

It is not known how hyphophysectomy mediates the observed alterations in branched-chain a-keto acid decarboxylase activity. The changes appear to be related to the hormonal milieu and not to the lower weight of hypophysectomized rats since mitochondria prepared from normal 150- and 300-g rats have similar enzyme activities. Moreover, measurements of branched-chain a-keto acid decarboxylases have demonstrated that, after hypophysectomy, the enzymes increase in activity and fall to a new steady state different from normal (manuscript in preparation) . Wohlhueter and Harper (5) reported a concordant increase in the activity of all three branched-chain a-keto acid dehydrogenases in liver mitochondria prepared from rats fed increased amounts of dietary amino acids. The increase in enzyme activity was mediated by feeding various combinations of amino acids and was not specific for dietary branched-chain amino acids. These investigators also demonstrated that food utilization and circadian oscillations of enzyme activity were important factors in the “induction” by dietary amino acids. These complications were avoided or minimized in our experiments by sacrificing rats between 7:00 and 10:00 AM which is the peak of enzyme activity in normal animals (5). Finally, the activity increases observed by Wohlhueter and Harper were always concordant for all three branched-chain a-keto acids whereas hypophysectomy causes differential increases with the three substrates. There is evidence that the hypophysis plays an important role in mitochondrial metabolism (15). Following an injection of growth hormone into hypophysectomized rats, greater than 40% of the total taken up by the liver localized in mitochondria. Hypophysectomy decreases mitochondrial protein and RNA synthesis and alters the activity of mitochondrial NADH oxidase (increased) and cytochrome oxidase (decreased). The morphology of hepatocyte mitochondria is changed in hypophysectomized rats, and these changes are reversed by treatment with growth hormone (15). Our findings of differential alterations in

233

DECARBOXYLASES

branched-chain a-keto acid decarboxylase activities in mitochondria from hypophysectomized rats further strengthen the relationship between the hypophysis and mitochondrial function. REFERENCES 1. LINN, T. C., PELLEY, J. W., PETTIT, F. H., HuCHO, F., RANDALL, D. D., AND REED, L. J. (1972) Arch. Biochem. Biophys. 148, 327-342. 2. BARRERA, C. R., NAMIHIRA, G., HAMILTON, L., MUNK, P., ELEY, M. H., LINN, T. C., AND REED, L. J. (1972) Arch. Biochem. Biophys. 148, 343-358. 3. SCHWARTZ, E. R., AND REED, L. J. (1970) J. Biol. Chem. 245,183-187. 4. SEARLS, R. L., AND SANADI, D. R. (1960) Biothem. Biophys. Res. Commun. 2, 226-229. 5. WOHLHUETER, R. M., AND HARPER, A. E. (1970) J. Biol. Chem. 245, 2391-2401. 6. JOHNSON, W. A., AND CONNELLY, J. L. (1972) Biochemistry 11, 1967-1973. 7. JOHNSON, W. A., AND CONNELLY, J. L. (1972) Biochemistry 11, 2416-2421. 8. GUBLER, C. J. (1961) J. Biol. Chem. 236, 31123120. 9. KANZAKI, T., HAYAKAWA, T., HAMADA, M., FuKUYOSHI, Y., AND KOIKE, M. (1969) J. Biol. Chem. 244, 1183-1187. 10. DREYFUS, P. M., AND PRENSKY, A. L. (1967) Nuture (London) 214, 276. 11. HOGEBOOM, G. H. (1955) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 1, pp. 16-19, Academic Press, New York. 12. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 13. DANNER, D. J., AND BOWDEN, J. A. (1966) Fed. Proc. 25, 747. 14. SANADI, D. R., LANGLEY, M., AND WHITE, F. (1959) J. Biol. Chem. 234, 183-187. 15. MADDAIAH, V. T., AND COLLIPP, P. J. (1975) in Regulation of Growth and Differentiated Function in Eukaryote Cells (Talwar, G. P., ed.), pp. 453-459, Raven Press, New York. 16. CONNELLY, J. L., DANNER, D. J., AND BOWDEN, J. A. (1968) J. Biol. Chem. 243, 1198-1203. 17. BOWDEN, J. A., AND CONNELLY, J. L. (1968) J. Biol. Chem. 243, 3526-3531. 18. GARLAND, P. B., AND RANDLE, P. J. (1964) Biothem. J. 91, 6c-7c. 19. SMITH, C. M., BRYLA, J., AND WILLIAMSON, J. R. (1974) J. Biol. Chem. 249, 1497-1505. 20. COOPER, R. H., RANDLE, P. J., AND DENTON, R. M. (1975) Nature (London) 257, 808-809. 21. ELSAS, L. J., PRIEST, J. H., WHEELER, F. B.,

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