Biochem. J. (1969) 111, 565 565 Printed in Great Britain

Metabolism of in Protein-Calorie-Deficient Rats

By I. G. McFARLANE* AND C. VON HOLTt Department of Biochemi8try, Univer8ity of the West Indie8, Kingston, Jamaica (Received 21 October 1968)

1. Protein-calorie-depleted rats exhibit a decreased ability to oxidize leucine as compared with control animals. 2. The block in degradation of L-leucine is due to decreased activities of L-leucine and D-amino acid oxidase associated with liver mitochondria. 3. Cytoplasmic L-leucine transaminase in liver is not affected by the protein depletion. 4. The specific activities of the mitochondrial can be increased by a single oral dose of 1g. of Bacto-Peptone (meat hydrolysate). The effect ofBacto-Peptone can be inhibited by prior administration of dosages of 10mg. of puromycin/lOOg. body wt., suggesting that the increased specific activities may be due to new synthesis of enzyme protein.

The overall oxidative degradation oftwo essential stage of decarboxylation of 4-methyl-2-oxopen- amino acids (leucine and phenylalanine) is markedly tanoate. decreased in rats fed on a low-protein diet whereas To gain further insight into the observed changes that of two non-essential amino acids (glutamate of leucine degradation, the activities of the enzymes and alanine) is virtually unaffected. The decreased concerned had to be determined. Existing pro- degradation of leucine is due to a block in the cedures for the determination of enzymic trans- catabolic pathway at some stage before the forma- amination involve either measurement of the tion of 3-methylbutyryl-CoA (isovaleryl-CoA) transfer of an amino group to an acceptor a-oxo acid (McFarlane & Holt, 1969). or measurement of the a-oxo acid formed from the In animal tissues amino acids are oxidatively amino acid (Bergmeyer & Bernt, 1963). Both these deaminated to their respective a-oxo acid analogues, methods require separation of the reaction products the L-isomers by the action of a transaminase in the from the reactants. However, losses incurred presence of an amino group acceptor and the during the separation procedures impose limitations D-isomers by the action of D-amino acid oxidase on the accuracy, sensitivity and reproducibility of (Meister, 1965a). The a-oxo acids are then de- the techniques. Other methods, such as measure- carboxylated to give the next lower homologous ment of glutamic semialdehyde formed by oc-trans- fatty acid. It is doubtful whether any other path- amination of ornithine (Peraino & Pitot, 1963), are way contributes significantly to the degradation of based on physicochemical properties peculiar to the leucine in animal tissues. A leucine decarboxylase reactants and are applicable for these trans- has been found in plants and micro-organisms, but amination reactions only. Therefore a rapid not in animals (Meister, 1965b). Although an sensitive routine method had to be developed for L-amino acid oxidase has been purified from rat the simultaneous determination of oc-oxo acid kidney, its activity is very low, and it is unlikely decarboxylase and transminase (or D-amino acid that this enzyme plays a major role in the con- oxidase) activities in crude tissue preparations. version of amino acids into a-oxo acids (Krebs, 1964; Meister, 1965b). Previous observations (McFarlane & Holt, 1969) MATERIALS AND METHODS showed that rats deprived of protein exhibit an increased urinary excretion of 4-methyl-2-oxo- Chemicats. Labelled compounds were obtained from pentanoate (a-oxoisocaproate) and a decreased urin- The Radiochemical Centre (Amersham, Bucks.) or from ary excretion of leucine. This suggested that the Calbiochem (Los Angeles, Calif., U.S.A.), or were prepared from materials obtained from one of these sources. primary block in leucine catabolism occurs at the starting All other chemicals, unless otherwise stated, were A.R. * Present address: Department of Nutrition, Sir John grade or equivalent and were obtained from various Atkins Laboratories, Queen Elizabeth College, London, commercial sources. W. 8. Animals and diets. Animals, diets and fee(ling regimen t Present address: Department of Biochemistry, Univer- were as described in the preceding paper (' cFarlane & sity of Cape Town, Cape Town, South Africa. Holt, 1969). The term 'protein-depleted rat refers to an 5;66 I. G. McFARLANE AND C. VON HOLT 1969 animal that had been fed on the protein-deficient diet for remaining in the beaker rinsed into the counting vial with 8 weeks. two 0-5ml. lots of ethanol. The radioactivity of the sample Measurement of radioactivity. All determinations of was then measured to determine the amount of 14CO2 radioactivity were conducted with a Packard Tri-Carb produced. model 314E liquid-scintillation spectrometer. The scintil- The incubation tube was centrifuged to remove pre- lator system used contained 0-3g. of 1,4-bis-(4-methyl-5- cipitated protein and a 1-Oml. sample of the supernatant phenyloxazol-2-yl)benzene and 5-0g. of 2,5-diphenyloxazole was incubated as above for lhr. with 3-Oml. of saturated in 11. of toluene. Ce(SO4)2 in 4N-H2SO4 for quantitative decarboxylation of Tissue preparations. Animals, kept without food for carboxyl-group-labelled a-oxo acid. The 14CO2 arising 14hr. overnight, were killed by decapitation and the livers from this treatment was trapped in Hyamine and its were excised, chilled and weighed. Mitochondria were radioactivity measured as above to determine the amount prepared, by the method of Hogeboom (1955), from a 10% of 4-methyl-2-oxopentanoate produced by the tissue (w/v) liver homogenate in 0-25M-sucrose, pH7-4. Mito- preparation. The particular conditions of the reaction were chondria-free supernatant fractions were obtained by determined with known quantities of sodium 4-methyl-2- centrifuging lOml. portions of the liver homogenates at oxo[l-14C]pentanoate. 15000g at 0-3° for 15min. in a Sorvall RC-2B refrigerated Determination of DNA and protein. The DNA content of centrifuge. liver homogenates was determined by the method of Assay of enzyme activities. Amino acids are oxidatively Schneider (1957). The protein concentration in tissue deaminated and decarboxylated according to the general preparations was determined by the method of Lowry, reaction: Rosebrough, Farr & Randall (1951). Re-feeding experiments. Animals were kept without food Transaminase L-Amino acid (a)+ a-oxo acid(b) - -si overnight and, between 8.00 a.m. and 9.00 a.m. the following morning, were given either 75mg. of DL-leucine or 1g. of a-oxo acid(a)+ L-amino acid(b) Bacto-Peptone (meat hydrolysate) in 2-5ml. of water by oral intubation. Animals were killed 3hr. later and enzyme D-Amino acid activities were assayed. Some animals were given 10mg. oxidase of puromycin/lOOg. body wt. (Pitot & Peraino, 1963), by intraperitoneal injection, lhr. before the Bacto-Peptone a-Oxo acid Decarboxylase* fatty acid+ CO2 was administered. Statistical tests. The significance ofthe difference between Amino acids labelled in the carboxyl group will result in the means of two sets of values was determined with the t labelled a-oxo acids. Subsequent decarboxylation by a test for small samples (Bancroft, 1965). decarboxylase will yield 14CO2. Therefore the amount of 14CO2 plus any labelled a-oxo acid not decarboxylated will be a measure of the transaminase or D-amino acid oxidase activity. a-Oxo acids undergo quantitative decarboxylation RESULTS in the presence of Ce(SO4)2 (Meister, 1952). At a given Enzymes of leucine catabolism. Initial experi- concentration the enzymically produced 14CO2 ments revealed that (a) a boiled homogenate of liver from amino acids labelled in the carboxyl group reflects the decarboxylase activity and the sum ofenzymically liberated showed no leucine-catabolic activity and (b) the plus Ce(SO4)2-liberated 14CO2 the transaminase activity utilization of leucine was linear with respect (with L-leucine as substrate) or D-amino acid oxidase (with to time and homogenate concentration. This D-leucine as substrate). established that the 14CO2 and 4-methyl-2-oxo- The apparatus used was a 25ml. wide-mouthed round- [1-14C]pentanoate produced during the incubation bottomed centrifuge tube stoppered with a 'needle- were the result of enzyme activity. puncture' rubber cap, of the type used in preparation of Table 1 gives the intracellular distribution of the solutions for hypodermic injection. At the top of the tube, enzymes deaminating and decarboxylating D- and suspended from the stopper, was a 2ml. plastic beaker L-leucine. The activities related to unit fresh containing a strip of Whatman no. 4 filter paper (0-8 cm. x 8-0cm.), accordion-pleated to fit inside the beaker and weight of liver show that approx. 30% of the moistened with 0-2ml. of 1-Om-Hyamine hydroxide in L-leucine catabolism occurs in the supernatant and methanol (Rapkin, 1961). The incubation system (total 70% in the mitochondria. Over 90% of D-leucine volume 2-Oml.), containing [1-14C]leucine, the necessary oxidation occurs in the mitochondrial fraction. cofactors and tissue preparation, was placed in the bottom The remainder, which was found in the super- of the centrifuge tube, which was then stoppered and natant, can probably be attributed to contamination incubated in a Warburg apparatus, with continuous shaking of the latter by mitochondrial fragments. In a at 370, for 1 hr. The enzymic reaction in the incubation separate experiment, with 1 mM-sodium 4-methyl-2- mixture was stopped by injection of 0-5ml. of lON-H2SO4 oxo[I-14b]pentanoate (50 000disintegrations/min.)in to liberate enzymically produced 14CO2. Incubation was continued for a further lhr. to ensure that all 14CO2 in place of labelled leucine, it was determined that over the incubation mixture was released from solution and 90% of the 4-methyl-2-oxopentanoate-decarboxyl- trapped in the Hyamine. The plastic beaker was removed, ating activity is found in the mitochondria also. the filter-paper strip transferred to a counting vial con- Maximal 4 - methyl - 2 - oxopentanoate - decarb - taining lOml. of toluene scintillator and any Hyamine oxylating activity is achieved when the incubation Vol. III LEUCINE AND PROTEIN DEFICIENCY 567 Table 1. Intracellular di8tribution of leucine-catabolic activity in mitochondrial and 8upernatant fractions of liverfrom control rats The incubation system contained: 1-OmM-D- or -L-[1-14C]leucine (l00000disintegrations/min.), 1-OmM-sodium a-oxoglutarate, 25mmi-Sorensen's phosphate buffer, pH7-4, and mitochondria or supernatant equivalent to 100mg. of fresh tissue. Values given are the means of duplicate determinations. The variation from any quoted mean does not exceed + 21/%. Leucine degraded (mfimoles/hr./mg. of protein)

D-Leucine L-Leucine

Liver To CO2 To a-oxo Total To CO2 To a-oxo Total fraction (decarboxylase) acid (oxidase) (decarboxylase) acid (transaminase) Mitochondria 42-00 0-96 42-96 7-72 Nil 7-72 Supernatant 0-18 1-22 1-40 0-22 2-72 2-94

Table 2. Effect ofadded ac-oxoglutarate on the degradation of leucine by mitochondrial and 8upernatantfraction8 of liverfrom control rats The incubation system contained I-OmMe-D-[1-14C]leucine (50000disintegrations/min.) or -L-[1-14C]leucine (l00000disintegrations/min.), 1-OmM-sodium a-oxoglutarate, 0-2mM-thiamine pyrophosphate, 0-1mM-CoA, 2-OmM-MgCl2, 50mM-KCl, I-Omm-ATP (dipotassium salt), O-l mM-cytochrome c, 25mM-Sorensen's phosphate buffer, pH7-4, and mitochondria or supernatant equivalent to 100mg. of fresh tissue. When cx-oxoglutarate was omitted, the incubation system was made up to volume with 0-25m-sucrose, pH7-4. Results are the means of duplicates. The variation from any quoted mean does not exceed + 3-2%. Leucine degraded (m,moles/hr./mg. of protein)

D-Leucine L-Leucine To C02 To a-oxo Total To C02 To a-oxo Total (trans- Liver fraction oc-Oxoglutarate (decarboxylase) acid (oxidase) (decarboxylase) acid aminase) Mitochondria Omitted 47-40 1-18 48-58 3-16 Nil 3x16 Mitochondria Added 45-90 1-20 47-10 8-05 Nil 8-05 Supernatant Omitted 0-16 Nil 0-16 Supernatant Added 0-23 2-81 3-04

system contains 0-2mM-thiamine pyrophosphate, 4 - methyl - 2 - oxopentanoate - decarboxylating 2-0mM-Mg2+, 0-lmM-CoA and I-OmM-ATP. Addi- enzyme(s), in contrast with the deaminating tion of NAD+ or lipoic acid or both did not signific- enzymes, operates at much lower substrate satura- antly influence the reaction. tion, between 0-5 and 1-OmM. The reaction L-Leucine utilization is enhanced by the presence sequence could therefore not be measured at of a-oxoglutarate in the incubation system, saturation concentrations of the amino acids, but whereas D-leucine catabolism is not (see Table 2). only at such substrate concentration that allowed This indicates the operation of a transaminase in a linear reaction rate during the incubation period. the catabolism of the former, whereas that of the Comparison of D- and L-leucine cataboli8m in latter is probably catalysed by D-amino acid control and protein-depleted rat8. The leucine- oxidase. transaminating, leucine-deaminating and 4-methyl- Substrate saturation for the transamination of 2-oxopentanoate-decarboxylating abilities of L-leucine in mitochondria with excess of oc-oxo- control and protein-depleted rats are shown in glutarate present is not reached at 20mM, at which Tables 3-8. The results are expressed in terms of concentration the amino acid becomes insoluble in the DNA content of the liver. Reference to DNA the test system. ac-Oxoglutarate saturation is permits direct comparison of enzyme activities/cell, reached at 10mM. The D-amino acid oxidase, since the liver DNA content is unaffected by changes similarly, cannot be saturated with D-leucine up to in dietary protein intake (Mendes & Waterlow, a concentration of 18mM, The mitoehondrial 1958; Mariani, Migliaccio, Spadoni & Ticca, 1966). 568 I. G. McFARLANE AND C. VON HOLT 1969 Table 3. Catabolism of L-[1-140]leucine by mitochondrial and 8upernatantfraction8 of liverfrom control and protein-depleted rat8 Results are the mean values obtained from six control and four protein-depleted animals. Duplicate measure- mentsweremadeforeachanimal. Theincubationsystemcontained: 1 mM-L-[1-14C]leucine (400000disintegrations/ min.), 10mM-sodium ao-oxoglutarate, 0-2mM-thiamine pyrophosphate, O-lmm-CoA, 2 0mM-MgCI2, 50mms-KCI, 1Omm-ATP (dipotassium salt), Olmm-cytochrome c, 25mm-Sorensen's phosphate buffer, pH7-4, and mito- chondria or supernatant equivalent to 100mg. of fresh tissue. Significance of differences: P < 0 001 for a-b, c-d and e-f; other differences between the two groups ofanimals are not significant (i.e. P> 0 05). L-Leucine degraded (m,moles/hr./mg. of DNA) To C02 To a-oxo Total Liver fraction Animal group (decarboxylase) acid (transaminase) Mitochondria Control 220.3a 27.1l 247.4e Mitochondria Depleted 82.6b 48.9d 131-5f Supernatant Control 10-5 74.4 84*9 Supernatant Depleted 7-0 72-0 79-0

Table 4. Catabolim of D-[1-14C]leucine by mito- Table 5. Effect of added 4-methyl-2-oxopentanoate on chondria of liver from control and protein-depleted L-leucine tran8amina8e activity of the mitochondrial rats fraction of liverfrom a protein-depleted rat

Results are the mean values obtained from six control Each value is the mean of a duplicate measurement. The and four protein-depleted rats. Duplicate measurements variation from any quoted mean does not exceed + 2.4%. were conducted for each animal. The incubation system The composition of the incubation system was the same as was the same as that used in Table 3 except that labelled in Table 3. Sodium 4-methyl-2-oxopentanoate was added L-leucine was replaced by 10-OmM-D-[1-14C]leucine at a final concentration of 5-0mm. (400000disintegrations/min.), no a-oxoglutarate was added L-Leucine degraded (m,umoles/hr./mg. and mitochondria equivalent to only 25mg. of fresh tissue of DNA) were used. Significance of differences: P<0O01 for a-b; P< 04001 for and e-f. To C02 Total 4-Methyl-2- (decarb- To cc-oxo (trans- D-Leucine degraded (m,umoles/hr./mg. oxopentanoate oxylase) acid aminase) of DNA) Omitted 95.7 51-0 146-7 Added 28-0 222-2 250-2 Animal To C02 To a-oxo Total group (decarboxylase) acid (oxidase) Control 573.0a 2327c 29006 Depleted 382.8b 9484 1331f Table 6. Decarboxylation of4-methyl-2-oxopentanoate by the mitochondrialfraction of liverfrom control and protein-depleted rats

Results are the mean values obtained for three animals - As shown in Tables 3 and 4, the activities of in each group. Duplicate measurements were conducted for mitochondrial enzymes for both L- and D-leucine each animal. The composition of the incubation system was the same as in Table 4 except that D-leucine was re- degradation are significantly decreased in protein- placed by 5-Omm-sodium 4-methyl-2-oxo[1-14C]pentanoate depleted animals. This is true for the L-leucine (40000disintegrations/min.). Values in parentheses denote transaminase, the D-leucine oxidase and the the ranges ofthe results. 4 - methyl - 2 - oxopentanoate - decarboxylating C02 produced enzyme(s). The cytoplasmic (supernatant) L- Animal group (m,umoles/hr./mg. of DNA) leucine transaminase is, however, not affected. Control 1448 (1398-1527) WithL-leucine as substrate (Table 3) the decreased Depleted 1363 (1329-1387) decarboxylation of 4-methyl-2-oxopentanoate is accompanied by an accumulation of a-oxo acid in the system. There is also a marked decrease in L-leucine transamination. To exclude the pos- accumulated oc-oxo acid, liver mitochondria from a sibility ofonly an apparent decrease oftransaminase protein-depleted animal were incubated with activity owing to a mass-action effect exerted by the L-[1-14C]leucine in the presence and absence of Vol. 111 LEUCINE METABOLISM AND PROTEIN DEFICIENCY 569 Table 7. Effect offeeding with DL-leucine or Bacto-Peptone on-letucine cataboli8m by the mitochondrialfraction of liverfrom protein-depleted rat8 Each value is the mean obtained from the number of animals shown in parentheses for each group. Duplicate measurements were made for each animal. The composition ofthe incubation medium was the same as in Table 3. DL-Leucine (75mg.) or Bacto-Peptone (1g.) was force-fed at 3hr., and puromycin (lOmg./lOOg.) was injected intraperitoneally at 4hr., before the rats were killed. Significance of differences: for a-c, P< 0001; for a-d, P< 0-05; for a-e, P< 0-02; for b-d, P< 0-05; for d-e, P< 0-01; forf-h, P< 0-001; forf-i, P< 0-05; forf-j, P< 0-01; for g-i, P= 0-05; for k-n, P< 0-01; for k-o, P< 0-02; for I-n, P< 0-02; for n-o, P< 0-05; differences between other pairs, such as b-c, b-e, g-h, gj, i-j, k-n, I-n and I-o, are not significant (i.e. P> 0-05). L-Leucine degraded (mumoles/hr./mg. of DNA) To C02 To oc-oxo Total (trans- Animal group Treatment (decarboxylase) acid aminase) Control (6) 220-3a 27-if 247.4k Depleted (4) 82.6b 48-9" 131-51 Depleted (3) Leucine-fed 94.6c 54-0h 148-6m Depleted (3) Peptone-fed 147-7d 35-5f 183-2n Depleted (3) Puromycin, peptone-fed 110.08 41-41 151.40

Table 8. Effect of feeding with DL-leucine or Bacto-Peptone on D-leucine catabolism by the mitochondrial fraction of liverfrom protein-depleted rats Each value is the mean obtained from the number of animals shown in parentheses for each group. Duplicate measurements were made for each animal. The composition ofthe incubation system was the same as in Table 4. Treatment with DL-leucine, Bacto-Peptone and puromycin was the same as indicated in Table 7. Significance of differences: for a-c, P< 0-01; for b-e, P=0-05; for f-h, P< 0-001; for f-j, P< 0-001; for g-i, P< 0-01; for ij, P< 0-05; for k-n, P< 0-001; for k-o, P< 0-01; for 1-n, P< 0-001; for n-o, P< 0-05; differences between other pairs,. such as a-d, a-e, b-c, b-d, d-e,f-i, g-h, g-j, k-n, i-n and l-o, are not significant (i.e. P> 0-05). D-Leucine degraded (m,umoles/hr./mg. of DNA) To CO2 To a-oxo Total Animal group Treatment (decarboxylase) acid (oxidase) Control (6) 573-0a 23271 2900k Depleted (4) 382-8b 9489 13311 Depleted (3) Leucine-fed 321-5c 972h 1294m Depleted (3) Peptone-fed 483-5d 2095i 2568n Depleted (3) Puromycin, peptone-fed 529-40 10061 15360

added 4-methyl-2-oxopentanoate. The addition of has no significant effect on any of the enzymes 4-methyl-2-oxopentanoate does not decrease the concerned with the early stages ofleucine catabolism transamination rate (see Table 5). On the contrary, in the liver. Feeding with Bacto-Peptone, however, there is an increase in the rate ofleucine degradation produces significant increases in the specific for which no satisfactory explanation has been activities of all the mitochondrial enzymes. The found. latter effects can be inhibited by prior administra- The above suggests that enzymic decarboxylation tion of puromycin. of 4-methyl-2-oxopentanoate produced endogen- ously, i.e. from D- or L-leucine, is decreased in the DISCUSSION livers ofprotein-depleted rats. The decarboxylation of added sodium 4-methyl-2-oxo[jl14C]pentanoate, The metabolic pathway from L-leucine to however, is not diminished (Table 6). acetoacetate and acetyl-CoA has long been Effect of including leucine or a mixture of amino established (Meister, 1965a). However, until acid8 in the diet on the cataboliwm of leucine in recently, details ofthe first two steps in the pathway protein-depleted rate. As shown in Tables 7 and 8, have remained unclear. Although it was known for feeding protein-depleted animals with DL-leucine some time that L-leucine transaminates with 570 I. G. McFARLANE AND C. VON HOLT 1969 a-oxoglutarate, only recently has a specific enzyme centration was obviously sufficiently high, so that been isolated that catalyses reversible transamina- added NAD+ had no stimulatory effect. tion between the three branched-chain amino acids, These, therefore, were the enzymes investigated: L-leucine, L-valine and L-isoleucine, and a-oxo- L-leucine (cytoplasmic and mito- glutarate (Taylor & Jenkins, 1966a,b). In addition, chondrial), D-amino acid oxidase (mitochondrial) in heart muscle and in liver there are two leucine and 4 - methyl - 2 - oxopentanoate decarboxylase transaminases that are isoenzymes (Ichihara & (mitochondrial). The last-named enzyme is so Koyama, 1966). One of these is localized in the called tentatively; it is recognized that no enzyme mitochondria and the other in the cytoplasm has been described that specifically decarboxylates (Ichihara, Takahashi, Aki & Shirai, 1967). The 4-methyl-2-oxopentanoate and only circumstantial cytoplasmic activity can be separated into two evidence is presented here to suggest that such an fractions by DEAE-cellulose chromatography, one enzyme may exist. of which catalyses the transamination of all three Of the enzymes concerned with L-leucine catabol- branched-chain amino acids, whereas the other is ism, only the cytoplasmic leucine transaminase is active only with leucine. unaffected by protein depletion. The mitochondrial D-Leucine is deaminated by D-amino acid oxidase leucine transaminase and 4-methyl-2-oxo- (Meister, 1965b). This enzyme has been found in the pentanoate decarboxylase (when L-leucine is used kidney and liver of a large number of mammals as substrate) are significantly decreased in activity. (Meister, 1965b; Krebs, 1964), localized in cyto- The decrease in mitochondrial leucine transaminase plasmic particles containing also urate oxidase and activity cannot be explained by a mass-action catalase (de Duve, Wattiaux & Baudhuin, 1962). effect exerted by the accumulated a-oxo acid on the These particles sediment with the mitochondrial enzyme (Table 5). The accumulation of x-oxo acid fraction, but can be separated by subfractionation. in the system suggests that there may also be a D-Amino acid transaminases have so far been found genuine decrease in activity of the 4-methyl-2- only in micro-organisms and it is doubtful whether oxopentanoate decarboxylase. The increase in they exist in mammalian tissues (Meister, 1965b). 4-methyl-2-oxopentanoate excretion in protein- The conversion of 4-methyl-2-oxopentanoate into depleted rats (McFarlane & Holt, 1969) supports 3-methylbutyryl-CoA is assumed to involve a this view. However, no such decrease was found decarboxylation reaction analogous to the con- when 4-methyl-2-oxopentanoate was used as sub- version of pyruvate into acetyl-CoA (Meister, strate (Table 6). From this and the results in 1965a). Tables 4 and 3 one may deduce that the rate of In the current investigation, conversion of a-oxo acid generation is the rate-limiting factor in L-leucine into 4-methyl-2-oxopentanoate was found the 4-methyl-2-oxopentanoate decarboxylase re- to be enhanced by the addition of a-oxoglutarate, action and the decreased activity ofdecarboxylation indicating the operation of transamination. The in depleted animals is only apparent but does not distribution of enzyme activity between the mito- reflect a decrease in enzyme concentration. How- chondrial and supernatant fractions suggests the ever, this would not account for the accumulation of existence of two enzymes. It would appear that a-oxo acid in vitro (Table 3) and the increased these enzymes correspond to the leucine trans- excretion (McFarlane & Holt, 1969). A satisfactory aminase isoenzymes described by Ichihara et al. answer to this discrepancy has to await further (1967). a-Oxoglutarate did not enhance the investigation. deamination of D-leucine, suggesting that the D-Amino acid oxidase activity is decreased conversion of the latter into 4-methyl-2-oxo- significantly per cell, in confirmation ofthe observa- pentanoate is catalysed by D-amino acid oxidase tions of other authors (Knox, Auerbach & Lin, associated with the mitochondrial fraction. As 1956). compared with the L-leucine transaminase the The results given in Tables 7 and 8 show that D-amino acid oxidase activity is roughly tenfold leucine-catabolizing enzymes in protein-depleted higher. This is in good agreement with the observa- animals increase in activity after feeding with tion by Greenberg (1961) that a greater amount of Bacto-Peptone. It had been previously observed ketone bodies is produced from D- or DL-leucine that tryptophan pyrrolase can be induced by than from the L-isomer. tryptophan (Knox & Mehler, 1951) and that The decarboxylation of 4 - methyl - 2 - oxo - tyrosine-a-oxoglutarate transaminase can be in- pentanoate requires Mg2+, thiamine pyrophosphate duced by tyrosine (Lin & Knox, 1958). These and CoA for maximal activity, and is associated enzymes operate at the beginning of the respective with the mitochondrial fraction. These catabolic pathways. A block in the early stages of requirements point to an enzyme similar to the degradation of leucine could be located in this pyruvate oxidase complex. Under the experimental investigation, in vivo as well as in vitro, as occurring conditions the intramitochondrial NAD+ con- before the formation of 3-methylbutyryl-CoA. The Vol. 111 LEUCINE METABOLISM AND PROTEIN DEFICIENCY 571 reappearance of leucine transaminase, amino acid oxidase and to a smaller extent 4-methyl-2-oxo- REFERENCES pentanoate decarboxylase activity after the animals Bancroft, H. (1965). Introduction to Biostatistics, chapter 15. had been fed with Bacto-Peptone strongly suggests New York, Evanston and London: Hoeber Medical that enzyme induction might be the mechanism by Division, Harper and Row. which leucine-catabolizing activity is controlled. Bergmeyer, H. U. & Bernt, E. (1963). In Methods of Enzy- The fact that the Bacto-Peptone effect can be matic Analysis, p. 837. Ed. by Bergmeyer, H. U. New inhibited by prior administration of puromycin York and London: Academic Press Inc. weight to this view. de Duve, C., Wattiaux, R. & Baudhuin, P. (1962). Advanc. adds Enzymol. 24, 291. Although the feeding with Bacto-Peptone caused Greenberg, D. M. (1961). In Metabolic Pathways, vol. 2, a significant increase in the activity of all the chapter 14. Ed. by Greenberg, D. M. New York and enzymes concerned with the first two steps of London: Academic Press Inc. leucine catabolism, the activities of the L-leucine- Hogeboom, G. H. (1955). In Methods in Enzymology, converting enzymes did not return to normal vol. 1, p. 16. Ed. by Colowick, S. P. & Kaplan, N. 0. (Table 7). However, the induction of D-leucine New York: Academic Press Inc. degradation by Bacto-Peptone led to a return of Ichihara, A. & Koyama, E. (1966). J. Biochem., Tokyo, 59, D-leucine oxidase activity to normal values (Table 160. The equivocal results on the decarboxylating Ichihara, A., Takahashi, H., Aki, K. & Shirai, A. (1967). 8). Biochem. biophys. Res. Commun. 26, 674. activity may indicate the presence of more Knox, W. E., Auerbach, J. H. & Lin, E. C. C. (1956). than one enzyme decarboxylating 4-methyl-2-oxo- Physiol. Rev. 36, 164. pentanoate. Knox, W. E. & Mehler, A. H. (1951). Science, 113, 237. It is difficult to find an explanation for the fact Krebs, H. A. (1964). In Mammalian Protein Metabolism, that the Bacto-Peptone effect could not be vol. 1, chapter 5. Ed. by Munro, H. N. & Allison, J. B. duplicated by feeding the rats with DL-leucine. It New York and London: Academic Press Inc. may be that, owing to the slight solubility of Lin, E. C. C. & Knox, W. E. (1958). J. biol. Chem. 233, 1186. leucine, the amount of the L-isomer given was only Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, about one-third of that present in the Bacto- R. J. (1951). Life Sci. 5, 325. McFarlane, I. G. & Holt, C. von (1969). Biochem. J. 111, Peptone used and may not have been adequate to 557. produce a detectable increase in enzyme activity. Mariani, A., Migliaccio, P. A., Spadoni, M. A. & Ticca, M. One point of particular interest, namely the (1966). J. Nutr. 90, 25. difference in response of the mitochondrial and Meister, A. (1952). J. biol. Chem. 197, 309. cytoplasmic leucine transaminases to dietary Meister, A. (1965a). Biochemistry of the Amino Acids, protein restriction, has emerged from these studies. vol. 1, chapter 4. New York and London: Academic Differences in responses of the two isoenzymes to Press Inc. various stimuli have also been observed by Ichihara Meister, A. (1965b). Biochemistry of the Amino Acids, et al. (1967). Because there is no 4-methyl-2-oxo- vol. 2, chapter 6. New York and London: Academic Press Inc. pentanoate decarboxylase activity in the cytoplasm, Mendes, C. B. & Waterlow, J. C. (1958). Brit. J. Nutr. 12,74. leucine cannot be further degraded without the aid Peraino, C. & Pitot, H. C. (1963). Biochim. biophys. Acta, of the mitochondrial enzymes. The mitochondrial 73, 222. leucine transaminase, perhaps because of the close Pitot, H. C. & Peraino, C. (1963). J. biol. Chem. 238, proximity to the 4-methyl-2-oxopentanoate de- cl910. carboxylase, plays the key role in the overall Rapkin, E. (1961). Packard Technical Bulletin, no. 3 degradation of the amino acid and appears to be (revised). La Grange, Ill.: Packard Instrument Co. Inc. controlled independently from the cytoplasmic Schneider, W. C. (1957). In Methods in Enzymology, enzyme. vol. 3, p. 680. Ed. by Colowick, S. P. & Kaplan, N. 0. New York: Academic Press Inc. We acknowledge the support received from the Medical Taylor, R. T. & Jenkins, W. T. (1966a). J. biol. Chem. 241, Research Council (Grant no. G.964/108/T). This work has 4396. been presented as part of a Thesis submitted by I. G.McF. Taylor, R. T. & Jenkins, W. T. (1966b). J. biol. Chem. 241, for the Ph.D. degree of the University of London in 1967. 4407.