Leucine catabolism in human term placenta

BIOCHEMICAL

MEDICINE

30, 141-145 (1983)

Leucine Catabolism in Human Term Placenta P. W. D.

%XLOWSKI,

Depurtment

J.

S.~OLNIEROWICZ. AND

of Biochemistry. 80-21 I Gdarisk,

L.

SWIERCZYNSKI,

I?LEWSKI

I. B. M. Medical School Dqbinki I, Poland

of GdaI‘1sk.

Received July 15. 1982

The placenta is known to concentrate nearly all amino acids intracellulary mainly for the transport to the fetus (l-3). However little is known about the metabolism of amino acids in the placenta. According to Schneider ef al. (4) and Lemons and coworkers (5) glutamate is the only amino acid actively metabolized by the trophoblast. It was also shown that some other amino acids like aspartate and alanine (6), phenylalanine and DL-leucine (7) are oxidized to CO* by placenta. Dancis et al. (8) demonstrated that term placental tissue is able to degradate valine to the corresponding oxoacid. Stimulatory effect of oxoglutarate on ammonia from branched-chain amino acids formation by human term placental mitochondria (9) suggests that this tissue is able to metabolize the branchedchain amino acids. The initial step in the metabolism of the branchedchain amino acids is a transamination to produce the respective oxoacids. This reaction occurs mainly in the cytosol of all the tissues examined (10-12). The resulting oxoacids are subsequently decarboxylated and converted to the corresponding acyl-CoA derivatives via branched-chain oxoacids dehydrogenase present in mitochondria (12,13). The purpose of the present study is to investigate the rate of the r.-leucine transamination and decarboxylation reactions and their subcellular distribution in human term placenta. MATERIALS AND METHODS

The NAD, dithiothreitol, pyruvic acid, or-carnitine, potassium salt of 2-oxoglutarate, and dilithium salt of CoA were obtained from Sigma Chemical Company (St. Louis, MO.). L-Leucine was purchased from Fluka AG-Buchs, Switzerland. L-[l-‘4C]Leucine of a specific activity 59 mCi/mmole was from Radiochemical Centre, Amersham, England. Other 141 00062944/83 $3.00 Copyright 0 1983 by Academic Press. Inc. All rights of reproduction in any form reserved.

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&XLOWSKI

ET AL.

reagents were analytical grade products purchased from POCh. Gliwice (Poland). Preparation of homogenate. mitochondria, uui c~,~tosol. Human term placenta tissue was obtained and homogenized as described previously (14). This crude homogenate was centrifuged at 500~ for 10 min and the supernatant obtained is called homogenate. Mitochondria were prepared essentially as described previously (14) and suspended in 0.2s M sucrose + 2 mM EDTA, pH 7.8. + 10 mM Tris-HCI (pH 7.8) at about 5 mg protein/ml. Postmitochondrial supernatant was centrifuged at 105.000,~ for 60 min and the resulting supernatant is called cytosol. Protein concentration in all the fractions was determined by biuret method. Assays of decarboxylation and transamination of r-leucine were carried out essentially as described by Odessey and Goldberg (12). The dehydrogenase activity was estimated by measuring trapped CO? in the combined transaminase-dehydrogenase reaction. To assay the decarboxylation of leucine 1 ml (about 5 mg protein) of homogenate, mitochondria, and cytosol were added to 1 ml of medium containing: 100 mM Tris-HCl (pH 7.8), 20 mM potassium phosphate buffer (pH 7.8). 4 mM 2-oxoglutarate. and 1 mM L-leucine plus 0.25 PCi L-[l-‘4C]leucine. The incubation was carried out for 30 min. The rates of decarboxylation and transamination of leucine in human placental subcellular fraction were linear for 30 min. The incubation was performed at 30” C in the Warburg vessel containing in the central reservoir 0.2 ml of hyamine hydroxide. The reaction was terminated by the addition of 1 ml 1 N sulfuric acid. After shaking for 60 min 100 ~1 of the hyamine hydroxide was transferred from the central reservoir to the scintillation liquid and the radioactivity was measured. Transaminase activity for leucine was determined in the conditions described for the decarboxylation of leucine as the sum of the CO: trapped by hyamine after the addition of sulfuric acid and of the product of transaminase action (4-methyl-2-oxo[ l-‘4C]pentanoate) formed from [ l“Clleucine. The later compound formed was measured as 14C02 liberated after 0.5 ml of 30% H,O? had been added. The CO, formed after addition of hydrogen peroxide was collected for 30 min and the radioactivity determined as described above. RESULTS The results presented in Tables 1 and 2 indicate that transaminase and dehydrogenase activities distributed in a similar way as in other tissues (10-12). Most of the transaminase activity was soluble since about 60% of the activity remained in the supernatant obtained after centrifugation at 105,OOOg for 60 min. About 30% of the total activity was recovered in the mitochondrial fraction. The overall recovery of the homogenate activity in the subcellular fractions was about 90%. Relatively high specific

LEUCINE

CATABOLISM TABLE

SUBCELLULAR

DISTRIBUTION

Subcellular fraction

OF L-LEUCINE

1

AMINOTRANSFERASE

Total activity/g wet tissue (pmole x min-‘)

% of total activity

8200 2620 4890

100 31.9 59.6 91.5

Homogenate Mitochondria Cytosol Recovery

143

IN PLACENTA

IN HUMAN

TERM PLACENTA

Specific activity (pmole/min/mg protein) 570 2900 470

Note. Transaminase activity was measured as described under Materials and Methods. Each value represents the mean of three separate experiments. Differences between separate experiments were below 5%.

activity of mitochondrial transaminase has made it possible to use 1-14Clabeled L-leucine for the study of dehydrogenase activity in this subcellular fraction. Unlike the transaminase, dehydrogenase activity was present mainly in the mitochondrial fraction, since above 90% of the activity was found in this subcellular compartment. The overall recovery of the homogenate activity in the subcellular fractions was about 95%. It should be pointed out that transaminase activity decreased by about 92% in the absence of 2-oxoglutarate (not shown). This suggested that transamination is the major pathway for leucine deamination in the human term placenta homogenate. Pyruvate used at 2 mM concentration was only about 20% as effective as 2-oxoglutarate in stimulating transamination. This indicates that 2-oxoglutarate is the preferred amino acceptor for leucine transamination in human placenta. Exogenous CoA and NAD were necessary for a maximal activity of the decarboxylation of L-leucine in the isolated mitochondria. NAD was not required for the decarboxylation of L-leucine in nondialyzed homogenate (probably because of the presence of this cofactor in the homogenate). The addition of carnitine, a cofactor in TABLE DECARBOXYLATION

Subcellular fraction Homogenate Mitochondria Cytosol Recovery

OF L-LEUCINE

BY SUBCELLULAR

2 FRACTIONS

Total activity/g wet tissue (pmole X min-‘)

% of total activity

54.5 52.4 1.2

100 96.1 2.2 98.3

OF HUMAN

TERM PLACENTA

Specific activity (pmole/min/mg protein) 4.3 84.5 0.12

Note. Decarboxylation of L-leucine was measured as described under Materials and Methods. Each value represents the mean of five experiments. Differences between separate experiments were below 10%.

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TABLE COFACTORS

REQUIREMENT

FOR L-LEUCINE

3

DECARBOXYLATION

IN HUMAN

TERM PLACENTA

Activity of leucine decarboxylation (Q of control) Compound omitted ( - ) or added ( + ) None t NAD (1 mM) + CoA (1 mM) + oL-Carnitine (2 mM) + Arsenite (2 mM) - Oxoglutarate

Homogenate 100 IO0 I? 9

Mitochondria IO0 136 lh8 II’ II) 7

Note. For experimental conditions see Materials and Methods. Each value represents the mean of three separate experiments.

leucine oxidation subsequent to the decarboxylation step ( 151, had only a slight affect on the decarboxylation of leucine by mitochondria. Arsenite a known inhibitor of the reaction of oxidative decarboxylation of 2oxoacids inhibited leucine decarboxylation almost completely. These results are presented in Table 3. DISCUSSION

The present results clearly demonstrate the existence of aminotransferase specific for leucine and cY-ketoisocaproic dehydrogenase in human term placenta. Subcellular distribution of these enzymes in human term placenta is very similar to that previously reported in pig heart ( 1I), rat liver ( 10). and skeletal muscle (12). Essentially similar results were obtained with early placenta (not shown). The activity of leucine aminotransferase in human placenta amounted to about 8 nmoleiminlg fresh tissue. This value is only slightly higher than in the liver but much lower than in the other tissues of rat (16). The rate of leucine decarboxylation is extremely low as compared with the leucine transmination. The former amounted to about 50 pmole/min/g fresh tissue. Therefore it is reasonable to think that decarboxylation of cY-ketoisocaproic acid is the rate-limiting step in leucine degradation in human placenta. Similar results have been reported for skeletal muscle (12). Activity of aminotransferase specific for leucine many times higher than that of a-ketoisocaproic acid dehydrogenase would lead to the accumulation of a-ketoisocaproic acid in the placental tissue. Then the a-ketoisocaproate could be transferred to the fetus or mother liver for further oxidation. In this respect the placenta might act as a temporary “muscle” for the fetus with regard to branched-chain amino acids metabolism.

LELJCINE

CATABOLISM

145

IN PLACENTA

SUMMARY It has been shown that L-leucine is transaminated in the presence of 2-oxoglutarate and subsequently decarboxylated by human term placenta. About 60% of the transaminase activity was recovered in the cytoplasmic fraction and the remaining amount in the mitochondria. The dehydrogenase activity is localized almost exclusively in the mitochondrial fraction. The rate of the transamination of L-leucine is many times higher than the rate of decarboxylation of oxoacid. The possible physiological role of leucine degradation in human placenta is discussed. ACKNOWLEDGMENT This work was supported by the Ministry of Science Higher Education and Technology within the Project R. I .9.03.&l.

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