β-Aminoisobutyrate-α-ketoglutarate transaminase in relation to β-aminoisobutyric aciduria

374

BIOCHIMICA ET BIOPI-IYSICA ACTA

BBA 2 5 9 2 3

/3-AMINOISOBUTYRATE-e-KETOGLUTARATE TRANSAMINASE IN R E L A T I O N TO fi-AMINOISOBUTYRIC A C I D U R I A YASUO KAKIMOTO, A K I R A KANAZAWA, KAZUMI T A N I G U C I t I AXD ISAMU SANO

The Department of Neurology, The Institute of Higher Nervous Activity, Osaha University Medical School, Fukushima-ku, Osaka and The Department of Neuropsychiatry, Osaka University Medical School, Fukushima-ku, Osaka (Japan) (Received September ISth, I967)

SUMMARY

fl-Aminoisobutyrate-c~-ketoglutarate transaminase described b y I{UPIECKI AND COON was further purified from hog kidney. The enzyme catalyzed the transamination with its L isomer while the D isomer, the natural form, was practically inactive as the substrate. The activity of t h e enzyme in the kidney of an hereditary high excretor of/3-aminoisobutyric acid was not different from the activities of the low excretors. These findings indicate that the enzyme is not the principle enzyme catalyzing the degradation of the amino acid in vivo, and suggest the occurrence of another enzyme reaction b y which D-fl-aminoisobutyric acid is metabolized.

INTRODUCTION

fi-Aminoisobutyric acid (AIB) is excreted constantly in a large amount b y a certain percentage of the human population, and the trait probably results from the homozygosity of a single pair of recessive alleles. The rate of high excretors is about 4o % in Oriental populations while only a few percent of Caucasians are in this category. Previous investigations concerning the cause of excessive excretion generally, support the hypothesis that high excretors lack the ability to break down AIB l& The only enzyme which has been described as participating in the metabolism of AIB is AIB-~-ketoglutarate transaminase, reported b y KUPIECKI AND COON3, although the importance of the transamination in AIB catabolism in humans or in other mammals is not known. A question arising from the investigation of KUPIECKI AND COON3 is which optical isomer of AIB is the actual snbstrate of the enzyme since the substrates used in their experiments were racemic. I52AKIMOTOAND ARMSTRONG~ have isolated D(--)-AIB from the urine of excretors, and the same isomer was later isolated from human liver b y KAKIMOTO, KANAZAWA AND SANO5. Loading experiments b y ARMSTRONG et al. 2 showed that D(--)-AIB was quantitatively recovered in urine of excretors while the recovery of this optical isomer was lower in non-excretors. L ( + ) - A I B was catabolized in the high excretors as rapidly as in low excretors. This finding implies that D- and L-AIB are metabolized b y different enzymes. Abbreviation: AIB, fl-aminoisobutyric acid.

Bioehim. Biophys. Aeta, 156 (1968) 374-380

fi-AMINOISOBUTYRATE--~-KETOGLUTARATE TRANSAMINASE

375

The present paper describes the purification and properties of AiB-z-ketoglutaric acid transaminase from hog kidney and the activity of the enzyme in human kidney. METHODS AND MATERIALS

Material I)(--)-, and L ( + ) - A I B were prepared as described previously 4. D(--)-A:B was Mso isolated from human urine after thymine loading for the study of substrate specificity. The isolation method will be described in a later paper, Fresh hog kidney was supplied b y Nipponham Co., and acetone powder was prepared immediately. The powder was stored at --20 ° , Urine and autopsy specimens of human kidney were kindly supplied by the Department of Forensic Medicine, Osaka University Medical School.

Determination of amino acids Amino acids were determined with an automatic amino acid analyzer, Yanagimoto Model CL 5. AIB in urine was determined on a 7o-cm co!urnn with o.38 M sodium citrate buffer (pH 4.26). Determination of AIB could be completed within :oo rain and identity and purity of the peak of AIB were confirmed by paper chromatography of a fraction corresponding to this peak and b y its dose similarity to the one determined b y paper electrophoresis or b y a routine method of amino acid analysis% Glutamic acid was measured on a 7 o- or 3o-cm column with o.eo M sodium citrate buffer (pH 3.25). A 3o-cm column was used for purified enzyme preparations for which incubation did not show any detectable formation of amino acids other than glutamic acid.

Assay of the enzyme activity The standard reaction mixture contained Io #moles of L-AIB, Io #moles of ~-ketoglutaric acid solution neutralized with potassium bicarbonate to p H 7.5, 2o #moles of sodium borate buffer (pH 8.6) and enzyme solution. The final volume was adjusted to 0.5 mt with water. The mixture was incubated at 37 ° for 3o rain and the reaction was stopped b y the addition of 0.5 mi of Io % trichloroacetic acid. An aliquot was taken for the determination of glutamic acid. AIB was omitted from_ an incubation mixture which was used as a blank for crude enzyme preparations. Glutamic acid was not detected in a blank tube when the enzyme was purified beyond Step : (see below).

Preparation of AIB-c<-ketoglutarate tra~saminase All the procedures were performed at 0-2%

Step :. Preparation of crude extract of hog kidney. 24 g of acetone powder of hog kidney cortex were stirred with 15 vol. of o.o5 M potassium phosphate buffer (pH 7-5) for I h, and centrifuged at I o o o o x g for 2o rain. The supernatant solution was brought to 5o % saturation with solid ammonium sulfate, left for 3o rain and centrifuged. The supernatant solution was brought to 75 % saturation with ammonium_ sulfate, the precipitate was collected b y centrifugation after 3o rain, and dissolved in 24 ml of the phosphate buffer. The solution was desalted on a column of Sephadex G-25 equilibrated with the same buffer. Biochim. Biophys. Acts, :55 (:968) 374-380

Y. KAKIMOTOet al.

376

Step 2. Heat treatment and ammonium sulfate precipitation. To the above solution, 3 mM cysteine hydrochloride, 20 mM/5-alanifie and I mM pyridoxal phosphate were added. The solution was heated for 2 mill at 58° and cooled quickly to 2 o. Tile precipitate was removed b y centrifugation, a fraction of the supernatant solution which precipitated between 50 and 70 % saturation of ammonium sulfate was collected, the precipitate was dissolved in o.oz M potassium phosphate buffer (pH 7.5) and was desalted on a column of Sephadex G-25 equilibrated with the same buffer. Step 3. Treatment with DEAE-cdlulose. The solution was passed through a 2 cm × 24 cm column of DEAE-cellulose equilibrated with o.oi M phosphate buffer (pH 7.5) and protein was eluted with the same buffer. 18 ml of the effluent were collected immediately after the emergence of the front of the reddish colored band. Step 4. Treatment with CM-Sephadex and ammonium sulfate precipitation. The effluent was applied to a 2 cm × 2I cm column of CM-Sephadex equilibrated with o.oi M potassium phosphate buffer (pH 7.5). The column was washed with the same buffer, and a fraction of the effluent between 25 and 65 ml was collected. Ammonium sulfate was added to 6o % saturation and, after 3o-min stirring, the precipitate was removed b y centrifugation. The supernatant solution was brought to 70 % ammonium sulfate saturation and the precipitate was collected b y centrifugation. The solution of the precipitate was desalted with a Sephadex G-25 column equilibrated with 0.05 M potassium phosphate buffer (pH 7.5). This solution could be stored at --20 ° for at least 3 weeks without loss of activity and was used for most of the experiments reported here. D a t a on the purification are presented in Table I. TABLE I PURIFICATION OF AIB--C¢-I(ETOGLUTARATE TRANSAMINASE

Step

Total activity Recovery (#moles (%) glutamic acid per h}

Protein (mg)

Specific activity (#moles glutamic a~id per h per mg protein)

I. E x t r a c t of acetone p o w d e r 2. A m m o n i u m sulfate fractionation 3. H e a t t r e a t m e n t and a m m o n i u m sulfate fractionation 4- DEAE-cellulose 5- CM-Sephadex a n d a m m o n i u m sulfate fractionation

620 248

ioo 4°

9050 151o

0.069 o.164

544 554

88 90

606 204

o.9o 2.71

302

49

27.2

i i. i

Assay of A IB-ct-ketoglutarate transaminase in human kidney 50 g of kidney cortex were homogenized with 20o ml of 0.05 M potassium phosphate buffer (pH 7.5) and the homogenate was acidified to p H 6.o and centrifuged at ILOOO × g for 15 rain. The supernatant solution was brought to p H 7.5 with I M NaOH. A fraction of the solution which precipitated between 4 ° and 7 ° % saturation of ammonium sulfate was desalted with a 2 c m x 5o cm column of Sephadex G-25 which had been equilibrated with o.oi M potassium phosphate buffer (pH 7-5)- The desalted solution was passed through a 3 c m x 9 cm column of DEAE-cellulose equilibrated with the above buffer, and the proteins were eluted with IOO ml of the Biochim. Biophys. Acta, 156 (I968) 374-380

fi-AMINOISOBUTYRATE--o~-KETOGLUTARATE TRANSAMINASE

~77

same buffer. Proteins in the eluate which precipitated between 5o and 7o % saturation of ammonium sulfate were collected, desalted on Sephadex G-25 equilibrated with o.o5 M potassium phosphate buffer (pH 7-5)~ This procedure yielded 35 to 4 ° mt of a reddish solution, o.5 ml of this enzyme preparation was incubated at 37 ° for i h with io #moles of D- or L-AIB and zoo #moles of ~-ketoglutarate neutralized to p H 7.5 with potassium bicarbonate, in a final volume of o.8 ml. AIB was replaced with water in a blank tube. Glutamic acid was determined with an automatic amino acid analyzer, using a 7o-cm column. RESULTS

Pr@erties of purified A IB-c~-ketoglutamte tm~¢saminase (z) Substrate specificity KUPIECKI AND COON~ have reported that the enzyme catalyzes the transamination between e-ketoglutaric acid and several m-amino acids including DL-AIB. The present results shown in Table I I agree with their result, but most important is the finding that the transamination with AIB occurred almost exclusively with L-AIB. Activity for D-AIB was less than 1 % of t h a t of L-A[B. The activity against L-AIB was parallel to ~-AIB during the purification of the enzyme, There remained the possibility that a slight activity with regard to D-AIB is due to a contamination of T A B L E II S U B S T R A T ] ~ SPI~CIFICITY OF

A~I~-KI~TOGLUTARATI~ TRANSAIMINASE

Subslrate

Relative activity *

e)-Amino acids

L-AIB D-AIB (synthetic) D-AIB (natura!) fl-Alanine y - A m i n o b u t y r i c acid d-Amino-n-valeric acid e-Aminocaproic acid fi-Aminobutyric acid a-Ethyl-fl-alanine

•oo 0.8 o. 4 99 92 33 6 o.5 o.o

a - A m i n o acids

Glycine Alanine ~-Aminoisobutyric acid

o.o o.o o.o

~- a n d m-Diamino acids

Ornithine Lysine

4.6 o.o

O t h e r amino acids

Taurine O-Phosphoethanolalnine Homocarnosine

o.o o.o o.o

Amines

n-Butyramine Putrescine Spermine Ethanolamine

o.o o.o o.o o. o

* Ratio of the a m o u n t of glutamic acid formed in presence of a substrate to Jcha~ of L-AiB.

Biochim. Biophys. Acta, i56 (I968) 374-38o

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the D-AIB preparation by L-AIB, since the racemic form is more readily crystallized after the chemical resolution. In order to minimize contamination by L-AIB, I)-AIB was prepared from human urine. Production of glutamic acid was observed even with the natural material but in a significantly smaller amount. It may still be premature to conclude from this result that the transaminase is not the enzyme which catalyzes the catabolism of D-AIB in vivo since the experiment of ARMSTRONG et al. showed that D-AIB is degraded extremely slowly in comparison to L-AIB in the hmnan body. An interesting finding of the present study is that the enzyme preparation catalyzes transamination of co-amino acids having carbon chains between 2 (fl-alanine) and 5 (e-amino caproic acid), but not a-amino acids. Glycine, L-alanine and a-aminon-butyric acid did not serve as substrates. Even with the/5-amino acid structure, amino acids having a branched carbon chain, such as a-ethyl-3-alanine, r-aminobutyric acid (fi-ethylfl-alanine), were not metabolized by the enzyme. The presence of a carboxyl group seems obligatory: replacement of the carboxyl group with a sulfonate group (taurine), or a phosphate group (phospho-ethanolamine), the lack of a carboxyl group (n-butylamine, spermine and putrescine), and a peptide linkage with a carboxyl group (homocarnosine) all caused complete loss of activity. These findings imply that there is a specific enzyme which catalyzes the transamination between ~-ketoglutaric acid and co-amino acids. Pyruvic, oxaloacetic and glyoxylic acids were inactive.

(2) Properties of the enzyme Fig. I shows enzyme activities at various pH values. Maximal activity was observed at pH 9.I. A high concentration of a-ketoglutaric acid had an inhibitory effect. The addition of pyridoxal phosphate to the reaction mixture had a slight stimulatory effect at a low concentration (usually less than 5" Io-4 M), and was inhibitory at a higher concentration. But the behavior of the enzyme toward the cofactor differed from preparation to preparation. When enzyme activities were plotted against concentration of the enzyme, a linear relation was not obtained (Fig. 2). The activity per unit weight of protein differed with the enzyme concentration, as was observed in a preparation of y-aminobutyric acid-c~-ketoglutaric acid transaminase by WAICSMAN AND ROBERTSv. No attempt was made to elucidate the mechanism of this phenomenon.

Activity of the transaminase in human kidney An hereditary AIB non-excretor degrades D-AIB while the excretor does not. If indeed it is an enzyme which catalyzes the degradation of AIB present in the human body, then this enzyme is probably found only in the tissue of the nonexcretor. Urine was obtained from the urinary bladder of humans who had died without chronic disease, kidney affection, or intoxication. Kidney was simultaneously obtained and used immediately for enzyme purification and assay of the activity without an interval of storage. The results are summarized in Table III. In a separate experiment in our laboratory the concentrations of AIB were determined in about Iooo specimens of urine and a plot of the population against the concentration showed a clear bimodal distribution having two peaks at I I and 9°/~moles and the antimodal at 0.33 #mole/rag of creatinine. The analysis of families was consistent with the hypothesis that the characteristic of AIB excretion results from homozygosity of a single pair of recessive alleles using the above value to separate Biochim. Biophys. Acts, 156 (I968) 374-38o

fi-AI'ffINOISOBUTYRATE-c~-KETOGLUTARATE TRANSAMINASE

~7~

t h e e x c r e t o r s f r o m n o n - e x c r e t o r s . C a s e s i , 2 a n d 3 a r e t h u s classified as n o n - e x c r e t o r s a n d 4 as a n e x c r e t o r . T h e a c t i v i t i e s of A i B - a - k e t o g l u t a r i c a c i d t r a n s a m i n a s e "n t h e k i d n e y s d i d n o t differ s i g n i f i c a n t l y i n t h e s e f o u r cases.

c

~

4

/

3

u

L

e~

-I

2~

e~

E

""

G

K 2

4

/

•

0 6

7

8

9

I0

02

I[

D4 mg

PH

Q6

enzyme per rude

Fig. i. p H dependence of the AIB-~-ketoglutarate transaminase. The reaction mixtures consisted of 5o #moles cz-ketoglutaric acid, IO #moles L-AIB, o.i ml buffer solution, o.2 M potassium phosp h a t e (pit 6.1 to 8.45), o.2 M sodium borate (pH 8.6 to 9.4), o.2 M sodium b o r a t e - o . 5 Ni NaOH (pH 9.7 to io.8), and o.2 ml of enzyme solution (o.23 mg protein) in a final volume of 0. 5 mi, a n d incubated at 37 ° for 3 ° min. Fig. 2. Enzyme concentration and its activity. The reaction mixtures consisted of zo #moles of ~-ketoglutaric acid, IO ffmoles of L-AIB, o.I ml of 20 ffmoles sodium borate buffer (pH 8.6) and o.I ml of enzyme solution diluted with o.oi M phosphate buffer (pH 7.5) and were incubated for 3 ° min for 37 °. T A B L E Ill CONCENTRATION O F AIB IN ~IU~AN U R I N E AIVflNASE IN AUTOPSY SPECIMENS OF THE

AND THE ACTIVITY HUMAN KIDN]~Y

OF ~[B--
TRANS-

Case

Age

Sex

Cause of death

Conch. of A I B in urine (#mole of A I B per mg ereatinine)

Enzyme acgvily (#molesof glutamic acid formed per h per g of wet tissue)

i 2 3 4

8o 35 47 84

Male Male Male Male

Rupture of aorta Heart attack H e a r t attack Cerebral vascular accident

o.oo o.o2 o. 16 o.56

6. 9 26. 7 i4.6 23. 4

DISCUSSION I t is c l e a r t h a t A I B - a - k e t o g l u t a r a t e t r a n s a m i n a s e p l a y s n o i m p o r t a n t role i n t h e m e t a b o l i s m of D-AIB, t h e n a t u r a l f o r m of AIB, i n h o g a n d h u m a n k i d n e y , as i n d i c a t e d b y t h e f a c t s t h a t t h e e n z y m e is p r a c t i c a l l y i n a c t i v e a g a i n s t D - A I B (less t h a n 1 % of t h e a c t i v i t y a g a i n s t L - A I B ) , a n d t h a t t h e k i d n e y of a A I B e x c r e t o r w h o l a c k s t h e a b i l i t y t o m e t a b o l i z e D - A I B e x h i b i t s of A I B - ~ - k e t o g l u t a r a t e t r a n s a m i n a s e

BioeMm. Biophys. dcta, I56 (i968) 374-38o

380

¥. KAKIMOT0 et al.

activity similar to that found in non-excretors. Another unknown enzyme should occur which is involved in the metabolism of D-AIB. An important fact obtained from loading experiments by ARMSTRONG et al. ~ is that the genetic excretor excretes a load of AIB quantitatively. Previous studies and our recent study on inheritance of the excretion showed that the trait of excretion is determined by a single pair of alleles, indicating in turn that there is only one major pathway for the metabolism of D-AIB in human tissue. Our recent interest was directed to the discovery of the enzyme which catalyzes the degradation of D-AIB and an enzyme reaction was found which is almost exclusively specific for D(--)-AIB. The results will be published soon. Another problem of enzymological interest is whether AIB-a-ketoglutarate transaminase is the same as one of the other transaminases catalyzing the reaction between various a-amino acids and ~-ketoglutaric acid. The relations between the transaminases described by various authors are obscure either because the enzyme preparations were not pure enough or because the substrate specificity was not sufficiently investigated. The preparation examined in the present study showed a limited substrate specificity of AIB-a-ketoglutarate transaminase for several co-amino acids. Among the transaminases, 7-aminobutyrate-a-ketoglutarate transaminase has been studied most extensively from the neurochemieal standpoint, but no information is available about the identity of this enzyme with other enzymes occurring in liver and kidney. This problem is now being studied in our laboratory. ACKNOWLEDGEMENT

This work was supported by grant No. AM 1Io42-o2 from the U.S. Public Health Service. REFERENCES i S. M. GARTL~:R, Am. J. H u m a n Genet., II (1959) 257. 2 M. D. ARMSTRONG, K. YATES, Y. KAKIMOTO, I(. TANIGUCHI AND T, KAPPE, J. Biol. Chem., 238 (1963) 1447. 3 F. P. IKUPIECKI AND M. J. Cool;, J. Biol. Chem., 229 (I957) 743. 4 Y- I~AKIMOTO AND M. D. ARMSTRONG, dr. Biol. Chem., 236 (1961) 3283 . 5 Y. I~AKIMOTO, A. I4~ANAZAWAAND I. SANO, Biochim. Biophys. Acta, 97 (1965) 376. 6 D. H. SPACEMAN,W. H. STEIN AND S. MOOR, Anal. Chem., 3 ° (~958) 119I. 7 A. WAKSMAN AND E. ROBERTS, Biochemistry, 4 (1965) 2132-

Biochim. Biophys. Acta, 156 (I968) 374-380