Clin Biochem, Vol. 23, pp. 121-125, 1990 Printed in Canada. All rights reserved.

0009-9120/90 $3.00 + .00 Copyright o 1990 The Canadian Society of Clinical Chemists:

Electrophoretic and Kinetic Characterization of Three Variants of Soluble Cytoplasmic L-Alanine:2-Oxoglutarate Aminotransferase in Human Liver Tissue FUSAE KANEMITSU, 1 ISAMI KAWANISHI, 1 JUN MIZUSHIMA, 1 and TOHRU OKIGAKI 2 1Division of Clinical Laboratories, Kurashiki Central Hospital, 1-1-1 Miwa, Kurashiki, Okayama 710, Japan; 2Division of Cell Biology, Shigei Medical Research Institute, 2117 Yamada, Okayama 701-02, Japan Three common variants of soluble cytoplasmic L-alanine:2-oxoglutarate aminotransferase (ALT, EC 2.6.1.2), sALT 1, 2-1 and 2, were isolated from normal human liver, and characterized by electrophoretic and kinetic analyses. The isoelectric point of sALT 1 was pH 6.45. sALT 2-1 was focused into three bands with pl 6.1,6.2 and 6.45; sALT was focused into one band with pl 6.1. The electrophoretic mobilities of sALTs altered to the fast 13-globulin fraction after aging or papain treatment. Ammonia was produced during the latter, and the altered migration was considered to be caused by deamidation of sALT. The relative molecular mass of each of the enzymes was 110,000. Minor differences in the apparent Km values among the multiple forms for both L-alanine and 2-oxoglutarate were observed after incubation with 100 ~mol/L of pyridoxal phosphate (PALP). PALP stimulation of the enzyme activities was also different. sALT 1 was more stable than sALT 2-1 and 2 after heat and urea treatments. In human sera from 1065 adult Japanese, sALT 2-1, a heterozygote form of sALT 1 and 2, was dominant.

KEY WORDS: alanine aminotransferase, allele; electrophoresis; isoelectric focusing; L-alanine; LineweaverBurk plot; 2-oxoglutarate; pyridoxal phosphate. Introduction L

-alanine:2-oxoglutarate aminotransferase (ALT, EC 2.6.1.2) catalyzes the reversible reaction of L-alanine and 2-oxoglutarate to pyruvate and glutamate. Two isoenzymes of ALT are: a soluble cytoplasmic form and a mitochondrial form (1). Soluble cytoplasmic ALT (sALT) is a polymorphic enzyme (2), similar to cytoplasmic aspartate aminotransferase (3); 11 alleles including a silent one are known. The expressions of the alleles are designated as variants in the present study. More than 20 variants of sALT have been reported (2,4) and pedigrees of rare variants are also reported (2,5). sALT

Correspondence: F u s a e K a n e m i t s u , Division of Clinical

Laboratories, Kurashiki Central Hospital, 1-1-1 Miwa, Kurashiki, Okayama 710, Japan. Manuscript received March 31, 1989; revised August 29, 1989; accepted September 5, 1989. CLINICAL BIOCHEMISTRY, VOLUME 23, APRIL 1990

1 and 2, expressions of 2 autosomal alleles Gptl and Gpt2 (2), and their heterozygote, sALT 2-1, are the most common in humans (2,4). Although varied genetic data on sALT variants have been reported (2,4), their enzymatic characteristics have not been well studied. In the present work, sALT 1, 2-1 and 2 were isolated from normal human liver tissue and characterized. Materials a n d m e t h o d s CHEMICALS

Chemicals were purchased from the following sources: reagents for determination of sALT activity "GPT IFCC" and calibration proteins for molecular mass (Boehringer Mannheim, Mannheim, FRG); staining reagents for ALT activity (Iatron, Tokyo, Japan); Sephacryl S-300, DEAE Sephacel and pI markers (Pharmacia, Uppsala, Sweden); isogel and ampholytes (LKB, Bromma, Sweden); papain, pepsin, neuraminidase and concanavalin A (Sigma, St. Louis, MO, USA); glutathione and iodoacetamide (Wako, Osaka, Japan); and an ammonia determination kit "Determiner NH3" (Kyowa Medex, Tokyo, Japan). PARTIAL PURIFICATION OF

sALT

sALTs were partially purified from liver tissue obtained at autopsy from patients without liver diseases. Before purification, presence of variants had been confirmed by electrophoresis. Liver tissue was homogenized in four volumes of 50 mmol/L Tris-HC1 buffer, pH 7.4. The supernatant was separated by centrifugation at 100,000 x g for 30 min at 4 °C. The precipitate obtained by ammonium sulfate fractionation of the supernatant at saturation between 20-75% was dissolved in 200 mL of 500 mmol/L phosphate buffer, pH 7.4, and dialyzed against the Tris buffer overnight at 4 °C. The prepared 121

KANEMITSU, KAWANISHI, MIZUSHIMA, A N D 0KIGAKI

B

A

i

sALT 1

'

sALT 2-1

1

sALT 1" AA

sALT 2

: ' : " : ~ - ~ ; i ,~''~''~ "~" ~1 -"~ i -

-

r

0

+

I

Protein

6

a1 ab

-I-

Figure l--A: Electrophoretic patterns of three sALTs on Cellogel membrane. 0, sample application point. B: Changes of electrophoretic patterns of sALT 1 after storage at 4°C for 1 month (sALT 1').

material was chromatographed on a DEAE Sephacel ion-exchange column (5 x 50 cm) with a linear gradient of 0-300 mmol/L NaC1 at a flow rate of 100 mL/h. The effluent of sALT fractions was then applied on a Sephacryl S-300 column (5 x 50 cm). sALT was eluted with phosphate buffer at a flow rate of 20 mL/h and 5 mL fractions were collected. 100 t~mol/L of pyridoxal phosphate (PALP) was added to the enzyme preparation. The final specific activities were 26 U/mg, 20 U/mg and 24 U/mg for sALT1, 2-1 and 2, respectively. ASSAY METHODS

sALT activity was determined by a kinetic rate assay, "GPT IFCC" (6), on a GILFORD Impact 400E at 30 °C. sALT was electrophoresed at 130 V for 2 h on Cellogel (Chemetron, Milano, Italy) with 40 mmol/

pH 6.85 ]

6.45 6.2 6.1 5.85 !

!

l

L

sALT 1 sALT 2-I

i

L barbital-HC1 buffer, pH 8.4, and stained with reagents containing 800 mmol/L D,L-alanine, 11 mmol/L 2-oxoglutarate, 0.1 mmol/L methoxy-5-methylphenazinium methylsulfate, 1.0 mmol/L nitro blue tetrazolium, 32 mmol/L 13-nicotinamide adenine dinucleotide and 40,000 U/L glutamate dehydrogenase (GLDH) in 0.1 mol/L phosphate buffer, pH 7.4 at 37 °C for 30 min. Isoelectric focusing was done on supporting matrices, using an isoelectric focusing system (Joko, Tokyo, Japan). Agarose (124 x 258 x 0.5 mm) consisted of 1% (w/v) isogel and 2% ampholytes (1% of pH 3.5-5 and 5-8.0); 15 mU of sALT was applied per plate. As the catholyte and anolyte, 0.5 mol/L ethanolamine and 0.2 mol/L citric acid, respectively, were used. Focusing was done at 500 V for 10 min at 4 °C, 800 V for 15 min, 1200 V for 60 min and finally at 1300 V for 30 min. ALT bands were stained with reagents for ALT isoenzymes at 37 °C for 30 min. Relative molecular mass was estimated using 8-30% polyacrylamide gradient gel slabs; 15 mU of sALT was applied to the gel and electrophoresed at 100 V for 60 min, 300 V for 120 min, 400 V for 60 min and, finally, at 500 V for 60 min with 12.5 mmol/L Tris-EDTA buffer, pH 9.18. Separated sALT and calibration proteins were electrophoretically blotted on a nitrocellulose membrane at 30 mA for 30 min with 25 mmol/L Tris-HC1 buffer, pH 8.8, at 4 °C and stained for sALT activity, and for proteins using 0.025% coomassie brilliant blue. Ammonia was determined with an enzymatic method (7) at 340 nm. Results

-

+

Figure 2--Isoelectric focusing patterns of sALTs on agarose plates. Fifteen mU of sALTs and 1-2.5 Ixg of each marker protein were applied on the plate. Myoglobinbasic band (pI 6.85), human carbonic anhydrase B (pI 6.55) and bovine carbonic anhydrase (pI 5.85) were used as markers.

122

and discussion

Electrophoretic patterns of three sALTs from liver extracts on a Cellogel membrane are shown in Figure 1A. sALT i was electrophoresed to a sharp band and sALT 2-1 to a broad band in the [3-globulin region, sALT 2 ran to a single band anodal to sALT 1. The identical electrophoretic patterns were con-

CLINICAL BIOCHEMISTRY, VOLUME 23, APRIL 1990

VARIANTS OF SOLUBLE CYTOPLASMIC L-ALANINE:2-OXOGLUTARATE AMINOTRANSFERASE

A sALT

B sALT I

I

i

B

Papain 25 gg/mL

Papain sALT 2-i Papain sALT 2

75 gg/mL

Papain

d

100gg/mL

÷

6

0

+

Figure 3 - - A : A l t e r a t i o n of electrophoretic p a t t e r n s of sALT 1 after papain t r e a t m e n t at 37 °C for 1 h. B: A l t e r a t i o n of electrophoretic p a t t e r n s of sALTs after 100 ~g/mL papain t r e a t m e n t (Papain).

sistently obtained from fresh extracts of the liver. Therefore, the patterns were not due to modification of sALTs which occured during the purification processes. The isoelectric point of sALT 1 was pH 6.45. sALT 2-1 was divided into three bands with pI 6.1, 6.2 and 6.45; sALT 2 moved as one band with pI 6.1 (Figure 2). The results indicate that sALT 1 and 2 are molecularly homogeneous, and sALT 2-1 is heterogeneous because it is a heterozygote form of sALT 1 and 2. The electrophoretic mobility of sALT 1 altered in vitro and an anodal band appeared after storage at 4 °C for one month (sALT 1' of Figure 1B). One of the most frequent causes of enzyme mobility alteration toward the anode is deamidation due to aging (811). To confirm this, sALTs were treated with papain. Electrophoretic patterns of sALT 1 after incubation with 25-100 ~tg/mL papain at 37 °C for 60 min are shown in Figure 3A. As the amount of papain increased, additional bands appeared at the anodal

side of sALT 1, and the original enzyme band finally migrated anodal to native sALT 1. In this process, ammonia was produced in the sALT 1 preparation (Table 1). The altered migration was similarly observed in sALT 2-1 and 2 with papain treatment (Figure 3B); but, the alteration was not observed after incubation of papain at 60 °C for 30 min with inhibitors of proteolysis: 1 mmol/L benzamidine, 1 mmol/L phenylmethyl-sulfonyl fluoride, 10 mmoUL N-ethylmaleimide and 25 mmol/L EDTA (12). The electrophoretic mobilities of the three sALT variants did not alter after treatment with neuraminidase, concanavalin A, glutathione or iodoacetamide, sALT activities were completely inactivated by pepsin. Papain is an endopeptidase, although it has deamidase activity (13). Inhibition of the proteolytic activity of papain rendered it incapable of altering sALT migration; this suggests that this alteration might be due to the proteolytic activity of papain.

TABLE 1 A m m o n i a Production After P a p a i n T r e a t m e n t of sALT 1. Mean ± SD of Five E x p e r i m e n t s A m m o n i a Concentrationa Determined Produced Before p a p a i n t r e a t m e n t After p a p a i n t r e a t m e n t c 50 ~g/mL 75 ~g/mL

sALT 1 Activity b

144.0 - 0.6

0

2215 - 5

149.9 -- 0.6 151.8 -+ 0.5

5.7 ± 0.9 7.6 - 0.8

2211 _ 5 2174 ± 5

a~mol/L; bU/L; CsALT was incubated with 50 ~g/mL or 75 ~g/mL o f p a p a i n at 37 °C for 60 min.

CLINICAL BIOCHEMISTRY, VOLUME 23, APRIL 1990

123

KANEMITSU, KAWANISHI, MIZUSHIMA, AND OKIGAKI

H o w e v e r , the i n h i b i t i o n condition was severe. T h e r e fore, d e a m i d a t i o n activity m i g h t also be i n h i b i t e d d u r i n g t h e i n h i b i t i o n process. Anodal m i g r a t i o n of both a m y l a s e and c r e a t i n e k i n a s e are due to deamidation by d e a m i d a s e activities of p e p t i d o g l u t a m i nase (10) a n d c a r b o x y p e p t i d a s e (11), respectively. Table 1 shows t h a t u n d e r conditions of p a p a i n treatm e n t leading to m a r k e d a l t e r a t i o n in electrophoretic mobility, v i r t u a l l y no loss of e n z y m e activity was detected, s u g g e s t i n g t h a t proteolysis is unlikely. B y contrast, significant a m m o n i a production was noted, consistent w i t h the finding t h a t d e a m i d a t i o n is the principal cause of a l t e r e d mobility. A f t e r 100 ~g/mL p a p a i n t r e a t m e n t , the t h r e e v a r i a n t s were focused into two b a n d s w i t h pI 6.1 a n d 6.25. T h e s e isoelectric points differed from those of the original sALTs. Thus, sALT 2-1 and 2 w e r e not considered to be d e a m i d a t e d forms of sALT 1. F i g u r e 4 shows blotting p a t t e r n s of sALTs w i t h m a r k e r proteins on a nitrocellulose m e m b r a n e a f t e r electrophoresis on a 8 - 3 0 % p o l y a c r y l a m i d e g r a d i e n t gel. T h e sALTs of different origins r a n as a single b a n d b e t w e e n a l b u m i n (Mr 67,000) a n d lactate deh y d r o g e n a s e (Mr 140,000). T h e r e l a t i v e m o l e c u l a r mass of t h e e n z y m e s was e s t i m a t e d to be 110,000 +_ 5,000 ( m e a n _+ SD). Thus, the v a r i a n t s of s A L T were of identical m o l e c u l a r mass. sALT 1 and 2 were a c t i v a t e d to 1.5 t i m e s by PALP; however, the a c t i v a t i o n of sALT 2-1 was 1.1 t i m e s (Table 2). Thus, a c t i v a t i o n of sALT 2-1 by

MI" r

1232k -140k 67k

llOk •

+

Figure 4--Blotting patterns of sALTs on nitrocellulose membrane following 8-30% polyacrylamide gradient gel electrophoresis. Fifteen mU of sALTs and 5-7 ~g of each marker protein were applied on the gel. Albumin (Mr 67,000), lactate dehydrogenase (Mr 140,000) and catalase (Mr 232,000) were used as the calibration standards. P A L P was s m a l l e r t h a n of the o t h e r two v a r i a n t s . A p p a r e n t K m v a l u e s of sALTs for the s u b s t r a t e s a f t e r i n c u b a t i o n w i t h 100 ~mol/L P A L P at 37 °C for 30 m i n are also listed in T a b l e 2. U n d e r this condi-

TABLE 2 Apparent Km Values of Three sALT V a r i a n t s for L-Alanine and 2-Oxoglutarate, Activation by Pyridoxal Phosphate (PALP) and Residual Activities After Heat and Urea T r e a t m e n t s sALT 1

sALT 2-1

sALT 2

(mean +- SD) Apparent K m values" (mmol/L) L-alanine

43.1

+- 0.6

39.5

I

2-oxoglutarate Activation by added PALP b (%)

Residual activity (%) Heating ~

0.44 ± 0.01 I

154.1

- 0.6

39.2 - 0.2 I

Urea a

40.3 -+ 0.5 I

-+ 1.1

34.0

,

!

0.56 - 0.01 II

,

113.7

- 0.3

23.3 --- 0.4 ,

30.5 -+ 0.7 ,

- 0.8

0.65 -+ 0.01 I

,

148.5

-+ 0.5

19.6 - 1.0 I

27.9 - 0.8 I

*p < 0.05. "Lineweaver-Burk plots were drawn with data of five experiments with different samples at 30 °C. bData of four experiments. Initial enzyme activities were 197 U/L, 324 U/L and 202 U/L for sALT 1, 2-1 and 2, respectively, and preparations were incubated with 100 ~mol/L PALP for 30 min at 37 °C. CData of four experiments, sALTs were heated at 56 °C for 60 min. 100% activities were 463 U/L, 481 U/L and 338 U/L for sALT 1, 2-1 and 2, respectively. dData of four experiments, sALTs were incubated in 4 mol/L urea at 27 °C for 120 min. 100% activities were 524 U/L, 527 U/L and 504 U/L for sALT 1, 2-1 and 2, respectively.

124

CLINICAL BIOCHEMISTRY, VOLUME 23, APRIL 1990

VARIANTS OF SOLUBLECYTOPLASMICL-ALANINE:2-OXOGLUTARATEAMINOTRANSFERASE tion, apo-sALT is completely altered to holo-sALT (6). Differences in the K m values between sALT 1 and 2 for L-alanine were revealed (p