Characteristics of pancreatic branched chain amino acid aminotransferases from rat, man and dog

Camp.Biochem.Physiol.Vol. 778,No. I, pp. 175-180, 1984 0

Printed in Great Britain

0305-0491/84 $3.00 + 0.00 1984 Pergamon Press Ltd

CHARACTERISTICS OF PANCREATIC BRANCHED CHAIN AMINO ACID AMINOTRANSFERASES FROM RAT, MAN AND DOG MINORU MAKINO, YOHSUKEMINATOGAWA,ETSUOOKUNO and RYO KIDO Departmentof Biochemistry,WakayamaMedical College,Wakayama640, Japan (Received24 June 1983) Abstract-l. Branchedchain amino acid aminotransferase (EC 2.6.1.42)from pancreasof rat, man and dog was highly purified and characterized. 2. Rat pancreascontainedonly one type of branchedchain amino acid aminotransferase, but human and dog pancreascontainedtwo types.Thesewere namedEnzyme I and EnzymeIII accordingto the classificationby Aki et al. (1969). 3. Five enzymespurified were activatedby the addition of 2-mercaptoethanol and glutathione. 4. EnzymeIII has lower K, valuesfor amino acids and 2-0~0 acids than Enzyme I. 5. Some other propertiesof purified enzymesare described.

SeikagakuKogyo, Tokyo, Japan, hydroxyapatite;Boehringer AG, Germany,Tris (hydroxymethyl)-aminomethane, from beefliver; Sigma,St. Louis, Branched chain amino acid aminotransferase glutamatedehydrogenase (EC 2.6.1.42)catalyzingtransaminationbetweenva- U.S.A., glutathione (reducedform). The other chemicals line, leucine, or isoleucineand 2-oxoglutaratewas usedwere of the highestgradecommerciallyavailable. INTRODUCHON

first isolatedfrom hog heart (Ichihara and Koyama, 1966;Taylor and Jenkins,1966a).It is elutedfrom a DEAE-cellulose column with 0.02M phosphate buffer (Enzyme I). In hog brain, however,there is anothertype of enzymewhich has similar properties to the heart enzyme,being eluted from the DEAEcellulosecolumn by 0.2 M phosphatebuffer (Enzyme III) (Aki et al., 1969).Hog heart and brain contain only one type of enzyme,while liver and kidney containtwo types.Aki et al., (1968)havereportedthat branched-chainamino acid aminotransferasehas three types (I-III), which are distinguishableby DEAE-cellulosechromatography.EnzymesI and III are active for leucine, isoleucine and valine, but EnzymeII is activeonly for leucinewhich existsin rat liver. They have reported also that branchedchain amino acid aminotransferase activity in pancreasand stomachis 8-10 timeshigherin comparisonwith that in heart and kidney (Ichiharaet al., 1975).They have demonstratedalso that rat pancreascontainedone enzymeand human pancreastwo enzymes(Goto et al., 1977).So far hog and rat enzymeshave been extensivelystudied(Ichihara et al., 1975;Goto et al., 1977;Taylor and Jenkins, 1966b,1966~;Aki et al., 1967,1968,1973).Branchedchainamino acid aminotransferasefrom other speciesand rat pancreatic enzyme,however,has not beenelucidated. In order to understandthe physiologicalrole of the enzymein pancreas,its characterizationis required. In the presentpaper,we describethe purification and characterizationof pancreaticbranchedchain amino

Animals Human pancreasewas obtainedas soon as possibleafter

autopsyfrom theDepartment of Pathologyof this school. MaledogsCanisfamiliurislinnaus(mixed breed,10-12kg), male rats Rattusnoruegicus albus(Wistar strain, IO&120g) weremaintainedat about 20°C in a room. Food and water were availablead libirum. Enzymeassay The assaymixture contained,unlessspecifiedotherwise; 40mM L-aminoacid, IOmM 2-oxoglutarate, 4OpM pyridoxal 5’- phosphate, 0.1 M potassium phosphatebuffer, pH 8.0 and enzymepreparation.Transaminationbetween L-branchedchain amino acid (leucine,isoleucineand valine) and 2-oxoglutaratewas in principle assayedby the method of Taylor and Jenkins(1966b).The assavof transamination betweenaromatic L-amino acids (Noguchi et al., 1976)or other L-amino acids (Nakatani et al., 1970) and 2-oxoglutarateweredeterminedas cited reference.A unit of enzymeactivity is defined as the amount of enzymethat catalysesa formation product or a decreasein substrateof 1 pmol/min at 37°C. PuriJicationof enzymes All manipulationswerecarried out at 0-4”C exceptheat treatment.Distilled and deionizedwater was used.Potassium phosphatebuffer, pH 7.5, containing 2OOyM pyridoxal 5’- phosphate,10mM 2-mercaptoethanoland 1 mM 2-oxoglutaratewasusedthroughoutunlessstatedotherwise. L-Leucine-2-oxoglutarateaminotransferaseactivity was monitored through the enzymepurification.

Rat enzyme One hundred and twenty wistar strain male rats acid aminotransferase from rat, man and dog. (200-250g body wt) wereused.The pancreaswas homogenized with 5~01 of 5mM buffer by Waringblenderand MATERIALS AND METHODS sonicated at 10kHz for 4min. After centrifugation at Materials 105,000 g for 30min, the resultingsupematantsolution was The following sourcessupplied chemicals: Pharmacia, added to 2OOml of DEAE-Sepharoseequilibrated with Uppsala,Sweden,DEAE-Sepharoseand SephadexG-l 50; 5 mM buffer and gently stirred for 30min at 4°C. Through 175

176

MINORU

MAKINO

the treatment of DEAE-Sepharose, the buffer without pyridoxal 5’-phosphate and 2-oxoglutarate was used because of adsorption of them to DEAE-Sepharose. The resulting DEAE-Sepharose suspension was centrifuged at 3000g for 15 min. The precipitate was resuspended in 1000 ml of 5 mM buffer and packed to a column (5 x 10 cm). The column was washed by 300 ml of 5 mM buffer before the elution with a linear gradient of 1OOOml of 5 mM buffer and lOOOm1 of 250mM buffer. Fractions (20ml) were collected and the enzyme was eluted from the column at the buffer concn of 150 mM as a single activity peak. The enzyme solution was applied to the hydroxylapatite column (2.1 x 5 cm) equilibrated with 5mM buffer. The column was washed with 50ml of 5mM buffer and then it was eluted by a linear gradient of 500ml of 5 mM buffer and 500ml of 250mM buffer. After active fractions were concentrated by ultrafiltration with a Diaflo apparatus (Amicon Far East Ltd., Tokyo, Japan), the enzyme solution (3.7 ml) was added to a Sephadex G-l 50 column (2.5 x 100 cm) which had been equilibrated with 50 mM buffer. The column was eluted with the same buffer of the equilibration. The active fractions were collected and concentrated by ultrafiltration as described above. PuriJication of Enzymes I and III from human and dog pancreas The same procedure was applied to human and dog pancreas. The homogenization, sonication and centrifugation were the same as described for that of rat. The resulting supernatant solution was heated rapidly to 65°C in a boiling water bath and maintained at this temp for 1 min with constant stirring, and then it was quickly chilled in an ice bath. After centrifugation at 10,000 g for 30 min, solid (NH&SO, was added to the resulting supematant solution to 30% saturation with constant stirring in an ice bath. After 30min, the precipitate was removed by centrifugation at 10,OOOgfor 30 min and discarded. Further solid ammonium sulphate was added to the resulting supematant solution to 75% saturation and stirred for 30 min. The resulting precipitate was collected by centrifugation as above and dissolved in 5mM buffer before dialysis against the same buffer overnight. The non-diffusible solution was centrifuged at 10,OOOg for 30min and the resulting supematant solution was applied to a column (3.3 x 8 cm) of DEAE-Sepharose equilibrated with 5 mM buffer omitting pyridoxal 5’-phosphate and 2-oxoglutarate during this column chromatography. The column was washed with 500 ml of 5 mM

Details of the purification and assayare given in the text

et al.

buffer and then it was eluted by a linear gradient of 1000 ml of 5 mM buffer and 1000 ml of 300 mM buffer, and 20 ml fractions were collected. Two activity peaks were obtained from the DEAE-Sepharose column of each preparation of human pancreas and dog pancreas. The faster eluted enzyme was designated as Enzyme I and the slower one was designated as Enzyme III. Human pancreatic Enzymes I and III were eluted from the DEAE-Sepharose column at the buffer concn of 20mM and 230mM, respectively and dog pancreatic isoenzymes of 60 mM and 110 mM respectively, were eluted. Enzyme I and Enzyme III of each species were separated and purified individually with the same procedures as follows. Each enzyme solution separated by DEAESepharose column was diluted with 2 mM buffer to adjust the buffer concn to about 5 mM and concentrated by ultrafiltration. The concentrated enzyme solution was applied to a hydroxylapatite column (2.0 x 8 cm) equilibrated by 5 mM buffer. The column was washed with 100 ml of 5 mM buffer and eluted with a linear gradient of 500 ml of 5 mM buffer and 500 ml of 250 mM buffer. The effluent was collected in 10 ml fractions. The active fraction was pooled and concentrated by ultrafiltration and the enzyme solution was applied to a Sephadex G-l 50 gel filtration. A single and symmetric activity peak was obtained with each enzyme preparation. The active fractions were pooled .and concentrated by ultrafiltration. Other methodr Polyacrylamide-disc-gel electrophoresis in the presence or absence of sodium dodecyl sulphate (SDS), isoelectric focusing, determination of protein, approximate molecular weight by sucrose gradient centrifugation and Sephadex G-150 gel filtration and kinetics were carried out as described previously (Okuno et al., 1980). RESULTS

As shown in Table 1, the rat enzyme was purified 60-fold with a recovery of 23%. Human Enzymes I and III were purified 47-fold with a 2.1% recovery and 973-fold with a 5.7% recovery, respectively. Dog Enzymes I and III were also purified 89-fold with an 8.7% recovery and 602-fold with a 2.9% recovery, respectively. On polyacrylamide disc gel electrophoresis, the purified pancreatic enzyme from rat and

177

Pancreatic branched chain amino acid transaminase

a)c

Enzymes I and III from dog were stained as a single protein band (data not shown). Heat stability

of the purified

Km= 11.1mM

0.3

enzymes

/

x

Rat pancreatic branched chain amino acid aminotransferase was labile to heat. The activity was completely lost by heating at 65°C for 1 min. By heating at 5O”C, about 30% of the enzyme activity was lost within 1 min and about 70% within 10 min. Purified Enzymes I and III from human and dog pancreas were not inactivated by heating at 65°C for 10min. The branched chain amino acid aminotransferase

QJ0.2 2 2 =01

k

^ 0, i3

0 0.25 1 I ILeuanel CmM)

05

h E

activity in crude pancreatic supernatant in dog was somewhat activated by heating at 65?C for 1 min as shown in Table 1.

2 - 0.;

pH optimum

The rat enzyme had the pH optimum between 7.5 and 8.0, the human

and dog Enzyme

I had 9.0, the

human Enzyme III had 8.5 and dog Enzyme III had between 8.5 and 9.5. The rat enzyme, human and dog Enzymes I were obviously inhibited by Tri-HCl buffer, but Enzymes III of human and dog were not inhibited by the buffer. Molecular

0.5 1 I (2-Oxoglutarote]

1.I ( mM )

Km=4-OmM

0

weight and isoelectric point

Molecular weights were estimated by Sephadex G-l 50 gel filtration, sucrosedensity-gradient centrifugation and SDS-polyacrylamide disc gel electrophoresis (Table 2). The molecular weights of rat and dog enzymes suggest that all branched chain amino acid aminotransferases of pancreas examined consisted of two identical subunits. Isoelectric points of these purified enzymes were shown also in Table 2.

1.0 mM)

0 0.5 l/ [2-Oxoglutorotej(

Kinetic properties

2-Oxoglutarate was removed by a Sephadex G-25 column (2.5 x 20 cm) for each purified enzyme solution. Michaelis constants of the purified enzymes to

branched chain amino acids and 2-oxoglutarate were measured. The reciprocal values of V,,,,, for branched chain amino acid and 2-oxoglutarate at several concns of the co-substrate were estimated. A secondary plot of these values against the reciprocal of the co-substrate concentrations permits the estimation of V,,,,, at infinite co-substrate concn and calculation of the absolute K,,, values for L-leucine-2-oxoglutarate aminotransferase activity of rat enzyme suggesting a Ping Pong Bi Bi mechanism of this reaction as shown in Fig. 1. Similar parallelism of double-reciprocal plots of activity and co-substrates was also obtained Table 2. Approximate

mol. wt and isoelectric

0-

Fig. I. Kinetic properties of branched chain amino acid aminotransferases. purified from rat pancreas. Doublereciprocal plot of V,,, of the leucine-2-oxoglutarate aminotransferase activity of the enzyme against the concn of L-leucine: (a) 20 mM (A), 10 mM (A); 4.5 mM (e), 2.2 mM (0) and 2-oxoglutarate (b), 10 mM (A), 5 mM (A), 2 mM (a), and 1 mM (0) are shown. Assay conditions were as described in the text, except L-amino acid concn and 2-oxoglutarate concn.

point estimations of rat, human and dog pancreatic amino acid aminotransferases Rat

Human Enzyme

Molecular Sepnadrx Suc,,ise

weipht

irt,matcd

68000 cer.trlfugatlon

SDS polyacrylamlde electrnphoresls Iscelectric

Experimental

polrt,

dlS'2

gel

pH

details are given

I

Enzyme

branched

Enzyme

I

Enzyme

80000

96000

79000

80000

90000

35000

39000

43000

5.6

4.6

5.6 in

the text

80000

6.6

90000

4.2

chain

DOE III

by

G-Ii<, gradlent

O-4 1 /LLeucinel(mM)

III

MINORU

178

Table 3. Absolute

K, values of branched

MAKINO

chain amino acid aminotransferase

l72.r

( mi,,) With

z-oxog1utarate

et al.

Enzyme I 171N,

Enzyme

Enzyme

I

Enzyme

(mM)

11.1

25.0

6.8

22.2

10.3

3 0

30.X

8.3

"Zillne

143

3.6

2.3

22.2

4.?

7.4

3.u

wltn

Leuclne

4.0

7.6

1.0

3.2

1.4

ISOleUClne

14.2

4.8

1.1

10.0

1.0

"allne

12.5

5.0

1.7

2.7

Experimental concn.

III

(mMj

ior

ISOleUClne

2-oxoglutarate

DOE

III

(mM)

Leucine

For

from rat, human and dog

Human

details are given in the text, except branched

I

7

chain amino acid concn and Z-oxoglutarate

from other co-substrates examined and other purified enzymes. The absolute K,,, values of the purified enzymes for branched chain amino acids and 2-oxoglutarate are shown in Table 3. The absolute K,,, values for branched amino acids and 2-oxoglutarate of Enzyme III showed somewhat lower than those of rat enzyme and Enzyme I.

nicotinic acid hydrazide and semicarbazide. Dog Enzymes I and III were similarly affected by hydroxylamine and hydrazine but little or no effect of isonicotinic acid hydrazide and KCN was observed. Dog Enzyme III was somewhat inhibited by semicarbazide but no inhibition of dog Enzyme I by semicarbazide was observed. Each branched chain amino acid aminotransferase was similarly affected Substrate spec$citie.y by hydroxylamine and hydrazine, but variously Branched chain L-amino acids (40 mM), leucine affected by isonicotinic acid hydrazide, KCN and isoleucine, and valine, were active to the purified semicarbazide. enzymes with 2-oxoglutarate (10mM) at pH 8.0. Relative activities for branched chain amino acids are Effect of PCMB and suljhydryl compounds summarized in Table 4. Rat enzyme showed the Enzyme preparations were dialysed overnight at highest activity to leucine, and dog Enzyme III 4°C against the buffer without 2-mercaptoethanol. similar activity to leucine and isoleucine, but human p-Chloromercuribenzoate (PCMB) (0.5 mM) comEnzymes I and III and dog Enzyme I had the highest pletely inhibited the rat enzyme, human Enzyme I activity to isoleucine. Each enzyme activity for valine and dog Enzyme I, but not completely the Enzyme III showed 4Ck50°%of leucine. of man and dog (Table 6), suggesting the presence of essential sulfhydryl group in these enzymes. These ESfect of carbonyl reagents inactivations were protected l-5 mM by Effect of carbonyl reagents on the purified 2-mercaptoethanol. branched chain amino acid aminotransferases are summarized in Table 5. Rat enzyme was affected by DISCUSSION semicarbazide, hydroxylamine and hydrazine but it Branched chain amino acid aminotransferases were was little affected by isonicotinic acid hydrazide and KCN. Human Enzymes I and III were similarly highly purified from pancreas of rat, human and dog inhibited by carbonyl reagents used except iso- with high purification ratio and high recovery rate. Table 4. Substrate

Amino

acids

specificity

of branched chain amino acid aminotransferases rat. human and don aancreas

Rat

Human Enzyme

I

Enzyme

from

Ijog III

Enzyme

I

Enzyme

III

Enzyme activities were assayed at pH 8.0. The final concn of L-amino acids was 40 mM but that of tyrosine was 6.5 mM because of the low solubility and that of 2-oxoglutarate was 10 mM.

Pancreatic

branched

Table 5. Effect of carbonyl Reagrrf

chain

Rat (%)

Enzyme I i '6 j

100

100

84

179

transaminase

84

Isonicotinic acid hydrazine KCN

Human Enzyme (%)

III

Enzyme (%)

I

Dog Enzyme (%)

100

100

08

71

67

,?1

71

69

83

HK

116

68

86

116

'17

107

75

77

114

93

62

76

91

101

86

nydroxylnm~nr Hydrazine

acid

reagents on branched chain amino acid aminotransferases from rat, human and dog pancreas

added

NO"e

amino

HC;

Semicarbazide

III

100

Leucine-Z-oxoglutarate aminotransferase activity was assayed by the condition in the text, except with the additional of I mM carbonyl reagent.

as cited

Table 6. Effect of sulfhydryl reagents and p-chloromercuribenzoic acid (PCMB) on branched amino acid aminotransferases from rat, human and dog pancreas Reagent

added

COllC. (mMi

None

Rat (%I

Enzyme (%I

I

HUW3.ll Enzyme (%I

III

Enzyme (%I

I

chain

Dog Enzyme (%I

100

100

100

1(JO

100

G:utathione

5

130

111

162

127

145

Cysteine

5

151

89

123

107

117

2-MeXaptOetha"Ol

5

134

173

177

161

158

PCMB

0.5

5

14

7

21

i+)2-merca~,toethanol

1

102

67

43

86

78

i+)Z-mercaptoethanol

5

113

143

149

146

106

2.2

III

Leucine-Z-oxoglutarate aminotransferase activity was assayed by the condition as described in the text, except with the addition of sulfhydryl reagent and PCMB. The reaction containing PCMB (0.5 mM) was also carried out by the concomitant addition of 2.mercaptoethanol (I and 5 mM).

During purification, 2-mercaptoethanol and 2-oxoglutarate stabilized these enzymes. Rat pancreas contained one branched chain amino acid aminotransferase and human pancreas contained two types of the aminotransferase. These finding agree with those of Ichihara et al. (1975) and Goto et al. (1977). Dog pancreas also contained two types, as human pancreas. Rat enzyme was heat labile but crude enzymes of dog pancreas were activated by heating (Table 1). Rat enzyme is referred to Enzyme III from DEAE-cellulose column elution, but has similar properties (K, values and inhibition by Tris) of Enzyme I from dog and man. Rat enzyme and Enzyme I showed mol. wts of approx. 80,000 probably consisting of two identical subunits (approx. 40,000). Rat enzyme and dog Enzyme I had the same isoelectric point, pH 5.6, but human Enzyme I had a higher one (~16.6). Absolute X, values of rat enzyme, and human and dog Enzyme I were somewhat higher for L-branched chain amino acids and 2-oxoglutarate than those of Enzyme III. Kinetic properties of pancreatic branched chain amino acid aminotransferase and the concentration of these substrates may be important to regulate the branched chain 2-oxoacids formation. Postprandial level of branched chain amino acids in

portal vein is higher than in other vessels(Aikawa et al., 1973; Elwyn et al., 1968) and the level in venous plasma is transiently elevated at the beginning of starvation (Felig et al., 1969). The different kinetic properties of Enzyme I and Enzyme III may contribute to a variety of concentrations of branched chain amino acid in pancreas. It is known that insulin releasefrom isolated mouse islets is highly stimulated by the simultaneous addition of 2-oxoisovalerate and 2-oxoisocaproate (Panten et al., 1981). Thus, high activity of pancreatic branched chain amino acid aminotransferase might be related to the release of insulin. Glutathione and 2-mercaptoethanol activated the pancreatic branched chain amino acid aminotransferase (Table 6). The activation ratios of Enzyme III by glutathione were to some extent higher than those of enzymesI and rat enzyme. It is assumed that cysteine affects the aminotransferase activity in km. Enzyme III, possessinglow K,,,values for substrate and high activation ratio by glutathione, may contribute to the formation of branched chain 2-oxoacids in the steady state, even in low concentration of branched chain amino acids. Otherwise, Enzyme I might play an important role with increase in concentration of branched chain amino acids.

180

MINORU MAKINO r~ al. REFERENCES

Aikawa T., Matsutaka H., Yamamoto H., Okada T.. Ishikawa E., Kawano T. and Matsumura E. (1973) Gluconegenesis and amino acid metabolism II. Interorganal relations and roles of glutamine and alanine in the amino acid metabolism of fasted rats. J. Biochem. (Tokyo) 74, 100331017. Aki K., Ogawa K.. Shirai A. and lchihara A. (1967) Transaminase of branched chain amino acids III. Purification and properties of the mitochondrial enzyme from hog heart and comparison with the supernatant enzyme. J. Biochem. (Tokyo) 62, 6l(t617. Aki K., Ogawa K. and Ichihara A. (1968) Transaminasesof branched chain amino acids IV. Purification and properties of two enzymesfrom rat liver. Biochim. Bioph,vs.Acra 159, 276284. Aki K.. Yokojima A. and Ichihara A. (1969) Transaminase of branched chain amino acids VI. Purification and properties of the hog brain enzyme. J. Biochem. (Tokyo) 65, 539-544. Aki K., Yoshimura T. and lchihara A. (1973) Transaminase of branched chain amino acids. IX. Conformational change of Isoenzyme I in vitro. J. Biochrm. (Tokyo) 74, 779-784. Elwyn D. H., Parikh H. C. and Shoemaker W. C. (1968) Amino acid movements between gut, liver, and periphery in unanesthetized dogs. Am. J. Physiol. 215, 126s 1275. Felig P.. Owen 0. E., Wahren J. and Cahill G. F.. Jr. ( 1969) Amino acid metabolism during prolonged starvation. J. Clin. Inoest. 48, 584-594. Goto M., Shinno H. and lchihara A. (1977) Isoenzyme patterns of branched-chain amino acid transaminase in human tissues and tumors. Gunn 68, 663-667.

Ichihara A. and Koyama E. (1966) Trdnsaminase 01 branched chain amino acids. 1. Branched chain amino acids-a-ketoglutarate transaminase. J. Biochem. ( Tokyo) 59, 16&169. Ichihara A., Noda C. and Goto M. (1975) Transaminase of branched chain amino acids X. High activity in stomach and pancreas. Biochem. hiophys. Res. Commun. 67, 1313~1318. Nakatani Y.. Fujioka M. and Higashino K. (1970) r-Aminoadipate aminotransferase of rat liver mitochondria. Biochim. Biophys. Actu 198, 219-228. Noguchi T. and Kido R. (1976) Identity of kynurenine:pyruvate aminotransferase with histidine: pyruvate aminotransferase. Hoppe-Seykr’s Z. phpsiol. Chem. 357, 6499656. Okuno E., Minatogawa Y., Nakamura M., Kamoda N., Nakanishi J., Makino and Kido R. (1980) Crystallization and characterization of human liver kynurenineglyoxylate aminotransferase: identity with alanineglyoxylate aminotransferase and serine-pyruvate aminotransferase. B&hem. J. 189, 581-590. Panten U., Bierman J. and Graen W. (1981) Recognition of insulin-releasing fuels by pancreatic B-cells. a-Ketoisocaproic acid is an appropriate model compound to study the role of B-cell metabolism. M&c. Phurmuc. 20, 7682. Taylor R. T. and Jenkins W. T. (1966a) Leucine aminotransferase II. Purification and characterization. .I. hiol. Chem. 241, 43964405. Taylor R. T. and Jenkins W. T. (1966b) Leucine aminotransferase 1. Calorimetric assays. .I. hiol. Chem. 241, 4391.4395. Taylor R. T. and Jenkins W. T. (1966~) Leucine aminotransferase III. Activation of fi-mercaptoethanol. J. hid. Chem. 241, 44064410.