Differences of adenosine kinases from various mammalian tissues

Comp. Biochem. Physiol. Vol. 71B, No. 3, pp. 367 to 372. 1982

0305-0491/82/030367-061i03.00/0 © 1982 Pergamon Press Ltd

Printed in Great Britain.

DIFFERENCES OF ADENOSINE KINASES FROM VARIOUS MAMMALIAN TISSUES YASUKAZU YAMADA, HARUKO GOTO and NOBUAKI OGASAWARA Department of Biochemistry, Institute for Developmental Research, Aichi Prefectural Colony, Kasugai, Aichi 480-03, Japan

(Received 20 July 1981) Abstract--1. Adenosine kinases purified to homogeneity from various mammalian tissues have a monomeric structure, and their molecular weights were estimated around 40,000. 2. The enzyme activity per wet weight of tissue appears to vary from source to source, but the specific activities of the final enzyme preparations were alike, which were 6.3-7.8 ,umol/min/mg protein. 3. No or small difference was observed in the kinetic properties among all seven adenosine kinases. 4. Some differences in structure were observed among five liver enzymes from human, rabbit, rat, mouse and Mongolian gerbil, but no difference was observed between the enzymes from human placenta and liver, or the enzymes from rat liver and brain.

INTRODUCTION Adenosine kinase (ATP: adenosine 5'-phosphotransferase E C 2.7.1.20) catalyzes the phosphorylation of adenosine to A M P according to the following reaction: Adenosine + ATP

of the same animals, the enzymes from human placenta and liver, or the enzymes from rat liver and brain.

Mg2+ ' A M P + ADP.

Decreased levels of adenosine kinase have been associated with resistance to the antiproliferative effects of a number of adenosine analogs (Caldwell et al., 1967), increased purine excretion ( C h a n e t al., 1973), and primary gout (Nishizawa et al., 1976). In mammalian cells, adenosine is removed by deamination to inosine or phosphrylation to AMP. The phosphorylation of adenosine and deoxyadenosine, the substrate of adenosine deaminase, is important in adenosine deaminase deficiency associated with immunodeficiency (Carson et al., 1977). Adenosine kinase was first discovered in yeast (Caputto, 1951; Kornberg & Pricer, 1951) and in mammalian tissues (Caputto, 1951), and exists at relatively high levels in tissue (Andres & Fox, 1979; Ho et al., 1968; Krenitsky et al., 1974). It has been purified to homogeneity from brewer's yeast (Leibach et al., 1971), from yellow lupin seeds (Guranowski, 1979), and recently from a number of mammalian sources (Andres & Fox, 1979; DeJong et al., 1980; Miller et al., 1979a; Yamada et al., 1980, 1981) by affinity chromatography on AMP-Sepharose or AMP-agarose. The enzyme properties and their molecular weights were very alike in mammalian tissues and also in plant or yeast. We have obtained apparently homogeneous adenosine kinase from various mammalian tissues by the same purification procedure as we previously described (Yamada et al., 1980). In a part of recent study (Yamada et al., 1981), we reported some differences in structure of three liver enzymes from human, rabbit and rat. In this paper, we report the differences among five liver enzymes from human, rabbit, rat, mouse and Mongolian gerbil, and furthermore, we compare the enzymes from different organs 367

MATERIALS AND METHODS

Materials [8-14C] adenosine was purchased from The Radio Chemical Centre, Amersham. ATP, phosphoenolpyruvate, pyruvate kinase (rabbit muscle) and trypsin (bovine pancreas) were obtained from Boeringer Mannheim. AMPSepharose 4B, Sephadex G-100 and electrophoresis calibration kit were obtained from Pharmacia. Staphylococcus aureus V8 protease was purchased from Miles Laboratories. Other reagents were commercial preparations of the highest purity available. Enzyme assay and protein determination Adenosine kinase was assayed radiochemically as previously described (Yamada et al., 1980). One unit of the enzyme activity is defined as the amount catalyzing the phosphorylation of 1 ,umol of nucleoside for 1 min. Specific activity was defined as units per mg protein. Protein concentration was determined by the method of Lowry et al. (1951), using bovine serum albumin~as a standard. Purification of enzymes From human liver, rabbit (Oryctolaous cunieulus) liver, and rat (Rattus norveyicus) liver and brain, adenosine kinases have been already purified to homogeneity in our laboratory (Yamada et al., 1980, 1981), By the same purification procedure, the enzymes were also purified to homogeneity from human placenta, CF No. 1 mouse (Mus musculus musculus) liver and Mongolian gerbil (Meriones unouiculatus) liver, by ammonium sulfate fractionation (45-80~o), affinity chromatography on AMP-Sepharose 4B, Sephadex G-100 gel filtration and DEAE-cellulose (Whatman, DE-52) column chromatography. Each final enzyme preparation was stored at -70°C. Amino acid analysis. Purified enzymes (50 #g each) were hydrolyzed in 0.5 ml of 6 N HC1 in a vacuum hydrolysis-tubes (Pierce Chem. Co., Rockford) at 110°C for 24, 48 and 72 hr. The hydrolysate was analyzed with an automatic amino acid analyzer (Hitachi, model 835). Values for threonine and serine were

YASUKAZUYAMADAet al.

368

obtained from the samples hydrolyzed for 24 hr, and those for valine and isoleucine were from the samples hydrolyzed for 72 hr. For all other amino acids, values were obtained by averaging the results. Contents of half-cystine and tryptophan were not determined.

Peptide mapping analysis Peptide mapping by limited proteolysis in sodium dodecyl suflate (SDS) and analysis by gel electrophoresis were performed according to the method described by Cleveland et al. (1977)~ Purified enzymes were dissolved at approximately 0.01 mg/ml in the buffer containing 0.1 M Tris-HC1 (pH 6.8), 0.5% SDS and 10% glycerol. About 50 #l (0.5 #g) of each sample was heated at 110°C for 2 rain and, after cooling, placed into a gel well with a protease. Proteolytic digestions proceeded directly in the stacking gel during the subsequent electrophoresis. Slab gel electrophoresis was performed in 15% polyacylamide using the system described by Laemmli (1970), and the stacking gel was longer than usual gel, at about 4 cm, in order to obtain the best results. Electrophoresis was carried out with current of 10 mA in the stacking gel (about 2 hr), and 50 mA in the separation gel (about 2 hr). Gels were fixed in a solution of 50% methanol and 10% acetic acid and then peptide bands in gels were detected by an ultrasensitive silver stain described by Oakley et al. (1980). RESULTS AND DISCUSSION

Purification of enzymes All seven enzymes from various mammalian tissues, human placenta and liver, rabbit liver, rat liver and brain, mouse liver, and Mongolian gerbil liver, were purified by the same method previously described (Yamada et al., 1980) with the yield of 40-60%. Each final enzyme preparation appeared to be homogeneous, since only a single protein band was observed by SDS polyacrylamide gel electrophoresis (Fig. 1). The stepwise purification of adenosine kinase

from human placenta is summarized in Table 1, and the purifications from other tissues showed the similar results. However, in the elution pattern from DE-52, the enzymes from mouse and Mongolian gerbil livers were different from other five enzymes. The enzyme from mouse liver was eluted with higher concentration of KCI (about 60mM) than others (40mM) (Yamada et al., 1980, 1981), and the enzyme from Mongolian gerbil liver was eluted with lower concentration of KCI (about 25 raM). The enzyme activities per wet weight of tissue appeared to vary from source to source (Table 2). The activities of rabbit or rat, mouse, and Monogolian gerbil livers were 5-6, 15, and 30 fold, respectively, over the activity of human liver. The ratios of enzyme activities of human liver to placenta, and those of rat liver to brain were about 3:1, and 20:1, respectively. However, specific activities of the final enzyme preparation were alike, at 6.3-7.8#mol/min/mg protein (Table 2).

Molecular weight Each adenosine kinase purified from various mammalian tissues is a monomer of molecular weight of about 40,000 from the results obtained by Sephadex G-100 gel filtration and SDS polyacrylamide gel electrophoresis (Fig. 1). The values of the molecular weight obtained were similar to the values of previous studies on mammalian tissues (Andres & Fox, 1979; Miller et al., 1979a; Yamada et al., 1980, 1981), and also to the values of studies on yellow lupin seeds (Guranowski, 1979) and yeast (Leibach et al., 1971).

Enzyme properties Apparent Km values for adenosine, substrate inhibition, and pH optimum of seven adenosine kinases

,

94,000 67, O00

43,000

..~,~. ,, ~

,

30,000

20,100 14.,400

A

B

C

D

E

F

G

H

I

Fig. 1. SDS gel electrophoresis of adenosine kinase from various mammalian tissues. The samples prior to electrophoresis, were incubated for 3 min in 0.1 M Tris HC1 (pH 6.8) containing 1% SDS and 5% mercaptoethanol. Electrophoresis was carried out in 10% polyacrylamide by the method of Laemmli (1970). A and I: standard proteins; phosphorylase b (94,000), bovine serum albumin (67,000), ovalbumin (43,000), carbonic anhydrase (30,000), soybean trypsin inhibitor (20,100), and ~-lactalbumin (14,400). B: adenosine kinase from human placenta. C: from human liver. D: from rabbit liver. E: from rat liver. F: from rat brain. G: from mouse liver. H: from Mongolian gerbil liver.

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Differences of mammalian adenosine kinases Table 1. Purification of adenosine kinase from human placenta. One unit equals 1 ~mol of [8-14CLAMP formed per rain under standard assay conditions. Purification was initiated from 80 g of human placenta Fraction

Total vol. (ml)

Total activity (U)

250 50 5 30 9

2.95 2.80 2.21 1.92 1.44

Crude extract (NH4)2SO4 (45-80~o) AMP-Sepharose 4B Sephadex G-100 DE-52 cellulose

Total protein (rag) 2880 912 3.52 0.72 0.22

Specific activity (U/mg protein)

Yield (~o)

0.001 0.003 0.627 2.67 6.55

100 95 75 65 49

Table 2. Activity of adenosine kinase from various mammalian tissues. The activity was assayed at 1 #M [8-1*C]adenosine, 1 mM ATP and 0.5 mM MgCI2 Tissue Human placenta Human liver Rabbit liver Rat liver Rat brain Mouse liver Mongolian gerbil liver

Activity/ wet wt

Specific activity of the final preparation

nmol/min/g 37 121 541 657 29 1840 3420

ttmol/min/mg protein 6.55 6.32 6.85 7.44 7.78 6.62 6.86

Table 3. Comparison of adenosine kinases in properties. The activity was assayed under standard condition Tissue Human placenta Human liver Rabbit liver Rat liver Rat brain Mouse liver Mongolian gerbil liver

K,. for adenosine (/~M)

Inhibition by 10#M adenosine (Vo control)

pH optimum (pH)

0.15 0.15 0.16 0.18 0.20 0.10 0.09

58 60 55 62 62 52 54

5.5, 7.5-8.5 5.5, 7.5-8.5 5.5, 7.5-8.5 5.5, 7.5-8.5 5.5, 7.5-8.5 5.5, 7.5 8.5 5.5, 7.5-8.0

Table 4. Amino acid composition of adenosine kinase from liver. The values (number/mol) were calculated from a tool. wt of about 40,000 by averaging two series of experiments. Contents of half-crystine and tryptophan were not determined Amino acid Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine

c.13.e. 71/3B

c

Human Rabbit 32 16 18 37 6 20 29 16 6 19 21 7 18 26 8 10

31 16 18 39 9 20 28 16 5 18 20 7 18 26 7 8

Rat

Mouse

Mongolian gerbil

35 18 17 35 12 21 29 19 5 14 21 7 19 22 5 11

36 15 18 36 12 20 31 20 3 15 21 8 18 21 6 11

34 16 22 38 10 23 34 19 5 12 23 7 18 23 2 10

370

YASUKAZUYAMADAet al. 67,000 48,000

30,000

20,100

14,400

A

B

C

D

E

F

G

H

I

Fig. 2. Peptide maps of adenosine kinase from various mammalian tissues digested with S. aureus V8 protease. The enzymes were digested with 0.01 pg S. aureus V8 protease. A: digestion of adenosine kinase from human placenta. B: from human liver. C: from rabbit liver. D: from rat liver. E: from rat brain. F: from mouse liver. G: from Mongolian gerbil liver. H: 0.01 #g of S. aureus V8 protease. I: standard proteins described in the legend to Fig. 1. are compared in Table 3. Effects of adenosine concentration on the activity of adenosine kinase from various tissues, were alike. An apparent K,, value of each enzyme was 0.1q?.2 pM, and the maximum activities were observed at 0.5/~M in all enzymes. At higher concentrations of adenosine, the activities were strongly inhibited; 50-60~o inhibition at 10/~M adenosine in all enzymes. At optimal concentration of adenosine, the pH optimum profile was biphasic, a sharp pH optimum at pH 5.5 and a broad pH optimum at 7.5-8.5, in the

----

A

w

D

B

C

enzymes from rat brain (Yamada et al., 1980) and human liver (Yamada et al., 1981). The enzymes from other tissues also showed biphasic pH optimum at pH 5.5 and pH 7.5-8.5, except the enzyme from Mongolian gerbil liver which showed the optimum at pH 5.5 and pH 7.5 8.0. Previous reports (Andres & Fox, 1979; Guranowski, 1979; Leibach et al., 1971; Miller et al., 1979a,b; Palella et al., 1980; Yamada et al., 1980, 1981) supported the existence of small differences in properties of enzymes from various species including plant and yeast. Indeed, there are some dif-

m

D

D

E

e

F

G

H

Fig. 3. Peptide maps of adenosine kinase from various mammalian tissues digested with trypsin. The enzymes were digested with 0.02/~g trypsin. A: digestion of adenosine kinase from human placenta. B: from human liver. C: from rabbit liver. D: from rat liver. E: from rat brain. F: from mouse liver. G: from Mongolian gerbil liver. H : 0.02 pg of trypsin.

Differences of mammalian adenosine kinases ferences in nucleoside triphosphate donor specificity, or in metal ion requirement. However, they cannot be simply compared, since adenosine kinase activity is complicated in that it is influenced by pH and by concentrations of nucleosides, ATP and Mg 2+ (Miller et al., 1979a; Yamada et al., 1980, 1981). Amino acid composition

In order to examine differences in structure of the enzymes from various tissues, firstly, amino acid analysis was carried out. The amino acid composition of five liver enzymes from human, rabbit, rat, mouse and Mongolian gerbil, are summarized in Table 4. The amounts of pure enzyme preparations of human placenta and rat brain were too little to analyze. The amino acid compositions of five liver enzymes were alike, and especially those of human and rabbit, or those of rat and mouse resemble each other closely. The differences between human group (human, rabbit) and rat group (rat, mouse, Mongolian gerbil) were obtained in the numbers of proline, valine, isoleucine and histidine residues. In the numbers of serine, alanine, isoleusine and histidine residues, Mongolian gerbil liver was different from the others. Peptide mapping analysis

In a recent study (Yamada et al., 1981), we reported some differences in structure of three liver enzymes from human, rabbit and rat, by amino acid analysis and peptide mapping analysis. In these studies, difference between human and rabbit was not clear by amino acid analysis, but clear by peptide mapping analysis since some extra peptide bands were observed in the rabbit enzyme. Therefore, peptide mapping analysis was then performed. The amounts of pure enzyme preparations from human placenta and rat brain were so little that the highly sensitive silver staining technique for polyacrylamide gel described by Oakley et al. (1980) was used. This technique permits detection of polypeptides in polyacrylamide gels at concentrations 50-100 fold lower than those required for Coomassie brilliant blue staining. But the proteolysis in test tubes was not suitable, since substrate proteins were low in concentration and the peptide bands from protease disturbed the peptide maps of substrate proteins. Therefore, poroteolytic digestions were proceeded directly in the stacking gel during subsequent electrophoresis. The amounts of protease used in this method were 100 fold less than those used for digestion in test tubes. Firstly, the enzymes were digested with S. aureus V8 protease and secondly with trypsin. The results of peptide mapping of seven enzymes are shown in Figs 2 and 3. Comparison of peptide maps of five liver enzymes showed that each enzyme had some specific peptide bands in digestion with both S. aureus V8 protease and trypsin, while several peptide bands were common to the five liver enzymes. Peptide maps of human and rabbit liver were alike, but in digestion with S. aureus V8 protease some extra bands were observed in the rabbit enzyme as described in recent study (Yamada et al., 1981). In digestion with trypsin, however no difference was observed. The same or very similar peptide maps were observed in the enzymes from different organs of the same animals, the enzyme from human placenta and

371

liver, and those from rat liver and brain, in digestions with both S. aureus V8 protease and trypsin. S. aureus V8 protease cleaves the polypeptide band at the COOH-terminal side of aspartic and glutamic acid residues, and trypsin cleaves at the COOH-terminal side of lysine and arginine residues. Therefore, adenosine kinases from human placenta and liver, or those from rat liver and brain seem to be the same proteins. Acknowledgements--We are grateful for the help given us by Mr. Takayoshi Shionoya at Central Hospital in Aichi Prefectural Colony in performing amino acid analysis. This work was supported by grants from the Ministry of Education, Japan. REFERENCES

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