Properties of human hepatoma adenylate kinases

PROPERTIES

OF HUMAN

HEPATOMA

ADENYLATE

KINASES

EFTIHIA CAYANIS Porphyrin Research Unit, Department of Haematology, The South African Institute for Medical Research. PO Box 1038, Johannesburg. South Africa

(Receiced 5 January 1978) Abstract-l. Three adenylate kinase isoenzymes, designated AK I, II and III in order of their increasing electrophoretic mobility towards the anode were partially purified from human hepatomas. 2. When AK III was passed through phosphocellulose, multiple forms were again obtained. 3. Starch gel electrophoresis revealed that AK I and AK III C. as well as AK II and AK III B had similar electrophoretic mobilities. 4. A study of the properties, which included pH optima, acid. alkali and temperature stability studies, substrate analogue utilization and Michaelis constant estimations. showed certain similarities as well as differences between the various adenylate kinases.

INTRODUCTION Adenylate kinase (ATP:AMP phosphotransferase 2.7.4.3) catalyses the interconversion of the energy compounds ATP and ADP as follows:

EC high

ATP + AMP G+ 2 ADP The enzyme has been purified and characterized from a variety of sources, some of which indude, rabbit (Noda & Kuby, 1957). porcine (Schirmer ef al., 1970) and human muscle (Thuma et al., 1972), bovine liver mitochondria (Markland & Wadkins, 1966 a & b), rat (Criss et al., 1970; Sapico et al., 1972), and porcine (Chiga & Plaut, 1960) livers, rat hepatomas (Criss et al., 1974), rat brain (Pradhan & Criss, 1976), human erythrocytes (Tsuboi & Chervenka, 1975) and yeast (Chiu & Russel, 1967). At least four adenylate kinase isoenzymes were reported in rat normal liver and hepatoma and the mitochondrial adenylate kinase III and cytoplasmic AK II purified from these tissues (Sapico et al., 1972; Criss et al., 1974). The occurrence of adenylate kinase in multiple molecular forms, which are species and tissue specific (RusseI et al., 1974) prompted a study on the separation and characterization of the adenylate kinase isoenzymes from human hepatoma tissue. The results obtained showed the presence of three adenylate kinase isoenzymes in human hepatoma tissue. They also indicate that these isoenzymes, as well as the multiple forms obtained, when AK III was rechromatographed on phosphocellulose, have different properties. MATERIALS AND METHODS Materials The auxiliary enzymes, lactate dehydrogenase (LDH) from pig heart, pyruvate kinase (PK) from rabbit muscle and hexokinase (HK) and glucose-6-phosphate dehydrogenase (G6PD) from yeast, the coenzyme NADH (disodium salt) as well as the disodium salts of the substrates ATP, AMP, ADP, dATP, dAMP and CMP were obtained from Biochemical Boehringer, Mannheim, Germany. The reduced form of nicotinamide adenine dinu~leotide (NADH). the sodium salts of type I, GTP and UTP from

equine muscle, type 5. CTP from yeast. grade I, dCMP. dGTP, IDP, ITP, type I, UDP, from yeast, dUMP. TMP, TDP, the disodium salt of CMP and adenosine 3’:5’ cyclic monophosphoric acid (CAMP) were obtained from Sigma Chemical Co., St. Louis, MO. U.S.A. CM-Sephadex C-50, DEAE-Sephadex A-50, Sephadex G-25 and Sephadex G-75 were obtained from Pharmacia, Uppsala, Sweden; and phosphocellulose Whatman PI I from the Whatman Biochemicals Ltd., Springfield Mill, Maidstone, Kent, England. Phenazine methosulphate was obtained from Sigma, St. Missouri, U.S.A. 2-p-(iodophenyl-3-(p-nirroLouis, phenyl)-S-nitrophenyl tetrazolium salt (INT) was from the National Biochemical Corp., Cleveland, Ohio, U.S.A. and Bacto-Agar from the Difco Laboratories, Detroit, Michigan, U.S.A. Connaught hydrolysed starch was purchased from the Connaught Medical Research Laboratories, Toronto, Canada. Dithiothreitol (DTT) was obtained from Calbiochem, San Diego, California, U.S.A., and Carbowax (polyethylene glycol 20M) from British Drug Houses, Poole, England. All other reagents were obtained from E. Merck, Darmstadt, Germany or British Drug Houses, Poole, England and were of analytical grade. Assay procedures All assays were performed on a Unicam SP 800 recording s~ctrophotometer at the controlled temperature of 30°C by following the rate of oxidation of NADH or reduction of NADP’ at 340 mm. The enzyme was assayed by using a slightly modified method of Sapico et al. (1972). For the assay of the enzyme in the forward direction (production of ADP) the standard reaction mixture contained in a final volume of 3 ml, 100 mM triethanolamineHCI buffer (pH 7.5), 5.0 mM KC], 2 mM MgCl,, 1.2 mM ATP, 0.5mM AMP, 2mM PEP, 0.15 mM NADH, 14U pyruvate kinase, IOU LDH and a suitable amount of the adenylate kinase preparation to be assayed. For the assay of adenylate kinase in the reverse direction (production of ATP), the standard reaction mixture contained in a final volume of 3 ml; 100 mM triethanolamineHCI buffer (pH 7.5), 3 mM ADP, 0.36 mM NADP, 1.5 mM glucose, 4 U G6PD, 4 U HK, 1.5 mM MgCI, and suitable amounts of the adenylate kinase preparation. A unit of activity -is defined ai thk amount of enzyme catalysinx the reduction of 1 umole NADP or oxidation of 4; mole NADH/min, assuming a molar absorptivity of NADPH and NADH at 34Onm of 6.22 x IO3 (Horecker & Kornberg, 1948). Specific activity is the a~ivity/mg pro-

EFTIHIA

618

0

40

0

CAYANIS

50

Fractton

100

number

Fig. 1. Column chromatography of human hepatoma adenylate kinases on CM-Sephadex. Following application of 36,000 g supernatant fraction to a column (14 x 3 cm) of CM-Sephadex, approximately 150 ml of 1OmM potassium phosphate buffer, pH 6.5 containing 207; glycerol and 0.1 mM DTT was passed through. Elution of the adenylate kinase isoenzyme was carried out using a linear gradient of I I of OXl.2 M KC1 in IO mM potassium phosphate buffer, pH 6.5 containing 20% glycerol and 0.1 mM DTT. Fractions of 7 ml were collected.

tein. The protein content was determined of Warburg & Christian (1942).

by the method

Horizontal starch gel clecrrophoresis The procedure for separating the various adenylate kinase isoenzymes and the method used to stain them were similar to those of Fildes & Harris (1966). Isolarion

of the different adenylate kinase isoenzymes

Human hepatoma tissue was obtained at autopsy, l-6 hr after death and stored at -20°C until required. All operations were performed at 4°C unless otherwise stated. Extraction Approximately 30-50 g of hepatoma tissue was used for the isolation of AK I and II, whereas 1OOg of hepatoma tissue was used for the partial purification of AK III. These were weighed and homogenized with ai Ultra-Turrax homogenizer in 2 vol of IOmM potassium phosphate buffer pH 6.5 containing 20% glycerol and 0.1 mM DTT. The homogenate was then centrifuged at 36,OOOg for 30min and the supernatant filtered through glass-wool to remove lipids. This was designated the 36,OOOg supernatant fraction. (A) Partial purijication of AK I and II CM-Sephadex. The 36,000 g supernatant fraction was applied to a CM-Sephadex column (14 x 3cm) that had previously been equilibrated with 10 mM potassium phosphate buffer, pH 6.5 containing 20% glycerol and 0.1 mM DTT. Following application of the protein, the CMSephadex column was washed with equilibrating buffer until all unadsorbed protein was eluted. (The eluate fraction contained a mixture of adenylate kinase isoenzymes). Elution of the adsorbed protein (AK I and II), was achieved by using a linear gradient of 1 I of &0.2 M KCI in 10 mM potassium phosphate buffer, pH 6.5 containing 20% glycerol and 0.1 mM DTT. Fractions of 7 ml were collected and assayed for adenylate kinase activity. The active fractions, which generally eluted between 0.025-0.1 M KC1 (Fig. l), were pooled and concentrated by dialysing against Carbowax overnight. The concentrate (approximately 3040 ml) was applied to a Sephadex G-25 column of dimensions 20 x 5.5 cm that had previously been equilibrated with IO mM imidazole_HCI buffer. pH 7.0 containing 0.1 mM DTT and 20% glycerol.

Phosphocelhdose. The effluent from the Sephadex G-25 column was applied to a phosphocellulose column of dimensions 24 x 2cm that had previously been equilibrated with 10 mM imidazole-HCI, pH 7.0 containing 0.1 mM DTT and 20% glycerol. After washing with 50ml equilibrating buffer to remove unadsorbed protein, AK I and II were separated by elution of the protein with 1 I pf a linear gradient of O-O.2 M KCI in 0.1 M imidazoleHCl buffer pH 7.0 containing 0.1 mM DTT and 20% glycerol. Fractions of 6ml were collected and assayed for adenylate kinase activity. The most active fractions of the two peaks eluted were pooled separately and concentrated using an Amicon Cell and PM 10 membrane. The peak eluting between 0.04-0.095 M KC1 was confirmed by electrophoresis to be AK II, whereas the one eluting between 0.10-0.15 M KCI was found to be AK I (Fig. 3). Preparations having a mixture of AK I and II isoenzymes on electrophoresis were re-chromatographed again on a phosphocellulose column, after passing the concentrated preparation through a Sephadex G-25 column equilibrated with 10 mM imidazole buffer, pH 7.0 containing 200/i glycerol and 0.1 mM DTT. Sephadex G-75. The concentrate was then applied to a Sephadex G-75 column of.dimensions 86 x 3 cm that had previously been equilibrated with 10 mM potassium phosphate buffer, pH 7.0 containing 0.1 mM DTT and 2p% glycerol. Fractions of 5 ml were collected and assayed for enzyme activity. The active fractions were pooled and frozen. These were used for kinetic studies. (B) Partial purification of AK III CM-Sephadex (batchwise). AK III was separated from the other AK isoenzymes by treatment of 36,000 g supernatants with CM-Sephadex, to which AK III did not adsorb under the experimental conditions used. The CMSephadex previously equilibrated with 10 mM potassium phosphate buffer, pH 6.5 containing 0.1 mM DTT and 20% glycerol, was sucked dry on a Buchner funnel and added (150 g wet weight) to the 36,OOOg supernatant fraction to form a thick suspension. This was stirred intermittently for 20min. The suspension was sucked dry on a Buchner funnel and the ion exchanger washed with a small volume of equilibrating buffer until no further. adenylate kinase activity could be detected in the eluate. The filtrate was again added to the CM-Sephadex. The process was repeated until the specific activity of filtrate remained constant. The filtrate was then concentrated by dialysing

619

of adenylate kinase isoenzymes obtained with (a) 36,000 g super rnatz mts Fig. St2uch gel electrophoresis of t mn nan hepatoma and partially purified (b) AK I, (c) AK II, (d) AK III obtained after DE :AE ellulose treatment, (e) AK III A-obtained after phosphocellulose chromatography. Fig. 4. St,arch gel electrophoresis of human hepatoma adenylate kinase III after partial after phosphocellulose treatment, (a) 4K . II I A, (b) AK III B, (c) AK 111 C. obtained A obtained after treatment with DEAE-Sephadex.

purii ficati on: (d) AK III

Human

hepatoma

Fraciion

adenylate

kinases

number

Fig. 2. Column chromatography of AK III on Phosphocellulose. Following application of the CMSephadex: Sephadex G-25 sample to a column (6 x 3.5 cm) of phosphocellulose, approximately 150 ml of IO mM imidazole buffer, pH 7.0 containing 2Opd glycerol and 0.1 mM DTT was washed through. Elution of the adenylate kinase isoenzymes was carried out using a linear gradient of 1,600ml of 04.2 M KC1 in 100 mM imidazole buffer pH 7.0 containing 20% glycerol and 0.1 mM DTT.

against Carbowax overnight. Starch gel electrophoresis of this fractiorr at this stage revealed the presence of AK III (Fig. 3). The concentrated fraction was applied to a Sephadex G-25 column of dimensions 20 x 5.5cm that had previously been equilibrated with 1OmM imidazaleHCI buffer, pH 7.0 containing 0.1 mM DTT and 20% glycerol. Phosphocellulose. The effluent from the Sephadex G-25 was applied to a phosphocellulose column (dimensions, 6 x 3.5cm) that had been equilibrated with the same buffer as that used to equilibrate the Sephadex G-25 column in the previous step. The phosphocellulose column was washed with equilibrating buffer until all the unadsorbed protein was eluted. This fraction, which contained considerable adenylate kinase activity, and which was found to migrate similarly to AK III on electrophoresis, was designated AK III A. (In experiments where the AK III fraction was rechromatographed onto a second phosphocellulose column all the protein and adenylate kinase activity was recovered on washing the column with eluting buffer. This confirmed that AK III A did not adsorb onto phosphocellulose). This fraction was concentrated down by dialysing against Carbowax overnight. The concentrate was dialysed against 2 changes of 2 I of 10 mM Tris-HCI buffer, pH 8.0 containing 0.1 mM DTT and 20% glycerol for 5 h and frozen until required. Elution of the adsorbed protein was achieved by using a linear gradient of increasing ionic strength consisting of 1,600 ml of &0.2 M KCI in 100 mM imidazole-HCI buffer pH 7.0, containing 0.1 mM DTT and 20% glycerol. Fractions of 1Oml were collected and assayed for adenylate kinase activity. The adenylate kinase fractions which eluted between 0.04-0.10 M KCI, was designated AK III B. Those eluting between 0.114.15 M KCI, was designated AK III C (Fig. 2). The effluents from the phosphocellulose column were pooled separately, concentrated down using an Amicon cell and PM 10 membrane and frozen till required. Preparations which were found to contain more than one form of adenylate kinase on electrophoresis were rechromatographed on phosphocellulose again. DEAE-Sephadex Further purification of AK III A was achieved by thawing the frozen partially purified AK III A preparation and applying it to a Sephadex G-25 column (dimensions, 20 x 5.5 cm) that has been equilibrated with 1OmM Tris-HCI buffer, pH 8.0 containing 20% glycerol and

0.1 mM DTT. The eluate from this column was applied to a DEAE-Sephadex column equilibrated with the same buffer. After washing the column with equilibrating buffer to remove unadsorbed protein, the adsorbed protein together with the enzyme was eluted with 1 I of IOmM Tris-HCI buffer, pH 8.0 containing 0.1 mM DTT, 20% glycerol and 0.05 M KCI. Fractions of 6 ml were collected and assayed for adenylate kinase activity. The active fractions were combined, concentrated down using an Amicon cell and a PM 10 filter and frozen, until required for kinetic studies. Determination of kinetic parameters Reciprocal velocities were plotted vs reciprocal substrate concentrations at various fixed levels of the second substrate. Data which gave linear plots were fitted to equation

(1).

VA t’ = K+A

(1)

where L’represents the observed velocity. V the maximum velocity, A the substrate concentration and K the apparent Michaelis constant (K,app) for substrate A at one fixed concentration of B. Data which conformed to a sequential initial velocity pattern (i.e. all lines meeting in a point) were fitted to equation (2). V ’ = 1 + K,/A

+ K,/B

+ K,,K,/AB

(2)

where K, and K, represent the limiting Michaelis constants for substrates A (AMP) and B (ATP) respectively. Ki, the dissociation constant of the first substrate to attach, if the mechanism is ordered. For a random mechanism. K,, the dissociation constant for B. is obtained from the relationship K,,Kb = KibK,. The limiting Michaelis constants are the Michaelis constants for each substrate at infinite concentration of the other. The data were analysed using the computer programmes of Cleland (1963a) which are based on the least-squares fit of the data points to the equations and give values for the constants and their standard errors. The lines on the figures were drawn according to the results of the computer analysis. The weighted means of the Michaelis constants and their standard errors were obtained as described by Morrison & Uhr (1966).

622

EFTIHIA CAYANIS Table

1. Partial

purification

Total activity (units)

Fraction

Crude homogenate CM-Sephadex chromatography Sephadex G-25 Phosphocellulose chromatography AK II AK I Sephadex G-75 AK II AK 1

516 410 361

Total

of human

protein (mg) 7668 369 326

I96 114 100 13.5

8.7 8.9

16.0 18.7

6.3 5.2

15.9 14.0

Electrophoretic behauiowr Starch gel electrophoresis at pH 7.0 of 36,000 g supernatants of human hepatoma revealed the presence of three adenylate kinase isoenzymes. These were designated AK I, II and III in order of their increasing electrophoretic mobility towards the anode (Fig.

2. Partial

36,000 g supernatant CM-Sephadex batch eluate (1) CM-Sephadex batch eluate (2) CM-Sephadex batch eluate (3) CM-Sephadex batch eluate (4) CM-Sephadex batch eluate (5) Concentration in carbowax Sephadex G-25 Phosphocellulose chromatography AK III A (unadsorbed) AK III B (adsorbed) AK III C (adsorbed) purification

of human

Total activity (units)

Fraction

Further

purification

Purification factor

22.5 12.6

2 show

Table

Specific activity (units/mg of protein)

I 14.7 14.5

typical results obtained with purification procedures used. Overall purification factors of 211- and 187-fold and yields of 17.3 and 12.8% were obtained on partial purification of human hepatoma AK II and I respectively. With human hepatoma AK III, no overall purification was obtained for AK III A, but overall purification factors of 4.48and 13.5-fold and yields of 0.8 and 1.3 were obtained for AK III B and III C respectively. From the results in Table 2, it can be seen that AK III, and in particular AK III A; lost considerable activity when concentrating these preparations by dialysing against Carbowax. The Amicon filtration technique generally .used to concentrate were not initially used, because the high protein content of these preparations made the filtration process very slow. 1 and

AK I and II

0.075 1.1 I.1

RESULTS Tables

hepatoma

1127 358 190 117 70.6 69.4 35.96 43.7

Yield (“J 100 71 64 34.0 16.7

211 187

17.3 12.8

3a). Figure 3 shows how the human hepatoma adenylate kinase isoenzymes were resolved into their separate components by using the purification technique described above. AK III was separated from AK I and II by means of CM-Sephadex chromatography. The latter two isoenzymes were then resolved by phosphocellulose chromatography. Although the electrophoretic mobilities of partially purified AK II and III were similar to those found in the 36,000g supernatant fraction, those of AK I were found to differ. Partially purified AK I, was found to have a slightly faster electrophoretic mobility towards the anode (in the front of origin) when compared to AK I of 36,000 g supernatants (behind the origin). Although AK III appeared as a single isoenzyme after CMSephadex treatment (Fig. 3d) three forms were later resolved on phosphocellulose chromatography. Figure 4 shows the electrophoretic mobility of these three forms of AK III, with AK III A migrating the fastest and III B and III C showing decreased electrophoretic mobility towards the anode. Both the electrophoretic mobilities and elution profiles from phosphocellulose of AK III B and AK II as well as those of AK III C and partially purified AK I were similar.

AK III and its secondary

products

Total protein (mg)

Specific activity’ (units/mg protein)

16,380 7176 6318 5760 5300 5256 5ooo 5ooo

0.07 0.05 0.03 0.02 0.013 0.013 0.0070 0.0087

10.0 9.9 14.7

2160 28.3 15.4

0.0046 0.314 0.943

4.75 8.2 6.2

2160 1472 124

0.0022 0.0056 0.05

Purification factor 1

4.48 13.41

Yield (S’,) 100

0.8 0.8 I.3

of AK III A

Dialysis and concentration in carbowax Sephadex G-25 chromatography DEAE-Sephadex chromatography

-

0.4 0.7 0.5

623

Human hepatoma adenylate kinases

0

5

IO

f 0

I

[MgC(,l

[MQCI,] mM

I

5

IO

mM

Fig. 5. (a) Effect of variation in the total magnesium concentration (MgCfCI,)on the initial velocity of AK 11 at various fixed ADP levels. Reaction mixtures contained 1OOmM imidazole-HCl buffer pH 7.0, 0.36mM NADP. 4 U G6PD. 4 U HK in 3.0ml in addition to MgC12. ADP and AK Il. Fig. 5. (b) EfTect of variation in the total magnesium concentration (MgCl,) on the initial velocity of AK II at various fixed ATP levels. Reaction mixtures contained IOOmM imidazole-HCl buffer pH 7.0. 5.0mM KCI. l.OmM AMP, 0.15 mM NADH, 3mM PEP. 14 U PK. IOU LDH in 3.0ml in addition to MgC12, ATP and AK II. The electrophoretic mobilities of AK III found in crude extracts of human hepatoma, AK III obtained after CM-Sephadex treatment and AK III A obtained after phosphocellulose treatment were identical. However, on DEAE-Sephadex chromatography of AK III A, an isoenzyme having an efectrophoretic mobility similar to AK II was obtained (Fig. 4). Cata/~tic

properties

~ffg~les~u~ r~qa~re~ents. In order to optimize the assay conditions, the activity of AK I when assaying in the reverse direction, was estimated as a function of the total magnesium chloride level in the presence of various constant levels of ADP. When assaying the enzyme in the forward direction, constant amounts of AMP and various fixed levels of ATP were used (Fig. Sa & b). In the reverse direction, maximum activity was obtained between 1 and Table 3. Stability of adenylate kinase isoenzymes to acid and alkali Isoenzyme AK AK AK AK AK

I Ii III A III B III C

Percentage activity” pH 10.5 pH 2.5 57 69 36 28 22

76 70 90 80 60

The various adenylate kinases were incubated in 5 mM glycine-HCl buffer pH 2.5 or 5 mM glycine-NaOH buffer pH 10.5 for 20 min at 25°C and then assayed. The reaction mixture contained 100 mM Imidazole-HCI buffer pH 7.0, 1SmM glucose, 4U G6PD, 4U HK, 1.5 MgC12 1SmM ADP and rate limiting amounts of enzyme. a Percentage activity = AK activity immediately after addition of enzyme to acid or alkali/AK activity after 20 min incubation.

1.5 mM MgC&. In the forward direction, optimum activity was obtained at 1 mM MgClz for concentrations of ATP below 0.1 mM. In the presence of 1 mM ATP, optimum activity was obtained at 2 mM MgCI,. As the nucleotide levels used were in the same range as those used for the kinetic studies the magnesium chloride concentration was maintained at a constant level of 1SmM and 2mM when assaying for

adenylate kinase in the reverse and forward directions respectively. pH Stability studies. that with the exception in both acid and alkali kinases were less stable more, the various AK

From Table 3, it can be seen of AK II which was stable solutions, the other adenylate at pH 2.5 than 10.5. Further-

isoenzymes responded differently to acid treatment, AK II being the most stable at pH 2.5 with AK I, AK III A, AK III B and AK III C showing decreased stability respectively. At

pH 10.5, AK III A, and AK III B were more stable than AK I, II and AK III C respectively. ~her~osfabi~~ry s~ad~es. Studies at 55°C showed the

adenylate kinase isoenzymes responded differently to heat inactivation. As can be seen from Fig. 6, AK I was the most stable, AK III A and III B showing intermediate stability and AK II and III C being the most labile. Utilization of substrate analogues

Substrate analogue utilization was determined by replacing the substrate in the assay mixture with an equimolar amount of the analogue. Na~ieot~de diphosphutes. Al1 the adenylate kinase isoenzymes exhibited no activity with UDP, TDP or dCDP (1 mM). However, with 1 mM IDP, a low activity that was 6.5x, 9.6x, 19.5% and 22% of that obtained with the normal substrate (1 mM ADP) was

found for AK II, III A, III B and III C respectively. 1 mM IDP did not serve as a substrate for AK I.

624

EFTIHIACAYANIS

Table 5. Michaelis constants hepatoma adenylate kinases AK isoenzyme I II III III III III

mins

incubated

Nucleotide monophosphates. Of all the nucleotide monophate analogues, such as dAMP, CMP, dUMP, TMP, dCMP, IMP and cyclic AMP (1 mM) tested, the adenylate kinases could only utilize dAMP as a phosphoryl acceptor. With 1 mM dAMP, 54.4%, 36x, 50.6%, 50% and 22% of the activity obtained with the natural substrate (1 mM AMP) was obtained with AK I, II, III A, III B and III C respectively. Nucleotide triphosphates, Table 4 shows the percent activity of the various adenylate kinases obtained, when ATP in the assay mixture was replaced by different nucleotide triphosphates. Higher activities were obtained for all the adenylate kinases in the presence of 1 mM dATP when compared to those obtained with 1 mM ATP. Very low activities were found for all the adenylate kinases in the presence of 1 mM dGTP and 1 mM dCTP. In the presence of GTP, CTP, ITP and UTP (1 mM), AK III B and AK II Table

4. Utilization

Adenylate

kinase

of

nucleotide

isoenzyme

Nucleotide triphosphate:-m 1 mM ATP 1 mM dATP 1 mM dGTP 1 mM dCTP 1 mM GTP 1 mM CTP 1 mM ITP 1 mM UTP 1 mM TTP

K, (ADP) 0.36 0.17 0.18 0.15 0.15 0.48

A.’ A” B c

mM

+ 0.070 i 0.006 f 0.005 + 0.018 + 0.009 _+ 0.10

Reaction mixtures contained 100 mM Imidazole-HCI buffer pH 7.0. 0.36 mM NADP. 1.5mM MgCl,. 4 U G6PD. 4 U HK. and enzyme in 3.0ml in addition to ADP. ,’ AK III A obtained after Phosphocellulose chromatography. “AK III A obtained after DEAESephadex chromatography.

at 55°C

Fig. 6. Effect of heat on the stability of the adenylate kinase isoenzyme. Activity is expressed as percentage of initial activity AK I (H), AK II (O-O), AK III A (A-A), AK III B (A---A) and AK III C ( x __ x ). The reaction mixture contained 100 mM imidazole-HCI buffer pH 7.0, 0.36mM NADP, 4 U G6PD, 4U HK 1.5 mM ADP in 3.0ml in addition to 1.5 mM MgCl,, enzyme.

of human for ADP

were more active than the other adenylate kinases. Apart from dATP, UTP also appeared to act as a good phosphoryl donor for AK II and AK III B. In its presence, 72y0 (AK II) and 80% (AK III B) of the activity of the enzyme was obtained when compared to that obtained with ATP. No activity was obtained in the presence of TTP. Michaelis

constants

The Michaelis constant obtained from double reciprocal plots of initial velocity vs ADP levels for the various adenylate kinases are listed in Table 5. The K, values for ADP of AK II, AK III B, as well as those of AK III A, after phosphocellulose, and DEAE-Sephadex treatment were similar (0.15-0.19 mM) whereas those of AK I (0.36 + 0.07 mM) and AK III C (0.48 + 0.10 mM) were somewhat higher, In the direction of ADP synthesis, reciprocal plots of initial velocity vs AMP concentration at 1 mM ATP gave a series of straight lines. With AK II and III A, the lines curved up at levels of AMP above 0.1 mM, indicating inhibition by high AMP levels. High levels of AMP did not inhibit AK I, III B and III C (Fig. 7). By replacing the ATP with 1 mM

triphosphate isoenzymes

I

100 150 1.9 7.2 3.0 4.3 10.0 13.6 -

analogues

by

adenylate

Percentage activity” III A III B II

loo I68 9,s 5.6 18.0 16.9 47.7 72.0

100 142 5.4 2.3 5.9 10.7 18.2 37.0

100 I61 8.3 5.8 30 24.8 85 80.0 -

kinase

III c

loo 119 8.1 6.3 8.8 3.0 18 21.2

The assay mixture cantained 100 mM imidazole-HCI buffer pH 7.0, 5.0 mM KCI, 2mM MgC12, l.OmM AMP, 0.15mM NADH, 2mm PEP, 14U PK. 1OU LDH, suitable amounts of enzyme preparation in 3.0ml in addition to 1 mM nucleotide triphosphosphate. activity in presence of nucleotide triphosphate X 100. il Percentage activity = activity in presence of ATP

625

Human hepatoma adenylate kinases

late kinases were found to be similar (0.26-0.41 mM).

With the exception of AK I, all the AK isoenzymes had a higher affinity for AMP when assayed at constant levels of ATP than with dATP. Higher apparent Michaelis constants were obtained for dAMP than AMP when assaying in the presence of 1 mM ATP; However, no significant variations were obtained in the apparent Michaelis constants for ATP and dATP

when assaying in the presence of 1 mM AMP. and dissociation constants for from double reciprocal plots of initial velocity vs substrate concentration at various constant levels of the second substrate of the type shown in Fig. 8 for AK III C. The lowest dotted line in Fig. 8 represents a secondary plot of intercepLimiting

Michaelis

ATP and AMP were obtained

Fig. 7. Double reciprocal plots of initial velocity, YSAMP concentration. The reaction mixture contained 100 mM imidazole-.HCl buffer pH 7.0, 5.0 mM KCI. 2 mM MgCIZ, l.OmM ATP.O.15mM NADP,2mM PEP, 14U PK. IOU LDH, rate limiting amounts of enzyme and AMP in 3.0 ml. AK I (G----O), AK II (a----O). AK IIIA (A---+Q. AK III B (A-A), AK III C ( x ~ x ). dATP, no inhibition levels was obtained.

of AK II and III A by high AMP

Double reciprocal plots of initial velocity vs dAMP levels at 1 mM ATP were also linear even at the high concentration of dAMP for all the adenylate kinase &enzymes, indicating no substrate inhibition by high levels of dAMP. Table 6 lists the apparent Michaelis constant obtained for AMP and dAMP when assaying in the presence of 1mM ATP of dATP. In the presence of 1mM ATP, the apparent Michaelis constants for AMP were diferent for the various adenylate kinase isoenzymes; that of AK I being the highest (0.20 + 0.013 mM) with those of AK III C (0.068 f 0.006 mM) and the other adenylate kinase isoenzymes being much lower (0.02~.~ mM). On the other hand, when assaying in the presence of 1 mM dATP, the apparent Michaelis constants for AMP of the different adeny-

tion vs reciprocal ~oncentratio1~ of the second substrate (Florini & Vestling, 1957). Similar linear plots with the lines meeting at a point above the abscissa were obtained with all the other adenylate kinase isoenzymes. The Michaelis and dissociation constant for ATP and AMP of the adenylate kinase isoenzymes thus obtained are summarized in Table 7. The Michaelis constants for AMP of AK II were the lowest (0.012 & 0.002 mMf: those of AK III A (0.0213 + 0.003 mM), AK III B (0.035 & 0.008 mM) and AK III C (0.047 & 0.003 mM) slightly higher whereas that of AK I (0.195 + 0.027 mM) was considerably higher. With the Michaelis constants for ATP, a similar trend was found, namely that the I(, value for ATP of AK 11, III A, III B and III C increased progressively from 0.06 to 0.1 mM. The K, value for ATP of AK I was again much higher (0.213 k 0.054mM) than those found for the other adenylate kinase isoenzymes. In all cases the dissociation constants for ATP and AMP were found to be higher than their respective Michaelis constants.

DISCUSSION

The separation and characterization of AK I, II and III from human hepatoma tissue confirms that these are distinct &enzymes with unique and differing properties, as has been found for these isoenzymes from other mammalian tissues (Noda & Kuby. 1957;

Table 6. Apparent Michaelis constants for AMP, dAMP, ATP and dATP of partially purified adenylate kinases Isoenzyme AK AK AK AK AK AK

I II III III fit III

A,’ A” B C

App I<, AMP mM’ App K, AMP mM” App K, dAMP mM’ 0.200 0.023 0.032 0.040 0.028 0.068

The reaction mixture 2 mM PEP, 14 U PK. amounts of enzyme. ’ AK II1 A obtained ’ AK III A obtained ’ Assayed at 1.OmM ” Assayed at l.OmM ’ Assayed at 1.0 mM ’ Assayed at l.OmM %Assayed at l.OmM

+ 0.031 f 0.004 i: 0.004 & 0.001 & 0.011 &-0.006

App K, ATP’

App K, dATP”

0.26 & 0.015 0.27 * 0.04Q

1.18 + 0.190 0.33 2 0.036

0.147 i: 0.0027

0.127 & 0.096 0.118 k 0.007

0.41 f 0.043 0.37 I: 0.054 0.28 rfr 0.003

0.80 + 0.149 0.56 It 0.060 0.77 + 0.017

0.08 + 0.002 0.060 * 0.003 0.144 + 0.020

0.126 i: 0.0125 0.120 * 0.007 0.278 k 0.125

contained 1OOmM imidazole-HCl buffer pH 7.0, 5.0 mM KCI, 2 mM MgC&, 0.15 mM NADH, 10 U LDH 1 mM ATP or 1 mM dATP, or 1 mM AMP or 1mM AMP and rate limiting after Phosphoceilulose after DEAE-Sephadex ATP. dATP. ATP. AMP. AMP.

treatment. treatment.

EFTIHIA CAYANIS

626 200

[AMP]~M p

0040

150 I

-10

-5

0

5

IO

15

[ATP]mM

[X&F] mM-' Fig. 8. Double reciprocal plots of initial velocity vs substrate concentration at various fixed levels of the second substrate. Reaction mixtures contained 100 mM imidazoleHCI buffer DH 7.0. 5.0 mM KCl. 2 mM M&L,. 0. I5 mM NADP. 2 mM PEP, 14 U PK, 10 U LDH in 3.0ml in addition to rate limiting amounts of AK III C, ATP and AMP. Chiga & Plaut, 1960; Markland & Wadkins, 1966; Chiu & Russel, 1967; Criss et al., 1970; Schirmer er a/., 1970; Sapico et al., 1972; Thuma et al., 1972; Criss et al., 1974; Russel et al.. 1974; Tsuboi & Chervenka, 1975; Pradhan & Criss, 1976). In the present study, the various adenylate kinase isoenzymes were separated by means of ion exchange chromatography techniques. Apart from studies showing the separation of the adenylate kinase isoenzymes from human erythrocytes (Tsuboi & Chervenka, 1975), ion exchange chromatography techniques involving phosphocellulose, DEAE and CM-

Table

7. Limiting

Michaelis

0.195 0.35 0.213 0.244

f * f *

constants

0.027 0.06 0.054 0.018

0.012 0.099 0.060 0.248

+ + + f

of human

hepatoma

III A

II

I

AK isoenzyme Kinetic constants” K, (mM) &, (mM) & (mM) Kih (mM)

and dissociation

Sephadex. have mainly been used to isolate and purify only the predominant forms of the enzyme from rabbit (Noda & Kuby. 1957). porcine (Schirmer et al.. 1970) and human muscle (Thuma er a/.. l972), swine (Chiga & Plaut. 1960) and bovine liver (Markland & Wadkins, 1966) and yeast (Chiu & Russel, 1967). Generally, isoelectric focusing and subcellular fractionation techniques have been used to isolate the various adenylate kinases from various rat tissues (Criss er ul., 1970; Sapico et al., 1972; Criss et ~1.. 1974). As the human hepatoma tissue used was frozen, the subcellular fractionation techniques used for the isolation of rat adenylate kinases could not be applied in this study. Furthermore, it was felt that any subcellular enzyme distribution results obtained with fresh hepatoma tissue would not be valid as the tissue was often cirrhotic and showed variable degree of necrosis. The AK III fraction was obtained after exhaustive adsorption of the original crude extract onto CMSephadex which removed 94”: of the total AK activity. This minor component had approximatelv & the specific activity of the crude fraction. Phosphocellulose chromatography of AK III gave three fractions namely AK III A (non-adsorbed) III B and III C which not only eluted but also migrated electrophoretically like isoenzymes, III, II and I respectively. However, a second passage of AK III A over phosphocellulose did not give a new redistribution of the isoenzymes which should have resulted if an equilibrium existed between forms III, II and I. It is possible the original CM-Sephadex treatment left traces of forms I and II unadsorbed in the fraction III preparatior. and these for some reason did not show up on electrophoresis. Perhaps the starch-gel electrophoresis technique used was not sensitive enough or some impurities in the crude preparation may have affected the activity of AK I and II. These impurities may also have interfered in some way with the removal of the last traces of forms I and II by the CMSephadex. When the bulk of the protein impurities were removed with fraction III A by passing the unadsorbed protein through phosphocellulose the remaining enzymes eluted from phosphocellulose (III B and III C) could perhaps be identified as types II and I respectively. However, from Table 8 it can be seen that slight differences in the properties of AK I and III C as well as AK II and AK III B are apparent indicating that they are similar but not identical. On the other hand, the Michaelis constants obtained for AK III A after various stages of purification were found to be similar, even though the elec-

0.002 0.018 0.009 0.035

0.02 13 + 0.072 + 0.062 * 0.225 +

0.003 0.011 0.009 0.042

adenylate

kinases

III B 0.035 + 0.008 0.176+ 0.042 0.078 f 0.01 0.316 & 0.009

for ATP

and AMP

III c 0.047 0.129 0.10 0.236

+ k + +

0.003 0.009 0.006 0.0219

a K,, Ki,, K,, Kib represent the Michaelis and dissociation constants for AMP, and Michaelis and dissociation stants for ATP respectively assuming the reaction is random. If it is ordered, K,, has no meaning.

con-

i AK III A obtained after DEAE-Sephadex

Phospho~liuiose eiution profite pH Optimum (forward direction) (reverse direction) Stability at pH 2.5 Stability at pH 10.5 Thermostability % substrate anaio_gue -~-__ -. utilization ---. IDP dAMP GTP CTP ITP UTP Effect of High AMP levels Apparent K, AMP at 1 mM dATP Apparent li;, dAMP at I mM ATP K, AMP L (ADP) 22.0 22.0 8.8 3.0 18.0 21.2 0.28 mM 0.77 mM 0.047 mM 0.48 mM

0.0 54.4 3.0 4.3 10.0 13.6 0.26 mM 1.18 mM 0.195 mM 0.36 mM

column chromatography.

0.10-0.15M KCI 6.5 7.5-8.5 22.0 60.0 Unstable

AK III C

0.10-0.15 M KCI 5.5 7.0 57.0 76.0 Unstable

AK I

16.9 47.7 72.0 Inhibits 0.27 mM 0.33mM 0.012 mM 0.17mM

18.0

19.5 50.0 30.0 24.8 85.0 80.0 0.37 mM 0.56 mM 0.035 mM 0,15 mM

O.WO.10 M KCI 5.5-6.0 65-7.0 28.0 80.0 Intermediate

0.04-0.095 M KC1 5.5 7.0 69.0 70.0 Stable 6.5 36

AK III B

AK II

Table 8. A comparison of some of the properties of human hepatoma adenyiate kinases

9.6 50.6 5.9 IO.7 18.2 37.0 lnhibits 0.41 0.80 0.021 0.15-0.18

5.5-6.0 6.5-7.5 36.0 90.0 Intermediate

AK III A

EFTIHIA CAYANIS

628

trophoretic mobility of AK III A towards the anode was reduced and resembled that of AK II after DEAE-Sephadex chromatography. In spite of this finding, it can be seen from Table 8 that AK III and II had different acid, alkali and temperature stabilities showed marked variation in ITP and UTP utilization and had different K, values for AMP and dAMP. The initial specific activities of 0.075 U/mg protein obtained for 36,000 g supernatants fractions of human hepatoma tissue were approximately an order of magnitude lower than the value of 0.45 U/mg protein, reported for rat liver supernatant adenylate kinases (Criss ef al., 1970; Sapico et al., 1972). The substrate analogue studies showed that human hepatoma adenylate kinases were relatively specific for the nucleoside di- and mono-phosphate and nonspecific for the nucleoside triphosphate. Apart from ADP, human hepatoma adenylate kinases could react only with IDP. This is contrary to the findings obtained with rabbit muscle adenylate kinases, which was shown to react only with ADP and CDP and not with IDP, GDP or UDP (Noda, 1958). Of the nucleotide monophosphates tested, the human hepatoma adenylate kinases could react only with AMP and dAMP. In this respect they resembled human erythrocyte AK, and AK, but differed from rat liver adenylate kinases II and III which could only utilize AMP as a substrate (Sapico et al., 1972). As in the present study, rabbit muscle adenylate kinase has been shown to react (in order of decreasing activity) with ATP > 2’dATP > GTP > ITP > UTP (Noda, 1958; O’Sullivan & Noda, 1968). Although human hepatoma adenylate kinases were not very specific for the nucleoside triphosphate substrate, rat liver adenylate kinase isoenzymes II and III have been shown to utilize only ATP and dATP and not dGTP, GTP, UTP or ITP as substrates (Sapico et al., 1972). However. human hepatoma adenylate kinases resembled rat liver adenylate kinase isoenzymes II and III

Table 9. Comparison

of Michaelis

(Sapico et al., 1972) in that they were more active in the presence of dATP than ATP. The higher activity noted with dAMP than AMP with human hepatoma AK II and III A could partly be attributed to the fact that these isoenzymes are inhibited by I mM AMP. However activation by dAMP was also obtained with AK I, III B and III C, which are not affected by high AMP levels. In fact, Criss et al. (1970) demonstratedthat dAMP increased the maximum velocity of rat liver adenylate kinase III, though the K, value for dAMP was higher than that obtained with AMP. The kinetics in both the forward and reverse directions of all the adenylate kinase preparations were found to be similar in that they all displayed Michaelis Menten kinetics. In this respect they resemble rabbit muscle (Noda & Kuby, 1957; Callaghan, 1957), human muscle (Thuma et al., 1972), bovine liver mitochondria (Markland & Wadkins, 1966), rat liver (Criss et al., 1970), erythrocyte (Tsuboi & Chervenka, 1975) and yeast (Chiu & Russel, 1967) adenylate kinases. When assaying in the forward direction. the double reciprocal plots of initial velocity versus substrate concentration at various constant levels of the second substrate were linear and met at a point. This suggests that all the human hepatoma adenylate kinases tested have a sequential mechanism whereby both substrates must add to the enzyme before either product is released (Cleland, 1963b). These findings are comparable to those obtained for human erythro,cyte adenylate kinase (Tsuboi & Chervenka. 1975). The kinetic data are however not detailed enough to distinguish between a random mechanism and one in which the substrates add onto the enzyme in a given order. Although similar kinetics were obtained for all the human hepatoma adenylate kinases, the constants found for the various human hepatoma adenylate kinases differed not only among themselves. but also

constants Appartent

Adenylate Human

kinase

type

[ATP]

hepatoma

Human muscle” Rat Morris heoatoma Adult rat liver’

I 11 111 A III B III c 3924Ah II’ III’ III”

Foet?l Adult Adult Bovine Rabbit Yeasth

rat liver rat musc‘le h rat brain’ liver mitochondria’ muscle”

Data taken from ~’Thuma et al. (1972). b Criss et al. (1974). F Criss et al. (1970). ’ Sapico et al. (1972).

0.15 0.08 0.06 0.14 -I 0.27 33.0 7.0 0.39 0.43 0.125 40.0 10.0 15.0 1.80 0.33

of various

[AMP]

3.0mM AMP 60mM AMP 22.5 mM AMP 0.25 mM AMP 0.25 mM AMP 2.0mM AMP

at 22.5mM at 6.0mM

kinases

K, for

mM

at 1 mM AMP

at at at at at at

adenylate

AMP AMP

’ Pradhan and Criss (1976). ’ Markland and Wadkins (1966). e Noda, L. (1957). h Chiu, C. et al. (1967).

mM

0.20 0.02 0.03 1 at 1 mM ATP 0.03 0.07 0.32 at 3.0mM ATP 18.0 at 60mM ATP 1.9 at 22.5mM ATP 0.07 at 3.0 mM ATP 0.13 at 3.0mM ATP 0.13 at 3.0mM ATP 20.0 6.2 at 22.5 mM ATP 7.7 2.7 at 12mM ATP 0.26

K, [ADP]

mM

0.36 0.17 0.15 0.48 0.35

0.30 0.18 0.18 14.30 12.30 1.80 K, = 0.33 0.27

Human hepatoma adenylate kinases

from those obtained for adenylate kinases from human muscle (Thuma ef al., 1972). rat tissues (Criss et al., 1970; Sapico et al., 1972; Criss et a!., 1974; Pradhan & Criss, 1976), bovine liver (Markland 8r Wadkins, 1966). rabbit muscle (Noda & Kuby, 1957) and yeast (Chiu & Russel, 1967). As can be seen in Table 9, the apparent K, values for ATP vary from 0.06 to 40mM. for the various adenylate kinases; those for AMP from 0.02 to 20mM and those for ADP from 0.1514.3mM. The wide variation noted in the apparent K, value for ATP may partly be attributed to the assaying in the presence of high AMP levels which could be inhibitory. In spite of the variation, the apparent K, values for ATP obtained for the various human hepatoma adenylate kinases are of the same order of magnitude as those noted for human muscle (Thuma et al., 1972) adult rat liver isoenzyme II and III (Criss et al., 1970; Sapico er al., 1972) and rabbit muscie (Noda & Kuby, 1957) but differ from the rat Morris hepatoma 3924A (Criss et ul., 1974) foetal rat (Pradhan & Criss, 1976) rat muscle (Criss et al., 1974). rat brain (Pradhan & Criss, 1976) and bovine mjto~hondria (Markiand & Wadkins, 1966) adenylate kinases. As regards the apparent K, value for AMP, the human hepatoma AK resembles those of human muscle (Thuma et a/., 1972) adult rat liver (Criss et al., 1970; Sapico et al., 1972) and rabbit muscle (Noda & Kuby, 1957). Although the apparent K, value for AMP of AK I was approximately an order of magnitude higher than those obtained for the other human hepatoma adenytate kinases (AK III C being an exception), it is still considerably lower than the I(,,, value for AMP reported for rat Morris hepatoma 3824A (Criss et al., 1974), adult rat liver (Criss et al., 1974), foetal rat liver (Pradhan & C&s, 1976) adult rat muscle (Criss et al., 1974), adult rat brain (Pradhan & Criss, 1976) and bovine liver mito~hondria (Markland & Wadkins, 1966) adenylate kinases. The Michaelis constants for ADP obtained for the different human hepatoma adenylate kinases did not differ significantly from each other (0.15-0.48mM) and resembled those obtained for human muscle (Thuma er al., 1972), rat liver (Criss et al., 1970; Sapico et aL, 1972). rabbit muscle (Noda & Kuby, 1957) and yeast (Chiu et at., 1967). These were however considerably lower than those reported for foetal rat liver (Noda & Kuby, 1957) adult brain (Pradhan & Criss, 1976) and bovine liver mitochondria (Markland & Wadkins, 1966). ~~k~o~~e~ge~e~rs-I would like to thank Dr D. Balinsky for helpful suggestions and the South African Medical Research Council for providing support for part of this work.

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