Threonine Aldolase and Allothreonine Aldolase in Rat Liver

Transcription

1 European J. Biochem. 8 (1969) Threonine Aldolase and Allothreonine Aldolase in Rat Liver G. RIARIO-SFORZA, R. PAGANI, and E. MARINELLO Istituto di Chimica Biologica, UniversitA di Siena (Received July 8/November 12, 1968) 1. The aldol breakdown of L-threonine and L-allothreonine in rat liver is catalyzed by systems localized in the soluble fraction of the homogenate. 2. Evidence in presented that two different enzymes, indicated as threonine aldolase and allothreonine aldolase, are involved in the breakdown of the two amino acids. Braunstein and Vilenkina [l], during studies on the formation of glycine from serine, threonine and other b-hydroxy-a-amino acids in animal tissues, demonstrated the presence of an enzymatic system which cleaves L-threonine and L-allothreonine into acetaldehyde and glycine. These investigations were extended by Karasek and Greenberg [2] using sheep liver, and by Gilbert 1131, Lin and Greenberg [4], Malkin and Greenberg [5], in rat liver. Two different enzymes were demonstrated in sheep liver, namely threonine aldolase which catalyzes reversibly the reaction : L-threonine f acetaldehyde + glycine and allothreonine aldolase which is implicated in another reversible reaction : (I) L-allothreonine + acetaldehyde + glycine (2) On the other hand, in rat liver, Malkin and Greenberg [5] showed a unique enzyme, which they called threonine or allothreonine aldolase, capable of catalyzing the breakdown of both L-threonine and L-allothreonine, and the synthesis of only L-allothreonine [3-51. We have studied the aldol breakdown of L-threonine and L-allothreonine in rat liver, in order to define the intracellular distribution and properties of the system catalyzing these reaction and to establish whether a single enzyme is responsible for two activities or not. Our experiments gave evidence for the existence of two different enzymes, as in the case of sheep liver. For this reason we prefer the use of the terms threonine aldolase and allothreonine aldolase instead of the terminology proposed by Malkin and Greenberg [5]. MATERIALS AND METHODS Adult Wistar strain albino rats weighing about 200 g and fed on a standard laboratory diet were used. The animals were killed by decapitation, the Enzymes. Threonine aldolase (EC ); allothreonine alclolase (EC ). livers were washed in 5 volumes of the appropriate solution and homogenized using Potter-Elvehjem apparatus. The pestle operated at 1,000 rev./min. The technique proposed by DeDuve and coworkers [6] was used to separate the subcellular fractions. In some experiments liver was homogenized in 0.25 M sucrose, containing I mm EDTA. By differential centrifugation, nuclear, mitochondrial, lysosomal, microsomal and soluble fractions were separated. The nuclear, mitochondrial and lysosomal fractions were washed with 4 ml of the above solution. Nuclei and microsomes were resuspended in 4 volumes of the homogenizing medium per g of fresh liver, while mitochondria and lysosomes were suspended in two volumes per g of liver. In other experiments the 20 /0 homogenate was prepared in isotonic KCI. In this case the homogenate was first centrifuged at 20,000 x g for 20 min; the sediment was discarded ; the supernatant was centrifuged at 100,000 x g for 60 min. The supernatant thus obtained, was brought up to the initial volume of the homogenate and submitted to differental fractionation procedure with (NH,),SO,. (NH,),SO, fractionations were performed at 0 by the addition of a saturated solution of (NH,),SO,: Kunitz s [7] formula was employed to reach the desired saturations. After the addition of (NH,),SO,, the precipitate was spun down at 20,000xg for 10 min. Precipitates were resuspended in 0.01 M phosphate buffer, ph 6.8 (1 g of tissue per ml). In some experiments the soluble fraction was submitted to ammonium sulphate fractionation between Olio saturation (A), and /, saturation (B), in other experiments between 0-40 saturation, Ol0 saturation, Ol0 saturation and Ol0 saturation. Threonine aldolase and allothreonine aldolase activities were measured by following the production of acetaldehyde by two different methods. In some experiments the semicarbazide method of Lin and Greenberg [4] was employed. The incu-

2 Vo1.8, No. 1, 1969 G. RIARIO-SFORZA, R. PAGANI, and E. MARINELLO 89 bation mixtures (1 ml) had the following composition : 4 mm m-allothreonine or 60 mm L-threonine, 10 pm pyridoxal phosphate, 50 mm phosphate buffer, ph 7.5, samples from the various fractions in quantities corresponding to 10 mg tissue. In some experiments, NaF was added to prevent phosphatase activities, at concentrations which will be indicated in the tables. At the end of theincubation, acetaldehyde, which had been trapped by the semicarbazide reagent of Lin and Greenberg [a], was measured according to the method of Burbridge and coworkers [8]. In other experiments the NADH dependent enzymatic method of Malkin and Greenberg was employed; the incubation mixtures (1 ml) had the following composition : 4 mm m-allothreonine or 60 mm L-threonine, 10 pm pyridoxal phosphate, 10 mm phosphate buffer, ph 7.5, 0.1 mm NADH, supernatant or various fractions in concentrations which will be indicated in the different tables and figures, 5 p1 alcohol dehydrogenase. The decrease in absorbance at 340 my was followed for 10 min. With all fractions control experiments were run, in which substrate was omitted, in order to measure NADHoxidase activity, the values of which were then substrated for the activity recorded in the tests with substrates. Spectrophotometric readings were taken in a Optica CF, spectrophotometer. Centrifugations were carried out in Lourdes Betafuge and in Spinco L50 centrifuges at 0". One unit of enzyme was defined as the quantity which forms 1 pmole of acetaldehyde in 1 min. Buffers were prepared according to Gomori [9]. m-allothreonine was purchased from the Koch- Light Company, L-threonine from BDH, pyridoxal phosphate and NADH from the Sigma Chemical Company, alcohol dehydrogenase from the C.F.Boehringer & Soehne (Mannheim). RESULTS From the results of Table 1 it is evident that both L-threonine aldolase and L-allothreonine aldolase activities are present only in the soluble fraction. The threonine aldolase and allothreonine aldolase activities of rat liver are very probably pyridoxal phosphate dependent, as in the case of the sheep enzyme [2]. Since in the crude extracts the coenzyme could de dephosphorylated, NaF was added to the incubation mixture (the same addition was done to the soluble fraction and to the ammonium sulphate precipitates even though they are known to be free of phosphatase activities). The determination of the enzyme activity in the soluble fraction was measured both by the semicarbazide and by the enzymatic method. The results obtained by the two procedures were found to be in good agreement. Table 1. Intracellular distribution of dlothrwnine aldolme and thrwnine aldolase Allothreonine aldolase and threonine aldolase were measured by the semicarbazide method[4] as described in the text. The reaction mixtures contained aliquots of the single fract,ion corresponding to 10 mg of fresh tissue, in a final volume of 1 ml; 0.2 M NaF was always added to inhibit phosphatase activities. The semicarbazide method was chosen in order to avoid the interference of NADH-oxidases present in some fractions Total activity Recovery Fractions Volume Allothreo- Threo- Allothreo- Threonine nine nine nine aldolase aldolsse aldolase aldolase ml units units "0 "0 Homogenate Nuclear Nitochondrial Lysosomal Microsomal Soluble Time'(rnin) Fig.l. Time course of threonim aldolaae and dlothrwnine aldolase activity. Allothreonine aldolaae and threonine aldolase activities were measured by the enzymatic method of Malkin and Greenberg [ti]. The composition of the reaction mixtures is indicated in the text : the supernatant was added in a final concentration corresponding to 10mg of tissue per ml. The pmoles of acetaldehyde formed at the different times in 1 ml of the assay mixture are reported in the ordinate. 0, allothreonine aldolase; 0, threonine aldolase activity The course of the reaction as assayed by the enzymatic method was linear with time (Fig.1). In all subsequent experiments the latter method was adopted, for routine purposes. The ph optimum in the soluble fraction was found to be 7.6, for both threonine aldolase and allothreonine aldolase. The relative rates of the aldolic breakdown of allothreonine and threonine in the homogenate and in the different ammonium sulphate fractions were followed in order to establish whether the activities shown were linked to one or to two enzymes.

3 90 Threonine Aldolase and Allothreonine Aldolase in Rat Liver Europcan J. Biochem. Table 2. Threonine aldolase and allothrwnine aldolase activity in supernatant and in the fraction (A) and (B); effecta of various treatments Aldolase activities were measured with the enzymatic method of Malkin and Greenberg [5], as described in Material and Methods. Reaction mixtures contained supernatant or fraction (B) in concentrations corresponding to 10 mg tissue; fraction (A) in concentrations corresponding to 130 mg/ml. (NH,),SO, was added only to the incubation mixtures and values in parentheses represent the (NH4),S0, saturations thus realized PH Fig.2. ph optimum of allothreonine aldolaae and thrwnine aldolase. The enzymatic activity was measured by the semicarbazide method of Lin and Greenberg [4], since the enzymatic method cannot be used below ph 6. The experimental conditions are indicated in the text; the supernatant was added in final concentrations corresponding to 10mg tissue per ml. -, allothreonine aldolase; ----, threonine aldolase activity Fractions Total activity Al,othreo- nine Volume $ ; 2 nldolase: 8 = +z2 a4 threonine aldolase in1 units units ratio Supernatant A A + (NH4XS04 (0.003) A + (NH4),S0, (0.006) A + (NH&.SO4!0.009) Alrept for 5mm at 100" B (NH.&SO, (0.003) B + (NH&SO, (0.006) B + (NH4hSO4!0*009) I B kept for 5mm at 100" Tablc 3. Threonine aldolaae and allothrwnins aldolase activity in fractions obtained at different (NH4),S04 saturations- from the supernatant Aldolase activity was measured with the enzymatic method of Malkin and Greenberg [5] as described in the text Fraction: (xh,),so, satrlration 'Oncn. Total activity Recovery Allothreonine aldolase : Volume Allothreonine Threonine Allothreonine Thrconine threonine aldolasc aldolase aldolase aldolase aldolase mg tissuelrnl ml units units % "1. ratio Soluble to 40 /, to 450/, to 50 /, to 60 /,, The ratio allothreonine aldolaae activity : threonine aldolase activity differed in the various ammonium sulphate fractions. This ratio was about 1 in the soluble fraction, 0.2 in the fraction (A) precipitating between 26 and 35O/, saturation and 2.1 in the fraction (B) precipitating between 35 and 60 saturation. As shown in Table 2 these differences in the ratio were not due to the differences in ammonium sulphate concentrations in fractions A and B. Heat inactivation at 100" for 5 min. showed that the activities were entirely enzymatic. All these experiments supported the conclusion that the inversion of the ratio was due to the presence of two Merent enzymes, one specfic for L-threonine more abundant in fraction A than in fraction B, the other specific for L-allothreonine, mainly present in fraction B and almost absent from fraction A. In order to obtain further evidence for this hypothesis the ammonium sulphate fractionation was carried out under more selective conditions. Table 3 shows that allothreonine aldolase activity increased gradually with increasing concentrations of ammonium sulphate up to 50 /,, saturation. The opposite behavior was shown by threonine aldolase, the activity of which gradually decreases with increasing ammonium sulphate saturation. The ratio allothreonine aldolase activity : threonine aldolase activity underwent a gradual, clear cut inversion, going from a value of 0.15 at 4001, saturation to a value of 13 at 60 /, saturation. The values at 4501, saturation and in the supernatant were respectively 0.86 and 1.&. The hypothesis that threonine aldolase and allothreonine aldolase are two distinct enzymes was

4 VoI.8,?Jo.l, 1069 G. RIAELIO-SFORW, R. PACANI, and E. MARINELLO 91 1 LI 1 5 K) [HGhlbM) Fig.3. Threonine aldolase and allothreonine aldolase activities in the presence of different wncentrdiona of HgCh. Threonine aldolase and allothreonine aldolase activities were measured by the enzymatic method of Makin and Greenberg[5]. The incubation had the same compositions as in Fig. 1, and contained fraction (A) in concentrations corresponding to 130 mg of fresh tissue per ml, fraction (B) in concentrations corresponding to 10mg of fresh tissue per ml; HgCl, was added at the concentrations indicated in abscissae. The activity values are corrected for NADH-oxidese activity, which had been determined in separate experiments. 0, allothreonine aldolaee activity in fraction precipitated between 0 and 350/,, saturation of ammonium sulphate; 0, threonine aldolaae activity in the same fraction; A, allothreonine aldolase activity in fraction precipitating between 50 and 60 /o saturation of ammonium sulphate; A, threonine aldolase activity in the same fraction confirmed by their different sensitivity to EDTA and HgCb. Fig. 3 shows the behavior of threonine aldolase and allothreonine aldolaae in fractions (A) and (B) after a 30 min incubation in the presence of different concentrations of HgCl,. Both enzymes were inhibited by HgCl, indicating the involvement of -SH groups. In both fractions, decay of threonine aldolase activity, however, is much more marked since, at a concentration of 20 HgCl,, it was only 15 /0 of the original, while at the same HgC4 concentrations, the activity of allothreonine aldolase was 50 /, of the original. The addition of 2 mm EDTA to the incubation mixtures, completely restored both activities. Both activities were sensitive to higher concentrations of EDTA, aa shown by Fig.4. At 10 mm EDTA allothreonine aldolase activity in fractions (A) and (B) was reduced to 50 /,, of its original value, while under the same conditions threonine aldolase activity fell to 200/0. x) [EOTA!(rnM) Fig.4. inhibition of threonim a2dolaae and allothreonine aldohe by different wncedrdiona of EDTA. Threonine aldolaee and allothreonine aldoltlse activitiee were measured by the enzymatic method of Malkin and Greenberg [5]. The incubation mixtures had the same composition as indicated in Fig.1, and contained fraction (A) in a concentrations corresponding to 130 mg of freeh tissue per ml, fraction (B) in a concentration correaponding to 1Omg of fresh tissue per ml; EDTA was added at the concentrations indicated in the abscissae. The activity valuw were corrected for the NADH-oxidaee activities determined in separate experiments. Symbols aa in Fig. 3 The experiments with EDTA as well as those with HgCI, indicate that allothreonine aldolase is more stable towards these reagents than threonine aldolase. CONCLUSIONS The present experiments suggest that two different enzymes, one of which is active on L-threonine and the other on L-allothreonine, are present in rat liver. Supporting evidence is provided by ammonium sulphate fractionation of the soluble fraction liver homogenate which initially contains all of the two activities. The ratio allothreonine aldolase : threonine aldolase in the different fractions showed an antithetical behavior. Inhibition experiments with EDTA and HgCl, confirm the hypothesis. Both enzymes are sensitive to EDTA and HgCI,, but threonine aldolase is inhibited by both EDTA and HgC1, to a greater extent than allothreonine aldolase. These conclusions are different from those of Malkin and Greenberg [5] who claimed that a single enzyme, called by them threonine or allothreonine aldolase could split both substrates. A very important problem which arises from our research is the physiological role of L-allothreonine

5 92 G. RIARIO-SFORZA et al. : Threonine Aldolase and Allothreonine Aldolase in Rat Liver European J. Biochcm. aldolase. This is difficult to understand, since L-allothreonine is not a natural amino acid. However, the possibility of an enzymatic isomerization of L-threonine to L-allothreonine cannot be excluded; allothreonine aldolase could be involved in L-threonine metabolism, and in the regulation of the aldol breakdown of L-threonine. This work was supported by grants from the Consiglio Nazionale delle Ricerche of Italy, Impresa Enzimologia. REFERENCES 1. Braunstein, A. E., and Vilenkina, G. Y., Dokl. AM. Nauk. SSSR, 66 (1949) Karasek, M. A., and Greenberg, D. M., J. BioZ. Chem. 227 (1957) Gilbert, J. B., J. Am. Chem. 8oc. 76 (1954) Lin, S. C. C., and Greenberg, D. M., J. Gen. Physiol. 38 (1954) Malkin, L., and Greenberg, D. M., Biochim. Biophys. Acta, 85 (1964) De Duve, C., Pressman, B. C., Gianetto, R., Wattiaux, R., and Applemans, F., Biochem. J. 60 (1955) Kunitz, M., J. Qen. Physiol. 35 (1952) Burbridge, T.N., Hine,C. H., and Schick,A. F., J. Bacterial. Clin. Med. 35 (1950) Gomori, E., Methods in Enzymology (edited by S. P. Colowick and N. 0. Kaplan), Academic Press, New York, London 1955, Vol. I, p G. Riario-Sforza, R. Pagani, and E. Marinello Istituto di Chimica Biologica dell'univemita Via Sallustio Bandini, 54, Siena, Italy