Assay of phosphoenolpyruvate car☐ykinase in crude yeast extracts

ANALYTICAL

BIOCHEMISTRY

74, 576-584 (1976)

Assay of Phosphoenolpyruvate Carboxykinase Crude Yeast Extracts ROBERT J. HANSEN,'HELGAHINZE,

in

ANDHELMUTHOLZER~

Biochemisches lnstitut der Universitiit Freiburg im Breisgau und Institut ftir Biochemie der Gesellschaft ftir Strahlen- und Umweltforschung, Hermann-Herder-Strasse 7, D-7800 Freiburg im Breisgau, Federal Republic of Germany Received February 23, 1976; accepted April 27, 1976 The applicability of a spectrophotometric assay of phosphoenolpyruvate carboxykinase to crude yeast extracts has been studied. The assay measured oxalacetate production by coupling to the malate dehydrogenase reaction (phosphoenolpyruvate + ADP + bicarbonate + oxalacetate + ATP; oxalacetate + NADH + malate + NAD). Disappearance of NADH depended strictly on the presence of phosphoenolpyruvate, bicarbonate, ADP, and Mn*+. Furthermore, the disappearance of NADH was shown to be accompanied by stoichiometric accumulation of malate. Addition of 10 mM quinolinate, which is a known inhibitor of liver phosphoenolpyruvate carboxykinase, completely prevented phosphoenolpyruvate-dependent NADH disappearance. These observations demonstrated that the assay provides a quantitative measure of phosphoenolpyruvate carboxykinase activity in crude extracts. The assay could be applied to crude extracts from yeast cells grown under laboratory conditions but not to extracts from commercially produced baker’s yeast, because of an extremely high rate of endogeneous oxidation of NADH in the latter extracts. With the spectrophotometric assay, optimal activity was observed at pH 7.0 with both crude extracts and a 15-fold-purified preparation.

During the course of studies on the effects of glucose on the activities of yeast proteinases and proteinase inhibitors, it became necessary to compare changes in the activities of these enzymes and inhibitors with changes in the activities of enzymes known to be repressed or/and inactivated by the presence of glucose in the medium (l), such as phosphoenolpyruvate carboxykinase (EC 4.1.1.49) (2,3). Previous measurements of phosphoenolpyruvate carboxykinase activity in unpurified cell-free extracts have used modifications of the method of Cazzulo er al. (4). That method measures the incorporation of 14C from NaH14C0, into oxalacetate or its more stable product, malate. The use of a spectrophotometric assay using the NADH-dependent malate dehydrogenase, r Present address: Department of Physiological Sciences, School of Veterinary cine, University of California, Davis, Calif. 95616. * To whom correspondence should be addressed. 576 Copyright All rights

0 1976 by Academic Press, Inc. of reproduction in any form reserved.

Medi-

PHOSPHOENOLPYRUVATE

phosphoenolpyruvate

+ HC03-

oxalacetate

577

CARBOXYKINASE

+ ADP

+ NADH

Mn2+ e oxalacetate

Z$ malate + NAD+,

+ ATP,

Ul

PI

for measuring phosphoenolpyruvate carboxykinase in crude extracts was ruled out in the earlier studies of Cannata et al. (5,6) because very high levels of endogenous NADH-oxidizing activities were always obtained. We have reexamined the use of the simpler and more rapid spectrophotometric test for assaying phosphoenolpyruvate carboxykinase in crude extracts. It is demonstrated that his spectrophotometric assay, called the “kinetic assay,” can be used to measure the activity of phosphoenolpyruvate carboxykinase in crude extracts of yeast grown under a variety of laboratory conditions. It is also demonstrated that commercially available baker’s yeast, such as used by Cannata and Stoppani (6) for their original studies, contained high levels of NADH-oxidizing activity but that, when the same yeast cells were grown in the laboratory in synthetic medium, very low levels of endogenous NADH-oxidizing activity were present. The pH optimum of the enzyme in the assay presented here is 7.0. MATERIALS

AND METHODS

Glutathione, ADP, phosphoenolpyruvate, NADH, and malate dehydrogenase were obtained from Boehringer Mannheim, GmbH (Mannheim, Germany); DEAE-Sephadex A-50 from Pharmacia (Uppsala, Sweden); KHC03, MnCl,, and imidazole from Merck (Darmstadt, Germany); and avidin from Sigma (Munchen, Germany). The major yeast strain used in these experiments was the haploid wildtype X 2180-1B originally isolated by R. Mot-timer (University of California, Berkeley). Others, used for comparison with X 2180-lB, were the triploid strain S740, originally isolated by R. R. Fowell (Surrey, England), and the haploid strain H4 isolated by H. Betz (Biochemisches Institut, Freiburg i.Br., Germany). The commercial yeast types Pleser (Darmstadt), Weingarten (Weingarten), Fala (Achern-Gamshurst), and Sinner (Karlsruhe) were purchased from BAKO Backer-Einkauf EGmbH (Freiburg i.Br., Germany). All yeasts were grown for 48 hr on YEPD (1% yeast extract, 2% Bactopeptone, and 2% glucose) with shaking at 28°C. Samples of the commercial yeast types were grown in YEPD medium with 20 pg of tetracycline/ml. Cell-free extracts were prepared by suspending washed cells in 0.1 M potassium phosphate buffer, pH 7.0 (1: 1, wet weight of cells: volume) and homogenized in a French pressure cell (American Instrument Company, Inc. Silver Spring, Md.) by passing the suspension through the cell twice with a pressure of 20,000 psi. The homogenate was centrifuged for 20 min at 34,000g at 0-4°C. The clear supernatant was removed and used as the source of phosphoenolpyruvate carboxykinase in the crude extract.

578

HANSEN,

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AND HOLZER

Partial puri$cation of phosphoenolpyruvate carboxykinase . A partially purified preparation of phosphoenolpyruvate carboxykinase was obtained by the following scheme (all operations at 0-4°C): (i) X 2180-1B yeast cells were grown with shaking for 48 hr at 28°C and a cell-free extract prepared as described above using 0.01 M potassium phosphate, 0.001 M disodium ethylenediaminetetraacetic acid, and 0.001 M 2-mercaptoethanol, pH 7.0, as buffer. (ii) The cell-free extract was subjected to (NH&SO, fractionation. The precipitate from the 44-60% fraction was collected by centrifugation, dissolved in a small volume of 0.01 M potassium phosphate, pH 7.0, and dialyzed overnight against three changes of the same buffer. (iii) The dialyzed sample was adsorbed to a DEAE-Sephadex column (5 x 15 cm) that was previously equilibrated to pH 6.2 in 0.01 M potassium phosphate buffer. The enzyme was eluted from the gel by a linear KC1 gradient (O-O.3 M KC1 in 0.01 M phosphate buffer; total volume, 1000 ml). (iv) The fractions containing phosphoenolpyruvate carboxykinase activity were pooled and subjected to (NH&SO4 fractionation. The precipitate from the 44-60% fraction was dissolved in a small amount of 0.01 M potassium phosphate buffer and dialyzed overnight against three changes of the same buffer. The final specific activity was 15 times that of the crude extract. Some characteristics of the enzyme were studied using this preparation and compared to the same characteristics of the enzyme in the crude extract. Kinetic assay. The activity of phosphoenolpyruvate carboxykinase was measured spectrophotometrically at 366 nm and 25°C. The assay mixture, expressed as final concentration in the 2-ml assay mixture was 100 mM imidazole/HCl, pH 6.6, 50 mM NaHC03, 2.5 mM phosphoenolpyruvate, 1.25 mM ADP, 2 mM MnCI,, 2 mM glutathione, 0.45 mM NADH, and 3 IU of malate dehydrogenase. The final pH of the assay mixture was 7.0. At the beginning of each test the endogenous rate of NADH oxidation in the enzyme preparation was determined so that it could be subtracted from the total rate of NADH oxidation. The reaction was then started by the addition of phosphoenolpyruvate. The reaction was linear for at least 15 min and was proportional to enzyme concentration up to a level of 0.03 IU of carboxykinase. Malate assay. The amount of malate produced during an assay was determined as follows: (i) An assay was started by the addition of phosphoenolpyruvate as described above. After 10 min the reaction was stopped by the addition of 0.1 ml of 70% perchloric acid and allowed to stand 10 min in ice. (ii) The pH of the mixture was then adjusted to 9.0 by the addition of small amounts of 5 N KOH, and the mixture was placed in ice for 30 min. The precipitate was pelleted by centrifugation. (iii) The amount of malate present in 1 ml of supernatant was determined using the L-malate uv test kit of Boehringer Mannheim. The amount of malate formed in the phosphoenolpyruvate carboxykinase assay was then calculated. Data are presented in Table 2 as change inAsG6 nm in order to be com-

PHOSPHOENOLPYRUVATE

579

CARBOXYKINASE

TABLE

1

REQUIREMENTSFOR PHOSPHOENOLPYRUVATE CARBOXYKINASECATALYZEDCARBOXYLATION REACTION

AA 366nm (% of complete systeml’l Partially purified enzyme

Assay system

Crude extract

1. Complete system* 2. Phosphoenolpyruvate carboxykinase deleted 3. Phosphoenolpyruvate deleted 4. ADP deleted 5. HCO,- deleted 6. MnCl, deleted 7. Malate dehydrogenase deleted 8. Glutathione deleted 9. ADP deleted, 1.25 mM IDP added 10. ADP deleted, 1.25 mM GDP added 11. MgCI, deleted, 50 mM MgCIZ added 12. Phosphoenolpyruvate deleted, 2.5 mM pyruvate added

100

100





100 100

12 100

<1








n Endogenous AAsG6,,,,,subtracted. * See Materials and Methods for composition of the complete system.

parable to the change in absorbance observed in the phosphoenolpyruvate carboxykinase-catalyzed kinetic assay. RESULTS AND DISCUSSION

The data presented in Table 1 show that the known properties of the phosphoenolpyruvate carboxykinase reaction (4-6) are demonstrable using crude extracts and the spectrophotometric assay described above. An absolute requirement for phosphoenolpyruvate, HC03-, manganese ion, and ADP was observed. The requirement for ADP excludes participation of phosphoenolpyruvate carboxylase (EC 4.1.1.3 1) in the observed disappearance of NADH. Substitution of GDP or IDP for ADP, pyruvate for phosphoenolpyruvate, or magnesium for manganese did not produce a measurable reaction. In assays of the crude extract the omission of malate dehydrogenase did not affect the reaction; its omission from assays of the partially purified enzyme produced a drastic reduction of the measured

580

HANSEN,

HINZE

FIG. 1. Phosphoenolpyruvate carboxykinase purified enzyme (0) as a function of pH.

AND HOLZER

activity of crude extract (x) and partially

activity. Doubling the amount of malate dehydrogenase produced no further increase in the reaction rate (experimental data not presented). The omission of glutathione from the assay mixture was without effect on the enzyme reaction when either the enzyme in the crude extract or the partially purified form was used. The pH optimum of the partially purified phosphoenolpyruvate carboxykinase-catalyzed reaction was 7.0 (Fig. 1). The pH optimum of the reaction catalyzed by the enzyme in the crude extract was similar (Fig. 1). To show that the NADH oxidation being measured in assays of the crude extract was accompanied by a simultaneous and equimolar formation of malate, the amount of malate produced during the assay was determined by enzymic assay (Table 2, column 4). A comparison of columns 1 and 4 in Table 2 shows that 90 to 100% of the malate expected from the change in absorbance was actually found by assay after stopping the reaction with perchloric acid. The maximal rate of malate formation was found at pH 7.0, as was observed from the rate of NADH oxidation in the kinetic assay. Addition of lactate dehydrogenase causes a substantial increase in the AA in the kinetic assay. Presumably this results from reduction of pyruvate which was formed from phosphoenolpyruvate by pyruvate kinase. When malate was determined enzymically after addition of perchloric acid, about 20% less malate was found in reaction mixtures with added lactate dehydrogenase as compared to controls (Table 2, column 4 vs column 5). Possible causes of this disturbance by lactate dehydrogenase are the formation of pyruvate from malate by the malic enzyme and removal of ADP by the pyruvate kinase reaction. The experiments with added lactate dehydrogenase show that the kinetic assay is not usable in crude extracts that contain lactate

PHOSPHOENOLPYRUVATE TABLE EFFECT

OF PH ON THE CARBOXYKINASE

581

CARBOXYKINASE 2

ACTIVITY OF PHOSPHOENOLPYRUVATE IN CRUDE YEAST EXTRACTS

AA 366.,/ 10 min Calculated from malate determination

Kinetic assay PH

Standard

LDH added

-PEP”

Standard

LDH added

5.0+

0.017 0.074 0.18 0.28 0.42 0.14 0.049

0.27 0.38 0.48 0.66 0.60 0.42 0.37

0.040 0.020 0.018 0.018 0.017 0.007 0.008

0.017 0.077 0.15 0.25 0.39 0.14 0.059

0.049 0.060 0.11 0.19 0.27 0.11 0.055

5.9 6.0 6.5 7.0 7.5 8.0

a “Endogenous” AA,, “,,,. * pH change during test: With test adjusted to pH 5.0, the pH after 3 min was 5.3 and after 10 min, pH 5.4; with test adjusted to pH 5.5, the pH after 3 min was 5.6 and after 10 min. pH 5.7.

dehydrogenase, as is the case for most mammalian tissues. From Table 2 it can also be seen (column 3) that “endogenous” NADH oxidation without addition of phosphoenolpyruvate is quite small. At pH values above 5.5, the endogenous NADH oxidizing activity is much less than the phosphoenolpyruvate carboxykinase activity. Further evidence that the rate-limiting reaction being measured in the kinetic assay was in fact catalyzed by phosphoenolpyruvate carboxykinase was provided through the use of the inhibitors quinolinic acid and avidin. As previously reported for phosphoenolpyruvate carboxykinase in rat liver (7), quinolinic acid is a very effective inhibitor of the phosphoenolpyruvate carboxykinase reaction. Addition of 10 mrvr quinolinic acid to an ongoing reaction, catalyzed by either crude extract or partially purified enzyme, reduced the rate of NADH oxidation in less than 15 set to 4% of its original value, which indicates that yeast phosphoenolpyruvate carboxykinase is also inhibited by quinolinate (cf. Fig. 2). The combined action of pyruvate kinase and pyruvate carboxylase might also mimic the phosphoenolpyruvate carboxykinase reaction in this assay. To test this possibility, avidin (2 IU/ml) was mixed 1: 1 with the crude extract and incubated 1 hr in an ice bath, as suggested for inhibition of yeast pyruvate carboxylase by Haarasilta and Oura (3). Aliquots of avidin-treated and untreated crude extract were then assayed in the kinetic assay. No significant difference in the rate of NADH oxidation was observed.

582

HANSEN,

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Quindii

AND HOLZER

[mM]

FIG. 2. Inhibition of partially purified phosphoenolpyruvate carboxykinase by quinolinate. Test conditions and enzyme were as described in Materials and Methods under Kinetic assay. For preparation of the enzyme, see Partial purification of phosphoenolpyruvate carboxykinase.

The following findings (data not presented) lend further support to the conclusion that pyruvate carboxylase does not interfere with the assay described here: (i) ATP, necessary for the pyruvate carboxylase reaction could not be substituted for ADP in the reaction mixture. (ii) No reaction was observed when pyruvate was substituted for phosphoenolpyruvate in the reaction mixture. (iii) The specific activity of phosphoenolpyruvate carboxykinase, as measured by the test described herein, decreased when glucose was added to 48-hr cultures, in agreement with the earlier findings of Haarasilta and Oura (3) and Gancedo et al. (2). Experiments were also performed to determine whether the reported inability to assay phosphoenolpyruvate carboxykinase in crude extracts of baker’s yeast with the kinetic (spectrophotometric) assay (5,6) might be due to the yeast strain used or to growth conditions. In the crude extracts of commercially produced baker’s yeasts from Pleser, Weingarten, Fala, or Sinner (see Materials and Methods) the endogenous rate of NADH oxidation was generally three to five times greater than the phosphoenolpyruvate-dependent oxidation. Under these conditions it was indeed impossible to determine phosphoenolpyruvate carboxykinase activity accurately. When samples of these yeasts were grown for 48 hr on YEPD medium with added tetracycline, the rate of endogenous NADH oxidation was reduced to only 5- 10% of the phosphoenolpyruvate carboxykinase activity. For example, crude extracts of Weingarten yeast purchased from BAKO had an endogenous change in AsG6 ,,,,, of 0.023/min/0.005 ml and a change of 0.027 after the addition of phospho-

PHOSPHOENOLPYRUVATE

CARBOXYKINASE

583

enolpyruvate. After growth of the same yeast for 48 hr on YEPD medium plus tetracycline, the endogenous change in Ase6 nm in equivalent crude extracts was 0.005/min/0.005 ml and 0.105 after the addition of phosphoenolpyruvate. After growth on YEPD medium for 48 hr, all of the commercial yeast types and the isolated strains H4 and S740 had specific phosphoenolpyruvate carboxykinase activities between 0.2 and 0.4 U/mg of protein, which compared well with an average of 0.3 U/mg of protein obtained from strain X 2180-1B. These data suggest two reasons for the inapplicability of the kinetic assay to commercially produced baker’s yeast: (i) the high endogenous NADH oxidation in extracts from this yeast and (ii) our culture conditions lead to a higher phosphoenolpyruvate carboxykinase specific activity than is found with commercial baker’s yeast. A serious discrepancy exists between the data of Cannata and Stoppani (6) and the data presented here with regard to the pH optimum of the phosphoenolpyruvate carboxykinase reaction. The pH optimum reported here for the enzyme in crude extracts, as well as for the partially purified enzyme, was pH 7.0; that reported by Cannata and Stoppani (6) for the NaH14C0, assay, and also used for the spectrophotometric assay, was pH 5.4. As seen in Fig. 1, the activity observed at pH 5.5 in these experiments was only one-fifth of that at pH 7.0. In a recent paper by Cannata and Stoppani (8) the pH optimum for the reaction catalyzed by crystalline baker’s yeast phosphoenolpyruvate carboxykinase in the direction of oxalacetate formation was reinvestigated and found to be pH 6.5. The differences in composition of the reaction mixtures, mainly with regard to the buffers employed in each case, could account for the discrepancy with the value of pH 7.0 reported in the present paper (see Fig. 1 and Table 2). To determine if there were other discrepancies between the two sets of data, some kinetic parameters of the enzyme were measured. In essential agreement with Cannata and Stoppani (6) it was found that high amounts of ADP inhibited phosphoenolpyruvate carboxykinase and that the enzyme had K, values for ADP of 2.2 x 1OW M, for phosphoenolpyruvate of 8 x 10e5 M, and for MnCl, of 1 x 1O-3 M. According to Cannata and Flombaum (8) the true substrate for the reaction is very likely the ADP-Mnlcomplex. The K, values for ADP and Mn therefore strongly depend on the conditions in which they are obtained. At pH values between 5 and 5.5, the endogenous rates of NADH oxidation are relatively high as compared to the activity of phosphoenolpyruvate carboxykinase. This fact, coupled with the fact that yeast obtained commercially always has abnormally high rates of endogenous NADH oxidation probably led previous authors to the conclusion that the kinetic (spectrophotometric) assay could not be used to assay phosphoenolpyruvate carboxykinase in crude extracts. The data presented in

584

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AND HOLZER

this report suggest that the simpler kinetic (spectrophotometric) assay can be used to assay the enzyme in crude yeast extracts, provided the yeast is not commercially prepared and provided that the enzyme is assayed at pH 7.0 instead of 5.4. ACKNOWLEDGMENTS Major support for these studies was supplied by the Bundesministerium fur Forschung and Technologie, Bereich Biotechnologie; partial support was supplied by Grants AM 15059 and AM 16648 to R. .I. H. from the National Institute for Arthritis, Metabolism, and Digestive Diseases. R. J. H. was the recipient of a Forschungsstipendium from the Alexander von Humboldt-Stiftung during these studies.

REFERENCES 1. Holzer, H. (1975) in Proceedings of the 9th FEBS Meeting, Vol. 32, pp. 181-193, Budapest Akademiai Kiado, Budapest. 2. Gancedo, C., Schwerzmann, N., and Molano, J. (1974) in “Proceedings Fourth International Symposium on Yeasts,” Vienna, Austria, 8-12 July 1974 (H. Klaushofer and U. B. Sleytr, eds.), printed by Hochschiilerschaft a.d. Hochschule fur Bodenkultur, Wien 1974, Part I, Paper Sessions-Abstracts, p. 9, AS. 3. Haarasilta, S., and Oura, E. (1975) Eur. J. Biochem. 52, l-7. 4. Cazzulo, J. J., Claisse, L. M., and Stoppani, A. 0. M. (1%8) J. Bacterial. 96, 623628. 5. Cannata, J. J. B. (1970) J. Biol. Chem. 245, 792-798. 6. Cannata, J. J. B., and Stoppani, A. 0. M. (1%3) J. Biol. Chem. 238, 1196-1207. 7. Veneziale, C. M., Walter, P., Kneer, N., and Lardy, H. A. (1967) Biochemistry 6, 2129-2138. 8. Cannata, J. J. B., and Flombaum, A. C. de (1974) J. Eiol. Chem. 249, 33563365.