Purification and Properties of Two Forms of 6-Phosphogluconate Dehydrogenase from Candida utizis

Transcription

1 European J. Biochem. 1 (1967) Purification and Properties of Two Forms of 6-Phosphogluconate Dehydrogenase from Candida utizis M. RIPPA, M. SIGNORINI, and S. PONTREMOLI Istituto di Chimica Biologica, UniversitA di Ferrara (Received November 8, 1966) Crude extracts of Candida utilis contain two types of 6-phosphogluconate dehydrogenase. One can be obtained in the crystalline form and has already been described and studied. In the present paper the purification of the second one, together with the differences in the properties between the two purified proteins, is reported. The two types of enzyme have identical ph optimum, same K, for the substrates and same specificity. They differ in amino acid composition, molecular weight, electrophoretic mobility, stability to ph and heat treatment, sensitivity to chlorodinitrobenzene and proteolytic treatment. All evidence indicates a greater instability of the crystalline enzyme in respect to the non-crystalline one and seems to exclude that the two forms of enzyme are an artefact due to the extraction or the purification procedures. A method for the purification of the 6-phosphogluconate dehydrogenase from Candida utilis has been described by Horecker and Smyrniotis [I]. The enzyme has been obtained in a homogeneous crystalline form by Pontremoli et al. [2]. We have previously shown that, in the active center of the enzyme, there is at least a cysteine residue, a lysine residue and a phosphate attracting group. The enzyme can be inactivated by the binding of iodoacetate [3] or chlorodinitrobenzene [4] to a single cysteine residue, or by the binding of pyridoxal-5 -phosphate to a single lysine residue [5] of the enzyme molecule. The crystalline enzyme does not account for the total 6-phosphogluconate dehydrogenase activity of the crude extract of Candida utilis. In the present paper we present evidence which indicates that another protein, with 6-phosphogluconate dehydrogenase activity, is present in our extracts and can be purified. A method has been worked out for the purification of this form of enzyme. This protein does not crystallize in the conditions used for the crystallization of the other type of enzyme. The crystallizable 6-phosphogluconate dehydrogenase will be indicated as enzyme typei, the non-crystalline as enzyme type 11. Enzymes. 6-phosphogluconic dehydrogenase, or phosphogluconate dehydrogenase, or 6-phospho-~-gluconate: NADP oxidoreductase (decarboxylating) (EC ) ; glycerophosphate dehydrogenase, or ~-glycerol-3-phosphate: (acceptor) oxidoreductase (EC ); ~-Fructose-l,B-diphosphate 1-phosphohydrolase, or Hexosediphosphatase (EC ); ribosephosphate isomerase, or D-ribose-5- phosphate ketol-isomerase (EC ). From the amino acid composition, molecular weight determinations and from other parameters it appears that the enzyme type1 could be derived from proteolytic digestion of the enzyme type 11. The enzyme type I exhibits a greater instability to the chemical and enzymatic action of several agents. EXPERIMENTAL PROCEDURES Materials Candida utilis, dried at low temperature, was supplied by the Lake States Yeast Gorp., Rhinelander, Wisconsin, and kept at 4. 6-phosphogluconate, TPN, pyridoxal-5 -phosphate, glutathione and p-hydroxymercuribenzoate were purchased from Sigma Chem. Co., St. Louis, Missouri. Protamine sulfate was obtained from Lilly Co. Indianapolis, Indiana. Glycerophosphate dehydrogenase, from rabbit muscle, was purchased from Boehringer, Germany. Fructose 1,6-diphosphatase was prepared as previously described [6]. This enzyme has a molecular weight of 127,000 [7]. Twice crystallized trypsin was a Worthington product. Chemicals used for disc-gel electrophoresis were obtained from Canal Industrial Co., Bethesda, Maryland. All other chemicals were reagent grade. Methods The ammonium sulfate concentration was measured with a Barnstead purity meter. Whatman DEAE-cellulose and phospho-cellulose were freed from fines by allowing them to settle several times after suspension in water. The resins were then suspended in 0.5M NaOH, filtered and washed with

2 Vol. 1, No.2,1967 M. RIPPA, M. SIGNORINI, and S. PONTREMOLI 171 distilled water until neutrality. The adsorption of the enzyme on the resin was carried out batchwise and was checked by analyzing, for enzymatic activity, samples of the supernatant collected after removal of the resin by centrifugation. Spectrophotometric determinations were carried out in a Zeiss PMQII spectrophotometer. All the purification procedures were carried out at 0-4", unless otherwise stated. Enzyme Assay The enzymatic activity of both forms of 6-phosphogluconate dehydrogenase was determined spectrophotometrically at 22", following the initial rate of TPN reduction in an assay mixture (1 ml) containing 0.3 mm 6-phosphogluconate, 0.3 mm TPN and 1OmM phosphate buffer, ph 7.4. The reaction was started by the addition of the enzyme and readings were taken at 5 sec intervals at 340 mp. One unit of enzyme activity was defined as the quantity that would produce a change in absorbance of 1.0 per min. RESULTS Purification of the Two Forms of Enzyme Extraction, Protamine and Heat Treatment. The dry yeast (200 g) was suspended in 1.2 liters of distilled water. After 4 hours at 37" the autolyzed suspension was centrifuged in the cold for 30min at 20,000 x g. The supernatant (crude extract, Table 1) was adjusted to ph 6.2 with 1 M NaOH and treated with 2 g of protamine, previously dissolved in 1 ml of water. The precipitate was discarded by centrifugation. The supernatant (protamine fraction) was adjusted to ph 5.5 with 1 M CH,COOH, heated in a water bath at " for 6 min, with continuous stirring, and cooled to 4". The precipitate was discarded as above. Column Chromatography. The supernatant obtained after centrifugation (heat fraction) was diluted 5 fold with distilled water and the ph of the solution was adjusted to 5.8. Wet phosphocellulose was added to the solution batchwise, until no more enzymatic Table 1. Purification procedure of the two forms of 6-phosphoyluconate dehydroyenase Enzyme Step Volume Activity Proteins Specific activity Purification Yield ml units/ml total units mg/ml units/mg "1. Crude extract , Protamine 1, , Heat , Phosphocellulose , First ammonium sulfate , Type I First crystals Second crystals Third crystals Fourth crystals Type I1 Supernatant fist crystals Second ammonium sulfate DEAE-cellulose , , , ,800 I.o , , , Protein concentration was determined from the absorbance at 280mp, based on dry weight determination. A solution containing 1.0 g per ml of pure enzyme (type1 or typeii) has an absorbance of at 280 mp. In the early steps of purification the turbidimetric method of Bucher [8] was used. The specific activity of the enzyme was defined as units per mg of protein. The specific activity of the purified crystalline 6-phosphogluconate dehydrogenase was approximatively 160. One mg of purified crystalline enzyme catalyzes the oxidation of 25.6 pmoles of 6-phosphogluconate per min. All studies of a given kinetic or physical parameter were performed in a similar fashion with enzyme preparations of similar age and treatment in order to minimize all differences other than those specihally due to the enzyme type studied. activity was present in the supernatant (see Methods). During this procedure the ph of the suspension was kept constant at 5.8. The resin suspension was then filtered through a Buchner funnel and the filtrate discarded. The wet cake of resin was suspended in 0.5 liters of mm phosphate buffer, ph 6.2, and the suspension poured into a 4.5 x cm chromatographic column. The resin was washed on the column with 2 liters of mm phosphate buffer, ph 6.2, containing 0.1 mm EDTA. No enzymatic activity was detected in the washings. The average volume of the resin packed in the column was 200 ml. The enzyme was then eluted with 0.4 M phosphate buffer, ph 7.4, containing 0.1 mm EDTA and fractions of 10 ml each were collected. The fractions containing enzyme activity were pooled and adjusted to ph 6.2 (phosphocellulose column).

3 172 Two Forms of 6-Phosphogluconate Dehydrogenase European J. Biochem. Ammonium Sulfate Precipitation and Crystallization. The resultant solution was treated with an amount of solid ammonium sulfate sufficient to give a 01, saturation. During the addition of ammonium sulfate, the ph of the solution was kept at 6.2. The precipitate was discarded by centrifugation. To the supernatant (first ammonium sulfate) a cold saturated ammonium sulfate solution was added dropwise, with continuous stirring, keeping the ph at 6.2, until a slight turbidity appeared. At this point the ammonium sulfate saturation was approximately 51,. Crystal formation began in a few minutes and was complete after hours. The suspenrion was stored overnight at 4". Separation of the two Forms of Enzyme. In order to separate the two forms of 6-phosphogluconate dehydrogenase, the crystalline suspension was centrifuged and the precipitate dissolved in ml of IOmM phosphate buffer, ph6.2, containing 0.1 mm EDTA. This fraction (first crystals) contained essentially only the crystallizable enzyme (6-phosphogluconate dehydrogenase, type I), while the supernatant (supernatant first crystals) contained predominantly the non-crystallizable enzyme (6-phosphogluconate dehydrogenase, type 11). Each enzyme was further purified as described below. Purification of the 6-Phosphogluconate Dehydrogenase Type I. For recrystallization, the solution containing the enzyme type1 was treated with a saturated ammonium sulfate solution, added dropwise, until a slight turbidity appeared. After 4 hours at 4", the crystalline suspension was centrifuged and the supernatant discarded. The precipitate, was again dissolved in 10 mm phosphate buffer, ph 6.2, containing 0.1 mm EDTA (second crystals). The enzyme was subjected to further crystallizations using the same procedure. Usually after 4 crystallizations the enzyme appeared to be homogeneous as judged by disc-gel and starch-gel electrophoresis, sucrose density gradient centrifugation, and no change in specific activity after column chromatography on DEAE- CM- and phosphocellulose (see below). The enzyme was kept as a crystalline suspension at 4". Purification of the 6-Phosphoglucomte Dehydrogenme Type II. The supernatant (supernatant first crystals) was treated with a saturated ammonium sulfate solution, keeping the ph of the solution at 6.2, until a turbidity appeared. At this point the ammonium sulfate saturation was approximately 61,. The suspension was kept at 4" for 2 days during which the turbidity increased. The suspension was then centrifuged and the inactive precipitate discarded. The supernatant was brought to ph 4.7 with 5 M CH,COOH and the inactive precipitate discarded. The supernatant was adjusted to ph 6.2 with 1 M NaOH and the enzyme precipitated on raising the ammonium sulfate saturation to 85,lo. The precipitate, collected by centrifugation, was dissolved in a small volume (usually 20ml) of 10 mm phosphate buffer, ph 7.2, containing 0.1 mm EDTA, and dialyzed 5 hours against the same buffer (second ammonium sulfate fraction). The dialyzed enzyme was then diluted to 2 ml with water and adsorbed on DEAE cellulose at ph 7.4; the wet resin was added batchwise to the solution as described before. The resin suspension was then poured into a chromatographic column (2 x 30 cm) and washed with 10 mm phosphate buffer, ph 7.4, until no more protein was detected in the effluent. The average volume of the resin packed in the column was 60 ml. The enzyme was eluted from the resin with a solution containing 0.5 Ol0 saturated ammonium sulfate in the same buffer. The fractions containing the enzyme activity were pooled (DEAE cellulose fraction) and were kept at 4" in 80 saturated ammonium sulfate, 10 mm phosphate buffer, ph 6.2, containing 0.1 mm EDTA. Remarks on the Purification Procedure. For the crystallization of the enzyme type I the enzymatic activity must be at least 7 unitslmg. In the case of the enzyme typeii, crystalline material was not obtained at any step of the purification, under the conditions used for the type1 enzyme. Properties of the Two Forms of Enzyme Stability. The two types of enzyme, stored as described, did not show any appreciable loss of enzymatic activity after several months. Alternative Extraction Methods. The possibility that the two forms of enzyme represent an artefact due to the extraction procedure, was ruled out by using different methods of extraction of the enzyme from yeast. Crude extracts were prepared as follows : (a) grinding the dry yeast with silica powder in a mortar, at 4", with water; (b) autolyzing the dry yeast for 20min at 4"; (c) autolyzing at 37" for a period of 30 min, 4 and 8 hours; (d) autolyzing the dry yeast for 20 min at 4", and incubating the supernatant and the precipitate (collected after centrifugation and dissolved in water) separately at 37" for 4 hours. All extracts were submitted to the same purification procedure described in detail above. In all cases (see Table2) the ratio of the two forms of enzyme, as calculated from the ratio of crystallization, did not vary appreciably, altough the total starting units were different, depending on the different type of extraction. Alternative Purification Procedure. In order to eliminate the possibility of an artefactual modification of the protein, during the purification, as a cause of the occurrence of the two forms of enzyme, a modification to the present method of purscation was followed, which eliminates the heat and the phospho-

4 Vol.1, No.2, 1967 M. RIPPA, M. SIaNoRmr, and S. PONTREMOLI 173 Table 2. Effect of different methods of extraction on the ratio of the levels of the two forms of 6-phosphogluconate dehydroqenase Extraction method Crude extract Before enzyme crystallization Enzyme crystallized total units total units total units 1. Mechanical rupture of cells 4,900 3,6 1, Autolysis for 20 min at 4" 1,600 1, Autolysis for 30 min at 37" 5,0 3,800 1, Autolysis for 4 hours at 37" 5. Autolysis for 8 hours at 37O 5,1 5,130 3,600 3,5 1,260 1, Incubation for 4 hours at 37" of the supernatant obtained after autolysis for 20 min at 0" 1,600 1, Incubation for 4 hours at 37" of the precipitate collected after autolysis for 20 min at 0" 3,400 2, Table 3. Alternative purification procedure of the two forms of 6-phosphogluconate dehydrogenase Step Volume Activity Protein Specific activity TypeI Type I1 ml unitsiml total units mgbl unitsimg Crude extract , Protamine 1, , First ammonium sulfate , Phosphate gel, eluate , Second ammonium sulfate , First crystals , Supernatant fist crystals , cellulose column steps. The modified purification procedure was carried out as follows: the protamine fraction was treated with solid ammonium sulfate, keeping the ph at 6.2, and the fraction, precipitated at an ammonium sulfate saturation of between and 7/,, was collected, dissolved in 100ml of 0.1 M phosphate buffer, ph 6.2, containing 0.1 mm EDTA, and dialyzed for 6 hours against the same buffer (ammonium sulfate fraction, Table 3). The dialysate was diluted to 400 ml with 0.1 M phosphate buffer, ph 6.2, and treated with calcium phosphate gel. This was added in an amount to allow the adsorption of inactive proteins. To the supernatant, collected after centrifugation, more calcium phosphate gel was added, sufficient to adsorb 90 Oi0 of the active enzyme protein. The gel was centrifuged and the enzyme eluted with a 20OIO saturated ammonium sulfate solution, containing 0.1 mm EDTA. The resulting solution was submitted to further treatment as indicated under "ammonium sulfate precipitation and crystallization," and the crystallizable form separated from the non-crystallizable one. The results obtained (Table 3) indicate that the ratio of the two forms of enzyme is identical, following the two different purification procedures. Purity of the Two Forms of Enzyme. Both purified forms of 6-phosphogluconate dehydrogenase contained no detectable glucose-6-phosphate dehydrogenase or ~-ribose-5-phosphate isomerase activities. The disc-gel and starch-gel electrophoresis patterns of both types of enzyme, shown in Fig. 1, indi- 12 European J. Biochem., Vol. 1 TYPE I DISC GEL + STARCH GEL Fig. 1. Gel electrophoresis of the two purified forms of 6-phosphogluwnate dehydrogenase. Disc-gel electrophoresis was carried out in 7O/, standard polycrylamide gel, ph 8.6, at 6 ma per tube. The samples contained 0.1 mg of each type of enzyme. Starch-gel electrophoresis was carried out in 0.03M phosphate buffer, ph 7.0, at 30mA, 200volts for 8 hours cate that each type of enzyme was obtained in a homogeneous form. EDTA was present in the buffer used for the electrophoresis, owing to the instability of the enzyme type I at ph 8.6. The data reported in Big.1 indicate that at ph 7 the two forms of enzyme have a different electrophoretic mobility as could be expected by the differences in amino acid composition. - 0

5 174 Two Forms of 6-Phosphogluconate Dehydrogenase European J. Biochem TUBE NUMBER (MENISCUS AT TUBE 99) Fig. 2. Sucrose gradient centrifugation analyses of the two forms of 6-phosphogluconate dehydrogenase (GPGdH). The method employed was that of Martin and Ames [9]. The purified enzyme (type I or type 11) was placed (0.5 mg in 0.05 ml) at the top of the sucrose gradient [5 to ZOO/, sucrose (w/v) in 0.01 phosphate buffer, ph 6.21 together with fructose diphosphatase (FDPase) and glycerophosphate dehydrogenase (GPdH) used as internal standards. After centrifugation at 37,000 rev./min in the rotor SW 39 of the Spinco model L preparative centrifuge, for 19 hours at 4", fractions were collected and analyzed for the three enzyme activities. Ordinate represents enzyme activities in arbitrary units f I I I // I I I I // TIME (mid Fig. 3. Spectrophotometric titration of the sulfhydryl groups of the two forms of 6-phosphogluconate dehydrogenase with p- hydroxymercuribenzoate (phhb). The ratio of moles of phmb bound per mole of enzyme has been calculated from the increment in absorbance at 2 mp and was standardized with a sample of glutathione. The reaction mixture contained 0.05 mg of enzyme (type I or type 11), 0.1 mm phmb, mm phosphate buffer, ph 7.5 and, where indicated, 20/o (w/v) sodium dodecyl sulfate. The temperature was 22" The homogeneity of each form of 6-phosphogluconate dehydrogenase was also tested by sucrose density gradient centrifugation. Five mg of each form of enzyme, dissolved in 0.3 ml of 10 mm phosphate buffer, ph 6.2, containing 0.1 mm EDTA, were placed at the top of a 5 to ZOO/, (w/v) sucrose gradient in the same buffer. The tubes were subjected to centrifugation at 25,000 rev./min in the rotor SW 25.1 of a Spinco L preparative centrifuge, at 4" for 39 hours. After the run the tubes were punctured and the fractions collected and analyzed for protein concentration and enzymatic activity. One symmetrical peak was obtained for each type of enzyme. The specific activity throughout the peak was equal to that of the sample before centrifugation. No protein contamination was detected using column chromatography on DEAE- CM- and phosphocellulose. Molecular Weight of the Two Forms of Enzyme. The approximate molecular weight of both forms of enzyme was determined by sucrose density gradient centrifugation, according to the method of Martin and Ames [9]. Fructose 1,6-diphosphatase and glycerophosphate dehydrogenase were used as markers in each tube (Fig.2). Calculations of molecular weights, based on several runs, were in good agreement. Assuming spherical proteins, the molecular weight values were 101,000 for the enzyme type I and 111,000 for the enzyme type 11. Amino Acid Composition. The amino acid analysis of each purified form of 6-phosphogluconate dehydrogenase was carried out with the Beckman model 120 B analyzer [lo]. The data reported in Table 4 indicate that the percentages of the amino acid residues present in the two proteins are different. On the basis of the molecular weight reported above it appears (Table4, last column) that the enzyme type I1 contains, in addition to all the amino acid residues present in the enzyme type I, 30 basic amino acids (14 lysines, 4 histidines, 12 arginines), 46 hydrophobic amino acids (12 threonines, 10 prolines, 6 methionines, 16 leucines and 2 tryptophans) and 3 cysteine, 4 serine and 6 aspartic acid residues. Spectrophotometric Titration of Sulfhydryl Groups. In agreement with the amino acid analysis, the number of sulfhydryl groups, determined according to the procedure of Benesch and Benesch [13], based on the work of Boyer [14], was determined to be 8 for the enzyme type 1 and 11 for the enzyme type 11. The same values were obtained if the titrations were carried out in 2 O/,, (w/v) sodium dodecylsulfate. While p-hydroxymercuribenzoste titration was complete in 2min in the case of the enzyme typei, two hours were required for the complete titration in the case of the enzyme typeii, as it appears from Fig. 3. Absorption Spectrum. The absorption spectra of the two forms of enzyme, in 10 mm phosphate buffer,

6 Vo1.1, No.2, 1967 M. RIPPA, M. SIGNORINI, and S. PONTREMOLI 175 TabIe 4. Amino acid composition of the two forms of 6-phosphogluconate dehydrogenasea Hydrolysis time Nearest Amino acid Average integer 24 hours 24 hours 48 hours 72 hours per 101,000 6-phosphogluconate dehydrogenase type I pmoles 0 Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine HaIf cysteinec Valine Methionine d Isoleucine Leucine Tyrosine Phenylalanine Tryptophane phosphogluconate dehydrogenase type I1 Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Gly cine Alanine Half cysteinec Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophane a A sample of each form of enzyme was dialyzed two days against distilled water and then evaporated to dryness. The dry material was dissolved in 5.7 N HC1, divided into three equal parts each containing 1.0 mg of protein and hydrolyzed in vacuum for 24, 48, and 72 hours. Cysteine was determined as cysteic acid and mcthionine as methionine sulfone in samples oxidized with performic acid [ll]. Each analysis was carried out in duplicate. Corrections were made for physical loss, destruction or incomplete hydrolysis. The values reported in the table arc expressed as micromoles of amino acids per 1.0 mg of protein. The amino acid composition reported in the last column was based on the molecular weight of 101,000 for the enzyme type I and 111,000 for the enzyme type 11. b Samples oxidized with performic acid. C Determined as cysteic acid. d Determined as methionine sulfone. e Determined spectrophotometrically ph 7.0, are identical. There is only one peak of absorption, with a maximum at 280mp and no significant absorption at 340 mp, also in the presence of 6-phosphogluconate. The ratio between the absorbancies at 280 and 260 mp is 1.9 for both enzymes. ph Optimum, Substrate Affinities and Specificity. The ph optimum for the activity of both forms of enzyme is the same (Fig.4). The ph curve shows half maximal activity at p1-i 6.34 and 7.94 for the enzyme type I and 6.44 and 8.4 for the enzyme type 11. The more rapid decrease of activity at high ph, for the enzyme type I, with respect to the type 11, might be due to the greater instability of this form of enzyme at high ph (see below). The apparent K, values for 6-phosphogluconate were determined to be 52 pm for the enzyme type I and 31 pm for the enzyme type 11. The apparent K, value for TPN was 20 pm for both forms of enzyme. Both forms are highly specific with respect to coenzyme, showing no detectable activity with DPN. 12.

7 176 Two Forms of 6-Phosphogluconate Dehydrogenase European J. Biochem. >- k > g 60 4c 2c A TYPE1 0 A TYPE1 I I I PH Fig.4. Enzymatic activities of the two forms of 6-phosphogluconate dehydrogenase as a function of ph. 0.1 M phosphate buffer (0,o) or 0.1 M Tris-HC1 buffer (A,A) were used Inhibition of Enzymatic Activity by Different Reagents. Studies on the kinetics of inhibition or of inactivation of the catalytic activity of the two forms of enzyme were undertaken to determine if differences existed between the two forms. The data reported in Table 6 show that the treatment with chlorodinitrobenzene caused complete loss of the catalytic activity of the enzyme type I, while it had no effect on the catalytic activity of the enzyme type 11. The two forms of enzyme behaved in an identical manner with respect to the inhibitory action of pyridoxal-5'-phosphate, which was previously shown to form a Schiff base derivative with the crystalline enzyme [5]. The catalytic activity of both forms of enzyme is inhibited by incubation with p-hydroximercuribenzoate. The data reported in Fig.5 show that the rate of inhibition is greater for the enzyme type I with respect to the enzyme type 11. This is in agree- Table 5. Effect of heat and ph on the stability of the two forms of 6-phosphogluconate dehydrogenase Treatment Enzyme type I Enzyme type I1 Duration of treatment Inhibition Duration of treatment Inhibition min "0 min "0 Incubation at 60"a Incubation at ph 4.lb Incubation at ph 8.0c a A sample containing 0.2 mg per ml of enzyme (type I or type 11) in 20 mm phosphate buffer, 0.1 mm EDTA, ph 6.2, was heated at 60". Samples were taken at intervals and analyzed for enzymatic activity. b Solutions containing 0.2 mg per ml of enzyme in 100 mm acetate buffer, 0.1 mm EDTA, ph 4.1, were incubated at 22". Samples were taken at intervals and analyzed. Solutions containing 0.2 mg per ml of enzyme in 1 M Tris-HC1 buffer, ph 8.0, were incubated at 22". Samples were taken at intervals and analyzed for enzymatic activity. Table 6. Effect of chlorodinitrobenzene and pyridoxal-5'-phosphate on the catalytic activity of the two forms of 6-phosphogluconate dehydrogenase Samples containing the type I or type I1 form of enzyme, at a concentration of 0.2 mg per ml, were incubated at 22" with the reagents indicated in the table under the conditions described below; at intervals samples were taken and analyzed for the enzymatic activity. Reagent mm chlorodinitrobenzene, I mm EDTA, I M Tris-HC1 buffer, ph 8. Reagent mm pyridoxal phosphate, 10 mm phosphate buffer, ph 7.4 Reagent Chlorodinitrobenzene Pyridoxal-5'-phosphate Enzyme type I Duration of treatment Inhibition Duration of treatment min min Enzyme type I1 Inhibition "io 0 Stability to Heat, Acid and Alkaline ph. The two forms of enzyme showed significant differences in the sensitivity to inactivation by heat treatment or by exposure to acid or alkaline medium. The data reported in Table 5 show that the catalytic activity of the enzyme type I1 is less sensitive than that of the enzyme type I, to the action of these agents. ment with the titration data reported above. 6-phosphogluconate protects both forms of enzyme against the inhibition by p-hydroxymercuribenzoate. Inactivation by Dodecybulfate and Trypsin Bigestion. The catalytic activity of the two forms of enzyme was also affected in a different way by the treatment with sodium dodecylsulfate and with

8 Vol.1, No.2, 1967 M. RIPPA, M. SIGNORINI, and S. PONTREMOLI 177 trypsin. As shown in Fig. 6A, loss of catalytic activity of both types of enzyme was observed after incubation with dodecylsulfate, but the rate of inactivation was significantly higher for the enzyme typei. The proteolytic digestion with trypsin (Fig6B) caused almost complete loss of the catalytic activity of the enzyme type I in 10 min, while only 200/, of the original activity of the enzyme type I1 was lost in 40 min. enzyme type 11. The rate of titration of the sulfhydryl groups with p-hydroxymercuribenzoate and the consequent loss of the catalytic activity, follow different kinetics with the two enzyme proteins as indicated by the fact that the reaction, in order to be complete, requires a longer time in the case of the enzyme type 11. Thus Candida utilis 6-phosphogluconate &hydrogenase can be added to the increasing number of 100,C. tf TYPE1 t TYPEE I 0 c TYPE I TYPE I /"-'- -I- 1 I V I ' TIME (rnin) Fig.5. Inhibition of the enzymatic activity of the two forms of 6-phosphogluconate dehydrogenase by p-hydroxymercuribenzoate (phmb). 0.1 mg of enzyme (type 1 or type 11) was incubated at 0" in a reaction mixture (0.5ml) containing 0.01 mm phmb, 20 mm phosphate buffer, ph 7.4, and, where indicated, 1.5 mm 6-phosphogluconate (6PG). At intervals aliquots were taken and analyzed for activity DISCUSSION Two types of 6-phosphogluconate dehydrogenase have been obtained in a homogeneous form from extracts of Candida utilis. The separation of the two forms of enzyme has been obtained taking advantage of a different property of the two forms with respect to crystallization : the enzyme type I crystallizes, while, in the same conditions, the enzyme type11 does not crystallize. The approximate value of the molecular weight is io1,ooo for the enzyme type I and iii,ooo for the enzyme typeii. From the amino acid composition and assuming the reported molecular weight values, it appears that the enzyme type I1 contains, in addition to all the amino acid residues present in the enzyme type I, 89 other amino acid residues. The two forms of enzyme differ in several respects in the response to physical, chemical and enzymatic treatments. The catalytic activity of the enzyme type11 is less affected, than that of the enzyme type I, by heat treatment and exposure to acid and alkaline ph,and is more resistant to the proteolytic digestion with trypsin and to the action of dodecylsulfate. The treatment with chlorodinitrobenzene produces a complete inactivation of the enzyme type I but has no effect on the catalytic activity of the TYPElI _. A n K B " TIME (rnin) Fig. 6. Effect of dodecylsulfate and trypsin treatment on the activity of the two forms of 6-phosphogluconate dehydrogenase. Solutions containing 0.14 mg per ml of enzyme (type I or type 11) were incubated at 22' with sodium dodecylsulfate or trypsin under the conditions described below. At intervals aliquots were taken and assayed for enzymatic activity. Fig.6, left: 0.010/, (w/v) sodium dodecylsulfate, 1 mm EDTA, 20mM phosphate buffer, ph 6.2. Fig.6, right: 0.01 mg per ml trypsine, 1 mm EDTA, Tris-HC1 buffer, ph 8 enzymes which have been demonstrated to exist in multiple molecular forms, although the molecular basis which could explain this phenomenon is still unknown. They would not appear to result from different polymeric states of a single enzyme, inasmuch as the molecular weight of the two forms seems to differ by only i0,ooo. The multiple forms would also not appear to result from extraction or purification artefacts, since the two forms are present in different conditions of autolysis and after different procedures of purification. The presence of the two forms does not depend upon the particular batch of yeast used, since two forms have been detected in 6 different batches of yeast obtained in a 2 year interval. It is known that in Candida utilis two forms of fumarase [i5] and at least 2 forms of transaldolase [is] exist. The presence of multiple forms of an enzyme might result from proteol ytic digestion during the purification [ 171. Attempts to produce interconversion of the two forms of 6-phosphogluconate dehydrogenase by the intrinsic proteolytic activity of the crude extract or by the addition of trypsin have been so far unsuccessful. Although trypsin failed to transform one form into the other, the differences in molecular weight,

9 178 RIPPA, SIGNORINI, and PONTREMOLI : Two Forms of 6-Phosphogluconate Dehydrogenase European J. Biochem. amino acid composition and the properties of the two forms might suggest that the enzyme type I could be a product of a partial proteolytic digestion of the enzyme type 11. The enzymatic properties of the two forms are essentially the same, and some of the different properties observed can be attributed to different structural forms. Other explanations for the presence of two forms, based on the existence in our starting material of different strains of yeast, or on different intracellular localization of the two enzymes are still possible. The presence of two forms of 6-phosphogluconate dehydrogenase exhibiting differences in their properties may help future studies on the structure and mechanism of action of this enzyme. This work was supported by grants from the Italian C.N.R. (Impresa di Enzimologia), the National Institutes of Health (GM and TW ) and N.A.T.O. (Grant 218). REFERENCES 1. Horecker, B. L., and Smymiotis, P. Z., J. Biol. Chem. 193 (1951) Pontremoli, S., De Flora, A., Grazi, E., Manaiarotti. G., Bonsignore, A., and Horecker, B. L., J. Biol. Chem: 236 (1961) Grazi, E., Rippa, M., and Pontremoli, S., J. Biol. Chem. 240 (1965) Rippa, M., Grazi, E., and Pontremoli, S., J. Biol. Chem- 241 (1966) Rippa, M., Spanio, L., and Pontremoli, S., Arch. Biochem. Biophys. 118 (1967) Pontremoli, S., Traniello, S., Luppis, B., and Wood, W. A., J. Biol. Chem. 240 (1965) Pontremoli, S., Luppis, B., Traniello, S., Rippa, M., and Horecker, B. L., Arch. Biochem. Biophys. 112 (1965) Bucher, T., Biochim. Biophys. Acta, 1 (1947) Martin, R. G., and Ames, B. N., J. Biol. Chem. 236 (1961) Spackman, D. H., Stein, W. H., and Moore, S., Anal. Chem. 30 (1958) Hirs, H. W., J. Biol. Chem. 219 (1956) Goodwin, T. W., and Morton, R.A., Biochem. J. 40 (1946) Benesch, R., and Benesch, R. E., Methods Biochem. Anal. 10 (1962) Boyer, P. D., J. Am. Chem. Xoc. 76 (1954) Hayman, S., and Alberty, R. A., Ann. N. Y. Acad. Xci. 94 (1961) Horecker, B. L., and Tchola, O., personal communication. 17. Kaji, A., Trayser, K. A., and Colowick, S. P., Ann. N.Y. Acad. Xci. 94 (1961) 798. M. Rippa, M. Signorini, S. Pontremoli Istituto di Chimica Biologica, UniversitB degli Studi di Ferrara Via Fossato di Mortara 25 Ferrara, Italy