CASEI' which catalyzes the oxidation of D-sorbitol-6- phosphate to fructose-6-phosphate.

Transcription

1 D-SORBITOL-6-PHOSPHATE DEHYDROGENASE FROM LACTOBACILLUS CASEI' THOMAS E. SHOCKLEY AND HAROLD S. PRIDE2 Departent of Microbiology, Meharry Medical College, Nashville, Tennessee Received for publication Noveber 3, 1958 Several different systes for the conversion of sorbitol to hexose have been described in icroorganiss. In the pseudoonads, sorbitol ay be oxidized to fructose (Shaw, 1956) or to sorbose (Sebek and Randles, 1952). The latter investigators concluded that the conversion of sorbitol to fructose or sorbose did not involve phosphorylation since carbon dioxide was not liberated fro bicarbonate buffer upon incubabation of whole cells, adenosine triphosphate, and hexitol. Cell-free preparations of Acetobacter suboxydans have been shown to dissiilate sorbitol by three different pathways. In addition to a dehydrogenase, presuably a flavoprotein which catalyzes the oxidation of sorbitol to hexose (Wider et al., 1956; Arcus and Edson, 1956) two alternate enzye systes found in the soluble portions of the cells have been described (Cuins et al., 1957a). Separation of these systes revealed the existence of a DPN3- and a TPN-linked sorbitol dehydrogenase (Cuins et al., 1957b). The product of the DPN-linked oxidation was found to be fructose whereas the latter fored sorbose. Studies by Wolff and Kaplan (1956a) deonstrated that D-sorbitol-6-phosphate is dehydrogenated by cell-free extracts of Escherichia coli strain B. The phosphorylated route was indicated as the ajor pathway of sorbitol dissiilation since the specific activity of extracts of sorbitol-grown cells was approxiately 6-fold greater for sorbitol-6-phosphate as copared to sorbitol. The results presented in this paper describe the isolation, purification, and properties of a DPN-linked enzye fro Lactobacillus casei which catalyzes the oxidation of D-sorbitol-6- phosphate to fructose-6-phosphate. MATERIALS AND METHODS Organis and culture conditions. L. casei strain ATCC 4646 was eployed in this study. Stock cultures were aintained in ilk. Cells for the preparation of cell-free extracts were cultivated in a ediu containing per L: trypticase, 2 g; MgSO4-7H2O,.5 g; MnSO4,.5 g; Tween 8, 1 l; sodiu dihydrogen phosphate, 1 g; sorbitol, 1 g. Three-liter capacity Fernbach flasks containing 1 L of the above ediu were inoculated with 1 l of a 24-hr culture of the organis and incubated for 18 hr. Sonic disruption. Crude cell-free extracts were prepared by subjecting washed cells suspended in twice their volue of.67 M phosphate buffer at ph 7. to the sonic field of a Raytheon 1 kc oscillator for 4 in. Unbroken cells and cell debris were reoved by centrifuging at 2, x G for 3 in in a Servall refrigerated centrifuge. Purification of enzye. All operations were conducted at 4 C. Fifty-five l of alkaline aoniu sulfate (adjusted to ph 8. with aoniu hydroxide) were added to 55 l of crude extract. The precipitate was discarded and the supernatant was brought to.75 saturation. The resulting precipitate was dissolved in 3 l of distilled water and the extract adjusted to ph 4.1 with cold 2 N acetic acid. After standing for 9 in, the extract was centrifuged for 1 hr at 25, X G. The supernatant fluid (ph 4.1) was ade.52 saturated by the addition of aoniu sulfate and allowed to stand overnight. The supernatant 1 This investigation was supported by a grantin-aid fro the Gustavus and Louise Pfeiffer fluid was discarded and the precipitate was dissolved in 15 l of cold distilled water. The further Research Foundation. 2 Lederle Medical Student Research Fellow. addition of an equal volue of distilled water resulted in the foration of a heavy precipitate 3 The following abbreviations are used: diphosphopyridine nucleotide, oxidized and reduced, which was discarded. The supernatant fluid was DPN and DPNH; triphosphopyridine nucleotide. treated with aoniu sulfate and the fraction TPN; tris(hydroxyethyl)ainoethane, Tris. obtained between.5 and.7 saturation was 695

2 696 SHOCKLEY AND PRIDE [VOL. 77 dissolved in 12 l of.5 M Tris buffer, ph 7., and dialyzed overnight against 2 L buffer, ph 7.. Further purification of the enzye was achieved by gel adsorption. To 13 l of the dialyzed extract were added 26 l of a suspension of calciu phosphate gel containing approxiately 12 g of solids per l (gel to protein ratio = 4:1). Alost all the activity was reoved fro the supernatant fluid. The gel was washed successively with 1-l quantities of.5 M and.1 M phosphate buffer, ph 6.8. A sall aount of the enzye was lost during these washings. The enzye was eluted fro the gel by treating with two 1 l portions of.5 M phosphate buffer, ph 6.8. The first eluate contained high concentrations of phosphohexose isoerase and was discarded. The second eluate was dialyzed against.67 M phosphate buffer, ph 7., and stored until ready for use. This procedure routinely gave a 12- to 2-fold purification; however, soe preparations were purified as uch as 4-fold. Analytical ethods. Absorption easureents were ade with a Beckan odel DU spectrophotoeter with corex cuvettes, all having a 1.-c light path. For colorietric procedures, a Klett-Suerson colorieter was eployed. Fructose and fructose-6-phosphate were estiated by the ethod of Roe (1934) or by the cysteine-carbazole ethod of Dische and Borenfreund (1951). Fructose-6-phosphate was identified as the reaction product of sorbitol-6-phosphate oxidation spectrophotoetrically with TPN, Zwischenferent, and crude phosphohexose isoerase. Reducing sugar was estiated by the ethod of Soogyi (1952). For the estiation of the hexitol phosphates, the procedure of West and Rapoport (1949) as odified by Wolff and Kaplan (1956b) was eployed. The concentration of reduced DPN was estiated by using the value of 6.22 X 16 (c2 X oles-) for the extinction coefficient of DPNH at 34,u (Horecker and Kornberg, 1948). Protein was deterined by the ethod of Lowry et al. (1951). lmaterials. The following substances were coercial preparations: DPN, TPN, DPNH, lactic acid dehydrogenase (specific activity, 2, Racker units per g protein), annose-6-phosphate, fructose-6-phosphate, and glucose-6-phosphate (bariu salts), Nutritional Biocheicals Corporation; Zwischenferent (specific activity of.8 units per g protein), sodiu glucose-6- phosphate, and crude rabbit uscle phosphohexose isoerase, Siga Cheical Copany. Sorbitol-6-phosphate and annitol-1-phosphate were prepared by borohydride reduction of glucose-6-phosphate and annose-6-phosphate respectively, as described by Wolff and Kaplan (1956b). The bariu salts of glucose-6- phosphate, fructose-6-phosphate, and annose- 6-phosphate were converted to the sodiu salts by dissolving the in a dilute solution of hydrochloric acid and adding an equivalent aount of Na2SO4. Calciu phosphate gel was prepared according to a ethod described by Tsuboi and Hudson (1957). Enzye assay. Absorbance was read at 34,u in the Beckan odel DlU spectrophotoeter before and at 3-see intervals after the addition of enzye to the reaction ixture. The reaction ixture for enzye assay consisted of: DPN, 1,ole; sorbitol-6-phosphate, 2.28,uoles; enzye, 1 to 3 units; glycine-naoh buffer to a total volue of 3. l. One unit of enzye is defined as that aount which effects an increase in optical density (A A34) of.1 per in at roo teperature. The reaction was usually linear for the first 2 in; therefore, this portion of the curve was used for deterinations. Specific activity is defined as units per g of protein. RESULTS A suary of the purification procedure is presented in table 1. The purified preparation showed no detectable lactic acid dehydrogenase, DPNH oxidase. glucose-6-phosphate dehydrogenase, or activity with fructose-6-phosphate. Phosphohexose isoerase activity was found to vary with the preparation. The enzye could be stored at -2 C for several onths with no appreciable loss of activity. Figure 1 shows the reaction rate as a function of enzye concentration. Identification of product of sorbitol-6-phosphate oxidation. The accuulation of hexose could be easily deonstrated by the Roe reaction or the eysteine-carbazole ethod. The product of the oxidation was identified in an experiental syste siilar to that eployed by Marur and Hotchkiss (1955). The enzyatic conversion of sorbitol-6-phosphate to fructose-6-phosphate was followed by coupling the syste with phosphohexose isoerase, Zwischenferent, and TPN. Eliination of the absorbance by DPNH fored fro the oxidation of sorbitol-6-phosphate was

3 1959] SORBITOL DEHYDROGENASE FROM L. CASEI 697 TABLE 1 Suwnary of sorbitol-6-phosphate dehydrogenase purification procedure Fraction Total Volue Total Protein Total Units Specific Activity Recovery l g unius/g protein % 1. Crude , Aoniu sulfate saturation , Acetic acid , After addition of distilled H , Aoniu sulfate saturation , nd.5 M phosphate buffer eluate fro calciu phosphate gel , E zca en LIA cc Ji zvl C- ui z.2.1 I PROTEIN pug) Figure 1. Effect of enzye con reaction rate. Cuvettes contained: sorbitol-6-phosphate, I j,ole of I concentrations of sorbitol-6-phosph enase, and glycine buffer to a tol 3. l. phate + DPNH + H+ Pyruvic acid + DPNH + H+ DPN+ tion ixtures containing varying concentrations -* Fructose-6-phosphate glucose-6-1phosphate of sor6itol-6-phosphate and a nonliiting con- Glucose-6-phosphate + TPN+ 6-phosphoglu- centration of DPN. The K8 for DPN was likewise conate + TPNH + H+ deterined by varying the concentration of DPN Net reaction: sorbitol-6-phosphat e + pyruvic and aintaining a constant nonliiting concen- acid + TPN+ -) 6-phosphogluconate + lactic acid + TPNH + H+ As seen in table 2, reduction of TPN was not observed when sorbitol-6-phosphate was oitted. The sall aount of TPN reduction observed when sorbitol-6-phosphate dehydrogenase or DPN was oitted was probably due to trace aounts of glucose-6-phosphate in the sorbitol- 6-phosphate preparation. Glucose-6-phosphate, however. could not be detected by the reducing sugar test. Slight activity was also observed when phosphohexose isoerase was oitted, indicating the presence of this enzye in the partially purified preparation. Reversibility of reaction. Evidence for the reversibility of the reaction was obtained when the enzye was incubated with fructose-6-phosphate and DPNH. A rapid oxidation of DPNH was obcentration on served as shown in figure poles of Effect of ph. The initial rate of DPN reduc- )PN, varying tion was arkedlv affected by ph. Maxial rate ~ate dehydrog-.. v. tat dehydrog- of oxidation of sorbitol-6-phosphate was observed to occur between ph 9 to 1 in Sorensen's glycine-naoh buffer (figure 3). As a result of finding the optial ph 9.5, glycine buffer ph 9 to 1 accoplished by coupling with pyri uvate and lac- was routinely used in ost assays. tic acid dehydrogenase. The reactio: ns of the co- fmichaelis constants. Substrate and DPN con- centrations giving half-axial velocity were plete syste are suarized as folllows: deterined for the enzye bv following the re- Sorbitol-6-phosphate + DPN+ -* fri sctose-6-phos- duction of DPN. The dissociation constant for lactic acid + sorbitol-6-phosphate was deterined fro reac-

4 698 SHOCKLEY AND PRIDE [VOL. 77 tration of substrate. Graphic analysis of the data by the ethod of Lineweaver and Burk (1934) showed the K> for sorbitol-6-phosphate to be 2.9 X 1-5 M and for DPN, 5.4 X 1-5 M. Results of these experients are shown in figures 4a and 4b. Equilibriu constant. Assuing that one ole of DPNH is fored per ole of fructose-6- phosphate, the equilibriu constant for the oxi- TABLE 2 Enzyatic conversion of sorbitol-6-phosphate to 6-phosphogluconate AA34 ia at End of 7 Min Coplete syste Sorbitol-6-phosphate... -TPN... - Sorbitol-6-phosphate dehydrogenase DPN Phosphohexose isoerase Zwischenferent Coplete syste contained 15,oles of Tris buffer, ph 8.; sorbitol-6-phosphate dehydrogenase, 2 units; pyruvic acid, 1,uoles; lactic acid dehydrogenase, 1 units; DPN, 1,uole; TPN,.2,uole; MgCl2, 5.4.uoles; Zwischenferent..1 unit; sorbitol-6-phosphate, 1.2,.toles. Total volue, 3. l; at roo teperature. E z < ( J z Q t a MINUTES Figure 2. Enzyatic reduction of fructose-6- phosphate. Measureents were ade in a syste containing: 4 units of partially purified enzye,.3 jaole of DPNH, 15,ioles of Tris buffer, ph 8.1, and 4,uoles of substrate. Total volue 3. l at roo teperature. Fructose-6-phosphate (); control (v). Figure 3. Activity of sorbitol-6-phosphate dehydrogenase as a function of ph. Experiental syste contained: 2 units of enzye, 1,uole of DPN, 1.1,uoles of sorbitol-6-phosphate, and glycine buffer to total voltue of 3. l at roo teperature. -1> a (A) SORBITOL-6-PHOSPHATE CONCENTRATION (S) X Ȧ 1 ( S)S X 1 Figure 4a. Effect of sorbitol-6-phosphate concentration on reaction velocity. Cuvettes contained: 2 units of enzye, 1,uole of DPN, varying concentrations of sorbitol-6-phosphate, and glycine buffer to total volue of 3. l at roo teperature. dation of sorbitol-6-phosphate was calculated fro the equation: D-Sorbitol-6-phosphate + DPN+ > n ;o t9 _: D-fructose-6-phosphate + DPNH + H+ (fructose-6-phosphate) (DPNH) (H+) Ke = (sorbitol-6-phosphate) (DPN+)

5 1959] SORBITOL DEHYDROGENASE FROM L. CASEI v1> - 4 (A) DPN CONCENTRATION (S) X I I z 'A.12 C_ () X lo-3 Figure 4b. Effect of DPN concentration on reaction velocity. Cuvettes contained: 4 units of enzye, 2.28,oles of sorbitol-6-phosphate, varying concentrations of DPN, and glycine buffer to a total volue of 3. l at roo teperature. Fro experients where the relative concentrations of DPN and sorbitol-6-phosphate were varied, the Ke for the reaction at ph 9.31 was deterined to be 1.7 X 1-9. Activators and inhibitors. The inhibition of the enzye by heavy etals and by p-chloroercuribenzoate suggested the presence of sulfhydryl groups. For the tie interval of to 2 in, 6.6 X 13 M ZnSO4, CuSO4, HgC12, and p-chloroercuribenzoate effected 45, 4, 7, and 5 per cent inhibition, respectively. A constant aount of inhibition (15 to 2 per cent) was observed for concentrations of Versene (disodiu ethylenediainetetraacetate) ranging fro 6.6 X 1-4 to 6.6 X 1-3 M. This inhibition was not reversed by Mg-. The requireent for a etal could not be deonstrated. No significant stiulation was observed with 1.6 X 1-2 M MgCl2, MnCl2, or CaCl2. TPN did not replace DPN as coenzye. Specificity. The reverse or forward reaction was not deonstrable when glucose, fructose, sorbitol, annitol, dulcitol, glucose-6-phosphate, annose-6-phosphate, xylitol, or arabitol were tested at a concentration of 1.2 X 14 M. A sall aount of activity was observed when annitol-1-phosphate was tested at a concentration of 1 X 1-3 M. This activity was presued to be the result of containation of annitol-1- phosphate with sorbitol-6-phosphate..-i DISCUSSION In bacteria, sorbitol ay be oxidized directly to fructose or it ay first be phosphorylated, then oxidized. Fro available reports, a phosphorylative route in anials has not been detected (Blakely, 1951; Willias-Ashan and Banks, 1954; McCorkindale and Edson, 1954; Hollann and Touster, 1947). One of the chief differences which has been observed between the polyol dehydrogenases obtained fro anials and those obtained fro bacteria is the broader specificity possessed by the enzyes of anial origin. As in the case of the annitol-1-phosphate dehydrogenase fro Diplococcus pneuoniae (Marur and Hotchkiss, 1955) and E. coli (Wolff and Kaplan, 1956), which catalyzes the interconversion of annitol- 1-phosphate and fructose-6-phosphate, sorbitol- 6-phosphate dehydrogenase appears to be highly specific. However, a ore extensive study of various phosphorylated alcohols should be ade before attepting to define its specificity requireents. On coparing the equilibriu constant of sorbitol-6-phosphate dehydrogenase, 1.7 x 1-9, with that obtained for the sorbitol dehydrogenase (obtained fro rat accessory sexual organs) studied by Willias-Ashan and Banks (1954), 2.5 x 1-9, a close siilarity is noted. A rise in ph which displaces the equilibriu towards the coplete oxidation of sorbitol was noted by these workers. A siilar effect was observed with sorbitol phosphate dehydrogenase. The high degree of affinity of sorbitol phosphate dehydrogenase for substrate and DPN is indicated fro exaination of the dissociation constants. The values obtained in this study reveal that the enzye has a soewhat higher affinity for substrate and DPN when copared with the annitol phosphate dehydrogenase. Additional siilarities were observed of sorbitol phosphate dehydrogenase when copared with the annitol-1-phosphate dehydrogenase fro E. coli; these include a ph optiu near 1, lack of stiulation by divalent etals, and lack of coplete inhibition by Versene. SUMMARY An enzye fro Lactobacillus casei which interconverts D-sorbitol-6-phosphate and D-fructose-6-phosphate has been isolated and purified approxiately 14-fold. The ph optiu lies

6 7 SHOCKLEY AND PRIDE [VOL. 77 between 9 and 1. Diphosphopyridine nucleotide is required for activity; triphosphopyridine nucleotide is inactive. The inhibiting action of sulfhydryl-binding agents suggest the necessity of free SH groups for full activity. Dissociation and equilibriu constants have been deterined. REFERENCES ARCUS, A. C. AND EDSON, N. L Polyol dehydrogenases 2. The polyol dehydrogenases of Acetobacter suboxydans and Candida utilis. Bioche. J., 64, BLAKLEY, R. L The etabolis and antiketogenic effects of sorbitol. Sorbitol dehydrogenase. Bioche. J., 49, CUMMINS, J. T., KING, T. F., AND CHELDELIN, V. H. 1957a The biological oxidation of sorbitol. J. Biol. Che., 224, CUMMINS, J. T., CHELDELIN, V. H., KING, T. E. 1957b Sorbitol dehydrogenases in Acetobacter suboxydans. J. Biol. Che., 226, DISCHE, Z. AND BORENFREUND, E A new spectrophotoetric ethod for the detection and deterination of keto sugars and trioses. J. Biol. Che., 192, HOLLMANN, S. AND TOUSTER, The L-xylulose-xylitol enzye and other polyol dehydrogenases of guinea pig liver itochondria. J. Biol. Che., 225, HORECKER, B. L. AND KORNBERG, A The extinction coefficients of the reduced bands of pyridine nucleotides. J. Biol. Che., 175, LINEWEAVER, H. AND BURK, D The deterination of enzye dissociation constants. J. A. Che. Soc., 56, LOWRY,. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J Protein easureent with the folin phenol reagent. J. Biol. Che., 193, MARMUR, J. AND HOTCHKISS, R. D Mannitol etabolis, a transferable property of pneuococcus. J. Biol. Che., 214, MCCORKINDALE, J. AND EDSON, N. L Polyol dehydrogenases 1. The specificity of rat liver polyol dehydrogenase. Bioche. J., 57, ROE, J. H A colorietric ethod for the deterination of fructose in blood and urine. J. Biol. Che., 17, SEBEK,. K. AND RANDLES, C. I The oxidative dissiilation of annitol and sorbitol by Pseudoonas fluzorescens. J. Bacteriol., 63, SHAW, D. R. D Polyol dehydrogenases 3. Galactitol dehydrogenase and D-iditol dehydrogenase. Bioche. J., 64, SOMOGYI, M Notes on sugar deterination. J. Biol. Che., 195, TSUBOI, K. K. AND HUDSON, P. B Enzyes of the huan erythrocytes. I. Purine nucleoside phosphorylase; isolation procedure. J. Biol. Che., 224, WEST, C. D. AND RAPOPORT, S Modification of colorietric ethod for deterination of annitol and sorbitol in plasa and urine. Proc. Soc. Exptl. Biol. Med., 7, WIDMER, C., KING, T. E., AND CHELDELIN, V. H Particulate oxidase systes in Acetobacter suboxydans. J. Bacteriol., 71, 737. WILLIAMS-ASHMAN, H. G. AND BANKS, J The ketose reductase of rat liver and accessory sexual organs. Arch. Bioche. Biophys., 5, WOLFF, J. B. AND KAPLAN, N a Hexitol etabolis in Escherichia coli. J. Bacteriol., 71, WOLFF, J. B. AND KAPLAN, N b D-Mannitol-1-phosphate dehydrogenase fro Escherichia coli. J. Biol. Che., 218,