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Isomerases from Three Clostridial Species YUEN WAN SHING, JAMES M

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Isomerases from Three Clostridial Species YUEN WAN SHING, JAMES M JouRNAL oF BACToOLOGY, Apr. 1975, p. 177-184 Vol. 122, No. 1 Copyright 0 1975 American Society for Microbiology Printed in U.SA. Psychrophilic, Mesophilic, and Thermophilic Triosephosphate Isomerases from Three Clostridial Species YUEN WAN SHING, JAMES M. AKAGI,* AND RICHARD H. HIMES Departments of Biochemistry and Microbiology,* University of Kansas, Lawrence, Kansas 66045 Received for publication 27 December 1974 Triosephosphate isomerase was purified to homogeneity as judged by analyti- cal gel electrophoresis from Clostridium sp. strain 69, Clostridium pasteurianum, and C. thermosaccharolyticum, which grow optimally at 18, 37, and 55 C, respectively. Comparative studies on these purified proteins showed that they had the same molecular weight (53,000) and subunit molecular weight (26,500). They were equally susceptible to the active site-directed inhibitor, glycidol phosphate. However, their temperature and pH optima, as well as their stabilities to heat, urea, and sodium dodecyl sulfate, differed. The proteins also had different mobilities in acrylamide gel electrophoresis. This difference in ionic character was also reflected in the elution behavior of the enzymes from hydroxyapatite and in the isoelectric points determined by isoelectric focusing in acrylamide gel. The amino acid composition of these proteins showed that the thermophilic enzyme contains a greater amount of proline than the other enzymes. The ratio of acidic amino acids to basic amino acids was 1.79, 1.38, and 1.66 for the thermophilic mesophilic and psychrophilic enzymes, respectively. This is consistent with the relative iscelectric point values of these three enzymes. The biochemical basis for the loss in viability Enzyme assay. Triosephosphate isomerase activ- of psychrophiles at moderate temperatures is ity was routinely measured in the direction from now generally accepted to be protein denatura- DL-glyceraldehyde-3-phosphate to dihydroxyacetone tion (4, 8, 12). We found that a phosphate by coupling the product to a-glycerophos- recently phate dehydrogenase and following the oxidation of psychrophilic Clostridium contains a triose- reduced nicotinamide adenine dinucleotide at 340 phosphate isomerase (EC5.3.1.1) that is very nm. The assay mixture (1 ml) contained 0.14 nM heat labile and is probably responsible for the reduced nicotinamide adenine dinucleotide, 1.5 mM low maximum growth temperature of this orga- DL-glyceraldehyde-3-phosphate, 10 Ag of a-glycero- nism (17). The same enzyme from a thermoph- phosphate dehydrogenase (Sigma Chemical Co.), 5.4 ilic Clostridium was previously found to be very mM ethyenediaminetetraacetate, and 20 mM trietha- stable to heat and the one from a mesophilic nolamine hydrochloride (pH 7.9). One unit of activity Clostridium showed a thermostability some- is defined as the amount required to convert 1 Mmol of where between the two extremes (10). To gain D-glyceraldehyde-3-phosphate into dihydroxyacetone phosphate per min at room temperature. The protein more information about the physical and chem- concentration was determined either by a modified ical properties of these proteins and the reasons phenol reagent method (14) or, in the case of purified for their different thermostabilities, we purified proteins, by measuring the absorbance at 280 nm. The this enzyme from the psychrophilic, meso- E17 of purified triosephosphate isomerase was de- philic, and thermophilic clostridial species. termined by performing amino acid analysis on sam- Comparisons of some of the properties of the ples of known absorbancy at 280 nm. Assuming 95% purified enzymes show that the main difference recovery after hydrolysis and chromatography, an is in the ionic character of the proteins. E2%0 value of 13.8 was obtained for all three proteins. Rabbit muscle triosephosphate isomerase is reported to have an El%? value of 13 (13). MATERIALS AND METHODS Chromatography. Diethylaminoethyl (DEAE) - Organisms. Clostridium sp. strain 69 (18), Clos- cellulose (Schleicher and Schuell) was washed with tridium pasteurianum ATCC 6013 (6), and C. ther- 0.5 N KOH and 1 N HCL and after several water mosaccharolyticum strain 3814 (27) were grown at 18, washes was suspended in 0.2 M phosphate buffer, pH 37, and 55 C, respectively, in the media previously 7. Hydroxyapatite was prepared as described by described. All organisms were cultivated anaerobi- Bernardi (2). Sephadex G-100 (Sigma Chemical Co.) cally, harvested by certrifugation at their respective was allowed to swell and to degas overnight in 0.05 M mid-exponential growth phase, and lyophilized. tris(hydroxymethyl)aminomethane (Tris)-hydrochlo- 177 178 SHING, AKAGI, AND HIMES J. BACTERIOL. ride buffer, pH 7.5. Columns of these materials were Amino acid composition. A known amount, ap- packed by gravity and stored at 4 C. proximately 1.5 ml, of the sample solution containing Electrophoresis. Preparative disc electrophoresis 0.2 to 1.0 mg of the protein in 6 N HCl was incubated was used in the final step for the purification of at 110 C in an evacuated sealed tube for 24 h. At the triosephosphate isomerase. A separating gel (6 cm) end of the incubation, the tube was opened and containing 7.5% acrylamide, 0.12% methylene bisac- evacuated overnight in a desiccator containing NaOH rylamide, 0.03% (vol/vol) N,N,N',N'-tetrame- pellets. The dried residue in the tube was dissolved in thylenediamine, 0.07% ammonium persulfate, and 0.4 a known volume of 0.1 M lithium citrate buffer, pH M Tris-hydrochloride buffer, pH 9.0, was first formed 2.2. Samples (1 ml) were used for column chromatog- in a Canalco column PD-2/230. Then a stacking gel (2 raphy according to the method described by Spack- cm) containing 3.5% acrylamide, 0.06% methylene man et al. (21), using a Beckman Spinco model 120 C bisacrylamide, 0.06% (vol/vol) N,N,N',N'-tetrame- automatic amino acid analyzer. The combined con- thylenediamine, 0.5 mg of riboflavin per 100 ml, and tent of cystine and cysteine in triosephosphate isom- 0.06 M Tris-hydrochloride buffer, pH 6.8, was formed erase was determined by estimation of the amount of on the top of it by exposure to fluorescent light. The cysteic acid formed by the performic acid oxidation of electrode buffer contained 0.025 M Tris and 0.19 M these amino acids (9). The content of tryptophan was glycine, pH 8.2. One millilter of the sample solution determined by estimation of the tryptophanyl fluores- containing approximately 20 mg of protein, 5% su- cence obtained from the SDS-treated and mercapto- crose, and 15 gl of tracking dye (0.05% bromophenol ethanol-reduced protein (16). blue in water) was applied to the column. Separation Purification of triosephosphate isomerase. Tri- was performed at a constant current of 20 mA with the osephosphate isomerase was purified to homogeneity cathode on top. The elution was carried out with 0.4 as judged by analytical gel electrophoresis from the M Tris-hydrochloride buffer, pH 9.0, containing. psychrophile, Clostridium sp. strain 69, the meso- 0.03% (vol/vol) N,N,N',N'-tetramethylenediamine phile, C. pasteurianum, and the thermophile, C. at a flow rate of 1 ml/min. Each fraction contained 1.5 thermosaccharolyticum. The following procedure was ml of eluate. devised. All operations were performed at 2 to 4 C Analytical gel electrophoresis was used to deter- unless otherwise stated. mine the purity and relative mobility of the triose- (i) Extraction. Dried cells (100 g) were added to phosphate isomerase. The electrode buffer, separating 0.05 M Tris-hydrochloride, pH 8, containing 1.6 mM gel (1.2 ml), and stacking gel (0.3 ml) were as ethylenediaminetetraacet ate to make a 10 to 12% described above except that 10% acrylamide was used (wt/vol) suspension. This suspension was homoge- in the separating gel. Ten to 100 gl of the sample nized twice with a Manton-Gaulin model 15 M-8 TA solution containing approximately 50 gg of protein, homogenizer and centrifuged for 90 min at 30,000 x g. 10% glycerol, and 5 ,ul of tracking dye (0.05% bromo- The residue was discarded. phenol blue in water) was applied. Electrophoresis (ii) DEAE-cellulose column. The cell-free extract was run at a constant current of 2 mA per tube. The was applied to a DEAE-cellulose column (35 by 5 cm). gels were stained with 0.5% napthol blue black in 7% The column was eluted with 0.05 M Tris-hydrochlo- acetic acid solution for at least 1 h and destained ride, pH 7.6. The enzyme did not adhere to the electrophoretically in 7% acetic acid solution. DEAE-cellulose column under these conditions, and Polyacrylamide electrophoresis in sodium dodecyl the effluent containing triosephosphate isomerase sulfate (SDS) was used to determine the subunit activity was collected. molecular weight of triosephosphate isomerase by the (iii) Ammonium sulfate fractionation. The efflu- method of Weber and Osborn (26). ent from the DEAE-cellulose column was adjusted to Electrofocusing. Gel electrofocusing (28) was used pH 9 with 2 N KOH, brought to 60% saturation with to determine the isoelectric point of the triosephos- solid ammonium sulfate, stirred for 2 h, and cen- phate isomerase. The gel (1.4 ml) containing 7.5% trifuged for 90 min at 30,00 x g. The supernatant was acrylamide, 0.2% methylene bisacrylamide, 1% car- adjusted to pH 6.5 with 6 N HCl and then brought to rier ampholytes (LKB; pH 3 to 6), and 0.06% potas- 85% saturation with solid ammonium sulfate. The sium persulfate was prepared in each tube. The upper residue was collected by centrifugation, dissolved in a chamber (cathode) and the lower chamber (anode) minimal volume of 0.001 M potassium phosphate contained 0.4% triethanolamine and 0.2% sulfuric buffer, pH 7.5, and dialyzed against the same buffer acid, respectively.
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