Dehydrogenase Obtained from Heated Spores of

Transcription

1 JOURNAL OF BACTERIOLOGY, Aug. 1971, p Copyright 1971 American Society for Microbiology Vol. 17, No. 2 Printed in U.S.A. Reactivative Action of Ethylenediaminetetraacetic Acid or Dipicolinic Acid on Inactive Glucose Dehydrogenase Obtained from Heated Spores of Bacillus subtilisi Y. HACHISUKA AND K. TOCHIKUBO Department of Microbiology, Nagoya City University, Nagoya, Japan Received for publication 3 April 1971 Partially purified inactive glucose dehydrogenase obtained from spores which were heated at 87 or 9 C for 3 min is converted to an active form by the addition of ethylenediaminetetraacetic acid, dipicolinic acid, or some salts. The molecular weight of the inactive glucose dehydrogenase in the heated spores is about one-half of that of the active glucose dehydrogenase in the intact resting spores. The possibility is discussed that the active glucose dehydrogenase in the intact resting spores divides into subunits and is converted to stable and inactive form during heating of spores at a particular range of temperature (87 to 9 C). One of the remarkable characters of bacterial spores is heat resistance. Since spores are able to maintain their viability after being subjected to high temperature, some enzyme activities in spores should remain intact. The mechanisms of heat resistance of spore enzymes have been considered from two aspects. One of them is that spore enzymes are different from vegetative cell enzymes and are intrinsically heat-stable proteins. The other is that enzymes in spores and vegetative cells are the same proteins, but spore proteins are stabilized by some mechanism in spores. Many workers (5, 6, 14, 15) have reported that several enzymes in spores cannot be distinguished from those in vegetative cells. Therefore, the latter opinion has gained acceptance. This report presents evidence concerning the mechanism of heat resistance of glucose dehydrogenase of spores. MATERIALS AND METHODS Spore preparation. Spores of Bacillus subtilis PCI 219, prepared on meat extract-agar at 37 C for 5 days, were harvested and washed six times with distilled water by centrifugation at 1,4 x g. Each spore suspension was checked with an Olympus phase-contrast microscope; it was confirmed that the preparations did not contain debris or vegetative cells. Clean spore suspensions were pooled and stored at -2 C. Heating of spore suspension. Spore suspensions (2 ml, containing about 1' spores/ml) were heated in a water bath at the indicated temperature. Heating was stopped by chilling in ice water. Chilled spores were collected by centrifugation and suspended in 2 ml of 1 mm phosphate buffer (ph 6.8). Crude cell-free extract. Spores suspended in phosphate buffer were disrupted with 5 g of.45- to.5- mm diameter glass beads (B. Braun Melsungen Apparatebau, Frankfurt/M, West Germany) by using a 1- kc Kubota sonic oscillator for 1 hr under circulation of ice water. The mixture was centrifuged at 1,5 x g for 3 min, and the supernatant solution was used as the crude cell-free extract. Crude cell-free extracts were pooled and clarified by centrifugation at 1, x g for I hr to remove particulate reduced nicotinamide adenine dinuclotide (NADH) oxidase. The supernatant solution was used as material for the gel filtration experiments. Partial purification of enzymes. Supematant solution (3.5 ml) was applied to a column (2 by 3 cm; bed volume 94 ml) containing Sephadex G-2 suspended in 5 mm phosphate buffer (ph 6.8). Fractions of 7 ml were collected. The fractions containing the majority of the glucose dehydrogenase activity were pooled, concentrated in a collodion bag, and used as partially purified glucose dehydrogenase. Enzyme assays. Glucose dehydrogenase activity was assayed in a Hitachi Perkin-Elmer spectrophotometer at 34 nm at 37 C, by using a cuvette of I-cm light path. The assay cuvette contained 6 Amoles of glucose, 1.2,umoles of nicotinamide adenine dinucleotide (NAD), 3 Mmoles of phosphate buffer (ph 6.8), various concentrations of ethylenediaminetetraacetic acid ' Portions of this paper were presented at the Joint Meeting on Genetic and Biochemical Regulation of Dormancy of the U.S.-Japan Cooperative Science Program, 1 to 14 November (EDTA) or other chemicals, enzyme solution, and 197, Kyoto, Japan. water to make up a total volume of 3. ml. In the ac- 442

2 VOL. 1 6, ACTION OF EDTA OR DIPICOLINIC ACID 443 tivity of glucose dehydrogenase from intact resting spores, the time course of the increase in optical density at 34 nm was found to be almost linear in a short time after the reaction was started. The specific activity was expressed as the value for nanomoles of NAD reduced per minute per milligram of protein obtained from the initial velocity. On the other hand, in the activity of inactive glucose dehydrogenase reactivated by EDTA or other chemicals, it was impossible to obtain a straight line just after starting of reaction. Therefore the specific activity was expressed as the value for nanomoles of NAD reduced per minute per milligram of protein obtained from the maximal inclination in a sigmoid curve. NADH oxidase activity was assayed by the reduction in absorbance at 34 nm. Measurement of protein. Protein was estimated by the method Lowry et al. (8). Molecular weight estimation by gel filtration. Assays were made by the method of Andrews (1). Partially purified enzyme solution (2. ml) containing.1 ml of 1% blue dextran 2, and.1 ml of 3 mg of bovine serum albumin per ml was applied to a column (1.35 by 8 cm; bed volume 114 ml) containing Sephadex G- 2 suspended in 5 mm phosphate buffer (ph 6.8). Elution was accomplished by using the same buffer solution, and fractions of 3 ml each were collected. A reference run was also made by using blue dextran 2,, catalase, and bovine serum albumin; the elution volume was estimated from the absorption at the wavelength of 63 nm for blue dextran, 28 nm for bovine serum albumin, and from the decreased absorption of H22 at 24 nm for catalase activity (3). Chemicals. EDTA *4Na was purchased from Katayama Kagaku Industrial Co., Osaka, Japan, dipicolinic acid and its chemical analogues were from Tokyo Kasei Industrial Co., Tokyo, Japan, and NAD was from Sigma Chemical Co., St. Louis, Mo. The chemical analogues of dipicolinic acid tested were isocinchomeronic acid, lutidinic acid, and quinolinic acid. EDTA was dissolved in distilled water and adjusted to ph 6.8 with I N acetic acid, and dipicolinic acid and its analogues were dissolved in distilled water with 5 N NaOH and adjusted to ph 6.8 with I N acetic acid. All inorganic salts used were of reagent grade. RESULTS Activities of glucose dehydrogenase in the crude cell-free extract obtained from spores which were heated at various temperatures. The temperature of heating at which glucose dehydrogenase in spores loses its activity completely was determined. The results are shown in Table 1. From these results it is clear that glucose dehydrogenase in the crude cell-free extract obtained from spores which were heated at 87 C for 3 min had lost its activity. Reactivative action of EDTA on the inactive glucose dehydrogenase obtained from heated spores. As shown in Fig. 1, it was found that EDTA promotes the activation of the inactive glucose dehydrogenase in the crude cell-free extract obtained from the spores which were heated TABLE 1. Activities ofglucose dehydrogenase in the crude cell-free extracts obtained from spores which were heated at various temperatures Temp of heating Heating time Specific (C) (min) act ivitya No heating a Values expressed as nanomoles of reduced nicotinamide adenine dinucleotide per minute per milligram of protein. I-. -4 C EDTA (mm) FIG. 1. Reactivative action of EDTA on the inactive glucose dehydrogenase in the crude cell-free extract obtained from heated spores. The assay cuvette contained 6,umoles of glucose, 1.2 jsmoles of nicotinamide adenine dinucleotide (NA D), 3,moles ofphosphate buffer (ph 6.8), various concentrations of EDTA, enzyme solution (about.5 mg ofprotein), and water to make up a total volume of 3. ml. Values of EDTA are shown as final concentrations. Nanomoles per minute per milligram in Fig. I to 4 are expressed as nanomoles of reduced NAD (NADH) or NADH oxidized per minute per milligram ofprotein.

3 444 HACHISUKA AND TOCHIKUBO J. BACTERIOL. at 87 C for 3 min. When recovery of the enzyme activity was calculated on the basis of the activity obtained from the nonheated spores (Table 1), it was also found that about 9% of the enzyme activity was recovered by the addition of 3 mm EDTA. EDTA also activated the inactive glucose dehydrogenase obtained from the spores which were heated at 9 C for 3 min, but it did not show any action on that from spores heated at 1 C for 3 min. It was clear from the result that EDTA has no effect on completely inactivated enzyme. Since we used the crude cell-free extract as an enzyme solution in these experiments, a question arose concerning NADH oxidase activity in the crude cell-free extract. To resolve this question, the partially purified inactive glucose dehydrogenase was treated with EDTA. Effect of EDTA on the partially purified inactive glucose dehydrogenase obtained from heated spores. Partial purification procedures were performed tentatively on the crude cell-free extract obtained from the intact resting spores. The crude cell-free extract was centrifuged at 1, x g for 1 hr to remove the particulate NADH oxidase, and the supematant solution was applied to a column containing Sephadex G-2 as reported in Materials and Methods. As shown in Fig. 2, glucose dehydrogenase was separated from soluble NADH oxidase by the procedures described. Fractions containing most of the glucose dehydrogenase were pooled, condensed, and )_ CD C) -.. c - I E 3::-E.Iu w 2 a 1 I FRACTION NUMBER FIG. 2. Separation of reduced nicotinamide adenine dinucleotide oxidase and glucose dehydrogenase in a crude cell-free extract obtained from intact resting spores. The crude cell-free extract obtained from intact resting spores was centrifuged at 1, x g for I hr. The supernatant solution was applied to a column containing Sephadex G-2. z used as a partially purified glucose dehydrogenase. The glucose dehydrogenase was purified about 11-fold. EDTA had no effect on the partially purified active glucose dehydrogenase. The same procedures were performed on the cell-free extract obtained from the spores which were heated at 87 C for 3 min (Fig. 3). Assay of the fractions indicated that there are two kinds of activity of glucose dehydrogenase; one is active without EDTA. and the other is active after the addition of EDTA. As already shown in Fig. 1, the former was not seen in the crude cellfree extract. The origin of this enzyme will be discussed later. Effects of various salts on the partially purified inactive glucose dehydrogenase. To further clarify the action of EDTA, the minimal effective concentration of EDTA on the partially purified inactive glucose dehydrogenase was determined (Fig. 4). It was clear from these results that the greater the concentration of EDTA, the greater the activity of glucose dehydrogenase, and that relatively large amounts of EDTA are necessary E 2 z "-11 1-flC a. 5 INACTIVE GDH / (REACTIVATED Im 3 GDH BY EDTA) E /!T.,-PROTEIN NADH oxadasenb.. ACTIVE* GDH 4c - cn E 3 E 2 _- (a 1< ~ FRACTION NUMBER FIG. 3. Separation of reduced nicotinamide adenine dinucleotide oxidase and inactive glucose dehydrogenase in a crude cell-free extract obtained from spores which were heated at 87 C for 3 min. The crude cellfree extract obtained from the heated spores was centrifuged at 1, x g for I hr. The supernatant solution was applied to a column containing Sephadex G- 2. Inactive glucose dehydrogenase was reactivated by 3 mm EDTA which was added as a final concentration in the assay cuvette containing 6 Mmoles of glucose, 1.2,moles of nicotinamide adenine dinucleotide, 3,umoles of phosphate buffer (ph 6.8), 1 ml of enzyme solution, and water to make up a total volume of 3. ml. (-*) Active glucose dehydrogenase was shown without EDTA. The enzyme elutes at the same position with the inactive enzyme, and therefore the active form probably results from activation during assay method. O. i

4 VOL. 16, ACTION OF EDTA OR DIPICOLINIC ACID 445 CD E E 5 O 5 1 EDTA (mm) FIG. 4. Reaclivative action of EDTA on the partially purified inactive glucose dehydrogenase. Experimental conditions were the same with those in Fig. I except that the concentration of enzyme protein was about.1i mg. Values of EDTA are shown as final concentrations. for significant reactivation of the inactive glucose dehydrogenase. From these results we considered the possibility that EDTA would not act as a chelating agent, but as a high concentration salt on the inactive glucose dehydrogenase, because it is known that high salt concentration has a positive effect on the enzyme activity (4). To investigate this possibility, sodium chloride and other salts were used instead of EDTA. Results (Table 2) indicate that sodium chloride and other salts have a reactivative action on the partially purifi'ed glucose dehydrogenase but are remarkably less effective than EDTA. Therefore, EDTA may act as a chelating agent on the inactive glucose-dehydrogenase and remove metals which are combined with glucose dehydrogenase; consequently, the inactive glucose dehydrogenase is converted to active form. Effects of dipicolinic acid and its analogues on the partially purified inactive glucose dehydrogenase. Since it seemed possible that the action of EDTA on the inactive glucose dehydrogenase was due to a chelating action, we examined other chelating agents, dipicolinic acid and its analogues, in the same way as EDTA. As shown in Table 2, it is clear that dipicolinic acid and its analogues have a reactivative action on the inac- TABLE 2. Reactivative action of various salts, dipicolinic acid, and its analogues on the partially purified inactive glucose dehydrogenase Chemicals Specific activitya None mm EDTA" mm NaCI 48 1 mm KCI mm LiCl mm (NH4)2SO mm Dipicolinic acid 4 1 mm Isocinchomeronic acid mm Lutidinic acid 58 1 mm Quinolinic acid 384 a Values expressed as nanomoles of reduced nicotinamide adenine dinucleotide per minute per milligram of protein. b Values of ethylenediaminetetraacetic acid (EDTA) or other chemicals are shown as final concentrations. tive glucose dehydrogenase obtained from the spores which were heated at 87 C for 3 min. However, they are not as active as EDTA, making it unclear whether the reactivative action of EDTA is due to chelation. Since dipicolinic acid causes activation of the inactive glucose dehydrogenase, it seemed possible that the active glucose dehydrogenase in partially purified enzyme solution obtained from heated spores (Fig. 3) was derived from the inactive glucose dehydrogenase, which was activated by dipicolinic acid during rupturing of spores. If this hypothesis is true, the peak of elution pattern must be the same as that of the active form. However, the active and inactive enzymes in Fig. 3 elute at the same position, and therefore the active form probably results from activation during assay method. Differences between glucose dehydrogenase obtained from intact resting spores and the inactive glucose dehydrogenase obtained from the heated spores. It was shown that EDTA causes the activation of the inactive glucose dehydrogenase obtained from the spores heated at 87 or 9 C for 3 min only. It has no effect on the active glucose dehydrogenase obtained from intact resting spores. This fact led us to assume that there would be some differences between both dehydrogenases. A few characteristics of them were compared. Partially purified active and inactive glucose dehydrogenases were applied to a Sephadex G- 2 column (1.35 by 8 cm) equilibrated with 5 mm phosphate buffer (ph 6.8). In both dehydrogenases the enzyme activities gave different peaks in the elution patterns (Fig. 5). After the preincubation of the inactive glucose

5 446 HACHISUKA AND TOCHIKUBO J. BACTERIOL BOVINE SERUM AIIBUMIN (7) 1.5[ 1.3 INACTIVE GOM AACTIVE GDH CATALASE ( 25.) FRACTION NUMBER FIG. 5. Elution patterns on Sephadex G-2 of the partially purified active glucose dehydrogenase, the partially purified inactive glucose dehydrogenase, and the preincubated mixture of EDTA and the partially purified inactive glucose dehydrogenase. The mixture of the partially purified active glucose dehydrogenase (or the partially purified inactive glucose dehydrogenase), blue dextran, and bovine serum albumin was filtrated through Sephadex G-2. (*) Inactive glucose dehydrogenase was reactivated by 6 mm EDTA. (* *) EDTA solution was added to the partially purifled inactive glucose dehydrogenase at a final concentration of 25 mm and preincubated at 37 C for 1 min. Then, the mixture was filtrated through Sephadex G-2 with blue dextran and bovine serum albumin. Enzyme activity was shown without EDTA. A reference run was also made using blue dextran, catalase, and bovine serum albumin. Nanomoles per minute per milliliter are expressed as nanomoles of nicotinamide adenine dinucleotide reduced per minute per milliliter of each fraction. dehydrogenase with EDTA at 37 C for 1 min, the mixed solution was filtrated through Sephadex G-2 column by the same method mentioned above. As shown in Fig. 5, only the active glucose dehydrogenase appears and elutes at the same position of the active enzyme obtained from the resting spores. Molecular sizes of both dehydrogenases were determined by gel filtration through Sephadex G- 2 column by using blue dextran 2, to determine the exclusion volume; catalase and bovine serum albumin were used to calibrate the column. From the results (Fig. 6), the molecular weight of active glucose dehydrogenase in intact resting spores was calculated by the method of Andrews (I) to be about 1, and that of the inactive glucose dehydrogenase in heated spores to be about 4,. DISCUSSION There are many opinions about the basis of heat resistance of enzymes in spores. In the relationship between spore structure and heat resistance, Murrell (1) reported that catalase in intact spores of B. subtilis is heat stable but is no LOG ( MOLECULAR WEIGHT x 15) FIG. 6. Calibration curve for Sephadex G-2 column and locations of active glucose dehydrogenase and inactive glucose dehydrogenase. Assays were made by the method of Andrews (1). The column was calibrated by using the following values of molecular weights: 25, for catalase and 7, for bovine serum albumin. more stable than the vegetative cell enzyme when in an extract. He suggested the possibility that spore coats were involved in contributing to heat resistance. Halvorson and Church (7) proposed that the binding of the enzyme to large structures could render them heat resistant. On the other hand, several investigators suggested that changes of molecular size of enzyme protein are the basis of heat resistance. Nakata (11) found that the ribosidase in spore-free extract is particulate and heat stable. Militzer et al. (9), Steward and Halvorson (13), and Black and Gerhardt (2) had suggested the aggregation of macromolecules as the basis for thermal stability in spores. Recently, Sadoff et al. (12) found that glucose dehydrogenase from B. cereus spores shows a remarkable increase of heat resistance which is due to dimer-monomer interconversion. In these experiments no studies were done with the enzyme itself in the heated spores. The inactive enzyme in the heated spores did not become a subject of experimentation. Our experiments indicate that glucose dehydrogenase in the intact resting spores changes to the inactive form during heating of spores at 87 or 9 C for 3 min, and the molecular size of glucose dehydrogenase changes to about onehalf. Moreover, the inactive glucose dehydrogenase obtained from heated spores changes to an active form by the addition of EDTA, salts, or dipicolinic acid. From these results we suggest that an interconversion of glucose dehydrogenase occurs in spores during heating at a particular range of temperature and consequently the enzyme changes to an inactive form. Reactivation of the

6 VOL. 16, 1971 ACTION OF EDTA OR DIPICOLINIC ACID 447 inactive form could be enhanced by dipicolinic acid in spores. ACKNOWLEDGMENTS We thank T. Murachi, Department of Biochemistry, Nagoya City University Medical School, for his useful suggestions in the purification of the enzyme and the molecular weight determination by Sephadex column chromatography. LITERATURE CITED 1. Andrews, P The gel-filtration behaviour of proteins related to their molecular weights over a wide range. Biochem. J. %: Black, S. H., and P. Gerhardt Permeability of bacterial spores. IV. Water content, uptake, and distribution. J. Bacteriol. 83: Chance, B., and A. C. Maehly Assay of catalases and peroxidases. In S. P. Colowick and N.. Kaplan (ed.), Methods in enzymology, vol. 2. Academic Press Inc., New York. 4. Dixon, M., and E. C. Webb Enzymes, 2nd ed., p Longmans, Green and Co. Ltd., London and Colchester. 5. Falaschi, A., and A. Komberg Biochemical studies of bacterial sporulation. 11. Deoxyribonucleic acid polymerase in spores of Bacillus subtilis. J. Biol. Chem. 241: Gardner, R., and A. Kornberg Biochemical studies of bacterial sporulation and germination. V. Purine nucleoside phosphorylase of vegetative cells and spores of Bacillus cereus. J. Biol. Chem. 242: Halvorson, H., and B. Church Biochemistry of spores of aerobic bacilli with special reference to germination. Bacteriol. Rev. 21: Lowry,. H., N. J. Rosebrough, A. L. Fare, and R. J. Randall Protein measurement with the Folin phenol reagent. J.. Biol. Chem. 193: Militzer, W., T. B. Sonderegger, L. C. Tuttle, and C. E. Georgi Thermal enzymes. II. Cytochromes. Arch. Biochem. 26: Murrell, W. G The bacterial endospore. University of Sydney, Australia. 11. Nakata, H. M Discussion, p In H.. Halvorson (ed.), Spores. American Institute of Biological Sciences, Washington, D.C. 12. Sadoff, M. L., J. A. Bach, and J. W. Kools Significance of multiple forms of glucose dehydrogenase in relation to its heat resistance, p In L. L. Campbell and H.. Halvorson (ed.), Spores III. American Society for Microbiology, Ann Arbor, Michigan. 13. Stewart, B. T., and H.. Halvorson Studies on the spores of aerobic bacteria. II. The properties of an extracted heat-stable enzyme. Arch. Biochem. Biophys. 49: Tono, H., and A. Kornberg Biochemical studies of bacterial sporulation. III. Inorganic pyrophosphatase of vegetative cells and spores of Bacillus subtilis. J. Biol. Chem. 242: Tono, H., and A. Kornberg Biochemical studies of bacterial sporulation. IV. Inorganic pyrophosphatase of vegetative cells and spores of Bacillus megaterium. J. Bacteriol. 93: Downloaded from on September 23, 218 by guest