SORBIC ACID INHIBITION OF ENOLASE FROM YEAST AND LACTIC ACID BACTERIA Thain fo: the Deer» of Ph. D. MICHIGAN STATE UNIVERSITY John .I. Azuku 1962 “H5515 Glyceraldehyde-S-phosphate Isomerase l DPN, PO: Glyceraldehyde-SP-dehydrogenase l,3-Diphosphoglyceric acid l.ADP, Mg++ 3-phosphoglycerate-l-kinase SFPhosphoglyceric acid H ;”9 2,3-Phosphoglyceric mutase ZrPhosphoglyceric acid ++ Mg Enolase 27Phosphoenol pyruvic acid ADP, Mg++ Pyruvate kinase Lactic acid< Pyruvic acid DPNH ‘ ThPP. MgH Lactic ICarboxylase \. Dehydrogenase .Acetaldehyde + CO _ 2 DPNH ..Alcohol Dehydrogenase Ethanol Scheme 1. Pathway of anaerobic glucose metabolism in yeast. i2 alcoholic fermentation but it does not have the enzymes carboxylase and alcohol dehydrogenase. The heterolactic fermentation by Leuconostoc mesenteroides was elucidated by DeMoss, Bard and Gunsalus (1951) and by Gunsalus and Gibbs (1952) upon the following evidence. Equimolar quantities of C02, ethanol, and lactate were produced and the ratio of these was constant under varying conditions. Aldolase could not be demonstrated. Also, CO2 arises from carbon 1 of glucose, the lactate from carbons h, 5, and 6 and the methyl carbon of ethanol from carbon 2 of glucose. In this fermentation glucose goes to glucose-b-phosphate and then to 6-phosphogluconic acid. The phosphogluconic acid is oxidized and decarboxylated to yield C02 and a phosphorylated five-carbon sugar, ribulose phos- phate. Ribulose phosphate is then split to yield one mole- cule of triose phosphate and a two-carbon fragment that becomes reduced to alcohol. Triose phosphate is oxidized via the Embden-Meyerhof pathway (EMF) to pyruvic acid and the pyruvate goes to lactic acid. In this pathway only one molecule of lactic acid is formed from the hexose substrate, while CO2 and alcohol are also formed. More recently, Eltz and Vandemark (1960) demonstrated a similar system in Lactobacillus brevis. Thus, the heterofermentative lactic acid bacteria are also dependent on the enzymes of the lower fraction of the EMP scheme. 13 Compagative enzyme biochemistry. Are the preparations of an enzyme obtained from different sources identical? Before this question can be answered, one must define what is meant by identical. Is it identity merely of the active center and its immediate surroundings, so that the name of an enzyme is merely the name of a particular active center, which may be attached to different proteins; or does it mean that the whole protein is identical? If it is the latter, a study of only the catalytic properties will not answer the question. One must study the protein itself, and in particular the amino acid composition and sequence. Jolles and Promageot (1956) prepared lysozymes from the spleen and kidney of dog and rabbit. They were all similar in properties, but not identical even when prepared from two tissues of the same animal. Henion and Sutherland (1957) found that phosphorylases from different organs in the same animal as well as from different animals were immunologically different; and Antoni and Keleti (1957) found quantitative immunological differences between alco- hol dehydrogenase from closely related yeast species. The observation that a single organism may contain more than one enzyme catalyzing the same biochemical re- Kornberg and Pricer (1951) found One was DPN action is not uncommon. yeast to contain two isocitric dehydrogenases. Ebesuzaki and Barron (1957) Stadtman and the other TPN dependent. found yeast to contain two alcohol dehydrogenases. 1h et a1. (1961) demonstrated that cell-free extracts of Escherichia coli contain two different aspartokinases that catalyze the phosphorylation of aspartate by.ATP. These enzymes have been separated from each other by ammonium sulfate precipitation. It is postulated that the biologi- cal significance of the presence of more than one enzyme catalyzing the same biochemical reaction is part of a cellular regulatory control mechanism. EXPERIMENTAL METHODS The sorbic acid used was refined sorbic acid (water content, 5.5%) provided by the Carbide and Carbon Chemicals Co., New York. This was recrystallized three times from distilled water before use. Fresh stock solutions (2.0 x 10-5) were maintained in the refrigerator during the experiments. These were adjusted to the pH level desired for each experiment and diluted to give the final concen- tration necessary for the specific study. Enolase activity was determined by the method of Warburg and Christian (l9hl) as modified by Wold and Ballou (1957). With 2-phosphoglyceric acid (2P0) as a substrate, the appearance of phosphoenolpyruvate (PEP) is followed by measuring the increase in Optical density (OD) at ZLIQnu. A Beckman model DU spectrophotometer was used for all spec- trophotometric work. The crystalline enolase used was prepared from yeast and kindly supplied by Dr. Finn Wold, Department of Chemistry and Chemical Engineering, university of illinois, Urbana, illinois. The glucose used was "Difco" Bacto-dextrose. Pyruvic acid solution was prepared by dissolving sodium pyruvate and adjusting the pH. The 2-phosphoglyceric acid solution was made from the barium salt by dissolving it in 1N HCL and 15 16 then adding sodium sulfate equivalent to the barium ion. The precipitate of barium sulfate was removed by centrifuga- tion, the supernatant made up to volwme, and the pH adjusted. 3-Phosphoglyceric acid (3P0) was also prepared from its barium salt by the procedure Just described. Phosphoenol- pyruvate was prepared from its silver barium salt. The barium was removed as previously described and the silver was removed as silver chloride. The cultures Lactobacillus plantarum, Lactobagillus brevis, and Padlococcus cerevisiae used in this study were grown in a medium of the following composition: 1% yeast extract, 0.5% dextrose, and 0.5% dibasic potassium phosphate. They were stock cultures which had been originally isolated from cucumber fermentations. Twenty liters of medium minus the dextrose were sterilized in carboys. The dextrose was sterilized separately and added aseptically. The inoculum was started in a test tube, and was transferred 18 hours later to a 1 liter flask, which in turn was transferred to a 3 liter flask after 18 hours. This was transferred to the carboys. The carboys were incubated for 18 hours at 30C, the cells harvested with a Sharples Super Centrifuge (the Sharples Specialty Co., Philadelphia, Pa.) and washed twice with distilled water. These cells were used for the intact cell Warburg studies, the acetone dried cell Warburg studies, the cell-free extract studies, and the enzyme purifications. 17 The yeast used was baker's yeast. The yeast was air dried and frozen for storage. The cell-free extracts were prepared from this dried and frozen yeast. The effects of sorbic acid on the fermentation of glucose by intact yeast and lactic acid bacteria were measured by the conventional Warburg technique. Cells, buffer, sorbic acid, and water were placed in the main com~ partment of the Warburg flask. The substrate was put in one of the side arms. The total volume after tipping in of the substrate was 3.0 ml. The flasks were flushed with nitrogen in experiments with yeast cells, while in those with lactic acid bacteria they were flushed with a mixture of 5% CO2 and 95% nitrogen. They were shaken until thermal equilib- rium was reached, readings taken and the substrate tipped into the main compartment. After this, readings were taken at definite time intervals. In both instances CO2 evolution was measured, but in the case of the lactic acid organisms this was an indirect measurement of lactic acid production. The homofermentative lactic organism produces two moles of lactic acid per mole of glucose. One mole of acid will liberate one mole of C02 from a bicarbonate buffer system. The acetone dried cells were prepared in the following manner from cells grown in carboys. The aqueous cell sus- pension was added slowly with stirring to ten volumes of acetone previously cooled to -20 C. After stirring briefly, the cells were allowed to settle, the solvent removed by 18 filtration, and the ftner cake of cells washed with 2 to 5 volumes of -20 C acetone. The filter cake was then trans- ferred to a desiccator in the presence of paraffin to absorb the solvent. The resulting powder was used for Warburg studies. For preparing cell-free extracts, 159 (wet weight) of lactic acid bacteria or air dried yeast cells and hSQ of glass beads were combined with 50ml of water and blended in a Servall Omnimixer (ivan Sorvall, inc., Norwalk, Conn.) for five minutes in an ice bath to disrupt the cells. The beads were pavement marking beads, average size 0.2mm., manufac- tured by the Minnesota Mining and Manufacturing Co., Minneapolis. Before blending in the Cmnimixer, the mixture of cells and beads was cooled to a few degrees above 0 C. The cup was filled to the top to prevent foaming and the resulting surface denaturation. The beads, cell debris, and intact cells were removed after blending by centrifugation in the cold. The supernatant contained the enzymes of interest. The extracts were dialyzed for 2h hrs against distilled water before use in the various assays. The protein concentration in the extracts was deter- mined by the turbidimetric trichloroacetic acid method of Stadtman, Novelli, and Lipmann (1951). The extract contain- ing 0.1 to 2.0 mg of protein was made up with water to a volume of 2 ml, and 3 ml of 5% trichloroacetic acid added. The resulting suspension was allowed to stand for 30 sec and 19 the turbidity measured in a calorimeter at a wavelength of suo mu. Crystalline egg albumin was used as a standard. Enolase activity in the cell-free extracts of lactic acid bacteria was measured in several different ways. One of these ways was by coupling enolase to pyruvate kinase and lactic dehydrogenase. With 2-phosphoglyceric acid as sub- strate in the presence of MgSOh and adenosine diphosphate (ADP), the oxidation of reduced diphosphopyridine nucleotide (DPNH) was followed spectrophotometrically at ShO mu. Eno- 1ase activity in cell—free extracts was also measured by acid labile phosphate (that which is hydrolyzed completely in 1N acid at 100 C in seven minutes). With 2-phosphoglyceric acid as substrate and MgSOu as cofactor, the amount of acid labile phosphate formed in three minutes was measured. The total volume of the reaction mixture was 3.0 ml. The reac- tion was started by the addition of substrate and stopped at the end of three minutes by the addition of 3 ml of 10% tri- chloroacetic acid. This also served to deproteinize the reaction mixture. The mixture was then centrifuged to re- move the precipitated protein. Sulfuric acid was added to the supernatant fluid to a concentration of 1N, the tubes heated in a boiling water bath for seven minutes, and cooled. Phosphate was determined by the procedure of Fiske and SubbaRow (1925). This procedure was slightly modified by use of monomethyl~para-aminophenol sulfate in place of l-amino-z-naphthol-h-sulfonic acid in the reducing reagent. 20 The estimation of free sulfhydryl (SH) groups was accomplished by use of the p-chloromercuribenzoate procedure based on the method of Boyer (l9Su). This is based on the fact that p-chioromercuribenzoate reacts stoichiometrically with SH groups to form mercaptides which can be quantitated by measurement of their strong light absorption at 255mu. Crystalline glutathione was used as a reference standard. Enolase in extracts of lactic acid bacteria was par- tially purified by the following procedure. The cell-free extracts were prepared as previously described and then were canbined into one batch. The extract was cooled in an ice bath and cold acetone was added slowly with stirring until a 33 1/3% acetone concentration was reached. The precipi- tate formed was centrifuged and discarded. Additional cold acetone was added to the supernatant fluid until a concen- tration of 50% acetone was reached. The precipitate was centrifuged and the supernatant fluid discarded. The pre- cipitate was then taken up in distilled water and the in- soluble material was centrifuged and discarded. The solu- tion was then brought to pH u.8 with lN acetic acid, cold ethanol added cautiously with stirring until 33 1/3% con- centration of ethanol was reached, and the precipitate cen- trifuged and discarded. Additional cold ethanol was added to the supernatant fluid until 50% ethanol concentration was reached, the precipitate centrifuged, and the superna- tant fluid discarded. The precipitate was then suspended 21 in distilled water and dialyzed overnight against distilled water. After dialysis, insoluble material was centrifuged, and the solution put through a column that was prepared in the following manner. Twenty-five grams of cellex-P cation exchange cellulose (California Corporation for Biochemical Research, Los Angeles, Calif.), exchange capacity 1.0 milliequivalents per gram, which consists of phosphoric acid exchange groups on a Whatman cellulose lattice, were made into a slurry with distilled water and packed into a column 3 cm in diameter and 16 cm long. The column was charged with magnesium by passing 100 ml of 1M MgSOLL through it. it was then washed with distilled water. The solution con- taining the enzyme was then applied to the column and washed in with distilled water. The enzyme was then eluted with 1M KCl. Five ml fractions were collected with an automatic fraction collector and these were assayed for activity of enolase spectrophotometrically at 2h0 mu with 2-phospho- glyceric acid as substrate. A Shandon electrophoresis (Consolidated Laboratories, Inc., Chicago Heights, ill.) apparatus was used for the starch block electrophoretic separation of the extracts of cells of lactic acid bacteria. The buffer used was tris- (hydroxymethyl)aminomethane (tris) 0.05M, pH 8.6. The time of the run was three hours at 100 volts, lS milliamps. .A well was cut in the center of the starch block and the sample placed in it and then the starch was replaced. After the completion of the run, the starch block was cut into strips about 1 cm in width. These were placed in 3 ml of 0.05M imidazole buffer, pH 7.h, and the protein eluted from the starch by shaking and allowing to settle. The eluates were assayed for enolase and lactic dehydrogenase. Enolase activity with 2-phosph091yceric acid as substrate was measured spectrophotometrically. Lactic dehydrogenase was determined by the method of Kubowitz and Ott (l9h3). With pyruvate as substrate the oxidation of DPNH was followed spectrophotometrically at 3u0 mu. Extracts of cells of E. plantarum were fractionated with the addition of solid ammonium sulfate in the following manner. The solid ammonium sulfate was added to the extract slowly with stirring to the point of desired saturation. The precipitate was then centrifuged and redissolved in dis- tilled water. The supernatant fluid was used for further fractionatiai. The mixture was maintained throughout the fractionation process at u C. The assays for enolase and lactic dehydrogenase were run on the precipitates of the particular fractions that were redissolved in distilled water. Crystalline lactic dehydrogenase was purchased from General Biochemicals, Chagrin Falls, Ohio. it was origin- ally isolated from animal muscle tissue and had been crystallized 2x. RESULTS Mechanism of Yeast Enolase inhibition by §orbic Acid In determining enolase activity in the presence of sorbic acid, it was noted that there was an initial decrease in optical density at 2h0 mu on the addition of enolase to the reaction mixture. This masked the initial increase in optical density due to enzymatic action and created an ap- parent lag. In the inhibition studies this was eliminated by adjusting the instrument to zero 5 sec after adding enzyme to the reaction mixture. This decrease in absorbancy prob- ably results from a combination of the sorbate with the enzyme, since the sorbate absorbs light strongly at this wavelength. The extent of the decrease in optical density (00) was directly related to sorbic acid concentration when "IOM (Fig.1). the enzyme concentration was constant at 6.0 x 10 The same relationship was observed when the sorbate concen- tration was held constant and the enzyme concentration varied. However, the lag was independent of substrate concentration (Table 1). Similar results were obtained when phosphoenolpyruvate was added to the enzyme and the 0D loss measured at a wave- length of 2&0 mu. Since the sorbate and phosphoenolpyruvate 23 24 .0 8‘? A V .07 o o '0 a on m —A 00 at 250 my 2) . . ‘1‘ 1 L l l l l _l V T‘ I y —j l . 0 2.0 3.0 4.0 5.0 6.0 7.0 Sorbic Conc. x [0’8 Moles Fiquro I. The change in OD due to addition of sorbate to enolase and magnesiumq Cuvotte contents: 0.0 x 10--10 H enolase, 8.0 x 10" M M3804. 25 TABLE 1 EFFECT OF SUBSTRATE CCNCENTRATION CN TIME BETWEEN ADDITION OF SUBSTRATE AND APPARENT ENZYMATIC ACTIVITY (LAG) Substrate (2-phosphoglyceric acid) conc. Time of lag in sec 2 x 10"3 M 30 2 x 10"LL M 30 b x lO-LL M 30 must be bound to amino acid residues on the enzyme, a number of amino acids were added to these two compounds along with Mg++ and the loss in optical density measured. it was found that the addition of individual amino acids to a solution containing magnesium and sorbic acid, and also the addition of an amino acid to a solution containing magnesium and phosphoenolpyruvate would result in a similar loss of opti- cal density (Table 2). Similar losses in optical density were observed on mixing other alpha, beta-unsaturated acids (crotonic and cinnamic acids) with magnesium and enolase. The inhibition of yeast enolase activity by sorbic acid was measured in a spectrophotometer at room temperature. As can be seen in Fig. 2 sorbic acid concentratims of 1.5 x 10""L M and higher resulted in definite inhibition of enolase activity with a substrate concentration of 2 x lO-h'M. 00 at 240 m}! .l2‘ .ll‘ .IO‘ 26 0 Control 1: l.5xl0‘4Msorbic acid 0 2.0xl0'4M sorbic acid 2.5xl0'4M sorbic OCid . Di "\ h D - IO 20 30 40 50 60 Time in sec. Fiqure 9. Inhibition of V0 st 0n01“se activitv by sorbic acid. Hnrh ruvotto contoinod 3 P‘1 of n rewction mixtnrn of {Mr Foliowinq: G x 10-10 d onolnso, 2 x 10‘” fl substrate (T—nhosn'oqucoric acid), 2 x 10‘? W H380 , and S x 10‘“ M imidnzole buffer at o” 7.4. \nnronrlnth solutions of sorbic acid adjusted to pH 7.4 were added to give the sorbate concentrations indiCnted. 2? Concentrations of sorbic acid below 1.5 x lO'u'M resulted in very little or no inhibition of the enzyme at this substrate level, and when the sorbic acid level was raised much above 2.5 x 10-” N no significant enzyme activity could be detected. TABLE 2 REDUCTICN IN OPTICAL DENSITY ON ADDING VARIOUS AMINO.ACIDS TO SOLUTIONS OF SORBATE AND PHOSPHOENOLPYRUVATE Amino Acid Net Loss in CD3 of solutions of: Added (0.1 M) Sorbate Phosphoenolpyruvate Glycine .150 .090 L-Histidine .IuS .080 LeArginine .150 .085 L-Alanine .th - L-Lysine .150 - L-Methionine .1h5 - “ aThe losses in OD rith sorbate (l x 10"h M) and phos- phoenolpyruvate (2 x 10‘4 M) were measured at 250 and 2&0 mu respectively. The inhibition of enolase was also demonstrated with phosphoenolpyruvate as substrate. The same procedure was followed as previously described except that phosphoenol- pyruvate (1 x 10"3 M) was used as substrate and the decrease in optical density at 2&0 mu followed. .As illustrated in Fig. 3, 1.0 x 10'“ M and 2.0 x lO’u'M sorbic acid concen- trations significantly inhibited enolase activity. 00 at 240m}: 0.4 0.3” .0 '1’ 0.l‘ PEP lxlO’3M \:\ 0 Control X lxlO'4M sorbic acid A 2x10'4M sorbic acid 1 L 1 I I T Figure 9. In' i_bi1ion of ye sf onolnso A J l I l I I5 30 45 60 75 90 Time in sec. activity by i1‘ 1 whosniioonoinvruv”1(~ ES substrate, sorbic void 1, 1c 4(1 i1ed in blq. 2 oxcopt R'WPbinn mix1nrtq "orr FOT‘ tiu- STU)FtT“ViC. 29 Crotonic and cinnamic acids also inhibited enolase aCtIVItY (F19. h). However, a x lo'h'M cinnamic acid was no more effective than 2 x lo'h'M sorbic, and crotonic acid was even less effective. Thus, the chain length may influence the relative effectiveness of these acids. The presence of the unsaturated bond is important since the addition of 6.6 x 10'3 M of either glycine, acetic or butyric acid had no effect on the purified yeast enolase. The results of inhibition studies run as described in Fig. 2 utilizing substrate concentrations of 5.0 x 10'”5 M, 2.0 x 10-h M, and h.0 x 10'” M were used in analyzing the type of inhibition occurring by use of a Lineweaver-Burk plot. The reaction velocities were calculated in terms of the change in optical density per min for each sorbic acid- substrate combination, and the reciprocals of the velocities plotted versus the reciprocals of the substrate concentra- tions for each sorbic acid concentration indicated in Fig. 5. it can be seen that the substrate concentration had a pro- nounced effect on the degree of enolase inhibition by a given level of sorbic acid. From this plot it appears that the inhibition is competitive at sorbic acid concentrations up to 2.0 x lO-u'M and is non-competitive at higher inhibitor levels. Thus, the intercept is constant and the slope in- creases with increasing inhibitor concentrations up to 2.0 x lo'h'M, whereas both the slope and intercept change at higher concentrations. However, plots of reciprocal 00 at 240 my .l3‘ ll‘ 0 .071 .031 .0|‘ - ---— — - - -- -.—~u—-_- _—.—- —--—-—u—. -_.n . -I—u— 30 2 PG 2xl0" 4 M 0 Control 1: 5X10-3M crotonic acid A 4le0‘4 M cinnamic acid / . / / / ./ \\ T / ./ Time in sec. Fiquro 4. Inhibition of ve~st enolase activity by crotonic ”nd cinnamic acids. 500 Fig. 2 for composition of reaction mixtures. QPG 2 Q—nhosnhoglycoric acid. 91 unawpanpcuocoo one pd c nvnm D d .2260 ny...muk._ my .2 «soled e‘ d. o. xfi l _I e \ s. Texan a\\\‘n. .eeemoaeea wow cannon an cowpfinfincfi onaaoco Ho poao xuomuna>mmzacaq om ahsmfih oo— no “N. d A A A °U.IUI/ 00 v x7. .5 — 32 velocities versus inhibitor concentrations (Fig. 6) for the various substrate concentrations do not yield straight lines. This indicates that the inhibition is partially competitive and partially non-competitive (Dixon and Webb, 1958). Effect of Sorbic Acid on Enolase From Lactic Acid Bacteria The effect of sorbic acid on fermentation by intact yeast cells was compared to its effect on fermentation by intact cells of L, plantarum. The conventional Whrburg technique was used with glucose as the substrate. At pH 6.2 the yeast was significantly inhibited by 6.0 x 10"3 M sorbic acid, but 5, plantarum_was not affected by this con- centration of sorbic acid (Fig. 7). Since the lactic fermentation by intact cells of L. plantarum was not inhibited by sorbic acid, the question arose as to whether the sorbate was getting to the site of action. in order to eliminate the permeability barrier acetone dried cells were prepared as described in Materials and Methods. With glucose as substrate, the production of lactic acid was measured in the Warburg from a bicarbonate buffer pH 6.2. As can be seen from Fig. 8, sorbic acid at a concentration of 6.0 x 10"3 M had no effect on the lactic acid production of the acetone dried preparation of L. plantarum. uni—I- R3 901' o 5xI0’5M 80% 7o» 60-» \ 50+ XZxIO‘4M It H 5 404 E \ s 8 <1 30'" O o/ 4xl0‘4M 20v- '().» C) O 1.0 1.5 2.0 2.5 Sorbate x 10‘4M Figure 0. Slot of reciprocal velocities versus inhibitor concentr~tions with the substrwte levels i.r‘(]-icq-+I(\(1 . 1!! of 602 34 1701' 0—0 Yeast control / 150+ x—x 6x|0"3 M sorbic acid o O--0 Lactic control 1304L x--x 6xl0'3 M sorbic acid a I )0“ so x x’ 1 ’Q/ // 70v ’9 / 9/ 50v / 304» I011 5 IO 15 20 25 30 Time in min. Figure 7. Rf”ert of sorbic acid on glucose fermentation by ingnct crlls of yrnst and g. plantarum. The cups contained the Followinq mixture: 0.5 m1 of 0.1 H qincose, 0.? ml of 0.093 T NnHCOP, 30 me of cells. 35 0—0 Lactic control 804) x—x 6xI0‘3 M sorbic acid 70-1.- 01 C) *0 J» C) o\ / o/ [I’X X x / /9 3 4 5 6 Time in min. Uffnrf of sorbic HCid 0“ ““01”S° ”CtiVity _ ZAOimL m; turn ponl-inid: 9 x 10 H g—nhosnhoq;yceric x liY-3fd imiflnznie bufFiW'ifi1'7.A, 8 X 10-. M [u4b 39 To obtain a more reliable measure of the effect by high sorbate concentrations on the enolase activity in the cell-free extracts, they were assayed by determination of the acid labile phosphate produced on conversion of 2-phos- phoglyceric acid to 2-phosphoenolpyruvate. .A concentration of s x 10’3 M sorbic acid inhibited the activity of the enolase in the crude extract of yeast by over 50%, but failed to affect the reaction rate observed with the extract of E. plantarum cells (Table 3). TABLE 3 THE hFFECT OF SORBIC ACID CN ENOLASE ACTIVITY OF YEAST AND L. PLANTARU'i CELL-FREE EATRACTS AS MEASUREUHEYFACID LABILE PHOSPHATE Acid Labile P Minus Preparationa on at 660 mu endogenous Yeast extract endogenous .035 ‘— ~- Yeast extract control .080 .0h5 Yeast extract + sorbic (S x 10-3 M) .055 .020 Lactic extract endogenous .OhO -- Lactic extract control .085 .OhS Lactic extract + sorbic (5 x 10-3 M) .085 .ohs 8The reaction mixture contained the following: extract 1.0 ml, 8 x 10'” M MgSOu, 5 x 10-2 M imidazole buffer (pH 7.h), l x 10-3 M substrate 2-phosphoglyceric acid, and water to a total volume of 3.0 ml. The reaction was started by the addition of substrate and stopped at the end of 3 min by the addition of 3 mi of 10% trichloroacetic acid. After hydrolysis of the acid labile phosphate the total phosphate was measured by the procedure of Fiske and SubbaRow (1925) modified as described in Materials and Methods. The color formed was measured at 660 mu. hO The enolase from L, plantarum was partially purified by organic solvent fractionation and passage through a phos- phorylated cellulose column, and then tested for sensitivity to sorbic acid. Fig. ll shows that it was inhibited by 1.0 x 10’“ M sorbic acid, 7.0 x lO'h'M crotonic acid and 3.0 x 10-h M cinnamic acid. The order of effectiveness of these acids was the same as observed with the yeast enolase; sorbic being the most effective followed by cinnamic and then crotonic. These data indicate the presence of a fac- tor(s) in extracts of L. plantarum_which protect the enolase idwuiinhibition by alpha, beta-unsaturated acids. Thus, a search was initiated for such factors. Protection of Enolase in Extracts of Lactic Acid Bacteria from Sorbic.Acid inhibition Sorbic acid has been reported to combine with free sulfhydryl (SH) groups (Morgan and Friedmann, 1938). This suggested that an abundance of free SH groups might be re- sponsible for the protection of the lactic extract. There- fore, the free SH levels in extracts from both yeast and L, plantarum cells were analyzed for free SH by use of the p- chloromercuribenzoate method. The results indicated that the yeast extract contained 1.2 microequivalents per mg of protein, and the Lactobacillus extract contained 1.1 00 at 240 mp l8“ 0 .l6‘ .l4'* .l2‘ .lO'l .08‘ .064 .041 41 Figure 11. cinnamic purified From extracts of E. "s in Fig. mixture acids on Time in min. The effect of sorbic, crotonic, and "ctivitv of enolase nnrtinlly 10. plantarum. Reaction , L 0 Control x l.0x|0‘4Msorbic acid 0 A/ A 7.0xl0'4M crotonic acid ' 0 3.0xl0“4M cinnamic acid A / 0 t G _ a / x o / r- A:/ . o A L G) 0 x A + Q . x . l 2 3 4 5 6 7 II Liz microequivalents per mg of protein. While these values are believed to be unrealistically high due to impurities in the glutathione standard, they are satisfactory for compara- tive purposes. Certainly, the extract from the lactic acid bacterium did not contain high levels of free SH as compared to the yeast extract. The addition of either cysteine or crystalline alcohol dehydrogenase did not protect yeast enolase from sorbic acid inhibition; in fact high levels of cysteine were inhibitory. Therefore, the protection of the enolase in the extract of the lactic acid bacteria from sorbate did not seem to be due to an unusually high concen- tration of free SH groups. Attempts were made to demonstrate protection of puri- fied yeast enolase by addition of crude extracts from cells of L, plantarum but without success. When the crude extract of E, plantarum was added to pure yeast enolase in the pre- sence of sorbic acid it was found to be additive to the inhibition of sorbic acid, and the bacterial extract by it- self was inhibitory to the yeast enolase. This is probably due to free SH groups since iodoacetate reduced the inhibi- tion. The protection of the enolase is not due to non- Specific protein since the addition of albumin had no effect. Next an attempt was made to find a protective factor by adding back various fractions from a Mg++ activated phos- phorylated cellulose column to a fraction containing sorbic acid sensitive enolase. The enolase in the bacterial 43 extracts was separated from its protective factors by a direct passage of the crude extract through the phosphory- lated cellulose column. Then the other fractions were checked for protective activity by addition back to the un- protected enolase. No protection could be demonstrated by any of the fractims from the column. Either the material(s) reSponsible for the protection did not come through the column, were too dilute in the eluates, or the process is irreversible. The above work led to a different approach to the fractionation of the extracts. The crude extract was sub- Jected to starch block electrophoresis, various eluates tested ftr enolase activity, and active fractions tested for sensitivity to sorbic acid. This technique yielded both sorbic acid sensitive and protected enzyme fractions (Table h). Since the lactic acid bacteria in general are rela- tively insensitive to sorbic acid and since high lactic de- hydrogenase activities are common to this group of organisms, the possibility of this enzyme protecting the enolase system was considered. This experiment offered some support of this hypothesis. Thus, the fraction in tube 9 (Table h) was protected from inhibition and also contained the largest amount of lactic dehydrogenase activity. Also, the addition of the contents of tube 9 to the contents of tube 8 resulted nu TABLE. h SEPARATION OF ENOLASE 1N g. PLANTARLM EXTRACTS FRCM THE PROTECTIVE PW)! STARCH BLOCK ELECTROPHORESIS Lactic Tube Enolase dehydrogengse Inhibifiion by Number activity activity 1.0 x 10 M sorbic 1 002w A 003m 1 --- --- --- 2 --- --- --- 3 _-- --- --- l. _-_ --- --- S .005/min --- yes 6 .eso/min .OSO/min yes 7 .OSO/min .060/min yes 8 .020/min .lOO/min yes 9 .020/min .ZSO/min no 10 --- .080/min --- ll --- .OSO/min --- 12 ~-- --- --- 13 --- --- --- 1h --— --- --- aThe tubes contained 5 x 10'2 M imidazole buffer PH 7.h, and the protein in that particular fraction. bEnolase activity wasflassayed by taking 0.5 ml of each fraction, adding 8 x 10'“ M MgSOh, 2-phosphoglyceric acid as substrate and measuring the increase in OD at 2&0 mu. CLactic dehydrogenase was assayed with pyruvate as substrate, and measuring DPNH oxidation at 3u0 mu. MS in the protection of the enolase activity in tube 8 from inhibition by sorbic acid. Fractions of crude extracts of E, plantarum cells in which enolase was inhibited by sorbic acid and other frac- tions in which no inhibition was evident were also obtained by fractionation with ammonium sulfate (Table 5). The fractions with the highest levels of lactic dehydrogenase were those in which the enolase was protected from sorbic acid inhibition. However, the specific activity of enolase was quite low in the protected fractions. Finally the effect of purified lactic dehydrogenase (obtained from General Biochemicals Corporation, Chagrin Falls, Ohio, from muscle) was tested on the inhibition of enolase from L, plantarum by sorbic. 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