‘ a... m 5 R n a. f... 8, E.. cw: . ma. mum up. 8 AU. 1.5. PC. nu.- ”up“ .8 ... m. . .. . . wk": Q“. 1“». I t ‘c. ‘ ..:. 3.. {u A a a...» EON £3»? " 633:; 3 9‘99?” a? $25.. 5, is? A WW: ”on” an” "be. Gum" its. a‘ ‘ n ,. MU M.” 3m“ .5 at. .a .. m. 1 AU. IL pan 9“ w; - h ‘FERET" F . . G si H T Ri- ' P: c. ‘ 0‘6 a“: ”,P Mp-. _: i=2:223;:32;;E mm arias-we. 62/ EAST LANblNu, MICHIGAN HICAN STATE UNIVERSETY | '3’: A l‘";\ ‘ )’\Ju\‘.‘\ ISOLATION, PURIFICATION, AND PARTIAL CHARACTERIZATION OF SEVERAL PLANT PHOSPHOMONOESTERASES By RUTH MARIE ALLEN A THESIS Submitted to the College of Science and Arts of Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Chemistry 1961 ACKNOWLEDGMENT The author wishes to express her deep gratitude and profound indebtedness to Dr. Gordon L. Kilgour for assistance and guidance throughout the course of this work. This work has been supported by a National Insti- tute of Health research grant (HG-5517). To Dalton and Marie TABLE OF CONTENTS Introduction. . . . . . . . . . . . . . . . Historical. . . . . . . . . . . . . . . . . Results and Discussion. . . . . . . . . . . Ammonium Sulfate Fractionations. . . . DEAE—Cellulose Column Separations. . . A. Sunflower Column I. . B. Sunflower Column II . C. Wheat Bran Column I . I D. Wheat Bran Column II. . EXperimental. . . . . . . . . . . . . . . . Reagents . . . . . . . . . . . . . . . Preparation of Crude Tissue Extracts . Routine Phosphatase Assay Conditions . Enzyme Purifications . . . . . . . . . A. Ammonium Sulfate Fractionations . . . . B. DEAE-Cellulose Column Separations . . Sunflower Column I . Sunflower Column II. Wheat Bran Column I. Wheat Bran Column II Characterization of the Various Enzyme A. Metal Ion Studies . . B. Temperature Optima. . C. pH Optima . . . . . . D. Phytase Studies . . . Bibliography. . . . . . . . . . . . . . . . Fractions Page . 15 l5 17 23 29 31 31 32 32 33 33 33 LIST OF TABLES Table Page I Summary of Some Previous Work on Phosphatases. . 5 II Ammonium Sulfate Fractionation of Sunflower EXtraCts O O O O O O O O O O O O O O O 0 O O O 0 14 III Refractionation of O to 30% Ammonium Sulfate Precipitate. . . . . . . . . . . . . . . . . . . 14 IV A Comparison of the Activity of Dialyzed and Undialyzed Ammonium Sulfate Fractions of Sun- flower Seedling Extracts . . . . . . . . . . . . 15 V Metal Ion Studies on the Peak Tubes from Sun- flower Column I. . . . . . . . . . . . . . . . . 17 VI Metal Ion Studies of Peak Tubes from Sunflower COlumn II. o o o o o o o o o o o o o o o o o o o 19 VII Metal Ion Studies on Fractions from Wheat Bran COlumn I o o o o o o o o o o o o o o o o o o o o 23 LIST OF FIGURES Figure Page I Separation of Phosphatase Activity from Sun- flower Seedlings on DEAE-Cellulose: Column I. 16 II Separation of Phosphatase Activity from Sun- flower Seedlings on DEAE-Cellulose: Column II 18 III Influence of Temperature on Activity of Phos- phatases Obtained from Sunflower Column II . . 20 IV Influence of pH on Activity of Phosphatases Obtained from Sunflower Column II. . . . . . . 21 V Separation of Phosphatase Activity from Wheat Bran on DEAE-Cellulose: Column I. . . . . . . 22 VI Separation of Phosphatase Activity from Wheat Bran on DEAE—Cellulose: Column II . . . . . .. 24 INTRODUCTION Phosphatases are enzymes which act on a variety of phosphate esters liberating inorganic phosphate. They are important in glucose metabolism, in bone formation, and a variety of disease conditions. Serum phosphatase levels in relation to cancer have been studied, in addition to the serum phosphatase assays routinely carried out to determine extent of liver or heart damage. Precise and definitive studies on the phosphatases are rare and this type of work on this class of enzymes is only beginning to develop. The substrates of this group of enzyme are all esters of orthOphosphate. Among the substrates which have been used for assay of phosphatase activity are sodium 0(- glycerOphosphate and sodium p-glycerophosphate, p-nitro- phenyl phosphate, o-carboxyphenyl phosphate, phenyl phos- phate, and a(-napthyl phosphate. One of the greatest problems in work on the phos- phatases has been that of attempting to classify the various types of phosphatase activity that have been found. One attempt at classification has been based upon specificity for'substrate. The phosphatases have been classified as 1) general, that is acting on several substrates or 2) Specific, acting on one compound or series of compounds only. However, since the specificities of many of the en- zymes vary with the cofactor used in the assay of the en- zyme activity, this classification is not too precise. Another classification attempt has been based upon the pH optimum, but this can be carried out effectively only on highly purified enzyme preparations. There are in the literature many contradictory results arising from the use of preparations of a wide variety of purities. The majority of phosphatases of animal origin have pH optima in the alkaline range, while most of the phosphatases from plant sources have their optimum on the acid side. Michaelis constants and competitive and noncom- petitive inhibitors have also been used as a means of clas- sification. Roche (I) has classified phosphomonoesterases into four different classes: Group I or alkaline phospha- tases (found mostly in animals): inactivated after 30 minutes in an alkaline solution, pH optimum 9.2 to 9.6, activated by magnesium and inhibited by calcium, sulfhydryl compounds, cysteine, and fluoride ions. Group II or acid phosphatases (mainly occurring in plants): pH Optimum 5.2 to 5.6, inhibitors are the most distinguishing character- istic of this group. Oxalate ions, fluoride, and molybdate ions inhibit while amino acids, magnesium and other divalent cations have no effect. Group III (a rather poorly char- acterized group, found generally in animals, unstable in neutral solution): pH optima 3.4 to 4.2, inhibited by magnesium ions. Group IV (yeast phosphatase): pH optimum 5.2 to 6.2, activated by magnesium and manganese ions (un— like phosphatases of Group II). The Group IV phosphatases are not well characterized. Besides catalyzing the hydrolysis of the esters of phosphoric acid, alkaline phosphatases have been known to catalyze the synthesis of inorganic pyrophosphate from orthOphosphate as described by Roche (2). The purpose of this present work has been to iso- late, purify, and somewhat characterize some phosphatases from convenient plant sources. A number of distinct, dif- ferent acid phosphatases have been isolated from sunflower seedlings and wheat bran and have been partially character— ized with respect to metal ion requirements, pH specifici- ties, and temperature characteristics. The ability of these partially purified fractions from sunflowers and wheat bran to liberate inorganic phosphate from inositol polyphosphates has also been studied. HISTORICAL Much work has been done in this field by various workers who have approached this problem from many differ- ent angles. Some have considered the action of phosphatases with respect to substrate, such as °( or p glycerophos- phate, p-nitrOphenyl phosphate, and some of the phosphory- lated carbohydrates; some have considered inhibitors and activators as well as pH specificities and temperature re- quirements, while others have only attempted to purify par- tially this class of enzyme from a variety of sources, both plant and animal. Some of the work done in recent years is summarized in Table I. The references mentioned will be found in the Bibliography. In addition to these studies on the characteris- tics, requirements, and sources of phosphatases, other papers have been presented which deal with the mechanism of action of this class of enzymes. Harary (36) has sug- gested that phosphatases may also act as transferases as in the following set of reactions: ADP + glyceric acid- (1) ATP + 3 PGA Wer°kinase+ 1,3-diphosphate . _ _ acyl L . (2) glyceric acid 1,3 diphosphate phosphatase” 3 PGA + P1 hos ho l cerokinase (1)+(2) ATP acy phosp atase )- ADP + Pi HISTORICAL Much work has been done in this field by various workers who have approached this problem from many differ- ent angles. Some have considered the action of phosphatases with respect to substrate, such as °( or F3 glycerophos- phate, p-nitrophenyl phosphate, and some of the phosphory- lated carbohydrates; some have considered inhibitors and activators as well as pH specificities and temperature re- quirements, while others have only attempted to purify par- tially this class of enzyme from a variety of sources, both plant and animal. Some of the work done in recent years is summarized in Table I. The references mentioned will be found in the Bibliography. In addition to these studies on the characteris- tics, requirements, and sources of phosphatases, other papers have been presented which deal with the mechanism of action of this class of enzymes. Harary (36) has sug- gested that phosphatases may also act as transferases as in the following set of reactions: ADP + glyceric acid- (1) ATP + 3 PGA h°3 h° 1 °er°k1nase 1,3-diphosphate . _ _ acyl L . (2) glyceric acid 1,3 diphosphate phosphatase” 3 PGA + P1 hos ho l cerokinase (1)+(2) ATP acy phosp atase ADP + Pi HISTORICAL Much work has been done in this field by various workers who have approached this problem from many differ- erit angles. Some have considered the action of phosphatases with respect to substrate, such as °( or @ glycerOphos- Innate, p-nitrophenyl phosphate, and some of the phosphory- lsxted carbohydrates; some have considered inhibitors and axztivators as well as pH specificities and temperature re- qiiirements, while others have only attempted to purify par- tzially this class of enzyme from a variety of sources, both Iilant and animal. Some of the work done in recent years 1J3 summarized in Table I. The references mentioned will be found in the Bibliography. In addition to these studies on the characteris- 'tics, requirements, and sources of phosphatases, other papers have been presented which deal with the mechanism cif action of this class of enzymes. Harary (36) has sug- ggested that phosphatases may also act as transferases as in.the following set of reactions: ADP + glyceric acid- (1) ATP + 3 PGA hos ho l cerokinase 1,3-diphosphate . . _ _ acyl L . 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It has been pointed out that the acyl phosphatase will not hydrolyze ATP, ADP, or AMP, nor will it transfer a phosphate group to glucose or creatine. The mechanism of inhibition by oxalate has also been studied. There are three possible complexes which may be formed in the presence of enzyme, oxalate and inorganic phosphate; 1) An ES-complex, 2) An E-S-inorganic phosphate complex, and 3) An enzyme-oxalate complex. This mechanism does not appear to be supported by all of the available data (37). The mechanism of cleavage of the phosphate esters has also been studied (38). Studies with several different substrates has shown the cleavage to occur between the oxygen-phosphate bond rather than between the carbon-oxygen bond. This has been determined using oxygen-18 water and has been confirmed by the fact that no inversion of con- figuration takes place on phosphatase action (39). A num- ber of phosphatases of plant origin have been shown (40) to be able to transfer the phosphate group from phenyl phosphate or p-nitro phenyl phosphate to various alcohols; sugars and inositol did not act as acceptors in this system. Studies on some of the alkaline phosphatases (41) have shown that the optimum pH varies with the concentra- tion of substrate; the higher the concentration, the higher the pH. By dilution of the substrate, the pH Optimum can be reduced almost to neutrality, and it is felt that this is in some way related to the action of the metal ion which is bound to the enzyme. Chemical composition studies have also been car- ried out on some of the more highly purified phosphatases. Binkley (42) has reported glucose to be an active constitu- ent of the alkaline phosphatase of hog kidney. This enzyme was also reported to contain a pyrimidine moiety as part of its structure. Portmann (43) has reported that during purification of the alkaline phosphatases of rat, swine, and bovine intestine, the increase in specific activity of the enzyme was paralleled by an increase in hexoseamine content. When the proteins of human semen were separated by starch electrophoresis (44) 11 fractions were found, of which one consistently represented the acid phosphatase activity. This band was found between the F-alpha2 and albumin fractions. Highly purified preparations of alka- Iine phosphatase have been prepared from dog intestine and from ox kidney and attempts have been made to crystal- lize these (45). What was at one time believed to be crys- tals of enzyme was later shown to be crystals of magnesium phosphate bound to the enzyme; further attempts at purifi- cation of alkaline phosphatases have been claimed (46) when the crude enzyme is incubated with trypsin for several hours at 37° C. Electrophoretic studies of intestinal alkaline and emkiphosphatases have also been carried out by Harris and thl (47). They achieved some degree of purification by 12 repeated separations on starch-gel electrophoresis. As a result of electro-dialysis studies on intestine, kidney, and liver alkaline phosphatases (48) it is claimed that normal alkaline phosphatases contain three components: (I) the protein apoenzyme, (2) an organic dialyzable co- enzyme, (3) magnesium. It is claimed that the differences between the enzymes from various sources largely represent differences in binding between the apoenzyme and coenzyme involved as well as slight differences in the apoenzyme portions from different sources. A number of reviews are available having reference to the phosphatases: Altman and Dounce (49) have included many phosphatases in their review of non-oxidative and non-proteolytic enzymes. Akamatsu (50) has given special emphasis to inhibition constants, Michaelis-Menten values, The chemical structure of the and activation energies. Other phosphatase molecule has also been reviewed (51). reviews are those of Axelrod (52) and Burnham (53). RESULTS AND DISCUSSION In the work done in this paper, p-nitrophenyl phosphate has been used as the substrate for determining the activity of the enzyme fractions. Since this was the compound used in the majority of routine screening proced- ures, phosphatases which are not capable of catalyzing the hydrolysis of this compound would not be apparent. The reactions involved are: p-nitrophenyl phosphate £11m p—nitrophenol + H3PO4 p-nitrophenol EL} p-nitrophenoxide' + H2O It must also be noted that magnesium ion was used also in the routine screening procedures. Again some of the en- zymes may not have been detected due to inhibition of these enzymes by magnesium. However, when one of the sunflower separations was re-assayed using zinc as the metal ion, no activity in any of the fractions was detected. Ammonium Sulfate Frac tionations As a result of the ammonium sulfate fractiona- tions, there was little separation of activity found. The activity seemed to concentrate in the 0-33% fraction as concluded from Table II. 13 Weaning; .334 14 Table II .Anunonium Sulfate Fractionation of Sunflower Extracts (Acid) Enzyme Fraction Optical Density Units Ratio (Alkaline) 0-33% pH 9.6 .145 109 33-66% pH 5.0 .29 70 19.4 pH 9.6 .015 3.6 66-100% pH 5.C) .03 0 0 pH 9.6 .00 0 Note: A unit of activity is defined as that amount of en- zyme which causes an increase in optical density of 0.100 per hour at 35° C. It may be noted that most of the activity was found below 80% saturation which influenced further separa- tion attempts. Since the 0-33% fraction seemed to contain the most activity, it was refractionated into 0-10, 10-20, and 20-30% fractions, and found to contain the distribution of1uuts described in Table III. Table III Refractionation of 0 to 30% Ammonium Sulfate Precipitate Fraction 280m}: 260 111}: Protein Total Units Concentration Acid Alkaline 040% 1.25 1.48 8.37 mg./m1. 2520 540 10-20% .377 .400 280 mg./ml. 6300 3150 20-30% .540 .562 4.1 mg./ml. 4350 330 As a result of these data, a second series of fisfiflmmtion attempts was carried out on a new batch of seedling extrac t s . In Table IV dialyzed and undialyzed 15 fractions are compared for activity. extracts is described in the Experimental section. A Comparison of the Activity of Dialyzed and Undialyzed Ammonium Sulfate Fractions of Sunflower Seedling Extracts Fractnnz 280mp 260mP Protein % Nucleic Total Units Concentration Acid Acid Alka- line 0-10% .385 .475 2.35 mg./m1. 6.25 2272 288 0—10% D .137 .168 0.837 mg./ml. 6.12 1760 0 10-20% .357 .445 21.5 mg./m1. 6.50 5440 4640 10-20% D .610 .790 31.5 mg./ml. 7.60 4800 3840 20-30% .325 .405 19.5 mg./m1. 6.50 4480 2336 20-30% D 2.03 2.39 13.5 mg./m1. 5.37 4480 2336 30-1003 1.43 1.65 9.72 mg./m1. 5.15 4000 2944 30-100% D .620 .630 4.78 mg./ml. 3.65 4160 768 Table IV Preparation of the There was, however, still no satisfactory separa- tion of acid activity from alkaline activity. DEAE-Cellulose Column Separations A. Sunflower Column I .As Figure I shows, the sunflower seedlings, after being eluted from a column of DEAE-cellulose with an in- creasing concentration of tris buffer, pH 7.5, (see Experi- mental section), were separated into several active frac- tions, having as their peaks tubes 37, 45, and 53. As a 931111: of this work, a more extensive separation was car- ied out as described in the Experimental section. When different metal ions, other than magnesium, re used in the assays of the peak tubes, differing results 16 Absorbance 1.00 F .90 . M 3.50 r A 080 ’ .01 ~ 0.5 1.4 MM '70 " M M MIN .60 n ~ .50 - 42 A .01 M .40 P 1 L 030 . .20 1's .‘ E“. '10 " :r': “- .33. . 9. z. . 3': .3. J» """" u. '..............jr° ‘2 ; i ““0 v I l I‘7 I l T I O 10 20 3O 40 5O 60 70 80 90 100 110 Fraction Number Figure I. Separation of Phosphatase Activity from Sunflower Seedlings on DEAE-Cellulose: Column I Note: The dotted line represents absorbance at 280my. 17 were obtained depending upon the ion used. A typical re- sult is expressed in Table V. Table V Metal Ion Studies on the Peak Tubes from Sunflower Column I Opti 1 Dens y with Tube No. No Metal Mgfi Znn Mn“ Mg+++Zn++ 37 pH 5.0 0.00 0.097 0.000 0.192 0.07 37 pH 9.6 0.00 0.012 0.000 0.110 0.005 45 pH 5.0 0.032 0.160 0.010 0.210 not studied 45 pH 9.6 0.010 0.065 0.005 0.172 not studied 53 pH 5.0 0.375 0.480 0.030 0.500 0.060 53 pH 9.6 0.00 0.085 0.000 0.160 0.005 At this point it was decided to repeat the pro- cedure on a larger column in order to get larger amounts of the enzymes for more extensive characterization. B. Sunflower Column II The results of this second column are summarized in Figure II. Again the column was eluted with increasing concentrations of trisbuffer, pH 7.5, and the tubes were assayed at pH 5.0 according to the procedure described in the Experimental section. Again several peaks were found which will be designated as peak numbers 305, 440, 480, and 625. Metal ion studies were carried out on these peak fractions. A typical result is expressed in Table VI. 18 .iwomm pm womanhompm mpcmmopmma mafia coppoe one “0902 HH sadaoo "mmoasaaoo Imapo< mmwpwnmmonm mo soapmhmmmm .HH pmnssz :oapomnm oov com com OOH madmam l 3. . A-.. 1 00¢. oow. oom.H oooé eoueqxosqv 19 Table VI Meufl.Ion Studies of Peak Tubes from Sunflower Column II Optical Density with Peak No. EDTA Mg” Zn++ Mn” Mg+++Zn++ 305 0.025 0.250 0.115 0.175 0.050 440 0.86 0.84 0.31 0.85 0.32 480 0.02 0.24 0.00 0.19 0.00 625 0.38 0.44 0.175 0.435 0.155 From these results it is probable that tubes 440 and 625 are the same enzyme, while tubes 305 and 480 contain two other distinct enzymes. Thus further studies involving temperature characteristics and pH specificities were undertaken. The results of this work are summarized in Figures III and IV respectively. These provide addi- tional support for the conclusion that tubes 440 and 625 represent the same enzyme while the other peaks represent different enzymes. C. Wheat Bran Column I Similar studies were carried out on another source of enzyme, this time one which is known to contain phytase activity; A commercial grade of wheat bran was extracted ‘with tris buffer as described in the Experimental section. The activity of these fractions was ascertained and the results are expressed in Figure V. Metal ion studies were again carried out on the two peak tubes, 32 and 38. Again a typical example of the results is given in Table VII. 20 .03» an cmofi>flc soon o>ws o>aso oee on» non mosamb mocmnaomnm one “0902 HH nasaoo Hosoaqum Scam cocamppo momwpmnmmozm Ho hpfl>wpo< so oMSPMAmQame mo mocmsaecH .HHH oazmwm manpmnmmaoe om o> 0m 0m oe Om om _ JT 4 4 A J as. OOH. CON. 2;. eoueqmosqv cow. 1 .1 1 1 .33 .1... ‘ 21 II .03» mp omcfi>fic noon o>m£ o>H50 oee map How mosam> womanhompm one ”opoz HH :EsHoo nosofimssw scam cocflmpno mommpmnmmonm mo hawbapod no mm 90 ooqosHMGH .>H ohsmwm mm 0.8 3. 0; me 08 Tm Rm 3. o... n I q u q q d d 2;. 3s . 02. . V G. 8 O J O. 1. oom. m 0 9 2;. .. oom. +03. 22 .Ar omm pm oozmnaomnm on» mpcmmoammn mafia wouvoc one Honadz soapomam 0m umpoz H mafiaoo HomOHsHHooumfipo< omwpmnmmonm mo soapmnmmom A. 4 2M. .2. cu. 00m. 00¢. 000. .> oasmflm eousqxosqv 1.11.104 1 23 Table VII Ifletnal Ion Studies on Fractions from Wheat Bran Column I Optical Density with Peak No . EDTA Mg++ Zn++ Mn++ Mg+++Zn++ '32 0.01 0.035 0.00 0.81 0.015 139 --- 0.685 0.205 0.92 0.190 One may suppose the existence of two separate enzyme fractions from this study with little certainty. Further work remains yet to be done in this respect. It should be noted that on the basis of Specific activities (enzyme units per mg. of protein) of the solu- tion put on the column and of tube 32, a 145 fold purifi- cation was achieved. Phytase studies, that is the ability of these enzyme fractions to catalyze the hydrolytic release of inorganic phosphate from phytic acid, have been undertaken but yield little conclusive evidence for the presence of this activity in either of the phosphatase peaks. One cannot be sure if the activity measured is actually phytase activity or merely experimental error in the readings for the activity measured is very low. IL Wheat Bran Column II A second attempt to fractionate a wheat bran extant has been carried out, but with poor results. As Figne VI indicates there is little separation of activity. ihisfractionation was carried out by means of a gradient elufion technique which did not give completely satisfactory remflts. More work remains to be done on this fractionation. 24 .AF 00m as womanhomnw macaw mafia emppoc one no?» hp coew>fie coop o>m£ m>pzo hpwbapow on» now mosaw> cosmnnompw one ”opoz HH maddoo "omoasaaooamwpo< ommpmnmmonm mo cowpmnwmom .H> oasmwm nonasz nofipownm on mm om ma 0a m a .4 ooa. / . +08. _ ooh. I | _ , Too... ’ \ (FIOOm. ,. eousqxosqv / A 1 1608. a... 00. com. 000.H 25 In summary, it may be concluded that three dis- tinct phosphatases have been separated after one pass on a column of DEAE cellulose and that these enzymes have very little, if any, phytase activity. Preliminary re— sults indicate at least two different phosphatases are present in the extracts of wheat bran. Phytase activity pg. of these enzymes, if present at all, is at a very low level. ;1 EXPERIMENTAL Reagents Sunflower seeds (Helianthusggiganteum), a product of the Olds Seed Co. graciously supplied by Dr. Dalton Allen If? Diethylamino ethyl cellulose from Eastman Organic Chemicals (abbreviated: DEAE) p-Nitrophenyl phosphate, sodium; (13 104 grade) from Sigma 7 i j Chemical 00. Wheat bran, commercial grade Molybdate Reagent 2 (A solution of ammonium molybdate in 3 normal sulfuric acid) from the Hartman-Leddon Company Vermiculite, Terra-Lite brand from the Zonolite Co. Tris (hydroxy methyl) aminomethane from the Sigma Chemical Co. under their trade name of Sigma 7-9 (abbrevi- ated: tris) p-Methyl amino phenol sulfate produced by the Eastman Kodak 00. under the trade name of Elon Preparation of Crude Tissue Extracts The sunflower seeds were soaked overnight in water, then planted in Vermiculite and harvested after time intervals varying from 3 to 7 days. The two inch 26 27 seedlings, after removal of the roots, were homogenized in 0.04 molar tris buffer, pH 7.5 for 2 minutes in a Waring Blendor and centrifuged for 30 minutes at 5° C at 40,000 RCF, filtered through glass wool (to yield a cloudy yellow solution) which was then further fractionated. If the seedlings were allowed to grow to a height of six inches the supernatant solution after centrifugation was a clear greenish yellow. The commercial wheat bran was extracted by ho- mogenizing a sample in 10 volumes of water in a Waring j 5 j Y" . blendor for five minutes, stirring the resulting material overnight with a magnetic stirrer, filtering through cheese— cloth and then through glass wool. The solution was then centrifuged at 8,000 RCF at 5° C to yield a clear, light yellow supernatant. This solution was then adjusted to 80% ammonium sulfate saturation and centrifuged for 10 minutes at 4,000 RCF. The resulting pellet was dissolved in less than 20 ml. of water and dialyzed overnight against water at 0° C. Routine Phosphatase Assay Conditions 3.0 ml. acetate buffer (pH 5.0, 0.1 molar) 1.0 ml. p-nitrophenyl phosphate, sodium salt (50 mg./100 ml.) 0.1 ml. MgCl2 (0.1 molar) 0.5 ml. enzyme solution Incubated the mixture for one hour at 35° C, then added 5.4 ml. NaOH (0.1 molar) to stop the reaction. 28 Read at 400mu using a Beckman Model B spectrophotometer. Note: For assay of alkaline phosphatases, 3.0 ml. acetate buffer was changed to 3.0 ml. of glycine buffer, pH 9.6. Enzyme Purifications FE A. Ammonium Sulfate Fractionations 51" The solution after centrifugation was adjusted 1 to 1/3, 2/3, and complete saturation with ammonium sulfate. 5 ‘_ Each of the precipitates was spun down at 3,000 RCF for éj 20 minutes at 5° C and the resulting pellets suspended in 0.005 molar phosphate buffer, pH 7.0 and frozen. The clear yellow supernatant solution from the 100% saturation procedure was assayed for both acid and alkaline activity and showed no measurable activity. Then each of the dis- solved precipitates as well as the 100% supernatant was dialyzed against 0.005 molar phosphate buffer, pH 7.0 at 5° C for sixty hours. Each of the solutions was then as- sayed for acid and alkaline activity by the standard assay method. The 0-33% saturation fraction seemed to be the ;most active fraction and contained only 4% nucleic acid. It was refractionated to give precipitates corresponding to 0-10%, 10-20%, and 20-30% saturation. Again the pre- cipitates were suspended in 0.005 molar buffer and dialyzed and.stored in the refrigerator. Each fraction was then .read at 280 and 260mu and a standard assay for phosphatase 29 activity was run. Another preparation was carried out and the 0-10%, 10-20% and 20-33% fractions were split into two portions, one of which was dialyzed and the other was not. Each of the eight fractions as well as the original solu- tion was read at 280 and 260m». Again each of these frac- tions was checked for acid and alkaline activity by the standard assay procedures. B. DEAE-Cellulose Column Separations Sunflower Column I An extract of six-inch sunflower seedlings was prepared as outlined above. Ammonium sulfate was added to 70% saturation and the precipitate was collected by centrifugation for 20 minutes at 9750 RCF at 0° C. The pellet was dissolved in distilled water and after dialysis against running distilled water overnight, the solution was read at 280 and 260mp (after a 1:500 dilution): 280 .280 . 260%fi = 7555 = 0.97 or 106 mg. protein/ml. (The values for protein concentration were obtained from tables by Warburg and Christian (54).) Approximately 50 mg. (3 ml.) was put onto a 12 mm. diameter column containing 1.25 g. DEAE, and eluted with a discontinuous concentration gradient of tris buffer (pH 7.5) according to the following program: . "‘—',='F m En‘mm—an‘numfl I ‘ a! he... _ room at 2° 10.0 mls.) was read at 280 and 260mp and every second tube was assayed according to the routine assay method for both P The entire procedure was carried out in the cold C. Each of the resulting fractions (approximately Eluent mls. mls. mls. mls. mls. mls. mls. mls. mls. water 0.01 0.02 0.04 0.08 0.20 0.50 1.00 1.00 molar molar molar molar molar molar molar molar acid and alkaline activity. 30 tris tris tris tris tris tris tris tris Tubes 1-9 10-17 18-30 31-42 43-49 50-82 83-93 94-98 (stopped overnight) 99-112 (pH 10.6) _ 1 b was. A modified procedure was used subsequently on the fractions eluted with the 0.50 molar, and the 1.0 molar tris buffers at pH 7.5. pH 10.6 buffer. as well as the fractions eluted with the Modified procedures: 0.50 molar 1.0 molar Incubated 2.75 mls. buffer (0.1 molar acetate pH 5.0) 0.25 mls. 0.5 1.0 H00“) OUIU'IU'I all 2 mls. NaOH (0.1 normal) mls. mls. mls. mls. mls. mls. HCl 1.0 molar enzyme solution p-nitrophenyl phosphate at zero time buffer (0.1 molar acetate pH 5.0) HCl 1.0 molar enzyme solution p-nitrophenyl phosphate at zero time tubes 5 hour at 35° C, then added to all tubes 3.5 mls. water to give a total volume of 10.0 mls. 31 Read on a Beckman Model B spectrOphotometer at 400mp. In view of the results of these assays, tubes 84, 88, 92, 96, 100, 106, and 109 were dialyzed against running distilled water overnight and re-assayed according to the routine procedure for both acid and alkaline activity. Sunflower Column II A second crop of sunflower seedlings was prepared 1.44 Therefore, the original solution contained 69 mg. protein/ml. and found to give gagqp = 1112 (for a 1:100 dilution). E 1 Approximately 15 mls. of solution (1 g.) were applied to j‘ a similar DEAE column which was then eluted with tris buffer L pH 7.5 of increasing concentration as follows: Eluent Tubes 1500 mls. 0.02 molar tris 1-129 (123 Let stand overnight) 1500 mls. 0.04 molar tris 130-288 (263 Let stand overnight) 2000 mls. 0.08 molar tris 289-508 (379 Let stand over weekend) (499 Let stand overnight) 1000 mls. 0.20 molar tris 509-610 (519, 587, 596 Let stand overnight) 1000 mls. 1.00 molar tris 611-706 The resulting fractions were assayed by taking a one m1. aliquot from each of five consecutive tubes and combining them. From this combined solution a one ml. sample was taken for determination of acid activity and protein con- centration. Wheat Bran Column I An extract from wheat bran was prepared by taking a portion of the filtered solution previously described and adding ammonium sulfate to 80% saturation. The result- ing precipitate was dialyzed against running distilled water 32 at 5° C overnight. A 3 ml. aliquot of the resulting solu- tion was diluted to 50 mls. and read at 280m _ .104 - . . 53535 — 75§7 - 1.20 Therefore, there is 98 mg. protein/ml. One-half ml. of the dialysate was then put on a 12 mm. diameter column containing 0.5 gm. DEAE which had been prewashed with 0.1 molar tris, pH 7.5, to neutral effluent g which required about 250 ml. The column was then eluted Ti with tris buffer pH 7.5 according to the following scheme: Eluent Tubes l I 10 mls. water 0-1 1,] 50 mls. tris 0.01 molar 2-10 a. 100 mls. tris 0.02 molar 11-23 50 mls. tris 0.04 molar 24-29 50 mls. tris 0.08 molar 30-35 150 mls. tris 0.2 molar 36-50 Assays were run on the fractions for both acid and alka- line activity and protein concentration. Wheat Brgn Column 11 A second identical column was prepared through which was passed 10 mls. of the dialysate (equivalent to 60 mg. of protein). The column was then eluted with tris buffer pH 7.5 by the gradient elution technique starting with 125 mls. of water in the single mixing flask and add- ing 0.5 molar buffer. Each fraction was assayed for acid phosphatase activity as well as protein concentration. Characterization of the Various Enzyme Fractions A. Metal Ion Studies Assays were run on some of the peak tubes from 33 the Sunflower Column II fractions to see if any two or more of the peaks represented identical enzymes. Five of the tubes near and including the peak tube were combined and used as the enzyme source. Assays were then run according to the routine assay procedure except that various metals were substituted for magnesium, such as the chlorides of zinc, manganese, and the disodium salt of ethylene diamine tetraacetic acid (EDTA). Any precipitates that were formed were Spun down before the tubes were read. Similar experiments were carried out on the peak tubes from the wheat bran fractionations. B. Temperature Optima Studies on the temperature optima of several peaks from the second fractionation of sunflower seedling extracts were run. Routine assay procedure was followed except that instead of incubation at 35° C for one hour, assays were run for one-half hour at 20°, 30°, 40°, 50°, 55°, 60°, 65°. and 70° C. C. ppH Optima Studies on the pH optima of four of the fractions from the second fractionation of sunflower seedlings were also undertaken. Buffers at each 0.5 pH unit were used from 4.0 to 9.0 in the routine assay scheme. The buffers, each 0.1 molar, were succinate from pH 4.0 to 7.0, and tris from pH 7.5 to 9.0. D. Phytase Studies A further attempt at characterization was also _ 1! .._ L -K;n-"-m 34 made by studying the phytase activity (that is the ability of the enzyme fractions to split phosphoric acid from phy- tic acid). The assay (a determination of inorganic phos- phate) was run as follows: 3.8 mls. acetate buffer (0.1 molar, pH 5.0) 0.4 mls. phytic acid solution 0.1 mls. MgCl2 (0.1 molar) 0.2 mls. water 0.5 mls. enzyme sample An aliquot of 1.0 ml. was taken at zero time and at one hour. These samples were immediately added to tubes containing 03 7 mls. of water and 1 ml. of Molybdate Reagent 2. Color was produced upon the addition of 1 m1. of bisulfite-Elon reducing solution (1% Elon-3% sodium bisulfite) and allowed to develop for 20 minutes before being read at 660mm in the Beckman Model B spectrophotometer. The readings were referred to a standard curve for determination of amount of phosphate released. These assays-were run on the solu- tion prepared for addition to Sunflower Column II as well as both the wheat bran preparations. Phytase assays were also run on every fifth tube from the first wheat bran fractionation. The phytic acid used in the assay was prepared by adding a sample of barium phytate to IR 120 (H). The resin removed the barium giving soluble phytic acid. The solution was then assayed for total phosphate and inorganic phosphate according to the following procedure (which is 35 described in Carter, "Experimental Biochemistry", Second Reprint, page 66 (55): Totalgphosphate: Two mls. of the sample and 0.5 mls. of 10 normal sulfuric acid are heated to l30°-l60° C for thirty minutes and cooled. Then 2 drOps of 30% H202 are added and the mixture is again heated to l30°-160° C for 15 minutes. After cooling, 2 mls. of water are added and the resulting solu- tion is heated to boiling in a water bath for 10 minutes and cooled. The assay for inorganic phosphate can now be run on this solution and the amount of total phosphate determined by com- parison to a standard curve. Inorganic‘phosphate: Run assay according to above procedure. The difference between total phosphate and inorganic phos- phate can be used as a measure of the concentration of phytic acid in the sample. A!“ p I. l. 11. 12. 13. 14. 15. 16. BIBLIOGRAPHY J. Roche, in "The Enzymes 1," part 1, 473 (J. B. Sum- ner, and Myrbéck, Academic Press, Inc., New York, New York, 1361 pp., 1951). J. Roche, Nguyen-van-Thoai, and E. Danzas, Bull. soc. chim. biol., 21, 599 (1945). HE J. E. Courtois, C. Anagnostopoulos, and M. Khorsand, “11 Bull. soc. chim. biol., 33, 1813 (1951). G. R. Nakamura, and E. L. Becker, Arch. Biochem. Bio- PhyS-o 219 78 (1951)- J. Courtois, and M. Khorsand, Biochem. et Biophys. Acta, 6, 175 (1950). B. K. Joyce, and S. Grisolia, J. Biol. Chem., 235, 2278 (1960). D. W. A. Roberts, J. Biol. Chem., 212, 711 (1956). M. Gibbs, J. M. Earl, and Ritchie, J. Plant Physiol., 29’ 463 (1955). W. S. Pierpont, Biochem. J., 65, 67 (1957). D. H. Turner, and J. F. Turner, Biochem. J., 14, 486 (1960). M. Andreau, et al., Anales real soc. espan. fis. y quim., 2§_. 67—TI960)- t} E. I. Ratner, and S. A. Samoilova, Fiziol. RasteniI, ‘2, No. 1, 30 (1955). E. B. Brown, Jr., and E. R. Stadtman, J. Biol. Chem., 235, 2928 (1960). S. Belfanti, A. Contardi, and A. Ercoli, Biochem. J., 22’ 842 (1935). K. K. Tsuboi, Biochem. Biophys. Acta, Q, 173 (1952). o. Bodansky, J. Biol. Chem., 165, 605 (1946). 36 37 17. S. Belfanti, A. Contardi, and A. Ercoli, Biochem. J., 29, 517 (1935). 18. N. Tomlinson, and R. A. J. Warren, Can. J. Biochem. Physiol., 38, 605 (1960). 19. N. Tomlinson, Can. J. Biochem. Physiol., pg, 633 (1958). 20. N. Tomlinson, Can. J. Biochem. Physiol., 31, 945 (1959). 21. A. R. Armstrong, Biochem. J., 29, 2020 (1935). 22. Nguyen-van-Thoai, J. Roche, and M. Roger, Biochim. et BiOphys. Acta, 1, 61 (1947). 23. P. Portmann, R. Rossier, and H. Chardonnens, Helv. Physiol. et Pharmacol. Acta, 18, 414 (1960). 24. C. A. Zittle, and E. W. Bingham, Arch. Biochem. Bio- phys-..§é. 25 (1960)- 25. G. Schmidt, and S. J. Tannhauser, J. Biol. Chem., 149, 369 (1943). 26. E. V. McCollum, and E. B. Hart, J. Bio. Chem., 4, 497 (1908). 27. V. Sadasivan, Nature, 170, 421 (1952). 28. 0. Bodansky, J. Biol. Chem., 174, 465 (1948). 29. M. Martland, and R. Robison, Biochem. J., 21, 665 (1927). 30. L. Raijman, S. Grisolia, and H. Edelhoch, J. Biol. Chem., 235, 2340 (1960). 31. S. Grisolia, J. Carovaca, and B. K. Joyce, Biochim. et BiOphys. Acta,.ga, 432 (1958). 32. Z. F. Ahmed, and E. J. King, Biochim. et Biophys. Acta, 49, 320 (1960). 33. K. K. Tsuboi, and P. B. Hudson, Arch. Biochem. and Biophys...£2. 339 (1953). 34. W. N. Valentine, K. R. Tanaka, and R. E. Fredericks, J. Lab. Clin. Med., 25, 303 (1960). 35- S- P- Colowick. and N. Kaplan, "Methods in Enzymology," Academic Press, Inc., New York, New York, vol. II, 523 1955-7 . 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49- 50. 51. 52. 53. S40 55. 38 1. Harary, Biochim. et Biophys. Acta, 16, 434 (1957). S. Belfanti, A. Contardi, and A. Ercoli, Biochem. J., .29. 1491 (1935)- S. S. Stein, and D. E. Koshland, Jr., Arch. Biochem. Biophys., 29, 229 (1952). M. Dixon, and E. C. Webb, "Enzymes," Academic Press, New York, New York, 333 (1958). C. Anagnostopoulos, J. E. Courtois, and J. Servant, Bull. soc. chim. biol., 26, 1581 (1954). E F. Cacioppo, E. Quagliariello, M. Coltori, and G. D. : Pietra, Arch. sci. biol., 21, 563 (1953). F. Binkley, J. Am. Chem. Soc., 81, 1507 (1960). P. Portmann, Z. Physiol. Chem., 292, 87 (1957). H B. Estborn, and B. Swedin, Scand. J. Clin. and Lab. Invest., 11, 235 (1959). M. A. M. Abdul-Fadl, E. J. King, J. Roche, and Nguyen- van-Thoai, Biochem. J.,‘ii, 428 (1949). A M. A. M. Abdul-Fadl, and E. J. King, Biochem. J., 11, 434 (1949). E. S. Harris, and J. W. Mehl, Proc. Soc. Exptl. Biol. Med., 99, 521 (1955). . M. A. NI. AdeIl-Fadl , and E. J. King, BiOChemo Jo ’ K. I. Altman, and A. L. Dounce, Ann. Rev. Biochem., 11, 29 (1952). S. Akamatsu, J. Japan Biochem. Soc.,‘12, 93 (1951). M. L. Tamayo, Bull. soc. chim. biol., 8, 983 (1956). B. Axelrod, Ann. Rev. Biochem., 11, 45 (1955). S. W. Burnham, H. M. Lemon, M. M. Davison, and M. K. Schwartz, Am. J. Clin. Pathol., 11, 807 (1954). 0. Warburg, and W. Christian, Biochem. Z., 310, 384 (1942). H. E. Carter, "Experimental Biochemistry," Second reprint, Stipes Publishing Co., Champaign, Illinois, P. 66 (1959). ||Hlllillllll(IllMHWIIHHHII|||||||||ll|||||H||H|H(I 31293 03037 972