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The enzymic hydrolysis of phosphoric acid esters by barley extracts Brink, Vernon Cuthbert 1936

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THE EETZYMIC HYDROLYSIS OF PHOSPHORIC ACID ESTERS 'BY BARLEY EXTRACTS." "by Vernon C. Brink, BcS.A. (Brit ish ColumbiaJ Thesis Submitted i n Candidacy,, for the Degree, of Master of Science i n Agriculture. The University of Br i t i sh Columbia. September 1936 2 ACMOWLEDGEMEITS I express my gratitude to Dr. A. H. Hutchinson, Head of the Botany Department, for M s assistance and interest in the development of this problem. My sincere thanks are also extended to Mr. W. Jack and Mr. B. Hi l lary for valuable help in obtaining materials and apparatus. For taking discouragement from many an evening with her midnight luncheons, I am obliged to Miss Y. Mizuno. s TABLE OF CONTENTS. .1 . Experimental Work* Pg» 4 1. Introduction Pg» 4 2. Materials and Methods Pg. 7 5. Experimental Results Pg.21 4. Discussion Pg.36 .II . Discussions Pg.39 1. Introduction Pg.39 2. Enzymic Kinetics Pg.41 3. Activation and Inhibition Pg.45 4. Methods of Determining Orthophosphoric Acid".49 5. The Role of Phosphatases in Metabolism Pg«,5Q 6. Problems Pg.54 .III , Bibliography! Pg.57 EXPERIMENTAL WORK 4 DTTRODUCTIOII Phosphoric acid esters and the enzymes hydrolyzing them have assumed a very considerable importance, bath/ian applied and purely scienti f ic one, i n the study of mammal and yeast physiology. In the higher plants l i t t l e has been determined about their functions* Recognizing this , we commenced work on plant phosphatases choosing as our representative spermatophyte-barley. The occurrence and necessity of the element phosphorus in the l iv ing ce l l was established by de Saussure (1804), Sachs (1860) and other investigators early in the 19th century. The discovery of i t s essentiality to l i f e and i t s close association with carbohydrates, fats and proteins has led to the belief that i t s study would be a key to disclose much of the mechanism of l iv ing organisms. Progress in the study of phosphorus metabolism has, however, been conditioned by a lack of knowledge of the chemistry of the element and i t s compounds as they occur in organisms. In recent years this state has been altered. Kay [X] has reviewed our present knowledge of the phosphorus compounds of plants and animals. Phosphoric acid esters f i r s t assumed importance in physiology following the important discoveries of Harden and his associates (8) in 1905 on the general effect of the addition of sodium phosphate increasing the total fermentation produced by yeast juice on sugar. With these researches and the long series following the phosphorus metabolism of yeasts wa s intimately linked with the fermentation process through the esters of the mono- and di-saccharides. ^ 1 3 sufficient to say here that other phosphoric esters, the phospho-'lipides, phosphoproteins, meta-, ortho- and pyro-phosphates etc. have assumed an import in many metabolic processes. Prior to Harden's researches, Buchner (1897) had succeeded in isolating from l iv ing yeast zymase, which, when freed from the last t r a c e 0 f organized ce l l material was able to bring about identical fermentation processes as had before deemed to be possible only in the presence of active yeast ce l l s . Enzyme chemistry developed rapidly in the ensuing years and several observations appeared on enzymes effecting the cleavage of.organic phosphoric acied esters (e.g. C. leuberg and L. Earczag (2)). Their wide distribution i n the plant and animal kingdoms was soon indicated. Phosphatases and their substrates have now, in addition to their importance in fermentations, become significant in the study of muscle contraction, ossif ication, i n the work of the kidney and in many other processes in mammal physiology; In plants we know l i t t l e or nothing of their relation to metabolic processes. Conceivably they might be of importance in the transformation of sugars apart from the photosynthetic process,— in the germination of malting barley, in sugar storage by sugar beets. If in mammals they are intimately linked with the calcium compounds, might not the same be true in plants, Many questions might be asked. The writer while real izing the many problems presented by phosphatases and their substrates in plants has sought only to 6 -5-prepare the way for their future study. The work accomplished while not far reaching i s fundamental. Something has been gained about the preparation of phosphatase extracts, and something determined of their distribution through the development of the barley plant. 7 MATERIALS AND METHODS (a) The Plant Materials Seed of a pure line of Duckbill barley (a six-rowed variety of Hordeum vulgare L.) was obtained from the Department of Agronomy and sown, 4 seeds in each, in 8" clay pots in phosphorus free sand. One series received weekly Hoagland's complete nutrient solution and a second series received a similar solution in which potassium dihydrogen phosphate was replaced by potassium chloride.' The complete and phosphorus deficient solutions were made up as follows! ) ) in 1 L*aq. d is t . ) in 1 L.aq. d i s t . i n 1 L.aq. d i s t . i n 1 L.aq. d is t . 22 c . c . , 26 c .c . and 12 c .c . of reserve solutions 1, 2 and 3 respectively added to sufficient aq. d is t . to make 2 1. gave the phosphorus deficient nutrient solution. Since the water requirements of the plants at different stages of growth varied so greatly and since the object in view was only to obtain normal and phosphorus deficient plants, fixed quantities of solution were not given the pots after the f i r s t few weeks. Bather, the amoTint required became a matter of judgement to see that the sand was uniformly moist in a l l the pots. D i s t i l l ed water likewise was given as required. Reserve Solution 1. KbI03 67 g. MQS04 100 g. »* ' " 2 . Ca(N03)2.4HgO 208 g. » " 3. KHgP04 50 g. " " 4. KCh. 26 g. 8 -2-Firs t sowings were made November 2, 1935 and similar series 'were set up during the experimental period (until March 15, 1936^  every three weeks. The photoperiodic effects on the barley were very marked. Plants sown November 2, 1955, for example, came into head but a few days earlier than plants sown the second week i n January. As a result where enzyme act ivi t ies at different growth stages were to be compared, plants of a single sowing were used. Plants of the second series showed marked phosphorus deficiency within five weeks. At eight weeks the deficiency was very pronounced with typical reddening of the foliage (from the leaf tips and edges back) with eventual necrosis of the lower leaves and a low dry weight * " The photo belov/ indicates the difference between plants of the two series; Plants receiving phosphate weekly grew rapidly and were highly succulent as a result no doubt of the combination of the high green-house temperatures and short photoperiods. Such a condition was favorable for aphis attacks for which pests the plants had to be continually sprayed. Normal and deficient plants came into head about the same date. Accordingly, i t was assumed that on a given date such plants of the same sowing were at the same developmental stage. When germinating seed was required, dormant seed was placed between blotting paper in a dark room at 50° + 3° CT f o r five flays. (b) Materials and Methods Required for Enzyme Action i n v i t r o : Phosphatases effect the cleavage of phosphoric acid esters l iberating free phosphoric acid. To determine their presence and act iv i ty the usual conditions for enzyme action in vitro must be set up. To a substrate (some given phosphoric acid ester] in a solution buffered to a pH. suitable for reaction and set at a desirable temperature, is added a colloidal solution containing the enzyme previously extracted from the l iv ing plant tissue. At the end of a known period the phosphoric acid liberated is determined, assuming the amount of acid freed i n unit time is indicative of the act ivity of the phosphatase. In greater detail our procedure w.as as followss To 8 c .c . of phthalate, veronal, or glycine buffer, a trace of 10 toluene and 1 c .c . of Z% substrate in a test tube was added 1 c .c . "of enzyme extract (about 6 mgs. of dried extract) and the reaction set at the desired temperature. At the end of a definite period, usually 1 hour, 1 c .c . of trichloacetic acid was added, the mixture centrifuged and the liberated phosphoric acid determined by the Zing ( 5 colorimetric method. Series were run in duplicate. After the conditions for enzyme action in vitro were set up and a reaction liberating phosphoric acid shown, i t was necessary to show that the action is enzymic and not an ordinary catalyzed chemical reaction. Certain characteristics of enzyme preparation and their action are used to determine the difference. In brief some of those are: (1) the thermal ins tabi l i ty of enzyme i enzymes are inactivated at certain temperatures. (2) their sensit ivity to 0(H) and C(OH). (5) their activation and inhibit ion by certain substances. (4) their colloidal and protein nature as exhibited in extracts. (5) their ab i l i ty to be specif ical ly absorbed, etc. (6) their sensit ivity to maceration, etc. In addition to demonstrating the enzymic nature of a reaction, i t i s sometimes desirable, though d i f f icu l t of proof, to establish the specificity of the enzyme involved—i.e. whether more than one enzyme was acting on the substrate and i f one, whether i t i s capable of acting on other substrates. This is pertinent to phosphatase investigations for i t is not known just how many different phosphatases act on phxosphoric esters. Again, an enzymic estract might conceivably act on a substrate in different ways—e.g. barley extract on a hexose •ester. In this case glucolysis i . e . enzymic degradation of the hexose unit to lact ic acid might liberate free phosphate, or, on the other had a true phosphatase might act yielding free phosphate but leaving the hexose unit intact . Which i s true may be determined by following the quantitative relations of the reaction. Further complications may be readily introduced into the question of speci f ic i ty of enzymes. (1) Preparation of Standard Buffer Solutionst Sorenson 1909 (10) and Michael i s 1909 (H) recognized and emphasized the fact of the profound influence of C(H) and C(0H) on the act ivi t ies of enzymes. As "regulators" for the production and maintenance of a definite hydrogen ion concentration the standard buffer solutions recommended by these men are widely employed in enzyme chemistry. The choice of buffers which can be employed in phosphatase reactions are l imited. Since free phosphate would interfere with the presumably reversible reaction in which phosphate is one of the end products phosphate buffers cannot be used. Additional free phosphate would also decrease the r e l i a b i l i t y of i t s colorimetric determinations. Farther, many buffers have been found to inhibit certain enzyme reactions and only those, found by t r i a l to permit suitable act iv i ty , can be considered. IE ~6~ We have found several standard buffers to be suitable for our barley phosphatase reactions. (See also W.Jack (12).) (a) M Potassium acid phthalate and M HC1 buffer mixtures by 5 5 Sorenson (10); range pH 2.2—3.8; prepared as given in table by Olark (13). (b) M Potassium acid phthalate and M KaOH buffer mixtures by 5 5 " Sorenson (10) ; range pH 4.0—6.2; prepared as given in table by c l a r k (13) . t'c) 7.505 g. glycine +• 5.85 g, sodium chloride in 1 l . * 0.1 N Sodium hydroxide; pH r a n g e n . 2 —8 . 3 ; prepared as given in table D y Clark (13); buffer mixtures by Sorenson (10). (d) .1 M glycines .1 M NaCh + 0.1 11 HCh; range pH 1.1—3.6 prepared as goven i n table by Clark (13); buffer mixtures by Sorenson (10). (e) Michaelis ' (14) veronal buffer. (2) Protectants: Enzyme chemists generally employ some protectant in reaction tubes and during autolyses to prevent the growth of, and hence the possible interference by, microorganisms. Anaesthetics and electro-lytes, euch as sodium chloride, in high concentrations, are frequently employed since reactions of an enzymic nature are not greatly inhibited by them. In our experiments we used C P . toluene at a drop per tube. Chloroform, and concentrated sodium chloride were used with equally good results. 15 -7-(5) Substrates; Since esterases vary i n their ab i l i ty to hydrolyse their substrates, i t was necessary for us to use several phosphoric acid esters. .1 . Sodium glycerophosphate—British Drug Houses " C e C . " quality, and phosphate free. .2. Sodium pyrophosphate—"CP." ^ phosphate free obtained from "Physician's Pharmacy," Vancouver, B. C. .3. Calcium hexose diphosphate obtained from Brewing Co., Ontario, was an impure product with much insoluble matter and with a high percentage of Phosphoric acid. .4. Hexose monophosphoric acid. (Robison ester.) This ester was f i r s t isolated by Robison and co-workers (77) and is found along with the di-acid during yea3t fermentation. Several methods have been used for i t s i solat ion. We used the following procedure by Raymond and Levene (7&) . To l ive yeast and an excess of glucose, sodium phosphate was slowly added to maintain an optimum concentration and was followed by frequent colorimetric analyses for phosphate. While fermentation was s t i l l proceeding vigorously i t was interrupted by adding trichloracetic acid and the mixture was centrifuged. The diphosphate and inorganic phosphate were precipitated by adding a solution of BaC/g equivalent to the phosphate used and then Ba(0H)g to pH 9. After being centrifuged the solution was c lar i f ied with charooal and concentrated at reduced pressure to a small volume. ^ e m o n o ~ phosphate was precipitated by adding an equal amount of 95$ alcohol and purified by repeated solution and precipitation. Pryde (#) gives Robison's Method for the preparation of hexose d i - and mono-phosphoric acids by yeast fermentation. This procedure has an advantage in i t s exactness and production of higher yie lds . It i s , however, much less rapid. The preparation of the Itfeuberg (?•?) ester by hydrolysis of yeast diphosphoric acid is also given by Pryde. Pasternak %ej) has recently prepared the Robison ester from wheat flour and we prepared a small amount of i t according to his method. Wheat flour was hydrolyzed by 2% H2SO4 for five hours . at boiling temperatures, cooled and the hydrolysate neutralized to phenolphthalein with baryta. Following f i l t r a t i o n , the f i l t ra te was treated with two volumes of alcohol and purefied by further precipitation from equal volumes of alcohol; from rotations of the Barium salt and free acid and from the melting points of the phenylhydrazine salt , Pasternak found his preparation to be identical with Robison's ester. Sodium phenyl phosphate, Sodium metaphosphate, hydrogy-ge-r-miolitte phosphate, etc. are obtainable in pure form, and are suitable substrate for t r i a l . Sodium hexosediphosphate may be obtained in relat ively pure form under the trade name "candiol in." (4) Preparation and Purification of Enzyme Extracts; The method used i n obtaining an extract of phosphatase with i t s associated materials depends essentially on i ts solubil i ty in water and insolubi l i ty in protein precipitants such as alcohol, 15 -9~ acetone, saturated ammonium sulphate solution, e t c To transfer the enzyme from the plant source to an aquaeous infusion some method of opening the c e l l structure must be found. We tr ied autolysis ( i » e « automatic dissolution of cells by unchecked degrading enzymes such as the erepsins) and mechanical fragmentation. The act iv i ty of extracts obtained with different autolyses and with different periods of maceration in a ha l l m i l l , in a hand mincer and in a mortar with sand and with glass wool, i s given in TABLE I; TABLE 1 Extract Sources germinating seed—at 6 mgs. a tube. Buffer; veronal—pH 5 . 6 . Timet 1 hour. Substrate; 2% Sodium glycerophosphate. Temp.t gg" C. TUBE 1 - . • EXTEACTIOH t 1 TOTAL"p INITIAL PINAL FREE P f EROGEDUEE. pH iMGS. | FREE P \ PER C.G.f MGS.PEE READING p ! • • \ j c* 0 • 1 i ; • Bal l m i l l — 4 hours. . 4 4 j .08 • 22 .02 : 2 Bal l mill—1/2 hour. . 4 4 .06 55 .00 i - 5 - Ball mill—1/4 hour. - . 4 4 . . i .06 35 .00 \ 4 Mortar—sand—15 mins. 5 . 5 . 4 4 a l i t t l e a l i t t l e ; 1 5 Mortar—glass wool— - •1 53 .01 ! 15 mins* .05 E Mincer-autolysis— 5*5 . 4 4 .06 18 4 hours. _ I The method adopted in subsequent work was to grind the plant 16 -10-plant material twice in a hand mincer and followed by 10-15 minutes maceration in a mortar; water was then added to 90$ (only in the case of seed had much water to be added) and using toluene as a protectant autolysis allowed for 4-8 hours at constant temperature. The autolysate was centrifuged for 5 minutes on a small e lectr ica l ly driven centrifuge, f i l tered through a coarse f i l t e r paper and from the f i l t ra te by twice precipitating from 25% a i cohol the enzyme extract was obtained. The extract, dried over concentrated HgS04 had later to be ground finely, "dissolved" a n f j the weight of insol-uble matter determined. 4 mgs. of extract were generally added to each tube, which amount gave a satisfactory reaction. TABLE II Extract Sources germinating seed. Buffer: veronal. Time: 1 hour. Substrate: 2% sodium glycerophosphate. Temp.s 50° 0. TUBE 10. 1 j TOTAL PS .INITIAL STANDARD P ENZYME j OF pH.JMGS.PEEl FREE P JIGS. | ENZYME! I O.C, j LIBERATED P READING| MGS.PER I O.C. READING MGS.PERf I C*Co I 1 2 3 4 5 6 8 Active 15.11 . 4 4 8 " [5.7 I . 4 4 4 . " | 5 . l | . 4 4 4. " ' |5i7] v.44-2 V J5.1 I . 4 4 2 " J5.7l . 4 4 4 Inactive |5.1 \ . 4 4 4 I " ;15.7.| . 4 4 i l i t t l e - 4' I " 1 » -trace Uittle 20 20 20 20 20 20 20 20 .1 .1 .1 .1 .1 .1 .1 .1 11 8 28 25 il8 S .25 j .07 .08 { l i t t l e ' ti j tt i it i 21 .30 o c i CQ £** ' D Q»7 r o 8 1T19S. e x t r a c t Fiy.1: Enzyme Concentration Extract Source^>erminatin9 seed S t o J r a f e : 2 S o d i u m glycerophosphate .Buffar: veronal Ophs.l Oph 57 l i m e . : f hr. T e m p e r a t u r e : 3 0 ° C . > e n e s 17 -11-Precipitantst Methyl alcohol was used as a precipitant chiefly because i t was available inconsiderable quantity. Generally 8-10 times the f i l t ra te volume of alcohol brought down a flocculent proteinaceous precipitate. It was noted, from general observation the more rapid the precipitation the more active was the extract. We were unable to develop a uniform precipitation time even with the addition of coagulating agents. Dialysis as a further means of purification was not employed because i t led to a decrease in activity of the extract. However, the extract was dialyzed to remove electrolytes when i t s activation by magnesium ions was attempted. Collodion membranes as prepared by Cole (<ff) were used for semi-permeable membranes. Acetone and ammonium sulphate used as precipitants yielded extracts as active as, or more active than methyl alcohol. TABJEE III Enzyme Extract; A— a c e tone precipitation. B—alcohol precipitation. C—ammonium sulphate precipitation. Buffer: Potassium Acid Phthalate. Time: 11/2 hours. Substrate; Sodium glycerophosphate. Temp.: 32° C# TUBE pH ENZYME EXTRACT TOTAL Pf I N I T I A L l J T ^ A E L ^ B E S ^ ^ MGS.PEEfPEEE P 1 I I E A D I N G T P ~ M G S i . C.C. | M G S , P E R { . . • P E E C O . I X P E P ^ I E N T A L ^ T O T E S I E S D I N G ~ T ' P M G S T " P E E C . C . Ge C« ) i 2 A t! .44 .44 '.44 20 20 20 •J 13 .15 7 .29 11 .18 fTablg T i l conolmxgat 18 -12-2!ABLE III (Cont.) K I B E | _ j ENZYME j TOTAL p| > INITIAL 1 |STANDARD KIBES • . EXPERIMENTAL TUBES 10.. JVM EXTRACT! JIGS. PES! FREE P j READING | P MGS. READING P\MGS. r MGSgPES) PER C.C. PER C.C. c. c. i j *!' - j-f 1 5.0 I s . - •; V44 | trace 20 j « i 20 .10 2 5.6 i " .44 j ti 20 j .1 25 !• .08 3 6.5 . "• . • [ • •« .44 ] H 20 j .1 18 I- •. .11 ,1 5.0 0 . . j . .44 1 .05 20 . J .1 .- ' . 14 a 4 2 ' 5.6 j " .44 J .05 20 i ' .1 10 120 3 6.3 1 tt >44 j .05 . ; 20 I. ,1 '. 20 .10 4 : 5.6 1 ' .44 ' 1 20 1 • a " : " " In the study of mammal phosphatases, tissues in a solution of substrate have been found to spl i t off phosphoric acid. While in tissue reaction the unknown factors are increased in number, they have an advantage in rapidity of preparation. We tried several reactions with tissues of the barley plant. (5) Trichloracetic Acidt This acid is a general precipitant of proteins, congulating them in such a way as to render enzymes in the proteinaceous extract, inactive. Trichloracetic acid does not interfere with deter-mination of free phosphorus by the King (f5") method to any appreciable extent and i t prevents the interference in this determination by protein through their precipitation. Hinsburg (tf) using Fiske and Subarrow (31) colorimetric method reports that the use of trichloracetic acid may cause an error as high as 38$. (6) Method of Determining Free Phosphoric Acidt Since Bell and Doisy (/) 19 -13-proposed their method for the colorimetric determination of phosphoric acid in 1920 many modifications of i t have appeared. In our work, we used a recent modification of Kin© (•%') . Materials required were as follows: .1 . 72-60$ perchloric ac id . .2. 5% ammonium molybdate (phosphate free). .3 . 0.2$ 1:2:4 aminonaphtholsulfonic acid (.5 gms. of the 1:2:4 of the acid, 30 gms. sodium bisulphite and 6gms. sodium sulphate dissolved in aq. d i s t . to make a volume of 250 c .c . j le f t stand overnight, f i l tered and prepared every 2 weeks.) .4. Standard Phosphate 2.1955 g. KHgP0A i n 500 c . c . aq. d i s t . to give 1 mg. phosphorus per c .c . To 10 c . c . of reaction mixture obtained after centrifuging the trichloracetic acid precipitate and similarly to tube containing 10 c .c . of standard phosphate or some di lut ion of i t , was added. 1 c .c . perchloric acid followed by gentle agitation. 1 c . c . ammonium molyhdate " " " " 0.5 c .c . 1:2:4 acid " " " " 5. minutes was allowed for the blue color of the phosphomolybdic complex to develop and the reaction tube read against the standard tube in the colorimeter. According to de-Beer's Law the absorption of l ight by solutions i s directly proportional to the concentration of the coloring substance. The amount of phosphate in the reaction tube may thus be computed from the equation Cg:Ci z I a : L 2 where Cg i s the phosphate concentration of the standard and L ? the tube length of the standard (in m.m.) as read -14- -on the colorimeter, and were 0^ is the phosphate concentration of the reaction tube (the unknown) and i t s tube lengiiih. Our colorimetric determinations were made with a Kleth-Bio colorimeter used and cared for in the manner described by Coles {ft) . The accepted reading on a tube was the average of three. Total Phosphorus was also determined by the King ('/*') method. (7) pH Determinations t With few exceptions determinations of pH were made electrometrically using a Leeds and Northrup Co. quinhydrone pH indicator. Possible "dr i f t " and "salt error" were minimized by fre-quently checking the pH determined electrometrically with La Motte color standards. Temperature corrections were made from the table provided. pH mearsurements above 8 were determined by the La Motte set. Leeds and lorthrup Co. (f>) in their valuable notes have described the use and care of the quinhydrone pH indicator. Coles Iff) also deals with i t s use.; Though only small amounts of extract are available, l/z generally goes into pH determination tubes. B.D.H. capil lator, colorimetric system of pH determination requires only small amounts of mixture, as also do several recently developed micro-electrodes for use on the gurnhydrone. (Pierce and Montgomery (T«) 21 EXPEREIENTAL RESULTS (a) To demonstrate the existence of an enzymic reaction, with phosphoric acid esters as substrates, i n which one of the end products is free phosphoric acid: • Free phosphoric acid is not liberated except in the presence of the active enzyme extract. From the data in Table IV i t is seen that combinations of buffer and substrate without active extract, as of buffer and active extract without the substrate yield no phosphoric acid; that the buffer plays l i t t l e part in the reaction other than regulation of pH is indicated i n the l iberation of phosphoric acid by a combination of enzyme and substrate with buffer absent. TABLE IT Extract Source: germinating seed. Time: 1 hour. Substrate: 2% Sodium glycerophosphate. Temp.: 26° C* Buffer: Potassium Acid Phthalate; pH 5.5. TUBE NO. BUFFER 10 C.C. • j I SUBSTRATE j 1 C.C. 1 1 1 ••""•] EXTRACT 1 C.C. - • " — : EXTRACT BOILED X C ft c« TOTAL P MGS.PER C.C. LIBERATED P MGS. PER Q» C e • 1 ' ,•' •H- 1 I 'I - t - • .44 .27 2 •f- 1 -+- — • .44 trace •. •f- I ~ -r .00 •00 '• 4 <r— . r + . + " ' *~- •44 •04 • 5 1 -** • — — .- .44 .00 22 -2-The thermal ins tabi l i ty of an ester cleaving substance is shown in a consideration of Table V and Figure 2. The data, while not very satisfactory from the point of view of completeness is sufficient to demonstrate the progressive inactivation of the enzyme at temperatures above and below a broad optimum around 26-57° 0. TABLE 7 Conditions as in Table I V » Extract Sourcei A-germinating seed. B-leafy tops. I ( T U B E N O . 1 T I M P E M T U B E ° C . I E X T E A C T ! 8 • L • T O T A L P M G S . P E R : I N I T I A L P M G S . P E R C« C 8 L I B E E A T E D P M G S . P E B C « C e 1 7° • • f i 1 A .44 •' . — 2 • • 26° 1 1 A »44 — • .22 3 37 ° A .44 — .25 4 60-65° 1 A .44 „ 0 4 ; 5 . . 57° \ B • .44 - .05 . .10 •'• . ! 6 " " 65'° I f ; .44 .05 • ., \ "' - ' • • i 7 100° • f B & A I .44 .05 i *05 1 (a) * 37 ° A • • • • ,'•' • .05 .15 ^ ^ * Tube 1 (a) is Tube 1 after 1 hour at 7 ° C. The Signifigance of pH: The hydrogen and hydroxyl ion concen-tration of a medium is a fundamental influence in most enzyme reactions. Activity pH curves comparing relative ect ivity with the pH may be constructed to demonstrate a characteristic optimal region 33 25 .20 KJ t / v s 1 cS <4 i \ \ \ \ i § \ \ \ \ \ > i i t i i i V V ! US o § » 40 60 • % % t o* \ \ » \ \ % . E19 2: Activity-Temperature Series ExTmct Source: (a) ferminaf/np seed (b) fops Subsfrafe ; 2 ^sodium 9lycerophosphate Buffer: Phthalate T/me.-l hr of action. (Figures 5 and 4). With crude extracts and often with highly purified ones the position of the reaction optimum in relation to the pH scale may vary considerably with the source of the extract. Thus with our crude phosphatase extracts the optimal reaction zone with root extracts {pH 6.5-6.8) than that with the extract from green tops (pH 5.0-5.6). Different buffers with identical extracts may influence the position of the pH zone of optimal reaction. In a comparison of Tables V I and VII and figures, i t can be noted that act ivity pH curves are somewhat different where conditions are equal except for the buffers employed. Other substances and varying conditions of temperature may charge the optimal pH zone. TABLE VI Extract Source: germinating seed—6 mgs. per tube. Substrate: 2% Sodium glycerophosphate. Time: 1 hour. Buffer: veronal. Temp.: 26° C. HO. TDBEIIITIALTIIABEIZYMTOTAL PJNITIALj 1 2 3 4 5 6 pH PH 3.2 4.4 5.2 5.8 6.8 7.5 7 i 8.0 8 I 5.8 3 »B 4.5 +• 5..2::; j 5.8 6*7 7.4 7.8 | + 5.7 -f T--f I .44 .44 | .44 .44 I .44 j .44 .44 .44 .02 .02 .02 STANDARD MGS. PERPEEE P C.C. jffiGS . PERJREJDIMGJ MGS. PteEADING-t MGS .iREADING! P LIBERATED PER j 0 . C . j I PER 1 20 20 20 20 20 20 20 20 .1 «x ,»i 9 X .1 .1 .1 .1 30 27 22 18 18 25 37 a XX .11 .08 trace — MGS. PER 0 • C e 31 .06 27 .074 25 .09 17 .10 19 .11 29 .050 27 .054 — trace 24 -4-TABLE VII Extract Sources A - germinating seed ( acetone precipitation )—6 mgs. ~ B - dormant seed—6mgs. per tube. Substrate; 2% Sodium glycerophosphate. Time; 1 hour. Temperature* 26° 0. iTUBEjEKITIALf IMlpiZTJfflTOTAL ppiTIALJ jMGS.PEE P | c.c. P G S . P E E N A D I K G 0« G« pH ! pH STANDARDS; P LIBERATED I MGS .jPREADINMGS .SEALING! P E E C» G« jPEE G* Ot M G S . PEE C.C. 1 2  3  4 5 I 7 1 8 5,1 4.0 5.5 6.5 7.0 8.0 10.0 5.5 3.1 j 4.1 J 5.4 { 6.5 I 7.0 | 7.7 9.5 I 5.5 A A A A A A A .44 .44 .44 .44 .44 .44 .44 20 .20 20' 20 20 20 •20 20 .1 .1 .1 .1 .1 i .05 ! .05 i .05 35 21 9.' 11 25 25 26 1.05 i . l 1*24' [.18 U08 i.05 1.05 38 18 11 11 53 28 26 — it trace — .05 1 .11 I ,18 I .18 ! .06 i .07 j .08 it race 1 2 3 5.5 1 5.5 6.5 | 6.5 5.5 5.5 B B .44 .44 .44 l i t t l e | 20 — j 20" I 20 .1 a . i i i 12 1,18 1.16 The optimal reactionzones for a l l our phosphatase extracts l i e on the acid side of neutrality. Correlated with this is the fact that the reaction of the crude ce l l sap of barley is acid . (pH 5.0-6.8). This does not preclude the poss ibi l i ty of barley phosphatases with optimal reaction zones on the basic side. A l l our pH act ivi ty curves (figures 3 and 4) possess relatively broad zones of optimal reation. This i s in striking contrast to the act iv i ty pH curves for mammal phosphatases where .2 pH may cause large differences. 25 -5-Suitable salt mixtures whose acidity does not alter appreciably over a certain range by the addition of acids, bases or amphoteric substances are used i n reaction tubes to prevent an alteration of acidity due to products already present or products formed in the course of enzyme reaction. The addition of a substrate such as sodium glycerophosphate of alkaline reaction w i l l tend to raise the pH. Similarly other substrates and enzyme solutions may alter the i n i t i a l pH (W. Jack (M ) . Over most of their range the buffers we selected were very suitable, changing but l i t t l e during the course of reaction. Tables YI and VII. With the question of the influence of materials concomitant with the enzyme reaction are to be considered specific activators and inhibitors . Lohmann and others have shown that a specific activator of yeast and mammal phosphatases i s magnesium in the ionic form. By dialyzing our extracts to remove electrolytes then adding a magnesium chloride solution we were able to activate barley phosphatase. Table VIII and Figure 5. TABLE VIII Extract Source: A - germinating seed (dialyzed)—6 mgs. B - normal root (dialyzed)—6mgs. C - germinated seed (not dialyzed)—6 mgs. Buffer: Phthalate. Time: 1 hour. Substrate: 2% Sodium Glycerophosphate. Temp.: 26° C. 26 -6-TABLE VIII - Continued: M G C L 2 [ E X T R A C T 6 mgs. P E E 0 • 0 A T O T A L Pi F R E E P i STANDARD M G S . P E R J M G S . P E E ! R E A D I N G ! M G S | P E E ! R E A D I N G I M G S . P E 3 JJlBJEAJSaLIL C» 0» I I Co Ce 1 15.6 i & 3 J5.S | + I ' ' ;- 4 5.6 5 J5.1 6 J5.1 "i 7 :s.5 A A B 3 A A C .44 .44 .44 .44 .44 .44 frrace ! 20 I : 20 I i ! 20 I j'.20 | I 20 .1 .1 ,1 «& X o X 0 X 9 . : 18 14 33 15 33 12 .11 .14 .06 .13 .06 .17 1* i'5.6 2* 5.6 A A .37 .37 20 [ .1 20 20 35 .10 .06 * Substrate: Sodium pyrophosphate. Prom several series, dialysis of dormant seed extracts seemed to increase their act ivity and i t was thought that a dialyzable, thermostable inhibitor of barley phosphatases had been found. A later series did not confirm these results and the use of impure toluene as a protectant cast doubt on the former findings. The question of activators other than magnesium and inhibitors was left in abeyance. (a) Distribution of Phosphatases in Barley: A substance i n barley precipitated as a protein sensitive to maceration and capable of catalyzing a reaction in which free phosphate p 1 8. 1 \ • r V \ \ \ X X \ $ / f ( • >3' ./a .20 .26' liberated F?mp& perac EiqA-. Activity-pH Series Mw£JMMm: germinating seed Substrate•• Naglycerophosphate Time-.ihr Bufer: Phthalate Im^tm.: 26°0. / / • • _ fflSB <*s 0 XJ" 1 I \ < X • '> V 4 > * *0 to a C D P C O © > s e CD cd 27 -7-was spl i t from phosphoric acid esters, a substance with act ivity optima at definite pH and temperature zones and activated by magnesium chloride, we believed to be a true phosphatase. (Its determination as such, however, was not quite complete—discussion, page ) . The following tables IX-XIV i l lustrate the qualitative distribution of phosphatases through the development of normal and phosphorus deficient barley from seed to heading. While a few short series not given, were run at other stages of development the date is sufficient to indicate the distribution of the enzyme, in a gross way at least, throughout growth and in a l l parts of the plant. Table XII summarizes this data. Many omissions are. obvious, particularly in regard to the pyrophosphatases. TABLE IX Extract Sources A - germinating seed. B— dormant seed. Buffer; Potassium acid phthalate. Time; 1 hour. Substrates Sodium pyrophosphate .06 M. Temp.s gg ° C. I T T J B E E X T R A C T pH T O T A L P F R E E P •... S T A N D A R D S i L I B E R A T E D P : N O . ! S O U R C E M G S . P E R M G S . P E R I B E A D B I G | M G S . P 1 R E A D I N G j M G S . P E R I 1 ! !•• ' 1 ' 1 0» Ce C . 0. | PER C* Ce I j 1 p n i -- ! ! l i A 5.0 .37 20 1 ' • - i , i ; 3.5 j . 0 6 j j 2 i .' A " 5.4 .37 20 .1 ! 25 j .08 1 I •.'•'.A.' ' 6.1 .37 trace 20 ' 1 °1 18 : ' . l l 4 . - A - 6*7 .57 • 20 . .1 1 18 | .11 5 A 7.2 .37 trace 20 1 .1 •' 28 1 .07 6 j 5.4 • 57 20 1 .1 ! . , ~ ~ l B 6.1 .37 • -s- , 20 I 35 *055 2 B 6.7 .37 20 1 • X . 35 .06 3 B 5*4 .37 20 1 ©X 59 .05 .4 _ 5*4 .37 20 .1 1 1 28 -8-TABLE X Extract Source: A _ germinating seed—6 mgs. per tube. ' B - dormant seed—S mgs. per tube. Substrate: 2% HA glycerophosphate* ^ime8 hour. -Buffers Potassium Acid Phthalate. T e m p . : 26 ° C. SUBSTRAW, ENZ-TME TOTAL P MGS.PEB 0.0 INITIAL FREE P " STANLY USD LIBERATED 'P . READING MGS.P PER C.C, READING MGS.PER C.C. Sodium glycero- 5.5 .44 20 a ,7 .29 phosphate tt it + 5.9 .44 —— 20 - a 10 .20 Calcium hexose™ 5.6 .52 .25? 20 : a 6? .08? diphosphate it u ; -t 5.9 .52 • .25? 20 .a * * . 6? .08? Sodium hexose- 5.5 .34 20 a . 9 a ? diphosphate tt tt 5.7 ,34 .05 20 . i 9 .17 Sodium hexose- 5.6 .22 " i04 20 a 20 .10 mononopho sphate (Pasternak) * •* • " » tt . : t 5.9 .22 i04 20 a 20 .10 Sodium hexose- 5.6 .34 .05 20 ' a •=•-9 •i-8 diphosphate 12 ' ,11 »t tt 6*0 .34 .05 20 a Calcium hexose-diphosphate tt tt ' -5i5 5.9 .32 ,32 .25? s,25? '20 20 a a . 6.5? .08 .06 CD O • * CZ CjJ O 3 o e > * < \* £ .0) ^ CD o CD CD CD O o | s c 3 O W i TABLE XI Extract Source: A - Boots of Normal Plants—6 mgs. per tube. B - Tops of Normal Plants—6 mgs. per tube. Substrate' Sodium glycerophosphate. Time: 1 hour. Buffers Potassium acid phthalate. Temp.t 26° 0. TUBE EXTBAOTipH TOTAL P INITIAL STANDARD FINAL P No.. 1.:-i }. MGS.PEB G. G. FREE P MGS.PER Co Ce BEADING | MGS.P PER G a C o jBEADING { ' [ MGS. P -PEB C.C. ;'' LIBERATED i I. A ' • j ; 5.0 ! | . 4 4 20 .1 S 1 { j .08 | 2 i |. A 1-J5.3 l ' . 4 4 20 a J ' 1 . 22 j .,09 , 3 - A;' 1 15.8 j . , 4 4 — 20 a ! 12.5 1 .16 4 A j6.5 . 4 4 20 a 15 j .15 A i7.0 {• . 4 4 . — : 20 1 1 4 . 6 — • • I \ 5.8 i • { i . 4 4 • **— 20 . 1 i .. : • .... - . ' 1 B , 5.0 ' [ . 4 4 20 9 1 • 15 .13 2 B ; 5 .5 I . 4 4 • 20 13 .15 3 B ' 5*8 | . 4 4 — 20 • 20 .10 B 6.5 ; . 4 4 — 20 20 . .10 5 B 'w.of . 4 4 20 a j 35 .06 6 B - ' i 5.8! I 1 1 . 4 4 — 20 a 30 -10-TABLE XII  Buffer; Potaggi^m Acid Phthalate. Temp.: 26 ° C. Times 1 hour. EXTRACT I :SUBSTRASE t 1 | OPT. TOTAL'P ] INITIAL P LIB- $ OF ESTER SOURCE ; . . :v, '•' • ! PH PEE C.C. 1 FREE P ERATED HTDRO-1 • MG. j MG. . LYZED iDormant %• 2%Sod.iim glycero-• i i j ' ' • 40%* ; phosphate 5.5-6.0 .44 f • .18 jSeed. 2$ Sodium hexose- j • 1 • 1 1 • f'" ' ' ' diphosphate i5.5-6.0 j .34 1 : .18 j 53%' \ iDormant 2$Calcium hexose-!' ' ' ! ! • f* -i • • ! diphosphate :5.5-6.0 .08 , 2 5 $ • ] Seed. !.06M Sodium pyro- I ; 1, 27$ i • ' . • ,: 1 s phosphate • 1 6.0-6.5 i .37 • — — .10 jGerrax-- • 2 $ Sodium glycero-i : • • • -! phosphate i5.5-6.0 .44. > .29 66$ | nating 2$ Sodium hexose- i' • • i j •'' ' : jSeed. diphosphate 5.5-6.0 . - . .54 — e 22 65$ 2% Calcium hexose- X \ diphosphate 15.5-6.0 ;•• .52 • - • — • .08 25$ \ 2% Robison \ ester 5.5 • 22 — .10 45$ '.06M Sodium pyro-i I J phosphate 6.0-6.5 a 37 — .11 J 50% f . . ;Roots o f 2% Sodium glycero-Phos- phosphate 6.0 .44 — . . .08 18$ phorus g$ Calcium hexose-Defic- diphosphate 6.0-6.5 .34 .05 14$ ient .06M Sodium pyro-Plants phosphate •'6.5. .37 .07 18$ (Table XII Continued) 51 -11-TABLE XII (Continued) JEXTRACT jsUBSTRASE SOURCE 1 1 (.1 ' """ • • OPT. j TOTAL P pH | PER C.C. MG, INITIAL FREE P P LIB- ['$ OP ESTER ERATED j HYDRO-MG. LYZED - i • • 1 -|. • - j ' iRoots of |2$ Sodium glycero-} j phosphate INormal i 2% Calcium hexose-i i diphosphate jPlants I.06M Sodium pyro-| | phosphate i V- .1 • . ' ! 6.0 ] .44 • ' { • • • 6.5 I .52 J 6.5 ! .37 ' 1 • • k .16 .) 36$ f .08 j 25$ .11 ] 50$ i . •• ! . . I • ' • • r-(Tops of jjPhos-jphorus ^Deficient Iplants r $ A 2% Sodium glycero-phosphate 5.5 f i • .44 ' i • • , . . . . . ! • • . , ! .07 1 • • -15$ (' iTops of iNormal plants 2% Sodium glycero-phosphate 5.7 .44 : trace .15 34$ TABLE XIII Tissue; A - Normal Roots—15 c .c . Time: 1 1/2 hours. B - Normal Roots inactivated—15 c .c . Temp.: 30° C. [TUBE (NO. 2 3 4 pH : T I S -rsuE 6.7 6.7 6.5 6.5 A A B B STANDARD INITIAL FREE P SUBSTRATE ._. 10 C O . j READING] MGS.PJ READING} MGS. |READING •I" i 1 C.C LIBERATED P I C.C.f. MGS. PER ! c. c. I i Glucose 4$ Sodium glycero-phosphate 10$ Glucose 4$ Sodium glycero-phosphate 20 20 20 20 i i-I j I » 1 ; i 15 11 30 SO .15; .18? .07;: .07! 20 12 22 21 .00 .08 .00 .00 32 -12-• Maccerated tissues where inorganic phosphorus was not too high nad to which substrate was added, gave reactions similar to those given by extracts. The use of tissues introduces more unknown factors and their only advantage is in speed of preparation. In mammals where, usually, highly specialized parts are more definitely set apart, tissue reactions have proved of value (Bodansky (f) ) . For what they are worth then the data in the Tables XIV-XVII are presented: TABLE XI7 Tissues: A - roots of Normal plants. B - roots of Normal plants previously heated to 100° C. for C - roots of phosphorus deficient plants. (5 minutes. D - roots of phosphorus deficient plants previously heated to 100° C. for 3 minutes. Substrate: 2% Sodium glycerophosphate. Time: 1 l/2 hours. Temperature: 50° C. |TUBE pfi TISSUES j SUBSTRATE! I N I T I A L P j. STANBAEDS / : LIBERATED P : . , |io. I io c.bj. 2 c.c. |1EAD1N&TSG57] HEADING|MGS7| R E A D I N G ! M G S . ! j . • • • • • ' ! • • I PER I PER i j PER 1 i ! .1- ' 6.8 • A • -I- 20 ! >1-\ 20 .08 j - 6.6 B •+ 40 .05 20 ,1 ; 40 .00 6.0 0 .00 . — 20 a j 22 ,09 1 4 ; "6.0 D .00 — . . . 20 a i trace f 6.8 A .20 .1 20 a | i ! 24 some i 33 .^.13-TABLE XV Tissue? A - active root tissue of Normal plants* B - inactivated root tissue of normal plants. C - active root tissue of P deficient plants. D - inactivated root tissue of normal plants. Substrates 2% Sodium glycerophosphate. Times 1 hour. Temperatures 30° 0. STUBS | pH TISSUE SUBSTBATEj STANLABDS INITIAL P LIBEBATED P jNQ. 10 O . C i 2 C • C« 1 READING MGS.P[BEADING |MGS. j BEADING IMGS. PEB i - . { PEB PEE C.C. $ 1 } Co C e Ce C*> i i i. 6; 8 A • i -20 a 20 o X • 11 .08 2 I 6.6 B \ 20 .1 40 .05 40 ,00 i 3 i 6.0 0 + . . ; 20 «x 00 © — 22 .09 1 * •" I • • • • 6.0 D -20 ' a ; 00 trace 00 i 5 6.8 A 20 a 20 a 22 00 TABLE; XVI " Tissues; Substrates 2% Sodium glycerophosphate. A - Pi" deficient j tops active, B - P deficient tops; inactivated by boi l ing . Times 1 1/2 hours, C - Normal plants; tops active. D - Normal plants; tops inactivated. Temp.s 26° C. buBE pH 15 C.C. " ' | SUBSTBATEJTOTAL P 1 INITIAL STANDARDS LIBEBATED P iNO. TISSUE 4 C.C. iPEE C.Cj PEEE P BEADING MGS.P;.BEADING MGS. S; ! MG. • • PEE PEE • i I'- -i ! • / C oC- • C.C. ( u i x 5.6 A - ' trace 20 • a -25 .08 • 5.4 B + trace 20 a 40 ,05 3 5.4 C -t 10 .20 20 a 7 .07 4 5.5 D ,20 20 a 18 .19 5 5.5, A aq.dist . trace 20 - 1 .02 54 -14-TABLE XVII -Tissue: A - normal barley kernel—15 c .c . Time: 1 l / E hours. B - P deficient barley kernel—15 c .c . Buffer: Phthalate. Temp.: 50° 0. TUBE NO. TISSUE SUBSTRATE STANDARDS READING MGS.P • PER Co Co INITIAL FREE P MGS. PER c« c« LIBERATED P MGS. PER C • 0 • 1 1. Sodium glycero-phosphate 2$ 20 .1 ,05 .48 2 A Sodium pyro-phosphate .06M 20 .1 .05 .59 5 None 20 .1 .05 trace 4 B Sodium glycero-phosphate 20 .1 traces l i t t l e Several points are worth noting. Free phosphate, for an example, did not increase markedly unti l the ester substrate had been added. Again heating tissues to temperatures of 60° and over caused a decrease in phosphate. Just at what temperatures this process occurred were not determined. In view of the doubt cast on the occurence inorganisms of a synthesizing "phosphatase," should this process be a constant thing in tissues i t s further investigation might be warranted. Several attempts were made by addition of glucose and phosphate to extracts to obtain an enzymic synthesis of phosphoric acid esters but they were not successful. The only encouraging results we obtained in this direction were with green leaf tissues containing considerable free 55 -16-esters phosphate or to which dextrose and phosphate had been added a r t i f i c i a l l y at a temperature of 60° C. or oter. Such conditions led to a disappearance of free phosphate. The nature of the reaction was not determined. Tables XIY-XYII. 56 DISCUSSION Different preparations from the same part of the barley plant, using the same procedure, gave extracts of satisfactory but not uniform act iv i ty . More active extracts might be prepared using precipitating agents such as acetone or ammonium sulphate or using sodium chloride solution, rather than water in extraction. Lohmann ("t*) has prepared highly pure extracts of phosphatases using absorption methods. Harden and Macfarlane («r) have found the mechanism supplying inorganic phosphate in yeast to be highly sensitive to grinding and usually nearly destroyed while the zymase and co-enzyme system passed with at least l/2 of i t s original act iv i ty . Sensitivity to grinding may have been an explanation for the lack of uniformity in different extracts. It was noted too that act ivity varied with the length of time taken by precipitates to fluctuate in the MeOBV •;' We found Sorenson's glycine and potassium acid phthalate and Michaelis* veronal (Sodium diethylbarbiturate) buffers to be suitable for our use. Of our substrates sodium glycerophosphate and sodium pyrophosphate were the only " C P . " products. The others were of untested purity. Davies [Z'f) and other investigators have found the King (w) color i-metric determination of inorganic phosphorus to be a suitable method i n phosphatase investigations. Unless some c r i t i c a l comparison of current colorimetric methods is undertaken, and, unless i t i s shown to be at fault i t s use, because of i t s convenience, w i l l be wide. 3 7 -2-In several cases our enzyme extract hydrolyzed i t s own weight of substrate in one hour. With phosphatases, never with a high active content i n extract, this i s satisfactory. From our tests we concluded that the barley plant possessed true phosphatase systems liberating phosphoric acid from ester substrates. The catalyzer we showed to be thermally unstable above 65° C. with optimal reactions about 2 6 ° - 5 7 ° C. pH act ivity curves were determined showing broad optimal zones. While optimal act ivity was always in acid media the poss ibi l i ty of alkaline phosphatases is not precluded for no enzyme extractions were made in alkaline media. Precipitating by usual protein precipitants indicated the protein and col loidal nature of the extract. In common with other phosphatase extracts ours was sensitive to maceration and activated by magnesium ions. One point in proof of the identity of our enzymes is lacking. The hexose unit i n the sugar phosphates might be attacked glucolytical ly to yie ld lact ic acid and free phosphate. By following the products of the reaction quantitatively, the proof would be complete. In another manner a similar end could be achieved. Olucolysis occurs only in the presence of a dialyzable thermostable co-enzyme. If following dialysis phosphoric acid esters were cleaved glucolysis as a possible factor would be removed. Demonstration of specific phosphatases in our extracts i s lacking. Loftmann (f/) by differential absorption was able to separate pyro-phosphatase and sugar phosphatases in muscle. Levene by reactions in point of time was able to establish the identity of three separate phosphatases acting on phospholipides. Hotto M believing the 38 - 3 -specif ic i ty of phosphatases was determined not alone by the nature of "the bond uniting the phosphoric acid but also by the nature of the alcoholic residue divided phosphatases into (a) phosphodiesterases (b) phosphomonoesterases {c) pyrophosphatases (d) amidophosphatases. The presence of a metaphosphatase in corn has been suggested by Menjdahl and Weissflag if/} . ¥ a l d s c h m i d t - L e i t z (;*») upholds the determination of Levine and Lohmann only* The distribution of phosphatases has been shown to be very wide and their functions are just becoming known. In mammals and invertebrates (Roche) different phosphatases are found in many organs and tissues, muscle, bone, kidney, blood, brain, intestinal mucose, and tumours. In yeasts and Aspergillus oryzeae, the source of takadiastase, phosphatases hydrolyzing many substrates are found. Phosphatases are found i n rice and jackbean. From our tests i t would seem that i n barley, also, their distribution i s wide, and are to be found in roots, tops and seeds and at a l l stages i n development, CONCLUSION The results of our work must be regarded as essentially qualitative. Because of the very considerable variations in conditions of extraction and reaction quantitative results cannot be deduced from the data. Many refinements in technique are obviously necessary for further work, We do, however, submit the following conclusions: 1. The barley plant possesses true phosphatase systems, some of which, at least, are activated by magnesium ions. 2. Phosphatases are widely distributed throughout the developmental cycle from seed to seed and occur in phosphorus deficient as well as in normal plant, though possibly less active in the former. DISCUSSION 39 . 1 . INTRODUCTION In view of the rapidly increasing numbers of researches on phos-phatases and their related metabolic exchanges, recent pertinent literature is d i f f icul t to appraise. The following discussion is, as a consequence, very limited both in general outlook and in the number of papers reviewed. "Enzymes may be defined as definite material catalyzers of organic nature with specific powers of reaction formed indeed by l iv ing cel l s , but independent of the presence of the latter i n their operation." {Waldschmidt-Leitz) With some important differences they are much in keeping with Ostevald's (f ) definition of a catalyst. (a) their action consists of accelerating reactions probably already i n progress and which are, theoretically reversible (Tan't Hoff.) (b) the enzyme is not a product, i s required in small amounts only, and does not act in molecular quantities, (c) i n a general way the rate of reaction depends on the concentration of the enzyme * {d) most enzyme reactions are hydrolytic. Unlike most catalysts of the usual type (a) their action is specif ic , (b) some particular optimum pH and temperature is required for their action. {c) enzymes are usually slowly destroyed in the process of reaction, and optimum conditions are those in which the enzyme reacts most quickly, (d) enzymes are readily subject to inactivation or acceleration by concomitant substances, (e) while the primary function of enzymes relates essentially to energy transfer the accompanying heat effect is s l ight . Although at the present time differences of opinion as to the mode of 40 . . ~2- • • • enzyme action exist only in details as Waldschmidt-Leitz states the foundations for another important phase of the above definitions are much less assured, namely in regard to the assumption of a definite material nature for these catalyzers. Crystalline enzyme preparations have been prepared (Summers.' urease (#0 , Northrop's pepsin P) ,) Levene and Halberger fa) , however, have concluded that the chief components of the crystals are extraneous proteins. Waldschmidt-Leitz and Purr (P7) have separated "crystal l ine" trypsin into four by special adsorption agents. The prevailing concept of the material nature of enzymes is that of the Wilstatter school. This school considers that enzymes are composed of a colloidal bearer and a specific active group which enables them to be bound to the substrate and the composition of which at the same time conditions the col loidal nature of the entire complex. Evidence in support of the Wilstatter theory has been gained chiefly from well known and verif ied absorption experiments where enzymes in a high state of purity have been obtained through use of absorbents such as kaolin and alumina. In support of this theory that enzymes are col loidal in character with an active specific group are the recent observations of Bredig and Gerstner {«) wherein a diphenylamine group ad'ded.':to'cotton.made/atcatyst sp l i t t ing carbonic acid from B-heto-carbonic acid, and those of Langenbeck [P] on activating groups within the active enzyme proper. Pischgold's and Amnion's theory of esterase act ivi ty through displacement of Hg0 on the enzyme surface depends on the Wilstatter theory. » 2 . fflZYMIQ KIHETIOS Enzymic reactions while essentially catalytic do not necessarily follow the laws of chemical dynamics as formulated for catalysts of the usual sort. The complicated end imperfectly understood composition of enzymic reaction systems together with our ignorance of the chemical nature of the effective catalyzers and the influence of adventitious impurities upon their af f inity often results in preventing a classif ication of even the simpler transformations along basic principles . The va l id i ty of older kinetic-reaction research applying the mass low in one form or another is in many cases very limited because of the many unknown factors disregarded. Waldschmidt-Leitz { ) has reviewed examples best supported by experimental evidence. Metabolic exchange systems in v/hich phosphatases are involved are complex and new factors and relationships are being discovered from time to time and for this reason the kinetics of phosphatase system have not been worked upon to any extent. The kinetics of glycerophosphate cleavage by kidney phosphatase has been examined i n detail by Jacobsen M . The determining factors he found to be the liberation of free phosphate and the concentration of the substrate. The usual curves indicating formation of substrate enzyme complex through induction periods were given. Martland and Eobison («) studied the course of sodium glycero-phosphate hydrolysis by bone phosphatase in the absence and presence of inorganic phosphate. They found that even without the addition of phosphates the velocity constant calculated according to the equation for unimolecular reactions decreased more rapidly than could be 42 accounted for by inactivation of the enzyme; they advanced the hypothesis that a resynthesis between phosphoric acid and glycerol was occurring. They demonstrated the influence of phosphate i n the reaction, but were unable to demonstrate the poss ib i l i ty of a resynthesis working with glyceral and phosphate in consentration employed i n previous hydrolysis. Erdtman (2?) using kidney extract from hogs found the velocity constant i n absence of activating agents to decrease rapidly. He attributed the fact at least i n part to enzyme destruction and to i n -activating products of hydrolysis. Belfanti , Contardi and Erco l i [t>) found a similar rapid decrease i n the velocity-constant and believed i t to be i n agreement with the " Schutz Rule, which implies the reaction velocity was not only proportional to the substrate concentration but inversely to the quantity already transformed. These workers postulate that i n addition to an enzyme-substrate complex there is an enzyme-phosphate complex capable of hydrolyzing action. , • . The. dependence of the act ivity of most enzymes upon pH was f i r s t brought out by Sorenson (1909) and Michaelis (1909). A characteristic optumal reaction region is generally apparent and the activity pH ratios can be expressed i n pH activity curves. Many factors as has been previously pointed out disturb or distort the measurement. In this regard concomitant substances such as buffers, activators, inhibitors are important as well as, in addition, the s tabi l i ty of the enzyme i t s e l f , at a given pH. The theoretical significance attached to activity—pH curves for enzymes themselves are to be regarded as amphoteric electrolytes, 43 ~5» which according to the pH action varying capacities on anions, kations a undissociated Molecules . At f i r s t , Michaelis implied that the undis-sociated material was to be considered as the catalytically active portion. These results, however, have been strongly questioned by many workers including Michaelis himself, Euhfc (?«") finds that the H ions themselves influence the decomposition velocity of the enzyme substrate combination. Horthrop p") working with proteolytic enzymes found the dissociation of the substrate to be influenced by the H ions£ his data showed activity-pH carves to be coincident with dissociation curves of the substrate. Michaelis {si) also used the behaviour of enzymes in an electr ical f i e ld and their absorption af f ini t ies to explain their electro-chemical nature. In acid medium i n an e lectr ica l f i e l d he showed the amphoteric enzyme particles moved to the cathode, in alkaline medium to the anode, and in the intermediate region corresponding to their isoclective point no migration at a l l . His work on electro-chemical absorption i . e . on the enrichment of dissolved materials on the surface of solid bodies, has been fundemental in absorption methods of enzyme purification so extensively used by the Wilstattler school. if In principle every reaction is reversible (Hernst) and according to the mass law the concentration of reacting substances determines not only the velocity but also the direction of the reversible process. Theoretically these enzymes may catalyze reactions not only in the direction of hydrolysis but also i n the direction of synthesis. Numerous examples of synthetic action by enzymes are known (Baylies ['/) 44 -6-on enzymic syntheses.) The recognition of the synthesizing action of enzymes has led to the question as to whether or not enzyme equilibrium is to be regarded as identical with that effected by other catalysts. "According to the laws of thermodynamics i t was expected that this would be so, i f the enzyme were incapable of binding an essential portion of the reacting components. The results obtained with enzymic equil ibria have shown that this requirement i s not f u l f i l l e d in a l l enzymic reactions even though the point of the enzymic equilibrium cannot be definitely determined. According to Euler [w] the equilibrium position of enzymic reaction is not determined as an ideal catalysis by the concentration of the substances concerned but by the relation of their af f init ies to the enzyme i t s e l f . Bayliss [f ) emphasizes the non-necessity of assuming special synthesizing enzymes and that enzymes must accelerate both hydrolytic and synthesizing aspects unless they carry the reaction to completion under the conditions present. Several references i n the literature are found on enzymic syntheses of phosphoric acid esters. Martland and Eobison («) have explained abnormal drops i n velocity constant during hydrolyses to resymthesis of glycerol and H 3 P O 4 . lagai {^ ') has found the ab i l i ty of kidney and l iver of pigeons to esterify HgPC>4 to be 14-78$ greater in a condition of B-avitatninosis and attributes this to acceleration of a phosphatase Hemini K) and Tsukitari (J7) were unable to demonstrate the presence of a phosphatase i n Phizopus (Mucor) t r i t i c i , a fungus grown under a variety of conditions. Rumstrom, Linnesstrand and Bori i ( ) obtained esterification of 45 -7-HgFO^ in haemolyzed blood i n presence of co-zymase. Pett and Wynne were unable to demonstrate synthesis of the esters by bacteria. According to Wal d schmidt-Leitz [f°) the differentiation of a separate phosphatese is not to be accepted. Rather i t seems attention should be paid to the conditions necessary for synthesis on the part of phosphatases—a complex of conditions probably, involving the presence or absence of definite activators, inhibitors and accompanying reactions. Lohmann \f') found phosphorylation of glycogen in muscle to occur only i n the presence of a complete co-ferment system (adenylpyrophosphate MQ) . . 3 . ACTIVATION AND INHIBITION In addition to the influence of hydrogen and hydroxyl ions, substrates, etc . , activation of enzymes by specific activators, inhibit ion by inhibitors of a specific nature can be demonstrated. Waldschmidt-Leitz classes these in four ways. (1) Activation by Inorganic Ions: e.g. the s tabi l i ty of enzyme-substrate complex appears, for example, using saccharan, to decrease in the following sequence i n the presence of these ions - NOg , CI , Bi , SO^ . The mode of activation is not clear. (2) Specific Activation is characterized by the fact that i t is incapable of expressing i t s e l f except with respect to definite particular af f init ies of an enzyme or else with a definite stage of the reaction which i t accelerates. One of the best demonstrated examples is the activation of pancreatic trypsin by enterokinase from the intestinal mucous. Waldschmidt-Leitz believes there is formed in 46 -8-this case a.compound of enzyme and activation, Co-zymase, dialyzable, and thermotable i s an activator of this class (Harden and Young fo) .) ' Myrbach {C3\ and Kyrbach and Euler {(A believe co-zymase to be a nucleotide closely related to adenylic acid and capable of being hydrolyzed by phosphatases. Anti-enzymes, about -which there is l i t t l e known, might be recognized as attributible to specific activators, (5) Non-specific activation and Inhibition: It has been shown by Wil3tatt/Ler, Waldschmidt and Memmen {^ \ that activation phenomenae in solutions of lipase are based merely on specially favorable conditions for the contact of water soluble enzyme with i t s insoluble substrate, The activating effect of ga l l salts ,etc. i s due to the production of col loid particles which exert and absorbent action with respect to enzyme and substrate thereby fac i l i ta t ing the reaction. (4] Toxic Influence of Heavy Metal Salts i s common to a l l enzymes to a greater or lesser degree. Euler and co-workers (^ have demonstrated the "poisoning" of saccharase to be accompanied by a corresponding decrease in heavy metal ions and further that act ivity could be renewed by addition of HgS to precipitate the metals. The effect is apparently upon dissociation of enzyme-substrate complex. (5) Inhibition and Activation of Enzymes by Definite Salts on Organic Addition Substances: Many reports on this class of enzyme activation and inhibition occur in the l i terature. Only a few of these, pertinent to phosphatases w i l l be mentioned, (a) Magnesium Activation of Phosphatases: Erdtman (-?;% using kidney extracts, f i r s t reported magnesium activation of phosphatase; later work found i t to be an activator of phosphatases generally. 4 7 -9-Lohmann H found that hexosediphosphate loses no phosphate when added to dialyzed muscle extracts; when magnesium ions are present one molecule is lost , thereby becoming E/nbden ester, and equilebrium mixture of aldo-hexose with a small amount of keto-hexose. Embden ester i s then dephosphorylated i n the presence of adenylpyrophosphate. Further the enzymic change of morganic pyrophosphate to orthophosphate i s , according to Lohmann, carried out only i n the presence of magnesium, (b) Activation of Phosphatases by Arsenates Pett and Wynne H investigating the oft reported influence of arsenate and arsenite on the enzymatic breakdown of phosphoric esters could find no act ivity increase for phosphatase in general, but did find activation by arsenate in special cases. Wherever arsenate activation occurred arsenate less rapidly gave a similar result* Harden and Young ( 3 £ ) showed arsenate and arsenite ions accelerated the liberation of inorganic phosphorus from hexosediphosphate by yeasts. Meyerof H working with heart and muscle extracts, Macfarlane k'A and Harden ¥'\ with yeast believed the effect to ba exercised on the glycolytic and not the phosphatase system. Braunstein and Lewitow (?) observed progressive diminution of inorganic arsenate i n a mixture of yeast, sugar, arsenate, toluene and aq, dist . and suggested the formation of labi le hexosearsenates. (c) Activation and Inhibition by Potassium Cyanide: Warburg M concluded hydrogen cyanide in the presence of phosphate stowed down alcoholic fermentation by yeasts and suggested i t affected chiefly the mechanism for l iberation of phosphate from hexosediphosphate. Kiss Patterson {v\ has i n some detail investigated the effects of KCN and HC1T on alcoholic fermentations generally but has not as yet determined the systems upon which they might act. (&) Influence of Oxalates! Several workers mention the effects of oxalates on phosphatase systems, noting chiefly the inhibit ing action. B l l f an t i , Ercol i and Oontardi (&) found that when extracts of l i v e r , kidney or hone phosphatase are allowed to act on glycerophosphate i n alkaline medium with oxalate, the hydrolysis at f i r s t proceeds slowly then l i t t l e by l i t t l e , accelerates and f ina l ly proceeds as i f the oxalate were no longer present unt i l the hydrolysis of the substrate is complete. In the presence of phosphate, added a r t i f i c i a l l y , sodium oxalate does not manifest andy inhibitory action. It was suggested that the explanation of these phenomena was that the inorganic phosphate set free from glycerophosphate gradually displaces the oxalate ions from the inactive enzyme—oxalate complex giving rise to an active enzyme—• phosphate complex capable l ike the free enzyme of uniting with substrate and hydrolyzing i t . Bodansky (f) and others mention the inhibitory effects of oxalates on phosphatase. (e) Fluorides Fluoride has been known as an activator of lipase ', and several other enzymes for some time and recently phosphatase has been added to this l i s t , Loevenhardt and Pierce (-), Smith and Lantz <f3\ • Auhagen and Grzycki (') found kidney phosphatase to be unaffected by sodium fluoride: found yeast phosphatase to be highly sensitive, and takaphosphatase from Aspergillus oryzeae less so. (f) Sulfhydryl groups: Phosphatases generally are inhibited by sulfhydryl groups at their pE optimum. Schaffner and Bauer (^ ) . Cysteine inhibits yeast phosphatase most at pH 6.1 and at pH 8.8 with kidney phosphatase. Dislysis renders the enzyme more susceptible. 49 -11-Reactivation with iodoacetic occurred with kidney phosphatase. Mowatt ., and Stewart ( ) find on the other hand that iodoacetic acid prevents gluolysis, and destroys glutathione—the activator of glyoxalase. .4. Methods of Determining Orthophosphate Acid. Jack has at some length discussed the various methods used in the determination of micro quantities of orthophosphoric acid and their mention w i l l suffice here. Bel l and Doisy ( r ) , Briggs ( ), Fiske and Sub arrow , Martland and Eobison f3) and Kinsp («-} have proposed colorimetric methods. Kirk [*•) proposes a very convenient gasometric method. A micro-gravimetric method has been suggested by Plimmer (A) . Titrametric methods have been put forward by Neumann (/>') rf°z-and Monasteric {(°) . Davies and Davies ( 2 3 ) . , Hinsberg and Pett ^) have examined the nature of interference by extraneous substances to reaction in the colorimetric methods and have suggested modifications. Valuable service would be rendered in a c r i t i c a l comparison of these various micro-methods. Hinsberg (Jf) has done something towards this end. Emmert (•*•*') and Litynski (*/) has put forward colorimetric methods suitable for use with vegetable tissue. Total phosphorus and organic P, by f i r s t combusting materials, may be determined by the above colorimetric methods. Elek and H i l l K ) , Pulcher (-), Garell i ( -) have proposed estimation of organic P by f i r s t combusting materials in a micro-bomb. Lohmann (•*'<>) , Boyland (") and Hinsberg give micro-methods for determination of pyrophosphoric acid. 50 -18-Menjdahl fr) for metaphosphoric acid and for phosphorores and hypo-phosphoror«3 acids. Microdeterminations, gravimetric and colorimetric, are given through the literature on phosphatases. Harden (3t) , Pryde fa), Raymond and Levene (m) : for determination of hexosemonophosphoric acid see Cori and Cori (x/) . Determination of small quantities of phosphatides may he made by methods of Bloor and Luider (•>-*) , Backlin (- ) , Jewett , Morberg and Leavi l l ['?) , Jordan and Chibnall , levene and associates («•?) . Hexoses may be determined by the Hagedvorn and Jensen method or by Hanes t52) modification of i t . Several colorimetric methods for lact ic acid determinations are available. .5 . THE BOLE OF PHOSPHATASES I I METABOLISM The role of phosphatases in metabolism is a multiple one as might be gathered from i t s wide occurrence in organisms. The chief current investigations on the part they play i n metabolic exchanges may be resolved as they concern (a) yeast fermentation (b ) the chemistry of muscle (c) the chemistry of ossification (d) the work of the kidney, (a) Their significance in yeast fermentation: Harden and co-workers i n a series of researches have been able to show that the addition of soluble phosphate to a yeast juice, hexose fermentation produces an equivalent amount of carbon dioxide and alcoho quantitatively expressed in the equation, thus: I. 2 C 6 H l g 0 6 + 2 E2HP04—> 2 C02 + 2 CgE50E + 2 HgO + C 6 H 1 0 0 4 (P0 4E 2} 2 . 51 -13-It has been shown that the phosphate is indispensable to the process and that at least three stages occur in the process. (1) A period coincident with the increased fermentation during which free phosphate rapidly diminishes. (2) A period of uniform activity where only small amounts of free phosphate occur. 15) A period of lessened act iv i ty and rapid increase of free phosphate. An enzymic hydrolysis similar to the last stage is effected after removal of the co-enzyme of zymase indicating the presence of a phosphatase, the action of which might be represented as: I I . C 6 H 1 Q 0 4 (P04E2) 2 + 2 HgO—? C 6 H 1 2 0 4 + 2 EgHP04 Harden ( ) gives the following simple explanation of the sequence of events during fermentation: "The rapid diminution in the amounts of free phosphorus during stage (1) corresponds with the occurrence of reaction I. During the whole period of fermintation the enzymic hydrolysis of the hexose proceeds according to equation II. Up to the end of stage (2) the phosphate thus produced enters into the equation according to equation II, with the sugar which is present in excess and is thus reconverted into hexose phosphate, so that, as long as alcoholic fermentation is proceeding freely no accumulation of free phosphate can occur." As soon as alcoholic fermentation ceases, however, phosphate accumulates, there being no hexose present with which i t might react. . Harden has shown recently three types of fermentation in yeasts, (a) the relat ively slow fermentation, without addition of P0 4 . (b) the more rapid process by adding P04« (c) rapid fermentation by addition of Arsenate by either stimulating phosphatase activity or by the formation of more labile hexosearsenates. 52 -14-The rate of hexose monophosphoric and pyrophosphoric acids in yeasts has largely to be worked out, (b) Muscle Contration: That phosphates play a part in muscle contraction is evidenced by the facts: (1) that free phosphate and lact ic acid are liberated in equimolecular amounts. (2) that by adding phosphate a l l glycogen is converted to lact ic acid. (5) that during muscular work ther is an increase i n the secretion of phosphate into the urine. (4) that performance of muscular work is augmented by addition of phosphate. In a long series of researched Embden, Meyerhof and their associates have established an intimate connection between the conversion of carbohydrates into lact ic acid in the contracting muscle and the presence of phosphates in the la t ter . Experiments were made indicating that a hexosemonophosphate (the lactocidogen) i s found as an intermediate compound in the breakdown of glycogen to l ac t ic acid. The changes may be represented in two phases (a) the anaerobic and (b) aerobic. anaerobic (contactile) 5 G 6 H 1 0 0 5 + 5 H20 + 4 K2HH>4 4 CgHjjOgtPO^) t- ' C 6H 1 20g + RgO glycogen. hexosemonophosphate. glucose. 4c0 6 H n O 5 tPO 4 K 2 ) —> 8 0 3 H 6 0 5 + 4 K2HP04 + CgH12Gg lact ic acid. glucose. aerohic (recovery) 8 C 5 H 5 0 5 + 4 glucose -t- K2HP04 + 602 —?4 C 6 H U 0 5 ( P04K2) t 6 C 0 2 + 10 H 2 0 The glucose freed during the contractile period is oxidized during the recovery period; then l / s of the glycogen is burned. 5 3 -15-That there are Enzymes in muscle capable of bringing about the changes outlined above has been demonstrated by Lohmann { ), Meyerhof ( ), etc. They have shown i t possible to separate completely the lactic acid producing enzyme free from muscle carbohydrate. A dialyzable thermostable co-enzyme has been discovered. The preparations split hexose under special conditions and readily act on starch and glycogen. In the com-plex i s an enzyme splitting hexose mono-and di-phosphoric acids. Embden and Zimmermann (*/} , Lohmann have found in addition to lactocldogen in muscle other phosphorus containing compounds, adenylic acid, and pyrophosphoric acid occur. Eggleton and Eggleton {<4 reported the presence of phosphagens. Lohmann has shown a definite pyro-phosphatase may act on adenylpyrophosphoric acid and pyrophosphoric acid, . to convert them to the orthophosphoric form. Meyerhof and Lohmann {s>\ investigating phosphagens {unstable compounds of inoyanic phosphate and creative or orginine) have found muscle immersed in sodium salts of monacetic acid and fatiqued, contract without utilization of glycogen or formation of lactic acid; ammonia was produced, phosphagen decreased, phosphoric acid increased, creature phosphate ^creature •+- HgPQ^creature — 7 » 1 H 5 + x. H 3 P Q 4 •+- hexose — h e x o s e phosphate —-7 H 3 F O 4 lactic acid. lactic acid—-9 COg -f HgO. Eggleton and Eggleton (ibid) demonstrated aerobic resynthesis of phosphogens. jLchmanschen anaerobic resynthesis after relaxation. (c) Chemistry of Ossification: « Me-Marrowless, dry bone consists of some 60-70$ of Ca.ly. phosphates 54 -16-and carbonates deposited in a protein matrip. Of these mayamic consti-tuents calcium phosphate is most important and investigations have chiefly revolved around the problem of i t s deposition. Several theories have been advanced and considerable experimental work done on the problem. The chief of theories involve the presence of born phosphatases. (Tell and Robison (w) .) (d) Significance of Phosphatases in the Kidney: Eichholtz, Robison and Bruell (*?) have attached special significance to phosphatases of the kidney in regard to the excretion of phosphate in the urine and some of the most recent work on phosphatases is on kidney phosphatase. Davies has investigated phosphatases of the spleen. Kagai [(>') and other Japanese workers are studying the importance of Phosphoric acid esters and their hydrolyzing and synthesizing enzymes in diet—in B-avitaminasis particularly. .6. PROBLEMS Prom the study of work being done on mammal and yeast phosphatases and from our own brief investigation we are confronted with numerous questions concerning the role of these enzymes and their substrates in the higher plants. The r&le of these enzymes in yeast fermentation and in muscular work has been closely linked with carbohydrate metabolism particularly with the ut i l i za t ion of carbohydrates in the production of free energy. 55 -17-Might there not be a close relationship i n the higher plants? With the , process of carbon assimilation i t has been suggested that phosphorus is not necessary. ( ) . In the transformation and synthesis of sugars apart from the photosynthetic process e.g. as occurs in malting barley, in sugar beet storage, etc . , phosphatases might play a part. Indirect evidence i n support of this suggestion is the universal occurrence of traces of phosphate in starch of plant origin. W i l l i s , Seland and Gray p1) working with soyabean have determined an intimate connection between Calcium, magnesium and phosphate, both i n absorption and processes within the plant. Demonstration of natural substrates in the higher plants, on which phosphatases might act i s l imited. Burkard and Keuberg (^ ) have shown hexosediphosphate to be present in sugar beet leaves, and Oockefair [*°\ in other plants; Msnjdahl and Weissflag (5J) have indicated the presence of meta- and pyro-hosphates. Phosphagens have not been reported for plants. Phosphorus metabolism in general, and phosphorus absorption more particularly i n the higher plant i s notably influenced by changes in radiant energy. (Barton-Wright {ff) .) Might not phosphatases and their substrates, directly or indirectly, be involved in this relationship? Some enzymes are l ight sensitive. Are plant phosphatases similarly constituted? The study of phosphatases might have a directly practical value in the malting of barley. Quality in malting barley is determined to a great extent by the ab i l i ty of the seed to hydrolyze i t s starch reserves to y ie ld sugars. Could an intimate relationship be established between quality and phosphatase act ivity a ready test for malting quality might 56 -18-be devised. This brief speculation may at least indicate the variety and number of problems relating to plant phosphatases. BIBLIOGRAPHY 57 llBiaO&ffiPEI 1. Auhagen, E . and Grogychi, Biochem. J . 265; 217-22 (1955) 2. Bakwin, H. and Bodansky, A. J . Bid. Ghem. 101: 641 (1933) 3. Barrenscheen, H. K. and Beneschowsky, H. Bicchem. Z. 265: 159-168 (1933) 4. Bayliss, W.M. "The Nature of Enzyme Action" Longmans, London 1919 5. Bayless, ¥ . M. "Principles of General Physiology" Longmans, London . " 1924 . 6. Belfanti, L . } Contardi, A . , and E r c o l i , A . Biochem. J . 29: 1491 (1935) 7. Bel l and Doisy J . B i o l . Chem. 44: 55 (1920) 8. Bloor, W.C. and Snider, E . H. J . B i o l . Chem. 107: 459 (1934) 9. Bodansky,, A. J . B i o l . Chem. 99_: 197-206 (1932) 10. Bodansky, A . , Hallman, L .P . and Bonoff, E . J . B i o l . Chem. 104: 475-8"5Tl934) 11. Boyland, E . (a) Biochem. J . 25: 219 (1929( (b) Biochem. J.24; 350 (1950) 12. Boyland, E . and Mawson, C.A. Biochem. J . 28: 1409 (1934) 13. 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