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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. ( B r i t i s h ColumbiaJ  Thesis Submitted i n Candidacy,, for the Degree, of Master of Science i n Agriculture.  The University of B r i t i s h Columbia. September 1936  2  ACMOWLEDGEMEITS  I express my gratitude to D r . A . H. Hutchinson, Head of the Botany Department, for M s assistance and interest i n the development of t h i s problem.  My sincere thanks are also extended to Mr. W. Jack and Mr. B. H i l l a r y for valuable help i n 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* 1.  .II.  .III,  Pg» 4  Introduction  Pg» 4  2. Materials and Methods  Pg. 7  5.  Experimental Results  Pg.21  4.  Discussion  Pg.36  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 i n Metabolism  Pg«,5Q  6.  Problems  Pg.54  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 s c i e n t i f i c 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 t h i s , we commenced work on plant phosphatases choosing as our representative  spermatophyte-barley.  The occurrence and necessity of the element phosphorus i n the l i v i n g c e l l was established by de Saussure (1804), Sachs (1860) and other investigators  early i n the 19th century.  The discovery of  its  e s s e n t i a l i t y to l i f e and i t s close association with carbohydrates, fats and proteins has led to the b e l i e f that i t s study would be a key to disclose much of the mechanism of l i v i n g  organisms.  Progress i n 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 i n organisms. years t h i s state has been a l t e r e d .  In recent  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 i n physiology following the important discoveries of Harden and his associates (8) i n 1905 on the general effect  of the addition of sodium phosphate  increasing the t o t a l 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' l i p i d e s , phosphoproteins, meta-, ortho- and pyro-phosphates  etc.  have assumed an import i n many metabolic processes. Prior to Harden's researches, Buchner (1897) had succeeded i n i s o l a t i n g from l i v i n g yeast zymase, which, when freed from the last t r a c e  0  f organized c e l l material was able to bring about  identical fermentation processes as had before deemed to be possible only i n the presence of active yeast c e l l s .  Enzyme chemistry  developed rapidly i n 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  d i s t r i b u t i o n i n the plant and animal kingdoms was soon indicated. Phosphatases and their substrates have now, i n addition to t h e i r importance i n fermentations,  become significant i n the study of  muscle contraction, o s s i f i c a t i o n , i n the work of the kidney and i n many other processes i n mammal physiology;  In plants we know  l i t t l e or nothing of their r e l a t i o n to metabolic processes. Conceivably they might be of importance i n the transformation of sugars apart from the photosynthetic process,— i n the germination of malting barley, i n sugar storage by sugar beets.  I f i n mammals  they are intimately linked with the calcium compounds, might not the same be true i n plants,  Many questions might be asked.  The writer while r e a l i z i n g the many problems presented by phosphatases and their substrates i n plants has sought only to  6  -5prepare the way for their future study. not far reaching i s fundamental.  The  work accomplished while  Something has been gained  about the preparation of phosphatase extracts, and something determined of their d i s t r i b u t i o n through the development of the barley p l a n t .  7  MATERIALS AND METHODS  (a)  The Plant Materials Seed of a pure l i n e of Duckbill barley (a six-rowed variety of  Hordeum vulgare L.) was obtained from the Department of Agronomy and sown, 4 seeds i n each, i n 8" clay pots i n phosphorus free sand. One series received weekly Hoagland's complete nutrient solution and a second series received a similar solution i n which potassium dihydrogen phosphate was replaced by potassium chloride.' The complete and phosphorus deficient solutions were made up as follows! Reserve Solution 1.  »*  '  "  2  .  KbI03  67 g.  MQS04  100 g .  ) i n 1 L*aq. d i s t . )  Ca(N0 3 ) 2 .4HgO 208 g.  i n 1 L.aq. d i s t .  )  »  "  3.  KHgP04  50 g .  i n 1 L.aq. dist.  "  "  4.  KCh.  26 g.  i n 1 L.aq. dist.  22 c . c . , 26 c . c . and 12 c . c . of reserve solutions 1, 2 and 3 respectively added to sufficient aq. d i s t . to make 2 1. gave the phosphorus deficient nutrient s o l u t i o n . Since the water requirements of the plants at  different  stages of growth varied so greatly and since the object i n 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 i n a l l the pots. water likewise was given as required.  Distilled  8  -2F i r s t sowings were made November 2, 1935 and similar series 'were set up during the experimental period ( u n t i l Ma r ch 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 e a r l i e r than plants sown the second week i n January.  As  a result where enzyme a c t i v i t i e s at different growth stages were to be compared, plants of a single sowing were used. Plants of the second series showed marked phosphorus within five weeks.  deficiency  At eight weeks the deficiency was very pronounced  with t y p i c a l 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 greenhouse temperatures and short photoperiods. favorable  for aphis  Such a condition was  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 wa s required, dormant seed was placed between b l o t t i n g paper i n a dark room at 50° + 3° CT f o r  (b)  five  flays.  Materials and Methods Required for Enzyme Action i n v i t r o : Phosphatases effect  the cleavage of phosphoric acid esters  l i b e r a t i n g free phosphoric a c i d .  To determine t h e i r presence and  a c t i v i t y the usual conditions for enzyme action i n v i t r o must be set up. To a substrate (some given phosphoric acid ester] i n a solution buffered to a pH. suitable for reaction and set at a desirable temperature,  is added a c o l l o i d a l solution containing the enzyme  previously extracted from the l i v i n g plant t i s s u e .  At the end of a  known period the phosphoric acid liberated is determined, assuming the amount of acid freed i n unit time i s indicative of the a c t i v i t y of the phosphatase. In greater d e t a i l 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 i n 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 t r i c h l o a c e t i c acid was added, the mixture centrifuged and the liberated phosphoric acid determined by the Zing ( 5 colorimetric method.  Series were run i n duplicate.  After the conditions for enzyme action i n v i t r o were set up and a reaction l i b e r a t i n g phosphoric acid shown, i t was necessary to show that the action i s enzymic and not an ordinary catalyzed chemical reaction. Certain characteristics  of enzyme preparation and their action  are used to determine the difference.  In b r i e f some of those are:  (1) the thermal i n s t a b i l i t y of enzyme i enzymes are inactivated at certain temperatures. (5)  (2)  their s e n s i t i v i t y to 0(H) and C(OH).  their activation and i n h i b i t i o n by certain substances.  c o l l o i d a l and protein nature as exhibited i n extracts. a b i l i t y to be s p e c i f i c a l l y absorbed, to maceration,  etc.  (6) t h e i r  (5)  (4)  their  their  sensitivity  etc.  In addition to demonstrating the enzymic nature of a reaction, i t i s sometimes desirable,  though d i f f i c u l t of proof, to  establish  the s p e c i f i c i t y 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. investigations  This i s pertinent to phosphatase  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 i n different ways—e.g. barley extract on a hexose •ester.  In this case glucolysis i . e . enzymic degradation of the  hexose unit to l a c t i c acid might liberate free phosphate, or, on the other had a true phosphatase might act y i e l d i n g free phosphate but leaving the hexose unit i n t a c t .  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 s p e c i f i c i t y of enzymes.  (1) Preparation of Standard Buffer Sorenson 1909 (10)  Solutionst  and Michael i s 1909 ( H ) recognized and  emphasized the fact of the profound influence of C(H) and C(0H) on the a c t i v i t i e s  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 i n enzyme chemistry. The choice of buffers which can be employed i n phosphatase reactions are l i m i t e d .  Since free phosphate would interfere with  the presumably reversible reaction i n which phosphate i s 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 i n h i b i t  certain enzyme reactions and only those, found by t r i a l to permit suitable a c t i v i t y , 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 i n 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 i n table by c  l a r k (13) . t'c) 7.505 g. glycine +• 5.85 g, sodium chloride i n 1 l . * 0 . 1  N Sodium hydroxide; pH r a n g e n . 2 — 8 . 3 ; prepared as given i n table D y  Clark (13); buffer mixtures by Sorenson (d)  (10).  .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) M i c h a e l i s ' (14) veronal buffer.  (2)  Protectants: Enzyme chemists generally employ some protectant i n 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, i n high concentrations, are frequently employed since reactions of an enzymic nature are not greatly inhibited by them. at a drop per tube.  In our experiments we used C P . toluene  Chloroform, and concentrated sodium chloride  were used with equally good r e s u l t s .  15  -7(5)  Substrates; Since esterases vary i n t h e i r a b i l i t y to hydrolyse  t h e i r 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 .2.  free.  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 a c i d . .4. ester was f i r s t  Hexose monophosphoric a c i d .  (Robison ester.)  This  isolated by Robison and co-workers (77) and i s found  along with the d i - a c i d during yea3t fermentation. have been used for i t s i s o l a t i o n .  Several methods  We used the following procedure  by Raymond and Levene (7&) . To l i v e 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 wa s interrupted by adding t r i c h l o r a c e t i c 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 l a r i f i e d 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 p u r i f i e d by repeated solution and p r e c i p i t a t i o n . Pryde (#)  gives Robison's Method for the preparation  of  hexose d i - and mono-phosphoric acids by yeast fermentation.  This  procedure has an advantage i n i t s exactness and production of higher y i e l d s .  It i s , however, much less r a p i d .  The preparation of  the Itfeuberg (?•?) ester by hydrolysis of yeast diphosphoric acid i s also given by Pryde. Pasternak % j) has recently prepared the Robison ester from e  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  filtrate  was treated with two volumes of alcohol and purefied by further precipitation from equal volumes of alcohol; from rotations Barium s a l t and free acid and from the melting  of the  points of the  phenylhydrazine s a l t , Pasternak found his preparation to be i d e n t i c a l with Robison's  ester.  Sodium phenyl phosphate, phosphate, for t r i a l .  Sodium metaphosphate, hydrogy-ge-r-miolitte  etc. are obtainable i n pure form, and are suitable substrate Sodium hexosediphosphate may be obtained i n r e l a t i v e l y  pure form under the trade name " c a n d i o l i n . "  (4)  Preparation and P u r i f i c a t i o n of Enzyme Extracts; The method used i n obtaining an extract of phosphatase with  i t s associated materials depends essentially on i t s s o l u b i l i t y i n water and i n s o l u b i l i t y i n protein precipitants  such as alcohol,  15  -9~ acetone,  saturated ammonium sulphate solution,  To transfer  etc  the enzyme from the plant source to an aquaeous  infusion some method of opening the c e l l structure must be found. We t r i e d autolysis  ( i » e « automatic dissolution of c e l l s by unchecked  degrading enzymes such as the erepsins) and mechanical fragmentation. The a c t i v i t y of extracts obtained with different autolyses and with different periods of maceration i n a h a l l m i l l , i n a hand mincer and i n a mortar with sand and with glass wool, i s given i n TABLE I;  TABLE 1 Extract Sources Buffer;  germinating seed—at 6 mgs. a tube.  veronal—pH 5 . 6 .  Substrate;  2% Sodium glycerophosphate.  1 hour.  Temp.t  gg" C.  t  1  TUBE  Timet  1  - . • EXTEACTIOH EROGEDUEE. •  •  TOTAL"p INITIAL pH iMGS. | FREE P \ PER C.G.f MGS.PEE j c* 0 •  PINAL FREE P READING  f  !  p  1  \  • •  i 2  5  22  .02  .44  .06  55  .00  i  -  .44  . . i.06  35  .00  \  5.5  .44  .44  B a l l mill—1/2 hour.  - 5 - B a l l mill—1/4 hour. 4  .08  ; B a l l m i l l — 4 hours.  Mortar—sand—15 mins. Mortar—glass wool— 15 mins* Mincer-autolysis— 4 hours.  5*5  j  a little;  a little  1  •1  .44  :  .06  53  .01  18  .05  The method adopted i n subsequent work was to grind the plant  ! E  _  I  16  -10plant material twice i n a hand mincer and followed by 10-15 minutes maceration i n a mortar; water was then added to 90$ (only i n 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 l e c t r i c a l l y driven centrifuge, f i l t e r e d through a coarse f i l t e r paper and from the f i l t r a t e by twice p r e c i p i t a t i n g from 25%a i c o h o l the enzyme extract was obtained.  The extract,  dried over concentrated  HgS04 had later to be ground f i n e l y , "dissolved" uble matter determined.  anf  j the weight of insol-  4 mgs. of extract were generally added  to  each tube, which amount gave a satisfactory reaction. TABLE II Extract Sources Buffer:  germinating seed. Time:  veronal.  Substrate:  2% sodium glycerophosphate.  Temp.s  1 hour. 5 0 ° 0.  1  TUBE 10.  j TOTAL PS .INITIAL STANDARD P LIBERATED P ENZYME j OF pH.JMGS.PEEl FREE P READING| MGS.PER READING MGS.PERf JIGS. | ENZYME! I O.C, j I O.C. I C*Co I  1 2  8 8  3 4  4  5 6  4.  2 2 4 4  Active 15.11 . 4 4 " [5.7 I . 4 4 . " | 5 . l | .44 " ' |5i7] v.44V J5.1 I . 4 4 " J5.7l . 4 4 Inactive |5.1 \ . 4 4 I " ;15.7.| . 4 4  ilittle - 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 { little' ti j tt  i  it  i  21  .30  D  o ci  CQ £**'  Q»7r  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.  Temperature:30°C.  >enes  17  -11Precipitantst  Methyl alcohol was used as a precipitant chiefly  because i t was available i n c o n s i d e r a b l e quantity.  Generally 8-10  times the f i l t r a t e volume of alcohol brought down a flocculent proteinaceous p r e c i p i t a t e .  It was noted, from general observation  the more rapid the p r e c i p i t a t i o n the more active was the extract. We were unable to develop a uniform p r e c i p i t a t i o n time even with the addition of coagulating agents. D i a l y s i s as a further means of p u r i f i c a t i o n was not employed because i t led to a decrease i n a c t i v i t y 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 a l c o h o l . TABJEE III  Enzyme Extract;  Buffer:  Potassium Acid Phthalate.  Substrate;  TUBE pH  A—acetone precipitation. B—alcohol p r e c i p i t a t i o n . C—ammonium sulphate p r e c i p i t a t i o n .  Sodium glycerophosphate.  2  1 1 / 2 hours.  Temp.:  3 2 ° C#  ENZYME TOTAL Pf I N I T I A L l J T ^ A E L ^ B E S ^ ^ I X P E P ^ I E N T A L ^ T O T E S IESDING~T'P M G S T " EXTRACT MGS.PEEfPEEE P 1 I I E A D I N G T P ~ M G S i . C.C.  i  Time:  A t!  .44 .44  '.44  |MGS,PER{ . .•  Ge C« )  20 20 20  PEE  PEE CO.  •J  13 7 11  C.C.  .15 .29 .18  fTablg T i l conolmxgat  18  -122!ABLE III  KIBE|_  10..  JVM  (Cont.) j j 1 ENZYME TOTAL p| INITIAL |STANDARD KIBES • . EXPERIMENTAL TUBES EXTRACT! JIGS. PES! FREE P j READING | P MGS. READING P\MGS. r MGSgPES) PER C.C. PER C.C. i c. c. j j>  *!'  f  1  5.0 I s . 5.6 i " 6.5 [ • •«  2 3  •; V44 .44 .44  | j ]  20 j 20 j 20 j  trace  ti  H  «i  20 25 18  .1 .1  .10 .08 I- •. .11 !•  . "• . •  ,1 5.0 0 2 ' 5.6 j " 3 6.3 1 tt 4 5.6 :  1  '  . . j . .44 1 .44 J >44 j .44 '1  .05 .05 .05  20 . J .1 . - ' . 20 i ' .1 . ; 20 I. ,1 '. 20 1 • a  14 10  a4  120 .10  20 "  " "  :  In the study of mammal phosphatases, tissues i n a solution of substrate have been found to s p l i t off phosphoric a c i d .  While i n tissue  reaction the unknown factors are increased i n number, they have an advantage i n rapidity of preparation.  We t r i e d several reactions with  tissues of the barley plant. (5) Trichloracetic Acidt  This acid i s a general precipitant of proteins,  congulating them i n such a way as to render enzymes i n the extract, i n a c t i v e .  proteinaceous  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 i n t h i s determination by protein through their p r e c i p i t a t i o n .  Hinsburg (tf) using Fiske and  Subarrow (31) colorimetric method reports that the use of t r i c h l o r a c e t i c acid may cause an error as high as 38$. (6) Method of Determining Free Phosphoric Acidt  Since B e l l and Doisy (/)  19  -13proposed their method for the colorimetric determination of phosphoric acid i n 1920 many modifications of i t have appeared. used a recent modification of Kin© (•%') . as  In our work, we  Materials required were  follows:  the 1:2:4  .1.  72-60$ perchloric a c i d .  .2.  5% ammonium molybdate (phosphate f r e e ) .  .3.  0.2$ 1:2:4 aminonaphtholsulfonic acid (.5 gms. of  of the acid, 30 gms. sodium bisulphite and 6gms. sodium  sulphate dissolved i n aq. d i s t . to make a volume of 250 c . c . j  left  stand overnight, f i l t e r e d and prepared every 2 weeks.) .4.  Standard Phosphate 2.1955 g. KH g P0 A 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 t r i c h l o r a c e t i c acid precipitate and s i m i l a r l y to tube containing 10 c . c . of standard phosphate or some d i l u t i o n 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 i n the colorimeter. According to de-Beer's Law the absorption of l i g h t by solutions i s d i r e c t l y proportional to the concentration of the coloring substance. The amount of phosphate i n 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^ i s 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 i n 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 " d r i f t " and " s a l t 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>) i n their valuable notes have described the use and care of the quinhydrone pH indicator. use.;  Coles Iff) also deals with i t s  Though only small amounts of extract are available,  goes into pH determination tubes.  l/z  generally  B.D.H. c a p i l l a t o r , 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  To demonstrate the existence of an enzymic reaction, with  (a)  phosphoric acid esters as substrates, i n which one of the end products i s free phosphoric a c i d :  •  Free phosphoric acid i s not liberated except i n the presence of the active enzyme extract.  From the data i n Table IV i t is seen that  combinations of buffer and substrate without active extract, as of buffer and active extract without the substrate y i e l d no phosphoric a c i d ; that the buffer plays l i t t l e part i n the reaction other than regulation of pH i s indicated i n the l i b e r a t i o n of phosphoric acid by a combination of enzyme and substrate with buffer absent. TABLE IT Extract Source: Substrate: Buffer:  TUBE NO.  •.  2% Sodium glycerophosphate.  EXTRACT 1 C.C.  I  -t-  •H-  5  'I  2 6 ° C*  1  -+-  •f-  I  ~  1  — :  EXTRACT TOTAL P LIBERATED P MGS.PER MGS. PER BOILED C.C. Q» C e X Cftc«  .44  .27  .44  trace  .00  •00  •44  •04  .44  .00  •  •f-  . r  • "  ••""•]  1 1 1 1  r  4  1 hour.  Temp.:  -  •j BUFFER I SUBSTRATE 10 C.C. j 1 C.C.  <— '•  Time:  Potassium Acid Phthalate; pH 5.5.  •• 1 ' ,•' 2  germinating seed.  +  —•  -r .  +  "  '  *~-  •  -**  •  —  —  .-  22  -2The thermal i n s t a b i l i t y of an ester cleaving substance is shown i n a consideration of Table V and Figure 2.  The data, while not very  satisfactory from the point of view of completeness i s sufficient demonstrate the progressive inactivation of the above and below a broad optimum around 2 6 - 5 7 °  to  enzyme at temperatures  0.  TABLE 7 Conditions as i n Table I V » Extract Sourcei  A-germinating seed. B-leafy tops.  I  •  8  P  MGS.PER:  !  1  INITIAL P MGS.PER  LIBEEATED P MGS . P E B C « Ce  C« C 8  •  L  1  f i  7° • •  2  1  26°  3  .44  11  • •  A  4  60-65°  5 . .  57°  100° •  1 (a) *  37°  B  —  .22  .44  —  .25  .44  .05  .44  f B & A I • •  •  ,'•'  „04 .05  A  Tube 1 (a)  »44  • .44 ;  •  —  .44  A  I f  " " 65'°  7  1 \  •' .  •  A A  37°  6  *  TOTAL  T I M P E M T U B E I EXTEACT ° C.  (TUBE NO.  . "'  ;  .10 • .,  •'•  .05  *05  .05  .15  of pH:  The hydrogen and hydroxyl ion concen-  t r a t i o n of a medium i s a fundamental influence i n most enzyme reactions.  A c t i v i t y pH curves comparing relative e c t i v i t y with the  pH may be constructed to demonstrate a characteristic  i  ^ ^  •  optimal region  ! \  - '• •  is Tube 1 after 1 hour at 7 ° C.  The Signifigance  .  i  33  KJ t  25  /  v <4  s 1  .20  cS i  \ \ \ \ \ \  i  \ \ \  §  i i  i t i  >  i V VUS!  §  o •  %  %  t  o* \  »  40  .  60  » \  \ \ %  E19 2: Activity-Temperature Series  ExTmct Source: (a) ferminaf/np seed (b) fops Subsfrafe ; 2 ^sodium 9lycerophosphate Buffer: Phthalate  T/me.-l hr  of a c t i o n .  (Figures 5 and 4 ) .  With crude extracts and often with highly purified ones the position of the reaction optimum i n r e l a t i o n 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 i d e n t i c a l 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  a c t i v i t y 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: Substrate: Buffer:  germinating seed—6 mgs. per tube.  2% Sodium glycerophosphate.  veronal.  TDBEIIITIALTIIABEIZYMTOTAL PJNITIALj MGS. PERPEEE P HO. pH PH C.C.  1 2 3 4 5  3 »B 3.2 4.5 +• 4.4 5.2 5..2::; f 5.8 j 5.8 T6.8 6*7 -f 6 7.5 7.4 7 i 8.0 7.8 | + 8 I 5.8 5.7 -  I .44 .44 | .44 .44 I .44 j .44 .44 .44  Time:  1 hour.  Temp.:  2 6 ° C.  STANDARD  P LIBERATED  jffiGS . PERJREJDIMGJ MGS. PteEADING-t MGS .iREADING!MGS. PER PER j I PER 1 0. C. j 0• Ce  .02 .02 .02  20 20 20 20 20 20 20 20  .1  «x  ,»i 9  X  .1 .1 .1 .1  30 27 22 18 18 25 37  XX  31 27 25 17 19 29 27  trace  —  a  .11 .08  —  .06 .074 .09 .10 .11 .050 .054 trace  24  -4TABLE VII Extract Sources ~ Substrate; Time;  A - germinating seed ( acetone p r e c i p i t a t i o n )—6 mgs. B - dormant seed—6mgs. per tube.  2% Sodium glycerophosphate.  1 hour.  Temperature*  iTUBEjEKITIALf IMlpiZTJfflTOTAL ppiTIALJ jMGS.PEE P pH ! pH | c.c.  1 2 3 4  5  I7 18  1 2 3  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  5.5 6.5 5.5  1 5.5 | 6.5 5.5  B B  P LIBERATED  STANDARDS;  I  PGS.PEENADIKG  0« G«  .44 .44  2 6 ° 0.  .44 .44 .44 .44 .44  20 .20 20' 20 20 20 •20 20  .44 .44 .44  l i t t l e | 20 — j 20" I 20  MGS .jPREADINMGS .SEALING! M G S . PEE PEE jPEE C.C. C» G« G* Ot  .1  1.05 i.l  .1 .1 .1 .1 i .05 ! .05 i .05  35 21 9.' 11 25 25 26 —  [.18 U08 i.05 1.05 ittrace  .1  ii  1,18  a  .i  12  1*24'  38 18 11 11 53 28 26  —  .05 1 .11 I ,18 I .18 ! .06 i .07 j .08 it race  1.16  The optimal reactionzones for a l l our phosphatase extracts l i e on the acid side of n e u t r a l i t y . Correlated with this i s the fact that the reaction of the crude c e l l sap of barley i s a c i d .  (pH 5.0-6.8).  This does not preclude the p o s s i b i l i t y of barley phosphatases with optimal reaction zones on the basic side. A l l our pH a c t i v i t y curves (figures 3 and 4) possess r e l a t i v e l y broad zones of optimal reation.  This i s i n s t r i k i n g contrast to the  a c t i v i t y pH curves for mammal phosphatases where .2 pH may cause large differences.  25  -5Suitable salt mixtures whose a c i d i t y 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 a c i d i t y due to products already present or products formed i n 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 a l t e r 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 i n 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:  Buffer:  A - germinating seed (dialyzed)—6 mgs. B - normal root (dialyzed)—6mgs. C - germinated seed (not dialyzed)—6 mgs.  Phthalate.  Substrate:  2% Sodium Glycerophosphate.  Time: Temp.:  1 hour. 2 6 ° C.  26  -6TABLE VIII - Continued:  M CL G  2  6 mgs.  STANDARD [ E X T R A C T T O T A L Pi F R E E P i JJlBJEAJSaLIL M G S . P E R J M G S . P E E ! READING! M G S | P E E ! R E A D I N G I M G S . P E 3  C»  PEE  0 • 0A 1  15.6 i  &  I  A  .44  B  .44  I  3  .44  i  5 J5.1  A  .44  J5.1  A  .44  7 :s.5  C  .44  1* i'5.6  A  .37  20  2* 5.6  A  .37  20  3 J5.S | ; - 4 I5.6 ' '  6  +  ! 20 I  j'.20  "i  *  Substrate:  20  :  frrace  | I  Co Ce  9. : 18  .11  .1  14  .14  ,1  33  .06  «& X  15  .13  oX  33  .06  X  12  .17  20  .10  35  .06  0  20  I  I  .1  ! 20  A  0»  [ .1  Sodium pyrophosphate.  Prom several series, d i a l y s i s of dormant seed extracts seemed to increase t h e i r a c t i v i t y and i t was thought that a dialyzable, inhibitor of barley phosphatases had been found.  thermostable  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 l e f t i n abeyance. (a)  D i s t r i b u t i o n of Phosphatases i n Barley:  A substance i n barley precipitated as a protein sensitive to maceration and capable of catalyzing a reaction i n which free  phosphate  1 8. 1  \  • r V  \ \  \  p  X  X  \  $ /  f  •  ( >3'  .20  ./a  liberated  F?mp&  .26' perac  EiqA-. Activity-pH Series Mw£JMMm:  germinating seed Substrate•• Naglycerophosphate Time Bufer: Phthalate Im^tm.: 26°0.  *0  to a •  CD  / /  CO  0  •  P ©  _ fflSB <*s  XJ" 1 I  \  <  > s  X •  '>  e CD  4> V  *  cd  27  -7was s p l i t from phosphoric acid esters, a substance with a c t i v i t y 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, The following tables IX-XIV i l l u s t r a t e  page  ).  the qualitative  distribution  of phosphatases through the development of normal and phosphorus barley from seed to heading.  deficient  While a few short series not given, were  run at other stages of development the date i s sufficient  to indicate the  d i s t r i b u t i o n of the enzyme, i n a gross way at least, throughout growth and i n a l l parts of the plant.  Table XII summarizes this data.  Many  omissions are. obvious, p a r t i c u l a r l y i n regard to the pyrophosphatases. TABLE IX Extract Sources  A - germinating seed. B—  Buffer;  Potassium acid phthalate.  Substrates  !•• '  !  pH  2  i  .' A  •.'•'.A.' ' 4 .-A5 A 6 j  "  5.0 5.4 6.1 6*7 7.2 5.4  C.  .37 .37 .37 .57 .37 • 57  1  B B B _  6.1 6.7 5*4 5*4  .37 .37 .37 .37  1 '  trace •  trace  -s-  g g ° C.  LIBERATED P i R E A D ING j MGS. P E R 1  | PER C* Ce I j  0.  20 ,i 20 .1 20 ' 1 °1 20 . .1 20 .1 20 1 .1  1  •  l 2 3 .4  Temp.s  P F R E E P •... S T A N D A R D S MGS.PER MGS.PERI BEADBIG|MGS.P  1  A  1 hour.  TOTAL  0» Ce  ' 1 - ! l i  Time;  Sodium pyrophosphate .06 M.  ITTJBEEXTRACT : NO.!SOURCE !  dormant seed.  ,  20 20 20 20  • -  !  1  I 1  •X . 1 ©X  1  .1  i ;  •'  1 i  3.5 25 18 18 28  35 35 59  p n  j  .06  j | 1  .08 '.ll .11 .07  !.,  ~~  :  *055 .06 .05  I1 -  j j 1 I  28  -8TABLE X Extract Source: ' Substrate: -Buffers  A _ germinating seed—6 mgs. per tube. - dormant seed—S mgs. per tube.  B  2% HA glycerophosphate*  Calcium hexose™ diphosphate it u Sodium hexosediphosphate tt  Sodium hexosemononopho sphate (Pasternak) •" »  tt .  :  Sodium hexosediphosphate tt  Calcium hexosediphosphate tt  Temp.:  2 6 ° C.  tt  '  -  5.5  .44  5.9  .44  5.6  .52  ; -t5.9  .52  5.5  .34  5.7  ,34  5.6  .22  +  LIBERATED 'P . READING MGS.PER C.C.  STANLYUSD TOTAL P INITIAL MGS.PEB FREE P " READING MGS.P PER 0.0 C.C,  ENZTME  Sodium glycerophosphate tt it  »t  hour.  Potassium Acid Phthalate.  SUBSTRAW,  tt  ^ime8  •  20  a  ,7  .29  ——  20 -  a  10  .20  .25?  20  .25?  20  .05 " *  i04  a  6?  .08?  .a**.  6?  .08?  9  a?  :  20  a  20  . i  9  .17  a  20  .10  20  .10  •=•-9  •i-8  20  .  •*  t 5.9  .22  i04  20  a  5.6  .34  .05  20  ' a  6*0  .34  .05  20  a  5i5 5.9  .32 ,32  .25? s,25?  '20 20  a a  12  .  6.5?  '  ,11  .08 .06  CD  O  • *  CZ O o  CjJ 3  e £  > *  <  \* CD CD  ^  .0)  o  CD  CD O  O W  o|  s  c 3  i  TABLE XI Extract Source: Substrate' Buffers  A - Boots of Normal Plants—6 mgs. per tube. B - Tops of Normal Plants—6 mgs. per tube.  Sodium glycerophosphate.  Time:  Potassium acid phthalate.  TOTAL P INITIAL MGS.PEB FREE P G. G. MGS.PER Co Ce  TUBE EXTBAOTipH  No..  1.:i  1 hour.  Temp.t  26° 0.  FINAL P STANDARD BEADING| MGS.P jBEADING MGS. P -PEB C.C. PER ;'' LIBERATED [ Ga Co {  }.  i 2  I. i |.  S  j A ' • ; 5.0 A  3 -  A;'  4  A A  1  .1  20  .44  ! |  1J5.3 1 l 15.8 j . j6.5  —  j  ' .44 ,44  —  .44  i7.0  .44  .  —  :  20  a  20  a  20  a  J'  1 !  • • I \ 5.8  .44  • **—  . 22  1  .,09 ,  j  .16 .15  14.  .1  20  j  12.5 1 15  20  .08  |  {  {•  6  '  i ..  i  : •  • ....  {  -  i •  B ,  2  B  5.0 ' [ ; 5 .5  3  B  ' 5*8 |  .44  —  20  B  6.5 ;  .44  —  20  1  5  B  'w.of  6  B  5.8! I  - '  20  .44  1  15  .13  13  .15  20  .10  20 .  .10  35  .06  •  I  20  .44  .44  i  1 1  9  .44  —  •  20  a  20  a  j  .  '  30  -10TABLE XII Buffer;  Potaggi^m Acid Phthalate.  Temp.:  2 6 ° C.  Times t  I  EXTRACT SOURCE  :SUBSTRASE  ;..:v,  1  1 hour.  1  '•'  •  | OPT. ! PH  TOTAL'P ] INITIAL P LIB- $ OF ESTER PEE C . C . 1 FREE P ERATED HTDROMG. j MG. . LYZED  •  •  i  iDormant  2%Sod.iim glycero- i phosphate 5.5-6.0 2$ Sodium hexosediphosphate i5.5-6.0  %•  jSeed.  1 1 1  •  •  .34  j  f'" ' ' '  !'  iDormant f* i • •  '  jGerrax-  •  \ \  \ i  I  J f  : .18  j  ;  53%'  •  \  !  .37  •  •  .08  , 2 5 $ 27$  .10  ——  •  • ] i ' . • ,: 1  i : •  $ Sodium glycero• • phosphate i5.5-6.0 .44. > 2$ Sodium hexose- i' diphosphate 5.5-6.0 .54 2% Calcium hexosediphosphate 15.5-6.0 ;•• .52 • 2% Robison ester 5.5 • 22 '.06M Sodium pyrophosphate 6.0-6.5 a 37 2  nating j •'' ' : jSeed.  40%*  .18  •  j 1  i  •  !  ' ' •  f  !  ;  -  j  '  2$Calcium hexose: ! diphosphate 5.5-6.0 I !.06M Sodium pyro1, 1 s phosphate 6.0-6.5  Seed.  ;Roots o Phosphorus Deficient Plants  .44  -  .29  66$  e 22  65$  .08  25$  —  .10  45$  —  .11  —..  .08  18$  .34  .05  14$  .37  .07  18$  • •  .  —  - .  2% Sodium glycerophosphate 6.0 g$ Calcium hexosediphosphate 6.0-6.5 .06M Sodium pyrophosphate •'6.5.  .44  •• —  •  (Table XII Continued)  J f  50% . .  | i X  51  -11-  TABLE XII (Continued)  1  •  JEXTRACT  SOURCE  |. • iRoots of } INormal i jPlants | i VI  •  '  OPT. pH  jsUBSTRASE  1  •  -j' |2$ Sodium glyceroj phosphate 6.0 i 2% Calcium hexosei diphosphate 6.5 I.06M Sodium pyro| phosphate 6.5 .1 • . ' •  •  r(Tops of jjPhos2% Sodium glycerojphorus phosphate ^Deficient Iplants r(' $ AiTops of 2% Sodium glyceroiNormal phosphate plants  (. ' """ • 1  TOTAL P INITIAL P LIB- ['$ OP ESTER PER C.C. FREE P ERATED j HYDROMG. LYZED MG, - i •  j |  •1 k  ! ]  .44  .16  .52  .08  J  i .  f i •  .44 '  i  !  !  50$ .  .  15$  1•• 34$  .15  trace  .44 :  ] ••  .07  • • , . . . . . ! • • . ,  5.7  25$  j  .11  •  ! .37 '1 •  5.5  36$  f  ' { • • •  I  .)  TABLE XIII Tissue;  [TUBE  pH  (NO.  2  A - Normal Roots—15 c . c . B - Normal Roots inactivated—15  :TIS-  rsuE  SUBSTRATE 10 C O .  A  i Glucose  20  6.7  A  4$ Sodium glycerophosphate 10$ Glucose  20  4$ Sodium glycerophosphate  20  6.5  B  4  6.5  B  Time:  1 1/2 hours.  Temp.:  3 0 ° C.  INITIAL LIBERATED P STANDARD ._. FREE P j READING] MGS.PJ READING} MGS. |READING MGS. PER ! c. c. I •I" I C.C.f. i 1 C.C  6.7  3  c.c.  i  iI j  15  .15;  20  .00  11  .18?  12  .08  30  .07;:  22  .00  SO  .07!  21  .00  I 20  »1 ; i  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 i s i n speed of preparation.  In mammals where,  usually, highly specialized parts are more d e f i n i t e l y set apart, tissue reactions have proved of value (Bodansky (f)  ).  For what they are  worth then the data i n the Tables XIV-XVII are presented: TABLE XI7 Tissues:  Substrate: Time:  A B C D  -  roots roots roots roots  of of of of  Normal plants. Normal plants previously heated to 1 0 0 ° C. for phosphorus deficient plants. (5 minutes. phosphorus deficient plants previously heated to 100° C. for 3 minutes.  2% Sodium glycerophosphate.  1 l/2 hours.  Temperature:  5 0 ° C.  |TUBE pfi TISSUES j SUBSTRATE! I N I T I A L P j. STANBAEDS / : LIBERATED P |io. I i o c.bj. 2 c.c. |1EAD1N&TSG57] H E A D I N G | M G S 7 | R E A D I N G ! M G S . !  j. • • • • • ' !  I PER I  • •  PER  i  j  A  •  i  .08  -I-  20  ! >1- \  20  •+  40  .05  20  ,1 ;  40  .00  . —  20  a j  22  ,09  20  a  20  a  6.6  B  6.0  0  .00  14;  "6.0  D  .00  f  6.8  A  .20  -  —...  .1  . ,  j PER  1  6.8 • ! .1- '  :  trace i  | i  !  24  some i  33  ^..13TABLE XV Tissue?  A B C D  Substrates Times  -  active root inactivated active root inactivated  tissue of Normal plants* root tissue of normal plants. tissue of P deficient plants. root tissue of normal plants.  2% Sodium glycerophosphate.  1 hour.  Temperatures  3 0 ° 0.  INITIAL P LIBEBATED P STUBS | pH TISSUE SUBSTBATEj STANLABDS jNQ. 10 O . C i 2 C • C« 1 READING MGS.P[BEADING |MGS. j BEADING IMGS. PEB { . i PEB PEE C.C. Ce C*> Co C e 1 $  }  i  6; 8  i  • i  A  i  2 3  i  1 * •" I  20  a  20  oX  11  .08  •  i.  I  -  • • ••  i 5  6.6  B  6.0  0  6.0  D  \  ..  +  ;  20 20  «x  20  ' a  20  a  -  6.8  A  .1  ;  40  .05  40  ,00  00  ©  22  .09  trace  00  22  00  —  00  a  20  TABLE; XVI " Tissues; Substrates 2% Sodium glycerophosphate. A - Pi" deficient j tops a c t i v e , B - P deficient tops; inactivated by b o i l i n g . Times 1 1/2 hours, C - Normal plants; tops a c t i v e . D - Normal plants; tops inactivated. Temp.s 2 6 ° C. buBE iNO.  S;  1 " ' | pH 15 C.C. SUBSTBATEJTOTAL P INITIAL 4 C.C. iPEE C.Cj PEEE P TISSUE • ! MG. •  i  •  i  u  ! •  I'- -  i  STANDARDS LIBEBATED P BEADING MGS.P;.BEADING MGS. PEE PEE C oC- • C.C.  /  A  5.6  x  •  - '  5.4  B  +  3  5.4  C  -t  4  5.5  D  5  5.5,  A  aq.dist.  10  -  trace  20  •a  25  .08  trace  20  a  40  ,05  .20  20  a  7  .07  ,20  20  a  trace  20  -  18 1  .19 .02  (  54  -14TABLE XVII -Tissue:  A - normal barley kernel—15 c . c . B - P deficient barley kernel—15 c . c . Phthalate.  Buffer:  TUBE TISSUE NO.  1 l / E hours.  Temp.:  5 0 ° 0.  Co Co  INITIAL FREE P MGS. PER c« c«  STANDARDS READING MGS.P • PER  SUBSTRATE  Time:  LIBERATED P MGS. PER C• 0•  1  1.  Sodium glycerophosphate 2$  20  .1  ,05  .48  2  A  Sodium pyrophosphate .06M  20  .1  .05  .59  None  20  .1  .05  trace  Sodium glycerophosphate  20  .1  traces  little  5 4  B  Several points are worth noting.  Free phosphate, for an example,  did not increase markedly u n t i l the ester substrate had been added. Again heating tissues to temperatures of 6 0 ° and over caused a decrease i n 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  i n 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 i n  this direction were with green leaf tissues containing considerable free  55  -16esters 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 6 0 ° C. or oter. a disappearance of free phosphate. determined.  Tables XIY-XYII.  Such conditions led to  The nature of the reaction was not  56  DISCUSSION  Different preparations from the  same part of the barley plant, using  the same procedure, gave extracts of satisfactory but not uniform a c t i v i t y . More active extracts might be prepared using precipitating agents such as acetone or ammonium sulphate or using sodium chloride solution, rather than water i n 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 o r i g i n a l a c t i v i t y . S e n s i t i v i t y to grinding may have been an explanation for the lack of uniformity i n different extracts.  It was noted too that a c t i v i t y  varied with the length of time taken by precipitates to fluctuate i n 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 p u r i t y .  Davies [Z'f) and other investigators have found the King (w) c o l o r 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 i s 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.  37  -2In several cases our enzyme extract hydrolyzed i t s own weight of substrate i n one hour. i n extract,  this i s  With phosphatases, never with a high active content  satisfactory.  From our tests we concluded that the barley plant possessed true phosphatase systems l i b e r a t i n g phosphoric acid from ester substrates.  The  catalyzer we showed to be thermally unstable above 6 5 ° C. with optimal reactions about 2 6 ° - 5 7 ° C. broad optimal zones.  pH a c t i v i t y curves were determined showing  While optimal a c t i v i t y was always i n acid media  the p o s s i b i l i t y of alkaline phosphatases i s not precluded for no enzyme extractions were made i n alkaline media.  Precipitating by usual protein  precipitants indicated the protein and c o l l o i d a l nature of the extract. In common with other phosphatase extracts ours was sensitive to maceration and activated by magnesium ions. One point i n proof of the identity of our enzymes i s l a c k i n g .  The  hexose unit i n the sugar phosphates might be attacked g l u c o l y t i c a l l y to y i e l d l a c t i c acid and free phosphate.  By following the products of  the reaction quantitatively, the proof would be complete. manner a similar end could be achieved.  In another  Olucolysis occurs only i n the  presence of a dialyzable thermostable co-enzyme.  If following d i a l y s i s  phosphoric acid esters were cleaved glucolysis as a possible  factor  would be removed. Demonstration of specific phosphatases i n our extracts i s l a c k i n g . Loftmann (f/) by d i f f e r e n t i a l absorption was able to separate pyrophosphatase and sugar phosphatases i n muscle.  Levene by reactions i n  point of time was able to establish the identity of three separate phosphatases acting on phospholipides.  Hotto M  believing the  38  -3-  s p e c i f i c i t y 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) (b) phosphomonoesterases {c)  phosphodiesterases  pyrophosphatases (d) amidophosphatases.  The presence of a metaphosphatase i n corn has been suggested by Menjdahl and Weissflag if/} .  ¥aldschmidt-Leitz  (;*») upholds the determination  of Levine and Lohmann only* The d i s t r i b u t i o n 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 i n many organs and tissues, muscle, bone, kidney, blood, brain, i n t e s t i n a l mucose, and tumours.  In  yeasts and Aspergillus oryzeae, the source of takadiastase, phosphatases hydrolyzing many substrates are found. and jackbean.  Phosphatases are found i n rice  From our tests i t would seem that i n barley, also, their  d i s t r i b u t i o n i s wide, and are to be found i n 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 i n conditions of extraction and reaction quantitative results cannot be deduced from the data.  Many  refinements i n 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 i n phosphorus deficient as well as i n normal plant, though possibly less active i n the former.  DISCUSSION  39  .1.  INTRODUCTION  In view of the rapidly increasing numbers of researches on phosphatases and their related metabolic exchanges, recent pertinent l i t e r a t u r e i s d i f f i c u l t to appraise.  The following discussion is, as a  consequence, very limited both i n general outlook and i n 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 i v i n g c e l l s , but independent of the presence of the l a t t e r i n their operation." {Waldschmidt-Leitz)  With some important differences they are much i n  keeping with Ostevald's (f ) d e f i n i t i o n 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 i s  not a product, i s required i n small amounts only, and does not act i n molecular quantities,  (c) i n a general way the rate of reaction depends  on the concentration of the enzyme * hydrolytic. specific,  {d) most enzyme reactions are  Unlike most catalysts of the usual type (a)  their action is  (b) some particular optimum pH and temperature is required  for their a c t i o n . {c)  enzymes are usually slowly destroyed i n the  process of reaction, and optimum conditions are those i n 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  slight.  Although at the present time differences of opinion as to the mode of  40  . .  ~2- • • •  enzyme action exist only i n details as Waldschmidt-Leitz states the foundations for another important phase of the above definitions are much less assured, namely i n 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 proteins.  extraneous  Waldschmidt-Leitz and Purr (P7) have separated " c r y s t a l l i n e "  trypsin into four by special adsorption agents. The p r e v a i l i n g concept of the material nature of enzymes i s that of the Wilstatter school.  This school considers that enzymes are composed  of a c o l l o i d a l 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 c o l l o i d a l nature of the entire complex. Evidence i n support of the Wilstatter theory has been gained chiefly from well known and v e r i f i e d absorption experiments where enzymes i n 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  c o l l o i d a l i n character with an active specific group are the recent observations of Bredig and Gerstner { « ) wherein a diphenylamine group ad'ded.':to'cotton.made/atcatyst  s p l i t t i n g carbonic acid from B-heto-  carbonic a c i d , and those of Langenbeck [P] on activating groups within the active enzyme proper.  Pischgold's and Amnion's  theory of  esterase a c t i v i t y through displacement of H g 0 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 s o r t .  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 a f f i n i t y often results i n preventing a of even the simpler transformations  along basic p r i n c i p l e s .  classification The  v a l i d i t y of older kinetic-reaction research applying the mass low i n one form or another i s i n many cases very limited because of the many unknown factors disregarded.  Waldschmidt-Leitz { ) has reviewed  examples best supported by experimental evidence. Metabolic exchange systems i n 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 d e t a i l by Jacobsen M .  The determining factors  he found to be the l i b e r a t i o n 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 glycerophosphate hydrolysis by bone phosphatase i n the absence and presence of inorganic phosphate.  They found that even without the addition of  phosphates the v e l o c i t y constant calculated according to the equation for unimolecular reactions decreased more rapidly than could be  42  accounted for by i n a c t i v a t i o n 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  p o s s i b i l i t y of a resynthesis working with  glyceral and phosphate i n 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 r a p i d l y .  He  attributed the fact at least i n part to enzyme destruction and to i n activating products of hydrolysis. B e l f a n t i , Contardi and E r c o 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 v e l o c i t y 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 i s an enzyme-phosphate complex capable of hydrolyzing action. , • . The. dependence of the a c t i v i t y of most enzymes upon pH was f i r s t brought out by Sorenson (1909) and Michaelis (1909).  A characteristic  optumal reaction region i s generally apparent and the a c t i v i t y pH ratios can be expressed i n pH a c t i v i t y curves.  Many factors as has been  previously pointed out disturb or distort the measurement. concomitant substances such as buffers, important as  well as,  activators,  In this regard  inhibitors are  i n addition, the s t a b i l i t y 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, a undissociated M o l e c u l e s .  kations  At f i r s t , Michaelis implied that the undis-  sociated material was to be considered as the c a t a l y t i c a l l y 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 v e l o c i t y of the enzyme substrate combination.  Horthrop p") working with proteolytic enzymes found the  dissociation of the substrate to be influenced by the H i o n s £ his data showed a c t i v i t y - p H carves to be coincident with dissociation curves of the substrate. Michaelis {si) also used the behaviour of enzymes i n an e l e c t r i c a l f i e l d and t h e i r absorption a f f i n i t i e s to explain their electro-chemical nature.  In acid medium i n an e l e c t r i c a l f i e l d he showed the amphoteric  enzyme p a r t i c l e s moved to the cathode, i n alkaline medium to the anode, and i n the intermediate region corresponding to t h e i r 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 s o l i d bodies, has been fundemental i n absorption methods of enzyme p u r i f i c a t i o n so extensively used by the W i l s t a t t l e r school. if In p r i n c i p l e every reaction is reversible  (Hernst) and according  to the mass law the concentration of reacting substances determines not only the v e l o c i t y but also the direction of the reversible process. Theoretically these enzymes may catalyze reactions not only i n the direction of hydrolysis but also i n the direction of  synthesis.  Numerous examples of synthetic action by enzymes are known (Baylies  ['/)  44  -6on enzymic syntheses.) The recognition of the synthesizing action of enzymes has led to the question as to whether or not enzyme equilibrium i s to be regarded as i d e n t i c a l 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 e q u i l i b r i a have shown  that t h i s requirement i s not f u l f i l l e d i n a l l enzymic reactions even though the point of the enzymic equilibrium cannot be d e f i n i t e l y determined.  According to Euler [w] the equilibrium position of enzymic  reaction i s not determined as an ideal catalysis by the concentration of the substances concerned but by the r e l a t i o n of their to the enzyme i t s e l f .  affinities  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 l i t e r a t u r e 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  H3PO4.  l a g a i {^')  has found the a b i l i t y of kidney and l i v e r  of pigeons to esterify HgPC>4 to be 14-78$ greater i n a condition of Bavitatninosis 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 B o r i i ( ) obtained e s t e r i f i c a t i o n of  45  -7HgFO^ i n 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 d i f f e r e n t i a t i o n of a separate phosphatese i s 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 i n muscle to occur only i n the presence of a complete  .3.  co-ferment system (adenylpyrophosphate  MQ) .  ACTIVATION AND INHIBITION  In addition to the influence of hydrogen and hydroxyl ions, substrates, e t c . , activation of enzymes by specific activators, i n h i b i t i o n by i n h i b i t o r s of a specific nature can be demonstrated. Waldschmidt-Leitz (1)  classes these i n four ways.  Activation by Inorganic Ions:  e.g.  the s t a b i l i t y of enzyme-  substrate complex appears, for example, using saccharan, to decrease i n the following sequence i n the presence of these ions - NOg , CI , Bi , SO^ . (2)  The mode of activation i s not c l e a r .  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 a f f i n i t i e s of an enzyme or else with a definite stage of the reaction which i t accelerates.  One of the best demonstrated examples i s  the activation of pancreatic t r y p s i n by enterokinase from the i n t e s t i n a l mucous.  Waldschmidt-Leitz  believes there i s formed i n  46  -8this 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 i s l i t t l e known, might  be recognized as a t t r i b u t i b l e to specific (5)  activators,  Non-specific activation and I n h i b i t i o n :  Wil3tatt/Ler, Waldschmidt and Memmen {^\  It has been shown by  that activation phenomenae i n  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 g a l l s a l t s , e t c . i s due to the production of c o l l o i d p a r t i c l e s which exert and absorbent action with respect to enzyme and substrate thereby (4]  f a c i l i t a t i n g the reaction.  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 i n heavy metal ions and further that a c t i v i t y could be renewed by addition of HgS to precipitate the metals. apparently upon dissociation of enzyme-substrate (5)  The effect  is  complex.  Inhibition and Activation of Enzymes by Definite Salts on Organic  Addition Substances:  Many reports on this class of enzyme activation  and i n h i b i t i o n occur i n the l i t e r a t u r e .  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.  47  -9Lohmann H  found that hexosediphosphate loses no phosphate when added  to dialyzed muscle extracts; when  magnesium ions are present one  molecule i s l o s t , 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 a c t i v i t y increase for phosphatase i n general, but did find activation by arsenate i n special cases.  Wherever arsenate activation occurred arsenate less  rapidly gave a similar result*  Harden and Young (3£)  showed arsenate and  arsenite ions accelerated the l i b e r a t i o n 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 g l y c o l y t i c and not the  Braunstein and Lewitow ( ? )  phosphatase system.  observed progressive diminution of inorganic  arsenate i n a mixture of yeast, sugar, arsenate, toluene and aq, d i s t . and suggested the formation of l a b i l e hexosearsenates. (c) Activation and Inhibition by Potassium Cyanide:  Warburg M  concluded hydrogen cyanide i n the presence of phosphate stowed down alcoholic fermentation by yeasts and  suggested i t affected chiefly the  mechanism for l i b e r a t i o n of phosphate from hexosediphosphate.  Kiss  Patterson {v\ has i n some d e t a i l 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 i n h i b i t i n g action.  B l l f a n t i , E r c o l 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 i n a l l y proceeds as i f the oxalate were no longer present u n t 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 i n h i b i t o r y action. the explanation of these phenomena was  It was suggested that  that the inorganic phosphate  set  free from glycerophosphate gradually displaces the oxalate ions from the inactive enzyme—oxalate complex giving r i s e to an active enzyme—• phosphate complex capable l i k e 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  and several other  Fluoride has been known as an activator of lipase ', enzymes for some time and recently phosphatase has  been added to this l i s t , Auhagen and Grzycki (')  Loevenhardt and Pierce (-),  Smith and Lantz <f \ • 3  found kidney phosphatase to be unaffected by  sodium f l u o r i d e : found yeast phosphatase to be highly sensitive, and takaphosphatase from Aspergillus oryzeae less so. (f)  Sulfhydryl  groups:  Phosphatases  by sulfhydryl groups at t h e i r pE optimum.  generally are inhibited  Schaffner and Bauer (^)  .  Cysteine i n h i b i t s yeast phosphatase most at pH 6.1 and at pH 8.8 with kidney phosphatase.  D i s l y s i s renders the enzyme more susceptible.  49  -11Reactivation 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  i n the determination of micro quantities of orthophosphoric acid and their mention w i l l suffice here. Fiske and Sub arrow  , Martland and Eobison f 3 )  proposed colorimetric methods. gasometric method. Plimmer (A) .  B e l l and Doisy ( r ) , Briggs ( ),  Kirk [*•)  and Kinsp («-} have  proposes a very convenient  A micro-gravimetric method has been suggested by  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 i n the colorimetric methods and have suggested modifications. Valuable service would be rendered i n 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 (*/) suitable  has put forward colorimetric methods  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. K),  Pulcher (-),  Elek and H i l l  G a r e l l i ( -) have proposed estimation of organic P  by f i r s t combusting materials i n a micro-bomb. Lohmann (•*< ' >) , Boyland (")  and Hinsberg  determination of pyrophosphoric a c i d .  give micro-methods for  50  -18Menjdahl fr) for metaphosphoric acid and for phosphorores and hypop h o s p h o r o r « 3 acids. Microdeterminations, gravimetric and colorimetric, are given through the l i t e r a t u r e 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  L e a v i l l ['?) , Jordan and Chibnall  , levene and associates («•?) .  Hexoses may be determined by the Hagedvorn and Jensen or by Hanes t52) modification of i t . acid determinations are  .5.  , Morberg and  method  Several colorimetric methods for l a c t i c  available.  THE BOLE OF PHOSPHATASES I I METABOLISM  The role of phosphatases i n metabolism i s a multiple one as might be gathered from i t s wide occurrence i n 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 o s s i f i c a t i o n (d) the work of the kidney, (a) Their significance i n 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 i n the equation, thus: I. 2 C 6 H l g 0 6 + 2 E 2 HP0 4 —> 2 C0 2 + 2 CgE50E + 2 HgO + C 6 H 1 0 0 4 ( P 0 4 E 2 } 2 .  51  -13It has been shown that the phosphate i s indispensable to the process and that at least three stages occur i n the process. (1) A period coincident with the increased fermentation during which free phosphate rapidly diminishes. (2) A period of uniform a c t i v i t y where only small amounts of free phosphate occur.  15) A period of  lessened a c t i v i t y and rapid increase of free phosphate.  An enzymic  hydrolysis similar to the l a s t stage i s effected after removal of the co-enzyme of zymase indicating the presence of a phosphatase, the action of which might be represented II.  C 6 H 1 Q 0 4 (P0 4 E 2 )  2  as:  + 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 i n 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 I I .  Up to the end of stage (2)  the phosphate thus produced enters into the equation according to equation II, with the sugar which i s present i n excess and is thus reconverted into hexose phosphate,  so that, as long as alcoholic  fermentation i s 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 i n yeasts, (a)  the r e l a t i v e l y slow fermentation, without addition of P 0 4 .  (b)  the more rapid process by adding P04« (c) rapid fermentation by addition of Arsenate by either stimulating phosphatase a c t i v i t y or by the formation of more l a b i l e hexosearsenates.  52  -14The rate of hexose monophosphoric and pyrophosphoric acids i n yeasts has largely to be worked out, (b) Muscle Contration: That phosphates play a part i n muscle contraction is evidenced by the facts:  (1)  that free phosphate and l a c t i c acid are liberated i n  equimolecular amounts.  (2)  that by adding phosphate a l l glycogen is  converted to l a c t i c a c i d . (5) that during muscular work ther is an increase i n the secretion of phosphate into the urine.  (4)  that  performance of muscular work i s 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 l a c t i c acid i n the contracting muscle and the presence of phosphates i n the l a t t e r . hexosemonophosphate  Experiments were made indicating that a  (the lactocidogen)  i s found as an intermediate  compound i n the breakdown of glycogen to l a c t i c a c i d .  The changes  may be represented i n two phases (a) the anaerobic and (b)  aerobic.  anaerobic (contactile) 5 G 6 H 1 0 0 5 + 5 H 2 0 + 4 K2HH>4 glycogen.  4 CgHjjOgtPO^) t- ' C 6 H 1 2 0g + RgO hexosemonophosphate. glucose.  4 c 0 6 H n O 5 t P O 4 K 2 ) —> 8 0 3 H 6 0 5 + 4 K 2 HP0 4 + CgH12Gg lactic acid. glucose.  aerohic (recovery) 8 C 5 H 5 0 5 + 4 glucose -t- K 2 HP0 4 + 60 2 —?4  C 6 H U 0 5 ( P0 4 K 2 ) t  6 C 0 2 + 10 H 2 0  The glucose freed during the contractile period i s oxidized during the recovery period; then l / s of the glycogen i s burned.  53  -15-  That there are Enzymes i n 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 l a c t i c acid  producing  enzyme free from muscle carbohydrate. A dialyzable thermostable  co-enzyme has been discovered. The preparations s p l i t hexose under special conditions and readily act on starch and glycogen.  In the com-  plex i s an enzyme s p l i t t i n g hexose mono-and di-phosphoric acids. Embden and Zimmermann (*/} , Lohmann  have found i n addition to  lactocldogen i n 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 i n sodium salts of monacetic acid and fatiqued, contract without u t i l i z a t i o n of glycogen or formation of l a c t i c acid; ammonia was produced, phosphagen decreased, phosphoric acid increased, creature phosphate — 7 » 1 H  H3PQ4  5  +  ^creature •+- HgPQ^creature  x.  •+- hexose — h e x o s e phosphate —-7  H3FO4  l a c t i c acid.  l a c t i c 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  -16and carbonates deposited i n a protein matrip.  Of these mayamic consti-  tuents calcium phosphate i s 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.  ( T e l l and Robison (w) (d)  .)  Significance of Phosphatases i n the Kidney:  Eichholtz, Robison and Bruell (*?) have attached special  significance  to phosphatases of the kidney i n regard to the excretion of phosphate i n the urine and some of the most recent work on phosphatases i s 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 i n d i e t — i n B-avitaminasis p a r t i c u l a r l y .  .6.  PROBLEMS  Prom the study of work being done on mammal and yeast phosphatases and from our own b r i e f investigation we are confronted with numerous questions concerning the role of these enzymes and their substrates i n the higher plants. The r&le of these enzymes i n yeast fermentation and i n muscular work has been closely linked with carbohydrate metabolism p a r t i c u l a r l y with the u t i l i z a t i o n of carbohydrates i n the production of free energy.  55  -17Might there not be a close relationship i n the higher plants?  With the  , process of carbon assimilation i t has been suggested that phosphorus i s not necessary.  (  ).  In the transformation and  synthesis of sugars apart from the photosynthetic process e.g.  as  occurs i n malting barley, i n sugar beet storage, e t c . , phosphatases might play a p a r t .  Indirect evidence i n support of t h i s suggestion  is  the universal occurrence of traces of phosphate i n 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 i n the higher plants, on which phosphatases might act i s l i m i t e d .  Burkard and Keuberg (^)  have shown  hexosediphosphate to be present i n sugar beet leaves, and Oockefair [*°\ i n other plants; Msnjdahl and Weissflag (5J) of meta- and pyro-hosphates.  have indicated the presence  Phosphagens have not been reported for  plants. Phosphorus metabolism i n general, and phosphorus absorption more p a r t i c u l a r l y i n the higher plant i s notably influenced by changes i n radiant energy. (Barton-Wright {ff) .) Might not phosphatases and their substrates, d i r e c t l y or i n d i r e c t l y , be involved i n this relationship? Some enzymes are l i g h t sensitive.  Are plant phosphatases similarly  constituted? The study of phosphatases might have a d i r e c t l y p r a c t i c a l value i n the malting of barley.  Quality i n malting barley i s determined to a  great extent by the a b i l i t y of the seed to hydrolyze i t s starch reserves to y i e l d sugars.  Could an intimate relationship be established between  quality and phosphatase a c t i v i t y a ready test for malting quality might  56  -18-  be devised. This b r i e f speculation may at least indicate the variety and number of problems r e l a t i n g 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 . B i d . Ghem. 101:  3.  Barrenscheen, H. K. and Beneschowsky, H. Bicchem. Z .  4.  B a y l i s s , W.M. "The Nature of Enzyme Action"  5.  Bayless, ¥ . M. "Principles of General Physiology"  641 (1933)  265: 159168 (1933) Longmans, London 1919  7.  Longmans, London . " 1924 . Belfanti, L . } Contardi, A . , and E r c o l i , A . Biochem. J . 29: 1491 (1935) B e l l and Doisy J . B i o l . Chem. 44: 55 (1920)  8.  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