ACYLPHOSPHATASE STUDIES on ACYLPHOSPHATASE of Vigna Catjang

ACYLPHOSPHATASE STUDIES ON ACYLPHOSPHATASE OF Vigna Catjang

A Thesis submitted to the UNIVERSITY OF POONA for the degree of DOCTOR OF PHILOSOPHY

by Mrs. V. V. DESHPANDE; M. Sc.

Division of Biochemistry National Chemical Laboratory Poona

October 19 7 3 ACaCWDWLiSDCilEMT

1 take this opportnnitjr te ezpreas my de«p sense of gratitude to Dr. V. Jagannathany National Cheaieal Laboratory,

Poona for his assisiame and gaidaneo throaghovt the coarse of this investigation*

I wish to record ogr gratefal thanks to Ifr H. 6. Y a x U k ,

Dr. S. ▼. Paranjpe and Mr A. M. Bodhe for help in preparing large batches of ensjnae and Hr Mazhar Hnsain in estimation of snlflqrdrjrl groups.

I gratefully acknowledge the help given by Dr. C.SivsBanan in carrying oat the altracentrifagal stndies.

Ify thaAs are also dae to the Council of Scientific and

Industrial Besearch for the award of a fellowship and the

Director, National Cheaieal Laboratory, ?oona for pemission to subait this work in the fora of a thesis for the degree of

Doctor of Philosophy.

cl^ CONTENTS

Ghapttr Page

I INTROKJCTION 1-39

Section It Inirodaoiory 1

II: TTiatorical 2 - 4

III: Occurrcnc* 5 - 7

IV: Types of aeylphosphatM* 8 - 10

V: Methods of estimation 11 - 13

VI: Parifioation of aoylphosphatas* 14 - 18

VII: Properties of aejlphesphatase 19 - .30

V I I I : Effset of thjrrozine 31-33

IX: Role of aejrlphosphatMe 34 - 39

X: Ifeehanisii of aetien 5 t ' 39

XI: Present wofk 39

I I MATBOALB AND METBODB 40-50

I I I RXPHIIMENTAL AND RBSOLTS 61-88

rV PBOPHtTIBS AND KINETICS 89 - 119

V DISCUSSION 120 - 131

VI SUMMASr AND CONCLUSIONS 132 - 138

B I B L I 0 G R A P H T 138 - 138 CHAPTER I

INTRODUCTl 0 N INTRODUCTION

SFXJTION I

Aejrlphoaphatass phosphohydrolftM (H .C .3.6.1,7) (aeylphospbatase) oataljrz** th* hydrolysis of aeyl phosphatss. This siuiyas was diseoversd by Lipnann during th« eonrs* of bis stttdlss on aeatyl pbosphat*. Tbs

•nzyme is specific for aeylphospbatss and doss not act on other pbospbats anhydrides or esters. Sereral workers hare purified the enzyae from animal tissaes. It has been obtained in highly purified form fro* horse muscle, boYine brain, poric heart and hnnan erjrthrocytes and the

enzyme from rabbit auscle has been crystallized. Its occurrenee and role in plants have, howerer, not been studied. The present work deals with the isolation in pure form of a specific aeylphosphatase froa the

seeds of Vigna catjang and a study of its properties, kinetics and biological role. 2

SBCTION II

HISTORICAL

AcylphoBirfiatas* waa diaeovered by Llpaiann in 1945. Ha found that aeatyl phoaphata waa rapidly daatroyed by tha action of an unknown anzyna from animal tiaauaa* Latar the anzysa waa identified aa a apaoifie acylfihoaphataae, which could be readily obtained in aoluble form from animal tiaauea. Muacle waa ahown to have high aeylphoaphataae activity, whereaa the conmon phoaphataaea are abaent or preaent only in email amonnta in thia tiaaue. The enzyme waa found to have no action on phoaphate eatera. Ita enzymic nature waa eata^ bliahed by the fact that it waa nondialysable and waa completely deatroyed by the action of p«pi»in. The enzyme waa found to be heat

atable, eapecially in acid aolution. It waa alao not denatured by trichloroacetic acid. Theae propertiea of the enzyme were uaed for ita partial purification and by heating, trichloroacetic acid preei->

pitation and acetone fractionation a preparation waa obtained which

ahowed the propertiea of a baaic protein. Thia enzyme waa inhibited by aeveral polyvalent aniona. Inorganic phosphate mia a potent inhibitor and leaa stronf[ inhibition waa obaerved with aulphate, citrate and oxalate. Fluoride had very little effect on ita activity.

Shapiro and Wertheimer (1940) alao demonatrated rapid enzymic hydrolyaia of acetyl phoaphate by ertracta and homogenatea of liver. brain, Kascle and kidmjr. All thea* tiaaaaa had approzinataly tha aama aetirity. Thara waa no enzymie aetirity in aertm. Tha hydrolyaia of acetyl phoaphate waa ahown to ba inbibitad by phoaphata but not by fluoride or cyanide. The actirity waa totally deatroyed by heating to

100® for 5 oiin.

The enzyme from horae muscle waa purified by Koahland (1 9 5 5 ),

Harary (1963) and Ranponi, Guarritore, Trevea, Naaai and Baecari

(1 9 6 9 ). Extenaive purification of the enzyme from bovine brain waa reported by ftaijman, Griaolia and lildelhoch (i9 60 ) and IHederich and

Griaolia (1 9 6 9 ). The latter have alao obtained the pork heart enzyme in highly purified form (Diederich and Griaolia, 1971). Other animal tissue enzymes which have been purified are thoae from chicken muacle

(Pechere, 1957) and human erythrocytes (Bakitzia and Mills, 1969).

Shiokawa and Moda (1970) obtained the rabbit muacle enzyme in cryatalline form.

The metabolic function of this enzyme is not well understood*

Bacteria, which synthesize and metabolize acetyl phoaphate generally contain very little or no acylphosphatase. Animal tissues, on the other hand, do not form acetyl phosphate. (Acetyl phosphate and fonqrl phosi^iate can be formed by the action of carbaayl (^osphate synthetase but they are not important metabolic intermediates in animal tissues).

Possible substrates for this enzyme are the glycolytic intermediate,

1,3 diphosphoglyceric acid and carbaa^l phosphate. The observation that acylphosphatase is inhibited by a very low concentration of thyroxine is of considerable interest. The iaelation of the •vasymt i n higblj purified form and ita w ery low aolecalar weight hare etianlated considerable interest in acylphosphatase. In recent jears studies on nltracentrifugation and electrophoresis, amino acid composition, molecular weight and other properties and kinetics of the enzyme hare been described. 5

SECTIOW III

OnCTTRllENCE

Acylphosptiataae ia widalj diatributad in animal tiaauaa. It

haa been reported (Harary, 1063) to be preaent in guinea pig, rabbit

and bovine retina, brain, kidney, apleen, bone aarrow and teatea.The

occarrence of thia enzyme in planta haa not hitherto been reported.

Animal tiaaaea?

l) Muacle. The demonatration of the enzyme in horae maaole

(Lipmam, 1945) and ita purification (Koahland, 1955; Harary, 1963;

Tlanponi, Guerritore, Treres, Naaai and Haccari, 1969) hare been

described earlier. Reference has alao been made to the isolation of crystalline acylphosphatase from rabbit muscle (Shiokawa and Noda,

1 970). Pechere (1967) purified the enzyme from chicken muacle and

obtained three different fractions with enzyme activity.

Heart. Diederich and Grisolia (l97l) purified the enzyme from pork heart.

Brain. The enzyme from thia tissue has alao been obtained in highly purified form (Diederich and Griaolia, 1969).

Erythrocytes. The enzyme was ahown to be present in human erythrocytes and waa purified by Rakitzis and Mills (1 9 6 9 ).

Placenta. The acylphoaphataae of human placenta was studied by Guerritore and Dellepiane (1955). The acetylphosphataae content of placenta waa less than that of other organs. ()

TuMOurs. Dinescu and Kfctaloea (1964) obs«rv*d that there was a higher acetylphosphatase actlTi-ty in Jensen sareona, Guerin eareinoiaa. Walker earciiioaa 256 and Yoshida sarcoma than in normal rat kidnejr, muscle, spleen and lirer and negligible aeti-vlty in ascitic Ehrlich carcinima. There was no difference in the levels of the enzjnae in whole blood of healthy and cancer hearing individuals.

Acylphosphatase of house-fly

An extract of acetone powder of house-flies hydrolyzed acetylphosphate anil was shown to be a mixture of acetyl phosphatase and phosphotransacetylase^s^^^^^^

Acylphosphatase and Na*-K* ATPase.

A stisHilated acetylphosphatase of brain nicrosomes was described (Sachs, Ilose and Ilirschowitx, 1967). Microsomal Na’*‘-K‘*’

ATPase was compared with K*** acetyl phosphatase (Yedy Israel and

Slwood Titus, 19B7). On the basis of differences in the Inhibitory effects of ouabain, oligomycin and N-methylaaleimide it has been suggested that the K‘*’-acetyl phosphatase activity of beef brain may represent an entity different from Ma‘*’-!C‘*’ ATPase.

This surrey will not deal with the extensive literature on

Na^-K^ ATPase which lies outside the scope of the present woric,

2) Localization of acylphosphatase

Very little work has been done on the distribution of the enzyme between nuclei, mitochondria and other cell components* 7

Zanobini, Ramponi and Gaerritor* (1962) ob»err«d that whan mito- ohondria are kept for 16 ain in 0.3 M aacroae or in hypotonic

Bolution there is a parallelism between the release of acetylphos-

phatase actirity and the swellii^ effect.

The distribution of carbaogrl fiAiosphatase aotiritx in cells differs with the tissue studied (Melani, Hamponi and Gnerritore,

1961). Of the total actirity of hoaogenates of ^eletal muscle and brain of rats was in the soluble fraction, whereas from the homogenates of liver and kidney about 45^ was in the soluble fraction.

The percentage of soluble enzyme in liver and kidney appears to be

less than that in muscle and brain. But no conclusive evidence has been presented for the occurrence of particulate acylphosphatase. SECTION IV

Typ«« of acylpho«phata»>

(1 ) Acid-atabl» and aeid-»an»table types

The acylphosphatase actiTities obtaiaed from lirer nito-

ehondria and ■ascl* differ from each other in their behaTionr to heat at low pH and in their reaetions to anions (Zanobini, Raaponi

and Gaerritore, 11)61). Apparently aeylphoephataee oecar* in both heat-stable and heat-labile foras. The heat-labile form predominates

iu liver while the heat-stable form is preponderant in masele. The

heat-labile form is not inhibited by phosphate in contrast to the

inhibition of the heat-stable form by phosphate as well as by a

number of polyralent anions. Further work is needed to establish that both forms are true acylphosphatases.

(2) Carbamyl phosphatase and acylphosphatase

A phosphatase which hydrolyzes carbasqrl phosphate has been

demonstrated in a number of tissues. It was noted by Grisolia (i960)

that a purified preparation from bovine brain acted both upon acetyl-

phosphate and carban^l phosphate. Thus carbamylphosphatase was shown to be the same enzyme as acetylphosphatase previously described by

Lipmann (1946). The enzyme was found to be widespread in animal tissues. Until recently very little was known about the physiological

function of acylphosphatase. The fact that carbamyl phosphate and

1 ,3-diphosphoglyceric acid could be the naturally occurring substrates y

for this •nzymc euggeete a possibl* role for aeylphoaphata>«.

Purified preparations of acylphosphatase fro* imscle,brain and heart act upon earbasQrl phosphate. The ratio of the aetlTitjr with acetyl phosphate to that with earbaayl phosphate for parified preparations of aeylphosphatase from horse Bnscle, beef brain, pig heart and human erythrocytes were 11, 10, 11 and 0 respectirely.

The ratio is the same for the first three enzymes whereas acylphos-

phatase from human erythrocytes does not act upon carbamyl phosphate.

An extract of liver mitochondria has been reported to hare acetylphosphatase actiTity which is twice that of carban^lphosphatase activity (Hfelani et ( ^ , 1961).

(3) Isoemymes

During the purification of acylphosphatase from rabbit muscle (Shiokawa and Noda, 1970) the ensymic activity was separated

into three fractions A, B and C by chromatography on carboxymethyl cellulose at pH 4«8. The fractions B and C were obtained by eluting the enzyme at 0 .1 6 II and 0 .2 5 U NaCl respectively. The specific acgivities of these two fractions were nearly the same. Fraction A was obtained by eluting the enzyme at 0 .1 M NaCl and its specific activity was about half that of fractions B and C. The crystalline enzyme was obtained from fraction B which contained most of the activity. The activities represented by peaks A and C were postulated to be isoenzymes of the crystalline enzyme. 10

The ezistene* of a second aeetylphosphata.«e waa alae reported in himan erythrocytes by Rakitzis and Mills (1969). The final specific actirity of acylphosphatase purified fron huaan erythrocytes ranged froa

700 to 6,800 ^^oles of acetylphosphate hydrolysed per ag protein per hoar at pH 6.4). Preparations of low specific activity asually had traces of hemoglobin. The enzyne obtained after the CU-cellnlose step could be adsorbed on DFAE-eellalose at pH 9.4 but at least one fourth of the original activity of the crude hemolysate vas retaiiMid on a DEAE^ellulose coluan at pH 6.9. These obserrations suggested the presence of an enzyme which is different from the more basic purified acylphosphatase. The former enzyme was not further investigated.

Pechere (1957) obtained three different fractions with enzyme activity from chicken muscle but whether they are isoenzymes or not is not known.

These reports suggest the existence of isoenzymes of acylphos> phatase in different tissues but none of them is well characterized. 11

SECTION V

MTTTtrODS OF ESTIMATION

(1) Differential precipitation aethod

Lipmann and Tuttle in 1944 introduced a method based on the

differential precipitation of calcium acetylphosphate and inorganic

phosphate. With the aolybdate reagent of Fiske and Subbarow complete

decomposition of acetylphosphate occurs at room t«iperature in less

than 10 ain. Hence direct colorimetry yields an apparent phosphate

▼alue which represents the sum of acetylphosphate and inorganic phosphate.

True inorganic phosphate is estiaiated after precipitation with alcoholic

calcium chloride solution which leaves the soluble calcium acetylphos-

phate in the supernatant liquid. The difference between apparent and

true inorganic phosphate is regarded as acetylphosphate. This method

has not been widely used.

(2 ) Lipaann and Tuttle*s hydroxylamine method (1945)

This method is based on the measurement of residual acetyl-

phosptiate. On treatment with hydro:qrlaaine acyl phosphates are converted

into hydroxamic acids as follows:

RCOOPOg ♦ NH2OH ---- R.CONHOH + HPO^

acylphosphate hydroayl- hydroxa> amine mate

The hydroxaraic acid forms a colored complex with trivalent iron. 12

The color varies from orange-brown to purplish brown depending on the concentration of acylphosphate. The main absorption of the

purple iron complex is between 480 to 640 nm in which region ferric chloride does not absorb. A mmber of anions such as fluoride, phosphate, oxalate, sulphate and citrate depress the intensity of the color by forming iron complexes, but t h ^ do not interfere with

the reaction between acylphosphate and hydroxylamine.

This method is widely used because it is rapid and conrenient and applicable over a wide range of conditions such as (£, enzyme concentration and different buffer systems.

( 3) Spectrophotometrie method

Ramponi et a l . (1966) introduced this method for the rapid estimation of acylphosphatase and the study of its properties. It makes use of aromatic substrates such as benzoyl phosphate or jg-nitrobenzoyl phosphate, since the phosphorylation of these compounds

is accompanied by marked change in absorption in the ultraviolet. This method is rapid and sensitive and, since the progress of the reaction can be followed without stopping it as in the hydroxaraate method, it

is particularly suitable for kinetic studies.

( 4) Uicromethod for acetylphosphate determination

Hecently Satchell and White (1070) have developed a new method based partly upon Lijxaann's method (194S) and partly upon that intro duced by Mission (1908) for the determination of inorganic phosphate.

The basis of the new micromethod is to convert acylphosphate to hydro 13

procedure and then to estiaate colorimetricalljr not the hydroxamic acid but the inorganic phosphate. This vas accomplished by forming the phosphomolybdoTanadate complex, extracting it with butanol and measuring the colour of the butanol layers at 310 am against a reagent blank (Parrin and Smith, 1969). 14

slx;t io n VI

1>1/R1FIC\TI0N

Animal tiwsues

Hor»» Mg»el»

Lipmann (1045) partially pnrified th* enzyme from horse muscle by beating, trichloroacetic acid precipitation and aestone fractionation and Koshland (19SS) obtained a 37-fold preparation by a slightly modified procedure from the same source. Harary (1963) reported a 635-fold purification of acylphosphatase from horse muscle.

His purification procedure consists of heating the mzyme in acid, trichloroacetic acid precipitation and ammonium sulphate fractionation.

The final specific actirity of the enzyme was 3,810 units* per milli gram (at 37“ and (rfl 5,4), lUmponi et (i960) have described a procedure for the purification of acylphosphatase from horse muscle, which consists of extraction with acid and two successire chromato graphic separations on CU-Sephadex C-25. They used benzoyl phosphate as substrate. The final specific activity of this highly purified preparation from horse muscle was 72,000 units/mg. The homogeneity of the final product was established by starch gel electrophoresis, acrylamide gel electrophoresis, gel filtration on Sephadex G^7S and u 1 trac ent ri f ugati on*

(*€nzyme activities have been calculated in terms of/umoles of

acetylphosphate hydrolyzed per hour). 15

Rabbit au«el»

A crystalline aeylphosphataa* was obtained froa rabbit muscl* by Shiekawa and Neda (1970). The crystalline enzyae had a

specific activity of 51,600 units per mg (at 26* and pH 5 .4 ) . This

purification was achieved by acid extraction, ehrooatography on

CM-cellulose and ammoniua sulphate fractionation. The enzyme was

homogeneous by ultracentrifugation and disc electrophoresis on acrylamide gel.

Brain

Bovine brain. Raijman, Grisolia and IMelhoch (i960) purified the enzyne fron bovine brain. Their purification procedure involved acidification to pii 4, precipitation with sodiun picrate, precipita^

tion with acetone, removal of impurities with sodium sulfosalicylate, acet

7,800 units per mg. Diederich and Grisolia (1069) obtained a highly purified preparation of the enzyme from the same source. Their puri

fication procedure involved the following steps: extraction of the tissue at pH 4 , acetone precipitation, removal of proteins by sulfosalicylate and two subsequent fractionations on columns of Bio-Rex-70 resin. The specific activity nf the purified preparation was 75,000 units per mg. Acrylamide gel electrophoresis of the purified enzyme indicated a single band at pH 8 ,3 and pH 4 .5 .

Erythrocytes

A specific acylphosphatase was purified by Rakitzis and IB

Uilla (1969) from human erythrocytes. Blood was eentrifnged and the plasma and baffy coat were removed. The paeked cells were lysed and the hemolysate was applied to a CM-cellalose eoluon in 0 .0 0 5 U phosphate baffer at pH 7*5 and elated with 0.5 M NaCl. It was then transferred to a DEAE-^ellulose colonn at pH 9.4 in 0.01 M Na^HPO^ solution and elated with 0.01 U phosphate buffer at pH 6.0. The specific activity of the final preparation ranged froa 700 to 6,800 units per mg. Preparations of low specific activity usually had traces of hemoglobin as the major protein contaminant. The variation in activity was attribated to contamination with hemoglobin, bat no data on inhibition of the enzyme by hemoglobin were presented.

Heart

Pork heart. Acylphosphatase from pork heart was purified by

Piederich and Grisolia (l97l). The purification procedure involved heating the acid extract of the enxyme at 70*, acetone fractionation and chromatography on two successive columns of Bio-Kez-70 resin.

The final specific activity of the purest fraction was 36,000 units per mg and it appeared as a single homogeneous band when subjected to acrylamide disc electrophoresis at pH 8.3 and 4.5.

The results on the purification of acylphosphatase from animal tissues are siaunarized in Table i . It w ill be seen that the maximum specific activity of the enzyme varies with the tissue. It is highest for tiie enzyme from brain and the lowest for the enzyme from erythrocytes and vaoes from 700 to 7 5 ,0 0 0 . The reason for

this variation is not known, especially in the case of the erythrocyte 17 enzyne the actirity of which differs by one to two orders of magni-> tude from that of the other enzjrraes. Bat it should be noted that the erythrocyte enzyme also differs in its specificity and, unlike the other enzymes, has no action on carbamyl phosphate. 18

TABLE 1

SIMIAHY OF PimiFICATTON OF ACYLPH9SPH\TASR FROM ANIMAL TISSUES

Maximoa Natare of p H Temp. specific the tissue activity

units/n£ protein

Horse auscle 5 .3 2 6 “ 30,000

Rabbit muscle 5.4 26* 51,600

Bovine brain - 27® 7 5,000

Human erythrocytes 5 .4 37* 700 - 7,000

Pork heart 27* 36,000

Aetivities are expressed as yomoles of aeetylpbosphate

hydroljrzed per hour. 19

SKCTION V II

PROPERTIES

(l) Effect of tempwator*

Acylphosphataat from muscl* is heat-atable. At aeid pHa the enzyme withatands heating to 60* for 20 min without loss in activity.

Heating under the same conditions at pH 8.6 in tris buffer leads to a loss of approximately 33 to 50 per cent of the activity.

Partially purified preparations of acylphosphatase from brain are also very heat-stable. The partially {mrified brain enzyme could be heated to 80° for 15 min between pH 1 to 7 with only little (lO to

20^) loss in activity. The thermal stability of purer fractions is

less than that of the crude fraction. Heating of the dialyzed purified preparation at 37* for 20 min results in 27 per cent loss in activity.

In contrast to the muscle and brain acylphosphatases, the

enzyme from erythrocytes is thermolabile. The stability of erythrocyte acylphosphatase was tested with the crude hemolysate as well as with

the purified enzyme* Heating for 10 min at 60* over a pH range of 5-9 completely destroyed the activity of the crude hemolysate. Heating of the purified preparation at 70* for 5 min destroyed 40$ of its activity.

Thus stability decreases for the muscle and brain enzymes on

purification but increases for the erythrocyte enzyme. The enzyme is more stable to acid than to alkaline conditions. 20

(2) 8ttb»trat« «p«cificity

Aejlphosphatase of animal tiaaaes does not eatalyza the hydro

lysis of pyrophosphate, ATP, ADP, AMP, acetyl adeiqrlate, acetylcholine, acetyl CoA or glycerol phosphate. It catalyzes the hydrolysis of propionyl, batyryl, succinyl phosphate as well as acetylphosphate.

Muscle

Horse muscle (Ramponi a l , 1969)

Pure acylphosphatase from horse muscle catalyzes the hydrolysis of compounds, which are anhydrides of carboxyl and phosphate groups.

However, compounds of the type acy1-AMP (such as amino acid-AMP anhydrides foraed by amino acid activation during protein synthesis) are not hydrolyzed. The phosphate group should presumably be unsubsti

tuted. The enzyme showed activity with jg-nitrobenzoyl phosphate, benzoyl

phos(4iate, 3-phosphoglyceryl phosphate, acetylphosphate and carbamyl

phosphate. There is obviously less specificity regarding the nature of the group attached to the carboxyl group. The enzyme possesses very little activity towards phosphocreatine, adenosine-S'-triphosphate,

pyrophosphate, and ^nitropheigrlphosphate at pH 5.3. It does not split

2-nitrophenylphosphate at pll 10.4, phosphoenolpyruvate, acetyl AMP or phosvitine. The enzyme i s , therefore, specific for compounds of the type R.CO.O.PO^Hg. Further work i s , however, needed to clarify the effect of chain length and substituents in The relative rates of hydrolysis of £>nitrepheryl phosphate, benzoyl phosphate, 3-phosphoglyceryl phosphate, acetylphosphate and carbamyl phosphate were 21, 6,

1 .2 , 1 and 0 .0 9 respectively. Relative activities varjring from 21

6,8 X 10 - 7,6 X 10 (relatir* to 1 for acetylphosphate) were reported for phoephoereatlne, ATP, pjrrophoephate and f-nitropheiqrl

phosphate, but theee aotlvltiee are reiy lew Hnd be d a e to

impuritiee.

Bovine brain (Diederich and Griaolia, 1969)

Aeylphosphataae from bovine brain hydrolyzes acetylphosphate, carban^l phosphate and 1:3 diphosphoglyeerate. It was noted that the ratios of activity with acetyl phosphate and carbaaiyl phosphate were essentially the same daring 100-fold parification. The same enzyae, therefore, catalyzes the hydrolysis of all the three compounds. The

acetylj^osphate/carbamyl phosphate activity ratio for the purified

«axym t was 10 and the activity ratio of acetylphosphate/diphosphoglycerate was found to be 9 , The enzyme has no effect on ATP, AMP and i^osphoenol pyruvate.

Human erythrocytes (Rakitzis and Mills, 1969)

Acylphosphatase purified from human erythrocytes hydrolyzes acetylphosphate and 1 ,3 diphosphoglyeerate but not carbamyl phosphate.

Moreover no enzyme activity could be detected with 3-phosphoglycerate,

phosphoenol pyruvate, fructose-6-phosphate, ATP, ADP, 3 ',5 ' cyclic AMP,

6-phosphogluconate, phosphoserine, inorganic pyrophosphate and j^nitro-

phenyl phosphate.

Pork heart (Diederich and Grisolia, 1971)

Purified pork heart acylphosphatase acts on acetylphosphate,

carbamyl phosphate and 1,3 diphosphoglyeerate. The ratio of activity 22 with acetylphosphate to that with carbaoqrl phosphate or diphosphoglycerate was 11.

It will be seen that all animal tissue acylphosphatases act

both on acetylphosphate and 1,3 diphosphoglyeeric aeid and that with

the exception of the erythrocyte enzyme they also act on carbamyl

phosphate.

( 3 ) Activators and inhibitors

(i) Horse ■nscle (Raaponi et al. 1969)

Acylphosphatase from muscle does not seem to require a metal

for its activity. This was shown by the lack of effect of metal ions

and metal-binding inhibitors such as EDfA or -dij^ridyl. lodo-

benzoate, iodoacetate and jgCMB do not inhibit the enzyme indicating

that -SH groups are not necessary for activity. The enzyme is compe

titively inhibited by ortho- and i^rophosphate and irreversibly by

-5 preincubation with 10 ii thyroxine,

(ii) Bovine Brain (Diederich and Grisolia, 1989)

Caffeine. Up to 2 .6 x 10 caffeine had no effect on enzyme

activity.

Adrenaline, Adrenaline at 1.35 x 10 inhibited the enzyme

Inosinic acid. lansinic acid had no effect upto 2.3 x 10 -•3 33<^ activation was noted at 4.6 x 10 U but further increase in —3 concentration (upto 7 x 10 M) did not produce farther activation. 23

2.4-Dinitroph>nol« DNP at 2,6 z 10"^M when pr«incabat*d with the enzyme inhibited 50^ of the aetirity, but it was approximately only half a* effeetiTe when added directly to the inonbation mixture.

Phoephate. With 2 x 10 ^ phosphate 50 to 60^ inhibition was obserred. 4 x 10~^ phosphate inhibited 70 to 80^ of the activity.

Urea and guanidine. When acyl phosphatase was dissolved in 5 M area or 5 M guanidine and assayed, there was complete inhibition of ensyme actirity. This type of inhibition was, however, reversible since 95^ of the original activity was recovered upon lowering the concentration of the denatarant by dilution. Any unfolding of the peptide chain caused by urea or guanidine is, therefore, completely reversible.

Proteolytic enaymes

Preincubation of the enzyme with 2^ pepsin at pH 2 and at 37* for 10 min inactivated the enzyme entirely. Trypsin and papain at

O.K and concentration respectively, when preineubated with the enzyme at pH 7 and 37* for 10 min, had no effect on enatyme activity.

A slight activation of about 15^ was sometimes observed after papain digestion. However, when the papain digested acylphosphatase was dialyzed for 30 min at 3-4* against veiy small volumes of deionized water, approximately 10^ of the activity was found in the diffusate whereas none appeared in the controls. No further work has been reported on this interesting observation.

Mercuric chloride and iodoacetate

Mercuric chloride at 4 x lO^^M and iodoacetate at 4 x 10**^U in 24 the assay systsm showed no inhibition.

KCl and MgCl^

16 aM KCl and 5 aM UgCl^ were found to enhance enzyme aetiTity when tris-aeetyl phosphate was used as a substrate. This is the only report of metal aeti-vatien of this enzyme.

(iii) Human erythrocytes (llakitzis and Mills, 1969)

The effect of several metabolic intermediates on the erythro cyte enzyme was determined using acetyl phosphate and 1,3 diphosphoglyceric acid as substrates.

At S bM inhibition by phosphoeiral pyruvate, 6-phosphogluconate, fructose-6-phosphate, fructose 1:6 diphosphate and glucose-6-phosphate was 12, 23, 32, 44 and 50 per cent respectively. Inhibition in the

presence of either acetyl phosphate or l,3->diphosphoglyeeric acid was nearly the same with ATP and phosphoenolpyravate, 3-Phosphoglyceric acid showed 20 per cent inhibition with 1,3 diphosphoglycerate and only 5 per cent with acetylphosphate. MAIM showed negligible effect on activity. Further work is needed to assess the significance of these inhibitions at physiologically occurring concentrations of these compounds.

jgCMB and iodoacetate do not inhibit the enzyme appreciably.

Thyroxine and triiodothyronine are both inhibitory (74, 64 and 57 per cent inhibition by 0.1 adif D, L-thyroxine and triiodothyronine). Sodiosi fluoride and EDTA inhibit significantly. Mg at 10 nm concentration did not have any effect on erythrocyte acylphosphatase at pH 7.5.

Further work is needed on the reason for EOTA inhibition. The ery throcyte enzyme differs from other animal tissue enzjrmes not only in 25 its lack of action on earbamyl phosphate and low sp«clficity but also in its inhibition by NaF and RDTA.

(iv) Perk heart (Diederich and Grisolia, 1971)

The effect of phosphate on pork heart acylphosphatase was studied using acetyl phosphate as substrate. P i, *TP and 3-phospho~ glycerate showed 37^, 11^ and 8^ inhibition respectirely when 1,3~ diphosphoglyceric acid was the substrate. HgClg (2 aM ) j»CMB (l hM) and iodoacetate (10 nM) showed n ^ l ig i b l e effect on acylphosphatase activity.

(4) Optianm pH

The optianm pH for the brain enzyme was 7.4 - 7,6 as against

5.3 for the horse muscle enayne. Ilakitsis and Mills (1969) found the optimum pH of the human erythrocyte enasyme to be 5. The optimum pH raqge for the hydrolysis of acetyl phosphate by heart acylphosphatase as determined by Diederich and Grisolia was also 5,4 to 5.6.

(6) Isoelectric pH

Harary (1963) found an isoelectric point of pH 8.6 for horse muscle acylphosphatase by paper electrophoresis. HowsTer, Ramponi

et al, (1967) by the method of thin layer gel filtration on Sephadex

G-76 obtained a value of 11.4 for the isoelectric pH of muscle acyl phosphatase. The isoelectric pH for the erythrocyte enzyme as determined by llakitzis and M ills (1969) was 8 .9 and that for the heart enzyme was 7 .2 5 to 7 .3 as determined by electrofusing. 26 - (6) Ultracentrlfagation (Table 2)

Hor»» muscle (TUutponi »t al* 1069)

The •edimentation constants at 5.4 ag/ml and 8.R mg/ml wer«

SgQ 1.23 X 10 and 1.32 z 10 and the diffusion constants were 10.71 x 10 ^ and 12.38 x 10 ^ respectirely. The molecular weights as determined the Archibald method were 8,450 and 10,300 respectively at the abore two concentrations.

Rabbit muscle

The acyl phosphatase from rabbit muscle crystallised by Shiokawa and Noda (1970) sedimented as a single peak in the analytical ultra>- centrifuge. The sedimentation coefficient ^ was calculated to be 2.1 and the molecular weight as determined by the sedimentation equilibrium method was 2 3 ,5 0 0 .

Bovine brain (Diederich and Qrisolia, 1969)

The borine brain ensyme sedimented as a single peak in the ultra> centrifuge with S.. of 1.25 at a concentration of 0.1 and 0.5^ m U f V protein in 0.1 M acetate at pH 4.7. The molecular weight was cal culated to be 13,000.

( 7 ) Amino acid composition

Amino acid analyses have been reported for the purified enzymes from ^ r e e sources (Table 3 ).

Horse muscle (Ram^eni wt a j. 1 9 6 9 ).

From the specific volumes of constituent amino acid residues the partial specific volume V was calculated to be 0.725 ml/g. The ratio of polar to apolar amino acid residues was found to be 2.05, which places the enzyme in the group of proteins with a large proportion of polar residues. 27

TABLE 2

SEPIMRNTATION CONST.MTS AND MOLECUL.^R WEIGHT OF

ACYLWIOSPHATASE FROM DIFFEllENT SOURCES

Sedimentation Molecular Tisaa* constant weight

S20,. ^ Daltons

Horse nusele 1.32 10,300

Rabbit mascl* 2.1 23,500

Borinc brain 1.25 13,000

Pork heart - 11,096 2 8

TABLE 3

Amt^O ACID ANALYSIS; DATA FOR ANIMAL TISSUE ACYLPtfOSPtfATASES

Residues per mole of enzyme Amino acid Rabbit muscle Horse aascle BoTine brain

Cy8t«ic aeid - 2 -

Aspartic acid 17 0 7

Threonine 15 S S

Serine 24 10 4

Glutamic acid 17 9 11

Proline 7 3 3

Glycine 20 7 7

Alanine 6 3 4

Valine 22 8 7

leoleucine 8 2 3

Leucine 7 3 5

Tyrosine 8 3 2

PheiQrlalanine 8 3 4

Lysine - 8 7

Histidine 1 1 2

Arginine 10 6 3

Tryptophan 3 - 2

Methionine 4 2 1

Ammonia 7

Calculation on 29 # » Rabbit Mawele

The aalno acid eomposition of crystalline aejlphosphatase

from rabbit nusele «as detcmined by Shiokaira and Noda. It vas

noted that only one histidine residue is present in a moloeale of

the enzyme (Shiokawa and Noda, 1 9 7 0 ).

BoTine brain (Diederich and Grisolia, 1969)

The molecular weight of bovine brain acylphosphatase determined

on the basis of amino acid composition was found to be 8 ,7 3 2 . The

tryptophan content was calculated to be 2.1 aoles/mole of the enzyme. -6 -6 The sulfhydryl content was 1 x 10 mole of groups per 8 z 10

mole of enzjme which is equivalent to only about 0.01 mole of -SB

groups per mole of enzyme. It was not possible to detect an 1^2’*

terminal amino acid deriwatiTe by either the fluorodinitrobenzene or

the fluoresceinisothioeyanate method indicatiiig that this group is

substituted*

(8) Kinetics norse Ml sole

Harary (1963) found that the Uiehaelis-4fenten constant for acetyl phosphatase from muscle was 8 z 10 if for acetylphosphate and about 10 ^11 for 1,3-diphosphoglyceric acid.

Human erythrocytes

A rather broad range of values from 7 .4 to 1 2 .7 was noted with acetyl phosphate as substrate (Rakitzis and Mills, I960). This variation in Km values may be attributed to the presence of different proportions of isoenzymes in different enzyme preparations or to the presence of small and variable amounts of inorganic phosphate aa So iaparity in different samplea of aeetylphoaphate. Determination of

with a crude beaolysate ufliof; aeetylpbosphate aa •ubatrate gave a value of 8.6 mM and the maximal velocity was 300 ;moles/nil/h at ^ 7.5.

A of 117 joM vas obtained for aeylphoephatase from erythrocytes uaiE|(

1:3-0PGA arkl the maximal velocity with 1:3-0PGA was 47*6 jomolee/ml/h.

The inhibition of acylphosphataae by ATP at {di 7.5 waa atudied uaing acetyl{rfioaphate aa Bubatrate. ATP at the level of 10 mU did not affect the maxioam velocity, but the waa increaaed in the preaenee of ATP. A purely competitive type of inhibition waa obaerved with a

Ki of 4.4 mM. Inorganic phoaphate waa alao a competitive inhibitor of acylphoaphataae with a Ki of 3 adf with acetylphoaphate aa snbatrate.

Carbaaqrl phoaphate waa alao found to inhibit competitively with a Ki of 8.9 aM with acetylphoaphate aa aubatrate. The K^ and Ki for erythrocyte acylphoaphataae are aumaarized in Table 4.

TABLE 4

KINITTIC PROPERTIES OF EHYTMllOCYTB ACYLPHOSPHATASE

Subatrate V K Ki (ATP) Ki(Pi) Ki(carbafflyl max m phoaphate)

Acetylphoaphate 300 10.3 mU 4.4 mU 3 mil 6.9 mM

1 :3 Diphoapho- 45 0 .1 2 my glycerate

From the Table 4 it will be aeen that the ia much lower (by

2 ordera of magnitude) for 1,3-diphoaphoglycerate than for acetylphoaphate, though ia greater with the latter aubatrate. The phyaiological conceit tration of l,3~diphoaphoglycerate ia very low and the low with thia substrate ia hence of significance for the enzyme to be effective in regulating glycolyaia. 51

SKCTION VIII

W B C T OF THYllOXINE

The tiasue levels of acylphosphatase are increased by ii^eetion of thyroxine althout^h the enzyoe itself is inhibited by tfajroxine in vitro. There was no inhibition by 1 x iO” ®M thjrroxine at 36* or by

1 X at 16* but nearly 50 per cent inhibition was observed at

5 X 10”*M thyroxine at 18* and B x 10*^M at 35*.

Harary (1957) studied the inhibition of horse nuscle aeylphosphatase by L-thyroxine. The enzyne used for this purpose was a partially purified preparation from horse skeletal Buscle. The effect of 5 X lO'^iif thyroxine on enzyme activity was only slight i f both the substrate and L-thyroxine were added at the same time*

However, upon preincubation of the enzyme with 5 x 10 thyroxine complete inhibition of the hydrolysis of acetylphosphate was observed.

Further experiments with varying concentrations of enzyme and

L-thyroxine indicated an api>arent stoicheiometric inhibition of the enzyme. Both DIr-3-3'-5 triiodothyronine and Dti-3-5-di-iodo-thyronine at the same concentration inhibited the enzyme but were 60^ as effective as thyroxine. No inhibition was observed with 5 x 10~’^ sodium iodide. Benzoic acid, DNP, L-thyroxine and DL-phenylalanine were not inhibitory at 5 x 10“*^!, but at 6 X 10~^ an inhibition resulted with these compounds which was 80^ of ^ a t observed with

S X thyroxine. Except in the case of benzoic acid this inhibition did not depend upon preincubation with the enzyme. 52

Attempts m»r« made to inveetigate the awehanism of thie

inhibition (Harary, 1957). It ia known that thyroxine haa metal

complexing propertiea. It was aoggeated that the inhibition of the

enzyme may be due to the binding of thyroxine with the metal rather

than due to the inactiTation of enzyme by thyroxine. However, all attempts to show a metal requirement for this enzjrme were unsuccessful,

EDTA, NaF, NbCN,o( ,e<-dipyridyl, Na-diethyldithiocarbamate or j^phenanthroline did not iidiibit the enzyme. Dialysis of acetyl-

phosphatase a^rainst distilled and demineralized water resulted in a

loss of 20^ of the activity and this loss in activity could not be

restored by WgCl^, CaClg, MnCl^, Cu(N0g)2, CoClg, ^®Clg, Zn(acetat«)2.

FeSO^ or by the addition of concentrated dialysate. Thus billing of

thyroxine with an essential metal is unlikely to be the cause of

thyroxine inhibition.

•3 Addition of zinc acetate (10 U) to the preincubation mixture

at zero time prevented inhibition by thyroxine, but addition after

the preincubation period did not reverse the already established

inhibition. It is possible that zinc protects the enzyme by binding with thyroxine and prevents it from binding with enzyme.

Additions of npto 40 ^ o l e s of acetyl phosphate after pre

incubation of thyroxine and enzyme without substrate had no effect

on reversing the already established inhibition. However, addition

of 5 ;imoles of acetyl phosphate in the preincubation mixture was

effective in preventing 50^ of the inhibition. The substrate

therefore protects the enzyme, perhaps by combining with the suscep

tible site. 33

Grisolia (i960) stadisd the effect of preineubation of thyroxine with brain acylphosphatase at two temperatures. He found that thyroxine was more inhibitory iriten preineubation was carried out at 16° than at 25**,

It is not clear whether in all the experiments cited above, thyroxine caused inhibition or inactivation. As has been noted earlier, eryUirocyte acylphosphatase is inhibited 50 per cent or more by D and Ir-thyroxine and triiodothyronine. Very little is known about the physiological role of the inhibition in vitro. Further elucidation of the aechanism of the inhibition may throw some light on i h * possible role of acylphosphatase in regulating metabolic processes. 3?

SECTION IX

PHYSIOLOGICAL HOLE OF ACYIfHOSPHATASE

It la not known whether phoapbatasea have a rale in the cell

other than the hydrolyaia of phoaphate eompoonda. It haa been poatn-

lated that theae enzymea aay in aone eaaea function alao aa tranafera-

aea. Bat iaotope and eheaiieal atadiea indicated that acylphoaphataae

doea not eatalsrze the transfer of acetate from acetyli^oaphate to a

groap of acetyl acceptora or the tranafer of phoaphate to glucoae or

creatine. Thna acylphoaphataae doea not poaaeaa acetyl tranaferaae

or phoaphotranaferaae activity and it appeara to act in the cell only

aa a hydrolaae with apecificity for acylphoaphatea*

Since there ia no evidence for the occurrence of acetyl-

phoaphate in manmalian tiaauea, the role of acylphoaphataae remaina

in doubt. There are a few theoriea poatulated to explain the role

of acylphoaphataae.

(l) The hydrolyaia of 1;3 dipheaphoglyceric acid by acylphoBphataae

(larary (1957) ahowed that acylphos{rfiataae catalyzea the

hydrolyaia of l:3>-diphoaphoglyeeric acid. 1|3~DP((A can be generated

by the phoaphorylation of 3-phoaphoglyceric acid by ATP catalyzed by

phoaphoglyceric acid kinase. It3-DPGA in turn ia hydrolyzed by

acylphoaphataae to yield phoaphoglyceric acid and inorganic phoaphate.

(i) ATP + 3 phoaphoglyceric acidADP + 1:3-DPGA

(ii) ii3-D!'6A— — *------> 3 phoaphoglyceric acid ♦ PI

The auB of equationa ( i ) and ( i i ) girea 35

Equation ( i l l ) would aean a PGA dopendant h7drol7 ai« of ATP eatalycad by PGA kinaaa and aeylphoaphataa*.

At low lerala of tlaaue inorcanie phoaphat* tha rata of glycolyaia nay ba limited by tha inorganic phoaphata eontant. At thia ataga acylphoaphataaa nay anhanca tha rata of hydrolysing i:3-diphoaphoglyearie acid and tharaby uneoupling glyeolyaia from phoaphorylation.

(ii) Acylphoaphataaa in Na*-K* tramport

Data from aevaral laboratoriaa auggaat that ATPaaa which haa acetylphoaphataaa la involved in tha tictlTa tranaport of aodium and potaaaium. It ia poaaible that aeylphoiphataae in erythrocytea ate. may have a role in tranaport of Iona acroaa cell membranea. There ia, howeTer, no evidence to aupport thia auggeation.

The difference in aubatrate apeeificity of the erythrocyte enzyme from that of enqrme from vtier animal tiaauea auggeat the poaaibility that their rolea may be different.

(ill) Acylphoaphataae aa a chemotropic effector

A Tery intereating and novel suggestion has been made by

Grisolia (1068) regarding the phyalological role of acylphoaphataaa.

It was shown that a non-enzymic carbamylation of glutamic dehydro genase occurs when carbanqrl phoaphata ia incubated with the enzjrmef thereby inactivating the enzjrme. This may have an important bearing on regulation of enxymic activity making proteina more susceptible to degradation by cathepsins or lysozomal enzymes. The function of acylphosphatase may be to regulate the concentration of the acylphosphates (carbanqrl phosphate, fomyl phosphate and acetylphosphate. 36

which are all known to be proiiuced by carbamyl phosphate tynthctase

and 1:3-DP6A) to protect onduly high levels of these compounds being

fomed, which would have a harmful effect. Prerention or regulation

of non-enzymic acylation of enzymes (and possibly other proteins)

would be a function of acylphosphatase which is termed a chemotropic

effector.

Acylphosphatase of erythrocytes acts on 1:3-DPGA but not on

carbamyl phosphate, since the latter is not formed or is present only

in negligible amounts in erythrocytes. It would be interesting to

determine whether avian erythrocytes, which are micleated, contain

enzymes which hydrolyze both 1:3-DPGA and carbangrl phosphate. 37

SKCTION X

MECHMIHM OF ACTION

AeylphoBph.atas* catalyzes th* hydrolysis of a rariety of

acylphospbatss such as aoetylphosphats, l:3>diphosphoglyceric acid

and carbauyl phosphate. Whether the enzyme elearage takes place

between carbon and oxygen or between phosphorus and oxygen was

studied by Bentley (1949) with water enriched with the ^^0 isotope.

18 Since an analytical method for the estimatioa of 0 in phosphate

was not readily awailable only the acetate portion of the molecule

was studied. At the end of the reaction the products were analyzed. 18 Only a rery small amnunt of 0 was found in the isolated acetic acid

indicating that the enzymic hydrolysis proceeds with ttie splitting

of the phosphorusoo^gen bontd as shown in the following scheme.

0 ' 0 0 0 CHg-C-O ^ CHg-C-0 + IT O-P-0 ♦ IT ' 0 " 0 “ I

H I ^®0H i iKoshland (1052) studied the nonenzymic hydrolysis of acetylphosphate

catalyzed by acid and base, metal ions and pyridine. In these experi

ments it was obsenred that at the extremes of the dominant pathway

was a nucleophilic attack by water on the carbonyl carbon, whereas 4t

neutrality the major reaction was an attack on the phosphorus atom.

The enzymic reaction resem‘

Koshland suggested that the accelerated rate of the enzjrmic reaction 58

ia due to polarization of tho •loetrons in the •ubatrat* molecnle at the active site of the enzyne*

Uecently Satchell and White (1972) have studied the ehemieal mechanism of the reaction in detail and have supported the earlier studies of Bentley and Koshland, They observed that the enzynie catalysis differs significantly from the nonenzyoic hydrolysis of acylphosphates. The substrate is held primarily by the phosphate group. During the surface reaction the phosphorus atom is uniquely

located by bonds to three of the phosphate oaygen atoas and suffers a slow nucleophilic attack by an adjacent water molecule. The >2 mechanism explains the observed unsuitability of species BOPO U itCO.OPOgH and HCO.OPOgR' as substrates for muscle acylphosphatase* 39

SECTION XI

PILESENT ffOnK

The work reported in this thesia deals with the isoliitlon in pure form of acyl phosphatase froa the seeds of Vigna cat.jang ai^ the study of its properties and kinetics*

The enzyme was separated from non-specific phosphatases and purified by fractionation with aaimonium sulphate and chromatography on Dl*l4l!!-cellulose, CM-cellulose and Sepbadex<-G-100. The purified enzyme was homogeneous by ultracentrifugation and gel electrophoresis.

The maximum specific activity obtained by this procedure was 300 to

1200 umoles of aoetylphosphate hydrolyzed per mg protein per hour at

30*- at pH 6 .7 .

The study of the properties and kinetics of the enzyme include the effects of pH, temperature, substrate concentration and inhibitors, ultracentrifugation, acrylamide gel electrophoresis and amino acid composition*

Chapter I I of the thesis deals with the materials and experimental methods used in these studies.

Chapter III deals with the isolation of acylphosphatase, its

8c>|>aration from other phosphatases and its fwirificHtlon*

Chapter IV describes properties and kinetics of purified acylphospbatase*

Chapter V deals with the discussion of the results of these studies.

Chapter VI contaios a summary of the results and conclusions of this work*

A bibliography is presented at the end* C H A P T E R I I

MATERIAL S A N D METHODS CHAPTKR 11

MATERIALS AND METHODS

Materialei

Seedat Vigna cat.lang seeda were purchased from tha local aarket and

8tor«d at 0® till u««.

Chwdeala; All comtnon chemicals a« well as Tris were of analytical grade. The following chemicals were obtained from Sigma Chemical

Company, U .S .A . t ATP, diisopropylfluorophosphate, dithiothreitol, oxidized glutathione, reduced glutathione, 5~6* dithio-bis-2-nitro- bemsoic acid, j^hydroxymercaribenzoate. Sodium borohydride and cysteine hydrochloride were obtained from Floka (A6).

Aeetylphosphate was either obtained from Sigma Chemical

Compaiqr or from Biochemicals Unit, Delhi or prepared in this laboratory (Stadtean, 1967(a) ). Carbanyl phosphate was obtained from Sigma Chemical Company and recrystallised before use (Metzenberg,

Marshall and Cohen, I9 6 0 ).

Amberlite IRC-60 ( X & 4 4 ) (mesh 200 to 400) was obtained from Rohm and Haas. DEAE-cellalose (lOO to 200 mesh 0 .5 meq per g ),

CM-cellulose (0.7 meq per g) and cellulose phosphate were obtained either from Bio-ttad Laboratories or from Sigma Chemical Company.

Calcium phosphate gel was prepared according to the procedure of

Swingle and Tiselius (l96l). Celluloses were mshed according to the method described by Peterson and Sober (1956). CU-Sephadez,

Se(rfiadex G-75 and Sephadex-G-lOO were obtained from Pharmacia Fine

U 353G 41

Cbeaieala, Sweden. Sephadex was aaspended in water, kept on a boiling water bath for 5 hour*, cooled and deaerated before use.

All the chroaatographie coluiine were prepared with flow of liquid under grarity without application of external pressure. Stepwise change in «olarity of the buffer was used for the elution of the

enzyne from the column.

The chemicals used for acrylanide gel electrophoresis, acrylamide, N-N'-methylene-bis-acrylMiide and tetranethyl-methyl-'

ethylene-dianine and Amido Black lOB were obtained from Eastman

Kodak Company, U .S .A . 42

M • t h o d ■;

D»finition of unit of actiYity and ■peclfic aetirltr

The unit of acylphosphatase activity ia defined as the amount

of enzyme that hydrolyze* onejomole of acetylphosphate per hour at 30*

at pH 5 .7 . The specific actirity of the enzyme is defined as the

activity per mg of protein.

Estimation of acylphosphatase activity

H estrin's (104Q) colorimetric method was followed for assaying acyl phosphatase . The details of the method are as follows.

The assay system consisted of 100 ^o le s of K-aeetate buffer

pH 9 .7 , acetylphosphate (9 /m oles) and enzyme in a final volume of

1 .5 ml. The final pH of the reaction mixture was 5 .7 and the tempera ture was 30**. The reaction was started by adding enzyme and the refustion mixture was incubated for 30 min. The amount of acetylphosphate hydrolyzed was not greater than 25 to 30^ of the initial quantity. The reaction was stopped by adding 3 ml of alkaline hydroxylamine (prepared by mixing equal volumes of 3 .5 tl NaOH and

2 M hydroxylamine hydrochloride). After 5 min this was followed by the addition of 1.5 ml of hydrochloric acid (2 volumes of concentrated

HCl mixed with 3 volumes of water) and 1.5 ml of a 10 per cent solution

(w/v) of ferric chloride in 0,1 N UCl. The colour was read at 540 na.

An optical density change of 0.120 for a 1 cm light path was taken as equivalent to one /imole of acetylphosphate hydrolyzed. A blaidc with substrate alone without enzyme was always run. Blanks with en^me 43 vere negligibl« «xc«pt with erudc cztraet*. While determining the effects of some compounds on enzyme activity controls were run to ensure that the corapoand did not interfere with the color given by acetylphosphate. In the ease of reaction mixtures conr> taining more than 100 yUg of protein, the

In some of the experiments acylphosphatase actirity was determined by Lipmann’ s method (1 9 4 5 ). There was no difference in the actiwities obtained by the two methods. Hestrin's method was used for most of this work.

Estiaiation of ATPase, glneose-6-phosphatase and P-glycerephosphatase activity ai^ units of activity

Fiske and Subba Row's (1926) colorimetric method was followed for assaying ATPase, G-^-Pase and ^glycerophosphatase activities.

The reaction mixture consisted of 10 ^o le s of ATP, G-6-P or DL- f^glycerophosphate, 100 ^moles of K-acetate buffer pH 5.7 and enzyme in a final volume of 1 .5 ml. The reaction was stopped by adding

1.5 ml of 10^ trichloracetic acid and the amount of inorganic phos phorus formed was estimated. Blades without enzyme and without substrate were also run. The unit of ATPase and glucose-6-phosphatase was defined as the amount of enzyme that liberates 1 yUsole of inorganic phosphorus from the corresponding substrate per hour at pH 5.7 at 30*.

Estimation of protein

Protein was usually determined by the following method based on a modification of that of Warburg and Christian (1941). A correction for nucleic acid and other ultraviolet absorbing impurities 44

is mad* by th« following eqaation (Jagannathan et »1. 1956). It waa aaaoned that a 0.1 per c«nt aolution of protein haa an optieal density of 1 at 280 am for a light path of 1 ca.

I X 2.3 X (0.0. 280 na > O.D. 340 na) - (O.D. 260 ns-O.O. 340 m)

m ng protein per ml

If necessary the enzyme solutions were diluted with 0.01 M K-aeetate buffer, pH 0.7 and the optical densities at 260, 280 and 340 nm were determined. A buffer of the saffle coaposition was used as the blank*

Protein was also detemined by the method of Lowry et al (iOSl) using borine serun albnnin as the standard. It was found that protein values as determifld by Lowry's (l9 6 l) method were twice those obtained by the speotrophotonetrie method (with purified enzyme preparatiom ).

For the accurate estimation of protein in the final purified preparation only Lowry's method was used. The spectrophotometric method «as rou~ tinely used during purification of the enosyme.

Centrifugations were carried out at 0" in an international cen trifuge (Model I'U^l and PA-2), Sorrall (Model SS-i), Spinco (Model L) or Sharpies supereentrifuge (AS 12 c la r ifie r ). Chroraatographio fractions were collected on a Technicon automatic fraction collector.

All glassware was routinely washed with sodium carbonate and then with nitric acid, rinsed successirely with tap water, distilled water and glass distilled wat<«r and dried. No jrease or silicone was used for ground glass joints and stopcocks in chromatographic columns. 45

Glass-dktilled water was as«d for the preparation of all solatioiui*

Speetrophotoaetric determinations were carried out eitiier witii

a Model DU Beekaan •peetrophotoaeter or a Unieaa »pectrophotoaeter

SP 500 model with cuvettes with a light path of 1 ea.

pH eetimatioae were carried out with the glass eleetrode.

The |di of concentrated salt solutions was detemined after dilating

the solution four times with water.

.\nmoniua snlphate precipitations

Aofflonium sulphate saturations were carried out at 0* and were

calculated according to Jagannathan ^ (1956), The following

equations were used for calculating amraoniom sulphate saturations*

For solid aaraonium sulphate -

60 (Sg - Sj) X m 1 - 0 .2 8 X Sg

For saturated amaonioa sulphate solution

100 (Sj - Sj) Y - l - S g where X is the g of solid a m o n iia sulphate to be added for ererjr

100 ml of emsyme solution and Y is the nl of saturated SMBonius

sulphate to be added for 100 u l of solution* in the initial

saturation and 8^ is the required saturation of aaaoniim sulphate at

0 ».

Solid aanoniuffl sulphate was added slowly over a period of

30 min with gentle stirring. Foraation of froth was avoided* The

precipitates were dissolved in a known volume of buffer and the

final volume was then noted. The increase in volume was assumed to 4B

be due to ammoniam sulphate at the aaturation at which the preci

pitate was obtained and a correction was nade for the aanoniam sulphate

concentration of the enzyme solution*

Dltraeentrifuitation

The Molecular weight of the enzyme was determined by the

Archibald (1947) aethod. Runs were carried out on a Spinco Uodel *E*

analytical instnwent equipped with a phase plate and a rotor tea^ra-

ture indicator and control system capable of ■aintaining a constant

tenperature duripg the m n . The phase plate was used at an angle of

80*. The speeds of centri ftigation for linear extrapolation of the

gradient curre were calculated according to La Bar (1966). A synthetic

boundary cell was used and readings at the Beniscus were taken. All

solutions were routinely spun at 13,000 x £ for 30 min before analysis

by ultracentrifugation.

Photographic plates were meamred on a Hilger L-50 two-way

measuring micrometer with a sensitivity of 1 u. Photographic plates

were read at 0.1 mm intervals for molecular weight determinations and

areas determined by trapezoidal analysis. The partial specific Tolnme

of the enzyme was assumed to be 0 .7 2 5 .

Polyacrylamide gel electrophoresis

Acrylamide gels were prepared by following tiie method of

Keisfeld rt al. (1962) with a slight modification . 15^ acrylamide

gels were used. The pH of the gel and the running buffer was 4.6.

0.35 II K-acetate buffer, pll 4.6 was used. The current applied was

4 milliamps per tube and the period of mn was 6 h. Protein was 47

■tained bjr uaiiig 0 .5 ^ Aside Blaek 10 B in 7% acctie acid for 30 Bin

and it waa dastainad by kaaping oyam ight with 3^ aeatie acid.

The phoapbataaa banda wtra loeatad by ataining tha gal with

Lowry-Lopac (1946) raaganta* After the run the gel vaa incubated for

30 ain vith 6 ml of a reaetion aixture eoniaining 00 uaolea of aeetyl-

phoaphate and 400 jaaolea of acetate buffer, pH 5»7* It waa then

removed and placed in a reaetion mixture eontainipg 6 ml of 0 ,1 If

acetate buffer pH 4.0, 1 ml of 1% aaeorbie acid and 1 ml of 1%

afflfflonium molybdate and kept for 30 min. Only the phoaphataaea which

hydroljrze acetylphoaphate gave banda by thia method*

Bjr staining the gel by the above two methoda it waa poaaibla

to dietinguiah the enzyme from other > protein impuritiea*

Determination of free -SH groapa

DTNB titrations for the determination of free >SH groupa wera

carried out according to the procedure of Bllman (1 0 5 8 ). The protein

was treated with an exceas of reagent and the net abaorption at 412 nm

waa employed to calculate the aulfhydryl content. A molar extinction 4 2 -1 coefficient of 1.36 x 10 cm x mole was aasumed for the free thiol

ion of the reagent at pH 8.0. A reagent blank waa uaed to correct for

the absorption of the reagent. The accuracy of the method waa checked with glutathione*

Determination of total aulfhydryl and diaulfide groups

Total -SH groups and -S-S- linkages in acylphosphatase wera determined by two different procedures.

1) Reduction of -S-S- linkages using Na£H^ in 8 U urea followed by

DTNB titrationa after removal of excess of H a W ^* 4S

2) Oxidation of -SH and -S-S- groapa to eyatele aeld iha ataodard

proeadura of parfontie aeid oxidation followed bjr acid hydrolyaia

and ejataic aeid aatimation uaing an amino aeid analyser.

1 ) Redaction of e n a y a by NaBH^ in urea

The method of C arallini, Graziani and Dupre (1966) waa uaed.

The enzjnae waa redueed with borohjrdride in the preaenee of urea, EDTA, and phoaphate buffer, fdi 7*6 at 38*. After incubation for 45 min exeeaa borohydride waa deatroyed by the addition of potaaaiun dihydro gen {riioaphate containing acid. The color waa then developed with DTNB in an atraoaphere ' of nitrogen and read at 412 na. The nuiber of

sulfhydryl groupa (N) waa calculated uaing the following fonmla.

II Mw X A X V " " 12,000 X ■ where Mw - molecular wei^t of the protein

A ■ abaorbaney

V ■ rolnne of the final aolution

■ ■ weight in ag of the protein aample analysed

The aceuraey of the aaaay waa cheeked with bovine aeraa albumin aa atandard.

Amine acid analyaia

Afflino aeid analyaia of perform!c acid oxidised aample of aeylphoaphataae waa carried eat according to the method deaeribed by

Hire (1967).

Betimation of tyroaine and tryptophan

Tyrosine and tryptophan in the enzyme were determined by

( 1) Goodwin and tforton'a (1946) method and (2) Bencze and Sclnid'a method (1957). The yalues of tryptophan and tyrosine obtained by 49

the two Mthods were in good agreement*

Goodwin and >lorton*e method (lQ46)

Proteina show aeleotive abaorption in the ultraviolet region

and the poaition of the abaorption maxiaum rariea with pH. The majority of the constituent amino acida do not show any absorption in the region 250-320 nm and it ia known that phenylalanine, tyroaine and tryptophan are responsible for the obserred ultraviolet absorption of protein solutions. In 0 .1 N NaOH the absorption by tyrosine and by tryptophan is much stronger and that by pheiqrlalanine is negligible.

Under these conditions the protein solutions may be treated as two- component system for spectrophotometric analysis. The intensity of absorption at the point where the curves for tyrosine and tryptophan intersect is a direct measure of the total molar solute coneentration and will be the same however the proportions are varied. At other wave lengths the intensity of absorption will vary with the relative propor tions of the components. Using 0 .1 N NaOH as solvent the two absorp tion curves intersect at 294.4 n ( (^>2376) and 257.15 nm ( e « 2748),

By determinii^ the absorption of the protein in 0 .1 N NaOH at the above two wavelengths and at one other wavelei%th (e.g. at 280 nm) it is possible to determine the relative pvoportions of tyrosine and trypto* phan in the protein.

Thus, if X « total mol / 1 in solution

y - g fflol / 1 of tyrosine

* “ 7 ” g mol / 1 of tryptophan at any wavelength other than the point of intersection 50

let ^ tyroain* b« A and € tryptophan be fi and the obserred intensity

of absorption be E

then,

B - yA ♦ (x-y)B

E- xB - r r i

j ^ valae at an intersection ^ tyr. at an interseetion

(2) Beneze and Scb«id*s ■etbod (graphical aethod)

This aethod is based upon aeasarinf the absorbance of the protein in

0.1 N NaOH in the range between 278 and 204 nsi at 2 nsi interrals. The

readings are plotted against the warelength and a line is drawn trn- gentially to the two characteristic peaks. From the slope of the tangent, the maxima absorption between 270 and 200 na and the aole- cnlar weight of the protein the tyrosine and tryptophan content can be deterained* CHAPTER III

EXPERIMENTAL AND RESULTS 51

s E c n o s I

Acylpbo«phata»« of plant ealtur««

PrsllBinarjr work on acyl phosphatase was carriad out with tisaaa

eultarea of plants. Aeylphosphatase was found to bs pressnt both in

xMnal and tuaor tissue eultures of Parthenocissas. In addition ATPase

and glueose-6-phosphatase were also present in the tissue and by extrae-

tion with 0,2 N HCl-0.2 N KCl, followed by aanoniw sulphate fractional

tien an acylphosphatase preparation nas obtained free froa the other

phosphatases* These results, which will not be described in detail,

showed the presence of a true acylphosphatase in plant tissues free

fron phosphatase activity with other substrates.

AcyliAosphatase of seeds

Since plant tissue eultures take a rery long time to grow

in Titro and are difficult to obtain in large quantities, extraction of

the enzyme from a plant source, such as seeds, which is available

throughout the year, was tried*

In the ease of Vigna catjang (black eyed peas), naize, and

sorghum 1 g of handgroandseeds was extracted with a aixture of 5 al

of 0 .0 1 U K-acetate buffer pH 5 .7 , 1 al of 0*1 M HCl and 75 mg of solid KCl. The extract was allowed to stand for 30 ain and neutralized

to pH 6.7 by 0*1 H KHCO^. The supernatant liquid obtained after centri fugation was assayed for acylfriiosphatase aetirity. In the case of aung beans instead of 0*1 U HCl, 0*2 U HCl was used for extraction. Acyl- phosphatase was present in all the different seeds which were tested 52

(Table 5), but the actirity varied considerably. The highest aotiTity waa found in seeds of Vigna eat.1aog«

TAffluE 5

ACYLPHOSPHATASE ACTIVITY OF SEEDS

Seed ActiTity

(tt/g dry seed)

Cholai (Vigna cat.1ang) 243

Soya beans (Glycine so.1a or Glycine max) 90

ilaize (Zea mays) 80

Sorghum (SorgtKim ralgare) 18

Vfung beans (Phaseolus mango or 36 Phaseolus radiatus rar. muige)

The results are not corrected for other non-specific phosphatases which aay act on acetylphosphate.

Acylphosphatase of cholai seeds

(The local name cholai is used for Vigna cat.1ang)

Since cholai was the richest source of acylphosphatase, cholai

seeds were chosen for further work on the ensyoie.

An extract of cholai seeds was prepared as before and the acylphosphatase actirity as well as the liberation of inorganic phos phate from ATP, glucose-6-pfaosphate and ^glycerophosphate were deter mined* 53

TABLE 6

C n O U I SEED PHOSPHATASES

Actirity

( « /g seed)

Aoylphosphatase 284

ATPase 108

Glaoose>4-phosphatase 14

^-Gljre ero phos phat as e 16

The •xtraet contain* high ATPase in addition to aejlphos* phataao as well as rslativsly low aetivitlss with glaeoss-4- phosphate and ^-glyesrophosphate « Tho rolatiro aaoants of acylf^osphatase and ATPaso varied with different lots of seeds and different methods of extraction. 5?

SECTION I I

SEPARATION OF ACYLPHOSniATASE FllOM OTHER PHOSPHATASES

Th* following experinents wor* earriod oat to establlsfai wfaother th« acylphosphataae «a« apacific or whather it aaa a nonapaeifie phoaphataaa.

Haating

Acjrlphoaphataae waa found to be fairly atabla towarda haat and acid. Haating of the acid extract of the aeeda at SO* for 6 rain reaulta in narked destruction of ATPaae, whereas only 40 to BOjt of aeyl-

(^oapbataae is lost (Table 7).

TABLE 7

EF’FBCT OF Hl'ATING ON AcPasi? AND ATP'a~se

AcPaae ATPase

o /g « / f

Before heating 243 302

After heating 117 31 at 5 0 ” for S nin

Effect of fluoride

The effect of fluoride on the different phosphataaea was tested. The phosphatase activities with different substrates with and without fluoride are shown in Table 8. 55

TABLE 8

EFFECT OF FLUOBIDE ON CllOLAI SRED PHOSl>HATASES

AcPase ATPase G-6-Pase units/g anits/g units/g

Without fluoride 56 36 9

With 10 mM fluoride 38 3 3

Inhibition ( f ) 33 92 66

It vaa fonnd that 10 mU fluoride inhibited aeylphoapbataa* aetivitjr

only 30;( whereas G-6-Pa«e was more strongly inhibited and ATPase

almost completely inhibited. These results also suggested that aoyl-

phosphatase is different from the other phosphatases.

Acid extraction

The amounts of different phosphatases extracted when the co»>

centration of acid used for extracting the seeds was varied were

examined. In three different experiments 10 g of seeds were extracted with 5U ml of 0.01 M K-acetate buffer, pU 5.7 containing 0.75 g KCl

and 10 ml of 0.2 U, 0.4 M or 0.6 If HCl. The extracts were centrifuged

for 30 ain at 3,000 x £. The supernatant liquids were neutralised to

pH 6.7 by the addition of 2M KHCO^. The precipitates were centrifuged

off and the clear supernatant liquids were assayed for different phos

phatase activities. Table (9). 5B

TABLE 0

EFFECT OF ACID ON CHOLAI SEED PHOSPHATASES

Concentration of AcPaae ATPaae G-^—Paae acid for extraction

u/g tt/g

0 .2 U HCl 192 144 42

0 .4 M HCl 96 32 8

0 .6 M HCl 72 16 2

I t w ill be a«en that with ii^reaaing amouuta of acid the amounts of ATPase and G-6-Pa»e extracted are more narkedly reduced than in the case of acyl phosphatase. With 0*6 U HCI, rery little G>-6-Pase was present in the extract. The differences in the activities of the enxTaes in the extracts were not due to differences in salt content after neutralization since the relatiTe actiYities with the different substrates were unchanRed even after precipitation of the enzyaes with anoniua sulphate followed by dialysis.

The above results alse indicate that a specific aeylphosphatase is present in these seeds. Extraction with 0.6 if HCl-KCl was used in all further experiments. The amounts of ATPase and

G>6-Pase were, however, variable with different batchea and in aome eaaea much higher amounta of ATPaae relative to AcPaae were preaent in the extract. 57

SECTION III

T»uni FI CATION

Preliminary «xp«rim«nts on th* extraction of the enzyae indi

cated that aoylphoaphataBe was extracted best with a aolution consis

ting of five ml of 0 .0 1 U K-acetate buffer pH 5«7*- 0 .2 U KCl and one ml of 0.8 M KCl per g of seed. Umler these conditions the extraction

of nonspecific phosphatases was aarkedljr redaced.

Extraction

600 g of seeds were washed repeatedly first with tap water and

then with distilled water and soaked in glass-distilled water at rooa temperature for 30 giin. Washing of the seeds was necessary for the removal of preserratiTes which were sometimes added to the seeds. All

sabsequent operations were at 0* unless otherwise stated. Soaked

seeds (equivalent to ISO g of dry seeds) were blendorized at a time with 300 ml of 0.01 II K-acetate buffer, pH 5.7, for 3 min. 1,8000ml of 0.01 M K-acetate buffer, pH 6.7, 600 ml of 0.6 U HCl and 4 .5 g of

KCl were then added to the homogenate. The mixture was stirred for

30 min and squeezed through oaislin cloth. The filtrate was centri

fuged for 30 min at 3,000 x £. The supernatant liquid was brought to a pll of 5.7 by the addition of 2 U KHCO^. The precipitate formed was collected by centrifugation for 30 min at 3,000 x £ and discarded and the clear sapernatant liquid was used for purification. 58

SECTION IV

AMMONIPM SULPHATE FRACTIONAnON

Preliminaiy experiaents shoved that when th« *ozjm 9 extract prepared as described in the prerious section was fractionated with afflRionium sulphate and the fractions obtained at 0-0.5, 0.5-0.7 and

0.7-0.9 saturation, tested for actiTitjr, aost of the activity was present in the 0.5- 0.7 fraction. Other experinents showed that the precipitate obtained between 0.4 and 0.7 saturation contained alisost all the acylphosphatase activity. Based on these results the following procedure was used for aamonium sulphate fractionation.

0 to 0.4 saturation. The crude enxyne was fractionated between

0 to 0.4 saturation. To every 100 ml of enzyae solution 22.5 g of powdered aamonium sulphate were added with stirring and after 30 a in at 0*, the solution was centrifuged at 3,000 x for 30 ain« The sediment was discarded*

0.4 to 0.7 saturation. To every 100 ml of the supernatant liquid 19 g of anmoniun sulphate were added to increase the satura tion to 0.7* The precipitate was collected by centrifugation at

13,000 X £ for 30 nin.

Washing with 0«7 saturated anmoniua sulphate. The precipitate obtained by 0.4 to 0.7 saturation was washed with 0.7 saturated aamo nium sulphate solution to remove the adhering aother liquor.

Fractionation at pll 8.5 (0.4 - 0.9 saturation). Amaonina sulphate precipitation at an alkaline pH was carried out, since the enzyme precipitated at pH 6,7 was unstable on storage in some experiments. * 59 This fraction also showed lack of linearity of activity with «sz]nM

concentration. Precipitation at alkaline pff increased the purity

only 2 to 3>fold bat it gave an enzyme preparation, which could be

stored for several weeks without loss of activity. The activity of

the preparation was also proportional to ensyme concentration due

possibly to the reaoval of inhibitory iapurities.

The precipitate obtained after washing with 0.7 saturated

anffloniua sulphate was suspended in 0.1 M Tris-HCl buffer, pH 8.5, which was 0.4 saturated with respect to anaoniua sulj^te. It was centrifuged at 13,000 z £ and the sediment was discarded. To every

100 ml of the above supernatant liquid 33 g of aanioninm sulphate were added in order to increase the saturation from 0.4 to 0.0. The pre cipitate obtained by centrifugation at 13,000 z £ for 30 min was dissolved in 0.01 U K»acetate buffer pH 6.7 and dialyzed against two or three changes of 100 volumes of the same buffer.

The results of a typical ammonium sulphate fractionation are presented in Table 10.

It is evident from the Table that about 7~fold purification is eichieved in this fractionation. The final specific activity with different batches ranged between 30 to 50. fiO

5 t CO S a

ac 8 » I- PSg

•3-C o o •«» *H r - o e •*» m « H O 00 CO « CM

M9 -H 4L ► m r - •H 4* I

o lO lO o» o C4 « e fHI 0 >

g 1 I ■S. I 0 e •*» « « 00 • § o o -P 5 % Ik s Cm - 'f 4 * m CQ O © I 61

SECTION V

Ml«e«llaneoaB ad»orb»nt« tRC-50-XE>64

The •nzyme obtained after amaonitui aalphate preelpitailon was found to be adeorbed completely by the cation exehanga reain IRC<-50-

XE-64 in 0 ,0 1 M K-aoetate buffer, pH 5 .7 , 20 ml of the enzyme con taining 10,000 units were treated with 10 g of reain in 0.01 U

K»aeetate buffer, ^ 5.7. The mixture was stirred for 30 min and centrifuged. The residue was washed suecessiTely with 20 ml of 0.03 II

K-acetate buffer, pU 5.7, 200 ml of 0.08 M K-aeetate buffer, 16 ml of

0.15 U K>acetate buffer, 20 ml of 0.2 U K-acetate buffer and 40 ml of

0.3 M K-acetate buffer, pU 5.7. It was then eluted with 40 ml of 0.5 II

K-acetate buffer, pH 5.7 in two lots. The enayme was present in the

0,5 U eluates. Table 11 presents the summary of results on adsorption and elution of the enssyne from IRC-50-XB-fi4.

I t will be seen from the Table 11 that eniyme of vexy high purity was obtained merely by batchwise chromatography of the enzyme on IRC. These results were obtained in three successiTe experiments, but it was not possible to reproduce them subsequently. Sereral experiments were tried to ascertain the reason for this lack of repro ducibility. These include washing of IliC at different {^s and with phosphate buffer, washing the seeds to remove any preservatives added to the seeds, passing the enzyme through Sephadex-G-25 before IIIC- treatment etc. The results of all these experiments were negative and it was not possible to reproduce the earlier results. The reasons 62

« K •H •+» •H ► « O :S - g 0 1 ^ 8 fl »-l

d 8 * « • Jkl £f • o o H rH m.. a . ^ so<

fl •H fH cm W ■*» o ^ s =■ £

X ■*» ■ w w 3 S e -p 1 § ? H g 0 O O (B

> O O •M 2 g s Cl s

i *2 S $

fl wA •H a «w . ®^ e •*>

B ■S 5 e e ••■1- —« •f* S >*• s ^ lO £ i o 63

for thl* lack of r«prodaolbility is not known. It may b« da* to the

Chang* in th* atrain of s*cda us*d for these experiments. This nethod

was not furtiier used.

Cellulose phosphate

It was found that 1 ag (dry weight) of eellalose phosphate was

required to adsorb 5 units of crude ensyne in O.Oi II K-aeetate buffer,

pii 5 .7 and at pH 4 .8 . The enzyme could be elated with 0 .1 M K-acetate

buffer, pH 5.7 or with 0.1 M K-acetate buffer, pH 4.8. But there was no

significant increase in specific activity. This adsorbent was not used

for further work.

Calcium phosphate gel

The partially purified enzyme (i.e ., the enzyne obtained after

CM-^ellulose chroaatography) \ras used for these experiments. 50^ adsor

ption was observed when 4 mg of the adsorbent was used per unit of enzyne

in 0.01 K-aeetate buffer, pH 5.7 containing 0,01 If NaCl. Elution with

0.1 M K-aeetate buffer, pH 5.7 did not result in any increase in specific

activity.

SE-Cellulose

50^ adsorption of the partially purified enzyme (the enzyme obtained after CM-cellulose ehronwtography) was observed on SE-eellulose in two to three batchwise experiments in O.Oi If K-aoetate buffer, pH 5.7 and about 4-fUd increase in the specific activity of the nnadsorbed enzyme was observed. Hut it was not possible to reproduce these results. 61

Alumina -» Cy

Adsorption of partially purified «nzyn« on alunina-Cy was tried using two different amounta of adsorbent (l tag of alumina per

40 units and 1 mg of alumina per unit of enzyme) in 0.01 M tris, pH 7.S.

Only 50^ adsorption was observed in both the cases. There was no increase in the specific activity of the unadsorbed enzyme. The enzyme could not be eluted with 0.2 11 tris, pH 7.5. This method was not used for further purification*

CM-Sephadex

Preliminary experiments with this adsorbent (l mg per 10 units,

5 units or 2 units) showed complete adsorption of the partially purified enzyme in all the three cases* But no enzyme could be eluted with 0 .2 If

K->aeetate buffer, pH 5 .7 and o n ^ 30^ of the enzyme could be eluted with

0 .5 U K<-acetate buffer, {A 6 .7 . In view of the low recovery of enzyme and absemse of any significant purification column chromatography nas not tried with CM-Sephadex.

Sephadex G-75

Chromatography of enzyme obtained after CM-cellulose chromato graphy on columns of Sephadex G-75 in 0.05 M K-acetate buffer, pH 5.7 and in 0.05 U K-acetate buffer, pH 5.7, containing ammonium sulphate

(7 g/lOO ml > 0.1 saturation) did not result in any increase in the specific activity of the enzyme in any of the fractions* 65

SECTION VI

CHmytATOGRAPHY ON CARn0X!gMy.T!IYL CEUinjOSE

The •ncym* obtained after anmoniuB ealphate fractionation vas used for atudiea with CM>eelluloae. Preliainazy batchwise experiments indicated that at pii 5.7, 10 anita of the enzyne were adsorbed completely by 1 mg of CM-«ellaloae in 0.01 If K-aeetate buffer at pH 5.7. The activity could be eluted by 0.3 M K>acetate buffer, pH 5 ,7 . At pH 7 .5 , 5 unite of enzyve were adsorbed conpletely by 1 ng of CU-cellulose and the enzyae could be eluted with

0,2 II Tris buffer pil 7.5. About two to three-fold purification was achiered in both the cases. Since at pH 7.5, tiie aaount of iapnri- ties that were left in the unadsorbed fraction was more than at pH 5.7, chromatography at pH 7.5 was preferred. ATPase and 6-6-Pase were completely removed in the unadsorbed fraction. Separation of acyl phosphatase from other no»>speeific phosphatases as well as significant purification was obtained by chromatography of the enzyme on CM-cellulose.

The adsorbent was washed according to the method of Petersen ani Sober (1962) and finally washed with 0.001 M EDTA, pH 7.5. A column (3.8 x 10 cm) was prepared and washed with 0,01 U Tris buffer, pH 7.5, 1 mg of adsorbent being used for 1 unit of enzyme. 19 ml of the enzyme (corresponding to 5,700 units) obtained after ammonium sulphate fractionation and dialysis were loaded on the column. The column was washed with about 400 ml of 0.01 M Tris buffer, pH 7,5 and with 250 ml of 0.1 M Tris buffer, pH 7.5. The column was then 6B

«lut«d with aboat 300 ml of 0.2 M Tria buffer, pH 7.5. 20 al fractions wer« eolleet«d and th* activity and {urotoin in «a«h fraction were determined. The reealta are presented in Table 12 and the actiTity and protein patterns are rtievn in F ig , 1 ,

Fractions 6 to 10 were pooled and precipitated with aisfflonium sulphate at 0.9 saturation by adding 60 g of aomoniuas sulphate for 100 ml of liquid. The actiTity and protein of the dialyzed enzyme were deterained. About 26 to 50 per cent of the initial actirity was recorered with about 35-fold increase in specific actiTity. Since other non-specific enzymes were remored here, the actual recoTery of true acylphosphatase is higher.

While precipitating the enzyme from dilute solutions Teiy high losses in recoTery were obserTed. About 30 to 40 per cent of the activity was lost in this step. This was aToided by con centrating the enzyme two to four times by lyophilization before precipitation at 0,9 saturation with aimoninm sulphate. 80 to 00 per cent of the actirity was recorered by this procedure*

During this chromatography, ATPase and other nonspecific phosphatases remain in the unadsorbed supernatant fraction and

0.1 M buffer washii^s. 0.2 M buffer eluates are completely free from ATPase and G-6-Pase. Only true acylphosphatase is present after Q(-cellulase chromatography (Table 13). 67

sr OI •t) -p > a V 03 (d •a lO ooo'4*Mnooaooo 00 • •••••••• • eti<4«noi«^^«-«oo lO -I ir I

38S;38SSSS• •••••••• CO i IS o o o o o o o o o

a 3•♦» *H ► O •»» I § I s in n M

M CM C4 • • o s s g s g s * ” - •** 0 I < 9

to 9 •2* »-i®

So I ■•» ■** ”o, O -H —' o B 0 so e * •H e o. ■P U t 2 ■p 1 114 O SP O* -*» » 1 1 s • >» • -H ^ 4> •e oi n u o u »H *H ^ «t M ^ tk,

ELUTION PATTERf >HOSPH 68

TABLE 13

SEPARATION OF ACYU^HOSPHATASE FROM OTHER PHOSPIUTASBS By CHROMATOGRAPHY ON CM-CRLLULOSE COUTMN AT pH 7 ,5

Fraction AcPass ATPass G-d-Pass

units units units

Before CU*>oellaloBe 9,600 5 ,500 1,000 chromatography

0.01 If washings 1,300 1,100

0.1 If washings 3,860 3,100

0.2 M eluates 2,300 0 0 69

SBCTIOM VII

SEPHADFX-O-lOO

The cnaiya* obtained after CM-cellalose ehrooati^raphy showed three

bands (and in some eases a few other faint bands) on disc electrophoresis when the gels were stained with Aaido Schwarz. However, when the gels were stained for acylphosphatase (by first incnbating the gel with aoetyl-

phosphate and then treating with a mixture of 1^ ascorbate and ammoniiui

molybdate at pH 4,5, Chapter II) only one phosphatase bands was observed.

This indicated that the inpurities present in the enzyne were protein

impurities other than phosphatases. From the position of the bands on

the gel it could be seen that the imparities were higher molecular weight

imfmrities. Due to this difference in molecular weights of the three

components, it appeared to be worthwMle to attempt the separation of the

enzyme from the imparities by ehroraatogra;phy on Sephadez G-iOO. 0 .0 6 U

K-acetate buffer at pR 4.5 was used because at this pH the protein showed good separation on gel electrophoresis. The stability of the enzyme at

this pH was determined separately. The enzyme was found to lose no activity in 24 hr at 0* at pH 4*5.

16 g of Sephadex-6-100 were allowed to swell in a sufficient volume of 0.05 M K-acetate buffer, pH 4.5 at room temperature for about three to four days and a column ( 1 .8 x 100 cm) was prepared. The column was equili brated with the same buffer at 0°.

3 ml (2,250 units and 9 mg) of enzyme (in 0.05 II K-acetate buffer, pH 4.5) obtained after Cll-cellulose chromatography were loaded on the column. 0.05 M K-acetate buffer at pH 4.5 was passed thrnagh the column.

1.5 ml fractions were collected and the protein in all the fractions was 7n

deterained. The fractions baring {orotein were assayed for enzyme activity. The protein was present in fractions 24 to 40. The activity was also present in fractions 24 to 40. The purity of the active

fractions was also deterained by gel electrophoresis. Fractions 28 to

40 showed a single band. They were pooled (18 a l) and precipitated with amnonium sulphate at 0*9 saturation. The precipitate was dissolved

in 3 b1 of 0.01 U K-acetate buffer, pH 5.7 and dialyzed against two

chaiiges of 500 al of the same buffer. The voluae after dialysis was

4 a l . 55^ of the In itial activity was recovered in this ehromatographgr.

The results are shown in table 14 and Fig. 2 .

Froa the Table 14 it will be seen that there is relatively

little increase in purity in this fractionation, but the enzyne obtained

after 01f->cellulose chronatography showed several bands on acrylaaide gel

electrophoresis, whereas after sephadex chrooatografihy only one enzyae band was obtained* 71

■i i « ^ Cu I s s s s s s s 2 ^ a Z HOOOOOOO o • e ■*» %» o • * H

8 e* «o o o ^ I i n ss R N cn a 1

fl M9 lO lO « 00 35 r t m (M o o Ok ot (Ms « ? IC A •* eo o • aw ^ s. o o o cs o o o o o

a CO le lO o 5 s « s so § sei 8C9 CO CVJ CM O I VA • i I o o o o e o o o o •p►> CO 00 >J s « s s§ C9 Ms s »H s I o -♦»•»* •a a- 9 S c"

n CM ■ s o s » O g -P s ? « s s 5 ■a t - 0

lO lo lo le lO M3 lO lA 5 CO ►

e A . ^ ■s t 2 m w ♦ ^ A /Mk W (M O. « 0 0 . - « 3 o Vm% at10 f t CO g s ■♦» o O i-l 4> • o •M « 2 j . > £ O U s s (S £ fa •<»

SBCTION VIII

MODIFIED PURIFICATION PROCEDURE

Th« •ztraetion procedure deaeribed in the beginning of thie chapter waa suitable for the extraction of en^gme on a email scale.

However, for aore rapid work and to get larger anounte of enzyme it was easential to modify the earlier procedure. The following changes were made in the new purification procedure*

1) Seed extraction

Only 150 g of seeds could be blendorized at a time in the

Waring blendor. For processing big batches of 3 kg of seeds blendorizing was too time-consuming. The blendor was, therefore, replaced by a motorized meat mincer. The soaked seeds were passed through a meat mincer and the minced material was suspended in buffer. The mincing operation required 30 min whereas blendorizing would have taken a mnch longer period. Uoreorer, the enzyme is unstable in strongly acid solution if the temperature is not low and it was difficult to carry out blendorizing without undue increase in temperature.

2) Ammonium sulphate fractionation

In the preTious procedure the extract was first precipitated at 0*4 annonium sulphate saturation. The solution was centrifuged and the precipitate was discarded. The ammoniun sulphate saturation of the supernatant liquid was then raised to 0.7. This inrolred two centrifugations of a large Tolume of liq u id . It was easier directly to precipitate the enzyme at 0 .7 saturation and then to refractionate 73

it at 0.3-0.9 saturation in a smaller volume rather than fractionate

a large volume between 0.4*>0.7 aaturation.

In the nodified parifieation procedure the fractionation <

at pH 8.5 was deleted and chromatography on DEAB-celluleae was

introduced.

Chromatography on DEA.B~celluloae

In some of the experiments, even after aomoniiui sulphate

fractionation, the ATPase content of the extract was high. If a

direct chromatography on CM-cellulose was performed with such enzyme

preparations, the specific activity of the enzyme obtained after CM-

cellulose chromatography was rather low (120-150). Further purifi cation of this enzyme was d iffic u lt. To overcome this diffic u lty ,

negative adsorption of the enzyme on DEAE-cellulose was introduced before chromatography on CM-cellulose.

In a preliminary batchwise experiment it was found that when

20 mg of OEAE^cellulose were used for 1 mg of protein, acylphosphatase

remained unadsorbed while almost all the AT?ase was adsorbed on the cellulose. Column chromatography of the enzyme on DKAE-cellulose nas

then carried out.

30 g of DEAB-cellulose were washed and equilibrated with 0.01 U potassium phosptiate buffer, p!I 7.5. A column (6 x 10 cm) was prepared and wasned with 0.01 U phosphate buffer, pH 7 .5 . 30 ml of the annonium sulphate precipitated and dialyzed enzyme corresponding to 21,000 units and 1 .5 g of protein were loaded on the coloan. The column was washed with 600 ml of 0.01 M K-phosphate buffer, pH 7.5. 50 ml fractions 74

w«re collect«d and the actirity and protein of eaeh fraction wore detemined. (Phosphate is inhibitory to acyl phosphatase aetirity

and dialysis is required to obtain true acylphosphatase actirity;

but for routine work dialysis of each fraction was not carried out.)

The results are presented in Table 16 and Fig. 3. The results are not corrected for inhibition by phosphate. Host of the ATPase is revored in this step. Fractions 4 to 8 were pooled and precipitated with ammonium sulphate at 0*9 saturation. The precipitate was dissolved and dialyzed against 0.01 U K~acetate buffer, pH 6.7. About

50^ of the initial activity was recovered and about two to three-fold increase in the specific activity was observed. The specific activity of the enasyme obtained after fractionation on DBAE-cellulose ranged from 40 to 70 units per mg protein.

For large-scale preparation of the enzyme three such eoluams were run simultaneously.

On the basis of the experiments described in previous sections the purification procedure was modified and is described in the next

Section. 75

I lO 00 n n •t) -p +> n CO •a a e « w i 2 f" S S

^ fCii e .

« t« et] o to s iO A.

o 10 o OI s s O -M o S B en § N PS

t o c n •p CO •H-P o •a s

o o 35 M I g slO scO scp s s s

SP T» O D. N n 00 ^fl 2w o r1 O -♦» «» •*» O I o In s (S 7ti

SECTION IX

FINAL PtIRIFICATION PROCEDURE

Step I , Extraction

Thr*« kg batches were usually processed. The seeds were washed with glass-distilled water and soaked in water at room tempera

ture for 20 rain. All further operations were carried out at 0*. The

seeds were passed through a meat mincer and the minced material was

suspended in the extraction medium consisting of 16,000 ml of 0.0-1 If

K-acetate buffer, pH 5.7, 225 g of KCl, aad 3,000 ml of 0.6 If HCl.

The mixture was allowed to stand for 30 min with occasional stirring

and then squeesed through m slin. The liquid was neutralised to

pH 5.7 by the addition of 2 U fCHCO^. It was passed through a Sharpies centrifuge and the precipitate was discarded (Fraction I). All

Derations upto neutralization of the extract to pH 5,7 should be carried out as rapidly as possible.

Step I I . Ammonium sulphate fractionation

0 to 0.7 saturation. The supernatant liquid from Step I

(14,200 ml) was precipitated with ammonium sulphate by addii^; 6177 g

(43.5 g for every 100 ml solution). It was passed thrmigh a Sharpies centrifuge. The precipitate was collected and the supernatant liquid was discarded.

Washing with 0.7 saturated aaBnonium sulphate. precipitate was suspended in 0.7 saturated annonium sulphate. It was thoroughly mixed to break up lumps. (All operations upto this st(^;e should be carried out on the same day). The next day the b A C T : . ;

-A P R O T F I N

u "T

^RACTIO

FIG.3 ACTIVITY AND PROTEIN PATTERN OF ACYL PHOSPHATASE ON

OEAE-CELLULOSE CHROMATOGRAPHY 77

saapcnsion was e«ntrifug«d for 30 ain at 13,000 x £. The prceipltat*

was disaolTAd in 500 al of 0.01 H K-aeetat* buffer, pH 5.7. The final

volaae of the solutioii waa noted (540 ml). (Fraction II). The final

aauBoniam sulphate eaturation was calculated to be about 0.06.

0.05 to 0.3 saturation. The anmoniun sulphate saturation of

the above solution was raised fron 0.05 to 0.30 by adding 13.6 g of

ammoniuffl sulphate to ereiy 100 ml of the solution. It was allowed to

stand for 30 min and centrifuged for 30 min at 13,000 x £. The preci

pitate was discarded and the supernatant liquid (Tolume 625 ml -

Fraction III) was collected.

0.3 to 0.9 saturation. Fraction III was precipitated with

aomonium sulphate bjr increasing the saturation from 0.3 to 0«9 by

adding 40 g of ammonium sulphate for every 100 ml of solution. The

precipitate was dissolved in 11^ ml of 0.01 M K-acetate buffer, pH 5.7

and dialyzed against three changes of 2 1 each of the same buffer.

The volume after dialysis and centrifugation was 165 ml (Fraction I V ) .

Step II I. Chromatography on DEAE-cellulose.

30 g of DEA19-cellulose were washed and equilibrated with

0.01 y K-phosphate buffer, pH 7.5. A (6 x 10 cm) column was prepared

and washed with 0.01 M phosphate buffer, 7.5. 30 ml of aaraonium

sulphate precipitated and dialyzed enzyme (Fraction IV) were loaded

on the column. The column waa waahed with 600 ml of 0.01 M phoaphate

buffer, pH 7,5. 50 ml fractiona were collected and the activity and

protein of the fractiona were determined. The active fractiona were

pooled and precipitated at 0*9 saturation. The precipitate waa

diasolved and dialyzed against 0.01 M K-acetate buffer, pH 5.7

(Fraction V ). Three such columns were usually run simultaneously. 78

About 50^ of tho initial activity wa* reeoTorod and about 2->3 fold incroas* in the a pacific aetiTi^ waa obtained. The specific activity of the enzyM obtained after fractionation on DEAE-cellulose ranged from 30 to 70 unite per mg.

Step IV. Chromatography on CM-celluloee.

14 g of CU-celluloae were washed and equilibrated with 0.01 U

Tris buffer, pH 7.5. A (15 z 4«4 ca) column was prepared and washed with the same buffer. Two such columns were usually run simultaneously.

35 ml of the enzyme from Step III (Fraction V) were loaded on the column.

The column was washed with 400 ml of 0.1 M Tris buffer, pU 7*5 and eluted with 400 ml of 0.2 M Tris buffer, pll 7*5. 50 ml fractions were collected and estimated for activity and protein. The active fractions with specific activity higher than 200 units/mg were pooled (total volume

275 ml) and concentrated by lyophilisation to less than one-fourth of the in it ia l volume (60 m l). The enzyme was then precipitated with amraoniun sulphate at 0«9 saturation by adding 60 g of ammonium sulphate for every 100 ml solution. The precipitate was dissolved in 3 ml and dialyzed overnight against i litre of 0.01 If K-acetate buffer, pH 5.7

(Fraction VI). About 10-fold purification was achieved by this fractio nation and 40^ of the activity was recovered. The specific activity of the enzyme after fractionation ranged from 300 to 1 ,500 units per mg.

This step was carried out with 10 batches and the fin al enzyme was free from other phosphatases. 79

Step V. Chromatography on S«phad«x-^-100.

Aerylamide gel eleetrophoresia of th« enzya* obtainad after

CU-callaloa* chromatography ahowad that it naadad further parifieatlon.

Aa atatad pravioualy ataining the gala with Aaido Sehwari and for aeylf^oaphataaa ahowad that tha anzyaa from Stap IV (Fraction VI) eontainad at laaat two pretain impuritiaa other than aoylphoaphataae.

Chromatography on Saphadaz-G-'lOO waa carried oat for farther purifica tion. It ahould be noted that the main criterion for parity of the eluate fractiona waa not merely apecific activity bat the absence of any ether protein impuritiea on acrylaaida gel electrophoreaia.

A (loo X 1 .8 cm) colamn waa prepared with 15 g of Sephadex-G-100 and equilibrated with 0.05 M K-acetate buffer, pH 4.5. 4.6 ml of

Fraction VI containing 2,700 unite and 10 mg protein were loaded on the column. The column waa waahed with 0.05 M K>acetate buffer, pH 4.5 and

1.5 ml fractiona were collected. The fractiona containing protein were aaaayed for acylphoaphataae actirity and the fractiona having acylt^oa-

[^atase activity were tested for their purity by acrylamide gel electro- pjioresis. The fractiona having acylphoaphataae activity and ahowii^ a single band on gel by both the staining techniqaes were pooled and precipitated at 0.0 ansonium sulphate saturation* The precipitate waa dissolved in 3 to 4 ml of 0.01 If K-acetate buffer, pH 6*7 and dialyzed overnight againat two changea of 1 litre each of 0.01 U K-acetate buffer, pH 6.7 (Fraction VIl).

Four such (3 kg) batches were processed upto the last step of purification. The final specific activity of the ensyme ranged from so

300 to 1200 units per ug and the final yield of the enzyme was

The yield is higher if correction is aade for other phosphatases present in tiie initial extract. The purified enzyae (Fraction VII) showed a single band on gel electrophoresis and was hoaogeneous in the altra- centrifuge.

The results of a typical fractionation procedure are giren in

Table 16. 81

** ■*» •H ^ I ei n «t •** P5 -s CO 0

5 5 e» 00 *H -I

CM 00 CO I g s

O -o O o o tt •*> 0 o o§ ao ■d • -p -s • 00m § 0 1 s s 'g u «H•f o •#» *a s o n 4? 0 «D «

«10 OI

fl -He +*« •PI “S I 8 «M *2 n N O *-« e0 •H •H■*» Ish o e « ■*» -pe « 1 e 82

SECTION X

LOCALIZATION OF EN7YME

Acylphotphataae aetirity of th« saad anbryoa was •atiaatad in order to detarmine whether the aetlTily iraa localized mainly in the embryoa. The actirity of the root nodalea on the plant waa alao eati- mated in order to determine whether the bacteria are reaponaible for the high aetiTity of enzyme in thia legume,

Embryoa

Seeda of V ,cat.iane were waahed with water to remove preaerratirea and aoaked in glaaa-diatilled water for 30 min at room temperature. The aeedcoata were removed and the embryoa were aeparated from the aeeda*

1 g of embryoa waa obtained from 20 g of aeeda. The embryoa were ground and extracted with a mixture of 5 ml of 0.01 U K-aeetate buffer, pil

7& iqg of KCI (ao lid ) and 1 ml of 0 .6 y HCl. The extract waa allowed to atand for 30 min and centrifuged at 13,000 x j; for 30 min. The auper- natant liquid waa neutralized to pH S.7 with 2 VI KHCOg and waa teated for acylphoaphataae aetiTity. It waa obaerred that the acylphoaphataae activity of embryoa waa 43 unita per g of embryoa. 20 g ef aeeda which correaponded to 1 g of embryoa had a total acylphoaphataae activity of

1 ,0 0 0 unita, Thia ahowed ^ a t the embryo haa negligible acylphoaphataae activily. Theae reaulta are not corrected for other phoaphataaea, aince true acylphoaphataae activity ia not leaa than 25^ of the total activily whereaa the activity of the embryoa ia only ef the total activity.

Nodulea

300 mg of nodules from a one month old plant were waahed tho roughly with glaaa^diatilled water and were extracted with a mixture of 83

1.5 ml of 0.01 M K-ac«tate buffer p i ! 5.7, 0.3 nl of 0.6 U HCl atd 22

of solid KCl. The mixtore was thoroughly ground uaing a pestle and mortar till the plant cells appeared broken under the microscope (only

the plant cells and not the Rhizobia were tested for the oizyne). The

extract was allowed to stand for 30 ndn and centrifnged at 13,000 x jj'

for 30 min. The supernatmt liquid was neutralized to pU 5.7 with 2 U

KHCOg and was tested for acyl phosphatase activity. It was found that

nodules of V.cat.iang possess no acyl phosphatase actirity.

Localization of enzyme in the cell

SO g of seeds were washed and soaked in water for 30 min. The

soaked seeds were extracted with 500 ml of 0.06 U Tris buffer, pH 7,5

(containing 0.061 M Mg^, O.OOi M thioethanol and 0.25 M sucrose) by blendorizing for 3 min. The homogenate was allowed to stand for 30 min and centrifuged at 800 x The residue contained nuclei, cell debris and unbroken seeds. It was suspended in 125 nl of 0.01 U R->acetate buffer, pH 5.7 and tested for acylphosphatase actiTity. Only 20^ of

the total activity was found to be present in the "800 x £ residue".

The supernatant liquid was further centrifuged at 20,000 x

The mitochondria sedimented in the residue. The residue was suspended in 40 ml of 0.05 M Tris buffer, pH 7.5 and tested for acylphosphatase activity. Mitochondria had negligible acylphosphatase activity. The residue was sonicated to see whether they release enzyme upon breaking.

There was no difference in activity before and after sonication.

The supernatant liquid obtained after centrifugation at 20,000 x £ contained microsomes and the soluble fraction of the seeds. It was assayed for acylphosphatase activity and it was found to have 80^ of the 8?

total aetirity. Aeylphosphataa* and ATPase aetlTitles of th* difforont fraction* are ■anuirized in Table 17.

In a separate experinent the extract of the seeds was eentri> fuged at 100,000 x All the aotiTity was found to be present in the sapematant liquid. The aierosomes are, therefore, inactive and the aeylphosphatase of V.catjang is a soluble enzyme. 85

«4 M wS ^ i O§

t i el ■**

0) a &

fl m O rs ■** f4 5-s «o eS .5 u ■a o CM i 5 w ei 53

•O0 •H ■ s e •i- i § I? « r- 0 El a a •H 0o fl g 0 o VI eg CM •* 'I'

lO 5.-S ^ §

e •O0 0 •H « » tf Ol CM CM

« « & 8 «

SECTION XI

ATPase FRACTION WITH ACYLPH0SPHATA8E ACTIVITY

The crude extract of V .catjang contained besides aeylphosphatase other non-specific phosphatases sach as ATPase and 6-6->Pase vhich also had aeylphosphatase actiTity. Duriqg the purification of aeylphosphatase on CM-cellulose colium (in 0,01 U Tris-HCl buffer, pH 7.5) the unadsorbed

fraction contained ATPase actirity (which also had phosphatase activity) while "true" aeylphosphatase could be adsorbed conpletely and was eluted with 0 .2 M Tris-HCl buffer, pH 7 .5 . The latter was free from ATPase.

The effect of some compounds on ATPase and "aeylphosphatase" activity of the ATPase fraction was detemined. The ATPase fraction obtained during CM-cellulose frcuitionation was used for these experiments.

It is not known whether it contained fl>nall amounts of true aeylphosphatase.

The ATPaee fraction was tested for its activity with ATP and acetylphosphate in the presence of different inhibitors.

Effect of p-CMB and iodoacetate

6.8 X 10”Sl jgCMB inhibited true aeylphosphatase activity by 66jt and 6 .6 X 1 0 ~ \ iodoacetate inhibited it by 4 0 ,gCl(B and iodoacetate at the same concentrations had no effect on ATPase actirity ef the ATPase fraction whereas the "aeylphosphatase** activity of this fraction was inhibited 26^ by both the compounds*

DFP

Diisopropylfluorophosphate at 6.8 X 10"*M had no effect on true

AcPase or on the "AePase" activity of the ATPase fraction whereas ATPase activity was inhibited 25J(. 87

HfClj and NijSjOj

HgClg at 6,6 z lo”^ inhibited true acylphosphatase 80JJ and "acylphosphatase" activity of the ATPase fraction 100^. There was only 16^ inhibition of the ATPaae activi^. Siailarly eodium netabiaulphite inhibited true AcPaee aid "AcPaee" of the ATPaee fraction 66^ and 75^ respectively. ATPase was inhibited 40^. The inhibition of true aeylphosphatase by and by sodiaa ■etabisulphite was irreversible, whereas the inhibition of "acylphosphatase" activity of the ATPase fraction conld be reversed by dialysis or by treatment with cysteine or thioethanol

(6 aoles of reducing agent per nole of Hg).

Effect of heat and HgCl^ on the ATPase fraction

The following experiaent was carried out in order to detentine whether the two activities of ATPase (with ATP and acetylphosphate) could be separated by selective destruction of one. The enzyme was treated with

0.002 M HgClg at pH 5.7 for 15 min. After the treatment the pH was brought down to 3.5 and the enzyme was heated at 60* for 5 ain at this {A. The heated enzyme was neutralized to pH 5 .7 , centrifuged and the supernatant liquid was dialyzed against one litre of 0.01 II K-acetate buffer, pH 5.7 containing 0.01 U EDTA. In the control experiment the enzyae was given exactly the same treatment but without treatment with mercuric chloride.

The results are presented in Table 18.

TABLE 18

HEATING OF llgCl^ TRF^ATB) AcPase/ATPase FTiACTION

HgCl2 treated Untreated Before heating After heating Before heating After heating u/ml u/al u/al npal

AcPase 2.6 2.6 5.4 4.2

ATPase 8.8 6.8 18.4 16.0 8S

From Tabl« IB It will be se«n that acylphosphatase is not destroyed completely by mercarie chloride treatment and by heating it at 50* for

5 min at pH 3*5. It was not possible to separate ATFase from AePase by this method.

Effect of fluoride, phosphate and BDTA

True acylphosphatase actirity is unaffected by fluoride (6.6 x 10 and 1.32 x 10 ^ M) but there is 80^ inhibition in the "acylphosphatase"

activity of the ATPase fraction. 1.3 z 10 li phosphate inhibited "acyl-

phosphatase” activity of the ATPase fraction by 30^ whereas true acylphos- “ 3 phatase was unaffected at this phosphate concentration. 6,6 x 10 If

RDTA inhibited "AePase” activity of the ATPase fraction by 35^. EDTA had

no effect on true aeylphosphatase activity.

These experiments show that the seeds of Vigna cat.1ang contain an enzyme which hydrolyses both ATP and acetyl phosphate. The two activities could not be separated and possibly reside in the same enzyme though more woik is needed to establish this. The "aeylphosphatase" activity of this

fraction is different from true aeylphosphatase.

No studies were carried out on the glucose-6-phosphatase which also had a fluoride sensitive acylfriiOBphatase activity. The wide variation

in the ratios of G-6-Pase and ATPase activities of different fractions

showed that they are different enzynms. CHAPTBlt V

P I 8 C C a 8 r 0 N 120

DISCDSSION

Parifiaatlon

A«7 lphoaphatM« trwm T.eatjag§ m » paiifitd »boat aOO-fold. la

th« orud* •xtrMt, aejlphAcpkatM* totmai oalj * part of tiM

total aetlTity vhioh vaa also da* to ATPaa* ota. flonao tha actual pwri-

fieatioB la alaaar to 000 ta 900 fald. Tha poriflad praparatias vaa

heaogaaaoaa hy altraaaatrifagatiaa aad gava a ali^la baad an gal alaaira-

pheraaia. flM final apaalfia aatlTltjr af 900 ta 1200 maita par ng abiainai

bjr thia praaadara la tha lawaat whaa eoaparad to tha apaaifia aatiritiaa

abtaiaad f o r tha aaajraa fro a M daal aoaraaa. flia praparatiaaa fraa

anlaal aoaraaa hava apaaifia aatiritiaa af 80,000 (haraa aaaala) ta

76,000 aaita/«g (boTiaa braia) and onljr tha boMui arjrthraajrta aograw haa

a apaaifia aativitf af T00«7000 aaita/ag.

Tha aaparatioa of aajlphoaphataaa from othar aompaaifia ^aphai-

taaaa offarad diffiaaltj dariag parifiaatioii. Haatiag af tha arada aztraat

vaa to a aartaia axtaat aaafal ia daatrojriag {riioaphataaaa athar thaa aajl*

phoaphataaa, bat tha raaalta vara aot rapradaaibla. Extraatiaa af aaada

vith atroi^t aaid vaa aara aaafal ia kaapiag tha aMaata of aoaapaaifia

^oaphataaaa to a aiaiaaa. iaaoaioa a a l^ta fraatioaatioa aad ahraaata-

g r a ^ on OSAS-aaUaloaa raaavad tha aoaapaaifia phoaphataaaa aaah aa

AfPaaa and fi-6-Paaa, A a aajar parifiaatioa vaa abtaiaad bf GM-aallalaaa

ahroaatagraplqr. Tha amqraa abtaiaad aftar Qf-aallalaaa ahroMtograplqr vaa

aoaplatal7 frao fraa ATPaaa aad *-€»Faaa, bat aaataiaad tva high

aolaaalar vai^t iaparitiaa m A v n Iqr aarylaaida gal alaa^ro~

I^raaia, Thaaa ii^paritiaa vara aaparatad fraa tha amjaa bgr

ahroaatograilij' aa 8aphadax-0>i00« I t ia aotavarttgr that tha aaqnaa 121

has no mobility at ^ 5,7 or 8.6 ia poljraeiylaaido gol at 7 par eont gol, vhoroaa it waa sobilo in 1ft par eaoi gal at pH 4.B. Tbia appaara

to ba eharaatarlatie of baaie protalna of lew mlaeular walght

(Raiafiald, 1062).

Aa nnaxplaioad finding daring purification «aa tba lade of

raprodaeibilitgr of the reaulta during fraationation on IRC-00 XE 64

and SB-cellttloae. In the earlier ezperiBonta on IRC, ensyme of high

parity (1,000 anita/ag) waa obtained in ene atop. But later it waa

not poaaible to reprodoee theae reaalta. Siailarly with S13-eellaloae

in aoae experiaenta 50J( of the ensyne waa adaorbed on eellaloae and thi

parity of the ensyae whioh remained anadaorbed waa very high (1,000

anita/i^), Thia reaalt alao could not be reprodaeed* The reaaon for

theae diacrepaneiea ia not known.

Another pozaling feature about the enzyae ia the wide variation

in the apeoifie aetiTity of the final parified ensyae. Thoagh it waa

hoaogeneoua in all oaaea by gel eleetrophoreaia, different lota of

aeeda and even the aaae lo t gare ensyae of aaziaaa ap eo ifie a c t iv it y raiying froa 300 to 1,200 unita/ag. The emyae ia quite atable and it

ia iaprobable that the variation in apeoifie activity ia due to the

preaenee of denatured ensyae. In the caae of the ery^roeyte ensyae alao the final apeeific activity variea froa 700 to 7000 unita/ag*

The ensyae with low apecific activity waa found to contain tracea of

heaoglobin aa an iaparity. No each explanation ia available for the variation in apeeific activity of aeylphoa^ataae froa V.eatjang. It ia poaaibly due to genetic inhoaogneiety of the aeeda need. 122

S t a b ilit y

Th« •nsyM (l mg/al) «M ramarkably stabls at higher l«T«la of pnritj whan atorad at >20*, pB 5.7• It waa not Taiy atabla in tha e a r lie r atagea of p a rifie a tio n , probably beeauae of the preaenee af proteolytie enssynea in the emde extract.

Molecular weight

The aolecalar weight of aejrlphoaphataae aa detemined by altraaentrifngatien ia 0,000 daltona. It ia lower than that reported

for the ensyve from other aoarceat 10,000 (for horae anaale), 23,000

(rabbit ■naele), 13,000 (borine brain)and 11,000 (pork heart). The

acylphoaphataae of Y .aat,1ang appeara to be one o f the aflMtlleat ensyaea deaeribed hitherto.

Sttbatrate concentration

The ia abore 4.5 tM and a t 90* a t pH 5.7 ia 0.85 wM,

Thia Talae ia lower than the ralaea obtained far the enq«e froa muacla (8 alf) and erythrocytea (7.4 > 12.7 aif). Inorganic phoaphata

inhibited acylphoaphataae activity, the Ki of 2.2 nlf being conparabla w ith the K i of 3 iril fo r the erythro cyte Mosyae. The in h ib itio n ia of

the competitiTe type in both the caaea.

Sulphate waa found to inhibit acylphoaphataae actiTity non- coapetitiTely with a Ki of 1.7 all.

Effect of teaperatnre

There ia no aignificant inactivation of the nuyae under the conditiona of aaaay at teaperaturea ranging froa 2-30*C. The increaae 123

in MtlTitjr for a 10* ria* in tMiparatar* waa only 30J( in eontraai to an ararac* of 100}( for aararal ansyma. Tha anargjr of aetiratian vaa oaloalatad to ba aboat 6200 cal. Tha anayaa froa V«catjan» raaamblaa tha nnaela and brain ansyaa in baing thamoatabla, wharaaa tha arythroeyta ansym ia aora tharaolabila,

Bffact of pH

A aharp {ril optiana of 5.6 waa obaanrad for aeylphoaphataaa of

V.a4tjang. Thia ia aiailar to tha pH optiaoa raportad for tha aaaela, arythroeyta and haart ansyaa. Tha optiaoa jdl of tha aiayaa froa brain waa, howarar, raportad to ba 7.6.

Aaino acid analyaia

Aeylphoaphataaa froa V .oatJana: eontaina 1 aola aa«h of tyraaina and tryptophan par aola of ansyaa wharaaa 2 aolaa of tryptophan and

3 aoles of tyroaiiM par aola of ansyaa hara baan raportad far bawina brain and rabbit aasela anzyaaa raapaetifaly* Aeylphoaphataaa of

V .catjang waa found to hawo ana anlphida and ana diaalphida greap par aola of ansyaa. Aa tha dawalopaant of eolor with DTNB waa wary alow th« aulfhydryl group appaara to ba burlad inaida tha aolaenla and ia ralativaly inaoeaaaibla. Aa atatad earlier the aulfhydryl eontent of the ensyae froa bovine brain (Diederieh and Griaolia, i960) waa only

0 .0 1 aola of -8H groups per aola of ensyae. Witii DTNB there waa no difference in eolor of the blaidc and experiaental aaaple after 16 and

30 ain auggaating that there waa no -SR group. Howarer, in wiew of the reaulta obtained for aeylf^oaphataae froa V. eat Jang, the abore reaulta with the brain ensyae will hawa to be reeheeked and it would 124

b« B««aasar]r to dstsrains whether the en^Bw froa brain alao poaaeaaes

a baried aalfhjdryl groap.

The preaenoe of a peptide of lew aeleealar weight whieh ia bo*ad

by a diaalfide link to the eniTae waa reported (Raaponi ot a|^. 197l)for

aejlphoaphataae froa herae anaele. Thia peptide waa later reeognised to

be glutathioae. There ia no aueh evidenee of a nixed diaalfide for aejl-

phoaphataae froa V.eat.lang. If aneh a aaall peptide ia attached to the

enayne by an -8-S> link and ia reqaired for aetiTity, rednetion with

borohydride aheald hare inaetiTated the enzyne, whereaa ensyae aetirilgr

la anaffeeted by borohydride. It ia alao noteworthy that in apite of

the preaenee of an -8~S- link in the emyae it ia anaffeeted by dithio>

threitol and borohydride. It waa, howerer, not eatabliahed whether

reozidation of -SB to fora -S-S- takea place readily and ia appreciable

daring the period of aaaay.

In h ib ito ra

pCMB and ioA> acetate

The plant enzyne ia different froa the aniaal tiaaae enayaea in

being inhibited hj jfHIB and iodoaeetate. HowoTer atteapta to ahow

binding of ^QIB to the emyae by a apectrojAotoaetria aethod (Bayer,

1954) were onaucceaafal* It ia donbtfal whether ^IfB binda with the

aalfhydryl of the ensyae. Aa atated earlier, inhibition of the enayae by jgOCB aay be ai#llar to the ii^b itio n by jfM B of pancreatic ribo-

mieleaae, whieh haa no ->SH gronp.

Cyateine. thioettaael and rednced glntathioaa

Cyateine, thioethanol and reduced glatathione had no effect on 125

•n>y«« aetirily , Ineabation of th« ensjra* with qrstciiw or thiooihanol

or CoA togothor with HgOg for M h at 0* had no offeet on onzyBO aetlTity.

HgOg b7 itself haa no effect on enajnae aetiritjr.

Oaidised glutathione

Addition of oxidized glatathione to the encjnae inhibited enxyse actirity by 80}( inhibition «aa obtained by incubating the ensyne with oxidised glutathione and ,£ClfB, the inhibition being nearly additiTe.

Biaulphite

Incubation of the ensyae with biaulphite resulted in 60J( loss in

actirity. Incubation of cysteine, thioethanol, CoA or sodiua borohydride with bisulphite treated and dialysed erntyne did not result in the roTorsal

of the inhibition caused by bisulphite.

A possible explanation for these results is that Q-S-S-G acts on the ->8H group of the ensyae to fora a deriTatiTO of the type &-S-S>Enz which has only half tiM actiTity of the ensyae. The lack of partial inactiration with cysteine and thioethaml ouiy be due to the fact that the deriratiTos of the ensyme with these coapounds hawe the saaw activity as the ensyae. Airther work is needed, preferably with labeled compounds to deteraine whether the glutathione is liaked to the enzyae. But this supposition does not explain why reduction with excess cysteine or boro hydride followed by dialysis does not restore the actiwity of the GSS6 treated enayae.

The effect of bisulphite in inhibiting the enzyae and of H g^ in inactiwation of the enzyae aay be related to breaking of the S-8 bond 126

of th « •Bsym*,

In thia eonncetioa it is int«r«atiog to not* roeont work on tho rogttlatioB of onzyBO aetlTity by linkioK tha ansjnaa aalffagrdryl group with anothar thio-darlTatiwa by a diaulphida liakaga. Fnietoaa

1,6-diphoaphataaa whiah haa baan atodiad by Nadiaahiaa, Horaakar,

Traniallo and Pontraaoli (1970) ia altarad in aetiwity and valua by ineubation with CoA or aoyl earriar protain nndar aneh eonditiona aa to link ^aaa eoapoanda by diaulphida bonda to a aulfhydryl group

in tha anzyaa. It waa raeantly raportad by Raaponi, Cappugi, Traraa and Naaai (i07l) that tha horaa anaola ansyaa containa glutathiana linkad by a diaulphida group to tha anzyaa* Tha altaration of aetirity and by linkaga of anothar a u lfh yd ryl ooapound or p ro ta in by a diaulphida link to tha anzyaa appaara to ba a naw aaehaniaa for tha ragulation of anzyaa aetirity, which aay ba of phyaiologiaal iapor- tanea. f’urth a r woik ia naadad to aluoidata tha natura of tha eoapound linkad to V«catJang acylphoaphataaa.

Matala and aatal chelating aganta

Tha anqraa doaa not appaar to raquira a aatal for ita aatirity aa datarainad by the lack of affect of added aatal iana and aatal binding agenta auch aa KCN, - -diiqrridyl and EDTA. ExhauatiTa dialy~ aia of tha enzyae againat 0*01 If KOTA d id not ra a u lt in aqgr loaa in a e t ir it y . Na'*’, Mg**, Ca**, Mn** and Co** hare no e ffe c t on enzyae aetirity. Acylphoaphataaa froa brawer'a yeaat was reported to require

Mg** for ita aetirity (Harary, 1957). No eridence eauld be obtained for a Mg** requirMwnt for acylphoaphataaa froa Y.eatjaag. Howerer 127

th« possibility of trae* aaoanta of Ifg or othor motal being prosont in tho wibatrato or buffer was not ezcladad. Ifora rigoroaa porifiea- tion of all roaganta vaj b« naadad to aatablisb unaquiToeally that this anzym baa no aatal raqairaaant. Wbatbar different aaaplea of aeatyli^osphata contain traeea of U g ^ aa iaparitj wbieb aetiratea the ensyae is not known. MgCl^ (5 sM) waa found to enhance the actlwity of the enajTsie froK bovine brain when triB>^etylphosphate aerred aa a substrate (Diederieh and Grisolia, I960)

The erTthroeyte ensTM ia the only one which haa been reported to be inhibited by EDTA. Big’^ atroqgly inhibited the plant eaayve and the inhibition waa irreToraible whereaa none of the aniaal tiame enay«ea haa been reported to be inhibited by Hg'*"^.

Aniona

Sulphate, oxalate and dtrate inhibited the onayM. Howerer, chloride and tartrate had no effect on enzyne activity. Phoaphate inhibita competitiToly and Ktlphate noncoapetitively. Phosphate inhi bition ia characteristic of animal tiaaue aoylphoaphataaea.

Miscellaneoua coapounda

A yariety of other conpounds such aa nucleotidea, coenxyMa, amino acids and sugars had no effect on enayme actirity. Thyroxine , -5 . (3 *3 X 10 M) had no effect when it waa added to the system but preincubation with ensyme ahowed 20^ i nhibition. The plant enEyme reaembles the animal tiaaue enaymea in showing inhibition on ineubatiom with thyroxine. DFP, K l, alcohol, abscisic acid, benaaldefayde, cadaTorine and 2 ,4>dinitrophenyl hydraaine had no effect on ensyme a ctirity . 128

Th« plant •nsym* r*a«mbl«a tha arytiuroeyta ancyata im baiog inhibitad by earbaiqrl ^oaphata, bni diffara frwa it in ahawing na inhlbitian by flnorida*

Sttbatrata apacificity

Aeylphaaphataaaa fren anlaal tiaaaaa, azaapt tha axythroeyta ansjma, aet both upon aeatylphoaphsta and earbttqrl phoaphata. Tha ratia af aetirity with aeatylphoaphata to that with earbuqrl phoaphata ia tha aama (lOti) for tha anzynaa purified frma berina brain, poilc haart, horaa maela and baaf livar. Only tha arythroeyta anssyse doaa not act on earbaayl phoaphata* HowoTer, all enzynaa from animal tiaaaaa taatad so far wara foand to hydrolyse 1,3-diphosphoglyoerate,

The enzyne from V .catjang dees not aet either on earbaayl phosphate or on l,3->diphoaphoglyeerate. It acta both on acetylphosphate and propionyl phoaphata. Thas it appears to aet on fatty aeylphoaphatea unaubstitated in the acyl group.

The noM aeylphosphatase rather than acetyl phosphatase has been used since it acts on both acetyl and propionyl phosphates. Other aeyl- phosphates aast be teated aa aabatratea before a awre specific na«e is given for the ensyae. In any case in riew of the wide difference ia specificity of known acylphosphatases» their noaenclatare requirea reconaideratien. The acylphoaphatasea (excluding nonapecific j^ospha- tases) act (a ) on acetylphosphate, 1 ,3 DPCA and earbaayl {dioaphata,

(b) aeatylphoaphata and 1,3 DPGA and (c) aeatylphoaphata only. The apacificity of the Vigna catjang ensyae for acetyl phoaphata and propionyl phoaphata and lade of aetirity either with ATP, 6-6-P etc. or with 129

Mjlphoaphai** Mflh »• earbMjl phesphat* aad l,8-diphM ^glj«M rm t«

■how that it la diffaraat fr«a »11 othar knawii phaapliataaaa and aa^l- phoaphataaaa.

Bela af aeylnhoaiihataaa

Tha rola of aejrlfAoaphataaa ia not known, bat it haa baan poa** talatad that tha fane ti on af aajrl^oaphataaa wqr ba to proTont or rogalato tho aanaantration of aajlphoaphataa and tharabj aat aa a shoBotrapia affaator. It haa alao baan poatalatad that it ragalataa tho rata of glyooljraia hjdralyainf 1,8 DPQA and ttorobj onaoaplii^t

(iTOoljraia fro« phoaphorylation.

Tha anionaa froa planta aata naithar an 1,S diphaaphagljaarata nor aa oarbaagrl [^aphata. Ita pfagraiologiaal aabatrata and a»da af aatian a ra , thorofora, oaknawn and ra a a ia to bo datandnad* Th» oaoorranaa of aaotjlphoaphato, aaotokinaaa and phoaphatraoaaaatjlaaa

In planta raqoiroa ta ba raaxaainad. Thara ia aa oridaaaa far tka oeoorronaa af aaotyl phaaphata axaapt ia ■iaraargaaiaaa, bat it ia paaaibla that aeatjrlphaaphata dooa oaaar ia plaata, poaaiblj bgr tha aatiaa of aaat^inaaa (froai aaotata and ATP) ar phoaphatraaaaea^laaa

(fra* aaatjl-CaA and phoaphato). Tho faaatiMi af tha onajnaa aagr th«a ba ta rogalata tha eonaaatration of aaal^lphaaf^ta. Bat if aaatgfl-

^oaphato ia not famad in planta, farthar waik w ill ba naadad ta iataraina whiah aabatrata ia aatad upon bjr aaa^lphoairiMitaaa af r.aayage.

Laaaliaation

Tho aejlphoaphataaa a a tiTitj of root nodaloa of Y.aatiang waa

B^ligibla. niora waa nagliglbla — tiritj in tho aaad nbrjaa. n* 50

M jor MtlTitj wM faoBdl to b« ettiM«ntr*t«d in tiM eoiylsdona. frftetio^

DAtioB «f ill* M«d cxtrMt bjr dl ffarMiiiAl ahev«d that

tta« aielaar, aito«h*adrial and mloreaoMil fraeilona had r *r j l l i i l a

aailTitgr, vharaaa iha 100,000 x g MparnataBt liqvid «Miaiiitd all tlw

aejrlphaaphaiaaa aatiritj. Tfaoa aajl^aai^taa* af ^t.aatiaag la a

aalahla aaqnaa laaatad 1 b tha ajrtaplaMU

AtPaaa fractlea with aaylnhaaphataaa aail^lty

Tha anida aziraata af aaada af T.aatiaag aaatalaal ATPaaa aad

e-e>Faaa aa vail aa aa/lphoaphataaa. Saw af tha ^ p a r t l a a af tha

ATPaaa vara atsdlad 1b datall. AfPaaa la Bat Idaatlaal vlth aay^lphaa-

phataaa nar waa It darlTtd froa aajlphaaidiataaa bj aeld traataaat

darlBf axtraetias. Tha pariflad aejrlf^oaphataaa haa bo ATPaaa aetlTlty.

Havarar, attanpta to ebtala ATPaaa without aejlphoaphataaa aatlTltj wara

aaanceaaafal. Tha propartiaa of traa aejljdioaphataaa aad af ATFaaa vara

qalta dlffaraat. jGI0 and ladoaeatata iBhihltad aaylphaaphataaa but aat

ATPaaa, DFP haa b o affaat aa tha AaPaaa aotlTitj af althar af tha aaajaaa

wharaaa ATPaaa waa iahlbltad (t5^). waa iahlbltarx ta «ae7 lphaa>

phataaa aatlrlty” of tha ATPaaa fraatlaa, bat had llttla affaat aa Ita

ATPaaa aatlTlI^-. Tha iatdbitiaa af traa aajrlphaaphataaa bgr aad

blaalphlta waa IrraToraibla, wharaaa iahibltlaa 1^ thaaa aMipaaada af

"aajrlphaqphataaa* af ttia A T ^ a fraatlaa waa rawaralbla. H)TA aad

flBorlda do aot lahlblt tha apaaifia a«]rlphoaphataaa bat iahlblt tha

"aajlphaai^ataaa* aatlwltgr af tha ATFaaa fraatlaa. Tha aatlrltlaa

towarda aaatflfdtaaphata aad ATP af tha ATPaaa prabahljr azlat l a tha aaaa aalaaala bat at dlffaraat aatlva aaatraa. 131

l a •onelaaien MylphesphatM* of ▼.eating iriiieh h M iMlatcd in h«M(MMOBS fom is a teaie protain of utsaallj l«v

■ «lM «lar m^ighct vltleh la apaaifia for aaot/lj^oaplukia and prapioagrl phoaphata and luui «» aatioa oa aarbaagrl phoaptata or l,a>dipkaaplM- gljaarata and ^ pfafaialagiaal aabairaia aad rala af whiah ara udmava« B I B L I 0 G R A P H Y 136

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