Occurrence of trehalose-6-phosphatase in Phormia regina Meig

ARCHIVES

OF

BIOCHEMISTRY

Occurrence

AND

BIOPHYSICS

88,

(1960)

339-%3

of Trehalose-6-phosphatase

in Phormia

regina

Meig.

S. FRIEDMAN’ From

the Department of Entomology, National Institutes Received

Purdue of Health,

University, Bethesda,

November

Lafayette, Maryland2

Indiana;

and

the

12, 1959

An enzyme capable of hydrolyzing trehalose g-phosphate has been purified 56fold from a homogenate of Phormia regina Meig. using a DEAE-cellulose column, The enzyme is quite specific, exhibiting a low activity toward glucose B-phosphate and undetectable activity against a number of other phosphate-containing compounds. Other properties and its function in the metabolism of the insect are discussed.

its properties are t’he subjects of this publiThe mechanism of synthesis of trehalose cation. has been studied in great detail in yeast by MATERIALS AND METHODS Cabib and Leloir (1) who have found that it is apparently formed through a condensation The substrate of major significance, trehalose of uridinediphosphate glucose with glucose 6-phosphate, was a sample of synthetic material 6-phosphate forming an intermediate, treha- prepared and furnished as the barium salt by Dr. lose 6-phosphat,e, which is then split to yield D. MacDonald of the National Institutes of trehalose and inorganic phosphate. Some Health, Bethesda, Maryland, and was converted recent work of a preliminary nature in in- to the potassium salt before use. The other comare commercially sects has shown that a similar mode of syn- pounds used in the investigation available. thesis may occur in the locust (2). The enzyme was prepared in essentially the The present author, investigating this same manner as trehalase (3), and, in fact, became same phenomenon in Phormia regina, found a by-product of trehalase production while those that crude preparations of insect tissue st.udies were being carried out. Whole flies (Phorwould split the terminal phosphate from mia regina Meig.) were homogenized 1.5 min. in a adenosine triphosphate (ATP) much more Waring blendor at 3°C. with 2.5 vol. w/v Tris rapidly in the presence of t’rehalose than [tris(hydroxymethyl)aminomethane]-HCl buffer when glucose was added to the reaction mix- (pH 7.0, 0.1 M). The homogenate was pressed ture. It was postulated that through some through cheesecloth and its volume measured I). It was then centrifuged at 12,800 X g unknown mechanism trehalose 6-phosphate (Fraction for 15 min. at O”C., the supernatant filtered was formed and subsequently hydrolyzed. glass wool, and the filtrate (Fraction II) The existence of a phosphatase was con- through used for further purification. firmed, and t#he results of some studies on INTRODUCTION

The diethylaminoethyl (DEAE) cellulose columns used in the purification procedure were prepared as already described (3). Enzymic activity was assayed as follows unless ot,herwise noted: The reaction mixture, consisting of trehalose 6-phosphate (Kc) (0.02 M) 0.2 ml., MgC12 (0.1 M) 0.05 ml., Tris-HCl buffer (pH 7.2, 0.2 M) 0.3 ml., enzyme 0.2 ml., and water to give a final volume of 1.5 ml., was incubated at 32°C. for 15 min., and the reaction was stopped by addition of an equal volume of 10% trichloroacetic

1 This work was partially supported by Grant No. E-2440 from The National Institutes of Health, Bethesda, 14, Maryland. 2 Some of this work was initiated and carried on while the author was employed in the Laboratory of Physical Biology at the National Institute of Arthritis and Metabolic Diseases, N.I.H., Bethesda, 14, Maryland. Purdue University Agricultural Experiment Station, Journal No. 1518. 339

340

FRIEDMAN

acid. Inorganic phosphate was determined method of Sumner (4). Trehalose was measured by the enzymic described by Friedman (3) and protein technique of Lowry et al. (5).

by the method by the

RESULTS

PURIFICATION

OF THE ENZYME

The purification procedure followed that used for the purification of trehalase (3). The filtrate described above (Fraction II) was subjected to an ethanol fractionation at -3”C., and the 40-60 % fraction was disTABLE SUMMARY

I

OF TREHALOSE-6-PHOSPHATASE PURIFICATION

Conditions of assay in the Methods section.

are

the

same

as described

-

Fraction

PROPERTIES Total units

Total proteir

Specific activity

Total IKOV-

erY

mg.

Crude homogenate (Frac I) Supernatant (Frac II) 40-60 alcohol ppt. (Frac III) Column eluate

solved in Tris-maleate buffer (pH 8.0, 0.005 M). This was then dialyzed overnight against the same buffer (Fraction III), the dialyzed solution was placed on the washed DEAE cellulose column, and gradient elution was begun. The phosphatase was removed from the column during the first step, i.e., a pH change from 8.0 to 6.0, which involved introducing 300 ml. of Tris-maleate buffer, pH 6.0, 0.005 M, into a mixing vessel containing 100 ml. of Tris-ma1eat.e buffer, pH 8.0, 0.005 M, at the same rate the contents of the mixing vessel were being introduced onto the column. The purification procedure is summarized in Table I. One unit of activity corresponds to 1 pmole of inorganic phosphate released in 15 min. at 32°C.

% -

4950 7020 4550

1880

2.4

92

1650

221

7.5

33

39.5

11

13.1 I

510

OF THE ENZYME

1. Stoichiometry of the Reaction Incubation of a purified enzyme preparation with trehalose 6-phosphate resulted in the complete releaseof inorganic phosphate, accompanied by an equivalent increase in trehalose (Table II). 2. Speci$city

Since no disaccharide phosphates except the nominal substrate were available for testing, the specificity of the enzyme cannot TABLE II be truly assessed.However, in its present STOICHIOMETRY OF TREHALOSE 6-PHOSPHATE state of purity, the enzyme is almost comHYDROLYSIS pletely inactive against a large variety of Reaction mixture contained : Trehalose B-phosmonosaccharide phosphates and nucleoside phate (K+, 0.02 M) 0.1 ml.; MgClz (0.1 M) 0.05 phosphates (Table III). ml.; Tris-HCl (pH 7.2, 0.2 M) 0.3 ml.; purified enThere is no satisfactory evidence that the zyme 0.1 ml.; water up to 0.9 ml. Incubated 30 enzyme itself does not attack glucose 6-phosmin. at 32°C. phate. In crude preparations, the activity QllkUP toward this compound is approximately 12 % Substrate Time Analysis tity formed of that toward trehalose 6-phosphate. Table III illustrates the fact that the activity in min. pmoles a BO-fold purified preparation is 8 % of that Trehalose 6-P (2pM) 0 Trehalose 0 against trehalose B-phosphate. Do. 0 Glucose 0 -

Do. DO. Do. Do.

0 30 30 30

Inorg. phosphate Trehalose Glucose Inorg. phosphate

0 1.88 0 1.93

3. Effect of pH The purified enzyme was tested for activity at various pH’s using a number of buffers, and was found to be maximally active at pH 7.0 (Fig. 1).

341

TREHALOSE-GPHOSPHATASE

TABLE SPECIFICITY

III

OF TREHALOSE-GPHOSPHATASE

React.ion mixture contained: Substrate 0.2 ml., MgClz (0.1 M) 0.05 ml., purified enzyme 0.05 ml., Tris-HCl buffer (0.2 &f pH 7.2) 0.3 ml., water 0.2 ml. Incubated 15 min. at 32°C. Pi liberated

Substratea

pmoles

Trehalose 6-P (Ii+) Glucose 6-P (K+) RIannose 6-P (K+) Fruct,ose 6-P (K+) Ribose 5-P (K+) Fructose 1,6-di P (Ii+) Glucose 1-P (K+) 3-Adenylic acid (K+) 5-Adenylic acid (K+) Uridylic acid (K+) Flavin mononucleotide Adenosine triphosphate Na pyrophosphat,e

1.1 0.08 <.05* <.05 <.05 <.05 <.05 <.05 <.05 <.05 <.05 <.05 <.05

(Xa+) (K+)

u Four micromoles of each substrate was b Quantity undectectable by this method

activity when held at 57°C. for 15 min. (Fig. 2). In a frozen state it has suffered no loss after 2 months at - 12°C. 5. Cofactor Requirements

and Inhibitors

The enzyme is inactive in the absence of a bivalent metal, and as may be seen in Table IV, Mg++ serves best at the concemrations tested. Co++, Fe++ , and Mn++ are much less effective. The Mg++-substrate ratio for maximum activity is approximat’ely 1: 1. This is shown

used.

oO 0

IO

20

30 TEMP (‘Cl

40

50

t

FIG. 2. Temperature-activity curve of trehalose-6-phosphatase. Conditions of assay were as described in Methods section under Delerlnination of Enzymic Activity. 01

1

I 5

6

7

0

9

1

TABLE

I0

PH

Reaction mixture contained: Trehalose 6phosphate (K+, 0.02 fil) 0.2 ml.; Tris-HCl buffer (pH 7.2, 0.2 M) 0.3 ml.; cofactors: MgCl? , CoCIZ , FeSOd , MnCl2 , (0.1 fif) 0.05-0.2 ml.; enzyme (Frac II) 0.2 ml.; water to give 1.0 ml. Incubated 15 min. at 32°C. Cofactor

,$. Temperature

ox INORGANIC LIBERATION

PHOSPHATE

FIN. 1. pH-activity curve of trehalose&phosphatase. Conditions of assay were as follows: Trehalose g-phosphate (K+, 0.02 iM) 0.2 ml.; buffer (0.1 Jf) 0.6 ml.; MgCl, (0.1 M) 0.05 ml.; purified enzyme 0.05 ml.; water to give 0.9 ml. Incubated 15 min. at 32°C.

E$ects

Maximal activity is attained by the puritied preparation at 46°C. The enzyme, as we have prepared it! is stable at somewhat higher temperatures, ret,aining 85% of its

IV

OF Co~acTo~~3

INFLUENCE

1 x 10-s M Micromoles

hlg++ co++ Fe++ Mn++

2.0-l 0.71 0.58 0.39

Concentration .5 x 10-s M inorganic

2.06 0.90 0.39

1 x 10-s M P

1 x 10-4 M

liberafei

1.46 1.69 0.49 0.43

1.56 O.li 0.35

342

FRIEDMAN

in Fig. 3 in which inorganic phosphate released is plotted against the Mg+--trehalose 6-phosphate ratio. The figure is a composite of a number of experiments in which the absolute concentrations of the substrate were varied. In the presence of 5 X 10e3 M Mg++, 6 X 1O-4 M Zn++ inhibits hydrolysis 80 %, while

TABLE OCCURRENCE

V

OF TREHALOSE IN HEMOLYMPH

PHOSPHATASE

Reaction mixture contained: Substrate 0.2 ml., MgClz (0.1 M) 0.05 ml., Tris-HCl (pH 7.2 0.2 M) 0.3 ml., water 0.2 ml., blood (15 ~1. diluted to 1 ml.) 0.2 ml. Incubated 30 min. at 32°C. Substrate

Pi liberated pmoles

Trehalose 6-P (4 PM) Glucose 6-P (4 &f)

IO-

0.25 0.00

9s-

Cu++ at the same concentration inhibits 50% Arsenate inhibits 10% at 1OP M and fluoride 70% at 2.5 X 10e2M.

?s fl E5 m -I

6-

a”

3

n ~!JM T-6-P A SyM T-6-P 0 12 JIM T-6-P

4-

6. Substrate Concentration Effects Figure 4 illustrates the relation between hydrolysis rate and substrate concentration. Calculation of the data obtained from this curve leads to an estimated value of 1.4 X 10h3M for the Michaelis constant (K,) of the enzyme-substrate complex.

2IOL

’ 05

I 3

I 2

’ IO

Mg*‘:

I 4

T-6-P

I 5

RATIO

3. Relationship between eneymic activity and Mg++-substrate ratio. Conditions of assay were as follows: Trehalose B-phosphate (K+, 0.02 34) 0.2-0.6 ml., MgC12 (0.1 M) 0.02-0.04 ml.; Tris-HCl buffer (pH 7.2, 0.2 M) 0.3 ml.; enzyme (Frac II, 1:lO dilution) 0.2 ml.; water to give 1.5 ml. Incubated 15 min. at 32°C. FIG.

7. Localization The enzyme has not been localized, obtained as it is from a homogenate of large numbers of flies, but experiments have been run in an effort to determine whether or not there is a phosphatase capable of splitting trehalose 6-phosphate in the blood. Table V illustrates an experiment of this type and shows that there is, indeed, such an enzyme. There is no detectable splitting of glucose 6-phosphate within the sametime, due, probably, to the small quantity of enzyme available in these experiments.

(s) V

DISCUSSION

01 1 ’ 0123456789

’

’

’ (S)

’

’

r

’

’ IO

’ II

1 12

x I03

FIG. 4. Lineweaver-Burk plot of a concentration-activity curve. Conditions of assay were as follows: Trehalose B-phosphate (K+) 0.4-16.0 &/ml. assay medium; MgCL 0.620 &/ml. assay medium; Tris-HCl buffer (pH 7.2, 0.2 M) 0.3 ml.; purified enzyme 0.05 ml.; water to give 1.5 ml. Incubated 15 min. at 32°C.

The knowledge already extant concerning the synthesis of trehalose in yeast, namely that it is formed through the intermediation of trehalose B-phosphate, and the fact that a phosphatase capable of splitting this ester has also been demonstrated in yeast (l), leads one to the hypothesis that a somewhat specific trehalose-6-phosphatase in Phormia may have a similar function. The information recently presented by Candy and Kilby (2) showing a similar mode of trehalose synthesis in the locust would tend to verify this

343

TREHALOSE-6-PHOSPHATASE

line of reasoning. The results which led to the initial finding of the phosphatase in Phormia, i.e., an apparent formation of trehalose 6-phosphate when a fly homogenate is incubated with trehalose and ATP, take a somewhat different direction from what is already known of the synthesis of the compound, and are being studied furt’her at present. The enzyme exhibits no peculiar properties as far as the investigations have t.aken us with t,he exception of its apparent specificity, and even this, in the absence of other disaccharide substrates, is questionable. The pH optimum of the enzyme is very close to the pH of Phormia regina hemolvmph (6); so in the absence of other limany trehalose 6-phosphate iting factors, formed in the animal, which reaches the

blood, should be hydrolyzed to free trehalose and inorganic phosphat’e. Investigations are presently in progress in an effort to find the phosphate ester in the tissues of Phormia. ACKNOWLEDGMENTS The Amir-Fazli

assistance

of Miss C. Mecca is great,ly appreciated.

and

Mrs.

G.

REFERENCES 1. CABIB, E., AND LELOIR, L. F., J. Bid. Chem. 231, 259 (1958). 2. CANDY, J., AND KILBY, A. B., Mature 183, 1594 (1959). 3. FRIEDMAN, S., Arch. Riochem. Biophys. 87, 252 (1960). 4. SUMNER, J. B., Science 100, 413 (1944). 5. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J., J. Biol. Chem. 193, 265 (1951). 6. FRIEDMAN, S., J. Insect Physiol. 3, 118 (1959).