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The purification and properties of trehalase isolated from Phormia regina, Meig
ARCHIVES
OF
BIOCHEMISTRY
The
AND
Purification
BIOPHYSICS
87,
252-258
(1960)
and Properties of Trehalase from Phormia regina, Meig.’
Isolated
S. FRIEDMAN From the Department Institute of Arthritis
of
Entomology, and Metabolic
Purdue University. Diseases, National Received
October
Lafayette, Institutes
Indiana;2 Health,
of
and from the National Bethesda, Maryland3
2, 1959
An enzyme responsible for the hydrolysis of a,ol-trehalose has been isolated from Phormia regina, Meig. and purified approximately 1000 times using a diethylaminoethylcellulose column. The properties of the enzyme are described and its possible role in the blood of the insect discussed. Through use of the enzyme a sensitive, specific method for the assay of trehalose has been effected. INTRODUCTION
Investigations of carbohydrate metabolism in insects have confirmed the idea that, in general, the degradative pathways are similar to those found in other animals (1, 2). Certain important differences have been called to our attention, however, and it is with one of these that this report is involved. Wyatt and Kalf (3), in a recent survey of insect hemolymph, have shown that a nonreducing disaccharide, a!,a-trehalose [(WDglucosido)-a-n-glucoside], is the major blood sugar in a number of genera, occurring in some casesin concentrations ten times that of the omnipresent component n-glucose. This disaccharide, originally isolated from a mold (4), can be split to two moles of glucose by a hydrolytic enzyme, trehalase, which has been described in a number of organisms, fungi (5) as well as insects (6). Kalf and Rieder (7) have lately obtained a somewhat purified preparation of the enzyme from a i This work was partially supported by Grant No. E-2440 from the National Institutes of Health, Bethesda, 14, Maryland. 2 Journal Paper No. 1502 of the Purdue University Agricultural Experiment Station. 3 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. 252
homogenate of larvae of Galleria wtellonella and have described some of its properties. An investigation of the blood of adult Phormia regina has shown that both glucose and. trehalose are present, as is the hydrolytic enzyme trehalase. The questions raised by this finding have led to an examination of the enzyme in this insect. Purification of the enzyme and its subsequent utilization as a tool in the measurement of minute quantities of trehalose and glucose are the subjects of this paper. MATERIALS
AND
METHODS
Enzymic activity was obtained from two sources : 1. From Blood. Measured quantities (up to 2 ~1.) of hemolymph were removed from the dorsal sinus of adult Phormia regina using a modification of the micro injection apparatus of Buck (8). The modification permits direct measurements of 0.6 ~1. i 770, (or less, although this has not been determined) using a Starrett gauge (No. 25-611, L. S. Starrett Co., Athol, Mass.) as a direct reading device coupled between a micrometer and a hypodermic syringe to which is attached a glass needle. The syringe and needle are filled with mercury to the point of involvement with the hemolymph, thus providing a system with almost complete rigidity (see Fig. 1). This instrument was used for all experiments involving removal or delivery of blood samples. The blood samples were used without further purification as sources of enzyme.
ISOLATION
OF
2. From Whole Insects. Wild adults of Phormia regina were trapped and used as the nucleus of a laboratory culture which was reared by the usual methods. Five-day-old flies, having been fed on 5% glucose solutions from emergence, were anesthetized with CO2 and homogenized for 1.5 min. in a Waring blendor at 3°C. with 2.5 vol. (w/v) [tris (hydroxymethyl) aminomethane] Tris - HCl (pH 7.0, 0.1 iM). The homogenate was pressed through cheesecloth to remove large pieces of cuticle, and thle total volume was measured (Fract#ion 1) and centrifuged at 12,800 X g for 15 min. at 0°C. The supernatant was poured through glass wool to remove a fatty accumulation, and the turbid solution (Fraction II) was used for further purification. Activity of the enzyme was determined as follows: 0.3 ml. of enzyme solution was mixed with 0.5 ml. of trehalose (20 rmoles/ml.) and 0.6 ml. of citrate buffer (0.1 M, pH 5.6). The mixture was incubated 15 min. at 32”C., and the reaction was stopped by addition of 1 ml. of Ba(OH)z and 1 ml. of ZnSOl standardized according to Somogyi. The Nelson-Somogyi method (9) was then used to measure reducing sugar. Protein was measured by the method of Lowry et al. (10). Preparation of the diethylaminoethylcellulose (DEAE-cellulose) column followed the directions outlined by Peterson and Sober (11). The DEAEcellulose was purchased from Eastman Organic Chemicals and washed with 1 N NaOH until it was no longer colored. This was followed by washing with 1 N HCl and then successive washings with the two alternately. The cellulose was finally suspended in 1 N NaOH, vacuum filtered until almost dry, and washed a number of times in distilled water. It was suspended in Tris-maleate buffer, pH 8.0,0.005 M, poured into a column 18 X 270 mm., allowed to settle, and packed under 10 lb./sq. in. pressure without permitting the column to dry. The column was then placed in a cold room (3°C.) and permitted to reach temperature equilibrium, after which it was again packed under the same pressure. At this point it was ready for use. Five-milliliter fractions were collected at a rate of ca. 15 ml./hr. The sugars used in the investigations were commercially available grades and were recrystallized from alcohol when necessary. Trehalose was checked against a sample of the chromatographitally pure compound obtained through the courtesy of Dr. D. MacDonald of the National Institutes of Health. Trehalose g-phosphate was obtained as the barium salt from Dr. MacDonald and converteld to the potassium salt. The assay method developed for micro quantities of trehalose uses the purified enzyme and involves a mod.ification of the calorimetric glucose
253
TREHALASE
LI
7
6
4 32
I
Fm. 1. Micro injection apparatus numbers designate the following parts: 1. Micrometer spindle without bow. 2. Ball bearing coupling, inner race force coupled to micrometer spindle, outer race joined to 3. Lucite coupling locked by screw to one side of Starrett depth gauge. 4. Spring for maintaining pressure against coupling, permitting minimum play in ball bearing. 5. Face of Starrett gauge, reading directly to horizontal movement of 0.001 in. (total movement 0.2 in.). 6. Lucite ring, coupling gauge and plunger of syringe directly. 7. One-milliliter syringe, barrel and plunger lubricated with silicone high vacuum grease. 8. Glass needle, made from No. 421-B Baketel glass-adapter (Becton, Dickinson & Co., Rutherford, N. J.). The whole unit is mounted firmly on a steel plate, permitting very little warping along its entire length. The syringe is mounted so that it may be easily removed and replaced or refilled with mercury. oxidase technique developed by Keston (12) for measurement of glucose. Glucose oxidase, obtained from Worthington Biochemicals, Freehold, N. J., was made up as a 20% suspension in 0.01 M phosphate buffer pH 8.0 and centrifuged at 2°C. for 10 min. at 3000 X g. Twenty milliliters of the supernatant was transferred to the top of a DEAE-cellulose column prepared as above except that the buffer used for suspending and packing the column was phosphate 0.01 M pH 8.0. The enzyme was then eluted from the column with 100 ml. of phosphate buffer of the same pH and molarity, and came out in the first 50-60 ml. as a tan solution. This was dialyzed against 2 1. of water and lyophilized or concentrated to 20 ml. under vacuum. Peroxidase and o-dianisidine were obtained from Worthington and made up in concentrations ten times that normally used for glucose analyses. Thus, 50 mg. of o-dianisidine was dissolved in 1 ml. methanol and poured into 49 ml. of 0.05 M phosphate buffer pH 7.0 containing 25 mg. peroxidase. The solutions were mixed and centrifuged at 2°C. for 10 min. at 5000 X g. For determinations, the quantitatively unknown substrate was added in a volume not to exceed 0.3 ml., followed by 0.4 ml. of the peroxidase-o-dianisidine mixture, 0.05 ml. of the purified trehalase or 0.05 ml. water if only glucose was to be measured, and 0.04 ml. of purified glucose oxidase. This mixture was incubated for 30 min. at 32”C., the reaction was stopped by addition of 0.02 ml. of 4 N HCl, and measurements were made at 401 rn,u in a Beckman spectrophotometer. The
254
FRIEDMAT;
30,
I
25-
8
I
20
i
LOO-
I 0 ..--. I\ 9
FRACX ml
\
.._.._.-
/\
-.-.-.-.-.-.-.-.-.--.-c-.
2%
t
H
I
.-..o.-.-
200
1000
FIG. 2. Effluent diagram of 4&60 alcohol fraction of Phormia regina: 40 ml. (containing 148 mg. dialyzed protein) placed on an 18 X 270 mm. column of DEAE-cellulose. Effluent collected in ca. 5-ml. fractions. Buffers used: I. 300 ml. of 0.005 M Tris-maleate pH 6.0 siphoned into mixing chamber containing 100 ml. of 0.005 M Tris-maleate pH 8.0 at same rate that mixture leaves chamber and enters column for elution. II. 100 ml. of 0.005 M pH 6.0 Tris-maleate put directly on column. III. 300 ml. of 0.05 M Tris-maleate pH 6.0 into 100 ml. 0.005 M Tris-maleate pH 6.0. IV. 100 ml. of 0.05 M Tris-maleate pH 6.0 put directly on column. V. 200 ml. of 0.05 M Tris-maleate pH 6.0 containing 0.1 M NaCl into 100 ml. of 0.05 M Tris-maleate pH 6.0. VI. 100 ml. of 0.05 M Tris-maleate pH 6.0 containing 0.2 M NaCl put directly on column. TABLE SUMMARY
I
OF TREHALASE
Conditions
of assay under
Methods section Activity.
PURIFICATION
were as specified Determination of
Total protein
activity _____
.nits/?lzg. protein
mg. homoge(Fr. I)
Total reCOVUY
SPCCifiC
Crude nate
the
-
Total units
Fraction
in
Enzyme
TO
8250
7020
1.17
-
Supernatant (Fr. II)
6900
1880
3.7
84
4&60 alcohol cipitate
2675
221
12.1
pre (Fr.
32.5
III) Column
blank strate,
elution
consisted for which
2530
-
2.1
-
1200
of all reagents except water was substituted.
31
the
sub-
RESULTS PURIFICATION
OF
TREHALASE
The solution designated in the Methods section as Fraction II was subjected to an
ethanol fractionation at -3°C. TOI 130 ml. of Fraction II was added slowly with stirring 87 ml. of absolute ethanol, bringing the alcohol concentration up to 40 %. After standing for 15 min., the resulting suspensionwas centrifuged at 6000 X g for 10 min. and the precipitate was discarded. The supernatant was treated with 108 ml. ethanol iti the same manner, brining the concentration up to 60 %. After centrifugation the supernatant was discarded, the precipitate was dissolved in 60 ml. of Tris-maleate buffer, 0.005 M pH 8.0, and the solution was dialyzed overnight against 2 1. of the samebuffer (Fraction III). Forty millilit.ers of the dialyzed solution was placed on top of the previously described DEAE-cellulose column (see Methods section), and after washing it in with one-third volume of the same buffer, gradient elution of the proteins was begun. Figure 2 showsthe results of the elution and is self-explanatory. The protein peak arising immediately after point II contains trehalose-6-phosphatase, which will be discussed in a subsequent paper.
ISOLATION
OF TREHALASE
Table I contains a resume of the steps in the purification procedure. In all experiments, one unit of activity is equivalent to 1 pmole glucose released in 15 min. at 32°C. PROPERTIES
OF THE ENZYME
255 TABLE
II
ACTIVITY OF TREHALASE TOWARD VARIOUS SUBSTRATES Conditions of assay were as follows: Substrates in solution in 0.3-0.4 ml. of water, 0.6 ml. citrate buffer 0.1 M pH 5.6, 0.1 ml. of a partially purified preparation of enzyme.
1. Specijicity
As can be seen in Table II, the enzyme, even in a semipurified condition, is fairly specific for trehalose, having no activity toward any of the selected carbohydrates tested. The splitting of trehalose is not inhibited by the addition to a reaction mixture of any of the sugars noted in Table II at those concentration levels. Maltose, not shown on the table, is split by a crude preparation, but preparations taken off the column show no activity toward this disaccharide. As will be seen later, concentrations of glucose up to 20 times that of trehalose do not inhibit the enzyme in the assay system used.
Klett rdg at 540i3q.P
Substrate Trehalose, Sucrose, Melibiose, Lactose, Raffinose, Turanose, a Corrected
6 pmoles 20 pmoles 8 rmotes 8 rmoles 10 rmoles 8 rmoles for
blank
’ 5
’ 6
118 0 0 0 0 0 readings.
f?. pH E$ect The effect of pH upon the hydrolysis of trehalose was determined by the use of buffers of suitable values. Figure 3 indicates that the rate of hydrolysis is a maximum at a pH of 5.6. The maximum rate is attained in citrate buffer, with no increase noted in the presence of phosphate. Tris-HCl buffer appears to be inhibitory to the enzyme, a fact which has been confirmed by some experiments involving the buffer used in the column elution. 3. Temperature E$ects The maximum rate of splitting occurs when the purified enzyme is incubated with substrate at ,45’C. Inactivation proceeds rapidly above t,his temperature, so that under standard assay conditions, activity is decreased by 50% at 55°C. The enzyme appears to be rather stable in the frozen state, no decrease in activity having been noted after 2 months at - 12°C. .J. Substrate Concentration E$ects Figure 4 illustrates a Lineweaver-Burk plot of a co:ncentration activity curve. Calculating from the data obtained through
0’
3
’ 4
pIi
’ ’ 7 8 UNITS
’ 9
’ IO
FIG. 3. pa-Activity
curve of trehalase. Buffer concentrations are 0.1 M. Other conditions of assay are the same as described in the Methods section under Determination of Enzyme Activity. X, citrate; 0, citrate PO, ; A PO, ; 0, Veronal; A, glycine NaOH; IX, Tris-HCl.
this device, the K, for trehalase is 6.7 X 10-d M. UTILIZATION OF TREHALASE THE PRESENCE OF SMALL OF TREHALOSE
IN TESTS FOR QUANTITIES
The coupling of trehalase with glucose oxidase and peroxidase was easily attained, but certain difficulties in the over-all assay method immediately presented themselves. The most important of these was concerned with the fact that the commercial glucose
FRIEDMAN
256
FIG. 4. Lineweaver-Burk plot of a concentration activity curve. Conditions of assay are the same as described in the Methods section under
Determination
of Enzyme
Activity.
TABLE REMOVAL
III
FROM PREPARATION
OF TREHALASE
OXIDASE
GLUCOSE
Conditions of assay are the same as those described in the Methods section under Assay Method for Micro Quantities of Trehalose. Glucose
oxidase
preparation
oxidase preparation contained measurable quantities of trehalase and other glucosidases. Trehalase was easily removed from the preparation through the use of a DEAEcellulose column. At a pH of 8, the column holds trehalase, while glucose oxidase and certain other impurities are easily eluted. A description of the procedure for this purification may be found in the Methods section, and Table III shows the results of passage of glucose oxidase through the column. The slight residual activity cannot be removed by this method. The ability of this enzyme complex to measureglucose and trehalose occurring concurrently can best be demonstrated by reference to Table IV in which is summarized a typical experiment. When determinations are made on small quantities of blood, there appears to be no constituent which interferes with the materials used in the assay. An example of such a determination made in the presence and absence of added glucose and trehalose may be seen in Table V. DISCUSSION
Rdg. at 401 mfl
Trehalose
The discovery of the existence of trehalase in the blood of Phormia regina and the findings regarding the quantitative distribution of the substrate and product of the reaction prompted a further look at the enzyme in an effort to determine what mechanisms might
Irk=.
Pre column (0.08 ml.) Post column-concentrated (0.04 ml.)
2X
20 20
0.990 0.030
TABLE ENZYMIC
MEASUREMENT
IV
OF GLUCOSE
Conditions of assay are the same as those described in Methods Quantities of Trehalose. Substrate
Glucose oxidase
Do.
20 pg.
Do.
Do. Do. Do.
4 pg. 20 pg.
4 pg. + trehalose
20 Mg.
Glucose 4 pg. + Do. 0.8 fig. 2 pg. 4 pg. 20 pg. 4 pg. 4 pg. 4 pg. 4 pg.
0 Values obtained
Do. Do. Do.
+ Do.
Do. Do. Do. Do. Do. Do. Do. Do.
+ + + + + + + +
ObseyIt
0.258 1.289 0.277 0.260 1.262 0.304
+ trehalase Do. Do. Do. Do. Do. Do. Do. Do.
using 20 rg. glucose and trehalose
TREHALOSE
section under Assay Method
Enzyme(s)
Glucose 4 pg.
Do. 4 pg. + Do. Do. 4 pg. + Do. Do. 4 pg. + Do. Do. 2 pg. + Do. Do. 4 pg. + Do. Do. 8 pg. + Do. Do. 20 pg. + Do.
AND
0.394
0.485 1.502 0.397
0.482 0.758 1.528 as standards.
rdp.
for Micro Calcd.”
rdg.
0.258 0.258 0.252 0.309 0.388 0.516 1.528 0.388 0.516 0.774 1.528
Thus, 1.289/20 = O.O64/pg.
ISOLATION
OF
TABLE ASSAY
Conditions of assay tities of Trehdose.
OF GLUCOSE
are the same
as described
AND
V
TREHALOSE
in Methods
Substrate
Glucose (4 Trehalose Trehalose Blood (1.2 Blood (1.2 Blood (1.2
IN
PRORMIA
section
under
(4pg.) (4 pg.) (4 pg.) +
tre-
Glucose Glucose Glucose Glucose Glucose Trehalose tion) Glucose Glucose Glucose
be operative in controlling its activity. Although the blood enzyme was of major interest, the microquantities of this fluid which could be drawn from adult Phormia (maximum of 2-3 ~1.) necessitated an approach through the enzyme obtained from homogenates. Trehalase was, therefore, purified about lOOO-fold from whole fly homogenat,es, and its properties were studied. With somevery minor exceptions, they were found to be quite similar to those described for Galleria mellonella by Kalf and Rieder (7). However, contrary to the findings of Baba (13), that trehalose B-phosphate is a substrate for trehalase, the enzyme from Phormia, would not, after purification, attack the ester. As will be reported at a later date, trehalose 6-phosphate is actually split by a specific phosphatase also found in Phormia.
The fact that the pH optimum of trehalase is 5.6 and the pH of the blood is 6.8-7.0 led to experiments reported elsewhere (14) to determine whether muscular activity would lower blood. pH sufficiently to permit the enzyme to act more efficiently on trehalose, thus bringing about an increase in reducing sugar. The pH of the blood is unchanged, however, even after sustained flight, leading to the conclusion that this type of control is ineffective. An interesting observation which is currently under study is the finding that when blood is diluted and incubated for 30 min.
BLOOD
Assay
Glucose Observed
0.184 0.005 0.177 0.113 0.470
+ trehalose + trehalose (by calcula-
0.305 0.660 0.850
+ trehalose + trehalose
TABLE APPEARANCE BLOOD
for Micro Quan-
Method
Beckman rdg. at 401 m/I
Determination
pg.) (4 pg.) (4 pg.) pl.) ,.,l.) rl.)
Blood (1.2~1.) + glucose Blood (1.2 ~1.) + trehalose Blood (1.2 ~1.) + glucose halose (4 pg.)
257
TREHALASE
Irg.
fig.
4 0 4 2.4 10.2 -
7.8
6.6 14.3 18.4
6.4 14.2 18.2
VI
OF GLUCOSE UPON
Calculated
DILUTION
IN IN
PHORMIA VITRO
Undiluted blood withdrawn, incubated, heated for 2 min. @ lOO’C., then diluted and assayed as in Methods section under Assay Method for Micro Quantities of Trehalose. Diluted blood withdrawn, diluted l:lOO, incubated, heated for 2 min. @ lOO”C., then assayed as in Methods section under Assay Method for M~CTO Quantities of Trehalose. Substrate
Blood (1 pl. undil.) Do. Blood (1 pl. diluted) Do. Centrifuged blood (1 ~1. undil.) Do. Centrifuged blood (1~1. diluted) Do.
;,T$;&
Glucose
min.
Pg.
0 30 0 30 0 30 0 30
4.8 5.4 8.5 13.4 6.1 5.6 8.4 13.4
at 32°C. there is a marked increase in reducing sugar levels compared to the negligible change which takes place when incubated without dilution (seeTable VI). Centrifugation of blood at 8200 X g, which will remove the larger blood elements, does not lead to a reduction in formation of free glucose during incubation of diluted blood. The possibility of the presence of a naturally occurring inhibitor of trehalase which becomes ineffective upon dilution, permitting the appearance of activity, is not being ignored. In regard to the assay method, a few
258
FRIEDMAN
things have become evident which were inibially overlooked. The first concerns the fact t#hat the glucose oxidase preparat’ion, as commercially available, is impure to the extent of containing both trehalase and maltase. It is possible to remove the trehalase activity by the judicious use of a column, but maltase remains and is not removed by any of a number of methods. For gross analysis these contaminating enzymes present no great problem, but when one is attempting to work at levels from 1 to 20 pg., any minute contamination has a significant effect. Another problem encountered is the inhibition of trehalase by tris(hydroxymethyl)aminomethane to the extent of 30 % at 2.5 X 1O-2 M. The maltase found in the glucose oxidase preparation is even more sensitive to this compound, being inhibited 92 % at the same concentration. There are, however, no concentrations at which trehalase is unaffected and maltase is completely inhibited. Glucose oxidase retains full activity in the presence of this salt at all concentrations tested. Phormia blood has not been analyzed to the extent which would permit stating with certainty that there are no compounds present that could react with peroxidase to simulate glucose in t,he assay system. The
blood has, however, been checked by other methods for reducing sugars, and the analysis appears to be quit,e similar to that obtained by the glucose oxidase method. ACKNOWLEDGMENTS The assistance of Miss C. Mecca Amir-Fazli is greatly appreciated.
and
Mrs.
G.
REFERENCES 1. CHEFCRKA, W., Enzymologia 17, 73 (1954). R. W., AND CHELDELIN, V. H., 2. NEWBURGH, J. Biol. Chem. 214, 37 (1955). 3. WYATT, G. R., AND KALF, G. F., J. Gen. Physiol. 40, 833 (1957). 4. WIGGERS, H. A. L., Ann. 1, 129 (1832). E., Bull. sot. mycologic France 5. BOURQUELOT, 9, 189 (1893). 6. FR~GREJACQUE, M., Compt. rend. 213, 88 (1941). G. F., AND RIEDER, S. V., J. Biol. 7. KALF, Chem. 230, 691 (1958). 8. BUCK, J. B., Rev. Sci. Irastr. 20, 676 (1949). 9. SOMOGYI, M., J. Biol. Chem. 196, 19 (1952). 0. H., ROSEBROUGH, N. J., FARR, A. 10. LOWRY, L., AND RANDALL, R. J., J. Biol. Chem. 193, 265 (1951). 11. PETERSON, E. A., AND SOBER, H. A., J. Am. Chem. Sot. ‘78, 751 (1956). 12. KESTON. A. S., ABSTRACTS, p. 31~. A.C.S., Div. Biol. Chem., April 1956. 13. BABA, T., Biochem. 2.273, 207 (1934). 14. FRIEDMAN, S., J. Insect Physiol. 3, 118 (1959).
OF
BIOCHEMISTRY
The
AND
Purification
BIOPHYSICS
87,
252-258
(1960)
and Properties of Trehalase from Phormia regina, Meig.’
Isolated
S. FRIEDMAN From the Department Institute of Arthritis
of
Entomology, and Metabolic
Purdue University. Diseases, National Received
October
Lafayette, Institutes
Indiana;2 Health,
of
and from the National Bethesda, Maryland3
2, 1959
An enzyme responsible for the hydrolysis of a,ol-trehalose has been isolated from Phormia regina, Meig. and purified approximately 1000 times using a diethylaminoethylcellulose column. The properties of the enzyme are described and its possible role in the blood of the insect discussed. Through use of the enzyme a sensitive, specific method for the assay of trehalose has been effected. INTRODUCTION
Investigations of carbohydrate metabolism in insects have confirmed the idea that, in general, the degradative pathways are similar to those found in other animals (1, 2). Certain important differences have been called to our attention, however, and it is with one of these that this report is involved. Wyatt and Kalf (3), in a recent survey of insect hemolymph, have shown that a nonreducing disaccharide, a!,a-trehalose [(WDglucosido)-a-n-glucoside], is the major blood sugar in a number of genera, occurring in some casesin concentrations ten times that of the omnipresent component n-glucose. This disaccharide, originally isolated from a mold (4), can be split to two moles of glucose by a hydrolytic enzyme, trehalase, which has been described in a number of organisms, fungi (5) as well as insects (6). Kalf and Rieder (7) have lately obtained a somewhat purified preparation of the enzyme from a i This work was partially supported by Grant No. E-2440 from the National Institutes of Health, Bethesda, 14, Maryland. 2 Journal Paper No. 1502 of the Purdue University Agricultural Experiment Station. 3 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. 252
homogenate of larvae of Galleria wtellonella and have described some of its properties. An investigation of the blood of adult Phormia regina has shown that both glucose and. trehalose are present, as is the hydrolytic enzyme trehalase. The questions raised by this finding have led to an examination of the enzyme in this insect. Purification of the enzyme and its subsequent utilization as a tool in the measurement of minute quantities of trehalose and glucose are the subjects of this paper. MATERIALS
AND
METHODS
Enzymic activity was obtained from two sources : 1. From Blood. Measured quantities (up to 2 ~1.) of hemolymph were removed from the dorsal sinus of adult Phormia regina using a modification of the micro injection apparatus of Buck (8). The modification permits direct measurements of 0.6 ~1. i 770, (or less, although this has not been determined) using a Starrett gauge (No. 25-611, L. S. Starrett Co., Athol, Mass.) as a direct reading device coupled between a micrometer and a hypodermic syringe to which is attached a glass needle. The syringe and needle are filled with mercury to the point of involvement with the hemolymph, thus providing a system with almost complete rigidity (see Fig. 1). This instrument was used for all experiments involving removal or delivery of blood samples. The blood samples were used without further purification as sources of enzyme.
ISOLATION
OF
2. From Whole Insects. Wild adults of Phormia regina were trapped and used as the nucleus of a laboratory culture which was reared by the usual methods. Five-day-old flies, having been fed on 5% glucose solutions from emergence, were anesthetized with CO2 and homogenized for 1.5 min. in a Waring blendor at 3°C. with 2.5 vol. (w/v) [tris (hydroxymethyl) aminomethane] Tris - HCl (pH 7.0, 0.1 iM). The homogenate was pressed through cheesecloth to remove large pieces of cuticle, and thle total volume was measured (Fract#ion 1) and centrifuged at 12,800 X g for 15 min. at 0°C. The supernatant was poured through glass wool to remove a fatty accumulation, and the turbid solution (Fraction II) was used for further purification. Activity of the enzyme was determined as follows: 0.3 ml. of enzyme solution was mixed with 0.5 ml. of trehalose (20 rmoles/ml.) and 0.6 ml. of citrate buffer (0.1 M, pH 5.6). The mixture was incubated 15 min. at 32”C., and the reaction was stopped by addition of 1 ml. of Ba(OH)z and 1 ml. of ZnSOl standardized according to Somogyi. The Nelson-Somogyi method (9) was then used to measure reducing sugar. Protein was measured by the method of Lowry et al. (10). Preparation of the diethylaminoethylcellulose (DEAE-cellulose) column followed the directions outlined by Peterson and Sober (11). The DEAEcellulose was purchased from Eastman Organic Chemicals and washed with 1 N NaOH until it was no longer colored. This was followed by washing with 1 N HCl and then successive washings with the two alternately. The cellulose was finally suspended in 1 N NaOH, vacuum filtered until almost dry, and washed a number of times in distilled water. It was suspended in Tris-maleate buffer, pH 8.0,0.005 M, poured into a column 18 X 270 mm., allowed to settle, and packed under 10 lb./sq. in. pressure without permitting the column to dry. The column was then placed in a cold room (3°C.) and permitted to reach temperature equilibrium, after which it was again packed under the same pressure. At this point it was ready for use. Five-milliliter fractions were collected at a rate of ca. 15 ml./hr. The sugars used in the investigations were commercially available grades and were recrystallized from alcohol when necessary. Trehalose was checked against a sample of the chromatographitally pure compound obtained through the courtesy of Dr. D. MacDonald of the National Institutes of Health. Trehalose g-phosphate was obtained as the barium salt from Dr. MacDonald and converteld to the potassium salt. The assay method developed for micro quantities of trehalose uses the purified enzyme and involves a mod.ification of the calorimetric glucose
253
TREHALASE
LI
7
6
4 32
I
Fm. 1. Micro injection apparatus numbers designate the following parts: 1. Micrometer spindle without bow. 2. Ball bearing coupling, inner race force coupled to micrometer spindle, outer race joined to 3. Lucite coupling locked by screw to one side of Starrett depth gauge. 4. Spring for maintaining pressure against coupling, permitting minimum play in ball bearing. 5. Face of Starrett gauge, reading directly to horizontal movement of 0.001 in. (total movement 0.2 in.). 6. Lucite ring, coupling gauge and plunger of syringe directly. 7. One-milliliter syringe, barrel and plunger lubricated with silicone high vacuum grease. 8. Glass needle, made from No. 421-B Baketel glass-adapter (Becton, Dickinson & Co., Rutherford, N. J.). The whole unit is mounted firmly on a steel plate, permitting very little warping along its entire length. The syringe is mounted so that it may be easily removed and replaced or refilled with mercury. oxidase technique developed by Keston (12) for measurement of glucose. Glucose oxidase, obtained from Worthington Biochemicals, Freehold, N. J., was made up as a 20% suspension in 0.01 M phosphate buffer pH 8.0 and centrifuged at 2°C. for 10 min. at 3000 X g. Twenty milliliters of the supernatant was transferred to the top of a DEAE-cellulose column prepared as above except that the buffer used for suspending and packing the column was phosphate 0.01 M pH 8.0. The enzyme was then eluted from the column with 100 ml. of phosphate buffer of the same pH and molarity, and came out in the first 50-60 ml. as a tan solution. This was dialyzed against 2 1. of water and lyophilized or concentrated to 20 ml. under vacuum. Peroxidase and o-dianisidine were obtained from Worthington and made up in concentrations ten times that normally used for glucose analyses. Thus, 50 mg. of o-dianisidine was dissolved in 1 ml. methanol and poured into 49 ml. of 0.05 M phosphate buffer pH 7.0 containing 25 mg. peroxidase. The solutions were mixed and centrifuged at 2°C. for 10 min. at 5000 X g. For determinations, the quantitatively unknown substrate was added in a volume not to exceed 0.3 ml., followed by 0.4 ml. of the peroxidase-o-dianisidine mixture, 0.05 ml. of the purified trehalase or 0.05 ml. water if only glucose was to be measured, and 0.04 ml. of purified glucose oxidase. This mixture was incubated for 30 min. at 32”C., the reaction was stopped by addition of 0.02 ml. of 4 N HCl, and measurements were made at 401 rn,u in a Beckman spectrophotometer. The
254
FRIEDMAT;
30,
I
25-
8
I
20
i
LOO-
I 0 ..--. I\ 9
FRACX ml
\
.._.._.-
/\
-.-.-.-.-.-.-.-.-.--.-c-.
2%
t
H
I
.-..o.-.-
200
1000
FIG. 2. Effluent diagram of 4&60 alcohol fraction of Phormia regina: 40 ml. (containing 148 mg. dialyzed protein) placed on an 18 X 270 mm. column of DEAE-cellulose. Effluent collected in ca. 5-ml. fractions. Buffers used: I. 300 ml. of 0.005 M Tris-maleate pH 6.0 siphoned into mixing chamber containing 100 ml. of 0.005 M Tris-maleate pH 8.0 at same rate that mixture leaves chamber and enters column for elution. II. 100 ml. of 0.005 M pH 6.0 Tris-maleate put directly on column. III. 300 ml. of 0.05 M Tris-maleate pH 6.0 into 100 ml. 0.005 M Tris-maleate pH 6.0. IV. 100 ml. of 0.05 M Tris-maleate pH 6.0 put directly on column. V. 200 ml. of 0.05 M Tris-maleate pH 6.0 containing 0.1 M NaCl into 100 ml. of 0.05 M Tris-maleate pH 6.0. VI. 100 ml. of 0.05 M Tris-maleate pH 6.0 containing 0.2 M NaCl put directly on column. TABLE SUMMARY
I
OF TREHALASE
Conditions
of assay under
Methods section Activity.
PURIFICATION
were as specified Determination of
Total protein
activity _____
.nits/?lzg. protein
mg. homoge(Fr. I)
Total reCOVUY
SPCCifiC
Crude nate
the
-
Total units
Fraction
in
Enzyme
TO
8250
7020
1.17
-
Supernatant (Fr. II)
6900
1880
3.7
84
4&60 alcohol cipitate
2675
221
12.1
pre (Fr.
32.5
III) Column
blank strate,
elution
consisted for which
2530
-
2.1
-
1200
of all reagents except water was substituted.
31
the
sub-
RESULTS PURIFICATION
OF
TREHALASE
The solution designated in the Methods section as Fraction II was subjected to an
ethanol fractionation at -3°C. TOI 130 ml. of Fraction II was added slowly with stirring 87 ml. of absolute ethanol, bringing the alcohol concentration up to 40 %. After standing for 15 min., the resulting suspensionwas centrifuged at 6000 X g for 10 min. and the precipitate was discarded. The supernatant was treated with 108 ml. ethanol iti the same manner, brining the concentration up to 60 %. After centrifugation the supernatant was discarded, the precipitate was dissolved in 60 ml. of Tris-maleate buffer, 0.005 M pH 8.0, and the solution was dialyzed overnight against 2 1. of the samebuffer (Fraction III). Forty millilit.ers of the dialyzed solution was placed on top of the previously described DEAE-cellulose column (see Methods section), and after washing it in with one-third volume of the same buffer, gradient elution of the proteins was begun. Figure 2 showsthe results of the elution and is self-explanatory. The protein peak arising immediately after point II contains trehalose-6-phosphatase, which will be discussed in a subsequent paper.
ISOLATION
OF TREHALASE
Table I contains a resume of the steps in the purification procedure. In all experiments, one unit of activity is equivalent to 1 pmole glucose released in 15 min. at 32°C. PROPERTIES
OF THE ENZYME
255 TABLE
II
ACTIVITY OF TREHALASE TOWARD VARIOUS SUBSTRATES Conditions of assay were as follows: Substrates in solution in 0.3-0.4 ml. of water, 0.6 ml. citrate buffer 0.1 M pH 5.6, 0.1 ml. of a partially purified preparation of enzyme.
1. Specijicity
As can be seen in Table II, the enzyme, even in a semipurified condition, is fairly specific for trehalose, having no activity toward any of the selected carbohydrates tested. The splitting of trehalose is not inhibited by the addition to a reaction mixture of any of the sugars noted in Table II at those concentration levels. Maltose, not shown on the table, is split by a crude preparation, but preparations taken off the column show no activity toward this disaccharide. As will be seen later, concentrations of glucose up to 20 times that of trehalose do not inhibit the enzyme in the assay system used.
Klett rdg at 540i3q.P
Substrate Trehalose, Sucrose, Melibiose, Lactose, Raffinose, Turanose, a Corrected
6 pmoles 20 pmoles 8 rmotes 8 rmoles 10 rmoles 8 rmoles for
blank
’ 5
’ 6
118 0 0 0 0 0 readings.
f?. pH E$ect The effect of pH upon the hydrolysis of trehalose was determined by the use of buffers of suitable values. Figure 3 indicates that the rate of hydrolysis is a maximum at a pH of 5.6. The maximum rate is attained in citrate buffer, with no increase noted in the presence of phosphate. Tris-HCl buffer appears to be inhibitory to the enzyme, a fact which has been confirmed by some experiments involving the buffer used in the column elution. 3. Temperature E$ects The maximum rate of splitting occurs when the purified enzyme is incubated with substrate at ,45’C. Inactivation proceeds rapidly above t,his temperature, so that under standard assay conditions, activity is decreased by 50% at 55°C. The enzyme appears to be rather stable in the frozen state, no decrease in activity having been noted after 2 months at - 12°C. .J. Substrate Concentration E$ects Figure 4 illustrates a Lineweaver-Burk plot of a co:ncentration activity curve. Calculating from the data obtained through
0’
3
’ 4
pIi
’ ’ 7 8 UNITS
’ 9
’ IO
FIG. 3. pa-Activity
curve of trehalase. Buffer concentrations are 0.1 M. Other conditions of assay are the same as described in the Methods section under Determination of Enzyme Activity. X, citrate; 0, citrate PO, ; A PO, ; 0, Veronal; A, glycine NaOH; IX, Tris-HCl.
this device, the K, for trehalase is 6.7 X 10-d M. UTILIZATION OF TREHALASE THE PRESENCE OF SMALL OF TREHALOSE
IN TESTS FOR QUANTITIES
The coupling of trehalase with glucose oxidase and peroxidase was easily attained, but certain difficulties in the over-all assay method immediately presented themselves. The most important of these was concerned with the fact that the commercial glucose
FRIEDMAN
256
FIG. 4. Lineweaver-Burk plot of a concentration activity curve. Conditions of assay are the same as described in the Methods section under
Determination
of Enzyme
Activity.
TABLE REMOVAL
III
FROM PREPARATION
OF TREHALASE
OXIDASE
GLUCOSE
Conditions of assay are the same as those described in the Methods section under Assay Method for Micro Quantities of Trehalose. Glucose
oxidase
preparation
oxidase preparation contained measurable quantities of trehalase and other glucosidases. Trehalase was easily removed from the preparation through the use of a DEAEcellulose column. At a pH of 8, the column holds trehalase, while glucose oxidase and certain other impurities are easily eluted. A description of the procedure for this purification may be found in the Methods section, and Table III shows the results of passage of glucose oxidase through the column. The slight residual activity cannot be removed by this method. The ability of this enzyme complex to measureglucose and trehalose occurring concurrently can best be demonstrated by reference to Table IV in which is summarized a typical experiment. When determinations are made on small quantities of blood, there appears to be no constituent which interferes with the materials used in the assay. An example of such a determination made in the presence and absence of added glucose and trehalose may be seen in Table V. DISCUSSION
Rdg. at 401 mfl
Trehalose
The discovery of the existence of trehalase in the blood of Phormia regina and the findings regarding the quantitative distribution of the substrate and product of the reaction prompted a further look at the enzyme in an effort to determine what mechanisms might
Irk=.
Pre column (0.08 ml.) Post column-concentrated (0.04 ml.)
2X
20 20
0.990 0.030
TABLE ENZYMIC
MEASUREMENT
IV
OF GLUCOSE
Conditions of assay are the same as those described in Methods Quantities of Trehalose. Substrate
Glucose oxidase
Do.
20 pg.
Do.
Do. Do. Do.
4 pg. 20 pg.
4 pg. + trehalose
20 Mg.
Glucose 4 pg. + Do. 0.8 fig. 2 pg. 4 pg. 20 pg. 4 pg. 4 pg. 4 pg. 4 pg.
0 Values obtained
Do. Do. Do.
+ Do.
Do. Do. Do. Do. Do. Do. Do. Do.
+ + + + + + + +
ObseyIt
0.258 1.289 0.277 0.260 1.262 0.304
+ trehalase Do. Do. Do. Do. Do. Do. Do. Do.
using 20 rg. glucose and trehalose
TREHALOSE
section under Assay Method
Enzyme(s)
Glucose 4 pg.
Do. 4 pg. + Do. Do. 4 pg. + Do. Do. 4 pg. + Do. Do. 2 pg. + Do. Do. 4 pg. + Do. Do. 8 pg. + Do. Do. 20 pg. + Do.
AND
0.394
0.485 1.502 0.397
0.482 0.758 1.528 as standards.
rdp.
for Micro Calcd.”
rdg.
0.258 0.258 0.252 0.309 0.388 0.516 1.528 0.388 0.516 0.774 1.528
Thus, 1.289/20 = O.O64/pg.
ISOLATION
OF
TABLE ASSAY
Conditions of assay tities of Trehdose.
OF GLUCOSE
are the same
as described
AND
V
TREHALOSE
in Methods
Substrate
Glucose (4 Trehalose Trehalose Blood (1.2 Blood (1.2 Blood (1.2
IN
PRORMIA
section
under
(4pg.) (4 pg.) (4 pg.) +
tre-
Glucose Glucose Glucose Glucose Glucose Trehalose tion) Glucose Glucose Glucose
be operative in controlling its activity. Although the blood enzyme was of major interest, the microquantities of this fluid which could be drawn from adult Phormia (maximum of 2-3 ~1.) necessitated an approach through the enzyme obtained from homogenates. Trehalase was, therefore, purified about lOOO-fold from whole fly homogenat,es, and its properties were studied. With somevery minor exceptions, they were found to be quite similar to those described for Galleria mellonella by Kalf and Rieder (7). However, contrary to the findings of Baba (13), that trehalose B-phosphate is a substrate for trehalase, the enzyme from Phormia, would not, after purification, attack the ester. As will be reported at a later date, trehalose 6-phosphate is actually split by a specific phosphatase also found in Phormia.
The fact that the pH optimum of trehalase is 5.6 and the pH of the blood is 6.8-7.0 led to experiments reported elsewhere (14) to determine whether muscular activity would lower blood. pH sufficiently to permit the enzyme to act more efficiently on trehalose, thus bringing about an increase in reducing sugar. The pH of the blood is unchanged, however, even after sustained flight, leading to the conclusion that this type of control is ineffective. An interesting observation which is currently under study is the finding that when blood is diluted and incubated for 30 min.
BLOOD
Assay
Glucose Observed
0.184 0.005 0.177 0.113 0.470
+ trehalose + trehalose (by calcula-
0.305 0.660 0.850
+ trehalose + trehalose
TABLE APPEARANCE BLOOD
for Micro Quan-
Method
Beckman rdg. at 401 m/I
Determination
pg.) (4 pg.) (4 pg.) pl.) ,.,l.) rl.)
Blood (1.2~1.) + glucose Blood (1.2 ~1.) + trehalose Blood (1.2 ~1.) + glucose halose (4 pg.)
257
TREHALASE
Irg.
fig.
4 0 4 2.4 10.2 -
7.8
6.6 14.3 18.4
6.4 14.2 18.2
VI
OF GLUCOSE UPON
Calculated
DILUTION
IN IN
PHORMIA VITRO
Undiluted blood withdrawn, incubated, heated for 2 min. @ lOO’C., then diluted and assayed as in Methods section under Assay Method for Micro Quantities of Trehalose. Diluted blood withdrawn, diluted l:lOO, incubated, heated for 2 min. @ lOO”C., then assayed as in Methods section under Assay Method for M~CTO Quantities of Trehalose. Substrate
Blood (1 pl. undil.) Do. Blood (1 pl. diluted) Do. Centrifuged blood (1 ~1. undil.) Do. Centrifuged blood (1~1. diluted) Do.
;,T$;&
Glucose
min.
Pg.
0 30 0 30 0 30 0 30
4.8 5.4 8.5 13.4 6.1 5.6 8.4 13.4
at 32°C. there is a marked increase in reducing sugar levels compared to the negligible change which takes place when incubated without dilution (seeTable VI). Centrifugation of blood at 8200 X g, which will remove the larger blood elements, does not lead to a reduction in formation of free glucose during incubation of diluted blood. The possibility of the presence of a naturally occurring inhibitor of trehalase which becomes ineffective upon dilution, permitting the appearance of activity, is not being ignored. In regard to the assay method, a few
258
FRIEDMAN
things have become evident which were inibially overlooked. The first concerns the fact t#hat the glucose oxidase preparat’ion, as commercially available, is impure to the extent of containing both trehalase and maltase. It is possible to remove the trehalase activity by the judicious use of a column, but maltase remains and is not removed by any of a number of methods. For gross analysis these contaminating enzymes present no great problem, but when one is attempting to work at levels from 1 to 20 pg., any minute contamination has a significant effect. Another problem encountered is the inhibition of trehalase by tris(hydroxymethyl)aminomethane to the extent of 30 % at 2.5 X 1O-2 M. The maltase found in the glucose oxidase preparation is even more sensitive to this compound, being inhibited 92 % at the same concentration. There are, however, no concentrations at which trehalase is unaffected and maltase is completely inhibited. Glucose oxidase retains full activity in the presence of this salt at all concentrations tested. Phormia blood has not been analyzed to the extent which would permit stating with certainty that there are no compounds present that could react with peroxidase to simulate glucose in t,he assay system. The
blood has, however, been checked by other methods for reducing sugars, and the analysis appears to be quit,e similar to that obtained by the glucose oxidase method. ACKNOWLEDGMENTS The assistance of Miss C. Mecca Amir-Fazli is greatly appreciated.
and
Mrs.
G.
REFERENCES 1. CHEFCRKA, W., Enzymologia 17, 73 (1954). R. W., AND CHELDELIN, V. H., 2. NEWBURGH, J. Biol. Chem. 214, 37 (1955). 3. WYATT, G. R., AND KALF, G. F., J. Gen. Physiol. 40, 833 (1957). 4. WIGGERS, H. A. L., Ann. 1, 129 (1832). E., Bull. sot. mycologic France 5. BOURQUELOT, 9, 189 (1893). 6. FR~GREJACQUE, M., Compt. rend. 213, 88 (1941). G. F., AND RIEDER, S. V., J. Biol. 7. KALF, Chem. 230, 691 (1958). 8. BUCK, J. B., Rev. Sci. Irastr. 20, 676 (1949). 9. SOMOGYI, M., J. Biol. Chem. 196, 19 (1952). 0. H., ROSEBROUGH, N. J., FARR, A. 10. LOWRY, L., AND RANDALL, R. J., J. Biol. Chem. 193, 265 (1951). 11. PETERSON, E. A., AND SOBER, H. A., J. Am. Chem. Sot. ‘78, 751 (1956). 12. KESTON. A. S., ABSTRACTS, p. 31~. A.C.S., Div. Biol. Chem., April 1956. 13. BABA, T., Biochem. 2.273, 207 (1934). 14. FRIEDMAN, S., J. Insect Physiol. 3, 118 (1959).