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Solubilization, purification, and some properties of trehalase from honeybee (Apis mellifera)
Solubilization,
Purification,
and
Honeybee
some (Apis
T. A. LEE’EBVIIE
cvr)
Properties
of Trehalase
from
mellifera) K. E:. HUBEIt
The insoluble fraction of trehalase (EC S.2.1.28) from holqybre (Apis j,lellijeru) was solubilixed by raising the pH of the enzyme suspension to 10.0. The soluble ellzpme in the suspension was not deactivated by this procedure and the solution rontaitling both the solubilized trehalase and the soluble trehalase was then purified at least BHO-fold. Only one batid was observed on disc gel a11d the trehalase activity migrated with this band. No contaminating srlcrase act.ivity was folmd in the purified preparation. The molecldar weight of the trehalase as estimated by Sephadex chromatography was llO,OOO-120,000. The purified enzyme had a K,,, of 1.6 mu and a pH optimum of 6..i. Tris and stlcrose strongly inhibited the enzyme; maguesirim and ethylenediamine tetraacetic acid had slight inhibitory effects, while sodimn borate a11t1 mercaptoet hanol had no effects. ATP and AMP did not alter the enzymatic hydrolysis rate. Enxymat ic activity was rapidly lost at temperatures greater t hall 40’. at pH vallies less t hall 4.0, and ill high ccjllcentratiolls of IIre:,.
The presence of the enzyme, trehulast (EC 321.28; a,a’-glucoside-l-glucohydrolase), which hydrolyzes a ,a-trehalose to 2 molecules of glucose has been reported in microorganisms, in plants, and in animals (1-3). However, its occurrence is most widespread in insects (4). The enzyme has been purified to various degrees but, as yet, the preparation of a homogeneous form has not been demonstrated, possibly because large quantities of most insects are difficult to obtain and also because a large fraction of tile trehnlase in insects is naturally insoluble. The present study entailed the solubilization of the insoluble portion of the trehalase of tile honeybee (Apis mellijwa) snd the purification of this fraction together with the soluble fraction for further studies of the properties of trelinlase in 3 l~omogenfwus state. RIAT~:I:TAT,S
ANI)
METIIOI)S
Adrdt honeybees were obtained it1 large qllarltit.ies from it loral tiotley farm. C:yatlidc gas was
IIsed to kill bees. with 110 loss of the level of trrhalasc activity. Immediately after death the bees were sllspended ilr 0.1 M sodirlm citrate bufer ($1 5.6). Honeybees stored it1 this brtffer at - 15’ for it period of at did not lose trehalasc activity 1f:ast (i months.
Aliqllots of etqme were added to at, assay mixture (fillal volume 2 ml) which cotltained 0.03 hr sodium citrate brlffrr (pH 6.5) and 0.005 51 LY,CYtrehalose dihydratc (Sigma Chemical Company). ,4ppropriate blanks :ttld glucose standards were used. Assay mixtlues were illcrlbated at 30” for 15 mitt alld the reactiotl was stopped by placing the solutions illto :I boilitlg water bath. 111order to bluffer ihesoll,tiotl at pll 7.0, 2 ml of aO.l M sodium potassium phosphate solritioll (pII 10.5) were‘ added. Two milliliters of (;lrlcostat I!eagrnt (Worthingtolr Biochemical Company) ill 0.1 M phosphate buffer (pII 7.0) were added to 2 ml of the solution and the mixtlvr was incubated at 30” for 10 mill. This reactiolr was stopped by the, addition of 3 drops of 3 x TICI atld the absorbance at 420 nm (.442(I) was measured. One Itnit was defitled as the arnol,llt of enzymr’ which catalyzed the hydrolysis of 1 rmole of trehalose per mill at 30” a11d pII Ci.5.
PURIFICATION
OF HONEYBEE
&muse Activity
Xolubilixation Trehalase in honeybees was found to consist of approximat,ely 50y0 soluble arId 50yC insoluble enzyme. Several techniques were used on suspensions of the insoluble fraction in an attempt to solubilize it.
It. has been reported (7) that sucrase activit,y is very dificult to separate from t.rehalase activit.y. The sucrase activity in enzyme samples was determined by adding 0.03 1%sucrose to the assay mixture instead of the trehalose (7).
lhzymatic One liter of bees in suspension (286 g of whole bees) was homogenized for 15 set it1 a Waring Blendor. The homogenate was filtered through cheeseclot,h and brought to G5y0 satklration with ammonium sulfate. The pellet after centrifugatioil at 10,000 g for 1 hr was suspended in 0.1 M sodillm citrate buffer (p1E 56) and dialyzed against large volumes of t,his bntl’er. Sodium hydroxide solution (3 N) was then added to the enzyme preparation to bring the pT1 to 10.0. Aft,er stirring for 2 hr the pII 10.0 mixture was centrifuged at 10,OOOgfor 1 hr alld the srlpernatstnt fluid was extensively dialyzed against 0.005 M sodium borate briffer (pH 8.0). All furt.her purificatioti was carried out in this buffer. The dialyzed supernatant fraction was applied to a 5 X 100.cm l>l~:hli:-celhilose (Whatman 1)Ell) colrmm aud eluted with 2 liters of a O-47; linear sodirim chloride gradient. Thr most active fractions were pooled, dialyzed, and then concentrat,ed by lpophilization. The enzyme solution was then applied t.o a 5 X loo-cm Sephades (;-ZOO column and eluted with 2 liters of the borate buffer. rlctive fractions were pooled, concclutrated, and reapplied to a 2.5 X 45.cm I)EAE:cxellr1lose col~imn. ,4ctive fractions elrkted using 2 liters of a O-37& linear sodium chloride gradient were conceutrated with Aquacide II (Calbiochcmj. Flvt.her fractionation of t.he enzyme solution was achieved by two passes through a preparative disc-gel electrophoresis apparatus (Buchler “1’01,~ Prep”) using a 3.0-cm column of 7.5’; acrylamide gel for the first pass and a 6.0.cm colurn~l for the sccolld. Aquacidc II was used to concentrate the ellzgme. Protein collcelltrat.ioils wrre estimated by the ~(215-225) method (5).
Anal&al
Disc-Gel Electrophoresis
Acrylamide disc-gel (7.57,) electrophoresis (6) was performed in duplicate at 2’ in a Tris-glycille brlffer system with a rmming pII of 0.3. One was st.ained wit,h amido schwarz sollltiotl ar~l t,hen washed with 77” acetic acid to remove the excess stain. (+el patterns were traced with a Dellsicord 5+2 (Photovolt, Corporation) densitometer. The other gel of the pair was sliced into $ie-in. slices and each of t,he slices was separately assayed. The assay results of the slices were sllperimposed upon the densi tometer t ratings.
513
TREIIALASIC
Properties
The K,, was det.ermirled by methods recommended by Dowd and Riggs (8). Opt,imum pH studies and the effect of temperature on the trehalase reaction rates were observed. The effects of various added constituents were also studied. The stability of the enzyme at various temperatures, pH values, and urea rolicelLtrations was determilled.
Estimation of Size The molerular wpight of the purified enzyme was estimated with a previously calibrated column (Sephadex K25/100) of Sephadrx (j-150 using Pharmac.ia stalldards. RESULTS
A1?;1) 1>1SCUYS10N
Solubilizafio?2, Activation, and Stability Table achieved
I compares the solubilizations by the different methods. The greatest solubilizstion WLSnoted when 0.5 % sodium dodecgl sulfate ~-as added to the suspension either at pH (i..T or at pH 10.0. TABLk:
I
No treatment, Octanol (.jfc) Freezing and thawing (.iX) Sodium dodecyl sulfate (0.3C;) Adjustment to pII 8.0 (3 N NaOII) Adjustment, to pH 9.0 (3 N KaOH) Adjustment to pH 10.0 (:S N SaOIL) Adjustment to pII 10.0 (3 N NaOH) in the presence of sodium dodecyl sulfate (O..ii;)
natant (%i
Super-
Precipitate m
2 1:; 2 96 50
08 87 98 4 .io
8.;
1-j
94
6
96
4
516
LEFEBVRE
ANI)
The pH 10.0 treatment without addition of detergent was almost as effective. Table 11 shows the percentage of original activity observed 2 hr and 24 hr after treatment by the different solubilization techniques. Raising the pH to 8.0, to 9.0, or to 10.0, or adding 0.5 % sodium dodecyl sulfate resulted in marked activation after 2 hr. However, after 24 hr the enzyme treated with detergent had lost more activity than the enzyme at pH 9.0 or 10.0. Also detergents are known to be very difhcult to remove from protein molecules. Thus, it was decided to solubilize the enzyme by raising the pH to 10.0 followed by extensive dialysis against 0.005 M sodium borate buffer (pH 8.0) to prevent the inactivation which results after a prolonged spell at pH 10.0. TABLE
II
ACTIVITY
OF SUSPENSIONS OF INS~LCBLE TREHALASIC 2 hr lack 24 hr AFTER V.\RIOUS PHYSIc.11, .IR‘D CHEMICAL TIILITMENTS. THE TOTAL SUSPENSION ACTIVITY OF THE UNTRL~TIXI AFTER 2 hr Was I)IGSIGNATI~W AS 100C;A No. hr
2 (5%) 24(%) h’o treatment
100
Octanol (:jCy ) Freezing and thawing (5X) Sodium dodecyl sulfate (0.5%) Adjustment to pH 8.0 (3 N N&H) Adjustment to pH 9.0 (3 N NaOll) Adjustment to pH 10.0 (3 N NaOll) Adjustment to pH 10.0 (3 pi NaQII) in the presence of sodium dodecyl sldfate (0.3%)
107 87 184 201
105 118 113 156 201
220
200
201
177
201
160
PURIFICATION
Purification step 1 2 3 4 5 6 7
65rj, (NH4)?SQh precipitation pH 10.0 supernatant First DEAE-cellulose Sephadex G-200 Second DEAE-cellulose First preparative disc gel Second preparative disc gel
HUBER
Purification Activities and protein concentrations determined at each step of the isolation of the enzyme are shown in Table III. The activit) of the enzyme in the original crude bee cxtract could not be determined because of the high natural content of glucose in the bee which interferes with the assay. The enzynle eluted from the DEAE columns at N&l concentrations between 1 and 2%. It eltited through the Sephadex G-200 column after about 800 ml of eluent had passed. A final yield of over 1 mg of purified enzyme having a specific activity of approximately 76 units/ mg was obtained. This represented a fold purification of at least 680. Homogeneity of the Protein by Analytical Disc-Gel Electrophoresis Analytical disc-gel electrophoresis performed after the enzyme solution had been eluted from the second DEAE-cellulose column indicated the presence of several protein bands which could not be clearly resolved because of their proximity to each other. Only one of these was active although the active band could not be specified. After the first preparative electrophoresis two protein bands of similar mobilities were observed. Only one was active. Figure 1 is a densitometer tracing of the analytical disc gel obtained with enzyme from the second preparative electrophoresis pass. Only one band was observed and the trehalase activity migrated with this band. Although the soluble and insoluble forms of honeybee trehalase were purified together, the fact that only one active band resulted suggests that the soluble and insoluble enzyme forms are
TABLE III OF IIOXEYBEE
TAEH.IL.ISI.:
Solution (ml)
Total units
Total protein (w)
Specific activity
Yield (%)
Purification factor -__.
380 330 210 73 130 10 ;j
640 901 753 532 363 248 104
5714 3726 987 139 .x 5.17 1.36
0.112 0.242 0 763 3.827 6.640 47.99 76.47
100 141 118 x3 57 :!I9 16
1.0 2.2 6.8 34.2 .i9 439 683
517 80 -
-16
70
- 14
-
z - 1.2
= 60z IfJl 50k c z 5 5
-10
4030-
-
20-
z L -
-06
: Q
-06
d 5
.. 04
E
? 10 -,,‘I 0
2
4
6
I 6
10
12
14
16
GEL
SLICE
20
16
22
24
26
26
30
32
34
NUMBER
FIG. 1. Trehalase activit,y of slices of unstained gel superimposed upon a densitometer tracing of the duplicate-stained gel obtained after the second preparative disc-gel electrophoresis. The trehalase activity is designat,ed by the bars, each bar representing the trehalase activity in one slice (&o/E min). Gel slices Were numbered from the cathode end of the gel to the anode end. A .X-/*1 sample containing about 25 Mg of protein was placed on the gel. I
I
11 -
10 OS
.I0 -
A
09 06 07 06 2 OS r
0
1
2
3
4
5
6
7
6
9
10
$1
,2
PH
FIG. 2. Ilofstee plot for the activity of trehalase. The best-fit line was drawn ignoring the deviations at higher substrate concentrations. Assays were carried out on 10-J samples of the purified enzyme (21 units/ml). The initial velocit,y is expressed as .4.&O pl/mln.
the same except for their physical state. Alternatively, one form of the enzyme may be more sensitive to the purification procedure. Since there were no large losses of activity during any one step this possibility is not as likely. Further work must be done to firmly establish whether the same enzyme is present in each of the two physical states.
FIG. 3. Activity of dpis melliferu trehalase as a function of pH. Bssays were conducted on lo-p1 samples of the purified enzyme at each of the pH values indicated and are expressed as ,4&E ~1/15 min.
Determination of Sucrase Decreasing amounts of sucrase were found after each succeeding step of the purification and no sucrase activity could be detected after the second preparative electrophoresis. The removal of this closely related activity is indicative of the high degree of purity achieved (7). Enxynatic
Properties
Figure 2 is the plot used to determine the K, . There is a tendency for the line to curve
518
LEFEBVRE TABLE
ANI)
IV
OF V.\morrs COMPOUNDS ON TREH~L~ISE ACTIVITY (Bpis nzellifera). COMPOI.NDS WICRI: ASSLY ;Llrx~YIcl~: IN THE ADDED TO THIS
EFFECTS
CONCENTRATIONS
Compound
INDICATIW
added
Percentage of original activity remaining (%I
to the assay mixture
hibitory effects while magnesium ions and sodium borate had no effect on the enzymatic activity. AMP and ATP assayed in the presence of K, concentrations of the substrate had no effect on the activity, suggesting that AMP and ATP are not allosteric effecters of the purified enzyme. Stability
10 33 74
Tris 0.025 M Sucrose 0.00% M Ethylenediamine tetraacetic acid 0.002 M Mercaptoethanol 0.001 3~ MgClz lO-5 M Sodium borate 0.005 M AMP (10-3 M trehalose) 0.003 11 ATP (lOA M trehalose) 0.003 M
HUBER
The enzyme lost activity rapidly at temperatures exceeding 40” and at pH values less than 4.0. High concentrations of urea caused a noticeable drop in activity of the enzyme (Fig. 4). A preliminary disc-gel experiment showed that the urea-treated enzyme did not migrate differently than the untreated enzyme and there was no evidence to suggest breakdown of the enzyme to subunits.
82 100 105 100 100
Estimation of Molecular Weigll t b!y Gel Filtration The molecular weight was llO,OOO-120,000. Since the were globular, this molecular upon the assumption that globular protein.
estimated standards weight is trehalase
to be used based is a
ACKNOWLETIGMENTS I
I
I 2M
I
I 4M
I
CONCENTRATION
I
I
6M
I 8M
OF
I
I
I
10M
UREA
FIG. 4. Effect of urea concentration on t.rehalase stability. The purified enzyme was incubated for 30 min at each of t.he cotmentrations of urea indicated and then was diluted for assay. Results are expressed as i14?o/50 ~I/15 min.
downward at high [Xl suggesting substrate inhibition. When these points were ignored and the line of best fit drawn the K, w-as found to be 1.6 mN. The pH optimum of the purified honeybee enzyme was about 6.5 (Fig. 3). The optimum temperature for a 15-min assay was 55”. Table IV compares the activity of the enzyme in the presence of various added constituents. Tris and sucrose strongly inhibited the enzyme. Ethylenedia,mine tetraacetic acid and mercaptoethanol had slight in-
This investigation National Research .4-4691.
1. 2.
was supported in part b,v Council of Canada Grant
M. A.> C:UILLOUX, E., .\XD Coou’rors, .J. E., C. R. H. r~cad. Sci. 267, 1779 (1968). (:L.NXOT:, K. T., .\sn G.\YLOR, K. IL., Plan/n 86, 299 (1969).
AIXIL.\,
4. WY.\TT, G. It., ;lttvun. Inse~f Physiol. 4, 287 (1967). 5. hIURPHy, J. B., .,R;u KIM, &I. W., Biwhiw. Bi0ph~y.s. ilcla 46, 382 (1960) 6. ORNS.ITIN, L., .LND lhm, B. I,., Awn. S. 3’. .lcatl. Sri. 121, 321 (1964). 7. M~KZLVF. (;. A., .Jrch. Biorhetu. Biophys. 134, 8 (1969). 8. l>omm, J. E., AND I~ICGS, 1). s., J. Viol. Chew. 240, 8% (1905)
Purification,
and
Honeybee
some (Apis
T. A. LEE’EBVIIE
cvr)
Properties
of Trehalase
from
mellifera) K. E:. HUBEIt
The insoluble fraction of trehalase (EC S.2.1.28) from holqybre (Apis j,lellijeru) was solubilixed by raising the pH of the enzyme suspension to 10.0. The soluble ellzpme in the suspension was not deactivated by this procedure and the solution rontaitling both the solubilized trehalase and the soluble trehalase was then purified at least BHO-fold. Only one batid was observed on disc gel a11d the trehalase activity migrated with this band. No contaminating srlcrase act.ivity was folmd in the purified preparation. The molecldar weight of the trehalase as estimated by Sephadex chromatography was llO,OOO-120,000. The purified enzyme had a K,,, of 1.6 mu and a pH optimum of 6..i. Tris and stlcrose strongly inhibited the enzyme; maguesirim and ethylenediamine tetraacetic acid had slight inhibitory effects, while sodimn borate a11t1 mercaptoet hanol had no effects. ATP and AMP did not alter the enzymatic hydrolysis rate. Enxymat ic activity was rapidly lost at temperatures greater t hall 40’. at pH vallies less t hall 4.0, and ill high ccjllcentratiolls of IIre:,.
The presence of the enzyme, trehulast (EC 321.28; a,a’-glucoside-l-glucohydrolase), which hydrolyzes a ,a-trehalose to 2 molecules of glucose has been reported in microorganisms, in plants, and in animals (1-3). However, its occurrence is most widespread in insects (4). The enzyme has been purified to various degrees but, as yet, the preparation of a homogeneous form has not been demonstrated, possibly because large quantities of most insects are difficult to obtain and also because a large fraction of tile trehnlase in insects is naturally insoluble. The present study entailed the solubilization of the insoluble portion of the trehalase of tile honeybee (Apis mellijwa) snd the purification of this fraction together with the soluble fraction for further studies of the properties of trelinlase in 3 l~omogenfwus state. RIAT~:I:TAT,S
ANI)
METIIOI)S
Adrdt honeybees were obtained it1 large qllarltit.ies from it loral tiotley farm. C:yatlidc gas was
IIsed to kill bees. with 110 loss of the level of trrhalasc activity. Immediately after death the bees were sllspended ilr 0.1 M sodirlm citrate bufer ($1 5.6). Honeybees stored it1 this brtffer at - 15’ for it period of at did not lose trehalasc activity 1f:ast (i months.
Aliqllots of etqme were added to at, assay mixture (fillal volume 2 ml) which cotltained 0.03 hr sodium citrate brlffrr (pH 6.5) and 0.005 51 LY,CYtrehalose dihydratc (Sigma Chemical Company). ,4ppropriate blanks :ttld glucose standards were used. Assay mixtlues were illcrlbated at 30” for 15 mitt alld the reactiotl was stopped by placing the solutions illto :I boilitlg water bath. 111order to bluffer ihesoll,tiotl at pll 7.0, 2 ml of aO.l M sodium potassium phosphate solritioll (pII 10.5) were‘ added. Two milliliters of (;lrlcostat I!eagrnt (Worthingtolr Biochemical Company) ill 0.1 M phosphate buffer (pII 7.0) were added to 2 ml of the solution and the mixtlvr was incubated at 30” for 10 mill. This reactiolr was stopped by the, addition of 3 drops of 3 x TICI atld the absorbance at 420 nm (.442(I) was measured. One Itnit was defitled as the arnol,llt of enzymr’ which catalyzed the hydrolysis of 1 rmole of trehalose per mill at 30” a11d pII Ci.5.
PURIFICATION
OF HONEYBEE
&muse Activity
Xolubilixation Trehalase in honeybees was found to consist of approximat,ely 50y0 soluble arId 50yC insoluble enzyme. Several techniques were used on suspensions of the insoluble fraction in an attempt to solubilize it.
It. has been reported (7) that sucrase activit,y is very dificult to separate from t.rehalase activit.y. The sucrase activity in enzyme samples was determined by adding 0.03 1%sucrose to the assay mixture instead of the trehalose (7).
lhzymatic One liter of bees in suspension (286 g of whole bees) was homogenized for 15 set it1 a Waring Blendor. The homogenate was filtered through cheeseclot,h and brought to G5y0 satklration with ammonium sulfate. The pellet after centrifugatioil at 10,000 g for 1 hr was suspended in 0.1 M sodillm citrate buffer (p1E 56) and dialyzed against large volumes of t,his bntl’er. Sodium hydroxide solution (3 N) was then added to the enzyme preparation to bring the pT1 to 10.0. Aft,er stirring for 2 hr the pII 10.0 mixture was centrifuged at 10,OOOgfor 1 hr alld the srlpernatstnt fluid was extensively dialyzed against 0.005 M sodium borate briffer (pH 8.0). All furt.her purificatioti was carried out in this buffer. The dialyzed supernatant fraction was applied to a 5 X 100.cm l>l~:hli:-celhilose (Whatman 1)Ell) colrmm aud eluted with 2 liters of a O-47; linear sodirim chloride gradient. Thr most active fractions were pooled, dialyzed, and then concentrat,ed by lpophilization. The enzyme solution was then applied t.o a 5 X loo-cm Sephades (;-ZOO column and eluted with 2 liters of the borate buffer. rlctive fractions were pooled, concclutrated, and reapplied to a 2.5 X 45.cm I)EAE:cxellr1lose col~imn. ,4ctive fractions elrkted using 2 liters of a O-37& linear sodium chloride gradient were conceutrated with Aquacide II (Calbiochcmj. Flvt.her fractionation of t.he enzyme solution was achieved by two passes through a preparative disc-gel electrophoresis apparatus (Buchler “1’01,~ Prep”) using a 3.0-cm column of 7.5’; acrylamide gel for the first pass and a 6.0.cm colurn~l for the sccolld. Aquacidc II was used to concentrate the ellzgme. Protein collcelltrat.ioils wrre estimated by the ~(215-225) method (5).
Anal&al
Disc-Gel Electrophoresis
Acrylamide disc-gel (7.57,) electrophoresis (6) was performed in duplicate at 2’ in a Tris-glycille brlffer system with a rmming pII of 0.3. One was st.ained wit,h amido schwarz sollltiotl ar~l t,hen washed with 77” acetic acid to remove the excess stain. (+el patterns were traced with a Dellsicord 5+2 (Photovolt, Corporation) densitometer. The other gel of the pair was sliced into $ie-in. slices and each of t,he slices was separately assayed. The assay results of the slices were sllperimposed upon the densi tometer t ratings.
513
TREIIALASIC
Properties
The K,, was det.ermirled by methods recommended by Dowd and Riggs (8). Opt,imum pH studies and the effect of temperature on the trehalase reaction rates were observed. The effects of various added constituents were also studied. The stability of the enzyme at various temperatures, pH values, and urea rolicelLtrations was determilled.
Estimation of Size The molerular wpight of the purified enzyme was estimated with a previously calibrated column (Sephadex K25/100) of Sephadrx (j-150 using Pharmac.ia stalldards. RESULTS
A1?;1) 1>1SCUYS10N
Solubilizafio?2, Activation, and Stability Table achieved
I compares the solubilizations by the different methods. The greatest solubilizstion WLSnoted when 0.5 % sodium dodecgl sulfate ~-as added to the suspension either at pH (i..T or at pH 10.0. TABLk:
I
No treatment, Octanol (.jfc) Freezing and thawing (.iX) Sodium dodecyl sulfate (0.3C;) Adjustment to pII 8.0 (3 N NaOII) Adjustment, to pH 9.0 (3 N KaOH) Adjustment to pH 10.0 (:S N SaOIL) Adjustment to pII 10.0 (3 N NaOH) in the presence of sodium dodecyl sulfate (O..ii;)
natant (%i
Super-
Precipitate m
2 1:; 2 96 50
08 87 98 4 .io
8.;
1-j
94
6
96
4
516
LEFEBVRE
ANI)
The pH 10.0 treatment without addition of detergent was almost as effective. Table 11 shows the percentage of original activity observed 2 hr and 24 hr after treatment by the different solubilization techniques. Raising the pH to 8.0, to 9.0, or to 10.0, or adding 0.5 % sodium dodecyl sulfate resulted in marked activation after 2 hr. However, after 24 hr the enzyme treated with detergent had lost more activity than the enzyme at pH 9.0 or 10.0. Also detergents are known to be very difhcult to remove from protein molecules. Thus, it was decided to solubilize the enzyme by raising the pH to 10.0 followed by extensive dialysis against 0.005 M sodium borate buffer (pH 8.0) to prevent the inactivation which results after a prolonged spell at pH 10.0. TABLE
II
ACTIVITY
OF SUSPENSIONS OF INS~LCBLE TREHALASIC 2 hr lack 24 hr AFTER V.\RIOUS PHYSIc.11, .IR‘D CHEMICAL TIILITMENTS. THE TOTAL SUSPENSION ACTIVITY OF THE UNTRL~TIXI AFTER 2 hr Was I)IGSIGNATI~W AS 100C;A No. hr
2 (5%) 24(%) h’o treatment
100
Octanol (:jCy ) Freezing and thawing (5X) Sodium dodecyl sulfate (0.5%) Adjustment to pH 8.0 (3 N N&H) Adjustment to pH 9.0 (3 N NaOll) Adjustment to pH 10.0 (3 N NaOll) Adjustment to pH 10.0 (3 pi NaQII) in the presence of sodium dodecyl sldfate (0.3%)
107 87 184 201
105 118 113 156 201
220
200
201
177
201
160
PURIFICATION
Purification step 1 2 3 4 5 6 7
65rj, (NH4)?SQh precipitation pH 10.0 supernatant First DEAE-cellulose Sephadex G-200 Second DEAE-cellulose First preparative disc gel Second preparative disc gel
HUBER
Purification Activities and protein concentrations determined at each step of the isolation of the enzyme are shown in Table III. The activit) of the enzyme in the original crude bee cxtract could not be determined because of the high natural content of glucose in the bee which interferes with the assay. The enzynle eluted from the DEAE columns at N&l concentrations between 1 and 2%. It eltited through the Sephadex G-200 column after about 800 ml of eluent had passed. A final yield of over 1 mg of purified enzyme having a specific activity of approximately 76 units/ mg was obtained. This represented a fold purification of at least 680. Homogeneity of the Protein by Analytical Disc-Gel Electrophoresis Analytical disc-gel electrophoresis performed after the enzyme solution had been eluted from the second DEAE-cellulose column indicated the presence of several protein bands which could not be clearly resolved because of their proximity to each other. Only one of these was active although the active band could not be specified. After the first preparative electrophoresis two protein bands of similar mobilities were observed. Only one was active. Figure 1 is a densitometer tracing of the analytical disc gel obtained with enzyme from the second preparative electrophoresis pass. Only one band was observed and the trehalase activity migrated with this band. Although the soluble and insoluble forms of honeybee trehalase were purified together, the fact that only one active band resulted suggests that the soluble and insoluble enzyme forms are
TABLE III OF IIOXEYBEE
TAEH.IL.ISI.:
Solution (ml)
Total units
Total protein (w)
Specific activity
Yield (%)
Purification factor -__.
380 330 210 73 130 10 ;j
640 901 753 532 363 248 104
5714 3726 987 139 .x 5.17 1.36
0.112 0.242 0 763 3.827 6.640 47.99 76.47
100 141 118 x3 57 :!I9 16
1.0 2.2 6.8 34.2 .i9 439 683
517 80 -
-16
70
- 14
-
z - 1.2
= 60z IfJl 50k c z 5 5
-10
4030-
-
20-
z L -
-06
: Q
-06
d 5
.. 04
E
? 10 -,,‘I 0
2
4
6
I 6
10
12
14
16
GEL
SLICE
20
16
22
24
26
26
30
32
34
NUMBER
FIG. 1. Trehalase activit,y of slices of unstained gel superimposed upon a densitometer tracing of the duplicate-stained gel obtained after the second preparative disc-gel electrophoresis. The trehalase activity is designat,ed by the bars, each bar representing the trehalase activity in one slice (&o/E min). Gel slices Were numbered from the cathode end of the gel to the anode end. A .X-/*1 sample containing about 25 Mg of protein was placed on the gel. I
I
11 -
10 OS
.I0 -
A
09 06 07 06 2 OS r
0
1
2
3
4
5
6
7
6
9
10
$1
,2
PH
FIG. 2. Ilofstee plot for the activity of trehalase. The best-fit line was drawn ignoring the deviations at higher substrate concentrations. Assays were carried out on 10-J samples of the purified enzyme (21 units/ml). The initial velocit,y is expressed as .4.&O pl/mln.
the same except for their physical state. Alternatively, one form of the enzyme may be more sensitive to the purification procedure. Since there were no large losses of activity during any one step this possibility is not as likely. Further work must be done to firmly establish whether the same enzyme is present in each of the two physical states.
FIG. 3. Activity of dpis melliferu trehalase as a function of pH. Bssays were conducted on lo-p1 samples of the purified enzyme at each of the pH values indicated and are expressed as ,4&E ~1/15 min.
Determination of Sucrase Decreasing amounts of sucrase were found after each succeeding step of the purification and no sucrase activity could be detected after the second preparative electrophoresis. The removal of this closely related activity is indicative of the high degree of purity achieved (7). Enxynatic
Properties
Figure 2 is the plot used to determine the K, . There is a tendency for the line to curve
518
LEFEBVRE TABLE
ANI)
IV
OF V.\morrs COMPOUNDS ON TREH~L~ISE ACTIVITY (Bpis nzellifera). COMPOI.NDS WICRI: ASSLY ;Llrx~YIcl~: IN THE ADDED TO THIS
EFFECTS
CONCENTRATIONS
Compound
INDICATIW
added
Percentage of original activity remaining (%I
to the assay mixture
hibitory effects while magnesium ions and sodium borate had no effect on the enzymatic activity. AMP and ATP assayed in the presence of K, concentrations of the substrate had no effect on the activity, suggesting that AMP and ATP are not allosteric effecters of the purified enzyme. Stability
10 33 74
Tris 0.025 M Sucrose 0.00% M Ethylenediamine tetraacetic acid 0.002 M Mercaptoethanol 0.001 3~ MgClz lO-5 M Sodium borate 0.005 M AMP (10-3 M trehalose) 0.003 11 ATP (lOA M trehalose) 0.003 M
HUBER
The enzyme lost activity rapidly at temperatures exceeding 40” and at pH values less than 4.0. High concentrations of urea caused a noticeable drop in activity of the enzyme (Fig. 4). A preliminary disc-gel experiment showed that the urea-treated enzyme did not migrate differently than the untreated enzyme and there was no evidence to suggest breakdown of the enzyme to subunits.
82 100 105 100 100
Estimation of Molecular Weigll t b!y Gel Filtration The molecular weight was llO,OOO-120,000. Since the were globular, this molecular upon the assumption that globular protein.
estimated standards weight is trehalase
to be used based is a
ACKNOWLETIGMENTS I
I
I 2M
I
I 4M
I
CONCENTRATION
I
I
6M
I 8M
OF
I
I
I
10M
UREA
FIG. 4. Effect of urea concentration on t.rehalase stability. The purified enzyme was incubated for 30 min at each of t.he cotmentrations of urea indicated and then was diluted for assay. Results are expressed as i14?o/50 ~I/15 min.
downward at high [Xl suggesting substrate inhibition. When these points were ignored and the line of best fit drawn the K, w-as found to be 1.6 mN. The pH optimum of the purified honeybee enzyme was about 6.5 (Fig. 3). The optimum temperature for a 15-min assay was 55”. Table IV compares the activity of the enzyme in the presence of various added constituents. Tris and sucrose strongly inhibited the enzyme. Ethylenedia,mine tetraacetic acid and mercaptoethanol had slight in-
This investigation National Research .4-4691.
1. 2.
was supported in part b,v Council of Canada Grant
M. A.> C:UILLOUX, E., .\XD Coou’rors, .J. E., C. R. H. r~cad. Sci. 267, 1779 (1968). (:L.NXOT:, K. T., .\sn G.\YLOR, K. IL., Plan/n 86, 299 (1969).
AIXIL.\,
4. WY.\TT, G. It., ;lttvun. Inse~f Physiol. 4, 287 (1967). 5. hIURPHy, J. B., .,R;u KIM, &I. W., Biwhiw. Bi0ph~y.s. ilcla 46, 382 (1960) 6. ORNS.ITIN, L., .LND lhm, B. I,., Awn. S. 3’. .lcatl. Sri. 121, 321 (1964). 7. M~KZLVF. (;. A., .Jrch. Biorhetu. Biophys. 134, 8 (1969). 8. l>omm, J. E., AND I~ICGS, 1). s., J. Viol. Chew. 240, 8% (1905)