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Purification of α-amylase from C3H mouse submaxillary gland by gel filtration
NOTES
547
&ROM. 4637 Purification
of ac-amylase from
C,W
mouse submaxillary
gland
by gel flltration
In previous studies on the marker-enzymes of the mouse submaxillary glandf, these were compared to those of a spontaneous tumor derived from the submaxillary gland of a female mouse of the CaH strain2, and particular attention was given to proteolytic enzymes and amylase, which are partially maintained in the tumor tissue. It seemed interesting to purify these enzymes, in order to achieve a complete, comparative analysis of their chemico-physical and kinetic properties. The present paper reports a simple procedure for the isolation of amylase from -the submaxillary gland, The method is partially based on the fact that amylsse is retarded when eluted through dextran gels, such as Sephadex. This property was already described by FLODIN~ and by GELOTTE~ for pancreatic or-amylase and by AURICCWIO~~~ for an acid a-glucosidase from rat liver and human kidney. Materials and methods Adult male mice of the C,H strain were sacrificed by cervical dislocation and the submaxillary glands promptly excised, freed of any connective tissue and frozen. Pools of IOO glands were homogenized in a Waring Blendor with 3 volumes of cold 5 mM Tris-HCl buffer, pH 7.4, and subsequently centrifuged at 15ooo x g for 20 min in a refrigerated centrifuge. The supernatant was recentrifuged at IOOOOO x g for go min in a Spinco Model Lz-50 centrifuge, The sediment was discarded and protein concentration in the supernatant was estimated by the method of LOWRY~,using bovine albumin as standard, while total amylase activity was determined according to the method of STREET AND CLOSE’.
Sephadex
G-100
cm \75r 94 Flow rate =ml 21th
Fig. I. Chromatography dcx G-100.
of a. crude extract from male C,H mouse submaxillary
J.
glands on Sepha-
Ckromalog., 48 (1970) 547-550
NOTES
548
Two ml of The supernatant (protein concentration: 75 mg/ml) were filtered through a Sephaclex G-100 (Pharmacia, Uppsala, Lot. No. 9262) chromatographic column;,(I.75 ..x”g4’crn); equilibrated,and eluted‘ with ~‘mM.Tris-WC1 buffer; pH 7:4; Elution flow was kept constant by a peristaltic pump and all the operating procedures were made in. a cold room.. Bio-g&l cm
Flow
-I
P 100
1.75x96 rate= ml 24/h
! I
i 2
0.7 -
I_
Ii
O.l-,
I
I1
1 50
I
.I
Fig. 2. Rechrotiatography
4 5 11
1
I1
1
100
on Bio-Gel
I?-100
I. 150
I
r :
11 1 I, 200 ml &fluent volume
of amylase’pooled
$
fractions eluted from SephacJex
Fig. ,3. Disc eleotrophoretic analysis of purified amylase (right), of male C,H mouse submaxillary gland extract (middle), and of C&H mouso serum (left). IJ. Chvomatog.,
48 (1970) 547-550
NOTES
549
The proteins were estimated directly on the column effluent by means of a LKR Uvicord at a wavelength of 254 rnp. Amylase activity was determined on the effluent fractions. Fractions containing amylase activity were pooled, dialysed overnight against cold distilled water, lyophilised, redissolved in a minimal volume of buffer and filtered through a Bio-Gel P-100 (Eio Rad, Richmond, Calif. Lot No. 2880) chromatographic column 1.75 x 94 cm), equilibrated and eluted as before. Fractions containing amylase activity were pooled, dialysed overnight against cold distilled water, lyophilised and used for disc electrophoresis and ultracentrifuge analyses.
Fig. 4. Ultracentrifuge pattern of purified enzyme preparation. Protein concentration : IO mg/ml; picture taken 4560 see after reaching the full speed of 59 780 r.p.m.
Results When the crude extract of submaxillary gland is filtered through a Sephadex G-100 column the amylase activity is eluted with the fourth protein peak, corresponding to a molecular weight of about 16000, as calculated according to the method of AMBREWS” (Fig. I). When the fractions containing amylase are refiltered through Bio Gel P-100, which is a molecular sieve very similar to Sephadex G-100, amylase activity is eluted earlier in a distinct and symmetrical peak, corresponding to a molecular weight of about 60 000 (Fig. 2.). TABLE
I
PURIFICATION
OF
MOUSE
SURMAXILLARY
GLAND
AMYLASE
-. ml
Protein mglml
Total mng firotein
Specific activity*
Total activityt’
Yield yO
I so0
100
-
Submaxillary gland homogenate 2 75.0 150 I2 G-roo Amylase pool 42 0.42 17.6 92 P- IOO Amylase pool 20 .0.06 1.2 1300 -a Specific activity = mg starch hydrolyscd/h/mg sample at 37’. b Total activity = smylase activity x total mg sample.
1620 1560
90 87
$1.Clbronralog., 48 (x970) 547-550
NOTES
550
Electrophoretic anaJ.ysisof this peak on polyacrylamide gel shows a single band corresponding to the al proteins; the same band is also present in the serum of C3H strain mice and in the crude extract of the’submaxillary glands (Fig. 3). As observed in Fig. 4 a single symmetrical peak is also detectable in the ultracentrifuge pattern, with a sedimentation coefficient of S:&= 2.24 x 10-18. Amylase was purified about LOOfold with a final yield of about go y0 of the starting activity (Table I) by the method described. It is noteworthy that when the Sephadex chromatography is performed after the Bio-Gel chromatography the recovery of the amylase activity is less. This phenomenon is probably due to the presence of a relevant amount of proteolytic enzymes, which are eluted together with amylase. The authors thank Dr. VJNCENZOM&co, Director of the Animal Genetics Laboratory, Radiobiology Department, CNRV Casaccia, Roma, for the kind supply of selected inbred mice. >,’ ,.
lnstitztte of Gemral Pathology, University & Pevugia, and Regina Elena Institute for Cancer Aese&ch, ,' Rome (Italy)
L. FRATI
A. CAFUTO
:;:. I
2 3 4 3 6 7 8 g
M. L. SALVI,~?. U. ANGELETTI AND L.FRATI,F&zw~ L. FRATI AND A. CAPUTO,BOZZ.SOC. Ital. Biol:Spev., P. F. F. 0. N. P. B.
(Pavia) Sci. Ed., 20 (1965) 12. 44 (1968) fast. 20, 21. FLODIN, Dsxtvan Gels- and theiv Applicationd’in,Gel FiZtvation, Pharmacia, Uppsala, 1962. AURICCHIO AND C.B. BRUNI, Biocltem.J., 105 ‘(1g67).35. AURICCIIIO, C. B. BRUWI AND V. SICA, Biocirem.J., 108 (1968) 161: PI.Lowizy. N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL,J. BioE. Cirem., 193 (igsr) 265. V. STREET AND J_ R. CLOSE, CZiur. Chim. Acta, I (1936) 404. ANDREWS,B~~~I~~WZ.J.,~I (1964) 222. GELOTTE, ActaChem. &and., 18 (1964) 1283.
Received January zgth, 1970 J.
Ciwomatog.,
48 (1970)
547-550
547
&ROM. 4637 Purification
of ac-amylase from
C,W
mouse submaxillary
gland
by gel flltration
In previous studies on the marker-enzymes of the mouse submaxillary glandf, these were compared to those of a spontaneous tumor derived from the submaxillary gland of a female mouse of the CaH strain2, and particular attention was given to proteolytic enzymes and amylase, which are partially maintained in the tumor tissue. It seemed interesting to purify these enzymes, in order to achieve a complete, comparative analysis of their chemico-physical and kinetic properties. The present paper reports a simple procedure for the isolation of amylase from -the submaxillary gland, The method is partially based on the fact that amylsse is retarded when eluted through dextran gels, such as Sephadex. This property was already described by FLODIN~ and by GELOTTE~ for pancreatic or-amylase and by AURICCWIO~~~ for an acid a-glucosidase from rat liver and human kidney. Materials and methods Adult male mice of the C,H strain were sacrificed by cervical dislocation and the submaxillary glands promptly excised, freed of any connective tissue and frozen. Pools of IOO glands were homogenized in a Waring Blendor with 3 volumes of cold 5 mM Tris-HCl buffer, pH 7.4, and subsequently centrifuged at 15ooo x g for 20 min in a refrigerated centrifuge. The supernatant was recentrifuged at IOOOOO x g for go min in a Spinco Model Lz-50 centrifuge, The sediment was discarded and protein concentration in the supernatant was estimated by the method of LOWRY~,using bovine albumin as standard, while total amylase activity was determined according to the method of STREET AND CLOSE’.
Sephadex
G-100
cm \75r 94 Flow rate =ml 21th
Fig. I. Chromatography dcx G-100.
of a. crude extract from male C,H mouse submaxillary
J.
glands on Sepha-
Ckromalog., 48 (1970) 547-550
NOTES
548
Two ml of The supernatant (protein concentration: 75 mg/ml) were filtered through a Sephaclex G-100 (Pharmacia, Uppsala, Lot. No. 9262) chromatographic column;,(I.75 ..x”g4’crn); equilibrated,and eluted‘ with ~‘mM.Tris-WC1 buffer; pH 7:4; Elution flow was kept constant by a peristaltic pump and all the operating procedures were made in. a cold room.. Bio-g&l cm
Flow
-I
P 100
1.75x96 rate= ml 24/h
! I
i 2
0.7 -
I_
Ii
O.l-,
I
I1
1 50
I
.I
Fig. 2. Rechrotiatography
4 5 11
1
I1
1
100
on Bio-Gel
I?-100
I. 150
I
r :
11 1 I, 200 ml &fluent volume
of amylase’pooled
$
fractions eluted from SephacJex
Fig. ,3. Disc eleotrophoretic analysis of purified amylase (right), of male C,H mouse submaxillary gland extract (middle), and of C&H mouso serum (left). IJ. Chvomatog.,
48 (1970) 547-550
NOTES
549
The proteins were estimated directly on the column effluent by means of a LKR Uvicord at a wavelength of 254 rnp. Amylase activity was determined on the effluent fractions. Fractions containing amylase activity were pooled, dialysed overnight against cold distilled water, lyophilised, redissolved in a minimal volume of buffer and filtered through a Bio-Gel P-100 (Eio Rad, Richmond, Calif. Lot No. 2880) chromatographic column 1.75 x 94 cm), equilibrated and eluted as before. Fractions containing amylase activity were pooled, dialysed overnight against cold distilled water, lyophilised and used for disc electrophoresis and ultracentrifuge analyses.
Fig. 4. Ultracentrifuge pattern of purified enzyme preparation. Protein concentration : IO mg/ml; picture taken 4560 see after reaching the full speed of 59 780 r.p.m.
Results When the crude extract of submaxillary gland is filtered through a Sephadex G-100 column the amylase activity is eluted with the fourth protein peak, corresponding to a molecular weight of about 16000, as calculated according to the method of AMBREWS” (Fig. I). When the fractions containing amylase are refiltered through Bio Gel P-100, which is a molecular sieve very similar to Sephadex G-100, amylase activity is eluted earlier in a distinct and symmetrical peak, corresponding to a molecular weight of about 60 000 (Fig. 2.). TABLE
I
PURIFICATION
OF
MOUSE
SURMAXILLARY
GLAND
AMYLASE
-. ml
Protein mglml
Total mng firotein
Specific activity*
Total activityt’
Yield yO
I so0
100
-
Submaxillary gland homogenate 2 75.0 150 I2 G-roo Amylase pool 42 0.42 17.6 92 P- IOO Amylase pool 20 .0.06 1.2 1300 -a Specific activity = mg starch hydrolyscd/h/mg sample at 37’. b Total activity = smylase activity x total mg sample.
1620 1560
90 87
$1.Clbronralog., 48 (x970) 547-550
NOTES
550
Electrophoretic anaJ.ysisof this peak on polyacrylamide gel shows a single band corresponding to the al proteins; the same band is also present in the serum of C3H strain mice and in the crude extract of the’submaxillary glands (Fig. 3). As observed in Fig. 4 a single symmetrical peak is also detectable in the ultracentrifuge pattern, with a sedimentation coefficient of S:&= 2.24 x 10-18. Amylase was purified about LOOfold with a final yield of about go y0 of the starting activity (Table I) by the method described. It is noteworthy that when the Sephadex chromatography is performed after the Bio-Gel chromatography the recovery of the amylase activity is less. This phenomenon is probably due to the presence of a relevant amount of proteolytic enzymes, which are eluted together with amylase. The authors thank Dr. VJNCENZOM&co, Director of the Animal Genetics Laboratory, Radiobiology Department, CNRV Casaccia, Roma, for the kind supply of selected inbred mice. >,’ ,.
lnstitztte of Gemral Pathology, University & Pevugia, and Regina Elena Institute for Cancer Aese&ch, ,' Rome (Italy)
L. FRATI
A. CAFUTO
:;:. I
2 3 4 3 6 7 8 g
M. L. SALVI,~?. U. ANGELETTI AND L.FRATI,F&zw~ L. FRATI AND A. CAPUTO,BOZZ.SOC. Ital. Biol:Spev., P. F. F. 0. N. P. B.
(Pavia) Sci. Ed., 20 (1965) 12. 44 (1968) fast. 20, 21. FLODIN, Dsxtvan Gels- and theiv Applicationd’in,Gel FiZtvation, Pharmacia, Uppsala, 1962. AURICCHIO AND C.B. BRUNI, Biocltem.J., 105 ‘(1g67).35. AURICCIIIO, C. B. BRUWI AND V. SICA, Biocirem.J., 108 (1968) 161: PI.Lowizy. N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL,J. BioE. Cirem., 193 (igsr) 265. V. STREET AND J_ R. CLOSE, CZiur. Chim. Acta, I (1936) 404. ANDREWS,B~~~I~~WZ.J.,~I (1964) 222. GELOTTE, ActaChem. &and., 18 (1964) 1283.
Received January zgth, 1970 J.
Ciwomatog.,
48 (1970)
547-550