Structure and function of amylases

ARCHIVES

OF

BIOCHEMISTRY

AND

BIOPHYSICS

Structure I. The Subunit JOHN

F. ROBYT,

Department

and

Structure

CAROL

of Biochemistry Received

144, 160-167

(1971)

Function

of Amylases

of Porcine

Pancreatic

G. CHITTENDEN, and Biophysics,

December

Iowa

8, 1970;

AND

State

accepted

cu-Amylase’ CATHERINE

University, February

Ames,

T. LEE Iowa

50010

1, 1971

The isozymes of porcine pancreatic or-amylase were reduced with dithiothreitol to two enzymatically active subunits which had molecular weights of 25,000 daltons each. These subunits could be isolated and separated from each other by chromatography on DEAE-cellulose. Subsequent treatment of the subunits with EDTA, DTT, and iodoacetamide gave derivatives that emerged in identical elution volumes from DEAE-cellulose columns and that migrated very rapidly and identically on disc-gel electrophoresis. These latter experiments suggested that the subunits might have similar primary structures. This hypothesis was tested by preparing tryptic peptide maps of the subunits. The results indicated that the subunits had very similar, if not identical, primary structures. It was concluded that the porcine pancreatic a-amylase molecule is composed of two very similar peptide subunits held together by intermolecular disulfide linkages and that the differences between the subunits might be attributable to differences in their tertiary structures.

Until recently porcine pancreatic (Yamylase (PPA)2 was thought to be a homogeneous protein of about 50,000 daltons (1). Amino acid composition analyses gave a minium molecular weight of 45,000 (a), and sedimentation studies indicated a single protein (3). However, investigators in two laboratories (4,5) independently isolated two isozymes by DEAE-cellulose chromatography and gel electrophoresis. Equilibrium sedimentation gave identical molecular weights of 52,600 =t 1,200 for each isozyme (6).

An indication that PPA may be composed of subunits was provided by the observation of Levitzki et al. (7) that the enzyme formed multimolecular complexes with glycogen. The formation of these complexes was dependent on the relative ratios of enzyme to glycogen. The precipitated complex could be dissolved by adding either excessenzyme or excess glycogen. These experiments suggested that the format’ion of the complex was analogous to the antibody-antigen reaction. If the enzyme was associating with the glycogen exclusively at the active site, a basic principle of polymer chemistry would demand that the enzyme have at least two active sites per molecule. Using equilibrium dialysis with maltotriose, Loyter and Schramm (8) strengthened this hypothesis by determining that PPA had two maltotriose binding sites per 50,000 daltons. In the present study, we obtained active subunits of PPA by treating the enzyme with an efficient reducing agent, dithiothreitol, at pH 8.5. We were able to separat,e

r Journal Paper No. J-6786 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Projects 1116 and 1485. Supported by grants from the Corn Industries Research Foundation, the National Institutes of Health (GM-08822), and the Agricultural Research Service, U.S. Dept. of Agriculture 12-14100-9887 (71). 2 Abbreviations used: PPA, porcine pancreatic a-amylase; DEAE, diethylaminoethyl; EDTA, ethylenediaminetetraacetic acid; DTT, dithiothreitol; SDS, sodium dodecylsulfate. 160

SUBUNIT

STRUCTURE

OF

PORCINE

three subunit,s on DEAE-cellulose; two had molecular weights of 25,000 and the third had a molecular weight of 12,500. Additional treatment of t’he 23,000-dalton subunits with EDTA, DTT, and iodoacetamide gave derivatives that emerged in identical positions from DEAEPcellulose columns and gave identical mobilities on disc-gel electrophoresis. Pept,ide maps of the subunits demonstrated that they have similar primary struct,ures and that they could differ from each other in their tertiary structures. ESPERI1lENTAL

A. Jtaterials The following were purchased from Worthington Biochemical Corporation, Freehold, New Jersey: porcine pancreatic ol-amylase3, 2X cryst,allized; lysozyme, 2X crystallized; bovine TPCKtrypsin, IX crystallized. The following were purchased from Sigma Chemical Co., St. Louis, MO.: cyt.ochrome c, type II; pepsin, 2X crystallized; chymotrypsinogen, 3X crystallized; 2-mercaptoethauol; iodoacetamide; l)l
PANCREATIC

contentj

was analyzed

a-amylase enzyme (1).

is well

knowtl

The

results

are

Separation of subunits. A reducing DEAEcellulose was prepared by equilibrating DEAEcellulose in 10 mM sodirlm glycerophosphate buffer (pH 8.5) containing 50 mM Z-mercaptoethanol. The column was packed and washed with 10 rnM sodium glycerophosphat,e buffer (pII 8.5) until 2-mercaptoethallol co\lld Ilot be detected at 280 Ilrn. Crystalline PPA (5 mg) was dissolved in l-2 ml of 10 m%t sodium glycerophosphate buffer (pH 8.5), that was 10 m%1 in 1)TT. The solution was allowed to stand a minimum of 4 hr at room temperature; it was then applied to the top of a reducing I>ErZE-cellulose column. The protein fractions were eluted with 10 m>r sodium glycerophosphate buffer (pII 8.5); the flow rate was 15 ml/hr. Fractions of 3 ml were collected, and the protein was located by determining the absorbante at 280 nm or by enzyme assay (11).

0.2 A

p1280

30

3 Porcine pancreatic as a calcirnn-metallo

at 280 nm.

show11 in Fig. M.

B. Methods Pomation of subztnils. Crystalline PPA (3 mg) . ^ was dissolved 111 1 ml of 10 my sodium glycerophosphate buffer (pH 8.5). The solution was made 1.5 mM ill l)TT aud allowed to incubate at room temperature for 2 hr. The solution was then passed over a Sephadex C-100 column (1.5 X 60 cm) that previously had been equilibrated wit,h 10 rnM sodium glycerophosphate buffer (pH 7.0) ; 3-ml fractions were collected, and the protein

161

a-AMYLASE

40

50 Elution

I

60

I

70

Volume

I

SO

90

I

100

8

110

(ml)

1. A. Gel filtration ou Sephadex G-100 of porcine pancreatic ol-amylase treated with 1.5 mM dithiothreit,ol at pH 8.5. B. Molecular weight determination of t,he two forms on Sephadex G-100 (pH 7). The molecular weights of the standards are those given in the references: B. sub. A, Bacillus szlbtilis a-amylase dimer (1); BSA, bovine serum albumin (14); Pepsin (13); Chym., chymotrypsinogen (13); Lys., lysozyme (13); I, fraction I of Fig. 1A and II, fraction II of Fig. 1A. FIG.

162

ROBYT,

CHITTENDEN,

Molecular weight determinations. The molecular weights of the fractions were estimated by gel filtration on Sephadex G-100 and Bio-Gel P-150 by using proteins of known molecular weights as standards (12). The molecular weights were also determined by using SDS-polyacrylamide gel electrophoresis (13, 14). Sephadex G-100 and Bio-Gel P-150 were swollen and washed in 10 mM sodium glycerophosphate buffer (pH 7.0) and packed to give 1.5 X 60-cm column beds. Protein standards were selected to encompass the range of molecular weights expect,ed for the unknowns. Each standard was applied separately to the same column and det,ected by absorbance at 280 nm. Low concentrations of active amylases also were detected by a starch-agar plate assay (11). Peak volumes were used in the molecular weight plots (Figs. 1B and 2). Molecular weight determination by SDS-gel electrophoresis was executed as described by Weber and Osborn (13) with the addit,ion of 170 disodium EDTA to the gel and buffer. Electrophoresis was achieved wit,h 10 mA per gel until the dye marker was 1 cm from the bottom of the gel. The proteins were stained by the method of Duncker and Rueckert (14), and the mobilities were calculated by the formula distance

of protein

migration

length

of gel after

staining

length x

. distance

of gel before

of dye migration

staining before

staining

Polyacrylamide gel electrophoresis. The electrophoresis cell was purchased from Canalco Corp. (Bethesda, Md.), and the chemicals were purchased from Eastman Kodak (Rochester, N. Y.). The procedures of Davis (15) were used as modified in the Canalco formulation sheet? of 1965. The gels were cast in tubes (11 X 6 mm) with 2 ml of separating gel and 0.2 ml each of spacer and sample gels. Approximately lo-50 pg of protein were applied for each sample. Fixing and staining were accomplished by the method described by Duncker and Rueckert (14). Mobilities were calculated as for SDS-gel electrophoresis. Derivatization with iodoacetamide. Iodoacetamide was used to derivatize the available sulfhydryl groups in the PPA subunits. Aliquots of the subunit fractions (ca. 1 mg) were made 10 mM in sodium glycerophosphate buffer (pH 8.5), 10

4 “Chemical Formulation phoresis”: Mimeographed Canalco Corp., Bethesda,

for Sheet Maryland

Disc Electrodistributed by (1965).

AND

LEE

mM in disodium EDTA, and 10 mu in DTT; the solution was incubated for 16-20 hr at room temperature. The solutions were then made 50 mM in iodoacetamide and allowed to react for 1 hr. The derivatized fractions were dialyzed 15-20 hr at 4” against 1000 vol of 10 mM sodium glycerophosphate buffer (pH 8.5) and chromatographed on DEAE-cellulose using fresh 10 my, pH 8.5 buffer. The derivatized fractions that were subjected to gel electrophoresis were first dialyzed against 1 mM sodium glycerophosphate buffer (pH 8.5) and concentrated to 0.1 vol on a rotary vacuum evaporator. rlmylase assay. The procedure was that described by Robyt and Whelan (16), except that the reducing value was determined by an alkaline ferricyanide-cyanide procedure on a Technicon AutoAnalyzer. Peptide mapping. The peptide maps were obtained by a combination of methods (17). Essentially, the subunits [fractions were cut, Fig. 2: A (75-100 ml); B (110-130 ml)] were oxidized with performic acid, treated with trypsin, and the peptides separated by paper chromatography and high-voltage electrophoresis. The subunit fractions were dialyzed against 10 mM disodium EDTA in 1 mM ammonium carbonate buffer (pH 8) for 12 hr and then dialyzed an additional 12 hr against 1 mM buffer without EDTA. The fractions (2-3 mg each) were freeze-dried and then dissolved in 1 ml of freshly prepared performic acid (18); oxidation was allowed to proceed for 15 min at 50”. The oxidized fractions were diluted to 5 ml with distilled water and freeze-dried. The fractions were then dissolved in 1 ml of 100 rnhf ammonium carbonate buffer (pH 8), and TPCK-bovine trypsin was added until the ratio of amylase to trypsin was 1OO:l. The tryptic digestion was allowed to proceed for 8 hr at 24”; after this time, a second aliquot of trypsin was added for an additional 4 hr of reaction. The digests were then freeze-dried, dissolved in 100 ~1 of water, and applied to Whatman 3 MM paper for chromatography and electrophoresis. The peptides were detected by a ninhydrin-collidine spray. RESULTS

Treatment of crystalline PPA with DTT (1.5 rnxx) at pH 8.5 gave two fractions, I and II, on Sephadex G-100 (Fig. 1A). The molecular weights of t’hese fractions, as determined by gel filtration on Sephadex G-100 by using proteins of known molecular weight, were 50,000 and 25,000, respectively

SUBUNIT

STRUCTUlLE

OF

POIWINI’,

PANCREATIC!

Elution

.(mll

163

ol-AMYLASR

0.8

FIG. 2. Separation lose at pH 8.5 after

Volume

of porcine pancreatic or-amylase subunits on reduction of the enzyme with 10 mM dithiothreitol

IO 9

IO 9 8 7

I-3 6-

6

5MW X I04

reducing-DEAFrcelluat pH 8.5.

5 MW X I04

4-

4

3-

3

*t

2

B-

20

30 Elution

Volume

50 (ml)

1

6C

FIG. 3. Molecular weight determination of porcine pnncreatic ol-amylase subunits by gel filtration on Bio-Gel P-150. The molecular weights of the standards are those given in the references: BSA, bovine serum albumin (14) ; Ao, Aspergillus oryzae a-amylase (1); Ova., ovalbumin (14); cyt. c, cytochrome c (14); A, fraction (75-100 ml), B, fraction (110-130 ml), and C, fraction (140-165 ml) of Fig. 2.

(Fig. 1B). Passage of the untreated PPA over Sephadex G-100 gave only a single peak corresponding to 50,000. The treatment of PPA with an efficient reducing agent, DTT, presulnably cleaved one or more intermolecular disulfide linkages between subunits of 25,000 daltons each.

.I

.2

FIG. 4. Molecular porcine and C)

pancreatic by SIX-gel

.3

4 .5 MOBILITY

.6

.7

.8

.9

weight determination a-amylase subunits electrophoresis.

(A,

of B,

To increase t,he yield of the subunit,, the concentration of DTT was increased to 10 rnM and the sample was passed over a reducing-DEAE-cellulose column at, pH 8.5 (Fig. 2). Three enzymically active fractions (A, B, and C) were obtained. The molecular weights of t,hese fractions were det>ermined by gel filt#ration on Bio-Gel P-150 (Fig. 3) and SDS-gel electrophoresis (Fig. 4). With both techniques, the molecular weights of A and B were 25,000 and that of C was 12,500. The specific activities of A, B, and C were

164

ROBYT,

CHITTENDEN,

1.27, 1.54, and 1.76 ~~41of bond cleaved per minute per pg of protein, respectively. Subunits A and B were treated with 10 rnN1 EDTA and 10 rnM DTT at pH 8.5 for 16 hr and then derivatized with iodoacetamide. The derivatized subunits, A’ and B’, were each chromatographed on DEAEcellulose. Figure 5 shows that A’ and B’ have identical elution volumes which are very close to the elution volume of the original A subunit. Exposure of the subunit’s to EDTA and DTT and derivatization with iodoacetamide rendered A and B chromatographically equivalent. Table I gives the gel-electrophoresis mobilities of subunits A and B, which had been treated in various ways. The original, untreated frjactions gave mobilities for A and B that were distinctively different. Treatment with EDTA and DTT converted A and B into rapidly migrating proteins with identical mobilities. Treatment with EDTA and DTT, followed by derivatization with iodoacetamide, convert,ed A and B into very slowly migrating proteins with ident,ical mobilities.

AND

LEE

The molecular weights of subunits A’ and B’ were determined by gel filtration on BioGel P-150. Both A’ and B’ had molecular weights close to 25,000. Because of the conversions of subunits A and B into apparently identical forms, (A’ and B’), peptide maps were made to determine if the primary structures of A and B were the same. Figure 6 is a comparison of the peptide maps of performic acid-oxidized subunits treated with trypsin. The peptide maps show that identical peptides are produced from both A and B subunits. DISCUSSION

Treatment of crystalline PPA with DTT at pH 8.5 gave two fractions on Sephadex G-100 (Fig. IA). Passage of the untreated enzyme over Sephadex G-100 gave a single fract,ion corresponding to fraction I. Calibrat,ion of the Sephadex column with proteins of known molecular weight indicated that the slower-moving fraction (II) had a molecular weight of 25,000, and the faster moving fraction (I) a weight of 50,000. Increasing the DTT concentration eliminated fraction I. TABLE

t

I

GEL ELECTR~PHORETIC PANCRE.~TI~ WAMYLISE WITH

Subunit

I MOBILITIES SUBUNITS

Vz\RIOUS

OF PORCINE TREATED

RE.\GENTS

-

Treatment

-.

10 mM 10 mM 10 mM

DTT DTT DTT

+

0.72” 0.536 0.9gb 0.976 0.11

10 mM

DTT

+

0.12

None

None 10 rn~ EDTA + 10 IIIM EDTA + 10 mM EDTA + 50 mM IAcNHP 10 mM EDTA + 50 my IAcNHec

(1 Mobility calculated according distance of protein migration length of gel after staining length of gel before x distance of dye migration Elution

FIG.

subunit El)TA,

Volume

(ml)

5. DEAE-cellulose chromatography fractions A and B that were treated DTT, and iodoacetamide.

of with

MobilitieP

to the formula

staining before

staining

* Sample applied to separating gel after electrophoresis of the gel for 60 min to remove the oxidizing agent, ammonium persulfate. c IAcNH2 is the abbreviation for iodoacetamide.

SIXIJNIT

STRUCTUI:I’

OF

I’ORCINI~:

I’ANCI:I~;ATIC

a-ARIYLASF:

1%

FIG. 6. l’eptide maps of porcine pancrcat,ic a-amylase snhlmits .4 and B. Both subunits were oxidized with performic acid prior to hydrolysis with trypsin. I)irection 1 was high-volt,age electrophoresis (2300 V for 1 hr) using plI -1.0 pyridine:acet,ic acid:water buffer (1:3.1:10!) parts by vollune) in the Gilson high-voltage electrophoresis apparatus; direct ioll 2 was descending paper chromatography rtsing ~,lltarlol-1:pyridirre: acet,ic acid: water (W:(iD: 18:72 parts by volume) as irrigation solvent.

It has been claimed that amylases interact with the Sephadex gel and are thereby ancmalously retarded (19, 20). Treatment, of the enzyme with DTT produced a slower moving fract,ion that, wau not, previously present. Increasing the concentration of DTT increased the amount of t)he slower moving fraction and decreawd the amount> of t)he faster moving fraction. Our calibrat,ion of the molecular weight,s did not indicate an anomalous retardation of fractions I. To avoid anomalous molecular weight determinations, however, we used Rio-Gel P-150 and SDS-gel electrophoresis methods in subsequent. molecular weight determinat,ions. The reduced enzyme could be resolved into t,hree components (,4, B, and C) on a reducing-DEAE-cellulose column (Fig. 2). The molecular weights of the t,hree fractions, as determined by gel filtration on Bio-Gel l’-150 (Fig. 3) and by SDS-gel electrophoresis (Fig. 4), were 25,000 for ,4 and B and 12,500 for C. The reaction of DTT with t>he original enzyme evidentl!- reduces an int,ermolecular disulfide bond bet \veen two subunits of 25,000. The mode of formation of fraction C is unknown. It was always produced in

smaller amounts than \vas A or B, and in some experiment,s was completely absent. All three of these fractions were enzymatitally active, each having a specific activity of approximately 1.5 P-\I of bond cleaved per minute per pg of; protein. Icractions A and B migrated on electrophoresis gels at1two distinct posit(ions (Table I). Treat,ment of ,4 and B with EDTA and DTT gave forms that migrated very rapidly and identically on electrophoresis gels. Reaction of these forms with iodoacetamide gave derivatives that behaved identically on electrophoresis gels and on DEAEcellulose (E‘ig. 5). Further, this treatment did not, convert’ A and B into C, as judged by a molecular weight] determination on Rio-Gel I’-150. These experiments indicated t]hat A and B could be converted into very similar, if not, identical, forms by treatment’ w&h EDTA and DTT. In confirmation of this hypothesis, t,he tryptic peptide maps showed t’hat A andB have very similar, if not identical primary structures. The final proof will have t’o await amino acid analyses and sequence st>udies of subunit,s A and B. Initially DTT reduces an intermolecular

166

ROBYT,

CHITTENDEN,

disulfide bond or bonds between two subunits, A and B, of 25,000 daltons each. These subunits behave differently on DEAEcellulose and on gel electrophoresis, and thereby permit their separation. The subunits may differ from each other in the extent to which they have been reduced, or they may have different tertiary structures that depend on the permutations of intramolecular disulfide bonds. That is, the subunits could be sulfhydryl-disulfide conformers. These types of isomers have been observed in plasma albumin (21). All of the intramolecular disulfide bonds of A and B are not initially reduced by DTT, possibly because of structural burying. The disulfide masking may be due to a tight protein structure formed by calcium chelation.3 An analogous situation occurs in A. oryzae ol-amylase in which a sulfhydryl group is buried and unreactive until the essential calcium is removed (22). This hypothesis for the masked disulfide conformers of A and B is supported by the observation that the simultaneous treatment of A and B with EDTA and DTT gave compounds that migrated very rapidly and identically on electrophoresis gels (cj. Table I). Presumably, the EDTA removes the calcium and opens the structure so that the DTT is able to reduce the buried disulfide linkages. The resulting structures have sulfhydryl groups that are ionized under the pH conditions of the electrophoresis. This ionization imparts a substantial negative charge to the molecules t,hat was not possible before reduction and thus facilitates their rapid and identical migration toward the anode. Likewise, the derivatization of the sulfhydryl groups with iodoacetamide converted A and B into forms that were not ionized and hence t,hey migrat,ed very slowly and identically toward the anode (cf. Table I). In the characterization of porcine pancreatic amylase-isozymes, Cozzone et al. (6) stated that the isozymes have molecular weights in the range of .51,OOOG54,000 and consist of a single peptide chain. The conclusion that the amylase isozymes have a single peptide chain was based on reduction and SDS-gel electrophoresis experiments.

AND

LEE

Cozzone et al. initially treated the isozymes with 2-mercaptoethanol at pH 7.1. SDS-gel electrophoresis of the reduced isozymes gave molecular weights of around 50,000 and did not indicate the presence of any lower molecular weight fractions. Hence, it, was concluded that the individual isozymes consisted of a single peptide chain with a molecular weight of 50,000. In contrast, we have found that the isozymes are made up of at least two associated peptide chains with molecular weights of 25,000 each. The differences between our conclusions and those of Cozzone et al. are probably because of the greater reducing efficiency of mercaptans at pH 8.5 than at pH 7.1 and because DTT is a much more efficient reducing agent than 2-mercaptoethanol (23). In addition, the polyacrylamide-gel system for electrophoresis contains a powerful oxidizing agent, ammonium persulfate, that could readily reoxidize sulfhydryl groups unless proper precautions are taken to remove the persulfate by prior electrophoresis of the separating gel before sample application. If t,he persulfate was not removed, we have observed the conversions of each of our A and B fractions into two fract’ions; in both cases the new fraction migrated slower than t’he original subunit. Thus, under proper conditions, the PPA isozymes can be reduced to act’ive subunits of 25,000 daltons, which differ from each other in their tertiary structure. The individual isozymes have at least two active sites per 50,000 and perhaps four, if subunits A and B are further composed of two C subunits. Recently PPA has been found to have three isozymes5 instead of two (6). The differences in the three isozymes may be due to the various combinations of A and B subunits: viz., A-A, A-B, and B-B ACKNOWLEDGMENTS We critical Dexter se1 .

thank reading French

5 Robyt,, results.

J.

Professor Paul Hartman for his of the manuscript and Professor for his sustained int,erest and coun-

F.,

and

Lee,

C.

T.,

unpublished

SUBUNIT

STRUCTURE

OF

PORCINE

REFERENCES 1. FISCHER, E. II., .IND STEIN, E. A., a-Amylases in “The Enzymes,” (P. D. Boyer, II. Lardy, and K. Myrbtick, eds.), Vol. 4, 2 Ed., p. 333. Academic Press, New York (19GO). 2. ChLDwVELL, M. L., DICIW,Y, E. S., ITBNH.\H.\N, V. M., KUNG, H. C., KUNG, J. T., AND MISKO, M., J. =Imer. Chem. Sot. 76, 143 (1954). 3. I~~NIELSSON, C. IX., Nature London 160, 899 (1947). 4. MARCHIS-MOCREN, G., AND PGIS~~O, I,., Biochim. Biophys. Acta 140, 36G (1967). 5. Itow~, J., W~KIM, J., AND THOMA, J., Anal. Biochem. 26, 206 (1968). 6. COZZONE, P., PISERO, L., BEXUPOIL, B., AND M.~RcHIs-MOUREN, G., Biochim. Biophys. Ada 207, 490 (1970). 7. LEVITZKI, A., IIELLER, J., .~ND SCHR.\MM, M., Biochim. Biophys. Acta 81, 101 (1964). 8. LOYTER, A., AND SCHRAMM, M., J. Biol. Chem. 241, 2611 (1966). 0. FISCHER, E. H., BND STEIN, E. A., Biochrm. Prep. 8, 34 (1961). 10. B~BHAR, I. J., POWER, V. K., AND JOGANNATHAN, V., Biochim. Biophys. Ada 66, 347 (1962). 11. HARTMAN, P. A., “Miniaturized Microbio-

12. 13. 14. 15. 16.

17.

18.

19. 20.

21. 22. 23.

PANCREATIC

ol-AMYLASE

167

logical Methods,” p. 112. ilcademic Press, 1New York (1968). ANDREWS, P., Biochem. J. 91, 222 (1964). WEHJGR, K., .\ND OSWRN, hr., J. Biol. Chem. 244, 4406 (1969). I)UN~!IWR, A. K., -\ND I