Kamaryt, Zemek and Kuniak: -Amylase molecular weight determination 451

J. Gin. Chem. Clin. Biochem.Vol. 20, 1982, pp. 451-455

Determination of the Molecular Weight of -Amylase, äs the Enzyme-Inhibitor Complex,using Thin Layer Gel Filtration on Sephadex

By /. Kamaryt, J. Zemek and L'. Kuniak

Pediatric Research Institute (Head: As. Prof. Dr. M. Stavratjev, DrScJ, Brno, ÖSSR and

Institute ofChemistry, Slovak Academy of Sciences (Head: Ing. M. RepäS, CSc.) Bratislava,

(Received July 30/December 29,1981)

Summary: Thin layer gel filtration on Sephadex was perfbrmed äs a simple method for recognition of the smallmolecular weight differences of human -amylases from different sources. Activity was located with dry chromogenicSubstrate, using a replica technique. Undesirable interaction between the gel matrix and Substrate binding sites on theenzyme, which causes an anomalous decrease in the migration rate of the enzyme protein, was suppressed by prein-cubation of the enzyme with appropriate inhibitor. Gradual masking of Substrate binding sites of the enzyme byincreasing concentrations of amyläse inhibitor resulted in two distinct migration rates for the enzyme in thin layer gelfiltration. This suggests the existence of two Substrate binding sites in the enzyme molecule. Together with thin layergel affinity chromatography on a mixture of Sephadex and ConA-Sepharose, the method yielded useful data on themolecular weight of amyläse and its glycosylated forms and served äs a valuable tool for the differential diagnosis ofmacroamylasaemia.

Bestimmung des Molekulargewichtes von ot-Amylase als EnzymJnhibitor-Komplex mit Dünnschichtgelfiltrationan SephadexZusammenfassung: Die Dünnschichtgelfiltration an Sephadex^Gel wurde für das einfache Erkennen der geringenMolekulargewichtsdifferenzen der menschliehen a^Amylasen verschiedener Herkunft verwendet. Die Enzymaktivi-täten wurden mit einem chromogenen Substrat und nachfolgender „Replica-Technik" lokalisiert. Die unerwünschteInteraktion zwischen den Substratbindungsstellen von Amyläse und Gel-Matrix, welche die anomale, reduzierteBeweglichkeit des Enzymproteins im Laufe der Entwicklung des Chromatogramms verursachte, wurde durch dieVorinkubation der Amylase mit Inhibitor unterdrückt. Die schrittweise Maskierung der Substratbindungsstellen desEnzyms durch steigende Sättigung mit dem Amyläse Jnhibitpr resultierte in zwei verschiedenen Beweglichkeiten desEnzym-Moleküls während der Entwicklung des Chromatpgranims, was die Existenz von zwei Substratbindungsstellendes Amyläse-Moleküls andeutet. Die Methode der Dünnschichtgelfilträtion zusammen mit Dünnschichtgel-Affinitäts-chromatographie an Sephadex und Con A-Sepharose-Gel führt zu nützlichen Angaben über das unterschiedliche Mole-kulargewicht von Amyläse und ihrer glykosylief ten Formen, und die Methode zeigt sich auch als ein wertvolles Ver-fahren für die Differentialdiagnose der Makroamylasämie.

Introduction pancreatic amyläse cannot be differentiated with amyläseThe -amylases (a-1,4-glucan 4-glucanohydrolase, B.C. antibodies; the antigenicities of the two isoenzymes are3.2.1.1) from salivary glands and pancreas, which may be completely identical (3). The biosynthesis of both iso-distinguished by means of various analytical techniques, amylases is controlled by two separated gene loci AMY lparticularly by electrophoresis, are very similär enzymes. and AMY 2, which are closely linked on chromosome lCompärison of the peptide maps of both amylases shows (4,5). The molecular weights of salivary and pancreaticmore similarities than differences (1). Also efforts to amylases are slightly different. This may be due to post-show immunological differences between salivary and transcriptional glycosylation especially of the enzymepancreatic amyläse remain unsuccessful (2). Salivary and molecules originating from salivary glands (6). It is

0340-076X/82/0020-0451 $02.00© by Walter de Gruyter & Co. · Berlin · New York

452 Kamaryt, Zemek and Kuniak: -Amylase molecular weight determination

possible that the different renal clearances of these twoisoamylases are a consequence of these molecular ältera-tions. The salivary amylase of the blood passes throughthe glomeruli with roughly the half velocity of thepancreatic enzyme (7). The inolecular weight estimationof amylases yields useful data for the characterization ofenzymes originating from various sources and for thediagnosis of macroamylasaemias.

Thin layer gel filtration on Sephadex, commonly used forthe molecular weight determination of proteins,represents a simple and versatile rnethod. Unfortunately,the -amylase interacts with the Sephadex matrix, sothat the migration rate of the enzyme is decreasedduring chromatography and the apparerit molecularweights are lower than those obtained by other techni-ques (8, 9). Using Sephadex G-100,Minamiura et al. esti-mated the molecular weight of -amylase in humanurine to be less than 12000 (10). The enzyme wasadsorbed on this gel so strongly that the elution volumewas larger than that of lysozyme. Similarly on columnsof -Gel P-100 and P-l 50 human and rat amylasewas eluted much later than proteins of approximatelythe same molecular weight, even though the anomalousretardation was unexpected since Bio-Gel does notsimulate the natural Substrate for amylase (11, 12).These troubles may be avoided and thin layer gelfiltration on Sephadex used successfully, if the Substratebinding sites of the enzyme are masked by an appro-priate amylase inhibitor and a simple and sensitivedetection technique for the residual enzyme activity isused.

for 10 minutes. The replica with reference proteins was stainedwith Coomassie Brilliant 'Blue R-250 (Serva Biochemica, Heidel-berg). The -amylase-inhibitor complex was separated on thinlayer gels of Sephadex under the above deseribed conditions.Increasing concentrations (5.4-540 /ig) of inhibitor proteinwere added to 450 mU of enzyme in l ml. The a?amyiase ihhi-bitor isolated from potatoes is a protein with a relatively lowmolecular weight ofabput 15 000-17 000 (product of Instituteof Chemistry, Slpvak Academy of Sciences, Bratislava, CSSR)and it exhibits a higher degjree of Inhibition against salivaryamylase (83 %> than against the pancreatic enzyme (62%).

Results and Discussiön

The suitability of the replica detection technique usingchromogenic Substrate for the visualisation of amylaseactivities (after development on thin layer gels ofSephadex) is demonstrated by the Separation of theenzyme from human rnilk (fig. 1). The chromätögräm ·=-actually the paper reprint öf the thin layer gel chromato-

. gram — öf various dilutions of one human milk samplewas evaluated reflectometrically. The relationshipbetween gradually increasing amylase activities, Spotsize and its colour intensity expressed in Integrationunits is plotted in figure 2. The thin layer gel filtrationrevealed two dinstinct breast milk aniylase spots,differing in their molecular weights. Similarly, äs in thecase of salivary amylase, a part of the breast milkamylase is glycosylated.

Masking the Substrate binding sites of -amylase by in-creasing concentrations of appröpriate inhibitor gradu-ally diminishes the anomalous binding effect of

-amylase tö Sephadex and contributes to the normal-ization of the migration rate of the enzyme protein

Materials and Methods

The -amylases of blood serum, urine, saliva, duodenal juice,and breast milk were analyzed. Only fresh biological materialwas examined. The samples were diluted to the catalytic con-centration ränge 200-500 U/l (3.3-8.4 Mkat/l); but the serumfrom the individual with macroamylasaemia was not diluted,because dilution causes dissociation of the macroarriylase com-plex. Thin layer gel filtration was performed with the apparatusfrom Pharmacia Fine Chemicals, Uppsala. For the required Separa-tion efficacy, the Standard plates 20 X 20 cm or 20 X 40 cm wereused. Sephadex G-100 and G-150 Superfine grade served äs thestationäry phase (thickness of layer 0.6 mm). The mobile phasewas Tris/HCl buffer 100 mmol/1, pH 8.0. After overnight equi-libration at an angle of 10°, 10 biological fluid samples ofenzyme catalytic concentration 200-500 U/l were applied tothe gel layer. Reference marker proteins for calculation of rela-tive migration distance were applied simultaneously to the layer.After 4-7 hours development (depending on the plate size,20 X 20 or 20 X 40 cm respectively) at an angle of 15° and atroom temperature, the chromatograms were covered with drypowdered chromogenic Substrate (cross-linked blue starch poly-mer, product of Slovakofarma Hlohovec, CSSR) (75 mg per20 X 20 cm plate) and incubated at 37 °C for the time necessaryfor the enzyme spots to become visible. a-Amylase hydrolyzespolymer into water-soluble blue starch frägments. The replieatechnique was then used for preparing permanent records. Thewater-soluble blue starch frägments were transferred from thegel layer to filter paper Whatman 3MM by a contact with the gelfor l minute. The paper chromatogram was then dried at 100 °C

Sample ffaction0.05 0.067 0.1 0.2 0.4

i Start

.*(* i

Fig. 1. Thin layer gel filtration of various dilutions of humanmilk amylase ori Sephadex G-100 Superfinei The volumefractions of the originally undiluted milk sample (2280U/l) are given, Thin4ayer plate: 20 X 20 .cm. Layer thick-ness: 0.6 mm. Time: 5 h. Angle: 15°. Buffer: 100 mmol/1

° Tris/HCl, pH BiO containing 0.2 g/l sodium azide. Twodistinct amylase spots are evident (sample volümes 10 ).The glycosylated amylase shows the.jgaster migration rate.

J. din. Chem. Clin. Biochem. / Vol. 20,1982 / No. 6

Kamaryt, Zemek and Kuniak: α-Amylase molecular weight determination 453

(fig. 3). The uninhibited residual enzyme activity issufficient to be detected during prolonged incubationwith chromogenic Substrate.Thin layer gel filtration on Sephadex permits the esti-mation of the relative small molecular weight differ-ences between salivary and pancreatic amylases. Part

600

400

200

200 400 600 ΘΟΟ<K-Amylase [U/l]

1000

Fig. 2. The relationship between increasing araylase activities ofgradually diluted human milk sample and Integration unitsobtained by reflectometric scanning (at 600 nm) of pre-vious chrom togram. The sum of glycosylated andunglycosylated enzyme Integration units was plotted.

Referenceproteins F 6

5 Start

of the salivary amylase is glycosylated and thereforehas a higher molecular weight. Pancreatic amylasedoes not show such a high degree of posttranscriptionalglycosylation. Using amylase inhibitor for masking theenzyme binding sites permits the estimation of the realmolecular weight by thin layer gel filtration on Sephadex.Separation of the salivary amylase of saliva and the pan-creatic amylase of duodenal Juice, with or withoutprevious preincubation with amylase inhibitor, areshown in figure 4; the small molecular weight differ-ences between the various isoamylases originating fromboth glands are expressed.Thin layer gel affinity chromatography of human breastmilk on a mixture of Sephadex G-150 and Con A-Sepharose (2:1) revealed two isoamylases differing inthe amount of sugar residues (fig. 5). The carbohydraterieh amylase showed a considerably retarded migrationdue to the interaction with the incorporated affinityligand concanavalin A. The isoamylase with low carbo-hydrate content showed the usual migration rate.Takeuchi similarly separated the salivary amylases byineans of concanavalin A Sepharose chromatographyinto two fractions. The adsorbed fraction with affinityfor concanavalin A showed a higher carbohydrate con-tent than the unadsprbed one without affinity for con-

S S*I P P+I S S+IReference

P+I proteins'r* * i '

4 . — ^ Stuft

Rfbo- inucleaseA

Bovineserumalbumin

Fig. 3. Masking of Substrate binding sites of amylase molecules(urine amylase) with gradually increasing concentrationsof amylase inhibitor.A = urine sample without inhibitor;B = urine with 5.4 Mg;C = 16.2 Mg;D = 32.4 Mg;E = 54.0 Mg;F = 270.0 Mg;G = 540.0 Mg of inhibitor protein to 450 mU of enzyme in

1ml.RP = reference proteins:RI A = ribonuclease A, Μτ 13700BSA = bovine serum albumin, Mt 670000 - indicates the uninhibited amylase spot migration rate.1 - the f rst Step andII - the second Step of enzyme Substrate binding sitemasking with amylase inhibitor.

Ribo-nuclease A

Bovineserumalbumin

Fig. 4. The molecular weight differences between salivary (S)and pancreatic (P) amylase from saliva and duodenaljuice. Each sample, native and inhibited (+1 = with inhi-bitor) was doubled in order to avoid migration irregulari-ties. Two salivary amylase families are evident: unglycosy-lated (U) and glycosylated (G) s in human milk. Thepancreatic amylase from duodenal juice did not show thisphenomenon.Thin-layer plate: 20 X 40 cm. Gel: Sephadex G-150 Super-fine. Layer thickness: 0.6 mm. Time: 6 h 35 min.Angle: 15°.

J. Clin. Chem. Clin. Biochem. / Vol. 20» 1982 / No. 6

454 Kamaryt, Zemek and Kuniak: α-Amylase molecular weight determination

Urine Milk,sample f raction

0.4 0.2 0.1 0,067. Start

0 oFig. 5. Thin layer gel affinity chromatography of human milk

amylase. U = urine from healthy control; M = human milksamples with indicated dilution coefficients. The glycosy-lated amylase revealed decreased enzyme activity andreduced migration rate due to the interaction with theaffinity ligand concanavalin A.Thin-layer plate: 20 X 20 cm. Gfel: Sephadex G-150 Supef-iine and Con A-Sepharose (2:1). Layer thickness: 0.6 mm.Time: 5 h 30 mm. Angle: 15°. Buffer: Tris/HCl 100mmol/1, pH 8.0.

canavalin A. The data indicated that the salivary amylasethat binds to concanavalin A is that with the higher mole-cular weight (3). The isoamylases in breast milk and salivashowed identical electrophoretic mobility. Isoelectricfocusing also revealed four identical bands in breastmilk and saliva: one main band with a pl of 6.4 and threeweaker bands with pl of 5.9, 5.6 and 5.4 respectively(own unpublished results).

The use of amylase inhibitor in combin tion with anenzyme detection technique with powdered chromo-genic Substrate makes it possible to easily distinguish themacroamylase, which is usually the complex of serumamylase with the large globulins preventing clearanceby the kidney (fig. 6). The binding substance evidentlymasks the binding sites of a portion of the blood serumamylase. The migration distances of unhibited andinhibited macroamylase during thin layer gel filtrationare the same. Hence it follows that the binding sites ofthe macroamylase are occupied by the binding sub-stance — and thus do not interact with the matrix ofthe gel. After the preincubation of macroamylasaemicblood serum with amylase inhibitor, the originally slowlymigrating portion of normal amylase exhibited a migra-tion distance corresponding to the correct molecularweight.The detection technique using the dry powderedchromogenic Substrate followed by the replicatechnique was the key to the successful applicationof thin layer gel filtration on Sephadex or thin layer gelaffinity chromatography. The technique makes itpossible to follow the development of blue colour dueto the α-amylase action in the gel layer during the in-cubation period.

The anomalous migration rate of amylase on Sephadexgel and thus the impossibility of using this method formolecular weight determination was observed earlier byseveral authors. Wilding (9) ascertained by means of gelfiltration on Sephadex that the amylase was eluted fromthe column at a point which suggests that amylase has anapparent molecular weight f less than 20000. Mutzbaueret al. (13) using the value of the partial specific volumecalculated from the amino acid composition gives a value55 200 for the molecular weight of human salivary amyl-ase. The studies of Keller et l. (6) yielded a molecularweight of salivary amylase of 61 900 and 56 100. Stiefelet al. (1) reported a moleeular weight of 54 O forhuman pancreatic amylase. Flodin suggested that theanomalous behaviour of α-amylase in gel filtration onSephadex is due to the interaction betweeri the enzymeand the gel matrix. He Claims this is a reasonable hypo-thesis in view of the similarity in structure of starch anddextran (8). According to Takeuchi, chromatography ofmammalian amylases on Sephadex G-100 and Sepharpse

Start

Referenceproteihs A„ 0, CN D, EN f, GN H,

Ribo-nucleose A

;̂ :V.

Ι ι

i

. " ' · Jj

ftChymotfyp- j f |sinogen

[ί

Ovalbumia ι 1 1 ,,! " ι \

Bovine i .̂ i ! rseruma lbumin

m

';ΐϋ

fl © φ¥ ·]%.a "' » · i

f r ^ ' fl l\/ '"

; '̂ r

Fig. 6. Thin layer gel filtration of the serum amylase from a .subject with macroamylasaemia.A = native undiluted serum;B = macroamylasaemic serum preincubated with amylase

inhibitor;C and D = Sfoid diluted macroamyl saemic serum native (N)

and inhibited (I);£ and F = macroamylasaemic urine native and inhibited;G and H = urine frorh healthy control native and inhibited.RP = reference proteins:ribonucle se A,Mr 13700;chymotrypsinogen A, M^ 25 000;ovalb min, Μτ 43 000;bovine serum albumin, MT 67 000.thm4ayer plate: 20 X 40 cm. Gel: Sephadex G-150superfine. Layer thickness: 0.6 mm. Time: 6 h 50 niin.Angle: 15°. Buffer: tris/HCi 100 mmol/1, pH 8.0.

J. Clin. Chem. Clin. Biochem. / Vol. 20,1982 /No. 6

Kamaryt, Zemek and Kuniak: a-Amylase molecular weight determination 455

4B is affinity chromatography, not gel filtration (3). Theamylase appears to be held on the columns by inter-actions other than simple gel filtration effects (l 1).Minamiura et al. supposed that the very strong adsorp-tion of -amylase on Sephadex may be due to trypto-phan and/or tyrosine residue(s) protruding from theenzyme molecule (10). The amylases are rieh in hydroxy-arnino acids or their amides and have a high capacity forhydrogen bonding both intramolecularly to give a partic-ularly compact structure, and extramolecularlyj withmaterials such äs Sephadex and Bio-Gel (12). The inter-action of the amylase with the dextran matrix, whichcauses the decreased migration rate during thin layer gelfiltration on Sephadex, can be diminished by means of thepreincubation of enzyme-containing biological fluidwith inhibitor before the sample is applied to the gellayer. The increasing eoncentrations of inhibitor influ-ence the enzyme migration rate during gel-layer develop-ment in two Steps. In the first step it seems that oneSubstrate binding site of the enzyme is masked, and themolecules behave with an apparent molecular weight ofabout 34 000. In the second step during fufther inhibitorSaturation the second enzyme binding site is masked andthe migration rate of the complex enzyme-inhibitoraccelerates dramatically and cprresponds to the expectedmigration rate fpr a component of molecular weight70 000-75 000 (fig. 7). Thus oür findings agree withthese ofLoyter et al. who supposed the existence of twoSubstrate binding sites in pancreatic amylase (14). Ön theother band Mora et al. consider three independentbinding sites (15), The preincubation of biologicalsämples to be anälyzed before thin layer gel filtrationwith amylase inhibitor enables the estimation of thedifferences in molecular weight of -amylases originatingfrom various glands and tissues and offers a particularlysimple ässäy for the differentiäl diagnosis of macro-arnyläsaemiä. The findings ofKitämuraet al. (16) thatthe amylase binding substance of the rnacroamylase is

1 2.0

0,1.5

1.0

\Ribonuclease A

• Chymotrypsinogen

vOvalbumin

vßovine serum a lbumin .

04 105 106

Fig. 7. Relationship between migration distance (relative tobovine serum albumin) and logA/r for some Standardproteins (the black circles).The open circles with arrows:top — spot of unhibited urine amylase (apparent MTsimilar to that of ribonuclease A);middle — spot of urine amylase with incompletely maskedSubstrate binding sites of the enzyme with inhibitor(apparent MT 34 000);bottom — the migration rate of the complex amylase-inhibitor with completely masked Substrate binding sitesof the erizyme ( 75 000).Thin-layer plate: 20 X 40 cm. Gel: Sephadex G-150Superfine. Layer thickness: 0.6 mm. Time: 6 h 50 min.Angle: 15°. Migration distance for bovine serum albuminwas 260 mm. Buffer: Tris/HCl 100 mmol/1, pH 8.0.

capable of binding to the substrate-binding site agree withpur present results obtained by thin layer gel filtration onSephadex.Thin layer gel filtration of amylase and amylase-inhibitor complex, accompanied by the appropriatedetection of the enzyme activities with chromogenicSubstrate, represents a simple and reliable method per-rnitting determination of enzyme molecular weight andproviding an insigjit into the catalytic mechanism of theenzyme. The method gives results comparable with thoseobtained with other möre complicated and more timeconsuming procedures.

References1. Stiefel, D. J. & Keller, P. J. (1973) Biochim. Biophys. Acta

302, 345-361.2. Karn, R. C., Rosenblum, B. B., Ward, J. C:, Merritt, A. D.

& Shulkin, J. D. (1974) Biochem. Genet. 12,485-499.•" 3. Takeuchi, T. (1979) Cto. Chem. 25,1406-1410.

4. kamaryt, J., Adämefc, R. & Vrba, M. (1971) Humangenetik77,213-220.

5. Merritt, A. D., Lovrien, E. W., Rivas, M. L. & ConneaUy,P. M. (1973) Am. J. Hum. Genet. 25,523-538.

6. Keller, P. J., Kauffman, D. L., Ailan, B. J. & Williams, B. L.(1971) Biochemistry 70,4867-4874.

7. Karnaryt, J. (1969) Z. Klin. Chem. JCün. Biochem. 7,51-52.8. Flodin, P. (1962) Dextran gels and their application in gel

filtration. AB Pharmacia, Uppsala.9. Wilding, P. (1963) din. Chim. Acta 8, 918-924.

10. Minairiiura, N., Kimura, Y., Tsujino, K. & Yamamoto, T.(1974) J. Biochem. 77,163-169.

11. Alian, B. J., Zager, N. I. & Keller, P. J. (1970) Aren. Biochem.Biophys. 136, 529-540.

12. Kauffman, D. L., Zager, N., Cohen, E. & Keller, P. J.(1970), Aren. Biochem. Biophys. 137, 325-339.

13. Mutzbauer, H. & Schulz, G. V. (1965) Biochim. Bio-phys. Acta 102,526-532.

14. Loyter, A. & Schramm, M. (1966) J. Biol. Chem. 241,2611-2617.

15. Mora, S., Simon, I. & Elödi, P. (1974) Mol. Cell. Biochem.4,205-209.

16. Kitamura, T., Yoshida, K., Ehara, M. & Akedo, H. (1977)Gastroenterology 73,46 -51.

J. Kamaryt, Ph. D.Pediatric Research InstituteCernopoini 9CS-66262Brno

J. Clin. Chem. din. Biochem. / Vol. 20,1982 / No. 6