Amylas aktivitets lab 1) Spotta ner i ett vågskeppet, bara en halv mL saliv behövs till labben Hur många micro (u) liter är en halv milliliter? Märk 10 glasrör enligt punkt 5, samt ett gruppnamn 2) Pipettera 500 uL saliv till ett annat glasrör och späd med 10 mL buffert, blanda noga genom att vända röret upp och ner ett par gånger, detta blir er ”salivspädning” som ni kommer använda i fortsättningen. 3) De märkta 10 glas provrören ska tillsättas 750 uL buffert och 500 uL Stärkelselösning. Rör 9 och 10 tillsätts Inhibitor (koncentration 1mg/mL) efter överenskommelse

4) Inkubera rör 1-5 och och 9 och 10 i vattenbad 37C 3 min, rör 6 på is, rör 7 i

rumstemperatur (RT) och rör 8 i vattenbad 60C Låt gärna rören stå kvar i sina respektive temperaturer vid steg 5 Steg 5 och 6 är kritiska med tiden, tänk igenom i förväg hur just du vill arbeta för att reaktionen i alla provrören ska gå i exakt 3 minuter. 5) Tillsätt olika volymer av salivspädningen till glas provrör 2-10:

Rör Volym salivspädning (uL) Volym inhibitor (uL)

Temperatur för inkubering (Cᴼ)

1 0 37

2 50 37

3 250 37

4 500 37

5 500 37

6 500 4

7 500 22

8 500 60

9 500 10-150 37

10 500 -||- 37

6) Inkubera 3 minuter i vattenbad 37C (rör 1-5 och rör 9)

Rör 6 på is, 7 i RT, 8 i vattenbad 60C 7) Sätt till 1000 uL salicylat till varje provrör För rör 1 sätts 500 uL salivlösning till efter salicylatet! DVS nu. Varför? Vad har salicylat lösningen för egenskap utöver sin färg? 8) Ställ glas provrören i kokande vatten i 5 minuter Varför?

9) Tillsätt 10 mL vatten till alla glas provrören 10) Mät proverna i Spektrofotometern vid 540 nm. Rör 1 är Nollan/Referensen och mäts först. En mL av provet sätts i åt rätt håll i sin kuvett och sedan trycker man på ”set ref” Sedan kan rör 2-8 Absorbans mätas utan att man trycker på någon knapp.

Provrör Absorbans 540 nm

1

2

3

4

5

6

7

8

9

10

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

1,8

0 1 2 3 4 5 6 7 8 9 10

Absorbans 540 nm

Absorbans 540 nm

Provrör

Amylaslaboration gabriela.danielsson@scilifelab.se

2

Stärkelse

• Viktig energikälla i människans kost

• Finns i t ex potatis, ris, och mjöl

– Produceras av gröna växter för energiförvaring

• Stärkelse är en kolhydrat som består av långa,

grenade kedjor av sockret glukos

3

4

Maltos

α-Dextrin

Maltotrios Amylas

Enzym

Produkt

Stärkelse

Substrat

Proteinet amylas

• Enzym som bryter ner stärkelse

– Katalysator, effektiv, specifik, reglerbar

• Finns i saliv och bukspott

• Humant (mänskligt) -amylas

Ramasubbu, N., Paloth, V., Luo, Y., Brayer, G.D., Levine,

M.J.Journal: (1996) Acta Crystallogr.,Sect.D 52: 435-446

Inhibitor

• Produkt: A1520 sigma

• Kompetitiv

– Att den tävlar mot

stärkelsen att binda till

amylas

• Affinitet

– Hur väl den binder till amylas

6

Inhibition

Substrat

E=Enzym

Inhibitor

Inhibition

Katalys

Katalys

7

Laborationens syfte

Vi ska undersöka hur effektivt amylas kan bryta ner

stärkelse med olika förutsättningar

Temperatur (T)

Koncentration

Inhibitor

Fosfat buffert

• Vattenlösning med salter

• För spädning av saliv

– BLANDA ordentligt

• Inhibitor sätts till under 37C inkubering

• Temperatur: Likadant förberedda

provrör sätts i OLIKA T

– is, RT, vattenbad 37C, 60C

9

Inkubering

• Under inkuberingen sker reaktion 1

• Börja ta tid precis när ni droppat i substratet, stärkelsen i

provrören

• Blanda noga!

• Inkubering i 3 minuter

• Under tiden:

förbered för tillsättning av salicylatlösning

Maltos

α-Dextrin

Maltotrios Amylas

Enzym

Produkt

Stärkelse

Substrat

Reaktion 1

Klyvning av stärkelse med amylas

Maltos

α-Dextrin

Maltotrios Amylas

Enzym

Produkt

Stärkelse

Substrat

Temperatur

Δ

pH

OH-

H+

11

12

• Handskar och Labbrock!

• Salicylat har pH 12 pga tillsatt OH- så amylas slutar fungera

• 0-prov/Blank.

– !OBS! tillsätt först salisylatet och sedan stärkelsen

• Koka alla provrör och 0-provet

Reaktion 2, Salicylatreagens

Kokning=∆

Reduktion

Minskat antal bindningar till

elektronegativ atom

NH2

Aldehyd oxidation under basiska förhållanden Karboxylsyra (COO-)

+ +

Spektrofotometer

• Späd alla rör

• Mät 0-provet

• Absorbansen är proportionell mot

hur mycket salicylat som reagerat

med socker...

• ... som i sin tur är proportionell

mot hur mycket stärkelse som

bröts ned

Absorbans

• Vi mäter absorbans av ljus vid

våglängden 540 nm

• Absorbansen är proportionell mot

hur mycket salicylat som reagerat

med socker...

• ... som i sin tur är proportionell mot

hur mycket stärkelse som bröts ned

16

Rapporten

• Skriv:

– Inledning, förklara och diskutera med egna ord :

• Reaktion 1 och reaktion 2

– Resultat, presentera objektivt

• Ett diagram för Koncentration och ett för Temperatur

• Mätvärden i en tabell

– Diskussion, förklara och diskutera med egna ord:

• Hur amylas aktivitet beror av miljön det befinner sig i

– Koncentration

– Kallt, kroppstemperatur, varmt, hur påverkar detta lösningen samt amylas struktur och därmed funktion?

• Ord att lära sig att använda:

• Denaturera, Rörelseenergi, Aminosyra, Struktur (primär,

sekundär, tertiär), 3,5-dinitrosalicylat, 3-nitro-5-

aminosalicylat, Substrat, Produkt, Katalytisk, Kvantifiering,

Reduktion

17

Aminosyrors olika karaktär

• Alifatiska, Aromatiska

• Polära

• Negativt laddade, Positivt laddade

-

-

18

Protein struktur

• Primär

• Sekundär

• Tertiär

• Kvartenär

19

Tänk efter före, vad förväntar ni er

för resultat?

Am ylasakt ivitet

0

0,2

0,4

0,6

0,8

1

0 10 20 30 40 50 60 70

T

Ab

so

rb

an

s

Amylasförberedelser

Med labgrupp avses 12 personer med 6 labpar. Om inget annat nämns ska lösningarna förvaras i kylskåp. Om kommentar finns under lösningstabell, läs den först.

Dagsfärsk lösning

Stärkelselösning

volym 1

labgrupp

mängd 1

labgrupp

H2O kokande 200 ml

Stärkelse, vattenlöslig 2 g

Aliquotera 5 ml i ett 15 ml falconrör/labpar

Salicylatreagens

volym

1 labgrupp

mängd

1 labgrupp

volym

5 labgrupper

mängd

5 labgrupper

di-nitrosalicylat 3,5 g 15,5 g

NaOH 5,6 g 28 g

K-Na tartrat 105 g 525 g

tot volym 350 ml 1750 ml

Lös i vatten under omrörning och värmning på platta. Förvara färdig lösning över 50C. Innan lab

aliquotera 10 ml salicylatreagens i ett 15 ml falconrör/labpar

Buffert

3 olika lösningar:

slutkonc MW (g/mol)

volym

1 labgrupp

Mängd

1 labgrupp

volym

5 labgrupper

mängd

5 labgrupper

Citronsyra 0,1M 210,14 25 ml 0,53 g 125 ml 2,65 g

Glycin 0,2 M 75,07 20 ml 0,3 g 100 ml 1,5 g

NaOH 0,2M 40 10 ml 0,08 g 50 ml 0,4 g

slutkonc

MW

(g/mol)

volym

1 labgrupp

mängd

1 labgrupp volym 5 labgrupper

Mängd 5 labgrupper

Buffert A Na2HPO4 0,2 M 179,99 200 ml 7,2 g 1 L 35,6 g

NaCl 0,1 M 58,44 100 ml 0,58 g 500 ml 2,9 g

Citrat-fosfat buffer/Glycinbuffert (räcker till 5 labgrupper)

citronsyra 0,1

M Buffer A NaCl 0,1M

Glycin

0,2M

NaOH

0,2M H2O Total Volym

pH3 8 ml 2 ml 10 ml 80 ml 100 ml

pH5,6 4,2 ml 5,8 ml 10 ml 80 ml 100 ml

pH7,0 25.5 ml 124,5 ml 150 ml 1200 ml 1500 ml

pH8,6 10 ml 9,4 ml 0,6 ml 80 ml 100 ml

pH10,6 10 ml 5,4 ml 4,6 ml 80 ml 100 ml

Använd 50 ml falconrör.

Av pH 7 behövs 40 ml/labbpar

Av pH 3; 5,6; 8,6 och 10,6 behövs 2 ml/labpar så 2mL*30grupper=60 mL

2012-10-02 21:43α-Amylase Inhibitor from Triticum aestivum (wheat seed) Type I, lyophilized powder | Sigma-Aldrich

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Description

Unit Definition

One unit will reduce the activity of two units of α-amylase (A0521) by 50% after pre-incubation at 25 °C.

Physical form

Lyophilized powder containing buffer salts as sodium phosphate.

Biochem/physiol Actions

Competitive inhibitor of human salivary α-amylase. KI = 2.9 nM, compared to a KM of 5.9 mM (calculated per mole of α-1,4-linked maltose residues).

PropertiesRelated Categories Biochemicals and Reagents, Enzyme Inhibitors,

Enzyme Inhibitors by Type, Enzymes, Inhibitors, andSubstrates, Proteins More...

type Type I

form lyophilized powder

activity ≥1000 inhibitor U/mg protein (using humansalivary α-amylase)

≥200 inhibitor U/mg protein (using porcinepancreatic α-amylase)

composition Protein, 35-65% biuret

storage temp. −20°C

Price and Availability

SKU-Pack Size Availability Price(EUR/SEK)Quantity

A1520-1MG Estimated Delivery 02.10.2012 - FROM 681.03 0

A1520-5MG Estimated Delivery 02.10.2012 - FROM 2,706.52 0

A1520-25MG Estimated Delivery 02.10.2012 - FROM 9,294.27 0

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A1520 SIGMA

α-Amylase Inhibitor from Triticum aestivum (wheat seed)Type I, lyophilized powder

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2012-10-02 21:43α-Amylase Inhibitor from Triticum aestivum (wheat seed) Type I, lyophilized powder | Sigma-Aldrich

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Technical information & documentation associated with this product is available in the Safety & Documentation tab.References

Silano, V. Biochim. Biophys. Acta 391, 170, (1975)

O'Donnell, M. and McGeeney, K. Biochim. Biophys. Acta 422, 159, (1976)

Goff, D.J., and Kull, F.J., The inhibition of human salivary α-amylase by type II α-amylase inhibitor from Triticum aestivum is competitive, slow and tight-binding J. Enzym. Inhib. 9, 163-170,(1995) Abstract

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February 2006: Alpha-amylases Glucose is a major source of energy in your body, but unfortunately, free glucose is relatively rare in our typical diet. Instead, glucose is locked up in many larger forms, including lactose and sucrose, where two small sugars are connected together, and long chains of glucose like starches and glycogen. One of the major jobs of digestion is to break these chains into their individual glucose units, which are then delivered by the blood to hungry cells throughout your body. Attacking Starch Alpha-amylase begins the process of starch digestion. It takes starch chains and breaks them into smaller pieces with two or three glucose units. Two similar types of amylase are made in your body--one is secreted in saliva, where it starts to break down starch grains as you chew, and the other is secreted by the pancreas, where it finishes its job. Then, these little pieces are broken into individual glucose units by a collection of enzymes that are tethered to the walls of the intestine. Amylase in Action Since amylase needs to perform its job in the unpleasant environment of the intestine, it is a small, stable enzyme resistant to unfavorable conditions. The amylase shown here (PDB entry 1ppi) is made by the pancreas in pigs. A small chain of five sugars (colored yellow) is bound in the active site, which is found in a large cleft on the enzyme. Structures for the two human enzymes (which look very similar) are available in PDB entries 1smd and 1hny. As you look through the PDB, you will also find many structures of alpha-amylases and other starch-digesting enzymes from bacteria and plants.

February 2006: Alpha-amylases Industrial Strength

Alpha-amylase is used in large quantities in the production of high fructose corn syrup, a mixture of sugars created from corn that is similar in taste and sweetness to the sucrose obtained from sugar beets and sugar cane. The process requires three steps, each performed by a different enzyme. Amylase performs the first step of breaking starch into small pieces. Bacterial amylases, like the one shown on the left from PDB entry 2taa, are typically used since they are easy to obtain in large quantities. The second step is performed by a fungal glucoamylase, shown here in the center from PDB entry 1dog. It breaks the small chains into individual glucose units. Unfortunately, glucose does not have a particularly palatable taste, so a third step must be added. This is performed by glucose isomerase, also known as xylose isomerase, as shown on the right from PDB entry 4xia. This enzyme converts some of the glucose into fructose, creating a tasty mixture that is used to sweeten everything from soft drinks to power bars. However, this cheap and widely available sweetener may come with some disadvantages: a quick search on the WWW will reveal a whirlwind of controversy about the role of high fructose corn syrup in obesity and diabetes.

February 2006: Alpha-amylases Exploring the Structure

The active site of alpha-amylase contains a trio of acidic groups (colored white and red) that do most of the work. In the amylase shown here (PDB entry 1ppi), glutamate 233, aspartate 197, and aspartate 300 work together to cleave the connection between two sugars in a starch chain. This structure contains a short chain of five sugar units (colored yellow and orange) bound in the active site. The site of cleavage is shown in pink. A calcium ion, shown as the large gray sphere, is found nearby where it stabilizes the structure of the enzyme. A chloride ion, shown as a green sphere, is bound underneath the active site in many amylases, where it may assist the reaction. This picture was created with RasMol.

J. Enzyme Inhibition, 1995, Vol. 9, pp. 163-170 Reprints available directly from the publisher Photocopying permitted by license only

@ 1995 Harwood Academic Publishers GmbH Printed in Malaysia

THE INHIBITION OF HUMAN SALIVARY a-AMYLASE

TRITICUM AESTIVUM IS COMPETITIVE, BY TYPE I1 a-AMYLASE INHIBITOR FROM

SLOW AND TIGHT-BINDING

DAVID JUDSON GOFF and FREDRICK J. KULL*

Department of Chemistiy, Dartmouth College Hanovel; New Hampshire 03755, USA

(Received 7 October 1994; in final form I7 December 1994)

A kinetic analysis of the inhibition of human salivary a-amylase (EC 3.2.1.1) by wheat seed (Triticum aestivurn) type I1 a-amylase inhibitor revealed the inhibition was slow and tight-binding. The inhibition was competitive with an inhibition binding constant of the a-amylase inhibitor for a-amylase of 0.29 nM. The KM of a-amylase for soluble starch (calculated per mole of a-1,4 linked maltose residues) was 5.87 mM.

KEY WORDS: Human salivary a-amylase; Tn’ticum aestivum type I1 a-amylase inhibitor; Competitive, slow, tight-binding inhibition

INTRODUCTION

Three different proteins in the 20-25,000 M, range have been isolated from wheat seed (Tn’ticum aestivum) that have much greater specificity for human salivary a-amylase (EC 3.2.1.1) than pancreatic a-amylase.14 Although not rigorously quantified, the type I1 inhibitor (M,, 21,400) appears to be more than 6 times a more effective inhibitor of human salivary a-amylase than type I (M,, 22,500) whereas type I has a greater specificity for human salivary a-amylase relative to human pancreatic a- amylase by about the same factor. On the other hand, type I1 is more than an order of magnitude more specific for human salivary a-amylase relative to human pancreatic a-amylase than type I11 (MI, 25,000).3 Although the mode of inhibition of human salivary a-amylase (Mr> 98,000) by any of these three inhibitors has not been defined, it has been noted that the inhibition is strong and that the order of addition of reaction components is imp~rtant.”~ Because these observations mirror characteristics of other more defined hydrolase : protein hydrolase inhibitor including the inhibition of porcine pancreatic a-amylase by a red kidney bean a- amylase inhibitor,* we were prompted to test the question as to whether or not the

*Correspondence: Telephone: (603) 646-1552; Fax: (603) 6463946.

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164 D.J. GOFF and F.J. KULL

mode of inhibition operating in the human salivary a-amylase : wheat germ type I1 a-amylase inhibitor system is also slow and/or tight-binding.

In instances of slow binding inhibition there is a discernible lag period before inhibition is manifested. In tight-binding inhibition the effective concentrations of inhibitor are in the same order as that of the enzymes they inhibit, thus, tight-binding systems cannot be analyzed using classical Michaelis-Menten inhibition kinetics.22527-" In fact, Goldstein12 pointed out in 1944 that in instances where the ratio of [enzyme]: Ki is greater than 0.001 the Michaelis-Menten assumptions do not hold.

We report here experiments demonstrating that the mode of inhibition of the human salivary a-amylase: wheat germ type I1 a-amylase inhibitor system is of the competitive, slow, tight-binding type (Ki, 0.29 nM). This is in contrast to the porcine pancreatic a-amylase : kidney bean a-amylase inhibitor system which, although slow and tight-binding, is non-competitive (K,, 30 PM) ,~

MATERIALS AND METHODS

Human salivary a-amylase (Type IX-A), Type I1 a-amylase inhibitor from wheat germ (T'ticurn aestivum), maltose (Grade I), and soluble starch (ACS reagent grade) were obtained from the Sigma Chemical Company, St. Louis, MO, USA. The activity of a-amylase was measured using the method of Bernfield13 in which the reducing groups of maltose units liberated from starch are measured by the production of a chromophore (maximum absorbance at 540 nm) that results from reduction of 3,5-dinitrosalicylic acid (Worthington Biochemical Corp., Freehold NJ, USA).

Solutions (1%) of 3,5-dinitrosalicylic acid were prepared weekly as follows. First, 1.0 g 3,5-dinitrosalicylic acid was dissolved in 50 ml water. To this 30 g sodium potassium tartrate were slowly added while stirring. Next, 20 ml of 2.0 M NaOH were added and the mixture was then diluted to 100 ml with water.

A standard curve for detection of reducing equivalents was prepared using a variety of maltose concentrations (0.58-5.84 pmol ml-') all in 1.0 ml. To each tube 1.0 ml of the 3,5-dinitrosalicylic acid reagent (above) was added, the resulting mixtures covered with marbles and then heated in a boiling water bath for 5 min. After cooling to 25"C, 10.0 ml of water was added to each tube and after mixing, absorbances at 540 nm were determined versus a water blank. Using the slope of this linear standard curve, the pmol of reducing equivalents produced when soluble starch was incubated with a-amylase could be estimated by dividing the ASM values by 0.172. Several concentrations of maltose were routinely assayed each time enzymatic assays were performed as assay controls.

Soluble starch solutions for enzymatic assays were prepared as follows. 2 g of starch were suspended in a final volume of 100 ml20 mM sodium phosphate buffer (pH 6.9, 6 mM NaCl) and gently boiled. After cooling to 25"C, the volume was readjusted to 100 ml with water. Prior to assay, the stock starch solutions were incubated at 25°C for 4-5 min. Because molar concentrations of soluble starch solution cannot be determined, the concentration of a-amylase's hydrolytic substrate, a-maltose residues,14 was calculated assuming the starch was composed of 100% a-1,4

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INHIBITION OF HUMAN SALIVARY a-AMYLASE 165

linked glucan units. Thus, the starch stock solution translated to 58.6 pmol maltose residues ml-'.

The standard starch solutions were used in enzymatic assays (all done at 25OC) in varying amounts depending on the particular experiment. For example, to determine both the linearity of the assay with respect to time and velocity with respect to a-amylase concentration, matrix-type experiments were performed using saturating amounts of starch (29.3 mM maltose residues; K,, 5.87 mM). For such an experiment 0.5 ml of starch was added to each tube including the non-enzymatic controls. Next 0.25 ml water was added and then reactions were begun in 0.5 min intervals by addition of 0.25 ml of various concentrations of a-amylase (2.9-8.6 nM final concentrations). After incubating for different times (0.5-7.0 rnin), the reactions were stopped by addition of 1.0 ml of the 3,5-dinitrosalicylic acid reagent (44 mM in 0.4 M NaOH). Plots of velocity (pmol reducing equivalent produced m i d ) as a function of a-amylase concentration were linear for 5.0 rnin at all a-amylase concentrations and for 7.0 rnin up to 6.45 nM.

When reaction mixtures contained soluble starch, a-amylase inhibitor and a- amylase, they were also in a final volume of 1.0 ml that was 10 mM in sodium phosphate buffer, pH 6.9 and 3 mM NaCI and were done at 25°C.

A unit of a-amylase activity is defined as the amount of a-amylase necessary to release one pmol of reducing equivalent min-' at 25°C and pH 6.9.

RESULTS

Slow Binding

Wheat germ type I1 a-amylase inhibitor was determined to be a slow-binding inhibitor of human salivary a-amylase by carrying out two types of preincubation experiments. First, starch and a-amylase inhibitor were preincubated for 15 rnin and then the reaction was started with the addition of o-amylase. An initial steady-state velocity changed to a slower steady-state velocity after 2-3 min. Next, amylase and the inhibitor were preincubated (also for 15 min) after which the reaction was initiated by the addition of soluble starch. After a 2-3 rnin lag a steady rate of reaction was observed that was essentially identical to the above steady-state velocity. Both results indicated that a slow type of binding had occurred between inhibitor and enzyme.I5

Tight Binding

Preliminary experiments showed that the molar concentration of a-amylase inhibitor necessary to effectively inhibit a-amylase was of the same order of concentration as a-amylase, a result suggesting that the inhibition was tight-binding. An experiment was then performed to measure the steady state velocities obtained using a range of inhibitor or concentrations at each concentration of a-amylase. As shown by Figure 1, this experiment generated a series of curves with approximately linear and curved segments. At low inhibitor concentrations the linear regions had slopes

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[a-amylase] (nM)

FIGURE 1 Tight binding between a-amylase and a-amylase inhibitor. 0, no inhibitor; +, 1.7 nM inhibitor; ., 2.5 nM inhibitor; 0, 5.3 nM inhibitor. In the presence of 10.5 nM inhibitor no activity was observed. The line for ‘no inhibitor’ was “best fit”; the other lines were approximated. Velocities are jmol reducing equivalents produced min? .

roughly parallel to that of the uninhibited case. (At greater inhibitor concentrations the linear segments are not apparent because the concentration of free inhibitor is significant).” By replotting the data of Figure 1 as well as other data not shown (where the inhibitor concentration relative to enzyme concentration was sufficient for complete inhibition) as depicted in Figure 2, it is apparent that inhibition of a-amylase by a-amylase inhibitor is sigmoidal with 50% inhibition at equal molar concentrations of enzyme and inhibitor and that inhibition was essentially complete when the [a-amylase inhibitor] : [a-amylase] was 5:l. This does not represent a stoichiometric ratio, but rather a ratio which represents the steady-state situation of the various equilibria involved in the overall system.

Determination of the type of tight-binding inhibition

To determine the type of inhibition and the K, of the inhibitor for a-amylase, velocities were determined at several substrate concentrations in the absence and presence of varied amounts of a-amylase inhibitor, each at a fixed concentration of a-amylase. The data generated by this experiment were then analyzed as described by Dixon.’ Figure 3 is for a single concentration of substrate (14.6 mM) and is representative of those obtained at all other concentrations of substrate.

A “I(average” (Kave) was thus obtained for each concentration of starch. Aplot of these K,’s versus substrate concentration produced a straight line with ascending slope that is characteristic of competitive tight-binding inhibition (Figure 4). The ordinate

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INHIBITION OF HUMAN SALIVARY a-AMYLASE 167

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loo 90

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.- - -

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[Inhibitor - Enzyme] (nM)

-

- ” “

FIGURE 2 Inhibition of a-amylase by a-amylase inhibitor. Data from Figure 1 and other data not shown in Figure 1 are plotted to show the relationship between the concentration of free a-amylase and the extent of inhibition. The abscissa shows the nM excess of inhibitor to enzyme.

0.40 k

\ I....I

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[a-amylase Inhibitor] (nM)

FIGURE 3 A plot at one substrate concentration (14.6 mM a-1,4 maltose residues) showing how velocity changed with inhibitor concentration after steady-state between a-amylase inhibitor and a-amylase was established. Following a 15 min preincubation of a-amylase and a-amylase inhibitor, reactions (2.5 min) were begun by addition of substrate. The curved line represents a curve generated by the “exponential” fit of empirical velocity measurements at various concentrations of inhibitor and in the absence of inhibitor (vo, the ordinate value of 0.381 pmol m i d . Points on the curve were selected at %, 3, 2, and I and straight lines were generated from v, through these points to the abscissa. The distance values between the straight lines’ intersections with the abscissa were determined and averaged to obtain the Kve at the particular concentration of substrate; in the case illustrated, 0.97 nM.

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[a-maltose Residues] (mM)

FIGURE 4 Determination of wheat germ, type I1 a-amylase inhibitor’s inhibition binding constant (K,) for human salivary a-amylase (0.29 nM), a-amylases KM for soluble starch (as calculated for a-1,4 maltose residues, 6.20 mM) and the mode of inhibition (competitive). Kve values for each concentration of substrate were obtained as explained in the legend to Figure 3. The line is “best fit”.

intercept is Ki (0.29 nM) and the abscissa intercept yields a value for -KM (6.20 mM). This KM was in good agreement with that calculated from a Lineweaver-Burk plot of the uninhibited system (5.54 mM). The K M ’ s found by these two different kinetic methods were averaged to obtain the KM we report here; 5.87 mM a-maltose units in the soluble starch.

DISCUSSION

Analysis of the mode of inhibition of human salivary a-amylase by wheat seed type I1 a-amylase inhibitor was performed following procedures developed by Goldstein,I2 M o r r i ~ o n , ~ ~ ~ ~ ~ Cha,I7,l8 and Dix~n.’~’~

Formation of the a-amylase: a-amylase inhibitor complex was found to be slow by first preincubating substrate and inhibitor and initiating reaction by addition of a-amylase. As significant amounts of a-amylase became complexed with a- amylase inhibitor, the initial steady-state composed of free enzyme and productive enzyme : substrate complex was perturbed and a new steady-state situation of free enzyme, productive enzyme : substrate complex, and non-productive enzyme: inhibitor complex was established. The length of time necessary to establish this latter, slower steady-state velocity was in the order of 2-3 minutes. When enzyme and inhibitor were preincubated for 15 minutes preceding addition of substrate, a lag of about 2-3 minutes was noted prior to production of product. This lag was

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INHIBITION OF H U I " SALIVARY a-AMYLASE 169

consistent with establishment of a steady-state among free enzyme, enzyme: inhibitor and enzyme : substrate, with the latter leading to product. Therefore, both binding (first trial) and release (second trial) of a-amylase by a-amylase inhibitor is slow, i.e., in the order of 2-3 minutes. It might be relevant to note that steady-state production of product in the typical uninhibited enzymatic reaction is in the order of msec."

Tight-binding should be suspected if an inhibitor is effective at about the same concentration as that of the enzyme. Tight-binding can be confirmed or rejected by observing the effect of covarying enzyme and inhibitor under conditions of saturating substrate." A plot of the data produced by a tight-binding inhibitor produces a series of curves each having linear and curved regions. The curved regions can be extended to the 0:O coordinate. Extrapolation of the linear portions, actually asymptotes, to the abscissa yields information about the moles of inhibitor bound per mole of enzyme."

Tight-binding can be further classified as to its mode: i.e., whether it is non- competitive, competitive, etc. Non-competitive and competitive can be distinguished by determining the K,, values obtained by geometric measurements of data produced by covarying substrate and inhibitor at a fixed enzyme concentration. If the same K,,, is obtained at different substrate concentrations, the inhibition is non-competitive, but if K,,, increases with increasing substrate concentration, the inhibition is competitive. In the latter instance the line extended to the abscissa yields -KM.9

The dimensionless ratio of K, : K, is another indicator of tight-binding if values over 100 are seen.'' In our case the KM : K, ratio, 5.87 mM : 0.29 nM, yielded a value of 20.2 x lo6. Finally, the [enzyme] : K, ratio of our system was in the 28 to 103 range; clearly much greater than the 0.001 value arrived at by Goldstein'* as the upper limit for application of Michaelis-Menten assumptions and kinetic analyses.

Both the human salivary a-amylase : wheat germ type I1 a-amylase inhibitor system studied here and the porcine pancreatic a-amylase : kidney bean a-amylase inhibitor system studied by Wilcox and Whitaker' are slow and tight-binding. However in contrast to the latter system, which follows a non-competitive inhibitory mechanism, the one studied here demonstrated competitive inhibition.

There have been too many reports distinguishing between salivary and pancreatic a-amylases and various categories of a-amylase inhibitors to review here. (See references 3, 4 and 8 for some of these). Thus, it was not surprising that the type of inhibition reported here (competitive) for the human salivary a-amylase : wheat germ type I1 a-amylase inhibitor system differed from the porcine pancreatic a- amylase : kidney bean @-amylase inhibitor system studied by Wilcox and Whitaker.' While the difference between non-competitive and competitive inhibition has some mechanistic implications the significance of this difference between the two systems is largely speculative at this point. The only other difference between these two systems, that perhaps should be mentioned, is in the substrates used for the kinetic analyses : p-nitrophenyl a-D-maltoside' as opposed to soluble starch.

Similar to the a-amylase : a-amylase inhibitor system reported on here and the system studied by Wilcox and Whitaker,' some proteinases and ribonucleases have been shown to be inhibited by slow, tight-binding proteinaceous In all these cases the proteinaceous inhibitors seem to have evolved to serve two different types of functions : regulatory and defensive.

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170 D.J. GOFF and F.J. KULL

At least two different mammalian ribonucleases are inhibited by mammalian ribonuclease inhibitor : pancreatic ribonuclease A, a secretory (extracellular) enzyme and latent alkaline ribonuclease, an intracellular enzyme.' Although the ribonuclease A system can be considered artificial in the sense that there is no evidence of ribonuclease A ever encountering the inhibitor in normal cells, the latter system seems to be involved in intracellular regulation of RNA metabolism.m~21

In contrast to the mammalian ribonuclease system that exists within the same cell, it's assumed that the tight-binding mode of inhibition has evolved in the inter-kingdom proteinase : proteinase inhibitor systems because it confers some sort of selective advantage to plants whose seeds contain the inhibitor. That is, predators would be less likely to benefit from consumption of the seeds containing the inhibitors. The same sort of evolutionary logic could be applied to the a-amylase : a-amylase inhibitor system studied here.

References 1. Silano, V., Furia, M., Gianfreda, L., Macri, A., Palescandolo, R., Rab, A., Scardi, V., Stella, E. and

Valfre, E (1975) Biochim. Bbphys. Acta, 391,170. 2. O'Donnell, M. and McGeeney, K. (1976) Biochim. Biophys. Acta, 422,159. 3. OConnor, C.M. and McGeeney, K.E (1981) Biochim. Biophys. Acta, 658,387. 4. OConnor, C.M. and McGeeney, K.E (1981) Bwchim. Biophys. Acta, 658,397. 5. Green, N.M. and Work, E. (1952) Biochem. J . , 54,347. 6. Wynn, R. and Laskowska, M. Jr. (1990) Biochim. Biophys. Actu, 166,1406. 7. Turner, P.M., Lerea, KM. and Kull, F.J. (1983) Biochem. Biophys. Res. Commun., 114,1154. 8. Wdcox, E.R. and Whitaker, J.R. (1984) Biochemistry, 23,1783. 9. Dixon, M. (1972) Biochem. J . , 129,197.

10. Dixon, M. and Webb, E.C. (1979) Enzymes (3rd Edition). Academic Press; New York. 11. Williams, J.W. and Morrison, J.E (1979) Meth. Enzymol., 6 3 4 437. 12. Goldstein, A. (1944) J. Gen. Physiol., 27,529. 13. Bemfield, €! (1951)Adv. Enzymol., 12,379. 14. Takeshita, M. and Hehre, E.J. (1975)Arch. Bwchem. Biophys., 169,627. 15. Momson, J.E (1982) Pen& Bwchem. Sci., 7,102. 16. Morrison, J.E (1969) Biochim. Biophys. Acta., 185,269. 17. Cha, S. (1975) Biochem. Phamacol., 24,177. 18. Cha, S. (1975) Biochem. Phamacol., 25,2695. 19. Cornish-Bowden, A. (1979) Fundamentals of Enzyme Kinetics., Buttenvorths; London. 20. Blackburn, P. and Moore, S. (1982) The Enzymes, 15,317. 21. Crute, B.E., Markstein, J.A., Kull, EJ., unpublished data; Markstein, J.A., Crute, B.E., Kull, F.J.,

unpublished data.

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