J. Biol. Chem. 1959 Tomizawa 307 10

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HalseyHenry H. Tomizawa and Yadviga Dowmontfrom Beef LiverIsolation of an Insulin-degrading EnzymeARTICLE:

1959, 234:307-310.J. Biol. Chem.

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Isolation of an Insulin-degrading Enzymefrom Beef Liver*

HENRY H. TOMIZAWA AND YADVIGA DOWMONT HALSEYFrom the Departments of Medicine and Biochemistry, University of Washington School of Medicine, Seattle, Washington

(Received for publication, August 25, 1958)It has been shown that insulin can be rapidly degraded byliver in vitro and that insulin-I131 is useful for studying this process(l-3). Mirsky and Perisutti (4) have presented the hypothesisthat a single enzyme with a relatively high degree of specificity

for insulin is present in liver. Degradation by liver prepara-tions of other hormones, such as glucagon (5,6) and corticotropin(7)) has been reported. Results from earlier work in this labora-tory suggested that a liver enzyme system may degrade severalprotein and peptide hormones (8). Since the number of enzymescomprising this system was not known, a program of purifica-tion was initiated.This report concerns the isolation of a beef liver enzyme whichpromotes the degradation of insulin.

EXPERIMENTAL

Assay SystemPurification of the enzyme was followed with the use of in-

sulin-1131 as substrate and by means of a modification of apreviously described assay (2). Each reaction was carried outin a 12-ml. centrifuge tube in a system containing a buffer of0.1 M potassium phosphate: pH 7.5, with 5 X lO-3 M Vcrsene(the disodium salt of ethylenediaminetetraacetic acid). A quan-tity of phosphate buffer and 0.1 ml. o f 0.02 itt GSH werecombined such that the addition of 5 to 100 ~1. of a liver prep-aration brought the volume to 1 ml. A ml. of substrate solu-tion, 0.4 to 0.5 KC. (


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308 Isolation of Insulin-degrading Enzyme Vol. 234, No. 2TABLE I

Evidence for adequacy of insulin-1131 assay systemA

PreparationDialyzed extract...........Dialyzed extract.Dialyzed extract.Dialyzed extract...........12-18yo Ethanol fraction.12-18yo Ethanol fraction.12-18% Ethanol fraction.12-18% Ethanol fraction.

B

PreparationCollected proteins..Fraction of collected pro-teins precipitating withammonium sulfate at:o-0.50 saturation..0.504.60 saturation..0.60-0.85 saturation.. .Total.

-v-

III

,///

-

Veight ofproteinmg.

-

1-

Supernatantxdioactivity70 of fold

0.15 1.50.30 3.20.60 5.41.20 8.40.00120.00240.00480.0096Specificactivity

2.5 2,0804.7 1,9609.1 1,90012.8 1,330-- Total weightof proteinw.

155

35122335

1,870

936431532

1,899102% ofcollectecproteins

--1

1-

Specificactivityt

101197

?&al activityt

289 ) 800

32,75052,640178,308263,69891% of col-lectedproteins

* Corrected for approximately 1 per cent trichloroacetic acid-nonprecipitable radioactivity.t Specific activity = corrected percentage of supernatantradioactivity per mg. of protein.Total activity = specific activity X total mg. of protein.then dialyzed against 18 1. of distilled water for 18 hours withone change of water. 1.3 1. of reddish brown dialyzed extractcontained 12 mg. of protein per ml.Treatment with Amberlite XE-64--Ion exchange resin withsettling-time in water between 2 and 15 minutes was used.The resin was treated with 4 N HCl for 1 day, washed withwater, and then adjusted to a pH o f 5.6 with 2 N NaOH. Resinequilibrated after treatment with several changes of 0.05 Mcitrate, pH 5.63, containing 5 X 1O-3 M Versene, was stored at5 with thymol as preservative.

For reactivation, used resin from 2 columns was left over-night in 800 ml. of 1 M NaCl adjusted to pH 8 with NaOH.This was followed by overnight treatment with 10 gm. of BRIJ(polyoxyethylene lauryl alcohol)4 in 2 1. of hot water adjustedto pH 8. After washing until the odor of BRIJ was no longernoticeable, 3 volumes of 4 N HCl were added to the resin andthe mixture was heated on a steam bath for 1 day with occa-sional stirring. Treatment with BRIJ and HCl was repeateduntil the color of the resin matched that of unused resin.The dialyzed extract was mixed with 0.5 volume of 1 M citrate,pH 5.63, and 0.5 volume of 0.1 M Versene. This was passedat a rate of 3 ml. per minute through one of two 7-cm. diameter

columns, each filled to a depth of 18 cm. with equilibrated resin.Buffer, 5 X 1O-2 M citrate, pH 5.63, with 1 X 1O-3 M Versene,was then added in excess of the hold-up volume. Ammon iumsulfate was added to the 1.6 1. of protein-containing eluate to asaturation of 0.85. After centrifugation after 30 minutes, theprecipitate was dissolved in 100 ml. of phosphate buffer andwas frozen.Ammonium Sulfate Fractionation-After thawing, the in-crease in volume over that of the 100 ml. of buffer was assumedto be due to residual 0.85~saturated ammonium sulfate. Upondilution with buffer to an ammonium sulfate saturation of 0.1,the resultant 180 ml. of brownish yellow so lution contained 10mg. of protein per ml. After the addition of ammonium sulfateto 0.5 saturation, the mixture was centrifuged. The procedurewas repeated at 0.60 and 0.85 saturation with ammonium sul-fate, and the final precipitate was brought to 50 ml. with phos-phate buffer.Ethanol Fractionation-The solution containing protein prc-cipitating between 0.60 and 0.85 saturation with ammoniumsulfate was dialyzed overnight against 4 1. of deionized watercontaining 1 X 10-a M Versene, with one change of dialyzingmedium. The pale brownish yellow solution contained 4 mg.of protein per ml. after adjustment of the volume to 126 ml.with deionized water. After addition of 1.4 gm. of MgC12.6H20and 14 ml. of 0.25 M citrate buffer, pH 5.8, 19.1 ml. of chilledredistilled absolute ethanol were stirred into the solution whichwas placed in a bath at -2. After additional stirring for 3minutes, the mixture was centrifuged at -5. Upon the addi-tion o f 11.63 ml. of absolute ethanol to the supernatant fractionand centrifugation, the precipitate, protein-insoluble in ethanolconcentrations between 12 and 18 per cent, was dissolved in 4

FIG. 1. Zone electrophoretic purification of the insulin-degrad-ing enzyme from ethanol-fractionated liver protein. O--O,protein; A---A, activity.TABLE II

Purijication and recovery of isolated enzymePreparation Totalweight ofprotein

Dialyzed extract*. . 15,600Collected proteins. 1,7750.69-0.85 saturationwith (NHI)&SO~. 4580.12-0.18 ethanol...... 57Active peak.. 8

* 90 gm. of liver powder.4 BRIJ 35, polyoxyethylene lauryl alcohol, obtained from theAtlas Powder Company, Wilmington, Delaware.

;f 70343,200 22257,400 145 6.5 75137,280 30085,800 1,50041,184

i5,148

I 4 i 068 25

234 12

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February 1959 H. H. Tomizawa and Y. D. Halseyml. of 0.01 phosphate uffer, pH 7.5, with 5 X 1O-3 Versene.After overnight dialysis against 500 ml. of the same bufferwith one change, he 6 ml. of pale yellow solution contained10mg. of protein per ml.

Zone Etectrophoretic Purification-Washed technical grade ofpotato starch 11) was used in an electrophoretic apparatusrecently describedby Goldsworthy and Volwiler 12). Thesolution containing 12 to 18 per cent ethanol-precipitablepro-tein was lyophilized to 3 ml. One-half of this volume wasapplied 9 cm. from a short sideof eachof two blocks contain-ing potassiumphosphate, pH 8.0, ionic strength, 0.075. Acurrent of 25 ma. per block 500 to 600 volts) was applied for16 to 17 hours. A lengthwisestrip 1 cm. wide was cut fromeach block, and this in turn was sliced into 5-mm. segmentswith a multiblade cutter. After elution of protein within eachsegmentwith 1 ml. of water, aliquots were taken for proteinanalysis as well as for enzymatic activity. Fig. 1 shows heresults from a representative starch block. The portion ofeach block containing material of a constant specific activityenzyme units per mg. of protein) was eluted and the eluatecombined with the remainsof the correspondingsegments fthe lengthwisestrip. The 60 ml. of enzyme solution containing0.15 mg. of protein per ml. were essentiallycolorlessat thishigh dilution. The constant specificactivity of the eluatesofa portion of the starch block of approximately 0.1 of its entirelength indicates that the activity has been solated n a highlypurified form.Protein which migratesconsiderablymoreslowly in the buffersystemusedcontainsactivity which degrades lucagon.6 Cur-rently, efforts to purify this activity further are n progress.Table II shows n general he extent of purification and re-covery of the isolated insulin-degradingenzyme at each step

of the process. The values for both purification and recoverymay be low since the presenceof more than one insulin-Im-degradingactivity in liver powder s not unlikely.

FIG 2. Sedimentation diagram of the purified insulin-degrad-ing enzyme in 0.01 M potassium phosphate, pH 7.5. This patternwas obtained after 88 minutes at 59,160 r.p.m. with the Spincomodel E ultracentrifuge.

Further Evidence of Homogeneity of Isolated EnzymeSedimentation Diagram-Enzyme isolated by several runsthrough the preparatory procedure was combined and ly-ophilizcd. The sedimentationpattern of this material at aconcentrationof 11.7mg. per ml. in 0.01 potassium hosphate,pH 7.5, was determined with the use of an analytical ultra-centrifuge Fig. 2 showsa resultant pattern which gives noindication of inhomogeneity. The sedimentation constant~3.2~)as 3.0 SPaper Electrophoretic Patterns-Since not enough materialwas available for a moving boundary electrophoresis,paperelectrophoreticpatterns were determined n two buffer systems.These esultsshown n Fig. 3 demonstrate he effective separa-tion of the enzyme from contaminating proteins present subsc-quent to ethanol fractionation.

DISCUSSION

FIG 3. Paper electrophoretic patterns of the ethanol-frac-tionated liver protein (A and C) and of the purified enzyme subse-quently isolated by zone electrophoresis (B and D). Upperpatterns were obtained with the use of potassium phosphate, pH8.0, ionic strength 0.1. Lower patterns were obtained with theuse of diethylbarbiturate, pH 8.6, ionic strength 0.05.The high degreeof purity of the isolatedenzyme s indicatedby the constant specificactivity of the active peak after starch6The authorsare ndebted o Dr. P. D. Goldsworthy for useofthis equipment and for his kind assistance.6 Unpublished results.7 The authors are grateful to Mr. R. D. Wade of the Depart-.

block electrophoresis. Results rom the determination of paperelectrophoretic and ultracentrifugal patterns are corroborativeevidence indicating a highly effective separation from otherproteins.Although insulin-113i as the measurable ubstrate used for

ment of Biochemistry for this determination following the purification of this enzyme, it is certain that in-

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J. Biol. Chem. 1959 Tomizawa 307 10

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