ARTICLE: Isolation of an Insulin-degrading Enzyme from Beef Liver Henry H.

Tomizawa and Yadviga Dowmont Halsey

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

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Isolation

of an Insulin-degrading from Beef Liver*

Enzyme

HENRY

H. TOMIZAWA AND YADVIGA University

DOWMONT HALSEY School of Medicine, Seattle, Washington

From the Departments

of Medicine

and Biochemistry,

of Washington

(Received for publication,

It has been shown that insulin can be rapidly degraded by liver in vitro and that insulin-I131 is useful for studying this process (l-3). Mirsky and Perisutti (4) have presented the hypothesis that a single enzyme with a relatively high degree of specificity for insulin is present in liver. Degradation by liver preparations of other hormones, such as glucagon (5,6) and corticotropin (7)) has been reported. Results from earlier work in this laboratory suggested that a liver enzyme system may degrade several protein and peptide hormones (8). Since the number of enzymes comprising this system was not known, a program of purification was initiated. This report concerns the isolation of a beef liver enzyme which promotes the degradation of insulin.
EXPERIMENTAL

August 25, 1958)

in mg., which will, in 5 minutes at 37, render 1 per cent of the radioactivity of insulin-1131 soluble in trichloroacetic acid. Protein values were determined by the method of Lowry et al. (9). Ability of liver preparations to degrade insulin-I131 was rapidly determined under these conditions. In the lower range of values for percentage supernatant radioactivity, the extent of degradation of insulin-1131 was in direct proportion to the concentration of liver proteins, as shown in Table I, A. In addition, upon fractionation of a given liver preparation under mild conditions, satisfactory recovery of total activity in the resultant fractions could be demonstrated by this assay (Table I, B). Purification Procedure

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Assay System Purification of the enzyme was followed with the use of insulin-1131 as substrate and by means of a modification of a previously described assay (2). Each reaction was carried out in a 12-ml. centrifuge tube in a system containing a buffer of 0.1 M potassium phosphate: pH 7.5, with 5 X lO-3 M Vcrsene (the disodium salt of ethylenediaminetetraacetic acid). A quantity of phosphate buffer and 0.1 ml. of 0.02 itt GSH were combined such that the addition of 5 to 100 ~1. of a liver preparation brought the volume to 1 ml. A ml. of substrate solution, 0.4 to 0.5 KC. (<5 pg.) of dialyzed insulin-I131 with 0.1 mg. of amorphous insulin in phosphate buffer, was added and the mixture immediately placed in a 37-bath. 15 seconds before terminating the 5-minute incubation with 3 ml. of 10 per cent trichloroacetic acid, 1 ml. of a 2 per cent solution of dried human plasma was added as carrier protein. 10 incubations begun at 30-second intervals could be run within a lominute period. The precipitates and supernatant fractions were prepared and counted in the manner described previously (2). Enzymatic activity was expressed as the percentage of radioactivity in the supernatant fraction per mg. of liver protein. Thus, a unit of activity is the amount of liver protein, * This investigation was supported by Research Grants A-575 and A-761, from the National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, United States Public Health Service. 1 Crystalline zinc insulin (Lilly), assaying 27 units per mg., was radio-iodinated by Abbott Laboratories. Amorphous insulin, assaying 21 units per mg., was a gift of Eli Lilly and Company. 2 0.1 M potassium phosphate, pH 7.5, containing 5 X 10-a M Versene (ethylenediaminetet,raaceta.te) will henceforth be designated as phosphate buffer.

Liver Powder-This was prepared from fresh beef liver by a procedure similar to one used to prepare pancreatic powder (10). Immediately after removal of a 5-kg.3 liver from a steer, the organ was kept on ice and brought to the laboratory cold room for processing. Unless otherwise noted, purification was carried out at 5. The liver was perfused with cold water to remove blood from the main vessels. All fibrous matter was removed before the remaining 4 kg. of liver tissue were passed through an electric meat grinder with a sieve containing 3-mm. holes. This grinding was repeated four more times to yield a mixture with a mushy consistency. Four volumes of cold acetone were added to the paste, and the mixture was stirred for 5 hours. The suspension was filtered in two IO-inch diameter Buchner funnels through Whatman No. 1 filter paper, and the precipitate was washed with acetone. 3 volumes of acetone were added to the precipitate and the mixture was stirred another 5 hours, filtered, and washed. The procedure was repeated after stirring for 10 hours. The precipitate was then shaken for 12 hours with 2 volumes of acetoneether (1: 1) in a 10-l. bottle. The precipitate was collected by filtration and washed and then was shaken for 12 hours with 2 volumes of ether. After a second shaking with 2 volumes of ether for 12 hours, the filtered precipitate was allowed to air-dry at about 5, preferably in open air. When the odor of ether was no longer evident, the tan powder was placed in vacuum desiccators over silica gel. The yield of powder from 4 kg. of processed liver tissue was 900 gm. gm. of liver powder were Extraction of Liver Powder-90 ground with mortar and pestle and stirred with 1.4 1. of 0.1 M tris(hydroxymethyl)aminomethane, pH 7.5, for 2.5 hours. After centrifuging, the extract was passed through glass wool, 3 Approximate values are given in the description fication procedure. of the puri-

307

308

Isolation of Insulin-degrading
TABLE I adequacy of insulin-1131 assay system -

Enzyme

Vol. 234, No. 2

Evidence
A

for

vVeight of protein mg. I0.15 I0.30 I0.60 1.20 ,0.0012 /0.0024 /0.0048 /0.0096 Specific activity

1 -

Supernatant xdioactivity 70 of fold 1.5 3.2 5.4 8.4 2.5 4.7

Specific activityt

Preparation Dialyzed Dialyzed Dialyzed Dialyzed 12-18yo 12-18yo 12-18% 12-18%

extract........... extract. extract. extract........... Ethanol Ethanol Ethanol Ethanol

B

10 11 9
7 2,080

fraction. fraction. fraction. fraction.

9.1 12.8
Total weight of protein w.

--

1,960 1,900 1,330

1 ?&al activityt

Preparation Collected proteins.. Fraction of collected proteins precipitating with ammonium sulfate at: o-0.50 saturation.. 0.504.60 saturation.. 0.60-0.85 saturation.. . Total.

155

1,870

289 ) 800

35 122 335

936 431 532 1,899 102% of

32,750 52,640 178,308 263,698

collectec 1 proteins
-

91% of collected proteins

* Corrected for approximately 1 per cent trichloroacetic acidnonprecipitable radioactivity. t Specific activity = corrected percentage of supernatant radioactivity per mg. of protein. $ Total activity = specific activity X total mg. of protein. then dialyzed against 18 1. of distilled water for 18 hours with one change of water. 1.3 1. of reddish brown dialyzed extract contained 12 mg. of protein per ml. Treatment with Amberlite XE-64--Ion exchange resin with settling-time in water between 2 and 15 minutes was used. The resin was treated with 4 N HCl for 1 day, washed with water, and then adjusted to a pH of 5.6 with 2 N NaOH. Resin equilibrated after treatment with several changes of 0.05 M citrate, pH 5.63, containing 5 X 1O-3 M Versene, was stored at 5 with thymol as preservative. For reactivation, used resin from 2 columns was left overnight 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 adjusted to pH 8. After washing until the odor of BRIJ was no longer noticeable, 3 volumes of 4 N HCl were added to the resin and the mixture was heated on a steam bath for 1 day with occasional stirring. Treatment with BRIJ and HCl was repeated until 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 passed at a rate of 3 ml. per minute through one of two 7-cm. diameter 4 BRIJ 35, polyoxyethylene lauryl alcohol, obtained from the Atlas Powder Company, Wilmington, Delaware.

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. Ammonium sulfate was added to the 1.6 1. of protein-containing eluate to a saturation of 0.85. After centrifugation after 30 minutes, the precipitate was dissolved in 100 ml. of phosphate buffer and was frozen. Ammonium Sulfate Fractionation-After thawing, the increase in volume over that of the 100 ml. of buffer was assumed to be due to residual 0.85~saturated ammonium sulfate. Upon dilution with buffer to an ammonium sulfate saturation of 0.1, the resultant 180 ml. of brownish yellow solution contained 10 mg. of protein per ml. After the addition of ammonium sulfate to 0.5 saturation, the mixture was centrifuged. The procedure was repeated at 0.60 and 0.85 saturation with ammonium sulfate, and the final precipitate was brought to 50 ml. with phosphate buffer. Ethanol Fractionation-The solution containing protein prccipitating between 0.60 and 0.85 saturation with ammonium sulfate was dialyzed overnight against 4 1. of deionized water containing 1 X 10-a M Versene, with one change of dialyzing medium. 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 MgC12.6H20 and 14 ml. of 0.25 M citrate buffer, pH 5.8, 19.1 ml. of chilled redistilled absolute ethanol were stirred into the solution which was placed in a bath at -2. After additional stirring for 3 minutes, the mixture was centrifuged at -5. Upon the addition of 11.63 ml. of absolute ethanol to the supernatant fraction and centrifugation, the precipitate, protein-insoluble in ethanol concentrations between 12 and 18 per cent, was dissolved in 4

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

Purijication
Preparation

and

recovery

of

isolated

enzyme

Total weight of protein

$;f
Dialyzed extract*. . Collected proteins. 0.69-0.85 saturation with (NHI)&SO~. 0.12-0.18 ethanol...... Active peak.. 15,600 1,775
458 57 8 343,200 257,400 137,280 85,800 41,184

70
75 40 25 12

i I i
6.5 300 1,500 5,148 14 68 234

22 145

* 90 gm. of liver powder.

February

1959

H. H. Tomizawa

and Y. D. Halsey

ml. of 0.01M phosphate buffer, pH 7.5, with 5 X 1O-3 M Versene. After overnight dialysis against 500 ml. of the same buffer with one change,the 6 ml. of pale yellow solution contained10 mg. of protein per ml. Zone Etectrophoretic Purification-Washed technical grade of potato starch (11) was used in an electrophoretic apparatus recently describedby Goldsworthy and Volwiler (12). The solution containing 12 to 18 per cent ethanol-precipitableprotein was lyophilized to 3 ml. One-half of this volume was applied 9 cm. from a short sideof eachof two blocks containing potassiumphosphate, pH 8.0, ionic strength, 0.075. A current of 25 ma. per block (500 to 600 volts) was applied for 16 to 17 hours. A lengthwisestrip 1 cm. wide was cut from each block, and this in turn was sliced into 5-mm. segments with a multiblade cutter. After elution of protein within each segmentwith 1 ml. of water, aliquots were taken for protein analysis as well as for enzymatic activity. Fig. 1 showsthe results from a representative starch block. The portion of each block containing material of a constant specific activity (enzyme units per mg. of protein) was eluted and the eluate combined with the remainsof the correspondingsegments of the lengthwisestrip. The 60 ml. of enzyme solution containing 0.15 mg. of protein per ml. were essentiallycolorlessat this high dilution. The constant specificactivity of the eluatesof a portion of the starch block of approximately 0.1 of its entire FIG. 2. Sedimentation diagram of the purified insulin-degradlength indicates that the activity has been isolatedin a highly ing enzyme in 0.01 M potassium phosphate, pH 7.5. This pattern purified form. was obtained after 88 minutes at 59,160 r.p.m. with the Spinco Protein which migratesconsiderablymoreslowly in the buffer model E ultracentrifuge. systemusedcontainsactivity which degrades glucagon.6 Currently, efforts to purify this activity further are in progress. Table II showsin generalthe extent of purification and recovery of the isolated insulin-degradingenzyme at each step of the process. The values for both purification and recovery may be low since the presenceof more than one insulin-Imdegradingactivity in liver powder is not unlikely.
Further Evidence of Homogeneity of Isolated Enzyme Sedimentation Diagram-Enzyme isolated by several runs through the preparatory procedure was combined and lyophilizcd. The sedimentationpattern of this material at a concentrationof 11.7mg. per ml. in 0.01M potassium phosphate, pH 7.5, was determined with the use of an analytical ultracentrifuge Fig. 2 showsa resultant pattern which gives no indication of inhomogeneity. The sedimentation constant (~3.2~) was 3.0 S Paper Electrophoretic Patterns-Since not enough material was available for a moving boundary electrophoresis,paper electrophoreticpatterns were determinedin two buffer systems. FIG. 3. Paper electrophoretic patterns of the ethanol-fracTheseresultsshownin Fig. 3 demonstratethe effective separa- tionated liver protein (A and C) and of the purified enzyme subsetion of the enzyme from contaminating proteins present subsc- quently isolated by zone electrophoresis (B and D). Upper quent to ethanol fractionation. patterns were obtained with the use of potassium phosphate, pH
DISCUSSION

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8.0, ionic strength 0.1. Lower patterns were obtained use of diethylbarbiturate, pH 8.6, ionic strength 0.05.

with the

The high degreeof purity of the isolatedenzyme is indicated by the constant specificactivity of the active peak after starch block electrophoresis. Resultsfrom the determination of paper electrophoretic and ultracentrifugal patterns are corroborative 6The authorsare indebtedto Dr. P. D. Goldsworthy for useof evidence indicating a highly effective separation from other this equipment and for his kind assistance. proteins. 6 Unpublished results. Although insulin-113i was the measurable substrate used for 7 The authors are grateful to Mr. R. D. Wade of the Depart. ment of Biochemistry for this determination. following the purification of this enzyme, it is certain that in-

310

Isolation of Insulin-degrading

Enzyme
SUMMARY

Vol. 234, No. 2

Upon incubation of insulin in phossulin itself is a substrate. phate buffer with a sulfhydryl compound such as GSH or 2-mercaptoethanol, the addition of this enzyme greatly increa.ses the rate at which the solution becomes turbid. Along with this is an increase in the amount of trichloroacetic acid-soluble material from insulin.6 Although the isolated enzyme may promote the cleavage of peptide bonds of insulin, enzymatic reductive cleavage of disulfide bonds, a possibility investigated by Racker (13) and by Narahara and Williams (14), must also
be considered.

A highly purified enzyme which promotes the degradation of insulin has been isolated from beef liver. The usefulness of an 1131-labeled protein as substrate for developing a procedure for enzyme purification has also been demonstrated. Acknowledgments-The authors wish to thank Dr. R. H. Williams for his encouragement and support, and Mrs. Ann Harmon, Mrs. Adclc Hopkins, and Mrs. Ellen Larson for excellent technical assistance.

REFERENCES
1. VAUGHAN, M., Biochim. et Biophys. Acta, 16, 432 (1954). 2. TOMIZAWA, H. H., NUTLEY, M. L., NARAHARA, H. T., AND WILLIAMS, R. H., J. Biol. Chem., 214, 285 (1955). 3. MIRSKY, I. A., PERISKJTTI, G., AND DIXON, F. J., J. Biol. 8. TOMIZAWA,

9. 10. 11. 12. 13. 14.

4. 5. 6. 7.

Chem., 214, 397 (1955). MIRSKY, I. A., AND PERISUTTI, G., J. Biol. Chem., 228, 77 (1957). KENNY, A. J., Am. J. Physiol., 186, 419 (1956). NARAHARA, H. T., AND WILLIAMS, R. H., Endocrinology, 60, 285 (1957). GESCHWIND, I. I., AND LI, C. H., Endocrinology, 60,226 (1952).

H. H., AND WILLIAMS, R. H., J. Biol. Chem., 217, 685 (1955). LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J., J. Biol. Chem., 193, 265 (1951). FISCHER, E. H., AND STEIN, E. A., Arch. xi. Geneva, 7, 131 (1954). PAIGEN, K., Anal. Chem., 28, 284 (1956). GOLDSWORTHY, P. D., AND VOLWILER, W., J. Biol. Chem., 230, 817 (1958). RACKER, E., Federation Proc., 12, 711 (1953). NARAHARA, H. T., AND WILLIAMS, R. H., J. BioZ. Chem., 233, 1034 (1958).

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