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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of the puri- 307 . of a liver preparation brought the volume to 1 ml.raaceta. 1 filter paper. 1 Crystalline zinc insulin (Lilly). (<5 pg. which will. the tan powder was placed in vacuum desiccators over silica gel. for 2. assaying 27 units per mg. After centrifuging. of a 2 per cent solution of dried human plasma was added as carrier protein. 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. The precipitate was collected by filtration and washed and then was shaken for 12 hours with 2 volumes of ether. the filtered precipitate was allowed to air-dry at about 5”.6) and corticotropin (7)) has been reported. a program of purification was initiated. gm. 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). Thus. Protein values were determined by the method of Lowry et al. 3 Approximate values are given in the description fication procedure. 2013 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). 10 incubations begun at 30-second intervals could be run within a lominute period.1 mg. * This investigation was supported by Research Grants A-575 and A-761. Unless otherwise noted. with 5 X lO-3 M Vcrsene (the disodium salt of ethylenediaminetetraacetic acid). in 5 minutes at 37”. and washed. A quantity of phosphate buffer and 0.4 to 0. of processed liver tissue was 900 gm. 0.4 1. The suspension was filtered in two IO-inch diameter Buchner funnels through Whatman No. EXPERIMENTAL August 25. of substrate solution. After a second shaking with 2 volumes of ether for 12 hours.1 ml. preferably in open air. was radio-iodinated by Abbott Laboratories. Liver Powder-This was prepared from fresh beef liver by a procedure similar to one used to prepare pancreatic powder (10). 2 0. assaying 21 units per mg. All fibrous matter was removed before the remaining 4 kg.5 hours. Immediately after removal of a 5-kg. The liver was perfused with cold water to remove blood from the main vessels. The yield of powder from 4 kg. Results from earlier work in this laboratory suggested that a liver enzyme system may degrade several protein and peptide hormones (8). 15 seconds before terminating the 5-minute incubation with 3 ml. purification was carried out at 5”. Purification Procedure Downloaded from http://www. Amorphous insulin. of amorphous insulin’ in phosphate buffer.. Since the number of enzymes comprising this system was not known.02 itt GSH were combined such that the addition of 5 to 100 ~1. Four volumes of cold acetone were added to the paste. Degradation by liver preparations of other hormones. National Institutes of Health. A ml. TOMIZAWA AND YADVIGA University DOWMONT HALSEY School of Medicine. This grinding was repeated four more times to yield a mixture with a mushy consistency.1 M potassium phosphate. This report concerns the isolation of a beef liver enzyme which promotes the degradation of insulin. the extent of degradation of insulin-1131 was in direct proportion to the concentration of liver proteins. In the lower range of values for percentage supernatant radioactivity.5. United States Public Health Service.org/ by guest on November 25. of liver tissue were passed through an electric meat grinder with a sieve containing 3-mm. upon fractionation of a given liver preparation under mild conditions. as shown in Table I. of liver protein.5. 1 ml. bottle. a unit of activity is the amount of liver protein. filtered.1 M tris(hydroxymethyl)aminomethane. of 0. was a gift of Eli Lilly and Company.3 liver from a steer.5. such as glucagon (5. the extract was passed through glass wool. A. of liver powder were Extraction of Liver Powder-90 ground with mortar and pestle and stirred with 1. In addition.1 M potassium phosphate: pH 7. Each reaction was carried out in a 12-ml. pH 7. the organ was kept on ice and brought to the laboratory cold room for processing. The precipitate was then shaken for 12 hours with 2 volumes of acetoneether (1: 1) in a 10-l.te) will henceforth be designated as phosphate buffer. When the odor of ether was no longer evident. of Washington (Received for publication.) of dialyzed insulin-I131 with 0. Ability of liver preparations to degrade insulin-I131 was rapidly determined under these conditions. 1958) in mg..Isolation of an Insulin-degrading from Beef Liver* Enzyme HENRY H. satisfactory recovery of total activity in the resultant fractions could be demonstrated by this assay (Table I. was added and the mixture immediately placed in a 37”-bath. and the precipitate was washed with acetone. containing 5 X 10-a M Versene (ethylenediaminetet. Enzymatic activity was expressed as the percentage of radioactivity in the supernatant fraction per mg. from the National Institute of Arthritis and Metabolic Diseases. 3 volumes of acetone were added to the precipitate and the mixture was stirred another 5 hours. (9). and the mixture was stirred for 5 hours. Seattle. of 10 per cent trichloroacetic acid. centrifuge tube in a system containing a buffer of 0. of 0. Washington From the Departments of Medicine and Biochemistry.jbc. render 1 per cent of the radioactivity of insulin-1131 soluble in trichloroacetic acid. holes..5 KC. The precipitates and supernatant fractions were prepared and counted in the manner described previously (2). The procedure was repeated after stirring for 10 hours. B). pH 7.

8. fraction. fraction.4 gm.504. . 0. pH 5.6H20 and 14 ml.. Fraction of collected proteins precipitating with ammonium sulfate at: o-0..775 458 57 8 343. After washing until the odor of BRIJ was no longer noticeable.400 137. 15..870 289 ) 800 35 122 335 936 431 532 1.080 fraction.. Buffer.63 ml.. and then adjusted to a pH of 5. of buffer was assumed to be due to residual 0.6 1.. pH 5. of 1 M NaCl adjusted to pH 8 with NaOH. with equilibrated resin... obtained from the Atlas Powder Company.12-0.2 5. 1.. After the addition of ammonium sulfate to 0.. of liver powder. with deionized water. washed with water.800 41.698 collectec 1 proteins - 91% of collected proteins * Corrected for approximately 1 per cent trichloroacetic acidnonprecipitable radioactivity. of distilled water for 18 hours with one change of water. with phosphate buffer.6 with 2 N NaOH.0048 /0.5 volume of 1 M citrate. Ethanol Ethanol Ethanol Ethanol B 10 11 9 7 2. and the final precipitate was brought to 50 ml..8 Total weight of protein w. was dissolved in 4 Downloaded from http://www.20 . of protein-containing eluate to a saturation of 0. I0.. the mixture was centrifuged.330 ‘1 ?&al activityt Preparation Collected proteins.15 I0. of deionized water containing 1 X 10-a M Versene.. activity. was stored at 5” with thymol as preservative.5 saturation..7 Specific activityt Preparation Dialyzed Dialyzed Dialyzed Dialyzed 12-18yo 12-18yo 12-18% 12-18% extract. The resin was treated with 4 N HCl for 1 day. the precipitate was dissolved in 100 ml.jbc. Ammonium sulfate was added to the 1.308 Isolation of Insulin-degrading TABLE I adequacy of insulin-1131 assay system - Enzyme Vol.0096 Specific activity 1 - Supernatant xdioactivity’ 70 of fold 1. For reactivation. TABLE II Purijication Preparation and recovery of isolated enzyme Total weight of protein $. 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 precipitate. 0. 234. This was passed at a rate of 3 ml.1. 19. 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..4 2..0024 /0. Total.. 2 Evidence A for vVeight of protein mg.0.60 1. protein. Resin equilibrated after treatment with several changes of 0.. Ethanol Fractionation-The solution containing protein prccipitating between 0. No.63. of absolute ethanol to the supernatant fraction and centrifugation. of reddish brown dialyzed extract contained 12 mg.85 saturation with ammonium sulfate.184 70 75 40 25 12 i I i 6.69-0. the resultant 180 ml..899 102% of 32. pH 5.280 85. was then added in excess of the hold-up volume.960 1. 5 X 1O-2 M citrate. . 0. pH 5. per minute through one of two 7-cm.. each filled to a depth of 18 cm.5 4.5 volume of 0. Treatment with BRIJ and HCl was repeated until the color of the resin matched that of unused resin.5 300 1. 0. of protein per ml. then dialyzed against 18 1. After addition of 1. The dialyzed extract was mixed with 0.f Dialyzed extract*.. columns.85 saturation with (NHI)&SO~.600 1. of protein. Active peak. This was followed by overnight treatment with 10 gm.5 3. A---A.. $ Total activity = specific activity X total mg. Wilmington. Upon the addition of 11. Upon dilution with buffer to an ammonium sulfate saturation of 0.60 and 0. Collected proteins.60 and 0. O--O.85~saturated ammonium sulfate.85.. diameter 4 BRIJ 35. -- 1. - 9. Delaware.640 178. The procedure was repeated at 0.148 14 68 234 22 145 * 90 gm.63. used resin from 2 columns was left overnight in 800 ml. of MgC12.50 saturation. of 0.200 257. of protein per ml.1 M Versene.85 saturation with ammonium sulfate was dialyzed overnight against 4 1. . containing 5 X 1O-3 M Versene.900 1.25 M citrate buffer.3 1.63.. protein-insoluble in ethanol concentrations between 12 and 18 per cent. 155 1. the mixture was centrifuged at -5”.308 263.1 ml. After additional stirring for 3 minutes.0012 /0.1 12. extract.30 I0.18 ethanol.05 M citrate. Ammonium Sulfate Fractionation-After thawing. Zone electrophoretic purification of the insulin-degrading enzyme from ethanol-fractionated liver protein.org/ by guest on November 25. of phosphate buffer and was frozen.500 5. After centrifugation after 30 minutes. 2013 FIG. and 0. fraction. of BRIJ (polyoxyethylene lauryl alcohol)4 in 2 1. of protein. after adjustment of the volume to 126 ml.60 saturation. 1. the increase in volume over that of the 100 ml. of chilled redistilled absolute ethanol were stirred into the solution which was placed in a bath at -2”.85 saturation.4 8... with one change of dialyzing medium. of hot water adjusted to pH 8. of brownish yellow solution contained 10 mg. t Specific activity = corrected percentage of supernatant radioactivity per mg. with 1 X 1O-3 M Versene. polyoxyethylene lauryl alcohol.. extract. extract..60-0.. The pale brownish yellow solution contained 4 mg.750 52..

Wade of the Depart. and this in turn was sliced into 5-mm. FIG. from a short sideof eachof two blocks containing potassiumphosphate. The 60 ml. of protein per ml.the 6 ml. Although insulin-113i was the measurable substrate used for 7 The authors are grateful to Mr. The solution containing 12 to 18 per cent ethanol-precipitableprotein was lyophilized to 3 ml.0. The sedimentationpattern of this material at a concentrationof 11. 2. 3. Resultsfrom the determination of paper electrophoretic and ultracentrifugal patterns are corroborative 6The authorsare indebtedto Dr. were essentiallycolorlessat this high dilution. pH 8. R.075. was obtained after 88 minutes at 59.February 1959 H.1 of its entire FIG. 6 Unpublished results. ment of Biochemistry for this determination.jbc. following the purification of this enzyme. proteins.160 r. in 0. Tomizawa and Y. This pattern purified form. Goldsworthy for useof evidence indicating a highly effective separation from other this equipment and for his kind assistance.6.paper electrophoreticpatterns were determinedin two buffer systems. efforts to purify this activity further are in progress. pH 7. P. with 5 X 1O-3 M Versene.7mg.01 M potassium phosphate. was determined with the use of an analytical ultracentrifuge’ Fig.org/ by guest on November 25.m. 1 showsthe results from a representative starch block.quently isolated by zone electrophoresis (B and D). pH DISCUSSION Downloaded from http://www. 3 demonstratethe effective separa. 0. of water. H. pH 8. Lower patterns were obtained use of diethylbarbiturate. D. The sedimentation constant (~3. segments with a multiblade cutter.1. of protein per ml. Upper quent to ethanol fractionation.6 Currently. patterns were obtained with the use of potassium phosphate.p. The values for both purification and recovery may be low since the presenceof more than one insulin-Imdegradingactivity in liver powder is not unlikely.0. 2 showsa resultant pattern which gives no indication of inhomogeneity. ionic strength 0. One-half of this volume was applied 9 cm. per block (500 to 600 volts) was applied for 16 to 17 hours. A lengthwisestrip 1 cm. After elution of protein within each segmentwith 1 ml. with the Spinco Protein which migratesconsiderablymoreslowly in the buffer model E ultracentrifuge. of protein) was eluted and the eluate combined with the remainsof the correspondingsegments of the lengthwisestrip. The constant specificactivity of the eluatesof a portion of the starch block of approximately 0. ionic strength. Halsey ml. pH 7. Fig. Sedimentation diagram of the purified insulin-degradlength indicates that the activity has been isolatedin a highly ing enzyme in 0. with the The high degreeof purity of the isolatedenzyme is indicated by the constant specificactivity of the active peak after starch block electrophoresis. ionic strength 0.5.5. D. The portion of each block containing material of a constant specific activity (enzyme units per mg. of pale yellow solution contained10 mg. Zone Etectrophoretic Purification-Washed technical grade of potato starch (11) was used in an electrophoretic apparatus recently describedby Goldsworthy and Volwiler (12). Further Evidence of Homogeneity of Isolated Enzyme Sedimentation Diagram-Enzyme isolated by several runs through the preparatory procedure was combined and lyophilizcd.05. wide was cut from each block. D.5. it is certain that in- . pH 7.01M phosphate buffer.2~) was 3. of the same buffer with one change. After overnight dialysis against 500 ml. Table II showsin generalthe extent of purification and recovery of the isolated insulin-degradingenzyme at each step of the process.01M potassium phosphate. systemusedcontainsactivity which degrades glucagon. A current of 25 ma. aliquots were taken for protein analysis as well as for enzymatic activity.tionated liver protein (A and C) and of the purified enzyme subsetion of the enzyme from contaminating proteins present subsc. of 0. 2013 8.0 S Paper Electrophoretic Patterns-Since not enough material was available for a moving boundary electrophoresis. Paper electrophoretic patterns of the ethanol-fracTheseresultsshownin Fig. per ml.15 mg. of enzyme solution containing 0.

R. J. NUTLEY. R. 28. 193. TOMIZAWA. J. ROSEBROUGH. AND WILLIAMS. I. M. 285 (1955)..... KENNY. 214. GOLDSWORTHY... TOMIZAWA. L.. H. I. A highly purified enzyme which promotes the degradation of insulin has been isolated from beef liver. R. J. Williams for his encouragement and support.. 77 (1957).. 14. J... MIRSKY..ses the rate at which the solution becomes turbid. J. Endocrinology. REFERENCES 1. H. 9. 265 (1951). R. AND PERISUTTI. H. BioZ. A.6 Although the isolated enzyme may promote the cleavage of peptide bonds of insulin. Chem. Downloaded from http://www. D. Chem. Biol. 214. MIRSKY. Biochim. 1034 (1958). FISCHER. 234. Along with this is an increase in the amount of trichloroacetic acid-soluble material from insulin.. K... H. Am. 6. 4. AND WILLIAMS. 60. AND WILLIAMS. 5. Chem. J. H. M. A. NARAHARA. E. Federation Proc.. 233. 13. enzymatic reductive cleavage of disulfide bonds.. 8.. H.... 432 (1954). G. AND STEIN. The usefulness of an 1131-labeled protein as substrate for developing a procedure for enzyme purification has also been demonstrated. R. Chem.org/ by guest on November 25. E. Chem. H. 11. T. J. 284 (1956).. J.. Geneva. P. 685 (1955). must also be considered. a possibility investigated by Racker (13) and by Narahara and Williams (14). T. T.. Biol. N. NARAHARA. Biol... Chem. 711 (1953). PERISKJTTI. A. AND DIXON. the addition of this enzyme greatly increa.. NARAHARA. J. GESCHWIND. Endocrinology. xi. 285 (1957). AND LI.. RACKER.. Biol. 397 (1955). Anal. PAIGEN. J. W. Ann Harmon. Physiol. J.. VAUGHAN.. AND WILLIAMS. R. 419 (1956). Biol. 131 (1954). AND RANDALL. FARR. H. F. 228. et Biophys. A. 7. 2... 230. LOWRY. 12. Biol. 186.226 (1952). J. Chem.... 12. 16.310 Isolation of Insulin-degrading Enzyme SUMMARY Vol. Acknowledgments-The authors wish to thank Dr. Chem. I. 2013 . G. 7. L. Acta. No. 3. H.. Ellen Larson for excellent technical assistance. and Mrs. H. 817 (1958). 2 Upon incubation of insulin in phossulin itself is a substrate. H. H.. E. Arch. H. I. Mrs. 60. Adclc Hopkins.. AND VOLWILER. 0. 217. C. A. and Mrs.jbc. 10. H. H. phate buffer with a sulfhydryl compound such as GSH or 2-mercaptoethanol.