SUMMARY. Insulin-degrading enzyme (IDE), which proteolytically degraded insulin with a high degree of specificity, was purified from pig skeletal muscle by ...
Immunochemical Studies on the Insulin-degrading Enzyme from Pig and Rat Skeletal Muscle KOICHI YOKONO, YOSHIMICHI IMAMURA, KOZUI SHII, NOBUHIKO MIZUNO, HIDEYO SAKAI, AND SHIGEAKI BABA
SUMMARY Insulin-degrading enzyme (IDE), which proteolytically degraded insulin with a high degree of specificity, was purified from pig skeletal muscle by ammonium sulfate precipitation, chromatography on Bio-Gel P-200 and DEAE-cellulose, and finally rechromatography on Sephadex G-200 (rechromatography fraction). The enzyme was also purified by affinity chromatography (affinity fraction). Both fractions migrated as a single component at the same position on polyacrylamidegel disc electrophoresis. Antiserum against pig muscle IDE was obtained by immunization of rabbits using the rechromatography fraction. By means of antiserum, it was shown that pig muscle IDE (affinity fraction), rat muscle cytosol-, and membrane-IDE gave a precipitin band of identity in Ouchterlony double-immunodiffusion systems. Quantitative immunoprecipitin data demonstrated that the antiserum inhibited the activities of the above three IDEs compared with normal rabbit serum. These data suggest that the insulindegrading enzyme from porcine muscle and that from rat muscle have similar immunologic properties. The antiserum described here should be a useful tool for the examination of subcellular distribution and the quantitative analysis of insulin-degrading enzyme. It may also be helpful in determining the physiologic Significance Of IDE. DIABETES 29:856-859, October 1980.
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any reports are currently available concerning the insulin-degrading enzyme that proteolytically degrades insulin with a high degree of specificity. The insulinase reported by Mirsky was heat-labile,1 restricting the procedures available for enzymatic purification. However, insulin protease was purified from rat skeletal muscle using a procedure that in-
From The Second Department of Internal Medicine, Kobe University School of Medicine, Kobe, Japan. Address reprint requests to Prof. Shigeaki Baba, The Second Department of Internal Medicine, Kobe University School of Medicine, Ikuta-ku, Kobe, Japan. Received for publication 31 July 1980.
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eluded affinity chromatography.2'3 We reported that insulin-degrading enzyme (IDE) could be highly purified from pig skeletal muscle by the isoelectrofocusing technique. Porcine IDE prepared in this way appeared to be identical to rat insulin protease in most of its biochemical properties.4 These enzymes have been extensively studied in terms of enzymatic characteristics1-4-5 and biologic significance.6"8 On the other hand, a number of questions remain unanswered, such as whether these enzymes exist in blood and how they might interact with insulin receptors in the process of insulin degradation. Immunochemical studies using antiserum against these enzymes should be a useful tool for the examination of these problems. However, there is no report concerning the immunochemical study of these enzymes since it has been difficult to obtain enough pure enzyme protein to form an antiserum. In the present study, IDE purified from pig skeletal muscle by Sephadex G-200 rechromatography was utilized to produce IDE antibodies in rabbits. The immunologic purity of the antiserum was demonstrated in the Ouchterlony double-immunodiffusion system, and quantitative antigen-antibody titrations of pig and rat muscle IDEs with the antiserum were carried out. MATERIALS AND METHODS
Materials. 125l-labeled porcine insulin (specific activity: 150-200 fiCUfig) was purchased from Dinabott Rl Laboratories, Tokyo. Pork monocomponent insulin was obtained from Novo Research Institute, Copenhagen. Bovine serum albumin (BSA) was from Sigma Chemical Co., St. Louis, Missouri. Sephadex G-25, G-200, and CNBr-activated Sepharose 4B were manufactured by Pharmacia Fine Chemicals, Inc., Piscataway, New Jersey. Bio-Gel P-200 was bought from Bio-Rad Laboratories, Richmond, California, and DEAE-cellulose from Brown Co., Berlin, New Hampshire. Freund's complete adjuvant was obtained from Difco Laboratories, Detroit, Michigan. Other chemicals were of reagent grade. Enzyme purification. Freshly prepared pig leg muscles were trimmed of fat and connective tissue, minced, and ho-
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mogenized in 0.35 M sucrose in a Waring blender for five 15-sec intervals. The homogenate was centrifuged at 10,000 x g for 20 min. The supernatant was fractionated by ammonium sulfate precipitation, Bio-Gel P-200, and DEAEcellulose column chromatography as previously described.4 The enzyme protein of the DEAE-cellulose fraction was lyophylized and then further purified by Sephadex G200 column rechromatography or affinity chromatography. For the former, the lyophylizate, dissolved in 25 mM potassium phosphate buffer (KPB), pH 7.5, containing 1 mM dithiothreitol (DTT), was chromatographed on a Sephadex G200 column (3.2 x 93 cm) equilibrated with the same buffer containing 1 mM DTT. Fractions comprising the peak enzyme activity were pooled, salted out with 80% saturation of ammonium sulfate solution, desalted by passage through a Sephadex G-25 column, and concentrated with a collodion bag. This preparation was rechromatographed on a Sephadex G-200 column, following the exact procedure of the first chromatography. The final concentrate was lyophilized with 1% sucrose and stored at -80°C until used (rechromatography fraction). A second batch of the DEAE-cellulose fraction was purified by affinity chromatography according to Duckworth et al.2 Cyanogen bromide-activated sepharose 4B was reacted with insulin in 0.2 M sodium citrate buffer at pH 5. The enzyme was adsorbed to the insulin-Sepharose column (1.8 x 18 cm), washed with 20 mM acetate buffer (pH 6.2) containing 1 mM DTT, and eluted with 0.2 M NaCI in the same buffer. The purified enzyme was dialyzed against 50 mM KPB (pH 7.5), lyophylized with 1 % sucrose, and stored at -80°C until used (affinity fraction). Insulin-degrading enzymes from the cytosol fraction (cytosol-IDE) and the plasma membrane fraction (membraneIDE) of rat skeletal muscle were prepared as previously described.8 Immunologic studies. Five New Zealand white female rabbits (2-2.5 kg body weight) were bled before the immunization to provide control serum. Each rabbit was injected subcutaneously with 0.6 ml of an emulsion prepared from equal volumes of the rechromatographed fraction of pig muscle IDE (1 mg protein in 0.1 M KPB, pH 7.0) and Freund's complete adjuvant. The injections were repeated at 3-wk intervals and bleedings were carried out 8 days after each injection. All serum was stored at -20°C until used. Ouchterlony double-immunodiffusion studies were performed according to the method of Stollar and Levine.9 Diffusion was allowed to proceed at 4°C in a moist chamber for about 48 h. Quantitative antigen-antibody titrations were carried out as described by Varandani.10 Solutions of IDE from pig muscle (affinity fraction) or rat muscle were incubated with antiserum, normal rabbit serum, or 0.1% BSA in 0.1 : M KPB (pH 7.0) for 1 h at 25°C. After centrifugation aliquots of supernatant were assayed for insulin-degrading activity. Analytic procedures. Insulin-degrading activity was estimated by determining the quantity of trichloroacetic acid (TCA)-soluble radioactivity produced after incubation with 125 l-insulin.8 Protein concentrations were measured by the Lowry method,11 with BSA used as standard. RESULTS AND DISCUSSION
As previously reported,2i4>5 insulin-degrading activity of the ammonium sulfate fraction was eluted at the front shoulder of the protein peak on Bio-Gel P-200 filtration; maximum en-
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10
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40 50 60 FRACTION NUMBER
FIGURE 1. Elutlon pattern of the DEAE-cellulose fraction on a Sephadex G-200 column (3.2 x 93 cm) eluted with 25 mM phosphate buffer (pH 7.5) containing 1 mM DTT. The upper panel shows the first chromatography and the lower panel shows the rechromatography on a Sephadex G-200 column. Shaded areas Indicate the fractions used for additional studies.
zyme activity appeared in the 0.25 M NaCI fraction on DEAE-cellulose chromatography. Figure 1 shows the elution pattern of DEAE-cellulose purified enzyme after passage over the Sephadex G-200 column. The upper panel shows the first chromatography on the Sephadex G-200 column. The highest enzyme activity appeared in the front protein peak. The fraction indicated by the shaded area was rechromatographed on the same column (lower panel). Fractions comprising the maximum enzyme activity after the second chromatography, indicated by shaded area, were pooled (rechromatography fraction). With pig muscle homogenate as starting material, an 1100-fold purification was obtained. The DEAE-cellulose fraction was also purified by affinity chromatography and a 3000-fold purification was obtained by this method (data not shown). This value was of the same order reported by Duckworth and Kitabchi in rat skeletal muscle.2*3 The enzyme purified by DEAE-cellulose chromatography showed several bands on polyacrylamide-gel disc electrophoresis. However, the enzyme protein obtained from Sephadex G-200 rechromatography migrated as a single component at the same position as the fraction from affinity chromatography (Figure 2). Thus, IDE was highly purified not only by affinity chromatography but also by Sephadex G-200 column rechromatography of the DEAEcellulose fraction. Since the procedure of affinity chromatography is more difficult and not suitable for handling large quantities of enzyme protein, the method of rechroma-
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IDE FROM PIG AND RAT SKELETAL MUSCLE
•—•
Pig IDE
o—o Rat Cytosol IDE
100
Rat Membrane IDE
+ NORMAL RABBIT o SERUM
ANTI-PIG IDE SERUM 0 50 100
200
300 450 SERUM
FIGURE 4. Antigen-antibody titratlons obtained with anti-pig muscle IDE serum and the enzymes isolated from three sources. Insulindegrading activities were measured In the supernatants after removing the specific precipitates.
(1) FIGURE 2. Polyacrylamlde-gel disc electrophoretic pattern of various enzyme preparations: (1) DEAE-cellulose fraction, (2) rechromatography fraction, and (3) affinity fraction.
tography on Sephadex G-200 was used for immunization. It has the advantage of being simple, and it yields a fairly pure enzyme preparation. As shown in Figure 3, double-immunodiffusion of the affinity fraction with antiserum to the rechromatography fraction showed a single precipitin band. Quantitative precipitin
data (Figure 4) showed that insulin-degrading activity of the affinity fraction was almost entirely inhibited by the antiserum in contrast to normal rabbit serum. We previously reported that enzymatic characteristics of insulin-degrading activity in the cytosol (cytosol-IDE) and plasma membranes (membrane-IDE) partially purified from rat skeletal muscle were similar to those of IDE from pig skeletal muscle.8 Rat cytosol and membrane-IDEs showed a single precipitin band continuous with the precipitin band obtained with the affinity fraction in the Ouchterlony double-immunodiffusion. Enzyme activities of both rat IDEs were inhibited equally by FIGURE 3. Ouchterlony double-immunodiffusion with antiserum to pig muscle IDE. The antiserum (10 fi\) to pig muscle IDE was placed in the central well. The peripheral wells received 10 MI of IDEs in the following order, starting from the arrow In a clockwise direction: pig muscle, rat muscle cytosol fraction, and rat muscle plasma membrane fraction. The remaining wells were empty.
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the antiserum but not as efficiently as was the pig IDE. The reason for this difference is not clear. However, these data do suggest that pig muscle insulin protease is both biochemically and immunologically similar to the rat enzyme. The procedure described in this paper should allow widespread use of antibodies to insulin-degrading enzyme. Hopefully it will help to dissect the physiologic role played by IDE in normal and pathologic states. ACKNOWLEDGMENTS
We acknowledge the valuable advice and the excellent technical assistance of Dr. Shigenori Emi, Toyobo Co. REFERENCES
1 Mirsky, I. A.: Insulinase, insulinase-inhibitors, and diabetes mellitus. Recent Prog. Horm. Res. 73:429-72, 1957. 2 Duckworth, W. C, Heinemann, M. A., and Kitabchi, A. E.: Purification of insulin-specific protease by affinity chromatography. Proc. Natl. Acad. Sci. USA 69:3698-3702, 1972.
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3 Duckworth, W. C, and Kitabchi, A. E.: Insulin and glucagon degradation by the same enzyme. Diabetes 23:536-43, 1974. 4 Baba, S., Sakai, K, Imamura, Y., Yokono, K., and Emi, S.: Metabolism of insulin and proinsulin. In Proinsulin, insulin, C-peptide. Baba, S., Ed. Amsterdam, Excerpta Medica, 1979, pp. 270-282. 5 Burghen, G. A., Kitabchi, A. E., and Brush, J. S.: Characterization of a rat liver protease with specificity for insulin. Endocrinology 91:633-42, 1972. 8 Morgan, C. R., Spah, J., Frazier, V., and Fleitz, S.: Insulin, a possible inducer of the biosynthesis of rat liver insulinase. Proc. Soc. Exp. Biol. Med. 728:795-97, 1968. 7 Kitabchi, A. E., Stentz, F. B., Cole, C , and Duckworth, W. C : Accelerated insulin degradation: an alternate mechanism for insulin resistance. Diabetes Care 2:414-17, 1979. 8 Yokono, K., Imamura, Y., Sakai, H, and Baba, S.: Insulin-degrading activity of plasma membranes from rat skeletal muscle. Its isolation, characterization, and biologic significance. Diabetes 28:810-817, 1979. 9 Stollar, D., and Levine, L: In Methods in Enzymology, Vol. VI. Colowick, S. P., and Kaplan, N. 0., Eds. New York, Academic Press, 1963, pp. 848-54. 10 Varandani, P. T.: Insulin degradation. I. Purification and properties of glutathione-insulin transhydrogenase of rat liver. Biochim. Biophys. Acta 286:126-35, 1972. 11 Lowry, H. 0., Rosebrough, N. J., Farr, A. L, and Randall, R. J.: Protein measurement with the Folin phenol reagent. J. Biol. Chem. 53:265-75, 1951.
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