Biochimica et Biophysica Acta, 490 (1977) 235-246 © Elsevier/North-Holland Biomedical Press BBA 37529

STUDIES ON BIOTRANSFORMATION OF LYSOZYME III. COMPARATIVE STUDIES ON BIOTRANSFORMATION OF EXOGENOUS AND ENDOGENOUS LYSOZYME IN RATS

TERUAKI YUZURIHA, KOUICHI KATAYAMA and TAKESHI FUJITA

Department of Drug Metabolism, Section of Experimental Therapeutics Research, Research and Development Division, Eisai Co., Ltd., Koishikawa, Bunkyo-Ku, Tokyo (Japan) (Received July 6th, 1976)

SUMMARY

Exogenous hen lysozyme or endogenous rat lysozyme labeled with 131I was intravenously injected to rats with the same dosage, respectively, and the uptake and degradation of injected 131I-labeled rat lysozyme in liver and kidney were studied in comparison with those of 131I-labeled hen lysozyme. 1. Although the serum levels of both enzymes injected were almost identical during the first 6 h, the liver uptake of 131I-labeled hen lysozyme was 2.2-fold more than that of 13q-labeled rat lysozyme at the peak time of 5 min after injection. The uptake and clearance of 13q-labeled rat lysozyme in the kidney were exclusively slow as compared with those of ~3q-labeled hen lysozyme. 2. The intracellular distribution in the liver and kidney were examined by the differential centrifugation after injection of each lysozyme. The protein-bound radioactivity of each subcellular fraction was found to be the highest in the 12 000 x g (10 min) fraction in the liver and the 19 600 x g (20 min) fraction in the kidney. The relative specific activity of 12 000 x g fraction of the liver after injection increased with the time lapse. On the other hand, the relative specific activity of 105 000 x g (1 h) fraction of the liver attained a maximum within 5 rain after injection and thereafter decreased. It was assumed that the mechanism of the uptake of injected ~3q. labeled rat lysozyme in the liver and kidney was similar to that of 13q-labeled hen lysozyme. 3. The degradation of exogenous or endogenous lysozyme in subcellular particles was examined. From the effect of pH, activator and inhibitor on the degradation, the proteolytic enzyme to degrade the injected 131I-labeled hen lysozyme was indicated to be mainly cathepsin B1, with the optimal pH of about 5.0, and the injected 131I-labeled rat lysozyme was mainly degraded by cathepsin D, with the optimal pH of about 3.5. The in vitro degradation of exogenous and endogenous lysozymes showed a tendency similar to the in vivo clearance from the liver and kidney.

236 INTRODUCTION Lysozyme has attracted special interest recently as a diagnostic tool in hematology and nephrology, and the knowledge on biotransformation of this ehzyme has been required. Hansen et al. [1], in the studies of the turnover of lysozyme in men and rats employing the radioactivity labeled enzyme, documented the roles of granulocytes in the production of lysozyme and of the kidney in the elimination of lysozyme. Christensen and Maunsbach [2] provided the evidence that 12Sl-labeled egg-white lysozyme injected intravenously in rats was taken up in lysosomes of renal proximal tubule cells from the observation by electron microscopic autoradiography and could be digested within. The tissue distribution of 13q-labeled hen lysozyme and degradation in the kidney were reported previously [3]; the injected 13q-labeled hen lysozyme was mainly taken up into kidney heterolysosomes, and then the enzyme was degraded into ~31I-labeled tyrosine by cathepsin B1. In recent years, the numerous studies on the biotranstormation of proteins have been reported in correlation with the mechanism of the uptake and degradation of foreign or altered soluble macromolecules [4-10]. However, the reports on the uptake and degradation of endogenous proteins are scanty, and the degradation of both endogenous and exogenous lysozymes in the liver and kidney has not been clarified. Therefore, the present study was carried out to investigate the difference in the biotransformation between endogenous and exogenous lysozyme in rats, especially the uptake and degradation of two enzymes in the liver or the kidney in detail. MATERIALS AND METHODS

Hen egg-white lysozyme. Hen egg-white lysozyme-hydrochloride (abbreviated as hen lysozyme) recrystallized five times was used for 131I-labeling. 131I-labeled hen lysozyme with the specific radioactivity of 100-113 ffCi/mg was prepared by the method of McConahey and Dixon [11] and Greenwood et al. [12] as described previously [13]. Rat lysozyme. Rat lysozyme was purified from the urine of lysozymuria induced by sodium chromate, a nephrotoxic agent, as follows. Single dosage of 20 mg sodium chromate (Na2CrO4) per kg in male rats of Wistar strain (350-480 g) was administered intraperitoneally according to the method of Balazs and Roepke [14], and then the urine of 10 rats was collected for 6 days. After NaC1 was added to the urine (1 1) at the final concentration of 1 M, the urine was centrifuged and the resulting supernatant was dialysed in distilled water. The urine stirred with 6 g of CM-Sephadex C-50 (Pharmacia) for l h at room temperature and the suspension was packed in three columns (3 × 10 cm). Each column was exclusively washed with water, and then was developed with a stepwise procedure of 0.05 M Tris. HC1 (pH 7.0) buffer containing NaC1 from 0.1 to 0.4 M. The fraction with lysozyme activity was then loaded on Sephadex G-75 column (1.5 x 30 cm), and eluted with 0.15 M NaCI. The active fraction was dialysed in distilled water and lyophilized. In this procedure, 10.4 mg of rat lysozyme was obtained from the urine of ten rats. 13q-labeled rat lysozyme with the specific radioactivity of 180 ffCi/mg was prepared by the same method as that of hen lysozyme. The purity of rat lysozyme preparation was checked with the specific enzyme activity, ultraviolet absorption

237 spectra and sodium dodecyl sulfate-polyacrylamide gel electrophoresis according to the method of Weber and Osborn [15]. Reagents. Cytochrome c (Type VI, Sigma Chemical Co.), glucose 6-phosphoric acid (B.D.H. Chemicals), fl-glycerophosphate (Merck), iodoacetamide (Tokyo Chemical Industry Co.), fl-phenylpyruvic acid (Merck), Micrococcus lysodeikticus (Washington Biochemical Co.), Triton X-100 (Wako Pure Chemical Industries), sodium dodecyl sulfate (Kaso Chemical Co., Tokyo) and Na131I (Daiichi Radioisotope Lab.) were used. Animal experiments. Male rats of Wistar strain (180-280 g) were fasted for 24 h prior to the experiments. The rat was injected into the femoral vein with a dose of 0.4 mg of 131I-labeled hen lysozyme or 13q-labeled rat lysozyme per kg. Rats were killed by decapitation 1 min, 5 min, 15 min, 30 min, 1 h, 3 h and 6 h after the injection. The liver and kidney were perfused in situ with cold 0.15 M NaC1 and then removed. The tissue was homogenized in three volumes (v/w) of 0.25 M sucrose as described previously [3]. Blood samples collected were centrifuged and the supernatants were assayed as follows. The radioactivity in each homogenate and serum was assayed by the precipitation in a final concentration of 5 ~ trichloroacetic acid. The biliary excretion of radioactivity after injection of 13q-labeled hen lysozyme was also examined in rats with bile fistula. Subcellular fractionation of liver and kidney. The liver was homogenized in three volumes (v/w) of 0.25 M sucrose/0.2 M KC1 and the kidney was in three volumes (v/w) of 0.2 M KC1, at 1200 rev./min with four strokes. The liver homogenate was centrifuged at 56 × g for 5 min and the resulting pellet was discarded. The 56 x g supernatant was centrifuged at 900 × g for 10 min and the resulting pellet was washed once with 0.25 M sucrose/0.2 M KCI by resuspension and recentrifugation under the same condition. After the washing had been added to the 900 x g supernatant, the combined supernatant was centrifuged at 12 000 x g for 10 min and this pellet was also washed once. The final supernatant was centrifuged at 105 000 x g for 1 h. The radioactivity in each fraction was analyzed by trichloroacetic acid precipitation and the protein was measured. The kidney homogenate was fractionated by the differential centrifugation according to the method of Straus [16] as described previously [3]. In the differential centrifugation of kidney, the 19 600 × g (20 min) fraction corresponded to the mitochondria-lysosomal fraction. Determination of protein and enzyme activity. Protein, acid phosphatase, cytochrome oxidase and glucose-6-phosphatase were measured by the methods described previously [3]. Enzyme activity of lysozyme was determined using hen lysozyme as a standard and M. lysodeikticus as a substrate according to the method of Gorin et al. [17]. Lysozyme activity in subcellular fractions of the liver was determined using the supernatant added to a final concentration of 0.25 ~ Triton X-100 (0 °C, 1 h). Triton X-100, 0.25 ~ (w/v), had no significant effect on lysozyme activity. Determination of degradative activity. The 12 000 x g fraction of liver 5 min after intravenous injection of ~s~I-labeled hen lysozyme or a3q-labeled rat lysozyme was homogenized in two volumes (v/w) of 0.25 M sucrose/0.2 M KC1 and then an equal volume of 0.1 M buffer (see below) containing 0.25 M sucrose/0.2 M KC1 was added to the homogenate. The 19 600 × g fractions of kidney 30 min after the injection of ~3q-labeled hen lysozyme and 1 h after the injection of 13q-labeled rat

238 lysozyme were homogenized in two volumes (v/w) of 0.25 M sucrose, and then an equal volume of 0.1 M buffer containing 0.25 M sucrose was added to the homogenates. After the incubation at 37 °C for an appropriate time, the equal volume of the 10 ~o (v/w) trichloroacetic acid solution was added to the homogenate and the mixture was centrifuged. The degradative activity for each protein injected was expressed as trichloroacetic acid-soluble radioactivity corrected using that before the incubation. Effect of pH and reagents on degradative activity. The homogenate of the 12 000 × g fraction of liver or the 19 600 × g fraction of kidney was added to 0.1 M buffer at various pH values and then the mixture was incubated at 37 °C for an appropriate time. The 0.1 M buffers used were as follows, glycine. HC1 (pH 2-2.8), citrate (citric acid/sodium citrate, pH 3-5.4) and phosphate (NaHzPO4" 2H20/NazHPO412H20, pH 5.5-7.4). The pH values of the mixtures were measured. Iodoacetamide or fl-phenylpyruvic acid was added to the assay mixture in order to determine their effects on the degradative activity of the 12 000 x g fraction at pH 3.5. R ES U LTS

Criteria for purification of rat lysozyme The isolated rat lysozyme had about 83-fold purity as compared with that obtained before loading on CM-Sephadex C-50. The specific activity was calculated as 2.49 from the enzyme activity using hen lysozyme as a standard per protein of rat lysozyme (A280nm -- 1.17, 1 mg/ml). This result was in good agreement with the report of Gordon et al. [18]. The ultraviolet absorption spectra and sodium dodecyl sulfate-polyacrylamide gel electrophoretic pattern of rat lysozyme labeled with 134 are shown in Fig. 1 as compared with those of hen lysozyme. The ultraviolet spectra of rat lysozyme in Fig. 1 was in good agreement with that of rat lysozyme isolated from rat kidney nuclear fraction [19]. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 13aI-labeled rat lysozyme, the enzyme moved as a single band with the mobility of 0.82, different from that of hen lysozyme, [

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242 in the 12000 x g fraction. The relative specific activity of 12000 x g fraction following injection increased with the time lapse. On the other hand, the relative specific activity of 105 000 x g fraction attained a maximum within 5 rain after injection and then declined rapidly. The distribution of injected 13q-labeled rat lysozyme in liver (Fig. 4E) showed a pattern similar to that of '3'l-labeled hen lysozyme in Fig. 4D. Effect of pH and reagents on the degradation of injected '311-labeled hen lysozyme or 131I-labeled rat lysozyme in the 12 000 × g fraction of liver and in the 19 600 × g fraction of kidney The degradative activity in the 12 000 × g fraction prepared freshly from the liver of the rat 5 min following injection of ~3q-labeled hen lysozyme or ~3q-labeled rat lysozyme was determined within the range from pH 2.5 to 7.5. The results are presented in Fig. 5. Trichloroacetic acid-soluble radioactivity formed by the degradation of ~311labeled hen lysozyme was observed to have the biphasic maxima at pH 3.4 and 4.7 (Fig. 5A), while that of 13q-labeled rat lysozyme was found to have a main peak at pH 3.5-3.7 (Fig. 5B). The acid proteinases with those optimal pH values were suggested to correspond to those of cathepsin B1 and cathepsin D. The effects of iodoacetamide as an inhibitor for cathepsin B I [20-22] and of /~-phenylpyruvic acid as an inhibitor for catepsin D [20-22] on the degradative activity, were investigated using the 12 000 x g fraction of liver 5 rain after injection of a3'I-labeled rat lysozyme. The degradative activity at pH 3.5 was inhibited approximately to the same degree by each 10 mM of iodoacetamide and /3-phenylpyruvic acid, as well as 5 mM iodoacetamide or 20 mM /3-phenylpyruvic acid. The degree of degradation by the presence of the inhibitors was about 14 ~o during the first 1 h, and that by the absence of the inhibitors was 26.2~. The degradation of injected 1311labeled hen lysozyme by the 12 000 x g fraction at the optimal pH was also determined in comparison with that of '3'l-labeled rat lysozyme. The initial rate in the

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Fig. 6. Effect of pH and reagents on the degradative activity of injected z31I-labeled rat lysozyme (0) by the 19 600 × g fraction of kidney. (A) Effect of pH; incubation time at 37 °C was 2 h in 0.05 M buffer containing 0.25 M sucrose. (B) Effect of reagents; the 19 600 × g fraction was incubated at 37 °C in 0.05 M buffer (pH 3.5) containing 0.25 M sucrose with 10 m M fl-phenylpyruvic acid (A) or 10 mM iodoacetamide (T), Degradation of injected 13tI-labeled hen lysozyme (O) or by the 19 600 x g fraction was also examined in 0.05 M buffer (pH 5.0) containing 0.25 M sucrose. Vertical bars represent the S.E. with the mean of three experiments.

degradation of 13q-labeled rat lysozyme during the first 1 h was 26.2~ at pH 3.5, and that of 131I-labeled hen lysozyme was 16.3 ~ at pH 3.4 and 11.2 ~ at pH 4.7. The effect of pH and reagents on the degradation of ~3q-labeled rat lysozyme in the 19 600 × g fraction of kidney 1 h after injection also was determined and the results are shown in Fig. 6. Trichloroacetic acid-soluble radioactivity formed by the degradation of injected ~3q-labeled rat lysozyme within the range of pH tested was found to have the biphasic at pH 3.6 and 5.4 (Fig. 6A). As well as the case of liver, the optimal pH value of 3.6 in the degradation corresponded to that of cathepsin D. The effects of 10 mM iodoacetamide and of 10 mM fl-phenylpyruvic acid on the degradation at pH 3.5 were determined (Fig. 6B). The degradative activity was inhibited by fl-phenylpyruvic acid and was not completely inhibited by iodoacetamide. The release of trichloroacetic acid-soluble radioactivity at pH 3.5 on the degradation of ~aq-labeled rat lysozyme was 18.3 ~ with the initial rate during the first 2 h, and that at pH 5.0 on the degradation of 13q-labeled hen lysozyme was 38.7 ~ (Fig. 6B). Therefore, the degradative activity of injected 13q-labeled rat lysozyme by the 19 600 × g fraction of kidney was indicated to be less than that of injected 131I-labeled hen lysozyme. DISCUSSION

The comparison of biotransformation between exogenous and endogenous lysozyme in rats, especially the uptake and degradation of the two enzymes in the liver or the kidney were investigated in detail. There was not a significant difference in the clearance from the liver, but in

244 the hepatic uptake between both enzymes injected (Fig. 2, Table 1). This fact suggested that the degradative rate of injected 13q-labeled hen lysozyme and J31I-labeled rat lysozyme in the liver was the same to each other in vivo. The isoelectric point of hen lysozyme is pH 10.5-11.0 [23]. Based on the preliminary observation of cellulose acetate e!ectrophoresis, it was suggested that the isoelectric point of rat lysozyme slightly differs from that of hen lysozyme. In addition, as shown in Fig. 1, the molecular weight of rat lysozyme was somewhat larger than that of hen lysozyme, 14 307 [24]. The difference in physicochemical properties between 131I-labeled hen lysozyme and ~31I-labeled rat lysozyme might have an influence on the hepatic uptake of both enzymes injected. The pattern of trichloroacetic acid-precipitable radioactivity in the kidney after injection of ~3q-labeled rat lysozyme was in good agreement with the result described by Hansen et al. [I]. It can be noted that 13q-labeled rat lysozyme injected has a tendency to accumulate and degrade more slowly than 13q-labeled hen lysozyme in the kidney (Fig. 2, Table I). Foreign proteins absorbed into cells by the process of pinocytosis, enter pinocytotic vesicles. The vesicles than become incorporated into lysozyme, which are the site where these foreign substances undergo degradation [4, 25, 26]. The radioactivity of exogenous and endogenous lysozymes injected was the highest in the 12 000 x g fraction in the liver and in the 19 600 × g fraction in the kidney (Fig. 4, ref. 3), with a pattern similar to a lysosomal marker enzyme (Fig. 3, ref. 3). As seen from the time course of intracellular distribution of injected 13q-labeled hen lysozyme and 131Ilabeled rat lysozyme in the liver (Fig. 4), the relative specific activity of the 12 000 < g fraction increased with the time lapse, while that of the 105 000 × g fraction decreased. These results suggest that the injected enzymes are taken up into the 105 000 ~< g fraction or endocytotic vesicles at first and then are transferred to the 12 000 ~: g fraction or heterolysosomes accompanied by an increase of particle size, as discussed by Mego and McQueen [27] and previous report [7]. In the kidney, it is suggested that the enzyme taken up to the 19 600 × g fraction presented as phagolysosomes (Straus [25]) or heterolysosomes (Maunsbach [4]). Only cathepsins B and D are known to be capable of attacking intact proteins and then degrade it to the level of trichloroacetic acid-soluble materials [28, 29]. The acid proteinase with the optimal degradative activity near pH 5.0 seems to be mainly cathepsin B1 based on the papers [3, 8, 20-22]. The other acid proteinase with the optimal activity at p H 3.4-3.6 is mainly identified to be cathepsin D [8, 30, 31 ]. These findings indicate that cathepsin BI plays the main role in the degradation of injected 13q-labeled hen lysozyme (Fig. 5A, ref. 3), whereas J311-1abeled rat lysozyme is mainly degraded by cathepsin D (Figs. 5B and 6A). The in vitro degradation of injected enzyme at the optimal pH values was observed to be almost similar in the 12 000 × g fraction of liver between ~3q-labeled hen lysozyme and 131I-labeled rat lysozyme, but the former was rapidly degraded by the 19 600 ~. g fraction of kidney (Fig. 6B). This result seems to correspond to the in vivo findings in the elimination of trichloroacetic acid-precipitable radioactivity in the liver and kidney, as shown in Fig. 2. Based on the present data and previous report [3], ~3q-labeled hen lysozyme injected intravenously is rapidly taken up in the liver and kidney as the 105 000 x g particles or endocytctic vesicles. The vesicles can fuse with lysosomes as described

245 by De Duve and Wattiaux [26], and m a y develop into heterolysosomes in the liver and kidney. In heterolysosomes, 13tI-labeled hen lysozyme are mainly degraded into 13q-labeled tyrosine by cathepsin B1, and then 13q-labeled tyrosine is deiodinated in microsomes [32], followed by excretion in bile and urine as inorganic 13112. On the other hand, 13q-labeled rat lysozyme injected intravenously was rapidly taken up in liver and slowly in kidney as endocytotic vesicles. In heterolysosomes, 13q-labeled rat lysozyme was degraded mainly by cathepsin D and the degradative rate was fast in liver and slow in kidney. Klockars and Reltamo [33] have shown in h u m a n liver that endogenous lysozyme could not be detected in the parenchymal cells, but in Kupffer cells as intensive activity using immunohistochemical technique. In the present study, we were not able to elucidate whether the injected ~3q-labeled hen lysozyme or 1311labeled rat lysozyme was taken up into parenchymal cells or Kupffer cells. However, on the uptake and degradation of proteins, whether proteins used were exogenous or endogenous in experimental animals it m a y also be necessary to pay attention to whether proteins used were native or denatured. ACKNOWLEDGMENTS The authors thank Dr. S. Ohtake, Director, Section o f Experimental Therapeutics Research, for his valuable advice and Mr. A. Yamagishi, Director o f Research and Development Division, for his support of our research. REFERENCES 1 Hansen, N. E., Karle, H. and Andersen, V. (1974) in Lysozyme (Osserman, E. F., Canfield, R. E. and Beychok, S., eds.), pp. 307-319, Academic Press, New York 2 Christensen, E. I. and Maunsbach, A. B. (1974) Kidney Int. 6, 396-407 3 Yuzuriha, T., Katayama, K. and Fujita, T. (1975) Chem. Pharm. Bull. (Tokyo) 23, 1315-1322 4 Maunsbach, A. B. (1969) in Lysosomes in Biology and Pathology (Dingle, J. T. and Fell, H. B., eds.), Vol. 1, pp. 115-154, North-Holland Publ. Co., Amsterdam 5 Tappel, A. L. (1969) in Lysosomes in Biology and Pathology (Dingle, J. T. and Fell, H. B., eds.), Vol. 2, pp. 207-244, North-Holland Publ. Co., Amsterdam 6 Mego, J. L. (1973) in Lysosomes in Biology and Pathology (Dingle, J. T., ed), Vol. 3, pp. 138-168, North-Holland Publ. Co., Amsterdam 7 Katayama, K. and Fujita, T. (1974) Biochim. Biophys. Acta 336, 178-190 8 Katayama, K. and Fujita, T. (1974) Biochim. Biophys. Acta 336, 191-200 9 Huisman, W., Lanting, L., Doddema, H. J., Bouma, J. M. W. and Gruber, M. (1974) Biocbim. Biophys. Acta 370, 297-307 10 Buys, C. H. C. M., De Jong, A. S. H., Bouma, J. M. W. and Gruber, M. (1975) Biochim. Biophys. Acta 392, 95-100 11 McConahey, P. J. and Dixon, F. J. (1966) Int. Arch. Allergy 29, 185-189 12 Greenwood, F. C., Hunter, W. M. and Glover, J. S. (1963) Biochem. J. 89, 114-123 13 Yuzuriha, T., Katayama, K. and Fujita, T. (1975) Chem. Pharm. Bull. (Tokyo) 23, 1309-1314 14 Balazs, T. and Roepke, R. R. (1966) Proc. Soc. Exp. Biol. Med. 123, 380-385 15 Weber, K. and Osborn, M. (1969) J. Biol. Chem. 244, 4406--4412 16 Straus, W. (1962) J. Cell Biol. 12, 231-246 17 Gorin, G., Wang, S. F. and Papapavlou, L. (1971) Anal. Biochem. 39, 113-127 18 Gordon, S., Todd, J. and Cohn, Z. A. (1974) J. Exp. Med. 139, 1228-1248 19 Raghunathan, R. and Gurnani, S. (1974) Ind. J. Biochem. Biophys. 11,273-278 20 Barrett, A. J. (1972) Anal. Biochem. 47, 280-293 21 Barrett, A. J. (1973) Biochem. J. 131,809-822

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Franklin, S. G. and Metrione, R. M. (1972) Biochem. J. 127, 207-213 Alderton, G., Ward, W. H. and Fevold, H. L. (1945) J. Biol. Chem. 157, 43-58 Canfield, R. E. (1963) J. Biol. Chem. 238, 2698-2707 Straus, W. (1964) J. Cell Biol. 20, 497 507 De Duve, C. and Wattiaux, R. (1966) Ann. Rev. Physiol. 28, 435 492 Mego, J. L. and McQueen, J. D. (1965) Biochim. Biophys. Acta 100, 136 143 Barrett, A. J. (1969) in Lysosomes in Biology and Pathology (Dingle, J. T. and Fell, H. B., eds.), Vol. 2, pp. 245-312, North-Holland Publ. Co., Amsterdam Snellman, O. and Sylven, B. (1967) Nature 216, 1033 Barrett, A. J. (1967) Biochem. J. 104. 601-608 Barrett, A. J. (1970) Biochem. J. 117, 601-607 Stanbury, J. B. (1957) J. Biol. Chem. 228, 801 811 Klockars, M. and Reltamo, S. (1975) J. Histochem. Cytochem. 23, 932-940

Studies on biotransformation of lysozyme. III. Comparative studies on biotransformation of exogenous and endogenous lysozyme in rats.

Biochimica et Biophysica Acta, 490 (1977) 235-246 © Elsevier/North-Holland Biomedical Press BBA 37529 STUDIES ON BIOTRANSFORMATION OF LYSOZYME III. C...
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