LIPOPOLYSACCHARIDE OF PSEUDOMONAS ARRUGINOSA WITH SPECIAL REFERENCE TO PYOCIN R RECEPTOR ACTIVITY' KAYOKO IKEDA2 AND FUJIO EGAMI'

J. Gen. Appl. Microbiol., 19, 115-128 (1973) LIPOPOLYSACCHARIDE OF PSEUDOMONAS ARRUGINOSA WITH SPECIAL REFERENCE TO PYOCIN R RECEPTOR ACTIVITY' KAYOKO IKEDA2 AND FUJIO EGAMI' Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Hongo, Tokyo, Japan (Received September 14, 1972) 1) Lipopolysaccharide with pyocin R receptor activity was isolated from Pseudomonas aeruginosa P14 by the phenol method. Lipopolysaccharide was dissociated and fractionated into amino-sugar-rich fraction and lipopolysaccharide subunits by Sephadex G-100 gel filtration after heat treatment in the presence of sodium deoxycholate. 2) The lipopolysaccharide subunits (mol. wt. 12,000-16,000) had no receptor activity in the presence of sodium deoxycholate, but they were reassociated in the absence of sodium deoxycholate and the activity was recovered. Therefore, the subunits may be regarded as the chemical entity of the receptor activity. 3) Chemical analysis of the reassociated lipopolysaccharide and the carbohydrate fragment (mol. wt. 1,300-1,500) after acid treatment for the elimination of "lipid A" was carried out. The former contained glucose, rhamnose, heptose, glucosamine, galactosamine, quinovosamine, fucosamine, and a 2-keto-3-deoxy-sugar acid. The latter contained only glucose, rhamnose, heptose, galactosamine, 2-keto-3-deoxy-sugar acid, and phosphate. Lipopolysaccharide of Pseudomonas aeruginosa has been studied by HOMMA, one (F.E.) of the present authors, and their co-workers (1-4), and by GRAY, WILKINSON, and their co-workers (5-7) with different strains. The latter has studied mainly its chemical nature, and the former mainly biological activities such as toxicity, pyrogenicity, Schwartzman activity, and recently pyocin R receptor activity. Pyocin R is a bacteriocin produced by Pseudomonas aeruginosa P15 and 1 This constitutes Part II of a series entitled "Studies pyocin R." Part I: J. Biochem. (Tokyo), 65, 603 (1969). 2 Present address : Department of Neuropharmacology Niigata University, Niigata 951, Japan. s Present address : Mitsubishi-Kasei Institute of Life Machida, Tokyo 194, Japan 115 on receptor substance for, Brain Research Institute, Sciences, 11 Minamiooya,

116 IKEDA and EGAMI VOL. 19 kills some strains of the same species (8). In a previous paper (4), we described the preparation of lipopolysaccharide with pyocin R receptor activity from a pyocin R-sensitive strain by trichloroacetic acid extraction method. In the present paper, the preparation of lipopolysaccharide from Pseudomonas aeruginosa P14 by the phenol method and further studies on the relationship between the chemical nature and pyocin R receptor activity of the lipopolysaccharide are described. MATERIALS AND METHODS Bacterial strains. Pseudomonas aeruginosa P14 (wild type, sensitive to pyocins R, R2, R3, and R4) was used for the preparation of lipopolysaccharide. Culture medium. Either nutrient broth or glutamate broth was used. The former contained 10 g of polypeptone, 10 g of meat extract, 1 g of NaCI in 1 liter of water, and ph was adjusted to 7.0. The latter contained 20 g of sodium glutamate, 5 g glucose, 0.1 g MgSO4.7H2O, 5.63 g Na2HPO4.12H2O,, 0.25 g KH2PO4, and 1 g yeast extract in 1 liter of water. Cultivation. One milliliter of preculture was added to 200 ml of culture medium in a 500-ml round bottle and incubated at 37 with shaking. Preparation of lipopolysaccharide. Cells were harvested at the late logarithmic phase and washed with cold saline. Lipopolysaccharide was prepared essentially by the same procedure as described by WESTPHAL and JANN (8 ). Fifty grams of packed cells was suspended in 250 ml of distilled water and 250 ml of 90% phenol was added. This mixture was stirred for 45 min at 67-70. The mixture was cooled in an ice bath and two layers were separated by centrifugation. The phenol layer and insoluble middle layer were further extracted with 125 ml of water under the same conditions. The aqueous layers were combined, dialyzed against water for 48 hr, and concentrated to about 100 ml by lyophilization. The thawed solution was adjusted to 0.05 M with 1 M Tris-HC1 buffer (ph 7.5) and incubated with 10 mg of RNase A and 1 mg of DNase I at 37 for 24 hr. Insoluble material was centrifuged at low speed and the supernatant was centrifuged at 130,000 x g for 3 hr to precipitate the lipopolysaccharide. Resulting pellet of clear gel was resuspended in 40 ml of water and centrifuged again under the same conditions. When the precipitate revealed absorbancy at 260 nm, RNase digestion and the centrifugation process were repeated. The resulting material was lyophilized. The yield of this lipopolysaccharide varied from 100 to 300 mg. Trichloroacetic acid extraction method was also carried out as previously described (4). Pyocin. Pyocin R was prepared from the Mitomycin-C lysate of P. aeruginosa P15 by the method of KAGEYAMA (9). Pyocin activity was assayed as described previously (10).

1973 Pseudomonas aeruginosa Lipopolysaccharide 117 Assay o f receptor activity. Receptor activity was estimated as the activity inactivating pyocin R as described previously ( 4). Analytical method. Total neutral sugar was determined by the phenol H2SO4 method (11) with glucose as a standard. In the presence of sodiumm deoxycholate, total neutral sugar was measured by the anthrone method (12 ) with a slight modification (5-min boiling instead of 15-min boiling). Reducing power was measured by the method of PARK and JOHNSON (13). D-Glucose was determined with glucose oxidase system (14 ) after hydrolysis of the sample at 100 for 4 hr with 1 N HCI. Heptose was determined by the cysteine-h2so4 method (15 ) as modified by OSBORN (16 ). Rham nose was also determined by cysteine-h2so4 method from the absorbancy difference between 369 and 427 nm (17 ). The amount of glucose and rham nose was determined by quantitative paper chromatography as described previously (4). Total hexosamine was determined by the RANDLE and MORGAN method (18) after hydrolysis in 4 N HCl for 4 hr at 100 in a sealed tube, with gluco-- samine as a standard. The amount of glucosamine, galactosamine, quinovo samine, and fucosamine was determined by an amino acid analyzer (Hitachi. KLA-3B). 2-Keto-3-deoxy-sugar acid, presumably 3-deoxy-D-octulosonic acid, was determined by the thiobarbituric acid method (19) as modified by OSBORN (16), with N-acetylneuraminic acid as a standard. Protein was determined by the method of LOWRY (20) and phosphorus by the method of ERNSTER (21). Enzymes. RNase A (EC 2.7.7.16.) and DNase I (EC 3.1.4.5.) were purchased from Worthington Biochem. Co., and Seikagaku Kogyo Co. Glucose oxidase (EC 1.1.3.4.), peroxidase (EC 1.11.1.7.) and a-chymotrypsin (EC 3.4.4.5.) were the products of Sigma Chemical Co. Chemicals. Quinovosamine-HC1 and fucosamine-hc1 were kindly supplied by Dr. N. Suzuki of the Institute of Medical Science, University of Tokyo. Di-N-acetylneuraminyl-lacto-N-tetraose was a gift from Dr. K. Tsurumi of Fukushima Prefectural Medical College. Sepharose 2B, Sephadex G-100, and Sephadex G-75 were the products of Pharmacia Co., and Biogel P-300 and Biogel P-2 were those of Bio-Rad Co. Sodium deoxycholate was purchased from Nakarai Chemical Co., Ltd. Basal features o f lipopolysaccharide RESULTS In the previous paper (4), we described the preparation of lipopolysac charide from the strain P11 by the trichloroacetic acid method, because the phenol method was not successful in this strain (4). In the present work, the phenol method was applied to another pyocin R-sensitive strain, Pseudo-

118 IKEDA and EGAMI VOL. 19 Table 1. Composition and receptor activity of various lipopolysaccharide preparations. Preparation I was prepared from a culture in nutrient broth by the combination of trichloroacetic acid extraction and phenol treatment. Preparation II was prepared from the culture in nutrient broth by the phenol method. Preparation III was prepared from the glutamate medium by the phenol method. Analytical methods were described in the text. Amounts of glucose and rhamnose were determined by quantitative paper chromatography. Samples of 200 igf 200 pl were used for the receptor activity assay. monas aeruginosa P14. Three lipopolysaccharide preparations were obtained from this strain by the use of different media and extraction procedures (Table 1). Preparation I was obtained by trichloroacetic acid extraction followed by the phenol method. Preparations II and III were obtained by the phenol method. Chemical composition and pyocin receptor activity of these preparations are shown in Table 1. Glucose, rhamnose, and heptose were detected as neutral sugars and four amino sugars were found. Large difference in chemical composition, especially in hexosamine and neutral sugar, was observed with different preparations. In the following experiments, preparation III was used, as this preparation showed the highest activity. Preparation III in Table 1 was applied to the Sepharose 2B column with or without sodium deoxycholate (Fig. 1). It is generally known that lipopolysaccharide with a molecular weight of 106,..,107 from a gram-negative bacterium prepared by the phenol method is an aggregated particle and is dissociable by alkali or detergent such as sodium dodecylsulfate or sodium deoxycholate (22). In 0.05 M Tris-HCI without sodium deoxycholate, lipopolysaccharide

1973 Pseudomonas aerugcnosa Lipopolysaccharide 119 Fig. 1. Sepharose 2B gel filtration with or without 0.5% sodium deoxycholate. The crude lipopolysaccharide (7.4 mg) was dissolved in 1.5 ml of 0.05 M Tris-HCl buffer (ph 7.5) and charged on a column (lx 60 cm). Elution was done with the same buffer and 0.9 ml portions were collected ( - ). Gel filtration in the presence of sodium deoxycholate was done in the solution of 0.5% sodium deoxycholate and 0.01 M Tris-HCI. Lipopolysaccharide (7.3 mg) was dissolved in 1.5 ml of the same buffer and eluted with it (0-0). A solution of pyocin R (1.5 ml) with 4 x 104 activity was charged on the same column. Equilibration and elution were done with pyocin dilution buffer (0.1 M NaCI, 0.01 M Tris-HC1 buffer, and 0.001 M MgCl2) (x -- x). Samples, 200 pl each were used for the total neutral sugar assay. showed a very broad peak around the peak of native pyocin R preparation used as a standard, which had the molecular weight of about 107 daltons. In the experiment with sodium deoxycholate, lipopolysaccharide was dissolved and developed in a solution of 0.5% sodium deoxycholate and Tris-HCl buffer (ph 7.5). Lipopolysaccharide appeared at the late stage of elution and dissociation into smaller molecules was suggested. This fraction was excluded by Sephadex G-100 chromatography and slightly included by Biogel P-300 column chromatography, in the experiments with the same sodium deoxycholate concentration. Dissociation of lipopolysaccharide into subunits by sodium deoxycholate Lipopolysaccharide was further treated with the solution of increased sodium deoxycholate concentration. Preparation III was dissolved in 1 % sodium deoxycholate and 0.05 M Tris-HCl (ph 7.5) solution and developed on Sephadex G-100 column, in the presence of the same solution of sodium de-.oxycholate. Two major peaks were revealed by the anthrone reaction (Fig.

120 IKEDA and EGAMI VOL. 19 Fig. 2. Sephadex G-100 chromatography in the presence of 1% sodium deoxycholate. Lipopolysaccharide (20 mg) was dissolved in 2 ml of 1% sodium deoxycholate and 0.05 M Tris-HC1 (ph 7.5), and applied to Sephadex G-100 column (lx 62 cm). Equilibration and elution were done with the same buffer. From a fraction (3 ml each), 200 pl was assayed for neutral sugar (0-0), and pyocin receptor activity per 200,1 was measured by the serial dilution method, both in the presence (O---- A) and absence (x -- x) of 0.5% sodium deoxycholate. 2). Receptor activities were assayed both in the presence and absence of sodium deoxycholate. The first peak with pyocin R receptor activity corresponded to the position of void volume. The second peak had activity only in the absence of sodium deoxycholate. This means that the large part of lipopolysaccharide was dissociated into smaller subunits which had no receptor activity, but in the absence of sodium deoxycholate it recovered the activity. In order to find the conditions for complete dissociation, lipopolysaccharide was treated with 1 % or 2% sodium deoxycholate at different temperature (Table 2) and residual receptor activity after dilution or dialysis was measured. In the presence of 2% sodium deoxycholate, receptor activity was lost almost completely with or without heating and a little activity was recovered after dialysis. In 1% sodium deoxycholate solution, the activity remained even after heating at 90, although the recovery of activity was not high (about 1/5). Then dissociation condition was settled to 5-min heating at 90 in 1% sodium deoxycholate solution. After this treatment, lipopolysaccharide was chromatographed on Sephadex G-100 column in the presence of 0.5% sodium deoxycholate and 0.05 M Tris-HC1 (Fig. 3). Receptor activity was found only in the second peak. Most of amino sugars were found in the first peak, and thus the amino sugar-rich fraction, which had no receptor activity,

1973 Pseudomonas aeruginosa Lipopolysaccharide 121 Table 2. Change in receptor activity by heat and sodium deoxycholate treatment. Lipopolysaccharide (7 mg) was dissolved in 3.5 ml of 1% or 2% sodium deoxycholate solution. A portion of 0.5 ml was heated for 5 min at different temperature. After cooling 100 it of sample was withdrawn and diluted with 400 ~cl of a dilution buffer and the receptor activity was measured. Remaining 400 pl of the sample was dialyzed over 1 day against 0.05 M Tris-HC1 buffer and the activity was measured. was separated from the second lipopolysaccharide peak with the receptor activity. Properties of lipopolysaccharide subunit. In order to determine the molecular weight of lipopolysaccharide subunit, the fraction of the second peak in Fig. 3 was charged on Sephadex G-75 column, in the presence of 0.5% sodium deoxycholate (Fig. 4). a-chymotrypsin and RNase A were used as standard molecules. Lipopolysaccharide subunit peak showed the same R f value as RNase A (mol, wt. 12,640) in this experiment, but the gel filtration in the presence of sodium deoxycholate was not quite reproducible, and the molecular weight of 16,000 and 14,800 was obtained in other experiments. Therefore, the molecular weight of lipopolysaccharide subunit might be regarded as about 12,000-16,000, or might be smaller, considering the standard substances used are globular particles of protein nature. Sedimentation experiment carried out in the presence of 0.5% sodium deoxycholate in 0.05 M Tris-HC1 solution gave the S value of 0.82 for the subunit. Fractions of the second peak in Fig. 4 were collected and dialyzed against Tris-HC1 buffer to remove sodium deoxycholate. Complete removal of sodium deoxycholate was difficult and dialysis in a water bath at 40-50 was effective. The dialyzed sample was concentrated and charged on Sephadex G-100 column (Fig. 5). Lipopolysaccharide was found at exclusion area showing the reassociation of subunits by removal of sodium deoxycholate. In this experiment, the reassociated lipopolysaccharide had practically no receptor activity. However, in other experiments, in which subunits were dialyzed and chromatographed in the solution of buffered saline, without warming or concentration process, an appreciable receptor activity was obtained in reas-

122 IKEDA and EGAMI VOL. 19 Fig. 3. Sephadex G-100 chromatography after heat treatment in 1% sodium deoxycholate. Lipopolysaccharide (32 mg) was dissolved in 3 ml of 1% sodium deoxycholate and 0.05 M Tris-HCI, and heated 5 min at 90. This sample was applied to the Sephadex G-100 column (1.5x 54 cm) and the column eluted in the presence of 0.5% sodium deoxycholate and 0.05 M Tris-HCI. From a portion of 3.2 ml each, 100 p1 was used for neutral sugar assay (a---0), 100,1 for amino sugar after hydrolysis (z-- and 200 p1 for protein estimation (x -- x). Receptor activity was measured with 200 pl of the sample in the presence (x-x) or absence (Q-o) of sodium deoxycholate. sociated Lipopolysaccharide, though recovery of the activity was below 1/3 and the activity decreased rapidly on standing. Receptor activity in the solution of 0.5% sodium deoxycholate was also unstable. For example, peak fraction in Fig. 3 had 26 activity per 0.2 ml just after chromatography and which decreased to 2 after 10 days at room temperature. Analytical data of sugar component of reassociated lipopolysaccharide are shown in Table 3. Glucose was the dominant sugar. Hexosamine components were glucosamine, galactosamine, quinovosamine, and fucosamine, and their molar ratio was about 2 : 2: 1:1. There was no absorption peak at 260 nm. Phosphorus (5.7%) and protein (4.2%) were detected.

1973 Pseudomonas aeruginosa Lipopolysaccharide 123 Fig. 4. Sephadex G-75 chromatography of lipopolysaccharide subunit. A sample (2 ml) from Fig. 3 second peak was applied to the Sephadex G-75 column (1.4 x 65 cm) bufferized with 0.5% sodium deoxycholate and 0.05 M Tris-HCI. Fractions (2.0 ml each) were collected and 200 ~cl portions were used for anthrone reaction (0-0). RNase A (x -- x) and a-chymotrypsin (z-- 0 ), each 1.5 mg, were used as standardd substances. Properties o f polysaccharide moiety Lipopolysaccharide is degraded to "lipid A" and a polysaccharide by dilute acid hydrolysis. The reassociated lipopolysaccharide was hydrolysed in 0.1 N HCl for 30 min at 100, (In other experiments 1% acetic acid was used instead of 0.1 N HC1). Insoluble residue produced, probably "lipid A", was removed and the supernatant was evaporated to dryness. This polysaccharide moiety was dissolved in water and charged on the Sephadex G-50 column (Fig. 6). A polysaccharide peak appeared slightly ahead of the standard sugar, di-n-acetyl neuraminyl-lacto-n-tetraose (mol. wt. 1,289). In another experiment with Biogel P-2, a polysaccharide was eluted a few tubes behind the peak of blue dextran and in the same position as di-n-acetylneuraminyl-lacto-n-tetraose. Therefore,, the molecular weight of the polysaccharide part was estimated as 1,300-1,500.. Reducing power of this polysaccharide was measured before and after hydrolysis. The ratio of reducing end to total was 1 : 7.8, using glucose as a standard. From these results the number of sugar moiety was estimated to be about 8. The result of sugar analysis is shown in Table 4. Galactosamine was the only amino sugar found in this preparation. As neutral sugars,

124 IKEDA and EGAMI VOL. 19 Fig. 5. Sephadex G-100 chromatography of reassociated lipopolysaccharide. Fraction Nos. 16-20 shown in Fig. 3 were collected and dialyzed against 0.05 M Tris-HC1 and concentrated to 2 ml by evaporation. The solution was charged on a Sephadex G-100 column (1.2 x 50 cm) in 0.05 M Tris buffer. From the fraction 1.6 ml each, 100 u1 was used for neutral sugar assay ( -~). Receptor activity was assayed with 200 pl of the sample (~-0). Blue dextran was measured at 600 nm (x--x). Another experiment was done in the presence of 0.1 M NaCI and 0.01 M Tris- HCl during dialysis and gel filtration. Lipopolysaccharide was charged without concentration after dialysis and the receptor activity was measured (0-0). Table 3. Carbohydrate composition of reassociated lipopolysaccharide. Reassociated lipopolysaccharide was analyzed after dialysis and lyophilization. Analytical methods were described in the text.

1973 Pseudomonas aerugtnosa Lipopolysaccharide 125 Fig. 6. Sephadex G-50 gel filtration of polysaccharide moiety. Reassociated lipopolysaccharide (9 mg) was hydrolyzed in 1 ml of 0.1 N HCl for 30 min at 90. Insoluble residue was eliminated by centrifugation and washed with 0.5 ml H2O. Combined supernatant was evaporated to dryness and dissolved in 1 ml of 0.05 M citrate-phosphate buffer (ph 6.6). The carbohydrate fragment was charged on the Sephadex G-50 column (1.2x 54.5 cm) and developed with the same buffer. Fraction (1.5 ml) was collected and neutral sugar was measured ( - ). Blue dextran (x - x), di-n-acetylneuraminyl-lacto-n-tetraose (0--a), and glucose (D -- 0 ) were developed on the same column. Table 4. Composition of carbohydrate fragment. Carbohydrate fragment was prepared as shown in Fig. 6. But hydrolysis of lipopolysaccharide was done in 1% acetic acid solution and the carbohydrate fragment was chromatographed on Biogel P-2. The result of two experiments is described. Amount (pg) of the constituents was calculated as the content per 100 pl of the peak fraction. The molar ratio was calculated by taking galactosamine as 1.

126 IKEDA and EGAMI VOL. 19 Table 5. Summarized scheme of the results. glucose, rhamnose 2:1:1: rhamnose, and heptose were detected. The molar ratio heptose, 2-keto-3-deoxy-sugar acid, and galactosamine 1:1. These results are summarized in Table 5. of glucose, was about DISCUSSION Lipopolysaccharide with pyocin R receptor activity was isolated by the phenol method from Pseudomonas aeruginosa P14, a strain sensitive to pyocin R. It was dissociated by sodium deoxycholate into an amino sugar-rich fraction and lipopolysaccharide subunits without the receptor activity. The former might correspond to "glycosaminopeptide" described by KEY et al. (6). The subunits were reassociated by removal of sodium deoxycholate, accompanied with a partial recovery of the receptor activity. It means that reassociation or polymerization of subunits is required for the recovery of receptor activity. The size of receptor site required for receptor activity was estimated to be 105-106 (unpublished data). The receptor activity of reassociated lipopolysaccharide decreased rapidly. The amino sugar-rich fraction might involve stabilization of the activity. The molecular weight of completely dissociated lipopolysaccharide subunits was estimated to be about 12,000 by Sephadex G-75 gel filtration. It was

1973 Pseudomonas aeruginosa Lipopolysaccharide 127 smaller than the endotoxin (mol, wt. 20,000) of E, coli ( 23 ) dissociated by 2% sodium deoxycholate and much smaller than lipopolysaccharide of E, coli in 0.1% sodium deoxycholate ( 24, 25 ). Further studies will be required for the exact estimation of molecular weight of the subunit by different methods. According to RIBI ( 23 ), the endotoxin subunits of E. coli reassociated by dialysis formed uniform particles with molecular weight of 500,000-1,000,000. In our experiments, reassociated particles were rather insoluble in water and buffer solution, and sedimented more rapidly than the native lipopolysaccharide. Perhaps the elimination of amino sugar-rich fraction might increase the hydrophobic property of lipopolysaccharide and induce the formation of a large aggregate. It may be the reason for the decrease of receptor activity. The molecular weight of carbohydrate moiety isolated from the watersoluble fraction after acid treatment for the elimination of "lipid A" was estimated to be 1,300-1,500. It was much smaller than that of Shigella dysenteriae (26) and Salmonella groups (27). It remains to be elucidated whether 0-antigen specific side chain of Pseudomonas aeruginosa was indeed much shorter than others or if it was due to acid degradation after sodium deoxycholate treatment. The carbohydrate moiety contained glucose, rhamnose, heptose, and galactosamine, but neither glucosamine, fucosamine, nor quinovosamine, unlike native lipopolysaccharide. The results of chemical analysis of lipopolysaccharide are not quite consistent with those carried out long ago by Homma, Egami, and their coworkers or with those carried out by Gray, Wilkinson, and their co-workers with different strains. HOMMA, EGAMI and others identified arabinose and ribose as minor components besides glucose, rhamnose and heptose as neutral sugars (2, 3). It should be reinvestigated if the apparent disagreement is due to the difference in strains. The present authors identified fucosamine and quinovosamine besides common hexosamines in lipopolysaccharide. These less common amino sugars had been identified in lipopolysaccharide of Pseudomonas aeruginosa by SUZVKI (28, 29)0 FENSOM and GRAY (5) identified fucosamine, but not quinovosamine. NAOI et al, found alanine firmly bound to the polysaccharide moiety (3). The same was observed by FENSOM and GRAY (5). We thank Dr. M. Kageyama for valuable discussion, and thank Dr. N. Suzuki Dr. K. Tsurumi for their gift of precious chemicals. and REFERENCES 1) F. EGAMI, M. SHIMOMURA, H. ISHIHARA, J.Y. HOMMA, K. SAGEHASHI, and S. HOSOYA, Bull. Soc. Claim. Biol., 36, 779 (1954). 2) J.Y. HOMMA, N. HAMAMURA, M. NAOI, and F. EGAMI, Bull. Soc. Chirn. Biol_, 40, 647 (1958). 3) M. NAOI, F. EGAMI, N. HAMAMURA, and J.Y. HOMMA, Biochem. Z., 330, 421 (1958). 4) K. IKEDA and F. EGAMI, J. Biochem. (Tokyo), 65, 603 (1969).

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