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Characterization of Membrane-bound Spermidine Dehydrogenase of Citrobacter freundii a

a

a

ab

Tomohiro Hisano , Kousaku Murata , Akira Kimura , Kazunobu Matsushita , Hirohide Toyama a

ab

& Osao Adachi

ab

Research Institute for Food Science, Kyoto University, Uji, Kyoto 611, Japan

b

Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753, Japan. Published online: 12 Jun 2014.

To cite this article: Tomohiro Hisano, Kousaku Murata, Akira Kimura, Kazunobu Matsushita, Hirohide Toyama & Osao Adachi (1992) Characterization of Membrane-bound Spermidine Dehydrogenase of Citrobacter freundii, Bioscience, Biotechnology, and Biochemistry, 56:12, 1916-1920, DOI: 10.1271/bbb.56.1916 To link to this article: http://dx.doi.org/10.1271/bbb.56.1916

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Biosci. Biotech. Biochem., 56 (12), 1916-1920, 1992

Characterization of Membrane-bound Spermidine Dehydrogenase of Citrohacter freundii Tomohiro HISANO, Kousaku MURATA, Akira KIMURA, Kazunobu MATSUSHITA,* Hirohide TOYAMA,* and Osao ADACHI* Research Institute for Food Science, Kyoto University, Uji, Kyoto 611, Japan Faculty of Agriculture, Yamaguchi University, Yamaguchi 753, Japan. Received April 24, 1992

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* Department of Biological Chemistry,

Spermidine dehydrogenase found in the membrane fraction of Citrohacter freundii IFO 12681 was solubilized with Triton X-tOO and further purified to homogeneity. The properties of the membrane enzyme were almost identical to those obtained from the soluble fraction of the organism with respect to molecular and catalytic properties. Thus, binding properties of the enzyme to the bacterial membrane were checked. The ratio of enzyme activity found in the soluble fraction to the membrane fraction was dependent on salt concentration during cell disruption. A hydrophobic interaction was largely involved in anchoring the enzyme to the membrane fraction. Purified spermidine dehydrogenase from the soluble fraction was readily adsorbed into the membrane fraction in the presence of salt. Spermidine dehydrogenase appeared to be a membrane-bound enzyme localized in the cytoplasmic membranes in a manner that makes a partial release of the enzyme possible during mechanical cell disruption. When spermidine oxidation was done with the resting cells of C. freundii, a stoichiometric formation of two reaction products, t ,3-diaminopropane and y-aminobutyraldeyde, was observed without any lag time. These facts indicate that the enzyme is localized on the outer surface of the cytoplasmic membranes or in the periplasmic space of the organism.

In our previous papers 1 - 3) on spermidine dehydrogenase from C. freundii IFO 12681, enzyme formation, purification, and examination of the purified enzyme were described. The enzyme was found to be a single polypeptide including two prosthetic groups, heme b and a covalently bound carbonyl compound. 3 ) The carbonyl compound has been thought to be a covalently bound PQQ. The location of the enzyme in the organism is still unknown, though it catalyzes an important oxidative splitting reaction of spermidine into 1,3-diaminopropane and y-aminobutyraldehyde. About a half of the total enzyme activity remains in the membrane fraction when separated the cell homogenate into the soluble fraction and membrane fraction. 3 ) Solubilization and further examinations of the membrane-bound spermidine dehydrogenase are essential in relation to the location of the enzyme. Thus, in this paper, the enzyme in the membrane fraction was solubilized and further purified to homogeneity. It is indicated that the enzymatic properties of the purified enzyme are almost identical to those from the soluble fraction, suggesting the identity of the two enzymes. Localization of spermidine dehydrogenase in C. freundii is also discussed.

Materials and Methods Preparation ofmembrane fraction. C.freundii IFO 12861 was cultured on a basal medium in which spermidine trihydrochloride was added to 0.5% as described prevously. 1.3) The cell suspension of I g wet cells per 5 ml of 5 mM potassium phosphate, pH 7.2, was passed through a French pressure cell press for 2 times at 1000 kg/cm 2 and exposed to sonic oscillation for 30 min. Temperature was kept below 4°C during cell disruption. Topologically, the bacterial cell membranes prepared under these conditions were a mixture of inside out membranes and right side out membranes as described previously.6) The cell homogenate was centrifuged at 5000 x g for 10 min to remove undisrupted cells. The resulting cell-free extract was further fractionated into the membrane fraction and soluble fraction by ultracentrifugation at 68,000 x g for 90 min. The precipitated membrane fraction was re-homogenized by a glass homogenizer in the presence of 0.25 M potassium phosphate, pH 7.2. The protein content

was adjusted to 10 mg/ml with 0.25 M potassium phosphate, pH 7.2, and the membrane suspension was further exposed to a sonic oscillator as above to facilitate enzyme dissociation if any in the presence of high salt concentration. The homogenate was then centrifuged at 68,000 x g for 90 min. The precipitate at this stage was designated as the membrane fraction.

Assays. The measurement of enzyme activity, definition of enzyme activity, and measurement of protein content were done as described in our previous paper. 3) Amino acid composition. Analysis of amino acid composition was done with the purified enzyme (about 0.2/lmol). After hydrolysis in 6 N HCl for 72 h at 110°C, amino acid analysis was done with a Shimadzu amino acid analyzing system, model LC-6A. Estimation ofhydrophobic interaction ofspermidine dehydrogenase to the membrane fraction. A crystalline enzyme (0.2 mg, 180 units/mg) prepared from the soluble fraction 3 ) was mixed with the membrane fraction (10 mg) in the presence or absence of 3 M KCI in 10 ml of total volume. The membrane fractions used had been extensively washed with diluted buffer solutions or water until no appreciable amount of enzyme activity was observed in the supernatant. Thus, it had also been confirmed before use that, unless treated with detergent, the membrane fractions gave no liberation of the enzyme activity even though they were treated with a diluted buffer solution or water. Such membrane preparations showed 0.14 to 0.16 units/mg of specific activity. The first supernatant solutions were obtained after the mixtures were centrifuged at 68,000 x g for 90 min. The resulting precipitates were homogenized in IOml of I mM potassium phosphate, pH 7.2, and separated into the second supernatant solutions and the membrane precipitates. Estimation ofdissociable and nondissociable spermidine dehydrogenase by lowering hydrophobicity. A ratio of dissociable spermidine dehydrogenase to the nondissociable one from the membrane fraction was measured by lowering KCl concentrations during cell fractionation. First, KCl was added to I M to the bacterial cell suspension including 109 of cells and the total volume was adjusted to 40 ml. The cell-free extract obtained after cell disruption and removal of intact cells was centrifuged at 68,000 x g for 90 min to separate the supernatant (I MKCl sup.) from the membrane. The precipitated membrane fraction was homogenized in 10 mM potassium phosphate, pH 7.2, containing 1M KCl, and the total volume was adjusted to 40 ml with the same buffer and centrifuged to collect the supernatant

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Spermidine Dehydrogenase of Citrobacter freundii

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(I M KCl wash). The resulting precipitate (washed membrane) was homogenized in 0.5 mM potassium phosphate, pH 7.2, and centrifuged again to separate the soluble fraction from the membrane fraction.

Measurement of reaction products. y-Aminobutyraldehyde, one of the reaction products of spermidine oxidation was identified and measured by colorimetry. Spermidine was incubated with resting cells at 25°C under shaking. A smapie of the incubation mixture (0.1 ml) was spun down in a flash centrifuge. L11~Pyrroline, a spontaneous cyclization product of y-aminobutyraldehyde, was measured in the supernatant by adding o-aminobenzaldehyde as in a similar method described by Holmstedt et al. 4 ) The intensity of the yellow color was estimated by measuring the absorbance at 435 nm. The millimolar extinction coefficient of 1.86 was used for calculations. Incubation of spermidine with resting cells was done until no further increase in intensity at 435 nm was observed. The incubation mixture was finally centrifuged to separated the supernatant. The precipitated cells were once homogenized in a small volume of water and centrifuged. The second supernatant was combined with the first supernatant and adsorbed onto a small column (1 x 15 cm) of Dowex 50 (H + -form). Development was done with gradient elution of 2.5 N HCl under essentially the same conditions described by Tabor et af. 5) Another reaction product, 1,3-diaminopropane, was measured colorimetrically with ninhydrin after the column was developed. Other analyses. Polyacrylamide gel electrophoresis, SDS-polyacrylamide gel electrophoresis (SDS-PAGE), analytical ultracentrifugation, and measurement of cytochrome were done under essentially the same conditions as described in our previous paper. 3)

1917

used for the enzyme from the soluble fraction,3) except that all buffer solutions had Triton X-IOO added to 0.1 %. In a preliminary examination, some detergents including Triton X-IOO had been shown to solubilize the enzyme from the membrane fraction. 7) After column chromatographies using DEAE-cellulose and w-aminooctylagarose, the enzyme was finally purified by gel filtration with a Sephadex G-200 column. A symmetric elution peak accompanying enzyme activity came out separately from most of impurities which were eluted fast at the void volume. Judging from the elution profile (data not shown), the enzyme seemed to be almost pure and gave a constant specific activity of spermidine dehydrogenase throughout the elution peak. One example of enzyme purification is summarized in Table II.

Properties of purified enzyme The purified enzyme showed a single protein band in both polyacrylamide gel electrophoresis and analytical ultracentrifugation. The sedimentation constant, Szo,w, of the enzyme was 4.3 S, which is similar to that of the enzyme obtained from the soluble fraction. In Fig. I, sedimentation patterns and SDS-PAGE are shown as the evidence for purity of the preparation.

Results and Discussion Distribution and location of spermidine dehydrogenase in C. freundii Fractionation of the bacterial cells was done in a conventional manner such as that used previouslyl,3) and distribution of the enzyme activity was examined (Table I). Nearly 50% of the total enzyme activity was always found in the supernatant after the first ultracentrifugation. About 3% of the total enzyme activity was usually found in the second supernatant and about 45% to 500/0 of the total enzyme activity was detected in the membrane fraction. When the precipitate from the first ultracentrifugation was treated with I M KCI as above, no appreciable amount of the enzyme activity was detected in the resulting supernatant. Thus, it was suggested that the enzyme is membrane-bound and some of which is dissociated partly from the cytoplasmic membranes during mechanical cell disruption.

Table II. Summary of Purification of Membrane-bound Spermidine Dehydrogenase from C. freundii

Step

Total protein (mg)

Cell-free extract 14,920 Membrane fraction 8,680 Triton X-IOO sup. 2,380 DEAE-cellulose 1,500 10 Aminoagarose Gel filtration 5.7

Total Specific activity activity (units) (units/mg) 2,675 1,435 850 850 805 745

0.18 0.16 0.35 0.56 81.31 130.70

Purification (fold)

Recovery (%)

1.0 1.0 2.0 3.1 451.7 726.1

100 54 32 32 30 28

Purification of membrane-bound spermidine dehydrogenase The membrane fraction obtained from 75 g of wet cells of C. freundii was used. Preparation of the membrane fraction was done as described in Methods. Purification of the enzyme was done under essentially the same procedure Table I. Distribution and Localization of Spermidine Dehydrogenase in C.freundii Fractionation of cell-free extract was done with 75 g of fresh cells as described in Materials and Methods.

Step

Total activity (units)

Total protein (mg)

Specific activity (units/mg)

Distribution of activity (%)

Cell-free extract 1st Supernatant 2nd Supernatant Membrane fraction

2,675 1,240 80 1,435

14,920 6,240 440 8,680

0.18 0.19 0.18 0.18

100 49 3 47

Fig. l. Sedimentation Patterns and SDS-PAGE of Spermidine Dehydrogenase Purified from the Membrane Fraction of C. freundii. (A) Sedimentation patterns were measured by an absorption scanning measured at 420nm with the protein concentration of about I mg per ml of IOmM potassium phosphate, pH 7.2, containing 0.1% Triton X-IOO. The scanning was set for 15-min intervals. Temperature was kept at 20 e throughout the measurement. (B) SDS-PAGE was done as shown in our previous paper l ) except that the gel concentration was increased to 15.5%. Lane I is marker proteins consisting of phosphorylase b (M, 94,000), bovine serum albumin (M r 68,000), ovalbumin (M, 43,000), carbonic anhydrase (M, 31,000) and lysozyme (M r 14,000) from top to bottom. Lanes 2 and 3 are the purified enzyme of 5 pg and 10 Ilg. respectively. G

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T. HlsANo et al.

1918

Table III.

Amino Acid Composition of Spermidine Dehydrogenase Number of residues/mole enzyme

Amino acids

I I I I

0.4

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j

I

, ;' I 0.2

1\ I \ J

581 ~ II

I

\\ I

\

\ \

530 /1

','

360

440

Soluble fraction

73.0 21.0 33.0 62.0 37.0 62.0 71.0 0.1 36.0 11.0 25.0 55.0 10.0 24.0 26.0 17.0 29.0

72.9 21.2 32.6 61.9 37.0 61.9 71.0 0.4 35.6 10.7 25.1 54.9 10.4 24.1 26.0 16.7 28.8

1\ I \

'",j "'\\ 0 280

Membrane fraction Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cysteine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine

0.8

i

Spermidine dehydrogenase from

520

800

Wavelength (nm)

Fig. 2. Absorption Spectra of Spermidine Dehydrogenase Purified from the Membrane Fraction of C. freundii. The oxidized enzyme of spermidine dehydrogenase (I mg/ml in 10 mM potassium phosphate, pH 7.2, containing 0.1% Triton X-lOO) is traced by a solid line. The absorption spectrum of the reduced enzyme (broken line) was prepared immediately after addition of 2/11 (2/1mol) of spermidine to the enzyme solution.

Absorption maxima of the enzyme were found at 561, 530, 428, and 330 nm in the visible region with the reduced enzyme, while a sole absorption peak at 416 nm was obtained with the oxidized enzyme (Fig. 2). The heme b involved was autooxidizable as well. This was convincing evidence that a b type of cytochrome was involved in the enzyme as has been identified with the enzyme from the soluble fraction. 3) One mole of cytochrome b was measured per mole of the enzyme as well. Absorption spectrum in the ultraviolet region could not be measured due to the presence of Triton X~100 in the enzyme solution. However, it could be deduced that the absolute absorption spectrum of the enzyme is identical to that from the soluble fraction of the organism. The molecular weight of the enzyme was estimated to be about 65,000 by dual methods using SDS-PAGE and gel filtration on Sephadex G-200 column. Amino acid composition of the enzyme is compared with that from the soluble fraction in Table III. It is clear that the amino acid composition of the enzyme is also identical to that from the soluble fraction, confirming the identity of the both enzymes, although the immunochemical cross reaction of the enzymes has not been checked. Although the corresponding data were not shown here, no appreciable difference in substrate specificity, electron acceptor specificity, and inhibitor specificity was observed between the two enzymes. Binding of spermidine dehydrogenase to the membrane fraction by hydrophobic interaction A crystalline spermidine dehydrogenase from the

The values given here are the average of dual analyses of pure spermidine dehydrogenase from C. freundii recalculated for a molecular weight of 63,000. Table IV. Hydrophobic Interaction of Spermidine Dehydrogenase to the Membrane Fraction A crystalline enzyme prepared from the soluble fraction 3 ) was incubated with the membrane fraction in the presence or absence of 3 M KCl as described in Materials and Methods. Enzyme activity was measured with the individual supernatants, where the total volume was taken to be 10 mI.

Step

Total activity (units)

Total protein (mg)

Incubation in the absence of KCL 1st Supernatant 37.5 2nd Supernatant 0.1 Precipitate 1.6 Incubation in the presence of KCI 1st Supernatant 23.4 2nd Supernatant 10.9 1.5 Precipitate Q

b

0.19

Specific activity (units/mg)

Distribution of activity (%)Q

197.37

94.9 0.2 4.0

NOb 9.58

0.16

0.15 0.05 9.80

157.06 227.08 0.15

59.2 27.5

3.8

The total enzyme activity was calculated to be 39.5 units, which came from 38.0 units of crystalline enzyme (0.2 mg protein) and 1.5 units of membrane fractions (l0 mg protein). Not detected.

soluble fraction 3 ) was mixed with the membrane fraction to see whether the membrane fraction is able to bind the spermidine dehydrogenase found in the soluble fraction and to see whether the enzyme from the soluble fraction is an identical species from that from the membrane fraction. As clearly shown in Table IV, in the absence of KCI, no appreciable adsorption of spermidine dehydrogenase into the membrane fraction was observed and about 95% of the original enzyme activity was recovered in the 1st supernatant. On the other hand, a smaller amount of enzyme acivity was detected in the 1st supernatant prepared in the presence of KCl. The adsorbed enzyme appeared in the 2nd supernatant upon treatment of the NII-Electronic Library Service

Spermidine Dehydrogenase of Citrobacter freundii 1.2r-----~--------.1.2

Table V. Estimation of Dissociable and Nondissociable Enzyme Activity by Lowering Hydrophobicity Cell disruption was done in the presence of I M KCI. After separation of the membrane fraction from the soluble fraction, the resulting membrane fraction was treated with a diluted buffer solution as described in Materials and Methods.

:::I

Step

Total protein (mg)

Il-Pyrroline

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"0 0.8

~.

E

20.6

.S "0 ~ 0.4·

Specific Distribution activity of activity (units/mg) (%)

u

Cl. I

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Characterization of membrane-bound spermidine dehydrogenase of Citrobacter freundii.

Spermidine dehydrogenase found in the membrane fraction of Citrobacter freundii IFO 12681 was solubilized with Triton X-100 and further purified to ho...
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