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Agricultural and Biological Chemistry

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Characterization of Betaine Aldehyde

Dehydrogenase from Cylindrocarpon didymum

Nobuhiro Mori, Bunsei Kawakami, Kimiaki Hyakutome, Yoshiki Tani &

Hideaki Yamada

To cite this article: Nobuhiro Mori, Bunsei Kawakami, Kimiaki Hyakutome, Yoshiki Tani
& Hideaki Yamada (1980) Characterization of Betaine Aldehyde Dehydrogenase from
Cylindrocarpon didymum M-1, Agricultural and Biological Chemistry, 44:12, 3015-3016, DOI:

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Published online: 09 Sep 2014.

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Agric. Bioi. Chern., 44 (12), 3015~3016, 1980

Note C. didymum M-I was grown in a medium containing

1.0 g of choline chloride, 0.1 g of meat extract, 0.1 g of
Characterization of Betaine Aldehyde K 2 HPO., 0.1 g of KH 2 PO., 0.1 g of NaCl and 0.05 g of
Dehydrogenase from Cylindrocarpon MgSO. 7H 20 in 100 ml of tap water at pH 7.0. The
cultivation was carried out at 28C for 60 hr with
didymum M-l
reciprocal shaking. The harvested cells were suspended in
100 mM potassium phosphate buffer (pH 8.0) containing
Nobuhiro MORI,* Bunsei KAWAKAMI, 0.5 mM dithiothreitol (DTT)*I) and disrupted with an
Kimiaki HYAKUTOME, Yoshiki TAN! ultrasonic oscillator. The cell-free extract was added to
solid ammonium sulfate to 30% saturation and the
and Hideaki YAMADA
precipitate was discarded. The precipitate formed by the
Department of Agricultural Chemistry, further addition of ammonium sulfate to 60% saturation
was collected and dissolved in 40 mM buffer. The enzyme
Kyoto University, Kyoto 606 Japan
solution was desalted by Sephadex G-2S equilibrated with
Received July 21, 1980 the 40 mM buffer containing 20% glycerol. Then, the
enzyme solution was applied to DEAE-Sephacel column
In the previous papers, we reported the purification and chromatography. The enzyme which was eluted with a
characterization of choline oxidase,I,2) dimethylglycine linear gradient of the buffer concentration from 40 mM to
oxidase') and sarcosine oxidase4 ) which are involved in 150 mM. Active fractions were combined and concentrated
choline metabolism by Cylindrocarpon didymum M-l. In by ultrafiltration in a collodion bag apparatus. The
addition, we found betaine aldehyde dehydrogenase in the concentrated enzyme solution was applied to a Sephacryl
cell-free extract of fungus grown on choline medium. S-200 column (2.5 x 80 cm) equilibrated with a 100mM
Betaine aldehyde dehydrogenase from rat liverS) was buffer containing 20% glycerol. Repeated gel filtration
partially purified and characterized to require NAD+ as gave single and nearly symmetrical peaks of protein and
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coenzyme. Highly purified betaine aldehyde dehydro- activity. Result of the purification is summarized in Table
genase from Pseudomonas aeruginosa A-166 ) required both I. The purified enzyme gave many protein bands on
NADP+ and NAD+ as coenzyme. On the other hand, acrylamide gel electrophoresis but one 'predominant band
choline oxidase purified from C. didymum M-I oxidized and one faint band on SDS-acrylamide gel electrophoresis.
betaine aldehyde in addition to choline. 2) The apparent Km The molecular weight of the enzyme was estimated to be
value for betaine aldehyde (5.8 mM) was about four times about 220,000 by gel filtration on Sephacryl S-200. The
the Km for choline (1.3 mM). This was also found in molecular weight of the subunit of the enzyme was
choline oxidase from Arthrobacter globiformis. 7 ) The estimated to be 58,000 by SDS-acrylamide gel elec-
present report deals with the partial purification and trophoresis. Enzyme activity was completely lost when
characterization of betaine aldehyde dehydrogenase from dialyzed aginst the 10 mM buffer (pH 8.0). However,
C. didymum M-1. addition of glycerol at a concentration of 20% stabilized


FROM Cylindrocarpon didymum M-I
The reaction system for the determination of betaine aldehyde dehydrogenase activity consisted of 250
limol of potassium phosphate buffer (pH 8.0), llimol of betaine aldehyde, llimol of NAD+ and a suitable
amount of enzyme solution in a total volume of 3.0 m!. The increase of absorbance at 340 nm was measured
spectrophotometrically at 30C. One unit of the enzyme activity was defined as the amount of enzyme which
catalyzed the formation of llimol of NADH per min. Protein concentration was determined by the absorbance
at 280nm, where Ej:;;. value of 10.0 was used. Specific activity was defined as unit per mg protein.

Total Total Specific

Fraction protein activity activity
(mg) (U) (U/mg) (%)

Cell-free extract 8,564 521 0.06 100

Ammonium sulfate
(30 ~ 60% sat.) 1,666 508 0.30 98
D EAE-Sephacel 42.7 214 5.03 41
Sephacryl S-200 6.6 135 20.5 26

* Present address: Department of Agricultural

Chemistry, Tollori University, Koyama, Tollori 680, Japan. *1) This buffer was used throughout this work.

the activity. The enzyme was most active at pH 8.5 ~ 9.0. -c

The enzyme activity was completely inhibited by Cu 2 +, --,

Zn 2 +, Hi' +, p-chloromercuribenzoate, iodoacetate, 0-

phenanthroline and partially by Co2+, IX,IX'-dipyridyl. The
enzyme was highly specific for betaine aldehyde. Aliphatic o 2 4 6
aldehydes and a variety of aldehydes such as' crotonal- A

dehyde, benzaldehyde and D-glucose were not dehydro-

genated. The enzyme required NAD+ as coenzyme but .,E o
NADP + was inert. Double reciprocal plots of v against , 10
betaine aldehyde gave parallel straight lines for various ->-~

o 5 10 15
1/Betaine aldehyde(mM-1)
o 2 4 6 8
l/Betaine aldehyde (mM-1}
A FIG. 2. Double Reciprocal Plots of the Reaction
Catalyzed by Betaine Aldehyde Dehydrogenase.
-.S 15 C
Experimental conditions were the same as those in Fig. 1.
.,-E The concentrations of NAD + were: A, 0.66; B, 0.33; C,
,10 0.23; D, 0.17 mM, respectively.
choline was oxidized to betaine aldehyde by choline
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oxidase and further oxidized to betaine by NAD-

dependent betaine aldehyde dehydrogenase in C. didymum
o 5 10 15
1/NAD+( mW1}
Acknowledgments. We wish to thank Professor
FIG. 1. Double Reciporcal Plots of the Reaction
Yoshio Ichikawa, Tottori University, for his encourage-
Catalyzed by Betaine Aldehyde Dehydrogenase.
ment during the course of this study.
Experimental conditions were described in Table 1. The
concentrations of betaine aldehyde were: A, 0.33; B, REFERENCES
0.23; C, 0.17; D, 0.13 mM, respectively.
I) Y. Tani, N. Mori, K. Ogata and H. Yamada, Agric.
BioI. Chern., 43, 815 (1979).
concentrations of NAD + (Fig. I). Parallel lines were also 2) H. Yamada, N. Mori and Y. Tani, Agric. Bioi.
observed for various concentrations of betaine aldehyde in Chem., 43, 2173 (1979).
double reciprocal plots of v against NAD + concentration 3) N. Mori, B. Kawakami, Y. Tani and H. Yamada,
(Fig. 2). These results suggest that the reaction proceeds by Agric. Bioi. Chern., 44, 1383 (1980).
a "Ping-Pong" mechanism as designated by Cleland. B ) 4) N. Mori, M. Sano, Y. Tani and H. Yamada, Agric.
Michaelis constants for betaine aldehyde and NAD + were, Bioi. Chern., 44, 1391 (1980).
respectively, calculated from secondary plots of the 5) H. A. Rothschild, O. Cori and E. S. G. Barron, J.
intercept of the parallel lines at the ordinate versus the BioI. Chem., 208, 41 (1954).
concentration of NAD + or betaine aldehyde (inserted in 6) T. Nagasawa, Y. Kawabata, Y. Tani and K. Ogata,
Figs. 1 and 2). Km values of 0.31 mM for betaine aldehyde Agric. Bioi. Chem., 40, 1743 (1976).
and 0.38 mM for NAD+ were obtained. Thus, the affinity 7) S. Ikuta, S. Imamura, H. Misaki and Y. Horiuti, J.
for betaine aldehyde of the enzyme was about twenty times Biochem., 82, 1741 (1977).
that of choline oxidase. Therefore, it can be concluded that 8) W. W. Cleland, Ann. Rev. Biochem., 36, 77 (1967).