J. Biol. Macromol., 10 (3), 73-82 (2010)

Regular Paper

Expression of recombinant human histidine decarboxylase with full length andC-terminal truncated forms in yeast and bacterial cells
Yoko Nitta,* Juri Ohshita, Hu Liu, Yoshie Kuronuma and Hiroshi Ueno
Laboratory of Applied Microbiology and Biochemistry, Nara Women’s University,
Nara 630-8506, Japan

Received October 19, 2010; Accepted October 21, 2010

Histamine-synthetic enzyme (HDC) from human basophilic leukemia cell line
KU-812-F was expressed by yeast, cell-free, and bacterial expression systems. A
full length form of 74-kDa HDC was expressed in S. cerevisiae and purified byusing ion exchange and gel filtration chromatographies. In the purification
process, the 74-kDa HDC was found in an enzymatically inactive fraction, while
degraded HDCs were detected in an enzymatically active fraction. The 74-kDa
HDC expressed in the wheat germ cell-free system was also found in an inactive
fraction. When a C-terminal truncated form of HDC expressed in E. coli waspurified, the enzyme had high specific activity. Our results suggest that the
enzyme activity exhibited by the degraded HDCs expressed in S. cerevisiae could
be due to the C-terminal truncation.
Key words: histamine, HDC, recombinantHistidine decarboxylase (HDC, EC:

amine, that influences physiological reactions

4.1.1.22) catalyzes a decarboxylation reaction

including allergy, gastric acid secretion,of histidine to produce histamine, a bioactive*To whom correspondence should be
addressed: School of Human Science andEnvironment, University of Hyogo,
1-1-12 Shinzaike-honmachi, Himeji,
Hyogo 670-0092, Japan. E-mail:nitta@shse.u-hyogo.ac.jp, Phone:+81-79-292-9337

Abbreviations: AroDC, aromatic aminoacid decarboxylase; DTT, dithiothreitol;E. coli, Escherichia coli; GAD, glutamate
decarboxylase; HDC, histidine
decarboxylase; OPA, o-phthalaldehyde;
PEG, polyethylene glycol; PLP,
Pyridoxal 5'-phosphate; S. cerevisiae,
Saccharomyces cerevisiae

Characterization of human histidine decarboxylase

74

capillary dilatation, neurotransmission, andinitiallysmooth muscle contraction.

solubilization, the enzymatic activity was

In human,insoluble

where,

after

theseveral cell types capable of producing

detected [12].

histamine have been identified.

Since therecombinant 74-kDa HDC expressed by the

HDC expression pattern in individual cells

baculovirus system was concluded to be

are not entirely understood, it is possible thatinactive [13] and rat recombinant 74-kDa

the histamine production may be found in

HDC expressed by the reticulocyte cell lysate

other cell types.

system was also inactive [7].

Human HDC cDNA was isolated

On the other hand, mouse

In the bothcases, the solubility of the enzyme was notand characterized from basophilic leukemia

documented.

cells [1], and rat and mouse HDC cDNAs

recombinant 74-kDa HDC remain unclear,

were isolated from fetal liver [2] andwe need to obtain a soluble 74-kDa HDC

mastocytoma P-815 cells [3], respectively.

protein in homogeneous preparation in order

Generally, the HDC cDNAs encode a protein

to carry out biochemical studies.

with a molar mass of 74-kDa. Taguchi et al.

In our previous studies, we were able to

and Ohmori et al. purified HDC from fetal rat

successfully express human recombinantliver [4] and from mouse mastocytoma P-815

74-kDa HDC as a soluble form in yeast,

cells [5], respectively. The molecular mass of

however the enzyme was not purified [14].

the both HDCs was estimated to be

In the present study, the purification of the

53-54-kDa, that is about 20-kDa smaller than

74-kDaHDC produced by thisyeast

the size estimated from the full length

expression system was carried out.

As a

cDNAs.

reference, we also examined the 74-kDa

The difference in the molecular

Since the properties of themass is explained by the post-translational

HDC

processing that truncates C-terminal about

expression system, since this in vitro

20-kDa region [6].

expression

Several attempts made

by

using

wheat

systemgerm

supposedcell-freetobeto understand the detailed mechanism of theprotease-free and the full-length productpost-translational processing did not clear thewould be expected.

activity profile on 74-kDa HDC [6-11]; thus

expression system of 54-kDa HDC, theit is important to closely examine whether or

C-terminal truncated form, was attempted

not the full length form of HDC exhibitswith E. coli． We evaluated the enzyme

enzyme activity.

activity of the purified 74-kDa HDC forms

Furthermore, an

There were several attempts madeproduced by the yeast and wheat germto produce recombinant 74-kDa HDC; such

systems as well as the 54-kDa HDC obtained

as baculovirus system, reticulocyte cell lysate

by the bacterial system.

system and yeast system.

HDC protein

expressed by the baculovirus system was

MATERIALS AND METHODSNitta, Y. et al.

75

Construction of the expression vectors - The

system and 54-kDa HDC cDNA for bacterial

plasmid pWIH31, previously constructed forexpression system.

the expression of human HDC [14], was used

was used for the expression of 74-kDa HDC

forin the wheat germ cell-free expression systemthe74-kDaHDC

Saccharomyces cerevisiae.

expression

inpWIH31 was

The plasmid pEU-E01(CellFree Sciences, Ehime, Japan).

Thealso used as a template to obtain 74-kDa

plasmid pET21a (Novagen, Madison, WI),HDC cDNA for wheat germ expression

was

SalI

HDC

XhoI

SalI
GTCGACCATG

used

for

expression

of

XhoI
TAGACTCGAG

HDC

20 kDa54 kDapWIH31

the74 kDa

EcoRI

SalI

KpnI

pBluescript-HDC

SalIGTCGACCATG

TAG

KpnI
GGTACCEcoRI
GAATTC

XhoI
CTCGAG

ATG

Histidine tag

pEU-74HDC

Fig 1.

pET-54HDC-His6

Structure and construction of the recombinant expression vector pEU-74HDC andpET-54HDC-His6.

Characterization of human histidine decarboxylase76

polyhistidine-tagged

54-kDaHDC

in

Escherichia coli strain BL21(DE3).

yeast extract, 1% peptone, and 2% glucose
for overnight and was cultured in a medium

A DNA fragment containing HDC

containing 0.67% yeast nitrogen base without

coding region was obtained from plasmidamino acid, 2% histidine, 2% leucine, 2%

pWIH31 after digestion with SalI and XhoI.

adenine sulfate, 2% uracil, and 2% sodium

It was inserted into pBluescript at the SalI

acetateand XhoI sites to give pBluescript-HDC.

late-exponential phase were harvested by

The plasmid vector, pBluescript-HDC, wascentrifugation. The precipitated cell pellets

then digested with SalI and KpnI to give awere suspended into 60 mM potassium

fragment containing HDC coding region, andphosphate buffer, pH 7.1, containing 0.1 mM

then the fragment was inserted into pEU-E01

DTT, 0.5 mM PLP and 1 mM EDTA, then,by using the SalI and KpnI sites to constructapplied for the cell disruption.

pEU-74HDC.

suspended cells were placed in Bead-Beater

at

28

°C.

Cells

in

the

The

For C-terminal truncated HDC,

(Biospec Products, Bartlesville, OK, U.S.A.)

54-kDa HDC cDNA was amplified by PCR

that was kept cold with ice and one-minutewith two primers, 5’- GAG AAT TCG ATA

disruption was repeated for seven times.

TCA AGC TTA TCG ATA CCG -3’ and 5’-

The suspension was centrifuged at 10,000 × g

TCC TCG AGT GAC TCA GGA TGA GAG

for 30 min and the supernatant was collected-3’, and pBluescript-HDC was used as a

as a cell lysate.

template. Those primers were designed to

In vitro protein expression was

introduce XhoI site at downstream of Ser476

performed

codon. The PCR product was digested withexpression system by using wheat germ

EcoRI and XhoI and inserted into the same

expression premium kit (CellFree Sciences,

sites

Ehime,

ofthe

pET21atoconstruct

with

wheat

Japan).

cell-freeTranscription

and

were

out

pET-54HDC-His6, so that the product,translation

54-kDa HDC-His6, was designed to have aaccording to the manufacture’s protocol.

poly-His tag at its C-terminus end.

The

The

plasmid construction was summarized in Fig.

reactionsgerm

reaction

products

carriedobtained

were

subjected for biochemical analysis.

1. The constructed plasmid was confirmed

E. coli strain transfected withby the restriction enzyme digestion analysis

pET21a-54HDC-His6 was grown at 37 °C for

and DNA sequencing.

overnight in LB medium containing 50 µg/ml

Preparation of cell lysate - Cell

ampicillin. This pre-culture was transferred

growth and cell lysate preparation of S.

into the fresh LB medium containing 50

cerevisiae were carried out as describedµg/ml ampicillin to make an initial OD600 of

previously

were

0.05, which was then inoculated at 37 °C

pre-cultured in a medium containing 1%

until OD600 reached above 0.6. The HDC

[14].

Yeast

cells

77

Nitta, Y. et al.

protein production was carried out without

sulfate was completely dissolved and an

any induction substances such as IPTG. The

additional 20-min stirring was completed, theculture was continued to inoculate at 30 °C

suspension was centrifuged at 10,000 × g forfor another 15 h.

After the centrifugation,

30 min to obtain a supernatant.

the

were

sulfate was then added to the supernatant tocellpellets

collected

andAmmonium

re-suspended with an equal amount of PBS

be 55% saturation.

The suspension was

which was constituted with 0.2 g/l KCl, 0.2

centrifuged at 10,000 × g for 30 min and theg/l KH2PO4, 8 g/l NaCl and 1.15 g/lpellets were dissolved with about 5 volumes

Na2HPO4.

The cell lysate was obtained byof 20 mM potassium phosphate buffer, pH

treating the suspension with ultrasonication

6.8, and then the solution was dialyzed

and centrifugation at 10000 × g for 20 min at

against the same buffer for overnight in the

4 °C.

cold room.

The dialyzed solution was

Enzyme activity assay - HDC

centrifuged at 10,000 × g for 30 min and theactivity was determined by quantitatingsupernatant containing the enzyme washistamine on an HPLC equipped withcollected.

o-phthalaldehyde

was applied on a Fractgel EMD DEAE

(OPA)

derivatization

system as described previously [15].

The obtained enzyme solutionAn

650(S) HPLC column (1.5 × 13 cm) that was

assay mixture contained 0.1 mM DTT, 1%

pre-equilibrated with 20 mM potassium(v/v) PEG#300, 10 µM PLP, 3.2 mM

phosphate buffer, pH 6.8, containing 0.2 mM

L-histidine and the enzyme solution in 50

DTT, 1% (v/v) PEG#300, 10 µM PLP andmM potassium phosphate buffer, pH 6.8,was

0.5 mM EDTA at a flow rate of 1.0 ml/min.

prepared as described by Ohmori et al. [5].

The enzyme fraction was eluted within a 30

The assay mixture was incubated at 37 °C formin by application of a linear gradient of

30 or 60 min and the reaction was terminatedpotassiumby adding 60% perchloric acid at the amountcontaining 0.2 mM DTT, 1 v/v % PEG#300,

of 5% of the assay volume.

10 µM PLP and 0.5 mM EDTA from 20 to

The protein

phosphatebuffer,pH

6.8,

concentration was estimated by using BioRad

500 mM.

The fractions containing HDC

protein assay with bovine serum albumin as

activity were pooled and applied on astandard.

Sephacryl S-400 gel filtration HPLC column
Enzyme purification procedure -(2.5 × 45 cm) that was pre-equilibrated withpurificationout

200 mM potassium phosphate buffer, pH 6.8,

according to the procedure described bycontaining 0.2 mM DTT, 1% (v/v) PEG#300,

Ohmori et al. [5].

10 µM PLP and 0.5 mM EDTA at a flow rate

EnzymewascarriedAmmonium sulfate

precipitation was carried out by adding solidof 0.7 ml/min.

ammonium sulfate slowly into the solution to

the same buffer and aliquots of each of thereach 25% saturation.

fractions were examined for HDC activity,

After ammonium

The column was eluted with

Characterization of human histidine decarboxylase

78

SDS-PAGE and Western blot analysis.

treatment with ECL reagent, the membrane

The cell lysate of E. coli cells wasapplied on a column packed with cobalt
based

immobilized

metal

cassette (Invitrogen, CA, U.S.A.).

affinity

chromatography resin (Clontech, Mountain
View, CA, U.S.A.).

was exposed to Polaroid film (Type 667) in a

The column was

RESULTS AND DISCUSSION
Expressionofrecombinantequilibrated with 50mM sodium phosphatefull-length

buffer, pH 6.8, containing 0.3 N NaCl, 1%

purification of HDC – In our previous study,

PEG#300, and 10 µM PLP, and washed withhuman HDC was expressed in S. cerevisiae

30 mM imidazole containing buffer.

Theand exhibited enzymatic activity of 0.2

HDC containing fraction was eluted with 150

nmol/min/mg [14]. The crude extract gavemM imidazole in 50mM sodium phosphatea major band at around 74-kDa in Western

buffer, pH 6.8, containing 0.3 N NaCl, 1%

blot analysis; however, we did not perform

PEG#300, and 10µM PLP.

any extensive purification on the 74-kDa

The eluted

HDC inS. cerevisiae

andprotein was concentrated by an Amicon

HDC.

In this study, we applied the crude

ultrafiltration with YM 30 membrane.

extract on a DEAE HPLC column to purify
onthe 74-kDa HDC, after confirming the

SDS-PAGE gel was electrically blotted to

74-kDa band on the electrophoretic analysis

nitrocellulose(Fig. 2).

Westernblot

analysis–membrane

Proteins

ona

BioRadFor each fractions containing thesemi-dry blotter for 1 hr with the use of

enzymatic activity in the DEAE column

blotting

purificationbufferconstituted

with

20%

methanol, 0.3% Tris and 1.44% glycine.

specific

After the blotting, the membrane was soaked

nmol/min/mg.

wereactivitycollected,

where

the

was

around

0.7

at

The SDS-PAGE andin skim milk solution for 1 hr, washed with
TTBS buffer, constituted with 20 mM Tris,
154 mM NaCl, 0.05% Tween-20, pH 7.4, andincubated with rabbit polyclonal antibody
against HDC (Progen, CA, U.S.A.).

74-kDaThe

membrane was washed with TTBS and thenincubated with secondary antibody, donkey
anti-rabbit IgG antibody with HRP (GEHealthcare,CA,

U.S.A.).

The

immuno-positive bands were visualized with
ECL Western blotting detection reagent (GEHealthcare, CA, U.S.A.) according to the
manufacturer’s

instruction.

After

the

Fig. 2. Western blot of crude extract ofS. cerevisiae transfected with pWIH31.
Rabbit polyclonal antibody was usedagainst HDC.

79

Nitta, Y. et al.

Western blot analysis on each pooled

(a)

(b)

74-kDa74-kDafractions gave several bands smaller than
74-kDa (data not shown), where thosefractions exhibiting 74-kDa band did notshow any significant enzymatic activity.
The crude extract was also applied on gel
filtration chromatography and the fractions
containing the 74-kDa HDC were found to be
enzymatically inactive (data not shown).

1

Although the sequential purification with
DEAE and gel filtration chromatography
gave a fraction containing 74-kDa HDC
protein, our further purification attempts
were unsuccessful because of the low yield of
the 74-kDa HDC in the fraction.

Before212Fig. 3. (a) CBB-stained SDS-PAGE gel and(b) Western blot of reacted mixture ofwheat germ cell-free expression system.
Lane 1; pEU-74HDC, lane 2; pEU-E01
empty. Antibody was rabbit polyclonal
antibody against HDC.

attempting the further purification, we needmore proteins to treat with. For this reason,

cerevisiae system.

Although the wheat

development of expression system with

germ cell-free expression system is supposed

higher protein expression levels is needed.

to be proteolysis-free, several bands appeared

Also desired would be the expression system

on Western blot analysis (Fig. 3), and theythat is capable to prevent the unnecessary

are apparently degraded HDC proteins. The

proteolytic degradation in the host cells.

supplier noted that the cell-free protein

recombinant

synthesis system is not completely devoid offull-length HDC by using wheat germ

wheat endosperm which could be the sourcecell-free expression system - Proteolysis-freeof proteases.

wheat germ cell-free expression system waspossibility that human 74-kDa HDC isemployed for the expression of 74-kDa HDC.

enzymatically

SDS-PAGE and Western blot analysis of therecombinant mouse 75-kDa HDC [13];reaction products indicated that the wheat

however,germ cell-free expression system gave higher

conclusive.

Expressionofprotein expression level than the S. cerevisiae

Our results implicate ainactivetheresults

Expression

as
were

and

was
not

the
totally

purificationofAliquots of the reaction

recombinant C-terminal truncated HDC –products were measured for HDC activity

HDC belongs to the same family withand protein concentration, where the specificglutamatedecarboxylase(GAD)

activity of 0.3 nmol/min/mg was obtained,

L-aromatic

amino

decarboxylase

the value being nearly the same as that of S.

(AroDC).

Their amino acid sequencesystem (Fig. 3).

acid

and

Characterization of human histidine decarboxylase

80homology

indicatesthat

PLP-dependent enzymes.

they

are

(kDa)

When primary97

structure alignment was examined among
66

these three decarboxylases, HDC showed an
extended C-terminal of about 150 aminoacids.

45

We have been puzzling over why

HDC has this extended C-terminal region of
HDC.

31

While working on rat HDC,

Ichikawa’s group raised a possibility that
74-kDa HDC was a precursor form andremoval of the C-terminal 150 amino acids
could activate the enzyme.

Hence, we have

1

2

Fig. 4. CBB-stained SDS-PAGE gel ofprotein purified from E. coli transfectedwith pET-54HDC-His6 (lane 2). Lane
1; marker.

re-designed our expression system so that
C-terminal

truncated

HDC

expressed in E. coli system.

would

be

An XhoI sitenear 54-kDa.

Overall yield was about 5 mg

was introduced to the HDC cDNA, where

HDC per 10 L culture (about 50 g wet cells).

C-terminal 20-kDa truncated protein was

In order to evaluate the C-terminal andproducedwasN-terminal parts of the eluted protein,corresponding to the residues from 477 toWestern blot and protein sequence analyses

622 (Fig. 1). The EcoRI-XhoI fragment waswere performed.

insertedmakeanalysis, the band was visible with polyclonal

After theantibody against HDC but it was not visible

and

into

the

deletionpET

vectorpET-54HDC-His6 plasmid vector.

size

to

a

In the Western blot

transformation of E. coli host cells, thewithmonoclonal

antibody

againsttransformant cells were inoculated without

polyHis-tag that supposed to be locating at

IPTG since no induction substances were

the C-terminal end (data not shown). The

necessary to induce HDC synthesis.

Thesample was blotted on PVDF membranegrown cells were harvested, and lysed to give

which was then used for protein sequencea soluble fraction, in which the specificanalysis. The analysis gave a sequence ofactivity of 2.9 nmol/min/mg was found.

MEPEEY, which is the exact match with the

The lysate was loaded on a metal affinity

N-terminal six amino acids as expected forchromatography that specifically binds tohuman HDC based upon cDNA sequence.

His-tag protein.

Homogeneous protein

The E. coli system expressed a soluble form

fraction having an apparent size of 50-kDa

of HDC with its size of 50-kDa andand specific activity of 1,200 nmol/min/mg

enzymatic activity.

was obtained (Fig. 4). SDS-PAGE analysis

has its C-terminal end truncated.

indicated that there is a single band at the size

a question remained why the monoclonal

The expressed protein
However,

81

Nitta, Y. et al.

antibody failed to detect polyHis-tag andderived from human basophilicneeds to be clarified in the future research.

leukemia cell line, KU-812-F.

In conclusion, the full length
recombinant human HDCs expressed by S.

Nucleic Acids Res., 1990. 18:5891.
[2]

Joseph, D.R., P.M. Sullivan, Y.M.

cerevisiae and wheat germ cell-free systems

Wang, C. Kozak, D.A.

were enzymatically inactive.

The residual

Fenstermacher, M.E. Behrendsen,

enzyme activity exhibited in the S. cerevisiae

and C.A. Zahnow, Characterization

and wheat germ cell-free systems probably

and expression of the complementaryarose from the degraded HDCs.

DNA encoding rat histidine

The role ofC-terminal 150 amino acids in 74-kDa HDC

decarboxylase. Proc. Natl. Acad. Sci.

is still unclear; however, HDC mutant protein

U.S.A., 1990. 87:733-737.

that lacks the C-terminal 150 amino acids
clearly gave high enzyme activity.

[3]

The

Yamamoto, J., K. Yatsunami, E.
Ohmori, Y. Sugimoto, T. Fukui, T.

present results support an assumption that

Katayama, and A. Ichikawa,

post

cDNA-derived amino acid sequencetranslational

processing

could

be

involved for the activation of human HDC.

of L-histidine decarboxylase from

However, since a possibility of misfolding ofmouse mastocytoma P-815 cells.

the protein cannot be excluded, a further

FEBS Lett., 1990. 276:214-218.

study is warranted to clarify if the full length[4]

Taguchi, Y., T. Watanabe, H. Kubota,

recombinant human HDC has the enzymatic

H. Hayashi, and H. Wada,

activity.

Purification of histidine
decarboxylase from the liver of fetal
rats and its immunochemical and

ACKNOWLEDGEMENTS
Authors

indebted

toimmunohistochemical

Kieikaicharacterization. J. Biol. Chem., 1984.

Research Foundation for the kind supports.
Authors also thank to Yumiko M. Ueno forher critical reading of the manuscript.

259:5214-5221.
[5]

Ohmori, E., T. Fukui, N. Imanishi, K.
Yatsunami, and A. Ichikawa,
Purification and characterization of
L-histidine decarboxylase from

REFERENCES[1]

mouse mastocytoma P-815 cells. J.

Yamauchi, K., R. Sato, Y. Tanno, Y.
Ohkawara, K. Maeyama, T.
Watanabe, K. Satoh, M. Yoshizawa,S. Shibahara, and T. Takishima,
Nucleotide sequence of the cDNA
encoding L-histidine decarboxylase

Biochem., 1990. 107:834-839.
[6]

Furuta, K., K. Nakayama, Y.
Sugimoto, A. Ichikawa, and S.
Tanaka, Activation of histidine
decarboxylase through

82

[7]

Characterization of human histidine decarboxylase

post-translational cleavage by

and L. Persson, Histidine

caspase-9 in a mouse mastocytoma

decarboxylase in rat stomach ECL

P-815. J. Biol. Chem., 2007.

cells: relationship between enzyme

282:13438-13446.

activity and different molecular

Fleming, J.V., I. Fajardo, M.R.

forms. Regul. Pept., 1999. 81:41-48.

Langlois, F. Sanchez-Jimenez, andKamada, K. Hori, and T. Higuchi,

L-histidine decarboxylase

Comparative studies of humanspecifically inhibits enzymic activity

reconbinant 74- and 54-kDa

and disrupts pyridoxal

L-histidine decarboxylases. J. Biol.

phosphate-dependent interactions

Chem., 1995. 270:30813-30817.
Yamamoto, J., T. Fukui, K. Suzuki, S.
Tanaka, K. Yatsunami, and A.

3):769-778.

Ichikawa, Expression andFleming, J.V., F. Sanchez-Jimenez,characterization of recombinant

A.A. Moya-Garcia, M.R. Langlois,

mouse mastocytoma histidine

and T.C. Wang, Mapping of

decarboxylase. Biochim. Biophys.

catalytically important residues in theActa, 1993. 1216:431-440.
[14]

Ueno, H., T. Kanai, H. Atomi, M.

using bioinformatic and site-directed

Ueda, A. Tanaka, H. Ohtsu, K.

mutagenesis approaches. Biochem. J.,

Yamauchi, and T. Watanabe,

2004. 379(Pt 2):253-261.

Expression of human histidine

Fleming, J.V. and T.C. Wang, The

decarboxylase in Saccharomyces

production of 53-55-kDa isoforms iscerevisiae. J. Biochem. Mol. Biol. &not required for rat L- histidine

Biophys., 1998. 2:141-146.

decarboxylase activity. J. Biol.
[10]

[13]

Biochem. J., 2004. 381(Ptrat L-histidine decarboxylase enzyme[9]

Yatsunami, K., M. Tsuchikawa, M.

T.C. Wang, The C-terminus of rat

with L-histidine substrate analogues.

[8]

[12]

[15]

Nitta, Y., H. Kikuzaki, and H. Ueno,

Chem., 2003. 278:686-694.

Food components inhibiting

Olmo, M.T., J.L. Urdiales, A.E. Pegg,

recombinant human histidine

M.A. Medina, and F.

decarboxylase activity. J. Agric.

Sanchez-Jimenez, In vitro study ofFood Chem., 2007. 55:299-304.

proteolytic degradation of rat
histidine decarboxylase. Eur. J.
Biochem., 2000. 267:1527-1531.
[11]

Dartsch, C., D. Chen, R. Hakanson,

Communicated by Ohnishi Masatake