Natural occurrence of guanidinooxypropylamine in Wistaria floribunda and the sword bean Canavalia gladiata

Vol.

129,

No.

May

31,

1985

1, 1985

BIOCHEMICAL

BIOPHYSICAL

AND

COMMUNICATIONS

RESEARCH

Pages

NATURAL OCCURRENCE OF GUANIDINOOXYPROPYLAMINE IN Wistaria AND THE SWORD BEAN CanavaZia gladiata Koei

Hamana and Shigeru

46-51

fZoribunda

Matsuzaki*

College of Medical Care and Technology and *Department of Physiology, Institute of Endocrinology, Gunma University, Maebashi 371, Japan Received

April

16,

1985

SUMMARY: We found a new guanidinooxyamine in Wistaria ftoribunda seeds and seedlings of the sword bean, Canavalia gladiata. This amine was not only ninhydrin-positive but also gave a positive alkaline nitroprussideferricyanide reaction. It was characterized as y-guanidinooxypropylamine [H2N(NH=)CNHO(CH2)$H21 by comparison with the authentic compound on column and thin-layer chromatograms visualized with specific reagents and by reductive cleavage. Evidence for the occurrence of another unusual guanidino amine, homoagmatine in W. floribunda seeds was also presented. 0 1985 Academic Press, Inc.

Polyamines molecular

have

biosynthesis

have

been found

have

been detected

cadaverine

in higher

plants

have

also

course

have

observed

gave

a positive

corresponded

of

several

plants,

in Lathyrus

unknown

on our column

nitroprusside-ferricyanide to either

leguminous

plants

contain

canavanine

(6-8),

we speculated

it

amino

This

products ation

of these product

several

agmatine

acids.

of L-canavanine.

guanidino would

The present

0006-291X/85 $1.50 Copyr&ht All rights

0 1985 by Academic Press, of reproduction in any form

Inc. reserved.

46

them.

Several

bean,

seedlings

for

(5).

in leguminous

be one of

plants,

chromatograms. though

acids the

compound communication

example,

CanavaZia

it

or homoagmatine. amino

unknown

and spermine

plants,

reaction,

L-canavanine,

of

in sword

sativus

polyamines

1,3-diaminopropane,

number

on polyamines

peaks

of macro-

spermidine

in particular

canavalmine

control

common and "unusual"

while

been found

investigations

in the

Putrescine,

in a limited

(3),

(4) and homoagmatine

Both

(2).

are found

in sandalwood

In the

(1).

in many different

polyamines

homospermidine

to be implicated

and growth

and agmatine

"unusual"

gZadiata

been shown

One of did

we them

not

Since

including

L-

decarboxylation was indeed deals

a decarboxylwith

the

Vol.

129,

No.

1, 1985

BIOCHEMICAL

and characterization

isolation

in Wistaria

AND

BIOPHYSICAL

of a hitherto

RESEARCH

unrecognized

COMMUNICATIONS

guanidinooxyamine

and C. gladiata.

floribunda

MATERIALS AND METHODS Chemicals_L-ArginineeHCl, L-homoarginine.HCl, L-canavanine.H2SOh, putrescine*2HCl, cadaverines2HC1, spermidines3HC1, spermines4HC1, arcaine.H2SOk hirudonine*H2SOh, agmatineeH2S04, octopine, o-methylisourea sulfate and palladium-barium sulfate were purchased from Sigma (St. Louis, MO). 3-Hydroxypropylamine was obtained from Wako Pure Chemicals (Osaka). Ten aliphatic pentaamines which contain aminopropyl and/or aminobutyl moieties were kindly supplied by Dr. K. Samejima of Josai University. Homospermidine and aminobutylhomospermidine (homospermine) were synthesized according to Okada et al The synthesis of homoagmatine (monoamidinocadaverine) and audouine (9). (diamidinocadaverine) was achieved by guanidation of cadaverine with ocadaverinee2HCl was treated with o-methylisoureamethylisourea (5). Briefly, HCl in 2M NaOH at pH 10.5 for 5 days at 25'C. Homoagmatine and audouine were separated from unreacted cadaverine with a column (9 x 120 mm) of ion-exchange resin (Hitachi Custom 2612). The conversion of sulfate forms of L-canavanine, agmatine and o-methylisourea to hydrochloride forms was achieved by passing through a column of Dowex l-X8 before use. Guanidinooxypropylamine was enzymatically synthesized from L-canavanine. L-CanavanineeHCl was incubated with acetone-dried Escherichia coli 7020 (IF0 3544) in 0.2M acetate buffer (pH 5.25) at 3O'C for 1 hr (10). After the termination of the reaction with cold 0.5N HClOt+, the decarboxylation product of L-canavanine was purified by column chromatography as described above.

Canavalia gladiata seeds were fZoribunda seeds were collected in water imbibition for 12 hr, the seeds in the dark on moistened filter paper

Materials

Wistaria After grow

purchased from a local seed dealer. botanical gardens in Maebashi. were germinated and allowed to discs.

Polyaminc analysis Seeds and seedlings were homogenized in 3 volumes of 0.5N HC~OL,. After centrifugation of the homogenates, the supernatants were passed through a column (2.5 x 2 cm) of Dowex-50 W to concentrate polyamines Polyamines were analyzed by highand to eliminate amino acids (11). performance ion-exchange column chromatography as described previously (12) The identity of polyamines was also established with slight modifications. by thin-layer chromatography on cellulose and silica gel (13). Amines on chromatograms were visualized by spraying either ninhydrin, Sakaguchi or alkaline nitroprusside-ferricyanide (pentacyanoferrate) reagent. Large

scale isolation of guanidinooxypropylamine Approximately 1 kg of seeds and CanavaZia seedings were homogenized in 10 1 of 0.5N Hc101+. Polyamines and unknown compounds in HC104 extracts were concentrated by the and then separated by a column of Hitachi Custom use of a Dowex-50 W column, 2612 (13).

Wistaria

RESULTS When analyzed chromatography, were

detected

appeared

the use of our highly

with putrescine,

in the

in seedlings

seed of

spermidine,

Wisttia

of W. floribunda

sensitive

homospermidine, f~orib~nda

(1B)

(Fig.

high-performance spermine 1A).

liquid

and agmatine

Cadaverine

and Canava~ia gZadiata

(li).

Vol.

129,

No.

BIOCHEMICAL

1, 1985

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

1 2 ‘7 /3 1

50

5 LL- d 1

’

:‘I

IF-

60

o”o-

ELUTION

Fig.

Besides

polyamines

This

identical

to

peak

such

The retention including

tested

such

eluted

both

authentic systems

prior

not

seeds

norepinephrine, tryptamine,

and Canavalia

not

were (Fig.

any of

seedlings whenever

shown). 48

the

10 penta-

compounds

(diamidinoputrescine),

also lD),

was not

Other

behaved different

[N2-(l-

octopamine,

histamine,

excluded. though

audouine

octopine

cystamine,

and

and homospermine.

normetanephrine,

tyramine,

seedlings

(data

of

(diamidinospermidine),

guanidinooxypropylamine used

to that

spermine

It

thermospermine

arcaine

hirudonine

in C. gladiata

between

and homocaldopentamine.

and N-carbamylputrescine

were

0

to canavalmine.

correspond

as aminopropylcadaverine,

serotonin,

Wistaria

60

(min)

as norspermine,

caldopentamine

carboxyethyl)-L-arginine],

appeared

7x2

peak Xl was found

a little

of Xl did

(diamidinocadaverine),

histamine,

TIME

a prominent

tetraamines

time

amines

dopamine,

40

1. Elution profiles of polyamines found in Wistaria f~or'hndu and Camvalia ghfiata. Polyamines were separated by highperformance ion-exchange chromatography from the seeds (A and C) and seedlings (B and D) of W. fZoribu?da and C. ghdiata, Elution patterns of the polyamines were followed respectively. The numbers above the peaks correspond to: by o-pthalaldehyde. (1) putrescine; (2) cadaverine; (3) spermidine; (4) homospermidine; (5) spermine; (6) canavalmine; (7) agmatine and (8) homoagmatine. Xl and X2, unknown compounds. Dotted lines indicate the curves at 25 times the standard sensitivity.

these

agmatine.

X1 6

2’

8

J I;+4 40

l-methyl-

The same peak

not

in the

seeds.

chromatographically columns

and solvent

as Xl Xl from like

Vol.

129,

No.

1, 1985

BIOCHEMICAL

When pooled were

analyzed

is

A-I

by thin-layer

propylamine which

fractions

(Fig.

3).

specific

homoagmatine,

which

produced.

All

of which

COMMUNICATIONS

contained

Xl

just

like

(Fig.

compounds.

Unlike

to Natelson

show that

(14),

reaction,

agmatine

When reduced

and

in the presence

Xl disappeared

is indeed

of

and new

and 3-hydroxypropylamine Xl

2)

guanidinooxy-

nitroprusside-ferricyanide

to guanidine

findings

RESEARCH

Xl behaved

a positive

according

corresponded these

both

Sakaguchi-positive.

sulfate

compounds

gave

guanidinooxy

Xl was not

palladium-barium

and B-I

BIOPHYSICAL

chromatography, It

for

AND

were

identical

to guanidinooxy-

propylamine. A-II

was identified

techniques

(Fig.

and seed.Lings amount A minor

of this

3).

of

Agmatine

of W. ftoribunda guanidino

peak which

seedlings

as agmatine is

amine

but

the most

(0.6-0.8

not

and thin-layer

abundant

pmoles/g

was detected

corresponded

Wistaria,

by column

polyamine weight),

to homoagmatine

other F

F

VOLUME

(ml)

03 O

only

a small (Fig.

in both

5

minor

peaks

3 Gua 0

0

B

O:H-Agm

0

H-A~Q ACQ Call

andard

/ B-I

A-l A-II

0

andard

6-I

A-I A-II

Fig.

separation of polyamines and Xl from W. fZoribunda 2. Large-scale (A) and C. gZa&iata (B). Polyamines were detected by ninhydrinreagent at 570 nm. Pooled fractions A-I, A-II and B-I were used for further analysis.

Fig.

chromatographic separation of A-I, A-II and B-I 3. Thin-layer on cellulose (A) and silica gel G (B). Arginine (Arg), homoarginine (H-Arg), canavanine (Can), agmatine (Agm), homoagmatine (H-Amg) and guanidinooxypropylamine (Gus) were run Spots on chromatograms were visualized by stimulataneously. spraying alkaline nitroprusside-ferricyanide (open circle) or Sakaguchi (dotted circle) reagent. Solvent systems used were isopropanol-ammonia (7:3, V/V) (A) and chloroform-methanol-ammonia (2:2:1, V.V) (B). 0, origin; F, solvent front. 49

1D).

seeds

A

:Q,. H-Agm .Q.Agm .e. 0,. - GM 0

ELUTION

seeds

seedlings

was observed Several

in both

while

in CanavaZia

in CanavaZia.

chromatographic

and

were

Vol.

129,

No.

found

1, 1985

BIOCHEMICAL

in Canavalia

identified

seeds

from

formula,

the

AND

BIOPHYSICAL

and seedlings.

retention

times

RESEARCH

The unknown as pentaamine

H~N(CH~)~NH(CH~)I+NH(CH~)~NH(CH~)~NH~

peak

of

X2 was tentatively

the

among the

COMMUNICATIONS

constitutional 10 pentaamines

examined,

DISCUSSION L-Canavanine acid

first

(a-amino-y-guanidinooxybutyric

found

in the

to occur

in a number

especially

abundant

(8).

It

these

plants.

jack

of

in the

can be converted

this

by E. coZi

contain

experiments

It

is

detoxicated

metabolite

of interest

to note

not

a decarboxylase amino

acid

amounts

known,

this

for

the

for

content

for

arginine

substrate

but

an inhibitor

(18).

Thus,

it

formed

by arginine

remains

that

is

it

no

amine

occurs

L-canavanine

in these

plants

(15).

of Leguminosae

which

as a normal is merely

an inert

active

increases induced

L-

contains

Possibly

species

in

from

this

gladiata.

is

acid

and Wistaria

normally

has shown

whether

L-canavanine

amine.

in CanavaZia during

or It

is

seedlings.

germination

like

(16).

seeds

contain

enormous it

formation

substrate

of Canavalia

or a biologically

amine

amino

by decarboxylation

other

however,

of guanidinooxypropylamine,

responsible

This

guanidinooxypropylamine

decarboxylases

W. fZoribunda

Since

contain

of L-canavanine that

8).

as by arginase

that

amino

(7) and has been shown

bacterium

and CanavaZia as well

a nonprotein

shown to be formed

this study

revealed

also

constituent.

other

though

floribuda

L-canavanine

Probably

(lo),

by decarboxylation

Our preliminary

(6, species

has been

The present

in Wistaria

is metabolized

of various

amine

7020

guanidinooxypropylamine. naturally

ensifomis

of Leguminosae

seeds

is

to guanidinooxypropylamine

In fact,

canavanine

Canavalia

bean,

species

acid)

of these

decarboxylase for

is possible amines.

decarboxylase

that

(17),

decarboxylase

whether

of agmatine

or not

and large

a common enzyme

Although

of E. co2-i

argine

unknown

amounts

L-canavanine it

does

purified

not from

is serve oat

decarboxylase

a as a

seedlings

guanidinooxypropylamine

or by a specific

is

is in leguminous

plants. Our present in Wistaria

study

seedlings.

has provided This

amine

evidence was first

50

for

the

reported

occurrence in Lathyms

of homoagmatine sativus

Vol.

129,

No.

seedlings

1, 1985

(5).

BIOCHEMICAL

We also

found

other

the hitherto

recognized

amines.

corresponded

to some of

the

these

compounds

are

under

AND

minor

RESEARCH

peaks

corresponded

Our preliminary

10 aliphatic

way in our

ACKNOWLEDGMENTS: We are indebted the kind supply of 10 different

BIOPHYSICAL

which

experiments

pentaamines.

Attempts

COMMUNICATIONS

to none

showed

that

of

they

to characterize

laboratories.

to Dr. K. Samejima pentaamines.

of Josai

University

for

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

U. (1973) Function of Naturally Occurring Polyamines. Academic Bachrach, Press, New York. Smith, T. A. (1975) Phytochemistry 14, 865-890. Kuttan, P., Radhakrishnan, A. N., Spande, T. and Witkop, B. (1971) Biochemistry 10, 361-365. Fujihara, S., Nakashima, T. and Kurogochi, Y. (1982) Biochem. Biophys. Res. Commun. 107, 403-410. Ramakrishna, S. and Adiga, P. R. (1974) Phytochemistry 12, 2691-2695. Bell, E. A. (1958) Biochem. J. 70, 617-619. Kitagawa, M. and Tomiyama, T. (1929) J. Biochem. (Tokyo) 11, 265-271. Nakatu, S., Matugaki, Z., Simada, H., Kasida, T., Motomura, T., Oshima, M. and Sakai, N. (1962) Seikagaku 34, 253-257. Okada, M., Kawashima, S. and Imahori, K. (1979) J. Biochem. (Tokyo) 85, 1235-1243. Makisumi, S. (1961) J. Biochem. (Tokyo) 49, 292-296. Inoue, H. and Mizutani, A. (1973) Anal. Biochem. 56, 408-416. Matsuzaki, S., Hamana, K., Imai, K. and Matsuura, K. (1982) Biochem. Biophys. Res. Commun. 107, 307-313. Hamana, K. and Matsuzaki, S. (1984) J. Biochem. (Tokyo), 95, 1105-1110. Natelson, S. (1985) Experientia 41, 257-259. G. A. and Cohen, W. S. (1983) Plant Physiol. Downum, K. R., Rosenthel, 73, 965-968. Altman, A., Friedman, R. and Levin, N. (1982) Plant Physiol. 69, 876-879. E. A. and Snell, E. E. (1968) 3. Biol. Chem. 243, Blethen, S. L., Boeker, 1671-1677. Smith, T. A. (1979) Phytochemistry 18, 1447-1452.

51