Purification and characterization of human urine-derived digitalis-like factor

Vol.

154,

August

No. 15,

3, 1988

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

1988

Pages

847-853

PURIFICATION AND CHARACTERIZATION OF HUMAN URINE-DERIVED DIGITALIS-LIKE FACTOR

A. Goto'), K. Yamada'), M. Ishii'), M. Yoshioka') T. Ishiguro3), C. Eguchi3), and T. Sugimotol) 1)

Second

Department of Internal Medicine, University of Tokyo, Tokyo, 2)

Faculty Japan

of Medicine,

Faculty of Pharmaceutical Sciences, Setsunan University, Hirakata, Japan 3) Central Research Laboratories, Ajinomoto Co., Inc., Kawasaki, Japan

Received

June

24,

1988

a digitalis-like factor to apparent homoSUMMARY: We were able to purify geneity from human urine based on the inhibitory effect on [3H]-ouabain This ouabain displacing compound closely binding to intact human erythrocytes. resembles ouabain in its polarity, molecular weight, non-peptidic nature and mode of action except for its UV absorbance spectrum. This compound sharing many biological activities of ouabain may be the endogenous ligand for the c 1988 Na+,K+-ATPa,se and serve as a specific regulator of the sodium pump. Rcadrmlc Press, klc.

The plasma protein Since

the

sides,

it

has been that

factors

in plasma, in

urine,kidney

recently

dog plasma erythrocytes [3H]-ouabain cultured

based

on the

binding, vascular

capacity

This

cells.

cardiac

ligands (l-4).

glyco-

for

Indeed,

of such

of different

glycosides

and the

pathogenesis

these experi-

endogenous

mammalian

to cardiac

excretion

muscle

urine.

Furthermore,

species

(5-7).

may be impliof human

of regulating to

sodium

apparent

the chemical

in comparison

847

with

human urine

binding

to intact

and competitive

In the present

compound

from

[3H]-ouabain

a reversible

cells.

we investigated compound

factor

to inhibit

was capable smooth

digitalis-like

compound,

the cmuabain displacing of this

endogenous

the existence

counterparts

a polar

purify activities

for

activity

membrane

in animal

(8).

isolated

(9,lO).

are

for

of sodium

hypertension

We have

there

and brain

endogenous

an integral

receptors

Na+,K+-ATPase

accumulated

the regulation

essential

high affinity that

regulate

is

Na+ and K' gradients

contains

has been

Such physiological cated

maintaining

postulated

might

evidence

(E.C.3.6.1.3)

Na +,K'-ATPase for

Naf,K+-ATPase

receptors mental

membrane

responsible

inhibitor

pump activity study,

we were

homogeneity properties

and human of

in able

to

from human

and biological

ouabain. 0006-291X/88 $1.50 Copyright 0 1988 by Academic Press. Inc. All rights of reproduction in any form reserved.

Vol.

154,

No.

3, 1988

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MATERIAL AND METHODS: Purification of ouabain displacing comound (ODC). Approximately 1000 liters of human urine collected from normal male volunteers were processed as described previously with minor modifications (9,lO). First, human urine was treated with Amberlite XADZ preequilibrated with distilled water. ODC was eluted with methanol and evaporated to dryness. The residue was suspended in distilled water and applied to the reverse phase high performance liquid chromatography (HPLC) on a preparative Cl8 column (70~; 2.0x25cm). ODC was eluted with 20% acetonitrile and lyophilized. The active material was fractionated successively by the reverse phase HPLC run at a flow rate of 10 ml/min on a D-ODS-5 column (51.1; 2.0x25cm) eluted, first, with a gradient of acetonitrile (O-30%) over 60 min and, second, with 8% acetonitrile. ODC The active fraction was further emerged at 43 min and 150 min, respectively. analysed by the gel filtration HPLC on a diol-60 column (0.08x5Ocm) in O.lM phosphate buffer, PH 7.4/acetonitrile (4/l) at 1 ml/min, and successively by the reverse phase HPLC on an A-402 phenyl column (0.46x15cm) with a gradient of acetonitrile (lo-20%) over 40 min at 1 ml/min. ODC eluted at 17 min and 36 min, respectively. The purification was accomplished by the rechromatography on an A-402 phenyl column under the identical conditions just described. All of the HPLC columns were obtained from YMC, Kyoto, Japan. Through the separation steps, an inhibitory effect on [3H]-ouabain binding to intact human erythrocytes was used as the principal assay to follow the digitalis-like activity. One unit of ODC was defined as that amount required to inhibit [3H]ouabain binding to human erythrocytes by 50%. Chemical properties of ODC. Polarity of ODC was estimated from the elution position on a D-ODS-5 reverse phase HPLC column in comparison with ouabain. Solubility of ODC in water, methanol and chloroform was examined. Molecular weight of ODC was estimated by gel filtration on a diol-60 column in comparison with ouabain (MW=584) and taurine (125). Molecular mass of ODC was analysed by fast atom bombardment mass spectrometry using Nihon Densi DX-300 mass spectrometer. The UV absorbance spectrum of ODC was compared to that of ouabain using Hitachi U-3200 spectrophotometer. Protease sensitivity of ODC was determined as follows. ODC was incubated for 2h at 37'C with one of the following peptidases : trypsin, carboxypeptidase A and leucineaminopeptidase (2x10v4M). The reaction was stopped by boiling for 5 min. After centrifugation, the supernatants were assayed. Biological and immunological properties of ODC. The purified ODC was tested for its effects on isolated dog kidney Na+,K+-ATPase (Sigma, St. Louis, U.S.A), [3H]-ouabain binding to the isolated rat brain synaptosome and ouabainsensitive s6Rb uptake into human erythrocytes according to the method detailed elsewhere (9). Further, ODC was tested for its digoxin-like immunoreactivity using an antidigoxin antiserum (Miles, Elkhart, U.S.A) (11). Selectivity of action. The effect of ODC on Na+,@-ATPase, Ca'+-ATPase and Mg2+-ATPase activities in erythrocyte membranes were determined. Red blood cell membranes were prepared according to the method of Jarret and the ATPase activities were measured by the rate of hydrolysis of [v-~~P] ATP (12). Human lymphocytes contain specific receptor for atria1 natriuretic peptide (ANP) as well as high affinity binding site for ouabain (sodium observations). LLC-PK1 cells, pump) (9, unpublished the established cell line derived from porcine kidney tubular cells, retain receptor for ANP in addition to sodium pump (13,14). The effects of ODC on the binding of [1251]-human ANP and [3H]-ouabain to these cells were compared according to the method described previously (9,15).

RESULTS:

ODC emerged

absorption diol-60 molecular Preliminary

peak gel

(Fig

filtration

weight

at a retention 1).

HPLC column

of ODC was estimated

analysis

time

The retention

by a fast

of 36 min under

times were

16,

of ouabain, 17 and 17 min,

to be less

atom bombardment 848

than

the

single

UV

ODC and taurine that

respectively. of ouabain.

mass spectrometry

revealed

on a The

Vol.

154,

Nlo.

3, 1988

,L---28 01

32

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

36 40

(“m)

time(min)

Retention

profile of ouabain displacing compound (ODC) from human --FIGURE 1: Elution urine on an A-402 phenyl column. The active fraction obtained from previous step was analysed by reverse phase HPLC using a gradient of acetonitrile (O20%) over 40 min at 1 ml/min. The optical density was monitored at 210nm.Apart of the actual chromatogram is illustrated because the baseline gradually rose due

to

the

increasing

concentration

of

acetonitrile.

Chart

speed

was

5mm/min.

--FIGURE 2:

Comparison of the LJV absorbance spectra of ouabain (A; final and OD'C (B; approximately 0.7 units). Both substances were diluted with distilled water and monitored. Distilled water alone showed no distinct

that

it

has a molecular

from

D-ODS-5

indicating

column

These

res-ults

Ouabain

tion

ODC showed

ODC for

these

affect

were

of 0.5-50

eluted

respectively,

were

cell

Ca

In contrast,

ODC had a sharp peak

UV

at longer

wave-

to enzyme

diges-

(Fig

parallel

Fig.4

Na+,K+-ATPase

human erythrocytes

depicts

3).

to those

of ouabain

for

the

2+-ATPase

and Mg2+-ATPase 849

of ODC and immuno-

concentration.

When the

inhibitory

5).

Na+,K+-ATPase.

Ten units activities

of

at the

and ouabain-sensi-

5mM to 2.5mM or 0.5mM, (Fig

curves

a week digoxin-like

on potassium

from

activity, [3H]-ouabain

the crossreactivities

ODC exhibited

dependent

and

The dose-response

of ODC on Na +,K+-ATPase

potentiated

of ODC was specific red blood

intact

almost

was reduced markedly

ODC in dog plasma.

UV absorption

synaptosome

effects

concentration

in chloroform.

from

of dog kidney

into

antiserum. strongly

insoluble

A and leucineaminopeptidase.

units.

antidigoxin

of ODC were

The action not

brain

but

of ODC was refractory

inhibition

The inhibitory

potassium effects

rat

activities

*6Rb uptake

consistently

in water,

obtained

220~11.

The action

86Rb uptake

to isolated

tive

2).

a dose-dependent

concentrations

those

peak at

carboxypeptidase

binding

with

with

at 187nm and no apparent

ouabain-sensitive

reactivity.

and methanol,

in accordance

2OOnm (Fig

by trypsin,

ouabain

in water

a W absorption

peak

were

peak.

polarity.

are

than

ODC and ouabain

18% and 19% acetonitrile

soluble

showed

absorption length

with

similar

ODC was highly

mass of 343.

lo-'M)

of ODC did at all

(Fig

Vol.

154,

No.

BIOCHEMICAL

3, 1988

AND

BIOPHYSICAL

Ouabain

IO 9

-log

8

7

RESEARCH

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ODC

6

5

(Ouabain(M))

FIGURE 3: Effects of ODC on canine kidney Na+,K+-ATPase activity (A), ouabain-sensitive 86Rb uptake into human erythrocytes (B) and [3H]-ouabain binding to rat brain synaptosome (C). Dose response curves of ODC were compared with those of ouabain. Data are mean of triplicates from representative experiments and expressed as percent of the maximal activities or binding.

OOV

-log

100

ODC

[Ouabaln(M)]

(units)

FIGURE 4: Crossreactions of ouabain (A) and ODC (B) with antidigoxin antiserum. Ouabain (low3 -10e6M) and ODC (0.5-50 units) were tested for their digoxin-like immunoreactivities and data are mean of triplicates from representative experiments.

850

Vol.

154,

No.

3, 1988

BIOCHEMICAL

K concentration

AND

BIOPHYSICAL

(mM)

RESEARCH

K concentration

(mM)

Effect of potassium on the inhibitory activities 5: --FIGURE for canine kidney Na+,K+-ATPa se (A) and ouabain-sensitive human erythrocytes (B). Other assay conditions were exactly the potassium concentration (n=4).

6A).

ten

Moreover,

binding

units

of ODC possessed

to human lymphocytes

and LLC-PKl

The present

describes

DISCUSSIOJ:

displacing

compound

paper

(ODC) from

glycosides

anticipated

include

j:nhibition

of Na+,K+-ATPase,

tition

at: the

motion

of

receptor

site,

natriuresis

digitalis-like

for

activity

in

inhibition

that

it

showed

on ['251]-hANP

purification

of ouabain

6B).

important

endogenous of the

of cardiac

Human urine-derived

for

effect

Some of the

a physiological

enhancement

(l-4).

(Fig

the complete

human urine.

cardiac

of ODC (1 unit) 86Rb uptake into the same except

no inhibitory cells

COMMUNICATIONS

actions

sodium

pump,

contractility

compe-

and pro-

ODC actually

a dose-dependent

possessed inhibition

A

the of

SE

T

Na’, K’ATPase Red

Ca’+ATPase blood

cell

+-waba~n

Mg’+ATPase

Human

ghosts

tz5t.hANP

lymphocytes

‘Ii-ouabaln

LLC-PK,

“‘1-hANP

cells

A: Effects of ODC (10 units) on Na+,K+-ATPase, Ca'+-ATPase and FIGURE 6: ___These activities are expressed Mg2+-ATPase activities in erythrocyte membranes. as percent of the basal activities in the absence of ODC (n=4). B: Effects of ODC (10 units) on the binding of [3H]-ouabain and [lz51]-hANP to human lymphoData are expressed as percent of the maximal binding cytes and LLC-PK1 cells. in the absence of ODC (n=4).

851

of

counterpart

Vol.

154,

No.

canine

BIOCHEMICAL

3, 1988

kidney

Na+,K+-ATPase

human erythrocytes ODC shared similar

and

activity,

to human lymphocytes

and LLC-PK1

the

specific

Overall,

of ODC is the

purified

ODC is

with

on Ca'+-ATPase

cells.

effect

These

brain

from

indicating

and Mg'+-ATPase on [lz51]-hANP

and

indistinguishable

into

synaptosome.

ouabain

observations

Na +,K+-ATPase

COMMUNICATIONS

*6Rb uptake rat

by potassium

no inhibitory

for

RESEARCH

to isolated

ODC had no effect ODC showed

Moreover,

action

binding

of inhibition

mode of action.

BIOPHYSICAL

ouabain-sensitive

[3H]-ouabain

the antagonism

activities.

AND

binding

demonstrate

[3H]-ouabain ouabain

that

binding.

in

its

functional

in its

polarity,

characteristics. As to the

chemical

non-peptidic

properties

nature,

preliminarily

of ODC, ODC resembles

and molecular

determined

weight.

by FAB-mass

distinct

from

ouabain

in

its

possible

that

the

concentration

report

documents

ouabain

The molecular

spectrometry

LTV absorption

spectrum,

of ODC was too

mass of ODC was

to be 343 Da. although

it

low to permit

its

ODC was is

still

definite

detection. Some recent digitalis-like which

substance

shows

already

that

(16).

ODC is

cornfirmed

the

actually

the presence

importance

At present

we do not

produced

agent.

from human urine Its

structural

inotropic)

actions

ACKNOWLEDGEMENTS: assistance. Research

This from

may be the

features are

We thank study

existence strongly

direct

body.

of

evidence

However, plasma

we have (10).

of similar compound in suggest that ODC we endogenous

in vivo

sources

digitalis-like

(natriuretic

and

positive

investigation.

Ms. Eri

Baba and Ms. Tamako Kubota

was supported

the Ministry

the

ODC in mammalian

long-sought

and its

now under

exogenous

have

in mammalian

of identical

Moreover, Tamura et al have reported the These findings bovine adrenal gland (17). isolated

of excluding

in part

of Education,

by a Grant-in-Aid

Science

and Culture,

for

technical

for

Scientific

Japan

(No.

62570382).

REFERENCES: 1. 2. 3. 4. 5. 6. 7. 8. 9.

Haddy, F.J. (1987) N. Eng. J. Med. 316, 621-623. L. (1987) Clin. Sci. 72, 647-655. Poston, Graves, S.W., and Williams, G.H. (1987) Ann. Rev. Med. 38, 433-444. Haber, H., and Haupert, G.T.Jr. (1987) Hypertension. 9, 315-324. Cloix, J-F. (1987) Hypertension, 10 (Suppl I), 1-67-I-70. Hamlyn, J.M., Schenden,J.A., Zyren, J., and Baczynskyj, L. (1987) Hypertension. 10 (Suppl I), 1-71-I-77. Kelly, R.A. (1987) Hypertension, 10 (Suppl I), 1-87-I-92. de Wardener, H.E., and Clarkson, E.M. (1985) Physiol. Rev. 65, 658-759. Goto, A., Yamada, K., Ishii, M., Yoshioka, M., Ishiguro, I., and Sugimoto, T. (1988) Hypertension. in press. 852

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13. 14. 15. 16. 17.

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Goto, A., Yamada, K., Ishii, M., Yoshioka, M., Ishiguro, I., Eguchi, C., and :Sugimoto, T. (1988) Biochem. Biophys. Res. Comm. 152, 322-327. Gruber, K.A., Whitaker, J.M., and Buckalew, V.M. (1980) Nature. 287, 743745. Jarrett, H.W., and Penniston, J.T. (1978) J. Biol. Chem. 253, 4676-4682. Kennedy, B.G., and Lever, J.E. (1984) J. Cell. Physiol. 121, 51-63. Inui, K., Saito, H., Matsukawa, Y., Nakao, K., Morii, N., Imura, H., Shimokura, M., Kiso, Y., and Hori, R. (1985) Biochem. Biophys. Res. Comm. 132, 253-260. Goto, A., Yamada, K., Ishii, M., Yoshioka, M., Ishiguro, I., Eguchi, C., and !jugimoto, T. (1988) J. Hypertension. submitted for publication. Kelly, R.A. (1986) Am. J. Physiol. 251, H205-H209. Tamura, M., Lam, T-T., and Inagami, T. (1987) Biochem. Biophys. Res. Comm. 149, 468-474.

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