Vol. 66, No. 4,1975

BIOCHEMICAL

SALINOMYCIN

Mitsuaki

: A NEW MONOVALENT CATION IONOPHORE

Mitani,

Research

Tadashi

Division,

.July

21,

Yamanishi

and Yukio

Kaken Chemical

Honkomagome, Received

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Bunkyo-ku,

Miyazaki

Company,

Tokyo,

Limited

113 Japan

1975

SUMMARY: The cation discriminations of salinomycin and its derivatives have been studied by measuring complexability with cations and transport rate of them across organic phase. Salinomycin exhibited a great preference for K+ over other monovalent and divalent cations in migrating cations into organic phase in two phase systems. The antibiotic mediated the transport of Na+ and Rb+ as effectively as that of K+ across CC14 bulk phase, but not those of Cs+, Mg’+, Ca2+ Sr2+. From the above results, salinomycin is concluded to act as an alkali ion carrier. The OH-acylated salinomycins retained the activity of parent compound, but the COOH-esterified salinomycins lost the activity. INTRODUCTION It ability

has been known

that

to

cations

transport

biological

membranes

electrically

neutral

the membranes

(4,5)

Salinomycin tricyclic

(1,2,3)

In this derivatives cations

from

barriers

.

These

lipid

ionophorous

exchange-diffusion

is (6).

polyether

across

type

antibiotics

have the

of artificial antibiotics

of cation

and

mediate

transport

an

across

. a monocarboxylic

spiroketal

the molecule

monocarboxylic

ring It

systems

has both

paper,

we wish

as alkali

ion

carriers

aqueous

phase

into

polyether

antibiotic

and an unsaturated

antimicrobial

to report

unique

six-membered

ring

in

and anticoccidialactivities

the properties by measuring

and through

with

of salinomycin

their

organic

abilities

(7). and its to transport

phase.

MATERIALS AND METHODS The cation discrimination patterns of salinomycin and its derivatives were determined by measuring their abilities to complex with various cations in two phase distribution systems. The antibiotics were mixed vigorously with organic solvent, i.e. n-butanol-toluene, and aqueous buffer containing isotopically labeled metal ions. The cation contents migrated from aqueous phase into organic phase were determined by counting the radioactivity of metals in an aliquot of the latter phase. The association constants, KA, of the antibiotics for monovalent and divalent cations were calculated according to the following equations recommended by Pressman (5): for

Copyrighr All righa

monovalent

C.I I9 7-i h-v Acaden~ic Prrsc. of’repmdtrctim itI arz,v jiwm

cation,

1~. rcsenyrl.

KA =

[Complex orgl [ Ionophore1232

orgl

. [“+aqueousl

Vol. 66, No. 4,1975

for

BIOCHEMICAL

divalent

cation,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

[Complex

KA =

orgl

1Ionophore-orgl ’ * [M2+queous1 The antibiotic-mediated cation transport across a bulk phase was measured in two systems both described by Pressman (5). In the first system, a vessel with a septum sealed across the top was used. Into the vessel 12 ml of CC14 was poured, and then 3 ml of aqueous buffer containing 25mM tris-glycin, pH 9.8 and 1OmM test cation was added on the CC14 layer, so that the aqueous layer is entirely separated into two parts by the septum. The labeled cation solution was added to one part of the aqueous layer, and the time course of the cation transport to the other part was measured. The second system we employed was that which consists of three layers (bottom phase: 3 ml of 50 % sucrose soluction containing 25 mM tris-glycin, pH 9.8 and 1OmM isotopically labeled cation, middle phase: 2 ml of CClbCroH22 mixture (l:l), upper phase: 3 ml of 25mM tris-glycine, pH 9.8 and 1OmM cation). For the determination of Mg2+ concentration was determined by atomic absorption analysis. In both barrier systems, the antibiotics were added to the organic phase and the lower layer was stirred with magnetic stirring bar. Crystalline salinomycin and its derivatives used in this study were prepared in our laboratory and nigericin was generously supplied by 22Na+, s6Rb+, 13rCs+, 47Ca2’ and Dr. H. Lardy of Wisconsin University. s5Sr2+ were purchased from the Radiochemical Centre, Amersham, England and 42K+ from Japan Atomic Energy Research Institute, Tokyo, Japan. RESULTS Salinomycin cations

and its

from aqueous

toluene

.

derivatives

buffer

The association

into

formation, parent

group with

more preference The loss

carboxyl

group

still for

of said

indicates

to migrate

organic

of these

Salinomycin cations.

of salinomycin

compound.

terminal

less-polar

constants

cations were shown in Table I. monovalent cations over divalent of hydroxyl

have the abilities

solvent,

antibiotics exhibited

for

retained

the ability

Na+ and less

preference

induced

the critical

i.e.

n-butanol-

various

great preferences and propionate

Acetate

ability

metal

of complex for

Cs+ than

by esterification

role

for

of this

group

of the in complex

formation. Fig.

1 shows

determined

by the

from the

respective

42K+ or

for

ions

affinities dissociation

ion

discrimination

ability

alkali constants

profiles

of unlabeled

alkali

*‘Rb+ -complexes were

given

ion

of the antibiotics

to displace

for

values

42K+ or 86Rb+ The relative

of the reciprocal

K+ or Rb+ obtained

given alkali ions to those for Salinomycin and nigericin for complexation. for K+ over the other alkali ions and the ion

antibiotics determined by *6Rb+ displacement.

and nigericin

of antibiotics.

as relative

the competition with cation ences

of salinomycin

by

K+, the most favourable showed strict preferselectivities

of both

were similar to those obtained by 42K+ displacement These results gave a good agreement with the ion

1232

Vol. 66, No. 4,1975

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Table I KA of Salinomycin and Its Derivatives

for

Various Cations

Salinomycin

Acetyl Salinomycin

Propionyl Salinomycin

Methyl Ester

Bromophenacyl Ester

Na+

1.7

2.0

2.7