Salinomycin: A new monovalent cation ionophore

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

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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


<1o-4

K+ cs+

3.2

2.4

3.0

0.47

0.05

0.06


<1o-4 <1o-4

0.026

0.007

0.008

0.030

0.004

0.010

<1o-4 <1o-4

<1o-4 <1o-4

0.374

0.050

0.063

0.004

0.006

Mg2+ Ca2+ Sr2+

The antibiotics dissolved in 0.1 ml of 25 % dimethylformamide plus 75 % ethanol were mixed vigorously with 1 ml of a 30 %n-butanol-70 % toluene mixture and 0.5 ml of aqueous buffer containing 0.1 to 20 mM radioactive metal salts and 40 mMtris- HCl, pH 8.5. The antibiotics were used at a concentration of 2 x 10s4M and the KA values were determined by the procedures described in Materials and Methods. For KA multiply above values by 102.

(A)

0.60 095 IA' Nd

Ionic

(B)

Salinomycin

1.33 166 1.69 K' Rb' Cs'

Radius

of

Alkali

0.60 LI'

Metal

Nigericin

0.95 No'

133 146 169 K' Rb' Cs'

Cation

A*

Fig. 1. Ion selectivity patterns of salinomycin and nigericin. Relative affinities for various alkali ions were determined by their abilities to displace 42K+(a) and *'Rb+(b) from the respective ionophore-complexes under the conditions similar to those of Table I.

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Vol. 66, No. 4,1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Antlblotlc

0

2

4

6

8

10

Hours Fig. 2. The bulk phase transport of alkali ions mediated by salinomyicin. The transport system consisted of two layers (the upper one was aqueous phase and the lower one was the organic phase of Cc11+). The upper layer was separated into two parts by a vertical septum. The test antibiotic was added to CC14layer at a concentration of lo-"M. The labeled ions were added to one part of the aqueous layer and the time course of the apperance of radioactivity in the other part of aqueous layer was monitored.

4

6

8

10

12

Hours Fig. 3. The net transport of 4zK+ mediated by salinomycin and its derivatives. The reaction mixture the sameas Fig. 2. SL:Salinomycin.

discrimination

pattern obtained by direct

measurementsof KA of salinomycin.

Salinomycin mediated the bulk phase transport of alkali ions across the lipid barrier of CC14as shown in Fig. 2. After equilibration for two hours, the antibiotic was added to organic phase and the time course of alkali ion transport was monitored. Salinomycin transported Na+, Rb+, K+ preferentially but failed to transport Cs+. Fig. 3 gives the time course of net K+ transport mediated by salinomycin, its derivatives and nigericin. Acetyl and propionyl derivatives of salinomycin catalyzed net transport of K+ to the sameextent of that

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BIOCHEMICAL

Vol. 66, No. 4,1975

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

obtained with the parent compoundbut methyl and bromophenacyl esters were unable to transport K+. Salinomycin exhibited nigericin under these experimental conditions.

greater efficiency

over

The results obtained by another barrier system consisting of three layers were given in Table II. These data indicated that the sequence for cation transport activities of salinomycin and its derivatives was Rb+, Na+ > K+B cs+, Mg2+,Ca2+, Sr'+. The activity of salinomycin as an ion carrier was lost by esterification of the terminal carboxyl group.

Table II Bulk Transport of Metal Cations Mediated by Salinomycin and Its Derivatives

Initial

Rate (nanomolesper hour)

Salinomycin

Acetyl Salinomycin

Na+ K+

95

58

114

1

67

81

Rb+

105 11 6

70 8 5

92 138

1 0.5

17 33

0.5 0.6

11

2

12

3

2 4

0.1 0.1

cs+ Mg2+ Ca2+ sr2+

Propionyl Salinomycin

Methyl Ester

Bromophenacyl Ester 1 1 0.5 0.5 0.6 0.1 0.1

The initial rate of cation transport across the lipid barrier of CCl4-CroHaa was determined by the procedure described in Materials and Methods. The antibiotics were dissolved in organic solvent at a concentration of 2 x lo-'M.

DISCUSSION Numerousstudies on the mode of action of polyether antibiotics revealed that these antibiotics form lipid-soluble complexes with cations presumably by ion-dipole interactions and carry cations through the lipid barriers of membranesby passive diffusion processes (l-5,8). These properties of the antibiotics have been applied as useful tools for studying dynamic processes of ion carrier systems in biological membranes(9,lO).

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Vol. 66, No. 4, 1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Salinomycin migrated more effectively

monovalent cations than divalent

cations from aqueous buffer into organic solvent and the OH-acylated salinomycin retained the activity of parent compound(Table I). The sequence of ion selectivities of the antibiotics was as follows: K+ > Na+ 2+ > cs+ > Sr > Ca2*, Mg2T The similar patterns of ion discrimination were obtained with the experiments of 42K' and s6Rb+ displacements (Fig. 1). Although salinomycin exhibited a greater affinity for K+ than Na+ and Rb' in complexation, the antibiotic transported Na+ and Rb+ more effectively than K+ across the bulk phase of CC14 (Fig. 2). Similar results were obtained in case of acetyl and propionyl derivatives (Fig. 3 and Table II). As described by Pressman, the rates of complexation and decomplexation with ions limit the efficiency of complexing agent to act as an ion carrier of complexation and cation transport induced (5) * The loss of the abilities by esterification of the terminal carboxyl group indicates the critical role of this group for the ionophorous activity of salinomycin. These results obtained by two phase distribution systems and the model systems of lipid barrier indicate that salinomycin and its derivatives are the ionophores with strict selectivities for alkali ions. Our preliminary data on the effects of salinomycin on rat liver mitochondria showedthat the antibiotic caused the net efflux of alkali ions accumulated by valinomycin and monazomycin in mitochondria. The study of the effect of salinomycin on mitochondria will be published elsewhere. The authors are indebted to Dr. H. A. Lardy of Wisconsin University for the gift of nigericin and wish to thank Dr. S. Shirato for valuable discussions. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Pressman, B. C. (1968) Federation Proc. 27, 1283-1288. Pressman, B. C. (1970) Antimicrobial Agents and Chemotherapy 1969, 28-34. Ashton, R., and Steinrauf, L. K. (1970) J. Mol. Biol. 49, 547-556. Reed, P. W., and Lardy, H. A. (1972) J. Biol. Chem. 247, 6970-6977. Pressman, B. C. (1973) Federation Proc. 32, 1698-1703. Kinashi, H., and Otake, N., and Yonehara, H. (1973) Tetrahedron Letters 49, 4955-4958. Miyasaki, H., Shibuya, M., Sugawara, H., Kawaguchi, O., Hirose, C., 27, 814-821. Nagatsu, J., and Esumi, S. (1974) J. Antibiotics Rottenberg. Rottenberg, Harold, F. Acad. Sci.

H., and Scarpa, A. (1974) Biochemistry 13, 4811-4817. H. (1973) J. MembraneBiol. 11, 117-137. K. H., and Hirata, H. (1974) Ann. NewYork M., Altendorf, 235, 149-160.

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