JOURNAL OF RECEPTOR RESEARCH, 8 ( 1 - 4 ) , 3 2 3 - 3 4 3 (1988)

RADIORECEPTOR ASSAY FOR a-MSH USING MOUSE 816 MELANOMA CELLS+

Walter Siegrist, Marc Oestreicher, Sibylla Stutz, Jurg Girard and Alex N. Eberle"

Laboratory of Endocrinology, Department of Research University Hospital and University Children's Hospital

CH-4031 Basel, Switzerland

Key words: a-MSH, f3-MSH, ACTH, radioreceptor assay, Scatchard analysis, tyrosinase assay, melanin assay, Anolis skin assay, pituitary extracts

ABSTRACT

A radioreceptor assay for a-MSH is described which is based on cultured llpouse B16 melanoma cells and bioactive monoiodinated [Nle 1-a-MSH tracer. The assay was used (1) to study the binding characteristics of a-MSH to 816 cells, (2) to determine the relative binding activity of MSH peptides, and ( 3 ) to measure MSH in tissue extracts. The association of a-MSH to 816 cells reached a stable plateau after 3 h at 15OC. At 2 5 O or 37OC, the binding was transient and at O-lOC, the association was very slow. The hormone-receptor complex was relatively stable between O o and 15OC whereas a 50% dissociation was reached after 90 min at 2 5 O C and after 35 min at 37OC. The mean KD for a-MSH of four saturation experiments was 1.3 nM and the number of receptors 9 5 7 0 per cell. 1,lO- Phenanthroline had a stabilizing effect in the binding assay when used at a 0.3 mM concentration. From the MSH peptides tested in the binding assay, some showed simi- lar potencies in three bioassays (tyrosinase, melanin and Anolis skin), whereas others displayed considerably

+ In memoriam Gaby Schmitt, Strasbourg

323

Copyright @ 1988 by Marcel Dekker, Inc.

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324 SIEGRIST ET AL.

lower bioassay values than expected from the binding data. This shows that binding and bioactivity can be dissociated in some of the MSH peptides. The biological activity of MSH from the neurointermediate lobe of the rat pituitary as measured by its binding to B16 cells corresponds fairly well with RIA results; in the anteri- or lobe, a-MSH values are overestimated because of the large amount of ACTH present.

INTRODUCTION a-Melanotropin (a-melanocyte-stimulating hormone, a-

MSH) is well known to induce pigment-dispersion in melanophores of lower vertebrates as well as melanogene- sis in mammalian melanocytes and melanoma cells. The peptide also functions as neuromodulator, affecting various parameters such as behaviour, nerve regeneration and temperature control in fever (reviewed in [l]). At the cellular level, a-MSH has been studied extensively with melanophore bioassays by determination of struc- ture-activity relationships [ 2 , 31, or with mammalian melanoma cells by measuring adenylate cyclase activa- tion, cyclic AMP production or tyrosinase stimulation [ 4 , 51. More recently, a very sensitive melanin assay has been developed with which MSH-induced melanogenesis in cultured melanoma cells can be determined in situ [6]. This assay correlates well with the melanophore assays both in terms of sensitivity and in measurement of the final response of the cell (melanin production). L i k e melanophore assays, the melanin assay can be used for the determination of MSH bioactivity in biological fluids and tissue extracts, but it does not yield infor- mation concerning receptor-binding activities of MSH peptides. This aspect, however, is of great importance in the search for MSH binding sites in different types of melanoma and other tissues.

Mouse 816 and Cloudman S91 melanoma cells are the systems of choice for an MSH receptor-binding assay

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RADIORECEPTOR ASSAY 325

because the cells are easily grown and known to respond to the hormone. The first quantitative binding analysis using S91 cells in combination with tritiated a-MSH was performed many years ago but was hampered by a high degree of non-specific binding and hence only permitted an approximation of receptor numbers [7]. A subsequent study using radioiodinated 6-MSH gave more precise results on receptor numbers and affinities [8]. However, biologically active monoiodinated @-MSH is difficult to prepare and is less active in most bioassays than a-MSH. Moreover, it is not present in several vertebrate species (e.g. rats and mice) so that a radioreceptor assay for MSH should be based on a-MSH. The higher sensitivity of 816 cells as compared to S91 cells to a-

MSH suggests the use of B16 cells for a receptor-binding assay,

A prerequesite for such an assay is the availability of bioactive a-MSH tracer. Bioactive tritiated a-MSH has been prepared to fairly high specific radioactivity (>lo0 Ci/mmol) [ 9 , 101 but is less suitable for the detection of low numbers of high-affinity binding sites than [ 1251 1-monoiodinated a-MSH with approximately 2000 Ci/mmol. However, the preparation of radioiodinated a- MSH with chloramine T is thought to lead to inactivation of the peptide, particularly when a-MSH with the natural sequence is used because Met4 is oxidized to the S-oxide (111. Although an equimolar chloramine T method has been described with which bioactive radioiodinated a-MSH can be prepared [9], the experiment needs considerable expertise and is not always reproducible. [Nle 1-a-MSH has a better resistance to chloramine T [12] and hence is a better choice for radiolabelling.

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326 SIEGRIST ET AL.

We describe an a-MSH receptor-binding assay based on cultured mouse B16 melanoma cells and monoiodinated [Nle 1-a-MSH tracer with which receptor affinities and numbers can be determined, the relative binding activity of MSH analogues measured, and which can also be used as radioreceptor assay for the estimation of MSH binding- activity in biological tissue.

4

MATERIALS AND METHODS Pept i de s

[Tyr( I ) 2 1-a-MSH ( 3r-iodotyrosine2-a-MSH) , [Met(O) 4 1-

a-MSH (oxidized a-MSH) and desacetyl-a-MSH were synthe- sized in our own laboratory by a classical solution approach [9]. [Nle 1-a-MSH, [Nle4,D-Phe 1-a-MSH and 8- MSH (porcine) were obtained from Bachem (Bubendorf, Switzerland). a-MSH, ACTH(1-24) and ACTH(1-39) was a gift from CIBA-GEIGY AG (Basel, Switzerland).

4 7

4 Iodination of [Nle 1-a-MSH [Nle 1-a-MSH was radioiodinated using the chloramine

T procedure: 1.5 mCi Na1251 was centrifuged in a 1.5 ml Eppendorf tube and diluted with 20 p1 of 0.25 M Na- phosphate buffer, pH 7 . 4 . The peptide (1.5 pg in 3 ,411 5 mM H3P04) was added followed by chloramine T (10 pg in 10 p1 of water). After 30 sec, the reaction was stopped by addition of 600 pl of 50 mM Na-phosphate buffer con- taining 0.25% BSA. The solution was applied to a small reversed-phase column, consisting of a 1 ml Cyringe filled with Spherisorb ODS (10 pm) up to 0.4 ml of its volume, primed with a solution of Polypep (Sigma). The product was eluted with an i ~ c r e a c i n g methanol gradient containing 1% trifluoroacetic acid. Reversed-phase HPLC was used f o r further purification in some experiments.

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RADIORECEPTOR ASSAY 327

Rat pituitary extracts The neurointermediate lobes of rat pituitaries were

separated from the anterior lobes, homogenized in 0.2 ml 10 mM HC1 at O°C using a small potter. The suspension was heated to 100°C for 5 min in a water bath and neu- tralized by addition of 0.1 ml 100 mM Tris base contain- ing 3 mM 1,lO-phenanthroline. Insoluble material was removed by centrifugation and the clear supernatants stored at -2OOC.

Tissue culture 2 B16-Fl Mouse melanoma cells [13] were grown in 75 cm

Falcon tissue culture flasks using modified Eagle's medium (MEM) with Earle's salts (GIBCO, Paisley, U.K.), supplemented with 10% heat-inactivated fetal calf serum (Amimed, Basel), 2 mM L-glutamine, 1% MEM nonessential amino acids (loox; GIBCO), 1.5% MEM vitamin solution (1OOx; GIBCO), 5 0 units/ml of penicillin, and 50 pg/ml of streptomycin, at 37OC in a humidified atmosphere of 95% air and 5% C02. Subculturing was done every 2 to 3 days using 0.02% EDTA in phosphate-buffered saline ( 8 g NaC1, 0.2 g KC1, 0.2 g KH2P04, 1.44 g Na2HP04 2H20 per liter) for detaching the cells. The cells used for each binding experiment (-2x106 cells), were grown in a Costar 900 cm2 roller bottle for 2-3 days, using an initial cell number of - 3 ~ 1 0 ~ cells (two 75 cm2 flasks) and 250 ml of medium.

Binding experiment For the binding experiment, the MEM medium did not

contain the additives mentioned above, but was supple- mented with 25 mM Hepes (+MEM-Hepes), 0.2% bovine serum albumin, and 0.3 mM 1,lO-phenanthroline (except where indicated). The binding reaction was started by adding 0.5 ml of cell suspension (0.5-2x107 cells/ml) to 12x75

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328 SIEGRIST ET A L .

mm polystyrene tubes containing 50 gl radioligand and 50 g1 unlabeled peptide. The final concentration of [1251]- a-MSH was usually 200,000 cpm/ml, except f o r saturation experiments. For equilibrium binding, the cells were incubated during 3 h at 15OC and resuspended occasional- ly by gentle mixing. Triplicate 150 p l aliquots were layered on top of 150 g1 silicon oil in 0.4 ml polyethy- lene microtubes. The oil was prepared to a density of 1013 kgm-3 by mixing equal volumes of AR 20 and AR 200 (Wacker Chemie GmbH, Munich, FRG). After centrifugation at 4OC the tubes were cut with a scalpel through the oil layer and the radioactivities of the cell pellet and the supernatant were measured in a LKB y-counter.

Bioassays, Radioimmunoassays Tyrosinase activity was determined using a modifi-

cation [ 6 ] of the original method of Pomerantz 1141. Melanogenesis was measured by the in situ melanin assay described by Siegrist & Eberle [6]. Melanin-dispersing activity was assayed using skin pieces of Anolis carolinensis, a s described by Eberle & Girard [15]. Radioimmunoassays for a-MSH, ACTH and 6-LPH were carried out by solid-phase and conventional techniques estab- lished in our laboratory.

Data analysis

analyzed with LIGAND, an iterative nonlinear regression program established by Munson et a1 [16] for IBM personal computers.

Data from competition and saturation experiments were

RESULTS Iodination of [Nle J-a-MSH 4

4 Iodination of [Nle 1-a-MSH by chloramine T followed by rapid purification on C18 reversed-phase material Jo

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RADIORECEPTOR AS

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40 m

30 m

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4 F I G . 1 HPLC elution pattern of [Nle I-u-MSH (P) after iodination. Peak I: mono-iodinated compound; peak 11: di-iodinated compound. The HPLC elution buffers consisted of 0.1% trifluoroacetic acid ( A ) and 70% aqueous acetonitrile containing 0.1% trifluoroacetic acid (B). The column (0.4~25 cm) was from Knauer and contained Spherosil ODS (5 pm). An expontential gradient was used and the flow rate was 1 . 2 ml/min.

produced mainly monoiodinated tracer with a minor por- tion of the diiodinated form, as illustrated by the HPLC tracing shown in Figure 1 (in some experiments, the pro- portion of diiodinated tracer was higher depending on how the elution from the minicolumn was performed). For complete homogeneity, the tracer was purified by HPLC and the monoiodinated fractions were selected for the

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330 SIEGRIST ET A L .

100

7 80 3 0 n I VJ t

60

- 20 s

0

total: 15°C 0-1°C

+ 37°C A 25°C

non - specific : 0 15°C

0-1°C 0 37°C

0 1 2 3 4 5 6 7 8 Time (hours)

F I G . 2 Association of a-MSH to 816 cells at O-loC, 15OC, 25OC, and at 37OC. Closed symbols: total binding; open symbols: non-specific binding.

assay. The biological activity of monoiodinated tracer, as measured with the Anolis skin assay, corresponded approximately to that of non-radioactive [Tyr(I) l-a- M S H .

2

Rate of association and dissociation 4 Binding of [Nle 1-a-MSH to 816 melanoma cells is

time- and temperature-dependent. As shown in Figure 2, association at 37O and 25OC was rapid but transient, i.e. the specific binding decreased immediatly after reaching its maximal value. At 0-1 OC binding was considerably slower and did not reach a peak even after 8 h of incubation. At 15OC a stable plateau occurred after 3-4 h. The non-specific binding was virtually in- dependent of temperature. This shows that for the

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RADIORECEPTOR ASSAY 331

100 - 7

0-1°C

0 1 I I I I 1 0 1 2 3 4 5

Time (hours)

FIG. 3 Dissociation of receptor-bound U-MSH from B16 cells at O-lOC, 15OC, 2SoC, and at 37°C.

binding assay using a - M S H tracer and mouse B16 melanoma cells, a 3 h incubation at 15°C should be used for reaching equilibrium.

Specific binding to 816 cells is reversible (Figure 3 ) . At 37"C, half of the bound ligand dissociated after 35 min and after 4 h, the binding was reduced to the non-specific values. At 25OC, a 50% dissociation occur- red after 90 min. At lower temperatures, the dissocia- tion was much slower. For example, 23% of the receptor- bound ligand dissociated after 4 h at 15°C and only 7% of the label was released after 5 h at 0-1OC.

Effect of 1,lO-phenanthroline 1,lO-Phenanthroline is a chelating agent with the

ability to inhibit metallo-endopeptidase activities [cf 171. In a first series of experiments with high cell

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332 SIEGRIST ET A L .

- m 9 x

specific [ phenant hrolinel 15

non-specific

0 1 1 I I I I 1

0 1 2 3 4 5 6 Time (hours)

F I G . 4 Effect of 1,lO~phenanthroline on the binding of iodinated [Nle 1-a-MSH in an early binding experiment (see text).

concentrations, 1,lO-phenanthroline proved to be useful t o reach equilibrium in specific binding and to suppress high levels of non-specific binding (Figure 4). However, in later experiments when the cell number was reduced and the homogeneity of the MSH tracer increased, the effect of 1,lO-phenanthroline was less clear (Figure 5). At high concentration (3 mM), 1,lO-phenanthroline even reduced specific binding by almost 5 0 % . This could be due to the chelating properties of the compound which at high concentrations also chelates divalent cations known to be essential f o r MSH binding in melanoma cells [18]. Nevertheless, f o r routine experiments 1,lO-phenanthro- line is added in a 0.3 mM concentration because it has a stabilizing effect and does not interfere with the binding.

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RADIORECEPTOR ASSAY 333

I I I I I I

0 0.1 0.3 1 3 nM Concentration of 1 , l O - phenant hroline

F I G . 5 Effect of different concentrations of 1,lO- phenanthrgline on the specific binding of iodin- ated [Nle 1-m-MSH in 7 different experiments.

Saturation of binding The data of a typical saturation experiment are shown

in Figure 6. The tracer was diluted 15-fold by addition of unlabelled a-MSH, resulting in a specific radioacti- vity of 230 cpm/fmol. Binding in the presence of 4 flt4 a-

MSH was linear and considered as non-specific. Scatchard analysis pointed to a single population of 10,390 binding sites per cell with a dissociation constant ( K D ) of 1.02 nM. The following mean values were obtained from 4 similar experiments: K D = 1.31 nfi ( + 0.31 nM) and Bmax = 9570 ( + 1750) sites per cell.

Competitive binding Receptor affinities of MSH peptides were assayed in

competitive binding experiments. Binding was measured in

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334 SIEGRIST ET AL.

0.2 W W oc LL - n Z 3 g 0.1

FREE InM)

0 100 200 PM

I I I I

0 10 20 30 40 BOUND ( fmoles/2xlO6 cells)

F I G . 6 Demonstration of a typical saturation binding experiment. Equilibrium was achieved after incubation for 3 hours at lS°C. The figure shows the Scatchard plot of the binding data presented in the insert. For details see text.

the presence of a constant amount of tracer and varying concentrations of each analogue. Competition curves obtained from a typical experiment with a-MSH, desace- tyl-a-MSH, [Tyr(I) 1-a-MSH, [Met(O) 1-a-MSH, [Nle l-a- MSH, [Nle ,D-Phe 1-a-MSH, B-MSH, ACTH(1-39) and ACTH(1- 24) as well as the mean binding affinities from 2-3 experiments are shown in Figure 7. The KD for a-MSH was slightly higher in the competitive binding assay (1.9 nM) as compared to the saturation assay (1.3 nM). The

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RADIORECEPTOR ASSAY 33 5

IJ, 100 - C U .- 5 80-

.G 60- u .- L

al Q

- 40- .- i ; 20- X

[ Nle4, D - P ~ ~ ~ I - M S H L, [Tyr(l)*J-MSH 0 [Nle41-MSH o Desac-MSH

a-MSH 7 ACTHq-24 + B,-MSH

ACTH,.,g [ Met (014 I -MSH

K, +SD ( n M ) 0.32 t 0.13 1.3 2 0.3 1.8 2 0.1 1.8 + 0.2 1.9 2 0.4 2.0 t 0.3

22.0 2 4.2 6.2 0.8

218 + 33 IJ, 100 C U .- 5 80

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al Q

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a p 0

X

c 0

10 9 a 7 6 5 Concentration ( log M )

1 10 9 a 7 6 5

Concentration ( log M )

F I G . 7 Log dose-response curves from a competition binding experiment with several a-MSH analogues and related peptides. The KD-values for each peptide are the mean of 2-3 experiments.

binding activities as compared to that of u-MSH ranged from 6-fold higher, such as for [Nle4,D-Phe 1-a-MSH, to 100-fold lower, such as for oxidized a-MSH. All a-MSH analogues with modifications in the N-terminal part displayed about the same affinity, showing that this portion is not essential for binding to the receptor. B- MSH has a 3-fold lower affinity than U-MSH. ACTH(1-24) displayed almost the same KD as a-MSH whereas ACTH(1-39) had a 10-fold lower binding constant.

7

Comparison between binding assay and bioassays When the relative potencies of the different MSH

peptides in the tyrosinase, melanin and Anolis skin

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336 SIEGRIST ET A L .

Table - 1 Relative potencies in the competitive binding assay as well as in the tyrosinase, melanin and melanophore assays for a-MSH and structural analogues.

Peptide Relative potencies Binding Tyrosinase Melanin Anolis

a-MSH 4 [Nle 1-a-MSH

[ 3 -1odo-Tyr ] -u-MSH De Sace t yl- a-MSH [ Nle4 , D-Phe ] -a-MSH 6-MSH (porcine)

2

7

ACTH(1-24) ACTH( 1-39 )

4 [Met(o) 1-a-MSH

1 1 1 1.1 0.071 0.046 1 . 5 1.0 0.79 1.1 0.19 0 . 0 5 9

6.0 12.3 12.5 0.31 0 . 2 5 0.64 0.97 0.24 0.048 0.088 0.052 0.074 0.0087 - 0.010

1 0.66 1 . 5

0.75 10.0 0.30 0.013 0.013 0.01

assays are compared with their apparent binding con- stants (Table l), the peptides can be separated into two groups. Peptides such as [Tyr(I) I-u-MSH, [Nle4,D-Phe 1- a-MSH, f3-MSH and ACTH(1-39)) have similar relative potencies in all assays whereas peptides such as desacetyl-a-MSH, [Nle 1-a-MSH and ACTH(1-24) show lower bioactivity than would be expected from their binding constants.

2 7

4

Determination of MSH in pituitary extracts

vity in biological samples. Extracts from the anterior and neurointermediate lobe of rat pituitaries were able to displace radiolabelled a-MSH from B16 binding sites in the same dose-related manner as a-MSH. The amount of MSH-like activity in the lobes of 8 animals was deter-

The binding assay was tested to measure MSH bioacti-

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RADIORECEPTOR ASSAY 337

Table 2 Comparison of the radioreceptor assay with radioimmunoassays in the determination of the MSH content of rat anterior and neurointermedi- ate lobe extracts (n = 8 animals).

Animal Radioreceptor Radioimmunoactivity assay (ng)

(ng f SEM) a-MSH ACTH LPH

Anterior lobe 1 3 4 . 8 t 1 . 0 2 4 1 . 5 + 3 . 5 3 3 0 . 4 T 0 . 3 4 3 8 . 1 T 2 . 7 5 3 4 . 5 t 1 . 3 6 4 5 . 2 + 2 . 0 7 4 0 . 5 0 .8 8 3 9 . 2 - + 0 . 5

Neurointermediate lobe 1 1 9 3 . 0 + 9 . 7 2 5 5 4 . 6 T 2 3 . 8 3 3 7 8 . 2 T 1 9 . 7 4 2 7 8 . 9 7 1 9 . 7 5 1 8 2 . 5 T 6 . 6 6 6 0 4 . 8 T 3 6 . 0 7 6 7 2 . 6 T 1 1 . 2 8 2 9 1 . 8 t 1 2 . 8

< 2 5 < 2 5 t 2 5 t 2 5 t 2 5 < 2 5 t 2 5 t 2 5

7 3 4 . 7 8 0 3 . 7 4 0 8 . 9 3 2 0 . 0 2 0 4 . 4 9 0 0 . 0 6 9 5 . 1 2 6 5 . 8

1 9 0 . 0 2 6 0 . 0 1 8 4 . 8 1 5 9 . 8 2 1 5 . 0 2 7 0 . 9 2 3 2 . 0 2 3 2 . 0

1 0 8 . 3 1 2 6 . 0 1 5 8 . 4 1 4 9 . 3

6 2 . 6 9 2 . 5

2 1 4 . 7 1 3 4 . 7

2 . 2 4 2 . 5 6 3 . 3 5 2 . 7 2 1 . 8 8 2 . 0 7 1 . 9 5 2 . 1 6

2 . 5 1 2 .89 2 .56 1 . 8 4 1 . 9 2 3 . 1 3 3 . 3 5 2 . 5 6

mined by comparison of the displacement values with an a-MSH standard curve. The minimal detectable concentra- tion in extracts using this method was 1 ng/ml a-MSH. Table 2 presents the data in comparison with radio- immunological determinations of a-MSH, ACTH and LPH in the same lobes. The MSH-activity in extracts of neuro- intermediate lobes determined with the a-MSH-receptor assay corresponds fairly well with that measured by the a-MSH radioimmunoassay. The presence of relatively high amounts of ACTH in anterior lobe extracts may be

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338 SIEGRIST ET A L .

the reason for the slight overestimation of the MSH content in this tissue using the a-MSH-receptor assay.

DISCUSSION The radioreceptor assay for a-MSH described in this

paper has a broad range of applications, namely for studying the characteristics of the MSH receptor of B16 mouse melanoma cells as well as structure-activity relationships of MSH peptides and for the determination of NSH bioactivity in tissue extracts. In combination with B16 cells, monoiodinated [Nle I-a-MSH appears to be an ideal tracer since it retains approximately the same binding characteristics as natural a-MSH. Both [Nle ]-a- NSH and its iodinated derivative retain almost full bio- logical activity in the Anolis skin assay when compared with natural a-NSH and [Tyr(I) 1-a-MSH respectively [l, 191. However, the potency of [Nle I-u-NSH (and presum- ably of its iodinated analogue) in the tyrosinase and melanin assays using B16 cells is considerably lower

4

4

2

4

than that of a-NSH. This may indicate that ties of iodinated [Nle 1-a-MSH in other me systems must be carefully investigated.

4

The rate of association of a-MSH to B16 similar to that observed by Lambert et a1

the proper- anoma cell

cells was 81 for 6-NSH-

receptor binding to Cloudman S91 cells. For both assays, a temperature of 15°C was found to be ideal. At higher temperatures, binding of the radioligand was lower and reached a peak of relatively short duration because of degradation of the radioligand by tissue proteases (unpublished) resulting in a rapid loss of radioactivity from the cell surface. At very low temperatures, a steady-state was not reached within a reasonable time. In 816 cells, the binding affinity for U-MSH was about

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RADIORECEPTOR A S S A Y 339

3-fold higher ( K D = 1.9 nM; competition experiment) than that for &ASH (KD = 6.2 nM) which is almost identical with that found for 6-MSH in Cloudman S91 cells ( K D = 5 nM). The receptor number in the two biological system was also similar (approximately lo4 receptors/cell).

1,lO-Phenanthroline was the only one of several protease inhibitors tested which did not interfere with the assay and had a positive effect. Bacitracin which has been frequently used in binding assays (e.9. for ACTH [20]) considerably reduced the specific binding of a-MSH to 816 cells (data not shown). In view of the rapid degradation of MSH tracers at higher temperatures, it is not surprising that in the recently described binding assay for f3-MSH and B16-M2R cells performed in tissue culture flasks at 37'C for 30 min, the affinity constant was only 20 n M [2l]. Rapid degradation of 6-MSH at 37OC in the absence of inhibitors had also been shown by Lambert & Lerner [22].

The difference of the KDs for a-MSH in the saturation and competition experiments may reside in the uncertain- ty of the correct value f o r the specific radioactivity of the INle 1-a-MSH tracer and in its slightly higher affinity as compared to normal a-MSH. The KD values reported above are based on the assumption of a -90% homogeneity of the tracer, i.e. a specific acitivity of -1900 Ci/mmol. Since the determination of this value after each iodination is cumbersome (see [ 9 ] ) , the values reported here for KD and receptor numbers will have to be verifyed with tritiated a-MSH of precisely known specific radio-activity.

4

Structure-activity data show that binding and bio- assay results for some of the a-MSH peptides correspond

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3 40 SIEGRIST ET AL.

well whereas with other analogues, the relative binding affinity is higher than their biological activity. This dissociation of binding and bioactivity indicates that the latter cannot be predicted from the potency of a peptide in the binding assay, even if the peptide does not show any antagonistic or partial agonistic proper- ties. This is not surprising because the binding experiment is of 'short-term' character whereas a bio- assay requires 2-3 days. Therefore, comparisons between binding data and potencies obtained in the tyrosinase and melanin assays should be made with caution.

The radioreceptor assay can be useful for the deter- mination of binding activity to MSH receptors of tissue extracts, particularly in situations where RIA results alone are not sufficiently reliable. Since the receptor binding activity does not differentiate between a-MSH,

desacetyl-u-MSH and ACTH-peptides, such as ACTH(1-24), it is not possible to determine a-MSH in the presence of large amounts of any of these peptides.

ACKNOWLEDGEMENTS The authors are grateful to Dr R. Andreatta and Dr.

B. Kamber (CIBA-GEIGY AG) for the gift of ACTH peptides, and to MS V. Jaggin and G. Gambon for the RIA results. This work was supported by grants from the Schweizeri- sche Krebsliga, the Krebsliga beider Basel and the Swiss National Science Foundation.

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