Fluorescence studies on the interaction of the tumor promoter phorbol myristate acetate and related compounds with rat liver plasma membranes

Chem.-Biol. Interactions, 15 (1976) 233--246 233 © Elsevier/North-Holland Biomedical Press, Amsterdam -- Printed in The Netherlands

FLUORESCENCE STUDIES ON THE INTERACTION OF THE TUMOR PROMOTER PHORBOL MYRISTATE ACETATE A N D RELATED COMPOUNDS WITH RAT LIVER PLASMA MEMBRANES *

BENJAMIN L. VAN DUUREN~ SIPRA BANERJEE and GISELA WITZ

Laboratory of Organic Chemistry and Carcinogenesis, Institute of Environmental Medicine, New York University Medical Center, New York, N.Y. 10016 (U.S.A.) (Received May 7th, 1976) (Revision received July 19th, 1976) (Accepted July 29th, 1976)

SUMMARY

The interaction of the potent tumor-promoting agent phorbol myristate acetate (PMA) with purified rat liver plasma membranes suspended in phosphate-buffered saline (PBS), pH 7.4, was studied by fluorescence spectrophotometry. Exposure of membranes to PMA caused up to 21% decrease of the native membrane emission, i.e. the fluorescence of both tryptophan and tyrosine, compared to non-treated membranes. The decrease in the membrane emisssion varied with both the PMA and the membrane concentration. Treatment of rat liver plasma membranes with biologically less active analogs of PMA, phorbolol myristate acetate (PHMA) and 4a~-phorbol didecanoate (4a~-PDD), resulted in a 5--10% decrease of the native membrane emission. These studies suggest that PMA causes alterations in membrane structure which are due, at least in part, to conformational changes in the membrane proteins.

1' This work was reported in part at the 66th annual meeting of the American Association for Cancer Research, May 1975 (G. Witz, B.L. Van Duuren and S. Banerjee, Proc. Amer. Assoc. Cancer Res., 16 (1975) 30). Supported by USPHS under grants CA 14211, ES 00260 and CA 13343. Abbreviations: PBS, phosphate-buffered saline; 4aa-PDD, 4a0~-phorbol didecanoate; PHMA phorbolol myristate acetate; PMA, phorbol myristate acetate (trivial name for Chemical Abstracts Registry Number 20839-11-6; the numbering of carbon atoms used in this report is the same as that used by Chemical Abstracts)•

234 INTRODUCTION PMA is the most p o t e n t k n o w n tumor-promoting agent for two-stage carcinogenesis on mouse skin [1]. Since the isolation of this c o m p o u n d from croton oil [2,3] and its structural elucidation [1,4,5], a number of biochemical studies have been carried out in order to determine its possible mode of action. In addition to its tumor-promoting activity, PMA induces cell division in BALB/c-3T3 cells [6,7] and thymic lymphoblasts [8] and is a p o t e n t aggregator of blood platelets [9]. PMA rapidly raises intracellular cyclic GMP levels in BALB/c-3T3 cells [10] and stimulates the early synthesis of phosphatidylcholine and phosphatidylethanolamine in mouse skin [11]. Based on the lipophilic-hydrophilic nature of PMA, it was suggested by us that a primary interaction of this c o m p o u n d is at the plasma membrane [1]. Such a physical binding is expected to cause structural changes in the cell membrane, including changes in membrane permeability. The resulting alterations could in turn signal a variety of intracellular processes, some of which may be related to the mode of action of the t u m o r promoter. Several observations indicate that the binding of PMA to cell membranes causes alterations in functional membrane-associated processes. PMA was found to inhibit the Mg 2÷-, Ca 2÷-, and (Na*--K÷)ATPases of rat liver plasma membranes [12] and the (Na*--K*)ATPase of beef brain microsomes [13]. In addition, PMA altered the permeability and cell shape of BALB/c-3T3 cells [14] and caused a 15% decrease in the electrophoretic mobility of hyperdiploid Ehrlich--Lettr~ ascites t u m o r cells [ 1 3 | . It must be borne in mind that to date PMA has been shown to be active only on mouse skin and not in liver. However, valuable information on the biological effects of PMA have come out of the studies cited above [7,12--14], none of which involved use of mouse skin. Some of the findings reported in these earlier studies may well bear directly on the mode of action of PMA in mouse skin. It should also be emphasized that aromatic hydrocarbons are carcinogenic to skin and lung but not to liver. Nevertheless, many valuable studies on the metabolism of aromatic carcinogens have been carried out on rat liver homogenates or microsomal preparations from liver. Therefore, the use of rat liver plasma membranes in the present study is considered by us relevant to one or more of the various in vivo biological effects of PMA. In the present work, the interaction of PMA with rat liver plasma merebranes was examined by use of fluorescence spectrophotometry, which is a sensitive tool for detecting structural changes in membranes [15]. For comparison purposes, the binding of PHMA, a metabolite of PMA on mouse skin [16], and 4a~-PDD, an analog of PMA, was also examined. Bioassays from this laboratory indicate that the metabolite PHMA is a moderate t u m o r promoter (B.L. Van Duuren et al., unpublished results), while 4aa-PDD was reported to be inactive [17] ; the duration of the latter experiment may have been inadequate for a clearcut evaluation of tumor-promoting activity. The tumor-promoting activity of 4aa-PDD and related compounds are currently being examined in this laboratory.

235 MATERIALS AND METHODS

Membrane isolation Rat liver plasma membranes were isolated from 8--11-week-old female Sprague--Dawley rats (A.R. Schmidt, Madison, Wis.). A 10% homogenate of 4--6 g liver was prepared in cold 0,25 M sucrose containing 0.01 M Tris--HC1, pH 8.6, and centrifuged at 10 000 g for 10 min in a Sorvall RC2 centrifuge at 4 ° C. The pellet was washed twice with the same buffer. The supernatants were combined and centrifuged at 131 0 0 0 g for 60 min at 4°C (Beckman L5 ultracentrifuge, Spinco rotor SW 27). The 131 000 g pellet containing plasma membranes and endoplasmic reticulum was washed first with 0.25 M sucrose, 0.01 M Tris--HC1, pH 8.6, and then with 0.001 M Tris--HC1, pH 8.6. The pellet was suspended in 0.001 M Tris--HC1, pH 8.6, containing 0.001 M MgSO4, passed through a gauge No. 21 needle and dialyzed for 60 min at 4°C against the same buffer. The plasma membranes were isolated by using a Dextran 110 (Pharmacia) gradient (d --- 1.09--1.10) according to the method of Wallach and Kamat [18]. The membranes were washed once in 0.001 M Tris--HC1, pH 8.6, followed by PBS, pH 7.4. The membrane isolation described above was also carried o u t in' the presence of calcium, e.g. all buffers contained 0.5 • 10 -3 M Ca 2.. The membrane pellets were suspended in PBS, pH 7.4, containing 0.5 • 10 -3 M Ca 2÷ and stored at 4°C in an ice bucket. E n z y m e assays The following enzymes were assayed to determine the purity of the plasma membranes: (i) the plasma membrane marker enzymes phosphodiesterase [19] and 5'-nucleotidase [20,21]; (ii) glucose-6-phosphatase [22] and NADH oxidoreductase [18] for contamination by endoplasmic reticulum; (iii} succinic dehydrogenase [23] for mitochondrial contamination. These enzymes were routinely determined for the homogenate, the 10 000 g pellet, the 10 0 0 0 g supernatant, the soluble supernatant and the plasma membranes. Protein was measured according to the method of Lowry [24] using crystalline bovine serum albumin (Sigma, fraction No. 5) as standard. Fluorimetric studies The membrane pellet from 4--6 g rat liver was suspended in PBS, pH 7.4, containing 0.5 • 10 .3 M Ca 2., and dispersed by passing several times through a 21-gauge needle attached to a disposable plastic 10-ml syringe. Aliquots of this membrane suspension were diluted with cold PBS to the desired membrane concentration (t~g membrane protein per ml). Samples of the diluted membrane suspension were pipetted into plastic vials, followed by addition of the agent to be tested dissolved in PBS and incubated at 37°C for 30 min. For each membrane sample containing PMA or a PMA analog, a corresponding control membrane sample without added agent was also incubated. After incubation, the samples were cooled to room temperature for 5 min in a water bath, and transferred to 1 cm quartz cuvettes. Corrected fluorescence emission spectra in quantum units and corrected excitation spectra

236 in energy units were recoded on an automatically corrected luminescence s p e c t r o p h o t o m e t e r [25]. The m e m b r a n e samples were excited at 280 or 295 nm and 5 nm slits were used bot h on the exciter and analyzer m o n o c h r o mators. An ultraviolet polarizer (105 U.V., Polacoat, Inc.) was inserted on the exciter side in experiments with polarized excitation. In order to minimize the effects of ultraviolet irradiation on cell membranes only one spectr u m was r eco r de d per sample.

Determination o f concentration of PMA and analogs in PBS The PMA used in these studies was isolated by the procedure described by us earlier [26]. An excess of pure pow de red PMA was stirred in 500 ml PBS, pH 7.4, for 24 h at r o o m t e m p e r a t u r e and filtered. The c o n c e n t r a t i o n of PMA in PBS could n o t be det er m i ned directly by m e a s u r e m e n t of the ultraviolet absorption spectrum because of the low solubility and extinction coefficient of PMA. Th er ef or e, the saturated PMA-PBS solution was ext ract ed repeatedly with freshly distilled m e t h y l e n e chloride. The m e t h y l e n e chloride ext ract was dried with a nhydr ous MgSO4, filtered, and flash-evaporated. The residue was dissolved in absolute ethanol and absorption spectra recorded on an ultraviolet absorption s p e e t r o p h o t o m e t e r (Cary Model 14, Varian Associates). Comparison with a solution of PMA in ethanol of k n o w n concentration gave a value of 2.3 mg PMA/1 PBS (3.72 • 10 -6 M). This saturated PMAPBS solution was used in the m e m b r a n e experiments. 4aa-PDD was synthesized and fully characterized in this l aborat ory (B.L. Van Duuren and S.-S. Tseng, unpublished results). 5.8 mg 4aa-PDD was dissolved in anhydrous ether (2.9 ml) and an aliquot of this solution was added to 250 ml PBS, pH 7.4. This PBS solution was stirred for 16 h at r o o m t e m p e r a t u r e , and N2 was co n tin u o u s ly bubbled through in order to eliminate the ether. The 4aa-PDDPBS solution was extracted repeatedly with freshly distilled m e t h y l e n e chloride and the c o n c e n t r a t i o n of 4aa-PDD was det erm i ned as described above for PMA. Comparison with a solution of 4aa-PDD in m e t h y l e n e chloride of k n o w n c o n c e n t r a t i o n gave a value of 1.8 mg 4aa-PDD/1 PBS (2.67 • 10 -6 M). PHMA was identified as a metabolite of PMA and synthesized in this labo r a t o r y [16]. This c o m p o u n d is more polar than PMA since it contains a h y d r o x y l rather than a carbonyl group at C-5. PHMA was dissolved in PBS, pH 7.4, by stirring at r o o m t e m p e r a t u r e for 24 h at a c o n c e n t r a t i o n of 2 rag/1 (3.23 • 10 -6 M). RESULTS

Membrane purification The m e m b r a n e yield varied bet w e e n 4--6 mg m e m b r a n e protein per four g liver. The e n z y m e activities of purified plasma membranes prepared in calcium-containing buffers are shown in Table I. Phosphodiesterase and 5'nucleotidase, the plasma m e m b r a n e marker enzymes, were enriched 78- and 2.8-fold, respectively, in the m e m br a ne fraction com pared with the homogenate. The membranes were virtually free of mitochondria, as seen by the specific activity of 0.1 for succinic dehydrogenase for the membranes

237 TABLE I ENZYME ACTIVITIES OF PURIFIED RAT LIVER PLASMA MEMBRANES a E n z y m e assayed

Homogenate, specific activity

Plasma membranes, specific activity

Phosphodiesterase b 5'-Nucleotidase c Glucose-6-phosphatase d NADH oxidoreductase e Succinic dehydrogenase f

0.94 200 995 0.19 5.6

74 565 315 0.1 0.1

a b c d

Average of seven m e m b r a n e preparations. p m substrate h y d r o l y z e d / h / m g protein. /.tg Pi r e l e a s e d / m i n / m g protein. /~g Pi released/15 m i n / m g protein. e p m N A D H oxidized/rain/rag protein. f A A b s o r b a n c e / 3 0 m i n / m g protein.

compared with 5.6 for the homogenate. The plasma membranes did contain endoplasmic reticulum, as indicated by the activity of glucose-6-phosphatase and NADH oxidoreductase, marker enzymes for endoplasmic reticulum. The plasma membrane: homogenate ratio of the specific activity of glucose-6phosphatase obtained in the current membrane fractionation was 0.316, which compares favorably with ratios of 0.38 and 0.59 for the same enzyme obtained for purified liver cell plasma membranes isolated by Boyer and Reno [27] and Gavard et al. [28l, respectively. For membranes prepared in calcium-free buffers, the specific activity of the plasma membrane marker enzyme phosphodiesterase was lower, e.g. 40 (average of six membrane p.reparations) than for membranes isolated in the presence of calcium. The specific activities of 5'-nucleotidase and NADH oxidoreductase, as well as the membrane yield, remained the same when calcium was omitted from the membrane isolation procedure. The specific activity of glucose-6-phosphatase was about twice that obtained for membranes isolated with calcium.

Intrinsic fluorescence characteristics o f rat liver plasma m e m branes Rat liver plasma membranes suspended in PBS, pH 7.4, have an emission maximum at 330 nm and a shoulder at ~ 3 4 5 nm upon excitation at 280 nm. The emission m a x i m u m or shoulder did not shift when a different wavelength of excitation, e.g. 295 nm or 270 nm, was used. At membrane concentrations less than ~ 20 pg membrane protein/ml, a Raman scatter peak appeared at 310 nm and at still lower membrane concentrations, < 5 pg membrane protein/ml, a second Raman scatter peak appeared at ~ 3 4 7 nm when 280 nm was used as the excitation wavelength. The Raman scatter peak at 310 nm was also present in PBS, pH 7.4. The excitation spectrum showed maxima at 222 nm, 278 nm, and a shoulder at 260 nm. Fluorescence calibration curves in terms o f fluorescence intensity at 330 nm (h~ x 280 nm) vs. pg membrane protein/ml were linear, at least up to 125

238 pg m e m b r a n e protein/ml. Comparison of fluorescence calibration curves for different mem br ane batches stored for less than 8 h at 4°C showed t hat the slopes varied from batch to batch. Storage of rat liver plasma membranes at 4°C in PBS, pH 7.4, containing no divalent cations resulted in a progressive decrease of the native emission. After 6 days of storage at 4 ° C, the membranes had lost up to 40% of their original fluorescence. This fluorescence decay was most rapid for the first 3 days of storage and occurred regardless of w het her the membranes were isolated in Ca2÷-free or Ca2+-containing (0.5 • 10 -3 M) buffers. The emission spectra of membranes stored in PBS at 4°C up to 8 days did n o t show any abnormalities, e.g. red-shifted emission, but had the same general shape and emission characteristics as those of fresh membranes stored for less than 2 h. Ca 2÷ or Mg 2÷ (0.5 • 10 -3 M) in PBS considerably stabilized the isolated membranes as indicated by negligible or at most a 10% decay of the native fluorescence u p o n storage up to 3 days.

Effect of PMA on the native membrane fluorescence These experiments were carried out with rat liver plasma m em branes isolated in calcium-containing buffers and stored at 4°C in PBS, pH 7.4, containing 0.5 • 10 -3 M Ca ~'÷. Membranes were active for at least 3 days after isolation and gave reproducible results within that time period. The in vitro exposure o f rat liver plasma m e m br a ne s to PMA resulted in a decrease of the native m e m b r a n e fluorescence c o m p a r e d with control samples, i.e. membrane samples incubated w i t h o u t PMA. The percent decrease was concentrat i o n - d e p e n d e n t both with respect to PMA and membranes, as shown in Table II. At 1.7 pg PMA/ml, the pe r cent fluorescence decrease was 9, 16, and 6 at 3.7, 7.5 and 15.0 pg m e m b r a n e pr ot e i n / m l , respectively. Incubation with lower concentrations of PMA, 1.2 pg/ml, resulted in decreased quenching of the m e m b r a n e fluorescence, e.g. 11% at 7.5 pg m e m b r a n e prot ei n/ m l as opposed to a 16% decrease observed for 1.7 pg PMA/ml at this m e m b r a n e c o n c e n t r a t i o n . Thus, at a given m e m b r a n e c o n c e n t r a t i o n , the degree of fluorescence quenching was PMA c o n c e n t r a t i o n - d e p e n d e n t , and vice versa. Generally, all m e m b r a n e batches showed c o n c e n t r a t i o n - d e p e n d e n t fluorescence quenching bot h with respect to PMA and m em brane c o n c e n t r a t i o n . The m a x i m u m fluorescence quenching varied from 15--21% for different m e m b r a n e batches incubated with 1.7--2.0 pg PMA/ml and the optimal m e m b r a n e c o n c e n t r a t i o n varied between 7.5--22.0 pg m e m b r a n e prot ei n/ m l f o r memb r an e s from di f f e r ent batches, i.e. the effect o f PMA and/or membrane protein concentrations on quenching of fluorescence emission occurred only within certain concentration ranges. The latter s t a t e m e n t applies also to the data presented in Table III which is discussed below. In order to determine w he t he r the fluorescence quenching resulted from an aggregation or disaggregation of m e m branes in the presence of PMA, the intensity of the scatter peak at 280 nm, which coincides with the excitation wavelength, was routinely measured. The relative intensity of the 280 nm scatter peak for the PMA-treated m e m b r a n e samples was f o u n d to be the

239 TABLE II EFFECT OF PMA ON THE NATIVE FLUORESCENCE OF RAT LIVER PLASMA MEMBRANES Membranes were incubated with PMA and spectra recorded as described under MATERIALS AND METHODS. The results from three membrane preparations were pooled and the % fluorescence change was calculated from two to five separate measurements. The % fluorescence change at 330 nm (kex 280 nm) refers to the decrease of the membrane emission of the PMA-treated samples with respect to control membrane samples incubated without PMA. The final calcium concentration in all samples was 6.25 - 10 -5 M. Membrane concentration, pg protein/ml

PMA concentration, gg/ml

% Fluorescence change at 330 nm

3.7 3.7

1.7 (2.7 • 10 -6 M) 2.0 (3.2 • 10 -6 M)

-- 9 --10

7.5 7.5 7.5

1.2 1.7 2.0

--11 --16 --16

15.0 15.0

1.7 2.0

-- 6 -- 5

same as t h a t o f the m e m b r a n e c o n t r o l samples. In o r d e r t o r e d u c e the excitation wavelength scatter peak and in o r d e r t o ascertain t h a t the m e m b r a n e spectra were free o f artifacts [ 2 9 ] , a series o f emission spectra was r e c o r d e d using an ultraviolet polarizer {105 UV} o r i e n t e d h o r i z o n t a l l y on the excitation side. The f l u o r e s c e n c e characteristics o f t h e P M A - t r e a t e d m e m b r a n e samples and c o n t r o l s were identical t o t h o s e o b t a i n e d w i t h o u t the polarizer. The t y r o s i n e c o n t r i b u t i o n to the m e m b r a n e emission was e x a m i n e d bY r e c o r d i n g s p e c t r a using ~ x 2 8 0 n m and ~ex 2 9 5 n m , and s u b t r a c t i n g the emission spectra o b t a i n e d at }~ex 295 n m f r o m t h o s e o b t a i n e d at ~e~ 2 8 0 nm. In these e x p e r i m e n t s 7.5 pg m e m b r a n e p r o t e i n / m l was i n c u b a t e d with 2.0 pg P M A / m l and the spectra were r e c o r d e d with an ultraviolet polarizer (105 UV) o r i e n t e d h o r i z o n t a l l y on the e x c i t a t i o n side. The polarizer was used since it c o n s i d e r a b l y r e d u c e d the scatter at 2 8 0 n m and 347 n m w h e n the sample was excited at 2 8 0 n m , in a d d i t i o n to r e d u c i n g the c o r r e s p o n d i n g scatter peaks w h e n 295 n m was used as the e x c i t a t i o n wavelength. The s p e c t r a were c o r r e c t e d for the b a c k g r o u n d due to PBS and the transmission characteristics o f the polarizer and r e p l o t t e d in terms o f c o r r e c t e d emission intensity in q u a n t u m units vs. wavelength. A t ~¢x 2 8 0 rim, where b o t h the t y r o s i n e and t r y p t o p h a n residues o f m e m b r a n e p r o t e i n s absorb, PMA q u e n c h e d the native m e m b r a n e emission by 17% c o m p a r e d with the c o n t r o l . A 16% decrease was observed for the P M A - t r e a t e d sample c o m p a r e d with t h e c o n t r o l at ~.e× 295 n m w h e r e t r y p t o p h a n and n o t t y r o s i n e absorbs ultraviolet light. The calculated d i f f e r e n c e spectra indicated an 18% decrease in t h e native emission at 3 3 0 n m for t h e P M A - t r e a t e d m e m b r a n e sample c o m p a r e d with m e m b r a n e s i n c u b a t e d w i t h o u t PMA. Similar results were o b t a i n e d f r o m e x p e r i m e n t s with a d i f f e r e n t m e m b r a n e batch. These d a t a indicate t h a t PMA

240 quenches b o t h the t r y p t o p h a n and tyrosine emission of rat liver plasma m emb r an es to equal extents.

Effect o f PHMA and 4aa-PDD on the native membrane fluorescence Rat liver plasma m e m br a ne s isolated in calcium-containing buffers and stored in PBS, pH 7.4, in the presence of 0.5 • 10 -3 M Ca 2+, were incubated 1 for ~ h at 37°C with PHMA, a metabolite of PMA in mouse skin, and 4aaPDD. The latter c o m p o u n d , which was n o t fully characterized, did not result in t u m o r s in a short-term e x p e r i m e n t [17]. Additional m e m b r a n e aliquots with and w i t h o u t PMA were incubated for comparison. The results of these experiments are shown in Table III. The concent rat i ons used for these measurements were those t ha t resulted in optimal fluorescence changes and hence were n o t equimolar for the three c o m p o u n d s . With this in mind equimolar c o n c e n t r a t i o n s could n o t be used because of differences of solubility of these c o m p o u n d s . These results are from one m e m b r a n e preparation, and similar results were obtained from four ot her m e m b r a n e batches. Similarly to PMA, the metabolite PHMA and 4aa-PDD also decreased the native membrane fluorescence and the e x t e n t of fluorescence quenching was d e p e n d e n t b o t h on the m e m b r a n e c o n c e n t r a t i o n and the c o n c e n t r a t i o n of the agent added. At 7.5 pg m e m b r a n e protein/ml, PMA (3.2 • 10 -(~ M), PHMA (2.8 • 10 .6 M), and 4aa-PDD (2.3 • 10 -~ M), quenched the native m em brane fluorescence by

T A B L E III EFFECT OF PMA, PHMA AND 4a~-PDD ON THE NATIVE FLUORESCENCE LIVER PLASMA MEMBRANES M e m b r a n e s w e r e i n c u b a t e d w i t h t h e a g e n t t o h e t e s t e d a n d s p e c t r a r e c o r d e d as under MATERIALS AND METHODS. The % fluorescence change at 330 nm nm) refers to the decrease of the native emission of membranes treated with t e s t e d c o m p a r e d w i t h c o n t r o l m e m b r a n e s a m p l e s i n c u b a t e d in t h e a b s e n c e a g e n t . T h e f i n a l c a l c i u m c o n c e n t r a t i o n in all s a m p l e s w a s 6 . 2 5 . 10 -s M.

OF RAT described (he× 2 8 0 the agent of added

Compound tested

Concentration of compound tested pg/ml

% Fluorescence change at 330 nm

7.5 7.5 7.5

PMA PHMA 4aa-PDD

2 . 0 ( 3 . 2 • 10 .6 M ) 1.7 ( 2 . 8 . 10 -6 M ) 1 . 6 ( 2 . 3 - 10 .6 M)

--21 --10 --13

7.5 7.5 7.5

PMA PHMA 4aa-PDD

1.2 ( 1 . 9 • 10 .6 M ) 1 . 0 ( 1 . 6 . 10 .6 M ) 0 . 9 ( 1 . 3 - 10 -6 M )

--11 -- 6 -- 4

15.0 15.0 15.0

PMA PHMA 4aa-PDD

2.0 1.7 1.6

--21 --12 -- 6

22.5 22.5 22.5

PMA PHMA 4a(~-PDD

2.0 1.7 1.6

--21 --17 --14

Membrane concentration, /.tg p r o t e i n / m l

241 21, 10 and 13%, respectively, compared with the control. The spectra for these results are shown in Fig. 1. At the same membrane concentration (7.5 pg membrane protein/ml) but lower PMA (1.9 • 10 -6 M), PHMA (1.6 • 10 -6 M), and 4aa-PDD (1.3 • 10 -6 M) concentrations, the extent of fluorescence quenching decreased and was found to be 11, 6, and 4%, respectively. These data indicate that PMA quenches the native fluorescence by about twice as much compared with PHMA and 4a~-PDD at 7.5 /~g membrane protein/ml. At 15 pg membrane protein/ml, the decrease in native fluorescence caused by PMA was still two times that caused by PHMA or 4aa-PDD. At 22.5 pg membrane protein/ml, however, the quenching caused by PMA was not significantly different from that due to PHMA. At these membrane concentrations, 4aa-PDD still quenched the native membrane fluorescence significantly less than PMA. The data in Table IH indicate that binding of PMA to rat liver plasma membranes always results in a greater quenching of the native fluorescence compared with 4aa-PDD. Except at very low membrane concentrations (3.7 pg membrane/protein/ml), PMA quenches the native membrane fluorescence to a greater extent than PHMA or 4aa-PDD.

80 A

j~

~.~__

70

Membranes

+ 4 a ~ - PDD

Membranes

+ PHMA h~ ~ _ ~

+ PHMA

I A'~-~ -

+

.,v _ \ ,,~

~

60

PMA

_ + PMA

,j

r., == 5o

L 40

30

20

fO

I

I

275

325

I

I

I

375 Wavelength (nm)

l

I

425

275

I

I

325

I

I

375

I

l

425

Fig. 1. Fluorescence emission spectra of rat liver plasma m e m b r a n e s treated with PMA (2.0 pg/ml ), PHMA (1.7 pg/ml) and 4a0~-PDD ( 1.6 pg/ml), as described under MATERIALS A N D METHODS; e x c i t a t i o n at 2 8 0 nm. A, 7.5 pg m e m b r a n e protein/ml; B, 15.0 ktg m e m b r a n e protein/ml.

242

Effect o f PMA on the native fluorescence o f membranes isolated and stored in calcium-free buffers The effect of PMA on the fluorescence o f rat liver plasma membranes isolated and stored in calcium-free buffers varied with (i) the length o f storage o f the m e m b r a n e suspension at 4°C; (ii) the PMA-membrane concent rat i on ratio, and (iii) the particular m e m b r a n e batch. Generally, freshly prepared membranes used immediately after isolation exhibited a fluorescence enh a n c e m e n t at 330 nm o f 15--30% after incubation with PMA com pared with control m e m b r a n e samples incubated w i t h o u t PMA. The m e m b r a n e concentration for m a x i m u m fluorescence e n h a n c e m e n t varied from 10--22 pg membrane p r o t e i n / m l depending on the particular m em brane batch. A m a x i m u m e n h a n c e m e n t of the native emission at o p t i m u m m e m b r a n e concentrations was generally observed after incubation with 0.05 pg PMA/ml (8.1 • 10 -s M). Thus, fresh membranes isolated and stored in the absence of calcium respond to 40-fold lower PMA concent r a t i ons compared to membranes containing calcium. Incubation of fresh membranes with higher PMA concentrations, up to 2.26 pg PMA/ml, at optimal m e m b r a n e concent rat i ons did n o t significantly alter the e x t e n t of the fluorescence enhancement. After storage for 24 h at 4 ° C, the membranes generally exhibited up to a 20% decrease of the native emission when incubated with PMA. To observe a m a x i m u m fluorescence decrease after storage for 24 h at 4 ° C, the PMA concent rat i on usually had to be increased to 0.11 pg/ml (1.6 • 10 -~ M) or greater, using m e m b r a n e concentrations corresponding to o p t i m u m c o n c e n t r a t i o n for freshly isolated membranes. Membranes stored for 48 h at 4°C generally did not exhibit fluorescence changes when incubated with PMA com pared with control m e m b r a n e sam~)les incubated w i t h o u t PMA. DISCUSSION The experiments described in this r e p o r t indicate that the t u m o r p r o m o t e r PMA quenches the native fluorescence of rat liver plasma membranes by 15--20%. This response to PMA was consistently observed for membranes isolated and stored in calcium-containing buffers. Calcium-free rat liver plasma membranes, however, showed a t i m e-dependent variation in the direction of the fluorescence change caused by PMA. Fresh membranes exhibited up to a 30% increase in native fluorescence while membranes stored for one day or more showed up to 20% decrease in native emission after exposure to PMA. Aging of membranes has been report ed in some instances to affect biochemical m e m b r a n e properties. Bos and E m m e l o t [12] observed an increase in the specific activity of Mg~*-ATPase and a decrease for (Na÷--K~) ATPase u p o n aging of rat liver plasma membranes stored in d o u b l y distilled water or isotonic sucrose. The same workers also showed that the inhibition of (Na÷--K*)ATPase by PMA was markedly less for cold-stored membranes than that obtained with fresh membranes. Postel-Vinay et al. [ 15] , who showed that the interaction of bovine growth h o r m o n e with rat liver plasma membranes resulted in a quenching of

243 the native emission, noted that the direction of the change in fluorescence became reproducible when membranes were isolated in the presence of calcium (0.5 mM) and when magnesium (4 • 10 -4 M) was added to the membrane suspension. Inorganic cations, specifically divalent cations such as calcium and magnesium, are known to be a necessary requirement for the maintenance of structural membrane integrity. This was shown by Reynolds and Trayer [30] who demonstrated that 90% of the proteins from erythrocyte ghosts are soluble in aqueous media after removal of inorganic cations by chelating agents. In the present studies, calcium-free membranes exhibited a progressive decrease in native fluorescence upon cold-storage at 4°C which was not observed in the presence of calcium. Thus, the decay in native fluorescence upon storage and the variation of the PMA effect in terms of direction of the fluorescence change for calcium-free membranes is most likely related to the instability of these membranes in the absence of calcium. The decrease in the native emission at 330 nm caused by PMA was observed at 280 nm excitation, where both tryptophan and tyrosine absorb, and also at 295 nm excitation where only tryptophan absorbs. The relative degree of fluorescence quenching at 330 nm due to PMA was the same at 280 and 295 nm excitation. In addition, difference spectra indicated that the tyrosine contribution to the fluorescence of rat liver plasma membranes was quenched by PMA to an extent similar to that of tryptophan. These results indicate that the interaction of PMA with rat liver plasma membranes quenches both the t r y p t o p h a n and tyrosine emission which might be expected as a result of structural changes in the membrane. T h e present results are in contrast to those of Sonenberg on the interaction of human growth hormone with human erythrocyte membranes [31]. In the latter studies, a quenching was observed at 282 nm excitation and not at 295 nm excitation, indicating that only the tyrosine emission was quenched. In order to relate the fluorescence effects of PMA to biological activity, two closely related compounds, PHMA and 4a~-PDD, were studied. Reduction of PMA at the C-5 carbonyl to the corresponding alcohol yields PHMA, a metabolite of PMA on mouse skin [16]; this c o m p o u n d is a moderate t u m o r p r o m o t e r (B.L. Van Duuren, unpublished results). 4a~-PDD has decanoate side chains at C-9a and C-9 in addition to an inverted h y d r o x y group at C-4a. This compound has been found in the present work to be much less soluble in water than PMA and hence, is probably more lipophilic in nature. Examination of molecular models also shows that the 4a~-stereoisomer shows a marked difference in stereochemical conformation compared with PMA. 4a~-PDD may be inactive as a t u m o r promoter [17] although these results were inconclusive. The metabolite PHMA and the PMA analog 4a~-PDD both quenched the native fluorescence at 330 nm, but to a significantly lesser extent than PMA. The degree of fluorescence quenching varied with the concentration of each agent and the membrane concentration. At comparable PMA, PHMA and 4a~-PDD concentrations, the differences in fluorescence quenching caused by PMA compared with PHMA and 4a~-PDD were significant at intermediate membrane concentrations. At high mere-

244 brane concentrations 4a~-PDD quenched the native fluorescence to a lesser extent than PMA or PHMA. In general, 4a~-PDD quenched the native membrane fluorescence less than PHMA. The above results indicate that differences exist between the interaction of PMA with rat liver plasma membranes compared with PHMA or 4a~-PDD. The quenching of the native fluorescence could result from changes in membrane structure, as has been shown for other biologically active agents interacting with functionally active mammalian plasma membranes [ 15,32]. Many biological studies have been carried out concerning the mode of action of tumor promoters [1,321. The lipophilic-hydrophilic nature of PMA suggested that interactions with the plasma membrane may be involved in early events necessary for tumor promotion [1]. The experiments carried o u t to date do n o t pinpoint the nature of the PMA binding site. PMA may cause conformational changes by binding directly to membrane proteins, resulting in the observed decrease of the tryptophan and tyrosine emission of rat liver plasma membranes. Conversely, PMA may indirectly cause protein conformational changes by binding to and causing perturbations in the lipid bilayer, which in turn could affect membrane protein conformation. Since 4aa-PDD did not affect the native membrane fluorescence to an extent similar to that of PMA, it may be concluded that the t u m o r promoter binds to a greater extent or at different membrane receptor sites compared with 4aaPDD. Long-term two-stage carcinogenesis experiments are currently underway with 4ac~-PDD and related analogs. The nature and relevance of structural changes in PMA and their relationship to t u m o r promotion will require further exploration by a combination of biochemical and physical studies. Finally, we do not wish to imply from the findings reported in this paper that there is a direct correlation between the fluorescence changes described in this report and two-stage carcinogenesis on mouse skin. Rather, the major goal of the initial studies reported here are aimed at increasing our knowledge on possible modes of action of tumor-promoting agents. ACKNOWLEDGEMENT

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