212

Biochimica et Biophysica Acta, 538 (1978) 212--225 © Elsevier/North-Holland Biomedical Press

BBA 28410 E F F E C T OF H Y D R O L Y T I C ENZYMES AND PRO T E IN -MO D IFY IN G R E A G E N T S ON G O N A D O T R O P I N R E C E P T O R S IN BOVINE CORPUS LUTEUM CELL MEMBRANES

Ch.V. RAO Departments of Obstetrics-Gynecology and Biochemistry, University of Louisville, School of Medicine, Louisville, Ky. 40202 (U.S.A.)

(Received May 10th, 1977) Summary Preincubation of membranes with various concentrations of pronase, trypsin, lipase, phospholipase A from Vipera russelli and from Crotalus durissus terrificus, phospholipase C from Bacillus cereus and from Clostridium welchii, acetic anhydride, 2,4-dinitrofluorobenzene and t e t r a n i t r o m e t h a n e resulted in a dose-dependent inhibition of ~2SI-labeled hu m an c h o r i o g o n a d o t r o p i n binding. At the submaximal concentrations of enzymes and at bot h submaximal and maximal concentrations of protein-modifying reagents, the losses were always greater with ~2SI-labeled human c h o r i o g o n a d o t r o p i n than with 12SI-labeled h u m an lutropin. The inhibition of binding was a consequence of changes in the membranes rather than changes in the h o r m o n e caused by the agents being carried over to the final incubation. Inhibition of binding was non-competitive and irreversible. In u n tr eated membranes, the ~2SI-labeled human c h o r i o g o n a d o t r o p i n binding was h o m o g e n e o u s (K d = 1.7 • 10 -~° M; N = 60 fm ol / m g protein). T r e a t m e n t of membranes with various enzymes and protein-modifying reagents e x c e p t tetran i t r o m e t h a n e resulted in heterogeneous binding. The n u m b e r of available high affinity receptors was greatly reduced in every case. However, the affinity of these sites were either unchanged (trypsin, lipase, phospholipase A from V. russelli, d i n i t r o f l u o r o b e n z e n e and the t e t r a n i t r o m e t h a n e ) or decreased (pronase and acetic anhydride). The newly appeared second recept or site had a K d which varied from 3.2 • 10 -l° to 7.1 • 10 -9 M depending on the agent used, and the r e c e p t o r numbers were low in all cases e x c e p t acetic anhydride. R e c e p t o r o c c u p a n c y conferred the receptors with marked p r o t e c t i o n against various h y d r o l y t i c enzymes, d i n i t r o f l u o r o b e n z e n e and t et rani t rom et hane. These data suggest t ha t inhibition o f binding by the above agents was primarily a consequence o f changes in the r e c e p t o r molecules themselves.

213 Introduction Receptors which bind 12SI-labeled human choriogonadotropin and 12SI-labeled lutropin are present primarily, but not exclusively, in the outer cell membranes of bovine corpora lutea (refs. 1--3, and unpublished). However, human lutropin appears to bind to these receptors with a lower affinity compared to human choriogonadotropin. These receptors in bovine corpus luteum [ 4--8] and in luteal tissue from other species (reviewed in ref. 9) were characterized by the use of various hydrolytic enzymes and/or protein-modifying reagents. In very few or none of these studies, however, were experiments directed toward establishing dose vs. response relationships with various enzymes and protein-modifying reagents, the influence of using 12SI-labeled human choriogonadotropin or ~2SI-labeled lutropin to assess the binding losses, mechanism of inhibition of binding (decreased receptor affinity or number) or the effect of receptor occupancy. In a traditional *approach to assess the macromolecular nature of membrane gonadotropin receptors the possibility exists that the inhibition of binding due to enzymes and protein modifying reagents could reflect changes in the receptors themselves and/or a consequence of changes in other membrane proteins whose structural and functional integrity secondarily influence receptor recognition phenomena. We have undertaken these studies to resolve the abovementioned issues in probing the macromolecular nature of membrane gonadotropin receptors by the use of various enzymes and protein-modifying reagents. Experimental procedure Materials. Unlabeled human choriogonadotropin ( C R l l 9 ; 11 600 I.U./mg) was a gift from Center for Population Research, National Institute for Child Health and Human Development, National Institutes of Health. Unlabeled human lutropin (LER-960; 4620 I.U./mg) was generously donated by Dr. Leo Reichert of Emory University. The following items were purchased from indicated commercial sources: carrier-free Nal2SI from New England Nuclear Inc., or Amersham/Searle; Phospholipase A from Vipera russelli (10.7 units/mg protein), and Crotalus durissus terrificus (230 units/mg protein), phospholipase C from Clostridium welchii (6 units/mg solid) and tetranitromethane from Sigma Chemical Co.; lipase from porcine pancreas (270 units/mg protein), lactoperoxidase (20.3 I.U./mg at 30°C), phospholipase C from Bacillus cereus (145 units/mg protein), and pronase (89 000 P.U.K./g at 40°C) from Calbiochem; soybean trypsin inhibitor and trypsin (.twice crystalized, 189 units/mg) from Worthington Biochemical Corp.; 2,4-dinitrofluorobenzene from Nutritional Biochemical Corp. and Gelman Metricel filters of pore size 0.45 pm from Scientific Products Co. Methods. Unlabeled human choriogonadotropin and human lutropin were iodinated by t h e lactoperoxidase technique essentially as described by Miyachi et al. [10]. The specific activities were determined by the trichloroacetic acid precipitation of crude reaction mixture prior to gel filtration. In our experience, the specific activities calculated by the trichloroacetic acid precipitation method and by radioreceptor assay using bovine corpus luteum cell membranes, are in excellent agreement. Specific activities varied from 36.8 to 88.6

214

pCi/pg for '25I-labeled human choriogonadotropin and 47.7 to 83.0 pCi/pg for '2SI-labeled lutropin on different occasions. Labeled gonadotropins were separated from unreacted free Na '2sI by gel filtration on a Sephadex G-100 (1 × 30 cm) column using physiological saline containing 1% bovine serum albumin as the eluant. The biological activities of human choriogonadotropin and human lutropin iodinated by the lactoperoxidase technique have been shown to be fully retained [10]. Each of the eluted fractions were checked for binding to bovine corpus luteum cell membranes. The fractions that exhibited the best binding, usually those around the peak portion of the first radioactivity peak, were pooled for further use. The pooled fraction was diluted in saline/bovine serum albumin solution, frozen and stored in aliquots at --20°C until used. The pooled fraction showed binding up to 72.7 and 36.5% of added (1 • 10 -'° M) '2SI-labeled human choriogonadotropin and ':SI-labeled h u m a n lutropin respectively, in the presence of excess membranes. The procedures for the collection of bovine corpora lutea and isolation of cell membrane fractions were the same as described earlier [11]. Cell membrane fractions were stored frozen in aliquots at --20°C. The protein content in an aliquot of the membranes was determined according to Lowry et al. [12], using bovine serum albumin as the standard. The binding studies were conducted at 38°C for 2 h (unless specified otherwise) with 200--250 pg membrane protein and approx. 1 • 10 -'o M 12"~I-labeled human choriogonadotropin and '2SI-labeled human lutropin. We have previously shown that '2SI-labeled human choriogonadotropin binding reaches equilibrium under these incubation conditions [13]. Procedures for the separation of bound and free hormones were the same as described earlier [13] except that the Metricel filters were soaked in 2% bovine serum albumin in saline solution prior to their use. Non-specific binding was determined in each experiment using the same a m o u n t of membrane protein and '2SI-labeled gonadotropin as were used in total binding tubes but in the presence of 1.3 • 10-TM unlabeled human choriogonadotropin. We could not use unlabeled human lutropin to assess '2SI-labeled human lutropin non-specific binding because of lack of sufficient hormone. Furthermore, use of unlabeled human choriogonadotropin for this purpose appears to be valid primarily because '2SI-labeled human lutropin, at the concentration (approx. 1 • 10 -'° M) used in these experiments, appears to bind to the same sites as '2SI-labeled human choriogonadotropin [ 14 ]. Nonspecific binding for both '2~I-labeled human choriogonadotropin and '2SI-labeled human lutropin was essentially the same as binding to the filters in the absence of membrane fractions (blanks) suggesting that it was quite insignificant in these preparations. However, the non-specific binding as expressed as percentage of total cpm added was about 1% for '2SI-labeled human choriogonadotropin and 5--7% for '2SI-labeled human lutropin. The higher non-specific binding with '2SI-labeled human lutropin appears to reflect filter binding of '2SI-labeled human lutropin. Specific binding, which was obtained by subtracting non-specific binding from total binding, was presented in all the tables and figures. Fixed amounts of untreated and treated membrane protein (see Figs. 2A and 2B) were incubated with increasing concentrations of '2SI-labeled human choriogonadotropin. The resulting specific binding data were analysed accord-

215 ing to Scatchard [15]. When a single line described the binding function, the data were simply subjected to least squares curve fit analysis by the computer in order to determine the slope and intercept. When the Scatchard plots were curvilinear, the last points (varied from 3 to 6 in different experiments) were used to obtain the line representing a least squares curve fit. The line for the first data points (varied from 3 to 7) was then determined by least squares curve fit after subtracting the a m o u n t of binding attributable to line with the least slope. The slopes and intercepts were obtained from the above analysis by the computer. The apparent dissociation constants (Kd) were calculated from the reciprocal of the slopes. The x-axis intercepts of the lines with least and highest slope, gave the numbers for total and high affinity receptors, respectively. The difference between total and high affinity receptors gave the value for the number of low affinity receptors. The graphic display of the data was done b e f o r e c o m p u t e r calculations, therefore apparent slopes and intercepts of some of the data do not correspond to computer calculated values. This was done to avoid confusion from having too m a n y lines in graphs (see Figs. 2A and 2B). All the enzyme solutions were made up and diluted in 10 mM Tris. HC1 buffer of pH 7.0. In the case of phospholipases, 1 mM Ca 2+ was included in this buffer. Phospholipases A and C were heated for 5 min at 80°C and subsequently centrifuged at 6000 X g for 15 min to remove any precipitate that might have been formed during heating. The clear supernatants containing phospholipases were saved and used in the experiments. Protein-modifying reagents were diluted and added in ethanol so that the final ethanol concentration was 9--10% (v/v). The membranes were treated with various enzymes and protein-modifying reagents'in one of the following ways: (a) Membranes were preincubated with enzymes (30 min at 38°C) and protein modifying reagents (60 min at 22°C). The tubes were then centrifuged for 15 min at 6000 X g, and the supernatants were aspirated. The membrane pellets were washed twice (6000 X g for 15 min) with 1.0 ml of 10 mM Tris. HC1 buffer of pH 7.0. The washed pellets were resuspended to original volume with homogenizing buffer (10 mM Tris" HC1 buffer of pH 7.0 containing 250 mM sucrose, 1 mM Ca 2+, 1 mM dithiothreitol and 0.1% gelatin) and aliquots were then incubated with 12SI-labeled gonadotropin under the conditions described before in this section (referred to hereafter as preincubation with enzyme and protein-modifying reagents). (b) Membranes were preincubated with '2SI-labeled h u m a n choriogonadotropin to reach an equilibrium and then various enzymes and protein-modifying reagents were added and incubation continued for 30 min at 38°C (enzymes) or for 60 min at 22°C (protein-modifying reagents) (referred to hereafter as final incubation with enzymes and protein-modifying reagents). In both pre- and final incubations with enzymes and protein-modifying reagents, the membranes for controls were handled similar to treatment tubes in every respect except for the addition of enzymes and protein-modifying reagents. In these tubes, however, volumes were made up with Tris buffer (enzymes) or ethanol (protein-modifying reagents). The binding in control tubes was expressed as fmol/mg membrane protein using 40 000 and 30 000 as molecular weights for human choriogonadotropin and h u m a n lutropin, respec-

216 tively. The percentage inhibition of binding in treatment tubes was calculated assuming 0% loss of binding in control tubes. Wherever data on comparative binding losses for h u m a n choriogonadotropin and human lutropin due to enzymes and protein-modifying reagents were presented, these data were obtained simultaneously on the same membrane fractions in order to avoid variations due to doing experiments at different times, on different membrane fractions and with different batches of enzymes and protein-modifying reagents. In preincubation experiments with enzymes and protein-modifying reagents, centrifugation and washing steps were included to remove enzymes and protein-modifying reagents from the membranes as much as possible prior to incubations with ~2SI-labeled gonadotropins. The efficacy of the above experimental protocol in accomplishing this goal will be evident later in the manuscript (see Table II). Results

Dose dependency o f enzymes and protein-modifying reagents inhibition o/ ~2SI-labeled human choriogonadotropin binding. Figs. 1A--1C show that preincubation of membranes with increasing concentrations of pronase, trypsin, lipase, phospholipase A from V. russelli, and Cr. durissus terrificus, phospholipase C from B. cereus and C. welchii, acetic anhydride, dinitrofluorobenzene and tetranitromethane resulted in a dose-dependent inhibition of ~2SI-labeled human choriogonadotropin binding to the membranes. The complete or near complete inhibition of binding was obtained with every enzyme (except phospholipases C) and protein-modifying reagent used. The order of potency of enzymes in inhibiting binding was: Pronase > trypsin > lipase; phospholipase A from V. russelli ~ and from Cr. durissus terrificus ~ phospholipase C from B. cereus ~ and from C. welchii. Unlike enzymes, protein-modifying reagents at the same concentrations did not differ (except possibly 1 mM tetranitromethane) in inhibiting ~2SI-labeled human choriogonadotropin. Comparative losses o f ~2SI-labeled human choriogonadotropin and ~2sIlabeled human lutropin binding due to various e n z y m e s and protein-modifying reagents. When ~2SI-labeled human choriogonadotropin and ~2SI-labeled human lutropin were used to measure the binding losses due to preincubation of membranes with submaximal concentrations of various enzymes and protein-modifying reagents, the losses were always greater with the former than with the latter ~2SI-labeled gonadotropin (Table I). The binding losses as measured by ~2sIlabeled human lutropin were only 56.4--88.4% of the losses as assessed by l~SI-labeled human choriogonadotropin. When the maximal concentrations of enzymes were used, the binding losses were nearly complete regardless of ~2sIlabeled gonadotropins used (Table I). However, with maximal protein-modifying reagent concentrations, differential losses were still maintained. Differential 12SI-labeled human choriogonadotropin and 12SI-labeled human lutropin binding losses due to enzymes and protein-modifying reagents could be an artifact due to carry over of these agents from pre- to final incubation resulting in selective modification of one hormone over the other. This possibility was explored with trypsin and tetranitromethane (Table II). In the

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Fig. 1. (A--C) Effect of preincubating m e m b r a n e s with various e nz yme s and prot e i n-modi fyi ng reagents on 125 I-labeled h u m a n c h o r i o g o n a d o t r o p i n (12 S I-hCG) binding. A l i quot s of m e m b r a n e fractions were preincubated with increasing c o n c e n t r a t i o n s of enzymes and prot e i n-modi fyi ng reagents, and t he n tested for binding with 125 I-labeled h u m a n choriogonadotropin. The binding m c ont rol tubes varied from 16.5 to 20.1 fmo l/mg protein. Each value represents the mean and its standard error of twelve observations in three experiments.

event t h a t trypsin carried over to final incubation, the addition of excess soybean trypsin inhibitor during final incubation should have resulted in m uch lower binding losses. T he results in Table II show t hat regardless of the addition o f soybean trypsin inhibitor, the binding losses were similar. If tetranitro-

218 TABLE

I

EFFECT OF PREINCUBATING MEMBRANES WITH VARIOUS ENZYMES ING REAGENTS ON 125I-LABELED GONADOTROPIN BINDING

AND PROTEIN-MODIFY-

Membrane fractions were preincubated with various enzymes and protein-modifying reagents. Aliquots of the treated membranes were assayed for 125Iqabeled human choriogonadotropin and 125I-labeled human lutropin binding simultaneously ( s e e M e t h o d s ) . In t h e c a s e o f t r y p s i n , p r o n a s e a n d l i p a s e , t h e a m o u n t s added were expressed as mg enzyme per mg membrane protein. For phospholipases this was in units per mg membrane protein. The above enzymes and protein-modifying reagents had essentially no effect on non-specific binding, therefore binding decreases reflect decreases in specific binding. The binding in control tubes was 16.3--20.6 fmol/mg protein for 1251-1abeled human choriogonadotropin and 8.0--10.1 f m o l / m g p r o t e i n f o r ! 25 I - l a b e l e d h u m a n l u t r o p i n . E a c h o b s e r v a t i o n r e p r e s e n t s t h e m e a n a n d i t s s t a n d a r d error of twelve determinations in three experiments. Percent inhibition

Addition

of binding

125 I-labeled human choriogonadotropin

Trypsin 1.0.10 4.0-10

-3 -2

58.3±1.6 97.1±1.0

Pronase 1.0 • 10 -3 3.5 " 10 -3 Lipasc 1.0.10 1.0.10

81.8 ± 1.5 97.2 ± 1.2

-2 -1

PhospholipaseAfrom 1.0-10 -3 4 . 0 . 1 0 -1

44.4±3.6 100.0±2.0

I 25 I - l a b e l e d h u m a n

lutropin

33.7±2.4 100.0±1.5

53.2 + 0.9 97.5 ± 1.3

26.6± 100.0±

1.9 1.0

V. russelli 61.1±1.2 100.0±0.9

39.4±4.1 98.6±1.1

67.0±2.3 94.3±2.3

39.4±4.1 89.1±2.5

91.4±0.7

80.8±1.0

Aceticanhydride lmM 10mM

74.0±1.6 94.1±0.9

41.7±3.0 76.3±3.2

Dinitrofluorobenzenc lmM 10mM

71.7±5.9 94.3±1.0

44.2±3.5 70.2±3.1

Tetranitromethane 1 mM

89.9 + 1.9

62.0 ± 3.1

PhospholipaseAfromCr. 1.0.10 -I 7.5 PhospholipaseC fromB, 1

durissuste~ificus

cereus

m e t h a n e w a s carried over t o t h e final i n c u b a t i o n , t h e n r e i n c u b a t i o n o f fresh membrane with hormone recovered from tetranitromethane-treated membrane t u b e s s h o u l d have e x h i b i t e d p o o r binding. T h e results in Table II s h o w that there was n o d i f f e r e n c e in binding ability b e t w e e n u n i n c u b a t e d h o r m o n e s or h o r m o n e s that w e r e p r e v i o u s l y i n c u b a t e d w i t h t e t r a n i t r o m e t h a n e - t r e a t e d m e m branes. T h e significant feature o f these results w a s that t h e d i f f e r e n c e s in I25Ilabeled h u m a n c h o r i o g o n a d o t r o p i n and I2SI-labeled h u m a n l u t r o p i n binding

219 T A B L E II LACK OF CARRY OVER OF TRYPSIN FINAL INCUBATION

AND TETRANITROMETHANE

FROM PREINCUBATION

TO

Membrane fractions were preineubated w i t h t r y p s i n (1 • 1 0 - 4 m g / m g m e m b r a n e p r o t e i n ) . D u r i n g f i n a l i n c u b a t i o n , s o y b e a n t r y p s i n i n h i b i t o r (~[ . 1 0 - 4 m g / m g m e m b r a n e p r o t e i n ) w a s a d d e d t o h a l f o f t h e t u b e s a n d t o t h e o t h e r h a l f a n e q u i v a l e n t v o l u m e o f b u f f e r p r i o r t o i n c u b a t i o n w i t h 12 S I - l a b e l e d g o n a d o t r o p i n . Membrane fractions w e r e also p r e i n c u b a t e d with tetranitromethane and then tested for binding with 12SI-labeled gonadotropin. At the end of the second incubation, bound and free hormones were separated by eentrifugation ( 6 0 0 0 X g, 15 m i n ) and free hormone (0.5 • 10 - 1 0 M) was then rcincubated with fresh membranes f o r 2 h at 3 8 ~ C a l o n g w i t h t u b e s t h a t r e c e i v e d a n e q u i v a l e n t c o n c e n t r a t i o n o f u n i n cubated hormone. E a c h v a l u e r e p r e s e n t s t h e m e a n a n d its s t a n d a r d e r r o r o f e i g h t o b s e r v a t i o n s in t w o experiments. 12 S I - l a b e l e d human choriogonadotropin

Description

(Percent Trypsin-pretreated

membranes

only

Trypsin-pretreated membranes + soybean trypsin inhibitor addition during final incubation

Incubation of fresh membranes ously unineubated hormone

with previ-

Incubation of fresh membrane with hormone that was previously incubated with membranes pretreated with 1 mM tetranitromethane

125 I - l a b e l e d human lutropin

inhibition of binding)

24.0±0.6

4.2±1.6

21.4±2.5

6.2±0.5

(Percentofaddedhormonebound) 14.8±0.6

13.7±0.9

10.9±0.2

9.5±0.8

losses were not narrowed by the above approaches. Although the above experiment was not run on every enzyme (it is not even necessary for enzymes such as lipase and phospholipases) and protein-modifying reagent used, it appears quite safe to suggest that differential 12SI-labeled human choriogonadotropin a n d 125I-labeled human lutropin binding losses due to enzymes and proteinmodifying reagents were real. Furthermore this data also points out that the centrifugation and washing procedures employed either completely removed or at least reduced them to a level such that these enzymes and protein-modifying reagents could no longer destroy or modify the hormone. Scatchard analysis o f 12SI-labeled human choriogonadotropin binding to untreated and treated membranes. The data in Figs. 2A and 2B show that 12sIlabeled human choriogonadotropin binding to untreated membranes was homogeneous. In all the membranes that were preincubated with enzymes and protein-modifying reagents, except tetranitromethane, the binding became heterogeneous. The number of available receptors and the apparent Kd values were calculated from the Scatchard plots and presented in Table III. This table shows that the number of available high affinity receptors with all the above agents were greatly reduced. However, the K d of these sites were either unchanged (trypsin, lipase, phospholipase A from V. russelli, dinitrofluorobenzene and tetranitromethane) or increased (pronase and acetic anhydride). The newly appeared second set of receptor sites had a K d which varied from 3.2 • 10 -l° to

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125I-hCG BOUND, pM Fig. 2. (A,B) Seatchard analysis o f 125 I-labeled human choriogonadotropin (125 I-hCG) binding to contro] membranes and to membranes preincubated w i t h various enzymes and p r o t e i n - m o d i f y i n g reagents. The incubations w i t h 125 I-labeled human e h o r i o g o n a d o t r o p i n were conducted f o r 2 h at 22°C. The fo|]owing are the amounts o f enzymes used: Pronase and trypsin, I - 10 -3 rag/rag membrane protein; ]ipase, 5 • 10 -2 rag/rag membrane protein; phosphoIipase A f r o m V. russelll, 1 • 10 -3 units/rag membrane protein. Each data p o i n t represents the mean and its standard error o f f o u r observations in one experiment.

7.1 • 10 -9 M d e p e n d i n g o n t h e agent. T h e n u m b e r o f t h e s e n e w l y appeared sites w a s l o w in all cases e x c e p t for acetic a n h y d r i d e . T h e n e w l o w a f f i n i t y receptors c o u l d p o s s i b l y be t h e m o d i f i e d pre-existing sites. In order t o avoid congest i o n in inset o f Fig. 2 A , t h e data for p h o s p h o l i p a s e A f r o m Cr. durissus terrifu-

221

T A B L E III THE APPARENT T O R S (N) F O R

DISSOCIATION 125I-LABELED

BATED WITH VARIOUS

CONSTANTS (Kd) AND THE NUMBER OF AVAILABLE RECEPHUMAN CHORIOGONADOTROPIN IN MEMBRANES PREINCU-

ENZYMES

AND PROTEIN-MODIFYING

REAGENTS

These values were calculated from the Scatchard plots presented in Figs. 3A and 3B; therefore, the experim e n t a l d e t a i l s are t h e s a m e as t h o s e i n F i g s . 3 A a n d 3 B l e g e n d s . Membranes preincubated

with

Kdl

(M)

Kd2(M)

N1

N2

(fmol/mg protein) None * Pronasc Trypsin Lipase P h o s p h o l i p a s e A f r o m V. russelli N o n e ** Acetic anhydride Dinitrofluorobenzene Tetranitromethane

1.1 • 6.3 • 1.2" 1.0" 1.5 " 1.7 • 3.3 • 1.9 • 1.0 •

10 -10 10 -10 10 -10 10 -10 10 -10 10 -10 10 -10 10 -10 10 -l 0

-1.9 • 1 0 - 9 3.8" 10 -10 6.3" 10 -10 1.8 - 10 -9 -7.1 • 1 0 - 9 3.2 - 10 -10 _

45.0 33.0 13.5 6.9 4.7 28.5 3.8 4.2 8.8

-3.0 16.5 7.1 8.3 -44.2 10.3 --

S e r v e d as c o n t r o l s f o r e n z y m e a d d i t i o n s , t h e r e f o r e r e c e i v e d e q u i v a l e n t v o l u m e o f T r i s b u f f e r during preincubation. ** S e r v e d as c o n t r o l s f o r p r o t e i n - m o d i f y i n g r e a g e n t a d d i t i o n s , t h e r e f o r e r e c e i v e d e q u i v a l e n t v o l u m e o f ethanol during preincubation. *

cus and phospholipase C from B. cereus was not shown. However, these data were very similar to those presented for phospholipase A from V. russelli. Effect o f Ca 2÷ on protein-modifying reagents inhibition of 12SI-labeled human choriogonadotropin binding. Addition of Ca 2÷ 15 min before proteinmodifying reagents addition during preincubation had no effect on tetranitromethane inhibition of binding but binding losses by acetic anhydride and dinitrofluorobenzene were further increased (Table IV). These increased binding losses may reflect Ca2+-induced conformational changes in membrane proteins resulting in exposure of previously unexposed modifiable protein functional groups. The lack of such an effect with tetranitromethane may suggest that tyrosyl groups, for which this reagent is extremely specific [16]. were all avail-

TABLE IV EFFECT

O F C A 2+ O N P R O T E I N - M O D I F Y I N G

CHORIOGONADOTROPIN

REAGENTS

INHIBITION

OF 125I.LABELED

HUMAN

BINDING

M e m b r a n e f r a c t i o n s w e r e p r e i n c u b a t e d w i t h a n d w i t h o u t a d d e d C a 2+ f o r 1 5 m i n a t 2 2 ° C . P r o t e i n - m o d i f y ing reagents were then added and preincubation was continued for another 1 h at the same temperature. F o l l o w i n g t h e p r e i n e u b a t i o n , m e m b r a n e s w e r e t e s t e d f o r b i n d i n g w i t h l 25 I - l a b e l e d h u m a n c h o r i o g o n a d o tropin. The binding in control tubes was about 16.9 fmol/mg protein. Each value represents the mean and its standard error of twelve observations in three experiments. Protein reagent added (mM)

Acetic anhydride, 0.9 D i n i t r o f l u o r o b e n z e n e , 0.9 T e t r a n i t r o m e t h a n e , 0.9

Percent inhibition of 125I-labeled human choriogonadotropin - - C a 2+

+ C a 2+, 1 0 0 m M

5 8 . 8 t 1.0 32.1 t 3.2 8 7 . 0 -+ 1 . 0

6 9 . 1 +- 3.1 6 0 . 0 +- 2 . 0 8 8 . 3 +- 1 . 3

binding

222 TABLE V EFFECT OF ENZYMES AND PROTEIN-MODIFYING REAGENTS ADDITION DURING PRE- AND FINAL INCUBATION, ON INHIBITION OF 12SI-LABELED HUMAN CHORIOGONADOTROPIN BINDING V a r i o u s a d d i t i o n s w e r e m a d e d u r i n g p r e - o r f i n a l i n c u b a t i o n (see M e t h o d s ) . T h e e n z y m e s a d d e d w e r e p e r mg membrane protein. Appropriate controls were run of both pre- and final incubation and the binding in these t u b e s varied f r o m 13.2 to 19 f m o l / m g m e m b r a n e p r o t e i n . T h e -- sign i n d i c a t e s t h e i n h i b i t i o n , whereas the + sign indicates increase of the binding with respect to corresponding controls. Each value represents the mean and standard error of twelve observations in three experiments. Addition

Pronase, 4 • 10 -2 m g T r y p s i n , 4 - 1 0 -1 m g L i p a s e , 1 • 1 0 -1 m g P h o s p h o l i p a s e A f r o m V. russelli, 1 - 1 0 - 1 u n i t s P h o s p h o l i p a s e A f r o m Cr. d u r i s s u s t e r r i f i c u s 2 u n i t s P h o s p h o l i p a s e C f r o m B. c e r e u s , 1 0 u n i t s Acetic anhydride, 9.1 mM D i n i t r o f l u o r o b e n z e n e , 9.1 m M Tetranitromethane, 9.1 mM

P e r c e n t c h a n g e o f 125 I - l a b e l e d h u m a n choriogonadotropin binding Preincubation

Final incubation

--100.0 --100.0 --100.0 --100.0 --100.0 --75.0 --94.0 --96.0 --88.0

--57.2 --32.6 --26.9 --36.6 +8.7 --2.0 --100.0 +29.2 --23.2

_+ 4 . 0 _+ 1 . 0 + 1.0 + 2.0

+ 2.4 -+ 4 . 4 + 3.6 + 1.0 ± 1.0 +- 1 . 0 + 4.6 + 2.9

able for nitrosylation regardless of conformational changes. Di- and monovalent cations induced conformational changes resulting in alterations in protein-modifying reagents inhibition of binding was reported earlier for prostaglandin F2~ and opiate receptors in their appropriate target tissues [17,18]. Effect o f receptor occupancy on enzyme and protein-modifying reagents inhibition o f 12SI-labeled human choriogonadotropin binding. The concentrations of enzymes and protein-modifying reagents that drastically inhibited 12sIlabeled human choriogonadotropin binding when added during preincubation, inhibited binding only a little to moderate extent when added during final incubation (Table V). The exception to the above findings was acetic anhydride. Regardless of acetic anhydride addition either during pre- or final incubation, the binding losses were complete. This observation may indicate that binding losses may reflect changes primarily in other membrane macromolecules and/or that receptor occupancy had no protective effect on receptor and/or hormone from the acetylation (see Discussion). Addition of dinitrofluorobenzene or phospholipase A from Cr. durissus terrificus during preincubation resulted in a drastic inhibition of binding, while on the other hand, when they were added during final incubation there was a small to moderate increase in binding. The receptor occupancy could still be considered to have a protective effect against these agents despite binding increases. Discussion Preincubation of membranes with increasing concentrations of enzymes and protein-modifying reagents resulted in a dose-dependent inhibition of 12sIlabeled human choriogonadotropin binding. This inhibition of binding was a consequence of changes in the membranes rather than changes in the hormone.

223 The loss of binding was non-competitive and irreversible because the addition of increasing amounts of 125I-labeled human choriogonadotropin during final incubation did not overcome the inhibition (see Figs. 2A and 2B) and removal of these agents by centrifugation and washing prior to incubation with ~2sIlabeled gonadotropin did not restore the binding activity. The rational interpretation of differing enzyme potencies in inhibiting 12SI-labeled human choriogonadotropin binding was not possible because of the differences in their intrinsic specificities. However, the data should clearly indicate that quantitative results within a group of enzymes, for example proteolytic enzymes and phospholipases A and C, vary not only with the concentration but also which of the enzymes was selected for use. Our data on phospholipases A (from either V. russelli or Cr. durissus terrificus) being more effective than phospholipases C (from either B. cereus or C. welchii) in inhibiting binding was at variance with the findings of Azhar and Menon [7]. These authors showed that phospholipase C was more effective than phospholipase A. The reasons for the above discrepency are n o t known at the present time. Proteolytic breakdown of membrane proteins was perhaps responsible for pronase and trypsin inhibition of binding. Removal of membrane lipids was most likely responsible for lipase inhibition of binding. Azhar et al. [8] have elegantly demonstrated that phospholipase A inhibition of binding was due to membrane-associated lysophosphatides, whereas phospholipase C inhibition of binding was attributable to true release of phospholipids from the membranes. Therefore inhibition of binding by phospholipases A and C observed in these studies could be attributable to the above-described phenomena. Scatchard analysis of 12SI-labeled h u m a n choriogonadotropin binding to untreated and treated membranes suggest that both decreased receptor number and affinity, depending on the agent used, were responsible for decreased gonadotropin binding. The observations on differential losses of 12SI-labeled human choriogonadotr0pin and 125I-labeled human lutropin binding, due to preincubation of membranes with submaximal concentrations of enzyme and protein-modifying reagents and maximal concentration of protein-modifying reagents, are difficult to reconcile if human choriogonadotropin and human lutropin bind with identical affinity to the same receptors. Recent studies from our laboratory in fact demonstrate that although human choriogonadotropin and human lutropin share c o m m o n receptor sites, human lutropin binds to them with lower affinity (ref. 14, manuscript in preparation). In addition, human lutropin binds to sites to which h u m a n choriogonadotropin has no affinity. Therefore, in view of the above information, as well as Scatchard analysis of 12SI-labeled human choriogonadotropin binding to untreated and treated membranes, it could be suggested that differential human choriogonadotropin and human lutropin binding losses were due to an increase in the K d of high affinity sites and/or to the appearance of new low affinity receptors to which human lutropin but not human choriogonadotropin can more readily bind. Differential losses in tetranitromethane-treated membranes (where there was no appearance of new low affinity receptors) and the membranes treated with maximal concentrations of all three protein-modifying reagents may suggest that protein func-

224 tional groups modification was relatively more detrimental to human choriogonadotropin-receptor compared to human lutropin-receptor interaction. Drastic decrease in the numbers of all available receptors and/or marked decrease in receptor affinities could explain 100% losses of binding, regardless of ~2sIlabeled gonadotropin used, in membranes preincubated with maximal concentrations of enzymes. Among the few studies that examined enzyme and protein-modifying reagent effects on gonadotropin receptors in bovine corpus luteum cell membranes [4--8], only one study investigated whether phospholipase A and C inhibition of binding was due to decrease in receptor number or affinity [7]. Their findings i.e. decreased receptor number but not affinity was in agreement with our data only in so far as high affinity receptors were concerned. The above discussion quite clearly suggests that not only the type and a m o u n t of agents used but also which of the 125I-labeled gonadotropins selected for use to follow the losses, could influence the results of enzyme and proteinmodifying reagents effects on membrane gonadotropin receptors. While there is a lot of data available in the literature on the enzyme effects (reviewed in ref. 9), there are only three studies to date, on protein-modifying reagents effects on membrane gonadotropin receptors [4,19,20]. All three of these studies agree on the lack of the need for intact SH groups in binding interaction. Two of these studies also agree on the need for intact tyrosyl, histidyl, t r y p t o p h a n and amino groups (any one or all of them) for binding interaction [4,20]. Since enzymes and protein-modifying reagents used in these studies may not have selectivity towards any particular macromolecule within the membrane structure, both receptors and other membrane macromolecules should be considered as targets for the changes induced by these agents. Therefore, interpretation of results is complicated by the possibility that the loss of binding may be a consequence of the changes in the receptors themselves and/or a consequence of changes in other membrane proteins whose structural and functional integrity secondarily influence receptor-hormone interaction. The above possibilities should be delineated in order to gain insight into the true macromolecular nature of membrane gonadotropin receptors. We have used the approach of receptor occupancy effect on the binding losses to discriminate between the above possibilities. If the binding loss was a consequence of changes in other membrane proteins alone, then receptor occupancy should have little or no protective effect against these agents. On the other hand, if the receptor occupancy has a protective effect, it would indicate that binding losses were primarily a consequence of changes in the receptors themselves. The above logic was based on the reasonable assumption that the other membrane molecules were available all the time, whereas receptors were primarily available only in the unoccupied state, for enzymatic attack and protein functional group modification. The results presented in this paper indeed demonstrate the marked protective effect of receptor occupancy on binding losses caused by various enzymes, dinitrofluorobenzene and tetranitromethane. Therefore, conclusions drawn in this study and in others [4--8,19,20] on the macromolecular nature of membrane gonadotropin receptors using various enzymes and protein-modifying

225 reagents appear to be valid. Furthermore, some of the data obtained using enzymes and protein-modifying reagents on solubilized receptors are similar to those with membrane receptors [ 19,21].

Acknowledgements This work was supported by a grant HD09557, from National Institute of Child Health and Human Development, National Institutes of Health. The excellent technical assistance of Mr. Fred Carman, Jr. throughout these studies is gratefully acknowledged.

References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

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