An hydroxylapatite batch assay for the quantitation of 1α,25-dihydroxyvitamin D3-receptor complexes

ANALYl

ICAI

BIOCHEMISTRV

92, 314-323

( 1979)

An Hydroxylapatite Batch Assay for the Quantitation Icr,25-Dihydroxyvitamin D,-Receptor Complexes] WAYNE

R.

WECKSLER

AND

ANTHONY

W.

of

NORMAN”

A versatile hydroxylapatite batch assay for l&25-dihydroxyvitamin D,-receptor complex from chick intestinal mucosa has been developed. The assay has been characterized with respect to time and temperature of incubations, protein concentration, amount of hydroxylapatite required to bind receptor-steroid complexes, pH, and effects of KCI and phosof nonspecifically phate. Triton X-100 (0.5%, v/v) was found to be essential for the removal bound ligand. The hydroxylapatite was shown to bind the la.25.dihydroxy-vitamin D:, receptor as demonstrated by the specificity and high affinity for la,25-dihydroxyvitamin D:, and the sedimentation properties of the phosphate-extracted hydroxylapatitebound complex on sucrose density gradients. Binding appears to be nearly quantitative. The efficient separation of bound from free ligand utilizing this assay makes it possible to examine a number of aspects of the binding of this steroid hormone to its cytoplasmic receptor that has not previously been possible.

It is now well known that vitamin D:{ must be metabolized to la,2Sdihydroxyvitamin D:, [lcu,25(OH),DJ3 before it can exert its biological actions on intestinal mucosa (l-3). This steroid facilitates the translocation of calcium across the intestinal mucosal cell and into the blood by what is now recognized as a classical steroid hormone mechanism. Upon entering the mucosal cell, la,2S(OH),D, binds with high affinity and specificity to a cytoplasmic receptor (4-6). As first shown in this laboratory (7) and subsequently confirmed by others (5,8). this 3.7 S lol,25(0H),D,,-receptor complex is transferred to the chromatin fraction of ’ This is the 15th paper in a series entitled “Studies on the Mode of Action of Calciferol.” 1 To whom all inquiries should be addressed. ‘( Abbreviations used: 1cu.25tOH),DZ,, lu,?Sdihydroxyvitamin D,: PPO. polyphenylene oxide: POPOP, 1.4.bis[2-(5-phenyloxazolyl)]-benzene; PBD. I .3,4phenylbiphenylyloxadiazole: ZS-OH-5,6-1rrrr?.\-D:,, 25. hydroxy-5.6-rrtrns-vitamin D:,: In-OH-D:,. lol-hydroxyvitamin D,: 25.OH-D,. 25.hydroxyvitamin D:,: la.24R.25.(OH):,D.,. lu.24R.25.trihydroxyvitamin D.,; 24R,2S(OH),D,. 24R.25.dihydroxyvitamin D.,: RCI. relative competitive index. OOO3-2697/79/0203 Cop)rlght All ,lghf\

14- 10$02.00/O

(( lY7Y hy Acxlemtc Pur\\. Ins 01 rc,voJuct~on I” any Iurn rr,e,veJ

314

the cell. This appears to be a temperaturedependent process (9- 11). Following chromatin localization, transcriptional events are altered (12) and the de novo synthesis of a vitamin D-dependent calciumbinding protein is induced ( 13). It is possible to devise a number of kinetic and equilibrium binding studies for steroid-receptor systems utilizing an assay that can accurately quantitate the steroidreceptor complexes and efficiently remove nonspecifically bound ligand. Of the several techniques that have been used (e.g., charcoal-dextran, ammonium sulfate, protamine sulfate, DEAE-cellulose filters, ion;exchange or molecular-sieve resins, DNA-cellulose, etc.), only charcoaldextran (4.8.14) and DEAE-cellulose filters (5.15) have been adapted to the la,25(OH)+,,-receptor system. To date, these assays have only been used to determine equilibrium binding constants. A reconstituted chromatin-cytosol assay ( I I, 16) and a polyethyleneglycol precipitation assay (17) have been used for competitive-

ASSAY

OF

lt~.?S-DIHYDROXYVITAMIN

binding radioligand assays. However, neither of these assays has either been tried or used successfully to examine other types of binding processes. Hydroxylapatite has previously been used in a column ( 18) and batch (19) assay for steroid-receptor complexes. The batch assay developed by Williams and Gorski ( 19). while being excellent for the estrogen receptor. is incapable of separating receptorbound from free ltu,25(OH),D:,. We wish to report a modification of the hydroxylapatite batch assay (19) which allows the quantitation of lcu..2S(OH),D,,-receptor complexes from rachitic chick intestinal mucosa and its application to a number of different kinds of kinetic and equilibrium binding studies.

D, RECEPTORS

315

carrying out the equilibration in a graduated flask. a 50% slurry was made by adding a volume of buffer equivalent to the settled volume of the resin. Prior to each use the settled hydroxyapatite must be resuspended by gentle swirling. Cytosol (0.5 ml) was added to L:‘H]la,25(OH),D,, at the desired concentration (5-20 nM) in the presence or absence of > 100X molar excess nonradioactive Itr.25 (OH),D:, (0.05 ml of ethanol) and vortexed to begin the binding reaction. The incubation was routinely carried out for 45 min at room temperature and terminated by removing the incubation tubes ( I .5 x 9.5 cm, polypropylene) to an ice bath and immediately adding 0.5 ml of the hydroxylapatite slurry.’ The tubes were vortexed and left on ice for I5 min with vortexing every 5 min. The samples were then centrifuged at EXPEIRIMENTAL PROCEDURE 12,000,r: for 5 min in a Sorvall RC-2 refrigerAll animals used in these studies were ated centrifuge (SM-24 rotor). The hywhite Leghorn cockerels obtained locally on droxylapatite pellets were washed three the day of hatching and placed on a rachitotimes with 10 mM TrisiHCI-0.5% Triton genie diet containing no vitamin D:,. 0.6$% X-100, pH 7.5, by vortexing and centrifuging. as above. The final washed hydroxylcalcium. and 0.42 phosphorus (20). Animals had access to deionized water rrtl apatite pellet was quantitatively extracted with 4 ml of 2:l methanol:chloroform and lihirrrm and were raised under incandescent the solvent was transferred to scintillation lighting on a 16-h light/8-h dark cycle. vials and evaporated to dryness. ScintillaAfter 4 to 6 weeks the rachitic animals were sacrificed by decapitation. tion cocktail was added (9 ml of butylIntestinal cytosol was prepared as previously PBD: 5.25 g of 2-[4’-tert-butylphenyl-5described ( il.5). Some cytosol preparations (4’.biphenyl-3.4-oxadiazole)] per liter of were lyophilized and stored at -20°C under toluene) to the vials and the samples were argon until use when they were reconcounted for 5 min at 47% counting stituted by the addition of distilled water to efficiency. When desired, counts per minute their original volume. were converted to disintegrations per A 50% slurry of hydroxylapatite was minute using an external standard. prepared by adding IO g of Bio-Gel HTP After initial characterization. the assay (Bio-Rad Laboratories, Richmond. Caiif.) ’ Incubations TV form (,‘HI la.?tOH),D.,-receptor to 60 ml of 10 mM TrisiHCl-0.1 M KCI, complex were carried out for various times at either pH 7.5. wit.h gentle swirling. The suspen0 to 4’C or room temperature. These difference\ sion was allowed to settle for 10 min and were B matter of expedience for the particular experiment and their only effect was on the amount the supernatant was decanted off. Fresh of complex formed at the time of ashy. Thuh. buffer was added and the hydroxylapatite short incubations (~4-6 h) at 4-C. although not at was resuspended and allowed to settle two equilibrium. in no way alter the ability of the assay to more times. The final slurry was allowed measure those Itu.~.ctOH),D.,-receptor complexe\ to equilibrate overnight at 0 to 4°C. By which have formed.

316

WECKSLERANDNORMAN

was scaled down to allow the use of a Beckman Microfuge B (1 .&ml tubes) to facilitate the centrifugation steps. In this assay 0.2 ml of cytosol is incubated with steroids in 0.02 ml of ethanol, 0.4 ml of the hydroxylapatite slurry is added, and the centrifugation steps are carried out for 2 to 3 s in the microfuge (14,000- 15,000 rpm). The hydroxylapatite pellets were extracted twice with 0.9 ml of 2:l methanol:chloroform to assure complete removal of radioactive ligand. This miniassay greatly reduces the amount of time necessary to carry out the washing and extraction steps. Intestinal chromatin was prepared as previously described (11). Briefly, homogenates were centrifuged at 4300~ for 10 min. The resulting pellet was resuspendedin 10 mM Tris/ HCl-0.5% Triton X-100, pH 8.5, and crude chromatin was harvested by centrifugation at 12,004r: for 15 min. This crude chromatin preparation was resuspended in 50 mM TrisiHCl, pH 7.5. to remove the Triton X-100 and reisolated by centrifugation at 12,000~ for 15 min. Reconstituted chromatin-cytosol competitive-binding assays were carried out as described in detail elsewhere (11,21). Chromatin and cytosol fractions were prepared as above and reconstituted by homogenization. Aliquots (0.5 ml) were incubated for 45 min at 23°C with 10 nM [“HI lcu,25(OH),D:, and the analog of choice dissolved in 0.05 ml of ethanol. The ability of varying concentrations of competitor to decrease the amount of [:‘H] ln,25(OH),D:, bound to the reisolated chromatin after three washes with 10 mM TrisiHCl-0.5% Triton X-100, pH 8.5, was measured. The competitive binding assay using the hydroxylapatite assay was carried out in an exactly analogous manner. Aliquots (0.5 ml) of cytosol were incubated with 10 nM [:JH] la,25(OH)2D,, and the analog of choice for 45 min at room temperature. The ability of the analog to decrease the amount of [:‘H] lo1,25(OH),D,, bound to the washed hydroxylapatite pellet was then measured.

The ability of an analog to compete with [:‘H] ln,25(OH),D,, for binding to either its cytosol receptor (measured by the hydroxylapatite assay) or to chromatin (measured by the reconstituted chromatin-cytosol assay) can be expressed on a linear scale of relative competitive index (RCI) where lar,25(OH),D,, is defined as 100 (21,22). A plot of , 1 I

percentage maximum bound [:‘H]lu,25(OH),D, [competitor] ”

I [“HI ln,25(OH),

D:,

will yield a straight line with ay-intercept of 1.0. The slope of the line equals the competitive index (cy). Data are normalized to a lcu,25(OH),D, standard curve which is run each day by the following relationship:

Linear 4.0 ml of 5 to 20% sucrose density gradients in 0.3 M KCI- 10 mM TrisiHCl- 1 mM EDTA-12 mM thioglycerol, pH 7.5. were made using a Buchler gradient former and peristaltic pump (Buchler Instruments, Fort Lee, N. J.). Cytosol (0.2 ml) was incubated for 30 min at room temperature with 4.9 nM [BH]1,,25(0H),D,J (33 Ciimmol) in the presence or absence of a 500-fold molar excess of nonradioactive lcu,25(OH),D,,. This incubation was terminated by the addition of 0.4 ml of hydroxylapatite slurry. After washing, as above, the hydroxylapatite pellets were extracted for 15 min at room temperature with 0.5 ml of 0.4 M potassium phosphate, pH 7.5. Aliquots (0.15 ml) from the cytosol incubations or hydroxylapatite extract were then layered on top of gradients. Gradients were run for 20 h at 50,000 rpm in a Beckman L5-50 (SW 60 rotor) ultracentrifuge. Fractions (6 drops) were collected by hand from the bottom directly into scintillation vials using a peristaltic pump and 10 ml of

ASSAY

OF

lt~.?.S-DIHYDROXYVITAMIN

6r

x I

3

z

2 1

001 %

0 I% 0 05 % Trlton X-100 cone

0 5 % in HAP

I4 %

5 0 o/0

Washes

FIG. 1. The effect of Triton X-100 concentration on binding of [:lH] la.ZS(OH),D~,-receptor complex to hydroxylapatite. Cytosol was isolated as described in the text and incubated with I8 nM [“H]Ia,25(OH),D,, (hatched bars) or with 18 flM [:‘H]la.?S(OH),D,, in the presence of ?40-fold excess nonradioactive la.25(OH),D, (open bars). Incubations were carried out for 45 min at 23°C. The indicated concentration (v/v) of Triton X-100 was included in the wash buffer. Results are expressed as mean _f SD of duplicate samples.

scintillation cocktail was added (8 g of PPO, 0.4 g of POPOP per liter of 33%, Triton X-100 in toluene). Samples were counted at 35% counting efficiency for 5 min. [‘“ClOvalbumin (3.7 S) was run in a parallel gradient and used as a molecular weight marker. The [‘Ylovalbumin was prepared by the meth’od of Rice and Means (23). Crystalline 1~~,25(0H),D,,. la,24R,25(OH),,D,,, and 25-OH-5,6-t~~,zs-D:, were generous gifts of Dr. M. Uskokovic of Hoffmann-LaRoche (Nutley. N. J.). and crystalline 25-OH-D,, and Icr-OH-D,, were kindly provided by Dr. J. Babcock of Upjohn (Kalamazoo. Mich.). All compounds were chemically pure as assessed by uv-spectroscopy and thin-layer chromatography. 25 - Hydroxy[26(27) - nlrfhyl:sH]vitamin D:, (3-12 Ciimmol) was obtained from Amersham/Searle. High specific activity 25hydroxy[:‘H]vitamin D:, (33 Ciimmol) was a gift of Dr. N. Miller of Hoffmann-LaRoche (Nutley. N. J.). Tritiated lcu,25(OH),D, was biosynthesized in \Ytro (20) from the tritiated 25-OH-D,, and either used directly or di-

317

D:, RECEPTORS

luted with synthetic 1cu.25(0H),DZ, to a lower specific activity. The radiochemical purity of the tritiated steroids was examined by chromatography on Sephadex LH-20 (1 x 60 cm, 35:65 hexane:chloroform) and determined to be at least 95%, for [:‘H]25-OH-D,, and greater than 99% for [:‘H]lcu,25(OH),D:,. [“C]Formaldehyde (10 mCi/ mmol) was obtained from New England Nuclear and used to synthesize [‘“Clovalbumin. RESULTS Preliminary experiments using hydroxylapatite to bind la,25(0H),D,s-receptor complexes clearly demonstrated that a wash buffer lacking Triton X-100 would not remove nonspecifically bound lcu,25(OH),D,, which became adsorbed to the resin. Similar

1

I-

‘: -0 X 2 a 0

0

I

2

Number

3

of

4

5

Washes

FIG. 2. The effect of the number of Triton X-100 washes on the specific binding of [“HI la,25(OH),D,,receptor complexe\ to hydroxylapatite. Cytosol was isolated as described in the text and incubated with IO nM [“H]la.?510H),D:, in the presence (nonspecific) and absence (total) of 125.fold excess nonradioactive Itu.?S(OH),D,, for 30 min at 23°C. The hydroxylapatite was washed the indicated number of times in IO mM Tris!HCI-0.5% Triton X-100. pH 7.5. Specific binding is taken as the difference between the total and the nonspecific binding. Samples were run in duplicate and the results are expressed as the mean i- SD.

318

WECKSI.ER

observations have been made from this laboratory with respect to lt~,25(OH)~D:, adsorption to chromatin (1 I) and DEAEcellulose filter disks (Wecksler and Norman, unpublished observations). Accordingly, both the number of Triton X-100 washes and the optimal concentration of Triton X-100 in the wash buffer were examined. Figure 1 shows the effect of Triton X-100 concentration in the hydroxylapatite wash buffer upon the amount of bound [:jH]la,25(OH),D, after a standard incubation and three washes. The number of 0.5% Triton X-100 washes (in 10 mM TrisiHCl. pH 7.5) needed to reduce nonspecific binding to a constant level was also determined (Fig. 2). The amount of specific binding appeared to be constant after two washes with 10 mM Tris/HCl-0.5% Triton X-100, pH 7.5, and three washes were adopted as part of the standard assay conditions. The volume of hydroxylapatite slurry that was required to obtain optimal binding of

2.5

2.0 m bx

I.5

H s

1.0

0.5

Volume

of

HAp

Slurry

(ml)

FIG. 3. The volume of hydroxylapatite required to bind r1H]Icu.25(0H),D,,-receptor complexes. Cytosol was incubated for 5 h at 0°C with 10 nM [:rH]ltu.25(OH)& in the presence (at or absence (A) of excess nonradioactive Iu.?S(OH),D,,. The indicated volume of 50% hydroxylapatite slurry was added and the samples were washed as described in the text.

AND

NORMAN

5 Protein

Concentrotlon

IO

15 (mg/ml)

FIG. 4. The effects of protein concentration of the amount of [“HI Io.25(OH)&-receptor complex measured by the hydroxylapatite assay. A 40’; (w/v) homogenate prepared as described previously and cytosol was isolated by centrifugation as described elsewhere (IS). The cytosol was diluted into 0.25 M sucrose-10 mM TrisiHCI-25 rnbt K,HPO,-5 mM MgCI,. pH 7.5. and incubated (23°C. 45 mint with IO mM [:‘H]lu,?S(OH),D~, in the presence or absence of 500-fold excess nonradioactive la.25(OH),D,,. The amount of specifically bound [:‘H]ln,?S(OH),D,, was then assayed.

[:‘H] ln,25(0H),D,-receptor complexes was also determined (Fig. 3). Preliminary experiments utilized 0.3 ml of slurry for a 0.5 ml cytosol (6-10 mg of cytosol protein/ml) incubation but 0.5 ml of slurry was subsequently utilized in all assays carried out in 0.5-m] cytosol aliquots. The effect of protein concentration in the 0.5-m] aliquot of cytosol was examined. The total cytosol protein concentration was varied over a range from 0.5 to 15 mgiml (Fig. 4). Binding of [“H]la.25(OH),DZ,receptor complex to hydroxylapatite was found to be linear over this range of cytosol protein concentration. The effects of pH and KC1 concentration in the hydroxylapatite equilibration buffer were also tested. KC1 concentration in IO mM TrisiHCl. pH 7.5. had no apparent effect on binding over a range of 0 to 0.2 M. The pH of a IO ITIM TrisiHCl-0.1 M KCI equilibration buffer was varied from 7 to 8. again with no apparent adverse effects on binding. Three different batches of Bio-Gel HTP (hydroxylapatite) were equilibrated in two

ASS.4Y

IO

Time

20

30

OF

la.5DIHYDROXYVITAMIN

40

of Incubation

50

examined and found to be constant from 5 to 25 min. The time course for saturation of the cytoplasmic receptor at room temperature is shown in Fig. 5. Some incubations were carried out for 30 min but 45 min was adopted as part of the standard assay conditions. In order to determine what proportion of the steroid-receptor complexes was being bound by the hydroxylapatite. the assay was compared to the well-characterized reconstituted chromatin-cytosol binding assay ( 11). Cytosol from a 20% homogenate was incubated with 10 nM [“H]lcu,25(OH),D,, in the presence or absence of excess nonradioactive lcu.25(OH),D,, for 45 min at 23°C and 0.5-m] aliquots of the incubation were assayed for binding to either hydroxylapatite or chromatin. The amount of specific binding to hydroxylapatite in quadruplicate samples was at least 95% of that which bound to chromatin demonstrating a near equivalence in binding. Table 1 shows the results of a series of competitive-binding experiments in which several analogs of lcu,25(OH),D,, were compared in both the hydroxylapatite assay and the reconstituted chromatin-cytosol assay. It should be pointed out that the hydroxylapatite assay measures steroid-receptor complexes while the reconstituted

60

(min)

FIG. 5. Time course for binding of [“H]la.?(OH)& to cytosol receptor as measured by hydroxylapatite assay. Cytosol was incubated with 10 nM (“HI la,2S(OH),D., and stopped at the indicated time by addition of 0.3 ml of hydroxylapatite slurry. Samples were processed as described in the text. Results are expressed as mean i SD of duplicate determinations.

different buffer systems t 10 mM Trisi HCI-0.1 M KCI, pH 7.5, and 10 mM Tris/ HCI-IO rnvr K,HPO,. pH 7.5) and found to have equivalent binding capacities under the standard assay conditions. The quantitative binding of receptorsteroid complexes to the resin at 0°C was TABLE COMPEI

ITIVF

BILGING

wtrrt

91 UDIFS

I

STRU~TURAI.

Relative

Analog 25-OH-5.6.trans.D., lu-OH-D:,” Z-OH-D,

24R,25(OH),D,,”

Reconstituted chromatin-cytosol assay 1.97 0.33 0.34 0.77

-t 2 c 2

319

D:, RECEPTORS

0.30 0.04 0.14 0.05

” The relative competitive index for ltu.25tOH),D,, is defined independent determinations. h Significantly different at P < 0.01 (Student’s f-teat).

ANRI

competitive

:v 4 3 4 3

OGS OF Itu.?.S(OH),D,

index”

Hydroxylapatite assay 1.67 0.52 0.32 0. IO

as 100. Numbers

k t i -+

A’

0.18 0.02 0.07 0.02

r-epresent

4 3 3 3 mean

i SD of jr

320

WECKSLER

chromatin-cytosol assay measures only those complexes that subsequently bind to chromatin. The first process is strictly a binding event while the second may be much more complicated than simply steroid-receptor complex binding to chromatin. Since there is some evidence for an “activation” step involved in chromatin binding (9- I I), this would mean at least one more process required for chromatin binding of receptor-steroid complex that is not required for ligand binding to receptor. Accordingly, relative competitive index (RCI) values were determined for the same set of steroids in both the hydroxylapatite assay in the steroid-receptorchromatin assay. Two of the four analogs had significantly different RCI values in the two assays (P < 0.01); 24R,25(OH),D,, which is a side-chain analog that does not possess the important ICY-OH on the A-ring (I I) and la-OH-D,, which is missing the critical 25OH on the side chain (1 I). The significance of this observation is not yet known but it is interesting to note that neither assay consistently gave higher or lower RCI values than the other, thereby suggesting that there are no methodological biases in the new assay. In addition, preliminary studies with lcu,24R,25(OH):,D:,, a much better competitor [RCI = 41, (2l)]. showed no significant difference in the RCI as determined by the two assays (results not shown). In other experiments. the amount of receptor-steroid complex remaining in solution after the incubation with hydroxylapatite was determined. After a standard incubation of steroid with cytosol; addition of hydroxylapatite and centrifugation to pellet the resin. the resulting supernatant was combined with fresh hydroxylapatite. In two separate experiments it was found that the amount of complex bound in the second hydroxylapatite incubation was less than 3% of that bound in the first. The steroid-receptor complex appeared, therefore, to be tightly associated with the

AND

NORMAN

I2

IO

T 08 x 5 0 4

2

I 4

Bottom

Fraction

FIG. 6. Sucrose-gradient phosphate-extracted

[:‘H]la.?S(OH),D:,

the

resin

was

I 20

I 24

Number

complexes. was (33

23°C in the presence excess nonradioactive was terminated by

I 16

Top

sedimentation hydroxylapatite-bound

la.?S(OH),D:,-receptor from a 40% homogenate nM

I 12

I 8

analysis

Cytosol incubated

Ciimmol)

for

of [:‘HJ-

(0.2 with 30

min

ml) 4.9 at

IA) or absence (A) of 500.fold la,2S(OH),D,,. The incubation addition of hydroxylapatite and

washed

to

remove

nonspecifically

bound ligand as described in the text. Phosphate (0.4 M. pH 7.5) was added and the samples were extracted for 15 min at 23°C. Aliquots (0.15 ml) of the extract were density gradient text. The large

layered on linear 5 to 203 and centrifuged as described peak in the center of the

represents receptor-steroid at approximately 3.7 S when humin marker. The peak

complexes compared appearing

17 through

binding.

19 is nonspecitic

sucrose in the gradient

migrating to a (“C]ovalin fractions

hydroxylapatite. KCI concentrations up to 0.5 M only removed 10 to 15% of the [“HIln,25(OH)2D:, from the washed hydroxylapatite. High concentrations of phosphate (0.3-0.5 M). pH 7.5. removed greater than 70%, of the complexes from the washed resin. To verify the identity of the extracted [“H]l(u,25(OH),D:,-receptor complexes. aliquots of a 0.4 M phosphate extract of hydroxylapatite-bound lLu,25(0H),D,,-receptor complex were run on 5 to 20% linear sucrose gradients (Fig. 6). There was a distinct peak of specifically bound radio-

ASSAY

OF

“2

la.?-DIHYDROXYVITAMIN

510

I00 I

,

/

150

/

200

250

l,25(OH)2D3

320

FIG. 7. Standard competition pg of [:‘H]lu,25(OH)PD.,

WC. mean

The miniassay was i SEM saf quadruplicate

and

binding increasing

carried samples.

out

for as

321

D., RECEPTOKS

activity migrating at approximately 3.7 S (mean of live determinations) on these gradients. This also suggested that binding to hydroxylapatite does not significantly alter the sedimentation properties of the steroid receptor complex. an observation previously reported for the chromatinextractable lcomplex (5,8,24). A Scatch,lrd analysis of the binding of la,2S(OH).,D,, to its intestinal mucosa receptor at OC utilizing the hydroxylapatite assay yielded a K,, of 5 x IO-!’ M. This is in good agreement with that previously determined using a DEAE-cellulose filter assay (5,1_0 and a charcoal-dextran assay (8). The hydroxylapatite assay has also been scaled down, as described under Experimental Procedures, and is well suited for use in a clinical assay for lcu,2S(OH),D,,. A standard dilution curve of nonradioactive Itu,25(0H)ZD:, with a fixed concentration of [“H]I(-Y,?~(OIH),D;~ is shown in Fig. 7. This curve is exactly comparable to that previously reported (16) and confirmed in our laboratory (Vdecksler. Bishop, and Norman. unpublished observations) using the reconstituted chromatin-cytosol assay. As little as 30 pg of ln,2S(OH),D,, could

I

350

(pg)

hydroxylapatite concentrations described

I

300

of in

assay. Cytosol nonradioactive

the

text

and

(O.? ml) was Itu.Z(OH),D, the

results

are

incubated for 45

with min

expressed

at as

easily be detected using this assay and the commercially” available specific activity (3- 12 Ci/mmol) [“HI lcu.25(OH),D,, (after i/z )‘ifr’o conversion from [“HI25-OH-D,,). The coefficient of variation with duplicate samples in the hydroxylapatite assay was found to be approximately 5% with a background nonspecific binding of 10to 20%. DISCUSSION Of the several well-known potential assay methods that have been utilized to assay steroid-receptor complexes (charcoaldextran, ammonium sulfate, protamine sulfate, DEAE-cellulose filters, ion-exchange resins, Sephadex G-25 chromatography. DNA-cellulose) very few have been successfully adapted for use with the ltu.25 (OH),D:, receptor from chick intestinal mucosa. Charcoal-dextran has been attempted with (8) or without (14) success and only for estimation of the equilibrium dissociation constant (K,,). The DEAEcellulose filter assay (5,15) has been used i It ia now vitamin D., at \prcific activity sensitivity (5-10

possible to obtain 5hydroxy[:‘H]60 to 90 Cii’mmol. This increase allows for an even greater level pg) using this assay.

in of

322

WECKSLER

AND NORMAN

with success but, again, only for determination of K,,. More recently, an assay utilizing polyethyleneglycol (17) has been used to precipitate steroid-receptor complexes for a competitive-binding radioligand assay. To date the most useful assay has been the reconstituted chromatin-cytosol binding assay (11.16). This assay, however. because of its potential complications in terms of binding steps is not suited for use in binding assaysother than for competition-type studies. The use of an hydroxylapatite batch assay provides for the first time a facile method for assaying la,25 (OH),D,-receptor complex under a variety of conditions. Of particular interest is the apparent insensitivity to ionic strength suggesting that the assay might be used directly on fractions eluting from an ionexchange column. This would be preferable to following macromolecular-bound tritium during purification of the complex since the ligand continuously dissociates from the receptor. The addition of Triton X-100 to the hydroxylapatite wash buffer was found to be the key element in allowing the removal of nonspecifically adsorbed [“H]la,25(OH),D, from the resin. A standard set of conditions has been found that optimizes the assay; OS-ml aliquots of a 20% cytosol (6-10 mg of cytosol protein/ml) from rachitic chick intestinal mucosa are incubated with 10 nM C:‘H]lcr.25(0H)2DR for 45 min at 23°C; the binding reaction is terminated by addition of 0.5 ml of a 50% slurry of hydroxylapatite (equilibrated in IO mM TrisiHCl-0.1 M KCI, pH 7.5) at 0°C followed by a 15min incubation (0°C) to allow steroid-receptor complexes to bind to the resin and subsequent washing with a TrisTriton X-100 buffer to remove nonspecifically bound ligand. The validity of the assay was assessed by comparison to a DEAE-cellulose filter assay and a reconstituted chromatin-cytosol assay. The characteristics of binding in terms of specificity (Table 1) and quantity

of complex (>95%) measured agreed very well when compared to the reconstituted chromatin-cytosol assay. The assay appears to bind 197% of the complexes in solution under the conditions employed. The values of K,! determined from the hydroxylapatite assay agreed well with previous reports using different assay techniques (5,8,15). The washing procedure which was used to remove nonspecifically bound ligand does not appear to reduce the amount of complexes specifically bound to the resin (Fig. 2). In addition, when the hydroxylapatite-bound complexes are extracted with a phosphate buffer of high concentration (0.4 M) and sedimented on sucrose gradients (Fig. 6), they appear to migrate as 3.7 S macromolecules, the sedimentation coefficient for both cytoplasmic (6,8,9,10,15,24) and chromatinextractable (5,15,24) complexes. We feel that by these criteria the hydroxylapatite assay gives an accurate quantitation of la,25(0H),D,,-receptor complex. The hydroxylapatite assay is easy to manipulate and adaptable to a number of types of binding assays. It can be used to (a) assess structure-binding relationships (Table l), (b) quantitate la,25(OH),D,, (Fig. 7) making it useful for a clinical assay (Norman er (71.. manuscript in preparation), (c) determine equilibrium binding constants, (d) apply to kinetic binding studies for estimation of on-rates and offrates (Wecksler and Norman, manuscript in preparation), and (e) assay C”H]la.25(OH),D,,-receptor complexes during purification of the receptor. It also appears to be useful for quantitating receptor- lcu.25(OH),D, complex from chick parathyroid gland (15) and studies are currently in progress to assess its general applicability to mammalian lcu,25(0H),D,,-receptor systems. ACKNOWLEDGMENTS The authors wish to thank Dr. J. Gorski at whose suggestion studies on this assay were initiated. The

ASSAY

OF

lo.5DIHYDROXYVITAMIN

expert technical assistance of Ms. J. Bishop is greatly appreciated. (Gifts of vitamin D analogs (Dr. M. Uskokovic and Dr. J. Babcock) and high specitic activity I”HJ~~.?SIOH)~D:, (Dr. N. Miller) were indispensible to these studies.

( 1977) PY~w. Not. Ac.trd. Sri. L’SA 71, ?3372341. 13. Emtage. J. S.. Lawson, D. E. M.. and Kodicek. K. t 1973) Ntr~rrrc~ (l,,&~~r) 246. lOO- 101. 14. Haddad, J. G.. Hahn, T. J.. and Birge. S. F. ( 1973) Bi~rltim. Bi~~p/t~.c. Ar,ttr 329, 93-97. IS. Wecksler, W. R.. and Norman. A. W. (1978)

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4. Tsai. H. C.. and Norman. A. W. (1973) ./. Hirll. C‘llc~nl. 248, 5967-5975. 5. Brumbaugh. P. F.. and Haussler. M. R. (1974)

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Liao. S.. and Liang, T. (1975) in Methods in Enzymology (O’Malley. B.. and Hardman. Joel G.. eds.), Vol. 36A. pp. 313-319, Academic Press. New York. 23. Rice, R. H.. and Means. G. E. (1972) J. Bird. Chrrtr. 24.

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