125I-Labeling and Functional Evaluationof [Tyr36]Parathyroid Hormone-RelatedPeptide(1-36)

Michael Babich,*,1 Faustino M. Gonzales,*and Ramendra N. Pandey†

*Department of Biomedical Sciences, University of IllinoisCollege of Medicine, Rockford, Illinois 61107; and†Robert Wood Johnson Pharmaceutical ResearchInstitute, Raritan, New Jersey 08869

Received January 2, 1997

Several methods to radiolabel and purify ligands areroutinely employed to study receptors or protein bio-chemistry. Radioiodination is often used as a relativelyrapid method to develop ligands with high specific activ-ity for receptor research. However, damage to the labeledmolecules and altered effects on biological systems aresome of the inherent drawbacks to radioiodination. Anal-yses of the parathyroid hormone (PTH2) receptor hasgenerally involved utilization of PTH analogs 125I-labeledby various means and then purified by HPLC or otherchromatography techniques (e.g., 1–4), but verification ofthe radioligand activity other than receptor binding isnotably lacking. In addition, the advantage of using acompetitive ligand chemically distinct from the radioli-gand (to increase the probability of determining specificreceptor binding; 5) was not available until the discoveryof PTH-related peptide (PTHrP). In the context of theabove, the present work describes a revised, convenientmethod for mild radiolabeling and purification of 125I-labeled [Tyr36]PTHrP(1-36) with subsequent analyses ofreceptor binding and biochemical effects on a processdistal to the hormone–receptor complex.

Methods

Radioiodination. N-Chloro-benzenesulfonamide(IODO-BEADS; Pierce Chemical Co., Rockford, IL) wasselected as the iodination reagent due to ease of use(e.g., rapid and complete reaction start/stop) andmilder oxidation than the common chloramine-Tmethod. The sequential steps were (i) centrifuge 0.1%BSA/10 mM acetic acid (0.5 ml; 0.45-mm filtered)through a micropartition system (MPS-1, 1000 MWCO;Amicon Division of W. R. Grace & Co., Beverly, MA) at1900g, 4°C, 3 ;45 min; retain ;100 ml residual vol-

ume to preserve filtration membrane; (ii) during step 1,prerinse bead with iodination buffer (50 mM sodiumphosphate, pH 7.2) and place into a siliconized glassreaction vial (polypropylene tube is also acceptable)containing buffer 1 2 mCi 125I (17 Ci/mg, carrier-freestock in 10 mM NaOH) 3 2 min at room temperature;(iii) add [36Tyr]PTHrP(1-36) (20 mg from a 1 mg/mlstock solution in 10 mM acetic acid) 3 3 min, terminateiodination by bead removal; (iv) transfer reaction vol-ume (100 ml total) into MPS-1 and q.s. to 1 ml with a 10mM acetic acid rinse of reaction vial 1 bead; (v) cen-trifuge 3 2 h and again bring retentate to 1-ml volume;(vi) repeat centrifugation/wash cycle 3 5 total times;the ultrafiltrate cpm verified that steady-state elutionof free 125I was achieved by the fourth wash and pro-duced no detectable specific binding (discussed below);and (vii) collect and q.s. final retentate to 0.5 ml with10 mM acetic acid rinse of MPS-1 sample reservoir.The specific activity of recovered peptide was 119 6 37mCi/mg (mean 6 SE; N 5 6 preparations).

Receptor binding assays. Receptor binding wasperformed as described (2) with intact UMR 106-H5rat osteosarcoma cells grown in 24-well plates. Thesteps were basically (i) minimum essential medium(MEM)/0.1% BSA/20 mM Hepes buffer (pH 7.4) 3 1 hat 37°C; (ii) change to 225 ml MEM/BSA/Hepes/bac-itracin (100 mg/ml)/chymostatin (50 mg/ml) (3; anti-biotics led to a nonstatistical increase in total andspecific binding); (iii) add 25 ml vehicle or bPTH(1-84) 1 25 ml 125I-labeled [36Tyr]PTHrP(1-36) 3 1 h atroom temperature; (iv) 33 rapid wash with 1 mlice-cold phosphate-buffered saline (pH 7.4); and (v)scrape and collect with 0.5 ml of 0.5 M NaOH 1 0.5ml H2O rinse for g-counting.

Measurement of intracellular free calcium (Cai21).

UMR 106-H5 cell Cai21 was measured in suspended

cells loaded with the indicator dye fura-2 AM(Molecular Probes, Eugene, OR) as previously de-scribed (6).

Statistical analyses. Linear regression analysis,one-way ANOVA, and Student–Newman–Keuls test ofcomparison between means were calculated by handand/or performed with a computer program (DataMostCorp., Salt Lake City, UT).

Results and Discussion

Both saturation and competition binding isotherms (tocharacterize the labeled and unlabeled compounds, re-spectively) are necessary, but not included in many re-ports on PTH-receptor binding in target cells (e.g., boneand kidney). Therefore, a limited saturation binding ex-periment was performed to estimate the affinity (Kd) andspecific binding site density (Bmax) of 125I-labeled [36Tyr]-PTHrP(1-36) for intact UMR 106-H5 cells (Fig. 1). The

1 To whom correspondence should be addressed. Fax: 815-395-5666. E-mail: michaelb@uic.edu.

2 Abbreviations used: PTH, parathyroid hormone; PTHrP, PTH-related peptide; BSA, bovine serum albumin; MEM, minimum es-sential medium.

158 NOTES & TIPS

ANALYTICAL BIOCHEMISTRY 258, 158–160 (1998)ARTICLE NO. AB9725470003-2697/98 $25.00Copyright © 1998 by Academic PressAll rights of reproduction in any form reserved.

results indicate that saturation of specific sites is ap-proached with the addition of .100,000 cpm of 125I-la-beled [36Tyr]PTHrP(1-36) (i.e., .0.86 nM), with esti-mates of Kd (0.1 nM) and Bmax (14.2 pmol/mg protein) ingeneral agreement with other reports utilizing differentradiolabeling procedures (e.g., 2, 4). However, the presentKd estimate is on the lower side (i.e., a higher affinity) ofthose reported in the literature, which may reflect theimportance of Scatchard analysis of saturation bindingisotherms rather than competition binding experimentsonly; it may also demonstrate the influence and interplayof total receptors (Rt), radioligand concentrations, andinitial estimates of radioligand on the derivation of trueKd (5).

The total and nonspecific binding of 125I-labeled[36Tyr]PTHrP(1-36) (Fig. 2) was compared to a radio-ligand generated by the well-established chloramine-T

labeling/HPLC purification method (kindly provided byDr. Robert A. Nissenson, Veterans Affairs MedicalCenter, San Francisco, CA; 1, 2). The total and nonspe-cific bindings of both radioligands were statistically thesame, suggesting they interact with the same popula-tion of specific receptors and that the present radioio-dination and purification method is comparable to ex-isting standards.

Competition binding experiments (5, 7) were per-formed to test if 125I-labeled [36Tyr]PTHrP(1-36) is alsosuitable for estimating the affinity of unlabeled hor-mones that interact with a population of receptors oc-cupied by the radioligand (Fig. 3). The amount of un-labeled bPTH(1-84) required to displace 125I-labeled

FIG. 1. Saturation binding of 125I-labeled [Tyr36]PTHrP(1-36) to siteson intact UMR 106-H5 cells. (A) Labeled peptide (3–100 3 103 cpm;300 3 103 cpm not shown) 6 10 mM bPTH(1-84) (to define nonspecificbinding). (B) Scatchard plot derived from the saturation binding exper-iments (Kd 5 0.1 nM; Bmax 5 14.2 pmol/mg protein). Means 6 SE(contained within the symbols) of $triplicate determinations from atleast three different labeling and cell preparations shown.

FIG. 2. Comparison of total and nonspecific binding between[Tyr36]PTHrP(1-36) radiolabeled and purified by different methods.Approximately 15,000 cpm were added to wells containing vehicle(total bound) and 1 mM bPTH(1-84) (nonspecific bound). Means 6 SEof at least triplicate determinations from seven different intact cellpreparations.

FIG. 3. Displacement of 125I-labeled [Tyr36]PTHrP(1-36) bybPTH(1-84). Means 6 SE shown of triplicate determinations fromthree or four experiments, each on a different labeling and cellpreparation. IC50 5 30 6 5 nM; Ki 5 13 6 2.2 nM for bPTH(1-84).

159NOTES & TIPS

[36Tyr]PTHrP(1-36) is within the range of values ob-tained for binding of PTH analogs to osteoblastic cellreceptors (2, 3) and further validates the currentmethod of generating a PTH/PTHrP receptor probe.

Radioiodination often renders certain damage to theligand. The viability of current radioiodinated PTH orPTHrP analogs has been difficult to test because manyof the key assays (e.g., adenylate cyclase) require otherradioactive substances which are difficult to separateout or count from the energy of the radioligand. How-ever, the ability to raise Cai

21 in UMR 106-H5 cells isan alternative method to confirm that 125I-labeled[36Tyr]PTHrP(1-36) is viable because the radioactivitywill not interfere with the Cai

21 analysis (Fig. 4). Al-though it remains possible that the radioiodinationprocedure produced some peptide damage, it was not

significant enough to alter the apparent affinity (EC50),efficacy (ECmax), or conformation of the Cai

21 response(i.e., onset, peak, duration, return to basal). Thus, theradiolabeling procedure is sufficiently mild to render afully viable peptide. The results further indicate that.95% of the peptide is recovered during the radioiodi-nation and purification procedure, which agrees withbiochemical protein determinations (not shown). Itshould be noted, however, that a significant amount ofradioligand was used (up to ;6 mg/sample point); it istherefore suggested that duplicate or triplicate pointswith one lower concentration (e.g., # EC50) be per-formed for verification.

Summary

The present work describes a convenient methodfor generating 125I-labeled [36Tyr]PTHrP(1-36) thatis biochemically active and suitable for receptorbinding studies. Although this labeling and purifica-tion approach should not supplant existing methods,there are, however, the following advantages: (i) onlycommon laboratory equipment and techniques famil-iar to most investigators are required; (ii) the finalproduct is ready for use within ;15 h; (iii) there isrelatively less hands-on time, thereby decreasing thetotal work and radioactive exposure; (iv) less radio-active waste is generated and it is well contained;and (v) other modifications or equipment substitu-tions (e.g., tabletop centrifuge, various stop points,other ligands of choice) can be applied to accommo-date individual laboratories.

Acknowledgments. The excellent technical assistance of LisaR. P. Foti was appreciated. This work was supported by NationalInstitutes of Health Research Grant DK45675.

REFERENCES

1. Nissenson, R. A., Teitelbaum, A. P., and Arnaud, C. D. (1985)Methods Enzymol. 109, 48–56.

2. Nissenson, R. A., Diep, D., and Strewler, G. J. (1988) J. Biol.Chem. 263, 12866–12871.

3. Orloff, J. J., Ribaudo, A. E., McKee, R. L., Rosenblatt, M., andStewart, A. F. (1992) Endocrinology 131, 1603–1611.

4. Chorev, M., Caulfield, M. P., Roubini, E., McKee, R. L., Gibbons,S. W., Leu, C-T., Levey, J. J., and Rosenblatt, M. (1992) Int. J.Pept. Protein Res. 40, 445–455.

5. Bennett, J. P., and Yamamura, H. I. (1985) in NeurotransmitterReceptor Binding (Yamamura, H. I., Ed.), pp. 61–73, RavenPress, New York.

6. Babich, M., Alford, G. E., and Nissenson, R. A. (1994) J. Phar-macol. Exp. Ther. 269, 172–177.

7. Cheng, Y-C., and Prussoff, W. H. (1973) Biochem. Pharmacol. 22,3099–3108.

FIG. 4. Cai21 response of UMR 106-H5 cells to radioiodinated

and unlabeled [Tyr36]PTHrP(1-36). (A) Dose–response curve (1–100 nM; mean of peak/basal 6 SE; N $ 3). (B) Representativetracings of the Cai

21 response to the ECmax (100 nM) of eachpeptide.

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