Preparation and characterization of radioactive monoiodotyrosine and diiodotyrosine derivatives of parathyroid hormone

ANALYTICAL

BIOCHEMISTRY

Preparation

214-222 (1984)

14,

and Characterization of Radioactive Monoiodotyrosine Diiodotyrosine Derivatives of Parathyroid Hormone’

and

JAMESE.ZULLANDJACINTACHUANG Department of Biology, Case Institute of Technology, Case Western Reserve University, Cleveland, Ohio 44106 Received January 4, 1984 Highly purified native parathyroid hormone was iodinated by the enzymatic method and separated from unlabeled hormone by isocratic HPLC. The separation system used also resolved iodohistidine, monoiodotyrosine, and diiodotyrosine forms of the hormone from one another. A simplified procedure for direct bioassay of the carrier-free, high specific activity, mono- and diiodinated parathyroid hormone (PTH) by the renal membrane adenylyl cyclase method was also developed. Both labeled forms of the hormone are very potent in this assay,but the iodinated forms appeared to give a lower V,, than the native hormone. The methods for iodination, separation and biological characterization of this PTH tracer are exceptionally facile, inexpensive, and convenient. KEY WORDS: parathyroid hormone; k&nation; adenylyl cyclase; HPLC, radioimmunoassay.

The preparation of a radioactive tracer of native parathyroid hormone (PTH)’ with high specific activity and full biological activity has been a difficult problem which has occupied the efforts of several groups of investigators. Although iodination of PTH with chloramineT has been used for many years to produce both ‘3LI-PTH and “‘1-PTH of high specific activity (1,2), the oxidizing conditions of this method are too rigorous for PTH, which is readily oxidized at methionine residue, with the loss of biological activity. This difficulty, and that of separation of iodinated from unmodified PTH, have led to the development of other approaches. In 1973, Sutcliffe et al. (3) prepared iodinated PTH using lactoperoxidase and 75Se-labeled hormone for use in receptor studies, and Sammon et al. (4) described an electrolytic method of iodide oxidation to prepare iodinated PTH with appar* Supported by NIH Grant AM 28426. ’ Abbreviations used PTH, parathyroid hormone; BSA, bovine serum albumin; DTT, dithiothreitol; QUSO, microtine silica; TFA, trifluoroacetic acid, SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

0003-2697184 $3.00 Copyright Q 1984 by Academic Press. Inc. All rights of reproduction in any form reserved.

214

ent biological activity. In neither case was separation of the labeled Corn unlabeled hormone attempted. Furthermore, direct bioassay of high-specific-activity iodo-PTH was difficult because (a) it either required handling of exceptionally large amounts of radioactivity in single experiments, or (b) it involved assays which themselves utilize radioisotope methods, and which were thus technically difficult, or impossible, with a radioactive form of the hormone. At this time, we developed the use of modification of lysine residues using tritiated methyl acetimidate to produce a labeled derivative of native parathyroid hormone with full biological activity which was used for studies of PTH receptors (5-7). This material is labeled at seven to nine sites in the PTH molecule, and its content of unlabeled PTH is essentially zero, but still the specific activity was not as high as desirable for studies at physiological concentrations of PTH. Furthermore, its properties were somewhat unpredictable, and preparations of higher specific activities were less stable than was desirable.

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PARATHYROID

HORMONE

In a different approach, Rosenblatt et al. (8,9) utilized the preparation of methioninefree analogs of the l-34 fragment of PTH (which is known to be fully biologically active), with a tyrosine in place of phenylalanine at residue 34, so that iodination could be achieved. Although unlabeled hormone was not separated from the tracer, this material has been useful in studies of PTH receptors ( 10). Recently, however, these problems have been overcome to some extent by the use of improved methodology. Kremer et al. (11) utilized lactoperoxidase-catalyzed iodination of native PTH and HPLC separation of iodoPTH from native hormone to obtain an iodinated tracer which activated rabbit kidney membrane adenylyl cyclase. Also, Rosenberg et al. ( 12) described iodination of native PTH by an electrolytic method, separation of iodoPTH from unlabeled hormone by isoelectric focusing, and direct in viva bioassays of carrierfree high-specific-activity hormone. Their results strongly suggest that the tracer is fully active when prepared by these methods. In this paper, we describe our work with enzymatic iodination of PTH and separation of iodinated from native hormone by isocratic HPLC. The data confirms that high-specificactivity iodinated PTH prepared by this readily available method retains high levels of biological activity (Kn < 1O-9 M), and that monoiodotyrosine PTH can be easily separated from unlabeled hormone as well as from diiodotyrosine PTH and iodohistidine forms of the hormones by HPLC. We also describe a method which allows straightforward bioassay of high-specific-activity PTH in the adenylyl cyclase bioassay without dilution with stable isotope. MATERIALS

AND

METHODS

Chemicals and reagents. All chemicals and reagents were obtained from standard supply houses and were of the highest quality avail-

IODOTYROSINE

DERIVATIVES

215

able. Acetone powder of parathyroid glands was from Sigma. Antisera against CAMP (Rabbit) and goat anti-rabbit antisera were from Shadrack Biologic& (Cleveland, Ohio). l-34 PTH was from Penninsula. Parathyroid hormone. PTH was purified to homogeneity, starting with acetone powder of bovine parathyroid glands, by methods described elsewhere( 12,14). Oxidized forms were separated from the hormone prior to iodination by HPLC (15). The concentrations of the hormone stock solutions used in biological characterization were determined by uv absorption and by amino acid analysis. The two methods gave identical results. Zodination. Generally, the enzymobead (Bio-Rad) methodology was used according to the directions supplied by the manufacturer. PTH (2-5 pg) was iodinated at one time. Iodination was terminated by the addition of sodium metabisulfite, and 1 ml of microfine silica (QUSO) suspension (5 mg) in 0.2 M Na,HPO, containing 2 mg BSA was added. The QUSO was pelleted by centrifiigation, and the pellet containing adsorbed iodinated PTH was resuspended in 1 ml of 20% acetone, 1% HAc, containing 300 mg of Dowex AGl-X8 resin. The sample was then centrifuged, and the supematant containing the eluted PTH was lyophilized. The sample was then either stored in the dry form, or redissolved in 50 InM HAc at a final concentration of 10e6 M for storage or further experiments. Adenylyl cyclase assay. Adenylyl cyclase activity was measured by previously described methods (16), with the exception that CAMP was assayed by radioimmunoassay instead of using radioactive ATP. The incubation mixture contained, in a final volume of 100 ~1, 50 IIIM Tris-HCl (pH 7.5), 4.5 mM MgClz, 30 InM KCl, 9 mM theophylline, 4 InM creatine phosphate, 0.1 mg/ml creatine phosphokinase, 1.1 mM ATP, 0.08% BSA. Hormone samples were diluted in 10 mM HAc, 10 mM DTT, 0.1% BSA, and added in lo-p1 aliquots. Incubations were initiated by addition of 20 pl(80 pg) of partially purified plasma

216

ZULL

AND CHUANG

membrane preparation from bovine kidney cortex (6) and allowed to proceed at room temperature for 15 min. Reactions were stopped by boiling for 5 min. Each sample was diluted lo-fold with 50 mM Tris (pH 7.5), containing (5 mg) QUSO to adsorb out iodinated PTH, and centrifuged for 5 min. In the case of assays with unlabeled PTH, this step was also included to correct for any possible artifacts introduced by QUSO. The amount of CAMP in the supernatant was then determined directly by radioimmunoassay, using a double-antibody method. Aliquots of 50 ~1 of sample + 50 ~1 Tris were added to 100 ~1 of 1% normal rabbit serum in 0.05 M NaAc, pH 5.8, containing [iZ51]c-AMP and 0.3% Triton X- 100; finally, 100 ~1 of 5% goat anti-rabbit y-globulin and 1:30,000 dilution of rabbit anti-CAMP antiserum in 0.05 M NaAc, pH 5.8, were added. Incubation proceeded in the cold overnight, the immune precipitate was centrifuged out, and the pellet was counted. Interference by ATP present in the incubation mixture was found to be negligible in control experiments. In the bioassays of [*251]pTH, to determine the radioactivity carried over to the Ab-Ag complex from the iodinated hormone, control tubes were run with all reagents except [‘2SI]cAMP, and any radioactivity present in the controls was subtracted from the experimental values. Since earlier studies had shown that double-recip rocal and Scatchard plots deviate from linearity at low hormone concentrations (16), V,, values were estimated using double-reciprocal plots, but Kn values were then determined from Hill Plots. Isocratic high-performance liquid chromatography. All chromatography was done on C-l 8 PBondapak columns (Waters) with acetonitrile-water mixtures containing 0.1% TFA. Normally 5-10 pg of labeled PTH in 0.1% TFA was injected into the isocratic flow at 27% acetonitrile, and the eluted radioactivity was collected in glass tubes previously rinsed with 0.1% BSA. The elution position is very sensitive to small changes in acetonitrile

concentrations, and changes approximately 10 ml with each 0.5% concentration difference. Thin-layer chromatographic identification of iodotyrosine, diiodotyrosine, and iodohistidine. Iodinated hormone samples were digested with a mixture of Pronase and leucine aminopeptidase M for 24 h. Ahquots of the digest were spotted on Redi-Plate (Analtech) Silica gel G thin layer plates (20 X 20 cm). The developing solvent was 100 parts chloroform, 60 parts 1-butanol, 20 parts methanol, 20 parts glacial acetic acid, and 15 parts water. All radioactivity on the plates was found in the iodotyrosine and iodohistidine region; i.e., no undigested or partially digested material was detected. RESULTS

HPLC-purified PTH is completely free of oxidized forms of the hormone (15) and, in general, is a higherquality preparation than any we have obtained previously. Following iodination, this hormone still appeared exceptionally clean by conventional techniques. As illustrated in Fig. 1, SDS-PAGE of such material gave a remarkably sharp single peak of radioactivity, which suggested essentially no size heterogeneity in the labeled hormone. This is in marked contrast to our earlier ex-

8Ot

QEL SLICE

NO.

FIG. 1. Polyacrylamide gel electrophoresis of iodinated PTH in denaturing gels. PTH was iodinated as de-scribed under Materials and Methods, and electrophoresed, as described elsewhere, on 12% polyacrylamide gels (18).

LABELING

OF PARATHYROID

HORMONE

perience with PTH labeled either with ‘25I or other methods, where some contamination with small amounts of apparent fragments was nearly always observed (18). Despite its apparent electrophoretic and size homogeneity (Fig, I), we found that iodination generated several forms of the hormone which could be resolved by isocratic HPLC, and further confirmed that iodinated hormone was resolved from unlabeled PTH by this technique. Figure 2 shows a typical HPLC profile obtained following iodination of PTH. Five prominent radioactive peaks (A-E) were routinely observed, with the major iodinated peak (peak D) eluting considerably later than unlabeled PTH. Occasionally, as noted in Fig. 2, a small peak eluting between D and E was observed (D’). This material was not studied further.

6-

E

0’ II

I

I

20

A c.

1

I

I

ti

40 TIME

I

60

I

I

60

(mid

FIG. 2. HPLC elution profile of iodinated PTH. Hormone was iodinated as described under Materials and Methods, and the redissolved hormone was injected onto the C-l 8 @on&p& column. The amlws mark the elution position of unlabeled hormone. The HPLC flow rate was 1 ml/min.

IODOTYROSINE

DERIVATIVES

217

-.05

::. iiii::: j:i;i: :Iiif!, :1:

-.04

! L z z a

-.03

z -.02

2 (”

-.o

1

A 20

40 TIME

60

60

(min)

FIG. 3. &chromatography of purified peak D iodinated PTH (- - -) and HPLC-pure native bPTH ( - * - ). Chromatography conditions were as described in Fig. 2, except that the flow rate was 1.5 ml/min.

Although the amount of unlabeled hormone present in the iodination mixture was not sufficient to produce a clear peak of uv absorbing material in the usual iodination procedure, the separation of unlabeled PTH from peak D (iodinated PTH) was illustrated clearly by cochromatography of purified peak D and the native hormone, as shown in Fig. 3. The total volume separation between the peak tubes for unlabeled PTH and peak D under the conditions described in this experiment was 15-20 ml, and this extent of separation has been observed in numerous repeated experiments. Three of the major radioactive peaks (C, D, and E) from HPLC were isolated and examined for their content of monoiodotyrosine, diiodotyrosine and iodohistidine, with the results shown in Table 1. Peak C contained significant amounts of iodohistidine, monoiodotyrosine, and diiodotyrosine, while the primary peak (Peak D) contained greater than 90% of its radioactivity as monoiodotyrosine. Peak E consisted primarily of a PTH derivative containing diiodotyrosine. Thus, the major labeled form of the hormone (peak D) ap

218

ZULL TABLE

AND

CHUANG

1

T

IOWHISTIDINE, MONOIOD~TYROSINE, AND DIIODOTYROSINECONTENTOF HPLC-SEPARATED PEAKS FROM IODINATED FTH ’ Peak C D E

Iodohistidine

Monoiodotyrosine

Diiodotyrosine

22 +I 0.3 + 0.6 0

60 f I 93 f 3 822

18 +O 7+3 92 + 2

’ Data are from three separate iodinated PTH preparations, and represent the means + SE of the percentage of the total cpm in each sample.

80

TIME

peared to represent the purified monoiodotyrosine derivative, and the latest-eluting form (peak E) was the diiodotyrosine derivative. However, peak C contains a mixture of several forms, including iodohistidine PTH and forms of monoiodotyrosine and diiodotyrosine labeled hormone which differ chemically from peaks D and E. Since enzymatic iodination utilizes H202 which is known to oxidize methionine residues, it seemed likely that both the chemically modified forms of iodotyrosine PTH present in peak C and the earlier-eluting peaks (A-B) represented forms of iodo-PTH oxidized at methionine residues similar to those described by us elsewhere (15). Indeed, as indicated in Fig. 4, when the purified monoiodotyrosine PTH (peak D) was treated further with HzOz, three major new peaks were generated which eluted in positions very similar to those seen in iodinated native hormone (Fig. 2). In addition, when isolated Peak C from iodinated native PTH was reduced by treatment with sulfhydryl reagents, it was partially, but not totally, converted to Peak D (Fig. 5). This suggested that some fully reduced forms of the hormone were already present in the peak C region (iodohistidine forms), but that some sulfur-oxidized forms were also present. It therefore appears that peaks A-C represent a mixture of iodohistidine forms of PTH, metoxidized iodotyrosine derivatives, and possibly met-oxidized iodohistidine forms as well.

(mini

FIG. 4. Effect of H202 oxidation of purified peak D. Purified peak D (2 ng), which gave the elution profile shown (-), was treated with 0.000 1% Hz4 for 45 min at room temperature, and then frozen and lyophilized prior to rechromatography ( + . . ).

We found that the high-specific-activity iodinated PTH could be assayed directly in the renal membrane adenylyl cyclase by using ra::

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(

::.’ :I i i/ g I 4

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\ 20

40 TIME

80

80

(min)

FIG. 5. Effect of reduction of purified peak C on its HPLC elution profile. Purified peak C, which gave the elution profile shown (-), was treated with either 2.5 M cysteine (. - * - *) or 2.5 M mercaptoethylamine (. . *) for 1 h at 80DC prior to HPLC.

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HORMONE

dioimmunoassay methods to measure cyclic AMP. Despite the fact that the aliquots of the incubation mixture used for CAMP assay contain 125I from the hormone sample, which at high hormone concentration represents more than lo- 100 times the amount of ‘25I from the CAMP itself, less than 1% of the ‘251-PTH contaminates the final pellet when the immune precipitation is done in the presence of 0.1% Triton X- 100. (Without the T&on, contamination was nearly 20%.) However, although this amount of contamination was relatively insignificant at lower hormone concentrations, at concentrations where the radioactivity from the hormone exceeded 95% of the total, artifacts were observed, resulting in underestimation of the amount of cyclic AMP. This difficulty was eliminated by the inclusion of microfine silica in the dilution buffer used preceeding the immune precipitation steps which effectively removed PTH from the incubation residues prior to CAMP analysis (See Materials and Methods). Control experiments showed that this step did not alter the CAMP content, but reduced the nonCAMP radioactivity (PTH) in the assay to less than 0.002% of the total. Thus, its inclusion allowed full dose-response curves of both mono- and diiodotyrosine PTH to be obtained. Isolated peak D and E material from several separate iodinations was obtained and bioassayed using this method. For these assays the concentration of the iodinated hormone was based directly on radioactivity, since it is carrier free, with correction for the slight amount of the diiodinated derivative. Dose-response curves for preparations of native, monoiodotyrosine, and diiodotyrosine hormone are shown in Fig. 6. Results similar to these were seen in all hormone preparations, and they suggested that the iodinated forms may have a lower V,, than the native hormone in the assay utilized. This was confirmed by extrapolation of double-reciprocal plots to estimate Vmax7and a comparison of the mean V,, and KH values for multiple preparations of high-

IODOTYROSINE

DERIVATIVES

J

-9

-10 LOG

219

-0 ~P1l-l~

FIG. 6. Bioassay of native, monoiodotyrosine, and diiodotyrosine PTH. Comparison of the activation of renal membrane adenylyl cyc1a.wby native (0) monoiodotyrosine (0), and diiodotyrosine PTH (A). The assay conditions are described under MateriaIs and Methods. All data are expressed as the percentage of the maximal stimulation so that comparisons can be made between assays conducted with different membrane preparations. Absolute values of picomoles CAMP are presented in Table 2.

specific-activity iodinated derivatives and the native hormone are shown in Table 2. Although the means of the V,.,,, values for the different preparations did not differ from one another in a statistically significant manner, this was due to the substantial variation in the absolute value of V,,,, obtained from different membrane preparations. In experiments where direct comparison of the hormone derivatives was assessed in the same experiment, the V,,.,, values for the mono- and the diiodo derivatives were always less than that of the native hormone. In a paired t test, this difference was significant at the 98% confidence level, and it can be concluded that, in the assay used, iodination of PTH does lead to a reduction on the apparent maximal stimulation of adenylyl cyclase as determined by extrapolation of double-reciprocal plots. On the other hand, estimation of potency by determination of KH values indicated that only with the diiodo derivative was the KH

ZULL AND CHUANG

220 TABLE 2

DISCUSSION

COMPARLWN OF NATIVE FTH, MONOIOD~TYR~~INE FTH, AND DIIOIXWYR~~INE FM-I IN ACIWATION OF RENAL MEMBRANE ADEIWLYL C~LASE KH

Preparation Native PTH (5)* Monoiodotyrosine PTH (10) Diiodotyrosine PTH (5)

(molarity X lOlo)

Vmax”

7.0 + 4

47.6 + 11

6.0 f 1.3

38.2 + 9

3.4 + 0.89

27.0 + 8

’ pmol c-AMP min-’ mg-‘; basal activity was 3.5 f 1 pmol CAMP min-’ mg-‘. * Numbers in parentheses represent the number of different hormone samples assayed.

value significantly different from that of the native hormone, and in this case the value is lower, i.e., diiodinated derivative appears more potent. Whether this is due to the fact that the V,, is lower, thus reducing the concentration of hormone required to reach V,,/2, or to a truly greater affinity of the diiodinated form for the PTH receptors is not known. Thus, biological characterization of the iodinated derivatives leads to the conclusions that, although they may differ somewhat in the shape of the dose-response curve and the apparent V,, values, both high-specific-activity forms of the hormone are very active in the assay used, and achieve ha&maximal activation of the enzyme at concentrations equal to or lower than those required of the native hormone. Finally, although long-term storage has not yet been studied, a stability study of 8 days’ duration was also conducted with the monoiodotyrosine form of PTH. The results, shown in Fig. 7, indicated that when this high-spec&activity PTH was stored in dilute acetic acid solution there was little apparent loss of biological activity (Fig. 7) or alteration in the HPLC profile (insert, Fig. 7) over this period of time. However, some damage has been observed when the high-specific-activity hormone wan stored a~ a solid (data not shown).

We believe that the evidence obtained in our work and that of others has now clearly established the biological activity of carrierfree, iodinated PTH, both of the monoiodotyrosine and the diiodotyrosine forms. The primary contribution in the present work is the more complete development of rapid, inexpensive, and convenient techniques to obtain and characterize these useful tracer forms of PTH (both biologically and chemically), and the characterization of the diiodotyrosine form. As regards the methods, it should hrst be noted that hormone labeling is achieved with the exceptionally simple and widely used enzymatic method, which is clearly preferable to the electrolytic method since the latter requires special material and apparatus. Second, the use of isocratic conditions for HPLC is very convenient (e.g., it can be achieved with a single pump) and reproducible. Further-

IAl 20408080 1

1

t

-10

-9 LOG

TIME

WlIfj)

-8 (PTH)

FIG. 7. Storage of high-specifk-activity monoiodotyrosine PTH. Comparison of the biological activity of purified monoiodotyrosine PTH 1 day (0) and 8 days (0) following preparation. The hormone was stored at 10d M concentration in 50 mM HAC at 4°C. The insert is a comparison of the HPLC profile of the hormone preparations after (-) 1 and (. - - ) 8 days.

LABELING

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more, the resolution obtained is superior to previously published methods, leading to sep aration of the iodotyrosine forms of PTH completely from oxidized forms and from iodohistidine forms generated during labeling. It also completely resolves the mono- and diiodotyrosine derivatives from the unlabeled hormone in the mixture and from one another. The elution position of PTH on isocratic HPLC is sensitive to small changes in aceto&rile concentration so that it is necessary to confirm the elution position exactly in each HPLC run with fresh solvents, but in our experience excellent separation can be achieved with isocratic conditions which vary from 25 to 28% acetonitrile. Finally, the method is also clearly preferable to isoelectric focusing both in speed and convenience. The use of radioimmunoassay rather than the 32P-labeled ATP method for assay of CAMP also proved to be a significant improvement. This method has advantages with regard to time, effort, and convenience. Since aliquots of the reaction mixture are measured directly, no corrections for CAMP recovery are required and no double-isotope methodology is involved. The manipulations are simpler and less subject to error than those required with the labeled ATP method, and the restrictions on timing and convenience of assays due to the short half-life of “P are reduced. Furthermore, the time and energy commitment to preparation and regeneration of the numerous columns required for separation of ATP .and CAMP is eliminated. As indicated by the HPLC profiles, chemical modification of the hormone other than iodination of tyrosine does occur during labeling, and only about half the labeled material following iodination is recovered as the monoiodotyrosine derivatives. In other studies we have shown that early eluting peaks are generated by oxidation of PTH with H202, and these represent hormone oxidized at methionines 8 and 18 (15). It is not possible to obtain reliable bioassay data for these early eluting iodinated forms since they are not well

IODOTYROSINE

DERIVATIVES

221

separated from the unlabeled hormone. However, analogous forms of unlabeled PTH all have activity, but are of different potency (D>CBB>A)(15).Inthispaperwealso show that oxidation of iodinated hormone leads to earlier-eluting peaks (Fig. 3) similar to those generated by the iodination conditions, and that reduction of one of these peaks regenerated material eluting in the region of the biologically active major iodinated hormone form, peak D. (Fig. 4). Finally, it should be noted that, by use of the radioimmunoassay method for CAMP measurements, we have been able to obtain the bioactivity of iodinated PTH undiluted with rz71in the adenylyl cyclase system. Thus, it is not necessary to infer that high-specific-activity preparations remain undamaged through secondary radiation effects, since they could be assayed directed. Indeed, our experience indicates that iodinated PTH stored lyophilized in carrier-free form does undergo radiation damage, although the biologically active form can be separated from damaged forms by HPLC prior to use. However, as shown in this paper, high-specific-activity hormone undiluted with 127I is fully biologically active and, when stored in dilute acid solution in the cold, retains its chromatographic and biological properties for at least short periods of time (1 week) following preparation. Therefore, it should be a very useful tracer for PTH in studies requiring high specific activity. REFERENCES 1. Yallow, R. S., and Berson, S. A. (1964) kfethoak Anal. Biochem. 12,69-79. 2. Berson, S. A., and Yallow, R. S. (1968) J. Clin. Endocrinol. Metab. 28, 1037-1046. 3. Sutclifle, H. S., Martin, T. J., Eisman, J. A., and Pilezyk, R. (1973) Biochem. J. 134,913-921. 4. Sammon, P. J., Brand, J. S., Neuman, W. F., and Raist, L. G. (1973) Endocrinology 92, 1586-1603. 5. Zull, J. E., and Repke, D. (1972) J. Biol. Chem. 247, 2183-2188.

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AND CHUANG

J. E., Malbon, C. e., and Chuang, J. (1977) J. Biol. Chem. 252, 1071-1078. Zull, J. E., and Chuang, J. ( 1975) J. Biol. Chem. 250, 1668-1675. Rosenblatt, M., Goltzman, D., Keutmann, H. T., Treagear, G. W., and Potts, J. T., Jr. ( 1976) J. Biol. Chem. 251, 159-164. Rosenblatt, M., and Potts, J. T., Jr. (1977) Endo. Res. Commun. 4, 115-133. Segre, C. V., Rosenblatt, M., Reiner, B. C., Mahaffey, J. E., and Potts, J. T., Jr. (1979) J. Biol. Chem.

6. Zull, 7.

8. 9. 10.

254, 6980-6986.

11. Kremer, R., Bennett, H. P., Mitchell, J., and Goltzman, D. (1982)J. Biol. Chem. 23, 14048-14054. 12. Rosenberg, R. A., MuzafEar, S. A., Heenche,

13. 14. 15. 16.

J. N. U., Jez, D., and Murray, T. M. ( 1983) Anal. Biochem. 128, 331-338. Keutmann, H. T., Dawson, B. F., Aurbach, G. D., and Potts, J. T., Jr. (1972) Biochemistry 11, 1973. Zull, J. E. and Malbon, C. M. (1976) in Methods in Receptor Research (Bletcher, M. Ed.), pp. 533564, Dekker, New York. Frelinger, A. L., and Zull, J. E. (1983) J. Biol. Chem. In Press. Heath, E., and Za J. E. (1980) Endo. Res. Commun. 1, 87-94.

17. Coltrera, M. D., Potts, J. T., Jr., and Rosenblatt, M. (1981) J. Biol. Chem. 256, 10555-10559. 18. Heath, ,E. B., Chuang, J., Botti, R., Whitely, B., and Zull, J. E. (1980) J. Biol. Chem. 255, 1577-1585.