Large-scale preparation and biological activity of recombinant human parathyroid hormone

journal of

biotechnology ELSEVIER

Journal

of Biotechnology

39 (1995) 129-136

Large-scale preparation and biological activity of recombinant human parathyroid hormone J.

Paulsen

*,

D. Ochs, M. Harder, C. Duvos, H. Mayer, E. Wingender

Gesellschaft fiir Biotechnologische Forschung mbH, Mascheroder Weg I, 38124 Braunschweig, Germany Received

26 September

1994; accepted

26 December

1994

Abstract Human parathyroid hormone (hPTH) has been bacterially expressed in bioreactors as cro-@-galactosidase-hPTH fusion protein. We have developed a large-scale purification scheme that exploits the pH-dependent differential solubility of hPTH and a two-step chromatographic procedure. We demonstrate that in a number of assay systems, the recombinant material obtained by this procedure is biologically active. Keywords:

Parathyroid

hormone; Recombinant

expression; Large-scale purification

1. Introduction Parathyroid hormone (PTH) is one of the main regulators of calcium homeostasis. It acts on calcified bone matrix where it gives rise to enhanced bone resorption and thus increases the calcium level in the serum. In addition to this bone catabolic effect, PTH also stimulates a number of anabolic events in the skeletal system such as enhanced DNA synthesis of chondrocytes or osteoblasts in cultivated cells or organs as well as in vivo (Schliiter et al., 1989; Somjen et al., 1990, Somjen et al., 1991; Lewinson et al., 1992). Because of its anabolic activities, PTH has been administered to osteoporotic individuals and revealed positive effects on their bone mass (Slovik

* Corresponding

author.

0168-1656/95/$09.50 0 1995 Elsevier SSDI 0168-1656(95)00002-X

Science

et al., 1986). However, most previous studies have been conducted using PTH fragments due to their availability and for cost reasons. To study the pleiotropic biological effects of intact PTH, we established an effective bacterial expression system for human PTH as [Pro- ‘I-hPTH(l-84) (Wingender et al., 1989). The corresponding Escherichiu coli strain has successfully been grown in bioreactors on complex and synthetic media (Harder et al., 1993, Harder et al., 19941. Here we describe the procedure for cultivations to high cell densities on complex media and purification of the recombinant hormone. The resulting material has been assayed for some biological activities such as stimulation of CAMP synthesis by isolated renal cortical plasma membranes, calcium release from long bones in organ culture, and enhancement of DNA synthesis in osteoblastoid cells in culture.

B.V. All rights reserved

2. Materials and methods Medium Cells were cultivated on YF medium which comprises 48 g I _ ’ technical yeast extract (Ohly) and 12 g I-’ fructose (Merck). The technical yeast extract differs from topgrade quality in a higher NaCl concentration (2% instead of about 0.1%). Fructose and yeast extract were autoclaved separately with 100 ~1 of the polyethyleneglycol derivative Ucolub N38 (Brenntag) to prevent foaming. Sterile filtrated ampicillin (100 mg I-‘) and glycine betaine (140 mg I-‘> were added to the medium for the selection of transformed cells and as an osmoprotectant (Le Rudulier et al., 1984). Bioreactor A 12-I stirred bioreactor (Giovanola, Switzerland) was used in this study. Temperature was monitored and controlled. The pH was measured by a glass electrode (Ingold) and maintained at 7.0 through addition of either 1 mol I-’ H,PO, or NaOH. Filter sterilized air and oxygen was supplied to the medium. The impeller speed was regulated to keep the oxygen partial pressure in the medium at 20% of air saturation. Cultil:ation procedure Single colonies picked from an agar plate were grown overnight in shake flasks on YF medium at 30°C. Each reactor culture of 6 1 was inoculated with 50 ml and cultivated at 30°C to cell densities of 2.3-3.3 g I-‘. Induction was performed by shifting the temperature within 10 min to 38°C. After 3 h the culture was fed at a rate of 0.3 1 hh ’ with concentrated medium (320 g I- ’ yeast extract, 80 g I-’ fructose). Cell disruption For cell disruption the ceil suspension was passed twice through a high pressure homogenizer Lab 60 (APV-Gaulin, Liibeck, Germany) at 600 bar and a flow rate of 20 1 h- ‘. Separation of inclusion bodies from the soluble protein and part of the cell debris was performed by continuous centrifugation in a SA 1 separator from

Westfalia (Oelde, was 18 I hh’.

Germany),

the flow rate used

Extraction and cleallage of fusion protein The inclusion bodies were solubilized with 20 mM Tris-HCI, pH 7.4, 9 M urea and 4 mM D’IT at room temperature and a protein concentration of 30-40 mg ml-‘. After 1 h the same volume of concentrated formic acid was added and the suspension was incubated for 3 d at 37°C. Part of the formic acid was neutralized dropwise (during 2 h) with 1.25 ~01s. of 2.5 M NaOH. The resulting precipitate was separated by centrifugation for 20 min at 10000 X g. Reversed phase chromatography 400 ml of the supernatant was loaded with a flow rate of 50 ml min-’ on a 54 x 300 mm C6 Spherisorb column (Latek, Eppelheim, Germany) connected to HPLC equipment from Knauer (Bad Homburg, Germany). The column was previously equilibrated with 30 mM phosphoric acid, pH 3.2, 150 mM NaCl and after sample application and extensive washing with the same buffer hPTH was eluted with an increasing, discontinuous gradient from 0 to 70% isopropanol. The hPTH containing fractions were pooled, diluted ‘L-fold with 0.1% TFA and loaded on a 10 X 250 mm C6 Spherisorb column (Phase Separation, Queensferry, UK). hPTH was eluted with an increasing, discontinuous gradient from 0 to 80% acetonitrile. Fractions containing hPTH were lyophilized and stored at -20°C. Concentration of hPTH The hormone was concentrated by ultrafiltration (cut-off limit 3 kDa) or by precipitation with 3 M ammonium sulphate. For analytical trials hPTH was concentrated by precipitation with 5 ~01s. of cold acetone. Analytical procedures Biomass was determined gravimetrically by centrifuging, decanting and drying of 1 ml culture medium in balanced caps. The product hPTH was determined by harvesting, purifying and quantifying the insoluble fusion-protein as described elsewhere (Harder et al., 1993).

J. Paulsen et al. /Journal

of Biotechnology 39 (1995) 129-136

Protein concentration was measured according to the method of Lowry et al. (1951) with bovine serum albumin as standard. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) was carried out with the Pharmacia Phast system (Freiburg, Germany) using 20% homogeneous gels. Protein bands were visualized by silver staining (Butcher and Tomkins, 1985). Determination of cyclic AMP synthesis

Stimulation of CAMP production by hPTH was determined in sterna from 17-day-old embryonic chicken. The organs were cultivated in 1 ml FCS-free DME medium for 2-3 days. The medium was changed when cultures were pretreated for 25 min with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX, 1 mM1 followed by incubation with hPTH for 15 min. Sterna were returned to the incubator during this procedure. The reaction was stopped by transferring the sterna quickly into ice-cold PBS washing solution. Cyclic AMP was extracted overnight at -20°C with 95% ethanol acidified with 1% HCl. The samples were subsequently dried under vacuum and dissolved in 200 ~1 PBS. 50-~1 aliquots were removed and cyclic AMP was measured by a binding competition assay (Amersham) and expressed as pmol CAMP per sternum.

dine was added to each well and cultures were incubated for another 20 h. The sterna were subsequently washed in ice-cold phosphatebuffered saline (PBS), transferred into cold 10% TCA and homogenized at 4°C. After centrifugation precipitates were washed several times with 10% TCA and dissolved in 300 ~1 of methylbenzothoniumhydroxide. After neutralization with 1 M acetic acid, 4 ml scintillation cocktail were added and radioactivity was determined.

3. Results and discussion 3.1. High-density cultivation

Time courses of three cultivations are shown in Fig. 1. After 15 h, cell densities of approx. 45 g 1-i dry cell mass were obtained. This corresponds to a yield coefficient of 0.23 g g- ‘, which is about 25% lower than using yeast extract with top-grade quality (Harder et al., 1993). Also, the pattern in product formation was similar during the first 9 h of the induction period. 492 & 2 mg 1-l hPTH were expressed by E. coli N483O:pEX-PPTH. Volumetric product concentrations decreased due to an increase of the liquid volume by feeding. Specific product concentrations after 9 h were in the

-W

Determination of calcium mobilization

Tibiae of 17-day-old embryonic chicken were cultivated in FCS-free DME supplemented with 2 mM glutamine, 50 pg ml-’ ascorbate and 20 pg ml-’ gentamycine for 24 h (Duvos, Scutt and Mayer, unpublished data). After supplementing the organs with fresh ascorbate, PTH was added. The other tibia of each animal served as untreated control. After 3 days, calcium concentrations were determined by atomic absorption spectrometry (AAS).

DNA synthesis rate was determined in sterna of embryonic chicken by measuring the incorporation of [ 3Hlthymidine into trichloroacetic acid (TCAl-insoluble material. Following an incubation period of 20 h with hPTH, 5 PCi [3H]thymi-

Start feeding

lBlomass /

0

2

4

nme after Assay for 13H/thymidine incorporation

131

6

8

10

12

14

induction from 30” to 38°C (h)

Fig. 1. Fed-batch cultivation of E. coli N483O:pEX-PPTH (standard deviation of three cultivation experiments as error bars). Feeding started 3 h after induction. Biomass was quantified gravimetrically, hPTH was analyzed after quick smallscale purification of the fusion protein and densitometric evaluation of Coomassie-stained SDS-PAGE (Harder et al.. 1993).

.I. Paulsen et al. /Journal

132

of Biotechnology 39 (1995) 129-136

range value batch 1993) 3.6% tively,

of 18 + 4 mg hPTH per g dry cells. This is twice as much already estimated during growth on the same medium (Harder et al., and corresponds to a product expression of ChPTH) or 21% (fusion protein), respecof the total cellular protein.

3.2. Separation of inclusion bodies from cell debris

Flow

After sedimentation of the particulate material of the cell homogenate on analytical scale, 67% of the total protein, including the fusion protein, was found in the pellet, 33% in the soluble fraction. If the inclusion bodies were separated by continuous centrifugation with a SA 1 separator, increasing flow rates resulted in increasing protein concentration in the supernatant, whereas the hPTH-containing fusion protein was still found quantitatively in the pellet fraction (Fig. 2). 20% of the contaminating host proteins could

rate (1 h-t)

Fig. 2. Amount of sedimented protein and percent of total hPTH (as fusion protein) in sediment as a function of the flow rate during centrifugation of the cell homogenate with the SA 1,The cell homogenate has a protein concentration of 3 g I _ ‘. 0 I hh’ represents a sedimentation with an Eppendorf centrifuge.

kDa

12

3

4

5

6

7

8

9

c

12.5

C

6.5

10

Fig. 3. SDS-PAGE of samples during neutralization of the hydrolysate with sodium hydroxide and of the homogeneous hPTH after the second reversed phase chromatography. The hydrolysate was neutralized over a time period of 90 min with 2.5 M NaOH. Every 30 min samples were taken and centrifuged. Lane 1, hydrolysate before neutralization; lanes 3, 5 and 7, precipitates after 30, 60 and 90 min; lanes 4, 6 and 8, soluble protein after 30, 60 and 90 min. Lane 9, molecular mass markers (kDa): 6.5 trypsin inhibitor (bovine lung), 12.5 cytochrome c, 21 trypsin inhibitor (soy bean), 29 carbonic anhydrase. Lane 10, hPTH after second reversed phase column.

J. Paulsen et al. /Journal

of Biotechnology 39 (1995) 129-136

133

thus be separated at this step by increasing the flow rate of the centrifuge to 18 1 hh’.

tation of a significant amount of hPTH as well (data not shown).

3.3. Extraction and cleavage of the fusion protein

3.4. Reversed phase chromatography

The fusion protein was quantitatively extracted after 60 min of incubation with 9 M urea. For cleavage of the fusion protein, the suspension was incubated with 50% formic acid. The amount of free hPTH increased during the first 3 days of hydrolysis until a plateau was reached (data not shown). According to this result, a hydrolysis time of 3 days was chosen to achieve optimal hormone release. The formic acid was partially neutralized by slow addition of 1.25 ~01s. of 2.5 M sodium hydroxide. During this neutralization procedure a large amount of protein precipitated which could be separated by centrifugation, whereas hPTH was retained completely in the soluble fraction. Gel electrophoretical analysis of this fraction revealed mainly one protein of 9.5 kDa corresponding to hPTH (Fig. 3). It should be pointed out that the renaturation of the hormone takes place during the neutralization step. Therefore, it is extremely important to follow exactly the neutralization conditions. Adding the equivalent amount of sodium hydroxide as a 10 M solution or neutralizing the hydrolysate in a shorter period of time leads to precipi-

The acidic supernatant (pH 2.71, containing still 2 M urea and 20% formic acid, could be loaded directly on a reversed phase column. The capacity of the column turns out to be 0.6-l ml supernatant per ml gel corresponding to 1 mg protein per ml gel. After extensive washing with the equilibration buffer, hPTH eluted with an isopropanol gradient at 30% of the eluent (Fig. 4). The peak at 24% isopropanol plateau was a coloured fraction containing almost no protein. The small peaks flanking the main hPTH peak also contained hPTH (Fig. 4). The reason for this different elution pattern may be microheterogeneities resulting from the harsh purification conditions. However, over 95% of the hPTH was found in the main peak. For further purification and separation of the salt, this peak was diluted 2-fold with 0.1% TFA and loaded on an 80-ml reversed phase column (0.6 mg hPTH per ml gel). hPTH was eluted as a nearly homogeneous peak with an increasing acetonitrile gradient at 47% of the solvent as judged by silver-stained SDS-PAGE (Fig. 3). After th e second reversed phase column, 260 mg of homogeneous hPTH were obtained per

hPTH I

60

40

60

120

Time (min) Fig. 4. Semipreparative HPLC of hPTH on a 80 ml reversed phase column. 50 ml of partially the column. Application and elution were performed at a flow rate of 8 ml min-‘.

neutralized

hydrolysate

was applied

to

J. Paulsen et al. /Journal

134

Table 1 Stimulation of CAMP synthesis in porcine renal plasma brane fraction and in embryonal chicken sterna Concentration

CAMP synthesized

(M)

renal plasma membrane (pm01 mg-’ min-‘)

sterna (pmol/sternum)

Control I x lo-’ 3x IO_” 1x10 x 3x lomx

2.14kO.06 1.84 * 0.05 2.41 k 0.07 6.21 io.41 6.65 * 0.41 7.16 & 0.24 7.54 * 0.36

2.25 * 0.50 2.57i_0.17

I x Ior’ 3x10-7

of Biotechnology 39 (1995) 129-136

mem-

(pmol)

2.58 * 0.33 7.34 f 1.07 12.30+ 1.34 15.90& 1.90

Stimulation of CAMP synthesis either in porcine renal plasma membrane fraction or in embryonal chicken sterna has been determined as described in Materials and methods. Given are pmol CAMP synthesized per min by 1 mg renal membrane protein (n = 3) or by a single sternum in organ culture (n = 4).

liter of acidic supernatant. This preparation was used for the further investigations. The described production process for hPTH offers the possibility to produce large amounts of the peptide hormone. 2.9 g of fusion protein, corresponding to 492 mg of hPTH, was produced per liter of medium. After purification, 260 mg of the homogeneous material was obtained corresponding to an overall yield of 53%. 3.5. Stimulation of CAMP synthesis by recombinant hPTH We have shown previously that the recombinant [Pro-‘I-hPTH(l-84) stimulates CAMP synthesis of porcine renal cortex membranes in virtually identical manner as ‘native’ (i.e., synthetic) hPTH(l-84). To ascertain the biological activity of the material which has been obtained from the above large-scale purification protocol, we applied the same assay and found the recombinant hPTH described here active with an identical K,,, as reported previously (5 nM; Table 1; Wingender et al., 1989). This value has been repeatedly determined for recombinant hPTH from different preparations. To approach the in vivo situation, we also assayed for CAMP stimulation in whole sterna from embryonic chicken, whose cells in primary cell culture have been shown earlier to respond with increased CAMP synthesis to PTH (Schliiter et al., 1989). The sterna organ culture

also displayed enhanced CAMP production when treated with recombinant hPTH, although about 1 order of magnitude higher concentrations were required (Table 1). Thus, the recombinant [Pro-‘I-hPTH(l-84) exerts the classical biological activity of PTH in stimulating cellular adenylate cyclase at a concentration previously reported for the authentic hPTH(l-84) sequence (Goldman et al., 1988). 3.6. Calcium resorption by long bones in organ culture Tibiae responded with enhanced CAMP synthesis in a manner similar to the isolated cortex membrane fractions (data not shown). Using this system, we now addressed the question whether a more complex effect such as the release of calcium from the whole organ can also be provoked using the recombinant hormone. This effect is known to require CAMP synthesis (Rasmussen and Tenenhouse, 1968; Wong et al., 1977; Kfeen et al., 1988). We found that recombinant hPTH was able to stimulate bone resorption in this organ culture system in a dose-response relationship which parallels the CAMP enhancement in this and other assay systems (Table 2). 3.7. Stimulation of cell proliferation by recombinant hPTH It is well-established that hPTH also exerts CAMP-independent effects which may reside in Table 2 Calcium mobilization Concentration 1 x 10-u Ixlo-q

I x lomx IX lo-’ 1x10-”

from tibiae by recombinant

(M) Mobilized

calcium &g ml-’

hPTH supernatant)

0.8 f 0.8 3.8+0.6 9.0+ 1.6 12.4 + 0.7 10.4* 1.8

Tibiae of embryonal chicken were kept in organ culture as described in Materials and methods and were treated with hPTH fn = 5). The increase in calcium concentration in the culture medium was measured by AAS after 72 h and is given with SEM. The control culture had 0.4 k 0.9 pg calcium in the supernatant. The agonist fragment hPTH(I-34) released 9.68 + 1.3 pg ml-’ calcium at a concentration of 1 x lo-’ M.

J. Paulsen et al. /Journal Table 3 [3H]Thymidine incorporation onic chicken sterna Concentration

E/C

(MJ

UMR-106

into UMR

106 cells and embry-

+ SEM sterna

recombinant [Pro-‘I-hPTH(l-84) 1x10-‘” 1x10-Y 1 x10-x 3x 10-s 1x10~’ 3x 10-7 1x10-h

of Biotechnology 39 (1995) 129-136

1.25 f 0.09 1.18+0.11 1.52kO.18 2.89 f 0.25 5.66 + 0.47 6.98 + 0.60

synthetic hPTH(l-84) 1.45f0.27 2.43kO.22 4.00*0.17

1.5OkO.25 3.60+0.31 4.07+0.13

4.96 + 0.28 4.74 f 0.32

Stimulation of DNA synthesis in UMR 106 cells or in embryonic chick sterna has been assayed by measuring [3H]thymidine incorporation into perchloric acid-precipitable material. Each concentration has been tested with five cell or organ cultures, respectively. The untreated control cell cultures revealed an incorporation of 1076+ 109 cpm, the basal incorporation rate of the organ cultures was 76795+4748 cpm (C). Incorporation by cultures treated with hPTH is given as experimental per control value (E/C) with the standard error of the mean (SEMI.

different functional domains than the CAMP-dependent activities and which may act through the PLC/PKC and/or calcium pathway (Schliiter et al., 1989; Siimjen et al., 1990, Somjen et al., 1991; Jouishomme et al., 1992; Whitfield et al., 1992). It was therefore of interest to assay for these hormonal effects as well. Using either UMR 106 cells or embryonic chicken sterna organ culture, we found recombinant hPTH highly active, although both systems respond with different sensitivity (Table 3). It revealed virtually the same biological activity as synthetic hPTH(l-84).

4. Conclusion It thus turned out that in a number of assay systems, the recombinant human parathyroid hormone described here, [Pro- ‘I-hPTH(l-841, exerts biological activity which is indistinguishable from that of hPTH(l-84) despite the extra proline residue at the N-terminus. This proline extension may specifically disturb the induction of adenylate cyclase (Potts et al., 1982, and refer-

135

ences cited therein). However, at least in those systems investigated here, we did not obtain hints on such a perturbing influence. But, such influences might become obvious on other biological cell or organ systems, which tend to differentially respond to this hormone. Moreover, preliminary results in the analysis of the molecular structure by ‘H-NMR spectroscopy revealed that it is nearly identical with that of hPTH(l-84). Having previously resolved the three-dimensional structure of individual PTH fragments (Klaus et al., 1991; Wray et al., 19941, we now approach the solution structure of the whole hormone molecule by preparation of “N-1abelled recombinant [Pro-‘I-hPTH(l-84).

Acknowledgements

The authors wish to thank Mrs. Ute Willig, Mrs. Ina Schulz and Mrs. Gunda Sellenriek for expert technical assistance.

References Butcher, L.A. and Tomkins, K.T. (1985) A comparison of silver staining methods for detecting proteins in ultrathin polyacrylamide gels on support film after isoelectric focussing. Anal. Biochem. 148, 384-388. Goldman, M.E., Chorev, M., Reagan, J.E., Nutt, R.F., Levy, J.J. and Rosenblatt, M. (1988) Evaluation of novel parathyroid hormone analogs using a bovine renal membrane receptor binding assay. Endocrinology 123, 14681475. Harder, M.P.F., Sanders, E.A., Wingender, E. and Deckwer, W.-D. (1993) Studies on the production of human parathyroid hormone by recombinant Escherichia cd. Appl. Microbiol. Biotechnol. 39, 329-334. Harder, M.P.F., Sanders, E.A., Wingender, E. and Deckwer, W.-D. (1994) Production of human parathyroid hormone by recombinant Escherichia coli TGl on synthetic medium. J. Biotechnol. 32, 157-164. Jouishomme, H., Whitfield, J.F., Chakravarthy, B., Durkin, J.P., Gagnon, L., Isaacs, R.J., MacLean, S., Neugebauer, W., Willick, G. and Rixon, R.H. (1992) The protein kinase-C activation domain of the parathyroid hormone. Endocrinology 130, 53-60. Klaus, W., Diekmann, T., Wray, V. and Schomburg, D. (1991) Investigation of the solution structure of the human parathyroid hormone fragment (l-34) by ‘H NMR spectroscopy, distance geometry, and molecular dynamics calculations. Biochemistry 30, 46-52.

136

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of Biotechnology 39 (1995) 129-136

Kleen, R.F., Nissenson, R.A. and Strewler. G.J. (lY88) Forskolin mimics the effects of calcitonin but not parathyroid hormone on bone resorption in vitro. Bone Miner. 4. 247-256. Le Rudulier, D., Strom, A.R., Danekar, A.M., Smith, L.T. and Valentine, R.C. (1984) Molecular biology of osmoregulation. Science 224, 1064-1068. Lewinson, D., Shurtz-Swirski, R., Shenzer, P., Wingender, E., Mayer, H. and Silbermann, M. (1992) Structural changes in condylar cartilage following prolonged exposure to the human parathyroid hormone fragment (hPTH) 1-34 in vitro. Cell Tissue Res. 268, 257-266. Lowry, O.H., Rosebrough, N.J., Farr. A.C. and Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. Potts, J.T., jr., Kronenberg, H.M. and Rosenblatt, M. (1982) Parathyroid hormone: Chemistry, biosynthesis and mode of action. Adv. Protein Chem. 35. 323-396. Rasmussen, H. and Tenenhouse. A. (1968) Cyclic adenosine monophosphate, Ca “, and membranes. Proc. Natl. Acad. Sci. USA 59, 1364-1370. Schliiter, K.-D., Hellstern, H.. Wingender, E. and Mayer, H. (1989) The central part of parathyroid hormone stimulates thymidine incorporation of chondrocytes. J. Biol. Chem. 264, 11087-11092. Slovik. D.M., Rosenthal, D.I., Doppelt, S.H., Potts Jr., J.T., Daly, M.A., Campbell. J.A. and Neer, R.M. (1986) Restoration of spinal bone in osteoporotic men by treat-

ment with human parathyroid hormone (l-34) and 1,25dihydroxyvitamin D. J. Bone Mineral Res. 1, 377-381. Somjen, D., Binderman, I., Schliiter, K.-D., Wingender, E., Mayer, H. and Kaye, A.M. (1990) Stimulation by defined parathyroid hormone fragments of cell proliferation in skeletal derived cell cultures. Biochem. J. 272, 781-785. Somjen, D., Schliiter, K.-D., Wingender, E., Mayer, H. and Kaye, A.M. (1991) Stimulation of cell proliferation in skeletal tissues of the rat by defined parathyroid hormone fragments. Biochem. J. 277, 863-868. Whitfield, J.F., Chakravarthy, B.R., Durkin, J.P., Isaacs, R.J., Jouishomme, H.. Sikorska, M., Williams, R.E. and Rixon, R.H. (1992) Parathyroid hormone stimulates protein kinase C but not adenylate cyclase in mouse epidermal keratinocytes. J. Cell Physiol. 150, 299-303. Wingender. E., Bercz, G., Blocker, H., Frank, R. and Mayer, H. (1989) Expression of human parathyroid hormone in Escherichia cob. J. Biol. Chem. 264, 4367-4373. Wong, G.L., Luben, R.A. and Cohn, D.V. (1977) 1,25-Dihydrocholecalciferol and parathormone: effects on isolated osteoclast-like and osteoblast-like cells. Science 197, 663665. Wray, V., Federau, T., Gronwald, W., Mayer, H., Schomburg, D.. Tegge, W. and Wingender, E. (1994) The structure of human parathyroid hormone from a study of fragments in solution using ‘H NMR spectroscopy and its biological implications. Biochemistry 33, 1684-1693.