Biochimica et Biophysica Acta, 1010 (1989) 177-183 Elsevier

177

BBA 12406

Parathyroid hormone degradation by chymotrypsin-like endopeptidase in the clonal osteogenic UMR-106 cell

Toru Yamaguchi, Masaald Fukase, Masashi Nish/kawa, Tadao Fujimi and Takuo Fujita Third Division, Department o/ Medicine, Kobe University School of Medicine, Chuo.ku, Kobe (Japan)

(Received 21 June 1988)

Key words: Parathyroid hormone; Peptide degradation; UMR-106 cell; Chymotrypsin; Endopeptidase; (Rat)

Parathyroid hormone (PTH) -degrading activity was studied using osteoblast-like UMR-106 ceils. P'IH-degrading activity was assessed by the amount of PTH fragments produced in the medium after exposure of intact human P T H - ( | - ~ ) to UMR-106 cells. PTH immunoreactiv|ty recovered in trichloroacetic acid-so|uble products of the medium and in fractions uluted from reverse-phase high-pedormance liquid chromatography (HPLC) was measured by radioimmunoassay using an antibody specific for the mid-region and C-terminus of ~ ' H . |n this study, intact UMR-106 on|is but not extraceHular enzymes cleaved human P T H ( | - ~ ) into fragments which were released into the medium (in a time- and temperatm'e-dependent fashion). HPLC analysis of the PTH fragments depicted three immunoreactive peaks (peaks | , 2 and 3) besides intact PTH, indicating a limited l~rH-hydrolyzing activity of the cells. Furthermore, a |000-fold molar excess of either hPTH-(3-34) or [NleS,NleaS,Tyr~]hPTH-(3-34)amide inhibited PTH-degrading activity by 63% and 80% of control, respectively, whereas neither calcitonin, vasopressin nor growth hormone suppressed it. Additionally, HPLC analysis of the samples treated with [NleS,NleiS,Tyr~ ]hPTH-(3-34)amide showed a reduction of the three peaks, suggesting an invo|vement of I ~ H receptor in the production of PTH fragments. This ~'H-degrading activity was strongly inhibited by phenyimethyisulfonyl fluoride and chymostatin, but not by soybean trypsin inhibitor, elastatinal or inhibitors of cysteine, aspartic or metalloproteinases, indicating that it is due to a seryl chymotrypsin-like endopepfidase. Chymotrypsin-like activity seems to be solely responsible for PTH-degrading activity in intact UMR-106 cells, since all three PTH fragments were predominantly suppressed in the presence of chymostatin. Further analysis of chymotrypsin-digested products of hPTH-(|-g4) eluted from HPLC exhibited five fragments detected by ultraviolet absurbance at 210 nm, three of which were measurable by PT~ radioimmunoassay, each corresponding to the three PTH fragments produced by UMR-|06 cells. To explore the cleavage sites of PTH further, amino acid analysis of chymotrypsin-deaved products was performed. The ¢~dts stTong|v support the view that the chymotrypsin-like enzyme in UMR-106 cells cleaved the hormone between residues 23-24 and 34-35, to produce, at leash hPTH-(24-~) and -(35-~). Our present study indicates that a chymotrypsin-like endopeptidase is solely responsible for limited hydrolysis of PTH by intact UMR-106 cells.

Introduction

The mechanism of immunoheterogeneity of para- thyroid hormone (PTH) in the blood [1] has been inten- sively investigated in vivo, and the cleavage of PTH into N-terminal and C-terminal fragments was demonstrated in the parathyroid gland [2,3], liver [4,5] and kidney

Abbreviations: (h)PTH, (human)parathyroid hormone; PMSF, phen- ylmethylsulfonyl fluoride; DMEM, Dulbecco's modified Eagle's medium.

Correspondence: T. Yamaguchi, Third Division, Department of Medicine, Kobe University School of Medicine, 7-5-2, Kusunoki-cho, Chuo-ku, Kobe 650, Japan.

[6,7]. However, previous studies on characterization of the PTH-degrading enzymes in these organs had been limited to tissue homogenate in vitro: cathepsin B-like PTH-degrading activity was rep~,,ted to split intact PTH into N-terminal and C-terminal fragments in the parathyroid gland and liver [8], and cathepsin D-like PTH-degrading activity in the parathyroid gland [3] and kidney [7]. However, recently, in the intact parathyroid cells and parathyroid slices [9,10], structures of secreted PTH fragments have been shown to differ markedly from those produced by cathepsin B- or D-like activities in the parathyroid homogenate, suggesting the involve- ment of non-cathepsin B- or D-like PTH-degrading activity in the production of PTH fragments released from intact cells.

0167-4889/89/$03.50 © 1989 Elsevier Science Pubhshers B.V. (Biomedical Division)

178

Compared to these organs, studies on degradation of PTH by bone cells have been limited until now. Freitag et al. [11] found that intact bovine PTH was degraded by isolated cell preparations of fetal rat calvaria. How- ever, the nature of the degrading activity has not yet been characterized. In this study, we investigated PTH- degrading activity in intact UMR-106 cells, which have some characteristics of mature osteoblasts, including PTH receptors coupling to a cAMP response [12] and alkaline phosphatase activity [13], and we explored whether or not cathepsin B or D would be responsible for PTH-degrading activity in intact bone cells.

Materials and Methods

Materials. The following chemicals were purchased: hPTH-(1-84), [NleS,Nlen,Tyr 34]hPTH-(3-34)amide, leupeptin, pepstatin A, chymostatin, elastatinal and phosphoramidon (Peptide Institute Inc., Minoo, Japan); soybean trypsin inhibitor, phenylmethylsulfonyl fluo- ride (PMSF), monensin, chloroquine and a-chymotryp- sin (Sigma Chemical Co., St. Louis, MO, U.S.A.). Ep475 was a generous gift from Dr. K. Tanaka (the Institute for Enzyme Research, Tokushima, Japan). hPTH- (35-84) was prepared using hPTH-(1-84) cleaved by cathepsin D [7]. hPTH-(3-34) and other synthetic frag- ments were kind gifts from Toyo Jozo Co. Ltd. (Shizuoka, Japan). Other reagents were of the highest quality available from standard suppliers.

Cell culture. UMR-106 cells were kindly donated by Dr. T.J. Martin (University of Melbourne, Australia). The cells were cultured using Dulbecco's modified Ea- gle's medium (DMEM) containing 1070 fetal calf serum.

PTH assay. PTH content in the samples was mea- sured by radioimmunoassay (RIA) using a Yamasa PTH-RIA kit (Yamasa Shoyu Co. Ltd., Choshi, Japan). This kit consists of th~ chicken PTH antiserum (CK9) raised by Slatopolsky et al. [14], nSl-[Tyr43]hPTH- (44-68) as a radioligartd and synthetic hPTH-(1-84) as standard. The concentration of hPTH-(1-84) required for half-maximal supression of radioligand binding to antiserum was 70 pmol/l, and those of hPTH-(39-68), hPTH-(44-68) and hPTH-(39-84) were each 44 pmol/l, whereas hPTH-(1-34), hPTH-(1-44) and hPTH-(69-84) caused no inhibition of radioligand binding even at 1000.fold molar excess [15]. Accordingly, this assay recognizes the intact PTH and its fragments containing at least the amino acid sequence of 44-68 in the intact PTH molecule.

Measurement of PTH fragments and cAMP production by intact UMR-106 cells. After reaching confluence, the monolayers of UMRo.106 cells (approx. 4.0.106 cells per 9.6 cm 2 dish) were washed three times with ice-cold phosphate-buffered saline and were incubated at 37 °C in 1 ml of serum-free DMEM containing 50 ng/ml (--5.3 nM) hPTH-(1-84). As previously described [16],

after an appropriate incubation period, PTH fragments produced by degradation of hPTH-(I-84) in 1 ml of medium were separated from intact PTH by an addition of 1 ml of 1070 trichloroacetic acid and 50/t l of 1070 bovine serum albumin as carrier to yield final con- centrations of 570 and 0.2570, respectively. After centri- fugation at 3000 rpm for 10 min, the supernatant (i.e., the trichloroacetic acid-soluble fraction) containing PTH fragments was washed three times with diethyl ether to remove residual trichloroacetic acid and PTH content was measured by RIA. Cyclic AMP in the medium was measured by RIA according to the method of Steiner et al. [17], using the antiserum kindly provided by Dr. K. Martin [18]. PTH and cAMP in the cells were also measured after washing with ice-cold phosphate- buffered saline three times and extraction by 1 ml of 670 trichloroacetic acid as described elsewhere [19].

High-performance liquid chromatography (HPLC) sys- tem. HPLC was performed using a Synchropak RP-P reverse-phase column (C18, 250 × 4.1 mm, SynChron, Inc., Linden, IN, U.S.A.) and a 0.170 trifluoroacetic acid-acetonitrile gradient system. Each sample was eluted under a linear gradient (10-27.570 of acetonitrile) during 35 min at a flow rate of 1 ml/min as illustrated in the figures. The eluates were collected in 1 ml frac- tions and freeze-dried. PTH content was measured by RIA after reconstituting with 0.1 M borate buffer at pH 8.4 containing 470 BSA.

Determination of chymotrypsin-induced products of hPTH-(1-84). 20 /tg hPTH-(1-84) was injected into HPLC after incubation with 0.2 U a-chymotrypsin in 1 ml of 0.1 M potassium phosphate buffer (pH 8.0) for 10 min at 37 ° C. Each degradation product of PTH was collected in a test tube and freeze-dried. These samples were prepared for amino acid analysis as follows. After dissolving in 1 ml of water and adding 40 /tl 2.5 /tmol/ml norvaline (as an internal standard) and 2 ml 6 M HCI, samples were hydrolyzed in evacuated sealed tubes with constantly boiling HCI for 24 h at 110°C. Amino acid analysis was then performed on a Biotronik LC-5000 automated analyzer using a Lithium-citrate buffer and Ninhydrin reaction.

Cell counts. The cells were dispersed with 0.0570 trypsin/0.0270 EDTA and were counted by a hemocy- tometer. Viable cells were determined by the Trypan blue dye exclusion method.

Statistical analysis. Statistical significance was as- sessed by Duncan's new multiple range test [20].

Results

Degradation of hPTH-(1-84) in UMR.106 cells As shown in Fig. 1, when UMR-106 cells (approx.

4.106 cells per 9.6 cm e dish) were incubated with 5.3 nM hPTH-(1-84) at 37°C, the cells caused a time-de- pendent increase in immunoreactive PTH levels in a

4

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500

o ~ 0 120 120

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Fig. 1. (A) Time course of PTH degradation by intact UMR-106 cells. Confluent UMR-106 cells in 9.6 cm 2 dish were incubated with 50 n g / m l ( = 5.3 nM) hPTH-(1-84) in 1 nd of serum-free DMEM at 37 o C. After incubation for the times indicated, the PTH content in the trichloroacetic acid-soluble fraction of the medium (o) and in the cell extract ( o ) was measured as described in Materials and Methods. For the control study, 1 ml of serum-free DMEM was incubated for 2 h with cells and the conditioned medium was transferred to another dish. The medium was incubated with 50 ng/ml hPTH-(I-84) in the absence of cells, then the PTH content in the trichloroacetic acid-solu- ble fraction of the conditioned medium (~} was measured. (B) Time course of cAMP content in the cell extract (o ) and medium (0). The cells were incubated with 50 n g / m l hPTH-(1-84) in 1 ml of serum-free DMEM containing 1 mM isobutyimethylxanthine at 37°C. The cyclic AMP content was measured as described in Materials and

Methods. Values are mean + S.E. of three different cultures.

I79

Effect of PTH antagonists on PTH-degrading activity To evaluate whether a receptor-mediated process was

hwolved in ~he PTH=degrading activity, UM;~-106 cells were incubated with 5.3 nM hPTH-(1-84) plus increas- ing amount of PTH antagonists. As shown in Fig. 2, a 1000-fold molar excess (5.3/tM) of either hPTH-(3-34) or [NleS,NlelS,Tyr34]hPTH.(3_34)amide markedly in- hibited PTH degradation. These peptides also sup- pressed the hPTH-(1-Sg)-stimulated increase in cAMP content. The inhibitory potency of hPTH-(3-34) was less effective than [NleS,NlelS,Tyr34]hPTH.(3_34)amide. In contrast, 1000-fold molar excesses of eel calcitonin, arginine vasopressin and human growth hormone did not suppress PTH-degrading activity. Further analysis for the samples from this experiment by HPLC showed that [Nlea,Nle ts,Tyr 34 ]hPTH-(3- 34)amide markedly re- duced the height of peaks 1, 2 and 3, compared to the control (Fig. 3C).

Effects of endopeptidase mhibitors on PTH-degrading ac- tivity

To determine whether this PTH-degrading activity was related to some kind of endopeptidases, UMR-106 cells were incubated with various endopeptidase inhibi- tors. 1-h pretreatments of the cells with 0.1 and 1 mM PMSF (a serine proteinase inhibitor) and 50 /tg/ml chymostatin (a chymotrypsin-like serine proteinase

trichloroacetic acid-soluble extract of the incubation medium. Degradation of immunoreactive PTH was completely prevented at 4 ° C. The conditioned medium itself preincubated with cells did not exhibit PTH-de- grading activity, indicating that the measured PTH frag- ments in the medium were not produced by extracellu- lar enzymes, but by the cells themselves. PTH content in the cell extract remained undetectable throughout the incubation period. Cyclic AMP levels in both cell ex- tract and medium increased in response to hPTH-(1-84) as shown in Fig. 1.

tligh-performance liquid chromatography (HPLC) analy- sis of degraded PTH in UMR-106 cells

Using HPLC, we further analyzed the tdchloroacetic acid-soluble products from the above experiment. In- creasing amounts of three immuroreactive intermediate peaks (peaks 1, 2 and 3) in fractions appeared in 60 and 120 rain incubations prior to the hPTH-(1-84) peak (Fig. 3A), compared to no such peaks at zero-time. The retention times of peaks 1, 2 and 3 were 15, 17, and 20 rain, respectively, with that of hPTH-(1-84) being 33 rain. Small amounts of immunoreactive PTH corre- sponding to hPTH-(1-84) in the tdchloroacetic acid- soluble fraction were found only at zero-time, but were not not found in either the 60 or 120 rain incubation.

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Fig. 2. Dose-dependent effects of hPTH-(3-34) ( @ ~ @ ) or [NleS,NlelS,Tyr34]hPTH-(3-34)amide ( o - - - - - o ) on PTH degradation (A) and cAMP production (B) by intact UMR-106 cells. Confluent UMR-106 cells in 9.6 cm 2 dish were incubated with 50 ng /nd ( = 5.3 nM) hPTH-(I-84) plus increasing concentrations of hPTH-(3-34) or [NleS,NlelS,Tyr34]hPTH-(3-34)amide, or with the addition of 5.3/tM of eel calcitonin (m), arginine vasopressin (<~) or human growth hormone fin) in 1 ml of serum-free DMEM containing 1 mM isobutylmethylxanthine. After a 60-min incubation, PTH-de- grading activity was assessed by measuring the PTH content in the trichloroacetic acid-soluble fraction of the medium. The cyclic AMP content in the cell extract and medium was also measured and the total cAMP content was calculated, hPTH-(3-34) (A- - A} and [NleS,NleiS,Tyr34]hPTH-(3-34)amide ( z x - - - - - z x ) alone showed no increase in the basal cAMP level. Values are mean+S.E, of five

separate incubations.

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Fig. 3. (A) HPLC analysis of PTH content in the trichloroacetic acid-soluble fraction of the medium after incubation of cells with hPTH-(1-84). 2.5 ml of the trichloroacetic acid-soluble fractions from Fig. 1A were fractionated by HPLC, and the PTH content in the eluant was measured by RIA as described in Materials and Methods. Arrows at the top show retention times of the indicated synthetic fragments. (13) HPLC analysis of the fragments of hP'I'H-(1-84) produced by purified a-chymotrypsin. 20 /~g hPTH-(1-84) was injected into HPLC after incubation with 0.2 U a-chymotrypsin in 1 ml of 0.1 M potassium phosphate buffer (pH 8.0) for 10 rain at 37°C. (C) Effect of [NleS,NlelS,Tyr34]hPTH-(3-34)amide on FrH fragmentation by UMR-106 cells. 2.5 ml of the trichloroacetic acid-soluble fractions from Fig. 2 were analyzed by HPLC, and the PTH content in the eluant was measured by RIA as described in Materials and Methods. o - - - - too , without PTH antagonists; O O, with 5.3 sM [NleS,NietS,Tyr~]hFrH.(3_34)amide. (D) Effect of chymostatin pretreatment on PTH fragmentation by UMR-106 cells. 3 ml of the trichloroacetic acid-soluble fractions from Table I were analyzed by HPLC, and the PTH content in the eluant was measured by RIA as described

in Materials and Methods. o - - ~ - - o , without inhibitors; e o, with 50 ltg/ml chymostatin.

inhibitor), respectively, reduced PTH-degrading activity (Table I). In contrast, even 6-h pretreatments of the cells with other inhibitors, including Ep475 (a cysteine

proteinase inhibitor), ~eupeptin (a cathepsin B inhibitor), soybean trypsin h,~:bitor, elastatinal (an elastase-like serine proteinase inhibitor), EDTA, phosphoramidon

181

~ B L E I

Effect o f various inhibitors on PTHDA in intact UMR-I06 cells

The results are the means of four separate incubations. Intact UMR- 106 cells (monolayers of 4.106 cells per 9.6 cm 2 dish) were prein- cubated for 1 h or 6 h at 37 o C with 2 ml of DMEM containing 10~ fetal calf serum and various inhibitors before the medium was replaced with 1 ml of serum-free DMEM containing 50 ng /ml hPTH-(1-84) and in~'bitors. PTH-dcgrading activity was assessed in the trichloro- acetic acid-soluble fraction after 60 rain of incubation at 37 o C.

Inhibitors Inhibition (~)

l h 6 h

Ep475 (50 ttg/ml) 0 0 Leupeptin (50 ffg/ml) 0 0 PMSF (0.01 mM) 0 0

(0.1 mM) 17 (1 raM) 78

Chymostatin (50/tg/ml) 81 Soybean trypsin inhibitor (1 mg/nd) 0 0 Elastatinal (50 pg/ml) 0 0 EDTA (1 raM) 0 0 Phosphoramidon (50 pg/ml) 0 0 Pepstatin A 00/~8/ml) 0 0 Chloroquine (300/tM) 0 0 Ammonium chloride (25 :,aM) 0 0 Moncnsin (10ttM) 0 0

(metalloproteinase inhibitors) or pepstatin A (an inhibi- tor of aspartic proteinase such as cathepsin D), did not inhibit PTH degradation. To examine the inhibitory effects of chymostatin on PTH-degrading activity fur- ther, the 1-h pretreated samples with chymostatin from the above experiments were chromatographed on HPLC

AsD Thr

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ii

Peak 6 j .

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Peak 8

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Fig. 4. Amino acid compositions of peaks 5, 6, 7 and 8 compared to the theoretical amino acid composition of hPTH-(35-84), -(24-84), -(1-23) and -(1-34). Peaks 5, 6, 7 and 8 were obtained after incuba- tion of hFrH-(1-84) with purified a-chymotrypsin (Fig. 3B). These peaks were prepared for amino acid analysis as described in Materials and Methods. Solid lines show the amino acid compositions of peaks 5, 6, 7 and 8, and dotted lines show the corresponding theoretical amino acid compositions of hPTH-(35-84), -(24-34), -(1-23) and

-(1-34).

and immunoreactive PTH was measured in each frac- tion. Chymostatin caused a marked reduction in the height of all three peaks (peaks 1, 2 and 3) (Fig. 3D).

Direct effect of chymotrypsin on cleavage of hPTH-(1-84) Since chymostatin inhibited the PTH-degrading ac-

tivity in UMR-106 cells, we further studied the direct effect of chymotrypsin on cleavage of hPTH-(1-84) using HPLC. As shown in Fig. 3B, estimated by ultra- violet absorbance at 210 nm, a-chymotrypsin cleaved hPTH-(1-84) into five metabolite peaks, designated 4, 5, 6, 7 and 8. Retention times of which were 15, 17, 20, 29 and 31 rain, respeetivdy. Peaks 4, 5 and 6 but not 7 and 8 were detectable by the PTH assay used in this study (data not shown). Both retention times and immunoreactivities of peaks 4, 5 and 6 corresponded to peaks 1, 2 and 3 produced by intact UMR-106 cells, respectively.

Amino acid analysis of chymotrypsin-digested products of hPTH-(1-84)

Amino acid analysis of peaks 5, 6, 7 and 8 are presented in Fig. 4 as well as the theoretical composi- tions for the hormone fragments 35-84, 24-84, 1-23 and 1-34. It is clear that peaks 5, 6, 7 and 8 closely match the composition of PTH-(35-84), -(24-84), -(1-23) and -(1-34), respectively. Peak 4 could not be identified.

Effects of lysosomotropic agents on PTH-degrading acti, ity

To determine whether this PTH-degrading activity would be related to a lysosomal process, we examined the effects of lysosomotropic agents. Chloroquine, am- monium chloride or monensin did not affect PTH-de- grading activity (Table I).

Discussion

In this study, we utilized osteoblast-like UMR-106 cells to determine more precisely the mechanism of PTH degradation in the bone. UMR-106 cells retain PTH-degrading activity in the intact state, since hFI 'H- (1-84) was shown to be cleaved into three discrete immunoreactive fragments in a time- and temperature- dependent manner, not by extraceUular enzymes but by the cell itself (Figs. 1 and 3A). This proteolytic activity of UMR-106 cells induced the release of PTH frag- ments into the medium. Since it is known that the lining osteoblast exposes its surface to the circulating blood in vivo, it seems possible that a part of PTH fragments in the circulating blood could be released from the bone, after cleavage of intact PTH by osteoblasts.

In another osteoblast-like cell line, ROS 17/2.8 [21], Rizzoli et al. [22] have shown that PTH antagonist

182

([NleS Nlelg,Tyr34]bovine PTH-(3-34)amide) prevented the binding of bovine PTH-(1-84) to the cell receptors. In this study, PTH antagonists (hPTH-(3-34) and [NleS NlelS,Tyr34]hPTH-(3-34)amide) prevented PTH- degrading acttvity in UMR-106 cells, but large amounts of calcitonin, vasopressin or growth hormone did not (Figs. 2 and 3C). These results from the two osteoblast- like cells suggest that a receptor-mediated process is possibly involved in PTH-degrading activity in UMR- 106 cells. Moreover, this PTH-degrading activity was likely to be of non-lysosomal origin, since lysosomal blockers (chloroquine, ammonium chloride or monen- sin) failed to inhibit PTH degradation (Table I).

This PTH.degrading activity appears to be due to a seryl chymotrypsin-like endopeptidase, since it was strongly inhibited by PMSF and chymostatin, but not by soybean trypsin inhibitor, elastatinal or inhibitors of aspartic, cysteine or metalloproteinases (Table I). In addition, all of the three immunoreactive PTH frag- ments produced by UMR-106 cells were exclusively suppressed by chymostatin pretreatment (Fig. 3D). Fi- nally, in the HPLC analysis (Fig. 3A and 3B), the coincidence of retention times of PTH fragments pro- duced by UMR-106 cells (peaks 1, 2 and 3) with those by purified chymotrypsin (peaks 4, 5 and 6), respec- tively, also supports the chymotrypsin-like nature of the enzyme.

Although direct amino acid analysis of the UMR-106 cell-generated fragments would be better, PTH-cleaved products sufficient for this analysis could not be ob- tained. However, the amino acid analysis of the corre- sponding chymotrypsin-generated fragments (Fig. 4) strongly suggests that the chymotrypsin-like enzyme in UMR-106 cells cleaved PTH between residues 23-24 and 34-35, to produce, at least, hPTH-(24-84) and -(35-84). Since hPTH-(1-84) contains tryptophan at position 23 and phenylalanine at position 34, these cleavage sites are compatible with the character of chymotrypsin, known to hydrolyze preferentially the peptide bonds in which the carboxyl group is contrib- uted by the aromatic amino acids phenylalanine, tyro- sine or tryptophan [23].

Recently, we reported [24] that opossum kidney (OK) cells, which possess characteristics of proximal renal tubules such as PTH receptors [25,26], cleaved hPTH- (1-84) between residues 23-24 and 34-35 by a non- iysosomal chymotrypsin-like enzyme. In addition, clea- vage of PTH between Trp-23 and Leu-24 was also reported by MacGregor et al. [9,10] in a study using intact parathyroid cells, and non-cathepsin B- or D-like activity was thought to be involved in this cleavage. In this regard, a similar mechanism of PTH degradation could exist in the three intact cells. Furthermore, the fact that PTH degradation in these three cells resulted ~a the release of fragments into the medium raises the possibility that circulating PTH fragments in the blood

could be produced by non-cathepsin B-or D-like activ- ity.

Cleavage of PTH between Trp-23 and Leu-24 is likely to be characteristic of a chymotrypsin-like endo- peptidase, since cathepsin B or D have been reported to degrade bovine PTH between Phe-34 and Val-35, but not between Trp-23 and Leu-24 [3,7]. To comfirm the role of a chymotrypsin-like endopeptidase in the pro- duction of circulating PTH fragments in vivo, we are presently trying to discover whether hPTH-(24-84) or hPTH-(1-23) exist in the human blood circulation.

Acknowledgements

We are thankful to Yamasa Shoyu Co., Ltd. for supplying us with PTH-RIA kits. This study was sup- ported by a Research Grant from the Japanese Ministry of Education, Science ai~d Culture.

References

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3 Hamilton, £W., Jilka, R.L. and MacGregor, R.R. (1983) Endo- crinology 113, 285-292.

4 Martin, K.J., Hruska, K.A., Greenwalt, A., Klahr, S. and Slatopol- sky, E. (1976) J. Clin. Invest. 58, 781-788.

5 Bringhurst, F.R., Segre, G.V., Lampman, G.W. and Ports, J.T. (1982) Biochemistry 21, 4252-4258.

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7 Zull, J.E. and Chuang, J. (1985) J. Biol. Chem. 260, 1608-1613. 8 MacGregor, R.R., Hamilton, J.W., Kent, G.N., Shotstall, R.E. and

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(1979) End~Jcrinology 104, 510-516. 12 Partridge, N.C., Alcorn, D., Michelangeli, V.P., Kemp, B.E., Ryan,

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