Calcium-binding Proteins in the Parathyroid Gland - jbc.org · Calcium-binding Proteins in the Parathyroid Gland 2793 tary cells. PSP (or Chromogranin A in the adrenal medulla), an

THE JOURNAL 0 1989 hy The American Society for Biochemistry

OF BIOLOGICAL CHEMISTRY and Molecular Biology, Inc.

Val. 264, No. 5, Issue of February 15, pp. 2792-2600,1989 Printed in U. S. A.

Calcium-binding Proteins in the Parathyroid Gland DETAILED STUDIES OF PARATHYROID SECRETORY PROTEIN*

(Received for publication, September 21, 1988)

Margarita LeiserS and Louis M. SherwoodS5T From the Departments of $Medicine and §Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461

In order to identify calcium (Ca2+)-binding proteins in the parathyroid gland, we used electrophoretic blots of proteins separated by a two-dimensional nondena- turingldenaturing gel system and incubated them with 4aCa2+. Parathyroid secretory protein (PSP) and proteins with approximate molecular weights of 98,000, 88,000, 58,000, and 30,000 were noted to bind Ca” in cytosolic fractions from bovine parathyroid, adrenal, and pituitary glands. However, differences in the binding affinity and capacity of the various proteins were observed. PSP displayed a low affinity and high binding capacity for Ca”. In the presence of 5 mM MgClz and 60 mM KC1, native PSP (immobilized on nitrocellulose filters) bound 7.5 mol of Ca2+/mol of protein monomer with an apparent K d of 1.1 mM.

Immunoblotting identified the association of PSP with parathyroid cell membranes in a Ca2+-dependent manner. This property, together with its heat stability, distinguished PSP from other cytosolic Ca2+-binding proteins which were identified. There was also evi- dence for a Ca2+-dependent protein-protein interaction (aggregation) of PSP present in a Nonidet P-40 extract of cell membranes. The high Ca2+ binding capacity of PSP and its Ca2+-dependent membrane association may be features that make PSP a potentially important protein in secretory cells.

Intracellular calcium (Ca”) homeostasis is maintained by proteins that bind Ca2+ preferentially and reversibly in the presence of other cations (1). Although the role of Ca2+- binding proteins as intracellular Ca2+ regulators through their Ca2+ buffering capacity is a general one, some are more efficient than others. Minute amounts of Ca2+-binding proteins in membranes acting as Ca2+ transporters are able to facilitate movement of large amounts of Ca2+ across the plasma membrane or the membrane of one of the organelles, i.e. Ca2+ channel (2), Na+/CaZ+ exchanger (3-5), or Ca2+- ATPase (6-8). A significant portion of intracellular Ca2+ is complexed by soluble (cytosolic) Ca2+-binding protein(s), although the limiting factor is the total amount of protein and its Ca2+ binding capacity. These proteins may be activated or change activity after binding Ca2+. Their major role seems to be the processing of the cytosolic Ca2+ signal, i.e. calmodulin present in all eukaryotic cells (9) and troponin C (10) in muscle are recognized as major mediators of cytosolic Ca2+ action.

* This work was supported in part by Grants DK-28556 and DK- 34822 from the United States Public Health Service. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “uduer- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

ll Current address: Merck, Sharp, and Dohme, Rahway, NJ 07065.

Of particular interest, in view of their localization, are proteins whose association with the membrane is enhanced by Ca2+ (11). Examples are protein kinase C (12), calmodulin (13-15), calcimedins (16), synexin (17), chromobindins (13, 18), lipocortin (19-21), and a number of other related proteins that have been detected in diverse systems. Geisow and Walker (11) described a group of Ca”-phospholipid-dependent membrane-associatedproteins that differ from calmodulin in that they lack an EF hand type calcium binding site; however, they share a consensus amino acid sequence of 17 amino acids. Their subcellular function is unknown, but they could have a specific role in mediating the transduction of the Ca2+ signal from the plasma membrane to the interior of the cell and in mediating the interaction between cytoskeletal and membrane components.

Some insight into the regulatory properties of Ca2+ through Ca2+-binding proteins could be accomplished by their identification in secretory cells. In most secretory cells, exocytosis is related to an increase in the free intracellular Ca2+ through stimulus-secretion coupling (22). Among secretory cells, the parathyroid has shown certain unique characteristics such as sensitivity to changes in extracellular Ca”. Increases in the concentration of extracellular Ca2+ within a narrow range elicit large increases in intracellular Ca2+ (22-28), while other secretory cells maintain a constant level of cytosolic Ca2+, even in the face of much larger changes. The mechanism of secretion in the parathyroid cell seems to be the reverse of stimulus-secretion coupling (29,30), in that more parathyroid hormone (PTH)’ is secreted in response to a decrease in extracellular Ca2+. These observations have led to the sugges- tion that there may be unique Ca2+-binding protein(s) on the parathyroid cell surface (“Ca2+ receptor”). Such a receptor could modulate parathyroid function and be the initial event in regulating secretion from the cell (31). However, the role of cytosolic Ca2+ in the secretory process of the parathyroid cell is unclear, and the activation of a putative receptor may lead to changes in the level of other intracellular signal(s) that activate exocytosis, i.e. the calcium-independent pathway (32). It remains to be determined if receptor activation (or occupancy) results in the formation of an inhibitory intracellular signal or the loss of a stimulatory one.

It was therefore of interest to identify Ca2+-binding proteins in the parathyroid and related secretory cells. In the present study, we have shown the presence of common Ca2+-binding proteins in the cytosol of the parathyroid, adrenal, and pitui-

The abbreviations used are: PTH, parathyroid hormone; PBS, phosphate-buffered saline; HEPES, N-2-hydroxyethylpiperazine-N’- 2-ethanesulfonic acid EGTA, [ethylenebis(oxyethylenenitrilo)] tetraacetate; PMSF, phenylmethanesulfonyl fluoride; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; PSP, parathyroid secretory protein; Stains All, l-ethyl-2-[3(1-ethyl- naphtol[l,2d]thiazolin-2-ylidene)-2-methylpropenyl]naphtho[l,2d]- thiazolium bromide.

2792

Calcium-binding Proteins in the Parathyroid Gland 2793

tary cells. PSP (or Chromogranin A in the adrenal medulla), an acidic glycoprotein cosecreted with PTH by the parathyroid gland (33-40) and with catecholamines (ie. epinephrine) in adrenal medulla (41), was identified as one of them. This protein was studied in detail and showed unique characteristics in relation to other cytosolic Ca2+-binding proteins, in- cluding heat stability, Ca2+-dependent membrane association, low Ca" binding affinity, and high Ca2+ binding capacity.

The translocation of PSP from the cytosol to the membrane could be an important event in signal transduction, leading to the association of the protein with specific polypeptides. Another Ca2+-dependent membrane-related event was observed with a portion of cellular PSP which is linked to the membrane in a state that requires detergent for solubilization, independent of the presence of Ca2+ or chelator. This fraction was shown to be sensitive to Ca2+ and to undergo protein- protein interaction (aggregation).

EXPERIMENTAL PROCEDURES

Preparation of Tissue Fractions-Fresh bovine parathyroid glands were trimmed of fat, washed in 0.3 M sucrose, and frozen in liquid NI. Frozen bovine parathyroid, adrenal, and pituitary glands were thawed, washed in 0.3 M sucrose, minced, and homogenized in 2 volumes of 0.15 M NaCl, 10 mM HEPES (pH 7.4) containing 2 mM CaC12 and protease inhibitors (10 pg/ml leupeptin, 5 pg/ml pepstatin A, and 0.2 mM phenylmethanesulfonyl fluoride (PMSF)) (buffer A), using 30-s pulses of a Polytron unit (Brinkmann Instruments) for 5 min at 4 "C and a Potter glass Teflon homogenizer (two strokes). The homogenate was centrifuged at 750 X g for 10 min; the pellet fraction was resuspended in buffer A and subjected to another burst with a Polytron and centrifuged as before. Both supernatants were collected, combined, and centrifuged at 30,000 X g for 30 min. The supernatant was centrifuged further at 100,OO X g for 60 min. The high speed supernatant obtained from the spin was dialyzed against 10 mM HEPES (pH 7.4) containing 1 mM EGTA, 50 mM NaCl, and 0.2 mM PMSF.

The 30,000 X g pellet was utilized for the extraction of Ca2+- dependent membrane-associated proteins by the following procedure. The 30,000 X g pellet was resuspended in buffer A using a motor- driven Teflon pestle homogenizer and then centrifuged again at 30,000 X g for 30 min. This step was repeated once to complete the washing of the particulate fraction in Caz+-containing buffer. The final pellet was resuspended (1 ml/g of original weight) in the same buffer containing 5 mM EGTA instead of Ca2+ (buffer B), incubated for 20 min on ice, and centrifuged at 30,000 X g for 30 min. The supernatant (EGTA extract) was recentrifuged at 100,OO X g for 60 min. The pellet was washed twice with buffer B and resuspended at 3 mg/ml in a solution of 1% Nonidet P-40 in 10 mM sodium phosphate, pH 7.2, 0.17 M NaC1, 3 mM KC1, 1 mM MgC12, 1 mM EGTA, and protease inhibitors (10 pg/ml leupeptin, 5 pg/ml pepstatin A, 0.2 mM PMSF) in order to prepare a detergent-soluble extract. When indicated, 2 mM CaCL was added instead of EGTA. After incubation for 30 min on ice, the suspension was centrifuged at 30,000 X g for 30 min to separate the insoluble (pellet) and soluble material (Nonidet P-40 extract). The extract was centrifuged further at 100,000 X g for 60 min.

The sample was concentrated by placing Sephadex G-100 outside a dialysis bag containing the dilute protein solution. The sample was then dialyzed further against a solution of 50 mM NaCl and 10 mM HEPES, pH 7.4 (buffer C). Nonidet P-40 extract was dialyzed against buffer C, before and after sample concentration. All steps were performed at 4 "C.

Preparation of Heat-stable Fraction-Following tissue fractiona- tion, 5 mM EGTA, 5 mM (3-mercaptoethanol, and l mM PMSF were added to the cytosol (100,000 X g supernatant) before heating for 5 min at 100 "C in a dry bath. The heated sample was then centrifuged for 30 min at 40,000 X g. The supernatant containing the heat-stable proteins was subjected to gel electrophoresis.

Preparation of Purified PSP-PSP was purified from fresh bovine parathyroid glands by our published method (40).

Gel Electrophoresis-The analysis of proteins by SDS-polyacrylamide gel electrophoresis was performed using the discontinuous buffer system of Laemmli (42) in 1.5-mm-thick 10% acrylamide slab gels.

Electrophoresis under nondenaturing conditions was carried out in detergent-free gels using an SE-200 vertical minislab unit (Hoefer Scientific Instruments). A gel of 0.75-mm thickness with Gel-bond PAG film (FMC) backing was used. One-fifth volume of a solution containing 50% glycerol, 150 mM Tris-HC1, pH 6.8, and 0.01% bromphenol blue was added to protein samples. The samples were applied to the lanes of a 3.5% polyacrylamide stacking gel (0.75 cm) that was prepared using the nondenaturing buffer system of Davis (43). The 10% polyacrylamide resolving gel (5 cm) was also prepared as described by Davis (43). When included, CaClz was present at a concentration of 2 mM in the gel and electrode buffer. Electrophoresis was carried out a t room temperature at a constant voltage of 120 V until the dye front migrated to the bottom of the gel.

Two-dimensional gel electrophoresis was performed using as first dimension the nondenaturing system described above. At the end of the run, the gel was divided into vertical strips and placed in equilibration buffer (0.5 ml/cm2) of 125 mM Tris-HCI, 0.1% SDS, and 1% (v/v) (3-mercaptoethanol for 30 min at room temperature. The cut lane was placed horizontally on top of the stacking gel of a 10% SDS- polyacrylamide slab gel (1-1.5 mm thick) for the second dimension electrophoresis. It was kept in place by the 1% warm agarose solution in equilibration buffer containing 0.01% bromphenol blue. The second dimension was a 10% acrylamide gel containing 0.1% SDS as described by Laemmli (42). Apparent molecular weights (M,) were calculated from a graph of relative mobility uersus log M, of standard proteins.

Western Blot Analysis-Protein samples were first separated by electrophoresis as described above. The resolved polypeptides were transferred electrophoretically to nitrocellulose filters (0.45 pm, Schleicher and Schuell) by the Western blotting procedure of Bur- nette (44). Transfers were carried out at room temperature for 16-18 h at a constant current of 0.1 A (30 V) in transfer buffer containing 25 mM Tris base, 192 mM glycine, and 20% methanol at pH 8.3. Proteins bound to the nitrocellulose were stained with 0.05% Coo- massie brilliant blue in 25% methanol, 10% acetic acid, and destained with 50% methanol, 3% acetic acid. The nitrocellulose sheet was incubated for 30 min at 40°C in a solution of 10 mM potassium phosphate buffer, pH 7.4, 0.15 M NaCl (PBS), containing 5% bovine serum albumin (buffer D). The sheet was then transferred to fresh buffer D containing antiserum to PSP diluted 1:lOOO at 4 "C for 16- 18 h (40). The filters were then washed for 10 min with PBS, twice for 10 min with PBS containing 0.1% Nonidet P-40, and for 10 min with PBS. The nitrocellulose sheet was then incubated with buffer D containing rabbit anti-guinea pig IgG (1:lOOO) for 30 min at room temperature. The filters were again washed as described above and incubated for 30 min at room temperature with fresh buffer D containing 12'I-protein A (2-5 X lo5 cpm/ml). The radioactive solution was aspirated and the nitrocellulose strips again washed as described above. The nitrocellulose strips were blotted with filter paper, dried, and exposed to Kodak XAR-5 film at -75 "C.

Ooerlrty Techniqw for Detectiorz of &Ca2+-binding Proteins-Label- ing of proteins with '%a2+ on nitrocellulose blots was carried out as described by Maruyama et al. (45). Proteins from cytosolic fractions or EGTA extract were electrophoretically transferred from polyacrylamide gels to nitrocellulose filters (0.1 pm, Schleicher & Schuell) as described above. Western blots were incubated in a solution containing 5 mM MgC12,60 mM KC1, and 10 mM imidazole HCl (pH 6.9) for 2 h with three changes of buffer. The nitrocellulose sheet was then transferred to new buffer containing 2 mCi of "CaClz/liter (1.7 mCi/ pmol) and incubated for 15 min at room temperature. The nitrocellulose sheet was washed with 50% ethanol, dried, and subjected to autoradiography. The proteins providing 45Ca2+ were visualized by exposure to Kodak XAR-5 film or Hyper film p-max (Amersham Corp.) at room temperature.

Solid Phase 45Ca2+-binding Assay-"'Ca2+ binding activity of crude and purified preparations of PSP was measured by "Ca2+ overlay on nitrocellulose paper as described above. Crude protein preparations of PSP were immobilized on nitrocellulose after two-dimensional gel electrophoresis (first dimension, nondenaturing gel; second dimension, SDS-PAGE) and electrophoretic transfer. Purified PSP (7-10 mg) in 50 pl of buffer (25 mM Tris, 192 mM glycine, pH 8.3) was applied to the wells of a Bio-slot microfiltration apparatus (Bio-Rad) and allowed to filter through nitrocellulose paper (0.45 mm, Schleicher & Schuell) prewet in the same buffer. After filtration, the wells were washed twice with 200 $1 of buffer. The nitrocellulose sheet was incubated further in a solution containing 5 mM MgC12, 60 mM KC1, and 10 mM imidazole HCl (pH 6.9) for 2 h with three changes of buffer. Rectangles of 1 X 1.5 mm, each corresponding to

2794 Calcium-binding Proteins in the Parathyroid Gland one well, were cut and incubated with the indicated concentrations of "Ca" in incubation buffer for 15 min at room temperature. The filters were treated further as described for the 45Ca2+ overlay technique. After autoradiography, the Ca2+ binding to PSP was quantified by excising the spots or slots corresponding to PSP from the nitrocellulose filter and determining the radioactivity by liquid scintillation counting.

Overlay Technique for the Detection of PSP-binding Proteins- Western blots of proteins from various fractions were incubated in 10 mM potassium phosphate buffer, pH 7.4, 0.15 M NaCl (PBS) containing 5% fraction V bovine serum albumin (buffer D) for 45 min at 40 "C. The nitrocellulose strips were transferred to fresh buffer D containing the indicated concentrations of PSP and incubated for 4 h at room temperature. The strips were washed with 10 changes of PBS (5-10 min). Binding of PSP was determined by incubation with anti-PSP antibody and '251-protein A, as described above.

Stains All Staining of Ca2+-binding Proteins-Staining with the cationic carbocyanine dye Stains All was carried out as described (46).

Radioiodination of Protein A-lZ5I-Labeled protein A (3 X 10' cpm/ pg) was prepared by the procedure of Schubart and Fields (47).

Protein concentration was determined bv the method of Peterson

Materials-Fresh bovine parathyroids were obtained from a local slaughterhouse and transported to the laboratory on ice. Bovine adrenals and pituitaries were purchased from Pel-Freez. '5CaC12 was purchased from Amersham Corp. Purified calmodulin and the cationic carbocyanine dye Stains All were obtained from Sigma. The prestained protein standards used to calibrate the SDS-polyacrylamide gels, myosin H-chain (MI = 206,000), phosphorylase b (Mr = 100,000), bovine serum albumin (Mr = 68,000), ovalbumin (MI = 42,000), a-chymotrypsinogen (M. = 25,000), p-lactoalbumin (Mr = 18,000), and lysozyme (Mr = 15,000) were purchased from Bethesda Research Laboratories. Sephadex G-100 was purchased from Phar- macia LKB Biotechnology Inc.

RESULTS

Identification of Cytosolic Ca2+-binding Proteins in Parathy- roid, Adrenal, and Pituitary Cells-The presence of Ca2+- binding proteins in the high speed supernatant (100,000 x g ) of cells disrupted in the presence of 2 m M CaC12 was studied by 45Ca2+ overlay on nitrocellulose blots. Several proteins showed the capacity to bind Ca2+ in the presence of 5 mM

A.

PARATHYROID

6.

ADRENAL

C.

PITUITARY

98 - 88 - 72 - 58 -

30 -

98 - 88 75 58 -

30 -

98 - 88 - 75 - 58 -

MgCl2 and 60 m M KC1 following separation by SDS-PAGE.

COOMASSIE BLUE

30 -

4 5 ~ a AUTORADIOGRAPHY IMMUNOBLOT -

I I

FIG. 1. Identification of common cytosolic Ca2+-binding proteins in parathyroid, adrenal, and pituitary. Soluble proteins (100,000 X g supernatant, 100 pg) from parathyroid (A), adrenal ( B ) , and pituitary (C) were separated by two-dimensional electrophoretic analysis (first dimension, nondenaturing gel; second dimension, SDS-PAGE) transferred to nitrocellulose and analyzed for 45Ca2+ binding as described under "Experimental Procedures." Panels a-c are nitrocellulose blots stained with Coomassie Blue. Arrowheads indicate the spots that are at the same position as the autoradiogram in the middle panel. Panels d-f are "Ca2+ autoradiograms of nitrocellulose blots. The major 45Caz+-binding proteins are marked 1, 2, 3, 4, and 5. Panels g-i are immunoblots after reactions with antiserum to PSP. The protein detected with antiserum corresponds to protein 3 in the 45Ca2+ autoradiogram. The numbers on the left are interpolated values in kilodaltons.


A B ,

4SC02*BOUND(pmoll ‘5Ca2*BOUNDlnmol/nmolSP-I)

FIG. 2. Ca” binding to PSP and P-88/P-98. The experiments were carried out by “CaZ+ overlay on nitrocellulose blots as described under “Experimental Procedures.” A, a crude preparation of soluble Caz+-binding proteins (88 pg) was separated on nondenaturing gel electrophoresis. The 1-cm section was cut out from the top of the gel and subjected to SDS-PAGE, followed by electrophoretic transfer. Identical nitrocellulose blots were incubated with various “Ca2+ concentrations. Panel a, autoradiogram of “Caz+ overlay. Lane 1, 100 pM; lane 2,300 pM; lune 3, 500 pM; lane 4, 1000 pM. Scatchard plot (56) of data derived from “Ca2+ binding to PSP (0) and P88/98 (0) was obtained as follows. Each point was obtained by quantification (liquid scintillation counting) of the “Ca2+ radioactive spot and subtraction of background. Background values were obtained from quantification of an equal-size nitrocellulose section without blotted proteins. Background was 35-67% of total binding with the highest value occurring at the highest Ca” concentration. The data presented are consistent with the results of three separate experiments. The data for PSP were fit to a straight line by least square analysis and had a slope indicating a K d of 0.92 mM and an X intercept of 92 pmol of rsCa2+. The amount of PSP present in cytosol was determined by radioimmunoassay with an antiserum raised against PSP (40). As- suming a molecular weight of 50,000 for the peptide backbone of PSP (57, 58) and for the cytosolic fraction of PSP of 2.1%, we calculated a stoichiometry of 2.5 mol of Ca2+/mo1. B, purified PSP (8.3 pg) was immobilized on nitrocellulose by filtration (slot blot microfiltration apparatus) and incubated with 45Caz+ as described under “Experimen- tal Procedures.” Panel b, autoradiogram of %a2+ overlay. Lane numbers correspond to lane numbers in panel a. Scatchard plot (56) of data obtained as described in panel a. Background was 35-70% of total binding. The data were fit to a straight line by least square analysis and had a slope indicating a K d of 1.1 mM and an X intercept of 7.5 mol of *Ca2+/mol of PSP. The data represent the average of two separate experiments.

These proteins were separated clearly by a two-dimensional gel electrophoresis system (first dimension, nondenaturing gel; second dimension, SDS-PAGE) and identified as proteins with molecular weights of 30,000, 58,000, 72,000 (parathyroid)/75,000 (adrenal), 88,000, and 98,000, respectively (Fig. 1).

It was anticipated that PSP/Chromogranin A should be present in the cytosolic fraction from parathyroid, adrenal, and pituitary cells (49). Indeed, immunoblots in Fig. 1 identified the 72,000 and 75,000 molecular weight Ca2+-binding proteins as PSP and Chromogranin A, respectively. The amount of PSP present in pituitary cytosol was below the sensitivity of detection with the 46Ca2’ binding assay.

We also examined the parathyroid samples with Stains All, a stain that has been used to identify Ca2+-binding proteins (46). Proteins with molecular weights of 30,000, 58,000, and 72,000 stained positively (blue), while the 100,000 region stained purple (Fig. 5A) . An 18,000-dalton blue-staining spot which had the same mobility as calmodulin was also seen. Adrenal and pituitary fractions showed the same blue-staining pattern observed for parathyroid, whether the second dimension was 10% SDS-PAGE or a system that resolves low molecular weight polypeptides (50). Although the pituitary form of PSP was not shown with the 45Ca2+ overlay technique,

45Ca Auto- Immuno- radiography blot ~n

1 L 2‘

f

- PSP

1 2 3 FIG. 3. Ca” binding properties of cytosolic proteins sepa-

rated on nondenaturing gels. 88 pg of parathyroid cell cytosol was analyzed by a nondenaturing gel system containing 1.6 mM CaC12. Lanes 1 and 2, autoradiogram of nitrocellulose blots after “Ca2+ incubation as described under “Experimental Procedures.” Lane 1, incubation with 1.6 p~ 45Ca2+ (2 mCi/liter); lane 2, incubation with 1.6 p M 45Ca2+ in the presence of 1 mM cold CaC12; lane 3, immunoblot of a third gel identical to that in lunes 1 and 2, after reaction with antibody against PSP. Numbers on the left point to the most prominent 45Ca2+-binding proteins.

it was clearly present with Stains All (not shown). Characterization of Ca2+ Binding Properties-Densitomet-

ric analysis (not shown) of the 46Caz’ autoradiograph (Fig. 1, panel d ) as well as protein stain (Fig. 1, panel a) estimated a higher ratio of Ca2+ bound to protein for proteins with molecular weights of 30,000 (P-30) and 58,000 (P-58) than for PSP and proteins with molecular weights of 88,000 (P-88) and 98,000 (P-98). The ratios were 1.7,3.0, 3.6,5.7, and >200 for P-98, P-88, PSP, P-58, and P-30, respectively. This observation suggested that the present method for detection of Caz+- binding proteins in crude samples might be sensitive to differences in affinity for Ca”. The inset in Fig. 2A (panel a) shows a direct correlation between 46Ca2+ concentration in the overlay solution and the intensity of the stain on the autoradiogram. Scatchard analysis of the data obtained by quantification of the radioactive spots (Fig. 2 A ) revealed that the K d for Ca2+ binding for P-88/P-98 was 0.16 mM and for PSP 0.92 mM in the presence of 5 mM MgClz and 60 mM KC1 at pH 6.9.

A shortcoming of this method is that the proteins being analyzed have been exposed to SDS and heat treatment. After gel electrophoresis, they were transferred to a nitrocellulose membrane without any renaturing process. Return to the original conformation of proteins might be difficult, and the degree of renaturation may vary for different proteins. There- fore, some Ca2+-binding proteins might lose Caz+ binding ability. The question was whether partial denaturation accounted for the lower Ca2+ binding affinity shown by PSP. Fig. 2B shows the binding of 46Ca2+ to nondenatured purified PSP, immobilized on nitrocellulose through filtration. Scat- chard analysis of data obtained under these conditions showed a K d for Ca2+ of 1.1 mM (Fig. 2B). While the Caz+ affinity values determined by both methods agreed (Fig. 2, A and B), the stoichiometry of Caz+ binding to PSP revealed 7.5 mol of Ca2+/mol of protein for native PSP, instead of 2-3 mol of Ca2’/mol of protein, suggesting that some 46Ca2+-binding sites might have been masked or not able to reform. Therefore, the next experiment compared the Ca2+ binding capacity of cy-


A. Coomassie Blue

M~ x

100 - 68 -

--a 42-

25 - 18 -

100 - 68 -

+Ca 42 -

25 -

18 -

3 2 1 Fraction No.

FIG. 4. Relative mobility of 4"Ca2+-binding proteins on nondenaturing gels as a function of Ca2+. Cytosolic proteins from parathyroid (150 pg) were subjected to nondenaturing PAGE in the presence or absence of 2 mM CaC12. Three 0.4-cm segments were cut from the top of each gel (fractions 1-3, fraction 1 being closest to the origin) and placed on the second dimension for separation on SDS- PAGE. Panel A , Coomassie Blue-stained blot. Arrowheads indicate the polypeptides that changed their position when Ca2+ was added to the first dimension. Open arrowheads correspond to the densities seen in the 45Ca2+ autoradiogram in panel B. Panel B, autoradiogram of nitrocellulose blots incubated with 45Ca2+ as described under "Exper- imental Procedures." Numbers on the left represent the molecular weight standards.

tosolic proteins from parathyroid cells under nondenaturing conditions. Nitrocellulose blots of proteins separated on nondenaturing gel electrophoresis and incubated with 4sCa2+ (2 mCi/liter) showed the presence of various Ca2+-binding proteins (Fig. 3, lane 1 ) . Two protein bands (Bands 1 and 2) bound 4sCa2+ prominently. Band 2 could be attributed to PSP (Fig. 3, lane 3) . On addition of 1 mM cold CaClz to a 4sCa2+ overlay solution, only PSP still showed 4sCa2+ binding (Fig. 3, lane 2). These results confirmed the selectivity of Ca2+ binding of all proteins with 4sCa2+ binding activity as well as the high Ca2+ binding capacity of PSP.

Relative Mobility of Caz+-binding Proteins as a Function of Ca2+-We next investigated the possibility that Ca2+ binding would alter the mobility of the proteins. A protein-protein or protein-polypeptide interaction (whether Ca2+-induced aggregation or an interaction with a target protein) could lead to an alteration in molecular weight. Such changes would be detected upon separation of proteins on nondenaturing gel electrophoresis in the presence and absence of Ca2+ (Fig. 4). To assess the possible change in relative mobility on Ca2+ binding, 0.4-cm segments were cut (starting from the top of the gel) and the proteins separated further on SDS-PAGE. In the absence of Ca2+, Ca2+-binding proteins PSP, P-88, and P- 98 were found in fraction 3 (Fig. 4B). However, in the presence of 2 mM CaC12, P-88 and P-98 moved to fraction 2, while PSP

B. 45Ca Autoradiography

d P- 98

- PSP - P-88

~ P -98

- PSP - P-88

3 2 1

remained in fraction 3 (Fig. 4B). Fig. 4A shows the corresponding changes in Coomassie Blue protein stain pattern. In addition to P-88 and P-98, a n~n-~'Ca'+-binding protein of molecular weight 55,000 also changed its relative mobility on addition of Ca2+ (Fig. 4A, fractions 2 and 3).

We also found that the addition of Ca2+ changed the mobility of P-30, detected as a Stains All blue-staining protein and as a 4sCa2+-binding protein (Fig. 5). Under the same conditions, the blue-staining 18,000 molecular weight protein (calmodulin) only redistributed partially (Fig. 5A) . Calmo- dulin was not visualized in the 4sCa2+ autoradiograph (Fig. 5B). Therefore, the Stains All staining appears to be a more sensitive procedure than 4sCa2+ overlay for calmodulin detection.

Whether a charge alteration after Ca2+ binding (51) or a protein-protein or protein-polypeptide interaction caused the changes in mobility of P-88, P-98, and P-30 is not known. The possibility of detecting a charge alteration had been minimized by the use of a gel buffer system that separates proteins at pH 9.5 (see "Experimental Procedures").

Immunologic Detection of PSP/Chromogranin as a Ca2+- dependent Membrane-associated Protein-Detection of PSP/ Chromogranin in the lysate and membrane fraction has been reported earlier (36,52). Therefore, it was of interest to study the ability of PSP/Chromogranin to interact with membranes in a Ca2+-dependent manner. This was accomplished by test- ing the immunoreactivity of the membrane extract obtained after solubilization with EGTA (Fig. 6).

When cell membranes (30,000 X g pellet) were homogenized in 0.15 M NaCl, 10 mM HEPES, pH 7.4, and 2 mM CaClz and washed twice, subsequent treatment with the same buffer containing EGTA led to the solubilization of PSP/Chromo- granin in the three secretory cells tested parathyroid, adrenal, and pituitary. The enrichment of this fraction in PSP/Chro- mogranin (Fig. 6, lane 3) eliminated the possibility that detection of this protein in membrane extracts was due to insufficient washing. Separation of the proteins on a two- dimensional gel electrophoresis system (nondenaturing SDS- PAGE) showed PSP as one of the proteins migrating in the 70-75-kilodalton range (Fig. 7, panels a and b). Moreover, a 4sCa2+ overlay on a nitrocellulose blot (Fig. 7, panel c ) showed PSP as the most prominent Ca2+-binding protein in the parathyroid fraction. None of the other cytosolic Ca2+-binding proteins were identified in the EGTA membrane extract. Consistent with this was the detection of only two blue- staining proteins with Stains All, PSP, and a 60,000 molecular weight polypeptide (data not shown).

Calcium-dependent PSP Interaction with Membrane-A fraction of the PSP found in the cell associates with the membrane in a Ca2+-dependent manner (Fig. 6). It was of interest to determine whether the interaction was through direct binding to membrane proteins. The approach used to detect binding of PSP to cellular proteins included separation on SDS-PAGE and transfer to nitrocellulose (53).

Nitrocellulose blots were incubated sequentially with media containing PSP, antisera against PSP, and '"I-protein A. Comparison of these autoradiograms with controls containing identical samples and processed through the same steps, with the exception of PSP overlay, allowed the identification of PSP-binding proteins.

Fig. 8 compares the binding of PSP to cytosolic and membrane polypeptides. In the presence of Ca2+, PSP bound three membrane-associated polypeptides of 27,000, 25,000, and 19,000 daltons, respectively. These polypeptides are tightly associated with the membrane, since they remain a component of the particulate fraction after detergent extraction.


A. STAINS A L L B. 4 5 ~ 0 AUTORADIOGRAPHY ~ ~ ~ 1 0 - 3 - - - - 206 -

- ”

100 - ,P-98

68 - 42 -

7 P-88 - (*)PSP - PSP - WP-5E - P-58

a b

I

25 - 18 -

I ,* .* -P-30

Ca - + - + FIG. 5. The effect of Ca2+ on the staining (Stains All) and radioactivity (.‘%a2+) patterns of cytosolic

Ca2+-binding proteins separated by two-dimensional gel electrophoresis. Two-dimensional gels of cytosolic proteins from parathyroid (162 pg) were carried out as described under “Experimental Procedures.” The first dimension (nondenaturing gel) was run in the absence (a) or presence ( b ) of 2 mM CaCL At the end of the run, a strip of each gel was placed on the same slab gel (10 X 14 cm) to undergo separation in the second dimension. Panel A , Stains All staining (46). The asterisks (*) on the right indicate a blue-staining protein. The uertical series of bands at the extreme left are the molecular weight standards, which were separated by electrophoresis in the SDS gel dimension. Panel B, autoradiogram of nitrocellulose blots incubated with 4sCa2+ (50 pM, 80 cpm/pmol) as described under “Experimental Procedures.” Arrowheads, indicate the spots that changed their position when Ca2+ was present in the first dimension. Arrows in panel A highlight the streaking of calmodulin (CaM) toward the top of the nondenaturing calcium containing first dimension.

1 2 3 A

72 -

B 75 -

C 72 -

Cyt + - - Mem - + - Ext - - +

FIG. 6. Ca2+-dependent cellular distribution of PSP/Chro- mogranin immunoreactivity. Protein fractions (15 pg) from parathyroid (panel A ) , adrenal (panel B ) , and pituitary (panel C ) separated on SDS-PAGE were transferred to nitrocellulose for immunoblot analysis with antisera to PSP (see “Experimental Procedures”). Lane 1, cytosolic proteins after high speed centrifugation (Cyt); lane 2, particulat,e fraction (30,000 X g ) washed with buffer A containing 2 mM CaCI2 (see “Experimental Procedures”) and resuspended in buffer A containing 5 mM EGTA (Mem); lane 3, EGTA extract after high speed centrifugation (Ext ) . Numbers on the left correspond to interpolated values from molecular weight standards in kilodaltons.

The identity of these polypeptides is unknown. Similar results were obtained when Western blots were incubated with purified PSP or a crude preparation (100,000 x g supernatant). PSP did not bind to polypeptides present in the cytosol. These results, combined with those shown in Fig. 4, demonstrate that the PSP present in the lysate has only membrane- associated protein targets. Calmodulin, on the other hand, mediates the Ca2+ action through the association with soluble and membrane-associated target proteins (51, 54).

Effect of Ca2+ on Nonidet P-40-extracted Membrane PSP- Immunoblot analysis of membranes after EGTA extraction is shown in Fig. 9. A significant fraction of PSP remained associated with the membrane after this procedure (Fig. 9, lanes 3 and 6). However, PSP could be extracted further from membranes using the nonanionic detergent Nonidet P-40. Fig. 9 shows the extraction to be independent of the chelation of Ca2+.

45Co - COOMASSIE BLUE IMMUNOBLOT AUTORADIOGRAPHY

206 - 100 - 1

* 68 - - PSP b x 42 - I L

25 - 18 -

FIG. 7. Ca2+-dependent membrane-binding proteins: Ca2+ binding properties. Ca2+-dependent membrane-binding proteins extracted from parathyroid membranes (31 pg) were subjected to two- dimensional analysis and electrophoretic transfer as described under “Experimental Procedures.” Panel a, Coomassie Blue-stained blots; panel b, immunoblot after antisera to PSP; panel c, autoradiogram of blots incubated with 45Ca2+. Arrowhead represents the protein corresponding to the density seen in the 45Ca2+ autoradiogram (panel c) and to the densities seen in the immunoblots (panel b). Numbers to the left represent the molecular weight standards.

To determine whether Ca2+ had an effect on protein-protein interaction of the membrane-associated PSP, the same approach as in Fig. 4 was used. Immunoblots of PSP extracted by Nonidet P-40 in the presence of EGTA showed various electrophoretic forms on a two-dimensional gel system (nondenaturing/SDS-PAGE) (Fig. 10, panel a). The lower molecular weight forms could be accounted for as degradation products or as species with lower degrees of glycosylation (36).

On incubation with Ca2+ at a final concentration of 2 mM and with Ca2+ present in the gel system, the most prominent electrophoretic form of PSP appeared as an entity with slower relative mobility (Fig. 10, panel b) . These results suggest that Ca2+ promoted a protein-protein interaction (aggregation) of PSP or, alternatively, a protein-polypeptide interaction. In contrast, under the same conditions, neither cytosolic PSP (Figs. 4 and 5) nor PSP present in EGTA-membrane extract (not shown) showed any apparent change in relative mobility.

Heat Sensitivity of Cytosolic Ca2+-binding Proteins-As a soluble Ca2+-binding protein, PSP/Chromogranin was unique in its Ca2+-dependent association with membranes (Fig. 7). Another unique characteristic was its heat stability. Chro- mogranin has been reported to be heat-stable (55), and it was therefore of interest to determine whether this property was shared by other cytosolic Ca2+-binding proteins. Fig. 11 shows PSP/Chromogranin as the only common heat-stable 45Ca2-

2798 Calcium-binding Proteins in the Parathyroid Gland a PSP

I I

PSP<Crude - + Purified - - + - + - "

_I__. . Y "4: -

68 - A2 -

-PSP

25 -

18 - E M C E M C E M E M E M - U

Co EGTA

FIG. 8. Calcium-dependent binding of PSP to membrane proteins. Western blots of cytosol ( C , 100 pg), particulate fraction remaining after EGTA and nonidet P-40 extraction (M, 100 pg), and nonidet P-40 extract (E, 36 pg) were incubated in the presence or absence of 11 pg of crude PSP or 7 pg of purified PSP (28 nM). The nitrocellulose filters were then incubated with antiserum against PSP and "T-protein A and visualized by autoradiography. When indicated, 1 mM CaClz or 5 mM EGTA was added to all steps of the procedure.

1 2 3 4 5 6 ' - "K." .%n"""paT " l C

- PSP

Ca - - - + + + EGTA + + + - - -

FIG. 9. Nonidet P-40 extraction of PSP immunoreactivity from EGTA-extracted membranes. Membranes after extraction with EGTA were resuspended in buffer containing 1% Nonidet P-40 in the presence of Caz+ (lane 6) or EGTA (lane 3 ) . After centrifugation (30,000 X g), PSP was almost absent from the pellet (lanes 2 and 5) and enriched in the supernatant: Nonidet P-40, EGTA extract (lane 1); Nonidet P-40, Ca2+ extract (lane 4 ) .

- Ca 206 -

h 100 -

' 68 - 9 2 x

42 -

4 + Ca b

FIG. 10. Separation of multiple electrophoretic forms of membrane-associated PSP on nondissociating polyacrylamide gel. Comparison of the two-dimensional electrophoretic pattern of PSP present in Nonidet P-40 extract from the parathyroid (60 pg) in the absence (panel a ) and presence (panel b) of 2 mM CaC12. Nonidet P-40 extract was dialyzed against 10 mM HEPES, pH 7.4, containing 50 mM NaCl before and after concentration of the protein sample. Panels a and b, immunoblots after antisera to PSP. Arrowheads point to the major isoelectric variants of PSP. The unmarked densities on the right occur at the position of aggregated protein that failed to enter the nondenaturing gel (first dimension).

binding protein in parathyroid, adrenal, and pituitary cells. The 30-kilodalton 45Ca2+-binding protein present in the parathyroid fraction (Fig. l lA, lune 1 and Fig. 11B) is apparently

A. B. 4 5 ~ a AUTO- RADIOGRAPHY I MMUNOBLOT I- 4 5 ~ a AUTORADIOGRAPHY I"- s"(, -V.?rI.I i 5: , .

72 - 72 -

30 - 30 -

1 2 3 4 5 6 FIG. 11. Heat stability of Chromogranin. Cytosolic fraction

(10-20 mg/ml) was heated for 5 min at 100 "C before centrifugation at 40,000 X g for 30 min. Proteins remaining in the supernatant were subjected to SDS-PAGE (panels a and b) or two-dimensional electrophoresis (nondenaturing/SDS-PAGE) (panel c) . Panel a, autoradiogram of nitrocellulose blots incubated with "Ca"; panel b, immunoblot after antisera to PSP. Lane numbers represent supernatants from parathyroid ( I and 4 ) , adrenal (2 and 5), and pituitary (lanes 3 and 6). Panel c, 45Caz+ autoradiography of heat-stable proteins from parathyroid.

unique to this cell since it was not detected in the other secretory cells, either by 45Ca2+ binding assay (Fig. 1lA) or by Coomassie Blue staining (not shown). The identity of this 30- kilodalton polypeptide is not known at this time.

DISCUSSION

These studies were prompted by a possible role for Ca2+- binding proteins in regulating the secretion of PTH by the parathyroid gland. Calcium-binding proteins appear to regu- late many cell functions via their interaction with subcellular targets after they bind Ca2+, i.e. calmodulin modulates the activity of its cytosolic targets phosphodiesterase, calcineurin (59), and membrane-bound Ca2+/Me-ATPase (54), while protein kinase C is translocated from the cytosol to the membrane upon activation with Ca2+ (60). Of particular interest are proteins whose association with the membrane is regulated by Ca2+ ( l l ) , i.e. chromobindins (18). Synexin, one of the chromobindins which has been characterized, has been suggested as a promoter of membrane fusion (17). An approach to determining the role of Ca2+-binding proteins in exocytosis is to seek a better understanding of the relationship between such proteins and their subcellular targets.

This report describes the presence of five common cytosolic proteins that bind Ca2+ in bovine parathyroid, adrenal, and pituitary cells (molecular weights 30,000, 58,000, 72,000/ 75,000, 88,000 and 98,000). Furthermore, immunochemical studies demonstrated the following: 1) PSP/Chromogranin corresponds to proteins with a molecular weight of 72-75,000; 2) cytosolic PSP translocates to the membrane on interaction with Ca2+; and 3) membrane-associated PSP undergoes a CaZf-dependent alteration in apparent molecular weight.

Membrane-associated Ca2+-binding proteins may play an important role in Caz+-mediated processes. Of the five Ca2+- binding proteins we identified, only PSP/Chromogranin showed Ca2+-dependent binding to membrane in vitro. This observation disagrees with that of Settleman et al. (52) who showed no release of protein from membranes with chelating agents.

The ability of PSP to bind Ca2+ may be involved in the Ca2+ requirement for the association of this protein with membranes (Figs. 6 and 8). PSP shows significant Ca2+ binding activity in the presence of 5 mM MgClz and 60 mM KCl. This result confirms previous reports on Ca2+ binding activity of Chromogranin A (61) and PSP (62), using a Ca2+ electrode


for estimating binding affinity and stoichiometry. Our studies reflect the Ca2+ binding properties of PSP in vitro at pH 6.9 in the presence of 5 mM MgClz and 60 mM KCl, factors that have been shown to affect Ca2+ affinity but not maximal Ca2+ binding (61). Under these conditions, a K d of 0.92 mM for PSP was compatible with values obtained for Ca2+-mediated function of other proteins that associate with the membrane in a Ca2+-dependent manner (i.e. synexin). The properties of self-association and membrane aggregation shown by synexin are regulated by low affinity sites (-200 PM) (13, 17). Fur- thermore, self-association is a property shown by the detergent-soluble fraction of PSP (Fig. 10). The physiologic signif- icance of such high values is uncertain. However, Ca2+ binding affinities in vitro may differ from the values in vivo (63); furthermore, the concentration of Ca2+ at the endoplasmic face of the plasma membrane may be significantly higher than the measured value for cytosolic Ca2+ (64). The lower affinity may then be a reflection of the fluctuations of Ca2+ concentration at the membrane and not in the cytosol.

PSP/Chromogranin is known to be associated with membranes (36, 52). In this report, we describe the extraction of PSP from membranes with a nonionic detergent (Nonidet P- 40) and the detection of various electrophoretic forms. Alter- ation in the molecular weight of PSP was detected when nondenaturing gel electrophoresis was carried out in the presence of 2 mM CaC12. It is of interest that 2 mM ca2+ promoted a possible protein-protein interaction (i.e. aggregation) or protein-polypeptide interaction of detergent-extracted PSP, while it did not have similar effects on PSP that was associated with the membrane (Ca2+ enhanced) or present in the cytosol. Gorr et al. (62) reported aggregation of purified soluble PSP at high Ca2+ concentration (5-10 mM). It has been suggested that PSP plays a role in the condensation of secretory granule content in parathyroid (62) and other endocrine cells such as the chromograffin cell of the adrenal medulla (49, 65). Such granules are known to have a high calcium concentration.

The question is whether the Ca2+-dependent association of PSP with membrane is a determining factor for its internal- ization within the membrane and further aggregation or vice- versa. It is possible that PSP (by going from cytosol to the membrane surface) promoted a change in the Ca2+ requirement for protein-protein interaction (i.e. aggregation). Pos- sible effects of membrane localization of PSP would include the following: 1) producing an effective concentration of PSP on the surface of the membrane; 2) facilitating PSP as a substrate for membrane enzymes; or 3) facilitating the interaction of PSP with specific substrates in the membrane, either proteins or lipids.

The experiments we have described in this study show PSP as a Ca2+-binding protein with unique characteristics. As a Ca2+-dependent membrane-associated protein PSP has utilized its calcium-binding sites for its association with specific membrane polypeptides (Fig. 8). Membrane-bound PSP did not appear to be associated with components of the membrane cytoskeleton (detergent-insoluble network underlaying the plasma membrane). Direct binding of PSP to nitrocellulose- immobilized polypeptides from partially purified microtubules and fodrin preparations was investigated. No bound PSP was detected immunochemically with antisera to PSP (not shown). In contrast, other Ca2+-binding proteins have been reported to associate with the membrane cytoskeleton in a Ca2+-dependent manner, i.e. a 68,000-dalton polypeptide (11, 63). They become a component of the Nonidet P-40-insoluble membrane fraction in the presence of free Ca2+. We have shown the solubilization of PSP with nonionic detergents

independently of the presence or absence of Ca2+ (Fig. 9). Heat sensitivity can further differentiate PSP from other Ca2+- and phospholipid-dependent membrane-associated proteins. Heat stability is a common property of PSP/Chromo- granin A and calmodulin (9). However, the Ca2+ binding affinity of PSP/Chromogranin is less than that reported for calmodulin but in the same range as the other Ca2+-binding membrane-associated proteins (13), which in turn have shown heat sensitivity (66).

The information presented in these studies contributes further to our understanding of secretory cells through Ca2'- binding proteins and suggests that the function of PSP in endocrine cells could occur on association with membranes. One postulated mechanism would be by Ca2+-induced changes in subcellular localization, which could mean a difference in accessibility to modification (e.g. phosphorylation or sulfation (67,68). If a modification caused changes in the Ca2+ affinity for protein-protein interaction and/or for protein-peptide interaction this would be reflected in the state of aggregation or in cellular distribution, respectively. However, the function of PSP/Chromogranin still remains obscure. Elucidation of the function of a specific target for PSP in the membrane is required, and it will be important to evaluate PSP as a potential substrate for membrane-associated protein kinases. Since PSP is found widely in secretory cells and granules, it could play a role in the exocytic process.

Acknowledgments-We wish to thank Jennifer B. Sherwood for excellent technical assistance and Luciana Lesch for preparation of the manuscript.

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Calcium-binding Proteins in the Parathyroid Gland - jbc.org · Calcium-binding Proteins in the Parathyroid Gland 2793 tary cells. PSP (or Chromogranin A in the adrenal medulla), an