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Neurobiology Unit and John Holt Protein Structure Laboratory, St. Vincent’s Institute of Medical Research, Fitzroy, Victoria, 3065, Australia
Address all correspondence and requests for reprints to: Dr. Patrick M. Sexton, Neurobiology Unit, St. Vincent’s Institute of Medical Research, 41 Victoria Parade, Fitzroy, Victoria, 3065, Australia. E-mail: pms{at}rubens.its.unimelb.edu.au
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In addition to human CT-like immunoreactivity (hCT-I), salmon CT-like immunoreactivity (sCT-I) occurs in human brain extracts (8). Similarly, a biologically active sCT-like peptide is present in extracts of rat diencephalon (9). A role for a sCT-like peptide as a neurotransmitter/neuromodulator is supported by the presence of sCT-I within the CNS of lower vertebrates. In pigeon (10), the levels and distribution of sCT-I are consistent with our findings in rat brain (9), with the highest amount detected in the hypothalamus, followed by the midbrain, and low levels in brain stem. Immunohistochemical studies in lizard brain localized sCT-I to varicosities and terminals in the diencephalon, although not in other brain regions (11). Furthermore, the rat C1b CT receptor isoform has very little interaction with the thyroid-derived form of CT (12, 13). This receptor isoform is abundant in rat brain, exhibiting an almost parallel distribution to the C1a receptor (14). For this receptor to be physiologically meaningful, a CT-like ligand, distinct from the thyroid-derived form (such as the sCT-I molecule) must exist in rat brain.
Salmon CT-I is also released from cultured rat anterior pituitary cells
(15), and intense sCT-I staining occurs in the mouse pituitary
carcinoma cell line, 
-TSH (16). Although the physiological
significance of CT-like material in the pituitary remains to be fully
determined, CT receptors occur in the intermediate pituitary (14, 17)
and in -TSH cells (18), suggesting a possible paracrine role.
Accordingly, Shah et al. (15) demonstrated that anti-sCT
serum stimulates PRL release in cultured anterior pituitary cells,
suggesting that the sCT-like peptide in the anterior pituitary gland
exerts an inhibitory paracrine influence on PRL secretion.
The existence of multiple types of CT within one species is supported by the immunological detection of at least two forms of CT in fish, reptiles, amphibia, mammals and birds (19, 20). The existence of a gene expressing a sCT-like peptide in humans is also supported by hybridization of chicken CT cDNA to Northern blots of human medullary thyroid carcinoma tissue (21).
This paper describes the purification and partial characterization of CT-like activity from rat pituitary and diencephalon.
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Materials and Methods |
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Cell culture
Human embryonic kidney (HEK)-293 cells and HEK-293 cells stably
transfected with either the rat C1a or rat C1b CT receptor were
maintained in DMEM containing 5% FBS, 80 mg/liter gentamicin, 1
mg/liter minocycline, and 15 mM HEPES, at 37 C in a
humidified atmosphere of 5% carbon dioxide in air. The stable C1a CT
receptor expressing (D11; 
600,000 receptors/cell; passage 50–60)
and C1b CT receptor expressing (B8-H10; 6,000,000 receptors/cell;
passage 90) cell lines were generated as previously described (13)
with selection pressure maintained with 200 µg/ml geneticin.
sCT RIA
Synthetic sCT was iodinated using a modified chloramine-T method
as previously described (22) and purified by reverse-phase high
pressure liquid chromatography (HPLC) to a specific activity of 2000
Ci/mmol. The anti-sCT antisera (1464) was raised in rabbits using a
synthetic sCT analog R11,18K14 sCT (23)
conjugated to BSA as antigen. It was used in the RIA, described below,
at a final titer of 1:30,000, with a detection limit of 10 pg/tube.
The antibody is 100% cross-reactive to sCT and less than 0.1%
cross-reactive to hCT or rat CT (rCT). During the course of this work,
Hanna et al. (16) demonstrated, immunohistochemically,
presence of sCT-I in mouse -TSH cells (derived from a pituitary
carcinoma), using a commercial anti-sCT antibody (Peninsula, Belmont,
CA). As a consequence, this antibody (designated PAb) was also used for
RIA. PAb was used at a final titer of 1:24,000, with a detection limit
of approximately 3 pg/tube. The antibody is reported by the supplier to
be 100% cross-reactive with sCT, eel CT, and chicken CT but does not
cross-react with hCT, rCT, PDN-21, human and rat amylin, or human CT
gene-related peptide. Subsequent analysis of antibody specificity using
chimeric sCT/hCT peptides (ACT15, ACT19, ACT20, ACT27) indicated that
both antisera were C-terminally directed, with the primary epitope
located between residues 22 and 32 (not shown). Before assay, salt or
detergent was removed from samples by application to C18 Sep-Pak
cartridges, and elution using 3 ml 80% acetonitrile/0.1% TFA. The
eluates were frozen on dry ice, lyophilized, and stored at -20 C until
assayed.
For RIA, lyophilized samples were reconstituted in RIA buffer (0.1
M sodium phosphate, pH 7.2, containing 0.1% BSA and 0.02%
(wt/vol) sodium azide) and were assayed in duplicate. A standard curve
for sCT was constructed using 10–100,000 pg sCT. Nonspecific binding
tubes contained excess (100 ng) sCT. Tubes containing sample or sCT
standard were vortexed and incubated for approximately 16 h at 4
C. 125I-sCT (10,000 ± 2,000 cpm) was then added, the
tubes vortexed, and incubated at 4 C overnight. Separation of bound
ligand from free was achieved by the addition of 0.5 ml bovine
-globulin (3.0 mg/ml; Sigma) and 1.5 ml of 20% polyethylene glycol
(average molecular weight 8000; Sigma). The tubes were vortexed, and
incubated at 4 C for 15 min, before centrifugation for 15 min at
5,000 x g in a Beckman J-6 M/E centrifuge (Palo
Alto, CA) at 4 C. Supernatants were aspirated and the pellets counted
on a Packard -counter (70% efficiency; Packard, Downers Grove,
IL).
cAMP assay
Cells in 24-well plates were preincubated for 20 min, at 37 C,
in a humidified incubator with 5% CO2, in growth medium
containing 0.1% (wt/vol) BSA and 1 mM
isobutylmethylxanthine (this step was omitted for C1b receptor
expressing cells due to their high basal levels of cAMP; 13). Cells
were subsequently incubated for 20 min either with increasing
concentrations of sCT (sCT standard curve) or with lyophilized samples
from chromatography runs that had been reconstituted in the incubation
media. Samples were assayed in duplicate. Following incubation, cells
were washed with PBS (140 mM NaCl, 2 mM KCl, 1
mM KH2PO4, 8 mM
Na2HPO4) and the cAMP extracted with 0.5 ml of
absolute ethanol. Levels of cAMP were assayed using a specific RIA as
previously described (24).
Peptide purification
For small-scale studies, Sprague-Dawley rats (200–250 g) were
killed by decapitation and rat pituitaries and rat brain diencephalon
(principally hypothalamus) were rapidly removed and frozen by immersion
into isopentane cooled with dry ice. For large-scale purification, 3000
fresh frozen rat pituitaries and 4000 rat diencephalons were obtained
from Harlan Bioscience Products (Indianapolis, IN). Tissues were stored
at -70 C until processing. The procedures used for peptide
purification are similar to those developed by Bennett, Browne and
Solomon for the purification of pituitary peptides (25, 26).
A number of different extraction media and conditions were examined
before selecting a protocol of tissue-homogenisation, at 4 C, in an
acid mix media comprising 1 M HCl, containing 5% formic
acid, 1% TFA, 1% NaCl, 1 mM
phenylmethylsulfonyl-fluoride, 10 µg/ml pepstatin, 1 mM
EDTA, 10 mM benzamidine and 20 µg/ml leupeptin, as the
most efficient. Following homogenization, crude extracts were
centrifuged at 30,000 x g for 30 min at 4 C, the
supernatant filtered through Miracloth and then lyophilized. This
protocol was used for all tissue extractions in this study.
Protein levels were monitored by Bradford protein microassay (27) using
bovine -globulin as the standard. For efficient extraction in
large-scale purification, tissue was divided into batches of 500
diencephalon/250 ml or 1000 pituitaries/100 ml of acid mix media and
homogenized for approximately 1 min, at 4 C. Following centrifugation,
supernatants were pooled and filtered through Miracloth. The
supernatant was again divided into aliquots of 250 ml for diencephalon
or 100 ml for pituitary, placed in glass 1 liter round-bottom flasks,
frozen, and lyophilized.
Preparative chromatography
Vydac C18 chromatography. Vydac C18 resin was swollen in
20% acetonitrile/0.1% TFA and packed into a HR column (16/5)
(Pharmacia). This column was used as an initial step in protein
purification to desalt the tissue extract. The Vydac C18 column was
connected to a fast protein liquid chromatography (FPLC) system
(Pharmacia). Lyophilized sample was reconstituted in buffer A (10%
acetonitrile/0.1% TFA), passed through a 0.45-µm filter, and loaded
onto the column at a flow rate of 1 ml/min. The column was washed with
at least 30 ml buffer A at a flow rate of 2 ml/min. Batch elution was
achieved with 10–20 ml buffer B (90% acetonitrile/0.1% TFA), and
eluates were frozen and lyophilized.
Preparative cation exchange (CEX) chromatography was performed using a SP Sepharose HP (16/10) column connected to the FPLC system. Batch elution of tissue extract was performed. The CEX chromatography was performed at pH 5. For large-scale purification, lyophilized sample was reconstituted in buffer A (20 mM ammonium acetate, 40% acetonitrile, 0.1% TFA, 0.02% (vol/vol) Tween-20), passed through a 0.45-µm filter, and loaded onto the column at a flow rate of 1 ml/min. The column was preequilibrated and washed with 60-ml buffer A at a flow rate of 2 ml/min. Sample was batch eluted with at least 30 ml buffer B (buffer A + 1 M guanidinium hydrochloride [GuHCl], pH 5.0) and collected into TFA at a final concentration of 0.5%, to reduce the pH and thus prevent degradation. The eluate was frozen and lyophilized.
Sephadex G-50 size exclusion chromatography
To prevent noncovalent aggregation between CT-like peptides or
with other proteins, size exclusion chromatography (SEC) was performed
under denaturing conditions, in 6 M GuHCl. Sephadex G-50
resin was swollen in 6 M GuHCl, pH 3, for at least 3 h
at room temperature (22 C). The resin was degassed for 30 min and a
glass column (100 x 1.5 cm; Pharmacia) packed, and allowed to
settle for 1–2 days at room temperature. Blue Dextran 2000 (2 mg/ml)
was applied to the column to determine the void volume and to check the
homogeneity of the column bed before sample application. Lyophilized
sample was reconstituted in the SEC buffer (6 M GuHCl, pH
3) and glycerol added to a final concentration of 10% (vol/vol). The
sample was passed through a 0.45-µm filter before application. For
brain extract, an additional step of immersion into a boiling water
bath for 10 min and rapid cooling on ice was also included before
loading onto the column. The column was run under gravity. Fractions
were collected every 30 min into polypropylene tubes.
Preparative C8 reverse-phase HPLC
A Brownlee C8 reverse-phase column (100 x 10 mm) was
connected to a Waters 600 series HPLC (Waters, Milford, MA). Samples
were reconstituted in buffer A (20% acetonitrile/0.1% TFA), unless
otherwise stated, passed through a 0.45-µm filter, and loaded onto
the column at a flow rate of 1 ml/min. Elution was achieved using a
linear gradient of 20–60% acetronitrile/0.1% TFA at a flow rate of 3
ml/min over a period of 60 min. Fractions were collected every minute
and lyophilized before assay.
Analytical C8 reverse-phase HPLC
A Brownlee C8 reverse-phase column (250 x 4.6 mm) was
connected to the Waters HPLC system. Sample was reconstituted in buffer
A (10% acetonitrile/0.1% TFA), passed through a 0.45-µm filter, and
loaded onto the column. The flow rate was 1 ml/min. Elution was
achieved with a linear gradient of 20–50% acetonitrile/0.1% TFA over
60 min.
Narrowbore reverse-phase HPLC
Narrowbore columns were connected to a HP1090 series II liquid
chromatograph (Hewlett Packard Company, Waldbronn, Germany). Unless
otherwise stated, the narrowbore reverse-phase HPLC was performed as
follows. Sample was loaded onto the column at a flow rate of 250
µl/min, and elution was achieved using a linear gradient of 0–60%
acetonitrile/0.1% TFA at a flow rate of 100 µl/min. Fractions were
collected manually into 1.5 ml polypropylene tubes, frozen and stored
at -70 C.
Aminopeptidase digestions
Aliquots of peptide were diluted 1:5 with the appropriate buffer
(see below) before addition of enzyme. Trypsin (1.0 µg) digests were
performed for 18 h, at 37 C, in 0.1 M Tris-HCl, pH 8,
containing 10% (vol/vol) acetonitrile. Asp-N (0.2 µg) digests were
performed for 18 h, at 37 C, in 50 mM sodium
phosphate, pH 8, containing 10% (vol/vol) acetonitrile. Lys-C (1.0
µg) digests were performed for 18 h, at 37 C, in 25
mM Tris-HCl, pH 8.5, containing 1 mM EDTA and
10% (vol/vol) acetonitrile. Glu-C (0.2 µg) digests were performed
for 18 h, at 25 C, in 25 mM ammonium carbonate, pH
7.8, containing 5% (vol/vol) acetonitrile. Pyroglutamate digestion was
performed for 24 h, at 4 C, using 1 µg enzyme, in 0.1
M sodium phosphate, pH 8, containing 10 mM
EDTA, 5 mM DTT, and 5% (vol/vol) glycerol. Peptide digests
were chromatographed using the narrowbore hypersil C18 reverse-phase
column, as described above, with fractions manually collected into 1.5
ml polypropylene tubes containing 50 µl 0.2% Tween-20.
Reduction and alkylation of extracts
Small-scale extract was Seppaked and chromatographed on Mono S
(CEX), pH 5.0 and active fractions pooled and lyophilized. Half the
material was resuspended in 1 M Tris, pH 8.5/6
M GuHCl, containing 100 mM DTT. This sample was
incubated at 45 C for 2 h, then alkylated by addition of
iodoacetamide (0.3 M) for 1 h at 22 C. The material
was acidified to 2% TFA and chromatographed on C8 reverse-phase HPLC.
Control material was resuspended in 0.1% TFA.
Peptide analysis
Protein sequencing was performed on reverse-phase HPLC purified
peptides which were subjected to pulsed liquid phase protein sequencing
on an Applied Biosystems 471A Protein Sequencer (PE-Applied Biosystems,
Foster City, CA) or on a Hewlett Packard G1000A Protein Sequencer
(Hewlet-Packard, Palo Alto, CA).
For amino acid analysis, the sample was hydrolyzed under vacuum, at 110 C for 24 h in 6 M HCl, containing 0.2% (vol/vol) phenol and 0.2% (vol/vol) thioglycolic acid. Quantitative analysis was performed on a Beckman 6300 amino acid analyzer.
Mass spectrometry was performed on reverse-phase HPLC purified peptides on a PE Sciex (API3+) triple quadrupole mass spectrometer (PE-Sciex, Toronto, Canada) or on a Voyager-DE matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF) mass spectrometer (Perspective Biosystems, Framingham, MA).
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Results |
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While sCT-I was not detected using either of the anti-sCT antisera (not
shown), bioactivity was observed in the cAMP accumulation assay with
C1a transfected cells (Fig. 1
, B and C),
with the predominant activity peak occurring at approximately 31%
acetonitrile. No corresponding activity was seen when material was
assayed in untransfected cells, although a minor peak of activity,
eluting at approximately 29% acetonitrile, was detected in both cell
lines (Fig. 1, B and C). To confirm that the activity monitored by the
cAMP assay, in C1a expressing cells, was specifically generated by the
CT receptor, the material was assayed in the presence and absence of
the CT receptor antagonist AC512. Using 10-6 M
AC512, the dose-response curve to sCT was shifted approximately 1.5
orders of magnitude to the right (Fig. 1A). In the presence of this
concentration of antagonist, the major activity peak at 31%
acetonitrile was abolished (Fig. 1B). However, the minor activity peak,
detected in both transfected and untransfected cells, was not blocked
by the antagonist (Fig. 1C), indicating that stimulation of cAMP, in
this case, occurs by activation of unrelated receptors. Throughout the
remainder of this paper, the specific CT receptor-mediated activity
will be referred to as pituitary CT.
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For large-scale purification of pituitary CT, 3000 pituitaries were
homogenised in acid mix media, centrifuged and the supernatant filtered
through Miracloth and lyophilized as described. The first two steps in
the purification involved sequential batch application to and elution
from a Vydac C18 (5 x 1 cm) reverse-phase and a SP Sepharose HP
(16 x 1 cm) CEX column, respectively. Recoveries are listed in
Table 1.
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10 ml) than was optimal
for the column, due to the combined high concentration of protein and
GuHCl. Nonetheless, a significant purification (36-fold) was
achieved (Fig. 2; Table 1
). Active
fractions were pooled and diluted 1:1 with dH20, to reduce
the GuHCl concentration, and then fractionated on a preparative C8
reverse-phase column. Activity eluted in a single peak, corresponding
to approximately 31.5% acetonitrile (Fig. 3; Table 1). These fractions were then
applied to a Hypersil C18 column using HFBA as the ion-pairing agent.
Activity corresponded to the peak with a retention time of 34 min,
which was well separated from the bulk of the protein (Fig. 4A). This peak was subsequently applied
to a Hypersil C18 narrowbore column. The activity co-eluted with a
single, apparently homogeneous peak, with a retention time of 38.1 min
(Fig. 4B). An aliquot (40 µl; 1/10) of the active peak was
subjected to N-terminal sequencing by Edman degradation. However, no
sequence was obtained, indicating that the peptide was N-terminally
blocked. The remaining material was analyzed as described below.
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). No
digestion occurred with Asp-N, whereas only partial digestion occurred
with Glu-C. Novel, overlapping sequence was obtained from peptide peaks
from each of the trypsin [(K/R)S(E/Q)SP], Lys-C [KSQXP] and Glu-C
[EKSQXP] digestions. The consensus sequence for this fragment was
EKSQSP. In each case, the sequence ended abruptly with the proline
residue, independent of the level of peptide in the preceding cycle,
consistent with termination of the sequence. Comparison of the sequence
with the EMBL, GenBank, and SwissProt databases indicated no exact
matches to known protein and peptide sequences. An alignment of the
pituitary CT with known sequences for CT, amylin and CT gene-related
peptides sequences is present in Fig. 6.
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To further characterize the pituitary CT, aliquots of material were
subjected to amino acid composition and mass spectrometric analyses.
Although the amino acid analysis used does not detect cysteine or
tryptophan residues (due to degradation) and does not discriminate
between aspartic acid/asparagine (Asx) or glutamic acid/glutamine (Glx)
residues, the resulting composition revealed a peptide of at least 29
amino acids (Table 2). No methionine or
histidine residues were present; however, a low level signal for
tyrosine (0.45, Table 2) was present, and while this was called as
absent in Table 2, it is possible that one of the remaining residues is
a tyrosine. The presence of two residues designated Asx, combined with
the observation of lack of digestion by Asp-N, indicated that 2
asparagines were present. A search of the amino acid composition
against the SwissProt protein database
(http://expasy.hcuge.ch/ch2d/aacompi.html) did not identify any known
protein. Electrospray mass spectrometric analysis indicated two
molecular weight components: a major component of 3267 Da and a minor
component of 3540 Da; however, time-of-flight analysis revealed only a
single mass of 3264 Da, suggesting that the minor peak in the
electospray analysis may have been a contaminant. Based on the
protein concentration determined from the amino acid analysis and the
mass of 3267 Da, approximately 1434 ng of peptide was calculated to be
present following the initial narrowbore C18 separation. This is three
times greater than the level of peptide calculated from the cAMP
bioassay, assuming a specific activity equivalent to that of sCT (Table 1).
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Large-scale purification of sCT-like peptide from rat brain
diencephalon
We have previously shown that the rat brain diencephalon contains
sCT-I (9). In this study, we have carried out large-scale purification
of extracts from 4000 diencephalons. Preliminary analysis of material
separated by C8 reverse-phase HPLC, using RIA with either the 1464 or
PAb antibodies, indicated that the primary recognition products of the
two antibodies differed (not shown). Consequently, fractionated
material was assayed for sCT-I using both antibodies.
As with pituitary extracts, extractions were carried out in acid mix
media and initial purification used batch application to and elution
from the Vydac C18 reverse-phase and SP Sepharose CEX columns.
Recoveries for each step in the purification are listed in Table 3.
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). Interestingly, the RIA profiles
reveal three distinct peaks, with the level of activity in each peak
differing markedly depending upon the anti-sCT sera used. The
immunoreactivity in the first peak (SEC I) eluted in the void volume of
the column (mol wt > 13,700), whereas the immunoreactivity in the
second peak (SEC II) eluted just after ribonuclease A (mol wt 13,700),
and the immunoreactivity in the third peak (SEC III) eluted just before
aprotinin (mol wt 6,500). The 1464 antiserum principally detected the
two peaks of higher molecular weight (Fig. 7A). The PAb antiserum
detected these two peaks and also the lower molecular weight species,
SEC III (Fig. 7B).
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). In C1a
receptor-expressing cells, SEC I had no significant activity (Fig. 8A),
whereas undiluted SEC II appeared to give a small stimulation (Fig. 8B). No stimulation was observed in the untransfected cells. However,
SEC III stimulated significant cAMP production in C1a
receptor-expressing cells, in a dose-dependent manner (Fig. 8C).
Comparison of the activity levels in the cAMP assay and the RIA, with
sCT as standard, indicated approximately three times greater activity
in the bioassay (Table 3). A small response was also observed in the
untransfected cell line. This was most likely due to activity of non-CT
peptides, as the extract was still crude at this stage. In the C1b
receptor-expressing cells, which exhibit a high basal level of cAMP,
the cAMP levels were unaffected. Because the SEC III fraction was
biologically active and exhibited greatest separation from
contaminating protein, this material was used for subsequent
purification, with activity monitored by RIA with the PAb
antiserum.
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, Fig. 9). However, recoveries
were relatively poor, and no sCT-I activity was detected after
application of postnarrowbore material to a microbore SGE column
(250 x 1 mm) (Table 3).
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Discussion |
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Lack of recognition of the pituitary peptide by the anti-sCT antisera
was surprising, in particular, because the PAb antibody reveals,
immunohistochemically, sCT-I in the mouse pituitary carcinoma cell line
-TSH (16). The basis for this discrepancy is unclear but may be
related to different reactivities in immunohistochemistry
vs. RIA. Nonetheless, other anti-sCT sera have detected
sCT-I release from anterior pituitary cells (15). Immunohistochemical
staining of rat pituitaries with anti-hCT and anti-rCT sera has also
been reported, although the pituitary CT cross-reacted with only 2 of
15 anti-hCT/rCT sera (4). Together, the data indicate that, whereas the
pituitary CT shares structural epitopes with CT peptides, it is not
identical to either sCT or rCT. This was confirmed by the amino acid
composition analysis in Table 2 and by the unique molecular weight of
3267 Da. Recently, Shah et al. (31) reported that the
anti-sCT serum used in their studies may recognize the N-terminal
region of the sCT molecule containing the disulfide-bridged loop. This
suggests that the pituitary CT and sCT may have similar N-terminal
residues. As the N-terminus is highly conserved, it may also explain
the cross-reactivity of the limited number of antirat/human CT antisera
in the early immunohistochemical studies (4). If the principal site of
sequence convergence between pituitary CT and sCT-like peptides is in
the N-terminus, this would explain the lack of cross-reactivity with
the 1464 and Peninsula anti-sCT sera, which are C-terminally
directed.
Conservation of sequence in the N-terminus is not surprising as this region of CT is both highly conserved across species and is crucial for the agonist activity of CT peptides (32). Structure-activity studies demonstrate that residues 3–6 of sCT are particularly important for its biological activity (32). In the current study, the pituitary CT activated adenylate cyclase in cell lines with heterologously expressed CT receptors. The specificity of the response was demonstrated by 1) the stimulation of cAMP production in CT receptor-containing cells but not in the CT receptor-negative cells, and 2) abolition of activity by the CT receptor antagonist, AC512. Thus, conservation within the N-terminal region of the peptide is likely. In contrast, considerable divergence in the mid to C-terminus of the peptides can be tolerated without major effect on peptide activity. Most of the amphiphilic core of sCT can be replaced with other residues of similar hydrophobicity without markedly impairing either the biological activity or the ability to bind CT receptors (33). Similarly, AC413, a chimeric analog of sCT(8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32) and amylin, which is approximately 50% substituted by amylin residues, maintains high affinity for CT receptors (34). Therefore, the lack of cross-reactivity with the C-terminally directed anti-sCT anti-sera used in this study is not necessarily surprising.
Using conventional chromatography, an approximately 900-fold purification of the pituitary CT was achieved. Activity analyzed on the narrowbore reverse-phase HPLC column eluted as a homogenous peak, with a principal mass of 3267 Da. This differs from the molecular mass of both rat CT (3400 Da) and sCT (3432 Da). Based on the assumption that the purified peptide remained fully active, the pituitary CT is approximately 3-fold less potent than sCT. However, minor structural modifications during extraction and purification, which affect biological activity, cannot be ruled out.
The procedure used for the current amino acid analysis was unable to resolve all the component residues. However, based on the existing composition, the 3267-Da mass predicts a 32-amino acid peptide. As cysteine residues are degraded by this procedure, it is likely that the remaining residues include cysteines. Reduction and alkylation of the pituitary CT altered its retention time in reverse-phase HPLC, suggesting that disulfide-bridged cysteines are indeed present. This is consistent with the conservation of the cysteine-bridged N-terminal loop that occurs in all known CT peptides. The abrupt termination of sequence at proline, for each of the protease digestion peaks yielding sequence, implies that this sequence may represent the C-terminal end of the peptide. This would be consistent with the invariant proline at the end (position 32) of all known CTs. In CT, the proline is amidated and is also essential for biological activity (35).
The exact physiological role of endogenous pituitary CT is not resolved. Nonetheless, CT receptors are present in both intermediate pituitary (14) and anterior pituitary (15, 16, 31). Furthermore, release of endogenous CT-like peptides from cultured anterior pituitary cells has been demonstrated (15), and inhibition of PRL release occurs in response to both exogenous and endogenous CTs (15, 31). More recently, Shah and colleagues (31) demonstrated a marked in vivo increase in PRL secretion following injection of specific anti-sCT antisera. This antisera did not cross-react significantly with rat CT, suggesting that the action was mediated by binding to and inhibiting nonthyroidal CTs. Together, as proposed by Shah and colleagues, this data suggests that CT may be a physiological regulator of PRL secretion and that, in this action, endogenous pituitary CT may be acting in a paracrine or autocrine fashion.
Although purification to homogeneity was not achieved for the sCT-like peptide from diencephalon, a number of important observations were made. Diencephalon extract fractionated on a Sephadex G-50 column revealed multiple immunoreactive peaks. The 1464 antisera detected primarily the higher molecular weight peaks (SEC I and SEC II), whereas the PAb anti-sera detected both of the two peaks seen with the 1464 antisera, as well as a third form (SEC III), which had the highest biological activity. Multiple immunoreactive forms of CT also occur in tissue and blood samples, where 2–7 peaks are observed (36, 37, 38, 39, 40). While the number of peaks seen in these studies is dependent on antisera specificity, both antisera used in the current study are C-terminally directed and demonstrate similar general specificity for peptide recognition. The difference between the anti-sCT sera used in this study may relate to the sCT peptide used to raise the antisera as the 1464 was raised against an R11,18K14 modified sCT (23) conjugated to BSA, rather than native sCT. The identities of the higher molecular mass forms are unclear, although it is probable that they derive from precursor forms of the peptide (thyroid-derived procalcitonin is approximately 11,000 Da), or polymers of CT resulting from intermolecular disulfide bridge formation and/or high molecular weight complexes formed by disulfide bridge linkage of the monomer to an unrelated protein (36, 37). It is possible that SEC I and SEC II represent such high molecular mass forms of CT. Consistent with previous studies, the high molecular mass forms of SEC I and SEC II were essentially biologically inactive (36, 37).
Although the brain and pituitary CT-like peptides appear to be different, both are likely released from precursor forms. It is not surprising that high molecular mass forms of CT were not detected in the pituitary as they tend to be biologically inactive. However, bands of molecular mass of approximately 23 and 6.5 Da, along with a band equivalent to monomeric CT of 3.5 Da, were immunoprecipated with anti-sCT and anti-hCT sera from anterior pituitary cell lysates (15). This data suggest that precursor forms of the CT-like peptide also occur in pituitary. Whether or not both peptides derive from a common precursor remains to be resolved and relies on either full sequencing of the brain and pituitary peptides or isolation of their cDNAs.
In summary, conventional chromatography was used to partially purify CT-like peptides from both rat brain and pituitary, during which different assay systems were used to monitor their activity. Although the pituitary CT shares features of commonality with characterized CT peptides, determination of the amino acid composition and molecular weight indicated the purification of a novel peptide which may function as an endogenous regulator of pituitary function.
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2 Research Fellow of the Australian Research Council.
Received August 27, 1997.
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