(Received for publication, November 28, 1994; and in revised form, February 17, 1995)
From the
Neuropeptide Y (NPY) receptors consist of three subtypes,
designated NPY
Neuropeptide Y (NPY) ( NPY receptors have been identified throughout the brain.
Two classes of NPY receptors with high and low affinity have been found
in the porcine brain(17, 18) , but not in the rat
brain(19, 20, 21, 22) . Functional
and binding studies have indicated three distinct NPY/PYY receptor
subtypes, Y NPY and PYY seem to have similar receptors in the brain and other
tissues (35) and have been shown to be highly conserved
throughout evolution(20) . NPY receptor subtype Y
Degradation of
Lectin chromatography was not used during
purification of NPY receptor because of its poor adsorption, as well as
the inadequate recoveries from the lectin affinity columns. For
assessment of the quantity of the glycosylation component, solubilized
cross-linked receptors and the purified receptors were deglycosylated
(37 °C for 3 h) with protease-free endoglycosidase F (0.5
milliunit) and O-glyconase (2 milliunits) enzymes
(Glycoanalysis, MA). Control samples were treated in an identical
fashion, but without enzymes. These samples were then concentrated by
chloroform-methanol precipitation (41) and subjected to
SDS-PAGE as described above. The cross-linked NPY receptor was
visualized with autoradiography and purified NPY-Y
Figure 1:
Effects of detergent concentrations on
the solubilization of NPY receptors and membrane protein by 1% octyl
glucoside. Membranes were diluted with buffers containing various
concentrations of phosphates and centrifuged at 30,000
NPY and PYY
displaced bound
Figure 2:
The specificity of NPY receptors:
membrane-bound (hatched bars) and solubilized receptors (shaded bars) incubated with
Figure 3:
A, the solubilized receptor preparation
was loaded onto a hydrophobic interaction chromatographic (HIC) column
at a high salt concentration (1% octyl glucoside in 1 M K
Figure 4:
A,
Autoradiography of cross-linked NPY-Y
NPY was readily coupled to Affi-Gel 10 in 50
mM sodium acetate buffer (pH 7). The coupling efficiency of
NPY to Affi-Gel gradually increased with the pH of the incubation
medium: 80% coupling was achieved at pH 8.0, but only 45% at pH 5.0.
The highest binding capacity of solubilized NPY receptors and the
highest yield of NPY receptors recovered from the NPY affinity columns
was found at the first application. This value was taken as 100% to
compare the receptor yields obtained during subsequent usage. The
efficiency of Affi-Gel columns in purifying NPY receptors was
dramatically decreased after repeated use. After being used 10 and 20
times, the efficiency of the column in isolating NPY receptors
decreased to 60% and 20%, respectively. Therefore, a newly prepared
NPY-specific Affi-Gel column could be used effectively only up to
It
was not possible to generate enough purity of NPY receptors from the
solubilisate by HIC, IEC, and lectin affinity chromatography, as
compared with specific affinity gel chromatography (Table 3).
However, the specific NPY-affinity chromatography step was highly
efficient in purifying NPY receptors as demonstrated by a considerable
increase in specific activity and the finding of a single protein band
corresponding to a molecular mass of 60 kDa on the SDS-PAGE and silver
staining (Fig. 5). Affinity cross-linking of
Figure 5:
SDS-PAGE of isolated and purified NPY
receptor. Proteins were visualized with silver stain, and molecular
weights were estimated against known molecular weight markers. Lane
a, affinity-purified NPY receptors after 1 cycle; lane b,
affinity-purified NPY receptors after 2
cycles.
Figure 6:
Autoradiogram of
When the purified NPY receptors were cross-linked with
Figure 7:
Autoradiogram of
Figure 8:
A,
This report demonstrates the first quantitative isolation,
purification, and biochemical characterization of NPY-Y A gradual loss of affinity of C-terminal
NPY fragments was observed with the progressive N-terminal truncation
of the NPY ligand (Table 2), which is characteristic of the
Y Affinity
chromatography has been used extensively to purify neurotransmitter
receptors(48, 49) . It is especially useful for the
purification of detergent-solubilized membrane proteins. The decreasing
ability of the NPY-Affi-Gel column to purify NPY receptors after usage
could be explained by hydrolysis of the ligand attached to the
insoluble support of agarose and/or the poor mechanical and chemical
stability that caused ``bleed off'' of ligand or
ligand-receptor conjugates. The efficiency of affinity gel was also
decreased by lipids building up on the gel matrix and by being clogged
by other particular matter, which could be expected after several
chromatograms. However, efficiency of the column was not improved by
using several methods of cleaning. Although this problem was minimized
by prior use of other separation methods such as HIC or IEC, the best
performance of the NPY-Affi-Gel columns was achieved only in the first
few runs. HIC, IEC, and lectin affinity chromatography (WGA and ConA)
by themselves were insufficient to purify the NPY receptor to
homogeneity, but, after the specific affinity chromatographic step, NPY
receptors appeared as a single band in SDS-PAGE (Fig. 5). The
purity of the isolated NPY receptor was further increased, and the
receptors were concentrated by a second cycle of specific affinity
chromatography. Highly purified NPY receptors retained their ability to
bind specifically to The molecular mass of the
NPY receptor obtained after protein purification was 60 kDa, similar to
that obtained after chemical cross-linking ( Fig. 4to 7) and by
HPLC gel chromatography (Fig. 8). The previously reported
molecular mass of the ``NPY-binding protein,'' after
cross-linking studies with bifunctional reagents, varied from 37 kDa to
70 kDa. A common receptor for NPY, PYY, and PP has been shown to be
When NPY receptors in the brain are similar to APP and PYY
receptors, in that they also bear carbohydrate moieties, including
mannose. The lectin adsorption studies and change of the M
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
, NPY
, and NPY
. The
Y
receptor has been cloned. The present study reports the
purification of the NPY-Y
receptor from porcine brain and
its biochemical characterization. NPY receptors were solubilized and
purified by sequential hydrophobic interaction, ion exchange, and
NPY-affinity chromatography. By use of SDS-polyacrylamide gel
electrophoresis, high performance liquid chromatography gel permeation
chromatography, and chemical cross-linking studies, the
affinity-purified brain NPY-Y
receptor was identified as a
monomeric glycoprotein with a molecular mass of 60 kDa. Following
deglycosylation, the molecular mass of the Y
receptor was
decreased to 45 kDa. Although the
I-NPY binding to the
purified NPY receptor was considerably decreased by N-ethylmaleimide, guanine nucleotides had no effect.
Therefore, the purified NPY-Y
receptor is probably not
associated with G-proteins, but may have intramolecular-free sulfhydryl
groups. The specific activity of the isolated NPY-Y
receptor is 15.8 nmol/mg of protein. The isolated receptor
retained its capacity to bind to
I-NPY, specific to NPY
and peptide YY, and showed no cross-reactivity with any other peptides.
Highly purified (10
-fold purification) NPY receptor from
the brain was identified as the Y
subtype as demonstrated
by its affinity to C-terminal fragments of NPY, including
NPY-(13-36).
)is a 36-amino-acid peptide (1, 2) and shares amino acid sequence homology with
pancreatic polypeptides (PP) (
50% homology) and peptide YY (PYY)
(69% homology)(1, 3) . It is a member of the
superfamily of peptides consisting of NPY, PP, and
PYY(1, 2, 3) . Human NPY and porcine NPY are
structurally identical except for the amino acid residue 17 (methionine
in human-NPY and leucine in porcine-NPY)(3) . NPY is abundant
in the central nervous system of all mammalian
species(3, 4, 5) , including
human(6) . Although the physiological functions of this peptide
are incompletely understood, many pharmacological and behavioral
effects of centrally administered NPY and homologous peptides have been
reported(3) . These effects include increases in fluid and food
intake(7, 8, 9) , changes in sexual behavior (9) and body temperature, decreased motor activity and induced
catalepsy, and increased muscle tone(10) . NPY is a potent
vasoconstrictor peptide and is present in the heart around the coronary
arteries and in the sinoatrial and atrioventricular
nodes(11, 12, 13) . NPY is often co-localized
and co-released with noradrenaline (14) and modulates
catecholaminergic responses in various
tissues(11, 13, 15) . As with the other
members of the NPY/PP family, NPY alters circadian rhythms when
injected into the suprachiasmatic nucleus (16) and elicits
numerous physiological responses by specific pre- and postsynaptic
receptors.
, Y
, and
Y
(15, 18, 23, 24, 25, 26, 27, 28, 29) .
Pharmacological characterization of NPY-Y
and -Y
receptor subtypes is based on the affinity of C-terminal NPY
fragments (e.g. NPY-(13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) )
to the Y
receptor, whereas Y
receptors interact
only with the intact NPY/PYY-(1-36)(26) . Quantitatively,
Y
receptors are the most abundant NPY receptor subtype in
the brain(18, 29, 30, 31) . NPY and
its receptors are highly concentrated in the limbic and olfactory
systems, hypothalamo-neuro-hypophyseal tract, corpus striatum, and in
cerebral cortex(17, 18, 32, 33) .
Based on electrophysiological and competitive binding data, NPY-Y
receptors have been found in the olfactory bulb, superficial
layers of the cortex, and hippocampus, localized to prejunctional sites
of neurons(31) , and Y
receptor in the lateral
septum, hypothalamus, thalamic nuclei, and area
postrema(4, 6, 19, 32) . In the
vasculature, Y
receptors are found at a prejunctional site
on sympathetic nerves(15) , whereas the postjunctional NPY
receptors are either the Y
or Y
subtype(26, 27, 28, 29, 34) .
,
but not Y
, was cloned and
sequenced(23, 36) . Because of the involvement of the
Y
receptors in neurophysiology (e.g. fluid and
food intake, sexual behavior, control of body temperature) and the
cardiovascular system, we decided to extend the studies with this
particular receptor subtype to further understand its involvement in
pathophysiology and to aid in possible future drug development.
Solubilization and analytical scale purification of NPY receptors have
been reported previously(18, 37) . The present study
was undertaken to isolate and purify the NPY-Y
receptor
with a view to raising monoclonal antibodies and to obtain partial
amino acid sequences for cloning the Y
receptor gene to
understand the actions of NPY at the molecular level. Pig brain was
chosen as the starting material because it is rich in NPY-Y
receptor and we could obtain unlimited supplies of fresh
material. This paper reports the first quantitative isolation,
purification, and biochemical characterization of NPY-Y
receptors from porcine brain.
Materials
NPY-(1-36) and its fragments
were synthesized using t-butoxycarbonyl chemistry in
solid-phase technology on an ABI-430A automated peptide synthesizer
(Applied Biosystems). Synthetic NPY was purified by ion exchange
chromatography, followed by reversed phase-HPLC and fully characterized
by fast atom bombardment mass spectrometry, amino acid sequencing, and
amino acid analysis. Monoiodinated I-Tyr-NPY was prepared
by a modified chloramine T method and purified by reversed phase-HPLC.
Bolton-Hunter-labeled [
I-Lys
]NPY
was purchased from Amersham International (specific activity 74
TBq/mmol (
2000 Ci/mmol)). Affi-Gel 10 and polyacrylamide gel
electrophoresis reagents were from Bio-Rad, disuccinimidyl suberate
from Pierce, molecular weight markers and protease inhibitors from
Sigma, and wheat germ agglutinin-agarose (WGA) and concanavalin A
(ConA) from Boehringer Mannheim. All other chemicals and reagents were
of reagent grade and were from Sigma. For the final purification
procedure, 1000 pig brains weighing
250 kg were collected in
batches from a slaughterhouse.
Preparation of Neural Membranes
Pig brains were
removed from decapitated heads, immediately placed in dry ice, and
stored at -70 °C. Experiments were carried out by thawing six
pig brains at a time. Spinal cord, meninges, cerebellum, and some white
matter were removed because these tissues are not rich with NPY-Y receptors. Brain tissues were dissected on ice and quickly
homogenized after addition of 5 volumes of freshly prepared ice-cold
homogenization buffer containing 50 mM HEPES, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 320
mM sucrose (pH 7.4). The dissection time and temperature were
kept to a minimum to prevent receptor losses due to enzyme activation.
Homogenized membranes were centrifuged at 1000
g for
10 min at 4 °C, and the supernatants were collected. The pellet was
then rehomogenized in 3 volumes of fresh homogenization buffer, and the
supernatants were pooled and centrifuged at 30,000
g for 45 min at 4 °C. The membrane-rich pellets were kept for
solubilization and assay for receptor activity. Identical results were
obtained on
I-NPY receptor binding activity with the
100,000
g microsomal pellet and with the cerebral
membranes prepared on a discontinuous sucrose gradient to give a
synaptosomal membrane preparation compared with 30,000
g crude membrane pellet. Therefore, for convenience and for the
handling of a large quantity of material, 30,000
g crude membranes were used for the NPY receptor purification.
Solubilization of Membrane-bound Receptors
Optimum
conditions for solubilization of active NPY receptors were assessed by
treating cerebral membranes with different detergents of various
concentrations. Cerebral membranes were resuspended at 2 mg/ml in 50
mM HEPES, 1 mM MgCl in the presence of 1
mM PMSF, and aprotinin, 100 units/ml. After addition of
detergent, the mixtures were gently rocked for 60 min at 4 °C. The
resultant mixture was then centrifuged at 30,000
g for
90 min at 4 °C in an MSE cooling centrifuge. The supernatant was
removed and assayed for
I-NPY binding and for protein
content. Several widely used detergents, including Triton X-100,
digitonin, sodium cholate, Zwittergent 3-14, Lubrol PX, CHAPS, and n-octyl-
-D-glucoside, were examined for
efficiency of solubilization of NPY receptors. The ability of NPY
receptors to bind to
I-NPY was measured under various
storage conditions with protease inhibitors, such as PMSF, bestatin,
and aprotinin, and other protectants, such as glycerol. The stability
of solubilized NPY receptors were assessed because the solubilized
receptors are more prone to degradation and denaturation than are
membrane-bound receptors.
NPY was iodinated with a modified
chloramine T method, purified by reversed phase-HPLC, and assayed as
described previously(18) . NPY receptor activity in the
cerebral membrane was measured by a binding assay with I-NPY Binding Assay for Membrane-bound
and Solubilized Receptors
I-NPY. In brief, 100-µl membrane suspensions (2.0 mg
of protein/ml) were incubated with 30 pM
I-NPY
in a 50 mM HEPES buffer (pH 7.0), containing 5 mM KCl, 1 mM PMSF, 200 kallikrein inactivating units of
aprotinin/ml in the presence of 0.2% (w/v) heat-inactivated bovine
serum albumin, in triplicate for 120 min at 4 °C, while they rocked
in a total volume of 200 µl. The label-bound membranes were
separated by centrifugation at 11,000
g for 2 min at 4
°C in a microcentrifuge.
I-NPY-bound solubilized
receptors were separated by adding 125 µl of 0.5% human
-globulin and then 500 µl of 30% polyethylene glycol. The
assay tubes were vortexed and incubated for an additional 10-min period
at 4 °C; 1 ml of 15% polyethylene glycol was then added, vortexed,
and centrifuged at 5000
g for 20 min at 4 °C. The
tubes were decanted, and the pellet was counted for the bound
I-NPY activity. Identical incubations were carried out
omitting the detergent extracts to obtain background counts, and these
were subtracted from the total and the nonspecific binding before
analysis. The difference in count in the absence and presence of excess
unlabeled NPY (0.5 µM) was taken as the specific binding
activity.
I-NPY was assessed by
precipitation with trichloroacetic acid and was routinely <5% at the
end of the incubation. Competitive binding data were analyzed by
computerized Scatchard analysis(38) . Specificity of
I-NPY binding to membranes and solubilized receptors was
examined by competition of NPY, NPY fragments, and various other
peptides in the presence of
I-NPY. In the experiments in
which competitive binding was assessed, concentration of unlabeled NPY
and related peptides varied from 0 to 1 µM. Membrane- and
octyl glucoside-solubilized NPY receptors were incubated with
increasing concentrations of
I-NPY in assay buffer, in
triplicate for 120 min at 4 °C to assess the kinetics of
I-NPY receptor binding.
Protein Concentration
The influence of protein
concentration and the detergent:protein ratio in the efficiency of
solubilization of NPY receptors were examined to determine the optimum
membrane protein:detergent concentration. Protein was estimated using a
dye-binding method (39) and a microplate reader to facilitate
measurement of a large number of samples of small volume (e.g. 50 µl).Hydrophobic Interaction (HIC) and Ion Exchange (IEC)
Chromatography
The solubilized NPY receptor solution was loaded
onto a phenyl-Sepharose CL-4B HIC column. The bound receptors were
eluted with an inverse salt gradient, and the fractions containing NPY
receptors were pooled and applied to an ion exchange column (IEC).
Preliminary experiments suggested that NPY receptors with a net
positive charge can be separated on cation exchange columns below their
isoelectric point. We decided to use the HIC and IEC columns because of
the capacity of these columns to handle a large quantity of solubilized
material, although the recovery of NPY receptors was not optimal. The
chromatographic fractions (11 to 18) from HIC containing NPY receptor
activity were pooled and dialyzed against HEPES buffer (10 mM,
pH 7.5). The dialyzed material from 2 to 3 HICs was pooled and applied
to a QAE-Sephadex A-25 ion exchange column. Bound NPY receptors were
eluted with an increasing ionic strength of NaCl up to 1 M.
The fractions (11 to 16) containing specific NPY binding activity were
pooled and dialyzed before being used in specific NPY-affinity
chromatography.Specific Affinity Chromatography of NPY
Receptor
Affinity columns were prepared by covalently coupling
synthetic NPY-(1-36) to activated agarose (Affi-Gel 10) (1 mg of
NPY/ml of Affi-Gel) because this was suitable for peptides having their
isoelectric points at neutral to basic pH, as in the case of NPY. The
optimum pH facilitating a higher coupling efficiency of NPY to Affi-Gel
was investigated by carrying out the coupling reaction at 4 °C for
4 h and using 50 mM acetate buffer of pH values from 5 to 8. A
known amount of I-NPY was also added to the unlabeled NPY
during coupling to Affi-Gel, and the percentage of incorporation was
determined by counting the radioactivity of the NPY-Affi-Gel complex
after washing the matrix three times. Pooled, dialyzed fractions
containing NPY receptors from 3 to 4 IECs were loaded onto the specific
NPY-affinity column. Columns were washed thoroughly with 10 mM HEPES buffer (pH 7.4), followed by glass-distilled water, and then
eluted with 1 mM acetic acid (pH 3.0). Eluted material was
neutralized immediately with 1 M NH
HCO
, and an aliquot was taken out to
estimate
I-NPY binding activity. The remainder was then
reloaded onto a second specific NPY-affinity column and eluted as
described above. Fold of purification at each step was calculated by
the improvement obtained in the quantity of the NPY receptor to protein
ratio.
Chemical Cross-linking of
Crude cerebral membranes (30,000
I-NPY to
Cerebral NPY Receptors
g pellet) were incubated at 1.0 mg of protein/ml in 50
mM HEPES buffer (pH 7.4), containing 5 mmol of KCl, 1 mM MgCl
, and 1 mM PMSF with 50 pM
I-NPY, for 120 min at 4 °C in the presence or absence
of 50 nM unlabeled NPY. Cross-linking experiments were also
performed in the presence of 0.5 µM calcitonin,
katacalcin, amylin, and calcitonin gene-related peptide (CGRP), 50
nM NPY-(1-36), PYY, PP, and NPY-(13-36). The
receptor-bound radioligand was separated by centrifugation, washed, and
resuspended in 1.0 ml of the same buffer. Disuccinimidyl suberate was
freshly dissolved in dimethyl sulfoxide, added to give a final
concentration of 1 mM, and was cross-linked for 30 min at 4
°C. The reaction was then quenched with 1.0 ml of ice-cold Tris/HCl
buffer (pH 7.4). The cross-linked receptor-ligand complex was pelleted
by centrifugation, washed, and solubilized with 1% octyl glucoside, 50
µl of 10% sodium dodecyl sulfate (SDS), and 20 µl of 20%
glycerol in the presence or absence of 2-mercaptoethanol. Similar
experiments were conducted with the affinity-purified receptor with the
exception of inclusion of additional C-terminal NPY fragments
NPY-(16-36), NPY-(22-36), and PYY-(1-36).
Cross-linked
I-NPY-receptor complexes were concentrated
using Ultrafree C3 (Millipore) and subjected to electrophoresis on slab
gels containing 10% polyacrylamide (SDS-PAGE) at a constant voltage of
140 V for 2 h(40) . The gels were stained with Coomassie
Brilliant Blue, destained, and dried. Autoradiographs were obtained by
exposing the dried gel to Kodak X-Omat AR film with enhancing screens
at -70 °C.
Lectin Affinity Chromatography
Fresh cerebral
membranes and I-NPY cross-linked membranes were
solubilized with octyl glucoside. Solubilized receptors were examined
for adsorption on immobilized lectins. Samples were loaded onto WGA or
ConA lectin columns pre-equilibrated with binding buffer. Columns were
washed, and the adsorbed lectin-bound material was eluted by adding the
appropriate competitive
monosaccharide-
-methyl-D-glucoside on ConA columns and N-acetyl-D-glucosamine (1 M) on WGA columns,
respectively. In the case of receptors cross-linked to
I-NPY, the radioactivity was counted on the eluted
material after precipitation by polyethylene glycol, whereas with a
solubilized uncross-linked receptor, binding activity was assessed with
I-NPY.
receptor
by Western blotting, with NPY-Y
-specific anti-receptor
monoclonal antibodies.
HPLC-Gel Permeation Chromatography
HPLC-gel
permeation chromatography was used to obtain an index of the molecular
size/configuration of the NPY receptor. Highly purified NPY receptors
were loaded onto a SW3000G column and eluted with pH 5.5, 0.2 M NHHCO
buffer containing 0.2% octyl
glucoside at room temperature. The chromatographic fractions were then
assayed for
I-NPY binding activity. Solubilized
cross-linked NPY-receptor complexes (in the absence of NPY and in the
presence of NPY, PYY, NPY-(13-36), or CGRP) were also studied in
a similar fashion in separate chromatographs, and radioactivity in the
fractions was counted. Molecular weight markers were chromatographed
separately on the same column, and the relative elution positions of
the receptor were calculated (K
)(42) .
The Effect of Different Detergents on the
Solubilization of NPY Binding Activity
The criterion of optimal
solubilization was judged as the one that yielded the highest I-NPY specific binding activity while releasing the least
amount of total protein (i.e.
I-NPY binding/mg
of protein). The specific binding of solubilized NPY receptors and the
percentage of the total protein solubilized with each detergent were
0.5% and 45%, 2.6% and 49%, 4.2% and 40%, and 8.5% and 38% for Triton,
digitonin, CHAPS, and octyl glucoside, respectively. Solubilization
with octyl glucoside was superior to that obtained with Triton, CHAPS,
and cholate. Among the various concentrations of octyl glucoside
examined, a 1% final concentration was selected because it solubilized
the maximum quantity of intact NPY receptors and the least proteins. In
a similar fashion, we examined the effects of various concentrations of
K
HPO
and the octyl
glucoside:K
HPO
ratio on NPY receptor
solubilization and found that optimum solubilization of NPY receptors
was achieved with 1% octyl glucoside and 125 mM
K
HPO
(Fig. 1). Concentrations below this
dramatically decreased the solubilization efficiency. The maximum
specific activity was detected at the detergent:protein ratio of 6:1
(mg/mg).
g for 90 min at 4 °C. The resulting supernatant and pellet were
tested for their ability to bind to
I-NPY and compared
with that of the membrane-bound receptors (n = 6).
Concentrations of K
HPO
used were: 1000
mM, 500 mM, 250 mM, 125 mM, and
62.5 mM, whereas octyl glucoside concentration remained at 1%.
The yield of solubilized protein (shaded bars) and NPY
receptors (hatched bars) is expressed as a percentage of the
amount of proteins or receptors in the fresh membrane preparation, as
determined by protein assay and
I-NPY binding,
respectively, as described under ``Experimental
Procedures.''
Stability of NPY Receptor
Pilot experiments
carried out after optimization of the solubilization procedure
indicated that approximately 200 separate solubilization experiments (6
brains/experiment) were required to acquire an adequate quantity of NPY
receptor for obtaining partial amino acid sequences of the NPY-Y receptor. Therefore, it was important to maintain the stability
and the integrity of the solubilized NPY-Y
receptor during
storage as well as throughout the purification procedure (i.e. to be able to utilize a specific NPY-affinity purification step).
When the stability of NPY receptors was tested at -20 °C for
a period of 1 month with different detergent concentrations, it showed
that the receptors were most stable in 1% octyl glucoside, 125
mM K
HPO
buffer than in higher
concentrations of the detergent. Loss of activity was proportional to
the increase in concentration of the detergent. However, a combination
of protease inhibitors (PMSF, bestatin, and aprotinin) and 20% glycerol
preserved
I-NPY binding activity and permitted storage of
the samples for 2 to 3 days at 4 °C or up to 35 days at -70
°C. This finding was crucial because HIC and IEC steps each took
several hours (usually overnight) to complete. The presence of 1 mM dithiothreitol increased the stability of NPY receptors during
freezing and thawing. Irrespective of temperature of storage, the
activity of the solubilized NPY receptor tends to decrease with time
and also is affected by temperature and ionic concentration of the
buffer.
Receptor Binding
I-NPY binding to
both crude membranes and the solubilized receptors was specific and
time- and temperature-dependent. At 4 °C, the binding reached a
steady state after 120 min and was maintained up to 3 h. Binding
properties of octyl glucoside-solubilized receptors were determined and
compared with those of membrane-bound receptors with competitive
binding of
I-NPY. Scatchard analysis of the data obtained
after increasing the concentration of
I-NPY suggested
that the binding of NPY to its receptors was saturable and disclosed a
curvilinear plot with upward concavity that suggested receptor
heterogeneity as previously reported(18, 21) . The
specificity of solubilized receptors was unchanged, and the binding
data for solubilized receptors were similar to the curvilinear plots
obtained for membrane-bound receptors(18) . Although the
detergent itself may interact with the ligand-receptor complex and
affect the Scatchard data, the dissociation constants (K
) obtained from the membrane-bound and
solubilized NPY receptors are comparable (Table 1)(18) .
However, the maximum binding capacity (B
)
expressed as femtomoles/mg of protein is doubled in the solubilized
fraction, but retained the same pharmacological specificity as those of
membrane-bound receptors ( Table 1and II).
I-NPY from the solubilized receptors with
half-maximal concentrations (ED
) of 1.1
10
and 1.6
10
,
respectively. Structurally related peptides porcine-, avian-, and
bovine-PP inhibited
I-NPY receptor binding only over 800,
2,500, and 15,000 molar excess, respectively. A number of other
peptides, such as vasoactive intestinal peptide, cholecystokinin-8,
insulin, growth hormone, calcitonin, amylin, and CGRP were totally
ineffective. The C-terminal fragments of NPY (e.g. NPY-(13-36)) displaced bound
I-NPY from
membrane-bound, solubilized, and purified NPY receptors in a
dose-dependent manner (Table 2). However, the N-terminal
fragment(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22) and
the
mid-molecule(18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28) of NPY
were ineffective.
Specificity of NPY Receptor
The radioactivity
remaining on the membrane-bound and solubilized cross-linked
ligand-bound receptor is shown in Fig. 2. The total binding was
found to be 30% of the total counts, which may represent both
cross-linked and specifically bound
I-NPY. The
nonspecific binding was <5% in both solubilized and membrane-bound
receptors. The addition of 0.5 µM (10
-fold
excess) salmon calcitonin, CGRP, katacalcin, amylin, porcine-PP, and a
number of other peptides to the solubilized receptor had no effect on
I-NPY binding (Fig. 2).
I-NPY and
cross-linked using disuccinimidyl suberate. The free unreacted tracer
was separated by centrifugation or precipitation of the
I-NPY-receptor complex by polyethylene glycol, and the
radioactivity of the pellet (total binding) was counted. In determining
the nonspecific binding and cross-reactivity, various unlabeled ligands
such as NPY, porcine pancreatic polypeptide (PP), katacalcin (KC), calcitonin (CT), CGRP, and insulin were
incubated with
I-NPY, and the radioactivity of the
resulting pellets was determined (n = 5 for each
column). Total binding of
I-NPY after cross-linking to
fresh membrane receptors (hatched bars) and solubilized
receptors (shaded bars). The figure shows that displacement of
I-NPY was determined in the presence of 50 nM NPY (i.e. nonspecific binding) and of 0.5 µM katacalcin (KC), calcitonin (CT), and calcitonin
gene-related peptide (CGRP).
I-NPY
binding to the membrane-bound and solubilized NPY receptors was
inhibited in a dose-dependent fashion by guanine nucleotides Gpp(NH)p,
GTP, and GDP, but not by GMP, as has previously been
reported(21, 34, 43) .
I-NPY
receptor binding was decreased by 80% in the presence of N-ethylmaleimide. With the exception of GMP, these compounds
increased the rate of dissociation of the receptor-bound
I-NPY and decreased their affinity for the high affinity
sites. Similar effects have been reported previously for NPY receptor
preparations from porcine brain (44) and rabbit
kidney(45) . These data suggest that the solubilized
NPY-receptor complex includes a guanine nucleotide-binding protein
(G-protein), but the guanine nucleotide effect was lost after the IEC
step during purification. The lack of effect of these compounds on the
I-NPY binding to the final isolated and purified NPY
receptors suggests that either G-protein may not be a part of the
purified receptor or that structural change of the protein had
occurred. Furthermore, C-terminal fragments of NPY competed with the
purified NPY receptor and indicated that the identity was the Y
receptor.
Isolation and Characterization of the NPY
Receptor
The solubilized receptor preparation was purified
sequentially by HIC and IEC. I-NPY binding activity was
eluted as a single, but broad, band of several chromatographic
fractions in both methods (Fig. 3). That this band coincided
with a large quantity of protein suggested that receptors were far from
pure. Nevertheless, sequential use of HIC and IEC allowed elimination
of the majority of lipids and a large proportion of unwanted proteins,
thus permitting the use of the resultant material directly on the
specific NPY-affinity columns. Lectin affinity chromatography showed
NPY receptors were readily adsorbed by WGA agarose. Of WGA
agarose-bound NPY receptors, 30% were eluted by the addition of 0.2 MN-acetylglucosamine. Receptors were also partially
adsorbed by ConA, and 39% of the receptors were desorbed with 1 M
-methyl glucoside. Deglycosylation of NPY-Y
receptor with endoglycosidase F and O-Glyconase enzymes
revealed an apparent molecular mass shift of approximately 15 kDa (Fig. 4).
HPO
(pH 7.4)) to maximize binding. The
concentration of the mobile phase ionic strength was then gradually
decreased with an inverse gradient up to 1% octyl glucoside in 10
mM K
HPO
to promote elution.
Chromatographic fractions were assayed for
I-NPY binding
activity (
) and for protein content (
). Mean data from 6
experiments are shown. The broken line shows the salt
gradient. B, the ion exchange column (IEC) findings: pooled
chromatographic fractions from HIC, containing NPY receptor activity,
were subjected to overnight dialysis against Tris-HCl buffer (10
mM, pH 7.5) at 4 °C, then loaded onto an IEC (QAE-Sephadex
A-25) at low ionic strength and eluted with increasing ionic strengths
of NaCl up to 1 M with 1% octyl glucoside. NPY receptor
activity (
) and protein content (
) of the chromatographic
fractions were determined. Mean data from 6 experiments are shown. The broken line shows the salt
gradient.
receptor. Lane
a, control; lane b, following treatment with
endoglycosidase F and O-Glyconase deglycosylating
enzymes.
10 times (e.g. to purify only
60 pig brains).
I-NPY
to brain membranes, before and after solubilization, was performed to
determine whether the solubilized NPY receptor retained its structural
integrity. Fig. 6shows an autoradiogram of SDS-PAGE after
cross-linking
I-NPY to membrane proteins and subsequent
solubilization. The figure shows a specifically labeled protein with an
apparent M
of
64,000 (Fig. 6, lane
2), which is comparable to the protein band with silver staining (M
60,000) as shown in Fig. 5. The
incorporation of
I-NPY into this M
= 64,000 band was completely inhibited in the presence of
50 nM unlabeled NPY and partially with NPY-(13-36) (Fig. 6, lanes 1 and 3) and suggested that the
labeled protein did indeed represent the solubilized NPY receptor.
I-NPY
cross-linked to brain NPY receptors. SDS-PAGE was performed under
reducing conditions (in the presence of 2-mercaptoethanol) on 10%
acrylamide gels. Each lane represents NPY-Y
receptors extracted from approximately 4 to 5 pig brains. The
gels were stained with Coomassie Brilliant Blue to visualize molecular
weight markers, destained, and dried. Autoradiographs were obtained by
exposing dried gels to Kodak X-Omat AR films with enhancing screens at
-70 °C. Lane 1, cerebral membranes cross-linked in
the presence of 50 nM unlabeled NPY-(1-36); lane
2, in the absence of NPY; and lane 3, in the presence of
50 nM NPY-(13-36). a, 97 kDa; b, 66
kDa; c, 58 kDa; and d, 42
kDa.
I-NPY, one could demonstrate the specificity further with
the competition with various NPY fragments and confirm the identity of
the isolated receptor as the Y
subtype (Fig. 7). In
the presence of 0.5 µM NPY,
I binding to
membrane-bound, solubilized, and purified receptor was completely
inhibited (Fig. 6, lane 1). After two cycles of
specific affinity purification, the final yield of NPY receptors was
3.0 nmol, and the purification efficiency was 0.8 µg of NPY
receptors/kg of porcine brain resulting in 10
-fold
purification (Table 3). The total recovery for the purification
procedure was 1.4%. The specific activity of the purified NPY-Y
receptor was 15.8 nmol/mg of protein, being 16.6 nmol/mg as the
calculated theoretical values for the pure NPY-Y
receptor.
I-NPY
cross-linked to affinity-purified NPY receptor (1 cycle). Cross-linked
purified NPY receptors were chromatographed on SDS-PAGE and
autoradiographed as described in Fig. 6. Each lane represents NPY-Y
receptors purified from approximately
15 to 20 pig brains. Lane 1, in the presence of
NPY-(16-36); lane 2, NPY-(22-36); lane 3,
PYY-(1-36); lane 4, NPY-(1-36); lane 5,
control; lane 6, NPY-(13-36); lane 7,
NPY-(1-36); lane 8,
NPY-(1-22).
Molecular Weight Assessment of the Y
Fig. 8A demonstrates the
size-exclusion HPLC profile (SW3000G column) of the affinity-purified
YReceptor by Size-exclusion
HPLC
receptor. The peak of specific binding activity with
I-NPY was eluted around M
=
66,000. Fig. 8B illustrates the profiles of
cross-linked preparations with
I-NPY in the presence and
absence of NPY-(1-36) or PYY and Fig. 8C, in the
presence of CGRP and NPY-(13-36). That the
I-NPY
binding was completely abolished in the presence of 50 nM NPY/PYY and partly by 50 nM NPY-(13-36), whereas
CGRP and other peptides were completely ineffective, suggested the
specificity and identity of the purified NPY-Y
receptor.
I-NPY binding activity
following gel permeation chromatography (SW3000G) of purified NPY
receptors. Specific binding activities of the chromatographic fractions
were detected by
I-NPY binding activity (
). B, solubilized cross-linked
I-NPY-receptor
complex was chromatographed, and radioactivity of each chromatographic
fraction was counted. Chromatographic profiles in the absence of
(
) and in the presence of (
) 50 nM NPY or
PYY-(1-36). C, chromatographic profiles of
I-NPY-cross-linked NPY receptors, in the presence of 0.5
µM CGRP, or amylin (
) and 50 nM NPY-(13-36) (
). A solution containing molecular weight
markers was chromatographed separately on the same column with a UV
detector set at 220 nm to assess the elution positions. Molecular
weight markers: a, fructose 6-phosphate (84,000); b,
bovine serum albumin (66,000); c, chicken albumin (45,000); d, carbonic anhydrase (29,000); e, cytochrome c (12,400); f, aprotinin (6,400); g,
KCl.
receptor. During purification, it was important to retain the
capacity of solubilized receptors to bind specifically to the ligand
for subsequent use in the specific affinity column because some
detergents denatured receptors with detergent-polypeptide chain
aggregates and caused a loss of receptor activity. The binding of
I-NPY to solubilized NPY receptors was specific and
saturable and exhibited high affinity. The kinetics and optimal
conditions for binding of
I-NPY to solubilized receptors,
such as pH, temperature, and ionic strength, were similar to those of
membrane receptors(18, 20, 22) . That N-ethylmaleimide significantly affected the
I-NPY binding to the solubilized as well as the isolated
NPY receptor suggested the existence of free sulfhydryl groups or
intramolecular disulfide bridges or both. Indeed, in previous studies
using N-ethylmaleimide and benextramine, the presence of
sulfhydryl groups in the Y
receptor has been
demonstrated(46) .
receptor(18, 31) . Previous studies have
shown that the brain NPY receptor did not distinguish between NPY and
PYY(26, 27, 28, 29) . NPY is found
in high concentrations in the
brain(2, 3, 4, 5, 6, 29) ,
whereas PYY is located mainly in the gastrointestinal
tract(1, 5, 14, 29) . Thus, the
Y
receptors in the brain (e.g. hippocampus and
cerebral cortex) where a high concentration of NPY is found are
probably ``NPY
receptors''(26, 30, 37, 47) .
Y
receptors on the renal tubular cells, where no NPY
innervation is found, may be PYY receptors, which responds to
circulating PYY(43, 45) . Taken together, that
purified NPY receptors specifically interact with C-terminal fragments
of NPY gives further evidence that the receptor isolated in this study
is the Y
subtype of the NPY receptor.
I-NPY.
65 kDa(24) , whereas the molecular mass of NPY receptors
has been reported between 52-59 kDa and 37-39
kDa(37, 50, 51) . Cross-linked studies have
shown that NPY/PYY receptors have two components, with molecular masses
of 70 kDa and 50 kDa representing Y
and Y
receptor subtypes(18, 26, 51) . There
seem to be differences in the size of the Y
receptor
(between 50 and 67 kDa) as demonstrated by cross-linking studies in
various mammalian
species(18, 20, 37, 51) . However,
the molecular mass of another member of this family, avian pancreatic
polypeptide receptors, in chicken and pigs was 67 kDa, whereas the
avian-PP receptor in dogs was 85 kDa(44) . These data suggest a
possible diversity of this family of NPY receptors in the brain and of
species differences. Whether this difference is due to major
differences in the amino acid sequences of these proteins, or simply
due to various degrees of glycosylation (Fig. 4), is yet to be
determined.
I-NPY was covalently attached to
membrane proteins, solubilized, and subjected to PAGE or cross-linked
to solubilized proteins, the same M
=
60,000 protein band was observed as in the native cerebral membranes.
This finding indicates that the solubilized NPY receptor retained its
structural integrity after solubilization and gives further evidence
that
I-NPY binds to the functional NPY receptor. Using
nondenaturing solvents in HPLC gel permeation chromatography revealed a
slightly higher molecualr mass of the purified NPY receptor (about 66
kDa), in contrast to that seen on SDS-PAGE-protein staining (60 kDa),
and by
I-NPY cross-linking studies with disuccinimidyl
suberate (64 kDa) ( Fig. 5to 8). We have used this isolated
NPY-Y
receptor for raising monoclonal antibodies.
Therefore, in the future, isolation of the Y
receptor can
be achieved with immunoaffinity purification using this specific
antibody.
after deglycosylation (Fig. 4) suggested
the glycoprotein nature of the brain NPY receptor, as in NPY-Y
and many other peptide receptors(49, 52) .
Deglycosylation experiments suggested that glycosylated products are
responsible for approximately 25% of the M
of the
intact NPY-Y
receptor. That receptor binding was markedly
decreased in the presence of the alkylating agent N-ethylmaleimide suggests the importance of free sulfhydryl
groups for binding within the Y
receptor (46) . A
similar effect was previously reported with avian PP receptors (53) . The M
remains the same in the
presence of reducing agents such as 2-mercaptoethanol and indicates
that the receptor consists only of one subunit. These results give
added evidence of the nature, identity, and integrity of both the
solubilized and purified NPY-Y
receptor. In summary, using
a preparative scale purification, NPY-Y
receptor was
quantitatively isolated and then biochemically characterized. The use
of receptor autoradiography, protein staining of purified NPY
receptors, and gel permeation chromatography of NPY receptors revealed
that the NPY-Y
receptor is a monomer of the 60-kDa
glycoprotein. This isolated 60-kDa protein had a specific activity of
15.8 nmol/mg of protein which approached the theoretical maximum of
16.6 nmol/mg, giving further evidence of the purity of the isolated
NPY-Y
receptor.
I am grateful to Dr. Richard Bukoski and Dr. Yogesh
Awasthi at the University of Texas Medical Branch at Galveston for
critical review of this manuscript. The technical assistance of F.
Zhang is greatly appreciated.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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Copyright © 1995 by the American Society for Biochemistry and Molecular Biology.
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Molecular and Cellular Proteomics
Journal of Lipid Research
Biochemistry and Molecular Biology Education