From the
Chromogranins A and B have been known to undergo pH- and
Ca
Chromogranins A and B are found in the secretory vesicles of
virtually all neuronal and endocrine cells (Simon and Aunis, 1989;
Winkler and Fischer-Colbrie, 1992). They are co-stored with hormones,
ions, neurotransmitters, and other proteins and peptides in the
secretory vesicles (Winkler et al., 1986). Both chromogranins
are acidic proteins with isoelectric points of 4.5-5.5
(Fischer-Colbrie and Schober, 1987; Simon and Aunis, 1989; Winkler and
Fischer-Colbrie, 1992), and both bind Ca
In addition, chromogranins A and B are known to aggregate at acidic
pH levels in the presence of Ca
Although
purified CGB has previously been obtained either by elution from
SDS-polyacrylamide gels (Fischer-Colbrie and Schober, 1987; Benedum
et al., 1987; Gorr et al., 1989; Gill et
al., 1991) or by employing a boiling step (Fischer-Colbrie and
Schober, 1987), which is known to change the native conformation of
chromogranin A (Yoo and Albanesi, 1990b), chromogranin B has not been
purified in its native state so far. As part of efforts to elucidate
the biochemical property of CGB, purified native CGB was studied using
fluorescence and circular dichroism spectroscopy, gel filtration
chromatography, and pH- and Ca
The purified native CGB was obtained by subjecting the
secretory vesicle lysates of bovine adrenal medullary chromaffin cells
to several chromatographic and electrophoretic steps
(Fig. 1)
As had been observed with chromogranin A (Yoo and Albanesi,
1990b), the present results demonstrate that chromogranin B has
hydrophobic regions and also undergoes pH- and
Ca
Analogous to
chromogranin A, which had been shown to contain 60-65% random
coil with 25-40%
The aggregation of CGB was highly dependent on pH and
Ca
Furthermore, by comparing the amount of protein needed
to induce a given level of protein aggregation at a fixed
Ca
Given that pH- and Ca
I thank Dr. James Ferretti for use of the CD
spectrometer.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-dependent aggregation, and this property is
considered essential for the proper sorting of the vesicular matrix
proteins. In the present study, purified native chromogranin B (CGB)
from bovine adrenal medulla was used to study the pH- and
Ca
-dependent conformational changes and aggregation
property. Similar to chromogranin A (CGA), which had been shown to
undergo pH- and Ca
-dependent conformational changes
and to be composed of 60-65% random coil with 25-40%
-helicity, chromogranin B was also shown to consist of
65-70% random coil, 15-25%
-helix, and 10-15%
-sheet structures. The high percentage of random coil suggests
that CGB behaves hydrodynamically as an asymmetric molecule, thus
explaining its anomalous migration on SDS-polyacrylamide gels. Further,
CGB eluted from a gel filtration column in the volume indicative of a
globular protein with molecular weight of
200,000 at both the
intravesicular pH of 5.5 and a near physiological pH of 7.5.
Considering that dimeric CGA eluted from a gel filtration column in the
position suggestive of a 300-kDa protein, this result indicated that
CGB exists in a monomeric state at both pH levels. Like CGA, which
exhibited greater aggregation at pH 5.5 than at pH 7.5 upon
Ca
binding, CGB also aggregated much more readily at
pH 5.5 than at pH 7.5. However, there was a marked difference in the
aggregation properties of CGA and CGB with regard to their sensitivity
to Ca
: CGB was at least 2 orders of magnitude more
sensitive to Ca
than CGA. This suggested that, in
spite of the low concentration of CGB (approximately one-tenth that of
CGA) in bovine adrenal chromaffin cells, CGB would start to aggregate
well ahead of CGA in the trans-Golgi network. In view of the
proposed importance of the pH- and Ca
-induced
chromogranin aggregation in vesicle biogenesis, the extreme sensitivity
of CGB aggregation to Ca
appears to underline the
potential importance of CGB aggregation in the early stages of vesicle
biogenesis.
(Bulenda and
Gratzl, 1985; Reiffen and Gratzl, 1986). Chromogranin A is a high
capacity, low affinity Ca
-binding protein, binding up
to 32-55 mol of Ca
/mol of protein with
dissociation constants of 2-4 mM (Yoo and Albanesi,
1991), and is suggested to be responsible for the inositol
1,4,5-trisphosphate-sensitive intracellular Ca
store
role of the secretory vesicles of adrenal chromaffin cells (Yoo and
Albanesi, 1990a). Furthermore, chromogranins A and B were shown to
interact with the secretory vesicle membrane at the intravesicular pH
of 5.5 and to be released from it at a near physiological pH of 7.5
(Yoo, 1993a, 1993b). In view of the fact that chromogranin A was shown
to exist in a dimeric state at pH 7.5 and in a tetrameric state at pH
5.5 (Yoo and Lewis, 1992), it appears that tetrameric CGA
(
)
interacts with the protein component(s) of the vesicle
membrane at the intravesicular pH of 5.5 (Yoo, 1993a). Subsequent
studies indicated that CGA interacts with the integral membrane
proteins of the vesicle membrane, and one of the CGA-interacting
membrane proteins was tentatively identified to be the inositol
1,4,5-trisphosphate receptor/Ca
channel (Yoo, 1994).
(Gerdes et
al., 1989; Gorr et al., 1989; Yoo and Albanesi, 1990b;
Chanat and Huttner, 1991), and this pH- and
Ca
-dependent aggregation property of chromogranins
has been postulated to be essential for a proper sorting of vesicle
matrix proteins (Gerdes et al., 1989; Gorr et al.,
1989; Yoo and Albanesi, 1990b; Chanat and Huttner, 1991). The
aggregation of CGA was shown to be highly dependent on both pH and
Ca
concentration; at a given Ca
concentration, the extent of CGA aggregation was very high at pH
5.5, whereas there was considerably less aggregation at pH 7.5 (Yoo and
Albanesi, 1990b). The rate and extent of aggregation were directly
dependent on Ca
concentration at pH 5.5, demonstrated
by the fact that the rate of aggregation was faster and the extent
greater at higher Ca
concentrations (Yoo and
Albanesi, 1990b). The aggregation of CGA at pH 5.5 appeared to be so
nearly instantaneous that the aggregation of CGA was virtually complete
in the first few seconds after the introduction of Ca
(Yoo and Albanesi, 1990b). Moreover, circular dichroism data
indicated that CGA undergoes pH- and Ca
-dependent
conformational changes (Yoo and Albanesi, 1990b) and that the protein
is comprised largely of random coil (60-65%) with some
-helix (25-40%) (Yoo and Albanesi, 1990b).
-dependent aggregation
study. Unlike chromogranin A, which was found to exist in a dimeric or
tetrameric state depending on pH (Yoo and Lewis, 1992), chromogranin B
appears to exist entirely in a monomeric state. Moreover, chromogranin
B exhibited a pH- and Ca
-dependent aggregation
property quite different from that of chromogranin A (i.e. CGB
started to aggregate at protein or Ca
concentrations
that were at least 2 orders of magnitude lower than those shown with
CGA). Although the aggregation property of CGB is in line with the
proposed roles of chromogranins A and B in vesicle biogenesis (Huttner
et al., 1988 and 1991; Yoo, 1993a and 1993b), the difference
in the aggregation property appears to underline the subtle but crucial
differences in the roles CGA and CGB might play during vesicle
biogenesis.
Materials
4,4`-Bis(1-anilinonaphthalene
8-sulfonate) (bis-ANS) was from Molecular Probe. Sephacryl S-300 and
molecular size markers (except calmodulin) were obtained from Pharmacia
Biotech Inc. Chelex 100 and acrylamide were from Bio-Rad. Sodium
acetate, EGTA, and MOPS were from Sigma.
Chromogranin and Calmodulin
Preparation
Chromogranin A was purified using the vesicle
lysates of bovine adrenal medullary chromaffin cells as the starting
material as described previously (Yoo and Albanesi, 1990b), and
chromogranin B was also purified from the vesicle lysates utilizing
several chromatographic and electrophoretic steps.(
)
Calmodulin from bovine brain was purified according to the
method of Dedman and Kaetzel(1983).
Fluorescence Spectroscopy
Steady-state
fluorescence measurements were carried out using a DMX-1000
spectrofluorometer (SLM-AMINCO). Excitation and emission bandpasses
were set at 8 nm. All fluorescence measurements were made at 22 °C
using a dual path length ``T-cuvette'' with the short path
length (2 mm) oriented toward the excitation side to minimize inner
filter effects.
CD Spectroscopy
CD spectra were recorded using a
Jasco (Easton, MD) 600 circular dichroism spectrophotometer using a
cell with a path length of 0.2 mm and a protein concentration of 0.08
mg/ml in either 7.5 mM sodium acetate, pH 5.5, or 7.5
mM MOPS, pH 7.5. All spectra were taken at 22 °C and are
the averages of at least four scans. Analysis of the spectra was
carried out using the polylysine spectra of Greenfield and Fasman(1969)
as a standard.
Aggregation Experiments
Chelex 100-treated
chromogranin B or chromogranin A in either 15 mM sodium
acetate, pH 5.5, or 15 mM MOPS, pH 7.5, was titrated with
concentrated CaCl. Aggregation was monitored by measuring
the turbidity change at 320 nm with a Beckman DU 70 spectrophotometer.
All the measurements were done at 22 °C.
Other Methods
Concentration of bis-ANS in ethanol
was determined spectrophotometrically at 385 nm using 16,670
mol cm
as the extinction
coefficient (Farris et al., 1978). Protein determination was
according to Bradford(1976).
and was used in the present experiments.
Although chromogranins are highly charged acidic proteins, CGA was
shown to possess hydrophobic regions, as demonstrated by the increase
in fluorescence emission upon binding the fluorescence probe, bis-ANS
(Rosen and Weber, 1969). bis-ANS is known to bind to the hydrophobic
regions of proteins and to increase its fluorescence emission markedly
upon binding to the hydrophobic regions (Rosen and Weber, 1969). In
order to determine whether CGB also has hydrophobic regions, the
changes in bis-ANS fluorescence were measured upon introduction of
purified native CGB. As shown in Fig. 2, the fluorescence
emission of bis-ANS shows a marked enhancement and blue shift upon
introduction of CGB. The emission due to 2 µM bis-ANS,
shown in spectrum A, exhibits a maximum near 498 nm. However,
the emission maximum shifts to 480 nm (spectrum B) upon
introduction of 1 µM CGB, and the yield (calculated from
the areas under the spectra) increased approximately 50-fold
(the ordinates for spectra A and B are accordingly
different). The inset shows the titration of 1 µM
CGB with bis-ANS, demonstrating a marked enhancement of fluorescence
intensity and a saturation effect. Unlike CGA, which had shown the
increase in fluorescence signal upon the introduction of Ca
at the saturating level of bis-ANS (40 µM) (Yoo and
Albanesi, 1990b), it was not readily apparent what effect
Ca
has on the fluorescence change of bis-ANS-CGB due
to the extreme sensitivity of the Ca
-induced
aggregation of CGB to Ca
(see below).
Figure 1:
SDS-polyacrylamide gel electrophoresis
of the vesicle lysates and purified chromogranin B. The vesicle lysate
proteins and purified CGB from bovine adrenal medullary chromaffin
cells were visualized on a 10% SDS-polyacrylamide gel. LaneA, vesicle lysate protein (14 µg); laneB, purified intact CGB (0.6 µg). Chromogranins A and
B are indicated by arrows.
Figure 2:
Fluorescence emission spectra showing the
increase in quantum yield upon binding of bis-ANS to chromogranin B.
Fluorescence emission spectra of either 2 µM bis-ANS only
(A) or 2 µM bis-ANS plus 1 µM
chromogranin B (B) are shown. SpectrumA was
drawn on an expanded scale. Excitation was at 400 nm, and the buffer
used was 20 mM sodium acetate, pH 5.5. Inset,
titration of 1 µM chromogranin B with bis-ANS until the
fluorescence signal plateaued. Excitation, 420 nm; emission, 480
nm.
To determine
the secondary structure of CGB, CGB was studied by CD spectroscopy. The
CGB spectra in Fig. 3show that CGB exhibits different CD spectra
at pH 5.5 and 7.5, indicating that CGB undergoes a pH-dependent
conformational change. In the absence of Ca, the
change of pH from 7.5 to 5.5 increased the
-helicity from 15 to
20% based on the Greenfield and Fasman approximation(1969), although
the random coil content remained the same at 70% (). As had
been observed with CGA, Ca
has also changed the CD
spectra of CGB at pH 5.5. Addition of 0.2 mM Ca
at pH 5.5 further increased the
-helicity from 20 to 25%,
whereas the random coil decreased from 70 to 65%. Because the estimated
-helicity of 15-25% for CGB is lower than that of CGA
(25-40%) and the random coil content of 65-70% for CGB is
higher than that of CGA (60-65%) (Yoo and Albanesi, 1990b), CGA
was subjected to CD spectroscopy in the presence of 0.2 mM
Ca
at pH 5.5 in order to obtain the secondary
structure information under identical conditions. The results in
Fig. 4
show a marked difference in the spectra of CGB and CGA
under identical conditions. Analysis of the CGA spectra by the same
method (Greenfield and Fasman, 1969) shows that CGA has 40%
-helicity, 0%
-sheet, and 60% random coil in the presence of
0.2 mM Ca
at pH 5.5 (),
agreeing with the previous results obtained in the absence of
Ca
at pH 5.5 (Yoo and Albanesi, 1990b). The absence
of the effect of submillimolar Ca
on CGA secondary
structure has already been observed in our previous study in which
Ca
was able to induce the conformational changes of
CGA only at millimolar Ca
concentrations (10
mM or higher) (Yoo and Albanesi, 1990b).
Figure 3:
CD
spectra of chromogranin B at pH 5.5 and 7.5. A, chromogranin B
in 7.5 mM MOPS, pH 7.5, and 0.2 mM EGTA; B,
CGB in 7.5 mM sodium acetate, pH 5.5, and 0.2 mM
EGTA; C, CGB in 7.5 sodium acetate, pH 5.5, and 0.2
mM CaCl. Chromogranin B concentration was 0.08
mg/ml.
Figure 4:
CD
spectra of chromogranin A and chromogranin B at pH 5.5. Chromogranin B
(solid line) and chromogranin A (dotted line) in 7.5
mM sodium acetate, pH 5.5, and 0.2 mM CaCl are shown. Chromogranin B and chromogranin A concentrations were
0.08 mg/ml (1.1 µM) and 0.11 mg/ml (2.3 µM),
respectively.
In light of the
fact that CGA exists in a dimeric or tetrameric state (Yoo and Lewis,
1992) and dimeric CGA elutes from a gel filtration column in the
elution volume indicative of a globular protein with a molecular weight
of 300,000 (Yoo and Albanesi, 1990b, 1991), chromogranin B was also
subjected to gel filtration chromatography to estimate the
oligomerization state of the molecule. As shown in Fig. 5, CGB
eluted in the volume characteristic of a globular protein with
molecular weight of either 220,000 at pH 7.5 or 170,000 at pH 5.5.
These estimated values are far below that found for dimeric CGA
(300,000). Therefore, considering the fact that monomeric CGA and
CGB migrate as proteins with masses of 75 and 110 kDa, respectively, on
SDS-polyacrylamide gel electrophoresis (Fig. 1), the estimated
CGB mass of
200 kDa by gel filtration chromatography
(Fig. 5) may simply reflect the highly random, markedly
asymmetric structure of CGB and strongly suggests that CGB exists in a
monomeric state at both pH 5.5 and 7.5.
Figure 5:
Gel filtration on Sephacryl S-300. Chelex
100-treated chromogranin B was chromatographed on a calibrated
Sephacryl S-300 column (0.9 47 cm) equilibrated with either 15
mM Tris-HCl, pH 7.5, or 15 mM sodium acetate, pH 5.5,
both in 0.1 M KCl, 1 mM dithiothreitol, and 2
mM EGTA and eluted at 7.2 ml/h. The column was calibrated with
molecular markers, blue dextran for void volume
(V
, 2
10
Da), ferritin (440
kDa), aldolase (158 kDa), and calmodulin (17 kDa). The elution
locations of chromogranin B at pH 5.5 (A) and at pH 7.5
(B) are indicated by
arrows.
In view of the pH- and
Ca-dependent aggregation of CGA, the pH- and
Ca
-dependent aggregation property of CGB has also
been studied by measuring Ca
-induced turbidity
changes. As shown in Fig. 6, Ca
induced
aggregation of CGB at various protein concentrations, and the
aggregation pattern was dependent upon the protein concentration; a
lower concentration of Ca
was required to induce
aggregation as CGB concentration was increased. The extent of
aggregation was also strongly dependent on Ca
concentration at each CGB concentration. When the chromogranin B
concentration was low, the maximal degree of aggregation was also low,
thus indicating a critical dependence of the aggregation on the CGB
concentrations. Moreover, the rate of aggregation at a given CGB
concentration was directly dependent on Ca
concentration (Fig. 7).
Figure 6:
Aggregation of chromogranin B as a
function of Ca concentration. Chromogranin B
concentrations are 0.4 (
), 0.15 (
), 0.06 (
), and
0.02 mg/ml (
). The A
values shown are
those taken 2 min after the addition of Ca
. The
dashedportion of each curve indicates that
the aggregation peaked and the aggregated protein started to
precipitate out of the solution.
Figure 7:
Time course of chromogranin B aggregation.
The aggregation was monitored after adding 10 (), 3 (
), or
1 mM CaCl
(
) to 0.06 mg/ml CGB in 20
mM sodium acetate, pH 5.5.
Because CGA aggregation was
highly dependent on pH (Yoo and Albanesi, 1990b), the effect of pH on
CGB aggregation was also studied. As shown in Fig. 8, the extent
of aggregation was much higher at pH 5.5 than at pH 7.5; even at a
maximal level of aggregation, the extent of aggregation at pH 7.5 was
less than half that at pH 5.5, clearly demonstrating the favorable
aggregation of CGB at the intravesicular pH of 5.5. Analogous to the
results obtained with CGA (Yoo and Albanesi, 1990b), chromogranin B
also exhibited a markedly different Ca sensitivity at
the two pH levels; at pH 5.5 the aggregation was completed at 8
mM Ca
, but, at the same Ca
concentration (8 mM), the aggregation at pH 7.5 was only
half of the maximal aggregation, which occurred at 16-17
mM Ca
. The extent and Ca
dependence of CGB aggregation at pH 5.5 appeared to be
significantly different from those of CGA (Yoo and Albanesi, 1990b).
Hence, in order to directly compare the aggregation property of CGA and
CGB, the aggregation of CGA as a function of Ca
was
measured. As shown in Fig. 9, 0.4 mg/ml CGB started to aggregate
upon introduction of submillimolar Ca
at pH 5.5, and
the aggregation was completed at 3-4 mM
Ca
, whereas the aggregation of the same amount of CGA
virtually did not even start at 10 mM Ca
.
However, CGA aggregation became appreciable at 20 mM
Ca
and peaked at 38-40 mM
Ca
, agreeing with the previous results, in which 0.6
mg/ml CGA aggregated maximally at approximately 38 mM
Ca
(see Fig. 5of Yoo and Albanesi, 1990b).
Even at its maximal aggregation, the extent of CGA aggregation was less
than one-sixth of that observed with the same amount of CGB.
Figure 8:
Effect of pH on chromogranin B
aggregation. Aggregation of CGB (0.15 mg/ml) at pH 5.5 () and 7.5
(
) was measured as a function of Ca
concentration. The buffers were either 20 mM sodium
acetate, pH 5.5, or 20 mM MOPS, pH 7.5. The A
values are those taken 2 min after the addition of
Ca
.
Figure 9:
Comparison of chromogranin A and
chromogranin B aggregation as a function of Ca
concentration. Protein concentration was 0.4 mg/ml in 15 mM
sodium acetate, pH 5.5. The A
values for CGA
(
) and CGB (
) are those taken 2 min after the addition of
Ca
.
Further, in order to determine whether the presence of both CGA and
CGB affects the aggregation property of each chromogranin, the
Ca-induced aggregation of the mixture of CGA and CGB
was studied. As shown in Fig. 10, the CGA/CGB mixture aggregated
maximally at a Ca
concentration that lies between the
two Ca
concentrations needed for the maximal
aggregation of CGA and CGB, respectively; when 0.06 mg of CGB/ml was
mixed with 0.4 mg of CGA/ml, a CGB/CGA ratio similar to that found in
secretory vesicles, the mixture exhibited a maximal aggregation at
30 mM Ca
, which is higher than the 12
mM Ca
needed for a maximal aggregation of
0.06 mg of CGB/ml but lower than the 40-45 mM
Ca
needed for a maximal aggregation of 0.4 mg of
CGA/ml. In another experiment, when a larger amount of CGB (0.15 mg of
CGB/ml) was mixed with the same amount of CGA (0.4 mg/ml), the
aggregation of the CGB/CGA mixture showed a maximal aggregation at
13 mM Ca
, which is higher than the 8
mM Ca
needed for the maximal aggregation of
0.15 mg of CGB/ml (Fig. 6) but significantly lower than the
30 mM Ca
needed for the maximal
aggregation of the mixture of 0.06 mg of CGB/ml and 0.4 mg of CGA/ml
(Fig. 10), suggesting that the Ca
sensitivity
of the aggregation of the CGB/CGA mixture is related to the relative
ratio of CGB over CGA in the mixture.
Figure 10:
Aggregation of chromogranin A and
chromogranin B mixture as a function of Ca
concentration. Aggregation of the mixture of CGB (0.06 mg/ml) and CGA
(0.4 mg/ml) in 15 mM sodium acetate, pH 5.5, was measured as a
function of Ca
concentration. The A
values for CGB alone (
), the CGA-CGB mixture (
),
and CGA alone (
) are those taken 2 min after the addition of
Ca
.
-dependent conformational changes. However, unlike
CGA, which had been shown to exist in a dimeric state at a near
physiological pH of 7.5 and in a tetrameric state at the intravesicular
pH of 5.5 (Yoo and Lewis, 1992), CGB appeared to exist only in a
monomeric state at both pH 5.5 and 7.5 (Fig. 5).
-helix and 0-10%
-sheet
structures (Yoo and Albanesi, 1990b), CGB was also shown to consist
largely of random coil (65-70%) with 15-25%
-helix and
10-15%
-sheet structures. The change of pH from 7.5 to 5.5
in the absence of Ca
increased the
-helicity
from 15 to 20%, whereas the random coil content remained the same at
70%. The addition of 0.2 mM Ca
to CGB at pH
5.5 further increased the
-helicity from 20 to 25% while
decreasing the random coil content from 70 to 65%. Although the
introduction of Ca
to CGA at pH 5.5 has also changed
the conformation of CGA (Yoo and Albanesi, 1990b), there was no
detectable conformational change of CGA in the low range of
Ca
concentration; the conformational change of CGA
was only seen at Ca
concentrations of 10 mM
or higher (Yoo and Albanesi, 1990b). However, in the case of CGB,
submillimolar Ca
was sufficient to cause
conformational changes of CGB ( Fig. 3and ). In fact,
higher concentrations of Ca
caused aggregation of CGB
(Fig. 6-8). Considering that CGB binds to the vesicle
membrane at pH 5.5 (Yoo, 1993b), the increase of
-helicity at pH
5.5 might contribute to the pH-dependent membrane binding of CGB.
Further, the presence of a high percentage of random coil
(65-70%) suggests that CGB is probably a markedly asymmetric
molecule, contributing to its anomalous migration on SDS-polyacrylamide
gel electrophoresis in that it migrates significantly more slowly than
globular molecules of its size. In our previous study, dimeric CGA
eluted on gel filtration chromatography in the volume suggestive of a
300-kDa protein (Yoo and Albanesi, 1990b, 1991), which is significantly
larger than its actual mass of 96 kDa (2
48 kDa). Therefore, in
view of the fact that the molar mass of bovine CGA is 48 kDa (Iacangelo
et al., 1986; Benedum et al., 1986) and that of
bovine CGB is 71 kDa (Bauer and Fischer-Colbrie, 1991), it is
reasonable to assume that CGB exists in a monomeric state based on its
elution position on gel filtration chromatography, where it eluted in
the volume indicative of a globular protein with a molecular weight of
200,000.
as was the case with CGA. However, there were
large differences in the extent of aggregation and the sensitivity to
Ca
concentrations between CGA and CGB. When an equal
amount of protein was used, 10-fold higher Ca
was
needed to induce a maximal aggregation of CGA than was needed for a
maximal aggregation of CGB (Fig. 9) (i.e. approximately
40 mM Ca
was needed to induce a maximal
aggregation of 0.4 mg/ml CGA (A
reading of
0.27), whereas only approximately 4 mM Ca
was needed to induce a maximal aggregation of the same amount of
CGB (A
reading of 1.72)). Nevertheless, the
extent of maximal CGA aggregation was only one-sixth that of CGB under
identical conditions (Fig. 9). The difference in Ca
sensitivity of aggregation between CGA and CGB can be
demonstrated more clearly by comparing the concentrations of
Ca
needed to induce the same degree of aggregation
with an equal amount of each protein. For example, to induce the CGB
aggregation with an A
reading of 0.2, 0.4 mg/ml
CGB required 0.2 mM Ca
at the intravesicular
pH of 5.5, whereas an equal amount of CGA required 33 mM
Ca
to induce the same level of aggregation
(Fig. 9). This result suggests that the aggregation of CGB is
approximately 165-fold more sensitive to Ca
than CGA.
The difference in Ca
sensitivity between CGA and CGB
aggregation might be due to the fact that 1) a lower Ca
concentration can cause larger conformational changes in CGB than
possible in CGA (Tables I and II) (Yoo and Albanesi, 1990b), thereby
possibly exposing more hydrophobic regions in CGB, which may
participate in the aggregation of CGB, or 2) CGA exists in a tetrameric
state at pH 5.5 (Yoo and Lewis, 1992) compared with the monomeric
existence of CGB (Fig. 5), which will severely restrict the
orientation of CGA molecules toward each other, thus reducing the
possibility of the potential interacting regions from interacting with
each other.
concentration, the degree of sensitivity of each
CGA and CGB aggregation can also be determined. As shown in
Fig. 6
, 0.02 mg/ml CGB required 12 mM Ca
to induce its aggregation to the extent that A
= 0.1, whereas 3.2 mg/ml CGA was needed at the same
Ca
concentration (12 mM) to induce the same
level of aggregation (see Fig. 5of Yoo and Albanesi (1990b)). In
other words, 160-fold more CGA was required at a given Ca
concentration to induce the same degree of aggregation that was
achieved with CGB. In light of the observation that a higher degree of
chromogranin aggregation is achieved in the presence of a larger amount
of chromogranin at a given Ca
concentration
(Fig. 6) (Yoo and Albanesi, 1990b), the above results clearly
demonstrate the fact that CGB aggregation at the intravesicular pH of
5.5 is at least 2 orders of magnitude more sensitive than that of CGA
at a given Ca
concentration. This result in turn
suggests that CGB would start to aggregate in the low pH and high
Ca
environment of the intravesicular milieu even at a
concentration less than one-hundredth that of CGA, although aggregation
of CGB will be affected by the presence of CGA and other proteins
(Fig. 10). In particular, in view of the observation that CGA and
CGB bind to each other at the intravesicular pH of 5.5 (Yoo, 1995), the
intermediate Ca
sensitivity of the aggregation of the
CGA/CGB mixture (Fig. 10) appears to be an inevitable result of
two interacting proteins, each with a different Ca
sensitivity. Nevertheless, in spite of the fact that the amount
of CGB in the secretory vesicles of bovine adrenal chromaffin cells is
approximately one-tenth that of CGA (Fischer-Colbrie and Schober, 1987;
Winkler and Fischer-Colbrie, 1992), the exceedingly
Ca
-sensitive nature of CGB aggregation would more
than compensate for the low CGB concentration, resulting in the
aggregation of CGB ahead of CGA in the trans-Golgi network.
-induced aggregation of
chromogranins A and B has been proposed to play vital roles in sorting
the vesicle matrix proteins in the trans-Golgi network (Gerdes
et al., 1989; Gorr et al., 1989; Yoo and Albanesi,
1990b; Chanat and Huttner, 1991), the present results provide strong
evidence in support of such proposals. In particular, given that the
sorting of the potential vesicular matrix proteins is considered an
essential step during vesicle biogenesis, the aggregation of CGB,
probably well ahead of CGA in the trans-Golgi network where
secretory vesicles are formed, may prove to be a critical first step in
the sorting of the vesicular matrix proteins. Recently, chromogranin B
was also shown to bind to the vesicle membrane at the intravesicular pH
of 5.5 and to be released from it at a near physiological pH of 7.5
(Yoo, 1993b). Further, chromogranin B was shown to interact with the
vesicle membrane significantly more tightly than CGA.
Therefore, in light of the present results, which suggest the
aggregation of CGB prior to that of CGA in the trans-Golgi
network, it is likely that the CGB aggregate would begin to interact
with the vesicle membrane well ahead of CGA during vesicle biogenesis,
thereby setting the stage favorably for the subsequent participation by
CGA for their potential joint work in vesicle biogenesis. In this
regard, the present results appear to underline the critical roles that
CGB might play during the early stages of vesicle biogenesis and bring
a new dimension to our perception of the potential functions accorded
CGB.
Table:
Effects of pH and Ca on
chromogranin B conformation
Table:
Comparison of conformations between
chromogranin A and chromogranin B
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.