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(Received for publication, September 22, 1994; and in revised form, November 2,
1994) From the
Thyroid follicular cells coordinate several oppositely located
surface enzyme activities. Recent studies have raised questions about
the basic mechanisms used to achieve thyroid surface polarity. We
investigated these mechanisms in primary thyroid epithelial monolayers
cultured on porous filters. In the steady state, most
Na Like other epithelial cells, thyroid follicular cells maintain a
highly polarized surface organization (Wollman, 1989). For example, the
driving force for active uptake of iodide by the thyroid epithelium is
provided by a basolateral Na In
some instances, epithelial surface asymmetry is regulated by
intracellular sorting of newly synthesized membrane proteins at the
level of the trans-Golgi network (Fuller et al.,
1985; Rindler et al., 1985) (for review see Simons and
Wandinger-Ness(1990)). Interestingly, the site of this sorting may vary
for different protein-epithelial cell combinations (Pathak et
al., 1990), since some proteins may be delivered bi-directionally
(Haller and Alper, 1993) but may be selectively stabilized or degraded
at one surface (Contreras et al., 1989; Hammerton et
al., 1991) or may be relocated to the contralateral membrane
surface (Bartles et al., 1987; Matter et al., 1990)
(for reviews see Mostov et al. (1992) and Nelson(1992)).
Moreover, the same cells may exhibit different surface protein
distributions during the development versus subsequent
maintenance of epithelial polarity (Balcarova-Stander et al.,
1984; Herzlinger and Ojakian, 1984; Wollner et al., 1992) (for
review see Rodriguez-Boulan and Powell(1992)). It is well
established that thyroid follicular cells in culture form highly
polarized epithelial monolayers (Chambard et al., 1983; Gerard et al., 1985; Mauchamp et al., 1987). However,
reports of the Fischer rat thyroid (FRT) (
Figure 1:
Surface distribution of thyrocyte
surface proteins after metabolic labeling to steady state.
Filter-polarized thyrocytes were labeled with
Figure 2:
Quantitation of the surface tagging of APN
and NKA. Thyrocytes were labeled, biotinylated, lysed,
immunoprecipitated, and reprecipitated with avidin-agarose as described
in the legend to Fig. 1. Densitometric scanning of fluorographs
was used to calculate apical:basolateral (APN) or
basolateral:apical (NKA) distribution ratios, consistent with
the known physiological distributions of these proteins. The data shown
are from a representative experiment (of
two).
Figure 3:
Apical
immunofluorescent staining of APN. Filter-grown thyrocytes were
immunostained for APN according to ``Materials and Methods.''
The monolayers were optically divided into eight X-Y sections by
confocal microscopy. The apical-most section is shown; lateral and
basal layers, as well as the filter itself, revealed only background
fluorescence (not shown).
Figure 4:
Rapid arrival of newly synthesized plasma
membrane proteins at the thyroid cell surface. Filter-polarized
thyrocytes were pulse-labeled for 30 min with
Figure 5:
Quantitation of the surface arrival of
newly synthesized APN, NKA, and TPO. After polarized cell surface
biotinylation of pulse-labeled thyrocytes, APN, NKA, and TPO
immunoprecipitates were reprecipitated with avidin-agarose. The
avidin-bound fractions quantified by SDS-PAGE and fluorography were
normalized to the full recovery of these antigens by
immunoprecipitation in order to calculate the fraction of newly
synthesized APN and NKA delivered to the cell surface over a 3-h chase.
Each curve is representative of two independent
experiments.
Figure 6:
Polarized surface delivery of a fraction
of newly synthesized TPO. Filter-polarized thyrocytes were
pulse-labeled for 30 min with
Figure 7:
Apically polarized distribution of
GPI-anchored surface proteins in primary thyroid epithelial cells.
Unlabeled filter-grown thyrocytes were vectorially biotinylated and
probed by
There is little doubt that establishment and maintenance of
epithelial surface polarity is essential to normal thyroid function
(Mauchamp et al., 1987). However, to our knowledge, there are
no previous studies examining polarized delivery of new thyroid plasma
membrane proteins except in the FRT cell line, which has lost many
features of differentiated thyroid function (Nitsch et al.,
1985) and which behaves in a highly atypical manner (Zurzolo et
al., 1992a, 1992b, 1993). For this reason, we have examined the
well established system of primary thyroid epithelial monolayers
cultured on porous filters (Arvan and Lee, 1991; Prabakaran et
al., 1993; Kuliawat and Arvan, 1994). We find that all three
proteins studied in the reconstituted thyroid epithelium exhibit
polarized surface distributions like that expected in vivo (Fig. 1). Specifically for APN and NKA, a large fraction of
these enzymes arrives at the cell surface (Fig. 5) shortly after
the acquisition of Golgi-specific posttranslational processing (Fig. 4), indicating direct delivery of these newly synthesized
proteins predominantly to the apical and basolateral plasma membranes,
respectively. While a modest fraction of newly synthesized proteins may
be missorted or mistargeted to the contralateral epithelial surface and
additional mechanisms (Nelson, 1992) may fine tune A/B ratios to those
observed at steady state (Fig. 2), the data strongly support the
idea that in normal thyroid follicular cells, epithelial surface
asymmetry is regulated largely by intracellular sorting of newly
synthesized membrane proteins at the level of the trans-Golgi
network. Thus, the targeting mechanism is not unique for the thyroid as
has been implied from studies of FRT cells (Zurzolo et al.,
1992b) but is in fact consistent with conventional models of epithelial
surface protein targeting such as in Madin-Darby canine kidney cells
(Fuller et al., 1985; Rindler et al., 1985; Wessels et al., 1990). Moreover, the data suggest that, like most
epithelia but unlike FRT cells, GPI-anchored proteins are heavily
distributed to the apical surface of filter-polarized thyrocytes (Fig. 7). These data indicate that the behavior of the FRT cell
line is not representative of normal thyroid epithelial cells. Such a
conclusion is further supported by evidence indicating that FRT cells
are deficient in caveolin (Zurzolo et al., 1994), a membrane
protein implicated in the apically polarized distribution and delivery
of GPI-anchored proteins (Lisanti et al., 1993), which is
found in primary thyrocytes at levels comparable with other epithelial
cells. ( To our knowledge, the present report represents
the first study of biosynthetic targeting of TPO. Surprisingly in
filter-grown epithelial monolayers, a large fraction of
immunoprecipitable TPO was inaccessible to surface labeling (e.g.Fig. 5and Fig. 6) and hence was presumably
intracellular. Such an observation is very unlikely to represent a
methodological artifact for the following reasons. First the ability to
tag a high fraction of plasma membrane proteins was well preserved in
these monolayers, as judged by the efficient biotinylation of other
surface molecules (Fig. 2). Second, despite the fact that
overall surface biotinylation was much greater basolaterally (Fig. 7B), there was no defect in apical vesicle
trafficking or in apical-specific labeling in filter-grown thyrocytes,
as judged by the apically polarized delivery and distribution of APN ( Fig. 1and Fig. 4), GPI-linked proteins (Fig. 7),
and polarized delivery of thyroglobulin (Arvan and Lee, 1991;
Prabakaran et al., 1993; Kuliawat and Arvan, 1994). Third, it
is unlikely that intrinsic structural features prevent TPO from being
efficiently biotinylated, since this type 1 membrane protein contains a
large extracytoplasmic domain that includes 14 lysine residues
(Magnusson et al., 1987). Indeed, it is not that TPO failed to
be tagged or exhibited defective kinetics of surface delivery ( Fig. 1and 6); rather, only Unfortunately, the present data cannot distinguish whether only
Finally, it is tempting to speculate,
as have others (Wessels et al., 1990), that the continuous
mistargeting of a small fraction of TPO to the basolateral surface can
account for a modest basolateral presence of TPO under steady-state
conditions (Fig. 1). Moreover, this missorted fraction could
contribute to the presentation of TPO to the circulating immune
surveillance system and might help to explain the reproducible
antigenicity of this protein in autoimmune thyroiditis (Czarnocka et al., 1986; Kotani et al., 1986). More work will be
needed to investigate such a possibility.
Volume 270,
Number 6,
Issue of February 10, 1995 pp. 2478-2482
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
/K
-ATPase and aminopeptidase N were
available for surface biotinylation, and these proteins exhibited
physiological distributions (basolateral and apical, respectively).
Glycosylphosphatidylinositol-anchored proteins were also apically
distributed. By pulse-chase, newly synthesized transmembrane proteins
exhibited polarized surface delivery that was oriented similarly to
that observed at steady state. Little time elapsed between acquisition
of Golgi-specific processing and cell surface arrival. Interestingly,
when either newly synthesized or steady state-labeled thyroid
peroxidase was similarly analyzed, only
30% of the enzyme was ever
detected at the cell surface. Of this, the majority was localized
apically. The data suggest that most thyroid peroxidase remains
intracellular in these monolayers, consistent with the possibility of
intracellular iodination activity in addition to apical extracellular
iodination. Nevertheless, in filter-polarized thyrocytes, most newly
synthesized plasma membrane proteins appear to be sorted in the Golgi
complex for direct delivery to apical and basolateral domains.
gradient generated by
Na
/K
-ATPase (Chow et al.,
1986; Vilijn and Carrasco, 1989; Golstein et al., 1992). Upon
transport to the opposite end of the follicular cell (Nakamura et
al., 1990; Nilsson et al., 1992), iodide becomes
available to the catalytic domain of thyroid peroxidase. In the
presence of H
O (Bjorkman and Ekholm, 1988),
thyroid peroxidase catalyzes iodide organification predominantly in the
apical extracellular space (Ofverholm and Ericson, 1984; Gruffat et
al., 1991) where the substrate protein (thyroglobulin) is
concentrated to extraordinary levels (Herzog et al., 1992). A
smaller degree of initial iodination of thyroglobulin may also occur
intracellularly (Matsukawa and Hosoya, 1979; Kuliawat and Arvan, 1994).
Nevertheless, an asymmetric distribution of epithelial cell surface
enzymes is thought to be required for thyroid hormonogenesis.
)cell line (Nitsch et al., 1985), a recently developed model of thyrocyte
polarity (Zurzolo et al., 1992a), have described significant
differences from many other epithelial cells such as the (apical)
polarity of Semliki Forest virus budding and (basolateral) polarity of
transfected CD8 lymphocyte antigen (Zurzolo et al., 1992b). In
addition, despite the normal basolateral targeting of
Na/K
-ATPase (Zurzolo and
Rodriguez-Boulan, 1993), FRT cells display a preferential distribution
of glycosylphosphatidylinositol-anchored proteins (which in most
epithelia reside apically) on the basolateral surface (Zurzolo et
al., 1993). Such findings have been interpreted to suggest unique
mechanisms for protein targeting in the thyroid (Zurzolo et
al., 1992b). To investigate this question further, we have
examined filter-polarized epithelial monolayers comprised of normal,
primary thyrocytes, in which polarized sorting of secretory proteins
has already been demonstrated (Arvan and Lee, 1991; Prabakaran et
al., 1993). Using this cell culture system, a biotin tag has been
introduced onto surface proteins in order to examine the distribution
and delivery of three endogenous plasma membrane proteins:
aminopeptidase N (APN, an apically polarized enzyme),
Na
/K
-ATPase (NKA), and thyroid
peroxidase (TPO), the latter two exhibiting basolateral and apical
activities, respectively.
Cell Culture
Primary porcine thyrocytes were
isolated and seeded at high density onto Millicell HA (Millipore Corp.,
Bedford, MA) 0.4-µm pore filters and cultured in the presence of 5
milliunits/ml bovine TSH (Sigma) and 5% calf serum added basolaterally,
as described previously (Kuliawat and Arvan, 1994). This approach
allowed monolayers to achieve electrical resistances 
1000
ohm-cm. Experiments were carried out on day 7.Cell Labeling, Biotinylation, and Lysis
Confluent
monolayers were radiolabeled with ExpreS
S
(DuPont NEN) either for 30 min in Met/Cys-deficient medium (for
pulse-chase analysis) or for 2 days in complete medium (for
steady-state labeling). Before biotinylation, the cells were washed 3
times in ice-cold phosphate-buffered saline. Monolayers were then
exposed either apically or basolaterally to 0.6 mg/ml sulfo-NHS-biotin
(Pierce) for 30 min at 4 °C in a buffer containing 0.25 M sucrose, 2 mM CaCl
, 0.5 mM MgCl, 10 mM triethanolamine, pH 8.5. The
biotin reagent was quenched by subsequent incubation with 50 mM NH
Cl in phosphate-buffered saline for 10 min at 4
°C. The cells were again washed in phosphate-buffered saline and
lysed either in boiling 1% SDS to inhibit proteolysis (Rolland and
Lissitzky, 1976) or in 1 ml of immunoprecipitation buffer containing
0.15 M NaCl, 25 mM Tris, pH 7.5, 10 mM iodoacetamide, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton
X-100, 5 mM EDTA, and a mixture of protease inhibitors as
described previously (Kim et al., 1992). Cells lysed directly
in SDS were then diluted into immunoprecipitation buffer lacking SDS to
yield the same final detergent concentrations prior to
immunoprecipitation.Antibodies, Precipitations, and
Immunofluorescence
A rabbit antiserum to TPO was obtained
through the laboratory of the late Dr. S. Ingbar (Beth Israel Hospital,
Boston, MA). Antiserum to porcine aminopeptidase N (Hansen et
al., 1987) was obtained from Drs. O. Noren and H. Sjostrom (Panum
Institute, Copenhagen, Denmark). A rabbit antiserum to lamb kidney
Na/K
-ATPase, cross-reacting with the
pig enzyme, was obtained from Dr. W. J. Ball (University of Cincinnati,
OH). Immunoprecipitations were typically overnight at 4 °C, using
protein A-agarose (Sigma) as a secondary reagent. Immunoprecipitates
were treated with 1% SDS and then diluted 10-fold into
immunoprecipitation buffer lacking SDS. UltraAvidin-agarose (Leinco)
was then used for precipitation of surface-labeled proteins.
Avidin-biotin conjugates were dissociated by boiling in SDS-gel sample
buffer and analyzed by SDS-PAGE. After absorption of biotinylated
proteins on immobilized avidin, nonbiotinylated proteins were
concentrated by a 1:3 (v/v) addition of Pro-Cipitate according to the
manufacturer (Affinity Technology) and were similarly analyzed by
SDS-PAGE. Indirect immunofluorescence localization of aminopeptidase N
was performed on nonpermeabilized filter-grown thyroid monolayers fixed
in 2% formaldehyde prior to incubation with a fluorescein
isothiocyanate-conjugated goat-antirabbit serum, using Bio-Rad confocal
optical analysis coupled to a Zeiss Axiophot microscope.
Analysis of Surface Polarity of Glycophosphatidylinositol
(GPI)-anchored Membrane Proteins
Sets of four thyrocyte cultures
grown on porous filters were biotinylated from apical or basolateral
sides before lysis, as above. Hydrophobic membrane proteins in the
lysate were extracted by Triton X-114 phase partitioning (Bordier,
1981). The detergent phase was then either treated with
phosphatidylinositol-specific phospholipase C (PI-PLC) to release the
lipid anchor or mock-digested (Lisanti et al., 1988). Triton
X-114 phase separation was then repeated for both the mock digests and
PI-PLC-treated samples; proteins released into the aqueous phase were
collected and run on SDS-PAGE. Proteins were then electrotransferred
from the gel to nitrocellulose, and the filter was blotted with I-streptavidin before autoradiography.
Steady-state Distributions of APN and NKA
In
physiologically polarized thyrocytes, APN (M
160,000) resides nearly exclusively at the apical plasma
membrane (Feracci et al., 1981; Barriere et al.,
1986; Nilsson et al., 1987) while NKA (
-subunit, M 100,000;
-subunit M 60,000) is predominantly localized to the basolateral
surface (Gerard et al., 1985). We examined these distributions
in confluent porcine thyroid epithelial monolayers cultured on porous
filters. In monolayers radiolabeled to steady state with
S-amino acids, surface proteins were tagged by vectorial
biotinylation, using a reagent that reacts with the
-amino groups
of lysine residues that are extracytoplasmically disposed (see
``Materials and Methods''). The cells were then lysed and
immunoprecipitated for specific antigens. After release from antibody
by denaturation in SDS, biotinylated proteins were recovered by binding
to avidin-agarose and were analyzed by SDS-PAGE and fluorography (Fig. 1). Finally, immunoprecipitable proteins that were not
precipitated with avidin-agarose were recovered (see ``Materials
and Methods''); presumably, these nonbiotinylated proteins were
intracellular and hence inaccessible to the tagging reagent. However,
as expected in the steady state, the preponderance of
immunoprecipitable APN and NKA (as quantitated from the
-subunit()) were indeed at the cell surface. Further,
APN was detected with markedly greater abundance apically, while NKA
exhibited an opposite pattern (Fig. 2). Although other methods
employed were not readily quantified, the apical localization of APN
was visually confirmed by confocal immunofluorescence microscopy (Fig. 3), and the distributions of both plasmalemmal proteins
were supported by blotting of tagged, unlabeled proteins with
avidin-peroxidase, followed by enhanced chemiluminescence detection
(not shown).
S-amino
acids for 2 days. The cells were then biotinylated at either the apical (A) or basolateral (B) cell surface, lysed, and
immunoprecipitated for APN (M
160,000), NKA
(seen best for the
-subunit,M 60,000), or TPO (M
105,000). The
immunoprecipitates were solubilized as described under ``Materials
and Methods,'' and the surface-tagged proteins were recovered by
precipitation with avidin-agarose and analyzed by SDS-PAGE and
fluorography.
Delivery of Newly Synthesized APN and NKA to the
Follicular Cell Surface
Next, the biotinylation protocol was
employed to measure surface delivery of newly synthesized proteins in
pairs of thyrocyte monolayers tagged apically and basolaterally,
respectively, at different chase times after pulse labeling with S-amino acids. At 5 min of chase, most APN was devoid of
terminal carbohydrate and could not be detected at the cell surface
(not shown). However, at 30 min of chase, some of the newly synthesized
APN exhibited an electrophoretic mobility shift indicative of Golgi
sugar processing; at this time, cell surface arrival was already under
way (Fig. 4). As measured for NKA, the situation was quite
similar (Fig. 4). Thus, it was apparent that cell surface
delivery occurred shortly after arrival of newly synthesized proteins
in the Golgi complex, and after 3 h a majority of these proteins were
found at the cell surface (Fig. 5), approaching steady-state
levels. Importantly, at all chase times tested, delivery of newly
synthesized APN and NKA to the plasma membrane exhibited apical and
basolateral predominance, respectively (Fig. 4). Thus, the data
indicated that to a large extent, the proteins were already sorted as
the newly synthesized molecules exited the Golgi complex.
S-amino
acids. At the different chase times shown, the monolayers were
vectorially biotinylated and newly synthesized APN and NKA
immunoprecipitated. The avidin-bound (Surface Tagged) and
avidin unbound (Intracellular) fractions were analyzed by
SDS-PAGE and fluorography. Note that at 30 min of chase, while
considerable amounts of each newly synthesized enzyme had not yet
received Golgi sugar processing as detected by electrophoretic
mobility, cell surface arrival was nevertheless already under way. The
data are representative of three
experiments.
Distribution and Delivery of TPO
In thyrocytes
cultured on porous filters, TPO (M 105,000)
exhibited important differences in distribution and delivery from the
other surface proteins studied. After radiolabeling to steady state,
reproducibly not more than 30% of immunoprecipitable TPO was detected
at the cell surface. Of the surface-tagged TPO fraction (Fig. 1), the distribution was predominantly apical, with an A/B
ratio of 4.4. When newly synthesized proteins were examined, surface
delivery of TPO exhibited similar behavior, achieving a plateau with
30% at the cell surface (Fig. 5). Since pulse radiolabeling
was less extensive than steady-state labeling and the fraction of
surface-tagged molecules was relatively low, it was difficult to state
with confidence the precise polarity of new TPO delivery, although in
all experiments the A/B ratio appeared roughly comparable with that
observed for TPO at steady state (Fig. 6). Nevertheless, this
did not appear quite as apically polarized as that observed for APN (e.g.Fig. 1).
S-amino acids. At the
different chase times shown, the monolayers were vectorially
biotinylated, and newly synthesized TPO was immunoprecipitated. The
avidin-unbound (Intracellular) and avidin-bound (Surface
Tagged) fractions were analyzed by SDS-PAGE and fluorography.
Similar to steady state-labeled thyrocytes, the majority of newly made
TPO at all chase times up to 4 h was inaccessible to surface
biotinylation. Of the surface-tagged fraction, the majority exhibited
an apically polarized delivery. The results from two experiments
showing different levels of protein radiolabeling are
shown.
Distribution of GPI-linked Proteins in Primary Thyroid
Epithelial Monolayers
Clustering within membranes of the trans-Golgi network has been proposed to play an important
role in the surface delivery of newly synthesized GPI-linked proteins,
resulting in their apical polarity in many epithelia (Rodriguez-Boulan
and Powell, 1992; Hannan et al., 1993). We wished to see if
this behavior was characteristic of normal thyroid epithelial
monolayers cultured on porous filters. For this, proteins from
thyrocyte monolayers (biotinylated apically or basolaterally) extracted
with Triton X-114 were either digested with PI-PLC or mock digested.
These samples were then re-extracted to identify molecules that had
lost their lipid anchor and now partitioned to the aqueous phase as a
specific consequence of PI-PLC digestion (Fig. 7A). A
number of proteins recovered in the aqueous phase were not specific to
PI-PLC digestion; these nonspecific bands were recovered exclusively
from cells biotinylated basolaterally (Fig. 7A, lane 3) where the degree of overall surface tagging was
greater by manyfold (Fig. 7B). However, bands released
selectively by PI-PLC digestion were found largely in the M 30,000 range and were obviously apically
polarized (Fig. 7A, lane 2). Thus, unlike the
thyroid-derived FRT cell line, which displays a surface distribution of
GPI-anchored proteins that is preferentially basolateral (Zurzolo et al., 1993), the distribution of such proteins in normal
thyrocytes is predominantly apical.
I-streptavidin as described under
``Materials and Methods.'' A, Triton
X-114-extractable proteins from these monolayers were treated with or
without PI-PLC as described in the text. Proteins released
nonspecifically and as a specific consequence of PI-PLC cleavage are
shown. Only two bands in the range of M
30,000 were specifically released (asterisks); both
bands were clearly apically polarized. B, profile of all
vectorially biotinylated surface proteins reveals that overall tagging
of surface proteins is heavily weighted toward the basolateral side;
apically labeled bands were only detected upon further exposure (not
shown).
)30% of TPO could ever be
detected at the plasma membrane (e.g.Fig. 5). Further,
the enzymatic activity of TPO is well known to be responsible for the
iodination of thyroglobulin, and in independent experiments, we have
recently reported that filter-grown thyrocytes exhibit intracellular
iodination activity in addition to that which is apically polarized
(Kuliawat and Arvan, 1994). Since the N-linked glycans of
porcine TPO undergo little or no modification by terminal sugars
(Rawitch et al., 1992) and remain very sensitive to digestion
with endoglycosidase H (Long et al., 1991), one might think
that the intracellular TPO pool is in the endoplasmic reticulum.
However, in filter-grown thyrocytes, intracellularly iodinated
thyroglobulin is exclusively resistant to digestion with
endoglycosidase H, suggesting that both the substrate and enzyme are
contained in Golgi/post-Golgi compartments (Kuliawat and Arvan, 1994).
This is compatible with a major intracellular distribution of TPO in
the apical cytoplasm of thyroid epithelial cells in vivo, as
determined by immunocytochemistry (Watanabe et al., 1991).
30% of newly synthesized TPO ever arrives at the cell surface or
whether TPO is efficiently delivered to the plasma membrane but is
rapidly internalized, rendering it inaccessible to surface tagging.
Regardless of which of these is the correct explanation, the surface
delivery data are clearly concordant with the data regarding
steady-state distribution of TPO. The large intracellular pool of TPO
and the fact that surface TPO appears less apically polarized than APN
indicate that thyrocytes handle these apical marker proteins
differently. This is not surprising since there is already strong
support for this fact, given that different apical membrane proteins
reside in discrete apical subdomains (Barriere et al., 1986;
Alquier et al., 1989). Thus, an important goal must be to try
to identify the structural features among different apical membrane
proteins that account for the rather substantial differences in their
intracellular distributions.
)/K
-ATPase; TPO, thyroid
peroxidase; TSH, thyrotropin; PAGE, polyacrylamide gel electrophoresis;
GPI, glycosylphosphatidylinositol; PI-PLC,
phosphatidylinositol-specific phospholipase C.
)-subunit was biotinylated far more
efficiently than the -subunit.)
We gratefully acknowledge Dr. W. Ball (University of
Cincinnati) for the antiserum to NKA and Drs. O. Noren and H. Sjostrom
(Panum Institute, Copenhagen, Denmark) for the antiserum to APN. We
thank the animal facility of Beth Israel Hospital (Boston, MA) for
assistance in access to fresh porcine tissue. We also thank members of
the Arvan laboratory for helpful discussions during the course of this
work.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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