(Received for publication, December 8, 1995; and in revised form, January 18, 1996)
From the
Stem cell factor (SCF) is a cytokine that is active toward
hematopoietic progenitor cells and other cell types, including germ
cells, melanocytes, and mast cells, which express its receptor, the
tyrosine kinase, Kit. SCF exists as noncovalently associated dimer at
concentrations where it has been possible to study its quaternary
structure; it stimulates dimerization and autophosphorylation of Kit at
the cell surface. We have used recombinant versions of human SCF and
human Kit extracellular domain (sKit) to study SCF-Kit interactions. By
size exclusion chromatography, plus various physical chemical methods
including light scattering, sedimentation equilibrium, and titration
calorimetry, we demonstrate the formation of complexes containing a
dimer of SCF (unglycosylated SCF) plus two
molecules of sKit. The concentrations of SCF and sKit in these studies
were in the range of 0.35-16.2 µM. The data are
analyzed and discussed in the context of several possible models for
complex formation. In particular, the sedimentation data are not
consistent with a model involving cooperative binding. The K
estimate for SCF-sKit interaction,
obtained by sedimentation equilibrium, is about 17 nM at 25
°C. With glycosylated SCF
, the K
is considerably higher.
Stem cell factor (SCF) ()is encoded by the steel (Sl) locus in the mouse(1, 2, 3) ,
and is the ligand for the tyrosine kinase type receptor,
Kit(1, 3, 4) . Kit in turn is encoded by the
dominant white spotting (W) locus in the
mouse(5, 6) . From the phenotypes of Sl and W mice, roles for SCF in hematopoiesis, gametogenesis, and
melanogenesis could be inferred(7, 8) . These
inferences have been supported by many in vitro and in
vivo studies in the last few years; it is clear that SCF
influences hematopoietic progenitors, gametes, and melanocytes both
developmentally and in the adult animal as well (reviewed in (8) ). SCF influences other cell types also, including mast
cells (reviewed in (8) ), and possibly certain intestinal cell (9, 10) and liver cell (11) subtypes.
SCF is expressed in different forms, determined by alternate splicing. A longer form, including extracellular domain, transmembrane domain, and cytoplasmic domain, is initially expressed at the cell surface; a presumed proteolytic cleavage can then lead to release of soluble SCF corresponding to amino acids 1-165, derived from the extracellular domain(12, 13, 14) . The alternate splicing leads to a shorter form which lacks the exon 6 amino acids including the proteolytic cleavage site and therefore tends to remain membrane bound(12, 13) . From several different approaches, there have been indications of functional differences between soluble and membrane-bound SCFs(12, 13, 15, 16, 17) . Soluble SCF is found at substantial levels in human serum(18) .
Soluble SCF exists as noncovalently associated homodimer at concentrations where it has previously been possible to study its quaternary structure(19) , although monomer dissociation/reassociation is rapid(20) . Whether or not membrane-bound SCF is dimeric has not been determined.
Kit is a
member of the type III tyrosine kinase type receptor family, which also
includes the platelet-derived growth factor receptor, the macrophage
colony-stimulating factor receptor, and
FLK2(21, 22, 23) . It should be noted that
platelet-derived growth factor and macrophage colony-stimulating factor
are both covalently associated dimers (24, 25, 26, 27, 28) . The
human Kit cDNA encodes a signal sequence (amino acids 1-23)
followed by extracellular domain (amino acids 24-520; five
Ig-like domains), hydrophobic transmembrane region (amino acids
521-543), and cytoplasmic domain (residues 544-976; split
kinase domains)(29) . The major expression product at the cell
surface is a glycoprotein with a molecular weight of about
145,000(29) . In comparison, the calculated molecular weight of
the polypeptide without signal sequence and without glycosylation is
109,740. As with other tyrosine kinase type receptor families, members
of the type III family undergo obligatory dimerization and
transphosphorylation as the initial event in ligand-induced signal
transduction(21) . Subsequent signaling events initiated by
SCF/Kit have been extensively studied in various cell types (see
introduction to (30) ). Varying somewhat with the system
studied, K values reported for SCF-Kit
interaction have generally been in the range of 0.5-65
nM, although additional higher affinity (16-310
pM) sites of SCF binding have been described for some cell
types(31, 32, 33, 34, 35, 36, 37, 38) .
Most of these affinity determinations have been arrived at using
recombinant Escherichia coli-derived soluble SCF
(nonglycosylated). The affinity of CHO cell-derived soluble SCF
(glycosylated) has been shown to be relatively lower by a factor of
5-10, and its mitogenic potency is lower also(14) .
Two forms of Kit have been reported. They differ in that one (designated Kit A) has a 4-amino acid insert just prior to the transmembrane domain(39) . The two forms arise as a result of alternative mRNA splicing(39) . In addition, soluble Kit (sKit; i.e. Kit extracellular domain) has been found in the conditioned medium of various cells that express Kit (37, 38, 40) and, at high levels, in human serum(30) .
For the growth hormone receptor, it has been shown that a monomeric ligand can mediate receptor dimerization(41, 42) . Because the ligands that associate with the type III tyrosine kinase type receptors appear to be dimeric, it is reasonable to suggest that this property may be important for mediating receptor dimerization within this family. However, this has not been definitively shown. Lev et al. (31) have proposed and discussed a model for SCF-mediated Kit dimerization in which it is not essential that SCF be dimeric when bound to Kit, and the same group has shown that Kit-Kit interactions play a role in Kit dimerization(43) .
In the present work, we have used recombinant human soluble SCFs and recombinant human sKit (Kit extracellular domain) to study the stoichiometries and affinities of SCF-Kit interactions by a variety of physical chemical methods. We demonstrate the formation of a complex containing two molecules of SCF monomer plus two molecules of Kit extracellular domain.
I-labeled E. coli-derived human
SCF of specific radioactivity 61 mCi/mg was prepared by the
lactoperoxidase method(46) . 12.5 µCi (about 16 million
cpm) of the labeled SCF was mixed with 2.4 mg of unlabeled E.
coli-derived human SCF such that the volume of the mixture was 0.4
ml. This solution was applied to a Superose 6 size exclusion column (1
29 cm; Pharmacia Biotech Inc.) equilibrated with PBS. Eluting
fractions were collected, and those corresponding to the single peak of A
-absorbing material, representing SCF, were
pooled to give a solution of SCF at 1.71 mg/ml and about 6 million
cpm/ml.
CHO cell-derived recombinant human sKit was expressed and
purified as described(38) . Based on the expressed sequence and
the expected processing of the signal sequence, the material represents
amino acids 24-520 according to the numbering of Yarden et
al.(29) , i.e. it corresponds to the entire
extracellular domain of Kit. Note that the 4-amino acid insert
(residues 510-513) characteristic of the Kit A isoform is
present. The material is heavily glycosylated, with mostly N-linked carbohydrate but some O-linked carbohydrate as
well(30) . Mass/volume concentrations for the sKit were
determined using an of 1.185
liters/g.cm, which is a value calculated from Trp, Tyr, and cystine
composition (45) ; thus these concentrations represent the sKit
polypeptide only, exclusive of carbohydrate. Molar concentrations of
sKit were determined from mass/volume values using a molecular weight
value of 55,815 (calculated from amino acid sequence of the
polypeptide).
The sedimentation data on the mixtures were fitted to various heterogeneous association models. The fitting procedure accounts for the different molar extinction coefficients of the various species at the measurement wavelength. During the fitting, the reduced molecular weights of sKit and CHO cell-derived SCF were held fixed at the values determined when they were run alone; the molecular weight calculated from sequence for E. coli-derived SCF was used because its sedimentation data were consistent with that value. The data for up to 18 samples were globally fitted to determine the dissociation constants. During fitting, the fitted parameters were constrained to values that correctly accounted for the total concentrations of sKit and SCF placed into the cell.
Figure 1:
Size exclusion chromatography of
incubated mixtures of sKit and E. coli-derived SCF. sKit at 40
µg/ml (0.72 µM) was incubated with levels of SCF
ranging from 6.6 to 52.4 µg/ml (0.18-1.40 µM of
SCF dimer). These represented molar ratios of sKit to SCF dimer of 4,
2, 1, and 0.5 (chromatograms 6, 5, 4, and 3, respectively). Chromatogram 2 represents SCF alone
(in the amount used in chromatogram 3), and chromatogram 1 represents sKit alone. Incubations were carried out at room
temperature for 30 min, and aliquots (200 µl) were then subjected
to size exclusion chromatography using a Superose 6 column as described
under ``Experimental Procedures.'' The amounts of eluting
materials were quantitated by integration of areas under peaks (data
not shown). The arrows at the top of the figure
represent (from left to right) void volume of the
column and then the elution positions of bovine thyroglobulin (M 670,000), bovine
-globulin (M
158,000), chicken ovalbumin (M
44,000), horse myoglobin (M
17,000), and
cyanocobalamin (M
1350). (Note that, as reported
previously (19) , the SCF elutes unusually early, probably
because of an elongated shape.)
This
equimolar stoichiometry was confirmed with the use of I-labeled SCF.
I-labeled SCF, prepared as
described under ``Experimental Procedures,'' was mixed with
unlabeled SCF (E. coli-derived) to give a preparation having
specific radioactivity of 163 cpm/µg. A mixture of sKit (at 40
µg/ml) and the SCF preparation having molar ratio of sKit to SCF
dimer of 1 was prepared, incubated, and subjected to size exclusion
chromatography as in Fig. 1. Fractions containing complex were
collected and pooled. Values of 2916 cpm/ml for radioactivity and 0.071
for absorbance at 280 nm were obtained for the pool. From the specific
radioactivity of the SCF (163 cpm/µg), it could be calculated that
SCF was present in the pool at 17.9 µg/ml or 0.96 µM (for SCF monomer). Because
for the SCF is 0.536, it could also be calculated that absorbance
due to SCF was 0.010. Thus absorbance due to Kit was 0.061 and, using
of 1.185, sKit was present in the
pool at 51.3 µg/ml or 0.92 µM, and the molar ratio of
sKit to SCF monomer determined in this way was 0.96 for the complex.
A priori, the equimolar amounts of sKit to SCF monomer in
the complex could represent 1:1 or 2:2 complex. (Note that all
references to stoichiometries for the complex, e.g. 1:1, 1:2,
2:2, will indicate the ratio of sKit to SCF monomer). The
elution positions of marker proteins of known molecular weights are
indicated by arrows in Fig. 1. However, because
molecular shape as well as molecular size influences elution position,
molecular weight values for SCF, sKit, and SCFsKit complex cannot
be reliably determined relative to marker proteins, and we did not
attempt to determine stoichiometry of the complex on the basis on
molecular weights estimated from elution positions.
As we have described
previously(49) , to calculate the polypeptide molecular weight
of the complex we need its extinction coefficient, which depends on the
(assumed) stoichiometry. We therefore used an approach in which the
calculated molecular weight is deemed valid only if it is consistent
with the stoichiometry assumed. If we assume the complex contains one
sKit plus two SCF monomers (one SCF dimer), the data imply M = 163,000, which is inconsistent with the
theoretical value of 93,128. With an assumed stoichiometry of two sKit
plus four SCF monomers (two SCF dimers) for the complex, the light
scattering data give M
= 163,000, again
inconsistent with the theoretical value of 186,256. In contrast, when
we assume a stoichiometry of two sKit plus two SCF monomers (one SCF
dimer), the calculated M
of 149,000 agrees very
well with the theoretical value of 148,943. We therefore conclude that
the complex indeed contains two sKit and two SCF monomers (one SCF
dimer).
Figure 2: Size exclusion chromatography of incubated mixtures of sKit and CHO cell-derived SCF. Experiments were done as for Fig. 1, except that CHO cell-derived SCF was used instead of E. coli-derived SCF.
The sKit behaved as an ideal single monomeric species, with no appreciable self-association at concentrations up to about 1 mg/ml. Using the polypeptide formula weight of 55,815 and partial specific volume of 0.732 ml/g, the data give a carbohydrate weight for sKit of 18,900 [15,900-21,200], and a total molecular weight of 74,800 [71,700-77,000]. A second, independent set of data for sKit gave total weight of 75,200 [72,400-77,200], showing excellent reproducibility of the results.
Figure 3:
Sedimentation equilibrium of mixtures of
sKit and E. coli-derived SCF. Rotor speed was 8000 rpm. Diamonds, sample made with 3 µM sKit plus 1.5
µM SCF dimer; squares, sample made with 3
µM sKit plus 3 µM SCF dimer. The natural log
of the absorbance at 280 nm is plotted versus (r - r
)/2, where r is the
radius and r
is the radius at the center of the
sample. The solid line illustrates the slope calculated for a
2:2 complex (two sKit per two SCF monomers (one SCF dimer)). The dashed line and the dotted line illustrate the slopes
calculated for a 1:2 complex.
Further quantitative analysis of these sedimentation equilibrium
data requires fitting to an appropriate binding model. Our size
exclusion chromatography, light scattering, sedimentation equilibrium,
and titration calorimetry (next section) studies all indicate that the
fundamental interaction stoichiometry is one sKit per SCF monomer and
that the largest complex is the 2:2 complex, so we need only consider
models that are consistent with this stoichiometry. Fig. 4schematically illustrates four such binding models.
Because at the protein concentrations used in these studies SCF will be
essentially completely dimeric, in the first three of these models SCF
dimer is treated as a single species with two binding sites for Kit
(these are the models used previously to treat the interactions of
neurotrophins with the receptors TrkB and TrkC(49) ). In Model
1, these two binding sites per SCF dimer have fixed, equivalent
affinities. In Model 2, the two binding sites have fixed inequivalent
affinities. Model 2 is included for completeness, but because there is
no reason to expect that a symmetric SCF dimer will present
inequivalent sites, this was not considered an appropriate model for
this system. In Model 3, the two binding sites are initially
equivalent, but they interact with either positive or negative
cooperativity to alter the affinity for binding the second receptor. It
is important to note that in this model the actual mechanism of
cooperativity is irrelevant; for example, it could arise either through
creation of a receptor-receptor interaction site, as has been
proposed(31, 43) , or through changes in SCF
conformation or other mechanisms. Lastly, in Model 4 we allow for a
finite dissociation of SCF into monomers, while keeping fixed the
intrinsic interaction between Kit and an SCF monomer. ()That
is, there is no cooperativity in this model, and the monomer
dimer equilibrium of SCF is assumed to be independent of whether Kit is
bound.
Figure 4:
Models for receptor-ligand interactions.
In all models the shapes of the receptor (R) and ligand (L or L) are schematic and not intended
to imply any specific structural features (the models are completely
defined by the association reactions and their dissociation constants).
In Models 1-3, the SCF dimer is treated as a nondissociable
molecule with two binding sites. In Model 4, SCF dimer is allowed to
reversibly dissociate into monomers, and this dissociation is assumed
to be independent of receptor binding.
Fig. 5shows the results of a simultaneous fit of Model 1 to data for sKit plus E. coli-derived SCF mixtures made up at both the two sKit per SCF dimer and one sKit per SCF dimer molar ratios, and at three different rotor speeds. This fit is excellent and returns an intrinsic dissociation constant for each site of 17 [12-21] nM. As implied by Fig. 3, these data fit poorly (with about 10-fold higher variance (not shown)) to a model having only one sKit binding per two SCF monomers (one SCF dimer). Indeed, the fitted parameters imply that, averaged across the cell, the sample made at a molar ratio of two sKit per SCF dimer shown in Fig. 3has 83% of its absorbance from the 2:2 complex, 11% from the 1:2 complex, 6% from free sKit, and 0.2% from free SCF, whereas the sample made at a molar ratio of one sKit/SCF dimer has 52% of its absorbance from the 2:2 complex, 43% from the 1:2 complex, 1% from free sKit, and 4% from free SCF. A second, independent set of experiments gave equivalent results, returning an intrinsic dissociation constant of 17 [14-20] nM.
Figure 5: Sedimentation equilibrium data, fitted curves, and residuals for sKit plus SCF mixtures. The data are for samples run at 8000, 11,000, and 14,000 rpm and loaded at the following concentrations (SCF concentrations are the concentrations of SCF dimer): 3 µM sKit plus 3 µM SCF; 1.5 µM sKit plus 1.5 µM SCF; 3 µM sKit plus 1.5 µM SCF; 430 nM sKit plus 215 nM SCF; and 375 nM sKit plus 375 nM SCF. The upper panel shows the raw data points and the fitted curves; for the sake of clarity, the data sets at 11,000 and 14,000 rpm have been displaced upward by 0.3 and 0.7 absorbance, respectively. All 2548 data points were globally fitted to Model 1 (Fig. 4), in which each SCF dimer has two equivalent sites to which sKit molecules bind independently. The best fit dissociation constant from this analysis is 17 nM. The bottom panel shows that the residuals from this fit do not exhibit large systematic deviations.
We have also examined fits to the cooperative binding model, Model 3. Because an examination of model data showed that the distribution of species is more sensitive to cooperativity when there is a larger excess of SCF in the samples, these fits included samples made at molar ratio of 0.5 sKit/SCF dimer. With these data sets, the cooperative model does not improve the fit over a fit to Model 1, and it can be concluded with 95% confidence that the dissociation constant for the second sKit on an SCF dimer is between 0.87 and 1.26 times that of the first. We therefore conclude that there is no binding cooperativity in this system, and we emphasize that this conclusion is based on data at high protein concentration and thus is free of any influence from possible dissociation of SCF dimers.
Model 4, which
incorporates the possibility of appreciable dissociation of SCF dimer
to monomers, can also be considered. Although our sedimentation data
show no evidence for dissociation of SCF dimers, they also cannot rule
out a monomer dimer K
of the order of a few
nanomolar or less. We therefore used model 4 to verify that such a K
value would not significantly affect our
estimates of the K
for SCF association with sKit.
Lastly, we have also used sedimentation equilibrium to compare the
affinities of sKit for glycosylated and nonglycosylated SCFs. Analysis
of 12 data sets for sKit plus CHO cell-derived SCF at molar ratios of
two sKit per SCF dimer and at one sKit per SCF dimer using Model 1 gave
a K of 590 [540-630] nM,
which implies a 35-fold weaker binding than that for E.
coli-derived SCF. The sedimentation data therefore confirm the
weaker binding suggested by the size exclusion chromatography studies.
Because SCF molecules with different glycosylation have different
affinities(14) , the 590 nMK
value determined here represents an average value. However, it is
likely that the data analysis does not weight molecules of different
affinity equally, so this value probably does not represent a true
overall average affinity.
Figure 6:
Titration calorimetry of SCF binding to
sKit. The upper panel shows the heat evolved (negative
signals) as a 500 µM solution of SCF dimer was titrated
(20 injections of 3 µl spaced 8 min apart) into the calorimeter
cell containing 1.36 ml of a 16.2 µM solution of sKit. The
integrated heats from each injection are shown as the data points in
the lower panel, after subtraction of the small constant heat
per injection apparent in injections 10-20. The solid curve in the lower panel is the best fit to a binding model in
which SCF has an unknown number of identical binding sites for sKit.
The best fit values imply that there are 2.03 sites/two SCF monomers
(one SCF dimer) with a dissociation constant of 60 nM and
H of -13.0 kcal/mol.
By a variety of physical chemical methods, including size exclusion chromatography, light scattering, sedimentation equilibrium, and titration calorimetry, we have clearly shown that SCF and sKit can form a stable 2:2 complex (i.e. two sKit per two SCF monomers or two sKit per SCF dimer). Our studies have utilized recombinant soluble forms of human SCF and human Kit.
The interactions that we describe and the conclusions that we reach provide insight into the possible nature of SCF-Kit interactions in vivo. The 2:2 complex formation clearly provides a mechanism for Kit dimerization, which is known to occur in vivo and to be required for initiation of signal transduction(21, 31, 51) . However, because all of our data is obtained in vitro, we cannot definitely state that such a complex would transmit or initiate biological signaling in vivo, and, as always, there are differences between in vitro and in vivo situations. As mentioned in the Introduction, both SCF and Kit are present in membrane-bound as well as soluble (extracellular domain) forms in vivo, and interactions involving the former may not be very well represented by our studies; the kinetics and energetics of interactions at surfaces will differ from those in solution, and our studies obviously would not reflect any interactions that might involve transmembrane or intracellular portions of SCF and Kit. In addition, even for soluble SCF and soluble Kit in vivo, concentrations are much lower than those in our in vitro studies (mean values in human serum, respectively, are 3.3 ng/ml (177 pM, for SCF monomer) and 324 ng/ml (5.8 nM)(18, 30) ). Clearly, SCF is mostly dimeric at the concentrations used in our in vitro studies, but we cannot exclude the possibility that SCF could be significantly dissociated into monomers at the low concentrations found in vivo.
Nevertheless, our studies suggest that the dimeric nature of SCF is important to the mechanism of Kit dimerization. This would differ from cases of monomeric ligands that can mediate dimerization of their receptors(41, 42) . Note that macrophage colony-stimulating factor and platelet-derived growth factor, whose receptors are members of the Type III tyrosine kinase family along with Kit, are covalently held dimers (24, 25, 26, 27, 28) .
It is important to ask whether our results are in accord with other proposed models for interaction of SCF and Kit and for Kit dimerization. A sequential model for this process was proposed by Lev et al.(31) . In this model, the initial binding of Kit to an SCF dimer to form a 1:2 complex is monovalent (i.e. it involves only one of two binding sites/SCF dimer) and has relatively low affinity. This SCF binding triggers a conformational change in Kit that exposes or creates a receptor-receptor interaction site (a ``receptor dimerization site''). Consequently a second Kit associates, giving rise to an unstable 2:2 complex in which the receptors are held together only by the dimerization site, and only one of the receptors interacts with SCF. Next, the second Kit molecule binds to the second site on the SCF dimer, giving rise to a stable, high affinity 2:2 complex held together both via the receptor dimerization site and via the SCF dimer. This model also proposes that when SCF is in great excess over Kit, a single Kit dimer may bind two SCF dimers (each held by only a single interaction with a receptor), forming a 2:4 complex. A key feature of this model, i.e. that receptor dimerization can occur independently of the bivalent nature of the SCF ligand, was invoked primarily to account for the observation that human SCF is able to heterodimerize mouse and human Kit on cells that express both receptors, even though the affinity of human SCF for mouse Kit is very low. This group has proposed that a Kit dimerization site is present in immunoglobulin-like domain 4 of the Kit extracellular domain, because recombinant Kit truncations that lack immunoglobulin-like domain 4 are unable to dimerize(43) .
However, this sequential binding model is inconsistent with the
behavior we observe for sKit and SCF in significant ways. The model
predicts that binding will be highly cooperative, because the addition
of a second sKit molecule to a 1:2 complex to form the stable 2:2
complex adds the receptor-receptor binding energy in addition to a
second receptor-ligand bond. As discussed above, we find no evidence
for cooperativity. In other words, the sequential model predicts that
2:2 complex will persist when ligand is in great excess, whereas we
find that even a 2-fold excess of SCF (in terms of monomer) over sKit
substantially reduces the proportion of 2:2 complex in favor of 1:2
complex (Fig. 3). ()Note that reported bell-shaped
SCF dose-response curves (43, 51) can be interpreted
as being consistent with a lack of cooperativity, i.e. if the
binding of a second Kit is not favored relative to binding of the
first, Kit molecules will tend to be associated singly with SCF dimers
when SCF is in excess over Kit. Another way in which our data are
inconsistent with the sequential model is that we find no evidence by
sedimentation equilibrium for formation of the 2:4 complexes predicted
by this model when SCF is in great excess.
The binding affinities
determined here by sedimentation equilibrium can be compared with
reported K values determined by techniques
involving the binding of radiolabeled SCF. We have previously reported
a value of 1.5 nM for the same sKit used in these
studies(38) , whereas others have reported values of 0.7 nM and 0.3 nM, respectively, for recombinant Kit
extracellular domain (32) and for a fusion between Kit
extracellular domain and an antibody Fc fragment(34) . For the
full-length receptor recombinantly expressed on cells, values of
0.5-2 nM have been reported(31, 33) .
For various cell types naturally expressing the full-length receptor,
high affinity (K
16-310 pM) and low
affinity (K
1.7-65 nM) classes of
receptor have been reported, with both being present together on some
of the cell types(35, 36, 37) . However, in
making comparisons between values from different laboratories or
techniques, it is important to note that the affinity of sKit for SCF
must be strongly temperature-dependent, and the data cited were
obtained at either 4 (34, 38) or 22
°C(32) . The fairly large
H for this
interaction seen by titration calorimetry (which matches that reported
for human growth hormone and its receptor(41) ), implies a
4.6-6.2-fold increase in affinity between 25 and 4 °C
(assuming
H and
S are
temperature-independent). For example, our estimate of 17 nM for the K
from sedimentation equilibrium at
25 °C implies a K
of about 3 nM at 4
°C, in reasonable agreement with the value of 1.5 nM from
the binding of radiolabeled SCF(38) . Further, in comparing the
present results to those obtained by other methods, it is important to
ask whether the stoichiometry of the interactions and multivalent
nature of SCF were explicitly considered, particularly with regard to
binding to cell surfaces, where the dimerization of Kit should produce
nonlinear Scatchard plots that overestimate the true intrinsic
affinity(52) .
By size exclusion chromatography and by
sedimentation equilibrium, we find that glycosylated SCF has a lower
affinity for Kit than does nonglycosylated SCF. This conclusion is
consistent with previous data (14) showing lower in vitro biological response potency and weaker binding in a competition
type receptor binding assay for glycosylated SCF in comparison with
nonglycosylated SCF; the differences were shown to be caused
particularly by glycosylation at Asn and at Asn
(14) and presumably represent steric effects of the
glycosylation. As mentioned above, we have also shown that soluble SCF
and soluble Kit are both present in human serum (mean levels of 177
pM (for SCF monomer) and 5.8 nM, respectively) and,
using a K
value of 1 nM, we had
calculated that much (about 85%) of the SCF would be associated with
soluble Kit(30) . Because native SCF is
glycosylated(18) , the higher K
values
would lead to a calculation of considerably less SCF association with
soluble Kit in the serum. The temperature dependence of K
noted above would affect such calculations
similarly.