(Received for publication, February 5, 1997, and in revised form, April 9, 1997)
From Src homology 3 (SH3) domains are conserved
modules which participate in protein interaction by recognizing
proline-rich motifs on target molecules. To identify new SH3-containing
proteins, we performed a two-hybrid screen with a proline-rich region
of human SOS-1. One of the specific SOS-1 interacting clones that were
isolated from a mouse brain cDNA library defines a new protein that
was named amphiphysin 2 because of its homology to the previously reported amphiphysin. Amphiphysin 2 is expressed in a number of mouse
tissues through multiple RNA transcripts. Here, we report the amino
acid sequence of a brain form of amphiphysin
2 (BRAMP2) encoded by a 2.5-kilobase mRNA. BRAMP2
associates in vitro with elements of the endocytosis
machinery such as A limited set of protein modules mediates molecular
interactions and underlies the diversity of intracellular signaling
pathways. Among these modules, Src homology 3 (SH3)1 domains were first identified as
non-catalytic regions of 55-70 AA, present in signaling and
cytoskeletal proteins (1). A common feature of SH3 domains is their
ability to bind proline-rich sequences on target molecules. A SH3
domain is composed of a hydrophobic binding pocket with finely
positioned aromatic residues and charged loops outside of this pocket
that determine the specificity of the interaction with proline-rich
motifs. Phage display or peptide library screenings with individual SH3
domains demonstrated the absence of a strict exclusivity for SH3 and
proline-rich partners (2, 3). This feature may be the basis for the
identification of new SH3-containing proteins.
Human SOS-1 C terminus contains a multitude of intermingled
proline-rich motifs. It was used as a "pseudo-degenerated" bait in
search for new SH3 domains in a two-hybrid screen. Several SH3 domains
were isolated, and one of them allowed us to identify a new protein,
homologous to, but different from, amphiphysin (4), that was named
amphiphysin 2. Amphiphysin is a strictly neuronal protein that plays a
crucial role in the endocytosis of synaptic vesicles (5). A current
hypothesis suggests that a ubiquitous homologue involved in general
endocytosis should exist. We show here that this hypothesis is correct
and incomplete. Amphiphysin 2 is expressed under several forms: a
2.0-kb transcript which is expressed almost ubiquitously and a 2.5-kb
transcript that is brain-specific. The latter form was further
characterized in vitro and in vivo. We show that
the brain form of amphiphysin 2
(BRAMP2) retains the same interactive abilities as amphiphysin itself,
i.e. it binds dynamin and Two-hybrid Screen and Full-length cDNA Cloning
Briefly, a fusion between the GAL4-binding domain and the
C-terminal portion of human SOS-1 (hSOS-1, AA 1131-1333 into the pGBT10 vector) (6) was used as bait to screen a Balb/c mouse brain
cDNA library constructed in the pGAD1318 vectors (7, 8). The
cDNA inserts from His+/LacZ+ specific
clones were sequenced with an automatic sequencer (Applied Biosystems
Inc., model 373A). One of them was hybridized to a mouse brain Northern Blot Analysis
Hybridizations were performed sequentially with three
random-primed 32P-labeled probes corresponding to bp
1283-1581, bp 1824-2374 of the full-length BRAMP2, and Recombinant Proteins, Purified Proteins, and Peptide
A central fragment of BRAMP2 cDNA corresponding to AA
178-511 (bp 784-1784) was subcloned into pGEX4T1 (Pharmacia Biotech, Uppsala, Sweden) and pMalC2 (New England Biolabs, Beverly, MA) to
produce a glutathione S-transferase (GST) and a
maltose-binding protein (MBP) fusion protein, respectively. The full
coding sequence of BRAMP2 (bp 250-2506) was subcloned into pGEX4T1.
After production in Escherichia coli, GST fusion proteins
were purified on glutathione-Sepharose beads, and MBP fusion proteins
were purified on amylose resin. Rabbit Ab were produced against
GST-BRAMP2 (AA 178-511) and affinity-purified over a HiTrap
NHS-activated column (Pharmacia) to which MBP-BRAMP2 (AA 178-511) had
been covalently linked, before use in biochemical experiments. Rat
brain dynamin was purified according to Gout et al. (9). A
dynamin I peptide, GPPPQVPSRPNR, was synthesized in a solid-phase
method on an Applied Biosystems apparatus and purified by reverse phase
high performance liquid chromatography. The anti-dynamin monoclonal
antibody (mAb) recognizing dynamin I and II was purchased from
Transduction Laboratories (Lexington, KY). The anti- In Vitro Interactions
Human SOS-1 and SOS-2
subcloned into pBluescriptSK Mouse fibroblast 3T3 cells or
rat pheochromocytoma PC12 cells were lysed in 10 mM Tris,
pH 7.5, 150 mM NaCl, 1 mM EDTA, 1.0% Triton
X-100. After centrifugation at 10,000 × g for 30 min,
supernatants were incubated with either GST-BRAMP2 (full coding
sequence) or GST, both of them purified on glutathione-Sepharose beads.
After washing in lysis buffer, proteins bound to beads were separated by electrophoresis, transferred to nitrocellulose, and immunoblotted with anti-dynamin (1/1000) or - Cell Transfections
BRAMP2 (bp 250-2506) was subcloned into pRK5. Subconfluent 3T3
cells were transfected with 5 µg of pRK5-BRAMP2 or pRK5 plasmids per
100-mm dish, using a modified calcium phosphate coprecipitation method
as described previously (10). After transfection, cells were grown for
36 h and lysed in Triton X-100-containing buffer. After
centrifugation at 10,000 × g for 30 min, supernatants were used as cell extracts, next to PC12 cell extracts and mouse brain extracts for immunoblotting with anti-BRAMP2 Ab (0.5 µg/ml).
Immunoprecipitations
Serum-deprived PC12 cells were incubated with 50 ng/ml NGF
(Sigma) for 10 min at 37 °C, before lysis in 10 mM Tris,
pH 7.5, 150 mM NaCl, 1 mM EDTA, 1.0% Chaps.
After centrifugation at 10,000 × g for 30 min, the
supernatant was precleared on protein A-Sepharose beads (Pharmacia) and
incubated with anti-BRAMP2 Ab previously bound to protein A beads.
After washing in lysis buffer, proteins bound to beads were separated
by electrophoresis and transferred to nitrocellulose before immunoblot
analysis, as mentioned above.
Fluorescence in Situ Hybridization Analysis
Fluorescence in situ hybridization to metaphase
chromosomes prepared from a normal human male was carried out according
to a usual technique, using R-banding (11). The probe was a BRAMP2 DNA
fragment from Surface Plasmon Resonance (SPR)
SPR experiments were carried out on a Biosensor BIAcore 2000 apparatus (Pharmacia Biosensor). Dynamin or GST-BRAMP2 in 10 mM potassium acetate buffer, pH 4.0 and pH 5.25, respectively, were covalently bound to the dextran matrix of a CM5
sensor chip, according to a method previously described (12). Binding
experiments were performed at 25 °C with a flow rate of 20 or 30 µl/min, in HBS running buffer (20 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM dithiothreitol, 0.005%
surfactant P20). Control experiments were performed with a blank cell
allowing us to measure the resonance units due to injected proteins
that were subtracted from the specific binding values. After each
binding experiment, the sensor chip was regenerated with 10 µl of 3 M guanidinium chloride. Estimation of kinetic parameters
was done by repetitive injections of a range of protein concentrations
over different densities of immobilized partner. Data analysis was
performed with the interactive software BIA evaluation version 2.0.
To identify new SH3-containing
molecules, we performed a two-hybrid screen with a proline-rich region
of hSOS-1 and a mouse brain cDNA library. One of the specific SOS-1
interacting clones was subsequently used to screen a mouse brain
A mouse multi-tissue RNA blot was hybridized with a
first BRAMP2 probe (bp 1824-2374) possibly recognizing multiple
amphiphysin 2 isoforms and with a second probe (bp 1283-1581)
corresponding to the 108-AA central insert. As shown in Fig.
2A, amphiphysin 2 mRNAs are expressed in
a number of tissues tested: brain, muscle, liver, lung, spleen, and
kidney, as 2.0- and 2.5-kb messengers. In contrast to amphiphysin,
amphiphysin 2 expression does not appear to be restricted to neuronal
cells. Nonetheless, the splicing isoform corresponding to the BRAMP2
cDNA shows a more restricted pattern of expression and is mostly
expressed in brain as a 2.5-kb RNA (Fig. 2B). A control
The
BRAMP2 cDNA was first isolated in a two-hybrid screen with the
proline-rich region of SOS-1. To confirm the yeast genetic data, we
tested in vitro binding of GST-BRAMP2 to
35S-labeled SOS-1 and -2. As shown in Fig.
3A, GST-BRAMP2 bound SOS-1 and -2, whereas
GST did not. The specificity of the interaction was further confirmed
by using 35S-luciferase, unable to interact with either
GST-BRAMP2 or GST. These in vitro data are consistent with
the yeast two-hybrid result and further show that the interaction
between brain amphiphysin 2 and SOS is direct.
Thereafter, we tested the ability of brain amphiphysin 2 to interact
with previously reported partners of amphiphysin: dynamin and
To further study
BRAMP2/dynamin interaction, we have used a SPR assay with a BIAcore
Biosensor. This method allowed us to detect molecular interactions in
real time and to estimate their kinetic parameters. Fig.
4A shows the direct binding of increasing concentrations of GST-BRAMP2 to immobilized dynamin (from 0 to 300 s), followed by a dissociation phase (300 to 600 s). Control GST
did not give any specific binding on the dynamin sensor (data not
shown). The association rate for GST-BRAMP2, ka = 1.5 × 104 M
We have defined a new SH3-containing molecule, highly homologous
to amphiphysin. It is encoded by a gene located in human chromosome
2q21, a locus different from the 7p13-14 identified for amphiphysin.
Based on its similarity to amphiphysin, the product of this gene was
named amphiphysin 2. Unlike amphiphysin, it has a broad expression in
several mouse tissues and may be seen as a ubiquitous amphiphysin
homologue. In patients with stiff-man syndrome associated with breast
cancer (15), it is a potential target outside the brain for
anti-amphiphysin autoantibodies. From one mouse tissue to the other,
the expression of amphiphysin 2 seems to be dependent on multiple
transcripts, generated either by different sites of
initiation/polyadenylation or by alternative splicing. One of these
transcripts encodes the BRAMP2 sequence that we report herein. Shorter
transcripts should be related to the previously reported SH3P9 (13) and
BIN1 (14) sequences which are identical to BRAMP2 in their N- and
C-terminal ends. BRAMP2, SH3P9, and BIN1 are probably three splicing
variants of the same genomic sequence that may give rise to
functionally diverse molecules. For example, BIN1 was reported to have
a short nuclear localization signal absent in the other isoforms and a
Myc-binding domain maintained in the other isoforms (14). BRAMP2 has a
long central insert absent in SH3P9 and BIN1 that makes it structurally closer to amphiphysin and correlates with a brain-restricted
expression. It remains to be determined whether the same brain cell
types express amphiphysin and this form of amphiphysin 2.
We have shown that BRAMP2 associates in vitro with key
elements of the endocytic pathway: The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U86405[GenBank].
INSERM U248, Institut Curie, 26 rue d'Ulm,
75231 Paris Cedex 05, France, ¶ INSERM U363,
INSERM
U301,
-adaptin and dynamin. On a biosensor surface, the
BRAMP2/dynamin interaction appeared to be direct and partly dependent
on a proline-rich sequence of dynamin. Association with dynamin was
also observed in PC12 cells after cell stimulation with nerve growth
factor, suggesting that amphiphysin 2 may be connected to
receptor-dependent signaling pathways. This hypothesis is
strengthened by the ability of BRAMP2 to interact with the
p21ras exchange factor SOS, in vitro, as a possible
point of interconnection between the endocytic and signaling
pathways.
-adaptin in vitro.
In vivo, PC12 amphiphysin 2 co-immunoprecipitates with
dynamin when cells have been stimulated with NGF. Thus, the interaction
of PC12 amphiphysin 2 with key players of the endocytosis machinery is
dependent on the stimulation of a signaling pathway initiated by NGF
binding to its membrane receptor. The interconnection between
endocytosis and membrane receptor signaling is further supported by the
ability of brain amphiphysin 2 to interact with SOS in vitro
as well as within the yeast two-hybrid system. This result brings
evidence for an additional level of molecular interaction between
endocytosis and signal transduction pathways, supporting the idea that
both pathways may be mutually regulated.
gt10
cDNA library (CLONTECH, Palo Alto, CA). The cDNA inserts were subcloned into pBluescriptSK
for automatic sequencing.
-actin,
using mouse multi-tissue RNA blots from CLONTECH,
according to the manufacturer's instructions.
-adaptin mAb was
from Alexis Corp. (San Diego, CA).
were used as DNA templates in a
transcription/translation reaction with reticulocyte lysates and T7 RNA
polymerase. Luciferase DNA provided by the manufacturer was used as
control (Promega, Madison, WI). [35S]Methionine-labeled
proteins were incubated with either GST-BRAMP2 (full coding sequence)
or with GST, bound to glutathione beads. After washing in 20 mM Tris, pH 7.5, 100 mM NaCl, 0.5 mM dithiothreitol, 0.2% Nonidet P-40, samples were
separated by electrophoresis and autoradiographed.
-adaptin mAb (1/1000). Bound Ab were
detected by peroxidase-labeled anti-mouse Ab and the ECL system
(Amersham, Alesbury, UK).
gt10 screening (bp 305-2530).
Primary Structure of BRAMP2
gt10 cDNA library, and positive overlapping inserts were
sequenced. The 2.5-kb full-length sequence, BRAMP2, contains an open
reading frame of 1765 bp (bp 250-2014, data not shown) encoding a
protein of 588 AA. The first methionine following an in-frame stop
codon is within a good Kozak consensus sequence. Analysis of the
predicted AA sequence revealed an SH3 domain in the C-terminal part of
the molecule (AA 520 to 588) and two proline-rich motifs (AA 297-306
and AA 340-346) that could be seen as internal SH3-binding sites.
Comparison of BRAMP2 with data base sequences demonstrated significant
homology to human amphiphysin (4) (Fig. 1), with a 42%
identity at the AA level. The BRAMP2 DNA fragment allowed us to
localize the corresponding gene in human 2q21 (data not shown), a locus
different from 7p13-p14 defined for amphiphysin. Based on its sequence
similarity to amphiphysin, we named the product of this gene,
amphiphysin 2, another member of the "amphiphysin family." Data
base analysis also revealed that BRAMP2 is similar to recently reported
sequences, murine SH3P9 (13) and human BIN1 (14), with portions of
almost complete identity in the N- and C-terminal ends of the
molecules. This suggests that BRAMP2, SH3P9, and BIN1 are three
splicing isoforms of the same genomic sequence. With a 108-AA central
insert absent in the latter two molecules, BRAMP2 is structurally
closer to amphiphysin.
Fig. 1.
BRAMP2 cDNA encodes a novel member of the
amphiphysin family. The predicted sequence of full-length BRAMP2
protein is shown. Comparison between murine BRAMP2 and the previously reported murine SH3P9, human BIN1, and human amphiphysin was carried out by using Clustal V software. The SH3 domain (dark) and
the proline-rich sequences (clear) are
boxed.
[View Larger Version of this Image (43K GIF file)]
-actin probe indicates that variations in the detection of
messengers cannot be due to differences in RNA loading (Fig.
2C). As detected by Western blotting, the expression of
pRK5-BRAMP2 in NIH-3T3 transfected cells produces a doublet of
polypeptides migrating at 88/96 kDa, an apparent molecular mass
different from the theoretical 64.5 kDa (Fig. 2D). This
difference is probably due to post-translational modifications that may
also account for the protein doublet since no particular cleavage site has been localized in the sequence. Such a doublet can also be detected
in a PC12 cell extract as well as in a mouse brain extract, reinforcing
the idea that this amphiphysin 2 isoform is expressed in neuronal cell
types. Anti-BRAMP2 Ab were able to recognize other polypeptides in
NIH-3T3 cells, probably because the portion of BRAMP2 used for Ab
production is common to multiple isoforms. These 57- and 67-kDa
polypeptides could represent different splicing isoforms of amphiphysin
2 such as BIN1, which was reported to be a 70-kDa molecule (14).
Fig. 2.
Amphiphysin 2 expression. A Northern
blot containing mouse poly(A)+ RNA from the indicated
tissues was hybridized with BRAMP2 DNA probes corresponding to bp
1824-2374 (A), bp 1283-1581 (B) or with a
-actin DNA (C) to control for the presence of RNA.
D, cell or tissue extracts were separated by SDS-PAGE,
transferred to nitrocellulose, and blotted with anti-BRAMP2 Ab.
1, 3T3 cells; 2, 3T3 cells transfected with
control pRK5 vector; 3, 3T3 cells transfected with
full-length BRAMP2 in pRK5; 4, PC12 cells; 5, mouse brain extract.
[View Larger Version of this Image (43K GIF file)]
-Adaptin
Fig. 3.
Interaction of BRAMP2 with SOS, dynamin, and
-adaptin. A, in vitro synthesized
35S-labeled SOS1 or SOS2 or control luciferase was
incubated with purified GST (central panel) or GST-BRAMP2
(right panel). Samples were run on SDS-PAGE in parallel with
an aliquot of the 35S-labeled starting material (left
panel) and autoradiographed. B, 3T3 cell extracts were
incubated with GST or GST-BRAMP2. Samples were run on SDS-PAGE in
parallel with an aliquot of the starting cell lysate. After transfer to
nitrocellulose, protein blots were incubated either with anti-dynamin
(left panel) or with anti-
-adaptin (right
panel). In the central panel, PC12 cells were either
unstimulated or stimulated shortly with NGF before lysis in 1.0%
Chaps. Immunoprecipitations were performed with anti-BRAMP2 Ab or a
control rabbit Ig. Samples were run on SDS-PAGE, in parallel with an
aliquot of unstimulated or stimulated PC12 cell lysates, transferred to
nitrocellulose, and blotted with an anti-dynamin mAb.
[View Larger Version of this Image (45K GIF file)]
-adaptin. 3T3 cell extracts were incubated with GST-BRAMP2 or GST,
and bound proteins were detected by immunoblot. As shown in Fig.
3B (left panel), anti-dynamin mAb was able to
specifically recognize the 100-kDa dynamin in GST-BRAMP2 samples.
Identical results were obtained with PC12 cell extracts (data not
shown). Similarly, a 110/115-kDa doublet of
-adaptins was
specifically retained by GST-BRAMP2 beads (Fig. 3B,
right panel) and detected by anti-
-adaptin mAb. For both
immunodetections, the specificity of the interaction was confirmed by
using Sepharose beads loaded with equivalent amounts of GST as negative
control. The in vitro interaction between BRAMP2 and dynamin
was strengthened by coimmunoprecipitation experiments. After lysis of
PC12 cells in mild conditions (1.0% Chaps), an anti-BRAMP2
immunoprecipitate was shown to contain the 100-kDa dynamin when cells
had previously been stimulated with NGF. In the same lysis conditions,
amphiphysin 2 immunoprecipitated from unstimulated PC12 cells was not
associated with dynamin (Fig. 3B, central panel).
Taken together, these results strongly link the BRAMP2 form of
amphiphysin 2 to the endocytic pathway, as was previously reported for
amphiphysin.
1
s
1, was calculated from the slope of a plot of
ks against GST-BRAMP2 concentration (Fig.
4B). The dissociation rate in buffer flow was near 0 and
could not be studied by dynamin injection because of the
oligomerization of the molecule. To prevent this problem, the
dissociation rate was measured after binding of 0.5 µM
dynamin to a GST-BRAMP2-coated membrane by injection of 1 µM GST-BRAMP2 (Fig. 4C). Repetitive
experiments gave two dissociation rate constant values on the order of
kd1 = 1.6 × 10
1 s
1
and kd2 = 3.5 × 10
3
s
1. However, when low concentrations of dynamin were
used, the faster dissociation rate was not observed, suggesting that it
could represent dynamin aggregation on the sensor. The equilibrium
dissociation constant was thus calculated with kd2
as kDa = kd/ka
240 nM. As shown in Fig. 4D, BRAMP2/dynamin
interaction could be inhibited partially by the GPPPQVPSRPNR peptide
from dynamin, involving this particular proline-rich motif and the SH3
domain of BRAMP2 in the association between both molecules.
Fig. 4.
Interaction of BRAMP2 with dynamin studied by
surface plasmon resonance. A, increasing concentrations of
GST-BRAMP2 (40, 80, 120, 160, 240, 320, and 400 nM) were
injected over immobilized dynamin. The arrows indicate the
beginning and the end of injection. B, the
ka value was obtained from the slope of
ks (calculated by using the BIAcore software)
versus concentration of injected GST-BRAMP2. C,
real dissociation value (kd) was determined by
injection of a high concentration of GST-BRAMP2 (1 µM)
after injection of a high concentration of dynamin (0.5 µM) over immobilized GST-BRAMP2. D,
specificity of BRAMP2/dynamin interaction was determined by the
decrease of GST-BRAMP2 (0.4 µM) binding preincubated with
a proline-rich peptide (PRP, 500 µM) from
dynamin.
[View Larger Version of this Image (23K GIF file)]
-adaptin and dynamin from 3T3 as well as from PC12 cells. The interaction with dynamin is direct and
likely to rely on the SH3 domain of amphiphysin 2 on one side and on a
proline-rich sequence of dynamin on the other side. This conclusion is
based on molecular interactions studied on a BIAcore biosensor where a
proline-rich peptide of dynamin I corresponding to one of the
previously described Grb2-binding sites is partially inhibitory (9,
16). This suggests that BRAMP2 interacts at least in part with this
proline-rich sequence. The affinity of BRAMP2/dynamin interaction on
the biosensor membrane was estimated in the 240 nM range,
revealing an affinity similar to other SH3 domain/protein ligand
interactions (17) and much higher than what have been described with
proline-rich peptide ligands (18). The association of brain amphiphysin
2 with dynamin was also detected in vivo after stimulation
of PC12 cells with NGF. This result shows that interaction of
amphiphysin 2 with a protein of the endocytosis machinery is dependent
on a signaling event coming from a cell surface receptor. A number of
previous reports demonstrated that the organization of the
clathrin-dependent endocytic machinery is tightly
controlled by the activation of the surface receptors that are going to
be endocytosed, as part of signal attenuation. First, endocytosis is
dependent on the tyrosine kinase activity associated with the receptor
(19). Second, ligand binding stimulates the interaction of activated
receptors with the AP2 complex on one hand (20) and with dynamin
through signal transduction molecules on the other hand (21). Grb2 is
an important element in this cascade that was reported to interact with
dynamin upon ligand binding and stimulate dynamin GTPase activity,
possibly by promoting dynamin self-association (9, 21, 22). It is
conceivable that upon receptor stimulation, amphiphysin 2 joins the
multimolecular complex located at the cytoplasmic end of the receptor.
The possible competition between amphiphysin 2 and Grb2 for dynamin
binding can be reconciled in a sequential scheme involving
phosphorylation- or nucleotide binding-dependent changes of
conformation. Inside the multimolecular complex depicted above,
elements of the endocytic pathway are intimately associated with
elements of the signaling pathway. Cumulative evidence suggests that
both pathways regulate each other, at multiple levels (23). Even though
we need to confirm it in vivo in mammalian cells, our
ability to detect an interaction between the brain form of amphiphysin
2 and the p21ras exchange factor SOS, brings further support to
the interconnection between both pathways, at the site of receptor
stimulation. We are currently testing whether a ternary complex of
amphiphysin 2/SOS/dynamin exists in vivo and defines a new
point of functional interaction, regulating endocytic function as
well as signaling function.
*
This work was supported in part by grants from Association
pour la Recherche contre le Cancer (ARC), Ligue Nationale contre le
Cancer (Comité de Paris), and Groupement de Recherche et
d'Etudes sur les Génomes.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed: INSERM U248, Institut
Curie, 26 rue d'Ulm, 75231 Paris Cedex 05, France. Tel.: 33-1-42-34-66-43; Fax: 33-1-42-34-66-50; E-mail:
leprince{at}curie.fr.
Supported by a postdoctoral European Community
fellowship.
1
The abbreviations used are: SH, Src homology;
AA, amino acid; GST, glutathione S-transferase; mAb,
monoclonal antibody; MBP, maltose-binding protein; NGF, nerve growth
factor; PAGE, polyacrylamide gel electrophoresis; SPR, surface plasmon
resonance; bp, base pair(s); kb, kilobase(s); Chaps,
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.