Enhanced solubilization of membrane proteins by alkylamines and polyamines

Article abstract of DOI:10.1002/pro.326

Around 25% of proteins in living organisms are membrane proteins that perform many critical functions such as synthesis of biomolecules and signal transduction. Membrane proteins are extracted from the lipid bilayer and solubilized with a detergent for biochemical characterization; however, their solubilization is an empirical technique and sometimes insufficient quantities of proteins are solubilized in aqueous buffer to allow characterization. We found that addition of alkylamines and polyamines to solubilization buffer containing a detergent enhanced solubilization of membrane proteins from microsomes. The solubilization of polygalacturonic acid synthase localized at the plant Golgi membrane was enhanced by up to 9.9-fold upon addition of spermidine to the solubilization buffer. These additives also enhanced the solubilization of other plant membrane proteins localized in other organelles such as the endoplasmic reticulum and plasma membrane as well as that of an animal Golgi-localized membrane protein. Thus, addition of alkylamines and polyamines to solubilization buffer is a generally applicable method for effective solubilization of membrane proteins. The mechanism of the enhancement of solubilization is discussed. Published by Wiley-Blackwell.

Full text of DOI:10.1002/pro.326

Enhanced solubilization of membrane
proteins by alkylamines and polyamines
Kazutoshi Yasui,1 Masamichi Uegaki,1 Kentaro Shiraki,2 and Takeshi Ishimizu1*
1Department of Chemistry, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan
2Institute of Applied Physics, University of Tsukuba, Tsukuba, Ibaraki 305-8573, Japan
Received 22 July 2009; Revised 18 December 2009; Accepted 20 December 2009
DOI: 10.1002/pro.326
Published online 6 January 2010 proteinscience.org
Abstract: Around 25% of proteins in living organisms are membrane proteins that perform many
critical functions such as synthesis of biomolecules and signal transduction. Membrane proteins
are extracted from the lipid bilayer and solubilized with a detergent for biochemical
characterization; however, their solubilization is an empirical technique and sometimes insufficient
quantities of proteins are solubilized in aqueous buffer to allow characterization. We found that
addition of alkylamines and polyamines to solubilization buffer containing a detergent enhanced
solubilization of membrane proteins from microsomes. The solubilization of polygalacturonic acid
synthase localized at the plant Golgi membrane was enhanced by up to 9.9-fold upon addition of
spermidine to the solubilization buffer. These additives also enhanced the solubilization of other
plant membrane proteins localized in other organelles such as the endoplasmic reticulum and
plasma membrane as well as that of an animal Golgi-localized membrane protein. Thus, addition of
alkylamines and polyamines to solubilization buffer is a generally applicable method for effective
solubilization of membrane proteins. The mechanism of the enhancement of solubilization is
discussed.
Keywords: alkylamine; membrane protein; polyamine; polygalacturonic acid synthase; solubilization
preparation of solubilized membrane proteins and
for purification of membrane proteins from living
cells. These experimental conditions must be opti-
mized for every membrane protein, and it often
takes a great deal of time and effort. Therefore,
more effective, standardized methods of handling
membrane proteins are still actively sought.
Introduction
Membrane proteins function in critical biological
processes such as synthesis of biomolecules, energy
metabolism, the transduction of signals, and the
transport of solutes across membranes. Around 25%
of naturally occurring proteins are predicted to be
membrane proteins1,2; however, they have been less
studied than soluble proteins mainly because of low
abundance in cells and their poor recovery in solu-
tion. In addition, it is difficult to produce recombi-
nant membrane proteins in heterologous cells. Fur-
thermore, there are no standardized methods for
The procedure for the purification of a mem-
brane protein begins with its solubilization. Mem-
brane proteins have poor recovery in aqueous buffer
due to their being embedded in a lipid bilayer and
due to their hydrophobic nature. Solubilization of
membrane proteins has been achieved in appropri-
ate buffers with mild detergents. Numerous deter-
gents for solubilization of membrane proteins have
been investigated.3,4 This solubilizing step is an em-
pirical technique and the selection of detergent and
optimization of solubilization conditions are deter-
mined by trial and error experiments that depend
on the protein of interest. Despite laborious experi-
ments, sufficient levels of solubilization of a mem-
brane protein are sometimes not accomplished.
Grant sponsor: Grants-in-Aid for Special Research on Priority
Areas; Grant number: 21024006; Grant sponsors: Grant-in-Aid
for Scientific Research for Plant Graduate Students from Nara
Institute of Science and Technology. Both grants are from the
Ministry of Education, Culture, Sports, Science, and Technology
of Japan.
*Correspondence to: Takeshi Ishimizu, Graduate School of
Science, Osaka University, 1-1 Machikaneyamacho, Toyonaka,
Osaka 560-0043, Japan. E-mail: txi@chem.sci.osaka-u.ac.jp
C
V
486 PROTEIN SCIENCE 2010 VOL 19:486—493
Published by Wiley-Blackwell. 2010 The Protein Society

Polygalacturonic acid synthase localized at the plant
Golgi membrane5 is one such example. Although its
purification has been attempted since detection of its
activity from plant sources in the 1960s,6 this has
not been achieved because the enzyme activity in
the solubilized solution is too low.
The effects of various additives on the solubiliza-
tion of membrane proteins have been investigated.7
Polyols such as glycerol, polyethylene glycol, and su-
crose enhance the stability of membrane proteins in
solution presumably by preventing unfavorable hydro-
phobic interactions. The addition of sufficient EDTA
and EGTA is useful for inhibiting proteolysis to stabi-
lize membrane proteins in solution. Protease inhibi-
tors such as phenylmethylsulfonyl fluoride, leupeptin,
aprotinin, E-64, and pepstatin are also added to buf-
fers to increase stabilization. Additives for enhancing
solubilization of membrane proteins can give a signifi-
cant advantage in the purification of membrane pro-
teins. Nondetergent sulphobetaines were reported to
enhance the recovery of some kinds of membrane pro-
teins.8 Other than those listed above, no other addi-
tives have been reported to be generally effective for
enhancing solubilization of membrane proteins.
Figure 1. Structure of alkylamines and polyamines used in
this study.
gent was used for its solubilization, insufficient
amounts of the enzyme were obtained to allow purifi-
cation. Thus, more effective solubilization conditions
are required. We investigated the effects of three
kinds of alkylamines (ethylammonium chloride, eth-
ylammonium nitrate, and propylammonium chloride)
and three polyamines (putrescine, spermidine, and
spermine) (the structures of which are shown in Fig.
1) on solubilization of this enzyme. We prepared
microsomal fractions from azuki bean epicotyls.
When the enzyme was solubilized from the microso-
mal fraction with a solubilization buffer (containing
20 mM CHAPS) supplemented with each additive at a
concentration of 100 mM, enhanced solubilization of
the enzyme was observed in all cases. The specific ac-
tivity of the enzyme increased from 2.2- to 8.2-fold,
while the total activity also increased from 2.3- to 6.3-
fold (Table I). Of the additives, spermidine was the
most effective in the solubilization of the enzyme. A
notable difference in the effect of the counter anion
(ethylammonium chloride and ethylammonium ni-
trate) was not observed. Such enhanced solubilization
was also observed when Triton X-100 or sodium chol-
ate was used as the detergent instead of CHAPS. In
the presence of 0.5% Triton X-100, ethylammonium
nitrate (100 mM) and spermidine (100 mM) enhanced
the solubilization of polygalacturonic acid synthase
by 1.8- and 1.6-fold, respectively. In the presence of
1% sodium cholate, both two additives enhanced the
solubility of this membrane enzyme by 1.6-fold.
In this study, we investigated alkylamines or
polyamines as additives for enhancing solubilization
of membrane proteins. Polyamines such as spermine
and spermidine, which are abundantly synthesized
in the cells of all living organisms, are involved in a
number of processes, but their specific molecular
mechanism is largely unknown.9,10 Interestingly,
polyamines are important modulators of some mem-
brane proteins including a variety of channels and
receptors.10 In addition, they have been shown to
prevent thermal inactivation and aggregation of pro-
tein.11,12 Ethylammonium nitrate, an ionic liquid, is
also known to prevent aggregation of denatured pro-
teins.13 This solvent has also been used as a protein
crystallization reagent.14 The moderate hydrophobic-
ity and cationic character of alkylamines and poly-
amines are expected to assist in the solubilization of
membrane proteins. In addition, the positive charge
of these molecules has the potential to interact with
the negatively charged lipid bilayers and is thus
expected to further help solubilization of membrane
proteins. Here, we demonstrate that the addition of
alkylamines and polyamines to
a solubilization
buffer containing a detergent enhances the solubili-
zation of membrane proteins.
Dependence of solubilization of
polygalacturonic acid synthase on alkylamine
and polyamine concentration
Once we had shown that various alkylamines and
polyamines enhance the solubilization of a membrane
enzyme, the relationship between enhancement of the
total activity of the enzyme solubilized and concentra-
tion of ethylammonium nitrate and spermidine was
investigated. Enhancement was observed at a concen-
tration of ethylammonium nitrate as low as 10 mM
[Fig. 2(A)]. Under optimum conditions (100 mM), a
Results
Enhanced solubilization of polygalacturonic acid
synthase by alkylamines and polyamines
Polygalacturonic acid synthase localized at the plant
Golgi membrane has previously been solubilized
from a microsome fraction with a solubilization
buffer containing CHAPS.15–17 Even when a deter-
Yasui et al.
PROTEIN SCIENCE VOL 19:486—493 487

Table I. Effects of Alkylamines and Polyamines on Solubilization of Polygalacturonic Acid Synthase
Polygalacturonic acid synthase activity
Additive
Specific activity (mU/mg protein)
Total activity (mU/g fresh weight)
No additive
0.17 (1.0)
0.54 (3.2)
0.37 (2.2)
0.36 (2.2)
0.41 (2.4)
1.4 (8.2)
0.032 (1.0)
0.14 (4.4)
0.11 (3.4)
0.11 (3.4)
0.12 (3.8)
0.20 (6.3)
0.074 (2.3)
Ethylammonium chloride
Ethylammonium nitrate
Propylammonium chloride
Putrescine
Spermidine
Spermine
0.94 (5.5)
Each additive was added to solubilization buffer at a concentration of 100 mM. The parenthetic values indicate activities
relative to those of the enzyme solution prepared without additive.
3.4-fold increase of total activity was observed. How-
ever, as the concentration increased, total activity of
the enzyme solubilized gradually decreased. The rela-
tionship between solubilization and concentration for
spermidine was similar to that for ethylammonium
nitrate [Fig. 2(B)]. Thus, enhancement of solubiliza-
tion was observed even at a concentration of 10 mM.
Maximum enhancement (9.9-fold) was observed at 50
mM spermidine. As was the case with ethylammo-
nium nitrate, as the spermidine concentration was
increased, the amount of enzyme solubilized
decreased drastically.
zation of mitochondrial localized cytochrome c oxi-
dase was not observed to the same extent (1.1-fold)
because most of the mitochondrial fragments were
not contained in the microsome fractions. The solu-
bilization of c-glutamyl transpeptidase localized at
the plasma membrane was enhanced 3.8- and 3.4-
fold by ethylammonium nitrate and spermidine,
respectively. Table II summarizes how the addition
of alkylamines and polyamines to solubilization
buffer is applicable for effective solubilization of
these plant membrane proteins. The values of rela-
tive specific activity are not much different from
those of total activity, indicating that the solubiliza-
tion of microsomal proteins other than the mem-
brane enzymes tested was also enhanced by ethyl-
ammonium nitrate and spermidine.
Enhanced solubilization of other membrane
enzymes by alkylamines and polyamines
The alkylamines and polyamines used in this study
were shown to enhance the solubilization of polyga-
lacturonic acid synthase from microsome fractions.
Subsequently, we investigated whether these
reagents enhance the solubilization of other mem-
brane enzymes. Polygalacturonic acid synthase is
localized at the Golgi membrane. We therefore chose
three other plant enzymes localized at membranes
other than the Golgi. The solubilization of NADH-
dependent cytochrome c reductase localized at the
endoplasmic reticulum was 2.2- to 2.5-fold enhanced
by addition of 100 mM ethylammonium nitrate or 50
mM spermidine (Table II). Enhancement of solubili-
The effects of these additives were also investi-
gated for an animal membrane protein, namely bo-
vine liver b-glucoside a1,3-xylosyltransferase.18 The
relationship between solubilization of this enzyme
from a bovine liver microsomal fraction and the con-
centration of the additives is shown in Figure 3. In
the absence of additives the enzyme activity was 1.37
U/g fresh weight. The solubilization of this enzyme
was enhanced by ethylammonium nitrate and spermi-
dine up to 7.1- and 4.7-fold, respectively. The opti-
mum concentrations of the additives (500 mM ethyl-
ammonium nitrate and 100–500 mM spermidine) are
Figure 2. Relationship between solubilization of polygalacturonic acid synthase and concentration of ethylammonium nitrate
and spermidine in solubilization buffer. (A) Ethylammonium nitrate and (B) spermidine.
488 PROTEINSCIENCE.ORG
Enhanced Solubilization of Membrane Proteins

Table II. Effects of Ethylammonium Nitrate and Spermidine on Solubilization of Various Plant Membrane
Enzymes
Specific activity
Ethylammonium
Enzyme
Total activity
No additive
nitrate
Spermidine
Polygalacturonic acid synthase
(Golgi)
1.0
1.0
1.5
2.8
6.8
9.9
NADH-dependent cytochrome c
reductase
(endoplasmic reticulum)
1.0
1.0
1.0
1.9
2.2
2.2
2.8
2.5
2.5
Cytochrome c oxidase
(mitochondria)
1.0
1.0
1.1
1.1
1.5
1.1
c-Glutamyl transpeptidase
(plasma membrane)
1.0
1.0
3.7
3.8
4.4
3.4
Ethylammonium nitrate (100 mM) or spermidine (50 mM) was added to solubilization buffer. Upper and lower values in a
column represent specific activity and total activity of the enzymes, respectively. The values indicate activities relative to
those of the enzyme solution prepared without additive.
around fivefold higher than that for plant membrane
proteins. The higher concentration of additives
decreased the recovered yield of the enzyme.
tle stronger than that of ethylammonium nitrate.
These results argue against the idea that the
enhanced solubilization of membrane proteins by
alkylamines and polyamines is due to stabilization
of the membrane proteins.
Alkylamines and polyamines do not stabilize
polygalacturonic acid synthase
Alkylamines and polyamines do not activate
polygalacturonic acid synthase
How do alkylamines and polyamines enhance the
solubilization of membrane proteins? We investi-
gated possible explanations by analyzing the solubi-
lization of polygalacturonic acid synthase as a model
membrane protein. First, we investigated the contri-
bution of these additives for stabilization of the pro-
tein in aqueous buffer. The membrane enzymes solu-
bilized with various concentrations of additives were
incubated at 4ꢀC for 24 h. Subsequent enzyme activ-
ities were compared with that of the enzyme solubi-
lized without additives. The results showed that the
additives did not stabilize polygalacturonic acid syn-
thase but rather destabilized it [Fig. 4(A)]. Thus, the
greater the concentration, the lower the enzyme ac-
tivity. This indicates that ethylammonium nitrate is
a weak protein denaturant. Spermidine also destabi-
lized the enzyme solubilized in aqueous buffer [Fig.
4(B)]. The denaturing effect of spermidine was a lit-
Subsequently, it was investigated whether the addi-
tives contribute to the activation of the enzyme. Eth-
ylammonium nitrate or spermidine (100 mM) was
added to the enzyme solution prepared without addi-
tives during the assay. Neither reagent activated pol-
ygalacturonic acid synthase but in fact rather inhib-
ited it (Table III). This indicates that the increased
values of enzyme activity in Tables I and II obtained
upon addition of alkylamines and polyamines are not
due to the activation of the enzyme by these reagents.
Alkylamines and polyamines do not act as
alternatives to detergent
We checked whether alkylamines and polyamines
could act as alternatives to detergents and thereby
enhance the solubilization of membrane proteins.
Figure 3. Relationship between solubilization of b-glucoside a1,3-xylosyltransferase and concentration of ethylammonium
nitrate and spermidine in solubilization buffer. (A) Ethylammonium nitrate and (B) spermidine.
Yasui et al.
PROTEIN SCIENCE VOL 19:486—493 489

Figure 4. Stability of polygalacturonic acid synthase solubilized with a buffer containing (A) ethylammonium nitrate or (B)
spermidine at various concentrations. Enzyme activity was measured after incubation of the solubilized enzyme at 4ꢀC for 24
h. Remaining activities relative to the enzyme prepared without additives are plotted.
When the proteins were solubilized from the micro-
some fraction, 100 mM ethylammonium nitrate or
100 mM spermidine was added to the solubilization
buffer instead of 20 mM CHAPS. These reagents
worked for solubilization of polygalacturonic acid
synthase to a small extent (Table IV) but this does
not explain the reason for enhanced solubilization of
membrane proteins by these reagents.
Thus, upon purification of a membrane protein solu-
bilized by this method, it will be necessary to remove
the additive from the protein solution as soon as pos-
sible. When these reagents were used instead of a
detergent (CHAPS), some solubilization of a mem-
brane protein was observed (Table IV). But this ob-
servation does not explain the size of the enhance-
ment of the solubilization of membrane proteins by
these reagents.
This enhanced solubilization of membrane pro-
teins was not specific to those solubilized with
CHAPS. Alkylamines and polyamines in the pres-
ence of detergents other than CHAPS also enhanced
solubilization of membrane enzymes. Thus,
enhanced solubilization of bovine xylosyltransferase
upon addition of additives was also observed with
the use of Triton X-100 (Fig. 3). When Triton X-100
or sodium cholate was used as a detergent for solubi-
lization of polygalacturonic acid synthase, enhanced
solubilization upon addition of additives was also
observed.
Discussion
Alkylamines and polyamines were found to be effec-
tive additives for solubilization of polygalacturonic
acid synthase from microsome fractions. Of the addi-
tives, spermidine was the most effective for plant
membrane proteins (Table I). With polygalacturonic
acid synthase, which has not previously been puri-
fied, addition of 50 mM spermidine to solubilization
buffer enhanced the solubilization by 9.9-fold (Fig.
2). Because one of the reasons for the lack of purifi-
cation of the enzyme is low enzyme activity in solu-
bilized microsomal fractions, this method opens the
way for its purification. These additives also
enhanced the solubilization of other plant membrane
enzymes (Table II) and an animal membrane
enzyme (Fig. 3). This implies that these additives
are effective for solubilization of membrane proteins
from various sources. This solubilization method
may also be applicable to the biochemical characteri-
zation of especially low abundance membrane pro-
teins such as glycosyltransferases.
The reagents used in this study have a number
of common structural features as they have rela-
tively simple structures that are composed of multi-
valent amines. Because of their cationic nature,
alkylamines and polyamines possibly bind to anionic
sites of organic compounds. In fact, the polyamines
have been reported to interact with microsomes or a
lipid
bilayer
membrane
and
cause
their
Although the present report does not provide a
precise mechanism to explain the enhanced solubili-
zation of membrane proteins by alkylamines and
polyamines, we would like to discuss possible mecha-
nisms. Based on the data described here, the higher
enzyme activity of the enzyme solubilized with the
additives was shown to not be caused by the addi-
tives working as stabilizers (Fig. 4) or activators (Ta-
ble III) of the enzymes. Indeed, it has been reported
that spermidine slightly denatures proteins.11,19
Table III. Activating Effects of Ethylammonium
Nitrate or Spermidine on Polygalacturonic Acid
Synthase Activity
Additive
Enzyme activity (lU/mL)
No additive
Ethylammonium nitrate
Spermidine
372 (1.00)
43.2 (0.12)
9.4 (0.03)
Each additive was added to reaction buffer at a concentra-
tion of 100 mM. The parenthetic values indicate activities
relative to those of the enzyme solution without additive.
490 PROTEINSCIENCE.ORG
Enhanced Solubilization of Membrane Proteins

Table IV. Solubilization Effects of Ethylammonium Nitrate or Spermidine Acting as Detergents
Polygalacturonic acid synthase activity
Additive
Specific activity (mU/mg protein)
Total activity (lU/g fresh weight)
CHAPS (20 mM)
Ethylammonium nitrate (100 mM)
Spermidine (100 mM)
0.28 (1.0)
0.11 (0.4)
0.15 (0.5)
69 (1.0)
21 (0.3)
22 (0.3)
Each additive (100 mM) was added to solubilization buffer instead of detergent. The parenthetic values indicate activities
relative to those of the enzyme solution prepared in the presence of 20 mM CHAPS.
destabilization.20–22
Furthermore,
they
were
This study shows that alkylamines and poly-
amines are effective additives for the solubilization
of some plant membrane proteins and animal Golgi-
localized proteins. Further application of this
method to membrane proteins from other organisms
may allow this method to be generally applied for
the effective solubilization of membrane proteins.
observed to bind to microsomes but not to microso-
mal proteins.20 These results suggest the possibility
that these reagents interact with phospholipids and
enhance delipidation of membrane proteins. Thus,
they may assist in the release of membrane proteins
from lipid bilayers, so that solubilization of mem-
brane proteins is enhanced.
When a membrane enzyme from bovine liver
was solubilized from microsomes, around a fivefold
higher concentration of ethylammonium nitrate and
spermidine was needed for optimum solubilization
compared to that used for microsomal proteins of
azuki bean epicotyls. The compositions of phospholi-
pids in lipid bilayers of plants and animals are
Materials and Methods
Materials
Ethylammonium nitrate was prepared by adding
equimolar amounts of ethylamine (70%) to nitric
acid (61%) drop-by-drop with stirring and cooling in
an ice bath.30 The resulting salt solution was con-
centrated in vacuo on a rotary evaporator. The con-
centrated solution was dried by lyophilization for 2
days. The density (1.20 g cmꢁ3) was close to the lit-
erature value.31,32
totally different.23–25 Plant microsomes have
a
higher content of phosphatidylethanolamine (PE)
and phosphatidylinositol (PI) and a lower content of
phosphatidylcholine (PC) than animal liver micro-
somes. The binding strength of polyamines to phos-
pholipids has been reported to be in the order PI >
PE > PC.20 This probably explains the difference in
the observed optimum concentration of additives for
membrane protein solubilization from animals and
plants. Thus, higher concentrations of polyamines
may be required for effective solubilization of mem-
brane proteins of animal liver microsomes in which
the PC content is higher than in plant microsomes.
Furthermore, binding of spermidine to rat liver mi-
crosomes is reported to be stronger than that of
spermine.26 This report is consistent with our data,
which demonstrate that spermidine is a more effec-
tive reagent for enhancing the solubilization of
microsomal proteins (Table I). The phospholipid com-
positions of lipid bilayers are not constant between
organisms. However, the addition of alkylamines
and polyamines for enhancing solubilization of mem-
brane proteins appears to be applicable to various
organisms, although the optimum reagent and con-
centration may need to be determined for each
sample.
Ethylammonium chloride, propylammonium
chloride, and putrescine dihydrochloride were pur-
chased from Wako Pure Chemical Industries (Osaka,
Japan). Spermidine trihydrochloride and spermine
tetrahydrochloride were from MP Biomedicals
(Irvine, CA). UDP-galacturonic acid was enzymati-
cally synthesized as reported previously.33 UDP-
xylose was chemically synthesized as described pre-
viously.34 Fluorescent-labeled oligogalacturonic acids
were prepared as described previously.16,35 2-[(2-Pyr-
idyl)amino]ethyl b-glucoside was prepared by trans-
glycosylation activity of b-glucosidase as described.18
All other chemicals used were of the highest grade
commercially available.
Preparation of the solubilized membrane
enzyme
Azuki beans (Vigna angularis) were germinated and
grown at an ambient temperature in the dark for
around 10 days. Their epicotyls (50 g) were pulver-
ized with a mortar and a pestle under liquid nitro-
gen. The ground powder was stirred at 4ꢀC for 20
min with 50 mL of a homogenizing buffer (50 mM
HEPES-KOH, pH 7.0, 25 mM KCl, 50% (v/v) glyc-
erol, 2 lg/mL aprotinin, 5 lg/mL leupeptin, 0.7 lg/
mL pepstatin, and 1 mM phenylmethylsulfonyl fluo-
ride). To the supernatant (20,000g, 10 min), MgCl2
(final 25 mM) was added and incubated at 4ꢀC for
It has been frequently reported that membrane
enzymes are activated by polyamines.27–29 In most
reports, enzyme activities were measured using mi-
crosome fractions. Based on this study, polyamines
may not activate membrane enzymes but in most
cases enhance their solubilization from microsomes.
Yasui et al.
PROTEIN SCIENCE VOL 19:486—493 491

30 min. This solution was centrifuged at 30,000g for
30 min to obtain the microsomal fraction as pel-
lets.36 This fraction was solubilized with 1 mL of a
solubilization buffer (20 mM HEPES-KOH, pH 7.0,
25 mM KCl, 25% (v/v) glycerol, 2 mM EDTA, 20 mM
CHAPS) at 4ꢀC for 20 min. The additives (alkyl-
amines or polyamines) were added to a solubilization
buffer before solubilization. The supernatant
(30,000g, 10 min) was used as a solubilized enzyme
solution.
buffer, pH 7.2, 0.01% digitonin, 0.02 mM reduced bo-
vine heart cytochrome c, and the enzyme solution.38
The reduced cytochrome c was prepared by chemical
reduction with 20 mM sodium dithionite on ice for
10 min. The enzyme activity was quantified by
reduction of absorbance at 550 nm.
c-Glutamyl transpeptidase activity was per-
formed with c-glutamyl-p-nitroanilide (GPNA) as a
donor substrate and glycylglycine as an acceptor
substrate.39 The reaction was carried out in a mix-
ture of 1 mM GPNA, 40 mM glycylglycine, 0.1M
Tris-HCl, pH 7.5, and the enzyme solution at 30ꢀC
for 1 h. Liberated p-nitroaniline was quantified at
405 nm.
b-Glucoside a1,3-xylosyltransferase activity18 in
the solubilized enzyme solution prepared from bo-
vine liver was assayed using 2-[(2-pyridyl)ami-
no]ethyl b-glucoside18 as an acceptor substrate and
UDP-xylose34 as a donor substrate. The reaction
mixture (20 mM HEPES-NaOH, pH 7.2, 150 mM
NaCl, 0.1% Triton X-100, 20 mM MnCl2, 1 mM 2-[(2-
pyridyl)amino]ethyl b-glucoside, and 1.3 mM UDP-
xylose) was incubated at 37ꢀC for 12 h. The sub-
strate and product were analyzed on a Cosmosil
5C18-P reversed-phase column (4.6 mm ꢂ 150 mm,
Nacalai Tesque) with isocratic elution in 50 mM am-
monium acetate buffer, pH 4.5, at a flow rate of 2
mL/min. They were quantified by their fluorescence
(excitation wavelength, 320 nm; emission wave-
length, 400 nm).
Bovine liver (20 g) was homogenized with 180
mL of 20 mM HEPES-NaOH buffer, pH 7.4, contain-
ing 250 mM sucrose and 5 mM MgCl2. The homoge-
nate was centrifuged at 10,000g at 4ꢀC for 25 min.
The supernatant was further centrifuged at
105,000g at 4ꢀC for 1 h. The pellets were collected
and the protein was solubilized with 30 mL of a sol-
ubilization buffer (20 mM HEPES-NaOH buffer, pH
7.2, containing 20 mM MgCl2, 150 mM NaCl, 10%
(v/v) glycerol, and 0.5% Triton X-100). The additives
(alkylamines or polyamines) were added to a solubi-
lization buffer before solubilization. The supernatant
obtained after centrifugation (105,000g for 1 h) was
used as a solubilized enzyme solution.18
Enzyme assay
Enzyme activities were measured just after solubili-
zation of membrane proteins from microsomal frac-
tions. Polygalacturonic acid synthase activity was
measured using pyridylaminated oligogalacturonic
acids and UDP-galacturonic acid as substrates.16,35
A reaction mixture containing 0.5 mM UDP-galactu-
ronic acid, 5 lM pyridylaminated oligogalacturonic
acid with degree of polymerization of 12, 33 mM
HEPES-NaOH, pH 7.0, 17 mM KCl, 5 mM MnCl2,
0.13M sucrose, 0.033% bovine serum albumin, 10
mM CHAPS, and the enzyme solution was incubated
at 30ꢀC for 10 min. Transferred galacturonic acid
was quantified from the chromatogram peak area
obtained from anion-exchange chromatography with
fluorescence detection. One unit of enzyme activity
was defined as the amount of the enzyme that trans-
ferred 1 lmol of galacturonic acid onto oligogalactur-
onic acid per min under the conditions described
earlier.
We performed at least two independent experi-
ments for each table and figure. The values of rela-
tive enzyme activities in tables and figures are typi-
cal data from a single experiment. The values are
reproducible values, although the actual enzyme ac-
tivity differed between each experiment because of
the use of different biological sources.
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