全 文 ：Received 5 Apr. 2004 Accepted 22 Jul. 2004
Supported by the State Key Basic Research and Development Plan of China (G1998010100).
* Author for correspondence. E-mail: .
http://www.chineseplantscience.com
Acta Botanica Sinica
植 物 学 报 2004, 46 (10): 1186-1191
Isolation and Characterization of the Cytoplasmic Membrane
from the Terrestrial Cyanobacterium—Nostoc flagelliforme
HUANG Hui, ZHONG Ze-Pu, WANG Ke-Bin, BAI Ke-Zhi, LI Liang-Bi, KUANG Ting-Yun*
(Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany,
The Chinese Academy of Sciences, Beijing 100093, China)
Abstract: Cytoplasmic membrane of Nostoc flagelliforme Born. et Flah. was isolated for the first time
with a new method, the unique feature of which is the combined use of French pressure cell and digitonin
to disrupt cells. After passed twice through French pressure cell (at 80 MPa), cells in sample (20 mg of dry
weight/mL) were disrupted effectively by digitonin (3 mg/mL), and then the cytoplasmic membrane was
isolated by density gradient centrifugation. The membrane contained carotenoids with absorption peaks at
458, 487 and 524 nm and a precursor of chlorophyll a with a minor peak at 673 nm. The fluorescence
emission peaks of the membrane were emitted from the precursor of chlorophyll a. More than 30 polypep-
tides were detected in the membrane, in which the most obvious corresponded to the polypeptides with
molecular mass of 80, 28, 19 and 17 kD. The membrane contained four types of glycerolipids: MGDG
(62.4%), DGDG (18.9%), SQDG (16.7%) and PG (2.0%). 16:0, 16:1 [9], 18:0, 18:1 [9], 18:2 [9, 12] and 18:3
[9, 12, 15] fatty acids were determined in the membrane, in which 16:1 and 18:3 fatty acids were the main
components, representing 32.3% and 34.4% of the total fatty acids respectively. High proportion of 18:3
fatty acid in the cytoplasmic membrane may be an important factor of N. flagelliforme in its remarkable
drought-tolerant ability.
Key words: Nostoc flagelliforme ; cytoplasmic membrane; isolation; digitonin
Nostoc flagelliforme is a filamentous terrestrial
cyanobacterium, well-known for its remarkable drought and
chilling tolerance (Gao, 1998). Its morphology is distinctive:
in hair-shape thallus, filaments of torulose cells are embed-
ded in a common mucoid sheath (Wang and Gu, 1984);
besides, every filament has a film of mucilage coating the
surface of cells (Wang et al., 1988).
Three types of membranes exist in cyanobacterial cells:
outer and inner (cytoplasmic) membranes of cell envelopes
and thylakoid membranes (Stanier and Cohen-Bazire, 1977).
Cytoplasmic membrane is involved in the transport of an-
ions such as bicarbonate (Ogawa, 1991), nitrate (Omata et
al., 1989) and sulfate (Green and Grossman, 1988). In
addition, it is significant in chilling susceptibility of
cyanobacterium. Nevertheless, the biochemical nature of
the cytoplasmic membrane of N. flagelliforme has not been
studied.
After lysozyme treatment, cells of aquatic cyanobacteria
and cyanobacteria cultivated in solutions (such as N. com-
mune UTEX 584) can be disrupted effectively by French
pressure cell. Sometimes, the treatment of freezing and thaw-
ing enhances the susceptibility of cells to lysozyme.
However, because of the protection by mucilage and sheath,
cells of N. flagelliforme show pronounced resistance to
the effects of lysozyme and many techniques for physical
disruption. Therefore, isolation of the cytoplasmic mem-
brane from N. flagelliforme is not successful with the meth-
ods described before (Omata and Murata, 1983; 1984; Olie
and Potts, 1986; Chauhan et al., 2000).
During our experiments, digitonin was found to be an
effective chemical disruptor to cells of N. flagelliforme
which had been passed through French pressure cell. In
this paper, a new method is described for isolation of the
cytoplasmic membrane from N. flagelliforme.
1 Materials and Methods
1.1 Organism and rewetting conditions
Nostoc flagelliforme was collected from Sunitezuoqi,
Inner Mongolia (Nei Mongol), and stored at -20 ℃ under
dry conditions. Sample was rewetted in BG-11 medium
(without any nitrogen source) with the steps described by
Scherer and Zhong (1991). It was rewetted for 16 h in the
white light of 100 mmol photon.m-2.s-1 at 20 ℃.
1.2 Isolation and purification of the cytoplasmic mem-
brane
Rewetted sample (1 g of dry weight) was frozen in liquid
HUANG Hui et al.: Isolation and Characterization of the Cytoplasmic Membrane from the Terrestrial Cyanobacterium—
Nostoc flagelliforme 1187
nitrogen, grinded into tiny powder and suspended in 50 mL
20 mmol/L N-Tris (hydroxymethyl) propane-1, 3-diol (TES)-
NaOH buffer (3 mg/mL digitonin, 400 mmol/L sucrose, 1
mmol/L phenyl methyl sulfonyl fluoride (PMSF), pH 7.0).
The following procedures were carried out at about 4
℃.
The suspension was passed twice through a French
pressure cell at 80 MPa and then stirred for 40 min. The
homogenate was centrifuged at 30 000g for 30 min to elimi-
nate unbroken cells, debris of sheath and cell walls. The
supernatant was centrifuged at 160 000g for 60 min to
collect membranes. The pellet was suspended in 3 mL 20
mmol/L TES-NaOH buffer (15 mmol/L NaCl, 5 mmol/L MgCl2,
5 mmol/L EDTA; pH 7.0) (Buffer A). Nought point five mL
of the suspension was loaded onto the surface of a 10 mL
linear sucrose density gradient (5%-40% (W/V) in Buffer
A). The gradient was centrifuged at 135 000g (RPS40T rotor,
Hitachi 70P-72) for 8 h. An orange band at the sucrose
concentration of 12%-15% (W/V) and a dark-green band
at 18%-20% (W/V) were identified. The orange band was
removed from the gradient and dialyzed against Buffer A
with stirring for 3-4 h. The cytoplasnic membrane was col-
lected as pellet after centrifugation of the dialysate at
180 000g for 60 min and the pellet was suspended in Buffer
A. This procedure was repeated several times for
purification.
1.3 Spectrophotometric measurements
Absorption spectra were measured with an UV-2550
spectrophotometer (Shimsdzu). Fluorescence emission
spectra were measured at 20 ℃ and -196 ℃ (77 K) with an
F-4500 spectrofluorometer (Hitachi).
1.4 Analysis of membrane proteins
Purified cytoplasmic membrane was solubilized as de-
scribed by Olie and Potts (1986). Samples after solubiliza-
tion were analyzed on 13.5% (W/V) polyacrylamide gel.
Electrophoresis was performed with the buffer system of
Laemmli (1970). Gels were stained with Coomassie brilliant
blue.
1.5 Analysis of glycerolipids and fatty acids
The procedures of isolation, separation and identifica-
tion of glycerolipids from purified cytoplasmic membrane
by TLC (thin-layer chromatography) were according to the
methods of Makewicz et al. (1997), Xu and Eichenberger
(1999). The individual glycerolipid separated by TLC or the
total glycerolipids of the cytoplasmic membrane were
transesterified with 5% H2SO4 in methanol (V/V) for 60 min
at 85 ℃ (Wang et al., 2000). The fatty acid methyl esters
were separated by gas chromatography (HP 6890 Series GC
System).
2 Results
After density gradient centrifugation, cytoplasmic mem-
brane formed a narrow orange band at the sucrose concen-
tration of 12%-15%, and a dark-green band of thylakoid
membrane stayed at 18%-20% (Fig.1). Purification and char-
acterization of the thylakoid membrane will be discussed in
another paper.
Fig.1. The result of the separation of the membranes by floata-
tion centrifugation and the absorption spectrum of the orange
band.
Acta Botanica Sinica 植物学报 Vol.46 No.10 20041188
As shown in Fig.1, the absorption at 620–660 nm of the
orange band was strong, which indicated that
phycobiliproteins mixed in the orange band. During the
purification by centrifugation, phycobiliproteins, as water-
soluble protein-complexes, stayed in supernatant and the
cytoplasmic membrane formed pellet, so that the cytoplas-
mic membrane could be purified from the contamination of
phycobiliproteins. The absorption spectrum of the cyto-
plasmic membrane after purification was identical to that of
the orange band, except that the absorption at 620-660 nm
almost disappeared (Fig.2).
The main absorption peaks of the cytoplasmic mem-
brane appeared at 458, 487 and 524 nm, which were due to
carotenoids, and there was a minor peak at 673 nm (Fig.2).
The minor peak wavelength was shorter by 5 nm than that
of chlorophyll a in thylakoid membrane (Murata et al., 1981).
Therefore, the minor peak indicated the existence of a pre-
cursor of chlorophyll a (Wada and Murata, 1998), which
could not be deleted during the purification by
centrifugation.
The emission spectrum at 20 ℃ showed one peak at 675
nm (Fig.3A). The fluorescence emission peak at -196 ℃
was at 671 nm (Fig.3B). These fluorescence peaks were
ascribed to the emissions from the precursor of chlorophyll
a (Murata et al., 1981).
Figure 4 shows the polypeptide composition of the cy-
toplasmic membrane, in which there were more than 30
bands discernible. The molecular weights of most polypep-
tides ranged from 40 to 70 kD, and the polypeptides with
molecular weight of 80, 28, 19 and 17 kD were most obvious.
Fig.2. The absorption spectra of the cytoplasmic membrane
after purification (solid line) and its second derivative (dash line).
Table 1 Composition of glycerolipids in the cytoplasmic mem-
brane
Glycerolipid (mol%)
MGDG 62.4
DGDG 18.9
SQDG 16.7
PG 2.0
DGDG, digalactosyl diacylglycerol; MGDG, monogalactosyl
diacylglycerol; PG, phosphatidylglycerol; SQDG, sulfoquinovosyl
diacylglycerol.
Fig.4. SDS (sodium dodecyl sulfate)-PAGE (polyacrylamide
gel electrophoretic analysis) of the cytoplasmic membrane of
Nostoc flagelliforme. A, cytoplasmic membrane; B, molecular-
mass standard.
Fig.3. Fluorescence emission spectra of the cytoplasmic mem-
brane at 20 ℃ (A) and at -196 ℃ (B), with the excitation
wavelength of 436 nm.
As shown in Table 1, the cytoplasmic membrane con-
tained four types of glycerolipids: MGDG, DGDG, SQDG
and PG. The content of PG is lower than that from whole
cells of N. flagelliforme (Wang et al., 2000). The fatty acid
HUANG Hui et al.: Isolation and Characterization of the Cytoplasmic Membrane from the Terrestrial Cyanobacterium—
Nostoc flagelliforme 1189
compositions of individual glycerolipids in the membrane
(Table 2) are similar to those from whole cells (Wang et al.,
2000). Six types of fatty acids were detected in the cyto-
plasmic membrane: palmitic (16:0), palmitoleic (16:1 [9]),
stearic (18:0), oleic (18:1 [9]), linoleic (18:2 [9, 12]) and lino-
lenic (18:3 [9, 12, 15]) acids (Table 2). 16:1 [9] and 18:3 [9, 12,
15] fatty acids were the main components, accounting for
32.3% and 34.4% of the whole fatty acids, respectively.
use of French pressure cell and digitonin (at 3 mg/mL) was
adopted in the procedure of disruption.
It is the first scientific attempt to isolate the cytoplasmic
membrane from a terrestrial cyanobacterium with filaments
of cells embedded in a common sheath. In our study, the
cytoplasmic membrane of N. flagelliforme was isolated free
of thylakoid membrane. Because of different kinds of caro-
tenoids contained, absorption spectra of cytoplasmic mem-
branes from various species of cyanobacteria are different
with each other, except the minor peak at about 673 nm. The
polypeptide composition of the cytoplasmic membrane of
N. flagelliforme is similar not only to that of its ecotype—
N. commune (Olie and Potts, 1986), but also to those of
Synechocystis PCC 6714 (Omata and Murata, 1984) and
Anacystis nidulans (Omata and Murata, 1983), indicating
the same basic functions carried out by cytoplasmic mem-
branes of cyanobacteria living in various kinds of
circumstances.
Our previous reports on the glycerolipid and fatty acid
compositions of N. flagelliforme have been obtained from
whole cells (Wang et al., 2000). In the cytoplasmic mem-
brane of N. flagelliforme, the proportion of 18:3 fatty acid
is higher than other cyanobacteria which have been char-
acter ized to data . In another drought-tolerant
cyanobacterium—N. commune UTEX 584, 20:3w3 is the
major component of the fatty acid of cytoplasmic mem-
brane (Olie and Potts, 1986). In cyanobacteria, the substi-
tution of 18:2 for 18:3 fatty acid has practically no effect on
phase transition and the tolerance to low-temperature
photoinhibition (Gombos et al., 1992; Tasaka et al., 1996).
The distinctive functions of 18:3 fatty acid in cyanobacteria
have not been studied. It may be assumed that high pro-
portion of 18:3 fatty acid in cytoplasmic membrane is an
important factor of N. flagelliforme in its pronounced
drought-tolerant ability.
Acknowledgements: We sincerely thank Prof. XU Yi-
Nong for his critical reading of the manuscript and his valu-
able suggestions.
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