Vol.

70 391

The Chemical Composition and Structure of the

Cell Wall of Chlorella pyrenoidosa
By D. H. NORTHCOTE, K. J. GOULDING
Department of Biochemistry, University of Cambridge
AND R. W. HORNE
Cavendish Laboratory, University of Cambridge
(Received 21 January 1958)
 The wall is composed into the sediment, leaving
The distribution of been correlated with a clear colourless
of two distinct phases: supernatant.
chemical substances in the cell-wall structure
an organized All the analyses were
a com- in
microfibrillar structure carried out on material
plex organized structure
embedded in a continuous certain dried at
such as a plant-cell 0 01 mm. Hg over P205 at
wall matrix, and it has been species (Nicolai
room temperature. Total N
can be studied by possible to separate & Preston, was determined by micro-
correlating a chemical these more or less 1952-53;
Kjeldahl digestion
analysis intact one from the (Chibnall, Rees &
with a microscopical other. Treatment of the Kreger, 1957). Williams, 1943) followed
examination of the
wall with dilute sodium by distillation and
material. titration.
With green plants it is or potassium hydroxide EXPERIMENTA Total P was determined
solutions removed the
necessary to obtain a
matrix and bundles of
L AND according to Fiske &
Subbarow
homo-geneous tissue and
microfibrils were RESULTS (1925).
to separate the cells
isolated. Incuba-tion Material used and Chromatography and
before electrophoresis of sugars.
with an enzyme general analytical Descending
isolating the walls, and
preparation made from method8
these are difficult chromatograms were run on
the Alga. Chlorella Whatman no. 1 papers with
experi-mental procedures.
intestinal tract of the pyrenoidosa was grown under pyridine-ethyl acetate-water
In the present work they (1:2:2, by vol.) for 14 hr.
snail removed the micro- sterile conditions on a
have been avoided by (Jermyn & Isherwood, 1949).
fibrillar elements and completely defined salt
using a unicellular Electrophoreses were run at
left a large part of the medium (Emerson &
green alga, 210v in an apparatus
Lewis, 1939) in large flasks similar to that described
matrix
Chlorella. It has been (1-3 1.). A current of by Consden
possible to isolate from material still in the shape air+C02
of the original cell wall. & Stanier (1952), with
this (95:5) was passed through
the medium (600 ml./min.) borate buffer, pH 9-2.
plant a cell-wall In this way not only the
preparation which and The sugar spots were
chemical composition of growth was maintained for coloured in both
appears to be
the various parts of the 10-14 days in daylight in procedures with aniline
free from cell contents,
and by using the wall were determined a window facing west. The hydrogen phthalate
electron but cells were harvested by (Partridge, 1949).
microscope much of the also a more detailed centri- Chromatography of amino
fine detail of its knowledge of the fuging (1500g for 20 acids. Two-dimensional
min.), and washed with chro-
structure distribution
water and matograms were run in
has been of the recentrifuged. Yield was butanol-acetic acid-water
shown. components was approx. 0 7 g. dry (4:1:5, by vol.) and phenol
obtained. wt./I. buffered at pH 9-3 with
Isolation of the cell- borate accord-ing to Levy &
However, no The chemical wall material by mechanical
histochemical Chung (1953). The spots
composition of the cell breakage of the cell.
stains, even of the limited were coloured with
walls of algae by Chlorella cells (50 mg. dry
specificity of those used
wt.) were suspended in 10 ml. 0 3 %
in optical microscopy, direct isolation and triketohydrindene
of water with 4 g. of fine
are available for use chemical analysis has glass beads (Ballotini no. hydrate in butanol.
with the electron not previously been 12, 0-15 mm. diam., Chance
microscope and it was studied, although some Bros. Ltd., Birmingham) and
the mixture was placed in a
difficult to assess the indirect staining
vertical cup (internal
chemical nature of the reactions and X-ray measurements 5 cm. x 2*2 cm.)
structures observed. We studies have of a Mickle cell disintegrator
have attempted to (Mickle, 1948). Vibrations
isolate the structural were continued for 90 min.,
components from the wall after which the resultant
and analyse them suspension was treated as

directly. To do this shown in Fig. 1, where the

yields of the various
the wall has been
fractions are also given. The
degraded by chemical
temperature of the mixture
and enzymic methods during the breakage
which have destroyed one
rose 15° from the
or more components but initial room
have left a discrete temperature.
part ofthe wall which Washed whole Chlorella
cells centrifuged at 800g
could be seen with the for
aid of the electron
10 min. and at 190g for
microscope. 30 min. came down
completely
392 D. H. NORTHCOTE, K. J. GOULDING AND R. W. HORNEI958
Chromatography of amino 8ugars. These were investigated 2-5 hr., and centrifuging and
siphoning off the alcoholic
by the methods described above for both sugars and amino supernatant from the walls, which were then redried at acids. The
spots were coloured by the spray reagents used 0-01 mm. Hg over P205. The material was re-extracted with for sugars and amino

acids and also by the Elson-Morgan boiling dry ether for 2-5 hr. Yield of extracted cell walls
reagent described by Partridge (1948). was 50-1 mg. The alcohol and ether solutions were evapor-
Enzyme preparationfrom Helix pomatia. The snails were ated and the fat was weighed directly. Yield was 4-9 mg.
dissected according to Keilin (1956) and the digestive (8-9 %) and the wt. loss of the walls was 5-0 mg. (9-2 %).
tracts removed and cooled in ice. They were mixed with an Acid hydrolysis. (i) For monosaccharide constituents.
equal volume ofice-cold water and crushed in a homogenizer The whole cell
walls (4 mg.) were hydrolysed by 2 ml. of
with a Perspex pestle (Potter & Elvehjem, 1936). The 2N-H2SO4 for 6 hr. at 1000,
then neutralized with BaCO3
resultant extract was dialysed against distilled water at 40 and filtered and
the filtrate evaporated to dryness. Glucose,

for 24 hr. and any precipitate which formed was removed galactose, mannose, arabinose, xylose and rhamnose were
by centrifuging at 15 000g at 40 for 20 min. identified in the hydrolysate by chromatographic and
This preparation has been quantitatively analysed and electrophoretic investigations.
shown to contain only weak proteolytic activity but active (ii) For amino acids. Whole cell walls (4 mg.) were
lipases and carbohydrases. Some twenty different carbo- hydrolysed by boiling in 5 ml. of a mixture (1:1, v/v) of
hydrases were examined, including cellulase, xylanase and conc. HCI and formic acid for 24 hr. The hydrolysate was
mannanase (Myers & Northcote, 1958). evaporated at 250 under vacuum, and dissolved in water
and re-evaporated several times. Serine, glycine, glutamic
Chemical investigation of the cell wall acid, threonine, arginine, lysine, histidine, alanine, proline,
Elementary analysis and mineral content. The total N was tyrosine, valine, methionine, phenylalanine and leucine
were identified in the hydrolysate.
4-6%, and the total P 0-67% of the dry weight. When (iii) For amino sugars. Whole cell walls (4 mg.) were
maintained at red heat (approx. 8000) in a platinum boat in hydrolysed by 1 ml. of 3N-HC1 at 1000 for 6 hr. The hydro-
a stream of clean dry air for 1 hr., 2-11 mg. of walls yielded lysate was evaporated at 250 under vacuum, dissolved in
a light yellow ash; 0-11 mg., 5.2%. This ash was dissolved water and re-
evaporated several times. The hydrolysate
in dil. HNO3 and investigated chromatographically on was finally dissolved in 5 ml.
of 0-5N-HCI and put through
Whatman no. 4 paper run with butanol saturated with N- an ion-exchange column
(Dowex 50), which was washed
HOl at 350 for 48 hr. The spots were detected by spraying with 10 ml. of water, and then the amino sugars were
with kojic acid and 8-hydroxyquinoline (Lederer & eluted with 2N-HCI and estimated according to Boas
Lederer, 1955) and examination of the paper, before and (1953). Calculated as glucosamine the yield was 3.3%.
after exposure to NH3 vapour, under ultraviolet light. Iron, A neutral solution of the amino sugar was investigated
calcium and possibly aluminium were detected; no other chromatographically and electrophoretically and was
spots were visible. identified as glucosamine.
Lipid analysis. The lipid was determined by boiling the Poly8accharides of the cell wall. Walls (50-1 mg.) from
cell walls (55.1 mg.) with aq. 95% (v/v) methanol for which fat had been extracted were treated with 10 ml. of

Chlorella
(100 mg., dry wt.)
90 min. vibration in cell disintegrator

Dark-green suspension of broken cells

centrifuged at 800g for 10 ma.
Dark-green supernatant Residue,
(soluble material, dark-green ringed
chloroplasts and other with white
cell particles) (whole cells + walls)
(81.4 mg.)
Resuspended in 10 ml. of water,
centrifuged at 190g for 30 min.
 OPalescent supernatant Dark-green residue
(cell walls and some with a white ring
cell particles) (mainly whole cells
Centrifuged at 800g + some walls)
(10-4 mg.)
for 10 min.
Residue Pale-green
white cell walls supernatant
(3-8 mg.) (2-8 mg.)
Fig. 1. Differential centrifuging of mechanically disintegrated Chlorella cells. The total recovery of material was 98-4 mg.
VoI. 70 CELL WALL OF CHLORELLA 393
 galactose was relatively added in small portions
24% (w/v) KOH at room until precipitation was
precipitate of the soluble
temperature under N2 for 2 much more intense than that polysaccharides was
complete.
hr. of the other sugars. A obtained.
The mixture was
The solution was siphoned hydrolysate of the This was collected, washed
centrifuged and the
off after centrifuging and hemicellulose contained with ethanol and dried.
supernatant liquid
the Yield
galactose, mannose, was added to 21. of
residue re-extracted with arabinose, xylose and ethanol, whereupon a 4-34 g., 8-7% (N,
a further 10 ml. of KOH
rhamnose. The colour of the
white flocculent 0-1%; ash,
for 2 hr. The residue from
galactose spot was 0.05%).
this extraction was
Insoluble
relatively much more
thoroughly washed
with polysaccharides. These were
intense that of the
than
water by suspension and hydrolysed with acid and
other sugars. A solution of
centrifuging. then investigated by
the hemicellulose gave no
Normally six to eight chromatography and
colour with dilute iodine
washings were required to electrophoresis, which
solution and contained no
give a showed the presence of
starch.
supernatant with the same glucose and galactose. This
pH value as the original
wash Separation of fraction probably
water. The washings and
alkali-soluble and corresponded to the o-
-insoluble
alkali extracts were cellulose isolated
combined and from the cell
the residual a-cellulose was
polysacchari
dried over P205 at 0 01 mm. Hg. des from the walls.
Soluble polysaccharides.
Yield of white preparation whole cell
A solution of this fraction
was 8-5 mg., 15-4% (N, Freeze-dried Chlorella
gave a blue colour with
0.0%; cells (50 g.) were
dilute iodine solution. Acid
ash, 0-05 %). extracted with
hydrolysis followed by
The alkaline solution of 1-2 1. of methanol on a
hemicellulose was boiling-water bath for 6 chromatography and
neutralized at hr. and electrophoresis showed
4° with cooled acetic acid filtered, and the pale- the presence of galactose,
and then evaporated under green residue washed
was glucose, arabinose,
reduced pressure to half its with methanol. The cells were mannose,
volume. It was poured into a then re-extracted with 600 xylose and rhamnose. A
volume of ethanol so that the ml. of boiling acetone for 6 solution of the
polysaccharides
final concentration of hr. and filtered, and
(100 mg. in 10 ml. of borate
alcohol was 85%, and the washed with acetone and
dried. The cells, which were buffer, pH 9.2) was
precipitate which formed was
still coloured a investi-gated in a Tiselius
allowed to settle for 24 hr.
This precipitate was
light green, were treated electrophoresis apparatus
with 500 ml. of 3% NaOH at (Perkin-
centrifuged, 1000 Elmer Co.,
and U.S.A.;
washed with ethanol and under N2 for 6 hr. The Model by the method
38)
dried. Yield, 34-1 mg. residue was centrifuged and
washed described by Northcote
From a previous
twice with water and then (1954). Three peaks were
experiment it was known
re-extracted at room observed with mobilities of
to contain mineral matter
(approx. 27%) and N tempera-ture with 200 ml. 2-5 x 10-5 cm.2v-1 sec.-',
(approx. 3%) and a hydro- of 24% KOH, centrifuged 7-48 x 10-5 cm.2v-sec.-l and

lysate contained both amino and washed six times with 12-5 x 10-5 cm.2v-1 sec.-'.
acids and the amino sugar. water. The residual Calculated from the areas
It was therefore redissolved material was dried. under the curves the relative
in water and reprecipitated amounts of the com-
Yield, 1-05 g., 2-1%
with a mixture (1:4, v/v) (N, 0-02%; ash, ponents present were
approximately 2:4: 1
of acetic acid and 85% 0.8%). respectively.
ethanol. This The 3% NaOH extract and The solution of the
precipitate was well the washings were polysaccharides was
washed with ethanol and combined, investigated by
dried. Yield cooled in ice and brought to zone electrophoresis on
of white powder was 17-1 pH 4-2 with acetic acid. A
glass paper with borate
mg., 31-0% (N, 0-2%; ash, precipitate formed, which
buffer, pH 9-2 (Fuller &
0.3%). was centrifuged off. After
Northcote, 1956). Three
The oa-cellulose was evaporation to small bulk
spots were ob-
hydrolysed and the sugars under reduced pressure, the
was tained. The distances of the
were in-vestigated by supernatant liquid
spots from the starting line
chromatograms and poured into 21. of ethanol,
towards the cathode were 0-0,
electrophoresis. Glu-cose, where-upon a precipitate
3 0 and 5.0 cm.; 2:3:6-tri-0-
galactose, arabinose, formed. This was separated
methyl-D-glucose moved 13
mannose, xylose and rhamnose by centri-fuging, dried and
dissolved in 130 ml. of cm. Starch used as a marker
were identified. The colour of
water. The solu-tion was gave a spot at 5B0 cm. from
the spots of glucose and
cooled in ice and 100 ml. of the origin. The spot from
trichloroacetic acid the mixture which moved to 5
0 cm. was shown to be starch borate buffer (pH 9.2) was put
by its blue-staining on aglass-powder
reaction with iodine vapour. electrophoresis column
A solution of the (Hocevar & Northcote, 1957)
hemicellulose isolated from and
the cell wall gave a spot at a separation of the
components obtained. A
3 0 cm. from the polysaccharide
origin. ([o] + 17-1 in water c, 1-0;
Purification of a soluble
polysaccharide from the 1, 2.0) was obtained which
whole cell. gave one peak in the Tiselius
The alkali-soluble electrophoresis apparatus
polysaccharide (1 g.) was
dissolved in with a mobility of 9-9 x 10-
50 ml. of water to give a 5 cm.2v'1 sec.-'. This
clear solution. To this was corresponded
added a solution (2-5 ml.)
containing amylase
(prepared from
human saliva by adding 10
vol. of water and
centrifuging)
and the mixture was kept at
250 for 30 hr. under
toluene.
A control solution
containing 0-5 g. of
soluble starch in 50 ml. was
also treated in the same way
and this, after
incubation, gave a light-red
colour with iodine solution. The
solution of the
polysaccharides from
Chlorella was dialysed
against running tap water for
12 hr. and then against 101.
of distilled water for 6 hr. A
light precipitate formed in
the polysaccharide solution
and this was removed by
centri-fuging. The clear
supernatant was precipitated
with
350 ml. of 80% (v/v)
ethanol. The precipitate was
col-lected and dried; yield,
0-4 g. A solution of this
material gave no colour with
iodine. In the Tiselius
apparatus it gave two peaks
with mobilities of 8-7 x 10-5
cm.2v-1 sec.-' and 12-5 x
10-5 cm.2v-1 sec.-'; the
relative amounts of the
components present were
approximately 3-5:1. Thus
the peak with the mobility
of 2-5 x 10-5 cm.2v-1 sec.-'
in the original alkali-
soluble polysaccharide
fraction was identi-
fied as starch.
Electrophoresis on glass
paper showed one
spot corresponding to the
substance moving at 3-0 cm.
in
the original
material.
A solution of 0-1 g. of the
alkali-soluble
polysaccharides in 2 ml. of
394 D. H. NORTHCOTE, K. J. GOULDING AND R. W. HORNE I958
 original walls the material osmium tetroxide soln. (1%,
lost was made up of lipid, w/v) for
with the component with a butyl methacrylate and 15
4.0 %, hemicellulose, 2 hr. (Palade, 1952); they
mobility of 7-48 x JO-l
4.0%, and a-cellulose, 10- % (v/v) of methyl
cm.2v-1 were then washed with water
8%, which methacrylate which was
sec.-' in the original and dehydrated with ethanol
mixture or that with a accounts for a total loss of polymerized with 1.5%
in the normal manner. The
mobility of material of 18-8%. Very (w/v) of benzoyl peroxide
specimens were embedded in
8-7 x 10- Cm.2v-1 sec.-' in little protein was removed, at 50°. The sections were
the amylase-treated a mixture of 85% (v/v) of
probably because of the weak cut with glass knives in a
material.
proteo-lytic activity of the microtome described by
On glass-paper
enzyme preparation. In terms Porter & Blum (1953).
electrophoresis it gave of the individual
Unsectioned material. The
one spot correspond-ing to constituents of the wall the
microfibrils of the cell wall
the substance moving at 3 digestion had removed 70 % of
could be seen in the
0 cm. in the original the total x-cellulose, 43 % untreated preparations (Plate
mixture and to the of the lipid but 1, Fig. 2), but were more
hemicellulose isolated
from the cell wall. An
only 13 % of the apparent when the walls were
hemicellulose. treated with dilute NaOH
examination of the acid
soln. at room temperature
hydrolysate of this poly- Microscopical (Plate 2, Fig. 3a-e).
saccharide showed the examination of the
The NaOH soln. used was
presence of galactose, cell wall
either 0 5 or 3 % (w/v), and
arabinose, mannose, xylose The cell walls were the time of exposure to the
and rhamnose. The colour examined by the optical and alkali varied from 5 min. to
of the galactose spot on electron microscope. Whole 24 hr.
the chromatogram was
cells can easily be With the stronger solution
relatively much more
distinguished under the and times of over 30 min. the
intense than that of the
optical microscope. Many wall lost its shape and
other sugars.
fields of numerous prepara. eventually disintegrated into
tions were examined and few bundles of isolated fibres
Analyses of whole cells were detected. (Plate 1, Fig. 4). The milder
the cell The electron-microscope
treatment revealed the
walls examinations showed that in
microfibrils as structures
treated nearly all cases few small
having a diameter of approx.
with cell particles contaminated
30-50A existing as a complete
snail the
net-work over the wall and
digestiv preparations. lying in two principal
A comprehensive
e enzyme examination of the cell wall
directions at right angles to
one another (Plates 1 and 2,
Cells walls (32-7 mg.) was made under the electron
were incubated at 250 at Figs. 5 and 3).
microscope with a Sieman's
pH 6-0 In some of the specimens
Elminskop and with a high-
with snail digestive-enzyme (Plate 1, Fig. 2) the inner
tension voltage of 80 kv.
preparation and control surface of the wall could be
Both unsectioned and
experi-ments were carried out seen at the characteristic
sectioned material was
concurrently with enzyme in cleavage
examined. The preparations
the absence of cell walls which occurred when the cells
were supported on
and with cell walls in the nitrocellulose films were broken in the cell
absence of enzyme. After 14 stabilized with carbon, which disintegrator. The
hr., the cell walls were were mounted on Athene copper orientation of the
centrifuged, and grids (New microfibrils in this region
washed with water and dried; 200, diam. 2-3 mm.; was the same as that on the
yield, 25-8 mg. (loss of wt. Smethurst, High-Light outer surface. Generally the
21-1%). No precipitate Ltd., Bolton,
microfibrils were irregularly
was observed in the Lancs.). interwoven throughout the
incubation without cell Unsectioned material entire wall and covered the
walls and no loss in was examined as follows. wall completely (Plates 1 and
weight was apparent in The walls were suspended 2, Figs. 2 and 3). The
the incubation without in water and allowed to microfibrils could also be
enzyme. dry at room temperature in
exposed by treating the walls
The digested walls were air on to the specimen
with ethanolamine (Wise,
then analysed by the grids. These were shadowed
Peterson &
procedure already described. with uranium or chromium
Harlow, 1939) for 4
Found: lipid, 17 mg., 5-2%; in an experimental
hr. at 25° (Plate 1,
hemi-cellulose, 8.8 mg., 27 % evaporator (Coslett &
Home, 1955). The shadowing
Fig. 5). The charac-
(N, 0.5%; ash, 0.2%); a-
angle teristics of these
cellulose
preparations were the
1-5 mg., 4-6 % (N, 0.0 %; was 600.
ash, 0.2 %). Compared with Sectioned material was same as those described
the prepared by fixing the walls above.
in buffered. (pH 7.3)
 The intermicellar material preparations.
was revealed as a granular The section ofthe whole
substance packed around the cell (Plate 3, Fig. 9)
microfibrils and could be showed that the preparation
seen in situ in the as isolated (Plate 4, Fig.
preparations which were 8) did represent the outer
treated with NaOH soln. for membranes of the cell. The
short times (Plate 2, Fig.
thickness of the wall
3). When the walls were measured from these sections
treated with snail
(Plates 3 and 4, Figs. 9 and
digestive enzyme (see
8)
above) they retained their
shape but no microfibrils
could be seen.
Instead the surface of the
wall appeared rough and made
up of a granular substance
(Plate 3, Fig. 6). The
walls under these
conditions were quite
mechanically sound and could
be centrifuged, placed on
the supporting grids and
em-
bedded in the acrylate
without disintegration.
However, if these
preparations were treated
with 0.5 % NaOH soln. for
5 min. or longer they
completely fell apart and
very little
insoluble material could be
recovered. Material
previously treated with 0.5
% NaOH soln. for 25 min. and
then with the enzyme
preparation still retained
the shape of the cell wall
and although very thin and
fragile could be recovered.
When examined
microscopically it had a
granular appear-
ance (Plate 1,
Fig. 7).
Sectioned material. The
difficulty in the
interpretation of
the sectioned material was
that of distinguishing
definite
layers in the wall from a
layer-like appearance due to
the
representation by a two-
dimensional photograph of
a
three-dimensional section
in which the top and bottom
edges of the section could
appear as layers in the
photo-graph. In some cases
(Plate 4, Fig. 8) sections of
the wall could easily be
recognized as ribbon-like
structures having a top and
bottom edge. But generally
our interpretation of the
sectioned material is of
necessity cautious and
depends upon a close study
of a very large number of
such
Vol. 70 CELL WALL OF CHLORELLA 395
 3- The material isolated is soluble in
was approx. 210L. The overall diameter of the cells was about
uronic acid constituents
has a fairly dilute sodium
41. In the section shown in Plate 3, Fig. 9, the outer wall appeared to of the plant-cell wall
consist of at least two membranes and after treatment with the digestive enzyme constant composition hydroxide. are associated with the
and when examined
this division into membranes became much more apparent (Plate 4,

Fig. 10). Each membrane

No uronic acids were
micro- middle lamella or
indicated by the dark line, detected in the chemical
scopically it resembles intercellular material
i.e. electron-dense or the wall of the intact analyses and if these
which is absent in this
osmium-stained material, cell. are present they are
unicellular alga.
was approx. 501 thick and The cell wall has a pre. sumably in very The oa-cellulose of the
the space between them was thickness of
low concentrations. wall as isolated by the
approx. 100l. approximately
Thus the section at this 210k whereas the Normally the an-alytical procedure is
region was in fact approx. diameter of the whole composed of
200A deep, which represents cell is polysaccharide(s)
the thickness of the intact 3-4 ,u. made up of
wall. The general analysis galactose,
An interesting
characteristic of these
of the cell wall shows arabinose, mannose,
sections is the pronounced an approximate xylose and rhamnose
curling which takes place at composition of in addition to
the breakage cleft protein, 27 %; lipid, glucose.
(Plate 4, Figs. 8 and 10). 9-2 %; a-cellulose, The hemicellulose
It was not ascertained at fraction of the cell wall
what
15-4%; hemicellulose,
31-0%; on hydrolysis gives rise
stage after to galactose in relatively
glucosamine, 3.3 %;
breakage this ash, 5-2% (containing large amounts and also to
occurred. iron mannose, arabinose, xylose
The preparations treated
and calcium), which and rhamnose. The
with dilute NaOH soln.
accounts for over 90 % alkali-soluble
showed no definite lamellae polysaccharides
but the walls seemed to
of the material. The
protein content of the from the whole cells
have swollen (Plate 3, Fig.
wall is high compared have been investigated
11). The reticulate nature
with the few analyses electrophoretically and
of the microfibrils was
continuous throughout the that have been made on shown to contain at
section with no obvious other plant-cell walls least
lamellae due to local from soft tissues three components. One of
concentrations or (Thimann & Bonner, these has similar
variations in orientation. 1933; Tripp & Rollins, characteristics to that
The microfibrils when 1952; obtained from the cell
stained with osmic acid Wirth, 1946). It has wall
appeared as a rather been calculated in and another is starch.
fluffy haze.
this analysis from the An electrophoretically
Sections
of walls nitrogen content of the pure polysaccharide has
treated first with
walls after correcting been obtained from this
dilute NaOH soln. and
then with the enzyme for the amount of amino mixture which
preparation were sugar resembles the material
difficult to stain present. This protein isolated from the
with osmic acid and may be structural in
function wall. The purification
showed no appearance was achieved and
or metabolically active
of membranes. followed analytically
as part of the synthetic
by using
DIS system of the cell-wall
constituents, or both. electrophoretic
CUS procedures. The
The presence ofthe amino
SIO sugar is of interest in hemicellulose has a
N relation mobility of
to the high protein
9-9 X 10-5 cm.2v-1
The cell wall of sec.-' in 0-05M-borate
Chlorella pyrenoidosa content of the wall and
buffer,
has been might
indicate the presence pH 9-2, 00 and
obtained by mechanical
breakage and
of a glycoprotein. It [ox]7 of + 17-
differential seems unlikely that an 10.
insoluble The yield of
centrifuging. It is not
polysaccharide such as hemicellulose and a-
contaminated by whole
chitin is present since cellulose from the whole
cells or cell debris.
the glucosamine cell together with the
component analytical amounts of
these constituents in the plants rather than the
isolated wall enables the secondary wall. In some
percentage (w/w) of wall primary walls, how-
in the whole cells to be ever, differences in
calculated. If the ac- orientation of the
cellulose yield from the microfibrils
whole cell is taken to from the outside to the

have been 2-1 % then the

inside of the cell have
cell wall been observed (Scott,
represents 13-6 % (w/w) Hamner, Baker & Bowler,
of the dry weight of the 1956).
cell. Thus in the Chemically the
isolation procedure microfibrils
employed in this work
correspond to the
about 28 % (w/w) of the
total available wall
material is isolated.
The yield of walls was
undoubtedly lowered by
the differential centri-
fuging, as indicated
in Fig. 1, but this
loss is necessary if
the preparation
obtained is to be free
from cell debris and
whole cells.
The electron-
microscope studies have
shown the wall to consist
of the usual two-phase
system which is known to
occur in the cell walls
of most higher plants
(Northcote, 1958). This
consists of a micro-
fibrillar structure
embedded in a continuous
matrix. The microfibrils
have a diameter of 30-
50A and are irregularly
interwoven in a
continuous network over
the wall. Two main
directions of the
microfibrils are
apparent, lying at right
angles to one another.
There seems to be no
difference in the general
arrangement and
orientation of the micro-
fibrils on the inner and
outer surfaces of the
wall and sections of wall
treated with dilute
sodium hydroxide solution
showed no local
concentrations of
microfibrils. In these
respects the wall
resembles the primary
cell wall of higher
396 D. H. NORTHCOTE, K. J. GOULDING AND R. W. HORNE I958
 be seen, one near the Glucosamine has been
isolated x-cellulose 30-50k in diameter are
outer edge and one near estimated.
fraction and thus enclosed in a
the inner edge, 4. The structure of continuous
contain poly-
separated by a space of the wall has been
saccharides composed matrix.
100A. This would studied by the electron 5. The protein, some
of monosaccharides
indicate local microscope and shown to of which may exist as
other than glucose. It
concentrations of some a
is possible that some be a two-phase system in
of the materials of the glycoprotein, is
of the material which microfibrils
matrix in these outer associated with the
containing these other approximately hemicellulose
and inner lamellae
sugars may be adsorbed and with these
although it is not
on to the microfibrils polysaccharides
implied that these
from the continuous
matrix materials are
makes up the greater
matrix during their part of the
completely localized
preparation. The continuous matrix.
in these regions. The
matrix does contain a 6. The
sections of the wall show
polysaccharide microfibrillar
an interesting
composed of these non- structure, which is
characteristic curling at
glucosidic sugars. nearly all
the breakage cleft, and
However, treatment of polysaccharide,
although several
the wall with 3 % corresponds to the a-
explanations of this
solutions of sodium cellulose of the
are possible it might
hydroxide seems to well indicate chemical analysis. It
give microfibrils differences in is composed of poly-
which are free from molecular mers of glucose,
the matrix substance composition of galactose, mannose,
when these are the outside and arabinose and
examined microscopic- inside regions of rhamnose.
ally. the cell wall. 7. The hemicellulose
The matrix has a can be isolated as an
granular appearance and electrophoretically
is continuous over the
SU pure polysaccharide
cell surface. Chemically MM from the whole cell and
it is related to the AR some of its
characteristics have
substances soluble in Y
dilute sodium hydroxide. been studied
These are the
1. A cell-wall quantitatively. An
hemicellulose, protein, fraction of Chliorella acid hydrolysate con-
amino sugar and possibly pyrenoido8a has been tains galactose,
the lipid. The impure isolated after mannose, arabinose,
hemicellulose contains a disintegration of the xylose and rhamnose.
high proportion of the whole cells in a Mickle 8. The microfibrils
nitrogen present in the cell disintegrator are present as a
original cell wall. The followed by continuous irregular
walls digested with the differential network over the cell
snail enzyme lose 70 % of centrifuging. wall and throughout its
their oc-cellulose, 43 % thickness. They lie
2. The isolated
of their lipid and only 13 wall has been shown approximately in two
% of their hemicellulose. to be free of whole directions at right
This accoimts for nearly cells and debris. It angles to one another.
all the material digested is approximately 210k 9. The continuous
by the enzyme, so that thick and represents matrix is granular in
very little of the protein 13X6 % of the dry appear-ance.
of the wall is removed. weight of the whole Microscopical
The cell. examination and chemical
material left shows no 3. A quantitative
analysis of cell walls
microfibrillar structure chemical analysis of the treated with snail
but resembles the granular wall has been made with digestive enzyme show
matrix, and when sectioned respect to the following that some of the
these digested walls have frac-tions: a-cellulose,
a laminated appearance.
substances of which it
hemicellulose, protein, is composed are
Two distinct layers
lipid and ash.
partially localized in
approximately 50A thick
can
two regions. One of Mickle, H. (1948). J.
these is near the outer B. micr. Soc. 68, 10.
Myers, F. L. & Northcote, D.
surface and the other H. (1958). J. exp. Biol.
near the inner surface 35,639.

of the wall. Nicolai, E. & Preston, R. D.

(1952-53). Proc. Roy. Soc. B,
One of us (R.W. H.) 140, 244.
wishes to thank the Northcote, D. H.
Agricultural
(1954). Biochem. J.
Research Council for 58, 353.
financial aid. The Sieman's
Elminskop
was purchased by a
generous financial grant
from the
Nuffield Foundation. We
wish to thank Miss F. L.
Myers for providing the
enzyme preparation from
Helix pomatia.

REFE
RENC
ES
Boas, N. F. (1953).
J. biol. Chem. 204,
553.
Chibnall, A. C., Rees, M.
W. & Williams, E. F.
(1943).
Biochem. J. 37,
354.
Consden, R. & Stanier, W.
M. (1952). Nature, Lond.,
170,
1069.
Coslett, V. E. & Home, R. W.
(1955). Vacuum, 5, 109.
Emerson, R. & Lewis, C. M.
(1939). Amer. J. Bot. 26,
808.
Fiske, C. H. & Subbarow, Y.
(1925). J. biol. Chem. 66,
375.
Fuller, K. W. & Northcote,
D. H. (1956). Biochem. J.
64,
657.
Hocevar, B. J. &
Northcote, D. H. (1957).
Nature, Lond.,
179, 488.
Jermyn, M. A. & Isherwood,
F. A. (1949). Biochem. J.
44,
402.
Keilin, J. (1956).
Biochem. J. 64,
663.
Kreger, D. D. R. (1957).
Nature, Lond., 180, 914.
Lederer, E. & Lederer,
M. (1955).
Chromatography, 1st
ed., p. 318. London:
Elsevier Publishing
Co.
Levy, A. L. & Chung, D.
(1953). Analyt. Chem. 25,
396.
BIOCHEMICAL JOURNAL, VOL. 70, NO. 3 PLATE 1
 *-oj-W-mlv

I,

D. H. NORTHCOTE. K. J. GOULDING AND R. W. HORNE-THE CHEMICAL COMPOSITION

AND STRUCTURE OF THE CELL WALL OF CHI OREl,LA PI'RENOIDOSA
(Facing p. 396)
PLATE 2 BIOCHEMICAL JOURNAL, VOL. 70, NO. 3
D. H. NORTHCOTE, K. J. GOULDING AND R. W. HORNE-THE CHEMICAL COMPOSITION
AND STRUCTURE OF THE CELL WALL OF CHLORELLA PYRENOIDOSA
\ w,,, ; 8 1 11

IE t: R. 1# 118
1 ,92 s }t:
i
 le WIE|WW sSllIW;P' a e';,
:,, :X

[;'* !! 4,> - !:
"''

| '
RI|||
.... .................................... .................................... .................................... ...

-9112lrt1!:s,,
|tR: ''''''",:,,,,...:s
F m ok
s C

j |N .
,i; I

l-ss $:v:!
Fra,

..1llul e,
a;;
,j,;

t 1 . %
*0<4gg0 - \; -X
,

z]
!8Sk
M
s
[

Y4sII 't |
- 2;, wRs., .sR
-d..F;f

:;g

, ,, 1!

.. s'sj ::

|is' ,f,,, *,Fii..................... - WYC t ':

::r iFt t!:]L,: ,

-
\...... :,''';w'Sr'""... .. . XL ...... _;.

Lw s
wU'' |1

.a ....g.
;,...............................-
A:Wj, fR'

''' a
s
t
3 PLATE 3
X *'''s
x
BIOCHEMICAL JOURNAL, VOL. 70, NO.
lil , .; ,4

M.

I
1|1_
wofiedr:t,0r
t '! S , !'9
.,,, , ., s;' ; '"'"s'
.,ut.., , i:'' g<t
:,

,.
.''t' R:

, , ,,,, Jbt ikGASF.. : ' '

,R, ,,
F'

p a F
,:.g,,,.,; Wa.. .
................
;-a ,,-U ,jL,# .W4S

'utflL * +
5 .c:ri'

\ ''' a
D. H. NORTHCOTE, K. J. GOULDING AND R. W. HORNE-THE CHEMICAL COMPOSITION
AND STRUCTURE OF THE CELL WALL OF CHLORELLA PYRENOIDOSA
PLATE 4 BIOCHEMICAL JOURNAL, VOL. 70, NO. 3
 8

yI.
$Wt

I
.1-I
10

D. H. NORTHCOTE, K. J. GOULDING AND R. W. HORNE THE CHEMICAI,

COMPOSITION
AND STRUCTURE OF THE CELL XVALL OF CHLORELLA PYRENOIDOSA
Vol. 70 CELL WALL OF CHLORELLA 397
Northcote, D. H. (1958). Biol. Rev. 33, 53. Scott, F. M., Hamner, K. C., Baker, E. & Bowler, E. (1956).
Palade, G. E. (1952). J. exp. Med. 95, 285. Amer. J. Bot. 43, 313. Proc. Roy. Soc. B,
Partridge, S. M. (1948). Biochem. J. 42, 238. Thimann, K. V. & Bonner, J. (1933).
Partridge, S. M. (1949). Nature, Lond., 164, 443. 113, 126.
Porter, K. R. & Blum, J. (1953). Anat. Bec. 117, Tripp,V. W. & Rollins,M. L. (1952). Analyt. Chem.24, 1721.
685. Wirth, P. (1946). Ber. 8chweiz. bot. Ge.8. 56, 175.
Potter, V. R. & Elvehjem, C. A. (1936). J. biol. Chem. 114, Wise, L. E., Peterson, F. C. & Harlow, W. M. (1939).
495. Industr. Engng Chem. (Anal.), 11, 18.

EXPLANATION OF PLATES 1-4

PLATE 1
Fig. 2. Untreated cell walls of Chlorella.
Microfibrils can be seen and the undersurface
of the wall is visible at the breakage cleft.
Fig. 4. Cell walls treated with 3% NaOH for 14 hr.
at room temperature. The cell wall has completely
disinte-grated, giving bundles of microfibrils
with very little matrix material visible.
Fig. 5. Cell walls treated with ethanolamine for 4 hr. at
250, showing microfibrils.
Fig. 7. Cell walls treated with 0.5% NaOH for 25
min. at room temperature and then snail
digestive en-zyme at 250 for 14 hr. No
microfibrils are visible and although the wall
is very thin it still retains the cell shape.
PLATE 2
Fig. 3. Cell walls treated with NaOH at room temperature: principal directions at right angles to one
(a) 05% NaOH for 5 min.; (b) 05% NaOH for 25 min.; another can be clearly seen. The granular material
(c) 0.5 % NaOH for 25 min.; (d) 0.5 % NaOH for 30 min.; of the matrix is visible. In (e) the wall has lost
(e) 3 % NaOH for 30 min. Microfibrils lying in two its form and is disinte-grating into microfibrils.
PLATE 3
Fig. 6. Cell walls treated with snail digestive enzyme for Fig. 11. Section of cell walls treated similarly to those

14 hr. at 25°, showing rough granular shown in Plate 2, Fig. 3c. The walls have swollen and
surface and absence of microfibrils. the microfibrils are visible as a diffuse material
Fig. 9. Section of whole cell. The appearance of layers in the throughout the thickness of the sections.

cell wall is visible. Compare with Plate 4, Fig. 8.

PLATE 4
Fig. 8. Sections of untreated cell walls showing ribbon-like Fig. 10. Section of cell walls treated similarly to
appearance and characteristic curling at the breakage those shown in Plate 3, Fig. 6. The appearance of
cleft. Compare with Plate 1, Fig. 2 and Plate 3, Fig. 9. two layers in the wall is very apparent.
 Phosphorolysis of Citrulline by Mammalian Liver:
the Effect of a Bacterial Activator
BY H. A. KREBS, P. K. JENSEN* AND L. V. EGGLESTON
Medical Research Council Unit for Research in Cell Metaboli8m, Department of Biochemnistry,
University of Oxford
(Received 8 April 1958)
In a previous paper to the removal of
(Krebs, Eggleston & ornithine, which acted
Knivett,
as an inhibitor of the
1955) it was shown that
phosphorolysis.
washed suspensions of
However, an
E8cherichia coli N.C.I.B.
8571 accelerate the phos- accelerating
phorolysis ofcitrulline effect remained
by mammalian liver
extracts.
even when the
activity of ornithine
The bacterial cells
decarboxylase was
contained ornithine
abolished by
decarboxyl-ase, and most
hydroxylamine. From
of the acceleration could
this it was concluded
be ascribed
*
that this strain of E.
Present address: The
University Institute for coli contained an
Experi- additional substance (or
mental Endocrinology,71 'factor') which promotes
NorreAlle, Copenhagen,
Denmark. the phos-

phorolysis of
citrulline.