c-phycocynin as storage protein
TRANSCRIPT
-
8/11/2019 C-phycocynin as Storage Protein
1/5
Arch . Microbiol. 125, 14 3- 147 1980)
rchives
o
i c r o b i e l o g y
@) by Springer-Verlag 1980
C Ph y co cy a n i n a s a S to ra g e Pro te i n i n th e B l u e G reen Al g a p i r u l i n a p l a t en si s
S a m y B o u s s i b a a n d A m o s E . R i c h m o n d
he Institu ie for Desert Research, Ben-Gurion University of the Negev, Sed e Boqer Camp us, P.O . Box 2053, Bee r Sheva 84120, Israel
A b s t r a c t .
The p oss ib i l i ty tha t c -phy cocya n in serves as a
n i t rogen source in
Spirutina platensis
dur ing n i t rogen
s ta rva t ion was s tud ied . The fo l lowing ev idence was
o b t a i n e d i n s u p p o r t o f t hi s i d ea . 1. U n d e r f a v o u ra b l e
cond i t ions fo r g rowth , c -phycocyan in ex i s ted in l a rge
excess in the a lga l ce l ls . 2 . W hen the sup p ly o f n i t rogen
w a s l o w , a b o u t 3 0 - 5 0 o f t h e c -p h y c o c y a n i n d i s a p -
p e a re d w i t h o u t a n y e f f e c t o n t h e m a x i m a l g ro w t h r a te .
3 . A c u l t u re w h i c h w a s d e p r i v e d o f n i t r o g e n c o n t i n u e d
t o g ro w u n a f f e c t e d l y fo r a p e r i o d , t h e d u ra t i o n o f w h i c h
d e p e n d e d o n t h e c -p h y c o c y a n i n c o n t e n t i n t h e c e l l
befo re n i t rogen s ta rva t ion was in i t i a ted . 4 . c -phyco-
c y a n i n w a s t h e o n l y n i t r o g e n o u s c o m p o u n d t h a t w a s
d e p l e t e d d u r i n g t h e c o u r s e o f n i t r o ge n s t a rv a t i o n
w h e n
g ro w t h w a s y e t u n a f f e c te d . 5 . W h e n p ro t e i n s y n t he s i s
was inh ib i t ed e i ther by n i t rogen s ta rva t ion o r by
m e t h i o n i n e s u l fo x i m i n e (M S O ) , p h y c o c y a n i n c o n t e n t
b e g a n t o d e c li n e i m m e d i a t e l y a n d g ro w t h c o n t i m l e d a t
no rmal ra tes as long as c -phycocyan in d id no t dec l ine
be low 50 . 6 . The decrease in c -phyc ocyan in con te n t
d u r i n g n i t r o g e n s t a rv a t i o n w a s a c c o m p a n i e d b y a n
increase in p ro teo ly t i c ac t iv i ty .
K e y w o r d s : N i t ro g e n s t a rv a t i o n - - c -p h y c o c y a n i n
B l u e g r e e n
alga -
Spirutina p latensis .
The p igmen t c -phycocyan in i s p resen t in a l l b lue-
g reen a lgae (Ch apm an , 1973 ; Glazer , 1976 ; S tan ier and
Cohen Bazi re , 1977) . I t s concen t ra t ion in the ce l l
d e p e n d s o n e n v i ro n m e n t a l c o n d i t i o n s (M y e r s a n d
Kra tz , 1955) . c -phyc ocyan in i s genera l ly be l i eved to be
a n accesso ry p igmen t w i th wh ich l igh t energy i s cap tu red
a n d
i s t rans fered i t to ch lo rophy l l a (Bogoarad , 1975) .
H o w e v e r , t h e e x i s te n c e o f m u t a n t s l a c k in g t h e p i g m e n t ,
o r par t o f it , bu t s ti ll hav ing the sam e g row th ra te a n d
pho to syn the t ic e f f ic iency (S tevens and M yers , 1976 ;
Rog ers e t a l. , 1977) sugges t tha t th i s b i l ip ro te in p igm en t
may have o ther func t ions in add i t ion to i t s recogn ized
ro le in pho tosyn thes i s . Abel iov ich and Sh i lo (1972)
s u g g e s t e d t h a t p h y c o c y a n i n m a y t h n c t i o n a s a n a n t i -
ox idan t an d M i l le r and H ol t (1977) p rop ose d i t to se rve
as a carbon-s to rage mater ia l .
Al len and Smi th (1969) were the f i r s t to po in t ou t
t h e p o s s i b i l i t y t h a t p h y c o c y a n i n m a y b e a n i t r o g e n
s t o r a g e c o m p o u n d i n t h e c e l l . O t h e r w o rk s b ro u g h t
somewhat inconclus ive f ind ings fo r th i s poss ib i l i ty
(V a n G o rk o m a n d D o n z e , 1 9 7 1 ; S t e w a r t a n d Le x ,
1970; Van Liere et al . , 1977).
Ownby e t a l . (1979) recen t ly repor ted on exper i -
m e n t s w i t h Anabaena in wh ich pu l se l abe l ing a n d
t r e a t m e n t w i t h m e t h i o n i n e s u l fo x i m i m e (M S O ) w e re
u s e d t o t r a c e t h e c o u r s e o f p ro t e i n d e g ra d a t i o n d u r i n g
n i t ro g e n s t a rv a t io n . Th e y c o n c l u d e d t h a t c y a n o b a c -
te r ia rap id ly degrade p ro te ins in response to n i t rogen
s ta rva t ion as wel l as in response to t rea tm en t w i th MS O
ut il i z ing the am ino ac ids thus re leased fo r the syn thes i s
o f n e w p ro t ei n s . Th e y h y p o t h e s iz e d t h a t t h e m a j o r f o rm
of p ro te in hydr o lyze d in n i t rogen s ta rved cel ls - o r
MSO t rea ted ce l l s was phycocyan in . Lau e t a l . (1977)
w e re t h e f i r s t t o p r e s e n t e v i d e n c e t h a t s p e c t ro p h o t o -
met r ic de te rm ina t ions o f phyc ocya n in re f lec t (a t l as t in
the case o f n i t rogen s ta rva t ion ) even ts a t the l eve l o f
a p p o p ro t e i n d e g ra d a t i o n .
In t i l e p resen t s tudy , we sough t to b r ing n e w
phys io loN cal ev idence fo r the thes is tha t c -phyc ocyan in
a c t s as a n i tr o g e n - s to r a g e c o m p o u n d . O u r e x p e r im e n t s
were d i rec ted a t es tab l i sh ing a co rre la t ion be tween
n i t rogen supp ly a n d t h e c o n c e n t r a t i o n o f c -p h y c o-
c y a n i n , a s r e f l e c t e d b y g ro w t h p a ra m e t e r s . C o n -
s e q u e n t l y , i n m o s t o f o u r e x p e r i m e n t s w e u s e d a
c o n t i n u o u s c u l t u r e of Spirulinaplatensis , w h i c h a l l o w e d
us to con t ro l a l l g rowth fac to rs .
0302-8933/80/0125/0143/ 01.00
-
8/11/2019 C-phycocynin as Storage Protein
2/5
144 Archl Micro biot. , Vol. 125 (1980)
M a t e r i a l s a n d M e t h o d s
Organism an d G rowth C onditions. Spirulina platensis
(Lb 1475/4a)
was ob ta ined f rom the Cambr idge Cu l tu re Co l lec t ion . Zarouk ' s
med ium (1966) was used as the g rowth m ed ium wi th the fo l lowing
modif ica t ions : the concen t ra t ion o f NaHC O3 was 4 .2 g / l, and th a t o f
K2H PO4 was 0 .185 g / l , NaNO3 was used a t va r ious concen t ra t ions .
(See Results a nd Discussion.)
The a lgae were g rown a t 35~ e i the r in con t inuous cu ltu re in a
tu rb idos ta t o r in ba tch cu ltu res . The pH was ma in ta ined a t 8 .5 -9 .0 ,
and the cu l tu res were s t i r red by m eans o f a i r s t ream (4 l / ra in )
con ta in ing 1 .5 % CO2 . I l lumina t ion a t va r ious in tens i tie s was p ro -
vided by cool white lamps, as described in the text. Cultures were
allowed to grow for at least 10 dou bling t imes at which stage,
regarded to be s teady s ta te , the exper imen ts began .
Nitrogen Starvation Experiments and Methionine Suljbximime
M SO ) Trea tmen t o f Ce l ls .
Cells were harvested by centrifug ation,
then re -suspended in n i t rogen- f ree med ium, cen t r i fuged aga in and
washed thorough ly wi th d is ti lled wa ter . For t rea tmen t wi th M SO,
ce ll s were resuspended in fu l l med ium and MSO was added to a f ina l
concen t ra t ion o f 5 gM.
Concentration of Pigments.
For the de te rm ina t ion o f c -phycocyan in ,
the ce ll s were f i rs t d is rup ted by one o f two m ethods : 1 . Son ica t ion in
0 .1 M Na-phosph a te buf fe r , pH 7 .0 , o r 2 . t rea tmen t wi th a so lu t ion o f
lysozyme, 100 gg/ml of 0 .1 M Na- phos phate buffer, con taini ng
t 0 0 m M N a E D T A . T h e a b s o r b a n c e o f t h e r e s u l ti n g s u s p e n si o n w a s
read spec t ropho tometr ica l ly a t 620 nm (Myers an d K ra tz , 1955) , and
the conce n t ra t ion o f c -phycocyan in was then ca lcu la ted f rom the
specific abso rptio n coefficient E 1% = 73 (Boussiba and Rich mo nd,
1979).
Ch lo rophy l l a was ex t rac ted by son ica t ion n 80 ~ ace tone and i t s
concen t ra t ion was de te rmined f rom i t s absorbance a t 663 nm and an
extinctio n coefficient of 0 .82 (M cK inney , 1941).
Nucleic Acid Determination.
R N A a n d D N A w e r e d e t e r m i n e d
according to the methods of Burton (1956) and Schneider (1957)
respectively~
Measurements of Oxygen Evolution. Cells were suspended in fresh
me dium an d the suspen sion was transferred to a 3 .0-ml Perspex cell .
The ra te o f oxygen evo lu t ion was measured a t 30~ by the use o f a
Clark-ty pe Oxygen Electrode (YS14004, Yellow Springs Ins trum ent
Co. , Yellow Springs, Ohio) conn ected to a recorder (Servogor 310).
The light intensi ty at the surface of the cell was I05 ergs nr 2 , s ~ t .
Specif ic growth rate was ca lcu la ted as ln2 9 - I (P i t t , 1975). The
ou tp u t ra te was ob ta ined by m ul t ip ly ing the speci f ic g rowth ra te by
the cell density (mg/ml).
Cell density
was main ta in ed a t 0 .5 mg d .wt /ml in a l l ~ e con t inuou s
culture experiments.
Protein and Free Amino Acid Measurem ents:
Pro te in was de te rmined
accor d ing to the p rocedure o f Lowry e t a l . (1955) . F ree am ino ac ids
a n d o t h e r n i t r o g e n c o n t a in i n g co m p o u n d s , s u c h a s a m i d e s a n d
amm onia , were es t ima ted by the n inh ydr in es t (Rosen , 1957). Cel ls
were disrupte d by sonication, which was followed by precipitatio n in
10 % trichloroacetic acid (TCA). Leucine was used as the sta ndard .
Dry we igh t
(d.wt) was measured by fi l tering culture samples throu gh
8-m g mem bran e fi l ters . The fi lters were weighed, then d ried at 80~
for 1 h and reweighed.
Determination of P rotease Activ i ty During Nitrogen Starvation. Six-
d a y - o l d c u lt u re s g r o w i n g i n Z a r o u k ' s m e d i u m c o n t a i n i n g N a N O 3 ,
2 .5 g / t, were ha rves ted a nd re -suspended in n i t rogen- f ree med ium
after thoro ugh w ashing with distil led water. Tw o-liter samples were
taken at interwfls. Each sample was centrifuged, and the sediment
was re -suspended in a smal l vo lume o f 0 .1 M Na-phospha te buf fe r,
pH 7 .0 . The ce il s were then d is rup ted in a mechan ica l ce t t homoge-
n ize r (B rawn Mode l M SK) fo r 3 ra in , and the suspens ion was
centrifuged at 10,000 x g for 15 min . The precipitate , which inc luded
cell wails an d debris, was discarded. Protease activity was deter m ined
in the resu lting crude extract.
Protease Assay.
Accord ing to FoUlds and Car t (1977) pure
(620/280> 4) [14C]c-phyc ocyan in (7 x 105 cpm /ml), 324 gg/mt, was
used as the s tandard so lu t ion fo r the assay . Each assay so lu t ion
usua l ly con ta ined 20g l o f the s tandard c -phycocyan in so lu t ion ,
0 .9 mt o f the c rude ex t rac t and 80 ~ tl o f 0 .1 M Na phospha te buf fe r ,
p i t 7 .0 . The te s t tubes con ta in ing the assay so lu t ions were incuba ted
in a shak ing ba th a t 30~ tb r 1 .5h . The reac t ion was te rmina ted by
add i t ion o f an equa l vo lume o f 10 % T CA and t ile p rec ip i tate
was
removed by cen t r i fuga t ion . Al iquo ts o f the supern a tan t , con ta in ing
degra dation pro ducts of c-phycocy anin, were placed in 5-ml vials
c o n t a i n i n gscinti l la tio n luid [0.4 g of 2 .5-diphenyloxazole (PPO ) and
50 mg of 1,4-bis-(5-phenyloxazoly) benze ne (POPO P) pe r l i ter in
to luene] . Rad ioac t iv i ty was coun ted in a Packard l iqu id sc in t i l la t ion
spectrometer (model3320). The c oun ting efficiency for [14C]c-
phyc ocya nin was 85 %. Protease activity was expressed as counts
released per mg protein per h .
Re s u l t s a nd i s c us s i o n
E f Jb c t o f N O ~ C o n c e n tr a t io n
a t D i f f e r e n t L i g h t I n t e n s i t i e s
C o n t i n u o u s c u l tu r e s o f
S . p l a t e n s i s
w e r e g r o w n a t t w o
d i f f e re n t c o n c e n t r a t i o n s o f N a N O 3 , 2 9 . 4 m M a n d
3 .7 m M e a c h a t h i g h a n d l o w l i g h t i n t en s i t ie s , 1 75 a n d
8 0 m i c r o E i n s t e i n 9 m - 2 9 s - 1 , r e s p e c t i v e l y ( T a b l e 1 ).
T h e r e s p o n s e s o f t h e g r o w t h p a r a m e t e r s a t th e t w o
l i g h t r e g i m e s w e re s i m i l a r - g r o w t h r a te a n d o u t p u t
r a t e a s w e l l a s t o ta l p r o t e i n c o n t e n t a n d c h l o r o p h y l l
c o n c e n t r a t i o n w e r e m a i n t a i n e d a t t he s a m e l e v el a t b o t h
n i t r o g e n c o n c e n t r a t i o n s - b u t t h e c - p h y c o c y a n i n
c o n t e n t w a s m a r k e d l y r e d u c e d a t t h e l ow e r c o n c e n -
t r a t i o n o f N O 3 , i .e . 5 0 a n d 3 0 % o f t h e c - p h y c o -
c y a n i n d i s a p p e a r e d a t t h e h i g h a n d l o w i n t e n s it i e s ,
r e s p ec t iv e l y . N e v e r th e l e s s , t h e a b s o l u t e a m o u n t o f c-
p h y c o c y a n i n w h i c h d e c l i n e d w a s t h ~ s a m e , i. e. 1 4 -
1 5 g g p e r 5 0 0 t ~ g d r y w e i g h t , u n d e r t h e t w o l i g h t
r e g i m e s . I t w a s , t h e r e f o r e , c o n c l u d e d t h a t c -
p h y c o c y a n i n i s p r e s e n t i n ex c e ss a n d c a n b e d e p l e t e d
w i t h o u t i m p a i r i n g th e m a x i m a l g r o w t h r a t e u n d e r t h e
c o n d i t i o n s o f t h e e x p e r i m e n t .
E f f e c t o f N i t r o g e n S t a r v a t i o n
a t D i f f e r e n t L i g h t I n t e n s i t i e s
W h e n c o n t i n u o u s c u l t u re s g r o w n a t t h e h i g h n i t r o g e n
( 2 9 A t o M ) , u n d e r e i t h e r h i g h o r l o w l i g h t i n t e n si t i e s,
w e r e d e p r i v e d o f n i t r o g e n , t h ey c o n t i n u e d t o g r o w f o r
o n e d o u b l i n g cy c l e a t t h e g r o w t h r a t e t h e y h a d d i s-
p l a y e d p r i o r t o t h e d e p r i v a t i o n ( F i g . t a , b ) . T h e p h o t o -
s y n t h e t i c p o t e n t i a l o f t h e c el ls , a s i n d i c a t e d b y t h e r a t e
o f n e t O z p r o d u c t i o n , d e c r e a s e d o n l y w h e n t h e l e v el o f
c - p h y c o c y a n i n f e ll b e l o w 1 4 .5 o r 3 5 .7 1 ag 9 m l - t , i n
-
8/11/2019 C-phycocynin as Storage Protein
3/5
S . B o u s s i b a a n d A . E . R i c h m o n d : C - P h y c o c y a n i n a s a S t o r a g e P r o t e i n 1 4 5
T a b l e 1 . E ff e c t o f N O ~ c o n c e n t r a t io n o n g r o w t h p a r a m e t e r s a n d c o n t e n t o f p r o t e in , c h l o r o p h y l l a n d c - p h y c o c y a n i n i n c o n t i n u o u s c u l t u re s o f
p i r u li n a p l a t e n s i s
L i g h t i n t en s i ty N O ; c o n c . S p e c if i c O u t p u t r a t e T o t a l C h l o r o p h y l l c - p h y c o c y a n i n
g E i n s t e i n s i n m e d i u m g r o w t h r a t e ( m g d . w t . 9ml - 1 p r o t e i n ( b tg - ml i )
9m - Z ' S - 1 ( m M ) ( h - 1 ) . h - ~ ) ( g g - m l - ~ ) ( t ag . m 1 - 1 )
H i g h
175
L o w
80
29.4 0.073 3.5 250 2.85 28.4 100
3.7 0.073 3.5 245 2.80 14.5 51
29 . 3 0 . 049 2 . 5 250 5 . 70 50 100
3 . 7 0 . 050 2 . 6 250 5 .90 35 70
~ -30
O - c 0
8
\ -25
21
,,,.
20 ?
x
~) T -15
W Oa
4 - 3 0
3" 2 0
; 1 4
T i M E ( h )
F i g . l . E f f e c t o f n i t r o g e n s t a r v a t i o n o n o x y g e n e v o l u t i o n a n d
c - p h y c o c y a n i n c o n t e n t i n c u l t u re s g r o w i n g u n d e r h i g h N O ~
( 2 9 .4 m M ) a t h i g h (a ) o r lo w l i g h t i n t e n s i t y ( b ). , - - O O x y g e n
e v o l u t i o n , a t . . . . . a t c - p h y c o c y a n i n c o n t e n t
cultures grown under high or low light intensity,
respectively.
We tested the hypothesis that the length of the
period of normal growth and development before the
effect of nitrogen deprivation is manifested, is related to
the cellular content in c-phycocyanin. For this purpose,
cultures were grown in continuous culture with either
high or low nitrogen and were exposed to high light
intensity. These cells, which contained either high or
low c-phycocyan in concentrations, were transferred to
a nitrogen free medium 9As expected, the growth rate o f
cells with the relatively low concentration of c-
phycocyanin was reduced immediately on exposure to
nitrogen deficiency (Fig. 2).
The role of c-phycocyaninas a storage substance for
nitrogen is further supported by analysis of the kinetics
of dry weight, protein and c-phycocyanin content in
T
r
E
7
r
E
ID
"8
fi
g
2
o
l a l
I 0
8
6:
4 -
2 -
0
O
\ X "x~,
N Z ~ , . ( b ) x
\
\
o
{ a
b J 2 i 6
T I M E ( h )
F i g . 2 . E f f e c t o f n i t r o g e n s t a r v a t i o n o n o x y g e n e v o l u t i o n i n c u l t u re s
g r o w i n g a t h i g h l i gh t i n t e n si t y u n d e r h i g h N O ~ - 2 9 . 4 m M ( a)
O - ~ o r l o w N O ~ - 3 , 7 m M ( b ) A - -- Z~
batch culture when transferred in the logarithmic phase
of growth to a nitrogen-free medium (Fig. 3). After the
transfer, the cells continued to grow at the initial
growth rate for one generation (Fig. 3a). The am ount
of total protein did no t increase (Fig. 3b) while c-
phycocyan in decreased continuously during the course
of the experiment (Fig. 3c). The amount of c-phyco-
cyanin which disappeared during starvation was quan-
titatively sufficient to maintain the concentration of
non-c-phycocyanin protein at a cons tant level: at the
beginning of starvation, protein content was 50gg
9ml - i, of which 11 btg. ml i were c-phycocyanin, while
after 72 h of growth in a nitrogen-free medium, the
concentration o f protein was the same (50 btg. ml-1),
but that of c-phycocyanin had fallen to as little as 2 btg"
ml - 1. In parallel with the decline of c-phycocyanin, a
marked increase o f proteolytic activity was observed
(Fig. 4).
Still further support for this role of c-phycocyanin
as a storage compound was obtained in experiments
that tested the effect of MSO on cell growth and
-
8/11/2019 C-phycocynin as Storage Protein
4/5
1 4 6 A r c h . M i c r o b i o L , V o l . 1 2 5 ( 1 9 8 0 )
7
~. 2oo-
oo o b t c
o / 7 '
/ o / 3 ~ I
3 / 1 / t, ~ , /
A
, [ 20-
/ /
ioo" ] : /
o
/
5 @ / ~ ~
o l
IOC ~ ~l. ~8 i a O0 d4 4e 72 ~4 48 I l~
T I M E ( h )
F i g . 3 . E f f e c t o f n i t r o g e n s t a r v a t i o n o n ( a ) d r y w e i g h t ( b ) p r o t e i n
c o n t e n t a n d ( e ) c - p h y c o c y a n i n c o n t e n t . F u l l s y m b o l s - c o n t r o l
t r e a tm e n t , e m p t y s y m b o l s - n i t r o g e n s t a r v a t i o n t r e a t m e n t
--
o
O .
4 - / k .
/ - - , ,
z - / \ a
o T
o
, 2 14 3 6,~ a d o
T I M E
h)
P 1 3
- 1 2
?
2o g
.B g
6
~"
3
4 . :
F i g . 4 . E f f e c t o f n i t r o g e n s t a r v a t i o n o n p r o t e a s e a c t i v i t y a n d c -
p h y c o c y a n i n c o n t e n t . O - . - P r o t e a s e a c t i v it y , A - A c - p h y c o -
c y a n i n c o n t e n t ,
N
o
. 2 .
T
o
. I 0
.9 a ~
.8
.7
.6
.5
4
.3.
i
6 12 IB
N
~ .2 9
, , 7 , . - - m
T , I'2 '
0 6 18
~ o f c
T i , , i i
0 6 1 2 1 8
H g . 5
T h e e f f e c t o f M S O o n ( a ) d , w t, ( b ) p r o t e i n c o n t e n t
( e ) c - p h y c o c y a n i n c o n t e n t . E m p t y s y m b o l s -
c o n t r o l t r e a t m e n t , f u l l s y m b o l s - . c e ll t r e a t e d w i t h
5 g M M S O
T I M E ( h )
T a b l e 2 . E ff e c t o f n i t r o g e n s t a r v a t i o n o n t h e c o n t e n t o f n i n h y d r i n -
r e a c t a n t s u b s t a n c e s u n d e r h i g h a n d l o w l i g h t i n t e n s i t i e s
S t a r v a t i o n
(h)
C o n t e n t o f n i n h y d r i n -r e a c t a n t
s u b s t a n c e s ( p m o l e - m l - 1)
H i g h i n t e n s i t y
L o w i n t e n s i t y
0 0 . 1 8 8 0 . 1 8 0
1 0 . 2 t 4
2
3 0 . 2 0 0 0 . 1 9 6
4
5 0 . 1 8 8
6 0 . 1 7 5
8
1 0 0 . 1 8 0 0 . 1 8 5
1 2
t 6
2 4 0 . 1 8 5 0 . 1 8 5
L e u c i n e e q u i v a l e n t s
phyc ocya n in dep le t ion . In c lose s imi la r ity to the e f fec t
o f n i t ro g e n s t a rv a t io n o n p h y c o c y a n i n c o n t e n t, M S O
c a u s e d a n i m m e d i a t e d e c l i n e i n p h y c o c y a n i n c o n t e n t
b u t g ro wt h wa s n o t a l t e r e d u n l e s s p h y c o c y a n i n f e l l
be lo w 50 ~ (F ig . 5 ) .
The con ten t o f n inhydr in rea c tan t sub s tances , i. e.
f r e e a m i n o a c id s , a m i d e s a n d a m m o n i a , wa s v e ry sm a l l
a n d d i d n o t c h a n g e s i g n if i ca n t ly t h ro u g h o u t t h e c o u r s e
o f n i t ro g e n d e p r i v a ti o n (T a b l e 2 ) . T h e a m o u n t o f
nuc le ic ac ids (7 - 8 ~o o f d ry weigh t ) a l so rem ained the
s a m e . C l e a r l y , t h e o n l y n i t ro g e n c o m p o u n d wh i c h
decreased s ign i f ican tly du r ing the course o f the de-
p r iva t ion and which cou ld , therefo re , have served as a
n i t rogen-s to rage molecu le , was c -phycocyan in .
I t m a y b e w o r t h n o t i n g t h a t i n t h e b l u e -g re e n a l g a e,
c -p h y c o c y a n i n d i f f e r s f ro m o t h e r k n o wn s t o r a g e m a -
terials in that i t has a d ual role in the cel l and th at i ts
syn thes i s occurs l a rge ly du r ing the logar i thmic pha se o f
-
8/11/2019 C-phycocynin as Storage Protein
5/5
S. Bouss iba and A. E . R ichm ond: C -Phycocyan in as a S to rage P ro te in 147
g r o w t h a n d p r a c t i c a l l y c ea s e s d u r i n g t h e s t a t i o n a r y
p h a s e ( S i m o n , 1 9 7 3 ) . T h i s i s i n c o n t r a s t t o o t h e r s t o r a g e
p r o d u c t s w h i c h a r e u s u a l ly a c c u m u l a t e d a t t h e e n d o f
t h e g r o w t h p h a s e o r i n t h e f i n a l s ta g e o f t h e l if e c y c l e
( L e h m a n n a n d W o b e r , 1 97 6) .
T h e a d v a n t a g e o f c - p h y c o c y a n i n a s a s to r a g e f o r m
o f re d u c e d n i t r o g e n s k e le t o ns m a y b e s p e c u l a t e d : W h e n
c e ll s w h i c h g r o w l o g a r i t h m i c a l l y w i t h a n a b u n d a n t
n i t r o g e n s o u r c e a r e e x p o s e d t o a t e m p o r a r y s h o r t a g e o f
n i t r o g e n , t h e y w o u l d b e a b l e to g r o w u n h i n d e r e d f o r u p
t o a n o t h e r g e n e r a t i o n b e f o r e t h e ir g r o w t h c e a s es . A l s o ,
w h e n c e l l s o f t h e s e a l g a e s o m u l t i p l y a s t o c a u s e d e n s e
b l o o m s , a p o r t i o n o f c - p h y c o c y a n i n b e c o m e s p o t e n -
t i a l ly a s t o r a g e m a t e r i a l , w h i c h f a c i l it a t e s i m m e d i a t e
r e s u m p t i o n o f a c c e n t u a t e d g r o w t h w h e n c el ls f r o m s u c h
b l o o m s a r e d i s p e r s e d a n d l i g h t c e as e s t o b e l i m i t i n g t o
g r o w t h .
Acknowledgements We wish to thank Miss Doro t Imber and Mrs .
Inez Mu re in ik fo r va luab le comments and fo r ed i t ing the manuscr ip t .
e f e r enc e s
Abe liovich, A. , Shilo, M.: Photo-o xidative reactions of c-
phyc ocyan in. Biochem. Biophys. A cta. 283, 483--4 91 (1972)
Allen, M . M ., Smith, A . J . : Nitr ogen chlorosis in blue-g reen algae.
Arch . Mikrob io l . 69 , 114-120 (1969)
Bogorad , L . : Phycob i l ip ro te ins and complem enta ry ch romat ic adap-
ta t ion . Ann . Rev . P lan t Phys io l . 26 , 369 -40 1 (1975)
Bouss iba , S . , R ichmond , A . : I so la t ion an d pur i f ica t ion o f phyco-
cyan ins f rom the b lue -g reen a lga SpiruIina platensis Arch.
Microb io l . 120 , 15 5-1 59 (1979)
Bur ton , K . : A s tudy o f the cond i t ions and mechan ism o f the
d ipheny lamine reac t ion fo r the ca lo r imet r ic e s t ima t ion o f de -
oxyribo nucleic acid. Biochem. J . 62, 315 -3 23 (1956)
Chapm an , D. J . : B i l ip ro te ins and b i le p igments. In : The b io logy o f
blue-gree n algae (N. G. Carr, B. A. W hitton , eds.) , pp. 162 -18 5.
Oxford: Blackwell 1973
Fou lds , I . J . , C ar r , N. G. : A p ro teo ly t ic enzyme degrad ing phyco-
cyan in in the cyanobac te r ium Anabaena cylindrica F E M S .
Microb io l . Le tt . 2 , 117 -11 9 (1977)
Glazer , A. N. : Phycocyan ins : S t ruc tu re and func t ion . Pho tochem.
Pho tobi ol. Rev. 1, 71 - 115 (1976)
Lau, R. H . , Mackenzie, M. M ., Doolit t le , W. F. : Phyc ocyan in
syn thes is and degrada t ion in the b lue -g reen bac te r ium Anacystis
nidulans J. Bacteriol. 1 32, 77 1- 77 8 (1977)
Lehm ann , M. , Wober , G . : Accum ula t ion , mobi l iza t ion and tu rno ver
o f g lycogen in the b lue -g reen bac te r ium Anacystic nidulans
Arch . Microb io l . 111 , 93 -9 7 (1976)
Lowry, O. H. , Ro sebroug h, N. J . , Farr , A. L. , Rand all , B. J . : Prote in
mea surem ents with the folin phen ol reagent. J . Biol. Chem. 193,
2 6 5 - 2 7 5 ( 1 9 5 1 )
McK inney , G. : Abso rp t ion o f l igh t by ch lo rophy l l so lu tions . J. B io l.
Chem. 140 , 31 5-3 22 (1941)
Miller, L. S. , Ho lt , S . C. : Effect of carb on dioxide on p igm ent and
m e m b r a n e c o n t e n t i n Syneehococcus lividus Arch . Microb io l .
115 , 105-198 (1977)
Myers, J . , K ra tz , K . A. : Re la t ion be tween p igment con ten t and
photo synth etic characteristics in blue-green alga. J . Gen.
Physiol. 39, 11-22 (1955)
Ownby , J . D. , Shannahan , M. , Hood , E . : P ro te in syn thes is and
degrada t ion in Anabeana dur ing n i t rogen s ta rva t ion . J . Gen .
Microb io i . 110 , 255 -26 1 (1979)
Rogers, R. L. , And ersen, W. R. , Bradshaw, W . H. : A mu tan t of
Aphanocapsa 6308, lack ing phycocyan in . Ann . Mee t ing . Am er .
Soc. Micro biol. 170 (1977)
Rosen , H . : A m odif ied n inhydr in co lo r imet r ic analysis fo r amino-
acids. Arch. Biochem. Biophys. 67, 10-15 (1957)
Schneider, W . C. : Determ inati on of nucleic acids in t issues by pentose
analysis . In : M ethods in entom ology, Vol. III (Colowick, P. S. ,
Kap lan , N. N . , eds .) , pp . 68 0- 684 . New Y ork : Acad . P ress 1957
Simon , R . D. : Measurem ent o f the cyanophyc in g ranu le po lypep t ide
con ta ined in the b lue -g reen a lga Anabaena cylindrica J
Bacteriol. 114, 12 13- 121 6 (1973)
Stanier, R. Y. , Cohen-Brazire, G. : Photo troph ic protokar yotes: T he
cyanobac te r ia . Ann . Rev . Microb io l . 31 , 22 5- 27 4 (1977)
Stevens, C. L. R. , Myers, J . : Chara cterizatio n of pigm ent muta nts in
blue-green algae Anaeystis nidulans J . Phyco l . 12 , 99 -1 05
(1976)
Stewart, W . D. P. , Lex, M . : Nitrogana se activity in the blue-green
algae Plectonema boryanum strain 594. Arch. Microbiol. 73,
2 5 0 - 2 6 0 ( 1 9 7 0 )
Van G orkom , H . J . , Donze , M . : Loca l iza t ion o f n i t rogen fixa t ion in
Anabaena N a t u r e 2 3 4 , 2 3 1 - 2 3 2 ( 19 7 1)
Van L ie re , L . , Zavenbo om, W. , M ur , L . B . : Ni t rogen as a l imi t ing
fac to r fo r the g rowth o f the b lue -g reen a lga Oscillatoria agandhi
Prog. Wat. Theo. 8 , 301-312 (1977)
Zarouk , C . : Con t r ibu t ion a l e tude d une cyanophycee in f luence de
divers facteurs physique et chimiques sur la croissance et
pho tosyn these de Spirulina marina Geitler , Ph .D . Thesis (1966)
Received September 3, 1979