Walsby, 1966; Thomas, 1970; Thomas and David,. 1972; Haystead et al., 1970; Stewart, 1974). Vegetative cells are susceptible to sonic disruption, whereas the ...
Archives of
Micrnbiology
Arch. Microbiol. 108, 35-40 (1976)
9 by Springer-Verlag 1976
Properties of Heterocysts Isolated with Colloidal Silica R. B. PETERSON and R. H. BURRIS Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53706, U.S.A.
Abstract. A method is described for the isolation of heterocysts that are virtually free of contaminating cell debris after sonication of aerobically grown Anabaena 7120. Isolated heterocysts reduced acetylene in a light-dependent process in the absence of exogenously provided ATP; heterocysts supplied with ATP and NazS204 reduced acetylene slowly in the dark but still showed a marked light activation. Nitrogenase activity was greatest in fractions containing intact heterocysts. Up to 13 ~ of the activity of the intact filaments was accounted for in the isolated heterocyst preparation. Isolated heterocysts took up 02 in a light-independent process; Oz uptake with added NADP + was enhanced by pyruvate, isocitrate and intermediates of the oxidative pentose pathway. Key words: Acetylene reduction - Nitrogenase Heterocyst - Colloidal silica - Blue-green algae Anabaena.
When certain filamentous, Nz-fixing blue-green algae are grown in the absence of a utilizable source of combined nitrogen, 5 - 7 ~ of the cells differentiate into heterocysts. Kulasooriya et al. (1972) showed that the appearance of heterocysts was accompanied by an increase in the cellular C : N ratio, and that as the first fully differentiated heterocysts appeared in the culture, nitrogenase activity was detectable. As the percentage of fully differentiated heterocysts increased, nitrogenase activity also increased. Addition of NO~-N or NH2-N to an N2-fixing culture produces a gradual loss ofnitrogenase activity and a decrease in heterocyst frequency (Stewart etal., 1968; Fogg, 1949). Such results have led investigators to speculate that the heterocyst is the major site of N2 fixation in certain filamentous blue-green algae (Fay et al., 1968; Wolk
et al., 1974; Fay and Kulasooriya, 1972; Fay and Walsby, 1966; Thomas, 1970; Thomas and David, 1972; Haystead et al., 1970; Stewart, 1974). Vegetative cells are susceptible to sonic disruption, whereas the bulk of the heterocysts remain intact. Stewart et al. (1969) and Wolk and Wojciuch (1971a) used differential sonic disruption to demonstrate partial recovery of nitrogenase activity in a heterocystenriched fraction from Anabaena cylindrica. A rigorous demonstration that the heterocyst is the sole site of fixation in aerobically grown filaments demands nearly full recovery of nitrogenase in a heterocyst preparation essentially devoid of extraneous vegetative cell material. Morgenthaler et al. (1974) have described the use of Ludox (colloidal silica) for preparing spinach chloroplasts. We have succeeded in isolating heterocysts relatively free from contaminating vegetative cell material by sonication followed by centrifugation into colloidal silica. Although nitrogenase specific activity is greater in the isolated heterocysts than in intact filaments, we have not yet recovered the full activity relative to intact filaments. Heterocysts also possess an active respiratory metabolism. Fay and Walsby (1966) reported appreciable O2 uptake by a preparation of isolated heterocysts. Leach and Cart (1969, 1970) demonstrated NADH oxidase and NADPH oxidase in several species of blue-green algae, and they coupled oxidative phosphorylation to NADPH oxidase from Anabaena variabilis. Heterocysts isolated with colloidal silica exhibit significant uptake of 02, and this is enhanced by adding certain tricarboxylic acid or pentose phosphate pathway intermediates. MATERIALS AND METHODS Algae and Conditions for Culture. A culture of Anabaena 7120 was obtained from Dr. Rosmarie Rippka of the Institut Pasteur. The alga was supplied 0.5 ~ C Q in air and the medium of Allen and
36 Arnon (1955), and was grown in a volume of 16 1 with continuous illumination from an immersed incandescent lamp. The minimum generation time was about 12 h. Algae were harvested with a small de Laval separator and were stored in liquid N2 until used.
Isolation of Heteroeysts. To isolate heterocysts, 1 - 2 g of cell paste was suspended in 20 ml of buffer [30 mM HEPES (N-2-hydroxyethylpiperazine-N'-2'-ethanesulfonic acid), 30 mM PIPES (piperazine-N-N'-bis-2-ethane sulfonic acid), 1.0 mM MgC12, pH 7.2]; this buffer was used for all operations. The suspension then was sonicated with a Branson Sonifier (Branson Ultrasonic Corp.) at setting 8 (60 watts input) for a total of 75 s in 10-15 s bursts followed by 30-60 s intervals for cooling. The suspension then was centrifuged (angle centrifuge) for 5 min at 1100 x g, the supernatant was removed, and the heterocyst-rich pellet was resuspended in 5.0-7.0 mt of buffer. Ludox LS was obtained from E. I. du Pont de Nemours and Co. as a 30~ sol. Inhibitory substances can be removed by shaking with 10 mg of the Na + form of Chelex 100/ml and 5 gM Na2EDTA followed by filtration through Whatman No. 2 paper. Prior to use the Ludox was diluted with buffer to 22~o (1.04 specific gravity at 23.5~ A 2.0 ml aliquot of the resuspended 1100 x g pellet was carefully layered on top of 10 ml of 22 ~ Ludox contained in a 50 ml centrifuge tube, and the tube was centrifuged (swinging bucket centrifuge used with Ludox) at 15 x g for 5 min at 10~ The upper phase was enriched in cell debris, whereas the heterocysts moved into the Ludox (lower phase). The upper phase was removed carefully with a hypodermic syringe. The heterocysts were pelleted by eentrifugation of the lower phase at 700 x g for 5 min, the supernatant was removed, the heterocysts were resuspended in 2.0ml of buffer and the centrifugation into 22~o Ludox was repeated. When nitrogenase activity was to be assayed, all operations were performed anaerobically under H2, and best results were obtained when 1.0 mM NazSzO4 was present in the buffer to scavenge Oz. Acetylene Reduction Assays. Assays were performed in triplicate in 6 ml serum bottles each containing 1 ml of liquid. The gas phase consisted of 17 ~ C2Hz and 83 ~o Hz and the temperature was 30~C. When addition of an ATP-generating system is indicated, it was composed of 5.0 mM ATP, 7.0 mM MgC12, 20 mM creatine phosphate and 0.05 mg creatine kinase. Heterocyst-containing fractions were incubated for 20 rain and then were inactivated by injection of 1.0 ml of 7.5 N H2SO~. A light intensity of 700 footcandles was provided by tungsten-filament ftoodlamps. Ethylene determinations were done on an Aerograph Model 600-D gas chromatograph equipped with a flame ionization detector and a 1.2 m long by 2 mm diameter column of Porapak R operated at 50~C ; N 2 w a s the carrier gas. Ethylene standards were prepared from ethylene (C.P.) purchased from Matheson Gas Products.
Cell-Free Extracts. The 1100 • g supernatant obtained from sonicated preparations was centrifuged at 40000xg for 1 h, and the supernatant was concentrated with an Amicon Diaflo ultrafiltration device with a PM30 filter under 60 psi of argon. The extract was incubated for 30 min in the presence of acetylene, an ATPgenerating system and 3.0 mM N%SzO4; the gas phase then was assayed for ethylene as described.
Uptake of Oxygen. Change in oxygen concentration was monitored in suspensions of isolated heterocysts with an oxygen electrode purchased from Rank Brothers, Bottisham, Cambridge, England. Chemicals. Creatine phosphate was purchased from Pierce Chemical Co,, Rockford, Illinois; malic acid from Aldrich Chemical Co., Milwaukee, Wisconsin; and succinic acid from J. T. Baker Chemical Co,, Phillipsburg, New Jersey. All other chemicals were furnished by Sigma Chemical Co., St. Louis, Missouri.
Arch. Microbiol., Vol. 108 (1976) 0
I
I
I
t2
I
o ~
:D"
x
4oo
g ~~
r~
2
300
X
200
100
I 0
20
I 40
I 60
I 80
I 1 100
Seconds of sonication
Fig. t.
Effect of increasing sonication time on heterocysts of
Anabaena 7120.4.0 g of algal paste was suspended in a final volume of 20 ml with buffer. After each 15 s sonication, 0.1 ml of the suspension was removed, diluted, and the number of "intact, mature" heterocysts was counted (see text). Chlorophyll a was extracted from the supernatant with 80~ acetone after removal of the heterocysts by centrifugation. Chlorophyll a was determined spectrophotometrically at 665 rim; the extinction coefficient given by Vernon (1960) was used. Each point is a mean of duplicate determinations
Assayfor Protein. Protein was determined by the method of Lowry et al. (1951), and bovine serum albumin was used as the standard.
RESULTS
Isolation and Nitrogenase Activity T o assess t h e effect o f s o n i c a t i o n o n h e t e r o c y s t s , a suspension of algae was subjected to sonic oscillation f o r a m a x i m u m o f 2 m i n ; small, a l i q u o t s w e r e r e m o v e d at i n t e r v a l s a n d t h e n u m b e r o f " m a t u r e , i n t a c t " h e t e r o c y s t s w a s d e t e r m i n e d (Fig. 1). H e t e r o c y s t s w e r e s c o r e d as " m a t u r e , i n t a c t " if (a) t h e y h a d t h i c k w a l l s a n d p o l a r b o d i e s , (b) t h e r e w a s n o a p p a r e n t m e c h a n i cal d a m a g e to t h e cell e n v e l o p e , a n d (c) little o r n o loss o f c y t o p l a s m i c c o n t e n t s w a s a p p a r e n t . V i r t u a l l y all o f t h e v e g e t a t i v e cells w e r e d e s t r o y e d a f t e r 75 s, b u t c h l o r o p h y l l a still w a s b e i n g s o l u b i l i z e d . Photomicrographs o f s o n i c a t e d Anabaena 7120 b e f o r e a n d a f t e r t w o c e n t r i f u g a t i o n s i n t o 22 ~o L u d o x a r e p r e s e n t e d (Fig. 2 A a n d B). T h e s o n i c a t i o n a p p e a r e d t o d i s r u p t t h e v e g e t a t i v e cells c o m p l e t e l y l e a v i n g o n l y free, d e t a c h e d h e t e r o c y s t s p l u s cell debris. T h e 1100 • g p e l l e t c o n t a i n e d c o n s i d e r a b l e cell w a l l m a t e r i a l , m e m brane fragments, and mucilage. However, two centrifugations into 22~ L u d o x effected a s u b s t a n t i a l p u r i f i c a t i o n . V i r t u a l l y all o f t h e cells r e c o v e r e d w e r e heterocysts and were identifiable by their refractile p o l a r b o d i e s . G e n e r a l l y , 50 ~ o f t h e c h l o r o p h y l l a in t h e 1 1 0 0 x g p e l l e t w a s r e c o v e r e d in t h e i s o l a t e d h e t e r o c y s t f r a c t i o n ( T a b l e 1). Sonicated preparations reduced acetylene readily u n d e r H2 ( T a b l e 1). A c t i v i t y w a s e n h a n c e d b y a d d i t i o n
O I /3
R. B. Peterson and R. H. Burris: Isolation of Heterocysts
37
Fig. 2. (A) Photomicrograph of resuspended 1i00 x g pellet shows detached heterocysts with associated cellular debris. The average length of the heterocyst is approximately 5 gin. (B) Photomicrograph of l l 0 0 x g pellet after two centrifugations into 22~ Ludox shows removal of most of the cellular debris. The volumes of the two suspensions were adjusted to give approximately the same heterocyst concentrations
Table 1. Nitrogenase activity in fractions of sonicated Anabaena 7120. See the section on "Materials and Methods" for assay conditions
Table 2. Effect of 700 ft-c light and substrates on nitrogenase activity in isolated heterocysts of Anabaena7120. Note that the buffer already contains 1.0 mM NazS204
Fractions and conditions
Treatment
Total ~tg chl. a
nmolesC2H4 nmoles C2H4 ktg chl. a x 20 rain
Intact filaments 1100 x g pellet 154 Cell debris 39.4 Isolated heterocysts 70.2 No additions + 5.0 mM ATP and its generating system
mg N • 20 rain 1535
21.2 a 1.79" 23.7
2110
36.8
3260
" Assayed in presence of 5.0 mM ATP and its generating system plus 1.0 mM Na2S204.
o f a n A T P - g e n e r a t i n g system. L o w activity in the cell d e b r i s f r a c t i o n likely can be a t t r i b u t e d to heterocysts t r a p p e d at the L u d o x interface a n d r e m o v e d with the u p p e r phase. Specific activities were highest in the i s o l a t e d b e t e r o c y s t fraction, a n d r e d u c t i o n o f acetylene was linear for at least 20 min. T h e cells used for the e x p e r i m e n t o f T a b l e I were n o t f r o z e n b u t were h a r v e s t e d i m m e d i a t e l y b e f o r e use; lower recoveries o f activity were o b t a i n e d f r o m frozen cells. A f t e r the I100 x g s u p e r n a t a n t ( o b t a i n e d after 75 s o f s o n i c a t i o n ) was c e n t r i f u g e d at 4 0 0 0 0 x g for I h, it h a d d e t e c t a b l e n i t r o g e n a s e activity in the supern a t a n t b u t n o t in the pellet. This soluble f r a c t i o n
No additions, light No additions, dark ATP gen. system, light + 1.0 mM added Na2S204 ATP gen. system, dark, + 1.0 mM Na2S204
nmoles C 2 H 4
nmoles Cfl-I4
~tgchl. a • 20 rain
mg N • 20 min
2.67 0.18 14.7
435 29.5 2390
5.95
970
s h o w e d a s t r o n g d i l u t i o n effect a n d h a d a m a x i m u m specific activity o f 0.55 n m o l e C2H4/(mg p r o t e i n x min) at a p r o t e i n c o n c e n t r a t i o n o f 5 - 1 0 m g / m l [this is a p p r o x i m a t e l y 200 n m o l e s C2Hg/(mg N x h)]. T a b l e 2 indicates the effect o f light o n acetylene r e d u c t i o n b y i s o l a t e d h e t e r o c y s t s with a n d w i t h o u t a d d e d A T P a n d N a z S 2 0 4. Effects o f c o n c e n t r a t i o n o f A T P a n d N a 2 S 2 0 4 were i n v e s t i g a t e d ; no f u r t h e r increases in activity were n o t e d a b o v e 1.0 m M A T P a n d 2.0 m M Na2S204.
Uptake of Oxygen H e t e r o c y s t s i s o l a t e d with c o l l o i d a l silica exhibit cons i d e r a b l e r e s p i r a t o r y activity. T a b l e 3 shows t h a t h e t e r o c y s t s possess N A D P H - o x i d a s e activity, a n d O2
38
Arch. Microbiol., Vol. 108 (1976)
Table 3. Effect of various substrates plus N A D P + on 02 uptake by isolated heterocysts - Indicates no addition (endogenous rate) Substrate
gl 02 uptake/ (rag N x h)"
Cofactor
4.6 m M glucose 6-phosphate 4.2 m M glucose 6-phosphate
12.1 69.5 250 g M N A D P + 135.0
-: -4.6 m M DL-Na isocitrate
272 p.M N A D P + 250 g M N A D P +
2.7 m M fructose 6-phosphate 2.5 m M fructose 6-phosphate
68.2 109.0 250 g M N A D P + 195.0
4.6 m M 6-phosphogluconate 4.2 m M 6-phosphogluconate
66.7 121.0 250 g M N A D P + 177.0
-
-
-
-
71.2 106.0
-
4.2 m M D-glucose, 4.2 m M ATP, 5.8 m M MgC12 3.8 m M D-glucose, 3.8 m M ATP, 5.4 m M MgC12
33.5 39.6 79.0
-
231 g M N A D P + 154.0
273 ~tM N A D H
--
13.3 39.5
273 g M N A D P H
--
49.8 95.3
-
-
Variations in endogenous rates result from the use of different batches 'of cells in the various experiments.
a
Table 4. C o m p a r i s o n of 02 uptake by intact filaments assayed in the dark with heterocysts isolated and assayed in the presence of 1.0 m M dithiothreitol. For conditions see the section on "Materials and M e t h o d s " Reaction conditions
gl Q / ( m g N x h)
Intact filaments (dark) Isolated heterocysts: N o additions + 273 IxM N A D P + + 417 g M pyruvate 250 g M N A D P §
280 40.1 186 295
uptake is enhanced when intermediates of the oxidative pentose phosphate pathway plus NADP § are added to the reaction mixture. N A D H addition resulted in a modest enhancement of Oz uptake, but NAD § was not very effective as a cofactor in oxidation of pyruvate, glucose 6-phosphate or isocitrate. Added succinate and malate plus NADP § did not influence Oa uptake and white light (2400 ft c) had little effect on 02 uptake either in the presence or absence of an
oxidizable substrate such as glucose-6-phosphate plus NADP § Pyruvate supported an increase in respiratory activity in the presence of NADP + (Table 4). Heterocysts used in this experiment were isolated in the presence of 1.0 mM dithiothreitol, and this appeared to increase rates of 02 uptake. For comparison, the table records the rate of dark respiration for intact filaments of Anabaena 7120 growing at its maximum rate. DISCUSSION Nitrogenase activity from a blue-green alga in a particulate fraction rich in heterocysts first was reported by Stewart et al. (1969); however, the rates were quite low. Wolk and Wojciuch (1971 a) recovered up to 20 of the activity of the intact, aerobically grown algal suspension after virtually complete sonic disruption of the vegetative cells. Activity was associated with the pellet sedimented at 8000 x g for 5 min. Aerobically grown Anabaena 7120 has a heterocyst frequency of about 6.2~. The maximum specific activity obtained in our experiments was 3260 nmoles CzH4/(mg total N x 20 min), whereas the activity in the intact filaments was 1535. About 13~o of the activity of the intact filaments was recovered in the isolated heterocyst fraction; in other experiments there was a 1 0 - 1 2 ~ recovery. Although these results are not definitive for the problem of localization of nitrogenase in aerobically-grown, heterocystous, bluegreen algae, they indicate clearly that the heterocysts have a higher specific activity for nitrogenase than do the vegetative cells. The question of localization of nitrogenase in filamentous heterocystous blue-green algae is complex and has been approached in a variety of ways. Early reports indicated that the supernatant obtained after disruption of vegetative cells by sonication and complete removal of the heterocysts by centrifugation was devoid of nitrogenase activity (Wolk and Wojciuch, 1971a; Stewart et al., 1969). Although this might be interpreted to mean that vegetative cells lack nitrogenase, other reports (including this one) indicate that some nitrogenase is present in the soluble fraction (Wolk and Wojciuch, 1971b; Haystead et al., 1970). Smith and Evans (1970, 1971) indicated that most nitrogenase activity detected after disruption of anaerobically grown Anabaena cylindrica was associated with a 40000xg supernatant. Fay and Lang (1971) have found that disruption of heterocysts either by sonication or with a French press ruptures the terminal pores, breaks the plasmalemma and causes some loss of cytoplasmic material from the heterocysts. It is difficult to distinguish between soluble nitrogenase originating from the vegetative cells and from damaged
R. B. Peterson and R. H. Burris : Isolation of Heterocysts
heterocysts. In this study, 2 5 - 30 ~o of the heterocysts after 75 s of sonication were destroyed or showed some damage (Fig. 1). The relatively low specific activity of our soluble fraction may reflect milder than usual disruption conditions and limited solubilization ofnitrogenase from heterocysts (Haystead et al., 1970; Smith and Evans, 1970, 1971). Heterocysts appear to have photosystem I and the associated capacity to photophosphorylate and photoreduce (Donze et al., 1972; Wolk and Wojciuch, 1971 a). This could account for enhancement of nitrogenase activity by light in the absence of added cofactors. However, light activation persists when an ATP-generating system and NazS204 are provided. These results suggest that isolated heterocysts do retain photophosphorylation activity. However, the fact that an exogenous ATP-generating system enhances activity in the light indicates the endogenous system is incapable of maintaining maximal nitrogenase activity. The low activity in the dark in the presence of the ATP-generating system possibly stems from an impermeability of the heterocyst to the substrate. 2.0 mM N a 2 S 2 0 4 and 1.0 mM ATP nearly saturate the system for acetylene reduction. Winkenbach and Wolk (1973) have reported high levels of enzymes of the oxidative pentose pathway in isolated heterocysts of Anabaena cylindrica. Wolk (1968) has shown that carbon recently fixed in the vegetative cells, migrates down the filament and into the heterocyst. Fay and Walsby (1966) reported substantial 02 uptake in isolated heteroeysts from Anabaena cylindrica. We found that key intermediates of the oxidative pentose pathway, such as glucose 6-phosphate, fructose 6-phosphate and 6-phosphogluconate, markedly enhance 02 uptake in isolated heterocysts. Among Krebs cycle intermediates tested, only isocitrate enhanced 02 uptake. Much evidence suggests that heterocysts lack an O2-evolving photosystem II (Thomas, 1970; Donze et al., 1972). We find no 02 evolution from heterocysts even under high light intensity. Nor does increasing light intensity increase the rate of O2 uptake in the presence of a readily utilizable substrate such as glucose 6-phosphate. The highest rates of O2 uptake in heterocysts occurred when 1.0 mM dithiothreitol was present during their isolation and assay (Table 4). A substantial enhancement in O2 uptake was observed with added NADP + or pyruvate only when heterocysts were isolated in the presence of dithiothreitol. Oxidation of pyruvate or pentose phosphate intermediates may provide reductant for nitrogenase while ATP is generated via photophosphorylation or oxidative phosphorylation (Bothe and Falkenberg, 1973; Codd et al., 1974; Smith et al., 1971).
39
Our results support the hypothesis that much of the nitrogenase is localized in the heterocysts of bluegreen algae. We hope that the simple method described for recovering metabolically competent heterocysts relatively free from contaminating cell debris will aid further investigations of the role of heterocysts in N2 fixation. Acknowledgements. This investigation was supported by the College of Agricultural and Life Sciences, University of Wisconsin-Madison, by Public Health Service Grant AI-00848 from the National Institute of Allergy and Infectious Diseases, by National Science Foundation Grant GB-21422, and by Public Health Service Training Grant no. GM-00236BCH from the National Institute of General Medical Sciences.
REFERENCES Allen, M. B., Arnon, D. I. : Studies on N-fixing blue-green algae. I. Growth and N-fixation by Anabaena cylindrica Lemm. Plant Physiol. 30, 366- 372 (1955) Bothe, H., Falkenberg, B. : Demonstration and possible role of a ferredoxin-dependent pyruvate decarboxylation in the nitrogenfixing blue-green alga Anabaena cylindriea. Plant sci. Lett. 1, 151 - 156 (1973) Codd, G. A., Rowell, P , Stewart, W. D. P. : Pyruvate and N2ase activity in cell free extracts of the blue-green alga Anabaena cylindrica. Biochem. biophys. Res. Commun. 61,424-431 (1974) Donze, M., Haveman, J., Schiereck, P.: Absence of PSII in heterocysts of the blue-green alga Anabaena. Biochim. biophys. Acta (Amst.) 256, 157-161 (1972) Fay, P., Kulasooriya, S. A. : Tetrazolium reduction and nitrogenase activity in heterocystous blue-green algae. Arch. Mikrobiol. 87, 341 - 352 (1972) Fay, P., Lang, N. J. : The heterocysts of blue-green algae. I. Ultrastructural integrity after isolation. Proc. roy. Soc. B 178, 185-192 (1971) Fay, P., Stewart, W. D. P., Walsby, A. E., Fogg, G. E.: Is the heterocyst the site of nitrogen fixation in blue-green algae? Nature (Lond.) 220, 810-812 (1968) Fay, P., Walsby, A. E. : Metabolic activities of isolated heterocysts of the blue-green alga Anabaena eylindrica. Nature (Lond.) 209, 94-95 (1966) Fogg, G. E.: Growth and heterocyst production in Anabaena cylindriea Lemm. II. In relation to carbon and nitrogen metabolism. Ann. Bot. 13, 241-259 (1949) Haystead, A., Robinson, R., Stewart, W. D. P. : Nzase activity in extracts of heterocystous and non-heterocystous blue-green algae. Arch. Mikrobiol. 74, 235-243 (1970) Kulasooriya, S. A., Lang, N. J., Fay, P. : The heterocysts of bluegreen algae. III. Differentiation and nitrogenase activity. Proc. roy. Soc. B 181, 199-209 (1972) Leach, C. K., Cart, N. G. : Electron transport and oxidative phosphorylation in the blue-green alga Anabaena variabilis. J. gen. Microbiol. 64, 5 5 - 7 0 (1970) Lowry, O. H., Rosebrough, N. J., Farr, A., Randall, R. J.: Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275 (1951) Morgenthaler, J. J., Price, C. A., Robinson, J. M., Gibbs, M.: Photosynthetic activity of spinach chloroplasts after isopycnie centrifugation in gradients of silica. Plant Physiol. 54, 532- 534 (1974) Smith, R. V., Evans, M. C. W. : Soluble N2ase from vegetative cells of the blue-green alga Anabaena cylindrica. Nature (Lond.) 225, 1253-- 1254 (1970)
40 Smith, R. V., Evans, M. C. W. : Nzase activity in cell-free extracts of the blue-green alga Anabaena cylindrica. J. Bact. 105, 913 - 914 (1971) Smith, R. V., Noy, R. J., Evans, M. C. W. : Physiological electron donor systems to the Nzase of the blue-green alga Anabaena cylindrica. Biochim. biophys. Acta (Amst.) 253, 104-109 (1971) Stewart, W. D. P. : Blue-green algae. In: The biology of nitrogen fixation (A. Quispel, ed.), pp. 202-237. Amsterdam-Oxford: North-Holland 1974 Stewart, W. D. P., Fitzgerald, G. P., Burris, R. H. : Acetylene reduction by nitrogen-fixing blue-green algae. Arch. Mikrobiol. 62, 336-348 (1968) Stewart, W. D. P., Haystead, A., Pearson, H. W. : Nitrogenase activity in heteroeysts of blue-green algae. Nature (Lond.) 224, 226- 228 (1969) Thomas, J. : Absence of the pigments of photosystem II of photosynthesis in heterocysts of a blue-green alga. Nature (Lond.) 228, 181 - 183 (1970) Thomas, J., David, K. A. V. : Site of nitrogenase activity in the bluegreen alga Anabaena species L-31. Nature (New Biol.) 238, 219-221 (1972)
Arch. Microbiol., Vol. 108 (1976) Vernon, L. P. : Spectrophotometric determination of chlorophylls and pheophytins in plant extracts. Analyt. Chem. 32, 1144-1150 (1960) Winkenbach, F., Wolk, C. P. : Activities of enzymes of the oxidative and the reductive pentose phosphate pathways in heterocysts of a blue-green alga. Plant Physiol. 52, 480-483 (1973) Wolk, C. P. : Movement of carbon from vegetative cells to heterocysts in Anabaena cylindriea. J. Bact. 96, 2138-2143 (1968) Wolk, C. P., Austin, S. M., Bortins, J., Galonsky, A. : Autoradiographic localization of 13N after fixation of 13N-labelled nitrogen gas by a heterocyst forming blue-green alga. J. Cell Biol. 61, 440-453 (1974) Wolk, C. P., Wojciuch, E. : Photoreduction of acetylene by heterocysts. Planta (Berl.) 97, 126-134 (1971 a) Wolk, C. P., Wojciuch, E. : Biphasic time course of solubilization of nitrogenase during cavitation of aerobically grown Anabaena cylindrica. J. Phycol. 7, 339 - 344 (1971 b)
Received November 4, 1975
