Planta (Berl.) 113, 143--155 (1973) 9 by Springer-Verlag 1973
Cation Regulation in Anacystis nidulans* Maureen A. Dewar** a n d J. B a r b e r Department of Botany, Imperial College, Prince Consort Road, London SW7 2BB, U. K. Received May 8, 1973
Summary. Anacystis nidulans accumulates K + in preference to Na +. The majority of the internal K+ exchanges with ~2K by a first order process at rates of about 1.3 pequiv, em 2. sec-a in the light and 0.26 pequiv, cm 2. sec-1 in the dark. Although the K+/K + exchange was stimulated by light and inhibited by 10-4 M CCCP and 10-s M DCMU there are several indications that this cation is passively distributed in Anaeystis. Inhibition of the exchange by CCCP and DCMU occurred at concentrations greater than those required to inhibit photosynthesis and the K + fluxes were stimulated by low temperatures. Moreover, although valinomycin stimulated the exchange this compound did not induce a net K + leak. Assuming K + is passively distributed and in free solution within the cytoplasm, as indicated by osmotic studies, would imply that there is an active Na+ extrusion pump operating in this organism. As yet there are no firm conclusions about the nature of the energy source for this efflux pump. Introduction Most organisms seem to e x h i b i t some degree of s e l e c t i v i t y between s o d i u m a n d potassium. I n lower organisms t h e m e c h a n i s m of s e l e c t i v i t y seems to be either an electrogenic o u t w a r d l y d i r e c t e d s o d i u m p u m p , where K + m a y function as a counterion (Harold, B a a r d a , a n d P a v l o s o v a , 1970) or a t i g h t l y coupled exchange m e c h a n i s m which operates to accum u l a t e p o t a s s i u m w i t h i n t h e cell ( S l a y m a n a n d T a t u m , 1965 a, b; E p s t e i n a n d Schultz, 1966; Shieh a n d Barber, 1971). The energy source for this preferential r e t e n t i o n of p o t a s s i u m m a y be A T P d e r i v e d from r e s p i r a t i o n ( H a r o l d a n d B a a r d a , 1969; I t a r o ] d et al., t969) or p h o t o s y n t h e s i s (MacRobbie, 1965; Barber, 1968 a; R a v e n , 1970) or occasionally a d i r e c t link w i t h electron t r a n s p o r t has been p o s t u l a t e (Saddler, i970). T h e Na+ a n d K + relations of a wide v a r i e t y of micro-organisms h a v e been i n v e s t i g a t e d including t h e fungi, Neurospora crassa ( S l a y m a n a n d S l a y m a n , 1968) a n d y e a s t (Rothstein, 1964), t h e alga, Chlorella
Abbreviations: CCCP, earbonyl cyanide m-chlorophenylhydrazone; DCMU, 3(3,4-dichlorophenyl)-l,l-dimethylurea. ** Present address: Chemistry Department, The Polytechnic of North London, Holloway, London. N7 8DB.
144
M.A. Dewar and J. Barber
pyrenoidosa (Barber, 1968 b; Shieh and Barber, 1971) and the bacterium Streptococcus/aeealis (Harold, Baarda and Pavlosova, 1970). The bluegreen algae have so far been neglected and for this reason a study of the ionic relations of Anacystis nidulans was undertaken.
Anacystis is a procaryotic organism with its thylakoid membranes arranged in series parallel to the surface of the cell (Allen, 1968). These membranes are believed to be the sites of both respiration and photosynthesis and there is evidence that they contain electron carriers common to both processes (Carr and Hallaway, 1965; Bisalputra, Brown and Weier, 1969). This represents a simple system with respect to ion transport compared with euearyotic ceils where respiration and photosynthesis occur in the mitochondria and chloroplasts respectively and there are other membrane enclosed compartments to consider such as the nucleus and vacuoles. I t has been suggested (Lang, 1968) t h a t unicellular blue-green algae m a y be the ancestors of chloroplasts, symbiotic associations such as Nostoc sphaericum in Geosyphon pyri/orme being cited as possible intermediates in the invasionary process. If this theory is accepted then Anacystis m a y be thought of as analogous to an isolated chloroplast with the cell plasmalemma representing the intact chloroplast envelope. Although this analogy should not be carried too far, Anacystis does have a complex two photosystem mechanism of photosynthesis differing from t h a t of higher plants only in detail (Amesz and Duysens, 1962; Rurainski, Randles and Hoch, 1970). The main accessary pigment is phycocyanin rather than chlorophyll b and the cytochrome components of the electron transport chain tend to have different redox potentials; one strongly negative in keeping with its proposed dual role in photosynthesis and respiration (Holton and Myers, 1967 a, b). Having two photosystems operating within a relatively non-compartmentalised cell Anacystis m a y be compared with both isolated chloroplasts and bacteria and seems an ideal organism to study photoinduced ion movements. Material and Methods
General. Anacystis nidutans was obtained from the Biophysical Laboratory of the State University, Leiden, The Netherlands (originating from the Cambridge Culture Collection). Ceils were grown at 25~2~ either in liquid "Medium C" (Kratz and Myers, 1955) or in a slight modification of this where equimolar substitution of sodium for potassium had been made (Na+-medium). They were gassed with 4% carbon dioxide 96% air at a light intensity of 500 foot candles using incandescent lamps. During the late log phase of growth the cells were harvested by centrifugation and resuspended in fresh culture medium. Densities of cell suspensions were estimated using haematecrit tubes and the packed cell volumes obtained converted into
Cation t~egulation in Anacystis
145
units of surface area of cells and cell water. Conversion factors were obtained from measurements of cell dimensions and numbers and from measurement of water content of cell pellets. Correction for extracellular space in the pellets was obtained using [14C]inulin. It was found that a litre of packed cells had a surface area of 2.6 • 107 cm -2 and contained 710 g cell water. Internal Ion Analyses. Samples of about 100 ~l packed ceils were digested with concentrated nitric acid and analysed for Na + and K + using an EEL flame photometer calibrated with standard solutions. The overall internal osmolarity of the cell was estimated using the method of Mitchell and Moyle (1957). To obtain spheroplasts of Anacystis the cell walls were degraded with lysozyme as outlined by Vance and Ward (1969). Oxygen and Fluorescence Measurements. Oxygen uptake and evolution were measured using a Rank oxygen electrode calibrated with air saturated distilled water and sodium dithionite solution. Changes in current, which were proportional to changes in oxygen tension, were measured as the voltage across a variable resistor and recorded using a Rikadenki twin-channelled chart recorder. The cells were illuminated at an intensity of 1000foot candles using a Rank Aldis 2000 projector in conjunction with a broad band red filter. Chlorophyll fluorescence was measured using a laboratory constructed fluorimeter. The blue exciting light transmitted by Schott cut-off filters BG 18 (4 ram) and BG 38 (2 ram) was focused on the cell suspension contained in a 1 cm cuvette. The resulting red chlorophyll fluorescence was detected with an EMI $20 photomultiplier (type 9558) which was shielded from the exciting light by aBalzer interference filter transmitting at 689 nm coupled with Schott cut-off filters RG665 (6 ram) and RG 695 (4 ram). The signals were amplified and displayed on a Honeywell chart recorder (Electronik 194).
Results ~Va+ and K+ Levels. As T a b l e 1 shows Anacystis like m o s t o t h er organisms an d cells, a c c u m u l a t e s K+ r a t h e r t h a n N a +. I n t h e case of this blue-green alga t h e m e c h a n i s m of controlling t h e s e l e c t i v i t y seems to be h i g h l y efficient. I t was f o u n d v e r y difficult to replace i n t e r n a l K + w i t h N a + e v e n u n d e r conditions when e x t e r n a l K + was limiting. F o r example, growing Anacystis cells in culture m e d i u m low in K +, a t e c h n i q u e successfully e m p l o y e d w i t h y e a s t (Conway an d Moore, 1954), Neurospora ( S l a y m a n a n d S l a y m a n , 1968), and Chlorella (Shieh and Barber, 1971) to o b t a i n cells rich in N a + and low in K +, was n o t effective w i t h this o r g a n i s m (see Fig. 1). I n ~act, w h e n t h e r e was no e x t e r n a l K + t h e cells ceased to grow and t h e i n t e r n a l K + / N ~ + ratio always r e m a i n e d g reat er t h a n unity. One possible m e c h a n i s m of d i s c r i m i n a t i o n b et w een these two cations is selective bi n d in g to intracellular macromolecules (Ling, 1962). F o r example, D a m a d i a n (1969) has suggested t h a t intracellular K + - b i n d i n g proteins are responsible for t h e high i n t e r n a l levels of this cation in Eseherichia coll. I t o w e v e r , d e t e r m i n a t i o n of i n t e r n a l o s m o l a r i t y of Anacystis using t h e m e t h o d of Mitchell and Moyle (1957) g a v e values of 10 Planta (Berl.), Vol. 113
146
M.A. Dewar and J. Barber
~ . !20 _. 100 o
8() ~ 6O 49 ~ 2O o)
E o
9
0
/
i
i
10
20
30
40
External K+ (raM) 40
30 20 10 + External Na (raM)
0
Fig. 1. Effect of different external concentrations of Na + and K + on the internal concentrations of these cations. Closed triangles are K + and closed squares are Na +. The cell cultures were inoculated at the same initial density and grown for six days in various media containing different levels of Na + and K+ where the total external concentration of these two ions was 40 mM
Table 19 Internal and external Na+ and K + concentration
K+ Na +
External (raM)
Internal (raM)
Equilibrium potential (E) (mV)
20.5=t=0.3 (22) 12 9 (22)
149g-3.7 (45) 4.6• (39)
--50.0 -~25.6
a Data is presented ~ S9E.M 9 (No. of determinations) and the internal concentrations are expressed in terms of cell water. Cells were suspended in culture medium 9 b Equilibrium potentials have been calculated using the Nernst Equation E = 58 log Co/Ci mV where C o and Ci are external and internal concentrations respectively.
a b o u t 0.46 M s t r o n g l y i n d i c a t i n g t h a t t h e m a j o r i n t e r n a l c a t i o n K + is o s m o t i c a l l y a c t i v e . I f t h i s is so t h e n t h e K + / N a + s e l e c t i v i t y is l i k e l y to be c o n t r o l l e d b y t h e cell m e m b r a n e , as s e e m s t o be t h e case for m o s t a n i m a l a n d algal cells w h i c h h a v e b e e n s t u d i e d in a n y detail. F o r t h i s r e a s o n we h a v e i n c l u d e d c a l c u l a t e d e q u i l i b r i u m p o t e n t i a l s in T a b l e 1, a s s u m i n g t h a t t h e i n t e r n a l a n d e x t e r n a l a c t i v i t y coefficients are a p p r o x i m a t e l y t h e same.
Cation Regulation in Anacystis
147
0.8
0.6
o
0.4
~ o 0.2
o
0 0
015
:o
1 Time (h)
1
15
2.0
Fig. 2. Semilog plots of the uptake of K+ by Anacystis cells in the light (open circles) and dark (closed circles). A s was the final internal radioactivity at tracer equilibrium and A i was the internal radioactivity at any particular time during the uptake. The cells were maintained at 25 ~ 0.5 oC. in normal culture medium and the light was supplied by two 40 W fluorescent tubes at a light intensity of 750 ft-candles
Table 2. Average light and dark K + influxes into Anacystis Condition
K + influx (p- equivs. K+cm -e- sec-1)
Light Dark
1.30~ 0.14 (38) 0.26• (32)
a The results are given • S. E. M. (No. of determinations) and the ceils were suspended in culture medium.
Potassium Exchange Fluxes and Kinetics. Using 42K it was f o u n d t h a t all t h e cell K + was r e a d i l y e x c h a n g e a b l e w i t h e x t e r n a l K+. Moreover, semilog plots of t h e t y p e shown in Fig. 2 y i e l d e d s t r a i g h t lines showing only small i n t e r c e p t s a n d i n d i c a t i n g t h a t Anacystis like o t h e r n o n - v a c u o l a t e d organisms such as E. coli (Sehultz et al., 1962), Chlorella (Barber, 1968a, d) a n d Neurospora ( S l a y m a n a n d T a t u m , 1965a) bring a b o u t exchange of t h e m a j o r i t y of t h e i n t e r n a l K + b y a first o r d e r process. A v e r a g e values for t h e r a t e of K + / K + exchange m e a s u r e d from t r a c e r influx e x p e r i m e n t s are shown in Table 2 for b o t h light a n d d a r k t r e a t e d cells a n d correspond with values o b t a i n e d , w i t h i n e x p e r i m e n t a l error, b y m e a s u r i n g 42K effluxes into i n a c t i v e m e d i u m . I n f l u x e s were u s u a l l y e s t i m a t e d from t h e initial slopes of t h e t i m e course of 42K u p t a k e while r a t e constants, o b t a i n e d from plots of log i n t e r n a l r a d i o a c t i v i t y a g a i n s t t i m e were used for calculating effluxes (Barber, 1968d). F l a m e p h o t o m e t r y analyses of 10"
148
M.A. Dewar and J. Barber
t 140 r
a~
-
120 I
I001
140 12Q
,~
0
100 8O +cd Z
+~ 60 > 40 E
>
E
o o
~-
I
Z
Z
'0
'0
0
c
:z:s c
LO
~
40
~
20
=,
!
Nz'~i ,
Cation Regulation in Anacystis nidulans* Maureen A. Dewar** a n d J. B a r b e r Department of Botany, Imperial College, Prince Consort Road, London SW7 2BB, U. K. Received May 8, 1973
Summary. Anacystis nidulans accumulates K + in preference to Na +. The majority of the internal K+ exchanges with ~2K by a first order process at rates of about 1.3 pequiv, em 2. sec-a in the light and 0.26 pequiv, cm 2. sec-1 in the dark. Although the K+/K + exchange was stimulated by light and inhibited by 10-4 M CCCP and 10-s M DCMU there are several indications that this cation is passively distributed in Anaeystis. Inhibition of the exchange by CCCP and DCMU occurred at concentrations greater than those required to inhibit photosynthesis and the K + fluxes were stimulated by low temperatures. Moreover, although valinomycin stimulated the exchange this compound did not induce a net K + leak. Assuming K + is passively distributed and in free solution within the cytoplasm, as indicated by osmotic studies, would imply that there is an active Na+ extrusion pump operating in this organism. As yet there are no firm conclusions about the nature of the energy source for this efflux pump. Introduction Most organisms seem to e x h i b i t some degree of s e l e c t i v i t y between s o d i u m a n d potassium. I n lower organisms t h e m e c h a n i s m of s e l e c t i v i t y seems to be either an electrogenic o u t w a r d l y d i r e c t e d s o d i u m p u m p , where K + m a y function as a counterion (Harold, B a a r d a , a n d P a v l o s o v a , 1970) or a t i g h t l y coupled exchange m e c h a n i s m which operates to accum u l a t e p o t a s s i u m w i t h i n t h e cell ( S l a y m a n a n d T a t u m , 1965 a, b; E p s t e i n a n d Schultz, 1966; Shieh a n d Barber, 1971). The energy source for this preferential r e t e n t i o n of p o t a s s i u m m a y be A T P d e r i v e d from r e s p i r a t i o n ( H a r o l d a n d B a a r d a , 1969; I t a r o ] d et al., t969) or p h o t o s y n t h e s i s (MacRobbie, 1965; Barber, 1968 a; R a v e n , 1970) or occasionally a d i r e c t link w i t h electron t r a n s p o r t has been p o s t u l a t e (Saddler, i970). T h e Na+ a n d K + relations of a wide v a r i e t y of micro-organisms h a v e been i n v e s t i g a t e d including t h e fungi, Neurospora crassa ( S l a y m a n a n d S l a y m a n , 1968) a n d y e a s t (Rothstein, 1964), t h e alga, Chlorella
Abbreviations: CCCP, earbonyl cyanide m-chlorophenylhydrazone; DCMU, 3(3,4-dichlorophenyl)-l,l-dimethylurea. ** Present address: Chemistry Department, The Polytechnic of North London, Holloway, London. N7 8DB.
144
M.A. Dewar and J. Barber
pyrenoidosa (Barber, 1968 b; Shieh and Barber, 1971) and the bacterium Streptococcus/aeealis (Harold, Baarda and Pavlosova, 1970). The bluegreen algae have so far been neglected and for this reason a study of the ionic relations of Anacystis nidulans was undertaken.
Anacystis is a procaryotic organism with its thylakoid membranes arranged in series parallel to the surface of the cell (Allen, 1968). These membranes are believed to be the sites of both respiration and photosynthesis and there is evidence that they contain electron carriers common to both processes (Carr and Hallaway, 1965; Bisalputra, Brown and Weier, 1969). This represents a simple system with respect to ion transport compared with euearyotic ceils where respiration and photosynthesis occur in the mitochondria and chloroplasts respectively and there are other membrane enclosed compartments to consider such as the nucleus and vacuoles. I t has been suggested (Lang, 1968) t h a t unicellular blue-green algae m a y be the ancestors of chloroplasts, symbiotic associations such as Nostoc sphaericum in Geosyphon pyri/orme being cited as possible intermediates in the invasionary process. If this theory is accepted then Anacystis m a y be thought of as analogous to an isolated chloroplast with the cell plasmalemma representing the intact chloroplast envelope. Although this analogy should not be carried too far, Anacystis does have a complex two photosystem mechanism of photosynthesis differing from t h a t of higher plants only in detail (Amesz and Duysens, 1962; Rurainski, Randles and Hoch, 1970). The main accessary pigment is phycocyanin rather than chlorophyll b and the cytochrome components of the electron transport chain tend to have different redox potentials; one strongly negative in keeping with its proposed dual role in photosynthesis and respiration (Holton and Myers, 1967 a, b). Having two photosystems operating within a relatively non-compartmentalised cell Anacystis m a y be compared with both isolated chloroplasts and bacteria and seems an ideal organism to study photoinduced ion movements. Material and Methods
General. Anacystis nidutans was obtained from the Biophysical Laboratory of the State University, Leiden, The Netherlands (originating from the Cambridge Culture Collection). Ceils were grown at 25~2~ either in liquid "Medium C" (Kratz and Myers, 1955) or in a slight modification of this where equimolar substitution of sodium for potassium had been made (Na+-medium). They were gassed with 4% carbon dioxide 96% air at a light intensity of 500 foot candles using incandescent lamps. During the late log phase of growth the cells were harvested by centrifugation and resuspended in fresh culture medium. Densities of cell suspensions were estimated using haematecrit tubes and the packed cell volumes obtained converted into
Cation t~egulation in Anacystis
145
units of surface area of cells and cell water. Conversion factors were obtained from measurements of cell dimensions and numbers and from measurement of water content of cell pellets. Correction for extracellular space in the pellets was obtained using [14C]inulin. It was found that a litre of packed cells had a surface area of 2.6 • 107 cm -2 and contained 710 g cell water. Internal Ion Analyses. Samples of about 100 ~l packed ceils were digested with concentrated nitric acid and analysed for Na + and K + using an EEL flame photometer calibrated with standard solutions. The overall internal osmolarity of the cell was estimated using the method of Mitchell and Moyle (1957). To obtain spheroplasts of Anacystis the cell walls were degraded with lysozyme as outlined by Vance and Ward (1969). Oxygen and Fluorescence Measurements. Oxygen uptake and evolution were measured using a Rank oxygen electrode calibrated with air saturated distilled water and sodium dithionite solution. Changes in current, which were proportional to changes in oxygen tension, were measured as the voltage across a variable resistor and recorded using a Rikadenki twin-channelled chart recorder. The cells were illuminated at an intensity of 1000foot candles using a Rank Aldis 2000 projector in conjunction with a broad band red filter. Chlorophyll fluorescence was measured using a laboratory constructed fluorimeter. The blue exciting light transmitted by Schott cut-off filters BG 18 (4 ram) and BG 38 (2 ram) was focused on the cell suspension contained in a 1 cm cuvette. The resulting red chlorophyll fluorescence was detected with an EMI $20 photomultiplier (type 9558) which was shielded from the exciting light by aBalzer interference filter transmitting at 689 nm coupled with Schott cut-off filters RG665 (6 ram) and RG 695 (4 ram). The signals were amplified and displayed on a Honeywell chart recorder (Electronik 194).
Results ~Va+ and K+ Levels. As T a b l e 1 shows Anacystis like m o s t o t h er organisms an d cells, a c c u m u l a t e s K+ r a t h e r t h a n N a +. I n t h e case of this blue-green alga t h e m e c h a n i s m of controlling t h e s e l e c t i v i t y seems to be h i g h l y efficient. I t was f o u n d v e r y difficult to replace i n t e r n a l K + w i t h N a + e v e n u n d e r conditions when e x t e r n a l K + was limiting. F o r example, growing Anacystis cells in culture m e d i u m low in K +, a t e c h n i q u e successfully e m p l o y e d w i t h y e a s t (Conway an d Moore, 1954), Neurospora ( S l a y m a n a n d S l a y m a n , 1968), and Chlorella (Shieh and Barber, 1971) to o b t a i n cells rich in N a + and low in K +, was n o t effective w i t h this o r g a n i s m (see Fig. 1). I n ~act, w h e n t h e r e was no e x t e r n a l K + t h e cells ceased to grow and t h e i n t e r n a l K + / N ~ + ratio always r e m a i n e d g reat er t h a n unity. One possible m e c h a n i s m of d i s c r i m i n a t i o n b et w een these two cations is selective bi n d in g to intracellular macromolecules (Ling, 1962). F o r example, D a m a d i a n (1969) has suggested t h a t intracellular K + - b i n d i n g proteins are responsible for t h e high i n t e r n a l levels of this cation in Eseherichia coll. I t o w e v e r , d e t e r m i n a t i o n of i n t e r n a l o s m o l a r i t y of Anacystis using t h e m e t h o d of Mitchell and Moyle (1957) g a v e values of 10 Planta (Berl.), Vol. 113
146
M.A. Dewar and J. Barber
~ . !20 _. 100 o
8() ~ 6O 49 ~ 2O o)
E o
9
0
/
i
i
10
20
30
40
External K+ (raM) 40
30 20 10 + External Na (raM)
0
Fig. 1. Effect of different external concentrations of Na + and K + on the internal concentrations of these cations. Closed triangles are K + and closed squares are Na +. The cell cultures were inoculated at the same initial density and grown for six days in various media containing different levels of Na + and K+ where the total external concentration of these two ions was 40 mM
Table 19 Internal and external Na+ and K + concentration
K+ Na +
External (raM)
Internal (raM)
Equilibrium potential (E) (mV)
20.5=t=0.3 (22) 12 9 (22)
149g-3.7 (45) 4.6• (39)
--50.0 -~25.6
a Data is presented ~ S9E.M 9 (No. of determinations) and the internal concentrations are expressed in terms of cell water. Cells were suspended in culture medium 9 b Equilibrium potentials have been calculated using the Nernst Equation E = 58 log Co/Ci mV where C o and Ci are external and internal concentrations respectively.
a b o u t 0.46 M s t r o n g l y i n d i c a t i n g t h a t t h e m a j o r i n t e r n a l c a t i o n K + is o s m o t i c a l l y a c t i v e . I f t h i s is so t h e n t h e K + / N a + s e l e c t i v i t y is l i k e l y to be c o n t r o l l e d b y t h e cell m e m b r a n e , as s e e m s t o be t h e case for m o s t a n i m a l a n d algal cells w h i c h h a v e b e e n s t u d i e d in a n y detail. F o r t h i s r e a s o n we h a v e i n c l u d e d c a l c u l a t e d e q u i l i b r i u m p o t e n t i a l s in T a b l e 1, a s s u m i n g t h a t t h e i n t e r n a l a n d e x t e r n a l a c t i v i t y coefficients are a p p r o x i m a t e l y t h e same.
Cation Regulation in Anacystis
147
0.8
0.6
o
0.4
~ o 0.2
o
0 0
015
:o
1 Time (h)
1
15
2.0
Fig. 2. Semilog plots of the uptake of K+ by Anacystis cells in the light (open circles) and dark (closed circles). A s was the final internal radioactivity at tracer equilibrium and A i was the internal radioactivity at any particular time during the uptake. The cells were maintained at 25 ~ 0.5 oC. in normal culture medium and the light was supplied by two 40 W fluorescent tubes at a light intensity of 750 ft-candles
Table 2. Average light and dark K + influxes into Anacystis Condition
K + influx (p- equivs. K+cm -e- sec-1)
Light Dark
1.30~ 0.14 (38) 0.26• (32)
a The results are given • S. E. M. (No. of determinations) and the ceils were suspended in culture medium.
Potassium Exchange Fluxes and Kinetics. Using 42K it was f o u n d t h a t all t h e cell K + was r e a d i l y e x c h a n g e a b l e w i t h e x t e r n a l K+. Moreover, semilog plots of t h e t y p e shown in Fig. 2 y i e l d e d s t r a i g h t lines showing only small i n t e r c e p t s a n d i n d i c a t i n g t h a t Anacystis like o t h e r n o n - v a c u o l a t e d organisms such as E. coli (Sehultz et al., 1962), Chlorella (Barber, 1968a, d) a n d Neurospora ( S l a y m a n a n d T a t u m , 1965a) bring a b o u t exchange of t h e m a j o r i t y of t h e i n t e r n a l K + b y a first o r d e r process. A v e r a g e values for t h e r a t e of K + / K + exchange m e a s u r e d from t r a c e r influx e x p e r i m e n t s are shown in Table 2 for b o t h light a n d d a r k t r e a t e d cells a n d correspond with values o b t a i n e d , w i t h i n e x p e r i m e n t a l error, b y m e a s u r i n g 42K effluxes into i n a c t i v e m e d i u m . I n f l u x e s were u s u a l l y e s t i m a t e d from t h e initial slopes of t h e t i m e course of 42K u p t a k e while r a t e constants, o b t a i n e d from plots of log i n t e r n a l r a d i o a c t i v i t y a g a i n s t t i m e were used for calculating effluxes (Barber, 1968d). F l a m e p h o t o m e t r y analyses of 10"
148
M.A. Dewar and J. Barber
t 140 r
a~
-
120 I
I001
140 12Q
,~
0
100 8O +cd Z
+~ 60 > 40 E
>
E
o o
~-
I
Z
Z
'0
'0
0
c
:z:s c
LO
~
40
~
20
=,
!
Nz'~i ,
