- Email: [email protected]
Light-dark transients in oxygen exchange of blue-green algae
436
VOL. 24 (1957)
P R E L I M I N A R Y NOTJ¢:S
Light-clark transients in oxygen exchange of blue-green algae The dark-light transition curves (DLTC) of p h o t o s y n t h e t i c gas exchange (induction phenomena) have been investigated by several authors],2, a as well as the related induction p h e n o m e n a in chlorophyll fluorescenceI, 4. On the other hand, the light-dark transition curves (LDTC) hav~ received m u c h less attention. During investigations b o t h of the general course and of the fine details of oxygen exchange in assimilating organisms5 some remarkable characteristics of the LDTC were observed. The m e t h o d of BLINKS AND ,~KOW6, 7, with some modifications, was used for registration ot the oxygen exchange rates. Some of the details have been given previously 5, and a f u r t h e r description will be published later. Blue-green t h e r m a l algae (Oscillatoria, Symploca, Mastigocladus), cultivated in a t h e r m o s t a t on mineral agar media, were used as the m o s t convenient experimental material. Nearly identical results were also obtained with Chlorella and other algae. I n Fig. i some representative records of p h o t o s y n t h e t i c oxygen exchange are reproduced. T h e y were chosen to d e m o n s t r a t e the following salient points: i. On d a r k e n i n g - - u n d e r aerobic c o n d i t i o n s - - t h e oxygen line moves from the light steady s t a t e to the dark one only gradually, often taking several minutes. This is not due to the hysteresis of the method. The LDTC curves are of various shapes, b u t t h e y nearly always have definite characteristic features, e.g., two inflections or minima, which are m o s t clearly seen on records 24ob, 474 and 996 (Fig. i), also on R 478 and 844 (in the latter case with a third m i n i m u m ) . The perfectly exponential course of LDTC, as seen in R 643b, was observed only exceptionally, with low light intensities, and could not be reproduced at will. RmSt
R240 l
2' 'tO
- -
~
2,6. t0
Rm
R ~,74.
2- t0 z
m
i
m
2' t0 z
R ~78
mm
t. t0~'
~o0
f
m
2.10~
~
.
~
i
11928 a
I. I0~
Fig. i. Records of o x y g e n exchange. (For m e t h o d , see previous paperS.) On the lower m a r g i n s of the records, d a r k strips are found representing intervals of darkness and figures indicating light intensity in lux. O t h e r m a r k s as in previous paper. R 643b - Symploca, 25 ° C, p H 7, air, sensitivity s 2. 7. lO-2; R 24ob - Oscillatoria, 25 ° C, p H 5, a i r + i % CO~, sensitivity 6.75. lO-2; R 996 - Oscillatoria, 5 ° C, p H 7, air, sensitivity 2. 7. io-2; R 474 - Symploca, 15 ° C, p H 7, air + 1% CO s, sensitivity 6.75. lO-2; R 478 - Symploca, 15 ° C, p H 7, air, sensitivity 4.o5. lO-2; R 844 - Oscillatoria, 35 ° C, p H 7, air, sensitivity 9-5" lO-1; R 50o - S y m p l o c a , 15 ° C, p H 7, nitrogen + i % CO2, sensitivity 4.o5. io-Z; R 928a - Oscillatoria, IO ° C, p H 7, nitrogen + i % COs, sensit i v i t y 4.o5. i o -~. 2. U n d e r anaerobic conditions the oxygen line on darkening follows a m u c h simpler course (Fig. i, R 5oo and 928). No irregularities are observed on the falling leg of the oxygen production curve and it a t t a i n s zero within a relatively short time. 3. The DLTC a n d L D T C on aerobic records are often found to be symmetrical. This s y m m e t r y is sometimes nearly perfect w i t h curves obtained at low or m e d i u m intensities of illumination (Fig. i, records 643b, 24ob, 474, 996; w i t h curves of the R 643b t y p e the p a r a m e t e r s of the exponential functions for the ascending and descending leg are equal). Striking s y m m e t r y is occasionally observed even on curves t a k e n at high light intensities (R 844), and b y comparative
VOL. 24 (1957)
PRELIMINARY NOTES
437
analysis, analogous sections m a y be found on DLTC and LDTC in practically all records under aerobic conditions (even, for example, on R 478). U n d e r nitrogen, the LDTC has a character oi a rapid m o n o p h a s i c decay and it is never found to be s y m m e t r i c a l with the DLTC. I n a previous communication, the a u t h o r has suggested t h a t the shape of the aerobic LDTC records results from the (pseudo)monomolecular liberation of oxygen from an intermediary p r o d u c t p r e s e n t at the m o m e n t the light is s h u t off. To explain the two inflections or minima c o m m o n l y observed on the LDTC, two o x y g e n - c o n s u m i n g processes were a s s u m e d to take place at the beginning of the d a r k period. This t y p e of explanation, however, seems r a t h e r inadequate, if the striking s y m m e t r y of the LDTC and DLTC is taken into account. I t would be strange if b y mere chance similar time c o n s t a n t s were to govern b o t h the s t a r t of the complicated reaction chain in p h o t o s y n t h e s i s and the single reaction of oxygen liberation from an intermediary product. I t seems more plausible that, in some w a y or other, the LDTC reflect changes in reservoir sizes of the same intermediates and variations in rates of the same reversible reactions, which are reflected by the DLTC w h e n the opposite transition from dark to light takes place. Before a reasonable i n t e r p r e t a t i o n of the p h e n o m e n a observed can be a t t e m p t e d , more data m u s t be collected. I n particular, parallel d e t e r m i n a t i o n s of oxygen exchange b y a dropping m e r c u r y electrode and b y the m e t h o d of BLINKS AND SKOW are planned. I t is the disadvantage of the latter m e t h o d t h a t it fails to give a clear discrimination between net oxygen production a n d uptake. Consequently, the p o i n t of basic fnterest c a n n o t be established, i.e., which sections of the records fall above the c o m p e n s a t i o n point and which are below it. To k n o w this is obviously of great i m p o r t a n c e for an a d e q u a t e i n t e r p r e t a t i o n of the LDTC and for the correct evaluation of interactions existing between p h o t o s y n t h e s i s and respiration. T h a t such interrelations influence the shape of aerobic LDTC records is suggested b y the striking difference between aerobic and anaerobic light-dark transition curves. IVAN ~ETLfK*
Institute o~ Plant Physiology, Charles U~iversity, Prague (Czechoslovakia) 1 j . FRANCK, Ann. Rev. Plant Physiol., 2 (1951) 53; Arch. Biochem. Biophys., 45 (1953) 19o; H. GAFFRON AND J. ROSENBERG Naturwissenscha/ten, 42 (1955) 53; E. C. WASSINK AND C. J. P. SPRUIT, VIIIe Congr~s Intern. de Bot., Rappts. et Communs., Sec. i i et I2, p. 3. 2 K. A. CLENDENNING AND F. T. HAXO, Can. J. Botany, 34 (1956) 214. s C. P. WHITTINGHAM, J. Exptl. Botany, 7 (1956) 273. 4 E. C. WASSINK, Advances in Enzymol., i i (195o) 91. s I. ~ETLfK, Rozpravy ~SAV, ~ada MPV, 64 (1954) segit 3. e L. R. BLINKS AND R. K. SKOW, Proc. Natl. Acad. Sci. U.S., 24 (1938) 413 . 7 F. T. HAXO AND L. R. BLINKS, J. Gen. Physiol., 33 (195 o) 389 • 8 I. ~ETLfK, Biochim. Biophys. Acta, 24 (1957) 434. Received J a n u a r y 2ist, 1957 • P r e s e n t address: Botanick~ zAhrada SAV, K o m e n s k 6 h o 67, Kogice (Czechoslovakia).
Inhibition of citrate oxidation by glyoxylate in rat liver homogenates The m e c h a n i s m of the inhibition produced b y glyoxylate on a respiring tissue suspension, first s h o w n b y KLEINZELLER 1, has been investigated b y s t u d y i n g the action of v e r y small a m o u n t s of glyoxylate on the oxidative b e h a v i o u r of the m o s t i m p o r t a n t 16 metabolites related to the tricarboxylic acid cycle. F o r this p u r p o s e o.ooi M glyoxylate was added to a rat liver h o m o g e n a t e incubated in the presence of citrate, cis-aconitate, isocitrate, a-ketoglutarate, succinate, fumarate, malate, oxalu acetate and p y r u v a t e , each at a final concentration of o.oo2M. ~s The final v o l u m e of the incubation m i x t u r e s was always 4.o ml; the concentration of the h o m o g e n a t e was I : IO (prepared in K r e b s ' phospho-saline m e d i u m free of Ca ++ and with the addition of o . o 2 5 M Mg ++ and O . o l M F - ) . The incubation was for 60 min at 38°C in W a r b u r g vessels of a b o u t 15 ml capacity; 02 gas phase; p H 7.4. A n o t h e r experiment was carried out u n d e r the same U r n o l e s oxalacetate conditions with o.oo1 M glyoxylate and differing concentrations Fig. I. R a t liver h o m o g e n a t e of oxalacetate as s h o w n in Fig. I. i : io; glyoxylate o.ooi M ; I n all the e x p e r i m e n t s the oxygen u p t a k e was followed manoN a F o.oi M. metrically, and at the end of the incubation citrate was determined _
29
7
VOL. 24 (1957)
P R E L I M I N A R Y NOTJ¢:S
Light-clark transients in oxygen exchange of blue-green algae The dark-light transition curves (DLTC) of p h o t o s y n t h e t i c gas exchange (induction phenomena) have been investigated by several authors],2, a as well as the related induction p h e n o m e n a in chlorophyll fluorescenceI, 4. On the other hand, the light-dark transition curves (LDTC) hav~ received m u c h less attention. During investigations b o t h of the general course and of the fine details of oxygen exchange in assimilating organisms5 some remarkable characteristics of the LDTC were observed. The m e t h o d of BLINKS AND ,~KOW6, 7, with some modifications, was used for registration ot the oxygen exchange rates. Some of the details have been given previously 5, and a f u r t h e r description will be published later. Blue-green t h e r m a l algae (Oscillatoria, Symploca, Mastigocladus), cultivated in a t h e r m o s t a t on mineral agar media, were used as the m o s t convenient experimental material. Nearly identical results were also obtained with Chlorella and other algae. I n Fig. i some representative records of p h o t o s y n t h e t i c oxygen exchange are reproduced. T h e y were chosen to d e m o n s t r a t e the following salient points: i. On d a r k e n i n g - - u n d e r aerobic c o n d i t i o n s - - t h e oxygen line moves from the light steady s t a t e to the dark one only gradually, often taking several minutes. This is not due to the hysteresis of the method. The LDTC curves are of various shapes, b u t t h e y nearly always have definite characteristic features, e.g., two inflections or minima, which are m o s t clearly seen on records 24ob, 474 and 996 (Fig. i), also on R 478 and 844 (in the latter case with a third m i n i m u m ) . The perfectly exponential course of LDTC, as seen in R 643b, was observed only exceptionally, with low light intensities, and could not be reproduced at will. RmSt
R240 l
2' 'tO
- -
~
2,6. t0
Rm
R ~,74.
2- t0 z
m
i
m
2' t0 z
R ~78
mm
t. t0~'
~o0
f
m
2.10~
~
.
~
i
11928 a
I. I0~
Fig. i. Records of o x y g e n exchange. (For m e t h o d , see previous paperS.) On the lower m a r g i n s of the records, d a r k strips are found representing intervals of darkness and figures indicating light intensity in lux. O t h e r m a r k s as in previous paper. R 643b - Symploca, 25 ° C, p H 7, air, sensitivity s 2. 7. lO-2; R 24ob - Oscillatoria, 25 ° C, p H 5, a i r + i % CO~, sensitivity 6.75. lO-2; R 996 - Oscillatoria, 5 ° C, p H 7, air, sensitivity 2. 7. io-2; R 474 - Symploca, 15 ° C, p H 7, air + 1% CO s, sensitivity 6.75. lO-2; R 478 - Symploca, 15 ° C, p H 7, air, sensitivity 4.o5. lO-2; R 844 - Oscillatoria, 35 ° C, p H 7, air, sensitivity 9-5" lO-1; R 50o - S y m p l o c a , 15 ° C, p H 7, nitrogen + i % CO2, sensitivity 4.o5. io-Z; R 928a - Oscillatoria, IO ° C, p H 7, nitrogen + i % COs, sensit i v i t y 4.o5. i o -~. 2. U n d e r anaerobic conditions the oxygen line on darkening follows a m u c h simpler course (Fig. i, R 5oo and 928). No irregularities are observed on the falling leg of the oxygen production curve and it a t t a i n s zero within a relatively short time. 3. The DLTC a n d L D T C on aerobic records are often found to be symmetrical. This s y m m e t r y is sometimes nearly perfect w i t h curves obtained at low or m e d i u m intensities of illumination (Fig. i, records 643b, 24ob, 474, 996; w i t h curves of the R 643b t y p e the p a r a m e t e r s of the exponential functions for the ascending and descending leg are equal). Striking s y m m e t r y is occasionally observed even on curves t a k e n at high light intensities (R 844), and b y comparative
VOL. 24 (1957)
PRELIMINARY NOTES
437
analysis, analogous sections m a y be found on DLTC and LDTC in practically all records under aerobic conditions (even, for example, on R 478). U n d e r nitrogen, the LDTC has a character oi a rapid m o n o p h a s i c decay and it is never found to be s y m m e t r i c a l with the DLTC. I n a previous communication, the a u t h o r has suggested t h a t the shape of the aerobic LDTC records results from the (pseudo)monomolecular liberation of oxygen from an intermediary p r o d u c t p r e s e n t at the m o m e n t the light is s h u t off. To explain the two inflections or minima c o m m o n l y observed on the LDTC, two o x y g e n - c o n s u m i n g processes were a s s u m e d to take place at the beginning of the d a r k period. This t y p e of explanation, however, seems r a t h e r inadequate, if the striking s y m m e t r y of the LDTC and DLTC is taken into account. I t would be strange if b y mere chance similar time c o n s t a n t s were to govern b o t h the s t a r t of the complicated reaction chain in p h o t o s y n t h e s i s and the single reaction of oxygen liberation from an intermediary product. I t seems more plausible that, in some w a y or other, the LDTC reflect changes in reservoir sizes of the same intermediates and variations in rates of the same reversible reactions, which are reflected by the DLTC w h e n the opposite transition from dark to light takes place. Before a reasonable i n t e r p r e t a t i o n of the p h e n o m e n a observed can be a t t e m p t e d , more data m u s t be collected. I n particular, parallel d e t e r m i n a t i o n s of oxygen exchange b y a dropping m e r c u r y electrode and b y the m e t h o d of BLINKS AND SKOW are planned. I t is the disadvantage of the latter m e t h o d t h a t it fails to give a clear discrimination between net oxygen production a n d uptake. Consequently, the p o i n t of basic fnterest c a n n o t be established, i.e., which sections of the records fall above the c o m p e n s a t i o n point and which are below it. To k n o w this is obviously of great i m p o r t a n c e for an a d e q u a t e i n t e r p r e t a t i o n of the LDTC and for the correct evaluation of interactions existing between p h o t o s y n t h e s i s and respiration. T h a t such interrelations influence the shape of aerobic LDTC records is suggested b y the striking difference between aerobic and anaerobic light-dark transition curves. IVAN ~ETLfK*
Institute o~ Plant Physiology, Charles U~iversity, Prague (Czechoslovakia) 1 j . FRANCK, Ann. Rev. Plant Physiol., 2 (1951) 53; Arch. Biochem. Biophys., 45 (1953) 19o; H. GAFFRON AND J. ROSENBERG Naturwissenscha/ten, 42 (1955) 53; E. C. WASSINK AND C. J. P. SPRUIT, VIIIe Congr~s Intern. de Bot., Rappts. et Communs., Sec. i i et I2, p. 3. 2 K. A. CLENDENNING AND F. T. HAXO, Can. J. Botany, 34 (1956) 214. s C. P. WHITTINGHAM, J. Exptl. Botany, 7 (1956) 273. 4 E. C. WASSINK, Advances in Enzymol., i i (195o) 91. s I. ~ETLfK, Rozpravy ~SAV, ~ada MPV, 64 (1954) segit 3. e L. R. BLINKS AND R. K. SKOW, Proc. Natl. Acad. Sci. U.S., 24 (1938) 413 . 7 F. T. HAXO AND L. R. BLINKS, J. Gen. Physiol., 33 (195 o) 389 • 8 I. ~ETLfK, Biochim. Biophys. Acta, 24 (1957) 434. Received J a n u a r y 2ist, 1957 • P r e s e n t address: Botanick~ zAhrada SAV, K o m e n s k 6 h o 67, Kogice (Czechoslovakia).
Inhibition of citrate oxidation by glyoxylate in rat liver homogenates The m e c h a n i s m of the inhibition produced b y glyoxylate on a respiring tissue suspension, first s h o w n b y KLEINZELLER 1, has been investigated b y s t u d y i n g the action of v e r y small a m o u n t s of glyoxylate on the oxidative b e h a v i o u r of the m o s t i m p o r t a n t 16 metabolites related to the tricarboxylic acid cycle. F o r this p u r p o s e o.ooi M glyoxylate was added to a rat liver h o m o g e n a t e incubated in the presence of citrate, cis-aconitate, isocitrate, a-ketoglutarate, succinate, fumarate, malate, oxalu acetate and p y r u v a t e , each at a final concentration of o.oo2M. ~s The final v o l u m e of the incubation m i x t u r e s was always 4.o ml; the concentration of the h o m o g e n a t e was I : IO (prepared in K r e b s ' phospho-saline m e d i u m free of Ca ++ and with the addition of o . o 2 5 M Mg ++ and O . o l M F - ) . The incubation was for 60 min at 38°C in W a r b u r g vessels of a b o u t 15 ml capacity; 02 gas phase; p H 7.4. A n o t h e r experiment was carried out u n d e r the same U r n o l e s oxalacetate conditions with o.oo1 M glyoxylate and differing concentrations Fig. I. R a t liver h o m o g e n a t e of oxalacetate as s h o w n in Fig. I. i : io; glyoxylate o.ooi M ; I n all the e x p e r i m e n t s the oxygen u p t a k e was followed manoN a F o.oi M. metrically, and at the end of the incubation citrate was determined _
29
7