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Simultaneous viscosity changes in chloroplast suspensions upon illumination
PRELIMINARY NOTES
211
extracts the absorption maximum between 670 and 680 m/, shifts with time to shorter wavelengths. After 2 days storage at 4 °, the peaks will be at about 673 m/~ for the first extract and 671 m~ for the second. Although we have referred to the solubilizing action of the sodium dodecyl sulfate, the formation of a colloidal form is more correct. Centrifugal studies indicate that the pigment particles in the first extract are 5-1o times heavier than those in the second. Correspondingly there is about twice as much protein for each chlorophyll in the first extract. Most of the pigment complexes in both extracts can be precipitated with 15 % (NH,)~SO 4. Kjeldahl analyses of these precipitates and of the total extracts indicate that essentially all of the detectable protein is bound with the pigments in both cases. We have been unable to solubilize the pigment particles again in buffer after (NH4) 2S04 treatment. The total xanthophyll and carotene contents were determined separately by a modification of GOODWlN'S alkaline hydrolysis method e. The ratios of chlorophyll to carotene were not significantly different in the two extracts. There were always higher proportions of the xanthophylls and chlorophyll b in the second extract containing more Ca 670. First experiments indicate that spinach chloroplasts yield the same results as tobacco. A partial separation of the two maior forms of chlorophyll a absorbing at 670 and 680 m~ has been obtained with sodium dodecyl sulfate. The two extracts also have different amounts of chlorophyll b, xanthophyll and protein.
Department of Plant Biology, Carnegie Institution of Washington, Stanford, Calif. (U.S.A.)
JEANETTE
S, BROWN
Commissariat ~ l'Energie Atomique, Ddpartement de Biologie, JACQUESG. DURANTON Centre d'Etudes Nucldaires de Saclay, Gif-sur- Yvate (France) 1 j . S. BROWN, Photoehem. Photobiol., 2 (1963) 159. 2 M. B. ALLEN, J. C. MURCHIO, S. W. JEFFREY AND S. A. BENDIX, in Japan. Soc. Plant Physiol., Microalgae and Photosynthetic Bacteria, Univ. T o k y o Press, 1963, p. 407 . 3 W. L. BtYTLER AND J. E. BAKER, Biochim. Biophys. Acta, 66 (1963) 2o6. 4 j . S. BROWN, Carnegie Inst. Wash. Yearbook, 58 (1959) 33 o. 5 E. L. SMITH, J. Gen. Physiol., 24 (194.1) 583 • 8 T. W. GOODWIN, in K. PAECH &NO M. V. TRACEY, Modern Methods of Plant Analysis, Vol. 3, Springer Verlag, Berlin, 1955, p. 272.
Received November 22nd, 1963 Biochim. Biophys. Acta, 79 (1964) 2o9-211
PN 41oo1 Simultaneous viscosity changes in chloroplast suspensions upon illumination Recent reports from two laboratories 1, ~ have brought a new dimension to the study of the role of the chloroplast as an organized energy-transducing system. Chloroplasts incubated under conditions of either ATP synthesis or hydrolysis, manifest upon Biochim. Biophys. Acta, 79 (1964) 211-213
212
PRELIMINARY NOTES
illumination remarkable changes in their physical properties which are demonstrable by measurements of light-scattering intensity 1, rectilinear attenuance s, size as determined from readings in a Coulter counter s, and by changes in fine structure as seen with the electron microscope s. The results with the Coulter counter and electron microscope give direct evidence on the nature of the morphological changes occurring in the chloroplast, but provide static information on the physical change. These considerations led us to design an apparatus capable of detecting continuous changes in the viscosity of chloroplast suspensions since this technique affords a rapid recording of size changes. A Couette-type-viscometer with cylindrical tubings fabricated of transparent materials was constructed. The outer rotating cylinder was of 3.8 cm inside-diameter precision glass tubing; this was surrounded by a cylindrical water jacket fabricated of polystyrene through which water was circulated to maintain constant temperature. An inner rod of stainless steel (io cm height and 3.6 cm in diameter) was suspended within the outer cylinder by a phosphor-bronze wire. The chloroplast suspension filled the space in the annular gap between the rod and the outer rotating cylinder. Illumination was provided by locating several lamps symmetrically around the center of rotation. The outer cylinder was driven by a synchronous motor which provided rotation at a constant velocity. Rotation velocity could be adjusted by a transmission system. The velocity gradient produced in the I - m m annular gap was calculated to be in the range of 2o to 33o s e c -1, and within this range no turbulent flow could be detected. A differential transformer made with three commercial high-frequency choke coils, was employed as a transducer to detect the angular deviation produced by the torque on the inner rod. The transformer was driven by an audiofrequency generator at 15 kcycles. A phase-sensitive amplifier produced a d.c. voltage which was amplified and then recorded (Varian GI 4, I-V. full-scale) at a m a x i m u m sensitivity of approximately o.I centipoise full-scale. A typical experiment is shown in Fig. I. It is seen that the viscosity of the chloroplast suspension in the dark does not change with time. Upon illumination, a remarkable and simultaneous decrease of the apparent viscosity reading is observed. The viscosity decrease continues until it reaches a steady level, being approximately 27 % of the viscosity difference between the suspension in darkness and the medium.
130 D.
IOO
?
90 u
125
80 o_ < 70
I
I
I
0
5
I0
[ 15 Minutes
L
i
20
25
1.20
Fig. I. T i m e r e c o r d i n g of v i s c o s i t y c h a n g e s a c c o m p a n y i n g i l l u m i n a t i o n of s p i n a c h chloroplasts u n d e r c o n d i t i o n s of p h o t o p h o s p h o r y l a t i o n . Chloroplasts were isolated f r o m s p i n a c h leaves 1 a n d s u s p e n d e d in t h e d a r k in a r e a c t i o n m e d i u m (15 ml) c o n t a i n i n g 35 m M NaC1, 5 m M MgCI~, 5 m M s o d i u m a s c o r b a t e , 5 m M Tris buffer (pH 8.o), 5 m M p h o s p h a t e (pH 8.o), i m M A D P , 20/~M p h e n a z i n e m e t h o s u l f a t e , a n d c h l o r o p l a s t s (5 ml, 1.45 m g ] m l chlorophyll). T h e c h l o r o p l a s t susp e n s i o n was t r a n s f e r r e d to t h e a n n u l a r g a p in darkness. T h e e x p e r i m e n t w a s carried o u t a t 28 ° a t a v e l o c i t y g r a d i e n t of 288 sec -1. c.p. d e n o t e s centipoise.
Biochim. Biophys. Acta, 79 (1964) 2 I I - 2 t 3
PRELIMINARY NOTES
213
Following removal of actinic light, the decreased state of viscosity recovers to the original dark level seen at the outset of the experiment. Control experiments showed no detectable orientation of the chloroplast particles over the range of velocity gradients employed; deviation of temperature upon illumination was also negligible. Furthermore, in support of the notion 1, ~ that the physical changes triggered by light are the result of the activity of the photophosphorylation system, it is found that, (a) chloroplasts suspended in a medium devoid of phosphate do not respond, and (b) chloroplasts in a complete medium with NH4C1 (2 mM) added also fail to produce light-dependent viscosity decreases. It was generally observed that the rates of viscosity decrease and recovery depend somewhat on the duration of pre-incubation in the dark. Longer pre-incubation generally led to slower rates of change especially in the dark recovery process. However, the duration of the pre-incubation period was independent of the final steady-state level of decreased viscosity achieved after illumination. These results suggest that the metabolic reactions which drive the viscosity changes may be impaired by a long pre-incubation time. While interpretations other than size or volume are possible from the apparent viscosity changes observed here, the following considerations suggest that this is not the case. (A) The time course of the viscosity changes are similar to those reported by ITOH, IZAWA AND SHIBATA2 with the Coulter counter, and (B) observations of packed chloroplast volume made in light and darkness which substantiate the occurrence of size changes. These results provide strong evidence that chloroplast particles under conditions of photophosphorylation truly manifest shrinkage in the light, which is fully reversible in the dark. It may be that the incomplete recovery of shrinkage observed with the Coulter counter 2 may have arisen from the effects (cited above) of pre-incubation time on recovery. The transparent-wall viscometer may find wide application for biophysical studies of light-dependent processes in organized systems such as chloroplasts, since it permits the continuous observation of a structural parameter by rates of electron transport, which can be precisely controlled by variation of the nature and intensity of the actinic light. This research was supported by the National Science Foundation.
Department of Physiology, University of California, Berkeley, Calif. (U.S.A.)
YASUO MUKOHATA* LESTER PACKER
1 L. PACKER, Biochim. Biophys. Acta, 75 (1963) 12. M. ITOH, S. IZAWAAND K. SHIBATA, Biochim. Biophys. Acta, 66 (1963) 319 .
Received October 26th, 1963 * Visiting from Department of Biology, Faculty of Science, Osaka University, Osaka, Japan.
Biochim. Biophys. Acta, 79 (1964) 2II-213
211
extracts the absorption maximum between 670 and 680 m/, shifts with time to shorter wavelengths. After 2 days storage at 4 °, the peaks will be at about 673 m/~ for the first extract and 671 m~ for the second. Although we have referred to the solubilizing action of the sodium dodecyl sulfate, the formation of a colloidal form is more correct. Centrifugal studies indicate that the pigment particles in the first extract are 5-1o times heavier than those in the second. Correspondingly there is about twice as much protein for each chlorophyll in the first extract. Most of the pigment complexes in both extracts can be precipitated with 15 % (NH,)~SO 4. Kjeldahl analyses of these precipitates and of the total extracts indicate that essentially all of the detectable protein is bound with the pigments in both cases. We have been unable to solubilize the pigment particles again in buffer after (NH4) 2S04 treatment. The total xanthophyll and carotene contents were determined separately by a modification of GOODWlN'S alkaline hydrolysis method e. The ratios of chlorophyll to carotene were not significantly different in the two extracts. There were always higher proportions of the xanthophylls and chlorophyll b in the second extract containing more Ca 670. First experiments indicate that spinach chloroplasts yield the same results as tobacco. A partial separation of the two maior forms of chlorophyll a absorbing at 670 and 680 m~ has been obtained with sodium dodecyl sulfate. The two extracts also have different amounts of chlorophyll b, xanthophyll and protein.
Department of Plant Biology, Carnegie Institution of Washington, Stanford, Calif. (U.S.A.)
JEANETTE
S, BROWN
Commissariat ~ l'Energie Atomique, Ddpartement de Biologie, JACQUESG. DURANTON Centre d'Etudes Nucldaires de Saclay, Gif-sur- Yvate (France) 1 j . S. BROWN, Photoehem. Photobiol., 2 (1963) 159. 2 M. B. ALLEN, J. C. MURCHIO, S. W. JEFFREY AND S. A. BENDIX, in Japan. Soc. Plant Physiol., Microalgae and Photosynthetic Bacteria, Univ. T o k y o Press, 1963, p. 407 . 3 W. L. BtYTLER AND J. E. BAKER, Biochim. Biophys. Acta, 66 (1963) 2o6. 4 j . S. BROWN, Carnegie Inst. Wash. Yearbook, 58 (1959) 33 o. 5 E. L. SMITH, J. Gen. Physiol., 24 (194.1) 583 • 8 T. W. GOODWIN, in K. PAECH &NO M. V. TRACEY, Modern Methods of Plant Analysis, Vol. 3, Springer Verlag, Berlin, 1955, p. 272.
Received November 22nd, 1963 Biochim. Biophys. Acta, 79 (1964) 2o9-211
PN 41oo1 Simultaneous viscosity changes in chloroplast suspensions upon illumination Recent reports from two laboratories 1, ~ have brought a new dimension to the study of the role of the chloroplast as an organized energy-transducing system. Chloroplasts incubated under conditions of either ATP synthesis or hydrolysis, manifest upon Biochim. Biophys. Acta, 79 (1964) 211-213
212
PRELIMINARY NOTES
illumination remarkable changes in their physical properties which are demonstrable by measurements of light-scattering intensity 1, rectilinear attenuance s, size as determined from readings in a Coulter counter s, and by changes in fine structure as seen with the electron microscope s. The results with the Coulter counter and electron microscope give direct evidence on the nature of the morphological changes occurring in the chloroplast, but provide static information on the physical change. These considerations led us to design an apparatus capable of detecting continuous changes in the viscosity of chloroplast suspensions since this technique affords a rapid recording of size changes. A Couette-type-viscometer with cylindrical tubings fabricated of transparent materials was constructed. The outer rotating cylinder was of 3.8 cm inside-diameter precision glass tubing; this was surrounded by a cylindrical water jacket fabricated of polystyrene through which water was circulated to maintain constant temperature. An inner rod of stainless steel (io cm height and 3.6 cm in diameter) was suspended within the outer cylinder by a phosphor-bronze wire. The chloroplast suspension filled the space in the annular gap between the rod and the outer rotating cylinder. Illumination was provided by locating several lamps symmetrically around the center of rotation. The outer cylinder was driven by a synchronous motor which provided rotation at a constant velocity. Rotation velocity could be adjusted by a transmission system. The velocity gradient produced in the I - m m annular gap was calculated to be in the range of 2o to 33o s e c -1, and within this range no turbulent flow could be detected. A differential transformer made with three commercial high-frequency choke coils, was employed as a transducer to detect the angular deviation produced by the torque on the inner rod. The transformer was driven by an audiofrequency generator at 15 kcycles. A phase-sensitive amplifier produced a d.c. voltage which was amplified and then recorded (Varian GI 4, I-V. full-scale) at a m a x i m u m sensitivity of approximately o.I centipoise full-scale. A typical experiment is shown in Fig. I. It is seen that the viscosity of the chloroplast suspension in the dark does not change with time. Upon illumination, a remarkable and simultaneous decrease of the apparent viscosity reading is observed. The viscosity decrease continues until it reaches a steady level, being approximately 27 % of the viscosity difference between the suspension in darkness and the medium.
130 D.
IOO
?
90 u
125
80 o_ < 70
I
I
I
0
5
I0
[ 15 Minutes
L
i
20
25
1.20
Fig. I. T i m e r e c o r d i n g of v i s c o s i t y c h a n g e s a c c o m p a n y i n g i l l u m i n a t i o n of s p i n a c h chloroplasts u n d e r c o n d i t i o n s of p h o t o p h o s p h o r y l a t i o n . Chloroplasts were isolated f r o m s p i n a c h leaves 1 a n d s u s p e n d e d in t h e d a r k in a r e a c t i o n m e d i u m (15 ml) c o n t a i n i n g 35 m M NaC1, 5 m M MgCI~, 5 m M s o d i u m a s c o r b a t e , 5 m M Tris buffer (pH 8.o), 5 m M p h o s p h a t e (pH 8.o), i m M A D P , 20/~M p h e n a z i n e m e t h o s u l f a t e , a n d c h l o r o p l a s t s (5 ml, 1.45 m g ] m l chlorophyll). T h e c h l o r o p l a s t susp e n s i o n was t r a n s f e r r e d to t h e a n n u l a r g a p in darkness. T h e e x p e r i m e n t w a s carried o u t a t 28 ° a t a v e l o c i t y g r a d i e n t of 288 sec -1. c.p. d e n o t e s centipoise.
Biochim. Biophys. Acta, 79 (1964) 2 I I - 2 t 3
PRELIMINARY NOTES
213
Following removal of actinic light, the decreased state of viscosity recovers to the original dark level seen at the outset of the experiment. Control experiments showed no detectable orientation of the chloroplast particles over the range of velocity gradients employed; deviation of temperature upon illumination was also negligible. Furthermore, in support of the notion 1, ~ that the physical changes triggered by light are the result of the activity of the photophosphorylation system, it is found that, (a) chloroplasts suspended in a medium devoid of phosphate do not respond, and (b) chloroplasts in a complete medium with NH4C1 (2 mM) added also fail to produce light-dependent viscosity decreases. It was generally observed that the rates of viscosity decrease and recovery depend somewhat on the duration of pre-incubation in the dark. Longer pre-incubation generally led to slower rates of change especially in the dark recovery process. However, the duration of the pre-incubation period was independent of the final steady-state level of decreased viscosity achieved after illumination. These results suggest that the metabolic reactions which drive the viscosity changes may be impaired by a long pre-incubation time. While interpretations other than size or volume are possible from the apparent viscosity changes observed here, the following considerations suggest that this is not the case. (A) The time course of the viscosity changes are similar to those reported by ITOH, IZAWA AND SHIBATA2 with the Coulter counter, and (B) observations of packed chloroplast volume made in light and darkness which substantiate the occurrence of size changes. These results provide strong evidence that chloroplast particles under conditions of photophosphorylation truly manifest shrinkage in the light, which is fully reversible in the dark. It may be that the incomplete recovery of shrinkage observed with the Coulter counter 2 may have arisen from the effects (cited above) of pre-incubation time on recovery. The transparent-wall viscometer may find wide application for biophysical studies of light-dependent processes in organized systems such as chloroplasts, since it permits the continuous observation of a structural parameter by rates of electron transport, which can be precisely controlled by variation of the nature and intensity of the actinic light. This research was supported by the National Science Foundation.
Department of Physiology, University of California, Berkeley, Calif. (U.S.A.)
YASUO MUKOHATA* LESTER PACKER
1 L. PACKER, Biochim. Biophys. Acta, 75 (1963) 12. M. ITOH, S. IZAWAAND K. SHIBATA, Biochim. Biophys. Acta, 66 (1963) 319 .
Received October 26th, 1963 * Visiting from Department of Biology, Faculty of Science, Osaka University, Osaka, Japan.
Biochim. Biophys. Acta, 79 (1964) 2II-213