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[30] Preparation and properties of phosphorylating subchloroplast particles
302
CELLULAR AND SUBCELLULAR PREPARATIONS
[30]
rated were proved 27 to be from photochemical systems I and II, respectively. The pigment compositions of the two pigment proteins obtained from spinach chloroplasts with different solubilizing agents are listed in the table, where pigment fractions are normalized to 100 molecules of total chlorophyll. The light and heavy particles solubilized with digitonin ~,29 and with Triton X-10029,~° were separated by differential or density gradient centrifugation. Components I and II of Ogawa et al. and complexes I and II of Thornber et al. solubilized with SDS and DBS, respectively, were separated by the above procedure of electrophoresis. High chlorophyll a:b ratios and high fl-carotene contents were found for the light particle, or component I or complex I, whereas low a:b ratios and high xanthophyll contents were found for the heavy particle, or component II or complex II. The same trend of uneven distributions of carotene and xanthophylls was found for the two pigment proteins prepared similarly by electrophoresis from Anabaena variabilis, Porphyra yezoensis, and Phaeodactylum tricornutum. ~5 2~B. Ke, C. Seliskar, and R. Breeze, Plant Physiol. 41, 1081 (1966). ~L. P. Vernon, B. Ke, S. Katoh, A. San Pietro, and E. R. Shaw, Brookhaven Syrup. Biol. 19, 102 (1967).
[30] Preparation and Properties of Phosphorylating Subchloroplast Particles By RICHARD E. MCCARTY
The rates of photophosphorylation and photoreduction catalyzed by chloroplasts vary considerably from preparation to preparation. Further, these activities are generally rapidly lost on storage of the chloroplasts. In view of these facts, it was of interest to develop a procedure for the preparation of subchloroplast particles capable of catalyzing photophosphorylation which could be stored for extended periods without loss of activity. Vambutas and Racker 1 reported the preparation of phosphorylating subchloroplast particles by exposure of chloroplasts to sonic oscillation in the presence of phosphatides. The following procedure is essentially a modification of that used by Vambutas and Racker.
Preparation Chloroplasts were prepared from washed, market spinach leaves from which the midribs had been removed. All operations described below were V. K. Vambutas and E. Racker, J. Biol. Chem. 240~ 2660 (1965).
[30]
PHOSPHORYLATING SUBCHLOROPLAST PARTICLES
303
performed at 0°-4 °. A total of 200 g of leaves were homogenized for 15 sec with 300 ml of a medium (STN) containing 0.4M sucrose, 10 mM NaC1, and 20 mM tris (hydroxymethyl)methylglycine-NaOH (pH 8.0) in a Waring Blendor at top speed. The chloroplasts in the homogenate were isolated as described by Avron and Jagendorf 2 and were washed once with 150 ml of STN. The chloroplasts were resuspended by gentle homogenization in a minimal volume of STN, and the chlorophyll concentration of the suspension was adjusted to 2-2.5 mg/ml by the addition of STN. Twenty milliliters of the chloroplast suspension was transferred to a 50-ml stainless-steel beaker which was immersed in an ice bath. A Branson 20-kHz Sonifier equipped with a 0.5-inch solid step horn was used to expose the chloroplasts to sonic irradiation. The probe was immersed about 1 cm into the chloroplast suspension and the Sonifier was run for 15 seconds at full output. The temperature rose to 8 ° during the sonication. After the temperature of the suspension fell to 4 °, another 15-second period of sonication was given. This procedure was repeated once more, and the suspension was centrifuged at 6500 g for 10 minutes. The pellet which contained less than 10% of the total chlorophyll of the suspension was discarded. The supernatant fluid was centrifuged at 104,000 g for 45 minutes. The supernatant fluid was discarded and the pellet resuspended in 20 ml of STN by gentle homogenization. This suspension was centrifuged at 104,000 g for 45 minutes and the pellet resuspended in a small volume of STN. The chlorophyll concentration of the suspension was adjusted to 4-6 mg/ml by the addition of STN. Aliquots of 0.15 ml of the suspension were distributed to small tubes which were flushed for a few seconds with prepurified nitrogen and tightly stoppered. The tubes were stored at --80 ° in a Revco freezer. Yield From 200 g of leaves, subchloroplast particles equivalent to about 30 mg of chlorophyll were isolated. This recovery represents 60-70% of the chlorophyll contained in the original chloroplast suspension. Properties Under the electron microscope, subchloroplast particles negatively stained with phosphotungstic acid appeared to be vesicular. The diameter of the vesicles varied from about 0.08 to 0.5 ~, with an average diameter of 0.2 ~. Subchloroplast particles catalyze noncyclic phosphorylation as well as cyclic phosphorylation (see the table). The rates of ferricyanide-dependent phosphorylation and pyocyanine-catalyzed cyclic phosphorylation 2M. Avron and A. T. Jagendorf,J. Biol. Chem. 234, 967 (1959).
304
CELLULAR
AND
SUBCELLULAR
[30]
PREPARATIONS
SOME ACTIVITIES CATALYZED BY SUBCHLOROPLAST PARTICLESa
Activity measured Ferricyanide reduction and coupled phosphorylation NADP reduction and coupled phosphorylation Pyocyanine-dependent cyclic phosphorylation Light-induced H + uptake Acid-base induced ATP synthesis Postillumination ATP synthesis
Reduction (t~moles reduced/hr/mg chlorophyll)
Phosphorylation (ttmoles ATP formed/ hr/mg chlorophyll)
P/e2
H + uptake (~eq H + accumulated/mg chlorophyll)
202-556
25-79
0.19-0.59
--
32-98
32-48
O.3-0.7
--
--
125-490
--
--
--
--
--
O. 3-0.63
--
35-60b
--
--
--
25-40b
--
--
a Phosphorylation assays were carried out at room temperature in a reaction mixture which contained in a volume of 1.0 mh 50 mM Tricine-NaOH (pH 8.0), 50 mM NaC1, 3 nu]// ADP, 2 mM potassium phosphate buffer (pH 8.0), 5 mM MgC12, 1 mg of defatted bovine serum albumin, subchloroplast particles equivalent to about 50 t~g of chlorophyll and about 106, cpm ~2Pi. For phosphorylation coupled to ferricyanide reduction, 1 mM K3Fe(CN)~ wss present; for NADP reduction, 0.25 mM NADP was added; and for pyocyanine-dependentphosphorylation, 0.05 raM pyocyanine was used. The light intensity was 2 X 106 ergs/cm2/sec, and the gas phase was nitrogen. Ferricyanide and NADP reduction were determined spectrophotometrically, and 32Pi esterification was assayed as previously described, Acid-base induced ATP formation and postillumination ATP synthesis were assayed as described elsewhere in this volume. Light-induced H + uptake was measured in the presence of 5 ml of a solution containing 50 mM NaC1, 50 ~M pyocyanine, 0.66 mM Tris-HC1, and subchloroplast particles equivalent to 100 t~g of chlorophyll. The initial pH was 6.5 and the temperature, 5°. b Nanomoles 3~Piesterified per milligram of chlorophyll. in s u b c h l o r o p l a s t particles a v e r a g e d a b o u t half of those observed for these r e a c t i o n s in chloroplasts, whereas the rates of f e r r i c y a n i d e reduct i o n were c o m p a r a b l e to those in chloroplasts. I n the presence of ferredoxin a n d f e r r e d o x i n - N A D P reduetase, N A D P r e d u c t i o n with w a t e r as the electron d o n o r a n d associated p h o s p h o r y l a t i o n in s u b c h l o r o p l a s t particles also occurred. M a x i m a l rates of p h o s p h o r y l a t i o n were observed o n l y in the presence of d e f a t t e d b o v i n e s e r u m a l b u m i n (1 m g J m l ) a n d of a n i t r o g e n a t m o s p h e r e .
~1]
SUBCHROMATOPHORE FRAGMENTS
305
The extent of the light-dependent H + uptake 3 in subchloroplast particles was about half that of chloroplasts. Further, ATP synthesis in the dark resulting from either a rapid charge in the pH of a subehloroplast particle suspension 4 or from a prior illumination;~ could be readily detected. A major difference between chloroplasts and subchloroplast particles is the relative insensitivity of phosphorylation in the latter preparation to uncoupling by NH4C1 and amines. 6 Whereas 2 mM NH~C1 inhibited phosphorylation in chloroplasts by over 50%, it had only a slight effect on phosphorylation in subchloroplast particles. The activities catalyzed by subchloroplast particles are quite stable to storage of the particles at --80 ° . For example, no decrease in either the rate of ferrieyanide reduction or coupled ATP synthesis was observed after a year's storage. 3j. S. Neumann and A. T. Jagendorf, Arch. Biochem. Biophys. 107, 109 (1964). 4A. T. Jagendorf and E. Uribe, Proc. Nat. Acad. Sci. U.S. 55, 170 (1966). G. Hind and A. T. Jagendorf,Proc. Nat. Acad. Sci. U.S. 49, 715 (1963). R. E. McCarty, Biochem. Biophys. Res. Commun. 32, 37 (1968).
[31] S u b c h r o m a t o p h o r e F r a g m e n t s : C h r o m a t i u m , Rhodospirillum rubrum, a n d R h o d o p s e u d o m o n a s palustris By AUGUSTO GARCIA, J. PHILIP THORNBER, and LEO P. VEaNO• The photosynthetic membrane systems of some purple bacteria are readily cleaved by both nonionie and ionic detergents to yield distinct subchromatophore fragments which are characterized by the unique forms of baeterioehlorophyll contained in the particles. Other differences are also found, primarily in the cytochrome content and the presence of specific enzymes. The types of subchromatophore fragments obtained depend to a large extent upon the nature of the detergent, the presence or absence of carotenoids in the membrane, and the conditions used for growing the bacteria. In those bacteria examined to date, at least two fragments are produced, one containing the reaction center bacteriochlorophyll (BChl) and varying amounts of other associated BChl, while the other one contains only light-harvesting BChl (except for R. rubrurn). In the ease of Chromatium, fragmentation with sodium dodecyl sulfate (SDS) produces three fragments, but two of the three are quite similar in their general properties. In v~vo the BChl a (the form of BChl contained in the bacteria under
CELLULAR AND SUBCELLULAR PREPARATIONS
[30]
rated were proved 27 to be from photochemical systems I and II, respectively. The pigment compositions of the two pigment proteins obtained from spinach chloroplasts with different solubilizing agents are listed in the table, where pigment fractions are normalized to 100 molecules of total chlorophyll. The light and heavy particles solubilized with digitonin ~,29 and with Triton X-10029,~° were separated by differential or density gradient centrifugation. Components I and II of Ogawa et al. and complexes I and II of Thornber et al. solubilized with SDS and DBS, respectively, were separated by the above procedure of electrophoresis. High chlorophyll a:b ratios and high fl-carotene contents were found for the light particle, or component I or complex I, whereas low a:b ratios and high xanthophyll contents were found for the heavy particle, or component II or complex II. The same trend of uneven distributions of carotene and xanthophylls was found for the two pigment proteins prepared similarly by electrophoresis from Anabaena variabilis, Porphyra yezoensis, and Phaeodactylum tricornutum. ~5 2~B. Ke, C. Seliskar, and R. Breeze, Plant Physiol. 41, 1081 (1966). ~L. P. Vernon, B. Ke, S. Katoh, A. San Pietro, and E. R. Shaw, Brookhaven Syrup. Biol. 19, 102 (1967).
[30] Preparation and Properties of Phosphorylating Subchloroplast Particles By RICHARD E. MCCARTY
The rates of photophosphorylation and photoreduction catalyzed by chloroplasts vary considerably from preparation to preparation. Further, these activities are generally rapidly lost on storage of the chloroplasts. In view of these facts, it was of interest to develop a procedure for the preparation of subchloroplast particles capable of catalyzing photophosphorylation which could be stored for extended periods without loss of activity. Vambutas and Racker 1 reported the preparation of phosphorylating subchloroplast particles by exposure of chloroplasts to sonic oscillation in the presence of phosphatides. The following procedure is essentially a modification of that used by Vambutas and Racker.
Preparation Chloroplasts were prepared from washed, market spinach leaves from which the midribs had been removed. All operations described below were V. K. Vambutas and E. Racker, J. Biol. Chem. 240~ 2660 (1965).
[30]
PHOSPHORYLATING SUBCHLOROPLAST PARTICLES
303
performed at 0°-4 °. A total of 200 g of leaves were homogenized for 15 sec with 300 ml of a medium (STN) containing 0.4M sucrose, 10 mM NaC1, and 20 mM tris (hydroxymethyl)methylglycine-NaOH (pH 8.0) in a Waring Blendor at top speed. The chloroplasts in the homogenate were isolated as described by Avron and Jagendorf 2 and were washed once with 150 ml of STN. The chloroplasts were resuspended by gentle homogenization in a minimal volume of STN, and the chlorophyll concentration of the suspension was adjusted to 2-2.5 mg/ml by the addition of STN. Twenty milliliters of the chloroplast suspension was transferred to a 50-ml stainless-steel beaker which was immersed in an ice bath. A Branson 20-kHz Sonifier equipped with a 0.5-inch solid step horn was used to expose the chloroplasts to sonic irradiation. The probe was immersed about 1 cm into the chloroplast suspension and the Sonifier was run for 15 seconds at full output. The temperature rose to 8 ° during the sonication. After the temperature of the suspension fell to 4 °, another 15-second period of sonication was given. This procedure was repeated once more, and the suspension was centrifuged at 6500 g for 10 minutes. The pellet which contained less than 10% of the total chlorophyll of the suspension was discarded. The supernatant fluid was centrifuged at 104,000 g for 45 minutes. The supernatant fluid was discarded and the pellet resuspended in 20 ml of STN by gentle homogenization. This suspension was centrifuged at 104,000 g for 45 minutes and the pellet resuspended in a small volume of STN. The chlorophyll concentration of the suspension was adjusted to 4-6 mg/ml by the addition of STN. Aliquots of 0.15 ml of the suspension were distributed to small tubes which were flushed for a few seconds with prepurified nitrogen and tightly stoppered. The tubes were stored at --80 ° in a Revco freezer. Yield From 200 g of leaves, subchloroplast particles equivalent to about 30 mg of chlorophyll were isolated. This recovery represents 60-70% of the chlorophyll contained in the original chloroplast suspension. Properties Under the electron microscope, subchloroplast particles negatively stained with phosphotungstic acid appeared to be vesicular. The diameter of the vesicles varied from about 0.08 to 0.5 ~, with an average diameter of 0.2 ~. Subchloroplast particles catalyze noncyclic phosphorylation as well as cyclic phosphorylation (see the table). The rates of ferricyanide-dependent phosphorylation and pyocyanine-catalyzed cyclic phosphorylation 2M. Avron and A. T. Jagendorf,J. Biol. Chem. 234, 967 (1959).
304
CELLULAR
AND
SUBCELLULAR
[30]
PREPARATIONS
SOME ACTIVITIES CATALYZED BY SUBCHLOROPLAST PARTICLESa
Activity measured Ferricyanide reduction and coupled phosphorylation NADP reduction and coupled phosphorylation Pyocyanine-dependent cyclic phosphorylation Light-induced H + uptake Acid-base induced ATP synthesis Postillumination ATP synthesis
Reduction (t~moles reduced/hr/mg chlorophyll)
Phosphorylation (ttmoles ATP formed/ hr/mg chlorophyll)
P/e2
H + uptake (~eq H + accumulated/mg chlorophyll)
202-556
25-79
0.19-0.59
--
32-98
32-48
O.3-0.7
--
--
125-490
--
--
--
--
--
O. 3-0.63
--
35-60b
--
--
--
25-40b
--
--
a Phosphorylation assays were carried out at room temperature in a reaction mixture which contained in a volume of 1.0 mh 50 mM Tricine-NaOH (pH 8.0), 50 mM NaC1, 3 nu]// ADP, 2 mM potassium phosphate buffer (pH 8.0), 5 mM MgC12, 1 mg of defatted bovine serum albumin, subchloroplast particles equivalent to about 50 t~g of chlorophyll and about 106, cpm ~2Pi. For phosphorylation coupled to ferricyanide reduction, 1 mM K3Fe(CN)~ wss present; for NADP reduction, 0.25 mM NADP was added; and for pyocyanine-dependentphosphorylation, 0.05 raM pyocyanine was used. The light intensity was 2 X 106 ergs/cm2/sec, and the gas phase was nitrogen. Ferricyanide and NADP reduction were determined spectrophotometrically, and 32Pi esterification was assayed as previously described, Acid-base induced ATP formation and postillumination ATP synthesis were assayed as described elsewhere in this volume. Light-induced H + uptake was measured in the presence of 5 ml of a solution containing 50 mM NaC1, 50 ~M pyocyanine, 0.66 mM Tris-HC1, and subchloroplast particles equivalent to 100 t~g of chlorophyll. The initial pH was 6.5 and the temperature, 5°. b Nanomoles 3~Piesterified per milligram of chlorophyll. in s u b c h l o r o p l a s t particles a v e r a g e d a b o u t half of those observed for these r e a c t i o n s in chloroplasts, whereas the rates of f e r r i c y a n i d e reduct i o n were c o m p a r a b l e to those in chloroplasts. I n the presence of ferredoxin a n d f e r r e d o x i n - N A D P reduetase, N A D P r e d u c t i o n with w a t e r as the electron d o n o r a n d associated p h o s p h o r y l a t i o n in s u b c h l o r o p l a s t particles also occurred. M a x i m a l rates of p h o s p h o r y l a t i o n were observed o n l y in the presence of d e f a t t e d b o v i n e s e r u m a l b u m i n (1 m g J m l ) a n d of a n i t r o g e n a t m o s p h e r e .
~1]
SUBCHROMATOPHORE FRAGMENTS
305
The extent of the light-dependent H + uptake 3 in subchloroplast particles was about half that of chloroplasts. Further, ATP synthesis in the dark resulting from either a rapid charge in the pH of a subehloroplast particle suspension 4 or from a prior illumination;~ could be readily detected. A major difference between chloroplasts and subchloroplast particles is the relative insensitivity of phosphorylation in the latter preparation to uncoupling by NH4C1 and amines. 6 Whereas 2 mM NH~C1 inhibited phosphorylation in chloroplasts by over 50%, it had only a slight effect on phosphorylation in subchloroplast particles. The activities catalyzed by subchloroplast particles are quite stable to storage of the particles at --80 ° . For example, no decrease in either the rate of ferrieyanide reduction or coupled ATP synthesis was observed after a year's storage. 3j. S. Neumann and A. T. Jagendorf, Arch. Biochem. Biophys. 107, 109 (1964). 4A. T. Jagendorf and E. Uribe, Proc. Nat. Acad. Sci. U.S. 55, 170 (1966). G. Hind and A. T. Jagendorf,Proc. Nat. Acad. Sci. U.S. 49, 715 (1963). R. E. McCarty, Biochem. Biophys. Res. Commun. 32, 37 (1968).
[31] S u b c h r o m a t o p h o r e F r a g m e n t s : C h r o m a t i u m , Rhodospirillum rubrum, a n d R h o d o p s e u d o m o n a s palustris By AUGUSTO GARCIA, J. PHILIP THORNBER, and LEO P. VEaNO• The photosynthetic membrane systems of some purple bacteria are readily cleaved by both nonionie and ionic detergents to yield distinct subchromatophore fragments which are characterized by the unique forms of baeterioehlorophyll contained in the particles. Other differences are also found, primarily in the cytochrome content and the presence of specific enzymes. The types of subchromatophore fragments obtained depend to a large extent upon the nature of the detergent, the presence or absence of carotenoids in the membrane, and the conditions used for growing the bacteria. In those bacteria examined to date, at least two fragments are produced, one containing the reaction center bacteriochlorophyll (BChl) and varying amounts of other associated BChl, while the other one contains only light-harvesting BChl (except for R. rubrurn). In the ease of Chromatium, fragmentation with sodium dodecyl sulfate (SDS) produces three fragments, but two of the three are quite similar in their general properties. In v~vo the BChl a (the form of BChl contained in the bacteria under