Effect of DNAase on fluorescent properties of pigment-protein complexes, contained in photosystem II

It was found that chlorophyll fluorescence spectra and spectra of fluorescence excitation of pigment-protein complexes of photosystem II are affected by treatment with DNase. Pigment-protein complexes were isolated from pea thylakoid membranes. Spectra were measured at room temperature. It was shown that the treatment with DNase leads to a 30% increase in fluorescence yield at excitation in chlorophyll absorption bands in the fraction containing CP47, CP43, and CP29, and also in the fraction containing reaction center complexes with minor contaminations of light-harvesting complexes. Upon excitation at 260-300 nm and in the region of 500 nm, a diminishing of fluorescence yield takes place. These results suggest that pigments and/or pigment-protein complexes are bound to nucleic acids. This association, by influencing the pigment properties, can participate in the photoregulation of biochemical reactions through changes in the thermal dissipation of excited chlorophyll molecules.     

Correlation of the 680 to 672 nm Spectral Shift and the Halving of the Apparent Molecular Weight for Chlorophyll(ide) Holochrome from Barley

Protochlorophyll(ide) holochrome was isolated from dark-grown barley (Hordeum vulgare L.) leaves and photoconverted. When the chlorophyll(ide) absorption maximum had decreased from 680 to 676 nm the preparation was chromatographed on a Sephadex-gel column under conditions which strongly inhibited a further decrease in the absorption maximum. The absorption properties of the column fractions and the shape of the chlorophyll(ide) elution-profile indicated the presence of two distinct chlorophyll(ide)-bearing molecular species with apparent molecular weights of c. 74,000 and 29,000 and absorption maxima at 680 and 672 nm, respectively. It is concluded that: (1) no long-lived species with intermediate absorption maximum is formed during the 680 to 672 nm shift of the absorption maximum of newly photoconverted holochrome; (2) no long-lived pigment-protein complexes with intermediate molecular weights are formed during the approximate halving of the molecular weight; (3) the shift in the absorption maximum and the decrease in molecular weight are closely correlated.     

A single step separation of PS 1, PS 2 and chlorophyll-antenna particles from spinach chloroplasts

After solubilization of photosynthetic membranes by digitonin, three main protein pigment complexes were isolated by electrophoresis with deoxycholate as detergent.The band with the slowest mobility, fraction 1, had PS 1 activity and was devoid of PS 2 activity. This fraction was four times enriched in P700 when compared with chloroplasts. Fraction 1 had little chl b, a long wavelength absorption maximum in the red, a maximum of low temperature emission fluorescence at 730nm, and a circular dichroism spectrum characteristic of PS 1 enriched fraction.Fraction 2 exhibited a PS 2 activity and no PS 1 activity. It was enriched five times in PS 2 reaction centre and had little chl b and carotenoids. The absorption maximum was at 674 nm and the low temperature fluorescence emission maximum was at 700 nm. Fraction 2 might be useful PS 2 enriched particle because of the great stability of this fraction with regard to photochemical activity and also rapidity and simplicity of its preparation.Fraction 3, which had the fastest migration, was devoid of photochemical activities; It was rich in chl b and had the fluorescence and the circular dichroism spectrum characteristic of an antenna complex.Abbreviations PS 1 (2) photosystem 1 (2) - chl chlorophyll - car carotenoid - Q primary plastoquinone electron acceptor - P700 primary electron donor of PS 1 - P680 primary electron donor of PS 2 - K₃Fe(CN)₆ potassium ferricyanide - DCMU dichlorophenyldimethylurea - DCPIP dichlorophenolindophenol - DPC diphenyl-carbazide     

Treatment of the thylakoid membrane with surfactants : assessment of effectiveness using the chlorophyll a absorption spectrum

Treatment of higher plant (Nicotiana tabacum L. var. Samsun) chloroplast thylakoid membranes with surfactants results in a shift of the chlorophyll a absorption maximum in the red spectral region from its in vivo value of 678.5 nanometers to shorter wavelengths. The magnitude of this shift is correlated with membrane disruption, and is not necessarily due to the release of pigment from pigment-protein complexes present in the membrane. Membrane disruption has been measured by the amount of pigment in the supernatant fraction after centrifugation of surfactant treated membranes. For an equivalent amount of disruption, the extent of the blue-shift is influenced by the ionic nature of the surfactant: anionic surfactants cause small shifts, cationic surfactants cause the largest (~10 nanometers) shifts, and nonionic surfactants produce intermediate shifts. The wavelength of maximum absorbance of chlorophyll a in the red region is a convenient criterion for assessing the potential utility of different surfactants for studies on the structure, composition and function of higher plant thylakoid membranes.     

Structural-functional organization of chloroplasts in leaves of xantha-702 mutant of Gossypium hirsutum L

For cotton mutant xantha (Gossypium hirsutum L.), it has been established that synthesis of 5-aminolevulinic acid was blocked in the light. In the light this mutant accumulates chlorophyll by 30 times lower as compared to the parent type. In mutant xantha, a very few pigment-protein complexes of PS-I and PS-II are formed in chloroplasts, and formation of membrane system in these is blocked at the early stages, in most cases, at the stage of bubbles and single short thylakoids. Functional activity of reaction centers of PS-I and PS-II is close to zero. Only light-harvesting chlorophyll-a/b protein complexes of the two photosystems are formed in mutant xantha plastid membranes with maximum chlorophyll fluorescence at 728 and 681 nm, respectively. It has been concluded that in mutant xantha genetic block of 5-aminolevulinic acid biosynthesis in the light disturbs the formation and functioning of the complexes of reaction centers of PS-I and PS-II, hindering the development of the whole membrane system in chloroplasts, causing a sharp decrease in productivity.     

Organization and role of the long-wave chlorophylls in the photosystem I of the Cyanobacterium spirulina.

The data on the organization and function of the photosystem I pigment-protein complexes of the cyanobacterium Spirulina and the characteristics of pigment antenna of the photosystem I monomeric and trimeric core complexes are presented and discussed. We proved that the photosystem I complexes in the cyanobacterial membrane pre-exist mainly as trimers, though both types of complexes contribute to the photosynthetic electron transport. In contrast to monomers, the antenna of the photosystem I trimeric complexes of Spirulina contains the extreme long-wave chlorophyll form absorbing at 735 nm and emitting at 760 nm (77 K). The intensity of fluorescence at 760 nm depends strongly on the P700 redox state: it is maximum with the reduced P700 and strongly decreased with the oxidized P700 which is the most efficient quencher of fluorescence at 760 nm. The energy absorbed by the extreme long-wave chlorophyll form is active in the photooxidation of P700 in the trimeric complex. The data obtained indicate that the long-wave form of chlorophyll originates from interaction of the chlorophyll molecules localized on monomeric subunits forming the photosystem I trimer. Kinetic analysis of the P700 photooxidation and light-induced quenching of fluorescence at 760 nm (77 K) allows the suggestion that the excess energy absorbed by the antenna monomeric subunits within the trimer migrates via the extreme long-wave chlorophyll to the P700 cation radical and is quenched, which prevents the photodestruction of the pigment-protein complex.     

Evidence of monomeric photosystem I complexes and phosphorylation of chlorophyll a/c-binding polypeptides in Chroomonas sp. strain LT (Cryptophyceae)

Thylakoid membranes of the cryptophyte Chroomonas sp. strain LT were solubilized with dodecyl-beta-maltoside and subjected to sucrose density gradient centrifugation. The four pigment protein complexes obtained were subsequently characterized by absorption and fluorescence spectroscopy, SDS-PAGE, and Western immunoblotting using antisera against the chlorophyll a/c-binding proteins of the marine cryptophyte Cryptomonas maculata and the reaction-center protein D2 of photosystem II of maize. Band 1 consisted mainly of free pigments, phycobiliproteins, and chlorophyll-a/c-binding proteins. Band 2 represented a major chlorophyll a/c-binding protein fraction. A mixture of photosystem II and photosystem I proteins comprised band 3, whereas band 4 was enriched in proteins of photosystem I. Western immunoblotting demonstrated the presence of chlorophyll a/c-binding proteins and their association with photosystem I in band 4. Phosphorylation experiments showed that chlorophyll a/c-binding proteins became phosphorylated. Negative staining electron microscopy of band B4 revealed photosystem I particles with dimensions of 22 nm. Our work showed that PSI-LHCI complexes of cryptophytes are similar to those of Chlamydomonas rheinhardtii, the diatom Phaeodactylum tricornutum, and higher plants.     

The origin of the long-wavelength fluorescence emission band (77 degrees K) from photosystem I

Isolated photosystem I (PSI)-110 particles, prepared using a minimal concentration of Triton X-100 [J. E. Mullet, J. J. Burke, and C. J. Arntzen (1980) Plant Physiol. 65, 814-822] and further subjected to short-term solubilization with sodium dodecyl sulfate (SDS), were resolved into four pigment-containing bands on polyacrylamide gel electrophoresis (PAGE). We have identified these in order of increasing electrophoretic mobility as being (a) CPIa, (b) CPI, (c) the light-harvesting complex of photosystem I (LHC-I), and (d) a free pigment-zone. LHC-I had an absorption maximum in the red at 668-669 nm and a shoulder at 650 nm, which was resolved by its first-derivative spectrum to indicate the presence of chlorophyll b. LHC-I exhibited a 77 degrees K fluorescence emission maximum at 729-730 nm. The 77 degrees K fluorescence emission maxima of CPIa and CPI, excised from the gel, were at 729 and 722 nm, respectively. The LHC-I band, excised from the gel and rerun on dissociating SDS-PAGE, was resolved into two polypeptide doublets of 24-22.5 and 21-20.5 kDa. The CPIa band under similar conditions was resolved into polypeptides of 68, 24, 22.5, 21, 20.5, 19, 15, and 14 kDa; on the contrary, CPI contained only the 68-kDa polypeptide. When intact thylakoids were subjected to "nondenaturing" SDS-PAGE, LHC-I comigrated with an oligomeric form (dimer) of the light-harvesting chlorophyll a/b pigment-protein that preferentially serves photosystem II (LHCP-II). When this combined LHC-I/LHCP-II pigment-protein band was prepared by SDS-PAGE from isolated stroma lamellae, it exhibited a long-wavelength fluorescence band near 730 nm at 77 degrees K. When a similar preparation was obtained from sucrose density gradients containing SDS [J. Argyroudi-Akoyunoglou and H. Thomou (1981) FEBS Lett. 135, 171-181], it was found to be enriched in a 21-kDa polypeptide. The data suggest that the 21-kDa polypeptide of LHC-I is the chlorophyll-containing polypeptide responsible for the long-wavelength fluorescence of LHC-I; other polypeptides in the complex (20.5, 22.5, and 24 kDa) presumably bind chlorophyll and also serve an antennae function.     

Orientation of pigments and pigment-protein complexes in the diatom Cylindrotheca fusiformis. A linear-dichroism study

The orientation of pigments and pigment-protein complexes of the marine diatom Cylindrotheca fusiformis was studied by linear dichroism at 77 K. The technique of polyacrylamide gel squeezing was used to orient the diatom intact cells, their isolated thylakoid membranes and the three pigment-protein complexes: chlorophyll ac-fucoxanthin, chlorophyll ac and PS I complexes. The data indicate that specific orientation of various pigments exists at all structure levels. Tentative assignments of various features of the linear-dichroism spectra to the major photosynthetic pigments are presented. The orientation of the three pigment-protein complexes with respect to the thylakoid membrane plane and the major axis of the cell is also discussed.     

A diatom light-harvesting pigment-protein complex : purification and characterization

A light-harvesting pigment-protein complex was isolated from the diatom Phaeodactylum tricornutum using the zwitterionic detergent CHAPS (3-[3-cholamidopropyl)dimethylammonio]-1-propanesulfonate). Detergent-solubilized membranes were fractionated by sucrose density gradient centrifugation into three components. The medium density fraction contained chlorophyll a, chlorophyll c, and fucoxanthin. This fraction was purified by DEAE-ion exchange chromatography, and contained chlorophyll a, chlorophyll c, and fucoxanthin in a molar ratio of 2.4:1.0:4.8. Fluorescence emission and excitation spectra of the isolated complex demonstrated that light energy absorbed by chlorophyll c and fucoxanthin was coupled to chlorophyll a fluorescence. Upon denaturation, the apoprotein yielded a polypeptide doublet at 17.5 to 18.0 kilodaltons which accounted for 30 to 40% of the toal membrane protein. These findings indicate that this pigment-protein complex is a major component of the diatom photosynthetic lammellae. The quantitative amino acid composition of the apoprotein was very similar to those reported for other membrane-bound pigment-protein complexes. Based on the protein to chlorophyll a ratio of 7700 grams protein per mole chlorophyll a for the complex, each apoprotein molecule contains, to the nearest integer, two chlorophyll a, one chlorophyll c, and five fucoxanthin molecules. Polyclonal antibodies raised against the 17.5 to 18.0 kilodaltons apoprotein showed a monospecific reaction with only the 17.5 to 18.0 protein zone from denatured P. tricornutum membranes as well as to the nondenatured pigment-protein complex. It appears that this complex is common to other diatom species.     

Quantum yield and rate of formation of the carotenoid triplet state in photosynthetic structures

The formation of the triplet state of carotenoids (detected by an absorption peak at 515 nm) and the photo-oxidation of the primary donor of Photosystem II, P-680 (detected by an absorption increase at 820 nm) have been measured by flash absorption spectroscopy in chloroplasts in which the oxygen evolution was inhibited by treatment with Tris. The amount of each transient form has been followed versus excitation flash intensity (at 590 or 694 nm). At low excitation energy the quantum yield of triplet formation (with the Photosystem II reaction center in the state Q⁻) is about 30% that of P-680 photo-oxidation. The yield of carotenoid triplet formation is higher in the state Q⁻ than in the state Q, in nearly the same proportion as chlorophyll a fluorescence. It is concluded that, for excited chlorophyll a, the relative rates of intersystem crossing to the triplet state and of fluorescence emission are the same in vivo as in organic solvent. At high flash intensity the signal of P-680⁺ completely saturates, whereas that of carotenoid triplet continues to increase.

The rate of triplet-triplet energy transfer from chlorophyll a to carotenoids has been derived from the rise time of the absorption change at 515 nm, in chloroplasts and in several light-harvesting pigment-protein complexes. In all cases the rate is very high, around 8 · 10⁷ s⁻¹ at 294 K. It is about 2–3 times slower at 5 K. The transitory formation of chlorophyll triplet has been verified in two pigment-protein complexes, at 5 K.     

Absorption and fluorescence spectra of chlorophyll-proteins isolated from Euglena gracilis

A spectroscopic study of chlorophyll-protein complexes isolated from Euglena gracilis membranes was carried out to gain information about the state of chlorophyll in vivo and energy transfer in photosynthesis. The membranes were dissociated by Triton X-100 and separated into fractions by sucrose gradient centrifugation and hydroxyapatite chromatography. Four different types of chlorophyll-protein complexes were distinguished from each other and from detergent-solubilized chlorophyll in these fractions by examination of their absorption, fluorescence excitation (400–500 nm) and emission spectra at low temperature. These types were: (1). A mixture of antenna chlorophyll a- and chlorophyll ab-proteins with an absorption maximum at 669 and emission at 682 nm; (2) a P-700-chlorophyll a-protein (chlorophyll: P-700 = 30 : 1), termed CPI with an absorption maximum at 676 nm and emission maxima at 698 and 718 nm; (3) a second chlorophyll a-protein (CPI-2) less enriched in P-700, with an absorption maximum at 676 nm and emission maxima at 680, 722 and 731 nm; (4) a third chlorophyll a-protein (CPa₁) with no P-700, absorption maxima at 670 and 683 nm, and an unusually sharp emission maximum at 687 nm. Treatment of CPa₁ with sodium dodecyl sulfate drastically altered its spectroscopic properties indicating that at least some chlorophyll-proteins isolated with this detergent are partially denatured. The results suggest that the complex absorption spectra of chlorophyll in vivo are caused by varying proportions of different chlorophyll-protein complexes, each with different groups of chlorophyll molecules bound to it and making up a unique entity in terms of electronic transitions.     

Differential distribution of pigment-protein complexes in the Thylakoid membranes of Synechocystis 6803

Thylakoids in Synechocystis 6803, though apparently uniform in appearance in ultrastructure, were found to consist of segments which were functionally dissimilar and had distinct proteomes. These thylakoid segments can be isolated from Synechocystis 6803 by successive ultracentrifugation of cell free extracts at 40,000×g (40?k segments), 90,000×g (90?k segments) and 150,000×g (150?k segments). Electron microscopy showed differences in their appearance. 40?k segments looked feathery and fluffy, whereas the 90?k and 150?k thylakoid membrane segments appeared tiny and less fluffy. The absorption spectra showed heterogeneous distribution of pigment-protein complexes in the three types of segments. The photochemical activities of Photosystem I (PSI) and Photosystem II (PSII) showed unequal distributions in 40?k, 90?k and 150?k segments which were substantiated with low temperature fluorescence measurements. The ratio of PSII/PSI fluorescence emission at 77?K (λ(ex)?=?435?nm) was highest in 150?k segments indicating higher PSII per unit PSI in these segments. The chlorophyll fluorescence lifetimes in the membranes, determined with a time-correlated single-photon counting technique, could be resolved in three components: τ(1) (=)?<40?ps, τ(2) (=)?425-900?ps and τ(3) (=)?2.4-3.2?ns. The percentage contribution of the fastest component (τ(1)) decreased in the order 40?k?>?90?k?>?150?k segments whereas that of the other two components showed a reversed trend. These studies indicated differential distribution of pigment-protein complexes in the three membrane segments suggesting heterogeneity in the thylakoids of Synechocystis 6803.     

The Chlorophyll-Protein Complexes Resolved from Ultrasonically Broken Thylakoid Memb-ranes of Blue-Green Algae

When the thylakoid membranes of blue-green algae were broken by ultrasonic vibrations and subjected to polyacrylamide gel electrophoresis at 4℃, six green zones were resolved. They were designated as CPIa, CPlb, CPI; CPal, CPa2, and FC. The absorption spectrum of CPI had a red maximum at 674 nm and a peak in the blue at 435 nm. It was identified as PS chlorophyll a-protein Complex, but was contaminated with minor PSⅡ which was implied by the appearance of fluorescence emission peak at 680 nm besides the main one at 725 nm at 77 K. The spectral properties of CPIa and CPlb were similar to that of CPl. The absorption spectra of CPa1 and CPa2 were similar, both having red maxima at 667 nm and peaks in the blue at 431.5 nm. Their fluorescence emission had the same peaks at 684 nm at 77 K indicating that they belonged to PSⅡ. It was recognized that CPal of 47 kD is the reaction center complex of photosystem Ⅱ and CPa2 of 40 kD is the internal antenna complex of photosystem Ⅱ. The spectral characteristics of the chlorophyll-protein complexes resolved by ultrasonic method were similar to those of the same complexes resolved by SDS solubilization, except the absorbance positions of CPa1 and CPa2 in the blue peak and the red one which shifted to blue about 3–5 nm. It was calculated that in thylakoid membranes of blue-green algae 40.93% chlorophyll was in PSⅠ, while 38.78% of chlorophyll in PSⅡ. The difference of chlorophyll contents between PSⅠ and PSⅡ was only 2.15%. Concerning the fact that minor PSⅡ compound remained in the part of PSⅠ zones, it might be concluded that the distribution of chlorophyll between PSⅠ and PSⅡ in blue-green algae was equal. This result was in agreement with the hypothesis that PSⅠ and PSⅡ operates in series in photosynthetic electron transport.     

Comparative Study of Chlorophyll-Protein Complexes of Thylakoids of Bryopsis and Spinacia

properties, pigment compositions, Chl a/b ratios and apparent molecular weights of chlorophyll-protein complexes were compared between spinach and a marine green alga, Bryopsis corticulans. The results are as follows: 1. Ten chlorophyll-protein complexes were resolved from spinach thylakoid membranes solubilized by SDS in a final SDS/Chl weight ratio of 10:1, and subjected to SDS-PAGE with 11% resolution gel. CPIa 1–3 and CPI belonged to photosystem Ⅰ, and the rest to phorosystem Ⅱ. The maximum absorption of CPIa2, CPIas and CPI were all at 674nm, but that of CPIa1 at 670nm, and those of LHCII and D2 at 670 and 673nm, respectively. Chlorophyll ia PSⅡ was 63% of the total. In PSⅡ, most of chlorophyll was in LHCII which contained 86% of the chlorophyll in PSⅡ. In PSⅠ, chlorophyll in CPla was 72% of the total. Chlorophyll a was the main pigment in PSⅠ components which have Chl a/b ratio over 15. 2. Eight chlorophyll-protein complexes were isolated from B. corticulans with a SDS/Chi weight ratio of 8:1 and 8% resolution gel. The maximum absorption of CPIa, CPI, LHCII and D2 were respectively at 671nm, 673nm, 669nm and 664nm. PSⅡ contained 77% of the total chlorophyll. LHCII chlorophyll was 95% of the PSⅡ chlorophyll. CPI held 77% of PSⅠ chloro~ phyll. There was more chlorophyll b in Bryopsis complexes, especially in LHCI1 (Chl a/b< 0.8). The molecular weights of Bryopsis complexes were higher than those of the spinach complexes. Bryopsis LHCII contained siphoxanthin and siphothin, the marked pigments of Siphohales, as functional pigments. The above results revealed three points of difference between these two plants. Firstly, Chl a is the main pigment in spinach, whereas in Bryopsis the main pigments are Chl b and siphoxanthin. This is in accordance with the suggestion that plants may change their pigment composition to adapt light regime in the environment during evolution. Secondly, in Bryopsis, chlorophyll is concentrated in photosystem Ⅱ, but in spinach chlorophyll is shared evenly by two photosystems. Finally, CPI in Bryopsis contained the major part of chlorophyll in PSⅠ, yet in spinach CPIa is the superior.     

The nature of a chlorophyll ligand in Lhca proteins determines the far red fluorescence emission typical of photosystem I

Photosystem I of higher plants is characterized by a typically long wavelength fluorescence emission associated to its light-harvesting complex I moiety. The origin of these low energy chlorophyll spectral forms was investigated by using site-directed mutagenesis of Lhca1-4 genes and in vitro reconstitution into recombinant pigment-protein complexes. We showed that the red-shifted absorption originates from chlorophyll-chlorophyll (Chl) excitonic interactions involving Chl A5 in each of the four Lhca antenna complexes. An essential requirement for the presence of the red-shifted absorption/fluorescence spectral forms was the presence of asparagine as a ligand for the Chl a chromophore in the binding site A5 of Lhca complexes. In Lhca3 and Lhca4, which exhibit the most red-shifted red forms, its substitution by histidine maintains the pigment binding and, yet, the red spectral forms are abolished. Conversely, in Lhca1, having very low amplitude of red forms, the substitution of Asn for His produces a red shift of the fluorescence emission, thus confirming that the nature of the Chl A5 ligand determines the correct organization of chromophores leading to the excitonic interaction responsible for the red-most forms. The red-shifted fluorescence emission at 730 nm is here proposed to originate from an absorption band at approximately 700 nm, which represents the low energy contribution of an excitonic interaction having the high energy band at 683 nm. Because the mutation does not affect Chl A5 orientation, we suggest that coordination by Asn of Chl A5 holds it at the correct distance with Chl B5.     

Structurally flexible macro-organization of the pigment–protein complexes of the diatom Phaeodactylum tricornutum

By means of circular dichroism (CD) spectroscopy, we have characterized the organization of the photosynthetic complexes of the diatom Phaeodactylum tricornutum at different levels of structural complexity: in intact cells, isolated thylakoid membranes and purified fucoxanthin chlorophyll protein (FCP) complexes. We found that the CD spectrum of whole cells was dominated by a large band at (+)698 nm, accompanied by a long tail from differential scattering, features typical for psi-type (polymerization or salt-induced) CD. The CD spectrum additionally contained intense (−)679 nm, (+)445 nm and (−)470 nm bands, which were also present in isolated thylakoid membranes and FCPs. While the latter two bands were evidently produced by excitonic interactions, the nature of the (−)679 nm band remained unclear. Electrochromic absorbance changes also revealed the existence of a CD-silent long-wavelength (∼545 nm) absorbing fucoxanthin molecule with very high sensitivity to the transmembrane electrical field. In intact cells the main CD band at (+)698 nm appeared to be associated with the multilamellar organization of the thylakoid membranes. It was sensitive to the osmotic pressure and was selectively diminished at elevated temperatures and was capable of undergoing light-induced reversible changes. In isolated thylakoid membranes, the psi-type CD band, which was lost during the isolation procedure, could be partially restored by addition of Mg-ions, along with the maximum quantum yield and the non-photochemical quenching of singlet excited chlorophyll a, measured by fluorescence transients.     

On the aggregational states of protochlorophyllide and its protein complexes in wheat etioplasts

The inner membranes from wheat ( Triticum aestivum L. cv. Walde) etioplasts were separated into membrane fractions representative of prolamellar bodies and prothylakoids by differential and gradient centrifugations. The isolated fractions were characterized by absorption-, low-temperature fluorescence-, and circular dichroism (CD) spectroscopy, by high performancy liquid chromatography and by sodium dodecyl sulphate polyacrylamide gel electrophoresis.
The prolamellar body fraction was enriched in NADPH-protochlorophyllide oxidoreductase (E.C. 1.6.99.1), and in protochlorophyllide showing an absorption maximum at 650 nm and a fluorescence emission maximum at 657 nm. Esterified protochlorophyllide was mainly found in the prothylakoid fraction. The carotenoid content was qualitatively the same in the two fractions. On a protein basis the carotenoid content was about three times higher in the prolamellar body fraction than in the prothylakoid fraction. The CD spectra of the membrane fractions showed a CD couplet with a positive band at 655 nm, a zero crossing at 643–644 nm and a negative band at 623–636 nm. These results differ from earlier CD measurements on protochlorophyllide holochrome preparations. The results support the interpretation that protochlorophyllide is present as large aggregates in combination with NADPH and NADPH-protochlorophyllide oxidoreductase in the prolamellar bodies.     

Events surrounding the early development of Euglena chloroplasts. 16. Plastid thylakoid polypeptides during greening.

Using sulfolipid to locate plastid thylakoid membranes in gradients from dark-grown resting cells it has been possible to study the plastid thylakoid membrane polypeptides of Euglena gracilis var. bacillaris undergoing light-induced chloroplast development. All plastid thylakoid bands seen in dark-growing wild-type cells and in mutant W3BUL in which plastid DNA is undetectable, are observed to increase in amount during plastid development. Others, which are undetectable in dark-grown wild-type and W3BUL increase greatly during plastid development and appear to be those associated with pigment-protein complexes. The data obtained from experiments where the polypeptides were labeled with 35S during development, either continuously or in pulses, were consistent with these findings. Cycloheximide strongly inhibited the increases in amount in all bands and chloramphenicol or streptomycin produced a lower level of inhibition in all bands indicating tight control of theformation of each plastid membrane constituent by the others. The formation of a polypeptide band of 25 000 molecular weight, thought to be a part of a pigment-protein complex of the thylakoid, and chlorophyll synthesis were inhibited identically by these antibiotics.