Effect of heat and 2-mercaptoethanol on intracytoplasmic membrane polypeptides of Rhodopseudomonas sphaeroides.

Solubilization at 75 degrees C of Rhodopseudomonas sphaeroides chromatophores in the presence of sodium dodecyl sulfate (SDS) and 2-mercaptoethanol (beta-ME) resulted in the selective absence of reaction center B and C polypeptides from SDS-polyacrylamide gel electrophoresis profiles. A newly identified, chromatophore-specific polypeptide, with a mass of 35.2 kdaltons, was also missing under these conditions of chromatophore solubilization. Solubilization at 27 degrees C in the presence of SDS and beta-ME also resulted in the disappearance of these three polypeptides, but at much slower rates. Disappearance of either endogenous or exogenously supplied reaction center polypeptides B and C during SDS solubilization of whole chromatophores at either 27 or 75 degrees C was shown to be entirely dependent upon the presence of beta-ME. After chromatophore solubilization in the presence of beta-ME and subsequent SDS-polyacrylamide gel electrophoresis, exogenously added reaction centers B and C could be localized in a complex of no less than 100 to 200 kdaltons. However, the precise size of the complex was influenced by the stoichiometry of the reacting components. The disappearance of the 35.2-kdalton polypeptide was neither dependent upon the presence of beta-ME nor dependent upon the presence of any additional chromatophore polypeptides. The 35.2-kdalton polypeptide underwent a heat-induced oligomerization to yield several high-molecular-weight species.     

Composition of a Photosystem I chlorophyll protein complex from Anabaena flos-aquae

The use of Triton X-100 to solubilize membrane fragments from Anabaena flos-aquae in conjunction with DEAE cellulose chromatography allows the separation of three green fractions. Fraction 1 is detergent-solubilized chlorophyll, and Fraction 2 contains one polypeptide in the 15 kdalton area. Fraction 3, which contains most of the chlorophyll and shows P-700 and photosystem I activity, shows by SDS gel electrophoresis varying polypeptide profiles which reflect the presence of four fundamental bands as well as varying amounts of other polypeptides which appear to be aggregates containing the 15 kdalton polypeptide. The four fundamental bands are designated Band I at 120, Band II at 52, Band III at 46, and Band IV at 15 kdaltons. Band I obtained using 0.1% SDS contains chlorophyll and P-700 associated with it. When this band is cut out and rerun, the 120 kdalton band is lost, but significant increases occur in the intensities of Bands II, III, and IV as well as other polypeptides in the 20–30 kdalton range.The use of 1% Triton X-100 coupled with sucrose density gradient centrifugation allows the separation of three green bands at 10, 25 and 40% sucrose. The 10% layer contains a major polypeptide which appears to be Band IV. The 25 and 40% layers show essentially similar polypeptide profiles, resembling Fraction 3 in this regard, except that the 40% layer shows a marked decrease in Band III. Treatment of the material layering at the 40% sucrose level with a higher (4%) concentration of Triton X-100 causes a loss (disaggregation) of the polypeptides occurring in the 60–80 kdalton region and an increase in the lower molecular weight polypeptides. Thus, aggregation of the lower molecular weight polypeptides accounts for the variability seen in the electrophoresis patterns. Possible relations of the principal polypeptides to the known photochemical functions in the original membrane are discussed.     

Triton X-114 phase fractionation of membrane proteins of the cyanobacterium Anacystis nidulans R2

The thylakoid polypeptides of the cyanobacterium Anacystis nidulans R2 were analyzed by Triton X-114 phase fractionation [C. Bordier (1981) J. Biol. Chem.256, 1604–1607, as adapted for photosynthetic membranes by T. M. Bricker and L. A. Sherman (1982) FEBS Lett.149, 197–202]. In this procedure, polypeptides with extensive hydrophobic regions (i.e., intrinsic proteins) form mixed micelles with Triton X-114, and are separated from extrinsic proteins by temperature-mediated precipitation of the mixed Triton X-114-intrinsic protein micelles. The polypeptide pattern after phase fractionation was highly complementary, with 62 of the observed 110 polypeptide components partitioning into the Triton X-114-enriched fraction. Identified polypeptides fractionating into the Triton X-114 phase included the apoproteins for Photosystems I and II, cytochromes f and b₆, and the herbicide-binding protein. Identified polypeptides fractioning into the Triton X-114-depleted (aqueous) phase included the large and small subunits of RuBp carboxylase, cytochromes c₅₅₀ and c₅₅₄, and ferredoxin. Enzymatic radioiodination of the photosynthetic membranes followed by Triton X-114 phase fractionation allowed direct identification of intrinsic polypeptide components which possess surface-exposed regions susceptible to radioiodination. The most prominent of these polypeptides was a 34-kDa component which was associated with photosystem II. This phase partitioning procedure has been particularly helpful in the clarification of the identity of the membrane-associated cytochromes, and of photosystem II components. When coupled with surface-probing techniques, this procedure is very useful in identifying intrinsic proteins which possess surface-exposed domains. Phase fractionation, in conjunction with the isolation of specific membrane components and complexes, has allowed the identification of many of the important intrinsic thylakoid membrane proteins of A. nidulans R2.     

Effect of linolenic acid on release of the 43 kDa polypeptide and manganese from photosystem II membranes depleted of the 33 kDa extrinsic polypeptide

The effect of linolenic acid (18:3) on release of the 43 kDa polypeptide and manganese from photosystem II ( PS II ) membranes depleted of extrinsic polypeptides was studied. In both control and NaCl-washed particles which were depleted of the extrinsic 23 and 16 kDa polypeptides, the 18:3 treatment caused a 20% release of the 33 and 43 kDa polypeptides. In CaCl₂, (or urea + NaCl)-washed particles, which were depleted of the 33 kDa polypeptide in addition to the 23 and 16 kDa polypeptides, the release of the 43 kDa polypeptide increased to 70%, whereas only 25% of the 47 kDa polypeptide was removed. These findings suggest (i) that the 33 and the 43 kDa polypeptides are neighbows in the photosynthetic membrane and (ii) that the 33 kDa polypeptide shields the 43 kDa polypeptide against the action of 18:3. Incubation of CaCl₂, or (urea + NaCI)-treated PSII particles in the presence or absence of 18:3 resulted in the loss of only 2 of the 4 Mn atoms present per reaction center. this indicates that the 2 Mn atoms more firmly associated with PSII are not affected by the removal of the extrinsic 16, 23 and 33 kDa polypeptides, and the intrinsic 43 kDa polypeptide. nor by the treatment with linolenic acid.     

Protein composition and architecture of the photosynthetic membranes from the cyanobacterium, Anacystis nidulans R2

The protein composition of the photosynthetic membrane from the cyanobacterium, Anacystis nidulans R2, was analyzed by acrylamide gel electrophoresis following solubilization with lithium dodecyl sulfate. Autoradiograms of ³⁵S-labelled membranes revealed over 90 bands by this procedure. The effect of solubilization conditions on protein resolution was analyzed by modifying temperature and sulfhydryl concentrations. Labelling cells with ⁵⁹Fe yielded nine iron-containing bands on these gels. Three of these bands, at 33, 19, and 14 kDa, were also heme proteins as determined by tetramethylbenzidine staining, and represent cytochromes f, b₆ and c-552, respectively. The remaining iron proteins are highly sensitive to solubilization conditions, especially the presence of 2-mercaptoethanol, and we suggest that these bands may be Fe-S proteins. Lactoperoxidase-catalyzed iodination of the membranes indicated that at least 41 proteins have surface-exposed domains. Some of the known proteins with external surfaces include cytochrome c-552 and the chlorophyll-binding proteins of Photosystems I and II. Neither cytochrome f nor b₆ appear to be accessible to external labelling. When this structural information was combined with the isolation of functional submembrane complexes, we constructed a topological model of the membrane. Using this model we have discussed the protein architecture of the cyanobacterial membrane.     

Phosphorylation of chlamydomonas reinhardi chloroplast membrane proteins in vivo and in vitro

Phosphorylation of thylakoid membrane proteins in the chloroplast of wild-type and mutant strains of Chlamydomonas reinhardi has been studied in vivo and in vitro. Intact cells or purified membranes were labeled with [32P]orthophosphate or [gamma-32P]ATP, respectively, and the presence of phosphorylated polypeptides was detected by autoradiography after membrane fractionation by SDS PAGE. The 32P was esterified to serine and threonine residues. At least six polypeptides were phosphorylated in vitro and in vivo, and corresponded to components of the photosystem II complex contributing to the formation of the light-harvesting-chlorophyll (LHC) a,b-protein complex, the DCMU binding site (32-35 kdaltons), and the reaction center (26 kdaltons). In agreement with previous reports (Alfonzo, et al., 1979, Plant Physiol., 65:730-734; and Bennett, 1979, FEBS (Fed. Eur. Biochem. Soc.) Lett., 103:342-344), the membrane-bound protein kinase was markedly stimulated by light in vitro via a mechanism requiring photosystem II activity. Phosphorylation of thylakoid membrane polypeptides in vivo was, however, completely independent of illumination. Similar amounts of phosphate were incorporated into the photosynthetic membranes of cells incubated in the dark, in white light with or without 3-(3,4- dichlorophenyl-1,1-dimethyl urea (DCMU), or in red or far-red light. Different turnovers of the phosphate were observed in the light and dark, and a phosphoprotein phosphatase involved in this turnover process was also associated with the membrane. Comparison of the amount of esterified phosphate per protein in vivo and the maximum incorporation in isolated membranes revealed that only a small fraction of the available sites could be phosphorylated in vitro. In contrast to the DCMU binding site, the LHC and 26-kdalton polypeptide were not phosphorylated in vivo when the reaction center II polypeptides of 44- 54 kdaltons were missing. The finding that all the phosphoproteins appear to be components of the photosystem II complex and are only partially dephosphorylated in vivo suggests strongly that protein phosphorylation might play an important role in the maintenance of the organizational integrity of this complex. The observation that the LHC is not phosphorylated in the absence of the reaction center lends support to this idea.     

Nitrate-induced polypeptides in membranes from corn seedling roots

The polypeptide composition of the membranes from corn (Zeamays L.) seedling roots upon nitrate induction was determinedby two-dimensional gel electrophoresis and silver-staining.The synthesis of five polypeptides (49, 48, 35, 33, and 32 kDa)in the tono-plast fraction and four polypeptides (50, 49, 38,and 33 kDa) in the plasma membrane fraction was induced by both2.5 mM Ca(NO₃)₂ and 5 mM KNO₃. Extensive washing of the membraneswith salt and NaOH demonstrated that three induced polypeptides(49, 48, and 35 kDa) in the tonoplast fraction and two inducedpolypeptides (49 and 33 kDa) in the plasma membrane fractionwere integral proteins. After incubation of seedlings in N-freemedium for 4 d, the 49 and 32 kDa polypeptides in the tonoplastfraction had disappeared. By the sixth day in N-free medium,the 35 kDa polypeptide had disappeared from the tonoplast fraction.The 50 kDa polypeptide of the plasma membrane fraction was nolonger detectable in seedlings incubated for 6 d in N-free medium.The size of the spots corresponding to the 33 kDa polypeptidesof both membrane fractions and to the 49 kDa polypeptide ofthe plasma membrane fraction was reduced following incubationof seedlings in N-free medium. The changes in nitrate-inducedpolypeptides in both membrane fractions following transfer toN-free medium correlated with a reduced capacity to take upnitrate in the treated seedlings. The results support the conclusionthat the nitrate-induced polypeptides may be involved in nitratetransport across the tonoplast and plasma membrane. Key words: Nitrate transport, induction, membrane peptides     

Structure of a bacterial photosynthetic membrane. Isolation, polypeptide composition, and selective proteolysis

A procedure for the isolation of highly purified bacterial photosynthetic membranes from Rhodopseudomonas viridis is described. The purity of the final membrane fraction has been confirmed by electron microscopy. Seven major polypeptide bands are associated with the photosynthetic membranes, and all seven are resistant to solubilization in Triton X-100 detergent. Two pigmented bands with apparent molecular weights of 44K and 41K are thought to be cytochromes. The three polypeptides with apparent molecular weights of 38K, 32K, and 28K have been reported in reaction center preparations of other laboratories. Two low-molecular-weight (16K and 11K) bands bind bacteriochlorophyll b and may represent light-harvesting bacteriochlorophyll-protein complexes. The structures that were isolated seem to represent complete photosynthetic membranes, consisting of reaction center, electron transport, and light-harvesting components, all arranged in the regular lattice characteristic of viridis. Selective proteolysis of these membranes indicates that all membrane components are accessible to digestion by trypsin and pronase, except for the light-harvesting complexes.     

Polypeptide composition of a Photosystem II core complex. Presence of a herbicide-binding protein

The polypeptide composition of a Photosystem II (PS II) core complex from higher plant chloroplasts has been characterized by subjecting the isolated complex to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two polypeptides in the 40–50 kDa size class, attributed to the chlorophyll a-binding apoproteins of PS II, were resolved when the urea concentration in the SDS-polyacrylamide gel electrophoresis was greater than 1 M. The two chlorophyll a-binding proteins were dissimilar in their primary structure based upon their different hydrolysis products on SDS-polyacrylamide gel electrophoresis following papain treatment. The core complex contained three additional polypeptides. Two polypeptides in the 30–34 kDa size class were resolved when the urea concentration in the gel system was increased to greater than 4 M. One of the polypeptides in this size class was identified as the herbicide-binding protein from azido[¹⁴C]atrazine labeling studies. The herbicide-binding protein displayed an anomalous electrophoretic migration behavior in SDS-polyacrylamide gel electrophoresis in the presence or absence of urea; its apparent molecular weight decreased when the urea concentration increased. The fifth protein component of the core complex was attributed to cytochrome b-559 which was found to consist of the ascorbate- and dithionite-reducible forms in the samples prior to SDS solubilization.     

Identification of a 32–34-kilodalton polypeptide as a herbicide receptor protein in Photosystem II

Photosystem II particles which retained high rates of herbicide-sensitive activity were used to examine the site(s) of action of various herbicides. A polypeptide of 32–34 kdaltons was identified as the triazine-herbicide binding site based upon: (a) parallel loss of atrazine activity and the polypeptide during either trypsin treatment or selective detergent depletion of protein in the Photosystem II complex, and (b) covalent labeling of the polypeptide by a ¹⁴C-labeled photoaffinity triazine.In Photosystem II particles depleted of the 32–34-kdalton polypeptide, electron transport was still active and was slightly sensitive to DCMU and largely sensitive to dinoseb (urea and nitrophenol herbicides, respectively). On the basis of this result it is proposed that the general herbicide binding site common to atrazine, DCMU and dinoseb is formed by a minimum of two polypeptides which determine affinity and/or mediate herbicide-induced inhibition of electron transport on the acceptor side of Photosystem II.     

Partial reconstitution of the photosynthetic oxygen evolution system by rebinding of the 33-kDa polypeptide

Treatment with 2.6 M urea of the Photosystem II particles depleted of two polypeptides of 24 kDa and 18 kDa completely released a polypeptide of 33 kDa and eliminated the oxygen-evolution activity. The 33-kDa polypeptide rebound to the urea-treated particles and partially reactivated the oxygen evolution. A quantitative analysis of the rebinding suggests tha there is a specific binding site for the 33-kDa polypeptide on the membrane surface.     

Cytochromes and prenylquinones in preparations of cytoplasmic and thylakoid membranes from the cyanobacterium (blue-green alga) Anacystis nidulans

The cytochrome and prenylquinone compositions were compared for cytoplasmic membranes and thylakoid membranes from the cyanobacterium (blue-green alga) Anacystis nidulans. Reduced-minus-oxidized difference absorption spectra at ?196°C indicated that the thylakoid membranes contained photosynthetic cytochromes such as cytochrome ?, cytochrome b-559 and cytochrome b₆, while cytochromes c-549 and c-552 were detected spectrophotometrically only after their release by sonic oscillation. The cytoplasmic membrane preparation contained one or two low-potential cytochrome(s) with α-band maxima at 553 and 559 nm at ?196°C, which differed from the cytochromes in the thylakoid membranes. A cytochrome specific to the cytoplasmic membranes was also found by heme-staining after lithium dodecyl sulfate-polyacrylamide gel electrophoresis. Both types of membranes contained the three prenylquinones plastoquinone-9, phylloquinone and 5′-monohydroxyphylloquinone, but in different proportions.     

Role of the 33-kDa polypeptide in preserving Mn in the photosynthetic oxygen-evolution system and its replacement by chloride ions

Treatment of Photosystem II particles from spinach chloroplasts with Triton X-100 with 2.6 M urea in the presence of 200 mM NaCl removed 3 polypeptides of 33 kDa, 24 kDa and 18 kDa, but left Mn bound to the particles. The (urea + NaCl)-treated particles could evolve oxygen in 200 mM, but not in 10 mM NaCl. Mn was gradually released with concomitant loss of oxygen-evolution activity in 10 mM NaCl but not in 200 mM Cl^?. The NaCl-treated particles, which contained Mn and the 33-kDa polypeptide but not the 24-kDa and 18-kDa polypeptides, did not lose Mn or oxygen-evolution activity in 10 mM NaCl. These observations suggest that the 33-kDa polypeptide maintains the binding of Mn to the oxygen-evolution system and can be functionally replaced by 200 mM Cl^?.     

Composition of a photosystem I chlorophyll protein complex from Anabaena flos-aquae.

The use of Triton X-100 to solubilize membrane fragments from Anabaena flos-aquae in conjunction with DEAE cellulose chromatography allows the separation of three green fractions. Fraction 1 is detergent-solubilized chlorophyll, and Fraction 2 contains one polypeptide in the 15 kdalton area. Fraction 3, which contains most of the chlorophyll and shows P-700 and photosystem I activity, shows by SDS gel electrophoresis varying polypeptide profiles which reflect the presence of four fundamental bands as well as varying amounts of other polypeptides which appear to be aggregates containing the 15 kdalton polypeptide. The four fundamental bands are designated Band I at 120, Band II at 52, Band III at 46, and Band IV at 15 kdaltons. Band I obtained using 0.1% SDS contains chlorophyll and P-700 associated with it. When this band is cut out and rerun, the 120 kdalton band is lost, but significant increases occur in the intensities of Bands II, III, and IV as well as other polypeptides in the 20-30 kdalton range. The use of 1% Triton X-100 coupled with sucrose density gradient centrifugation allows the separation of three green bands at 10, 25 and 40% sucrose. The 10% layer contains a major polypeptide which appears to be Band IV. The 25 and 40% layers show essentially similar polypeptide profiles, resembling Fraction 3 in this regard, except that the 40% layer shows a marked decrease in Band III. Treatment of the material layering at the 40% sucrose level with a higher (4%) concentration of Triton X-100 causes a loss (disaggregation) of the polypeptides occurring in the 60-80 kdalton region and in increase in the lower molecular weight polypeptides. Thus, aggregation of the lower molecular weight polypeptides accounts for the variability seen in the electrophoresis patterns. Possible relations of the principal polypeptides to the known photochemical functions in the original membrane are discussed.     

Triton X-100 as an effective surfactant for the isolation and purification of photosystem I from Arthrospira platensis

Surfactants play important roles in the preparation, structural, and functional research of membrane proteins, and solubilizing and isolating membrane protein, while keeping their structural integrity and activity intact is complicated. The commercial n-Dodecyl-β-D-maltoside (DDM) and Triton X-100 (TX) were used as solubilizers to extract and purify trimeric photosystem I (PSI) complex, an important photosynthetic membrane protein complex attracting broad interests. With an optimized procedure, TX can be used as an effective surfactant to isolate and purify PSI, as a replace of the much more expensive DDM. A mechanism was proposed to interpret the solubilization process at surfactant concentrations lower than the critical solubilization concentration. PSI-TX and PSI-DDM had identical polypeptide bands, pigment compositions, oxygen consumption, and photocurrent activities. This provides an alternative procedure and paves a way for economical and large-scale trimeric PSI preparation.     

Photosynthetic Membranes of Porphyridium cruentum: An Analysis of Chlorophyll-Protein Complexes and Heme-Binding Proteins

Three chlorophyll-protein complexes (CP I, CP III, CP IV) were electrophoretically separated from thylakoids of the eukaryotic red alga Porphyridium cruentum. CP I contained the primary photochemical reaction center of photosystem I as judged by its light-induced reversible absorbance change at 700 nanometers, by its fluorescence emission maximum at 720 nanometers (−196°C), and by the molecular weight of its apoprotein (68,000 daltons). CP III and CP IV appeared to belong with photosystem II as suggested by the absence of light-reversible absorbance at 700 nanometers, by their fluorescence maximum at 690 nanometers (−196°C), and by the presence of a chlorophyll-binding polypeptide with a molecular weight of about 52,000 daltons. CP IV when completely denatured had two additional polypeptides of about 40,000 and 48,000 daltons. All three chlorophyll-protein complexes contained carotenoids: the chlorophyll/carotenoid molar ratio of 15:1 for CP I, and 20:1 for CP III and CP IV. The thylakoid membranes of P. cruentum contained four cytochromes, detected by heme-dependent peroxidase activity, but there was no observed association with the electrophoretically separated chlorophyll-protein complexes.     

Dynamic aspects of phycobilisome structure: Modulation of phycocyanin content of Synechococcus phycobilisomes

Phycobilisomes of the cyanobacterium Synechococcus 6301 contain the phycobiliproteins phycocyanin, allophycocyanin, and allophycocyanin B, and four major non pigmented polypeptides of 75, 33, 30, and 27 kdaltons. The molar ratio of phycocyanin to allophycocyanin in wild type phycobilisomes can be varied over about a two-fold range by alterations in culture conditions with parallel changes in the amounts of the 33 and 30 kdalton polypeptides whereas the levels of the 27 and 75 kdalton polypeptides do not vary. Two nitrosoguanidine-induced mutants, AN112 and AN135, produce abnormally small phycobilisomes, containing only 35 and 50% of the wild type level of phycocyanin. AN135 phycobilisomes contain less 33 kdalton polypeptide than wild type and the 30 kdalton polypeptide is only detected in phycobilisomes from cultures grown under conditions favoring high levels of phycocyanin. AN112 lacks both the 30 and 33 kdalton polypeptides and produces phycobilisomes of constant size and composition, independent of growth conditions. Both mutant phycobilisomes have wild type levels of 27 and 75 kdalton polypeptides relative to allophycocyanin and have normal energy transfer properties. These results indicate that modulation of phycobilisome size involves concurrent regulation of the levels of phycocyanin and of both the 30 and 33 kdalton polypeptides with no change in the composition of the allophycocyanin-containing core.Abbreviations LP cells cells grown under conditions favoring low p phycobiliprotein levels - HP cells cells grown under conditions favoring high phycobiliprotein levels - SDS sodium dodecylsulfate - EDTA ethylenediamine tetraacetic acid - NaK-PO₄ NaH₂PO₄ titrated with K₂HPO₄ to a given pH A preliminary report of some of this work was presented at the 81st Annual Meeting of the American Society for Microbiology, Dallas, Texas, March 1981     

Effects of light intensity on membrane differentiation in Rhodopseudomonas capsulata

Cells of Rhodopseudomonas capsulata, strain 37b4, leu^?, precultivated anaerobically under low light intensity, were exposed to high light intensity (2000 W · m^?2). The cells grew with a mass doubling time of 3 h. The synthesis of bacteriochlorophyll (BChl) began after two doublings of cell mass. Reaction center and light-harvesting BChl I (B-875) were the main constituents of the photosynthetic apparatus incorporated into the membrane. The size of the photosynthetic unit (total BChl/reaction center) decreased and light-harvesting BChl I became the dominating BChl species. Concomitant with the appearance of the different spectral forms of BChl the respective proteins were incorporated into the membrane, i.e. the three reaction center polypeptides, the polypeptide associated with light-harvesting BChl I, the two polypeptides associated with BChl II. A polypeptide of an apparent molecular weight of 45 000 was also incorporated. A lowering of the light intensity to 7 W · m^?2 resulted in a lag phase of growth for 6 h. Afterwards, the time for doubling of cell mass was 11 h. The concentration of all three BChl complexes (reaction center, light-harvesting BChl I and II complexes)/cell and per membrane protein increased immediately. Also the size of the photosynthetic unit and the amount of intracytoplasmic membranes/cell increased.The activities of photophosphorylation, succinate dehydrogenase, NADH dehydrogenase and NADH oxidation (respiratory chain)/membrane protein are higher in membrane preparations isolated from cells grown at high light intensities than in such preparations from cells grown at low light intensities.     

Modulation of the Light-Stimulated Loss of Polypeptides from Isolated Wheat Thylakoids in vitro

Exposure of thylakoids free of vacuolar proteases to white light causes the loss of several thylakoid bound polypeptides. At a light intensity of 1,500 μE m^-2 s^-1, such loss is apparent within 5 min although this light intensity does not saturate the reaction. This degradation of thylakoid polypeptides proceeds most rapidly at a pH of 9.0. The rate of polypeptide degradation can be increased by incubation of thylakoids with low concentrations of the detergents Triton X-100 or SDS. Inclusion of an electron transport inhibitor or an uncoupler Of photosynthetic phosphorylation in the assay had no effect on the loss of thylakoid polypeptides in the light. Pre-digestion of thylakoids with trypsin or denaturing thylakoid proteins in a buffered solution of 2 % SDS, 6 M urea at 100 °C for five min prior to the assay did not prevent the loss of thylakoid polypeptides. These data strongly suggest that the light-stimulated loss of polypeptides is not mediated by a protease. The loss of thylakoid polypeptides could be prevented by a variety of reducing agents or by maintaining thylakoids in an anaerobic environment. These data suggest that a species of activated oxygen, probably singlet oxygen, is responsible for the loss of thylakoid polypeptides in the light.     

SDS-PAGE comparative studies on the polyhedral and viral polypeptides of the nuclear polyhedrosis viruses of Mamestra brassicae, Autographa californica, and Lymantria dispar

After solubilization of polyhedra of Autographa californica, Lymantria dispar, and Mamestra brassicae nuclear polyhedrosis viruses, PAGE showed at least eight distinct polyhedral polypeptide bands. Whereas the molecular weights of the major polypeptide were similar for the three NPVs (28.0–30.0 kdalton), characteristic differences between the species were found for the minor polypeptides having molecular weights in the range from 12.4 to 62.0 kdalton. It is assumed that these polypeptides are not generated by polyhedral alkaline protease since they are detected after protease inactivation. The data demonstrate that different baculoviruses can be distinguished from each other by SDS-PAGE of their polyhedral polypeptides.