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Biomedical subjects

I Ohad

Publications and source records attributed to I Ohad.

At least 73 records · Page 4Linked to original sources
PubMed1984

Membrane protein damage and repair: Selective loss of a quinone-protein function in chloroplast membranes.

A loss of electron transport capacity in chloroplast membranes was induced by high-light intensities (photoinhibition). The primary site of inhibition was at the reducing side of photosystem II (PSII) with little damage to the oxidizing side or to the reaction center core of PSII. Addition of herbicides (atrazine or diuron) partially protected the membrane from photoinhibition; these compounds displace the bound plastoquinone (designated as Q(B)), which functions as the secondary electron acceptor on the reducing side of PSII. Loss of function of the 32-kilodalton Q(B) apoprotein was demonstrated by a loss of binding sites for [(14)C]atrazine. We suggest that quinone anions, which may interact with molecular oxygen to produce an oxygen radical, selectively damage the apoprotein of the secondary acceptor of PSII, thus rendering it inactive and thereby blocking photosynthetic electron flow under conditions of high photon flux densities.

Journal Article↗
PubMed1984

Membrane protein damage and repair: removal and replacement of inactivated 32-kilodalton polypeptides in chloroplast membranes.

Incubation of Chlamydomonas reinhardii cells at light levels that are several times more intense than those at which the cells were grown results in a loss of photosystem II function (termed photoinhibition). The loss of activity corresponded to the disappearance from the chloroplast membranes of a lysine-deficient, herbicide-binding protein of 32,000 daltons which is thought to be the apoprotein of the secondary quinone electron acceptor of photosystem II (the QB protein). In vivo recovery from the damage only occurred following de novo synthesis (replacement) of the chloroplast-encoded QB protein. We believe that the turnover of this protein is a normal consequence of its enzymatic function in vivo and is a physiological process that is necessary to maintain the photosynthetic integrity of the thylakoid membrane. Photoinhibition occurs when the rate of inactivation and subsequent removal exceeds the rate of resynthesis of the QB protein.

Chlamydomonas↗
PubMed1982

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.

Chlamydomonas↗
PubMed1982

Functional Organization of the Chlorophyll-Containing Complexes of Chlamydomonas reinhardi: A Study of Their Formation and Interconnection with Reaction Centers in the Greening Process of the y-1 Mutant.

The stepwise synthesis and assembly of photosynthetic membrane components in the y-1 mutant of Chlamydomonas reinhardi have been previously demonstrated (Ohad 1975 In Membrane Biogenesis, Mitochondria, Chloroplasts and Bacteria, Plenum, pp 279-350). This experimental system was used here in order to investigate the process of formation and interconnection of the energy collecting chlorophylls with the reaction centers of both photosystems I and II. The following measurements were carried out: photosynthetic electron flow at various light intensities, including parts or the entire electron transfer chain; analysis of the kinetics of fluorescence emission at room temperature and fluorescence emission spectra at 77 K, and electrophoretic separation of membrane polypeptides and chlorophyll protein complexes. Based on the data obtained it is concluded that: (a) each photosystem (PSI and PSII) contains, in addition to the reaction center, an interconnecting antenna and a main or light harvesting antenna complex; (b) the formation of the light harvesting complex, interconnecting antenna, and reaction centers for each photosystem can occur independently. (c) the interconnecting antennae link the light harvesting complexes with the respective reaction centers. In their absence, energy transfer between the light harvesting chlorophylls and the reaction centers is inefficient. The formation of the interconnecting antennae and efficient assembly of photosystem components occur simultaneously with the de novo synthesis of chlorophyll and at least three polypeptides, one translated in the cytoplasm and two translated in the chloroplast. The synthesis of these polypeptides was found to be light dependent.

Journal Article↗
PubMed1981

Modulation of PSI and PSII Organization During Loss and Repair of Photosynthetic Activity in a Temperature Sensitive Mutant of Chlorella pyrenoidosa.

Photosynthetic activity and organization of chlorophyll(Chl)-protein complexes in a temperature sensitive mutant of Chlorella pyrenoidosa have been investigated. The mutant is practically indistinguishable from wild type cells when grown at 25 C. However, mutant cells grown at 33 C do not synthesize Chl and lose their ability to evolve O(2). O(2) evolution and Chl synthesis are restored upon incubation of the 33 C grown cells at 25 C in absence of cell division (repair).Based on polarographic measurements of photosynthetic activities, variable fluorescence, 77 K fluorescence emission, excitation spectra, analysis of Chl-protein complexes, membrane polypeptide pattern and radioactive labeling using sodium dodecyl sulfate-polyacrylamide gel electrophoresis techniques during growth at 33 C and/or under repair conditions, it is concluded that: a, polypeptides of chloroplastic translation required for H(2)O-splitting activity are absent from membranes of 33 C grown cells. Their synthesis and/or assembly during the repair process is light-dependent. b, Polypeptides required for the formation of photosystem II and photosytem I reaction centers continue to be formed during growth at 33 C in absence of Chl synthesis. These can be assembled into functional units following Chl synthesis and energization of the membranes during the repair process. c, The Chl-protein complex serving as an antenna of photosystem I is disorganized, and the Chl is used for the formation of functional reaction centers of photosystem I during growth at 33 C.These results show that Chl-protein complexes can be dissociated in vivo and reassembled in a different way; and formation of Chl-protein complexes can occur stepwise from previously synthesized and newly formed components including both polypeptides and Chl.

Journal Article↗
PubMed1980

Chloroplast-cytoplasmic interrelations involved in chloroplast development in Chlamydomonas reinhardi y-1: effect of selective depletion of chloroplast translates.

Chlamydomonas reinhardi y-1 cells grown in the dark in the presence of chloramphenicol (CD cells) are depleted of photosynthetic membranes and 70S translates. These cells were found to be unable to synthesize chlorophyll in the light until chloroplast protein synthesis was resumed. On the other hand, CD cells acquired the capacity to partially green in the presence of cycloheximide. This greening was characterized by the development of photosynthetic activity, as demonstrated by light-dependent oxygen evolution of whole cells and by measurements of ribulose-1,5-bisphosphate carboxylase and fluorescence kinetics. The chlorophyll synthesized de novo during greening in the absence of 80S ribosomal activity was organized in chlorophyll-protein complexes, as ascertained by low-temperature fluorescence-emission spectra. The morphology of these cells appeared to be normal. A model has been proposed as a working hypothesis, which could account for the phenomena described above and previously reported data pertaining to chloroplast development.

Chlamydomonas↗
PubMed1980

Light-induced Changes in Allophycocyanin.

Several lines of evidence indicate that allophycocyanin is the previously unidentified "phycochrome" observed in extracts of blue-green algae.Fractions containing phycoerythrin, phycocyanin, and allophycocyanin and exhibiting light-induced absorbance changes were prepared from extracts of Nostoc muscorum and Fremyella diplosiphon by isoelectric focusing. Illumination of such fractions with red light (650 nanometers) causes a reduction in absorbance at 620 nm ( approximately 1 to 2%) and an increase at 560 nm. The effect, (previously observed by Björn and Björn [1976 Physiol Plant 36: 297-304]) is reversible, upon illumination with green light (550 nm). Selective immunoprecipitation of the phycobiliproteins indicates that allophycocyanin is the photoresponsive pigment.At pH 4.0 to 4.2, allophycocyanin purified from the same algae or from Phormidium luridum exhibits a light-induced absorbance change at 620 nm, which coincides with its absorption maximum at this pH; the fluorescence emission of allophycocyanin under these conditions is at 647 nm and its S(20,w) is 2.28, compatible with an alpha(1)beta(1) polypeptide composition. At neutral pH (5.8 to 7.0), allophycocyanin aggregates have a sedimentation coefficient of 4.8 ( approximately alpha(3)beta(3)) and an additional absorption peak at 640 nm appears while that at 620 nm remains unaffected. The fluorescence emission maximum of the larger aggregate is at 667 nm and the light-induced change in its absorption is shifted to 650 nm. The effect of pH changes in the range 4.0 to 7.0 on the spectral and aggregation properties of allophycocyanin is completely reversible. Changes in pH which affect allophycocyanin aggregation have parallel effects on absorption and fluorescence maxima as well as on the light-induced absorbance changes of the biliprotein.No evidence is provided to resolve whether this phycochrome plays the role of an adaptochrome.

Journal Article↗
PubMed1979

Photoreversible absorbance changes in solutions of allophycocyanin purified from Fremyella diplosiphon: Temperature dependence and quantum efficiency.

Preparations of allophycocyanin isolated from the alga Fremyella diplosiphon show light-induced optical absorbance changes that suggest the presence of a photoconvertible component [Formula: see text] similar to the algal pigments described by J. Scheibe [(1972) Science 176, 1037-1039]. At pH < 4 the allophycocyanin has an absorption maximum at 620 nm. Red illumination causes a loss of absorbance in the red, centered at 620 nm, and subsequent green illumination restores the lost absorbance. We have studied this photoconversion at temperatures between 200 K and 307 K, analyzing the results in terms of photostationary states established under red (640 nm) and green (550 nm) light. As the temperature was lowered to 260 K, the state P(r) became progressively favored; the reaction P(r) --> P(g) induced by red light was attenuated but the reaction P(g) --> P(r) induced by green light was not. Decreasing the temperature from 260 K to 200 K had no further effect. Two distinct and simple models can account for this curious temperature dependence. By analyzing the kinetic and steady-state data, with reasonable estimates of the molar extinction coefficients of P(r) and P(g), we computed quantum efficiencies greater than 15% for the photoconversion at 300 K. We deduced that a conversion of "all P(r)" to "all P(g)" should produce a fractional absorbance change DeltaA/A at 620 nm equal to 0.1. If the chromatic adaptation response of intact F. diplosiphon shows the unusual temperature dependence reported here, the system P(r) right harpoon over left harpoon P(g) will be implicated in mediating this response.

Journal Article↗
PubMed1979

Control of Phycoerythrin Synthesis during Chromatic Adaptation.

Chromatic adaptation is the process by which blue-green algae alter the rates of biliprotein synthesis in response to changes in the color of available light. We have examined the control of phycoerythrin synthesis during the early stages of chromatic adaptation in Fremyella diplosiphon using fluorescence spectroscopy and (35)S-labeling of polypeptides. Phycoerythrin synthesis begins within 45 to 90 minutes after transfer of cells from red to green light, but is blocked by rifamycin. Transfer of cells from green to red light stops phycoerythrin synthesis with a t(1/2) = 45 minutes, as does the addition of rifamycin in green light. Transfer from green light to darkness slows but does not stop phycoerythrin synthesis. Gel electrophoresis of labeled polypeptides, both soluble and membrane-bound, shows that the synthesis of some polypeptides other than phycoerythrin are also affected by changes in light. These data suggest that chromatic adaptation involves gene regulation at the transcriptional level.

Journal Article↗
PubMed1979

Use of enzymatic and electron microscopy (freeze-etching) methods for studying ATP-dependent masking of erythrocyte membrane phospholipids.

Membrane phospholipids of ATP-depleted chicken, rat, and toad erythrocytes are more susceptible than fresh cells to hydrolysis by phospholipase C (Bacillus cereus), phospholipase A (bee venom), or the combination of these enzymes and sphingomyelinase. ATP depletion of chicken and rat erythrocytes greatly increased the membrane phospholipid fraction, which can be extracted by dry ether. Trinitrobenzene sulfonic acid attacked about 20% of the phosphatidylethanolamine of fresh chicken erythrocytes and 45% of ATP-depleted chicken erythrocytes. The intramembranous particles on the PF face of fresh chicken, rat, and toad erythrocytes are evenly distributed, while those in the same ATP-depleted erythrocytes are significantly clustered. The average distance between the intramembranous particles in the PF face of fresh chicken erythrocytes is about 13 nm, while in ATP-depleted erythrocytes it is about 30 nm. Studies with chicken erythrocytes revealed that ATP depletion is accompanied by dephosphorylation of certain membrane polypeptides. The correlation of dephosphorylation of membrane polypeptides, exposure of membrane phospholipids, and clustering of intramembranous particles is discussed.

Adenosine Triphosphate↗
PubMed1978

Chloroplast membrane biogenesis in Chlamydomonas: correlation between the formation of membrane components and membrane structure.

The y-1 mutant of Chlamydomonas reinhardi, when allowed to green in the presence of chloramphenicol (CAP), an inhibitor of protein synthesis on 70s ribosomes, form photosynthetic membranes which contain somewhat less chlorophyll than those of cells greened in the absence of the drug. Photosystem I and II activities are drastically reduced in the CAP-greened cells, and specific alterations in the polypeptide composition of the thylakoid membranes are also observed. We have examined the internal structure of the thylakoid membranes from cells greened in the presence and absence of CAP, and have found that the large particles observed on the exoplasmic fracture face (EF) are substantially reduced in size and number in the CAP-greened cells. This structural defect seems related to the absence of significant photo-system activities in the CAP-greened cells, despite the presence of most major membrane polypeptides. We suggest that CAP treatment results in a failure of the cell to organize functional reaction complexes, and is structurally reflected in the absence of large (EF) particles in such membranes. This defect can be repaired by allowing the affected cells to re-green in the absence of the drug, and the large particles reappear, paralleling an increase in photosynthetic activity.

Chlamydomonas↗
PubMed1977

Appearance and composition of chlorophyll-protein complexes I and II during chloroplast membrane biogenesis in Chlamydomonas reinhardi y-1.

The chlorophyll-protein complexes I and II have been isolated and anlyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis during greening and degreening of Chlamydomonas reinhardi y-1. At all stages of membrane formation, the complexes, when present, have a constant composition. Chlorophyll-protein complex I consists of a major polypeptide(s) of molecular weight 64,000 synthesized in the chloroplast, to which about 29 chlorophyll a molecules are bound. The complex is not detected when other polypeptides of chloroplastic origin, related to both Photosystem I and Photosystem II activities, are not synthesized. However, Photosystem I activity can develop in membranes in which chlorophyll-protein complex I is not detectable. Chlorophyll-protein complex II consists of two polypeptides of cytoplasmic origin, molecular weights 24,000 and 22,000, which bind 12 chlorophylls (a and b). The chlorophyll-protein complex II can be detected in membranes in which the development of photosystem II activity is prevented. Clipping of a Mr = 2000 fragment(s) from the Mr = 22,000 polypeptide following trypsin digestion of membranes, does not affect the complex. The detection of the complexes is possible only in membranes in which the simultaneous synthesis of both the chlorophyll and the corresponding polypeptides occurs. The 28,000 dalton polypeptide, reported to be present in the chlorophyll-protein complex II, comigrates with the complex but apparently is not part of the complex itself. The apparent molecular weight of the chlorophyll-protein complexes I and II are 88,000 and 28,000, respectively. The minimal true value for complex I is 89,000 or 154,000 and for complex II is 56,000.

Binding Sites↗
PubMed1977

Presence of Polypeptides of Cytoplasmic and Chloroplastic Origin in Isolated Photoactive Preparations of Photosystems I and II in Chlamydomonas reinhardi y-1.

Photoactive preparations were obtained by treatment of photosynthetic membranes with lipid micelles and deoxycholate.A photosystem I preparation can be extracted from the membranes by both lipid micelles and deoxycholate, whereas the photosystem II preparation remains in a particulate state.The photosystem II preparation exhibits electron transfer activity measured by photoreduction of 2,6-dichlorophenolindophenol with 1,5-diphenylcarbazide as an electron donor and has a ratio of chlorophyll a/b of 1.4. This preparation contains three major polypeptides of cytoplasmic origin and two of chloroplastic origin.The photosystem I preparation exhibits high rates of methylviologen photoreduction with ascorbate and 2,6-dichlorophenolindophenol as electron donors and does not contain chlorophyll b. This preparation contains two major polypeptides of chloroplastic origin and one of cytoplasmic origin. These results indicate that both cytoplasm and chloroplast contribute polypeptides for the formation of photosystems I and II in Chlamydomonas reinhardi.

Journal Article↗
PubMed1977

Preparation of Antibodies against Specific Chloroplast Membrane Polypeptides Associated with the Formation of Photosystems I and II in Chlamydomonas reinhardi y-1.

The major Chlamydomonas reinhardi y-1 chloroplast membrane polypeptides-I + II, IV, V(a + b)-have been isolated by use of preparative sodium dodecyl sulfate-gel electrophoresis.Rabbit antisera prepared against these polypeptides interact with intact membranes as well as membrane fractions containing these specific antigens. Antisera against polypeptides I + II partially inactivate photosystem I. Antisera against polypeptide IV prevent inactivation of the Hill reaction and inhibit photooxidation of diphenylcarbazide with dichlorophenolindophenol as an electron acceptor. The protection of the Hill reaction by IV antiserum, measured with whole membranes, is partially hindered by the presence of V antiserum, which, by itself, causes inhibition of diphenylcarbazide photooxidation.We concluded that parts of the primary structure of these polypeptides are exposed in situ and can be recognized in whole membranes by antisera prepared against sodium dodecyl sulfate-denatured antigens. Polypeptides I and II, and polypeptides IV, Va, and Vb are associated with photosystem I and photosystem II, respectively.

Journal Article↗