Comparative Immunological and Chemical Analysis of Lipids and Carotenoids of the D1-Peptide and of the Light-Harvesting-Complex of Photosystem II of Nicotiana tabacum

1999 ◽  
Vol 54 (3-4) ◽  
pp. 199-208 ◽  
Author(s):  
Anette Gasser ◽  
Stefan Raddatz ◽  
Alfons Radunz ◽  
Georg H. Schmid
Keyword(s):  
Fatty Acids ◽  
Nicotiana Tabacum ◽  
Photosystem Ii ◽  
Western Blot ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Light Harvesting Complex ◽  
Total Fatty Acids ◽  
Monospecific Antisera ◽  
Β Carotene

The light-harvesting-complex (LHCP) was isolated from photosystem II of Nicotiana tabacum var. John William’s Broadleaf by means of the detergent acetyl-β-ᴅ-glucopyranoside and fractionating centrifugation. The D1-peptide of photosystem II was isolated as a dimer with the molecular mass of 66 kDa from the chlorophyll-deficient tobacco mutant N. tabacum Su/su by means of sodium dodecyl sulfate polyacrylamide gel electrophoresis. Both preparations were characterized by means of the Western blot procedure using monospecific antisera to the proteins of photosystem II and monospecific antisera to lipids with which the lipids bound to peptides were determined. In parallel to this, lipids bound to the isolated LHCP-complex and to the isolated D1-peptide were determined by lipido-chemical methods. The extraction of the isolated core peptide D1 with a mixture of boiling methanol and chloroform and subsequent HPLC-chromatography showed that in the D1-peptide isolated via SDS-polyacrylamide gel electrophoresis, monogalactolipids, phosphatidylglycerol and sulfolipid molecules are bound in the molar ratio 1:3:17. By means of the immunological procedure of Western blotting we were able to show that the 66 kDa band of the isolated dimeric D1 reacts positively only with the antisera to monogalactolipid, sulfolipid, β-carotene and violaxanthin. With the antiserum to digalactolipid and that to phosphatidylglycerol a positive reaction is only observed if the preparation used in the Western blot is not the isolated D1-peptide but a ‘‘total” photosystem Il-preparation. The lipid extraction of the LHCP-complex and the subsequent analysis by thin-layer chromatography led to the result that the isolated LHCP-complex contained in bound form 3 molecules monogalactolipid, 1 molecule of digalactolipid, 1 molecule of phosphatidylglycerol and 1 molecule of lutein. Less than 1 molecule of sulfolipid, β-carotene, neoxanthin and violaxanthin are found. In the Western blot analysis only the antiserum to monogalactolipid and phosphatidylglycerol and among the carotenoid antisera only the antisera to β-carotene, violaxanthin and to neoxanthin reacted. With the antisera to the digalactolipid, to the sulfolipid and the antisera to the xanthophylls, namely to lutein and neoxanthin, a positive reaction occurred only if the material used in the Western Blot was the “total” photosystem Il-preparation. By gas chromatography of the fatty acids of the isolated peptide fractions it was shown that, compared to the lipids of photosystem II and of the thylakoid membrane, in lipids of the isolated D1-peptide and of the LHCP-complex the saturation degree of fatty acids is strongly increased. Whereas palmitic acid in chloroplast lipids makes up for only 11% of the fatty acids, this saturated fatty acid increases in the lipids of the LHCP to 20% and makes up for 74% of total fatty acids in the lipids of the D1-peptide. Linoleic and linolenic acids are completely absent and oleic acid makes up for 14% of total fatty acids. In contrast to the lipids of the thylakoid membrane, the lipids bound to proteins/peptides are characterized by a strongly saturated character.

Lipopolysaccharide and proteins of the cell envelope of Vibrio marinus, a marine bacterium

1973 ◽  
Vol 19 (10) ◽  
pp. 1211-1217 ◽  
Author(s):  
Carl F. Deneke ◽  
R. R. Colwell
Keyword(s):  
Fatty Acids ◽  
Gel Electrophoresis ◽  
Marine Bacterium ◽  
Lipid A ◽  
Cell Envelope ◽  
Polyacrylamide Gel Electrophoresis ◽  
Amino Sugar ◽  
High Ratio ◽  
Side Chain ◽  
Total Fatty Acids

Lipopolysaccharides isolated from the marine bacterium Vibrio marinus strain PS-207 were found to be similar to the lipopolysaccharides of R mutants of enteric organisms, with respect to extraction characteristics, percentage of lipid A (61%), and sugars of the polysaccharide side chain (glucose and heptose). A high ratio (2:1) of phosphate to amino sugar was found in the lipid A. Hydroxy fatty acids constituted only 14% of the total fatty acids of the lipid A fraction, whereas branched and straight-chain fatty acids were present in greater abundance. The major envelope proteins of V. marinus strain PS-207 fell into three molecular weight classes determined by SDS gel electrophoresis. Numerous protein species were observed in urea – acetic polyacrylamide gel electrophoresis preparations.

Binding of Lipids onto Polypeptides of the Thylakoid Membrane I. Galactolipids and Sulpholipid as Prosthetic Groups of Core Peptides of the Photosystem II Complex

1992 ◽  
Vol 47 (5-6) ◽  
pp. 406-415 ◽  
Author(s):  
R. Voß ◽  
A. Radunz ◽  
G. H. Schmid
Keyword(s):  
Photosystem Ii ◽  
Gel Electrophoresis ◽  
Light Harvesting ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Ps Ii ◽  
Light Harvesting Complex ◽  
Peptide Composition ◽  
Molecular Masses ◽  
Prosthetic Groups

Photosystem II complexes were prepared from chloroplasts of wild type tobacco Nicotiana tabacum var. John William’s Broadleaf and from two chlorophyll mutants derived from it, namely N. tabacum Su/su and N. tabacum Su/su var. Aurea. The hydrophobic peptides of these complexes were analyzed for bound lipid molecules by means of monospecific lipid antisera. A comparison of the peptide composition of the complexes of the three chloroplast types by means of polyacrylamide gel electrophoresis showed that the peptide composition was qualitatively identical. A major quantitative difference referred to a 66 kDa peptide which appeared to be much stronger in gels of photosystem II peptides originating from the yellowgreen and the yellow tobacco variety. Furthermore, we were able to show that different SDS polyacrylamide gel electrophoresis runs of the same PS II preparation yielded differences in the band strength of this peptide. Comparative densitometric measurements showed that an increase in this 66 kDa peptide was always correlated with a decrease in the D1 and D2 peptides. Obviously, the 66 kDa peptide is the heterodimer of D1 and D2. Differences in the peptide composition of photosystem II preparations from the 3 tobacco species refer above all to peptides of the light-harvesting complex with molecular masses of 28 and 26 kDa. After the transfer of the peptides from the polyacrylamide gel to nitrocellulose membranes, they were incubated with monospecific antisera to monogalactolipid, digalactolipid or sulfolipid. These experiments showed that the 66 kDa peptide reacted with antibodies to digalactolipid and with those to sulfolipid. The 66 kDa peptide reacts in the Western blot procedure also with an antiserum to a 66 kDa peptide prepared and characterized earlier and which was shown to inhibit electron transport reactions in the region of the reaction center of photosystem II. The monospecific antiserum to monogalactolipid reacts with the D1 and D2 peptide as well as with the chlorophyll-binding polypeptides of the masses 42 and 48 kDa, and also with the 26 and 28 kDa peptides of the light-harvesting complex as well as with the extrinsic peptides exhibiting the molecular masses, 33 ,21 -23 and 18 kDa. Whereas lipase treatment apparently destroys the lipids as antigenic determinants of the peptides on the nitrocellulose membrane, periodate treatment or treatment of the photosystem II preparations with organic solvents do not prevent the reaction of the 66 kDa peptide with the sulfolipid antiserum. These experiments show as the 66 kDa peptide appears to be the heterodimer of D1 and D2, that the galactolipids mono- and digalactosyldiglyceride as well as the sulfolipid are bound, much like prosthetic groups, onto the core peptides.

Immunological Evidence for the Binding of β-Carotene and Xanthophylls onto Peptides of Photosystem I from Nicotiana tabacum

1994 ◽  
Vol 49 (7-8) ◽  
pp. 427-438 ◽  
Author(s):  
A. Makewicz ◽  
A. Radunz ◽  
G. H. Schmid
Keyword(s):  
Nicotiana Tabacum ◽  
Photosystem Ii ◽  
Western Blot ◽  
Photosystem I ◽  
Light Harvesting ◽  
Wild Type ◽  
The Core ◽  
Ps I ◽  
Molecular Masses ◽  
Β Carotene

Photosystem I preparations were obtained from wild type tobacco Nicotiana tabacum var. John William’s Broadleaf (JWB) and from the two chlorophyll-deficient mutants N. tabacum Su/su and N. tabacum Su/su var. Aurea. The preparations were characterized with respect to the chlorophyll a/b ratio, their photosynthetic activity and their absorption spectroscopic properties. Peptides from these preparations were analyzed by SDS polyacrylamide gel electrophoresis and transferred for the detection of bound carotenoids according to the Western blot procedure to nitrocellulose or Immobilon membranes. The PS I preparation from the wild type JWB consisted of the core and the LHCP complex. The core complex contains the two core peptides with the same apparent MW of 66 kDa and several peptides with the lesser molecular masses of 22, 20, 19, 17, 16, 10 and 9 kDa. The light-harvesting protein complex consists of 4 subunits with the molecular masses 28, 26, 25 and 24 kDa. The PS I preparations of the yellow-green mutant Su/su and of the Aurea mutant Su/su var. Aurea contain as impurity traces of the D1 and D2 core peptides of photosystem II and also traces of the chlorophyll-binding photosystem II peptides with the molecular masses 42 and 47 kDa. The peptides of the photosystem I preparation were characterized by specific photosystem I antisera: An antiserum to the photosystem I complex reacts in the Western blot only with the homologous peptides of photosystem I. In comparative analyses with photosystem II preparations this antiserum (directed to photosystem I) reacts, as expected, only with the peptides of the light-harvesting complex. An antiserum to the CP 1 core peptides reacts only with the 66 kDa peptides of photosystem I and gives no cross reaction with heterodimer forms of the D1/D2 core peptides of photosystem II. In the Western blot procedure by means of polyclonal monospecific antisera to carotenoids it was demonstrated that β-carotene is bound in high concentration onto the core peptides CP 1 and to a lesser extent onto the two larger subunits of the LHCP complex, exhibiting the molecular masses of 28 and 26 kDa. Neoxanthin is bound onto the same peptides. In contrast to this, lutein was only identified on the core peptides CP 1 and violaxanthin only on the larger subunits of the LHCP complex. As the carotenoids are labelled with antibodies, even after SDS treatment in the electrophoresis, it is assumed, that the carotenoids are covalently bound via the ionon ring to the respective peptide

Partition of Phylloquinone Kx between Digitonin Particles and Chlorophyll-Proteins of Chloroplast Membranes from Nicotiana tabacum

1981 ◽  
Vol 36 (3-4) ◽  
pp. 276-283 ◽  
Author(s):  
E. Interschick-Niebler ◽  
H. K. Lichtenthaler
Keyword(s):  
Nicotiana Tabacum ◽  
Photosystem Ii ◽  
Chlorophyll A ◽  
Gel Electrophoresis ◽  
Protein Complexes ◽  
Sodium Dodecylsulfate ◽  
Total Chlorophyll ◽  
Photosynthetic Membrane ◽  
Chlorophylls A And B ◽  
Β Carotene

The partition of phylloquinone (vitamin K1), of chlorophylls a and b and of the two main carotenoids, β-carotene and lutein, in subthylakoid particles (digitonin treatment) and chlorophyll protein complexes (sodium dodecylsulfate polyamide-gel electrophoresis) isolated from tobacco chloroplasts (Nicotiana tabacum L.) is described. 1. The “light particle” fractions (S 90 000, S 150 000) of digitonin fragmented chloroplasts are enriched in CP I and contain a higher proportion of phylloquinone, chorophyll a and β-carotene as compared to whole chloroplasts. This is visualized by high values for the ratio a/b (6 -8) and for β-carotene/lutein (1.7) as well as about 3 mol of K1 per 100 mol of total chlorophyll. The “heavy digitonin particle” fraction (10 000 x g sediment), in turn, contains a higher proportion of chlorophyll b and lutein, but a lower level of phylloquinone than whole chloroplasts. 2. The chlorophyll a-protein CP I of pigmentsystem I, isolated by preparative gel electrophoresis using 0.5% and 4% SDS, is characterized by a stable level of phylloquinone (1 mol K1 per 100 mol of total chlorophyll), high chlorophyll a/b ratios (7 -10) and high values for β-carotene/lutein (~ 6.0). The light-harvesting chlorophyll a/b-protein LHCP of photosystem II (chlorophyll a/b = 1.1 - 1.5, β-carotene/lutein = < 0.1) contains either low amounts of phylloquinone (0.5% SDS) or only trace amounts of K1 (4% SDS). The free pigment fraction (FP) contains at 0.5% SDS 57% of the total phylloquinone of thylakoid membranes. At 4% SDS the K1 amount in the free pigment fraction increases to 84%. 3. The phylloquinone partition studies in digitonin particles and SDS chlorophyll proteins indicate that there exist at least two localization sites for phylloquinone K1 in the photosynthetic membrane. The CP I complex and a second site, presumably near photosystem II (CPa?)

scholarly journals The acylation of megakaryocyte proteins: glycoprotein IX is primarily myristoylated while glycoprotein Ib is palmitoylated

Blood ◽  
1996 ◽  
Vol 87 (4) ◽  
pp. 1377-1384 ◽  
Author(s):  
PK Schick ◽  
J Walker
Keyword(s):  
Fatty Acids ◽  
Myristic Acid ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Amide Bond ◽  
Endogenous Protein ◽  
Sds Page ◽  
Palmitic Acids ◽  
Protein Acylation

The acylation of megakaryocyte proteins was studied with special emphasis on the myristoylation and palmitoylation of the glycoprotein (GP) Ib complex. Guinea pig megakaryocytes were purified and separated into subpopulations at different phases of maturation. Cells were incubated with [3H]myristate, [3H]palmitate, or [3H]acetate to study endogenous protein acylation. Cycloheximide was used to distinguish between cotranslational and posttranslational acylation and hydroxylamine to distinguish between thioester and amide linkages. After incubations, delipidated proteins or GPIb complex subunits, immunoprecipitated with PG-1, AN-51 or FMC-25 monoclonal antibody, were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and assessed by fluorography. Radiolabeled fatty acids bound to GPIX and GPIb were also analyzed by high pressure liquid chromatography (HPLC) and scintillation spectrometry. With [3H]myristic acid and [3H]acetate, GPIX was found to be a major myristoylated protein in megakaryocytes and CHRF-288 cells. Myristic acid was linked to GPIX by an amide bond, and this process occurred cotranslationally. With [3H]acetate, GPIb was primarily palmitoylated, but with [3H]myristate, GPIb was acylated with about equal mounts of myristic acid and palmitic acids. Both fatty acids were linked to GPIb by thioester bonds, and acylation was posttranslational. The myristoylation of GPIX while the palmitoylation of GPIb occurred throughout megakaryocyte maturation. Myristoylation and palmitoylation may have different functions relevant to the assembly of the GPIb complex in megakaryocytes.

Phosphatidylglycerol and ß-Carotene Bound onto the D 1-Core Peptide of Photosystem II in the Filamentous Cyanobacterium

1994 ◽  
Vol 49 (1-2) ◽  
pp. 115-124 ◽  
Author(s):  
O. Kruse ◽  
A. Radunz ◽  
G. H. Schmid
Keyword(s):  
Fatty Acid ◽  
Photosystem Ii ◽  
Gel Electrophoresis ◽  
Lipid Analysis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Acid Mixture ◽  
Hexadecenoic Acid ◽  
Filamentous Cyanobacterium ◽  
Intact Cells ◽  
Oscillatoria Chalybea

Photosystem II-particles from the cyanobacterium Oscillatoria chalybea were isolated by fractionating centrifugation. Purification of these particles was achieved by a 22 hours centrifugation over a linear sucrose density gradient at 217.500xg. The obtained particle fraction exhibited an oxygen evolution activity which corresponded to three times the rate of intact cells and to five times the rate of intact thylakoids. The chlorophyll protein ratio was 1:10 and the ratio manganese/chlorophyll 1:34. SDS-polyacrylamide gel electrophoresis showed that the photosystem Il-fraction is composed of the core peptides D1 and D2, the chlorophyll-binding peptides CP 43 and CP 47, the extrinsic 33 kDa peptide (manganese stabilizing peptide, MSP) and phycobiliproteins with molecular masses between 16 to 20 kDa. Cyt b559 was not detected in our gel electrophoresis assay. Part of the peptides of the 30 kDa-region (D1, D2, MSP) occurred as aggregates with a molecular mass of 60 to 66 kDa. The D 1-peptide was isolated from the PS Il-preparation by SDS-gel electrophoresis. The intrinisic peptide reacts in the Western blot procedure with the antiserum to phosphatidylglycerol and with the antiserum to β-carotene. Incubation of the peptide with the antisera to monogalactosyldiglyceride, sulfoquinovosyldiglyceride and zeaxanthine resulted negatively. The binding of phosphatidylglycerol onto the D 1-peptide was confirmed by lipid analysis in HPLC and fatty acid analysis by gas chromatography. Only this lipid, respectively the typical fatty acid mixture of this lipid was detected. The lipid is characterized by the fact that the hexadecenoic acid does not exhibit trans-configuration, as is true for phosphatidylglycerol of higher plants and algae, but occurs in cis-configuration. With the antibody being directed towards the glycerol-phosphate residue and not towards the fatty acids, it can be concluded from the reaction of the antibodies with the bound lipid that the lipid is bound to the peptide via the fatty acid. The negatively charged phosphatidylglycerol increases the hydrophobicity of the peptide and leads to a negatively charged surface favouring binding of cations like calcium and magnesium. The fact that incubation of this PS Il-fraction with phospholipase inhibits photosynthetic activity by 25% which can be fully restored by addition of phosphatidylglycerol, shows that bound phosphatidylglycerol has a functional role.

scholarly journals Human erythrocyte antigens: II. The In(Lu) gene regulates expression of an antigen on an 80-kilodalton protein of human erythrocytes

Blood ◽  
1984 ◽  
Vol 64 (3) ◽  
pp. 599-606 ◽  
Author(s):  
MJ Telen ◽  
TJ Palker ◽  
BF Haynes
Keyword(s):  
Monoclonal Antibody ◽  
Western Blot ◽  
Erythrocyte Membrane ◽  
Human Erythrocyte ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Sodium Dodecyl ◽  
Two Dimensions ◽  
Antigen Activity

Abstract We have previously shown that a murine monoclonal antibody (A3D8) identifies a human erythrocyte protein antigen whose expression is regulated by the Lutheran inhibitor [In(Lu)] gene. In the present study, we demonstrated by immunoprecipitation and Western blot techniques that the antigen defined by A3D8 was on an 80-kD erythrocyte membrane protein. A second 170-kD protein was coprecipitated with the 80-kD protein but failed to show antigen activity by Western blot analysis. The 170-kD protein, when analyzed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis in two dimensions, was composed of 50- and 30-kD disulfide-linked subunits. In(Lu) Lu[a-b-) erythrocytes differed from Lu(a+b+) or Lu(a-b+) erythrocytes in that In(Lu) deoxycholate erythrocyte membrane extracts contained trace amounts of immunoprecipitable 80-kD protein compared with detergent-solubilized erythrocyte membrane extracts prepared from Lu(a+b+) or Lu(a-b+) subjects.

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