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.

Isolation, purification, and characterization of a peptide that contains the β-ketoacyl reductase, enoyl reductase, and β-hydroxyacyl dehydrase activities of the pigeon liver fatty acid synthetase

1985 ◽  
Vol 63 (1) ◽  
pp. 50-56
Author(s):  
Rajinder N. Puri ◽  
John W. Porter
Keyword(s):  
Fatty Acid ◽  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Fatty Acid Synthetase ◽  
Polyacrylamide Gel ◽  
Liver Fatty Acid ◽  
Dodecyl Sulfate ◽  
Pigeon Liver

Controlled proteolytic cleavage of pigeon liver fatty acid synthetase with elastase (4% w/w) for 5 h yields two peptides that are designated II and IV. After 5 h of proteolysis the incubation mixture containing these peptides retains all of the component enzyme activities of the fatty acid synthetase complex. The two peptides are then separated by chromatography on an Affi-Gel Blue column. Gel filtration of the fraction containing peptide II yields a homogeneous peptide as shown by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. The molecular weight of this peptide has been estimated to be 130 000 by sodium dodecyl sulfate – polyacrylamide gel electrophoresis, size exclusion chromatography, and amino acid analysis. The sedimentation coefficient for peptide II is approximately 7.4S. Peptide II contains the domains for the β-ketoacyl and enoyl reductases and β-hydroxyacyl dehydrase activities of the fatty acid synthetase complex.

scholarly journals Molecular weight and subunit size of fatty acid synthetase from rabbit mammary gland

1976 ◽  
Vol 159 (1) ◽  
pp. 181-184 ◽  
Author(s):  
N Paskin ◽  
R J Mayer
Keyword(s):  
Molecular Weight ◽  
Mammary Gland ◽  
Fatty Acid ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Fatty Acid Synthetase ◽  
Subunit Size

Fatty acid synthetase purified from the mammary gland of the rabbit has a mol. wt. of 968000 as determined by gel filtration. The enzyme gave one band, corresponding to a mol.wt. of approx. 35000, on polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate and phenylmethanesulphonyl fluoride.

Structural characteristics of growth hormone receptors on mouse 3T3 cells having different biological responses to growth hormone

1989 ◽  
Vol 3 (3) ◽  
pp. 239-245 ◽  
Author(s):  
E. Uchida ◽  
T. Hayakawa ◽  
S. Niimi ◽  
A. Tanaka ◽  
M. Morikawa
Keyword(s):  
Growth Hormone ◽  
Gel Electrophoresis ◽  
Structural Characteristics ◽  
Polyacrylamide Gel Electrophoresis ◽  
Cell Type ◽  
3T3 Cells ◽  
Intact Cells ◽  
Receptor Complexes ◽  
Gh Receptor ◽  
Adipose Conversion

ABSTRACT Cultured 3T3-F442A preadipocytes are able to undergo GH-promoted differentiation into adipocytes. The relationship between the structure and function of GH receptors on 3T3 cells (3T3-F442A preadipocytes, differentiated adipocytes and 3T3-C2 cells, which vary in susceptibility to adipose conversion or with respect to carbohydrate and lipid metabolism) was studied by the covalent cross-linking of 125I-labelled human (h) GH to intact cells with the bifunctional reagent disuccinimidyl suberate. When preadipocytes were cross-linked and analysed using sodium dodecylsulphate-polyacrylamide gel electrophoresis, a prominent 125I-labelled hGH-receptor complex of Mr 130 000 was observed along with minor complexes (Mr 300 000, 230 000 and 60 000) on autoradiography. Non-reducing—reducing two-dimensional gel electrophoresis revealed that the higher molecular weight complexes also contained the Mr 130 000 complex. Neuraminidase and tunicamycin treatment demonstrated that the GH receptor on F442A preadipocytes is a sialo-glycoprotein with N-linked carbohydrate chains. When the differentiated 3T3-F442A adipocytes and 3T3-C2 cells (a sub-line with no susceptibility to adipose conversion with GH) were examined in the same way as 3T3-F442A preadipocytes, no differences were observed in the specificity of GH binding and in the molecular size of the 125I-labelled hGH-receptor complexes and their glycosylation characteristics. This suggests that the structural characteristics of the GH receptor are closely related in each cell type, but that the hormonal signals produced after GH binding to the receptor may cause different effects according to the cell type.

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.

Discriminating between Cell Surface and Intracellular Plasminogen-binding Proteins: Heterogeneity in Profibrinolytic Plasminogen-binding Proteins on Monocytoid Cells

2000 ◽  
Vol 84 (11) ◽  
pp. 882-890 ◽  
Author(s):  
Michael Green ◽  
Lindsey Miles ◽  
Stephen Hawley
Keyword(s):  
Cell Surface ◽  
Peripheral Blood ◽  
Gel Electrophoresis ◽  
Binding Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Annexin Ii ◽  
Two Dimensional ◽  
Intact Cells ◽  
Carboxyl Terminal

SummaryWhen plasminogen binds to cell surfaces, its activation is markedly enhanced compared to soluble plasminogen. Although several distinct molecules may contribute to plasminogen binding to a given cell type, the subset of plasminogen receptors responsible for enhancing plasminogen activation expose a carboxyl-terminal lysine on the cell surface and are sensitive to proteolysis by carboxypeptidase B (CpB). To distinguish this subset of plasminogen receptors from plasminogen-binding proteins that are not profibrinolytic, we treated intact U937 monocytoid cells and peripheral blood monocytes with CpB to remove exposed carboxyl-terminal lysines, and subjected the membrane proteins to two-dimensional gel electrophoresis followed by ligand blotting with 125I-plasminogen. Western blotting was performed with antibodies against previously characterized candidate plasminogen receptors to identify plasminogen-binding proteins on the two-dimensional ligand blots. Densitometry of autoradiograms of the 125I-plasminogen ligand blots of U937 cell membranes revealed that membraneassociated α-enolase, actin and annexin II showed minimal changes in 125I-plasminogen binding following CpB treatment of intact cells, suggesting that these proteins are not accessible to CpB on the U937 cell surface and most likely do not serve as profibrinolytic plasminogen receptors on U937 cells. In contrast, densitometry of autoradiograms of 125I-plasminogen ligand blots of monocyte membranes revealed that 125I-plasminogen binding to α-enolase was reduced 71% by treatment of intact cells with CpB, while binding to annexin II was reduced 14%. Thus, a portion of membrane-associated α-enolase and annexin II expose carboxyl terminal lysines that are accessible to CpB on the peripheral blood monocyte surface, suggesting that these molecules may serve as profibrinolytic plasminogen receptors on monocytes. Our data suggest that U937 cells and peripheral blood monocytes have distinct sets of molecules that constitute the population of cell surface profibrinolytic plasminogen-binding proteins. Furthermore, our data suggest that while several plasminogen-binding proteins with carboxyl terminal lysines are associated with cell membranes, only a small subset of these proteins expose a carboxyl terminal lysine that is accessible to CpB on the cell surface. The abbreviations used are: 2D, two-dimensional; 2D-PAGE, two-dimensional polyacrylamide gel electrophoresis; BSA, bovine serum albumin; CpB, carboxypeptidase B; EACA, є-aminocaproic acid; HBSS, Hanks’ Balanced Salt Solution supplemented with 20 mM HEPES; HBSS-BSA, HBSS with 0.1% bovine serum albumin; HRP, horseradish peroxidase; IEF, isoelectric focusing; PBS, phosphate buffered saline; PMSF, phenylmethylsulfonyl fluoride; PVDF, polyvinylidene difluoride; SDS, sodium dodecyl sulfate; SDSPAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; TBST, Tris buffered saline with 0.1% Tween 20; uPAR, urokinase-type plasminogen activator receptor.

Mass Spectrometric Analysis of N2-Formation Induced by the Oxidation of Hydrazine and Hydroxylamine in Flash Illuminated Thylakoid Preparations of the Filamentous Cyanobacterium Oscillatoria chalybea

1991 ◽  
Vol 46 (7-8) ◽  
pp. 629-634 ◽  
Author(s):  
P. He ◽  
K. P. Bader ◽  
G . H. Schmid
Keyword(s):  
Mass Spectrometry ◽  
Photosystem Ii ◽  
Oxygen Uptake ◽  
Mass Spectrometric ◽  
Spectrometric Analysis ◽  
Filamentous Cyanobacterium ◽  
High Concentration ◽  
Single Turnover ◽  
Oscillatoria Chalybea ◽  
Light Flashes

In tobacco chloroplasts hydrazine-dependent dinitrogen formation measured by mass spectrometry as the consequence of short saturating light flashes is always linked to a substantial oxygen uptake (G. Renger, K. P. Bader, and G. H. Schmid, Biochim. Biophys. Acta 1015, 288, 1990). However, in thylakoids of the filamentous cyanobacterium Oscillatoria chalybea this dinitrogen formation is not linked to an apparent O2-uptake, even at the high concentration of 1 mм hydrazine. Whereas in tobacco chloroplasts Tris-treatment does not affect hydrazine dependent dinitrogen formation up to a concentration of 3 mм hydrazine, Tris-treatment of thylakoids of O. chalybea affects strongly both oxygen evolution and dinitrogen evolution under a single turnover flash as well as under ten flashes. In contrast to tobacco chloroplasts, the presence of hydrazine up to concentrations of 3 mм does not substantially affect photosynthetic O2-evolution. The observed dinitrogen evolution is affected by DCMU regardless whether induced by a single turnover flash or by ten flashes, whereas in tobacco dinitrogen evolution and the O2-uptake linked to it (which is not observed in the cyanobacterium) were clearly not affected by DCMU in the single turnover flash. In Oscillatoria the earlier described Photosystem II-mediated H2O2 formation and decomposition is influenced by hydrazine. In the presence of 300 μм hydrazine the usually present O2-uptake leading to H2O2 formation appears diminished.

scholarly journals Chemical Characterization and Pronase Susceptibility of the Na:K Pump-Associated Phosphoprotein of Human Red Blood Cells

1974 ◽  
Vol 63 (3) ◽  
pp. 305-323 ◽  
Author(s):  
Philip A. Knauf ◽  
Fulgencio Proverbio ◽  
Joseph F. Hoffman
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Chemical Characterization ◽  
Polyacrylamide Gel ◽  
Transport Parameters ◽  
Intact Cells ◽  
Human Red Blood Cells ◽  
Polypeptide Band ◽  
Pronase Treatment

The phosphoproteins formed by incubation of red cell ghosts with [γ-32P]ATP in the presence of Mg and Na + Mg have been characterized by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The 32P-labeled phosphoprotein was seen as a single peak confined to the region of the diffuse 90,000 dalton polypeptide band; labeling with Na + Mg considerably increased the quantity of 32P-phosphoprotein contained in this band relative to labeling with Mg alone. Treatment of intact cells with Pronase known to partially hydrolyze the glycoproteins and the 90,000 daltons polypeptide did not change either the amount or the position of the 32P-phosphoprotein present in the gels. The molecular weight of the 32P-phosphoprotein is estimated to be 103,000. Pronase treatment of intact cells also did not significantly alter any of the transport parameters of the membrane such as the K pump flux, ouabain binding, or Na,K-ATPase. In contrast, treatment of ghosts with Pronase not only resulted in drastic alteration of the transport parameters but also inhibited the formation of the phosphoprotein under all conditions. Thus, while the Na:K pump is not intrinsically resistant to Pronase, those elements of the pump which are susceptible are not accessible from the outside of the cell. Further, SDS-polyacrylamide gel electrophoresis after Pronase treatment of intact cells results in a substantial increase in the purification of the phosphoprotein relative to that which was previously possible in ghosts.

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