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Background In this study, lettuce roots and leaves were contaminated with enterohemorrhagic Escherichia coli O157:H− strain 431/97 under greenhouse conditions. Furthermore, the internalization of strain 431/97 in lettuce roots and leaves was examined. To track the inoculated bacteria during the experiments and to differentiate them from the autochthonous microbiota, a combined protocol including matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and PCR was established. Results At different time points after inoculation of Batavia lettuce with 5.5 × 106 cfu/mL (high inoculation level) or 3.0 × 101 cfu/mL (low inoculation level) E. coli O157:H− strain 431/97 as well as sterile 0.9% (w/v) sodium chloride solution (negative control), samples from the root and the leaf were taken and surface disinfected with gentamicin. After homogenization, microorganisms were isolated from the samples and analyzed by MALDI-TOF MS. Analysis of the root samples resulted in bacterial counts of 1.0 × 102–1.0 × 106 cfu/0.25 g depending on the inoculated viable counts and the incubation period. In the leaf samples, strain 431/97 was not detected. The investigation of the viable cell counts of E. coli O157:H− 431/97 following irrigation of the leaves resulted in bacterial counts of 102 cfu/0.25 g for the disinfected leaf samples. Conclusions Thus, the established protocol is suitable for detecting the investigated strain under greenhouse conditions in plant infection experiments. This strain may indeed survive in the soil, but did not enter the plant via the root in detectable numbers. Contrarily, viable counts exceeding the generally accepted infective dose of less than 100 cells for enterohemorrhagic E. coli were determined internalized after irrigation of the leaves. As this may pose a risk for the consumer, the present study provides a valuable set of tools for further research., Chemical and Biological Technologies in Agriculture, 6 (1)

Our previous work has established that the metabolic sensor AMP-activated protein kinase (AMPK) inhibits the epithelial Na(+) channel (ENaC) by promoting its binding to neural precursor cell–expressed, developmentally down-regulated 4-2, E3 ubiquitin protein ligase (Nedd4-2). Here, using MS analysis and in vitro phosphorylation, we show that AMPK phosphorylates Nedd4-2 at the Ser-444 (Xenopus Nedd4-2) site critical for Nedd4-2 stability. We further demonstrate that the Pak-interacting exchange factor β(1)Pix is required for AMPK-mediated inhibition of ENaC-dependent currents in both CHO and murine kidney cortical collecting duct (CCD) cells. Short hairpin RNA–mediated knockdown of β(1)Pix expression in CCD cells attenuated the inhibitory effect of AMPK activators on ENaC currents. Moreover, overexpression of a β(1)Pix dimerization–deficient mutant unable to bind 14-3-3 proteins (Δ602–611) increased ENaC currents in CCD cells, whereas overexpression of WT β(1)Pix had the opposite effect. Using additional immunoblotting and co-immunoprecipitation experiments, we found that treatment with AMPK activators promoted the binding of β(1)Pix to 14-3-3 proteins in CCD cells. However, the association between Nedd4-2 and 14-3-3 proteins was not consistently affected by AMPK activation, β(1)Pix knockdown, or overexpression of WT β(1)Pix or the β(1)Pix-Δ602–611 mutant. Moreover, we found that β(1)Pix is important for phosphorylation of the aforementioned Nedd4-2 site critical for its stability. Overall, these findings elucidate novel molecular mechanisms by which AMPK regulates ENaC. Specifically, they indicate that AMPK promotes the assembly of β(1)Pix, 14-3-3 proteins, and Nedd4-2 into a complex that inhibits ENaC by enhancing Nedd4-2 binding to ENaC and its degradation.

The influence of electrochemistry on the coagulation of blood on metal surfaces was demonstrated several decades ago. In particular, the application of cathodic currents resulted in reduced surface thrombogenicity, but no molecular mechanism has been so far proposed to explain this observation. In this article we used for the first time the quartz crystal microbalance with dissipation monitoring technique coupled with an electrochemical setup (EQCM-D) to study thrombosis at the blood-electrode interface. We confirmed the reduced thrombus deposition at the cathode, and we subsequently studied the effect of cathodic currents on adsorbed fibrinogen (Fg). Using EQCM and mass spectrometry, we found that upon applying currents Fg desorbed from the electrode and was electrochemically degraded. In particular, we show that the flexible N-terminus of the α-chain, containing an important polymerization site, was cleaved from the protein, thus affecting its clottability. Our work proposes a molecular mechanism that at least partially explains how cathodic currents reduce thrombosis at the blood-electrode interface and is a relevant contribution to the rational development of medical devices with reduced thrombus formation on their surface.

The vacuolar H+-ATPase (V-ATPase) in intercalated cells contributes to luminal acidification in the kidney collecting duct and nonvolatile acid excretion. We previously showed that the A subunit in the cytoplasmic V1sector of the V-ATPase (ATP6V1A) is phosphorylated by the metabolic sensor AMP-activated protein kinase (AMPK) in vitro and in kidney cells. Here, we demonstrate that treatment of rabbit isolated, perfused collecting ducts with the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) inhibited V-ATPase-dependent H+secretion from intercalated cells after an acid load. We have identified by mass spectrometry that Ser-384 is a major AMPK phosphorylation site in the V-ATPase A subunit, a result confirmed by comparing AMPK-dependent phosphate labeling of wild-type A-subunit (WT-A) with that of a Ser-384-to-Ala A subunit mutant (S384A-A) in vitro and in intact HEK-293 cells. Compared with WT-A-expressing HEK-293 cells, S384A-A-expressing cells exhibited greater steady-state acidification of HCO3−-containing media. Moreover, AICAR treatment of clone C rabbit intercalated cells expressing the WT-A subunit reduced V-ATPase-dependent extracellular acidification, an effect that was blocked in cells expressing the phosphorylation-deficient S384A-A mutant. Finally, expression of the S384A-A mutant prevented cytoplasmic redistribution of the V-ATPase by AICAR in clone C cells. In summary, direct phosphorylation of the A subunit at Ser-384 by AMPK represents a novel regulatory mechanism of the V-ATPase in kidney intercalated cells. Regulation of the V-ATPase by AMPK may couple V-ATPase activity to cellular metabolic status with potential relevance to ischemic injury in the kidney and other tissues.

Today, the application of MALDI-TOF MS in the analysis of whole cell microbial proteins is well established and widely used for categorization, differentiation, and identification of microorganisms. The power of this technique resides in the simple sample preparation and the fast software-assisted analysis of spectral data. Besides protein profiling, featuring also the possibility for characterization of microbes at the below-species level, MALDI-TOF and MALDI-TOF/TOF MS allow novel proteomics approaches using a classical cell extract. LC coupled to MALDI, but also ESI-based techniques offer the possibility for peptide profiling or top-down sequencing of intact proteins, which greatly increase the number of proteins detected and the discriminatory power. In this chapter we highlight applications of MALDI-TOF MS and ESI-MS including their use in tandem MS mode for characterization of microorganisms. We present novel MS-based approaches for protein and peptide biomarker identification and discuss future perspectives extending the application range of mass spectrometry in this area.

The Bacillus cereus group consists of eight very closely related species and comprises both harmless and human pathogenic species such as Bacillus anthracis, Bacillus cereus, and Bacillus cytotoxicus. Numerous efforts have been undertaken to allow presumptive differentiation of B. cereus group species from one another. However, methods to rapidly and accurately distinguish these species are currently lacking. We confirmed that classical matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) biotyping cannot achieve reliable identification of each type strain. We therefore assigned type strain-specific diagnostic peptides to the B. cereus group based on comparisons of their proteomic profiles. The number of diagnostic peptides varied remarkably in a type strain-dependent manner. The accuracy of the reference database was crucial to validate candidate diagnostic peptides and led to a noteworthy reduction of verified diagnostic peptides. Diagnostic peptides ranged from one for B. weihenstephanensis to 62 for B. pseudomycoides and were associated with proteins involved in diverse biological processes, e.g. amino acid biosynthesis, cell envelope, cellular processes, energy metabolism, and transport processes. However, 45.6% of all diagnostic peptides comprised currently unclassified proteins or proteins of unknown function. In addition, a phylogenetic tree based on clustering of theoretical precursor masses deduced from in silico-generated tryptic peptides was reconstructed., Journal of Proteome Research, 15 (9), ISSN:1535-3893, ISSN:1535-3907

International audience; AMP-activated protein kinase (AMPK) is emerging as a central cellular signaling hub involved in energy homeostasis and proliferation. The kinase is considered as a suitable target for pharmacological intervention in several energy-related pathologies like diabetes type II and cancer, although its signaling network is still incompletely understood. Here we apply an original two-dimensional in vitro screening approach for AMPK substrates that combines biophysical interaction based on surface plasmon resonance with in vitro phosphorylation. By enriching for proteins that interact with a specific AMPK isoform, we aimed to identify substrates that are also preferentially phosphorylated by this specific AMPK isoform. Application of this screen to full-length AMPK α2β2γ1 and soluble rat liver proteins identified the tumor suppressor fumarate hydratase (FH). FH was confirmed to interact with and to be preferentially phosphorylated by the AMPKα2 isoform by using yeast-two-hybrid and in vitro phosphorylation assays. AMPK-mediated phosphorylation of FH significantly increased enzyme activity in vitro and in vivo, suggesting that it is a bona fide AMPK substrate. In vivo, AMPKα2 is supposed to target the cytosolic/nuclear pools of FH, whose tumor suppressor function relies on DNA damage repair and inhibition of HIF-1α-signaling.

The sodium ion-translocating NADH:quinone oxidoreductase (Na(+)-NQR) from the human pathogen Vibrio cholerae is a respiratory membrane protein complex that couples the oxidation of NADH to the transport of Na(+) across the bacterial membrane. The Na(+)-NQR comprises the six subunits NqrABCDEF, but the stoichiometry and arrangement of these subunits are unknown. Redox-active cofactors are FAD and a 2Fe-2S cluster on NqrF, covalently attached FMNs on NqrB and NqrC, and riboflavin and ubiquinone-8 with unknown localization in the complex. By analyzing the cofactor content and NADH oxidation activity of subcomplexes of the Na(+)-NQR lacking individual subunits, the riboflavin cofactor was unequivocally assigned to the membrane-bound NqrB subunit. Quantitative analysis of the N-terminal amino acids of the holo-complex revealed that NqrB is present in a single copy in the holo-complex. It is concluded that the hydrophobic NqrB harbors one riboflavin in addition to its covalently attached FMN. The catalytic role of two flavins in subunit NqrB during the reduction of ubiquinone to ubiquinol by the Na(+)-NQR is discussed.

Identification of human pathogens by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry, based on profiling of mainly taxonomic relevant ribosomal proteins and comparison to a reference mass spectra database, has developed into a robust cutting-edge diagnostic technology and has revolutionized work in microbiological laboratories in recent years. This is due to the high speed of analysis allowing a short time to result and the streamlined protocol enabling an accurate and cost-effective identification within less than 20 min. Application fields include clinical and veterinary diagnostics, food safety control, outbreak tracking, environmental microbiology, biotechnology, and biodefense. A major challenge to MS-based identification has been to reliably increase the taxonomic resolution to the below-species level. This challenge originates from the fact that strains of one species exhibit substantial genetic overlap and thus high protein similarity. Two main approaches have been applied to resolve mass peak variations below the species level: the library-based and the proteomics-based approach. Within the library-based approach, both sample pre-treatment and data reduction strategies have been developed. Proteomics-based approaches comprise bottom-up and top-down characterization of biomarkers applying large databases available to the public.

We isolated the c rings of F-ATP synthases from eight cyanobacterial strains belonging to four different taxonomic classes ( Chroococcales , Nostocales , Oscillatoriales , and Gloeobacteria ). These c rings showed different mobilities on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), probably reflecting their molecular masses. This supposition was validated with the previously characterized c 11 , c 14 , and c 15 rings, which migrated on SDS-PAGE in proportion to their molecular masses. Hence, the masses of the cyanobacterial c rings can conveniently be deduced from their electrophoretic mobilities and, together with the masses of the c monomers, allow the calculation of the c ring stoichiometries. The method is a simple and fast way to determine stoichiometries of SDS-stable c rings and hence a convenient means to unambiguously determine the ion-to-ATP ratio, a parameter reflecting the bioenergetic efficacy of F-ATP synthases. AFM imaging was used to prove the accuracy of the method and confirmed that the c ring of Synechococcus elongatus SAG 89.79 is a tridecameric oligomer. Despite the high conservation of the c-subunit sequences from cyanobacterial strains from various environmental groups, the stoichiometries of their c rings varied between c 13 and c 15 . This systematic study of the c-ring stoichiometries suggests that variability of c-ring sizes might represent an adaptation of the individual cyanobacterial species to their particular environmental and physiological conditions. Furthermore, the two new examples of c 15 rings underline once more that an F 1 /F o symmetry mismatch is not an obligatory feature of all F-ATP synthases.

The ambition of systems biology to understand complex biological systems at the molecular level implies that we need to have a concrete and correct understanding of each molecular entity and its function. However, even for the best-studied organism, Escherichia coli, a large number of proteins have never been identified and characterised from wild-type cells, and/or await unravelling of their biological role. Instead, the ORF models for these proteins have been predicted by suitable algorithms and/or through comparison with known, homologous proteins from other organisms, approaches which may be prone to error. In the present study, we used a combination of 2-DE, MALDI-TOF-MS and PMF to identify 1151 different proteins in E. coli K12 JM109. Comparison of the experimental with the theoretical Mr and pI values (4000 experimental values each) allowed the identification of numerous proteins with incorrect or incomplete ORF annotations in the current E. coli genome databases. Several inconsistencies in genome annotation were verified experimentally, and up to 55 candidates await further investigation. Our findings demonstrate how an up-to-date 2-D gel-based proteomics approach can be used for improving the annotation of prokaryotic genomes. They also highlight the need for harmonization among the different E. coli genome databases.

Vitamin B6 is an essential metabolite in all organisms. De novo synthesis of the vitamin can occur through either of two mutually exclusive pathways referred to as deoxyxylulose 5-phosphate-dependent and deoxyxylulose 5-phosphate-independent. The latter pathway has only recently been discovered and is distinguished by the presence of two genes, Pdx1 and Pdx2, encoding the synthase and glutaminase subunit of PLP synthase, respectively. In the presence of ammonia, the synthase alone displays an exceptional polymorphic synthetic ability in carrying out a complex set of reactions, including pentose and triose isomerization, imine formation, ammonia addition, aldol-type condensation, cyclization, and aromatization, that convert C3 and C5 precursors into the cofactor B6 vitamer, pyridoxal 5'-phosphate. Here, employing the Bacillus subtilis proteins, we demonstrate key features along the catalytic path. We show that ribose 5-phosphate is the preferred C5 substrate and provide unequivocal evidence that the pent(ul)ose phosphate imine occurs at lysine 81 rather than lysine 149 as previously postulated. While this study was under review, corroborative crystallographic evidence has been provided for imine formation with the corresponding lysine group in the enzyme from Thermotoga maritima (Zein, F., Zhang, Y., Kang, Y.-N., Burns, K., Begley, T. P., and Ealick, S. E. (2006) Biochemistry 45, 14609-14620). We have detected an unanticipated covalent reaction intermediate that occurs subsequent to imine formation and is dependent on the presence of Pdx2 and glutamine. This step most likely primes the enzyme for acceptance of the triose sugar, ultimately leading to formation of the pyridine ring. Two alternative structures are proposed for the chromophoric intermediate, both of which require substantial modifications of the proposed mechanism.

Background Conventionally, human monocyte sub-populations are classified according to surface marker expression into classical (CD14++CD16−), intermediate (CD14++CD16+) and non-classical (CD14+CD16++) lineages. The involvement of non-classical monocytes, also referred to as proinflammatory monocytes, in the pathophysiology of diseases including diabetes mellitus, atherosclerosis or Alzheimer’s disease is well recognized. The development of novel high-throughput methods to capture functional states within the different monocyte lineages at the whole cell proteomic level will enable real time monitoring of disease states. Results We isolated and characterized (pan-) monocytes, mostly composed of classical CD16− monocytes, versus autologous CD16+ subpopulations from the blood of healthy human donors (n = 8) and compared their inflammatory properties in response to lipopolysaccharides and M.tuberculosis antigens by multiplex cytokine profiling. Following resting and in vitro antigenic stimulation, cells were recovered and subjected to whole-cell mass spectrometry analysis. This approach identified the specific presence/absence of m/z peaks and therefore potential biomarkers that can discriminate pan-monocytes from their CD16 counterparts. Furthermore, we found that semi-quantitative data analysis could capture the subtle proteome changes occurring upon microbial stimulation that differentiate resting, from lipopolysaccharides or M. tuberculosis stimulated monocytic samples. Conclusions Whole-cell mass spectrometry fingerprinting could efficiently distinguish monocytic sub-populations that arose from a same hematopoietic lineage. We also demonstrate for the first time that mass spectrometry signatures can monitor semi-quantitatively specific activation status in response to exogenous stimulation. As such, this approach stands as a fast and efficient method for the applied immunology field to assess the reactivity of potentially any immune cell types that may sustain health or promote related inflammatory diseases., BMC Biotechnology, 15 (1), ISSN:1472-6750

International audience; AMP-activated protein kinase (AMPK) and cytosolic brain-type creatine kinase (BCK) cooperate under energy stress to compensate for loss of adenosine triphosphate (ATP) by either stimulating ATP-generating and inhibiting ATP-consuming pathways, or by direct ATP regeneration from phosphocreatine, respectively. Here we report on AMPK-dependent phosphorylation of BCK from different species identified by in vitro screening for AMPK substrates in mouse brain. Mass spectrometry, protein sequencing, and site-directed mutagenesis identified Ser6 as a relevant residue with one site phosphorylated per BCK dimer. Yeast two-hybrid analysis revealed interaction of active AMPK specifically with non-phosphorylated BCK. Pharmacological activation of AMPK mimicking energy stress led to BCK phosphorylation in astrocytes and fibroblasts, as evidenced with a highly specific phospho-Ser6 antibody. BCK phosphorylation at Ser6 did not affect its enzymatic activity, but led to the appearance of the phosphorylated enzyme at the endoplasmic reticulum (ER), close to the ER calcium pump, a location known for muscle-type cytosolic creatine kinase (CK) to support Ca2+-pumping.

A well-accepted method for identification of microorganisms uses matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) coupled to analysis software which identifies and classifies the organism according to its ribosomal protein spectral profile. The method, called MALDI biotyping, is widely used in clinical diagnostics and has partly replaced conventional microbiological techniques such as biochemical identification due to its shorter time to result (minutes for MALDI biotyping versus hours or days for classical phenotypic or genotypic identification). Besides its utility for identifying bacteria, MS-based identification has been shown to be applicable also to yeasts and molds. A limitation to this method, however, is that accurate identification is most reliably achieved on the species level on the basis of reference mass spectra, making further phylogenetic classification unreliable. Here, it is shown that combining tryptic digestion of the acid/organic solvent extracted (classical biotyping preparation) and resolubilized proteins, nano-liquid chromatography (nano-LC), and subsequent identification of the peptides by MALDI-tandem TOF (MALDI-TOF/TOF) mass spectrometry increases the discrimination power to the level of subspecies. As a proof of concept, using this targeted proteomics workflow, we have identified subspecies-specific biomarker peptides for three Salmonella subspecies, resulting in an extension of the mass range and type of proteins investigated compared to classical MALDI biotyping. This method therefore offers rapid and cost-effective identification and classification of microorganisms at a deeper taxonomic level.

Early diagnosis of acute kidney injury (AKI) and accurate prognostic stratification is a prerequisite for optimal medical management. To identify novel prognostic markers of AKI, urine was collected on the first day of AKI in critically ill patients. Twelve patients with early recovery and 12 matching patients with late/non-recovery were selected and their proteome analyzed by gel electrophoresis and mass spectrometry. We identified eight prognostic candidates including α-1 microglobulin, α-1 antitrypsin, apolipoprotein D, calreticulin, cathepsin D, CD59, insulin-like growth factor–binding protein 7 (IGFBP-7), and neutrophil gelatinase–associated lipocalin (NGAL). Subsequent quantification by ELISA showed that IGFBP-7 was the most potent predictor of renal recovery. IGFBP-7 and NGAL were then chosen for further analyses in an independent verification group of 28 patients with and 12 control patients without AKI. IGFBP-7 and NGAL discriminated between early and late/non-recovery patients and patients with and without AKI. Significant upregulation of the urinary markers predicted mortality (IGFBP-7: AUC 0.68; NGAL: AUC 0.81), recovery (IGFBP-7: AUC 0.74; NGAL: AUC 0.70), and severity of AKI (IGFBP-7: AUC 0.77; NGAL: AUC 0.69), and were associated with the duration of AKI. IGFBP-7 was a more accurate predictor of renal outcome than NGAL. Thus, IGFBP-7 is a novel prognostic urinary marker that warrants further investigation.

The spectral effects of a number of endo- and exoproteases to the detergent-solubilized antenna complexes B802-858 (sphaeroides like) of strain Rhodopseudomonas acidophila 10050 and B802-824 of strain 7050 were analyzed. Upon protease digestion the time course of spectral modification between 250 and 900 nm was recorded. In addition, circular dichroism (CD) signals were measured between 450 and 950 nm. The endoprotease elastase as well as the exoprotease carboxypeptidase A (CP A) altered the B802-858 antenna complex, by changing the 858 nm band hypochromically (30-50%) with an additional small hypsochromic shift (3-6 nm)

AMPK is a metabolic stress-sensing kinase with important functions for red blood cell (RBC) survival. By using a proteomic approach, we identified putative AMPK targets in hemoglobin-depleted lysates of RBC, including metabolic enzymes, cytoskeletal proteins and enzymes involved in the oxidative stress response. These data tie in with the phenotypic observations of AMPKalpha1-deficient RBC and provide reference for future studies.

The vacuolar H(+)-ATPase (V-ATPase) is a major contributor to luminal acidification in epithelia of Wolffian duct origin. In both kidney-intercalated cells and epididymal clear cells, cAMP induces V-ATPase apical membrane accumulation, which is linked to proton secretion. We have shown previously that the A subunit in the cytoplasmic V(1) sector of the V-ATPase is phosphorylated by protein kinase A (PKA). Here we have identified by mass spectrometry and mutagenesis that Ser-175 is the major PKA phosphorylation site in the A subunit. Overexpression in HEK-293T cells of either a wild-type (WT) or phosphomimic Ser-175 to Asp (S175D) A subunit mutant caused increased acidification of HCO(3)(-)-containing culture medium compared with cells expressing vector alone or a PKA phosphorylation-deficient Ser-175 to Ala (S175A) mutant. Moreover, localization of the S175A A subunit mutant expressed in HEK-293T cells was more diffusely cytosolic than that of WT or S175D A subunit. Acute V-ATPase-mediated, bafilomycin-sensitive H(+) secretion was up-regulated by a specific PKA activator in HEK-293T cells expressing WT A subunit in HCO(3)(-)-free buffer. In cells expressing the S175D mutant, V-ATPase activity at the membrane was constitutively up-regulated and unresponsive to PKA activators, whereas cells expressing the S175A mutant had decreased V-ATPase activity that was unresponsive to PKA activation. Finally, Ser-175 was necessary for PKA-stimulated apical accumulation of the V-ATPase in a polarized rabbit cell line of collecting duct A-type intercalated cell characteristics (Clone C). In summary, these results indicate a novel mechanism for the regulation of V-ATPase localization and activity in kidney cells via direct PKA-dependent phosphorylation of the A subunit at Ser-175.

Comparative primary structural analysis of polypeptides from antenna complexes from species of the three families of Rhodospirillaneae indicates the structural principles responsible for the formation of spectrally distinct light-harvesting complexes. In many of the characterized antenna systems the basic structural minimal unit is an alpha/beta polypeptide pair. Specific clusters of amino acid residues, in particular aromatic residues in the C-terminal domain, identify the antenna polypeptides to specific types of antenna systems, such as B880 (strong circular dichroism (CD)), B870 (weak CD), B800-850 (high), B800-850 (low) or B800-820. The core complex B880 (B1020) of species from Ectothiorhodospiraceae and Chromatiaceae apparently consists of four (alpha 1 alpha 2 beta 1 beta 2) or three (2 alpha beta 1 beta 2) chemically dissimilar antenna polypeptides respectively. There is good evidence that the so-called variable antenna complexes, such as the B800-850 (high), B800-850 (low) or B800-820 of Rp. acidophila, Rp. palustris and Cr. vinosum, are comprised of multiple forms of peripheral light-harvesting polypeptides. Structural similarities between prokaryotic and eukaryotic antenna polypeptides are discussed in terms of similar pigment organization. The structural basis for the strict organization of pigment molecules (bacteriochlorophyll (BChl) cluster) in the antenna system of purple bacteria is the hierarchical organization of the alpha- and beta-antenna polypeptides within and between the antenna complexes. On the basis of the three-domain structure of the antenna polypeptides with the central hydrophobic domain, forming a transmembrane alpha helix, possible arrangements of the antenna polypeptides in the three-dimensional structure of core and peripheral antenna complexes are discussed. Important structural and functional features of these polypeptides and therefore of the BChl cluster are the alpha/beta heterodimers, the alpha 2 beta 2 basic units and cyclic arrangements of these basic units. Equally important for the formation of the antenna complexes or the entire antenna are polypeptide-polypeptide, pigment-pigment and pigment-polypeptide interactions.

Antenna polypeptides from two species of the family Ectothiorhodospiraceae have been investigated. By means of gel filtration and subsequent high-performance liquid chromatography, at least five polypeptides were isolated from each of Ectothiorhodospira halochloris and Ectothiorhodospira halophila. The majority of their primary structures was identified by Edman degradation. Comparison of these polypeptide sequences with the known primary structures of antenna polypeptides from various purple non-sulfur bacteria revealed interesting new aspects with regard to the structure of the core-peripheral antenna system. E. halochloris and E. halophila contain two pairs of alpha- and beta-polypeptides each with typical primary structure elements of core complexes, indicating a modified antenna complex organization.

The mobilization of metabolic energy from adipocytes depends on a tightly regulated balance between hydrolysis and resynthesis of triacylglycerides (TAGs). Hydrolysis is stimulated by beta-adrenergic signalling to PKA that mediates phosphorylation of lipolytic enzymes, including hormone-sensitive lipase (HSL). TAG resynthesis is associated with high-energy consumption, which when inordinate, leads to increased AMPK activity that acts to restrain hydrolysis of TAGs by inhibiting PKA-mediated activation of HSL. Here, we report that in primary mouse adipocytes, PKA associates with and phosphorylates AMPKalpha1 at Ser-173 to impede threonine (Thr-172) phosphorylation and thus activation of AMPKalpha1 by LKB1 in response to lipolytic signals. Activation of AMPKalpha1 by LKB1 is also blocked by PKA-mediated phosphorylation of AMPKalpha1 in vitro. Functional analysis of an AMPKalpha1 species carrying a non-phosphorylatable mutation at Ser-173 revealed a critical function of this phosphorylation for efficient release of free fatty acids and glycerol in response to PKA-activating signals. These results suggest a new mechanism of negative regulation of AMPK activity by PKA that is important for converting a lipolytic signal into an effective lipolytic response.

Background— Marfan syndrome (MFS) is a heritable disorder of connective tissue, affecting principally skeletal, ocular, and cardiovascular systems. The most life-threatening manifestations are aortic aneurysm and dissection. We investigated changes in the proteome of aortic media in patients with and without MFS to gain insight into molecular mechanisms leading to aortic dilatation. Methods and Results— Aortic samples were collected from 46 patients. Twenty-two patients suffered from MFS, 9 patients had bicuspid aortic valve, and 15 patients without connective tissue disorder served as controls. Aortic media was isolated and its proteome was analyzed in 12 patients with the use of 2-dimensional difference gel electrophoresis and mass spectrometry. We found higher amounts of filamin A C-terminal fragment, calponin 1, vinculin, microfibril-associated glycoprotein 4, and myosin-10 heavy chain in aortic media of MFS aneurysm samples than in controls. Regulation of filamin A C-terminal fragmentation was validated in all patient samples by immunoblotting. Cleavage of filamin A and the calpain substrate spectrin was increased in the MFS and bicuspid aortic valve groups. Extent of cleavage correlated positively with calpain 2 expression and negatively with the expression of its endogenous inhibitor calpastatin. Conclusions— Our observation demonstrates for the first time upregulation of the C-terminal fragment of filamin A in dilated aortic media of MFS and bicuspid aortic valve patients. In addition, our results present evidence that the cleavage of filamin A is highly likely the result of the protease calpain. Increased calpain activity might explain, at least in part, histological alterations in dilated aorta.

Objective Acute kidney injury is a well-known complication with high morbidity and mortality after cardiopulmonary bypass. Cardiopulmonary bypass–associated acute kidney injury is still poorly understood. Methods Thirty-six patients undergoing elective cardiopulmonary bypass were enrolled. Spot urine samples before and after cardiopulmonary bypass were collected. Acute kidney injury was defined according to the RIFLE classification. To identify differentially regulated proteins after cardiopulmonary bypass, we first analyzed the urinary proteome before and after cardiopulmonary bypass. To identify differentially regulated proteins in acute kidney injury, we next compared the urinary proteome obtained on the first postoperative day between patients with and without acute kidney injury. Difference fluorescence gel electrophoresis was used to compare protein profiles and mass spectrometry to identify individual proteins. Results After cardiopulmonary bypass, inflammation-associated (zinc-alpha-2-glycoprotein, leucine-rich alpha-2-glycoprotein, mannan-binding lectin serine protease 2, basement membrane-specific heparan sulfate proteoglycan, and immunoglobulin kappa) or tubular dysfunction-associated (retinol-binding protein, adrenomedullin-binding protein, and uromodulin) proteins were differentially regulated. Acute kidney injury developed in 6 of 36 patients. A modified urinary albumin was increased, and zinc-alpha-2-glycoprotein and a fragment of adrenomedullin-binding protein were decreased in patients with acute kidney injury. Decreased excretion of zinc-alpha-2-glycoprotein in patients with acute kidney injury was confirmed by Western blot and enzyme-linked immunosorbent assay in an independent cohort of 22 patients with and 46 patients without acute kidney injury. Conclusion Cardiopulmonary bypass leads to increased urinary excretion of inflammatory proteins and markers of tubular injury. Zinc-alpha-2-glycoprotein is a potentially useful predictive marker for acute kidney injury after cardiopulmonary bypass surgery.

Phosphoamino acid modifications on substrate proteins are critical components of protein kinase signaling pathways. Thus, diverse methodologies have been developed and applied to identify the sites of phosphorylated amino acids within proteins. Despite significant progress in the field, even the determination of phosphorylated residues in a given highly purified protein is not a matter of routine and can be difficult and time-consuming. Here we present a practicable approach that integrates into a liquid chromatography matrix-assisted laser desorption/ionization mass spectrometry (LC-MALDI MS) workflow and allows localization and quantification of phosphorylated peptides on the MALDI target plate prior to MS analysis. Tryptic digests of radiolabeled proteins are fractionated by reversed-phase LC directly onto disposable MALDI target plates, followed by autoradiographic imaging. Visualization of the radiolabel enables focused analysis of selected spots, thereby accelerating the process of phosphorylation site mapping by decreasing the number of spectra to be acquired. Moreover, absolute quantification of the phosphorylated peptides is permitted by the use of appropriate standards. Finally, the manual sample handling is minimal, and consequently the risk of adsorptive sample loss is very low. Application of the procedure allowed the targeted identification of six novel autophosphorylation sites of AMP-activated protein kinase (AMPK) and displayed additional unknown phosphorylated peptide species not amenable to detection by MS. Furthermore, autoradiography revealed topologically inhomogeneous distribution of phosphorylated peptides within individual spots. However, accurate analysis of defined areas within single spots suggests that, rather than such quantitative differences, mainly the manner of matrix crystallization significantly affects ionization of phosphopeptides.

To obtain information on the structural and functional role of highly conserved amino acid residues in the B870 alpha and beta light-harvesting polypeptides of Rhodobacter capsulatus, site-directed mutagenesis was performed. 18 mutants with single amino acid substitutions at nine different positions in the B870 antenna polypeptides were prepared in a B800-850-lacking strain. The characterization of the resulting phenotypes was based on a quantification of the core-complex elements (reaction center, light-harvesting polypeptides, bacteriochlorophyll a and carotenoid) and the core-complex spectral characteristics (absorption maximum, absorption coefficient and fluorescence intensity). These data generally showed that strong structural effects were caused by the amino acid substitutions. Thus, the three tryptophan exchanges at the position alpha 8 resulted in either the absence of a core complex (alpha Trp8----Leu), the absence of the core antenna (alpha Trp8----Ala) or a reduction in the carotenoid content (alpha Trp8----Tyr). Likewise, the mutants alpha Pro13Gly (i.e. alpha Pro13----Gly), beta Gly10Val and alpha Phe23Ala demonstrated an abnormal protein/pigment ratio in the core antenna, while a drastically reduced antenna size resulted from the amino acid exchange beta Arg45Asp. In contrast to the structural effects, the absorption maxima and the fluorescence intensities of the mutant antennae differed only slightly from the wild type. The strongest blue shift of the bacteriochlorophyll a (8-11 nm) was induced by substitutions of the Trp at position alpha 43 (alpha Trp43----Ala, Leu or Tyr). Contrary to the other spectral effects, the absorption coefficient of bacteriochlorophyll a was strongly influenced by the amino acid substitutions and varied by 1.6-times less (beta Arg45Asp) and 1.3-times greater (alpha Phe25Ala) than normal. The antenna-free mutant, alpha Trp8Ala, yielded a high rate of B800-850 revertants during phototrophic growth, indicating a direct energy transfer from the B800-850 antenna to the reaction center in these strains. Although conditions for growth were generally observed to influence phenotypic expression, the structural as well as spectral effects were demonstrated to differ to the greatest extent between chemotrophically grown and phototrophically grown cells.

The AAPS journal, 10 (2), ISSN:1550-7416, ISSN:1522-1059

International audience; AMP-activated protein kinase (AMPK) is a heterotrimeric serine/threonine kinase that is involved in the maintenance of energy homeostasis and recovery from metabolic stresses both at the cellular and whole body level. AMPK is found in all tissues examined so far, and a number of downstream targets have been identified. Recent work suggests that AMPK has specialized functions in the brain, such as involvement in appetite control. Nevertheless, brain-specific substrates of AMPK are unknown. Here, we performed a proteomic in vitro screen to identify new putative AMPK targets in brain. Prefractionation of murine brain lysates by liquid chromatography, utilizing four different, serially connected columns with different chemistries was found to be superior to a single column method. A pilot screen involving incubation of small volumes of individual fractions with radiolabeled ATP in the presence or absence of active AMPK, followed by one-dimensional SDS-PAGE and autoradiography, revealed the presence of potential AMPK substrates in a number of different fractions. On the basis of these results, several kinase assays were repeated with selected fractions on a preparative scale. Following separation of the radiolabeled proteins by two-dimensional electrophoresis and comparison of samples with or without added AMPK by differential autoradiography, 53 AMPK-specific phospho-spots were detected and excised. Thereof, 26 unique proteins were identified by mass spectrometry and were considered as new potential downstream targets of AMPK. Kinase assays with 14 highly purified candidate substrate proteins confirmed that at least 12 were direct targets of AMPK in vitro. Although the physiological consequences of these phosphorylation events remain to be established, hypotheses concerning the most intriguing potential targets of AMPK that have been identified by this search are discussed herein. Our data suggests that signaling by AMPK in brain is likely to be involved in the regulation of pathways that have not yet been linked to this kinase.

Membrane proteins play a central role in biological processes, but their separation and quantification using two-dimensional gel electrophoresis is often limited by their poor solubility and relatively low abundance. We now present a method for the simultaneous recovery, separation, identification, and relative quantification of membrane proteins, following their selective covalent modification with a cleavable biotin derivative. After cell lysis, biotinylated proteins are purified on streptavidin-coated resin and proteolytically digested. The resulting peptides are analyzed by high-pressure liquid chromatography and mass spectrometry, thus yielding a two-dimensional peptide map. Matrix assisted laser desorption/ionization-time of flight signal intensity of peptides, in the presence of internal standards, is used to quantify the relative abundance of membrane proteins from cells treated in different experimental conditions. As experimental examples, we present (i) an analysis of a BSA-spiked human embryonic kidney membrane protein extract, and (ii) an analysis of membrane proteins of human umbilical vein endothelial cells cultured in normoxic and hypoxic conditions. This last study allowed the recovery of the vascular endothelial-cadherin/actin/catenin complex, revealing an increased accumulation of beta-catenin at 2% O(2) concentration.

International audience

The modification effects on the absorption and cirular dichroic (CD) spectra of the isolated B800-860 antenna complex of Rhodocyclus tenuis by a number of proteolytic enzymes were investigated. The chymotrypsin modifications of the B800-860 complex led to an about 40% decrease of the 860-nm band and a blue-shift to 841 nm. The biphasic CD signal related to the B860 BChl disappeared and a new double CD signal with a zero-crossing point at 842 nm appeared. These absorption and CD spectral changes suggested that a B800-841 complex resulted after chymotrypsin digestion. The polypeptide components of the chymotrypsin-modified B800-860 complex were separated by reverse-phase chromatography, and their amino acid sequences determined by protein sequencing and mass spectrometry. Sequence analyses showed that the C-terminal 25 residues of the B800-860-α polypeptide and the C-terminal 8 residues of the B800-860-β polypeptide were cleaved by chymotrypsin, and the remaining α, β polypeptide fragments apparently form the structural basis for the newly-formed B800-841 complex. No significant spectral change was observed from exposing the isolated B800-860 complex to trypsin, carboxypeptidase A and the combination of carboxypeptidase A and carboxypeptidase B. Short-term proteinase K incubation of the B800-860 complex of Rc. tenuis led to a preferential decrease of the 860-nm absorbance band and its related CD signals, as compared to the 800-nm absorbance and CD bands, suggesting that the C-terminal portions of the antenna polypeptides are possibly exposed to the exterior of the B800-860 complex micelles. Whereas, long-term proteinase K digestion resulted in the spectral collapse of the B800-860 complex and the release of free BChls. Our proteolysis experiments support the hypothesis that the C-terminal portions of the antenna polypeptides play a key role in the redshift and strong molar extinction of the Qy band of the B850 BChls.

The photoreceptor complex (B885-RC) and the peripheral antenna complex (B800-860) were isolated from photosynthetic membranes of the purple non-sulfur bacterium Rhodocyclus tenuis DSM 109 using a detergent combination of Deriphate-160 and octyl glucoside and subsequent linear sucrose gradient centrifugation. The two complexes were characterized by room-temperature absorption, circular dichroism and fluorescence spectroscopy. The B800-860 complex has a more red-shifted B860 absorbance band. The alpha,beta-polypeptides were purified with a reverse-phase HPLC system and resolved at a ratio of 1:1 in the B800-860 complex and at an overall ratio of 1:1 for the B885-RC complex. The complete amino acid sequences of the alpha and beta polypeptides of the B800-860 and B885-RC complexes were determined by micro-sequencing analysis and mass spectrometry. The B800-860-alpha polypeptide possesses an identical N-terminal domain (the first 15 residues) to Rhodobacter sphaeroides B800-850-alpha polypeptide. The central hydrophobic and C-terminal domains of the B800-860-alpha,beta polypeptides show a number of B870/880-like structural elements in which, of special interest, is the WWSEF cluster in the C-terminal domain of the B800-860-alpha polypeptide which is very similar to the WWEF cluster in the same region of Rhodopseudomonas viridis B1015-alpha polypeptide. The more red-shifted absorption characteristic of the 860-nm bacteriochlorophylls could most probably be related to the B870/880-like polypeptide features in the central hydrophobic domains and the C-terminal domains of the B800-860-alpha,beta polypeptides. The hydropathy plot of the B800-860-alpha polypeptide exhibits an extended C-terminal hydrophobic segment indicative of a second membrane-contacting domain, which has not been found in the antenna polypeptides of the purple bacteria with intracytoplasmic membranes. Further sequence analysis revealed the existence of multiple forms of the B885-alpha,beta polypeptides, the B885-alpha 1,alpha 2 polypeptides, and the B885-beta 1,beta 2 polypeptides. The B885-Alpha 2 polypeptide shows an identical sequence to the B885-alpha 1 polypeptide, but it is 12 amino acid residues shorter than the B885-alpha 1 polypeptide at the C-terminal. The two species of the B885-beta polypeptides were identified as an identical sequence with only one amino acid residue variation at sequence position 34, where the B885-beta 1 has a valine residue and the B885-beta 2 polypeptide an isoleucine residue. The possible correlation between the intensity of the near-infrared circular dichroic signal and the specific structural features of the alpha and beta core antenna polypeptides is also discussed.

The core antenna/reaction-centre complex RC-B875 and the peripheral antenna complex B800-850 of the two strains DSM 149 and DSM 151 of the purple non-sulphur bacterium Rhodocyclus gelatinosus have been isolated from photosynthetic membranes by means of lauryl-N,N-dimethyl-amineoxide as a detergent and subsequent sucrose-gradient centrifugation. The two complexes were characterised spectroscopically by absorption and circular dichroism (CD) spectroscopy at room temperature. CD measurements revealed very weak signals for the core antenna B875 whereas for the peripheral antenna B800-850, a strong biphasic CD signal was observed, attributable to the B850 pigments. There is apparently no CD signal present for the B800 pigments. The core and the peripheral antenna complex are built up by a distinct alpha/beta-polypeptide pair. The pigment/protein ratio in the peripheral antenna complex is 3 bacteriochlorophyll/(alpha/beta)-polypeptide pair. The amino acid sequences of the alpha and beta polypeptides of both complexes from the two strains of Rc. gelatinosus were established by automated Edman degradation, chemical and enzymic digestion, amino acid composition analyses and carboxypeptidase digestion. In the case of the beta polypeptides, the amino acid sequence determination was confirmed by ion-spray MS of the isolated antenna apoproteins. The inter-strain (DSM 149 and 151) positional identity between the equivalent apoproteins is extremely large and varies in the range 90-100%. The B875-beta polypeptide from Rc. gelatinosus exhibits shortened C-termini, as detected for the analogous antenna apoproteins of Rhodobacter sphaeroides and Rhodobacter capsulatus, which can be correlated with weak core antenna near-infrared CD signals. However, the B800-850-alpha polypeptide of Rc. gelatinosus, with 71 amino acids, exhibits an extended C-terminal portion indicative of the formation of a second transmembrane domain, which so far has not been observed for bacterial antenna apoproteins. This part of the molecule is extremely rich in alanine and proline residues. All the sequenced antenna apoproteins of Rc. gelatinosus exhibit a characteristic membrane-buried histidine which is thought to ligate the B875 or the B850 pigments. In the B800-850-beta apoprotein, a second, so far beta-antenna-apoprotein-specific histidine, is replaced by a glutamine residue. A careful inspection of the determined antenna structures of Rc. gelatinosus revealed some remarkable structural similarities within presumed cofactor-binding sites of Fe-S-type-reaction-centre apoproteins, indicating possible basic structural motifs for complexing bacteriochlorophyll molecules.

The circular dichroism (CD) spectrum of isolated chlorosomes fromChloroflexus aurantiacus showed a conservative, S-shaped signal with a negative maximum at 723 nm, a positive maximum at 750 nm and a zero-crossing at 740 nm. Proteolytic treatment of chlorosomes with trypsin at 37°C did not change the CD signal or the absorption spectrum in contrast to treatment with proteinase K, where a twofold increase in rotational strength and a slight decrease of the absorption band at 740 nm were observed. Treatment with saturating 1-hexanol concentrations resulted in a blue shift of the absorption band at 740 nm as well as in changes of the CD spectrum. These changes reversed when the sample was diluted to half the saturating 1-hexanol concentration. In contrast to that, we observed an irreversible formation of a giant CD signal using the combination of 1-hexanol and proteinase K treatment. Electron micrographs of chlorosomes treated with both 1-hexanol and proteinase K showed large aggregates of multiple chlorosome size. By comparison of proteinase K induced effects with trypsin effects it appeared that the 5.7 kDa polypeptide has a structural role in the organisation of BChlc in the chlorosome.

Electron micrographs of photosynthetic membranes of the BChla-containing bacterium Rp, marina showed a quasi-crystalline structure. The photoreceptor units are arranged in a hexagonal lattice with a reaction center to reaction center distance of 102± A. Purified B880-complex was concentrated up to an OD? of 60 which induced the formation of large protein vesicles. The protein complexes within these vesicles were highly ordered and showed a hexagonal lattice with the same center to center distance of 102±3 A as was observed in the native membranes. Image processing of the micrographs revealed a ring-like structure of the B880-complex at 26 A resolution and suggests that the B880-complex consists of 5 or 6 subunits. For the first time it can be shown that an isolated core-complex is in a stable, ring-like structure even without the reaction center which is supposed to be located in the middle of the B880-ring. The data indicate that the isolated B880-complex exhibits the same structure as in the native membrane.

The BChla-containing B880-complex (core-complex) of Rhodopseudomonas marina (Rhodospirillaceae) was isolated with a new purification method. The isolation of the B880-complex was performed by solubilisation of the photosynthetic membranes with the detergent LDAO and subsequent fractionated ammonium-sulfate precipitation with about 50% recovery. The B880-complex retained its original spectral properties as revealed with absorption, fluorescence and circular dichroism spectroscopy. Furthermore, we dissociated the B880-complex with the detergent n-octyl-beta-glucoside (OG) and purified the developed subcomplex by the method of Miller et al. [1], which showed an absorption maximum at 820 nm (B820). The alpha- to beta-polypeptide ratio and the alpha- or beta-polypeptide to BChla ratio, respectively, were estimated to be 1:1 in both complexes. The molecular weights of the B880 and the B820-complexes, determined by gel filtration chromatography, were 181 and 32 kDa, respectively. Thus, it appears that the B880-complex of Rp. marina consists of 24 polypeptides and the B820-complex of four polypeptides. Six B820-complexes or possible subunits could form the B880-complex. On the basis of these data we propose a model for the structure of BChla containing core-complexes.

The B806-866 antenna complex was isolated from cytoplasmic membranes of the green thermophilic bacterium Chloroflexus aurantiacus. The membranes were treated with 7 M urea at 50 degrees C, the B806-866 antenna complex was solubilized with a mixture of Noni-fjdet P-40 (octylphenoxypolyethoxyethanol (Sigma)) and sodium dodecylsulphate (2:1) and isolated by sucrose density gradient centrifugation. This antenna complex was characterized by reversed-phase chromatography (fast polypeptide and polynucleotide liquid chromatography), amino acid and sequence analyses. The B806-866 antenna of Chloroflexus aurantiacus consists of two polypeptides: the B806-866-alpha and B806-866-beta polypeptides in an apparent stoichiometric ratio of 1:1, which may be equivalent to the structural elementary unit found in the antenna systems of many species of Rhodospirillaceae.

Proteins were solubilized selectively from chlorosomes of Chloroflexus aurantiacus by electrophoretic gel filtration according to Griebenow et al. Whereas the 11 kDa and 18 kDa proteins were extracted almost completely, the remaining modified chlorosomes contained high amounts of pigment and c-protein. It was concluded that the c-protein in contradiction to the publication by Griebenow et al. is indeed localized in the interior of Chloroflexus chlorosomes.

In addition to the previous isolated and sequenced polypeptides from green photosynthetic sulfur bacteria, which are presumably involved in binding BChl c and e, an analogous poly- peptide has been purified from the BChl d-containing bacterium Chlorobium vibrioforme f. thiosulfatophilum. The primary structure of this 6.15 kDa polypeptide was determined. It shows an extremely high homology (98.3%) to the corresponding polypeptide from Pelodictyon luteolum, indicative of an important functional role.

Some strains of Rp. palustris have been reported to exhibit similar cell-spectral features in the near-infrared as those of Rb. sphaeroides (wild-type) and Rb. capsulatus, indicative of a core antenna (B870) and one single peripheral antenna (B800–850) [1]. Further investigations on the antenna system of Rp. palustris revealed that certain strains may synthesize spectrally dissimilar intracytoplasmic membranes[2, 3], thus suggesting a similar situation as in Rp. acidophila [4]. In particular the B800–850 complexes from differently cultured cells (15000lux to 300lux) varied greatly in their absorption characteristics in the nearnfrared; i. e. high light cells synthesize a more standard, sphaeroides-like B800–850 complex, whereas low light induces a high 800 nm band and a low 850 nm band. From biophysical studies involving CD spectral analyses and resonance Raman spectroscopy it has been inferred that the B800–850 complex with a low 850 nm band differs from the ‘normaP B800–850 complex (high 850 nm) from Rb. sphaeroides or Rb. capsulatus regarding pigment organization [2, 3]. In addition, employing biochemical methods the two spectrally different peripheral antennae of Rp. palustris have been shown to vary 1.) in their polypeptide composition (only minor differences detected on SDS-gels) and 2.) in their pigment composition indicating a higher content of BChl in the low B850 complex [3]. The latter result has been confirmed by Cogdell et al. (paper in preparation).

Under anaerobic conditions purple photosynthetic bacteria are capable of phototrophic growth. They can convert light energy to chemical energy (to a proton gradient) with the help of membrane bound photochemical reaction centres and an electron recycling system (e. g. cytochrom bc, complex). In bright day light the absorption capacity of bacteriochlorophyll pigments of reaction centres is limited to 1 to 10 photons per second, although some orders more could be processed1. Therefore, it is inevitable that organisms increase the number of absorbing pigments in order to prevent light saturation of reaction centres even at optimal light conditions. Purple bacteria have solved this problem by the synthesis of light-harvesting pigment-protein-complexes which are specialized in absorbing light energy and delivering it to the photochemical reaction centre where charge separation takes place.

The light-harvesting complex B 880 from Rhodospirillum rubrum S 1 (wild type) and B 870 from the carotenoidless mutant G-9+ was shown to consist mainly of an organic solvent-(chloroform/methanol-) soluble and an organic solvent-insoluble polypeptide. The isolation and separation of these two low-molecular-mass polypeptides (Mr 6101 and Mr 6079) were achieved by a two-step extraction procedure of chromatophores using in the first step chloroform/methanol containing 0.1M ammonium acetate. Following Sephadex LH-60 chromatography of this first extract a light-harvesting polypeptide (B 870-alpha) was isolated and its complete amino acid sequence was determined (R. Brunisholz et al. (1981) FEBS Lett. 129/1, 150-154, B 880-alpha: G. Gogel et al. (1983) Biochim. Biophys. Acta 746, 32-39). Upon reextraction of the chromatophore pellet with chloroform/methanol/ammonium acetate containing in addition acetic acid a second low-molecular-mass polypeptide (B 880-beta of B 870-beta) was generated. The complete amino acid sequences of the chloroform/methanol-insoluble light-harvesting polypeptide of Rs. rubrum S 1 (B 880-beta) and of Rs. rubrum G-9+ (B 870-beta) were determined. They are identical and consist of 54 amino acid residues. The conserved histidine residue within the hydrophobic stretch raises more evidence for ligand complexation of bacteriochlorophyll to this specific histidine residue which therefore possibly plays the key role in pigment-protein interactions. Both polypeptides (B 880-alpha and B 880-beta) are part of the light-harvesting complex B 880 in an apparent ratio of 1:1. Based on the primary structure data a possible arrangement of both light-harvesting polypeptides within the membrane will be discussed.

The unspecific proteinase K and the specific proteases alpha-chymotrypsin, trypsin and S. aureus V 8 protease were used in order to determine the orientation of the polypeptides B 870-alpha and B 870-beta from the major antenna complex B 870 of Rs. rubrum G-9+ within the chromatophore membrane (inside-out vesicle). Although B 870-alpha exhibits cleavable peptide bonds, treatment with specific proteases yielded splitting only in B 870-beta within the N-terminal region. In the case of proteinase K, which was most effective, mainly 6 (B 870-alpha) and 16 (B 870-beta) amino acid residues were removed from their N-terminal parts as proved by means of Edman degradation of cleavage products. The major peptide bonds cleaved were identified as Gln6-Leu7 in B 870-alpha and as Lys16-Glu17 in B 870-beta. The central hydrophobic stretch regions and the relatively hydrophilic C-terminal parts of both light-harvesting polypeptides were not affected by proteinase K. On the basis of these degradation experiments a transmembrane orientation of B 870-alpha and B 870-beta is postulated, with their N-terminal towards the cytoplasm and their C-termini towards periplasm with regard to the photosynthetic membrane. This hypothesis is supported by the transmembrane model proposed by Brunisholz et al. (Hoppe-Seyler's Z., Physiol. Chem., (1984) 365, 675-688) in which the hydrophobic stretch of B 870-alpha and of B 870-beta forming an alpha-helix would span the membrane once. Organic solvent extraction of chromatophores treated with proteinase K yielded a fairly pure polypeptide fragment with an apparent molecular mass of 14000 Da. Its N-terminal amino-acid sequence is identical with the sequence within the N-terminal region of the reaction centre subunit L of Rs. rubrum G-9+. Thus it is most likely that as in the case of B 870-beta, proteinase K removed 16 amino acid residues from the N-terminal part of subunit L. This subunit therefore also seems to be exposed at the surface of the cytoplasmic side of the chromatophore membrane.

A polypeptide soluble in organic solvents was isolated from whole membrane fractions of the green thermophilic bacterium Chloroflexus aurantiacus by chromatography on Sephadex LH-60, Whatman DE-32 and Bio Gel P-10. The complete amino acid sequence of this 4.9 kDa polypeptide (44 amino acid residues) was determined. The polypeptide shows a 3-domain structure, similar to the domain structure of the antenna BChI polypeptides of purple photosynthetic bacteria, and sequence homologies (27–39%) to the light-harvesting α-polypeptides of the B870 (890) antenna complexes from purple bacteria. Therefore, the 4.9 kDa polypeptide is designated B(808-866)-α. The typical His residue (conserved His residue identified in all antenna polypeptides of purple bacteria as possible BChI binding site) is found within the hydrophobic domain, which extends from Asn 10 to Leu 30.

Three low molecular mass polypeptides have been isolated by using the technique of organic solvent extraction of thylakoid membranes or whole cells from Rhodopseudomonas viridis. Their primary structures were determined by long liquid phase sequencer runs, combined with the isolation and sequence analysis of the C-terminal o-iodosobenzoic acid fragment and carboxypeptidase degradation. The polypeptide which consists of 58 amino-acids and is 46% homologous to the antenna polypeptide B880-alpha from Rhodospirillum rubrum was designated as B1015-alpha (1 His residue). The sequence homology between the second polypeptide, named B1015-beta (55 amino acids, 2 His residues) and B880-beta from Rs. rubrum is 52%. For the third polypeptide consisting of 36 amino acids and exhibiting a high hydrophobicity, no equivalent polypeptide has so far been found in other purple bacteria. The molar ratio of these three organic solvent soluble polypeptides from Rp. viridis was estimated to be 1:1:1. Accordingly, the 36 amino-acid polypeptide is likely to be an additional constituent of the light-harvesting complex B1015, consequently termed as B1015-gamma. According to hydrophathy profiles, the transmembrane arrangement of B1015-alpha and B1015-beta within the thylakoid membrane is supposed to be similar. B1015-gamma, however, shows a somewhat different hydropathy profile. A particular feature of this polypeptide is its high amount of aromatic amino acids. It is postulated that B1015-gamma is involved in the formation of regular arrays of light-harvesting complexes.

The single polypeptide chain of native porcine plasminogen (molecular weight approx. 90000 after CNBr-cleavage and geiltration (Sephadex G-75) yielded a high molecular weight core fraction of fragments linked by disulfide bridges and three fragments of lower molecular weight (N-terminal and C-terminal CNBr-fragments and dodecapeptide). From the reduced and S-carboxamidomethylated core fraction an additional seven fragments with molecula5r weights between 2000 and 38000 were obtained. The CNBr-fragments were aligned in the porcine plasminogen polypeptide chain according to sequence homologies with the known primary structure of human plasminogen. Due to the lack of two methionine residues in kringle 1 and in the N-terminal part of the light chain region and to an additional methonine residue in kringle 2 the CNBr-fragment pattern differs from that of human plasminogen. Affinity chromatography of elastase-digested, native plasminogen yielded three fragments with intact lysine binding sites, originating from the heavy chain region and a non-adsorbable fragment, corresponding to human ‘mini’-plasminogen. This fragment was converted by urokinase into a proteolytically active protein which served for the isolation of the porcine plasmin light chain. With the aid of the fragments produced by the CNBr and elastase cleavage approx. 350 residues were sequenced, of which about 80% showed identity with the sequence of human plasminogen. This percentage varied depending on the region of the molecule, with the highest extent of identity (80 - 90%) found in the analyzed kringles 2 and 4.

The complete amino acid sequence has been obtained for a major light-harvesting polypeptide of wild-type Rhodospirillum rubrum. The peptide was isolated from lyophilized chromatophores by extraction with chloroform/methanol (1:1, v:v) and then purified by gel filtration chromatography using the same solvent with 0.1 M ammonium acetate added. The methods used for sequence determination were: (1) partial acid hydrolysis followed by separation of small peptides on a Beckman PA-35 column and then determination of their amino acid sequences by manual Edman degradation, (2) unblocking the N-terminus by mild acid treatment and subjecting the resulting polypeptide to automated Edman degradation, (3) cleavage by o-iodosobenzoic acid treatment, isolation of the C-terminal fragment by chromatography on a Bio-Rad P-2 column and sequencing by automated Edman degradation, and (4) determination of the C-terminus by carboxypeptidase Y treatment. The amino acid sequence obtained was: Nf-Met-Trp-Arg-Ile-Trp-Gln-Leu-Phe-Asp-Pro-Arg-Gln-Ala-Leu-Val-Gly-Leu-Ala-Thr- Phe-Leu-Phe-Val-Leu-Ala-Leu-Leu-Ile-His-Phe-Ile-Leu-Leu-Ser-Thr-Glu-Arg-Phe-Asn-Trp-Leu-Glu-Gly-Ala-Ser-Thr-Lys-Pro-Val-Gln-Thr-Ser-COOH. This is identical to the previously determined amino acid sequence of a light-harvesting polypeptide from the G-9 carotenoid-less mutant of R. rubrum. The long stretch of consecutive apolar amino acids suggests this polypeptide may span the membrane with an α-helical segment.

The antenna complex B 880 of Rp. marina has been isolated by applying ion-exchange chromatography on Whatman DE -52 resin and sucrose density centrifugation of LDAO-solubilized photosynthetic membranes. The antenna polypeptides B 880-α and B 880-β were pre­pared by organic solvent extraction of extensively dialyzed and freeze-dried B 880 antenna complex material or photosynthetic membranes. Gel filtration on Sephadex LH-60 and ion-exchange chromatography on Whatman DE -32 resin in the presence of organic solvents and an additional step on a C-8 reversed phase column yielded pure α-and β-apoproteins. Their complete primary structures have been elucidated using automated Edman degradation and carboxypeptidase diges­tion. According to quantitative Edman degradation the ratio of B 880-α and B 880-β has been determined as 1:1 in the isolated antenna complex as well as in the photosynthetic membrane. B 880-α of Rp. marina, presumably N -form ylated, consists of 52 amino acid residues and is 75, 56, 52 and 44% homologous to the corresponding core antenna polypeptides of Rs. rubrum, Rp. viridis, Rb. capsulatus and Rb. sphaeroides. In contrast, B 880-β (56 amino acid residues) is less homologous to the corresponding core β-antenna polypeptides of the same strains (57. 51. 41 and 42%). It shows an extended N-terminal domain as compared to the B 880-a polypeptide. Apart from the typical structural features of bacterial membrane-bound antenna polypeptides (three domain structure, His-residue in the hydrophobic stretch) the antenna polypeptides of Rp. marina are structurally related to polypeptides of core antenna complexes with strong near infra­red circular dichroism signals.