Phylogeny, topology, structure and functions of membrane-bound class III peroxidases in vascular plants

Peroxidases are key player in the detoxification of reactive oxygen species during cellular metabolism and oxidative stress. Membrane-bound isoenzymes have been described for peroxidase superfamilies in plants and animals. Recent studies demonstrated a location of peroxidases of the secretory pathway (class III peroxidases) at the tonoplast and the plasma membrane. Proteomic approaches using highly enriched plasma membrane preparations suggest organisation of these peroxidases in microdomains, a developmentally regulation and an induction of isoenzymes by oxidative stress. Phylogenetic relations, topology, putative structures, and physiological function of membrane-bound class III peroxidases will be discussed.     

Influence of urea calcium mixture supplementation on ruminal fermentation characteristics of beef cattle fed on concentrates containing high levels of cassava chips and rice straw

The purpose of this study was to evaluate the effects of various N sources in concentrates containing high levels of cassava chips, with rice straw as the basal forage, on rumen ecology, rumen microbial counts, microbial crude (CP) protein synthesis, and digestibility of nutrients. Four ruminally fistulated crossbred (Brahman × native) beef steers with initial body weight (BW) of 400 ± 40.2 kg were randomly assigned according to a 4 × 4 Latin square design. The dietary treatments were different sources of N in the concentrates and were: T1 = urea (control; urea); T2 = soybean meal (SBM); T3 = urea CaCl₂ mixture (U-Cal); T4 = urea CaSO₄ mixture (U-Cas). All steers were kept in individual pens and supplemented with concentrate at 5 g/kg of BW daily. The experiment was 4 periods, and each lasted 21 d. During the first 14 d, all steers were fed their respective diets ad libitum and for during the last 7 d, they were moved to metabolism crates for total urine and fecal collection. Dry matter intake ranged from 9.8 to 10.5 kg daily and was not altered by diet, while digestibility of NDF differed among treatments and was highest with U-Cas supplementation (P<0.05). Ruminal NH₃ N and plasma urea N with U-Cal, U-Cas, and SBM diets were lower compared with the urea supplemented group (P<0.05). Ruminal volatile fatty acid concentrations were not altered by treatments. Total viable, and cellulolytic bacteria, differed among treatments and were highest with U-Cas (9.1 × 10¹¹, and 4.0 × 10⁹ cfu/mL, respectively). In addition, efficiency of rumen microbial CP synthesis based on organic matter (OM) truly digested in the rumen was increased by SBM or U-Cal supplementation, and was highest with U-Cas supplementation (18.2 g of N/kg of OM truly digested in the rumen). Supplementation of U-Cas to a concentrate containing a high level of cassava chips improved rumen ecology and microbial CP synthesis in beef cattle, suggesting that urea calcium mixtures can replace soybean meal or urea in beef cattle diets without adverse affects on rumen fermentation and other rumen parameters.     

A decade of crystallization drops: Crystallization of the cbb3 cytochrome c oxidase from Pseudomonas stutzeri

The cbb₃ cytochrome c oxidases are distant members of the superfamily of heme copper oxidases. These terminal oxidases couple O₂ reduction with proton transport across the plasma membrane and, as a part of the respiratory chain, contribute to the generation of an electrochemical proton gradient. Compared with other structurally characterized members of the heme copper oxidases, the recently determined cbb₃ oxidase structure at 3.2 Å resolution revealed significant differences in the electron supply system, the proton conducting pathways and the coupling of O₂ reduction to proton translocation. In this paper, we present a detailed report on the key steps for structure determination. Improvement of the protein quality was achieved by optimization of the number of lipids attached to the protein as well as the separation of two cbb₃ oxidase isoenzymes. The exchange of n‐dodecyl‐β‐d ‐maltoside for a precisely defined mixture of two α‐maltosides and decanoylsucrose as well as the choice of the crystallization method had a most profound impact on crystal quality. This report highlights problems frequently encountered in membrane protein crystallization and offers meaningful approaches to improve crystal quality.     

An amphipathic polypeptide derived from poly‐γ‐glutamic acid for the stabilization of membrane proteins

Difficulties in the extraction of membrane proteins from cell membrane and their solubilization in native conformations have hindered their structural and biochemical analysis. To overcome these difficulties, an amphipathic polypeptide was synthesized by the conjugation of octyl and glucosyl groups to the carboxyl groups of poly‐γ‐glutamic acid (PGA). This polymer, called amphipathic PGA (APG), self‐assembles as mono‐disperse oligomers consisted of 4–5 monomers. APG shows significantly low value of critical micelle concentration and stabilization activity toward membrane proteins. Most of the sodium dodecyl sulfate (SDS)‐solubilized membrane proteins from Escherichia coli remain soluble state in the presence of APG even after the removal of SDS. In addition, APG stabilizes purified 7 transmembrane proteins such as bacteriorhodopsin and human endothelin receptor Type A (ET_A) in their active conformations. Furthermore, ET_A in complex with APG is readily inserted into liposomes without disrupting the integrity of liposomes. These properties of APG can be applied to overcome the difficulties in the stabilization and reconstitution of membrane proteins.     

Activation and Deactivation of F0F1-ATPase in Yeast Mitochrondia

The regulation of membrane-bound proton F₀F₁ATPase by the protonmotive force and nucleotides was studied in yeastmitochondria. Activation occurred in whole mitochondria and the ATPaseactivity was measured just after disrupting the membranes with Triton X-100.Deactivation occurred either in whole mitochondria uncoupled with FCCP, or indisrupted membranes. No effect of Triton X-100 on the ATPase was observed,except a slow reactivation observed only in the absence of MgADP. BothAMPPNP and ATP increased the ATPase deactivation rate, thus indicating thatoccupancy of nucleotidic sites by ATP is more decisive than catalyticturnover for this process. ADP was found to stimulate the energy-dependentATPase activation. ATPase deactivated at the same rate in uncoupled anddisrupted mitochondria. This suggests that deactivation is not controlled byrebinding of some soluble factor, like IF1, but rather by the conversion ofthe F₁.IF1 complex into an inactive form.     

An overview of Bacillus proteases: from production to application

Proteases have a broad range of applications in industrial processes and products and are representative of most worldwide enzyme sales. The genus Bacillus is probably the most important bacterial source of proteases and is capable of producing high yields of neutral and alkaline proteolytic enzymes with remarkable properties, such as high stability towards extreme temperatures, pH, organic solvents, detergents and oxidizing compounds. Therefore, several strategies have been developed for the cost-effective production of Bacillus proteases, including optimization of the fermentation parameters. Moreover, there are many studies on the use of low-cost substrates for submerged and solid state fermentation. Other alternatives include genetic tools such as protein engineering in order to obtain more active and stable proteases and strain engineering to better secrete recombinant proteases from Bacillus through homologous and heterologous protein expression. There has been extensive research on proteases because of the broad number of applications for these enzymes, such as in detergent formulations for the removal of blood stains from fabrics, production of bioactive peptides, food processing, enantioselective reactions, and dehairing of skins. Moreover, many commercial proteases have been characterized and purified from different Bacillus species. Therefore, this review highlights the production, purification, characterization, and application of proteases from a number of Bacillus species.     

Solubilization and partial purification of the rabbit parotid Na/K/Cl-dependent bumetanide binding site

Summary We demonstrate that the high affinity bumetanide binding site of the rabbit parotid acinar cell can be extracted from a basolateral membrane fraction using relatively low concentrations (0.07%, wt/vol; 1 mg membrane protein/ml) of the nonionic detergent Triton X-100. This extracted site cannot be sedimented by ultracentrifugation at 100,000 ×g × 1 hr. Bumetanide binding to this site retains the ionic characteristics of bumetanide binding to native membranes but shows a fivefold increase in binding affinity (K d=0.57±0.15 m vs.K d=3.3±0.7 m for native membranes). Inactivation of the extracted bumetanide binding site observed at detergent/protein ratios>1 can be prevented or (partially) reversed by the addition of exogenous lipid (0.2% soybean phosphatidylcholine). When the 0.07% Triton extract is fractionated by sucrose density gradient centrifugation in 0.24% Triton X-100, 0.2% exogenous lipid and 200mm salt, the high affinity bumetanide binding site sediments as a single band withS _20,w =8.8±0.8 S. This corresponds to a molecular weight 200 kDa for the bumetanide binding protein-detergent-lipid complex and represents a sevenfold purification of this site relative to the starting membrane fraction. In contrast to previous attempts to purify Na/K/Cl cotransport proteins and their associated bumetanide binding sites, the present method avoids harsh detergent treatment as well as direct covalent modification (inactivation) of the transporter itself. As a consequence, one can follow the still active protein through a series of extraction and purification steps by directly monitoring its bumetanide binding properties.     

Reinvestigation of the chlorophyll distribution among the chlorophyll-proteins and chlorophyll-protein complexes of Hordeum vulgare L.

Solubilization of barley (Hordeum vulgare L.) thylakoid membranes with sodium dodecylsulphate plus sodium deoxycholate with or without Triton X-100 and subsequent fractionation in the polyacrylamide gel electrophoresis system described in this paper resulted: (1) in the resolution of the chlorophyll-proteins and chlorophyll-protein complexes commonly known as CP1a, CP1, LHCP¹, LHCP², CPa and LHCP³; (2) in the highly increased stability of CP1 and CP1a, as judged by their chlorophyll content, (3) at the expense of the free pigment concentration (4) which could be reduced to a negligible amount. Some 40% of the total chlorophyll contained in the mature higher plant thylakoid membrane is associated with CP1 and CP1 a and as already suggested before [19] no significant amount of free chlorophyll occurs in vivo.Abbreviations chl chlorophyll - CP1 P₇₀₀-chla-protein - CPa P680-chla-protein - DOC sodium deoxychlolate - FC free chlorophyll - LHCP light-harvesting chlorophyll a/b-protein - PAGE(S) polyacrylamide gel electrophoresis (system) - SDS sodium dodecylsulphate - TX-100 Triton X-100     

Isolation of a membrane protein by chromatofocusing: Cytochrome b-561 of the adrenal chromaffin granule

Chromatofocusing, a form of ion-exchange chromatography in which proteins are separated on the basis of their differing isoelectric points, has been adapted for use with membrane proteins, solubilized by the non-ionic detergent Nonidet P-40. Using a two-step detergent extraction followed by chromatofocusing under high pressure, the highly hydrophobic protein cytochrome b-561 was isolated from chromaffin granule membranes and purified to near homogeneity in a functionally active form, in less than 5 h. Chromatofocusing conditions were optimized empirically since the behaviour of the chromaffin granule membrane proteins conformed less to the theory than that of soluble proteins, and the various factors affecting yield and resolution are discussed. The speed, high resolution and focusing effect could make this method particularly suitable for rapid isolation in a functionally active form of the many membrane proteins that are unstable in dilute solution and when removed from their lipid environment.     

Studies on detergent released choline acetyltransferase from membrane fractions of rat and human brain

The relationship between soluble and membrane choline acetyltransferase (ChAT) was studied. Differential solubilization of rat and human brain yielded ChAT in the soluble and membrane fractions. The addition of 1% Triton X-100 to membrane fractions resulted in a release of ChAT. A comparable release of lactate dehydrogenase was also observed. The Triton released ChAT and soluble ChAT from rat and human brain were efficiently purified by immuno-affinity chromatography. A single molecular weight of 68,000 was observed for both forms of rat and human brain ChAT. Epitope maps produced from both forms of human brain ChAT were identical. It is concluded that Triton release ChAT is identical to soluble ChAT and simply represents occluded soluble ChAT.