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Proline caused irreversible inhibition (involving reduction in V(max) without altering K(m) for RuBP) in Rubisco activity. Proline-induced suppression in Rubisco activity did not exceed beyond approximately 65% of the original activity even upon exposure to higher levels of proline for prolonged duration. However, NaCl-induced reduction in Rubisco activity was reversible. Native PAGE analysis of Rubisco-incubated with proline showed the presence of two distinct bands corresponding to approximately 430 and approximately 28 kDa, but that incubated with NaCl showed a single band. SDS-PAGE analysis revealed that the approximately 430- and approximately 28-kDa bands represent octamers of large subunits and dimers of small subunits, respectively. These results demonstrated for the first time that proline suppresses Rubisco activity by bringing about dissociation of the small subunits from the octamer core of large subunits, probably by weakening hydrophobic interactions between them.

Plasminogen undergoes a large conformational change when it binds 6-aminohexanoate. Using ultraviolet absorption spectroscopy and native PAGE, we show that hydrostatic pressure brings about the same conformational change. The volume change for this conformational change is −33 mL·mol−1. Binding of ligand and hydrostatic pressure both cause the protein to open up to expose surfaces that had previously been buried in the interior.

We have developed a modified blue native polyacrylamide gel electrophoresis (PAGE) protocol that can overcome aggregation of lipases seen in native PAGE. We have shown that two lipases, Pseudomonas aeruginosa lipase and Candida rugosa lipase, which aggregate in the native gel, can be resolved using our protocol. Activity staining was done to test for the functionality of the two lipases.

The different techniques described in purification protocols for pyridine nucleotide-dependent enzymes have been reviewed, covering mainly the papers published in the past six years. Chromatography was reported in 100% of reviewed papers and among the chromatographic techniques, affinity chromatography was the most used (ca. 92%), followed by ion-exchange chromatography (ca. 79%), size-exclusion chromatography (ca. 64%) and hydrophobic chromatography (ca. 24%). Other chromatographic techniques were used infrequently. Each chromatographic technique has a different specific capacity and chemical selectivity and, therefore, the order of selection should be based on a precise knowledge of the nature of the sample and the amount of the target enzyme that it contains. Analytical electrophoresis was used in about 95% of the reviewed papers, with denaturing polyacrylamide gel electrophoresis (PAGE) being the most widely used mode (ca. 92%), followed by native PAGE (ca. 48%). The use of isoelectric focusing was reported in 14% of the papers, while preparative gel electrophoresis was used in only 8% of the cases. The use of other electrophoretic techniques was reported in only a few papers. The use of continuous enzymatic activity assay methods (spectrophotometric) was found in most papers, while high-performance liquid chromatography-based methods (discontinuous assays) were reported in only 11% of the reviewed articles.

The Spanish and Portuguese working group of the ISFH comprises a total of 22 Laboratories from Spain, Portugal and some South American countries. Practically all the casework in forensic genetics in Spain (15 Labs) and Portugal (5 labs) is carried out in these laboratories. Since 1990 the group have organized collaborative exercises in DNA polymorphisms with the aim of progresion in standarization and for the discussion of technical and statistical problems as a first step towards a quality control program in Spain and Portugal.

A partial characterization of polyphenol oxidase (PPO) in papaya fruits is described in this work. Differences among papaya fruits from hermaphrodite and female flowers in terms of PPO activity are also presented. Total soluble PPO activity in female fruits was higher than in hermaphrodite fruits during all ripening proccesses, and greater PPO values were obtained from green papayas. In this ripening stage, female fruits exhibited 30% more PPO activity than hermaphrodite fruits. Specific PPO activity also underwent this evolution. Female green papayas showed a significantly higher PPO activity. This difference became insignificant in ripe fruits (10 days at 14 °C). Native PAGE experiments showed the presence of six PPO isoenzymes in hermaphrodite papayas whereas PPO from female fruits separated into only four bands (revealed with 0.003 M gallic acid). Assays of incubation with 0.003 M gallic acid followed by ethanolic washings produced a slightly different PPO pattern (five PPO active bands for hermaphrodite green fruits and four PPO active bands for female green ones). Through ripening, the PPO isoenzyme pattern did not undergo significant changes; only a band (Rf = 0.28) disappeared in the PPO extracts of mature-green, ripe, and overripe hermaphrodite fruits.

β -Lactoglobulin and α -Lactalbumin were isolated and crystallized by the classical method of Aschaffenburg and Drewry, and the purity of the crystals was assessed by native PAGE and SDS-PAGE. Although β -lactoglobulin was free of contaminants, α -lactalbumin was still contaminated after five recrystallization steps. Yields of crystals in this study were lower than reported by Aschaffenburg and Drewry. Further, it was observed that α -lactalbumin undergoes a Ca 2+ /Ca 2+ -free, dependent electrophoretic shift characteristic of many calcium-binding proteins.

Despite the ecological relevance of diatoms, many aspects of their photosynthetic machinery remain poorly understood. Diatoms differ from the green lineage of oxygenic organisms by their photosynthetic pigments and light-harvesting complex (Lhc) proteins, the latter of which are also called fucoxanthin-chlorophyll proteins (FCP). These are composed of three groups of proteins: Lhcf as the main group, Lhcr that are PSI associated, and Lhcx that are involved in photoprotection. The FCP complexes are assembled in trimers and higher oligomers. Several studies have investigated the biochemical properties of purified FCP complexes, but limited knowledge is available about their interaction with the photosystem cores. In this study, isolation of stable supercomplexes from the centric diatom Thalassiosira pseudonana was achieved. To preserve in vivo structure, the separation of thylakoid complexes was performed by native PAGE and sucrose density centrifugation. Different subpopulations of PSI and PSII supercomplexes were isolated and their subunits identified. Analysis of Lhc antenna composition identified Lhc(s) specific for either PSI (Lhcr 1, 3, 4, 7, 10-14, and Lhcf10) or PSII (Lhcf 1-7, 11, and Lhcr2). Lhcx6_1 was reproducibly found in PSII supercomplexes, whereas its association with PSI was unclear. No evidence was found for the interaction between photosystems and higher oligomeric FCPs, comprising Lhcf8 as the main component. Although the subunit composition of the PSII supercomplexes in comparison with that of the trimeric FCP complexes indicated a close mutual association, the higher oligomeric pool is only weakly associated with the photosystems, albeit its abundance in the thylakoid membrane.

The CIDE domain is a well known protein–protein interaction module that is initially detected at the apoptotic DNA fragmentation factor (DFF40/45). The interaction mechanism via the CIDE domain is not well understood. To elucidate CIDE domain mediated interactions in the apoptotic DNA fragmentation system, we conducted biochemical and mutational studies and found that the surface of CIDE domains can be divided into an acidic side and a basic side. In addition, a mutagenesis study revealed that the basic surface side of Drep2 CIDE is involved in the interaction with the acidic surface side of Drep1 CIDE and Drep3 CIDE. Our research supports the idea that a charge–charge interaction might be the general interaction mode of the CIDE:CIDE interaction. Structured summary of protein interactions Drep2 and Drep2 bind by molecular sieving (View Interaction: 1 , 2 ) Drep1 and Drep2 bind by molecular sieving ( View interaction ) Drep3 and Drep2 bind by molecular sieving ( View interaction ) Drep2 and Drep3 bind by blue native page ( View interaction ) Drep2 and Drep1 bind by blue native page ( View interaction )

Type IIa sodium-dependent phosphate transporter (NaPi-IIa) can be localized in the apical plasma membrane of renal proximal tubule to carry out a rate-limiting step of phosphate reabsorption. For the apical localization, NaPi-IIa is required to form a macromolecular complex with some adaptor proteins such as Na + /H + exchanger regula- tory factor 1 (NHERF-1) and ezrin. However, the detail of macromolecular complex con- taining NaPi-IIa in the apical membrane of the renal proximal tubular cells has not been clarified. In this study, we identified at least four different complexes (220, 480, 920, 1,100 kDa) containing NaPi-IIa by using blue-native polyacrylamide gel electrophoresis. In- terestingly, LC-MS/MS analysis and immunoprecipitation analysis reveal that megalin is a component of larger complexs (920 and 1,100 kDa). In addition, NaPi-IIa can be het- erogeneously co-localized with ezrin and megalin on the apical membrane of renal proxi- mal tubuler cells by fluorescence microscopy analysis. These results suggest that NaPi-IIa can form some different complexes on the apical plasma membrane of renal proximal tu- bular cells. J. Med. Invest. 58 : 140-147, February, 2011

Background Proliferating cell nuclear antigen (PCNA/POL30) an essential protein forms a homotrimeric ring encircling dsDNA and serves as a molecular scaffold to recruit various factors during DNA replication, repair and recombination. According to Candida Genome Database (CGD), orf19.4616 sequence is predicted to encode C. albicans PCNA (CaPCNA) that has not been characterized yet. Results Molecular modeling studies of orf19.4616 using S. cerevisiae PCNA sequence (ScPCNA) as a template, and its subsequent biochemical characterizations suggest that like other eukaryotic PCNAs, orf19.4616 encodes for a conventional homotrimeric sliding clamp. Further we showed by surface plasmon resonance that CaPCNA physically interacted with yeast DNA polymerase eta. Plasmid segregation in genomic knock out yeast strains showed that CaPCNA but not its G178S mutant complemented for cell survival. Unexpectedly, heterologous expression of CaPCNA in S. cerevisiae exhibited slow growth phenotypes, sensitivity to cold and elevated temperatures; and showed enhanced sensitivity to hydroxyurea and various DNA damaging agents in comparison to strain bearing ScPCNA. Interestingly, wild type strains of C. albicans showed remarkable tolerance to DNA damaging agents when compared with similarly treated yeast cells. Conclusions Despite structural and physiochemical similarities; we have demonstrated that there are distinct functional differences between ScPCNA and CaPCNA, and probably the ways both the strains maintain their genomic stability. We propose that the growth of pathogenic C. albicans which is evolved to tolerate DNA damages could be controlled effectively by targeting this unique fungal PCNA. Electronic supplementary material The online version of this article (doi:10.1186/s12866-015-0582-6) contains supplementary material, which is available to authorized users.

Metalloproteomics is a rapidly developing field of science that involves the comprehensive analysis of all metal-containing or metal-binding proteins in a biological sample. The purpose of this review is to offer an overview of the research involving Metal Isotope native RadioAutography in Gel Electrophoresis (MIRAGE), a powerful new method to visualize and study the proteome of a particular metal ion. MIRAGE involves four steps: i) labelling of target proteins with a radioisotope; ii) separation of intact holo-proteins using native isoelectric focusing (1D) combined with Blue Native PAGE (2D); iii) spot visualization and quantification using autoradiography; and iv) protein identification by tandem mass spectrometry. MIRAGE Investigations of the soluble Cu, Zn, and Fe metalloproteomes of Escherichia coli, and of the soluble Mo and W proteomes of the hyperthermophilic archaeon Pyrococcus furiosus are reviewed.

The mechanosensitive channel of large conductance (MscL) is a homooligomeric, stretch activated membrane protein responsible for regulating osmotic pressure in bacteria and archaea. Increasing membrane tension activates the protein resulting in a ∼2.9 nS non-selective pore. Two MscL crystal structures have been solved in distinct conformations and oligomeric states. M. tuberculosis MscL is a non-conducting pentamer while S. aureus MscL is a partially expanded tetramer. The primary sequences of these proteins are 38% identical and 57% similar. Given their high relatedness, the structures raise interesting questions regarding the assembly and activation of MscL homologs. We have been interested in understanding the molecular determinants responsible for the differences between the two structures, specifically the switch between tetrameric and pentameric species. Using a combination of multi-angle light scattering and mass tagging followed by Blue Native PAGE, we have characterized the oligomeric states of several MscL homologs and chimeras. We find that the two methods are in good agreement with each other and single molecule measurements. Potential reasons for the differences in oligomeric states will be discussed.