This is an RSS newsfeed from BioMed Central It is intended to be used with an RSS reader. For more information about RSS newsfeeds from BioMed Central, visit http://www.biomedcentral.com/info/about/rss/ http://www.biomedcentral.com/bmcbiochem/ The latest articles from BMC Biochemistry (ISSN 1471-2091) published by BioMed Central Background: The interconversion of two important energy metabolites, 3-hydroxybutyrate and acetoacetate (the major ketone bodies), is catalyzed by D-3-hydroxybutyrate dehydrogenase (BDH : EC 1.1.1.30), a NAD+-dependent enzyme. The eukaryotic enzyme is bound to the mitochondrial inner membrane and harbors a unique lecithin-dependent activity. Here, we report an advanced purification method of the mammalian BDH applied to the liver enzyme from jerboa (Jaculus orientalis), a hibernating rodent adapted to extreme diet and environmental conditions. Results: Purifying BDH from jerboa liver overcomes its low specific activity in mitochondria for further biochemical characterization of the enzyme. This new procedure is based on the use of polyclonal antibodies raised against BDH from bacterial Pseudomonas aeruginosa. This study improves the procedure for purification of both soluble microbial and mammalian membrane-bound BDH. Even though the Jaculus orientalis genome has not yet been sequenced, for the first time a D-3-hydroxybutyrate dehydrogenase cDNA from jerboa was cloned and sequenced. Conclusions: This study applies immunoaffinity chromatography to purify BDH, the membrane-bound and lipid-dependent enzyme, as a 31 kDa single polypeptide chain. In addition, bacterial BDH isolation was achieved in a two-step purification procedure, improving the knowledge of an enzyme involved in the lipid metabolism of a unique hibernating mammal. Sequence alignment revealed conserved putative amino acids for possible NAD+ interaction. http://www.biomedcentral.com/1471-2091/9/26 Driss Mountassif, Pierre Andreoletti, Zakaria El Kebbaj, Adnane Moutaouakkil, Mustapha Cherkaoui Malki, Norbert Latruffe and M'hammed Said El Kebbaj BMC Biochemistry 2008, 9:26 2008-09-30 doi:10.1186/1471-2091-9-26 BMC Biochemistry 1471-2091 9 26 2008-09-30 Background: Colocalization of Stk33 with vimentin by double immunofluorescence in certain cells indicated that vimentin might be a target for phosphorylation by the novel kinase Stk33. We therefore tested in vitro the ability of Stk33 to phosphorylate recombinant full length vimentin and amino-terminal truncated versions thereof. In order to prove that Stk33 and vimentin are also in vivo associated proteins co-immunoprecipitation experiments were carried out. For testing the enzymatic activity of immunoprecipitated Stk33 we incubated precipitated Stk33 with recombinant vimentin proteins. To investigate whether Stk33 binds directly to vimentin, an in vitro co-sedimentation assay was performed. Results: The results of the kinase assays demonstrate that Stk33 is able to specifically phosphorylate the non--helical amino-terminal domain of vimentin in vitro. Furthermore, co-immunoprecipitation experiments employing cultured cell extracts indicate that Stk33 and vimentin are associated in vivo. Immunoprecipitated Stk33 has enzymatic activity as shown by successful phosphorylation of recombinant vimentin proteins. The results of the co-sedimentation assay suggest that vimentin binds directly to Stk33 and that no additional protein mediates the association. Conclusions: We hypothesize that Stk33 is involved in the in vivo dynamics of the intermediate filament cytoskeleton by phosphorylating vimentin. http://www.biomedcentral.com/1471-2091/9/25 Bastienne Brauksiepe, Alejandro O Mujica, Harald Herrmann and Erwin R Schmidt BMC Biochemistry 2008, 9:25 2008-09-23 doi:10.1186/1471-2091-9-25 BMC Biochemistry 1471-2091 9 25 2008-09-23 Background: The efficient conversion of ammonia, a potent neurotoxin, into non-toxic metabolites was an essential adaptation that allowed animals to move from the aquatic to terrestrial biosphere. The urea cycle converts ammonia into urea in mammals, amphibians, turtles, snails, worms and many aquatic animals and requires N-acetylglutamate (NAG), an essential allosteric activator of carbamylphosphate synthetase I (CPSI) in mammals and amphibians, and carbamylphosphate synthetase III (CPSIII) in fish and invertebrates. NAG-dependent CPSI and CPSIII catalyze the formation of carbamylphosphate in the first and rate limiting step of ureagenesis. NAG is produced enzymatically by N-acetylglutamate synthase (NAGS), which is also found in bacteria and plants as the first enzyme of arginine biosynthesis. Arginine is an allosteric inhibitor of microbial and plant NAGS, and allosteric activator of mammalian NAGS. Results: Information from mutagenesis studies of E. coli and P. aeruginosa NAGS was combined with structural information from the related bacterial N-acetylglutamate kinases to identify four residues in mammalian NAGS that interact with arginine. Substitutions of these four residues were engineered in mouse NAGS and into the vertebrate-like N-acetylglutamate synthase-kinase (NAGS-K) of Xanthomonas campestris, which is inhibited by arginine. All mutations resulted in arginine losing the ability to activate mouse NAGS, and inhibit X. campestris NAGS-K. To examine at what point in evolution inversion of arginine effect on NAGS occured, we cloned NAGS from fish and frogs and examined the arginine response of their corresponding proteins. Fish NAGS were partially inhibited by arginine and frog NAGS were activated by arginine. Conclusions: Difference in arginine effect on bacterial and mammalian NAGS most likely stems from the difference in the type of conformational change triggered by arginine binding to these proteins. The change from arginine inhibition of NAGS to activation was gradual, from complete inhibition of bacterial NAGS, to partial inhibition of fish NAGS, to activation of frog and mammalian NAGS. This change also coincided with the conquest of land by amphibians and mammals. http://www.biomedcentral.com/1471-2091/9/24 Nantaporn Haskins, Maria Panglao, Qiuhao Qu, Himani Majumdar, Juan Cabrera-Luque, Hiroki Morizono, Mendel Tuchman and Ljubica Caldovic BMC Biochemistry 2008, 9:24 2008-09-18 doi:10.1186/1471-2091-9-24 BMC Biochemistry 1471-2091 9 24 2008-09-18 Background: YB-1 is a major regulator of gene expression in eukaryotic cells. In addition to its role in transcription, YB-1 plays a key role in translation and stabilization of mRNAs. Results: We show here that YB-1 interacts with tubulin and microtubules and stimulates microtubule assembly in vitro. High resolution imaging via electron and atomic force microscopy revealed that microtubules assembled in the presence of YB-1 exhibited a normal single wall ultrastructure and indicated that YB-1 most probably coats the outer microtubule wall. Furthermore, we found that YB-1 also promotes the assembly of MAPs-tubulin and subtilisin-treated tubulin. Finally, we demonstrated that tubulin interferes with RNA:YB-1 complexes. Conclusion: These results suggest that YB-1 may regulate microtubule assembly in vivo and that its interaction with tubulin may contribute to the control of mRNA translation. http://www.biomedcentral.com/1471-2091/9/23 Konstantin G Chernov, Alain Mechulam, Nadezhda V Popova, David Pastre, Elena S Nadezhdina, Olga V Skabkina, Nina A Shanina, Victor D Vasiliev, Anne Tarrade, Judith Melki, Vandana Joshi, Sonia Baconnais, Flavio Toma, Lev P Ovchinnikov and Patrick A Curmi BMC Biochemistry 2008, 9:23 2008-09-15 doi:10.1186/1471-2091-9-23 BMC Biochemistry 1471-2091 9 23 2008-09-15 Background: Autosomal dominant optic atrophy (ADOA), a form of progressive bilateral blindness due to loss of retinal ganglion cells and optic nerve deterioration, arises predominantly from mutations in the nuclear gene for the mitochondrial GTPase, OPA1. OPA1 localizes to mitochondrial cristae in the inner membrane where electron transport chain complexes are enriched. While OPA1 has been characterized for its role in mitochondrial cristae structure and organelle fusion, possible effects of OPA1 on mitochondrial function have not been determined. Results: Mitochondria from six ADOA patients bearing OPA1 mutations and ten ADOA patients with unidentified gene mutations were studied for respiratory capacity and electron transport complex function. Results suggest that the nuclear DNA mutations that give rise to ADOA in our patient population do not alter mitochondrial electron transport. Conclusion: We conclude that the pathophysiology of ADOA likely stems from the role of OPA1 in mitochondrial structure or fusion and not from OPA1 support of oxidative phosphorylation. http://www.biomedcentral.com/1471-2091/9/22 Vladimir I Mayorov, Angela J Lowrey, Valerie Biousse, Nancy J Newman, Susan D Cline and Michael D Brown BMC Biochemistry 2008, 9:22 2008-09-10 doi:10.1186/1471-2091-9-22 BMC Biochemistry 1471-2091 9 22 2008-09-10 Background: The human POB1/REPS2 (Partner of RalBP1) protein is highly conserved in mammals where it has been suggested to function as a molecular scaffold recruiting proteins involved in vesicular traffic and linking them to the actin cytoskeleton remodeling machinery. More recently POB1/REPS2 was found highly expressed in androgen-dependent prostate cancer cell lines, while one of its isoforms (isoform 2) is down regulated during prostate cancer progression. Results: In this report we characterize the central proline rich domain of POB1/REPS2 and we describe for the first time its functional role in receptor endocytosis. We show that the ectopic expression of this domain has a dominant negative effect on the endocytosis of activated epidermal growth factor receptor (EGFR) while leaving transferrin receptor endocytosis unaffected. By a combination of different approaches (phage display, bioinformatics predictions, peptide arrays, mutagenic analysis, in vivo co-immunoprecipitation), we have identified two closely spaced binding motifs for 14-3-3 and for the SH3 of the proteins Amphiphysin II and Grb2. Differently from wild type, proline rich domains that are altered in these motifs do not inhibit EGFR endocytosis, suggesting that these binding motifs play a functional role in this process. Conclusion: Our findings are relevant to the characterization of the molecular mechanism underlying the involvement of POB1/REPS2, SH3 and 14-3-3 proteins in receptor endocytosis, suggesting that 14-3-3 could work by bridging the EGF receptor and the scaffold protein POB1/REPS2. http://www.biomedcentral.com/1471-2091/9/21 Laura Tomassi, Anna Costantini, Salvatore Corallino, Elena Santonico, Martina Carducci, Gianni Cesareni and Luisa Castagnoli BMC Biochemistry 2008, 9:21 2008-07-22 doi:10.1186/1471-2091-9-21 BMC Biochemistry 1471-2091 9 21 2008-07-22 Background: Rpt6-1 is a thermosensitive yeast mutant with a deletion of a gene encoding a regulatory subunit of the 26S proteasome, RPT6, which is able to grow at 25°C but not at 37°C. In this study, peptidase activities, activation profiles, and the subunit composition of the 20S proteasome purified from the rpt6-1 mutant was characterized. Results: The 20S proteasome purified from rpt6-1 exhibited low levels of peptidase activities in the absence of activators, but nearly same activated activities in the presence of activators, suggesting a gating defect in the proteasome channel. Detailed analyses of the composition of the 20S proteasome through separation of all subunits by two-dimensional gel electrophoresis followed by identification of each subunit using MALDI-TOF-MS revealed that two subunits, α1 and α7, differed from those of wild-type cells in both electrophoretic mobility and pI values. The changes in these two α-subunits were apparent at the permissive temperature, but disappeared during stress response at the restrictive temperature. Interestingly, upon disappearance of these changes, the levels of peptidase activity of the 20S proteasome in the rpt6-1 mutant were restored as the wild-type. These results suggest that two different forms of the α-subunits, α1 and α7, block the proteasome channel in the rpt6-1 mutant. Conclusion: Two α-subunits (α1 and α7) of the 20S proteasome in the rpt6-1 mutant differed from their wild-type counterparts and peptidase activities were found to be lower in the mutant than in the wild-type strain. http://www.biomedcentral.com/1471-2091/9/20 Aktar Uzzaman Chouduri, Toshinobu Tokumoto, Hideo Dohra, Takashi Ushimaru and Shinpei Yamada BMC Biochemistry 2008, 9:20 2008-07-21 doi:10.1186/1471-2091-9-20 BMC Biochemistry 1471-2091 9 20 2008-07-21 Background: The renin-angiotensin-aldosterone system (RAS) cascade is a major target for the clinical management of hypertension. Although inhibitors of various components of this cascade have been developed successfully, development of renin inhibitors has proven to be problematic. The development of these inhibitors has been hindered by poor bioavailability and complex synthesis. However, despite the challenges of designing renin inhibitors, the enzyme remains a promising target for the development of novel treatments for hypertension. X-ray crystallographic data could greatly assist the design and development of these inhibitors. Here we describe the purification and characterization of recombinant human renin for x-ray crystallization studies. Results: A cDNA encoding the full length of native human preprorenin (406 amino acid residues) was introduced into the HEK-293 cell line. A clonal cell line expressing prorenin was generated and grown under serum free conditions in a hollow fiber bioreactor. Prorenin was constitutively secreted and purified directly from the conditioned medium. Concanavalin A chromatography effectively enriched and purified prorenin to 90% homogeneity in a single step. Prorenin was converted to active renin by trypsin digestion to remove the propeptide. Active renin was further purified using a cation exchange column followed by a gel filtration column. Biochemical characterization of the recombinant enzyme showed both binding and catalytic properties were essentially identical to previously reported activities for purified renin. Crystals were grown using this material in our X-ray structure studies, and high resolution diffraction was obtained. Conclusion: This present work describes a simple and efficient method for the generation and purification of active human renin. The protein is highly pure and is suitable for supporting structural biology efforts. http://www.biomedcentral.com/1471-2091/9/19 Zhongren Wu, Maria G Cappiello, Boyd B Scott, Yuri Bukhtiyarov and Gerard M McGeehan BMC Biochemistry 2008, 9:19 2008-06-26 doi:10.1186/1471-2091-9-19 BMC Biochemistry 1471-2091 9 19 2008-06-26 Background: A novel fluorescent cAMP analog (8-[Pharos-575]- adenosine-3', 5'-cyclic monophosphate) was characterized with respect to its spectral properties, its ability to bind to and activate three main isoenzymes of the cAMP-dependent protein kinase (PKA-Iα, PKA-IIα, PKA-IIβ) in vitro, its stability towards phosphodiesterase and its ability to permeate into cultured eukaryotic cells using resonance energy transfer based indicators, and conventional fluorescence imaging. Results: The Pharos fluorophore is characterized by a Stokes shift of 42 nm with an absorption maximum at 575 nm and the emission peaking at 617 nm. The quantum yield is 30%. Incubation of the compound to RIIα and RIIβ subunits increases the amplitude of excitation and absorption maxima significantly; no major change was observed with RIα. In vitro binding of the compound to RIα subunit and activation of the PKA-Iα holoenzyme was essentially equivalent to cAMP; RII subunits bound the fluorescent analog up to ten times less efficiently, resulting in about two times reduced apparent activation constants of the holoenzymes compared to cAMP. The cellular uptake of the fluorescent analog was investigated by cAMP indicators. It was estimated that about 7 μM of the fluorescent cAMP analog is available to the indicator after one hour of incubation and that about 600 μM of the compound had to be added to intact cells to half-maximally dissociate a PKA type IIα sensor. Conclusion: The novel analog combines good membrane permeability- comparable to 8-Br-cAMP – with superior spectral properties of a modern, red-shifted fluorophore. GFP-tagged regulatory subunits of PKA and the analog co-localized. Furthermore, it is a potent, PDE-resistant activator of PKA-I and -II, suitable for in vitro applications and spatial distribution evaluations in living cells. http://www.biomedcentral.com/1471-2091/9/18 Daniela Moll, Anke Prinz, Cornelia M Brendel, Marco Berrera, Katrin Guske, Manuela Zaccolo, Hans-Gottfried Genieser and Friedrich W Herberg BMC Biochemistry 2008, 9:18 2008-06-25 doi:10.1186/1471-2091-9-18 BMC Biochemistry 1471-2091 9 18 2008-06-25 Background: The pyridoxal-5'-phosphate (PLP)-dependent or vitamin B6-dependent enzymes that catalyze manifold reactions in the metabolism of amino acids belong to no fewer than four evolutionarily independent protein families. The multiple evolutionary origin and the essential mechanistic role of PLP in these enzymes argue for the cofactor having arrived on the evolutionary scene before the emergence of the respective apoenzymes and having played a dominant role in the molecular evolution of the B6 enzyme families. Here we report on an attempt to re-enact the emergence of a PLP-dependent protoenzyme. The starting protein was pancreatic ribonuclease A (RNase), in which active-site Lys41 or Lys7 readily form a covalent adduct with PLP. Results: We screened the PLP adduct of wild-type RNase and two variant RNases (K7R and K41R) for catalytic effects toward L- and D-amino acids. RNase(K41R)-PLP, in which the cofactor is bound through an imine linkage to Lys7, qualifies for a model proto-B6 enzyme by the following criteria: (1) covalent linkage of PLP (internal aldimine); (2) catalytic activity toward amino acids that depends on formation of an imine linkage with the substrate (external aldimine); (3) adjoining binding sites for the cofactor and amino acid moiety that facilitate the transimination reaction of the internal to the external aldimine and stabilize the resulting noncovalent complex of the coenzyme-substrate adduct with the protein; (4) reaction specificity, the only detectable reactions being racemization of diverse amino acids and β-decarboxylation of L-aspartate; (5) acceleration factors for racemization and β-decarboxylation of >103 over and above that of PLP alone; (6) ribonuclease activity that is 103-fold lower than that of wild-type RNase, attenuation of a pre-existing biological activity being indispensable for the further evolution as a PLP-dependent protoenzyme. Conclusion: A single amino acid substitution (Lys41Arg) and covalent binding of PLP to active-site Lys7 suffice to turn pancreatic ribonuclease A into a protein catalyst that complies with all plausible criteria for a proto-B6 enzyme. The study thus retraces in a model system what may be considered the committed step in the molecular evolution of a potential ancestor of a B6 enzyme family. http://www.biomedcentral.com/1471-2091/9/17 Rosa A Vacca, Sergio Giannattasio, Guido Capitani, Ersilia Marra and Philipp Christen BMC Biochemistry 2008, 9:17 2008-06-19 doi:10.1186/1471-2091-9-17 BMC Biochemistry 1471-2091 9 17 2008-06-19