Eukaryotic initiation factor 5 from calf liver is a single polypeptide chain protein of Mr = 62,000

Eukaryotic initiation factor 5 (eIF-5), which specifically catalyzes the joining of a 60 S ribosomal subunit to a 40 S initiation complex to form a functional 80 S initiation complex, has been purified from ribosomal salt wash proteins of calf liver. The purified factor exhibits only one polypeptide band of Mr = 62,000 following electrophoresis in 10% polyacrylamide gels in the presence of sodium dodecyl sulfate. The native protein has a sedimentation coefficient of 4.2 S and a Stokes radius of 33 A which is consistent with eIF-5 being a monomeric protein of Mr = 58,000-62,000. Less pure preparations of eIF-5 elute in gel filtration columns with an apparent Mr of 160,000-180,000 presumably due to association of eIF-5 with other high molecular weight proteins since eIF-5 activity present in such preparations can also be shown by gel electrophoretic separation under denaturing conditions to be associated with a 62,000-dalton protein. Furthermore, eIF-5 purified from calf liver extracts with or without a number of protease inhibitors is indistinguishable with regard to molecular weight and final specific activity of purified preparations. The purified factor catalyzes the hydrolysis of GTP present in 40 S initiation complexes in the absence of 60 S ribosomal subunits. The presence of 60 S ribosomal subunits neither stimulates nor inhibits the hydrolysis of GTP. However, the factor cannot mediate 40 S or 40 + 60 S ribosome-dependent hydrolysis of GTP in the absence of Met-tRNAf or other components required for 40 S initiation complex formation. It can be calculated that 1 pmol of eIF-5 protein can catalyze the formation of at least 10 pmol of 80 S initiation complex under the conditions of in vitro initiation reactions.     

Identification of ribosome-bound eukaryotic initiation factor 2.GDP binary complex as an intermediate in polypeptide chain initiation reaction

Studies on the formation and release of the eukaryotic initiation factor (eIF)-2.GDP binary complex formed during eIF-5-mediated assembly of an 80 S initiation complex have been carried out. Incubation of a 40 S initiation complex with eIF-5, in the presence or absence of 60 S ribosomal subunits at 25 degrees C, causes rapid and quantitative hydrolysis of ribosome-bound GTP to form an eIF-2.GDP binary complex and Pi. Analysis of both reaction products by Sephadex G-200 gel filtration reveals that while Pi is released from ribosomes, the eIF-2.GDP complex remains bound to the ribosomal initiation complex. The eIF-2.GDP binary complex can however be released from ribosome by subjecting the eIF-5-catalyzed reaction products to either longer periods of incubation at 37 degrees C or sucrose gradient centrifugation. Furthermore, addition of a high molar excess of isolated eIF-2.GDP binary complex to a 40 S initiation reaction mixture does not cause exchange of ribosome-bound eIF-2.GDP complex formed by eIF-5-catalyzed hydrolysis of GTP. These results indicate that eIF-2.GDP complex is directly formed on the surface of ribosomes following hydrolysis of GTP bound to a 40 S initiation complex, and that ribosome-bound eIF-2 X GDP complex is an intermediate in polypeptide chain initiation reaction.     

Differential membrane proteomics using 18O-labeling to identify biomarkers for cholangiocarcinoma

Quantitative proteomic methodologies allow profiling of hundreds to thousands of proteins in a high-throughput fashion. This approach is increasingly applied to cancer biomarker discovery to identify proteins that are differentially regulated in cancers. Fractionation of protein samples based on enrichment of cellular subproteomes prior to mass spectrometric analysis can provide increased coverage of certain classes of molecules. We used a membrane protein enrichment strategy coupled with 18O labeling based quantitative proteomics to identify proteins that are highly expressed in cholangiocarcinomas. In addition to identifying several proteins previously known to be overexpressed in cholangiocarcinoma, we discovered a number of molecules that were previously not associated with cholangiocarcinoma. Using immunoblotting and immunohistochemical labeling of tissue microarrays, we validated Golgi membrane protein 1, Annexin IV and Epidermal growth factor receptor pathway substrate 8 (EPS8) as candidate biomarkers for cholangiocarcinomas. Golgi membrane protein 1 was observed to be overexpressed in 89% of cholangiocarcinoma cases analyzed by staining tissue microarrays. In light of recent reports showing that Golgi membrane protein 1 is detectable in serum, further investigation into validation of this protein has the potential to provide a biomarker for early detection of cholangiocarcinomas.     

Bone grafts and bone morphogenetic proteins in spine fusion

Spinal fusions are being performed for various pathologies of the spine. Stabilizing vertebral segments by eliminating motion across those segments becomes critical in dealing with pathologies of the spine that lead to instability. The use of autograft has been the gold standard for spine fusion. However, due to complications such as donor site morbidity, increased operating time, and limited supply, the use of allograft as a graft extender has become an acceptable practice especially in fusions spanning multiple segments. The discovery and isolation of novel proteins (i.e., bone morphogenetic proteins, BMPs), which initiate the molecular cascade of bone formation, have experimentally been shown in numerous animal studies to be as effective as autografts. Although the use of BMPs has exciting applications in spine surgery, long-term clinical studies must be evaluated for its efficacy in various applications in humans. The use of biomimetic materials such as hydroxyapatite (HA), or tricalcium phosphate (TCP) has also been examined in several animal models as bone graft substitutes or carriers. Although these materials have shown some promise in specific site applications, more work remains in elucidating an efficacious combination of these materials and BMPs that can be as effective as autografts. This review will present the status of bone grafts, bone morphogenetic proteins, gene therapy, and work that has been done to facilitate spinal fusion and simultaneously eliminate the need for bone graft. This revised version was published online in July 2006 with corrections to the Cover Date.     

sll1981, an acetolactate synthase homologue of Synechocystis sp. PCC6803, functions as l-myo-inositol 1-phosphate synthase

l-myo-inositol 1-phosphate synthase (EC 5.5.1.4; MIPS) catalyzes the first rate limiting conversion of d-glucose 6-phosphate to l-myo-inositol 1-phosphate in the inositol biosynthetic pathway. In an earlier communication we have reported two forms of MIPS in Synechocystis sp. PCC6803 (Chatterjee et al. in Planta 218:989–998, 2004). One of the forms with a ~50 kDa subunit has been found to be coded by an as yet unassigned ORF, sll1722. In the present study we have purified the second isoform of MIPS as a ~65 kDa protein from the crude extract of Synechocystis sp. PCC6803 to apparent homogeneity and biochemically characterized. MALDI-TOF analysis of the 65 kDa protein led to its identification as acetolactate synthase large subunit (EC 2.2.1.6; ALS), the putatively assigned ORF sll1981 of Synechocystis sp. PCC6803. The PCR amplified ~1.6 kb product of sll1981 was found to functionally complement the yeast inositol auxotroph, FY250 and could be expressed as an immunoreactive ~65 kDa MIPS protein in the natural inositol auxotroph, Schizosaccharomyces pombe. In vitro MIPS activity and cross reactivity against MIPS antibody of purified recombinant sll1981 further consolidated its identity as the second probable MIPS gene in Synechocystis sp. PCC6803. Sequence comparison along with available crystal structure analysis of the yeast MIPS reveals conservation of several amino acids in sll1981 essential for substrate and co-factor binding. Comparison with other prokaryotic and eukaryotic MIPS sequences and phylogenetic analysis, however, revealed that like sll1722, sll1981 is quite divergent from others. It is probable that sll1981 may code for a bifunctional enzyme protein having conserved domains for both MIPS and acetolactate synthase (ALS) activities.Anirban Chatterjee and Krishnarup Ghosh Dastidar contributed equally.     

Identification and characterization of membralin, a novel tumor-associated gene, in ovarian carcinoma

Identification of new genes in cancer is the key to understand the molecular basis of tumor development as well as provide potential diagnostic markers and therapeutic targets. A novel gene, membralin (GeneBank accession number: DQ005958), was cloned from a human ovarian cancer cell line. Human membralin is unique and does not share significant sequence homology with other human genes, only membralins of other species. The gene contains 11 exons which encode at least two spliced variants in human cancer. The long form of membralin (membralin-1) comprises all 11 exons, encoding a protein of 620-amino acids long and the short form of membralin (membralin-3) contains all exons except for exon 10, encoding a protein of 408 amino acids. Expression of different membralin isoforms depends on tissue type. The long form, membralin-1, is expressed in ovarian and colorectal carcinomas but not in breast or pancreatic carcinomas, which express only the short splice form, membralin-3. Membralin-1-GFP fusion protein demonstrates exclusive cytoplasmic localization. Based on quantitative real-time PCR, in situ hybridization and Western blot analysis, membralin was highly expressed in ovarian serous carcinomas as compared to ovarian surface epithelium (P<0.001). Ovarian carcinomas in effusions demonstrated a significantly higher level of membralin expression than in solid tumors (P<0.001). In conclusion, these findings represent the first characterization of human membralin and suggest that membralin is a novel tumor-associated marker in ovarian serous carcinomas.