Title: PhaR, a protein of unknown function conserved among short-chain-length polyhydroxyalkanoic acids producing bacteria, is a DNA-binding protein and represses Paracoccus denitrificans phaP expression in vitro Maehara A, Doi Y, Nishiyama T, Takagi Y, Ueda S, Nakano H, Yamane T Ref: FEMS Microbiology Letters, 200:9, 2001 : PubMed
A putative regulatory protein, PhaR, which was identified in the polyhydroxyalkanoic acid synthetic locus (phaZCPR) in Paracoccus denitrificans, was investigated. The PhaR protein purified from a recombinant Escherichia coli was estimated to be 22 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, being consistent with the mass calculated from the nucleotide sequence. The molecular mass was determined to be 93 kDa by size-exclusion chromatography, suggesting that the protein formed a tetramer. A gel mobility shift assay showed that PhaR specifically bound to the intergenic region of phaC--phaP. In a cell-free protein synthesis system using E. coli S30 extract, the expression of the phaP gene was repressed by the addition of purified PhaR. These results suggest that PhaR is a DNA-binding protein and may play a role in the regulation of phaP gene expression.
Title: Effect of a hematopoietic-promoting factor derived from porcine kidney on megakaryocyte colony formation by murine bone marrow cells Kashiwakura I, Honda M, Hayase Y, Takagi Y Ref: Research Communications in Molecular Pathology & Pharmacology, 90:25, 1995 : PubMed
We investigated the effect of a hematopoietic-promoting factor (HPF) purified from porcine kidney on murine megakaryocyte colony formation. Murine bone marrow cells were cultured in agar in Iscove's modified Dulbecco's medium supplemented with 10% fetal calf serum in the presence of interleukin-3 (IL-3) as a source of megakaryocyte colony-stimulating factor. HPF alone exerted only a slight effect on megakaryocyte colony formation, and the combination of HPF with IL-3 enhanced colony formation compared with IL-3 alone. At days 11 and 13, 1.9-fold and 1.7-fold enhancements were observed, respectively. When erythropoietin (Epo) was added to the combinations of IL-3 plus HPF or IL-3 plus HPF plus SCF megakaryocyte colonies were enhanced compared with the cultures without Epo. As the result of determination the ploidy distribution, more than 20% of the megakaryocytes in IL-3 plus HPF showed 32N and above ploidy, but the number of 8N and under megakaryocytes was the same as those stimulated with IL-3 alone. These results suggest that HPF stimulates both the proliferation of megakaryocyte colony-forming units and the maturation of megakaryocytes in the presence of IL-3.
Title: An isozyme of microsomal carboxyesterases, carboxyesterase Sec, is secreted from rat liver into the blood Murakami K, Takagi Y, Mihara K, Omura T Ref: J Biochem, 113:61, 1993 : PubMed
It is generally believed that liver carboxyesterases are localized exclusively in the endoplasmic reticulum (ER), mostly in the lumen, loosely bound to the inner side of the membrane. A cDNA clone, clone (8-1/2-1) supposed to code for one of the isozymes, carboxyesterase E1, was isolated by Takagi et al. [J. Biochem. 104, 801-806 (1988)]. However, the protein coded by clone (8-1/2-1) had no consensus ER retention signal at its carboxy terminus, and the mechanism of its retention by ER lumen was unclear. When clone (8-1/2-1) was expressed in COS cells in this study, the plasmid-coded protein was secreted into the medium. When the carboxy terminal portion of the clone (8-1/2-1)-coded protein was replaced with the corresponding region of another carboxyesterase, pI 6.1 esterase, which had the HVEL sequence at the carboxy terminus, the chimeric protein was retained in the COS cells. We searched for a secretory form carboxyesterase in rat blood immunochemically using polyclonal antibodies to carboxyesterase E1, and detected a cross-reacting protein with a molecular weight of 68 kDa. The molecular weight was decreased by endoglycosidase F treatment but not by endoglycosidase H treatment, indicating that the protein carries complex type sugar chains. In addition, the cross-reacting protein was labeled with [3H] diisopropylfluorophosphate (DFP), suggesting that the protein has an esterase-type active center serine.(ABSTRACT TRUNCATED AT 250 WORDS)
A cDNA encoding human liver carboxylesterase and its gene were isolated. Nucleotide sequence analyses of the cDNA revealed that the predicted enzyme protein consists of 567 amino acids, including 18 amino acids of a putative signal peptide. Comparison of the deduced amino acid sequences of this enzyme with those of seven other carboxylesterases in various mammalian species, together with experimental data from several other laboratories, showed that these enzymes can be classified into three groups depending on the sequences at their carboxyl terminals and the presence or absence of one exon. A human carboxylesterase gene was found to span approximately 30 kb and to have 14 small exons. Alignments of this gene with those of human cholinesterase and rat cholesterol esterase indicated insertional sites at some introns and homologous amino acid sequences around them, although these genes have different numbers of exons. Thus the results supported the conclusion that these esterases evolved from a common ancestral gene.
Title: Molecular cloning and nucleotide sequence of cDNA of microsomal carboxyesterase E1 of rat liver Takagi Y, Morohashi K, Kawabata S, Go M, Omura T Ref: J Biochem, 104:801, 1988 : PubMed
cDNA clones of the mRNA for rat liver carboxyesterase E1, one of the carboxyesterases exclusively located on the luminal side of microsomal vesicles, were isolated. Sequence analysis of 2 kbp long cDNA revealed the primary structure of carboxyesterase E1, which consisted of 549 amino acids (Mr 60, 171.71) and contained an extra peptide of 18 amino acids at the NH2-terminus of the mature enzyme. Comparison of the deduced primary structure and sequences of some proteolytic fragments of the purified enzyme indicated the multiplicity of the enzyme. The extra peptide at the NH2-terminal had features in common with the signal peptides of most secretory proteins. However, no polar amino acid residues existed before the hydrophobic core of the signal peptide. A new interpretation is proposed to explain how the signal peptide without the NH2-terminal polar residues works. A tetrapeptide (KDEL) which was shown to keep a few microsomal proteins in the lumen of the endoplasmic reticulum was not found in the primary structure of carboxyesterase E1, which suggested the existence of another mechanism for retention of proteins in the lumen of endoplasmic reticulum. Carboxyesterase E1 showed significant homology with the COOH-terminal portion of thyroglobulin.
Title: Molecular cloning and nucleotide sequence of the lipase gene from Pseudomonas fragi Kugimiya W, Otani Y, Hashimoto Y, Takagi Y Ref: Biochemical & Biophysical Research Communications, 141:185, 1986 : PubMed
The gene coding for the lipase of Pseudomonas fragi was cloned into Escherichia coli JM83 by inserting Sau3A-generated DNA fragments into the BamH I site of pUC9. The plasmid isolated, pKKO, was restriction mapped and the position of the lipase gene on the 2.0 kb insert was pinpointed by subcloning. DNA sequencing revealed that the open reading frame comprises 405 nucleotides and gives a preprotein of 135 amino acids with a predicted Mr of 14643. By comparing the putative lipase amino acid sequence with porcine pancreatic, rat lingual and Staphylococcus hyicus lipases the amino acid sequence around the reactive serine was found to be common among the types of lipase which have been reported.
