ABSTRACT
We found a new protein haponin (an HLDF-alike protein) in promyelocyte HL-60 cells that is immunoreactive to polyclonal antibodies against HLDFbeta. Determination of the partial primary structure of the protein allowed us to reveal an immunogenic peptide of haponin and, on the basis of the amino acid sequence of this peptide, the degenerate primers were synthesized, which enabled us to clone the full-size cDNA of haponin. The stable heterologous expression of this cDNA in E. coli cells (Rosetta strain) was obtained. Preparations of natural and recombinant proteins exhibited antigenic cross-reactivity to polyclonal antibodies against this peptide.
Subject(s)
Mitochondrial Proteins/metabolism , Proteins/metabolism , Amino Acid Sequence , Cloning, Molecular , Escherichia coli/genetics , HL-60 Cells , Humans , Mitochondrial Proteins/analysis , Mitochondrial Proteins/genetics , Molecular Sequence Data , Proteins/analysis , Proteins/genetics , Recombinant Proteins/analysis , Recombinant Proteins/biosynthesisABSTRACT
PP7, a recently identified protein Ser/Thr phosphatase of the PPP family distantly related to phosphatases PP5/PPT and PPEF/rdgC, was purified from cauliflower extracts to apparent homogeneity. Purified cauliflower PP7 and recombinant PP7 expressed in Escherichia coli exhibit light absorption in the visible range with a maximum at approximately 430 nm. Under nonreducing conditions, native PP7 exists as a mixture of monomer with an intramolecular disulfide bridge, disulfide-linked homodimer, and possibly disulfide-linked complexes with potential partner proteins. The activity of recombinant Arabidopsis thaliana PP7 is reversibly regulated by redox agents. The results demonstrate the existence of PP7 protein in planta and suggest a possibility of redox regulation of this protein phosphatase.
Subject(s)
Brassica/enzymology , Phosphoprotein Phosphatases/isolation & purification , Arabidopsis/enzymology , Arabidopsis Proteins , Disulfides , Gene Expression Regulation, Enzymologic , Oxidation-Reduction , Phosphoprotein Phosphatases/metabolism , Plant Proteins/isolation & purification , Plant Proteins/metabolism , Protein Structure, QuaternaryABSTRACT
The C type natriuretic peptide (CNP) is a peptide hormone stimulating vasorelaxation and inhibiting cell proliferation. CNP activates the type B natriuretic peptide receptor (NPR-B), known as the guanylate cyclase B membrane enzyme, which results in the cGMP release. To study functional properties of NPR-B, its gene fragments were expressed in methylotrophic yeasts Pichia pastoris. Conditions were found providing for secretion of functionally active recombinant proteins NPR-Bs and NPR-B1 into the cultural medium in a yield of 25 mg/ml culture. Their specific activity was 0.97 and 0.93 mumol cGMP min-1 mg-1 protein, respectively. It was shown that NPR-B belongs to the family of Ser/Thr protein kinases and can be autophosphorylated at the serine residues.
Subject(s)
Guanylate Cyclase , Mutation , Receptors, Atrial Natriuretic Factor/genetics , Base Sequence , Cyclic GMP/metabolism , DNA Primers , DNA, Complementary , Pichia/genetics , Receptors, Atrial Natriuretic Factor/isolation & purification , Receptors, Atrial Natriuretic Factor/metabolismSubject(s)
Nucleoside-Diphosphate Kinase/genetics , Nucleoside-Diphosphate Kinase/metabolism , Retina/enzymology , Animals , Carbohydrates/analysis , Cattle , Cloning, Molecular , Isoenzymes/chemistry , Isoenzymes/genetics , Isoenzymes/metabolism , Nucleoside-Diphosphate Kinase/chemistry , Protein ConformationABSTRACT
Nucleoside diphosphate kinase (NDP kinase; ATP: NDP phosphotransferase; EC 2.7.4.6) was purified from bovine retina. The molecular mass of the native enzyme was found to be 72 kDa, and those of its subunits were 17.5 and 18.5 kDa. Kinetic characteristics of the enzyme were determined. It was shown that NDP kinase exists in retina in both soluble and membrane-bound forms.
Subject(s)
Nucleoside-Diphosphate Kinase/metabolism , Retina/enzymology , Animals , Catalysis , Cattle , Cell Membrane/enzymology , Kinetics , Nucleoside-Diphosphate Kinase/isolation & purification , Substrate SpecificityABSTRACT
The crystal structures of two isoforms of nucleoside diphosphate kinase from bovine retina overexpressed in Escherischia coli have been determined to 2.4 A resolution. Both the isoforms, NBR-A and NBR-B, are hexameric and the fold of the monomer is in agreement with NDP-kinase structures from other biological sources. Although the polypeptide chains of the two isoforms differ by only two residues, they crystallize in different space groups. NBR-A crystallizes in space group P212121 with an entire hexamer in the asymmetric unit, while NBR-B crystallizes in space group P43212 with a trimer in the asymmetric unit. The highly conserved nucleotide-binding site observed in other nucleoside diphosphate kinase structures is also observed here. Both NBR-A and NBR-B were crystallized in the presence of cGMP. The nucleotide is bound with the base in the anti conformation. The NBR-A active site contained both cGMP and GDP each bound at half occupancy. Presumably, NBR-A had retained GDP (or GTP) from the purification process. The NBR-B active site contained only cGMP.
Subject(s)
Isoenzymes/chemistry , Nucleoside-Diphosphate Kinase/chemistry , Retina/enzymology , Animals , Binding Sites , Cattle , Crystallography, X-Ray , Humans , Isoenzymes/metabolism , Models, Molecular , Nucleoside-Diphosphate Kinase/metabolism , Nucleotides/metabolism , Protein Conformation , SolventsABSTRACT
The biochemical and structural properties of bovine retinal nucleoside diphosphate kinase were investigated. The enzyme showed two polypeptides of approximately 17.5 and 18.5 kDa on SDS-PAGE, while isoelectric focusing revealed seven to eight proteins with a pI range of 7.4-8.2. Sedimentation equilibrium yielded a molecular mass of 96 +/- 2 kDa for the enzyme. Carbohydrate analysis revealed that both polypeptides contained Gal, Man, GlcNAc, Fuc, and GalNac saccharides. Like other nucleoside diphosphate kinases, the retinal enzyme showed substantial differences in the Km values for various di- and triphosphate nucleotides. Immunogold labeling of bovine retina revealed that the enzyme is localized on both the membranes and in the cytoplasm. Screening of a retinal cDNA library yielded full-length clones encoding two distinct isoforms (NBR-A and NBR-B). Both isoforms were overexpressed in Escherichia coli and their biochemical properties compared with retinal NDP-kinase. The structures of NBR-A and NBR-B were determined by X-ray crystallography in the presence of guanine nucleotide(s). Both isoforms are hexameric, and the fold of the monomer is similar to other nucleoside diphosphate kinase structures. The NBR-A active site contained both a cGMP and a GDP molecule each bound at half occupancy while the NBR-B active site contained only cGMP.
Subject(s)
Nucleoside-Diphosphate Kinase/isolation & purification , Nucleoside-Diphosphate Kinase/metabolism , Protein Conformation , Retina/enzymology , Amino Acid Sequence , Animals , Binding Sites , Carbohydrates/analysis , Cattle , Cloning, Molecular , Crystallography, X-Ray , Cyclic GMP/metabolism , Guanosine Diphosphate/metabolism , Molecular Sequence Data , Nucleoside-Diphosphate Kinase/chemistry , Nucleoside-Diphosphate Kinase/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Retina/chemistry , Retina/ultrastructure , Subcellular Fractions/enzymologyABSTRACT
Nucleoside diphosphate (NDP) kinase from bovine retina was found to contain carbohydrates. The subunits of NDP kinase were separated by SDS-PAGE, blotted onto an Immobilon-P membrane, and their carbohydrate content was determined. Both subunits contained equal amounts of Gal, Man, Fuc, Gal-NAc, and Glc-NAc. The total carbohydrate content was 2 to 3% of the protein weight.