V. M. Lipkin

Cyclic GMP phosphodiesterase from bovine retina Amino acid sequence of the α-subunit and nucleotide sequence of the corresponding cDNA

The primary structure of the γ‐subunit of cyclic GMP phosphodiesterase was determined by parallel analysis of the amino acid sequence of the protein and nucleotide sequence of the corresponding cDNA. The enzyme γ‐subunit contains 87 amino acid residues, its N‐terminal amino group being acetylated.

Primary structure of α-subunit of DNA-dependent RNA polymerase from Escherichia coli

Transcription of genetic information in bacterial cells is mediated by DNA-dependent RNA polymerase (ribonucleoside-triphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) [ 11. The enzyme from E. coli (mol. wt 500 000) has been shown to possess a complex structure [2] consisting of two large subunits, /? and 0’ (mol. wt 155 000 and 16.5 000, respectively), two a-subunits (mol. wt 40 000) and initiation factor, u (mol. wt 90 000). Its structural complexity is paralleled by the multistep nature of the transcription process. Only limited information on the role of the individual subunits in the functioning of RNA polymerase is available, owing in part to our lack of knowledge of its primary and spatial structure. We have, therefore, undertaken an investigation into the primary structure of DNA-dependent RNA polymerase from E. coli B. In the study reported here we have determined the complete amino acid sequence of the RNA poly merase a-subunit (see [3] ) using a variety of degradation methods and i4C-enriched amino acid residues for facilitating detection and isolation of the fragments. The polypeptide chain of the o-subunit has mol. wt 36 512 and consists of 329 amino acid residues. In a comparative study of the peptide compositions of various bacterial polymerase a-subunits it has been shown by peptide mapping that they are evolutionarily conservative proteins [4]. 2. Materials and methods

N-Myristoylation of recoverin enhances its efficiency as an inhibitor of rhodopsin kinase

Recoverin, a recently identified member of the EF‐hand superfamily of Ca2+‐binding proteins, is capable to inhibit rhodopsin phosphorylation by rhodopsin kinase at high but not at low free [Ca2+]. The N‐terminal glycine residue of retinal recoverin is heterogeneously acylated with myristoyl or related N‐acyl group. To clarify the role of the N‐terminal acylation of recoverin in its inhibitory action upon rhodopsin phosphorylation, we compared the efficiency of myristoylated and non‐myristoylated forms of recombinant recoverin as inhibitors of rhodopsin kinase activity. We have found that rhodopsin phosphorylation by purified rhodopsin kinase, which does not depend on free [Ca2+] in the absence of recoverin, is regulated by Ca2+ in the presence of both forms of the recombinant protein. EC50 values for Ca2+ are the same (2 μM) for the myristoylated and non‐myristoylated forms; the Hill coefficients of 1.7 and 0.9, respectively, indicate that the effect is cooperative with respect to Ca2+ only for myristoylated recoverin. In the presence of Ca2+, both forms of recoverin taken at saturated concentrations cause an almost equal inhibition of rhodopsin phosphorylation. However, the inhibitory action of the myristoylated form occurs at much lower its concentrations than that of the non‐myristoylated form (EC50 are 0.9 and 6.5 μM, respectively).

Complete amino acid sequence of γ‐subunit of the GTP‐binding protein from cattle retina

The complete amino acid sequence of the γ‐subunit of the GTP‐binding protein from cattle retina has been established. The polypeptide chain of the γ‐subunit consists of 69 amino acid residues and contains the unusual sequence Cys35‐Cys36. The M rof the γ‐subunit is 8008.7.

Active sites of the cyclic GMP phosphodiesterase γ‐subunit of retinal rod outer segments

Monoclonal antibodies were prepared to the γ‐subunit of the cGMP phosphodiesterase. One of them γp‐1, suppresses the activation of phosphodiesterase through the α‐subunit of transducin. The γ‐subunit fragment 24–45 rich in Arg and Lys residues is involved in γp‐1 binding and is essential for the γ‐subunit interaction with transducin. Carboxypeptidase Y cleaves off seven amino acid residues from the C‐terminus of the γ‐subunit resulting in phosphodiesterase activation. Thus, the C‐terminal fragment of γ‐subunit participates in phosphodiesterase inhibition.

Identification of a 28 kDa secretory protein from rat olfactory epithelium as a thiol-specific antioxidant

The 28 kDa secretory protein is one of the abundant water-soluble proteins in olfactory epithelium of mammals. Analysis of partial amino acid sequence of the 28 kDa protein strongly suggested that it belongs to a new family of highly conserved antioxidant proteins requiring thiol for their antioxidant activity (TSA/AhpC family). In the present study, we found the 28 kDa protein to have thiol-dependent antioxidant activity, thereby protecting radical-sensitive proteins such as glutamine synthetase and hemoglobin from oxidative modification caused by thiol-dependent metal ion-catalyzed oxidation system. The purified 28 kDa protein did not possess catalase or glutathione peroxidase activities, and required thiols to exhibit its antioxidant activity. The 28 kDa protein is the first member of the family of thiol-specific antioxidants identified in olfactory epithelium and the first secretory protein shown to be thiol-specific antioxidant.

Localization of 28-kDa peroxiredoxin in rat epithelial tissues and its antioxidant properties

Abstract. Peroxiredoxins are a novel family of antioxidant proteins that specifically prevent enzymes from metal-catalyzed oxidation. The localization of a member of the mono-cystein subfamily of peroxiredoxins, the 28-kDa protein, in different rat tissues and its antioxidant properties were investigated. By immunoblotting, the 28-kDa peroxiredoxin was found to be most highly concentrated in olfactory epithelium and present in all tissues tested (skin, lung, trachea, kidney, womb, and brain). Immunostaining with rabbit polyclonal antibody raised against the 28-kDa peroxiredoxin revealed the particularly high level of the 28-kDa peroxiredoxin immunoreactivity in air-contacting areas (apical regions and mucus of the olfactory and respiratory epithelium and skin epidermis), which are continually exposed to numerous air-borne reactive oxygen species. In the apical regions of the olfactory and respiratory epithelium, the 28-kDa-peroxiredoxin immunogold labeling outlined microvilli and cilia and was mainly located in sustentacular cells and in respiratory and goblet cells, as electron-microscopic analysis revealed. In skin epidermis, the 28-kDa peroxiredoxin immunoreactivity was confined to the granular layer and specifically concentrated in sebaceous glands of hair follicle. In situ hybridization with 33P-labeled antisense RNA probe revealed the expression of the 28-kDa peroxiredoxin mRNA in tissues with a high level of the 28-kDa peroxiredoxin immunoreactivity. Immunodepletion of the 28-kDa peroxiredoxin profoundly decreased the antioxidant activity of the olfactory tissue extract.

Primary structure of Escherichia coli RNA polymerase nucleotide substitution in the beta subunit gene of the rifampicin resistant rpoB255 mutant.

SummaryThe transducing phage λ dsupM814 and the plasmid pIB1830 containing the wild-type rpoB gene have been constructed and the primary structure of the genes central fragment has been established. In contrast with the wild-type, the gene of the rpoB255 mutant, whose primary structure has been published, was found to contain an A.T.→T.A. transversion entailing the substitution of a valine residue for the aspartic acid residue (516) of the wild-type β subunit.

A novel 45 kDa secretory protein from rat olfactory epithelium: primary structure and localisation

cDNA clones encoding the 45 kDa protein were isolated from a rat olfactory epithelium cDNA library and their inserts were sequenced. The reconstructed protein sequence comprises 400 amino acids with a calculated molecular mass of 46 026 Da. A homology was revealed between the amino acid sequence of the 45 kDa protein and the proteins involved in the transfer of hydrophobic ligands. Using in situ hybridisation, the 45 kDa protein mRNA expression was detected in the layer of supportive cells of olfactory epithelium, apical region of trachea, surface layer of the ciliated bronchial epithelium in lung and in skin epidermis.

Novel 28-kDa secretory protein from rat olfactory epithelium

We have isolated a novel secretory 28‐kDa protein which is an abundant component of the rat olfactory mucosa. The partial sequence of the 28‐kDa protein has been determined. The amino acid sequence of the 28‐kDa protein is similar to that of non‐selenium glutathione peroxidase from bovine ciliary body. The 28‐kDa protein catalyzed decomposition of the hydrogen peroxide as well as organic hydroperoxides by reduced glutathion and seems to be a member of the glutathion peroxidases family.