[Nucleotide sequence of cDNA and organization of the gene for alpha-subunit of photoreceptor phosphodiesterase of cyclic GMP in human retinal cones]. / Nukleotidnaia posledovatel'nost' kDNK i organizatsiia gena alpha'-sub''edinitsy fotoretseptornoi fosfodiésterazy tsiklicheskogo GMP kolbochek setchatki glaza cheloveka.

Five clones were isolated from a human retina cDNA library whose cDNA inserts allowed reconstruction of the total sequence of the human clone cGMP phosphodiesterase alpha'-subunit cDNA comprising 3455 bp. The protein's deduced sequence contains 858 amino acids residues with molecular mass 99,169 Da. A substantial homology was revealed between the amino acid sequence of the human cones cGMP-phosphodiesterase alpha'-subunit and the corresponding sequences of alpha, beta, and alpha' subunits of visual cGMP-phosphodiesterase of bovine, murine, chicken and human retinas. Four recombinant bacteriophages were isolated from a genomic library whose inserts made it possible to reconstruct a 32-kb fragment of the human cones cGMP-phosphodiesterase alpha'-subunit gene. 5'-Flanking region of the gene and first 14 exons, encoding an N-terminal segment of the protein, along with the adjacent intron segments were sequenced.

[Total solid phase synthesis of gamma-subunit of cGMP phosphodiesterase from bovine retina and physicochemical properties of synthetic protein]. / Polnyi tverdofaznyi sintez gamma-sub''edinitsy fosfodiesterazy cGMP setchatki glaza byka i nekotorye fiziko-khimicheskie svoistva sinteticheskogo belka.

The 87-membered polypeptide with the sequence of the gamma subunit of cGMP phosphodiesterase from bovine retina rods (PDE gamma) was synthesized by the solid phase method. Two synthetic approaches, which were based on the Boc/Bzl-strategy, were used; both syntheses were carried out in a continuous-flow reactor with swellographic monitoring. In the first approach, five Arg residues were coupled in the form of Boc-Arg(Z)2-OH and the final cleavage of the peptide from the support was effected by the mixture of CF3SO2SiMe3 and thionisole in trifluoroacetic acid. There resulted a heterogeneous, ornitine-rich, and absolutely inactive peptide material which was insoluble in aqueous alkali. In the second approach, Arg(Tos) and the HF low-high cleavage procedure were used, which resulted in a homogeneous polypeptide (according to HPLC and capillary electrophoresis) that manifested correct molecular mass under ion-spray mass spectrometry and the full functional activity characteristic of the native protein. The effect of zinc salts on the PDE gamma fluorescence in solutions and on its solubility was established. This demonstrated a significant PDE gamma affinity with Zn2+ ions and appeared to be connected with the functioning of the protein in the retina cells. For the first time, the dynamics of the peptidylpolymer swelling in different solvents was studied during the synthesis of peptides with very long sequences.

[Oligonucleotide-directed mutagenesis of inhibitory gamma-subunits of cGMP phosphodiesterase from bovine outer rod segments. A new hypothesis on mechanisms for inhibiting catalytic subunits by gamma-subunits and activation of a holoenzyme by transducin]. / Oligonukleotidnapravlennyi mutagenez ingibitornoi gamma-sub''edinitsy fosfodiésterazy cGMP iz naruzhnykh segmentov palochek setchatki byka. Novaia gipoteza o mekhanizmakh ingibirovaniia kataliticheskikh sub''edinitz gamma-sub''edinitsei ii aktivatsii kholofermenta transdutsinom.

Two mutants of the phosphodiesterase (PDE) gamma subunit (PDE gamma) from bovine retinal rods were synthesized by sequential transcription and translation in vitro. PDE gamma mutants R24E and H79L exhibited inhibitory properties similar to those of the wild-type PDE gamma (wtPDE gamma). At the same time, affinity to the rod outer segment (ROS) membranes is lower for R24E and higher for H79L in comparison with wtPDE gamma. The transducin alpha subunit (in a complex with the GTP non-hydrolyzable analogue, GTP gamma S) activates the trypsin-treated PDE (tPDE) inhibited by wtPDE gamma weaker than tPDE inhibited by R24E and stronger than tPDE inhibited by H79L. To explain the properties of these and earlier studied PDE gamma mutants, a new hypothesis on the mechanisms of inhibition of the PDE catalytic subunit dimer (PDE alpha beta) by PDE gamma and mechanism of the PDE holoenzyme (PDE alpha beta gamma 2) activation by the transducin alpha subunit in a complex with GTP (T alpha.GTP) is proposed: 1) two sites on PDE alpha beta for the PDE gamma binding (A- and the B-site) are different in structure. Sites on PDE gamma interacting with A- and the B-sites on PDE alpha beta are also different in structure. The site on PDE gamma interacting with the B-site partially overlaps with the T alpha.GTP binding site; 2) PDE gamma bound to the B-site provides the main contribution to inhibition of the enzyme catalytic activity; 3) T alpha.GTP first interacts with the PDE gamma bound to the A-site in the PDE holoenzyme and removes this PDE gamma in a PDE gamma.(T alpha.GTP) complex. This results in a slight increase of the catalytic activity of the PDE alpha beta gamma complex remaining bound to the ROS membranes; 4) after removal of PDE gamma from the A-site, another T alpha.GTP molecule is enabled to interact with both PDE alpha beta and PDE gamma bound to the B-site on PDE alpha beta. This interaction results in the formation of a ROS membrane-bound fully catalytically active triple complex PDE alpha beta.PDE gamma.(T alpha.GTP).

[Localization of segments in the Gs protein interacting with adenylate cyclase using a Gs/G0-chimera]. / Lokalizatsiia v belke uchastkov vzaimodeistviia s adenilattsiklazoi s pomoshch'iu konstruirovaniia Gs/G0-khimernykh form.

cDNAs coding for three types of alpha-subunits of GTP-binding proteins Gs and G0 (a short form of alpha s with Asp-Ser in positions 71 and 72, a long form of alpha s with the insertion of 16 amino acid residues instead of Asp-Ser (71-72)--both from bovine brain, and alpha 0 from bovine cerebellum as well as some chimeric alpha s/alpha 0 genes were cloned into a modified pGEM-2 plasmid vector under the control of the SP6 promoter. All the genes were in vitro transcribed and translated, and some functional properties of the resulting proteins were determined, such as adenylyl cyclase activation, ADP-ribosylation with pertussis toxin, limited nucleotide-dependent trypsin proteolysis. Parts of the alpha s polypeptide chain necessary for the activation of adenylyl cyclase were mapped. The alpha s domain interacting with adenylyl cyclase is formed by the alpha s polypeptide chain fragments 235-294 and 337-356 (numbering as of the alpha s long form).

[Cyclic GMP phosphodiesterase from bovine retina. Amino acid sequence of beta-subunit and nucleotide sequence of corresponding cDNA]. / Fosfodiésteraza tsiklicheskogo GMP iz setchatki byka. Aninokislotnaia posledovatel'nost' beta-sub''edinitsy i nukleotidnaia posledovatel'nost' sootvetstvuiushchei kDNK.

Primary structure of beta-subunit of the cyclic GMP phosphodiesterase has been determined by the parallel analysis of the protein amino acid sequence and the corresponding cDNA nucleotide sequence. The beta-subunit contains 852 amino acid residues, its molecular mass is 98291 Da. A significant homology is found between beta- and alpha-subunit of the cGMP phosphodiesterase.