[Total solid phase synthesis of gamma-subunit of cGMP phosphodiesterase from bovine retina and physicochemical properties of synthetic protein].
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.
Rodionova
L N
LN
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Pushchino Branch, Russian Academy of Sciences, Moscow, Russia.
Zagranichnyĭ
V E
VE
Rodionov
I L
IL
Lipkin
V M
VM
Ivanov
V T
VT
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000138:chemical synthesis* / Q000737:chemistry / Q000302:isolation & purification; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6; D019075:Electrophoresis, Capillary; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis* / Q000737:chemistry / Q000302:isolation & purification; D012160:Retina / Q000201:enzymology*; D016335:Zinc Fingers
D010455:Peptides; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6
Polnyĭ tverdofaznyĭ sintez gamma-sub''edinitsy fosfodiesterazy cGMP setchatki glaza byka i nekotorye fiziko-khimicheskie svoĭstva sinteticheskogo belka.