Stratification of chondroitin sulfate binding sites in 3D-model of bovine testicular hyaluronidase and effective size of glycosaminoglycan coat of the modified protein.

A 3D-model of bovine testicular hyaluronidase (BTH) was constructed based on established tertiary structure of human hyaluronidase Hyal1 using a molecular homological modeling method in silico. The analysis of the BTH 3D-model demonstrated lysine residue stratification during enzyme modification. The 3D-model of chondroitin sulfate (CHS)-modified hyaluronidase (BTH-CHS) was obtained by modeling covalent binding of lysine residues with benzoquinone-activated CHS. The degree of enzyme modification and the length of CHS chains were varied during 3D modeling. The importance of deep BTH modification degree for the formation of active and stable enzyme derivatives was shown, as determined earlier experimentally. The effective size of the CHS coat for productive BTH modification was confirmed. It is theoretically achieved at the increase in molecular mass of BTH-CHS derivative to approximately 140-180 kDa and can be practically obtained, according to experimental data, using CHS of different molecular mass (30-50 as well as 120-140 kDa).

Two-component ribonucleotidyl transferase from Escherichia coli. III. Effect of nucleoside diphosphates on synthesis and pyrophosphorolysis of polyribonucleotides by the enzyme.

1. The capacity of two-component ribonucleotidyl transferase to catalyze pyrophosphorolysis of polyribonucleotides is studied. 2. It is shown that nucleoside diphosphates (NDP), not being substrates for the enzyme, activate both the synthesis and pyrophosphorolysis of polynucleotides by the enzyme. The concentration of NDP is important for this effect: with an increase of NDP concentration the rate of synthesis increases and reaches a plateau at 10(-5) M NDP, while the rate of pyrophosphorolysis, attaining maximal values at 10(-5)--10(-3) M NDP, decreases with a further increase of NDP concentration. 3. The possible biological role of two-component ribonucleotidyl transferase is discussed.

Nucleoside 5'-triphosphates modified at sugar residues as substrates for DNA polymerase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius.

The ability of a wide variety of nucleoside 5'-triphosphates with modified sugar moiety to serve as substrates in DNA synthesis catalyzed by DNA polymerase A from the archaebacterium Sulfolobus acidocaldarius was studied. Most of the dNTP analogs tested are shown to be specific terminating substrates for the synthesis irreversibly blocking further elongation of a nascent chain. The most powerful inhibitors were found to be 3'-amino derivatives of deoxy and arabino nucleoside triphosphates, while specific reverse transcriptase inhibitors, 3'-azido and 3'-methoxy derivatives of dNTP, were found to be inactive.

A tw0-component ribonucleotidyl transferase from E. coli.

Some properties of an enzyme designated as a two component ribonucleotidyl transferase from E. coli are presented. The enzyme in the presence of magnesium ions catalyzes the synthesis of polyribonucleotide chains using all four nucleoside triphosphates as substrates. The enzyme consists of two components; component A in the presence of Mg2+ catalyzes the synthesis of homo- and heteropolymers using ATP, CTP and UTP but not GTP as substrates. Component B itself does not catalyze any synthesis at all, but its addition to component A affects this component in two ways: quantitatively- the activity of component A considerably increases, and qualitatively- both components together are capable of catalyzing the synthesis of polyribonucleotides consisting of all four ribonucleotides.

Specific termination of RNA polymerase synthesis as a method of RNA and DNA sequencing.

Termination of RNA synthesis with 3'-O-Methylnucleoside 5'-triphosphates have been studied using E. coli RNA polymerase holoenzyme and poly [d(A-T)] as well as unfractionated T7 D delta III DNA as templates. It was shown that the termination can be used for DNA sequencing. A sequence of a part of RNA synthesized from AI promoter of the DNA have been determined. Syntheses of four 3'-O-Methylnucleoside 5'-triphosphates are described.