PubMed Health⌕ Search

Biomedical subjects

L L Kisselev

Publications and source records attributed to L L Kisselev.

At least 19 recordsLinked to original sources
PubMed1979

An improved rapid enzymatic method of RNA sequencing using chemical modification.

A version of rapid gel sequencing procedure based on the analysis of partial endonuclease hydrolizates of chemically modified 5'-32P-labelled RNA is suggested. Complete and selective modification of cytidilic residues by a methoxyamine-bisulfite mixture leads to the unfolding of the RNA secondary structure and, due to this effect, to the generation of a more uniform set of fragments after partial RNAase hydrolysis. The position of cytidines in an RNA sequence can be determined by restricting the hydrolysis of phosphodiester bonds between the modified CMP residues and their 3'-neighbours with T2 and A RNAases. The method was verified with tRNATrp (yeast) and 5S RNA (rat liver and yeast).

Animals↗
PubMed1979

The effect of tRNA and tryptophanyl adenylate on limited proteolysis of beef pancreas tryptophanyl-tRNA synthetase.

Limited proteolysis of tryptophanyl-tRNA synthetase was used to detect changes in the enzyme molecule in the presence of substrates. Trypsinolysis of each of the two identical subunits occurs in succession from the N-terminus as follows: 60 leads to 51 leads to 40 leads to 24 kilodaltons. The transition 51 leads to 40 is hindered in tryptophanyl adenylate.enzyme complex. Yeast tRNATrp accelerates the first steps of hydrolysis and decelerates the transition 40 leads to 24. Once tRNATrp is added to the synthetase.adenylate complex, the protective effect of the adenylate disappears. The same effects are found also in the presence of tRNATrp oxidized with NaI04 and tRNATrp lacking the 3'-terminal adenosine. Oxidized tRNATrp (but not tRNATrp without the 3'-A) accelerates tryptophan-dependent hydrolysis of ATP catalyzed by the enzyme. A scheme is proposed for the interaction of yeast tRNATrp with beef pancreas tryptophanyl-tRNA synthetase involving the association of tRNA with a positively charged site(s) of the enzyme and the changes in the conformation of enzyme manifesting itself in unfolding of the acidic N-terminal fragment of the polypeptide chain and in the exposure of the adenylate.

Adenosine Monophosphate↗
PubMed1979

Immunochemical studies of beef pancreas tryptophanyl-tRNA synthetase and its fragments. Determination of the number of antigenic determinants and a comparison with tryptophanyl- tRNA synthetases from other sources and with reverse transcriptase from avian myeloblastosis virus.

The immunoglobulin G (IgG) fraction of the antiserum from rabbits immunized with homogeneous beef pancreas tryptophanyl-tRNA synthetase inhibits the enzyme activity in the reactions of both tRNATrp aminoacylation and tryptophan activation. Fab fragments of IgG act in a similar way. Common antigenic determinants have been detected in tryptophanyl-tRNA synthetases from beef, pig, chicken and rat livers using pure antibodies against beef pancreas tryptophanyl-tRNA synthetase. This observation indicates the evolutional stability of certain structural features of tryptophanyl-tRNA synthetases. The interaction of antibodies with the fragments of beef tryptophanyl-tRNA synthetase produced by endogenous and tryptic proteolysis of the enzyme has been studied. On third of the antiserum antibodies interacting with the C-terminal fragment of the enzyme (Mr approximately equal to 40000) inhibits its activity whereas the antibodies to the N-terminal fragment (Mr approximately equal to 20000) have no effect on the enzyme activity. The immunochemical identity of the two synthetase fragments differing in their enzymatic activity supports the assumption that the loss of enzymatic activity of the tryptic fragment is caused by lack of a small peptide which is retained in case of endogenous proteolysis; probably the amino acid residues of this peptide participate in formation of active centre of tryptophanyl-tRNA synthetase. A radioimmunochemical method is described for determining the number of antigenic determinants. One molecule of tryptophanyl-tRNA synthetase was found to bind 9 (+/- 1) molecules of Fab fragments. Antibodies against tryptophanyl-tRNA snythetase from beef pancreas do not inhibit noticeably the activity of reverse transcriptase from avian myeloblastosis virus. No antigenic determinants in common have been detected in reverse transcriptase and tryptophanyl-tRNA synthetase by radioimmunochemical assays.

Amino Acyl-tRNA Synthetases↗
PubMed1978

A method for purification of peptides from hydrolysates of proteins modified by chemically active analogues of substrates containing cis-diol groups.

A simple and rapid column procedure is described for the isolation from protein hydrolysates of peptides containing covalently bound substrate analogues with cis-diol groups. The method is based on complex formation between the cis-diol groups of peptide-bound compounds and dihydroxyborylic groups of a dihydroxyborylaminoethyl cellulose column. The method is useful for isolation of peptide(s) located in or near the active centre of enzymes after their affinity labelling by chemically active analogues of natural substrates like ribonucleotides, sugars, etc.

Adenosine Triphosphate↗
PubMed1977

DNA-polymerase inhibitors. Rifamycin derivatives.

Ten new derivatives of the antibiotic rifamycin with variable side chains at position 3 were synthesized. The inhibitory activity of these derivatives against DNA-polymerases isolated from avian myeloblastosis virus, E. coli and calf thymus were studied at various conditions. 3-(2,4,6-trinitrophenylhydrazone-(methyl) rifamycin SV is a strong inhibitor for all the polymerases tested and belongs to the C class inhibitors of reverse transcriptase. 3-(monoallylhydrazone-(methyl) rifamycin SV possesses a selective action on polymerases: at 0.1 mg/ml concentration it almost completely inhibits the reverse transcriptase and less than half of the bacterial and eukaryotic enzymes. A drug is found which strongly inhibits the DNA-polymerases from E. coli and calf thymus and weakly the viral enzyme. The inhibitory effect on reverse transcriptase is independent of the choice of template-primer; it could be overcome by the addition of excess enzyme but not of excess template-primer; the inhibition could be completely reversed by dilution of the drug-enzyme mixture. From Lineweaver-Burk analysis, the inhibition is noncompetitive with respect to the template-primer and, thus the drugs bind to the site different from the active site for the template-primer. From protective action of the template-primer and other data it might be suggested that the rifamycin derivatives act at an early step(s) in DNA synthesis catalyzed by reverse transcriptase. The obtained data are in agreement with the results for other derivatives of rifamycin SV described in literature.

Animals↗
PubMed1977

Reverse transcription of phage RNA and its fragment directed by synthetic heteropolymeric primers.

DNA synthesis catalysed by RNA-directed DNA-polymerase (reverse transcriptase) was found to proceed on the RNA template of an MS2 phage in the presence of heteropolymeric synthetic octa- and nonadeoxyribonucleotide primers complementary to the intercistronic region (coat protein binding site) and the region of the coat protein cistron, respectively. The product of synthesis consists of discrete DNA fractions of different length, including transcripts longer than 1,000 nucleotides. The coat protein inhibits DNA synthesis if it is initiated at its binding site, but has no effect on DNA synthesis initiated at the coat protein cistron. It has been suggested that, in this system, the initiation of DNA synthesis by synthetic primers is topographically specific. The MS2 coat protein binding site (an RNA fragment of 59 nucleotides) serves as a template for polydeoxyribonucleotide synthesis in the presence of octanucleotide primer and reverse transcriptase. The product of synthesis is homogenous and its length corresponds to the length of the template. The effective and complete copying of the fragment having a distinct secondary structure proves that the secondary structure does not interfere, in principle, with RNA being a template in the system of reverse transcription.

Avian Myeloblastosis Virus↗
PubMed1976

Interaction of aminoacyl-tRNA synthetases and tRNA: positive and negative cooperativity of their active centres.

The influence of tRNA on the kinetics of PP-ATP exchange and aminoacyl-tRNA formation catalysed by leucyl-, phenylalanyl-, and tryptophanyl-tRNA synthetases has been investigated. These enzymes were chosen because they belong to three main classes of quaternary structure alpha1, alpha2beta2 and alpha2, respectively. The present paper shows that the investigated synthetases manifest kinetic cooperativity of the active centres which is negative in the case of AAA formation and positive in the case of leucyl- and tryptophanyl-tRNA synthesis. The obtained data were interpreted with the aid of the trigger model of the enzyme.

Adenosine Triphosphate↗