Subject(s)
Bacillus/enzymology , Endoribonucleases/isolation & purification , Amino Acid Sequence , Bacterial Proteins/pharmacology , Chromatography, Ion Exchange , Endoribonucleases/antagonists & inhibitors , Endoribonucleases/metabolism , Enzyme Inhibitors/pharmacology , Kinetics , Mass Spectrometry , Molecular Sequence Data , Ribonucleases/antagonists & inhibitors , Ribonucleases/isolation & purification , Ribonucleases/metabolismABSTRACT
A comparative research of individual peptide structures obtained after hydrolysing of Bacillus circulans and B. amyloliquefaciens RNases by the Glu-specific staphylococcal protease was carried out by means of mass-spectrometry and Edman degradation methods. A complete amino acid sequence of B. circulans RNase was determined. Gln15, Gly65 and Gln104 residues in B. amyloliquefaciens RNase were found to be substituted by Leu, Ala and Lys residues in B. circulans RNase, respectively. Catalytic properties of the B. circulans RNase in transesterification reactions with poly- and oligonucleotides as substrates were investigated.
Subject(s)
Bacillus/enzymology , Ribonucleases/chemistry , Amino Acid Sequence , Catalysis , Chromatography, High Pressure Liquid , Hydrolysis , Kinetics , Mass Spectrometry , Molecular Sequence Data , Peptide Mapping , Ribonucleases/isolation & purification , Ribonucleases/metabolism , Serine EndopeptidasesABSTRACT
Intraspecific selection of Bacillus thuringiensis strains producing extracellular alkaline ribonucleases was carried out. Subtoxicus subspecies with increased expression of the enzyme was detected. A method was developed to isolate preparative amounts of homogeneous extracellular RNase of B. thuringiensis var. subtoxicus. The physico-chemical and catalytic properties of the enzyme was studied and compared with extracellular RNases of others Bacillus species. The conclusion about the structural and evolutional conservation of Bacillus extracellular RNases was drawn.
Subject(s)
Bacillus thuringiensis/enzymology , Ribonucleases/metabolism , Amino Acid Sequence , Amino Acids/analysis , Calorimetry , Catalysis , Chromatography, High Pressure Liquid , Electrophoresis, Polyacrylamide Gel , Mass Spectrometry , Molecular Sequence Data , Ribonucleases/chemistry , Ribonucleases/isolation & purificationABSTRACT
The extracellular ribonuclease (RNAse Bp) was isolated from the cultural medium filtrate of Bacillus pumilus by ammonium sulfate precipitation and two stages of ion-exchange chromatography on carboxymethyl- and phospho-cellulose columns. The amino acid composition and N-terminal amino acid residue have been determined. The kinetic parameters of cleavage reaction of synthetic polynucleotides have been measured. According to their structural homology RNAse Bp has been shown to be similar to RNAses Ba and Bi. Catalytic properties of the enzyme are very close to RNAse Bi.
Subject(s)
Bacillus/enzymology , Exoribonucleases/isolation & purification , Amino Acids/analysis , Catalysis , Enzyme Stability , Exoribonucleases/analysis , Exoribonucleases/metabolismABSTRACT
Phosphodiesterase stability of synthetic analogs of 2',5'-oligoadenylates, the mediators of antiviral and antiproliferative action of interferons was analysed. The analogs with a 3'-terminal acyclic nucleoside residue were prepared. These analogs were treated with NIH3T3 cell lysate, mice liver homogenate and snake venom phosphodiesterase. All analogs have demonstrated a high stability as compared with the natural 2',5'-oligoadenylate and its 3'-deoxyderivative. The possible biological activity of these stable analogs of 2',5'-oligoadenylates is discussed.
Subject(s)
Adenine Nucleotides/chemical synthesis , Oligoribonucleotides/chemical synthesis , Phosphoric Diester Hydrolases/metabolism , Adenine Nucleotides/metabolism , Adenine Nucleotides/pharmacology , Animals , Cells, Cultured , Chemical Phenomena , Chemistry , Drug Stability , Kinetics , Liver/enzymology , Mice , Oligoribonucleotides/metabolism , Oligoribonucleotides/pharmacologyABSTRACT
To get insight into the origin of pyrimidine specificity of ribonuclease A, a study of the enzyme interaction with the substrate analogs having a modified nucleobase was undertaken. Pyridine and pyrimidine cyclophosphates were obtained by phosphorylation of 2'(3')-O-dibutyl stannylidene derivatives of nonprotected nucleosides in high yields. The results of kinetic and NMR studies suggested that a substrate should be locked in anti-conformation in the productive enzyme-substrate complex as it was shown for the crystalline complexes of the enzyme with pyrimidine nucleotides by X-ray analysis. The interaction between carbonyl group in position 2 of substrate nucleobase and proton accepting group of the protein (NH of Thr45) was found to be a prerequisite for the specific recognition of a substrate by the enzyme. The rate constants for transformation of lactam form (slow) and lactim form (fast) of pyrimidine substrates were estimated.