ABSTRACT
Chicken intestine alkaline phosphatase was immobilized on seven carriers by adsorption or covalent binding. The efficiency of immobilization depended on the carrier, immobilization method, and initial enzyme concentration. The heat stability of the soluble and immobilized enzyme preparations, and the dependence of the enzyme activity on pH and Zn2+ and Mg2+ ions were studied. Alkaline phosphatase immobilized on magnetic carriers displayed lower heat stability than the soluble enzyme. The temperature optima of the immobilized and soluble enzymes were 80 and 60 degrees C; the pH optima were 9.0 and 8.0, respectively. Ions Mg2+ and Zn2+ were found to play an important role in the activation of both soluble and immobilized enzymes and their stabilization at high temperatures.
Subject(s)
Alkaline Phosphatase/isolation & purification , Enzymes, Immobilized/isolation & purification , Intestines/enzymology , Magnetics , Animals , Catalysis , Chickens , Enzyme Stability , Hot Temperature , Hydrogen-Ion Concentration , Magnesium , Solubility , ZincABSTRACT
To elucidate the role of the ribose moiety in substrate binding, various ATP derivatives modified in ribose moiety were studied as probes for the T4 RNA ligase first stage reaction. The kinetic parameters for competitive inhibition were determined. Inhibition experiments using substrate analogs demonstrated that the major binding determinants of ATP analogs were purine and triphosphate moieties of ATP; modification of the ribose moiety was not critical. Adenosine triphosphates attached to agarose were used as affinity adsorbent for purification of T4 RNA ligase. These derivatives had been successfully used in reversible binding of the enzyme. Best results were achieved with agarose coupled via N6 of the purine moiety of ATP.
Subject(s)
Adenosine Triphosphate/metabolism , Bacteriophage T4/enzymology , RNA Ligase (ATP)/metabolism , Ribose/metabolism , Adenosine Triphosphate/analogs & derivatives , Ribose/chemistry , Substrate SpecificityABSTRACT
The NTP binding site of bacteriophage T4 RNA-ligase (EC 6.5.1.3) was studied using several ATP analogs modified in the purine moiety of the nucleotide at positions 1, 2, 6 and 8: adenosine-N'-oxide 5'-triphosphate (I), 6-chloropurine riboside 5'-triphosphate (II), 6-mercaptopurine riboside 5'-triphosphate (III), 1,N6-ethenoadenosine 5'-triphosphate (IV), 8-sulphoadenosine 5'-triphosphate (V), 8-bromoadenosine 5'-triphosphate (VI), inosine 5'-triphosphate (VII) and guanosine 5'-triphosphate (VIII). For all of the ATP analogs under study a reversible inhibition was demonstrated. These analogs appeared to be competitive inhibitors of the T4 RNA-ligase adenylation reaction and only V, VI and VII were efficient as substrates. The kinetic parameters for the competitive inhibition, (I50, Ki) were determined. A putative structure for the T4 RNA-ligase active site was proposed.
Subject(s)
Purine Nucleotides/metabolism , RNA Ligase (ATP)/metabolism , T-Phages/enzymology , Adenosine Triphosphate/analogs & derivatives , Kinetics , Substrate SpecificityABSTRACT
The inhibiting effect of adenosine, AMP, ADP, ATP, gamma-thio ATP (I), beta,gamma-imine ATP (II), beta,gamma-methylene ATP (III), P1,P3-di(adenosine-5') triphosphate (IV), P1,P4-di(adenosine-5') tetraphosphate (V) and adenosine 5'-tetraphosphate (VI) on the first step of the T4 RNA ligase reaction was studied. All the compounds tested, with the exception of adenosine, appeared to be competitive inhibitors of the first step of the enzymatic reaction. The inhibition constants (Ki) for the ATP analogs were determined. The data obtained suggest that the efficiency of inhibition depends on the number of phosphate groups and on the structure of ATP analogs. All the compounds under study (I-VI), except for AMP and ADP, form covalent AMP-RNA ligase complexes.
Subject(s)
Adenine Nucleotides/metabolism , Adenosine Monophosphate/metabolism , Organophosphorus Compounds/metabolism , RNA Ligase (ATP)/metabolism , Catalysis , Electrophoresis, Polyacrylamide Gel , Kinetics , RNA Ligase (ATP)/antagonists & inhibitors , Substrate SpecificityABSTRACT
The data on the photodamage of biomolecules and experimental tumours by a hematoporphyrin derivative activated with laser radiation are presented. Inhibition of the tumour growth and prolongation of the life-span of treated tumour-bearing animals are observed.
Subject(s)
Hematoporphyrin Photoradiation , Neoplasms, Experimental/drug therapy , Nucleotides/analysis , Photochemotherapy , Animals , Female , Male , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Rats , Rats, Inbred StrainsABSTRACT
The conformation of human growth hormone (hGH) as monomer and aggregate as well as of those immobilized in the soluble polysaccharide matrix was investigated. The immobilization resulted in ten per cent decrease of the amount of alpha-helix and in the conformational mobility change of aromatic amino acid side groups. It did not, however, influence the biological activity of the hormone, thus suggesting that the above conformational variations did not affect functionally important portions of the molecule.
Subject(s)
Growth Hormone , Circular Dichroism , Humans , Protein ConformationABSTRACT
The biological mechanisms of photodynamic therapy, a new approach to the treatment of malignant lesions, are considered. The data on chemical composition of hematoporphyrin derivative are given, the photophysical and photochemical reactions caused by photosensitizer distribution in organism and cell, as well mechanisms of photodestruction of cells and cell organelles.
Subject(s)
Photochemotherapy , Animals , Hematoporphyrin Derivative , Hematoporphyrins/pharmacokinetics , Hematoporphyrins/therapeutic use , Humans , Neoplasms/drug therapy , Neoplasms/metabolism , Neoplasms, Experimental/drug therapy , Neoplasms, Experimental/metabolism , Photochemistry , Physical Phenomena , Physics , Radiation-Sensitizing Agents/pharmacokinetics , Radiation-Sensitizing Agents/therapeutic useABSTRACT
Behavior of the covalent [32P]- and [14C]AMP-RNA ligase complex under various conditions has been studied. The covalent structure is shown to be readily cleaved by acid and hydroxylamine and relatively stable to alkali and snake venom phosphodiesterase. Products of degradation of the AMP-RNA ligase and AMP-DNA ligase complexes were compared. The data obtained support the earlier assumption of a phosphoamide bond in the AMP-RNA ligase compound.
Subject(s)
Adenosine Monophosphate/metabolism , Polynucleotide Ligases/metabolism , RNA Ligase (ATP)/metabolism , Chemical Phenomena , Chemistry , Chromatography, Paper , Electrophoresis, Polyacrylamide Gel , Hydrolysis , Hydroxylamines/pharmacology , In Vitro Techniques , Phosphoric Diester Hydrolases/pharmacology , Snake Venoms/pharmacologyABSTRACT
A new method for isolation of polynucleotide phosphorylase from E. coli, including ion-exchange chromatography and gel-filtration has been developed. The method results in 300-fold purification of the enzyme, which being devoid of nuclease and phosphatase activities can further be utilized for oligonucleotide synthesis. It was shown that upon storage the enzyme loses the primer-independent activity and in the absence of NaCl can be used for further syntheses. An addition of NaCl stimulates the elongation of the oligonucleotide chain. Some advantages of polynucleotide phosphorylase from E. coli in comparison with the M. luteus enzyme are discussed.
Subject(s)
Escherichia coli/enzymology , Polyribonucleotide Nucleotidyltransferase/isolation & purification , Drug Stability , Enzyme Activation , Kinetics , Oligoribonucleotides/biosynthesis , Polyribonucleotide Nucleotidyltransferase/metabolism , Sodium Chloride/pharmacology , Structure-Activity RelationshipABSTRACT
The first and the third steps of the RNA-ligase reaction were studied. It was shown that the first step of the reaction consists in a formation of an enzyme-adenylate complex. The optimal conditions for this formation were established. Effects of acids, alkali, hydroxylamine and snake venom phosphodiesterase on the complex suggest that the linkage between the protein and adenylic acid may be of a phosphoester or phosphoamide type. Using synthetic adenylic acid pyrophosphates and mononucleotides (oligonucleotides) the RNA-ligase reaction was shown to involve intermediate pyrophosphates. It was found that the simplest pyrophosphates capable to bind to oligonucleotides in the absence of ATP are adenylic acid pyrophosphates, both of ribo- and deoxyribomononucleotides. The RNA-ligase reaction may be used for elongation of oligonucleotides by one definite mononucleotide or for incorporation of the label into the 3'-end of the polynucleotide chain.
