PURIFICATION AND CHARACTERIZATION OF A PROTEINASE FROM THE PROBIOTIC Lactobacillus rhamnosus OXY

A proteinase produced by the human gastrointestinal isolate Lactobacillus rhamnosus strain OXY was identified and characterized. The prtR2 gene coding for proteinase activity was detected in the examined strain. The PCR primers used were constructed on the basis of the sequence of the prtR2 proteinase gene from Lactobacillus rhamnosus GG. The enzyme was purified by fast protein liquid chromatography (FPLC) using CM-Sepharose Fast Flow and Sephacryl S-300 columns. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the enzyme had a relatively low molecular mass of 60 kD. Protease activity was observed at a pH range from 6.5 to 7.5 with optimum k _cat/K _m values at pH 7.0 and 40°C. Maximum proteolytic activity (59 U mL^?1) was achieved after 48 hr of cultivation. The activity of the enzyme was inhibited only by irreversible inhibitors specific for serine proteinases (PMSF and 3,4-dichloro-isocumarine), suggesting that the enzyme was a serine proteinase. Proteinase activity was increased by Ca²⁺ and Mg²⁺, and inhibited by Cu²⁺, Zn²⁺, Cd²⁺, and Fe^2+.     

Inhibition of jack bean urease by p-benzoquinone: elucidation of the role of thiols and reversibility of the process

p-Benzoquinone (pBQ) was studied as an inhibitor of jack bean urease in 20 mM phosphate buffer, pH 7.0, 1 mM EDTA, 25 °C. The inhibition was carried out by the use of a preincubation procedure in the absence of substrate. The influence of the inhibitor concentration and the preincubation time on the enzyme activity was elucidated. It was found that increase in pBQ concentration resulted in a linear decrease of urease activity. The dependence of the enzyme activity on the preincubation time showed that the rate of inhibition rapidly decreased at the beginning of the process in order to achieve the constant value. The inhibition became time independent in the studied time range. This observation is characteristic of a slow binding mechanism of inhibition. The protective experiment proved that the urease active site is involved in the binding of pBQ. High effectiveness of thiol protectors against pBQ inhibition indicates the strategic role of the active site sulfhydryl group in the blocking process. There were two methods used for reactivation of pBQ-inhibited urease. The dilution of the urease-pBQ complex in urea solution did not result in a regain of enzyme activity. Alternatively, the addition of dithiothreitol into the urease-pBQ mixture caused the instant and efficient reactivation of the enzyme. The experiments showed that the nature of the urease-pBQ complex is irreversible but the application of a specific thiol reagent can release the active enzyme from the complex.     

Inhibition of jack bean urease by tetrachloro-o-benzoquinone and tetrachloro-p-benzoquinone

Tetrachloro-o-benzoquinone (TCoBQ) and tetrachloro-p-benzoquinone (TCpBQ) were studied as inhibitors of jack bean urease in 20 mM phosphate buffer, pH 7.0, 1 mM EDTA, 25°C. The mechanisms of inhibition were evaluated by analysis of the progress curves obtained with two procedures: the reaction initiated by addition of the enzyme and the reaction initiated by addition of the substrate after preincubation of the enzyme with the inhibitor. The obtained results were characteristic of slow-binding inhibition. The effects of different inhibitor concentrations on the initial and steady-state velocities obeyed the relationships of two-step enzyme-inhibitor interaction, qualified as mechanism B. It was found that TCoBQ and TCpBQ are strong urease inhibitors. TCpBQ is more effective than TCoBQ with the overall inhibition constant of K_i* = 4.5 × 10^{? 7} mM. The respective inhibition constant of TCoBQ was equal to: K_i* = 2.4 × 10^{? 6} mM. The protective experiment proved that the urease active site is involved in the tetrachlorobenzoquinone inhibition process. High effectiveness of thiol protectors against inhibition by TCoBQ and TCpBQ indicates the strategic role of the active site sulfhydryl group in the blocking process. The stability of the complexes: urease-TCoBQ and urease-TCpBQ was tested in two ways: by dilution or addition of dithiothreitol. No recovery of urease activity bound in the urease-inhibitor complexes proves that the complexes are stable and strong.     

Iodine and Iodine Catalysed Phosphorylation of Nucleosides by Phosphorodiester Derivatives

Abstract

Sodium iodide was found to catalyse the phosphorylation of nucleosides by phosphorodiester derivatives such as chlorophosphates, pyrophosphates and triazolides. A similar catalytic effect, but on phosphorochloridates only, was also exerted by iodine.     

Studies on the Oxidation of Nucleoside Hydrogenphosphonates

Abstract

Oxidation of nucleoside H-phosphonate diesters has been investigated using dipyridyl disulphide, hexachloroacetone and iodine, under various reaction conditions.     

Preparation of Nucleoside H-Phosphonoselenoate Monoesters Via the Phosphinate Approach

An efficient entry to nucleoside 3′-H-phosphonoselenoate monoesters via phosphinate intermediates was developed. It involves a reaction of suitably protected nucleosides with triethylammonium phosphinate in the presence of pivaloyl chloride, followed by selenization of the intermediate nucleoside phosphinates with triphenylphosphine selenide, to produce the corresponding nucleoside H-phosphonoselenoates in 86–92% yields.     

Nucleotide Analogues Containing the P-F Bond. An Overview of the Synthetic Methods a

Abstract

In this paper a short review is given of synthetic methods for the preparation of nucleoside phosphorofluoridates, nucleoside phosphorofluoridothioates and nucleoside phosphorofluoridodithioates.

     

Stereochemistry of Internucleotide Bond Formation by the H-Phosphonate Method. 1. Synthesis and 31P Nmr Analysis of 16 Diribonulceoside (3′-5′)-H-Phosphonates and the Corresponding Phosphorothioates

Sixteen diribonucleoside (3′-5′)-H-phosphonates were synthesized via condensation of the protected ribonucleoside 3′-H-phosphonates with nucleosides, and the influence of a nucleoside sequence on the observed stereoselectivity was analyzed. ₃₁P NMR spectroscopy was used to evaluate a relationship between chemical shift and absolute configuration at the phosphorous center of the H-phosphonate diesters as well as of the corresponding phosphorothioate diesters. Although for the most cases such correlation was found, there was however several exceptions to the rule where the relative positions of resonances arising from R _P and S _P diastereomers were reversed.     

Synthesis and in vitro evaluation of antiviral and cytostatic properties of novel 8-triazolyl acyclovir derivatives

Abstract

As a part of the research aimed on identification of new nucleobase derivatives with improved biological properties, a series of novel 8-substituted acyclovir derivatives were synthesized. The 8-azidoguanosine 4 and novel 8-azidoacyclovir 9 were synthesized from commercially available guanosine 1 and acyclovir 6 which were transformed into 8-bromopurine derivatives 2 and 7 and hydrazine derivatives 3 and 8, respectively. 8-Triazolylguanosine 5 and 8-triazolylacyclovir analogs 10–12 were successfully synthesized via the Cu(I) catalyzed 1,3-dipolar cycloaddition reaction of azides 4 and 9 with propargyl alcohol, 4-pentyn-1-ol and 5-hexyn-1-ol. The novel 1,4-disubstituted 1,2,3-triazolyl compounds 5, 10–12 were evaluated for antiviral activity against selected DNA and RNA viruses and cytostatic activity against normal Madine Darby canine kidney (MDCK I) cells, and seven tumor cell lines (HeLa, CaCo-2, NCI-H358, Jurkat, K562, Raji and HuT78). While tested compounds exerted no antiviral activity at nontoxic concentrations, the 8-triazolyl acyclovir derivative 10, with the shortest alkyl substituent at the C-4 of triazole ring, was found to be the most active against the CaCo-2 cell line.     

The Influences of A Nitrogen Atom Position in Dinucleoside 2-,3-,4-Pyridylphosphonates on Fragmentation Patterns in Electrospray Ionization Multistage Tandem Mass Spectra

2-,3-,4-Pyridylphosphonates and their phosphonothioate congeners were analyzed by electrospray ionization multistage tandem mass spectrometry (ESI-MSⁿ). It was found that the fragmentation pathways of these compounds were not influenced to any detectable extent by the stereochemistry at the phosphorus centers but were sensitive to the position of a nitrogen atom in the pyridine ring of these compounds. Possible mechanisms for fragmentations of the investigated compounds are discussed in detail.