Characterization of heat‐labile uracil‐DNA glycosylase from Psychrobacter sp. HJ147 and its application to the polymerase chain reaction

Abstract

An affinity matrix containing the antimalarial drug target Plm II (plasmepsin II) as ligand was generated. This enzyme belongs to the family of Plasmodium (malarial parasite) aspartic proteinases, known as Plms (plasmepsins). The procedure established to obtain the support has two steps: the immobilization of the recombinant proenzyme of Plm II to NHS (N-hydroxysuccinimide)-activated Sepharose and the activation of the immobilized enzyme by incubation at pH 4.4 and 37 degrees C. The coupling reaction resulted in a high percentage immobilization (95.5%), and the matrices obtained had an average of 4.3 mg of protein/ml of gel. The activated matrices, but not the inactive ones, were able to hydrolyse two different chromogenic peptide substrates and haemoglobin. This ability was completely blocked by the addition of the general aspartic-proteinase inhibitor, pepstatin A, to the reaction mixture. The matrices were useful in the affinity purification of the Plm II inhibitory activity detected in marine invertebrates, such as Xestospongia muta (giant barrel sponge) and the gorgonian (sea-fan coral) Plexaura homomalla (black sea rod), with increases of 10.2- and 5.9-fold in the specific inhibitory activity respectively. The preliminary K(i) values obtained, 46.4 nM (X. muta) and 1.9 nM (P. homomalla), and the concave shapes of the inhibition curves reveal that molecules are reversible tight-binding inhibitors of Plm II. These results validated the use of the affinity matrix for the purification of Plm II inhibitors from complex mixtures and established the presence of Plm II inhibitors in some marine invertebrates.

Identification of Tight-Binding Plasmepsin II and Falcipain 2 Inhibitors in Aqueous Extracts of Marine Invertebrates by the Combination of Enzymatic and Interaction-Based Assays

“…Previous to the large-scale evaluation of more than 60 marine invertebrate extracts from Cuban coasts, we performed a validation experiment with five reference extracts, previously characterized in our lab as positives (+) or negatives (−) for inhibition against Plm II, FP2 and other related enzymes [46,47,53]: Plexaura homomalla (Plm II+/FP2+), Phallusia nigra (Plm II−/FP2−), Stichodactyla helianthus (Plm II+/FP2+), Xestospongia muta (Plm II+/FP2−) and Polyclinum constellatum (Plm II−/FP2−).…”

“…As expected, the clarified extracts of P. homomalla and S. helianthus showed high inhibitory activities against both enzymes, whereas P. nigra and P. constellatum showed only marginal inhibition. In the case of X. muta , we expected to find significant Plm II inhibitory activity in the heat-clarified extract [46]. Instead, a negative inhibition (which means increased substrate degradation in comparison with assay control) was observed.…”

“…Typical affinity profiles were obtained for the evaluation of P. homomalla (TCA) and X. muta (heat) clarified extracts on a Plm II-Sepharose matrix (Figure 4A,B), with the eluted fraction showing ~6-fold and 10-fold increase in the specific inhibitory activity, respectively [46]. Interaction of P. homomalla (heat) and S. helianthus (heat) positive extracts with clan CA family C1 enzymes were confirmed in the same way by the use of a Papain-Sepharose matrix (Figure 4C,D) [53].…”

“…The main analytical approach for the identification of protease inhibitors in natural extracts has been the evaluation of inhibitory activity by using typical enzyme-specific activity assays [42,44,46,47] and to a lesser degree, interaction-based assays which sense directly the binding to the target enzyme. Enzymatic activity assays are inexpensive, high-throughput capable and provide direct information about the inhibitory effect of the extract components on the activity of the target enzyme [48].…”

“…during screening of crude extracts. In contrast, interaction-based assays, such as affinity chromatography [46,49], Surface Plasmon Resonance (SPR)-biosensors [50] or Intensity Fading (IF) Matrix Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) [51,52], provide reliable information about the presence of target interactors even in the highly heterogeneous context of natural extracts. Nevertheless, they have been rarely used for the identification of protease inhibitors in these sources.…”