Generation of an affinity matrix useful in the purification of natural inhibitors of plasmepsin II, an antimalarial-drug target.
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.
Ramírez
Angel R
AR
Centro de Estudio de Proteínas, Facultad de Biología, Universidad de la Habana, Calle 25 #455 entre J e I, Vedado, CP 10400, La Habana, Cuba. aramirez@fam.cug.co.cu
Guerra
Yasel
Y
Otero
Anabel
A
García
Beatriz
B
Berry
Colin
C
Mendiola
Judith
J
Hernández-Zanui
Aida
A
Chávez
María de Los A
Mde L
D000818:Animals; D037421:Anthozoa / Q000737:chemistry; D000962:Antimalarials / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology*; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D001688:Biological Products / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D002846:Chromatography, Affinity / Q000379:methods*; D004791:Enzyme Inhibitors / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D004792:Enzyme Precursors / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D004800:Enzymes, Immobilized / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D010963:Plasmodium falciparum / Q000201:enzymology; D015800:Protozoan Proteins; D015203:Reproducibility of Results; D047131:Xestospongia / Q000737:chemistry
D000962:Antimalarials; D001688:Biological Products; D004791:Enzyme Inhibitors; D004792:Enzyme Precursors; D004800:Enzymes, Immobilized; D015800:Protozoan Proteins; D016282:Aspartic Acid Endopeptidases; C100637:plasmepsin II