Isolation and Characterization of a Cysteine Protease from the Latex of Araujia hortorum Fruits

Abstract

A new protease (araujiain h I) was purified to mass spectroscopy homogeneity from the latex of Araujia hortorum Fourn. (Asclepiadaceae) fruits by ultracentrifugation and ion exchange chromatography. The enzyme has a molecular mass of 24,031 (mass spectrometry) and an iso-electric point higher than 9.3. The optimum pH range for casein hydrolysis was 8.0-9.5. The enzyme showed remarkable caseinolytic activity at high temperatures, although its thermal stability decayed rapidly. The proteinase was activated by thiol compounds and inhibited by common thiol-blocking reagents, particularly E-64 and HgCl2, suggesting the enzyme belongs to the cysteine protease family. The concentration of active sites as determined by titration with E-64 was 3.3 microM. When assayed on N-alpha-CBZ-amino acid-p-nitrophenyl esters, the enzyme showed higher preference for the glutamine derivative, followed by those of alanine, asparagine, glycine, and leucine, in decreasing order. Partial homology (36-48%) with other plant cysteine proteinases was observed in an internal fragment obtained by Protease V8 treatment.

Study of phytoproteases stability in aqueous-organic biphasic systems using linear free energy relationships

“…Araujiain is the crude extract obtained from latex of fruits of Araujia hortorum Fourn. (Asclepiadaceae) [16,17]. Funastrain is the crude extract obtained from latex of stems of Funastrum clausum (Jacq.)…”

“…Each test tube was centrifuged at 3000 × g for 30 min and the absorbance of the supernatant was measured at 280 nm. An arbitrary enzyme unit (caseinolytic unit, Ucas) was defined as the amount of protease, which produces an increment of one absorbance unit per min in the assay conditions [17]. The initial total content of proteins from crude extracts was determined according to Bradford method [18].…”

Structure of the mexicain–E-64 complex and comparison with other cysteine proteases of the papain family

“…The optimal activity temperature with the same substrate is 338 K. Other cysteine proteases show a lower optimal activity temperature. For example, the proteases freesia protease B from Freesia reflacta and araujiain h I from Araujia hortorum show optimal activity temperatures of 323-328 and 333 K, respectively, using the same substrate (Kaneda et al, 1997;Priolo et al, 2000).…”

Purification, crystallization and preliminary X-ray analysis of mexicain

“…Cysteine proteases are widely distributed from viruses to mammals. In plants, cysteine proteases are by far the most abundant (Priolo et al, 2000). They are classi®ed into more than 30 families grouped into ®ve clans (CA to CE) according to the amino acids located in the active centre (Barrett & Rawlings, 2001).…”