title: New insights into the complex mixture of latex cysteine peptidases in Calotropis procera.
abstract: The latex of Calotropis procera is a rich source of proteolytic activity. This latex is known to contain two distinct cysteine peptidases: procerain and procerain B. In this study, new cysteine peptidases were purified from C. procera latex. The enzymes were purified by two sequential ion-exchange chromatography steps (CM-Sepharose plus Resource S(®)) at pH 5.0 and 6.0. The purified enzymes had molecular mass spectra corresponding to CpCP-1=26,213, CpCP-2=26,133 and CpCP-3=25,086 Da. These enzymes exhibited discrete differences in terms of enzymatic activity at a broad range of pH and temperature conditions and contained identical N-terminal amino acid sequences. In these respects, these three new proteins are distinct from those previously studied (procerain and procerain B). Circular dichroism analysis revealed that the new peptidases contain extensive secondary structures, α(15-20%) and β(26-30%), that were stabilized by disulfide bonds. The purified enzymes exhibited plasma-clotting activity mediated by a thrombin-like mechanism. The set of results suggest the three isolated polypeptides correspond to different post-translationally processed forms of the same protein.
authors.affiliation: Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, Fortaleza, CE, Brazil. vramos@ufc.br
authors.forename: M V
authors.initials: MV
authors.lastname: Ramos
authors.forename: E S
authors.initials: ES
authors.lastname: Araújo
authors.forename: T L
authors.initials: TL
authors.lastname: Jucá
authors.forename: A C O
authors.initials: AC
authors.lastname: Monteiro-Moreira
authors.forename: I M
authors.initials: IM
authors.lastname: Vasconcelos
authors.forename: R A
authors.initials: RA
authors.lastname: Moreira
authors.forename: C A
authors.initials: CA
authors.lastname: Viana
authors.forename: L M
authors.initials: LM
authors.lastname: Beltramini
authors.forename: D A
authors.initials: DA
authors.lastname: Pereira
authors.forename: F B
authors.initials: FB
authors.lastname: Moreno
mesh_terms: D000595:Amino Acid Sequence; D001777:Blood Coagulation; D031107:Calotropis / Q000201:enzymology*; D002852:Chromatography, Ion Exchange; D003029:Coagulants / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007840:Latex / Q000737:chemistry*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010940:Plant Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D017433:Protein Structure, Secondary; D059748:Proteolysis; D011517:Prothrombin Time; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid
chemical_list: D003029:Coagulants; D007840:Latex; D010940:Plant Proteins; D003546:Cysteine Endopeptidases; C474045:procerain