Biochemical characterization and immunolocalization studies of a Capsicum chinense Jacq. protein fraction containing DING proteins and anti-microbial activity.
The DING protein family consists of proteins of great biological importance due to their ability to inhibit carcinogenic cell growth. A DING peptide with Mr ∼7.57 kDa and pI ∼5.06 was detected in G10P1.7.57, a protein fraction from Capsicum chinense Jacq. seeds. Amino acid sequencing of the peptide produced three smaller peptides showing identity to the DING protein family. G10P1.7.57 displayed a phosphatase activity capable of dephosphorylating different phosphorylated substrates and inhibited the growth of Saccharomyces cerevisiae cells. Western immunoblotting with a custom-made polyclonal antibody raised against a sequence (ITYMSPDYAAPTLAGLDDATK), derived from the ∼7.57 kDa polypeptide, immunodetected an ∼ 39 kDa polypeptide in G10P1.7.57. Purification by electroelution followed by amino acid sequencing of the ∼39 kDa polypeptide yielded seven new peptide sequences and an additional one identical to that of the initially identified peptide. Western immunoblotting of soluble proteins from C. chinense seeds and leaves revealed the presence of the ∼39 kDa polypeptide at all developmental stages, with increased accumulation when the organs reached maturity. Immunolocalization using Dabsyl chloride- or Alexa fluor 488-conjugated antibodies revealed a specific fluorescent signal in the cell cytoplasm at all developmental stages, giving support to the idea that the ∼39 kDa polypeptide is a soluble DING protein. Thus, we have identified and characterized a protein fraction with a DING protein from C. chinense.
Unidad de Bioquímica y Biología Molecular de Plantas, Centro de Investigación Científica de Yucatán A.C., Calle 43 No. 130, Colonia Chuburná de Hidalgo, C.P. 97200, Mérida, Yucatán, Mexico.
Ligia
L
Brito-Argáez
Unidad de Bioquímica y Biología Molecular de Plantas, Centro de Investigación Científica de Yucatán A.C., Calle 43 No. 130, Colonia Chuburná de Hidalgo, C.P. 97200, Mérida, Yucatán, Mexico.
José A
JA
Tamayo-Sansores
Unidad de Bioquímica y Biología Molecular de Plantas, Centro de Investigación Científica de Yucatán A.C., Calle 43 No. 130, Colonia Chuburná de Hidalgo, C.P. 97200, Mérida, Yucatán, Mexico.
Dianeli
D
Madera-Piña
Unidad de Bioquímica y Biología Molecular de Plantas, Centro de Investigación Científica de Yucatán A.C., Calle 43 No. 130, Colonia Chuburná de Hidalgo, C.P. 97200, Mérida, Yucatán, Mexico.
Francisco J
FJ
García-Villalobos
Unidad de Investigación, Instituto Mexicano del Seguro Social, IMSS, T1, C.P. 97150, Mérida, Yucatán, Mexico.
Rosa E
RE
Moo-Puc
Unidad de Bioquímica y Biología Molecular de Plantas, Centro de Investigación Científica de Yucatán A.C., Calle 43 No. 130, Colonia Chuburná de Hidalgo, C.P. 97200, Mérida, Yucatán, Mexico.
Ángela
Á
Kú-González
Unidad Académica de Sistemas Arrecifales, Instituto de Ciencias del Mar y Limnología, Universidad Nacional Autónoma de México, Puerto Morelos, Quintana Roo, C.P. 77580, Mexico.
Marco A
MA
Villanueva
Unidad de Bioquímica y Biología Molecular de Plantas, Centro de Investigación Científica de Yucatán A.C., Calle 43 No. 130, Colonia Chuburná de Hidalgo, C.P. 97200, Mérida, Yucatán, Mexico. Electronic address: islasign@cicy.mx.
Ignacio
I
Islas-Flores
D000595:Amino Acid Sequence; D000935:Antifungal Agents / Q000302:isolation & purification / Q000494:pharmacology; D000972:Antineoplastic Agents, Phytogenic / Q000302:isolation & purification / Q000494:pharmacology; D002212:Capsicum / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D045744:Cell Line, Tumor; D006801:Humans; D007150:Immunohistochemistry; D007525:Isoelectric Focusing; D008970:Molecular Weight; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000254:growth & development; D017386:Sequence Homology, Amino Acid
D000935:Antifungal Agents; D000972:Antineoplastic Agents, Phytogenic; D010940:Plant Proteins; D010744:Phosphoric Monoester Hydrolases
Capsicum chinense Jacq. seeds; DING proteins; Immunolocalization; Phosphatase activity; Protein sequencing