title: Isolation and intracellular localization of insulin-like proteins from leaves of Bauhinia variegata.
abstract: Evidence based on immunological cross-reactivity and anti-diabetic properties has suggested the presence of insulin-like peptides in plants. The objective of the present study was to investigate the presence of insulin-like proteins in the leaves of Bauhinia variegata ("pata-de-vaca", "mororó"), a plant widely utilized in popular medicine as an anti-diabetic agent. We show that an insulin-like protein was present in the leaves of this plant. A chloroplast protein with a molecular mass similar to that of bovine insulin was extracted from 2-mm thick 15% SDS-PAGE gels and fractionated with a 2 x 24 cm Sephadex G-50 column. The activity of this insulin-like protein (0.48 mg/mL) on serum glucose levels of four-week-old Swiss albino (CF1) diabetic mice was similar to that of commercial swine insulin used as control. Further characterization of this molecule by reverse-phase hydrophobic HPLC chromatographic analysis as well as its antidiabetic activity on alloxan-induced mice showed that it has insulin-like properties. Immunolocalization of the insulin-like protein in the leaves of B. variegata was performed by transmission electron microscopy using a polyclonal anti-insulin human antibody. Localization in the leaf blades revealed that the insulin-like protein is present mainly in chloroplasts where it is also found associated with crystals which may be calcium oxalate. The presence of an insulin-like protein in chloroplasts may indicate its involvement in carbohydrate metabolism. This finding has strengthened our previous results and suggests that insulin-signaling pathways have been conserved through evolution.
authors.affiliation: Laboratório de Química e Função de Proteínas e Peptídeos, Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense, Campos dos Goytacazes, RJ, Brasil.
authors.forename: C R
authors.initials: CR
authors.lastname: Azevedo
authors.forename: F M
authors.initials: FM
authors.lastname: Maciel
authors.forename: L B
authors.initials: LB
authors.lastname: Silva
authors.forename: A T S
authors.initials: AT
authors.lastname: Ferreira
authors.forename: M
authors.initials: M
authors.lastname: da Cunha
authors.forename: O L T
authors.initials: OL
authors.lastname: Machado
authors.forename: K V S
authors.initials: KV
authors.lastname: Fernandes
authors.forename: A E A
authors.initials: AE
authors.lastname: Oliveira
authors.forename: J
authors.initials: J
authors.lastname: Xavier-Filho
mesh_terms: D000818:Animals; D001323:Autoantibodies / Q000097:blood; D036481:Bauhinia / Q000737:chemistry* / Q000166:cytology; D002417:Cattle; D002736:Chloroplasts / Q000737:chemistry* / Q000648:ultrastructure; D002851:Chromatography, High Pressure Liquid; D003921:Diabetes Mellitus, Experimental / Q000188:drug therapy*; D004591:Electrophoresis, Polyacrylamide Gel; D007004:Hypoglycemic Agents / Q000302:isolation & purification* / Q000627:therapeutic use; D007074:Immunoglobulin G / Q000097:blood; D018969:Insulin-Like Growth Factor Binding Proteins / Q000302:isolation & purification* / Q000627:therapeutic use; D051379:Mice; D046529:Microscopy, Electron, Transmission; D018515:Plant Leaves / Q000737:chemistry* / Q000166:cytology
chemical_list: D001323:Autoantibodies; D007004:Hypoglycemic Agents; D007074:Immunoglobulin G; D018969:Insulin-Like Growth Factor Binding Proteins