Isolation and intracellular localization of insulin-like proteins from leaves of Bauhinia variegata.
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.
Azevedo
C R
CR
Laboratório de Química e Função de Proteínas e Peptídeos, Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense, Campos dos Goytacazes, RJ, Brasil.
Maciel
F M
FM
Silva
L B
LB
Ferreira
A T S
AT
da Cunha
M
M
Machado
O L T
OL
Fernandes
K V S
KV
Oliveira
A E A
AE
Xavier-Filho
J
J
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
D001323:Autoantibodies; D007004:Hypoglycemic Agents; D007074:Immunoglobulin G; D018969:Insulin-Like Growth Factor Binding Proteins