In order to obtain more information's about the influence of membrane lipids composition on the action mechanism on the phophatidylcoline transfer protein from beef liver, this protein has spin labeled by means of iodoacetatamide spin label. The labeling was complete in 24h at 37°C 14 pH 5. 100% labeling has been confirmed by the use of C-iodoacetamide, and the proposed site of labeling is one of the five methionine residues of the protein. The EPR spectrum of the spin labeled protein shows an evident enhancement of the correlation time of the spin label as compared with one of the free species (8,9x 10-10 vs 0, 144x10-10) further demonstrating the binding of the probe to the protein. The protein's activity has proven to be fully retained after the labeling. Upon incubation of the spin-labeled PC-TP with phosphatidylcholine vesicles containing 2% or 10% phosphatidic acid, pH 5, "Strongly immobilized" EPR spectra (A=67 G) superimposed to original spectra (A =32 G ) appear. This feature reveals a strong PC-TP-vesicle interaction. This method proves most useful in the studying the PC-TP interactions whit both artificial and natural membranes. The possible applications of these spectra to the calculation of the protein-vesicle complex association constant are dealt with.
Spin labeling the phosphatidylcholine transfer protein from beef live
Lippolis R;
1984
Abstract
In order to obtain more information's about the influence of membrane lipids composition on the action mechanism on the phophatidylcoline transfer protein from beef liver, this protein has spin labeled by means of iodoacetatamide spin label. The labeling was complete in 24h at 37°C 14 pH 5. 100% labeling has been confirmed by the use of C-iodoacetamide, and the proposed site of labeling is one of the five methionine residues of the protein. The EPR spectrum of the spin labeled protein shows an evident enhancement of the correlation time of the spin label as compared with one of the free species (8,9x 10-10 vs 0, 144x10-10) further demonstrating the binding of the probe to the protein. The protein's activity has proven to be fully retained after the labeling. Upon incubation of the spin-labeled PC-TP with phosphatidylcholine vesicles containing 2% or 10% phosphatidic acid, pH 5, "Strongly immobilized" EPR spectra (A=67 G) superimposed to original spectra (A =32 G ) appear. This feature reveals a strong PC-TP-vesicle interaction. This method proves most useful in the studying the PC-TP interactions whit both artificial and natural membranes. The possible applications of these spectra to the calculation of the protein-vesicle complex association constant are dealt with.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
