Biotinylation of membrane proteins accessible via the pulmonary circulation in normal and hyperoxic rats. Am J Physiol 1997 Mar;272(3 Pt 1):L461-70
Date
03/01/1997Pubmed ID
9124603DOI
10.1152/ajplung.1997.272.3.L461Scopus ID
2-s2.0-0030977885 (requires institutional sign-in at Scopus site) 27 CitationsAbstract
It is well established that the phenotype of the pulmonary vascular surface can be affected by injurious stimuli, but the few proteins for which the expression and/or activity have been studied make up only a small fraction of the entire spectrum of luminal cell membrane proteins. To expand the capability for studying such proteins, we developed a method for biotinylating cell membrane proteins accessible via the vascular lumen in the isolated-perfused rat lung and examined the impact of hyperoxia on the spectrum of the biotinylated proteins. Labeling was carried out either by single-pass bolus injection of the cell impermeant biotinylation reagent sulfosuccinimidyl 6-biotin-amido hexanoate (NHS-LC-biotin) into the pulmonary artery cannula or by the addition of NHS-LC-biotin to a lung homogenate. Lung membrane fractions were prepared, and the proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose by electroblotting. The biotinylated proteins were visualized using a chemiluminescent substrate for streptavidin-linked horseradish peroxidase. The spectrum of proteins biotinylated via the vasculature was distinct from that of the biotinylated lung homogenate. Lectin affinity purification of biotinylated proteins from the lung membrane fractions of normal lungs biotinylated via the vasculature revealed characteristic spectra that were reproducibly different from those from rats exposed to hyperoxia for 48-60 h. These results demonstrate that biotinylation of membrane proteins accessible to an extracellular reagent during a single transit through the pulmonary vascular bed is feasible and that the spectrum of these labeled proteins reveals the effects of hyperoxic lung injury. The affinity of biotin for streptavidin makes this procedure potentially useful as a means of separating the labeled membrane proteins from the much larger population of membrane proteins that are not accessible via the vasculature, e.g., intracellular membrane proteins and plasma membrane proteins of cell types in luminally inaccessible regions of the intact lung. The consistent changes in the spectrum of labeled proteins seen with hyperoxia suggest that in itself the spectrum may be a useful encryption of certain aspects of vascular pathophysiology.
Author List
De La Fuente EK, Dawson CA, Nelin LD, Bongard RD, McAuliffe TL, Merker MPAuthor
Timothy L. McAuliffe PhD Professor in the Psychiatry and Behavioral Medicine department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
AnimalsBiotin
Chromatography, Affinity
Concanavalin A
Endothelium, Vascular
Glycoproteins
Hemodynamics
Hyperoxia
Male
Membrane Proteins
Molecular Weight
Precipitin Tests
Pulmonary Circulation
Rats
Rats, Sprague-Dawley
Ricin
Surface Properties
