Biotinylation of membrane proteins accessible via the pulmonary
circulation in normal and hyperoxic rats.
De, Elsa Kathleen, La Fuente, Christopher A. Dawson, Leif D. Nelin,
Robert D. Bongard, Timothy L. McAuliffe, and Marilyn P. Merker.
Departments of Anesthesiology, Pharmacology/Toxicology, Physiology,
Pediatrics and Biostatistics, Medical College of Wisconsin,
Milwaukee, Wisconsin 53226, Department of Biomedical Engineering,
Marquette University, Milwaukee, Wisconsin 53233, V.A. Medical
Center, Milwaukee, Wisconsin 53295
APStracts 3:0197L, 1996.
It is well established that the phenotype of the pulmonary vascular
surface can be affected by injurious stimuli, but the few proteins
whose 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 we examined the impact of hyperoxia on the spectrum of the
biotinylated proteins. Labelling was carried out either by single
pass bolus injection of the cell impermeant biotinylation reagent,
sulfosuccinimidyl 6-(biotinamido) 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
stretptavidin-linked horse radish 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 than those from rats exposed
to hyperoxia for 48-60 hours. 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 labelled 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
vasulature, e.g., intracellular membrane proteins and plasma membrane
proteins of cells types in luminally inaccessible regions of the
intact lung. The consistent changes in the spectrum of labelled
proteins seen with hyperoxia suggest that in itself the spectrum may
be a useful encryption of certain aspects of vascular
pathophysiology.
Received 13 June 1996; accepted in final form 18 October 1996.
APS Manuscript Number L177-6.
Article publication pending Am. J. Physiol. (Lung Cell. Mol.
Physiology).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 13 November 1996