Purification and biochemical characterization of pulmonary surfactant protein A of horses.
OBJECTIVE
To characterize surfactant protein isolated from bronchoalveolar lavage fluids of healthy horses.


METHODS
10 Thoroughbreds (5 males, 5 females; 26 to 40 months old) that did not have a history or clinical signs of respiratory tract disease.


METHODS
Bronchoalveolar lavage fluid (BALF) was obtained and centrifuged at 33,000 X g. Lipid was removed from precipitated fractions by means of extraction with 1-butanol, and organic solvent-insoluble protein precipitates were dialyzed against Tris buffer. The suspension was centrifuged, and supernatant was placed in a mannose-Sepharose affinity column, with calcium. The bound protein fraction was analyzed by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, western immunoblot analysis, and amino acid sequencing. A liposome-aggregation assay was also performed, using purified proteins.


RESULTS
Protein isolated by use of mannose-affinity matrices was identified as surfactant protein A (SP-A). It had carbohydrate-binding and phospholipid-aggregation properties characteristic of SP-A isolated from other animal species. The partial primary sequence of the isolated protein had high homology with rat and human SP-A. Furthermore, the equine SP-A reacted with anti-human and anti-rat SP-A specific antibodies.


CONCLUSIONS
Analysis of these findings indicated the existence of SP-A in pulmonary tissues of horses.


CONCLUSIONS
Measurement of SP-A concentrations may be useful for clinicians evaluating pulmonary disease of horses.
Clinical Science and Pathobiology Division, Equine Research Institute, Japan Racing Association, Tochigi.
S
S
Hobo
Y
Y
Ogasawara
Y
Y
Kuroki
T
T
Akino
T
T
Yoshihara
D000581:Amidohydrolases / Q000737:chemistry; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western / Q000662:veterinary; D018893:Bronchoalveolar Lavage / Q000662:veterinary; D001992:Bronchoalveolar Lavage Fluid / Q000737:chemistry*; D002847:Chromatography, Agarose / Q000662:veterinary; D017364:Collagenases / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel / Q000662:veterinary; D005260:Female; D006736:Horses / Q000502:physiology*; D008081:Liposomes / Q000502:physiology; D008168:Lung / Q000737:chemistry* / Q000502:physiology; D008297:Male; D008969:Molecular Sequence Data; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D010743:Phospholipids / Q000737:chemistry; D011510:Proteolipids / Q000737:chemistry / Q000302:isolation & purification* / Q000502:physiology; D037662:Pulmonary Surfactant-Associated Protein A; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants / Q000737:chemistry / Q000302:isolation & purification* / Q000502:physiology; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D008081:Liposomes; D010743:Phospholipids; D011510:Proteolipids; D037662:Pulmonary Surfactant-Associated Protein A; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants; D017364:Collagenases; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase