Purification and biochemical characterization of pulmonary surfactant protein A of horses

Seiji Hobo From the Clinical Science and Pathobiology Division, Equine Research Institute, Japan Racing Association, 321-4 Tokami-cho, Utsunomiya-shi, Tochigi 320-0856, (Hobo, Yoshihara) and the Department of Biochemistry, School of Medicine, Sapporo Medical University, South-1, West-17, Chuo-ku, Sapporo 060-0061 (Ogasawara, Kuroki, Akino), Japan.

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Yoshinori Ogasawara From the Clinical Science and Pathobiology Division, Equine Research Institute, Japan Racing Association, 321-4 Tokami-cho, Utsunomiya-shi, Tochigi 320-0856, (Hobo, Yoshihara) and the Department of Biochemistry, School of Medicine, Sapporo Medical University, South-1, West-17, Chuo-ku, Sapporo 060-0061 (Ogasawara, Kuroki, Akino), Japan.

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Yoshio Kuroki From the Clinical Science and Pathobiology Division, Equine Research Institute, Japan Racing Association, 321-4 Tokami-cho, Utsunomiya-shi, Tochigi 320-0856, (Hobo, Yoshihara) and the Department of Biochemistry, School of Medicine, Sapporo Medical University, South-1, West-17, Chuo-ku, Sapporo 060-0061 (Ogasawara, Kuroki, Akino), Japan.

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Toyoaki Akino From the Clinical Science and Pathobiology Division, Equine Research Institute, Japan Racing Association, 321-4 Tokami-cho, Utsunomiya-shi, Tochigi 320-0856, (Hobo, Yoshihara) and the Department of Biochemistry, School of Medicine, Sapporo Medical University, South-1, West-17, Chuo-ku, Sapporo 060-0061 (Ogasawara, Kuroki, Akino), Japan.

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Toyohiko Yoshihara From the Clinical Science and Pathobiology Division, Equine Research Institute, Japan Racing Association, 321-4 Tokami-cho, Utsunomiya-shi, Tochigi 320-0856, (Hobo, Yoshihara) and the Department of Biochemistry, School of Medicine, Sapporo Medical University, South-1, West-17, Chuo-ku, Sapporo 060-0061 (Ogasawara, Kuroki, Akino), Japan.

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Abstract

Objective

To characterize surfactant protein isolated from bronchoalveolar lavage fluids of healthy horses.

Animals

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

Procedure

Bronchoalveolar lavage fluid (BALF) was obtained and centrifuged at 33,000 × 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.

Conclusion

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

Clinical Relevance

Measurement of SP-A concentrations may be useful for clinicians evaluating pulmonary disease of horses. (Am J Vet Res 1999;60: 169-173)

Abstract

Objective

To characterize surfactant protein isolated from bronchoalveolar lavage fluids of healthy horses.

Animals

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

Procedure

Bronchoalveolar lavage fluid (BALF) was obtained and centrifuged at 33,000 × 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.

Conclusion

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

Clinical Relevance

Measurement of SP-A concentrations may be useful for clinicians evaluating pulmonary disease of horses. (Am J Vet Res 1999;60: 169-173)

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