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Purification and biochemical characterization of equine pulmonary surfactant protein D.
Abstract: To characterize surfactant protein D (SP-D) isolated from bronchoalveolar lavage fluid (BALF) of healthy horses. Methods: BALF from 10 Thoroughbreds (5 males, 5 females; 26 to 40 months old) without history or clinical signs of respiratory tract disease. Methods: BALF was obtained and centrifuged at 33,000 X g. The supernatant was applied to a mannose-Sepharose 6B affinity column in the presence of calcium, and the bound protein fraction was analyzed by use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblot analysis; amino acid composition was determined and partial sequencing was done. Phospholipid binding and liposome aggregation assay were performed, using purified proteins. Results: The protein isolated by use of mannose affinity matrices was SP-D. It bound carbohydrates and phosphatidylinositol, which are the characteristic features of SP-D isolated from other animal species. Amino acid analysis and partial primary sequence of the isolated protein indicated high homology with rat and human SP-D. Furthermore, immunoblot analysis indicated that equine SP-D reacted with human and rat SP-D-specific antibodies. Conclusions: SP-D exists in equine lungs; its measurement may be useful in evaluating equine lung disease.
Publication Date: 1999-04-03 PubMed ID: 10188822 The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
Summary
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The study presents the isolation and characterization of Surfactant Protein D (SP-D) from the bronchoalveolar lavage fluid (BALF) collected from healthy horses. By using various testing methods, researchers confirmed the presence of SP-D in horses’ lungs and their similarity with SP-D found in rats and humans.
Methods and Procedure
- The study started by collecting bronchoalveolar lavage fluid (BALF) from 10 healthy Thoroughbred horses of both sexes and ages 26 to 40 months. None of these horses had any history or signs of respiratory tract disease.
- This collected BALF was centrifuged at 33,000 X g, a high-speed process to separate different components of the fluid. The supernatant (the liquid lying above a solid residue after crystallization, precipitation, centrifugation, or other process) was taken for further analysis.
- The liquid portion was then exposed to a mannose-Sepharose 6B affinity column in the presence of calcium. Affinity columns are used to bind and isolate specific proteins.
- The bound protein fraction was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis to identify and characterize the proteins. The amino acid composition was determined, and a partial sequencing was performed for further verification.
- The phospholipid binding and liposome aggregation tests were also used to verify the protein’s characteristics.
Results and Findings
- Analysis confirmed that the isolated protein using mannose affinity matrices was indeed SP-D. This protein was found to bind with carbohydrates and a phospholipid called phosphatidylinositol, which are typical features of SP-D from other animal species.
- The amino acid analysis and partial primary sequence of the isolated protein revealed a high similarity to the SP-D found in rats and humans.
- Immunoblot analysis further confirmed this as the equine SP-D reacted with SP-D-specific antibodies from humans and rats.
Conclusions
- This research establishes that SP-D does exist in equine lungs, similar to that found in rats and humans.
- Testing for this protein can play a crucial role in evaluating equine lung disease.
Cite This Article
APA
Hobo S, Ogasawara Y, Kuroki Y, Akino T, Yoshihara T.
(1999).
Purification and biochemical characterization of equine pulmonary surfactant protein D.
Am J Vet Res, 60(3), 368-372.
Publication
Researcher Affiliations
- Clinical Science and Pathobiology Division, Equine Research Institute, Japan Racing Association, Tochigi.
MeSH Terms
- Amino Acids / analysis
- Animals
- Bronchoalveolar Lavage Fluid / chemistry
- Electrophoresis, Polyacrylamide Gel / veterinary
- Female
- Glycoproteins / chemistry
- Glycoproteins / isolation & purification
- Horses
- Humans
- Immunoblotting / veterinary
- Liposomes
- Male
- Phospholipids / metabolism
- Pulmonary Surfactant-Associated Protein D
- Pulmonary Surfactants / chemistry
- Pulmonary Surfactants / isolation & purification
- Rats
Citations
This article has been cited 2 times.- Soerensen CM, Nielsen OL, Willis A, Heegaard PM, Holmskov U. Purification, characterization and immunolocalization of porcine surfactant protein D. Immunology 2005 Jan;114(1):72-82.
- Kankavi O, Roberts MS. Detection of surfactant protein A (SP-A) and surfactant protein D (SP-D) in equine synovial fluid with immunoblotting. Can J Vet Res 2004 Apr;68(2):146-9.
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