Author
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Tabatabai, Louisa |
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Submitted to: Biochemical and Biophysical Research Communications
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 6/9/1995 Publication Date: N/A Citation: N/A Interpretive Summary: Pasteurella haemolytica is one of the organisms involved in causing pneumonia in cattle. The disease is also known as shipping fever. P. haemolytica secretes an enzyme, O-sialoglycoprotease, that degrades glycoproteins. The only method available for determining the presence of this enzyme is by using a radioisotope-labeled membrane protein from human red blood cells. This report describes a method for the assay of this enzyme using a readily available glycoprotein obtained from cow's blood, fetuin, that eliminates the need for radioisotopes. The method uses gel electrophoresis followed by staining of the gel. In addition, the specificity of the enzyme could be determined by amino acid sequencing of electroblotted fetuin fragments. Technical Abstract: A soluble bovine glycoprotein, fetuin, was used as an alternative substrate to identify O-sialoglycoprotease activity in culture supernatant protein fractions of Pasteurella haemolytica. An aliquot of a 24-hour incubation mixture containing fetuin and O- sialoglycoprotease was denatured and examined after gradient sodium dodecyl polyacrylamide gel electrophoresis. The Coomassie Brilliant Blue-stained gel was examined for the disappearance of the fetuin band at an apparent molecular mass of 64 KDa. Four major hydrolysis products were identified, an N-terminal fragment of 45 kDa, a 20 kDa fragment at Val/50, and two C-terminal fragments at Val/273 and His/287. |
