Abstract
Two major forms of native sheep plasminogen (SPg-a) have been isolated from plasma by affinity chromatography. These forms differ in molecular weight, charge characteristics, affinity for epsilon-aminocaproic acid (epsilon-Ahx), and carbohydrate content. Upon treatment of SPg-a with plasmin, lower molecular weight plasminogens can be isolated. A plasminogen (SPg-b) of molecular weight approximately 8,000 less than native plasminogen is rapidly produced when either major plasminogen form is treated with plasmin. The molecular weight differences found in the major SPg-a forms are retained in the SPg-b forms, derived from each SPg-a. Upon protracted treatment of either major form of SPg-a or SPg-b with plasmin, a plasminogen (SPg-c) or molecular weight approximately 32,000 less than SPg-b is produced. A single peptide (P) is also produced in this step. The SPg-c species produced from each original SPg-a major form possess essentially the same molecular weights and carbohydrate compositions; but the P cleaved retains the molecular weight and carbohydrate differences found in each major SPg-a or SPg-b form. A large decrease in the S20,w of SPg-a is observed upon the binding of epsilon-Ahx to this protein. A much smaller alteration in the S20,w of SPg-b and SPg-c is observed upon binding of epsilon-Ahx to these proteins.
Highlights
Two major forms of native sheep plasminogen (SPg-a) have been isolated from plasma by affinity chromatography
Each plasminogen exists as a single component in reduced DodSO, gels
In the pH 4.3 polyacrylamide gel system, each SPg-a major form migrates as a single component; the electrophoretic mobilities significantly differ for each form
Summary
Proteins - SPg-a was prepared by slight modifications of previously described procedures [1, 2] which are tailored to the sheep zymogen. Tris/HCl, 0.1 M lysine, pH 8.0, was incubated with urokinase-free sheep plasmin (10 plasminogen:l plasmin, mol/mol) for 15 min at 30”. The above sample was applied to a column (2.5 x 11 cm) of Sepharose-lysine, equilibrated with 0.18 M phosphate, pH 8.0. The only exception was that the gradient used to elute the SPg-b was changed to 250 ml of 0.1 M phosphate, 0.005 M l -Ahx, pH 8.0, as the starting solvent and 250 ml of 0.1 M phosphate, 0.0225 M e-Ahx, pH 8.0, as the limit solvent. The sample was applied at room temperature to a column (2.5 x 11 cm) of Sepharose/lysine, equilibrated with 0.3 M phosphate, pH 8.0. After application of the sample, the column was washed with the equilibrating buffer until the absorbance at 280 nm dropped to essentially zero. The samples were redissolved in a suitable amount of CHCl, and analyzed by gas-liquid chromatography
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