Per Wallén

Characterization of human plasminogen II. Separation and partial characterization of different molecular forms of human plasminogen

Abstract Purified preparations of human plasminogen often contain up to ten different forms of plasminogen, as demonstrated by zymographic analysis after starch-gel electrophoresis. Of these forms, only six have electrophoretic properties identical to those found in human plasma. The other forms, which move closer to the cathode, are probably altered by proteolytic degradation. By chromatography on DEAE-Sephadex using a pH and ionic gradient, a group separation was obtained of these plasminogen forms. One fraction (DE-A) contained four to six forms with electrophoretic properties consistent with the main plasminogen components of normal plasma. The other fraction (DE-B) contained the altered forms. On isoelectric focusing the isoelectric points of the components of Fraction DE-A range between 6.0 and 6.6, and those of Fraction DE-B between 7.3 and 8.8. The molecular weights of Fraction DE-A and Fraction DE-B were 90 000 and 105 000, respectively, as determined by gel filtration. There is evidence that the apparently higher molecular weight of Fraction DE-B is due to a partial degradation of the plasminogen molecules followed by conformational changes. As shown by gel-filtration experiments, the presence of e-aminocaproic acid in the solvent brings about an increase in molecular volume of plasminogen. This is much more pronounced for Fraction DE-A than for Fraction DE-B. The amino acid composition and the N-terminal dipeptide have been determined on the plasminogen forms of Fraction DE-A isolated by isoelectric focusing. No significant difference among the different forms were found. The amino acid composition is consistent with the rather low values of the isoelectric points.

Characterization of human plasminogen: I. On the relationship between different molecular forms of plasminogen demonstrated in plasma and found in purified preparations

Abstract Using starch gel electrophoresis in combination with a zymographic technique, up to six main plasminogen components have been demonstrated in normal fresh human plasma or serum from single individuals. Plasminogen components with the same electrophoretic mobilities as these are usually found in purified preparations. In addition, plasminogen forms with markedly deviating mobilities are often detected in significant amounts. The latter components, which probably are degraded forms of plasminogen, may be present in the plasma fractions used as starting materials. However they are especially abundant if spontaneous proteolytic activity is present or allowed to develop during the purification. A procedure for purification of human plasminogen from plasma fraction III is described. Great care is taken to remove contaminating proteolytic activity and to prevent partial activation, which easily occurs in later stages of the procedure. Prior to the last step, chromatography on DEAE-Sephadex, all traces of contaminating plasmin (EC 3.4.4.14) have to be removed. This is done by treatment with an inhibitor (Trasylol). The purified plasminogen obtained contains up to ten electrophoretic components all having plasminogen activity. Those components, which migrate differently from the plasminogen forms of plasma, may be removed by proper pooling of the effluent. Thus, preparations have been obtained containing 4–6 components, electrophoretically identical to the plasminogen forms of plasma. Glutamic acid constituted the major detectable N-terminal amino acid of these preparations (approx. 1 mole per mole plasminogen). Edman degradation in six steps showed the following N-terminal sequence: Glu-Pro-Leu-Asp-Asp-Tyr-.

Differential proteolysis and evidence for a residue exchange in tissue plasminogen activator suggest possible association between two types of protein microheterogeneity

The N‐terminal part of native one‐chain tissue plasminogen activator from melanoma cells is not homogeneous. The protein chain starts at two different postions, in all probability representing a processing difference in the N‐terminus. Both ‘long’ L‐chains and 3‐residue shorter S‐chains are present in the preparations. In addition, results compatible with a positional Ser/Gly microheterogeneity were obtained at a single position (positions L‐4 which is equal to S‐1). The N‐terminal tripeptide difference seems to be coupled to the possible microheterogeneity: L‐chains contain Ser in this position, while S‐chains appear to contain predominantly Gly.

Porcine tissue plasminogen activator: immunoaffinity purification, structural properties and glycosylation pattern

Tissue plasminogen activator was purified in high yield from pig heart by immunoaffinity chromatography and characterized by analysis of the glycosylation pattern and the N-terminal amino acid sequence. Comparisons with the human enzyme reveals residue exchanges in the A-chain at positions 3 (porcine Arg/human Gin) and 5 (Thr/Ile), and in the B-chain at positions 6 (Tyr/Phe), 10 (Thr/Ala) and 20 (Val/Ala). The glycosylation pattern for the porcine activator was determined by endoglycosidase treatment followed by gel electrophoresis. The A-chain contains a single high-mannose type of JV-linked glycan structure and the B-chain contains a complex type of oligosaccharide. A similar but not identical pattern has been observed for the human activator, purified from melanoma cells.Tissue plasminogen activator was purified in high yield from pig heart by immunoaffinity chromatography and characterized by analysis of the glycosylation pattern and the N‐terminal amino acid sequence. Comparisons with the human enzyme reveals residue exchanges in the A‐chain at positions 3 (porcine Arg/human Gin) and 5 (Thr/Ile), and in the B‐chain at positions 6 (Tyr/Phe), 10 (Thr/Ala) and 20 (Val/Ala). The glycosylation pattern for the porcine activator was determined by endoglycosidase treatment followed by gel electrophoresis. The A‐chain contains a single high‐mannose type of JV‐linked glycan structure and the B‐chain contains a complex type of oligosaccharide. A similar but not identical pattern has been observed for the human activator, purified from melanoma cells.