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Biomedical subjects

L Summaria

Publications and source records attributed to L Summaria.

At least 37 records · Page 2Linked to original sources
PubMed1977

Comparison of the esterase and human plasminogen activator activities of various activated forms of human plasminogen and their equimolar streptokinase complexes.

A comparison was made of the esterase and activator activities of the various activated forms of human plasminogen and their streptokinase complexes with Nalpha-Cbz-L-lysine-p-nitrophenyl ester as the substrate. The steady state kinetic properties of Glu- and Lys-plasmins, and Glu- and Lys-plasminogen-streptokinase complexes were identical, while the Lys-plasmin-streptokinase complex showed a 2-fold increase in Km with the same kcat and a 3-fold increase in Ki for the competitive inhibitor leupeptin. Lys-plasminogen (zymogen with an active site) was prepared which incorporated 0.7 mol of [3H]idisopropyl phosphorofluoridate and 0.43 mol of p-nitrophenyl-p'-guanidinobenzoate/mol of protein. The Km for Lys-plasminogen was 3-fold higher than that of Lys-plasmin, and its maximum velocity 10-fold lower. The steady state kinetic parameters of a plasmin-derived light (B) chain (CmCys)3, and a derived equimolar light (B) chain-streptokinase complex (CmCys)3, isolated from human plasmin and equimolar plasmin-streptokinase, or plasminogen-streptokinase, complexes, respectively, were determined. When the light (B) chain-streptokinase complex is isolated from its parent complexes, there is a complete retention of the original parent's esterase activities, with respect to Km and kcat, and interaction with the competitive inhibitors benzamidine and leupeptin. The plasmin-derived light (B) chain does not retain its parent esterase activities. This chain has very similar kinetic properties to Lys-plasminogen except that streptokinase, in an equal molar amount, does not impart full esterase activity to the light (B) chain whereas the zymogen can be completely activated by streptokinase. The kcat of the plasmin-derived light (B) chain, and its streptokinase complex can be enhanced by 50 and 30%, respectively, in the presence of 10(-4) M leupeptin, a competitive inhibitor of plasmin, attesting to the increased structural flexibility within the active site of this enzyme species. Urokinase hydrolyzes Nalpha-Cbz-L-lysine p-nitrophenyl ester efficiently with a kcat/Km of one-third that of plasmin. The human plasminogen activator activities of various activated forms of human plasminogen and their equimolar streptokinase complexes were compared in a kinetic assay. The Lys-plasmin-streptokinase complex, and streptokinase were the least active of the activator species and were approximately equal in their activator activities. Glu- and Lys-plasminogen-streptokinase complexes had approximately 1.5 times the activity of streptokinase, whereas the equimolar light (B) chain-streptokinase complexes had approximately 2- to 3-times the activator activity of streptokinase. Since the esterase activity remained unchanged, this indicates a greater degree of specificity in the active site of the equimolar light (B) chain-streptokinase activator complex. Urokinase proved to be a poor activator species...

Enzyme Activation↗
PubMed1976

Isolation of a human plasmin-derived, functionally active, light (B) chain capable of forming with streptokinase an equimolar light (B) chain-streptokinase complex with plasminogen activator activity.

A functionally active human plasmin light (B) chain derivative, stabilized by the streptomyces plasmin inhibitor leupeptin, was isolated from a partially reduced and alkylated enzyme preparation by an affinity chromatography method with a L-lysine-substituted Sepharose column. This light (B) chain derivative was found to be relatively homogeneous by electrophoretic analysis in both an acrylamide gel/dodecyl sulfate system and on cellulose acetate. It possessed approximately 3% of the proteolytic activity (casein substrate) of the original enzyme, and it incorporated 0.09 mol of [3H]diisopropyl phosphorofluoridate per mol of protein. It contained 3.1 +/- 0.3 carboxymethylated cysteines per mol of protein and can be designated as a CmCys5-light (B) chain (CmCys)3. When this isolated light (B) chain derivative was mixed in equal molar amounts with streptokinase, the mixture developed both human and bovine plasminogen activator activities; the bovine activator activity was approximately 66% of the bovine activator activity of the equimolar human plasmin-streptokinase complex. Although this complex now incorporated 0.50 mol of [3H]diisopropyl phosphorofluoridate per mol of protein, its proteolytic activity, on a molar basis, was the same as the proteolytic activity of the isolated light (B) chain derivative. It was shown by electrophoretic analysis in both an acrylamide gel/epsilon-aminocaproic acid system and on cellulose acetate that the light (B) chain derivative and streptokinase forms an equimolar light (B) chain-streptokinase complex, indicating that the binding site for streptokinase is located on the light (B) chain of the enzyme. A functionally active equimolar light (B) chain-streptokinase complex was also isolated from a partially reduced and alkylated equimolar human plasmin-streptokinase complex by the affinity chromatography method. The plasminogen activator activities (human and bovine) of this light (B) chain-streptokinase complex were similar to those of the plasmin-streptokinase complex from which it was derived. Although this complex incorporated 0.70 mol of [3H]diisopropyl phosphorofluoridate per mol of protein, its proteolytic activity, on a molar basis, was only 14% of proteolytic activity of the plasmin-streptokinase complex.

Amino Acids↗
PubMed1976

Isolation and characterization of the affinity chromatography forms of human Glu- and Lys-plasminogens and plasmins.

Affinity chromatography forms, 1 and 2, were each isolated from human Glu- and Lys-plasminogens by gradient elution from a L-lysine-substituted Sepharose column with a linear gradient of epsilon-aminocaproic acid. Although each of the two zymogen forms contains two affinity chromatography forms, the relative concentrattions of these forms in each of the zymogen preparations depended upon the plasma sample or enriched plasma fraction used for the preparation of the zymogen. Specific analytical acrylamide gel electrophoretic systems were used for the characterization of the zymogen and enzyme forms, and their component affinity chromatography forms, 1 and 2. The four zymogen affinity chromatography forms, Glu-1-plasminogen, Glu-2-plasminogen, Lys-1-plasminogen, and Lys-2-plasmingoen, show distinct stepwise differences in their molecular size and charge. The Glu-1-form is the largest in molecular size and the most acidic, and the Lys-2-form is the smallest in molecular size and the most basic. The proteolytically altered Lys-1- and Lys-2- forms appear to be specifically df the zymogen affinity chromatography forms showed a different distribution of isoelectric forms. The major isoelectric forms isolated from Glu-plasminogen with pI values of 6.2, 6.3, 6.4, and 6.6, and the major isoelectric forms isolated from Lys-plasminogen with pI values of 6.7, 7.2, 7.5, 7.8, and 8.1, (Summaria, L., Arzadon, L., Bernabe, P., Robbins, K. C., and Barlow, G. H. (1973) J. Biol. Chem. 248, 2984-2991) were shown to be mixtures of the Glu-1- and Glu-2- forms, or the Lys-1- and Lys-2- forms, respectively. Although the sialic acid contents of the Glu- and Lys- forms appear to be similar, the isolated affinity chromatography forms show distinct differences. The sialic acid contents of the Glu-1- and Lys-1- forms are identical, and are substantially higher than the sialic acid contents of the Glu-2- and Lys-2- forms which are also identical to each other. It is possible that the charge difference between the zymogen-1- and -2- forms may be related to the differences in their sialic acid content. Each of the four zymogen affinity chromatography forms, when activated by urokinase in the presence of the plasmin inhibitor, Trasylol, was converted to an apparently unique and different enzyme form. The four enzyme forms show distinct stepwise differences in molecular size; Glu-1-plasmin is the largest in size whereas Lys-2-plasmin is the smallest in size. Each plasmin-derived carboxymethyl heavy(A) chain was found to be different in molecular size, but the two carboxymethyl light(B) chains found in each of the four enzyme forms appeared to be identical and of the same molecular sizes. The four heavy(A) chains show a stepwise difference in molecular size; the Glu-1-heavy(A) chain is the largest in size whereas the Lys-2-heavy(A) chain is the smallest in size...

Chromatography, Affinity↗
PubMed1975

Physical and chemical properties of the NH2-terminal glutamic acid and lysine forms of human plasminogen and their derived plasmins with an NH2-terminal lysine heavy (A) chain.

Comparative physical and chemical data are described for the human NH2-terminal Glu-plasminogen and Lys-plasminogen forms in order to determine the exact relationship between these two types of the zymogen. The molecular weights of Glu-plasminogen and Lys-plasminogen were similar and were determined to be 83, 800 plus or minus 4, 500 and 82, 400 plus or minus 3, 300, respectively, by sedimentation equilibrium methods. The molecular weights were identical in dodecyl sulfate solutions, approximately 83, 000, by sedimentation equilibrium methods. The sedimentation coefficients, s-020, w of Glu-plasminogen and Lys-plasminogen were determined to be 5.0 S, and 4.4 S, respectively. These two plasminogen forms had different partial specific volumes, and calculations of the frictional coefficients from sedimentation coefficients and molecular weights indicated conformation differences. Glu-plasminogen appeared to be larger in size than Lys-plasminogen in acrylamide gel-dodecyl sulfate electrophoresis. The amino acid compositions of Glu-plasminogen and Lys-plasminogen, and their major isolated isoelectric forms, were found to be similar, but several amino acid residues (glutamic acid, alanine, isoleucine, phenylalanine, and lysine) were found to be significantly higher in the Glu-plasminogen forms. The derived plasmins from both the Glu- and Lys-plasminogens with an nh2-terminal Lys- heavy (A) chain were found to have identical molecular weights of 76, 500 plus or minus 2, 500, and sedimentation coefficients, s-020, w of 4.3 S.

Amino Acid Sequence↗