Kenneth C. Robbins

[22] Plasminogen and plasmin☆

Publisher Summary Plasmin is a serine protase with trypsin-like specificity. It cleaves or hydrolyzes proteins and peptides at arginyl and lysyl peptide bonds, and basic amino acid esters, and amides. Plasmin appears to have a preference for lysyl bonds in both protein and ester substrates. Assay methods for plasminogen and plasmin have been devised using both casein (caseinolytic) and fbrin (fibrinolytic) substrates; in the caseinolytic assay, the release of trichloroacetic acid, or perchloric acid, soluble peptides arc measured, and in the fibrinolytic assay, the time of dissolution of the fibrin clot is determined. This chapter describes specific active-site titration methods developed to determine the amount of enzyme in solution. Specific antibody and protein inhibitor assays have also been described. A method for the determination of human plasminogen and plasmin based on the interaction between the zymogen, or enzyme, and streptokinase has been described.

Comparative activation kinetics of mammalian plasminogens

Five native mammalian plasminogen species, namely, cat, dog, bovine, rabbit and horse, were studied and compared to native human plasminogen with respect to their substrate and enzymatic properties in various activated forms. These studies are an extension of previous work and were designed to confirm our previously proposed mechanism of plasminogen activation, using a series of native, but different, plasminogen substrates. The plasminogen activator species used were high molecular weight urokinase, streptokinase, human Glu-plasminogen-streptokinase complex, human plasmin-derived light(B)-chain-streptokinase complex, and the equimolar streptokinase activator complexes prepared from cat and dog plasmins. The peptidase parameters of the plasmins, plasmin-streptokinase and plasminogen-streptokinase complexes were determined with H-D-valyl-L-leucyl-L-lysyl-p-nitroanilide and Tos-glycyl-L-prolyl-L-lysyl-p-nitroanilide. Activation kinetics were measured with the same substrates. The peptidase parameters of all plasmin species were found to be similar, but with minor variations. The equimolar streptokinase mixtures of bovine, rabbit and horse plasminogens and plasmins did not form complexes and did not form active sites with plasminogen, under the conditions used. The second-order rate constants of activation revealed great differences (as much as 1400-fold), presumably expressing differences in the tertiary structure of the various plasminogen scissile bonds. The catalytic rate constants of activation, kplg, varied by as much as a 100-fold, while differences in Kplg were relatively small. The results of this study confirm the activation mechanism we have postulated previously, namely, that rapid-equilibrium rather than steady-state conditions prevail and that k2 (acylation) is the catalytic rate constant and the rate-determining step, while KS is a true dissociation constant. Calculations of the free energy of interaction of the peptidase and plasminogen activation reactions showed -4.4 to -5.6 kcal/mol for peptidase and -6.5 to -10 kcal/mol for the activation reaction. These values indicate 1-3 subsite binding interactions for the peptidase activity and 3-5 subsite binding interactions for the activation catalytic event. Streptokinase activator complexes have at least one more interacting subsite than the urokinase active site.

Studies on porcine elastase and proelastase

A partial purification of procine elastase is described. The important steps are precipitation of the enzyme by dialysis and adsorption on the substrate, elastin. Evidence is given for the adsorption of proelastase elastin, suggesting the concept of distinct combining and catalytic sites in the enzyme. Preparations of elastin, differing in source and initial alkali treatment, are compared for rate of elastolysis and adsorptive affinity for elastase and proelastase. Partially purified elastase preparations show strong proteolytic action on proteins other than elastin. The possibility that elastase is another pancreatic proteinase is discussed. Solubilizing action on thermally contracted collagen is a property of trypsin and chymotrypsin, as well as elastase.

[30] Human plasmin

Publisher Summary Various forms of human plasmin are described in this chapter, prepared, and isolated—namely, Glul-plasmin, Lys-plasmin, Val-plasmin. Glu-plasmin can be prepared from the native Glu-zymogen in the presence of plasmin inhibitors, Lys-plasmin can be prepared from either the native zymogen or the plasmin-degraded Lys-zymogen, and Val-plasmin can be prepared from the elastase-degraded Val-zymogen. Val-plasmin, the light (B) chain of Glu-plasmin, Lys-plasmin, and Val-plasmin, can be prepared by partial reduction of the two interchain-disulfide bonds connecting the heavy (A) chain and light (B) chain of the larger plasmins. Also, the afffinity-chromatography forms of each of the plasmins containing the heavy (A) chains with lysinebinding sites can be prepared. Recombinant plasmin can be prepared from the isolated sulfhydryl forms of the heavy (A) chain and light (B) chain.

Occurrence and Activation of an Elastase Precursor in Pancreas

Summary Dog pancreatic juice and extracts of hog pancreas contain elastase in an inactive form. Trypsin or a duodenal factor is capable of activating pro-elastase. The activation is blocked by pancreatic and soybean trypsin inhibitors, an indication that the duodenal factor is enterokinase.

Methods for studying fibrinolytic pathway components in human plasma

Methods have been developed to quantitatively measure the major plasma components of the human fibrinolytic system. Plasminogen is measured functionally with a 9M excess of streptokinase and immunochemically by rocket immunoelectrophoresis; the normal range was found to be 16.7-23.8 mg/dl and 17.4-21.6 mg/dl, respectively. Alpha 2-plasmin inhibitor is measured functionally and immunochemically; the normal range for the major plasma plasmin inhibitor was found to be 5.30-6.60 mg/dl by both methods. Plasminogen activator concentrations, as well as, free, and complexed, protease activities are measured along with plasmin generation rates by spectrophotometric assays with chromogenic substrates. Both activator and free protease activities are zero in plasma samples from normal human subjects. Plasmin generation rates are 0.25-0.47% with urokinase and 5.30-9.70% with streptokinase; these values are the percentages of the respective initial velocities of activation in purified systems.

Identification of the plasminogen activator(s) produced by the transformed liver cell line, SK-HEP-1.

The availability of a cell line derived from an adenocarcinoma of the liver has made it possible to study the plasminogen activator(s) (PA) biosynthesized in culture by liver cells. Conditioned cultured media purified on fibrin-celite, benzamidine-Sepharose and immunoabsorbent anti-urokinase columns have shown the presence of multiple plasminogen activators when separated on sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE). These PAs differ in molecular weight but all are urokinase-like based on their reaction with goat anti-urokinase and rabbit anti-tissue activator. Subcellular fractionation of the cultured cells shows the presence of activator in both the cytoplasmic and membrane fractions, but the higher molecular weight forms appear primarily in the cytoplasm.

Separation of Pertussal Toxin.

Summary Methods have been described for the separation of pertussal toxin from a 0.05 M CaCl2 extract of H. pertussis, strain No. 29, dried from the frozen state. The most active fraction contained 5260 LD50 per mg N and was found to be heterogeneous by electrophoretic analysis.

Studies on the Agglutination of Erythrocytes Treated with Human Plasmin and Some Other Enzymes

Summary The serological activity of human plasmin was compared with those of trypsin, bromelin, papain, and ficin. Human plasmin increased agglutination of erythrocytes with Rh-Hr antibodies, and to a lesser extent, with anti-Lea, anti-K, and anti-k (Cellano) antibodies. It did not decrease the agglutination of erythrocytes with anti-M, anti-N, anti-S, and anti-Fya antibodies. No increased agglutination could be observed with anti-P and anti-I antibodies. Bovine plasmin, human and bovine thrombin, and α-chymotrypsin did not alter the agglutinability of erythrocytes.

Preparation of Partially Purified Porcine Intrinsic Factor

Summary and Conclusions A simple method is described for preparation of highly purified intrinsic factor PI from porcine stomach pyloric mucosa. In pernicious anemia patients in relapse, 35 mg of intrinsic factor PI when given with 15 μg vit. B12 is defined as one U.S.P. oral unit. Dialysis of intrinsic factor PI gives a material with a potency of about 15 mg/U.S.P. unit. In the urinary excretion test, one-half U.S.P. unit of intrinsic factor PI gives significant and measurable urinary excretion of vit. B12 Co60 when given orally with 1 μg vit. B12 Co60. Approximately 1.5 U.S.P. units of intrinsic factor PI will give maximum urinary excretion of vit. B12 Co60. Intrinsic factor PI can be further purified on a DEAE-cellulose ion exchanger to give a material with an estimated potency of 9 mg/U.S.P. unit.