Biomedical subjects
Publications and source records attributed to N Toki.
Human urinary trypsin inhibitor (UTI) and (4-(2-carboxyethyl) phenyl trans 4-aminoethylcyclohexanecarboxylate hydrochloride (DV-1006) competitively inhibited the human acrosomal proteinase acrosin; Ki values were 1.2 x 10-8 M and 9.4 x 10-7, respectively.
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A new kallikrein inhibitor in human skin extract was further purified by successive column chromatography on DEAE-cellulose, hydroxylapatite-cellulose, and p-cellulose. By these procedures, 0.7 mg of purified preparation was obtained from 10 g of original skin. The purified material was homogeneous, as confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and ultracentrifugation. It had an S20,w value of 4.2 and an apparent molecular weight of 57,000 as measured by gel filtration on Sephadex G-200, and was heat unstable. It possessed an inhibitory activity towards not only human plasma kallikrein, but also human urinary and pancreas kallikrein. It also exhibits antiplasmin and antithrombin activity. This kallikrein inhibitor was found to be immunologically distinct from alpha 2-macroglobulin, alpha 1-antitrypsin, or Cl-inhibitor.
In a previous paper, the authors reported that the kallikrein-like activity was eluted in alpha 2-macroglobulin fractions when patient's plasma was fractionated with Sephadex G-200 gel filtration. In order to clarify the characteristics of the kallikrein-like enzyme, enzyme isolation methods were investigated. Success was attained by the addition of 1 % sodium-dodecyl-sulfate to alpha 2-macroglobulin fractions followed by G-200 chromatography. It was also confirmed that alpha 2-macroglobulin from which the enzyme was removed regained antiplasmin activity, and that some proteases other than the kallikrein-like enzyme were also bound to alpha 2-macroglobulin. The kallikrein-like enzyme isolated by sodium-dodecyl-sulfate-treatment was examined in respect of its molecular weight and its ability to be adsorbed on DEAE-cellulose and was found to possess a molecular weight approximating that of ovalbumin or human pancreatic kallikrein and a binding affinity for DEAE-cellulose. From these results, the authors speculate that during attacks of acute pancreatitis, the pancreas liberates kallikrein into the blood.
The fibrinolytic enzyme system in thirty-two patients suffering from cutaneous vasculitis was investigated. In five of seven patients with Behçet's disease and in five of thirteen patients with erythema nodosum a significant elongation of euglobulin lysis time was observed. On the other hand, in two patients with allergic vasculitis, a significant shortening of euglobulin lysis time was noticed. In the present study, factors responsible for the change of euglobulin lysis time were sought. It was confirmed that the significant elongation of euglobulin lysis time in patients with erythema nodosum was based on an increase of fibrinogen content in the euglobulin fraction. On the other hand, it was speculated that the significant elongation of euglobulin lysis time in patients with Behçet's disease might be due to an increase of antiactivators in the patients' plasma. In the present study, the authors further investigated the status of this antiactivator which is increased in plasma of patients with Behçet's disease, and proposed the possibility that the increased antiactivator might consist of at least two types of antiactivators, one of which is considered to be alpha 2-plasmin inhibitor.
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Two different molecular forms of urinary trypsin inhibitor, UTI-I and UTI-III, were highly purified from normal human urine. Their physicochemical properties were studied and are reported herein.
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A kinin-forming-enzyme in human skin extract was further purified by successive column chromatography on DEAE-cellulose, Hydroxylapatite-cellulose and Sepharose-4B. By these procedures, 2.7 mg of purified enzyme was obtained from 10 gm of original skin. The purified material was homogeneous as ascertained by cellulose acetate membrane electrophoresis, sodium dodecyl sulfate polyacrylamide gel disc electrophoresis and ultracentrifugation. It had an S20,w value of 4.3 and an apparent molecular weight of 104,000 as measured by gel filtration on Sephadex G-200. The purified enzyme was comparatively heat-stable, but was unstable below pH values of 5 and above pH 9. It possessed arginine or lysine esterolytic activity, but not tyrosine or tryptophane esterolytic activity and denatured proteolytic activity. This enzyme was not affected by metal ion, cystein, glutathion or rho-chloromercuribenzoate, but was strongly inhibited by alpha-N-rho-tosyl-L-lysine chloromethyl ketone or soybean-trypsin inhibitor. It was also inhibited by alpha 1-antitrypsin, but not by alpha 2-macroglobulin. This enzyme was confirmed to be immunologically distinct from human plasma, urinary or pancreas kallikrein.
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Six different plasmins were prepared by incubating human plasminogen with various amounts of streptokinase or urokinase. It was confirmed that the six different plasmins possessed similar caseinolytic activities, and the inhibitory effects of a alpha 1-antitrypsin on caseinolytic activities of the six different plasmins were all the same. On the other hand, interactions between the six different plasmins and alpha 2-macroglobulin were complicated. Plasmins activated by cleavage of plasminogen were almost immediately or effectively inhibited by alpha 2-macroglobulin. However, plasmin activated by complex formation of plasminogen with streptokinase was not so immediately or effectively inhibited by alpha 2-macroglobulin. It was supposed that the difference between these two results on the interaction between plasmin and alpha 2-macroglobulin might be due to the difference in molecular form of plasmin. In the present study, it was also confirmed that streptokinase or urokinase, in free form in the reaction mixture, interfered with the interaction between plasmin and alpha 2-macroglobulin. The cause for such interference was discussed.
The fact that esterolytic activity is significantly elevated in plasma of patients with acute pancreatitis and that it correlates with the serum amylase level was confirmed. No kinin activity, however, was detected in plasma. To characterize the esterolytic activity, patient and normal plasmas were chromatographed on Sephadex G-200 and the esterolytic and the kinin-forming activities of each fraction were examined. In the present study, it was speculated that during attacks of acute pancreatitis, kallikrein with amylase might be liberated into blood from the pancreas. It was confirmed that almost all of the kallikrein liberated was combined with alpha1-macroglobulin, and alpha2-macroglobulin-bound kallikrein itself possessed kinin-forming activity.
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The optimal salt concentration for extraction of plasma-kinin-forming enzyme from the human skin was investigated. It was confirmed that a salt concentration higher than 1.5 M in the extraction buffer was required for optimal extraction of this enzyme. An inhibitor of this enzyme was detected in the extract with buffer alone, and the coexistence of this enzyme with the inhibitor in buffer extract was also confirmed by using G-200 gel chromatography.