Naotika Toki

Urinary trypsin inhibitor and urokinase activities in renal diseases.

Using an improved method of determination, urinary trypsin inhibitor (UTI) activities were assayed in relation to urokinase (UK) activities in a large group of patients with various renal diseases. In normal subjects (n = 50), the mean 24-hour values of the UTI and UK activities were 4.29 +/- 1.44 U/ml and 9.80 +/- 3.81 IU/ml, respectively. Data for renal diseases such as renal stone, hydronephrosis, renal cancer, and chronic glomerulonephritis (UTI, 5.51 +/- 2.29 U/ml (p less than 0.005) and UK, 6.88 +/- 2.64 IU/ml (p less than 0.001); n = 40), and particularly uremia (UTI, 9.90 +/- 5.68 U/ml (p less than 0.001) and UK, 3.85 +/- 2.36 IU/ml (p less than 0.001); n = 30), showed that the UTI level was increased whereas the UK level was decreased. The UTI/UK ratio more clearly demonstrated the difference between these diseases.

Purification and partial characterization of two forms of urinary trypsin inhibitor

Crude urinary trypsin inhibitor was obtained by DEAE-cellulose column chromatography from normal fresh urine. By the purification of the crude urinary inhibitor on successive chromatography methods using Sephacryl S-200, DEAE-cellulose, CM-Sepharose CL-6B and Sephadex G-100, we detected two forms of urinary trypsin inhibitor: form I and form II. The specific activity of form I increased approx. 4-fold with a recovery of 60%, as compared to that of crude urinary trypsin inhibitor. N-terminal amino acids of form I and form II were determined to be alanine and valine, respectively. Molecular weights of forms I and II were estimated to be 67000 and 28000 by gel filtration on Sephadex G-100 and to be 43000 and 19000 by SDS-polyacrylamide gel electrophoresis. S-carboxymethylated form I migrated as a single band corresponding to a molecular weight of 59000 in SDS-polyacrylamide gel electrophoresis. From the results of the determination of a single N-terminal amino acid of form I and a single band of S-carboxymethylated form I, it is indicated that it is composed of single polypeptide chain. And the present study suggests that form I is a native form of trypsin inhibitor in normal human urine and form II is a fragmented product from form I in the purification steps.

Immunochemical studies of human urinary trypsin inhibitor.

Antisera against purified urinary trypsin inhibitor (UTI-I, molecular weight 67,000) and UTI-III (molecular weight 23,000) were first produced in rabbits. Both anti-UTI-I and anti-UTI-III sera formed a single immunoprecipitin line with human plasma inter-alpha-trypsin inhibitor (I alpha TI), whereas two immunoprecipitin lines were formed with crude urine. It was speculated that both UTI-I and UTI-II might be present in normal human urine. In the present study, the inhibitory effects of anti-UTI sera on UTI activity were examined by three different assay methods. The results indicated that the inhibitory effect was almost immediate. Although the inhibitory effect of anti-UTI-III serum on UTI-III was almost of the same degree of completeness for the three assay methods. UTI-I was partially inhibited by the anti-UTI-I serum when residual trypsin activity was measured by the caseinolytic or fibrinolytic assay method. This discrepancy was considered to be due to the difference in conformational change between UTI-I and UTI-III by antigen-antibody reaction.

Purification and some properties of a plasma protein which reacts with an antiserum against urinary trypsin inhibitor-1.

A plasma protein which forms a precipitation line with antiserum against urinary trypsin inhibitor was purified by ammonium sulphate fractionation, anti-urinary trypsin inhibitor rabbit IgG antibody coupled sepharose immunoadsorbent column chromatography and polyacrylamide-gel disc electrophoresis. By these procedures, 0.14 mg of purified material was obtained from 20 ml of plasma. It was homogeneous as ascertained by sodium dodecyl sulphate polyacrylamide-gel disc electrophoresis and had an apparent molecular weight of 90,000. It was found that this inhibitor was immunologically distinct from inter-alpha-trypsin inhibitor.

Inhibitors of acrosin and SH-protease in normal human urine.

Abstract Trypsin inhibitors isolated from human urine and highly purified by affinity chromatography displayed molecular weights of 43,000 (H-UTI) and 22,000 (L-UTI) during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These molecules not only inhibited trypsin [EC 3.4.21.4], chymotrypsin [EC 3.4.21.1], and plasmin [EC 3.4.21.7], but also showed strong inhibitory effects on the human sperm enzyme, acrosin [EC 3.4.21.10]. A novel protease inhibitor (anticin) which specifically reacted with SH-proteases, such as ficin [EC 3.4.22.3], papain [EC 3.4.22.2], and bromelain [EC 3.4.22.4], was also isolated from normal human urine.

Isolation of an Elastase-Like Enzyme from Skin Lesions with Sweet’s Syndrome

In the present study, an elastase-like enzyme which possessed elastolytic, proteolytic and Nα-Acetyl-L-tyrosine ethyl ester hydrolytic activities was extracted from skin lesions with Sweet’s syndrome.

Fibrinolytic and coagulation activity level during formation of experimental thrombus in dog's saphena vein

Abstract We could success in formation of the experimental thrombus by direct insertion of bovine fibrinogen into the lateral saphena vein of the dog. This method enabled us to clearly confirm the thrombus formation by X-ray micrograph. It was confirmed that this operation did not affect fibrinolytic and coagulation factors of experimental dogs. Reproducibility and stability of the result were also confirmed. So this experimental thrombus formation model is versatile for pharmacodynamic screening tests of the fibrinolytic enzyme system.

Studies on a kallikrein-kinin system in plasma of patients with acute pancreatitis: The preparation and characterization of a kallikrein-like enzyme in patient's plasma

In a previous paper, the authors reported that the kallikrein-like activity was eluted in alpha 2-macroglobulin fractions when patients 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.

Immunochemical determination of the serum protein reacting with antibody against human urinary trypsin inhibitor by single radial immunodiffusion: use of polyethylene glycol

A single radial immunodiffusion method is described for determining the serum protein which reacts with antibody against the urinary trypsin inhibitor, but does not react with anti-inter-alpha-trypsin inhibitor antibody. Since the amount of this protein could not be determined with an agar plate containing antibody alone, we first prepared an agar plate containing 4% polyethylene glycol 6 000 and 1 mg of anti-urinary trypsin inhibitor gamma-globulin and confirmed that the amount of this protein could be measured accurately from the sizes of precipitin rings after 72 h incubation at room temperature. Levels of this serum protein, alpha 1-antitrypsin, alpha 2-macroglobulin and inter-alpha-trypsin inhibitor were measured in normal human serum by the single radial immunodiffusion method, and it was confirmed that the level of this serum protein did not correlate with the levels of these 3 other proteins.

Multiple molecular forms of acid-stable plasmin inhibitor derived from human urinary trypsin inhibitor.

It was found that cyanogen bromide (BrCN) treatment of the highly purified human urinary trypsin inhibitors (H-UTI; specific activity 1,897 U/mg protein, and L-UTI; specific activity 1,850 U/mg protein) readily produced new plasmin inhibitors with almost no loss of UTI activity. Five multiple forms of chemically cleaved inhibitors (UTIB-I, UTIB-II, UTIB-III, UTIB-IV and UTIB-V) could be isolated from BrCN-treated L-UTI by isoelectric focusing and gel filtration. These inhibitors were very acid-stable and their isoelectric points (pI) were 4.5, 4.6, 4.9, 5.1 and 6.4, respectively. The molecular weights by SDS-polyacrylamide gel electrophoresis were almost the same at about 23,000 +/- 3,000. Although these inhibitors showed both anti-plasmin and anti-trypsin activities, much higher anti-plasmin/anti-trypsin activities were observed in the cleaved inhibitors than in the parent UTI. They competitively inhibited human plasmin with Ki values of 3.0-4.1 X 10(-8) mol/l (H-D-Val-Leu-Lys-pNA substrate).