Low Molecular Weight Trypsin-Plasmin Inhibitors Isolated from Papain Treated Urinary Trypsin Inhibitor

Abstract

SummaryPapain treatment of human urinary trypsin inhibitor (UTI67; mol. wt. 43,000 by SDS-polyacrylamide gel electrophoresis, specific activity 1,897 U/mg protein) produced four new protease inhibitors, which were highly purified by gel chromatography on Sephadex G-100 and isoelectric focusing. The purified inhibitors (UTI26, UTI9-I, UTI9-II, and UTI9-III) were shown to be homogeneous by polyacrylamide disc gel electrophoresis, and had apparent molecular weights of 26,000, 9,000, 9,000, and 9,800, respectively, by sodium dodecyl sulfate gel electrophoresis. During enzymatic degradation of UTI67, the amino acid compositions changed to more basic, and the isoelectric point increased from pH 2.0 (UTI67) to pHs 4.4, 5.2, 6.6, and 8.3 (UTI26, UTI9-I, UTI9-II, and UTI9-III), respectively. Both the parent and degraded inhibitors had anti-plasmin activity as well as antitrypsin and anti-chymotrypsin activities. Much higher anti-plasmin/anti-trypsin and anti-plasmin/anti-chymotrypsin activities were observed in the degraded inhibitors than in the parent UTI67. They competitively inhibited human plasmin with Ki values of 1.13 X 10-7 - 2.12 X 10-6 M (H-D-Val-Leu-Lys-pNA substrate). The reactions were very fast and the active site of the inhibitors to plasmin was thought to be different from that to trypsin or chymotrypsin.

Trinitrophenyl-Derivative of Urinary Trypsin Inhibitor: A Strong Antifibrinolytic Agent

“…Although we have previously prepared several modified UTIs [2,[6][7][8], none was found to be such a strong antifibrinolytic agent as TNP-UTI. The com pound 2,4,6-trinitrobenzenesulfonic acid was devel oped by Okuyama and Satake [16] as a reagent for the modification of the free amino acid groups of several proteins.…”

“…Urokinase MM 004, Japanese standard preparation, was employed as a standard. The amidolysis by plasmin, UK and TPA was estimated colorimetrically with 5xlO~4M H-D-Val-Leu-Lys-pNA, pyro-Glu-Gly-ArgpNA, and H-D-Ile-Pro-Arg-pNA, respectively, in 50mmol/l Tris-HC1 buffer containing 0.1 mol/l NaCI, pH 7.4 [6,12]. Protein con centration was determined by the method of Lowry et al [13] using bovine serum albumin (Armour Pharmaceutical Co.) as a standard.…”

“…Digestion of UTI with papain [6] and BrCN [7] readily changes the inhibitory spectrum; the active fragments pro duced reveal a stronger antiplasmin amidolysis than the intact UTI. However, with the natural substrate, fibrin, both UTI and its fragments showed much lesser antiplasmin activity [8].…”

Fibrin-Binding Urokinase (or Precursor Form of Urokinase) with a Molecular Weight of About 100,000 in Fresh Human Urine

“…), Sepharose 4B (Pharmacia), bovine thrombin, 90% pure (Mochida), bovine plas minogen-rich fibrinogen (Povite), urokinase (Otsuka) and agarose-L (Behringwerke). Lys-plasmin, prepared as described previously [27], revealed a specific activ ity of 25 CU/mg protein.…”

Multiple Molecular Forms of Acid-Stable Plasmin Inhibitor Derived from Human Urinary Trypsin Inhibitor

“…The weight modification was considered to be attribut able to a pepsin-like enzyme present in the urine which could be activated under acid Urinary trypsin inhibitor (UTI) is ex creted in increased amounts in various phys iological and pathological states, especially in shock, acute infectious diseases, advanced tu berculosis, rheumatic fever, renal stones, hy dronephrosis, chronic glomerulonephritis, and cancer [1][2][3][4][5][6][7], Recently, employing the conditions below pH 3.5. Enzymatic modifi cations of UTI were also possible in purified systems with papain and pronase [10,11], The properties of highly purified UTI-I (H-UTI), UTI-III (L-UTI) [12][13][14][15][16] and papain digested low molecular weight inhibitors [ 17] have been reported previously.…”