Gabriella Szabo

Investigation of the substrate-binding site of human plasmin using tripeptidyl-p-nitroanilide substrates

Abstract The hydrolysis of tripeptidyl-p-nitroanilides by human plasmin /EC 3.4.21.7./ was studied and the kinetic parameters were determined. The individual contributions of the amino acid side chains at the P 1 -P 4 subsites to the kinetic parameters were calculated by regression analysis. The highest contributions yielded the structure of an “optimum” substrate, D-Ile-Phe-Lys-pNA. Its predicted kinetic parameters, K m = 9.6 × 10 -6 M and k cat /K m = 284 789 M -1 s -1 , appeared to be about 40 times as good as those of H-D-Val-Leu-Lys-pNA /S-2251/ applied for the determination of the plasminogen and plasmin content of blood in various laboratories. At the S 2 -S 4 segment in the binding site, which interacts with the P 2 -P 4 moieties of the substrate, plasmin favoured uniformly hydrophobic substituents.

Mapping of the substrate-binding site of the human granulocyte elastase by the aid of tripeptidyl-p-nitroanilide substrates

The kinetic properties of the human granulocyte elastase /EC 3.4.21.11/ were investigated with 24 tripeptidyl-pNA substrates. By the regression analysis of the kinetic data obtained with 15 substrates a relatively hydrophobic compound, Boc-D-Phe-Ala-Nle-pNA, was predicted as the optimal substrate sequence. The compound was synthesized, assayed and the predicted Km = 4.2 uM was confirmed experimentally. The substrate-binding site of granulocyte elastase appeared to be hydrophobic and very much similar to that of the pancreatic enzyme at the S2–S4 subsites, but the S1 subsite, which determines the primary specificity, could accomodate bulkier residues and it was less selective than that in the pancreatic enzyme.

Disassembly of chromatin into ≅50 kb units by detergent

Abstract Nuclei isolated from higher eukaryotic cell lines were directly analyzed by field inversion gel electrophoresis. Brief incubation of nuclei with ionic detergents yielded a single band between 50–100 kb. The apparent fragment size decreased to ≅50 kb after proteinase digestion. The latter treatment alone induced less regular, ≦50 kb fragmentation. DNA extracted from detergent and proteinase-treated nuclei also appeared in a band of about 40 kb. Embedding into agarose plugs did not protect nuclei, as opposed to cells, from detergent-induced fragmentation. The phenomenon is strikingly analogous to the double-strand DNA cleavage reactions mediated by topoisomerase II. Our data are compatible with any of the following interpretations: 1.) regularly spaced protein bridges, probably involving topoisomerase II, maintain or control continuity of chromosomal DNA in certain states of higher eukaryotic cells. 2.) The DNA might become accessible to a putative endonuclease at regularly spaced sites upon detergent treatment of isolated nuclei.

Effect of some new thioglycosides on endotoxin-induced disseminated intravascular coagulation in rabbits ☆

Disseminated intravascular coagulation (DIC) is a systemic thrombohemorrhagic disorder seen in association with many clinical situations, e.g. sepsis, malignancy, obstetrical complications and intravascular hemolysis. In our model, disseminated intravascular coagulation was induced in rabbits by two consecutive intravenous bolus injections of endotoxin from Escherichia coli, 80 and 40 microg/kg. The control group was treated with 0.9% saline. The activity of thioglycosides was compared to unfractionated heparin (UFH) and efegatran with and without administration of endotoxin. Drugs were administered in the following doses: heparin 50 and 100 IU/kg/h i.v. infusion; efegatran 0.25 and 0.5 mg/kg/h i.v. infusion; GYKI 39521 (RGH-1875) as well as GYKI 39541 (RGH-1962) 12.5 and 25 mg/kg per os. Thioglycosides did not modify coagulation parameters in this model [prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT)] as compared with endotoxin/vehicle group. The changes in TFPI level after administration of thioglycosides and heparin were similar in the mentioned model to those without endotoxin. Endotoxin-induced changes of leukocyte count were not affected by GYKI 39521 and GYKI 39541 treatment in our model. Diminution of fibrinogen level and platelet count was prevented by GYKI 39521 and GYKI 39541. Fibrin degradation products and fibrinolysis were significantly decreased by GYKI 39521 and GYKI 39541. The thioglycosides may have a lower risk of bleeding in the treatment of disseminated intravascular coagulation than heparin.

Active Site-Directed Thrombin Inhibitors: α-Hydroxyacyl-Prolyl-Arginals, New Orally Active Stable Analogues of D-Phe-Pro-Arg-H

D-alpha-Hydroxyacyl-prolyl-arginals, a new type of analogues of D-Phe-Pro-Arg-H (R1), have been prepared and evaluated. Unlike R1, whose terminal group is NH2, the new analogues with a terminal OH group are stable, as are the N-substituted derivatives of R1, that is, D-MePhe-Pro-Arg-H (R2), the highly potent and selective thrombin inhibitor, and Boc-D-Phe-Pro-Arg-H (R3), the much less favorable analogue. The most notable of the new analogues corresponds to the general formula D-Xaa-Pro-Arg-H, wherein Xaa means the acyl residue of mandelic acid (Man, 1), diphenyllactic acid (Dpl, 2), hexahydrophenyllactic acid (Hpl, 3), or hexahydromandelic acid (Hma, 4). In plasma clotting assays, 1 to 4 appeared to inhibit thrombin as well as some other clotting enzymes involved in thrombin generation, whereas R1 and R2 seemed to produce anticoagulation through inhibition of thrombin only. In the fibrin plate assay, 1 to 4 possessed even more moderate antifibrinolytic activities than R2. In in vivo evaluation in rats and rabbits, 2 to 4 proved to be potent anticoagulants/antithrombotics even on oral administration in a dose of 5 mg/kg. In view of these findings with the alpha-hydroxyacyl-prolyl-arginals, it is very likely that the less favorable biologic properties of Boc-D-Phe-Pro-Arg-H are due to the hydrophobicity and bulkiness of the terminal Boc-NH rather than its neutrality.