Gerd Reinhardt

Human pancreatic secretory trypsin inhibitor (PSTI) produced in active form and secreted from Escherichia coli

As a basis for a protein design project, we decided to produce the human pancreatic secretory trypsin inhibitor (PSTI) in its active form. Total gene synthesis was carried out efficiently by (i) computer design of the gene fragments, (ii) synthesis of the oligodeoxynucleotides by the segmental support method, and (iii) assembly of double strands under optimized ligation conditions. Fusion to the ompA gene signal peptide led to secretion of processed PSTI in various constructions, with or without additional amino acids (aa) at the N-terminus. The secreted proteins (56 to 63 aa) were biologically active, suggesting that the three cysteine bridges were correctly formed. Surprisingly, after induction the product was found almost exclusively in the culture medium. Variants of PSTI with Asp or Asn at aa positions 21 and 29 [sequences published by Greene et al., Methods Enzymol. (1976) 813-825, and by Yamamoto et al., Biochem. Biophys. Res. Commun. (1985) 605-612] showed the same Ki for both human and porcine trypsin.

α‐HALOGENMETHYL CARBONYL COMPOUNDS AS VERY POTENT INHIBITORS OF FACTOR XIIAIN VITRO

Fibrin stabilization inhibitors can be subdivided grossly into two categories: one group blocks the crosslinking enzyme (F XIIIa) and the other one blocks the substrate (fibrin monomers). F XIIIa is known to be a sulfhydryl enzyme.’ Therefore, “sulfhydryl reagents” such as organomercurials, disulfides, and iodoacetic acid belong into the first category. They are able to form a covalent bond with the active site of the enzyme and thus inhibit its activity. These substances do not specifically react with F XIIIa and so high concentrations are necessary to obtain a considerable inhibition. To elucidate the action of the second group of substances, a schematic representation of the chemical events that occur during the crosslinking process is given in FIGURE 1. Initially, the active SH-group of the enzyme reacts with a glutamine residue of one fibrin molecule to form a thio ester (‘3-acyl enzyme”); ammonia is released in this step. The second step involves the aminolysis of the thio ester by a lysine residue of a second fibrin molecule releasing the free enzyme. This results in the covalent linkage of the two fibrin molecules. Since every fibrin molecule has several of such crosslinking sites, an indefinite network of covalently connected fibrin molecules is formed (fibrin polymer). Most of the compounds belonging to the second category of inhibitors have a primary amino group that is able to compete for the lysine residues during the aminolysis of the thio ester intermediate. These “competitive substrates” terminate the polycondensation reaction chain by rendering the crosslinking sites in the fibrin monomers unsusceptible to further reaction. Consequently, the formation of polymeric fibrin is prevented. As shown in FIGURE 2, the most efficient substrate competitors are structurally related to the lysine residue, which is the natural substrate. The aromatic nucleus and the -SO,-group are of additional importance. Obviously this combination provides a primary interaction between these molecules and the enzyme molecules, the exact nature of which is not known. A compilation of the molecular features of this class of substances has been given by Lorand and Nilsson.2 These compounds can be regarded as quite specific. Nonetheless, high concentrations must be available to produce a measurable inhibition, since they have to compete with the substrate, which itself is present in high concentration compared with that of the enzyme, To produce more effective inhibitors of the fibrin stabilization, the notion was had of combining the selectivity of the substrate-like amines with the chemical reactivity of the sulfhydryl reagents. As the result of these considerations, the general structure given in FIGURE 3 was designed. The group R’-X(with an aromatic nucleus in the position R’ and -X-=SO,-) represents

Fibrin oligomers of high molecular weight soluble in mca solutions: Implications on the time course of fibrinI formation

Abstract In gel filtration experiments, it was possible to demonstrate that the largest oligomers of fibrin still soluble in MCA solution contain at least 50 monomeric units. In the case of SDS-PAGE, it was also established that, after incubating fibrin s with F-XIIIa, the fibrin fraction, remaining soluble in MCA solution, has γ-chains which are largely dimeric. From these data we concluded that a critical minimal size is necessary to produce insoluble fibrin. On this basis, a quantitative approach has been attempted to describe the course of fibrin stabilization which finally yields a mathematical function connecting the amount of fibrini and the product of reaction time and enzyme concentration. The pattern of fibrini formation includes a latency phase which is followed by a relatively rapid increase in fibrini concentration. This phenomenon, already observed and interpreted by Loewy et al (6) in 1961, is easily explained by this function. Also, the influence of the critical minimal size of the oligomers, the substrate concentration, and the enzyme parameters (formation-constant and rate-constant) are discussed on the basis of this function.