Wolfgang A. Günzler

ASSAYS OF GLUTATHIONE PEROXIDASE

Publisher Summary To determine glutathione peroxidase reliably, some factors of potential pitfall have to be considered, for example, enzymatic side reactions of substrates (especially when crude tissue samples are assayed), high and variable spontaneous reaction rates of substrates, and the peculiar kinetics of the enzyme itself. With the best documented example, the enzyme of bovine red blood cells, ping-pong kinetics with infinite limiting maximum velocities, and Michaelis constants have been established. This means that the generally recommended conditions for determination of enzyme activity––that is, “saturating” concentrations of all substrates, cannot possibly be fulfilled. In consequence, compromises are inevitable in the choice of substrate concentration for the assay and in the definition of the unit of activity. Fixed-time assay measuring H 2 O 2 consumption and continuous monitoring of Glutathione disulfide (GSSG) formation are cited here. The main differences between the assay procedure described and those proposed by others are listed in the chapter. To compare the results obtained by different procedures, appropriate empirical converting factors are also given.

Cathepsin B efficiently activates the soluble and the tumor cell receptor-bound form of the proenzyme urokinase-type plasminogen activator (Pro-uPA).

Action of purified human cathepsin B on recombinant single-chain urokinase-type plasminogen activator (pro-uPA) generated enzymatically active two-chain uPA (HMW-uPA), which was indistinguishable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot from plasmin-generated HMW-uPA and from elastase- or thrombin-generated inactive two-chain urokinase-type plasminogen activator. Preincubation of cathepsin B with E-64 (transepoxysuccinyl-L-leucylamino- (4-guanidino)butane, a potent inhibitor for cathepsin B) prior to the addition of pro-uPA prevented the activation of pro-uPA. The cleavage site within the cathepsin B-treated urokinase-type plasminogen activator (uPA) molecule, determined by N-terminal amino acid sequence analysis, is located between Lys158 and Ile159. Pro-uPA is cleaved by cathepsin B at the same peptide bond that is cleaved by plasmin or kallikrein. Binding of cathepsin B-activated pro-uPA to the uPA receptor on U937 cells did not differ from that of enzymatically inactive pro-uPA, indicating an intact receptor-binding region within the growth factor-like domain of the cathepsin B-treated uPA molecule. Not only soluble but also tumor cell receptor-bound pro-uPA could be efficiently cleaved by cathepsin B to generate enzymatically active two-chain uPA. Thus, cathepsin B can substitute for plasmin in the proteolytic activation of pro-uPA to enzymatically active HMW-uPA. In contrast, no significant activation of pro-uPA by cathepsin D was observed. As tumor cells may produce both pro-uPA and cathepsin B, implications for the activation of tumor cell-derived pro-uPA by cellular proteases may be considered.

Effective activation of the proenzyme form of the urokinase‐type plasminogen activator (pro‐uPA) by the cysteine protease cathepsin L

Increased levels of both the cysteine protease, cathepsin L, and the serine protease, uPA (urokinase‐type plasminogen activator), are present in solid tumors and are correlated with malignancy. uPA is released by tumor cells as an inactive single‐chain proenzyme (pro‐uPA) which has to be activated by proteolytic cleavage. We analyzed in detail the action of the cysteine protease, cathepsin L, on recombinant human pro‐uPA. Enzymatic assays, SDS‐PAGE and Western blot analysis revealed that cathepsin L is a potent activator of pro‐uPA. As determined by N‐terminal amino acid sequence analysis, activation of pro‐uPA by cathepsin L is achieved by cleavage or the Lys158‐lle159 peptide bond, a common activation site of serine proteases such as plasmin and kallikrein. Similar to cathepsin B (Kobayashi et al., J. Biol. Chem. (1991) 266, 5147‐5152) cleavage of pro‐uPA by cathepsin L was most effective at acidic pH (molar ratio of cathepsin L to pro‐uPA of 1:2,000). Nevertheless, even at pH 7.0, pro‐uPA was activated by cathepsin L, although a 10‐fold higher concentration of cathepsin L was required. As tumor cells may produce both pro‐uPA and cathepsin L, implications for the activation of tumor cell‐derived pro‐uPA by cathepsin L may be considered. Different pathways activation of pro‐uPA in tumor tissues may coexist: (i) autocatalytic intrinsic activation of pro‐uPA; (ii) activation by serine proteases (plasmin, kallikrein. Factor XIIa); and (iii) activation by cysteine proteases (cathepsin B and L).

Adaptation of plasminogen activator sequences to known protease structures

The sequences of urokinase (UK) and tissue‐type plasminogen activator (TPA) were aligned with those of chymotrypsin, trypsin, and elastase according to their ‘structurally conserved regions’. In spite of its trypsin‐like specificity UK was model‐built on the basis of the chymotrypsin structure because of a corresponding disulfide pattern. The extra disulfide bond falls to cysteines 50 and 111d. Insertions can easily be accommodated at the surface. As they occur similarly in both, UK and TPA, a role in plasminogen recognition may be possible. Of the functional positions known to be involved in substrate or inhibitor binding, Asp 97, Lys 143 and Arg 217 (Leu in TPA) may contribute to plasminogen activating specificity. PTI binding may in part be impaired by structural differences at the edge of the binding pocket.

Effects of changing liver blood flow by exercise and food on kinetics and dynamics of saruplase

To investigate the influence of changes in liver blood flow on the pharmacokinetics and pharmacodynamics of single‐chain unglycosylated urokinase‐type plasminogen activator.

Activity and antigen of saruplase and two chain urokinase related plasminogen activator are stabilized by a combination of aprotinin and benzamidine in citrated plasma

Abstract Complete recovery of rscu-PA- and /or tcu-PA-activity or antigen as measured by Immune Activity Assay or by ELISA respectively, requires citrated plasma containing benzamidine and aprotinin.

Plasma markers of thrombin activity during coronary thrombolytic therapy with saruplase or urokinase: no prediction of reinfarction

Abstract One of the principal problems associated with thrombolytic therapy is rethrombosis of vessels which were initially patent. Although platelets as well as coagulation activation have been implicated in rethrombosis, the specific mechanisms leading to this complication are still unclear. Available evidence is limited to smaller studies using the current thrombolytic agents. Here we report on the multicentre SUTAMI trial comparing recombinant saruplase and urokinase in 543 patients with acute myocardial infarction, in 33 of whom early reinfarction was documented. Plasma from these patients and 33 matched patients without reinfarction was investigated for thrombin-antithrombin III complex and prothrombin activation fragments 1 + 2 as markers of activated coagulation, during 72h after starting the lytic therapy. Both drugs caused considerable systemic degradation of fibrinogen and the degree of systemic lysis was very similar. The median concentrations of both thrombin-antithrombin III complex and prothrombin fragments 1 + 2 significantly increased 3- to 6-fold after the therapy, indicating extensive activation of the coagulation system. Following heparin administration, both parameters returned towards normal in most patients. At no time points studied was there any significant difference in these coagulation parameters between the patients with and those without reinfarction. In contrast to other findings, thrombin-antithrombin III complex concentration was not a useful indicator of reinfarction in the patients studied and neither was the concentration of prothrombin activation fragments 1 + 2.

Computer Aided Protein Design: Three Dimensional Model Building of the Saruplase Structure

Modelling studies of the three-dimensional structures of the saruplase-domains are presented. The model of the N-terminal EGF-like domain highlights amino acids residues which might be involved in interactions with saruplase specific receptors. The distribution of charged residues on the surface of the kringle-model is different from other kringle- structures. The model structure of the catalytic serine-protease domain points to surface loops, which surround the active site and may participate in interactions with plasminogen. Starting from the structures of the isolated domains a model for the entire enzyme is constructed which is compatible with experimental results.

Enzymatic Defense Systems Against Hydroperoxides and Oxygen-Centered Radicals in Mammals: Glutathione Peroxidase and Superoxide Dismutases

Hydrogen peroxide, other hydroperoxides, superoxide radicals and further radicals derived therefrom are normally formed in the organism, but, in case of unbalanced production, they play a deleterious role. The most topical pathological events putatively attributed to hydroperoxides and oxygen-centered radicals are mutagenesis, inflammation and reperfusion injury. If produced simultaneously, hydroperoxides and superoxide interact with each other to give an aggressive mixture of pro-oxidant compounds including the hydroxyl radical. Overexposure to such pro-oxidant compounds is also referred to as “oxidative stress” (Sies, 1985).