Gene cloning and heterologous expression of a serine protease fromStreptomyces fradiaevar.k11

The gene sfp1, which encodes a predicted serine proteinase designated SFP1, was isolated by the screening of a gene library of the feather-degrading strain Streptomyces fradiae var.k11. The open reading frame of sfp1 encodes a protein of 454 amino acids with a calculated molecular mass of 46.19 kDa. Sequence analysis reveals that SFP1 possesses a typical pre-pro-mature organization that consists of a signal sequence, an N-terminal propeptide region, and a mature proteinase domain. The pre-enzyme of SFP1 was expressed in Escherichia coli and consequently purified. The 25.6 kDa fraction with protease activity separated by gel filtration chromatography indicated that the mature enzyme of SFP1 was formed by autolysis of the propeptide after its expression. The purified SFP1 is active under a broad range of pH and temperature. SFP1 has pH and temperature optima of pH 8.5 and 65 °C for its caseinolytic activity and pH 9 and 62 °C for its keratinolytic activity. SFP1 was sharply inhibited by the serine proteinase inhibitor phenylmethyl sulfonyl fluoride and exhibited a good stability to solvents, detergents, and salts. Comparison of the protease activity of SFP1 with other commercial proteases indicates that SFP1 has a considerable caseinolytic and keratinolytic activity as does proteinase K.

Identification and characterization of gelatin-cleavage activities in the apically located extracellular matrix of the sea urchin embryo

We have identified and partially characterized several gelatinase activities associated with the sea urchin extraembryonic matrix, the hyaline layer. A previously identified 41-kDa collagenase/gelatinase activity was generally not found to be associated with isolated hyaline layers but was dissociated from the surface of 1-h-old embryos in the absence of Ca2+ and Mg2+. While hyaline layers, freshly prepared from 1-h-old embryos, were devoid of any associated gelatinase activities, upon storage at 4°C for 4 days, a number of gelatin-cleavage activities appeared. Comparative analysis of these activities with the 41-kDa collagenase/gelatinase revealed that all species were inhibited by ethylenediamine tetraacetic acid but were refractory to inhibition with the serine protease inhibitors, phenylmethyl sulfonyl fluoride and benzamidine. In contrast, the largely Zn2+ specific chelator 1,10-phenanthroline had markedly different effects on the gelatinase activities. While several of the storage-induced, hyaline-layer-associated gelatinase activities were inhibited, the 41-kDa collagenase/gelatinase was refractory to inhibition as was a second gelatinase species with an apparent molecular mass of 45 kDa. We also examined the effects of a series of divalent metal ions on the gelatin-cleavage activities. In both qualitative and quantitative assays, Ca2+ was the most effective activator while Mn2+, Cu2+, Cd2+, and Zn2+ were all inhibitory. In contrast, Mg2+ had a minimal inhibitory effect on storage-induced gelatinase activities but significantly inhibited the 41-kDa collagenase/gelatinase. These results identify several distinct gelatin-cleavage activities associated with the sea urchin extraembryonic hyaline layer and point to diversity in the biochemical nature of these species.Key words: gelatinase, sea urchin, extracellular matrix.

Hemolytic and Elastolytic Activities Influenced by Iron in Plesiomonas shigelloides

The aim of this study was to determine the presence of hemolytic and elastolytic enzymes in several strains of Plesiomonas shigelloides in relation to the availability of iron in culture media. Hemolytic activity and elastolytic activity were detected in strains of P. shigelloides and were enhanced when the strains were grown in an iron-depleted medium and lost after thermal treatment at 100°C for 10 min. Also, elastolytic activity was inactivated by phenylmethyl sulfonyl fluoride, an inhibitor of serine proteases. Hemolytic activity was detected extracellularly in cell-free supernatants, whereas elastin degradation activity was cell associated. Both activities may be related to the virulence of P. shigelloides.

Protection of Platelet Surface Bound Factors IX a and VIII Against Specific Inhibitors

SummaryThe rate of inactivation of factors IX a and VIII as well as of the complex formed by them on the surface of platelets, induced by specific inhibitors has been studied. After the formation of complex both factors became more protected against high molecular weight inhibitors. As evidenced by the apparent first order inactivation rate constants, after complex formation the velocity of IX a – antithrombin III + heparin reaction fell to one-fourth, that of factor VIII – VIII : C antibody reaction decreased to 1/20. Enhanced resistance toward the high molecular weight inhibitors is presumably due to a steric change, since the low molecular weight phenylmethyl sulfonyl fluoride and diethyl pyrocarbonate inhibited free and complexed factor IX a to the same extent.

Defective Thrombin Binding by Abnormal Fibrin Associated with Recurrent Thrombosis

Studies of clotting activity and radioactivity (with 125I-thrombin) indicated that binding of human thrombin to fibrin depends upon the initial concentrations of the reactants. Scatchard analysis suggests two classes of binding site: a high affinity site with a Ka 5.8±0.9 x 105M-1 and a maximum molar binding ratio of thrombin to fibrin 0.4±0.2, and a low affinity site with a Ka 6.8±0.6 x 104M-1 and a maximum molar binding ratio of 1.6±0.5. The active site of thrombin is not required for binding since neutralization with phenylmethyl sulfonyl fluoride does not affect the binding. Thrombin also binds to fibrin formed in whole blood and can be recovered with full clotting activity when the clot is dissolved by plasmin, suggesting that binding of thrombin to fibrin is a potential mechanism for limiting thrombosis. Fibrinogen New York I, in a patient with recurrent venous and arterial thrombosis, was characterized by abnormalities in fibrinopeptide release by thrombin and fibrin polymerization. The patient’s plasma contained equal amounts of normal and abnormal fibrinogen. Thrombin was bound normally by fibrin derived from the normal fibrinogen but was not bound at all by fibrin from the abnormal fibrinogen. Fibrinogen New York II, in an asymptomatic patient, was characterized by delayed fibrinopeptide release and fibrin polymerization. Thrombin was bound normally by this fibrin. Thus defective binding of thrombin by fibrin is suggested as a new mechanism predisposing to recurrent thrombosis.