Prothrombin--substrate for blood plasma kallikrein and factor XIIa

A study was made of the interaction between prothrombin and enzymes: blood plasma kallikrein and factors alpha-XIIa and beta-XIIa immobilized on enzacryl-AH. Kallikrein-induced prothrombin proteolysis was accompanied by a decrease in prothrombin activity, appearance of BAME-esterase and poor clotting activity. As a result of fractionation of products on the column with DEAE-Sephadex A-50, some fractions that have thrombin amidase activity (splitting of the substrate S-2238) and high antithrombin activity were obtained. Antithrombin activity manifested in the inhibition of fibrinmonomer aggregation during fibrin formation. During incubation with prothrombin, factors alpha-XIIa and beta-XIIa also stimulated the appearance of BAME-esterase activity. None of the immobilized enzymes activated factor X.     

Use of a chromatographic method for determining kallikrein and prekallikrein in dog blood serum

The authors determined the conditions of chromatography to be used for determination of prekallikrein and kallikrein in the dog blood serum. The enzyme and its precursor do not adsorb on DEAE-Sephadex A-50 in a 0.02 M phosphate buffer, pH 7, containing 0.15 M NaCl, and thus their quantity can be determined in a non-adsorbed protein fraction from the rate of hydrolysis of N-benzoyl-L-arginine ethyl ester before and after exposure to trypsin. In 1 ml of the dog blood serum, kallikrein activity ranged from 0 to 60 mE, while prekallikrein activity from 127 to 187 mE.     

Method of simultaneous determination of kallikrein, plasmin and thrombin precursors and inhibitors in human blood plasma]

Prekallikrein, plasminogen and prothrombin of human blood plasma have been separately activated by caolin streptokinase and thromboplastin. By measuring the TAME-esterase (N-d Tozy-L-arginine methyl ester) activity of each enzyme and its changes in the course of plasma incubation with the activator, it was possible to estimate the values of precursors of kallikrein, plasmin, thrombin and their inhibitors. Evidence is given that under conditions described the activation is specific of each enzyme and does not affect the level of the two other percursors. The method has been developed in two modifications, permitting to obtain the value of seven parameters in 0.4--0.7 ml of blood plasma.     

The activation of pro-urokinase by plasma kallikrein and its inactivation by thrombin

Plasma kallikrein was found to be a good activator of pro-urokinase, the inactive zymogen form of urokinase. The complete activation of pro-urokinase by plasma kallikrein was obtained in 2 h with an enzyme/substrate weight ratio of 1/30. The rate of activation of pro-urokinase by plasma kallikrein was comparable to that catalyzed by plasmin and trypsin. The rate of activation of pro-urokinase by factor XIIa was approximately one-seventh of that by plasma kallikrein. The activation of the zymogen was due to the cleavage of a single internal peptide bond, resulting in the conversion of a single chain pro-urokinase (Mr = 55,000) into two-chain urokinase (Mr = 33,000 and 22,000), and these two chains were linked by a disulfide bond(s). These results indicate an important role of plasma kallikrein for the activation of pro-urokinase in the factor XII-dependent intrinsic pathway of fibrinolysis. Thrombin also converted pro-urokinase to a two-chain form that was not activatable by plasmin, plasma kallikrein, and factor XIIa. Thrombin specifically cleaved the Arg 156-Phe 157 bond which is located 2 residues prior to the activation site of Lys 158-Ile 159.     

Linkage between blood coagulation and inflammation: stimulation of neutrophil tissue kallikrein by thrombin

There has been major interest in the potential interaction between blood coagulation and inflammation. Most of the effort has focused on cellular interactions involving platelets and polymorphonuclear leukocytes (PMNS). The recent discovery of tissue kallikrein(TK) activity in PMNs prompted the study of the possible role of thrombin(IIa) in this process. Human PMNs were isolated by density gradient centrifugation. Human IIa was compared with fMLP with respect to chemotaxis and enzyme release. Results from the challenges by IIa and fMLP were compared to a NaCl control using Student's paired t-test. IIa was a potent chemotactic agent for PMNs (p less than or equal to 0.0121) and stimulated the release of TK (p less than or equal to 0.0001) as determined by hydrolysis of S-2266. FMLP significantly stimulated PMN chemotaxis (p less than or equal to 0.0028) but had no effect on TK release. Release of TK was confirmed by Western Blot analysis and 35S-methionine incorporation into a 35 KD protein after IIa challenge. These results demonstrate that IIa is chemotactic for PMNs and can cause release of tissue kallikrein demonstrating a direct role for blood coagulation in the regulation of the inflammatory response.     

Interaction of human plasma kallikrein and its light chain with C1 inhibitor

The light chain of human plasma kallikrein contains the enzymatic active site. The inactivation of kallikrein and of its isolated light chain by C1 inhibitor was investigated to assess the functional contributions of the heavy-chain region of kallikrein and of high molecular weight kininogen to this reaction. The second-order rate constants for the inactivation of kallikrein or its light chain were respectively 2.7 X 10(6) and 4.0 X 10(6) M -1 min -1. High molecular weight kininogen did not influence the rate of kallikrein inactivation. The nature of the complexes formed between kallikrein or its light chain and C1 inhibitor was studied by using sodium dodecyl sulfate (SDS) gradient polyacrylamide slab gel electrophoresis. Kallikrein as well as its light chain combined with C1 inhibitor to form stable stoichiometric complexes that were not dissociated by SDS and that exhibited apparent molecular weights (Mr's) of 185 000 and 135 000, respectively, on nonreduced SDS gels. Reduction of the kallikrein-C1 inhibitor complex gave a band at Mr 135 000 that comigrated with the complex seen for the light chain-C1 inhibitor complex. During the inactivation of both kallikrein and its light chain, a Mr 94 000 fragment of C1 inhibitor was formed which was unable to inactivate or bind kallikrein or its light chain. Kallikrein inactivated by diisopropyl phosphofluoridate did not form SDS-stable complexes with C1 inhibitor. These results demonstrate that the functional binding site for C1 inhibitor is localized in the light chain of kallikrein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of rat plasma kallikrein

Kallikrein enzyme initially was isolated from rat plasma by passage of citrated plasma through a DEAE-Sephadex column at pH 7.2. The active fraction was purified to electrophoretic apparent homogeneity by precipitation to 60% ammonium sulfate saturation, sequential passage through DE-52 cellulose, Sephadex G-200 and SP-Sephadex columns and finally by chromatofocusing on a PBE-94 column. The kallikrein content of each fraction during purification was monitored on the synthetic substrate N-alpha-tosyl-L-arginine methyl ester (TAME) and by its ability to form kinin from heat-treated rat plasma. The molecular weight was estimated by gel filtration to be 50,000 and by SDS-gel electrophoresis 41,000. Multiple isozymic forms were obtained with pI values ranging from 4.2 to 5.0. The enzyme has a pH optimum of 8.3. The Km and Vmax values for TAME, Bz-pro-phe-arg-pNA and H-D-val-leu-lys-pNA were 1.6, 0.16 and 1.7 mM and 3.09, 0.96 and 0.25 microM/mg/min respectively. The enzyme was inhibited by soybean trypsin inhibitor but not by lima bean trypsin inhibitor.     

Interaction of human plasma kallikrein and its light chain with alpha 2-macroglobulin

Human plasma kallikrein participates in the contact activation system of plasma. The light chain of kallikrein contains the enzymatic active site; the heavy chain is required for binding to high molecular weight kininogen and for surface-dependent activation of coagulation. This study has examined the functional contributions of the heavy chain of kallikrein and of high molecular weight kininogen in the inactivation of kallikrein and of its isolated light chain by alpha 2-macroglobulin (alpha 2M). Irreversible inhibition was observed for both kallikrein and its light chain, with the initial formation of a reversible enzyme-inhibitor complex. The second-order rate constants for these reactions were 3.5 X 10(5) and 4.8 X 10(5) M-1 min-1 for kallikrein and its light chain, respectively. When present in excess, high molecular weight kininogen decreased the rate of kallikrein inactivation by alpha 2M, whereas the rate of inactivation of the light chain was unaffected by high molecular weight kininogen. Although at a drastically reduced rate, high molecular weight kininogen was cleaved by alpha 2M-bound kallikrein. Sodium dodecyl sulfate gradient polyacrylamide gel electrophoresis was used to study complex formation between alpha 2M and kallikrein or its light chain. Under reducing conditions, four kallikrein-alpha 2M complexes were observed. Three of these complexes consisted of alpha 2M and the light chain of kallikrein (Mr 123 000, 235 000, and 330 000). Two alpha 2M-kallikrein light chain complexes incorporated [3H]diisopropyl fluorophosphate ( [3H]DFP) whereas the Mr 330 000 complex did not react with [3H]DFP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Estrogen-dependent expression of the tissue kallikrein gene (Klk1) in the mouse uterus and its implications for endometrial tissue growth

Tissue kallikrein mK1 is a serine protease involved in the generation of bioactive kinins for normal cardiac and arterial function in the mouse. In the present study, the tissue kallikrein gene Klk1, which codes for mK1, was shown to be one of the most prevalent of the Klk gene species in the uteri of adult mice, and its mRNA level was significantly higher at estrus than at diestrus. Klk1 mRNA expression was enhanced in the uteri of ovariectomized mice receiving estradiol-17beta treatment. Both endometrial epithelial and stromal cells isolated from the mice exhibited Klk1 expression at detectable levels when cultured in the presence of estradiol-17beta. mK1 was characterized using the recombinant active enzyme. mK1 had trypsin-like activity with a strong preference for Arg over Lys in the P1 position, and its activity was inhibited by typical serine protease inhibitors. Casein, gelatin, fibronectin, collagen type IV, and high-molecular-weight kininogen were degraded by mK1. The single-chain tissue-type plasminogen activator was converted to the two-chain form by mK1. In addition, mK1 degraded insulin-like growth factor binding protein-3. The present data suggest that mK1 may be implicated in the growth of uterine endometrial tissues during the proliferative phase.     

Isolation of an enzymatically active tissue kallikrein from human seminal plasma by immunoaffinity chromatography

A tissue kallikrein from human seminal plasma was isolated by immunoaffinity chromatography and characterized. Its molecular mass was determined by gel filtration to be approximately 40000 Da. The enzyme preparation liberates kinin from human HMW kininogen (specific activity: 0.594 HMW kininogen-U/mg), lowers the blood pressure of dogs after intravenous injection (specific activity: 1740 biol. kallikrein unit/mg) and is strongly inhibited by aprotinin but not by soybean trypsin inhibitor. N alpha-Acetyl-L-phenylalanyl-L-arginine ethyl ester, D-valyl-L-leucyl-L-agrine ethyl ester and N-benzyloxycarbonyl-L-tyrosine p-nitrophenyl ester are cleaved with identical rates by the enzyme from human seminal plasma and human urinary kallikrein.     

The origin of the coelom in Brachiopoda and its phylogenetic significance

The origin of the mesoderm and the subsequent formation of the coelom in the larvae of the brachiopod species Notosaria nigricans and Calloria inconspicua is documented in detail at the ultrastructural level. During gastrulation, the blastocoel is completely displaced by the invaginating archenteron. Initial mesoderm formation was observed in late wedge-shaped to early three-lobed stages in both species. Proliferation of mesodermal cells from the archenteral epithelium mainly occurs in the dorsolateral (C. inconspicua) and caudolateral (N. nigricans) parts of the archenteral wall. Thus, a compact mesodermal cell mass pushes its way towards the subepidermal basal lamina. During further development of the larva, the mesoderm is separated from the archenteral epithelium by an extracellular matrix secreted frontad from behind. As a result, a single coelomic anlage is formed. The initial mesoderm in both species is of archenteral/endodermal origin. Considering endodermal origin as the crucial character for enterocoely, coelom formation through proliferation of a compact, endodermally derived mesodermal cell mass in Brachiopoda is clearly identified as enterocoely. Endodermal origin of mesoderm and, therefore, of the coelomic epithelium is hypothesised as a synapomorphy of Brachiopoda and Deuterostomia. As a consequence: (1) Brachiopoda and Deuterostomia are considered sister groups, (2) Brachiopoda group within Radialia and (3) lophophorates (”Tentaculata”) remain as a paraphyletic grouping. Accepted: 26 November 1999     

Human tissue kallikrein. II. Isolation and characterization of human salivary kallikrein

Human salivary kallikrein was isolated from saliva using affinity chromatography on aprotinin-Sepharose and anti-human urinary kallikrein IgG-Sepharose followed by ion exchange chromatography on DEAE-Sepharose. The enzyme preparation had a specific activity of 950 U/mg protein towards the synthetic substrate Ac-Phe-Arg-OEt, a specific biological activity of 2000 KE/mg protein (measured in the dog blood pressure assay) and 0.64 HMW-kininogen-U/mg, corresponding to the liberation of 679 micrograms bradykinin equivalents per mg enzyme per min from HMW-kininogen (using the rat uterus test). In sodium dodecyl sulfate gel electrophoresis one protein band corresponding to a molecular mass of 32 kDa was obtained. The amino-acid composition was determined and isoleucin was found as the only N-terminal residue. The bimolecular velocity constant for the inhibition by diisopropyl fluorophosphate was determined as 8 l x mol-1 x min-1. The dissociation constant Ki of the human salivary kallikrein-aprotinin complex was calculated to be 0.7 x 10(-10)M. The Km and Vmax values for the hydrolysis of the synthetic substrates Ac-Phe-Arg-OEt and D Val-Leu-Arg-Nan were determined. In the enzyme immunoassay for human urinary kallikrein parallel binding curves were obtained.