Studies on human high molecular weight (HMW) kininogen. II. Structural change of HMW kininogen by the action of human plasma kallikrein

We have investigated in detail the cleavage of human high molecular weight (HMW) kininogen by human plasma kallikrein and revealed the formation of a nicked kininogen and a novel kinin-free protein (KFP) as intermediate cleavage products. The cleavage of a single chain HMW kininogen (Mr=120,000) by plasma kallikrein was a three-step reaction. The first cleavage yielded a nicked kininogen composed of two disulfide-linked 62,000 and 56,000 daltons chains. The second cleavage yielded kinin and an intermediate kinin-free protein, KFP-I, which was apparently of equal size to the nicked kininogen. The third cleavage yielded a stable kinin-free protein, KFP-II, composed of two disulfide-linked 62,000 and 45,000 daltons chains. The liberation of an 8,000 daltons fragment was identified when the 56,000 daltons chain isolated by SP-Sephadex C-50 chromatography of reduced and alkylated KFP-I was cleaved by plasma kallikrein into the 45,000 daltons chain. Although the antiserum against HMW kininogen cross-reacted with low molecular weight (LMW) kininogen, the antiserum against the 45,000 daltons chain was specific for HMW kininogen. These results suggest that the antigenic determinant groups common to HMW and LMW kininogens are located in the 62,000 daltons heavy chain, while those specific for HMW kininogen are located in the 45,000 daltons light chain, which is known to retain blood coagulation activity.     

Protein-protein interactions in contact activation of blood coagulation. Characterization of fluorescein-labeled human high molecular weight kininogen-light chain as a probe

Limited proteolysis of high molecular weight kininogen by kallikrein resulted in the generation of an inactive heavy chain of Mr = 64,000 and active light chains of Mr = 64,000 and 51,000 when analyzed by sodium dodecyl sulfate (SDS)-gel electrophoresis under reducing conditions. Starting with kininogen from outdated plasma, a light chain with an apparent molecular weight of 51,000 on 7.5% SDS gels was purified and characterized. Molecular weights of 28,900 +/- 1,100 and 30,500 +/- 1,600 were obtained by gel filtration of the reduced and alkylated protein in 6 M guanidine HCl and equilibrium sedimentation under nondenaturing conditions in the air-driven ultracentrifuge, respectively. The light chain stained positively with periodic acid-Schiff reagent on SDS gels indicating that covalently attached carbohydrate may be responsible for the anomalously high molecular weight estimated by SDS-gel electrophoresis. A single light chain thiol group reacted with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) in the presence and absence of 6 M guanidine HCl. Specific fluorescent labeling of the thiol group with 5-(iodoacetamido)fluorescein (IAF) occurred without loss of clotting activity. Addition of purified human plasma prekallikrein to the IAF-light chain resulted in a maximum increase in fluorescence anisotropy of 0.041 +/- 0.001 and no change in the fluorescence intensity. Fluorescence anisotropy measurements of the equilibrium binding of prekallikrein to the IAF-light chain yielded an average Kd of 17.3 +/- 2.5 nM and stoichiometry of 1.07 +/- 0.07 mol of prekallikrein/mol of IAF-light chain. Measurements of the interaction of prekallikrein with iodoacetamide-alkylated light chain using the IAF-light chain as a probe gave an average Kd of 16 +/- 4 nM and stoichiometry of 1.0 +/- 0.2 indicating indistinguishable affinities for prekallikrein.     

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)     

Identification of the binding site for plasma prekallikrein in human high molecular weight kininogen. A region from residues 185 to 224 of the kininogen light chain retains full binding activity

We studied the ability of fragments of the light chain of human high molecular weight kininogen to bind to plasma prekallikrein. In a competitive fluorescence polarization assay, kallikrein-cleaved light chain (light chain-2; residues 49-255), a cyanogen bromide fragment (residues 185-242), and a tryptic peptide (T-7; residues 185-224) had binding affinities of approximately 20 nM, equivalent to the value for the intact light chain (residues 1-255) of high-molecular-weight kininogen. In contrast, fragments consisting of residues 49-184 and 243-255 showed no binding activity (Kd much greater than 1,000 nM). Direct titrations of fluorescein-labeled derivatives of light chain-2 and peptide T-7 with prekallikrein confirmed that T-7 retained full binding activity for prekallikrein (Kd = 12 +/- 2 nM for labeled light chain-2; Kd = 7 +/- 1 nM for labeled T-7). These results localize the binding site of high molecular weight kininogen for prekallikrein within a region of 40 amino acids (residues 185-224) that resides in the near carboxyl terminus of the light chain of kininogen.     

Heavy chain of human high molecular weight and low molecular weight kininogens binds calcium ion

An antibody subpopulation, anti high molecular weight (anti-HMW) kininogen-Ca2+ antibody able to bind specifically to the HMW kininogen-Ca2+ complex, was isolated from anti-HMW kininogen antiserum. Partially purified anti-HMW kininogen antibody was applied to a HMW kininogen-Sepharose column equilibrated with 40 mM tris(hydroxymethyl)aminomethane hydrochloride buffer, pH 7.5, containing 1.0 M NaCl and 1 mM CaCl2, and anti-HMW kininogen-Ca2+ antibody was eluted with 5 mM ethylenediaminetetraacetic acid. As a result of characterization by enzyme-linked immunosorbent assay, this antibody specifically recognized the cyanogen bromide cleaved fragment 1 (CB-1) region (1-160 amino acid sequence) of the heavy chain of kininogen molecules in the presence of Ca2+ or Mg2+. Furthermore, circular dichroism (CD) experiments showed that the conformational changes of HMW kininogen and heavy chain were induced by metal ions such as Ca2+ and Mg2+ and that these changes were due to the conformational change of the CB-1 region of the heavy chain. The dissociation constant (Kd) for the heavy chain-Ca2+ measured by CD analysis at 214 nm was found to be 0.33 +/- 0.09 mM (mean +/- SD). The number of Ca2+-binding sites of heavy chain calculated from the Hill plot was 1.15 +/- 0.04 (mean +/- SD). Then, a possible Ca2+-binding site was found in the amino-terminal portion of the heavy chain of kininogen molecules.     

The role of bovine high-molecular-weight (HMW) kininogen in contact-mediated activation of bovine factor XII: interaction of HMW kininogen with kaolin and plasma prekallikrein

Previous studies from our laboratories (Sugo et al. (1980) Biochemistry 19, 3215-3220) have shown that bovine high-molecular-weight (HMW) kininogen remarkably accelerates the kaolin-mediated activation of Factor XII in the presence of prekallikrein, and that both fragment 1.2 and the light chain regions located in the COOH terminal half of the kininogen molecule are essential for the activation. In the present study, we demonstrate that the accelerating effect of HMW kininogen is mediated through its adsorption on the kaolin surface through the fragment 1.2 region and its complex formation with prekallikrein through the light chain region. The evidence is as follows: 1. HMW kininogen radio-labeled with 125I was adsorbed on kaolin and the adsorption was inhibited by the prior treatment of kaolin with fragment 1.2, fragment 1.2-light chain, kinin-free protein or HMW kininogen, but not with kinin- and fragment 1.2-free protein, light chain or low molecular-weight (LMW) kininogen. 2. The complex formation of HMW kininogen with prekallikrein in bovine plasma or in the purified system was examined by gel-filtration on a column of Sephacryl S-200 In bovine plasma, prekallikrein was eluted in the same fraction as HMW kininogen, showing an apparent molecular weight of 250,000, whereas purified prekallikrein was eluted in the fraction corresponding to an apparent molecular weight of 100,000. When purified prekallikrein was mixed with purified HMW kininogen in a mol ratio of 1 to 2, all prekallikrein was found to be associated with HMW kininogen. Furthermore, purified prekallikrein mixed with kininogen derivatives, such as kinin- and fragment 1.2-free protein, fragment 1.2-light chain or light chain, was eluted in the higher molecular weight fraction. HMW kininogen did not form a complex with prekallikrein. Using the same technique, it was shown that kinin- and fragment 1.2-free protein forms a complex not only with prekallikrein but also with kallikrein.     

Determination of the bifunctional properties of high molecular weight kininogen by studies with monoclonal antibodies directed to each of its chains

In normal human plasma two forms of kininogen exist, low molecular weight kininogen (LMWK) and high molecular weight kininogen (HMWK). When these proteins are cleaved they are found to have a common heavy chain and bradykinin, but each has a unique light chain. Monoclonal antibodies to the heavy and light chains of HMWK have been developed, and the effects of each on the function of this protein are defined. Initial studies showed that an antibody, C11C1, completely neutralized the coagulant activity of plasma HMWK whereas another antibody, 2B5, did not. On a competitive enzyme-linked immunosorbent assay (CELISA) the C11C1 antibody was consumed by kininogen antigen in normal plasma but not by kininogen antigen in HMWK-deficient plasma. On immunoblot, the C11C1 antibody recognized one kininogen protein in normal plasma and did not recognize any kininogen antigen in HMWK-deficient plasma. These combined studies indicated that the C11C1 antibody was directed to an epitope on the unique 46-kDa light chain of HMWK. In contrast, the 2B5 antibody on a CELISA was consumed by kininogen antigen in both normal plasma and HMWK-deficient plasma but not by total kininogen-deficient plasma. On immunoblot, the 2B5 antibody recognized both kininogens in normal plasma but only LMWK in HMWK-deficient plasma. These combined studies indicated that the 2B5 antibody was directed to the common 64-kDa heavy chain of the plasma kininogens. Utilizing direct binding studies or competition kinetic experiments, the 2B5 and C11C1 antibodies bound with high affinity (1.71 and 0.77 nM, respectively) to their antigenic determinants on the HMWK molecule. The 2B5 antibody did neutralize the ability of HMWK to inhibit platelet calpain. These studies with monoclonal antibodies directed to each of the HMWK chains indicate that HMWK is a bifunctional molecule that can serve as a cofactor for serine zymogen activation and an inhibitor of cysteine proteases.     

Evidence for binding of the ectodomain of amyloid precursor protein 695 and activated high molecular weight kininogen

To identify ligands that bind to the N-terminal portion of human amyloid precursor protein (APP), we sought binding partners for a fragment of the ectodomain of human APP695 (sAPP(695)T). The probe bound to fragments of high molecular weight kininogen (HK) in rat cortical membrane preparations in vitro. Laser confocal microscopy indicated that APP and HK colocalize near cerebral blood vessels, in the neuropil, and in many neurons of rat brain. sAPP(695)T bound to human activated kininogen (HKa) (K(d)=0.3+/-0.1 nM), but not to inactivated or low molecular weight kininogen. Binding was specific for the light chain sequence of HKa. Biotinylated human HKa also bound to sAPP(695) (K(d)=0.3+/-0.5 nM). sAPP(695) and HKa form tight complexes in solution that can be coimmunoprecipitated. These results support the hypothesis that forms of APP and kininogen can interact in brain tissue. Considering the implications of APP in neurite outgrowth, the APP-HKa interaction could modulate neurogenesis.     

The hyaluronan-binding serine protease from human plasma cleaves HMW and LMW kininogen and releases bradykinin

The influence of the hyaluronan-binding protease (PHBSP), a plasma enzyme with FVII- and pro-urokinase-activating potency, on components of the contact phase (kallikrein/kinin) system was investigated. No activation or cleavage of the proenzymes involved in the contact phase system was observed. The pro-cofactor high molecular weight kininogen (HK), however, was cleaved in vitro by PHBSP in the absence of any charged surface, releasing the activated cofactor and the vasoactive nonapeptide bradykinin. Glycosoaminoglycans strongly enhanced the reaction. The cleavage was comparable to that of plasma kallikrein, but clearly different from that of coagulation factor FXIa. Upon extended incubation with PHBSP, the light chain was further processed, partially removing about 60 amino acid residues from the N-terminus of domain D5 of the light chain. These cleavage site(s) were distinct from plasma kallikrein or FXIa cleavage sites. PHBSP and, more interestingly, also plasma kallikrein could cleave low molecular weight kininogen in vitro, indicating that domains D5H and D6H are no prerequisite for kininogen cleavage. PHBSP was also able to release bradykinin from HK in plasma where the pro-cofactor circulates predominantly in complex with plasma kallikrein or FXI. In conclusion, PHBSP represents a novel kininogen-cleaving and bradykinin-releasing enzyme in plasma that shares significant catalytic similarities with plasma kallikrein. Since they are structurally unrelated in their heavy chains (propeptide), their similar in vivo catalytic activities might be directed at distinct sites where PHBSP could induce processes that are related to the kallikrein/kinin system.     

Low molecular weight kininogen binds to platelets to modulate thrombin-induced platelet activation

The kininogens, high molecular weight kininogen (HK) and low molecular weight kininogen (LK), are multifunctional, single-gene products that contain bradykinin and identical amino-terminal heavy chains. Studies were performed to determine if LK would bind directly to platelets. 125I-LK specifically bound to gel-filtered platelets in the presence of 50 microM Zn2+. HK effectively competed with 125I-LK for the same binding site (Ki = 27 +/- 9 nM, n = 5). Similarly, the Ki for LK inhibition of 125I-LK binding was 12 +/- 1 nM (n = 3). Albumin, fibrinogen, factor XIII, and kallikrein did not inhibit 125I-LK binding to unstimulated platelets. 125I-LK (66 kDa) was not cleaved upon binding to platelets. The binding of 125I-LK to unstimulated platelets was found to be fully reversible by the addition of a 50 molar excess of unlabeled LK at both 10 and 20 min. LK binding to platelets was saturable with an apparent Kd of 27 +/- 2 nM (mean +/- S.E., n = 9) and 647 +/- 147 binding sites/platelet. Both LK and HK at plasma concentrations inhibited thrombin-induced platelet aggregation. LK and HK at about 5% of plasma concentration also inhibited thrombin-induced secretion of both stirred and unstirred platelets. Both kininogens were found to be noncompetitive inhibitors of proteolytically active thrombin binding to platelets. The kininogens did not inhibit D-phenylalanyl-prolyl-arginine chloromethyl ketone-treated thrombin from binding to platelets. These studies indicated that both kininogens have a region on their heavy chain which allows them to bind to platelets. Further, kininogen binding by its heavy chain modulates thrombin activation of platelets since it prevents proteolytically active thrombin from binding to its receptor.     

Protein-protein interactions in contact activation of blood coagulation. Binding of high molecular weight kininogen and the 5-(iodoacetamido) fluorescein-labeled kininogen light chain to prekallikrein, kallikrein, and the separated kallikrein heavy and light chains

Binding of the 5-(iodoacetamido)fluorescein (IAF)-labeled high molecular weight (HMW) kininogen light chain to prekallikrein and D-Phe-Phe-Arg-CH2Cl-inactivated kallikrein was monitored by a 0.040 +/- 0.002 increase in fluorescence anisotropy. Indistinguishable average dissociation constants and stoichiometries of 14 +/- 3 nM and 1.1 +/- 0.1 mol of prekallikrein/mol of IAF-light chain and 17 +/- 3 nM and 0.9 +/- 0.1 mol of kallikrein/mol of IAF-light chain were determined for these interactions at pH 7.4, mu 0.14 and 22 degrees C. Prekallikrein which had been reduced and alkylated in 6 M guanidine HCl lost the ability to increase the fluorescence anisotropy of the IAF-kininogen light chain, suggesting that the native tertiary structure was required for tight binding. The kallikrein heavy and light chains were separated on the basis of the affinity of the heavy chain for HMW-kininogen-Sepharose, after mild reduction and alkylation of kallikrein under nondenaturing conditions. Under these conditions, alkylation with iodo [14C]acetamide demonstrated that only limited chemical modification had occurred. Binding of the IAF-kininogen light chain to the isolated alkylated kallikrein heavy chain, when compared to prekallikrein and kallikrein, was characterized by an indistinguishable increase in fluorescence anisotropy, average dissociation constant of 14 +/- 3 nM, and stoichiometry of 1.2 +/- 0.1 mol of kallikrein heavy chain/mol of IAF-light chain. In contrast, no binding of the D-Phe-Phe-Arg-CH2Cl-inactivated kallikrein light chain was detected at concentrations up to 500 nM. Furthermore, 300 nM kallikrein light chain did not affect IAF-kininogen light chain binding to prekallikrein, kallikrein, or the kallikrein heavy chain. The binding of monomeric single chain HMW-kininogen to prekallikrein, kallikrein, and the kallikrein heavy and light chains was studied using the IAF-kininogen light chain as a probe. Analysis of the competitive binding of HMW-kininogen gave average dissociation constants and stoichiometries of 12 +/- 2 nM and 1.2 +/- 0.1 mol of prekallikrein/mol of HMW-kininogen, 15 +/- 2 nM and 1.3 +/- 0.1 mol of kallikrein/mol of HMW-kininogen, 14 +/- 3 nM and 1.4 +/- 0.2 mol of kallikrein heavy chain/mol of HMW-kininogen, and no detectable effect of 300 nM kallikrein light chain on these interactions. We conclude that a specific, nonenzymatic interaction between sites located exclusively on the light chain of HMW-kininogen and the heavy chain of kallikrein or prekallikrein is responsible for the formation of 1:1 noncovalent complexes between these proteins.     

High-molecular-weight kininogen from horse plasma. Isolation, characterization and comparison with bovine high-Mr kininogen

High-molecular-weight (high-Mr) kininogen was purified from horse plasma by chromatography on columns of DEAE-Sephadex A-50, CM-Sephadex C-50, p-chlorobenzylamine-Sepharose and Sephadex G-150. The yield was about 150 mg from 81 of fresh plasma. The purified material gave a single band on sodium dodecylsulfate/polyacrylamide gel electrophoresis and a single precipitin line on immunodiffusion and immunoelectrophoresis. The molecular weight of horse high-Mr kininogen was estimated to be 78000 by dodecylsulfate gel electrophoresis using the Ferguson plot. Its polypeptide content was determined to be 86% by amino acid analysis and there was a total of 581 amino acid residues/molecule of protein. The kininogen contained a total of 13.9% carbohydrates, consisting of hexoses (7.8%), glucosamine (1.9%), galactosamine (0.6%) and sialic acid (3.6%). On incubation of horse high-Mr kininogen with bovine and horse plasma kallikreins, several fragments which contained extremely high levels of histidine, were liberated, in addition to kinin. After the liberation of kinin and histidine-rich fragments, a protein free of kinin and its fragments was isolated. This protein consisted of two polypeptide chains, heavy chain and light chain, which are bridged by disulfide bonds. The molecular weight and amino acid composition of the heavy chain and the light chain from horse high-Mr kininogen were very similar to those of the heavy and light chains from bovine high-Mr kininogen, respectively. From these results, it was revealed that horse high-Mr kininogen is quite similar to bovine high-Mr kininogen in terms of their physicochemical and chemical properties, although they are immunologically distinguishable.     

Mapping of functional domains of human high molecular weight and low molecular weight kininogens using murine monoclonal antibodies

Thirty-four monoclonal antibodies directed against human high molecular weight (HMW) and low molecular weight (LMW) kininogens and their derivatives were obtained, and the specificities of the antibodies were assayed by enzyme-linked immunosorbent assay (ELISA). By use of HMW kininogen, kinin-free HMW kininogen, kinin-free and fragment 1.2 (fr 1.2) free HMW kininogen, fr 1.2-light chain of HMW kininogen, LMW kininogen, kinin-free LMW kininogen, heavy chain of LMW kininogen, and light chain of LMW kininogen, the monoclonal antibodies were characterized and classified into four groups: (A) 20 monoclonal antibodies reacting with only the heavy chain, a common region of HMW and LMW kininogens; each of these monoclonal antibodies possessed the specificity to domain 1 (2 monoclonal antibodies), domain 2 (2 monoclonal antibodies), domain 3 (7 monoclonal antibodies), and both domains 2 and 3 (7 monoclonal antibodies) of the heavy chain; (B) 7 monoclonal antibodies reacting with fr 1.2, a unique histidine-rich region; (C) 5 monoclonal antibodies reacting with the light chain of HMW kininogen; (D) 2 monoclonal antibodies reacting with the light chain of LMW kininogen. Two monoclonal antibodies in the first group (group A), designated HKG H7 and H12, effectively suppressed the thiol proteinase inhibitor activity of HMW kininogen to papain and calpains and of LMW kininogen to papain, but the others did not affect it. Further, all the monoclonal antibodies which recognized the fr 1.2 or light chain of HMW kininogen (groups B and C) suppressed the clotting activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of chemical modifications on the surface- and protein-binding properties of the light chain of human high molecular weight kininogen

The light chain of kallikrein-cleaved human high molecular weight kininogen is solely responsible for its cofactor activity in blood clotting. Sequencing of the NH2-terminal region of the light chain reported herein identified the third kallikrein cleavage site of high molecular weight kininogen as Arg-437. The co-factor activity of high molecular weight kininogen consists of the capacity to bind to negatively charged surfaces and to factor XI or prekallikrein. Chemical modification of the histidines by either photooxidation or ethoxyformic anhydride affected the equivalent of 14-16 of 23 histidines available and resulted in over 90% loss in procoagulant activity. The modified protein had drastically reduced surface- and zinc-binding capacity, but it bound successfully to either factor XI or prekallikrein. In contrast, modification of two carboxyl groups, which led to approximately 80-90% loss of procoagulant activity, seriously compromised protein binding but left surface binding unaffected. All 3 tryptophans were modified at pH 4.0 with N-bromosuccinimide with a 70% reduction in procoagulant activity, but only 1 tryptophan was available for reaction at pH 7.35, resulting in a 50% loss in activity. Tryptophan modification at acidic pH affected protein binding but did not modify surface or zinc binding. Modification of both available tyrosine and 9 of 18 available lysine residues did not have a significant effect on the procoagulant activity of the light chain. These studies indicate that histidines participate in surface binding and that free carboxyl groups and tryptophan side chains are involved in binding of high molecular weight kininogen to other clotting factors.     

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)     

Hypothesis. The molecular 'double-pivot' mechanism for water oxidation

High-molecular-weight (HMW) kininogen was purified from guinea-pig plasma by measuring its ability to correct the prolonged clotting time in human HMW kininogen deficient plasma (Fitzgerald trait). The purified HMW kininogen demonstrated a homogeneous band in disc gel electrophoresis in the presence of sodium dodecyl sulfate under reducing or non-reducing conditions with an apparent molecular weight of 100,000. Kinin released from HMW kininogen by treatment with guinea-pig plasma kallikrein was identified as bradykinin by reverse-phase HPLC and amino-acid analysis. The capacity of HMW kininogen as a thiol-proteinase inhibitor was realized by its dose-dependent inhibitory activity to papain. The Ki value for papain was estimated to be 42 pM. The kinin-free HMW kininogen maintained the inhibitor and clotting-factor activities with similar capacities to those of the HMW kininogen molecule. Heavy chain (H-chain) and light chain (L-chain) of HMW kininogen were prepared from reduced and alkylated kinin-free HMW kininogen by HPLC. The S-alkylated H-chain, but not L-chain, demonstrated the inhibitor activity with the Ki value 6.9 nM for papain, whereas the S-alkylated L-chain, but not H-chain, maintained the clotting activity one-third of the capacity of HMW kininogen. Specific antibodies recognized HMW kininogen, but also a probable low-molecular-weight kininogen(s) with an apparent molecular weight of 60,000 in the guinea-pig plasma. All of these properties are consistent with the reports on human, bovine and rat HMW kininogen.     

Determination of polypeptide chain molecular weights of human and bovine band 3 protein from erythrocyte membranes by low-angle laser light scattering combined with high-performance gel chromatography in the presence of sodium dodecyl sulfate

Polypeptide chain molecular weights of human and bovine band 3 proteins which are glycoproteins of the erythrocyte membrane were determined as 101,000 +/- 2000 for the former and 107,000 +/- 2000 for the latter by using the low-angle laser light scattering technique combined with a high-performance gel chromatography column, an ultraviolet spectrophotometer and a differential refractometer in the presence of sodium dodecyl sulfate. The advantage of this method is that, unlike the sedimentation equilibrium technique, neither information on the binding to proteins of all ligands present nor the partial specific volume is required to evaluate the polypeptide chain molecular weight of proteins in a multicomponent system.     

The fragments of bovine high molecular weight kininogen promote osteoblast proliferation in vitro

High molecular weight (HMW) kininogen is known to be a large plasma protein and cleaved by plasma proteinase kallikrein, then it generates four fragments in the blood coagulation cascade: heavy chain, bradykinin, fragment 1.2, and light chain. The fragment 1.2 has also been found in the basic protein fraction of bovine milk as a bioactive protein which promotes osteoblast proliferation. The milk basic protein has been shown to be a multi functional edible protein which promotes bone formation and inhibits bone resorption. In the present study, we purified the fragment 1.2 from bovine plasma and assessed it could promote osteoblast proliferation and posses the activity after pepsin digestion. Purified plasma HMW kininogen did not promote the proliferation, however, the kallikrein-cleaved HMW kininogen promoted the proliferation. The fragment 1.2, purified from the proteolysate, also promoted the proliferation. The pepsin digestion was performed according to the method of the assessment of allergenesity of genetically modified crops. After pepsin digestion, the fragment 1.2 generated resistant fragments and showed the promoting activity of osteoblast proliferation. These results suggest that the enzymatically-digested fragments of bovine HMW kininogen are able to be a naturally occurred active protein that promotes the bone formation by oral administration.     

High molecular weight kininogen as substrate for cathepsin B

We investigated the influence of pH and divalent cations (Zn2+, Mg2+ and Ca2+) on high molecular weight kininogen processing by cathepsin B. At pH 6.3, high molecular weight kininogen is hydrolyzed by cathepsin B at three sites generating fragments of 80, 60 and 40 kDa. Cathepsin B has kininogenase activity at this pH which is improved in the absence of divalent cations. At pH 7.35, high molecular weight kininogen is slightly cleaved by cathepsin B into fragments of 60 kDa, and cathepsin B kininogenase activity is impaired. Our results suggest that high molecular weight kininogen is a substrate for cathepsin B under pathophysiological conditions.