Neutralization of heparin-related saccharides by histidine-rich glycoprotein and platelet factor 4

Heparin and heparin oligosaccharides prepared by nitrous acid depolymerization were fractionated by affinity chromatography on immobilized antithrombin and by gel chromatography. The anticoagulant activities of high affinity heparin of Mr greater than or equal to 7,800 could be readily neutralized by the plasma protein histidine-rich glycoprotein (see also Lijnen, H.R., Hoylaerts, M., and Collen, D. (1983) J. Biol. Chem. 258, 3803-3808), whereas oligosaccharides falling below 18 saccharide units (Mr 5,400) became increasingly resistant to neutralization. An octasaccharide with characteristic marked ability to accelerate the inactivation of Factor Xa by antithrombin retained greater than 50% of its activity even at a histidine-rich glycoprotein/oligosaccharide molar ratio of 500:1. Histidine-rich glycoprotein, like the platelet-derived heparin neutralizing protein platelet factor 4 (Lane, D.A., Denton, J., Flynn, A.M., Thunberg, L. and Lindahl, U. (1984) Biochem J. 218, 725-732), therefore requires interaction with saccharide sequences in addition to the antithrombin-binding pentasaccharide of heparin in order to efficiently express its antiheparin activity. Heparan sulfate isolated from pig intestinal mucosa (HS I, Mr approximately 20,000) and from human aorta (HS II, Mr approximately 40,000) exhibited anti-Factor Xa activities of 180 and 20 units/micromol [corrected], respectively. A fraction corresponding to about 5% of HS I bound with high affinity to immobilized antithrombin and contained all of the anticoagulant activity of the starting material. While these heparan sulfates were readily neutralized by platelet factor 4, they were relatively resistant to neutralization by histidine-rich glycoprotein, although complete neutralization could be attained in the presence of molar excess of this protein. These findings may be of importance in relation (a) to the functional role of endogenous anticoagulant polysaccharides at the vascular wall and (b) to clinical situations in which heparin or heparin-related compounds are administered as exogenous anticoagulants.     

Low-angle neutron scattering analysis of Na/K-ATPase in detergent solution

Purified Na/K-ATPase from guinea pig renal outer medulla has been delipidated and solubilized in Brij 58 (polyoxyethylene ether; C-16, E-20). At a concentration of 2 mg of Brij 58/mg of protein, about one-half the enzyme complement was solubilized and almost 50% of Na/K-ATPase activity was retained by the enzyme-micelle complex. Guinier plots of the neutron scattering profiles yielded no evidence of heterogeneity with respect to subunit composition or the state of aggregation in the solubilized oligomers. Contrast matching with D2O used to obtain estimates of the molecular weight of the micellar form of Na/K-ATPase gave a mean value of 310,000 +/- 42,700, which corresponds to an alpha 2 beta 2 tetramer. A Stuhrmann plot of the neutron scattering data yielded an estimated radius of gyration of 67 A. The Stuhrmann plot also indicated an asymmetrical distribution of neutron scattering density. On the basis of the Stuhrmann plot parameters, the estimated molecular weight, and the radius of gyration, a low-resolution model was formulated of the oligomeric unit of Na/K-ATPase.     

Small-angle x-ray scattering of high- and low-affinity heparin

Solution characterization of heparin with high affinity (HA) and low affinity (LA) for antithrombin III was performed using the methods of small-angle x-ray scattering (SAXS), viscometry, and aqueous gel permeation chromatography (GPC). SAXS provided various topological parameters including the radius of gyration ([S2]1/2), radius of gyration of cross-section ([S2]q1/2), persistence length (a*), contour length (L), and mass parameters, e.g., overall molecular mass (Mr), and mass per unit length (Mq). The molecular weights of HA and LA pig mucosal heparins were found to be 14,900 and 11,500 and the respective radii of gyration were 40.1 and 33.6 A. The persistence lengths of HA and LA were 21.3 and 20.3 A, respectively. These parameters were compared to SAXS data of heparin [S. S. Stivala, M. Herbst, O. Kratky, and I. Pilz (1968) Arch. Biochem. Biophys. 127, 795-802] fractionated according to molecular weight only. It was found that the various experimental values of this heparin lie somewhere in between those of HA and LA heparins. It appears that there are no appreciable differences in the physico-chemical properties, including conformation, among the heparins in H2O at 25 degrees C.     

The acceleration of the inhibition of platelet prothrombinase complex by heparin.

The influence of heparin on the inhibition of factor Xa has been studied under conditions where factor Xa is bound to collagen-thrombin-stimulated platelets to form the prothrombinase complex. Unfractionated heparin was found to cause a concentration-dependent acceleration of the inhibition of the platelet prothrombinase complex up to a maximum rate constant of 4.1 X 10(7) M-1 X min-1 at heparin concentrations of 0.2 microM and above. This is equivalent to a 4800-fold acceleration over the rate constant for the inhibition in the absence of heparin, and is 6.8-fold lower than the rate constant for the inhibition of uncomplexed factor Xa in the presence of saturating concentrations of heparin which was determined as 2.8 X 10(8) M-1 X min-1. The effects of three Mr fractions of heparin were also studied. These were a gel-filtered heparin of Mr 15000, a gel-filtered heparin of Mr 6000 and a heparin oligosaccharide (primarily 8-10 monosaccharide units) prepared by nitrous acid depolymerization, each with high affinity for antithrombin III. These fractions all accelerated the rate of the antithrombin III inhibition of the platelet prothrombinase complex, with maximum rate constants of 6.8 X 10(7), 1.4 X 10(7) and 9.8 X 10(6) M-1 X min-1, respectively. On comparison with the effect of these heparin fractions on the rate of inhibition of uncomplexed factor Xa a progressively increasing disparity between the rate of inhibition of uncomplexed and complexed factor Xa was observed, rising from 1.7-fold with the oligosaccharide to 6.8-fold with the unfractionated heparin. A possible mechanism for this differential activity between uncomplexed and complexed factor Xa with the various heparin fractions is discussed in terms of an involvement of heparin binding to factor Xa.     

Neutron scattering studies of chromatosomes

Neutron scattering data establish that the radius of gyration of the DNA in chicken erythrocyte chromatosome particles is significantly higher, by about 0.3 nm, than the radius of gyration of the DNA in the core particle. Corresponding information of the radius of gyration of the protein component in the chromatosomes (3.75 nm) indicated an enlargement, compared to the radius of gyration of the octamer of histone proteins both in core particles and in the histone octamer stabilised in 2 M NaCl (3.25 nm). From the latter data, we could calculate the distance in the chromatosome between the centre of mass of the linker histone and the histone octamer as 5.5 nm. These results impose severe limitations for the organisation of the 22 bp extra DNA and the possible location of H1/H5 in the chromatosome, implying that the H1/H5 is close to the centre turn of the core particle DNA.     

An elongated model of the Xenopus laevis transcription factor IIIA-5S ribosomal RNA complex derived from neutron scattering and hydrodynamic measurements.

The precise molecular composition of the Xenopus laevis TFIIIA-5S ribosomal RNA complex (7S particle) has been established from small angle neutron and dynamic light scattering. The molecular weight of the particle was found to be 95,700 +/- 10,000 and 86,700 +/- 9000 daltons from these two methods respectively. The observed match point of 54.4% D2O obtained from contrast variation experiments indicates a 1:1 molar ratio. It is concluded that only a single molecule of TFIIIA, a zinc-finger protein, and of 5S RNA are present in this complex. At high neutron scattering contrast radius of gyration of 42.3 +/- 2 A was found for the 7S particle. In addition a diffusion coefficient of 4.4 x 10(-11) [m2 s-1] and a sedimentation coefficient of 6.2S were determined. The hydrodynamic radius obtained for the 7S particle is 48 +/- 5 A. A simple elongated cylindrical model with dimensions of 140 A length and 59 A diameter is compatible with the neutron results. A globular model can be excluded by the shallow nature of the neutron scattering curves. It is proposed that the observed difference of 15 A in length between the 7S particle and isolated 5S RNA most likely indicates that part(s) of the protein protrudes from the end(s) of the RNA molecule. There is no biochemical evidence for any gross alteration in 5S RNA conformation upon binding to TFIIIA.     

Antithrombin III-dependent anti-prothrombinase activity of heparin and heparin fragments

Heparin and heparin fragments in the molecular mass range 1,700-20,000 Da were examined for their ability to accelerate the antithrombin III (AT III)-dependent inhibition of human factor Xa and the prothrombin converting complex (prothrombinase) during human prothrombin activation. The prothrombinase reaction was modeled by a 3-parameter 2-exponential equation to determine the initial rate of prothrombin activation and the pseudo-first order rate constants of inhibition of prothrombinase and in situ generated thrombin activity. The catalytic specific activities of the heparins increased with increasing molecular size for both the inhibition of prothrombinase and factor Xa. A 10-fold increase over the entire Mr range was found. In contrast to results obtained by others (Ellis, V., Scully, M. F., and Kakkar, V. V. (1986) Biochem. J. 233, 161-165; Barrowcliffe, T. W., Havercroft, S. J., Kemball-Cook, G., and Lindahl, U. (1987) Biochem. J. 243, 31-37), all the heparins showed a 5-fold higher rate of inhibition of factor Xa when compared with the inhibition of prothrombinase, indicating that the factor Va-mediated protection of factor Xa from inhibition by AT III/heparin is independent of the molecular size of the heparin. Our original approach has also revealed a hitherto unrecognized phenomenon, namely, in addition to the accelerating effect of the heparins on the rate of formation of the inactive AT III-factor Xa complex, heparins with Mr greater than 4,500 reduce the initial rate of thrombin generation in the presence of AT III in a concentration-dependent way. We hypothesize that the formation of the dissociable ternary AT III-heparin-factor Xa complex results in a (partial) loss of factor Xa activity towards its natural substrate prothrombin.     

Statistical conformation of human plasma fibronectin

Fibronectin is a multifunctional glycoprotein (molecular mass, M = 530 kg/mol) of the extra cellular matrix (ECM) having a major role in cell adhesion. In physiological conditions, the conformation of this protein still remains debated and controversial. Here, we present a set of results obtained by scattering experiments. In "native" conditions, the radius of gyration (R(g) = 15.3 +/- 0.3 nm) was determined by static light scattering as well as small-angle neutron scattering. The hydrodynamic radius (R(H) = 11.5 +/- 0.1 nm) was deduced from quasi-elastic light scattering measurements. These results imply a low internal concentration compared to that of usual globular proteins. This is also confirmed by the ratio R(H)/R(g) = 0. 75 +/- 0.02 consistent with a Gaussian chain, whereas R(H)/R(g) = 1. 3 for spherical shaped molecules. However, adding a denaturing agent (urea 8 M) increases R(g) by a factor 2. This means that fibronectin "native" chain is not either completely unfolded. The average shape of fibronectin conformation was also probed by small-angle neutron scattering performed for reverse scattering vector q(-)(1) smaller than R(g) (0.2 < q(-)(1) < 15 nm). The measured form factor is in complete agreement with the form factor of a random string of 56 beads of 5 nm diameter. It rules out the possibility of unfolded chain as well as globular structures. These results have structural and biological implications as far as ECM organization is concerned.     

Binding of both Ca2+ and mastoparan to calmodulin induces a large change in the tertiary structure

The technique of small-angle X-ray scattering has been employed to examine the solution conformation of calmodulin and its complexes with Ca2+ alone, and with both Ca2+ and mastoparan. The radius of gyration decreased by 3.1 +/- 0.3 A upon binding of both 4 mol Ca2+/mol of protein and 1 mol mastoparan/mol of protein to form the ternary complex. A smaller increase was found for the separate binding of 4 mol Ca2+/mol of protein in the absence of mastoparan (0.6 +/- 0.3 A). The analyses of pair distance distribution function showed that the maximal pair distance in calmodulin complex with both Ca2+ and mastoparan decreased by 20-30% in comparison with calmodulin or its complex with Ca2+, and a shoulder near 40 A, which characterizes the dumbbell-shaped molecule of calmodulin, disappeared. These results indicate that the two globular domains of the calmodulin complex with Ca2+ and mastoparan come close together by 8.0-9.5 A on average, if the size and the overall shape of the globular domains are the same in Ca2+-calmodulin-mastoparan complex as in calmodulin or Ca2+-calmodulin complex.     

Structure of the proteolytic fragment F34 of calmodulin in the absence and presence of mastoparan as revealed by solution X-ray scattering.

The solution X-ray scattering technique has been applied to examine the conformations of the proteolytic fragment F34 (78Asp-148Lys) of calmodulin in the absence of both Ca2+ and mastoparan, in the presence of Ca2+ only, and in the presence of both Ca2+ and mastoparan. The radius of gyration and the molecular weight for the F34 fragment increased by 1.1 +/- 0.3 A and 19%, respectively, upon binding of both 2 mol of Ca2+/mol to the F34 fragment and mastoparan to form the tertiary complex. A smaller change was found for the Ca(2+)-saturated F34 fragment in the absence of mastoparan (0.3 +/- 0.3 A) without any change of the molecular weight. The analysis based on the small-angle scattering data showed that the F34 fragment in the presence of Ca2+ alone preserved the tertiary structure of the globular domain in the crystal to a great extent. Further analyses based on a two-domain model showed that the center-to-center distance between F34 and mastoparan is about 12.7 A, if the structure of the F34 fragment in the presence of mastoparan resembles that in the absence of mastoparan and if mastoparan in the complex retains an alpha-helical conformation. The modeling studies using their crystal structure coordinates have been made on the basis of the solution X-ray scattering data. The combined results support a model proposed by Persechini and Kretsinger [Persechini, A., & Kretsinger, R. H. (1988) J. Cardiovasc. Pharmacol. 12 (Suppl. 5), S1-S12], although the center-to-center distance between mastoparan and the F34 fragment is shorter by about 5 A than that in their model.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphorylation of elongation factor 1 (EF-1) and valyl-tRNA synthetase by protein kinase C and stimulation of EF-1 activity

A high Mr complex isolated from rabbit reticulocytes contains valyl-tRNA synthetase and the four subunits of elongation factor 1 (EF-1). Previously, valyl-tRNA synthetase and the alpha, beta, and delta subunits of EF-1 were shown to be phosphorylated in reticulocytes in response to phorbol 12-myristate 13-acetate (PMA). Phosphorylation of the complex was accompanied by an increase in both valyl-tRNA synthetase and EF-1 activity (Venema, R. C., Peters, H. I., and Traugh, J. A. (1991) J. Biol. Chem., 266, 11993-11998). To investigate phosphorylation of the valyl-tRNA synthetase EF-1 complex in vitro by protein kinase C, the complex has been purified to apparent homogeneity from rabbit reticulocytes by gel filtration on Bio-Gel A-5m, affinity chromatography on tRNA-Sepharose, and fast protein liquid chromatography on Mono Q. Valyl-tRNA synthetase and the beta and delta subunits of EF-1 in the complex are highly phosphorylated by protein kinase C (0.5-0.9 mol of phosphate/mol of subunit), while EF-1 alpha is phosphorylated to a lesser extent (0.2 mol/mol). However, the isolated EF-1 alpha subunit is highly phosphorylated (2.0 mol/mol). Phosphopeptide mapping of EF-1 alpha shows that the same sites are modified by protein kinase C in vitro and in PMA-treated cells. Phosphorylation of the valyl-tRNA synthetase.EF-1 complex results in a 3-fold increase in activity of EF-1 as measured by poly(U)-directed polyphenylalanine synthesis; no effect of phosphorylation is detected with valyl-tRNA synthetase and isolated EF-1 alpha. Thus, phosphorylation and activation of EF-1 by protein kinase C, which has been shown to occur in vitro as well as in reticulocytes, may have a role in PMA stimulation of translational rates.     

Expression of platelet glycoprotein Ib alpha in HEL cells

We have previously shown that platelet glycoprotein Ib is expressed in a minority of cells of the human leukemic cell line HEL (Tabilio, A., Rosa, J. P., Testa, U., Kieffer, N., Nurden, A. T., Del Canizo, M. C., Breton-Gorius, J., and Vainchenker, W. (1984) EMBO J. 3, 453-459). In this report, we have selected a stable HEL subclone with increased expression of glycoprotein (GP) Ib as assessed by 6 different monoclonal antibodies in order to investigate the biochemical characteristics of this glycoprotein. A single polypeptide chain of apparent Mr = 60,000 was precipitated under reducing and nonreducing conditions by a specific polyclonal anti-platelet glycocalicin antibody and two anti-GPIb alpha monoclonal antibodies (AN51 and AP1), both from surface-labeled and metabolically labeled HEL cells. We were unable to demonstrate the presence of a polypeptide corresponding to the beta subunit of GPIb or GPIX which is closely associated with GPIb. Competitive immunoprecipitation performed in the presence of an excess amount of cold platelet glycocalicin completely displaced the Mr = 60,000 polypeptide. Synthesis of N-linked oligosaccharide chains on this Mr = 60,000 polypeptide was inhibited by the antibiotic tunicamycin, and a shift of the apparent Mr from 60,000 to 48,000 was observed. O-Linked oligosaccharide chains identical to platelet GPIb hexasaccharides were deficient or incomplete since no peanut agglutinin binding to the Mr = 60,000 polypeptide was observed after neuraminidase treatment of HEL cells. Thus, our results provide evidence that the Mr = 60,000 polypeptide expressed on the surface membrane of HEL cells is closely related to platelet GPIb and corresponds to an incompletely or abnormally O-glycosylated GPIb alpha subunit.     

1H NMR studies of T4 gene 32 protein: effects of zinc removal and reconstitution

Gene 32 protein (g32P), the single-stranded DNA binding protein from bacteriophage T4, contains 1 mol of Zn(II)/mol bound in a tetrahedral ligand field. 113Cd NMR studies of Cd-substituted wild-type and mutant (Cys166----Ser166) g32Ps show Cys77, Cys87, and Cys90 to provide three sulfur donor atoms as ligands to the metal ion [Giedroc, D. P., Johnson, B. A., Armitage, I. M., & Coleman, J. E. (1989) Biochemistry 28, 2410]. Proton NMR signals from the His and Trp side chains of the protein have been followed as a function of pH and metal ion removal by biosynthesizing the protein with amino acids carrying protons at specific positions in a background of perdeuteriated aromatic amino acids. Only one of the two pairs of His resonances (from His64 and His81) titrates over the pH range 8.0-5.9. The nontitrating His side chain is most likely ligated to the metal ion. Upon Zn(II) removal, 1H NMR spectra of the fully protonated g32P-(A + B) exhibit substantial signal broadening in several regions of the spectrum, while the His 2,4-1H resonances are broadened beyond detection. The 1H NMR spectral characteristics of the original protein are restored by reconstitution with stoichiometric Zn(II). The broadening of the 1H NMR signals is not due to oligomerization of the protein, since small-angle X-ray scattering experiments show that the average radius of gyration of the apo-g32P-(A + B) is 25.0 A and that of the reconstituted Zn(II)-g32P-(A + B) is 31.2 A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Critical role of micelles in pancreatic lipase activation revealed by small angle neutron scattering

In the duodenum, pancreatic lipase (PL) develops its activity on triglycerides by binding to the bile-emulsified oil droplets in the presence of its protein cofactor pancreatic colipase (PC). The neutron crystal structure of a PC-PL-micelle complex (Hermoso, J., Pignol, D., Penel, S., Roth, M., Chapus, C., and Fontecilla-Camps, J. C. (1997) EMBO J. 16, 5531-5536) has suggested that the stabilization of the enzyme in its active conformation and its adsorption to the emulsified oil droplets are mediated by a preformed lipase-colipase-micelle complex. Here, we correlate the ability of different amphypathic compounds to activate PL, with their association with PC-PL in solution. The method of small angle neutron scattering with D(2)O/H(2)O contrast variation was used to characterize a solution containing PC-PL complex and taurodeoxycholate micelles. The resulting radius of gyration (56 A) and the match point of the solution indicate the formation of a ternary complex that is similar to the one observed in the neutron crystal structure. In addition, we show that either bile salts, lysophospholipids, or nonionic detergents that form micelles with radii of gyration ranging from 13 to 26 A are able to bind to the PC-PL complex, whereas smaller micelles or nonmicellar compounds are not. This further supports the notion of a micelle size-dependent affinity process for lipase activation in vivo.     

A Neutron Scattering Study of the Ternary Complex EF-Tu•GTP-valy1-tRNAVal 1A

Abstract

The complex formation between elongation factor Tu (EF-Tu), GTP, and valyl-tRNA^Val _1A has been investigated in a hepes buffer of “pH” 7.4 and 0.2 M ionic strength using the small-angle neutron scattering method at concentrations of D₂O where EF-Tu (42% D₂O) and tRNA (71% D₂O) are successively matched by the solvents. The results indicate that EF-Tu undergoes a conformational change and contracts as a result of the complex formation, since the radius of gyration decreases by 15% from 2.82 to 2.39 nm. tRNA^Val _1A, on the other hand, seems to mainly retain its conformation within the complex, since the radii of gyration for the free (after correction for interparticular scattering) and complexed form are essentially the same. 2.38 and 2.47 nm, respectively.     

Structure of subnucleosomal particles. Tetrameric (H3/H4)2 146 base pair DNA and hexameric (H3/H4)2(H2A/H2B)1 146 base pair DNA complexes

The tetrameric (H3/H4)2 146 base pair (bp) DNA and hexameric (H3/H4)2(H2A/H2B)1 146 bp DNA subnucleosomal particles have been prepared by depletion of chicken erythrocyte core particles using 3 or 4 M urea, 250 mM sodium chloride, and a cation-exchange resin. The particles have been characterized by cross-linking and sedimentation equilibrium. The structures of the particles, particularly the tetrameric, have been studied by sedimentation velocity, low-angle neutron scattering, circular dichroism, optical melting, and nuclease digestion with DNase I, micrococcal nuclease, and exonuclease III. It is concluded that since the radius of gyration of the DNA in the tetramer particle and its maximum dimension are very close to those of the core particle, no expansion occurs on removal of all the H2A and H2B. Nuclease digestion results indicate that histones H3/H4 in the tetramer particle protect a total of 70 bp of DNA that are centrally located within the 146 bp. Within the 70 bp DNA length, the two terminal regions of 10 bp are, however, not strongly protected from digestion. The optical melting profile of both particles can be resolved into three components and is consistent with the model of histone protection of DNA proposed from nuclease digestion. The structure proposed for the tetrameric histone complex bound to DNA is that of a compact particle containing 1.75 superhelical turns of DNA, in which the H3 and H4 histone location is the same as found for the core particle in chromatin by histone/DNA cross-linking [Shick, V. V., Belyavsky, A. V., Bavykin, S. G., & Mirzabekov, A. D. (1980) J. Mol. Biol. 139, 491-517]. Optical melting of the hexamer particle shows that each (H2A/H2B)1 dimer of the core particle protects about 22 base pairs of DNA.     

Stoichiometric analysis of the specific interaction of the glucocorticoid receptor with DNA

Purified preparations of activated glucocorticoid X receptor complex (GR) contain a Mr 94,000 hormone-binding polypeptide co-purifying together with a Mr 72,000 non-hormone-binding polypeptide (Wrange, O., Okret, S., Radojcic, M., Carlstedt-Duke, J., and Gustafsson, J.-A. (1984) J. Biol. Chem. 259, 4534-4541). GR binds selectively to discrete regions of DNA in mouse mammary tumor virus (Payvar, F., DeFranco, D., Firestone, G.L., Edgar, B., Wrange, O., Okret, S., Gustafsson, J.-A., and Yamamoto, K. R. (1983) Cell 35, 381-392). Such GR-binding DNA fragments were used to measure the stoichiometry of GR to DNA. Quantitative DNaseI protection "footprinting" analysis was used to ensure that saturation conditions for specific DNA-binding were achieved. Glycerol density gradient centrifugation was used to quantitate Mr 94,000 binding to specific and nonspecific DNA sites. One Mr 94,000 entity was bound per specific DNA site. A modified GR purification procedure resulted in increased amounts of Mr 72,000 polypeptide (1.6:1, 94,000:72,000 molar ratio), compared to previous GR preparations. Glycerol gradient centrifugation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the specific GR X DNA complex contained similar amounts of Mr 94,000 and Mr 72,000 polypeptide. It is as yet uncertain if the Mr 72,000 polypeptide is a functional subunit of GR or a co-purifying contaminant only.     

Antithrombin activity of fucoidan. The interaction of fucoidan with heparin cofactor II, antithrombin III, and thrombin

Fucoidan, poly(L-fucopyranose) linked primarily alpha 1----2 with either a C3- or a C4-sulfate, is an effective anticoagulant in vitro and in vivo (Springer, G. F., Wurzel, H. A., McNeal, G. M., Jr., Ansell, N. J., and Doughty, M. F. (1957) Proc. Soc. Exp. Biol. Med. 94, 404-409). We have determined the antithrombin effects of fucoidan on the glycosaminoglycan-binding plasma proteinase inhibitors antithrombin III and heparin cofactor II. Fucoidan enhances the heparin cofactor II-thrombin reaction more than 3500-fold. The apparent second-order rate constant of thrombin inhibition by heparin cofactor II increases from 4 x 10(4) (in the absence of fucoidan) to 1.5 x 10(8) M-1 min-1 as the fucoidan concentration increases from 0.1 to 10 micrograms/ml and then decreases as fucoidan is increased above 10 micrograms/ml. The fucoidan reaction with heparin cofactor II-thrombin is kinetically equivalent to a "template model." Apparent fucoidan-heparin cofactor II and fucoidan-thrombin dissociation constants are 370 and 1 nM, respectively. The enhancement of thrombin inhibition by fucoidan, like heparin and dermatan sulfate, is eliminated by selective chemical modification of lysyl residues either of heparin cofactor II or of thrombin. The fucoidan-antithrombin III reactions with thrombin and factor Xa are accelerated maximally 285- and 35-fold at fucoidan concentrations of 30 and 500 micrograms/ml, respectively. Using human plasma and 125I-labeled thrombin in an ex vivo system, the heparin cofactor II-thrombin complex is formed preferentially over the antithrombin III-thrombin complex in the presence of 10 micrograms/ml fucoidan. Our results indicate that heparin cofactor II is activated by fucoidan in vitro and in an ex vivo plasma system and suggest that the major antithrombin activity of fucoidan in vivo is mediated by heparin cofactor II and not by antithrombin III.     

Solubilization, purification, and characterization of an inositol trisphosphate receptor

Inositol 1,4,5-trisphosphate is a second messenger of the phosphoinositide system which can mobilize calcium from intracellular stores. Rat cerebellum is an abundant source of a receptor for inositol 1,4,5-trisphosphate (Worley, P. F., Baraban, J. M., Supattapone, S., Wilson, V. S., and Snyder, S. H. (1987) J. Biol. Chem. 262, 12132-12136). In this study we have solubilized and purified this receptor to apparent homogeneity from rat cerebellum. Crude membrane, detergent-solubilized, and purified receptor preparations display similar selectivity for inositol 1,4,5-trisphosphate over other inositol phosphates. The purified receptor is globular with a Stokes' radius of approximately 10 nm. Electrophoretic analysis reveals one protein band with an Mr of 260,000. While binding is reversibly inhibited by 300 nM calcium in particulate fractions and detergent-solubilized membranes, the purified protein is not inhibited by calcium concentrations up to 1.5 mM. Inhibition by calcium is reconstituted by addition of detergent-solubilized cerebellar membranes, but not by the cytosolic fraction of cerebellum.