erythro-β-hydroxyaspartic acid in bovine factor IX and factor X

To localize β-hydroxyaspartic acid in factor IX and factor X the two proteins were cleaved with cyanogen bromide and trypsin, respectively. Peptides containing β-hydroxyaspartic acid were isolated and subjected to Edman degradation. The phenylthiohydantoin derivative of β-hydroxyaspartic acid was identified by HPLC in position 3 in the factor IX fragment and in position 1 in the factor X fragment. This corresponds to position 64 in factor IX and position 63 in the light chain of factor X. The assignments were confirmed by subtractive Edman degradation and with the dansyl method.     

The action of defoliants on the energetics of rat liver mitochondria in vitro]

The effect of defoliants butyphos (I), dropp (II), butylcaptacs (III), hinazopin (IV), syhat (V), tetra-n-butylammonium bromide (VI), etrel (VII), gemetrel (VIII), allyl-4-methylpyridinium bromide (IX), 1-amino-cyclopropan-1-carbonate (ACPC) (X) at various concentrations (1 x 10(-5)-2 x 10(-4) M) on respiration, oxidative phosphorylation (OP) and permeability of the inner mitochondrial membrane from rat liver has been studied. It has been established that some of the compounds uncouple OP by increasing the inner mitochondrial membrane permeability for H+ (II) inhibit the respiration in V3 condition and induce less selective permeability for a number of ions (I, III). The other defoliants either induce respiration generally in metabolic states 3 and 4 (IV, VI, IX) or have no effect on the respiration and OP (V, VII, VIII, X). On the whole a good correlation between the common toxicity of the studied preparation (LD50) and their mitochondrial effect has been revealed, therefore the latter can be considered as intracellular "targets" involved in the realization of pesticide action.     

Synthesis of Some New α- and β-(l→2) Linked Disaccharides Containing L-Quinovose (6-Deoxy L-Glucose)

Hepta-O-acetyl-2-0-β-l-quinovopyranosyl-α-d-glucose (VI) and hepta-O-acetyl-2-O-α-l-quinovopyranosyl-β-d-gIucose (VIII) were prepared by the coupling of 2,3,4-tri-O-acetyl-α-l-quinovopyranosyl bromide (IV) with l,3,4,6-tetra-O-acetyl-α-D-glucose (V) in the presence of mercuric cyanide and mercuric bromide in absolute acetonitrile.

Similarly, hepta-O-acetyW-O-α-l-quinovopyranosyl-α-d-galactose (X) and hepta-O-acetyl-2-O-β-L-quinovopyranosyl-α-d-galactose (XI) were prepared by the reaction of IV with 1,3,4,6-tetra-O-acetyl-α-d-galactose (IX).

Removal of the protecting groups of VI, VIII, X and XI afforded the corresponding disaccharides. On treatment with hydrogen bromide, VI, VIII, X and XI gave the corresponding acetobromo derivatives.     

Synthesis of (±)-7,3′- and 7,4′-di-O-methyleriodictyol and of velutin and pilloin

Synthetic 2′-hydroxy-3,4′,6′-trimethoxy-4-benzyloxychalcone (I) affords (±)-7,3′-di-O-methyleriodictyol (II) and 7,3′-di-O-methylluteolin (or velutin, VII) identical with natural samples. Similarly synthetic 2′-hydroxy-4,4′,6′-trimethoxy-3-benzyloxychalcone (X) gives natural (±)-7,4′-di-O-methyleriodictyol (XI) and 7,4′-di-O-methylluteolin (or pilloin, IX). However, attempts to partially etherify II with one mole of prenyl bromide to obtain the natural prenyl ether failed; only the corresponding diprenyloxychalcone (IV) was obtained.     

The factor IX phospholipid-binding site is required for calcium-dependent activation of factor IX by factor XIa

To determine the functional role of the metal-dependent conformational changes in Factor IX, two populations of conformation-specific anti-Factor IX antibodies were prepared. Anti-Factor IX X Mg(II) antibodies bind to Factor IX in the presence of Mg(II) and other metal ions, but not in the absence of metal ions. Anti-Factor IX X Ca(II)-specific antibodies bind to Factor IX in the presence of Ca(II) and Sr(II), but not in the presence of Mn(II), Mg(II), and Ba(II). In the presence of a metal ion that induces the conformational transition recognized by the anti-Factor IX X Mg(II) antibodies, the concentrations of CaCl2 and SrCl2 needed for the half-maximal binding of the anti-Factor IX X Ca(II)-specific antibodies to Factor IX were reduced 3- and 20-fold, respectively. Factor IX binding to phospholipid vesicles was inhibited by the Fab fragments of the anti-Factor IX X Ca(II)-specific antibodies, but was not inhibited by the Fab fragments of the anti-Factor IX X Mg(II) antibodies. Factor XIa activation of Factor IX was also inhibited by the Fab fragments of the anti-Factor IX X Ca(II)-specific antibodies, but not by the anti-Factor IX X Mg(II) antibodies. These results support the hypothesis that Factor IX undergoes two metal-dependent conformational transitions: FIX----FIX'----FIX*. The first transition (FIX----FIX') is metal-dependent but cation-nonselective; the second transition (FIX'----FIX*) is metal-selective for Ca(II) or Sr(II). The second transition results in the expression of conformational determinants necessary for membrane binding and the Ca(II)-dependent activation of Factor IX by Factor XIa. These results suggest chemical similarity between a surface of a domain of Factor XIa and phospholipid vesicles, both of which interact with Factor IX in the presence of Ca(II).     

Acetylation and cleavage of purine nucleosides. Synthesis of 6-azauridine, 5-fluorouridine, and 5-methyluridine.

Inosine (I) when acetylated with acetic anhydride in the presence of acetyl chloride in acetic acid solution (the so called "acid acetylation"), affords an acetylated nucleoside III (75%) along with cleavage products of the nucleoside (hypoxanthine, 19%). The reaction of I with acetyl chloride (7 days) results in the formation of hypoxanthine (95%) and triacetylribofuranosyl chloride (IV) isolated in the form of tetraacetylribofuranose (47%). The acetylated purine nucleoside affords a similar result by reaction with acetyl chloride or acetyl bromide. 2'-Deoxyuridine gives a diacetyl derivative (80%) by reaction with acetyl bromide. On treatment with acetyl bromide, the nucleoside bond of purine nucleosides is quantitatively cleavaged (4 h, 20 degrees C) with the formation of tri-O-acetyl-D-ribofuranosyl bromide (X). The halogenose X affords pure beta-anomers, namely, 1,2,3,5-tetra-O-acetyl-beta-D-ribofuranose (75%), the triacetyl derivatives of 5-methyluridine (XVIIa; 75%, referred to guanosine), 6-azauridine (XVIII; 71%), and 5-fluorouridine (XIXa; 75%).     

Comparative interactions of factor IX and factor IXa with human platelets

Both factor IX and factor IXa were bound to gel filtered platelets in the presence of CaCl2 (2-20 mM) and human alpha-thrombin (0.06-0.2 units/ml) with maximal binding occurring in 10-20 min at 37 degrees C, and rapid reversibility was observed when unlabeled ligands were added in 100-fold molar excess. Competition studies with various coagulation proteins revealed that neither factor XI nor high molecular weight kininogen, at 300-fold molar excess, could compete with 125I-labeled factor IXa for binding sites on thrombin-activated platelets, whereas prothrombin and factor X, in 450-fold molar excess, could displace approximately 15 and 35%, respectively, of bound factor IXa in the absence of added factor VIII. Analysis of saturation binding data in the presence of CaCl2 and thrombin without factors VIII and X indicated the presence of 306 (+/- 57) binding sites per platelet for factor IX (Kd(app) = 2.68 +/- 0.25 nM) and 515 (+/- 39) sites per platelet for factor IXa (Kd = 2.57 +/- 0.14 nM). In the presence of thrombin-activated factor VIII (1-5 units/ml) and factor X (0.15-1.5 microM), the number of sites for factor IX was 316 (+/- 50) with Kd = 2.44 (+/- 0.30) nM and for factor IXa 551 (+/- 48) sites per platelet (Kd = 0.56 +/- 0.05 nM). Studies of competition for bound factor IXa by excess unlabeled factor IX or factor IXa, and direct 125I-labeled factor IXa binding studies in the presence of large molar excesses of factor IX, confirmed the conclusion from these studies that factor IX and factor IXa share approximately 300 low-affinity binding sites per thrombin-activated platelet in the presence of Ca2+ and in the absence of factor VIII and factor X, with an additional 200-250 sites for factor IXa with Kd(app) similar to that for factor IX. The presence of factor VIII and factor X increases by 5-fold the affinity of receptors on thrombin-activated platelets for factor IXa that participate in factor X activation.     

Binding of coagulation factors IX and X to the endothelial cell surface

Bovine coagulation factors IX and X bind to independent sites on bovine aortic endothelial cells. Binding studies with cells maintained serum-free showed that there are at least two classes of binding sites for factor IX and factor X with a dissociation constant of 4.9 x 10(-9) M and 2.1 x 10(-8) M for the respective high affinity sites. Ca+2 was required for specific binding and was reversed by addition of EDTA or EGTA. Competition experiments showed that factor IX and factor IXa bind to the same sites, which are different from the factor X binding sites. Neither binding of factor IX or factor X is inhibited by addition of prothrombin or protein C. Indirect immunofluorescence of factor IX indicated that binding was diffuse on the cell surface.     

The primary structure of coagulation factor IX/factor X-binding protein isolated from the venom of Trimeresurus flavoviridis. Homology with asialoglycoprotein receptors, proteoglycan core protein, tetranectin, and lymphocyte Fc epsilon receptor for immunoglobulin E

An anticoagulant protein, factor IX/factor X-binding protein (IX/X-bp), isolated from the venom of Trimeresurus flavoviridis, binds with factor IX and factor X in the presence of Ca2+ with a 1 to 1 stoichiometry (Atoda, H., and Morita, T. (1989) J. Biochem. (Tokyo) 106, 808-813). Analysis of S-pyridylethylated IX/X-bp by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a 16.0-kDa band (designated the A chain) and a 15.5-kDa band (designated the B chain). These two chains were separated by reversed-phase high performance liquid chromatography, and their complete amino acid sequences were determined by sequencing of the peptides obtained after digestion with lysyl endopeptidase, chymotrypsin, and V8 protease from Staphylococcus aureus and after chemical cleavage with cyanogen bromide. The A chain had an amino-terminal sequence of Asp-Cys-Leu-Ser-Gly- and consisted of 129 residues with Mr 14,830. The B chain has an amino-terminal sequence of Asp-Cys-Pro-Ser-Asp- and consists of 123 residues of Mr 14,440. There was 47% identity between the A and the B chain. The sequence of IX/X-bp showed 25-37% identity with that of the C-type carbohydrate recognition domain-like structure of acorn barnacle lectin, human and rat asialoglycoprotein receptors, the human lymphocyte Fc epsilon receptor for immunoglobulin E, proteoglycan core protein, pancreatic stone protein, and tetranectin. The sequences of the first 18 amino acid residues of both the A and B chains were also, to a certain extent, homologous to the partial amino acid sequence of the b subunit of factor XIII, a member of the beta 2-glycoprotein I-like family. In this region, some similarity with the amino-terminal amino acid sequence of botrocetin was also observed.     

Further biochemical and physicochemical characterization of minor disulfide-bonded (type IX) collagen, extracted from foetal calf cartilage

Minor disulfide-bonded collagen (previously termed X1-X7 and now called type IX collagen) was isolated from foetal calf cartilage after pepsin treatment. At least three native fractions, containing, respectively, the X1X2X3, X4, and X5X6X7 chains, were separated; and from further biochemical and physicochemical experiments (differential scanning calorimetry, electrical birefringence, rotary shadowing), we propose a tentative model for their organization within a parent molecule. X1 and X2 are molecules composed of three chains of apparent Mr 62,000 and 50,000 linked by interchain disulfide bonds and containing pepsin-sensitive regions. The cleavage of at least three of these sites, present within X2, gives rise to the X3 and X5X6X7 fractions composed of molecules 80-100 nm and 40-55 nm in length, respectively. The X5X6X7 fraction is not digested by pepsin at 30 degrees C owing to its high thermal stability (certainly explained by its high hydroxyproline + proline content). This organization is in good accordance with that proposed for chicken cartilage type IX collagen; differences could only exist in the number and (or) the location of the pepsin-sensitive sites.     

Formation of Glucose Isomerase by Streptomyces sp.

Sophoradin (I) [2′,4,4′-trihydroxy-3,3′,5-tris(3-methyl-2-butenyl)chalcone] which had been isolated from “Guang-Dou-Gen” (the root of Sophora subprostrata Chun et T. Chen) was synthesized through Claisen rearrangement. The reaction of p-hydroxybenzaldehyde and 3-chloro-3-methyl-1-butyne (III) gave 4-(1,1-dimethylpropargyloxy)benzaldehyde (VIII), which was catalytically hydrogenated over Lindlar catalyst to afford 4-(1,1-dimethylallyloxy)benzaldehyde (IX). IX was converted to 4-hydroxy-3-(3-methyl-2-butenyl)benzaldehyde (X) by Claisen rearrangement. The reaction of X and III gave 3-(3-methyl-2-butenyl)-4-(1,1-dimethylpropargyloxy)benzaldehyde (XI). Condensation of 2-hydroxy-4-(1,1-dimethylpropargyloxy)acetophenone (IV) and XI in alkaline solution gave a chalcone (XIII), which was catalytically hydrogenated over Lindlar catalyst to give 2′-hydroxy-4,4′-bis(1,-dimethylallyloxy)-3-(3-methyl-2-butenyl)chalcone (XIV). XIV was converted to I by Claisen rearrangement.     

In Vivo Imaging and Quantification of Carbonic Anhydrase IX Expression as an Endogenous Biomarker of Tumor Hypoxia

Carbonic anhydrase IX (CA IX) is a transmembrane protein that has been shown to be greatly upregulated under conditions of hypoxia in many tumor cell lines. Tumor hypoxia is associated with impaired efficacy of cancer therapies making CA IX a valuable target for preclinical and diagnostic imaging. We have developed a quantitative in vivo optical imaging method for detection of CA IX as a marker of tumor hypoxia based on a near-infrared (NIR) fluorescent derivative of the CA IX inhibitor acetazolamide (AZ). The agent (HS680) showed single digit nanomolar inhibition of CA IX as well as selectivity over other CA isoforms and demonstrated up to 25-fold upregulation of fluorescent CA IX signal in hypoxic versus normoxic cells, which could be blocked by 60%–70% with unlabeled AZ. CA IX negative cell lines (HCT-116 and MDA-MB-231), as well as a non-binding control agent on CA IX positive cells, showed low fluorescent signal under both conditions. In vivo FMT imaging showed tumor accumulation and excellent tumor definition from 6–24 hours. In vivo selectivity was confirmed by pretreatment of the mice with unlabeled AZ resulting in >65% signal inhibition. HS680 tumor signal was further upregulated >2X in tumors by maintaining tumor-bearing mice in a low oxygen (8%) atmosphere. Importantly, intravenously injected HS680 signal was co-localized specifically with both CA IX antibody and pimonidazole (Pimo), and was located away from non-hypoxic regions indicated by a Hoechst stain. Thus, we have established a spatial correlation of fluorescence signal obtained by non-invasive, tomographic imaging of HS680 with regions of hypoxia and CA IX expression. These results illustrate the potential of HS680 and combined with FMT imaging to non-invasively quantify CA IX expression as a hypoxia biomarker, crucial to the study of the underlying biology of hypoxic tumors and the development and monitoring of novel anti-cancer therapies.     

Hydrophobic contact between the two epidermal growth factor-like domains of blood coagulation factor IX contributes to enzymatic activity

The three-dimensional structure of activated factor IX comprises multiple contacts between the two epidermal growth factor (EGF)-like domains. One of these is a salt bridge between Glu(78) and Arg(94), which is essential for binding of factor IXa to its cofactor factor VIII and for factor VIII-dependent factor X activation (Christophe, O. D., Lenting, P. J., Kolkman, J. A., Brownlee, G. G., and Mertens, K. (1998) J. Biol. Chem. 273, 222-227). We now addressed the putative hydrophobic contact at the interface between the EGF-like domains. Recombinant factor IX chimeras were constructed in which hydrophobic regions Phe(75)-Phe(77) and Lys(106)-Val(108) were replaced by the corresponding sites of factor X and factor VII. Activated factor IX/factor X chimeras were indistinguishable from normal factor IXa with respect to factor IXa enzymatic activity. In contrast, factor IXa(75-77)/factor VII displayed approximately 2-fold increased factor X activation in the presence of factor VIII, suggesting that residues 75-77 contribute to cofactor-dependent factor X activation. Activation of factor X by factor IX(106-108)/factor VII was strongly decreased, both in the absence and presence of factor VIII. Activity could be restored by simultaneous substitution of the hydrophobic sites in both EGF-like domains for factor VII residues. These data suggest that factor IXa enzymatic activity requires hydrophobic contact between the two EGF-like domains.     

Zymogen factor IX potentiates factor IXa-catalyzed factor X activation

Intrinsic factor X activation is accelerated >10(7)-fold by assembly of the entire complex on the activated platelet surface. We have now observed that increasing the concentration of zymogen factor IX to physiologic levels ( approximately 100 nM) potentiates factor IXa-catalyzed activation of factor X on both activated platelets and on negatively charged phospholipid vesicles. In the presence and absence of factor VIIIa, factor IX (100 nM) lowered the K(d,appFIXa) approximately 4-fold on platelets and 2-10-fold on lipid vesicles. Treatment of two factor IX preparations with active-site inhibitors did not affect these observations. Autoradiographs of PAGE-separated reactions containing either (125)I-labeled factor IX or (125)I-labeled factor X showed that the increased factor X activation was not due to factor Xa-mediated feedback activation of factor IX and that there was increased cleavage of factor X heavy chain in the presence of factor IX in comparison with control reactions but only in the presence of both the enzyme and the surface. Since plasma concentrations of prothrombin, factor VII, protein C, or protein S did not by themselves potentiate factor Xa generation and did not interfere with the potentiation of the reaction of factor IX, the effect is specific for factor IX and is not attributable to the Gla domain of all vitamin K-dependent proteins. These observations indicate that under physiologic conditions, plasma levels of the zymogen factor IX specifically increase the affinity of factor IXa for the intrinsic factor X activation complex.     

A new method of chemical modification of N6-amino group in adenine nucleotides with formaldehyde and a thiol and its application to preparing immobilized ADP and ATP.

Reaction of AMP with formaldehyde and 3-mercaptopropionic acid at pH 11.7 gave a new AMP derivative, N6-[(2-carboxyethyl)thiomethyl]-AMP (I) in 91% yield and reaction at pH 3.1 gave another new derivative, N6,N6-bis[(2-carboxyethyl)thiomethyl]-AMP (II) in 57% yield. The structures were determined by their 13C and 1H nuclear magnetic resonance spectra coupled with those of the simple analogues, N6-[(2-carboxyethyl)thiomethyl]-9-methyladenine (III) and N6,N6-bis[(2-carboxyethyl)thiomethyl]-9-methyladenine (IV) which were synthesized from 9-methyladenine in the same way as for derivatives I and II. ADP and ATP were treated in the same way as AMP to afford the corresponding carboxyl derivatives, N6-[(2-carboxyethyl)thiomethyl]-ADP (V), N6-[(2-carboxyethyl)thiomethyl]-ATP (VI), N6,N6-bis[(2-carboxyethyl)thiomethyl]-ADP (X) and N6,N6-bis[(2-carboxyethyl)thiomethyl]-ATP (XI) in 71%, 75%, 53% and 40% yield, respectively. These compounds were coupled to 1,3-diaminopropane with a water-soluble carbodiimide to give the corresponding amino derivatives, N6-([N-3-aminopropyl)carbamoylethyl]thiomethyl)-ADP (VIII), N6-(N-(3-aminopropyl)carbamoylethyl]thiomethyl)-ATP (IX), N6,N6-bis([N-(3-aminopropyl)carbamoylethyl]thiomethyl)-ADP (XIII), and N6,N6-bis([N-(3-aminopropyl)carbamoylethyl]thiomethyl)-ATP (XIV), which were further bound to CNBr-activated dextran to give new polymer-bound derivatives of ADP and ATP. These free and bo-nd derivatives were tested for their coenzymic activities against several kinases. The activities of the ADP derivatives, V, VIII, X, XIII, dextran-bound VIII, and dextran-bound XIII against acetate kinase were 82%, 81%, 68%, 55%, 35%, and 15%, respectively, relative to ADP and those of the ATP derivatives, VI, IX, XI, XIV, dextran-bound IX, and dextran-bound XIV against hexokinase were 88%, 94%, 60%, 81%, 58%, and 49%, respectively, relative to ATP.     

Binding of Ca<Superscript>2+</Superscript> and Zn<Superscript>2+</Superscript> to factor IX/X-binding protein from venom of <Emphasis Type="Italic">Agkistrodon halys</Emphasis> Pallas: stabilization of the structure during GdnHCl-induced and thermally induced denaturation

Coagulation factor IX/coagulation factor X binding protein from the venom of Agkistrodon halys Pallas (AHP IX/X-bp) is a unique coagulation factor IX/coagulation factor X binding protein (IX/X-bp). Among all IX/X-bps identified, only AHP IX/X-bp is a Ca²⁺- and Zn²⁺-binding protein. The binding properties of Ca²⁺ and Zn²⁺ ions binding to apo-AHP IX/X-bp and their effects on the stability of the protein have been investigated by isothermal titration calorimetry, fluorescence spectroscopy, and differential scanning calorimetry. The results show that AHP IX/X-bp has two metal binding sites, one specific for Ca²⁺ with lower affinity for Zn²⁺ and one specific for Zn²⁺ with lower affinity for Ca²⁺. The bindings of Ca²⁺ and Zn²⁺ in the two sites are entropy- and enthalpy-driven. The binding affinity of AHP IX/X-bp for Zn²⁺ is 1 order of magnitude higher than for Ca²⁺ for either high-affinity binding or low-affinity binding, which accounts for the existence of one Zn²⁺ in the purified AHP IX/X-bp. Guanidine hydrochloride (GdnHCl)-induced and thermally induced denaturations of Ca²⁺–Ca²⁺-AHP IX/X-bp, Zn²⁺–Zn²⁺-AHP IX/X-bp, and Ca²⁺–Zn²⁺-AHP IX/X-bp are all a two-state processes with no detectable intermediate state(s), indicating the Ca²⁺/Zn²⁺-induced tight packing of the protein. Ca²⁺ and Zn²⁺ increase the structural stability of AHP IX/X-bp against GdnHCl or thermal denaturation to a similar extent. Although Ca²⁺ and Zn²⁺ have no obvious effect on the secondary structure of AHP IX/X-bp, they induce different rearrangements in local conformation. The Zn²⁺-stabilized specific conformation of AHP IX/X-bp may be helpful to its recognition of the structure of coagulation factor IX. This work suggests that in vitro, Ca²⁺ plays a structural rather than an active role in the anticoagulation of AHP IX/X-bp, whereas Zn²⁺ plays both structural and active roles in the anticoagulation. In blood, Ca²⁺ binds to AHP IX/X-bp and stabilizes its structure, whereas Zn²⁺ cannot bind to AHP IX/X-bp owing to the low Zn²⁺ concentration. AHP IX/X-bp prolongs the clotting time in vivo through its binding only with coagulation factor X/activated coagulation factor X.     

Blood clotting factor IX Niigata: substitution of alanine-390 by valine in the catalytic domain

Factor IX Niigata is a mutant factor IX responsible for the moderately severe hemophilia B in a patient who has a normal level of factor IX antigen with reduced clotting activity (1-4% of normal). We reported previously that the purified mutant protein could be converted to the factor IXa beta form by factor XIa/Ca2+ at a rate similar to that in the case of normal factor IX, but the resulting mutant factor IXa beta could not activate factor X in the presence of factor VIII, Ca2+, and phospholipids (Yoshioka, A. et al. (1986) Thromb. Res. 42, 595-604). In the present study, we analyzed factor IX Niigata at the structural level to elucidate the molecular abnormality responsible for the loss of clotting activity. Amino acid sequence analysis of a peptide obtained on lysyl endopeptidase digestion, coupled with subsequent SP-V8 digestion, demonstrated that the alanine at position 390 was substituted by valine in the catalytic domain of the factor IX Niigata molecule.     

Coordinate binding studies of the substrate (factor X) with the cofactor (factor VIII) in the assembly of the factor X activating complex on the activated platelet surface

The assembly of the factor X activating complex on the platelet surface requires the occupancy of three receptors: (1) enzyme factor IXa, (2) cofactor factor VIII(a), and (3) substrate factor X. To further evaluate this three-receptor model, simultaneous binding isotherms of (125)I-factor X and (131)I-factor VIII(a) to activated platelets were determined as a function of time and also as a function of the concentrations of both ligands in the presence of active site-inhibited factor IXa (45 nM) and 5 mM CaCl(2). In the presence of active site-inhibited factor IXa and factor VIIIa there are two independent factor X binding sites: (1) low affinity, high capacity (approximately 9000 sites/platelet; K(d) approximately 380 nM) and (2) low capacity, high affinity (1700 sites/platelet; K(d) approximately 30 nM). A single specific and selective factor X binding site was expressed (1200 sites/platelet; K(d) approximately 9 nM) when the shared factor X/factor II site was blocked by excess factor II (4 microM). In the presence of active site-inhibited factor IXa (4 nM) and factor II (4 microM), factor X binds to 3-fold more platelet sites than procofactor VIII with relatively low affinity (K(d) approximately 250 nM). The activation of procofactor VIII to factor VIIIa increases the affinity of binding to platelets of both factor VIIIa ( approximately 4-fold to K(d) approximately 0.8-1.5 nM) and factor X ( approximately 25-50-fold to K(d) approximately 5-9 nM). In the presence of excess zymogen factor IX, which blocks the shared factor IX/factor IXa binding site, the substrate, factor X, and the active cofactor, factor VIIIa, form a 1:1 stoichiometric complex. These coordinate binding studies support the conclusion that factor X initially binds to a high-capacity, low-affinity platelet binding site shared with prothrombin, which then presents factor X to a specific high-affinity site consisting of factor VIIIa bound to a high-affinity, low-capacity receptor on activated platelets.     

Thiol cross-linking of cytoplasmic loops in the lactose permease of Escherichia coli

The N- and C-terminal halves of lactose permease, each with a single-Cys residue in a cytoplasmic loop, were coexpressed, and cross-linking was studied in the absence or presence of ligand. Out of the 68 paired-Cys mutants in cytoplasmic loops IV/V and VIII/IX or X/XI, three pairs in loop IV/V and X/XI, (i) Arg135 --> Cys/Thr338 --> Cys, (ii) Arg134 --> Cys/Val343 --> Cys, and (iii) Arg134 --> Cys/Phe345 --> Cys, form a spontaneous disulfide bond, indicating that loops IV/V and X/XI are in close proximity. In addition, specific paired-Cys residues in loop IV/V (132-138) and loop VIII/IX (282-290) or loop X/XI (335-345) cross-link with iodine and/or the homobifunctional cross-linking agents N, N'-o-phenylenedimaleimide, N,N'-p-phenylenedimaleimide, and 1, 6-bis(maleimido)hexane. The results demonstrate that loop IV/V is close to both loop VIII/IX and loop X/XI. On the other hand, similar though less extensive cross-linking studies indicate that neither the N terminus nor loop II/III appear to be close to loops VIII/IX or X/XI. The findings suggest that the longer cytoplasmic loops are highly flexible and interact in a largely random fashion. However, although a Cys residue at position 134 in loop IV/V, for example, is able to cross-link with a Cys residue at each position in loop VIII/IX or loop X/XI, Cys residues at other positions in loop IV/V exhibit markedly different cross-linking patterns. Therefore, although the domains appear to be very flexible, the interactions are not completely random, suggesting that there are probably at least some structural constraints that limit the degree of flexibility. In addition, evidence is presented suggesting that ligand binding induces conformational alterations between loop IV/V and loop VIII/IX or X/XI.     

Type X collagen, a product of hypertrophic chondrocytes.

The synthesis of collagen types IX and X by explants of chick-embryo cartilages was investigated. When sternal cartilage labelled for 24h with [3H]proline was extracted with 4M-guanidinium chloride, up to 20% of the 3H-labelled collagen laid down in the tissue could be accounted for by the low-Mr collagenous polypeptides (H and J chains) of type IX collagen; but no type X collagen could be detected. Explants of tibiotarsal and femoral cartilages were found to synthesize type IX collagen mainly in zones 1 and 2 of chondrocyte proliferation and elongation, whereas type X collagen was shown to be a product of the hypertrophic chondrocytes in zone 3. Pulse-chase experiments with tibiotarsal (zone-3) explants demonstrated a time-dependent conversion of type X procollagen into a smaller species whose polypeptides were of Mr 49 000. The processed chains [alpha 1(X) chains] were shown by peptide mapping techniques to share a common identity with the pro alpha 1(X) chains of Mr 59 000. No evidence for processing of type IX collagen was obtained in analogous pulse-chase experiments with sternal tissue. When chondrocytes from tibiotarsal cartilage (zone 3) were cultured on plastic under standard conditions for 4-10 weeks they released large amounts of type X procollagen into the medium. However, 2M-MgCl2 extracts of the cell layer were found to contain mainly the processed collagen comprising alpha 1(X) chains. The native type X procollagen purified from culture medium was shown by rotary shadowing to occur as a short rod-like molecule 148 nm in length with a terminal globular extension, whereas the processed species comprising alpha 1(X) chains of Mr 49 000 was detected by electron microscopy as the linear 148 nm segment.