Localization of the high molecular weight kininogen binding site in the heavy chain of human factor XI to amino acids phenylalanine 56 through serine 86

We have previously demonstrated that a monoclonal antibody (5F7) directed against the heavy chain region of factor XI inhibits the binding of factor XI to high molecular weight kininogen (high Mr kininogen) and the surface-mediated proteolytic activation of factor XI by factor XIIa in the presence of high Mr kininogen. In order to identify the structural domain of factor XI that binds high Mr kininogen, CNBr-digested factor XI was passed over a 5F7 antibody affinity column. One of two CNBr peptides that bound to this 5F7 affinity column inhibited binding of 125I-factor XI to high Mr kininogen, as did intact factor XI. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate of an inhibitory peptide purified by high performance liquid chromatography revealed an Mr of 10,000-15,000. Gas-phase sequencing of this peptide revealed the following amino-terminal sequence: X-X-Val-Thr-Gln-Leu-Leu-Lys-Asp-Thr. These data together with the amino acid composition of the isolated peptide indicate that both the epitope recognized by antibody 5F7 and at least a portion of the high Mr kininogen binding site are contained within the amino-terminal portion of factor XI comprising residues Glu-1 through Met-102. Further cleavage of this peptide with o-iodosobenzoic acid at a tryptophanyl peptide bond revealed that an Mr 5,000 peptide (with the amino-terminal sequence Trp-Phe-Thr-Cys-Val-Leu) bound to a high Mr kininogen affinity column and inhibited binding of 125I-factor XI to high Mr kininogen. Finally, a synthetic peptide comprising residues Phe-56 through Ser-86 inhibited 125I-factor XI binding to high Mr kininogen. These experiments strongly suggest that the high Mr kininogen binding site is contained within the domain in the heavy chain region of factor XI comprising residues Phe-56 through Ser-86.     

Binding site for vitamin K-dependent protein S on complement C4b-binding protein

Half of the protein S in plasma is present as a complex with a C4b-binding protein (C4bp), a complement component (Mr 570,000). In this study, the protein S-binding site on C4bp was examined by using monoclonal anti-C4bp-IgGs. C4bp was cleaved by chymotryptic digestion into seven NH2-terminal arm fragments (Mr 48,000) and a COOH-terminal core fragment (Mr 160,000). The COOH-terminal fragment inhibited the cofactor activity of protein S and its binding to C4bp in a dose-dependent manner. A monoclonal anti-C4bp-IgG (MFbp16), which binds to the COOH-terminal fragment, inhibited the binding of protein S to C4bp. The chymotryptic digest of the reduced and carboxymethylated COOH-terminal fragment was subjected to MFbp16-Sepharose 4B column affinity chromatography, and a peptide of Mr 2,500 was obtained. Protein S bound to the Mr 2,500 peptide, and this binding was inhibited by C4bp in a dose-dependent manner. The sequence of this peptide corresponded to Ser447-Tyr467 near the COOH terminus of the C4bp subunit. MFbp16, which bound to Mr 570,000 C4bp (C4bp-high), did not bind to Mr 510,000 C4bp (C4bp-low) in human plasma that does not form a complex with protein S. This suggests that C4bp-low lacks the protein S-binding site present in the COOH-terminal region of C4bp-high. Since C4bp-low also dissociates into identical subunits when reduced, the interchain disulfide bond region that links the seven subunits of C4bp appears to be closer to the NH2-terminal end than the protein S-binding site.     

Hemoglobin binds to the amino-terminal 23-residue fragment of human erythrocyte band 3 protein

Hemoglobin, aldolase and glyceraldehyde 3-phosphate dehydrogenase are known to bind to the cytoplasmic domain of band 3 protein. Binding of glycolytic enzymes to band 3 protein is inhibited by its amino-terminal fragments. To precisely localize the sequence portion of band 3 protein to which hemoglobin binds and to see whether the same region of amino-acid sequence binds both hemoglobin and glycolytic enzymes, a simple, direct solid-phase binding assay was developed. Peptides generated from the 23-kDa fragment by trypsin, cyanogen bromide and mild acid hydrolysis were used as inhibitors to determine the minimal sequence structure involved in the binding of the 23-kDa fragment to hemoglobin. The shortest peptide which inhibits the binding of the 23-kDa fragment is an acid cleavage peptide containing the sequence positions 1 to 23. This sequence is unusual as 14 of its residues are negatively charged, it contains no basic residues and has its amino terminus blocked. Using aldolase, glyceraldehyde-3-phosphate dehydrogenase and hemoglobin as competitive inhibitors in the binding of 23-kDa fragment, the affinity of hemoglobin to this fragment appears several-fold weaker than that of both the enzymes. These findings demonstrate that glycolytic enzymes and hemoglobin bind competitively to the same polyanionic sequence region of band 3 protein.     

Degradation of fibronectin by human leukocyte elastase. Release of biologically active fragments

We have identified the biological activity of three polypeptides released by limited proteolysis of human plasma fibronectin by leukocyte elastase. A Mr = 140,000 peptide contains cell-spreading activity; a Mr = 60,000 peptide mediates binding to denatured collagen (gelatin), and a Mr = 29,000 peptide contains glutaminyl residues responsible for the transglutaminase (blood coagulation factor XIIIa)-catalyzed incorporation of amines. More extensive proteolysis yielded numerous peptides, including a Mr = 40,000 peptide derived from the Mr = 60,000 peptide which retains gelatin-binding activity. Quantification of the gelatin-binding peptides is consistent with two binding sites per dimeric fibronectin molecule of Mr = 440,000. Both Mr = 60,000 and 40,000 gelatin-binding peptides were enriched with half-cystine residues, containing 28 and 25, respectively, but devoid of cysteine. This, coupled with the electrophoretic behavior of both peptides, was consistent with the presence of intramolecular disulfide bonds in the gelatin-binding domain. Intact fibronectin contains 1 free cysteine residue/monomer, as recently described. This cysteine reacts with 5,5'-dithiobis(2-nitrobenzoic acid) very slowly under nondenaturing conditions but rapidly when fibronectin is denatured. The free cysteine is located in the Mr = 140,000 peptide. While the Mr = 40,000 and 60,000 gelatin-binding peptides bind to gelatin with an affinity about 30-fold and 5-fold less than intact fibronectin (based on a monomeric fibronectin Mr = 220,000), neither gelatin-binding peptide supports spreading of fibronectin-deficient test cells on gelatin or tissue culture plastic substrates. The purified Mr = 140,000 peptide supported cell spreading on plastic, retaining about one-half of the spreading activity of intact fibronectin on a weight basis. These data confirm recent results, suggesting multiple, protease- resistant domains with discrete biological functions within fibronectin. Our results, together with established data, suggest a model for the location of the transglutaminase-reactive glutaminyl residues, gelatin binding, and cell-adhesive domains in fibronectin. The release of univalent, biologically active fibronectin fragments by elastase, a major physiologically released inflammatory protease of human leukocytes, suggests a new potential mechanism for alteration of cell connective tissue interactions at sites of inflammation in vivo.     

Identification of the C3b receptor-binding domain in third component of complement

We report here that complement receptor type one (CR1) binds to a region of C3b that is contained within the NH2 terminus of the alpha' chain. In an enzyme-linked immunosorbent assay, CR1 bound to C3b, iC3b, and C3c but not to C3d, and this binding was inhibited by soluble C3b and C3c. Further attempts to generate a small C3 fragment capable of binding CR1 were unsuccessful. However, elastase degradation of C3 generated four species of C3c (C3c I-IV), two of which bound CR1. NH2-terminal sequence analysis and sodium dodecyl sulfate-gel electrophoresis of the C3cs indicated that the beta chains and the 40,000-dalton COOH-terminal alpha' chain fragments were identical; the NH2-terminal alpha' chain fragments of C3c I-IV varied from 21,000 to 27,000 daltons and accounted for the differential binding to CR1. C3c-I and II, which do not bind CR1, were missing 8 and 9 residues from the NH2 terminus of the alpha' chain when compared with the intact alpha' chain of C3b. C3c-III and IV, which bind CR1, had NH2 termini identical to the intact NH2-terminal alpha' chain of C3b. Using iodinated concanavalin A and endoglycosidase H, we showed that the NH2-terminal alpha' chains of C3c-I and III were glycosylated, while C3c-II and IV were not. Therefore, these data indicated that the amino terminus of the NH2-terminal alpha' chain fragment of C3c was responsible for binding CR1 while the COOH terminus of this fragment was not involved since the presence or absence of this region in C3c did not affect CR1 binding to C3c. Subsequently, two peptides were synthesized from the NH2-terminal alpha' chain fragment of C3c: X42, 42 residues in length from the NH2 terminus and C30, 30 residues in length from the COOH terminus. X42 inhibited binding of CR1 to C3b, and this effect was also observed with antipeptide antibodies against the X42 peptide. The C30 and other C3-derived peptides and antipeptide antibodies had no effect on the binding of CR1 to C3b.     

End-label fingerprintings show that the N- and C-termini of actin are in the contact site with gelsolin

Gelsolin was cleaved by chymotrypsin or thermolysin into an N-terminal Mr 45,000 fragment (45N) and a C-terminal Mr 38,000 fragment (38C). The N-terminal half was further cleaved into two fragments with Mr 17,000 (17N) and Mr 28,000 (28N). These fragments were complexed with actin and cross-linked with 1-ethyl-3-[3-(dimethylamino)prophyl]carbodiimide (EDC) to introduce covalent bonds into their contact sites. The location of these bonds was mapped along the actin sequence by end-label fingerprinting with highly sensitive probes for the N- and C-termini of actin. The mapping studies revealed that two gelsolin N-terminal fragments (17N and 28N) were cross-linked with the actin C-terminal segment. The result indicates that the actin N- and C-terminal segments are in the binding site of gelsolin.     

Mechanism-based fragmentation of coenzyme A transferase. Comparison of alpha 2-macroglobulin and coenzyme A transferase thiol ester reactions

The plasma proteins, alpha 2-macroglobulin and complement components 3 and 4, contain an internal thiol ester involving a glutamyl and cysteinyl residue. The thiol ester is susceptible to cyclization at greater than 37 degrees C and forms an unstable 5-oxyproline intermediate. The latter can be hydrolyzed to produce two peptide fragments. We propose that enzymes having activated glutamyl residues as part of their catalytic mechanisms may undergo an analogous cyclization and peptidyl cleavage. As a model, we have investigated pig heart succinyl-CoA:3-keto acid transferase. When the CoA-enzyme thiolester intermediate is heated at pH 7.4 and 70 degrees C for 1 h, approximately 60% of the Mr = 60,000 subunits are cleaved to give Mr = 40,000 and 20,000 fragments. We have shown that formation of the enzyme thiolester is an obligate precursor for the protein fragmentation. However, the reaction was incomplete with a maximum of approximately 65% cleavage at times greater than 60 min. These results suggest that there is a competing, deactivation reaction; namely, the thiol ester and oxyproline intermediates are hydrolyzed to regenerate the active site glutamic acid. Although the maximum rate of cleavage is at 70 degrees C, approximately 15% autolysis also occurs at 37 degrees C. The Mr = 40,000 fragment had the same amino terminal sequence as the Mr = 60,000 subunit, (Trp-Lys-Phe-Tyr-Thr-Asp-Ala-Val-Glu-Ala-). No amino terminal could be detected for the Mr = 20,000 fragment, even after digesting the fragment with pyroglutaminase. Peptide maps of the fragments and the uncleaved subunit indicate that the fragments are generated in parallel. The size of the fragments puts the active site about two-thirds of the way from the amino terminal of the protein.     

A discontinuous factor H binding site in the third component of complement as delineated by synthetic peptides

Factor H, a very important regulator of alternative pathway activation, exerts its effects by binding to the third component complement, C3. In this study we present evidence that factor H reacts with at least two sites in the third component of complement (C3), and we have mapped one of these sites within the C3d fragment of C3. By using direct binding assays of an anti-human H anti-idiotypic antibody (alpha alpha H) and of H to C3 fragments, it was shown that both bound to the C3b and C3d (but not to C3c) fragments of C3. Cleavage of C3d by CNBr generated two major fragments with Mr values of 12,500 (residues 997-1107) and 8,600 (residues 1178-1252). Binding studies with these two fragments showed that only the Mr 8,600 fragment bound to both H and alpha alpha H. Several synthetic peptides (A58, 1192-1249; P28, 1187-1214; P16, 1194-1209; P14, 1201-1214; B17, 1206-1222; J28, 1222-1249; and J16, 1234-1249) were synthesized according to the primary sequence of the Mr 8,600 fragment. Based on the differential binding of these synthetic peptides to H, their inhibitory effect on H binding to C3b or C3d, and their effect on H cofactor activity, we mapped the H binding site in C3 to a discontinuous site spanning residues 1187-1249 of the C3 sequence. By studying the inhibition of H binding to C3b or C3d by the different synthetic peptides, we also present evidence that a second binding site in C3b for H exists.     

Localization of the covalent C3b-binding site on C4b within the complement classical pathway C5 convertase, C4b2a3b

C5 convertase of the classical complement pathway is a trimolecular protein complex consisting of C4b, C2a, and C3b. In the complex there is an ester bond between C3b and C4b. We analyzed the C5 convertase formed on erythrocytes and localized the covalent binding site of C3b to a small region on C4b. The covalently linked C4b.C3b complex was purified from a detergent extract of the erythrocytes and digested with lysyl endopeptidase. An Mr 17,000 fragment containing the ester linkage between C4b and C3b was purified and its amino-terminal sequence was examined. Two amino acids were obtained at each cycle and identified with those in the sequences of C3 and C4. The sequence derived from C3 corresponded to the thioester region. The sequence derived from C4 started at Ala-1186. Alkali treatment of the fragment yielded an Mr 7,000 peptide derived from C4, which thus appeared to span the region of C4 from Ala-1186 to Lys-1259. Therefore, the covalent C3b-binding site on C4b is located within a 74-residue region of the primary structure. This finding supports the notion that after cleavage of C3 by the C4b2a complex, the covalent binding of metastable C3b to C4b is a specific reaction to form a trimolecular complex with a defined quaternary structure.     

Localization of thrombomodulin-binding site within human thrombin

A binding site for thrombomodulin on human thrombin (alpha-thrombin) was elucidated by identifying an epitope for a monoclonal antibody for thrombin (MT-6) which inhibited the activation of protein C by the thrombin-thrombomodulin complex by directly inhibiting the binding of thrombin to thrombomodulin. An 8.5-kDa fragment isolated by digestion of thrombin with Staphylococcus aureus V8 protease followed by reversed-phase high performance liquid chromatography (HPLC) and a peptide isolated by reversed-phase HPLC after reduction of the 8.5-kDa fragment, which was composed of three peptides linked by disulfide-bonds, bound directly to MT-6 and thrombomodulin. The amino acid sequence of the peptide coincided with the sequence of residues Thr-147 to Asp-175 of the B-chain of thrombin. A synthetic peptide corresponding to Thr-147 to Ser-158 of the B-chain inhibited the binding of thrombin to thrombomodulin. Elastase-digested thrombin, which was cleaved between Ala-150 and Asn-151, lost its binding affinity for both MT-6 and thrombomodulin. These findings indicate that the binding site for thrombomodulin is located within the sequence between Thr-147 and Ser-158 of the B-chain.     

Characterization of antisera to a synthetic carboxyl-terminal peptide of the glucose transporter protein

Peptides corresponding to amino acid residues 1-12 of the amino terminal and 480-492 of the carboxyl terminal of the deduced sequence of the glucose transporter were synthesized and used to produce site-specific polyclonal antipeptide sera. In a solid-phase radioimmunoassay, antiserum to the carboxyl terminal recognizes peptide 480-492 and purified human erythrocyte glucose transporter, but not peptide 1-12. Antiserum to the amino terminal recognizes peptide 1-12 but neither peptide 480-492 nor the erythrocyte transporter. The antiserum to the carboxyl terminal specifically immunoblots the Mr 55,000 glucose transporter in erythrocyte membranes and the purified erythrocyte transporter. It also recognizes a Mr 40,000-60,000 polypeptide in membranes of cells derived from different mammalian species and tissues including insulin-sensitive rat adipocytes as well as a Mr 20,000 tryptic fragment of the transporter which contains the site for photolabeling by cytochalasin B. Antiserum to the carboxyl terminal of the transporter binds specifically to leaky erythrocyte membranes but not to intact erythrocytes. This binding is saturable and competitively inhibited by peptide 480-492. Using immunofluorescence microscopy, this antiserum detects glucose transporter protein in permeabilized erythrocytes, but not in intact erythrocytes. These studies provide immunochemical evidence in support of the predicted cytoplasmic orientation of the carboxyl terminus of the glucose transporter, allow us to suggest a spatial relationship of the cytochalasin B binding site to the carboxyl terminal of the glucose transporter and suggest that antisera directed to the carboxyl terminal domain of the protein may be useful for the immunocytochemical localization of the glucose transporter.     

A binary complex of troponin-I and troponin-T from Akazara scallop striated adductor muscle.

A binary complex consisting of Mr 19,000 and Mr 40,000 components was co-purified with troponin from a crude troponin fraction of Akazara scallop (Chlamys nipponensis akazara) striated adductor muscle. This complex is incapable of conferring Ca(2+)-sensitivity to rabbit reconstituted actomyosin Mg-ATPase activity, rather strongly inhibiting it, but became capable on further complexing with Akazara scallop troponin-C. To examine the effects of the Mr 19,000 and Mr 40,000 components on the ATPase activity, they were separated from each other by CM-Toyopearl column chromatography. The Mr 19,000 component strongly inhibited the Mg-ATPase activity of actomyosin-tropomyosin and the inhibition was reversed by further addition of the Akazara scallop troponin-C. On the other hand, the Mr 40,000 component slightly increased it. On hybridization with the Akazara scallop troponin subunits, the Mr 19,000 and Mr 40,000 components were shown to be able to substitute for troponin-I and troponin-T, respectively. The amino acid compositions of the Mr 40,000 component and troponin-T were almost identical, and those of the Mr 19,000 component and Mr 17,000 C-terminal fragment of the troponin-I resembled each other fairly well. From these results, it may be concluded that the Mr 19,000-40,000 binary complex is the troponin-I-troponin-T complex.     

Multidomain binding of transforming growth factor alpha to the epidermal growth factor receptor.

Solubilized epidermal growth factor receptor (EGF-R) has been used in an extension of the Geysen epitope mapping protocol in order to provide additional insight into the amino acid residues in human transforming growth factor alpha (hTGF alpha) which are critical to recognition and binding. Overlapping heptapeptides which encompassed the 50 amino acid primary sequence of hTGF alpha were synthesized on a polyethylene solid phase, and the amount of detergent-solubilized EGF-R bound to each peptide was measured using ELISA. EGF-R appeared to bind reproducibly to four heptapeptides cognate to sequences in both the N- and C-domains of hTGF alpha (residues 22-28, 28-34, 36-42, and 44-50). Visualization of these four regions on three-dimensional solution phase structures of hTGF alpha, derived from 1H NMR measurements [Kline, T.-P., Brown, F.K., Brown, S.C., Jeffs, P.W., Kopple, K.D., & Mueller, L. (1990) Biochemistry 29, 7805-7813], indicated that the peptide segments are located on a single face of the protein and suggested the presence of a potential receptor binding cavity. If peptide segments within both the N- and C-domains of hTGF alpha are involved in binding to EGF-R, then this has direct consequences for possible molecular mechanisms by which receptor activation might take place. For example, the observed conformational flexibility in the six NMR-derived hTGF alpha structures due to variations in the main-chain torsion angles of Val-33, in combination with the involvement of residues from both domains in the proposed binding cavity, may imply that receptor activation results from interdomain reorientation in the protein ligand.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization and photoaffinity labeling of a calcitonin gene-related peptide receptor solubilized from human cerebellum.

Calcitonin gene-related peptide (CGRP) receptors were solubilized from human (h) cerebellum with use of the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS). Scatchard analysis of equilibrium binding data indicated that the soluble extract contained a single class of CGRP binding sites with apparent dissociation constants of 50 pM for the intact 125I-hCGRP-I(1-37) and 160 pM for the antagonist 125I-hCGRP-I(8-37). Unlabeled hCGRP-I and -II and hCGRP-I(8-37) displaced 125I-hCGRP-I from solubilized CGRP receptors with similar potencies (ID50 = 70-150 pM). Human CGRP-I(15-37), -(21-37), and -(28-37) were less potent (ID50 greater than or equal to 70 nM), suggesting that amino acid residues 8-14 may be important for maintaining high binding affinity. A novel photoreactive analogue of hCGRP-I, 125I-[C gamma-(4-azidoanilino)Asp3] hCGRP-I, was prepared by carbodiimide coupling of 4-azidoaniline to 125I-hCGRP-I. Photoaffinity labeling of soluble CGRP receptors with the photoreactive analogue and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed three specifically labeled binding proteins with apparent molecular weights (Mr) of 60,000, 54,000, and 17,000. Cross-linking of 125I-hCGRP-I and -II and 125I-hCGRP-I(8-37) to soluble CGRP binding sites using disuccinimidyl suberate revealed three specifically labeled binding proteins with the same Mr. The C-terminal fragment 125I-hCGRP-I(8-37), unlike the intact peptide, was, furthermore, cross-linked specifically to a 95,000 Mr protein. The CGRP receptor is N-glycosylated. Treatment with endoglycosidase F/N-glycosidase F converted the 60,000 and 54,000 to 46,000 and 41,000 Mr components.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural relationship, biosynthesis, and immunocytochemical localization of uteroferrin-associated basic glycoproteins

Uteroferrin, a progesterone-induced secretory protein of the pig uterus, can noncovalently associate with additional progesterone-induced glycoproteins (uteroferrin-associated glycoproteins or UfAP) to form a heterodimer. The UfAP were dissociated from uteroferrin by passage through an immunoaffinity column. The flow through material consisted of two immunologically related variants of different size (Mr = 47,000-50,000 and Mr = 39,000-40,000) forms. By using an antiserum to all molecular weight components of the UfAP, it was shown that these glycoproteins were localized in the glandular epithelium of the uterus. Amino acid sequence analysis of the higher molecular weight (Mr = 47,000-50,000) form indicated it had a common amino-terminal sequence which was distinct from that of the lower molecular weight (Mr = 39,000-40,000) form. Endoglycosidase F treatment converted the Mr = 47,000-50,000 form to a common product with Mr = 43,000. Tryptic peptide analysis showed that the Mr = 39,000-40,000 form was closely related in primary sequence to the larger species. When endometrial RNA was translated in vitro, a single major product (Mr = 45,000) was immunoprecipitated by using the UfAP antiserum. These results suggest that the different forms of the UfAP originate from a single precursor by differential glycosylation and peptide cleavage. Endometrial explant cultures released all forms of the glycoproteins. When [32P]orthophosphate was provided, label was incorporated into the 6-position of D-mannosyl residues on the oligosaccharide chains of the UfAP. Therefore the associated glycoproteins have a structural feature normally associated with lysosomal enzymes.     

Structural elements of human parathyroid hormone and their possible relation to biological activities.

Human parathyroid hormone (hPTH) and several deletion analogues were examined for the presence of secondary structure using circular dichroism spectroscopy. The spectra of hPTH and the deletion analogues 8-84, 34-53, 53-84, 1-34, 13-34, 1-19, and 20-34, in neutral, aqueous buffer, gave no evidence for extensive secondary structure. An alpha-helical-like spectral contribution was found to arise from a region within peptide 13-34. This spectral contribution was speculated to arise from partial stability of a helix consisting of residues 17-29. Molecular dynamics simulations of peptide 1-34 suggested that this peptide tends to fold with a bend defined by residues 10-14, with the amino-terminal and carboxyl-terminal residues tending to be in more extended forms and the other residues in helical-like conformations. The addition of trifluoroethanol promoted the formation of alpha-helix, mainly in the 1-34 region. The putative helix comprised of residues 17-29 was stabilized by the addition of 10-20% TFE, while a second putative helix proximal to the amino terminus, and comprised of residues 3-11, was stabilized by slightly higher concentrations of TFE. An amphiphilic sequence was identified within the 20-34 fragment. The development of alpha-helix on binding this fragment, and other analogues containing this sequence, to palmitoyloleoylphosphatidylserine vesicles provided experimental evidence for the potential role of this amphiphilic sequence in binding to membranes or to a membrane receptor. The relationships between these alpha-helical regions in 1-34, either potentiated by trifluoroethanol or lipid vesicles, are discussed in terms of different receptor-binding regions within hPTH.     

A major factor VIII binding domain resides within the amino-terminal 272 amino acid residues of von Willebrand factor

We have identified a Factor VIII (FVIII) binding domain residing within the amino-terminal 272 amino acid residues of the mature von Willebrand Factor (vWF) subunit. Two-dimensional crossed immunoelectrophoresis showed direct binding of purified human FVIII to purified human vWF. After proteolytic digestion of vWF with Staphylococcus aureus V8 protease (SP), FVIII binding was seen only with the amino-terminal SP fragment III and not with the carboxyl-terminal SP fragment II. A monoclonal anti-vWF antibody (C3) partially blocked FVIII binding to vWF and SP fragment III. FVIII also bound to vWF which had been adsorbed to polystyrene beads. This binding was inhibited in a dose-dependent manner by whole vWF, SP fragment III, and by monoclonal antibody C3. Binding could not be inhibited by SP fragment I, which contains the middle portion of the vWF molecule, or by reduced and alkylated whole vWF. SP fragment II caused only minimal inhibition. Trypsin cleavage of SP fragment III produced a monomeric 35-kDa fragment containing the amino-terminal 272 amino acid residues of vWF. This fragment reacted with monoclonal antibody C3 and inhibited the binding of FVIII to vWF in a dose-dependent manner. These studies demonstrate that a major FVIII binding site resides within the amino-terminal 272 amino acid residues of vWF.     

Human complement component C1s. Partial sequence determination of the heavy chain and identification of the peptide bond cleaved during activation

Human C1s proenzyme (Mr 83 000) was isolated by a rapid two-stage method involving affinity chromatography of C1 on IgG-Sepharose and isolation of subcomponent C1s by ion-exchange chromatography on DEAE-Sephacel. Single-chain C1s proenzyme was activated to two-chain C1s with self-activated C1r. After reduction and S-carboxamidomethylation the heavy chain of C1s (Mr 57 000) was isolated by ion exchange chromatography on DEAE-Sephacel. Cleavage of C1s heavy chain with CNBr yielded five fragments whose N-terminal sequences were determined. The alignment of the fragments within the heavy chain was established by tryptic peptides containing methionine. C1s heavy chain comprises about 470 amino acid residues and 42% of its sequence was determined. An intrachain sequence homology and a homology to the alpha 2 chain of human haptoglobin were identified. The C-terminal CNBr fragment comprising 44 amino acid residues was completely sequenced. From BNPS-skatole cleavage of reduced and alkylated C1s proenzyme a fragment was isolated which overlaps the C1s heavy and light chain parts and which contains the peptide bond cleaved during activation. The results show that this is an Arg-Ile bond and that under standard conditions of activation no peptide material is liberated from this portion of the molecule. The sequence data and homology to two-chain serine proteases indicate a single interchain disulfide bond in C1s.     

The primary structure of the mitochondrial energy-linked nicotinamide nucleotide transhydrogenase deduced from the sequence of cDNA clones

The amino acid sequence of the bovine mitochondrial nicotinamide nucleotide transhydrogenase, which catalyzes hydride ion transfer between NAD(H) and NADP(H) coupled to proton translocation across the mitochondrial inner membrane, has been deduced from the corresponding cDNA. Two clones were isolated by screening a bovine lambda gt10 cDNA library, using two synthetic oligonucleotides and a cDNA restriction fragment as probes. The inserts together covered 3,105 base pairs of coding sequence, corresponding to 1.035 amino acid residues. However, the reading frame at the 5' end was still open. N-terminal sequence analysis of the isolated enzyme indicated the presence of 8 additional residues. Thus, the mature transhydrogenase appeared to have 1,043 amino acid residues and a calculated molecular weight of 109,212. The deduced amino acid sequence of the transhydrogenase contained the sequences of four tryptic peptides that had been isolated from the enzyme. Two of these were the peptides that had been used for construction of the oligonucleotide probes. The other two were tryptic peptides isolated after labeling the NAD-binding site of the transhydrogenase once with [3H]p-fluorosulfonylbenzoyl-5'-adenosine (FSBA), and another time with [14C]N,N'-dicyclohexylcarbodiimide. The FSBA-labeled peptide was found to be located immediately upstream of the [14C]N,N'-dicyclohexylcarbodiimide-labeled peptide, about 230 residues from the N terminus. One of the tryptic peptides used for oligonucleotide probe construction was the same as that labeled with [3H]FSBA when the NAD-binding site was protected from FSBA attack. This peptide, which might be at the NADP-binding site of the transhydrogenase, was located very near the C terminus of the enzyme. The central region of the transhydrogenase (residues 420-850) is highly hydrophobic and appears to comprise about 14 membrane-spanning segments. By comparison, the N- and the C-terminal regions of the enzyme, which contain the NAD- and the putative NADP-binding sites, respectively, are relatively hydrophilic and are probably located outside the mitochondrial inner membrane on the matrix side. There is considerable homology between the bovine enzyme and the Escherichia coli transhydrogenase (two subunits, alpha with Mr = 54,000 and beta with Mr = 48,700), whose amino acid sequence has been determined from the genes (Clarke, D.M., Loo, T.W., Gillam, S., and Bragg, P.D. (1986) Eur. J. Biochem. 158, 647-653).     

Interaction of phenylisothiocyanate with human erythrocyte band 3 protein. II. Topology of phenylisothiocyanate binding sites and influence of p-sulfophenylisothiocyanate on phenylisothiocyanate modification

The two structurally related probes, the apolar phenylisothiocyanate and the polar, water-soluble p-sulfophenylisothiocyanate, were analysed for their topological interaction with human erythrocyte band 3 protein. Upon thermolytic and peptic digestion of labeled erythrocyte ghosts, the membrane-integrated segments of band 3 protein, the 17,000 and 10,000 dalton peptides, were isolated. At 2 mM initial label concentration, 90% of the hydrophobic probe phenylisothiocyanate was recovered in the 10,000 dalton peptide, the remaining amount of label being associated with the 17,000 dalton fragment. Pretreatment of the membranes with 5 mM p-sulfophenylisothiocyanate followed by labeling with 2 mM phenylisothiocyanate results in a consistent reduction in binding of phenylisothiocyanate by 1 mol/mol isolated band 3 protein. p-Sulfophenylisothiocyanate reportedly binds to the 17,000 dalton fragment (Drickamer, K. (1977), J. Biol. Chem. 252, 6909-6917). The interaction of the polar probe with the membrane protein affects binding of phenylisothiocyanate to the 10,000 dalton peptide by the equivalent of 1 mol/mol isolated peptide. The topological interrelation of the membrane-integrated segments is concluded.