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Biomedical subjects

W T Morgan

Publications and source records attributed to W T Morgan.

At least 37 records · Page 2Linked to original sources
PubMed1996

Experimental evidence against replication or dissemination of hepatitis C virus in mosquitoes (Diptera:Culicidae) using detection by reverse transcriptase polymerase chain reaction.

In 3 laboratory experiments, mosquitoes were fed hepatitis C virus (HCV)-RNA positive blood by using membrane feeders, separated into head, thorax, and abdomen, and tested by a reverse transcriptase polymerase chain reaction for HCV-RNA. HCV did not replicate or disseminate in mosquitoes that had ingested blood from patients that were HCV-viremic positive. When yellow fever mosquitoes, Aedes aegypti (L.), were held for 1, 3, 7, 14, and 21 d after feeding, HCV-RNA was detected in the abdomens of 5/5 mosquitoes at 1 d after feeding; remaining tissues were negative with the exception of a single positive head at 7 d. In agreement, HCV-RNA was detected in Asian tiger mosquito, Aedes albopictus Skuse, and Anopheles stephensi Liston abdomens at 1 d, but not 3 d after feeding no HCV-RNA was detected in heads or thoraces. In addition, HCV-RNA was detected in heads of Ae. aegypti at 10 and 20 min, but not at 30 min, after feeding. The latter results raise the possibility of HCV contamination of mouthparts and, theoretically, mechanical transmission of this virus.

Aedes↗
PubMed1995

MCD, EPR and NMR spectroscopic studies of rabbit hemopexin and its heme binding domain.

Heme binding to rabbit hemopexin and its domain I, obtained by proteolytic cleavage of intact hemopexin, was studied by EPR, MCD and 1H-NMR spectroscopies. The data obtained support the proposal that the heme Fe(III) is coordinated by two histidine ligands (Morgan et al. (1988) J. Biol. Chem. 263, 8220-8225; Muster et al. (1991) J. Protein Chem. 10, 123-128) and are inconsistent with recently reported mutagenesis studies indicating that bis-histidine ligation is unlikely (Satoh et al. (1994) Proc. Natl. Acad. Sci. USA 91, 8423-8427). Although the MCD data are consistent with both bis-histidine and histidine/lysine ligation, the EPR spectra are typical of bis-histidine ligation. Overall the magneto-optical spectra are characteristic for bis-histidine ligation. The EPR and NMR data indicate that there is a difference in the heme environments of the intact hemopexin and its domain I but overall the spectroscopic information suggests heme bound to domain I has the same ligands as intact hemopexin. The 1H-NMR studies indicate that heme binding to domain I perturbs at least 4 of the 5 histidines. This is consistent with axial ligation of the heme by two histidines, and a conformational change induced by heme binding affecting two more. Interestingly, resonances of the carbohydrate bound to intact hemopexin and domain I were also perturbed by heme binding. pH dependence studies showed that heme remained bound to intact hemopexin over the pH range 6.5-10.0 without any major change in the ligation or environment of the heme.

Amino Acid Sequence↗
PubMed1995

1.8 A crystal structure of the C-terminal domain of rabbit serum haemopexin.

BACKGROUND: Haemopexin is a serum glycoprotein that binds haem reversibly and delivers it to the liver where it is taken up by receptor-mediated endocytosis. Haemopexin has two homologous domains, each having a characteristic fourfold internal sequence repeat. Haemopexin-type domains are also found in other proteins, including the serum adhesion protein vitronectin and various collagenases, in which they mediate protein-protein interactions. RESULTS: We have determined the crystal structure of the C-terminal domain of haemopexin at 1.8 A resolution. The domain is folded into four beta-leaflet modules, arranged in succession around a central pseudo-fourfold axis. A funnel-shaped tunnel through the centre of this disc-shaped domain serves as an ion-binding site. CONCLUSIONS: A model for haem binding by haemopexin is proposed, utilizing an anion-binding site at the wider end of the central tunnel, together with an associated cleft. This parallels the active-site location in other beta-propeller structures. The capacity to bind both cations and anions, together with the disc shape of the domain, suggests that such domains may be used widely for macromolecular recognition.

Amino Acid Sequence↗
PubMed1995

Interaction of histidine-proline-rich glycoprotein with plasminogen: effect of ligands, pH, ionic strength, and chemical modification.

The association of plasma histidine-proline-rich glycoprotein (HPRG) with plasminogen (PLG) was examined using a sucrose density gradient assay in order to evaluate the effects of several relevant conditions on complex formation. Addition of PLG shifts the S-value of 125I-labeled HPRG from 4.8S to 6.8S, providing the first direct evidence that HPRG associates with the zymogen form of plasmin in solution. Complex formation is not sensitive to pH in the range of pH 6.5-8.5, but is abolished at high ionic strength (1 M NaCl). No species differences were found, as either rabbit or human HPRG bound readily to rabbit or human PLG. Of the ligands of HPRG tested, mesoheme (20 microM) but not heparin (M(r) 10,000, 10 microM) inhibits the formation of the HPRG-PLG complex. Modification of lysine residues of HPRG or competitive binding by lysine and anti-fibrinolytic agents containing primary amino groups also inhibits association. Conversely, modification of arginine or histidine residues of HPRG has no effect on complex formation. These results indicate that HPRG has independent binding sites for heparin and PLG and confirm that one or more lysine residues of HPRG are involved in its recognition by PLG. The protein-protein interaction was also quantitatively characterized at thermodynamic equilibrium by analytical ultracentrifugation. The stoichiometry and dissociation constant (KD) of the complex were determined from the equilibrium distribution of fluorescein-isothiocyanate-labeled PLG in the presence of HPRG. The experimental data were analyzed by nonlinear least-squares curve fitting and indicated that a heterodimer is formed.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Thermodynamics of heme-induced conformational changes in hemopexin: role of domain-domain interactions.

Hemopexin is a serum glycoprotein that binds heme with high affinity and delivers heme to the liver cells via receptor-mediated endocytosis. A hinge region connects the two non-disulfide-linked domains of hemopexin, a 35-kDa N-terminal domain (domain I) that binds heme, and a 25-kDa C-terminal domain (domain II). Although domain II does not bind heme, it assumes one structural state in apo-hemopexin and another in heme-hemopexin, and this change is important in facilitating the association of heme-hemopexin with its receptor. In order to elucidate the structure and function of hemopexin, it is important to understand how structural information is transmitted to domain II when domain I binds heme. Here we report a study of the protein-protein interactions between domain I and domain II using analytical ultracentrifugation and isothermal titration calorimetry. Sedimentation equilibrium analysis showed that domain I associates with domain II both in the presence and absence of heme with Kd values of 0.8 microM and 55 microM, respectively. The interaction between heme-domain I and domain II has a calorimetric enthalpy of +11 kcal/mol, a heat capacity (delta Cp) of -720 cal/mol.K, and a calculated entropy of +65 cal/mol.K. By varying the temperature of the centrifugation equilibrium runs, a van't Hoff plot with an apparent change in enthalpy (delta H) of -3.6 kcal/mol and change in entropy (delta S) of +8.1 cal/mol.K for the association of apo-domain I with domain II was obtained.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Probing protein-cofactor interactions in the terminal oxidases by second derivative spectroscopy: study of bacterial enzymes with cofactor substitutions and heme A model compounds.

Second derivative absorption spectra are reported for the aa3-cytochrome c oxidase from bovine cardiac mitochondria, the aa3-600 ubiquinol oxidase from Bacillus subtilis, the ba3-cytochrome c oxidase from Thermus thermophilis, and the aco-cytochrome c oxidase from Bacillus YN-2000. Together these enzymes provide a range of cofactor combinations that allow us to unequivocally identify the origin of the 450-nm absorption band of the terminal oxidases as the 6-coordinate low-spin heme, cytochrome a. The spectrum of the aco-cytochrome c oxidase further establishes that the split Soret band of cytochrome a, with features at 443 and 450 nm, is common to all forms of the enzyme containing ferrocytochrome a and does not depend on ligand occupancy at the other heme cofactor as previously suggested. To test the universality of this Soret band splitting for 6-coordinate low-spin heme A systems, we have reconstituted purified heme A with the apo forms of the heme binding proteins, hemopexin, histidine-proline-rich glycoprotein and the H64V/V68H double mutant of human myoglobin. All 3 proteins bound the heme A as a (bis)histidine complex, as judged by optical and resonance Raman spectroscopy. In the ferroheme A forms, none of these proteins displayed evidence of Soret band splitting. Heme A-(bis)imidazole in aqueous detergent solution likewise failed to display Soret band splitting. When the cyanide-inhibited mixed-valence form of the bovine enzyme was partially denatured by chemical or thermal means, the split Soret transition of cytochrome a collapsed into a single band at 443 nm.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Conformational analysis of hemopexin by Fourier-transform infrared and circular dichroism spectroscopy.

Hemopexin is a serum glycoprotein that binds heme with the highest known affinity of any characterized heme-binding protein and plays an important role in receptor-mediated cellular heme uptake. Complete understanding of the function of hemopexin will require the elucidation of its molecular structure. Previous analysis of the secondary structure of hemopexin by far-UV circular dichroism (CD) failed due to the unusual positive ellipticity of this protein at 233 nm. In this paper, we present an examination of the structure of hemopexin by both Fourier-transform infrared (FTIR) and circular dichroism spectroscopy. Our studies show that hemopexin contains about 55% beta-structure, 15% alpha-helix, and 20% turns. The two isolated structural domains of hemopexin each have secondary structures similar to hemopexin. Although there are significant tertiary conformational changes indicated by the CD spectra, the overall secondary structure of hemopexin is not affected by binding heme. However, moderate changes in secondary structure do occur when the heme-binding domain of hemopexin associates with heme. In spite of the exceptionally tight binding at neutral pH, heme is released from the bis-histidyl heme-hemopexin complex at pH 5.0. Under this acidic condition, hemopexin maintains the same overall secondary structure as the native protein and is able to resume the heme-binding function and the native structure of the heme-protein (as indicated by the CD spectra) when returned to neutral pH. We propose that the state of hemopexin identified in vitro at pH 5.0 resembles that of this protein in the acidic environment of the endosomes in vivo when hemopexin releases heme during receptor-mediated endocytosis.

Circular Dichroism↗
PubMed1993

Characterization of hemopexin and its interaction with heme by differential scanning calorimetry and circular dichroism.

Hemopexin is a plasma glycoprotein that has two structural domains (I and II) and binds and transports heme particularly to liver cells. Differential scanning calorimetry (DSC) studies show that hemopexin is largely stabilized by heme, which binds exclusively to domain I. The melting temperature (Tm) of heme-hemopexin is 66.4 +/- 0.7 degrees C as compared with 53.9 +/- 0.3 degrees C for apohemopexin, and this Tm increase is accompanied by a 100 kcal increase in molar enthalpy. Heme stabilizes hemopexin by stabilizing domain I. This is demonstrated by the 26 degrees C increase in Tm from 51.9 +/- 0.3 to 77.6 +/- 0.6 degrees C and the over 3-fold increase in molar enthalpy when domain I associates with heme. A moderate change in domain I secondary structure is indicated by an increase in negative molar ellipticity at 206 nm. However, there is no net effect on the secondary structure of holo-hemopexin caused by heme binding as indicated by both far-UV circular dichroism (CD) and Fourier-transform infrared spectra. The characteristic positive ellipticity of hemopexin at 233 nm, ascribed to tryptophan residues in domain II, is dramatically increased, suggesting a change in teritary structure for domain II of hemopexin. DSC and CD results show that isolated domain I and domain II interact both in the presence and absence of heme. Moreover, domain II destabilizes heme-domain I, which may be an important factor in facilitating heme release to the hemopexin receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Regulation of heme oxygenase and metallothionein gene expression by the heme analogs, cobalt-, and tin-protoporphyrin.

Two heme analogs, cobalt- and tin-protoporphyrin (CoPP and SnPP, respectively) have been used to probe the heme-hemopexin interaction, hemopexin receptor binding, and the mechanism of regulation of heme oxygenase (HO) and metallothionein-1 (MT-1) gene expression by hemopexin. Both CoPP and SnPP are HO inhibitors and hemopexin binds SnPP (Morgan, W. T., Alam, J., Deaciuc, V., Muster, P., Tatum, F. M., and Smith, A. (1988) J. Biol. Chem. 263, 8226-8231) and CoPP. The association of CoPP with hemopexin produces characteristic changes in the absorbance spectrum of CoPP and quenches the intrinsic fluorescence of hemopexin. Binding of CoPP is tight (Kd ca. 3 x 10(-7) M) although of lower affinity than heme itself (Kd < pM); and CoPP binding, like heme, produces conformational changes in hemopexin shown by an increase in the molar ellipticity at 233 nm and affords protection from proteolysis of the hinge region between the two structural domains of hemopexin. The coordination of the central cobalt atom is predicted to be similar to that of heme and to involve His56 and His127 of rabbit hemopexin. Furthermore, CoPP-hemopexin, like SnPP-hemopexin, binds to the hemopexin receptor as shown by competitive inhibition studies with radioactive heme-hemopexin. The effect of free heme analogs and their hemopexin complexes on HO and MT gene regulation was investigated and compared with the extent of induction by heme and heme-hemopexin. Free CoPP is a more effective inducer of HO steady state mRNA levels than free heme and produces a 5-fold increase within 1 h compared to only a 2-fold increase with heme, but free SnPP (up to 10 microM) produces no detectable increase in HO mRNA. In contrast, by 3 h heme-hemopexin and SnPP-hemopexin increase HO mRNA levels 11- and 6-fold, respectively; but the CoPP-hemopexin complex causes no detectable change in HO mRNA levels. The complexes of hemopexin with heme or either of the two heme analogs are effective inducers of metallothionein (MT) mRNA. Induction of MT mRNA by heme-hemopexin is rapid, increasing 4-fold within 1 h and 14-fold by 3-4 h. Strikingly, an even more rapid and slightly more extensive induction of MT mRNA is seen in response to either CoPP- or SnPP-hemopexin complexes, with MT mRNA rising 8-fold within 1 h. In contrast, free heme and the free analogs are far less effective inducers, increasing MT and mRNA levels and in vitro transcription rates only 3-4-fold and declining after 2-3 h.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

Identification of the histidine residues of hemopexin that coordinate with heme-iron and of a receptor-binding region.

Rabbit hemopexin cDNA was cloned from a rabbit liver lambda gt11 cDNA expression library using a mixture of five monoclonal antibodies raised against rabbit hemopexin, and the entire rabbit hemopexin sequence was determined. The heme-binding domain I of rabbit hemopexin (Smith, A., and Morgan, W. T. (1984) J. Biol. Chem. 259, 12049-12053) contains only 4 histidine residues which are conserved in rabbit, human, rat, and mouse hemopexin. The 2 axial heme-iron coordinating histidine residues, identified by Edman microsequencing and amino acid analyses of chemically modified domain I and isolated fragments of domain I, are the conserved histidine residues at positions 56 and 127 of the mature rabbit protein. The epitope recognized by JEN-14 (a monoclonal antibody which specifically reacts with domain I and blocks the hemopexin-receptor interaction (Morgan, W. T., Muster, P., Tatum, F. M., McConnell, J., Conway, T. P., Hensley, P., and Smith, A. (1988) J. Biol. Chem. 263, 8220-8225) was shown to lie between residues 122 and 142 by Western blotting of protease-digested domain I and transposon-insertion mutants of domain I expressed in a plasmid vector system. The location of this epitope near the heme-binding histidine residue 127 is compatible with a transport mechanism in which the release of heme from hemopexin is accompanied by a concomitant transfer of heme to the hemopexin receptor or the membrane heme-binding protein (Smith, A., and Morgan, W. T. (1985) J. Biol. Chem. 260, 8325-8329).

Amino Acid Sequence↗
PubMed1993

Crystallization of the C-terminal domain of rabbit serum hemopexin.

The C-terminal domain of rabbit serum hemopexin, comprising residues 215 to 435, has been crystallized following removal of the attached carbohydrate using the endoglycosidase Endo F. The crystals, grown by vapour diffusion from solutions containing polyethylene glycol 1500, are orthorhombic, with cell dimensions a = 41.0 A, b = 64.2 A, c = 85.2 A, space group P2(1)2(1)2(1), and one molecule in the asymmetric unit. The crystals diffract to 2.4 A resolution and are suitable for X-ray structure analysis.

Animals↗
PubMed1993

Induction of T-lymphocyte adhesion by histidine-proline-rich glycoprotein and concanavalin A.

Histidine-proline-rich glycoprotein (HPRG) is a plasma protein which binds to a specific receptor on T-lymphocytes and represses T-cell activation and proliferation. In the presence of Concanavalin A (Con A), HPRG causes human T-lymphoblastic MOLT-3 cells and a fraction of normal human peripheral blood lymphocytes to attach to the culture dish and significantly change morphology by either extending processes or becoming elongated at the poles, respectively. HPRG and Con A are just as effective at inducing MOLT-3 attachment in a soluble or an immobilized form. MOLT-3 cell attachment is specific for HPRG, dose-dependent, and reversible in 72 hr. Only certain T-cell mitogenic lectins, Con A and phytohemagglutinin, are effective at stimulating adherence. Mannose, glucose, fucose, and N-acetylglucosamine inhibit HPRG-induced MOLT-3 cell attachment to different degrees, with methyl alpha-D-mannopyranoside being the most potent inhibitor. We propose that in vivo HPRG together with a naturally expressed lectin induces T-lymphocyte adhesion to initiate cell migration to sites of inflammation.

Acute-Phase Proteins↗
PubMed1993

Possible genetical pathways for the biosynthesis of blood group mucopolysaccharides. Vox Sang 1959:4:97-119.

This paper put forward possible biosynthetic pathways for the formation of the blood group A, B, H and Lewis antigens based on the limited knowledge of their chemistry and genetics that was available in 1959. The schemes proposed that genes at four independent loci ABO, HH, Lele and Sese interacted to give the five specificities A, B, H, Lea and Leb found in secretions and that the primary products of the blood group genes were not the antigens but enzymes that catalysed the sequential addition of single sugars to complete the determinants.

ABO Blood-Group System↗
PubMed1992

Resonance Raman investigation of the effects of copper binding to iron-mesoporphyrin.histidine-rich glycoprotein complexes.

Histidine-rich glycoprotein (HRG) binds both hemes and metal ions simultaneously with evidence for interaction between the two. This study uses resonance Raman and optical absorption spectroscopies to examine the heme environment of the 1:1 iron-mesoporphyrin.HRG complex in its oxidized, reduced and CO-bound forms in the absence and presence of copper. Significant perturbation of Fe(3+)-mesoporphyrin.HRG is induced by Cu2+ binding to the protein. Specifically, high frequency heme resonance Raman bands indicative of low-spin, six-coordinate iron before Cu2+ binding exhibit monotonic intensity shifts to bands representing high-spin, five-coordinate iron. The latter coordination is in contrast to that found in hemoglobin and myoglobin, and explains the Cu(2+)-induced decrease and broadening of the Fe(3+)-mesoporphyrin.HRG Soret band concomitant with the increase in the high-spin marker band at 620 nm. After dithionite reduction, the Fe(2+)-mesoporphyrin.HRG complex displays high frequency resonance Raman bands characteristic of low-spin heme and no iron-histidine stretch, which together suggest six-coordinate iron. Furthermore, the local heme environment of the complex is not altered by the binding of Cu1+. CO-bound Fe(2+)-mesoporphyrin.HRG exhibits bands in the high and low frequency regions similar to those of other CO-bound heme proteins except that the iron-CO stretch at 505 cm-1 is unusually broad with delta nu approximately 30 cm-1. The dynamics of CO photolysis and rebinding to Fe(2+)-mesoporphyrin.HRG are also distinctive. The net quantum yield for photolysis at 10 ns is low relative to most heme proteins, which may be attributed to very rapid geminate recombination. A similar low net quantum yield and broad iron-CO stretch have so far only been observed in a dimeric cytochrome c' from Chromatium vinosum. Furthermore, the photolytic transient of Fe(2+)-mesoporphyrin.HRG lacks bands corresponding to high-spin, five-coordinate iron as is found in hemoglobin and myoglobin under similar experimental conditions, suggesting iron hexacoordination before CO recombination. These data are consistent with a closely packed distal heme pocket that hinders ligand diffusion into the surrounding solvent.

Animals↗
PubMed1992

Identification of histidine-rich glycoprotein in human colostrum and milk.

Histidine-rich glycoprotein (HRG) is a 74-kD glycoprotein, originally discovered in plasma, which contains an unusually large amount of histidine (13 mol%) and proline (13 mol%). The specific functions of this protein remain unclear, although it binds (reversibly) transition metal ions such as Cu(II) and Zn(II) with high capacity (10-13 equivalent) and moderate to high affinity (kd = 0.2-10 microM). Because the bioavailability of Cu(II) and Zn(II) ions in human milk is high, we have used specific antibodies from polyclonal antisera directed against purified human plasma HRG to investigate whether this or a related protein is a component of human colostrum and(or) mature milk. Fresh human colostrum (d 1-3) and milk (d 4-120) were collected in the presence and absence of multiple protease inhibitors and EDTA. Immuno "dot" blot analyses and ELISA were developed; HRG was present in both colostrum (0.13-10 micrograms/mL) and mature milk (0.1-10 micrograms/mL). Unidentified components in colostrum and milk, however, were found to depress HRG antigenicity in these assays. Western transfer and immunoblots of denatured colostrum and milk samples analyzed by SDS-PAGE revealed the presence of an immunoreactive band at 74-78 kD, with other bands at 47 and 24 kD under both reducing and nonreducing conditions; smaller immunoreactive fragments (12-14 kD) were detected in some samples. We observed at least one additional band of immunoreactivity of greater molecular mass (greater than 110 kD) in colostrum under nonreducing conditions; we did not observe these bands in plasma samples. Immunoaffinity and Zn(II) affinity isolation of HRG from colostrum and milk resulted in the copurification of several associated proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood Proteins↗
PubMed1991

The murine haemopexin receptor. Evidence that the haemopexin-binding site resides on a 20 kDa subunit and that receptor recycling is regulated by protein kinase C.

Haemopexin receptors from mouse hepatoma (Hepa) cells were affinity-labelled by cross-linking to haem-125I-haemopexin complexes using two homo-[disuccinimidyl suberate (DSS) and 3,3'-dithiobis(succinimidyl propionate) (DTSSP)] and one hetero-[sulphosuccinimidyl 4-(p-maleimidophenyl)butyrate (sulpho-SMPB)] bifunctional cross-linking agents. Analysis of the cross-linked products by SDS/PAGE in the absence of reducing agents revealed that 125I-haemopexin was cross-linked specifically to a protein of apparent molecular mass 85-90 kDa. Upon reduction, haemopexin remained cross-linked to a protein of 20 kDa, suggesting that the murine haemopexin receptor has a subunit structure. Two subunits were identified: alpha (p65) and beta (p20). Furthermore, because haemopexin was cross-linked by all three agents to p20, the shortest cross-linker arm being 1.1 nm (11 A), we propose that the haem-haemopexin-binding site resides on this subunit. In addition, a cysteine residue of p20 is located near the haemopexin-binding site, since haemopexin, which has no free thiol groups, is cross-linked to this subunit by the hetero-bifunctional agent sulpho-SMPB. Exposure of Hepa cells to the tumour-promoting phorbol ester 4 alpha-phorbol 12-myristate 13-acetate (PMA) causes a rapid redistribution of haemopexin receptors from the cell surface to the cell interior. Within 2-4 min of incubation with 100 nM-PMA, there was an approx. 50% decrease in cell-surface haemopexin receptors, as judged by ligand binding at 0 degrees C and affinity labelling of the receptor. This time- and dose-dependent down-regulation was fully reversible within 60-90 min after removal of PMA, and the affinity of the remaining receptors was unaltered by PMA. The specificity of PMA was demonstrated by comparison with the non-tumour-promoter 4 alpha-phorbol, which did not affect any of the parameters examined. The amine H-7, a specific inhibitor of protein kinase C, antagonised the receptor redistribution effect of PMA, suggesting that the down-regulation of haemopexin receptors on the cell surface was a consequence of protein kinase C activation. The PMA-induced decrease in surface haemopexin receptors was due to a 2-fold increase in the rate of internalization (from 0.73 min-1 to 1.32 min-1), whereas the rate of exocytosis (0.6 min-1) was unchanged. PMA treatment, like binding of the natural ligand, haem-haemopexin, results in a lower steady-state level of surface haemopexin receptors independent of receptor synthesis, and the receptors were not degraded but were recycled back to the cell surface.

Animals↗
PubMed1991

Further characterization of structural determinants of rabbit hemopexin function.

To further identify structural features of the hemopexin molecule important for its heme transport function, a fragment of the heme-binding domain (residues 1-213, Mr 35 kD, domain I) of rabbit hemopexin was obtained after digestion with subtilisin. Both apo- and heme-domain I were cleaved by subtilisin, and the subtilisin-digested form of domain I (called SD-DI) was shown by microsequencing to have been cleaved at Asp 22 forming a 30 kD subfragment lacking the conserved histidine residue at position 7 and the N-linked oligosaccharide at Asn 9. The 5 kD peptide cleaved from domain I is not disulfide linked to domain I and can be removed by membrane ultrafiltration. SD-DI retains the ability of domain I to bind heme, to associate with the other functional domain of hemopexin (domain II), and to interact with the hemopexin receptor on mouse Hepa cells. Moreover, although the heme complex of SD-DI is less thermostable than native heme-domain I, like heme-domain I, heme-SD-DI is stabilized to a large extent when associated with domain II. These results show that the conserved His 7 residue is not involved in heme binding by hemopexin and that residues 1-22 of hemopexin and the N-linked oligosaccharide at Asn 9 are not essential for either receptor binding or interdomain interactions. Nevertheless, these N-terminal residues of hemopexin do contribute significantly to the overall stability of the hemopexin molecule and the interdomain interactions necessary for receptor recognition.

Amino Acid Sequence↗