[Creative nursing. The "difficult patient"].
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P Muster
Publications and source records attributed to P Muster.
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↗
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↗
Use of hemopexin domains and monoclonal antibodies to hemopexin to probe the molecular determinants of hemopexin-mediated heme transport.
Plasmin cleaves rabbit serum apohemopexin (Mr = 60,000) at a single site producing a heme-binding domain (I, Mr = 35,000) and a second domain (II, Mr = 25,000) (W. T. Morgan and A. Smith (1984) J. Biol. Chem. 259, 12001-12005). The absorbance spectra of heme-domain I are indicative of a bis-histidyl coordination complex with the central heme iron atom. Chemical modification of the 5 histidine residues of apo-domain I with diethylpyrocarbonate abolished heme binding, supporting this assignment. Upon binding heme, domain I migrates more rapidly in sucrose gradients, and, in sedimentation velocity experiments, the s value of domain I increases from 3.17 +/- 0.04 to 3.71 +/- 0.09, a notably large increase which indicates that the domain becomes much more compact. This conformational change which plays a pivotal role in hemopexin function requires the bis-histidyl coordination with heme iron and leads to a tighter association between domain I and domain II shown by the co-migration of heme-domain I and domain II in sucrose gradients. In turn, the association of heme-domain I with domain II increases the thermal stability of the heme-domain I chromophore. Results of binding studies using mouse hepatoma cells and isolated domains indicate that domain I not only binds heme but also plays a vital part in the hemopexin-receptor interaction. The change in conformation of domain I upon heme binding and the association between domains I and II induced by heme are both notable determinants of the strength of the hemopexin-receptor interaction, but an intact "hinge region" between the domains is not necessary for receptor binding. The importance of both domains in bringing about the transport function of hemopexin is confirmed by the ability of three (two specific for domain I and one for domain II) of seven monoclonal antibodies raised against hemopexin to inhibit the hemopexin-receptor interaction.
Animals↗
Interaction of hemopexin with Sn-protoporphyrin IX, an inhibitor of heme oxygenase. Role for hemopexin in hepatic uptake of Sn-protoporphyrin IX and induction of mRNA for heme oxygenase.
Sn-protoporphyrin IX (SnPP), an inhibitor of heme oxygenase and a potential therapeutic agent for neonatal hyperbilirubinemia, is bound tightly by hemopexin. The apparent dissociation constant (Kd) at pH 7.4 is 0.25 +/- 0.15 microM, but estimation of the Kd for the SnPP-hemopexin complex is hampered by the fact that at physiological pH SnPP exists as monomers and dimers, both of which are bound by hemopexin. SnPP is readily displaced from hemopexin by heme (Kd less than 1 pM). The hemopexin-SnPP interaction, like that of heme-hemopexin, is dependent on the histidine residues of hemopexin. However, as expected from the differences in the coordination chemistries of tin and iron, the stability of the histidyl-metalloporphyrin complex is lower for SnPP-hemopexin than for mesoheme-hemopexin. Nevertheless, when SnPP binds to hemopexin, certain of the ligand-induced changes in the conformation of hemopexin which increase the affinity of the protein for its receptor are produced. Binding of SnPP produces the conformational change in hemopexin which protects the hinge region of hemopexin from proteolysis, but SnPP does not produce the characteristic increase in the ellipticity of hemopexin at 231 nm that heme does. Competition experiments confirmed that human serum albumin (apparent Kd = 4 +/- 2 microM) has a significantly lower affinity for SnPP than does hemopexin. Appreciable amounts of SnPP (up to 35% in adults and 20% in neonates) would be bound by hemopexin in the circulation, and the remainder of SnPP would be associated with albumin due to the latter's high concentration in serum. Essentially no non-protein-bound SnPP is present. Importantly, SnPP-hemopexin binds to the hemopexin receptor on mouse hepatoma cells with an affinity comparable to that of heme-hemopexin and treatment of the hepatoma cells with SnPP-hemopexin causes a rapid increase in the steady state level of heme oxygenase messenger RNA. These results show that hemopexin participates in the transport of SnPP to heme oxygenase and in its regulation by SnPP.
Animals↗
Importance of ligand-induced conformational changes in hemopexin for receptor-mediated heme transport.
Hemopexin alters conformation upon binding heme as shown by circular dichroism (CD), but hemopexin binds the heme analog, iron-meso-tetra-(4-sulfonatophenyl)-porphine (FeTPPS), without undergoing concomitant changes in its CD spectrum. Moreover, FeTPPS, unlike heme, does not increase the compactness of the heme-binding domain (I) of hemopexin shown by an increased sedimentation rate in sucrose gradients. On the other hand, like heme, FeTPPS forms a bishistidyl coordination complex with hemopexin and upon binding protects hemopexin from cleavage by plasmin. Competitive inhibition and saturation studies demonstrate that FeTPPS-hemopexin binds to the hemopexin receptor on mouse hepatoma cells but with a lower affinity (Kd 125 nM) more characteristic of apo-hemopexin than heme-hemopexin (Kd 65 nM). This provides evidence that conformational changes produced in hemopexin upon binding heme, but not upon binding FeTPPS, are important for increasing the affinity of hemopexin for its receptor. The amount of cell-associated radiolabel from 55FeTPPS-hemopexin increases linearly for up to 90 min but at a rate only about a third of that of the mesoheme-complex. As expected from the recycling of hemopexin, more iron-tetrapyrrole than protein is associated with the Hepa cells, but the ratio of 55Fe-ligand to 125I-hemopexin is only 2:1 for FeTPPS-hemopexin compared to 4:1 for mesoheme complexes. [55Fe]Mesoheme was associated at 5 min with lower density fractions containing plasma membranes and at 30 min with fractions containing higher density intracellular compartments. In contrast, 55FeTPPS was found associated with plasma membrane fractions at both times and was not transported into the cell. Although FeTPPS-hemopexin binds to the receptor, subsequent events of heme transport are impaired. The results indicate that upon binding heme at least three types of conformational changes occur in hemopexin which have important roles in receptor recognition and that the nature of the ligand influences subsequent heme transport.
Animals↗
Photosystem I reaction center from the thermophilic cyanobacterium Mastigocladus laminosus.
Photosystem I reaction center was isolated from the cyanobacterium Mastigocladus laminosus. It contained four different subunits with molecular masses (as determined by sodium dodecyl sulfate gels) of about 70,000 (subunit I), 16,000 (subunit II), 11,000 (subunit III), and 10,000 (subunit IV) daltons. The purified reaction center contained about 100 chlorophyll a molecules per P(700); however, they could be readily depleted down to about 50 chlorophyll a per P(700) without loss in the photochemical activities. The reaction center was active in cytochrome c photooxidation, but the photooxidation of an acidic cytochrome, like the Euglena cytochrome 552, required the presence of cations. The purified reaction center was found to be similar in several respects to the photosystem I reaction centers from higher plants and, especially, to the one isolated from green algae. Subunit I appeared on sodium dodecyl sulfate gels in the same position and possessed the same shape of an apparent double band as the corresponding subunits I of green plants and of algae. Subunits I and II of photosystem I reaction centers from Mastigocladus, higher plants, and green algae showed immunological crossreactivity. This observation might serve as biochemical evidence for the common evolution of the photosystem I reaction centers. In higher plants and green algae subunit II is a product of cytoplasmic ribosomes and therefore, a high degree of homology should have been preserved upon transfer of its gene from the prokaryote to the nucleus of the eukaryotes.
Journal Article↗