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Biomedical subjects

W T Morgan

Publications and source records attributed to W T Morgan.

At least 73 records · Page 4Linked to original sources
PubMed1985

Preferred heme binding sites of histidine-rich glycoprotein.

The heme binding sites of rabbit histidine-rich glycoprotein (HRG), 94 kDa, were studied with rose bengal (RB), a fluorescein derivative that associates with histidine residues. Difference absorbance spectra indicate that HRG binds RB at two thermodynamically preferred sites (Kd approximately 2 microM) that are spectroscopically equivalent. Up to 18-22 equiv of RB can also be bound by a set of lower affinity sites. Mesoheme is capable of displacing RB from the two preferred sites (Kd = 0.6 microM) and provides evidence that the two sites are not identical. Two peptides isolated from plasmin-digested HRG, one 35-kDa peptide rich in histidine (approximately 30 mol %) and one 15-kDa peptide relatively poor in histidine (approximately 4 mol %), also bind RB and mesoheme. The two preferred RB binding sites of HRG are located on the 15-kDa histidine-poor peptide and the lower affinity "class" of sites on the 35-kDa histidine-rich peptide. Mesoheme or RB quenches the tryptophan fluorescence of HRG and the histidine-poor peptide with an apparent binding stoichiometry near 2. Fluorescence quenching also indicates that 1-2 equiv of Cu(II) binds to the 15-kDa peptide, and absorbance spectroscopy provides evidence that Cu(II) is capable of displacing heme from the peptide. The fluorescence lifetimes of RB, determined by phase-modulation fluorometry, indicate that the two preferred sites in the histidine-poor domain are more apolar than the more numerous sites located in the histidine-rich region of the protein.

Animals↗
PubMed1985

Hemopexin-mediated heme transport to the liver. Evidence for a heme-binding protein in liver plasma membranes.

Isolated liver plasma membranes interact with heme-hemopexin and effect the removal of heme from the complex. This heme is rapidly accumulated by a previously undescribed heme-binding membrane component (HBC). This intrinsic membrane component can be solubilized from the membrane with Triton X-100 in a form that retains the ability to bind heme. Solubilized HBC was shown to be distinct from hemopexin itself, free heme, ligandin, globin, heme oxygenase, cytochrome P-450, and albumin. Since formation of the heme-HBC complex is effected by the interaction of heme-hemopexin with its receptor, HBC may either be a subunit of the heme-hemopexin receptor or a separate protein that interacts with the receptor. HBC can also bind heme (Kd apparent 200 nM) that is presented to it in a nonprotein bound form, showing true heme-binding activity. HBC is proteinaceous since treatment with proteases, heat, and disulfide bond reducing agents diminishes its ability to bind heme. HBC and any associated detergent elutes from Sephacryl S-200 with an apparent molecular weight of 115,000 and Stokes radius of 7.5 nm. This component, which may comprise 0.5% of liver plasma membrane protein, appears to have an acidic pI since it adsorbs to DEAE-cellulose at pH 7.4 but not to CM-cellulose at pH 6.4. In sucrose gradients, HBC migrates with S values of 1.69 and 4.02, suggesting that it has subunits or that it forms multimers under these conditions.

Animals↗
PubMed1985

The histidine-rich glycoprotein of serum has a domain rich in histidine, proline, and glycine that binds heme and metals.

Histidine-rich glycoprotein (HRG) from rabbit serum was digested with plasmin, reduced, and carboxymethylated, and the fragments produced were resolved by reverse-phase high-performance liquid chromatography. Several peptide fractions were obtained that contain unusually high contents of histidine, proline, and glycine. One His-Pro-Gly-rich peptide (apparent Mr 30 000) was obtained in sufficient yield and purity for further study. This peptide is 29 mol % histidine, 37% proline, and 16% glycine, indicating that most of these three amino acids are located in one region of HRG. The peptide contains 9% by weight carbohydrate and is devoid of tyrosine or tryptophan. The far-ultraviolet circular dichroism spectrum of the peptide has a minimum at 203 nm, indicating that the peptide contains polyproline II helical sections. The peptide represents a binding domain of HRG since it retains much of the ability of intact HRG to bind heme and metals including Zn2+, Ni2+, and Cu2+. As with the parent HRG molecule, interaction of the peptide with heme and metals is dependent on pH and intact histidine residues.

Animals↗
PubMed1984

Domain structure of rabbit hemopexin. Isolation and characterization of a heme-binding glycopeptide.

Plasmin preferentially cleaves rabbit hemopexin at a single site, generating two nondisulfide-linked carbohydrate-containing fragments. In contrast, heme-hemopexin is almost totally resistant to this enzyme and is more resistant than the apoprotein to digestion by trypsin, chymotrypsin, papain, subtilisin, and proteinase K as well. Plasmin digestion dramatically shortens the plasma clearance time of the molecule. The larger glycopeptide (I), shown to be derived from the amino terminus of the parent molecule by sequence analysis, has a molecular weight near 35,000 with a pI of 5.0. It binds 1 mol of heme per mol in a manner analogous to intact hemopexin, molecular weight near 60,000 and pI 5.8. The smaller glycopeptide (II) has a molecular weight near 25,000, a pI of 6.4, and does not bind heme. Of the four oligosaccharides of rabbit hemopexin, peptide I contains three oligosaccharides and peptide II contains one. At micromolar concentrations, the two peptides migrate together during centrifugation through sucrose gradients in the presence, but not in the absence, of heme. Peptide I has a far UV circular dichroism spectrum indicating it has some alpha-helical and extensive nonrepeating peptide structures whereas peptide II appears to be almost exclusively in a beta-sheet conformation. Peptide II is responsible for most of the positive ellipticity at 231 nm of native apohemopexin, but the increase in ellipticity at 231 nm characteristic of heme-hemopexin is not seen when peptide I binds heme, even in the presence of peptide II.

Amino Acid Sequence↗
PubMed1984

Hemopexin-mediated heme uptake by liver. Characterization of the interaction of heme-hemopexin with isolated rabbit liver plasma membranes.

Plasma membranes isolated from rabbit liver retain the ability to interact specifically with heme-hemopexin. In this system, apohemopexin does not compete effectively with heme-hemopexin for binding. The membranes bind heme-hemopexin complexes with high affinity (KD = 6.8 X 10(-7) M) and with an apparent capacity of 2.3 pmol/mg of membrane protein. These membranes also retain the ability to remove heme from heme-hemopexin. The release of heme reaches a plateau after 15-30 min at 30 degrees C and does not involve metabolic energy, proteolysis of hemopexin or pH gradients. The apohemopexin formed is rapidly released from the membranes. The accumulation of heme is saturable and is affected by pH and temperature with maximum uptake occurring between pH 5.5 and 6.5 and at 30 degrees C. Interestingly, much more heme (approximately 25 pmol/mg of membrane protein) is accumulated than hemopexin at saturation, implying that the receptor can turn over several times and that a heme-binding component exists in the rabbit liver plasma membrane.

Animals↗
PubMed1983

A blood group Sda-active pentasaccharide isolated from Tamm-Horsfall urinary glycoprotein.

Human Tamm-Horsfall urinary glycoprotein from an individual of the blood group Sd(a+) phenotype was tritium-labelled by treatment with galactose oxidase and sodium boro[3H]hydride and was then digested with endo-beta-galactosidase. A series of dialysable, labelled fragments was released from which a pentasaccharide was isolated that strongly inhibited the agglutination of Sd(a+) red cells by human anti-Sda serum and hence contained the Sda determinant structure. Reduction, methylation analysis and sequential exo-glycosidase digestion established the structure of the pentasaccharide as: GalNAc beta(1 leads to 4)[NeuAc(2 leads to 3)]Gal beta(1 leads to 4)GlcNAc beta(1 leads to 3)Gal

Blood Group Antigens↗
PubMed1983

Serum histidine-rich glycoprotein during pregnancy and hormone treatment.

The concentration of serum histidine-rich glycoprotein (HRG) was determined by radial immunodiffusion during weeks 27-42 of pregnancy in 110 pregnant women. HRG was also measured in serum from 11 lactating women 6 weeks post partum, from 11 women taking oral contraceptives, and from four women having progestin-releasing subcutaneous capsules used for contraception. The concentration of serum HRG decreased during the last trimester of pregnancy reaching a nadir at the 36-37th week (HRG 49 +/- 14 g/l, mean +/- SD). Thereafter serum HRG increased slightly towards term. In pregnancies complicated by hypertension the concentration of HRG was lower than in normal pregnancies at 32 weeks of pregnancy, but in other pathological pregnancies the values fell within the normal range. By 6 weeks postpartum normal non-pregnant HRG levels had been reached (107 +/- 13 g/l). The concentration of serum HRG was significantly lower in oral contraceptive users (74 +/- 22 g/l) than in controls (109 +/- 25 g/l, P less than 0.005). Low dose progestin treatment had no effect on serum HRG. The results show that serum HRG decreases during pregnancy and with oral contraceptive treatment and suggest that oestrogens are responsible for this increase.

Blood Proteins↗
PubMed1982

Characterization of zinc-binding proteins of plasma in familial hyperzincemia.

Plasmas from three brothers (aged 35 to 45) with chronic hyperzincemia (325 to 525 micrograms/dl Zn) were analyzed and compared with pooled control plasma (104 microgram/dl Zn). The levels of copper, iron, total protein, albumin, and amino acids were similar in normal and hyperzincemic plasmas. Distribution of zinc among plasma components was determined chromatographically. Zinc eluted quantitatively in two distinct peaks from Sephadex G-100 gel filtration resin. The amount of the metal in fractions containing species larger than 100,000 molecular weight (peak I) was similar (35 to 45 micrograms/dl Zn) in normal and hyperzincemic plasmas. The additional complement of zinc in hyperzincemic plasma was localized within fractions containing zinc-binding proteins such as albumin, transferrin and HRG. That zinc was not associated with transferrin was determined by Affi-Gel affinity chromatography. The amounts of HRG in hyperzincemic plasmas were similar to or below those in control plasma. Zinc and albumin were selectively retained by anti-human albumin IgG coupled to protein A-Sepharose. In contrast, anti-human HRG IgG coupled to CM Bio-Gel A failed to bind plasma zinc. The findings indicate that (1) most available, protein-associated zinc in normal plasma is bound to albumin and (2) the additional complement of zinc in familial hyperzincemic plasma is associated with albumin. The biochemical basis for the enhanced binding of zinc by albumin in hyperzincemic plasma is unknown.

Adult↗
PubMed1981

The interaction of heme-hemopexin with CO.

The equilibria and kinetics of the reaction of heme-hemopexin with CO were studied. A stoichiometry of one CO/heme was determined, and the affinity of heme-hemopexin for CO was found to be pH-dependent. At pH 8.0, the affinity constant was 4.5 X 10(5) M-1 compared with 4 X 10(6) M-1 at pH 6.1. The kinetics of CO binding were also pH-dependent. A biphasic reaction at neutral pH could be resolved into a faster phase (kon = 2.2 X 10(3) M-1 s-1) solely found at pH 6.0, and a slower phase (kon = 2.0 X 10(2) M-1 s-1) solely found at pH 8.0. The dissociation reaction on the other hand was found to be independent of pH in the range examined (koff = 5 X 10(-4) s-1).

Animals↗