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Biomedical subjects

W T Morgan

Publications and source records attributed to W T Morgan.

At least 55 records · Page 3Linked to original sources
PubMed1991

Primary structure and relative potency of an analog of beta-PDH (pigment-dispersing hormone) from the crayfish Procambarus clarkii.

A pigment-dispersing hormone (PDH) from eyestalks of the crayfish Procambarus clarkii was purified by gel filtration, cation-exchange chromatography, partition chromatography, and reversed-phase HPLC. Based on automated sequencing and by the identical chromatographic behavior of the native PDH and the synthetic amidated form of the deduced sequence, the primary structure of Procambarus PDH has been established as: Asn-Ser-Glu-Leu-Ile-Asn-Ser-Ile-Leu-Gly-Leu-Pro-Lys-Val-Met-Asn-Glu-Ala- NH2. This peptide differs from beta-PDH of the fiddler crab Uca pugilator at a single position, Glu17 in place of Asp17. Because of this substitution, Procambarus PDH was 4 to 7-fold less potent than beta-PDH in causing pigment dispersion in the erythrophores, leucophores, and melanophores of Uca. In contrast, Procambarus PDH was 4-fold more potent than beta-PDH in eliciting pigment dispersion in the erythrophores of Procambarus. These peptides displayed less marked differences in potency in triggering leucophore pigment dispersion and light-adaptational distal eye pigment movement in Procambarus. These findings indicate that the structural requirements for PDH-receptor interactions vary with the species and with the target cell type within a given species.

Amino Acid Sequence↗
PubMed1989

A heme- and metal-binding hexapeptide from the sequence of rabbit plasma histidine-rich glycoprotein.

Rabbit histidine-rich glycoprotein (HRG) binds low-spin heme and metals tightly at several sites that contain histidine. As part of an on-going effort to define and locate the binding sites for these and the other ligands of HRG, the sequence: NH2-Gly-His-Phe-Pro-Phe-His-Trp-... was found in a 16 kDa heme-binding peptide isolated from HRG. The spacing of the histidyl residues in this peptide, which contains the C-terminal 79 residues of HRG, together with molecular modeling suggested that this sequence might constitute one heme binding site of HRG by accommodating heme in a bis-histidyl linkage. Three peptides based on this sequence (I, HFPFHW; II, WHFPFH; and III, HFGFHW) were synthesized, and their ability to bind heme and metals examined. All three peptides bind heme as demonstrated by the changes produced in the absorbance of heme when mixed with the peptides. Substituting glycine for proline in the central position or moving the location of the tryptophan did not affect heme binding. The apparent Kd's of the mesoheme/peptide I, II and III complexes are 75 +/- 25 microM, indicative of heme binding approximately 100 times less avid than the mesoheme/HRG complex (Kd ca. 1 microM), but nearly 1000 times tighter than that of the mesoheme/histidine complex (Kd ca. 60 mM). The absorbance spectra of the mesoheme/peptide complexes, the loss of binding caused by modification of histidine residues, and the pH dependence of heme binding, all indicate that heme forms a low spin, bis-histidyl type of complex with these peptides, like that formed with HRG itself. Copper, but not cadmium or nickel, was an effective inhibitor of heme binding by the peptides. The sequence of HRG congruent with the sequence of peptide I is proposed to be one heme- and metal-binding site of rabbit HRG.

Amino Acid Sequence↗
PubMed1989

Enzymic synthesis, chemical characterisation and Sda activity of GalNAc beta 1-4[NeuAc alpha 2-3]Gal beta 1-4GlcNAc and GalNAc beta 1-4[NeuAc alpha 2-3]Gal beta 1-4Glc.

The tetrasaccharides GalNAc beta 1-4[NeuAc alpha 2-3]Gal beta 1-4Glc and GalNAc beta 1-4[NeuAc alpha 2-3]Gal beta 1-4GlcNAc were synthesised by enzymic transfer of GalNAc from UDP-GalNAc to 3'-sialyllactose (NeuAc alpha 2-3 Gal beta 1-4Glc) and 3'-sialyl-N-acetyllactosamine (NeuAc alpha 2-3Gal beta 1-4GlcNAc). The structures of the products were established by methylation and 1H-500 MHz NMR spectroscopy. In Sda serological tests the product formed with 3'-sialyl-N-acetyllactosamine was highly active whereas that formed with 3'-sialyllactose had only weak activity.

Animals↗
PubMed1989

A broad evaluation of B.t.i. for black fly (Diptera: Simuliidae) control in a Michigan river: efficacy, carry and nontarget effects on invertebrates and fish.

Efficacy for black fly control, carry and nontarget effects of B.t.i. (Teknar HP-D), applied in the Betsie River, Michigan, were studied in June 1988. Black fly mortality was high (approximately 100%) for a 2,200 m stretch downstream from the application site, declined to 30% at 3,200 m, and was nil at 4,500 m. Drift of black flies greatly increased after application at a downstream site, but did not change at an upstream site. There were no detectable nontarget effects of B.t.i. application on: 1) invertebrate macro- or micro-drift; 2) numbers of invertebrates in benthic Surber samples; 3) mortality or feeding of drifting and nondrifting insects; 4) growth or mortality of caged Stenomena sp. larvae; 5) invertebrate functional group composition; 6) mortality or weight change of caged rock bass; or 7) fish numbers, species composition, length-weight (rock bass only) relationships or rock bass diet. Sampling of Rheotanytarsus sp. midges on natural substrates indicated low (27%) mortality owing to B.t.i. at only 100 m downstream from the application site, with negligible mortality at all other downstream and upstream sites. This information, combined with no pronounced changes in numbers of midges in macro-drift after application, indicated that midge populations were not adversely affected by B.t.i. in the study.

Analysis of Variance↗
PubMed1988

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↗
PubMed1988

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↗
PubMed1988

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↗
PubMed1988

Paramagnetic probes of the domain structure of histidine-rich glycoprotein.

The interaction of Cu2+ and Fe3+-mesoporphyrin with histidine-rich glycoprotein (HRG) from rabbit serum was examined spectroscopically. The first equivalent of Cu2+ binds to HRG producing a type II electron paramagnetic resonance (EPR) spectrum with g[[ = 2.25, gm = 2.05, A[[ = 0.019 cm-1 (180 G), and superhyperfine along gm. These spectral parameters suggest moderately covalent coordination of Cu2+ to the protein by nitrogens. With increasing Cu2+ the superhyperfine disappears; however, the g and A values change only marginally. The increase in EPR signal amplitude throughout the addition of 1-15 equiv of Cu2+ is linear and thereafter maximizes, suggesting 18-22 equiv are bound. In contrast, changes in the circular dichroism spectrum at 280 nm appear sigmoidal and can be interpreted as the binding of Cu2+ to two structurally distinct regions of the protein. Evidence for two structurally distinct binding domains is found by comparing EPR spectra of Cu2+ complexes of HRG with spectra from complexes of two of its major proteolysis products (peptides). After binding 1 equiv of Cu2+, both the 30-kDa histidine-rich peptide and the native protein exhibit identical spectra including the pronounced superhyperfine. In contrast, the spectrum of the histidine-normal 45-kDa peptide with 1 equiv of Cu2+ bound lacks superhyperfine and parallels closely that of the native protein with 20 equiv bound. Finally, Fe3+-mesoporphyrin binds to HRG exhibiting both high-spin (g = 6.05) and low-spin (gz = 2.94, gy = 2.25, gx = 1.50) EPR resonances, and the latter imply bis(histidine) coordination.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Characterization of Cu2+ and Fe3+ -mesoporphyrin complexes with histidine-rich glycoprotein: evidence for Cu2+ -Fe3+ -mesoporphyrin interaction.

One equivalent of Fe3+ -mesoporphyrin (heme) is coordinated by two axial histidine ligands to a preferred site on histidine-rich glycoprotein (HRG). This study shows that titration of this stochiometric heme.HRG complex with 0-20 equivalents of Cu2+ produces a series of pronounced spectral changes indicative of multiple, sequential alterations of the heme environment. A monotonic low- to high-spin heme transition characterized by a decrease in resonance amplitude at g = 2.99, an increase at g = 6.0, and an increase in absorptivity at 620 nm is induced with the addition of the first 10 Cu2+ equivalents. Furthermore, optical absorption and circular dichroism spectra exhibit isosbestic and isodichroic points throughout the addition of the first 8 and 12 equivalents, respectively. The isosbestic points imply a transition between two optically well defined axial heme coordinations, and the isodichroic points suggest that these axial coordinations also represent two distinct protein conformations. A second isosbestic is formed during the addition of 14-20 equivalents of Cu2+, again suggesting well-defined coordinations; however, changes in the EPR spectra over this range are more complex. Whereas the amount of low-spin (g = 2.99) heme.HRG complex continues to decrease with the addition of 10-20 Cu2+ equivalents, the amount of the high-spin (g = 6.0) complex reaches a maximum near 14 equivalents and decreases markedly thereafter. Of potentially greater significance is the appearance of signals at g = 9.3 (maximum), 7.7 (maximum), 4.8 (crossover), and 1.61 (minimum) after addition of 10 or more Cu2+ equivalents. Some of these signals are similar to those exhibited by cardiac cytochrome c oxidase upon reduction and reoxidation. Thus, even without the addition of exogenous reductants and oxygen, the interaction of Cu2+ with the stoichiometric heme.HRG complex may produce structural features similar to those found in a mechanistically important but poorly understood form of cardiac cytochrome c oxidase.

Animals↗
PubMed1988

Accelerated autoxidation and heme loss due to instability of sickle hemoglobin.

The pleiotropic effect of the sickle gene suggests that factors in addition to polymerization of the mutant gene product might be involved in sickle disease pathobiology. We have examined rates of heme transfer to hemopexin from hemoglobin in dilute aqueous solution (0.5 mg of Hb per ml) at 37 degrees C. HbO2 S loses heme 1.7 times faster than HbO2 A, with apparent rate constants of 0.024 hr-1 and 0.014 hr-1, respectively. In contrast, Hb A and Hb S behave identically in their MetHb forms (very rapid heme loss) and their HbCO forms (zero heme loss). This indicates that the faster heme loss from HbO2 S is due to accelerated autoxidation (HbO2----MetHb) rather than to some other type of instability inherent in the relationship of sickle heme to its pocket in globin. This interpretation is supported by spectrophotometric measurement of initial rates of MetHb formation during incubation at 37 degrees C. This directly shows 1.7 times faster autoxidation, with apparent rate constants of 0.050 hr-1 for HbO2 S and 0.029 hr-1 for HbO2 A. While the participation of this process in the cellular pathobiology of sickle erythrocytes remains unproven, the present data are consistent with, and perhaps help explain, two prior observations: the excessive spontaneous generation of superoxide by sickle erythrocytes; and the abnormal deposition of heme and heme proteins on membranes of sickle erythrocytes.

Anemia, Sickle Cell↗
PubMed1988

Interference of oral phenytoin absorption by enteral tube feedings.

The medical records of 11 brain-injured patients, simultaneously on enteral tube feeding and phenytoin, were retrospectively reviewed for evidence of interference of phenytoin absorption. One case demonstrated no interference. Three cases suggested depression of serum phenytoin levels by enteral formula, but the contribution of other factors could not be ruled out. Seven cases did appear to reflect a direct alteration of phenytoin levels by enteral formula. To achieve adequate serum phenytoin levels, patients may need (1) large oral phenytoin doses above that recommended while on continuous enteral feedings, (2) administration of oral phenytoin between intermittent enteral feedings, or (3) a choice of alternative anticonvulsants which have been shown to be unaffected by enteral formula.

Absorption↗
PubMed1987

Further characterization of the interaction of histidine-rich glycoprotein with heparin: evidence for the binding of two molecules of histidine-rich glycoprotein by high molecular weight heparin and for the involvement of histidine residues in heparin binding.

Rabbit histidine-rich glycoprotein (HRG, 94 kDa) binds heparin with high affinity (apparent Kd 60-110 nM). Eosin Y (1 equiv) bound to HRG was used as a reporter group to monitor associations of HRG with heparins of molecular mass 10, 17.5, and 30 kDa. The stoichiometries of the heparin-HRG complexes were determined by fluorescence and absorbance measurements as well as by analytical ultracentrifugation. Two types of complex form: complexes of 1 heparin:1 HRG and of 1 heparin:2 HRG. The 1:2 complex formation requires a minimum heparin chain length since 17.5-kDa but not 10-kDa heparin binds two HRG molecules. The formation of the 1:2 complexes of the larger heparin fractions is enhanced by divalent copper or zinc (1-10 equiv) bound to HRG. However, metal is not required for complex formation since all sizes of heparin examined interact tightly with HRG in the presence of ethylenediaminetetraacetic acid. Between 0.1 and 0.3 M ionic strength, both 1:1 and 1:2 complexes of heparin with HRG are progressively destabilized. No heparin-HRG complex is found at ionic strengths of 0.5 M. Between pH 8.5 and pH 6.5 both 1:2 and 1:1 complexes are found with 17.5-kDa heparin, but at pH 5.5 only 1:1 complexes are formed. The heparin-HRG interaction is progressively decreased by modification of the histidine residues of HRG, whereas modification of 22 of the 33 lysine residues of HRG has little effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Histidine-rich glycoprotein modulation of the anticoagulant activity of heparin. Evidence for a mechanism involving competition with both antithrombin and thrombin for heparin binding.

Heparin binding to rabbit histidine-rich glycoprotein (HRG) was studied in a purified system, allowing for determination of a heparin dissociation constant of approximately 5.5 X 10(-8) M for the interaction with HRG at pH 7.0. The strong interaction between heparin and HRG was demonstrated to be competitive with the binding of both antithrombin and thrombin to the heparin chain. HRG was further tested as a modulator of the anticoagulant activity of heparin by comparing rates of the heparin-catalyzed reaction between antithrombin and thrombin in the presence and absence of added HRG. The heparin-antithrombin-thrombin reaction was modeled using the formalism of a two-substrate enzyme-catalyzed reaction with heparin as the enzyme and HRG analyzed as an enzyme inhibitor. HRG was shown to compete with both antithrombin and thrombin for binding to heparin by this kinetic analysis. Thus, both the kinetic and heparin-binding data indicate that the mechanism by which HRG modulates heparin anticoagulant activity involves competition for heparin with both the inhibitor and the protease. Inhibition by HRG of the heparin-catalyzed reaction was found to be highly dependent on pH, with a sharp increase in inhibition from about 15% to greater than 90% observed as pH was lowered from 7.4 to 7.0. Since little change in the rate of the heparin-catalyzed inhibition of thrombin by antithrombin occurs in this pH region, the dramatic change in HRG inhibition seen upon pH titration may reflect increasing ionic interaction between heparin and HRG due to the protonation of histidine residues which occurs in this pH region.

Antithrombins↗
PubMed1986

Serum histidine-rich glycoprotein levels are decreased in acquired immune deficiency syndrome and by steroid therapy.

Using radial immunodiffusion serum histidine-rich glycoprotein (HRG) levels were measured in acquired immune deficiency syndrome (AIDS) patients, in end-stage renal disease (ESRD) patients after renal transplantation and immunosuppressive steroid therapy, and in asthma and chronic obstructive pulmonary disease (COPD) patients treated with steroids. Compared with controls (12.5 +/- 3.0 mg/dl), HRG levels were significantly decreased in patients with AIDS (5.7 +/- 1.8 mg/dl, P less than 0.005): in patients with ESRD after renal transplantation with steroid therapy (4.4 +/- 1.1 mg/dl, P less than 0.005); and in asthmatic and COPD patients receiving steroids in acute (7.6 +/- 2.9 mg/dl, P less than 0.005) or chronic (7.4 +/- 3.0 mg/dl, P less than 0.025) high-dose regimens. In contrast, levels of hemopexin, another serum glycoprotein synthesized by the liver, were not lowered in these patients. These results show that serum HRG levels are selectively decreased in AIDS and in patients treated with immunosuppressive steroids.

Acquired Immunodeficiency Syndrome↗
PubMed1985

Proteolysis of histidine-rich glycoprotein in plasma and in patients undergoing thrombolytic therapy.

Plasmin and kallikrein but not thrombin cleave purified histidine-rich glycoprotein (HRG), and heparin binding inhibits the proteolysis of HRG. To assess the proteolysis of HRG in plasma, immunoaffinity chromatography was used to isolate HRG from human plasma samples and the extent of protein cleavage was determined after electrophoresis under reduced, denaturing conditions. In blood drawn into streptokinase or into urokinase, HRG (78 kDa) was degraded producing peptides ranging in apparent molecular weight from 67 to 9 kDa. In patients undergoing thrombolytic therapy almost no intact HRG remains after 30 minutes, but the levels of circulating HRG are unchanged, indicating that cleaved HRG is not quickly or extensively removed from the circulation.

Blood Proteins↗