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Biomedical subjects

W R Harris

Publications and source records attributed to W R Harris.

At least 55 records · Page 3Linked to original sources
PubMed1986

Purification and characterization of a glycogen phosphorylase analog missing the amino-terminal segment.

A glycogen phosphorylase analog missing only the amino-terminal 16 to 18 residues, which include the phosphorylation site, was produced by subtilisin Carlsberg cleavage of phosphorylase b in the presence of caffeine. The analog, named phosphorylase b's, was purified, and its enzymatic properties were compared with those of phosphorylase b. The KM's for glucose 1-phosphate are similar, but phosphorylase b's has a VM 43% higher than that of phosphorylase b. Also, phosphorylase b's is less sensitive to inhibition by glucose 6-phosphate and stimulation by sodium fluoride than is phosphorylase b. The subunit interactions in the two enzyme forms were also compared. The monomer-monomer interactions in phosphorylase b's are weaker than in phosphorylase b, as evidenced by a faster rate of resolution of the coenzyme, pyridoxal phosphate, from phosphorylase b's. The dimer-dimer interactions are also weaker in phosphorylase b's than in phosphorylase b, because phosphorylase b's does not form tetramers or crystals as readily as does phosphorylase b. Because removal of the amino-terminal segment changes the properties of the enzyme, this segment must be interacting with other parts of the protein. This statement conflicts with previous interpretation of X-ray crystallographic data that suggest that the amino-terminal region of phosphorylase b is freely mobile. Possible explanations for this contradiction are discussed.

Amino Acid Sequence↗
PubMed1986

Thermodynamics of gallium complexation by human lactoferrin.

Equilibrium constants for the successive binding of 2 equiv of Ga3+ to human lactoferrin have been measured by difference ultraviolet spectroscopy in 0.1 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid containing 5 mM bicarbonate at pH 7.4 and 25 degrees C. Ethylenediamine-N,N'-diacetic acid was used as the competing chelating agent. Values of the effective binding constants for the stated experimental conditions are log K1 = 21.43 +/- 0.18 and log K2 = 20.57 +/- 0.16. Comparison of these results with literature values for the gallium-transferrin binding constants indicates that lactoferrin binds gallium more strongly by a factor of approximately 90. The ratios of successive binding constants for the two proteins are essentially identical. A linear free energy relationship (LFER) for the complexation of gallium(III) and iron(III) has been prepared and used to estimate an iron(III)-lactoferrin binding constant for pH 7.4. The LFER prediction is compared with thermodynamic data on iron binding at pH 6.4 and gallium binding at pH 7.4. The results indicate that the ratio of iron binding constants for lactoferrin and transferrin is likely in the range of 50-90.

Gallium↗
PubMed1986

Estimation of the ferrous-transferrin binding constants based on thermodynamic studies of nickel(II)-transferrin.

The equilibrium constants for the binding of Ni2+ to human serum transferrin in 0.01 M hepes containing 5 mM sodium bicarbonate at 25 degrees C and pH 7.4 have been measured. The effective binding constants are log K1 = 4.10 +/- 0.15 and log K2 = 3.23 +/- 0.31 for the reactions Ni2+ + apoTr (K1) in equilibrium Ni2+-Tr. Ni2+ + Ni2+-Tr (K2) in equilibrium Ni2+-Tr-Ni2+ where the explicit terms for bicarbonate and hydrogen ion have been incorporated into the effective binding constants. Titration of both forms of mono(ferric)transferrin indicates that unlike other metal ions, Ni2+ binds preferentially to the N-terminal binding site, but that the site preference is rather small. A linear-free-energy relationship (LFER) for the complexation of Ni2+ and Fe2+ has been prepared. This LFER has been used to estimate effective binding constants of log K1 = 3.2 and log K2 = 2.5 for the ferrous-transferrin complex. These ferrous constants have been combined with the literature binding constants for ferric-transferrin to estimate formal reduction potentials of -340 mV vs. NHE for the C-terminal site and -280 mV for the N-terminal site.

Humans↗
PubMed1985

Thermodynamics of anion binding to human serum transferrin.

The binding of phosphate, bicarbonate, sulfate, and vanadate to human serum transferrin has been evaluated by two difference ultraviolet spectroscopic techniques. Direct titration of apotransferrin with bicarbonate, phosphate, and sulfate produces a strong negative absorbance near 245 nm, while titration with vanadate produces a positive absorbance in this region. Least-squares refinement of the absorbance data indicates that two anions of sulfate, phosphate, and vanadate bind to each transferrin molecule but that there is detectable binding of only a single bicarbonate anion. A second method used to study the thermodynamics of anion binding was competition equilibrium between anions for binding to the transferrin. The equilibrium constant for binding of the first equivalent of vanadate was determined by competition vs. phosphate and sulfate, while the equilibrium constant for binding of the second equivalent of bicarbonate was determined by competition vs. vanadate. Anion binding was described by two equilibrium constants for the successive binding of two anions per transferrin molecule: K1 = [A-Tr]/[A][Tr] and K2 = [A-Tr-A]/[A][A-Tr] where [A] represents the free anion concentration, [Tr] represents apotransferrin concentration, and [A-Tr] and [A-Tr-A] represent the concentrations of 1:1 and 2:1 anion-transferrin complexes, respectively. The results were the following: for phosphate, log K1 = 4.19 +/- 0.03 and log K2 = 3.25 +/- 0.21; for sulfate, log K1 = 3.62 +/- 0.07 and log K2 = 2.79 +/- 0.20; for vanadate, log K1 = 7.45 +/- 0.10 and log K2 = 6.6 +/- 0.30; for bicarbonate, log K1 = 2.66 +/- 0.07 and log K2 = 1.8 +/- 0.3.(ABSTRACT TRUNCATED AT 250 WORDS)

Anions↗
PubMed1985

Comparison of whole blood serotonin and platelet MAO in children with schizophrenia and major depressive disorder.

Whole blood serotonin and platelet monoamine oxidase (MAO) activity in boys with schizophrenia, schizotypal personality disorder, or major depressive disorder was compared with that of boys serving as controls. Boys with schizophrenia and schizotypal personality disorder had significantly higher platelet MAO than boys with major depressive disorder or controls. Boys with major depressive disorder had lower whole blood serotonin than boys with schizophrenia or schizotypal personality disorder.

Adolescent↗
PubMed1984

Contribution of nitropyrene to the mutagenic activity of coal fly ash.

Stack-collected coal fly ash from western low-sulfur coal was extracted with 60:40 benzene/methanol. This extract was fractionated by preparative-scale high performance liquid chromatography (HPLC) and the mutagenic activity of 14 fractions was evaluated by microbial assay with Salmonella typhimurium TA1538. A widespread distribution of direct-acting mutagens, which probably includes both mono- and dinitroaromatics, was detected. HPLC methods were also used to isolate 1-nitropyrene from the total benzene/methanol extract. The identification of 1-nitropyrene was based on gas chromatographic and HPLC retention measurements and mass spectral data. The concentration of 1-nitropyrene in the ash extract was determined by quantitative HPLC analyses. Mutagenicity assays of the total extract and an authentic 1-nitropyrene standard with Salmonella strains TA1538, TA100, and TA98 indicated that the 1-nitropyrene accounts for approximately 0.03-0.16% of the total mutagenic activity of the extract.

Chromatography, Gas↗
PubMed1984

Behavior of vanadate and vanadyl ion in canine blood.

Radiolabeled vanadium as either vanadyl ion or vanadate ion was injected intravenously into adult beagle dogs, and blood samples were collected at various times up to 48 hr post injection. For each sample, the distribution of vanadium between the cells and the plasma was determined, and the plasma was analyzed by electrophoresis to identify specific vanadium-binding proteins. Initially, vanadyl ion left the bloodstream more rapidly than vanadate, but the rates equalized after about 5 hr. A significant fraction of the vanadium in blood was associated with the cellular component following injection of both forms of vanadium. About 77% of the plasma vanadium was eventually bound by the serum iron transport protein transferrin, regardless of the vanadium species initially injected. For both vanadyl and vanadate, about 30 hr were required to reach the maximum degree of transferrin binding.

Animals↗
PubMed1984

Binding of vanadate to human serum transferrin.

Human serum transferrin specifically and reversibly binds 2 equiv of vanadate at the two metal-binding sites of the protein. The vanadium(V)-transferrin complex can be formed either by the addition of vanadate to apotransferrin or by the air oxidation of the vanadyl(IV)-transferrin complex. The formation of the vanadium complex can be blocked by loading the apotransferrin with iron(III), and bound vanadium can be displaced from the protein by the subsequent addition of either gallium(III) or iron(III). The binding constant for the second equiv of vanadate is 10(6.5) in 0.1 M hepes, pH 7.4 at 25 degrees C. The binding constant for the first equiv of vanadate is probably very similar, although no quantitative value could be determined. Although transferrin reacts with the vanadate anion, studies on the transferrin model compound ethylenebis(o-hydroxyphenylglycine) indicate that at pH 9.5, the vanadium is binding at the metal-binding site as a dioxovanadium(V) cation coordinated to two phenolic residues at each binding site. This bound cation appears to be protonated over the pH range 9.5-6.5, as shown by changes in the difference uv spectrum of the transferrin complex, to produce an oxohydroxo species. Further decreases in the pH lead to dissociation of the vanadium-transferrin complex.

Binding Sites↗
PubMed1984

Kinetics of the removal of ferric ion from transferrin by aminoalkylphosphonic acids.

The kinetics of ion removal at 25 degrees C in 0.1 M Tris, pH 7.4 by a series of phosphonic acids have been evaluated. The initial rate of iron removal is first order in ferric-transferrin, but shows a hyperbolic dependence on the concentration of the phosphonate ligand. At high ligand concentrations the reaction is clearly biphasic, and the data are interpreted in terms of nonequivalent rate constants for iron removal from the two transferrin iron-binding sites. Rate constants for three phosphonic acid ligands are approximately 0.025 min-1 and approximately 0.007 min-1 for the faster and slower binding sites. The results are discussed in relation to the conformational change mechanism for iron removal from transferrin proposed by Coward et al. [21].

Animals↗
PubMed1984

Mutagenicity in Salmonella of nitroorganic compounds in extracts of fly ash from a lignite-fired atmospheric fluidized-bed combustor.

The mutagenicity of a benzene/methanol extract of fly ash from an atmospheric fluidized-bed combustor burning Beulah, N.D., lignite was tested in Salmonella. Six strains were used, including three that were mutants in a nitroreductase gene locus. The numbers of revertants from his- to his+ as a function of the amount of fly ash extracted were determined. The results showed that the major mutagens in the crude extract were nitro compounds, from the fact that reversion rates in the nitroreductase-deficient strains were significantly lower than in the parent strains from which they were derived. The responses of the three parental strains, TA1538, TA98, and TA100, were quite similar; thus no conclusions could be made about frameshift versus base-substitution mutagens. Mutagenicity of 15 fractions of the extract was also tested, and one major peak of activity was detected. This activity eluted from a high-performance liquid chromatograph outside the range of retention times associated with mononitroaromatics. No further identification of specific nitroorganic compounds has been made.

Air Pollutants↗
PubMed1983

Thermodynamic binding constants of the zinc-human serum transferrin complex.

Serum transferrin is a mammalian iron-transport protein. It has two specific metal-binding sites that bind a variety of metal ions in addition to ferric ion. Equilibrium constants for the binding of zinc(II) have been determined by difference UV titrations using nitrilotriacetic acid and triethylenetetramine as competing ligands. The values are log K1* = 7.8 and log K2* = 6.4 in 0.10 M N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid and 15 mM bicarbonate, pH 7.4 at 25 degrees C. Titrations of the two forms of monoferric transferrin show that K1* corresponds to zinc binding to the C-terminal site and K2* corresponds to binding at the N-terminal site. These results indicate that at serum bicarbonate concentrations, transferrin should have a higher affinity for zinc(II) than serum albumin and therefore could play some role in zinc transport. A linear free-energy relationship has been constructed which relates the formation constants of a series of zinc(II) and iron(II) complexes. On the basis of the zinc-transferrin binding constants, this relationship has been used to estimate an iron(II)-transferrin binding constant of 10(7.4). Using this ferrous constant and literature values for the ferric transferrin binding constant, one calculates a ferric transferrin reduction potential of -140 mV, which is easily within the range of physiological reductants. Such a result tends to support mechanisms for iron removal from transferrin in which the ferric ion is reduced to the less tightly bound ferrous ion.

Apoproteins↗
PubMed1983

Thermodynamic binding constants for gallium transferrin.

Gallium-67 is widely used as an imaging agent for tumors and inflammatory abscesses. It is well established that Ga3+ travels through the circulatory system bound to the serum iron transport protein transferrin and that this protein binding is an essential step in tumor localization. However, there have been conflicting reports on the magnitude of the gallium-transferrin binding constants. Therefore, thermodynamic binding constants for gallium complexation at the two specific metal binding sites of human serum transferrin at pH 7.4 and 5 mM NaHCO3 have been determined by UV difference spectroscopy. The conditional constants calculated for 27 mM NaHCO3 are log K1 = 20.3 and log K2 = 19.3. These results are discussed in relation to the thermodynamics of transferrin binding of Fe3+ and to previous reports on gallium binding. The strength of transferrin complexation is also compared to that of a series of low molecular weight ligands by using calculated pM values (pM = -log [Ga-(H2O)6]) to express the effective binding strength at pH 7.4.

Edetic Acid↗
PubMed1983

Tyrosine-rich crystalloids in adenoid cystic carcinoma: histochemical and ultrastructural observations.

Tyrosine-rich crystalloids are described in an example of an adenoid cystic carcinoma arising in an oral minor salivary gland of the hard palate. This represents the first reported case of an adenoid cystic carcinoma of oral minor salivary gland in which tyrosine-rich deposits have been identified. The tyrosine deposits appear refractile but not birefringent at the light microscopic level; and histochemical findings indicate a relatively simple, homogeneous chemical composition rich in tyrosine. Their ultrastructural nature is that of a finely granular, but otherwise patternless, highly electron-dense material. The electron microscopic features of the neoplasm are similar to previous reports. Based upon the findings of this and previous investigations, it is not as yet possible to state whether the tyrosine-rich deposits truly represent a crystalline or amorphous solid.

Carcinoma, Adenoid Cystic↗