Fourier transform infrared spectroscopy and differential scanning calorimetry of transferrins: human serum transferrin, rabbit serum transferrin and human lactoferrin

Hadden, Jonathan M.; Bloemendal, Michael; Haris, Parvez I.; Srai, Surjit Kaila; Chapman, D.

doi:10.1016/0167-4838(94)90092-2

Cited by 53 publications

(47 citation statements)

References 33 publications

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“…Lf and Tf have been particularly well characterized in terms of their structural similarities (30), stable monomeric solution properties (44,45) and in vitro binding behavior (31)(32)(33)(34) (46,47)]. The reduction in D observed for Lf in the presence of H was, therefore, consistent with Lf-H complex formation and not Lf aggregation.…”

Section: Suitability Of Lf and Tf As In Vivo Diffusionmentioning

confidence: 55%

In vivo diffusion of lactoferrin in brain extracellular space is regulated by interactions with heparan sulfate

Thorne

Lakkaraju²,

Rodríguez-Boulan³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

120

135

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The intercellular spaces between neurons and glia contain an amorphous, negatively charged extracellular matrix (ECM) with the potential to shape and regulate the distribution of many diffusing ions, proteins and drugs. However, little evidence exists for direct regulation of extracellular diffusion by the ECM in living tissue. Here, we demonstrate macromolecule sequestration by an ECM component in vivo, using quantitative diffusion measurements from integrative optical imaging. Diffusion measurements in free solution, supported by confocal imaging and binding assays with cultured cells, were used to characterize the properties of a fluorescently labeled protein, lactoferrin (Lf), and its association with heparin and heparan sulfate in vitro. In vivo diffusion measurements were then performed through an open cranial window over rat somatosensory cortex to measure effective diffusion coefficients (D*) under different conditions, revealing that D* for Lf was reduced Ϸ60% by binding to heparan sulfate proteoglycans, a prominent component of the ECM and cell surfaces in brain. Finally, we describe a method for quantifying heparan sulfate binding site density from data for Lf and the structurally similar protein transferrin, allowing us to predict a low micromolar concentration of these binding sites in neocortex, the first estimate in living tissue. Our results have significance for many tissues, because heparan sulfate is synthesized by almost every type of cell in the body. Quantifying ECM effects on diffusion will also aid in the modeling and design of drug delivery strategies for growth factors and viral vectors, some of which are likely to interact with heparan sulfate.drug delivery ͉ extracellular matrix ͉ integrative optical imaging ͉ somatosensory cortex ͉ transferrin T he spread of diffusible signals in brain extracellular space (ECS) is influenced by the local environment, with clearance mechanisms and the ECS volume fraction, tortuosity, and width among the best appreciated factors (1-3). The role that brain extracellular matrix (ECM) components play in modulating diffusion is less understood. Normal brain ECM is composed mostly of hyaluronic acid, a nonsulfated glycosaminoglycan, and proteoglycans carrying either chondroitin sulfate (CSPG) or heparan sulfate (HSPG) glycosaminoglycan side chains, along with the more recently identified reelin and tenascin glycoproteins (4, 5). Although aging (6-8), pathological insults (9, 10), or genetic modifications (11) resulting in altered brain ECM content are often associated with changes in ECS volume fraction, the ability of ECM components to specifically bind and slow the migration of diffusing substances in the ECS remains an open question. A direct ECM effect on extracellular diffusion (e.g., sequestration or slowing of a diffusing substance) has been postulated for proteins capable of binding HSPG (12, 13), but little evidence exists for this phenomenon in vivo.HSPGs are thought to play essential roles in the physiology of all organ systems (14). They compris...

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Section: Suitability Of Lf and Tf As In Vivo Diffusionmentioning

confidence: 55%

In vivo diffusion of lactoferrin in brain extracellular space is regulated by interactions with heparan sulfate

Thorne

Lakkaraju²,

Rodríguez-Boulan³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

120

135

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“…However, the stabilizing effect of iron on the native conformation of hTH1 (see above) is further strengthened on dopamine binding to the holoenzyme. This effect is most likely on the monomer of the tetrameric enzyme, since each subunit binds equimolar amounts of the ligands, as found with other soluble proteins (24,31,32). Thus, inhibition of the enzyme by catecholamines is associated with a stabilization of its conformation and could be related to the proposed existence of two forms of the bovine enzyme (33), i.e.…”

Section: Discussionmentioning

confidence: 99%

Conformational Properties and Stability of Tyrosine Hydroxylase Studied by Infrared Spectroscopy

Martı́nez

Haavik

Flatmark

et al. 1996

Journal of Biological Chemistry

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The conformation and stability of recombinant tetrameric human tyrosine hydroxylase isoenzyme 1 (hTH1) was studied by infrared spectroscopy and by limited tryptic proteolysis. Its secondary structure was estimated to be 42% ␣-helix, 35% ␤-extended structures (including ␤-sheet), 14% ␤-turns, and 10% nonstructured conformations. Addition of Fe(II) or Fe(II) plus dopamine to the apoenzyme did not significantly modify its secondary structure. However, an increased thermal stability and resistance to proteolysis, as well as a decreased cooperativity in the thermal denaturation transition, was observed for the ligand-bound forms. Thus, as compared with the apoenzyme, the ligand-bound subunits of hTH1 showed a more compact tertiary structure but weaker intersubunit contacts within the protein tetramer. Phosphorylation of the apoenzyme by cyclic AMP-dependent protein kinase did not change its overall conformation but allowed on iron binding a conformational change characterized by an increase (about 10%) in ␣-helix and protein stability. Our results suggest that the conformational events involved in TH inhibition by catecholamines are mainly related to modifications of tertiary and quaternary structural features. However, the combined effect of iron binding and phosphorylation, which activates the enzyme, also involves modifications of the protein secondary structure.Tyrosine hydroxylase (TH, 1 EC 1.14.16.2) is a non-heme iron and tetrahydrobiopterin-dependent enzyme that catalyzes the conversion of L-tyrosine to L-dihydroxyphenylalanine (L-DO-PA), the first and rate-limiting step in the biosynthesis of catecholamines (1, 2). The catalytic activity of TH seems to be short-term regulated by feedback inhibition by catecholamines and by reversal of this inhibition by phosphorylation (1, 3).Until the recent cloning and overexpression of TH from different species (4 -6), the source of the enzyme has mostly been the bovine adrenal medulla or pheochromocytoma (PC12) cells. However, these enzymes are isolated partially inhibited by catecholamines bound at the active site and are activated severalfold by phosphorylation of Ser-40 by the catalytic subunit of cyclic AMP-dependent protein kinase (cAPK) (7-9). This activation has been related to the increased dissociation rate of catecholamines from the active site on phosphorylation (3).Human TH exists as several different isoforms generated by alternative splicing of pre-mRNA (10, 11); isoform 1 (hTH1) is the most abundant species in the adrenal medulla and in the substantia nigra of the brain (10). When expressed in Escherichia coli, human TH isoforms are isolated as homogeneous tetrameric apoenzymes composed of identical 56-59-kDa subunits (12) that are rapidly activated (up to 40-fold) by binding of 1 atom of Fe(II) per subunit (4). The recombinant human enzymes are inhibited by catecholamines that chelate iron at the active site, and this inhibition is partially reversed by phosphorylation of Ser-40 by cAPK (3, 12, 13).The three-dimensional structure of TH remains to be deter...

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“…and a Perkin-Elmer 7300 data station as described previously [25,261. All spectra were recorded at 20°C at a resolution of 4 cm-'.…”

Section: Methodsmentioning

confidence: 99%

A Fourier‐Transform Infrared Spectroscopic Investigation of the Hydrogen‐Deuterium Exchange and Secondary Structure of the 28‐kDa Channel‐Forming Integral Membrane Protein (CHIP28)

Haris

Chapman

Benga

1995

European Journal of Biochemistry

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Fourier-transform infrared spectroscopy (FTIR) has been employed to investigate the structural properties of the 28-kDa channel-forming integral membrane protein (CHIP28) present in phospholipid vesicles suspended in aqueous media. This study reports the FTIR spectra of this membrane protein present in H,O and *H,O. The secondary structure of the protein was determined and found to consist of 36% ahelical and 42% P-sheet structures. These results are in close agreement with the results of a previous CD study [Van Hoek, A. N., Wiener, M., Bicknese, S., Miercke, L., Biwersi, J. & Verkman, A. S. (1993) Biochemistry 32, 11 847-11 8561. However, the results differ from those given in an FTIR analysis by the same workers who recorded FTIR spectra of the CHIP28 protein in a dehydrated state. An unusually high extent of hydrogen-deuterium exchange of the peptide groups of this protein occurs. The magnitude of the spectral changes observed upon exposure of the protein to 'H,O is greater than has been observed with any other membrane protein previously studied. Thus, over 80% of the peptide groups exchange within 5 min and the amide I band maximum shifts to low frequency by approximately 20 cm-'. This high hydrogen-deuterium exchange observed with the CHIP28 protein is consistent with the presence of an aqueous pore within the protein structure.Keyvvords: membrane protein conformation ; 28-kDa channel-forming integral membrane protein (CHIP28) ; water channel : Fourier-transform infrared spectroscopy ; hydrogen-deuterium exchange.One of the main functions of the plasma membrane of living cells is the control of movement of water and solutes into and out of the cell. The availability, its simple structure (i.e. the lack of intracellular membranes), and the rather detailed knowledge of its biomembrane structure makes the red blood cell (RBC) ideally suited for studying plasma membrane permeability. A prominent feature of the RBC membrane is its high permeability to water [I]. Recognition of very high water transport across RBC plasma membranes, the low activation energy of this process [2, 31, and the inhibitory effects of mercurial SH blocking reagents [4-61 has led to the suggestion that water channels must be present within some of these membrane proteins 17-101.Consistent with this hypothesis, the first channel-forming integral membrane protein of molecular mass 28 kDa (called CHIP28 or aquaporin) from RBC was identified by Agre and coworkers [I 21. The CHIP-mediated water channel activity was demonstrated both with a Xenopus laevis oocyte expression system and also with CHIP28 reconstituted into liposomes 112-141. In both systems, CHIP28 markedly increases the water per- Abhreviutions. CHIP28, 28-kDa channel-forming integral membrane protein; FTIR, Fourier-transform infrared spectroscopy; RBC, red blood cell(s).Note. The permanent address of' G. Benga is: Department of Cell and Molecular Biology, 'Iulin Hatiagano' University of Medicine and Pharmacy, Cluj-Napoca, 6 Pasteur St., RO-3400 Cluj-Napoca, Romania. meabilit...

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Fourier transform infrared spectroscopy and differential scanning calorimetry of transferrins: human serum transferrin, rabbit serum transferrin and human lactoferrin

Cited by 53 publications

References 33 publications

In vivo diffusion of lactoferrin in brain extracellular space is regulated by interactions with heparan sulfate

In vivo diffusion of lactoferrin in brain extracellular space is regulated by interactions with heparan sulfate

Conformational Properties and Stability of Tyrosine Hydroxylase Studied by Infrared Spectroscopy

A Fourier‐Transform Infrared Spectroscopic Investigation of the Hydrogen‐Deuterium Exchange and Secondary Structure of the 28‐kDa Channel‐Forming Integral Membrane Protein (CHIP28)

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