Biomedical subjects

K C Ingham

Publications and source records attributed to K C Ingham.

At least 55 records · Page 3Linked to original sources

PubMed1990

Co-operative domains in fibronectin.

The melting of human plasma fibronectin and its proteolytic fragments has been studied by scanning microcalorimetry to reveal co-operative structural domains in the molecule. It has been established that each of the two similar polypeptide chains of fibronectin has at least 12 structural domains, which differ in stability, size and function. Many of the domains in the N-terminal half of the polypeptide chains appear to be composed of two homologous repeat modules that co-operate to form a single co-operative unit. In the intact fibronectin molecule, the C-terminal regions of both chains seem to interact forming a stable co-operative block.

Calorimetry, Differential Scanning↗

PubMed1990

Precipitation of proteins with polyethylene glycol.

Polyethylene glycol is a nondenaturing water-soluble polymer whose ability to precipitate protein from aqueous solution can be qualitatively understood in terms of an excluded volume mechanism. The increment in PEG concentration required to effect a given reduction in solubility is unique for a given protein-polymer pair, being insensitive to solution conditions and primarily dependent on the size of the protein and polymer. Selective manipulation of the solubility of specific proteins through control of their state of association or ligand environment can potentially remove some of the empiricism otherwise involved in fractional precipitation. Adequate methods for removing the polymer are available.

Chemical Precipitation↗

PubMed1989

Further localization of the gelatin-binding determinants within fibronectin. Active fragments devoid of type II homologous repeat modules.

Digestion of a 42-kDa gelatin-binding fragment (GBF) of fibronectin with pepsin followed by affinity chromatography on gelatin-Sepharose produces three fractions, a drop-through non-binding fraction, a retarded fraction that is dominated by a 13-kDa fragment whose NH2 terminus is identical to that of 42-kDa GBF, and a binding fraction that contains a homogeneous fragment of apparent mass 21 kDa with an NH2 terminus corresponding to Arg484. This 21-kDa GBF binds repeatedly to gelatin-Sepharose, eluting near 2.6 M in a urea gradient. It also binds in the fluid phase to a fluorescent-labeled collagen peptide with Kd = 10 microM and inhibits the binding of 42-kDa GBF to the same peptide with KI = 7.3 microM. Thus, major gelatin-binding determinants of fibronectin are located within a 21-kDa region that contains two type I homologous "finger" modules and is devoid of the type II "kringle-like" modules that were previously thought to be essential for this activity.

Amino Acid Sequence↗

PubMed1989

Calorimetric investigation of the domain structure of human complement Cl-s: reversible unfolding of the short consensus repeat units.

Cl-s is a multidomain serine protease that participates in Ca2+-dependent protein-protein interactions with other subcomponents of Cl, the first component of human complement. Proteolytically derived fragments that retain some of the functional properties of the parent protein have been isolated, and their thermal stability has been investigated by differential scanning calorimetry. Three endothermic transitions are observed in whole Cl-s near 37, 49, and 60 degrees C in 0.05 M Tris-HCl, pH 7.2, containing 0.22 M NaCl and 0.1 mM EDTA. The first (37 degrees C) and third (60 degrees C) transitions are also seen in Cl-s-A, a derivative comprised mainly of the intact nonenzymatic A chain. The second (49 degrees C) and third transitions are seen in Cl-s-gamma B, a fragment comprised of the intact B chain, disulfide linked to the C-terminal gamma region of the A chain. Thus, the first transition, which is alone stabilized by Ca2+, corresponds to the melting of the N-terminal alpha beta region of the A chain, the second to the melting of the catalytic B chain domain, and the third to the gamma region. The gamma region is comprised of two homologous short consensus repeat (SCR) motifs that are also found in several other complement and coagulation proteins. A new 24-kDa fragment, Cl-s-gamma, which contains these two SCRs, was isolated from plasmic and chymotryptic digests of Cl-s-A. Cl-s-gamma exhibits a reversible transition near 60 degrees C corresponding to the highest temperature peak in whole Cl-s and Cl-s-A.(ABSTRACT TRUNCATED AT 250 WORDS)

Calorimetry, Differential Scanning↗

PubMed1989

Reversible unfolding of the gelatin-binding domain of fibronectin: structural stability in relation to function.

Fibronectin, a large multidomain glycoprotein, binds denatured collagen (gelatin) and mediates cell attachment and spreading on collagen-coated surfaces. Despite the high affinity, binding to gelatin is disrupted by relatively mild conditions. We have examined the effects of denaturants on the structure and function of a 42-kDa gelatin-binding fragment (GBF) isolated from chymotryptic and thermolytic digests of the parent protein. Application of linear gradients to GBF-loaded gelatin-agarose columns resulted in peak elution of the fragment at pH 5.2 or 10.2, at 0.4 M dimethylformamide, 0.9 M GdmCl, or 2.0 M urea, conditions far short of those required to induce structural changes detectable by fluorescence or circular dichroism. Solvent perturbation, fluorescence quenching, and chemical modification experiments indicate that about half of the 8 tryptophans, one-third of the 21 tyrosines, and all of the 9 lysine residues are solvent-exposed in the native protein and that 1 or more of the latter are directly involved in binding to gelatin, most likely through a hydrogen-bonding mechanism. Titration with GdmCl produced a single unfolding transition centered near 2.5 M GdmCl as monitored by changes in fluorescence and circular dichroism. This transition was fully reversible with complete recovery of structural parameters and gelatin binding. Treatment with disulfide reducing agents caused rapid irreversible changes in structure similar to those produced by GdmCl with concomitant loss of gelatin binding. Thus, tertiary and secondary structures are important for binding, but binding can be disrupted without perturbing those structures.

Animals↗

PubMed1988

Domain structure, stability, and interactions of human complement C1s-: characterization of a derivative lacking most of the B chain.

A better understanding of the structure and function of C1 requires knowledge of the regions (domains) of the subcomponents that are responsible for Ca2+-dependent assembly. Toward this end, C1-s was digested with trypsin in the presence of Ca2+, a treatment that rapidly degraded the B chain, leaving a 56-kDa fragment comprised of a complete A chain disulfide linked to a small (less than 4-kDa) residual piece of the B chain. The purified fragment, referred to as C1-s-A, was shown by fast exclusion chromatography to be similar to C1-s in its ability to (1) reversibly dimerize in the presence of Ca2+, (2) substitute for C1-s in the formation of C1-r2-s2 tetramers, and (3) associate with C1-r and C1q to form macromolecular C1. Although C1-s-A was itself catalytically and hemolytically inactive, it competitively inhibited the expression of the hemolytic activity of C1-s in a reconstitution assay. When heated in the absence of Ca2+, C1-s exhibited a low-temperature transition (LTT) near 31 degrees C and a high-temperature transition (HTT) near 51 degrees C, similar to those previously observed in the homologous protein C1-r [Busby, T. F., & Ingham, K. C. (1987) Biochemistry 26, 5564-5571]. The midpoint of the LTT was shifted to 58 degrees C in 5 mM Ca2+ whereas the HTT was unaffected by Ca2+. C1-s-A exhibited only a LTT whose midpoint and Ca2+ dependence were similar to those of the LTT in C1-s. The HTT, which was accompanied by a loss of esterolytic activity, was reproduced in a plasmin-derived fragment representing the catalytic domain. These results provide strong support for the structural and functional independence of the catalytic and interaction domains of C1-s and strengthen current models regarding the role of these domains in various interactions. They also provide direct proof for the occurrence of Ca2+ binding sites on the A chain and demonstrate that all or most of the sites on C1-s that are responsible for its interaction with C1-r and C1q are located on the A chain.

Amino Acid Sequence↗

PubMed1988

Interaction of fibronectin and its gelatin-binding domains with fluorescent-labeled chains of type I collagen.

Fluorescent probes have been used to obtain dissociation constants for the fluid-phase interaction of human plasma fibronectin and several of its gelatin-binding fragments with purified alpha chains of type I rat tail collagen, as well as with a cyanogen bromide fragment (CB7) of the alpha 1 chain in 0.02 M Tris buffer containing 0.15 M NaCl at pH 7.4. Addition of fibronectin to fluorescein-labeled collagen chains caused a dose-dependent increase in the fluorescence anisotropy which continued over several logs of titrant concentration. Scatchard-type plots of the anisotropy response were biphasic indicating the presence of one or more weak sites (Kd greater than microM) along the collagen chain in addition to a strong site characterized by Kd = 1.3 X 10(-8) M at 25 degrees C. Gelatin-binding fragments with Mr = 42,000, 60,000, and 72,000 also produced a biphasic response with Kd values for the high affinity site being 10- to 20-fold greater than for intact fibronectin. Binding of fibronectin and its fragments to fluorescent-labeled CB7 was essentially the same as to the whole alpha 1 chain. In all cases, the anisotropy response could be reversed or prevented by addition of excess unlabeled gelatin or CB7, but not by synthetic peptides spanning the collagenase cleavage site of alpha 1 (I). Studies of the temperature dependence of Kd for binding of fibronectin to the high affinity site on alpha 1 produced a value of +16 kcal/mol for the enthalpy of dissociation below 30 degrees C. Above this temperature, fibronectin appeared to undergo a subtle conformational transition characterization by a reduced affinity for collagen. This transition occurred in whole fibronectin but not in the gelatin-binding fragments and may involve disruption of intramolecular interactions between different domains.

Binding Sites↗

PubMed1987

Calcium-sensitive thermal transitions and domain structure of human complement subcomponent C1r.

Fluorescent probes and other methods have been used to investigate the thermal stability of activated C1r and functionally intact fragments isolated from tryptic digests of the protein. This enzyme exhibits two irreversible transitions that differ with respect to their sensitivity to metal ions. The high-temperature transition occurs with a midpoint near 53 degrees C in 0.02 M tris(hydroxymethyl)aminomethane buffer and 0.15 M NaCl, pH 7.4. It is relatively insensitive to Ca2+ and ionic strength and is accompanied by a loss of catalytic activity. The low-temperature transition is most easily observed in the presence of ethylenediaminetetraacetic acid and is completely abolished by 100 microM Ca2+. Its midpoint varies between 26 degrees C at low ionic strength and 40 degrees C in the presence of 0.5 M NaCl. The low-temperature transition results in extensive polymerization of the protein without loss of the esterolytic activity or the ability to react with C1 inhibitor; however, the ability to reconstitute hemolytically active C1 or even bind to C1s in the presence of Ca2+ is destroyed. A highly purified N-terminal fragment generated by tryptic digestion of C1r in the presence of Ca2+ retained its ability to interact with C1s, disrupting the formation of C1s dimers in the presence of Ca2+. In the absence of Ca2+, this fragment displays only a low-temperature transition that is very similar to the one observed with the whole protein and that destroys its ability to bind to C1s. Addition of Ca2+ stabilizes this fragment, shifting the midpoint of its melting transition upward by more than 20 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Anilino Naphthalenesulfonates↗

PubMed1986

Kinetics of interaction of C1 inhibitor with complement C1s.

The kinetics of inhibition of the complement serine protease, C1s, by its only known inhibitor, C1 inhibitor, have been measured by a variety of methods. One method continuously monitors the loss of esterolytic activity with a synthetic substrate coupled to a chromogen while another monitors the formation of a stable (covalent) complex by high-pressure size-exclusion chromatography under dissociating conditions. Additional methods employ fluorescence probes to follow the formation of bimolecular complexes but are not expected to distinguish between covalent product and noncovalent (reversible) intermediates. There was good agreement between rate constants obtained by the various methods over a broad range of inhibitor concentrations, suggesting that noncovalent intermediates do not accumulate to a significant extent. The reaction appears to be pure second order with a bimolecular rate constant of 6.0 X 10(4) M-1 s-1 at 30 degrees C, independent of Ca2+, and an activation energy of 11.0 kcal/mol. The rate increases up to 35-fold in the presence of heparin which was shown to bind to all three components (enzyme, inhibitor, and complex) with similar affinity (Kd = 2.0-3.3 microM). The fluorescent probe 1,1'-bis(anilino)-4-,4'-bi(naphthalene)-8,8'-disulfonate [bis(ANS)] bound to the complex with Kd = 0.26 microM under conditions where the individual components had little affinity for the dye, consistent with the generation of one or more hydrophobic binding sites on the protein surface during complex formation.

Complement Activating Enzymes↗

PubMed1986

Thermal stability and ligand-binding properties of human plasma alpha 1-acid glycoprotein (orosomucoid) as determined with fluorescent probes.

The fluorescence of 1,8-anilinonaphthalene sulfonate is enhanced and blue-shifted upon binding to alpha 1-acid glycoprotein, a human plasma protein of uncertain function. Fluorescence titrations of delipidated protein indicate at least two classes of binding sites having dissociation constants of 0.33 microM and 12 microM at 25 degrees C in 0.02 M potassium phosphate/0.15 M NaCl, pH 7.4. Exclusion chromatography measurements indicate only 1 binding site per mol protein, suggesting that the heterogeneity is due to differences between protein molecules, the origin of which remains unclear. The fluorescence of a mixture of dye and protein is progressively diminished upon addition of ethanol and other organic solvents whose presence could be detected at concentrations as low as 100 mM. Addition of the adrenergic drug propranolol to a mixture of alpha 1-acid glycoprotein (2.5 microM) and 1,8-anilinonaphthalene sulfonate (4 microM) caused a hyperbolic decrease in dye fluorescence to 30% of the initial value, with half-maximal response near 1 microM propranolol. When the protein-dye mixture was heated, the fluorescence of the dye exhibited a reversible downward transition with midpoint near 65 degrees C, compared to a midpoint of 58.5 degrees C obtained by intrinsic fluorescence in the absence of dye. This stabilization was confirmed with fluorescein-labeled protein, whose fluorescence polarization revealed a melting transition at 58.8 degrees C in the absence of ligands which increased by 5-6 Cdeg in the presence of 1,8-anilinonaphthalene sulfonate or propranolol. The sensitivity of 1,8-anilinonaphthalene sulfonate fluorescence to changes in the conformation and ligand environment of alpha 1-acid glycoprotein should facilitate efforts to understand the structure and function of this acute-phase reactant.

Anilino Naphthalenesulfonates↗

PubMed1985

Interaction of gelatin with a fluorescein-labeled 42-kDa chymotryptic fragment of fibronectin.

A 42-kDa gelatin binding fragment of human plasma fibronectin was labeled with fluorescein and its fluid-phase interaction with gelatin was investigated. At 25 degrees C in 0.1 M Tris, 0.15 M NaCl, pH 7.3, a dissociation constant, Kd = 0.6 microM, was obtained from the dependence of fluorescence polarization on gelatin concentration. An identical value was obtained for the unlabeled fragment by competition. Binding was unaffected by higher concentrations of NaCl up to 1.0 M, but increased as much as 20-fold at low ionic strength. The dependence of Kd on temperature revealed that dissociation of the complex is accompanied by an increase in entropy. Thus, the interaction is not dominated by either hydrophobic or electrostatic forces; an important role for hydrogen binding is proposed.

Chymotrypsin↗

PubMed1985

Changes in protein conformation and stability accompany complex formation between human C1 inhibitor and C1-s.

The fluorescence spectrum of C1 inhibitor (C1-Inh) in aqueous buffer has a maximum at 324 nm which shifts to 358 nm in 6.0 M guanidinium chloride (GdmC1), indicating that fluorescent tryptophans are buried in the native protein. When titrated with GdmC1, the fluorescence intensity, polarization, and emission maximum of C1-Inh and C1-s exhibited clear transitions which were more prominent than those of the enzyme-inhibitor complex. Two of the variables (intensity and emission maximum) suggest biphasic unfolding of C1-Inh. Differential absorption measurements and sodium iodide quenching of intrinsic fluorescence were consistent with a net increase in the exposure of tryptophans and tyrosines upon complex formation. This reaction, i.e., complex formation, was also accompanied by an increase in the ability to enhance the fluorescence of the hydrophobic probe 8-anilino-1-naphthalenesulfonate. Fluorescence assays of heat denaturation showed transitions at 40 and 52 degrees C for C1-s and at 60 degrees C for C1-Inh whereas there was no detectable melting transition for the complex. Similarly, differential scanning calorimetric measurements revealed transitions at 42, 52, and 62 degrees C for C1-s and one transition at 60 degrees C for C1-Inh, with no major transitions detectable for the complex. The ratio of the calorimetric enthalpy to the apparent van't Hoff enthalpy for thermal unfolding of C1-Inh was 1.6. Taken together, these results suggest that C1-Inh and C1-s are each composed of at least two independently unfolding domains and that complex formation, which involves conformational change, yields a protein substantially more stable than either component alone.

Calorimetry↗

PubMed1985

Interaction of fibronectin with C1q and collagen. Effects of ionic strength and denaturation of the collagenous component.

By attaching native collagen and C1q to Sepharose, it was possible to test the binding of fibronectin (Fn) to the native and heat-denatured forms of these proteins without complications due to aggregation, precipitation, or fibril formation. Binding to the native proteins occurred only at low (sub-physiological) ionic strength whereas binding to the denatured proteins occurred even in 1 M NaCl. Thus both of these proteins possess one or more strong sites which are masked in the native state and become exposed during thermal denaturation. Fn did not bind to albumin-Sepharose or IgG-Sepharose either before or after heat-denaturation. C1q bound readily to native IgG-Sepharose but did not mediate the binding of Fn. Nor did Fn inhibit the reconstitution of C1 on antibody-coated erythrocytes. The fluorescence polarization of fluorescein-labeled collagen in 1 M NaCl displayed a downward transition at 38-40 degrees C consistent with unfolding of the triple helix. In the presence of Fn, the same material displayed an upward transition at slightly lower temperature suggesting that gross unfolding is not required to expose the strong binding site(s).

Affinity Labels↗

PubMed1985

Biological and physical properties of fibronectin pasteurized in the presence of stabilizers.

Interest in human plasma fibronectin (Fn) as a potential clinical product for replacement therapy in septic patients has prompted the search for stabilizers to protect the protein from heat denaturation during pasteurization designed to inactivate hepatitis viruses. Fn was pasteurized (60 degrees C, 10 h) in the presence of either citrate, tricarballylate, sucrose or four mixtures of lysine, glucarate, gluconate or citrate which had been found to increase the denaturation temperature of Fn by greater than or equal to 19 degrees C. All but a citrate/gluconate mixture were effective in preventing aggregation as measured by dye fluorescence, light scattering, gel filtration and electrophoresis. Binding to gelatin was retained and immunological activity was only slightly diminished compared to a sample heated without stabilizers. Opsonic activity was measured as ability to mediate the uptake of 125I-gelatin-coated polystyrene beads by attached human monocytes. Fn heated without stabilizers underwent a transient increase in activity which was traced to formation of aggregates having elevated specific activities. Pasteurized samples had slightly elevated opsonic activities with no detectable aggregates present, while the unstabilized control was inactive. These results indicate that the physical properties of Fn as well as the functional activities of the gelatin- and cell-binding domains can be protected against thermal denaturation by various compounds.

Blood Transfusion↗

PubMed1984

Thermal stability of human plasma fibronectin and its constituent domains.

Human plasma fibronectin undergoes thermal denaturation with a midpoint between 62 and 64 degrees C. The irreversible transition is characterized by an increase in the intensity and wavelength of intrinsic tryptophan fluorescence, by an increase in the ability to enhance the fluorescence of 1,8-anilinonaphthalene sulfonate, and by an increase in the fluorescence polarization of covalently attached fluorescein. Addition of molecules which bind to fibronectin with high affinity, e.g. gelatin or heparin, had no stabilizing effect. This was attributed to the presence of multiple domains, all of which must be stabilized to prevent denaturation and aggregation. Further support for this interpretation came from studies of six different proteolytic fragments of fibronectin which collectively span almost the entire molecule. Cell-binding fragments derived from the central regions of the chain were least stable, exhibiting behavior similar to that of the whole protein. Fragments derived from the C-terminal regions were more stable by 7-8 degrees C, and those derived from the N-terminal region showed no thermal transition by any of the fluorescence parameters up to 85 degrees C in some experiments. A fluorescein-labeled 60-kilodalton gelatin-binding fragment which had been heated to 70 degrees C produced an increase in polarization upon addition of gelatin with Kd = 1.3 X 10(-7) M, similar to that of an unheated control. The intrinsic fluorescence spectra of the fragments had maxima which decreased progressively from 335 nm at the N terminus to 313 nm at the C terminus. These observations further elaborate the multidomain structure of human plasma fibronectin and reveal significant differences between the tertiary structure and stabilities of the various domains.

Chymotrypsin↗

PubMed1984

Thermal stabilization of antithrombin III by sugars and sugar derivatives and the effects of nonenzymatic glycosylation.

A variety of neutral and acidic sugars and related compounds were evaluated in terms of their effect on the midpoint, Td, of the thermal denaturation curve of antithrombin III. The objectives were to determine which structural features of these molecules are responsible for their stabilizing properties and to identify more efficient stabilizers which combine the effects of lyotropic anions such as citrate with those of the polyols in a single molecule. The presence of one or more carboxylate groups in a sugar molecule invariably increased its stabilizing potency, whereas the number and position of hydroxyl groups appeared to have no influence on the molecules' stabilizing ability. Several compounds were shown to be effective in preserving antithrombin III activity during pasteurization for 10 h at 60 degrees C. However, the presence of reducing sugars invariably resulted in a decrease in activity following pasteurization, in spite of their ability to increase Td. In fact, when antithrombin III was pasteurized in the presence of 2 M glucose and 0.5 M citrate, it steadily lost its ability to inhibit thrombin even though Td under these conditions was 10 degrees C higher than in citrate alone where activity was preserved. This effect was shown to be coincident with the covalent incorporation of glucose into the protein molecule.

Anilino Naphthalenesulfonates↗

PubMed1984

Interaction and thermal stability of fluorescent labeled derivatives of thrombin and antithrombin III.

Derivatives of human thrombin and antithrombin III with fluorescent labels covalently attached to their carbohydrate moieties were prepared by reaction of periodate-oxidized proteins with amino derivatives of dansyl, fluorescein and pyrene. The labeled derivatives retained full biological activity, including their ability to form stable enzyme-inhibitor complexes, a reaction whose rate could be monitored by the increase in fluorescence polarization. When the dansyl-labeled derivatives were heated, they exhibited sigmoidal increases in polarization with midpoints near 50 degrees C for thrombin and 60 degrees C for antithrombin III. By contrast, a complex between antithrombin III and dansyl-thrombin showed no change in polarization up to 70 degrees C, suggesting that the individual components are more stable in the complex. These studies show that fluorescent labels attached to carbohydrate moieties of glycoproteins provide convenient probes for monitoring conformational changes and protein-protein interactions with minimum interference by the probe.

Antithrombin III↗

PubMed1984

Protein precipitation with polyethylene glycol.

Chemical Precipitation↗