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Biomedical subjects

C R Middaugh

Publications and source records attributed to C R Middaugh.

107 records · Page 6Linked to original sources
PubMed1980

Presence of spectrin tetramer on the erythrocyte membrane.

In order to examine the subunit structure of membrane-associated spectrin, human erythrocyte membranes were cross-linked with a variety of photosensitive heterobifunctional reagents by flash photolysis millisecond cross-linking (Kiehm, D.J., and Ji, T.H. (1977) J. Biol. Chem. 252, 8524-8531). Cross-linking of ghosts produced a series of new bands with apparent molecular weights of approximately 420,000, 710,000, and 910,000, as well as a band at the top of the gels. Evidence is presented that these three bands represent spectrin dimer, trimer, and tetramer, respectively. We propose that spectrin tetramer exists on the membrane as the predominant structural unit and that dimer is not a repeating structural unit.

Cross-Linking Reagents↗
PubMed1980

A photochemical crosslinking study of the subunit structure of membrane-associated spectrin.

The subunit structure of membrane-associated spectrin was investigated by crosslinking human erythrocyte membranes with a variety of cleavable photosensitive heterobifunctional reagents by flash photolysis millisecond crosslinking. Crosslinked complexes were analyzed on agarose-polyacrylamide gels whose high exclusion limit the resolution of molecular with Mr larger than 10(6). Crosslinking of membrane ghosts produced a series of new bands with apparent molecular weights of approximately 420 000, 170 000 and 910 000, as well as a band at the top of the gels. No significant bands were detectable between the 910 000-Mr band and the material at the gel top. The molecular weights of the three bands correspond to spectrin dimer, trimer, and tatramer, suggesting a limiting tetrameric stoichiometry for membrane-associated spectrin. The rate of formation of spectrin oligomers was examined by increasing crosslinking time. Dimers accumulate before trimer and tetramer, and as tetramer increased, dimer diminished. A simple kinetic model is employed which is also consistent with spectrin tetramer existing on the cytoplasmic surface of the erythrocyte membrane as the basic structural unit.

Azides↗
PubMed1979

Raman spectra and conformational structures of Fab mu and (Fc)5 mu fragments of cryoglobulin IgM-kappa McE.

Raman spectra have been obtained on aqueous solutions of the following human immunoglobulins: IgM-kappa McE, IgG-kappa Ger, IgM-kappa WSm and IgG-lambda Gui. The former two species exhibit the property of cryoprecipitation. Comparison of the spectra shows that all immunoglobulins have similar secondary structures, predominantly of the beta-sheet type. Fab mu and (Fc)5 mu fragments of IgM-kappa McE also yield Raman spectra which indicate closely similar secondary structures. Minor differences among the spectra can be explained by differences in amino acid compositions of the respective proteins.

Cryoglobulins↗
PubMed1979

Determination of the apparent thermodynamic activities of saturated protein solutions.

Although the solubility of a protein is a particularly informative solution parameter, little is known about the thermodynamics of protein solubilization. In these experiments, polyethylene glycol (PEG) is used to decrease the solubility of a number of proteins in a quantifiable manner. Simple thermodynamic considerations show that if the chemical potential of the PEG-induced solid phase is constant and plots of log protein solubility versus PEG concentration are linear, a valid extrapolation of the apparent solubility to zero PEG content can be made. Given the validity of these assumptions, extrapolated values should represent the activity of the protein in saturated solution. Evidence for the validity of this extrapolation includes (a) the experimentally observed linearity of log solubility versus PEG concentration plots, (b) the extrapolation of such plots to correct activities in the situation where protein activities can be experimentally determined, and (c) the independence of the extrapolated activities on protein concentration over a wide range. The utility of the PEG-determined activities, when applied in a comparative manner, is illustrated by application to various hemoglobin solutions. It is found that saturated solutions of the various hemoglobin forms, with the exception of deoxyhemoglobin S, manifest similar activities. In addition, all of the solutions demonstrate an apparent, surprising thermodynamic ideality.

Animals↗
PubMed1979

Role of bound water in biological membrane structure: fluorescence and infrared studies.

Bound water is a major component of biological membranes and is required for the structural stability of the lipid bilayer. It has also been postulated that it is involved in water transport, membrane fusion, and mobility of membrane proteins and lipids. We have measured the fluorescence emission of membrane-bound 1-anilino-8-naphthalenesulfonate (ANS) and the infrared spectra of membranes, both as a function of hydration. ANS fluorescence is sensitive to polarity and fluidity of the membrane-aqueous interface, while infrared absorption is sensitive to the hydrogen bonding and vibrational motion of water and membrane proteins and lipids. The fluorescence results provide evidence of increasing rigidity and/or decreasing polarity of the membrane-aqueous interface with removal of water. The membrane infrared spectra show prominent hydration-dependent changes in a number of bands with possible assignments to cholesterol (vinyl CH bend, OH stretch), protein (amide A, II, V), and bound water (OH stretch). Further characterization of the bound water should allow its incorporation into current models of membrane structure and give insight into the role of membrane hydration in cell surface function.

Anilino Naphthalenesulfonates↗
PubMed1978

Investigations of the molecular basis for the temperature-dependent insolubility of cryoglobulins. VI. Quenching by acrylamide of the intrinsic tryptophan fluorescence of cryoglobulin and non-cryoglobulin IgM proteins.

The acrylamide-quenching patterns of the intrinsic tryptophan fluorescence of six cold-soluble monoclonal immunoglobulin M (IgM) and two monoclonal IgM proteins possessing cryoglobulin properties (abnormal cold insolubility) have been compared. Static and dynamic components of quenching have been resolved by a modified form of the Stern-Volmer relationship. The unusual observation of static quenching seen with the multitryptophan containing IgM is determined to be a consequence of essentially homogeneous indole fluorescence arising from conserved tryptophan residues within each homologous immunoglobulin domain. Although the static component of the quenching of the two IgM cryoimmunoglobulins examined is similar to that of the non-cryoimmunoglobulin, IgM, some of the cryoglobulin's tryptophan residues appear to be more kinetically exposed to acrylamide than the tryptophans in the non-cryoglobulin IgM. An unusually large negative entropy of activation observed for the quenching process of both cryoimmunoglobulins suggests some abnormality in the dynamic (flexibility) properties of these proteins.

Acrylamides↗
PubMed1978

Hv(1), a variable-region genetic marker of human immunoglobulin heavy chains.

A new antigenic determinant was discovered with a hemagglutination-inhibition assay system. Designated Hv(1), it is located in the variable region of human immunoglobulin heavy chains of the G, M, and A classes. Pedigree and population analyses suggest that it has an autosomal dominant mode of inheritance. This represents the first description of an allotypic determinant in the variable region of human immunoglobulins.

Binding Sites, Antibody↗
PubMed1978

Physicochemical characterization of six monoclonal cryoimmunoglobulins: possible basis for cold-dependent insolubility.

The physical and chemical properties of five human and one canine monoclonal cryoimmunoglobulin have been compared. By many criteria, the proteins cannot be distinguished from the noncryoglobulin reference proteins analyzed in parallel; however, certain hydrodynamic and spectroscopic properties of the proteins indicate that cryoimmunoglobulins differ in tertiary structure relative to their cold-soluble counterparts. These differences seem to favor low-temperature-induced association between cryoglobulin molecules as an immediate consequence of increased intermolecular ionic or van der Waals forces. No evidence was found for the formation of cold-dependent antigen-antibody complexes or the ubiquitous presence of low-temperature-dependent conformation changes as a component of cryoprecipitation. Rather, the anomalous solution behavior of monoclonal cryoimmunoglobulins can be considered a direct result of the individual solubility properties of these proteins.

Amino Acids↗
PubMed1978

Localization of a conformational anomaly to the Fabmu region of a monoclonal IgM cryoglobulin.

The hydrodynamic (gel filtration and sedimentation) properties of an isolated monoclonal IgM-K cryoglobulin (McE.) and five non-cryoglobulin cold-soluble proteins, as well as their constituent monomeric subunits and (Fc)5mu and Fabmu fragments, are compared under both native and partially denaturing conditions. It is concluded that the cryoimmunoglobulin exhibits a significantly greater Stokes radius than the non-cryoglobulin reference proteins, and that this difference arises in the Fabmu region of the McE. molecule. When the proteins and their fragments are analysed by circular dichroism in the far u.v. region, an atypical conformation is again detected in the Fabmu region of the cryoglobulin. These findings are the first demonstration and partial structural localization of a conformation anomaly in a monoclonal cryoimmunoglobulin.

Circular Dichroism↗
PubMed1977

Effect of solutes on the cold-induced insolubility of monoclonal cryoimmunoglobulins.

The effects of a variety of compounds upon the cold-induced insolubility of the IgM-K cryoglobulin McE have been examined. Cryoprecipitation was found to be inhibited by certain neutral salts, ureas, amides, tetraalkylammonium salts, long chain sodium alkyl sulfates, sugars, and a number of other agents. Cryoprecipitation was enhanced by increasing the hydrophobicity of alcohols, ureas, amides, and tetraalkylammonium salts, as well as by low concentrations of many solutes. With the exception of the alkyl sulfates, inhibition was not accompanied by detectable changes in conformation of the cryoimmunoglobulin. These inhibitions and enhancements were also associated with changes in the temperature at which cryoprecipitation was initiated as well as the temperature at which a low temperature-induced conformation change occurs in the McE protein. The effects of solutes on McE are compared to results obtained with five other (two IgG, three IgM) cryoimmunoglobulins, and it is hypothesized that electrostatic and dispersion forces are primarily responsible for the cold insolubility of monoclonal cryoimmunoglobulins.

Alcohols↗
PubMed1977

Monoclonal cryoglobulinemia with macroglobulinemia in a dog.

A 9-year old female Doberman Pinscher had a 5-month history of epistaxis. On routine testing of the dog's serum, blockage of the intake tubes of the equipment resulted. At this point, cryoglobulinemia was suspected and was later confirmed to be due to a monoclonal cryoglobulin (IgM), by means of immunochemical studies. Clinical recovery was achieved by use of cytotoxic drugs.

Animals↗
PubMed1977

Comparison of proteins from thermophilic and nonthermophilic sources in terms of structural parameters inferred from amino acid composition.

The amino acid composition of 14 different proteins from thermophilic bacteria were compiled along with the amino acid compositions of 56 corresponding proteins from nonthermophilic sources. A comparison was made between proteins serving the same catalytic function, and significant differences in composition were noted for those proteins from thermophilic bacteria. However, no consistent pattern was evident and the differences were often small. The two data pools were treated as two distinct classes and a thermophilic versus non-thermophilic comparison of amino acid composition was made using the Student's t-test. Significant differences in composition were found for Asx (sum of Asp and Asn, if known), Ser, and Arg. Both classes of data have similar standard deviations for the mean of any single amino acid, suggesting a similar tolerance of variation in the two classes of proteins. This would argue against the hypothesis that thermophiles exhibit a greater frequency of errors in protein synthesis. The amino acid compositions were used to calculate structural parameters (% helix, % beta, % turn, hydrophobicity, and melting temperatures) for the two classes of proteins. Of these, only the predicted % beta content was significantly lower for proteins of thermophilic origin. No differences in hydrophobicity or predicted melting temperature were observed for the two classes of proteins. This study supports the hypothesis that while small differences may occur in the amino acid composition of thermophilic proteins, they are quite varied and often are very subtle.

Amino Acids↗
PubMed1976

The effect of temperature on ribose-5-phosphate isomerase from a mesophile, Thiobacillus thioparus, and a thermophile, Bacillus caldolyticus.

The enzyme ribose-5-phosphate isomerase [EC 5.3.1.6] was partially purified from a mesophilic organism, Thiobacillus thioparus, and from an extreme thermophile, Bacillus caldolyticus. The stability and kinetics of the two enzymes were compared with regard to temperature in the presence of a series of neutral salts and alcohols. The thermal stability of both enzymes was altered such that the salts (NH4)2SO4, NaCl, KCl, and LiCl increased stability, while LiBr, CaCl2, methanol, ethanol, and 1-propanol decreased stability. Ethylene glycol had little effect on the mesophilic enzyme, but increased the stability of the thermophilic protein. The kinetics of both enzymes were also affected by the salts and alcohols, and Arrhenius plots of two kinetic parameters, Km and Vmax, displayed discontinuities, or sharp changes in slope, at characteristic temperatures, TD. Neutral salts and alcohols altered the temperature of discontinuity in a sequence similar to that observed in studies of thermal stability. It is suggested that the slope change is due to temperature-dependent alterations in the enzymes at specific, but undefined, loci at the active site, although no evidence is offered for the absence of a larger conformation change in the entire enzyme.

Bacillus↗