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R L Coffey

Publications and source records attributed to R L Coffey.

9 recordsLinked to original sources
PubMed1995

Non-cooperative binding of the MAP-2 microtubule-binding region to microtubules.

Microtubule-associated protein (MAP)-2 is a multi-domain cytoskeletal protein that copurifies with brain microtubules (MTs) through repeated cycles of warm polymerization and cold disassembly. Recent equilibrium binding studies of high molecular weight MAP-2ab to taxol-stabilized MTs suggest that the interactions are highly cooperative, as indicated by sigmoidal binding curves, non-linear Scatchard plots, and an apparent all-or-none response in MAP binding in titration experiments (Wallis, K. T., Azhar, S., Rho, M. B., Lewis, S. A., Cowan, N. J., and Murphy, D. B. (1993) J. Biol. Chem. 268, 15158-15167). To learn more about the mechanism of MAP-2 binding to MTs, we investigated the binding properties of bacterially expressed MT-binding region (MTBR) of bovine brain MAP-2. Scatchard plots of the binding data showed no evidence of cooperativity, as reflected by the linear plots of v/[MTBR]free versus v. The stoichiometry was 1-1.1 mol of MTBR/mol of tubulin dimer, and the dissociation constant for the MTBR was 1.1 microM. Bovine brain tau protein competitively inhibited MAP-2 binding, as evidenced by an increased Kd value for MTBR binding to MTs. Although the second repeat peptide m2 (VTSK-CGSLKNIRHRPGGG) is thought to play a dominant role in MAP-2 binding to MTs, a MTBR mutant (with m2 replaced by the third octadecapeptide repeat m3) displays an Kd of 2.8 +/- 0.1 microM and stoichiometry of 0.9 +/- 0.05 mol of MTBR/mol of tubulin dimer. Another mutant with additional copies of the second repeat, designated by us as MTBR[m12m2m32], displayed noncooperative binding with a Kd of 0.53 +/- 0.05 microM and a stoichiometry of 2.2 +/- 0.2 mol of mutant MTBR/tubulin dimer. Equilibrium sedimentation experiments demonstrated that the wild-type MTBR is monomeric, whereas MTBR[m12m2m32] self-associates to a stable dimer over the concentration range used in our MT binding studies. This finding indicates that only one of the two MT-binding sites on the dimer is probably linked to a microtubule at any given time.

Amino Acid Sequence↗
PubMed1994

Exploring the microtubule-binding region of bovine microtubule-associated protein-2 (MAP-2): cDNA sequencing, bacterial expression, and site-directed mutagenesis.

A 1.1 kilobase fragment of bovine microtubule-associated protein-2 (MAP-2) cDNA coding for bovine MAP-2 microtubule-binding region (MTBR) was sequenced. Relative to mouse, rat, and human MAP-2, we observed striking preservation of primary structure, even beyond the sequence and spacing of the three nonidentical peptide repeats responsible for microtubule-binding interactions. For further analysis of microtubule-MAP interactions using site-directed mutagenesis, we developed a bacterial expression system coding for the MT-binding fragment of MAP-2 starting at the thrombin cleavage site (position 1629) and continuing to the C-terminus. This MT-binding fragment was purified to homogeneity by taking advantage of the unusual heat-stability and isoelectric properties of this cytomatrix component. We found that the MT-binding domain readily promoted tubulin polymerization, and the critical tubulin concentration was reduced in the presence of this recombinant protein. Because a second repeated sequence analogue can promote tubulin polymerization as well as displace the MT-binding region of MAP-2, this study was designed to learn more about the importance of each repeated sequence in MT binding. Accordingly, we mutated the first and third sequences to resemble the second repeated sequence, thereby generating the mutants designed m12-m2-m3, m1-m32, and m12-m2-m32. These recombinant proteins bound with an affinity comparable to or slightly better than equal concentrations of wild-type MT-binding fragment. Likewise, when the first or third sequence was replaced by an exact copy of the second octadecapeptide repeat, there was little, if any, increase in binding affinity, as reflected in the ability of mutant MT-binding fragments to promote tubulin polymerization.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1990

Cooperative interactions in the binding of ethidium ion to the self-complementary ribodinucleoside monophosphates CpG and GpC.

Complexes exhibiting the characteristics of cooperative interactions are formed by ethidium ion and the self-complementary dinucleoside monophosphates CpG and GpC. Complex formation, observed with an ethidium ion selective electrode, can be described by an equilibrium binding model in which complexes are formed with dinucleoside:ethidium combining ratios of 2:1, 2:2, and 2:3. The total amount of ethidium bound in 2:2 and 2:3 complexes, as calculated from the model, is proportional to a circular dichroism band in CpG-ethidium spectra near 305 nm. Van't Hoff analysis of the model equilibrium constants reveals that the addition of ethidium ion to the 2:1 and 2:2 species is exothermic and that the corresponding entropy changes are large and negative. Cooperative interactions in the binding of ethidium ion and of other ligands to some natural and synthetic polymeric nucleic acids have now been observed in several laboratories, but the present work shows that the effect can arise even with nucleic acid fragments as small as dinucleosides. Apparently, a macromolecular nucleic acid is not essential for cooperative interactions.

Animals↗
PubMed1983

Mechanisms of physiological dead space response to PEEP after acute oleic acid lung injury.

In acute increased-permeability edema, the Bohr physiological dead space (VD/VTCO2) can be influenced by changes in anatomic dead space, ventilation-perfusion (VA/Q) heterogeneity, shunt, and the Haldane effect. We used the multiple inert gas elimination technique to assess the effect of positive end-expiratory pressure (PEEP) on each of these components of VD/VTCO2 in 14 pentobarbital-anesthetized dogs with increased permeability edema induced by infused oleic acid. PEEP of 5, 10, 15, and 20 cmH2O was applied in random sequence. Following injury VD/VTCO2 increased. It decreased with 5 or 10 cmH2O PEEP but increased progressively at higher PEEP levels. The decrease in VD/VTCO2 at 5 or 10 cmH2O PEEP was due to reductions in shunt and midrange VA/Q heterogeneity. The increase in VD/VTCO2 that occurred with higher PEEP levels was due to increased ventilation to high VA/Q regions and a larger anatomic dead space. Haldane effect magnified the shunt component of VD/VTCO2 but reduced the influence of midrange VA/Q heterogeneity.

Acute Disease↗
PubMed1982

Respiratory and inert gas exchange during high-frequency ventilation.

Pulmonary gas exchange during high-frequency low-tidal volume ventilation (HFV) (10 Hz, 4.8 ml/kg) was compared with conventional ventilation (CV) and an identical inspired fresh gas flow in pentobarbital-anesthetized dogs. Comparing respiratory and infused inert gas exchange (Wagner et al., J. Appl. Physiol. 36: 585--599, 1974) during HFV and CV, the efficiency of oxygenation was not different, but the Bohr physiological dead space ratio was greater on HFV (61.5 +/- 2.2% vs. 50.6 +/- 1.4%). However, the elimination of the most soluble inert gas (acetone) was markedly enhanced by HFV. The increased elimination of the soluble infused inert gases during HFV compared with CV may be related to the extensive intraregional gas mixing that allows the conducting airways to serve as a capacitance for the soluble inert gases. Comparing as exchange during HFV with three different density carrier gases (He, N2, and Ar), the efficiency of elimination of Co2 or the intravenously infused inert gases was greatest with He-O2. However, the alveolar-arterial partial pressure difference for O2 on He-O2 exceeded that on N2-O2 by 5.4 Torr during HFV. The finding agrees with similar observations during CV, suggesting that this aspect of gas exchange is not substantially altered by HFV.

Animals↗
PubMed1981

Immunologic tests of value in diagnosis. 1. Acute phase reactants and autoantibodies.

Acute phase reactants are nonspecific indicators of tissue necrosis and/or inflammation but may be helpful in determining activity of disease. Rheumatoid factor is likewise rather nonspecific, but its presence is helpful in predicting the course, severity, and complications of rheumatoid arthritis. Numerous antinuclear antibodies have been identified in collagen vascular diseases; perhaps the most specific association is between anti-Sm antibody and systemic lupus erythematosus. Anti-smooth-muscle and antimitochondrial antibodies can aid in differential diagnosis of liver disease, while antithyroid antibodies can perform a similar function in diffuse goiter. Anti-parietal-cell and anti-intrinsic-factor antibodies are quite specific for pernicious anemia.

Acute Disease↗
PubMed1981

Immunologic tests of value in diagnosis. 2. Complement.

Laboratory tests are available to assess the function of the complement pathway and to measure levels of individual complement components. The pattern of complement abnormalities is often helpful in suggesting diagnostic possibilities. For example, when when total hemolytic complement, C3, and C4 are all decreased, one of the rheumatoid diseases is likely. In addition, complement levels in spinal and synovial fluid may provide helpful diagnostic clues.

Arthritis, Rheumatoid↗