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B G Barisas

Publications and source records attributed to B G Barisas.

72 records · Page 4Linked to original sources
PubMed1981

Changes in lectin receptor lateral mobilities accompany lymphocyte stimulation.

Changes in lateral mobilities of rabbit lymphocyte membrane components in response to succinyl concanavalin A (S Con A) have been studied by fluorescence photobleaching recovery (FPR). During hrs 0 to 3 after exposure to S Con A, lectin receptor mobilities on both T and B cells fall about 2-fold. Reduced mobility of T cell lectin receptors persists until hr 18. From hr 18 to 24 rapid recovery of original mobility occurs if and only if lectin is present. In contrast, nonresponding B cells recover original receptor mobility gradually over hr 4 to 48. Metabolic inhibitors added at hr 3 restore original receptor mobilities, but cytoskeletal disruptors have this effect on T cells only. From hr 0 to 15, washing lectin from the cell surface is decreasingly effective in restoring T cell receptor mobility. After hr 15, mobility cannot be enhance by lectin removal. Parallel DNA synthesis studies show that, for T cell stimulation, lectin must be present on the cell surface during hr 0 to 3 and 18 to 24. These are the periods when FPR measurements show lectin receptor mobilities being restricted and released, respectively. Stimulation of B cells by anti-Ig shows several interesting features. First, stimulation by intact anti-Ig fails to reduce the mobilities of Con A receptors in a manner similar to that produced by S Con A. Second, S Con A does reduce mobility of surface Ig. Thus, Con A receptors would appear to exert a unique anchorage modulation of mobilities of other membrane molecules.

Animals↗
PubMed1980

Calorimetric studies of carbon monoxide and inositol hexaphosphate binding to hemoglobin A.

Heats of CO and IHP binding to hemoglobin A have been determined under a variety of buffer and pH conditions. From these data heats of ion binding linked to hemoglobin oxygenation have been estimated. For IHP binding to deoxyhemoglobin the buffer-corrected enthalpies are surprisingly large, reaching -25 kcal/mol of IHP at pH 7.4. These values correspond to approximately -11 kcal/mol of proton absorbed upon IHP binding and may rise largely from the protonation of hitidine and NH2-terminal groups in the binding site (Arnone, A., and Perutz, M.F. (1974) Nature 249, 34-36). The decreased magnitude of delta HIHP observed at low pH parallels the decreased proton uptake at low pH. In 0.1 M chloride (pH 7.4) the reaction Hb(aq) + IHP leads to Hb x IHP(aq) has a standard free energy change (Edalji, R., Benesch, R.E., and Benesch, R. (1976) J. Biol. Chem. 251, 7720-7721) of -10 kcal and an enthalpy change of -25 kcal. Therefore, enthalpic forces provide the dominant driving force of this process. The origin of these large negative enthalpy changes is attributed to the exothermic protonation of protein basic groups induced by the proximity of phosphate negative charges. The importance of protonation in the binding of organic phosphates to hemoglobin may well extend to the specific binding of other phosphate substrates to enzyme reaction sites.

Calorimetry↗
PubMed1979

Thermodynamic analysis of carbon monoxide binding by hemoglobin trout I.

Calorimetric measurements at 25 degrees of the differential heat of CO binding by hemoglobin trout I have been examined together with the CO binding isotherms for the protein at 4 degrees and 20 degrees. Simultaneous treatment of these data sets by a statistically rigorous technique permits evaluation of all the thermodynamic parameters for both the Adair and the Monod, Wyman, Changeux (MWC) models. The results show the details of the unusual temperature dependent cooperativity which this hemoglobin exhibits. In the Adair formalism the increasingly favorable free energy change for successive steps of ligand binding are nearly linearly paralleled by increasingly negative enthalpy changes for these steps. This causes the enhanced cooperativity observed as the temperature is decreased. For the MWC case, lowering the temperature increases the stability of the unligated T state relative to the unligated R state since the enthalpy of the T leads to R transition is 29.4 kcal mol-1. Simultaneously, the favorability of ligating R forms relative to T is enhanced since R form ligation is 14.1 kcal (mol CO)-1 more exothermic than that of T. The balance between these opposing effects is to increase ligand binding cooperativity at low temperatures. The predicted temperature dependence of the Hill coefficient for the MWC and Adair models is identical at low and intermediate temperatures, but, interestingly, would show a strong divergence at high temperatures where negative cooperativity is suggested for the Adair case and positive cooperativity for the MWC case.

Animals↗
PubMed1979

Fluorescence photobleaching recovery measurement of protein absolute diffusion constants.

The technique of fluorescence photobleaching recovery [Axelrod et al., Biophys. J. 16 (1976) 1055] has been applied to the measurement of absolute diffusion constants of a number of fluoiescein isothiocyanate-labeled proteins. Measured diffusion constants agree to within +/- 7% of published values for the underivatized proteins. The method has sufficient sensitivity to reveal the concentration dependence at neutral pH of the diffusion constant of alpha-chymotrypsin. The rapidity with which the labelling and measurements can be performed and the small amount of material required suggest the technique may be useful in rapid characterization of small protein samples. Some developments in optical and electronic systems and in data processing for this technique are discussed.

Journal Article↗
PubMed1978

Analysis of ligand binding curves in terms of species fractions.

The ligand binding curve for a macromolecular system presents the average number of ligand molecules bound per macromolecule as a function of the chemical potential or the logarithm of the ligand concentration. We show that various observable properties of this curve, for example its asymptotes and derivatives, are expressible in terms of linear combinations of the mole fractions alphai of macromolecules binding i molecules of ligand. Whenever enough such properties of the binding curve are known, the linear equations in alphai can be solved to give the mole fractions of each of the various macromolecular species. An application of these results is that a Hill plot for hemoglobin-ligand equilibrium where the asymptotes approach unit slope can be made to yield the four Adair constants by a simple algebraic method. A second use is that a knowledge of the first and second derivatives of the binding curve at points along the curve can yield the species fractions as functions of the degree of saturation without direct knowledge of the ligand binding constants. These methods are illustrated by some numerical examples.

Binding Sites↗
PubMed1975

Heats of carbon monoxide binding by hemoglobin M Iwate.

The heat of reaction of CO gas with the alpha2Mmetbeta2 and alpha2Mbeta2 species of the alpha-chain mutant hemoglobin M Iwate has been studied in buffers with different heats of ionization of 25degrees and in the absence of organic phosphates. For the alpha2Mmetbeta2deoxy species we find a small Bohr effect (0.12 mol of H+/mol of CO) which is in correspondence with that found in equilibrium studies. The heat of reaction, when corrected for proton reaction with buffer, is -18.4 +/- 0.3 kcal/mol of CO at pH 7.4 At pH 9 the same value is observed within experimental error. This value compares closely with heats of reaction of CO with myoglobin and with van't Hoff determinations of the heat of oxygen binding to isolated hemoglobin alpha and beta chains after correction for the heat of replacement of O2 by CO. Furthermore, an analysis of the differential heat of ligand binding as a function of the extent of reaction indicated that, within experimental error, the heat of reaction with the first beta-chain heme in alpha2Mmetbeta2deoxy is the same as the second. Since the quaternary Tleads to R transition is blocked in this mutant hemoglobin, we compared it with Hb A to estimate the enthalpic component of the allosteric T leads to R transition in Hb A. The heats of reaction with CO(g) and Hb A are -15.7 +/- 0.5 and -20.9 +/- 0.5 kcal/mol at pH 7.4 and 9.0, respectively. In going from the T to the R state we find an enthalpy of transition of 9 +/- 2.5 kcal at pH 7.4 and -12 +/- 2.5 kcal at pH 9.0. From published free energies of transsition we conclude the T leads to R transition is enthalpically controlled at p/ 7.4 but entropically controlled at pH 9.0 A near normal Bohr effect is estimated from heats of reaction of CO with alpha2Mdeoxybeta2deoxy in various buffers. A large than normal heat of reaction (-21.6 +/- 0.5 kcal/mol of CO) is attributed to the abnormal alpha chains in Hb M Iwate.

Carbon Monoxide↗