Proton-linked subunit kinetic heterogeneity for carbon monoxide binding to hemoglobin from Chelidonichthys kumu

The pH dependence of CO binding kinetics to Chelidonichthys kumu hemoglobin (Hb) and human adult Hb has been investigated between pH 2.0 and 9.0 at 20 degrees C. For both Hbs, CO binding kinetics is characterized by two proton-linked transitions, with different pKa values for alpha- and beta-chains in C. kumu Hb, leading to a relevant functional kinetic heterogeneity at most pH values. On the other hand, in human adult Hb the CO binding does not display a functional heterogeneity. Lowering the pH from 9 to 6 brings about a decrease of the CO binding rate constants, to a different extent for human adult Hb and the two chains of C. kumu Hb. Further lowering the pH from 6 to 2 induces an enhancement of CO binding rate constants, probably related to the protonation of proximal HisF8 Nepsilon atom and the cleavage (or severe weakening) of the HisF8-Fe bond. The presence of physiological concentrations of ATP (approximately 3 mM) affects the pH dependence of CO binding kinetics to C. kumu. Moreover, the effect of temperature (between 8 degrees C and 38 degrees C) on CO binding kinetics has been investigated in the absence of ATP at different pH values. These results allow to interpret the functional kinetic heterogeneity of C. kumu Hb on the basis of different regulatory aspects in the alpha- and beta-subunits, as suggested by structural considerations

The Unique Hemoglobin System of Pleuragramma antarcticum, an Antarctic Migratory Teleost STRUCTURE AND FUNCTION OF THE THREE COMPONENTS

Pleuragramma antarcticum (suborder Notothenioidei, family Nototheniidae) is the most abundant fish in the antarctic shelf. This pelagic species has a circum-antarctic distribution and is characterized by spawning migration. This species displays the highest multiplicity of major hemoglobins (three); the other notothenioids have a single one (except one species, having two) with relatively low oxygen affinity regulated by pH and organophosphates. The hemoglobins of P. antarcticum display strong Bohr and Root effects; however, they reveal important functional differences in subunit cooperativity and organophosphate regulation and, above all, in the response of oxygenation to temperature. Despite the substitution ValbetaE11 --> Ile found in Hb 2, which decreases the affinity in human mutants, the hemoglobins have similar oxygen affinity, higher than that of the other notothenioids. Hb 1 has the alpha chain in common with Hb 2 and the beta in common with Hb 3. The amino acid sequence of all four chains has been established. Thus the hematological features of P. antarcticum differ remarkably from those of antarctic notothenioids. This unique and sophisticated oxygen transport system may adequately meet the requirements of the unusual mode of life of this fish

Hagfish Hemoglobins STRUCTURE, FUNCTION, AND OXYGEN-LINKED ASSOCIATION

Cyclostomes, hagfishes and lampreys, contain hemoglobins that are monomeric when oxygenated and polymerize to dimers or tetramers when deoxygenated. The three major hemoglobin components (HbI, HbII, and HbIII) from the hagfish Myxine glutinosa have been characterized and compared with lamprey Petromyzon marinus HbV, whose x-ray crystal structure has been solved in the deoxygenated, dimeric state (Heaslet, H. A., and Royer, W. E., Jr. (1999) Structure 7, 517-526). Of these three, HbII bears the highest sequence similarity to P. marinus HbV. In HbI and HbIII the distal histidine is substituted by a glutamine residue and additional substitutions occur in residues located at the deoxy dimer interface of P. marinus HbV. Infrared spectroscopy of the CO derivatives, used to probe the distal pocket fine structure, brings out a correlation between the CO stretching frequencies and the rates of CO combination. Ultracentrifugation studies show that HbI and HbIII are monomeric in both the oxygenated and deoxygenated states under all conditions studied, whereas deoxy HbII forms dimers at acidic pH values, like P. marinus HbV. Accordingly, the oxygen affinities of HbI and HbIII are independent of pH, whereas HbII displays a Bohr effect below pH 7.2. HbII also forms heterodimers with HbIII and heterotetramers with HbI. The functional counterparts of heteropolymer formation are cooperativity in oxygen binding and the oxygen-linked binding of protons and bicarbonate. The observed effects are explained on the basis of the x-ray structure of P. marinus HbV and the association behavior of site-specific mutants (Qiu, Y., Maillett, D. H., Knapp, J., Olson, J. S., and Riggs, A. F. (2000) J. Biol. Chem. 275, 13517-13528)

Migration and chaining of noncolloidal spheres suspended in a sheared viscoelastic medium. Experiments and numerical simulations

Migration and chaining of noncolloidal spheres in a worm-like micellar, viscoelastic solution under shear flow have been studied both experimentally and by numerical simulations. The microstructure dynamics have been experimentally investigated in the flow-gradient and in the flow-vorticity planes. 2D simulations in the flow-gradient plane have been performed for the same geometry, and with a proper selection for the constitutive equation of the suspending liquid. Experimental results show the formation of particle chains in the bulk, along with migration of a considerable fraction of spheres to the walls. At long times, chains in the bulk are stable, and cross-flow migration of individual spheres is suppressed. Numerical simulations with a standard viscoelastic constitutive equation (Giesekus fluid) reproduce the same phenomena observed experimentally, both in terms of fast particle migration to the wall and bulk chain stability. No alignment is, instead, found in simulations with a constant-viscosity, elastic fluid (Oldroyd-B model), in agreement with previous experimental results with Boger fluid

PECTIN METYLESTERASE INHIBITOR IN TOMATO FRUITS: CLONING AND MOLECULAR CHARACTERIZATION

Pectin is one of the main components of primary plant cell wall and its structure is critical for plant growth, intercellular adhesion, fruit texture and interaction with pathogens. Pectin is secreted in a highly methyl-esterified form and subsequently de-esterified by pectin methylesterases (PMEs) localized in the wall. PMEs take part in important physiological processes such as pollen growth and fruit ripening and are involved in the resistance response to fungal and viral pathogens. A mechanism of regulation of PME activity is played by specific proteinaceous inhibitors (PMEI). The structural bases of the interaction between PME from tomato and PMEI from kiwi fruit have been defined by solving the 3D structure of the PME/PMEI complex. We have identified and cloned a fruit-expressed cDNA of tomato with a significant sequence identity with kiwi PMEI. Structural alignment and modelling indicate that the encoded protein has the same fold of kiwi PMEI. Expression and functional characterization of the putative tomato PMEI will be presented