Thermodynamic modeling of internal equilibria involved in the activation of trypsinogen

The effect of activating dipeptides, sequentially homologous to the Ile16-Val17N-terminus of bovine beta-trypsin (beta-trypsin), on equilibria involved in the binding of strong ligands (i.e., n-butylamine, the bovine basic pancreatic trypsin inhibitor (Kunitz-type inhibitor; BPTI) and the porcine pancreatic secretory trypsin inhibitor (Kazal-type inhibitor, type I; PSTI)) to bovine trypsinogen (trypsinogen) was investigated at pH 5.51 (I = 0.1 M) and T = 21.0 +/- 0.5 degrees C; under the same experimental conditions, thermodynamics for the binding of strong ligands to beta-trypsin was also obtained. The equilibria involved in the binding of activating dipeptides and/or inhibitors to beta-trypsin and to its zymogen are described according to an induced-fit formalism, taking into account ligand-linked interaction(s) between different functional and structural domains of the (pro)enzyme possibly involved in the trypsinogen-to-beta-trypsin activation pathway. The analysis of data is focussed on parameters describing interactions between the so-called Ile-Val pocket (where the Ile16-Val17 N-terminus of beta-trypsin or activating dipeptides bind) and the primary and/or secondary recognition subsite(s) (where strong ligands associate) present in the (pro)enzyme. Such an analysis allows to dissect the contributions due to the primary recognition subsite, where small mono-functional ligands (e.g., n-butylamine) bind, from those of the secondary subsite(s), which are additional recognition clefts for macromolecular inhibitors (e.g., BPTI and PSTI).     

Evidence for a kinetic heterogeneity in ligand binding to R-state haemoglobin Kempsey [Asp-G1(99) beta----Asn].

Thermodynamic and kinetic properties of O2 and CO binding to haemoglobin (Hb) Kempsey [Asp-G1(99) beta----Asn] were investigated and the activation parameters for the two ligands were determined. At every temperature the O2-binding isotherms display a weak co-operativity, n ranging between 1.1 and 1.2, and dissociation kinetics show a single-exponential behaviour. O2-binding kinetics were studied at 25 degrees C by temperature jump and are characterized at each saturation (from Y = 0.31 to Y = 1.0) by two processes, a fast bimolecular one and a slow monomolecular one (tau -1 = 20 s-1), which contributes to approx. 30% of the whole relaxation amplitude at every Y. CO-binding kinetics to Hb Kempsey were followed at several temperatures by flash photolysis and stopped flow. The process is biphasic, as reported elsewhere [Bunn, Wohl, Bradley, Cooley & Gibson (1974) J. Biol. Chem. 249, 7402-7409], and the relative contributions of the two bimolecular rates to the whole process are only slightly affected by temperature. On taking account for the fraction of dimers at every protein concentration, the slow phase corresponds to approx. 50% of the ligand binding to tetramers. Correlation of these results with previous spectroscopic data leads to the hypothesis that the biphasic time course of CO binding may be attributed to alpha/beta heterogeneity of the R-state of tetrameric Hb Kempsey.     

A Novel Source of Cultured Podocytes

Amniotic fluid is in continuity with multiple developing organ systems, including the kidney. Committed, but still stem-like cells from these organs may thus appear in amniotic fluid. We report having established for the first time a stem-like cell population derived from human amniotic fluid and possessing characteristics of podocyte precursors. Using a method of triple positive selection we obtained a population of cells (hAKPC-P) that can be propagated in vitro for many passages without immortalization or genetic manipulation. Under specific culture conditions, these cells can be differentiated to mature podocytes. In this work we compared these cells with conditionally immortalized podocytes, the current gold standard for in vitro studies. After in vitro differentiation, both cell lines have similar expression of the major podocyte proteins, such as nephrin and type IV collagen, that are characteristic of mature functional podocytes. In addition, differentiated hAKPC-P respond to angiotensin II and the podocyte toxin, puromycin aminonucleoside, in a way typical of podocytes. In contrast to immortalized cells, hAKPC-P have a more nearly normal cell cycle regulation and a pronounced developmental pattern of specific protein expression, suggesting their suitability for studies of podocyte development for the first time in vitro. These novel progenitor cells appear to have several distinct advantages for studies of podocyte cell biology and potentially for translational therapies.     

Functional and structural roles of the N-terminal extension in Methanosarcina acetivorans protoglobin

Functional and structural properties of protoglobin from Methanosarcina acetivorans, whose Cys(101)E20 residue was mutated to Ser (MaPgb*), and of mutants missing either the first 20 N-terminal amino acids (MaPgb*-ΔN20 mutant), or the first 33 N-terminal amino acids [N-terminal loop of 20 amino acids and a 13-residue Z-helix, preceding the globin fold A-helix; (MaPgb*-ΔN20Z mutant)] have been investigated. In keeping with the MaPgb*-ΔN20 mutant crystal structure, here reported at 2.0 Å resolution, which shows an increased exposure of the haem propionates to the solvent, the analysis of ligand binding kinetics highlights high accessibility of ligands to the haem pocket in ferric MaPgb*-ΔN20. CO binding to ferrous MaPgb*-ΔN20 displays a marked biphasic behavior, with a fast binding process close to that observed in MaPgb* and a slow carbonylation process, characterized by a rate-limiting step. Conversely, removal of the first 33 residues induces a substantial perturbation of the overall MaPgb* structure, with loss of α-helical content and potential partial collapse of the protein chain. As such, ligand binding kinetics are characterized by very slow rates that are independent of ligand concentration, this being indicative of a high energy barrier for ligand access to the haem, possibly due to localized misfolding. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.     

Priming xylem for stress recovery depends on coordinated activity of sugar metabolic pathways and changes in xylem sap pH

Some plant species are capable of significant reduction of xylem embolism during recovery from drought despite stem water potential remains negative. However, the functional biology underlying this process is elusive. We subjected poplar trees to drought stress followed by a period of recovery. Water potential, hydraulic conductivity, gas exchange, xylem sap pH, and carbohydrate content in sap and woody stems were monitored in combination with an analysis of carbohydrate metabolism, enzyme activity, and expression of genes involved in sugar metabolic and transport pathways. Drought resulted in an alteration of differential partitioning between starch and soluble sugars. Upon stress, an increase in the starch degradation rate and the overexpression of sugar symporter genes promoted the efflux of disaccharides (mostly maltose and sucrose) to the apoplast. In turn, the efflux activity of the sugar‐proton cotransporters caused a drop in xylem pH. The newly acidic environment induced the activity of apoplastic invertases leading to the accumulation of monosaccharides in the apoplast, thus providing the main osmoticum necessary for recovery. During drought and recovery, a complex network of coordinated molecular and biochemical signals was activated at the interface between xylem and parenchyma cells that appeared to prime the xylem for hydraulic recovery.     

Nitrosylation of rabbit ferrous heme-hemopexin

Hemopexin (HPX) serves as a trap for toxic plasma heme, ensuring its complete clearance by transportation to the liver. Moreover, HPX-heme has been postulated to play a key role in the homeostasis of nitric oxide (NO). Here, the thermodynamics for NO binding to rabbit ferrous HPX-heme as well as the EPR and optical absorption spectroscopic properties of rabbit ferrous nitrosylated HPX-heme (HPX-heme-NO) are reported. The value of the dissociation equilibrium constant for NO binding to rabbit ferrous HPX-heme (i.e., H) is (1.4±0.2)×10^–7 M, at pH 7.0 and 10.0 °C; the value of H is unaffected by sodium chloride. At pH 7.0, rabbit ferrous HPX-heme-NO is a six-coordinate heme-iron species, characterized by an X-band EPR spectrum with an axial geometry and by =146 mM^–1 cm^–1 at 419 nm. At pH 4.0, rabbit ferrous HPX-heme-NO is a five-coordinate heme-iron species, characterized by an X-band EPR spectrum with three-line splitting centered at 334 mT and by =74 mM^–1 cm^–1 at 387 nm. The pK_a value of the reversible pH-induced six- to five-coordinate spectroscopic transition is 4.8±0.1 in the absence of sodium chloride and 4.3±0.1 in the presence of 1.5×10^–1 M sodium chloride. This result is in agreement with the effect of sodium chloride on rabbit HPX-heme stability. The present data have been analyzed in parallel with those of a related heme model compound and heme-protein systems.