Thermodynamic Modeling of Internal Equilibria Involved in the Activation of Trypsinogen

Abstract

The effect of activating dipeptides, sequentially homologous to the Ile 16-Val 17 N-terminus of bovine β-trypsin (β-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 5.5 (I = 0.1 M) and T = 21.0 ± 0.5°C; under the same experimental conditions, thermodynamics for the binding of strong ligands to β-trypsin was also obtained. The equilibria involved in the binding of activating dipeptides and/or inhibitors to β-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-β-trypsin activation pathway. The analysis of data is focussed on parameters describing interactions between the so-called Ile-Val pocket (where the Ile16-Val17/V-terminus of β-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).     

Molecular basis of α-thalassemia in Sicily

To evaluate the allelic frequency and genetic diversity of α-thalassemia defects in Sicily, both epidemiological and patient-oriented studies were carried out. For the epidemiological study, phenotypic data were collected on more than 1000 Sicilian individuals. Among them, 427 were explored at the molecular level for nine α-thalassemic variants known to be common in the Mediterranean region. Our data reveal an allele frequency of 4.1% for α⁺-thalassemia matching that of β-thalassemia in this region. The presence of α°-thalassemia (––^MEDI and ––^CAL) was observed only in the group of referred patients. Newly acquired nucleotide sequence data on the deletional breakpoint of ––^CAL allowed us to design a simple PCR-based procedure for exploring this allele. The data also provide additional information concerning the genetic mechanisms involved in such large deletions. Received: 8 August 1996 / Revised: 16 October 1996     

[3H]Ketanserin Binding in Human Brain Postmortem

This study aimed at comparing the binding characteristics of [³H]ketanserin, a high-affinity serotonin 2A (5-HT_2A) receptor antagonist, in the prefrontal cortex, hippocampus and striatum of human brain post-mortem. The results indicated the presence of a single population of binding sites in all the regions investigated, with no statistical difference in maximum binding capacity (B_max) or dissociation constant (K_d) values. The pharmacological profile of [³H]ketanserin binding was consistent with the labeling of the 5-HT_2A receptor, since it revealed a competing drug potency ranking of ketanserin = spiperone > clozapine = haloperidol > methysergide > mesulergine > 5-HT. In conclusion, the 5-HT_2A receptor, as labeled by [³H]ketanserin, would seem to consist of a homogenous population of binding sites and to be equally distributed in human prefronto-cortical, limbic and extrapyramidal structures.     

Do changes in the mechanical properties of articular cartilage promote catabolic destruction of cartilage and osteoarthritis?

Osteoarthritis (OA) is a joint disease characterized by cartilage degeneration, a thickening of subchondral bone, and formation of marginal osteophytes. Previous mechanical characterization of cartilage in our laboratory suggests that energy storage and dissipation is reduced in osteoarthritis as the extent of fibrillation and fissure formation increases. It is not clear whether the loss of energy storage and dissipation characteristics is a result of biochemical and/or biophysical changes that occur to hyaline cartilage in joints. The purpose of this study is to present data, on the strain rate dependence of the elastic and viscous behaviors of cartilage, in order to further characterize changes that occur in the mechanical properties that are associated with OA. We have previously hypothesized that the changes seen in the mechanical properties of cartilage may be due to altered mechanochemical transduction by chondrocytes. Results of incremental tensile stress-strain tests at strain rates between 100%/min and 10,000%/min conducted on OA cartilage indicate that the slope of the elastic stress-strain curve increases with increasing strain rate, unlike the reported behavior of skin and self-assembled collagen fibers. It is suggested that the strain-rate dependence of the elastic stress-strain curve is due to the presence of large quantities of proteoglycans (PGs), which protect articular cartilage by increasing the apparent stiffness. The increased apparent stiffness of articular cartilage at high strain rates may limit the stresses borne and prolong the onset of OA. It is further hypothesized that increased compressive loading of chondrocytes in the intermediate zone of articular cartilage occurs as a result of normal wear to the superficial zone or from excessive impact loading. Once the superficial zone of articular cartilage is worn away, the tension is decreased throughout all cartilage zones leading to increased chondrocyte compressive loading and up-regulation of mechanochemical transduction processes that elaborate catabolic enzymes.     

Hibernation and daily torpor in an armadillo, the pichi (Zaedyus pichiy).

Hibernation and daily torpor are physiological strategies to cope with energetic challenges that occur in many mammalian and avian taxa, but no reliable information exists about daily torpor or hibernation for any xenarthran. Our objective was to determine whether the pichi (Zaedyus pichiy), a small armadillo (Xenarthra, Dasypodidae) that inhabits arid and semi-arid habitats in central and southern Argentina and Chile, enters shallow daily torpor or prolonged deep hibernation during winter when environmental temperature and food availability are low. We studied body temperature changes during winter in semi-captive pichis by means of temperature dataloggers implanted subcutaneously. All individuals entered hibernation, characterized by torpor events of 75+/-20 h during which the subcutaneous temperature (T(sc)) decreased to 14.6+/-2.1 degrees C. These events were interrupted by periods of euthermia of 44+/-38 h with a T(sc) of 29.1+/-0.7 degrees C. After the hibernation season, daily torpor bouts of 4 to 6 h occurred irregularly, with T(sc) dropping to as low as 24.5 degrees C. We conclude that the pichi is a true hibernator and can enter daily torpor outside of the hibernation season.     

Functional properties of hemoglobin leiden (α2Aβ26 or7 Glu deleted)

Hemoglobin Leiden is an abnormal human hemoglobin in which a glutamic acid residue has been deleted from the β-chain at position 6 or 7. The α-amino groups of the β-chain N-termini in tetrameric hemoglobin A are thought to be directly involved in the binding of simple anions and organic phosphates (1). The deletion of the 4th or 5th residue of the A helix in hemoglobin Leiden shortens the N-terminus of the β-chain, and the results reported here show that the anion binding site has been affected. Hemoglobin Leiden shows a decreased response to inorganic phosphate, chloride, 2,3-diphosphoglycerate, and inositol hexaphosphate, both in equilibria and kinetics of ligand binding. Although hemoglobin Leiden shows an altered response to anions, neither the cooperativity of ligand binding nor the Bohr effect are significantly altered by the deletion. The decreased effect of cofactors seems to be due to a decrease in the strength of anion binding which may be attributed to the altered geometry of the anion binding site.     

Role of the phosphorolysis of deoxyadenosine in the cytotoxic effect of the combination of deoxyadenosine and deoxycoformycin on a human colon carcinoma cell line (LoVo)

In LoVo cells, phosphorolytic activity acting on deoxyadenosine plays a major role in the resistance to the cytotoxic effect of the combination of deoxynucleoside with deoxycoformycin. In fact, the observed dependence of toxicity on cell density appears to be related to the metabolic conversion of deoxyadenosine into adenine. The phosphorylation of the deoxynucleoside, which represents the first step towards the formation of the cytotoxic agent dATP, proceeds at a significantly lower rate as compared to the phosphorolysis of deoxyadenosine. The analysis of the levels of deoxyadenosine and its derivatives in the incubation media reveals that the rates of disappearance of deoxyadenosine and of formation of adenine increase in concert with the reduction of the effect on cell survival. J. Cell. Biochem. 80:241–247, 2000. © 2000 Wiley‐Liss, Inc.     

Characterization of sulfonylurea receptors in isolated human pancreatic islets

Current information on pancreatic islet sulfonylurea receptors has been obtained with laboratory animal pancreatic β cells or stable β-cell lines. In the present study, we evaluated the properties of sulfonylurea receptors of human islets of Langherans, prepared by collagenase digestion and density-gradient purification. The binding characterisitics of labeled glibenclamide to pancreatic islet membrane preparations were analyzed, displacement studies with several oral hypoglycemic agents were performed, and these latter compounds were tested as for their insulinotropic action on intact human islets. [³H]glibenclamide saturable binding was shown to be linear at ≤0.25 mg/ml protein; it was both temperature and time dependent. Scatchard analysis of the equilibrium binding data at 25°C indicated the presence of a single class of saturable, high-affinity binding sites with a K_d value of 1.0 ± 0.07 nM and a Bmax value of 657 ± 48 fmol/mg of proteins. The displacement experiments showed the following rank order of potency of the oral hypoglycemic agents we tested: glibenclamide = glimepiride > tolbutamide > chlorpropamide ≫ metformin. This binding potency order was parallel with the insulinotropic potency of the evaluated compounds. J. Cell. Biochem. 71:182–188, 1998. © 1998 Wiley-Liss, Inc.