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).     

X-ray crystal structure of the bovine alpha-chymotrypsin/eglin c complex at 2.6 A resolution

The crystal structure of the molecular complex formed by bovine alpha-chymotrypsin and the recombinant serine proteinase inhibitor eglin c from Hirudo medicinalis has been solved using monoclinic crystals of the complex, reported previously. Four circle diffractometer data at 3.0 A resolution were employed to determine the structure by molecular replacement techniques. Bovine alpha-chymotrypsin alone was used as the search model; it allowed us to correctly orient and translate the enzyme in the unit cell and to obtain sufficient electron density for positioning the eglin c molecule. After independent rigid body refinement of the two complex components, the molecular model yielded a crystallographic R factor of 0.39. Five iterative cycles of restrained crystallographic refinement and model building were conducted, gradually increasing resolution. The current R factor at 2.6 A resolution (diffractometer data) is 0.18. The model includes 56 solvent molecules. Eglin c binds to bovine alpha-chymotrypsin in a manner consistent with other known serine proteinase/inhibitor complex structures. The reactive site loop shows the expected conformation for productive binding and is in tight contact with bovine alpha-chymotrypsin between subsites P3 and P'2; Leu 451 acts as the P1 residue, located in the primary specificity S1 site of the enzyme. Hydrogen bonds equivalent to those observed in complexes of trypsin(ogen) with the pancreatic basic- and secretory-inhibitors are found around the scissile peptide bond.     

Binding of the recombinant proteinase inhibitor eglin c from leech Hirudo medicinalis to human leukocyte elastase, bovine alpha-chymotrypsin and subtilisin Carlsberg: thermodynamic study

The effect of pH and temperature on the apparent association equilibrium constant (Ka) for the binding of the recombinant proteinase inhibitor eglin c from leech Hirudo medicinalis to human leukocyte elastase (EC 3.4.21.37), bovine alpha-chymotrypsin (EC 3.4.21.1) and subtilisin Carlsberg (EC 3.4.21.14) has been investigated. On lowering the pH from 9.5 to 4.5, values of Ka for eglin c binding to the serine proteinases considered decrease thus reflecting the acid-pK shift of the invariant histidyl catalytic residue (His57 in human leukocyte elastase and bovine alpha-chymotrypsin, and His64 in subtilisin Carlsberg) from congruent to 6.9, in the free enzymes, to congruent to 5.1, in the enzyme:inhibitor adducts. At pH 8.0, values of the apparent thermodynamic parameters for eglin c binding are: human leukocyte elastase - Ka = 1.0 x 10(10) M-1, delta G phi = -13.4 kcal/mol, delta H phi = +1.8 kcal/mol, and delta S phi = +52 entropy units; bovine alpha-chymotrypsin -Ka = 5.0 x 10(9) M-1, delta G phi = -13.0 kcal/mol, delta H phi = +2.0 kcal/mol, and delta S phi = +51 entropy units; and subtilisin Carlsberg - Ka = 6.6 x 10(9) M-1, delta G phi = -13.1 kcal/mol, delta H phi = +2.0 kcal/mol, and delta S phi = +51 entropy units (values of Ka, delta G phi and delta S phi were obtained at 21 degrees C; values of delta H phi were temperature independent over the range explored, i.e. between 10 degrees C and 40 degrees C; 1 kcal = 4184J).(ABSTRACT TRUNCATED AT 250 WORDS)     

Somatostatin as a mediator of the effect of neurotensin on pentagastrin-stimulated acid secretion in rats

Neurotensin and somatostatin have both been shown to inhibit gastric acid secretion, but no interaction between these peptides has been demonstrated. To determine whether somatostatin might be a mediator of neurotensin's effect on pentagastrin-stimulated gastric acid secretion, we performed the following three experiments. First, we collected 0.2-ml samples of portal venous blood as frequently as every 5 min, and we confirmed a significant release of somatostatin-like immunoreactivity into portal venous blood during neurotensin-induced inhibition of acid secretion. This release of somatostatin-like immunoreactivity and inhibition of acid secretion were only seen in pentobarbital-anesthetized rats, but no sustained release of somatostatin-like immunoreactivity or inhibition of acid secretion occurred in urethane-anesthetized animals. In the second experiment, we analyzed portal plasma by high pressure liquid chromatography, and found that portal somatostatin-like immunoreactivity in blood collected during neurotensin infusion was composed of a single peak corresponding to somatostatin-14. In the third experiment, we found that infusion of antibody to somatostatin prevented neurotensin from inhibiting pentagastrin-stimulated acid secretion. Taken together, these data show that somatostatin, possibly from the stomach itself, is a necessary mediator of neurotensin's inhibitory effect in pentobarbital-anesthetized rats.     

Are prealbumin plasma levels linked to amino acid supply from peripheral tissues in liver cirrhosis?

Summary Prealbumin plasma level is considered a good index of liver function in liver cirrhosis. However, plasma protein levels depend not only on liver function, but also on amino acid supply which is consequent to nutritional status.In 12 cirrhotics we measured prealbumin plasma levels and the lower limb venous-artero difference of amino acid plasma levels in blood samples taken from femoral vein and femoral artery in post-absorptive conditions considered as a direct index of protein release from peripheral tissues and an indirect index of protein nutritional status.In arterial and in venous plasma amino acid sum was 1.86±0.40 (mean + sd) and 2.00 ± 0.04 mMol/l respectively.Prealbumin plasma levels were found directly correlated with the venousartero difference of amino acid plasma levels (r = 0.57p < 0.05) and of glutamate + glutamine levels (r = 0.73p < 0.007).In conclusions, these data suggest that prealbumin plasma levels are linked to amino acid supply from peripheral tissues in cirrhotics.     

A map of barley chromosome 2 using isozymic and morphological markers

The F₂ progeny of a cross between a chromosome 2 multiple marker stock and an adapted cultivar of barley were analyzed for four morphological markers and electrophoretic patterns of eight leaf isozymes. TheIdh-2 locus was linked to thePer-5 locus (27.96±5.07 cM) and to thee locus (10.26±3.13 cM). Also, thePer-5 ande loci were located on the short arm of chromosome 2. In additionIdh-2 was also located on barley chromosome 2 and was linked to thev locus (13.18±3.56 cM), which is located on the long arm of chromosome 2. Two other marker genes,li andwst,,B, were linked (26.50±5.24 cM) on chromosome 2 but segregate independently of the other loci evaluated. This project was supported by funds from the U.S.-Spain Joint Committee for Scientific and Technological Cooperation.     

Catalytic properties of bovine alpha-thrombin: a comparative steady-state and pre-steady-state study

Values of steady-state and pre-steady-state parameters for the bovine alpha-thrombin-catalyzed hydrolysis of ZArgONp and ZLysONp have been determined between pH 2.5 and 8 (I = 0.1 M) at 21 +/- 0.5 degree C. Kinetic properties of bovine alpha-thrombin have been analyzed in parallel with those of porcine pancreatic beta-kallikrein-B and bovine beta-trypsin, all acting on cationic substrates. The different primary specificity and catalytic behaviour of these three serine proteinases reflect subtle structural differences at their S1 subsite, especially at residue positions 190, 221 and 226 as well as in the 217-219 segment.     

Clinical importance of circulating immune complexes in human acute lymphoblastic leukemia

Summary A total of 122 sera from acute lymphoblastic leukemia (ALL) patients were analyzed for circulating immune complexes (CIC) by two methods: the ¹²⁵I-C₁q binding assay and the polyethylene glycol precipitation test (PEG). The results were correlated with induction, remission and relapse stages of the disease. Using the first method the levels of CIC in induction were 15.18±9.15, with 19/29 positive cases (65.50%), P<0.001 compared with controls. In the remission phase the levels were 9.02±5.62, 11/45 (24.49%) nonsignificant P value, and in relapse they were 16.14±11.17 28/48 (58.33%) P<0.001. The PEG precipitation test results were: 0.33±0.10, 8/22 (36.36%); 0.24±0.11, 10/48 (20.83%) and 0.28±0.10, 6/28 (21.42%), respectively. Thus the values of CIC as measured by PEG in the three clinical of phases ALL did not differ significantly from controls. This contrasts with results obtained by the radioiodinated C₁q binding assay, where the incidence of positive values was significantly higher in induction and in relapse and lower in the remission phase. These observations were extended in sequential vertical studies performed in a group of patients. These results suggest that raised CIC detected by the ¹²⁵I-C₁q method may reflect a progressive state in ALL and that quantitation of these immune complexes may provide an adequate biochemical marker for prognosis.     

Recycling of the asialoglycoprotein receptor and the effect of lysosomotropic amines in hepatoma cells

Receptor-mediated uptake and degradation of 125I-asialoorosomucoid (ASOR) in human hepatoma HepG2 cells is inhibited by the lysosomotropic amines chloroquine and primaquine. In the absence of added ligand at 37 degrees C, these amines induce a rapid (t1/2 5.5-6 min) and reversible loss of cell surface 125I-ASOR binding sites as well as a rapid decrease in 125I-ASOR uptake and degradation. There is no effect of these amines on the binding of 125I-ASOR to the cell surface at 4 degrees C or on the rate of internalization of prebound 125I-ASOR. The loss of 125I-ASOR surface binding at 37 degrees C is not attributable to altered affinity of ligand-receptor binding. In the presence of added ligand at 37 degrees C, there is a more rapid (t1/2 2.5-3 min) loss of hepatoma cell surface receptors. In addition, the amines inhibit the rapid return of the internalized receptor to the cell surface. We examined the nature of this loss of 125I-ASOR surface binding sites by following the fate of receptor molecules after biosynthetic labeling and after cell surface iodination. At 37 degrees C, chloroquine and primaquine induce a loss of asialoglycoprotein receptor molecules from the hepatoma cell surface to an internal pool.     

Inhibition of serine proteinases by tetra-p-amidinophenoxy-neo-pentane: thermodynamic and molecular modeling study

The inhibitory effect of the aromatic tetra-benzamidine derivative tetra-p-amidinophenoxy-neo-pentane (TAPP) on the catalytic properties of beta-trypsin (EC 3.4.21.4), alpha-thrombin (EC 3.4.21.5), factor Xa (EC 3.4.21.6), Lys77-plasmin (EC 3.4.21.7) and beta-kallikrein-B (EC 3.4.21.35) was investigated (between pH 2 and 8, I = 0.1 M; T = 37 +/- 0.5 degrees C), and analyzed in parallel with that of benzamidine, commonly taken as a molecular inhibitor model of serine proteinases. Over the whole pH range explored, TAPP and benzamidine show the same values of the dissociation inhibition constant (Ki) for beta-trypsin; at variance with the affinity of TAPP for alpha-thrombin, factor Xa, Lys77-plasmin and beta-kallikrein-B which is higher than that found for benzamidine association around neutrality, but tends to converge in the acidic pH limb. On lowering the pH from 5.5 to 3.0, values of Ki for TAPP binding to beta-trypsin as well as for benzamidine association to all the enzymes investigated decreased thus reflecting the pK-shift, upon inhibitor binding, of a single ionizing group. Over the same pH range, values of Ki for TAPP binding to alpha-thrombin, factor Xa, Lys77-plasmin and beta-kallikrein-B may be described as depending on the pK-shift, upon inhibitor association, of two equivalent proton-binding amino acid residues. Considering the X-ray three-dimensional structures and the computer-generated molecular models of serine proteinases: TAPP and :benzamidine adducts, the observed binding behaviour of TAPP and benzamidine to the enzymes considered has been related to the inferred stereochemistry of proteinase: inhibitor contact region(s).