Kinetic and proteolytic identification of heat-induced conformational changes in the urea herbicide binding site of isolated Phaseolus vulgaris chloroplast thylakoids

Kinetic parameters of 3-(3, 4-dichlorophenyl)-1, 1-dimethyl urea (DCMU)-induced inhibition of electron transport in chloroplast thylakoids isolated from Phaseolus vulgaris L. cv. Oregon 1604 were determined from analysis of a convergent, parallel electrical circuit. Through this analogue, the apparent affinity of the purported binding site for DCMU (K₁) and the relative amount of DCMU-insensitive electron transport (v^max₁/v_o) were obtained using a reiterative non-linear least squares curve-fitting procedure. Exposure of thylakoids to heat caused a gradual increase in K₁ (or decrease in the affinity of the thylakoid for DCMU) with an apparent activation energy of 134 kJ mol⁻¹. Tryptic susceptibility of a protein region regulating K₁ also decreased gradually with exposure to 45°C, suggesting that the heat-induced increase in K₁ might be due to a protein conformational change. On the other hand, thylakoid exposure to 45°C resulted in a rapid (<5 min) irreversible increase in v^max_I/v_o, which was also the apparent result of a conformational change in a region of the protein which regulates this function. These results are suggestive of the existence of differential thermal sensitivities of proteins within the thylakoids and, perhaps, of different regions within a single membrane protein.     

Limited proteolysis patterns of the B chain of insulin.

The oxidized B chain of insulin was used as a simple model for further consideration of limited proteolysis with low substrate:enzyme ratios. With low B chain:trypsin ratios, the ordinarily slower cleavage rate of the -Lys₂₉-Ala₃₀ bond essentially equaled the cleavage saturation rate of the -Arg₂₂-Gly₂₃ bond. This led to the disappearance of octapeptide which ordinarily forms most rapidly. Heptapeptide and alanine, formed mainly by cleavage of the octapeptide, decreased somewhat at high enzyme relative levels. Trypsin added to B chain formed a single chromatographic peak.     

Investigation and comparison of the binding between tolvaptan and pepsin and trypsin: Multi‐spectroscopic approaches and molecular docking

Tolvaptan (TF), a selective arginine vasopressin V2 receptor antagonist, was approved by the Food and Drug Administration in 2009. This study mainly investigated the differences between the binding of TF with pepsin and trypsin by using a series of spectroscopy and molecular modeling methods. Thermodynamic parameters and molecular docking results suggested that the binding of TF to pepsin and trypsin were both spontaneous but driven by different forces. For pepsin, the binding was driven by hydrogen bonds and van der Waals forces; but for trypsin, it was driven by electrostatic forces and hydrophobic forces. The quenching mechanism between TF and pepsin and trypsin was investigated by fluorescence experiments and time‐resolved fluorescence spectroscopy. Synchronous fluorescence and 3‐dimensional fluorescence were used to investigate the micro‐environmental and conformational changes of pepsin and trypsin after the insertion of TF. In addition, activity‐measurement results showed that both the pepsin and trypsin activities increased with increasing TF concentration, which may help to understand the possible effect of TF on the digestion and absorption of nutrients in vivo.     

Simultaneous Binding of Basic Peptides at Intracellular Sites on a Large Conductance Ca2+-activated K+ Channel : Equilibrium and Kinetic Basis of Negatively Coupled Ligand Interactions

The homologous Kunitz inhibitor proteins, bovine pancreatic trypsin inhibitor (BPTI) and dendrotoxin I (DTX-I), interact with large conductance Ca²⁺-activated K⁺ channels (maxi-K_Ca) by binding to an intracellular site outside of the pore to produce discrete substate events. In contrast, certain homologues of the Shaker ball peptide produce discrete blocking events by binding within the ion conduction pathway. In this study, we investigated ligand interactions of these positively charged peptide molecules by analysis of single maxi-K_Ca channels in planar bilayers recorded in the presence of DTX-I and BPTI, or DTX-I and a high-affinity homologue of ball peptide. Both DTX-I (K _d, 16.5 nM) and BPTI (K _d, 1,490 nM) exhibit one-site binding kinetics when studied alone; however, records in the presence of DTX-I plus BPTI demonstrate simultaneous binding of these two molecules. The affinity of BPTI (net charge, +6) decreases by 11.7-fold (K _d, 17,500 nM) when DTX-I (net charge, +10) is bound and, conversely, the affinity of DTX-I decreases by 10.8-fold (K _d, 178 nM) when BPTI is bound. The ball peptide homologue (BP; net charge, +6) exhibits high blocking affinity (K _d, 7.2 nM) at a single site when studied alone, but has 8.0-fold lower affinity (K _d, 57 nM) for blocking the DTX-occupied channel. The affinity of DTX-I likewise decreases by 8.4-fold (K _d, 139 nM) when BP is bound. These results identify two types of negatively coupled ligand–ligand interactions at distinct sites on the intracellular surface of maxi-K_Ca channels. Such antagonistic ligand interactions explain how the binding of BPTI or DTX-I to four potentially available sites on a tetrameric channel protein can exhibit apparent one-site kinetics. We hypothesize that negatively coupled binding equilibria and asymmetric changes in transition state energies for the interaction between DTX-I and BP originate from repulsive electrostatic interactions between positively charged peptide ligands on the channel surface. In contrast, there is no detectable binding interaction between DTX-I on the inside and tetraethylammonium or charybdotoxin on the outside of the maxi-K_Ca channel.     

Isolation and Characterization of Two Novel Nematicidal Depsipeptides from an Imperfect Fungus,Strain D1084

Two novel nematicidal cyclodepsipeptides, designated bursaphelocides A and B, were isolated from the culture filtrate of an imperfect fungus, strain D1084, belonging to Mycelia sterilia. Bursaphelocide A (1), containing 2-hydroxy-3-methylpentanoic acid, proline, isoleucine, N-methylalanine, N-methylvaline, and β-alanine in sequence, and bursaphelocide B (2), comprising 4-methylproline instead of proline in 1, are novel 2-hydroxy-3-methylpentanoic acid analogues of insecticidal destruxins.     

Isolation and Partial Characterization of the Major Seed Protein from Nicotiana tabacum,and Accumulation during Development

During the seed development of Nicotiana tabacum, appreciable accumulation of the soluble protein fraction started to occur at around the 6th day after anthesis and finally reached 12% on the basis of dry weight when seed maturation was accomplished. In the soluble fraction of mature seeds, four protein fractions were observed on analytical ultracentrifugation, and the protein having a sedimentation coefficient of 11.7S was the major one. The 11.7S protein was isolated and SDS-polyacrylamide gel electrophoresis indicated that the protein consisted of at least five subunits with molecular weights of 49,000, 31,000, 29,000, 21,000 and 19,000. The 11.7S protein was rich in glutamic acid or glutamine and arginine, and the presence of carbohydrate was confirmed.

During development, all of the five subunits started to appear during the period between the 12th and 15th day after anthesis.     

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).     

A trypsin and chymotrypsin inhibitor from seeds of Bauhenia purpurea

A trypsin and chymotrypsin inhibitor was partially purified from Bauhenia purpurea seeds and separated from a second inhibitor by Ecteola cellulose chromatography. The factor inhibited bovine trypsin and chymotrypsin as well as pronase trypsin and elastase. It formed a complex with trypsin and with chymotrypsin, but a ternary complex could not be detected. Differences were detected in the effect on trypsin and on chymotrypsin, although one enzyme interfered with the inhibition of the other. The results obtained point to two active centers on the inhibitor for the trypsin and chymotrypsin inhibition such that the one cannot complex with the inhibitor after this inhibitor had complexed with the other.     

白菜型油菜种子胰蛋白酶抑制剂纯化及部分性质研究

采用热变性、硫酸铵分步盐析及离子交换层析和分子筛层析等方法,从白菜型油菜种子中得到胰蛋白酶抑制剂(BNTI)。SDS-PAGE检测为单一条带,表明纯化的胰蛋白酶抑制剂电泳均一。SDS-PAGE测定其分子量约为14．4kD,等电聚焦测定其等电点约为4．7。BNTI具有较高的热稳定性。本文还考察了温度对溶液中BCH蛋白构象的影响,荧光光谱和测定抑制活力结果表明BNTI中的色氨酸和酪氨酸残基位于疏水部位。