Evidence that human α-thrombin is a monovalent cation-activated enzyme

The activity of human α-thrombin (EC 3.4.21.5) on small peptide substrates was enhanced by NaCl or KCl while tetramethylammonium chloride ((CH₃)₄NCl) or choline chloride (HO(CH₂)₂N(CH₃)₃Cl) which were used as ionic strength controls were without effect. The steady-state kinetic parameters of thrombin amidolysis of several peptidyl p-nitroanilide substrates were measured. Na⁺ enhanced thrombin activity by decreasing the K_m,app (0.2 to 0.7-fold) of all substrates, as well as increasing thombin turnover (3.4 to 4.5-fold) of some substrates. The average K_A for Na⁺for the four substrates examined was 3.5 × 10^?2m. A comparison of the effects of Na⁺ vs K⁺ on thrombin hydrolysis of a single substrate indicated that both cations similarly decreased the K_m,app (0.2 to 04.-fold) and increased thek_cat,app (3.1 to 3.4-fold) except that higher K⁺ concentrations (K_A = 2.8 × 10^?1M) were required. The rate of inactivation of thrombin by the active site-directed inhibitor N-p-tosyl-lysine chloromethyl ketone under pseudo-first-order conditions was enhanced 3-fold by saturating NaCl. Also, the fibrinogen clotting activity of thrombin was enhanced by NaCl compared to the choline chloride control. Spectral studies demonstrated that thrombin titration by Na⁺ caused a positive ultraviolet difference spectrum with maxima at 281.5 and 288.5 nm (Δ?_288.5 = +1067). The K_m for Na⁺ was 2.3 × 10^?2m which agrees with the kinetically determined K_A for Na⁺. The results are consistent with Na⁺ binding to thrombin causing a conformational change in the active site. It is concluded that human α-thrombin is a monovalent cation-activated enzyme.     

Evidence that expression of SpαI/65 hereditary elliptocytosis is compounded by a genetic factor that is linked to the homologous α-spectrin allele

Summary Many cases of hereditary elliptocytosis (HE) result from mutated spectrin -chains. It has repeatedly been observed that the amount of a mutant -chain is different in various affected individuals, resulting in clinical pictures of variable severity. The different levels are thought to result from different percentages of the spectrin allele in trans. Such percentages, in turn, could be under genetic control. We tested this hypothesis in a large Algerian family with Sp^I/65 HE. In an informative sibship, we found three persons with a distinctly high level of expression of the Sp^I/65 variant, suggesting the existence, in trans, of a low percentage -allele. The -spectrin gene haplotype associated with the latter was constantly – +–, based on the XbaI, PvuII and MspI polymorphic sites. In contrast, a basal level of expression of the Sp^I/65 variant in the same sibship indicated, in trans, the existence of a normal percentage -allele. The haplotype corresponding to this other -allele was + – +. Study of another generation of the family showed, however, that the – + – haplotype could also be linked to a normal percentage -allele. These results are consistent with the view that the expression level of ^I/65 spectrin (and of other types of -variants) is compounded by a genetic factor that is linked to the normal -allele in trans. The low percentage allele itself remains silent in the simple heterozygous state.     

Structural Analysis of Free N-Glycans in α-Glucosidase Mutants of Saccharomyces cerevisiae: Lack of the Evidence for the Occurrence of Catabolic α-Glucosidase Acting on the N-Glycans

Saccharomyces cerevisiae produces two different α-glucosidases, Glucosidase 1 (Gls1) and Glucosidase 2 (Gls2), which are responsible for the removal of the glucose molecules from N-glycans (Glc₃Man₉GlcNAc₂) of glycoproteins in the endoplasmic reticulum. Whether any additional α-glucosidases playing a role in catabolizing the glucosylated N-glycans are produced by this yeast, however, remains unknown. We report herein on a search for additional α-glucosidases in S. cerevisiae. To this end, the precise structures of cytosolic free N-glycans (FNGs), mainly derived from the peptide:N-glycanase (Png1) mediated deglycosylation of N-glycoproteins were analyzed in the endoplasmic reticulum α-glucosidase-deficient mutants. 12 new glucosylated FNG structures were successfully identified through 2-dimentional HPLC analysis. On the other hand, non-glucosylated FNGs were not detected at all under any culture conditions. It can therefore be safely concluded that no catabolic α-glucosidases acting on N-glycans are produced by this yeast.     

Evidence that the conformational stability of ‘aged’ organophosphate-inhibited cholinesterase is altered

In order to determine whether a structural modification at the active center of cholinesterase may alter the conformational stability of the enzyme we compared the urea-induced unfolding of the tetrameric form of non-inhibited and irreversibly inhibited human plasma cholinesterase (acylcholine acylhydrolase, EC 3.1.1.8). We studied enzyme inhibited by methanesulfonyl fluoride, diisopropylfluorophosphonate (DFP) and racemic soman. DFP- and soman-inhibited cholinesterases are converted spontaneously into non-reactivable forms called ‘aged’ enzymes through a process involving dealkylation of the bound organophosphate residue. The unfolding was followed by transverse urea-gradient polyacrylamide electrophoresis at various temperatures ranging from 0 to 60°C. Unfolding of cholinesterase appears to be a complex process. The denaturation patterns showed that partially unfolded states are thermodynamically unstable, but that several intermediates are involved; the lifetime of these depends on the temperature at which electrophoreses are carried out. Cholinesterase inhibited by methanesulfonyl fluoride behaved like the non-inhibited enzyme. On the other hand, small but significant differences in stability between non-inhibited and aged enzymes were observed. Whatever the temperature, the urea concentration at the mid-point of transition was always greater for aged enzyme than for the non-inhibited enzyme. In addition, aged enzymes showed more complex denaturation patterns at the lower temperatures (under 20°C). These findings suggest that the overall stability of aged-cholinesterases is slightly increased as compared with the stability of non-inhibited or methanesulfonyl fluoride-inhibited enzymes. The denaturation pattern obtained at 0°C for soman-inhibited cholinesterase under non-aging conditions (inhibition at 0°C, pH 10.7) was similar to that of non-inhibited enzyme at this temperature, although splitting in two of the denaturation curve over the transition zone reflects the heterogeneity of soman-inhibited enzyme. The slight difference in denaturation behavior between these species may be due to stereoisomerism in soman. The differences in electrophoretic behavior and apparent stability observed between non-inhibited and aged enzymes were interpreted as the result of a conformational change induced by the dealkylation reaction of enzyme-inhibitor conjugates.     

Actions of terazosin and its enantiomers at subtypes of α- and α2-Adrenoceptors in vitro

Abstract

Terazosin and its enantiomers, antagonists of α1-adrenoceptors, were studied in radioligand binding and functional assays to determine relative potencies at subtypes of α1- and α2-adrenoceptors in vitro. The racemic compound and its enantiomers showed high and apparently equal affinity for subtypes of α1-adrenoceptors with K values in the low nanomolar range, and showed potent antagonism of α1-adrenoceptors in isolated tissues, with the enantiomers approximately equipotent to the racemate at each α1-adrenoceptor subtype. At α2b sites, R(+) terazosin bound less potently than either the S(-) enantiomer or racemate. R(+) terazosin was also less potent than the S(-) enantiomer or the racemate at rat atrial α2B receptors. These agents were not significantly different in their potencies at α2a or α2A sites. Since the high affinity for α2B sites of quinazoline-type α-adrenoceptor antagonists has been used to differentiate α2-adrenoceptor subtypes, the low affinity of R(+) terazosin for these sites was unexpected. Because terazosin or its enantiomers are approximately equipotent at α1 -adrenoceptor subtypes, the lower potency of R(+) terazosin at α2B receptors indicates a somewhat greater selectivity for α1- compared to α2B adrenoceptor subtypes. The possible pharmacological significance of this observation is discussed.     

Distinct Mechanisms Underlying α1-Adrenoceptor and P2x Purinoceptor Operated ATP Release and Contraction in the Guinea-Pig Vas Deferens

The temperature-dependence of ATP release and contraction response evoked by different agonists were investigated in superfused guinea-pig vas deferens. -Adrenoceptor agonists, i.e. noradrenaline (300 M), and -methyl-noradrenaline (300 M), increased the basal ATP outflow, measured by the luciferin-luciferase assay, and induced biphasic contractile response. Cooling the bath temperature to 12°C almost completely inhibited ATP release and twitch contraction evoked by -adrenoceptor agonists, whereas the phasic contraction remained unaffected. In contrast, twitch contraction and subsequent ATP release induced by ,-methylene-ATP, a selective P2 receptor agonist (100 M), was not reduced by low temperature. The ectoATPase activity, measured by HPLC technique was not significantly different at 37°C and 12°C. Nifedipine (1 M), the voltage sensitive Ca²⁺ channel blocker eliminated ,-methylene-ATP evoked twitch contraction but not ATP release. In conclusion, -adrenoceptor and P2 receptor agonists utilize distinct mechanisms to elicit ATP release and contraction: -adrenoceptor-mediated ATP release and contraction is temperature-dependent, indicating the involvement of a carrier-mediated process in it, whereas P2x purinoceptor evoked ATP release and twitch is mediated by a different mechanism.     

Evidence that α-dihydrograyanotoxin II does not bind to the sodium gate

Summary The basis for the ability of -dihydrograyanotoxin II (-2HG-II) to promote Na⁺ conductance in axons was sought. The apparent binding of tritiated -2HG-II to neural and other preparations was studied, using equilibrium dialysis, with lobster axon membranes,Torpedo electroplax, housefly head, and rat brain, liver and kidney. In every case the binding was nonsaturating and was suggested to involve nonspecific partitioning into the tissue. Supporting evidence was the similarity of extent of binding in all tissues and its relative insensitivity to neuropharmacological agents. -2HG-II did not affect the Na⁺ conductance of phospholipid bilayers, nor did it permit transport of²²Na into a bulk organic phase. It was concluded that -2HG-II did not bind to the sodium gate, but possibly to a sodium permease present at a frequency of less than one per ² of cell membrane.     

A Ca-independent α-amylase that is active and stable at low pH from the Bacillus sp. KR-8104

Bacillus sp. KR-8104 was selected from a set of 18 bacteria strains isolated from soil samples and screened for production of amylase. The maximum productivity obtained at pH 5–6 and 60–65 h after cultivation in production medium. New extracellular Ca-independent α-amylase was highly purified using ion exchange and hydrophobic interaction chromatography, which showed a single band with an apparent molecular weight of 59 kDa by SDS-PAGE. This enzyme is active in a wide pH range with its maximum activity at low pH values (4.0–6.0) and has the 90% of its maximum activity at pH 3.5. The α-amylase is optimally active at 75–80 °C. The presence or absence of Ca²⁺ and EDTA did not affect enzyme activity and thermal stability.     

Vaccination against shigellosis: is it the path that is difficult or is it the difficult that is the path?

Following several decades of research, there is not yet a convincing vaccine against shigellosis. It is still difficult, in spite of the breadth of strategies (i.e. live attenuated oral, killed oral, subunit parenteral) to select an optimal option. Two approaches are clearly emerging: (i) live attenuated deletion mutants based on rational selection of genes that are key in the pathogenic process, and (ii) conjugated detoxified polysaccharide parenteral vaccines, or more recently conjugated synthetic carbohydrates. Some of these approaches have already undergone phase I and II clinical trials with promising results, but important issues have also emerged, particularly the discrepancy between colonization and immunogenic potential of live attenuated vaccine candidates depending upon the population concerned (i.e. non endemic vs. endemic areas). Efforts are needed to definitely establish the proof of concept of these approaches, and thus the need for clinical trials which should also soon explore the possibility to associate different serotypes, in response to serotype specific protection against shigellosis. More basic research is also required to improve what we can still consider as first-generation vaccines, and to explore possible new paradigms including the search for cross-protective antigens.     

Prejunctional α2-Adrenoceptors and Peroxide-Induced Potentiation of Norepinephrine Release from the Bovine Iris

Peroxides can enhance field-stimulated [³H]norepinephrine ([³H]NE) release in isolated irides from several mammalian species. In the present study, we investigated the role of prejunctional ₂-adrenoceptors in peroxide-induced potentiation of sympathetic neurotransmission in bovine isolated irides. Isolated hemi-irides were incubated in a Krebs buffered-solution containing [³H]NE and prepared for studies of neurotransmitter release using the superfusion method. ₂-Adrenoceptor agonists, oxymetazoline, UK-14304 and clonidine inhibited field-stimulated [³H]NE overflow without affecting basal tritium efflux. Pretreatment of tissues with H₂O₂ (300 M) had no effect on inhibition of evoked [³H]NE release caused by the ₂-adrenergic agonists. However, H₂O₂ (300 M) caused significant (P < 0.01) leftward shifts of excitatory concentration-response curves to yohimbine (10 nM–1 M). In contrast, yohimbine (1 M) did not prevent the enhancement of evoked [³H]NE overflow induced by H₂O₂ (300 M). In conclusion, excitatory effects of peroxides on sympathetic neurotransmission in bovine irides are not mediated by prejunctional ₂-adrenoceptors.     

Evidence of Native α-Synuclein Conformers in the Human Brain

α-Synuclein aggregation is central to the pathogenesis of several brain disorders. However, the native conformations and functions of this protein in the human brain are not precisely known. The native state of α-synuclein was probed by gel filtration coupled with native gradient gel separation, an array of antibodies with non-overlapping epitopes, and mass spectrometry. The existence of metastable conformers and stable monomer was revealed in the human brain.     

Evidence that αC region is origin of low modulus, high extensibility, and strain stiffening in fibrin fibers

Fibrin fibers form the structural scaffold of blood clots and perform the mechanical task of stemming blood flow. Several decades of investigation of fibrin fiber networks using macroscopic techniques have revealed remarkable mechanical properties. More recently, the microscopic origins of fibrin's mechanics have been probed through direct measurements on single fibrin fibers and individual fibrinogen molecules. Using a nanomanipulation system, we investigated the mechanical properties of individual fibrin fibers. The fibers were stretched with the atomic force microscope, and stress-versus-strain data was collected for fibers formed with and without ligation by the activated transglutaminase factor XIII (FXIIIa). We observed that ligation with FXIIIa nearly doubled the stiffness of the fibers. The stress-versus-strain behavior indicates that fibrin fibers exhibit properties similar to other elastomeric biopolymers. We propose a mechanical model that fits our observed force extension data, is consistent with the results of the ligation data, and suggests that the large observed extensibility in fibrin fibers is mediated by the natively unfolded regions of the molecule. Although some models attribute fibrin's force-versus-extension behavior to unfolding of structured regions within the monomer, our analysis argues that these models are inconsistent with the measured extensibility and elastic modulus.     

Thermodynamic Study of Iodocyanopindolol β-Adrenoceptors Interactions with Rat Lung and Cerebral Cortex

Abstract

(±)¹²⁵ I-cyanopindolol (±) I CYP) was used to characterize β-adrenoceptors on rat lung and cerebral cortex membranes. The affinity of (±) ICYP was higher for lung (K^d = 64.3 pM) at 37°C. The association reaction of (±) ICYP was faster with lung (k₊₁ = 1.52 × 10⁹ M^?1.min^?1) than with cerebral cortex β-adrenoceptors (k₊₁ = 1.75 × 10⁸ M^?1.min^?1). In both tissues, the dissociation reaction followed a biphasic process with a fast (t ½ = 15.4 min and 5.6 min for lung and cerebral cortex respectively) and a slow component (t ½ = 474 min and 255 min for lung and cerebral cortex respectively). The thermodynamic parameters for (±) ICYP - β-adrenoceptors binding have been determined from kinetics and equilibrium studies, for the two tissues, at several temperatures between 0° and 44° C. For lung and cerebral cortex, Arrhenius plots were linear with different energies of activation. Van't Hoff plot was not linear for lung and the standard enthalpy and entropy changes of (±) ICYP - β-adrenoceptors interaction decreased linearly with temperature : the binding occured with a negative heat capacity change (ΔCp° = -368.9 cal. moles^?1. K^?1) at 25° C. Thermodynamic and kinetic results show that binding of (±) ICYP to lung β-adrenoceptors could involve two successive equilibria with a conformational change of the β-adrenergic receptor.     

Evidence that the “waxy” protein of pea (Pisum sativum L.) is not the major starch-granule-bound starch synthase

The aim of this work was to identify the starch-granule-bound starch synthase of developing pea embryos. When starch-granule-bound proteins were solubilised by digestion of granules with α-amylase and fractionated on a Mono Q anion-exchange column, activity of starch synthase eluted as three peaks. The distribution of activity in fractions from the column coincided with that of a 77-kDa protein. An antibody to this protein inhibited starch-synthase activity both in solubilised, starch-granule-bound protein and on intact starch granules. Recoveries of activity through extraction, solubilisation and chromatography indicate that this protein is the major, if not the only, form of starch synthase on the starch granule. The major, 59-kDa protein of the pea starch granule is antigenically related to the product of thewaxy locus of potato, which has previously been identified as the starch-granule-bound starch synthase of the tuber. However, the distribution of the 59-kDa protein did not coincide with that of starch-synthase activity in fractions from the Mono Q column. An antibody to the 59-kDa protein did not inhibit starch-synthase activity. The results raise questions about the relationship between “waxy” proteins and starch-granule-bound starch synthases generally. I am grateful to my colleagues Kay Denyer, Ian Dry (CSIRO, Adelaide, Australia), Rob Ireland (Mount Allison University, New Brunswick, Canada), Cathie Martin and Steve Rawsthorne for useful discussions during the course of this work, Cliff Hedley for the gift of pea seeds, and Ian Bedford for preparing pea starch and gels of starch-granule-bound proteins. This work was supported by the Agriculture and Food Research Council via a grant-in-aid to the John Innes Institute.     

What is the shape of the distribution of protein conformations at equilibrium?

According to the thermodynamic hypothesis, the native state of proteins is that in which the free energy of the system is at its lowest, so that at normal temperature and pressure, proteins evolve to that state. We selected four proteins representative of each of the four classes, and for each protein make four simulations, one starting from the native structure and the other three starting from the structure obtained by threading the sequence of one protein onto the native backbone fold of the other three proteins. Because of their large conformational distances with respect to the native structure, the three alternative initial structures cannot be considered as local minima within the native ensemble of the corresponding protein. As expected, the initial native states are preserved in the .5?μs simulations performed here and validate the simulations. On the other hand, when the initial state is not native, an analysis of the trajectories does not reveal any evolution towards the native state, during that time. These results indicate that the distribution of protein conformations is multipeak shaped, so that apart from the peak corresponding to the native state, there are other peaks associated with average structures that are very different from the native and that can last as long as the native state.     

Evidence supporting the role of calpain in the α-processing of amyloid-β precursor protein

Amyloid plaques are a hallmark of the aging and senile dementia brains, yet their mechanism of origins has remained elusive. A central issue is the regulatory mechanism and identity of α-secretase, a protease responsible for α-processing of amyloid-β precursor protein (APP). A remarkable feature of this enzyme is its high sensitivity to a wide range of cellular stimulators, many of which are agonists for Ca(2+) signaling. This feature, together with previous work in our laboratory, has suggested that calpain, a Ca(2+)-dependent protease, plays a key role in APP α-processing. In this study we report that overexpression of the μ-calpain gene in HEK293 cells resulted in a 2.7-fold increase of the protein levels. Measurements of intracellular calpain enzymatic activity revealed that the calpain overexpressing cells displayed a prominent elevation of the activity compared to wild-type cells. When the cells were stimulated by nicotine, glutamate or phorbol 12,13-dibutylester, the activity increase was even more remarkable and sensitive to calpeptin, a calpain inhibitor. Meanwhile, APP secretion from the calpain overexpressing cells was robustly increased under both resting and stimulated conditions over wild-type cells. Furthermore, cell surface biotinylation experiments showed that μ-calpain was clearly detected among the cell surface proteins. These data together support our view that calpain should be a reasonable candidate for α-secretase for further study. This model is discussed with an interesting fact that three other deposited proteins (tau, spectrin and crystalline) are also the known substrates of calpain. Finally we discuss some current misconceptions in senile dementia research.