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PubMed1996

5-HT1D receptor agonists and human coronary artery reactivity in vitro: crossover comparisons of 5-HT and sumatriptan with rizatriptan and L-741,519.

1. Rizatriptan (MK-462, (N,N-dimethyl-2-[5-(1,2,4-triazol-1-ylmethyl)-1H-indol-3-yl] ethylamine)) and its structurally related analogue L-741,519 (N-methyl-4-[5-(1,2,4-triazol-4-yl)-1H-indol-3-yl]piperidine) are novel 5-HT1D-receptor agonists. Rizatriptan has shown efficacy as an anti-migraine agent in clinical trials. Since angiographic studies in patients have shown that sumatriptan (an established 5-HT1D-receptor agonist) can cause coronary artery vasoconstriction, we compared the effects of rizatriptan and L-741,519 with those of 5-HT and sumatriptan on endothelium-denuded segments of human coronary artery in vitro. 2. Coronary arteries were obtained from explanted hearts from patients undergoing cardiac transplantation (n = 16 viable arteries from 13 males, 3 females, aged 38-68 years) and arterial segments (5-6 mm in length) were mounted in organ baths for isometric tension recording. Each segment was first exposed to 45mM KCl and then to 5-HT (1 nM-100 microM). Concentration-effect curves to rizatriptan and sumatriptan (Study 1, n = 6 or 7 arteries) or sumatriptan and L-741,519 (Study 2, n = 8 arteries) were then performed in a consecutive and random manner. The response to repeated application of 5-HT was obtained in separate segments. 3. One artery showed severe atheroma and was not included in the analysis. ANOVA showed that 5-HT responsiveness varied significantly between arteries from different patients; but not between arterial segments from the same patient. Desensitization was seen consistently across all agonists but did not significantly affect inter-agonist comparisons. 4. There was graded effectiveness in the ability of the agonists to cause contraction with the rank order of Emax values being 5-HT >> sumatriptan > L-741,519 > rizatriptan. In terms of EC50 values, L-741,519 was significantly more potent than sumatriptan. 5. The present study (using a 'cross-over' experimental protocol) confirms our previous observation that rizatriptan is less effective than sumatriptan in causing contraction of human isolated coronary artery. Furthermore, it shows that the lower maximum contractile response to rizatriptan, compared with that of sumatriptan, is not merely the consequence of variability in response to 5-HT1D-receptor agonists between patients or between segments from the same artery.

Adult↗
PubMed2003

Identification of clinically relevant Trichosporon species.

A dichotomous identification key to pathogenic species of the basisiomycetous genus Trichosporon Behrend is provided. It is based on growth tests with carbon sources not traditionally used in yeast taxonomy, viz. uric acid, ethylamine, L-4-hydroxyproline, tyramine and L-phenylalanine as sources of carbon and nitrogen, and polygalacturonate, quinate, 4-ethylphenol, 2,3-dihydroxybenzoate and orcinol as sole carbon sources. Of the standard growth tests, assimilation of L-rhamnose and the maximum growth temperature proved to be useful. In addition to medically relevant species, other species able to grow at 37 degrees C were treated as well.

Animals↗
PubMed1994

Striatal transporter for dopamine: catechol structure-activity studies and susceptibility to chemical modification.

The apparent second-order association rate constant of dopamine binding to the striatal transporter (approximately 1 x 10(6) M-1 s-1) as well as the transporter turnover number (approximately 1.5 s-1) was estimated using rotating disk electrode voltammetry to monitor apparent zero trans entry of dopamine into striatal suspensions. The substrate specificity of the transporter was also assessed using catechol derivatives. Dopamine and norepinephrine were transported, whereas epinephrine and the acidic metabolites of dopamine were not transported. The metabolite, 3-methoxytyramine, was transported with a Km seven times greater than and a Vmax close to that for dopamine. 4-Methoxytyramine was transported more facilely than the 3-methoxy derivative. N-Alkylation of the amine side chain of dopamine reduced transport dramatically. 4-Ethylcatechol and 3,4-dihydroxybenzylamine were transported with velocities 79 and 91% less than that for dopamine, respectively. The rigid analogue 6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene was transported with a greater velocity than the 5,7-dihydroxy derivative. Finally, the apparent Km values for 4-ethylcatechol, 1-amino-2-phenylethane, tyramine, and m-tyramine as cosubstrates with dopamine were 1.1, 11, 17, and 2.6 microM, respectively. Pretreatments of striatal suspensions with chloroethylnorapomorphine, N-ethylmaleimide, Hg2+, 4,5-dihydroxy-4,5-dioxo-1H-pyrrolo[2,3-f] quinoline-2,7,9-tricarboxylic acid (a redox modulator of receptors in neuronal as well as other tissues), and neuraminidase reduced the velocity of transport of dopamine, whereas N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline had no effect. Thus, the dopamine transporter requires an intact catechol with a primary ethylamine side chain for optimal activity relative to shorter side chain derivatives (side chains longer than two carbons were not tested), the 3-hydroxyl group of dopamine is the more critical hydroxyl group, and the beta rotamer of the extended conformation of dopamine is transported preferentially. The catechol appears to mediate the recognition of the substrate, whereas the amine side chain apparently facilitates the conformational change of the transporter that results in movement of dopamine into or across the membrane.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2003

Effect of two new PBN-derived phosphorylated nitrones against postischaemic ventricular dysrhythmias.

Spin traps might exert antioxidant cardioprotective effects during myocardial ischaemia-reperfusion where free radicals are thought to be responsible for the occurrence of reperfusion injury. The aim of our study was to investigate the effects of two new alpha-phenyl N-tert-butylnitrone (PBN)-derived beta-phosphorylated nitrones: 2-N-oxy-N-[benzylidène amino] diéthyl propyl-2-phosphate (PPN) and 1-diethoxyphosphoryl-1-methyl-N-[(1-oxido-pyridin-1-ium-4-yl) methylidene] ethylamine N-oxide (4-PyOPN) compared with PBN on (1) the evolution of cardiovascular parameters and (2) the postischaemic recovery. Anaesthetized rats were injected with 120 micro mol/kg of the nitrones or 14 micro mol/kg of amiodarone, used as a reference antidysrhythmic drug. Ischaemia was induced in vivo through ligation of the left anterior descending coronary artery for 5 min followed by 15 min of reperfusion after release. Cardiovascular parameters and occurrence of ventricular premature beats (VPB), ventricular tachycardia (VT) and fibrillation (VF) were recorded throughout the experiment. Under nonischaemic conditions, none of the three spin traps was shown to modify cardiovascular parameters during the 25-min measurement period. Solvent-treated (NaCl 0.9%) animals challenged with ischaemia-reperfusion exhibited 39 +/- 10 VPB, 156 +/- 39 s of VT and 60% mortality caused by sustained VF. Nitrones improved slightly postischaemic recovery, reducing the occurrence of VF and mortality to 33% whereas amiodarone injection totally suppressed rhythm disturbances and mortality. Our study has shown only limited antidysrhythmic cardioprotective effects of PBN-derived beta-phosphorylated nitrones during reperfusion after a regional myocardial ischaemia but also minor antioxidant properties of these spin trapping agents.

Amiodarone↗
PubMed1979

The effect of (6-D-(O-TERT-B)-Ser)-gonadoliberin-(1--9) nonapeptide-ethylamide on gonadotropin release in prepubertal boys.

(6-D-(o-tert-B)-Ser)-gonadoliberin-(1--9) nonapeptide-ethylamine, (HOE 766), a highly active LH-RH analogue, was studied with regard to its effects on the release of follicle stimulating hormone (FSH) and luteinizing hormone (LH) in 29 prepubertal boys given different doses (1 microgram, 2,5 microgram, 5 microgram or 7,5 microgram respectively). The effect of HOE 766 is dose dependent for FSH, but not for LH. There are important differences in the reaction of these young subjects from those of adults. Whereas LH levels barely rose, FSH secretion was superior to that seen in adults.

Child↗
PubMed2002

Syntheses and stability studies of some Mannich bases of acetophenones and evaluation of their cytotoxicity against Jurkat cells.

Mannich bases, namely 1-aryl-3-dimethylamino-1-propanone hydrochlorides (Ia-f) as mono-Mannich bases (series I), bis(beta-aroylethyl)ethylamine hydrochlorides (IIa, IIb, IId, IIe) as bis-Mannich bases (series II), 3-aroyl-4-aryl-1-ethyl-4-piperidinol hydrochlorides (series III), which are structural isomers of bis derivatives and some representative quaternary salts (Ig, IIIf, IIIg), were synthesized to investigate the effect of chemical structure and ring substituents on cytotoxic activity in Jurkat cells. Stability studies of some representative compounds have also been realised. Compounds IIb, IId, IIe, and IIIe were reported for the first time. Id-g, IIa, IId, IIe, IIIf,g were 1.25-6.55 times more potent than 5-fluorouracil (CAS 51-21-8). However, the cytotoxic activity of the most potent compounds. Ig and IIIf, were one fifth of that of melphalan (CAS 148-82-3). The formation of compound IV during the stability studies of Ig, IIa, and IIIf suggested that they may be thiol alkylators. Bis-Mannich base IIa in nonsubstituted derivatives, piperidinol derivative IIIb in methyl substituted compounds, mono derivative Id in chloro substituted compounds were the most potent compounds when the cytotoxicity of the compound series which have the same substituents in benzene ring are compared. Replacement of the benzene with thiophene improved the cytotoxicity in both series I and II. Quaternization procedure also increased the cytotoxicity in both series I and III. Quaternary derivatives seem to be promising compounds for further studies to develop new anticancer drugs.

Acetophenones↗
PubMed2002

Antimicrobial evaluation of some Mannich bases of acetophenones and representative quaternary derivatives.

1-Aryl-3-dimethylamino-1-propanone hydrochlorides Ia-f (series I) as mono-Mannich bases bis(beta-aroylethyl)ethylamine hydrochlorides IIa, IIb, IId, IIe (series II) as bis-Mannich bases, 3-aroyl-4-aryl-1-ethyl-4-piperidinol hydrochlorides (structural isomer of bis derivatives IIIa-e, series III), and some of their representative quaternary salts (Ig, IIIf, IIIg) were synthesized. Antimicrobial activities of the compounds were evaluated against some bacteria and fungi. Series I and III showed antimicrobial activity against gram positive bacteria. All series demonstrated activity against fungi, however, they generally did not affect gram negative bacteria at the concentration range tested (2-64 micrograms/ml). Quaternisation procedure improved the bioactivity in compound IIIa for antibacterial activity and in compounds IIIa and IIIb for antifungal activity against Trichophyton rubrum and Mycosporium canis. There was no relationship between Hammett values of the aryl substituents and bioactivities in series III. The mono-Mannich bases of series I had better antimicrobial activities than bis-Mannich bases of series II. Compounds Ia, If, IIId had equal and compounds If and IIIf had higher antibacterial activities compared to the reference drug, streptomycin (CAS 57-92-1), against various gram positive bacteria. On the other hand, compounds Ia, IIIa, IIIc, IIIe, IIIf, and IIIg had equal and If, IIId, IIIf, IIIg had higher antifungal activity compared to the reference drug, amphotericin-B (CAS 1397-89-3), against various fungi. To conclude, the compounds of series III, having both marked antifungal and antibacterial activities, may serve as candidate compounds for further studies. Especially compound IIIf may serve as a model compound to develop new agents against dermatophytes.

Acetophenones↗
PubMed2006

Permanent electric dipole moments of four tryptamine conformers in the gas phase: a new diagnostic of structure and dynamics.

Rotationally resolved electronic spectroscopy in the gas phase, in the absence and presence of an applied electric field, has been used to determine the charge distribution of a cross section of the energy landscape of tryptamine (TRA). We report the magnitude and direction of the permanent electric dipole moments of the four TRA conformers GPyout, GPyup, GPhup, and Antiup in their S0 and S1 electronic states. Each dipole moment is unique, providing a powerful new tool for the conformational analysis of biomolecules in the gas phase. A comparison of the results for the different conformers of TRA reveals that the position and orientation of the ethylamine side chain play a major role in determining both the permanent and induced electric dipole moments of the different species in both electronic states.

Journal Article↗
PubMed1971

Specificity of the antibody receptor site to D-lysergamide: model of a physiological receptor for lysergic acid diethylamide.

Antibodies to D-lysergic acid have been produced in rabbits and guinea pigs and a radioimmunoassay for the hapten was developed. The specificity of this lysergamide-antilysergamide reaction was determined by competitive binding with unlabeled lysergic acid diethylamide (LSD), psychotomimetic drugs, neurotransmitters, and other compounds with diverse structures. LSD and several related ergot alkaloids were potent competitors, three to seven times more potent than lysergic acid itself. The N,N-dimethyl derivatives of several compounds, including tryptamine, 5-hydroxytryptamine, 4-hydroxytryptamine, 5-methoxytryptamine, tyramine, and mescaline, were only about ten times less effective than lysergic acid, even though these compounds lack some of the ring systems of lysergic acid. The pattern of inhibition by related compounds with various substituents suggests that the antibody receptor site recognizes structural features resembling the LSD molecule. In particular, the aromatic nucleus and the dimethylated ethylamine side chain in phenylethylamine and tryptamine derivatives may assume in solution a conformation resembling ring A and the methylated nitrogen in ring C of LSD. Among the tryptamine derivatives, a large percentage of the most potent competitors are also psychotomimetic compounds.

Amines↗
PubMed1982

T-cell suppression and contrasuppression induced by histamine H2 and H1 receptor agonists, respectively.

The intensity of Lyl+T helper and delayed type hypersensitivity effector cell activities is governed, in part, by an interplay between two classes of immunoregulatory T cells: suppressor cells and contrasuppressor cells. We asked whether histamine, at concentrations and duration of exposure that we calculated might be achieved at local sites of inflammation, could activate either or both of these classes of regulatory cells in vitro. To answer this question we used spleen cells from mice treated in vivo with the toleragen trinitrobenzenesulfonic acid as regulators of in vitro generation of primary anti-trinitrophenyl self-cytotoxic T lymphocytes. Under the conditions used, these spleen cells had no major regulatory effects. However, if these cells were preincubated with histamine at 0.1 mM for 30-60 min, suppressor activity was induced, but this occurred inconsistently and with nonstoichiometric results. The use of synthetic histamine agonists revealed that histamine may activate both suppressor and contrasuppressor cell subsets. A histamine H1 receptor agonist [2-(2-pyridyl)-ethylamine dihydrochloride] had a propensity to activate contrasuppression, whereas an H2 receptor agonist (dimaprit) tended to activate suppressor cells. Thus, histamine may have opposing actions that obscure suppression. This duality was shown by treatment of pyridylethylamine-induced contrasuppressor cells with complement and anti-I-J antibody that kills contrasuppressor cells. This treatment revealed a high level of suppressor cell activity that was not expressed until the opposing contrasuppressor cells were removed. Because histamine is released at local sites of delayed type hypersensitivity, these results indicate that histamine may serve as an inducer of microenvironmental immunomodulation by activating regulatory T cells at sites where immune responses are taking place.

Animals↗
PubMed1992

Short-chain analogs of luteinizing hormone-releasing hormone containing cytotoxic moieties.

Five hexapeptide and heptapeptide analogs of luteinizing hormone-releasing hormone (LH-RH) were synthesized for use as carriers for cytotoxic compounds. These short analogs were expected to enhance target selectivity of the antineoplastic agents linked to them. Native LH-RH-(3-9) and LH-RH-(4-9) containing D-lysine and D-ornithine at position 6 were amidated with ethylamine and acylated on the N terminus. The receptor-binding affinity of one hexapeptide carrier AJ-41 (Ac-Ser-Tyr-D-Lys-Leu-Arg-Pro-NH-Et) to human breast cancer cell membranes was similar to that of [D-Trp6]LH-RH. Alkylating nitrogen mustards (melphalan, Ac-melphalan), anthraquinone derivatives including anticancer antibiotic doxorubicin, antimetabolite (methotrexate), and cisplatin-like platinum complex were linked to these peptides through their omega-amino group at position 6. The hybrid molecules showed no LH-RH agonistic activity in vitro and in vivo but had nontypical antagonistic effects on pituitary cells in vitro at the doses tested. These analogs showed a wide range of receptor-binding affinities to rat pituitaries and cell membranes of human breast cancer and rat Dunning prostate cancer. Several of these conjugates exerted some cytotoxic effects on MCF-7 breast cancer cell line.

Amino Acid Sequence↗
PubMed1995

Detection and characterization of a transport system mediating cysteamine entry into human fibroblast lysosomes. Specificity for aminoethylthiol and aminoethylsulfide derivatives.

The uptake of [3H]cysteamine by Percoll-purified human fibroblast lysosomes was investigated to determine whether lysosomes contain a transport system recognizing cysteamine. Lysosomal cysteamine uptake is a Na(+)-independent process which rapidly attains a steady state within 1 min at pH 7.0 and 37 degrees C. A biphasic Arrhenius plot is observed for cysteamine uptake, giving a Q10 of 2.2 from 17 to 26 degrees C and a Q10 of 1.2 from 27 to 35 degrees C. The rate of lysosomal cysteamine uptake is maximal at pH 8.2, half-maximal at pH 6.8, and declines approximately 50-fold from the maximum to show very little transport at pH 5.0. Cysteamine uptake into fibroblast lysosomes displays complete saturability with a Km of 0.88 mM and Vmax of 1410 pmol of beta-N-acetylhexosaminidase/min at pH 7.0 and 37 degrees C. Analog inhibition studies demonstrated that all analogs recognized thus far by the cysteamine carrier are either aminothiols or aminosulfides and contain an amino group and sulfur atom separated by a carbon chain, 2 carbon atoms in length. The Ki constants for these analogs as competitive inhibitors of lysosomal cysteamine uptake are 2-(ethylthio)ethylamine (0.64 mM), 1-amino-2-methyl-2-propanethiol (0.74 mM), 2-dimethylaminoethanethiol (0.87 mM), thiocholine (1.6 mM), and bis(2-aminoethyl)sulfide (4.9 mM). L-Cysteine, D-penicillamine, and analogs lacking either a sulfur atom or amino group are not recognized by the cysteamine carrier including ethanolamine, choline, taurine, beta-mercaptoethanol, ethylenediamine, cadaverine, spermine, spermidine, histamine, dopamine, and 3-hydroxytyramine. In a cystine-depletion assay, a 2-h exposure of cystinotic fibroblasts to 1 mM 1-amino-2-methyl-2-propanethiol lowers cell cystine levels to the same low level obtained with cysteamine. Thus, all four aminothiols, known to deplete cystinotic fibroblasts of their accumulated cystine, are recognized as substrates by the lysosomal cysteamine carrier, suggesting the importance of this transporter in the delivery of aminothiols to the lysosomal compartment.

Biological Transport↗
PubMed1997

Critical role of asparagine 1065 of human alpha2-macroglobulin in formation and reactivity of the thiol ester.

It has been shown that the relative reaction preference of the C4 thiol ester toward oxygen and nitrogen nucleophiles upon activation by proteinase depends on whether residue 1106 is aspartate or histidine (Dodds, A. W., Ren, X.-D., Willis, A. C., and Law, S. K. A. (1996) Nature 379, 177-179). To determine if the equivalent residue in the related thiol ester-containing protein human alpha2-macroglobulin (alpha2M), asparagine 1065, plays a similar role, we have expressed and characterized four alpha2M variants in which this asparagine has been replaced by aspartate, alanine, histidine, or lysine. The change from asparagine resulted in an altered ability to form the thiol ester. This ranged from failure to form the thiol ester (Asn --> Asp) to a maximum extent of formation of about 50% (Asn --> Ala). For the three variants that were able to form the thiol ester, the rates of thiol ester cleavage by a given amine were found to be different from one another and slower in nearly all cases than plasma alpha2M, but with the same relative reactivity of methylamine > ethylamine > ammonia. The rate of conformational change that follows cleavage of thiol esters in a functional half-molecule was also found to differ between the variants and to be slower than plasma alpha2M. TNS emission spectra indicated that the conformations of the transformed variants differed measurably from transformed plasma alpha2M. These findings suggest that residue 1065 plays a critical role in human alpha2M, for formation of the thiol ester, for its subsequent reaction with nucleophiles, and for the conformational change induced by this reaction. By analogy with C4, where this residue influences the nucleophile preference through direct interaction with the thiol ester, residue 1065 in alpha2M is expected to be located in or very close to the thiol ester region in alpha2M.

Animals↗
PubMed1998

Cysteine scanning mutagenesis of the segment between putative transmembrane helices IV and V of the high affinity Na+/Glucose cotransporter SGLT1. Evidence that this region participates in the Na+ and voltage dependence of the transporter.

Site-directed mutagenesis and chemical modification of specific cysteine amino acid side chains by methanethiosulfonate (MTS) derivatives were combined to elucidate structure/function relationships of the cloned rabbit Na+/glucose cotransporter, SGLT1. Each amino acid in the region (residues 162-173) between putative transmembrane helices IV and V of SGLT1 was replaced individually with Cys. Mutant proteins were expressed in Xenopus laevis oocytes and studied using the two-electrode voltage clamp method. At certain key positions, Cys substitution resulted in 1) a change in the apparent affinity for sugar, 2) an alteration in the voltage dependence of the transient currents, and 3) a sensitivity to inhibition by either the ethylamine (MTSEA) or the ethylsulfonate MTS derivatives. For the three Cys mutants inhibited by MTSEA (F163C, A166C, and L173C), inhibition of steady state transport is related to changes in membrane potential-dependent transitions within the Na+/glucose transport cycle. MTSEA shifted the transient currents of these Cys mutants toward more negative membrane potentials (DeltaV0. 5 = -18 mV for F163C and A166C, -12 mV for L173C). When the mutations were combined to produce double and triple Cys mutants, the degree to which the transient currents were shifted along the membrane potential axis by MTSEA correlated with the number of cysteines. In this way it was possible to manipulate the voltage dependence of the transient currents over a range spanning 91 mV. Examination of the Na+ dependence of the transient currents indicates that a 91-mV shift is equivalent to that caused by a 10-fold reduction in the external Na+ concentration. We conclude that this region has a role in determining the Na+ binding- and voltage-sensing properties of SGLT1 and that it forms an alpha-helix with one surface possibly lining a Na+ pore within SGLT1.

Amino Acid Sequence↗
PubMed2000

Substrate specificity and inhibition studies of human serotonin N-acetyltransferase.

Arylalkylamine N-acetyltransferase (AANAT) catalyzes the reaction of serotonin with acetyl-CoA to form N-acetylserotonin and plays a major role in the regulation of the melatonin circadian rhythm in vertebrates. In the present study, the human cloned enzyme has been expressed in bacteria, purified, cleaved, and characterized. The specificity of the human enzyme toward substrates (natural as well as synthetic arylethylamines) and cosubstrates (essentially acyl homologs of acetyl-CoA) has been investigated. Peptide combinatorial libraries of tri-, tetra-, and pentapeptides with various amino acid compositions were also screened as potential sources of inhibitors. We report the findings of several peptides with low micromolar inhibitory potency. For activity measurement as well as for specificity studies, an original and rapid method of analysis was developed. The assay was based on the separation and detection of N-[(3)H]acetylarylethylamine formed from various arylethylamines and tritiated acetyl-CoA, by means of high performance liquid chromatography with radiochemical detection. The assay proved to be robust and flexible, could accommodate the use of numerous synthetic substrates, and was successfully used throughout this study. We also screened a large number of pharmacological bioamines among which only one, tranylcypromine, behaved as a substrate. The synthesis and survey of simple arylethylamines also showed that AANAT has a large recognition pattern, including compounds as different as phenyl-, naphthyl-, benzothienyl-, or benzofuranyl-ethylamine derivatives. An extensive enzymatic study allowed us to pinpoint the amino acid residue of the pentapeptide inhibitor, S 34461, which interacts with the cosubstrate-binding site area, in agreement with an in silico study based on the available coordinates of the hAANAT crystal.

Acyl Coenzyme A↗
PubMed2003

Critical role of Lys212 and Tyr140 in porcine NADP-dependent isocitrate dehydrogenase.

Lys212 and Tyr140 are close to the enzyme-bound isocitrate in the recently determined crystal structure of porcine NADP-specific isocitrate dehydrogenase (Ceccarelli, C., Grodsky, N. B., Ariyaratne, N., Colman, R. F., and Bahnson, B. J. (2002) J. Biol. Chem. 277, 43454-43462). We have constructed mutant enzymes in which Lys212 is replaced by Gln, Tyr, and Arg, and Tyr140 is replaced by Phe, Thr, Glu, and Lys. Wild type and mutant enzymes were each expressed in Escherichia coli and purified to homogeneity. At pH 7.4, the specific activity is decreased in K212Q, K212Y, and K212R, respectively, to 0.01-9% of wild type. The most striking change is in the pH-V(max) curves. Wild type depends on the deprotonated form of a group of pKaes 5.7, whereas this pKaes is increased to 7.4 in neutral K212Q and to 8.3 in K212Y. In contrast, the positive K212R has a pKaes of 5.9. These results indicate that (by electrostatic repulsion) a positively charged residue at position 212 lowers the pK of the nearby ionizable group in the enzyme-substrate complex. Lys212 may also stabilize the carbanion formed initially on substrate decarboxylation. The Tyr140 mutants have specific activities at pH 7.4 that are reduced to 0.2-0.5% of those of wild type, whereas their Km values for isocitrate and NADP are not increased. Most notable are the altered pH-V(max) profiles. V(max) is constant from pH 5.3 to 8 for Y140F and Y140T and increases as pH is decreased for Y140E and Y140K. These results suggest that in wild type enzyme, Tyr140 is the general acid that protonates the substrate after decarboxylation and that the carboxyl and ammonium forms of Y140E and Y140K provide partial substitutes. Relative to wild type, the Y140T enzyme is specifically activated 106-fold by exogenous addition of acetic acid and 88-fold by added phenol; and the K212Q enzyme is activated 4-fold by added ethylamine. These chemical rescue experiments support the conclusion that Tyr140 and Lys212 are required for the catalytic activity of porcine NADP-dependent isocitrate dehydrogenase.

Animals↗
PubMed2003

Methanethiosulfonate ethylammonium block of amine currents through the ryanodine receptor reveals single pore architecture.

The homotetrameric structure of the ryanodine-sensitive intracellular calcium (Ca2+) release channel (ryanodine receptor (RyR)) suggests that the four RyR subunits either combine to form a single pore or that each RyR subunit is an independently conducting pathway. Previously we showed that methanethiosulfonate ethylammonium (MTSEA+) covalently modifies the RyR to reduce current amplitudes in a time-dependent and stepwise manner. To ascertain the number of functionally conducting pores in the RyR, two approaches were combined: modification of the receptor by MTSEA+ and the use of different sized current carriers. Previous reports (Tinker, A., and Williams, A. J. (1993) J. Gen. Physiol. 102, 1107-1129) have shown that the organic cations methylamine, dimethylamine, ethylamine, and trimethylamine are permeant through the RyR but with reduced current amplitude depending upon the diameter of the respective amine. Experiments using the thiol reagent MTSEA+ to modify the channel protein showed that the current amplitudes decrease in steps leading to complete block of the channel when cesium (Cs+) is the current carrier. MTSEA+ modification decreased the number of channel substates as the diameter of the current carrier increased. Comparison of the degree of inhibition of MTSEA+-modified currents allows for differentiation between the two models for channel architecture. These results demonstrate that the conduction pathway for the RyR is comprised of a single central pore.

Amines↗
PubMedDate not supplied

Optical resolution of 5-alkyl-delta-valerolactones and synthesis of optically active 5-fluoroalkanols.

Optical resolutions of 5-alkyl-delta-valerolactones were carried out by derivatization to the diastereomeric amides, in which (R)-(+)-1-(1-naphthyl)ethylamine or (S)-(-)-1-phenylethylamine were used as resolving agents. Optically active 5-fluoroalkanols, useful intermediates for fluorinated ferroelectric liquid crystals, were derived from the resolved lactones in four steps without racemization.

Journal Article↗