Distinct Loci Mediate the Direct and Indirect Actions of the Anesthetic Etomidate at GABAA Receptors

Abstract: Most general anesthetics produce two distinct actions at GABA_A receptors. Thus, these drugs augment GABA-gated chloride currents (referred to as an indirect action) and, at higher concentrations, elicit chloride currents in the absence of GABA (referred to as a direct action). Because a β subunit appears to be required for the direct action of intravenous anesthetics in recombinant GABA_A receptors, site-directed mutagenesis of the β3 subunit was performed to identify amino acid residues that are critical for this action. In HEK293 cells expressing a prototypical GABA_A receptor composed of α1β3γ2 subunits, mutation of amino acid 290 from Asn to Ser dramatically reduced both etomidate-induced chloride currents and its ability to stimulate [³H]flunitrazepam binding. By contrast, the ability of etomidate to augment GABA-gated chloride currents and GABA-enhanced [³H]flunitrazepam binding was retained. The demonstration that the direct, but not the indirect, actions of etomidate are dependent on β3(Asn²⁹⁰) indicates that the dual actions of this intravenous anesthetic at GABA_A receptors are mediated via distinct loci.     

Different structural stability and toxicity of PrP(ARR) and PrP(ARQ) sheep prion protein variants

The polymorphisms at amino acid residues 136, 154, and 171 in ovine prion protein (PrP) have been associated with different susceptibility to scrapie: animals expressing PrP^ARQ [PrP(Ala136/Arg154/Gln171)] show vulnerability, whereas those that express PrP^ARR [PrP(Ala136/Arg154/Arg171)] are resistant to scrapie. The aim of this study was to evaluate the in vitro toxic effects of PrP^ARR and PrP^ARQ variants in relation with their structural characteristics. We show that both peptides cause cell death inducing apoptosis but, unexpectedly, the scrapie resistant PrP^ARR form was more toxic than the scrapie susceptible PrP^ARQ variant. Moreover, the α-helical conformation of PrP^ARR was less stable than that of PrP^ARQ and the structural determinants responsible of these different conformational stabilities were characterized by spectroscopic analysis. We observed that PrP toxicity was inversely related to protein structural stability, being the unfolded conformation more toxic than the native one. However, the PrP^ARQ variant displays a higher propensity to form large aggregates than PrP^ARR. Interestingly, in the presence of small amounts of PrP^ARR, PrP^ARQ aggregability was reduced to levels similar to that of PrP^ARR. Thus, in contrast to PrP^ARR toxicity, scrapie transmissibility seems to reside in the more stable conformation of PrP^ARQ that allows the formation of large amyloid fibrils.     

Neuritogenic determinant of bovine P2 protein in peripheral nerve myelin

Abstract: Experimental allergic neuritis (EAN) is an experimentally produced demyelinating disease of peripheral nervous system. Several peptides of bovine P₂ protein were tested for neuritogenic activity in Lewis rats. The hexacosapeptide CiT4 (residues 53-78 of bovine P₂ protein) showed the highest neuritogenic activity among the peptides tested. The nonapeptide (residues 70-78) and the tridecapeptide (residues 66-78) were synthesized using the liquid phase peptide synthesis technique. The tridecapeptide showed mild, but definite activity in inducing EAN in the rats, while the nonapeptide was inactive. The localization of the neuritogenic determinant of bovine P₂ protein in Lewis rats is discussed.     

EXPRESSION OF A BEE-VENOM PHOSPHOLIPASE A2 FROM APIS CERANA CERANA IN ESCHERICHIA COLI

Abstract  The venomous phospholipase A₂ (AcPLA₂) coding reading region of the Chinese honeybee ( Apis cerana cerana ), which is composed of 405 bp encoding a mature glycosylated peptide with 134 amino residues was transformed into the expression vector pETblue-1. Then the recombinant vector was introduced into Escherichia coli Tuner (DE3) plac I for expression. Analysis result of SDS-PAGE showed that the expression products had a protein band of about 15kD. Detection of western blot using ant-European honeybee ( Apis mellifera ) phospholipase A₂ (AmPLA₂) polyclonal serum as the first antibody showed that the expression products appeared a special blot same as the native AmPLA₂.The result demonstrated that the AcPLA₂ peptide had been expressed in E. coli and the AcPLA₂ has the similar antigenicity as the AmPLA₂.     

Regulation of the catalytic behaviour of L-form starch phosphorylase from sweet potato roots by proteolysis

Starch phosphorylase (SP) is an enzyme used for the reversible phosphorolysis of the α-glucan in plant cells. When compared to its isoform in an animal cell, glycogen phosphorylase, a peptide containing 78 amino acids (L78) is inserted in the centre of the low-affinity type starch phosphorylase (L-SP). We found that the amino acid sequence of L78 had several interesting features including the presence of a PEST region, which serves as a signal for rapid degradation. Indeed, most L-SP molecules isolated from mature sweet potato roots were nicked in the middle of a molecule, but still retained their tertiary or quaternary structures, as well as full catalytic activity. The nicking sites on the L78 were identified by amino acid sequencing of these peptides, which also enabled us to propose a proteolytic process for L-SP. Enzyme kinetic studies of L-SP in the direction of starch synthesis indicated that the K_m decreased during the proteolytic process when starch was used as the limiting substrate, but the K_m for the other substrate (Glc-1-P) increased. On the other hand, the maximum velocities (V_max) increased for both substrates. Mobility of the nicked L-SP was retarded on a native polyacrylamide gel containing soluble starch, indicating the increased affinity for starch. Results in this study suggested that L78 and its proteolytic modifications might play a regulatory role on the catalytic behaviour of L-SP in starch biosynthesis.     

Physical, immunological and kinetic properties of ribulose-1,5-bisphosphate carboxylase/oxygenase from fir (Abies alba) and spruce (Picea abies)

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) from fir ( Abies alba Mill.) and spruce ( Picea abies [L.] Karst.) needles was purified to homogeneity. The enzyme was isolated from crude extracts through quantitative precipitation in 40-55% and 40-60% (NH₄)₂SO₄ for fir and spruce. respectively, followed by linear sucrose gradient centrifugation. Using two dimensional gel electrophoresis, the isoelectric points were determined. For the large subunit (LSU) it was 6.7 for both species, and for the small subunit (SSU) it was 7.1 and 7.7 for fir and spruce, respectively. Very few differences in tryptic peptides and amino acid composition of Rubisco LSU were observed between fir and spruce. By contrast, marked differences characterized the same analyses for the Rubisco SSU of the two species. Moreover, substitution of residues was observed in the sequenced N-terminal region when comparing fir and spruce SSU. The Ouchterlony technique showed no immu-nochemical difference between Rubisco of fir and spruce when a rabbit antiserum to spinach Rubisco was used. The Eadie-Hofstee plots of carboxylase activity indicated that the apparent K_m(CO₂) were 31 and 36 μ M for the fir and spruce enzymes, respectively.     

Activating Mutations of the Serotonin 5-HT2C Receptor

Abstract: Site-directed mutagenesis was performed to create a mutant serotonin 5-HT_2C receptor that would mimic the active conformation of the native receptor. Structural alteration of receptor conformation was achieved by changing amino acid no. 312 from serine to phenylalanine (S312F) or lysine (S312K). After expression in COS-7 cells, the binding affinity of 5-HT for [³H]-mesulergine-labeled 5-HT_2C receptors increased from 203 n M (native) to 76 n M for S312F and 6.6 n M for S312K mutant receptors. 5-HT potency for stimulation of phosphatidylinositol (PI) hydrolysis increased from 70 n M (native) to 28 n M for S312F and 2.7 n M for S312K mutant receptors. The mutant receptors were constitutively active, stimulating PI hydrolysis in the absence of agonist. S312F and S312K mutations resulted in twofold and five-fold increases, respectively, in basal levels of PI hydrolysis. Mianserin and mesulergine displayed inverse agonist activity by decreasing basal levels of PI hydrolysis stimulated by S312K mutant receptors. [³H]5-HT and [³H]-mesulergine labeled the same number of S312K mutant receptors and 5'-guanylylimidodiphosphate had no effect on [³H]5-HT binding. These results indicate that serine → lysine mutation at amino acid no. 312 produces an agonist high-affinity state of the 5-HT_2C receptor that spontaneously couples to G proteins and stimulates PI hydrolysis in the absence of agonist.     

GM1 ELISA method for demonstration of Escherichia coli heat-stable enterotoxin

Abstract A GM1-ELISA for detection of the methanol soluble, heat-stable enterotoxin (ST_a) produced by many enterotoxinogenic E. coli strains has been developed. This ST-GM1-ELISA, which is based on inhibition of binding of anti-ST antibody to GM1-bound ST-cholera B subunit conjugates, is relatively simple and possible to perform with stable reagents and without any complicated equipment. By this method ST_a could be detected in culture filtrates of human E. coli isolates with 100% sensitivity and specificity. The sensitivity of the method for purified ST is considerably higher than that of the conventional infant mouse test and comparable to that of recently described radioimmunoassays for ST.     

Characterisation of a Myristoyl CoA:Glycylpeptide N-Myristoyl Transferase Activity in Rat Brain: Subcellular and Regional Distribution

Abstract: An enzyme activity in rat brain, capable of catalysing the transfer of myristic acid from myristoyl CoA to the amino terminus of synthetic peptides, has been characterised. The synthetic peptides used as substrates were one based on the N-terminal eight amino acids of cyclic AMP-dependent protein kinase and another hexadecapeptide based on the N-terminal sequence of p60^src. This N -myristoyl transferase (NMT) activity, which is both peptide dependent and heat labile, occurs in rat brain at levels at least three times those found in other rat tissues. In the presence of both ATP and CoA the enzyme catalysed the transfer of myristic acid, but not palmitic acid, specifically to the N-terminal glycine of the peptides. Both peptide substrates exhibited Mi-chaelis-Menten kinetics yielding K _m values of 100 μ M and 60 μ M , and V_max values of 5 and 14.8 pmol/min/mg for the cyclic AMP-dependent protein kinase peptide and sre-derived peptides, respectively. The majority of the NMT activity was present in the cytosol of the brain homogenates, and there was evidence of an NMT inhibitory activity in both the particulate fraction of brain homogenates and in brain cytosol. NMT activity could also be demonstrated in the 100,000 g supernatant of lysed synaptosomes, and the synaptosomal membranes also exhibited an inhibitory activity on the soluble enzyme. Different brain areas exhibited different levels of the N -myristoyl transferase activity and there was a fivefold difference in the activity found in the most active area, the hippocampus, compared to spinal cord.     

Genetic determination of essential residues of the Vibrio cholerae actin cross-linking domain reveals functional similarity with glutamine synthetases

Actin cross-linking domains (ACDs) are distinct domains found in several bacterial toxins, including the Vibrio cholerae MARTX toxin. The ACD of V. cholerae (ACD_Vc) catalyses the formation of an irreversible iso-peptide bond between lysine 50 and glutamic acid 270 on two actin molecules in an ATP- and Mg/Mn²⁺-dependent manner. In vivo , cross-linking depletes the cellular pool of G-actin leading to actin cytoskeleton depolymerization. While the actin cross-linking reaction performed by these effector domains has been significantly characterized, the ACD_Vc catalytic site has remained elusive due to lack of significant homology to known proteins. Using multiple genetic approaches, we have identified regions and amino acids of ACD_Vc required for full actin cross-linking activity. Then, using these functional data and structural homology predictions, it was determined that several residues demonstrated to be important for ACD_Vc activity are conserved with active-site residues of the glutamine synthetase family of enzymes. Thus, the ACDs are a family of bacterial toxin effectors that may be evolutionarily related to ligases involved in amino acid biosynthesis.     

Water relations of solute accumulation in Pseudomonas fluorescens

When Pseudomonas fluorescens was grown in a glucose salts medium adjusted with NaCl to a water activity (a_w) value of 0.980, the intracellular glutamic acid concentration increased 23-fold and comprised 90% of the total amino acid pool. This increase was not observed when the a_w of the medium was reduced to 0.980 with sorbitol. Sorbitol was taken up rapidly over a 30 min period and accumulated intracellularly to a level approximately two-fold greater than the concentration in the growth medium. In continuous culture, the specific rate of glutamic acid production and glucose uptake was greater at 0.980 (NaCl) than at 0.997 a_w. The maintenance coefficients for glucose uptake were similar at both a_w values but were 2.4-fold greater for glutamic acid production at 0.980 (NaCl) than at 0.997 a_w.     

Neutralization of activity of two different heat-stable enterotoxins (STh and STp) of enterotoxigenic Escherichia coli by homologous and heterologous antisera

Abstract Two distinct heat-stable enterotoxins (ST) produced by enterotoxigenic Escherichia coli , ST_p and ST_h, were purified and antisera against the purified ST_p and ST_h were prepared by immunizing rabbits with the purified ST's coupled with bovine serum albumin. Neutralizing activity of each antiserum was examined and it was found that both antisera neutralized not only homologous ST but also heterologous St. These data indicate that the two anti-ST antisera are raised against the region of common amino acid sequence of the two ST's.     

Characterization of a Novel Phospholipase A2 Activity in Human Brain

Abstract: Phospholipases A₂ (PLA₂) are a family of enzymes that catalyze the removal of fatty acid residues from phosphoglycerides. The enzyme is postulated to be involved in several human brain disorders, although little is known regarding the status of PLA₂ activity in human CNS. We therefore have characterized some aspects of the PLA₂ activity present in the temporal cortex of human brain. More PLA₂ activity was found in the membrane (particulate) fraction than in the cytosolic fraction. The enzyme could be solubilized from particulate material using 1 M potassium chloride, and was capable of hydrolyzing choline phosphoglyceride (CPG) and ethanolamine phosphoglyceride (EPG), with a preference (approximately eightfold) for EPG over CPG. When the solubilized particulate enzyme was subjected to gel filtration chromatography, PLA₂ activity eluted in a high molecular mass fraction (∼180 kDa). PLA₂ activity was weakly stimulated by dithiothreitol, strongly stimulated by millimolar concentrations of calcium ions, and inhibited by brief heat treatment at 57°C, bromophenacyl bromide, the arachidonic acid derivative AACOCF₃, γ-linolenoyl amide, and N -methyl γ-linolenoyl amide. Thus, whereas the human brain enzyme(s) characterized in our study displays some of the characteristics of previously characterized PLA₂s, it differs in several key features.     

l-[3H]Nitroarginine and l-[3H]Arginine Uptake into Rat Cerebellar Synaptosomes: Kinetics and Pharmacology

Abstract: Characteristics of the transport of the nitric oxide synthase substrate l -arginine and its inhibitor, N ^G-nitro- l -arginine ( l -NOARG), into rat cerebellar synaptosomes were studied. Uptake of both l -arginine and l -NOARG was linear with increasing amount of protein (up to 40 µg) and time of incubation (up to 5 min) at 37°C. Uptake of both compounds reached a steady state by 20 min. Maximal uptake of l -NOARG (650 pmol/mg of protein) was three to four times higher than that of l -arginine (170 pmol/mg of protein). l -NOARG uptake showed biphasic kinetics ( K _{m 1} = 0.72 m M , V _{max 1} = 0.98 nmol/min/mg of protein; K _{m 2} = 2.57 m M , V _{max 2} = 16.25 nmol/min/mg of protein). l -Arginine uptake was monophasic with a K _m of 106 µ M and a V _max of 0.33 nmol/min/mg of protein. l -NOARG uptake was selectively inhibited by l -NOARG, N ^G-nitro- l -arginine methyl ester, and branched-chain and aromatic amino acids. l -Alanine and l -serine also inhibited l -NOARG uptake but with less potency. Uptake of l -arginine was selectively inhibited by N ^G-monomethyl- l -arginine acetate and basic amino acids. These studies suggest that in rat cerebellar synaptosomes, l -NOARG is transported by the neutral amino acid carrier systems T and L with high affinity, whereas l -arginine is transported by the basic amino acid carrier system y⁺ with high affinity. These data indicate that the concentration of competing amino acids is an important factor in determining the rates of uptake of l -NOARG and l -arginine into synaptosomes and, in this way, may control the activity of nitric oxide synthase.     

Disrupted [Ca2+]i Homeostasis Contributes to the Toxicity of Nitric Oxide in Cultured Hippocampal Neurons

Abstract: Nitric oxide (NO) has been shown to be an important mediator in several forms of neurotoxicity. We previously reported that NO alters intracellular Ca²⁺ concentration ([Ca²⁺]_i) homeostasis in cultured hippocampal neurons during 20-min exposures. In this study, we examine the relationship between late alterations of [Ca²⁺]_i homeostasis and the delayed toxicity produced by NO. The NO-releasing agent S -nitrosocysteine (SNOC; 300 µ M ) reduced survival by about one half 1 day after 20-min exposures, as did other NO-releasing agents. SNOC also was found to produce prolonged elevations of [Ca²⁺]_i, persisting at 2 and 6 h. Hemoglobin, a scavenger of NO, blocked both the late [Ca²⁺]_i elevation and the delayed toxicity of SNOC. Removal of extracellular Ca²⁺ during the 20-min SNOC treatment failed to prevent the late [Ca²⁺]_i elevations and did not prevent the delayed toxicity, but removal of extracellular Ca²⁺ for the 6 h after exposure as well blocked most of the toxicity. Western blots showed that SNOC exposure resulted in an increased proteolytic breakdown of the structural protein spectrin, generating a fragment with immunoreactivity suggesting activity of the Ca²⁺-activated protease calpain. The spectrin breakdown and the toxicity of SNOC were inhibited by treatment with calpain antagonists. We conclude that exposures to toxic levels of NO cause prolonged disruption of [Ca²⁺]_i homeostatic mechanisms, and that the resulting persistent [Ca²⁺]_i elevations contribute to the delayed neurotoxicity of NO.     

Cloning and sequencing of the leucine dehydrogenase gene from Bacillus sphaericus IFO 3525 and importance of the C-terminal region for the enzyme activity

The structural gene (leudh) coding for leucine dehydrogenase from Bacillus sphaericus IFO 3525 was cloned into Escherichia coli cells and sequenced. The open reading frame coded for a protein of 39.8 kDa. The deduced amino acid sequence of the leucine dehydrogenase from B. sphaericus showed 76–79% identity with those of leucine dehydrogenases from other sources. About 16% of the amino acid residues of the deduced amino acid sequence were different from the sequence obtained by X-ray analysis of the B. sphaericus enzyme. The recombinant enzyme was purified to homogeneity with a 79% yield. The enzyme was a homooctamer (340 kDa) and showed the activity of 71.7 μmol·min⁻¹·mg⁻¹) of protein. The mutant enzymes, in which more than six amino acid residues were deleted from the C-terminal of the enzyme, showed no activity. The mutant enzyme with deletion of four amino acid residues from the C-terminal of the enzyme was a dimer and showed 4.5% of the activity of the native enzyme. The dimeric enzyme was more unstable than the native enzyme, and the K_m values for -leucine and NAD⁺ increased. These results suggest that the Asn-Ile-Leu-Asn residues of the C-terminal region of the enzyme play an important role in the subunit interaction of the enzyme.     

Ca2+/Calmodulin-Dependent Protein Kinase II and Protein Kinase C Phosphorylate a Synthetic Peptide Corresponding to a Sequence That Is Specific for the γ2L Subunit of the GABAA Receptor

Abstract: The γ₂ subunit of the GABA_A receptor (GABA_A-R) is alternatively spliced. The long variant (γ_2L) contains eight additional amino acids that possess a consensus sequence site for protein phosphorylation. Previous studies have demonstrated that a peptide or fusion protein containing these eight amino acids is a substrate for protein kinase C (PKC), but not cyclic AMP-dependent protein kinase A (PKA)-stimulated phosphorylation. We have examined the ability of PKA, PKC, and Ca²⁺/calmodulin-dependent protein kinase (CAM kinase II) to phosphorylate a synthetic peptide corresponding to residues 336–351 of the intracellular loop of the γ_2L subunit and inclusive of the alternatively spliced phosphorylation consensus sequence site. PKC and CAM kinase II produced significant phosphorylation of this peptide, but PKA was ineffective. The K _m values for PKC-and CAM kinase II-stimulated phosphorylation of this peptide were 102 and 35 μM , respectively. Maximal velocities of 678 and 278 nmol of phosphate/min/mg were achieved by PKC and CAM kinase II, respectively. The phosphorylation site in the eight-amino-acid insert of the γ_2L subunit has been shown to be necessary for ethanol potentiation of the GABA_A-R. Thus, our results suggest that PKC, CAM kinase II, or both may play a role in the effects of ethanol on GABAergic function.     

Purification and characterization of the major β-1,4-endoglucanase from Thermomonospora curvata

The major β-1,4-endoglucanase (EG) of the thermophilic actinomycete, Thermomonospora curvata , contributed over 80% of the total EG activity recovered from cell-free culture fluid after growth on cellulose. The enzyme was purified to electrophoretic homogeneity by ammonium sulphate precipitation, ion-exchange chromatography and size exclusion HPLC. This monomeric enzyme had a specific activity of 750 IU mg⁻¹ when assayed with 2.5% (w/v) carboxymethyl cellulose (CMC) at 70°C, pH 6.0. Highest activity was observed on CMC with a degree of polymerization of 3200. The EG was stable for 48 h at 60°C, pH 6.0 and had a half-life of 30 min at 80°C; temperature and pH optima were 70–73°C and 6.0–6.5, respectively. The mol. wt was 100000 and the pI was 4.0. The K _m and V _max values were 7.33 mg ml⁻¹ and 833 μmol min⁻¹, respectively. EG activity was inhibited by Fe^{2 +}, Hg^{2 +}, Ag⁺ and Pb^{2 +}, and enhanced by dithiothreitol and Zn^{2 +}. The first 12 amino acid residues at the N -terminus were: Asp-Glu-Val-Asp-Glu-Ile-Arg-Asn-Gly-Asp-Phe-Ser. Glutamic and aspartic acid constituted 24% of the total amino acid composition; no amino sugar was found.     

The amino acid sequence of the β-subunit: Of porcine enterotoxigenic Escherichia coli enterotoxin — Analysis and comparison with literature data

Abstract The amino acid composition and sequence of the β-subunit of heat-labile enterotoxin (LT) purified from a porcine (LT_p) strain, WT-1, of enterotoxigenic Escherichia coli was analysed, and the result was compared with that reported by Dallas and Falkow [Nature 288 (1980) 499-501] who deduced the amino acid sequence of LT_p from data on the DNA sequence of a porcine strain, EWD299. The purified β-subunit of the LT_p of WT-1 was carboxymethylated, succinylated, digested with chymotrypsin and subjected to high performance liquid chromatography (HPLC). The amino acid composition of the peptide peaks from the column were analysed and compared with the data reported by Dallas and Falkow. Only one fraction differed in amino acid composition from that reported, containing lysine instead of methionine. This fraction was found to consist of two peptides with the sequences Lys-Ser-Gly-Glu-Thr-Phe and Arg-Ile-Thr-Tyr. The former peptide is reported to have the sequence Met-Ser-Gly-Glu-Thr-Phe. Thus, the amino acid at position 43 from the N terminus of the β-subunit of LT_p is lysine, not methionine as reported. This is the first report which studied the amino acid sequence of LT_p analysed by protein toxin itself, not by DNA sequence analysis.     

Triiodothyronine Is a High-Affinity Inhibitor of Amino Acid Transport System L1 in Cultured Astrocytes

Abstract: The relationship between the transport of thyroid hormones and that of amino acids was examined by measuring the uptake of amino acids that are characteristic substrates of systems L, A, and N, and the effect of 3,3',5-triiodo-L-thyronine (T₃) on this uptake, in cultured astrocytes. Tryptophan and leucine uptakes were rapid, Na⁺-independent, and efficiently inhibited by T₃ (half-inhibition at ∼ 2 μ M ). Two Na⁺-independent L-like systems (L₁ and L₂), common to leucine and aromatic amino acids, were characterized kinetically. System L₂ had a low affinity for leucine and tryptophan ( K _m= 0.3–0.9 m M ). The high-affinity system L₁ ( K _m∼ 10 μ M for both amino acids) was competitively inhibited by T₃ with a K _i of 2–3 μ M (close to the T₃ transport K _m). Several T₃ analogues inhibited system L₁ and the T₃ transport system similarly. Glutamine uptake and α-(methylamino)isobutyric acid uptake were, respectively, two and 200 times lower than tryptophan and leucine uptakes. T₃ had little effect on the uptakes of glutamine and α-(methylamino)isobutyric acid. The results indicate that the T₃ transport system and system L₁ are related.