Insulin promoted decrease in the phosphorylation of protein synthesis initiation factor eIF-2

Insulin stimulates cellular protein synthesis in calf chondrocytes in suspension culture. This enhanced synthetic activity is seen in association with a decrease in phosphorylation of the α subunit of protein synthesis initiation factor eIF-2. [³²P] associated with the α subunit is reduced approximately 50% by insulin treatment of chondrocytes incubated in [³²P] containing media. Identical or closely located amino acids in the eIF-2 α subunit are phosphorylated by the chondrocyte kinase(s) and the rabbit reticulocyte hemin regulated kinase as indicated by comparative peptide fragment analysis of [³²P] labeled α subunits.     

Backbone resonance assignments for the ligand binding subunit of the histidine permease complex (HisJ) from Escherichia coli, under histidine-bound and unbound states

HisJ is a histidine binding subunit of the histidine permease, which exists in the outer membrane of Gram-negative bacteria. In order to incorporate the periplasmic histidine into the cell, HisJ captures histidine in the periplasm, and transfers the histidine to the transmembrane complex of histidine permease that is an ABC transporter. We established the backbone resonance assignments of ¹H/¹³C/¹⁵N-labeled HisJ from Escherichia coli, in the histidine-bound and unbound states.     

Cross-linking of transmembrane helices in proton-translocating nicotinamide nucleotide transhydrogenase from Escherichia coli: implications for the structure and function of the membrane domain

Proton-pumping nicotinamide nucleotide transhydrogenase from Escherichia coli contains an α and a β subunit of 54 and 49 kDa, respectively, and is made up of three domains. Domain I (dI) and III (dIII) are hydrophilic and contain the NAD(H)- and NADP(H)-binding sites, respectively, whereas the hydrophobic domain II (dII) contains 13 transmembrane α-helices and harbours the proton channel. Using a cysteine-free transhydrogenase, the organization of dII and helix-helix distances were investigated by the introduction of one or two cysteines in helix-helix loops on the periplasmic side. Mutants were subsequently cross-linked in the absence and presence of diamide and the bifunctional maleimide cross-linker o-PDM (6 Å), and visualized by SDS-PAGE.In the α₂β₂ tetramer, αβ cross-links were obtained with the αG476C-βS2C, αG476C-βT54C and αG476C-βS183C double mutants. Significant αα cross-links were obtained with the αG476C single mutant in the loop connecting helix 3 and 4, whereas ββ cross-links were obtained with the βS2C, βT54C and βS183C single mutants in the beginning of helix 6, the loop between helix 7 and 8 and the loop connecting helix 11 and 12, respectively. In a model based on 13 mutants, the interface between the α and β subunits in the dimer is lined along an axis formed by helices 3 and 4 from the α subunit and helices 6, 7 and 8 from the β subunit. In addition, helices 2 and 4 in the α subunit together with helices 6 and 12 in the β subunit interact with their counterparts in the α₂β₂ tetramer. Each β subunit in the α₂β₂ tetramer was concluded to contain a proton channel composed of the highly conserved helices 9, 10, 13 and 14.     

Activation of Type II Adenylyl Cyclase by the Cloned μ-Opioid Receptor: Coupling to Multiple G Proteins

Abstract: Opioid receptors are multifunctional receptors that utilize G proteins for signal transduction. The cloned δ-opioid receptor has been shown recently to stimulate phospholipase C, as well as to inhibit or stimulate different isoforms of adenylyl cyclase. By using transient transfection studies, the ability of the cloned μ-opioid receptor to stimulate type II adenylyl cyclase was examined. Coexpression of the μ-opioid receptor with type II adenylyl cyclase in human embryonic kidney 293 cells allowed the μ-selective agonist, [d -Ala², N-Me-Phe⁴,Gly⁵-ol]enkephalin, to stimulate cyclic AMP accumulation in a dose-dependent manner. The opioid-induced stimulation of type II adenylyl cyclase was mediated via pertussis toxin-sensitive G_i proteins, because it was abolished completely by the toxin. Possible coupling between the μ-opioid receptor and various G protein α subunits was examined in the type II adenylyl cyclase system. The opioid-induced response became pertussis toxin-insensitive and was enhanced significantly upon co-expression with the α subunit of G_z, whereas those of G_q, G₁₂, or G₁₃ inhibited the opioid response. When pertussis toxin-sensitive G protein α subunits were tested under similar conditions, all three forms of α_i and both forms of α_o were able to enhance the opioid response to various extents. Enhancement of type II adenylyl cyclase responses by the co-expression of α subunits reflects a functional coupling between α subunits and the μ-opioid receptor, because such potentiations were not observed with the constitutively activated α subunit mutants. These results indicate that the μ-opioid receptor can couple to G_i1–3, G_o1–2, and G_z, but not to G_s, G_q, G₁₂, G₁₃, or G_t.     

Specific labelling of the (Ca2+ + Mg2+)-ATPase of Escherichia coli with 8-azido-ATP and 4-chloro-7-nitrobenzofurazan

1. 8-Azido-ATP is a substrate for Escherichia coli (Ca²⁺ + Mg²⁺)-ATPase (E. coli F₁).2. Illumination of E. coli F₁ in the presence of 8-azido-ATP causes inhibition of ATPase activity. The presence of ATP during illumination prevents inhibition.3. 8-Azido-ATP and 4-chloro-7-nitrobenzofurazan (NbfCl) bind predominantly to the α subunit of the enzyme, but also significantly to the β subunit.4. The α subunit of E. coli F₁ seems to have some properties that in other F₁-ATPases are associated with the β subunit.     

Anthranilate synthase of Neurospora crassa: reaction and labeling with glutamine analogs

The multifunctional enzyme complex, anthranilate synthase from Neurospora crassa, irreversibly loses its glutamine-dependent anthranilate synthase activity on exposure to the reactive glutamine analogs DON and azaserine. Inactivation depends on the presence of the substrate chorismate, is enhanced by the cofactor Mg⁺², and is antagonized by glutamine. Inactivation correlates well with the incorporation of [¹⁴C]DON into the protein with modification localized to the β subunit (M_r 84,000) of the complex, demonstrating directly that the β subunit provides the glutamine binding site for the glutamine-dependent anthranilate synthase reaction. The slower and less extensive loss of ammonia-dependent anthranilate synthase activity indicates that maximum expression of the ammonia-dependent anthranilate synthase activity by the α subunit also depends on the interaction with an active glutamine amidotransferase domain of the β subunit.     

Bovine adrenocortical self-phosphorylating protein kinase,SPK 380: Purification and characterization

Recently, we described the partial purification and characterization of a novel adrenocortical cyclic nucleotide-independent protein kinase, PK 380, that catalyzes the phosphorylation of an endogenous peptide (120,000 daltons) and a serine residue(s) of the α subunit (38,000 daltons) of the eucaryotic initiation factor eIF-2 (Y. Kuroda, W. C. Merrick, and R. K. Sharma, 1982, Arch. Biochem. Biophys.213, 271–275). In the present communication we describe the purification to apparent homogeneity and characterization of this protein kinase (SPK 380). As shown by sucrose density sedimentation, the native enzyme has a molecular weight of 356,000. The protein is composed of three identical subunits of M_r 120,000. Polyacrylamide-gel isoelectric focusing electrophoresis revealed a single peak with pI 4.5. SPK 380 self-phosphorylated a histidine residue(s) of its 120,000-dalton peptide. This reaction utilized the terminal phosphate of ATP; GTP was inactive. Divalent cations (5 mm Mn²⁺ or 10 mm Mg²⁺) were essential for optimum activity. Thiol reagents (N-ethylmaleimide, p-chloromercuriphenylsulfonic acid) inhibited the kinase, indicating a sulfhydryl-group requirement for enzyme activity.     

Simultaneous measurement of intra- and intermolecular NOEs in differentially labeled protein-ligand complexes*

A new NOE strategy is presented that allows the simultaneous observation of intermolecular and intramolecular NOEs between an unlabeled ligand and a ¹³C,¹⁵N-labeled protein. The method uses an adiabatic ¹³C inversion pulse optimized to an empirically observed relationship between ¹ J _CH and carbon chemical shift to selectively invert the protein protons (attached to ¹³C). Two NOESY data sets are recorded where the intermolecular and intramolecular NOESY cross peaks have either equal or opposite signs, respectively. Addition and subtraction yield two NOESY spectra which contain either NOEs within the labeled protein (or unlabeled ligand) or along the binding interface. The method is demonstrated with an application to the B₁₂-binding subunit of Glutamate Mutase from Clostridium tetanomorphum complexed with the B₁₂-nucleotide loop moiety of the natural cofactor adenosylcobalamin (Coenzyme B₁₂).     

In vivo incorporation of [14C]tyrosine into the C-terminal position of the α subunit of tubulin

In vitro incorporation of [¹⁴C]tyrosine into the C-terminal position of the α subunit of tubulin was not affected by 4 mm cycloheximide. This inhibitor of protein synthesis was used for in vivo experiments. The in vivo incorporation of [¹⁴C]tyrosine into soluble brain protein of cycloheximide-treated rats was 10% of that of untreated rats. Treatment with vinblastine sulfate of the soluble brain protein showed that the incorporation of [¹⁴C]tyrosine into tubulin was higher in cycloheximide-treated than in untreated rats with respect to the incorporation into the total soluble protein. In the case of cycloheximide-treated rats, about 60% of the radioactivity incorporated into protein was released by the action of carboxypeptidase A, whereas 10% was liberated from the protein of untreated rats. The radioactive compound released by the action of carboxypeptidase A was identified as [¹⁴C]tyrosine. The α and β subunits of tubulin from animals that received [¹⁴C]tyrosine were separated by polyacrylamide gel electrophoresis. The radiosactivity ratio of $\text{α}\text{β}$ subunits of tubulin from cycloheximide-treated rats was threefold higher than that of untreated rats. When a mixture of [¹⁴C]amino acids was injected, the radioactivity ratio of $\text{α}\text{β}$ subunits of tubulin was similar for cycloheximide-treated and untreated rats. The results reported are consistent with the assumption that the α subunit of tubulin can be tyrosinated in vivo.     

Stable Isotope Labeled n-Alkanes to Assess Digesta Passage Kinetics through the Digestive Tract of Ruminants

We describe the use of carbon stable isotope (¹³C) labeled n-alkanes as a potential internal tracer to assess passage kinetics of ingested nutrients in ruminants. Plant cuticular n-alkanes originating from intrinsically ¹³C labeled ryegrass plants were pulse dosed intraruminally in four rumen-cannulated lactating dairy cows receiving four contrasting ryegrass silage treatments that differed in nitrogen fertilization level (45 or 90 kg nitrogen ha⁻¹) and maturity (early or late). Passage kinetics through the gastrointestinal tract were derived from the δ¹³C (i.e. the ratio ¹³C:¹²C) in apparently undigested fecal material. Isotopic enrichment was observed in a wide range of long-chain n-alkanes (C₂₇–C₃₆) and passage kinetics were determined for the most abundant C₂₉, C₃₁ and C₃₃ n-alkanes, for which a sufficiently high response signal was detected by combustion isotope ratio mass spectrometry. Basal diet treatment and carbon chain length of n-alkanes did not affect fractional passage rates from the rumen (K ₁) among individual n-alkanes (3.71–3.95%/h). Peak concentration time and transit time showed a quantitatively small, significant (p≤0.002) increase with carbon chain length. K ₁ estimates were comparable to those of the ¹³C labeled digestible dry matter fraction (3.38%/h; r = 0.61 to 0.71; p≤0.012). A literature review has shown that n-alkanes are not fermented by microorganisms in the rumen and affirms no preferential depletion of ¹³C versus ¹²C. Our results suggest that ¹³C labeled n-alkanes can be used as nutrient passage tracers and support the reliability of the δ¹³C signature of digestible feed nutrients as a tool to measure nutrient-specific passage kinetics.     

Production of Specifically Labeled Compounds by Methanobacterium espanolae Grown on H2-CO2 plus [13C]Acetate

Methanobacterium espanolae, an acidiphilic methanogen, required acetate for maximal growth on H₂-CO₂. In the presence of 5 to 15 mM acetate, at a growth pH of 5.5, the μ_max was 0.05 h^-1. M. espanolae consumed 12.3 mM acetate during 96 h of incubation at 35°C with shaking at 100 rpm. At initial acetate levels of 2.5 to 10.0 mM, the amount of biomass produced was dependent on the amount of acetate in the medium. ¹³C nuclear magnetic resonance spectra of protein hydrolysates obtained from cultures grown on [1-¹³C]- or [2-¹³C]acetate indicated that an incomplete tricarboxylic acid pathway, operating in the reductive direction, was functional in this methanogen. The amino acids were labeled with a very high degree of specificity and at greater than 90% enrichment levels. Less than 2% label randomization occurred between positions primarily labeled from either the carboxyl or methyl group of acetate, and very little label was transferred to positions primarily labeled from CO₂. The labeling pattern of carbohydrates was typical for glucogenesis from pyruvate. This methanogen, by virtue of the properties described above and its ability to incorporate all of the available acetate (10 mM or lower) from the growth medium, has advantages over other microorganisms for use in the production of specifically labeled compounds.     

Thermodynamic parameters of stabilization of the Yersinia pestis Caf113-149 subunit in compact form

Several experimental methods (circular dichroism, viscosity, intrinsic fluorescence, and fluorescence labeling) were used to study the conformational folding/unfolding transitions in a compact monomeric form of the Caf1_13-149 subunit under the action of guanidine hydrochloride in the temperature range 5–45°C. It has been shown that transitions always occur between two major states (unfolded and compact). This has made it possible to determine all the main thermodynamic functions that characterize the compact state of the Caf1_13-149 subunit: stability temperature T _m, free energy of stabilization ΔG _st, enthalpy ΔH _tr, and heat capacity jump ΔC in collapse of the structure. These data have been confirmed by an independent experiment on melting of fluorescently labeled protein.     

Sulphydryl groups of (Na+ +K+)-ATPase from rectal glands of Squalus acanthias: Titrations and classification

1. (Na⁺ +K⁺)-ATPase from rectal gland of Squlus acanthias contains 34 SH groups per mol (M_r 265000). 15 are located on the α subunit (M_r 106 000) and two on the β subunit (M_r 40 000). The β subunit also contains one disulphide bridge. 2. The reaction of (Na⁺ +K⁺)-ATPase with N-ethylmaleimide shows the existence of at least three classes of SH groups. Class I contains two SH groups on each α subunit and one on each β subunit. Reaction of these groups with N-methylmaleimide in the presence of 40% glycerol or sucrose does not alter the enzyme activity. Class II contains four SH groups on each α subunit, and the reaction of these groups with 0.1 mM N-ethylmaleimide in the presence of 150 mM K⁺ leads to an enzyme species with about 16% activity. The remaining enzyme activity can be completely abolished by reaction with 5–10 nM N-ethylmaleimide, indicating a third class of SH groups (Class III). This pattern of inactivation is different from that of the kidney enzyme, where only one class of SH groups essential to activity is observed. 3. It is also shown that N-ethylmaleimide and DTNB inactivate by reacting with the same Class II SH groups. 4. Spin-labelling of the (Na⁺ +K⁺)-ATPase with a maleimide derivative shows that Class II groups are mostly buried in the membrane, whereas Class I groups are more exposed. It is also shown that spin label bound to the Class I groups can monitor the difference between the Na⁺- and K⁺-forms of the enzyme.     

Histidine regulation in Salmonella typhimurium: XVI. A sensitive radiochemical assay for histidinol dehydrogenase

A sensitive radiochemical assay for measurement of histidinol dehydrogenase is presented. The method is based upon separation of the product of the reaction. [¹⁴C]histidine, from the substrate, [¹⁴C]histidinol, on small Dowex 50 columns. The assay can be performed on cell extracts or on toluenized cells and is approximately 100 times more sensitive than previously reported assays for this enzyme.[¹⁴C]histidinol is obtained in high yields through conversion of uniformly labeled ¹⁴C-glucose by a strain of Salmonella typhimurium derepressed for the histidine operon and blocked at the histidinol dehydrogenase step. Accumulated [¹⁴C]histidinol is purified from the culture supernatant by ion-exchange chromatography.This sensitive assay has facilitated measurement of reduced levels of histidine operon expression in promoter mutants, and has been adapted for study of histidine operon regulation in a cell free protein synthesizing system.     

Subunit-subunit interactions in TF1 as revealed by ligand binding to isolated and integrated α and β subunits

The binding of 3′-O-(1-naphthoyl)adenosinetriphosphate (1-naphthoyl-ATP), ATP and ADP to TF₁ and to the isolated α and β subunits was investigated by measuring changes of intrinsic protein fluorescence and of fluorescence anisotropy of 1-naphthoyl-ATP upon binding. The following results were obtained. (1) The isolated α and β subunits bind 1 mol 1-naphthoyl-ATP with a dissociation constant (K_D(1-naphthoyl-ATP)) of 4.6 μM and 1.9 μM, respectively. (2) The K_D(ATP) for α and β subunits is 8 μM and 11 μM, respectively. (3) The K_D(ADP) for α and β subunits is 38 μM μM and 7 μM, respectively. (4) TF₁ binds 2 mol 1-naphthoyl-ATP per mol enzyme with K_D = 170 nM. (5) The rate constant for 1-naphthoyl-ATP binding to α and β subunit is more than 5 · 10⁴ M⁻¹s⁻¹. (6) The rate constant for 1-naphthoyl-ATP binding to TF₁ is 6.6 · 10³ M⁻¹ · s⁻¹ (monophasic reaction); the rate constant for its dissociation in the presence of ATP is biphasic with a fast first phase (k^A₋₁ = 3 · 10⁻³s⁻¹) and a slower second phase (k^A₋₂ < 0.2 · 10⁻³s⁻¹). From the appearance of a second peak in the fluorescence emission spectrum of 1-naphthoyl-ATP upon binding it is concluded that the binding sites in TF₁ are located in an environment more hydrophobic than the binding sites on isolated α and β subunits. The differences in kinetic and thermodynamic parameters for ligand binding to isolated versus integrated α and β subunits, respectively, are explained by interactions between these subunits in the enzyme complex.     

Functional consequences of substitution of the active site (phospho)histidine residue of Escherichia coli succinyl-CoA synthetase

Succinyl-CoA synthetase (EC 6.2.1.5, succinate: CoA ligase (ADP-forming)) of Escherichia coli is an α₂β₂ tetramer, with the active site believed to be located at the point of contact between the two subunit types. It has been previously established that the reaction involves the intermediate participation of a phosphorylated enzyme form in the process of catalysis. The site of phosphorylation (His-246) and the binding sites for the substrates ADP and ATP are located in the α subunit, and the succinate and CoA binding sites are in β. A mutant form of this enzyme, with the active site histidine residue replaced by aspartate, has been produced in large quantities and purified to homogeneity. This form appears to be indistinguishable from the native enzyme with respect to its subunit assembly, but has no ability to catalyze the overall reaction. As expected, the His-246 α →Asp mutant is incapable of undergoing phosphorylation. We have developed an assay based upon the arsenolysis of succinyl-CoA that effectively isolates the partial reaction that occurs in the portion of the active site contributed by the β subunit; this reaction does not involve covalent participation of His-246 α. We have found that the His-246 α →Asp mutant is also devoid of activity in this arsenolysis reaction, indicating that an intact His-246 α is required for the establishment of the microenvironment in this portion of the active site that is required for the corresponding step of the overall reaction.     

Carbon-13 NMR studies of 13CO binding to human hemoglobin

In the ¹³C NMR spectrum of hemoglobin A carbonylated with ¹³CO, separate resonances can be distinguished at 207.04 ppm and 206.60 ppm (with respect to the ¹³C resonance of external tetramethyl-silane) for ¹³Co bound to the α and β chains of the hemoglobin tetramer. A study of the ¹³Co derivatives of the isolated α and β chains, and of the abnormal hemoglobin M_IWATE which contains α chains which are in the met [Fe(III)] form and do not bind CO, has permitted an assignment of the high field (206.60 ppm) resonance to the β chain ¹³CO and the low field one to the α chain ¹³CO. The identification of these ¹³Co resonances permits a study of the differences in the chemistry of the α and β heme units in intact hemoglobin. Some results on the differences in the redox behavior of these chains are included.     

Biosynthesis of methyl branched hydrocarbons of the German cockroach Blattella germanica (L.) (orthoptera,blattellidae)

The precursors and directionality of synthesis of the methyl branched cuticular hydrocarbons and the female contact sex pheromone, 3,11-dimethyl-2-nonacosanone, of the German cockroach, Blattella germanica, were investigated by radiotracer and carbon-13 NMR techniques. The amino acids [G-³H]valine, [4,5-³H]isoleucine and [3,4-¹⁴C₂]methionine labeled the hydrocarbon fraction in a manner indicating that the carbon skeletons of all three amino acids serve as the methyl branch group donor. The incorporation of [1,4-¹⁴C₂]- and [2,3-¹⁴C₂]succinates into the hydrocarbon and acylglycerol/polar lipid fractions indicated that succinate also served as a precursor to methylmalonyl-CoA. Carbon-13 NMR analyses showed that [1-¹³C]propionate labeled the carbon adjacent to the tertiary carbon, and, for the 3,x-dimethylalkanes, that carbon-4 and not carbon-2 was enriched. [1-¹³C]Acetate labeled carbon-2 of these hydrocarbons. This indicates that the methyl branching groups of the 3,x-dimethylalkanes were inserted early in the chain elongation process. [3,4,5-¹³C₃]Valine labeled the methyl, tertiary and carbon adjacent to the tertiary carbon of the methyl branched alkanes. Thus, the methyl branched hydrocarbon was formed by the insertion of methylmalonyl units derived from propionate, isoleucine, valine, methionine and succinate early in chain elongation.     

NMR structure of an intracellular third loop peptide of human GABA(B) receptor

GABA_B receptor is a G protein-coupled receptor for GABA and drug target for neurological and psychiatric disorders. From the analysis of GTPγS binding assay, we found that a synthesized peptide (GABAb: ETKSVSTEKINDHR) corresponding to the intracellular third loop region of metabotropic GABA_B receptor could activate G_i protein α subunit directly. The three dimensional molecular structure of the peptide in SDS-d₂₅ micelles was determined by 2D ¹H-NMR spectroscopy. GABAb peptide formed an α helical structure and a positive charge cluster at the C-terminal site. These structural features were also found in several other G protein activating peptides. From the comparison among these peptides, we found that peptides with high helical content show the high activity.