Isolation, crystallisation, and preliminary X-ray analysis of the bovine mitochondrial EF-Tu:GDP and EF-Tu:EF-Ts complexes.

Previous studies have shown that when bovine mitochondrial elongation factor Ts (EF-Ts) is expressed in Escherichia coli, it forms a tightly associated complex with E. coli elongation factor Tu (EF-Tu). In contrast to earlier experiments, purification of free mitochondrial EF-Ts was accomplished under nondenaturing conditions since only about 60% of the expressed EF-Ts copurified with E. coli EF-Tu. The bovine mitochondrial EF-Tu:GDP complex, the homologous mitochondrial EF-Tu:EF-Ts complex, and the heterologous E. coli/mitochondrial EF-Tu:EF-Ts complex were isolated and crystallised. The crystals of the EF-Tu:GDP complex diffract to 1.94 A and belong to space group P2(1) with cell parameters a=59.09 A, b=119.78 A, c=128.89 A and beta=96.978 degrees. The crystals of the homologous mitochondrial EF-Tu:EF-Ts complex diffract to 4 A and belong to space group C2 with cell parameters a=157.7 A, b=151.9 A, c=156.9 A, and beta=108.96 degrees.

Resistance of archaebacterial aEF-1 alpha.GDP against denaturation by heat and urea.

The elongation factor aEF-1 alpha, isolated as aEF-1 alpha.GDP from the thermoacidophilic archaebacterium Sulfolobus solfataricus, exchanges GDP for [3H]GDP at a rate which reaches a maximum at 95 degrees C. The rate constants at different temperatures of the heat inactivation of aEF-1 alpha.GDP are considerably lower compared to those referred to Escherichia coli EF-Tu.GDP. The Tm values determined for both aEF-1 alpha.GDP and EF-Tu.GDP are 97 and 53 degrees C, respectively. The addition of GDP during the heat treatment protects significantly EF-Tu.GDP but only slightly aEF-1 alpha.GDP. The ability of aEF-1 alpha.GDP to exchange GDP for [3H]GDP is impaired at 70 degrees C by urea at concentrations which are greater compared to those required to inactivate E. coli EF-Tu.GDP at 45 degrees C; apparently both factors are not protected by GDP against inactivation by urea.

2'-Deoxy-GTP in the microtubule cytoskeleton of neuronal cells cultured with nerve growth factor.

Tubulin, widely recognized as a GTP/GDP-binding protein, has been isolated in its polymerized state from rat PC12 cells and embryonic chick dorsal root ganglion neurons by Triton X-100 detergent extraction of the cytoskeletal fraction. Perchloric acid extraction and deproteinization of this fraction permitted subsequent analysis of nucleotide identity and content by high performance liquid chromatography. PC12 cells grown in the absence of nerve growth factor (NGF) contained ADP, ATP, GDP, and GTP at levels consistent with the actin and tubulin content of the cytoskeletal fraction. Microtubules from PC12 cells cultured in the presence of NGF contain an additional nucleotide that we have identified as dGTP. Analysis of whole cell nucleotide extracts from PC12 cells grown in the absence or presence of NGF revealed no evidence for the presence of dGTP at 4 and 14 days, respectively. We have determined that embryonic chick dorsal root ganglion neurons also contain this deoxyribonucleotide, and we found virtually no ADP or ATP in the extracted dorsal root ganglion cytoskeletal fraction. On the basis of metabolic labeling studies with [14C] guanine, we have inferred that the presence of dGTP in NGF-treated PC12 cells probably arises either from binding to the nonexchangeable nucleotide site of tubulin undergoing dynamic assembly/disassembly or from binding to the exchangeable site of tubulin subsequently incorporated into highly stabilized microtubules.

Direct assay method for guanosine 5'-monophosphate reductase activity.

A sensitive and simple micromethod for the accurate measurement of GMP reductase (EC 1.6.6.8) activity in crude extracts is described. The reaction product of [8-14C]IMP was separated from the substrate [8-14C]GMP by descending chromatography on Whatman DE81 ion-exchange paper. This separation method provides an analysis of the possible interfering reactions, such as the metabolic conversion of the substrate GMP to GDP, GTP, and/or guanosine, and guanine and the loss of the product IMP to inosine, hypoxanthine, and other metabolites. Low blank values (70-90 cpm) were obtained consistently with this assay because the IMP spot moves faster than the GMP spot. The major advantages of this method are direct measurement of GMP reductase activity in crude extracts, high sensitivity (with a limit of detection of < 10 pmol of IMP production), high reproducibility (< +/- 5%), and capability to measure activity in small samples (9 micrograms protein).

Stereochemistry and lifetime of the GTP hydrolysis intermediate at the active site of elongation factor Tu from Bacillus stearothermophilus as inferred from the 17O-55Mn superhyperfine interaction.

Electron paramagnetic resonance spectroscopy has been used to obtain information on the structure and stability of the products of GTP cleavage at the active site of elongation factor Tu (EF-Tu) from Bacillus stearothermophilus. Using stereospecifically labelled (Sp)-(Rp)-[beta-17O]GTP (prepared by modification of a previously published procedure which is now also suitable for guanine nucleotides), it was found that only one of the two possible diastereomers (Sp) led to detectable line-broadening of the EPR spectrum of Mn2+ at the active site of EF-Tu (linewidth 1.5 mT), whereas the Rp isomer caused the same linewidth as unlabelled nucleotide (1.3 mT). From our earlier work and from a demonstration that the lifetime of the state giving the broadened spectrum is too long to be assigned to the EF-Tu.GDP.Mn complex [the rate constant for decay as measured by displacement of GDP by the fluorescent 2'(3')-O-(N-methylanthraniloyl)-GDP is 6.2 x 10(-3) s-1 at 25 degrees C and pH 6.8], we conclude that the broadened signal arises from the EF-Tu.Mn.GDP.Pi complex, the predominant steady-state species. During the hydrolysis of GTP the Mn2+ remains bound to the beta-phosphate oxygen of GDP which arises from the beta pro-S oxygen of GTP, possibly until GDP dissociates and certainly until Pi dissociates. Addition of elongation factor Ts (EF-Ts) to this intermediate leads to rapid reduction of the linewidth to that expected for random distribution of interactions of one 17O and two 16O atoms of GDP with Mn2+, and is not distinguishable from that exhibited by (Rp)-[beta-17O]GTP in the corresponding complex in the presence of EF-Ts.

The quantitative determination of metabolites of 6-mercaptopurine in biological materials. VII. Chemical synthesis by phosphorylation of 6-thioguanosine 5'-monophosphate, 5'-diphosphate and 5'-triphosphate, and their purification and identification by reversed-phase/ion-pair high-performance liquid chromatography and by various enzymatic assays.

A fast and reliable two-step method has been established for the chemical synthesis of 6-thioguanosine 5'-monophosphate, 6-thioguanosine 5'-diphosphate and 6-thioguanosine 5'-triphosphate starting from the ribonucleoside. In the first step, 6-thioguanosine dissolved in triethyl phosphate, at high yield reacts with phosphorus oxide trichloride to 6-thioguanosine 5'-monophosphate which is purified by anion-exchange chromatography on DEAE-Sephadex using a step gradient of hydrochloric acid. In the second step, 6-thioguanosine 5'-monophosphate dissolved in water, reacts with phosphoric acid in the presence of pyridine/dicyclohexyl carbodiimide and is converted to 6-thioguanosine 5'-diphosphate and 6-thioguanosine 5'-triphosphate which are separated from each other and from the 6-thioguanosine 5'-monophosphate by anion-exchange chromatography on DEAE-Sephadex using a gradient of ammonium bicarbonate. Material from each step of the preparation procedure is separated by reversed-phase HPLC chromatography and analyzed for its free ribonucleoside content, 5'-monophosphate, 5'-diphosphate, 5'-triphosphate and small amounts of unidentified phosphorylated compounds. The purity of the final preparations and the identity of each 6-thioguanosine 5'-phosphate are proven by highly specific enzymatic peak-shifting/HPLC analyses using alkaline phosphatase, 5'-nucleotidase, pyruvate kinase, nucleoside diphosphate kinase and combined hexokinase/glucose 6-phosphate dehydrogenase.

Characterization of the aluminum and beryllium fluoride species bound to F-actin and microtubules at the site of the gamma-phosphate of the nucleotide.

Aluminum fluoride and beryllium fluoride complexes have previously been shown to bind tightly to F-ADP-actin and GDP-microtubules in competition with Pi and to mimic the XDP-Pi transient state of the polymerization. The structure of the bound complexes is investigated here in further detail. Using a fluoride ion-specific electrode, the number of fluoride atoms per aluminum or beryllium atom in the bound complex could be determined. The results indicate that AIF-4 and either BeF2(OH)-.H2O or BeF3-.H2O are the tightly bound species in both F-actin and microtubules. The dependences of the binding on pF and pH are consistent with this conclusion. The possible geometries of aluminum and beryllium fluorides in the gamma-phosphate subsite of the nucleotide are discussed in correlation with the catalytic mechanism of nucleotide hydrolysis.

The influence of bound GDP on the kinetics of guanine nucleotide binding to G proteins.

Purified guanine nucleotide-binding regulatory proteins, as either the oligomers or the isolated nucleotide-binding alpha subunits, display anomalous kinetics of nucleotide binding. This is due to the presence of tightly bound GDP in these preparations. The dissociation of bound GDP is the rate-limiting step for nucleotide binding. GDP can be removed by chromatography in the presence of 1 M (NH4)2SO4 and 20% glycerol, which yields preparations of G proteins that contain less than 0.1 mol of GDP/mol of guanosine 5'-(gamma-thio)triphosphate (GTP gamma S)-binding site. When the GDP is removed, the binding of GTP gamma S displays kinetics consistent with a bimolecular reaction.

Measurement of tissue purine, pyrimidine, and other nucleotides by radial compression high-performance liquid chromatography.

A high-performance liquid-chromatographic (HPLC) method for the rapid separation of purine and pyrimidine nucleotides, NAD+, NADP+, FAD, FMN, UDP-Glc, UDP-glucuronate, and ADP-ribose found in neutralized perchloric acid extracts of rat liver is described. Separation was achieved within 26 min on a radially compressed column of Partisil 10-SAX. The column was eluted with a gradient of sodium phosphate and sodium chloride. The sodium phosphate was purified by passage through tandem columns of anion- and cation-exchange resins to remove uv-absorbing impurities. The sensitivity of this procedure is such that an amount of ATP contained in 10 micrograms of liver can be measured. The recoveries of all nucleotides were between 87 and 107%. In extracts of rat liver interfering substances were found to elute with GDP, and UDP eluted with NADP. Consequently, the tissue contents of UDP and GDP were determined in a second run by measuring the increase in UTP and GTP, respectively, following sample pretreatment with pyruvate kinase (PK). The tissue level of NADP+ was calculated as the difference between the total UDP and NADP+ peak and the increase in UTP following PK treatment. In those nucleotides amenable to enzymatic analysis, namely NAD+, AMP, UDP-Glc, UTP, and ATP, the tissue contents measured enzymatically were not significantly different from those determined by HPLC. However, ADP as measured with PK was found to be 15% higher compared to the HPLC determination.

Isolation and characterization of Streptomyces aureofaciens protein-synthesis elongation factor Tu in an aggregated state.

The ability of EF-Tu to aggregate spontaneously was employed for the purification of homogeneous EF-Tu . GDP from Streptomyces aureofaciens. The formation of filamentous structures in the aggregated EF-Tu was demonstrated in a light microscope. The purified factor, with a specific activity of 19,100 +/- 1,000 units/mg in [3H]GDP exchange, was shown to be active in the translation of poly(U). Aggregated EF-Tu . GDP exhibited almost eight-times lower GDP-exchange capacity at 2 degrees C than at 30 degrees C. This suggests that GDP-binding sites are not freely accessible at lower temperatures in the aggregated factor, in contrast to Escherichia coli polymerized EF-Tu. Turbidimetric assays revealed that the solubilization of diluted aggregated S. aureofaciens EF-Tu is strongly dependent on temperature and causes an increase in the number of accessible GDP-binding sites.

The electrophoretic mobilities and activities of eleven enzymes of bloodstream and culture forms of Trypanosoma brucei compared.

Eleven soluble enzymes in the supernatant of bloodstream Trypanosoma brucei were compared for electrophoretic mobility and activity with those of T. brucei cultures grown in 3 different media. All bands of each enzyme found in the bloodstream form were also present in the cultured material, but extra bands of malate dehydrogenase (MDH) (EC 1.1.1.37), aspartate aminotransferase (ASAT) (EC 2.6.1.1), and in 2 to 6 cultures of isocitrate dehydrogenase (ICD) (EC 1.1.1.42) were present in culture forms but not in bloodstream forms. An interfering enzyme, peculiar to cultured T. brucei, which reacted with 2-oxoglutarate and possibly a trace amount of ammonium ions, ran with the fast-moving ASAT bands. Threonine dehydrogenase activity, high in cultured trypanosomes irrespective of the medium used but low in bloodstream trypanosomes, was markedly lower in Trypanosoma evansi and a much passaged T. brucei 8/18. Glucosephosphate isomerase activity on the other hand was high in bloodstream and low in cultured trypanosomes. Glutamate dehydrogenase activity was too low to record reliably in bloodstream trypanosomes, but could be clearly detected in cultured forms. As the differences point to some changes in gene expression between the two forms, culture material is likely to replace trypanosomes from living animals for electrophoretic characterization only when considerable comparative work has been done.

Endogenous components of the striatum confer dopamine-sensitivity upon adenylate cyclase activity: the role of endogenous guanyl nucleotides.

Repeated washing of the particulate material from rat striatum abolishes the dopamine-sensitivity of the adenylate cyclase activity. Readdition of the soluble fraction of the caudate homogenate restores the dopamine-sensitivity to the enzyme activity. Fractions, prepared with thin layer chromatography, containing endogenous GTP and GDP also restore dopamine-sensitivity to striatal adenylate cyclase activity. The effectiveness of GDP results from its conversion to GTP during the assay; when this conversion is eliminated, GDP can specifically block the coupling between the dopamine receptor and adenylate cyclase.