Evidence for conformational changes in elongation factor Tu induced by GTP and GDP

A comparative study of the rates of tritium-hydrogen exchange in three liganded states of the protein elongation factor Tu (EFTu) reveals a substantial conformational difference between the free (EFTu) or GTP-bound (EFTu·GTP) forms and when GDP is present (EFTu·GDP). This conformational difference is acentuated with short time tritiations. There are 25–35% more very slow hydrogens in EFTu·GDP than in EFTu·GTP, indicating that $\text{GDP}$ induces a $\text{tighter}$ conformation in EFTu than does $\text{GTP}$. Thus, a rationale is provided for the difference in reactivity of EFTu·GTP and EFTu·GDP for AA-tRNA and a conformational role in regulating protein biosynthesis may be proposed for GTP and GDP. Finally, we demonstrate that nucleoside polyphosphates may cause size-able conformational changes in proteins.     

Association of nucleosidediphosphate kinase with microtubules.

A nucleosidediphosphate kinase activity (EC 2.7.4.6) which phosphorylates GDP to GTP is present in bovine brain microtubule protein prepared by cycles of assembly-disassembly. This activity persists through 5 cycles of assembly-disassembly and sediments with microtubules in sucrose density gradients, but is not associated with the tubulin dimer. It is proposed that the kinase is an integral part of the microtubule and is therefore a microtubule associated protein (MAP). Several isozymes of nucleosidediphosphate kinase exist in our preparations with a pI 7.6 form predominant. It may be speculated that this enzyme affects tubulin assembly $\text{in vivo}$ by modulating the $\text{GTP}\text{GDP}$ ratio in the microtubule environment.     

Adenosine kinase from Trypanosoma cruzi

Adenosine kinase was demonstrated in the soluble fraction of $\text{Trypanosoma}\text{cruzi}$. Magnesium is required for activity. ATP and GTP are efficient phosphate donors while $\text{p}\text{-}\text{nitrophenylphosphate}$ is without activity. The pH optimum is high (8.0), it is heat labile and is stabile to freezing (?20° or ?80°). It is substrate inhibited, does not survive dialysis and its stability to gel filtration is enhanced by the presence of ATP or GTP. Time curves are parabolic only if the enzyme is preincubated with ATP (or GTP); sigmoid when preincubated with adenosine.     

Involvement of 50S ribosomal proteins L6 and L10 in the ribosome dependent GTPase activity of elongation factor G

CsCI-prepared 50S cores in the presence of groups of individual split proteins were tested for their capacity to support EF-G dependent GTP hydrolysis. The activity of cores prepared at 40 mM Mg²⁺ could be restored by adding $\text{L7}\text{L12}$ and L10 together, each in an amount of two copies per 50S particle, which abolishes the difference in activity between L7 and L12. In the range of 20-2 mM Mg²⁺, 50S cores lose the protein L6, which is also required for GTP hydrolysis. L10 cannot replace L6, or $\text{vice versa}$.     

Errata

Optimal conditions for activation of adenylate cyclase in membrane particles were studied. Enzyme activation with serotonin (5-hydroxytryptamine), NaF, and guanosine $\text{5′-(3-O-}\text{thio}\text{)-}\text{triphosphate}$ (GTPγS) was time- and temperature-dependent. Mg²⁺ was required for enzyme activation. Adenylate cyclase that was activated by NaF or GTPγS was gradually inhibited by $\text{N-}\text{methylmaleimide}$ while enzyme activated with serotonin and GTP responded faster to inhibition by the same sulfhydryl reagent. The enzyme responded in a similar fashion to a spin-labeled $\text{N-}\text{methylmaleimide}$ analog 3-(maleimidomethyl)-2,2,5,5-tetramethyl-1-pyrolidinyloxyl (i.e., $\text{N-}\text{methylmaleimide}$ nitroxide). Binding of the spin label was enhanced following enzyme activation by serotonin, NaF, or GTPγS in the presence of Mg²⁺. Activation of the enzyme was accompanied by an increase in the strong immobilization peaks in the EPR spectra. Both effects, the increase in binding and in the strong immobilization peaks, can be induced by Mg²⁺ alone. The results indicate that a general conformational change induced by Mg²⁺ may be essential for adenylate cyclase activation.     

Regulatory effects of purine ribonucleotides on Escherichia coli adenylosuccinate synthase

Adenylosuccinate synthase (EC 6.3.4.4.) ($\text{l-aspartate + GTP + IMP}\text{→}\text{Mg}{}^{\mathrm{2+}}\text{adenylosuccinate + GDP + P}{}_{\mathrm{i}}$) is an important site for the regulation of adenylate biosynthesis. A partially purified preparation of the enzyme from Escherichia coli B showed feedback inhibition by ADP and AMP, weak positive response to the adenylate energy charge, and weak positive response to the mole fraction of GTP in the GTP + GDP pool. These responses seem to ensure that the synthesis of adenine nucleotides will be controlled appropriately in response to the level of end products and to the energy state of the cell, and to avoid the potential difficulties arising from the fact that the end products of this sequence and the indicators of the energy state of the cell are the same compounds.     

Bioactive carbohydrates—In chemistry,biochemistry, and biology: By J. F. Kennedy and C. A. White,Halsted Press,New York, 1983, 331 pp. $79.95

A method for analysis of plasma adenosine which combines the principles of radioisotope dilution and enzymatic catalysis is presented. Plasma from venous heparinized blood containing the adenosine deaminase inhibitor 2′-deoxycoformycin is mixed with a small amount of [³H]adenosine and extracted with perchloric acid. Using highly purified enzyme and [γ-³²P]GTP as the phosphate donor, the neutralized extract then serves as substrate for adenosine kinase, and the AMP product is purified by high-performance liquid chromatography. Adenosine concentrations in plasma are linearly proportional to ${}^{32}\text{P}{}^3\text{H}$ ratios in the enzymatically synthesized AMP and are calculated from a standard curve. The advantages of the method are: ease of sample preparation; sensitivity of 20 nm in as little as 0.3 ml plasma; 20 samples per day can be analyzed by a single operator. Care must be used when obtaining plasma since cellular contamination will affect results. Using this assay, human plasma adenosine levels are 0.121 ± 0.054 μm for males and 0.101 ± 0.067 μm for females.     

Protein synthesis in rabbit reticulocytes: control of protein synthesis initiation by a met-tRNA Met f deacylase and peptide chain initiation factors

The 0.5M KCl wash of rabbit reticulocyte ribosomes (I fraction) catalyzes the deacylation of Met-tRNA_f^Met. Upon DEAE-cellulose column chromatography, the deacylase activity elutes with the 0.1M KCl wash of the column (f1) and is well-resolved from the peptide chain initiation factors (1–3). The deacylase activity is specific for Met-tRNA_f^Met (retic., $\text{E.}$$\text{coli}$). Other aminoacyl tRNAs tested including fMet-tRNA_f^Met (retic., $\text{E.}$$\text{coli}$), Phe-tRNA ($\text{E.}$$\text{coli}$), Val-tRNA (retic.), and Arg-tRNA (retic.) are completely resistant to the action of the deacylase. In the presence of the peptide chain initiation factor (IF1) and GTP, retic. Met-tRNA_f^Met forms the initiation complex Met-tRNA_f^Met:IF1:GTP (2), and in this ternary complex Met-tRNA_f^Met is not degraded by the deacylase. $\text{E.}$$\text{coli}$ Met-tRNA_f^Met binds to IF1 independent of GTP, and in this complex, this Met-tRNA_f^Met is degraded by the deacylase.Prior incubation of f1 with Met-tRNA_f^Met (retic.) strongly inhibited protein synthesis initiation, presumably due to deacylation of the initiator tRNA. This inhibition by f1 was completely prevented when Met-tRNA_f^Met (retic.) was pre-incubated with peptide chain initiation factors.     

Effect of guanine nucleotides on the assembly of brain microtubles: ability of 5'-guanylyl imidodiphosphate to replace GTB in promoting the polymerization of microtubules in vitro.

Polymerization of microtubule requires the presence of GTP, and the tubulin-bound GTP is hydrolyzed during microtubule formation. However, it was found that an unhydrolyzable analog of GTP, 5′-guanylyl imidodiphosphate (Gpp(NH)p), was able to replace GTP. The hydrolysis of the terminal phosphate group of GTP, therefore, does not seem to be a prerequisite to $\text{in vitro}$ assembly of microtubules. The microtubules formed in the presence of Gpp(NH)p were indistinguishable from those formed in the presence of GTP under electron microscopy, but a remarkable decrease was noted in their sensitivity to depolymerization by calcium ions.     

Enzymatic analysis of guanine nucleotides in tissues and cells

GTP and GDP concentrations can be determined by a simple and specific spectrophotometric assay that uses commercially available enzymes. The conversion of GTP to GDP catalyzed by nucleosidediphosphate kinase in the presence of ADP enables the subsequent use of guanylate kinase which is coupled with hexokinase and glucose-6-phosphate dehydrogenase as indicator enzymes. Guanylate kinase which is highly specific for GDP and 5′-GMP (Miech, R. P., and Parks, R. E., Jr. (1965) J. Biol. Chem.240, 351–357) is also used for the determination of 5′-GMP and of the sum of all acid-soluble guanine 5′-nucleotides. The latter are hydrolyzed by snake venom phosphodiesterase and assayed as 5′-GMP. The assays are highly reproducible with standard deviations of less than 2% when performed in the optimal range between 2 and 100 nmol of guanine nucleotide per cuvette. The sensitivity can be increased by use of dual wavelength measurements of fluorimetry or by following the generation of ATP with the luciferase-catalyzed luminescence. Contents of guanine nucleotides and of total nucleoside 5′-triphosphates were measured in liver, kidney, brain, and skeletal muscle of the rat. The effect of guanosine and of inhibitors of inosinate dehydrogenase (virazole and mycophenolate) on the level of GTP and GDP was examined in ascites hepatoma cells in suspension.     

Characterization of native 40S particles from krebs II mouse ascites tumor cells. I. Phosphorylation in vitro of non ribosomal proteins by a cAMP independent protein kinase.

In the presence of [γ³²-P] ATP or [γ³²-P] GTP 4 non ribosomal proteins (M_r 110,000; 105,000; 89,000 and 25,000) of the native 40S subunit became phosphorylated. The protein kinase responsible for this phosphorylation could be removed by treatment with 0.5$\text{M}$ KCl. Sucrose density gradient analysis showed that the endogenous enzyme activity sedimented with approx. 7.5S.     

Interaction of the low molecular weight form of elongation factor 1 with guanine nucleotides and aminoacyl-tRNA.

A low molecular weight form of the eukaryotic polypeptide chain elongation factor 1 (EF-1α) has been extensively purified from pig liver to give an apparently homogeneous preparation, which seemed to be analogous to the bacterial elongation factor, EF-Tu (Iwasaki, K., Nagata, S., Mizumoto, K., and Kaziro, Y. (1974) J. Biol. Chem. 249, 5008). Thus, the interaction of the purified EF-1α with guanine nucleotides as well as aminoacyl-tRNA has been investigated and the following results have been obtained. (1) EF-1α when kept in the absence of glycerol lost its activity to promote the binding of aminoacylt-RNA to ribosomes though it retained the ability to bind guanine nucleotides. However, the former activity could be stabilized by the addition of 25% ($\text{v}\text{v}$) glycerol to the solution. (2) EF-1α formed a binary complex with guanine nucleotides such as GTP, GDP, 5′-guanylyl methylenediphosphonate or 5′-guanylyl imidodiphosphate. The molar ratio of EF-1α to GTP or GDP in the binary complex was shown to be 1. (3) The presence of a ternary complex containing EF-1α, GTP and aminoacyl-tRNA was demonstrated by several methods, i.e., (i) an increased heat stability of EF-1α in the presence of GTP and Phe-tRNA, (ii) a decrease in the amount of the EF-1α·GTP complex in the presence of aminoacyl-tRNA, (iii) a protection of the ester linkage of Phe-tRNA from hydrolysis at alkaline pH by the presence of both EF-1α and GTP, and (iv) the isolation of the complex by gel filtration.     

Studies on the activity of brown adipose tissue in suckling,pre-obese, obob mice

The properties and activity of brown adipose tissue have been investigated in suckling, pre-obese, $\text{ob}\text{ob}$ mice in order to determine whether decreased thermogenesis in the tissue precedes the development of obesity in this mutant. At 14 days of age there was no difference between the $\text{ob}\text{ob}$ and normal animals in the total amount of interscapular brown adipose tissue, and the DNA content, protein content, and cytochrome oxidase activity of the tissue were similar in the two groups of mice. Respiration rates of brown adipose tissue mitochondria in the presence of albumin were, however, greater in the normal than the $\text{ob}\text{ob}$ animals, although after the addition of GDP to recouple the mitochondria there was no difference between the two groups. The mitochondrial membrane potential, measured with [³H]methyltriphenylphosphonium, was less affected by exogenous GDP in $\text{ob}\text{ob}$ mice than in normal animals. GDP binding to brown adipose tissue mitochondria, an index of the proton conductance pathway, was much greater in normal than in $\text{ob}\text{ob}$ mice at both 10 and 14 days of age; the decreased GDP binding in the mutant animals was found to result from a reduction in the number of binding sites. It is concluded that brown adipose tissue mitochondria of pre-obese $\text{ob}\text{ob}$ mice are more tightly coupled than those of normal siblings, and that the activity of the ‘thermogenic’ proton conductance pathway is lower in the mutant animals. A decrease in thermogenesis in brown adipose tissue is therefore an early event in the development of the $\text{ob}\text{ob}$ mouse and precedes the appearance of obesity.     

The heavy form of elongation factor 1 in Artemia salina embryos is functionally analogous to a complex of bacterial factors EF-Tu and EF-Ts

The purified heavy form of elongation factor 1 (EF-1) from cysts of $\text{Artemia salina}$ was found to catalyze the exchange of free GTP with a complex of EF-1_L (EF-1_α) and GDP. Furthermore, after heat treatment of EF-1_H in the presence of GTP, the factor, while inactive by itself, stimulated aminoacyl-tRNA binding to ribosomes as well as polyphenylalanine synthesis when combined with EF-1_α. These functional properties are similar to those reported recently for purified EF-1_β from pig liver [ Nagata,S., Motoyoshi,K., and Iwasaki,K. (1976) Biochem. Biophys. Res. Comm. $\text{71}$, 933–938 ]. We suggest that Artemia EF-1_H consists of a EF-1_α. EF-1_β complex which is functionally analogous to bacterial EF-Tu · EF-Ts.     

Human complex-forming glycoprotein, heterogeneous in charge: the primary structure around the cysteine residues and characterization of a disulfide bridge

L-929 cell surface membranes have been assayed in vitro and found to contain significant protein kinase activity. A steady-state kinetic analysis indicated that at least two distinct protein kinases were present. Plots of reaction velocity (v) against substrate (ATP) concentration were distinctly biphasic, as were Lineweaver-Burk plots of $\text{1}\text{v}$ versus $\text{1}\text{ATP}$. Michaelis constants of the two enzymes were calculated to be 22 and 173 μm, respectively. Sodium dodecyl sulfate polyacrylamide gel analysis of the phosphorylated membrane proteins provided additional support for the existence of more than one protein kinase. Different endogenous proteins were phosphorylated at 1 μm ATP compared to 1 μm ATP. Further studies of the low K_m (22 μm) enzyme suggested that it is a typical cyclic 3′,5′-AMP-independent protein kinase. Its activity was dependent on the presence of Mg²⁺, but it was not affected by cyclic 3′,5′-AMP, cyclic 3′,5′-GMP, or the heat-stable inhibitor of cyclic 3′,5′-AMP-dependent protein kinases. ATP and GTP, but not other nucleoside triphosphates, could serve as phosphoryl donor and maximum kinase activity was expressed at pH 7.0. Phosvitin and casein were superior to histones as exogenous substrates for the low K_m enzyme.     

Superoxide-dependent formation of hydroxyl radicals: Detection of hydroxyl radicals by the hydroxylation of aromatic compounds

A mixture of xanthine or hypoxanthine and xanthine oxidase generates the superoxide radical, O₂^$\text{?}$, and H₂O₂. In the presence of iron salts, O₂^$\text{?}$ and H₂O₂ can interact to produce the hydroxyl radical, OH·. Superoxide-dependent formation of OH· can be measured by its ability to hydroxylate salicylate as followed by an improved colorimetric assay described in this paper. A more accurate analysis of OH· can be obtained using its ability to hydroxylate phenol, the hydroxylated products being separated and measured after derivatization using gas-liquid chromatography and electron-capture detection. The derivatization and separation techniques are described.     

A radioimmunoassay for 11-oxotestosterone: Its application in the measurement of levels in blood sebum of rainbow trout (S. Gairdneri).

17β-Hyd.roxyandrost-4-ene-3,11-dione was linked via its 3-(O-carboxymethyl) oxime to bovine serum albumin to give a conjugate which was used to generate antiserum in rabbits. The antiserum, at an overall dilution of 1 in 16,000, together with [1,2-³H] 17β-hydroxyandrost-4-ene-3,11-dione synthesized from [1,2-³H] cortisone have been used to develop a radioimmunoassay for the parent steroid. The assay incorporates a purification step in which serum or plasma extracts are chromatographed on silica gel layers bound to plastic or aluminium sheets and the steroid, containing zones cut out and eluted directly with assay buffer. The cross-reactivities of several steroids with the antiserum and the specificity, sensitivity, accuracy and precision of the assay are described. Blood sera from Immature male rainbow trout contain $\text{ca}$ 0.2–0.4 μg/100 ml of 17β-hydroxyandrost-4-ene-3,11-dione. As male fish mature, serum levels rise sharply to reach values of 2 to >9 μg/100 ml. Levels in immature females rarely exceeded the assay sensitivity but serum from three ripe females showed low but detectable levels ($\text{ca}$ 0.2 μg/100 ml) of steroid. The assay has found application in sexing live fish for experimental purposes.     

Calcium stimualtion of protein kinase C in the absence of added phospholipids

The activity of protein kinase C as isolated and described by Inoue, M., Kishimoto, A., Takai, Y., and Nishizuka, Y. (1977) J. Biol. Chem. $\text{252}$, 7610–7616, can be markedly stimulated by Ca²⁺ in the presence of 4 mM Mg²⁺. This Ca²⁺ dependency does not require the presence of phospholipids or exogenous calmodulin. The increase in activity in the presence of Ca²⁺ is blocked by fluphenazine in the presence of 30 mM 2-mercaptoethanol. These results suggest that a calmodulin-like moiety may be a subunit of prokinase C.     

The preparation of a modified GTP-sepharose derivative and its use in the purification of dihydroneopterin triphosphate synthetase, the first enzyme in folate biosynthesis

The conditions for coupling periodate oxidized GTP to a hydrazide Sepharose derivative are described. Approximately 1 μmole of the ligand was bound per milliliter of settled gel. Gel columns prepared from this material bind D-$\text{erythrod}$ihydroneopterin triphosphate synthetase, the initial enzyme for folate biosynthesis in $\text{Lactobacillus}\text{plantarum}$. A yield of 28% and an overall enzyme purification of 765 fold were attained when the affinity technique was used with a conventional purification procedure.     

The measurement of cyclic GMP and cyclic AMP phosphodiesterases

Methods are described for measuring phosphodiesterases for cGMP and cAMP in the range of activity yielding 10⁻¹² to 10⁻⁸ mol of product. The 5′-GMP formed is measured by conversion to GDP with guanylate kinase. Amounts of GDP greater than 10⁻¹⁰ mol are measured directly with an enzyme system which results in stoichiometric oxidation of NADH. This is either determined by the decrease in fluorescence or the excess NADH is destroyed with acid and the NAD⁺ measured by its fluorescence in strong NaOH. With smaller amounts of GDP, sensitivity is amplified 1000-fold with the succinic thiokinase-pyruvate kinase cycle. In the case of cAMP diesterase, larger amounts of 5′-AMP are measured in the same way as 5′-GMP, except that adenylate kinase is substituted for guanylate kinase. With smaller amounts, the 5′-AMP is converted to ATP, and sensitivity is amplified with the adenylate kinase-pyruvate kinase cycle. As little as 20 ng dry weight of average brain is sufficient for accurate assay of the diesterase activity toward either cAMP or cGMP. When there is danger of significant destruction of AMP or GMP by tissue 5′-nucleotidase, this is prevented by adding GMP to the cAMP reagent, AMP to the cGMP reagent, or 5′-UMP to either reagent.