Analysis of Phosphorylation of Wheat Elongation Factor 1β and β′ by Casein Kinase II

The purified casein kinase II (CK II) from Arabidopsis thaliana phosphorylates wheat elongation factor Iβ (EF- Iβ), but not elongation factor Iβ’ (EF-lβ’), which lacks a serine residue in the conserved phosphorylation site. Both EF-1β and1β’ subunits, with similar functions, seem to undergo different regulation despite the partial amino acid sequence of EF-lβ being similar to that of EF-1β’.     

Crystal structure of the bifunctional ATP sulfurylase-APS kinase from the chemolithotrophic thermophile Aquifex aeolicus

The thermophilic chemolithotroph, Aquifex aeolicus, expresses a gene product that exhibits both ATP sulfurylase and adenosine-5'-phosphosulfate (APS) kinase activities. These enzymes are usually segregated on two separate proteins in most bacteria, fungi, and plants. The domain arrangement in the Aquifex enzyme is reminiscent of the fungal ATP sulfurylase, which contains a C-terminal domain that is homologous to APS kinase yet displays no kinase activity. Rather, in the fungal enzyme, the motif serves as a sulfurylase regulatory domain that binds the allosteric effector 3'-phosphoadenosine-5'-phosphosulfate (PAPS), the product of true APS kinase. Therefore, the Aquifex enzyme may represent an ancestral homolog of a primitive bifunctional enzyme, from which the fungal ATP sulfurylase may have evolved. In heterotrophic sulfur-assimilating organisms such as fungi, ATP sulfurylase catalyzes the first committed step in sulfate assimilation to produce APS, which is subsequently metabolized to generate all sulfur-containing biomolecules. In contrast, ATP sulfurylase in sulfur chemolithotrophs catalyzes the reverse reaction to produce ATP and sulfate from APS and pyrophosphate. Here, the 2.3 A resolution X-ray crystal structure of Aquifex ATP sulfurylase-APS kinase bifunctional enzyme is presented. The protein dimerizes through its APS kinase domain and contains ADP bound in all four active sites. Comparison of the Aquifex ATP sulfurylase active site with those from sulfate assimilators reveals similar dispositions of the bound nucleotide and nearby residues. This suggests that minor perturbations are responsible for optimizing the kinetic properties for the physiologically relevant direction. The APS kinase active-site lid adopts two distinct conformations, where one conformation is distorted by crystal contacts. Additionally, a disulfide bond is observed in one ATP-binding P-loop of the APS kinase active site. This linkage accounts for the low kinase activity of the enzyme under oxidizing conditions. The thermal stability of the Aquifex enzyme can be explained by the 43% decreased cavity volume found within the protein core.     

miR395 is a general component of the sulfate assimilation regulatory network in Arabidopsis

In plants, microRNAs play an important role in many regulatory circuits, including responses to environmental cues such as nutrient limitations. One such microRNA is miR395, which is strongly up-regulated by sulfate deficiency and targets two components of the sulfate uptake and assimilation pathway. Here we show that miR395 levels are affected by treatments with metabolites regulating sulfate assimilation. The precursor of cysteine, O-acetylserine, which accumulates during sulfate deficiency, causes increase in miR395 accumulation. Feeding plants with cysteine, which inhibits sulfate uptake and assimilation, induces miR395 levels while buthionine sulfoximine, an inhibitor of glutathione synthesis, lowers miR395 expression. Thus, miR395 is an integral part of the regulatory network of sulfate assimilation.     

The enzymatic sulphation of heparan sulphate by hen's uterus

A sulphotransferase preparation from hen's uterus catalysed the transfer of sulphate from adenosine 3′-phosphate 5′-sulphatophosphate to N-desulphated heparan sulphate, heparan sulphate, N-desulphated heparin and dermatan sulphate. Heparin, chondroitin sulphate and hyaluronic acid were inactive as substrates for the enzyme. N-desulphated heparin was a much poorer substrate for the enzyme than N-desulphated heparan sulphate suggesting that properties of the substrate other than available glucosaminyl residues influenced enzyme activity. N-acetylation of N-desulphated heparin and N-desulphated heparan sulphate reduced their sulphate acceptor properties so it was unlikely that the N-acetyl groups of heparan sulphate facilitated its sulphatiion. Direct evidence for the transfer of [³⁵S]sulphate to amino groups of N-desulphated haparan sulphate was obtained by subsequent isolation of glucosamine $\text{N-[}{}^{35}\text{S}\text{]}\text{sulphate}$ from heparan [³⁵S]sulphate product. This was made possible through the use of a flavobacterial enzyme preparation which contained “heparitinase” activity but had been essentially freed of sulphatases. Attempts to transfer [³⁵S]sulphate to glucosamine or N-acetylglucosamine were unsuccessfull.     

Adenosine-5′-phosphosulfate kinase is essential for Arabidopsis viability

In Arabidopsis thaliana, adenosine-5′-phosphosulfate kinase (APK) provides activated sulfate for sulfation of secondary metabolites, including the glucosinolates. We have successfully isolated three of the four possible triple homozygous mutant combinations of this family. The APK1 isoform alone was sufficient to maintain WT levels of growth and development. Analysis of apk1 apk2 apk3 and apk1 apk3 apk4 mutants suggests that APK3 and APK4 are functionally redundant, despite being located in cytosol and plastids, respectively. We were, however, unable to isolate apk1 apk3 apk4 mutants, most probably because the apk1 apk3 apk4 triple mutant combination is pollen lethal. Therefore, we conclude that APS kinase is essential for plant reproduction and viability.     

Characteristics of Cultured Tobacco Cell Strains Producing High Levels of Ubiquinone-10 Selected by a Cell Cloning Technique

Characteristic differences were examined between tobacco cell strains producing high levels of ubiquinone (UQ) and the original tobacco cell line (Nicotiana tabacum L. cv BY-2). The growth rate of strains producing high levels of UQ was about half of that of the original cells. The maximum yield in cell dry weight was about two-thirds of that of original cells. The time-course of UQ formation by selected strains and the respiratory rates were similar to those in the original cells. The UQ contents were much higher than those in original cells, not only per g-dry weight but also per cell. Most UQ in the selected strains were also localized in mitochondria, as well as in the original cells. On protein basis, the yield of purified mitochondria from strains producing high levels of UQ was 4.3 times as much as that from original cells. UQ formation per mg of mitochondrial protein and the molar ratios of UQ to the other electron transport components in selected cells were similar to those in original cells. The ratio of the mitochondrial protein yield in strains producing high levels of UQ to the yield in original cells correlated closely with the ratio of UQ content per g-dry weight in UQ-producing strains to UQ content in original cells.     

A quench-flow kinetic investigation of calcium ion accumulation by isolated cardiac sarcoplasmic reticulum. Dependence of initial velocity on free calcium ion concentration and influence of preincubation with a protein kinase,MgATP, and cyclic AMP

Ca²⁺ accumulation at pH 6.8 by isolated rabbit heart microsomes derived chiefly from sarcoplasmic reticulum was investigated by a quench-flow technique. The reaction was terminated at preset times by addition to the reaction mixture of an equal volume of 10 to 50 mM ethyleneglycol-bis-(β-aminoethyl ether)-N,N′-tetraacetic acid buffered at pH 6.0. The initial velocity of Ca²⁺ accumulation by microsomal preparations exhibiting a steady state Ca²⁺ accumulation of 25.6 nmol Ca²⁺/mg increased from 3.67 to 33.4 nmol Ca²⁺/mg · s as the free Ca²⁺ concentration was raised from 0.2 to 18.9 μM. Preincubation of the cardiac microsomes with a partly purified soluble cardiac cyclic AMP-dependent protein kinase, MgATP, and cyclic AMP lead to a significant increase in the initial Ca²⁺ accumulation rate. The amounts of Ca²⁺ that were found to accumulate in the first 200 ms of the reaction are comparable to the quantities of the ion that according to literature data need to be removed from the myofilaments and the myoplasm for induction of relaxation of the myocardial fibers.     

Phosphorylation of an actin · tropomyosin · troponin complex from human skeletal muscle

A human skeletal actin · tropomyosin · troponin complex was phosphorylated in the presence of [γ-³²P]ATP, Mg²⁺, adenosine 3′:5′-monophosphate (cyclic AMP) and cyclic AMP-dependent protein kinase (protein kinase). Phosphorylation was not observed when the actin complex was incubated in the absence of protein kinase or 1 μM cyclic AMP. In the presence of 10⁻⁷ M Ca²⁺ and protein kinase 0.1 mole of [³²P]phosphate per 196 000 g of protein was incorporated. This was two-fold higher than the [³²P]phosphate content of a rabbit skeletal actin complex but two-fold lower than that of a bovine cardiac actin complex. At high Ca²⁺, 5 · 10⁻⁵ M, little change in the phosphorylation of a human skeletal actin complex occurred. Phosphoserine and phosphothreonine were identified in the [³²P]phosphorylated actin complex. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate showed that 60% of the label was associated with the tropomyosin binding component of troponin. The inhibitory component of troponin contained 16% of the bound [³²P]phosphate. Increasing the Ca²⁺ concentration did not significantly decrease the [³²P]phosphate content of the phosphorylated proteins in the actin complex. No change in the distribution of phosphoserine or phosphothreonine was observed. Half maximal calcium activation of the ATPase activity of reconstituted human skeletal actomyosin made with the [³²P]phosphorylated human skeletal actin complex was the same as a reconstituted actomyosin made with an actin complex incubated in the absence of protein kinase at low or high Ca²⁺.     

Redox switching mechanism of the adenosine 5′-phosphosulfate kinase domain (APSK2) of human PAPS synthase 2

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Elucidation of the active conformation of the APS-kinase domain of human PAPS synthetase 1

Bifunctional human PAPS synthetase (PAPSS) catalyzes, in a two-step process, the formation of the activated sulfate carrier 3'-phosphoadenosine 5'-phosphosulfate (PAPS). The first reaction involves the formation of the 5'-adenosine phosphosulfate (APS) intermediate from ATP and inorganic sulfate. APS is then further phosphorylated on its 3'-hydroxyl group by an additional ATP molecule to generate PAPS. The former reaction is catalyzed by the ATP-sulfurylase domain and the latter by the APS-kinase domain. Here, we report the structure of the APS-kinase domain of PAPSS isoform 1 (PAPSS1) representing the Michaelis complex with the products ADP-Mg and PAPS. This structure provides a rare glimpse of the active conformation of an enzyme catalyzing phosphoryl transfer without resorting to substrate analogs, inactivating mutations, or catalytically non-competent conditions. Our structure shows the interactions involved in the binding of the magnesium ion and PAPS, thereby revealing residues critical for catalysis. The essential magnesium ion is observed bridging the phosphate groups of the products. This function of the metal ion is made possible by the DGDN-loop changing its conformation from that previously reported, and identifies these loop residues unambiguously as a Walker B motif. Furthermore, the second aspartate residue of this motif is the likely candidate for initiating nucleophilic attack on the ATP gamma-phosphate group by abstracting the proton from the 3'-hydroxyl group of the substrate APS. We report the structure of the APS-kinase domain of human PAPSS1 in complex with two APS molecules, demonstrating the ability of the ATP/ADP-binding site to bind APS. Both structures reveal extended N termini that approach the active site of the neighboring monomer. Together, these results significantly increase our understandings of how catalysis is achieved by APS-kinase.     

Crystal Structure of Yeast FAD Synthetase (Fad1) in Complex with FAD

Flavin adenine dinucleotide (FAD) synthetase is an essential enzyme responsible for the synthesis of FAD by adenylation of riboflavin monophosphate (FMN). We have solved the 1.9 Å resolution structure of Fad1, the yeast FAD synthetase, in complex with the FAD product in the active site. The structure of Fad1 shows it to be a member of the PP-ATPase superfamily. Important conformational differences in the two motifs involved in binding the phosphate moieties of FAD compared to the Candida glabrata FMNT ortholog suggests that this loop is dynamic and undergoes substantial conformational changes during its catalytic cycle.     

Differences in macromolecular binding between cyclic AMP and its dibutyryl derivative in vitro

Rat liver cytosol binds ³H-cAMP and ³H-DBcAMP $\text{in vitro}$. Fractionation of bound radioactivity by DEAE-Sephadex chromatography shows that ³H-cAMP is associated with a different cytosolic protein than is ³H-DBcAMP. The pI's of the cAMP-protein and the ³H-DBcAMP-protein complexes are 6.7 and 3.9, respectively. Competition studies between ³H-cAMP and its structural analogues have shown the following order of effectiveness in competing for binding sites in rat liver cytosol: cAMP > N⁶-MBcAMP > O²′-MBcAMP. No inhibition of ³H-cAMP binding was observed with 5′-AMP, adenosine, cGMP or DBcAMP. $\text{In vitro}$ binding experiments with rat serum has shown that only ³H-DBcAMP binds to any significant extent.     

A kinetic investigation of the aps-kinase from Chlamydomonas reinhardii CW 15

The reaction kinetics of APS-kinase from Chlamydomonas reinhardii showed that the enzyme formed PAPS from APS upon the addition of ATP. Evidence for a ³⁵S-labelled protein intermediate between APS and PAPS has been obtained. The APS-kinase activity could only be measured in the presence of low concentrations of APS (20 ± 10 μM) and of ATP (0.2 ± 0.05 mM) due to substrate inhibition. The inhibition was partially overcome by low concentrations of 3′,5′-PAP (10,μM). The rates of PAPS formation obtained with cell extracts from the alga varied from 2 to 6 nM PAPS/mg protein/min (33–100 × 10^?12 kat/mg).     

Formation of 3-(glutathion-S-yl)-N-methyl-4-aminoazobenzene and inhibition of aminoazo dye-nucleic acid binding in vitro by reaction of glutathione with metabolically-generated N-methyl-4-aminoazobenzene-N-sulfate

The reaction of glutathione (GSH) with metabolically-formed N-methyl-4-aminoazobenzene-N-sulfate (MAB-N-sulfate), a presumed ultimate carcinogenic metabolite of N,N-dimethyl-4-aminoazobenzene (DAB), was investigated using a hepatic sulfotransferase incubation mixture containing GSH and the proximate carcinogen, N-hydroxy-N-methyl-4-aminoazobenzene (N-HO-MAB). Under these conditions, 6–16% of the MAB-N-sulfate formed could be trapped as an aminoazo dye-GSH adduct. Upon subsequent purification, the adduct was shown to be chromatographically and spectrally identical to 3-(glutathion-S-yl)-N-methyl-4-aminoazobenzene (3-GS-MAB), a known biliary metabolite of DAB and a product of the reaction of the synthetic ultimate carcinogen, N-benzoyloxy-N-methyl-4-aminoazobenzene(N-BzO-MAB), with GSH. Neither 2′- nor 4′-GS-MAB, both products of the latter reaction, were detected in the sulfotransferase incubation mixture.GSH-S-transferases did not appear to be involved in the reaction of MAB-N-sulfate or N-BzO-MAB with GSH. The addition of triethyltin, a potent GSH-S-transferase inhibitor, had no effect on the yield of 3-GS-MAB in (N-HO-MAB sulfotransferase)-GSH incubations; and the addition of cytosol or purified GSH transferases A and B to a (N-BzO-MAB)-GSH reaction mixture did not increase the amount of 3-GS-MAB formed.GSH was shown to inhibit only partially the covalent binding of [³H]-MAB-N-sulfate to DNA and rRNA. At 10 and 100 mM GSH, the sulfotransferase-mediated binding of [³H]N-HO-MAB to both nucleic acids was reduced by 30% and 70%, respectively. The role of GSH in the detoxification of chemical carcinogens is discussed.     

In vitro studies of skeletal muscle membranes characterization of a phosphorylated intermediate of sarcolemmal (Na+ + K+)ATPase

The sarcolemmal membranes isolated from rat skeletal muscle are capable of incorporating ³²P from [γ?³²P]ATP. The membrane protein phosphorylation requires Mg²⁺. Cyclic AMP, cyclic GMP and their dibutyrul derivatives showed no marked effect on sarcolemmal phosphorylation.The Mg²⁺-dependent ³²P labeling was significantly enhanced by Na⁺. The rate of Na⁺ -stimulated ³²P incorporation was quite rapid reaching steady state levels within 5 s at 0 °C. K⁺ reduced the Na⁺ -stimulated ³²P-incorporation but enhanced the ³²P_i release. This inhibitory effect of K⁺ on Na⁺ -stimulated ³²P incorporation was prevented by the cardiac glycoside, ouabain.The Na⁺ -dependent ³²P labeling showed substrate dependency and the Na⁺ site was saturable. The apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for ATP was 2 · 10?5 M. The optimum pH for ³²P labeling was between 7 and 8.Na⁺ -dependent membrane phosphorylation showed a direct relationship with the (Na⁺ + K⁺ATPase activity. The high turnover rate of ³²P intermediate (12 000 min ?1) suggested its functional significance in the overall transport ATPase reaction sequence.The predominate portion (> 90%) of the phosphorylated membrane complex was sensitive to acidified hydroxylamine and to alkaline pH suggesting an acylphosphate nature of the phosphoprotein.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that ³²P incorporation occurred predominately into a 108 000 dalton subunit which is a major protein component of sarcolemmal membranes. A very low level of ³²P incorporation was also observed into a 25 000 dalton subunit and Ca²⁺ slightly enhanced the phosphorylation of this component.The size ($\text{M}{}_{\mathrm{<mtext>r</mtext>}}\text{108 000}$) and some properties of the sarcolemmal phosphoprotein are closely similar to other (Na⁺ + K⁺ATPase preparations reported so far.     

The X-ray crystal structure of APR-B,an atypical adenosine 5′-phosphosulfate reductase from Physcomitrella patens

Sulfonucleotide reductases catalyse the first reductive step of sulfate assimilation. Their substrate specificities generally correlate with the requirement for a [Fe₄S₄] cluster, where adenosine 5′-phosphosulfate (APS) reductases possess a cluster and 3′-phosphoadenosine 5′-phosphosulfate reductases do not. The exception is the APR-B isoform of APS reductase from the moss Physcomitrella patens, which lacks a cluster. The crystal structure of APR-B, the first for a plant sulfonucleotide reductase, is consistent with a preference for APS. Structural conservation with bacterial APS reductase rules out a structural role for the cluster, but supports the contention that it enhances the activity of conventional APS reductases.