P-fluorosulfonylbenzoyl Adenosine Research Articles

Effects of salicylic acid on protein kinase activity and chloroplast D1 protein degradation in wheat leaves subjected to heat and high light stress

In the north of China, wheat plants are often stressed by heat and high light during grain-filling stage, which leads to injury in photosynthetic apparatus and decline in photosynthetic rate. In order to develop a method to protect photosynthetic apparatus in wheat leaves subjected to heat and high light stress, the effects of SA (salicylic acid) and FSBA (5′- p-fluorosulfonylbenzoyl adenosine) on PK (protein kinase) activity, D1 protein degradation and the performance of PSII were investigated in present work. Our results showed that PK activity enhanced under heat and high light stress and declined when stress was removed. FSBA pretreatment resulted in marked decreases in PK activity and D1 protein level, suggesting a correlationship between degradation of D1 protein and phosphorylation. After 2 h of stress, D1 protein level in water-pretreated leaves decreased to 79% of control and then recovered to 81% after 3 h of recovery. This clearly indicated that the damage of D1 protein induced by heat and high light stress was reversible. Compared to the control, SA pretreatment could not only increase PK activity, retard the degradation of D1 protein during heat and high light stress, but also accelerate the recovery of D1 protein level when the stress was removed. Correspondingly, F v/ F m (maximum photochemical efficiency of PSII), Φ PSII (actual photochemical efficiency of PSII), ETR (electron transfer rate) and Pn (net photosynthetic rate) in SA-treated leaves were higher than that in leaves of control under both stress and non-stress conditions. Taken together, our results revealed that SA pretreatment could significantly alleviate damages of heat and high light stress on D1 protein and PSII of wheat leaves, and accelerate restoration of photosynthetic function.

Origins of Growth Factors: NGF and EGF

Growing up on the streets of Brooklyn, I remember being interested in how things worked: taking apart an old telephone and the gears of my new 4-speed bicycle was most enjoyable. As a biology major at Brooklyn College in 1945, I was fascinated by embryology. How does an egg turn into a chicken or a frog or a person? My only insight into the problem was the thought that it was necessary to understand the chemical reactions inside the egg and embryo and not simply observe biological structures. I, therefore, became a double major in chemistry and biology (also with the hope of earning a living as a laboratory technician). My first job after graduating was working nights as a bacteriologist in a milk processing plant. One of my professors at Brooklyn College wrote asking whether I was interested in going to graduate school. The college had been sending one student a year to the Biology Department at Oberlin to work as a laboratory assistant while studying for a master's degree. Because Oberlin offered to pay my tuition plus some $300 a semester for living expenses, I jumped at the chance. School and science were both interesting and fun. Upon graduating, I continued my education toward a Ph.D. working as an assistant in the Biochemistry Department of the University of Michigan under Howard B. Lewis. My research involved studying the Krebs urea cycle in the common earthworms that I collected from the university campus. I had been told at Oberlin that the yellow cells surrounding the gut in the worm corresponded to the mammalian liver. I decided to check whether the enzymatic reactions in worms were similar to those in mammals. They were (1). My first “real job” was in the Pediatrics Department at the University of Colorado studying creatine metabolism in premature infants under Harry Gordon. I think he hired me because he was impressed by my ability to stomach tube earthworms. After several years, I decided I needed to learn the then new technique of using radioisotopes in metabolic studies, and I obtained an American Cancer Society fellowship to work in the Radiology Department of Washington University under Martin Kamen where I combined this new knowledge with my interest in embryology. Although fertilized frog eggs and early embryos are impermeable to small molecules (amino acids, phosphates, etc.), sufficient amounts of C14O2 were metabolized by intact eggs and embryos to permit the identification of the radioactive compounds present at various stages of development (2). Upon completion of my fellowship, Dr. Kamen recommended me for a position in the Zoology Department of Washington University in the laboratory of Viktor Hamburger and Rita Levi-Montalcini. This move was critical in determining the direction of my research for the next 40-some odd years: the isolation, structure, and function of the first of the “growth factors,” nerve growth factor (NGF) and epidermal growth factor (EGF).

Structural characterization of the functional regions in the archaeal protein Sso7d

Sso7d from the extreme thermophilic crenarchaeon Sulfolobus solfataricus is a multifunctional protein in in vitro assays, whose in vivo role is still puzzling. Crystals of Sso7d in complex with DNA elucidated the protein surface involved in the binding to the nucleic acid, whereas the locations of the Sso7d regions responsible for a chaperone activity in renaturing protein aggregates (i.e., the protein-binding surface and the site of ATPase activity) are still unknown. We identified the regions of Sso7d involved in protein-binding by limited proteolysis experiments associated to advanced mass spectrometric procedures performed on isolated Sso7d and Sso7d in complex with the peptide melittin. By affinity labeling of Sso7d with the ATP analogue 5'-p-fluorosulfonylbenzoyl adenosine and characterization of the labeled tryptic peptides by tandem mass spectrometry, we found that Y7 and K39 are residues involved in ATP binding/hydrolysis. Insights into the positions of the ligands melittin and ATP were achieved by a molecular modeling study; the models obtained were in agreement with most experimental data. A comparison among the complexes of Sso7d with DNA, with melittin, and with ATP showed that the DNA-binding surface and the protein-binding surface overlap, whereas the ATPase site is mostly independent of the binding sites for the nucleic acid and melittin.

Potato tuber isoapyrases: Substrate specificity, affinity labeling, and proteolytic susceptibility

Apyrase/ATP-diphosphohydrolase hydrolyzes di- and triphosphorylated nucleosides in the presence of a bivalent ion with sequential release of orthophosphate. We performed studies of substrate specificity on homogeneous isoapyrases from two potato tuber clonal varieties: Desirée (low ATPase/ADPase ratio) and Pimpernel (high ATPase/ADPase ratio) by measuring the kinetic parameters K m and k cat on deoxyribonucleotides and fluorescent analogues of ATP and ADP. Both isoapyrases showed a broad specificity towards dATP, dGTP, dTTP, dCTP, thio-dATP, fluorescent nucleotides (MANT-; TNP-; ethene-derivatives of ATP and ADP). The hydrolytic activity on the triphosphorylated compounds was always higher for the Pimpernel apyrase. Modifications either on the base or the ribose moieties did not increase K m values, suggesting that the introduction of large groups (MANT- and TNP-) in the ribose does not produce steric hindrance on substrate binding. However, the presence of these bulky groups caused, in general, a reduction in k cat, indicating an important effect on the catalytic step. Substantial differences were observed between potato apyrases and enzymes from various animal tissues, concerning affinity labeling with azido-nucleotides and FSBA (5′- p-fluorosulfonylbenzoyl adenosine). PLP-nucleotide derivatives were unable to produce inactivation of potato apyrase. The lack of sensitivity of both potato enzymes towards these nucleotide analogues rules out the proximity or adequate orientation of sulfhydryl, hydroxyl or amino-groups to the modifying groups. Both apyrases were different in the proteolytic susceptibility towards trypsin, chymotrypsin and Glu-C.

Role of Light-harvesting Complex 2 Dissociation in Protecting the Photosystem 2 Reaction Centres Against Photodamage in Soybean Leaves and Thylakoids

The protective role of light-harvesting complex 2 (LHC2) dissociation from photosystem 2 (PS2) complex was explored by the 5′-p-fluorosulfonylbenzoyl adenosine (FSBA, an inhibitor of protein kinase) treatment at saturating irradiance (SI) in soybean leaves and thylakoids. The dissociation of some LHC2s from PS2 complex occurred after SI treatment, but FSBA treatment inhibited the dissociation as demonstrated by analysis of sucrose density gradient centrifugation of thylakoid preparation and low-temperature (77 K) chlorophyll (Chl) fluorescence. A significant increase in F0 and decrease in Fv/Fm occurred after SI, and the two parameters could largely recover to the levels of dark-adapted leaves after subsequent 3 h in the dark, but they could not recover in the FSBA-treated leaves at SI. Neither the electron transport activity of PS2 nor the D1 protein amount in vivo had significant change after SI without FSBA, whereas FSBA treatment at SI could result in significant decreases in both the PS2 electron transport activity and the D1 protein amount. When thylakoids instead of leaves were used, the PS2 electron transport activity and the D1 protein amount declined more after SI with FSBA than without FSBA. The phosphorylation level of PS2 core proteins increased, while the phosphorylation level of LHC2 proteins was reduced after SI. Also, the phosphorylation of PS2 core proteins could be greatly inhibited by the FSBA treatment at SI. Hence in soybean leaf the LHC2 dissociation is an effective strategy protecting PS2 reaction centres against over-excitation and photodamage by reducing the amount of photons transferred to the centres under SI, and the phosphorylation of PS2 core proteins plays an important role in the dissociation.

5′- p-Fluorosulfonylbenzoyl adenosine inhibits progesterone synthesis in human placental mitochondria

The human placental mitochondria have an ATP-diphosphohydrolase (apyrase) activity. In this paper we characterized the effect of 5′- p-fluorosulfonylbenzoyl adenosine (FSBA) on placental apyrase, and its repercussion on progesterone synthesis and oxygen consumption. Apyrase activity was inhibited by FSBA. Nucleosides tri- and diphosphates protected against FSBA inactivation, but divalent cations did not, indicating that FSBA attaches itself to an ATP-binding site of apyrase. In mitochondria, the inactivation of apyrase by FSBA was associated with inhibition of progesterone synthesis. Also, the oxygen consumption induced by ATP but not by ADP, was inhibited, clearly showing that FSBA exclusively inactivated the apyrase in human placental mitochondria. It is concluded that the apyrase activity is closely related to progesterone synthesis, probably associated with the cholesterol transport between mitochondrial membranes.

D1 protein phosphorylation/dephosphorylation alone has no effect on the electron transport activity of photosystem II in soybean leaves

In order to explore the relationship between the phosphorylation of D1 protein and the function of PSII reaction center, the changes in chlorophyll fluorescence parameters, PSII electron transport activity and D1 protein amount were observed after dephosphorylation of phosphorylated D1 proteins caused by FSBA (5′- p-fluorosulfonylbenzoyl adenosine, an inhibitor of protein kinase) treatment. Also, phosphorylated and non-phosphorylated D1 proteins (D1* and D1, respectively) were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western Blotting. The following results were obtained. (1) D1* could be up to 74% of the total D1 proteins in dark-adapted soybean leaves and could be completely dephosphorylated by FSBA (1 mM) treatment for 2 h. (2) Dephosphorylation of D1* resulted in neither substantial net loss of D1 proteins nor the significant changes in value of chlorophyll fluorescence parameters. (3) The electron transport activity of PS II (H 2O→1,4-BQ) was not changed significantly after D1* was completely dephosphorylated. Based on these results, it is concluded that D1 protein phosphorylation/dephosphorylation alone has no significant effect on the function of PS II reaction center in soybean leaves.

B-subunit of Phosphate-specific Transporter fromMycobacterium tuberculosis Is a Thermostable ATPase

The B-subunit of phosphate-specific transporter (PstB) is an ABC protein. pstB was polymerase chain reaction-amplified from Mycobacterium tuberculosis and overexpressed in Escherichia coli. The overexpressed protein was found to be in inclusion bodies. The protein was solubilized using 1.5% N-lauroylsarcosine and was purified by gel permeation chromatography. The molecular mass of the protein was approximately 31 kDa. The eluted protein showed ATP-binding ability and exhibited ATPase activity. Among different nucleotide triphosphates, ATP was found to be the preferred substrate for M. tuberculosis PstB-ATPase. The study of the kinetics of ATP hydrolysis yielded K(m) of approximately 72 microm and V(max) of approximately 0.12 micromol/min/mg of protein. Divalent cation like manganese was inhibitory to the ATPase activity. Magnesium or calcium, on the other hand, had no influence on the functionality of the enzyme. The classical ATPase inhibitors like sodium azide, sodium vanadate, and N-ethylmaleimide were without any effect but an ATP analogue, 5'-p-fluorosulfonylbenzoyl adenosine, inhibited the ATPase function of the recombinant protein with a K(i) of approximately 0.40 mm. Furthermore, there was hardly any ATP hydrolyzing ability of the PstB as a result of mutation of the conserved aspartic acid residue to lysine in the Walker motif B, confirming the recombinant protein is an ATPase. Interestingly, analysis of the recombinant PstB revealed that it is a thermostable ATPase; thus, our results highlight for the first time the presence of such an enzyme in any mesophilic bacteria.

Phosphorylation/dephosphorylation of D1 protein alone has no effect on the electron transport activity of photosystem II in soybean leaves

D1 protein, as a core component of the photosystem II (PSII) reaction center complex, can be reversibly phosphorylated/dephosphorylated in higher plants. The phosphorylated D1 protein (D1*) and non-phosphorylated D1 protein (D1), having slightly different mobilities on denaturing polyacrylamide gels, can be resolved by using SDS-PAGE and Western Blotting [Callahan et al, J Biol Chem 1990, 265: 15357-15360]. The relationship between the phosphorylation of D1 protein and the electron transport activity of PSII was investigated by changing the phosphorylation level of D1 proteins with an inhibitor of protein kinase, 5?-p-fluorosulfonylbenzoyl adenosine (FSBA). The following results were obtained. (1) D1* could be up to 74% of total D1 proteins in fully dark-adapted soybean leaves and could be completely dephosphorylated by FSBA (1 mM) treatment for 2 hours. (2) Dephosphorylation of D1* resulted in neither substantial net loss of D1 proteins nor the significant changes of chlorophyll a fluorescence parameters. (3) The electron transport activity of PS II (H2O ® 1,4-BQ) had no significant change after D1* was completely dephosphorylated. Based on these results, it is concluded that phosphorylation/dephosphorylation of D1 protein alone has no significant effect on the function of PS II reaction centers in soybean leaves.

Investigation of the ATP binding site of Escherichia coli aminoimidazole ribonucleotide synthetase using affinity labeling and site-directed mutagenesis.

Aminoimidazole ribonucleotide (AIR) synthetase (PurM) catalyzes the conversion of formylglycinamide ribonucleotide (FGAM) and ATP to AIR, ADP, and P(i), the fifth step in de novo purine biosynthesis. The ATP binding domain of the E. coli enzyme has been investigated using the affinity label [(14)C]-p-fluorosulfonylbenzoyl adenosine (FSBA). This compound results in time-dependent inactivation of the enzyme which is accelerated by the presence of FGAM, and gives a K(i) = 25 microM and a k(inact) = 5.6 x 10(-)(2) min(-)(1). The inactivation is inhibited by ADP and is stoichiometric with respect to AIR synthetase. After trypsin digestion of the labeled enzyme, a single labeled peptide has been isolated, I-X-G-V-V-K, where X is Lys27 modified by FSBA. Site-directed mutants of AIR synthetase were prepared in which this Lys27 was replaced with a Gln, a Leu, and an Arg and the kinetic parameters of the mutant proteins were measured. All three mutants gave k(cat)s similar to the wild-type enzyme and K(m)s for ATP less than that determined for the wild-type enzyme. Efforts to inactivate the chicken liver trifunctional AIR synthetase with FSBA were unsuccessful, despite the presence of a Lys27 equivalent. The role of Lys27 in ATP binding appears to be associated with the methylene linker rather than its epsilon-amino group. The specific labeling of the active site by FSBA has helped to define the active site in the recently determined structure of AIR synthetase [Li, C., Kappock, T. J., Stubbe, J., Weaver, T. M., and Ealick, S. E. (1999) Structure (in press)], and suggests additional flexibility in the ATP binding region.

On the binding of ATP to the autophosphorylating protein, Ptk, of the bacterium Acinetobacter johnsonii

The autophosphorylating protein, Ptk, of the bacterium Acinetobacter johnsonii was overproduced, purified to homogeneity and assayed for ATP binding by using the nucleotide analog 5′- p-fluorosulfonylbenzoyl adenosine. The ATP binding site of this bacterial autophosphorylating protein was found to be different from that generally used by eukaryotic protein kinases. It consists of two amino acid sequences that closely resemble the Walker motifs A and B. This observation was confirmed by site-directed mutagenesis experiments which showed, in addition, that the ATP molecule bound to these motifs is effectively employed by the bacterial protein to autophosphorylate on tyrosine. It is concluded that even though the overall autophosphorylation reaction is similar in eukaryotic and prokaryotic proteins, the mechanism involved is likely different.

Demonstration of an extracellular ATP-binding site in NCAM: functional implications of nucleotide binding.

A minor fraction of the total ecto-type (E-type) ATPase activity of rat synaptosomes has been detected in immunoprecipitates of the neural cell adhesion molecule, NCAM, indicating that this either is an intrinsic enzymatic activity of NCAM or of an ATPase tightly associated to NCAM [Dzhandzhugazyan & Bock (1993) FEBS Lett. 336, 279-283]. We here demonstrate ATPase activity in preparations of the lipid-anchored as well as the transmembrane NCAM isoforms immunoisolated from transfected L-cells. A fraction of the E-type ATPase activity is spontaneously released from synaptosomes. Released material was fractionated by various chromatographic procedures and an extracellular fragment of NCAM was shown to co-elute with the major part of the enzymatic activity. Furthermore, it was shown that agarose-coupled NCAM-antibodies retained 85% of the ATPase activity released from synaptosomes after treatment with phosphatidylinositol-specific phospholipase C. These findings restricted the association or expression of the enzymatic activity to the extracellular part of NCAM. An affinity reagent, 5'-p-fluorosulfonylbenzoyl adenosine, FSBA, was shown to inhibit ATPase activity of immunoisolated NCAM, and incorporation of FSBA was detected in all three major NCAM isoforms (A, B, and C). An excess of ATP prevented both inactivation of the enzyme and affinity labeling of NCAM. Thus, NCAM contains an ATP-binding site, and this site is localized extracellularly and probably has the catalytic function. Binding of the substrate or FSBA protected a proteolytic cleavage site in NCAM localized close to the membrane presumably by induction of a local conformational change in NCAM, indicating a mechanism by which ATP may regulate NCAM adhesion and adhesion-triggered processes. A possible role of this mechanism in synaptic plasticity and memory consolidation is proposed.

Activation of Ca 2+-Calmodulin-Dependent Protein Kinase Ia Is Due to Direct Phosphorylation by Its Activator

Ca 2+-Calmodulin-dependent protein kinase Ia (CaM kinase Ia) is phosphorylated, and its activity enhanced up to 50-fold, in the presence of a protein purified from pig brain termed CaM kinase Ia activator [Lee, J.C. and Edelman, A.M. (1994) J.Biol.Chem. 269, 2158-2164]. We report here that phosphorylation of CaM kinase Ia in the presence of the activator occurs primarily on threonine (87%) and slightly on serine (13%) residues. Treatment of CaM kinase Ia with the irreversible ATP affinity analogue, 5′-p-fluorosulfonylbenzoyl adenosine (FSBA), reduces its activity by 86% but has no effect on its ability to be phosphorylated, whereas FSBA-treatment of the activator reduces its ability to activate and phosphorylate CaM kinase Ia by 92 and 93%, respectively. Thus, CaM kinase Ia activator is a protein Thr/Ser kinase which activates by phosphorylating CaM kinase Ia rather than by enhancing the latter′s autophosphorylation.

Purification and characterization of glutamine synthetase from alkalophilic Bacillus No. 170.

Glutamine synthetase from an alkalophilic Bacillus No. 170 was purified to homogeneity from cells grown in synthetic medium at pH 9.0. The purified enzyme had a molecular weight of 600,000 and a subunit size of 50,000, similar to the enzymes from Bacillus subtilis and Bacillus cereus. Either Mg 2+ or Mn 2+ activated the enzyme ; however, the highest activity was obtained when 20mM Mg 2+ with 0.2mM Mn 2+ were added to the assay mixture. The peculiar divalent cation dependency of the glutamine synthetase was similar to that of other Bacillus enzymes but differed from that of the enteric bacteria enzymes. The kinetic properties of the enzyme, such as optimum pH, K m for each substrate, V max values, and inhibition by glutamine, were similar to those in the case of other Bacillus enzymes. The enzyme from this alkalophilic Bacillus was modified by iodoacetoamide or the ATP analog, 5'-p-fluorosulfonyl benzoyladenosine (FSBA). Only Mg 2+ -dependent activity was decreased by iodoacetamide, whereas each of the Mn 2+ -, Mg 2+ -, and Mg 2+ plus Mn 2+ -dependent activities was decreased with FSBA. The sequence of the NH 2 -terminal 20 amino acids of alkalophilic Bacillus glutamine synthetase was similar to those of other Bacillus enzymes.

The Polyphosphate- and ATP-Dependent Glucokinase from Propionibacterium shermanii: Both Activities Are Catalyzed by the Same Protein

The glucokinase (EC 2.7.1.63) from Propionibacterium shermanii phosphorylates glucose using inorganic polyphosphate (poly(P)) or ATP as the phosphate donor. In this investigation, we have purified the glucokinase to homogeneity, using two methods and show that the polyphosphate and ATP-dependent glucokinase activities eluted as a single protein. The protein peak is shown to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, reverse phase HPLC, and N-terminal sequence analysis. The purified protein eluted as a single peak from gel filtration and hydrophobic interaction HPLC columns and was found to display both the poly(P) and ATP glucokinase activities. Likewise, the two activities comigrated on a native isoelectric focusing gel. In addition, two analogues of ATP with different reactive groups displayed different inhibition patterns with respect to ATP and poly(P). The 2′,3′-dialdehyde of ATP, whose reactive group is the dialdehyde of the ribose ring, showed competitive and noncompetitive patterns with respect to ATP and poly(P), respectively. While, 5′-p-fluorosulfonylbenzoyl adenosine, whose reactive sulfonyl fluoride group is related to the γ-phosphoryl group of ATP, displayed competitive inhibition patterns with both ATP and poly(P). These observations provide evidence that the polyphosphate and ATP-dependent glucokinase activities of P. shermanii are the catalytic properties of a single enzyme and that the two substrates may have different binding sites on the enzyme with a common phosphorylating center.

Kinetic properties of type-II ATP diphosphohydrolase from the tunica media of the bovine aorta

The kinetic properties of type-II ATP diphosphohydrolase are described in this work. The enzyme preparation from the inner layer of the bovine aorta, mostly composed of smooth muscle cells, shows an optimum at pH 7.5. It catalyzes the hydrolysis of triand diphosphonucleosides and it requires either Ca 2+ or Mg 2+ for activity. It is insensitive to ouabain (3 mM), an inhibitor of Na + K + -ATPase , to tetramisole (5 mM), an inhibitor of alkaline phosphatase, and to Ap 5A (100μM), an inhibitor of adenylate kinase. In contrast, sodium azide (10mM), a known inhibitor for ATPDases and mitochondrial ATPase, is an effective inhibitor. Mercuric chloride (10μM) and 5′- p-fluorosulfonylbenzoyl adenosine are also powerful inhibitors, both with ATP and ADP as substrates. The inhibition patterns are similar for ATP and ADP, thereby, supporting the concept of a common catalytic site for these substrates. Apparent K m and V max, obtained with ATP as the substrate, were evaluated at 23 ± 3 μM and 1.09 μmol P i/min per mg protein, respectively. The kinetic properties of this enzyme and its localization as an ectoenzyme on bovine aorta smooth muscle cells suggest that it may play a major role in regulating the relative concentrations of extracellular nucleotides in blood vessels.

Stepwise phosphorylation of vesicular stomatitis virus P protein by virion-associated kinases and uncoupling of second step from in vitro transcription

Transcription-competent cores of vesicular stomatitis virus (VSV) contain two tightly bound protein kinase activities capable of phosphorylatiog the viral P protein (Beckes and Perrault, Virology 184, 383–386, 1991). We examined here the specificity of these kinases for the P protein substrate and their activity during the in vitro transcription process. Conditions favoring the VSVK1 kinase activity resulted in phosphorylation of the P1 species predominantly whereas conditions favoring VSVK2, or transcription conditions, led to an increase in the proportion of the faster migrating P2 and P3 species. A minimum of 2 mol phosphate/mol P protein was incorporated in 1 hr under optimal transcription conditions. Pulse-chase experiments revealed that the VSVK2 activity converted phosphorylated P1 to P2/P3 species. Most or all of the sites modified by VSVK1 (serines only) mapped to the 78 amino acid-long N-terminal fragment of the P protein; additional serine acceptor sites of undetermined location were also phosphorylated under VSVK2 conditions. Pretreatment of virion cores with 5′- p-fluorosulfonylbenzoyl adenosine had little or no effect on P1 phosphorylation but inhibited P1 to P2/P3 conversion nearly completely, with no effect on subsequent transcription. Likewise, the addition of cell extracts had relatively little effect on P1 phosphorylation but strongly inhibited the appearance of P2/P3, without affecting concurrent transcription. We conclude that phosphorylation of the P protein during transcription in vitro is a two-step process carried out by two distinct kinase activities, but only the first step may be essential for viral mRNA synthesis.

Analysis of 40 S and 80 S complexes with mRNA as measured by sucrose density gradients and primer extension inhibition.

The technique of primer extension inhibition has been adapted to analyze the eukaryotic ribosome-mRNA interaction. Formation of the ribosome-mRNA complex was performed in a nuclease-treated rabbit reticulocyte lysate. Before primer extension analysis, however, the complex is isolated by sucrose gradient centrifugation. Both 80 S- and 40 S-mRNA complexes can be individually analyzed because of this isolation step. 80 S ribosomes and 40 S ribosomal subunits could be localized at the initiation codon by a number of independent means where all complexes were formed in a manner consistent with the current understanding of the initiation pathway for translation in eukaryotes. Complexes were also isolated with the aid of the antibiotic edeine, where the 40 S ribosomal subunit was not located at the initiation codon, but 5' to the initiation codon. This extension inhibition assay was used to complement studies regarding the ATP dependence of the 40 S-mRNA interacting initiation steps that involve the mammalian RNA-interacting initiation factors eIF-4A, -4B, and -4F. A strong requirement for ATP was observed for 40 S-mRNA complex formation. A factor-mediated stimulation of complex formation by a combination of eIF-4A, -4B, and -4F was observed, and was one which required the presence of ATP. This factor-mediated ATP-dependent stimulation of complex formation was significantly inhibited by preincubating eIF-4A with the ATP analog 5'-p-fluorosulfonylbenzoyl adenosine. Finally, all complexes accumulated to a significant degree were analyzed by the primer extension assay. It was found that the 40 S ribosomal subunit was positioned at the initiation codon for all variations tested.

Activation mechanism of rabbit skeletal muscle myosin light chain kinase 5′-p-Fluorosulfonylbenzoyl adenosine as a probe of the MgATP-binding site of the calmodulin-bound and calmodulin-free enzyme

5′-p-Fluorosulfonylbenzoyl adenosine (FSBA), an ATP-like affinity labelling reagent, reacted with rabbit skeletal muscle myosin light chain kinase (skMLCK) and its calmodulin complex in a site-specific manner. Reaction was dependent upon the presence of the adenosine moiety of FSBA, saturated with increasing FSBA, was inhibited by MgATP, and was accompanied by stoichiometric incorporation of [14C]FSBA. The kinetic constants describing the reaction were similar for skMLCK and its calmodulin complex: k3= −0.040 min−1 and −0.038 mint-1, and Ki=0.18 mM and 0.40 mM, respectively. It is concluded that the MgATP-binding site on skMLCK remains accessible at all times and maintains a near constant conformation.

Irreversible inactivation of adenylyl cyclase by the “P”-site agonist 2',5'-dideoxy-,3'-p-fluorosulfonylbenzoyl adenosine.

2',5'-Dideoxy,3'-p-fluorosulfonylbenzoyl Adenosine (2',5'-dd3'-FSBA) was synthesized and found to be an agonist and affinity label for the "P"-site of adenylyl cyclase. This compound irreversibly inactivated both a crude detergent-dispersed adenylyl cyclase from rat brain and the partially purified enzyme from bovine brain. The irreversible inactivation by 100 to 200 microM 2',5'-dd3'-FSBA was blocked in a concentration-dependent manner by several established P-site inhibitors of adenylyl cyclase, 2',5'-dideoxyadenosine, 2'-d3'-AMP, adenosine, and 2'-deoxyadenosine, but not by inosine, N6-(phenylisopropyl)adenosine, adenine, 2'-d3':5'-cAMP, or 5'-AMP, agents known not to act at the P-site. Moreover, irreversible inactivation by 2',5'-dd3'-FSBA occurred in the presence of ATP at concentrations up to 3 mM, making it unlikely that inactivation was due to an effect on the enzyme's catalytic site. Adenylyl cyclase was also irreversibly inactivated by 5'-FSBA, although modestly (less than 20%) and apparently nonspecifically. Dithiothreitol protected the enzyme from irreversible inactivation by 2',5'-dd3'-FSBA, but reversible inhibition of the enzyme was still observed, although with reduced potency. When 2 mM dithiothreitol was added after a 30-min preincubation with 2',5'-dd3'-FSBA, the rat brain enzyme was partially (approximately 80%) reactivated. The data suggest that 2',5'-dd3'-FSBA may irreversibly inactivate adenylyl cyclase by reacting with a cysteinyl moiety in proximity to the P-site domain of the enzyme. These data together with results of studies of P-site inhibition kinetics published elsewhere (Johnson, R. A., and Shoshani, I. (1990) J. Biol. Chem. 265, 11595-11600) strongly suggest that the P-site and catalytic site are distinct domains on the enzyme. 2',5'-dd3'-FSBA, and especially its radiolabeled analog, should prove to be a useful probe for structural studies of adenylyl cyclase, particularly with regard to the P-site.