SAR (structure-activity relationship) studies of triazafluorenone derivatives as potent mGluR1 antagonists are described. The triazafluorenone derivatives are non-amino acid derivatives and noncompetitive mGluR1 antagonists that bind at a putative allosteric recognition site located within the seven-transmembrane domain of the receptor. These triazafluorenone derivatives are potent, selective, and systemically active mGluR1 antagonists. Compound 1n, for example, was a very potent mGluR1 antagonist (IC50 = 3 nM) and demonstrated full efficacy in various in vivo animal pain models.

Chloroplast fructose-i ,6-bisphosphatase (Fru-P2-ase) is an essential enzyme in the photosynthetic pathway of carbon dioxide fixation into sugars. The properties of the chloroplast enzyme are clearly distinct from cytosolic gluconeogenic Fru-P2-ases. Light-dependent activation by way of a ferredoxin/thioredoxin system and insensitivity to AMP inhibition are distinctive characteristics of the chloroplast enzyme. However, the chloroplast enzyme shows a high degree of amino acid sequence similarity to gluconeogenic Fru-P2-ases. Sequence data reported for a total of 285 residues (=75% of the structure) of the spinach chloroplast enzyme reveals a 46% amino acid sequence identity with pig kidney Fru-P2-ase. We now report the amino acid sequence of a region consisting of 46 additional residues. This region is located near the middle of the primary structure of the enzyme and it includes a 16-residue insert not present in other Fru-P2-ases. This sequence insert has two cysteines separated by only 4 amino acid residues (Cys-Val-Val-Asn-Val-Cys), a characteristic feature of at least three other enzymes containing redox-active cysteines. It appears likely that this region of chloroplast Fru-P2-ase is involved in light-dependent activation.Light plays an important role in the regulation of several enzymes involved in the synthetic or carbon-reduction phase of photosynthesis and in related biochemical processes. In most cases, light produces the activation of several chloroplast enzymes that are essentially inactive in the dark. The in vivo regulation of all these enzymes involves the ferredoxin/ thioredoxin system, which comprises ferredoxin, thioredoxin f or m, and ferredoxin/thioredoxin reductase. One of these light-regulated enzymes is chloroplast fructose-1,6-bisphosphatase , the enzyme that catalyzes the conversion offructose 1,6-bisphosphate to fructose 6-phosphate. The light-mediated activation Fru-P2-ase occurs in i'ivo by way of a ferredoxin/thioredoxin f system that converts an inactive oxidized form of the enzyme into reduced active Fru-P2-ase (see refs. 1 and 2). The activation process can be mimicked in vitro by reduction of the enzyme with dithiothreitol (3). In both cases, the activation reaction involves the reduction of a critical disulfide bond in the enzyme. This activation mechanism is a characteristic of chloroplast Fru-P2-ase that distinguishes the chloroplast enzyme from cytosolic Fru-P2-ase (4,5). In addition, the chloroplast enzyme is not sensitive to AMP inhibition (6, 7), a property of all cytosolic gluconeogenic Fru-P2-ases (4). Nevertheless, the chloroplast enzyme shows a high degree of amino acid sequence similarity with gluconeogenic Fru-P2-ases that suggests a common evolutionary origin for all Fru-P2-ases in spite of their different functions and modes of regulation (8, 9). Amino acid sequence data reported for a total of 285 residues (-75% of the structure) of the spinach chloroplast enzyme (9, 10) reveals a 46% sequence identity with pig kidney Fru-P2-ase. These studies have disclo...

We have identified a class of spinach plastid tRNA genes which do not require 5' upstream promoter elements for their expression in a chloroplast transcription system. The 5' DNA sequences flanking the trnR1 and trnS1 coding regions have little or no homology to previously characterized chloroplast promoter sequences. The deletion of the 5' DNA sequences from these genes to positions close to the start of the coding regions has little effect on their transcription in vitro. In addition, a synthetic DNA fragment homologous to the 5' region of trnS1 does not support the transcription of the promoter (-) trnM2 mutant 51 in a promoter/trnM2-51 fusion assay. In a dicistronic construct the wild type trnS1 gene does not support transcription of trnS1 transcription occurs immediately following the 3' end of the coding region. Both trnS1 and trnR1 compete with trnM2 for the same chloroplast RNA polymerase and/or common transcription factors.

On the basis of kinetic activation assays, the apparent affinity of muscle phosphofructokinase for fructose 2,6-bisphosphate was about 9-fold greater than that for fructose 1,6-bisphosphate, which in turn was about 10 times higher than that for glucose 1,6-bisphosphate. Equilibrium binding experiments showed that both fructose bisphosphates bind to phosphofructokinase with negative cooperativity; the affinity for fructose 2,6-bisphosphate was about 1 order of magnitude greater than the affinity for fructose 1,6-bisphosphate. Binding of fructose 2,6-bisphosphate to phosphofructokinase was antagonized by fructose 1,6-bisphosphate and glucose 1,6-bisphosphate and vice versa. Both fructose bisphosphates promoted aggregation of the enzyme to higher polymers as indicated by sucrose density gradient centrifugation. Other indicators of phosphofructokinase conformation such as thiol reactivity and maximum activation of in vitro phosphorylation by the catalytic subunit of cyclic AMP-dependent protein kinase gave identical results in the presence of fructose 2,6-bisphosphate, fructose 1,6-bisphosphate, or glucose 1,6-bisphosphate, indicating a common conformation is produced by all three ligands. It is concluded that the sugar bisphosphates bind to a single site on the enzyme.

No abstract

Two cysteine residues in phosphoenolpyruvate (PEP) carboxykinase from Saccharomyces cerevisiae [ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49] the modification of which leads to enzyme inactivation have been subjected to site-directed mutagenesis. PEP carboxykinase is inactivated by alkylation of Cys365 or Cys458; however, mutation of either or both of these residues to serine has little effect on the enzymatic activity. These results eliminate any possible catalytic function for these cysteinyl residues. In the course of this work, discrepancies in the published nucleotide sequence of the S. cerevisiae PEP carboxykinase gene were detected that alter the deduced amino acid sequence. Several of these discrepancies were verified through the sequencing of proteolytic peptides. Our results indicate that the protein corresponds to a 549 amino acid polypeptide and that the positions previously assigned to Cys364 and Cys457 correspond to Cys365 and Cys458. The individual reactivities and the microenvironment characteristics around these sulfhydryl groups were investigated by their selective modification with the fluorescent reagent N-(1-pyrenyl)maleimide (PyM). Our findings indicate that Cys458 is 7-fold more reactive toward the sulfhydryl-directed probe than Cys365, while quenching experiments of PyM-labeled mutant enzymes suggest that the former residue is located in a region more accessible to water than the latter.

A new class of agents with potential utility for the treatment of erectile dysfunction has been discovered, guided by the hypothesis that selective D4 agonists are erectogenic but devoid of the side effects typically associated with dopaminergic agents. The lead agent 2-(4-pyridin-2-ylpiperazin-1-ylmethyl)-1H-benzimidazole (1, ABT-724) was discovered by optimization of a series of benzimidazole arylpiperazines. This highly selective D4 agonist was found to be very potent and efficacious in vivo, eliciting penile erections in rats at a dose of 0.03 micromol/kg, with a positive response rate of 77% erectile incidence. Even at high doses, it was devoid of side effects in animal models of central nervous system behaviors, emesis, or nausea. The structure-activity relationship of the parent benzimidazole series leading to 1 is described, with the detailed in vitro and in vivo profiles described. Distinctive structural features were discovered that are associated with D4 selective agonism in this series of analogues.

Cytosolic fructose-l,6-bisphosphatase from spinach leaves was purified to homogeneity and characterized. The pure enzyme has a subunit mass of 38 kDa, its K,,, values for fructose 1,6-bisphosphate and Mg2+ are 1.5 pM and 260 mM, respectively, and its V,,,,, is 110-120 units/mg. It is inhibited by fructose 2,6-bisphosphate and AMP with Ki values of 0.07 pM and 120 pM, respectively. About 90% of the primary structure of the spinach cytosolic fructose-l,6-bisphosphatase has been determined by amino-acid sequencing. The sequence data demonstrate that the cytosolic enzyme lacks the sequence insert characteristic of chloroplast fructose-l,6-bisphosphatase. The data include also the sequences of peptides containing all seven cysteine residues. Only two of the seven cysteines are conserved between the two isozymes, none of which is believed to be involved with the light regulation of the chloroplast enzyme. Sequence comparisons between the spinach cytosolic enzyme and gluconeogenic fructose-l,6-bisphosphatases from other species reveal similarity ranging over 47 -%yo, which is higher than the 40 -45% similarity between the chloroplast enzyme and gluconeogenic fructose-l,6-bisphosphatases. However, similarity between these isozymes and Escherichia coli fructose-l,6-bisphosphatase are 44% and 47% for the cytosolic and chloroplast enzymes, respectively. Similarity between the cytosolic and chloroplast counterparts is 52%, indicating wide divergence between these two fructose-l,6-bisphosphatases.