L-myo-Inositol 1-phosphate synthase from Entamoeba histolytica.

L-myo-Inositol 1-phosphate synthase (I-1-P synthase) catalyses the primary reaction for the synthesis of inositol in a variety of prokaryotes, eukaryotes and in the chloroplasts of algae and higher plants. Inositol is a precursor of essential macromolecules like membrane phospholipids, GPI anchor proteins and lipophosphoglycans, which play a determinant role in the pathogenesis of protozoan parasites such as Leishmania and Entamoeba. However, there is no report of I-1-P synthase or its gene from these organisms. The gene INO1 coding for this enzyme was first cloned from Saccharomyces cerevisiae and subsequently from several plants. Using molecular cloning techniques we have isolated and characterised the INO1 gene coding for the enzyme I-1-P synthase from Entamoeba histolytica. Simultaneously, we have purified and characterised the native enzyme from E. histolytica trophozoites and the cloned gene product from Escherichia coli. The gene product and the purified enzyme were both shown to be recognised by a heterologous anti-I-1-P synthase antibody from the phytoflagellate Euglena gracilis. Phylogenetic analysis of I-1-P synthase sequences from different eukaryotes suggest that it is highly conserved across species and the origin of this enzyme precedes the evolutionary divergence of modern eukaryotes.

1L-myo-inositol-1-phosphate synthase.

1L-myo-Inositol-1-phosphate synthase catalyzes the conversion of D-glucose 6-phosphate to 1L-myo-inositol-1-phosphate, the first committed step in the production of all inositol-containing compounds, including phospholipids, either directly or by salvage. The enzyme exists in a cytoplasmic form in a wide range of plants, animals, and fungi. It has also been detected in several bacteria and a chloroplast form is observed in alga and higher plants. The enzyme has been purified from a wide range of organisms and its active form is a multimer of identical subunits ranging in molecular weight from 58,000 to 67,000. The activity of the synthase is stimulated by NH4Cl and inhibited by glucitol 6-phosphate and 2-deoxyglucose 6-phosphate. Structural genes (INO1) encoding the 1L-myo-inositol-1-phosphate synthase subunit have been isolated from several eukaryotic microorganisms and higher plants. In baker's yeast, Saccharomyces cerevisiae, the transcriptional regulation of the INO1 gene has been studied in detail and its expression is sensitive to the availability of phospholipid precursors as well as growth phase. The regulation of the structural gene encoding 1L-myo-inositol-1-phosphate synthase has also been analyzed at the transcriptional level in the aquatic angiosperm, Spirodela polyrrhiza and the halophyte, Mesembryanthemum crystallinum.

L-myo-lnositol 1-Phosphate Synthase from Plant Sources (Characteristics of the Chloroplastic and Cytosolic Enzymes).

L-myo-inositol 1-phosphate synthase (EC 5.5.1.4) from cyanobacterial (Spirulina platensis), algal (Euglena gracilis), and higher plant (Oryza sativa, Vigna radiata) sources was purified to electrophoretic homogeneity, biochemically characterized, and compared. Both chloroplastic and cytosolic forms of the enzyme were detected in E. gracilis, O. sativa, and V. radiata, whereas only the cytosolic form was detected in streptomycin-bleached or chloroplastic mutants of E. gracilis and in S. platensis. Both the chloroplastic and cytosolic forms from different sources could be purified following the same three-step chromatographic protocol. L-myo-inositol 1-phosphate synthases purified from these different sources do not differ significantly with respect to biochemical and kinetic parameters except for the molecular mass of the chloroplastic and cytosolic native holoenzymes, which appear to be homotetrameric and homotrimeric associations of their constituent subunits, respectively. Monovalent and divalent cations, sugar alcohols, and sugar phosphates are inhibitory to the enzyme activity. N-ethylmaleimide inhibition of synthase activity could be protected by the combined presence of the substrate glucose-6-phosphate and cofactor NAD+. Antibody raised against the cytosolic enzyme from E. gracilis immunoprecipitates and cross-reacts with both chloroplastic and cytosolic forms from the other sources studied.

Coordinate transcriptional induction of myo-inositol metabolism during environmental stress.

The pathway from glucose 6-phosphate (G 6-P) to myoinositol 1-phosphate (Ins 1-P) and myo-inositol (Ins) is essential for the synthesis of various metabolites. In the halophyte Mesembryanthemum crystallinum (common ice plant), two enzymes, myo-inositol O-methyltransferase (IMT1) and ononitol epimerase (OEP1), extend this pathway and lead to the accumulation of methylated inositols, D-ononitol and D-pinitol, which serve as osmoprotectants. This paper describes transcripts for the enzyme, Inps1, encoding myo-inositol 1-phosphate synthase (INPS1), from the ice plant. Two Inps-like sequences are present in the genome. The deduced amino acid sequences of the cloned transcript are 49.5% and 87-90%, respectively, identical to those of yeast and other higher plant sequences. Inps1 RNA amounts are upregulated at least fivefold and amounts of free Ins accumulate approximately 10-fold during salinity stress. Inps1 induction is by transcription, similar to the induction of Imt1. In contrast, Arabidopsis thaliana does not show upregulation of Inps1 or increased amounts of Ins when salt-stressed. The lack of Inps1 induction in Arabidopsis exemplifies differences in glycophytic and halophytic regulation of gene expression at the point of entry into a pathway that leads to osmoprotection. The stress-induced coordinate upregulation of this pathway and its extension by novel enzymes in the ice plant also highlights biochemical differences.

Divalent cations and chelators as regulators of brain fructose-1,6-bisphosphatase.

Purified fish and rat brain FruP2ase(s) are stimulated by a number of chelators, viz., histidine, EDTA, citrate, imidazole, and a number of histidine analogues. These also impart 5'-AMP sensitivity to the otherwise insensitive enzyme. Beyond 3 mM concentration, histidine inhibits the enzyme activity, which can be prevented by Mn2+. Atomic absorption spectrophotometry showed the presence of 5-6 mol of Mn2+ and Zn2+ bound to both fish and rat brain FruP2ase, which can be removed by exhaustive EDTA-dialysis. The EDTA-dialyzed brain FruP2ase records an absolute Mn2+ requirement and 5'-AMP sensitivity without any chelator treatment. The 5'-AMP sensitivity of such enzyme is abolished by prior incubation with Zn2+. The Zn(2+)-treated brain FruP2ase fails to bind to a Blue-Sepharose column, in contrast to that seen using the untreated enzyme. These results suggest that rat and fish brain FruP2ase(s) are actually Mn(2+)- and Zn(2+)-containing proteins with Zn2+ bound at or near the nucleotide-binding site.

Carboxymethylcellulase from Lenzites saepiaria, a brown-rotter.

Three wood-rotting fungi namely, Lenzites saepiaria, Polyporus xeranticus and Trametes gibbosa, were screened as cellulose-degraders from 20 different genera of both brown and white-rotters of Polyporaceae on the basis of their potential to degrade carboxymethylcellulose. The utilization of different carbon sources in the growth medium was studied with these fungi for identification of enzymes involved in saccharification. Carboxymethylcellulase and beta-glucosidase were identified as the two major enzymes involved in this process. Extracellular carboxymethylcellulase from L. saepiaria was purified to homogeneity and the enzyme partially characterized.

Beta-glucosidase of a white-rot fungus Trametes gibbosa.

Extracellular beta-glucosidase was purified from a white-rot fungus, Trametes gibbosa by 50% ammonium sulphate saturation and Sephadex G-100 column chromatography. It showed maximum activity towards p-nitrophenyl- beta-D- glucopyranoside (pNpG). The pH optimum was 3.5. Temperature optimum was 40 degrees C but shifted to 50 degrees C on preincubation with pNpG. Hg2+, Fe3+ and Cu2+ strongly inhibited the activity. The enzyme was competitively inhibited by glucose with a Ki of 5.2 mM. The apparent molecular mass as determined by gel filtration chromatography was 640 kDa.

Modulation of fish liver fructose-1, 6-bis phosphatase by ammonia stress.

Hepatic fructose 1, 6 bisphosphatase (Fru-P2ase) from Channa punctatus exhibits 100% and 60% elevations of its maximal catalytic rates (Vmax) at pH 7.5 and 9.5 respectively in summer over those in winter. Both the neutral and alkaline activities of the enzyme are enhanced remarkably (100%) in the winter-adapted fish due to exposure to 10 mg N/1 of unionized ammonia for three weeks. While the neutral activity of the purified enzyme is increased in vitro by NH+4 ions in dose-dependent manner upto about 150 mM NH4, the alkaline activity is found to be activated at a very low concentration of NH4+ (25 mM) and inhibited at 100-150 mM NH4+. The enzyme also shows a characteristics potentiation of 5'-AMP inhibition by NH4+ (125 mM).

Involvement of tyrosyl-tRNA synthetase in splicing of group I introns in Neurospora crassa mitochondria: biochemical and immunochemical analyses of splicing activity.

We reported previously that mitochondrial tyrosyl-tRNA synthetase, which is encoded by the nuclear gene cyt-18 in Neurospora crassa, functions in splicing several group I introns in N. crassa mitochondria (R. A. Akins and A. M. Lambowitz, Cell 50:331-345, 1987). Two mutants in the cyt-18 gene (cyt-18-1 and cyt-18-2) are defective in both mitochondrial protein synthesis and splicing, and an activity that splices the mitochondrial large rRNA intron copurifies with a component of mitochondrial tyrosyl-tRNA synthetase. Here, we used antibodies against different trpE-cyt-18 fusion proteins to identify the cyt-18 gene product as a basic protein having an apparent molecular mass of 67 kilodaltons (kDa). Both the cyt-18-1 and cyt-18-2 mutants contain relatively high amounts of inactive cyt-18 protein detected immunochemically. Biochemical experiments show that the 67-kDa cyt-18 protein copurifies with splicing and synthetase activity through a number of different column chromatographic procedures. Some fractions having splicing activity contain only one or two prominent polypeptide bands, and the cyt-18 protein is among the few, if not only, major bands in common between the different fractions that have splicing activity. Phosphocellulose columns resolve three different forms or complexes of the cyt-18 protein that have splicing or synthetase activity or both. Gel filtration experiments show that splicing activity has a relatively small molecular mass (peak at 150 kDa with activity trailing to lower molecular masses) and could correspond simply to dimers or monomers, or both, of the cyt-18 protein. Finally, antibodies against different segments of the cyt-18 protein inhibit splicing of the large rRNA intron in vitro. Our results indicate that both splicing and tyrosyl-tRNA synthetase activity are associated with the same 67-kDa protein encoded by the cyt-18 gene. This protein is a key constituent of splicing activity; it functions directly in splicing, and few, if any, additional components are required for splicing the large rRNA intron.

Chloroplast as a Locale of L-myo-Inositol-1-Phosphate Synthase.

Chloroplasts from 5 to 7 day old Vigna radiata seedling, grown under alternate light/dark conditions or from green Euglena gracilis Z. cells have been found to harbor L-myo-inositol-1-phosphate synthase (EC 5.5.1.4) activity. In contrast, dark-grown V. radiata seedlings, or streptomycin-bleached Euglena cells exhibit either reduced or no enzyme activity. An apparent enhancement of the chloroplastic inositol synthase by growth in presence of light is observed.

Modification of brain fructose-1,6-bisphosphatase activity by chelators: "induction" of 5'-AMP sensitivity.

Brain fructose-1,6-bisphosphatase (EC 3.1.3.11) from various sources are ordinarily insensitive to 5'-AMP. In addition to stimulation and conferring a "neutral" behaviour, prior treatment with histidine, EDTA or imidazole renders the brain enzyme sensitive to 5'-AMP. The histidine treated enzyme(s) bind to Blue-Sepharose CL-6B column and are specifically eluted by 5'-AMP in contrast to the untreated enzyme(s) which do not bind to the affinity column at all. The histidine effect in inducing 5'-AMP sensitivity was abolished by treatment of the native enzyme by subtilisin or by a number of divalent cations including Zn++.

Differences in thermal stability of the fetal and adult brain myoinositol-1-phosphate synthase. Probable involvement of NAD.

L-myo-inositol-1-phosphate synthase (EC 5.5.1.4) from mammalian fetal and adult brain differ considerably with respect to their stability towards different temperatures between 25-65 degrees C. This property has been found to be associated with the presence of the synthase co-factor, NAD, bound to the enzyme protein. The lower thermal stability of the fetal enzyme increases in presence of added NAD (0.8 mM) whereas the higher thermal stability of the adult brain enzyme declines when NAD is specifically removed from the enzyme.