Cyclic nucleotide phosphodiesterases in Drosophila melanogaster

Cyclic nucleotide PDEs (phosphodiesterases) are important enzymes that regulate intracellular levels of cAMP and cGMP. In the present study, we identify and characterize novel PDEs in the genetic model, Drosophila melanogaster. The Drosophila genome encodes five novel PDE genes in addition to dunce. Predicted PDE sequences of Drosophila show highly conserved critical domains when compared with human PDEs. Thus PDE-encoding genes of D. melanogaster are CG14940-PDE1C, CG8279-PDE6β, CG5411-PDE8A, CG32648-PDE9 and CG10231-PDE11. Reverse transcriptase–PCRs of adult tissues reveal widespread expression of PDE genes. Drosophila Malpighian (renal) tubules express all the six PDEs: Drosophila PDE1, dunce (PDE4), PDE6, PDE8, PDE9 and PDE11. Antipeptide antibodies were raised against PDE1, PDE6, PDE9 and PDE11. Verification of antibody specificity by Western blotting of cloned and expressed PDE constructs allowed the immunoprecipitation studies of adult Drosophila lysates. Biochemical characterization of immunoprecipitated endogenous PDEs showed that PDE1 is a dual-specificity PDE (Michaelis constant Km for cGMP: 15.3±1 μM; Km cAMP: 20.5±1.5 μM), PDE6 is a cGMP-specific PDE (Km cGMP: 37±13 μM) and PDE11 is a dual-specificity PDE (Km cGMP: 6±2 μM; Km cAMP: 18.5±5.5 μM). Drosophila PDE1, PDE6 and PDE11 display sensitivity to vertebrate PDE inhibitors, zaprinast (IC50 was 71±39 μM for PDE1, 0.65±0.015 μM for PDE6 and 1.6±0.5 μM for PDE11) and sildenafil (IC50 was 1.3±0.9 μM for PDE1, 0.025±0.005 μM for PDE6 and 0.12±0.06 μM for PDE11). We provide the first characterization of a cGMP-specific PDE and two dual-specificity PDEs in Drosophila, and show a high degree of similarity in structure and function between human and Drosophila PDEs.

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Soluble expression, purification and biochemical characterization of a C-7 cholesterol dehydrogenase from Drosophila melanogaster

Steroids ◽

10.1016/j.steroids.2019.108495 ◽

2019 ◽

Vol 152 ◽

pp. 108495

Author(s):

Zhangliang Zhu ◽

Chao Li ◽

Xiaotao Cheng ◽

Ying Chen ◽

Menglu Zhu ◽

...

Keyword(s):

Drosophila Melanogaster ◽

Biochemical Characterization ◽

Soluble Expression

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Genetic location and biochemical characterization of eye-colour mutants from natural populations of Drosophila melanogaster

Genome ◽

10.1139/g90-032 ◽

1990 ◽

Vol 33 (2) ◽

pp. 203-208 ◽

Cited By ~ 1

Author(s):

M. Luisa Aparisi ◽

Carmen Nájera

Keyword(s):

Drosophila Melanogaster ◽

Biochemical Characterization ◽

Natural Populations ◽

High Variability ◽

Genetic Location ◽

Eye Colour ◽

Different Seasons ◽

Allelism Tests ◽

Mutation And Selection

From six captures of Drosophila melanogaster carried out in three different habitats (cellar, vineyard, and pinewood) in two different seasons of the year (spring and autumn), 60 eye-colour mutations were isolated, which were reduced to 29 loci by means of allelism tests within and between populations. Forty-five of these mutations were analyzed genetically and biochemically; of these 33 turned out to be previously described mutants and mapped to a total of 17 loci. Twelve new mutants were discovered and they mapped to 12 new loci, distributed on chromosomes X, II, and III. The eye-colour mutants show large effects on the red and brown pigments. The high variability of the eye-colour loci is discussed in relation to the mutation and selection hypotheses.Key words: eye-colour mutants, variability, mapping, Drosophila melanogaster, pigment patterns.

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THE ANATOMY AND FUNCTION OF A SEGMENT OF THE X CHROMOSOME OF DROSOPHILA MELANOGASTER

Genetics ◽

10.1093/genetics/71.1.139 ◽

1972 ◽

Vol 71 (1) ◽

pp. 139-156

Author(s):

B H Judd ◽

M W Shen ◽

T C Kaufman

Keyword(s):

Drosophila Melanogaster ◽

X Chromosome ◽

Functional Group ◽

Deletion Mapping ◽

Mutant Type ◽

Functional Units ◽

Average Size ◽

And Function

ABSTRACT An average size chromomere of the polytene X chromosome of Drosophila melanogaster contains enough DNA in each haploid equivalent strand to code for 30 genes, each 1,000 nucleotides long. We have attempted to learn about the organization of chromosomes by asking how many functional units can be localized within a chromomere. This was done by 1) recovery of mutants representative of every cistron in the 3A2-3C2 region; 2) the characterization of the function of each mutant type and grouping by complementation tests; 3) the determination of the genetic and cytological position of each cistron by recombination and deletion mapping. The data clearly show one functional group per chromomere. It is postulated that a chromomere is one cistron within which much of the DNA is regulatory in function.

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ISOLATION AND BIOCHEMICAL CHARACTERIZATION OF BRUSH BORDERS FROM RABBIT KIDNEY

The Journal of Cell Biology ◽

10.1083/jcb.47.3.637 ◽

1970 ◽

Vol 47 (3) ◽

pp. 637-645 ◽

Cited By ~ 114

Author(s):

Sosamma J. Berger ◽

Bertram Sacktor

Keyword(s):

Brush Border ◽

Renal Cortex ◽

Biochemical Characterization ◽

Rabbit Kidney ◽

Renal Tubules ◽

Marker Enzymes ◽

Alkaline Phosphatases ◽

Brush Borders ◽

Specific Activities

A technique for the isolation of intact brush borders from rabbit renal cortex was evaluated. The procedure was monitored by phase and electron microscopy and marker enzymes, i.e. ATP:NMN adenylyl transferase, nuclear; cytochrome oxidase, mitochondrial; ß-glucuronidase, lysosomal; and glucose-6-Pase, microsomal; and indicated an essentially pure preparation of brush borders. The disaccharidase, trehalase, previously reported in renal tubules, was localized uniquely in brush borders. Maltase was also found; the specific activities of the two enzymes in the brush borders were increased 10- to 20-fold. Other disaccharidases, such as sucrase, isomaltase, lactase, and cellobiase, were absent. It is suggested that trehalase and maltase are appropriate candidates for marker enzymes of the renal brush border. Isolated brush borders possessed a ouabain-sensitive (Na+ + K+) ATPase, an oligomycin-insensitive Mg++ ATPase, and a Ca++-activated ATPase. Alkaline phosphatases, dephosphorylating ß-glycero-P, and trehalose-6-P were also present. The specific activities of these enzymes were increased three-to-five fold in the brush-border preparations; however, activities were found in other subcellular fractions of the renal cortex. Hexokinase, although evident in the isolated brush border, was found prominently associated with other membranous fractions. Phosphoglucomutase and UDPG pyrophosphorylase were localized in the soluble fraction of the renal cortex.

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Molecular cloning and biochemical characterization of rabbit factor XI

Biochemical Journal ◽

10.1042/bj20020232 ◽

2002 ◽

Vol 367 (1) ◽

pp. 49-56 ◽

Cited By ~ 13

Author(s):

Dipali SINHA ◽

Mariola MARCINKIEWICZ ◽

David GAILANI ◽

Peter N. WALSH

Keyword(s):

Human Factor ◽

Biochemical Characterization ◽

Protein A ◽

Size Exclusion ◽

Factor Xi ◽

Sds Page ◽

Exclusion Chromatography ◽

Intracellular Process ◽

And Function

Human factor XI, a plasma glycoprotein required for normal haemostasis, is a homodimer (160kDa) formed by a single interchain disulphide bond linking the Cys-321 of each Apple 4 domain. Bovine, porcine and murine factor XI are also disulphide-linked homodimers. Rabbit factor XI, however, is an 80kDa polypeptide on non-reducing SDS/PAGE, suggesting that rabbit factor XI exists and functions physiologically either as a monomer, as does prekallikrein, a structural homologue to factor XI, or as a non-covalent homodimer. We have investigated the structure and function of rabbit factor XI to gain insight into the relation between homodimeric structure and factor XI function. Characterization of the cDNA sequence of rabbit factor XI and its amino acid translation revealed that in the rabbit protein a His residue replaces the Cys-321 that forms the interchain disulphide linkage in human factor XI, explaining why rabbit factor XI is a monomer in non-reducing SDS/PAGE. On size-exclusion chromatography, however, purified plasma rabbit factor XI, like the human protein and unlike prekallikrein, eluted as a dimer, demonstrating that rabbit factor XI circulates as a non-covalent dimer. In functional assays rabbit factor XI and human factor XI behaved similarly. Both monomeric and dimeric factor XI were detected in extracts of cells expressing rabbit factor XI. We conclude that the failure of rabbit factor XI to form a covalent homodimer due to the replacement of Cys-321 with His does not impair its functional activity because it exists in plasma as a non-covalent homodimer and homodimerization is an intracellular process.

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Genetic and biochemical characterization of little isoxanthopterin (lix), a gene controlling dihydropterin oxidase activity in Drosophila melanogaster

MGG Molecular & General Genetics ◽

10.1007/bf00290656 ◽

1991 ◽

Vol 230 (1-2) ◽

pp. 97-103 ◽

Cited By ~ 6

Author(s):

Francisco J. Silva ◽

Baltasar Escriche ◽

Eugenio Ordoño ◽

Juan Ferré

Keyword(s):

Drosophila Melanogaster ◽

Oxidase Activity ◽

Biochemical Characterization

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N-linked glycosylation enzymes in the diatom Thalassiosira oceanica exhibit a diel cycle in transcript abundance and favor for NXT-type sites

Scientific Reports ◽

10.1038/s41598-021-82545-1 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Joerg Behnke ◽

Alejandro M. Cohen ◽

Julie LaRoche

Keyword(s):

Cell Biology ◽

Biochemical Characterization ◽

Solid Phase ◽

Transcript Abundance ◽

Growth Conditions ◽

Iron Starvation ◽

Glycosylation Sites ◽

Glycosylation Pathway ◽

And Function

AbstractN-linked glycosylation is a posttranslational modification affecting protein folding and function. The N-linked glycosylation pathway in algae is poorly characterized, and further knowledge is needed to understand the cell biology of algae and the evolution of N-linked glycosylation. This study investigated the N-linked glycosylation pathway in Thalassiosira oceanica, an open ocean diatom adapted to survive at growth-limiting iron concentrations. Here we identified and annotated the genes coding for the essential enzymes involved in the N-linked glycosylation pathway of T. oceanica. Transcript levels for genes coding for calreticulin, oligosaccharyltransferase (OST), N-acetylglucosaminyltransferase (GnT1), and UDP-glucose glucosyltransferase (UGGT) under high- and low-iron growth conditions revealed diel transcription patterns with a significant decrease of calreticulin and OST transcripts under iron-limitation. Solid-phase extraction of N-linked glycosylated peptides (SPEG) revealed 118 N-linked glycosylated peptides from cells grown in high- and low-iron growth conditions. The identified peptides had 81% NXT-type motifs, with X being any amino acids except proline. The presence of N-linked glycosylation sites in the iron starvation-induced protein 1a (ISIP1a) confirmed its predicted topology, contributing to the biochemical characterization of ISIP1 proteins. Analysis of extensive oceanic gene databases showed a global distribution of calreticulin, OST, and UGGT, reinforcing the importance of glycosylation in microalgae.

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