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E G Fragoulis

Publications and source records attributed to E G Fragoulis.

At least 19 recordsLinked to original sources
PubMed2001

L-DOPA decarboxylase association with membranes in mouse brain.

This work presents evidence on the association of active DDC molecules with membranes in mammalian brain. L-DOPA decarboxylase (DDC) is generally considered to be a cytosolic enzyme. Membrane-associated DDC was detected by immunoblotting and enzymatic assay experiments. DDC activity and immunoreactivity could be partially extracted from mammalian brain membranes by detergent. Fractionation of membranes by temperature-induced phase separation in Triton X-114, resulted in the recovery of membrane-associated DDC in separation phases where integral and hydrophobic membrane proteins separate. Treatment of membranes with phosphatidylinositol-specific phospholipase C or proteinase K, did not elute membrane-associated DDC activity, suggesting that a population of DDC molecules exist embedded within membranes. The elucidation of the functional significance of the enzyme's association with membranes could provide us with new information leading to the better understanding of the biological pathways that DDC is involved in.

Animals↗
PubMed2000

Isolation and sequencing of a cDNA encoding for a ribonuclease from the insect Ceratitis capitata.

Two overlapping clones encoding for a ribonuclease from six-day-old larvae of the insect Ceratitis capitata (Cc-RNase) have been isolated by immunoscreening a cDNA library and by 5' RACE. The sequence of the Cc-RNase cDNA contains an open reading frame of 414 nucleotides encoding for a precursor protein of 138 amino acids long with a putative signal peptide consisting of 19 amino acids. The calculated M(r) of the mature protein was found to be 13.7 kDa. Multiple alignments of the deduced amino acid Cc-RNase sequence with other ribonucleases revealed an approximate 25% average identity. Despite the low percentage of identity, histidine and lysine residues which are essential for its catalytic activity, were found to be completely conserved. Furthermore, expression of the clone in E. coli resulted in the production of a recombinant product that showed strong immunoreactivity with anti-RNase specific antibodies. These results support the hypothesis that the identified clone encodes for a protein which is a new member of the RNase superfamily.

Amino Acid Sequence↗
PubMed1998

Two different cDNAs code for L-DOPA decarboxylase in the white prepuparium and eclosion developmental stages of the insect Ceratitis capitata1.

In insects the DOPA decarboxylase gene (Ddc) is expressed in the epidermis where dopamine derivatives promote cuticle sclerotization and the central nervous system participating in the synthesis of serotonin and dopamine. The temporal course of Ddc enzyme activity during the white prepuparium and eclosion developmental stages primarily reflects this role. Here we report that a cDNA clone for l-DOPA decarboxylase (EC 4.1.1.28) has been isolated from the white puparium stage of the insect Ceratitis capitata, the cDNA sequence has been determined and it corresponds to an mRNA of 1341 base pairs. The comparison of this cDNA sequence with that of the eclosion stage which has been previously isolated in our laboratory revealed that the cDNA from the eclosion stage codes for protein consisting of 431 amino acid (aa) residues whereas that from the white prepuparium stage for protein of 425 aa residues. In addition there is a region of 33 aa residues close to the N-terminal region of the deduced protein in which marked differences in the aa composition are observed. This result was further confirmed by Northern blot analysis in which mRNAs isolated from both developmental stages were only hybridized with their respective polymerase chain reaction synthesized stage specific probes. These results suggest that during development of the insect C. capitata two different mRNAs are produced at the white prepuparium and the eclosion stages, giving rise to two protein isoforms presumably in response to different signals.

Amino Acid Sequence↗
PubMed1998

Characterization of acidic ribosomal proteins from three developmental stages of the medfly Ceratitis capitata.

Acidic proteins extracted with 0.5 M NH4Cl and 50% ethanol from ribosomes of the medfly Ceratitis capitata showed two major bands of MW 15 and 17 kD after SDS electrophoresis. Isoelectrofocusing of acidic proteins resolved two groups of bands at pH 4.5 and 3.5. Similar patterns were observed both from the acidic ribosomal protein fraction and from total ribosomes, treated with RNase. Treatment with alkaline phosphatase reduced the number of bands with a shift to a higher pI, indicating dephosphorylation. The phosphorylation pattern of the acidic proteins changed at three different stages of development, six day larvae, white pupae and 0-2 days old embryos. The two protein groups correspond to multi-phosphorylated forms of eucaryotic acidic ribosomal proteins P1 and P2. This was shown by immunoblotting with specific monoclonal antibodies.

Animals↗
PubMed1997

cDNA cloning of L-dopa decarboxylase from the eclosion stage of the insect Ceratitis capitata. Evolutionary relationship to other species decarboxylases.

The cDNA encoding the L-dopa decarboxylase (ddc) from the eclosion stage of the insect Ceratitis capitata was isolated by PCR and a molecular cloning strategy. The isolated cDNA clone encoded a protein of 431 amino acids with a calculated molecular weight of 47,843 Da. Northern blot analysis of poly(A)+ RNA showed an approximately 2 kb transcript. The deduced protein sequence shares a high percentage of homology with Ddc protein sequences of other species. Furthermore, the molecular weight of the deduced protein agreed well with that of the purified Ddc from the same insect. Data base search revealed significant and extensive sequence similarities among prokaryotic and eukaryotic PLP-dependent decarboxylases including Ceratitis capitata and bacterial histidine decarboxylase (HDC), strongly suggesting an ancient and common origin for all PLP-dependent decarboxylases.

Amino Acid Sequence↗
PubMed1997

Purification and characterization of a novel poly(U), poly(C) ribonuclease from Saccharomyces cerevisiae.

A new ribonuclease from Saccharomyces cerevisiae, specific for poly(U) and poly(C) substrate, was purified near to homogeneity by successive fractionation with DEAE-Sepharose, Heparin-Sepharose and CM-Sepharose chromatography. The purified molecule detected by SDS/polyacrylimide gel electrophoresis has a molecular mass of 29 kDa. The optimum pH for the enzyme activity is 5.5-7 and its isoelectric point is 7.5. The purified enzyme was able to degrade 26S, 18S and 5S rRNAs as well as mRNA obtained from in vitro transcription. No catalytic activity was observed when the RNase was incubated with tRNA and double stranded substrate. Our findings suggest that this novel RNase may play an important role in the processing of RNA in Saccharomyces cerevisiae.

Chromatography, Affinity↗
PubMed1996

Purification and characterisation of L-DOPA decarboxylase from pharate pupae of Ceratitis capitata. A comparison with the enzyme purified from the white prepupae.

In this paper we describe the purification of L-DOPA decarboxylase (DDC) to homogeneity from the developmental stage just before the eclosion (pharate pupae) of Ceratitis capitata. The enzyme was found to have a mol wt of approximately 100,000 and to be composed of two identical subunits (50,000 mol wt each). Polyclonal antibodies raised against the isolated enzyme reacted with the 50,000 dalton subunit and precipitated enzyme activity. Furthermore, properties of the enzyme isolated from the pharate pupa stage, were compared with those of DDC purified from the white prepupa stage with respect to substrate specificity, response to polyclonal antibodies, behaviour towards different cations and dependence of enzyme activity on the concentration of pyridoxal phosphate.

Animals↗
PubMed1995

Ribosome-associated ribonucleases from six-day larvae of the insect Ceratitis capitata.

A 34 kDa, poly(U) and poly(C)-specific ribonuclease, is shown to be tightly bound on purified polysomes from six-day-old larvae of the insect Ceratitis capitata. High salt treatment (400 mM KCl) is necessary to release it completely from the polysomes. Removal of the RNase does not disrupt the structure of the ribosomes, as shown by centrifugation on sucrose gradients and poly U directed polyphenylalanine synthesis.

Animals↗
PubMed1994

In vitro translation of an intact mRNA coding for a poly(U), poly(C) specific ribonuclease isolated from six-day-old larvae of Ceratitis capitata by a modified extraction procedure.

Intact ribonucleic acid was prepared from six-day-old larvae of Ceratitis capitata, by enriching the guanidinium thiocyanate extraction procedure with a specific mixture for the active ribonuclease inhibition. RNA obtained by this means was then used as a source for the identification of mRNA coding for poly(U), poly(C) specific ribonuclease. The isolated poly(A+) RNA was translated in a cell-free protein synthesizing system. The presence of a poly(U), poly(C) ribonuclease among the newly synthesized products was detected by immunoprecipitation with anti-rabbit polyclonal antibodies against poly(U), poly(C) ribonuclease.

Animals↗
PubMed1992

Preparation of monoclonal antibodies against a poly(U), poly(C) specific ribonuclease prepared from the insect Ceratitis capitata.

A poly(U), poly(C) specific RNase of apparent MW 34 kDa has recently been purified from 6 day old larvae of the insect Ceratitis capitata. Two monoclonal antibodies were obtained by immunizing mice with this protein. Immunoblot analysis of the RNase revealed that both antibodies recognize the 34 kDa protein. Furthermore, immunoprecipitation experiments show that both antibodies were capable of precipitating the ribonuclease without affecting its catalytic activity.

Animals↗
PubMed1990

Purification and characterization of L-dopa decarboxylase from human kidney.

L-dopa decarboxylase has been purified to homogeneity from post mortem removed human kidneys. Homogeneity was examined by polyacrylamide gel electrophoresis (PAGE) analysis both in the presence and absence of SDS. The enzyme has a molecular weight of 100,000 daltons estimated by gel filtration and 50,000 daltons determined after SDS-PAGE. Human L-dopa decarboxylase therefore is a dimer. Polyclonal antibodies produced against human L-dopa decarboxylase react with the 50,000 daltons enzyme subunit after immuno-blotting and also precipitates enzyme activity. Activity against L-dopa is partially inhibited by 5-hydroxytryptophan (5-HTP). The effect of various cations on L-dopa decarboxylase activity has also been tested.

Adolescent↗
PubMed1990

L-dopa decarboxylase in Ceratitis capitata white puparia and human: a comparative study.

1. L-DOPA decarboxylase (DDC) from Ceratitis capitata and from human kidney have been purified by the same methodology. 2. Both enzymes show mol. wts of 100,000, consisting of two identical mol. wt subunits and solely decarboxylate L-DOPA. 3. In the presence of 5-hydroxytryptophan (5-HTP) only the DDC activity from human kidney is remarkably reduced. 4. Addition of exogenous coenzyme is essential only for human DDC activity. 5. Polyclonal antibodies, raised against DDC purified from insects or humans, cross-react with both antigens.

Animals↗
PubMed1987

Purification and characterization of a ribonuclease specific for poly(U) and poly(C) from the larvae of Ceratitis capitata.

A specific ribonuclease was detected and purified to homogeneity from six-day-old larvae of the insect Ceratitis capitata and its homogeneity was checked by analysis in polyacrylamide gels in the presence of sodium dodecyl sulfate. The nuclease specifically degrades poly(U) and poly(C) whilst it fails to do so with other single-stranded homopolyribonucleotides. The enzyme has a pH optimum in the region 7-9 and relative molecular mass of about 25,000. The effect of this ribonuclease on the integrity of RNAs isolated from six-day-old larvae or rat liver was also studied.

Animals↗
PubMed1985

Comparison of native and KCl treated 40S ribosomal subunits from the silkmoth Antherea pernyi and mammals.

Native 40S ribosomal subunits and 18S ribosomal RNA from ovarian follicles of the silkmoth A. pernyi showed a lower sedimentation coefficient in comparison to ascites cells, in contrast to the KCl treated 40S ribosomal subunits where no difference was observed in both tissues. Moreover the silkmoth native 40S ribosomal subunits--in contrast to the KCl treated ones--could not reassociate with radioactive ascites cell 60S ribosomal subunits. These results, combined with the great similarities in the two dimensional electrophoretic patterns of 40S ribosomal proteins from silkmoth follicles and other mammalian cells lead to the possibility of the existence of a specific RNase associated with the 40S ribosomal subunit.

Animals↗
PubMed1975

Translation of mRNA for 3,4-dihydroxyphenylalanine decarboxylase isolated from epidermis tissue of Calliphora vicina R. -D. in a heterologous system. Dependence of mRNA concentration on the insect steroid hormone ecdysone.

RNA was isolated from the epidermis of Calliphora vicina larvae by phenol--chloroform extraction. The RN A sedimenting in sucrose gradients between 5 and 18 S was submitted to chromatography on oligo(dT)-cellulose columns. The fraction binding to the oligo(dT) is able to stimulate protein synthesis in a system consisting of mouse liverribosomal subunits, pH-5 factors from rat liver and initiation factors from rabbit reticulocytes. Optimal Mg2+ concentration for the translation of insect mRNA is 3.5 mM, that of K+ 76 MM. Initiation factors prepared from epidermis of Calliphora larvae are less efficient in the translation of insect mRNA than initiation factors isolated from reticulocytes. The pH-5 fraction from epidermis inhibits protein synthesis independent of the source of the mRNA fraction used. One of the proteins synthesized in the reconstituted system under the direction of insect mRNA has been identified as 3,4-dihydroxyphenylalanine (DOPA) decarboxylase by immunoprecipitation with specific antiserum against DOPA decarboxylase and comigration in dodecylsulphate-acrylamide electrophoresis with pure DOPA decarboxylase. Both mRNA from white prepupae and from 6--7-days-old larvae contain sequences coding for DOPA decarboxylase. However, white prepupae contains 3--4 times more DOPA decarboxylase-mRNA than 6--7-days-old larvae. The content of DOPA decarboxylase mRNA is proportional to the amount of active DOPA decarboxylase molecules present in the animals from which the mRNA was isolated.

Animals↗
PubMed1975

Induction of dopa (3,4-dihydroxyphenylalanine) decarboxylase in blowfly integument by ecdysone. A demonstration of synthesis of the enzyme de novo.

The activity of the enzyme dopa (3,4-dihydroxyphenylalanine) decarboxylase, present in the epidermis cells of blowfly larvae, increases during the late third instar under the influence of the steroid hormone, ecdysone. By using the double-labelling technique and immune precipitation with univalent antibody to dopa decarboxylase, we demonstrated that the increase in enzyme activity was due to a stimulation of synthesis of enzyme molecules de novo. In this respect, the action of ecdysone is similar to the action of other steroid hormones.

Animals↗