Purification of choline acetyltransferase from Drosophila melanogaster

Choline acetyltransferase (EC 2.3.1.6) from Drosophila melanogaster (Canton S, wild type) was purified 12,500-fold to a final specific activity of 500 mumol min-1 mg protein-1. The purification used homogenized fly heads and consisted of polyethylene glycol precipitation, DEAE-Bio-Gel A chromatography, Octyl-Sepharose chromatography, and affinity chromatography using solid phase Green A-agarose. The molecular weight of the native enzyme, as determined by molecular exclusion chromatography, was approximately 67,000 daltons. The final enzyme preparation showed two major protein bands at 67,000 and 54,000 daltons on polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS). After molecular exclusion chromatography, both SDS gel bands were present in the single symmetrical peak that contained the enzyme activity. Two-dimensional tryptic peptide maps prepared from the individual SDS gel bands indicated that they have very similar primary structures. Both SDS gel bands were precipitated by two different monoclonal antibodies derived against Drosophila choline acetyltransferase activity. The structural and immunological relatedness of the two SDS gel bands indicates that the enzyme is essentially homogeneous and that, in the native state, it may consist of more than one polypeptide chain.     

Purification and characterization of prophenoloxidases from pupae of Drosophila melanogaster

The effects of heparin and other polyanions on the myotoxicity of Bothrops jararacussu venom and purified bothropstoxin (BthTX) were investigated. The release rate of creatine kinase (CK) from isolated extensor digitorum longus muscle and the plasma CK activity of mice were used to quantify the results. The myotoxic effects of B. jararacussu venom or BthTX were inhibited by preincubation of these agents with one of the following: a heterogeneous heparin preparation (designated 'heparin'), low mol. wt heparin (H-4500) or dextran sulfates (DS-8000 and DS-500,000). Non-sulfated dextran (D-40,000) and two chondroitin sulfates were ineffective. The antimyotoxic effects of the polyanions are ascribed to their forming inactive acid-base complexes with the basic myotoxins of Bothrops venoms. Gel-filtration experiments in Sephadex provided direct evidence for complex formation between heparin and BthTX. Intravenous (i.v.) administration of H-4500 or DS-8000 opposed the increase in plasma CK activity induced by a subsequent i.m. injection of venom or BthTX. In contrast, pretreatment with i.v. heparin or DS-500,000 enhanced the venom-induced increase in plasma CK activity. This effect was not observed (1) when the animals were treated with a polyvalent antivenom, which inhibits the coagulation and local stasis induced by Bothrops venoms, and (2) when BthTX, which has no thrombotic or hemorrhagic properties, was the myotoxic agent. The potentiation of the venom-induced increase in plasma CK activity by heparin and DS-500,000 is ascribed to improved washout of the CK released from damaged fibers, because of the anticoagulant properties of the drugs.     

Mutational analysis of N-linked glycosylation of esterase 6 in Drosophila melanogaster

The primary sequence of the esterase 6 (EST6) enzyme of Drosophila melanogaster contains four potential N-linked glycosylation sites, at residues 21, 399, 435, and 485. Here we determine the extent to which EST6 is glycosylated and how the glycosylation affects the biochemistry and physiology of the enzyme. We have abolished each of the four potential glycosylation sites by replacing the required Asn residues with Gln by in vitro mutagenesis. Five mutant genes were made, four containing mutations of each site individually and the fifth site containing all four mutations. Germline transformation was used to introduce the mutant genes into a strain of D. melanogaster null for EST6. Electrophoretic and Western blot comparisons of the mutant strains and wild-type controls showed that each of the four potential N-linked glycosylation sites in the wild-type protein is glycosylated. However, the fourth site is not utilized on all EST6 molecules, resulting in two molecular forms of the enzyme. Digestion with specific endoglycosidases showed that the glycan attached at the second site is of the high-mannose type, while the other three sites carry more complex oligosaccharides. The thermostability of the enzyme is not affected by abolition of the first, third, or fourth glycosylation sites but is reduced by abolition of the second site. Anomalously, abolition of all four sites together does not reduce thermostability. Quantitative comparisons of EST6 activities showed that abolition of glycosylation does not affect the secretion of the enzyme into the male sperm ejaculatory duct, its transfer to the female vagina during mating, or its subsequent translocation into her hemolymph. However, the activity of the mutant enzymes does not persist in the female's hemolymph for as long as wild-type esterase 6. The latter effect may compromise the role of the transferred enzyme in stimulating egg-laying and delaying receptivity to remating.     

Cloning and sequence analysis of cDNA encoding a putative juvenile hormone esterase from the Colorado potato beetle

In the Colorado potato beetle, Leptinotarsa decemlineata, reproduction and diapause are mediated by the juvenile hormone (JH) titer in the hemolymph. This titer is controlled by JH synthesis in the corpora allata and by JH degradation. The main pathway of JH degradation is by JH esterase in the hemolymph. The native JH esterase appeared to be a dimer consisting of two 57 kDa subunits (Vermunt et al., 1997). The 57 kDa subunit of JH esterase was digested with endoproteinase Lys-C and the digestion products were separated by reversed phase HPLC. Three different peptides were collected and sequenced. The amino acid sequence of one peptide showed high similarity to fragments of other insect esterases. Based on the amino acid sequence of these peptides, degenerate primers were constructed for RT-PCR. A PCR product of 1.3 kb was obtained and sequenced. This product was used to screen a cDNA library for a complete cDNA copy and to analyze the messenger RNA from larvae and adult beetles. The size of the messenger RNA was 1.7 kb. The complete amino acid sequence of the protein was deduced from the nucleotide sequence of overlapping clones from a cDNA library and a 5'RACE product. An open reading frame (ORF) of 1545 base pairs encoded a 57 kDa protein with a predicted pI of 5.5. The ORF contained the sequence of the three peptides. It showed no significant homology to other proteins present in databases, but it did contain several functional esterase motifs.     

Disruption of lysosomal targeting is associated with insecticidal potency of juvenile hormone esterase

Juvenile hormone esterase (JHE; EC 3.1.1.1), which is intrinsically involved in regulation of development of some insect larvae, is rapidly removed from the hemolymph by the pericardial cells. Lys-29 and Lys-524, which are implicated in the degradation of JHE, were mutated to Arg. Neither the half-life of the modified JHE in the hemolymph nor the catalytic parameters were changed significantly, but when combined, these mutations resulted in apparent failure of lysosomal targeting in the pericardial cell complex. A hypothesis for the mechanism of reduced efficiency of lysosomal targeting is presented. Infection of larvae with a recombinant baculovirus expressing the modified JHE resulted in a 50% reduction in feeding damage compared with larvae infected with the wild-type virus, thus demonstrating improved properties as a biological insecticide. These data demonstrate that alteration of specific residues of JHE that disrupted lysosomal targeting, dramatically increased the insecticidal activity of this protein.     

Purification and characterization of a cephalosporin esterase from Rhodosporidium toruloides

A novel cephalosporin esterase (EC 3.1.1.41) from Rhodosporidium toruloides was purified to gel electrophoretic homogeneity. The enzyme is a glycoprotein with a molecular mass of 80 kDa. Upon deglycosylation, several forms of the enzyme were observed with a molecular mass range between 60 and 66 kDa. The isoelectric point of the enzyme is approximately 5.6, with the pH optimum for activity occurring at 6.0. The optimal activity of the enzyme occurred at 25 degrees C, with the enzyme rapidly losing activity at temperatures above 25 degrees C. The enzyme deacetylated a variety of cephalosporin derivatives, including cephalosporin C; the Km for this substrate is 51.8 mM, and the Vmax is 7.9 mumol/min/mg. In addition to cephalosporins, the enzyme hydrolyzed short-chain p-nitrophenyl esters, with the activity decreasing with increasing ester chain length. The enzyme also has the ability to acetylate desacetyl cephalosporins in high yields under mild conditions in the presence of various acetyl donors. A comparison of the physical properties of the esterase with those of other well-characterized cephalosporin esterases indicates that the enzyme is unique in this class.     

Purification and characterisation of a protein kinase from winged bean

A soluble, cytoplasmic protein kinase was purified from the developing seeds of winged bean (Psophocarpus tetragonolobus) following conventional methods of protein purification including anion-exchange chromatography, gel-filtration and Blue Sepharose chromatography. The purified enzyme consists of a single polypeptide of M(r) 45,000 as determined by SDS-PAGE and gel-filtration chromatography on Sephacryl S-200. The pH optimum of the protein kinase activity was 7.0, while the optimum concentration of Mg2+ was 5 mM. The enzyme utilised casein as an exogenous phosphate acceptor. The conventional modulators of protein kinases, including the cyclic nucleotides, Ca2+ and calmodulin, did not stimulate the purified enzyme. Heparin and spermine, too, had no effect on its activity. Phosphoamino acid analysis revealed that the enzyme transferred the gamma-phosphate of ATP only to serine residues of casein. All these characteristics, taken together, classifies the purified protein kinase as a member of the casein kinase I group of enzymes.     

Purification and characterization of a feruloyl esterase from the fungus Penicillium expansum

An extracellular phenolic acid esterase produced by the fungus Penicillium expansum in solid state culture released ferulic and rho-coumaric acid from methyl esters of the acids, and from the phenolic-carbohydrate esters O-[5-O-(trans-feruloyl)-alpha-L-arabinofuranosyl]-(1-->3)-O-beta- D-xylopyranosyl-(1-->4)-D-xylopyranose (FAXX) and O-[5-O-((E)-rho-coumaroyl)-alpha-L-arabinofuranosyl]- (1-->3)-O-beta-D-xylopyranosyl-(1-->4)-D-xylopyranose (PAXX). The esterase was purified 360-fold in successive steps involving ultrafiltration and column chromatography by gel filtration, anion exchange and hydrophobic interaction. These chromatographic methods separated the phenolic acid esterase from alpha-L-arabinofuranosidase, pectate and pectin lyase, polygalacturonase, xylanase and beta-D-xylosidase activities. The phenolic acid esterase had an apparent mass of 65 kDa under non-denaturing conditions and a mass of 57.5 kDa under denaturing conditions. Optimal pH and temperature were 5.6 and 37 degrees C, respectively and the metal ions Cu2+ and Fe3+ at concentrations of 5 mmol 1-1 inhibited feruloyl esterase activity by 95% and 44%, respectively, at the optimum pH and temperature. The apparent Km and Vmax of the purified feruloyl esterase for methyl ferulate at pH 5.6 and 37 degrees C were 2.6 mmol 1-1 and 27.1 mumol min-1 mg-1. The corresponding constants of rho-coumaroyl esterase for methyl coumarate were 2.9 mmol 1-1 and 18.6 mumol min-1 mg-1.     

New mutants of Drosophila melanogaster

Characteristics are given of 57 Drosophila melanogaster mutants catched in the South and Soeth-West Iran.     

Purification and characterisation of a secreted aminopeptidase from adult Ascaris suum

A metalloaminopeptidase was identified in culture fluids collected during in vitro cultivation of adult Ascaris suum. The enzyme was purified by anion-exchange and size-exclusion HPLC. The M(r) of the enzyme was estimated at 293 kDa and consisted of subunits with M(r)s of 153 and 142kDa. The isoelectric point of the aminopeptidase was 4.7. The aminopeptidase displayed a substrate preference for terminal arginyl residues. Aminopeptidase activity was also present in muscle, female reproductive tissue, pharynx, pseudocoelomic fluid and intestine. Among the various tissues, aminopeptidase activity was highest in the intestines; the highest activity was found in culture fluids (three-fold higher than intestinal tissue). The aminopeptidase released by adult A. suum was enzymatically and biochemically identical to an aminopeptidase released during in vitro development of A. suum third- to fourth-stage larvae.     

Purification and characterisation of a tripeptidyl aminopeptidase I from rat spleen

Proliferation of vascular smooth muscle cells (VSMC) is a principal event in neointima formation in saphenous vein-coronary artery bypass grafts. Since endothelin-1 (ET-1) promotes VSMC replication and ET-1 receptor antagonists inhibit neointima formation in arterial injury models, it is reasonable to propose that ET-1 may be involved in neointima formation in vein grafts. However, it is not known what alterations of ET-1 and its receptors (if any) occur in vein grafts. The objective of this study, therefore, was to investigate the distribution of ET-1 and ET-1 receptor subtypes (ET(A) and ET(B)) in porcine vein grafts. Unilateral interposition saphenous vein grafting was performed by end to end anastomosis after excision of a segment of carotid artery in Landrace pigs. One month after surgery, vein grafts, ungrafted saphenous veins and carotid arteries were excised, ET-1 immunoreactivity identified by immunocytochemistry and ET(A) and ET(B) receptor subtypes studied using autoradiography. In vein grafts, there was a greater density of ET(A) compared to ET(B) receptors in both the tunica media and neointima. ET(A) binding in the tunica media of ungrafted saphenous vein was greater than that in the carotid artery or vein grafts, but greater in the vein graft compared to the carotid artery. Immunoreactive ET-1 was located in endothelial cells and throughout the neointima of the vein graft. Dense ET-1 binding (to both ET(A) and ET(B) receptors) was also associated with microvessels in the adventitia within the graft. In vein grafts, there was strong ET(B) binding to neutrophils which were present in high numbers at the subendothelium and within the adventitia. It is concluded ET(A) receptors may play a role in vein graft thickening at the medial and neointimal VSMC level, whereas ET(B) receptors may play a role in microangiogenesis. The higher levels of ET(A) receptors in the tunica media of ungrafted saphenous vein relative to the carotid artery and vein graft may also render this conduit susceptible to neointima formation. These data indicate that studies on the effect of ET receptor antagonists on the pathobiology of vein graft disease is warranted.     

Identification and developmental characterization of a novel Y-box protein from Drosophila melanogaster

The Y-box proteins are a family of highly conserved nucleic acid binding proteins which are conserved from bacteria to human. In this report we have identified a new member of this family from Drosophila melanogaster. Degenerate-PCR was used to identify a conserved region within the highly conserved cold-shock domain (CSD) of Y-box proteins. Subsequently, the cDNA for this gene was sequenced, and the identified open reading frame was named ypsilon schachtel (yps). The expression pattern of yps indicates that this gene is expressed throughout development with the highest level of expression found in adult flies. In situ hybridization shows that the yps mRNA is maternally loaded into the egg cytoplasm. In addition, there appears to be expression of yps mRNA in mesodermal tissue during embryogenesis. YPS, while containing a conserved CSD, is novel in that it completely lacks the alternating acidic and basic regions found in the C-terminus of the other vertebrate eukaryotic Y-box proteins. The CSD of yps was purified and gel-shift analysis showed that this domain can interact with RNA. We predict that YPS would be an RNA-binding protein due to these results and the motifs which have been identified within the amino acid sequence.     

A comparative analysis of juvenile hormone metabolyzing enzymes in two species of Drosophila during development

The course of changes in the activities of enzymes degrading juvenile hormone (JH), epoxyde hydrolase (JHEH) and JH-esterase (JHE) was studied in two lines of Drosophila virilis (101 and 147) and in two lines of D. melanogaster (Canton-S and 921283). It was established for D. virilis that changes in the JH titre during pupal-adult development is determined by the activity level of JHE rather then JHEH, while in D. melanogaster developmental changes in JH titre are related to changes in the activity level of both JHE and JHEH. In adults of D. virilis, the high level of JH-hydrolysing activity is determined by JHE and in those of D. melanogaster by JHEH. Differences in the course of changes in the JHE activity level between adults of lines 101 and 147 of D. virilis were found, and also in the JHEH activity level between adults of lines Canton S and 921283 of D. melanogaster. It was shown that attainment of a definite JHE activity level in females of lines 101 and 147 agrees well with the onset of oviposition of fertilized eggs. The possible role of JHE in reproduction of D. virilis is discussed.     

The soluble alpha-mannosidases of Drosophila melanogaster

The alpha-mannosidases are implicated in both the catabolism of carbohydrates and the N-linked glycosylation pathway in insects, but little is known of the biochemistry of these glycosidases. In order to study the soluble alpha-mannosidases of Drosophila melanogaster we have used artificial fluorogenic substrates for detection of activity in situ following non-denaturing gel electrophoresis. This approach also permitted examination of the mannosidases present in different tissues and the sensitivity of the enzymes to known mannosidase inhibitors. Fluorogenic substrates were also used to determine the pH optima of partly purified mannosidases. We report that D. melanogaster contains several soluble alpha-mannosidase activities. Acidic mannosidases were detected in the gut, fat body and haemolymph of third-instar larvae. The major activity detected in larval guts was a neutral mannosidase presumed to be involved in digestion.     

Mutation and evolution of microsatellites in Drosophila melanogaster

Levels of nucleotide polymorphism in the Drosophila melanogaster genome are correlated with rates of recombination. This relationship may be due to hitchhiking of advantageous mutations (selective sweeps) or to continual removal of deleterious mutations from the genome (background selection). One test of the relative contributions of selective sweeps and background selection to the observed levels of variation in the genome of D. melanogaster is to compare levels of nucleotide variability (with a mutation rate on the order of 10(-9) per nucleotide per generation) with more rapidly evolving DNA loci such as microsatellites. This test depends critically on details of the mutational process of microsatellites. In this paper, we summarize our studies of microsatellite characteristics and mutation rates in D. melanogaster. We find that D. melanogaster microsatellites are short and have a mutation rate (6.5 x 10(-6) per locus per generation) several orders of magnitude lower than mammals studied to date. We further show that genetic variation at 18 dinucleotide repeat microsatellites in a population of D. melanogaster from Maryland is correlated with regional rates of recombination. These and other microsatellite data suggest that both background selection and selective sweeps may contribute to the correlation between DNA sequence variation and recombination in Drosophila.     

Isolation of protease-free alcohol dehydrogenase (ADH) from Drosophila simulans and several homozygous and heterozygous Drosophila melanogaster variants

The enzyme alcohol dehydrogenase (ADH) from several naturally occurring ADH variants of Drosophila melanogaster and Drosophila simulans was isolated. Affinity chromatography with the ligand Cibacron Blue and elution with NAD+ showed similar behavior for D. melanogaster ADH-FF, ADH-71k, and D. simulans ADH. Introduction of a second Cibacron Blue affinity chromatography step, with gradient elution with NAD+, resulted in pure and stable enzymes. D. melanogaster ADH-SS cannot be eluted from the affinity chromatography column at a high concentration of NAD+ and required a pH gradient for its purification, preceded by a wash step with a high concentration of NAD+. Hybrid Drosophila melanogaster alcohol dehydrogenase FS has been isolated from heterozygous flies, using affinity chromatography with first elution at a high concentration NAD+, directly followed by affinity chromatography elution with a pH gradient. Incubation of equal amounts of pure homodimers of Drosophila melanogaster ADH-FF and ADH-SS, in the presence of 3 M urea at pH 8.6, for 30 min at room temperature, followed by reassociation yielded active Drosophila melanogaster ADH-FS heterodimers. No proteolytic degradation was found after incubation of purified enzyme preparations in the absence or presence of SDS, except for some degradation of ADH-SS after very long incubation times. The thermostabilities of D. melanogaster ADH-71k and ADH-SS were almost identical and were higher than those of D. melanogaster ADH-FF and D. simulans ADH. The thermostability of D. melanogaster ADH-FS was lower than those of D. melanogaster ADH-FF and ADH-SS. D. melanogaster ADH-FF and ADH-71k have identical inhibition constants with the ligand Cibacron Blue at pH 8.6, which are two times higher at pH 9.5. The Ki values for D. simulans ADH are three times lower at both pH values. D. melanogaster ADH-SS and ADH-FS have similar Ki values, which are lower than those for D. melanogaster ADH-FF at pH 8.6. But at pH 9.5 the Ki value for ADH-FS is the same as at pH 8.6, while that of ADH-SS is seven times higher. Kinetic parameters of Drosophila melanogaster ADH-FF, ADH-SS, and ADH-71k and Drosophila simulans ADH, at pH 8.6 and 9.5, showed little or no variation in K(m)eth values. The K(m)NAD values measured at pH 9.5 for Drosophila alcohol dehydrogenases are all lower than those measured at pH 8.6. The rate constants (kcat) determined for all four Drosophila alcohol dehydrogenases are higher at pH 9.5 than at pH 8.6. D. melanogaster ADH-FS showed nonlinear kinetics.     

Phosphoenolpyruvate carboxykinase from Trypanosoma cruzi. Purification and physicochemical and kinetic properties

Phospho enolpyruvate carboxykinase (PEPCK) has been purified to homogeneity from epimastigotes of the Tul 0 strain of Trypanosoma cruzi. The physicochemical parameters determined allowed the calculation of an average molecular mass of 120 kDa; the subunit molecular mass, about 61 kDa, is in good agreement with the value of 58.6 kDa recently determined from the sequence by Sommer et al. (FEBS Lett. 359 (1994) 125-129). The PEPCK from T. cruzi presented, in addition to its molecular mass, typical properties of other ATP-linked PEPCKs, namely strict specificity for ADP in the carboxylation reaction and lower specificity in the decarboxylation and exchange reactions, and synergistic activation by CdCl2 or MgCl2 when added in addition to MnCl2. The enzyme presented hysteretic behaviour, shown by a lag period in the carboxylation reaction, which was affected by dilution and preincubation. The decarboxylation reaction catalyzed by the T. cruzi PEPCK was not inhibited by excess of ATP-Mn. The apparent Km values for the carboxylation reaction, including the low value for PEP (0.035 mM) are compatible with an important role of PEPCK, as suggested by previous NMR experiments, on the CO2 fixation in vivo which leads to succinate excretion during aerobic fermentation of glucose.