Alkaline phosphatase of Drosophila melanogaster. I. Partial purification and characterization

Two allelic forms (Aph-4 and Aph-6) of the alkaline phosphatase of late third instar larvae of Drosophila melanogaster have been partially purified and characterized. Both forms of the enzyme are inhibited by inorganic phosphate, cyanide ion, and cysteine. Aph-6 has a pH optimum of 8.0 and Aph-4 of 8.5. Zn²⁺ has been shown to be required for activity. Of the 22 phosphorylated compounds tested as substrates, only O-phosphotyrosine possesses a relative activity equal to that of the artificial substrate, p-nitrophenyl-phosphate. O-phosphothreonine, O-phosphoserine, and phosphoethanolamine are also good substrates. A majority of the compounds exhibits a relative activity of 0.5 or less.Paper No. 3446 of the Journal Series of the North Carolina State University Agricultural Experiment Station, Raleigh, North Carolina. This study was supported by Atomic Energy Commission Contract AT-(40-1)-3980.     

Alkaline phosphatase of Drosophila melanogaster. II. Biochemical comparison among four allelic forms

Biochemical analyses of partially purified preparations of APH-4 and -6 (common allelic forms) and APH-2 and -10 (rare allelic forms) of D. melanogaster reveal that the two common forms are similar in all properties investigated except for pH optimum (8.0 vs. 8.5). The common and rare forms share certain properties in common but differ in that the common forms are more stable to heat and more sensitive to inhibition by inorganic phosphate. With respect to such properties as substrate preferences and K _i values for inorganic phosphate, the common forms and APH-2 are similar to one another, whereas APH-10 is distinctly different. All four activities show preference for a phosphoaromatic compound as substrate, with O-phosphotyrosine being the best substrate of biological origin. Transphosphorylation, as related to these allelic forms of APH, is discussed.Paper No. 3892 of the Journal Series of the North Carolina State University Agricultural Experiment Station, Raleigh, North Carolina. This study was supported by Atomic Energy Commission Contract AT-(40-1-)-3980.     

Photoreceptor structures. III. Drosophila melanogaster

The eyes of three eye mutants of Drosophila melanogaster were fixed and thin sections studied for its structural detail in the electron microscope. Each ommatidium was found to have seven retinula cells with an equal number of rhabdomeres (visual units). The rhabdomeres average 1.2 micro in diameter and 60 micro in length. Each rhabdomere consists of osmium-fixed dense bands averaging 120 A in thickness, and with less dense interspaces 200 to 400 A. There is an average of 23 dense bands or 46 interfaces per micron within the rhabdomere. The rhabdomere as we have presented it is a single structure of packed rods or tubes. The "fine structure" within the rhabdomere is similar to that observed by electron microscopy for the retinula of the house fly, and to the retinal rods of the vertebrate eye, and to the chloroplasts of plant cells in a variety of animal and plant photoreceptor structures. In addition, the radial arrangements within the ommatidium of radially unsymmetrical units, the rhabdomeres, is probably related to the analysis of polarized light in the insect eye.     

Placental enzyme polymorphisms in Canadian populations. III. Alkaline phosphatase

Placental alkaline phosphatase phenotypes have been determined for a large Canadian population. The 'common' alleles, PL1, PL2, and PL3 have frequencies similar to those of European populations. Five new phenotypes are described and the incidence of rare phenotypes is compared for several populations.     

Inhibition of Drosophila melanogaster acetylcholinesterase by high concentrations of substrate.

Acetylcholine hydrolysis by acetylcholinesterase is inhibited at high substrate concentrations. To determine the residues involved in this phenomenon, we have mutated most of the residues lining the active-site gorge but mutating these did not completely eliminate hydrolysis. Thus, we analyzed the effect of a nonhydrolysable substrate analogue on substrate hydrolysis and on reactivation of an analogue of the acetylenzyme. Analyses of various models led us to propose the following sequence of events: the substrate initially binds at the rim of the active-site gorge and then slides down to the bottom of the gorge where it is hydrolyzed. Another substrate molecule can bind to the peripheral site: (a) when the choline is still inside the gorge - it will thereby hinder its exit; (b) after choline has dissociated but before deacetylation occurs - binding at the peripheral site increases deacetylation rate but (c) if a substrate molecule bound to the peripheral site slides down to the bottom of the active-site before the catalytic serine is deacetylated, its new position will prevent the approach of water, thus blocking deacetylation.     

Ultrastructural histochemical localization of acid phosphatase in salivary glands of Drosophila melanogaster.

The ultrastructural histochemical localization of acid phosphatase in salivary glands of third instar larvae of Drosophila melanogaster has been studied. Using Gomori's lead phosphate method for acid phosphatase detection, the optimal incubation time in the reaction medium was determined to be 30 min. When glands having wild-type acid phosphatase activity are incubated for this time, deposition of the final reaction product is observed in essentially every lysosome and artifactual staining is minimal.     

Acid phosphatase activity in the larval salivary glands of developing Drosophila melanogaster.

Both the biochemical profile and the optical and fine structural localization of acid phosphatase activity in the larval salivary glands of developing Drosophila melanogaster is described. Biochemically, acid phosphatase shows peak activity in the glands of feeding larvae, followed by a marked decline. Directly preceding the onset of cell histolysis however, enzyme activity increases 1.5 fold and is maintained at this level. Histochemically, acid phosphatase activity initially appears as discrete point or lysosomal sources. As development proceeds, an intense and diffuse form of enzyme is seen, accompanying an extremely vacuolated cytoplasm. Ultrastructurally, the enzyme is located in lysosomes, Golgi elements, multivesicular bodies and both within, and on the extracisternal surface of the rough endoplasmic reticulum. This extracisternal or cytosolic form appears directly preceding cell lysis and eventually shows a comprehensive cellular distribution. Large numbers of acid phosphatase positive haemocytes are attached to the basal glandular surface at all developmental stages. In morphologically intact gland cells, discrete extracisternal enzyme activity appears associated with local areas of degradation.     

Characterization of a functionally expressed dipeptidyl aminopeptidase III from Drosophila melanogaster.

A Drosophila melanogaster cDNA clone (GH01916) encoding a putative 723-residue long (82 kDa) protein (CG 7415) and displaying 50% identity with mammalian cytosolic dipeptidyl aminopeptidase (DPP) III was functionally expressed in Schneider S2 cells. Immunocytochemical studies using anti-(rat liver DPP III) Ig indicated the expression of this putative DPP III at the outer cell membrane and into the cytosol of transfected cells. Two protein bands (82 and 86 kDa) were immunologically detected after PAGE and Western blot of cytosol or membrane prepared from transfected cells. Western blot analysis of partially purified D. melanogaster DPP III confirmed the overexpression of these two protein bands into the cytosol and on the membranes of transfected cells. Despite the identification of six potential glycosylation sites, PAGE showed that these protein bands were not shifted after deglycosylation experiments. The partially purified enzyme hydrolysed the insect myotropic neuropeptide proctolin (Arg-Tyr-Leu-Pro-Thr) at the Tyr-Leu bond (Km approximately 4 micro m). In addition, low concentration of the specific DPP III inhibitor tynorphin prevented proctolin degradation (IC50 = 0.62 +/- 0.15 micro m). These results constitute the first characterization of an evolutionarily conserved insect DPP III that is expressed as a cytosolic and a membrane peptidase involved in proctolin degradation.     

Alkaline phosphatase in the thymus.

(1) Fetal thymuses, organs from patients who died from diseases that are not clinically known to be associated with concomitant lymphoid tissue involvement, as well as thymuses from patients dying from diseases which effect the lymphatic complex of the body, one way or another, have been investigated for their alkaline phosphatase activity, using Gomori technique and applying four different phosphate esters as substrates. (2) Three substrates (beta-glycerophophate, riboflavin 5-phosphate and adenosine triphosphate) showed essentially the same pattern of activity in which the cortex and Hassall's corpuscles were reactive, while the medulla was negative. A reversal of this pattern was demonstrated with 5-monophosphoric acid. (3) Before the age of 32-36 weeks of intra-uterine life there is no alkaline phosphatase activity in the thymus; therafter, the enzyme begins to make its first appearance. (4) There is a definite increase in the intensity of the reaction with advance of intra-uterine life. This increase in phosphatase content is continued postnatally, to reach its maximum at about the age of 10 years: after that, the enzyme activity gradually subsides. (5) There is a tremendous augmentation of phosphatase activity in the case of disease which are known to affect the lymphoid complex. (6) The phosphatase activity of the thymus has been discussed in relation to the prevailing concepts about the function of the thymus, with special emphasis on a possible association with 'lymphocyte-stimulating factor' production and/or secretion.     

Alkaline phosphatase activity of rumen bacteria.

Of the 54 strains of rumen bacteria examined for alkaline phosphatase (APase) production, 9 of 33 gram-negative strains and none of 21 gram-positive strains produced the enzyme. The APase of the cells of the three strains of Bacteroides ruminicola that produced significant amounts of the enzyme was located in the periplasmic area of the cell envelope, whereas the enzyme was located in the strains of Selenomonas ruminantium and Succinivibrio dextrinosolvens was associated with the outer membrane. The localization of APase production in the cells of natural populations of rumen bacteria from hay-fed sheep was accomplished by reaction product deposition, and both the proportion of APase-producing bacteria and the location of the enzyme in the cell envelope of the producing cells could be determined. We suggest that this procedure is useful in detecting shifts in the bacterial population and the release of cell-bound APase that accompany feedlot bloat and other sequelae of dietary manipulation in ruminants.     

Proteomics in Drosophila melanogaster.

To be able to understand cellular mechanisms, we require fully integrated data sets combining information about gene expression, protein expression, post-translational modification states, sub-cellular location and complex formation. Proteomics is a very powerful technique that can be applied to interrogate changes at the protein level. Studying this effectively requires specialised facilities within research institutes. Here, we describe the setting up and operation of such a facility, providing a resource for the Arabidopsis and Drosophila research communities.     

Terminal synthesis of xanthommatin in Drosophila melanogaster. I. Roles of phenol oxidase and substrate availability

Eye color mutants of Drosophila melanogaster are known which block the conversion of 3-hydroxykynurenine to xanthommatin. It has been proposed that this reaction depends on the presence of 3-hydroxykynurenine and a redox system maintained by phenol oxidase activity. The mutants st and ltd lack throughout development detectable amounts of 3-hydroxykynurenine or its metabolic derivatives. When the substrate is fed or injected, these mutants fail to form xanthommatin even though phenol oxidase activity is normal. The mutant cd accummulates excessive amounts of 3-hydroxykynurenine, has normal phenol oxidase activity, but is also deficient in xanthommatin formation. Mutants are also known which lack phenol oxidase activity but nevertheless form xanthommatin. It is concluded that the proposed relationship between 3-hydroxy-kynurenine and phenol oxidase activity is not sufficient to explain the in vivo synthesis and regulation of synthesis of xanthommatin in Drosophila. The bearing of these findings on the actual mode of synthesis is discussed.Supported by PHS 1029 and NSF GB-4539.     

A Comprehensive Study of Genic Variation in Natural Populations of Drosophila melanogaster. III. Variations in Genetic Structure and Their Causes between Drosophila melanogaster and Its Sibling Species Drosophila simulans

The natural populations of Drosophila melanogaster and Drosophila simulans were compared for their genetic structure. A total of 114 gene-protein loci were studied in four mainland (from Europe and Africa) and an island (Seychelle) populations of D. simulans and the results were compared with those obtained on the same set of homologous loci in fifteen worldwide populations of D. melanogaster. The main results are as follows: (1) D. melanogaster shows a significantly higher proportion of loci polymorphic than D. simulans (52% vs. 39%, P<0.05), (2) both species have similar mean heterozygosity and mean number of alleles per locus, (3) the two species share some highly polymorphic loci but they do not share loci that show high geographic differentiation, and (4) D. simulans shows significantly less geographic differentiation than D. melanogaster. The differences in genetic differentiation between the two species are limited to loci located on the X and second chromosomes only; loci on the third chromosome show similar level of geographic differentiation in both species. These two species have previously been shown to differ in their pattern of variation for chromosomal polymorphisms, quantitative and physiological characters, two-dimensional electrophoretic (2DE) proteins, middle repetitive DNA and mitochondrial DNA. Variation in niche-widths and/or genetic strategies of adaptation appear to be the main causes of differences in the genetic structure of these two species.     

Tarsus determination in Drosophila melanogaster.

Arista versus tarsus determination is well investigated in Drosophila, yet it remains unresolved whether Antennapedia (ANTP) cell autonomously or noncell autonomously determines tarsus identity and whether Sex combs reduced (SCR) is the HOX protein required for normal tarsus determination. Three observations rule out a cell autonomous role for ANTP in tarsus determination. (i) Clonal ectopic overexpression of ANTP did not repress the expression of the arista determining protein Homothorax (HTH) in early 3rd stadium antennal imaginal discs. (ii) Clonal ectopic expression of ANTP did not transform the arista to a tarsus. (iii) Ectopic overexpression of ANTP, Labial (LAB), Deformed (DFD), SCR, Ultrabithorax (UBX), Abdominal-A (ABD-A), or Abdominal-B (ABD-B), using the dppGAL4 driver, resulted in arista-to-tarsus transformations, and repressed HTH/Extradenticle (EXD) activity noncell autonomously in early 3rd stadium antennal imaginal discs. SCR may not be the HOX protein required for normal tarsus determination, because co-ectopic expression of Proboscipedia (PB) inhibited the arista-to-tarsus transformations induced by ectopic expression of DFD, SCR, ANTP, UBX, ABD-A, and ABD-B. The proposal that SCR is the HOX protein required for normal tarsus determination is dependent on SCR being the sole target of PB suppression, which is not the case. Therefore, the possibility exists that normal tarsus determination is HOX independent.