Insect Biochem., Vol. 10, pp. 535 to 541. 0020-1700/'80/1001-0535 $02.00/0 © Pergamon Press Ltd. 1980. Printed in Great Britain. ONTOGENY AND TISSUE DISTRIBUTION OF LEUCINE AMINOPEPTIDASE IN DROSOPHILA MELANOGASTER* VIRGINIA K. WALKER~ and JOHN H. WILLIAMSON Department of Biology, University of Calgary, Calgary, Alberta, Canada T2N 1N4 (Received 26 November 1979) Abstract--Drosophila melanogaster larvae contain two isozymes of leucine aminopeptidase that are detectable on starch gels. The more anodally migrating isozyme, LAP A, is first detected 12 hr after oviposition and is a soluble enzyme localized to the haemolymph. The specific activity of the other isozyme, LAP D, is highest in young larvae and associated with cell membranes, especially from the midgut and Malpighian tubules. It is probable that these two enzymes are both functionally and spatially unique in larvae of D. melanogaster. Key Word Index: Leucine aminopeptidase, Drosophila melanogaster, zygotic gene expression, cell fractionation, tissue specificity of enzymes INTRODUCTION DESPITE the presumptive physiological importance of peptidases their biochemical analysis in insects is fragmentary (LAW et al., 1977). The first report of peptidases in Drosophila melanogaster was that of BECKMAN and JOHNSON (1964). The two leucine aminopeptidase (LAP) isozymes observed in third- instar larvae (SAKAI et al., 1969) were found to be products of two genes Lap-A and Lap-D. These genes, on chromosome 3-position 98.3, are so closely linked that no recombination between them has been observed (BECKMAN and JOHNSON, 1964; FALKE and MACINTYRE, 1966; LINDSLEYand GRELL, 1968). It has been suggested that these enzymes are 'pupal enzymes' which are responsible for the histolysis of larval tissues (MUHS, 1975). Thus the presence of leucine aminopeptidases in larval stages of development assumes major interest. It is possible that these histolytic enzymes are synthesized early in development and stored in an inactive form prior to utilization. Therefore, the subcellular localization and tissue distribution of the LAP A and LAP D isozymes is also of interest. In this paper a biochemical assay is described for Drosophila LAP which was used to study the developmental expression and the cellular localization of this enzyme in Drosophila melanogaster. MATERIALS AND METHODS Drosophila stocks Wild type (Oregon-R) Drosophila melanogaster cultures were reared at 19-21°C on standard cornmeal medium (LEw~s, 1960). Eggs and embryos were collected on 3~ agar *Supported by the National Sciences and Engineering Research Council of Canada. ~'Present address: Department of Genetics, University of Cambridge, Cambridge, CB2 3EH, U.K. which was smeared with a yeast and vinegar paste containing a few drops of alcohol to stimulate oviposition (MITchELL and MXTCnELL, 1964). Unless specified, Oregon-R third- instar larvae were used for all experiments. Stocks homozygous for particular LAP electrophoretic variants were produced by homozygosis of third chromosomes from wild type stocks. Several lines from each stock were subjected to electrophoretic analysis for identification. A line homozygous for the fast variant of Lap-D (Lap-DF) and the fast variant ofLap-A (Lap-AF) was isolated from K~is 60. A line carrying the null allele for Lap-A (Lap-A °) and Lap.DF was isolated from Kiis 51. To determine the time of initial expression of the Lap-A gene, Lap-A o Lap-DF virgin females were mated with Lap-AF Lap-DF males, and progeny embryos were analyzed. To facilitate the collection of large numbers of parents for sp~ific crosses, stocks carrying C(1)RM, ts l(1)E-90 or XyLys, ts 1(1)E-90 were constructed. This temperature sensitive allele (kindly supplied by Dr. D. SuzuKI, Vancouver, Canada) with appropriately timed heat shocks (29°C) results in cultures of only one sex. Unfertilized eggs from Lap-A ° Lap-DF females were obtained by crossing virgins of this genotype with sterile X/O males. Enzyme assays Leucine aminopeptidase activity was measured by modification of the method for the determination of rat brain amidase activity (MARKS et aL, 1968). Samples were obtained by homogenizing whole animals in distilled water and centrifug- ing at 8000 g (4°C) for 3 rain. The assay solution contained 0.7 ml of 0.1 M Tris-maleate buffer, pH 6.7, and 0.3 ml of 1.8 mM L-leucyl-fl-naphthlamide--HCl (Sigma Chemical Co. St. Louis, MO, U.S.A.) in distilled water. The reaction was initiated by addition of the supernatant and incubated at 30°C for 20 min. Incubations were terminated by adding 0.5 ml of a solution containing 5% Tween-20 and 0.1% Fast Garnet GBC salt in 1.0 M acetate buffer, pH 4.2. Colour was allowed to develop for 10 rain at 22°C after which the samples were kept on ice until their absorption was measured at 520 nm. Reference blanks were prepared by the addition of the enzyme after inclusion of the coupling dye. Standard curves were prepared with fl-naphthylamine (flNA) (Sigma Chomcial Co.) dissolved in 40% ethanol. The sensitivity of the enzyme to the inhibitors EDTA and 1, 10-phenanthroline was tested by preincubating the enzyme 535