Michael M. Bentley

Characterization of a low-activity allele of NADP+-dependent isocitrate dehydrogenase from Drosophila melanogaster.

We have characterized biochemical effects of IdhGB1in Drosophila melanogaster. This is a “null”-activity allele for NADP+-dependent isocitrate dehydrogenase (NADP-IDH) isolated from a natural population. The homozygous mutant strain has 5% of the NADP-IDH specific activity found in controls and less than 24% of the immunologically cross-reacting material (CRM). This mutation maps to 27.2 on the third chromosome, to the right of h. The biochemical phenotype of this mutant strain includes a coordinate reduction in malic enzyme (ME) specific activity and CRM and an increase in specific activity for the pentose-phosphate shunt enzymes, 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase. The Kmvalues for purified NADP-IDH are not different from those found for the purified control enzyme for NADP+ or isocitrate. It is suggested that this allele may represent a cis-acting control mutation for one of at least two loci involved in the production of NADP-IDH in D. melanogaster.

Comparative properties of three forms of glucose-6-phosphate dehydrogenase in Drosophila melanogaster

Three alleles of the Zw locus of Drosophila melanogaster—ZwA, ZwB,and Zwlol—apparently code for dimeric, tetrameric, and monomeric forms of glucose-6-phosphate dehydrogenase (G6PD), respectively. The three forms of G6PD are characterized by different apparent Kmvalues for glucose-6-phosphate but similar apparent Kmvalues for NAPD+. When high concentrations of NAPD+ were added to enzyme preparations, the ZwAand Zwlolforms of G6PD assumed tetrameric and dimeric properties, respectively. Although Zwloladults exhibit little G6PD activity, they maintain levels of G6PD-antigen comparable to those in ZwAand ZwBadults. Thus the low level of G6PD activity in Zwlolindividuals cannot be explained as the consequence of lack of synthesis of the G6PD subunit.

Properties of Drosophila NADP+-isocitrate dehydrogenase purified on Procion Brilliant Blue-Sepharose-4B

1. 1. NADP+-dependent isocitrate dehydrogenase from Drosophila melanogaster has been purified to apparent homogeneity by chromatography on Procion Brilliant Blue-Sepharose-4B. 2. 2. Drosophila IDH† has a molecular weight of 110,000 and is a dimer of subunits with molecular weights of 60,000 and 50,000. 3. 3. The Km for isocitrate is 23 μM while the Km for NADP+ is 14.6 μM. 4. 4. Procion Brilliant Blue-Sepharose-4B will readily bind isocitrate dehydrogenase and glucose-6-phosphate dehydrogenase but will not bind NADP+-malic enzyme nor 6-phosphogluconate dehydrogenase.

Notizen: A New Mutant Affecting Aldehyde Oxidase in Drosophila melanogaster

Abstract A new locus, Aldox-2, which affects the activity and heat stability of aldehyde oxidase in D. melanogaster is described. The Aldox-2 locus is localized to map position 86 on chromosome 2, between c and px. Aldehyde oxidase activity in Aldox-2 homozygotes is approximately 25 - 30% that of the Oregon-R wild-type control strain. The enzyme from the mutant stock is much more heat labile than is the enzyme from the wild-type strain. Both the activity and heat phenotypes are completely recessive.

The isolation and characterization of a mutant allele at a new X-linked locus,mex, affecting NADP+-dependent enzymes inDrosophila melanogaster

The isolation and characterization of mutant alleles in a regulatory gene affecting NADP+-dependent enzymes are described. The locus,mex, is at position 26.5 ± 0.74 on the X chromosome ofDrosophila melanogaster. The newly isolated mutant allele,mex1, is recessive to either themex allele found in Oregon-R wild-type individuals or that found in thecm v parental stock in which the new mutants were induced. Themex1 mutant allele is associated with statistically significant decreases in malic enzyme (ME) specific activity and ME specific immunologically cross-reacting material (ME-CRM) in newly emerged adult males. During this same developmental stage in males, the NADP+-dependent isocitrate dehydrogenase specific activity increases to statistically significant levels. Females of themex1 mutant strain show statistically significant elevated levels of the pentose phosphate shunt enzymes, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. Isoelectric focusing and thermolability comparisons of the active ME from mutant and control organisms indicate that the enzyme is the same. Developmental profiles ofmex1 and control strains indicate that this mutant allele differentially modulates the levels of ME enzymatic activity and ME-CRM during development.

Analysis of Aldoxn alleles isolated from natural populations of Drosophila melanogaster

Aldox “null” alleles which were isolated from natural populations in Great Britain and North Carolina were analyzed for complementation. No complementation was observed between any combinations of “null” alleles for aldehyde oxidase (AO) specific activity in late third-instar larvae and newly emerged adults. AO immunologically cross-reacting material (AO-CRM) was quantitated in all homozygous stocks at both developmental stages as well as all allelic combinations in newly emerged adults. When the adult organism contains only Aldoxn alleles, the polypeptides are not immunologically recognizable or may be rapidly degraded. Larvae and adults have different abilities to degrade mutationally altered enzymatically inactive AO polypeptide or synthesize them differentially. This is indicated by easily measurable AO-CRM levels in late third-instar larvae of Aldoxn homozygotes, while newly emerged adult Aldoxn homozygotes have very little, if any, AO-CRM. Newly emerged adult heterozygotes of Aldoxn/Aldox+ do have increased AO-CRM, indicating that the Aldoxn alleles can code for a polypeptide which can be “rescued” if Aldox+ gene product is present. Heterozygotes containing an Aldox+ allele with a deficiency for the Aldox region produce 74.2% of the AO-CRM found in Aldox+ homozygotes. This may indicate the presence of trans-acting factors which serve to activate gene expression in a system in which each gene copy is not maximally expressed.

Sulfite sensitivity and sulfite oxidase actiivty inDrosophila melanogaster

The relationship between sulfite oxidase (SO) and sulfite sensitivity inDrosophila melanogaster is addressed. Significant improvements to the SO assay have provided an investigative tool which can be applied to further studies of this molybdoenzyme. Using the second-instar larval stage ofD. melanogaster, we have shown a direct relationship between measured levels of sulfite oxidase activity and the organisms ability to withstand a sulfite challenge. Implementation of a sulfite-testing procedure confirmed the documented instability of sulfite in solution and may explain some of the conflicting results reported in the SO literature. Results of the tungstate-addition experiments confirm thatDrosophila SO is a molybdoenzyme and its activity was shown to be governed by three of the four loci known to affect more than one molybdoenzyme. The ability ofD. melanogaster to withstand the application of exogenous sulfites is shown to be dependent on sulfite oxidase activity.

Thealdox-2 locus ofDrosophila melanogaster also affects sulfite oxidase and molybdenum metabolism

Mutation at thealdox-2 locus inDrosophila melanogaster affects the specific activities of four molybdoenzymes differentially during development. Sulfite oxidase activity is normal during late larval and pupal stages but is reduced during early adult stages inaldox-2 organisms. There was complete concordance among the effects ofaldox-2 on sulfite oxidase, aldehyde oxidase, xanthine dehydrogenase, and pyridoxal oxidase, when 38 stocks were analyzed which were derived from single recombination events betweenc andpx, markers which flankaldox-2. Several different biochemical analyses indicate that the active molybdoenzymes present in thealdox-2 strain are normal with respect to size, shape,pH-activity profile,Km, and molecular weight. Significant differences were found between thealdox-2 strain and the OR control strain in their responses to dietary Na2MoO4 and Na2WO4. The mutant strain is much more resistant to the effects of dietary Na2WO4 and much more responsive to the administration of Na2MoO4 than the OR control strain when these effects are quantitated by measurements of molybdoenzyme specific activities. This evidence suggests that thealdox-2+ gene product has a molybdenum binding site which can also bind tungsten and that this site is altered in the mutant strain. The hypothesis presented explains the observed effects of thealdox-2 mutation and relates them to the other mutations reported in this gene-enzyme system.

Genetic and developmental characterization of the aldox-2 locus of Drosophila melanogaster

The aldox-2 locus in Drosophila melanogaster has been shown to affect differentially three molybdoenzymes, aldehyde oxidase, pyridoxal oxidase, and xanthine dehydrogenase. These effects are most obvious at times surrounding the pupal-adult boundary, when the normal organism accumulates large amounts of these enzymes in their active form. This locus has been more precisely mapped genetically to 2–82.9±2.1, with complete concordance between the effects of all recombinant chromosomes on all three enzymes. The cytogenetic location has also been determined to be between 52E and 54E8, with the likelihood that it lies within the region 54B1-54E8. The aldox-2 mutant allele has no visible phenotype and is completely recessive for enzyme effects at all stages tested. Segmental duplication of this region, including the aldox-2+ allele, has no apparent effect on the visible phenotype or the enzymatic activity. The mutant aldox-2 allele has no effect on the developmental expression of two unrelated enzymes, 6-phosphogluconate dehydrogenase and NADP+-dependent isocitrate dehydrogenase. The effects of this locus on aldehyde oxidase, xanthine dehydrogenase, and pyridoxal oxidase suggest that this locus may code for a product involved in the synthesis of the molybdenum cofactor common to these enzymes.