Volume 29, number 1 MOLECULAR • CELLULAR BIOCHEMISTRY January 16, 1980

BIOCHEMICAL CHARACTERIZATION OF PHOSPHOGLUCOSE ISOMERASE AND GENETIC VARIANTS FROM MOUSE A N D DROSOPHILA M E L A N O G A S T E R

Daniel CHARLES* and Chi-Yu LEE~

Laboratory of Animal Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA

(Received August 27, 1979)

Summary

A simple and unique procedure was developed to purify phosphoglucose isomerase variants from the whole mouse body extracts and Drosophila homogenate. It involved the use of an 8-(6-aminohexyl)-amino-ATP-Sepharose column followed by a preparative isoelectric focusing. In each case, the enzyme in the homogenate was adsorbed by ionic interaction on the ATP-Sepharose column. Substantial purification was achieved by the affinity elution with the substrate-glucose-6-phosphate. Mouse and Drosophila phosphoglucose isomerase as well as the corresponding variants were shown to be dimers of similar molecular weight and to exhibit similar kinetic properties. The isoelectric points for the variants from DBA/2J and C57BL/6J mice were determined to be 8.4 and 8.7 respectively, while they were 6.8 and 6.3 respectively for Drosophila and 4/4 variants. Differential thermal stability was observed for the two mouse variants but not for the Drosophila ones. Amino acid composition analysis was performed for both mouse and Drosophila enzymes. Rabbit antisera for mouse (DBA/2J) and Drosophila (2/2) enzymes were

* National Institutes of Health Visiting Fellow. Present address: c/o DR. UDO H. EI-mlNG, Department of Genetics, Biological Institute for Radiation Research, Ingolstadter Landstrasse 1, 8042 Neuherberg bei Munchen, Federal Republic of Germany. t To whom the correspondence should be addressed. $ Abbreviations: CRM; Immunological cross-reacting mater- ials.

raised, Within each species, complete im- munological identity was observed between the variants. The antisera were used to characterize the null mutants of phosphoglucose isomerase identified in the mouse and Drosophila popula- tions. By rocket immunoelectrophoresis, the null allele of the naturally occurring heterozyg- ous null variant of Drosophila was shown to express no cross-reacting materials (CRM).

Introduction

Recently, our research interests have been focused on problems related to environmental mutagenesis and biochemical genetics 1-5. During the process of biochemical screening 5'6, numer- ous mutants carrying no apparent activity of certain enzymes have been detected among natural and y-irradiation-induced Drosophila lines or populations as well as the off-spring of the mutagenized mice 6. Among these confirmed mutants, some carrying the null allele of phos- phoglucose isomerase have been identified 7. Presumably, these null mutations were caused by either structural or regulatory gene muta- tions resulting in an allelic loss of apparent enzyme activity. Therefore, the identification of the cross-reacting materials (CRM) with an- tibodies specific to a given enzyme is an important step towards the understanding of gene mutations 3.

Phosphoglucose isomerase is known to catalyze the interconversion of glucose-6- phosphate and ffuctose-6-phosphate. Two elec-

Dr. W. Junk b.v. Publishers - The Hague, The Netherlands 11

trophoretic variants have been reported in in- bred strains of mice 8 and in Drosophila 7. The effects of charge variations on the biochemical properties of enzymes has been an important subject in biochemical genetics and mutation research 1,4,8.

Phosphoglucose isomerase has been exten- sively studied in many other species such as yeast 9 and humans 1°. Purifications of this en- zyme have been mainly based on either the conventional procedures 1~ or the substrate elu- tion from a phosphocellulose column ~-14.

In this study we employed an 8-(6- aminohexyl)-amino-ATP-Sepharose column as a weak ion exchange column for the purification of mouse and Drosophila phosphoglucose isomerase. Detailed biochemical, immunological and structural analyses were performed in order to characterize the genetic variants and the null mutants of phosphoglucose isomerase from the mouse and Drosophila.

Materials and Methods

Chemicals and enzymes ATP (disodium salt), NADP + (acid form), glucose-6-phosphate (sodium salt), fructose-6- phosphate (sodium salt) and glucose-6- phosphate dehydrogenase (from Torula Yeast) were purchased from Sigma Chemical Com- pany. Sepharose 4B was purchased from Phar- macia. Dithiothreitol was obtained from GIBCO, sodium dodecyl sulfate from BDH and ampholines from LKB. Urea, HC1, acetic acid, butanol, thioglycolic acid, iodoacetamide and trypsin were purchased from Pierce. All other chemicals were reagent grade.

Mouse and Drosophila melanogaster Two inbred strains of mice, DBA/2J and C57BL/6J were purchased from Jackson Laboratory at eight weeks of age. The animals were sacrificed by cervical dislocation, and after removing the skin and internal organs, the whole mouse bodies were frozen at -80 ° for later use in enzyme purification.

All the stocks of Drosophila melanogaster were supplied by Dr. Robert Voelker of our Institute. The fast (4/4) and slow (2/2) variants of phosphoglucose isomerase were purified re- spectively from Drosophila line with genotype

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of cn bw ; ri e and a stock of natural variant recovered in central North Carolina 7. A heterozygous null mutant of phosphoglucose isomerase (pgi n-Nc8°) was recovered from natural Drosophila populations in central North Carolina 7.

Enzyme assays Phosphoglucose isomerase was routinely assayed on a Beckman model 25 spectrophotometer at 25 °, following an increase in absorbance at 340 nm. The assay mixture contained in a total volume of 1 ml, 0.1 M tris-HCl, pH 8.0, 2 mu EDTA, 0.5 mM NADP +, i mM fructose 6- phosphate and 1 unit glucose-6-phosphate de- hydrogenase. One unit of enzyme activity is defined as the amount of enzyme catalyzing the formation of 1/zmole glucose-6-phosphate per minute.

Protein determination The protein concentration was determined by the procedures of B61-mEN et al. 15. Bovine serum albumin was used as the standard.

Sodium dodecyl sulfate acrylamide gel electrophoresis The purity of the enzyme at different stages of purification was routinely analyzed by ac- rylamide gel electrophoresis in the presence of 0.1% sodium dodecyl sulfate according to the procedure of LAEMML116. Seven and a half percent acrylamide slab gels were used for routine analysis.

For molecular weight determinations, 10% acrylamide tube gels were used in the presence of 0.1% sodium dodecyl sulfate. Bovine serum albumin (Mr 67,000), ovalbumin (Mr 45,000) and lactate dehydrogenase (Mr 36,000) were employed as standards.

A TP- Sepharose 8-(6-Aminohexyl)-amino-ATP-Sepharose was prepared according to the previously described procedure 17. The ligand density was determined to be 1.2/xmole per ml Sepharose. The ATP- Sepharose could be regenerated by washing the gel with 6 M urea and 2 M NaC1.

Sucrose density gradient centrifugation Centrifugations were performed in a Beckman model L2-65B ultracentrifuge using a SW 50.1

rotor at 49,000 rpm for 16 hours at 4 °. Linear sucrose density gradients of 5 to 20% were used and lactate dehydrogenase (7.2 S), enolase (5.9 S) and phosphoglycerate kinase (3.46 S) were employed as standards. The sedimentation coefficients were determined according to MAR- TIN and A3dES 18.

Gel filtration chromatography Sephadex G-200 superfine gel was employed for the determination of native molecular weight and Stoke's radius 19 of phosphoglucose isomer- ase. Aldolase (4.74 nm Stoke's radius), bovine serum albumin (3.70 nm), ovalbumin (2.76 nm), t~-chymotrypsinogen (2.26 nm) and ribonuclease (1.80 nm) were used as references. The column (1.6 x 76 cm) was equilibrated with phosphate buffer, pH 7.0 which was 1 mM in dithiothreitol and EDTA.

Isoelectric focusing Isoelectric focusing experiments were per-

formed with an LKB 8101 isoelectric focusing column (110 ml) 2°. For the determinations of isoelectric points, a 2% ampholine solution of pH 7 to 11 was used for the mouse variants and a solution of pH 5 to 8 was used for Drosophila variants. Fractions of 0.6 ml were collected for enzyme assays and pH measurements were performed with a Fisher Accumet 520 digital pH meter.

Thermal inactivation The thermal stability experiments were per- formed in a constant temperature water bath at 54 °. The test tubes which contained 0.1 M Tris-HC1, pH 8.0, 10 mM dithiothreitol and 1 unit phosphoglueose isomerase were incubated for various time intervals. The incubation was terminated by transferring the tubes to an ice bath. The residual enzyme activity was assayed immediately.

Immunology Pure phosphoglucose isomerase from DBA/2J mice and Drosophila (2/2) was employed for immunization. Approximately 30 to 50/~g of the enzymes in 0.5 ml phosphate buffer, pH 7.0 were mixed with 0.5 ml complete Freund's adjuvant. New Zealand white rabbits were injected subcutaneously on days 1, 14, 28 and 42. The rabbits were bled on day 49 to check

the antigenic response and boosted on day 56 and 70 with 100 p~g each of the antigens. The rabbits were bled on days 63 and 77 and sera were stored at - 2 0 ° .

Double immunodittusion experiments were performed according to OUCHTERLONY 21, in Petri dishes with 1% agarose in 0.046 M phosphate buffer, p H 7.1 containing 0.16 M NaC1 and 0.05% sodium azide.

Rocket immunoelectrophoresis experiments were performed in a LKB multiphor apparatus using 1% agarose gels in 0.088 M tris-acetate, pH 8.6, containing E D T A (1 mM) and antiserum (20 to 50/xl). The gels were stained for phos- phoglucose isomerase activity with the regular assay solution containing 0.03% phenazine methosulfate and 0.01% nitro-blue tetrazolium for 30 minutes at 37 ° . The area of the stained rockets was measured from photographs of the gels.

Inhibition of phosphoglucose isomerase by the raised antisera was performed by incubating the enzyme at 37 ° for 60 minutes with increas- ing amounts of antisera in 0.1 M phosphate buffer, pH 7.4. Normal nonimmune rabbit serum was used as a control. The endogeneous phosphoglucose isomerase activity in the origi- nal sera was eliminated by heating to 65 ° for 15 minutes.

Amino acid composition analysis Mouse and Drosophila phosphoglucose isomer- ase (250/xg each) were dialyzed extensively against 0.1% ammonium hydroxide and lyophilized. The enzyme samples were redissol- ved in 250/zl 6 N HC1 in sealed tubes under vacuum. After 24 and 48 hours at 110 ° , the hydrolysates were dried in a desiccator and redissolved in 100/xl of 0.2 M sodium citrate buffer followed by centrifugation at 9000 rpm for 5 minutes. The clear supernatant of the samples were analyzed with a Beckman model 118 Amino Acid Analyzer. Cysteine and tryp- tophan contents were determined according to the reported procedures 22'23.

Purification of phosphoglucose isomerase from mouse and Drosophila Identical procedures were employed for the purification of phosphoglucose isomerase from DBA/2J and C57BL/6J mice as well as its 2/2 and 4/4 variants of Drosophila. The purification

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of this enzyme from DBA/2J mice and 4/4 variant of Drosophila is presented here in details. Unless otherwise indicated, all the oper- ations were performed at 4 ° .

Step 1: Preparation of crude homogenate One hundred grams of whole mouse bodies

were homogenized with a meat grinder in 500 ml of 10 mM phosphate buffer, pH 6.5 which was 5 mM in dithiothreitol and i mM in E D T A (buffer A). After one hour, the solution was centrifuged at 27,000 x g for 30 minutes. The supernatant was filtered through glass wool.

For the purification of Drosophila enzyme, 10 grams of frozen flies were homogenized with a polytron homogenizer in 150 ml 10 mM phos- phate, pH 6.0 containing 5 mM dithiothreitol and 1 mM EDTA (buffer B). After centrifuga- tion at 27,000 x g for 30 minutes, the supernat- ant was filtered through glass wool.

The specific activity of phosphoglucose isomerase was determined to be 1.5 and 1.8 unit/mg for the extract from mouse and Drosophila respectively.

Step 2: Ammonium sulfate fractionation To the clear supernatant extract, ammonium

sulfate was added to bring about 40% satura- tion. One hour later, the precipitate was re- moved by centrifugation (27,000 x g, 20 mi- nutes). Ammonium sulfate was added to give 80% saturation to the supernatant. After one hour, the precipitate was collected by centrifug- ation as described. The pellet was redissolved in

20 ml of buffer A (for mouse enzyme) or buffer B (for Drosophila) and dialyzed overnight against 41 each of the appropriate buffer.

Step 3: 8-(6-Aminohexyl)-amino-ATP- Sepharose chromatography

After dialysis, the precipitate was removed by centrifugation and the supernatant was loaded on the ATP-Sepharose column (2.5 x 20 cm) equilibrated with buffer A or buffer B. After washing with one liter of the appropriate buffer, the mouse enzyme was eluted with a 0 to 10 mM glucose-6-phosphate linear gradient (300 ml by 300 ml), while the fly enzyme was readily eluted with 300 ml of 5 mM glucose-6-phosphate.

Step 4: Isoelectric Focusing Fractions containing phosphoglucose isomer-

ase activity were pooled and concentrated by ultrafiltration and dialyzed overnight with one liter of 1% glycine solution. The dialyzed enzymes were subjected to a preparative col- umn isoelectric focusing in the presence of 2% ampholyte generating a pH 3.5 to 10 gradient. After 18 hours at 1600 volts, fractions of 1.2 ml were collected and assayed for enzyme activity. Peak fractions containing pure enzyme were pooled and stored at - 2 0 ° .

Resalts

Enzyme purifications As shown in Table 1, genetic variants of phosphoglucose isomerase from mouse and

Table 1. Purification of mouse ~ and Drosophila b phosphoglucose isomerase

Purification step

Total Total Specific protein activity activity Yield

(mg) (Units) (Units/mg) (%)

i. Crude extract Mouse 13,500 20,500 Drosophila 2,500 4,400

2. 40-80% (NH4)2SO 4 fractionation Mouse 8,700 17,500 Drosophila 550 3,500

3. 8-(6-aminohexyl)-amino- ATP-Sepharose

Mouse 70 14,000 Drosophila 11 2,600

4. Isoelectric focusing Mouse 9.3 7,000 Drosophila 1.8 1,800

1.5 100 1.8 100

2 85 6.4 80

200 70 240 60

750 35 1,000 40

a 100 grams of whole mouse bodies from DBA/2J mice were used for this purification, b 20 grams of D. melanogaster cn bw; ri e (4/4) were used for this purification.

14

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Fig. 1. Elution profiles of phosphoglucose isomerase from an 8-(6-aminohexyl)-amino-ATP-Sepharose column (2.5 x 20 cm); Fractions of 13.5 ml were collected. A. The elusion of the enzyme from DBA/2J mice with a 0 to 10 nan linear gradient of glucose-6-phosphate (300 ml by 300 ml). B. The elution of the enzyme from Drosophila melanogaster (cn bw; rie, genotype 4/4) with 300 ml of 5 mM glucose-6- phosphate.

Drosophila melanogaster could be purified by a similar and straightforward procedure. Among the purification steps, 8-(6-aminohexyl)-amino- ATP-Sepharose column provided a major one for the enzyme purification. As indicated in Figure 1., the adsorbed enzyme from each species could easily be eluted biospecifically by glucose-6-phosphate from the ATP column with a high degree of purity. SDS acrylamide gel electrophoresis revealed a single protein band for the purified enzyme variants from either species (shown in Figure 3).

Biochemical characterizations Some biochemical properties of mouse and Drosophila phosphoglucose isomerase are pre- sented in Table 2. Among them are sedimenta-

tion coefficients, Stoke's radii, native and sub- unit molecular weight, isoelectric points, Km'S for fructose-6-phosphate, thermal stability as well as the specific activity. In between the two species, both enzymes exhibit quite similar molecular size but distinct differences in isoelectric points, Km's for fructose-6-phosphate and the thermal stability. In the case of mouse enzymes, the two genetic variants showed essen- tially identical biochemical properties except for isoelectric points (8.4 and 8.7 for DBA/2J and C57BL/6J variants respectively) as well as for the thermal stability. The activity remaining after 5 minutes of incubation at 54 ° was found to be 3 2 ± 2 and 1 7 + 2 % of the original, respectively for variants from DBA/2J and C57BL/6J mice. In the case of Drosophila variants, both showed identical biochemical properties except for the difference in isoelectric points (6.3 and 6.8 for 4/4 and 2/2 variants respectively).

Immunological studies A single precipitin line was observed when the crude extracts or the pure enzyme from DBA/2J and 2/2 Drosophila variant were tested with their respective antisera. No precipitin lines were observed (Fig. 2) between the mouse enzyme and the antiserum to Drosophila phos- phoglucose isomerase or vice versa. A complete fusion of the precipitin line was observed between the two mouse genetic variants or between the two Drosophila variants (Fig. 2B and Fig. 2C).

The mouse and Drosophila enzymes were inhibited specifically by their respective antisera when incubated at 37 ° for one hour. As shown in Figure 4, the enzyme variants showed identi- cal inhibition curves within each species.

Amino acid composition analysis The amino acid composition of mouse and Drosophila phosphoglucose isomerase is pre- sented in Table 3. The number of amino acid residues has been calculated based on a subunit molecular weight of 55,000. For comparative purpose, the amino acid composition of human and yeast enzymes is also presented 9'1°.

Within experimental errors, no significant differences in amino acid composition were observed between the two mouse or the two Drosophila variants (data not shown).

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A B C

Fig. 2. Ouchterlony plates to demonstrate the specificity and cross-reactivity of afftisera to mouse and_Drosophila phosphoglucose isomerase:

Plate A: Well a; 20 ~1 (1.1 mg protein) antiserum to DBA/2J phosphoglucose isomerase. Well b; 20 txl(5/~g) pure DBA/2J phosphoglucose isomerase. Well c; 20 t~l (1.3 mg protein) antiserum to 2/2 Drosophila phosphoglucose isomerase. Well d; 20/~1 (4 p,g) pure 2/2 Drosophila phosphoglucose isomerase.

Plate B: Well a; 20/xl antiserum to DBA/2J phosphoglucose isomerase. Well b; 20/xl pure DBA/2J phosphoglucose isomerase (5/~g). Well c; 20/xl nonimmune rabbit serum. Well d; 20/xl pure C57BL/6J phosphoglucose isomerase (5/xg).

Plate C: Well a; 20/xl antiserum to 2/2 Drosophila phosphoglucose isomerase. Well b; 20 t~l pure 2/2 Drosophila phosphoglucose isomerase (4 tzg)- Well c; 20/~1 nonimmune rabbit serum. Well d; 20/xl pure 4/4 Drosophila phosphoglucose isomerase (4/~g).

Immunological analysis of a Drosophila null mutant of phosphoglucose isomerase The heterozygous null mutant recovered from Drosophila population in Central North Carolina was analyzed in detail by rocket immunoelectrophoresis. Heterozygous 4/pgi NC80) and 2/pgi n-NcS° mutants were used for experiments, because the homozygous null mut- ant of phosphoglucose isomerase is not viable (R. VOELKER, personal communication). Typical gels of rocket immunoelectrophoresis are pre- sented in Figure 5 and Figure 6. The crude fly

homogenates with different genotypes were employed for analysis. The specific activity of phosphoglucose isomerase was determined prior to the analysis as follows: 0.85 (genotype 4/4); 0.59 (4/pgin-NC80); 1.19 (2/2) and 0.56 (2/pgi n-nc8°) unit/mg. The relative areas of the stained rocket under different dilutions of the homogenates were determined. Plots of the re- lative rocket areas vs. relative amounts of fly homogenate are performed for each genotype. Six independent determinations such as the one presented in Table 4 suggested that the null

Table 2 Biochemical properties of mouse and Drosophila phosphoglucose isomerase

Mouse Drosophila DBA/2J cn bw ; rie (4/4)

7.0+0.5 7.1±0.5 4.46 4.54

Sedimentation coefficient (S) Stoke's radius (rim) Molecular weight (dalton)

-native -denatured

Isoelectric point Michaelis constant (x 10 +5 M)

(fructose-6 -phosphate) Remaining activity (%)

Specific activity (units/mg)

120,000±7,000 127,000±10,000 55,000±2,000 55,000±2,000

8.4±0.1 6.3±0.2 4.0±0.4 10.0±0.8

32±2 8.5±1 750 1,000

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Fig. 3. SDS polyacrylamide slab gel to show the purity of purified phosphoglucose isomerase from DBA/2J mice (in slot A) and from Drosophila (4/4 genotype) (in slot B) (50 txg protein each).

allele of the mutant (pgi n-Nc8°) expresses no cross-reacting materials (CRM).

More recently, the heterozygous null mutant of phosphoglucose isomerase identified from the mutagenized mice was also subjected to a similar analysis. Preliminary results seem to indicate that it is also a mutant expressing no cross-reacting materials. Details of this study will be presented elsewhere.

Discussion

In this communication, we present a simple and unified procedure for the purification of phos- phoglucose isomerase and genetic variants from mouse and Drosophila. Previously published procedures 9-14 for the purification of this en- zyme from other species did not yield satisfac- tory results for the mouse and Drosophila enzyme. General ligand affinity chromatography utilizing 8-(6-aminohexyl)-amino-ATP as an affinity ligand has been extensively applied to the purifications of numerous kinases and de- hydrogenases 17"24'25. A novel result was recently reported, when a single column was employed for the co-purification of four enzymes from Drosophila 3 including alcohol dehydrogenase, cytoplasmic malate dehydrogenase, a - glycerolphosphate dehydrogenase and phos- phoglucose isomerase. In this study, 8-(6- aminohexyl)-amino-ATP-Sepharose column was mainly used as a weak ion exchange column for the purification of phosphoglucose isomerase, since little inhibition of this enzyme by 8-(6- aminohexyl)-amino-ATP was observed (CHaRI,ES, D. unpublished observation). Moreover, a slight change in pH (0.2 pH unit) could result in partial desorption of the enzyme from the ATP-column. This may suggest that the binding of phosphoglucose isomerase to ATP gel was mainly due to charge interactions and the substrate elution by glucose-6- phosphate simply resulted from the alterations of protein surface charges or conformations.

Biochemical studies of phosphoglucose isomerase showed that the mouse and Drosophila enzyme, in general, have quite simi- lar molecular properties which are similar to those reported for many other species 9-14. The antisera raised in rabbits were shown to have high specificity and no cross-reactivity was ob- served between the mouse enzyme and the antisera to Drosophila enzyme or vice versa. A higher antiserum titer was observed for the Drosophila enzyme. This is probably due to the fact that Drosophila is more distantly related to the rabbit than is the mouse in terms of evolution.

The results of amino acid composition analysis revealed that a certain degree of homology does exist among different species ranging from yeast to humans, especially the

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Fig. 4. Inhibition of phosphoglucose isomerase by antisera to mouse and Drosophila enzyme, A. DBA/2J enzyme + antiserum to DBA/2J phosphoglucose isomerase (Q--- -Q) , C57BL/6J enzyme + antiserum to DBA/2.1 phosphoglucose isomerase ( ~ , - - - k ) , DBA/2J enzyme + nonimmune serum ( © - - - O } , C57BL/6J enzyme + nonimmune serum ( ~ - - - A t . B. Drosophila 414 enzyme+antiserum to Drosophila ( i - - - - -m) , Drosophila 2]2 enzyme+Drosophila antiserum (V Y), 4[4 es~crrte+ nonimmune serum (C3 C3), 2/2 enzyme+nonirnmune serum (V ~7).

amino acid content of aspartic acid, glycine, histidine and lysine.

Electrophoretic variations of phosphoglue~se isomerase in mouse and Drosophila do not seem to alter their biochemical and immunological properties. However, differential thermal stabil- ity was consistently observed between the two mouse variants but not for Drosophila 2J2 and 414 variants.

The raised antisera were employed to charac- terize the null mutants idenitfied in mouse and Drosophila. Two main techniques have been previously developed and adopted for the analysis of Drosophila null mutants 3. Besides rocket immunoelectrophoresis, two-dimensional gel eleetrophoresis was also employed to analyze several null mutants of a- glycerolphosphate dehydrogenase, since its

18

Table 3. Amino acid composition of phosphoglucose isomerase

Mouse a D. melanogaster a Human b Yeasff Amino acid residue (DBA/2J) (cn bw; tie) (muscle) (Brewer)

Aspartic acid 52 58 58 60 Threonine 27 27 39 41 Serine 25 38 32 40 Glutamic acid 55 55 66 58 Proline 26 20 22 17 Glycine 44 47 44 44 Alanine 42 37 42 48 Valine 29 31 30 38 Methionine 4 9 13 7 Isoleucine 32 25 27 27 Leucine 56 46 55 47 Tyrosine 4 10 13 14 Phenylalanine 27 22 29 33 Histidine 20 19 21 18 Lysine 42 40 38 40 Tryptophan 14 d N.D. ~ 32 8 Arginine 22 14 32 10 Cysteine 4 f N.D. ~ 5 1

a Average of two hydrolysates at 24 and 48 hours. The values are normalized to a subunit molecular weight of 55,000 dalton. b From CARTER and YOSI-rI'DA 9. ~From KEMPE et al. 1°. d Determined in presence of 2% thioglyeolic acid during hydrolysis, assuming 85% recovery.

Not determined. f Determined separately as cysteie acid 22, assuming 85% recovery.

Fig. 5. Rocket immunoelectrophoresis. Ten flies were homogenized in 0.05 ml distilled water., 20 tzl of antiserum was mixed with 20 ml of 1% agarose. Gels were stained for enzyme activity after experiments. (a) 20~i Drosophila 4/pgi"-~cs°; (b) 20/~1 Drosophila 4/4 (c) 10 Izl Drosophila 4/pgin-Ncs°; (d) 10/~1 Drosophila 4/4 (e) 5 g,1 Drosophila 4/pgi n-NcS° (f) 5 p,1 Drosophila 4/4 (g) 2.5/zl Drosophila 4/pgP-Ncs°; (h) 2.5 ~1 Drosophila 4/4.

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Fig. 6. (a) to (h), same as Fig. 5 except that 2/2 and 2/pgP -Ncs° genotypes were used in each of the corresponding well.

Table 4. Determination of cross-reacting materials (CRAM) in phosphoglucose isomerase null

mutants from Drosophila

Relative Rocket area Calculated based on

Specific Genotype activity 100% 0%

ratio (unit/rag) CRM CRM Observed a

4/4 to 1.44 0.72 1.44 1.45 ± 0.15 4/pgi n-Nc80

2/2 to 2.1 1.05 2.1 1.98-4-0.18 2/pgi n-Ncs°

a The values of the area ratios were estimated Irom Figures 5 and 6.

protein spot can be readily detected from the two-dimensional gels of a single fly homoge- hate 1'3'26. However, this is not the case for mouse and Drosophila phosphoglucose isomer- ase. Furthermore, the homozygous null mutant of phosphoglucose isomerase in mouse and Drosophila is lethal. So the analysis can only be performed with the genotypes of 2/pgi n-Ncs° and 4]pgi n-NcS° for this Drosophila mutant. As de- monstrated in Figure 5, Figure 6, and Table 4, the null allele, pgp-Ucso appears to express no cross-reacting materials. The absence of cross- reacting materials in a null mutant could imply that the mutation has occurred through either a frame shift, deletion or a regulatory mutation.

To further pinpoint the nature of this null mu- tation, it is necessary to employ recombinant D N A techniques for future analysis 27-3°.

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