Glyceraldehydephosphate dehydrogenase: Crystallization from honeybees; Quantitative immunochemical and electrophoretic comparisons of the enzyme in other insects

3. Insect

Phyriol.,

1968,Vol. 14,pp. 317 to 333.

Pergamon Press. Printed in Great Britain

GLYCERALDEHYDEPHOSPHATE DEHYDROGENASE: CRYSTALLIZATION FROM HONEYBEES; QUANTITATIVE IMMUNOCHEMICAL AND ELECTROPHORETIC COMPARISONS OF THE ENZYME IN OTHER INSECTS RONALD

R. MARQUARDT,*

and RONALD

CHARLES

W. CARLSON,

W. BROSEMERf

Department of Chemistry, Washington State University, Pullman, Washington 99163 (Received 7 October 1967) Abstract-Glyceraldehydephosphate dehydrogenase (EC 1.2.1.12) was crystallized from honeybee (&is melliferu) thoraces. The yellow colour of the crystals is due to diphosphopyridine nucleotide bound to the enzyme. The sedimentation coefficient of the honeybee enzyme is the same as that reported for the enzyme from other species. A small amount of the dehydrogenase was also crystallized from bumblebee (Bombus nevadensis) thoraces. Rabbit antibodies directed against the pure honeybee enzyme were prepared, and the cross-reaction with extracts of other insects quantitatively measured with the micro-complement fixation technique. The order of decreasing crossreaction is : honeybee, leafcutting bee, bumblebee (three species)-lobstermining bee, flesh fly. The order: honeybee, leafcutting bee, bumblebee, was also observed in two-dimensional immunodiffusion and in a precipitin test. The relative cross-reaction of this enzyme, thus, varies considerably from the classical taxonomy of the species tested. The anomalous immunochemical properties of the glyceraldehyde-P dehydrogenases may be due to the relatively conservative changes in primary structure of this protein during evolution. Electrophoretic patterns of glyceraldehyde-P dehydrogenase activity reveal one band in extracts of honeybees and bumblebees, three bands in extracts of leafcutting bees and flesh flies, and up to five bands in extracts of mining bees. INTRODUCTION

IN COMPARATIVE taxonomic and phylogenetic studies, one of the most fruitful approaches is to isolate a pure protein from one species, prepare antibodies directed against this protein, and use the micro-complement fixation test (WASSERMAN and LEVINE, 1961) to quantitatively measure cross-reaction with extracts from the organisms (KAPLAN, 1965; SARICHand WILSON, 1966); this is especially true for studies within the class of insects (BROSJZMER et al., 1967). However, there has been only one such investigation on a single protein from several insects (BROSEMERet al., 1967). The micro-complement fixation technique was used to * Present address: Department of Animal Science, University of Manitoba, Winnipeg, Manitoba, Canada. t Send reprint requests to R. W. Brosemer. 317

318

RONALD R.

MARQUARDT, CHARLES W. CARLSON, ANDRONALD W. BRO~EMER

measure the cross-reaction of insect thoracic extracts with rabbit antibodies directed against pure honeybee glycero-P dehydrogenase (EC 1.1.1.8). The order of decreasing cross-reaction corresponded to the classical taxonomic order of the insects tested. Since this first study was so successful, attempts were undertaken to purify a second protein from honeybees in order to repeat another series of cross-reaction tests. The present report describes the purification of honeybee (Apti mellifera) glyceraldehyde-P dehydrogenase (EC 1.2.1.12) and the results of micro-complement fixation studies with rabbit immunoglobulins directed against this enzyme. In addition, parallel studies on electrophoretic patterns of glyceraldehyde-P dehydrogenase activity in various insects are described. METHODS

AND MATERIALS

Immunochemical methods

The injection schedule for obtaining rabbit antibodies was the same as that previously described (MARQUARDTand BROSEMER,1966), except that no subcutaneous injections were used. The 7s immunoglobulins were isolated (BAUMSTARKet al., 1964) and are termed immunoglobulins III. The microcomplement fixation technique was described by WASSERMAN and LEVINE (1961) and by SARICHand WILSON (1966). T wo-dimensional gel diffusion (Ouchterlony method) was described by CAMPBELLet aI. (1964). The method for precipitation of glyceraldehyde-P dehydrogenase activity from bee extracts (Fig. 7) is the following. Bee thoraces were homogenized in 0-I M tris, 10 mM EDTA, 1 mg/ml bovine serum albumin, O-14 M NaCl (pH 7-7). After centrifugation at 50,OOOg for 20 min, the supernatants were diluted to 1.9 enzyme units/ml; seven doubling dilutions of each extract were then prepared. To each dilution was added an equal volume of immunoglobulins III in saline. After incubation at 30°C for 20 m;n, and at 2°C for 14 hr, the precipitates were removed by ultrafiltration; glyceraldehyde-P dehydrogenase was assayed in each filtrate. Glyceraldehyde-P

dehydrogenase assay

The assay contained 29 mM pyrophosphate, 13 mM arsenate, 90 mM #I-mercaptoethanol, 0.33 mM DPN+, and 1.0 mM m-glyceraldehyde-3-P (pH 8.2). The reaction was started with glyceraldehyde-3-P; assay temperature was 30°C. The rate of change of DPNH concentration was measured at 340 rnp. One unit of enzyme activity is defined as the amount of enzyme catalysing the appearance of 1 pmole product/min under the assay conditions. Materials and other methods

have been previously described (MARQUARDTand BROSEMER,1966; 1967). Disk gel electrophoresis was performed using the standard system described by Canalco. These

BROSEMER et al.,

GLYCERAL.DEHYDBPHOSPHATE

DEHYDROGENAsE:

CRYSTALLIZATION

FROM

HONEYBEES

319

RESULTS

Enzyme purification Unless otherwise noted, all steps were carried out at 0 to 2°C. Fresh honeybee thoraces, including legs and wings, (567 g) were homogenized 1 min in a Waring blender with 2250 ml of 0.1 M trti, 1 mM EDTA, 0.3 M sucrose, 10 mM ,%mercaptoethanol (pH 7.6 at O’C). The homogenate was centrifuged 30 min at 10,000 g. Solid (NH&SO, was added; the l-4 M to 2.2 M (NH&SO, sediment was used for the isolation of glycerophosphate dehydrogenase (MARQUARLITand BROSEMER, 1966). The 2.2 M (NH,),SOI supernatant was saturated with solid (NH&SO, and the precipitate collected by filtration on Whatman No. 1 filter paper. This precipitate was dissolved in 5 mM trek, 2 mM EDTA, 10 mM p-mercaptoethanol, pH 8.3, and dialysed overnight against the same buffer. The solution (340 ml) was applied to a DEAE cellulose column (34 x 3 cm) and eluted at 22°C with the same buffer, using a flow rate of 2 ml per min. Glyceraldehyde-P dehydrogenase and a pink protein (probably cytochrome c) emerged with the void volume. The pink fractions provided an easy method for locating the dehydrogenase. Solid (NH&SO, was added to the pooled fractions until a slight turbidity was observed (about 2.3 M (NH,),SOp). Crystals formed overnight. Ammonium sulphate was added to 2.7 M concentration and the crystals harvested. The protein was recrystallized twice more. At this point the enzyme was probably pure. But since it was yellow, a further step was added in an attempt to remove the presumed coloured impurity. The twice recrystallized protein was placed on a Sephadex G-100 column (55 x 3.5 cm) at 22°C and eluted with 10 mM &is, 2 mM EDTA, 10 mM /%mercaptoethanol, dehydrogenase emerged pH 7.6; the flow rate was 1 ml/min. Glyceraldehyde-P with the void volume. The enzyme was crystallized from the pooled fractions by addition of solid (NH&SO,. The crystals were still yellow. TABLE I-PURIFICATION

Fraction Extract 14-2-2 M (NHJPSO, sediment DEAE cellulose eluate First crystallization Second crystallization Sephadex G-100 eluate Fourth crystallization

OFGLYCERALDEHYDEPHOSPHATEDEHYDROGENASE HONEYBEETHORACES

Vol. (ml)

Total enzyme activity (units)

Yield (%)

2044 340

91,000 88,500

100 97

409 130 92 59 65

74,500 63,000 55,600 48,400 44,800

82 69 61 53 49

* Ape0is absorbance at 280 rnp.

FROM

Specific activity (units/Aaao) * 2.0 10 14 32 60 77 74

320

RONALD R. MARQUARDT, CHARLES W. CARLSON, ANDRONALD W. BROSEMER

Table 1 summarizes the purification procedure. The overall yield was 49 per cent. The resulting protein was pure according to the following criteria: recrystallization to constant specific activity (Table l), cellulose acetate electrophoresis at pH 8.8 (limits of detection less than 1.0 per cent of applied protein), disk gel electrophoresis (Fig. l), and sedimentation velocity ultracentrifugation (Fig. 2). Three enzymes which utilize dihydroxyacetone-P or glyceraldehyde-3-P as substrates were assayed in the final crystalline preparation. Per cent contamination is expressed as the ratio of activity of contaminating enzyme to that of glyceraldehyde-P dehydrogenase, using assays described by MARQUARDTand BROSEMER (1966). The assayed enzymes and per cent contamination are : fructosediphosphate aldolase, less than 0.1 per cent ; triose-P isomerase, less than 0.02 per cent ; glycero-P dehydrogenase, less than 0.06 per cent. This isolation procedure is similar to those reported by ALLISON and KAPLAN (1964a) for the purification of glyceraldehyde-P dehydrogenases from several vertebrate, invertebrate, yeast, and bacterial sources; Dr. Allison has informed us that his group has also crystallized the enzyme from whole honeybees. The present procedure, starting with fresh honeybee thoraces, allows two DPN+dependent enzymes, glycero-P dehydrogenase and glyceraldehyde-P dehydrogenase, to be simultaneously prepared in high yield within 1 week. In addition, a small amount of glyceraldehyde-P dehydrogenase was crystallized from frozen thoraces of a single species of bumblebee (Bombus nevadensis) by essentially the same procedure as above. The bumblebee enzyme, however, is colourless. Ultracentrifugal sedimentation The sedimentation patterns of the honeybee and bumblebee enzymes are shown in Fig. 2. The sedimentation coefficient, sZow, for both is 7.3 under the conditions described in the figure; this is similar to the value for the enzyme from other organisms (ALLISONand KAPLAN, 1964a). Therefore, the molecular weights of both bee enzymes is probably around 140,000 (MURDOCKand KOEPPE, 1964). Enzyme-bound DPN+ The spectrum of the native dehydrogenase is shown in Fig. 3. The absorption maximum is at 276 rnp; A2s0/Aaao is 1.2. A broad shoulder with a maximum around 360 rnp extends into the visible region and is responsible for the yellow colour. This spectrum is similar to that of the rabbit enzyme (RACKERand KRIMSKY, 1952). In the presence of 1.6 mM nL-glyceraldehyde-3-P and 17 mM arsenate, the peak around 360 ml* disappears and the typical DPNH spectrum appears; addition of triose-P isomerase and glycero-P dehydrogenase to this mixture results in reoxidation of the DPNH. Iodoacetate (1.3 mM) also eliminates the peak around 360 rnp; precipitation of the enzyme commences about 1 min after iodoacetate addition at 30°C. Treatment of the native enzyme with charcoal removes the nucleotide, resulting in a

~m

U

Q

Fl~;. 1. Disk gel electrophoretograms ~,f honeybee vlyceraldehyde-P dehydro~enasc. l.eft ,,.~el: 35/*g of native yellow enzyme; centre ~2el: 35 tta of charcoaltreated enzyme ; right ~el : 17 t*g of each.

Fro. 2. Sedimentation pattern of honeybee 'and bumblebee glyceraldehyde-P dehydrogenases in 0.1 M phosphate, 7 mM ED'PA, 10 m M /~-mercaptoethanol (pH 7'0) at 22°C and 59,780 rev/min. The lower pattern is the honeybee protein, the upper pattern the bumblebee protein ; the concentration of both is 5"3 mg/ml. The picture was taken 48 min after top speed was attained; bar angle is 50 °. The small discontinuity to the right of the upper peak is an artifact of the wedge cell, not a protein impurity.

GLYCEIULDEHYDEPHOSPHATE DEHYDROGENASE : CRYSTALLIZATION FROMHONEYBEES321 typical simple protein spectrum with a maximum at 279 (Fig. 3). Addition of glyceraldehyde-3-P and arsenate enzyme results in very little increase in absorbance at 340 does not alter the migration pattern of the enzyme in (Fig. 1) ; enzyme specific activity is also not altered.

240

320

Wave

400

Length

rnp and A2sb/Aasoof 2.2 to the charcoal-treated mp. Charcoal treatment disk gel electrophoresis

400

560

(mp)

FIG. 3. Spectra of honeybee glyceraldehyde-P dehydrogenase in 10 mM tris, native enzyme; - - -, 1 mM EDTA, 10 mM p-mercaptoethanol at 30°C. -, charcoal-treated enzyme. The concentrations of the native and charcoal-treated enzymes are the same. The concentration of each above 320 my. is thirty times greater than that below 320 ny.

Most of the above properties have been reported for the rabbit enzyme (VELICK and FURFINE, 1963). After completing these studies, we were informed by Dr. W. Allison that the enzyme from several other species, including rabbit, is also yellow. The amount of bound DPN+ was estimated by reduction with substrate (ALLISON and KAPLAN, 1964a). The molar extinction coefficient of the DPN+-free enzyme was assumed to be the same as that of the rabbit enzyme, 0.815 cm2 mg-l (MURDOCK and KOEPPE, 1964); this is probably valid, because the tyrosine and tryptophan content of several animal glyceraldehyde-P dehydrogenases is remarkably constant (ALLISON and KAPLAN, 1964a). The molecular weight of the honeybee enzyme was assumed to be 140,000. With these assumptions, the amount 21

322

RONALDR. MARQUARDT, CHARLESW. CARLSON,ANDRONALDW. BROSEMER

of bound DPN+ is calculated to be 3.0 moles/mole of enzyme; because of a hypochromic shift of the DPNH spectrum, this value may be a slight underestimation (MURDOCK and KOEPPE, 1964). These data are consistent with the hypothesis that all glyceraldehyde-P dehydrogenases are tetramers with one DPN+-binding site per monomer (KAPLAN, 1965). Bee glyceraldehyde-P dehydrogenase is not always isolated as a yellow protein. During attempts to isolate another enzyme from honeybee thoraces, glyceraldehydeP dehydrogenase was accidentally crystallized; these crystals were colourless. The isolation procedure was similar to that described above, except that a 15-min heat step at 48” was inserted before chromatography. Since glyceraldehyde-P

.;

.-> +

so -

z 0

60

-

.-E :: 2 40-

FIG. 4. Effect of pH on honeybee glyceraldehyde-P dehydrogenase activity. The standard assay was used, except that pyrophosphate was 32 mM and arsenate 17 mM. The pH of the original buffer mixture (9.2) was altered by adding HCl or NaOH. The pH was measured after reaction.

dehydrogenase was not being assayed during this purification procedure, no yield data are available. As mentioned above, colourless glyceraldehyde-P dehydrogenase has also been crystallized from bumblebees. It is not clear what factors control the amount of DPN+ bound to the purified bee enzymes, but the rabbit enzyme can also be isolated with varying amounts of bound nucleotide (VELICK and FURFINE, 1963). pH Optimum

The pH optimum of honeybee glyceraldehyde-P dehydrogenase under the conditions of the standard assay is 9.1 (Fig. 4). The pH profile is dependent not

GLYCERALDEHYDEPHOSPH

DEHYDROGENASE:

CRYSTALLIZATION

FROM HONEYBERS

323

only on pH but also on ionic strength. Addition of 0.14 M NaCl to the standard assay results in 31 per cent inhibition of enzyme activity at pH 8.1 and 21 per cent at pH 8% No attempt was made to control the ionic strength in the pH profile shown in Fig. 4. The pH optimum of the crystalline bumblebee enzyme is also 9.1 under these assay conditions.

Immunod@sion The anti-honeybee glyceraldehyde-P dehydrogenase immunoglobulins were first checked for homogeneity in the Ouchterlony gel double-diffusion test (Fig. 5).

Hb Ex

0 HbYE

0

Bb Ex

HbCE

0

0

BbCE

0

FIG. 5. Two-dimensional imnmnodiffusion of bee glyceraldehyde-P dehydrogenases. In the centre well were immunoglobulins III diluted 1 : 2 from the concentration in the original anti-serum. In the side-wells were the following antigens : HbEx, honeybee thoracic extract; LCEx, leafcutting bee thoracic extract; BbEx, bumblebee (Bombus appositus) thoracic extract; BbCE, crystalline bumblebee @or&s rreuudensis)glyceraldehyde-P dehydrogenase; HbCE, white crystalline honeybee glyceraldehyde-P dehydrogenase; HbYE, yellow crystalline honeybee glyceraldehyde-P dehydrogenase. The minor bands with two of the wells were not always observed.

One major and one blurred minor band were usually observed when a honeybee extract or pure dehydrogenase was placed in the antigen well. The minor band was not always seen, although it appeared more often with the extract than with the pure enzyme. Since the crystalline dehydrogenase appeared to be homogeneous by the criteria mentioned above, it is possible that the minor band is due to an artifact. Since glyceraldehyde-P dehydrogenases contain several sulphydryl

324

RONALDR. MARQUARDT,CHARLESW. CARLSON,ANDRONALDW. BROSEMER

groups (ALLISON and KAPLAN, 1964a), it is possible that the enzyme was oxidized after injection into the rabbit and that antibodies directed against the oxidized protein were also produced. Attempts failed to increase the intensity of the minor gel diffusion band by oxidizing the crystalline enzyme with various oxidizing agents. The enzyme proved to be highly resistant to denaturation by oxidation. However, some oxidation certainly may occur in the rabbit, since the antigen circulates at about 37°C for several days. The minor band is not due to removal of bound DPN+ from the enzyme, since charcoal-treated dehydrogenase gives exactly the same immunodiffusion pattern as native enzyme. The lack of the minor band with the pure enzyme that had been isolated as colourless crystals (Fig. 5) is probably due to the lower amount of protein in the antigen well. Since only a few milligrams of these crystals had been obtained, it was used sparingly in all experiments. Micro-complement

fixation

Cross-reaction of heterologous antigens with antibodies as measured by the micro-complement fixation method is expressed in terms of the immunological distance (I.D., SARICH and WILSON, 1966). The amount of antibody required to obtain a certain percentage of complement fixation in the peak tube with the homologous system (i.e. antibodies plus honeybee extract) is first determined; the per cent complement fixed must be between 20 and 90 (&RICH and WILSON, 1966). Then the antibody titre required to fix the same amount of complement in the heterologous system (i.e. antibodies plus extract of another insect) is measured. The ratio of the latter titre to the former titre (heterologous/homologous) is the I.D.; the lower the cross-reaction, the greater is the I.D. Despite the presence of the minor band in the immuno-diffusion test, immunoglobulins III could be used for cross-reaction studies. In the micro-complement fixation test, immunoglobulins III react with honeybee thoracic extracts and with crystalline honeybee glyceraldehyde-P dehydrogenase in identical fashion; that is, a single peak is obtained with both the extract and crystalline antigen, and these peaks have the same shape and height (Fig. 6). The peaks also probably have exactly the same position, despite the slight apparent non-overlap shown in the figure. Since glycero-P dehydrogenase interferes with the glyceraldehyde-P dehydrogenase assay, estimation of enzyme activity in extracts is only approximate. Therefore, the amount of enzyme in the respective tubes of both dilution series is probably not identical but only similar. This test indicates that immunoglobulins III are reacting significantly only with glyceraldehyde-P dehydrogenase and thus can be used in cross-reaction studies. Six races of Apis mellifera were then compared. The glyceraldehyde-P dehydrogenase used for preparing immunoglobulins III had been isolated from workers of the Italian line. Extracts of Caucasian, Carniolan, Anatolian, Greek, and Hastings (Caucasian) workers all give I.D. values of 1.0. Therefore, there are no detectable immunological differences of the glyceraldehyde-P dehydrogenase proteins in these six races.

GLYCERALDEHYDEPHOSPHATE DEHYDROGENASE: CRYSTALLIZATIONFROM HONEYBEES 325

The cross-reaction of other insect extracts with immunoglobulins III is shown in Table 2. Several facets of the data are unexpected. First, the I.D. values for fixation bumblebees are very large; a value of 10 to 14 in micro-complement tests usually indicates quite a large taxonomic dissimilarity (KAPLAN, 1965). These 60

I

12s

64

32 Antigen

I

16

I

1

I

I

8

4

2

I

Dilution

FIG. 6. Micro-complement fixation of irnmunoglobulins III with honeybee thoracic extract (0) and with crystalline honeybee glyceraldehyde-P dehydrogenase (a). Enzyme activity was similar in the corresponding tubes of both dilution series.

values can be compared with I.D. values of 2.0 to 2.7 reported for micro-complement fixation cross-reaction of bumblebee extracts with anti-honeybee glycero-P dehydrogenase (BROSEMER et al.,1967). The high values are not due to interference by some factor in the thoracic extracts, because one of the I.D. values was obtained with the crystalline bumblebee enzyme (B. neaadeks). The high values are also probably not due to different amounts of DPN+ bound to the enzyme, since removal of the nucleotide from the honeybee enzyme does not alter the immunodiffusion pattern. The values for the three bumblebee species seem to differ somewhat from one another, but not sufficiently in order to be significant in comparative bumblebee taxonomy. The I.D. value (13) for the robust mining bee overlaps with the values (10 to 14) for bumblebees, although the former is definitely more distantly related to honeybees than are the latter. This may not be significant, however, since the error in I.D. values is greater when cross-reaction is low. The I.D. value (22) for the flesh fly is about the greatest that can be measured in this system and is consistent with its relative taxonomic dissimilarity from bees.

326

RONALD

R.MARQUARDT,CWARLBSW.CARLSON,AND

RONALDW.BROSEMER

The leafcutting bee, however, shows a completely anomalous result. The I.D. value (2.5) is much lower than that for bumblebees or mining bees, even though there is no valid doubt that the taxonomic order of these bees is: honeybee, bumblebee, mining bee, leafcutting bee. TABLE

%--CROSS-REACTION

OF INSECT THORAX EXTRACTS WITH HYDEPHOSPHATE DEHYDROGENASE

Lowest taxon common with honeybee *

Insect

ANTI-HONEYBEE

GLYCERALDE-

Immunological distance (I.D.)

Honeybee (Apk mellifera)

-

Bumblebee Bombus nevadensis (crystalline enzyme) B. appositus B. fervidus

Subfamily

Robust mining bee (Tetralonia sp .)

Family

13

Leafcutting bee (Megachile rotundata)

Superfamily

2.5

Flesh fly (Sarcophaga

Class

22

1.0

10 11 14

bulla ta)

* The classification is described by BORROR

and DELONG

(1964).

In order to determine if these anomalous results are due to an artifact of the micro-complement fixation test, two other immunological techniques were used. The formation and position of spurs in two-dimensional gel immunodiffusion (Fig. 5) indicate that the order of cross-reaction with immunoglobulins III is: honeybee, leafcutting bee, bumblebee. The precipitin line obtained with leafcutting bee extracts is also much darker than that obtained with bumblebee extracts. In a precipitin test at low relative concentrations of immunoglobulins III, the decreasing order of precipitation of enzyme activity from bee thoracic extracts is again: honeybee, leafcutting bee, bumblebee (Fig. 7). At high immunoglobulin titres, the total glyceraldehyde-P dehydrogenase activity is precipitated from all three extracts. This indicates that bumblebees or leafcutting bees lack an isoenzyme of glyceraldehyde-P dehydrogenase that does not cross-react with immunoglobulins III. This precipitin test is specific for glyceraldehyde-P dehydrogenase, since no glycero-P dehydrogenase activity is precipitated from any of the extracts by the highest immunoglobulin titres. Therefore, the anomalous cross-reaction pattern is not due to an artifact of any one immunochemical technique, since three methods give the same relative order

GLYCQZALDEHYDEPHOSPHATE

DEHYDROGENASE:

CRYSTALLIZATION

FROM

HONEYREES

327

of cross-reaction. A second rabbit was injected with crystalline honeybee glyceraldehyde-P dehydrogenase in order to determine whether the anomalous crossreactions are reproducible with another antiserum. Unfortunately, the antibody 100 ua2 G _z 80 Z ?! a %60 ..-z

t a 0, 4o

E 2

W 2

20

s t a

O0

100

lZLo+ive ArZbody CoEen+ro+iorY” FIG. 7. Precipitation of honeybee, bumblebee (B. uppositus),and leafcutting bee glyceraldehyde-P dehydrogenase from extracts by immunoglobulins III. The experiment is described under Materials and Methods. titre in the second rabbit remained low despite several subcutaneous and intragel immunodiffusion tests could venous injections of antigen. Two-dimensional be run, but no micro-complement fixation tests. Immunodiffusion shows strong reaction with honeybee extracts, a fairly strong band with leafcutting extracts, and a very faint band with bumblebee (Bombus uppositus) extracts. Therefore, the relative order of cross-reaction apparently is not an artifact produced by the antiserum of one particular rabbit.

Electrophoretic comparisons

One possible explanation of the anomalous order of cross-reaction is the presence of glyceraldehyde-P dehydrogenase isoenzymes. For example, honeybees and leafcutting bees may contain solely or mainly isoenzymes type I which strongly cross-react, whereas bumblebees and mining bees contain solely or mainly isoenzymes type II which weakly cross-react with the honeybee enzyme. Therefore, cellulose acetate electrophoresis was used to probe for possible isoenzymes. As is shown in Table 3, honeybee and bumblebee thoracic extracts contain only one electrophoretic band with glyceraldehyde-P dehydrogenase activity; the electrophoretic migration of each in phosphate buffer (pH 64) is similar. Mining bee, leafcutting bee, and flesh fly extracts all show three to five bands each; in each case the darkest band is the slowest moving component.

328

RONALDR. MARQUARDT, CHARLESW. CARLSON,ANDRONALDW. BROSEMER

TABLE 3-FLECTROPHORETIC PATTERNSOF GLYCEEALDEHYDEPHOSPHATE DEHYDROGENASE IN INSECTTHORAXEXTRACTS

Insect * Honeybee Bumblebee (B. appositus) Robust mining bee

Leafcutting bee

Flesh fly

Lobster 5

Per cent migration relative to honeybee enzyme t

Relative intensity of band:

100 90

-t+++ ++++

203 192 175 161 142 191 172 150 170 143 118 245

+ + ++ +++ ++++ ++ +++ ++++ ++ +++ ++++ ++-t-+

* The systematic names of these insects is given in Table 2. t Migration in each case is toward the anode. $ The major band for each insect is given the value + + + + . Bands indicated with + are faint and not always observed. 5 The purified lobster enzyme was used.

It has been suggested that multiple electrophoretic bands with dehydrogenase activity may be due to a carrier protein which binds more than one type of dehydrogenase enzyme or due to enzymes with more than one type of dehydrogenase activity (AGRELL and KJELLBERG, 1965). This was postulated, because certain electrophoretic bands of extracts may stain for more than one dehydrogenase activity. We therefore checked the thoracic extract electrophoretograms for the following activities : alcohol dehydrogenase (EC 1.1.1. l), glycerol dehydrogenase (EC 1.1.1.6), glycero-P dehydrogenase, lactate dehydrogenase (EC 1.1.1.27), and malate dehydrogenase (EC 1.1.1.37). With only one exception, none of the bands with glyceraldehyde-P activity stain for any of the above enzymes. The two slowest glyceraldehyde-P dehydrogenase bands of flesh fly do exhibit glycero-P dehydrogenase activity, but this probably reflects simply the identical migration rates of these separate proteins. In the absence of substrate and presence of DPN+, no staining activity at all was observed. These data do not completely eliminate the possibility that isoenzymes are responsible for the immunochemical anomalies ; however, they render this postulate improbable. Honeybees and leafcutting bees cross-react strongly, yet show completely different isoenzymic patterns; honeybees and bumblebees cross-react weakly, yet show very similar electrophoretograms ; honeybees and mining bees or

GLYCERALDEHYDEPHOSPHATE

DEHYDROGENASE:

CRYSTALLIZATION

FROM HONEYBEES

329

flesh flies show both weak cross-reaction and different electrophoretograms. Thus, there is no correlation between comparative immunochemical and electrophoretic properties of glyceraldehyde-P dehydrogenases from these insects.

Cross-reaction with lobster glyceraldehydephosphate dehydrogenase In our system, the first cross-reaction to be measured was that with bumblebee extracts. Since these I.D. values are 10 to 14, we believed this represented a surprisingly weak cross-reaction of proteins from two very closely related species. The next I.D. values measured were for mining bee (13) and flesh fly (22) ; although high, these values still are essentially consistent with classical taxonomy. It was not until leafcutting bee (I.D. value = 2.5) was investigated that the extent of the anomaly became apparent. This led us to measure the cross-reaction of antihoneybee glyceraldehyde-P dehydrogenase with the lobster enzyme in the microcomplement fixation test. ALLISON and KAPLAN (1964b) have reported that, when rabbit anti-lobster glyceraldehyde-P dehydrogenase cross-reacted with honeybee extracts in the micro-complement fixation test, an I.D. value of 11 was observed. SARICH and WILSON (1966) f ound that reciprocal cross-reactions in the micro-complement fixation test tend to be similar in the case of primate serum albumins; that is, the I.D. value for anti-X plus antigen Y is about the same as for anti-Y plus antigen X. If similar reciprocal cross-reaction holds for the glyceraldehyde-P dehydrogenase system, the I.D. value for anti-honeybee enzyme plus lobster antigen should be around 11. Lobster glyceraldehyde-P dehydrogenase was purified (ALLISON and KAPLAN, 1964a) in order to eliminate interfering substances ; micro-complement fixation with immunoglobulins III was measured. The I.D. value is indeed 11. It is striking that an identical reciprocal cross-reaction is observed even though the antibodies were prepared several years apart in different laboratories. Moreover, the cross-reaction of anti-honeybee glyceraldehyde-P dehydrogenase is essentially identical with either bumblebee or lobster extracts; honeybees and bumblebees share the same subfamily, honeybees and lobster only the same phylum. In addition, extracts of the flesh fly, which shares the same class with honeybees, shows less cross-reaction than the lobster enzyme. Unless one is ready to completely challenge classical taxonomy, it is obvious that the micro-complement fixation test with anti-honeybee glyceraldehyde-P dehydrogenase does not give meaningful taxonomic data. The electrophoretic migration of the lobster enzyme is included in Table 3. DISCUSSION Glyceraldehyde-P dehydrogenase is only about the sixth enzyme to be crystallized from insects. The isolation procedure and properties of the protein described in this report are similar to those of the enzyme in other widely differing organisms (ALLISON and KAPLAN, 1964a) ; this key glycolytic catalyst has apparently changed to a relatively small extent during the course of evolution.

330

RONALD R. MARQUARDT, CHARLES W. CARLSON, AND RONALD W. BROSEMER

The question has been raised whether insect glyceraldehyde-P dehydrogenase is not much more resistant to iodoacetate inhibition than is the mammalian enzyme (CHEFURKA,1954). As will be reported elsewhere, we have found that the inhibition by iodoacetate of the pure honeybee enzyme is very similar to its inhibition of the rabbit muscle enzyme. The micro-complement fixation technique proved quite useful in a quantitative immunochemical comparison of glycero-P dehydrogenases in various insects (BROSEMER et al., 1967). In that study, a direct correlation was observed between immunological cross-reaction and classical taxonomic dissimilarity of the insects. The I.D. values obtained with anti-honeybee glycero-P dehydrogenase antisera were such that these cross-reaction studies were limited largely to advanced Hymenoptera. Thi s system was most suitable for taxonomic comparisons of species within the bee superfamily, i.e. of species which are closely related to the reference insect, honeybee. When crystalline honeybee glyceraldehyde-P dehydrogenase was available, a new series of micro-complement fixation studies could be undertaken. ALLISON and KAPLAN(1964b) had reported that the I.D. value for cross-reaction of rabbit anti-lobster glyceraldehyde-P dehydrogenase with honeybee extracts was 11. This is a very strong cross-reaction for proteins from species which are so distantly related. We therefore expected that the I.D. values with rabbit anti-honeybee glyceraldehyde-P dehydrogenase would not significantly distinguish between species closely related to honeybees, but only between species of different taxonomic orders or even classes. This type of immunochemical system would complement the anti-honeybee glycero-P dehydrogenase system, where fine differences between closely related species could be measured. The results did not correspond to our expectations. The cross-reaction of anti-honeybee glyceraldehyde-P dehydrogenase with the lobster enzyme is the same as the reciprocal cross-reaction reported by Allison and Kaplan, but the cross-reaction with insect extracts does not fit any taxonomic pattern. Why does this antiserum exhibit anomalous cross-reactions ? As discussed above, it is apparently not an artifact produced by the antiserum of one particular rabbit, by the micro-complement fixation test itself, by varying amounts of enzymebound DPN+, or by the presence of isoenzymes. The artifacts are probably not due to the redox state of the antigen sulphhydryl groups for the following reasons. First, the honeybee enzyme is very resistant to denaturation by oxidizing agents, such as oxygen and oxidized glutathione. Secondly, when insect thoracic extracts were prepared in the presence or absence of /?-mercaptoethanol, no significant difference in I.D. values was observed. Thirdly, the specific enzymic activities of crystalline honeybee and bumblebee (B. nmadellsis) enzymes are almost identical; this suggests that the redox state of the sulphhydryl groups is similar or identical in both enzymes. Yet, the crystalline bumblebee enzyme, as w-e11as extracts of other bumblebee species, show very weak cross-reaction with anti-honeybee glyceraldehyde-P dehydrogenase. The immunochemical cross-reactions might indeed reflect the relative simi-

GLYCRRALDEHYDEPHOSPHATR

DEHYDROGRNASE:

CRYSTALLIZATION

FROM HONRYREES

331

larities in protein structure; that is, the descending order of primary structural similarities of glyceraldehyde-P dehydrogenases might be : honeybee, leafcutting bee, bumblebee-lobster-mining bee, flesh fly. This would be a highly complex example of convergent evolution at the molecular level. There are some examples where the comparative primary structure of a protein does not exactly correspond to the known taxonomic relationship of the organisms (FITCH and MARGOLIASH, 1967) ; however, these cases do not occur frequently and the discrepancies are not large. It seems most unlikely that the antigenic properties of the arthropod glyceraldehyde-P dehydrogenases in the present study actually correspond to the relative primary structures, since the discrepancies between evolutionary change at the protein level and at the organismal level would have to be far too great and complex. A possible explanation of the imrnunochemical anomalies was suggested to us by Dr. William Allison. He has found that the primary structure of glyceraldehyde-P dehydrogenases from a mammal and from a crustacean are remarkably similar; that is, this protein is very conservative in terms of structural changes during evolution. Since the structure of the honeybee and rabbit enzymes are probably also very similar, the number of antigenic determinant sites on the honeybee protein injected into rabbits may be relatively limited for a protein of this size. Each antigenic determinant may include sequences of the dehydrogenase protein that: (1) greatly differ between the honeybee and cross-reacting protein, (2) are essentially identical in the honeybee and cross-reacting protein, or (3) a combination of these. If, for example, the honeybee and bumblebee enzymes are indeed quite similar, but the few structural differences are concentrated at the limited number of antigenic determinant sites, the rabbit antibodies would preIf the protein structural differences are more sumably show low cross-reaction. concentrated in regions which do not include antigenic determinant sites (for example, leafcutting bee), the cross-reaction might be relatively strong. Although there is no direct evidence to indicate whether this hypothesis may actually account for the immunochemical anomalies, we believe it is the most reasonable explanation. Micro-complement fixation comparisons have been successfully made with anti-lobster glyceraldehyde-P dehydrogenase. ALLISON and KAPLAN (1964b) did observe cross-reactions consistent with the classical taxonomy of arthropods. However, strong cross-reaction with the protein of species which had diverged from the lobster a very long time ago is consistent with conservative evolutionary The reasons are not clear why the changes of glyceraldehyde-P dehydrogenase. micro-complement fixation test gives meaningful taxonomic comparisons in the case of anti-lobster glyceraldehyde-P dehydrogenase, but not in the case of antihoneybee enzyme. The present study is, therefore, a good example of the pitfalls that can be encountered, when any one criterion is applied to taxonomic comparisons. It also illustrates the fact that any particular immunochemical cross-reactions system does not necessarily give meaningful taxonomic and phylogenetic information.

332

RONALDR. MARQUARDT, CHARLESW. CARLSON,ANDRONALDW. BROSEMER

It is absolutely necessary to first standardize the cross-reactions with known taxonomic relationships before applying the technique to further studies. Electrophoretograms of glyceraldehyde-P dehydrogenase activity in insect extracts reveal only one band for honeybee and bumblebee, three bands for leafcutting bee and flesh fly, and possibly five bands for mining bee. Five bands would be consistent with a tetrameric structure of the enzyme, since two different polypeptide chains could give rise to a maximum of five molecular species. The number of insects investigated is far too limited in order to attempt conclusions concerning taxonomic relationships of the electrophoretic patterns. Whether the single bands in extracts from the two species of Apinae and multiple bands in the other three insect extracts have any biochemical or physiological significance cannot be determined without further studies. LEBHERZ and RUTTER (1967) have very recently reported a similar electrophoretic study on glyceraldehyde-P dehydrogenase variants in several species of animals, plants, and micro-organisms. Multiple dehydrogenase bands were detected in some organisms, only single bands in others. Only one insect, honeybee, was investigated ; as also reported in the present paper, a single form of glyceraldehyde-P dehydrogenase was observed. Acknowledgements-We wish to acknowledge the technical assistance of LOIS BARNES and DAVIDGROSSO. We wish to thank Dr. ALLAN C. WILSON for introducing one of us (R. W. B.) to the micro-complement fixation technique. The work was supported in part by grant GB-4863 from the National Science Foundation, by a development award to R. W. B. of the Public Health Service research career program (No. I K03 GM 11073-01 GMK) from the Institute of General Medical Sciences, and by funds provided for biological and medical research by the State of Washington Initiative Measure No. 171.

REFERENCES ACRELL I. and KJELLBERC B. (1965) A comparative analysis of the isozyme pattern of dehydrogenases. Camp. Biochem. Physiol. 16, 515-521. ALLISONW. S. and KAPLANN. 0. (1964a) The comparative enzymology of triosephosphate dehydrogenase. J. biol. Chem. 239, 2140-2152. ALLISONW. S. and KAPLANN. 0. (1964b) The comparative enzymology of triosephosphate dehydrogenase. In Taxonomic Biochemistry and Serology (Ed. by LEONE C. .4.), pp. 401-406. The Ronald Press, New York. BAUMSTARK J. S., LAFFIN R. J., and BARDAWILW. A. (1964) A preparative method for the separation of 7s gamma globulin from human serum. Archs biochem. Biophys. 108, 514-522. BORRORD. J. and DELONG D. M. (1964) An Introduction to the Study of Insects. Holt, Rinehart, and Winston, New York. BROSEMER R. W., GROSSOD. S., ESTESG., and CARLSONC. W. (1967) Quantitative immunochemical and electrophoretic comparisons of glycerophosphate dehydrogenases in several insects. J. Insect Physiol. 12, 1757-1767. CAMPBELLD. H., GARWY J. S., CREMERN. E., and SUSSDORFD. H. (1964) Methods in Immunology, pp. 143-149. Benjamin, New York. CHEFURKAW. (1954) Oxidative metabolism of carbohydrates in insects-I. Glycolysis in the housefly Musca domestica L. Enzymologia 17, 73-89.

GLYCERALDEHYDEPHOSPHATE DEHYDROCENASE: CRYSTALLIZATION FROMHONEYBEES 333 FITCH W. M. and MARGOLIASH E. (1967) Construction of phylogenetic trees. Science, N. Y. 155, 279-284. KAPLANN. 0. (1965) Evolution of dehydrogenases. In Evolving Genes and Proteins (Ed. by BRYSONV. and VOGELH. J.), pp. 243-277. Academic Press, New York. LEBHERZ H. G. and RUTTER W. J. (1967) Glyceraldehyde-3-phosphate dehydrogenase variants in phyletically diverse organisms. Science, N. Y. 157, 1198-1200. MARQUARDT R. R. and BROSEMERR. W. (1966) Insect extramitochondrial glycerophosphate dehydrogenase-I. Crystallization and physical properties of the enzyme from honeybee (A+ mellijera) thoraces. Biochim. biophys. Actu 128, 454-463. MURDOCKA. L. and KOEPPE 0. J. (1964) The content and action of diphosphopyridine nucleotide in triosephosphate dehydrogenase. J. biol. Chem. 239, 1983-1988. RACKER E. and KRIMSKY I. (1952) The mechanism of oxidation of aldehydes by glyceraldehyde-3-phosphate dehydrogenase. J. biol. Chem. 198, 731-743. SARICHV. M. and WILSON A. C. (1966) Q uantitative immunochemistry and the evolution of primate albumins: micro-complement fixation. Science, N. Y. 154, 1563-1566. VELICK S. F. and FURFINE C. (1963) Glyceraldehyde 3-phosphate dehydrogenase. In The Enzymes, 2nd ed. (Ed. by BOWR P. D., LARDY H., and MYRBACK K.) 7, 243-273. Academic Press, New York. WASSERMAN E. and LEVINE L. (1961) Q uantitative micro-complement fixation and its use in the study of antigenic structure by specific antigen-antibody inhibition. J. Immunol. 87, 290-295.