Comp. Biochem. Physiol., 1977, Vo/. 58 B. pp. 71 to 79. Pergamon Prcs& Prtnttd |n Great Brt~m

CHANGING DISTRIBUTION PATTERNS OF ct-GLUCOSIDASE ISOENZYMES IN EXTRACTS OF ANTENNAL SEGMENTS

AND IN THE HAEMOLYMPH OF THE COTTON STAINER DYSDERCUS INTERMEDIUS DIST.

K. P. GAFF^L Institut for Botanik und Pharmazeutische Biologie, Universit~t ~Erlangen-Niimberg,

8520 Erlangen, Schloflgarten 4, B.R.D.

(Received 7 January 1977)

Abatraet--l. The ",-glucosidase (E.C. 3.2.1.20) activities in extracts of the 2nd and 4th antennal segments (AS) and in the haemolymph of the cotton stainer were quantitatively examined by use of trehalose, p-nitrophenyi-,,-glucoside, sucrose, turanose, maltose, isomaltose and palatinose as substrateg

2. Three peaks of activity were isolated by gel filtration from the extracts of both AS, but only two peaks from the haemolymph.

3. The peaks Ill of both AS differ in their substrate specificities from those of the peaks I and II.

4. It was evident from experiments, which used fractional extraction and variation of pH in addition to gel filtration that more than a single enzyme contributed to the formation of each peak.

5. Results are discussed with reference to the hypothesized presence of sugar reception processes in insect gustatory cells.

I N T R O D U C T I O N

Up until now the digestive tracts and the glandular appendages of various animal species have been in- vestigated in regard to oligosaccharide splitting enzymes (GottschalL 1950; Nisizawa & Hashimoto, 1970). Particularly the coaction of these enzymes in the decomposition of nutritive polysaccharides has generated great interest. However, the existence of such enzymes in other tissues has also been demon- strated (van Handel, 1968a; Berger & Sacktor, 1970).

The present investigation deals with a-glucosidase activities in the antennai segments (AS) and the hae- molymph of the cotton stainer, and with their isola- tion by gel filtration. We wanted to determine whether or not the quantity of taste hairs on the antennal segments produced differences in the activity and pattern of their enzymes, as was evident from investigations of the tarsal, tibial and labellar seg- ments of flies (Morita, 1972; Hansen, 1975g Based on those results it was hypothesized that ~t-glucosi- dase acts as a receptor protein in the sugar receptor cells.

It is evident from the present results that the condi- tions in the cotton stainer are different from those of the flies in several aspects: The activities are much lower and antennal parts with many taste hairs con- tain a higher maltose-specific activity than antennal parts with fewer taste hairs.

M A T E R I A L A N D M E T H O D S

Cultivating the African cotton stainer Dysdercus interme- dius Dist. in the laboratory takes relatively little work. The animals thrive best at temperatures of 25--28°C and at 70% relative humidity. Cotton seed, soaked in water for 24 hr served as food. Immediately after the last moulting stage the adults were transferred to new cages and provided with food and water (soaked cotton plugs) for 4-5 days more.

The 2rid antennal segments (pedicels) and the 4th anten- nal segments (outermost flagellar segments) were dissected from the heads after decapitation. Neither of the antennal segments (AS) contain muscles (Weber, 1966). The 4th AS are crowded with sensory hairs (Grochol, 1970), through- out which 220-280 taste hairs are interspersed, whereas the 2nd AS are practically barren. In adults the ultra- structure of these sensory organs is similar to that of second stage nymphs (Gaffal, 1976). After washing the AS in 30 mM phosphate buffer (pH 7) which contained traces of serum albumin, the AS were frozen in liquid nitrogen and stored at -20°C until needed for experiments.

Preparation of the crude extract

After adding 1 ml of extraction buffer (l : 1 diluted Mc Ilvaine buffer, pH 7, 100raM KCI, I% Triton X-100), 1000-2000 AS were homogenized for 1 hr with a glass homogenizer (DuaU 21, Kontes Glasses Co.) which was cooled in an ice bath. Afterwards 5 ml buffer were success- ively added in 1 ml steps. After stirring with a magnetic stirrer for 18-24 hr at 4°C, the homogenate was centrifuged for 60min at 27,500 0. If <90% of the sucrose splitting activity was found in the supematant, the sediment was resuspended in 6ml extraction buffer and stirred for another 24 hr. Two to three such extractions were sufficient for at least 90% enzyme recovery.

Fractional extraction

Homogenization and extraction were carried out as above but with two different solvents: First extraction sol- vent: 1:1 diluted Mc llvaine buffer, pH 5.5; second extrac- tion solvent (and third, if necessary): 1:1 diluted Mc IIvaine buffer, pH 7, 100 mM KCI, 1~ Triton X-100. After- wards these two extract fractions were examined simul- taneously.

The volumes of the supematants (6-18 ml) were reduced to 400-700/tl with Collodion Bags (Sartorius Membrane Filter SM 13200) under low pressure with the aid of a water jet pump. During the concentrating process the Collodion Bags were surrounded with buffer cooled to 4°C (1:1 diluted Mcllvaine buffer, pH 5.5, 50mM KCI, 0.5% Triton X-100). The concentrated extracts were then run

71

72 K.P. GAFFAI.

on columns (polyacryl-amidgel P-200, Fa. BioRad; length 1.1 m; dia: 9 mm). 100 fractions of 300gl were collected at a flow rate of 800/~l/cm 2 hr.

In order to get exact information on differences in the glucosidase distribution of these two AS, the following points were heeded: (1) Only extracts from AS of the same animals were compared with each other. (2) As far as poss- ible the activity tests on the extracts to be compared were carried out simultaneously under the same experimental conditions.

Collection of haemolymph The antennae were cut off at the pedicel. The amount

of haemolymph, collected in ice-cooled centrifuge tubes could be increased by exerting slight pressure on the abdomens. Females yielded 2--3 times more haemolymph than males. 3(gb600 gl haemolymph were collected as fast as possible and centrifuged at 27,500 @ for 1 hr. The clear part of the liquid was drawn off with a syringe, mixed with elution buffer (1:1 diluted McIIvaine buffer, pH 5.5; 50 mM KCI, 1~ Triton-X-100) to a volume of 800-1000 jul and applied to the column.

Enzyme assays Enzymatic activity was estimated in the crude extract

at pH 6.1 and in the eluants at pH 5.5 at a temperature of 25°C. A 30-60 ~1 aliquot of crude extract or eluant was diluted 1:1 in substrate-buffer-solution for assay. Glucose liberated by enzymatic hydrolysis of the following di- saccharides (sucrose: fl-fructofuranosyl-~-glucoside; real- lose: 4-a-glucosylglucose; isomaltose: 6-a-glucosylglucose; trehalose: a-glucosyl-c~-glucoside; turanose: 3-a-glucosyl- fructose; palatinose: 6-a-glucosyifructose) was determined enzymatically at pH 7 with glucose oxidase, peroxidase and o-dianisidine as chromogen (KUhner & Hansen, 1975). Using p-nitrophenyl-~-glucoside (nipheglu) as a substrate, the liberated p-nitrophenol was measured spectrophotome- trically at 410 nm after addition of 200/A 10~o sodium car- bonate.

RESULTS

The total a-glucosidase (E.C. 3.2.1.20) activities in crude extracts of the 2nd and 4th antennal segments (AS) are listed in Table i. Several substrates were tested. On the substrate sucrose the 2nd AS, which are devoid of taste hairs, showed an average of 1.5 times more activity than the 4th AS which are rich in taste hairs. The difference is somewhat smaller----ca. 1.2 times more activity in the 2nd A ~ - w h e n nipheglu was used as a substrate. On the other hand maltose was hydrolyzed 2-3.5 times faster in the 4th AS than in the 2nd AS. With trehalose the activity in the 2nd AS was 3 times greater than in the 4th AS. This factor is then at least twice as high as the ratio of activity for the substrates sucrose or nipheglu. Trehalose was split fastest by the 2nd AS, whereas trehalose and

maltose were split at about the same rate by the 4th AS.

Isolation of enzymes by yel filtration

Typical isolations of the ~-glucosidases in extracts of the 2nd and 4th AS and of the haemolymph are demonstrated in Figs. l -3. Three main peaks resulted from the antennal extracts, but only two from the haemolymph. The substrate specificity, the solubiliza- tion by fractional extraction and the pH-dependence served as other criteria in the differentiation of indivi- dual enzymes.

2nd antennal segments

The first peak of activity appears together with dex- trane blue. This peak hydrolyzes sucrose, nipheglu and turanose at about the same rate, whereas maltose and isomaltose are hydrolyzed only at 60-80Vo and a mere 7~o, respectively, of this rate. Similar propor- tions of substrate specificity are also found in peak II. The existence of one more peak between the main peaks I and II can be derived from the asymmetric profile shape (at VJVo = 1.18), independent of the substrate tested. This intermediate peak hydrolyzes isomaltose better than both adjacent peaks.

The main peak III displays profiles of substrate hy- drolysis which are oriented asymmetrically to one another. The maxima of nipheglu-, turanose- and suc- rose-turnover appear 3--4 fractions earlier than those of the maltose-, isomaltose- and palatinose-turnover. Consequently two separate enzymes exist, namely III1 and III2. The enzyme III1 hydrolyzes nipheglu some- what faster than sucrose and turanose. Maltose is split likewise, but to what degree compared to the other substrates is unknown. The rate of hydrolysis of isomaltose seems to be especially slow.

Enzyme III2 which is eluted later is highly maltose- specific. The profiles of isomaltose and palatinose are oriented symmetrically to that of maltose (palatinosc profiles are not demonstrated in the Fig. at hand). The rate of isomaltose turnover amounts to 10~o of the maltose turnover. Another experiment revealed that palatinose is split half as well as isomaltose. The profiles recorded with nipheglu, turanose and sucrose are considerably steeper on the ascending than on the descending f lank--an indication that these sugars are also substrates of enzyme III 2.

4th antennal seyments

The three main peaks show the same behaviour in elution as the main peaks of the 2nd AS. The degrees of activity in peak I and II are nearly the same, but both peaks are smaller than the corre-

Table 1. Total activities in the 2nd and 4th antennal segments, related to 50 mg anten- nal tissue (dry weight). The supernatants contained an average of 90~o of the total

activity. Test conditions: pH 6.1; sucrose and maltose 80 raM; nipheglu 10 mM

Substrate Activity (mU/ml)

2nd antennal segments 4th antennal segments

sucrose(n = 8) 176 ± 72 116 ±69 nipheglu 510 432

380 350 maltose 510 1070

140 490

:~-Giucosidascs in Dysdercus 73

2.0

1.0

~th ANTENNAL SEGMENTS

• sucrOse (80 mMI

e, turonose {80raM)

o nipheglu (1C raM)

• mo[tose (80raM) •

peak I I~mk II

19 1.2 1..~ 1.6 1.8 2,0 22 2.4 MI/VO

j • I

• sucrose {80 mM) peak I ~ turanose (80.'~)

i / • Flrl(l|tose (80 raM) peok II ~ isomoLtose (80 raM)

peak Ill

1.0

1.0 1.2 I/- 1.6 1.8 2.0 2.2 2% ~/VO

Fig. I. Isolation of ,,-glucosidases by gel filtration of extracts got from the 4th AS (la) and 2nd AS (lb) of the same animals. The activities (mU/ml) in Figs I-7 relate to 50 nag of tissue (dry weight). Instead of the number of fractions, the ratio of elution volume (V,) to void volume (V,) is plotted

on the abscissa. VJV, = 1 is equivalent to the maximum of the dextrane blue peak.

sponding ones in the 2nd AS. The two peaks do not hydrolize maltose at a slower rate than sucrose and turanose, as is the case in the corresponding peaks of the 2nd AS, but rather equally fast. The nipheglu turnover exceeds that of sucrose. From these different splitting proportions in peak I and II of both anten- nal segments it was determined: 1. That each of the peaks is composed of at least two components, whose relative amounts are different in both antennal seg- ment~ 2. That because of the similarly changed split- ting proportions, each peak I is an associated product

of the following peak II. In addition both peaks have shoulders at VJV o = 1.1 and 1.4.

Peak III was collected later than that of the 2nd AS (Fig. 2). The proportions of substrate turnover are different from those of the 2nd AS. Only the turn- over of isomaltose and palatinose, which amounts to 6 and 3%, respectively, of the maltose turnover, is approximately the same in both AS. Sucrose and tur- anose are split at a considerably flower rote, whereas the rate of nipheglu hydrolysis is faster. The asym- metry seen in Fig. lb is nearly absent. It is deduced

74 K.P. GAFFAL

Fig.

ft. 4 th A N T E N N A L S E G M E N T S " • 8 0 r a m m a l t o s e + ' d ' ~ " ~

• 80t 'r IM t r e h o l o s e

12.S 2 nd A N T E N N A L SEGMENTS / : . :

:: 8 0 r a m m a l t o s e c 8 0 m M t r e h a l o s e /

• (,.C -- /

/

5 :.. - • . • " = . / \ / : ..-..',...

r ~ r L ' : ~ , ~ ¢ ~ " % "~,.,, s.~ ~,,, ,.. ",..\ • _ ~ - - - , : - , ! ~ , ~ ! . . , . ~ " , ~ ' . a ~ " ~

1.0 ' ? 1 t'. 1 ¢" 1.£ ~; " Z 2 2 ~ Ve/Vo

2. Distribution of trehalase and maltase activities after gel filtration of extracts from the 2nd and 4th AS of the same animals.

from these results that the component III1 is reduced to less than one third of its activity in the 2nd AS; its behaviour is similar to that in peaks I and II of the 4th AS. On the other hand the activity of com- ponent IIIz is considerably higher. The specificity of this extremely maltose-specific enzyme is evident from the steep slope of the flank of peak III, which is no longer influenced by the component III~. Relative to the maltose hydrolysis (100%), the hydrolysis of nipheglu amounts to 16% and that of sucrose and turanose to 5-10%. The percentage of component III1

in the total peak I l l maltose turnover remains un- clear. Correspondingly, the exact factor is unknown by which component III2 increases in the 4th AS compared to the 2nd AS.

Tests with trehalose revealed primarily two peaks, which were collected later than the enzymes II and III tested with maltose (Fig. 2). The second trehalose (E.C. 3.2.1.28) peak of the 2nd AS shows six times more activity than the corresponding maltose peak III, whereas about the same amount of activity in both substrates exist in the 4th AS. Trehalase activity

0 2 ~

H A E I 4 O L Y M PH

• s u c r o s e (SCrrt¢)

o n lpheg lu aCm~',

• m a l t o s e ~80"T'M}

r: i s o m a l t o s e (SCraM)

• t r e h a t o s e [B3mMI

~. m e h z l t o s e ( 8 0 m t c ]

,A , - ' \ ," \

Z /

P /. .

p e a k |[

0,1"-- \ p e a k I I ' ~ A I ~, I

' '~' d

~"I~"~-"F---T -- - r - q ~r .... T , 10 1,2 1,4 1.6 1.8 2.0 2.2 2.4 VelVo

Fig. 3. Isolation of glucosidases and trehalase by gel filtration of the haemolymph. On the supposition that haemolymph consists of 50?/0 of the difference between the dry and the live weight, the activity was related to the haemolymph content in 50 mg (dry weight) of antcnnal tissue. Notice that the

scale of the ordinate is c a . 10 times greater than in Fig. 1.

a-Glucosidases

near peak lI (related to the maltase activity) increases three to four times in both antennal segments. It is deduced from the asymmetry of the trehalose and maltose splitting profiles that two individual and specific trehalases exist.

H aemol ymph Figure 3 demonstrates the activity distribution of

the haemolymph glucosidases. The profile shape is different from that of the antennal segments. No ac- tivity is found in the zone of the AS peak II. Peak I is separated into two components of similar sub- strate specificity. Peak II which shows a higher ac- tivity contains several enzymes--at least three a-glu- cosidases and probably one trehalase. The maltose and trehaiose profiles are steeper than those of other

in Dysdercus 75

substrates~ In contrast to the antennal segments the peak maxima of the maltose and trehalose turnover coincide. The maximum of the sucrose turnover is not found, as in the AS, before but rather after that of maltose. The proportions of hydrolysis also differ from those of the AS; for instance the trehalose turn- over is only half as high as the maltose turnover.

The percentage of the haemolymph activity in the total activity is evaluated by the haemolymph content of the antennal segments. Based on the anatomical situation haemolymph is estimated to occupy 40% of the antennal volume. It is supposed further more, that the dry weight of the antennal tissues and of the cuti- cle amounts to 50% of the live weight. Hence it fol- lows that the haemolymph volume in #! is equivalent to about half of the difference between the live and

O8

G.6

peak I peak

/

/ o. •

|. • • " ee °%o eeeoe e

J I i i 1 C' 1.2 1L

4th ANTENNAL SEGMENTS

10 mM nipheglu

• 1 st ex t rac t

o 2 n d e x t r a c t

peak I[[

~ee° e

o' / • , . . ' /%\.

%'~1 • ooe°~ :_ I " " ~ I J 1 I I I

1.6 18 2.0 72 2J. ~/Vo

t/- l. tn ANTENNAL SEGMENTS

-E ! 80 rnM ma l tose

E ~, )st extract

3C ~ c 2 nd extract

, peak t

I I 1.0 12 1.~ 1.6 1.8 2.0 2.2 2.~ Ve/Yo

Fig. 4. Distribution of glucosidase activities after simultaneous gel filtration of both extracts, which originated from fractional extraction of the 4th AS. (a) tested with nipheglu; (b) tested with maltose.

76 K.P. GAFFAL

peak II

~0 ! O ~ ~ 2 ~'d ANTENNAL SEGMENT, S

i f \ . . . . o < , o . . ° . . o +1 t \ ~,'e..~, ,O-+n,..,. ~t I .. \ ,,.,+.,,oc, +o.~.o,,o.

t J x, 7°:,,, . . . . .

I I# ' '1"1

I [ . / X -" ,o, ~ ? '\~ ; .

: 7 ' h < - \ .- , , , ~ ! L,~ . : Y " ' " " - . - . .. • / . . ~ t ~ , ~ _ , - . , f - ' " "~ \ , . - " _ _ . " . . i ,

f 7 r " ....'.'.. ~ k .~...'" _ ~ 1 7 ~ , " , . I # ~ , i l ' l l l l l k i i ~ ~1~ # l I " ~ ' " " ~ ' ~ . I I I

" I ~ l i l l l e l i I l l I I I I l i l - - I I " I ~ I I ~ I ~ " ~ "--~'~-* "

' ? 12 '~- ' 6 IA 2[' $2 2- VelVo

Fig. 5. Distribution of glucosidase activities after simultaneous gel filtration of both extracts, which originated from fractional extraction of the 2nd AS.

the dry weight in mg. Seen this way, only a small percentage of the glucosidase activity found in the AS originates from the haemolymph. With maltose as substrate activity is maximally 10% of the AS. On the other hand it is shown that a high percentage of the glucosidases in the AS are localised in the hypodermal tissue, parts of which are the sensory and enveloping cells of the sensory organs•

Fractional extraction

The results of the stepwise extraction, carried out first with pure buffer only and then with addition of 1% Triton X-100 and 100mM KCi are demon- strated in Figs. 4 and 5. The :~-glucosidases differ in

their behaviour during extraction; one group (type I) is found in the supernatant after the first extraction, while the other group (type II) is transferred into it only after treatment with detergents. A third group (type III) is solubilized partially during the first and partially during the second extraction.

The main activity of the maltose and nipheglu turn- over appears in peak 1 of both AS only after treat- ment with detergents. On the whole the small portion of activity extracted from the 4th AS during the first extraction, quantitatively coincides with peak ! of the haemolymph, but it is eluted a little earlier (l ie/~ = 1.02 as compared to I/,./Vo = 1.1 1.2) (Figs. 3 and 4).

-.b _ ~,lh ANTENNAL SEGMENTS i,,,.-

~ : ~ ' ' I t 2 nd A NTEN NAL SEC4'4E N TS / ¢~,

' I -X peak , , J I

" I k c pH S.S 1 :~

l/a,\ i' ',,\ /! X_ \, / ! ., 0

• '~ 2.0 2.2 2 A " 8 2' ;; ? - 2 ,. VelVo

Fig. 6. Peak III glucosidase activities of both AS as changing with pH. Only simultaneously treated AS of the same animals were compared with one another.

:c-Giucosidases in Dysdercus 77

5.0

3 E

3.0

1o

' q t 4th ANTENNAL SEGMENTS

2nd ANTENNAL SEGMENTS

peak Ill 3.0

80 mM maltose

o DH S,S

c pH 4,4

2.C

j 6 8

1.0

I I I ] J F J I I I tB 2.0 2.2 2.~. Ve/Vo 1.8 2.0 2.2 ~/Vo

Fig. 7. Peak III giucosidase activities of both AS as changing with pH. The AS originated from the same animals.

Tested with nipheglu the profile of peak II is subdi- vided into two peaks after the second extraction. The existence of two peaks in the other substrates is also hinted in Figs. la and 4b. The first of these peaks belongs to type III, whereas the other seems to con- tain two components: one component hydrolyzes nipheglu but not maltose and a second component hydrolyzes maltose but not nipheglu (Fig. 4a,b~ Peak II of the 2nd AS tested with both substrates is desig- nated to type II (Fig. 5l It remains unclear, why the peak II profile of the 4th AS tested with maltose seems to belong to type III, whereas that of the 2nd AS seems to belong to type II.

In the main peak III of the 4th AS a double peak results from tests with maltose. The first peak is assigned to type III, the second to type I. Such a differentiation was not as distinct in the 2nd AS examined simultaneously (Fig. 5). The profiles of the nipheglu turnover belong to type III in both AS (Fig. 4a, 5).

pH-dependence of activity profiles Alterations in activity profiles of both antennal

main peaks III tested with maltose and nipheglu at various pHs are demonstrated in Figs 6 and 7. At pH 5.5 nipheglu is split in both AS at about the same rate, but faster than at pH 3.7 and 6.8. The extent of hydrolysis at pH 3.7 and 6.8 is more increased in the 4th AS than in the 2nd AS. The increase at pH 6.8 can be especially clearly seen on the left side of the curve (Fig. 6). Maximal activity in the 2nd AS is found at V,/Vo = 2.05 (pH 6.8); 2.08 (pH 5,5) and 2.15 (pH 3.7). A shoulder correlates with each maxi- mum in both of the other two curves. In the 4th AS the displacements of the maxima can not as convinc- ingly be seen, but several shoulders also coincide with each other in these curves.

The total activities of peaks III, tested at pH 4.4 with 80 mM maltose, are of the same value in both

AS; at pH 5.3 and 6.8 however they are essentially higher. The rate of hydrolysis is highest: at pH 5.5 in extracts of the 4th AS and at pH 6.8 in extracts of the 2nd AS. Several coinciding shoulders are also conspicuous in these curves (Fig. 7).

It is evident from these test series that the peaks III are formed by summated activities of several enzymes which differ in pH-optima. Further more it was once more shown that these enzymes are present in different amounts in the AS.

Determination of molecular weights The molecular weights of the enzymes were deter-

mined by the aid of reference standards (ferritin: tool. wt = 750,000; lactate dehydrogenase (rabbit): tool. wt = 130,000; alkaline phosphatase (E. coil): tool. wt = 80,000; chymotrypsinogen: mol. wt = 25,000; cytochrom c: mol. wt = 12,400). In the AS the peaks I range from about 770,000 to 2,000,000; the peaks II from 260,000 to 650,000 and the peaks III from 30,000 to 100,000. The two subunits of peak II (Fig. 4) have tool wt on an average of 340,000 and 430,000. The enzymes III~ and III2 of the 4th AS were esti- mated to have the tool. wt 38,000 and 56,000. The first peak of the haemolymph ranges from 660,000 to 1,500,000, the second from 35,000 to 76,000.

DISCUSSION

Number and properties of ~-glucosidases The extracts of the antennal segments (AS) and the

haemolymph of Dysdercus contain enzymes which are able to split disaccharides with ct-glucosidic bonds. Three peaks of ct-glucosidase activities were demon- strated in both AS, but only two in the haemolymph. Although the multiplicity of the enzymes in each of the peaks could be disclosed by use of additional separation tests, it unfortunately was impossible to completely isolate these subunits. For this reason we

78 K.P. GAFFAL

cannot give particulars on their exact number and properties. There is no doubt that at least 6-8 separ- ate units (two trehalase peaks included) exist in the AS, but their real number is roughly estimated to be on the same order (ca. nine) as in the tarses and labella of flies (Bfihrer & Hansen, cited in Hansen, 1975). In flies at first three separate ct-glucosidases could be discerned (Amakawa et al., 1972; Hansen & KiJhner, 1972), afterwards four (Kawabata et at., 1973) and later five (KiJhner & Hansen, 1975).

The three main peaks of both AS show correspond- ing behaviour in elution and obviously corresponding substrate specificities as well. Hence it follows that both AS probably contain the same stock of indivi- dual enzymes. Investigations on x-glucosidases in flies (Amakawa et al., 1972; Biihrer & Hansen, cited in Hansen, 1975) led to the same conclusion.

~t-Glucosidases m the blood of insects

The activity profile of the haemolymph lacks the corresponding peak II of the AS. It is the general consensus that the enzymes in the haemolymph originate from tissues (Wyatt, 1961; van Handel, 1968a,b; Friedman, 1975). Accordingly, the enzyme composition should be similar to that in the tissues. The greatest amount of "~t-glucosidase activity in the whole animal surely occurs in the digestive and mus- cular systems, which predominantly determine the enzyme composition of the haemolymph. Possibly these tissues lack peak II.

~t-Glucosidases and the receptor protein

The quantitative distribution patterns of the enzymes in these two AS distinctly differ from one another. Such differences in the extracts of the tarses, tibiae and labella of flies were used among other things to identify the receptor protein (Amakawa et al., 1972; Biihrer & Hansen, cited in Hansen, 1975). According to the working hypothesis on sugar recep- tion first presented by Hansen (1969), the taste hair density on body parts was correlated to the ct-glucosi- dase content. Subsequently this assumption was limited to one of the isoenzymes isolated by chroma- tographic prodecures (Amakawa et al., 1972; Hansen & Kiihner, 1972; Kiihner & Hansen, 1975). If one appfies this theory to the AS of the cotton stainer, then only enzyme III2 qualifies as a candidate for the receptor protein. All the other enzyme activities perceptible in these experiments are present in the 2nd AS to a higher or an equal extent. On the other hand investigations of enzyme activities at the site of sugar reception (tip of intact taste hairs of flies: Koizumi et al., 1973; Kijima et al., 1973) support the presumption which localizes the receptor protein in the dendritic membrane (Hansen, 1975). The frac- tional extraction with and without the membrane dis- integrating detergent Triton X-100 produces higher activity in the extract free of Triton X-100. Therefore it seems more plausible that predominantly enzymes which are not bound to the membrane are present in the first extracL which contains enzyme III2 for the most part. Hence the behaviour of solubilization of enzyme III2 seems to contradict its function as receptor protein.

The above experiments on Dysdercus have shown that under the given conditions the content of special ~t-glucosidases cannot bc correlated with the densit~ of taste hairs in an unrestricted manner. Nevertheless it cannot be ruled out, provided wc succeed in separ- ating all :t-glucosidases completely, that an unknown enzyme exists which satisfies all the claims of the sugar reception hypothesis

Acknowledyements- This study was supported b) research grants from the Deutsche Forschungsgemem- schaft. 1 would like to thank Dr. H. Biihrer. l)r. J. Ki.ihncr and Prof. Dr. K. Hansen for their help in this 'a, ork

REFERENCES

AMAKAWA T., KAWABATA K., KIJIMA H. & MORITA H. (1972) lsozymes of :t-glucosidases in the proboscis and legs of flies. J. Insect PIkvsiol. 18, 541 553.

BERGER S. & SACKTOR B. (1970) Isolation and biochemical characterization of brush borders from rabbit kidney J. Cell Biol. 47, 637-645.

FIEDMAN S. (1975) Multiple forms of trehalase m Phormia reoina. Partial purification, tissue specificities, and some kinetic properties of adult enzymes. Insect Biochem. 5. 151-164.

G^Fr^L K. P. (1976) Die Feinstruktur der Sinnes- und Hiillzellen in den antennalen Schmecksensillen yon Dys- dereus intermedius DIST. (Pyrrhocoridae, Heteroptera). Protoplasma 88, 101-115.

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