The β-glucuronidase content of Drosophila embryos

427 THE

/?-GLUCURONIDASE

CONTENT

F. BILLETT Institute

of Animal

OF DROSOPHILA

and S. J. COUNCE

Genetics, University Received

EMBRYOS

August

of Edinburgh,

Scotland

10, 1957

~SSERVAT~ONS on regenerating mammalian tissues [4] and on neoplastic tissues [3] have suggested that increased ,%glucuronidase activity is characteristic of normal and abnormal proliferations of cells. A study of the /3-glucuronidase content of Xenopus embryos during their early development, however, failed to reveal any marked increase of this enzyme at periods when cell proliferations would be a predominant feature [ 21. Compared with other sources the ,%glucuronidase content of Xenopus embryos is relatively low, being about ( of that of mouse liver. Drosophila embryos, on the other hand, appear to possess a glucuronidase activity which is comparable to that of mouse liver. The higher /3-glucuronidase activity of Drosophila embryos, compared with those of Xenopus, makes them a more favourable object in which to study the behaviour of this enzyme during development. In the case of Drosophila there is the added advantage of genetic control. The dry weight of 1000 Drosophila embryos is between 223 mg. This means that most biochemical determinations will require fairly large numbers of embryos, unless special micro techniques are employed. In the present case about 1000 embryos were required for each determination. The procedure, described below, was designed to yield between 50061000 eggs,./hr. Drosophila melanogaster (an Oregon K stock) was used throughout the experiments. The flies used were between S-14 days old and were stored in bottles containing food at 22°C. 15 bottles each containing about 150 pairs of flies were used. For egg collection the flies were transferred to empty bottles which were closed by watch glasses containing a layer of well moistened agar. At hourly intervals the watch glasses were removed and replaced by fresh ones. The eggs were removed from the lids, dechorionated with 1 per cent W,V sodium hypochlorite solution, washed with water, counted, placed on moist agar plates and allowed to develop at 22°C. Development was stopped after 1, 2 4, 4, 5, IO and 18 hrs by freezing the embryos ( - 20X). After a period in deep freeze (l-4 weeks) the embryos were homogenized in a small amount of distilled water (c. 1 ml) and the homogenate stood in a refrigerator overnight. The homogenate was centrifuged (5°C) at ca. 3000 x 9. The residue from the centrifuging was then resuspended in a small amount of distilled water and again centrifuged. The original suprrnatant and washing were combined and made up to a known volume, calculated so that each ml represented the extract from ca. 500 embryos. The residues were resuspended in the same volume of water. The enzyme activity of these preparations, extract and residue, was measured by the calorimetric determination of S-hydroxyquinoline formed by the enzymic hydrolysis of quinolyl S-glucuronide. Details of this method arc given elsewhere [l]. The activity of the preparations was expressed as the amount of 8.hydroxyquinoline 28-573705

Experimental

Cell Research 13

F. Billeff and S. J. Counce

428

TABLE Activities

expressed

I

/?-Glucuronidase activity of Drosophila as pg 8.0 H quinoline liberated/hi-/g

embryos. dry wt. of original

Extracts Stage of development

Age (hr.)

Residues % Activity n in extracts

Range

R

Mean activity

1800 2348

1045-2733 2154-2635

6 5

3590 3415

2540-4654 3008-3962

3 3

50 68

2030 2391

1500-2870 2070-2662

7 4

3051 4100

16544023 3462-4723

5 3

67 60

1913 2469

1133-2385 2283-2654

5 2

3710 4085

2510-4269 -

2 1

60 61

Mean activity

Cleavage 1 Syncytial blastoderm 2) Gastrulation and germ band extension 4 Primary histogenesis 7 Shortening of germ band; head involution 10 Hatching activity 18

material.

Range

liberated/hr/g dry wt. of the original material. The dry weight of the embryos was determined on separate samples. The results are summarized in Table I. Clearly extracts of Drosophila embryos possess an appreciable glucuronidase activity. This activity remains practically constant throughout development. The results also show that a large amount (ca. 40 per cent) of the enzyme remains bound to the residues from which the water extracts were prepared. Although cellular proliferation must be a marked feature of the initial stages of development of Drosophila, no change in the level of /Lglucuronidase activity can be associated with this phase. The idea that raised /Lglucuronidase activity is characteristic of cells undergoing division does not appear to apply to embryonic tissue. However, the presence of the enzyme in such tissues suggests that it is part of the normal enzymic equipment of relatively undifferentiated cytoplasm. We are indebted to Professor C. H. Waddington for the interest he has shown in our work. The Advisory Committee for Medical Research for Scotland provided a grant. One of us (S. J. C.) was the recipient of a Macaulay Fellowship. REFERENCES 1. 2. 3. 4.

BILLETT, I;., Hiochem. J. 57, 159 (1954). __ Proc. Roy. Phys. Sot. Edinburgh 25, 21 (1956). FISHMAN, W. M., The Enzymes, Pt. 1. Acad. Press, New York, 1950. LEVVY, G. A., KERR,L. M. H. and CAMPBELL, J. G., Biochem. J.42,462

Experimentnl

Cell Research 13

(1948).