053 ! -5565/78/1001-0343502.0010

Vol. 13, pp. 343-347. Pergamon Press Ltd., 1978. Printed in Great Britain.

xp. Geront.

LYSOSOMAL ENZYME ACTIVITY DURING IN DROSOPHILA MELANOGASTER

AGING

G. C. WEBSTERand S. L. WEBSTER Department of Biological Sciences, Florida Institute of Technology, Melbourne, FL 32901, U.S.A.

(Received 1 March 1978)

INTRODUCTION ON THE cellular level, aging appears to be accompanied frequently by the deterioration of organelles and other cellular structures (Timiras, 1972; Aune, 1976). These changes could be due to an age-related increase in degradative enzyme activity, or to a decrease in synthesis to an extent wkich could no longer counterbalance degradation. Although they may not be the only degradative enzymes of cells, lysosomal hydrolases have been reported to exhibit age-related increases in activity in mouse skeletal muscle (Youhotsky-Gore and Pathmanathan, 1968), mouse, hamster, and guinea pig liver (Goto et al., 1969), cultured human fibroblasts (Cristofalo, 1972; Cristofalo and Kabakjian, 1975), and houseflies (Sohal and McCarthy, 1973). Likewise, an age-related decrease in the capacity for protein synthesis has been observed in mouse liver microsomes (Mainwaring, 1969), rat liver microsomes (Hrachovec, 1969; Buetow and Gandhi, 1973) and mouse muscle ribosomes (Srivastava, 1969; Britton and Sherman, 1975). At present, it is not clear whether aging cells generally exhibit both an increase in hydrolytic, and a decrease in synthetic activities, or whether a decrease in the synthesis/degradation ratio may be characteristic of senescent cells. The fate of lysosomal hydrolase activity in aging Drosophila melanogaster is of interest, because deterioration of cellular structure and function has been observed in this organism (Takahashi et al., 1970; Vann and Webster, 1977), and protein synthesis declines markedly with age (Baumann and Chen, 1968; Chen, 1972). Thus, an increase in degradation in Drosophila cells could occur without an increase in hydrolase activity. However, if an increase in hydrolase activity is characteristic of aging cells, then increased lysosomal hydrolase activity in aging Drosophila melanogaster would be expected. The occurrence of lysosomes, and the presence of nine lysosomal enzymes in Drosophila melanogaster have been reported by Miquel et al. (1974). Our results confirm the presence of typical lysosomal hydrolases in Drosophila, and of an age-related increase in specific activity of these hydrolases, but little change in total activity. MATERIALS AND METHODS Drosophila melanogaster, strain Oregon R, was grown on banana agar medium (Demerec and Kaufmann, 1967) at 25 ° and 60% relative humidity. Groups of about 50 flies were housed in polycarbonate jars with plastic sponge stoppers. Cultures were transferred each week to fresh medium. For this investigation, a particulate fraction, similar to that used by Cristofalo and Kabakjian (1975), was prepared. Organisms of the desired age were cooled to about 5 ° for immobilization. Each 100 mg of flies were mixed with 10 ml of cold 0.25 M sucrose-0.01 M tris (hydroxymethyl) aminomethane-acetate (pH 7.5), and ground in a glass homogenizer with a motor-driven Teflon pestle. The resulting preparation was centrifuged at 15,000 g for 10 rain. The sediment was discarded, and the supernatant fluid was centrifuged at 15,000 g for I0 min. The supernatant fluid was separated from the sediment, and retained for assay 343

344

G. C. WEBSTER AND S, L. WEBSTER

of enzyme activity. The sediment was suspended, with the aid of a glass homogenizer, in 0"1 vol. of 0.25 M sucrose (pH 7"0), and likewise retained for the assay of enzyme activity. Assay of enzyme activities Acid phosphatase activity was assayed by the method described by Barrett (1972). The hydrolysis of 13-glycerophosphate was used as a measure of acid phosphatase activity, since p-nitrophenyl phosphate may not be specific (Singer and Fruton, 1957; Beaufay, 1972). The assay system contained 2 ml of 0"02 M sodium 13-gtycerophosphate in 0-01 M sodium acetate buffer of pH 5.0, and the enzyme preparation in a total volume of 3 ml. After incubation at 37°, the reaction was stopped by addition of 2 ml of cold 10% trichloroacetic acid. The solution was filtered through Whatman No. 1 paper. To the supernatant solution were added 0.8 ml of 2.5 % ammonium molybdate, and 0"2 ml of 0.5 % Elon (p-methylaminophenol sulfate) in I0~o sodium metabisulfite. Liberated orthophosphate was measured spectrophotometrically at 660 nm. Under the conditions of the experiment, the liberation of orthophosphate was a linear function of time for at least 60 minutes, One unit of activity was equal to one micromole of orthophosphate liberated from 13-glycerophosphate in 60 min. Acid ribonuclease activity was measured as described by Barrett (1972). The assay system contained 1"5 ml of 0.2% yeast RNA (Sigma) in 0"2 M sodium acetate buffer (pH 5"0), and the enzyme preparation, in a total volume of 2 ml. The mixture was incubated at 37°, and the reaction was stopped by adding 2 ml of a cold solution of 10% perchloric acid containing 0.25 ~ uranyl acetate. After centrifugation for 10 min at 2000 g, liberated nucleotides were measured spectrophotometrically at 260 nm. One unit of activity was equal to one mg of RNA hydrolyzed in 60 rain. Cathepsin D activity was measured by a modification of Method 1 of Barrett (1972). The assay system contained 0"5 ml of 4% hemoglobin, 0-25 ml of sodium formate buffer (pH 3'0), and 0.25 ml of the enzyme preparation. Following incubation at 37°, the reaction was stopped by addition of 5 ml of 3 % trichloracetic acid. The mixture was filtered through Whatman No. 1 filter paper, and the absorbance at 280 nm measured in a spectrophotometer. One unit of activity was equal to an absorbance change of 1.0 in 60 min. Protein was determined according to the biuret procedure of Gomall et al. (1949). RESULTS U n d e r the c o n d i t i o n s o f these experiments, the survival curve for the o r g a n i s m was that r e p o r t e d b y Biscardi a n d W e b s t e r (1978), with a m e a n life span o f a p p r o x i m a t e l y 40 days. In o r d e r to minimize ambiguity, which might occur f r o m assay o f only a single enzyme activity, the effect o f age was d e t e r m i n e d on the activities o f three enzymes (acid p h o s p h a tase, a c i d ribonuclease, a n d cathepsin D) t h a t have been f o u n d to be associated with lysosomes. In a g r e e m e n t with the results o f a n u m b e r o f investigators, the specific activity (unit/mg protein) o f acid p h o s p h a t a s e in the l y s o s o m a l fraction was f o u n d to be 5-5-times higher in 35-day-old Drosophila t h a n in 1-day-old organisms (Fig. 1). Similarly, the specific activities o f ribonuclease a n d cathepsin D increased f o u r - f o l d a n d 23-fold respectively in 35-day-old flies, c o m p a r e d with 1-day-old flies. I n c o n t r a s t to the large increases in specific activity o f the three enzymes in the l y s o s o m a l fraction, the t o t a l activities (units/fly) o f acid p h o s p h a t a s e , acid ribonuclease, a n d cathepsin D increased only 1.5-fold, 1.1-fold, a n d 6.1-fold respectively (Fig. 3a). The difference between the changes in specific activity a n d total activity a p p e a r s to be due to an agerelated decrease in the p r o t e i n level o f the isolated lysosomal fraction (Fig. 2). This decrease p r o d u c e d an increase in specific activity, while the total activity c h a n g e d relatively little. I n e x a m i n i n g the changes in total activities o f the three enzymes, it should be n o t e d that the cytosol c o n t a i n e d significant a m o u n t s o f total activities o f acid p h o s p h a t a s e a n d acid ribonuclease, a n d m u c h greater a m o u n t s o f cathepsin D t h a n occur in the l y s o s o m a l fraction (Fig. 3b). Since the activities in the cytosol might be due to leakage f r o m the lysosomes d u r i n g p r e p a r a t i o n , it was o f interest to examine the changes in t o t a l activities o f the c o m b i n e d l y s o s o m a l plus cytosol fractions, as a function o f age. As can be seen f r o m Fig. 3(c), the a c i d p h o s p h a t a s e a n d acid ribonuclease activities s h o w e d little change bet ween 1-day-old a n d 35-day-old flies, while the cathepsin D activity decreased m a r k e d l y .

LYSOSOMAL

ENZYME

ACTIVITY

DURING

AGING

IN

Drosophila melanogaster

345

Im YO

0.i6

0.14

D.7

0.12

0.6

z la

~

0.10

D.5

::::::;: .,...

~0.08 p.

~}.4

:i:!:i:i

0.06

O.5

U::i: i:~:i~: ::::'2:

0.04

0.2

0.02

:),1

ACID PHOSPHATASE

RIBONUCLEASE

CATHEPSIN D

FIG. 1. Specific activities of acid phosphatase, acid ribonuclease, and cathepsin D in the lysosomal fraction from Drosophilamelanogaster.Y = 1-day-old flies; 0 = 35-day-old flies.

2C

--,_1 16 u. o o

12

z_ t,l.I

g a.

8

Z< I

5

:o

,'5

i

2o

2;

i

3o

DAY

FIG. 2. Protein content of the lysosomal fraction from Drosophilamelanogasterof different ages.

DISCUSSION The results obtained in this investigation are consistent with the view that the total activity of three different lysosomal enzymes does not increase with age in Drosophila melanogaster. In this, Drosophila differs from the housefly, Musca domestica, where Sohal and McCarthy (1973) observed a significant age-related increase in the total activity of acid phosphatase. The lack of increase in total activity of the lysosomal enzymes is in marked contrast to the great increase in specific activity of the enzymes. The increase in specific activity is mis-

346

G. C. WEBSTER AND S. L. WEBSTER

li Y 0!0.5

LYSOSOME$ 0.008 0,004

33 CYTOSOL

~ 0.004 ~_ -

~'°'°°~

~ r

CYTO~OL

t

l~i !

LYSOSOMES+

0.01

0.5

0.3

0.006

0.002

~'~

~

O.I

ACID RIBONUCLEASECATHEPSlND PHOSPHATASE Flo. 3. Total activities of acid phosphatase, acid ribonuclease, and cathepsin D in the lysosomal fraction, and the cytosol, of 1-day-old, and 35-day-old Drosophila rnelanogaster. (a) Total activities of the three enzymes in the lysosomal fraction. (b) Total activities of the three enzymes in the cytosol. (c) Total activities of the combined lysosomal and cytosol fractions. Y . l-day-old flies; 0 = 35-day-old flies. leading, however, since it appears to be due entirely to a decrease in some non-lysosomal contaminant of the lysosomal fraction, which results in a marked decrease in the protein levels of the lysosomal fraction, with little decrease in the activities of the lysosomal enzymes. From the centrifugal force used for its preparation, it is likely that the lysosomal fraction used in these experiments is contaminated with mitochondria. Since there is evidence for an age-related decrease in mitochondria in some organisms (Herbener, 1976), the loss of mitochondria from a combined lysosomal-mitochondrial preparation would produce the observed increase in specific activity with little increase in total activity. Aging appears to be characterized by the degradation of many cellular components. This degradation could be caused by an increase in degradative enzyme activity, a decrease in synthetic activity, or some combination of both. Since the capacity for protein synthesis appears to decrease markedly during aging in a number of organisms, cellular component degradation is possible, even without an increase in degradative enzyme activity, if the synthetic ability decreased faster than a decrease in degradative enzyme activity. In Drosophila, protein synthesis has been reported to decrease markedly in adult organisms (Baumann and Chen, 1968; Chen, 1972; Maynard-Smith eta/., 1970). Thus, the constant level of lysosomal enzyme activity could become increasingly dominant in determining the levels and integrity of cellular organelles. It would be of considerable interest to know whether little change in hydrolase activity is characteristic of those cells in which the capacity for protein synthesis decreases markedly with. age, and whether an inverse ratio of degradation and synthesis is characteristic of all senescent cells. SUMMARY The activities of three lysosomal enzymes (acid phosphatase, acid ribonuclease, and

LYSOSOMAL ENZYME ACTIVITY D U R I N G AGING I N

Drosophila melanogaster

347

cathepsin D) were d e t e r m i n e d in l y s o s o m a l p r e p a r a t i o n s f r o m y o u n g a n d o l d Drosophila melanogaster. The specific activities o f all three enzymes were m a r k e d l y higher in old organisms. However, the total activities o f the three enzymes in the lysosomal plus cytosol fractions exhibited little change with age. Acknowledgement--This project was supported by U.S. Public Health Service Grant SO8 RR09032 awarded by the Biomedical Research Development Grant Program, Division of Research Resources, National Institutes of Health.

REFERENCES AUNE, J (1976) In: Cellular Aging: Concepts and Mechanisms (Edited by R. G. CUXLER), Part II, p. 44. Karger, Basel. BARRETt, A. J. (1972) In: Lysosomes--A Laboratory Handbook (Edited by J. DINGLE),p. 46. North-Holland, Amsterdam. BAUMANN,P. A. and CHEN,P. S. (1968) Rev. suisse Zool. 75, 1951. BEAUFAY,H. (1972) In: Lysosomes--A Laboratory Handbook (Edited by J. DINGLE),p. 33. North-Holland, Amsterdam. BISCARDI,H. M. and WEBSTER,G. C. (1978) Exp. Geront. In press. BRITTON, G. W. and SI~RMAN,F. G. (1975) Exp. Geront. 10, 57. BUETOW, D. E. and GANDHI,P. S. (1973) Exp. Geront. 8, 243. CHEN, P. S. (1972) In: Molecular Genetic Mechanisms in Development and Aging (Edited by M. ROCKSTEIN and G. T. BAKER,III), p. 199. Academic Press, New York. CRISTOFALO,V. J. (1972) In: Advances in Gerontological Research (Edited by B. L. STREHLER),Vol. 4, p. 45. Academic Press, New York. CRISTOFALO,V. J. and KABAKJIAN,J. (1975) Mech. Age. Devl. 4, 19. DEMEREC,M. and KAUFMANN,B. P. (1967) Drosophila Guide: Introduction to the Genetics and Cytology oJ Drosophila melanogaster, p. 10. Carnegie Institution, Washington. GORNALL,A. C., BARDAWILL,C. J. and DAVID, M. M. (1949) J. biol. Chem. 177, 751. GOTO, S., TAKANO,T., MIZUNO,D., NAKANO,T. and IMAIZUMI,K. (1969) J. Geront. 24, 305. HERBENER, G. H. (1976) J. Geront. 31, 8. HRACHOVEC,J. P. (1969) Gerontologia 15, 52. MAINWARING,W. I. P. (1969) Biochem. J. 113, 869. MAYNARD-SMITH,J., BOZCUK,A. N. and TEBBtrrr, S. (1970) J. Insect Physiol. 16, 601. MIQUEL,J., TAPPEL,A. L., DILLARD,C. J., HERMAN,M. M. and BENSCH,K. G. (1974) J. Geront. 29, 622. SINGER, M. F. and FRtrrON, J. S. (1957) J. biol. Chem. 229, 111. SOHAL,R. S. and McCARTHY, J. L. (1973) Exp. Geront. 8, 223. SRrVASTAVA,V. (1969) Archs Biochem. Biophys. 130, 129. TAKAHASI~,A., PrnLPOTr, D. E. and MIQUEL,J. (1970) J. Geront. 25, 222. TIMIRAS,P. S. (1972) Developmental Physiology and Aging, p. 429. Macmillan, New York. VANN, A. C. and WEBSTER,G. C. (1977) Exp. Geront. 12, 1. YotrnOrSKY-GORE, I. and PATHMANATHAN,K. (1968) Exp. Geront. 3, 281.