Comp. Biochem. Physiol., 1974, Vol. 47B, pp. 53 to 62. Pergamon Press. Printed in Great Britain

A L T E R A T I O N OF L-ALANINE A M I N O T R A N S F E R A S E , L-ASPARTATE AMINOTRANSFERASE AND

fl-HYDROXYACYL DEHYDROGENASE ACTIVITIES IN DROSOPHILA MELANOGASTER LARVAE BY NUTRITIONAL

MANIPULATION*

B. W. GEER and C. E. ZACHARIAS

Department of Biology, Knox College, Galesburg, Illinois 61401, U.S.A.

(Received 27 March 1973)

Abstract--1. Compared to glucose, pyruvate, acetate and ribose were less adequate but efficiently utilized dietary energy sources for Drosophila melano- gaster larvae; whereas malate, citrate, butyrate, L-alanine and L-aspartate were much less efficient than glucose in promoting larval development.

2. Relative to the enzyme activities observed in glucose-fed larvae, the activities of L-alanine aminotransferase (E.C. 2.6.1.2), L-aspartate amino- transferase (E.C. 2.6.1.1), and fl-hydroxyacyl dehydrogenase (E.C. 1.1.1.35) were increased in larvae by feeding carbohydrate-free diets high in L-alanine and L-aspartate in the case of the aminotransferases and high in butyrate in the case of fl-hydroxyacyl dehydrogenase.

INTRODUCTION

DROSOemLA MELANOGASTER is capable of utilizing several different carbohydrates as dietary energy sources for larval growth (Sang, 1956) and adult longevity (Hassett, 1948). Feeding pure solutions of test substances, Hassett (1948) found fructose, maltose, sucrose, glucose and galactose to be good promoters of adult longevity in that descending order of efficiency. Adults fed pentoses or organic acids common to metabolism survived for only brief life spans, but most test substances increased adult longevity beyond that observed when only water was supplied. Employing a defined diet, Sang (1956) observed that with but one exception, maltose, fructose and glucose and disaccharides containing these hexoses are utilized for larval growth. Nevertheless, elimination of carbohydrate from Sang's medium, which contained whole casein and yeast RNA, slowed larval growth only slightly; whereas D. melanogaster larval growth on a minimal amino acid diet, as evidenced in the current report, is more carbohydrate dependent.

In any event, D. melanogaster is able to adapt metabolically to a variety of dietary energy sources. Although dietary adaptation in D. melanogaster may be facilitated by regulation of enzyme activities, only the activity of tryptophan

* This work was supported in part by National Science Foundation Grant No. GB-35784.

53

54 B.W. GEaR AND C. E. ZACHARIAS

pyrrolase (Rizki & Rizki, 1963) has been shown to be susceptible to dietary influence. In contrast, many vertebrate enzymes have been found to fluctuate in activity in different tissues in response to changes in the composition of the diet (Schimke & Doyle, 1970).

In the current experimentation we assessed the capacity of D. melanogaster larvae to utilize pentose and different organic acids when fed a minimal amino acid diet and systematically screened for dietary influences upon a number of enzymes of energy-yielding metabolism.

MATERIALS AND METHODS Culture conditions

Throughout the experimentation, flies of the D. melanogaster Canton-S wild-type strain were cultured on a minimal amino acid diet like that previously employed (Geer, 1966) except L-cystine, glycine and L-tyrosine were omitted leaving the ten essential amino acids and L-glutamic acid as the primary dietary nitrogen sources. Unless otherwise indicated, sucrose was replaced in the diet by other carbohydrates and organic acids at a 0"029 M dietary concentration; the L-alanine-L-aspartate mixture containing 0"0145 M of each of the amino acids. Except for L-alanine, the organic acid supplements were administered as sodium salts. Filtration-sterilized carbohydrate and organic acid supplements were added to the medium after the other components had been mixed and sterilized by autoclaving. Routinely, eggs were collected from 4-day-old adult females, sterilized by methods previously described (Geer, 1963; Geer & Newburgh, 1970) and inoculated into 6-dram vials con- taining 5 rnl of test medium in numbers sufficient to yield forty to sixty larvae per culture. The larvae were cultured under aseptic conditions at 21"5°C with a 12-hr light-12 hr dark day. Cultures found to be contaminated with micro-organisms were discarded.

For growth determinations the number of larvae in each culture was scored 24 hr after the introduction of eggs. The time from egg inoculation to pupation, the growth period, was then measured by counting the number of larvae to pupate in each culture each day, and the total number of adults to eclose in each vial was subsequently scored. At different times during the larval feeding period, individuals were removed from cultures for protein and enzyme determinations. Thirty larvae of a test group were homogenized in 200/~1 of homogenizing solution using a ground glass homogenizer. The protein con- centration of the homogenate was determined on an aliquot by the method of Lowry et al. (1951) using crystalline bovine albumin as a standard. The remainder of the homogenate was retained for enzyme analysis.

Enzyme assay methods

Hexokinase was measured using homogenates of larvae prepared with 0"15 M KC1- 0"05 M KHCOs-0.006 M Na~H~ EDTA-0"005 M MgC12, pH 8"2 (Ward & Schofield, 1967). Aldolase, lactate dehydrogenase and malate dehydrogenase were assayed using preparations made with 0"15 M KCI-0"02 M KHCOs, pH 7"8 (Ward & Schofield, 1967). Larvae for L-aspartate aminotransferase and malic enzyme determinations were homo- genized in 0"15 M KCI-0.05 M KHCO3-0"01 M 2-mercaptoethanol, pH 8"2. /~-Hydroxy- acyl dehydrogenase was measured using tissue preparations made with 0-1 M KHzPO4- K2HPO4, pH 7'3 with 0"002 M Na2Hs E D T A (Beenakkers, 1969). L-Alanine amino- transferase and carnitine acetyltransferase determinations were made using tissue homo- genates prepared with 0"1 M KH2PO~-K2HPO4, pH 7"3 with 0"002 M Na2H2 EDTA and 0"01 M 2-mercaptoethanol.

The enzyme assays were conducted by measuring the oxidation of NADH or NADPH or the reduction of NAD + or NADP + in the reaction mixture at 30°C by following the change in extinction at 340 nm with a Beckman DU spectrophotometer equipped with a

DROSOPHILA NUTRITION AND METABOLISM 55

Gilford automatic cuvette positioner, Haacke constant temperature circulator and Honeywell recorder. Compositions of the individual reaction mixtures were as indicated in Table 1. Each enzyme was assayed using different amounts of enzyme preparation, 2-20/~g of

TABLE l--METHODS USED FOR ENZYME ACTIVITY DETERMINATIONS IN THE CURRENT STUDY

Systematic Name of enzyme E.C. number Reference

Hexokinase 2.7.1.1 Aldolase 4.1.2.6 Lactate dehydrogenase 1.1.1.27 Malate dehydrogenase 1.1.1.37 Malie enzyme 1.1.1.40 L-Alanine aminotransferase 2.6.1.2 L-Aspartate aminotransferase 2.6.1.1 fl-Hydroxyacyl dehydrogenase 1.1.1.35

DiPietro & Weinhouse (1960) Beizenherz (1955) Kornberg (1955) Bergmeyer & Bernt (1965a) Ochoa (1955) Mattenheimer (1970) Bergmeyer & Bernt (1965b) Geer et al. (1972)

homogenate protein, to be certain that an increase in enzyme preparation content was accompanied by a corresponding increase in enzyme activity. Specific enzyme activities are given as the number of nmoles of cofactor oxidized or reduced per mg of homogenate protein per min. Corrections for nonspecific changes in extinction were made when calculating enzyme activities by subtracting the rate of oxidation or reduction of cofactor during a 5-min preincubation period without substrate from that observed with the substrate.

Radioisotope procedures Radioisotope methods were used to examine the relative rates of entry of nutrients

from the medium into the test animals. Larvae that had been raised on the basal medium supplemented with glucose for 7 days were removed from culture vials, washed with distilled water and ten animals per test group were placed in a watchglass containing 1 ml of liquid basal medium (no agar) supplemented with 1"5/~Ci of either [U-x4c] glucose, [U-a4C] L-alanine, [2-1'C] sodium acetate, [2-14C] sodium pyruvate, [1,5-14C] citric acid or [ 1-14C] sodium butyrate plus sufficient unlabelled nutrient to bring the dietary level to 0"029 M. Larvae were allowed to feed on the labelled medium for 30 man and then trans- ferred to unlabelled medium for 1"5 hr. The purpose of the latter feeding period was to remove unabsorbed labelled material from the digestive tract. The ten animals of each test group were then placed in preweighed scintillation vials, dried at 90°C, weighed and then dissolved in 1 ml of Soluene. Ten ml of scintillation fluid (5 g PPO and 0"3 g dimethyl- POPOP/1. of toluene) was added and samples were counted with a Packard Tricarb 314 EX liquid scintillation spectrometer with 57-60 per cent efficiency. Sample counts were corrected for background. Larvae fed the different test diets for 2-hr periods were found not to differ significantly in dry weight.

Statistical analysis Protein contents, enzyme activities and growth periods of larvae of the test groups were

compared statistically by the least significant difference method of analysis. The percentages of larvae to pupate and to eclose were analyzed by the contingency chi-square method using larvae cultured on a glucose-supplemented diet as the standard for comparison. The level of significance was 0-05 per cent.

56 B. W. GEER AND C. E. ZACHARIAS

Chemicals Biochemicals and enzymes were the best available grade supplied by the Sigma Chemical

Company (St. Louis, Mo., U.S.A.). Materials for scintillation counting were purchased from the Packard Instrument Company (Downers Grove, Ill., U.S.A.). [2-uC] Sodium acetate (2 mCi/m-mole), [U-14C] L-alanine (8 mCi/m-mole), [2-14C] sodium pyruvate (4"04mCi/m-mole), [U-14C] D-glucose (4.8mCi/m-mole), [1-14C] sodium butyrate (2.5 mCi/m-mole) and [1,5-14C] citric acid (21-6 mCi/m-mole) were supplied by the New England Nuclear Corporation (Boston, Mass., U.S.A.). Other chemicals were the highest grade available.

RESULTS

Nutritional comparisons

When the energy-yielding substances were fed on an equimolar basis, glucose was the best promoter of larval development (Table 2), with pyruvate, acetate

T A B L E 2 - - G R O W T H OF D. melanogaster LARVAE ON SYNTHETIC MEDIUM SUPPLEMENTED

W I T H EQUIMOLAR AMOUNTS OF DIFFERENT ENERGY SOURCES

Compound No. of larvae

Larvae Larvae Growth to pupate to eclose period

(%) (%) (days _+ S.D.)

Glucose 1580 87.4* 78"6* 16"6 + 3"5* Pyruvate 1644 76"6t 47" 1 t 15-6 + 2"4t Acetate 661 70"9 51"9 16"9 + 2"1 Ribose 428 79"4 60"3 17"1 + 1"6 Citrate 595 67"3 43"2 18"4 + 2"5 Malate 250 54'9 36"2 17"6 + 2"1 L-Alanine-L-aspartate 652 55"9 31 "0 18"7 + 2"6 Butyrate 608 65"4 38"9 17-5 + 2"3 Unsupplemented 697 61 "7 34"7 19"6 + 4"1

* The values for all test groups differ from glucose-fed animals at the 0"01 per cent level of significance.

t The values for the pyruvate, acetate and ribose test groups differ from the citrate, malate, L-alanine--L-aspartate, hutyrate and unsupplemented test groups at the 0"05 per cent level of significance of the percentage of the larvae to pupate and eclose and at the 0"01 per cent level of significance for the growth period.

and ribose ranking as less adequate, but good, energy sources for develop- ment. T h e latter three substances ranked close together when either the length of the larval growth period, protein content of l 1-day-old larvae, survival to pupat ion or survival to eclosion were used as the criteria, though the order of efficiency varied somewhat depending upon the criterion employed. Compared to the aforementioned nutrients, citrate, malate, L-alanine, L-aspartate and butyrate were relatively inefficient energy sources. All of the dietary supplements, however, improved development above that observed when an unsupplemented diet was fed.

D R O S O P H I L A NUTRITION AND METABOLISM 57

Adjustment of the amount of the dietary supplements so that they were fed on an equal-carbon basis improved the growth of pyruvic acid-fed larvae so that the protein content of test larvae was close to that of their glucose-fed counterparts. (Table 3). Ribose and acetate remained less adequate promoters of larval growth than glucose when fed on at the same dietary carbon content.

TABLE 3 - - T H E PROTEIN CONTENTS OF D. melanogaster LARVAE FED A SYNTHETIC

MEDIUM SUPPLEMENTED W I T H DIFFERENT ENERGY SOURCES FOR 11 days

Protein Compound No. of determinations (~g per larva)

Equimolar amounts Glucose 16 70"6 Acetate 12 57"3 Pyruvate 12 56"7 Ribose 10 52-1 Citrate 13 40 '4 L-Alanine-L-aspartate 11 40"0 Butyrate 12 28.2

Equicarbon amounts* Glucose 4 68"1 Acetate 4 47"2 Pyruvate 4 66"1 Ribose 4 44"7 Unsupplemented 4 28 "4

* The dietary concentrations were 0"035 M ribose, 0"058 M pyruvate and 0"087 M acetate.

The development-promoting capacities of the test nutrients cannot be explained on the basis of their rate of uptake by test animals from the medium (Table 4). Though there are moderate differences in the rates of uptake for the test compounds, no direct correlation exists between the development-promoting capacities of and the dietary uptake rates of the nutrients. Thus, the stimulation of development by

TABLE 4 - - - U P T A K E OF LABELLED SUPPLEMENTS FROM THE MEDIUM BY 7-day-old LARVAE

DURING A 30-rain FEEDING PERIOD

Uptake Supplement (counts/min per la rva) Relative uptake

[U-14C] Glucose 282 1"00 [2-14C] Pyruvate 236 0-91 [U-~4C] L-Alanine 367 1"27 [2-~4C] Acetate 397 1"41 [1,5-14C] Citric Acid 240 0"85 [1-14C] Butyrate 345 1"22

58 B . W . GEER AND C. E. ZACHARIAS

the test nutrients appears to be based upon utilization of the nutrients at the intermediary metabolism level.

Enzyme comparisons

Of the enzymes examined in 11-day-old D. melanogaster larvae, only L-alanine aminotransferase, L-aspartate aminotransferase and fl-hydroxyacyl dehydrogenase activities were affected by the dietary energy source (Table 5). These three enzymes were higher in activity in larvae when substrates or compounds readily convertible

TABLE 5--ENZYME ACTIVITIES IN 1 1 - d a y - o l d D. melanogaster LARVAE RAISED ON A

SYNTHETIC MEDIUM SUPPLEMENTED WITH DIFFERENT ENERGY-YIELDING COMPOUNDS

L-Aspartate L-Alanine ~-Hydroxyacyl Supplement aminotransferase aminotransferase dehydrogenase

Glucose 319"0_+ 69"7 (15)* 341.3 ± 55.7 (7) 61"6 +_ 17.4 (11) Acetate 325"7 ± 63"6 (4) 359"1 ± 56-5 (5) 89"9 _+ 15"3 (5) Pyruvate 328"4 ± 74"6 (9) 366"9 ± 40"4 (7) 95-0 ± 13"8 (5) Citrate 351"4 ± 55"3 (5) 491"7 ± 58"5 (7)t 77-1 ± 14"7 (5) L-Alanine-L-aspartate 439"2 ± 57"3 (15)t 477"9 ± 58"4 (7)t 84-1 ± 18"1 (5) Butyrate 350"7 _+ 61"3 (4) 452"7 ± 50"3 (7)t 121"2 _+ 20-1 ( l l ) t

* Mean + S.D. The number of determinations is shown in parentheses. t Different at the 0"01 per cent level from the enzyme activity observed in

larvae fed a glucose-supplemented diet.

to a substrate were fed at high dietary concentrations. An L-alanine-L-aspartate mixture stimulated the activities of both L-aspartate aminotransferase and L-alanine aminotransferases and dietary butyrate raised the level of fl-hydroxyacyl dehydro- genase. In addition, L-alanine aminotransferase activity was higher in larvae raised on citrate and butyrate-containing diets than in glucose-fed larvae.

Since l 1-day-old larvae raised on diets supplemented with either citrate, butyrate or the L-alanine-L-aspartate mixture were much smaller than their glucose-fed counterparts (Table 3), the possibility that the enzyme level differences in test larvae were due to larval size differences was examined. Activities for L- aspartate aminotransferase, L-alanine aminotransferase and fl-hydroxyacyl dehydro- genase were determined for 4-, 7- and 11-day-old glucose-raised larvae (Table 6).

TABLE 6--ENZYME ACTIVITIES IN GLUCOSE-FED O. melanogaster LARVAE AT DIFFERENT AGES

Protein L-Aspartate L-Alanine fl-Hydroxyacyl Days (/,g per larva) aminotransferase aminotransferase dehydrogenase

4 6-6 255.9 + 36-8* 243.0 + 31.4 96.1 + 10.3 7 19.4 252.7 + 55.6 347.1 + 62.9 97.4 + 12.0

11 72.0 359.5 + 61.0 373.0 + 64.7 64.1 + 15.5

* Mean _ S.D.

D R O S O P H I L A N U T R I T I O N A N D M E T A B O L I S M 59

The sizes of these larvae spanned that observed for 11-day-old larvae raised on citrate, butyrate and L-alanine-e-aspartate supplemented diets. The thinking behind this approach was that if these enzymes vary in activity during the larval growth period, glucose-fed larvae of different ages should exhibit differences in r-alanine aminotransferase, e-aspartate aminotransferase and fi-hydroxyacyl dehydrogenase activities.

Enzyme activity differences were observed for different aged glucose-fed larvae. However, the activity differences for the aminotransferases in l 1-day-old larvae raised on glucose and the other test nutrients cannot be attributed to a size differ- ence in the test larvae. Both e-alanine aminotransferase and e-aspartate aminotrans- ferase increased in activity in glucose-fed larvae as the larvae increased in size; size-enzyme activity relationships opposite to that noted when 11-day-old glucose- fed and l 1-day-old e-alanine-e-aspartate larvae were compared. On the other hand, fl-hydroxyacyl dehydrogenase activity declined in glucose-fed larvae as they developed. Thus, the high level of fl-hydroxyacyl dehydrogenase in butyrate-fed larvae as compared to the level in glucose-fed larvae could, in part, be due to a size difference. Nevertheless, the enzyme was not significantly higher in l l -day- old citrate and e-alanine-e-aspartate-fed larvae than in glucose-fed l 1-day-old larvae, although these larvae were considerably smaller at 11 days of age than l 1-day-old glucose-fed larvae. Consequently, it is likely that fl-hydroxyacyl dehydrogenase activity is specifically affected by dietary butyrate.

The activities observed for the enzymes that did not respond to dietary mani- pulation--hexokinase, 70.0+11.0; aldolase, 38.1+7.7; lactate dehydrogenase, 33.4+ 8.3; malate dehydrogenase, 1166.7+ 183.6; malic enzyme, 127.6+ 16.0; and carnitine acetyltransferase, 371.5 _+ 52.1 (mean + S.D. for 11-day-old glucose- raised larvae)--were similar to the activities found in Drosophila in previous studies (Geer et al., 1972; Geer & Downing, unpublished).

DISCUSSION The capacity of D. raelanogaster larvae to utilize pentoses and organic acids for

development differs markedly from the ability of adults to employ these substances for adult maintenance. Whereas pyruvate, acetate and ribose are poor promoters of adult longevity (Hassett, 1948), they are readily used for larval development. On the other hand, malate, citrate, butyrate, e-alanine and e-aspartate are inefficient energy-yielding nutrients for both larvae and adults.

These observations suggest that D. melanogaster larval growth depends upon a nutrient that can be readily converted to glycolytic intermediates, whereas adult viability is dependent in part upon an energy-yielding pathway other than glycolysis and the Krebs cycle. An energy-yielding pathway much more prominent in adult D. melanogaster metabolism than larval metabolism is the ~-glycerophosphate cycle. In larvae the c~-glycerophosphate cycle enzymes are low and lactate dehydro- genase high in activity, but as the metabolism changes from a form dependent upon anaerobic glycolysis to the aerobic adult metabolism the relationship is reversed (Geer et al., 1972). Thus, one can speculate that the ~-glycerophosphate

60 B. W. GEER AND C. E. ZACHARIAS

cycle is critical for adult longevity and that nutrients such as pyruvate, acetate and ribose cannot be converted to a substrate suitable for the cycle at a rate sufficient to maintain adult life. O'Brien et al. (1972) have proposed that the a-glycerophosphate cycle is an important determinant of Drosophila fitness. This hypothesis, of course, will require further experimental testing.

That L-aspartate aminotransferase, L-alanine aminotransferase and/3-hydroxy- acyl dehydrogenase were the only D. melanogaster enzymes shown to be susceptible to dietary influences in the present investigation may be due to the experimental design. Because studies of larger animals have, by and large, indicated that diet- induced alterations of enzyme activities are tissue specific, certain tissue-specific enzyme fluctuations may have gone undetected in this study because of the whole animal procedures employed. Furthermore, diet-induced enzyme activity changes may have occurred in life stages different than that examined in the present study and, finally, the type of dietary manipulation applied in our experiments was only one of several which could be applied to trigger enzyme activity change. Thus , the results of the current study do not eliminate the possibility that enzymes other than the aminotransferases and fl-hydroxyacyl dehydrogenase are susceptible to nutritional influence in l). melanogaster.

L-Aspartate aminotransferase and L-alanine aminotransferase activities in D. melanogaster are a function of both the dietary nitrogen and carbon sources. This type of regulation of the aminotransferases is thoroughly documented for vertebrates. Blood serum levels of both L-aspartate aminotransferase and L-alanine aminotransferase are high in malnourished humans (Gupta & Santhanagopalan, 1962; DeFeo, 1963) compared to individuals with adequate diets, and in laboratory rats liver and serum activities of L-aspartate and L-alanine aminotransferase are negatively correlated to the biological value of the dietary protein (Nimni et al., 1962; Bergner et al., 1968). High levels of dietary protein in the absence of carbo- hydrate induce high L-aspartate aminotransferase and L-alanine aminotransferase activities in rat liver (Leveille, 1967; Szepesi & Freedland, 1967, 1968a) and muscle (Afar & Rogozkin, 1965), but moderate amounts of dietary carbohydrate suppress the induction of the enzymes (Szepesi & Freeland, 1968b).

L-Aspartate aminotransferase and L-alanine aminotransferase activities may be repressed in D. melanogaster by glycolytic intermediates, and nutrients readily converted to glycolytic intermediates. This being the case, in the absence of glycolysis-stimulating nutrients the aminotransferases would increase in activity to facilitate amino acid degradation for energy-yielding purposes. This hypothesis adequately explains the elevated L-alanine aminotransferase activities in D. melano- gaster fed citrate, butyrate and L-aspartate-L-alanine supplemented diets since glycolysis-stimulating nutrients are missing from these diets. As added evidence, the L-alanine aminotransferase activities observed in pyruvate and acetate-fed larvae, nutrients that can be readily converted to glycolytic intermediates, were similar to that found in glucose-fed individuals.

In contrast, D. melanogaster larvae fed butyrate, citrate or glucose-supplemented diets did not exhibit significantly different L-aspartate aminotransferase activities.

D R O S O P H I L A N U T R I T I O N AND METABOLISM 61

Thus , excess dietary amino acid rather than carbohydrate deficiency may be the primary inducer of higher L-aspartate aminotransferase activity in D. melanogaster larvae, f l-Hydroxyacyl dehydrogenase activity in D. melanogaster larvae responds to dietary butyrate but, in contrast to the aminotransferase response, at least part of the enzyme activity increase may be attributed to a size difference between l 1-day-old glucose and l 1-day-old butyrate-fed larvae fl-Hydroxyacyl dehydro- genase activity declines in Drosophila tissues during larval development, so that the butyrate-fed larvae might be anticipated to possess lower levels of the enzyme than their glucose-fed counterparts due to their smaller size. However, the dietary effect cannot be due to size alone, since larvae fed citrate and L-alanine-L-aspartate supplemented diets are smaller than glucose-fed larvae, yet they do not possess significantly different fl-hydroxyacyl dehydrogenase activities. Th e possibility that fl-hydroxyacyl dehydrogenase responds to dietary butyrate should be further examined in larvae exposed to butyrate under other dietary conditions.

In conclusion, utilization of the dietary carbohydrates and organic acids tested in the current study appears to be dependent upon D. melanogaster inter- mediary metabolic capabilities, and at least three enzymes are influenced by nutritional factors. Studies are underway in this laboratory to test the mechanisms by which the activities of the enzymes are influenced.

REFERENCES AFAR YA. & ROGOZKIN V. A. (1965) Aminotransferase activity and serum proteins in the

blood of rats after systemic muscular activity. Eksp. Med. Morfol. 4, 291-296. BEENAKKERS A. M. T. (1969) Carbohydrate and fat as a fuel for insect flight. A comparative

study, oV. Insect Physiol. 15, 353-361. BEIZENHERZ G. (1955) Triose phosphate isomerase from calf muscle. Meth. Enzym. 1,

387-391. BERGMEYER H. U. 86 BERNT E. (1965a) Malic dehydrogenase. In Methods of Enzymatic

Analysis (Edited by BERGMEYER H. U.), pp. 757-760. Academic Press, New York. BERGMEYER H. U. & BERNT E. (1965b) Glutamate-oxaloacetate transaminase. In Methods

of Enzymatic Analysis (Edited by BERGMEYER H. U.), pp. 837-842. Academic Press, New York.

BERGNER H., MUENCHOW H. & WIRTHGEN B. (1968) Correlations between protein nutrition, thyroid secretion rate, and the activities of certain enzymes. Acta biol. reed. germ 21, 683-686.

DEFEo V. (1963) Serum transaminases in malnutrition dystrophy of (nursing) infants. Riv. Clin. pediat. 72, 177-181.

DIPIETRO D. & WEINHOUSE S. (1960) Hepatic glucokinase in the fed, fasted and alloxan- diabetic rat. ft. biol. Chem. 235, 2542-2545.

GEER B. W. (1963) A ribonucleic acid-protein relationship in Drosophila nutrition, ft. exp. Zool. 154, 353-364.

G~R B. W. (1966) Utilization of ,-amino acids for growth by Drosophila melanogaster larvae, ft. Nutr. 90, 31-39.

GEER, B. W., MARTENSEN D. V., DOWNING B. C. & MUZYKA G. S. (1972) Metabolic changes during spermatogenesis and thoracic tissue maturation in Drosophila hydei. Devl. Biol. 28, 390-406.

GEER B. W. & NEWBURGH R. W. (1970) Carnitine acetyltransferase and spermatozoan development in Drosophila melanogaster..7, biol. Chem. 245, 71-79.

62 B. W. GEER AND C. E. ZACHARIAS

GUPTA S. & SANTHANAGOPALAN T. (1962) Serum transaminase activity in normal and malnourished children. 07. Indian Pediat. Soc. 1, 248-252.

HASSETT C. C. (1948) The utilization of sugars and other substances by Drosophila. Biol. Bull., Woods Hole 95, 114-123.

KORNBERO A. (1955) Lactic dehydrogenase of muscle. Meth. Enzym. 1,441-443. LEVEILLE G. A. (1967) Influence of dietary fat and protein on metabolic and enzymic

activities in adipose tissue of meal-fed rats. 07. Nutr. 91, 25-34. LowRY O. H., ROSEBROUGH N. J., FARR A. L. & RANDALL R. J. (1951) Protein measurement

with the Folin phenol reagent. 07. biol. Chem. 193, 265-275. MATTENHEIMER H. (1970) Micromethods for the Clinical and Biochemical Laboratory, pp.

137-138. Ann Arbor Science Publ., Ann Arbor, Michigan. NIMNI M. E., HOM n . & BARETTA L. A. (1962) Dietary composition and tissue-protein

synthes is- - I I . Tryptophan toxicity and amino acid deficiency in collagen synthesis. 07. Nutr. 78, 133-138.

O'BRIEN S. J., WALLACE B. & MAClNTYRE R. J. (1972) The c~-glycerophosphate cycle in Drosophila melanogaster--III. Relative viability of "null" mutants at the ~-glycero- phosphate dehydrogenase-1 locus. Am. Nat. 106, 767-771.

OCHOA S. (1955) "Malic" enzyme. Meth. Enzym. 1, 739-753. RIZKI T. M. & RIZKI R. M. (1963) An inducible enzyme system in the larval cells of

Drosophila melanogaster. 07. Cell Biol. 17, 87-92. SANG J. H. (1956) The quantitative nutritional requirements of Drosophila melanogaster.

07. exp. Biol. 33, 45-72. SCHIMKE R. T. & DOYLE D. (1970) Control of enzyme levels in animal tissues. Ann. Rev.

Bioehem. 39, 929-976. SZEPESl B. & FREEI)LAND R. A. (1967) Alterations in the activities of several rat liver

enzymes at various times after initiation of a high protein regimen. 07. Nutr. 93, 301-306. SZEPESI B. & FREEDLAND R. A. (1968a) Time-course of changes in rat liver enzyme

activities after initiation of a high protein regimen. 07. Nutr. 94, 463-468. SZEPESI B. & FREEDLAND R. A. (1968b) Dietary effects on rat liver enzymes in meal-fed

rats. 07. Nutr. 96, 382-390. WARD C. W. & SCHOFIELI) P. J. (1967) Glycolysis in Haemonchus contortus larvae and rat

liver. Comp. Biochem. Physiol. 22, 33-52.

Key Word Index--Drosophila melanogaster; nutrition; diet; enzyme regulation; L- asparate aminotransferase; L-alanine aminotransferase; ~-hydroxyacyl dehydrogenase