DEVELOPMENTAL

BIOLOGY

42, 181-187 (1975)

Energy Metabolism

in Developing

Ascaris

I. The Glycolytic

Enzymes

J. BARRETT’ Department

of Zoology, Accepted

AND I.

University

Eggs

BEIS

of Oxford,

September

lumbricoides

Oxford, England

16, 1974

The changes in the activities of the glycolytic enzymes were followed during the development of Ascaris lumbricoides eggs. The maximum catalytic capacities of the major catabolic pathways were estimated from the maximum activities of the nonequilibrium enzymes, and the results are compared with the changes in metabolic rate and the changes in carbohydrate and lipid utilization which occur during development. Although the onset of carbohydrate and lipid utilization was accompanied by an increase in the catalytic capacities of the corresponding pathways, there was no drop in the catalytic capacities of the pathways when the eggs became dormant, nor was there any change when the dormant egg was activited. INTRODUCTION

The adult

parasitic nematode Ascaris lives in the small intestine of its host and has an essentially anaerobic energy metabolism (Fairbairn, 1970). In contrast to the adult, the eggs of Ascaris are aerobic and develop in the soil. The eggs pass out of the host at the one-cell stage and the embryo develops inside the egg into an infective second stage larva. The fully developed infective Ascaris egg is dormant and becomes activated again only when swallowed by a suitable host. Ascaris eggs, therefore, form a convenient system in which to follow the biochemical changes associated with the onset and termination of dormancy as well as the changes associated with development. The lipid and carbohydrate content of developing Ascaris lumbricoides eggs and the changes in the rate of respiration of developing eggs have been extensively studied (Fairbairn, 1955; Passey and Fairbairn, 1955, 1957). The changes in the activities of the TCA cycle, P-oxidation, and glyoxylate cycle enzymes during development have been followed (Ward and Fairbairn, 1970; Barrett et al., 1970), and several correlations exist between the aclumbricoides

tivities of the enzymes of P-oxidation and glyoxylate cycle and the rate of lipid utilization. In this paper the changes in the activities of the glycolytic enzymes during development are reported, and from the data on the changes in the maximum catalytic activities of the nonequilibrium enzymes of lipid and carbohydrate metabolism the changes in the maximum catalytic capacit,ies of the major catabolic pathways in developing Ascaris eggs have been estimated, MATERIALS

0 1975 by Academic Press. of reproduction in any form

Inc. reserved.

METHODS

Ascaris eggs were prepared and embryonated as described by Ward and Fairbairn (1970). The eggs were incubated at 30°C and all times refer to development at this temperature. At 3O”C, the first cell division occurs after 36 hr, the morula is formed by 120 hr, and the blastula at 144 hr. The first stage larva is formed by day 10, and this moults at 15 days to give a second stage larva, which is fully infective from day 18 onwards. The dormant infective eggs were hatched (activated) as described by Fairbairn (1961).

’ Present address: Department of Zoology, University College of Wales, Aberystwyth, Wales. Copyright All rights

AND

Enzyme

Assays

The enzyme assays were performed 181

at

182

DEVELOPMENTAL BIOLOGY

30°C in a Gilford Model 240 recording spectrophotometer. The eggs were homogenized in a l-ml ground glass homogenizer, the homogenizer was cooled in ice and the progress of homogenization was checked microscopically. The homogenate was centrifuged for 5 min at 700 g, at 2”C, and the supernatant taken for enzyme assays. The homogenizing media used were 0.1 M Tris-Cl buffer pH 7.6,0.09 M KCl, 0.028 M MgSO,; for the dehydrogenase assays the eggs were homogenized in 20% glycerol (v/v), 0.025 M glycylglycine buffer, pH 7.4 (Bueding and Saz, 1968); for the phosphofructokinase assays, 0.005 M, Tris-Cl buffer pH 8.2, 0.001 M EDTA, 0.005 M MgSO, was used; and for the phosphorylase assays 0.035 M &glycerophosphate, pH 6.2, 0.001 M EDTA, 0.02 M NaF, 0.03 M mercaptoethanol (Cornblath et al., 1963). The enzymes were assayed by the following methods: hexokinase (EC 2.7.1.1), glucosephosphate isomerase (EC 5.3.1.9), glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), phosphoglycerate kinase (EC 2.7.2.3), and triosephosphate isomerase (EC 5.3.1.1) (Ward and Schofield, 1967); phosphoglucomutase (EC 2.7.5.1) (Abraham et al., 1961), pyruvate kinase (EC 2.7.1.40) and lactate dehydrogenase (EC 1.1.1.27) (Ward et al., 1969); the pyruvate kinase of Ascaris eggs was strongly activated by fructose-1,6-diphosphate, and the assays were all performed in the presence of 1 mM FDP; phosphofructokinase (EC 2.7.1.11) (Opie and Newsholme, 1967); glucose-6-phosphate dehydrogenase (EC 1.1.1.49) (Glock and McLean, 1953); glucose-6-phosphatase (EC 3.1.3.9) (Harper, 1963); phosphoglyceromutase (EC 2.7.5.3) and phosphopyruvate hydratase (EC 4.2.1.11) were assayed jointly (Barret and Beis, 1973); aldolase (EC 4.1.2.7) (Barrett and Beis, 1973); phosphorylase (EC 2.4.1.1) (Burleigh and Schimke, 1968); fumarate reductase activity was measured as described by Prichard (1973).

VOLUME42,1975

The enzyme activities (initial rates) are all expressed as nmoles/min/mg protein, at 30°C. The protein, in the 700 g supernatant was determined by the method of Lowry et al. (1951). The protein content of Ascaris eggs does not vary appreciably during development (Passey and Fairbairn, 1957); protein content can therefore be used as a basis for comparison of enzyme activities (Ward and Fairbairn, 1970). Standard errors were calculated as described by Dean and Dixon (1951). RESULTS AND DISCUSSION

The changes in the activities of the glycolytic and associated enzymes in developing Ascaris eggs are summarized in Table 1 and the percentage change in the enzyme activities in Figs. 1 and 2. The enzyme activities were measured using whole homogenates and this can be difficult to interpret in a complex developmental system, since differences between different tissues are lost. An enzyme may be increasing in one tissue while decreasing in another, and two pathways which appear to be interrelated may in fact occur in different tissues. The results from whole homogenates must, therefore, be treated cautiously, but they can provide useful information especially when taken in conjunction with other measurements such as oxygen uptake and substrate utilization. During development the activities of the glycolytic enzymes show no overall trend; however, two features can be distinguished. Firstly, several of the enzymes of glycolysis show oscillations in activity during the first 5 days of development, although the pattern of change during this time differs for different enzymes. Some enzymes show a peak of activity followed by a decline, while others show a decrease and then increase again. Secondly, a number of the glycolytic enzymes increase sharply in activity at about day 14. TWO corresponding “critical periods” also occur in the specific activities of the TCA cycle

(OO’T) (EZ’O)

(06’0)

CL.0 0'91 (O'P) (2

(09’0) 9L'E (9T'O) 06'0 (Z'O) Z'I (I'E)

P'L (LO'O) 86.0

CL.!,

9P’E (EZ’O) ZZ’I

(PZ’O)

(P.0) 9'Z WE)

OL'Z (LZ'O) PI.1

K%‘0)

OX.8

9L.6 (OZ'O) LZ'E (6'1) O'LZ WE) (0.2 ZEI (09) Z9LT (6'8Z) 98L (E99) S8'iX (E'L) L'LS (O'E) O'IZ (PZ) '289 (L'P) 9'IOT (9';'O) O'L (89'0) PP'Z

(OP’I)

OI’E (Z‘E) 0’8E (8.Z)

(6LP) 6T9P (S.8) E'LS (9'0) O'LI (LE) 289 (1.6) 9'101 (SZ'O) L'8 (9Z'O) II'Z

8

(8L.O) E9'9 (91'0) OP'Z (Z'O)

(L9.0) 08'6 (91.0) 09'8 (L'Z) 9'8E (Z'P) (9.:; 081 (19) PL91 (9‘01) LL9 (Z6E) PPPE (1'9) 0'19 (6'0) 0'91 (OE) 9P9 (1.L) O.L6 (OL.1) 1'6 (89'0) OZ'Z

01

(PE’O) GE.‘; (Pro) oo’z

09.6 (81'0) 19's (9.1) O'LE (O'ZI)

(Ei:. 901 (CP) (0% 991 (86) 9L91 (O'BZ 1 99L (09Z) EZOE (O'ZT) 0'6L (0.1) P.81 (IV) SE9 (0";) O'LH (08.1) P'OI &9'0) SE'E

PT

LV

S333

Sap!O3?JqwnlS~ID3SV

LLGE (0.6) E'09 (9'0) O'IZ (LI) ';6P (T'Z)

S3PlhZN3

E’P6 WO)

s

NI

I 318VtL.

6$+$x? : (sKep) aBv

PEL

.u!a?ord Bur/u!cupa~ouw LN3H3ddla

a3LVKIOSSV

E

(LZ) E09 (O'Z) 9'L9 (EL'O) L'9 (ZI'O) 98'0

E’EZ

(9'9) 8'89 (Z'I)

(Z ';Z91 (TPZ) 1901 (989) 6IZE

(97

6LLI (0%) LOL (8'21) ZI.LZ ('j'Z1) YL8 (O'Z) 9'81 (8E) P99 (9'9) O'LPT (09'1) L'OT (6P.O) 69'E

81

hY% P8T (19) EE91 (9'OZ)

G 991 (L9) 0891 (YZZ) IE8 (006) Z60E (E'II) 9'TTT (VZ) L'81 (Z1) 169 (9'9) O'OPI (06'1) 0'01 (OP'O) LE.8

zz

d0 S33V.LS

CL% 101 (89) z991 (E'ZT) 9ZOT (68p)

(ZZ 961 (09) OZ61 (E'sz) 098 (9LE) OOLZ (O'ZI) 0'66 (Z'Z) P'IZ (ZI) 999 (O'P) 0'9EI (OE'I) Z'II (09'0) 8'L

~UeuIrop ) 09

L.N3HdO'I3,,3a

(L9.0) 8Z'E &oO‘o) PZ'Z (9.0) O'Z (Z.Z) (SZ 061 (01) 69L (L'9T) 89t (009) 99LE (O'E) L'L9 (1.1) YZZ

(LE) 889 (L'Z)

O’PE (ZZ’O) P'L (LO'O) Z9'0

I

C,NV

(OE'O) It.1 (90'0) 91'0

COZ

(0% PLI (EI) OPZ (O'EZ) 9ZL (009 FSLP KT) 9'6P

(9'0) ';'PZ (PI) 8% (0'9) 0'99 (09'0) I'L (90.0) Oft'0

0

X,,h7OL,hlf)

3H1

aseua%arp -bqap altzqdsoqd-g-asoml3

aleANIq

aswlsqdsoqd-g-asom13

aseuq

x~e~~~p~q a~ean.xKdoqdsoqd + ase~ncuoraah[8oqdsoqd

aleqdsoqdaso!r&

aseu!q aJwa3b@oqdsoqd aseua%xpbqap aleqdsoqd-E-apl(qap[wa%Q)

awratuos!

awpw

aleqdsoqdasomlt)

3HA

aurdzu3

S3lU,,IL~v

asmxuos!

10

184

DEVELOPMENTALBIOLOGY

VOLUME 42,1975

Pnosptm$fcerom~~oie + pnosphopyruvote hydrctose

/ 25 $

0

I 5

I 10

I 15 Days

I 20

I / 25

embryonoted

Days embryoncted

FE. 1. Activities of the glycolytic enzymes relative to the maximum activities observed during the development of Ascaris lum bricoidei eggs. Calculated from the data in Table 1.

FIG. 2. Activities of the glycolytic enzymes relative to the maximum activities observed during the development of Ascaris lumbricoides eggs. Calculated from the data in Table 1.

enzymes (Ward and Fairbairn, 1970). Morphologically these two “critical periods” in Ascaris egg development correspond to the formation of the morula (day 5) and the second moult, which occurs at 12-15 days. The enzymes which show one or another of the different patterns of change do not seem to fall into any discernible groups. Aldolase, phosphoglucomutase, hexokinase, lactate dehydrogenase, and phosphorylase all show an increase in activity after day 14. Hexokinase and phosphorylase both control the entry of carbohydrates into the glycolytic pathway; phosfructokinase and pyruvate kinase, the other two key enzymes controlling the rate of flux through the glycolytic sequence, do not show an increase at day 14, but aldolase and lactate dehydrogenase, the enzymes immediately following phosphofructokinase and pyruvate kinase in catabolism, both increase sharply at about 14 days. The “reverse” TCA cycle also becomes evident after day 14. This is the pathway

from phosphoenolpyruvate via phosphoenolpyruvate carboxykinase, resulting in the production of succinate and pyruvate as the initial end products of carbohydrate catabolism, as in the adult Ascaris (Saz, 1971). The activity of phosphoenolpyruvate carboxykinase increases rapidly at this time (Barrett et al., 1970) and fumarate reductase activity can be detected in egg homogenates from day 14 onwards. Ascaris eggs contain appreciable amounts of trehalose, and some of it is metabolized during the first 3-4 days of development (Fairbairn and Passey, 1957; Passey and Fairbairn, 1957). The enzymes involved in trehalose metabolism have not been investigated, and in the infective egg the trehalose occurs almost exclusively in the perivitelline fluid (Fairbairn and Passey, 1957). The respiratory rate of Ascaris eggs alters considerably during development (Passey and Fairbairn, 1955). During the first day of development the rate of respira-

BARRETTANDBEIS

Energy Metabolism

tion drops, until about the time of the first cell division (which occurs after 38 hr); the respiratory rate then increases rapidly to reach a peak at 10 days, after which there is a slow decline in the rate of respiration, until by the time the infective egg is reached (18-19 days) respiration is proceeding at a very low rate. The infective egg is dormant and as such can survive for several years. However, on receipt of a suitable stimulus, usually provided by the hosts intestine, the infective larva is activated, its metabolic rate increases, and the larva hatches. Enzyme changes occurring during development are difficult to interpret unless they can be related to some other measurable overall change or process (Moog, 1965). The changes in the specific activities of the glycolytic enzymes during development do not correspond to changes in the rate of respiration of the egg, nor do they correspond to changes in the rate of carbohydrate utilization, which reaches a peak at 5 days (Barrett et al. 1970). Individual glycolytic enzymes decrease on day 1, while others show a maximum at day 5 or 10, but there is no general pattern. Nor is there any significant difference between the enzyme activities of dormant and activated infective eggs (Table 1). The enzymes of metabolism can be divided into two types; those enzymes which catalyze reactions that are near to their thermodynamic equilibrium, and those enzymes which catalyze reactions which are far displaced from their thermodynamic equilibrium. The nonequilibrium enzymes catalyze the rate-limiting steps in the catabolic pathways, and it is only through these enzymes that metabolic control can be 1964). exerted (Biicher and Riissman, Crabtree and Newsholme (1972) have suggested that the maximum in vitro catalytic activities of the nonequilibrium enzymes of catabolism would provide a reliable estimate of the maximum catalytic capacities of these pathways in viuo. Changes in the

in Ascaris Eggs. I

185

activities of the rate-limiting, nonequilibrium enzymes will alter the rate of flux through the pathway, whereas changes in the catalytic capacities of the near-equilibrium enzymes will have no affect on the rate of flux through the pathway, unless the activity of such enzymes are reduced to such an extent that they become rate limiting. How far it is possible to apply the results from in vitro enzyme assays to the situation in the developing Ascaris egg is difficult to assess. Nevertheless, an attempt can be made to estimate the changes in the maximum capacities of glycolysis in developing Ascaris eggs from the changes in the maximum catalytic capacities of the rate-limiting enzymes (hexokinase + phosphorylase; phosphofructokinase; or pyruvate kinase, depending on the developmental stage). Although the activities of the near-equilibrium enzymes of glycolysis vary widely during development, none of them appear to decrease in activity sufficiently to become rate limiting (Table l), and their activities are mostly an order of magnitude greater than the activities of the rate-limiting enzymes. This raises the question as to what is the significance of the changes in the specific activities of the near-equilibrium enzymes during development, since the change in specific activities of these enzymes does not affect the rate of flux through the pathway. The maximum catalytic capacities of the TCA cycle, P-oxidation sequence, and glyoxylate cycle can similarly be estimated from the maximum catalytic capacities of the rate-limiting enzymes (citrate synthase or isocitrate dehydrogenase; P-hydroxyacyl CoA dehydrogenase; malate synthase), and the results are shown in Fig. 3. The maximum capacities of glycolysis, P-oxidation, and the TCA cycle vary throughout development, and, although the onset of carbohydrate utilization, lipid utilization, and carbohydrate resynthesis is accompanied by a rise in the catalytic capacities of the corresponding pathways,

186

DEVELOPMENTAL 120

BIOLOGY

VOLUME 42, 1975

r

,.-.-.. ‘1 61”01”1.1. /. ‘\.\. C”Ill Li /’ ~-,*“odar,on ;; ,‘i’ : ‘.._,“‘(..........\.... ,..’,..’ ,’ ,’ ‘

Energy metabolism in developing Ascaris lumbricoides eggs. I. The glycolytic enzymes.

DEVELOPMENTAL BIOLOGY 42, 181-187 (1975) Energy Metabolism in Developing Ascaris I. The Glycolytic Enzymes J. BARRETT’ Department of Zoology,...
564KB Sizes 0 Downloads 0 Views