Certain key enzymes of alternative pathways of glucose metabolism, of amino acid metabolism and of redox systems have been measured in hydra and this profile compared with mammalian differentiated tissues with a view to locating pathways of specific importance in hydra. There was a marked constant proportionality in the major part of the enzymes investigated, the profile suggested a metabolic pattern geared to utilization of amino acids as a carbon source for biosynthesis and energy production and to the production and conservation of pyruvate. The importance of conversion to ionized forms was noted. The most notable specific proportion changes were the exceptionally low lactate dehydrogenase, malic enzyme and the relatively high citrate synthase. The proximal-distal gradients in hydra were examined and these gradients suggested a switch to a more anaerobic type of metabolism and an elevation of the pentose phosphate pathway as the basal region was approached. Measurements of the formation of 14CO2 from specifically labelled glucose provided additional evidence for the functional activity and polarity of the pentose phosphate pathway in hydra. The effect of oligomycin, which can reverse polarity in hydra, had a significant effect on gradients of enzymes eliminating all except that observed for G6P dehydrogenase. The profile suggested a movement towards a more anaerobic type of metabolism, in keeping with the known biochemical action of this inhibitor. It is suggested that redox states and/or phosphorylation states may be featured in the positional information of cells in hydra.

We have recently been investigating regeneration and pattern regulation in hydra within the framework of positional information (Wolpert, Hicklin & Hornbruch, 1971; Hicklin, Hornbruch, Wolpert & Clarke, 1973; Wolpert, Hornbruch & Clarke, 1974) which is essentially a gradient-type theory. It is suggested that head-end formation regeneration in a variety of different graft combinations can be explained in terms of the interaction between two gradients, both of which decrease in concentration from the head end, the one being of a diffusible morphogen and the other being more stable. A striking weakness of such models is our virtual total ignorance of the physiological or biochemical basis of such gradients. While the models can indicate what sort of data requires explanation, including how the gradients might change with time, they fail to provide an obvious biochemical approach. The main difficulty is that there is at present no assay for either gradient and so one is driven to less direct approaches such as trying to perturb the system with appropriate agents (Wolpert et al. 1974). We have found, for example, that a variety of agents applied to Hydra littoralis will cause a regenerating head end to form a foot end instead. Oligomycin is particularly potent in this respect and a foot will form at the head end even if intact animals are treated (Hornbruch & Wolpert, 1975). Another approach to the problem which we follow here is to measure biochemical parameters along the axis in the hope of finding differences which correspond with the postulated gradients. It is worth remembering that while the gradient concept has been around for nearly 75 years, no persuasive physiological correlates have yet been found in any system: those possibly giving a hint in this direction are reduction gradients in sea urchins (Gustafson, 1965) and RNA gradients in amphibian eggs (Brachet & Malpoix, 1971).

In order to try to gain insight into the biochemical control mechanisms regulating gradients in hydra, three lines of approach have been used. First, the enzyme profile of hydra has been compared with those of highly differentiated mammalian tissues in order to establish which enzymes are in constant proportion and which in specific proportion as a guide to enzyme systems of particular significance in hydra (Pette, Klingenberg & Bücher, 1962a;,Pette, Luh & Bücher, 1962b; Pette, 1966; Bass et al. 1969). Secondly, the specific activity of enzymes in hydra (head, gastric and foot regions) with and without exposure to oligomycin, have been measured and compared in order to determine the variant and invariant groupings and to relate these to linear changes in organization and function. Finally, the regional contribution of different pathways of glucose metabolism was studied in hydra using specifically labelled glucose.

Hydra littoralis were used for all experiments. Details with regard to culture methods, collection and section are given in Webster & Wolpert (1966). Hydra were starved for 18 h before collection. Head, gastric, budding and foot regions were prepared by microdissection and 100 regional segments were subjected to ultrasonic disintegration in 1 ml Hydra Medium ‘M’ 25 intact hydra in 1 ml medium were similarly treated. (The composition of hydra medium ‘M’ is KC1 10−4 M, NaHCO3 10−3 M, Tris 10−3 M, CaCl2 10−3 M, MgCl2 10−4 M.) These extracts were used for the assay of certain key enzymes of glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle and of enzymes concerned in the utilization of amino acids for energy and gluconeogenesis. In certain experiments hydra were treated with oligomycin 10 μ g/ml for 24 h.

The mammalian tissues were obtained from albino rats of the Wistar strain.

The preparation of homogenates and estimation of enzymes were essentially as previously described (Novello, Gumaa & McLean, 1969; Gumaa, Greenbaum & McLean, 1973; Baquer, McLean & Greenbaum, 1973 b). The enzymes measured, trivial names, abbreviations, enzymes nomenclature number and any special conditions are given below.

Hexokinase (HK), EC 2.7.1.1, was estimated with 25 mM fructose as substrate in order to evaluate total hexokinase isoenzymes (Gumaa & McLean, 1972). Phosphofructokinase (PFK), EC 2.7.1.11; phosphoglucoseisomerase (PG 1), EC 5.3.1.9 ; pyruvate kinase (PK), EC 2.7.1.40; lactate dehydrogenase (LDH), EC 1.1.1.27; glucose 6-phosphate dehydrogenase (G6P DH), EC 1.1.1.49; 6-phosphogluconate dehydrogenase (6PGDH), EC 1.1.1.44; citrate synthase (CS), EC 4.1.3.7; isocitrate dehydrogenase (ICDH), EC 1.1.1.42; malate dehydrogenase (MDH), EC 1.1.1.37; malic enzyme (ME), EC 1.1.1.40; glutamate dehydrogenase (GLDH), EC 1.4.1.2 ; this enzyme was measured with ADP as activator. Glutamate-pyruvate transaminase (GPT), EC 2.6.1.2; glutamate-oxaloacetate transaminase (GOT), EC 2.6.1.1; succinate dehydrogenase (SDH), EC 1.3.99.1 was measured with 2:6 dichlorophenolindophenol and phenazine methosulphate as the acceptor system essentially by the method of Veeger, Der Vartanian & Zeylemaker (1969); a-glycerophosphate oxidase (aGPOX), EC 1.1.99.5 was measured as described for succinate dehydrogenase with a-glycerophosphate as substrate and acceptor system as above.

The substrate and cofactors for these assay systems were obtained from Boehringer Corporation, London and Sigma Chemical Co., London.

Protein was estimated by the method of Lowry, Rosebrough, Farr & Randall (1951).

Pathways of glucose oxidation using specifically labelled glucose

Hydra were incubated in medium ‘M’ with 10 mM glucose containing 0·5 μ Ci specifically labelled glucose/ml of medium. Phenazine methosulphate was added to give a final concentration of 0·1 mM; 10 hydra (divided in half), or 20 separate head, gastric or foot regions were incubated in 1·0 ml medium for 30 min at 24 ° C with gentle shaking in stoppered tubes with centre wells. The reaction was stopped by addition of 0·2 ml of 5 N-HCl and the 14CO2 collected in 0·5 ml hyamine in methanol, added to the centre well, by further incubation for 1 h at 37 ° C. 14CO2 was determined by counting using the Packard Tricarb Scintillation counter. With the exception of [3,4-14C] glucose, obtained from NEN Chemicals, the specifically labelled glucoses were obtained from the Radiochemical Centre, Amersham, the low background [l-14C] glucose was used.

Production of 14CO2 from [l-14C] glucose was linear for 30 min under the conditions of the assay, thereafter there was some decrease in the rate. Values for 15,30 and 60 min incubation were respectively 23, 44 and 51 m μ g atoms glucose carbon/100 hydra.

The figures for mammalian tissues were obtained using Krebs Ringer Bicarbonate buffered medium with 20 mM glucose, final concentration as previously described (Lagunas, McLean & Greenbaum, 1970).

In view of the claims that nematocyst toxins can inhibit some enzymes (Kline & Waravdekar, 1960) we have tested the effect of extracts of hydra head and basal regions on the activity of cytosolic and mitochondrial enzymes of liver. We have found no effect for the following enzymes: hexokinase; G6P dehydrogenase; 6PG dehydrogenase; phosphofructokinase; phosphoglucoseisomerase; lactate dehydrogenase; malate dehydrogenase; glutamate-oxaloacetate transaminase; citrate synthase; succinate dehydogenase; a glycerophosphate oxidase; glutamate dehydrogenase; malate dehydrogenase; glutamate-pyruvate trans-aminase. It should be emphasized that the observations of Kline & Waravdekar (1960) on the inhibition of succinoxidase and our failure to find such inhibition with succinate dehydrogenase is most likely due to our assay not involving cytochrome C.

In each tissue enzyme activities are expressed relative to hexokinase which is assigned a fixed value of unity to facilitate comparison of enzymes of the major pathways of glucose utilization. For absolute values for hydra see Table 3. Hexokinase activity of hydra, rat liver, lactating rat mammary gland and brain are 7·8, 10, 35 and 54 m.u./mg protein respectively.

Constant and specific proportion enzymes in hydra

The systems relationships in hydra compared with a selection of differentiated mammalian tissues are given in Table 1 and Fig. 1.

Table 1.

Systems relationships in hydra – comparison with differentiated mammalian tissues.

Systems relationships in hydra – comparison with differentiated mammalian tissues.
Systems relationships in hydra – comparison with differentiated mammalian tissues.
Fig. 1.

Constant and specific proportion groups of enzymes in hydra -regional distribution. Values are given in milliunits/mg protein and represent the mean of 4 values, each comprising extracts from 100 hydra. Enzymes enclosed in the boxes and shown against horizontal arrows are in constant proportion in the three regions; those shown by the broken and solid arrows outside the boxes increase or decrease from the head to foot region as illustrated. MDH values have been divided by ten for representation on this figure.

Fig. 1.

Constant and specific proportion groups of enzymes in hydra -regional distribution. Values are given in milliunits/mg protein and represent the mean of 4 values, each comprising extracts from 100 hydra. Enzymes enclosed in the boxes and shown against horizontal arrows are in constant proportion in the three regions; those shown by the broken and solid arrows outside the boxes increase or decrease from the head to foot region as illustrated. MDH values have been divided by ten for representation on this figure.

Rat liver was selected for comparison because of its role in homeostasis and consequent ability to use and interconvert a wide range of substrates. Lactating mammary gland has the ability for rapid synthesis of protein, carbohydrate and fat, at the 14th day of lactation the growth phase is completed and a high rate of milk production is established so that this stage represents a predominantly synthetic state; developing brain (1-day post partum) is tissue in a rapid growth phase geared eventually to a high dependence upon glucose utilization.

In order to compare such a wide range of tissues one particular enzyme has been selected as the basis of expression and all other enzymes given in relation to this activity (McLean, Greenbaum & Gumaa, 1972; Gumaa et al. 1973; Baquer et al.1973 b). In this particular case, since pathways of glucose metabolism were primarily under consideration, hexokinase was selected. It is apparent from Table 1 that an essentially similar pattern would have emerged had phosphofructokinase, the rate limiting enzyme of the glycolytic pathway, been selected.

Pette et al. (1962 a,b, 1966) established the concept of constant and specific proportionality to delineate those enzymes of special functional significance in a tissue relative to those forming the basic framework of the metabolic pathways common to a wide range of tissues.

The results in Table 1 and Fig. 1 suggest that hydra is, in many essentials, similar in enzymic profile to a wide range of mammalian tissues. Many enzymes of the glycolytic route, pentose phosphate pathway, the tricarboxylic acid cycle and of deamination and transamination, are in constant proportion in hydra, liver, brain and mammary gland. These form the framework upon which are superimposed specific modifications of importance in the adaptation of the organism to its environment. It is particularly noteworthy that lactate dehydrogenase is extremely low in hydra, consistent with the need to conserve pyruvate for oxidative and synthetic reactions and with the need to prevent loss by diffusion of lactate in an aquatic environment. Correlated with this need to conserve pyruvate, is the low lactate dehydrogenase/pyruvate kinase quotient, pyruvate kinase being a regulatory enzyme in the formation of pyruvate from carbohydrate precursors.

Since protein comprises a substantial part of dietary intake, the conversion of amino acids to oxidizable substrates for energy production must be a pathway of major importance. The low lactate dehydrogenase relative to glutamate pyruvate transaminase is entirely in keeping with the need to conserve pyruvate. The relative importance of amino acids as opposed to carbohydrate in the formation of pyruvate is illustrated by the high glutamate pyruvate trans-aminase/pyruvate kinase quotient which is an order of magnitude higher than any of the other tissues examined here (Table 1).

Another aspect of the adaptation to conserve diffusible substrates is indicated by the high citrate synthase/pyruvate kinase ratio which is a key reaction in the entry of pyruvate into the tricarboxylic acid cycle and lipogenesis. The conversion of intermediates to highly ionized forms, phosphorylated derivatives, polycarboxylate anionic forms, is of particular importance in retaining substrates, establishing gradients and in metabolic compartmentation, as discussed in the early work of Davis (1958).

The pentose phosphate pathway in hydra is within the span of the range of tissues shown here (Table 1) being substantially less than in a tissue with highly active reductive synthetic reactions, e.g.mammary gland (Glock & McLean, 1958; Gumaa et al. 1973), and approximating more closely to liver (Novell© et al. 1969). The pentose phosphate pathway dehydrogenases and isocitrate dehydrogenase are the systems most closely geared to NADPH formation in hydra; there is an almost entire absence of malic enzyme.

Malic enzyme is linked to the process of lipogenesis from glucose where it is a component in the transhydrogenase sequence whereby NADH generated at the glyceraldehyde 3-phosphate dehydrogenase reaction is reoxidized and the hydrogen transferred to NADP+ for use in the reductive steps of processes such as lipogenesis (Shrago & Lardy, 1966; Rognstad & Katz, 1966; Williamson, Scholz, Thurman & Chance, 1969). In gluconeogenic situations and in conditions where there is a need to conserve dicarboxylic acids, malic enzyme would catalyse a futile cycle. The low level of malic enzyme in hydra is thus in keeping with an organism with a predominantly protein intake requiring carbon units for gluconeogenesis and energy.

There is a flexibility and range of options in systems for the generation and utilization of hydrogen and for the regulation of the redox state of the cell (Krebs & Veech, 1969). Thus, the pivot of the glycolytic sequence regulating the balance of glycolysis or gluconeogenesis is the glyceraldehyde 3-phosphate dehydrogenase reaction which is controlled by the redox state of free NAD+/ NADH and by the phosphorylation state of adenine nucleotides (Krebs & Veech, 1969; Veech, Raijman & Krebs, 1970). In a gluconeogenic situation hydrogens must be supplied for this reaction. The malate-aspartate shuttle (Borst, 1963) plays such a role in many tissues and the constancy of the malate dehydrogenase/glutamate-oxalacetate transaminase quotient (the component enzymes of this system) in hydra, mammary gland, liver and brain, suggest that this is an important feature of transfer of reducing equivalents between cell compartments in hydra.

When the glycolytic pathway has a flux in the direction of pyruvate then there is an imperative need to reoxidize the NADH generated at the glyceraldehyde 3-phosphate dehydrogenase stage (Rognstad & Katz, 1966; Shrago & Lardy, 1966; Flatt & Ball, 1966; Williamson et al. 1969; Katz & Wals, 1970). Four systems are normally available : (a) the lactate dehydrogenase reaction ; as already shown this is low in hydra : (b) the transhydrogenase system using malate dehydrogenase and malic enzyme; this is also low in hydra because of almost complete absence of malic enzyme: (c) the malate-aspartate shuttle, which, as discussed above, would appear to operate normally in hydra and (d) the a-glycerophosphate shuttle; the relatively high activity of a-glycerophosphate oxidase in hydra, which has an a-glycerophosphate oxidase/lactate dehydrogenase activity three orders of magnitude greater than liver, suggests that this may be an important route for hydrogen transfer in this organism.

This examination of the enzyme profile of hydra pinpoints areas of importance in consideration of linear patterns of organization.

Flux of glucose through alternative pathways

The operation of the pentose phosphate pathway in hydra receives further confirmation from the data in Table 2 which give the yield of 14CO2 from specifically labelled glucose. The quotient 14CO2 from [l-14C] glucose/14CO2 from [6-14C] glucose is frequently used as a broad indication of the extent of the pentose phosphate pathway relative to the combined glycolytic and tricarboxylic acid cycle activities. While there are many difficulties in the quantitative evaluation of pathways using 14CO2 data alone (Katz & Wood, 1960; Katz, Landau & Bartsch, 1966), quotients significantly above unity give a clear indication of significant contribution of the pentose phosphate pathway to glucose metabolism (Hollmann, 1964; Baquer, Cáscales, Teo & McLean, 1973 a).

Table 2.

Alternative pathways of glucose metabolism in hydra

Alternative pathways of glucose metabolism in hydra
Alternative pathways of glucose metabolism in hydra
Table 3.

Enzyme profile and gradients in hydra

Enzyme profile and gradients in hydra
Enzyme profile and gradients in hydra

The quotient of 7·6 for hydra is close to that for isolated liver cells (Baquer et al. 1973a) and falls between those of brain, a tissue in which the glycolytic pathway predominates, and lactating mammary gland which has a highly active pentose phosphate pathway geared to the high lipogenic rate (Glock & McLean, 1958; Gumaa et al. 1973).

In hydra, in common with many other tissues, the pentose phosphate pathway is not operating at full capacity. A rate limitation appears to be imposed by the availability of N ADP+ and/or by the inhibition from the high NADPH (McLean, 1960; Eggleston & Krebs, 1974). In the presence of the artificial electron acceptor, phenazine methosulphate, there is a sixfold increase in the oxidation of carbon-1 of glucose by hydra, an effect which may be ascribed to a combined action, this electron acceptor increasing NADP+ and in parallel decreasing NADPH providing a ‘push-pull’ system. Under these conditions the activity of the pentose phosphate pathway measured by decarboxylation of carbon-1 of glucose approximated closely to that of the in vitro activity of 6PG dehydrogenase measured with excess substrate and cofactors in the sonicated extracts; the figures are 380 mµg atoms [l-14C] glucose to 14CO2 and 360 milliunits of 6PG dehydrogenase per 100 hydra/h at 24° calculated from data in Tables 1 and 2.

The flux of glucose through the glycolytic pathway and decarboxylation by pyruvate dehydrogenase to yield acetyl CoA is estimated by the yield to 14CO2 from [3,4-14C] glucose. As shown in Table 2 the major part of the acetyl CoA is oxidized in the tricarboxylic acid cycle and the flux through the glycolytic pathway appears to be less than that via the pentose phosphate route.

Enzyme gradients in hydra

The results in Table 3 and Fig. 1 show the specific activity of enzymes in different regions of hydra, the data in Fig. 1 being presented on a logarithmic scale in order to place equal weighting on changes in low and high activity enzymes. The enzymes shown in the enclosed area are essentially in constant proportion in all regions of hydra, while enzymes placed outside this area are present in varying proportions, increasing or decreasing in activity from proximal to distal regions.

Of the glycolytic sequence hexokinase, phosphoglucose isomerase, and pyruvate kinase are in constant proportion while phosphofructokinase, the rate-limiting and allosterically controlled step, decreases towards the foot, possibly indicating a decreased glycolytic flux.

The enzymes of the pentose phosphate pathway increase as the basal region is approached, a change which is more clearly seen for the more highly active G6P dehydrogenase. The functional significance of this gradient in hydra remains obscure since it is generally found that there is a close gearing of the pentose phosphate pathway with RNA synthesis and reductive synthetic reactions (see Hollmann, 1964). It may be that closer examination of specialized regional functions of hydra will reveal a logical link with this gradient in the dehydrogenases of the pentose phosphate route.

The general picture that emerges is for a more anaerobic type of metabolism as the basal region is approached, the decrease in mitochondrial glutamate dehydrogenase, the rise in lactate dehydrogenase and the relative increase of enzymes of the malate-aspartate shuttle all point to this conclusion. Provided that a system for reoxidation of NADPH is present, the pentose phosphate pathway can function anaerobically so that an increase in this pathway in the basal region is not inconsistent with the profile.

The polarity in the activity of enzymes of the pentose phosphate pathway is confirmed by the measurements of 14CO2 production from [l-14C] glucose (Table 2 C). This gradient foot/head is 2·2, closely similar to that for 6PG dehydrogenase (2·8) but notably less than the gradient for G6P dehydrogenase of 4 (Table 3).

The rate of formation of 14CO2 from [l-14C] glucose in the presence and absence of phenazine methosulphate represents, as a first approximation, the maximum potential activity of the pentose phosphate pathway relative to the functionally expressed activity. It is of some interest that the differential of 5-fold stimulation is similar in all regions. The apparent excess potential activity of the pathway may suggest an intermittent high requirement for the products of this route of metabolism. The intense stimulation of the pentose phosphate pathway by processes such as phagocytosis come to mind in this context (Zatti & Rossi, 1965).

Effect of oligomycin on enzyme gradients in hydra

The effect of oligomycin at the end of 24 h treatment on enzymes, profiles and gradients in hydra are presented in Table 4 and Fig. 2. It is immediately apparent that gradients are largely eliminated, the only exception in the present series being G6P dehydrogenase. The effect of oligomycin in hydra is to convert a head end into a foot end but this is only fully achieved some 48 h after treatment (Hornbruch & Wolpert, 1975); biochemically it is known to inhibit respiration (NADH → O2 or succinate → O2) when coupled to phosphorylation.

Table 4.

Effect of oligomycin on enzyme activities and gradients in hydra

Effect of oligomycin on enzyme activities and gradients in hydra
Effect of oligomycin on enzyme activities and gradients in hydra
Fig. 2.

Effect of oligomycin on gradients of enzymes in hydra. Each value is the mean of 4 controls and 2 oligomycin experiments, each comprised the sonicated extracts of 100 hydra, head, gastric and foot regions. Figures across the control histogram represent Fisher’s P values for significance of difference from head-foot region. With the exception of G6P dehydrogenase, these gradients are lost following exposure to oligomycin (10 μ g/ml medium 24 h). The letters H, G and F at the foot of the histogram signify head, gastric and foot regions respectively.

Fig. 2.

Effect of oligomycin on gradients of enzymes in hydra. Each value is the mean of 4 controls and 2 oligomycin experiments, each comprised the sonicated extracts of 100 hydra, head, gastric and foot regions. Figures across the control histogram represent Fisher’s P values for significance of difference from head-foot region. With the exception of G6P dehydrogenase, these gradients are lost following exposure to oligomycin (10 μ g/ml medium 24 h). The letters H, G and F at the foot of the histogram signify head, gastric and foot regions respectively.

The modification in gradient under the influence of oligomycin is perhaps best illustrated by examination of the quotient of key mitochondrial and cytosolic redox systems, glutamate dehydrogenase and lactate dehydrogenase. In normal hydra this quotient is 13·5 to 3·8 for head and foot respectively, the corresponding values for hydra exposed to oligomycin are 6·9 and 7·9 (see Table 3).

The pentose phosphate pathway is sustained at normal values although phosphofructokinase declines in oligomycin-treated hydra, this is again illustrated by reference to the quotients in Table 4.

The effect of oligomycin in suppression of gradients and in the regional shift to a more anaerobic type of metabolism in the head region could be of considerable significance in the inhibition of normal regenerative procedures.

It seems possible that recognition factors involved in positional information may be mediated in part by redox systems and phosphorylation states, certainly the elimination of certain of these gradients by oligomycin leads to absence of appropriate signals. It will be important to see if those enzymes that are graded along hydra change in parallel with head and foot determination: this might tell us whether they are primary or secondary events in pattern formation.

We gratefully acknowledge the support of the Wellcome Trust and the Medical Research Council.

Baquer
,
N. Z.
,
Cáscales
,
M.
,
Teo
,
B. C.
&
McLean
,
P.
(
1973a
).
The activity of the pentose phosphate pathway in isolated liver cells
.
Biochem. biophys. Res. Commun
.
52
,
263
269
.
Baquer
,
N. Z.
,
McLean
,
P.
&
Greenbaum
,
A. L.
(
1973b
).
Enzymic differentiation in pathways of carbohydrate metabolism in developing brain
.
Biochem. biophys. Res. Commun
.
53
,
1282
1288
.
Bass
,
A.
,
Brdiczka
,
D.
,
Eyer
,
P.
,
Hofer
,
S.
&
Pette
,
D.
(
1969
).
Metabolic differentiation of distinct muscle types at the level of enzymatic organisation
.
European J. Biochem
.
10
,
198
206
.
Borst
,
P.
(
1963
).
In Funktionelle und Morphologische Organisation der Zelle
(ed.
P.
Karlson
), p.
137
.
Berlin
:
Springer-Verlag
.
Brachet
,
J.
&
Malpoix
,
P.
(
1971
).
Macromolecular syntheses and nucleocytoplasmic interaction in early development
.
Adv. Morphogen
.
9
,
263
316
.
Davis
,
B. D.
(
1958
).
On the importance of being ionized
.
Archs Biochem. Biophys
.
78
,
497
509
.
Eggleston
,
L. V.
&
Krebs
,
H. A.
(
1974
).
Regulation of the pentose phosphate cycle
.
Biochem. J
.
138
,
425
435
.
Flatt
,
J. P.
&
Ball
,
E. G.
(
1966
).
Studies on the metabolism of adipose tissue. XIV. An evaluation of the major pathways of glucose catabolism as influenced by acetate in the presence of insulin
.
J. biol. Chem
.
241
,
2862
2869
.
Glock
,
G. E.
&
McLean
,
P.
(
1958
).
Pathways of glucose utilization in mammary tissue
.
Proc. R. Soc. B
149
,
354
362
.
Gumaa
,
K. A.
,
Greenbaum
,
A. L.
&
McLean
,
P.
(
1973
).
Adaptive changes in satellite systems related to lipogenesis in rat and sheep mammary gland and in adipose tissue
.
European J. Biochem
.
34
,
188
198
.
Gumaa
,
K. A.
&
McLean
,
P.
(
1972
).
The kinetic quantitation of ATP: D-glucose 6-phospho-transferases
.
FEBS Lett
.
27
,
293
297
.
Gustafson
,
T.
(
1965
).
Morphogenetic significance of biochemical patterns in sea urchin embryos
.
In The Biochemistry of Animal Development
, vol.
1
(ed.
R.
Weber
), pp.
139
202
.
New York and London
:
Academic Press
.
Hicklin
,
J.
,
Hornbruch
,
A.
,
Wolpert
,
L.
&
Clarke
,
M.
(
1973
).
Positional information and pattern regulation in hydra: the formation of boundary regions following axial grafts
.
J. Embryol. exp. Morph
.
30
,
701
725
.
Hollmann
,
S.
(
1964
).
The pentose phosphate cycle
.
In Non-glycolytic Pathways of Metabolism of Glucose
, pp.
46
80
.
New York and London
:
Academic Press
.
Hornbruch
,
A.
&
Wolpert
,
L.
(
1975
).
Polarity reversal in hydra by oligomycin
.
J. Embryol. exp. Morph
.
33
,
845
852
.
Katz
,
J.
,
Landau
,
B. R.
&
Bartsch
,
G. E.
(
1966
).
The pentose cycle, triosephosphate isomerization and lipogenesis in rat adipose tissue
.
J. biol. Chem
.
241
,
727
740
.
Katz
,
J.
&
Wals
,
P. A.
(
1970
).
Effect of phenazine methosulphate on lipogenesis
.
J. biol. Chem
.
245
,
2546
2548
.
Katz
,
J.
&
Wood
,
H. G.
(
1960
).
The use of glucose C14 for the evaluation of the pathways of glucose metabolism
.
J. biol. Chem
.
235
,
2165
2177
.
Kline
,
E. S.
&
Waravdekar
,
V. S.
(
1960
).
Inhibitor of succinoxidase activity from Hydra littoralis
.
J. biol. Chem
.
235
,
1803
1808
.
Krebs
,
H. A.
&
Veech
,
R. L.
(
1969
).
Pyridine nucleotide interrelations
.
In The Energy Level and Metabolic Control in Mitochondria
(ed.
S.
Papa
,
J. M.
Tager
,
E.
Quagliariello
and
E. C.
Slater
), pp.
329
382
.
Bari
:
Adriatica Editrice
.
Lagunas
,
R.
,
McLean
,
P.
&
Greenbaum
,
A. L.
(
1970
).
The effect of raising the NAD content on the pathways of carbohydrate metabolism and lipogenesis in rat liver
.
European J. Biochem
.
15
,
179
190
.
Lowry
,
O. H.
,
Rosebrough
,
N. J.
,
Farr
,
A. L.
&
Randall
,
R. J.
(
1951
).
Protein measurement with the Folin Phenol reagent
.
J. biol. Chem
.
193
,
265
275
.
McLean
,
P.
(
1960
).
Carbohydrate metabolism of mammary tissue. III
.
Biochim. biophys. Acta
37
,
296
309
.
McLean
,
P.
,
Greenbaum
,
A. L.
&
Gumaa
,
K. A.
(
1972
).
Constant and specific proportion groups of enzymes in rat mammary gland and adipose tissue in relation to lipogenesis
.
FEBS Lett
.
20
,
277
281
.
Novello
,
F.
,
Gumaa
,
K. A.
&
McLean
,
P.
(
1969
).
The pentose phosphate pathway of glucose metabolism
.
Biochem. J
.
111
,
713
725
.
Pette
,
D.
(
1966
).
In Regulation of Metabolic Processes in Mitochondria
(ed.
J. M.
Tager
,
S.
Papa
,
E.
Quagliariello
and
E. C.
Slater
), pp.
28
50
.
Amsterdam
:
Elsevier Publishing Company
.
Pette
,
D.
,
Klingenberg
,
M.
&
Bücher
,
TH
. (
1962a
).
Comparable and specific proportions in the mitochondrial enzyme activity pattern
.
Biochem. biophys. Res. Commun
.
7
,
425
432
.
Pette
,
D.
,
Luh
,
W.
&
Bücher
,
TH
. (
1962b
).
A constant proportion group in the enzyme activity pattern of the Embden-Meyerhof chain
.
Biochem. biophys. Res. Commun
.
7
,
419
424
.
Rognstad
,
R.
&
Katz
,
J.
(
1966
).
The balance of pyridine nucleotides and ATP in adipose tissue
.
Proc. natn. Acad. Sci. U.S.A
.
55
,
1148
1156
.
Shrago
,
E.
&
Lardy
,
H. A.
(
1966
).
Paths of carbon in gluconeogenesis and lipogenesis
.
J. biol. Chem
.
241
,
663
668
.
Veech
,
R. L.
,
Raijman
,
L.
&
Krebs
,
H. A.
(
1970
).
Equilibrium reactions between the cytoplasmic adenine nucleotide systems and the nicotinamide adenine dinucleotide system in rat liver
.
Biochem. J
.
117
,
499
503
.
Veeger
,
C.
,
Der Vartanian
,
D. V.
&
Zeylemaker
,
W. P.
(
1969
).
Methods in Enzymology
, vol.
XIII
(ed.
J. M.
Lowenstein
), pp.
81
90
.
New York and London
:
Academic Press
.
Webster
,
G.
&
Wolpert
,
L.
(
1966
).
Studies on pattern regulation in hydra. I. Regional differences in time required for hypostome determination
.
J. Embryol. exp. Morph
.
16
,
91
104
.
Williamson
,
J. R.
,
Scholz
,
R.
,
Thurman
,
R. G.
&
Chance
,
B.
(
1969
).
Transport of reducing equivalents across the mitochondrial membrane in rat liver
.
In The Energy Level and Metabolic Control in Mitochondria
(ed.
S.
Papa
,
J. M.
Tager
,
E.
Quagliariello
and
E. C.
Slater
), pp.
411
429
.
Bari
:
Adriatica Editrice
.
Wolpert
,
L.
,
Hicklin
,
J.
&
Hornbruch
,
A.
(
1971
).
Positional information and pattern regulation in regeneration of hydra
.
In Control Mechanisms of Growth and Differentiation. Symp. Soc. exp. Biol
.
25
,
391
415
.
Wolpert
,
L.
,
Hornbruch
,
A.
&
Clarke
,
M. R. B.
(
1974
).
Positional information and positional signalling in hydra
.
Am. Zool
.
14
,
647
663
.
Zatti
,
M.
&
Rossi
,
F.
(
1965
).
Early changes of hexosemonophosphate pathway activity and of NADPH oxidation in phagocytizing leucocytes
.
Biochim. biophys. Acta
99
,
557
561
.