/. Embryol. exp. Morph. Vol. 33, 4, pp. 853-867, 1975
Printed in Great Britain
853
Positional information and pattern regulation
in hydra: enzyme profiles
ByNAJMA ZAHEER BAQUER1, PATRICIA McLEAN 1 ,
AMATA HORNBRUCH 2 AND L. WOLPERT 2
From the Courtauld Institute of Biochemistry, and Department of Biology as
Applied to Medicine, The Middlesex Hospital Medical School, London
SUMMARY
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.
INTRODUCTION
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
1
Authors' address: Courtauld Institute of Biochemistry, The Middlesex Hospital Medical
School, London WJP 5PR, U.K.
2
Authors' address: Department of Biology as Applied to Medicine, The Middlesex
Hospital Medical School, London W1P 6DB, U.K.
53-2
854
NAJMA ZAHEER BAQUER AND OTHERS
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 ah 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 & Biicher, 1962a; Pette,
Luh & Biicher, 19626; Pette, 1966; Bass etal 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.
METHODS
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 lO"4 M, NaHCO 3 10~3 M, Tris 10"3 M, CaCl2 10~3 M, MgCl2 10~* 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 /*g/ml for 24 h.
The mammalian tissues were obtained from albino rats of the Wistar strain.
Positional information and pattern regulation in hydra
855
The preparation of homogenates and estimation of enzymes were essentially as
previously described (Novello, Gumaa & McLean, 1969; Gumaa, Greenbaum &
McLean, 1973; Baquer, McLean & Greenbaum, 19736). 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 I), 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 /<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-HC1 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/*g atoms
glucose carbon/100 hydra.
The figures for mammalian tissues were obtained using Krebs Ringer
856
NAJMA ZAHEER BAQUER AND OTHERS
Table 1. Systems relationships in hydra- comparison with
differentiated mammalian tissues
Enzyme
Hexokinase
Glycolytic route
Phosphoglucose isomerase
Phosphofructokinase
Pyruvate kinase
Hydra
Lactating
rat
mammary Developing
gland
rat brain
Rat liver (14 days)
(1 day)
10
10
10
10
14
0-5
3-2
43
0-6
19
18
0-4
8
1-5
10
1-2
150
Lactate dehydrogenase
Pentose phosphate pathway and NADPH generating systems
1-6
0-8
G6P dehydrogenase
6PG dehydrogenase
0-4
1-9
Isocitrate dehydrogenase
71
10-8
Malic enzyme
<01
1-5
Tricarboxylic acid cycle and related enzymes
2-7
Citrate synthase
0-3
95
Malate dehydrogenase
191
Glutamate dehydrogenase
14
3
Glutamate-pyruvate transaminase
6-8
61
10
29
Glutamate-oxaloacetate transaminase
a-Glycerophosphate oxidase
003
0-3
Succinate dehydrogenase
0-64
0-35
4-8
27
10
8-2
0-4
0-9
0-5
2-3
0-2
1-3
0-2
2-1
35
—
0-7
3-5
—
—
11
45
0-7
0-4
4-7
007
018
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 transaminase. 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.
Positional information and pattern regulation in hydra
857
Table 1 (cont.)
Possible significance of specific proportion changes in hydra
Hydra
Rat liver
Mammary
gland
A. Generation and conservation of pyruvate
(i) Low conversion of pyruvate -> lactate
relative to glycolytic formation
LDH/PK
0-4
7-9
(ii) Low conversion of pyruvate —> lactate
relative to formation from alanine
LDH/GPT
0-2
25
(iii) High conversion pyruvate -» citrate
relative to glycolytic formation
CS/PK
0-8
002
(iv) High formation pyruvate from alanine
relative to glycolytic formation
GPT/PK
20
0-3
B. Transfer of reducing equivalents
(i) Normal cytosolic ^ mitochondrial
transfer via malate-aspartate shuttle
MDH/GOT
95
66
(ii) Raised a-glycerophosphate shuttle
aGPOX/LDH
2X10" 1
2xlO" 4
(iii) Depressed transhydrogenation
NADH -> NADPH ME/MDH
< 1 x 10~3
1 x 10"2
5-6
39
Brain
10
25
002
01
015
004
10
—
7 x 10"2
96
7xlO~ 3
4 x lO"3
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.
RESULTS AND DISCUSSION
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.
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-da.y 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
858
NAJMA ZAHEER BAQUER AND OTHERS
Head
PGI
MDH
Gastric
Foot
120
100
GLD
80
GOT
60
ICDH
40
o
(X
60
20 J
HK
10
L-i
LDH
.__JG6PD
"1PFK
PFKl
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.
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. (1962a, 6, 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,
Positional information and pattern regulation in hydra
859
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 glutamatepyruvate 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 transam inase/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 (Novello
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
860
NAJMA ZAHEER BAQUER AND OTHERS
(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 ah 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, Cascales, Teo & McLean, 1973 a).
The quotient of 7-6 for hydra is close to that for isolated liver cells (Baquer et
al. 1973 a) 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; G u m a a ^ o / . 1973).
Positional information and pattern regulation in hydra
861
Table 2. Alternative pathways of glucose metabolism in hydra
A. Conversion of specifically labelled glucose to "CO 2 :
m/tg atom glucose carbon/100 hydra/30 min/24°
14
(6)
35-6 ±3-5
[l- C]glucose
[l-14C]glucose +phenazine methosulphate
(5)
191 ±9
[3,4-14C]glucose
(1)
2-9
(2)
4-7
[6-14C]glucose
[u-14C]glucose
(1)
11-2
B. Relative contribution of pentose phosphate pathway
14
CO2 from [l-14C]glucose
14
CO2 from [6-14C]glucose
Hydra
7-6
Rat liver (isolated cells)
71
Lactating mammary gland (14 days)
15-6
Rat brain (1-day post partum)
21
C. Pentose phosphate pathway gradient in hydra 14CO2 from [l-14C]glucose:
m/ig atoms glucose carbon/mg protein/30 min/24°
[l-14C]glucose +
[l-14C]glucose phenazine methosulphate
Head
Gastric + budding region
Foot
(2) 5-4
(2) 7-2
(2) 121
25
66
50
Results are given as means ±S.E.M., figures in parentheses are the number of separate
experiments performed. The protein content of 100 hydra is 6mg. Values for isolated liver
cells are from Baquer et at. (1973 a) for rat mammary gland, from Glock & McLean (1958)
and for rat brain from N. Z. Baquer (unpublished observations).
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 NADP+ 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 m/ig 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
862
NAJMA ZAHEER BAQUER AND OTHERS
Table 3. Enzyme profile and gradients in hydra
Intact
hydra
Head
region
Enzymes
Gastric
region
Budding
region
Foot
Gradient
headfoot
milliunits/mg protein
Hexokinase
8-3 ±0-8
7-8 ±0-8
Glycolytic route
.107 ±26
Phosphoglucose
117±J9
isomerase
Phosphofructokinase
3-6 ±0-8
4-7 ±0-7
Pyruvate kinase
25 ±5
28 ±5
Lactate dehydrogenase 9-3 + 1-2 7-6±l-2
Pentose phosphate pathway
G6P dehydrogenase
12 8 ±2-9
5-2 ±0-5
10
6PG dehydrogenase
21 ±0-8
Tricarboxylic acid cycle and related enzymes
21 ±4
Citrate synthase
17 ±4
Isocitrate
55 ± 11
40 ±1
dehydrogenase
50
40
Succinate
dehydrogenase
Malate
743 ±104 648 ±110
dehydrogenase
2-1
1-9
a-Glycerophosphate
dehydrogenase
Glutamate
106 ±7
103 ±4
dehydrogenase
Glutamate-oxaloacetate 79±13
72±7
transaminase
33
53
Glutamate-pyruvate
transaminase
Protein/100 hydra
60 + 0-4
11 ± 0 1
7-4 ±0-6
8-8
9-9 ±1-7
0
98 ±13
0
+
106 ± 20
68
3-2 ±0-4
28 ±4
8-9± 1-8
3-2
31
6-4
2-8 ±0-2
27 ±4
14-6±2-8*
9-8±l-5*
101
2-8
19-8 ±5-4*
1-3
17 + 4
40±6
5-6
756 ±120
21
46
4-8
466
1-2
82 ±10
79
5-8
0
_
-
17 ±1
37±6
0
0
31
0
804 ±150
0
10
0
56±12*
+
80 ±16
110±12*
—
46
30
0
21 ±0-2
2-2
0-5 ± 0 1
The values are given as means ± S.E.M.; each value is the mean of four separate experiments;
where no S.E.M. value is given this is the mean of two separate experiments. Each experimental value was obtained from sonicated extracts from 100 segments of hydra. * Indicate
significant difference from the head region, P < 005.
The symbols in the column 'Gradient Head-Foot' signify that: 0, there is no change in
specific activity of the enzyme: + , that the specific activity of the enzyme is higher in the
head than the foot: - , that the specific activity is lower in the head than foot region.
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
Positional information and pattern regulation in hydra
863
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
864
NAJMA ZAHEER BAQUER AND OTHERS
Table 4. Effect of oligomycin on enzyme activities and
gradients in hydra
Intact
hydra
Head
region
Enzymes
Foot
Gastric
region
milliunits/mg protein
Gradient
head-foot
Hexokinase
Glycolytic route
Phosphoglucose isomerase
Phosphofructokinase
Pyruvate kinase
Lactate dehydrogenase
9-6
6-5
8-2
8-4
0
142
2-9
26
12-8
133
1-5
22
11-3
149
2-7
22
10-3
162
10
23
10-4
0
0
0
0
Pentose phosphate pathway
G6P dehydrogenase
16-5
4-8
12-2
17-4
13
45
5-9
921
107
113
14
40
6-2
826
83
117
0
0
0
0
0
0
51
37
0
Tricarboxylic acid cycle and related enzymes
Citrate synthase
26
17
53
34
Isocitrate dehydrogenase
7-2
4-3
Succinate dehydrogenase
942
815
Malate dehydrogenase
102
78
Glutamate dehydrogenase
100
100
Glutamate-oxaloacetate
transaminase
Glutamate-pyruvate
45
29
transaminase
(i) Effect of oligomycin on relative activity of mitochondrial and
cytosolic redox systems
GLDH/LDH Control
Oligomycin
111
7-9
13-5
6-9
9-3
10-3
3-8
7-9
+
0
(ii) Effect of oligomycin on relative activities of pentose phosphate
pathway and glycolysis
G6PDH/PFK Control
3-6
1-2
31
71
Oligomycin
5-7
3-2
4-5
17-4
The values are the means of two separate experiments; each experimental value was obtained from sonicated extracts of 100 segments of hydra treated with oligomycin 10/*g/ml
medium for 24 h. The change in gradient from head to foot is indicated by the symbols
0, + , - as given in Table 3.
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.
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).
Positional information and pattern regulation in hydra
25 r
865
- i 250
I
5c
Q
3
SC
Q
-
HGF HGF
Control Oligo
HGF HGF
Control Oligo
HGF HGF
Control Oligo
HGF HGF
Control Oligo
Lactate
. dehydrogenase
Glutamateoxaloacetate
transaminase
Glucose 6phosphate
dehydrogenase
Glutamate
dehydrogenase
50 g
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 /*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 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.
866
NAJMA ZAHEER BAQUER AND OTHERS
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MCLEAN,
{Received 3 August 1974)
5+
EMB
33