Enzymes of glucose catabolism in hydra

AHCHIVICY

OF

BIOCHISMISTRY

.\ND

Enzymes II. Application

133, 128-136 (1969)

of Glucose

of Microfhorometric

CHARLES Laboratory

HIOPHYSICS

Analyses

I,. RUTHERFORD*

for Quantitative Received

Biology, October

Catabolism

AND

University

in Hydra

to Patterns of Enzyme Localization’ HOWARD of Miami,

1, 1968; accepted

M. LENHOFF”

Coral Gables, Florida

83l2.j

May 4, 1969

The localization of two NADPH-producing enzymes was studied in Hydra littoralis by adaptation of the microfluorometric methods of Lowry et al. Individual hydra were frozen, dried, and dissected into 3&50 sections weighing from 0.1 to 0.8 pg dry weight, and were assayed for specific activity. In nonbudding, monopolar hydra glucose 6phosphate dehydrogenase (GGPDH) was 2@--K10~~more active in the areas of the hypostome and base than in the mid-portion of the animal. Conversely, isocitrate dehydrogenase (ICDH) activity was ZCrZOO% higher in the mid-portion than at the hypostome or base. A similar distribution of GGPDH and ICDH was found in hydra during the initiation of bud development. Likewise, this same distribution of GBPDH occurred in a bipolar mutant hydra, but, ICDH in this animal was not localized.

Hydroids, because of their rigid polarity and simple structure, have been used as a model system to study the relation between the localization of metabolic activity and patterns of development. These studies led to the metabolic gradient hypothesis which states that development is initiated and controlled by the region of an organism having the highest metabolic rate (1). However, whether or not metabolic gradients actually exist is still questionable because the methodology used to demonstrate gradients may contain analytical artifacts. Most of the studies on gradients have utilized histochemical assay (2, 3), susceptibility to 1 Supported by a grant from the Nutrition Foundation, New York, Grant GM 5491 from NSF, and Grants GM 12779 and 5-TOl-HD00187 from the USPHS. 2 Predoctoral fellow of NIH Training Grant 5-TOl-GMO0649. Present address: Retina Foundation, 20 Staniford Street, Boston, Mass. 02114. 3 PHS research career development award GM5011. Present address: School of Biological Sciences, University of California, Irvine, California 92664.

poisons (4) or oxidation and reduction of dyes (1). We are particularly interested in localization of enzymes, substrates, and coenzymes in hydra, a simple developmental system possessing a rigid polarity. In this paper we (a) adapt the ultramicrochemical methods described by Lowry (5) to the study of enzymes in hydra, (b) describe properties of hydra glucose 6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) and isocitrate dehydrogenase (ICDH, EC 1.1.1.42), (c) show the localization of these two NADPHgenerating enzymes in hydra, and (d) discuss their relation to reducing gradients and developmental processes. METHODS

AND

MATERIALS

Culture methods. Asexually reproducing Hydra littoralis (Hyman) were grown according to the mass culture methods of Loomis and Lenhoff (6) as modified by Lenhoff and Brown (7) in a culture solution of 10d3 M CaClx and 10s4 M NaHC08, pH 7.4. Clones of a mutant of Chlorohydra viridissima were grown as previously described (8). Hydra were fed freshly hatched -4rtemia nauplii once 128

ENZYMES

OF GLUCOSE

daily; on the day of the experiment they were not fed. Animals at specific stages of development were selected at random from the cultures. Budding animals were selected when buds were in the early stages of their development, and the bud tentacles had not yet begun to form [called “uniform hydra” in Lenhoft’ and Bovaird @)I. Preparation of sumples for assay. Hydra were prepared for enzyme assays by modifying the ultramicrochemical techniques developed by Lowry et al. (10). Hydra were frozen with liquid Freon-12 (Virginia Chemicals Inc., West Norfolk, t’irginia, 23501) which was cooled in a dry ieeacetone bath. The freezing chamber was 1 X 1 X f4 in. section of Plexiglas wit,h a M-in. diameter hole in the center attached to a 1 X l-in. section of a glass microscope slide. A drop of cult,ure solution containing a single hydra was placed on the microscope slide in the center of t,he hole. After the hydra relaxed to its normal lengt,h, the cold Freon was poured over the entire holder, freezing the hydra in less than 1 sec. Several of these holders were inserted into a drying tube and were placed under vacuum for 12 hr at -20”. After allowing the drying tubes to reach room t,emperatllre, the dry samples were removed to 2 X 4-in. glass slides for dissection. From these dry hydra of approximately 30 rg dry weight, bits of tissue from 0.1 to 0.6 pg were dissected. Cutting was carried out free-hand under a dissecting microscope. By cnreful dissection one hydra can be cllt into as many as 50 pieces. Measurement of dry weight. Specific enzyme activity was expressed per unit dry weight as measured by a quartz “fish pole” balance (11). The balances used varied from a sensitivity of 2.1250.0 pg per cm deflection of the quartz fiber. Once dissected and weighed, the dry tissue was used immediat.ely and was not refrozen. The freezedrying, dissection, and enzyme assays were all done within a 24.hr period Assay proc&ure. xach weighed section was placed in a 3 ml-Pyrex, conical-tipped centrifuge tube to which 15 ~1 of a reaction mixture were added. The reaction mixture for GGPDH contained 1 ml 0.1 M AMP buffer (2-amino-2-methyl-l,3 propanediol) (pII 9.0), 1 mM EDTA, 10 X~M MgC12, a mM G6P, and 0.1 mM NADPf. The reaction mixture for ICDH contained 1 ml 0.1 M Tris-HCl buffer (Sigma Chemical Co., St. Louis, Mo.)(pH 8.2), 0.01% BSA. 1 mM MnC12, 1 mM isocitrate, and 0.1 mM NADP+. The 3-ml conical-tipped tubes provided a convenient reaction vessel since sect.ions of hydra were easily placed through the large portion of the tube, yet the small reaction volume filled the tip of the tube. After incubation for 30 min at 37”, the tubes were placed in an ice bath, and 1 ml of

129

CATABOLISM

0.02 M carbonate-bicarbonate buffer (pH 10.0) was added to stop the reaction. The contents were transferred to a 3-ml Pyrex tube (10 X 75 mm) and the fluorescence due to NADPH formation was measured in a Farrand model A-3 fluorometer (Corning No. 5840 filter for t.he incident light and No. 3387 plus No. 4308 filters for the emitted light). Specific act,ivities of GGPDII and ICDH were expressed as moles NADP+ redaced/hr/kg dry tissue weight. After we recorded its original location on the animal, each section was placed in a separate rea&ion t.ube. The order of the tubes was then arranged randomly and assayed. RESULTS

Properties of enzymes. The characterization of GGPDH and ICDH was essential in order to determine the conditions that would give the maximum reaction rates. The K, of GGPDH for GBP and NADPf as determined by the Lineweaver-Burk plot (Fig. 1) was 3.05 X low4 M and 9.96 X 1O-5 WI respectively. The KS of ICDH for isocitrate was 2.5 X 10e4M and for NADPf ‘;vas 5.5 X 10-S M (Fig. 2). The Lineweaver-Burk plots (Figs. 1 and 2) showed further that there was no substrate or end product inhibition at the concentrations used in the enzyme assays. The optimum pH for ICDH activity was near S, while GGPDH had two optima, one at S and another at 9.2 (Fig. 3). The reaction rate of GGPDH and ICDH was linear over the 30-min incubation period. Therefore, we conclude that over this period the enzymes are stable and the concentrations of reagents are sufficient to maintain maximal activity. In addition, a single assay of the amount of NADPH produced by either enzyme at 30 min was a valid measurement of reaction rate. Relation between tissue size and erLzyme activity. By using tissue sections from 0.1 to 0.S /*g dry weight in a reaction volume of 15 ~1, the rate of NADPf reduction by GGPDH and ICDH was proportional to the dry weight of hydra tissue (Fig. 4). Therefore, the specific activities of tissue samples in this range reflected the true enzyme activities of the tissue. If, on the other hand, we used larger bits of tissue, from 2 to 5 pg dry

130

RUTHERFORD

AND LENHOFF

8$)G6P -4 6'/"

'1" -2

0

I

I 20

10

0

0

I 3

I 6

I 9

12O

'A

FIG. 1. Lineweaver-Burk plot of G6PDH from H. littoralis. The velocity of reduction of NADP+ (moles/min X 1W) is plotted as a function of the concentration of (a) G6P (moles X IO”), (b) NADP+ (moles X lo?). Saturating levels of G6P were 1 X 10e3 M; of NADP+ were 1 X 10-b M. Ten micrograms of hydra homogenate were used in all experiments.

0

I 0

4

I

a

I

I

12

16

I 0

3

I 6

I 9

12"

l/S

2. Lineweaver-Burk plot of ICDH from H. Zittoralis. The velocity of reduction of NADP+ (moles/min X leg) is plotted as a function of the concentration of (a) isocitrate (moles X lo’), (b) NADP+ (moles X 107). Saturating levels of isocitrate were 1 X 1W3 M; of NADP+ were 1 X 10-J M. Ten micrograms of hydra homogenate were used in all experiments. FIG.

weight, and a smaller reaction volume (5 /.A), the enzyme activities were not proportional to the dry weight of the tissue. Hence, in order to get valid and comparable specific activities, it was essential to use the conditions of Fig. 4. Localization of enzyme activity in nonbudding, monopolar hydra. Tables I and II show the distribution of G6PDH and ICDH activities along the vertical axis of hydra body columns. The first specific activity given in each column represents that of the hypostome; each subsequent value corresponds to the specific activity of the enzyme in the

next lower portion of the animal. G6PDH was 20-100 % more active in the area of the hypostome and base than in the mid-portion of the hydra. Conversely, the specific activity of ICDH was 20-200 % higher in the midportion of the animal than at the hypostome or base. In most hydra analyzed, the ICDH activity of the hypostome was lower than in any other part of the animal. The distribution of enzymes as described above was verified using a larger number of animals (Tables III and IV). In these experiments, enzyme activities of hypostomes and bases were compared with those of two sec-

ENZYMES

OF GLUCOSE

131

CATABOLISM TSBLE

-15

10 T

y 2

a-

c ;

6--

LOCa-of tion -10

;

z >

tissue samples

Specific 1

activity 2

dumoles/hr/mg 3

dry 4

weight) 5

6

; uQ

4-

0 ;

ACTIVITY

G

g

2

I

I,OC.~LIZ:YTIONOF GGPDH IN H. littoralis

-5

2-

;

2 v) 0

LL

I 9

I a

7

I 10

: v) -0

P”

3. Effect of pH on specific activities of GGPDH and ICDH from H. littoralis. Specific activities of both enzymes are in pmoles/hr/mg protein. Between pH 7.2 and 8.8 the buffer was 0.05 M-Tris-HCl and from pH 9.2 to 10.7 was 0.05 M-carbonate-bicarbonate. FIG.

I-

O

02

04 DRY

06

WEIGHT(/LGI

FIG. 4. GGPDH and ICDH activity in sections of hydra tissue of increasing dry weight. Tissue samples were weighed on a quartz fiber balance as described in Methods. NADPH concentration is expressed as moles X log. The dots indicate the GGPDH activity, the triangles, the ICDH activity.

tions from the mid-portions of the animals as shown in the drawing alongside Tables III and IV. The samples were in the same weight range (approximately 0.6 pg) as those samples described in Tables I and II. By sampling only the four critical areas of the hydra, however, rather than sectioning the entire animal, it was possible to get 10 replications of each area using hydra that

10.70 7.14 9 10.15 6.91 6.11 4.87 5.19 7.95 4.81 5.32 5.72 5.83 4.89 5.55 5.36 5.23 5.83 4.59 4.96 6.27 7.50 7.84

12.25 11.13 11.42 11.63 12.17 9.35 9.55 9.86 9.27 9.89 8.40 8.87 9.65 8.83 8.44 8.75 11.27 11.00 11.13

7.65 5.48 5.77 5.42 5.61 5.45 5.70 6.10 5.61 5.31 5.53 5.09 5.21 5.73 5.08 5.25 7.39 6.48 5.80 7.14 8.95 8.82

6.08

7.26

6.88

6;9 6.77 G.39 5.79 5.51 5.63 5.89 5.13 4.95 5.09 5.14 5.48 5.G9 5.19 0.07 7.96

:::: ;:;: 5.44 6.30 5.17 5.69 4.13 5.96 5.20 6.02 4.77 6.08 5.31 4.29 5.10 3.44 5.32 5.83 5.G7 5.60 5.26 5.45 5.52 5.77 5.45 5.81 5.32 5.66 5.02 5.62 4.8G 4.74 4.67 5.89 5.SG 5.24 6.34 G.19 8.17 F.84 8.54 9.32

were taken from the same mass culture at the same time, and assayed the same day. The specific activities in Tables III and IV were normalized in order to test the significance of differences between the activities of the hypostome, base, and mid-sections. All normalized activities were expressed in relation to the lowest activity of each replication which was set at 1.00. The GGPDH activities of both the hypostome and base were significantly different (p < 5 70) when tested by analysis of variance from the activity of either mid-section. There were also significant differences between the ICDH activity of the base and either mid-section, and between the activity of the hypostome and the mid-section nearest the base (p < 5 70). The differences between the activities of the midsections for either GGPDH or ICDH were not significantly different (p < 5 “;). The distribution of G6PDH and ICDH in Tables

132

RUTHERFORD TABLE

II

LOC.~LIZ.~TION OF ICDH H. littoralis

ACTIVITY

IN

Specific activity (,moles/hr/mg dry weight)

Location of tissue

t

AND LENHOFF

2.51 3.50 3.53 3.97 3.58 3.44 3.54 3.92 3.83 4.10 3.77 3.96 4.09 4.16

3.G5 4.92 4.57 4.04 3.72 6.07 2.96 3.92 5.48 6.28 5.75 5.01 6.33 6.80 6.86 5.41 8.16 3.79 4.84 4.05 4.54 4.34 4.88

4.29

5.93 6.50 6.67 5.96 G.30 6.72 G.23 7.45 5.40 0.68 5.83 G.G9 6.94 7.32 6.38 6.89 5.92 4.30 4.35

3.98 4.11 3.95 7.11 4.22 3.68 3.77 3.55

2.46 3.00 5.27 5.35 5.51 5.94 5.42 4.76 6.55 5.35 5.20 7.07 4.15 7.05 7.65 7.77 8.24 7.84 6.34 4.25

2.55 1.93 2.07 2.94 5.19 4.94 5.40 6.10 6.30 6.16 6.85 6.76 6.75 6.59 6.90 6.87 5.52 4.94 5.62

III and IV was the same as the distribution of specific activities described in Tables I and II; that is, GGPDH was more active at the extremes of the body column and ICDH was more active in the mid-portions. Localization of enzymes during the initiation of bud developme?zt.Preliminary to a detailed study in the distribution of GGPDH and TABLE COMP~HISON OF GBPDH Location of tissue sections

0 Normalized set at 1.00.

ACTIVITY

ICDH in hydra in different stages of development, we looked at the distribution of the specific activities of the enzymes in hydra having a bud in the earliest stage of development (Table V). The results show that the high activity of GGPDH and the low activity of ICDH found at the poles of the nonbudding, monopolar hydra (Tables I, II, III, and IV) are also retained by the animals during initiation of the budding process. In addition, the enzyme activities in the region of bud differentiation did not differ significantly from those of the surrounding tissue. Whether or not differences in enzyme activities occur during the entire sequence of bud development will be the subject of another study. Distribution of enzyme activity in a monopolar mutant hydra. The localization of NADPH enzymes was also measured in a mutant hydra (Chlorohydra viridissima, without algae) (8) containing a head at both ends (Table VI). The distribution of G6PDH in such bipolar mutant animals (Table VI) was similar to that found in the normal C. viridissima (without algae) and in H. littoralis (Tables I and III; i.e., the highest activity occurring at the extremes of the body column). The ICDH activities in the mutant, however, did not follow the pattern found in normal monopolar animals. Instead, there was more random distribution of activities without any apparent localization. III

FROM SELECTED AREAS ALONG THE BODY AXIS OF HYDRA Specific activity (normalized)a

1

2

3

1

5

6

7

8

9

10

MWlTl

1.24 1.00 1.08 1.65

1.42 1.23 1.09 1.49

1.61 1.17 1.00 1.55

1.11 1.01 1.00 1.88

1.18 1.00 1.07 1.61

1.13 1.00 1.25 1.48

1.29 1.00 1.08 1.57

1.09 1.00 1.02 1.38

1.34 1.07 1.00 1.72

1.45 1.00 1.05 1.21

1.29 1.05 1.05 1.55

activities

were expressed

in relation

of the lowest

activity

of each replication

which was

ENZYMES

OF GLUCOSE TABLE

COMPARISON OF ICDH Location of tissue

sections

ACTIVITY

133

CATABOLISM IV

FROM SELECTED AREAS ALONG THE BODY Specific

~.

activity

AXIS OF lI~01t.t

(normalized)n

2

3

4

5

6

7

X

9

10

Mean

1.00 1.35 1.70 1.07

1.33, 1.32 1.73 1.00

1.25 1.43 1.49 1.00

2.34 2.98 3.02 1.00

1.25 1.33 1.70 1.00

1.00 1.48 1.69 1.01

1.14 1.16 1.72 1.00

1.00 1.48 1.98 1.07

1.04 1.31 1.43 1.00

1.24 1.51 1.83 1.02

1

I

1.08 1.63 1.80 1.00

I

a Normalized activities was set at 1.00.

were expressed

in relation

TABLE LOC.~LIZATION OF GGPDH Location of tissue sections”

x

GhPDH 1

rk~~ ICDH

specific

2

to the lowest, act,ivity

of each replication

1’

IN HYDR.~ DURING E.\RLY BUD

activity’

Location of tissue sections”

3

which

ICDH ~---

1

DEVELOPMENT specik

activity’

2

3

13.39

12.17

12.35

4.76

3.46

4.62

14.33 15.82 14.92 10.26 11.86 11.10 10.79 12.18 11.73 10.74 12.50 12.83 11.79 12.99 12.45 11.71 10.70”

12.17 9.89 8.12 8.00 8.53 8.22 8.82 8.65 8.49 9.52 7.77 7.80 7.73 8.20 8.11” -

12.35 9.80

4.76

3.46

3.86 4.97 5.35 5.7V 4.62 5.78 6.57 6.51 6.73 6.03 G.43 5.61 5.01 5.32

11.05

10.32

11.37

a The location of tissue sections are indicated b Specific activity is expressed as rmoles/hr/mg c Area of developing bud.

by the numerals dry weight.

DISCUSSION

In this paper we have described: (a) the application of microfluorometric methods to the characterization of two enzymes producing NADPH in hydra, and (6) the adaptation of the micromethods developed by Lowry et al., (10) to the analysis of enzymes in specific morphological areas of this small metazoan.

in the figure at the left of each column.

Characterization of enzymes. To date, few enzymes from coelenterates have been characterized, [see Hammen, (12)]. The properties of hydra enzymes are of special interest to comparative biochemistry because coelenterates are thought to occupy a pivotal phylogenetic position in the evolution of multicellular organisms. Table VII compares the K,, pH optima, and specific

134

RUTHERFORD

AND

TABLE OF G6PDH

DISTRIBUTION

activity

ICDH specific activitya 1

2

3

7.07 5.93 4.98 4.81 4.51 3.79 4.22 4.16 4.02 4.46 4.51 4.15 5.18 3.54 3.81 4.55 4.22 4.46 3.75 4.07 4.49 4.39 6.65

9.81 8.25 6.61 6.77 -

9.81 7.35 6.21 6.62 5.43 5.25 -

0.86 0.99 0.86 0.88 0.72 -

1.03 1.11 1.04 0.66 0.71 -

0.76 0.78 0.95 0.88 -

1.22 0.71 0.94 0.66 -

0.98 1.00 0.86 1.38 1.00 -

5.81 6.15 6.17 -

6.32 6.42 6.23 5.62 6.24 5.62 5.84 5.46 -

6.64 6.21 6.25 5.68 6.32 5.99 6.34 5.69 5.67 6.10 5.57 7.36 8.43 as rmoles/hr/mg

OF GGPDH

AND

1.01 1.12 1.12 1.26 1.02 -

0.90 0.59 0.64 0.70 0.82

0.92 0.83 0.96 1.10 0.94

0.81 0.83 0.87 0.88 1.00 0.94 0.73 0.70 0.69 0.67 0.95

VII

ICDH

FROM VARIOUS

NADP+

SOURCES

pH Optima

1.2 x 10-S 2.6 X lW6 M

2.0 x

10-C

7.5-8.5 7.0-7.7

2.5 X lO+

5.5 x

10-C

8.0

Specific activity

11 moles/hr/k& 3.5 moles/hr/kgd

NADP+

3.5 x 10-s 4.0 x 10-5 1.3 x 10-s

3.08 x

1.08 0.89 0.83 0.90

dry weight.

Km Isocitrate

10-b

E. A. Noltman and S. A. Kuby, (18). 0. H. Lowry, (5). c From A. M. Burt and B. S. Wenger, (19). d Dry weight. b From

0.85 0.99 0.78 0.80 0.98 1.00 -

6.18 5.43 4.16 5.87 7.00 7.82 8.63

G6P

GGPDH Human erythrocytes” Yeast” Rat liver” Rat brain* Chick spinal cordc Hydra

OF

3

PROPERTIES

ICDH Guinea pig hear? Hog hearta Rat brainb Hydra

HYDRA

2

is expressed

Enzyme and source

MUT.~NT

1

TABLE

a From

VI

ICDH ACTIVITIES IN A DIPOL~R C. Viridissima (without algae)

AND

G6PDH specific activity’

Location of tissue sections

~1Specific

LENHOFF

4.2 X W6 2.0 x 10-b 1.3 x 10-b

9.9 x

10-b

8.1-8.6 7.7-8.6

and 9.0

8.0 and 9.5

1.8 moles/hr/kgd 1.3 moles/hr/kgd 7.3 moles/hr/kgd

ENZYMES OF GLUCOSE CATABOLISM activities of enzymes from hydra and from several other organisms. The K, values show that the hydra enzymes have less affinity for their subsbrates than do enzymes from the other tissues. These high K, values of hydra enzymes for their substrates, and in the case of GGPDH, for authentic NADP+, may support the view of Kaplan et al., (13) that the molecular forms of some enzymes from invertebrates differ from those of vertebrates. In contrast to the differences in K, between the hydra and vertebrate enzymes, the optimum pII for ICDH from hydra was similar to that of guinea pig heart (pH = 8). Similarly the GGPDH from hydra, like GGPDH of rat liver, had two pH optima. Despite the large K, values for GGPDH from hydra, the animal was rich in the enzyme, hydra being 6-fold greater in specific activity than either rat brain or chick spinal cord. This high activity of GGPDH is particularly interesting because of the absence in hydra (14) and other hgdroids (15) of the second enzyme of the hexose monophosphate pathway, GPGDH. Application, of Lo%wy micromethods to study localization of enzymes in a small metazoan. The micromethods used in this report were originally developed by Lowry et al., (10) for the study of the localization of enzymes, substrates, and pyridine nucleotides in various regions of mammalian brain. In those studies, pieces of tissue of approximately 50 mg wet weight were frozen, and microtome sections were prepared at 5-20 p in thickness. These sections were of uniform size, and when placed in a reaction mixture were thin enough to allow the enzymes to diffuse out. However, frozen hydra were difficult to section in a similar manner because of the lack of a mounting media which (a) could be instantly frozen, yet would not cause contraction of the animal, and (b) after lyophilization would leave no residue on the tissue sections which would add inert material to the dry weight of the tissue. If these analytical problems could be solved, the sampling techniques for hydra and similar organisms would be greatly simplified.

135

Because no satisfactory microtome sections of hydra could be obtained, we attempted manually to dissect tissue samples from frozen-dried animals. However, the first experiments were unsuccessful because we used tissue samples weighing l-5 pg for analysis in li-~1 reaction mixtures; the enzyme activities of such sections were not proportional to the dry weight of the tissue. Because specific activities of the smaller sections (ca. 1 pg) were 2 to 3 times higher than those of the larger sections (ca. 5 pg), we modified the enzyme assay by using smaller sections (0.1-0.8 pg) in a larger reaction volume (15 ~1). Samples of this size were small enough to release enzymes to the reaction mixture, yet generated enough NADPH during the 30-min incubation period to measure fluorometrically. Localization of NADPH-pyoduciny enzymes. Measurement of specific activity from extracts of whole animals, as usually is the case when studying small organisms, masks the effects associated with localization of the enzyme within definite regions of that organism. Even measurements made on homogenates of hydra dissected into 4 parts (8-10 pg dry weight each) did not show any localization of GGPDH or ICDH (p < 5 %). That the enzymes were localized in the hypostome and base was evident only after single organisms were dissected into 30-50 sections (Tables I-VI). Differences found in the specific activities of enzymes taken from different parts of a hydra are reminiscent of the classical metabolic gradients first described by Child and Hyman (4). They proposed that development was initiated and controlled by the region of an organism having the highest metabolic rate. The gradient theory, first proposed from experiments in regenerating Tub&aria, was supported by later experiments in which hydra showed an axial gradient in their susceptibility to poisons (4), and their ability to reduce methylene blue that had previously been taken up by their cells (1). In addition, the oxygen consumption of isolated pieces of living Tubu-

136

RUTHERFORD

laria indicated an apical to basal gradient of activity (16). The analytical methods used to gather information which supports the gradient theory are qualitative and fraught with analytical artifacts. For example, the results obtained by the use of dyes and poisons may be partially caused by the differential permeability of specific areas of the animals to those substances. Furthermore, differences in respiration observed in small sections of living tissue may result from increased respiration associated with a wound response to the cutting procedure. The methods used in this report overcome these artifacts since animals are frozen and then dried, thus stabilizing enzymes and substrates. Likewise, the actual maximum enzyme activity can be measured and expressed quantitatively. Our results show that the NADPHgenerating enzymes are localized in hydra, with GGPDH particularly high, and ICDH low, in the hypostome and base regions. Hence, specific areas along the body column have the potential for producing NADPH at different rates. For such a potential to be realized, however, the level of substrates and coenzymes must be present at saturating levels in all regions of the animal. Thus, differing rates of NADPH production along the body column could occur regardless of any enzyme localization, depending upon the concentration of substrates and coenzymes n each area of the hydra.

AND

LENHOFF REFERENCES

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