Succinic acid dehydrogenase activity in the gill epithelium of euryhaline fishes

ht. J. Bkdem.,

1970, I, 129-138

SUCCINIC GILL

129

ACID DEHYDROGENASE ACTIVITY IN THE EPITHELIUM OF EURYHALINE FISHES FRANK

P. CONTE

AND MARTHA

Department of Zoology, Oregon State Univmity, (Ratbed

20

J.

TRIPP*

Corvallis, Oregon 9733x, U.S.A.

May, xg6g)

ABsT%4cT I. Mitochondria were isolated from the gill filaments of chinook salmon (Oncorhyndurr ts~tsha), starry flounder (Pkwoncctcs stdatus), and staghom sculpin (Lapt0cDttu.t mmatuc) hy difFerential centr&gation. 2. The succinate+zytochrome c-rcductase activity from resuspended mitochondria was assayed. vmu for salt-water-adapted fish was found to he 3.5 for chinook salmon, 5. I for starry flounder, and 8.0 for staghom sculpin. In contrast, Vmu for freshwater-adapted fish was 4.8 for chinook salmon and 4. I for starry Sounder. No v+es were obtained for staghorn sculpin 3. Qu;mtitation of the mitochondria-rich cell population found in the gill filament was attempted. No significant dif%rencu could lx found for chinook salmon which were either in steady-state conditions or undergoing transitory exposures.

uLTRAsTRucTuRAL

studies ofgill

fiimxntsin

several species of euryhaline fishes have shown the presence of large numbers of mitoehondria-rich (MR)t cells in the cell population (Copeland, 1%~; Philpott and Copeland, 1963 ; Threadgold and Houston, 1964). These MR cells are considered to be the sites for salt secretion. An autoradiographic investigation of the filamental epithelium has shown that cell renewal is principally found in the region where the MR cells are located (Conte and Lin, 1967). When fish are adjusting from fresh to seawater there is rapid turnover of cells in the epithelium and their ability to stuvive in the

hyperosmotic environment is dependent upon this nplicative activity. Should this mitotic activity be inhibited either by X-radiation (Come, I 963) or by actinomycinD (Maetz, I g6g), there is loss of the ability to secrete salts and death will ensue. Few enzymatic changes have been reported which s Present address: Department of Chemistry, Florida State University, Tallahassee, Florida, U.S.A. ? Often referred to as the ‘chloride cell ‘.

correlate with these cellular events. The most consistent enzymatic change reported following adaptation to sea-water is the increase in Na-K-activated ATPase (Epstein, Katz, and Pickford, 1967; Kamiya and Utida, 1968; Zaugg, 1968). However, an earlier immunochemical study (Come and Morita, 1968) had shown that adaptation to sea-water causes an increase in several cellular proteins. Thus, the Na-K-activated ATPase may very well be one of these changes, but the others have not been identified. The purpose of the present study v as to pursue the problem of enzymatic adapl Ition during changes from fresh- to sea-wateA. In particular, the investigation was to determine if:I. Adaptation to sea-water would induce changes in a mitochondrial enzyme system, such as succinatexytochrome c-reductase. 2. Adaptation to sea-water would produce alterations in the MR cell population. LW~TERIALS AND h@IFIODS ExPERIsmNT~~ Juvenile spring chinook salmon, Onwrhphus tshawytshu, were reared from eggs to the juvenile

CONTEANDTRIPP

130

at the Oregon State Game Commission Fisheries Research Laboratory. Starry flounder, Pleuromctm stallatw, and staghom sculpin, Lrptocottus amatus, were captured by otter trawl in Yaquina Bay; Oregon. -All fish wUC maintained at the aauaria facilities at Orewn State Universitv. Wate; temperature was I o k ?’ C. and salinity was maintained at 307& in the closed-circulating sea-water system during the entire experiments. During storage the sea-water was condm~ally recirculated and filtered through diatomaceous stage

-L

CHINOOK SALMON

61

Int. J.

Bioch.

I : 10,000 w/v). The cokted blood was stored and diluted with cold filtered saline in a prechilled heparimzed test-tube. Analyses were made within I o minutes from the time of the blood collection. at

k4OLATION OF ~OXWUBONUCLEXC Aom (DNA)

The nucleoprotein fraction from both the homogenized gill brei and red blood-cells was isolated and analysed for the DNA content as previously reported (Conte and Lin, 1967). The method of addition techniaue (lovner and Filev. 1g66) was used to dcterm&e the’ amount of Dti;d; loss during isolation procedures. Salmon sperm DNA was the standard both for the spectrophotometric analysis and the method of additions. I~~M~ION OF M~T~~HONDSUAL F~cIIoN

i

STARRYFLOUNDER 1r9sNllohrs )

._ ‘; 20 f s 3 1.6 &

FIG. I.-H dependence of succinat+-cytochrome c-reductase for chinook salmon, 0. tstiazuyts~ and starry flounder, P. st&atur.

earth and calcium carbonate. 0xygen level measured in fresh water and sea-water was 1~6 and 7.3 p.p.m., respectively. Fish were fed daily either

choobed

frozen

souid

or wmmercial

t&h

pellets andphotoperiod was controlled so as to be nearly that of natural daylight hours. Ia OifrOCELL POPULATION Blood-cell population was determined by counting the cells in a particle counter.* A sodium -&oride saline solution (og per cent) was filtered three times through a o4 p Millipore membrane and used as a diluent i!or the blood. Aliquots of blood were collected by severing the caudal peduncle from anaesthetized fish (MS-222 l Coulter Particle Counter, Coulter Electronics, Florida, U.S.A.

The isolation of intact mitochondria from gill filaments was as follows:a. Gill fJaments were cxckd and cxccss biood removed by flushing with cold homogenizing

medium. b. The homogmizing medium consisted of 0.3 M sucrose and 0.01: M Tricene buffer at PH 7.4. . *. and was used in a tissue to medium ratio of I : IO (w/v). c. The tissue was homogenized for I minute in a Potter-Elvehjem glass-Teflon homogcnizr that was kept cold in an ice-bath. d. The total volwne of the homogenized brei was measured. At this time, two aliquots of brei were removed for assay of total DNA and total cellular protein. The remaming portion of the brei was set aside for differential centrifi~gation. c. The homogenate was centrif&sd at 755 g for 10 minutes to remove cell nuclei and cartilaginous debris. The remaining supematant was c&tIifuged at 12,100 g for 15 -minutes to obtain the mitochondrial pellet. The nellet was resuspended in a m&ured volume’ of hom+ g&zing media. Precise aliquots of the suspension were removed for rnitochondrial enzyme and mitochondrial protein assay. f. Three rnitochondrial enzyme reactions were examined: succinattcytodvo me c-reductase as based upon a moditiation of the procedure by Green and Ziegler (1963)) nicotinamide adenine diphosphopyridine nucleotide (NADH)-cytochrome c-reductase based on the procedure of Mahlcr (Ig55), and Bhydroxybutyrste dehydrogenase as reported by Lehninger, Sudduth, and Wise (rg6o). Enzyme activity was not stable for NADH-cvtochrome c-reductase svstem and there was little @-hydroxybutyrate . dehydmgenase activitv found in the niu mitochondria. However. succin&cytochrom< c-reductast was observed to be both stable and quite active when contrasted with a liver mitochondrial preparation. This mitochondrial system became the preferred enzyme assay. All reaction velocities were measured

SUCCXKIC ACID

*970, 1

~~c~ophotome~~y ti a GX5rd rccading spectrophotomcter. g. Protein content was asayed by using the Lowry technique for determination of soluble proteins (Lowry, Rosebrough, Farr, and Randall, 1951). RESULTS KINETICS OF &XINATE -cYrOcwRoME

c-

REDUCTASE

Fig. I describes the pH dependence of the succinate-cytochrome c-reductase system. For chinook salmon, 0. Gawytshq the pH optimum is 76 In contrast, the starry flounder, P. stdlatq has a much lower pH CNlNO5K SALMON O-O SbUNlTY =Qs%.

o-o

DEHYDROOENASE

*3r

of the reaction was directly proportional to the substrate and to the enzyme concentration. The range of K,,, (apparent) values for the succinate-cytochromc c-reductase for these species is quite similar (23-7” x I oe5 M) and appears to be independent of the environmental medium in which fish were found. Ivf.msm

OF c&u

POPULATION

IN GILL

EPITHELIUIU

To determine the number of c& in a given weight of gill tissue, it was assumed

P

!TARRI FlOUNDER O-0sALMTY.0.3~ A-ASALINITY* 30%

sALtNtrY*M%

/

0

.

.I:

FIG. 3.-Lineweaver-Burk double reciprocal plot showing relationship between sutmrate and enzyme concentration isolated &om fresh-waterand sea-water-adapted starry fiounder, P. sbunttu.

I

0’

I

-80

,

-40

.I.

. i ImY

I

40

,

60

.

,

(20

,

60

SUCCINATW

Fro. I.--Lineweaver-Burk double reciprocal plot showing relationship between subuate and enzyme concentration isolated from fresh-waterand sea-water-adapted chinook salmon, 0. tshauytsha. (5.8) which is quite similar to the staghorn sculpin, L. armatus. Figs. 2-4 represent the substrate dependency of the mitochondrial enzyme system as determined by a Lineweaver-Burk double reciprocaI plot. The data for the three species show that the rate

that a quantitative extraction of the deoxyribonucleic acid (DNA) from the tissue would provide an analytical method for estimating the cell population, providing that the DNA content per somatic cell could be established. Mirsky and FGs (1951) and Boivin, Vendrely, and Vendrely (1948) have shown that the DNA content is constant for certain somatic cells within a variety of species. Red bloodcells were chosen as representative of somatic cells because they are nucleated, diploid, and, in addition, could easily be counted by a ceil Heparinizcd blood-samples taken counter. from chinook salmon were diluted with physiological saline and counted. Following the counting procedure, they were collected

I

I

63.)

I

I

- .-

2:

57 4’ 74

;4

64 #fJ

:; 68 53 56 58

8.4

‘35

bg.)

PROTEIN*

( :ELLuLAP

TOTAL

0.070

0’075 oV9 o*o77

WOgg

o&o

o&34

0’090 0’079 **O&J 0’079

0,095 WI31 *ril3 0’107 0’094 a*049 @053 u.057 -__~ o&B

&

-_

-_

--

RATIOOF DNA to CELL PROTEIN I

-T-

3’3 3’3 6.3 4.8 4’4

(W)

PROTEINS

IbfITOQHONDRIAL

TOTAL

---

0.079

0’079

0’%7 O&O 0*0&j Oa$p

0’139

0.093 0’260 0’1 IO

0.096

wag I

29k

,_ .-. ,_~_. ._ ,. _ . . . _ ._ -

_ -. - _

11Omitted due to DNA value.

_ - - _

_.

.. -

I.3

_~._

-..-

.---

-

31.84 I I

22-g 49’4 49’4 IF8

22'0

x3-8 4 5

30’4 30’0 15’1

194

20-517

20’3 13-6 12.6 34’7

24'0 20'9

0’059 0’069 0.053 0’047 @%5 0’062

ki V

iii

ii

_ .-._ 0”

SPECIFIC \GTIvITY X IO-' PER CEI.LI[

’ 7’4 -

3’2 4’4 I.7 2-4

(Vnm,)

ENZYME kTMTyJ

CII~NOOR SALMON

0.055 0’01 I

MITOCHONDRIAI ~ROTl?.INTO CELL PROTEIN

RATM OF

AOTIVITY FOR GILL. BPITIIELIAL CELL PROM PR~sII-wA~~-ADAerEO AND THOSE EXPOSED FOR BRIEF PERIODS IN SEA-WATER

Average figures are means + I S.D. * Total cellular protein calculated from assay of unit volume of brei x total volume of brci. t Total DfJA calcu!ated f+n assay of unit volum: of brei x total volume of brei. i PtaI mIt~ond~l protern calculated from unrt volume pf suspended mito$Iondria x total volume of suspension. c reduced per mmute per ma. mItochondrIal nrotem. ---,I-x = MM cytochrome . Total m&ochondriaiprotein x V,,, * IJSpecific activity per cell = Total DNA x (No. ofcetfsfg. DNA) = pAf prduct vr minute per ““’

Average

sea-water 21.8 15’8 21.5 20’0 17.8

exposure in sea-water 82.2 21’0 81.6 20’0 66.1 18.2 5~6 I6.5

Transition : I z-hour exposrure in F -+SW-I 95-I F -+ SW-6 45’4 F -+ SW-7 ‘03.9 FjSW-8 99-8 F-+SW-9 659

Average

F : SW-3 F-SW-4 F + SW-5

TrarFGtirG:hour

-_-

77’9 87.0

YP4 77.2

* M-4

(cm.)

I

DEHYDROORNASE

WEICWT QJLLLRNOTH

Fresh-water steady-state 84-7

EXPERIMENTAL ENVIRONMENT

Tnbls &-SUCOINIC

SIJCCMIC

‘97% 1

ACID

and rhe DNA was extracted and assayed. Fig. 5 shows the linearity between cell number and the amount of DNA isolated. The amount of DNA per cell calculated from the graph is qual to 18 x xo-12 g., which is higher than has been reported. It was found neceSSary to remove the haemoglobin that caused interference with the diphenylamine The isolation 1 procedure was reaction. 0.0 $

-;,

2

1

paring the sea-water-adapted salmon to the marine starry flounder and staghom sculpin, there is a pronounced difference in that both of these marine species have much higher activities than salmon, as shown in TaHc III. Transition of juvenile salmon from fresh water into sea-water appears to induce an elevation of specific enzyme activity per cell

O-O SALINITY=30%

1

-00

0

-40 ‘IS

concentration

‘33

STAGHORN SCULPIN

01 ,

Fro. +-I&weaver-Burk

DEHYDROUENASE

40

00

120

160

(mMSUCClNATEr’

double reciprocal piot shcwing relationship between s.tbstratc arid enzyme from sea-water-adapted staghom scdpin, L. arm&u.

isolated

modified by swelling the cells with dilute saline which permitted removal of the haemoglobin. Fig. 6 shows the results from the three species of fish using the modified procedure. Values for the DNA content per cell is 5.7 x ro-l* g. for salmon, 6.8 x 10-l* g. for starry flounder, and 2.6 x IO-~*g. for staghom sculpin. Comparison to a haploid cell, such as the salmon spermatozoan, was quite good in that the DNA content was found to be 3.3 x to-i* g. per cell.

ENZYMATIC ADAPTATION AS A FUNCTIONOF ENWRONMENTALS.UJNITY T&es I and 1. show that the maximum enzyme activity for chinook salmon during steady-state fresh- (4’8 & 2.0) and sea-water (3.5 I: I-5) conditions is similar. One can note that little difference exists between the two environments. However, when com-

(2~5 4 7 + 31’8i I I), but there is a large degree of variation. However, the reverse transition does appear to produce an elevation in cnzymc activity per cell ( TubLs ?r,I.). This apparent increase in enzyme activity may be an art%act due to loss of DNA during isolation procedures as evidenced by the variation found in the DNA to cell protein ratio. The cell protein to mitochondrial protein ratio was nearly constant. In summary, it does not appear from these experiments that either the specific enzyme activity or the number of mitochondria-rich cells in the gill has changed during adaptation to sea-water or fresh water. DISCUSSION BIOCZUWAL FINIXNGS IN RELATION TO THE

ENZYMECYTOLOGY Copeland and colleagues Copeland, 1948; Copeland,

(Pettengill and rg5o) were the

* --

--

--

1 ,g

,

J ‘5

25’5

I Q‘2

,

c

: 1p-hour expose ‘e in fresh water

21’5 16.5 17.8 17’0

in fresh water

23’3 21.3 18.6 23.0 18.5 19.0 17.8

‘9’5

(cm.)

4’

;z

58

63

61

66

99

‘23 83 58

lo6

(mg.1

--

PROTEIN*

1P~JLL LENOTII CELLULAs

TOTAL

;:;

“7

2.6

2’1

4’2 2’7

4’9

9:;

8.2 6.2 7’4

154li

9’4

DNAt (mg.)

TOTAL

__-

0.053

*o54 0.042 o&q

~062

0.072 0.055 O.058 o&3

0.089

0.077

0.074 0.071 0.065

0.107

0'099

x:3

-

4’9

“7

1.8

4” 2.8 “5 “9

3’3 5’7 2.8 3’1 2’4

;:;

4’3

TOTAL R.4~10 0~ MITOCH~NDRIAL DNA TO PRO-IZIN~ ,CELL PROTEIN (mg.)

-

18.7 f 2 4.1+5

21’0

20.4

‘4’4 f 4

‘9’3

Il.7

‘7’7 8.9

12.gf6

14.8

-

-

12.6 18.0 25. i 6.8 12.4 6.7

8.6

\CTlVlTY X IO-’ SPECIFIC PER CELL

;:;

3.6

3’1 f I’5

;:g

3’2 “5

3’5 f “5

2’4

3’3 1.4 4’2 5’9 5’7 2’0 2.8

t Total DNA calculated from assay of unit volume of brei x total volume pf brei. f l$tal mitochondrial protein calculated frym unit volume pf suspen+d mltyhondria x total volume of suspension. ,,ml = @4 cytochrome c reduced per mmute per mg. mltochondrlal protein. Total mitochondrial protein x V,,, = 14M product per minute per cell. 11Specific activity per cell = Total DNA x (No. of cells/g. DNA) 1 Omitted due to DNA value.

0.043

0’054 0.037 0.036

0.053

0,070 0.056 o+oqr 0.046

0’047

0.057 0.057 0.042 0.05 I 0*040

0039 0.05 I

0.040

RATIO OF ENZYME V~IT~CH~NDRIAI kTlVlTY f PROTEIN To ( v,,*,) CELL PROTEIN

DEHYDROOENASEACTIVITY FOR GILL EPITHELIAL CELL PROM SEA-WATER-ADAPTED CHINOOK SALMON AND THOSE EXPOSEDFOR BRIEF PERIODS IN FRE~II WATER

Average figures are mean f I S.D. * Total cellular protein calculated from assay of uni It volume of brei x total volume of brei.

:z%e

Transition

Average

Transition: 4-hour exposur S+FW-I 97’4 S + FW-2 47’4 S + FW-3 54” S -+ FW-4 45”

Average

Salt-water steady-state SW-I 80.3 SW-2 I 28-8 SW-3 ‘04’7 ‘SW-4 62.3 120’0 SW-5 SW-6 72’5 SW-7 72.0 SW-8 57’7

EXPERIMENTAL WEIQIIT ENVIRONMENT (Be)

Table II.-SUCCINIC

k! +a

5

i5

8

SUCUNIC

‘970, 1

ACID

first to report the localization of alkaline phosphatases within the mitochondria-rich cells of the gill epithelium of the kilbfish, Fundulus hetmociitus. The cytochemical evidence derived from the substrates used in these experiments, i.e., glycerol phosphate, yeast nucleic acid (3'-nucleotidej , adenylic acid ( j’-nucleotide) , and hexose diphosphate, indicated that the activity of the alkaline phosphatase is probably due to an enzyme

‘35

DEHYDROGENASE

fksh water or sea-water. The enzyme activity was measured by using p-nitrophenyl phosphate as the substrate at pH g-5 and appears to be very similar in general properties to the enzyme in Gopeland’s experiment. It was observed that enzymatic activity was much higher in the intestinal cells of sea-water-adapted forms than in the fresh-water forms. However, comparison of the results obtained from the two types of gill 0 CHINOOK

CHINOOK SALMON la fsnawytsho

SALMON

0 COHO SALMON 0 STARRY FLOUNDER A STAGHORN

0

ov C

I

4 NUMBER

8

12

16

OF RED BLOOD CELLS

20

P

SCULPIN

I d

24

x 10’

FIG. J.-Linearity between red blood-cell population and quantity of deoxyribonucleic acid (DNA) isolated from the gills.

with a broad specificity. Recently it has been shown mainly by electron, as distinct from light, microscopy that many of the dephosphorylating enzymes have a membranous location within the cell, the exception being 5’-nucleotidase which appears restricted primarily to the plasma membrane rather than the &cyto-membranes (Gold&her, Eisner, and Novikoff, 1964). Utida (1967) and Utida and Isono (x967) biochemically isolated an alkaline phosphatase from the intestinal mucosa of the eel, Anguiila japonica, and the rainbow trout, Salmo gairdneri, which were adapted either to

0

k NUMW?

ri OF RED BLOOD

1’8 CELLS

is I IO’

FIG. 6.-Relationship ttetween quantity of DNA isolated and ceil number from four species of lish.

epithelium were questionable (Utida, Ig67j. Furthermore, comparison of acid phosphatase activity for the gill epithelium proved to be negative. At the moment it appears that the biochemical evidence does not support the original cytochemical observation in regard to induction of alkaline phosphatase during salt-water adaptation. Similarly, Natochin and Bocharov (I 962)) in a histochemical study on the sodiumexcreting cells in the gills of pink (0. gor6urcirct) and chum (0. kcta) salmon during

3 4 5

I 2

20’5 21’0 18.3

‘9’5

20’2 21.8 20’0

'9'3

20’9 21.6 21’1 18.4 18.9

2’1 “4 O.9ll

79 ;:

$I 46

45 45

~___ 0,023

0.027 0.026 0.018

0.056

0’059 0.058

0.066 ~048 0.050

4’5

7'9

44

5’2 3’9 7.6 6.7 7.6

7’4

0.052

2:;

0.074

5’0 7”

(w)

TOTAL LIIT~~H~NDRIAL PROTEINS

55

--

RATIO OF DNA TO CELL PROTEIN

g 6O

3’0 2’2 2.6 2.8 2’7

DNAt (mg.1

TOTAL

0’01 I O’MI 0.013 0*O6O

(mg.)

TOTAL :ELLULAR PROTEIN*

-____

-

-

0’101

0’53 0.099

O&l

0.130

0.139 0.165

0*114 0.086 ~146

oog3

(I.070 oo85 0.105 0.136

O.069

RATIO OF MITOCIIONDRIAI PROTEIN TO CELL PROTEIN ENZYME

-8.0f

a:;

8.0 IO.5 8.0

Total Total Total v,,,

1.7

5.1+0-g

6.5 5’4 5’4 4’4 4-o -___

-

104.8

61.9 ‘47’7 -

96.3

117.1 ‘44’7 74’4 68.9

86.6

75”

52.2 49’3

I24.0

__

___--

-

SPECIFIC X 10 ’ PER Cp.LL

4 kTlVlTY

( Vmr+J

ACTIVITY

+ I S.D. cellular rotein calculated from assay of unit volume of brei x total volume of brei. DNA ca Pculated from assay of unit volume of brei x total volume of brei. mitochondrial protein calculated from unit volume of suspended mitochondria x total volume of suspension. = @4 cytochrome c reduced per minute per mg. mitochondrial protein. Total mitochondrial protein x V,, 11Specific activity per cell = = f& product per minute per cell. TotalDNA x (No.ofceIls/g. DNA) 11Omitted due to DNA values.

* t t f

Average figures are mean

Fresh-water steady-state

c

106’2 IO&l

106.3 131.2

93’7

stead -state

Average

Salt-water Sculpin Sculpin Sculpin Sculpin Sculpin

Average

Salt-water steady-state Flounder I 9o-6 98.1 Flounder 2 Flounder 3 91’7 Flounder 4 54’3 Flounder 5 52’7

(cm.1

FULL LENOTII

Table III.---SWCCINIC DEIIYDROOENASEACTIVITY PER GILL EPITHELIAL CELL FROM STARRY FLOUNDF.RAND STAGHORN SCVLPIN

iif cd

5

3

::

w m

SUCCINXC ACID L IFXYDROGENAsE

197% 1

adaptation to life in sea water, showed that the activation of these cells was accompanied by an increase in succinic dehydrogenase activity. These investigators considered this enzyme system to be the most important component of the sodium-transporting structure of the gill epithelium. The biochemical results obtained Tom the present investigation do not support these cytochemical findings and lead us to be&eve, along with others (Copeland, rgso), that either the mitochondria-rich cells must indeed be very labile, or that this tissue is quite unique in its enzjmatic properties. Consideration should be given to the fact that we used a diEerent species of salmon in our study. None the less, we did not find significant changes in succinic dehydrogenase activity. j’-NUCLEOTIDE m

bf.mm~~ne

FUNCTION

It should be worth while to reiterate the fact that 5’-nucleotidase activity has been shown to be present within the plasma membrane from many types of cells (Goidf&her and others, 1964) and could possibly be contaminating the microsomal fi-action that give high ATPase levels (Kamiya and Utida, 1966). The microsomal pellet that was used in their experiment was prepared by the sodium iodide precipitation method as reported by Nakao, Nogano, Ada&i, and Nakao (x963). Since adenylic acid (AMP) was not assayed for release of inorganic phosphate (Pi), it could have participated through an alternate pathway as shown:I. ATP+ 2. AMP

AMP + PI?, e

Pi + adenosinc,

the first reaction being catalysed by a pyrophosphatase which would cleave adenosine triphosphate (ATP) to yield pyrophosphate (PPi) and AMP. The second step is the action of 5’-nucleotidase on AMP which liberates inorganic phosphate and acienosine. .kKNO~EM.ENT

Tbe research was sponsored by the U.S. Atomic &ergy Commission under Contract AT (45-I)2013-2.

137 REFERENCES

Born, A., V--Y, frw&f. ‘L’acide d-

R., and Vmxxw~, C. ‘bonudtiaue du nwau

&&&e, depositaire’des oaraci&es h&&itaires; arguments d’ordre asld~que’, c. 1.

hebd. Skanc.Amd. Sti., Pmir, 4, x061. F. (xg65), ’ Effects of icmizing radiation on Osmonauiation in fish Onwr~ kkutch’, Camp. i%och6m.P&&L, x5, 293. -_ and Lm. D. (10671. ‘Kinetics of ceh.Iar moxphogcrks in ‘& ’ &ithelium during sea water adaptation of 0~~~ kin&h’, Ibid., rr3¶945. -and Monrr~, T. (rQ68), ‘ ImmunOchcmical study of cell differentiation in gill @helium of eurybaiine oncLw&nchwkiwh’, Iti., y, ‘++5. COPELAND,D. (1947), ‘The cytoio@cal basisofsalt excretion from the gills of Fund&s h&m&us’, Eiol. B&i. mar. biol. &b., Woodr Ho&, 93, 192. Co=,

-

-

{X950),

chlorkie

ceil

‘pulaptive in

the

gill

bd-&ViOr

of

tilt

of Fimduiarh&m&us ‘, J.

Morph.3 8% 369. Epsrcpr, F., IL=, A., and PI-ORD, G. (1967)) ‘ !3odium and potassium-activated adenosine

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138

corn

AND

NA-, Y1., and Bommov, G. (1962)) ‘Acxivation afsodlum excreting cells in the gills of pink and chum salmon dukxg adaptation to life in sait water’, vop. Ikhtiol., 1, 687. PETTENOXLL, O., and COPE-, ti. (x948,, ‘Alkaline phosphatasc activity in the chloride cell of Fuultrirrr lictnacfittrr and its relation to motic work’, J. rxp. .+d., 108, 235. PHXLPO~, C., and COP-, D. (x4$53), ‘Fine structure of chloride cells from three sptcies of Fund&s’, 3. crll Biol., r8, $3g. Tmts~~oom~ L, and HOUSTON,A. (1964, ‘An ckctron mwoacope study of the “chloride cell” ofSalmos&L’,Eqi~WRes.,~ t. UTIDA, S. (1967)) ‘Effect of sodium chloride on

TRIPP

alkaline phcephatase activity in intestinal mucosa of the rainbow trout’, Proc. JapanAcad., at 783. UIIDA, s., and bON0, Er’. (1967), ‘Alkaline phosphatase activity in irstestiwl mucrxa of the ccl ;$pted to &ah water or sea water’, Ibid,, 43, ZmoC, W. (x968), Paper prescmed to the unitity of south Dakota symposium on Fii in Research, Vcrmillion, South Dakota. Xcy Word Index: Succinic dehycirogcwe, ti gill, sak-water adapwiQn, oBm0regulati0~ ONDriignukt l!haqtsha, Plau-, Lqxi?.

cot& afmatus.