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Changes in the ouabain-sensitive, sodium and potassium-activated adenosine triphosphatase of the gills… Giles, Michael Arthur 1969

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CHANGES IN THE OUABAIN-SENSITIVE, SODIUM AND POTASSIUM-ACTIVATED ADENOSINE TRIPHOSPHATASE OF THE GILLS OF COHO SALMON Oncorhynchus kisutch. DURING THE FRY TO SMOLT STAGES OF ITS LIFE HISTORY ANDJJPON EXPOSURE TO SEA WATER by MICHAEL ARTHUR GILES ' B.SC.(hons.), University of Manitoba, 1965 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Department of ZOOLOGY We accept this thesis as conforming to the required standard 'THE UNIVERSITY OF BRITISH COLUMBIA August, 1969 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and S t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s j s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . The U n i v e r s i t y o f B r i t i s h Co^ l V a n c o u v e r 8, Canada D e p a r t m e n t i ABSTRACT Some of the kinetic characteristics of the sodium and potassium-activated adenosine triphosphatase of the fragmented c e l l membranes of cells from the g i l l s of sea water adapted coho salmon Oncorhynchus kisutch, and changes in this enzyme upon exposure to sea water and during the fry to smolt stages of fresh water reared juvenile coho were investigated. Inhibition with 4 X 10~^ moles/liter ouabain was used to assay the activity of this enzyme since this ATPase i s specifically inhibited by ouabain (Skou, 1 9 5 7 ) . The following assay conditions were found to result in maximal hydrolysis of ATP in enzyme preparations from sea water adapted coho: pH, 7 . 4 ; incubation temperature, 40°C; NaCl and KC1 concentrations of 1 0 0 . 0 and 2 0 . 0 mmoles/liter, respective-ly, and Mg2+-ATP, 5 . 0 mmoles/liter. The Km for ATP was 0 . 2 mmoles/liter. The enzyme activity recorded with magnesium ions as the only cation present (Mg2+-ATPase) was not affected by any concentration of ouabain, although the addition of sodium ions (100 mmoles/liter) appeared to inhibit this activity slightly. The additional hydrolysis of ATP observed when sodium, potassium and magnesium ions were present was inhibited by ouabain. The Ki for ouabain was; 7 X 10~^ moles/liter when sodium and potassium ion concentrations were 1 0 0 . 0 and 2 0 . 0 mmoles l i t e r , respectively. The (Na*+ K*)- activated ATPase of sea water adapted coho was characterized by i t s high ouabain-sensitive activity and the large activating effect of potassium ions in the presence i i of magnesium and sodium ions compared to the a c t i v i t y observed with the l a t t e r two ions alone. This enzyme i n preparations from the g i l l s of fresh water reared f i s h was characterized by a high a c t i v a t i n g effect of sodium ions when present with magnesium ions. This sodium activation often comprised over 60% of the t o t a l ouabain-sensitive a c t i v i t y . Considerable increases i n the t o t a l a c t i v i t y , and act i v a t -ing e f f e c t s of potassium ions and decreases i n the ac t i v a t i n g effects of sodium ions alone were observed when fresh water reared coho were transferred d i r e c t l y to sea water. The changes i n the a c t i v a t i n g effect of the ions were noticable a f t e r 5 days exposure to sea water although no changes i n the t o t a l a c t i v i t y of the enzyme occurred u n t i l a f t e r 10 days exposure. On a seasonal basis changes i n enzyme a c t i v i t y occurred which were apparently linked to the stage of development of the parr-smolt transformation i n fresh water reared juvenile coho. A c t i v i t i e s during the period of October 1, 1968 to l a t e November, 1968 were generally quite low. A sharp peak i n a c t i v i t y occurred i n December, 1968 to l a t e January, 1969 which decreased to a low l e v e l by mid-February. Up to and including t h i s l a s t period the a c t i v i t y of enzymes from the g i l l s of both fresh water and sea water reared coho were q u a l i t a t i v e l y s i m i l a r although the seawater f i s h always had a higher enzyme a c t i v i t y . During the period of mid-February to l a t e April,1969 the enzyme from fresh water reared coho changed i n t o t a l a c t i v i t y and c h a r a c t e r i s t i c s of sodium activationaand potas-sium a c t i v a t i o n and became very s i m i l a r to that of sea water reared f i s h of the same age. . [ f i i i TABLE OF CONTENTS page ABSTRACT i TABLE OF CONTENTS i i i LIST OF FIGURES iv LIST OF TABLES v i ACKNOWLEDGMENTS v i i INTRODUCTION 1 MATERIALS AND METHODS General 2 Section I j Section II ^ RESULTS General Section I ^ Section II 2^ DISCUSSION 31 LITERATURE CITED 59 i v LIST OF FIGURES Facing Page 1. Mean weekly temperatures i n degrees Centigrade of sea water and fresh water. 4 2. Plot of mean weight and mean fork length of i n d i v i d u a l samples of juvenile coho from October 1, 1968 to A p r i l 21, 1969. 13 3. Relationship between mean weight and mean fork length of samples of juvenile coho from October 1, 1968 to A p r i l 21, 1969. 15 4. Plot of ouabain-sensitive hydrolysis of ATP against incubation time. 17 5. The eff e c t of pH and incubation temperature upon the ouabain-sensitive hydrolysis of ATP. 18 6. The effect of ATP concentration on the ouabain-sensit-ive hydrolysis of ATP. 19 7. The ef f e c t of potassium ion concentration upon the ouabain-sensitive hydrolysis of ATP at three concentra-tions of sodium ions. 20 8. Relationship between ouabain concentration and percent-age i n h i b i t i o n of the ouabain-sensitive hydrolysis of ATP. 23 Facing 9. Changes i n TS-a c t i v i t y , sodium a c t i v a t i o n , and P aS e potassium a c t i v a t i o n of coho g i l l s with time of exposure to sea water. 26 10. Changes i n TS-activity, sodium act i v a t i o n , and potassium a c t i v a t i o n of coho g i l l s from October 1, 1968 to May 8, 1969. 28 V I LIST OF TABLES Facing Page I. Composition of the incubation media used i n the determination of optimal assay conditions for ouabain-sensitive hydrolysis of ATP. B II. Composition of the various incubation media used in the study of seasonal changes and changes upon exposure to sea water in the ouabain-sensitive ATPase of the g i l l s of juvenile coho salmon. 10 III. Parameters and type 2 errors of the least-square f i t relationships of f i s h weight on fork length of sea water and fresh water reared juvenile coho salmon (log weight, g 5 3 log a + b log length, cm). 14 IV. The effect of various concentrations of ouabain on ATPases activated by magnesium ions and various concentrations of sodium and potassium ions. 22 ACKNOWLEDGMENTS I w i s h t o t h a n k D r . W. E. V a n s t o n e f o r h i s a s s i s t a n c e a n d g u i d a n c e d u r i n g t h i s i n v e s t i g a t i o n a n d a l s o f o r t h e p r o -v i s i o n o f t h e r e s e a r c h f a c i l i t i e s a t t h e F i s h e r i e s R e s e a r c h B o a r d o f C a n a d a , V a n c o u v e r , B r i t i s h C o l u m b i a . I am i n d e b t e d t o D r s . W. S . H o a r , P . W. H o c h a c h k a , a n d J. E. P h i l l i p s f o r t h e i r u s e f u l c r i t i c i s m d u r i n g t h e e x e c u -t i o n o f t h i s r e s e a r c h . I w o u l d a l s o l i k e t o e x p r e s s my g r a t i t u d e t o M r . J. R . M a r k e r t f o r h i s a d v i c e a n d a s s i s t a n c e i n t h e p r e p a r a t i o n o f t h i s m a n u s c r i p t . T h i s r e s e a r c h w a s s u p p o r t e d b y t w o g r a n t s f r o m t h e F i s h e r i e s R e s e a r c h B o a r d o f C a n a d a , h e l d d u r i n g t h e y e a r s o f 1967 t o 196S a n d 1968 t o 1969. INTRODUCTION The parr-smolt transformation of young salmon prior to and during their seaward migration i s accompanied by changes in form and by many biochemical, physiological and behavioral changes (Baggerman, I960; Hoar, 1965; Vanstone and Markert, 1968). Recently, changes in the sodium and potassium-activat ed, ouabain-sensitive adenosine triphosphatase enzyme of the g i l l s of fish transferred to sea water have been reported (Epstein, Katz and Pickford, 1967; Kamiya and Utida, 1968). The present investigation was undertaken to determine whether this enzyme changed in the g i l l s of juvenile coho salmon ex-posed to sea water and i f there were any preadaptive changes in the enzyme in freshwater f i s h during the parr-smolt trans-formation. Since the microsomal preparation from coho g i l l tissue contained phosphatases which do not require sodium and potas-sium ions for activity and are not inhibited by ouabain (Skou 1957), the sodium and potassium-activated ATPase was assayed in appropriate media with and without ouabain. The stage of development of the juvenile coho was deter-mined by visual observation of external changes (Houston and Threadgold, 1963), and by changes in the weight - length relationship (Vanstone and Markert, 1968). 2 MATERIALS AND METHODS General Methods Source and maintenance of fish Approximately 3500 coho f r y in fresh water were obtained from the Washington State Hatchery at Skaget, Washington, U.S.A. in May of 1968 and transported in large tanks to the laboratory in West Vancouver. Additionally, approximately 400 coho post smolts in sea water were obtained from the Bio-logical Station of the Fisheries Research Board of Canada at Nanaimo, B.C. and transported to West Vancouver. No morta-l i t i e s related to transportation were observed. The f i s h were placed in two fibreglass aquaria 1.8 m in diameter containing 5000 l i t r e s of water in which a circular flow was maintained by means of recirculating pumps. Chlorinated municipal water or sea water was supplied to each aquarium at a rate which re-placed the water in each tank at least four times in every 24 hr. These f i s h were fed once daily to satiation with a diet composed of beef li v e r , 4500 g; beef heart, 4500 g; canned salmon, 4500 g; salt, 110 g; and Pablum, (Meade-Johnson Canada Ltd.), 40 g. In August of 1968 approximately 1000 fry were placed in each of three 1.8 m aquaria and were designated seasonal fresh-water f i s h , freshwater adaptation f i s h and seawater adapta-tion fi s h respectively. Of the two freshwater groups of f i s h , which were maintained in chlorinated fresh water until April, 1969 one, the seasonal freshwater fi s h , was used to determine the seasonal changes in g i l l ATPase while the other, the fresh-3 water adaptation fi s h , was used for the sea water adaptation experiments. The seasonal seawater f i s h were maintained in chlorinated tap water for two days and then adapted to sea water over a 10 day period during which the salinity was in-creased approximately 3 °/ 0 0 P e r day resulting in a f i n a l salinity of 30 °/QQ equal to 100$ sea water. Mortality during this pro-cedure was less than 1%. These seasonal seawater f i s h served as "sea water controls" in the determination of seasonal changes in g i l l ATPase and were the source of f i s h used in the invest-igation of the effects of ouabain on this enzyme system. Length and weight records Length and weight data were obtained periodically on samples of seasonal freshwater and seasonal seawater f i s h . The f i s h were starved for 24 hours, anaesthetized lightl y with 2-phenoxyethanol, measured individually for fork length and wet weight (Mettler P-120 torsion balance) and returned to their respective aquaria. In some instances f i s h for the enzyme assays were measured in this manner and then maintained in their respect-ive aquaria, without food, for 24 hours before use. In other cases f i s h were starved for 24 hours and then measured immediately prior to use. These latter f i s h were immobilized by severing the spinal chord just posterior to the head region prior to measurement. Water temperature and salinity records Continuous temperature records of fresh and sea water were obtained with a recording thermograph (Taylor Instruments of 4-Legend for Figure 1. Mean weekly water temperatures in degrees Centigrade of sea water, (X), municipal fresh water, (0), and Cypress Creek water,(+). X X X + + X >© x O x © x o x + + O x X X X O O + O + c> + 8 + x X O O x X O O O x X X x o x© *> Ox O x O x O x O x O x O x O x O O X O x Ox © O x - i 1 1 1 1 1 -r r ^ CM O 0 0 00 oo CM 2 < 00 • a < CO o - s cr> CJ CD hoo cn CVJ 2} LJ CM 5> o O IO l-Q cn O o m o o CM 3 < o CO 10 5 Canada Ltd.). The salinity of the sea water was measured daily with a hydrometer. The mean weekly temperatures of fresh and sea water are presented in Figure 1. The sal i n i t i e s averaged 30°/ o o, range 28-32°/ o o, throughout the experimental period. Enzyme preparation Fish were decapitated at the level of the pectoral f i n insertion and the head pinned, ventral side up, to a disecting board. The heart was exposed and 2.0 ml of ice cold homogeniz-ing solution (composition in mmoles/liter; sucrose, 250; imidazol, 2.0; disodium ethylenediaminetetraacetate, 2.0; pH 7.4), was perfused through the g i l l s via the conus arterio-sus. Each g i l l arch was then disected out and placed in cold homogenizing solution. The g i l l s of two or more fish were pooled for each enzyme preparation. The g i l l s were then dried lightly on cellulose wipes to remove any external blood clots and extraneous moisture, weighed to the nearesrO.Ol g and placed in fresh homogenizing solution i n the proportion of 1 g g i l l to 10 ml solution. The tissue was then homogenized in a grinding tube with a loose f i t t i n g , motor-driven, teflon pestle for 45 sec, in an ice bath. A few glass beads (0.01 to 0.02 mm in diameter) or glass wool were added to f a c i l i t a t e grinding of the cartilage. This crude homoginate was then centrifuged in a Servall refrigerated centrifuge, SS 1 rotor, at 1-4°C for 10 min at 1000 X g, and then 2000 X g for an additional 10 min. The supernatant from this procedure was centrifuged at 10,000 X g for 30 min and the resulting supernatant was decanted and centrifuged at 37,000 X g for 60 min. The supernatant of this f i n a l c e n t r i f u g a t i o n was discarded and the p e l l e t resuspended i n 4-5 ml of i c e c o l d homogenizing s o l u t i o n . The p a r t i c l e s of membrane fragments thus obtained were stored i n capped tubes f o r 2-3 days at 0-2°C to f a c i l i t a t e . dissociation of the membra-ne fragments. A f t e r storage a vortex mixer was used t o uniform-l y suspend the fragments. With t h i s technique the c e l l membra-ne fragments d i d not n o t i c e a b l y s e t t l e out a f t e r standing 7 days. Enzyme assay Magnesium-ATP and disodium-ATP, (yeast c r y s t a l ) were ob-ta i n e d from Mann Research L a b o r a t o r i e s , New York, N.Y., and ouabain from N u t r i t i o n a l Biochemicals Corp., Cleveland, Ohio. A l l other reagents were purchased from F i s h e r S c i e n t i f i c Inc. In p r e l i m i n a r y experiments to determine optimum assay c o n d i t i o n s the i n c u b a t i o n media contained various concentrations of ATP, TRIS-HC1, NaCl and KCl. Magnesium concentration was always equimolar to ATP co n c e n t r a t i o n and ouabain concentration was 4 X 10"^ moles/l (Table I ) . The composition of the i n c u b a t i o n media used i n the long term i n v e s t i g a t i o n of the changes i n the o u a b a i n - s e n s i t i v e ATPase of coho g i l l t i s s u e during t h e i r f i r s t year of f r e s h water residence and upon exposure t o sea water are presented i n Table I I . Both the enzyme suspension and substrates were pre-incubated at 40°C f o r 5 minutes before 0.2 ml of the enzyme was p i p e t t e d i n t o 1.0 ml of the appropriate s u b s t r a t e . The r e a c t i o n was allowed to proceed f o r 10 minutes i n a constant temperature shaking water bath (Blue M E l e c t r i c Co.) and then stopped by the a d d i t i o n of 0.5 ml of c o l d 17.5% t r i -c h l o r o a c e t i c a c i d . Normally d u p l i c a t e or t r i p l i c a t e assays 7 were performed. The contents of each incubation tube were then assayed f o r inorganic phosphorus by the Fiske-SubbaRow method (Hawk, Oser, and Summerson, 1947), and the membrane nitrogen concen-t r a t i o n of the enzyme suspension determined, i n duplicate, by the microkjeldahl method (Association of O f f i c i a l A g r i c u l t u r a l Chemists, I960). Methods f o r Section I Optimal assay conditions The optimal assay conditions of incubation time, pH, temperature, ATP concentration and sodium and potassium ion concentrations were determined on enzyme preparations from the g i l l s of 20 month old post smolts which had been l i v i n g i n sea water f o r 3 months. Since these parameters were studied con-secutively no single substrate composition could be provided. The actual substrate composition f o r each experiment i s given i n Table I. A l l data are presented as the s p e c i f i c a c t i v i t y in^jimoles liberated/hour/mgN, of the ouabain-sensitive ATPase and have been corrected to the standard assay conditions presented i n Table I I . Effe c t of ouabain on the (Nst + K*)-activated adenosine- triphosphatase In A p r i l 1969, 16 seasonal seawater coho were used as a pooled source of g i l l t i s s u e . Only one enzyme preparation was made and each value presented represents the mean of t r i -p l i c a t e assays. Legend f o r Table I . Composition of the incubation media used i n the det mination of the optimal assay conditions f o r the ouabain se n s i t i v e hydrolysis of ATP. Concentration (mM/l) Variable Linearity with Time pH Incubation Temperature ATP Concentration Sodium and Potassium Ion Concentration Tris Mg-ATP Na+ K* 30.0 5.0 100.0 20.0 30.0 5.0 100.0 20.0 30.0 5.0 100.0 20.0 30.0 var. 100.0 20.0 30.0 5.0 var. var. Incubation Incubation Number Temperature Time of Ouabain pH ( C) (minutes) Replicates t 0.4 7.4 40 var. I i 0.4 var. 40 10 I -0,4 7.4 var. 10 2 - 0.4 7.75 45 5 2 - 0.4 7.75 45 10 I 9 Methods for Section II Changes in g i l l (Na* + K* )-activated ATPase upon transfer  to sea water In February 1969, 240 freshwater adaptated f i s h (10.6-11.5 cm in length), were randomly divided into 4 groups and placed in oval aquaria, 4&* X 110 cm containing 225 l i t r e s of water. These aquaria were provided with flowing Cypress Creek water but with no recirculating pumps. Two days later two of the groups were transferred directly to 100$ sea water, salinity 30 o/ Q O, while the remaining groups served as freshwater con-trols. The g i l l s of f i s h from both environments were assayed periodically for 50 days after transfer for ouabain-sensitive, (Na+ + K +)-activated, ATPase in the media outlined in Table II. Seasonal changes in (Na T + K )-activated ATPase The seasonal freshwater and seasonal seawater fish were sampled at intervals from October 15, 1968 to April 21, 1969 for analysis of the g i l l enzyme. Approximately equal sizes were taken from each stock to insure that the population densities remained equal. On April 21, 1969, 80 seasonal freshwater f i s h were divided equally between two oval aqua-r i a which were provided with flowing fresh water. On April 22, the fis h of one tank were adapted to sea water over an 18 hour period. The second aquaria was kept as a source of freshwater f i s h . Enzyme preparations from the g i l l s of a l l these fish were assayed in the media presented in Table II. Definitions of the enzyme components The following definitions faciliated the presentation of Legend for Table II. Composition of the various incubation media used in the study of seasonal changes and changes upon exposure to sea water in the ouabain-sensitive ATPase of the g i l l s of juvenile coho salmon. The values presented are f i n a l concentrations after the addition of G.2 ml of enzyme suspension. The pH of the media was 7 . 4 . F i n a l Concentration (mmoles/l.) Tris ATP MR2+ Ouabain Medium I 30.0 5.0 5.0 100.0 20.0 0.0 Medium 2 tr if lilt !Jtt 0.0 Medium 3 » ft If 20.0 0.4 the results of Section II and the discussion of the data: TS-ATPase activity i s the total ouabain-sensitive, sodium and potassium ion activated, magnesium-dependent, adenosine triphosphatase activity and was calculated as the specific activity, expressed in jumoles inorganic phosphorus released from ATP per hour per milligram nitrogen, measured in medium No.l minus the specific activity measured in medium No.3 of Table II. Ouabain-sensitive sodium activation is the portion of the TS-ATPase activated by sodium ions in the absence of potassium ions and was calculated as the specific activity measured in medium No.2 minus the specific activity measured in medium No. 3 of Table II. Ouabain-sensitive potassium activation i s the additional phosphatase activity recorded when both sodium and potassium ions are present in the incubation medium over that recorded when only sodium ions are present and i s calculated as the difference between TS-activity and sodium activation. RESULTS 12 General Growth i n length and weight The patterns of growth i n length and weight with time for sea water and fresh water reared f i s h are presented i n Figure 2. In both groups the rates of growth declined during the period of December 1, 1968 to mid-January 1969 which corresponded to the l a t t e r part of the period of declining water temperatures as shown i n Figure I. Growth was accelerated from mid-January to A p r i l 1969 as water temperature began to increase. Weight-length r e l a t i o n s h i p The parameter "a" and "b" of the weight-length formula W= aL D f o r sea water and fresh water reared f i s h are presented i n Table I I I . Although the "a" parameters varied widely i n both groups the "b" parameter tended to clus t e r around a value of 3.20. The plot of mean weights against mean lengths f o r sea wa-t e r and fresh water reared coho at each sampling time together with a short section of least square f i t r e l a t i o n s h i p of each sample i s presented i n Figure 3. The broken l i n e s are i n -cluded f o r reference purposes and have a slope of 3.2. In both groups a gradual decrease i n the "a" intercept with i n -creasing s i z e was observed while the slope "b" tended to re-main at 3.20. The t r a n s i t i o n from the upper broken l i n e to the lower broken l i n e f o r fresh water reared coho r e s u l t i n g i n a more streamlined f i s h occurred during the period November 29, 1968 to January 16, 1969 when the f i s h were experiencing 13 Legend for Figure 2. Plot of mean weight and mean fork length of individual samples of juvenile coho from October 1, 1968 to April 21, 1969. Solid circles refer to sea water reared f i s h and open circles to fresh water reared f i s h . WEIGHT (G) 0) 00 0 -fc • • I I • • • ro o * • L E N G T H (CM) GO O N • • . • . • 8 - . m g o ° 5 <o CD ro 00" ro o ° j> ro. z to m ro_ w oo -o ro a to 14 Legend for Table III. Parameters and type 2 errors of the least-square f i t relationships of f i s h weight on fork length of sea water and fresh water reared juvenile coho salmon (log weight, g = log a + b log length, cm). Sample si z e , Date (n) Least-square f i t of weight-length(¥=* aL ) a (mg) Error b Error Sea water reared Oct. 15, 1968 24 12.0 2.4 2.99 0.09 Nov. 4, tt 52 7.3 1.2 3.22 0.08 Nov. 29, it 65 7.2 0.8 3.23 0.05 Dec. 12, « 77 6.6 0.7 3.27 0.04 Jan. 17, 1969 66 9.0 1.1 3.12 0.05 Feb. Id , n 67 6.9 0.9 3.22 0.06 Mar. 11, n 53 5.7 1.0 3.31 0.07 Apr. 9, it 84 7.1 1.0 3.17 0.06 Apr. 21, tt 106 13.3 2.5 2.92 0.07 Fresh water reared Oct. 1, 1968 23 9.8 3.3 3.09 0.15 Oct. 15, w 24 5.8 1.0 3.34 0.08 Nov. 4, « 52 6.3 1.0 3.29 0.07 Nov. 29, tt 70 8.4 1.1 3.16 0.05 Dec. 12, tt 45 9.7 2.3 3.10 0.10 Jan. 16, 1969 58 5.2 0.9 3.34 0.07 Jan. 31, it 66 5.4 0.6 3.32 0.04 Feb. 18, « 67 8.5 1.9 3.13 0.09 Mar. 11, n 52 7.3 2.0 3.20 0.11 Apr. 10, w 71 8.3 1.6 3.13 0.08 Apr. 21, n 92 4.6 0.8 3.35 0.06 Legend f o r Figure 3* Relationship between mean weight and mean fork length of samples taken from October 1, 1968 to A p r i l 21, 1969. S o l i d c i r c l e s r e f e r to sea water reared coho and open c i r c l e s t o i r e s h water reared f i s h . The short s o l i d l i n e s are the slopes of the i n d i v i d u a l weight/length r e l a t i o n s ship of each sample. The broken l i n e s are included f o r reference purposes and have a slope of 3.20. a period of decreased rate of growth as demonstrated in Figure 2.. The length-weight relationship of sea water reared coho showed changes similar in form and time but smaller in magnitude in comparison to fresh water reared f i s h . Section I Optimal assay conditions The ouabain-sensitive and potassium-stimulated hydrolysis of ATP as a function of incubation time was found to be linear from 0 to 20 minutes as demonstrated in Figure 4. The effects of pH and incubation temperatures on this enzyme are shown in Figure 5. The optimum pH was found to be 7.4 and enzyme a c t i -vity declined sharply on either side of this value especially on the more alkaline side where activity at pH 8.0 was only 50% of that recorded at pH 7.4. The optimum incubation temp-erature was 40°C and a rapid decline in activity at higher temperatures was observed. The effect of increasing Mg-ATP concentration from 0.02 to 8.0 mmoles/1 was an increase in enzyme activity to a max-imum at 0.5 - 1.0 mmoles/1 and a slight decline in activity at higher concentrations. The apparent for ATP of the ouabain-sensitive ATPase was 0.2 mmoles/l. The specific activity a V f f l a x was 260. This data i s presented in Figure 6. Figure 7 demonstrates the effect of increasing potassium ion concentration at three concentrations of sodium ions. The TS-enzyme activity increased with increasing potassium ion concentration to a maximum activity which was dependent upon Legend for Figure 4« Plot of ouabain-sensitive hydrolysis of ATP against incubation time for the assay conditions presented in Table I. The data are presented as relative activities with no units. Membrane nitrogen concentration was 0.0588 mg/ml. R E L A T I V E A C T I V I T Y Legend for Figure 5 . The effect of pH (left scale), and incubation temperature (right scale), upon the ouabain-sensitive hydrolysis of ATP. The assay conditions are presented in Table I, and a l l data have been corrected to the standard assay conditions presented in Table II. The membrane nitrogen concentration for both parts of Figure 5 was 0.0719 mg/ml. i 1 r—i 1 1 1 1 1 1 1 1 1 i o o o o o o o ^" O 10 CVJ 00 CVJ CVJ — — A 1 I A I 1 0 V OldlOBdS 19 Legend for Figure 6, The effect of ATP concentration on the ouabain-sensitive hydrolysis of ATP. The assay conditions used are presented in Table I and the data corrected to the standard assay conditions presented in Table II. The membrane nitrogen concentration was 0.1607 mg/ml. 300H A T P CONCENTRATION ( M M O L E S / L ) 20 Legend for Figure 7» The effect of potassium ion concentration on the ouabain-sensitive hydrolysis of ATP at three concen-trations of sodium ions. Assay conditions used are presented in Table I and the data have been corrected to the standard asspy conditions presented in Table II. The open circles, triangles, and squares, refer to sodium ion concentrations of 10.0, 50.0, and 100.0 mmoles/1, respectively. The membrane nitrogen concentration was 0.1008 mg/ml. 2 8 0 sodium ion concentration, and then decreased at higher potassium ion concentrations. Higher sodium ion concentrations mitigated, somewhat, the inhibitory effect of high potassium ion concentrations as enzyme inhibition by potassium ions at sodium ion concentrations of 10.0, 50.0, and 100.0 mmoles/l f i r s t occurred at potassium ion concentrations greater than 12.5, 15.0 and 20.0 mmoles/l respectively. The latter values corresponded to the ion concentrations at which maximal hy-drolysis of ATP was observed. Half maximal activity for sodium ion concentrations of 10.0, 50.0 and 100.0 mm/l occurred at potassium ion concentrations of 2.0, 2.5 and 5.0 mm/1,respec-tively. Sodium ions in the absence of potassium ions also had a slight but measurable stimulating effect on the ouabain-sensitive ATPase. Effect of ouabain on Mgz+-ATPase and {He?-*+ Na4- + K*)- stimulated ATPase The results of the experiment to determine the effect of various concentrations of ouabain on the phosphatases activated by magnesium ions alone and those activated by sodium and po-tassium ions in the presence of magnesium ions are presented in Table IV. The incubation media consisted of MgATP, 5.0 mm/l; t r i s 30.0 mmoles/1; and various combinations of NaCl, KC1 and ouabain as indicated in the table. Each value presented re-presents the mean of three identical assays of the same enzyme preparation which had a nitrogen concentration of 0.0593 mg/ml. The data in the f i r s t row of Table II indicate that ouabain in concentrations of 10"^ to 10"^ moles/1 had no effect 22 Legend for Table IV The effect of various concentrations of ouabain on ATPases activated by magnesium ions and various con-centrations of sodium and potassium ions. A l l data are expressed as specific a c t i v i t i e s in micromoles P^/Hr./mg nitrogen. The percent inhibition by ouabain was calculated as follows: the (Na ++ K )-activated ATPase was considered to be 100% inhibited when incubated in a medium containing 5.0 mm Mg , 100 mm NaCl, and 10 M^ ouabain. The activity recorded with each concentration of KC1 with no ouabain was considered to be 100% activity for the enzyme under those conditions and a l l intermediate values are expressed as a percentage of 100% activity. Specific Activity-Specific Activity-Specific Activity-Percent Inhibition by Ouabain Specific Activity Percent Inhibition by Ouabain Cation Concentration (mmoles/1. )  Mg 2 + Na* K + 5.0 0.0 0.0 5.0 100.0 0.0 5.0 100.0 10.0 5.0 100.0 10.0 5.0 100.0 20.0 5.0 100.0 20.0 Ouabain Concentration (moles/1. )  0 10~9 10" 8 IO" 7 10~ 6 IO"5 IO" 4 10~3 126.5 128.3 — 123.3 ~ 135.4 — 130.0 112.2 106.9 110.4 110.4 108.7 103.3 105.1 99.3 169.2 172.3 165.7 155.0 133.9 119.3 110.4 99.3 0 0 7.5 22.5 45.0 72.5 35.0 100.0 199.5 190.6 183.3 131.7 169.2 142.5 119.3 105.1 0 3.9 10.7 17.9 30.4 57.1 30.4 94.6 23 Legend for Figure 8. Relationship between ouabain concentration and per-centage inhibition of the ouabain-sensitive hydrolysis of ATP in media containing Tris, 30.0; Mg-ATP, 5.0; NaCl, 100.0; and KC1, 10.0, (open ci r c l e s ) , or 20.0 mmoles/1, (squares), at a pH of 714 and incubation temperature of 40° Centigrade. Membrane nitrogen concentration was 0.0593 mg/ml. upon the phosphatases requiring magnesium ions alone, (Mg2 +-ATPase). Sodium chloride (100 mmoles/1) appeared to inhibit the Mg2"*"-ATPase by 13.6%. With sodium chloride, (100 mmoles/1), and ouabain (lO^moles/l) an additional 9.6% decrease in activity from that recorded with magnesium ions alone was observed. Potassium ions, when present together with magnesium and sodium ions exerted a stimulating effect on phosphatase activity much greater than that observed with the latter two cations alone. This activation by potassium ions was inhibited by increasing concentrations of ouabain and the inhibition by any concentration of ouabain, at constant concentrations of magnesium and sodium ions, decreased as potassium ion concentration increased. Since the media containing magnesium ions, 5.0 mmoles/l, sodium ions, 100 mmoles/1; potassium ions, 10mmoles/l; and ouabain, 10 moles/1 resulted in the same hydrolysis of ATP as the same media with no potassium ions, 99.8 jumoles P^  per hour per mg N, i t was considered that this value represented complete inhibition of the sodium and potassium-activated ATPase. Figure 8 presents graphically the ouabain inhibition data of Table IV for po-tassium ion concentration of 10.0 and 20.0 mmoles/l cal-culated with this assumption. The for ouabain was 2 X 10"^ and 7 X 10"^ moles/1 for potassium ion concentra-tions of 10.0 and 20.0 mmoles/l respectively. Section II Changes in ouabain-sensitive ATPase upon transfer to sea water The changes i n TS-ATPase, sodium ac t i v a t i o n and po-tassium a c t i v a t i o n with time of exposure of the f i s h trans-ferred d i r e c t l y from fresh water to 100$ sea water, s a l i n i t y 3 0 o / Q O , i n February, 1969 are presented i n Figure 9. Each point between 0 and 22 days exposure represents the mean, - 1 standard deviation, of 3 to 4 r e p l i c a t e preparations. Figure 9 demonstrates that although no s i g n i f i c a n t changes i n T S - a c t i v i t y occurred u n t i l 10 days a f t e r transfer, s i g n i f i c a n t changes i n sodium ac t i v a t i o n and potassium ac-t i v a t i o n occurred after 5 days exposure to sea water. Sodium act i v a t i o n declined s t e a d i l y at a rapid rate from day 2 to day 6 and continued declining at a slower rate thereafter to a value of 7 jjmoles P^hr/mg N at day 50. Potassium a c t i v a t i o n increased steadily a f t e r 4 days ex-posure to a maximum of 142 ,umoles P^/hr/mg N at day 14, then declined s l i g h t l y and l e v e l l e d o f f at approximately ^30 ;amoles P^/hr/mg N. The changes i n TS-activity p a r a l l e l l e d changes i n potassium a c t i v a t i o n a f t e r 8 - 1 0 days exposure as the l a t t e r comprised 80 - 90$ of the t o t a l ouabain-sensitive a c t i v i t y . The times i n sea water required f o r the i n i t i a l 50$ of the changes i n potassium and sodium activations to occur were 9 and 5 days respectively. Although changes i n the sodium activation and potassium acti v a t i o n of the fresh water controls were observed over the the experimental period these changes were much l e s s than those observed i n sea water exposed animals and may have represented normal changes observed during the smolting period (see Figure 26 Legend f o r F i g u r e 9* Changes i n T S - a c t i v i t y , (upper s c a l e ) , sodium a c t i -v a t i o n , (middle s c a l e ) , and potass ium a c t i v a t i o n , ( lower s c a l e ) , w i t h t ime o f exposure to sea w a t e r . The mean - 1 s t a n d a r d d e v i a t i o n i s p r e s e n t e d f o r sea water exposed f i s h , ( s o l i d c i r c l e s ) , and c o n t r o l s m a i n t a i n e d i n f r e s h w a t e r , (open c i r c l e s ) . 2 0 C H D A Y S A F T E R T R A N S F E R T O S E A W A T E R 10). No difference i n the degree of s i l v e r i n g of the skin or loss of parr marks was observed between f i s h exposed to sea water and the fresh water controls. Seasonal changes i n ouabain-sensitive ATPase of .juvenile  coho g i l l The changes i n the T S - a c t i v i t y , sodium act i v a t i o n , and potassium a c t i v a t i o n of the ouabain-sensitive ATPase during the f r y , presmolt and smolt stages of the l i f e cycle are pre-sented i n Figure 10. Although these l i f e stages are not well defined and tend to overlap i n time within a population of f i s h (Houston 1963), the following general descriptions may be v a l i d f o r the juvenile coho salmon used i n these experi-ments : Fry stage: parr marks dark and sharply delineated; no noticeable darkening of p e l v i c , anal, dorsal or caudal f i n s ; white coloration on the f i r s t few rays of the anal f i n ; weight-length r e l a t i o n s h i p approximate&ythe upper broken l i n e of Figure 3; was included i n the period ending i n l a t e November, 1968. Pre-smolt stage: parr marks dark but borders l e s s d i s -t i n c t , s l i g h t darkening of the t i p s of the p e l v i c , anal, dor-s a l and caudal f i n s ; noticeable s i l v e r i n g of the ventral and ventro-lateral skin; weight-length r e l a t i o n s h i p i n the t r a n s i -t i o n phase between the two broken l i n e s of Figure 3 ; and oc-curred i n the period of mid-December, 1968 to l a t e January, 1969. Smolt stage: parr marks v i s i b l e only i n oblique l i g h t , intense darkening of the extremities of the p e l v i c , anal, dor-s a l and caudal f i n s ; intense s i l v e r i n g of the ventral and 2 8 Legend f o r Figure 10. Changes i n TS-activity, (upper scale), sodium a c t i -vation, (middle scale), and potassium activation, (lower scale), from October 1, 1968 to May 8, 1969. The mean i 1 standard deviation i s presented f o r sea water reared f i s h , ( s o l i d c i r c l e s ) , and fresh water reared f i s h , (open c i r c l e s ) . The broken l i n e from A p r i l 21 to May 6 represents f i s h transferred from fresh to sea water on A p r i l 21. 0 J 20 9 29 19 8 28 17 9 29 18 8 OCT NOV DEC JAN FEB MAR APR MAY ventro-lateral skin; weight-length relationship approximates the lower broken line of Figure 3; and occurred i n the period of mid-February to mid-April, 1 9 6 9 . Sea water reared fis h also exhibited the characteristics of these stages but differentiation was less distinct than in fresh water reared coho. The data for sea water reared coho are included in Figure 1 0 as a reference to indicate whether or not the changes observed are the result of physiological changes occurring independently of the salinity of the aqua-t i c environment. The fry stage, ending in late November 1 9 6 8 , was charac-terized by relatively low, uniform levels of TS-activity, and potassium activation in both groups of f i s h although the va-lues were 3 to 7 times higher in the sea water reared f i s h . The high values of these activities observed in sea water reared coho on October 3, 1 9 6 8 may have been the result of adaptation of these f i s h to sea water as preliminary sea water adaptation experiments performed in this period showed a large i n i t i a l increase and subsequent daline in TS-activity, (data not presented). Sodium activation was also low in this period but was always higher in fresh water reared coho than in sea water reared f i s h . The f i r s t portion of the pre-smolt period (early December 1 9 6 8 , to mid-January 1 9 6 9 ) was characterized by 3-fold i n -crease in both TS-activity and potassium activation for sea water reared f i s h and 3.7 and 4 . 6 fold increases respectively for fresh water reared f i s h . These activities then declined from mid-January to mid-February i n both groups of f i s h . I n i t i a l l y sodium a c t i v a t i o n changed e r r a t i c a l l y i n fresh water reared coho but reached a low i n mid-January then increased sharply u n t i l mid-February. In sea water reared coho the so-dium a c t i v a t i o n was r e l a t i v e l y constant during the pre-smolt stage. In the smolt stage, mid-February to A p r i l 1969, the level s of T S - a c t i v i t y , sodium activation and potassium a c t i v a t i o n i n sea water reared f i s h did not change s i g n i f i c a n t l y from the values observed at the end of the pre-smolt stage. The fresh water reared coho, however, demonstrated marked decreases i n sodium a c t i v a t i o n and increases i n TS-a c t i v i t y and potassium a c t i v a t i o n u n t i l the f i r s t week i n A p r i l . Thus during t h i s period the c h a r a c t e r i s t i c s of the ouabain-sensitive ATPase of these f i s h tended to approach those of coho residing i n sea water. Freshwater f i s h transferred to sea water on A p r i l 18 (broken l i n e of Figure 10) showed the same increases i n TS-a c t i v i t y and potassium activation as seen i n Figure 9 while the former a c t i v i t y continued to decline i n f i s h held i n fresh water. This decline i n TS-a c t i v i t y i n the freshwater f i s h during the period of A p r i l 8 to May 6 was the re s u l t of de-creasing sodium a c t i v a t i o n which declined to a value near that observed i n f i s h during the f r y stage. DISCUSSION Section I As Skou (1965), points out, the assay conditions of pH, temperature, ATP concentration and sodium and potassium con-centrations which result in maximal hydrolysis of ATP by the ouabain-sensitive, (Na + +K"*")-activated ATPase vary widely in enzyme preparations from different tissue; moreover the pat-tern of inhibition of this enzyme by ouabain is also different in different preparations. Since the determination of optimal assay conditions in these experiments employed sea water ad-apted f i s h as an enzyme source, a question could be raised as to whether these assay conditions were optimal for enzyme pre-parations from fresh water f i s h . It would appear, however, that since the TS-activities of enzyme from f i s h adapted to both environments were very similar during the period of March and April, 1969 (Figure 10) the optimal assay conditions may be similar for enzymes from both sources. Only a detailed ana-ly s i s of the enzyme kinetics at closely spaced intervals during fresh water development would provide satisfactory answers to these problems. The optimal pH of 7.4 for the coho g i l l ouabain-sensitive ATPase was similar to that of the Japanese eel, reported as 7.5 by Kamiya and Utida (1968), although i t i s slightly different from that used by other workers, (Skou, 1957, Epstein et a l . 1967). Although the incubation temperature of 40°C has no physiological significance in fi s h i t was the temperature at which maximum ATP hydrolysis occurred. The Km of the enzyme f o r the ATP s u b s t r a t e , 0.2 mmoles/l i s very s i m i l a r to that recorded by Skou (I960), f o r the crab nerve (Na + K )-stimulated ATPase although the present ex-periments employed equimolar magnesium and ATP concentrations w h i l e Skou v a r i e d the magnesium/ATP r a t i o . Skou (1957) de-monstrated, however, that maximal ATP h y d r o l y s i s occurred when the magnesium and ATP concentrations were equal. The V m a x occurred at an ATP c o n c e n t r a t i o n of 0.5 mmoles/l which i s i n agreement w i t h the value of 0.75 mmoles/l reported f o r n a s a l gland homogenates of f r e s h water and s a l i n e adapted ducks ( F l e t c h e r et a l , 1967). In experiments u t i l i z i n g the g i l l s of sea water adapted Japanese eels as a source of enzyme Kamiya and Utida (1967), determined the concentrations of potassium and sodium ions which when present w i t h magnesium ions and ATP gave maximal h y d r o l y s i s of the ATP s u b s t r a t e . These authors found thafct maximal h y d r o l y s i s occurred at potassium ion concentrations of 15, 20 and 40 mmoles/l when the sodium concentrations were 10, 50 and 100 mmoles/l, r e s p e c t i v e l y . In sea water adapted coho, however, maximal o u a b a i n - s e n s i t i v e ATP h y d r o l y s i s occur-red at potassium i o n concentrations of 12.5, 15.0 and 20.0 mmoles/l r e s p e c t i v e l y f o r these sodium concentrations. These data i n d i c a t e that the o u a b a i n - s e n s i t i v e ATPase of coho g i l l s may have a higher a f f i n i t y f o r potassium ions than does t h i s enzyme i n Japanese ee]s. This suggestion i s supported by the f a c t that f o r c a t i o n concentrations of 100 mmoles Na and 20 mmoles K per l i t r e and ouabain concentrations of 10" , 5 X 10 and 10" ^  m o l e s / l r e s p e c t i v e i n h i b i t i o n s of 15, 30, and 55% of maximal ouabain-sensitive activity were recorded in Japanese eels (ibid) compared to 30, 44 and 56$ respectively in coho salmon (Figure 8). Thus, the sea water adapted coho g i l l en-zyme not only has a higher a f f i n i t y for potassium ions but this activation by potassium ions i s more sensitive to low concentrations of ouabain (Table IV) than this enzyme in the g i l l s of sea water adapted Japanese eels. The Mg^+-ATPase was not affected by any concentration of ouabain. This finding i s in agreement with the results on other tissues (Bonting and Caravaggio 1963, Kamiya and Utida, 1968, Post et a l I960 and Skou I960). The cause of the appa-rent inhibition by sodium ions of a fraction of the Mg2"^-ATP-ase (Table IV) is unknown. Since the enzyme preparation used in the present investigations was relatively impure i t can be speculated that sodium ions may act as a control mechanism which deactivates certain Mg2*-ATPases in order to insure adequate ATP substrate i s available for the (Na + + K +)-acti-vated enzyme. Another possible explanation suggested by Skou (1965) i s that the Mgz+-ATPase and the (Na + + K +)-activated ATPase may be part of the same enzyme system; the activity of this changes in different ionic environments of with different methods of purification. In general then the characteristics of the partially purified ouabain-sensitive (Na + + K +)-activated adenosine t r i -phosphatase prepared from the g i l l s of sea water adapted coho are similar i f not identical to those of other tissues and animals. Section II Relatively l i t t l e research into the effect of transfer from a hypotonic to a hypertonic environment on the (Na^t K )-activated ATPase of fis h g i l l s has been performed. Epstein et a l (1967) found that a 7-fold increase in the activity of g i l l microsomal preparations of this enzyme occurred when young k i l l i f i s h Fundulus heteroclitus were adapted to sea water as compared to fresh water controls. Kamiya and Utida (1968) reported 5-fold increases in activity of Nal treated microsomal preparations when Japanese eels Anguilla japonica were transferred from fresh to sea water. The latter authors also found that a rapid increase in the activity of this enzyme occurred in the f i r s t seven days of sea water exposure followed by a more gradual increase to the thirtieth day of exposure. In an analogous investigation, Fletcher et, a l , ( I 9 6 7 ) , using inhibition by 0 . 1 millimolar ouabain to measure the (Na+ + Re-activated ATPase in crude homogenates of duck nasal glands, found that the total ouabain-sensitive activity reached a maximum 8 to 10 days after a saline solution ( 284 mmoles/l NaCl) had been substituted for fresh water as the sole source of drinking water and that changes in the excretion of sodium and chloride by this gland were correlated to increases in the enzyme activity. The changes in TS-activity for coho trans-ferred directly from fresh to sea water (Figure 9) are gene-ral l y in agreement with these results from other f i s h g i l l s and duck nasal glands. The changes in sodium activation and in potassium activation presented in Figure 9 , however, have not been p r e s e n t e d e l sewhere . I f the h y p o t h e s i s t h a t i n t r a -c e l l u l a r sodium i s exchanged f o r e x t r a c e l l u l a r potas s ium (Skou 1957, I960, 1965, B o n t i n g and Caravagg io 1 9 6 3 , Post et a l . i 9 6 0 ) , and t h a t the e f f e c t o f potass ium i o n s i s t o a c t i v a t e t h i s t r a n s f e r o f sodium ( W i l l i s 1968 a , b , ), i s a c c e p t e d , then a measure o f t h e a c t i v a t i n g e f f e c t o f potass ium i o n s would d e s c r i b e the p o t e n t i a l f o r a c t i v i t y o f t h e enzyme s y s -tem. T h i s p r o p o s i t i o n i s suppor ted by the o b s e r v a t i o n t h a t sea water adapted coho which have an a b s o l u t e requ i rement t o e x c r e t e sodium i o n s and r e t a i n water have a h i g h potass ium a c t i v a t i o n which exceeds 90$ o f t h e T S / a c t i v i t y . F r e s h water adapted coho w i t h o u t t h i s requ i rement have a low potass ium a c -t i v a t i o n which may be as low as 22$ of t h e TS a c t i v i t y , ( F i g u r e 10, February 19, 1969) . V/hen coho a r e t r a n s f e r r e d from f r e s h to s ea water t h e i n i t i a l decrease \ , in sodium a c t i -v a t i o n and c o n c u r r e n t r i s e i n potass ium a c t i v a t i o n would p e r -mi t the enzyme system to more e f f e c t i v e l y remove i n t e r n a l so-dium i o n s to the e x t e r n a l medium l o n g b e f o r e t h e a c t u a l c o n -c e n t r a t i o n o f enzyme showed any i n c r e a s e . In t h i s r e s p e c t i t has been found (Conte and L i n , 1967) t h a t t h e t u r n o v e r t ime o f 50$ o f l a b e l l e d DNA, which i s a measure o f the r a t e o f de novo s y n t h e s i s o f DNA, was 15 .8 and 5.8 days f o r t h e g i l l c e l l s o f f r e s h and sea water adapted j u v e n i l e coho r e s p e c t i v e l y . In a d d i t i o n some s a l t - i n d u c i b l e changes i n a n t i g e n i c i t y have been found between g i l l microsomes o f f r e s h and sea water adapted chinook salmon which may r e f l e c t changes i n t h e ( N a + + Re-a c t i v a t e d ATPase (Conte and M o r i t a , 1968) . I t i s important to emphasize t h a t i n f i s h many p a s s i v e changes i n t h e d i s t r i -b u t i o n o f c h l o r i d e , e x t r a c e l l u l a r phase volume and p e r m e a b i l i -t y o f the body s u r f a c e t o i o n s , which t e n d t o decrease t h e r a t e o f change i n t h e i o n c o n c e n t r a t i o n s o f t h e body f l u i d s d u r i n g exposure to a h y p e r t o n i c environment but t h a t these changes can o n l y f u n c t i o n t o a l l o w r e g u l a t o r y proces se s s u f f i c i e n t t ime to become o p e r a t i v e (Houston, 1964) . The s e a s o n a l changes observed i n t h e l e v e l s and c h a r a c -t e r i s t i c s o f a c t i v a t i o n by sodium and potass ium i o n s o f t h e o u a b a i n - s e n s i t i v e ATPase o f coho g i l l s ( F i g u r e 10 ) , appear to be r e l a t e d to t h e s tage o f development o f the se f i s h . These s tages are r e l a t e d to changes i n the w e i g h t - l e n g t h r e -l a t i o n s h i p , d e p o s i t i o n o f guanine i n the b e l l y s k i n - (Van-s tone and M a r k e r t , 1968) , l o s s o f p a r r marks^ changes i n i n t e r -n a l i o n c o n c e n t r a t i o n s and d i s t r i b u t i o n (Houston and T h r e a d -g o l d , 1963; Conte and Wagner, 1965) , and to s e v e r a l endocr ine and b e h a v i o r a l changes (Hoar, 1965 ) . The p re sen t data i n d i -cate..; t h a t d u r i n g the p e r i o d o f O c t o b e r , 1968 to m i d - F e b r u a r y 1969 t h e changes observed i n enzyme l e v e l s were a response t o changes i n i n t e r n a l f a c t o r s independent o f t h e a q u a t i c e n v i -ronment, s i n c e the p a t t e r n , o f changes i n T S - a c t i v i t y were s i m i l a r i n f r e s h water and sea water r e a r e d f i s h . The c h a r a c -t e r i s t i c s o f a c t i v a t i o n by sodium i o n s and potas s ium i o n s , however, were d i f f e r e n t i n t h e two groups of f i s h (see p r e -v i o u s d i s c u s s i o n ) . -The peak i n T S - a c t i v i t y and potass ium a c t i v a t i o n d u r i n g the p e r i o d December 1968 and January 1969 may r e f l e c t the ob-s e r v a t i o n t h a t j u v e n i l e coho can osmoregulate i n a s h o r t e r time when transferred directly to sea water in this period than during the months immediately preceeding and following this interval (Conte, Wagner, Fessler and Gnose, 1966). Houston and Threadgold (1963) found significant decreases in plasma and tissue chloride during the parr to silvery-parr transition stage of Atlantic salmon which may reflect changes in sodium ion concentrates in this period. The smolt stage in fresh water f i s h was characterized by increasing levels of TS-ATPase, decreasing levels of sodium activation and increasing levels of potassium activation. These results agree with the findings of Conte et a l . (1966), that the time required for coho to in i t i a t e osmoregulation in sea water decreases to a minimum of 30 hours as the smolting period progresses. Thus (the/levels and characteristics of the activation components of the ouabain-sensitive ATPase of smolting fresh water coho as well as the,ability to osmore-gulate in sea water tend to approach those observed in sea water adapted, f i s h of the same age. It has been demonstrated that the g i l l s are the major site of ion excretion in seawater f i s h (Motais, Garcia Romeu, and Maetz, 1966). If the sodium and potassium activated adenosine triphosphatase of the g i l l s i s in fact an integral part of the osmoregulatory apparatus in seawater fish then the significance of preadaptive changes in the enzyme activity in f i s h migrating from fresh water to sea water i s obvious. The abil i t y to osmoregulate quickly upon exposure to sea water would avoid the .stresses associated with exposure of freshwater fi s h to a hypertonic environment. The preadapted f i s h could leave the estuarine environment and move directly to f u l l strength sea water, thus avoiding concentrations of potential predators and making available the abundant, food resources of the sea. 39 LITERATURE CITED Assoc ia t ion of O f f i c i a l A g r i c u l t u r a l Chemists. I960. O f f i c i a l methods of Ana lys i s . 9th ed. Baggerman, B . , I960. Factors i n the diadromous migrations of f i s h . Symposium of the Zoo log ica l Society London. 1: 33-58. Bonting, S . L . , and L . L . Caravaggio. 1963. Studies on sodium-potassium-activated adenosinetripnosphatase. V. Cor-r e l a t i o n of enzyme a c t i v i t y with cat ion f lux i n s i x t i s sues . Archives of Biochemistry and Biophys ics . 101: 37 - 46. Conte, F . P . , and D .H.Y . L i n . 1967. K ine t i c s of c e l l u l a r morphogenesis i n g i l l epithelium during sea water adapt-at ion of Oncorhynchus (Walbaum). Comparative Biochemis-t r y and Physiology. 23: 945 - 957. Conte, F . P . , and T . N . M o r i t a . 1968. Immunological study of c e l l d i f f e r e n t i a t i o n i n ' g i l l epithelium of euryhaline Oncorhynchus, (Walbaum)."Comparative Biochemistry and Physiology. 24: 445-454. Conte, F . P . , and H . H . Wagner. 1965. Development of osmotic and ion ic regulat ion i n juveni l e steelhead t r o u t , Salmon  g a i r d n e r i . Comparative Biochemistry and Physiology. 14: 603 - 619 Conte, F . P . , H .H. Wagner, J . Fess l er and C. Gnose. 1966. Development of osmotic and ion ic regulat ion i n juveni le coho salmon, Oncorhynchus k i su tch . Comparative Bioche-mistry and Physiology. 1*8: 1 - 1 5 . Epste in , F . H . , A . J . Katz, and G . E . P ick ford . 1967. Sodium and potassium-activated adenosine triphosphatase of g i l l s : r o l e i n adaptation of te leosts to sea water. Science. 156: 1245 - 1247. Fle tcher , G . L . , I . M . S ta iner , and W.N. Holmes. 1967. Sequen-t i a l changes i n the adenosinetripnosphatase a c t i v i t y and the e l e c t r o l y t e excretory capacity of the nasal glands of the duck (Anas platyrhynchos) during the period of adapt-at ion to hyper ton ic . sa l ine ." Journal of Experimental Bio logy. 47(3): 375 - 391. Hawk, P . B . , B . L . Oser, and W.H. Summerson. 1947. P r a c t i c a l Phys io log i ca l Chemistry. 12th ed. The Blackstone C o . , Toronto. Hoar, W.S. 1965. The endocrine system as a chemical l i n k between the organism and i t s environment. Transactions of the Royal Society of Canada. 4th s er i e s , V o l . I l l , Sec. I I I . -.:.175-200. 40 Houston, A.H. 1964. On passive features in the osmoregula-tory adaptation of anadromous salmonids to sea water. Journal of the Fisheries Research Board of Canada. 21: 1535 - 1538. Houston, A.H., and L.T. Threadgold. 1963. Body f l u i d regu-lation in smolting Atlantic salmon. Journal of the Fisheries Research Board of Canada. 20: 1355 - 1369. Kamiya, M., and S. Utida. 1968. Changes in activity of so-dium-potassium-activated adenosinetriphosphatase in g i l l s during adaptation of the Japanese eel to sea water. Comparative Biochemistry and Physiology. 26: 675 - 685. Motais, R., F. Garcia Romeu, and J. Maetz. 1966. Exchange diffusion effect and euryhalinity in teleosts. Journal of General Physiology. 50: 391 - 422. Post, R.L., C.R. Merritt, C.R. Kinsolving, and CD. Albright. I960. Membrane adenosine triphosphatase as a p a r t i c i -pant in the active transport of sodium and potassium in human erythrocyte. Journal of Biological Chemistry. 235(6): 1796 - 1302. Skou, J.C. 1957* The influence of some cations on an ade-nosine triphosphatase from peripheral nerves. Bioche-r mica et Biophysica Acta. 2 3 : 394 - 401. Skou, J.C. I960. Further investigations on a Mg + + + Na + -ac-tivated adenosinephosphatase, possibly related to the ac-tive, linked transport of Na and K+ across the nerve membrane. Biochemica et Biophysica Acta. 42: 6 - 22. Skou, J.C. 1965. Enzymatic basis for active transport of Na + AND"K+ across c e l l membranes. Physiological Reviews. 45: 596 - 617. Vanstone, W.E., and J.R. Markert. 1968. Some morphologi-cal "and biochemical changes in coho salmon, Oncorhynchus kisutch, during parr-smolt transformation. Journal of the Fisheries Research Board of Canada. 25: 2403 - 2417. Wil l i s , J.S., 1968, a. Ouabain inhibition of ion transport and respiration in renal cortical slices of ground squirrel and hampsters. Biochemica et Biophysica Acta. 163: 506 - 515. W i l l i s , J.S., 1968, b. The interaction of K+, ouabain and Na + on the cation transport and respiration of renal cortical cells of hampsters and ground squirrels. Bio-chemica et Biophysica Acta. 163: 515 - 530. 

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