UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

The mechanisms and energetics of nitrate uptake by marine phytoplankton Falkowski, Paul Gordon 1975

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1975_A1 F34_5.pdf [ 4.88MB ]
Metadata
JSON: 831-1.0100031.json
JSON-LD: 831-1.0100031-ld.json
RDF/XML (Pretty): 831-1.0100031-rdf.xml
RDF/JSON: 831-1.0100031-rdf.json
Turtle: 831-1.0100031-turtle.txt
N-Triples: 831-1.0100031-rdf-ntriples.txt
Original Record: 831-1.0100031-source.json
Full Text
831-1.0100031-fulltext.txt
Citation
831-1.0100031.ris

Full Text

THE MECHANISMS AND ENERGETICS OF NITRATE UPTAKE BY MARINE PHYTOPLANKTON by P a u l Gordon Falk o w s k i S. C i t y C o l l e g e o f the C i t y U n i v e r s i t y o f New York A. C i t y C o l l e g e o f the C i t y U n i v e r s i t y o f New York A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE. REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of BOTANY We accept t h i s t h e s i s as conforming to the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH June 1975 COLUMBIA In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h Co lumb ia , I a g ree that the 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 tudy . I f u r t h e r agree 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 purposes 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 tha 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 i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i thou t my w r i t t e n p e r m i s s i o n . Department o f Botany  The U n i v e r s i t y o f B r i t i s h Co lumbia Vancouver 8, Canada -6 i ABSTRACT The r e s u l t s of t h i s study suggest t h a t NO^ uptake i n many (but not a l l ) s p e c i e s of marine phytoplankton i s medi-ated by a membrane-bound (NO^, C l ~ ) - a c t i v a t e d adenosine t r i p h o s p h a t a s e . In the presence of N0~ and C l ~ , s e m i - p u r i f i e d membrane p r e p a r a t i o n s e x h i b i t enhanced adenosine t r i p h o s -phatase a c t i v i t y . The enzyme has c h a r a c t e r i s t i c s common to o t h e r membrane-bound p r o t e i n s : a break i n the A r r h e n i u s p l o t of 30.9 Kcal/mole a t 2.9 C, p a r a l l e l p u r i f i c a t i o n w i t h + + the (Na + K ) - a c t i v a t e d t r a n s p o r t adenosine t r i p h o s p h a t a s e , and a c t i v a t i o n of c a t a l y t i c a c t i v i t y by n o n - i o n i c and a n i o n i c d e t e r g e n t s . I t i s i n f e r r e d from p a r a l l e l p u r i f i c a t i o n of the (NO^/ C l ~ ) - a c t i v a t e d adenosine t r i p h o s p h a t a s e and the p h y s i o l o g i c a l k i n e t i c s of NO^ uptake by i n t a c t c e l l s , t h a t the enzyme t r a n s l o c a t e s NO^ a c r o s s the c e l l membrane, i n t o the cytoplasm, a g a i n s t the c h e m i c a l c o n c e n t r a t i o n g r a d i e n t of the i o n . The h a l f - s a t u r a t i o n c o n s t a n t s f o r a c t i v a t i o n of the adenosine t r i p h o s p h a t a s e by NO~ are l e s s than 1 uM f o r most s p e c i e s t e s t e d and c o r r e l a t e w i t h h a l f - s a t u r a t i o n c o n s t a n t s f o r NO^ uptake by whole c e l l s . The t h r e e d i n o f l a g e l l a t e s t e s t e d d i d not e x h i b i t any r e l a t i o n s h i p between NO^ c o n c e n t r a t i o n s and ATP h y d r o l y s i s , and i t i s i n f e r r e d t h a t the (NC^, CI ) - a c t i v a t e d adenosine t r i p h o s p h a t a s e i s probably absent from t h i s group. R e s u l t s of m e t a b o l i c i n h i b i t o r s t u d i e s ( i n c l u d i n g KCN, 2 , 4 - d i n i t r o p h e n o l , d i c h l o r o d i m e t h y l u r e a , and c a r b o n y l 11 cyanide m-chlorophenylhydrozone) imply t h a t the source of ATP f o r the NO^ t r a n s p o r t i s p r i m a r i l y c y c l i c photo-phosphorylation i n v i v o . These r e s u l t s are c o n s i s t e n t w i t h observations of s e l e c t i v e i n h i b i t i o n of NO^ uptake i n u n i a l g a l c u l t u r e s as w e l l as i n n a t u r a l p o p u l a t i o n s . F i e l d s t u d i e s w i t h n a t u r a l phytoplankton communities from Knight I n l e t , B. C , suggest a p h y s i o l o g i c a l adaptation to e x t e r n a l n i t r o g e n c o n c e n t r a t i o n s may occur. This adap-t a t i o n i s c h a r a c t e r i z e d by i n c r e a s e d i n t r a c e l l u l a r c h l o r o -p h y l l a s y n t h e s i s i n response to 10-1555 n i t r o g e n enrichment over a 6-8 hr p e r i o d . During the adaptive p e r i o d carbon f i x a t i o n i s t e m p o r a r i l y suppressed, apparently due to competition between i n o r g a n i c carbon and i n o r g a n i c n i t r o g e n f o r high-energy n u c l e o t i d e s from the l i g h t r e a c t i o n s . The r e s u l t s of t h i s study are r e l a t e d to previous proposals f o r the metabolic pathway of n i t r o g e n i n marine phytoplankton. In c o n c l u s i o n , a modified pathway i s proposed s t r e s s i n g (1) group d i f f e r e n c e s , i n t h a t n i t r o g e n a s s i m i l a t i o n i n d i n o f l a g e l l a t e s appears d i f f e r e n t from other groups, and. (2) the e n e r g e t i c s and b i o c h e m i c a l feed-back c o n t r o l s ^ c n i t r o g e n a s s i m i l a t i o n . i i i TABLE OF CONTENTS Page Abstract i Table of Contents i i i L i s t of Tables v L i s t of Figures v i Acknowledgements v i i i Introduction 1 Materials and Methods 7 A. Laboratory Studies 7 I. Cultures 7 I I . Preparation of membrane "ve s i c l e s " 8 I I I . Enzyme assays 11 a) ( N 03* c l )-activated adenosine 11 triphosphatase 1. Inorganic phosphate method 11 2. Coupled enzyme assay . 12 3. ( Y - P 3 2 ) - A T P method 12 b) Nitrate reductase 13 c) (Na + + K + ) - a c t i v a t e d adenosine 14 triphosphatase IV. Determination of protein and chlorophyll a 14 V. ATP determinations 14 B. F i e l d Studies i n Knight I n l e t , B. C. 16 Results 22 A. Laboratory Studies 22 I. C h a r a c t e r i s t i c s and k i n e t i c s of the (NO^, CI ) 22 -activated adenosine triphosphatase a) Ef f e c t s of hydrogen ion concentration 22 i v bl E f f e c t s of temperature on enzyme a c t i v i t y ^4 cl Determination of enzyme locus ^1 dj Divalent cation requirements OQ e) Interference from (Na+ + K+)-activated adenosine triphosphatase f) Anion requirements and s p e c i f i c i t y 32 g) Nucleotide s p e c i f i c i t y 39 h) V e l o c i t i e s of the reaction ( a c t i v i t y ) 39 i) A c t i v i t y of the ( N O 3 , C l )-activated 41 adenosine triphosphatase i n Skeletonema costaturn grown on NH+ I I . NO^ uptake and the i n t r a c e l l u l a r ATP pools i n 42 Skeletonema costatum 44 a) E f f e c t s of l i g h t and temperature on the 42 i n t r a c e l l u l a r ATP pool b) E f f e c t s of metabolic i n h i b i t o r s c) E f f e c t s of metabolic i n h i b i t o r s on NO^ 47 uptake d) E f f e c t s of e x t r a c e l l u l a r NH4, N0 3, and 49 P 0 | ~ on i n t r a c e l l u l a r ATP pools B. F i e l d Studies on Natural Phytoplankton Communities 51 i n Knight I n l e t I. Morphometric and hydrographic considerations 51 II . A b r i e f d e s c r i p t i o n on the phytoplankton 54 community I I I . Experimental r e s u l t s 55 a) E f f e c t s of nitrogen enrichment on 55 inorganic carbon f i x a t i o n b) E f f e c t s of nitrogen enrichment on 57 chlorophyll a / c e l l Discussion 60 A. Nitrate Uptake i n Marine Phytoplankton 60 B. A Model f o r N 0 3 Uptake Based on Bisubstrate Kinetics 76 References 83 Appendix I. Preliminary ..experiments on dinoflageHates 90 Appendix I I . Abbreviations 95 V LIST OF TABLES T a b l e Page 9 1. Medium " J " 2. Coordinates of sample stations i n Knight I n l e t 19 3. P a r a l l e l p u r i f i c a t i o n of the (NO^, C l ~ ) - 28 activated adenosine triphosphatase with the (Na+ + K + ) - a c t i v a t e d adenosine triphosphatase. 2+ 4- A c t i v i t y of the Mg -stimulated adenosine 31 triphosphatase 5. A c t i v i t y of the ( N O 3 , CI )-activated adenosine 35 triphosphatase i n nine species 6. Nucleotide s p e c i f i c i t y of the (N0~, C l ~ ) - 40 activated adenosine triphosphatase 7. Ef f e c t s of e x t r a c e l l u l a r nutrients on the 50 ATP pool 8. Nit r a t e and s a l i n i t y i n Knight I n l e t during 52 the sampling periods 9. Variations i n chlorophyll a / c e l l with 59 e x t r a c e l l u l a r nitrogen 10. I n t r a c e l l u l a r ion composition i n some marine 9 2 algae v i LIST OF FIGURES Page Fi g u r e 1. N i t r o g e n a s s i m i l a t i o n i n phytoplankton 3 Seasonal v a r i a t i o n s i n c h l o r o p h y l l a i n Knight I n l e t , B. C. 17 3. Knight I n l e t and a s s o c i a t e d waters, showing s t a t i o n p o s i t i o n s 18 4. pH p r o f i l e of the (NO^, CI ) - a c t i v a t e d adenosine triphosphatase 23 5. Arrhenius p l o t of the (NO^, CI ) - a c t i v a t e d adenosine triphosphatase without detergents 25 6. Arrhenius p l o t of the (NO^, C l ~ ) - a c t i v a t e d adenosine triphosphatase w i t h detergents 26 7. E f f e c t s of Na + on the (NO^, C l ~ ) - a c t i v a t e d adenosine triphosphatase 30 8a. Michaelis-Menten p l o t of the (NO^, C l ~ ) -a c t i v a t e d adenosine triphosphatase 33 b. Lineweaver-Burk p l o t of the (NO^, C l ~ ) - a c t i v a t e d adenosine triphosphatase 33 9. Michaelis-Menten and Woolf p l o t s f o r seven species 36 10. V a r i a t i o n s i n K f o r C l ~ w i t h NOl concentrations m 3 38 11. E f f e c t s of l i g h t and temperature on i n t r a c e l l u l a r ATP pools 43 12. E f f e c t s of metabolic i n h i b i t o r s on the ATP pool 46 12-2. E f f e c t s of.metabolic i n h i b i t o r s on NO^ uptake 48 13. Bottom topography of Knight I n l e t 5 3 14. V a r i a t i o n i n a s s i m i l a t i o n r a t i o s w i t h n i t r o g e n enrichments 56 v i i F i g u r e Page 15. A model of n i t r a t e metabolism i n phytoplankton 73 Ceoccept d i n o f l a g e l l a t e s ) 16. A proposed model of n i t r a t e metabolism 75 i n d i n o f l a g e l l a t e s 17. E f f e c t s of l i g h t and e x t r a c e l l u l a r N O 3 on NOZ 79 uptake v e l o c i t i e s v i i i Acknowledgements I would l i k e to express my sincerest thanks to Dr. F. J. R. Taylor, who served as thesis advisor, and from whom I could always receive thoughtful and uns e l f i s h encourage-ment. In addition, I would l i k e to thank Drs. T. R. Parsons, E. B. Tregunna and P. W. Hochachka f o r t h e i r h e l p f u l suggestions and c r i t i c i s m s , and R. F. Scagel, who served as chairman of the research committee. Most of a l l , I would l i k e to apologize to my wife, Donna, from whom I have stolen countless evenings and week-ends of companionship, and without whose love and support t h i s research would have been impossible. 1 "Every theory of the course of events i n nature i s necessari-l y based on some process of s i m p l i f i c a t i o n and i s to some extent, therefore, a f a i r y t a l e . " S i r Napier Shaw Introduction Photosynthetic marine organisms, l i k e t h e i r t e r r e s t r i a l counterparts, require l i g h t , carbon dioxide, c e r t a i n inor-ganic ions, and often organic compounds for growth and reproduction. In most marine environments, the inorganic nitrogen sources, ammonium, n i t r i t e , n i t r a t e (and possibly hydroxylamine) appear to be i n l i m i t e d supply to primary producers r e l a t i v e to the demand. Most natural o l i g o t r o p h i c conditions are encountered i n such areas as the Eastern T r o p i c a l P a c i f i c (Thomas, 1969, 19 70; Thomas and Owen, 19 71), the Sargasso Sea (Hulburt, 1960; Riley et a l , 1949), and perhaps the oceanic province i n general (Parsons and Takahashi, 1973). Even i n n e r i t i c waters, t o t a l inorganic -5 nitrogen i s usually less than 10 M (Harvey, 1960), and phy-toplankton appear to t h r i v e i n such a d i l u t e nutrient medium. Increasingly, i t i s becoming c l e a r , that the major l i m i t i n g factor c o n t r o l l i n g phytoplankton growth i n the seas i s most frequently a suitable nitrogen source (Corner and Davies, 19 71). Of the n a t u r a l l y occurring nitrogen sources, ammonium appears to be the most en e r g e t i c a l l y favorable form for amino acid anabolism. I t has been observed that more oxidized forms of nitrogen, such as n i t r i t e or n i t r a t e , must be e n z y m a t i c a l l y reduced to ammonium p r i o r to i n c o r p o r a t i o n i n t o amino a c i d s (Eppley et a l , 1969; L u i and Roels, 1972; Packard and Blasco, 1974). The stepwise r e d u c t i o n s to ammonium are endergonic r e a c t i o n s , r e q u i r i n g s p e c i f i c enzyme c o f a c t o r s (e.g. NAD(P)H; FADH; reduced f e r r e d o x i n ) . Thus, i t i s not s u r p r i s i n g t h a t most phytoplankton a s s i m i l a t e ammonium more r e a d i l y tha n i t r i t e or n i t r a t e . Despite t h i s apparent energy " b a r r i e r " to o x i d i z e d n i t r o g e n a s s i m i l a t i o n however, these o x i d i z e d n i t r o g e n molecules are more r e a d i l y a v a i l a b l e i n most oceanic environments. E c o l o g i c a l l y , n i t r a t e may represent the most important s i n g l e n i t r o g e n source i n the sea. In order to u t i l i z e n i t r a t e as a sub s t r a t e f o r c e l l u l a r anabolism, phytoplankton must t r a n s p o r t the i o n across the c e l l membrane, reduce the s u b s t r a t e i n a s e r i e s of r e a c t i o n s to ammonium, and i n c o r p o r a t e the ammonium i n t o primary amino acids (notably glutamate) ( f i g . 1). Of these three processes ( t r a n s p o r t , r e d u c t i o n , and i n c o r p o r a t i o n ) , t r a n s p o r t across the plasmalemma, presumably o f t e n a g a i n s t the negative chemical p o t e n t i a l of the i o n (Eppley and Rogers, 1970), i s the l e a s t understood. The mechanism of passage of n i t r a t e across the c e l l mem-brane has been hypothesized by some i n v e s t i g a t o r s . A l l u s i o n s to "permeases" and f a c i l i l i t a t e d d i f f u s i o n have been made by Packard and Blasco (1974) and P i a t t and SubbaRao (1973); F i g . 1. Nitrogen a s s i m i l a t i o n i n marine phytoplankton ( a f t e r Packard and Blasco, 1974). N i t r a t e , n i t r i t e , and ammonium are tra n s p o r t e d across the c e l l membrane v i a hypothesized permeases. Once i n the c e l l , n i t r a t e and n i t r i t e are reduced stepwise to ammonium, which i s then i n c o r p o r a t e d i n t o ct-keto g l u t a r a t e to form glutamic a c i d . This amino a c i d , at the major branch-p o i n t , may e i t h e r transaminate the amino n i t r o g e n to other carbon s k e l e t o n s , or undergo f u r t h e r mod-i f i c a t i o n s , to form new amino a c i d s . 3b F i g . 1 prol ine 3.arginine 4. glutamine proline dehydrogenase 1. glutamate N A D ( P ) + N A D ( P ) H H , 0 ornithine-citru Mine cycle gl utamine sythetase HC I HC H - C - C t transaminase 0\-keto acids O N H glutamate O' c*° To C H I dehyd rogenase 5. alanine 6. asparagine 7. aspartate 8 . cysteine 9. glycine lO.histadine H.isoleucine 12.leucine 13.lysine 14. methionine 15. phenylalanine -*.16.serine 17.threonine IS.tryptophane 19. tyrosine 20. valine N H 4 nitrite reductase  j O eya-keto glutarate N nitrate reductase N O ; eel — membrane N O : N O : A M I N O A C I D S Y N T H E S I S A M M O N I A A S S I M I L A T I O N N I T R A T E R E D U C T I O N E N V I R O N M E N T A L N U T R I E N T S N I T R O G E N A S S I M I L A T I O N however, no d i r e c t evidence has been p u b l i s h e d to support these s p e c u l a t i o n s . The p h o s p h o l i p i d b i l a y e r , thought to be the major c o n s t i t u e n t of the plasma membrane, i s not very permeable to charged molecules l i k e n i t r a t e , so abs o r p t i o n of the i o n must f o l l o w other routes i n t o the c e l l . An a l t e r n a t i v e p o s s i b i l i t y might.be a membrane-bound p r o t e i n i n the p h o s p h o l i p i d b i l a y e r t h a t mediates the t r a n s l o c a t i o n of n i t r a t e . This "ionophore" might simply a l l o w d i f f u s i o n , i n which case i t would be a tru e permease, f o l l o w i n g the o r i g i n a l d e f i n i t i o n of the term by Monod (1942); or i t may "pump" n i t r a t e against the negative gradient of the chemical p o t e n t i a l of the i o n . I f the n i t r a t e - a c c e p t i n g p r o t e i n simply allowed d i f -f u s i o n of n i t r a t e , the i n t e r n a l c o n c e n t r a t i o n of the i o n should equal the e x t e r n a l c o n c e n t r a t i o n , p r o v i d i n g no other process intervened. This hypothesis i s d i f f i c u l t to t e s t d i r e c t l y because i n the s t e a d y - s t a t e , i n t r a c e l l u l a r n i t r a t e i s c o n t i n u o u s l y being reduced to ammonium; d i m i n i s h i n g the i n t r a c e l l u l a r n i t r a t e c o n c e n t r a t i o n s . This type of d i f f u s i o n has been c a l l e d " t r a p p i n g d i f f u s i o n " (Wilson, 1962) , the d r i v i n g f o r c e being provided by the biochemical r e d u c t i o n of n i t r a t e w i t h i n the c e l l . This model i m p l i e s t h a t the n i t r a t e uptake r a t e i s a f u n c t i o n of n i t r a t e r e d u c t i o n , or s p e c i f i -c a l l y the enzyme n i t r a t e reductase (E.C. 1.6.6.1 and E.C. 1.6.6.3). N i t r a t e uptake by whole c e l l s can be des c r i b e d adequate-l y by Michaelis-Menten k i n e t i c s (e.g. Caperon and Meyer, 1972; Maclsaac and Dugdale, 1972). This type of k i n e t i c s relates the v e l o c i t y of uptake to the concentration of the nutrient by the expression: V -S _ max K + S s where: S i s the e x t r a c e l l u l a r nutrient concentration V i s the v e l o c i t y of nutrient uptake K i s the nutrient concentration supporting h maxi--mum uptake v e l o c i t y (%V„ ) . c -* max I f trapping d i f f u s i o n were responsible f o r n i t r a t e uptake by the c e l l , i t should follow that the h a l f - s a t u r a t i o n constant for n i t r a t e uptake should be of a s i m i l a r order of magnitude as the h a l f - s a t u r a t i o n constant f o r n i t r a t e reductase ( K ) . m Determinations of K for n i t r a t e reductase indicate that m t h i s value i s i n excess of the concentrations of n i t r a t e usually present i n natural seawater by about a hundredfold (Eppley and Rogers, 1970) . Thus i t i s improbable that n i t r a t e reductase serves to provide a chemical gradient across the c e l l membrane; i n f a c t , the c e l l must "pump" n i t r a t e into the cytoplasm i n order to reach e f f e c t i v e sub-strate concentrations for a high degree of reducing e f f i c i e n c y . Another possible mechanism for n i t r a t e uptake i s that of s o -called "active transport". This process requires that n i t r a t e uptake be coupled d i r e c t l y to an exergonic chemical r e a c t i o n . Here the i n t e r n a l n i t r a t e c o n c e n t r a t i o n may be r e l a t i v e l y i n s i g n i f i c a n t ; n i t r a t e may be accumulated against i t s c o n c e n t r a t i o n g r a d i e n t , p r o v i d i n g t h a t a s u i t - , able energy source i s a v a i l a b l e to d r i v e the i o n across the membrane. The obvious candidate f o r t h i s chemical energy i s ATP, which, through h y d r o l y s i s , l i b e r a t e s enough energy f o r the t r a n s l o c a t i o n of n i t r a t e . In t h i s model the n i t r a t e -a c c e pting p r o t e i n i s an adenosine triphosphatase (or some analagous enzyme), c a t a l y z i n g the h y d r o l y s i s of ATP i n the presence of e x t e r n a l n i t r a t e i o n s . An analagous s i t u a t i o n i s the (Na + + K + ) - a c t i v a t e d t r a n s p o r t adenosine t r i p h o s p h a -tase.(E.C. 3.6.1.3), f i r s t d e s c r i b e d by Skou i n 1957, and found i n many animal and p l a n t t i s s u e s ( c . f . A s k a r i , 19 74; Balke and Hodges, 1975; K a r l s s o n and K y l i n , 1974; Maslowski and Komoszynski, 19 74). At the o u t s e t t h i s t h e s i s was an attempt to determine some aspects of the p h y s i o l o g i c a l r o l e of ATP i n marine phytoplankton. This goal was not reached however, because of the s u r p r i s e d i s c o v e r y of the n i t r a t e - a c t i v a t e d adenosine triphosphatase. The r e s u l t was more involvement w i t h the k i n e t i c s and p h y s i o l o g i c a l r o l e of the enzyme, r a t h e r than w i t h ATP per se. I t i s hoped however, t h a t the research documented here w i l l c o n t r i b u t e t o knowledge of the physio-l o g i c a l r o l e of ATP i n n u t r i e n t a s s i m i l a t i o n processes, as w e l l as adding t o the understanding o f n i t r o g e n metabolism o f marine phytoplankton. M a t e r i a l s and Methods A. Laboratory Studies I . C u l t u r e s In a l l , nine species of marine phytoplankton were ex-amined f o r the presence of a (NO^, C l )-activated'adenosine triphosphatase. Most of the enzyme k i n e t i c s were determined from membrane pr e p a r a t i o n s from Skeletonema costatum (Grev) Cleve ( B a c i l l a r i o p h y c e a e ) , as t h i s diatom had the g r e a t e s t enzyme a c t i v i t y per u n i t carbon and was e a s i e s t to c u l t u r e . Caution should be s t r e s s e d however, about o v e r - g e n e r a l i z i n g the c h a r a c t e r i s t i c s and k i n e t i c s of the (NO^, C l ) - a c t i v a t e d adenosine triphosphatase from S_. costatum t o other s p e c i e s . The e i g h t other species were : Pity1urn b r i g h t w e l l i i (West) Grunow ( B a c i l l a r i p h y c e a e ) , D u n a l i e l l a t e r t i o l e c t a Butch CCholophyceae), E u t r e p t i e 1 1 a gymnastica S c h i l l e r (Eugleno-phyceae), Amphidinium c a r t e r a e Hulb. (Dinophyceae), Gonyaulax polyedra S t e i n (Dinophyceae), Gonyaulax tamarensis var. excavata Braarud (Dinophyceae), I s o c h r y s i s galbana Parke (Haptophyceae), Chroomohas s a l i n a (Crytophyceae). With the exceptions of I s o c h r y s i s galbana, Gonyaulax  pol y e d r a, and D u n a l i e l l a t e r t i o l e c t a , the c u l t u r e s were i s o -l a t e d i n t h i s l a b o r a t o r y by Ms. R. Waters and maintained as p a r t of the U n i v e r s i t y o f B r i t i s h Columbia Northwest P a c i f i c C u l t u r e C o l l e c t i o n . 1^ . galbana and D. t e r t i o l e c t a were o r i g i n a l l y obtained from Dr. M. Parke and the Woods Hole 8 Oceanographic I n s t i t u t i o n respectively. G. polyedra was obtained from R. W. Eppley at Scripps I n s t i t u t e of Oceanography. Each species was grown i n u n i a l g a l , but not axenic, batch cultures at 16-19 C i n s t e r i l e 2-6 1. Erlenmeyer f l a s k s . The two diatoms, Skeletonema costatum and Ditylum  b r i g h t w e l l i i were grown on medium " f " , d i l u t e d to "f/2" with autoclaved seawater (Gullard and Ryther, 1962). The other species were grown on medium " J " (Table 1), developed i n t h i s laboratory. Except where indicated, NaNO^ was the sole inorganic nitrogen source added to the culture media and atmospheric contamination of the media by ammonia was pre-vented by prebubbling a i r entering the culture vessels through a saturated s o l u t i o n of ZnC^ (Caperon and Meyer, 1972} . Light was supplied by banks of 40 Watt cool white f l u o r -escent tubes. The maximum l i g h t i n t e n s i t y was determined i n lux from a Photovolt Corporation l i g h t meter (Model 511 M) , and converted to langley/min using the conversion factor —6 5 x 10 ly/min = 1 lux (Westlake, 1965). Using t h i s conver-sion factor, the maximum l i g h t i n t e n s i t y was approximated at 0.12 ly/min. Cultures were maintained i n a 12:12 light/dark cycle. I I . Preparation of membrane " v e s i c l e s " After the cultures reached the stationary phase of growth Cas determined by d a i l y chlorophyll a determinations) TABLE 1 - Medium " J " 1,000 ml seawater ( f i l t e r e d through 0 . 4 5 y M i l l i p o r e ) 100 ml d i s t i l l e d water 2.5x10_6g Cu (as Cl") 0.25 g I r i s buffer 1x10"5g Na^toO^ 0.03 g Na 2Si0 3 1x10 g vitamin B ^ 0.1 g NaN03 _a 5x10 g thiamin HC1 0.01 g I^HPO^ -5 3x10 g nicotinic acid 6x10"3g Na2EDTA -5 3x10 g Ca pantothenate 2xl0" 3g FeCl3«6H20 -5 3x10 g p-arninobenzoate 2x10"4g Mn (as SO^ ) 1x10~6g biotin 2x10~5g Zn (as Cl") 5x10 g inosi t o l (meso) 2.5x10~6g Co (as Cl") - 7 6x10 g f o l i c acid 10 the c e l l s were harvested i n 250 ml centrifuge buckets at 5000 g i n a r e f r i g e r a t e d centrifuge at 5 C. The p e l l e t s were c o l l e c t e d i n 50 ml centrifuge tubes and sonicated i n an Ultrasonic T30C1 sonicator for ca. 10-20 min at 0-2 C. The exact length of time for the sonication period varied from species to species, depending upon the c e l l wall morphology, composition, and s i z e . When no i n t a c t c e l l s could be observed under phase contrast microscopy at 400 X magnifica-t i o n , the fragments were suspended i n 5 mM Tris-acetate buffer, pH 7.9 at 0 C. These were then centrifuged at 5000 g for 60 min. The supernatant was c o l l e c t e d , sonicated for 10 min, and centrifuged at 25,000 g for 30 min. This sonication/centrifugation procedure was repeated up to three times for those species with a thick wall (e.g. diatoms). A white, amorphorous, flocculent material was observed at the bottom of the tubes a f t e r the 25,000 g spin and the super-natant was clear and uncoloured. The supernatants were decanted and the p e l l e t s were then suspended i n a solution of 0.01% T r i t o n X-100 and 0.01% de-oxycholate (DOC) i n 5 mM Tris-acetate buffer. This suspen-sion was incubated i n a shaking water bath at 25-30 C for 15 min. The supernatant was treated i n the same manner as the p e l l e t . The p e l l e t suspension was then centrifuged at 40,000 g for 60 min and the supernatant was centrifuged at 100,000 g for 60 min. Supernatants from these l a t t e r two c e n t r i f u g a t i o n s were assayed f o r (NO^, CI ) - a c t i v a t e d adeno-si n e triphosphatase a c t i v i t y . I I I . Enzyme assays a) ( N O 3 " , C l ~ ) - a c t i v a t e d adenosine triphosphatase Three methods were used t o determine the a c t i v i t y of the (NO^/ C l ~ ) - a c t i v a t e d adenosine tr i p h o s p h a t a s e . They are described i n i n c r e a s i n g order of t h e i r s e n s i t i v i t y to detect adenosine triphosphatase a c t i v i t y . 1. Inorganic phosphate method (Fiske-SubbaRow) This method CFiske and SubbaRow, 1925 as adapted by Lindeman, 1958) has the advantage of being f a s t and r e l a t i v e -l y easy, although i t s s e n s i t i v i t y depends upon h i g h s p e c i f i c a c t i v i t y o f the enzyme p r e p a r a t i o n . I t was the assay o r i g i n -a l l y used here, and i s s u i t a b l e f o r s e m i - p u r i f i e d membrane prepa r a t i o n s from S_. cos t a t urn. . . • . The a c t i v i t y o f the enzyme was determined by f o l l o w i n g the h y d r o l y s i s of ATP and measuring the i n o r g a n i c phosphate re l e a s e d CFiske and SubbaRow, 1925). F i v e ug of p r o t e i n from the T r i t o n e x t r a c t s (ca. 200 u l ) were incubated i n 1.5 ml of s o l u t i o n c o n t a i n i n g 10 mM T r i s - a c e t a t e b u f f e r , pH 7.9; 10 ]iM NaN0 3; 50 uM MgCl 2; 50 uM Tris-ATP, and 10 uM . dit±iiothreitol (DTT). The r e a c t i o n mixture was incubated f o r 30 min at 20 C, a f t e r which 1.0 ml of 60% t r i c h l o r a c e t i c a c i d (TCC) was added. The p r e c i p i t a t e d p r o t e i n was c e n t r i -fuged at 10,000 g f o r 10 min. Two ml of the supernatant was • 12 c o l l e c t e d and assayed f o r ATP h y d r o l y s i s ; the p r e c i p i t a t e was assayed f o r p r o t e i n a g a i n s t bovine serum albumin standards (Sigma Chemical Co.) (Lowry e t a l , 1951). 2. Coupled enzyme assay (pyruvate kinase - l a c t a t e dehydrogenase) The adenosine t r i p h o s p h a t a s e may be coupled i n v i t r o to pyruvate kinase (E.C. 2.7.1.40} and l a c t a t e dehydrogenase (E.C. 1.1.1.271; the a c t i v i t y of the enzyme can be determined by f o l l o w i n g the change In abs o r p t i o n at 340 nm, due t o the s t o i c h i o m e t r i c o x i d a t i o n of NADH. Thus: The s u b s t r a t e c o n c e n t r a t i o n s used were the same as i n the Fiske-SubbaRow method (1 above) w i t h the a d d i t i o n o f 5 mM PEP, 0.2 mM NADH and excess l a c t a t e dehydrogenase (Sigma Chemical Co.; Type 1 w i t h pyruvate k i n a s e ) . The adenosine triphosphatase a c t i v i t y was f o l l o w e d from 0-0.2 OD u n i t s i n a r e c o r d i n g spectrophotometer. 32 3. (y-P )-ATP method ( I s o t o p i c d i s c r i m i n a t i o n assay) The most s e n s i t i v e assay f o r determining a c t i v i t y of the (NO^, C l ) - a c t i v a t e d adenosine triphosphatase was found 32 t o be w i t h the use of (y-P )-ATP as a su b s t r a t e (New England N u c l e a r ) . In t h i s procedure the f i n a l concentrations i n the assay mixture were: 2 mM Tris-ATP, pH 7.9, 2 mM MgCl 2, 10 uM NaNO-, 10 yM d i t h o t h r i t o l , 1.5 mM Tris-acetate buffer, 6 32 and 2 to 3 x 10 cpm C Y ~ p )-ATP. The mixture was preincu-bated for 10 min and the reaction was i n i t i a t e d by the addition of 5 ug protein. The f i n a l volume was 1.25 ml. After 15 min the reaction was terminated by the addition of a 4% solution of (NH.4)Mo7024-4H20 i n 10 N H 2S0 4 (Goldman and Albers, 19 73). Five hundred p i of isobutanol was added to each, tube and the solutions were mixed i n a vortex mixer for 60 sec. A 200 y l aliquot from the isobutanol phase was re-moved for counting i n a dioxane-naphthlene c o c k t a i l (Aqua-f l u o r ; New England Nuclear) i n a Nuclear Chicago Isocap/300 l i q u i d s c i n t i l l a t i o n counter. bl N i t r a t e reductase (NADH-dependent) Nit r a t e reductase was routinely assayed i n the Tr i t o n extracts p r i o r to determination of the (NO^, CI )-activated adenosine triphosphatase. If n i t r a t e reductase was detected, the extract was dialyzed for 2-4 hr (sometimes overnight) against 10 mM Tris-acetate buffer containing 5 mM EDTA and 1 mM d i t h i c t h r e i t o l . The NADH-dependent n i t r a t e reductase was assayed by following the NO^-stimulated oxidation of NADH from 0-0.2 OD units i n a recording spectrophotometer as described by Hageman and Hucklesby (19 71). 14 c) (Na + '+ K + ) - a c t i v a t e d adenosine triphosphatase The (Na + + K + ) - a c t i v a t e d adenosine triphosphatase was assayed as a marker for the c e l l meviibrane f r a c t i o n according to the methods of Maslowski and Komoszynski (1974). IV. Determination of Protein and Chlorophyll a Protein was determined as described by Lowry e t a_l (1951), using bovine serum albumin standards. Protein was used fo r determinations of the s p e c i f i c a c t i v i t y of the (NO^, C l ~ ) - a c t i v a t e d adenosine triphosphatase i n order that comparison between various ion-stimulated adenosine tr i p h o s -phatases could be made. In addition, the s p e c i f i c a c t i v i t y was used as a parameter of r e l a t i v e enzyme p u r i t y . Chlorophyll a was determined spectrophotometrically at 665, 645, and 630 nm respectively using the equations of Parsons and S t r i c k l a n d (Strickland and Parsons, 1972). C e l l s were f i l t e r e d on Whatman GF/C glass f i b e r f i l t e r s and coated with a few ml of a 1% MgCO^ suspension. The f i l t e r s were homogenized by hand i n an a l l - g l a s s t i s s u e grinder (Pyrex) with 10 ml of 90% acetone, centrifuged f o r 30 min at 2000 x g at 5 C. A c t i v i t y of the (NO^, C l ~ ) - a c t i v a t e d adenosine triphosphatase was calculated per mg chlorophyll a_ to compare r e l a t i v e s p e c i f i c a c t i v i t i e s between species. V. ATP determinations Parameters a f f e c t i n g the i n t r a c e l l u l a r ATP pool s i z e were investigated, to determine what, i f any, coupling 15 existed between NO^ uptake and ATP. One hundred ml aliquots of S_. costatum cultures were taken from log growth phase and incubated at 18 C i n 250 ml Pyrex b o t t l e s . The incident l i g h t , provided by banks of 40 Watt cool white fluorescent tubes, was attenuated by placing neutral density f i l t e r s (Kahlsico Internation Corp.) over the bottles to produce 60, 30, 15, 1 and 0% i l l u m i n a t i o n . One set remained at the maximum l i g h t i n t e n s i t y ( i . e . 100%) of ca. 0.12 ly/min. A f t e r a 2 hr incubation, the c e l l s were f i l t e r e d on Whatman GF/C glass f i b e r f i l t e r s and the ATP was extracted twice i n b o i l i n g Tris-HCl buffer as described by Holm-Hansen and Booth (1966). Using known amounts of ATP, the ef f i c i e n c y , of the extraction technique was estimated as ca. 80%. The samples were assayed f o r ATP with l u c i f e r i n -l u c i f e r a s e preparations (.Sigma Chemical Co.) and the photon emission was detected i n a Unicam l i q u i d s c i n t i l l a t i o n counter employing only one photomultiplier. Standards and background counts were performed i n conjunction with each sample. Samples were assayed i n t r i p l i c a t e and the mean of the three counts was used to calculate the net i n t r a c e l l u l a r ATP pools. To determine the e f f e c t s of temperature, the method was performed at 8, 18, and 28 C (±2 C) at each of the 6 l i g h t i n t e n s i t i e s . The e f f e c t s of metabolic i n h i b i t o r s and e x t r a c e l l u l a r 16 nutrients on the ATP pool was determined by the addition of the i n h i b i t o r s or nutrients at varying concentrations e i t h e r during the incubation period or immediately a f t e r , but always p r i o r to the ATP extractions. B. F i e l d Study at Knight I n l e t , B. C. To gain some understanding of the parameters c o n t r o l l i n g the p h y s i o l o g i c a l r o l e of ATP i n NO^ and NH^ uptake by whole, natural phytoplankton communities, two cruises were made on the C. S. S. Vector to Knight I n l e t , B. C. This i n l e t i s a gl a c i e r - f e d estuarine f j o r d ca. 250 miles north of Vancouver, B. C., and has been under intensive, continuous study by A. G. Lewis and D. P. Stone (Institute of Oceanography, University of B r i t i s h Columbia). The two cruises, i n July and September 1974, corresponded to the recession of the spring bloom and peak of the secondary f a l l bloom respective-l y , as determined by i n s i t u chlorophyll a concentrations ( f i g . 2). A control s t a t i o n was established i n the adjacent Queen Charlotte S t r a i t for comparison with the estuarine Inle t stations ( f i g . 3). Table 2 l i s t s the coordinates of the sample stations and t h e i r acronyms. To determine primary p r o d u c t i v i t y , hydrocast samples were made at each s t a t i o n during daylight hours from the 2 m depth with 2 1. Van Dorn b o t t l e s . Each sample was s p l i t into three equal parts. One part was enriched with 3 uM NO^/l and one with 3 uM NH*/1; the remaining sample was not en-17a F i g . 2. Seasonal v a r i a t i o n i n c h l o r o p h y l l a i n Knight I n l e t , B. C. i n 1973 at the s u r f a c e (A) and 5 m (•). Samples f o r the determination of primary p r o d u c t i v i t y were taken i n J u l y and September, F i g . 3. The west coast of B r i t i s h Columbia and northwest Washington showing the study area ( i n s e r t ) Above, Knight I n l e t , B. C , i n d i c a t i n g the p o s i t i o n s of the sample s t a t i o n s . 17 b 19 TABLE 2 Coordinates and depths of the s t a t i o n s sampled i n Knight I n l e t , B. C. S t a t i o n L a t i t u d e Longitude Depth (m) Q. C. 50°39.1'N 126°48.5'W 254 Kn 3 50°39.2'N 126°10.9'W 203 Kn 5 50°41.7'N 125°47.0'W 406 Kn 7 50°48.4'N 125°37.3'W 523 Kn 11 51°02.6'N 125°34.0'W 199 20 r i c h e d . The subsamples were preincubated i n a simulated-i n s i t u deck i n c u b a t o r f o r 0-6 hr i n d u p l i c a t e 125 ml Pyrex b o t t l e s equipped w i t h ground-glass stoppers. Banks of 40 Watt f l u o r e s c e n t tubes provided an I n c i d e n t l i g h t i n t e n s i t y of 0.09 ly/min at maximum. Flow-through seawater kept the samples a t approximately sea surf a c e temperature. The design of t h i s i n c u bator i s des c r i b e d by Doty and Oguri (1958). A f t e r the d e s i r e d p r e i n c u b a t i o n the samples were inocu-14 l a t e d w i t h 2 u C i Na 2C 0 3 and incubated f o r 2-4 h r . N e u t r a l d e n s i t y f i l t e r s were used t o attenuate the l i g h t t o 60, 30, 15, 1 and 0% of the i n c i d e n t i n t e n s i t y . One set remained at the maximum i n t e n s i t y . A f t e r the Incubation p e r i o d , the c e l l s were f i l t e r e d on 0.45 u M i l l i p o r e f i l t e r s a t l e s s than 12 cm vacuum pressure, fumed over concentrated HCl f o r 15 sec and d r i e d i n a d e s s i c a t o r c o n t a i n i n g 30% soda lime. The f i l t e r s were counted f o r r a d i o a c t i v i t y ashore u s i n g the channels r a t i o method. The i n o r g a n i c carbon f i x a t i o n was determined from the equations of S t r i c k l a n d and Parsons (1972). C h l o r o p h y l l a was determined i n each sample p r i o r t o . and a f t e r the i n c u b a t i o n p e r i o d as des c r i b e d i n sec. A-IV. Estimates of the 1% l i g h t depth were made w i t h a Secchi d i s c . In a d d i t i o n , v e r t i c a l p r o f i l e s of n i t r a t e , phosphate, d i s -s olved oxygen, c h l o r o p h y l l a, temperature and s a l i n i t y were made at each s t a t i o n . Whole water samples were taken from 21 the 2 rn hydrocast and preserved i n L u g o l 1 s s o l u t i o n f o r c e l l i d e n t i f i c a t i o n and counting (Banse, 1974). C e l l counts were made i n 10 cc chambers a f t e r a l l o w i n g the c e l l s to s e t t l e overnight. A Zeiss i n v e r t e d microscope, f i t t e d w i t h phase c o n t r a s t o b j e c t i v e s , was used t o i d e n t i f y and count each sample. Depending on the c e l l d e n s i t y , 50 to 100 random f i e l d s were counted a t 250 X and f i n a l c e l l counts were expressed on a per l i t e r b a s i s (Banse, 19 74). In a d d i t i o n , a l i q u o t s were preserved from the experimental samples a f t e r the i n c u b a t i o n p e r i o d and counted as d e s c r i b e d . Results A. Laboratory Studies I . C h a r a c t e r i s t i c s and k i n e t i c s of the (NO^, C l )-a c t i v a t e d adenosine triphosphatase a) E f f e c t s of hydrogen i o n c o n c e n t r a t i o n " The e f f e c t s o f hydrogen i o n c o n c e n t r a t i o n on the (NO^, C l ) - a c t i v a t e d adenosine triphosphatase are,shown i n f i g . 4 f o r Skeletonema costatum and Chroomonas s a l i n a . In both species the enzyme e x h i b i t e d a major a c t i v i t y peak at ca. pH 7.8-8.1 w i t h a secondary optimum at pH 6.9. Although bimodal pHL p r o f i l e s f o r a s i n g l e enzyme are not uncommon (Dixon and Webb, 19641, the p o s s i b i l i t y t h a t t h i s pH p r o f i l e represents the presence of more than one enzyme may be assumed, due both t o the heterogeneity of the p r e p a r a t i o n and the l a c k of u n i f o r m i t y between the pH 7.9/6.9 r a t i o i n both s p e c i e s . £Two major p r o t e i n s might be i n f e r r e d : one w i t h an optimum at pH 7.9 and a secondary enzyme w i t h an optimum at pH 6.9.3 As the hydrogen i o n c o n c e n t r a t i o n may a l t e r s u b s t r a t e b i n d i n g c h a r a c t e r i s t i c s of enzymes, k i n e t i c s t u d i e s were c a r r i e d out at pH 7.9 f o r a l l s p e c i e s . K a r l s s o n and K y l i n (19 74) have des c r i b e d the p r o p e r t i e s 2+ + + • of a Mg - s t i m u l a t e d (Na + K ) - a c t i v a t e d adenosine t r i p h o s -phatase from sugar beet cotyledons and i n d i c a t e d a s i m i l a r pH p r o f i l e f o r enzyme a c t i v i t y . The pH p r o f i l e was independ-ent of i o n i c s t r e n g t h and suggests the t i t r a t i o n of at Fig. 4. The pH profiles for the (NO^, CI -)-activated adenosine triphosphatase from Skeletonema costatum and Chrooinonas salina. Both profiles show a bimodal curve with maxima at pH 7.8 - 8.2 and a secondary peak at ca. 6.9 . 5.5 6.0 6.5 7.0 7.5 8.0 8.5 PH l e a s t two amino acid residues involved i n c a t a l y t i c a c t i v i t y . The p o s s i b i l i t y that ion transport adenosine triphosphatases may be conservative i n primary amino acid structure, and perhaps share a common mechanism, i f not s i m i l a r c a t a l y t i c binding s i t e ( s ) , i s suggested. b) E f f e c t s of temperature on enzyme a c t i v i t y Changes i n the l i p i d phase-state are known to a f f e c t the Arrhenius p l o t of membrane-bound enzymes i n v i t r o (La-brooke and Chapman, 1969; Raison et 'al, 19 71) . The l i p i d phase-state change i s r e f l e c t e d by a "break" i n the slope at a t r a n s i t i o n temperature c h a r a c t e r i s t i c of the l i p i d s as-sociated with the enzyme (Barnett and Grisham, 19 73a, 19 73b; Raison et al_, 19 71) . In f i g . 5 the Arrhenius p l o t for a crude enzyme preparation from Skeletonema costaturn i s shown from 0-25 C at pH 7.9 (the buffer pH was adjusted at each temperature to maintain a constant hydrogen ion a c t i v i t y ) . A difference i n the a c t i v a t i o n energy of 30.9 Kcal/mole was extrapolated to 2.9 C for the (NO^, C l )-activated adenosine triphosphatase when the preparation was assayed without the addition of e i t h e r T r i t o n X-100 or DOC to the membrane pre-paration. Upon addition of detergents however, the slope of the curve s h i f t e d to 16 Kcal/mole and was uniform through-out the temperature range examined (fig< 6). These r e s u l t s strongly suggest the (NO^, C l ~ ) - a c t i v a t e d adenosine triphos-phatase i s associated with a l i p i d moiety and at tempera-Fig. 5. Arrhenius plot of (N0~, Cl )-activated • adenosine triphosphatase from Skeletonema cos-, tatum membrane vesicles without the addition of detergents. A break i n the slope, correspon-ding to a change i n activation energy of 31.9 Kcal/mole, was extrapolated to 2.9 C. Fig; 6. Arrhenius plot of the (N03, Cl~)-activated adenosine triphosphatase from Skeletonema costatum. The enzyme was extracted from membrane vesicles with 0.1% Triton X-100 and 0.05% DOC. The slope corresponds to an activation energy of ca. 16 Kcal/mole. I 1 1 L L. .3.3 3.4 3.5 3.6 3.7 =f X10 3 °K t u r e s below c j i . 3 C the a c t i v a t i o n energy i s so great as to repress enzyme a c t i v i t y by about 50% i n v i t r o , c) Determination of the enzyme locus + + Data f o r the p a r a l l e l p u r i f i c a t i o n of the (Na + K )-a c t i v a t e d adenosine triphosphatase (Table 3 ) , and the Arrhenius p l o t s ( f i g s . 5 and 6} suggest the (N0 3, C l )-a c t i v a t e d adenosine triphosphatase i s bound t o , or a s s o c i -ated w i t h a membrane. As n i t r a t e reductase ( a s s o c i a t e d w i t h both the c y t o s o l and outer c h l o r o p l a s t membrane) was not detected when the (NO^, C l ~ ) - a c t i v a t e d adenosine t r i p h o s p h a -tase e x h i b i t e d the g r e a t e s t s p e c i f i c a c t i v i t y , a s s o c i a t i o n of the (NO^, C l ~ ) - a c t i v a t e d adenosine triphosphatase w i t h the c h l o r o p l a s t membrane was not probable. Although i t i s d i f f i c u l t to demonstrate a s p e c i f i c enzyme marker f o r the plasmalemma, the (Na + '+ K + ) - a c t i v a t e d adenosine t r i p h o s p h a -tase has not been demonstrated In endoplasmic r e t i c u l u m o r mitochondria ( c f . A s k a r i , 19 74) and may i n f a c t be a r e l i a b l e enzyme marker f o r the plasmalemma. To date, i t has been d i f f i c u l t t o o b t a i n p u r i f i e d membrane pre p a r a t i o n s of tonoplasts (vacuolar membranes); the p o s s i b i l i t y remains + + t h a t the (Na + K ) - a c t i v a t e d adenosine triphosphatase may be a s s o c i a t e d w i t h v a c u o l a r membranes as w e l l (Hodges, 1972). Despite t h i s p o s s i b i l i t y , i t i s thought probable t h a t the (NO-j, C l ) - a c t i v a t e d adenosine triphosphatase i s p r i m a r i l y a s s o c i a t e d w i t h the plasmalemma. TABLE 3 P a r a l l e l p u r i f i c a t i o n of the (NO^, Cl")-activated adenosine triphosphatase with the (Na + + K + ) - a c t i v a t e d adenosine triphosphatase. Fraction Nitrate Reductase + -1 (uM NAD /nig protein-hr ) (NO" Cl") ATPase -1 (yM POjj/mg protein-hr ) (Na+ + K+) ATPase _ i (yM PO^/rxj protein-hr ) 3000 x g spin supernatant 30.0 1.9 1.7 pellet 7.9 16.0 7.2 100,000 x g supernatant N.D. 210 19.0 pellet N.D. 15 2.1 The activities of three enzymes are indicated from a representative experiment from four fractions. The ( N O 3 , Cl)-activated adenosine triphosphatase appears to purify in parallel with the (Na + K)-activated adenosine triphosphatase, an enzyme marker of the plasma membrane. (ND=not detected). CO d) D i v a l e n t c a t i o n requirements . . 2+ 2+ Two d i v a l e n t c a t i o n s , Ca or Mg , were found to f u l -f i l the c a t i o n requirements f o r enzyme a c t i v i t y . Of these, 2+ Mg had a lower h a l f - s a t u r a t i o n constant (K = 17 yM as • m 2+ opposed to 52 pM f o r Ca ), and was used i n v i r t u a l l y a l l 2+ the assays. Mg s t i m u l a t e d adenosine triphosphatase a c t i v i t y without the a d d i t i o n of any a c t i v a t i n g anions 2+ however. In some enzyme pre p a r a t i o n s the Mg - s t i m u l a t e d adenosine triphosphatase represented over 90% of the t o t a l adenosine triphosphatase a c t i v i t y of the u n p u r i f i e d enzyme 2+ preparations (Table 4). As Mg was r e q u i r e d f o r f u l l (NO^, CI ) - a c t i v a t e d adenosine triphosphatase a c t i v i t y , and was added i n excess of the c a l c u l a t e d K m f o r the l a t t e r enzyme, 2+ the Mg - s t i m u l a t e d adenosine triphosphatase a c t i v i t y was subtracted from the (NO^, C l ) - a c t i v a t e d adenosine t r i p h o s -phatase. e) I n t e r f e r e n c e from (Na + + K + ) - a c t i v a t e d adenosine triphosphatase The (Na + + K + ) - a c t i v a t e d adenosine triphosphatase was de t e c t a b l e i n the membrane p r e p a r a t i o n s , and p u r i f i e d i n p a r a l l e l w i t h the (NO^, C l ) - a c t i v a t e d adenosine t r i p h o s p h a -tase (Table 3). As NO^ was u s u a l l y added as the Na + s a l t , the e f f e c t s of 1 mM Na + (as NaCl) on the (Na + + K + ) - a c t i v a t e d adenosine triphosphatase were determined without the ad d i -t i o n of K +. These data are shown i n f i g . 7, and suggest t h a t low concentrations of Na + have l i t t l e e f f e c t on ATP F i g . 7. The e f f e c t s of v a r y i n g Na on the a c t i v i t y o f the ( N O 3 , C l ~ ) - a c t i v a t e d adenosine t r i p h o s p h a t a s e from Skeletonema costatum. c E • c o Q. O) O > 4-1 u < u 0. CO 2 5 5 0 NaNOa, (^M) 75 100 100 75 NaCl 5 0 (pM) 2 5 u> o 31 TABLE 4 The per cent adenosine triphosphatase a c t i v i t y for three ion-stimulated adenosine triphosphatases i n s i x preparations. The enzymes were assayed from the 100,000 x g supernatant using the i s o t o p i c discrim-ination assay method.* Sample number Mg (NO", Cl") (Na + + K +) 1 81 9 10 2 90 4 6 3 71 16 13 4 79 11 10 5 84 13 3 6 67 21 12 * F i n a l ion concentrations were: Mg 2 + - 10 mM NO~ - 50wM; C l " - 1 mM Na + - 100 mM; K + 10 mM h y d r o l y s i s without the a d d i t i o n of K +. f) /Anion requirements and s p e c i f i c i t y The a d d i t i o n of C l " to the (N0~, C l " ) - a c t i v a t e d adeno-s i n e triphosphatase g r e a t l y enhances the a c t i v i t y of the enzyme i n v i t r o . The p o s s i b i l i t y t h a t a separate adenosine t r i p h o s p h a t a s e , s t i m u l a t e d by C l alone, and independent of NOg, was not i n f e r r e d because no s i g n i f i c a n t i n c r e a s e i n adenosine triphosphatase a c t i v i t y could be observed without the a d d i t i o n of NO^ ( i n the presence of excess Mg ). These data suggest a loose c o u p l i n g between C l and NO^, and are c o n s i s t e n t w i t h the hypothesis t h a t two separate anion b i n d -i n g s i t e s are present on the enzyme(s). The two anions (NO^ and C l ) a c t i v a t e adenosine triphosphatase according to Michaelis-Menten k i n e t i c s ( f i g . 8a); the c a l c u l a t e d K f o r m C l being 11 yM i n S. costatum a t ImM NO^ c o n c e n t r a t i o n . In a d d i t i o n , the Lineweaver-Burk p l o t (1/V vs. 1/S) ( f i g . 8b) i n d i c a t e s the two anions are not c o m p e t i t i v e , as the curves f o r each anion do not i n t e r c e p t at 1/V ^ • max N i t r a t e a c t i v a t i o n of the adenosine t r i p h o s p h a t a s e i s depic t e d g r a p h i c a l l y i n f i g . 9 f o r seven s p e c i e s . These curves are summarized i n Table 5 f o r a l l s p e c i e s . The dino-f l a g e l l a t e s , Amphidinium c a r t e r a e , Gonyaulax tamarensis var. excavata, and Gonyaulax polyedra f a i l e d t o demonstrate any NO^ a c t i v a t i o n of ATP h y d r o l y s i s ; the enzyme may not be present i n these s p e c i e s . The other species represented a l l 33a F i g . 8a. Michaelis-Menten p l o t of the (NO^, de-a c t i v a t e d adenosine triphosphatase from Skeletonema  costatum. The i n o r g a n i c phosphate method ( F i s k e -Subbarow) was used i n these assays. F i g . 8b. Lineweaver-Burk t r a n s f o r m a t i o n of the Michaelis-Menten p l o t (8a). The h a l f - s a t u r a t i o n constants f o r NO^ and C l ~ were 0.9 and 1 1 U M r e s p e c t i v e l y . 33.b F i g . 8a 15 (CD. 20 (uM) F i g . 8b. 1.5 O - NO3 • - c r 1.0 • V 0.5 • / 0 — ~ ~ 7 1.25 1.0.0 0 . 7 5 0 . 5 0 0 . 2 5 0 0 . 2 5 0 . 5 0 0175 1.00 1 .25 S S 34 had adenosine t r i p h o s p h a t a s e a c t i v i t y , a l t h o u g h the r e l a t i v e s p e c i f i c a c t i v i t y (ATP h y d r o l y s i s per mg c h l o r o p h y l l a) v a r i e d w i d e l y (Table 5 ) . The c a l c u l a t e d values f o r N0 3 a c t i v a t i o n of the adenosine t r i p h o s p h a t a s e a t 20 mM C l ( s a t u r a t i n g C l c o n c e n t r a t i o n ) were 0.37 uM f o r Skeletonema costatum, 0.7 uM f o r D i t y l u m b r i g h t w e l l i i , 0.2 3 uM f o r D u n a l i e l l a t e r t i o l e c t a , 0.13 U M f o r I s o c h r y s i s galbana, 0.12 uM f o r E u t r e p t i e l l a gymnastica, and 0.40 uM f o r Chro-omonas s a l i n a . These v a l u e s were c a l c u l a t e d from computer-i z e d l i n e a r - r e g r e s s i o n a n a l y s i s o f the Woolf p l o t s (S/V vs. S) and are s i g n i f i c a n t l y d i f f e r e n t from each o t h e r and from zero a t the 95% c o n f i d e n c e l e v e l . The b i n d i n g s p e c i f i c i t y o f the (N0 3, C l ) - a c t i v a t e d adenosine t r i p h o s p h a t a s e f o r o t h e r anions was i n t e r p r e t e d from a n a l y s i s o f h a l f - s a t u r a t i o n c o n s t a n t s . The assays f o r i o n s e l e c t i v i t y were made a t pH 7.9 and 20 C. H a l f - s a t u r a -t i o n c o n s t a n t s were c a l c u l a t e d from computerized l i n e a r -r e g r e s s i o n a n a l y s i s o f the Lineweaver-Burk p l o t s . I , Br and F~ (Na + s a l t s ) had K m v a l u e s Of 106 + 22, 74 + 16 and 92 + 25 r e s p e c t i v e l y (N = 3). Maximum enzyme a c t i v i t y was not suppressed by n o n - c h l o r i d e h a l i d e s , i n d i c a t i n g a com-p e t i t i v e type o f i n h i b i t i o n , o r common h a l i d e b i n d i n g s i t e ( s ) . N0*I d i d not a l t e r the K of C l " , up t o 1 mM NO-,. These 3 m r 3 r e s u l t s imply a s e p a r a t e b i n d i n g s i t e ( s ) f o r NO^ ( f i g . 10). Furthermore, the s p e c i f i c a c t i v i t y of the enzyme was not TABLE 5 A c t i v i t y o f the (N0~, C l " ) - a c t i v a t e d a d e n o s i n e t r i p h o s p h a t a s e Species V max -1, (pM PO^/mg protein-hr ) Vmax | _-L (uM PO^/mg chlorophyll a-hr ) j (pM) Gonyaulax polyedra Gonyaulax tamarensis var. excavata Amphidinium carterae dunaliella tertiolecta Eutreptiella gyrmastica Chroomonas salina Isochrysis galbana Skeletonema costatum Ditylum brightioellii 0 0 0 3.17 8.74 5.66 6.12 22.02 4.60 0 0 1.9 x 10' 3.1 x 10' 1.1 x 10' 4.4 x 10' 4.6 x 10' 6.1 x 10' 0.21 0.12 0.40 0.13 0.37 0.69 Grant and Turner (1969) " a l l others Eppley et a l , (1969) to tn F i g . 9. Michaelis-Menten and Woolf p l o t s f o r the (N0~, de-a c t i v a t e d adenosine t r i p h o s p h a -tase from seven s p e c i e s . Each p o i n t i s the mean of th r e e de-t e r m i n a t i o n s a t the correspond d i n g s u b s t r a t e c o n c e n t r a t i o n . Assays were done u s i n g the cr* i s o t o p i c d i s c r i m i r . o t i o n method. F i g . 10. E f f e c t s o f v a r y i n g N O l c o n c e n t r a t i o n s on the h a l f - s a t u r a t i o n c o n s t a n t f o r C l ~ i n the (N0~ C l a c t i v a t e d adenosine t r i p h o s p h a t a s e from Skeletonema  costatum a t 20 C and pH 7.9 . '. 12 2 " 3 o E 10 0 10 10 2 N O " Concentration (JL/M) 10 3 39 s t a t i s t i c a l l y a l t e r e d whether NaN0 3, KN0 3 or NH 4N0 3 was the source o f NO^ (P>0.05). I o n i c s t r e n g t h was kept c o n s t a n t w i t h i n each assay s e r i e s by the a d d i t i o n o f MgSO^; SO^ does not a f f e c t ATP h y d r o l y s i s under the c o n d i t i o n s o f the assay and was assumed not to be an a c t i v a t o r o f the enzyme, gl N u c l e o t i d e s p e c i f i c i t y D eterminations o f the n u c l e o t i d e s p e c i f i c i t y on the (NO^, C l ) - a c t i v a t e d , enzymatic h y d r o l y s i s o f the t e r m i n a l phosphate from v a r i o u s n u c l e o t i d e s were made u s i n g semi-p u r i f i e d membrane p r e p a r a t i o n s from S_. cos t a t urn. The r e s u l t s o f these experiments are summarized i n Table 6. GTP and ITP were h y d r o l y z e d i n the presence of 1 mM NO^ and 10 mM MgC^, however the a c t i v i t y o f the enzyme was o n l y 32% and 25% r e s p e c t i v e l y o f the h y d r o l y s i s observed when ATP was the s u b s t r a t e . CTP, TTP, PEP, and ADP d i d not show d e t e c t a b l e i n o r g a n i c phosphate ( F i s k e and SubbaRow technique) i n response t o NO^ a c t i v a t i o n , under i d e n t i c a l assay c o n d i t i o n s . These r e s u l t s suggest ATP i s the primary s u b s t r a t e under p h y s i o l o g i c a l c o n d i t i o n s ( i . e . the enzyme i s p r i m a r i l y an adenosine t r i p h o s p h a t a s e ) ; and GTP and ITP may support some enzyme a c t i v i t y , but the v e l o c i t y o f h y d r o l y s i s of these l a t t e r n u c l e o t i d e s (and presumably NO^ t r a n s p o r t ) i s much lower than w i t h ATP. h) V e l o c i t i e s o f the r e a c t i o n ( a c t i v i t y ) The s p e c i f i c a c t i v i t i e s o f the (NO^, C l ) - a c t i v a t e d 40 TABLE 6 The n u c l e o t i d e s p e c i f i c i t y of the (NO~, de-a c t i v a t e d adenosine triphosphatase from Skeletonema oostatum membrane p r e p a r a t i o n s . Enzyme a c t i v i t y was assayed w i t h the i n o r g a n i c phosphate technique. * Nucleotide % A c t i v i t y ( r e l a t i v e to ATP) ATP 100 ADP ND GTP 32 ± 11 ITP 25 ± 8 CTP ND TTP ND PEP ND * For l i s t of a b b r e v i a t i o n s see Appendix I I . (ND = not detected) 41 adenosine triphosphatase are shown i n Table 5. To compare i n t e r s p e c i f i c a c t i v i t i e s of the enzyme at saturating sub-str a t e concentrations (V ), independent of p u r i f i c a t i o n , max ' * . . * the s p e c i f i c a c t i v i t i e s were calculated on a unit chlorophyll a b a s i s . These data may not be p h y s i o l o g i c a l l y v a l i d ( i . e . they may not be the a c t i v i t y iri vivo) , as deter-gents are known to enhance the a c t i v i t y of some membrane-bound adenosine triphosphatases (Nakao et a l , 19 74). Despite the possible inadequacy of the technique, the r e l a t i v e s p e c i f i c a c t i v i t y may r e f l e c t V i f the degree of enzyme ac t i v a t i o n i s l i n e a r l y proportional to detergent concentra-tions (the f i n a l concentration of detergent was constant for , 7 each assay). The values ranged from 6.1 x 10 uM ATP/mg — 1 6 chl a - hr i n Ditylum b r i g h t w e l l i i to 1.9 x 10 uM ATP/mg chl a - hr ^ i n Du n a l i e l l a t e r t i o l e c t a . Experiments to de-termine the stoichiometric r a t i o of NO^ transported per ATP hydrolyzed were not successful primarily due to the d i f f i -c u l t y i n p u r i f y i n g the enzyme. The p o s s i b i l i t y remains that t h i s r a t i o may be species s p e c i f i c ; i t i s d i f f i c u l t to re l a t e ATP hydrolysis d i r e c t l y to NO^ transport at t h i s time. i) A c t i v i t y of the (NO^, C l )-activated adenosine triphosphatase i n Skeletonema costatum grown on NH* To determine whether the (NO^, C l )-activated adenosine triphosphatase was induced by N0 3 during c e l l growth or was a c o n s t i t u t i v e enzyme, Skeletonema costatum was grown on "f/2" with 28 ug-atoms NH^/l as the sole inorganic nitrogen source (substituting for 28 u g-atoms NO^/D . Under these conditions, neither n i t r a t e reductase nor n i t r i t e reductase were detectable. However, the (NO^, C l )-activated adenosine triphosphatase was found in-the c e l l membrane f r a c t i o n s , at a c t i v i t i e s as great (per mg chlorophyll a) as when NH* was excluded from the growth media. Under i d e n t i c a l assay con-+ 7 d i t i o n s , the NH^-grown c e l l s demonstrated 4.37 x 10 uM -1 7 ATP/mg chlorophyll a - hr as compared to 4.6 x 10 uM ATP/ mg chlorophyll a - hr when grown on NO~. The aberrance between these two data may be a t t r i b u t a b l e to experimental er r o r , and are the mean values of four determinations on each preparation. These r e s u l t s strongly imply that the (NO-j, C l )-activated adenosine triphosphatase i s c o n s t i t u t i v e . I I . NC>2 uptake and the i n t r a c e l l u l a r ATP pools i n Skeletonema costatum a) . E f f e c t s of l i g h t and temperature on the i n t r a c e l l u l a r ATP pool The r e s u l t s of the e f f e c t s of l i g h t and temperature on the net i n t r a c e l l u l a r ATP pool i n £. costatum cultures are shown i n f i g . 11. The i n t r a c e l l u l a r ATP pool varies d i r e c t -l y with l i g h t i n t e n s i t y , presumably due to the contributions of the l i g h t reactions. As the high-energy triphosphate pool s i z e was calcula t e d per mg chlorophyll a, the e f f e c t s of short-term l i g h t adaptation were minimized, s p e c i f i c a l l y F i g . 11. E f f e c t s o f l i g h t and temperature on the ATP p o o l i n Skeletonema costatum. 0 15 3 0 6 0 100 % Light Intensity those due t o i n c r e a s e d c h l o r o p h y l l a s y n t h e s i s per c e l l i n the l i g h t attenuated samples (J0rgensen, 1964; Ryther and Menzel, 1959; Steeman-Nielsen e t a l , _ 196 2; Uribe and L i , 19 73). In a d d i t i o n , due to the s h o r t i n c u b a t i o n p e r i o d before the ATP e x t r a c t i o n (2 h r ) , o n l y a 5% i n c r e a s e i n c h l o r o p h y l l a was detected i n the 100% l i g h t b o t t l e at 18 C. Increased ATP pools were observed at 8 and 18 C, r e l a t i v e t o the l e v e l observed i n c e l l s incubated at the h i g h e r tempera-t u r e , 28 C. As the ATP pool i s not a metabolic r a t e i t s e l f , but merely represents the d i f f e r e n c e between r a t e of i n p u t and r a t e of output of the n u c l e o t i d e , c a l c u l a t i o n s of the Q_10 (a r a t e dependent c o e f f i c i e n t ) would not be p h y s i o l o g i -c a l l y v a l i d . I t would seem however, t h a t decreased tempera-t u r e lowers the c a t a b o l i s m of ATP at a greater r a t e than the temperature independent s y n t h e s i s of ATP during photo-phosphorylation. This o v e r a l l d i f f e r e n c e (between enzymatic temperature dependent, ATP c a t a b o l i s m and photochemical, temperature independent ATP anabolism) i s r e f l e c t e d by a greater ATP pool at lower temperatures at corresponding l i g h t I n t e n s i t i e s . b) E f f e c t s of metabolic i n h i b i t o r s The ATP pool o f a p h o t o s y n t h e t i c organism i s f i l l e d from four d i s t i n c t sources, namely s u b s t r a t e phosphoryla-t i o n , o x i d a t i v e p h o s p h o r y l a t i o n ( i n c l u d i n g chemiosmotic g r a d i e n t s ) , n o n - c y c l i c photophosphorylation (photosystem II) and c y c l i c photophosphorylation (photosystem I ) . I t i s 45 possible to semi-selectively d i s t i n g u i s h the contributions of each of these ATP sources to the net i n t r a c e l l u l a r ATP pool by incubating the organisms with a " s e l e c t i v e " metabol-i c i n h i b i t o r p r i o r to ATP extraction. To determine the q u a l i t a t i v e contribution of oxidative phosphorylation to the ATP pool i n the l i g h t and dark, KCN and 2,4-dinitrophenol were added separately to 125 ml a l i -quots of Skeletonema costatum cultures. The samples were incubated i n duplicate for 2-4 hr at 18 C i n the l i g h t and dark and the ATP was extracted as described. In the l i g h t , neither i n h i b i t o r e f f e c t i v e l y reduced the ATP concentration per mg chlorophyll a by more than 10%, as indicated i n f i g . 12a, b. In the dark however, the ATP pool was depleted by 71% with KCN and over 88% with 2,4-dinitrophenol. These res u l t s imply the contributions of oxidative phosphorylation to the ATP pool i s r e l a t i v e l y minor i n the l i g h t , but not i n the dark, as suggested by Arnon (1963). Selective i n h i b i t i o n of non-cyclic photophosphorylation -4 with 10 M 3(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) (Boardman, 1971) was used to approximate the contributions of PS II to the ATP pool. The r e s u l t s of t h i s experiment are shown i n f i g . 12c, and indicate that DCMU reduced the ATP pool by about 47% i n the l i g h t and 12% i n the dark (N=3). In h i b i t i o n of c y c l i c photophosphorylation with meta-b o l i c i n h i b i t o r s i s not s e l e c t i v e . Raven (19 74) has i n t e r -F i g . 1 2 . E f f e c t s of 1 0 M KCN ( a ) , DNP (b), DCMU ( c ) , and CCCP (d) on the ATP p o o l of Skeletonema costatum a t 2 0 C i n the l i g h t and dark. 1 0 0 c o J3 c CL < 75 5 0 2 5 m i t m HI if • 1 & H Mi m W m m m I m m is •mm i t i t m m m m • i D preted the r e l a t i v e importance of c y c l i c photophosphoryla-t i o n i n phosphate uptake by i n h i b i t i n g the process with the uncoupler, carbonyl cyanide m-chlorophenylhydrozone (CCCP). Despite the possible multiple e f f e c t s of such an uncoupler (Boardman, 19 71) , attempts were made to determine' the role of PS I i n f i l l i n g the ATP pool. CCCP, at 10~4M, reduced the ATP pool by 84% i n the l i g h t and 29% i n the dark ( f i g . 12d), i n d i c a t i n g a major source of ATP i n the l i g h t i s due to c y c l i c electron flow i n PS I. Although i t i s not possible to p r e c i s e l y determine the rate of ATP output from each of the four possible sources from these data, the re s u l t s suggest that oxidative and sub-str a t e phosphorylations are r e l a t i v e l y minor sources of ATP i n the l i g h t , or at l e a s t the ATP pool i s not p r e f e r e n t i a l l y f i l l e d from these two processes. In the dark however, neither photosystem I or PS II i s operative, causing a reduc-t i o n i n the o v e r a l l ATP pool ( f i g . 11), and a dependence of the ATP pool on oxidative phosphorylation p r i m a r i l y ( f i g . 12a, b) . c) E f f e c t s of metabolic i n h i b i t o r s on NO^ uptake To determine the e f f e c t s of metabolic i n h i b i t o r s on NO^ uptake, 100 ml samples of S_. costatum (containing ca. 7 2 x 10 c e l l s ) were inoculated into 1 1. of a r t i f i c i a l sea-water (Kessler, 196 7) enriched with f/2 nutrients ( G u i l l a r d and Ryther, 1962). I n i t i a l NO-, concentrations were adjusted F i g . 1 2 - 2 . Effects of 1 0 M KCN, DCMU, and CCCP on nitrate uptake velocities i n Skeletonerra costatum at 2 0 C and 0.09 ly/rnin. 0 5 NO3 C o n c e n t r a t i o n ( / J M ) 00 49 to 28 ug-atoms/1. The c e l l suspension was subsampled into 125 ml Erlenmeyer f l a s k s , and incubated with eit h e r 10 KCN, 10~5M DCMU or 10~5M CCCP i n tb.fi l i g h t at 18 C. NO~ uptake was followed by measuring the decrease i n e x t r a c e l l u -l a r NO~ (Eppley et a l , 1971; Ahmad and Morris, 1967). Only CCCP i n h i b i t e d NO~ uptake i n the l i g h t ( f i g . 12-2), while KCN and DCMU had no appreciable e f f e c t on uptake per se. These results are consistent with those reported by Eppley and Coatsworth (196 8), and support the suggestion of Healy (1973) that c y c l i c photophosphorylation i s a major energy source for NO^ uptake i n the l i g h t . d) E f f e c t s of e x t r a c e l l u l a r NH*, NO~, and PO^~ on i n t r a c e l l u l a r ATP pools The addition of e x t r a c e l l u l a r NH^ or NO^ to cultures of S_. costatum (grown on NO^) within 60 sec of ATP extraction caused a marked reduction i n the ATP pool. The decrease i n ATP was non-linearly dependent upon the concentration of the ex t r a c e l l u l a r nitrogen source, and not demonstrable i n the dark (Table 7). These re s u l t s appear to be at variance with those reported by Ull r i c h - E b e r i u s (19 73) , who found NO^ had no appreciable e f f e c t on the ATP pool of Ankistrodesmus brau n i i (see discussion). In contrast to the e f f e c t of the 3-nitrogen ions however, 3 ug-atoms PO^ / l enrichments increas-ed the ATP l e v e l over a 60 sec period, suggesting the fl u x of PO^ into the nucleotide pool i s rapid. 50 Table 7 E f f e c t s of NO^, NHJ, and PoJ" on the ATP pool of Skeltonema costatum. C e l l s were grown a t 18 C and incubated f o r 1 min w i t h the n u t r i e n t c o n c e n t r a t i o n s i n d i c a t e d under i d e n t i c a l l i g h t c o n d i t i o n s (0.09 l y / m i n ) . Condition N nM ATP/mg chlorophyll a* Untreated 247 ± 34 900 yM NO~ 3 120 ± 19 9 yM NO^  6 163 ±28 2.5 yM NO~ 6 221 ± 30 900 yM NHj 3 149 ±31 9 yM NHj 3 194 ± 16 2.5 yM NHj 3 224 ± 23 8 yM PoJ" 3 284 ± 46 0.8 yM PO^" 3 261 ±17 * Mean of N determinations ± standard deviation I t seems reasonable to suggest that the addition of ei t h e r e x t r a c e l l u l a r NO^ or NH.^  causes increased hydrolysis of ATP and thereby reduces the i n t r a c e l l u l a r ATP pool. This process appears analagous to the addition of e x t r a c e l l u l a r + + + • K to the (Na + K )-activated transport adenosine triphos-phatase (Glynn, 1962) . B. F i e l d Studies on Natural Phytoplankton Communities i n Knight I n l e t I. Morphometric and hydrographic considerations Knight I n l e t i s divided into two main basins, separated by shallow s i l l s . One s i l l , at 63 m, separates s t a t i o n QC from Kn 3 and a second s i l l at 65 m separates Kn 3 from the headward stations ( f i g . 13). The consequences of t h i s bottom topography are complicated and not f u l l y understood, but e s s e n t i a l l y the s i l l s r e s t r i c t the deep water c i r c u l a -t i o n throughout most of the year (Stone, personal communica-tion) . Due to summer entrainment and offshore upwelling, deep water exchanges slowly from May to December with high s a l i n i t y water coming in t o the estuary from 10 m to the bottom. The less dense, low s a l i n i t y surface waters, con-tained i n the upper 10 m, flow seaward. The surface waters are separated from the subsurface water masses by a strong ha l o c l i n e from Kn 7 to the head (Table 8). The picno c l i n e does not appear to lead to nutrient impoverishment however, as both n i t r a t e and phos-52 TABLE 8 Salinity, nitrate and the estimated 1% l i g h t depths at the sample stations i n July and September i974. A pronounced halo-cline i s observable at a l l stations i n the Inlet, especially i n the upper 5 m. Station Depth (m) Salinity °/oo N O 3 (yg-at/1) \% lig h t depth July Sept. July Sept. July Sept. QC 0 31.06 31.02 20.21 15.92 16m 20m 5 31.08 31.09 20.21 20.45 10 31.27 31.39 19.49 21.45 30 31.68 31.73 19.97 24.17 Kn 3 0 16.70 22.19 16.64 17.03 10m 16m 5 24.48 27.62 13.91 18.42 10 29.23 29.73 . 15.18 18.81 30 30.22 20.44 19.28 -Kn 5 0 8.64 11.28 3.47 4.44 6m 5m 5 26.08 13.08 17.64 18.86 10 26.66 25.86 23.75 24.31 30 30.31 30.49 23.64 -Kn 7 . .0 3.27 3.82 1.09 2.38 4m 3m 5 20.92 24.59 14.08 6.86 10 26.99 27.91 19.62 19.03 30 29.86 30.11 24.11 24.41 Kn 11 0 0.57 0.89 1.36 1.33 0.2m 1m 5 20.43 20.59 12.55 7.04 10 27.87 26.97 19.30 20.18 30 29.93 30.03 22.20 22.55 -F i g . 13. Bottom topography and g e n e r a l i z e d c i r c u l a t i o n p a t t e r n i n Knight I n l e t , B. C. and adjacent Queen C h a r l o t t e S t r a i t s . U I CO 54 phorus are r e l a t i v e l y abundant i n the e u p h o t i c zone through-out most o f the y e a r . N i t r a t e averages ca. 25-30 ug-atoms/1 i n the e s t u a r y , f a r i n excess o f some r e p o r t e d v a l u e s of n i t r o g e n l i m i t a t i o n (Glooschenko and C u r l , 1971; Thomas, 1971) . I I . A b r i e f d e s c r i p t i o n of the phytoplankton community The s e a s o n a l v a r i a t i o n s i n c h l o r o p h y l l a ( f i g . 2) i n d i -c a t e a bimodal bloom, c h a r a c t e r i s t i c o f many temperate waters (Parsons and Takahashi, 19 73; Raymont, 1962; Smayda, 19 73). A spring.bloom i n M a r c h - A p r i l was observed i n 19 74 a t QC, Kn 3, Kn 5, Kn 7, and Kn 11 and a secondary f a l l bloom was seen i n August-September at QC, Kn 3, Kn 5, and Kn 11 f o r s u r f a c e waters (0-5 m). On a l l c r u i s e s , p h ytoplankton p o p u l a t i o n s were r e l a t i v e -l y low i n the i n l e t and h i g h i n the a d j o i n i n g S t r a i t , p o s s i b l y due to the steep s a l i n i t y g r a d i e n t s upstream. The major s p e c i e s r e p r e s e n t e d i n the S t r a i t were c h a r a c t e r i z e d by diatoms, e s p e c i a l l y Chaetoceros d e b i l i s , T h a l a s s i o s i r a  n o r d e n s k o l d i , Skeletonema costatum, A s t e r i o n e l l a j aponica, and Thalassionema n i t z s c h i o i d e s . The i n l e t s t a t i o n s were not dominated by a s i n g l e s p e c i e s , although a l l the net p l a n k t o n (>25 y) were marine, and undoubtedly were c a r r i e d i n t o the e s t u a r y from the a d j o i n i n g S t r a i t s . Skeletonema  costatum appeared abundant a t Kn 3 and Kn 5, but was v i r t u -a l l y absent from Kn 11 i n J u l y . At t h a t time nanophyto-55 plankton were predominantly observed i n the preserved samples. I I I . Experimental re s u l t s a) E f f e c t s of nitrogen enrichment on inorganic carbon f i x a t i o n F i e l d studies with whole, natural phytoplankton com-munities from Knight I n l e t indicate a b r i e f , t r a n s i t o r y decrease i n inorganic carbon f i x a t i o n per unit chlorophyll a_ ( i . e . a s s i m i l a t i o n ratio) at a l l l i g h t i n t e n s i t i e s tested with 3 ug-atoms N/1 enrichments ( f i g . 14). The enrichments (either NO^ or NH^ as the nitrogen source) only represent 10-15% of the dissolved i n s i t u nitrogen concentrations (Table 8); hence excess nutrient repression was not consider-ed to be a cause of the decreased pro d u c t i v i t y over a 2 hr incubation period (Maclsaac and Dugdale, 19 72). The a s s i m i l a t i o n r a t i o s ranged from 3 to 7 i n the un-enriched samples at maximum l i g h t i n t e n s i t y (i.e 100%). At Kn 11 however, l i g h t i n h i b i t i o n was observed at i n t e n s i t i e s over 60%, Implying the c e l l s were shade adapted (Ryther and Menzel, 1959). At t h i s l a t t e r s t a t i o n , p a r t i c u l a t e suspend-ed material decreased the l i g h t penetration so severely that the 1% l i g h t depth was estimated at less than 1 m. Enrich-ment with nitrogen during the f i r s t 2 hr resulted i n 60-70% decrease i n assimilation r a t i o s ( f i g . 14 A and E) . The inorganic carbon f i x a t i o n during t h i s period did not vary F i g . 14. Comparison of the e f f e c t s of NO^ (•) and NH^ (•) with unenriched samples (•) on photo-synthetic a s s i m i l a t i o n r a t i o s . At QC," a 2 hr incubation with e i t h e r nitrogen source resulted i n decreased a s s i m i l a t i o n r a t i o s (A), but t h i s e f f e c t was diminished a f t e r a 6 hr preincubation (B). At Kn 3 (C) and Kn 5 (D) 6 hr preincubation with NO-^  or NH^ indicated a d i f f e r e n c e i n the rate at ehich the two communities responded to nutrient conditions. Kn 3 showed less supression of carbon f i x a t i o n a f t e r 6 hr, possibly due to increased adaptation to external nutrients. At Kn 7 (E) a 2 hr incubation caused an altered l i g h t response; the enriched samples responded l i n e a r l y with l i g h t i n t e n s i t y , while the control described a hyperbola. At Kn 11 (F) i n h i b i t o n at i n t e n s i t i e s above 60% was observed i n a l l samples a f t e r a 2 hr incubation. 56 b F i g . 14. 0 50 100 0 50 100 % L i g h t Intens i ty h y p e r b o l i c a l l y w i t h l i g h t i n t e n s i t y , but remained l i n e a r , s u g g e s t i n g dark r e a c t i o n s were not l i m i t i n g (Qasim e t a l , 1969) . F o l l o w i n g a 6 hr p r e i n c u b a t i o n w i t h e i t h e r n i t r o g e n 14 source, a d d i t i o n o f 2 u C i Na 2C r e s u l t e d i n i n c r e a s e d a s s i m i l a t i o n r a t i o s compared t o unenriched samples ( f i g . 14 B, C, D, F ) . These r e s u l t s i n d i c a t e a p h y s i o l o g i c a l adapta-t i o n t o e x t e r n a l n u t r i e n t c o n c e n t r a t i o n s by the c e l l s . The ada p t i v e p e r i o d appears t o be between 2 and 6 h r and i s c h a r a c t e r i z e d by " r e v e r s e k i n e t i c " r e l a t i o n s h i p s between i n -o r g a n i c carbon f i x a t i o n and e x t e r n a l n i t r o g e n c o n c e n t r a t i o n s . These types o f k i n e t i c s imply d e c r e a s i n g carbon f i x a t i o n w i t h i n c r e a s i n g e x t r a c e l l u l a r n u t r i e n t c o n c e n t r a t i o n s . These data are c o n s i s t e n t w i t h unpublished o b s e r v a t i o n s by Dugdale ( H a r r i s o n , p e r s o n a l communication). b) E f f e c t s o f n i t r o g e n enrichment on c h l o r o p h y l l a / c e l l The p h y s i o l o g i c a l a d a p t a t i o n o f the phytoplankton com-munitie s t o 6 hr p r e i n c u b a t i o n s w i t h e i t h e r n i t r o g e n source was accompanied by i n c r e a s e d c h l o r o p h y l l a s y n t h e s i s p e r c e l l (Table 9 ) . At Kn 11 i n i t i a l c h l o r o p h y l l a / c e l l aver--7 . aged c a. 9.10 x 10 y g , r e p r e s e n t i n g 1.5 t o 2.3 times the i n t r a c e l l u l a r c h l o r o p h y l l a found i n c e l l s from the oth e r f o u r s t a t i o n s (Table 9 ) . T h i s r e l a t i v e l y h i g h l e v e l o f i n t r a c e l l u l a r c h l o r o p h y l l a i s c o n s i s t e n t w i t h u m b r o p h i l l i c a d a p t a t i o n (J^rgensen, 196 4; Ryther and Menzel, 19 59; 58 Steeman N i e l s e n e t a l , 1962). Upon a d d i t i o n of n i t r o g e n , c h l o r o p h y l l a / c e l l Increased by ca. 10 to 20% w i t h i n 6 t o 8 h r , and was accompanied by Increased a s s i m i l a t i o n r a t i o s Cfig. 14), at a l l s t a t i o n s . 59 TABLE 9 The effects of nitrate and ammonium i n intracellular chlorophyll a. These data are taken from Sept. samples from 2 m after 6 to 8 hr preincubation with either nitrogen source. C e l l counts were made on preserved samples within a week of collection. An inverted microscope was used to count 100 random fie l d s at 250 X. Chlorophyll a data are mean values and are intended for intrastation comparision only. Station Condition Cells/1(x10 b) Chi a/cell (x10~ ug) % change QC A 4.27 4.21 B 4.32 4.30 +2 C 4.51 4.93 +18 D 4.71 4.87 +13 Kn 3 A 2.11 8.06 B 2.19 7.66 -5 C 2.40 8.71 +8 D 2.61 8.63 +7 Kn 7 A 2.97 6.02 B 3.05 5.89 -2 C 3.09 6.48 +7 D 3.26 6.31 +5 Kn 11 A 0.33 9.10 — B 0.41 8.72 -4 C 0.37 10.88 +19 D 0.37 10.31 +13 A- original samples B- unenriched samples C- nitrate enriched D- ammonium enriched 60 Discussion A. Nitrate Uptake i n Marine Phytoplankton The r e s u l t s of the laboratory studies with membranes is o l a t e d from s i x species of marine phytoplankton (excluding the d i n o f l a g e l l a t e s 1 1 , indicate the presence of an enzyme 2+ - -that hydrolyzes ATP i n the presence of Mg , C l , and NO^. The p h y s i o l o g i c a l function of the (NO^, C l )-activated adenosine triphosphatase cannot be i n f e r r e d from the b i o -chemical studies alone because (a) the protein i s no longer oriented i n v i t r o on the plasmalemma, and (b) none of the a c t i v a t i n g ions are t r u l y substrates for the reaction ( i . e . the only true substrate apparently i s ATP). To date however, analagous membrane-bound enzymes, such as the (Na + + K + ) -2+ activated adenosine triphosphatase (Skou, 1957), the Ca activated adenosine triphosphatase (Schatzmann and Vincenzi, 1969) , the HCO^-activated adenosine triphosphatase (Narumi 2+ and Kanno, 1973), and the Si(OH)^-stimulated, Mg -activated adenosine triphosphatase (Hemmingsen, 19 71), have been implicated i n the tr a n s l o c a t i o n of t h e i r respective a c t i v a t -ing ions across various membranes. Mainly because of the d i f f i c u l t y i n understanding the molecular mechanism(s) of these enzymes (or more s p e c i f i c a l l y , the d i f f i c u l t y i n ex-p l a i n i n g the coupling between scalar energy l i b e r a t e d from 1 see Appendix I 61 ATP h y d r o l y s i s to the v e c t o r a l f l u x of i o n s ) , the r o l e s of membrane-bound adenosine triphosphatases i n r e g u l a t i n g i n t r a c e l l u l a r i o n co n c e n t r a t i o n s has to be determined by an examination of both p h y s i o l o g i c a l and b i o c h e m i c a l p r o p e r t i e s , s i n c e n e i t h e r i s adequate t o serve t h i s purpose alone. A major f e a t u r e of the (NO^, C l ) - a c t i v a t e d adenosine triphosphatase, i s o l a t e d from both Skeletonema costatum and Chroomonas s a l i n a , i s the bimodal pH p r o f i l e . I f the adenosine t r i p h o s p h a t a s e was s o l e l y r e s p o n s i b l e f o r NO^ t r a n s p o r t , NO^ uptake by whole c e l l s should e x h i b i t a s i m i -l a r hydrogen i o n dependence. This c o r r e l a t i o n i s d i f f i c u l t t o o b t a i n from the l i t e r a t u r e because of the l a c k of pub-l i s h e d data on the pH dependence of NO^ uptake by marine phytoplankton. U l l r i c h - E b e r i u s (197 3) has provided however, a pH p r o f i l e f o r NO^ uptake i n Ankistrodesmus b r a u n i i (Chlorophyceae) which e x h i b i t s a s i m i l a r bimodal pH depend-ence i n v i v o to t h a t observed here f o r the i s o l a t e d ( N 0 3 ' C l ) - a c t i v a t e d adenosine triphosphatase i n v i t r o . A n k i s t r o d e s - mus b r a u n i i was found to have an optimum NO^ uptake v e l o c i t y at ca. pH 8.0 and a secondary optimum at ca. pH 6.0. These r e s u l t s are comparable to the optima of 7.8-8.2 and 6.9 found f o r the (NO^, C l ) - a c t i v a t e d adenosine triphosphatase ( f i g . 4). I t may be p o s s i b l e to a t t r i b u t e the s l i g h t s h i f t i n the secondary pH peak to i n t e r s p e c i f i c v a r i a t i o n , or perhaps p h y s i o l o g i c a l c o n d i t i o n s t h a t were not d u p l i c a t e d (e.g. the a d d i t i o n of d e t e r g e n t s ) . A second p h y s i o l o g i c a l (and perhaps more e c o l o g i c a l l y s i g n i f i c a n t } f e a t u r e i m p l i c a t i n g the (NO^, C l ) - a c t i v a t e d adenosine triphosphatase w i t h NO^ uptake, i s the c o r r e l a t i o n between t h e i r h a l f - s a t u r a t i o n constants. Table 5 summarizes the comparison between K values c a l c u l a t e d f o r NO-, a c t i v a -m J t i o n of the (NO^, C l ) - a c t i v a t e d adenosine triphosphatase and K values reported f o r NO., a s s i m i l a t i o n by whole c e l l s (Eppley e t ' a l , 1969) . Skeletonema costatum, Ditylum b r i g h t -w e l l i i , and I s o c h r y s i s galbana h a l f - s a t u r a t i o n constants agree, w i t h i n experimental v a r i a n c e , w i t h reported K g values f o r NO^ uptake. The r e s u l t s f o r D u n a l i e l l a t e r t i o l e c t a are s i g n i f i c a n t l y d i f f e r e n t , although a c t i v a t i o n of the enzyme by a n i o n i c detergents d u r i n g the e x t r a c t i o n procedure (Nakao e t 'al_, 19 74) , c l o n a l v a r i a t i o n s (Hecky and Kilham, 19 74) , and v a r i a t i o n s i n c u l t u r i n g c o n d i t i o n s cannot be r u l e d out. 7 In a d d i t i o n , Grant and Turner (1969) , u s i n g 5 x 10 t o 8 — 5 x 10 c e l l s / m l , reported t h a t the NO^ uptake v e l o c i t y i n Amphidinium carterae was "too low t o measure", supp o r t i n g the observation t h a t the (NO^, C l ) - a c t i v a t e d adenosine t r i -phosphatase i s apparently absent from t h i s s p e c i e s . Eppley e t a l (1969) reported t h a t the K f o r NO~ uptake i n Gonyau-l a x polyedra i s ca. 10 yM. This value i s about t e n f o l d g r e a t e r than f o r species w i t h the (NO^, C l ) - a c t i v a t e d adeno-sine triphosphatase ( i . e . diatoms, euglenoids, e t c . ) . The apparent v a r i a t i o n s i n K , thought t o be a s s o c i a t e d w i t h c e l l s i z e and h a b i t a t (Eppley e t a l , 1969; Parsons and Takahashi, 1973} have not f u l l y e x p l a i n e d the a b i l i t i e s of d l n o f l a g e l -l a t e s t o r each the dominant p o p u l a t i o n d e n s i t i e s sometimes observed i n " r e d t i d e s " (e.g. Pincemin, 1969). Although the t h r e e d l n o f l a g e l l a t e s t e s t e d do not appear t o have the (NO^, C l ) - a c t i v a t e d adenosine t r i p h o s p h a t a s e to mediate the t r a n s p o r t o f NO^ across the plasmalemma, t h e i r undoubted c a p a b i l i t y t o u t i l i z e NO^ i n d i c a t e s t h e r e i s another mechan-ism f o r i t s uptake. For example, d i n o f l a g e l l a t e s may u t i l i z e a coupled t r a n s p o r t phenomenon: the maintenance of an e l e c t r o c h e m i c a l p o t e n t i a l a c r o s s the c e l l membrane, a l l o w i n g NO^ to be t r a n s p o r t e d a l o n g the e l e c t r o c h e m i c a l g r a d i e n t of the i o n (Katchalsky and Curran, 196 7). Ion uptake k i n e t i c s i n p h o t o s y n t h e t i c organisms are o f t e n c h a r a c t e r i z e d by c o m p l i c a t e d , m u l t i p h a s i c f u n c t i o n s (Nissen, 19 73, 19 74). E s s e n t i a l l y these k i n e t i c s can be r e s o l v e d i n t o two major components: a h i g h c a p a c i t y , low a f f i n i t y system ( i . e . h i g h V , h i g h K ) , and a low c a p a c i -in 3.x s t y , h i g h a f f i n i t y system ( i . e . low V , low K ) ( e . g . Azam and V o l c a n i , 19 74; H e l l e b u s t and Lewin, i n p r e s s ) . The a b i l i t y t o d i s t i n g u i s h between these two components k i n e t i c -l y i s a f u n c t i o n o f s u b s t r a t e c o n c e n t r a t i o n . The k i n e t i c s o f the (NO^, C l ) - a c t i v a t e d adenosine t r i p h o s p h a t a s e suggest t h a t the enzyme has a r e l a t i v e l y h i g h a f f i n i t y f o r NO^ uptake ( i . e . K^. < 1 uM NO^ f o r most s p e c i e s ) , however, due to the l a r g e energy of a c t i v a t i o n ( f i g . 5) and the d i r e c t dependence of NO^ t r a n s p o r t on ATP h y d r o l y s i s ( f i g . 8a), t h i s system may not have a h i g h c a p a c i t y . P h o t o s y n t h e t i c d i n o f l a g e l l a t e s , w h i l e capable of u t i l i z i n g NO^ f o r amino a c i d s y n t h e s i s (Packard and B l a s c o , 1974), have, on the whole, a lower a f f i n i t y f o r the i o n than t h a t of other groups. These observations suggest t h a t d i n o f l a g e l l a t . e s may possess a low a f f i n i t y , but h i g h c a p a c i t y system, f o r NO^ uptake, w h i l e other s p e c i e s , w i t h the (NO^, C l ) - a c t i v a t e d adenosine t r i p h o s p h a t a s e , u t i l i z e p r i m a r i l y a h i g h a f f i n i t y , low c a p a c i t y system. Eppley e t a l (1969) and Parsons and Takahashi (1973) have suggested the v a r i a t i o n s i n K f o r NO., uptake may be due t o v a r i a t i o n s i n c e l l s i z e , and perhaps h a b i t a t of the species (e.g. oceanic vs. n e r i t i c ) . Hecky and Kilham (1974) have d i s p u t e d the hypothesized r e l a t i o n s h i p between K and s c e l l s i z e , by p o i n t i n g out t h a t v a r i a t i o n s i n h a l f - s a t u r a -t i o n constants are sometimes observed i n v a r i o u s s t r a i n s of the tsame s p e c i e s , grown under i d e n t i c a l c u l t u r e c o n d i t i o n s . (Presumably c e l l s i z e s are uniform between s t r a i n s . ) As K values f o r NO^ uptake i n marine phytoplankton are c a l c u l a t e d over r e l a t i v e l y broad ranges of the e x t e r n a l NO^ concentra-t i o n s (e.g. NO^ may t y p i c a l l y vary from 0 to 50 yM), i t may be d i f f i c u l t to r e s o l v e the various components of the uptake k i n e t i c s . Thus, s p e c i e s , and perhaps even c l o n a l v a r i a t i o n s , may be due t o k i n e t i c i n t e r a c t i o n s between the h i g h c a p a c i t y , low a f f i n i t y system and the low c a p a c i t y , h i g h a f f i n i t y system, o p e r a t i n g i n d i f f e r e n t r e l a t i v e p r o p o r t i o n s under v a r i a b l e e x t e r n a l n u t r i e n t c o n c e n t r a t i o n s . In a d d i t i o n , i t should be s t a t e d t h a t both the h i g h c a p a c i t y , low a f f i n i t y system and the low c a p a c i t y , h i g h a f f i n i t y system a p p a r e n t l y e x h i b i t Michaelis-Menten k i n e t i c s i n d i v i d u a l l y (e.g. H e l l e -b u s t and Lewin, i n p r e s s ) . T h i s o b s e r v a t i o n makes i t e s p e c i a l l y d i f f i c u l t to r e s o l v e the two systems from k i n e t i c experiments where the c o n c e n t r a t i o n s range over two o r d e r s o f magnitude, and few d e t e r m i n a t i o n s o f n u t r i e n t uptake v e l o c i -t i e s are made a t h i g h c o n c e n t r a t i o n s . A t h i r d f e a t u r e i m p l i c a t i n g the (NO^r C l ) - a c t i v a t e d adenosine t r i p h o s p h a t a s e w i t h NO^ t r a n s p o r t i s the r e s u l t s of p h y s i o l o g i c a l experiments s u g g e s t i n g t h a t the primary energy source f o r the uptake process i s due to c y c l i c photo-p h o s p h o r y l a t i o n i n v i v o . As NO^ uptake i s not s e v e r e l y i n h i b i t e d by r e s p i r a t o r y i n h i b i t o r s , such as KCN or DNP, or by DCMU (an i n h i b i t o r of n o n - c y c l i c p h o t o p h o s p h o r y l a t i o n ) , but i s a f f e c t e d by CCCP, the c o r r e l a t i o n between NO^ uptake and c y c l i c p h o t o p h o s p h o r y l a t i o n i s a p p a r e n t l y not due to i n d u c t i o n of the c a r r i e r system, but r a t h e r to i n c r e a s e d s u b s t r a t e ( i . e . ATP) c o n c e n t r a t i o n s i n the l i g h t . These r e s u l t s are c o n s i s t e n t w i t h the o b s e r v a t i o n s t h a t i n c r e a s i n g l i g h t i n t e n s i t i e s r e s u l t i n i n c r e a s i n g ATP c o n c e n t r a t i o n s ( f i g . 11) . 66 Grant and Turner C1969) and Maclsaac and Dugdale C19 72) have demonstrated t h a t NO^ uptake i s l i g h t dependent and can Be d e s c r i b e d by Michaelis-Menten k i n e t i c s w i t h r e s p e c t to l i g h t i n t e n s i t y ( i . e . NO^ uptake v e l o c i t i e s vs_. l i g h t i n t e n -s i t i e s d e s c r i b e a r e c t a n g u l a r h y p e r b o l a ) . I f the (NO^, d e -a c t i v a t e d adenosine t r i p h o s p h a t a s e were s o l e l y r e s p o n s i b l e f o r NOg uptake, and p r i m a r i l y dependent on c y c l i c photophos-p h o r y l a t i o n f o r ATP, NOg uptake s h o u l d undergo d i e l p e r i o d i -c i t y i n s i t u . T h i s s u g g e s t i o n i s c o n s i s t e n t w i t h o b s e r v a -t i o n s made by Eppley e t a l (19 71), who r e p o r t e d t h a t NO^ uptake i n Skeletonema costatum e x h i b i t e d l i g h t / d a r k p e r i o d i -c i t y . Furthermore, not o n l y does NO^ uptake e x h i b i t d i e l p e r i o d i c i t y , but the s u b s t r a t e - i n d u c i b l e enzyme, n i t r a t e r e d u c t a s e , has been observed to be l i g h t dependent as w e l l (Eppley e t a l , 19 71; Packard, 19 73; Packard and B l a s c o , 19 74). The d i e l p e r i o d i c i t y observed f o r n i t r a t e r eductase may be a t t r i b u t e d t o l i g h t i n t e n s i t y per se (Beevers and Hageman, 1969), as w e l l as the i n d u c t i o n o f the enzyme by NO^. As the (NO^, C l ) - a c t i v a t e d adenosine t r i p h o s p h a t a s e i s appar-e n t l y c o n s t i t u t i v e (see R e s u l t s s e c t i o n A . I . i ) , NO^ uptake may occur a t low l e v e l s i n the l i g h t , even i n the presence of NH^ (a r e p r e s s o r o f n i t r a t e r e d u c t a s e ) . Under these con-d i t i o n s , the i n t r a c e l l u l a r NO^ c o n c e n t r a t i o n would not be a f f e c t e d by n i t r a t e r e d u c t a s e , as presumably the enzyme would be r e p r e s s e d by NH* (Rigano and V i o l a n t e , 19 73) . Consequent-l y , NO-j uptake, but not reduction or a s s i m i l a t i o n , may occur i n the presence of Ntljj. This phenomenon would cause a re-duction i n the e x t r a c e l l u l a r NO^ concentrations, but would not be r e f l e c t e d by NO^-dependent growth. This type of i n t e r a c t i o n may be termed "luxury consumption" (Ketchurn, 19 39), and i s suggested to be due to i n h i b i t i o n of NO^ re-duction and incorporation, but not NO^ uptake. In addition, ATP i s apparently the rate l i m i t i n g substrate i n the dark at non-limiting NO^ concentrations. Thus the induction of n i t r a t e reductase i n the l i g h t may be due to increased ATP supply to the (NO^, C l )-activated adenosine triphosphatase, r e s u l t i n g i n increased NO^ uptake. The increased NO^ uptake may stimulate the synthesis of n i t r a t e reductase, r e s u l t i n g i n a more c l a s s i c a l type of induction of n i t r a t e reductase. A fourth feature suggesting that NO^ uptake i s c o r r e l a t -ed to ATP hydrolysis may be i n f e r r e d from the r e s u l t s of e x t r a c e l l u l a r NO^ enrichments on the i n t r a c e l l u l a r ATP pool. These data (Table 7) imply that e x t r a c e l l u l a r NO^ reduces the ATP pool rapidly and non-linearly, presumably due to increased ATP hydrolysis. This phenomenon appears to be an-+ + + alagous to the addition of e x t r a c e l l u l a r K to the (Na + K )-activated adenosine triphosphatase (Glynn, 1962) and supports the hypothesis that the p h y s i o l o g i c a l role of the (N0~, de-activated adenosine triphosphatase i s to mediate the trans-port of external NO^ into the cytoplasm. In addition, the 68 data from Table 7 a l s o suggest t h a t e x t r a c e l l u l a r NH.^  s t i m u l a t e s ATP h y d r o l y s i s . As t h i s c a t i o n i s not an a c t i -v a t o r o f the (NO^, C l ) - a c t i v a t e d adenosine t r i p h o s p h a t a s e , these r e s u l t s suggest t h a t NH^ uptake may a l s o be due t o an a c t i v e t r a n s p o r t p r o c e s s , but v i a a d i f f e r e n t pathway than f o r N0~. U l l r i c h - E b e r i u s (19 73) has observed t h a t NO^ a d d i t i o n does not cause a r e d u c t i o n i n the ATP p o o l ( i n the l i g h t ) i n Ahki s t r o d e smus b r a u n i i . Although these r e s u l t s appear at v a r i a n c e w i t h the d a t a from T a b l e 7, the time i n t e r v a l be-tween a d d i t i o n o f NO^ and d e t e r m i n a t i o n o f the ATP p o o l i n A. b r a u n i i was about 10 min. T h i s r e l a t i v e l y l o n g i n t e r v a l may a l l o w the organism to r e s t o r e the ATP charge ( i . e . reach a new e q u i l i b r i u m ) (e.g. Holm-Hansen, 1973), p o s s i b l y l e a d -i n g t o the c o n c l u s i o n t h a t NO^ uptake was not d i r e c t l y ATP dependent. In 19 39 Ketchurn poxnted out the importance of PO^ i n - 3-NO-j uptake. The r e s u l t s of PO^ enrichments on the ATP p o o l 3-(Table 7) imply t h a t P 0 4 i s r a p i d l y i n c o r p o r a t e d i n t o o r g a n i c phosphates (notably ATP). I t i s suggested t h a t 3- - ' 3-PO^ may p a r t i a l l y l i m i t NO^ uptake i f PO^ c o n c e n t r a t i o n s 3_ are l i m i t i n g , o r the f l u x of PO^ i n the adenylate p o o l i s 3_ low. The i n d i r e c t e f f e c t o f PO^ l i m i t a t i o n under these c o n d i t i o n s i s suggested to be a r e d u c t i o n i n the ATP p o o l (but not n e c e s s a r i l y ADP or AMP), thereby l i m i t i n g such ATP dependent processes as NO^ uptake. This phenomenon has been observed by Sakshaug (personal communication) i n chemostat 3_ c u l t u r e s . In a d d i t i o n , Raven (19 74) has suggested t h a t PO^ uptake i s coupled t o ATP h y d r o l y s i s ( i . e . the anion i s a c t i v e l y t r a n s p o r t e d a c r o s s ' c e l l membranes). The' source of ATP f o r t h i s process i s apparently c y c l i c photophosphoryla-t i o n ; i t i s i n h i b i t e d by CCCP. As e x t e r n a l PO^ i s apparent-l y r e l a t e d to i n c r e a s e d ATP pools i n Skeletonema costatum, 3_ the s t o i c h i o m e t r i c r a t i o of PO^ per ATP hydrolyzed i s probably g r e a t e r than 1. ( I f the r a t i o were l e s s than 1 net i n t r a c e l l u l a r ATP would decrease i n the presence of i n c r e a s -ed P O 4 ~ . ) N i t r a t e uptake i n most species of marine phytoplankton i s apparently mediated by a c o n s t i t u t i v e , membrane-bound adenosine t r i p h o s p h a t a s e , and not i n h i b i t e d by NH^. F i e l d s t u d i e s w i t h n a t u r a l phytoplankton communities were c a r r i e d out t o determine what p h y s i o l o g i c a l feedback mechanisms c o n t r o l NO-j uptake i n v i v o . F i r s t l y , i t was observed t h a t NO^ enrichments (as w e l l as NH^ enrichments) i n h i b i t e d i n o r -ganic carbon f i x a t i o n f o r ca. 2 t o 6 hr ( f i g . 14). This suppression of carbon f i x a t i o n was not dependent on the previous l i g h t h i s t o r y of the c e l l s , as the samples were obtained from the same absolute depths at each s t a t i o n de-s p i t e extreme changes i n the l i g h t e x t i n c t i o n c o e f f i c i e n t s . As both. N0 3 uptake and i n o r g a n i c carbon f i x a t i o n are l i g h t dependent p r o c e s s e s , the i n h i b i t i o n of carbon f i x a t i o n i n the presence of i n c r e a s e d e x t e r n a l n i t r o g e n c o n c e n t r a t i o n s Implies a c o m p e t i t i o n between the two elements f o r e i t h e r l i g h t i t s e l f , o r a p r o d u c t of l i g h t r e a c t i o n s . A f t e r a 6 h r p r e i n c u b a t i o n w i t h e i t h e r n i t r o g e n source however, carbon f i x a t i o n was not a f f e c t e d by e x t e r n a l n i t r o g e n c o n c e n t r a t i o n s , although the l i g h t i n t e n s i t i e s were u n a l t e r e d . These r e -s u l t s suggest t h a t an a d a p t i v e p e r i o d of between 2 and 6 h r I s r e q u i r e d by n a t u r a l p o p u l a t i o n s of p h y t o p l a n k t o n a f t e r a l t e r a t i o n s i n the e x t e r n a l n i t r o g e n c o n c e n t r a t i o n s . The e c o l o g i c a l s i g n i f i c a n c e o f t h i s a d a p t a t i o n may not be c r u c i a l under most n a t u r a l c i r c u m s t a n c e s , because e x t r a c e l l u -l a r n i t r o g e n c o n c e n t r a t i o n s p r o b a b l y r a r e l y i n c r e a s e w i t h such r a p i d i t y . The a d a p t i v e p e r i o d observed i n K n i g h t I n l e t , f o l l o w i n g n i t r o g e n enrichments, was accompanied by i n c r e a s e d c h l o r o -p h y l l a / c e l l (Table 9 ) . As e x t e r n a l NO^ may i n c r e a s e ATP h y d r o l y s i s , thereby r e d u c i n g t h e i n t r a c e l l u l a r ATP p o o l (Table 7), i t i s i n f e r r e d t h a t i n c r e a s e d c h l o r o p h y l l a s y n t h e s i s r e l i e v e s t h i s energy d r a i n by p r o v i d i n g a g r e a t e r i n t r a c e l l u l a r l i g h t - t r a p p i n g a r e a . Thus ATP dependent p r o -c e s s e s , p r e f e r e n t i a l l y d r i v e n by l i g h t r e a c t i o n s , such as - + 3-carbon f i x a t i o n , NO^, NH^, and PO^ uptake, may be s u p p l i e d w i t h more s u b s t r a t e ( i . e . ATP) as the r e s u l t o f i n c r e a s e d 71 ligrit-dependent ATP synthesis. This suggestion has also been made, to some extent, by Bates (.1974) , who observed increased ch l o r o p h y l l a / c e l l following NO^ enrichments i n Skeletonema costatum (Bates, personal communication). The reverse k i n e t i c i n t e r a c t i o n between inorganic car-bon and inorganic nitrogen suggests that one major physio-l o g i c a l control of the u t i l i z a t i o n of these two elements involves chlorophyll a / c e l l . Biochemically however, chloro-p h y l l a / c e l l appears to be regulated, at l e a s t i n part, by feedback from ATP pools (or perhaps more p r e c i s e l y the 3-r e l a t i v e proportions of ATP, ADP, AMP, and P0 4 ). This reverse k i n e t i c pattern has been observed f o r other n u t r i -ents, including Si(OH) 4 and carbon, Si(OH) 4 and n i t r a t e , and NH4 and carbon (Dugdale, personal communication). In a l l cases where the reverse k i n e t i c phenomenon e x i s t s , the nutrients are apparently transported e i t h e r by an adenosine triphosphatase, or i n the case of carbon, coupled i n some way d i r e c t l y to ATP (e.g. i n the intermediary metabolism). In addition, the primary ATP source for the uptake and assimilation of these nutrients i s apparently from the l i g h t reactions, with the exceptions of the a p o c h l o r i t i c (colour-less) organisms, such as N i t z s c h i a alba, that require Si(OH) 4, but cannot u t i l i z e inorganic carbon (e.g. Goering, 1974; Hemmingsen, 1971). Based on the r e s u l t s of these experiments, a modified 72 pathway o f n i t r o g e n metabolism i n diatoms was c o n s t r u c t e d ( f i g . 151. The p r i n c i p a l m o d i f i c a t i o n i n t h e pathway l a s opposed t o the one pr e s e n t e d i n f i g . 11 i s the a d d i t i o n o f the (NO^f C l ) - a c t i v a t e d adenosine t r i p h o s p h a t a s e . I t sho u l d be noted however, t h a t the sources o f energy f o r NO^ uptake, NO^ r e d u c t i o n , and NH^ i n c o r p o r a t i o n are not i d e n t i c a l t o those proposed by Packard and B l a s c o (19 74) ( f i g . 1). Ahmad and M o r r i s (196 7) have i n d i c a t e d t h a t NO^ r e d u c t i o n i s s e n s i t i v e t o DCMU, s u g g e s t i n g t h a t the r e d u c t a n t s are d e r i v e d from n o n - c y c l i c p h o t o p h o s p h o r y l a t i o n . The r e d u c t a n t s ( e i t h e r NADH or NADPH, depending on the sp e c i e s ) are suggest-ed t o be s u p p l i e d s p e c i f i c a l l y from Photosystem I I , w h i l e the primary ATP source f o r NO^ uptake i s generated from Photosystem I . D i n o f l a g e l l a t e s appear t o d e v i a t e from t h i s proposed pathway i n a number of important a s p e c t s . F i r s t l y , the up-take o f NO^ i s not d i r e c t l y c o u p l e d t o ATP h y d r o l y s i s i n the sp e c i e s examined here. Hence, i t would appear t h a t NO^ up-take i n d i n o f l a g e l l a t e s i s mediated by some mechanism(s) other than the (NO^, C l ) - a c t i v a t e d adenosine t r i p h o s p h a t a s e . Secondly, o b s e r v a t i o n s o f n i t r a t e r e d u c t a s e a c t i v i t y i n a Gonyaulax p o l y e d r a bloom o f f the c o a s t o f Baj a C a l i f o r n i a by Packard and B l a s c o (19 74) i n d i c a t e t h a t t h i s enzyme does not appear t o undergo d i e l p e r i o d i c i t y , t y p i c a l l y observed i n other s p e c i e s . These workers have suggested t h a t NO^ reduc-73a F i g . 15. A g e n e r a l i z e d scheme of MO^ metabolism i n some marine phytoplankton. N i t r a t e uptake i s suggested t o be mediated by a membrane-bound (N0~, C l " ) - a c t i v a t e d adenosine t r i p h o s p h a t a s e . The primary (but not sole) ATP source f o r t h i s process appears to be c y c l i c photophosphorylation. Once i n s i d e the c e l l , N0~ may be sequestered i n a vacuole, or reduced v i a n i t r a t e reductase (NR) to N0~. N i t r i t e i s f u r t h e r reduced to ammonium, u t i l i z i n g f e r r e d o x i n as an e l e c t r o n donar. The ammonium i s probably i n c o r p o r a t e d i n t o a-keto g l u t a r a t e to form glutamate. Regeneration of a-keto g l u t a r a t e may occur i f the amino n i t r o g e n of glutamate i s transaminated to other keto a c i d s . I n d i r e c t evidence appears to suggest t h a t ammonium uptake i s ATP dependent as w e l l . 73b Fig. 15 A T P cy top la sm cr(?) NO; ADP N07 tt-i ,NAD(P)H-21 *NAD(P) + NO: ATP(?) ADP FDX DX •4 • tonoplast NO; vacuole -*NH4 ++C(-KG Q| -NADH ° | * N A D + m itochond r ion Glu. <l  1' a. a. +*-KG N I T R A T E M E T A B O L I S M ( G E N E R A L I S E D S C H E M E ) 74 t i o n may be coupled to r e s p i r a t o r y , r a t h e r than photosyn-t h e t i c , reductants. I t i s suggested t h a t n i t r a t e reductase In d i n o f l a g e l l a t e s i s s u b s t r a t e i n d u c i b l e (Eppley e t a l , 19 73) , i m p l y i n g t h a t even i n the dark NO^ uptake may occur i n t h i s group. T h i r d l y , the major enzyme i m p l i c a t e d i n NH^ a s s i m i l a t i o n , namely glutamate dehydrogenase, appears t o be absent from some s p e c i e s , notably Amphidiniurn c a r t e r a e (Falkowski, unpublished data; Ahmed, personal communication). In order to i n c o r p o r a t e NH^ i n t o amino a c i d s , at l e a s t some species must u t i l i z e an a l t e r n a t i v e pathway(s) than i n d i c a t e d i n e i t h e r f i g . 1 or f i g . 15. The proposed n i t r o g e n pathway i n d i n o f l a g e l l a t e s ( f i g . 16) i n d i c a t e s t h a t the probable route of NH^ i n c o r p o r a t i o n i s through glutamine synthetase. Transaminations from the amido-nitrogen of glutamine t o other amino acids may be c a t a l y z e d by glutamine(amide): 2-oxoglut-arate amino t r a n s f e r a s e oxido-reductase (also known as g l u t a -mate synthetase) (Dainty, 19 72). This pathway may a l s o be present i n blue-green algae (Dharmawadene e_t a l , 19 73; Haystead e t a l , 19 73). The enzyme glutamine synthetase r e q u i r e s ATP f o r NH^ i n c o r p o r a t i o n , but has a lower f o r NH^ than glutamate dehydrogenase. In a d d i t i o n , transamina-t i o n s from glutamine to a-keto g l u t a r a t e r e s u l t s i n the f o r -mation of 2 molecules of glutamate. This l a t t e r amino a c i d i s a s u b s t r a t e f o r glutamine synthetase. Thus, the s y n t h e s i s of 2 glutamate molecules may a l l o w one to act as an i n t e r -75a F i g . 16. A p o s t u l a t e d pathway f o r n i t r o g e n a s s i m i l a t i o n i n d i n o f l a g e l l a t e s . N i t r a t e uptake i s suggested t o be thermodynamically c o u p l e d t o a Na +/K + pump on the c e l l membrane. The r e d u c t i o n o f NO^ i s mediated by an NADH-dependent n i t r a t e r e d u c t a s e (NR), which probably d e r i v e s e l e c t r o n s from r e s p i r a t i o n . Once NO^ i s reduced to N0 2, n i t r i t e r e d u c t a s e ( e l e c t r o n source i n v i v o unknown) f u r t h e r reduces the n i t r o g e n t o NH^. The NH* i s incorpor-^' a t e d i n t o glutamate t o form glutamine. T h i s l a t t e r s t e p, mediated by glutamine s y n t h e t a s e (GS), i s p r o b a b l y f o l l o w e d by the a d d i t i o n o f the amido n i t r o g e n t o a-keto g l u t a r a t e t o form two moles o f glutamate. The r e a c t i o n i s c a t a l y z e d by glutamate s y n t h e t a s e (G1S),. The r e g e n e r a t i o n of * a-keto g l u t a r a t e i s achieved by the t r a n s a m i n a t i o n (TR) o f the amino n i t r o g e n from one glutamate to o t h e r keto a c i d s . In t h i s group ( d i n o f l a g e l l a t e s ) , amino a c i d metabolism i s suggested to o c c u r i n both the l i g h t and dark, as e l e c t r o n sources f o r r e d u c t i o n , and h i g h energy n u c l e o t i d e s do not appear t o be l i g h t dependent. I t i s suggested t h a t the major f u n c t i o n o f the c h l o r o p l a s t i s t o generate carbon s k e l e t o n s f o r amino a c i d s y n t h e s i s . 75 b F i g . 16. P O S T U L A T E D NO; NITROGEN ASSIMILATION in D INOFLAGELLATES tonoplast -•NO: * — • -ATP * *• NADH NO, O X TCA J mitochondrion c CO <u I J N R NOT NiR cytoplasm *• NH 4 + glutamate «*-*" A T P ^ f N A D H ADP NAD NADH NAD+ glutamine + a - K G — » glutamate f ^ •+ ' glutamate amino acids + a - K G TrA^  + ct-keto acids • » • 76 mediary n i t r o g e n source Gas i n f i g . 11, w h i l e the other i s r e c y c l e d through glutamine synthetase. This co n s e r v a t i o n of carbon skeletons may reduce the jarbon d r a i n from the t r i c a r b o x y l i c a c i d c y c l e . B. A Model f o r NO^ Uptake Based on B i s u b s t r a t e K i n e t i c s The i s o l a t e d (NO^, C l - ) - a c t i v a t e d adenosine t r i p h o s p h a -tase i s i d e n t i f i e d i n v i t r o by NO^-stimulated (and by C l , but t o a l e s s e r extent) ATP h y d r o l y s i s . While the enzyme i s b i o c h e m i c a l l y an adenosine t r i p h o s p h a t a s e , the p h y s i o -l o g i c a l r o l e of the membrane-bound p r o t e i n i s e s s e n t i a l l y the a c t i v e t r a n s p o r t of NO^. P r e s e n t l y NO^ uptake k i n e t i c s are determined d i r e c t l y by measuring the disappearance of NO-j from the medium, as i t i s not p o s s i b l e t o d i r e c t l y r e -l a t e the a c t i v i t y of the (NO^, C l ) - a c t i v a t e d adenosine triphosphatase to N0~ uptake at t h i s time. In v i t r o t h e r e -f o r e the anion (NO^l fu n c t i o n s as an enzyme a c t i v a t o r and may not be considered a tru e b i o c h e m i c a l s u b s t r a t e . From a p h y s i o l o g i c a l o r e c o l o g i c a l standpoint however, NO^ may be considered a tru e s u b s t r a t e i n v i v o . I t has g e n e r a l l y been accepted t h a t NO^ uptake k i n e t i c s may be approximated by the Michaelis-Menten expression (e.g. Caperon and Meyer, 1972; Eppley and Rogers, 1970). Grant and Turner (1969) and Maclsaac and Dugdale (19 72) have pointed out t h a t NO^ uptake v e l o c i t i e s a l s o appear t o be a f u n c t i o n of l i g h t i n t e n s i t y . The l a t t e r two authors have i n d i c a t e d t h a t l i g h t ( a c t i n g as a p h y s i o l o g i c a l s u b s t r a t e ) i s r e l a t e d t o NO-^ uptake v e l o c i t i e s by M i c h a elis-Menten k i n e t i c s as w e l l . The e f f e c t s of l i g h t are p r o b a b l y i n -d i r e c t ; the r e s u l t s of t h i s study suggest t h a t l i g h t i n c r e a s -es i n t r a c e l l u l a r ATP, a s u b s t r a t e necessary f o r the (NO^, C l " ) - a c t i v a t e d , a d e n o s i n e t r i p h o s p h a t a s e . F o r the purposes of a d e s c r i p t i v e model of NO^ uptake however, l i g h t may be c o n s i d e r e d to be a p h y s i o l o g i c a l s u b s t r a t e r e q u i r e d by many s p e c i e s f o r N0 3 uptake. B r i e f l y , b o t h e x t r a c e l l u l a r NO^ c o n c e n t r a t i o n s and l i g h t may be c o n s i d e r e d s u b s t r a t e s a f f e c t i n g the v e l o c i t y o f N0~ uptake i n s i t u . Both s u b s t r a t e s may obey M i c h a e l i s -Menten k i n e t i c s i n d i v i d u a l l y (when the o t h e r s u b s t r a t e i s s a t u r a t i n g ) . E c o l o g i c a l l y n e i t h e r l i g h t nor e x t r a c e l l u l a r NO^ i s o f t e n s a t u r a t i n g however. I t i s p robable t h a t NO^ uptake k i n e t i c s may not obey t r u e M i c h a e l i s - M e n t e n f u n c t i o n s when b o t h s u b s t r a t e s are l i m i t i n g s i m u l t a n e o u s l y . Before c o n s i d e r i n g the mathematical f u n c t i o n s necessary t o d e s c r i b e b i s u b s t r a t e uptake k i n e t i c s , a t t e n t i o n s h o u l d be drawn to the treatment o f l i g h t and e x t r a c e l l u l a r NO^ uptake as independent v a r i a b l e s a f f e c t i n g NO^ uptake v e l o c i t i e s . Three p o s s i b l e r e l a t i o n s h i p s between NO^ uptake v e l o c i t i e s (v) and both l i g h t (L) and e x t r a c e l l u l a r NO^ (N) are shown i n f i g . 17. These t h r e e - d i m e n s i o n a l p r o f i l e s may be o b t a i n -ed by s a t u r a t i n g the uptake system w i t h one s u b s t r a t e 78 ( e i t h e r l i g h t o r e x t r a c e l l u l a r NO^) and v a r y i n g the second s u b s t r a t e a c c o r d i n g l y . Consequently, two maximum uptake v e l o c i t i e s may be determined, one c o r r e s p o n d i n g t o l i g h t L - N (V } and the o t h e r t o e x t r a c e l l u l a r N0 o (V ). "max 3 max Of the t h r e e p o s s i b i l i t i e s , (aj r e p r e s e n t s the o p t i m a l s t e a d y - s t a t e system i n t h a t the ATP output generated from the l i g h t r e a c t i o n s i s bal a n c e d by the r a t e of ATP h y d r o l y -s i s s t i m u l a t e d by e x t r a c e l l u l a r NO^- The system d e p i c t e d i n (c) i s u n s t a b l e because l i g h t l i m i t a t i o n would e v e n t u a l l y l i m i t ATP, the energy source u l t i m a t e l y r e s p o n s i b l e f o r NO^ uptake (except i n d i n o f l a g e l l a t e s ) . As the system approaches the s t e a d y - s t a t e the k i n e t i c s would be more a c c u r a t e l y d e s c r i b e d by ( a ) . The c o n d i t i o n s r e p r e s e n t e d by (b) are s t a b l e , i n t h a t i n t r a c e l l u l a r ATP ( i . e . l i g h t r e a c t i o n s ) i s not l i m i t i n g NO^ uptake, alt h o u g h the NO^-stimulated ATP h y d r o l y s i s i s l e s s than i d e a l ( p o s s i b l y because o f l i m i t i n g uptake s i t e s ) . The t h r e e c o n d i t i o n s r e p r e s e n t e d i n f i g . 17 do not adequately r e s o l v e the NO^ uptake v e l o c i t i e s i n the maximum response p o r t i o n s of the curve ( i . e . the r e g i o n s where zero order k i n e t i c s are o p e r a t i v e ) . D e s c r i p t i o n s o f these r e g i o n s c o u l d be ob t a i n e d by i n c l u d i n g h a l f - s a t u r a t i o n c o n s t a n t s . As h a l f - s a t u r a t i o n constants cannot be s o l e l y d e r i v e d from V v a l u e s , and must be c a l c u l a t e d from e x p e r i m e n t a l d a t a , max a m a t r i x might be s e t up to i n c l u d e V and h a l f - s a t u r a t i o n • r max 79a F i g . 16. Three dimensional p r o f i l e s of NO^ uptake v e l o c i t i e s as a f u n c t i o n of l i g h t and e x t r a c e l l u l a r N O 3 . These p r o f i l e s may be o b t a i n e d by h o l d i n g one s u b s t r a t e c o n s t a n t ( e i t h e r l i g h t o r NO^) and v a r y i n g the second s u b s t r a t e a c c o r d i n g l y . I n d i v i d u a l l y , two f a m i l i e s of curves may be o b t a i n e d , one c o r r e s p o n d i n g to l i g h t and the o t h e r to e x t r a c e l l u l a r NO,. F i g . 17. (a) (b) ( c ) 80 c o n s t a n t s . Thus V max V N max K, CD where: i s the l i g h t I n t e n s i t y s u p p o r t i n g half-maximum NO-j uptake v e l o c i t i e s a t NO^ s a t u r a t i o n , i s the NO^ c o n c e n t r a t i o n s u p p o r t i n g half-maximum NO^ uptake v e l o c i t i e s a t l i g h t s a t u r a t i o n . F u n c t i o n a l l y i t i s p o s s i b l e t o c o n s t r u c t a curve f o r NO.J uptake from two p o i n t s , ^ m a x and the h a l f - s a t u r a t i o n c o n s t a n t , by f i t t i n g a l i n e through these p o i n t s i n a l i n e a r t r a n s f o r m a t i o n o f the Michaelis-Menten e x p r e s s i o n . The m a t r i x i n (1) can be e a s i l y r e a r r a n g e d t o f i t a l i n e a r t r a n s -f o r m a t i o n , f o r example the Lineweaver-Burk, double r e c i p r o -c a l r e l a t i o n s h i p . 1 - 1 Cla) When n e i t h e r s u b s t r a t e i s s a t u r a t i n g however, the a f -f i n i t y o f the phytoplankton c e l l s f o r NO^ may not be adequate-l y d e s c r i b e d by e i t h e r o r ( i . e . M i c h a elis-Menten k i n -81 e t i c s ) . I f the c e l l s are considered to be uptake s i t e s r e q u i r i n g two su b s t r a t e s C l i g h t and NO^), a h y p o t h e t i c a l t e r t i a r y Intermediate complex i s formed. k k (L) + (N e) + (C) , 1 • (L-N e-C) ^•(CN i) (2) k 2 The a f f i n i t y constant ( K L N ) i s def i n e d as: [L-N e-Cj k i ^ " [ L j [ N e J [ C J ". k 2 + k 3 C 3 ) In the stea d y - s t a t e c o n d i t i o n (e.g. f i g . 17a), K T X 7 i s a product of the su b s t r a t e c o n c e n t r a t i o n s (L and N g) support-i n g half-maximum uptake v e l o c i t i e s (determined when both su b s t r a t e s are s a t u r a t i n g ) . By i n c o r p o r a t i n g the m a t r i x given i n (1) i n t o b i s u b -s t r a t e enzyme k i n e t i c s (e.g. A l b e r t y , 1956) and s o l v i n g f o r K L N , the f o l l o w i n g equation i s generated: = -LN (1 + — + — - ZlH3 )^ (4) L N L N V In the steady s t a t e K T M i s a f u n c t i o n of V /2 ( i . e . ijiN max the f u n c t i o n Vmax may be approximated as 2). V K. LN represents a p h y s i o l o g i c a l parameter i n v e r s e l y pro-p o r t i o n a l t o the a f f i n i t y of the phytoplankton f o r N0~ but a l s o dependent on l i g h t , and may be u s e f u l i n p r e d i c t i n g the 82 i n t e g r a t e d e f f e c t s o f these two v a r i a b l e s on NO^ uptake. The v a l u e s of i n d i c a t e t h e r e l a t i v e a f f i n i t y o f a phyto-p l a n k t o n community f o r NO^ under e c o l o g i c a l l y r e a l i s t i c c o n d i t i o n s . In c o n c l u s i o n , the model p r e s e n t e d here attempts t o d e a l w i t h NO^ uptake as a two s u b s t r a t e system. The K L N v a l u e s t h a t may be c a l c u l a t e d attempt t o p r e d i c t the e f f e c -t i v e a s s i m i l a t o r y a b i l i t y i n s i t u from knowledge o f the l i g h t i n t e n s i t y and e x t r a c e l l u l a r N0 3 c o n c e n t r a t i o n s . The a p p l i c a -t i o n o f t h i s model t o f i e l d c o n d i t i o n s i s s t r a i g h t f o r w a r d , i f the k i n e t i c parameters i n (1) are determined. In a d d i t i o n t as l i g h t i n t e n s i t i e s are seldom r e p o r t e d a l o n g w i t h K s v a l u e s , c l a r i f i c a t i o n of r e a l or apparent h a l f - s a t u r a t i o n c o n s t a n t s may be r e s o l v e d through the use of K ^ . The v a l u e s o f K L N do not i n d i c a t e the r e l a t i v e importance o f l i g h t or e x t r a c e l l u l a r N0 3, but d e s c r i b e the e f f e c t i v e a f f i n i t y of the c e l l s f o r e x t r a c e l l u l a r NO^ at a g i v e n l i g h t i n t e n s i t y . T h i s l a t t e r i n f o r m a t i o n i s a p o t e n t i a l l y c r u c i a l parameter i n m o d e l l i n g primary p r o d u c t i v i t y i n the sea. 83 References Ahmad, J . and I . M o r r i s . 1967. I n h i b i t i o n of n i t r a t e and n i t r i t e r e d u c t i o n by 2,4 d i n i t r o p h e n o l i n A n k i s t r o d e s -mus. ^ r c h . M i c r o b i o l . 56_: 215-224. A l b e r t y , R. A. 1956. Enzyme k i n e t i c s . In Advances i n En zy mo logy. Ed. F. F. Nord. 17_: 1-64. I n t e r s c i e n c e P u b l i s h e r s , Inc. New York. /Anderson, L. W. J . and B. M. Sweeney. 1974. An a n a l y s i s of i n t r a c e l l u l a r ions and c e l l sedimentation r a t e s i n a marine c e n t r i c diatom, Ditylum b r i g h t w e l l i . J . Phycol. l O s u p p l . : 15-16 ( A b s t r . ) . Arnon, D. I . 1963. Photosynthetic phosphorylation and a u n i f i e d concept of photosynthesis. Proc. Of the F i f t h Cong, of Biochem. pp. 201-232. Pergamon Pre s s , Oxford. A s k a r i , A. (ed). 1974. P r o p e r t i e s and f u n c t i o n of (Na + + K + a c t i v a t e d adenosinetriphosphatase. Ann. N. Y. Acad. S c i v o l . 242. Azam, F. and B. E. V o l c a n i . 1974. Role of s i l i c o n i n diatom metabolism VI. A c t i v e t r a n s p o r t of germanic a c i d i n the h e t e r o t r o p h i c diatom N i t z s c h i a a l b a . Arch. Micro-b i o l . 101: 1-8. " Balke, N. E. and T. K. Hodges. 1975. Plasma membrane adeno-s i n e triphosphatase of oat r o o t s . A c t i v a t i o n and i n h i b i t i o n by Mg 2 + and ATP. P l a n t . P h y s i o l . 55: 83-86. Banse, K. 1974. A review of methods used f o r q u a n t i t a t i v e phytoplankton s t u d i e s . SCOR rep. 18. Ba r n e t t , R. E. and C. M. Grisham. 1973a. The r o l e of l i p i d -phase t r a n s i t i o n s i n the r e g u l a t i o n of the (sodium + potassium) adenosine triphosphatase. Biochem. 12: 2635-2637. Bar n e t t , R. E. and C. M. Grisham. 1973b. The e f f e c t s o_f long-chain a l c o h o l s on membrane l i p i d s and the (Na + K ) ATPase. Biochim. Biphys. Acta 311: 417-422. Bates, S. S. 1974. E f f e c t s of l i g h t and ammonium on n i t r a t e uptake by two species of e s t u a r i n e phytoplankton. M.A. Thesis. C i t y College of the C i t y U n i v e r s i t y of New York 84 Blinks, L. R. 1940. The r e l a t i o n of the b i o e l e c t r i c pheno-mena to i o n i c permeability and to metabolism i n large plant c e l l s . Cold Springs Harbor Symp. Quant. B i o l . 8_: 204-215. Boardman, M. K. 1970. Physical separation of the photosyn-t h e t i c photochemical systems. Ann. Rev. Plant. Physiol. 21: 115-140. Caperon, J. and J. Meyer. 1972. Nitrogen-limited growth of marine phytoplankton - I. Changes i n population charac-t e r i s t i c s with steady-state growth rate.;. Deep-Sea Res. 19: 601-618. Corner, E. D. S. and A. G. Davies. 1971. Plankton as a factor i n the nitrogen and phosphorus cycles i n the sea. Adv. Mar. B i o l . 9_: 101-204. Dainty, T. H. 1972. Glutamate biosynthesis i n Clostridium pasteurianum and i t s s i g n i f i c a n c e i n nitrogen metabolism. Biochem. J. 12 6: 1055-1056. Dharmawardene, M. W. N. , A. Haystead and W. D. P.. Stewart. 1973. Glutamine synthetase of the nit r o g e n ^ f i x i n g alga Anabaena cylindrothe'ca. Arch. Microbiol. 90: 281-285. Dixon, M. and E. C. Webb. 1964. Enzymes. Academic Press, New York. 950 pp. Doty, M. and M. Oguri. 1958. Selected features of the i s o -topi c carbon primary productivity technique. J. Cons. Int. Explor. Mer. 141: 47-55. Eppley, R. W. and J. L. Coatsworth. 1968. Uptake of n i t r a t e and n i t r i t e by Ditylum b r i g h t w e l l i i - k i n e t i c s and mechanisms. J . Phycol. 4_: 151-156. Eppley, R. W., J. N. Rogers and J . J . McCarthy. 1969. Half-saturation constants for uptake of n i t r a t e and ammonium by marine phytoplankton. Limnol. Oceanogr. L4: 912-920. Eppley, R. W. and J. N. Rogers. 1970. Inorganic nitrogen as s i m i l a t i o n of Ditylum b r i g h t w e l l i i , a marine plankton diatom. J . Phycol. 6_: 344-351. Eppley, R. W., J. N. Rogers and J . J. McCarthy. 1971. Light/ dark p e r i o d i c i t y i n nitrogen a s s i m i l a t i o n of marine phytoplankters Skeletonema costatum and Cc* c c o l i thus  huxleyi i n N-limited chemostat culture. J . Phycol. 7_: 150-154. 85 Fiske, C. H. and Y, SubbaRow. 1925. The col o r i m e t r i c deter-mination of phosphorus. J . B i o l . Chem. 66_: 375-400. Glooschenko, W. A. and H. C u r l . 1971. Influence of nutrient enrichment on photosynthesis and a s s i m i l a t i o n r a t i o s i n natural North P a c i f i c phytoplankton communities. J . Fish.Res. Bd. Can. 28: 790-793. Glynn, I. M. 1962. A c t i v a t i o n of adenosine triphosphatase a c t i v i t y i n a c e l l membrane by external K and i n t e r n a l Na. J . Physiol. 16_0: 18-19. Goering, J . J . 1974. Uptake of s i l i c i c a cid by diatoms. Tethys 6: 143-148. Goldman, S. S. and R. W. Albers. 1973. Sodium-potassium-activated adenosine triphosphatase IX. The role of phospholipids. J . B i o l . Chem. 248: 867-874. Grant, B. R. and I. M. Turner. 1969. Light-stimulated n i t r a t e and n i t r i t e a s s i m i l a t i o n i n several species of algae. Comp. Biochem. Physiol. 29: 995-1004. G u i l l a r d , R. R. L. and J . H. Ryther. 1962. Studies of marine planktonic diatoms I. Cycle-fell a' nana Hustedt, and Detonula confervacea (Cleve) Gran. Can. J . M i c r o b i o l . 8: 229-239. ~ Hageman, R. H. and D. P. Hucklesby. 1971. Nit r a t e reductase from higher plants. In Methods i n Enzymology. 23A: 491-503. Academic Press, New York. Harvey, H. W. 1960. The Chemistry and F e r t i l i t y of Sea  Waters. Cambridge University Press, 240 pp. Haystead, A., M. W. N. Dharmawardene and W. D. P. Stewart. 1973. Ammonia a s s i m i l a t i o n i n a n i t r o g e n - f i x i n g blue-green alga. Plant S c i . L e t t s . 1: 439-445. Healey, F. P. 1973. Inorganic nutrient uptake and deficiency i n algae. CRC C r i t i c a l Rev. M i c r o b i o l . Sept. 1973, Hecky, R. E. and P. Kilham. 1974. Environmental control of c e l l s i z e . Limnol. Oceanogr. 19: 361-365. Hellebust, J. A. and J . Lewin. Heterotropic N u t r i t i o n , submitted as a chapter i n Biology of Diatoms. Ed. D. Werner. (In press), 86 Hemmingsen, B. B. 1971, A mono- s i l i c i c acid-stimulated adeno-sine triphosphatase from protoplasts of the apochlori-t i c diatom, Nit z s c h i a alba. Ph.D. Thesis, Univer. C a l i f . Hodges, T. K. 1972. Energy coupling to ion transport i n plant roots. What's New i n Plant Physiol. 4_ Holm-Hansen, 0. and C. R. Booth. 1966. The measurement of adenosine triphosphate i n the ocean and i t s e c o l o g i c a l s i g n i f i c a n c e . Limnol. Oceanogr.. 1JL: 510-519. Hulburt, E. M. , J. H. Ryther and R. R. L. G u i l l a r d . 1960. The phytoplankton of the Sargasso Sea o f f Bermuda. J. Cons. Int. Explor. Mer. 25_: 115-128. J0rgensen, E. G. 1964. Adaptation to d i f f e r e n t l i g h t inten-s i t i e s i n the diatom C y c l o s t e l l a memeghin1ana Ktits. Physiol. Plant. 17: 136-145. 2+ Karlsson, J. and A. Kyl|.n. ^.974. Properties of Mg stimulated and (Na + K )—activated adenosine-5 1-triphos-phatase from sugar beet cotyledons. Physiol. Plant. 32: 136-142. Katchalsky, A. and P. F. Curran. 1967. Non-equ11ibriurn Thermodynamics i n Biophysics. Harvard University Press. 240 pp. Kessler, D. R. 1967. Preparation of a r t i f i c i a l seawater. Limnol. Oceanogr. 12: 176-179. Ketchum, B. H. 1939. The absorption of phosphate and n i t r a t e by illuminated cultures of Nitzschia closterium. Am. J. Bot. 2_6: 399-407. Ladbrooke, B. D. and D. Chapman. 1969. Thermal analysis of l i p i d s , proteins, and b i o l o g i c a l membranes. Review and summary of some recent studies. Chem. Phys. L i p i d s . 3_: 304-367. Lindeman, W. 1958. Observations on the behaviour of phos-phate compounds i n C h l o r e l l a at the t r a n s i t i o n from dark to l i g h t . Proc. Ilnd. Int. Conf. U. N. on the Peaceful Uses of Atomic Energy 24_: 8-15. Lowry, 0. H., N. J . Rosenbrough, A. L. Farr and R. J . Randall. 1951. Protein measurement with the F o l i n phenol reagent. J. B i o l . Chem. 193: 265-275. 87 Lui , N. S. T. and 0. A. Roels. 1972. Nitrogen metabolism of aquatic organisms. I I . The as s i m i l a t i o n of n i t r a t e , n i t r i t e and ammonia by Biddulphia a u r i t a . J. Phycol. 8_: 259-264. Maclsaac, J. J . and R. C. Dugdaie. 1972. Interactions of l i g h t and inorganic nitrogen i n c o n t r o l l i n g nitrogen uptake i n the sea. Deep-Sea Res. 19: 209-232. Maslowski, P. and M. Komoszynski. 1974. P u r i f i c a t i o n and properties of the adenosinetriphosphatase from Zea mays seedlings microsomes. Phytochem. 13_: 89-92. Monod, J. 1942. Reserches sur l a Croissance des Cultures Bacteriennes. 2nd ed. Hermann & Cie., P a r i s , 210 pp. Nakao, M., T. Nakao, Y. Hara, F. Nagai, S. Yagasaki, M. Koi, A. Nagagawa and K. Kawai. 1974. P u r i f i c a t i o n and properties of Na, K-ATPase from p i g brain. Ann. N. Y. Acad. S c i . 242: 24-35. Narumi, S. and M. Kanno. 1973. E f f e c t s of g a s t r i c a c i d stimulants and i n h i b i t o r s on the a c t i v i t i e s of the HCO^ -stimulated, Mg2+-dependent ATPase and carbonic anhydrase i n r a t g a s t r i c mucosa. Biochim. Biophys. Acta 311: 80-89. Nissen, P. 1973. Kinetics of ion uptake i n higher plants. Physiol.Plant. 2_8: 113-120 Packard, T. T. ' 1973. The l i g h t dependence of n i t r a t e reduc-tase i n marine phytoplankton. Limnol. Oceanogr. 18: 446-469. Packard, T. T. and D. Blasco. 1974. Nitrate reductase a c t i -v i t y i n upwelling regions. I I . Ammonia and l i g h t dependence. Tethys 6_: 269-280. Parsons, T. and M. Takahashi. 1973a. B i o l o g i c a l Oceanograph-i c Processes, Pergamon Press, New York, 186 pp. Parsons, T. R. and M. Takahashi. 1973b. Environmental con-t r o l of phytoplankton c e l l s i z e . Limnol. Oceanogr. 18: 511-515. Pincemin, J. M. 1969. Le problem de l'eau rouge. Rev. Int. Oceanogr. Med. 13-14: 181-203. 88 P i a t t , T. and D, V, Subba Rao. 1973, Some current problems i n phytoplankton p r o d u c t i v i t y . F i s h . Res. Bd. Can. Tech. Rep. 370, Qasim, S. Z., S. Wellerhaus, P. M. A. B h a t t a t h i r i and S.A.H. Abi d i . 1969. Organic production i n a t r o p i c a l estuary. Proc. Ind. Acad. S c i . 6_9: 51-94. Raison, J. K., J. M. Lyons and W. W. Thomson. 1971. The influence of membranes on the temperature-induced changes i n the k i n e t i c s of some respiratory enzymes of mito-chondria. Arch. Biochem. Biophys. 142: 83-90. Raven, J . A. 1974. Energetics of active phosphate i n f l u x i n Hydrodictyon afrleanurn . J . exp. Bot, __' 221-229. Raymont, J . E. G. 1963. Plankton and Productivity i n the  Oceans. Pergamon Press, Oxford, 660 pp. Rigano, C. and U. V i o l a n t e . 1973. E f f e c t of n i t r a t e , ammonia and nitrogen st a r v a t i o n on the regulation of n i t r a t e reductase i n Cyanidium caldarium. Arch. M i c r o b i o l . 90: 27-33. Riley, G. A., H. Stommel and D. F. Bumpus. 1949. Quantitative ecology of the Western North A t l a n t i c . B u l l . Bingham Oceanogr. C o l l . 12: 1-169. Riley, J. P. and R. Chester. 1971. Introduction to Marine  Chemistry• Academic Press, New York, 465 pp. Ryther, J. H. and D. W. Menzel. 1959. Light adaptation by marine phytoplankton. Limnol. Oceanogr. 4_: 492-497. Schatzmann, H. and F. F. Vincenzi. 1969. Calcium movements across the membrane of human red c e l l s . J . Physiol. 201: 369-395. Skou, J. C. 1957. The influence of some cations of adenosine triphosphatase from peripheral nerves. Biochim. Biophys. Acta 22: 394-401. Smayda, T. J. 1973. The growth of Skeletonema costatum during the winter-spring bloom i n Narragansett Bay, Rhode Island. Nor. J . Bot. 20: 219-247. Sprott, G. D., J. P. Drozdowski, E. L. Martin and R. A. MacLeod. 1975. Kin e t i c s of Na +-dependent amino acid transport using c e l l s and membrane v e s i c l e s of a marine pseudo-monad. Can. J . M i c r o b i o l . 21: 4 3-50. 89 Steemann Nielsen, E.,. V. Kr. Hansen and E. G. Jcirgensen. 1962, The adaptation to d i f f e r e n t l i g h t i n t e n s i t i e s i n C h l o r e l l a v u l g a r i s and the time dependence on trans-fer to a new l i g h t i n t e n s i t y . Physiol. Plant. 15: 505-517. Strickland, J . D. H. and T. R. Parsons. 1972. A p r a c t i c a l handbook of seawater analysis. F i s h . Res. Bd. Can. B u l l . 167, 2nd ed. 311' pp. Sweeney, B. M. 1974. The potassium content of Gonyaulax  polyedra and phase changes i n the cir c a d i a n rhythm of stimulated bioluminescence by short exposures of ethanol and valinomycin'. Plant Physiol. 5_3: 337-342. Thomas, W. H. 1969. Phytoplankton nutrient enrichment experiments o f f Baja C a l i f o r n i a and i n the eastern e q u i t o r i a l P a c i f i c Ocean. Limnol. Oceanogr. 2 6: 1133-1145. Thomas, W. H. 1970. On nitrogen deficiency i n t r o p i c a l P a c i f i c Ocean phytoplankton: Photosynthetic parameters i n poor and r i c h water. Limnol. Oceanogr. 15_: 380-385 . Thomas, W. H. and R. W. Owen. 1971. Estimating phytoplankton production from ammonium and chlorophyll concentrations i n nutrient-poor water i n the eastern t r o p i c a l P a c i f i c Ocean. F i s h . B u l l . U. S. 69_: 87-92. Ul l r i c h - E b e r i u s , C. I. 1973. Beziehungen der aufnahme von n i t r a t , n i t r i t und phosphat zur photosynthetischen reduction von n i t r a t und n i t r i t und zum ATP S p i e g e l bei Ankistrodesmus b r a u h i i . Planta 115: 25-36. Uribe, E. G. and B. L i . 1973. Stimulation and i n h i b i t i o n of membrane-dependent ATP synthesis i n chloroplasts by a r t i f i c i a l l y induced K gradients. Bioeng. 4_: 435-444. Wilson, T. H. 1962. I n t e s t i n a l Absorption. W. B. Saunders, Philadelphia, 263 pp. Westlake, D. F. 1965. Some problems i n the measurement of rad i a t i o n under water: A review. Photochem. Photobiol. 4: 849-868. Appendix I Preliminary Experiments on Di n o f l a g e l l a t e s 90b The apparent lack of the (NO^, C l )-activated adenosine triphosphatase i n the three d i n o f l a g e l l a t e s examined suggests that the mechanism for NO^ uptake may be d i f f e r e n t from the other organisms studied. The membrane fractions i s o l a t e d from the three species, e s p e c i a l l y Gonyaulax polyedra did y e x h i b i t (Na + + K + ) - a c t i v a t e d adenosine triphosphatase a c t i v i t y . This l a t t e r enzyme was assayed as a marker f o r the plasma-lemma. From the above i t was evident that there was no d i r e c t coupling between ATP hydrolysis and NO^ uptake. However, the accumulation of ions against t h e i r negative chemical gradients can occur without such d i r e c t coupling of the process to an exergonic chemical reaction. One such process, known as secondary transport, requires that the uptake of one nutrient (against i t s negative chemical gradient) be thermodynamically coupled to the active transport of some other ion. For example, Sprott et a l (1975) have recently suggested that a marine pseudomonad (recently designated as an Alteromonas haloplank- t i s ) transports some amino acids into the cytoplams by simul-taneously transporting Na + outward. In such a case the inward gradient for the uptake of the amino acids i s dependent upon an outward gradient for Na +. Here i t i s hypothesized that a s i m i l a r transport phenomena may occur i n d i n o f l a g e l l a t e s ; that NO^ uptake i s coupled to the energy expended i n maintain-ing an asymmetrical d i s t r i b u t i o n of monovalent ions. The following i s a b r i e f d e s c r i p t i o n of some preliminary experi-91 merits designed to t e s t t h i s hypothesis. To determine the i n t r a c e l l u l a r concentrations of Na +, K +, and C l i n Gonyaulax polyedra, 1 0 ml samples of culture were sedimented at 5000 x g In a r e f r i g e r a t e d centrifuge at 5 C, washed once with 0.5 M Tris-acetate b u f f e r (pH 7.9), sonicated for 5 min at 0 C, and the f i n a l suspension was centrifuged at 20,000 x g for 10 min. The supernatant was assayed for Na + and K + i n a Techtron AA 120 atomic absorption spectrophotometer at 589.0 my and 766.5 my respectively. Sea-water of 32°/oo was used as a reference and 0.5 M Tris-acetate buffer as the control blank. I n t r a c e l l u l a r C l ~ was measured by m i c r o - t i t r a t i o n with AgNO^ using a Radiometer (Model 25b) pH meter equipped with a scale expander. The res u l t s of these determinations are shown In Table A-10, along with re-ported ion compositions of Ditylum b r i g h t w e l l i i (Anderson and Sweeney, 1974), and sea water (Riley and Chester, 1971). The data from G. polyedra indicate that i n t r a c e l l u l a r Na + i s s l i g h t l y greater than K +, the opposite of the usual condition. Blinks (19 40) observed high i n t r a c e l l u l a r Na + r e l a t i v e to K + i n one species of Valonia. In the l a t t e r case the r e s t -ing p o t e n t i a l of the c e l l s was 10-20 mv p o s i t i v e to the outside. The unusual p o s i t i v e r e s t i n g p o t e n t i a l i n t h i s species, cor-rel a t e d with the d i s t r i b u t i o n s of Na + and K + suggests that the re s t i n g p o t e n t i a l may be p a r t i a l l y propagated by these ions. 92 TABLE A-10 The sodium, potassium, and chloride concentrations of seawater and some marine phytoplankters (mM) . Na K C l seawater (30u/oo) Ditylum brightwellii Light Dark Gonyaulax polyedra Amphidinium assym-etricum 414 101 118 317 148 90 126 64 119 78 482 156 117 408 190 93 Unfortunately, due to the small c e l l s i z e and thecal plates i n G. polyedra, i t i s d i f f i c u l t to measure the r e s t i n g poten-t i a l i n t h e i r species. Alriphidinium assymetricum Kof. et Swezy, a large (70 u length), naked d i n o f l a g e l l a t e was a v a i l -able for study. Individuals, taken from the middle of the l i g h t cycle (L/D:12/12), were placed on a Fuchs-Rosenthal hemocytometer s l i d e with c_a. 1 ml medium J (Table 1) i n a Faraday cage at room temperature. Glass microelectrodes, f i l l e d with 2 M KCl, and with a t i p diameter of less than 1 p, were inserted into apparently healthy, motile c e l l s . A s i l v e r - s i l v e r chloride reference electrode was placed i n the medium droplet. The membrane p o t e n t i a l was amplified 10 times by a D.C. amplifier and measured from a Techtronix D-15 storage o s c i l l o s c o p e . The mean of 10 i n d i v i d u a l recordings was calculated to be +7.6 mv r e l a t i v e to the medium. One i n d i v i d u a l had a negative p o t e n t i a l of 5.2 mv, but the remain-der of the recordings were a l l p o s i t i v e . These re s u l t s i n dicate that A. assymetricum can be p o s i t i v e l y charged r e l a t i v e to the e x t r a c e l l u l a r f l u i d , at l e a s t during part of i t s l i g h t cycle. This p o t e n t i a l i s probably correlated to a reversed d i s t r i b u t i o n of i n t r a c e l l u l a r Na + and K + (Table A-10). In the absence of d i r e c t recordings of membrane poten-t i a l s from other photosynthetic d i n o f l a g e l l a t e s , i t i s d i f f i c u l t to assess the consequences of the p o s i t i v e membrane pote n t i a l i n Amphidinium assymetricum from these data alone; i t i s especially; tenuous to generalize to other species of dinof lagellates -. In addition, d i e l p e r i o d i c i t y i n i o n i c composition may influence membrane p o t e n t i a l s . This l a t t e r complication has been found to influence the bioluminescence i n G_. polyedra (Sweeney, 1974). This process appears to be related to the membrane p o t e n t i a l . I f i t i s found that other species of d i n o f l a g e l l a t e s have s l i g h t p o s i t i v e membrane pote n t i a l s , i t would appear that the d r i v i n g force for the uptake of small negatively charged ions, such as NO^, N0 2, and PO^ would be propagated by the e l e c t r i c a l gradient. Such a model would further re-quire an ion receptor s i t e bound to the membrane to aid i n the permeation of the charged ions across the nonpolar phos-p h o l i p i d b i l a y e r , i n addition to enhancing ion s e l e c t i v i t y . These preliminary observations i n d i n o f l a g e l l a t e s suggest that NO^ uptake i n di n o f l a g e l l a t e s may not be a consequence of primary active transport, but i s due to a secondary coupled transport mechanism. Consequently, i t may be fea s i b l e to s e l e c t i v e l y i n h i b i t anion uptake i n d i n o f l a g e l l a t e s by absorb-ing a K + ionophore, such as valinomycin, onto the c e l l membranes, thereby hypopolarizing the membrane p o t e n t i a l and i n h i b i t i n g c e l l growth. Appendix II Abbreviations ATP, ADP, AMP - adenosine t r i , d i , and monoph.osph.ate CTP - cytoslne triphosphate DR - photosynthetic dark reactions ETS - electron transport system FDX , FDX - reduced and oxidized ferredoxin GTP - guanosine triphosphate ITP - inosine triphosphate a-KG - "alpha" keto glutarate (.= 2 oxoglutarate) LR - photosynthetic l i g h t reactions NADH - nicotinamide adenine dinucleotide NADPH - nicotinamide adenine dinucleotide phosphate PEP - phospho(enol)pyruvate P^ - inorganic phosphate TCA - t r i c a r b o x y l i c acid cycle TTP - thymidine triphosphate Pub!ications Falkowski, P. G. 1973. The respiratory physiology of hemocyanin in Limul us~ polyphemus. 0. exp. Zool .-£186: !U6. Falkowski, P. G. 1974. Facultaive anaeobiosis i n the horseshoe crab: Phosphoenolpyruvate carboxykinase and heart a c t i v i t i e s . Comp. Biochem. Physiol.- '49 B: 749-759. Falkowski, P. G. N i t ra te uptake in marine phytoplankton: (nitrate., . ch lor ide) -act ivated adenosine triphosphatase from Skeletonema - costatum (Baci l lar iophyceae). J . Phycol . (.in press). Falkowski, P. G. N i t rate uptake in marine phytoplankton: Comparison of ha l f - saturat ion constants from seven species. Limnol, Oceanogr. ( in press). • • r Falkowski, P. G. N i t rate uptake in marine phytoplankton: Energy sources and the interact ion with carbon f i x a t i o n . Mar.. B i o l . ( in press). Falkowski, P. G. A model for n i t r a te uptake based upon bisubstrate enzyme k ine t i c s . Limnol. Oceanogr. C submitted to press). 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0100031/manifest

Comment

Related Items