UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Studies on the p-nitrophenyl phosphate and adenosine monophosphate hydrolyzing activity of Dictyostelium… Bhanot, Pradeep 1986

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

Item Metadata

Download

Media
831-UBC_1986_A1 B52.pdf [ 6.21MB ]
Metadata
JSON: 831-1.0097217.json
JSON-LD: 831-1.0097217-ld.json
RDF/XML (Pretty): 831-1.0097217-rdf.xml
RDF/JSON: 831-1.0097217-rdf.json
Turtle: 831-1.0097217-turtle.txt
N-Triples: 831-1.0097217-rdf-ntriples.txt
Original Record: 831-1.0097217-source.json
Full Text
831-1.0097217-fulltext.txt
Citation
831-1.0097217.ris

Full Text

STUDIES ON THE P-NITROPHENYL PHOSPHATE AND ADENOSINE MONOPHOSPHATE HYDROLYZING ACTIVITY OF DICTYOSTELIUM DISCOIDEUM i • by Pradeep Bhanot B.Sc. Honours ( F i r s t C l a s s ) , Dalhousie U n i v e r s i t y , 19 M . S c , Dal h o u s i e U n i v e r s i t y , 1980 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY' OF GRADUATE STUDIES (Department of M i c r o b i o l o g y ) We accept t h i s t h e s i s as conforming to r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA ^ P r a d e e p Bhanot, 1986 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Department of )E-6 (3/81) i ABSTRACT Evidence i s presented that a s i n g l e enzyme i s respon-s i b l e f o r the h y d r o l y s i s of pNPP and AMP i n D i c t y o s t e l i u m  discoideum membranes. The two a c t i v i t i e s have i d e n t i c a l m o b i l i t i e s when su b j e c t e d to p o l y a c r y l a m i d e g e l e l e c t r o -p h o r e s i s under non-denaturing conditions.- The pH optima of h y d r o l y s i s of AMP or pNPP are h i g h l y dependent upon the c o n c e n t r a t i o n of the s u b s t r a t e and are i d e n t i c a l when s i m i l a r c o n c e n t r a t i o n s are used. pKm vs pH p l o t s gave i d e n t i c a l i n f l e c t i o n p o i n t s f o r AMP and pNPP and suggested that a f u n c t i o n a l group of pKa=8.5 i s i n v o l v e d i n the b i n d i n g of e i t h e r s u b s t r a t e to the enzyme. The p l o t s a l s o suggested t h a t while o n l y one such f u n c t i o n a l group i s r e q u i r e d f o r pNPP b i n d i n g , t h e r e are two i n v o l v e d i n the b i n d i n g of AMP. F u r t h e r evidence f o r the e x i s t e n c e of o n l y one enzyme was p r o v i d e d by the use of mutants with an a l t e r e d Km f o r both pNPP and AMP. Furthermore, both AMPase and pNPPase a c t i v i t i e s i n the mutant were unstable whereas both a c t i v i t i e s were s t a b l e i n the w i l d - t y p e s t r a i n . A p r e v i o u s demonstration t h a t AMPase a c t i v i t y was s e l e c t i v e l y r e l e a s e d from the membrane by c o - i n c u b a t i o n with a phospholipase C p r e p a r a t i o n was i n v a l i d a t e d by the f i n d i n g t h at the p h o s p h o l i p a s e C p r e p a r a t i o n contained an AMP s p e c i f i c h y d o l y t i c a c t i v i t y . The enzyme that h y d r o l y z e d both AMP and pNPP was shown t o be m e c h a n i s t i c a l l y an a l k a l i n e phosphatase. K i n e t i c evidence was obtained i m p l i c a t i n g T r i s as a c o - s u b s t r a t e i n the h y d r o l y s i s of pNPP, and a t r a n s p h o s p h o r y l a t i o n t o T r i s was demonstrated. Futhermore, b i p h a s i c k i n e t i c s of a double-displacement r e a c t i o n was d e t e c t e d by stopped flow spectrophotometry. The h y d r o l y s i s of s u b s t r a t e s v i a a double displacement mechanism and the consequent t r a n s p h o s p h o r y l a -t i o n t o a v a r i e t y of low molecular weight a l c o h o l s i s a d i s t i n g u i s h i n g f e a t u r e of a l k a l i n e phosphatases. The i n h i b i t i o n of the a l k a l i n e phosphatase with orthophosphate was c o m p e t i t i v e at low i o n i c s t r e n g t h but i r r e v e r s i b l e at h i g h i o n i c s t r e n g t h . The a l k a l i n e phosphatase a c t i v i t y i n D.discoideum i s developmentally r e g u l a t e d and i n c r e a s e s markedly d u r i n g c u l m i n a t i o n . Attempts were made to q u a n t i t a t e the l e v e l s of a l k a l i n e phosphatase by immunological methods and by a c t i v e s i t e l a b e l l i n g with 3 2 P i . Problems were encountered with both these methods and i t co u l d not be e s t a b l i s h e d with c o n f i d e n c e whether the i n c r e a s e i n a l k a l i n e phosphatase a c t i v i t y d u r i n g development i s due to the p r e v i o u s l y proposed h y p o t h e s i s that unmasking of p r e - e x i s t i n g enzyme may account f o r the developmental i n c r e a s e i n a c t i v i t y . The b i o l o g i c a l process that a c t i v a t e s a l k a l i n e phosphatase in v i v o i s unknown. However, two p l a u s i b l e methods of a c t i v a t i n g the enzyme were found. The v e g e t a t i v e c e l l enzyme i s a c t i v a t e d at pH 5.5 and by exposure to carbohydrate b i n d i n g p r o t e i n s . IV , TABLE OF CONTENTS Page ABSTRACT i TABLE OF CONTENTS i v LIST OF TABLES v i i LIST OF FIGURES v i i i LIST OF ABBREVIATIONS x ACKNOWLEDGEMENTS x i INTRODUCTION 1 MATERIALS AND METHODS 14 I. (a) Organisms 14 (b) C u l t u r e c o n d i t i o n s 14 I I . MATERIALS 15 I I I . METHODS 16 (a) Membrane p r e p a r a t i o n 16 (b) Membrane p r o t e i n s o l u b i l i z a t i o n 17 (c) P r o t e i n e s t i m a t i o n 17 (d) Enzyme assays 18 (e) Po l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s 20 (f) P u r i f i c a t i o n of pNPPase 21 ( i ) DEAE-Sephadex column chromatography 22 ( i i ) Phenyl-Sepharose column chromatography 23 ( i i i ) Concanavalin A-Sepharose column chromatography 23 V Page (g) I d e n t i f i c a t i o n o f O-phosphoryl-ethanolamine 24 (h) P r e p a r a t i o n of antiserum 27 ( i ) Immunodetection of APase on n i t r o c e l l u l o s e paper 27 ( j ) P i l a b e l l i n g of membrane p r o t e i n s 28 RESULTS SECTION 1: The r e l a t i o n s h i p between a l k a l i n e phosphatase and 5 ' - n u c l e o t i d a s e (a) Comparison of pNPP and AMP h y d r o l y z i n g a c t i v i t i e s f o l l o w i n g SDS-PAGE under non-denaturing c o n d i t i o n s 30 (b) pH optima f o r pNPP and AMP h y d r o l y s i s 36 (c) Dixon p l o t s of pNPP and AMP h y d r o l y t i c a c t i v i t i e s 36 (d) The p r o p e r t i e s of AMPase i n a mutant c e l l l i n e with a l t e r e d pNPPase a c t i v i t y 48 (e) Phosphohpase C treatment and s e l e c t i v e s o l u b i l i z a t i o n of AMPase 52 SECTION 2: St u d i e s on the mechanism of the pNPP and AMP h y d r o l y z i n g enzyme (a) AMP T h i o n u c l e o t i d e analog s t u d i e s 55 (b) T r a n s p h o s p h o r y l a t i o n s t u d i e s 56 (c) I d e n t i f i c a t i o n of the involvement of T r i s as a c o - s u b s t r a t e f o r the enzyme 58 (d) B i p h a s i c k i n e t i c s of pNPP h y d r o l y s i s 65 (e) I n t e r a c t i o n of pNPPase with orthophosphate 70 VI Page SECTION 3: Q u a n t i t a t i o n of APase i n crude membrane e x t r a c t s prepared from d i f f e r e n t developmental stages (a) Immunoquantitation of APase 80 (b) 3 2 p i l a b e l l i n g of pNPPase from v e g e t a t i v e phase and c u l m i n a t i o n phase c e l l s 86 SECTION 4: R e l a t i v e l y m i l d methods of a c t i v a t i o n of a l k a l i n e phosphatase (a) A c t i v a t i o n by m i l d l y a c i d i c c o n d i t i o n s a t 25 C 97 (b) A c t i v a t i o n by Concanavalin A 100 (c) A c t i v a t i o n by g l y c o s i d a s e mixture 104 DISCUSSION 113 REFERENCES 130 v i i LIST OF TABLES Table Page 1. E f f e c t of p-hydroxymercuribenzoate on pNPPase a c t i v i t y . 45 2. Km values f o r AMP and pNPP h y d r o l y s i s by s t r a i n s Ax-3 and HL101. 49 3. H y d r o l y s i s of AMP and t h i o n u c l e o t i d e analogues. 57 4. T r a n s p h o s p h o r y l a t i o n a c t i v i t y of D.discoideum e x t r a c t s u s i n g T r i s b u f f e r . 59 5. T r a n s p h o s p h o r y l a t i o n a c t i v i t y of D.discoideum e x t r a c t s u s i n g ethanolamine b u f f e r . 60 6. I n t e r a c t i o n of orthophosphate with pNPPase 77 7. P r o t e c t i o n by AMP a g a i n s t orthophosphate i n a c t i v a t i o n of pNPPase. 79 8. E f f e c t of a c i d treatment and g l y c o s i d a s e a d d i t i o n on pNPPase a c t i v i t y . 101 9. E f f e c t of a mixed g l y c o s i d a s e p r e p a r a t i o n on the a c t i v a t i o n of pNPPase. 109 v i i i LIST OF FIGURES F i g u r e Page 1. L i f e c y c l e of D i c t y o s t e l i u m discoideum. 3 2. SDS-polyacrylamide g e l e l e c t r o p h o r e s i s of p a r t i a l l y p u r i f i e d enzyme. 25 3. Na t i v e p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s of T r i t o n X-100 e x t r a c t s of v e g e t a t i v e and c u l m i n a t i n g c e l l membrane p r e p a r a t i o n s . 31 4. Native p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s of T r i t o n X-100 e x t r a c t of v e g e t a t i v e membrane p r e p a r a t i o n c o n t a i n i n g spontaneously generated lower molecular fragments of pNPPase a c t i v i t y . 34 5. pH Optima of pNPP and AMP h y d r o l y z i n g a c t i v i t i e s i n T r i t o n X-100 e x t r a c t of v e g e t a t i v e c e l l membranes. 37 6. Dixon p l o t s (pKm vs pH) of (a) pNPP and (b) AMP h y d r o l y z i n g a c t i v i t i e s . 39 7. Dixon p l o t s (pVmax vs pH) of (a) pNPP and (b) AMP h y d r o l y z i n g a c t i v i t i e s . 41 8. E f f e c t of p i c r y l s u l f o n a t e on the a c t i v i t y of pNPPase. 46 9. S t a b i l i t y at -20 C of pNPP and AMP h y d r o l y z i n g a c t i v i t i e s i n T r i t o n X-100 membrane e x t r a c t p r e -pared from v e g e t a t i v e c e l l s of s t r a i n Ax-3 and HL101. 50 10. N a t i v e T r i t o n X-100 p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s of AMP h y d r o l y z i n g a c t i v i t y i n v e g e t a t i v e c e l l membrane p r e p a r a t i o n s t r e a t e d with a phospholipase C p r e p a r a t i o n . 53 11. I d e n t i f i c a t i o n of O-phosphorylethanolamine produced d u r i n g the course of pNPPase r e a c t i o n i n ethanolamine b u f f e r . 61 12. pH P r o f i l e of t r a n s p h o s p h o r y l a t i o n a c t i v i t y of pNPPase. 63 13. Double r e c i p r o c a l p l o t s suggesting a double displacement mechanism f o r pNPPase. 66 i x F i g u r e Page 14. Stopped flow r a p i d spectrophotometric t r a c e of the r e a c t i o n between T r i t o n X-100 s o l u b i l i z e d pNPPase of v e g e t a t i v e c e l l s and pNPP. 68 15. Double r e c i p r o c a l p l o t s showing competive i n h i b i -t i o n of pNPPase a c t i v i t y by orthophosphate 71 16. pH Dependence of the i n h i b i t i o n of pNPPase a c t i v i t y by T r i s - C l . 73 17. pH Dependence of the orthophosphate induced i n a c t i v a t i o n of pNPPase i n T r i s - C l and the e f f e c t of AMP. 75 18. Western b l o t of T r i t o n X-100 s o l u b i l i z e d v e g e t a t i v e c e l l crude membrane p r e p a r a t i o n . 82 19. Western b l o t s of T r i t o n X-100 e x t r a c t s of crude membrane p r e p a r a t i o n s from d i f f e r e n t developmental sources ( u n b o i l e d p r e p a r a t i o n s ) . 84 20. Western b l o t of T r i t o n X-100 e x t r a c t s of crude membrane p r e p a r a t i o n s from d i f f e r e n t develop-mental sources ( b o i l e d p r e p a r a t i o n s ) . 87 21. J*Pi p h o s p h o r y l a t i o n of b u t a n o l e x t r a c t e d v e g e t a t i v e c e l l p r e p a r a t i o n . 90 22. J P i p h o s p h o r y l a t i o n of but a n o l e x t r a c t e d c u l m i n a t i o n phase c e l l p r e p a r a t i o n . 92 23. Autoradiography of 3 2 p i l a b e l l e d v e g e t a t i v e c e l l b u t a n o l e x t r a c t . 94 24. E f f e c t of pH on the a c t i v a t i o n of the v e g e t a t i v e c e l l pNPPase a t 25 C. 98 25. E f f e c t of Concanavalin A on the pNPPase a c t i v i t y of v e g e t a t i v e c e l l s 102 26. E f f e c t of Concanavalin A on the pNPPase a c t i v i t y of c u l m i n a t i o n phase c e l l s . 105 27. E f f e c t of mixed g l y c o s i d a s e on pNPPase of v e g e t a t i v e c e l l s 107 28. E f f e c t of membrane p r o t e i n s and d e g l y c o s y l a t e d membrane p r o t e i n s on the mixed g l y c o s i d a s e mediated a c t i v a t i o n of pNPPase. I l l X ABREVIATIONS AMP adenosine 5 1-monophosphate AMPase adenosine 5'-monophosphatase AMPS adenosine 5 1-monophosphorothioate APase a l k a l i n e phosphatase cAMP adenosine 3 ' , 5 ; - c y c l i c monophosphate dASMP 5'-deoxy 5'-thioadenosine 5'-monophosphate MES (2-N-morpholino) e t h a n e s u l f o n i c a c i d pHMB p-hydroxymercuribenzoate PMSF phenyl methyl s u l f o n y l f l u o r i d e PAGE po l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s P i orthophosphate pNPP p - n i t r o p h e n y l phosphate pNPPase p - n i t r o p h e n y l phosphatase SDS sodium dodecyl s u l f a t e T r i s T r i s (hydroxymethyl) amino methane ACKNOWLEDGEMENTS I wish t o express my a p p r e c i a t i o n to Dr. G. Weeks. In a d d i t i o n , I thank Dr. G. Mauk f o r the use of the stopped flow spectrophotometer, Dr. W.F. Loomis f o r p r o v i d i n g s t r a i n HL101 and Dr. E.F. Rossomando f o r suggesting the use of the t h i o n u c l e o t i d e s . The f i n a n c i a l h e l p from the N a t u r a l Sciences and E n g i n e e r i n g Research C o u n c i l , Canada and the U n i v e r s i t y of B r i t i s h Columbia i s a p p r e c i a t e d . 1 I n t r o d u c t i o n D i c t y o s t e l i u m discoideum i s a s p e c i e s of the A c r a s i a l a e d i s c o v e r e d i n 1935 (Raper, 1935) and has been s t u d i e d e x t e n s i v e l y as a model of e u c a r y o t i c c e l l d i f f e r e n t i a t i o n and morphogenesis (Loomis, 1982). The taxonomic c l a s s i f i c a t i o n of D i c t y o s t e l i u m i s somewhat pr o b l e m a t i c ; i t has been c o n s i d e r e d a fungus (Raper, 1973) as w e l l as a protozoan ( O l i v e , 1975). U n l i k e most protozoa, D i c t y o s t e l i u m has a m u l t i c e l l u l a r stage i n i t s l i f e c y c l e . When b a c t e r i a or d e t r i t u s are a v a i l a b l e , D. discoideum amoebae are f r e e - l i v i n g and reproduce by b i n a r y f i s s i o n . However, when n u t r i e n t s have been exhausted, a developmental process i s t r i g g e r e d t h a t r e s u l t s i n the formation of s pore-bearing m u l t i c e l l u l a r s t r u c t u r e s . The r e a c t i o n to s t a r v a t i o n i n v o l v e s chemotaxis t o cAMP. The amoebae stream to nodal p o i n t s i n response to cAMP p u l s e s and accumulate i n t o aggregates c o n t a i n i n g up to 10^ c e l l s . The center of the aggregate forms a t i p which r i s e s i n t o the a i r , forming a f i n g e r - l i k e p r o j e c t i o n . The p r o j e c t i o n assumes a h o r i z o n t a l o r i e n t a t i o n along the substratum, at which time i t i s c a l l e d a pseudoplasmodium or s l u g . The a n t e r i o r p a r t of the s l u g comprises p r e - s t a l k c e l l s , w h i le the p o s t e r i o r p a r t i s made up of pre-spore c e l l s . The e n t i r e s l u g i s ensheathed i n c e l l u l o s i c s l i m e . 2 The s l u g migrates i n response t o g r a d i e n t s of l i g h t and temperature. Upon c e s s a t i o n of m i g r a t i o n , the s l u g develops i n t o a f r u i t i n g body ( c u l m i n a t i o n phase). T h i s c o n s i s t s of a s t a l k composed of n e c r o t i z e d s t a l k c e l l s and a sorocarp c o n t a i n i n g condensed, encapsulated spores. T h i s l i f e - c y c l e i s summarized i n F i g u r e 1 and i s t r e a t e d more f u l l y i n s e v e r a l reviews (Loomis, 1982, 1975a; Raper, 1973; Bonner, 1967). Axenic s t r a i n s of D. discoideum (Watts and Ashworth, 1970) can be c o n v e n i e n t l y grown i n r i c h n u t r i e n t media and d i f f e r e n t i a t i o n can be induced by d e p o s i t i o n of washed amoebae onto n o n - n u t r i e n t agar (Sussman, 1966). The d i f f e r e n t i a t i o n process i n v o l v e s comprehensive b i o -chemical changes as w e l l as the morphologic changes out-l i n e d above. In a d d i t i o n t o e x t e n s i v e a l t e r a t i o n s i n gene e x p r e s s i o n (Loomis, 1982) p r o t e i n metabolism i s changed. S e v e r a l enzyme a c t i v i t i e s e i t h e r i n c r e a s e or decrease d u r i n g the d i f f e r e n t i a t i o n process (Loomis, 1975a). The changes i n enzyme a c t i v i t i e s are c h a r a c t e r i s t i c of d i s t i n c t morphologic stages and mutants blo c k e d at d i f f e r e n t developmental stages show corres p o n d i n g blockages i n enzyme a c t i v i t y a l t e r a t i o n s (Loomis, 1975a). A l k a l i n e phosphatase (APase) i n c r e a s e s i n a c t i v i t y d u r i n g the t e r m i n a l stages of development (Bonner et a l , 3 S l u g migrat ion Figure 1 L i fe cyc le of P ic tyos te l mm disccn 4 1955; Krivanek, 1956; Krivanek and Krivanek, 1958; G e z e l i u s and Wright, 1965; Loomis, 1969; Lee et. a l , 1975). The developmental r e g u l a t i o n of APase has been s t u d i e d u s i n g a number of techniques. Loomis (1969) observed an e i g h t - f o l d i n c r e a s e i n the s p e c i f i c a c t i v i t y of APase. His s t u d i e s with i n h i b i t o r s i n d i c a t e d t hat concomittant p r o t e i n s y s t h e s i s was e s s e n t i a l f o r developmental accumulation of a c t i v i t y . How-ever, RNA s y n t h e s i s c o u l d be i n h i b i t e d f o r up to e i g h t hours p r i o r t o the c u l m i n a t i o n phase without i n f l u e n c i n g the i n c r e a s e i n enzyme a c t i v i t y . Furthermore, mutants blocked i n the developmental sequence f a i l e d t o accumulate APase a c t i v i t y . Loomis (1969) a l s o d i s r u p t e d c u l m i n a t i o n s t r u c t u r e s t o generate s i n g l e c e l l suspensions and then p e r m i t t e d r e - a g g r e g a t i o n and subsequent development. When c e l l d i s s o c i a t i o n was done j u s t p r i o r to the i n c r e a s e i n APase a c t i v i t y , the r i s e i n a c t i v i t y was a b o l i s h e d . However, d i s s o c i a t i o n at an e a r l i e r stage had l i t t l e a f f e c t on the accumulation of APase s p e c i f i c a c t i v i t y . On the c o n t r a r y , A t r y z e k (1976) found that at no time d i d c e l l d i s s o c i a t i o n i n h i b i t a c c r e t i o n of APase a c t i v i t y when c e l l s were allowed to resume the developmental p r o c e s s . The resumed development of d i s s o c i a t e d c e l l s from s l u g s was b l o c k e d by treatment of the c e l l s with plasma membranes from c e l l s at the same stage of development (McMahon et. a l . , 5 1975). The APase a c t i v i t y i n the t r e a t e d c e l l s was prematurely i n c r e a s e d . The i n c r e a s e d i d not r e q u i r e p r o t e i n s y n t h e s i s . Tuchman et a_l. (1976) a l s o concluded that membrane-membrane con t a c t was e s s e n t i a l f o r the r i s e i n APase a c t i v i t y when they found t h a t treatment of c e l l s at the be g i n n i n g of the d i f f e r e n t i a t i o n sequence with aggregation phase c e l l membranes r e s u l t e d i n a p r e c o c i o u s i n c r e a s e i n APase a c t i v i t y . A requirement f o r cAMP has a l s o been i n d i c a t e d f o r the accumulation of APase a c t i v i t y . Rickenberg et a l . (1977) suspended s t a r v i n g D. discoideum c e l l s i n b u f f e r and a g i t a t e d them t o prevent aggregation. The APase a c t i v i t y i n c r e a s e d i n c e l l s which were exposed t o added cAMP but not when cAMP was omitted. These r e s u l t s i n d i c a t e d t hat c e l l - c e l l c o n t a c t (as i n normal development) or membrane-c e l l c o n t a c t (as i n the p r e v i o u s l y d e s c r i b e d experiments) was not a requirement f o r i n c r e a s e d APase a c t i v i t y when cAMP was p r e s e n t . Soloman e_t a_l. (1964) suggested that the i n c r e a s e i n s p e c i f i c a c t i v i t y d u r i n g d i f f e r e n t i a t i o n c o u l d be due to a s t a g e - s p e c i f i c isoenzyme of APase. Using s t a r c h g e l e l e c t r o p h o r e s i s , they observed one C(-napthyl a c i d phosphate r e a c t i v e band i n the v e g e t a t i v e and aggregation stages but two bands i n subsequent stages. In c o n t r a s t , McLeod and Loomis (1979) found that the APase a c t i v i t y of both 6 v e g e t a t i v e and c u l m i n a t i o n phase c e l l s migrated as s i n g l e bands when s u b j e c t e d to e l e c t r o p h o r e s i s i n T r i t o n X-100 p o l y a c r y l a m i d e tube g e l s . They a l s o found that a number of o t h e r p r o p e r t i e s i n c l u d i n g Km value, sedimentation c o e f f i c i e n t , thermal s t a b i l i t y , i s o e l e c t r i c p o i n t , pH optima +2 and Mg dependence of the a c t i v i t y from the two sources were n e a r l y i d e n t i c a l . A mutant with an APase with a h i g h e r Km f o r pNPP was a l s o i s o l a t e d by MacLeod and Loomis (1979). The h i g h e r Km was a p r o p e r t y of both the v e g e t a t i v e and the c u l m i n a t i o n phase enzyme, again s u g g e s t i n g that a s i n g l e enzyme expressed from the same gene was r e s p o n s i b l e f o r APase a c t i v i t y i n the d i f f e r e n t developmental stages. The i n c r e a s e d Km f o r the APase d i d not a d v e r s e l y a f f e c t the development of the organism. In a d d i t i o n , i n an e a r l i e r study (Loomis, 1975b), L - c y s t e i n e was found to i n h i b i t the accumulation of APase i n d e v e l o p i n g Ax-3 c e l l s , but t h i s had no e f f e c t on morphogenesis, i n d i c a t i n g t h at the e l e v a t e d l e v e l s of a c t i v i t y were not e s s e n t i a l to sorocarp formation. Mohan Das and Weeks (1980) showd that APase from v e g e t a t i v e c e l l s c o u l d be a c t i v a t e d 1 0 - f o l d by h e a t i n g membrane p r e p a r a t i o n s at 50 C. It was suggested that the accumulation of APhase a c t i v i t y d u r i n g the t e r m i n a l stages of 7 development c o u l d r e s u l t from the unmasking of p r e - e x i s t i n g v e g e t a t i v e c e l l enzyme. It was subsequently shown that the v e g e t a t i v e c e l l APase was i n h i b i t e d i n s i t u by a f a c t o r that c o u l d be removed by d i a l y s i s (Mohan Das, and Weeks, 1981). APase has been l o c a l i z e d w i t h i n s p e c i f i c c e l l s i n the m u l t i c e l l u l a r organism. Bonner et a_l. (1955) and Krivanek (1956) showed by h i s t o c h e m i c a l s t a i n i n g with ^ -glycerophos-phate as s u b s t r a t e that APase was l o c a l i z e d i n the a n t e r i o r p r e - s t a l k r e g i o n of the s l u g . Furthermore, i n t e n s e s t a i n i n g was observed i n the p r e - s t a l k r e g i o n adjacent to the p r e -spore r e g i o n of the d e v e l o p i n g f r u i t i n g body. S i m i l a r r e s u l t s were obtained when AMP r e p l a c e d ^-glycerophosphate as the s u b s t r a t e i n the h i s t o c h e m i c a l s t a i n (Krivanek and Krivanek, 1958; Armant et a l . 1980; Quiveger et a l . 1980). Hamilton and C h i a (1975) found that pNPP h y d r o l y z i n g a c t i v i t y i n c r e a s e d about 4 - f o l d i n a v a r i a n t of _D. discoideum that was b l o c k e d i n the spore-formation pathway and which produced o n l y s t a l k c e l l s . There have a l s o been s t u d i e s that i n d i c a t e a s p e c i f i c s u b - c e l l u l a r l o c a l i z a t i o n f o r the APase. Whole c e l l s , impermeable to pNPP, e x h i b i t e d APase a c t i v i t y when pNPP was used as the s u b s t r a t e ( P a r i s h and P e l l i , 1974). T h i s a c t i v i t y was about 15% of the t o t a l a c t i v i t y found i n T r i t o n 8 X-100 homogenates. The m a j o r i t y of APase a c t i v i t y was sedimentable and thus membrane bound. The s u r f a c e a c t i v i t y c o u l d be i n h i b i t e d by p r e - i n c u b a t i n g c e l l s with p-chloromercuribenzoate, a s u l f h y d r y l r e a c t i v e agent that a l s o f a i l s t o p e n e t r a t e whole c e l l s . When c e l l s t r e a t e d i n t h i s way were made permeable to s u b s t r a t e by adding Polymyxin B or T r i t o n X-100, more than h a l f of the t o t a l APase co u l d be d e t e c t e d . Thus a p a r t of the APase was l o c a l i z e d on the c e l l s u r f a c e with the remainder a s s o c i a t e d with membranes but not exposed t o the s u r f a c e . Rossomando and C u t l e r (1975) a l s o found h i g h Apase s p e c i f i c a c t i v i t y i n the plasma membrane f r a c t i o n prepared from c e l l s l y s e d u s i n g Amphotericin B. Green and Newell (1974) and G i l k e s and Weeks (1977) found APase a s s o c i a t e d with the plasma membrane i n t h e i r work on the p u r i f i c a t i o n of plasma membranes. AMP h y d r o l y z i n g a c t i v i t y was a l s o found to be a s s o c i a t e d with the plasma membrane i n a number of h i s t o c h e m i c a l s t u d i e s ( C u t l e r and Rossomando, 1975; Quivger et al. , 1980). The c e l l s u r f a c e l o c a l i z a t i o n of 5 ' - n u c l e o t i d a s e was confirmed by Armant et a_l. (1980) through the use of e l e c t r o n microscopy. E l e c t r o n dense s t a i n was l o c a l i z e d on the outer s u r f a c e of the c e l l membrane, i n d i c a t i n g that the enzyme f u n c t i o n e d on the outer s u r f a c e of the membrane. Furthermore, McMahon et a l . (1977) found AMP h y d r o l y z i n g a c t i v i t y a s s o c i a t e d with the 9 plasma membrane. However, i n an e a r l i e r study, Quivger et a l . , (1978) observed the m a j o r i t y of pNPP and AMP h y d r o l y z i n g a c t i v i t y , as r e v e a l e d by e l e c t r o n microscopy, to be l o c a l i z e d on the c o n t r a c t i l e vacuole membrane. A l k a l i n e phosphatases are r e l a t i v e l y n o n - s p e c i f i c enzymes t h a t are o p t i m a l l y a c t i v e at about pH 9.5, whereas 5 1 - n u c l e o t i d a s e s are u s u a l l y s p e c i f i c f o r mononucleotides and have a pH optimum of about 7.0 (McComb et a l . , 1979; Drummond and Yamamoto, 1971). There has been c o n s i d e r a b l e c o n t r o v e r s y over the q u e s t i o n of whether the D. discoideum a l k a l i n e phosphatase and 5 ' - n u c l e o t i d a s e a c t i v i t i e s are due to one enzyme or t o d i f f e r e n t enzymes. G e z e l i u s and Wright (1965) concluded that h y d r o l y s i s of pNPP, 5'-AMP, 5'dAMP and Ij -glycerophoshate was accomplished by one enzyme with a pH optimum of 9.0. They s t a t e d that 5'AMP and 51-dAMP were the o n l y probable in v i v o s u b s t r a t e s f o r the APase s i n c e a c t i v i t y with *Vj -glycerophosphate was low. They a l s o p o i n t e d out the d i s t i n c t i o n between the l i m i t e d s p e c i f i c i t y of the D. discoideum enzyme and the u n s p e c i f i c a l k a l i n e phosphatase from E s c h e r i c h i a c o l i which c a t a l y z e s the h y d r o l y s i s of a v a r i e t y of mono-, d i - and t r i -n u c l e o t i d e s , pyrophosphate and sugar-phosphates. A seven-f o l d i n c r e a s e i n enzyme a c t i v i t y was found d u r i n g development 10 r e g a r d l e s s of whether AMP, dAMP or pNPP were used as sub-s t r a t e s . However, there have been s e v e r a l r e p o r t s suggesting that pNPP and AMP h y d r o l y z i n g a c t i v i t i e s are due to d i s t i n c t enzymes, c o n t r a d i c t i n g the c o n c l u s i o n t h a t one p r o t e i n i s r e s p o n s i b l e f o r both a c t i v i t i e s . The work of Green and Newell (1974) suggested that while pNPPase was predominantly plasma membrane bound, 70% of the AMPase was s o l u b l e . Furthermore, Rossomando and Maldonado (1976) found that 90% of the AMPase was l o s t from the membrane f r a c t i o n as c e l l s e n tered s t a t i o n a r y phase while pNPPase was f i r m l y membrane bound. A s o l u b l e c y t o p l a s m i c f a c t o r was i n d i c a t e d i n the r e l e a s e of AMPase from membranes. Lee et. aJL. (1976) found pNPPase and AMPase to have s i m i l a r developmental p r o f i l e s . However, they r e p o r t e d s l i g h t l y d i f f e r e n t i n h i b i t o r s e n s i t i v i t i e s f o r the two a c t i v i t i e s as w e l l as d i f f e r e n t pH optima. Optimum AMPase a c t i v i t y was found at pH 7.6 w hile the pH optimum f o r pNPPase was 8.7. In a d d i t i o n , Rossomando and C u t l e r (1975) found h i g h e r AMP h y d r o l y z i n g a c t i v i t y at pH 7.5 than at pH 9.2 and suggested t h a t the a c t i v i t y was not an a l k a l i n e phosphatase. F i n a l l y , both Lee et a l . (1975) and Rossomando and C u t l e r (1975) r e p o r t e d the s e l e c t i v e r e l e a s e of AMPase from 11 membranes by c o i n c u b a t i n g membranes with phospholipase C. More r e c e n t l y , Mohan Das and Weeks (1984) found that although AMPase was masked i n v e g e t a t i v e c e l l membranes, as was the pNPPase (1980), i t was l e s s e a s i l y a c t i v a t e d than the pNPPase, f u r t h e r s u g g e s t i n g t h a t there were two d i s t i n c t phosphatases. The s e v e r a l r e p o r t s of p h y s i c a l s e p a r a t i o n of pNPPase and AMPase a c t i v i t i e s p r o v i d e s t r o n g evidence f o r two d i s t i n c t enzymes r e s p o n s i b l e f o r the h y d r o l y t i c a c t i v i t i e s . Compelling evidence i n favour of a s i n g l e p r o t e i n that h y d r o l y z e s both pNPP and AMP has been obtained r e c e n t l y . Armant and R u t h e r f o r d (1981) used a v a r i e t y of chromato-g r a p h i c and a f f i n i t y b i n d i n g techniques to p u r i f y a g l y c o -p r o t e i n t o apparent homogeneity. They found that pNPPase and AMPase a c t i v i t i e s c o p u r i f i e d and were r e s i d e n t on a M r 120,000 p r o t e i n p u r i f i e d from c u l m i n a t i o n s t r u c t u r e s . The enzyme was a c t i v e with pNPP and AMP as w e l l as 5'-dAMP but not with any of over a dozen other phosphate e s t e r s t e s t e d . The enzyme was determined to have a pH optimum of 9.5 and had a requirement f o r Zn"2 (Armant and Rutherford, 1983). The f u n c t i o n of the D. discoideum phosphatase i s as yet unknown, and the reasons f o r the i n c r e a s e i n a c t i v i t y and s p e c i f i c s p a t i a l arrangement i s u n c l e a r . Indeed, although e u c a r y o t i c a l k a l i n e phosphatases, p a r t i c u l a r l y the enzymes 12 from mammalian c e l l s , have been e x t e n s i v e l y s t u d i e d , no proven f u n c t i o n has yet been assig n e d to them. However, g i v e n t h e i r s u r f a c e l o c a l i z a t i o n (plasma membrane, m i c r o - v i l l i ) and t h e i r r e l a t i v e n o n - s p e c i f i c i t y , a popular h y p o t h e s i s i s t h a t they serve to dephosphorylate compounds p r i o r t o the uptake of the h y d r o l y t i c products (McComb et a l . , 1979). In summary, i t i s c l e a r that there i s c o n f l i c t i n g evidence i n the l i t e r a t u r e r e g a r d i n g the q u e s t i o n of the number of enzymes r e s p o n s i b l e f o r the h y d r o l y s i s of pNPP and AMP. Strong evidence has been presented i n favour of two d i f f e r e n t v e g e t a t i v e c e l l enzymes while, i n c o n t r a s t , e q u a l l y c o m p e l l i n g evidence i n d i c a t e s t h a t there i s o n l y a s i n g l e p r o t e i n r e s p o n s i b l e f o r the two a c t i v i t i e s i n c u l m i n a t i o n phase c e l l s . One of the aims of the present study was to r e s o l v e the problem of the number of enzymes r e s p o n s i b l e f o r the h y d r o l y s i s of pNPP and AMP. A number of approaches were f o l l o w e d to demonstrate t h a t there i s a s i n g l e v e g e t a t i v e c e l l enzyme r e s p o n s i b l e f o r the two a c t i v i t i e s . The nature of the developmental i n c r e a s e i n APase a c t i v i t y d u r i n g c u l m i n a t i o n i s a l s o c o n t r o v e r s i a l . In one study the i n c r e a s e was found to r e q u i r e p r o t e i n and RNA s y n t h e s i s (Loomis, 1969), while i n another, an i n c r e a s e i n 13 a c t i v i t y was ob t a i n e d without the requirement f o r p r o t e i n s y n t h e s i s (McMahon et a_l. , 1975). T h i s r e s u l t p r o v i d e d prima  f a c i e support f o r the hy p o t h e s i s of Mohan Das and Weeks that the developmental i n c r e a s e c o u l d be accounted f o r by unmasking of p r e - e x i s t i n g , i . e . v e g e t a t i v e c e l l , APase (Mohan Das and Weeks, 1980; 1981; 1984). In an attempt to r e c o n c i l e these d i f f e r e n c e s , two d i f f e r e n t methods were used i n the pr e s e n t study t o q u a n t i t a t e the amount of APase p r o t e i n at d i f f e r e n t stages of development. In a d d i t i o n , s t u d i e s were conducted on the mechanism of a c t i o n of enzyme, and i t s i n t e r a c t i o n with i n o r g a n i c phosphate. F i n a l l y , a d d i t i o n a l , r e l a t i v e l y m i l d methods were d i s c o v e r e d f o r the a c t i v a t i o n of APase from v e g e t a t i v e c e l l s . 14 M a t e r i a l s and Methods I. a) Organisms D. discoideum s t r a i n s Ax-2 and Ax-3 were obtained o r i g i n a l l y from Dr. J.A. Ashworth and Dr. W.F. Loomis, r e s p e c t i v e l y . S t r a i n HL101 was a g i f t of Dr. W.F. Lommis. S t r a i n Ax-2 was grown a x e n i c a l l y on HL-5 media (Weeks and Weeks, 1975) and s t r a i n s Ax-3 and HL101 were grown on n u t r i e n t agar i n a s s o c i a t i o n with E n t e r o b a c t e r aerogenes (Sussman, 1966). b) C u l t u r e C o n d i t i o n s The axenic medium HL-5 comprised the f o l l o w i n g : glucose (25%), 40 ml; y e a s t e x t r a c t , 10 g; b a c t e r i o l o g i c a l peptone, 10 g; KH 2P0 4(0. 5M) , 5.6 ml; Na 2HP0 4 (0. 5M) , 4.3 ml; d e - i o n i z e d water, 650ml. L o g a r i t h m i c phase c e l l s were i n o c u l a t e d i n 700 ml of HL-5 medium and incubated at 22 C on a g y r a t o r y shaker at 200 rpm. C e l l s were h a r v e s t e d at an approximate c e l l d e n s i t y of 5 X 10 6 c e l l s / m l by c e n t r i f u g a t i o n at 700 X g f o r 10 min. I f v e g e t a t i v e c e l l s were r e q u i r e d , c e l l s were washed twice with d e - i o n i z e d water. For d i f f e r e n t i a t i o n , the amoebe grown on HL-5 medium 15 were washed twice with Bonner's s a l t s s o l u t i o n (KCL, 0.02M; NaCl, 0.02M; CaCl, 0.002M) and p l a t e d on 2% non - n u t r i e n t agar c o n t a i n i n g Bonner's s a l t s . (Bonner, 1947) The c e l l s were suspended at a c e l l d e n s i t y of 2 X 10^ c e l l s / m l and p l a t e d on Bonner's s a l t s agar i n l a r g e t r a y s (23 X 57.5 cm) at a d e n s i t y of 1 X 10^ c e l l s / c m 2 . Aggregates formed w i t h i n 10-12 h, pseudoplasmodia at 20-25 h, and c u l m i n a t i o n s t r u c t u r e s at about 30 h. C e l l s at the d e s i r e d stages were h a r v e s t e d and washed t h r i c e with d e - i o n i z e d water. II M a t e r i a l s Adenosine-5'-monophosphorothioate, AMP, a s c o r b i c a c i d , 4 - c h l o r o naphthol, $\ -methyl-D-mannoside, MES, p - n i t r o p h e n y l phosphate, ty\ - n a p t h y l a c i d phosphate, n i t r o b l u e t e t r a z o l i u m , phenyl methyl s u l f o n y l f l u o r i d e , phospholipase C (type 1), T r i s , variamine b l u e and z i n c s u l f a t e were obtained from Sigma Chemical Co. Ammonium s u l f a t e and sucrose were from Schwarz-Mann; sodium dodecyl s u l f a t e was from BDH Chemicals; T r i t o n X-100 was from Amersham C o r p o r a t i o n ; l e a d n i t r a t e was from F i s h e r Chemical Co. L i q u i f l u o r , (U) "^C-AMP and 3 2 P i were from New England Nuclear. 5'-Deoxy-5'-thioaden-o s i n e - 5 ' -monophosphate was from Calbiochem. Acrylamide, b i s - a c r y l a m i d e , ammonium p e r s u l f a t e , g l y c i n e and TEMED were 16 o b t a i n e d from Bio-Rad L a b o r a t o r i e s . Sepharose-4B, Concana-v a l i n A-Sepharose, phenyl Sepharose, DEAE-Sephadex and Se p h a c r y l S-300 were obtained from Pharmacia Fine Chemicals. Mixed g l y c o s i d a s e p r e p a r a t i o n , Concanavalin A and monosac-c h a r i d e s were obtained from M i l e s L a b o r a t o r i e s . Ampholytes were obtained from LKB Company. Swine a n t i - r a b b i t a n t i b o d i e s were from Dako Immunoglobulins. N i t r o c e l l u l o s e paper was from S c h l e i c h e r and S c h u e l l Company. I l l Methods a) Membrane P r e p a r a t i o n The procedure of G i l k e s and Weeks (1977) was foll o w e d . Harvested c e l l s were washed twice with d e - i o n i z e d water and f i n a l l y with 5 mM T r i s - C l pH 7.4 c o n t a i n i n g 8.6% sucrose ( T r i s - s u c r o s e ) . The c e l l s were resuspended at a d e n s i t y of 10° c e l l s / m l i n T r i s - s u c r o s e c o n t a i n i n g f r e s h l y d i s s o l v e d PMSF. 3.3 g of g l a s s beads (0.45-0.50 mm diameter) were added per 2 X 10^ c e l l s and mechanical g r i n d i n g was accomplished by means of a magnetic s t i r r i n g bar f o r 10-15 min. Almost 100% c e l l breakage was ob t a i n e d as assessed by microscopy. The unbroken c e l l s and g l a s s beads were removed by c e n t r i -f u g a t i o n at 700 X g f o r 5 min. The supernatant was c e n t r i -fuged at 105,000 X g to o b t a i n p e l l e t which was resuspended wit h T r i s - s u c r o s e and r e c e n t r i f u g e d as b e f o r e . The f i n a l p e l l e t was resuspended i n 5 mM T r i s - C l pH 7.4 to g i v e a 17 p r o t e i n c o n c e n t r a t i o n of 25 mg/ml and was designated the crude membrane f r a c t i o n . b) Membrane P r o t e i n S o l u b i l i z a t i o n T r i t o n X-100 was added to a f i n a l c o n c e n t r a t i o n of 1% (v/v) t o the crude membrane suspension and incubated, with i n t e r m i t t e n t a g i t a t i o n , a t 4 C f o r 2 h. The supernatant f o l l o w i n g c e n t r i f u g a t i o n at 105,000 X g f o r 75 min. gave the s o l u b i l i z e d p r o t e i n f r a c t i o n . Butanol e x t r a c t i o n was a c c o r d i n g to the procedure of Ghosh and Fishman (1968). The crude membrane f r a c t i o n was t r e a t e d with 0.4 volume of c o l d b u t a n o l , mixed, and incubated a t 4 C f o r 2 h. with i n t e r m i t t e n t s t i r r i n g . C e n t r i f u g a t i o n at 15,000 X g f o r 3 0 min. gave two phases. The lower aqueous phase was a s p i r a t e d and f i l t e r e d through a smal l amount of g l a s s wool. The f i l t e r a t e was then c e n t r i f u g e d at 35,000 X g f o r 30 min. The supernatant was then d i a l y z e d overnight a g a i n s t 5 mM T r i s - C l pH 7.5. c) P r o t e i n E s t i m a t i o n The F o l i n procedure (Lowry et a l . , 1951) was followed except when T r i t o n X-100 was present i n the samples, i n which case the procedure of Sandermann and Strominger (1972) was used. 18 d) Enzyme Assays pNPPase was g e n e r a l l y assayed by the method of Lee et a l . (1975). Incubation mixtures contained 1 mM pNPP, 20 mM M gCl 2, 20 mM T r i s - C l pH 8.5, 30 mM NaF and p r o t e i n e x t r a c t (0.020 - 0.100 mg) i n a f i n a l volume of 1.0 ml. Incubations, u s u a l l y f o r 10 min at 30 C, were terminated by a d d i t i o n of 1.0 ml 1 M Na 2C03. Any r e s u l t i n g p r e c i p a t e was removed by c e n t r i f u g a t i o n at 2,500 rpm f o r 10 min. ( i n a S o r v a l G1C-2 t a b l e top c e n t r i f u g e ) . Absorbance at 410 nm p r o v i d e d a measure of amount of enzyme a c t i v i t y p r e s e n t . An e x t i n c t i o n c o e f f i c i e n t E ^ M = 1.62 X 1 0 4 was used to c a l c u l a t e the q u a n t i t y of p - n i t r o p h e n o l formed. AMPase a c t i v i t y was g e n e r a l l y assayed a c c o r d i n g to the p r o t o c o l r e p o r t e d i n Lee et al_. (1975), except that a 1 0 - f o l d h i g h e r c o n c e n t r a t i o n of AMP was used. Incubations contained 0.2 mM AMP (with 0.05 mCi (U) 1 4C-AMP) , 20 mM MgCl 2, 50 mM T r i s - C l pH 7.5, and p r o t e i n e x t r a c t (0.020 - 0.100 mg) i n a f i n a l volume of 1.0 ml. A f t e r 20-30 min. i n c u b a t i o n at 30 C, the r e a c t i o n was terminated by the p r e c i p i t a t i o n of AMP w i t h 0.2 ml of 0.25M ZnS0 4 and 0.2 ml of f r e s h l y prepared s a t u r a t e d s o l u t i o n of Ba(0H) 2. A f t e r c e n t r i f u g a t i o n at 2500 rpm f o r 10 min, r a d i o a c t i v i t y i n 0.6 ml of the super-natant was determined i n T o l u e n e - T r i t o n X-100 s c i n t i l l a t i o n c o c k t a i l c o n t a i n i n g L i q u i f l u o r (Lee et aJL. , 1975). T h i s gave 19 a measure of amount of enzyme a c t i v i t y p r e s e n t . These assay procedures were modified f o r some e x p e r i -ments; d e t a i l s are g i v e n i n the f i g u r e legends. In other experiments, the amount of i n o r g a n i c phosphate, measured by the procedure of Ames (1966), was compared d i r e c t l y t o the amount of dephosphorylated s u b s t r a t e . Butanol e x t r a c t e d enzyme was used i n these experiments as T r i t o n X-100 p r e c i p i -t a t e d the chromagen. D e t a i l s of the i n c u b a t i o n c o n d i t i o n s are g i v e n i n the r e l e v a n t f i g u r e legends. 0.3 ml P o r t i o n s were taken f o r the d e t e r m i n a t i o n of i n o r g a n i c phosphate. p - N i t r o p h e n o l was assayed s p e c t r o p h o t e m e t r i c a l l y a f t e r d i l u t i n g 0.15 ml of r e a c t i o n mixture to 1.5 ml with 1 M Na2C03« Adenosine was determined by the r a d i o i s o t o p e procedure d e s c r i b e d above. For the s t u d i e s on the h y d r o l y s i s of t h i o n u c l e o s i d e s , the amount of n u c l e o s i d e or t h i o n u c l e o s i d e formed was q u a n t i t a t e d f o l l o w i n g r e s o l u t i o n of 0.010 ml a l i q u o t s of the r e a c t i o n mixture on a Waters CIQ Bondapak HPLC column as d e s c r i b e d p r e v i o u s l y (Rossomando et a_l., 1983). D e t a i l s of the i n c u b a t i o n c o n d i t i o n s are given i n the r e l e v a n t t a b l e legend. The e l u t e d peaks were monitored at 254 nm u s i n g a Waters Model 441 d e t e c t o r and q u a n t i t a t e d by i n t e g r a t i n g the peak area. Amounts of e n z y m a t i c a l l y generated adenosine were 20 determined r e l a t i v e t o the d e t e c t o r response by known a l i q u o t s of a standard adenosine s o l u t i o n . A thioadenosine s t a n d a r d was generated from dASMP with E. c o l i a l k a l i n e phosphatase (Rossomando et a l . , 1983). e) P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s The Laemmli (1969) procedure and a m o d i f i c a t i o n of t h i s procedure were used. In the Laemmli procedure, 7% acrylamide g e l s were c a s t i n 0.15 M T r i s - C l pH 8.8 and 0.1% SDS. The s t a c k i n g g e l was 1% acrylamide i n 0.083 M T r i s - C l pH 6.8 and 0.1% SDS. The running b u f f e r contained 0.1% SDS i n 0.025 M T r i s - g l y c i n e . The sample b u f f e r contained 2% SDS and 0.083 M T r i s - C l pH 6.8, g l y c e r o l and d e - i o n i z e d water (1:1:6 v/ v / v ) . Bromophenol blue was used as the t r a c k i n g dye. In the g e l s used to demonstrate enzyme a c t i v i t y , t e n -f o l d lower c o n c e n t r a t i o n s of T r i s - C l b u f f e r were used i n the running and s t a c k i n g g e l s s i n c e T r i s - C l was found to i n a c t i v a t e the enzyme (See R e s u l t s ) . The lowered b u f f e r c o n c e n t r a t i o n d r a m a t i c a l l y i n c r e a s e d the s e n s i t i v i t y of the enzyme assays. Gels were s u b j e c t e d to 5mA f o r 1 h then 20 mA f o r 50-60 min. ( c f up to 4 h f o r Laemmli g e l s ) and the s h o r t e r running time f o r t h i s system was an a d d i t i o n a l advantage i n p r e s e r v i n g enzyme a c t i v i t y . The sample b u f f e r c o n t a i n e d 5 mM T r i s - C l pH 6.8, g l y c e r o l and d e - i o n i z e d water 21 (1:1:6 v / v / v ) . T r i t o n X-100 g e l s were prepared i d e n t i c a l l y but with 1% T r i t o n X-100 s u b s t i t u t e d f o r 0.1% i n the g e l . Gels were r o u t i n e l y s t a i n e d f o r 0^  -naphthyl a c i d phosphate h y d r o l y z i n g a c t i v i t y as d e s c r i b e d by Ghosh and Fishman (1968), except t h a t 0.1 M T r i s - C l pH 8.5 was used i n p l a c e of IM p r o p a n e d i o l b u f f e r . pNPP and AMP h y d r o l y z i n g a c t i v i t i e s were det e c t e d by i n c u b a t i n g the g e l s with 0.1% s u b s t r a t e and 0.1% l e a d n i t r a t e i n 0.1 M T r i s - C l pH 8.5 f o r 15 h at 30 C. Colour development of bands was then accomplished by the a d d i t i o n of 1% aqueous ammonium s u l f i d e . In the case of pNPP h y d r o l y s i s , the dark y e l l o w background was removed p r i o r to c o l o u r development by washing i n s e v e r a l changes of 50 mM T r i s - C l pH 7.5 b u f f e r over 2 h. The s t a i n e d g e l s sometimes r e t a i n e d a dark back-ground which was removed by immersing the g e l s i n tap water and exposing to s u n l i g h t f o r 2-3 days. Gels were s t a i n e d f o r p r o t e i n u s i n g the d i t h i o t h r e i t o l s i l v e r s t a i n i n g method of M o r r i s e y (1981). f) P u r i f i c a t i o n of pNPPase pNPPase was p u r i f i e d by m o d i f i c a t i o n of the method of Armant and Rut h e r f o r d (1981) t h a t had been used f o r the p u r i f i c a t i o n of a g l y c o p r o t e i n with pNPPase and AMPase 22 a c t i v i t i e s from D. discoideum c u l m i n a t i o n s t r u c t u r e s . The crude membrane f r a c t i o n was obtained from v e g e t a t i v e c e l l s and p r o t e i n s were s o l u b i l i z e d with 1% T r i t o n X-100 as d e s c r i b e d above. The ammonium s u l f a t e p r e c i p i t a t i o n and Sepharose 4-B g e l f i l t e r a t i o n steps d e s c r i b e d by Armant and Ru t h e r f o r d (1980) d i d not i n c r e a s e the s p e c i f i c a c t i v i t y of pNPPase and were thus omitted. I n c r e a s i n g the T r i t o n X-100 c o n c e n t r a t i o n t o 1% or s u b s t i t u t i n g 0.1% deoxycholate d i d not improve the r e s o l u t i o n of the g e l f i l t e r a t i o n . i ) DEAE-Sephadex column chromatography DEAE-Sephadex was t r e a t e d a c c o r d i n g t o the procedure of Himmelhock (1972). A f t e r washing the g e l s u c c e s s i v e l y with 0.1 M NaOH, 0.2 M HCL, water, 0.1 M NaOH, water and 25 mM T r i s - C l pH 8.5, the g e l was s t o r e d i n the c o l d f o r at l e a s t 2 weeks b e f o r e use. T h i s was done to minimize the amount of enzyme that bound i r r e v e r s i b l y t o the g e l . The g e l was poured i n t o a column (1.4 X 8.0 cm) and washed with 25 mM T r i s - C l pH 8.5 (about 200 mis ) . T r i t o n X-100 e x t r a c t s (3.0 -5.0 ml) were loaded onto the column and the column was washed wit h 25 mM T r i s - C l pH 8.5 b u f f e r (80 ml). The column was then washed s u c c e s s i v e l y with the same b u f f e r and 0.125 M NaCl (80 ml), b u f f e r and 0.25 M NaCl (80 ml), b u f f e r and 0.50 M NaCl (80 ml) and b u f f e r and 1.0 M NaCl (80 ml). F i n a l l y the pNPPase a c t i v i t y was e l u t e d with b u f f e r and 1.0 M NaCl 23 and 0.1% T r i t o n X-100. F r a c t i o n s (2 ml) were c o l l e c t e d and assayed f o r enzyme a c t i v i t y . The f r a c t i o n s were found to be too d i l u t e f o r p r o t e i n e s t i m a t i o n , thus p r o t e i n assays were not performed. i i ) Phenyl-Sepharose column chromatography Phenyl-Sepharose CL-4B was poured i n t o a column (1.4 X 8 cm) and washed s u c c e s s i v e l y with 200 mis each of water, e t h a n o l , b u t a n o l , e t h a n o l , water and 25 mM T r i s - C l pH 8.5. The a c t i v e f r a c t i o n s from the ion-exchange column were pooled and loaded onto the phenyl-Sepharose column. The column was then washed with 50 ml each of 25 mM T r i s - C l pH 8.5, b u f f e r c o n t a i n i n g 20% (v/v) dimethylformamide and b u f f e r and 0.1% T r i t o n X-100. The pNPPase a c t i v i t y was e l u t e d u s i n g b u f f e r and 0.3% T r i t o n X-100. F r a c t i o n s (2 ml) were c o l l e c t e d and assayed f o r a c t i v i t y . i i i ) C o ncanavalin A-Sepharose CL-4B column chromatography Concanavalin A-Sepharose CL-4B was packed i n a column (1.5 X 4 cm) and washed with 300 ml of 25 mM T r i s - C l pH 8.5 and 1.0% T r i t o n X-100 a t 20 ml/h. The pooled f r a c t i o n s from the hydrophobic column were loaded and the column was then washed with 50 ml each of 25 mM T r i s - C l i n 1.0% T r i t o n X-100 and then 0.3 M g a l a c t o s e s o l u t i o n . Enzyme was then e l u t e d by wit h 25 mM T r i s - C l pH 8.5 and 50 mM ^-methyl-D-mannoside. 24 v F r a c t i o n s (2 ml) were c o l l e c t e d and assayed f o r enzyme a c t i v i t y . The a c t i v e f r a c t i o n s were pooled and t r e a t e d with Bio-Rad SM-2 beads to remove the detergent (Holloway, 1973). The d e t e r g e n t - f r e e p r o t e i n s o l u t i o n was concentrated to a s m a l l volume u s i n g an Amicon u l t r a f i l t e r a t i o n u n i t . The f i n a l p u r i f i c a t i o n step used by Armant and R u t h e r f o r d (1981), S e p h a c r y l S-300 column chromatography, was omitted as t h i s s t e p d i d not enhance pNPPase p u r i t y . F i g u r e 2 shows the f i n a l product of the p u r i f i c a t i o n procedure s u b j e c t e d to SDS g e l e l e c t r o p h o r e s i s . A major band (arrow) corresponds to pNPPase a c t i v i t y , but there i s one other major band and a number of other minor contaminating components. The major band i n n a t i v e g e l s i . e . g e l s used to d e t e c t enzyme a c t i v i t y , had a molecular weight of 150,000. M a t e r i a l e l u t e d from t h i s r e g i o n of the n a t i v e g e l and s u b j e c t e d to SDS g e l e l e c t r o -p h o r e s i s gave one band at M r = 120,000 (data not shown). g) I d e n t i f i c a t i o n of 0-phosphorylethanolamine A m o d i f i c a t i o n of the procedure of Debruyne (1982) was used t o i s o l a t e and i d e n t i f y O-phosphorylethanolamine pro-duced as a t r a n s p h o s p h o r y l a t i o n product d u r i n g the course of the pNPPase a c t i o n i n ethanolamine b u f f e r . The r e a c t i o n was c a r r i e d out i n 10 mis of 200 mM ethanolamine-HCL pH 8.5 25 F i g u r e 2: SDS-polyacrylamide g e l e l e c t r o p h o r e s i s of p a r t i a l l y p u r i f i e d enzyme. P a r t i a l l y p u r i f i e d enzyme was o b t a i n e d by the use of i o n exchange column chromatography, hydrophobic column chromatography and l e c t i n column chromato-graphy as d e s c r i b e d i n the Methods. 0.010 mg of the p a r t i a l l y p u r i f i e d v e g e t a t i v e c e l l enzyme was e l e c t r o p h o r e s e d by the Laemmli (1970) procedure as d e s c r i b e d i n the Methods. The p a r t i a l l y p u r i f i e d enzyme as w e l l as molecular weight standards were b o i l e d f o r 3 minutes i n sample b u f f e r c o n t a i n i n g 0.083 M T r i s - C l pH 6.8, g l y c e r o l , d e - i o n i z e d water (1:1:6, v/v/v) and 2% SDS b e f o r e e l e c t r o p h o r e s i s . The g e l was s i l v e r s t a i n e d a c c o r d i n g t o the procedure of Morrisey (1981). The p o s i t i o n of the enzyme i s marked by the arrow. 500K 180K 158K 45K 17-8K 27 b u f f e r , 20 mM pNPP, 20 mM MgCl 2# and 30 mM NaF.1.0 ml of b u t a n o l e x t r a c t e d pNPPase (3.0 mg ml--'-) was added every 8-12 h over 2 days, at 30 C. At the end of i n c u b a t i o n , the pH was a d j u s t e d to 10.0 and the sample d i l u t e d 1 0 - f o l d and loaded on to a Dowex 1 x 4 column (1.5 cm x 35 cm). The p h o s p h o r y l a t e d a l c o h o l was recovered from the column and i d e n t i f i e d by paper e l e c t r o p h o r e s i s as d e s c r i b e d by Debruyne (1980). A u t h e n t i c O-phosphorylethanolamine was used as the standard. h) P r e p a r a t i o n of antiserum P u r i f i e d APase was obtained by u s i n g the column chrom-a t o g r a p h i c p u r i f i c a t i o n p r o t o c o l given i n f) and then s u b j e c t i n g the p a r t i a l l y p u r i f i e d APase to SDS p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s as d e s c r i b e d i n e) , f o r Laemmli (1970) procedure. The APase p r o t e i n band was e x c i s e d and mascerated by passage through a s y r i n g e . The mascerated g e l was mixed tho r o u g h l y with Freund's incomplete adjuvant at 4 C. A r a b b i t was immunized with the mixture by i n i t i a l i n t r a -muscular and subsequent weekly subcutaneous i n j e c t i o n s . Blood was c o l l e c t e d d u r i n g the f i f t h week by c a r d i a c puncture and the serum f r a c t i o n was obtained by c e n t r i f u g a t i o n at 700 x g f o r 20 min a f t e r c l o t t i n g was complete. i ) Immunodetection of APase on n i t r o c e l l u l o s e b l o t s P r o t e i n (Western) b l o t t i n g of T r i t o n X-100 e x t r a c t e d p r o t e i n s was done by a m o d i f i c a t i o n of the method of Towbin 28 et a l . (1979). T r a n s b l o t t i n g was done o v e r n i g h t at a constant v o l t a g e of 30 V. The excess b i n d i n g s i t e s on the n i t r o c e l l u l o s e paper were blocked with 2% BSA i n 25 mM T r i s -C l pH 7.5. The paper was then exposed t o 1:100 d i l u t i o n i n 25 mM T r i s - C l pH 7.5, 0.9% NaCl of the antiserum r a i s e d a g a i n s t p u r i f i e d APase f o r 2 h at room temperature. The excess antiserum was washed out i n s e v e r a l changes of 25 mM T r i s - C l pH 7.5 b u f f e r . Immunochemical d e t e c t i o n was done with swine a n t i r a b b i t antibody conjugated t o horse r a d i s h p e r o x i d a s e (1:200 d i l u t i o n i n 25 mM T r i s - C l pH 7.5, 0.9% NaCl) f o r 2 h f o l l o w e d a f t e r washing by 4 - c h l o r o - l - n a p h t h o l and hydrogen pe r o x i d e . j ) 3 2 P i l a b e l l i n g of membrane p r o t e i n s Crude membrane f r a c t i o n s prepared from v e g e t a t i v e c e l l s and c u l m i n a t i o n phase c e l l s were t r e a t e d with butanol t o e x t r a c t p r o t e i n s , as d e s c r i b e d above. Assays contained 0-0.080 mg of p r o t e i n , 200 mM T r i s - C l pH 7.5, 0.010 mCi 3 2 P i and 1 mM K 2 H P O 4 . The samples were incubated at room temperature f o r 10 min. 0.60 ml A l i q u o t s were taken and counted f o r t r i c h l o r o a c e t i c a c i d i n s o l u b l e r a d i o a c t i v e l y l a b e l l e d m a t e r i a l . The a l i q u o t s were absorbed onto Whatman No. 3 f i l t e r paper d i s k s (2.54 cm diameter) and washed f o r 1 h i n 10% t r i c h l o r o a c e t i c a c i d (twice) and 5% t r i c h l o r o a c e t i c 29 a c i d and then f o r 30 min. i n 100% e t h a n o l and f i n a l l y f o r 15 min. i n d i e t h y l ether. The f i l t e r d i s k s were d r i e d o vernight and then counted f o r r a d i o a c t i v i t y i n a l i q u i d s c i n t i l l a t i o n c o unter. 30 RESULTS SECTION 1 The r e l a t i o n s h i p between a l k a l i n e  phosphatase and 5 ' - n u c l e o t i d a s e . A number of approaches were taken to e s t a b l i s h whether pNPP and AMP were h y d r o l y z e d by a s i n g l e enzyme or by d i f f e r e n t enzymes. a) Comparison of pNPP- and AMP- h y d r o l y z i n g a c t i v i t i e s f o l l o w i n g SDS-PAGE under non-denaturing c o n d i t i o n s . The AMPase and pNPPase a c t i v i t i e s of c u l m i n a t i o n phase c e l l s were not d e t e c t a b l e f o l l o w i n g s e p a r a t i o n by the s t a n d a r d Laemmli SDS-PAGE procedure. The Laemmli procedure was t h e r e f o r e m o d i f i e d by reducing the b u f f e r c o n c e n t r a t i o n 1 0 - f o l d i n both the s t a c k i n g and running g e l s , s i n c e h i g h T r i s - C l c o n c e n t r a t i o n was found to be i n h i b i t o r y t o the enzyme a c t i v i t i e s (see S e c t i o n 2). E l e c t r o p h o r e s i s under these c o n d i t i o n s r e v e a l e d that ty^ -naphthyl a c i d phosphate as w e l l as pNPP and AMP were h y d r o l y z e d by the s i n g l e p r o t e i n component with a m o b i l i t y corresponding to an apparent molecular weight of 150K ( F i g u r e 3 ) . There was a l s o a s m a l l amount of a c t i v i t y a s s o c i a t e d with a low m o b i l i t y component (M r=350K) which perhaps i s due to an aggregation complex of the 150K p r o t e i n . In some samples that had been s t o r e d at 31 F i g u r e 3: Na t i v e p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s of T r i t o n X-100 e x t r a c t s of v e g e t a t i v e and c u l m i n a t i n g c e l l membrane p r e p a r a t i o n s . 0.070 mg of p r o t e i n was loaded i n each w e l l . The g e l system used was the modified Laemmli (1970) system c o n t a i n i n g t e n - f o l d lower c o n c e n t r a t i o n s of T r i s - C l b u f f e r as d e s c r i b e d i n the Methods. The sample b u f f e r c o n t a i n e d 5 mM T r i s - C l pH 6.8, g l y c e r o l and d e - i o n i z e d water (1:1:6,v/v/v). F o l l o w i n g e l e c t r o p h o r e s i s , a c t i v i t y was d e t e c t e d by vx -naphthyl a c i d phosphate h y d r o l y s i s as d e s c r i b e d i n the Methods. S t a i n i n g with e i t h e r pNPP or AMP showed the same bands as with b^-naphthyl a c i d phosphate. The p o s i t i o n s of s e v e r a l molecular weight standards are shown. The standard p r o t e i n s were d i s s o l v e d i n the sample b u f f e r g i v e n above but with i n c l u s i o n of 2% SDS. F u r t h e r -more the standard p r o t e i n s were b o i l e d f o r 3 minutes. (a) V e g e t a t i v e c e l l membrane (b) V e g e t a t i v e c e l l membrane that had been s t o r e d at -20 C f o r 2 weeks (c) C u l m i n a t i o n phase c e l l membrane 32 500K-1 180K-158K-45K 17-8K-a b c 33 -20 C f o r 2 weeks or more, two or three components of s i m i l a r m o b i l i t y were observed with the component of g r e a t e s t e l e c t r o p h o r e t i c m o b i l i t y b e i n g c o i n c i d e n t with the 150K p r o t e i n ( F i g u r e 3). It i s l i k e l y that these h i g h e r molecular weight a c t i v i t i e s a l s o r e s u l t from the aggregation of the 150K p r o t e i n . In some s t o r e d samples, low molecular weight s p e c i e s were observed ( F i g u r e 4) that were a c t i v e at both a l k a l i n e pH and a c i d pH whereas the 150K p r o t e i n was a c t i v e o n l y at a l k a l i n e pH. These low molecular weight s p e c i e s presumably r e s u l t from p r o t e o l y t i c h y d r o l y s i s d u r i n g storage. In a l l cases, the p r o t e i n s that were a c t i v e with pNPP as s u b s t r a t e were a l s o a c t i v e with AMP. These f i n d i n g s support the concept t h a t there i s o n l y one p r o t e i n r e s p o n s i b l e f o r the h y d r o l y s i s of both pNPP and AMP. S i m i l a r r e s u l t s were obtained with the a c t i v i t i e s from c u l m i n a t i o n s t r u c t u r e s . However, the major a c t i v i t y component had s l i g h t l y g r e a t e r m o b i l i t y than the 150K p r o t e i n from v e g e t a t i v e c e l l s ( F i g u r e 3). T h i s d i f f e r e n c e i n m o b i l i t y c o u l d perhaps be due to a lower l e v e l of g l y c o s y l a -t i o n i n the c u l m i n a t i o n phase enzyme. The m o b i l i t y of the v e g e t a t i v e c e l l enzyme c o u l d not be i n c r e a s e d , however, by treatment with p e r i o d a t e (50 mg T r i t o n X-100 e x t r a c t p r o t e i n , 16 mM p e r i o d i c a c i d , pH 7.0, 30 C, 30 min) or a mixed g l y c o s i d a s e p r e p a r a t i o n (50 mg T r i t o n X-100 e x t r a c t p r o t e i n , 1 mg mixed g l y c o s i d a s e p r e p a r a t i o n , 30 C, 30 min). 34 F i g u r e 4: Na t i v e p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s of T r i t o n X-100 e x t r a c t of v e g e t a t i v e membrane p r e p a r a t i o n c o n t a i n i n g spontaneously generated lower molecular weight s p e c i e s of pNPPase a c t i v i t y . 0.070 mg of p r o t e i n were loaded onto the g e l . E l e c t r o p h o r e s i s was c a r r i e d out us i n g the m o d i f i c a t i o n of the Laemmli (1970) system with 1 0 - f o l d lower c o n c e n t r a t i o n s of T r i s - C l b u f f e r as d e s c r i b e d i n Methods. The sample b u f f e r c o n t a i n e d 5 mM T r i s - C l pH 6.8, g l y c e r o l and d e - i o n i z e d water (1:1:6,v/v/v). pNPPase a c t i v i t y was de t e c t e d i n 50 mM T r i s - C l a d j u s t e d to pH 4.5. The n a t i v e enzyme i s not de t e c t e d under these c o n d i t i o n s . The standard p r o t e i n s were b o i l e d f o r 3 minutes i n the above sample b u f f e r with the a d d i t i o n of 2% SDS, p r i o r to e l e c t r o p h o r e s i s . 35 500K-180K-158K-45K-17.8K-36 b) pH Optima f o r pNPP and AMP h y d r o l y s i s . I t had been p r e v i o u s l y found that the pH optima f o r pNPPase and AMPase were d i f f e r e n t (Lee et a_l., 1975). However, d u r i n g t h i s study i t was found t h a t the pH optima f o r both pNPP and AMP h y d r o l y z i n g a c t i v i t i e s were h i g h l y dependent on the c o n c e n t r a t i o n of s u b s t r a t e ( F i g u r e 5 ) . In e i t h e r case, pH optima i n c r e a s e d by about 1 pH u n i t when s u b s t r a t e c o n c e n t r a t i o n s were i n c r e a s e d 1 0 - f o l d . When i d e n t i c a l c o n c e n t r a t i o n s of pNPP or AMP were used, the pH optima were s i m i l a r . These r e s u l t s are a l s o c o n s i s t e n t with the argument t h a t one p r o t e i n i s r e s p o n s i b l e f o r the h y d r o l y s i s of both pNPP and AMP. c) Dixon p l o t s of pNPP and AMP h y d r o l y t i c a c t i v i t i e s . The dependence of pH optima on s u b s t r a t e c o n c e n t r a t i o n suggested t h a t s u b s t r a t e b i n d i n g might be dependent on pH. Km and Vmax values were obtained f o r s e v e r a l pH values and the n e g a t i v e values (pKm and pVmax) were p l o t t e d a g a i n s t pH as suggested by Dixon (1965). For both AMP and pNPP, i n f l e c t i o n s were obtained at approximate pH 8.5, i n d i c a t i n g the p a r t i c i p a t i o n of an enzyme f u n c t i o n a l group with a pKa of 8.5 i n v o l v e d i n b i n d i n g of s u b s t r a t e ( F i g u r e s 6, 7). Another noteworthy f e a t u r e of the p l o t s was t h a t f o r the AMP the 37 F i g u r e 5: pH optima of pNPP and TAMP h y d r o l y z i n g a c t i v i t i e s i n T r i t o n X-100 e x t r a c t s of v e g e t a t i v e c e l l membranes. (a) TAMP h y d r o l y z i n g a c t i v i t y . Each assay contained i n 0.200 ml f i n a l volume 50 mM T r i s - C l b u f f e r at the i n d i c a t e d pH, 20 mM MgCl2» 0.100 mg p r o t e i n and the f o l l o w i n g c o n c e n t r a t i o n s of pNPP: ( O ) 0.25 mM, ( V ) ° - 7 5 m M > ( • ) 1-25 mM, ( Z\ ) 2.50 mM. C o n t r o l s c o n t a i n i n g no enzyme were performed at a l l pH values and s u b s t r a t e c o n c e n t r a t i o n s . (b) pNPP h y d r o l y z i n g a c t i v i t y . As f o r (a) except that pNPP was used as a s u b s t r a t e . The data were r e p r o d u c i b l e and are r e p r e s e n t a t i v e of those o b t a i n e d i n two experiments. E N Z Y M E ACTIVITY ( nmoles / min / mg protein) 39 F i g u r e 6: Dixon p l o t s (pKm vs pH) of (a) pNPP and (b) AMP h y d r o l y z i n g a c t i v i t i e s . Assay c o n d i t i o n s were i d e n t i c a l to those d e s c r i b e d i n the legend to F i g u r e 5 except that 10 and 5 c o n c e n t r a t i o n s of pNPP and AMP, r e s p e c t i v e l y were used at each pH value to determine Km va l u e s . The data were r e p r o d u c i b l e and are r e p r e s e n t a t i v e of those obtained i n two experiments. 40 E 41 F i g u r e 7 : Dixon p l o t s (Vmax vs pH) of a) pNPP and b) AMP h y d r o l y z i n g a c t i v i t i e s . Assay c o n d i t i o n s were d e s c r i b e d f o r F i g u r e 6 . 2 . 5 H X i 1 , 5 > Q_ l H 5 42 G —e © - - - " ' • 7. 5 6 B . 5 9 p H 1 1 9 . 5 1 0 43 4 4 magnitude of the slope was 2, whereas f o r pNPP i t was 1. A c c o r d i n g to the Dixon i n t e r p r e t a t i o n of such p l o t s (Dixon, 1965), t h e r e should be two i o n i z a b l e groups of pKa 8.5 i n v o l v e d i n b i n d i n g AMP but onl y one i n b i n d i n g pNPP. Lee et a l . (1975) had shown p r e v i o u s l y that the s u l f h y d r y l complexing agent p-hydroxymercuribenzoate at a c o n c e n t r a t i o n of 1 mM i n h i b i t e d both pNPPase and AMPase suggesting the p o s s i b i l i t y t h a t a s u l f h y d r y l group was r e s p o n s i b l e f o r the i n f l e c t i o n . The data i n Table 1 r e v e a l e d that even lower c o n c e n t r a t i o n s of p-hydroxymercuribenzoate i n a c t i v a t e d pNPPase a c t i v i t y c o n f i r m i n g the p o s s i b l e involvement of a s u l f h y d r y l group. C o - i n c u b a t i o n of AMP with p-hydroxy-mercur ibenzoate d i d not p r o t e c t the enzyme from i n a c t i v a t i o n T h i s r e s u l t may i n d i c a t e t h a t the e s s e n t i a l f u n c t i o n a l group m o d i f i e d by t h i s reagent i s not l o c a t e d i n the a c t i v e s i t e . However, i t i s p o s s i b l e that the f u n c t i o n a l group i s i n the a c t i v e s i t e and that i t s r e a c t i v i t y i s h i g h enough that f r e e enzyme, E, produced d u r i n g the e q u i l i b r i u m c o n d i t i o n of E + S ES, where S i n AMP, i s r e a d i l y i n a c t i v a t e d by p-hydroxymercur ibenzoate. Treatment with the amino group r e a c t i v e agent p i c r y l s u l f o n a t e d i d not i n a c t i v a t e the enzyme (Figure 8), sug-g e s t i n g that primary amine groups were not i n v o l v e d . 45 Table 1 E f f e c t of p-hydroxymercuribenzoate on pNPPase a c t i v i t y Treatment a pNPPase a c t i v i t y (nmoles/min/mg protein) None 107 ± 4.6 0.050 mM pHMB 11 + 5.1 0.050 mM pHMB and 2 mM AMP 13 + 2.5 0.05 0 mM pHMB and 4 mM AMP 8 + 4.6 a Assays contained dialyzed T r i t o n X--100 extracts (2.0 mg/ml, 30 in standard assay mixture with additions as indicated. Incubations were for 10 min at 30 C. The number of determinations was 3.The mean and the Standard Estimated Deviation are given. 46 F i g u r e 8: E f f e c t of p i c r y l s u l f o n a t e on the a c t i v i t y of pNPPase. T r i t o n X-100 of v e g e t a t i v e c e l l membrane pre p a r a -t i o n s were d i a l y z e d a g a i n s t 5 mM HEPES b u f f e r pH 7.5 t o remove p i c r y l s u l f o n a t e - r e a c t i v e T r i s present i n the T r i t o n X-100 e x t r a c t s . Samples with 1 mM p i c r y l s u l f o n a t e ( • ), or without p i c r y l s u l f o n a t e ( 0 ) were pr e - i n c u b a t e d at 30 C f o r the i n d i c a t e d times and 100 u l were assayed i n the standard assay mixture with the ex c e p t i o n that pNPP h y d r o l y s i s was determined by Pi r e l e a s e a c c o r d i n g to the procedure of Ames (1966) i n s t e a d of d i r e c t l y a t O.D.410 because of the y e l l o w c o l o u r of the r e a c t i o n product of T r i s and p i c r y l s u l f o n a t e . Egg yolk a l k a l i n e phosphatase was i n a c t i v a t e d by the same reagent as p r e v i o u s l y shown by Debruyne (1982), i n d i c a t i n g t h a t the p i c r y l s u l f o n a t e reagent was i n f a c t a c t i v e . 47 1 2 5 H 2 5 H o -f • i 1 r 1 5 3 0 4-5 T I M E C M I N D -~1 6 0 4 8 d) The p r o p e r t i e s of AMPase i s a mutant c e l l l i n e with a l t e r e d pNPPase a c t i v i t y . MacLeod and Loomis (1979) s e l e c t e d a mutant c e l l l i n e ( s t r a i n HL104) t h a t had pNPP h y d r o l y z i n g a c t i v i t y with i n c r e a s e d Km f o r pNPP compared to the parent s t r a i n (AX-3). They used t h i s mutant to demonstrate that the pNPPase i n v e g e t a t i v e and c u l m i n a t i o n stages were encoded by the same gene. I f pNPPase and AMPase a c t i v i t e s are due to the same p r o t e i n , i t f o l l o w s t h a t the KM f o r AMP should a l s o be i n c r e a s e d f o r the mutant s t r a i n enzyme. S t r a i n HL101, that a l s o has an a l t e r e d s u b s t r a t e b i n d i n g f o r pNPP, was k i n d l y p r o v i d e d by Dr. W.J. Loomis. The KM f o r pNPP f o r the h y d r o l y z i n g a c t i v i t y from s t r a i n HL101 was i n c r e a s e d seven-f o l d compared to t h a t of the AX-3 s t r a i n . The Km f o r AMP a l s o i n c r e a s e d although o n l y t h r e e f o l d (Table 2). These r e s u l t s a g a i n suggest the e x i s i t e n c e of a s i n g l e enzyme. Furthermore, both pNPPase and AMPase a c t i v i t i e s i n T r i t o n X-100 e x t r a c t s of the mutant were unstable d u r i n g storage at -20 C ( F i g u r e 9), whereas the a c t i v i t i e s of s i m i l a r e x t r a c t s from the p a r e n t a l s t r a i n were s t a b l e . The i n s t a b i l i t y of the a c t i v i t i e s from the mutant were not due to mutant s t r a i n s p e c i f i c proteases or i n h i b i t o r s s i n c e the pNPPase and AMPase a c t i v i t i e s i n T r i t o n X-100 e x t r a c t s of s t r a i n AX-3 were not reduced when mixed (1:1 v/v) with e x t r a c t s of s t r a i n HL101 49 Table 2 Km values f o r AMP and pNPP h y d r o l y s i s by s t r a i n s Ax-3 and HL101 Km values a St a i n pNPP h y d r o l y s i s AMP h y d r o l y s i s Experiment 1 Ax-3 0.18 mM 0.05 mM HL101 1.33 mM 0.14 mM Experiment 2 Ax-3 0.22 mM 0.06 mM HL101 1.23 mM 0.15 mM a Km values were ob t a i n e d under the f o l l o w i n g c o n d i t i o n s : Samples c o n t a i n i n g 0.020 ml of T r i t o n X-100 e x t r a c t (2.0 mg protein/ml) of e i t h e r s t r a i n Ax-3 or s t r a i n HL101 was incubated i n 50 mM T r i s - C l pH 8.5 b u f f e r ( f o r pNPP) or 50 mM T r i s - C l pH 7.5 b u f f e r ( f o r AMP) and 20 mM MgCl 2 and 30 mM NaF. The range of s u b s t r a t e c o n c e n t r a t i o n s used was 0.125 mM to 2.0 mM. 50 F i g u r e 9: S t a b i l i t y at -20 C of pNPP and AMP h y d r o l y z i n g a c t i v i t i e s i n T r i t o n X-100 membrane e x t r a c t prepared from v e g e t a t i v e c e l l s of s t r a i n HL101. T r i t o n X-100 e x t r a c t (at 2.0 mg p r o t e i n . m l - 1 ) was s t o r e d at -20 C. The sample was thawed and 0.05 ml p o r t i o n s were assayed at the times i n d i c a t e d . ( $ ), pNPP h y d r o l y z i n g a c t i v i t y ; ( Jtk ), AMP h y d r o l y z i n g a c t i v i t y . A f t e r storage of the mutant s t r a i n e x t r a c t at -20 f o r 20 days, such t h a t n e i t h e r a c t i v i t y was d e t e c t a b l e , the e x t r a c t was mixed with an i d e n t i c a l amount of e x t r a c t from Ax-3 and s t o r e d at -20 C. P o r t i o n s (0.05 ml) were assayed at the i n d i c a t e d times. ( 0 ), pNPP h y d r o l y z i n g a c t i v i t y ; ( A . ) , AMP h y d r o l y z i n g a c t i v i t y . 51 8 16 TIME ( D A Y S ) 52 t h a t had l o s t a l l pNPPase and AMPase a c t i v i t i e s on storage ( F i g u r e 9 ) . These r e s u l t s suggest that i n s t a b i l i t y of the mutant c e l l a c t i v i t i e s i s due to a mutated change i n a s i n g l e enzyme that c a t a l y s e s the h y d r o l y s i s of both AMP and pNPP. e) Phospholipase C treatment and s e l e c t i v e s o l u b i l i z a t i o n of AMPase In two p r e v i o u s r e p o r t s (Lee e_t a l . , 1975; C u t l e r and Rossomando, 1975) phospholipase C was used t o e f f e c t an apparent s o l u b i l i z a t i o n of 5' - n u c l e o t i d a s e a c t i v i t y . However, the AMPase a c t i v i t y t h a t was a p p a r e n t l y s o l u b i l i z e d by phospholipase C treatment was not d e t e c t a b l e f o l l o w i n g e l e c t r o p h o r e s i s i n the modified SDS-polyacrylamide g e l system. T h i s was determined to be due to the extreme SDS-s e n s i t i v i t y of t h i s a c t i v i t y . I t was, however, d e t e c t a b l e i n p o l y a c r y l a m i d e g e l s run i n the presence of T r i t o n X-100 and migrated with a g r e a t e r m o b i l i t y than the membrane bound AMPase (F i g u r e 10) . Subsequent a n a l y s i s of the commercial phosp h o l i p a s e C p r e p a r a t i o n s r e v e a l e d an AMPase a c t i v i t y that co-migrated with the p u t a t i v e s o l u b i l i z e d enzyme (Figure 10) i n d i c a t i n g t h at the p r e v i o u s l y r e p o r t e d s e l e c t i v e s o l u b i l i z a -t i o n of 5 ' - n u c l e o t i d a s e was probably an a r t e f a c t due to contamination of the phospholipase C p r e p a r a t i o n by an AMPase a c t i v i t y . 53 F i g u r e 10: N a t i v e T r i t o n X-100 p o l y a c r y l a m i d e g e l e l e c t r o -p h o r e s i s of AMP h y d r o l y z i n g a c t i v i t y i n v e g e t a t i v e c e l l membrane p r e p a r a t i o n s t r e a t e d with a phospholipase C p r e p a r a t i o n . Phospholipase C treatment was done as d e s c r i b e d by Lee et a l . (1975). E l e c t r o p h o r e s i s was c a r r i e d out u s i n g the m o d i f i c a t i o n of the Laemmli (1970) system with 1 0 - f o l d lower c o n c e n t r a t i o n s of T r i s - C l b u f f e r s and 1% T r i t o n X-100 i n s t e a d of 0.1% SDS as d e s c r i b e d i n the Methods. The sample b u f f e r c o ntained 5 mM T r i s - C l pH 6.8, g l y c e r o l and water (1:1:6, v/v/v) . The g e l was s t a i n e d f o r 7\MP h y d r o l y z i n g a c t i v i t y as d e s c r i b e d i n the Methods. (a) T r i t o n X-100 e x t r a c t of the p e l l e t obtained a f t e r phospholipase C treatment, 0.070 mg p r o t e i n loaded. (b) T r i t o n X-100 e x t r a c t of the p e l l e t o b t ained from a c o n t r o l which was not t r e a t e d with phospholipase C, 0.070 mg p r o t e i n loaded. (c) The supernatant o b t a i n e d a f t e r Phospholipase C t r e a t -ment, 0.050 mg p r o t e i n loaded. (d) Phospholipase C p r e p a r a t i o n , 0.050 mg p r o t e i n loaded. M o l e c u l a r weight markers are not shown because they smeared and migrated anomalously. 54 a b e d 55 SECTION 2 S t u d i e s on the mechanism of the pNPP and  AMP h y d r o l y z i n g enzme. The r e s t r i c t e d s u b s t r a t e s p e c i f i c i t y of the D. discoideum phosphatase (Armant and Rutherford, 1981; Mohan Das, 1983, G e z e l i u s and Wright, 1965) would suggest that the enzyme was a 5 ' - n u c l e o t i d a s e . The f o l l o w i n g s t u d i e s were conducted t o determine i f the enzyme had a 5 1 - n u c l e o t i d a s e or a l k a l i n e phosphatase type mechanism. a) AMP T h i o n u c l e o t i d e analog s t u d i e s The use of t h i o n u c l e o t i d e analogs of AMP t o d i s t i n g u i s h between a l k a l i n e phosphatases and 5'- n u c l e o t i d a s e s has been suggested r e c e n t l y (Rossomando et a l . , 1983). C a l f i n t e s t -i n a l mucosa APase was shown t o h y d r o l y z e dASMP at about 30% the r a t e of AMP (under s a t u r a t i n g c o n d i t i o n s ) , whereas t h i s a nalog was completely r e s i s t a n t t o h y d r o l y s i s by snake venom 5 1 - n u c l e o t i d a s e . Conversely AMPS was h y d r o l y z e d 2000 times l e s s r a p i d l y by APase, while 5 ' - n u c l e o t i d a s e h y d r o l y z e d AMPS at 1.9% the r a t e of AMP (Rossomando et a l . , 1983) T r i t o n X-100 e x t r a c t s of D. discoideum membranes c a t a l y z e d the h y d r o l y s i s of AMPS at 2.5% the r a t e of AMP h y d r o l y s i s and d i d not c a t a l y z e the h y d r o l y s i s of dASMP (Table 3 ) . Given the c r i t e r i a p r esented by Rossomando et aJL. (1983), t h i s r e s u l t i n d i c a t e d t h a t the a c t i v i t y was a 5 1 - n u c l e o t i d a s e - t y p e enzyme. However, when p a r t i a l l y p u r i f i e d APase was used, 56 n e i t h e r dASMP nor AMPS were h y d r o l y z e d (Table 3). As such, c l a s s i f i c a t i o n of the D. discoideum phosphatase c o u l d not be accomplished by AMP t h i o n u c l e o t i d e analog h y d r o l y s i s as suggested by Rossomando et a l . (1983). The enzyme r e s p o n s i b l e f o r the h y d r o l y s i s of AMPS i n crude e x t r a c t s c o u l d not be d e t e c t e d as a minor a c t i v e s p e c i e s on SDS g e l s a lthough i t s absence might be due to i n s t a b i l i t y of the enzyme under these c o n d i t i o n s . b) T r a n s p h o s p h o r y l a t i o n s t u d i e s . A f e a t u r e common to a l k a l i n e phosphatases from a v a r i e t y of sources i s the c a t a l y s i s of a t r a n s p h o s p h o r y l a t i o n r e a c t i o n i n v o l v i n g phosphoryl group t r a n s f e r to c e r t a i n low-molecular weight a l c o h o l s (MacComb et. al_. , 1979). In c o n t r a s t , b u l l t e s t i s 5 1 - n u c l e o t i d a s e c a t a l y z e s the h y d r o l y s i s of AMP without the a d v e n t i t i o u s p r o d u c t i o n of t r a n s p h o s p h o r y l a t i o n products (Morton, 1953). T h i s d i f f e r e n c e i n the r e a c t i o n s of the two enzyme types i s due to the involvement of an enzyme-phosphate co v a l e n t complex d u r i n g the double-displacement mechanism of a l k a l i n e phosphatases. The enzyme-phosphate in t e r m e d i a t e i s s u s c e p t i b l e to n u c l e o p h i l i c a t t a c k by a l c o h o l s to g i v e o r g a n i c phosphate compounds (McComb et a l . , 1979). In c o n t r a s t , the 5 ' - n u c l e o t i d a s e r e a c t i o n i s a s i n g l e -displacement type ( T s a i , 1980). 57 Table 3 H y d r o l y s i s of TAMP and t h i o n u c l e o t i d e analogues S u b s t r a t e T r i t o n X-100 e x t r a c t P a r t i a l l y p u r i f i e d enzyme (pmoles of n u c l e o s i d e p r o d u c e d ) 3 AMP 192 17 AMPS 5 0 dASMP 0 0 The r e a c t i o n mixture c o n t a i n e d 100 mM T r i s - C l pH 8.5, 20 mM mgCl2# 30 mM Naf, 0.100 mM s u b s t r a t e (AMP, AMPS, or dASMP). The r e a c t i o n was i n i t i a t e d by the a d d i t i o n of e i t h e r 0.010 ml d i a l y z e d T r i t o n X-100 e x t r a c t (1.5 mg/ml) ov v e g e t a t i v e c e l l s or 0.010 ml of p a r t i a l l y p u r i f i e d enzyme (1.0 mg p r o t e i n / m l ) . For the T r i t o n X-100 e x t r a c t , i n c u b a t i o n was f o r 10 min at 30 C, while a 60 min i n c u b a t i o n was used f o r the p a r t i a l l y p u r i f i e d enzyme. Nucleosides or t h i o n u c l e o s i d e s were q u a n t i f i e d by a n a l y z i n g 0.010 ml a l i q u o t s of the r e a c t i o n mixture by hi g h - p r e s s u r e l i q u i d chromatography as d e s c r i b e d i n Methods. 58 Both AMP and pNPP served as donors f o r the t r a n s f e r of a phosphoryl group to T r i s (Table 4) or ethanolamine (Table 5). The p r o d u c t i o n of o r g a n i c phosphate was h i g h l y dependent upon the c o n c e n t r a t i o n s of the acceptor T r i s or ethanolamine, a l l phosphate generated i n the presence of low T r i s or e t h a n o l -amine was accounted f o r as i n o r g a n i c phosphate, but i n c r e a s -i n g c o n c e n t r a t i o n s of acceptor r e s u l t e d i n p r o g r e s s i v e d e c r e a s i n g percentages of i n o r g a n i c phosphate. 0_-phosphor-ylet h a n o l a m i n e produced d u r i n g the course of r e a c t i o n of pNPPase i n ethanolamine b u f f e r was d i r e c t l y i d e n t i f i e d by paper e l e c t r o p h o r e s i s ( F i g u r e 11). These r e s u l t s demon-s t r a t e d t h a t the D. discoideum enzyme i s an a l k a l i n e phosphatase and not a 5 ' - n u c l e o t i d a s e . The pH dependence of t r a n s p h o s p h o r y l a t i o n was determined u s i n g pNPP as s u b s t r a t e and T r i s as a c c e p t o r . The r e s u l t s ( F i g u r e 12) show t h a t the t r a n s p h o s p h o r y l a t i o n i s optimal over the pH range 7.5 t o 9.0, d e c r e a s i n g s l i g h t l y at h i g h e r pH v a l u e s . c) I d e n t i f i c a t i o n of the involvement of T r i s as a co-s u b s t r a t e f o r the enzyme. K i n e t i c a n a l y s i s of a r e a c t i o n i n v o l v i n g the p a r t i c i p a -t i o n , of a c o - s u b s t r a t e r e v e a l s a s e r i e s of p a r a l l e l L i n e -weaver-Burke p l o t s where the Km i n the presence of cosub-s t r a t e i s lower than i t s absence (Dixon, 1965). 59 Tabl e 4 T r a n s p h o s p h o r y l a t i o n a c t i v i t y of D. discoideum e x t r a c t s u s i n g T r i s b u f f e r a T r i s - C l concent- Product formation from pNPP r a t i o n (mM) (nmoles) p - n i t r o p h e n o l Pi o r g a n i c phosphate* 5 5 160.7 + 8.0 162.7 + 4.7 50 317.7 + 7.4 258.7 + 7.5 59 (18.6%) 500 526.7 + 7.8 323.7 + 9.0 203 (38.5%) T r i s CI concent-r a t i o n (mM) adenosine Product formation from AMP (nmoles) P i o r g a n i c phosphate* 5 5 50 500 91.7 + 6.5 88.7 + 6.13 (3%) 180.7 + 7.6 127.7 + 4.6 53 (29.3%) 263.0 + 9.1 152.3 + 8.3 111 (42.2%) Samples c o n t a i n i n g 0.200 ml of but t a t i v e c e l l membrane p r o t e i n (3.0 b u f f e r of the a p p r o p r i a t e concentr o.3 M NaF and 4 mM s u b s t r a t e i n 1. incubated f o r 3 min at 30 C. The amount of a l c o h o l i c product and Pi i n Methods. The values given f o r mean of 3 determinations with the D e v i a t i o n . a n o l e x t r a c t e d vege-mg/ml), T r i s - C l pH8.5 a t i o n , 0.2 M MgCl 2/ 0 ml f i n a l volume were determinations of the were done as d e s c r i b e d these products are the Standard Estimated Determined as the d i f f e r e n c e of the amount of sub-s t r a t e c l e a v e d and the i n o r g a n i c phosphate produced. The value s g i v e n are the d i f f e r e n c e between the two means. 60 Table 4 T r a n s p h o s p h o r y l a t i o n a c t i v i t y of D. discoideum e x t r a c t s u s i n g ethanolamine b u f f e r 3 Ethanolamine-Cl- Product formation from pNPP c o n c e n t r a t i o n (mM) (nmoles) p - n i t r o p h e n o l P i o r g a n i c phosphate' 3 5 48.7 + 3.50 45.7 + 3.1 3 (6.1%) 50 80.3 + 6.04 66.3 + 4.6 14 (17.4%) 500 56.0 + 1.73 32.7 + 5.0 23 (41.6%) Ethanolamine-Cl Product formation from TAMP c o n c e n t r a t i o n (mM) adenosine 5 47.3 + 4.5 44.0 + 4.03 (7.0%) 50 139.0 + 9.2 106.3 + 6.1 33 (23.5%) 500 122.0 + 9.2 59.0 + 3.6 63 (51.6%) a Samples c o n t a i n i n g 0.200 ml of b u t a n o l e x t r a c t e d v e g e t a t i v e c e l l membrane p r e p a r a t i o n (3.0 mg p r o t e i n / m l ) , ethanolamine-Cl pH 8.5 b u f f e r of the i n d i c a t e d c o n c e n t r a t i o n , 0.2 M MgCl 2# 0.3 M NaF, and 4 mM s u b s t r a t e i n 1.0 ml f i n a l volume were incubated a t 30 C f o r 30 min. The determinations of the amounts of a l c o h o l i c product and Pi were done as d e s c r i b e d i n Methods. The values given f o r these products are the mean of 3 determinations with the Standard Estimated D e v i a t i o n s . k Determined as the d i f f e r e n c e of the amount of s u b s t r a t e c l e a v e d and the i n o r g a n i c phosphate produced. The values given are the d i f f e r e n c e between the two means. (nmoles) Pi o r g a n i c phosphate' 3 61 F i g u r e 11: I d e n t i f i c a t i o n of O-phosphorylethanolamine produced d u r i n g the course of pNPPase r e a c t i o n i n e t h a n o l a -mine b u f f e r . D i a l y z e d v e g e t a t i v e c e l l T r i t o n X-100 e x t r a c t was added to 200 mM ethanolamine-Cl pH 8.5 as d e s c r i b e d i n the Methods. O-Phosphorylethanolamine was i s o l a t e d by i o n -exchange column chromatography a c c o r d i n g to the procedure of Debruyne (1982). The i s o l a t e d m a t e r i a l was s u b j e c t e d to h i g h v o l t a g e paper e l e c t r o p h o r e s i s on Whatman #3 paper i n 1.66 M for m i c a c i d pH 1.58 f o r 45 min at 40 V/cm as d e s c r i b e d by Debruyne (1982). S t a i n i n g was done by d i p p i n g the water i n 0.25% n i n h y d r i n i n acetone and h e a t i n g at 100 C f o r 10 minutes. (A) m a t e r i a l produced d u r i n g the course of r e a c t i o n of the D. discoideum pNPPase. (B) a u t h e n t i c o-phosphorylethanolamine. anode o r i g i n cathode 63 F i g u r e 12: pH p r o f i l e of t r a n s p h o s p h o r y l a t i o n a c t i v i t y of pNPPase. Samples co n t a i n e d d i a l y z e d T r i t o n X-100 e x t r a c t s (2.0 mg.ml - 1, 0.100 ml), 200 mM T r i s - C l at the i n d i c a t e d pH, 20 mM MgCl 2» 30 mM NaF, and 4 mM pNPP i n a t o t a l volume of 1.0 ml. A f t e r 3 min i n c u b a t i o n at 30 C, p - n i t r o p h e n o l and P i formed were q u a n t i t a t e d as d e s c r i b e d i n Methods. P E R C E N T P H O S P H A T E A S T R I S - P H O S P H A T E • CD -1 TJ * 03 Ul (0 (0 U l o ro o o _ i _ cn • co o _J 65 F i g u r e 13 shows that such p l o t s are obtained when d i f f e r e n t c o n c e n t r a t i o n s of T r i s - C l are used as c o - s u b s t r a t e . The data i s s i m i l a r f o r 5 mM T r i s - C l pH 8.5 alone and 5 mM T r i s - C l pH 8.5 i n the presence of 200 mM KC1 (Figure 13) i n d i c a t i n g t h a t the i n c r e a s e d i o n i c s t r e n g t h i s not r e s p o n s i b l e f o r the a l t e r e d p l o t obtained f o r 200 mM T r i s - C l . T h i s data i s a l s o c o n s i s t e n t with the idea that the enzyme was an a l k a l i n e phosphatase which t r a n s f e r s a phosphoryl group t o a n u c l e o p h i l e acceptor, T r i s . d) B i p h a s i c k i n e t i c s of pNPP h y d r o l y s i s . Because the n u c l e o p h i l i c s u b s t i t u t i o n of enzyme f o r the a l c h o l i c p o r t i o n of s u b s t r a t e e s t e r occurs at a g r e a t e r r a t e than the subsequent turnover of the enzyme i n the a l k a l i n e phosphatase double-displacement mechanism (McComb et a_l. , 1979), i t should be p o s s i b l e t o measure b i p h a s i c k i n e t i c s d i r e c t l y . Although i n i t i a l s t u d i e s on E. c o l i APase f a i l e d t o r e g i s t e r an i n i t i a l b u r s t of a l c o h o l p r o d u c t i o n at pH values g r e a t e r than 7 (Block and S c h l e s i n g e r , 1973), t h i s was subsequently accomplished (Bale et a l . , 1980). F i g u r e 14 shows that the D. discoideum enzyme i n i t s r e a c t i o n with pNPP d i s p l a y s a s i g n i f i c a n t b u r s t phase at pH 7.5. An i n i t i a l l a g phase of about 30 msec was observed with the D. discoideum enzyme was w e l l as with a commercial p r e p a r a t i o n of E. c o l i enzyme, i t i s p o s s i b l e that the l a g 66 Figure 13: Double re c i p r o c a l plots suggesting a double displacement mechanism for pNPPase. Assays were done under the following conditions: ( O ) 0.02 ml dialyzed vegetative c e l l T r i t o n X-100 extract (2.0 mg/ml) in 5 mM T r i s - C l pH 8.5, 20 mM MgCl 2/ 30 mM NaF, and the indicated concentrations of pNPP i n a f i n a l volume of 1.0 ml; ( ^  ) -0.02 ml of dialyzed vegetative c e l l Triton X-100 extract, 20 mM mgCl 2, 30 mM NaF, 200 mM NaCl, and the indicated concentrations of pNPP i n a f i n a l volume of 1.0 ml; ( • ) 0.02 ml of dialyzed vegetative c e l l Triton X-100 extract, 20 mM MgCl 2, 30 mM NaF, 200 mM T r i s - C l pH 8.5, and the indicated concentrations of pNpp in a f i n a l volume of 1.0 ml. The samples were incubated at 30 C for 10 minutes. l/[S] i s the rec i p r o c a l of the pNPP concentration in millimolar and 1 / a c t i v i t y i s the reciprocal of the i n i t i a l enzyme a c t i v i t y i n the units 0.D.410/min/.040 mg protein. The l i n e s of best f i t were drawn using l i n e a r regression. 1/ACTIVITY 68 F i g u r e 14: Stopped flow r a p i d spectrophotometric t r a c e of the r e a c t i o n between T r i t o n X-100 s o l u b i l i z e d pNPPase (8 mg/ml) of v e g e t a t i v e c e l l s and pNPP (8 mM) . The mixing c u v e t t e p a t h l e n g t h was 0.5 cm. A dead time of 0.010 seconds was used b e f o r e i n i t i a t i n g data c o l l e c t i o n . The Km f o r i n o r g a n i c phosphate of pNPPase i n 5 mM T r i s - C l pH 7.5 (the b u f f e r p r e s e n t i n T r i t o n X-100 e x t r a c t s ) i s approximately 0.67 mM (from F i g u r e 15) while approximately 0.04 mM i n o r g a n i c phosphate has been produced d u r i n g the f i r s t 0.100 seconds of the r e a c t i o n . As such the decrease i n the enzyme r e a c t i o n r a t e i s not due to c o m p e t i t i v e i n h i b i t i o n by i n o r g a n i c phosphate produced under these c o n d i t i o n s . D e l t a absorbance i s at 410 nm at which the product p - n i t r o p h e n o l absorbs l i g h t maximally. The p l o t shown i s the average of f i v e experiments. 6 9 0 - . 6 I i j i i : I 0 .04 .08 .12 .16 .2 SECONDS 70 phase rep r e s e n t s an i n s t r u m e n t a l a r t e f a c t . e) I n t e r a c t i o n of pNPPase with orthophosphate. G e z e l i u s and Wright (1965) found that 50 mM ortho-phosphate i n h i b i t e d pNPPase by 90%. Most of the i n h i b i t e d a c t i v i t y c o u l d be recovered by d i l u t i o n . Mohan Das (1983) a l s o found that i n h i b i t i o n by P i was r e v e r s i b l e . In c o n t r a s t , Armant and R u t h e r f o r d (1981, 1979) found the i n h i b i t i o n t o be i r r e v e r s i b l e and suggested that P i was c o v a l e n t l y bound to the enzyme. These c o n f l i c t i n g data i n d i c a t e d that the i n t e r a c t i o n of P i with pNPPase was complex and that a s y s t e m a t i c study was r e q u i r e d . I t was found t h a t orthophosphate (lOmM) was a c o m p e t i t i v e i n h i b i t o r of pNPPase when the b u f f e r i o n i c s t r e n g t h was low ( F i g u r e 15). However, the i n t e r a c t i o n of P i w i t h the enzyme was d r a m a t i c a l l y d i f f e r e n t i n h i g h i o n i c s t r e n g t h b u f f e r s . T r i s - C l at 200 mM i n h i b i t e d the pNPPase i n a time-dependent and pH-dependent manner (F i g u r e 16). When 1 mM P i was i n c l u d e d i n h i g h i o n i c s t r e n g t h s o l u t i o n s , there was a marked enhancement of pNPPase i n h i b i t i o n which was a l s o s t r o n g l y dependent upon pH ( F i g u r e 17). The i n h i b i t i o n was i r r e v e r s i b l e (Table 6 ) . E . c o l i a l k a l i n e phosphatase has a l s o been found to be i n h i b i t e d i n T r i s b u f f e r s of h i g h pH (Roig et a l . , 1982). Roig et a l . (1982) observed i n h i b i t i o n 71 Figure 15: Double rec i p r o c a l plots showing competitive i n h i b i t i o n of pNPPase a c t i v i t y by orthophosphate. Assays were done under the following conditions: ( Q ) 0.20 ml dialyzed vegetative Triton X-100 extract (2.0 mg protein/ml), in 1.0 ml f i n a l volume of assay buffer; ( O ) < 0.020 ml dialyzed vegetative c e l l T r i t o n X-100 extract (2.0 : mg protein/ml), 10 mM k2HPC>4 i n 1.0 ml f i n a l volume of assay buffer. The assay buffer consisted of 5 mM T r i s - C l pH 7.5 buffer, 20 mM MgCl 2, 30 mM NaF and the indicated concentrations of pNPP. The samples were incubated at 30 C for 10 minutes. 1/[S] i s the re c i p r o c a l of the pNPP concentration in millimolar and 1 / a c t i v i t y i s the reciprocal of the i n i t i a l enzyme a c t i v i t y i n the units O. D.410/min/•040 mg protein. The l i n e s of best f i t were drawn using linear regression. Each point represents the average of two determinations. 1/ACTIVITY 73 F i g u r e 16: pH Dependence of the i n h i b i t i o n of pNPPase a c t i v i t y by T r i s - C l . Assays were done as f o l l o w s : ( O ) 0.020 ml of d i a l y z e d v e g e t a t i v e c e l l T r i t o n X-100 e x t r a c t (2.0 mg/ml), 200 mM T r i s - C l pH 7.0 i n a volume of 0.100 ml; ( • ) 0.020 ml of v e g e t a t i v e c e l l T r i t o n X-100 e x t r a c t (2.0 mg/ml), 200 mM T r i s - C l pH 9.5 i n a volume of 0.100 ml. A f t e r i n c u b a t i o n at 30 C f o r the i n d i c a t e d times, the samples were assayed f o r pNPPase a c t i v i t y i n 0.900 ml of assay mixture c o n t a i n i n g 500 mM T r i s - C l pH 8.5, 20 mM MgCl2/ 30 mM NaF and 4 mM pNPP. At pH values between pH 7.0 and pH 9.5, the i n h i b i t i o n of pNPPase was intermediate between the two p l o t s shown. The data from another experiment was s i m i l a r to that presented. 74 1 2 5 n hi o_ 2 5 H O -f • i 1 o —I 1 — 2 0 3 0 T I M E ( M I N ) — T - 1 4 0 75 F i g u r e 17: pH Dependence of the orthophosphate induced i n a c t i v a t i o n of pNPPase i n T r i s - C l and e f f e c t of AMP. D i a l y z e d v e g e t a t i v e c e l l T r i t o n X-100 e x t r a c t s (2.0 mg/ml, 0.020 ml) were incubated i n a f i n a l volume of 0.100 ml under the f o l l o w i n g c o n d i t i o n s : ( O ) 200 mM T r i s - C l at the i n d i c a t e d pH; ( A ) 200 mM T r i s - C l at the i n d i c a t e d pH, 1 mM K 2 H P 0 4 ; ( • ) 200 mM T r i s - C l a t the i n d i c a t e d pH, 1 mM K2HPO4, 1 mM AMP. The samples were incubated at 30 C f o r 10 min and then assayed i n 0.900 ml of assay s o l u t i o n c o n t a i n i n g 500 mM T r i s - C l pH 8.5, 20 mM MgCl2» 30 mM NaF and 4 mM pNPP. The number of measurements f o r each pH value was 5. The d i f f e r e n c e between phosphate t r e a t e d pNPPase and phosphate t r e a t e d pNPPase i n the presence of AMP was s i g n i f i c a n t a t pH value s 7, 7.5 and 8 as determined by Student's t - t e s t , p<0.01 a t each of the three pH v a l u e s . pNPPase a c t i v i t y i s expressed i n nmoles/min/mg p r o t e i n . pNPPase ACTIVITY 77 Table 6: I n t e r a c t i o n of orthophosphate with pNPPase Treatment 3 1 ) 5 mM T r i s - C l pH 9.0 2) 5 mM T r i s - C l pH 9.0 + 200 mM K C 1 3) 5 mM T r i s - C l pH 9.0 + 200 mM K C 1 + 1 mM K2HPO4 4) as 3) f o l l o w e d by d i a l y s i s * pNPPase a c t i v i t y " (nmoles/min/mg p r o t e i n ) 108.3 + 7.5 80.3 + 6.0 27.7 + 2.9 19.0 + 4.0 1 ) 5 mM Borate pH 9.0 9 6 . 3 + 4 . 2 2) 5 mM Borate pH 9.0 + 200 mM K C 1 98.0 + 7.0 D i a l y z e d T r i t o n X-100 e x t r a c t s (2.0 mg p r o t e i n , ml--'-) were incubated with the i n d i c a t e d a d d i t i o n s f o r 30 min a t 30 C. 0.100 ml A l i q u o t s were assayed f o r pNPPase a c t i v i t y u s i n g the standard assay mixture. The a c t i v i t y of pNPPase before the 30 C i n c u b a t i o n was 113 +5.5 nmoles/min/mg p r o t e i n . The mean of t r i p l i c a t e d eterminations and the Standard Estimated D e v i a t i o n s are gi v e n . A f t e r 30 min the sample was d i a l y z e d a g a i n s t 5 mM T r i s -C l pH 7.5 f o r 3 days. 78 of E. c o l i APase when T r i s c o n c e n t r a t i o n s exceeded 1.0M. It was found that the D. discoideum pNPPase was not i n h i b i t e d i n 5 mM borate b u f f e r pH 9.0 + 200 mM KC1 but i t was when T r i s - C l r e p l a c e d borate (Table 6 ) . T h i s suggested t h a t T r i s - C l i t s e l f was i n v o l v e d i n the i n h i b i t i o n p r o c e s s . However, as 5 mM T r i s - C l pH 9.0 d i d not i n h i b i t the pNPPase (Table 6), i t i s c l e a r t h at both h i g h i o n i c s t r e n g t h (200 mM KC1) as w e l l as T r i s - C l are r e q u i r e d to i n h i b i t pNPPase. At lower pH va l u e s , AMP c o u l d p a r t i a l l y p r o t e c t the enzyme a g a i n s t P i i n a c t i v a t i o n ( F i gure 17). Table 7 shows the c o n c e n t r a t i o n dependence of the p r o t e c t i o n by AMP. 79 Tabl e 7: P r o t e c t i o n by AMP of orthophosphate i n a c t i v a t i o n of pNPPase Treatment 3 pNPPase a c t i v i t y " (nmoles/min/mg p r o t e i n ) 1) None 105. 3 + 3.5 2) 1 mM K 2HP0 4 44. 7 + 4.0 3) 1 mM K 2HP0 4 + 1 mM AMP 69.0 + 5.0 4) 1 mM K 2HP0 4 + 2 mM AMP 76.0 + 5.0 5) 1 mM K 2HP0 4 + 4 mM AMP 82. 3 + 6.0 Assays c o n t a i n e d d i a l y z e d T r i t o n X-100 e x t r a c t s (2.0 Mg p r o t e i n / m l , 0.020 ml) and the i n d i c a t e d a d d i t i o n s i n 200 mM T r i s - C l pH 7.5, i n a f i n a l volume of 0.200 ml. A f t e r i n c u b a t i o n a t 30 C f o r 10 min, the samples were assayed f o r pNPPase a c t i v i t y u s i n g the standard assay mixture. The mean of t r i p l i c a t e d eterminations and the Standard Estimated D e v i a t i o n s are g i v e n . 80 SECTION 3 Q u a n t i t a t i o n of APase i n crude membrane e x t r a c t s  prepared from d i f f e r e n t developmental stages. I t has been p o s t u l a t e d that the developmental accumulation of APase c o u l d be due to the unmasking of pr e -e x i s t i n g v e g e t a t i v e c e l l APase by the removal of a low-molecular weight i n h i b i t o r (Mohan Das and Weeks, 1980, Mohan Das, 1983). I f t h i s h y p o t h e s i s i s c o r r e c t then i t f o l l o w s t h a t i n c r e a s e d l e v e l s of enzyme p r o t e i n should not be r e q u i r e d f o r the w e l l documented developmental i n c r e a s e i n a l k a l i n e phosphatase a c t i v i t y . a) Immunoquantitation of APase Antiserum t o p u r i f i e d APase was prepared as d e s c r i b e d under Methods. The antiserum d i d not i n h i b i t pNPPase (data not shown). It a l s o d i d not p r e c i p i t a t e pNPPase from T r i t o n X-100 e x t r a c t s , e i t h e r alone or i n c o n j u n c t i o n with Staphyloccocus aureus bound P r o t e i n A (data not shown). As the antiserum was r a i s e d to pNPPase i s o l a t e d from SDS p o l y a c r y l a m i d e g e l s , i t was p o s s i b l e that SDS treatment was r e q u i r e d f o r a n t i b o d y - a n t i g e n i n t e r a c t i o n . However, no p r e c i p i t a t i o n of pNPPase o c c u r r e d even i n the presence of 0.1% SDS (data not shown). However, when T r i t o n X-100 e x t r a c t e d membrane p r o t e i n s were separated on SDS 81 p o l y a c r y l a m i d e g e l s and subsequently t r a n s f e r r e d to n i t r o c e l l u l o s e paper, b i n d i n g of antibody was d e t e c t a b l e ( F i g u r e 18). The double banding p a t t e r n i s perhaps due to a g g r e g a t i o n of the enzyme ( c f . F i g u r e 3) under non-denaturing c o n d i t i o n s . C o n t r o l s t r e a t e d with normal r a b b i t serum i n s t e a d of immune serum d i d not show these bands ( F i g u r e 18). As F i g u r e 18 shows, other p r o t e i n s were r e a c t i v e , e s p e c i a l l y a h i g h molecular weight (Mj- = 500,000) component. T h i s may be due to c r o s s - r e a c t i v i t y between a n t i g e n i c determinants. The antibody s t a i n i n g technique was s e n s i t i v e enough to r e a d i l y d i s t i n g u i s h t w o - f o l d d i f f e r e n c e s i n amount of d e t e c t e d p r o t e i n ( F i g u r e 18). When T r i t o n X-100 e x t r a c t s prepared from four d i f f e r e n t stages of development were s u b j e c t e d to e l e c t r o p h o r e s i s and subsequently b l o t t e d , there was no i n c r e a s e found i n the amount of bound antibody as c e l l s proceeded through t e r m i n a l c u l m i n a t i o n ( F i g u r e 19). In f a c t , t h e r e was a d i s t i n c t decrease i n the i n t e n s i t y of s t a i n i n g with c u l m i n a t i o n e x t r a c t s ( F i g u r e 19). Furthermore, t h e r e was a s l i g h t decrease i n the apparent molecular weight of the p u t a t i v e pNPPase with development. A s i m i l a r i n c r e a s e i n m o b i l i t y of pNPPase i n c u l m i n a t i o n phase p r e p a r a t i o n s was a l s o observed i n n a t i v e g e l s where the enzyme had been s t a i n e d f o r a c t i v i t y ( F i g u r e 3 ) . The presence of two bands 82 F i g u r e 18: Western b l o t of T r i t o n X-100 s o l u b i l i z e d v e g e t a t i v e c e l l crude membrane p r e p a r a t i o n . The procedures used are d e s c r i b e d i n the Methods. The T r i t o n X100 e x t r a c t was not b o i l e d p r i o r to e l e c t r o p h o r e s i s . For ( 1 ) to ( 5 ) antiserum was used, while ( 6) was a c o n t r o l u s i n g normal r a b b i t antiserum. ( 1 ) 0.200 mg p r o t e i n ; ( 2 ) 0.100 mg p r o t e i n ; ( 3 ) 0.050 mg p r o t e i n ; ( 4 ) 0.025 mg p r o t e i n ( 5 ) 0.0125 mg p r o t e i n ; ( 6 ) 0.200 mg p r o t e i n . The molecular weight markers were t r a n s b l o t t e d to n i t r o -c e l l u l o s e paper along with the T r i t o n X-100 e x t r a c t e d p r o t e i n s and were s t a i n e d with India ink u s i n g the procedure of Hancock and Tsang (1983). T r a n s b l o t t i n g e f f i c i e n c y was almost 100% as determined by s i l v e r s t a i n i n g the p o s t - b l o t g e l . 83 50 OK 180K 15BK 45K 1 2 3 4 5 84 F i g u r e 19: Western b l o t of T r i t o n X-100 e x t r a c t s of crude membrane p r e p a r a t i o n s from d i f f e r e n t developmental sources. The p r e p a r a t i o n s were not b o i l e d p r i o r to e l e c t r o p h o r e s i s . 0.100 mg p r o t e i n was loaded onto each w e l l . ( 1 ) v e g e t a t i v e phase c e l l s ; ( 2 ) a g g r e g a t i o n phase c e l l s ; ( 3 ) s l u g phase c e l l s ; ( 4 ) c u l m i n a t i o n phase c e l l s . The Western b l o t t i n g procedures used are d e s c r i b e d i n the Methods. The M o l e c u l a r weight markers were t r a n s b l o t t e d to n i t r o c e l l u l o s e paper along with the T r i t o n X-100 e x t r a c t e d p r o t e i n s and were s t a i n e d with I n d i a ink u s i n g the procedure of Hancock and Tsang (1983). T r a n s b l o t t i n g e f f i c i e n c y was almost 100% as determined by s i l v e r s t a i n i n g the p o s t - b l o t g e l . 85 1 2 3 4 86 f o r pNPPase made i t d i f f i c u l t t o i n s p e c t t h e l a n e s f o r s m a l l d i f f e r e n c e s i n s t a i n i n g i n t e n s i t y . To overcome t h i s d i f f i c u l t y , t h e s a m p l e s were h e a t e d i n SDS-sample b u f f e r p r i o r t o e l e c t r o p h o r e s i s . As e x p e c t e d , t h e d o u b l e b a n d i n g p a t t e r n d i s a p p e a r e d and t h e r e was o n l y one major band p r e s e n t a t M j . = 120,000 ( f o r v e g e t a t i v e sample; F i g u r e 2 0 ) . The d e c r e a s e i n s t a i n i n g i n t e n s i t y and i n c r e a s e i n m o b i l i t y o f t h e p u t a t i v e pNPPase i n t h e c u l m i n a t i o n p h a s e was a g a i n a p p a r e n t ( F i g u r e 2 0 ) . However, t h e r e s u l t s o f t h e i m m u n o q u a n t i t a t i o n need t o be i n t e r p r e t a t e d w i t h c a u t i o n . A l t h o u g h t h e r e were good c o r r e l a t i o n s b etween t h e p o s i t i o n s o f a l k a l i n e p h o s p h a t a s e a c t i v i t y and t h e major i m m u n o r e a c t i v e components on t h e W e s t e r n b l o t s , t h e a n t i s e r u m d i d n o t i m m u n o p r e c i p i t a t e t h e pNPPase and i t c o u l d n o t be e s t a b l i s h e d w i t h c e r t a i n t y t h a t t h e major i m m u n o r e a c t i v e component on t h e n i t r o c e l l u l o s e p a p e r was i n f a c t pNPPase. b) 3 ^ P i l a b e l l i n g o f pNPPase from v e g e t a t i v e p h a s e and c u l m i n a t i o n p h a s e c e l l s . The t i m e d e p e n d e n t , i r r e v e r s i b l e i n a c t i v a t i o n o f pNPPase w i t h P i a t h i g h i o n i c s t r e n g t h s u g g e s t e d t h a t P i c o u l d be c o v a l e n t l y m o d i f y i n g t h e enzyme. S e v e r a l p i e c e s o f e v i d e n c e s u g g e s t e d t h a t c o v a l e n t m o d i f i c a t i o n would be a t t h e enzyme a c t i v e s i t e . The d o u b l e - d i s p l a c e m e n t mechanism o f t h e 87 F i g u r e 20: Western b l o t of T r i t o n X-100 e x t r a c t s of crude membrane p r e p a r a t i o n s from d i f f e r e n t developmental sources. The p r e p a r a t i o n s were b o i l e d i n e l e c t r o p h o r e s i s sample b u f f e r (0.083 M T r i s - C l pH 6.8, g l y c e r o l , water; 1:1:6,v/v/v) bef o r e e l e c t r o p h o r e s i s was done. The Western b l o t t i n g procedures used are d e s c r i b e d i n the Methods. ( 1 ) v e g e t a t i v e phase c e l l s ; ( 2 ) a g g r e g a t i o n phase c e l l s ; ( 3 ) s l u g phase c e l l s ; ( 4 ) c u l m i n a t i o n phase c e l l s . 0.100 mg p r o t e i n was used. A l s o shown i s a p r o t e i n s t a i n e d g e l of 0.100 mg of v e g e t a t i v e phase c e l l T r i t o n X-100 e x t r a c t , ( 5 ). The e l e c t r o p h o r e s i s of t h i s p r e p a r a t i o n was done under the same c o n d i t i o n s as f o r the Western b l o t and demonstrates that under these c o n d i t i o n s most of the p r o t e i n s migrate to the lower h a l f of the g e l . The developmental p r o f i l e of a l k a l i n e phosphatase i s g i v e n at the bottom of the f i g u r e . The molecular weight markers were t r a n s b l o t t e d t o n i t r o c e l l u l o s e paper along with the T r i t o n X-100 e x t r a c t e d p r o t e i n s and were s t a i n e d with India ink u s i n g the procedure of Hancock and Tsang (1983). T r a n s b l o t t i n g e f f i c i e n c y was almost 100% as determined by s i l v e r s t a i n i n g the p o s t - b l o t g e l . 88 Developmental stage vegetative aggregates slugs culmination APase activity (nrTKDles/min/mg protein) 18 44 81 89 enzyme, evidence f o r which i s p r o v i d e d i n S e c t i o n 2, r e q u i r e s p a r t i c i p a t i o n of a phosphorylated a c t i v e s i t e i n the h y d r o l y t i c r e a c t i o n . The f i n d i n g that Pi i s a c o m p e t i t i v e i n h i b i t o r at low i o n i c s t r e n g t h (Figure 15) a l s o i n d i c a t e s t h a t P i i n t e r a c t s with the a c t i v e s i t e . Furthermore, the p a r t i a l p r o t e c t i o n by TAMP a g a i n s t Pi induced i n a c t i v a t i o n (Table 7) s t r o n g l y i n d i c a t e s that Pi i n t e r a c t s with the a c t i v e s i t e . F i n a l l y , i t i s known that P i c o v a l e n t l y binds t o the a c t i v e s i t e s e r i n e r e s i d u e of APases from other sources (McComb et al., 1979). T h i s suggested that 3 2 P i l a b e l l i n g c o u l d be used as a measure of the number of a c t i v e s i t e s and hence as a measure of the amount of APase i n e x t r a c t s prepared from v e g e t a t i v e and c u l m i n a t i o n phase c e l l s . L a b e l l i n g of b u t a n o l e x t r a c t e d p r o t e i n s from v e g e t a t i v e and c u l m i n a t i o n phase c e l l s showed t h a t g r e a t e r amounts of 32pi were i n c o r p o r a t e d i n the v e g e t a t i v e c e l l p r e p a r a t i o n ( F i g u r e 21 and 22). However, SDS-PAGE of the phosphorylated p r e p a r a t i o n s f o l l o w e d by autoradiography f a i l e d to r e v e a l the expected Mj. = 120,000 band. Instead, a l l the l a b e l l e d m a t e r i a l was found w i t h i n the s t a c k i n g g e l ( F i g u r e 23). S i m i l a r r e s u l t s were obtained when SDS-PAGE was done u s i n g 32pi l a b e l l e d T r i t o n X-100 e x t r a c t e d p r o t e i n (data not shown). Although i t i s u n l i k e l y that any other p r o t e i n would 90 F i g u r e 21: J ^ P i p h o s p h o r y l a t i o n of but a n o l e x t r a c t e d v e g e t a t i v e c e l l p r e p a r a t i o n . Each assay contained 0.010 ml of 2 M T r i s - C l pH 7.5 b u f f e r , 0.010 ml K 2 H P O 4 , 0.010 mCi 3^Pi and enough but a n o l e x t r a c t e d p r e p a r a t i o n (10 mg/ml) t o g i v e the i n d i c a t e d amount of p r o t e i n i n a t o t a l volume 0.100 ml. The number of determinations f o r each p r o t e i n c o n c e n t r a t i o n was t h r e e . The samples were incubated at room temperature f o r 10 minutes. 0.060 ml P o r t i o n s were counted f o r r a d i o a c t i v i t y as d e s c r i b e d i n the Methods. The l i n e of be s t f i t was drawn u s i n g l i n e a r r e g r e s s i o n . When 0.010 mg p r o t e i n was l a b e l l e d i n the presence of 2 mM /AMP the amount of l a b e l i n c o r p o r a t e d was reduced by 72% (the number of dete r m i n a t i o n s was two). 91 14 -12 -100 M I C R O G R A M P R O T E I N 92 Fi g u r e 22: P i p h o s p h o r y l a t i o n of b u t a n o l e x t r a c t e d c u l m i n a t i o n phase c e l l membrane p r e p a r a t i o n ' . C o n d i t i o n s were as g i v e n f o r F i g u r e 3-1 • When 0.010 mg p r o t e i n was l a b e l l e d i n the presence of 2 mM AMP the amount of l a b e l i n c o r p o r a t e d was reduced by 6 7% (the number of d e t e r m i n a t i o n s was two) 94 F i g u r e 23: Autoradiography of •iZPi l a b e l l e d v e g e t a t i v e c e l l b u t a n o l e x t r a c t . Butanol e x t r a c t e d v e g e t a t i v e c e l l p r e p a r a t i o n (10 mg/ml) was r a d i o a c t i v e l y l a b e l l e d i n 0.100 ml c o n t a i n i n g 200 mM T r i s - C l pH 7.5, 1 mM K 2HP0 4, and 0.010 mCi of 3 2 P i . A f t e r i n c u b a t i o n f o r 10 minutes at room temperature, 0.060 ml of the s o l u t i o n was added t o 0.040 ml of e l e c t r o p h o r e s i s sample b u f f e r c o n t a i n i n g 0.083 M T r i s - C l pH 6.8, g l y c e r o l , d e - i o n i z e d water (1:1:6,v/v/v) and 2% SDS. The sample was b o i l e d f o r 3 minutes and then s u b j e c t e d to e l e c t r o p h o r e s i s i n the Laemmli g e l system as d e s c r i b e d i n the Methods. The g e l was then d r i e d and p l a c e d a g a i n s t r a d i o -g r a p h i c f i l m f o r 3 days b e f o r e being developed. S t a c k i n g g e l 96 be l a b e l l e d with orthophosphate i n the b u t a n o l e x t r a c t s , t h i s p o s s i b i l i t y cannot be completely r u l e d out and as such, these r e s u l t s were not unequivocal i n demonstrating that there was no i n c r e a s e i n APase p r o t e i n with development. 97 SECTION 4 R e l a t i v e l y m i l d methods of a c t i v a t i o n of  a l k a l i n e phosphatase The v e g e t a t i v e c e l l pNPPase a c t i v i t y i n membrane p r e p a r a t i o n s or T r i t o n X-100 e x t r a c t s can be a c t i v a t e d 10-12 f o l d by h e a t i n g i n 5 mM T r i s - C l pH 7.5 (50 C, up to 1 h) or by exhaustive d i a l y s i s (Mohan Das and Weeks, 1980; 1981). The enzyme from c u l m i n a t i n g c e l l s , however, i s onl y m a r g i n a l l y a c t i v a t e d , about 15-20% (Mohan Das and Weeks, 1980; 1981). The a c t i v a t i o n of the enzyme i n v i v o must be through a proc e s s t h a t has a b i o l o g i c a l c o u n t e r p a r t to the p h y s i c a l treatments of h e a t i n g or d i a l y s i s . Some b i o l o g i c a l l y p l a u s i b l e , a l b e i t unproven, methods f o r the a c t i v a t i o n of pNPPase in v i v o were found. a) A c t i v a t i o n by m i l d l y a c i d i c c o n d i t i o n s at 25 C F i g u r e 24 shows t h a t pNPPase c o u l d be a c t i v a t e d at 25 C i n MES-KOH b u f f e r (5 mM, pH 5.5) with k i n e t i c s and extent of s t i m u l a t i o n s i m i l a r t o that f o r a c t i v a t i o n by h e a t i n g (Mohan Das and Weeks, 1980, Mohan Das, 1973). Incubation at pH 6.0 or 6.8 was a l s o s t i m u l a t o r y although the extent of s t i m u l a -t i o n was l e s s pronounced. The p r e v i o u s l y d e s c r i b e d a c t i v a t i o n a t 50 C was r e v e r s i b l e (Mohan Das and Weeks, 1980); however, the a c t i v a t i o n produced at pH 5.5 was not. 98 F i g u r e 24: E f f e c t of pH on the a c t i v a t i o n of the v e g e t a t i v e c e l l pNPPase at 25 C. C e l l membrane p r e p a r a t i o n s (5.0 mg p r o t e i n . m l - 1 ) were incubated i n the f o l l o w i n g b u f f e r s at 25 C: ( 0 ), 5 mM MES-KOH, pH 5.5; ( Q ), 5 mM MES-KOH, pH 6.0; ( A ) 5 mM MES-KOH, pH 6.8. At times i n d i c a t e d 0.05 ml p o r t i o n s were removed and assayed f o r APase a c t i v i t y i n a 1.0 ml f i n a l assay volume. 100 Treatment a t pH 5.5 d i d not a c t i v a t e the pNPPase of e i t h e r d i a l y z e d v e g e t a t i v e c e l l membrane e x t r a c t s or u n d i a l y z i e d c u l m i n a t i n g c e l l e x t r a c t s (Table 8 ) . These r e s u l t s i n d i c a t e t h a t the a c i d a c t i v a t i o n of u n d i a l y z e d v e g e t a t i v e e x t r a c t s might be due to a s i m i l a r mechanism to t h a t i n v o l v e d i n the a c t i v a t i o n by 50 C treatment or prolonged d i a l y s i s i . e . the removal of a low molecular weight i n h i b i t o r from the enzyme. b) A c t i v a t i o n by Concanavalin A A marginal s t i m u l a t i o n of c e r t a i n membrane enzyme a c t i v i t i e s has been accomplished by c o i n c u b a t i o n with l e c t i n s (e.g. Riordan et al_. , 1977). The pNPPase was not s t i m u l a t e d by i n c u b a t i o n i n the presence of co n c a n a v a l i n A a t 25 C ( T r i t o n X-100 e x t r a c t p r o t e i n , 2.0 mg/ml; c o n c a n a v a l i n A, 1 mg/ml; 1 h) and any p o s s i b l e a c t i v a t i o n at 50 C i n 5 mM T r i s - C l pH 7.5 was a l s o not r e a d i l y apparent due to the autonomous a c t i v a t i o n produced by the 50 C treatment. How-ever, i n the presence of 50 mM T r i s - C l pH 7.5, there was a r a p i d d e n a t u r a t i o n of pNPPase a c t i v i t y when the i n c u b a t i o n temperature was r a i s e d to 53 C ( F i g u r e 25). P r e i n c u b a t i o n i n the presence of Concanavalin A at 30 C r e s u l t e d i n a dramatic a c t i v a t i o n when the i n c u b a t i o n temperature was subsequently r a i s e d to 53 C, an e f f e c t t h a t was prevented by the presence Table 8: 101 E f f e c t of a c i d treatment and g l y c o s i d a s e a d d i t i o n on pNPPase a c t i v i t y pNPPase A c t i v i t y Treatment D i a l y z e d T r i t o n X-100 e x t r a c t of v e g e t a t i v e c e l l membranes Undialyzed T r i t o n X-100 e x t r a c t of cu l m i n a t i n g c e l l membranes (nmoles.min •'•.mg p r o t e i n ^) No treatment 105 76 A c i d treatment- I l l 84 G l y c o s i d a s e a d d i t i o n 2 105 88 T r i t o n X-100 e x t r a c t s (2.0 mg.protein ml--'-) were incubated i n 5 mM MES-KOH, pH 5.5 b u f f e r f o r 30 min a t 25°C and 0.05 ml a l i q u o t s were assayed f o r pNPPase a c t i v i t y a t 30 C. 0.020 ml of mixed g l y c o s i d a s e s (5.0 mg.ml--'-) were added t o 0.010 ml of d i a l y z e d T r i t o n X-100 e x t r a c t s i n 0.070 ml of 50 mM MES-KOH b u f f e r pH 5.5. The samples were vortexed f o r 10 sec and immediately assayed f o r pNPPase a c t i v i t y at 30 C. 102 F i g u r e 25: E f f e c t of c o n c a n a v a l i n A on the pNPPase a c t i v i t y of v e g e t a t i v e phase c e l l s . Undialyzed T r i t o n X-100 e x t r a c t s (2.0 mg/ml) were p r e - i n c u b a t e d at 30 C f o r 30 minutes under the f o l l o w i n g c o n d i t i o n s : ( A ) 50 mM T r i s - C l pH 7.5; ( • ) 50 mM T r i s - C l pH 7.5, Concanavalin A (1 mg/ml); ( O ) 50 mM T r i s - C l pH 7.5, Concanavalin A (1 mg/ml), ^ -methyl-D-mannoside (0.1M). A f t e r p r e - i n c u b a t i o n , the samples were t r a n s f e r r e d to 53 C and 0.050 ml p o r t i o n s were assayed f o r pNPPase a c t i v i t y i n the standard assay mixture at the i n d i c a t e d times. 103 ^ 150 -Dl E ' \ C E \ CD C9 0 100 -c T I M E C M I N D 104 of O s^ -methyl-D-mannoside (F i g u r e 25). Concanavalin A p r o b a b l y a c t i v a t e s the v e g e t a t i v e c e l l pNPPase by b i n d i n g to the g l y c a n p o r t i o n of the p r o t e i n s i n c e -methyl-D-mannoside a b o l i s h e d the s t i m u l a t i o n . T h i s b i n d i n g may f a c i l i t a t e the removal of the p u t a t i v e i n h i b i t o r as Concanvalin A had no s t i m u l a t o r y e f f e c t on the pNPPase a c t i v i t y of c u l m i n a t i o n phase c e l l s ( F i g u r e 26), although i t d i d p a r t i a l l y s t a b i l i z e the pronounced i n a c t i v a t i o n of pNPPase that o c c u r r e d under these c o n d i t i o n s . c) A c t i v a t i o n by g l y c o s i d a s e mixture To f u r t h e r e l u c i d a t e the p o s s i b l e r o l e of the g l y c a n m o i e t i e s i n t h i s a c t i v a t i o n , T r i t o n X-100 e x t r a c t s ( i n 5mM T r l s - C l pH 7.5) were t r e a t e d with a mixture of g l y c o s i d a s e s t o attempt removal of the carbohydrate. Unexpectedly, there was an immediate t w o - f o l d i n c r e a s e i n pNPPase a c t i v i t y at optimum c o n c e n t r a t i o n s of g l y c o s i d a s e (Figure 27). In 5 mM MES-KOH, pH 5.5, the immediate a c t i v a t i o n was even more pronounced (Table 9). In view of the immediate i n c r e a s e i n a c t i v i t y , r e g a r d l e s s of g l y c o s i d a s e c o n c e n t r a t i o n (Figure 27), the a c t i v a t i o n i s probably due to b i n d i n g r a t h e r than enzymic a c t i o n , and i s t h e r e f o r e p o s s i b l y analogous to the a c t i v a t i o n by Concanavalin A ( F i g u r e 25). However, pre -i n c u b a t i o n of g l y c o s i d a s e mixture f o r 20 minutes with 0.2 M 105 F i g u r e 26: E f f e c t of Concanavalin A on the pNPPase a c t i v i t y of c u l m i n a t i o n phase c e l l s . U n d i alyzed T r i t o n X-100 e x t r a c t s (2.0 mg/ml) were p r e - i n c u b a t e d at 30 C f o r 30 minutes under the f o l l o w i n g c o n d i t i o n s : ) A ) 50 mM T r i s - C l pH 7.5; ( • ) 50 mM T r i s - C l pH 7.5, Concanavalin A (1 mg/ml); ( O ) 5 0 T r i s - C l pH 7.5, Concanavalin A (1 mg/ml), &V-methyl-D-mannoside (0.1 M) . A f t e r p r e - i n c u b a t i o n , the samples were t r a n s f e r r e d t o 53 C and 0.050 ml p o r t i o n s were assayed f o r pNPPase a c t i v i t y i n the standard assay mixture at the i n d i c a t e d times. 107 F i g u r e 27: E f f e c t of mixed g l y c o s i d a s e s on the pNPPase of v e g e t a t i v e c e l l s . U n d i a l y z e d T r i t o n X-100 e x t r a c t s (2.0 mg p r o t e i n , ml--1-) i n 50 mM MES-KOH, pH 5.5 were added to a mixed g l y c o s i d a s e p r e p a r a t i o n such that the f i n a l c o n c e n t r a t i o n of g l y c o s i d a s e was: ( f | ) , ° - 4 mg.ml-1, ( A ) , 1 mg.ml - 1, ( y ), 1.5 mg ml-"1-. C o n t r o l s ( ® ) contained no g l y c o s i d a s e . The samples were mixed by v o r t e x i n g f o r 10 sec and incubated a t 30 C. 0.05 ml p o r t i o n s were removed at the i n d i c a t e d times and assayed f o r pNPPase a c t i v i t y i n a f i n a l volume of 1.0 ml. Rates of r e a c t i o n were constant d u r i n g the 10 min assay p e r i o d . Under the c o n d i t i o n s used, the mixed g l y c o s i d a s e p r e p a r a t i o n s had no d e t e c t a b l e a l k a l i n e phosphatases a c t i v i t y . 1 0 8 1-o <109 Table 9: E f f e c t of a mixed g l y c o s i d a s e p r e p a r a t i o n on the a c t i v a t i o n of pNPPase G l y c o s i d a s e Concentration-*- pNPPase A c t i v i t y (mg/ml) (per cent c o n t r o l ) 0 100% 0.4 210% 1.0 500% 1.5 500% 80ul of u n d i a l i z d T r i t o n X-100 e x t r a c t s of v e g e t a t i v e c e l l membranes i n 5 mM MES-KOH, pH 5.5 (0.25 mg p r o t e i n ml--'-) were mixed with s u f f i c i e n t g l y c o s i d a s e p r e p a r a t i o n to g i v e the i n d i c a t e d g l y c o s i d a s e concent-r a t i o n s i n a f i n a l volume of 0.1 ml. The samples were vortexed f o r 10 sees and immediately assayed f o r pNPPase a c t i v i t y at 30 C. The experiment r e s u l t s were r e p r o d u c i b l e . 110 c o n c e n t r a t i o n s of one of mannose, g a l a c t o s e , fucose, N - a c e t y l galactosamine or N - a c e t y l glucosamine, f o l l o w e d by the a d d i t i o n of the T r i t o n X-100 e x t r a c t d i d not s i g n i f i c a n t l y i n h i b i t the a c t i v a t i o n . The immediate a c t i v a t i o n produced by g l y c o s i d a s e a d d i t i o n was s i m i l a r t o that obtained by 50 C or pH 5.5 treatment s i n c e d i a l y z e d T r i t o n X-100 e x t r a c t s were not a c t i v a t e d by the g l y c o s i d a s e p r e p a r a t i o n (Table 8). In a d d i t i o n the pNPPase of c u l m i n a t i o n phase c e l l s was o n l y m a r g i n a l l y a c t i v a t e d by g l y c o s i d a s e (Table 8), a r e s u l t that i s c o n s i s t e n t with s l i g h t a c t i v a t i o n by heat or by d i a l y s i s observed p r e v i o u s l y (Mohan Das and Weeks, 1980, 1981). Heat t r e a t e d T r i t o n X-100 s o l u t i o n s of butanol e x t r a c t s i n h i b i t e d the g l y c o s i d a s e a c t i v a t i o n of APase whereas d e g l y c o s y l a t e d membrane p r o t e i n s d i d not (Figure 28), f u r t h e r s u g g e s t i n g t h a t the g l y c o s i d a s e s b i n d to the carbohydrate m o i e t i e s of the APase. I l l F i g u r e 28: E f f e c t of membrane p r o t e i n s and d e g l y c o s y l a t e d membrane p r o t e i n s on the mixed g l y c o s i d a s e mediated a c t i v a t i o n of pNPPase. ( ) > buta n o l e x t r a c t e d p r o t e i n s i n 5 mM MES-KOH, pH 5.5 c o n t a i n i n g 1% T r i t o n X-100 (2.0 mg p r o t e i n . m l - 1 ) were incubated at 85 C f o r 40 min. The i n d i c a t e d amounts were added t o 0.2 ml of mixed g l y c o s i d a s e p r e p a r a t i o n (5.0 mg.ml -!) and the mixture made up to 700 u l t o t a l volume with 5 mM MES-KOH pH 5.5 c o n t a i n i n g 1% T r i t o n X-100. The mixtures were incubated at 30 C f o r 10 min. The experiment was i n i t i a t e d by the a d d i t i o n of 0.1 ml of an u n d i a l i z d T r i t o n X-100 e x t r a c t of v e g e t a t i v e c e l l membranes (2.0 mg protein.ml --'-) . The mixtures were incubated at 30 C f o r the i n d i c a t e d times and 0.1 ml assayed f o r pNPPase a c t i v i t y i n a f i n a l volume of 1.0 ml. ( © ), the bu t a n o l e x t r a c t e d p r o t e i n s were deglycosy-l a t e d by the procedure of Edge et a l . (1981), and then processed as d e s c r i b e d above. The immediate a c t i v a t i o n achieved by the a d d i t i o n of g l y c o s i d a s e i n the presence of 5 mM MES-KOH, pH 5.5 i s p l o t t e d as 100% a c t i v a t i o n . 112 D i s c u s s i o n P a r t of t h i s work has p r o v i d e d s t r o n g evidence that pNPPase and AMPase a c t i v i t i e s of D. discoideum are due to a s i n g l e p r o t e i n . pNPPase and AMPase a c t i v i t i e s migrated i d e n t i c a l l y i n 0.1% SDS p o l y a c r y l a m i d e g e l s . It i s c l e a r from the data t h a t the number of r e s o l v e d a c t i v e p r o t e i n s depends on the storage c o n d i t i o n s of membrane p r e p a r a t i o n s but every component c o u l d be det e c t e d with e i t h e r pNPP or AMP as s u b s t r a t e . Only one major band was d e t e c t e d i n f r e s h l y prepared crude membrane e x t r a c t s . T h i s f i n d i n g was c o n s i s t e n t with the r e s u l t of MacLeod and Loomis (1979) who concluded that there i s a s i n g l e gene product r e s p o n s i b l e f o r APase i n both v e g e t a t i v e and c u l m i n a t i o n phase c e l l s . They found a s i n g l e broad band of APase a c t i v i t y i n T r i t o n X-100 g e l s . However, i n the present study T r i t o n X-100 g e l s were found t o have poor r e s o l v i n g power: For some s t o r e d p r e p a r a t i o n s a s i n g l e broad band of a c t i v i t y c o u l d be r e s o l v e d i n t o three components. The 0.1% SDS g e l system c o n t a i n i n g lowered T r i s - C l c o n c e n t r a t i o n s , compared to the Laemmli (1970) g e l system, lends i t s e l f t o the s e n s i t i v e d e t e c t i o n of APase from D. discoideum and can be recommended f o r use i n f u r t h e r work on t h i s enzyme. The apparent d i s c r e p a n c i e s i n the l i t e r a t u r e over the pH 114 optima of pNPPase and AMPase a c t i v i t i e s have now been r e s o l v e d . Lee et al.. (1975) r e p o r t e d AMPase to have an optimum pH of 7.7 with 0.02 mM AMP as s u b s t r a t e , while pNPPase had a pH optimum of 8.5 but with 1.0 mM pNPP used as s u b s t r a t e . Furthermore, Mohan Das (1983) r e p o r t e d AMPase pH optimum of 8.0 wit h 0.2 mM AMP and pNPPase pH optimum of 9.0 with 1 mM pNPP. G e z e l i u s and Wright (1965) found a pH optimum of 9.0 f o r both pNPPase and AMPase at 10 mM s u b s t r a t e c o n c e n t r a t i o n . Armant and Ruther f o r d (1982) found pNPPase and AMPase o p t i m a l l y a c t i v e at pH 9.5 with 10 mM and 2.5 mM s u b s t r a t e , r e s p e c t i v e l y . Rossomando and C u l t e r (1975) found g r e a t e r AMP h y d r o l y z i n g a c t i v i t y at pH 7.5 than at pH 9.2 when 0.02 mM AMP was used. The d i f f e r e n c e s i n s u b s t r a t e c o n c e n t r a t i o n s used i n these s t u d i e s account f o r the d i f f e r e n c e s i n pH optima obtained. A s t r i k i n g s h i f t of pH optimum to more a l k a l i n e values was obtained with i n c r e a s i n g c o n c e n t r a t i o n s of e i t h e r s u b s t r a t e ( F i g u r e 5). When i d e n t i c a l c o n c e n t r a t i o n s of s u b s t r a t e were used, the pH optima f o r pNPP and AMP h y d r o l y s i s were the same. F u r t h e r -more, the i n f l e c t i o n p o i n t i n the Dixon p l o t was the same wi t h e i t h e r pNPP or AMP as s u b s t r a t e ( F i g u r e s 6, 7). These r e s u l t s a l s o support the concept that the two enzyme a c t i v i t i e s are due to the same p r o t e i n . AMP i n h i b i t s the D. discoideum enzyme at h i g h 115 c o n c e n t r a t i o n s while pNPP does not (Armant and Rutherford, 1982). The involvement of two groups of pKa 8.5 i n AMP b i n d i n g but o n l y one such group i n pNPP b i n d i n g , as determined from the Dixon p l o t s , may e x p l a i n the d i f f e r e n t i a l s e n s i t i v i t y of the enzyme t o s u b s t r a t e i n h i b i t i o n . I t i s p o s s i b l e t hat AMP forms a C l e l a n d - t y p e dead-end complex, ES2# at h i g h e r c o n c e n t r a t i o n s due to the requirement f o r two b i n d i n g s i t e s whereas pNPP does not undergo such non-p r o d u c t i v e b i n d i n g . In t h i s regard, i t i s i n t e r e s t i n g that the AMPase a c t i v i t y of v e g e t a t i v e c e l l s i s more r e s i s t a n t to a c t i v a t i o n and l e s s s t a b l e than the pNPPase (Mohan Das and Weeks, 1984). The i n h i b i t i o n of AMPase a c t i v i t y by 2.5 mM AMP occurs at pH values lower than 8.5 (Figure 5). T h i s r e s u l t suggests t h a t a charge dependent i n t e r a c t i o n between the enzyme and AMP leads to i n h i b i t i o n . Armant and Ru t h e r f o r d (1982), however, d i d o b t a i n i n h i b i t i o n of AMPase a c t i v i t y at pH 9.5, but at AMP c o n c e n t r a t i o n s g r e a t e r 2.5 mM. A mutant with a l t e r e d Km f o r pNPP was used by MacLeod and Loomis (1979) to show that the pNPPase from c u l m i n a t i n g c e l l s was the same enzyme as that present i n v e g a t a t i v e c e l l s . A d e r i v a t i v e of t h i s mutant was r e c r u i t e d to perform an experiment on the r e l a t i o n s h i p between pNPPase and AMPase 116 a c t i v i t i e s . The a l t e r e d Km f o r both AMP and pNPP and the pronounced r e d u c t i o n i n s t a b i l i t y of the two a c t i v i t i e s i n c e l l f r e e e x t r a c t s from the mutant argue s t r o n g l y that both pNPPase and AMPase a c t i v i t i e s are r e s i d e n t on the same p r o t e i n . The i n s t a b i l i t y of the mutant enzyme was not observed i n the e a r l i e r study of MacLeod and Loomis (1979). However, t h i s may be due to the use of a d e r i v a t i v e (HL101) of the o r i g i n a l mutant (HL104) i n the present study. Furthermore, u n l i k e s t r a i n HL104 which was grown a x e n i c a l l y , s t r a i n HL101 had to be grown i n a s s o c i a t i o n with b a c t e r i a . There i s no c o n c l u s i v e evidence that the mutation i s i n the s t r u c t u r a l gene f o r the enzyme p r o t e i n (MacLeod and Loomis, 1979), and i t i s c o n c e i v a b l e that the mutation might a f f e c t a p o s t - t r a n s l a t i o n a l m o d i f i c a t i o n of a number of g l y c o p r o t e i n s i n c l u d i n g d i s t i n c t s p e c i e s of pNPPase and AMPase. However, t h i s p o s s i b i l i t y i s u n l i k e l y s i n c e the mutation i s co-dominant with the w i l d - t y p e a l l e l e i n a d i p l o i d s t r a i n (MacLeod and Loomis, 1979). The f a c t t h at the Km f o r AMP i s not i n c r e a s e d by the same magnitude as the Km f o r pNPP again suggests s u b t l e d i f f e r e n c e s i n the b i n d i n g of the two s u b s t r a t e s . I t i s c l e a r that phospholiphase C d i d not s o l u b i l i z e membrane-bound AMPase a c t i v i t y . The AMPase i n the supernatant can be e n t i r e l y accounted f o r by AMPase present 117 i n the phospholipase C p r e p a r a t i o n . These c o n c l u s i o n s c o n t r a d i c t p r e v i o u s l y p u b l i s h e d s t u d i e s where phospholipase C from the same commercial source was used t o e f f e c t an apparent p h y s i c a l s e p a r a t i o n of pNPPase and /AMPase a c t i v i t i e s (Lee et a_l. , 1975; Rossomando and C u t l e r , 1975). These e a r l i e r r e s u l t s can now be probably e x p l a i n e d by the contamination of the phospholipase C p r e p a r a t i o n by an /AMPase and the f a c t t h a t the membrane bound AMPase a c t i v i t y i s l e s s s t a b l e than the pNPPase a c t i v i t y (Mahan Das and Weeks, 1984). These f i n d i n g s v i t i a t e a major o b j e c t i o n t o the concept of i d e n t i t y of pNPPase and AMPase a c t i v i t i e s . Evidence has been obtained i n the present study that the pNPP and AMP h y d r o l y z i n g a c t i v i t y of D. discoideum membranes i s m e c h a n i s t i c a l l y an a l k a l i n e phosphatase-type enzyme. A major problem i n the i d e n t i f i c a t i o n of a phosphoester h y d r o l y t i c a c t i v i t y as an a l k a l i n e phosphatase or a 5'-n u c l e o t i d a s e has been the d e f i n i t i o n of these terms. I t appears that by a g e n e r a l consensus, a l k a l i n e phosphatases have been regarded as those phosphohydrolases that d i s p l a y a h i g h pH optimum and a broad s u b s t r a t e s p e c i f i c i t y (McComb et a l . , 1979), while the term 5 1 - n u c l e o t i d a s e has been r e s t r i c t e d t o those phosphohydrolases that h y d r o l y z e AMP and a l l i e d compounds at n e a r - n e u t r a l pH (Drummond and Yammamoto, 118 1971). However, E. c o l i " 5 ' - n u c l e o t i d a s e " i s known to h y d r o l y z e ATP, ADP, UDP-glucose and b i s ( p - n i t r o p h e n y l ) phosphate (Neu, 1967), while one yeast " a l k a l i n e phosphate" seems to be s p e c i f i c f o r pNPP among the s u b s t r a t e s t e s t e d (Gorman and Hu, 1969). Rossomando et al. (1983) have r e c e n t l y suggested an e m p i r i c a l method to d i s t i n g u i s h a l k a l i n e phosphatase from 5 ' - n u c l e o t i d a s e enzymes. T h i s method i n v o l v e s the d i f f e r e n t i a l h y d r o l y s i s of t h i o n u c l e o t i d e analogs i n c l a s s i f i c a t i o n of phosphohydrolases. However, the p a r t i a l l y p u r i f i e d D. discoideum enzyme h y d r o l y z e d n e i t h e r dASMP nor AMPS, suggest i n g that the procedure i s not u n i v e r s a l l y a p p l i c a b l e . There i s evidence i n the l i t e r a t u r e to i n d i c a t e that a l k a l i n e phosphatases and 5 ' n u c l e o t i d a s e s d i f f e r fundamentally i n t h e i r r e s p e c t i v e mechanisms of h y d r o l y s i s . While the number of s t u d i e s i s not l a r g e , thus f a r , no e x c e p t i o n s have been r e p o r t e d . The mechanism of a l k a l i n e phosphatase h y d r o l y s i s allows ^-^O/phosphate i s o t o p e exchange (F e r n l e y , 1971). Furthermore, the r e a c t i o n proceeds w i t h net r e t e n t i o n of c o n f i g u r a t i o n about the phosphorous (Jones et aJL. , 1978). These r e s u l t s i n d i c a t e the p a r t i c i p a t i o n of a s t a b l e phosphoryl enzyme intermediate i n 119 a r e a c t i o n t h a t occurs v i a a double-displacement mechanism. Phosphoryl enzyme formation has been d i r e c t l y demonstrated from the s u b s t r a t e e s t e r s (Engstrom, 1961). A v a r i e t y of low-molecular weight a l c o h o l s can compete with water i n the breakdown of the phosphoryl enzyme int e r m e d i a t e t o generate o r g a n i c phosphate compounds (McComb et. a l . , 1979). In c o n t r a s t , snake venom 5 ' - n u c l e o t i d a s e c a t a l y z e s h y d r o l y s i s of i ft AMP i n E2 0 without i s o t o p e exchange, a r e s u l t t h a t excludes the involvement of a s t a b l e phosphoryl enzyme i n t e r m e d i a t e (Koshland and Springhorn, 1956). More recent r e s u l t s show that h y d r o l y s i s of AMP by snake venom 5' - n u c l e o t i d a s e leads t o i n v e r s i o n of c h i r a l i t y of the phosphorous c o n s i s t e n t with a s i n g l e - d i s p l a c e m e n t mechanism of h y d r o l y s i s ( T s a i , 1980). Moreover, b u l l t e s t e s 5'-nucleo-t i d a s e does not c a t a l y z e t r a n s p h o s p h o r y l a t i o n r e a c t i o n s (Morton, 1953), aga i n i n d i c a t i n g a s i n g l e - displacement mechanism. T r i s was shown to be a c o - s u b s t r a t e f o r the D. discoideum enzyme (Figure 13), suggesting a p o s s i b l e f u n c t i o n as a phosphoryl group a c c e p t o r . T r a n s p h o s p h o r y l a t i o n was d i r e c t l y demonstrated with e i t h e r pNPP or AMP as s u b s t r a t e and with e i t h e r T r i s or ethanolamine as acceptor, i n d i c a t i n g t h a t the enzyme had an a l k a l i n e phosphatase-type mechanism. The f i n d i n g t h a t AMP was a phosphoryl group donor strengthens 120 the c o n c l u s i o n that both pNPP and AMP are h y d r o l y z e d by a s i n g l e enzyme. The pH dependence of t r a n s p h o s p h o r y l a t i o n o b t a i n e d with the D. discoideum enzyme i s almost i d e n t i c a l t o t h a t found f o r the E. c o l i APase with pNPP and T r i s (Roig et a l . , 1982). However, Neuman (1969) found a t r a n s p h o s p h o r y l a t i o n maximum at pH 7.8 and n u l l at pH values g r e a t e r than 8.4 f o r the E. c o l i enzyme. The c o n t r a d i c t i o n between the r e s u l t s of Roig et a l . (1982) and Neumann (1969) may be e x p l a i n e d by the f a c t that while the former used pNPP as s u b s t r a t e , the l a t t e r used S - s u b s t i t u t e d phosphorothioate e s t e r s . I f t h i s i s so, the c o n t e n t i o n that t r a n s p h o s p h o r y l a t i o n i s independent of the type of s u b s t r a t e used (McComb e_t a l . , 1979) needs to be r e - e v a l u a t e d . The occurence of a double-displacement mechanism i n the a c t i o n of D. discoideum APase was confirmed u s i n g stopped-flow spectrophotometry. A h i g h - r a t e r e a c t i o n s i g n i f y i n g the b u r s t phase of p - n i t r o p h e n o l p r o d u c t i o n f o l l o w e d by a low-rate r e a c t i o n c o r r e s p o n d i n g to subsequent turnover were o b t a i n e d . In a d d i t i o n , a l a g phase was observed, but i t s s i g n i f i c a n c e i s q u e s t i o n a b l e . It was a l s o seen with the E. c o l i enzyme although i t has not been r e p o r t e d p r e v i o u s l y . As such, the l a g phase may r e p r e s e n t an i n s t r u m e n t a l a r t e f a c t a r i s i n g perhaps from incomplete mixing of enzyme and pNPP s o l u t i o n s d u r i n g the 30 msec dead-time b e f o r e instrument data c o l l e c t i o n . 121 Thus, the D. discoideum enzyme has been i d e n t i f i e d as an a l k a l i n e phosphatase-type enzyme, although, u n l i k e other enzymes of t h i s type i t has a more r e s t r i c t e d s u b s t r a t e s p e c i f i c i t y range. The f a i l u r e of the enzyme to c a t a l y z e the p r e d i c t e d h y d r o l y s i s of dAMP (Rossomando et. a l . , 1983) may perhaps be e x p l a i n e d by i t s r e s t r i c t e d s u b s t r a t e s p e c i f i c i t y . However, the p r o d u c t i o n of o r g a n i c phosphate d u r i n g the course of r e a c t i o n i n a s u i t a b l e b u f f e r i s a more r e l i a b l e m e c h a n i s t i c a l l y dependent probe t o d i s t i n g u i s h between a l k a l i n e phophatase and 5 ' - n u c l e o t i d a s e a c t i v i t i e s . As such, i t can be recommended f o r purposes of c l a s s i f i c a t i o n of these two enzyme types. The pH 8.5 value f o r the i n f l e c t i o n p o i n t i n the Dixon p l o t s i n d i c a t e s the p o s s i b l e involvement of a t h i o l or amine group i n the b i n d i n g of s u b s t r a t e t o the enzyme. The e x i s t e n c e of an e s s e n t i a l t h i o l group i s suggested by the ready i n a c t i v a t i o n of the enzyme by p-hydroxymercuribenzoate (Table 1 ) . Other authors expressed doubt over the presence of e s s e n t i a l t h i o l groups i n APases from other sources because h i g h c o n c e n t r a t i o n s of i n h i b i t o r s were r e q u i r e d f o r i n h i b i t i o n of a c t i v i t y (Debruyne, 1982; Van B e l l e , 1972; Fishman and Ghosh, 1967; Lasdunski and Q u e l l e t , 1962). In 122 c o n t r a s t , the D. discoideum enzyme i s extremely s e n s i t i v e to p-hydroxymercuribenzoate, although s i g n i f i c a n t i n h i b i t i o n was not observed with i o d o a c e t a t e , another s u l f h y d r y l r e a c t i v e agent. Thus the D. discoideum a l k a l i n e phosphatase may be fundamentally d i f f e r e n t i n s t r u c t u r e from other APases i n t h a t i t may possess a t h i o l group r e q u i r e d f o r a c t i v i t y . The c o r r e l a t i o n between the i n h i b i t i o n by t h i o l reagents and the i n f l e c t i o n i n the Dixon p l o t s must be t r e a t e d with c a u t i o n , however. Debruyne (1982) obtained an i n f l e c t i o n i n the Dixon p l o t f o r hen's egg yolk APase with pNPP at pH 8.22 but found the enzyme r e l a t i v e l y i n s e n s i t i v e t o e i t h e r p-hydroxymercur-ibenzoate or N-ethylmaleimide. Debruyne (1982) found a d d i t i o n a l i n f l e c t i o n s at pH 9.57 and 10.5. In an e a r l i e r study, Morton (1958) observed a s i n g l e i n f l e c t i o n at about pH 9.2 with phenylphosphate f o r c a l f i n t e s t i n a l mucosa APase. Krishnaswamy and Kenkare (1969), i n a comprehensive study of the k i n e t e i c parameters of E. c o l i APase, found a n e u t r a l a c i d group of pH of 9.08, a t 30 °C, i n v o l v e d i n enzyme-s u b s t r a t e b i n d i n g . T h i s group was t e n t a t i v e l y i d e n t i f i e d as a zinc-water c o r r d i n a t i o n complex. Mohan Das and Weeks (1980; 1981; 1984) have suggested t h a t the developmental i n c r e a s e i n APase a c t i v i t y c o u l d be accounted f o r by removal of an i n h i b i t o r from the v e g e t a t i v e 123 c e l l APase. T h i s model i m p l i e s that there i s no i n c r e a s e i n APase p r o t e i n to account f o r the i n c r e a s e i n APase a c t i v i t y d u r i n g the t e r m i n a l stages of development. An attempt was made to immunoquantitate APase p r o t e i n i n e x t r a c t s prepared from d i f f e r e n t developmental stages i n order to t e s t t h i s h y p o t h e s i s . Although the Western b l o t t i n g technique suggested that there was no i n c r e a s e i n the major band that was d e t e c t e d with development (Figure 20), there were some s e r i o u s problems encountered i n these s t u d i e s . The antiserum d i d not i n a c t i v a t e or immunoprecipitate the APase from T r i t o n X-100 e x t r a c t s . As such, there i s a doubt as to the s p e c i f i c i t y of the antiserum. I t i s a l s o p o s s i b l e that the immunoquantitation of APase leads to an u n d e r e s t i m a t i o n of the amount of enzyme i n c u l m i n a t i o n phase c e l l s . The c u l m i n a t i o n phase enzyme may l a c k c e r t a i n a n t i g e n i c components present on the v e g e t a t i v e c e l l enzyme. The enzyme might be m o d i f i e d d u r i n g development, l o s i n g a n t i g e n i c determinants that are r e c o g n i z e d by a n t i b o d i e s r a i s e d t o the v e g e t a t i v e c e l l p r o t e i n . Although MacLeod and Loomis (1979) presented good evidence i n favour of the v e g e t a t i v e c e l l and c u l m i n a t i n g phase enzymes being s p e c i f i e d by one gene, there i s some 124 evidence f o r m o d i f i c a t i o n of the enzyme du r i n g development. Crean and Rossomando (1977) r e p o r t e d t h a t about 50% of the c u l m i n a t i o n phase enzyme d i d not b i n d t o conca n a v a l i n A-Sepharose columns while more than 90% of the v e g e t a t i v e c e l l enzyme was bound. T h i s may i n d i c a t e a l o s s of gl y c a n m o i e t i e s d u r i n g development. Furthermore, Mohan Das (1983) found t h a t the c u l m i n a t i o n phase enzyme was more s e n s i t i v e to SDS than the v e g e t a t i v e c e l l APase. T h i s may a l s o be due to a lower l e v e l of g l y c o s y l a t i o n i n the c u l m i n a t i o n phase enzyme, as i t has been shown that n e o - g l y c o p r o t e i n s ( i . e . p r o t e i n s c h e m i c a l l y conjugated t o carbohydrate) are more r e s i s t a n t to d e n a t u r a t i o n by SDS ( M a r s h a l l , 1978). The APase from c u l m i n a t i o n s t r u c t u r e s ran with a s l i g h t l y g r e a t e r m o b i l i t y than the v e g e t a t i v e c e l l enzyme on n a t i v e p o l y a c r y l a m i d e g e l s ( F i g u r e 3), and the immunoreactive component from c u l m i n a t i o n phase s t r u c t u r e s a l s o had a g r e a t e r m o b i l i t y ( F i g u r e s 19 and 20). T h i s f i n d i n g can be e x p l a i n e d i n terms of a lower l e v e l of g l y c o s y l a t i o n i n the c u l m i n a t i o n phase enzyme. It has been shown r e c e n t l y t h a t some D. discoideum lysosomal g l y c o p r o t e i n s l o s e mannosyl phosphate immunode-terminants d u r i n g development (Knecht et a_l. 1985). Although 125 i t i s not known i f t h i s i s a g e n e r a l phenomenon a f f e c t i n g o t h e r g l y c o p r o t e i n s as w e l l , such a p o s s i b i l i t y c ould be r e s p o n s i b l e f o r the apparent decrease i n APase p r o t e i n from c u l m i n a t i n g s t r u c t u r e s i f c e r t a i n a n t i g e n i c components are l o s t . However, MacLeod and Loomis (1979) found the i s o l e c t r i c p o i n t s of APase from v e g e t a t i v e and c u l m i n a t i o n phase c e l l s t o be v i r t u a l l y i d e n t i c a l . As such, i t i s u n l i k e l y t h a t there are changes i n the s u l f a t i o n or p h o s p h o r y l a t i o n l e v e l s of APase g l y c a n d u r i n g development. The second approach used to attempt q u a n t i f i c a t i o n of APase d u r i n g development was the use of ^^Pi to s p e c i f i -c a l l y l a b e l the APase p r o t e i n . T h i s technique a l s o suggested t h a t there was no i n c r e a s e i n APase p r o t e i n with development. However, attempts to demonstrate A P a s e - s p e c i f i c phosphoryla-t i o n by autoradiography of l a b e l l e d e x t r a c t s e l e c t r o p h e r e s e d i n p o l y a c r y l a m i d e g e l s were f u t i l e . L a b e l l e d m a t e r i a l was observed i n the s t a c k i n g g e l but not i n the running g e l . The f a i l u r e t o observe a unique phosphorylated band i n SDS-PAGE g e l s compels c a u t i o n i n the i n t e r p r e t a t i o n of these r e s u l t s . G e z e l i u s and Wright (1965) and Mohan Das (1983) found t h a t orthophosphate was a r e v e r s i b l e i n h i b i t o r of pNPPase a c t i v i t y , whereas Armant and Rutherford (1979; 1981) found 126 t h a t phosphate i n h i b i t i o n was i r r e v e r s i b l e . Phosphate i n h i b i t i o n was found to be dependent on the i o n i c s t r e n g t h of the b u f f e r , b e i n g c o m p e t i t i v e at low i o n i c s t r e n g t h (Figure 15) but i r r e v e r s i b l e at h i g h i o n i c s t r e n g t h (Table 6 ) . A l k a l i n e phosphatase from D. discoideum (Armant and Rut h e r f o r d , 1981) and E. c o l i (Wilson et a l . , 1964; Roig et a l l , 1982) have been shown to be i n h i b i t e d by ammonia. Simpson and V a l l e e (1969) p o s t u l a t e d t h a t the Z n + 2 c o f a c t o r c o u l d be e x t r a c t e d i n the c r y s t a l l i z a t i o n process of E. c o l i APase by ammonium s u l f a t e i n the form of complex Zn ( N H 3 ) + 2 . Roig et a l . , (1982) demonstrated that i t was the NH 3 s p e c i e s and not NH 4 + that was r e s p o n s i b l e f o r the i n h i b i t i o n of h y d r o l y s i s observed at a l k a l i n e pH. Roig et a l . , a l s o observed i n h i b i t i o n of E. c o l i APase by T r i s at c o n c e n t r a t i o n s g r e a t e r than about 1.0 M at pH 8.1 but not at pH 5.75 and suggested that i n h i b i t i o n by T r i s was through the same mechanism as NH3 i n h i b i t i o n v i z Zn ( R N H 3 ) + 2 complex formation. The D. discoideum enzyme was a l s o i n h i b i t e d by h i g h c o n c e n t r a t i o n s of T r i s i n a pH dependent f a s h i o n ( F i g u r e 16) . I n t e r e s t i n g l y , low c o n c e n t r a t i o n s of T r i s a l s o i n h i b i t e d the enzyme as long as the i o n i c s t r e n g t h was h i g h (Table 6), but h i g h i o n i c s t r e n g t h by i t s e l f was not s u f f i c i e n t t o i n h i b i t the enzyme (Table 6 ) . I t i s now proposed that the i n h i b i t i o n by T r i s r e q u i r e s the exposure of 127 the enzyme to high io n i c strength solutions. It should be noted that, l i k e T r i s , orthophosphate i n h i b i t s the enzyme at high ionic strength and high pH (Figures 16 and 17). Thus i t i s l i k e l y that in solutions of high ionic strength and high pH, a l k a l i n e phosphatase assumes a conformation that makes the active s i t e accessible to T r i s and orthophosphate. Support for t h i s proposal w i l l have to await dir e c t conformational studies of a l k a l i n e phosphatases under appropriate conditons. Previous reports described the a c t i v a t i o n of D. discoideum vegetative c e l l APase by 50 C treatment (in 5mM T r i s - C l pH 7.4) or by d i a l y s i s (Mohan Das and Weeks, 1980; 1981) and evidence suggests that these treatments remove an otherwise t i g h t l y bound i n h i b i t o r (Mohan Das and Weeks, 1981). Two additional ways are now described i n which pNPPase can be activated. The enzyme i s markedly activated at pH 5.5 (Figure 24). The a v a i l a b l e evidence suggests that the pH 5.5 treatment also removed i n h i b i t o r (Table 8) and indicates a possible charge dependent interaction between enzyme and i n h i b i t o r involving a functional group with a pKa in the range of 6.0 -6.5. 128 pNPPase can a l s o be a c t i v a t e d by Concanavalin A and by a p r e p a r a t i o n of mixed g l y c o s i d a s e s ( F i g u r e 25, 27), although o t h e r l e c t i n s t r i e d produced no a c t i v a t i o n . The g l y c o s i d a s e a c t i v a t i o n would appear to be due to b i n d i n g r a t h e r than c a t a l y s i s . A n o n c a t a l y t i c b i o l o g i c a l r o l e f o r g l y c o s i d a s e s has a l s o been suggested by the work of Rauala et a l . (1981) who showed that g l y c o s i d a s e s had s i m i l a r f u n c t i o n s to l e c t i n s i n promoting the spreading of f i b r o b l a s t s on p r o t e i n - c o a t e d substratum. I t i s not known at the present time i f the a c t i v a t i o n by pH 5.5 treatment or by carbohydrate b i n d i n g p r o t e i n a d d i t i o n i s r e l e v a n t to the i n c r e a s e i n APase a c t i v i t y t h a t i s observed d u r i n g d i f f e r e n t i a t i o n . The two modes of a c t i v a t i o n are probably m e c h a n i s t i c a l l y d i s s i m i l a r , although the a v a i l a b l e evidence would i m p l i c a t e the r e l e a s e of the p u t a t i v e i n h i b i t o r under both c o n d i t i o n s . It i s noteworthy, however, t h a t the requirement f o r p r o t e i n s y n t h e s i s f o r the developmental accumulation of APase (Loomis, 1969) would be e x p l a i n e d i f a newly s y n t h e s i z e d carbohydrate b i n d i n g p r o t e i n was r e s p o n s i b l e f o r the unmasking of e x i s t i n g a c t i v i t y d u r i n g d i f f e r e n t i a t i o n (Mohan Das and Weeks, 1980; 1981). T h i s somewhat s p e c u l a t i v e s u g g e s t i o n that a carbohydrate b i n d i n g 129 p r o t e i n might r e g u l a t e the developmental a c t i v i t y of a g l y c o p r o t e i n enzyme adds an a d d i t i o n a l p o s s i b l e f u n c t i o n f o r the carbohydrate m o i e t i e s of g l y c o p r o t e i n s , an area of b i o c h e m i s t r y that i s o n l y p o o r l y understood ( M o n t r e u i l , 1982). 130 REFERENCES 1. Ames, B.N. (1966) In Methods i n Enzymology. V o l . 8, pp. 115-118. Academic Press, New York. 2. Armant, D.R. and C.L. Rutherf o r d (1982) P r o p e r t i e s of a 5'-AMP s p e c i f i c n u c l e o t i d a s e which accumulates i n one c e l l type d u r i n g development of D i c t o s t e l i u m discoideum. Arch. Biochem. Biopys. 216: 485-494. 3. Armant, D.R. and C.L. Rutherf o r d (1981) C o p u r i f i c a t i o n of a l k a l i n e phosphatase and 5'-AMP s p e c i f i c n u c l e o t i d a s e i n D i c t y o s t e l i u m discoideum. J . B i o l . Chem. 256: 12710-12718. 4. Armant, D.R. and C.L. Rut h e r f o r d (1979) 5'-AMP n u c l e o t i d a s e i s l o c a l i z e d i n the area of c e l l - c e l l c o n t a c t of prespore and p r e s t a l k r e g i o n s d u r i n g c u l m i n a t i o n o f D i c t y o s t e l i u m discoideum. Mechan. Aging Develop. 10: 199-217. 5. Armant, D.R., D.A. S t e t l e r and C.L. Rutherf o r d (1980) C e l l s u r f a c e l o c a l i z a t i o n of 5'-AMP n u c l e o t i d a s e i n p r e -s t a l k c e l l s of D i c t y o s t e l i u m discoideum. J . C e l l S c i . 45: 119-129. 131 6. Atryzek, V. (1976) D i s s o c i a t i o n of developing slime mold c e l l s does not i n h i b i t the developmentally r e g u l a t e d r i s e i n a l k a l i n e phosphatase a c t i v i t y . J . B a c t e r i o l . 126; 1005-1008. 7. Bale, J.R., C.Y. Huang and P.B. Chock (1980) T r a n s i e n t k i n e t i c a n a l y s i s of the c a t a l y t i c c y c l e of a l k a l i n e phosphatase. J . B i o l . Chem. 255; 8431-8436. 8. Bloch, N. and M.J. S c h l e s i n g e r (1974) K i n e t i c s of s u b s t r a t e h y d r o l y s i s by molecular v a r i a n t s of E s c h e r i c h i a c o l i a l k a l i n phosphatase . J . B i o l . Chem. 249: 1760-1768. 9. Bonner, J.T. (1947) Evidence f o r the formation of c e l l aggregates by chemotaxis i n the development of the slime mold D i c t y o s t e l i u m discoideum. J . Expt. Z o o l . 106; 1-26 10. Bonner, J.T. (1967) The c e l l u l a r slime molds. P r i n c e t o n U n i v e r s i t y Press, P r i n c e t o n , New J e r s e y . 11. Bonner, J.T., A.D. Chiquoine and M.Q. K o l d e r i e (1955) A H i s t o c h e m i c a l study of d i f f e r e n t i a t i o n i n the c e l l u l a r s lime molds. J . Exp. Z o o l . 130; 133-158. 12. Debruyne, I. (1982) Hen's egg yolk a l k a l i n e phosphatase: General c h a r a c t e r i z a t i o n and k i n e t i c study with i n h i b i t o r s . I n t . J . Biochem. 14: 519-528. 132 13. Dixon M. and E.C. Webb (1984) Enzymes, pp. 135-145. Academic Press Inc., New York. 14 Drummond G.I. and M. Yammamoto (1971) N u c l e o t i d e Phosphomonesterases i n P.D. Boyer, ed. The Enzymes. Academic Press Inc, New York. 15. Edge, A.S.B., C.R. Faltynek, L. Hof, L.E. R e i c h a r t , J r . , and P. Weber (1981 D e g l y c o s y l a t i o n of g l y c o p r o t e i n s by T r i f l o u r o m e t h a n s u l f o n i c a c i d . A n a l . Biochem. 18: 131-137. 16. Engstrom, L. (1961) F u r t h e r s t u d i e s on the i n c o r p o r a t i o n of i n o r g a n i c phosphate i n t o c a l f - i n t e s t i n a l a l k a l i n e phosphatase. Biochem. Biophys. A c t a . 54: 179-185. 17. Engstrom, L. (1961) S t u d i e s on c a l f - i n t e s t i a n l a l k a l i n e phosphatase I I . I n c o r p o r a t i o n of i n o r g a n i c phosphate i n t o a h i g h l y p u r i f i e d enzyme p r e p a r a t i o n . . Biochem. Biophys. A c t a . 52: 49-59. 18. F e r n l e y , H.N. (1971) Mammalian a l k a l i n e phosphatases. In P.D. Boyer, ed., The Enzymes Academic Press, N.Y. 19. Fishman, L. (1974) Acrylamide d i s c g e l e l e d t r o p h o r e s i s of a l k a l i n e phosphatase of human t i s s u e s , serum and a s c i t e s f l u i d u s i n g T r i t o n X-100 i n the sample and g e l matrix. Biochem Med. 9: 309-315. 20. Fishman, W.H. and N.K. Ghosh (1967) Influence of reagents r e a c t i n g with metal, t h i o l . a n d amino s i t e s on c a t a l y t i c a c t i v i t y and L - P h e n y l a l a n i n e i n h i b i t i o n with r a t i n t e s t i n a l a l k a l i n e phosphatase. Biochemical 105; 1163-1170. 21. Ghosh, N.K. and L. Fishman (1968) P u r i f i c a t i o n and p r o p e r t i i e s of molecular weight v a r i a n t s of homan p l a c e n t a l a l k a l i n e phosphatase. Biochem. J . 108; 779-792. 22. G i l k e s , N.R. and G. Weeks (1977) The p u r i f i c a t i o n and c h a r a c t e r i z a t i o n of D i c t y o s t e l l i u m discoideum plasma membranes. Biochem. Biophys. A c t a . 464; 142-156. 23. G e z e l i u s , K. and B.E. Wright (1965) A l k a l i n e phosphatase i n D i c t y o s t e l l i u m discoideum. J . Gen. M i c r o b i o l . 38; 309-327. 24. Green, A.A. and P.C. Newell (1974) The i s o l a t i o n and s u b - f r a c t i o n a t i o n of plasma membrane from c e l l u l a r slime mould D i c t y o s t e l l i u m discoideum. Biochem. J . 140: 313-322. 25. Hamilton, I.D. and W.K. C h i a (1975) Enzyme a c t i v i t y changes duri n g c y c l i c AMP-induced s t a l k c e l l d i f f e r e n t i a t i o n i n P4, a v a r i a n t of D i c t y o s t e l l i u m discoideum. J . Gen. M i c r o b i o l . 91: 295-306. 134 26. Hancock, K. and V. Tsang (1983) I n d i a ink s t a i n i n g of p r o t e i n s on n i t r o c e l l u l o s e paper. A n a l y t . Biochem. 133; 157-162. 27. Himmeloch, S.R. (1971) Chromatography on ion-exchange adsorbants. Methods Enzymol. 22.: 273-286. 28. Holloway, P.W. (1973) A simple procedure f o r removal of T r i t o n X-100 from p r o t e i n samples. A n a l . Biochem 53; 304-308. 29. Jones, S.R., L.A. Kindman and J.R. Knowles (1978) Stereochemistry of phosphoryl group t r a n s f e r u s i n g a c h i r a l ((16)0,(17)0,(18)0) s t e r e o c h e m i c a l course of a l k a l i n e phosphatase. Nature (London) 2 75; 564-565. 30. Knecht, D.A., E.D. Green, W.F. Loomis, R.L. Dimond (1985) Developmental changes i n the m o d i f i c a t i o n of lysosomal enzymes i n D i c t y o s t e l l i u m discoideum. Devel. B i o l . 107: 490-502. 31. Koshland, D.E., J r . and S.S. Springhorn (1956) Mechanism of a c t i o n of 5 ' - n u c l e o t i d a s e . J . B i o l . Chem 221: 469-476. 32. Krishnaswamy, M. and U.W. Kenkare (1970) The e f f e c t of pH, temperature and o r g a n i c s o l v e n t s on the k i n e t i c p a r a m e t e r s of E s c h e r i c h i a c o l i a l k a l i n e phosphatase. J . B i o l . Chem. 245: 3956-3963. 33 Krivenek, J.O. (1956) A l k a l i n e phosphatse a c t i v i t y i n the developing slime mold, D i c t y o s t e l i u m discoideum, Raper. J . Exp. Z o o l . 133; 459-480. 34. Krivanek, J.O. and R.C. Krivanek (1958) The h i s t o c h e m i c a l l o c a l i z a t i o n of c e r t a i n b i o c h e m i c a l in t e r m e d i a t e s and enzymes i n the developing slime mold, D i c t y o s t e l i u m discoideum, Raper. J . Exp. Z o o l . 137; 89-115. 35. Laemmli, U.K. (1970) Cleavage of s t r u c t u r a l p r o t e i n s d u r i n g the assembly of the head of bacteriophage T4. Nature (London) 227; 680-685. 36. Lazdunski, M. and L. Q u e l l e t (1962) I n h i b i t i o n de l a phosphatase a l c a l i n e i n t e s t i n a l e . Can. J . Biochem. P h y s i o l . 40_: 1619-1639. 37. Lee, A., K. Chance, C. Weeks and G. Weeks (1975) S t u d i e s on the a l k a l i n e phosphatase and 5 1 - n u c l e o t i d a s e of D i c t y o s t e l i u m discoideum. Arch Biochem. Biophys. 171: 407-417. 38 Loomis, W.F. (1969) Developmental r e g u l a t i o n of a l k a l i n e phosphatase i n D i c t y o s t e l i u m discoideum. J . B a c t e r i d . 100: 417-422. 39. Loomis, W.F. (1975a) D i c t y o s t e l i u m discoideum. A developmental system. Academic Press, New York. 40. Loomis, W.F. (1975b) Stage s p e c i f i c isozymes of Dictyostelium discoideum. In. Proc. Int. Cong Isozymes 3rd. Academic Press, New York. 41. Loomis, W.F.; ed. (1982) The development of Dictyostelium discoideum. Academic Press, New York. 42. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall (1951) Protein measurements with a F o l i n phenol reagent. J. B i o l . Chem. 193: 265-275. 43. MacComb, R.B., G.N. Bowers, J r . and S. Posen (1979) Structural features and reaction Mechanisms. In Alkaline Phosphatase Chapt 5 and 6, pp. 189-228, pp. 229-287. Plenum Press, New York. 44. MacLeod, C.L. and W.F. Loomis (1979) Biochemical and genetic analysis of a mutant with altered a l k a l i n e phosphatase a c t i v i t y in Dictyostelium discoideum. Develop. Genet. Is 109-121. 45. MacMahon, D., M. M i l l e r and S. Long (1977) The involvement of the plasma membrane i n the development of Dictyostelium discoideum I. P u r i f i c a t i o n of the palsma membrane. Biochem. Biophys. Acta. 46 5: 224-241. 137 46. MacMahon, D., S. Hoffman, W. Fry and C. West (1975) The involvement of the plasma membrane in the development of Dictyostelium discoideum, in D. MacMahon and C F . Fox, eds., Pattern formation and gene regulation i n development. W.A. Benjamin, Inc. Palo Alto. 47. Mohan Das, D.V. (1983) Ph.D. Thesis. Developmental regulation of al k a l i n e phosphatase in Dictyostelium  discoideum. Department of Microbiology, University of B r i t i s h Columbia. 48. Mohan Das, D.V., and G. Weeks (1980) Reversible heat ac t i v a t i o n of al k a l i n e phosphatase of Dictyostelium  discoideum and i t s developmental implication. Nature 288: 166-167. 49. Mohan Das, D.V., and G. Weeks (1981) The i n h i b i t i o n of Dictyostelium discoideum a l k a l i n e phosphatase by a low molecular weight factor and i t s implication for the developmental regulation of the enzyme. FEBS l e t t s . 130: 249-252. 50. Mohan Das, D.V. and G. Weeks (1984) Studies on the unmasking of membrane bound al k a l i n e phosphatase during the d i f f e r e n t i a t i o n of Dictyostelium discoideum. Can. J. Biochem. 62: 970-974. 138 51. Morrissey, J.H. (1981)Silver sta i n for proteins i n polyacrylamide gels: A modified procedure with enhanced uniform s e n s i t i v i t y . Anal. Biochem. 117: 307-310. 52. Montreuil, J . (1982) In, Neuberger, A. and L.L.M. Van Deenen, eds., Comprehensive Biochemistry 19B: 1-88. Elsevi e r S c i e n t i f i c Publishing Co. 53. Morton, R.K. (1953) Transferase a c t i v i t y of hydr o l y t i c enzymes. Nature 172: 65-68. 54. Murray, A.W. and M.R. Atkinson (1968) Adenosine 5'-phosphorothioate: A nucleotide analog that i s a substrate competitive i n h i b i t o r or regulator of some enzymes that interact with adenosine 5'-phosphate. Biochemistry, lj 4023-4029. 55. Neu, H.C. (1967) The 5'-nucleotidase of Escherichia c o l i 1 P u r i f i c a t i o n and properties. J. B i o l . Chem. 242: 3896-3906. 56. Newman, H. (1969) Phosphoryl transfer from S-substituted monoesters of phosphothioic acid to various acceptor catalyzed by E. c o l i . Eur. J . Biochem. 8: 164-173. 57. Olive, L.S. (1975) The Mycetezoans. Academic Press, New York. 139 58. Parish, R.W. and C. P e l l i (1974) Alk a l i n e phosphotase of Dictyostelium discoideum; C e l l surface location and colchicine e f f e c t on i n t e r n a l i z a t i o n during Phagocytosis. FEBS Lett. 48: 293-296. 59. Quiviger, B., J.C. Bemichou and A. Ryter (1980) Comparative cytochemcal l o c a l i z a t i o n of a l k a l i n e and acid phosphatases during starvation and d i f f e r e n t i a t i o n of Dictyostelium discoideum. B i o l . C e l l u l a i r e 32: 241-250. 60. Quiviger, B., C. de C h a s t e l l i e r and A. Ryter (1978) Cytochemical demonstration of a l k a l i n e phosphatase in the c o n t r a c t i l e vacuole of Dictyostelium discoideum. J. Ultrastruc. Res. 62_: 228-236. 61. Raper, K.B. (1935) Dictyostelium discoideum, a new species of slime mold from decaying forest leaves. J. Agr. Res. 50: 135-147. 62. Raper, K.B. (1973) Acrasiomycetes, In G.C. Ashworth, F.K. Sparrow and A.S. Sussman, Eds., The Fungi: An advanced t r e a t i s e , vol. 1VB: 9-36. Academic Press, New York. 140 63. Rauvala, H., W.G. Carter and S.I. Hakomori (1981) Studies on c e l l adhesion and recognition.1. Extent and s p e c i f i c i t y of c e l l adhesion triggered by carbohydrate reactive proteins (glycosidases and l e c t i n s ) and by fibr o n e c t i n . J. C e l l . B i o l . 88: 127-137. 64. Reid, T.W. and I.B. Wilson (1971) E. c o l i a l k a l i n e phosphatase. In P.D. Boyer, ed., The Enzymes, Academic Press, N.Y. 65. Rickenberg, H.V., C. Tihon and O. Guzel (1977) The effect of pulses of 3', 5' c y c l i c monophosphate on enzyme formation i n non-aggregated amoebae of Dictyostelium discoideum, i n P. Cappucineli and J.M. Ashworth, eds.; Developments in c e l l biology, Elsevier/North Holland. 66. Riordan, J.R. and M. Slavik (1974) Interaction of l e c t i n s with membrane glycoproteins: E f f e c t s of concanavalin A on 5'-nucleotidase. Biochem. Biophys. Acta 373: 356-360. 67. Roig, M.G., F.J. Burguillo, A. Del Arco, J.L. Usero, C. Izquierdo and M.A. Herraez (1982) Kinetic studies of the trans-phosphoylation catalyzed by a l k a l i n e phosphatase from E. c o l i . Int. J. Biochem. 14: 655-666. 141 68. Rossomando, E.F., G.A. Cordis and G.D. Markham (1983) 5'-Deoxy-5'-thioanalogs of adenosine and inosine 5'-monophosphate: Studies with 5'-nucleotidase and alkaline phosphotase. Arch. Biochem. Biophys. 220; 71-78. 69. Rossomando, E.F. and L.S. Cutler (1975) L o c a l i z a t i o n of adenylate cyclase in Dictyostelium discoideum 1. Preparation and biochemical characterization of c e l l f r actions and isolated plasma membrane v e s i c l e s . Exp. C e l l . Res. 95: 67-78. 70. Rossomando, E.F. and B. Maldonado (1976) Inhibitors of 5'-nucleotidase a c t i v i t y after growth of Dictyostelium  discoideum. Exp. C e l l Res. 100: 383-388. 71. Sanderman, H. and J.L. Strominger (1972) Biosynthesis of peptidoglycan of b a c t e r i a l c e l l wall, 27: P u r i f i c a t i o n and properties of C55-iso-prenoid alcohol phosphokinase from staphylococcus aureus. J. B i o l . Chem. 247: 5123-5131. 72. Simpson, R.T. and B.L. Vallee (1969) Zn and Co a l k a l i n e phosphatase. Ann. N.Y. Acad. S c i . U.S.A. 166: 670-695. 73. Soloman, E.P., E.M. Johnson and J.H. Gregg (1964) Multiple forms of enzymes in a c e l l u l a r slime mold during morphogenesis. Develop. B i o l . 9: 314-326. 142 74. Sussman, M. (1966) Biochemical and genetic methods i n the study of c e l l u l a r slime mold development. In Methods In C e l l Biology 2i 397-410. Academic Press, New York. 75. Tsai, M.D. (1980) Stereochemistry of the hydrolysis of adenosine 5 1-thiophosphate catalyzed by venom 5'-nucletidase. Biochemistry: Jj9: 5310-5316. 76. Towbin, H., T. Staehelin and J. Gordon (1979) Electrophoretic transferof proteins from polyachrylamide gels to n i t r o c e l l u l o s e sheets: Procedure and some applications. Proc. Natl. Acad. S c i . U.S.A. 76J 4350-4354. 77. Tuchman, J . , J.E. Smart and H.F. Lodish (1976) E f f e c t s of d i f f e r e n t i a t e d membranes on the developmental program of the c e l l u l a r slime mold. Dev. B i o l . 5_1: 77-85. 78. Van Belle, H. (1972) Kinetics and i n h i b i t i o n of alkaline phosphatases from canine tissues. Biochem. Biophys. Acta. 289: 158-168. 79. Watts, D.J. and J.M. Ashworth (1970) Growth of myxamoebae of the c e l l u l a r slime mould Dictyostelium  discoideum i n axenic culture. Biochem. J. 119: 171-174. 143 80. Weeks, C. and G. Weeks (1975) C e l l surface changes during the d i f f e r e n t i a t i o n of Dictyostelium discoideum. Exp. C e l l Res. 92_: 372-382 81. Wilson, I.B., J. Dayan and K. Cyr (1964) Properties of alk a l i n e phosphatase from E^ . C o l i . J. B i o l . Chem. 239: 4182-4185. 

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:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0097217/manifest

Comment

Related Items