UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Developmental regulation of alkaline phosphatase in Dictyostelium discoideum Mohandas, Devaki Velayudhan 1983

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

Notice for Google Chrome users:
If you are having trouble viewing or searching the PDF with Google Chrome, please download it here instead.

Item Metadata

Download

Media
831-UBC_1983_A1 M63.pdf [ 4.62MB ]
Metadata
JSON: 831-1.0095886.json
JSON-LD: 831-1.0095886-ld.json
RDF/XML (Pretty): 831-1.0095886-rdf.xml
RDF/JSON: 831-1.0095886-rdf.json
Turtle: 831-1.0095886-turtle.txt
N-Triples: 831-1.0095886-rdf-ntriples.txt
Original Record: 831-1.0095886-source.json
Full Text
831-1.0095886-fulltext.txt
Citation
831-1.0095886.ris

Full Text

DEVELOPMENTAL REGULATION OF ALKALINE PHOSPHATASE IN DICTYOSTELIUM DISCOIDEUM by Devaki Velayudhan Mohandas B.Sc. Un ivers i t y of Kera la , Ind ia , 1959 B.Sc. (App. Biochem. S c i . ) Saugar Un ive rs i t y , India , 1962 M.Sc..M.S. Un i ve r s i t y , India , 1971 M.Sc. Un ivers i t y of B r i t i s h Columbia, 1978 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Microbiology) We accept t h i s thes is as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA June 1983 (c) Devaki Velayudhan Mohandas, 1983 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e h e a d o f my d e p a r t m e n t o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f MICROBIOLOGY The U n i v e r s i t y o f B r i t i s h C o l u m b i a 1956 Main Mall V a n c o u v e r , Canada V6T 1Y3 Dat e i f t i n i i ABSTRACT The membrane bound a l ka l i ne phosphatase ac t i v i t y ' in vegetat ive c e l l s of Dictydste l ium discdideum was fdund td exh ib i t a 8 to 10 f o l d increase in s p e c i f i c a c t i v i t y when incubated at 50°C. This ac t i va t i on was reversed on t rans fer of the preparat ion to 0 °C . S imi la r a c t i va t i on of the vegetat ive enzyme was achieved by d i a l y s i ng the crude membrane prepara t ion , suggesting the removal of a low molecular weight i n h i b i t o r . A l ka l i ne phosphatase s o l u b i l i z e d from the membranes using T r i ton X-100 was s i m i l a r l y ac t i va ted by 50°C treatment or d i a l y s i s and the 50°C ac t i va t ion was reversed by incubat ion at 0 °C . Both the d ia l ysed vegetat ive membrane and the d ia l ysed Tr i ton X-100 extract were i nh ib i t ed by the addi t ion of concentrated d i a l y sa t e . This i n h i b i t i o n could be re l i eved by subsequent d i a l y s i s . , A 620 f o l d p u r i f i e d a lka l ine phosphatase preparat ion was obtained from crude vegetat ive membranes by a f f i n i t y and ion exchange chromatography a f t e r s o l u b i l i z a t i o n of the enzyme using Tr i ton X-100-5'-nucleot idase a c t i v i t y copu r i f i ed along with,the a l ka l i ne phosphatase a c t i v i t y in a l l the f r a c t i ona t i on steps employed. The membrane bound 5 '-nucleot idase a c t i v i t y was not ac t i va ted e i the r by incubat ion at 50°C or by d i a l y s i s and the a c t i v i t y was f a r less s tab le than the a l ka l i ne phosphatase. However, the T r i ton X-100 i i i extracted 5 '-nucleot idase was act ivated by d i a l y s i s to the same extent as the a l ka l i ne phosphatase and both a c t i v i t i e s in the p a r t i a l l y p u r i f i e d preparat ion were equal ly i nh ib i t ed by the d ia l ysa tes from vegetat ive membranes. These resu l t s suggest that both a l ka l i ne phosphatase and 5 '-nucleot idase are due to a s ing le prote in but the i n t e r a c t i on of the two subst ra tes , AMP and pNPP, with the enzyme are d i f f e r e n t and are markedly in f luenced by conformational changes induced by the i n h i b i t o r . Unlike the vegetat ive enzyme, the a l ka l i ne phosphatase a c t i v i t y in the culminat ing membrane was not markedly ac t i va ted by incubat ion at 50°C or by d i a l y s i s . Since the vegetat ive enzyme was ac t i va ted by both treatments to l eve l s s im i l a r to those found in culminat ing c e l l s , i t was proposed that the developmental increase in a l ka l i ne phosphatase in D.discoideum was due to the unmasking of already ex i s t i ng enzyme by the removal of i n h i b i t o r . However, the a l ka l i ne phosphatase a c t i v i t y of culminat ing c e l l s d i f f e r e d from that of vegetat ive c e l l s in chromatography on DEAE-Sephacel and conA-Sepharose and i t was less s table in low concentrat ions of T r i s-C l and in SDS. In con t ras t , the culminat ing enzyme was more stable in high concentrat ions o f T r i s - C l . These resu l t s suggest that the vegetat ive enzyme is modif ied during development. This modi f i ca t ion appears to be s l i g h t , s ince the vegetat ive and culminat ing enzymes migrate i d e n t i c a l l y in SDS polyacrylamide e l ec t rophore t i c gels and the enzymes from the two i v developmental stages were s im i l a r in pH optima, in i n h i b i t i o n by phosphate and in i n h i b i t i o n by concentrated d i a l y sa t e . V TABLE OF CONTENTS Page ABSTRACT 1 1 TABLE OF CONTENTS v LIST OF TABLES ix LIST OF FIGURES xi LIST OF ABBREVIATIONS xiv ACKNOWLEDGEMENTS xv INTRODUCTION 1 MATERIALS AND METHODS 14 A. Ma te r i a l s , Media and Solut ions 14 ( i ) Mater ia ls 14 ( i i ) Media 15 ( i i i ) Buffers and Solut ions 16 B. Methods 20 1. Organism and growth condi t ions 20 2. D i f f e r e n t i a t i o n experiments 20 3. Membrane ext rac t ion 21 4. Enzyme assays 22 5. Protein est imation 23 6. D i a l y s i s experiments 24 7. Arrhenius p lo ts 24 8. A lka l ine phosphatase p u r i f i c a t i o n procedures. 25 a. S o l u b i l i z a t i o n of a l ka l i ne phosphatase from 25 crude membrane oreparat ions by butanol or T r i ton X-100 vi b. Chromatographic procedures. 9. Sodium dodecyl sulphate-polyacrylamide gel e l e c t rophores i s . 10. Preparat ion of concentrated d i a l y sa te . RESULTS SECTION I Crypt i c existence of a l ka l i ne phosphatase in vegetat ive membrane of D.discoideum and i t s imp l i ca t ion on the developmental regu la t ion of th i s enzyme. a. Reversible heat a c t i v a t i on of the membrane bound a l ka l i ne phosphatase. b. S o l u b i l i z a t i o n of a l ka l i ne phosphatase from vegetat ive c e l l membranes. c. E f f ec t of 50°C treatment on a lka l ine phosphatase s o l u b i l i z e d with butanol and Tr i ton X-100. d. Ac t i va t ion of a l ka l i ne phosphatase by d i a l y s i s . e. E f f ec t of incubat ion at 50°C and d i a l y s i s on a l ka l i ne phosphatase in membranes of c e l l s from d i f f e r en t stages o f d i f f e r e n t i a t i o n . f. Reconst i tut ion of the d ia lysed vegetat ive membrane with the putat ive i n h i b i t o r . g. E f f ec t o f coincubat ion of vegetat ive and culminat ing membranes. h. E f f ec t of temperature and d i a l y s i s on the membrane bound 5 '-nuc leot idase . i . Summary. v i i Page SECTION II P u r i f i c a t i o n of a l ka l i ne phosphatase from vegetat ive membranes 56 of D.discdideum. a. P u r i f i c a t i o n using butanol ext rac t ion fo r s o l u b i l i z a t i o n . 56 b. P u r i f i c a t i o n using Tr i ton X-100 ext rac t ion fo r 59 s o l u b i l i z a t i o n . c. Summary. 78 SECTION i n : Propert ies o f a l ka l i ne phosphatase and 5 1 -nucleot idase 79 a c t i v i t i e s of the p a r t i a l l y p u r i f i e d preparat ion. a. E f f ec t of f l u o r i d e . 79 b. pH optima. 79 c. I nh ib i t i on of a l ka l i ne phosphatase by inorganic 79 phosphate. d. Inh ib i t ion by the putat ive i n h i b i t o r . ^3 e. Substrate s p e c i f i c i t y s tud ies . 86 f. Determination of Km fo r AMP and pNPP. 8 6 g. Summary. 90 SECTION IV Comparison between a l ka l i ne phosphatase from vegetat ive and 91 culminat ing c e l l s . a. P u r i f i c a t i o n of culminat ing enzyme. 91 b. Inh ib i t ion by sodium dodecyl sulphate. 91 c. S t a b i l i t y of vegetat ive and culminating enzymes 94 d. pH optima 97 e. Inh ib i t ion by inorganic phosphate 97 vi i i Page f. Arrhenius p l o t s . 97 g. Summary 101 DISCUSSION 1 ° 2 LITERATURE CITED 117 ix LIST OF TABLES Table Page I S o l u b i l i z a t i o n of a l ka l i ne phosphatase from crude 36 vegetat ive membranes of D.discoideum II Comparison of the e f f e c t s at 50°C and 0°C on the 37 a l ka l i ne phosphatase a c t i v i t y of in tac t membrane and butanol-extracted preparat ions. III E f f ec t o f d i a l y s i s and subsequent 50°C treatment on 40 a l ka l i ne phosphatase a c t i v i t y of vegetat ive membranes and T r i ton X-100 ex t rac t s . . - — --IV Comparison of the e f f e c t s of heat treatment and d i a l y s i s 42 on the a l ka l i ne phosphatase a c t i v i t y of membranes prepared from Ax-2 c e l l s at var ious stages of development. V E f f ec t o f add i t ion of d ia l ysa te on a l ka l i ne phosphatase 48 a c t i v i t y . VI E f f ec t of coincubat ion of precooled vegetat ive and 50 culminat ing membranes. VII E f f ec t of d i a l y s i s on membrane bound and T r i ton X-100 54 extracted 5 '-nucleot idase a c t i v i t i e s from vegetat ive membranes o f D.discoideum. VIII Ammonium sulphate f r a c t i ona t i on of butanol extracted 57 a l ka l i ne phosphatase from vegetat ive membranes of D.discoideum. IX A f f i n i t y chromatography of butanol s o l u b i l i z e d a l ka l i ne 52 phosphatase a c t i v i t y in three gel matr ices. X Octyl Sepharose Chromatography of butanol extracted 55 a l ka l i ne phosphatase from vegetat ive membrane of D.discoideum. XI P u r i f i c a t i o n of butanol s o l u b i l i z e d a l ka l i ne phosphatase 58 from vegetat ive membrane of D.discoideum. X Table Page XII P u r i f i c a t i o n of T r i ton X-100 s o l u b i l i z e d a l ka l i ne 76 phosphatase and 5 '-nucleot idase a c t i v i t i e s from vegetat ive membranes of D.discoideum. XIII E f f ec t of 30mM sodium f l uo r ide on the a l ka l i ne 80 phosphatase and 5 '-nucleot idase a c t i v i t i e s of vegetat ive c e l l s of D.discoideum. XIV Phosphatase a c t i v i t i e s of vegetat ive membrane and 84 p a r t i a l l y p u r i f i e d preparat ion on var ious phosphate es te r s . XV E f f ec t of 50°C treatment and d i a l y s i s on a l ka l i ne 87 phosphatase i nh ib i t ed by inorganic phosphate. xi LIST OF FIGURES Figure Page 1 L i f e cyc le of Dictydste l ium discdideum. 2 2. The e f f e c t of preincubat ion temperature on the 32 a c t i v i t y of membrane bound a l ka l i ne phosphatase. 3. The reve rs ib l e heat a c t i v a t i on o f a l ka l i ne phosphatase. 33 4. E f f ec t of 50°C and 0°C incubations on the a l ka l i ne 39 phosphatase a c t i v i t y of i n tac t membranes and T r i ton X-100 extracted membranes. 5. Reconst i tut ion of the d ia lysed vegetat ive membrane 46 with a concentrated d ia l ysa te preparat ion. 6. Comparison of the i n h i b i t i o n of a l ka l i ne phosphatase 47 a c t i v i t y by inorganic phosphate and the concentrated d i a l y sa t e . 7. Comparison of the e f f e c t of preincubat ion at 50°C 52 on the membrane bound a l ka l i ne phosphatase and 5 1 -nuc leot idase a c t i v i t i e s in vegetat ive c e l l s . 8. Comparison of the e f f e c t of long term incubat ion at 53 50°C on the membrane bound a lka l i ne phosphatase and 5!-nucleot idase a c t i v i t i e s in vegetat ive c e l l s . 9. Gel f i l t r a t i o n on Sephacryl-S-300 of butanol extracted 59 a l ka l i ne phosphatase from crude vegetat ive membranes. 10. Ion exchange chromatography on DEAE-Sephacel of 61 butanol extracted a l ka l i ne phosphatase from crude vegetat ive membranes. 11. A f f i n i t y chromatography on concanavalin A-Sepharose of 63 butanol extracted a l ka l i ne phosphatase from vegetat ive c e l l s a f t e r treatment with 1% sodium deoxycholate. 12. Gel f i l t r a t i o n on Sephacryl-S-300 of T r i ton X-100 70 extracted a l ka l i ne phosphatase from vegetat ive c e l l membranes. x i i Figure Page 13. A f f i n i t y chromatography on concanaval in A-Sepharose 71 of T r i ton X-100 extracted a l ka l i ne phosphatase from vegetat ive c e l l membranes. 14. Ion exchange chromatography on DEAE-Sephacel of 73 T r i ton X-100 extracted a l ka l i ne phosphatase from vegetat ive c e l l membranes a f te r a f f i n i t y chromatography. 15. SDS - polyacrylamide gel e lec t rophores is o f three 77 d i f f e r e n t steps in the p u r i f i c a t i o n of the a l ka l i ne phosphatase a c t i v i t y from the vegetat ive c e l l membranes of D.discdideum. 16. E f f ec t of pH on the a l ka l i ne phosphatase and 81 5 '-nucleot idase a c t i v i t i e s in the p a r t i a l l y p u r i f i e d preparat ion . 17. Inh ib i t ion of a l ka l i ne phosphatase a c t i v i t y in 82 vegetat ive c e l l s of D.discoideum by inorganic phosphate. 18. E f f ec t of concentrated d ia l ysa te on the a l ka l i ne 85 phosphatase and 5 '-nucleot idase a c t i v i t i e s in the p a r t i a l l y p u r i f i e d preparat ion. 19A. Lineweaver-Burk p lot fo r the a l ka l i ne phosphatase 88 a c t i v i t y in the p a r t i a l l y p u r i f i e d preparat ion. 19B. Lineweaver-Burk p lo t fo r the 5 '-nucleot idase 89 a c t i v i t y in the p a r t i a l l y p u r i f i e d preparat ion. 20. SDS-polyacrylamide gel e lec t rophores is of 92 a l ka l i ne phosphatase a c t i v i t i e s . 21. E f f ec t of sodium dodecyl sulphate on the 93 a l ka l i ne phosphatase a c t i v i t i e s of vegetat ive and culminat ing c e l l s . 22A. S t a b i l i t y of the' a l ka l i ne phosphatase a c t i v i t i e s 95 of vegetat ive and culminat ing c e l l membranes. 22B. S t a b i l i t y o f the a l ka l i ne phosphatase a c t i v i t i e s 96 of vegetat ive and culminating c e l l membranes at pH 8.0. x i i i Figure 23. E f fec t of pH on the a l ka l i ne phosphatase and the 5 '-nucleot idase a c t i v i t i e s in the culminat ing membrane preparat ions. 24. Inh ib i t ion of a l ka l i ne phosphatase a c t i v i t y in culminat ing c e l l membranes by inorganic phosphate. 25. Temperature dependence of the a l ka l i ne phosphatase a c t i v i t y . LIST OF ABBREVIATIONS ADP Adenosine 5 1-diphosphate AMP Adenosine 51-monophosphate ATP Adenosine 5'-triphosphate cAMP 3'51 - c y c l i c adenosine monophosphate ConA Concanavalin A EDTA Ethylene diamine te t ra ace t i c ac id GDP Guanosine 5'-diphosphate GMP Guanosine 5'-monophosphate O.D. Opt ica l densi ty PMSF Phenyl methyl sulphonyl f l uo r i de pNPP p-nitrophenyl phosphate pNPPASE p-nitrophenyl phosphatase SDS Sodium dodecyl sulphate XV ACKNOWLEDGEMENTS I wish to thank Dr. Gerald Weeks fo r his constant encouragement, suggestions and f r i endsh ip during the course of th i s work. I a lso wish to thank Drs. Geoffrey Webb, R.E.W. Hancock and B.D. Roufogal is fo r t he i r helpful suggestions and d i scuss ions . Thanks are a lso due to Mrs. Kathy LaRoy fo r ass istance during the i n i t i a l stages of laboratory work and Mr. Pradeep Bhanot fo r the modi f i ca t ions in the procedure fo r gel e lec t rophores is of a l ka l i ne phosphatase. I thank a l l my c lose f r iends fo r t h e i r encouragement and support. I a l so thank Mrs. Cel ine Gunawardene fo r typing th i s t hes i s . 1 INTRODUCTION Dictyoste l ium discoideum is a member of the c lass Ac ras i ae , comprising of species of f r e e - l i v i n g amoebae that lack a f l a g e l l a t e d stage and aggregate to form f r u i t i n g bodies. ( 8) This organism was f i r s t d iscovered by Raper in 1935 (49) and i s found in nature as a so i l organism in fo res t d e t r i t u s . The myxamoebae feed on bacter ia and decaying matter and d iv ide by binary f i s s i o n , l i k e many other amoeboid organisms. However, when the loca l environment i s depleted of food , they undergo a process of d i f f e r e n t i a t i o n . The ind i v idua l c e l l s c o l l e c t 5 in large streaming pat te rns . to form aggregates conta in ing up to 10 c e l l s (aggregation phase). In due course, each aggregate forms a migratory s lug or pseudoplasmodium (pseudoplasmodium phase) , which i s covered by a c e l l u l o s i c sheath. (34) At th i s s tage, c e l l u l a r d i f f e r e n t i a t i o n becomes c l e a r l y apparent*, the c e l l s at the f ront end of the migrat ing s lug are p re-s ta lk , the c e l l s at the rear are pre-spore. A f te r a period of migrat ion the pseudoplasmodium stops and develops into a f r u i t i n g body (culmination phase), composed of s p a t i a l l y separated s ta lk c e l l s and spore c e l l s . ( F ig . 1) The d i f f e r e n t i a t i o n of the i den t i ca l u n i c e l l u l a r myxamoebae in to two d i s t i n c t types of c e l l s provides a simple eucaryot ic model system fo r studies on c e l l d i f f e r e n t i a t i o n . D.discoideum can be grown in the laboratory on a wide va r i e t y of bac ter ia (50) and d i f f e r e n t i a t i o n i s induced by depos i t ion of the harvested vegetat ive c e l l s on to non-nutrient medium. Under laboratory cond i t i ons , th i s d i f f e r e n t i a t i o n process i s complete in about 24 hours. 2 Figure 1 L i f e cyc le of Dictyoste l ium discoideum 3 A large number of biochemical changes occur during the d i f f e r e n t i a t i o n process of t h i s organism. Several enzymes have been found to increase in a c t i v i t y at s p e c i f i c stages in d i f f e r e n t i a t i o n . ( 3 5 ) . These developmentally regulated enzymes do not accumulate in develop-mental mutants blocked at e a r l i e r morphological stages and accumulation continues to be c h a r a c t e r i s t i c fo r a p a r t i c u l a r morphological stage in temporal ly deranged developmental mutants. Hence many of these enzymes can be used as biochemical markers fo r p a r t i c u l a r stages of development. (36). A l ka l i ne phosphatase has been shown by several workers to be one of the developmentally regulated enzymes in th i s organism.(9, 30, 18). A lka l ine phosphatase i s almost un i ve r sa l l y found in most of the b i o l og i c a l specimens t e s t ed , a major exception being some higher p l an ts . Many bac te r ia l and mammalian enzymes have been studied in great detail- and character i sed with respect to t he i r enzymatic and physica l p roper t i es . These studies reveal considerable species - and even organ - s p e c i f i c d i f fe rences in a l ka l i ne phosphatase a c t i v i t y . Changes in a c t i v i t y have a lso been reported in r e l a t i on to growth and development and d i f fe rences in a c t i v i t y have been found in normal and malignant c e l l s - (39 ,59). Tissue - s p e c i f i c isoenzymes of a l ka l i ne phosphatase have been i d e n t i f i e d in mammalian sources and fur ther character i sed by standardized approaches (42). Most of the mammalian enzymes have been found to be plasma membrane - bound, (22 ) whereas, the bac te r i a l enzymes are found in the per ip lasmic space (40) 4 and the process of syn thes i s , processing and extrus ion of th i s enzyme prote in from the cytoplasm to the per ip lasmic space in ce r ta in bac ter ia has been studied in d e t a i l . (57). The phys io log ica l funct ion of a l ka l i ne phosphatase, which mostly occurs as a non-spec i f ic phosphatase ac t i ve on a va r ie ty of phosphate esters at a l ka l i ne pH, i s not yet understood, but the most genera l l y suggested funct ion i s the removal of phosphate from phosphorylated e x t r a c e l l u l a r components to al low a more rapid uptake of the cons t i tuents . The f i r s t report on the developmental regula t ion of a l ka l i ne phosphatase was by Bonner et a l . , who showed by histochemical s ta in ing that a l ka l i ne phosphatase in migrat ing pseudoplasmodia: was extremely ac t i ve in the an te r io r pre-sta lk region compared to the r e l a t i v e low a c t i v i t y in the pos te r io r pre-spore reg ion, ( 9 ) . Krivanek provided the f i r s t quant i t a t i ve data , showing that the s p e c i f i c a c t i v i t y of t h i s enzyme increased more than two-fold during ear l y culminat ion (30). He a lso conf i rmed, by histochemical s t a i n i n g , that the enzyme a c t i v i t y in migrat ing pseudoplasmodia was located fo r the most part in the pre-sta lk area. Krivanek and Krivanek demonstrated a phosphatase a c t i v i t y by histochemical s ta in ing using AMP as substrate , (31). They used a pH of 8 .3 , suggesting that the enzyme might be the prev ious ly descr ibed a l ka l i ne phosphatase. The maximum a c t i v i t y was found during pre-culmination and ea r l y culminat ion stages and again the a c t i v i t y was concentrated in the pre-sta lk region. (31). 5 Solomon and coworkers analysed a l ka l i ne phosphatase by s tarch gel e lec t rophores i s and found that there was only one band of enzyme a c t i v i t y in vegetat ive c e l l s (58), A second band of a l ka l i ne phosphatase a c t i v i t y appeared during the pseudoplasmodial stage. The s ta in ing i n t ens i t y of both isozymes was highest in culminat ion stage samples (58),. These resu l t s suggested that part of the increased a c t i v i t y was due to the expression of a d i f f e r e n t i a t i o n s p e c i f i c gene. A s i x - f o l d increase in the s p e c i f i c a c t i v i t y of a l ka l i ne phosphatase during culminat ion stages of D.discoideum was reported by Gezel ius and Wright (18) , They showed that the hydro lys is of adenosine monophosphate and deoxyadenosine monophosphate in add i t ion to the chromogenic t es t subs t ra te , p-nitrophenyl phosphate were a l l optimal at pH 9.0. A l l three a c t i v i t i e s were a lso found to be developmentally co-regulated and since a number of propert ies of the 5 '-nucleot idases and pNP Rase a c t i v i t i e s were iden t i ca l they concluded that a l l three a c t i v i t i e s res ided on the same prote in molecule, a non-spec i f i c phospha-tase . They a l so found a l ka l i ne phosphatase a c t i v i t y with 5'-GMP, GDP, ATP and UTP as subst ra tes , but s ince t he i r experiments were performed on crude c e l l - f r e e ex t r a c t s , i t was not poss ib le to t e l l i f a l l of these a c t i v i t i e s were due to a s ing le phosphatase ((18) • Loomis studied the k ine t i c s of accumulation of a l ka l i ne phosphatase in wi ld type and developmentally aberrant mutant s t ra ins of D.discoideum ( 37 ) . He observed an increase of about 8-fold in the 6 s p e c i f i c a c t i v i t y of th i s enzyme during culminat ion of the wi ld type s t r a i n . He found that concomitant prote in synthesis was essent ia l f o r the increase in a c t i v i t y , while RNA synthesis could be i nh ib i t ed during the 8 hours immediately preceding culminat ion without a f f e c t i ng the amount of enzyme accumulated. The developmental mutants showed no accumulation of a l ka l i ne phosphatase. He suggested that the expression of the d i f f e r e n t i a t i o n s p e c i f i c band of a l ka l i ne phosphatase, reported e a r l i e r by Solomon et a l . , ( 5 8 ) , might be blocked in these mutants. The f i r s t i nd i ca t i on that a l ka l i ne phosphatase (act ive on p-nitrophenyl phosphate as substrate) and 5 1 -nucleot idase (act ive on AMP) were due to d i s t i n c t enzymes in D.discoideum was the study by Green and Newell ( 24 ) . They found that only 30% of the 5 '-nucleot idase a c t i v i t y was membrane bound. In con t ras t , over 74% of the a l ka l i ne phosphatase a c t i v i t y was membrane bound and co-d is t r ibuted with the 125 lactoperoxidase - I l abe l l ed c e l l surface components. They concluded that a l ka l i ne phosphatase was a su i tab le plasma membrane marker, while 5 '-nucleot idase was not. Par ish and Pel 1i (47) estimated 40 to 50% of the to ta l a l k a l i n e phosphatase in D.discoideum to be located on the c e l l sur face . Further evidence fo r a c e l l surface l o c a l i z a t i o n of a l ka l i ne phosphatase came from the work of Rossomando and Cut ler (52). They reported high s p e c i f i c a c t i v i t y a l ka l i ne phosphatase in t he i r plasma membrane f r a c t i o n prepared from c e l l s lysed using the a n t i b i o t i c amphotericin-B. 7 Lee et a l . , (33 ) studied several proper t ies of a l ka l i ne phosphatase and 5 '-nucleot idase in the axenic s ta in AX-2 of D.discoideum. In contrast to Green and Newell ( 2 4 ) , they found both 125 enzymes to be t i g h t l y membrane bound co-sedimenting with I lactoperoxidase l abe l l ed components. The a c t i v i t i e s a lso fol lowed the same pattern of developmental inc rease , suggesting co-regulat ion during development. The enzymes showed s im i l a r responses towards various i n h i b i t o r s and temperature. However, there were two major d i f f e rences between the a c t i v i t i e s . The pH optimum fo r 5 1 -nucleot idase was 7.6, while in contrast a l k a l i n e phosphatase was opt imal ly ac t i ve at pH 8.7. Further -more, -the two a c t i v i t i e s could be phys i ca l l y separated by t rea t ing the crude membrane preparat ions with phospholipase - C. 90% of the 5'-nucleot idase a c t i v i t y was removed from the membrane, while in contrast only 10% of the a l k a l i n e phosphatase was s o l u b i l i z e d ( 33) . Furthermore, under ce r ta in condi t ions the two a c t i v i t i e s d id not change co-ord inate ly . Rossomando and Maldonado reported that the to ta l 5 1 -nuc leot idase a c t i v i t y of exponential phase c e l l s of D.discoideum Ax-2 decreased 10-fold when c e l l s passed into s ta t ionary phase, while the a l ka l i ne phosphatase a c t i v i t y was unchanged during th i s t r a n s i t i o n again suggesting that a lka l ine phosphatase and 5'-nucleotidase a c t i v i t i e s were due to two different-enzymes (53). They-found that 2.5% of the 5 '-nucleot idase of exponent ia l l y grown c e l l s was membrane bound, whereas only 10% of the enzyme from s ta t ionary phase c e l l s was membrane 8 bound, d i k e s and Weeks found that plasma membranes p u r i f i e d from exponent ia l l y growing c e l l s had both a l ka l i ne phosphatase and 5 '-nucleot idase a c t i v i t i e s t i g h t l y membrane bound and showed that both enzymes could be used as markers in plasma membrane p u r i f i c a t i o n ( 2 0 ) . However, when membranes were prepared from s ta t ionary phase c e l l s , a l ka l i ne phosphatase a c t i v i t y remained f i rm l y membrane bound, but 5 '-nucleot idase was p a r t i a l l y d i ssoc i a ted from the membrane, a r e su l t reminiscent of the data of Rossomando and Maldonado ( 5 3 ) . In order to understand more f u l l y the developmental regu la t ion of a l ka l i ne phosphatase, the e f f e c t s of a va r i e t y of developmental perturbat ions on enzyme accumulation have been s tud ied . D.discoideum c e l l s can be mechanical ly d i ssoc ia ted from aggregates or pseudoplasmodia at d i f f e r e n t stages of development. When they are redeposited on f i l t e r pads fo r d i f f e r e n t i a t i o n , these c e l l s reaggregate r ap id l y and r e c a p i -tu la te t he i r p r i o r morphogenesis and complete development (46). Loomis showed that a l ka l i ne phosphatase did not accumulate when culminat ing f r u i t i n g bodies were d isrupted and c e l l s redeposited on f i l t e r s ( 37) . However i t was not c l ea r from his experiments whether d i f f e r e n t i a t i o n d id continue fo l lowing d i s rup t i on . Moreover, Atryzek showed subsequently that a l ka l i ne phosphatase accumulation continued a f t e r disaggregated c e l l s had reassoc iated i r r e spec t i v e of the time of d i s s o c i a t i o n of the c e l l s (6). 9 A mutant of D.discoideum c e l l s produced only s ta lk c e l l s , -4 when deposited on d i a l y s i s membrane supports in the presence of 10 M cAMP (25). It was found that a l ka l i ne phosphatase accumulated in these c e l l s , where spore pathway s p e c i f i c enzymes d id not accumulate, suggesting again that a l ka l i ne phosphatase was s ta lk c e l l s p e c i f i c and that the normal process of m u l t i c e l l u l a r morphogenesis was not necessary fo r a l ka l i ne phosphatase expression ( 2 5 ) . MacMahon et a l . , found that the continued d i f f e r e n t i a t i o n of c e l l s from d i ssoc i a ted pseudoplasmodia could be i nh ib i t ed by t r ea t ing these c e l l s with plasma membranes of c e l l s from the same phase (44). They observed that a l ka l i ne phosphatase a c t i v i t y in these c e l l s , was t prematurely super-induced by the added plasma membranes. The e f f e c t was observed only on whole c e l l s and not on c e l l ex t r a c t s , was most s p e c i f i c when pseudoplasmodia! plasma membranes were used and was independent of prote in synthes is . They suggested that the ac t i v a t i on could be the d i r e c t r e su l t of a biochemical process such as phosphorylat ion occurr ing on the plasma membrane. Furthermore, Tuchman et a l . , found that exogenously appl ied aggregation phase membranes at the s t a r t of the d i f f e r e n t i a t i o n per iod induced the accumulation of a l ka l i ne phosphatase 9 hours before that found in control c e l l s (60 ) . They suggested that c e l l - c e l l contact was essent ia l f o r the accumulation of a l ka l i ne phosphatase during culminat ion and the treatment of the c e l l s with aggregation phase plasma membranes t r iggered th i s accumulation without actual morphogenesis. F i n a l l y , when D.discoideum c e l l s are 10 starved in buf fer and shaken to prevent normal morphogenesis, the a l k a l i n e phosphatase a c t i v i t y does not accumulate. (.51). However, pu ls ing the s tarv ing shaken c e l l s with 5 x 10~^M cAMP, produces a normal accumulation of a l ka l i ne phosphatase a c t i v i t y . Thus i t i s c l ea r from these studies that the normal m u l t i c e l l u l a r morphogenetic sequence i s not necessary fo r the expression of a l ka l i ne phosphatase a c t i v i t y . However, in the va r ious l y perturbed systems, e i t he r c e l l - c e l l contact , membrane-cell contact or the presence of c y c l i c AMP was necessary fo r a l ka l i ne phosphatase express ion. At the outset of the present work, a l ka l i ne phosphatase had been es tab l i shed as a membrane bound enzyme, which was developmentally regulated in D.discoideum, and a po t en t i a l l y exce l l en t marker fo r s ta lk c e l l pathway of d i f f e r e n t i a t i o n . Studies on developmental regula t ion had revealed the appearance of a new enzyme during development (58 ) and the requirement fo r prote in synthesis fo r the developmental increase (37 ) . Later studies showed that normal morphogenesis was not a requirement fo r the accumulation of a l ka l i ne phosphatase. The ava i l ab le evidence a lso suggested that 5 1 -nuc leot idase a c t i v i t y , which was developmental ly co-regulated with a l ka l i ne phosphatase, was due to another d i s t i n c t enzyme. The purpose of th i s study was to pu r i f y and charac ter ize the a l ka l i ne phosphatase enzyme and attempt to fur ther de l ineate i t s 11 developmental r egu l a t i on . Since th i s work was i n i t i a t e d there have been several papers publ ished on D.discoideuni a l ka l i ne phosphatase that have inf luenced the course of th i s study. MacLeod and Loomis i so l a ted a mutant of D.discoideum with low a l ka l i ne phosphatase a c t i v i t y and a l t e red substrate a f f i n i t y (41 ). The mutant exh ib i ted a pattern of developmental increase in a l ka l i ne phosphatase that was s im i l a r to the wi ld type. Comparison of several physica l proper t ies of enzymes from exponential and culminat ing phase c e l l s in the mutant and wi ld type s t ra ins showed that the enzymes from these two phases were i nd i s t i ngu i shab l e . Polyacrylamide gel e lec t rophores i s of t r i t o n s o l u b i l i z e d enzymes from vegetat ive and culminat ing c e l l s of these two s t ra ins revealed a s ing le band of a c t i v i t y and they concluded that the increased accumulation of a s ing le enzyme was respons ib le fo r the increase in a c t i v i t y during cu lminat ion. Using microtechniques fo r enzymatic analyses in segments of d i f f e r e n t i a t i n g slime molds, Armant and Rutherford found that., the a l ka l i ne phosphatase a c t i v i t y in the culminat ing pseudoplasmodium was l o c a l i z e d in the area of c e l l - c e l l contact of presumptive spore and s ta lk t i ssues (2). They gave fu r ther e lec t ron microscopic evidence ( 5 ) that th i s a c t i v i t y was present only in the prestalk, . c e l l s adjacent to the prespore region at cu lminat ion. They a lso found that the enzyme could be sta ined only on the outer surface of the plasma membranes of these c e l l s , suggesting that the enzyme funct ioned e x t r a c e l l u l a r l y . 12 Quiv iger et a l . , found that. the. al kai ine phosphatase .< • .. sta ined h is to-chemica l ly mainly in. the c o n t r a c t i l e vacuoles of vegetat ive D.discokleuinFY..< c e l l s during s tarvat ion (48 ). They suggested however that any plasma membrane l o c a l i z e d a l ka l i ne phosphatase might have been destroyed during t h e i r f i x i n g procedures. At cu lminat ion , they observed that the enzyme a c t i v i t y was concentrated in a group of pre-sta lk c e l l s located at the boundary with the prespore region and th i s a c t i v i t y sta ined h is tochemica l l y on the plasma membranes. They suggested that the loca t ion of the enzyme might change from the c o n t r a c t i l e vacuoles during s ta rva t ion to the plasma membranes during cu lminat ion . Recent ly , Armant and Rutherford provided evidence that 5 '-nucleot idase and a l ka l i ne phosphatase in D.discoideum were due to a s ing le enzyme (3). ' They found that both a c t i v i t i e s were l o c a l i z e d at the surface o f pre-sta lk c e l l s in the region adjacent to the pre-spore c e l l s during culminat ion of t h i s organism. They a lso p u r i f i e d the enzymes from culminat ing c e l l s by a combination of T r i ton X-100 s o l u b i l i z a t i o n and chromatography on DEAE-sephadex, phenyl-sepharose, concanaval in A-Sepharose and Sephacyl-S-300. The f i n a l product was p u r i f i e d to homogeneity as assessed by sodium dodecyl sulphate -polyacrylamide gel e l ec t rophores i s and had both a l ka l i ne phosphatase and 5 '-nucleot idase a c t i v i t y and the two a c t i v i t i e s co-pur i f i ed through a l l the steps in the p u r i f i c a t i o n procedure. They found that the p u r i f i e d prote in had a requirement fo r me ta l l i c Zn and a pH optimum 0 13 of 9.5 with both pNPP and AMP as substrates (4). The r e l a t i onsh ip between these more recent studies and the work presented i n t h i s thes is w i l l be discussed l a t e r . 14 MATERIALS AND METHODS Ma te r i a l s , Media and Solut ions ( i ) Mater ia l s . Bac te r io log i ca l peptone and yeast ext ract were obtained from Oxoid l abora tor ies and Bacto-agar was from Difco l abo ra to r i e s . •AMP, ADP, ATP, GMP, GDP, Cyc l i c AMP, e-glycerophosphate, glucose-6-phosphate, p-nitrophenyl phosphate, a-naphthyl ac id phosphate, phenyl methyl sulphonyl f l u o r i d e , L-ascorbic a c i d , a-methyl-D-mannoside, octy l g lucos ide , s i l v e r n i t r a t e , sodium deoxy cho la te , streptomycin sulphate ,Tr i s and Variamine blue were obtained from Sigma Chemical Co. Ammonium sulphate and sucrose were spec ia l enzyme grade from Schwarz-Mann. Sodium dodecyl sulphate was the s p e c i a l l y pure grade from BDH Chemicals and T r i ton X-100 was obtained from Amersham corporat ion . Acrylamide, b is-acry lamide, ammonium persu lphate , g lyc ine 14 and TEMED were obtained from Bio-Rad Laborator ies . C 5'-AMP and L i q u i f l u o r were from New England Nuclear. Sephacryl-S-300, DEAE-Sephacel, concanavalin A-Sepharose CL-4B and octy l Sepharose CL-4B were obtained from Pharmacia Fine Chemicals and AMP-Agarose and Reactive Blue B-Agarose were from Sigma Chemical Co. A l l other reagents used were the best ava i l ab le grade from F isher S c i e n t i f i c Co. or Sigma Chemical Co. 15 Media 1. Bonner's Sa l ts non NaC-1. K C H ; CaG l 2 . 2H 2 0 Agar Water nut r ient agar. 0.60g (10" 2M) 0.75g (10" 2M) 0.30g. (2 x 10" 3M) 20.00g 2% 1 1i t re 2. HL-5 medium (For large scale c u l t i v a t i o n of c e l l s in 2 l i t r e f l a sks ) Bac te r io log i ca l peptone lOg. Yeast Extract lOg, KH 2 P0 4 0.5M 5.6ml Na 2HP0 4 0.5M 4.3ml Deionised water 650ml. Dextrose 25% 40ml (Separately autoclaved) 16 ( i i i ) Buffers and So lut ions , Bonner's Sa l ts NaCl 0.60gms (10" 2M) KCl 0:.75gms (10" 2M) C a C l 2 ,.2H 20 0.30gms (2 x 10" 3M) Water 1.0 1 i t r e 2. Lower pad so lu t ion KCl. 1.5gms (2 x 10~2M) MgCl 2- 6H 20 0.5gms (2.5 x 10" 3M) Streptomycin Sulphate 0.5gms Phosphate buf fer , 50mM 1 l i t r e (pH 6.5) This so lu t ion was f i l t e r s t e r i l i z e d and stored a t-20 °C . 3. For sodium dodecyl sulphate - polyacrylamide gel e l e c t rophores i s . (a) Acry lamide/bis-acry l amide so lu t ion Acrylamide - 30gms Bis-acrylamide - O.Sgms Water to make up volume to - 100ml. F i l t e r e d through glass wool and stored at 4 °C . 17 (b) Running gel F inal Concentration Acrylamide/bis-acrylamide so lu t ion 7.0ml 7% SDS 10%(W/v) 0.15ml 0.05% Tr i s-C l 0.15M pH 8.8 8.65ml TEMED 0.1ml 0.33% Deionised water 14.05ml Ammonium persulphate 10% (w/v) 0.01ml 0.03% (c) Stacking gel Acrylamide/bis-acrylamide so lu t ion 1.0ml 3% SDS 10% (w/v) 0.05ml 0.05% T r i s-C l 50mM pH 6.8 1.25ml TEMED 0.005ml 0.05% Deionised water 7.55ml Ammonium persulphate 10%(w/v) 0.10ml 0.1% (d) Running buf fer Glycine 2.16gi. (19.2mM) T r i s 0.45g (2.5mM) SDS 0.75g (0.05% W/v) Deionised water 1.5 l i t r e s 18 4. Sta in fo r a l ka l i ne phosphatase a c t i v i t y A. Substrate so lu t ion Mg C l 2 0.03g, Zn S0 4 1.33g a-naphthyl ac id phosphate 0.108g T r i s - CI 0.1M (pH 8.5) 50ml B. Dye so lu t ion Variamine blue 0.2g (4 - amino diphenyl amine diazonium sulphate) Charcoal a pinch T r i s - CI 0.1M (pH 8.5) 50ml Mixed well and f i l t e r e d through a Buchner funne l . This so lu t ion i s always made fresh jus t before use. 5. S i l v e r s ta in ing method fo r prote ins Solut ion A. S i l v e r n i t r a t e 0.8g D i s t i l l e d water 4.0ml So lut ion B. Sodium hydroxide 0.36% 2.1ml Ammonium hydroxide 14.8M 1.4ml 19 Solut ion C Add so lu t ion A dropwise into so lut ion B with constant s t i r r i n g Make up volume to 100ml with d i s t i l l e d water So lut ion D C i t r i c ac id (1%) 2.5ml Formaldehyde (38%) 0.25ml Water 497.25ml 6. S c i n t i l l a t i o n f l u i d Toluene - 200ml T r i ton X - 1 0 0 - 100ml L i q u i f l u o r - 9ml 20 B. METHODS 1. Organism and growth condi t ions An axenic mutant s t r a in Ax-2 of Dictyoste l ium discoideum, capable of growing in a r i c h nut r ient l i q u i d medium was used throughout these s tud ies . The s t r a i n was obtained o r i g i n a l l y from Dr. J .M . Ashworth and has been maintained in our laboratory fo r several years . Logarithmic phase c e l l s were inoculated in 2 l i t r e f l a sks conta in ing 700mls of HL-5 medium ( 61) and incubated at 22°C on a gyratory shaker at 200 r .p.m. Ce l l growth was monitored by d i r e c t c e l l counts in a haemocytometer counting chamber. Ce l l s grew with a generation time of about 9 hours in th i s medium. The c e l l s were harvested when they at ta ined a densi ty of 5 x 10 c e l l s / m l . by cen t r i fuga t ion at 700 x g fo r 10 minutes. 2. Dif ferent iat ion experiments The harvested c e l l s were washed once with co ld deionised Q water and resuspended in the sameata cell! densi ty of 1.5 x 10 c e l l s per ml. A l iquots of 0.3ml were spread uniformly on Whatman Number 50 f i l t e r papers (4.25cm diameter) supported by pads soaked in lower pad so lu t ion ( 14 ) placed ins ide small (5.3cm.diameter) p l a s t i c pe t r i d ishes . The f i l t e r s were incubated at 22°C and observed p e r i o d i c a l l y using a stereo microscope (Olympus) at 60-800fold magn i f i ca t ion . D i s t i n c t migrat ing pseudoplasmodia were formed at 21 21-25 hours and f i n a l f r u i t i n g body construct ion s tar ted around 28 hours and was complete at 36 hours. A l t e r n a t i v e l y , in some experiments, where large quant i t i es of c e l l s were r equ i r ed , the harvested and washed myxamoebae were allowed to d i f f e r e n t i a t e on the surface of Bonner's sa l t s agar(. 7 ) in large p l a s t i c t r ays . (23 x 37.5cm)i . The developmental time-course was s im i l a r to that observed using f i l t e r papers. 3. Membrane ex t rac t ion Crude membranes from Dictyoste l ium c e l l s were prepared fo l lowing the method developed by Gi lkes and Weeks ( 2 0 ) . Harvested c e l l s were washed again in 5mM Tr i s-C l (pH 7.4) conta in ing 8.6% sucrose and resuspended in the same buffer saturated with PMSF at a c e l l o dens i ty of 10 c e l l s per ml. The c e l l s were disrupted by 10 minutes mechanical gr ind ing with g lass beads (0.45 - 0.50 mm diameter) using a magnetic s t i r r i n g bar and s t i r r e r . 3.3gs of beads was used o per 10 c e l l s . This procedure gave more than 90% d i s rupt ion of vegetat ive c e l l s . The culminat ion stage c e l l s were more d i f f i c u l t to d i s rupt by th i s method, and needed gr inding fo r 20 minutes to achieve in excess of 80% c e l l breakage. The unbroken c e l l s and g lass beads were removed by cen t r i fuga t ion at 700 x g fo r 5 minutes. The c e l l f ree supernatant was centr i fuged at 105,000 x g to p e l l e t the crude membrane f r a c t i on which was resuspended with 5mM Tr i s-C l 22 buf fe r (pH 7.4) conta in ing 8.6% sucrose and recent r i fuged . The f i n a l p e l l e t was resuspended in a small volume of the same buf fer to give a f i n a l prote in concentrat ion of about 25 mg , per ml. Membranes prepared in th i s way l o s t l i t t l e or no a l ka l i ne phosphatase a c t i v i t y on storage at -70°C fo r more than one year. 4. Enzyme assays A lka l i ne phosphatase was assayed as descr ibed by Lee et a l . ( 33) with s l i g h t mod i f i ca t i on . Incubation mixtures contained 1 ymole pNPP, 20 ymole MgC l 2 , 50 umoles T r i s-C l (pH 8 .5 ) , 30 ymoles sodium f l uo r i de and enzyme prote in made up to a f i n a l volume of 1.0 ml with deionised water. Sodium f l uo r i de was introduced into the assay as suggested by MacLoad and Loomis ( 41 ) to i n h i b i t any ac id phosphatase a c t i v i t y present. A f te r incubat ion at 30 °C , the react ion was terminated by addi t ion of 1.0ml: of 1M sodium carbonate. Amounts of enzyme and length of incubat ion were chosen c a r e f u l l y to ensure that the react ion was s t i l l l i n e a r with time at the t e rm i -nat ion of the r eac t ion . The r esu l t i ng p rec ip i t a t e was removed by cen t r i fuga t ion (2500 r .p.m. fo r 10 minutes) and the 410 nm absorbance of the supernatant provided a d i r e c t measure of the enzyme a c t i v i t y . A l l absorbances were measured using a Beckman DB-GT spectrophotometer. An ex t inc t i on c o e f f i c i e n t of E = 1.62 x 10 4 was used to ca l cu la te the quant i ty of p-nitrophenol produced. 23 5'- nucleot idase was assayed as descr ibed by Lee et a l . , ( 33) with a 10-fold increase in substrate concentrat ion. Incubations contained 0.2 ymole (0.05 yC i ) AMP, 20 ymoles Mg C l 2 , 50 ymoles T r i s-C l (pH 7.5) and enzyme prote in in a f i n a l volume of 1.0ml made up with de ionised water. A f te r incubat ion at 30 °C , react ions were terminated by p r e c i p i t a t i o n of the react ion mixture using 0.2ml of 0.25M z inc sulphate and 0.2ml of a f r e sh l y prepared saturated so lu t ion of barium hydroxide. The supernatant was separated by cen t r i fuga t ion of the above mixture in a Sorva l l GLC-2 cent r i fuge at 2500 rpm fo r 10 minutes. The r a d i o a c t i v i t y in the supernatant was determined and the rate of hydro lys is of AMP was ca l cu la ted from the non-precipi tab le r a d i o a c t i v i t y . In those experiments where the e f f e c t of pre incubat ion temperature on a l ka l i ne phosphatase or 5 '-nucleot idase was s tud i ed , the enzyme preparat ion was preincubated at the des i red temperature fo r the ind ica ted time. The react ion was i n i t i a t e d by the addi t ion of a small a l i quo t of enzyme(0.05ml to 0.1ml)to the remaining react ion mixture that had been prequi1ibrated at the assay temperature, 30°C. 5. Prote in est imat ion Prote in was determined by the Fo l in procedure (38 ). Prote in determination in T r i ton y;X -100 conta in ing samples was done by the procedure o f Sandermann and Strominger (56 ). 24 6. D i a l y s i s experiments D i a l y s i s of crude membrane preparat ions f o r a c t i v a t i on purposes were ca r r i ed out against more than 100 volumes of T r i s-C l buf fer (pH 7.5) f o r three consecutive days, rep lac ing the d ia l ysa te with f resh buf fer every 24 hours. The enzyme a c t i v i t y was monitored at each buf fer change and maximum a c t i v i t y was not obtained un t i l a f t e r 72 hours d i a l y s i s . Tr i ton ext rac t of crude membranes were d ia l ysed as descr ibed above e i the r against 5mM T r i s - CI buf fe r (pH 7.5) or the same buf fer conta in ing 1% T r i ton x -100. Both procedures gave maximum enzyme a c t i v i t y a f t e r 24 hours of d i a l y s i s . 7. Arrheinius p lots Enzyme a c t i v i t y at d i f f e r e n t temperatures between 10°C and 40°C was determined in a temperature gradient produced in an aluminium block conta in ing four p a r a l l e l rows of holes fo r tes t tubes. The block cooled by c i r c u l a t i n g 50% methanol at -10°C from one end, was e l e c t r i c a l l y heated at the other end. The holes in the aluminium block were p a r t i a l l y f i l l e d with water and the t es t tubes conta in ing the react ion mixtures were placed in these holes . Temperatures were monitored in corresponding tubes in adjacent ho les . Normally incubations were done fo r f ive-minutes and the temperature gradients were stable fo r considerably longer per iods . 25 8. A lka l i ne phosphatase p u r i f i c a t i o n procedures a. S o l u b i l i z a t i o n o f a l ka l i ne phosphatase from crude  membrane preparat ions by butanol or T r i ton x - 100 A f te r i n i t i a l experiments (see Table 1) demonstrated the supe r i o r i t y of butanol and T r i t on X - 100 fo r the s o l u b i l i z a t i o n of a l ka l i ne phosphatase from crude membranes of D.discoideum, a l l subsequent experiments involved e i the r of these two procedures. For T r i t on X - 100 s o l u b i l i z a t i o n , crude membrane preparat ions were suspended in 5mM T r i s - Cl buf fer (pH 7.5) at concentrat ions of . approximately 20mg protein/ml and incubated in the presence of 1% T r i t on X-100 fo r 2 hours at 4 °C . The incubat ion mixture was s t i r r e d occass iona l l y . At the end of the incubat ion the membrane suspension was cent r i fuged at 105,000 x g at 4°C fo r 75 minutes. Butanol ex t rac t ion was done fo l lowing the method used by Ghosh and Fishman ( 19) to s o l u b i l i z e a l ka l i ne phosphatase from human t i s sues . Crude membrane preparat ions in T r i s-C l buf fer conta in ing lOmg protein/ml were treated with 0.4 volumes of co ld butanol ( 4 ° C ) , mixed thoroughly and incubated at 4 °C . The incubat ion was continued fo r 2 hours with in termi t tant s t i r r i n g . This was cent r i fuged at 15,000 x g fo r 30 minutes at 4 °C . The lower aqueous layer was removed and f i l t e r e d through glasswool. Nitrogen was passed through the f i l t r a t e to remove res idual butanol and the f i l t r a t e was then cent r i fuged at 35,000 x g f o r 30 minutes. The resu l t an t supernatant was d ia l ysed against more than 50 volumes of 5mM Tr i s-C l 26 buf fer (pH 7.5) overnight with one change of buf fe r . b. Chromatographic procedures A l l chromatographic procedures were performed at 4°C in the co ld room. ( i ) Sephaciryl - S - 300 Sephaciryl - S - 300 was packed in columns (22 x 1.2cm) and equ i l i b r a t ed with 3 column volumes of 5mM Tr i s-C l buf fer (pH 7.5) conta in ing 1% T r i ton X - 100. In some experiments other detergents were subst i tu ted fo r T r i t on X - 100 and in other experiments no detergent was present . The columns were packed, equ i l i b r a t ed and e luted at a f low-rate of 10ml/hour. Sample volumes were always kept at or below 1% of the column volume. ( i i ) DEAE - 'Sephacel Chromatography DEAE - Sephacel (Pharmacia) was obtained as a preswollen g e l . lOgms (wet weight) of the preswollen material was washed th r i c e with column buf fer (5mM T r i s - C l , pH 7.5 conta in ing 0.1% T r i t on X-100) to remove storage buf fer and packed in a small column. (11 x 0.9 cm). The column was fur ther equ i l i b r a t ed with 3 column volumes of the same buf fe r . In some experiments no detergent was present in the bu f fe r . The flow-rate was adjusted to 10ml/hour and maintained throughout the f r a c t i o n a t i o n . Small volume samples were appl ied with pasteur p ipet tes and large volumes of d i l u t ed samples 27 were siphoned d i r e c t l y onto the column. A f te r app l i ca t i on of the sample, the column was washed with two volumes of column buffer to remove a l l unbound p ro te in . The column was normally e luted with a gradient of 0 to l.OvMNaCl in a tota l of 50ml. (i i i ) ConA - Sepharose Chromatography Pre-swollen concanavalin A - Sepharose 4B was- washed th r i ce with 5mM Tr i s-C l buf fer (pH 7.5) and packed in a column (8 x 2cm). The column was equ i l i b r a t ed with 3 column volumes of 5mM Tr i s-C l buf fer (pH 7.5) conta in ing e i t he r 1% sodium deoxy cholate or 1% T r i ton X - 100. In the absence of detergent there was no binding of enzyme to the column. Usual ly 30ml samples of T r i ton extracts conta in ing approximately 5mg protein/ml were appl ied to the column. The sample conta in ing e i the r ~\% T r i t on X - 100 or 1% sodium deoxy cholate was siphoned in to the column and the column was washed with three column volumes of the same buf fer to remove unbound pro te ins . The flow-rate was maintained at 6mls/hr. The a l ka l i ne phosphatase a c t i v i t y was e luted with two volumes of column buf fer conta in ing 0.2M a-methyl-D-mannoside. Both the prote in and a l ka l i ne phosphatase l eve l s in the washes and e luted f r a c t i ons were monitored. In some experiments a smal ler column (4 x 1cm) was used and propor t ionate ly less sample and buf fer were employed. 28 ( iv ) Other Chromatographic procedures used AMP-Agarose and Reactive blue - B - Agarose were packed in 5ml syringe columns and equ i l i b r a t ed with 3 column volumes of 5mM Tr i s-C l buf fer (pH 7.5). Samples were loaded with >pasteur p ipet tes and the unbound prote in was removed by washing with three column volumes of the same buffer . The columns were e luted with three column volumes of the same buf fer conta in ing 4mM AMP. The enzyme a c t i v i t i e s in the washes and e luted f r a c t i ons were monitored. 4 mis of preswollen Octyl-Sepharose was packed in a 5ml syr inge column and equ i l i b r a t ed with three column volumes of 25mM Tr i s-C l buf fer (pH 7.5) conta in ing 20% (w/v) ammonium sulphate. Prote in samples were t reated with 90% ammonium sulphate. The p rec ip i t a t ed prote in was resuspended in the column buf fer and loaded onto the column. The column was washed with 3 column volumes of the same buf fer to remove a l l unbound prote in and then e luted stepwise with two column volumes each of 10% ammonium sulphate in 25mM Tr i s-C l (pH 7:.5), 25mM Tr i s-C l buffer (pH 7.5) , lOmM T r i s - Cl (pH 7.5) , ImM T r i s - Cl (pH 7.5) , 1% T r i t on x -100 and f i n a l l y 50% ethylene g l y c o l . Enzyme a c t i v i t y was monitored in column washes and a l l e luted f r ac t i ons a f t e r d i a l y s i s of the f r a c t i ons overnight against 5mM T r i s - Cl (pH 7.5) buf fe r . 29 9. Sodium dodecyl sulphate - polyacrylamide gel e lec t rophores i s SDS-polyacrylamide s lab gel e l ec t rophore r i s in 7% acrylamide gels was done e s s e n t i a l l y fo l lowing the method of Laemmli ( 32) , with the fo l lowing mod i f i ca t ions . Only 0.05% sodium dodecyl sulphate was used in the gel and running buf fer s ince a l ka l i ne phosphatase in th i s organism is sens i t i ve to higher concentrat ions of th i s detergent, (eg. see F ig . 21 ), and the T r i s concentrat ion was reduced to 0.15M and 0.05M in the running and stacking gels r e spec t i v e l y , because of i n h i b i t o r y act ion of higher concentrat ions of T r i s . (eg. see F i g . 22 ). The samples were resuspended in sample buf fer conta in ing 5mM Tr i s-C l buffer (pH 6.8) and 20% g l y c e r o l . 0.001% bromophenol blue was present as a t rack ing dye. Usual ly 50 to 100ug prote in of T r i ton extracts of crude membranes in 20 to 50pl or 20 to 50ug prote in of p a r t i a l l y p u r i f i e d preparat ions in 10 to 20yl was loaded on to each well of the g e l . E lec t rophores is was ca r r i ed out fo r 1 hour at 5mA fol lowed by about 90 minutes at 25mA. The gels were removed from the apparatus and sta ined fo r a l ka l i ne phosphatase a c t i v i t y and pro te ins . A lka l ine phosphatase a c t i v i t y was stained by the method descr ibed by L. Fishman ( 16 ). Propanediol buffer used by Fishman was replaced by T r i s-C l buf fe r . The gel conta in ing a l ka l i ne phosphatase a c t i v i t y was immersed in the dye-substrate so lu t ion and incubated at 30°C. Usual ly bands appeared in the gels upon 15 to 20 minutes incubat ion . 30 Proteins were detected using the s i l v e r s ta in ing method as modif ied by Wray et a l . , ( 6 3 ). Gel to be stained was soaked in 50% methanol overnight . The methanol was removed and the gel was allowed to swell to normal s ize fo r about two hours in deionised water. The gel was sta ined with so lu t ion C fo r 15 minutes with constant gentle a g i t a t i o n . The gel was washed again in deionised water fo r 5 minutes and the s ta in was developed by soaking the gel in so lu t ion D unt i l bands appeared. Usual ly the bands appeared in about 10 minutes. 10. Preparation of concentrated d ia l ysa te 5 mis of vegetat ive c e l l membranes (containing approximately 20 mg protein/ml) was d ia lysed against about 50 mis of s t e r i l e 5 mM Tr i s-C l bu f f e r , pH 7 .5 . fo r 72 hours with buffer changes a f t e r every -24 hours. The d ia l ysa tes were pooled and concentrated in a f l a sh evaporator (Buchi) at 50°C to a f i n a l volume of 5 mis. 31 RESULTS  SECTION I CRYPTIC EXISTENCE OF ALKALINE PHOSPHATASE IN VEGETATIVE MEMBRANES OF D.DISCOIDEUM AND ITS IMPLICATION ON THE DEVELOPMENTAL REGULATION OF THIS ENZYME a) Reversible heat ac t i va t ion of the membrane bound a lka l i ne phosphatase. When crude membrane preparat ions of D.discoideum were stored at 4 ° C , the a l ka l i ne phosphatase a c t i v i t y was seen to decrease cons iderab ly . The a c t i v i t y was restored i f the membranes were incubated at room temperature, and a dramatic increase in a c t i v i t y was observed i f they were incubated at e levated temperatures. With increas ing temperature the rate and magnitude of ac t i va t ion increased, reaching a maximum at 50°C (F ig . 2 ) . The membranes were found to re ta in th i s e levated a c t i v i t y fo r as long as s ix hours at 50°C. The incubation at 60°C led to var iab le resu l t s and 70°C treatment resu l ted in tota l i n a c t i v a t i on of the enzyme (Data not shown). This heat ac t i va t i on was found to be t o t a l l y r e ve r s i b l e . A precooled membrane preparat ion (0 °C fo r 24 hours) was incubated at 50°C fo r 2 hours, and a l ka l i ne phosphatase a c t i v i t y increased markedly ( F ig . 3) . The preparat ion was then d iv ided and one port ion was stored at 0°C for 24 hours and the other port ion was rap id l y frozen in 32 20-1 c £ TIME (hours) Figure 2 The e f f e c t of preincubat ion temperature on the a c t i v i t y of membrane bound a lka l i ne phosphatase. Vegetative c e l l membranes were resuspended in 5mM T r i s - C l , pH 7.4 at 4mg.ml -and incubated at 13 .5 °C (o ) , 210c ( A ) , 33 .50C (x) and 50 °C ( • ). A f t e r the ind icated periods o f incubat ion , 0.1ml. a l iquots of the membrane were assayed fo r a l ka l i ne phosDhatase a c t i v i t v at 30 °C . 33 20-i •—» c a TIME (hours) Figure 3. The reve rs ib le heat ac t i va t i on of a lka l ine phosphatase. Crude vegetat ive membranes (4;mg.ml~l in 5mM Tr i s-C l , pH 7.5) were incubated at 50°C (o). A f te r 2 hours, the preparat ion was d iv ided into two and one port ion (•) was incubated fo r 24 hours at 0°C fol lowed by 1 hour at 50°C. The second sample ( A ) was r ap id l y cooled to -70°C and maintained at -70°C fo r 24 hours and then incubated at OOC for 24 hours, fol lowed by 1 hour at 50°C. At the ind icated time points (o, n , A ) 0.1 ml a l iquots were assayed, for a l ka l i ne phosphatase a c t i v i t y . 34 acetone/dry ice and stored at -70°C fo r 24 hours. The port ion stored at 0°C l o s t considerable a l ka l i ne phosphatase a c t i v i t y , but when reincubated at 50 °C , a c t i v i t y was rap id l y restored, ( F ig . 3 ) . This heat a c t i v a t i on and co ld i nac t i va t i on could be repeated in the same sample showing that the phenomenon was t o t a l l y r eve rs ib le , ( F ig . 3 ) . The port ion stored at -70°C reta ined the elevated heat-act ivated leve l of a c t i v i t y on thawing (F ig . 3 ) , but on subsequent t r ans fe r to 0 ° C , the a c t i v i t y dec l ined markedly over a 24 hour per iod . This could a lso be react iva ted by incubat ing at 50°C. b) S o l u b i l i z a t i o n of a l ka l i ne phosphatase from vegetat ive c e l l membranes. To inves t igate th i s temperature dependent r eve rs ib l e a c t i v a t i on fu r the r , attempts were made to s o l u b i l i z e a l ka l i ne phosphatase from crude vegetat ive c e l l membranes. Rossomando and Cut le r ( 52 ) reported the s o l u b i l i z a t i o n of 5 '-nucleot idase from the plasma membranes of vegetat ive c e l l s of D.discoideum using 0.1 - 0.2% Tr i ton X-100 and MacLeod and Loomis (41 ) used 0.1% T r i ton X-100 to s o l u b i l i z e a l ka l i ne phosphatase from both vegetat ive and culminat ing c e l l membranes. Armant and Rutherford ( 3 ) used T r i ton X-100 to s o l u b i l i z e th i s enzyme from l y o p h i l i z e d culminat ing c e l l s and found 0.5% T r i ton X-100 to give 80% s o l u b i l i z a t i o n of the enzyme. However, Lee et a l . , ( 33 ) found that sodium deoxycholate was a better s o l u b i l i z i n g agent than T r i ton X-100. Hence, a comparison 35 of several commonly employed procedures fo r s o l u b i l i z a t i o n of membrane-bound prote ins was made to determine the best method fo r s o l u b i l i z i n g a l ka l i ne phosphatase from crude membranes of vegetat ive c e l l s . Butanol and Tr i ton X-100 treatments s o l u b i l i z e d a large port ion of the a l ka l i ne phosphatase a c t i v i t i e s and both treatments increased the tota l enzyme a c t i v i t y . (Table I). Sodium deoxycholate and sodium dodecyl sulphate were less e f f i c i e n t in re leas ing a l ka l i ne phosphatase from the membrane and both o f these detergents decreased the to ta l enzyme a c t i v i t y . Treatments of the membrane with 3M KC1 or NaCT or 2.5% octy l g lucoside re leased only neg l i g i b l e amounts of enzyme a c t i v i t y in to the supernatant, while 20mM EDTA was found to be h igh ly i n h i b i t o r y f o r the enzyme (Table I). c) E f f e c t of 50°C treatment on a l ka l i ne phosphatase s o l u b i l i z e d with butanol and T r i ton X-100 The a l ka l i ne phosphatase a c t i v i t y in the butanol ext rac t showed higher s p e c i f i c a c t i v i t y than the heat ac t i va ted l e ve l s in the vegetat ive membrane (Table I I ) . When the butanol s o l u b i l i z e d enzyme was incubated at 0°C fo r 24 hours, there was no loss in a c t i v i t y , while incubat ion at 50°C fo r 60 minutes produced approximately 45% reduct ion in the enzyme a c t i v i t y , ( T a b l e . IT) behaviour very d i f f e r e n t from that of the enzyme of the in tac t membrane; suggesting that membrane i n t e g r i t y might be essent ia l fo r the heat-act ivat ion Table I Solubilization of alkaline phosphatase from crude vegetative membranes of D.discoideum Treatment Total activity Solubilization % Specific activity (A0.D., l n.min-l ) (percent original fc0D.min~l.mg~l ) Pellet activity Supernatant Pellet Supernatant Pellet Supernatant None 7.0 0.0 100% 0.0 0.14 0.00 1% Sodium deoxy cholate 2.2 2.0 31.0 28.5 0.12 0.04 0.1% Sodium dodecyl sulphate 4.9 0.0 69.3 0.0 0.11 0.00 1% Triton X-100 2.7 13.5 38.5 193 0.06 0.29 20mM EDTA 0.3 0 4.3 0.0 0.01 0.00 2.5% Octyl glucoside 48.4 1.9 690.0 26.7 0.96 0.17 3M NaCla 36.8 0.1 525.0 1.8 0.76 0.01 3M KCl a 40.7 0.3 581.0 4.3 0.89 0.04 28.5% Butanolb 8.0 21.0 114.0 300.0 0.27 1.20 a. Activities measured following dialysis b. Activity in the aqueous phase after butanol treatment Table II Comparison of the e f fec ts o f preincubation at 50 UC and 0°C on the a lka l ine phosphatase a c t i v i t y of in tac t membrane and butanol - extracted preparat ions sp. a c t i v i t y of a l ka l i ne phosphatase  Preparation No treatment Preincubated at Preincubated at 5QOC fo r 60 min. 0°C fo r 24 hours Intact membrane 9.2 Butanol extract 105 (n^moles. min.~ .mg p ro t e i n " ) 74.0 3.1 56.0 96.0 38 phenomenon. However, i t was subsequently found that the enzyme a c t i v i t y s o l u b i l i z e d by T r i ton X-100 behaved l i k e that in the in tac t vegetat ive membrane. Figure 4 shows the e f f e c t of 50°C treatment on a T r i ton X-100 ex t r ac t . The T r i t on X-100 ext rac t i s ac t i va ted by incubat ion at 50°C (F ig . 4). On coo l ing of the act ivated Tr i ton ext rac t f o r 24 hours at 0°C the preparat ion reaches o r i g i na l low l eve l s of a c t i v i t y . The a l ka l i ne phosphatase of th i s cooled ext rac t can be fu r ther heat ac t i va ted by re-incubat ion at 50°C ( F ig . 4). d) Ac t i va t ion of a l ka l i ne phosphatase by d i a l y s i s It was a lso found that d i a l y s i s o f crude membrane preparat ions of vegetat ive c e l l s fo r three days ac t i va ted the a l ka l i ne phosphatase by about 10-fold suggesting that the removal of a low molecular weight i n h i b i t o r ( s ) was responsib le fo r the a c t i v a t i o n . The d ia l ysed preparat ion could not be fur ther ac t iva ted by incubat ion at 50°C (Table I I I ) , suggesting that the a c t i v a t i on caused by heat treatment i s a lso due to the removal of an i n h i b i t o r from the enzyme. Table III a lso shows the e f f e c t of d i a l y s i s of a T r i ton X-100 extract of vegetat ive membranes. The s p e c i f i c a c t i v i t y of the T r i ton ext rac t increased 10-fold upon d i a l y s i s . The ac t i va t ion of the T r i ton ext rac t was achieved by overnight d i a l y s i s , in contrast to the extensive d i a l y s i s needed fo r ac t i va t i on of the enzyme in the in tac t membrane. The d ia l ysed T r i ton extract Was a lso stable to treatment at 50°C fo r 1 hour, in contrast to the butanol - s o l u b i l i z e d enzyme. These 39 300 0 .5 1 25 25.5 TIME (hours) 26 Figure 4. E f f ec t of 50 C and 0 C incubations on the a l ka l i ne phosphatase a c t i v i t y of i n tac t membranes and Tr i ton X-100 extracted membranes. Intact membranes at 4.0mg protein.ml"1 (o) and Tr i ton X-100 extracted membranes at 1.5 mg p r o t e i n . m l " 1 (A ) were incubated at 50°C in 5mM Tris-CT, pH 7.5. At the f i r s t arrow,.the samples were t rans fe r red to 0°C and incubated fo r 24 hours. At the second"arrow, the samples were t rans fer red back to 50°C. .A t the ind icated time po in t s , 0.1ml a l iquots were assayed fo r a l ka l i ne phosphatase a c t i v i t y . TABLE III E f fec t of d i a l y s i s and subsequent 50 C treatment on a l ka l i ne phosphatase a c t i v i t y of vegetative membranes and Tr i ton X-100 extracts Spec i f i c a c t i v i t y of a l ka l i ne phosphatase Preparation Before A f te r Incubation at 50°C for d i a l y s i s d i a l y s i s 1 h o u r „ a f t e r d i a l y s i s (nmoles. m i n - 1 . mg p r o t e i n - 1 ) Vegetative membrane 9.5 100 123.4 T r i ton X-100 extract 37.0 370 323.3 41 resu l t s suggest that the putat ive i n h i b i t o r i s extracted by T r i t on X-100 along with the enzyme. This i n h i b i t o r could then be removed from the enzyme by heat-treatment or d i a l y s i s . In con t ras t , the butanol s o l u b i l i z a t i o n procedure involves a d i a l y s i s step which i s essent ia l f o r a c t i v i t y which probably removes th i s i n h i b i t o r . Hence the butanol s o l u b i l i z e d preparat ion could not be fur ther ac t iva ted by 50°C treatment or fu r ther d i a l y s i s . e) E f f ec t of incubat ion at 50°C and d i a l y s i s on a l ka l i ne phosphatase in membranes of c e l l s from d i f f e r e n t stages of d i f f e r e n t i a t i o n Membranes from d i f f e r e n t stages of development were subjected to incubat ion at 50°C and d i a l y s i s , to determine i f th i s a c t i v a t i on phenomenon was exh ib i ted throughout the d i f f e r e n t i a t i o n process. Membranes from four d i f f e r e n t stages were used in th i s experiment namely vegeta t i ve , pseudoplasmodia!, ea r l y culminat ion and la te culminat ion stages, obtained a f t e r 0, 22, 30 and 34 hours of d i f f e r e n t i a t i o n , r espec t i ve l y . The a l ka l i ne phosphatase a c t i v i t y of f r e sh l y prepared membranes from c e l l s at l a t e r stages of development was cons iderably higher than that of vegetat ive c e l l s , reaching peak l eve l s at the culmination stages of development (Table IV) as shown by previous workers (18, 37). In the culminat ing c e l l membranes there was no decrease in a l ka l i ne phosphatase a c t i v i t y on storage at 0°C fo r 24 hours, and only a marginal e levat ion in a c t i v i t y on incubat ion at 50°C fo r 1 hour suggesting that they d id not respond to heat Table IV Comparison of the effects of heat treatment and dialysis on the alkaline phosphatase activity of membranes prepared from Ax-2 cells at various stages of development Developmental Stage Developmental time (hrs.) Specific activity of alkaline phosphatase (nmoles.min-1.mg-1) Freshly prepared membranes Preincubated at 0°C for 24 hrs. Preincubated at 50OC for 1 hr. After dialysis Vegetative 0 9.2 3.1 74.0 108.0 Pseudoplasmodium 22 43.0 71.0 105.0 86.0 Early culmination 30 108.0 105.0 139.0 129.0 Late culmination 34 105.0 92.5 133.0 126.0 43 ac t i v a t i on or co ld i n a c t i v a t i o n . A lka l ine phosphatase from pseudoplasmodial c e l l s , which was almost 5-times more act ive than the vegetat ive membrane enzyme, showed only pa r t i a l heat ac t i va t i on (Table IV). Incubation of the pseudoplasmodial enzyme at 0°C fo r 24 hours produced a s l i g h t increase in i t s s p e c i f i c a c t i v i t y . The peak a c t i v i t i e s of a l ka l i ne phosphatase in culminat ing membranes i s of the same order of magnitude as that of the a c t i v i t i e s in the heat-treated vegetat ive and pseudoplasmodial membranes. S imi la r r esu l t s were obtained in d i a l y s i s experiments (Table IV). Membranes from a l l four developmental stages were d ia l ysed extens ive ly to remove any low molecular weight i n h i b i t o r . Again, the culminat ing enzymes showed only marginal a c t i v a t i o n , while the pseudoplasmodial enzyme showed a two f o l d ac t i va t i on and the vegetat ive enzyme was ac t i va ted 12-fold (Table IV). The f i n a l a c t i v i t i e s a f t e r d i a l y s i s in a l l four stages of development were of the same order of magnitude. The above resu l t s ind icated that the increase in a l ka l i ne phosphatase a c t i v i t y during development of D.discoideum may be due to the removal of a low molecular weight i n h i b i t o r ( s ) , thus unmasking already ex i s t i ng enzyme. This phys io log ica l phenomenon might be mimiced by treatment of the vegetat ive membranes at 50°C or by extensive d i a l y s i s of the membranes. 44 f ) Reconst i tut ion of the d ia lysed vegetat ive membrane with the putat ive i n h i b i t o r Experiments were performed to t r y to reconst i tu te masked a l ka l i ne phosphatase. A preparat ion of the putat ive i n h i b i t o r was made as descr ibed under methods and added to a d ia l yzed ac t i va ted membrane preparat ion . Figure 5 shows the resu l t s of incubations of mixtures of d ia l yzed crude membranes and the putat ive i n h i b i t o r at r a t i os of 1:1, 2:1 and 4:1 and they gave about 85%, 72% and 42% i n h i b i t i o n of a l ka l i ne phosphatase respec t i ve l y a f t e r f i v e days incubat ion at 0 °C . The i n h i b i t i o n could be reversed by d i a l y s i s (Figure 5 ) . Since the i n h i b i t o r preparat ion contained a higher con-cent ra t ion of T r i s - C l , a separate control was employed to determine the e f f e c t of lOOmM Tr i s-C l on enzyme a c t i v i t y . About 90% of the o r i g i n a l a c t i v i t y of the enzyme was reta ined in th i s contro l a f t e r f i v e days of incubat ion (F ig . 5) . Inorganic phosphate is a known i n h i b i t o r fo r a l ka l i ne phosphatase and hence a l i k e l y candidate fo r the putat ive i n h i b i t o r . The i n h i b i t i o n of the a l ka l i ne phosphatase of a d ia lysed membrane preparat ion by phosphate was immediate and did not apprec iably increase during subsequent incubat ion (F ig . 6 ) , while i n h i b i t i o n with the putat ive i n h i b i t o r was only gradual ly acquired (F ig . 5, 6 ) . Attempts to ac t i va te the reconst i tu ted membranes by incubat ion at 50°C were not successful (Table V). The concentrated d ia l ysa te could a lso i n h i b i t enzyme a c t i v i t y in d ia l ysed Tr i ton extracts of vegetat ive 45 Figure 5. Reconst i tut ion of the d ia lysed vegetat ive membrane with a concentrated d ia l ysa te preparat ion. Mixtures of d ia l ysed vegetat ive membranes at approximately 1.5mg prote in m l - 1 in 5mM Tr i s-C l and the concentrated d ia l ysa te at r a t ios of 1:1 ( A ) , 2:1 (+) and 4:1 (•) were incubated at 0 ° C , One c o n t r o l , d ia l ysed vegetat ive membranes in 5mM T r i s - C l , pH 7.5 (o) and a second c o n t r o l , d ia lyzed vegetat ive membranes in 100 mM T r i s - C l , pH 7.5 (x) were a lso incubated at 0 ° C . A f te r 5 days (at the arrow) the 1:1 reconst i tu ted preparat ion and the 5mM Tr i s-C l control were d ia l yzed against 5mM T r i s - C l , pH 7.5 fo r 5 days, rep lac ing the d i a l y s i s buf fer every 24 hours. A lka l ine phosphatase a c t i v i t y in 0.1ml a l iquots was determined at the ind icated time per iods . Note: The data are from one representat ive experiment. S imi la r r esu l t s were obtained in three r e p e t i t i o n s . OPTICRL DEN5ITT( 4 1o n m)/10 min « » * Q ro -f^  cr> 47 TIME (Hours) Figure 6. Comparison of the i n h i b i t i o n of a l ka l i ne phosphatase a c t i v i t y by inorganic phosphate and the concentrated d i a l y sa t e . Dialysed vegetat ive membrane preparat ions at approximately 1.5mg protein.ml"1 were reconst i tu ted with concentrated d ia l ysa te at 1:1 r a t i o (o) or with 100 mM KH 2Po 4/Na 2HPo 4 buf fer (x) and incubated at 0 ° C , A l iquots (o.lmT) were removed at the ind icated time points and a lka l i ne phosphatase a c t i v i t y was determined. The values are expressed as percentages of a control d ia lyzed membrane suspended in 5mM Tr i s-C l bu f f e r , pH 7.5. 48 Table V E f f ec t of addi t ion of d ia l yza te on a l ka l i ne phosphatase a c t i v i t y . Sample a l ka l i ne phosphatase a c t i v i t y (n.moles, min-1.ml-1) Dialyzed vegetat ive membrane 153 Dialyzed vegetat ive membrane + d i a l y z a t e 9 23 Dialyzed vegetat ive membrane + d ia l yza te a f t e r 1 hour at 50OC 13 Dialyzed Tr i ton X-100 extract of vegetat ive membrane 175 Dialyzed T r i ton X-100 extract of vegetat ive membrane + d i a l y z a t e . 9 26 Dia lyzed T r i ton X-100 extract of culminat ing membrane 111 Dialyzed T r i ton X-100 extract of culminating membrane + d i a l y z a t e 9 16 a. Dia lysate preparat ion was added to extracts at a 1:1(v/v) r a t i o and incubated fo r 5 days at 0 °C . 49 or culminat ing membranes. (Table V). g) E f f ec t of coincubat ion of vegetat ive and culminating membranes Since the i n h i b i t o r in the d ia l ysa te c l e a r l y i nh ib i t ed both the vegetat ive and culminat ing phase enzymes, i t was of i n t e res t to determine the e f f e c t of coincubat ing vegetat ive membranes with culminat ing membranes on a l ka l i ne phosphatase a c t i v i t y . Membrane preparat ions from vegetat ive and culminat ing c e l l s that had been pre-incubated at 0°C fo r 24 hours were mixed and incubated at 0 °C . The a c t i v i t y of the mixture was monitored at i n t e r v a l s , along with unmixed vegetat ive and culminating membranes as cont ro ls (Table VI). It was found that the combined a c t i v i t y of the mixed membranes was reduced by 60% a f t e r 19 hours incubat ion. The a c t i v i t i e s of unmixed vegetat ive and culminat ing membranes did not change apprec iably during the incubat ion per iod . The experiment suggested that the i n h i b i t o r present in the vegetat ive membrane preparat ion can i n h i b i t the enzyme in the culminat ing membrane. h) E f f e c t of temperature and d i a l y s i s on the membrane bound 5 1 -nucleot idase Because of the co-pur i f i c a t i on and s im i l a r propert ies of a l ka l i ne phosphatase and 5 '-nucleot idase ( 3 ), the e f f e c t s of 50°C treatment and d i a l y s i s on membrane bound 5 '-nucleot idase was a lso s tud ied . When vegetat ive membrane preparat ions were 50 Table VI E f f ec t of coincubation of precooled vegetat ive and culminating membranes A lka l ine phosphatase a c t i v i t y a f t e r Sample incubation at 0 C ^ 0 D 4 1 0 / 1 0 m i n assay^ 0 hr. 7 hrs . 19 hrs . Vegetative membrane 0.05 0.08 0.09 Culminating membrane 0.35 0.36 0.39 Vegetative membrane plus culminat ing membrane 0.43 (0 .40 ) a 0.23 (0 .44 ) a 0.19 . ( 0 . 4 8 T a. Values in brackets show expected combined a c t i v i t i e s of the mixed membranes. Note The data are from.one representat ive experiment. S im i l a r resu l t s were obtained in three r e p e t i t i o n s . 51 incubated at 50 C, the 5 1 -nucleot idase a c t i v i t y decreased by about 30% in contrast to a l ka l i ne phosphatase measured in the same preparat ion which. increased 10-fold within an hour ( F ig . 7) . When a crude membrane was incubated at 50°C for prolonged per iods , the 5 '-nucleot idase a c t i v i t y was t o t a l l y e l iminated at the end of 10 hours whereas more than 55% of the heat ac t iva ted a l ka l i ne phosphatase a c t i v i t y was reta ined ( F ig . 8 ) . D i a l y s i s of vegetat ive membranes produced only a marginal increase in 5 1 -nucleot idase a c t i v i t y (Table VI I ) , whereas a l ka l i ne phosphatase a c t i v i t y increased 10 f o l d . However, when T r i ton X-100 s o l u b i l i z e d extracts were d i a l y z e d , 5 1 -nucleot idase showed a large increase in a c t i v i t y (Table VI I ) , s im i l a r to that observed fo r a l ka l i ne phosphatase a c t i v i t y in a s im i l a r T r i ton ex t rac t . (Table III). i ) Summary It was found that the a l ka l i ne phosphatase a c t i v i t y in crude vegetat ive membranes of D.discoideum exis ted in a masked form. Enzyme a c t i v i t y could be increased 8 to 10 f o l d by incubat ing the membranes at 50°C or by d i a l y s i s suggesting the removal of a low molecular weight i n h i b i t o r . The ac t i va t i on at 50°C was found to be reversed by incubat ion at 0 °C . When s o l u b i l i z e d from the membranes using Tr i ton X-100, the enzyme s t i l l exh ib i ted reve rs ib l e heat ac t i va t ion and ac t i va t ion by d i a l y s i s . The fac t that the culminat ing enzyme could not be ac t iva ted by d i a l y s i s or 50°C treatment suggested that the developmental increase 52 f 25-f l 1 1— 1 • 0 20 40 60 TIME (min) Figure 7. Comparison of the e f f e c t of preincubat ion at 50°C on the membrane bound a l ka l i ne phosphatase and 5 '-nucleot idase a c t i v i t i e s in vegetat ive c e l l s . A vegetat ive c e l l membrane preparat ion (4mq p r o t e i n . m l - 1 in 5mM T r i s - C l , pH 7.5) was incubated at 50°C fo r one hour and a lka l ine phosphatase (o) and 5 '-nucleot idase (A ) a c t i v i t i e s were determined at ind ica ted time po in ts . 53 Id h5 Ul *3 T J -P O <U U 3 c ID 6 8 TIME (Hours) Figure 8. Comparison of the e f f e c t of long term incubation at 50°C on the membrane bound a l ka l i n e phosphatase and 5 1 -nuc leot idase a c t i v i t i e s in vegetat ive c e l l s . A crude vegetat ive membrane preparat ion (about 4mg p r o t e i n . m l - ' in 5mM T r i s - C l , pH 7.5) was incubated at 50°C fo r 10 hours and a lka l ine phosphatase (0) and 5 1 -nuc leot idase ( A ) a c t i v i t i e s were determined at ind icated time po in ts . 54 Table VII E f f ec t of d i a l y s i s on membrane bound and T r i ton X-100 extracted 5 1 -nucleot idase a c t i v i t i e s from vegetat ive membranes of D-discdideum Sample Spec i f i c a c t i v i t y of 5 '-nucleot idase (nmoles. m i n - ! mg-1) Vegetative membrane 5.2 Dia lysed vegetat ive membrane 8.9 T r i ton X-100 ext rac t 2.7 Dia lysed T r i ton X-100 ext rac t 31.1 55 of a l ka l i ne phosphatase in th i s organism was brought about by the removal of the low molecular weight i n h i b i t o r . The addi t ion of concentrated d ia l ysa te to d ia l yzed membrane preparat ions or d ia l yzed T r i ton X-100 extracts resu l ted in enzyme i n h i b i t i o n . This i nh ib i t ed enzyme could be react ivated by d i a l y s i s . A second and s i m i l a r l y developmentally regulated enzyme a c t i v i t y , 5 '-nucleot idase d id not show ac t i va t ion e i the r on incubation at 50°C or d i a l y s i s of vegetat ive membrane preparat ions. However, when T r i ton X-100 extracts were d i a l y z e d , the 5 '-nucleot idase was act iva ted to the same extent as the a l ka l i ne phosphatase. 56 SECTION II PURIFICATION OF ALKALINE PHOSPHATASE FROM VEGETATIVE MEMBRANES OF D. discoideum a) P u r i f i c a t i o n using butanol ext rac t ion for s o l u b i l i z a t i o n . Treatments with e i t he r T r i ton X-100 or butanol s o l u b i l i z e d appreciable amounts of the to ta l al-kaline-phosphatase a c t i v i t y from crude membrane preparat ions. (Table I) The a c t i v i t y in the butanol ext rac t was f ree of detergents and f u l l y ac t iva ted while that in the T r i ton ext rac t was s t i l l masked. Hence i n i t i a l attempts at p u r i f i c a t i o n of a l ka l i ne phosphatase were performed using butanol extracts of crude vegetat ive membranes. Conventional ammonium sulphate p r e c i p i t a t i o n procedure gave only marginal p u r i f i c a t i o n and recovery of the enzyme (Table VI I I ) , despite the fac t that th i s procedure had been e f f e c t i v e in pu r i f y i ng the mammalian enzyme ( 19 ). When the butanol ext ract was chromatographed on a Sephacryl S-300 gel f i l t r a t i o n column, the enzyme a c t i v i t y and most of the prote in were excluded from the column suaqesting that the enzyme was in a h igh ly aggregated form. ( F ig . 9a). The butanol ext rac t was treated with detergents p r i o r to gel f i l t r a t i o n , to see i f the enzyme could be disaggregated. As shown in f igures 9b and 9c , treatment of the butanol extracted material with 1% Tr i ton X-100 or 0.1% sodium dodecyl sulphate d id not reduce the apparent molecular weight of the enzyme. However, when treated with 1% sodium deoxy cho la te , the enzyme was included in 57 Table VIII Ammonium sulphate f r ac t i ona t ion of butanol extracted a l ka l i ne phosphatase from vegetat ive membranes of D.discoideum Sample Spec i f i c a c t i v i t y of- a l ka l i ne phosphatase (AOD^Q.min-l.mg-" 1) Y i e ld a (per cent o r ig ina l a c t i v i t y ) Butanol ext rac t 1.2 100 80-90% ammonium sulphate so luble f r a c t i on 1.03 0.07 70-80% ammonium sulphate so luble f r a c t i on 2.5 1.1 60-70% ammonium sulphate so luble f r a c t i on 4.2 7.5 50-60% ammonium sulphate soluble f r a c t i on 0.98 4.5 40-50% ammonium sulphate soluble f r a c t i on 1.0 2.5 40% ammonium sulphate p rec ip i t a t e 1.05 23.5 a. Expressed as percentage of a c t i v i t y recovered in each f r a c t i o n . 58 Figure 9 Gel f i l t r a t i o n on Sephacryl-5-300 of butanol extracted a l ka l i ne phosphatase from crude vegetat ive membranes. 9.a . Butanol ex t rac t . 9.b. Butanol extract In 1% Tr i ton X-100; polumn equ i l i b r a t ed with 1% T r i ton X-100. 9.c. Butanol ext ract in b.1% sodium dodecyl su lphate ; column equ i l i b r a t ed with 0.1% sodium dodecyl: sulphate. 9.d Butanol ext ract t reated with 1% sodium deoxy cho la te ; column equl ibrated with 1% sodium deoxy cho la te . Protein concentrat ions are expressed as opt i ca l densi ty at 280nm (x) and the enzyme a c t i v i t y as op t i ca l dens i ty at 410nm per 0.1ml a l i quo t o f the f r ac t i ons fo r lOmin react ion time (o). The arrow shows the volume at which the Blue dextran peak was e lu ted . 59 FRACTION VOLUME (ML) 60 the gel f i l t r a t i o n column, well separated from the major prote in peak. (Figure 9d). Although the recovery of enzyme a c t i v i t y in a l l the gel f i l t r a t i o n experiments was greater than 75%, the sodium deoxy cholate t reated a c t i v i t y was unstable upon storage and upon subsequent p u r i f i c a t i o n procedures. The butanol ext rac t was a lso chromatographed using an ion exchange column of DEAE - sephacel . The enzyme a c t i v i t y was t o t a l l y bound to the column, and was then e luted by a 0 to l .OMNaCl gradient ( F ig . 10). The recovery of enzyme a c t i v i t y by th i s procedure was about 75%. A f f i n i t y chromatography of the butanol extracted enzyme on concanavalin A-Sepharose, AMP-Agarose and Reactive blue-B.-Agarose matrices were attempted. About 55% of the s o l u b i l i z e d a c t i v i t y d id not bind to a conA - Sepharose column (Table IX). No fur ther a c t i v i t y could be e luted by using 0.2M a-methyl-D-mannoside. None of the a c t i v i t y loaded onto e i t he r AMP-Agarose or Reactive blue-B-Agarose was bound. (Table IX). When the butanol extract was pretreated with 1% sodium deoxy cholate p r i o r to loading on to a conA-Sepharose column already equ i l i b r a t ed with buf fer conta in ing 1% sodium deoxy cho la te , the enzyme was found to bind to the column. The bound enzyme could be e luted from the column with 0.2M a-methyl-D-mannoside (Figure 11). The recovery of enzyme a c t i v i t y from th i s column was 27%. 61 FRACTION VOLUME (ML) Figure 10 Ion exchange chromatography on DEAE-Sephacel of butanol extracted a l ka l i ne phosphatase from crude vegetative membranes. Prote ins(x) and a l ka l i ne phosphatase a c t i v i t y (o) were e luted with a NaCl gradient of 0 to 1M. Protein i s expressed as opt i ca l dens i ty at 280nm and a l ka l i ne phosphatase a c t i v i t y as the opt i ca l density at 410nm per 0.1ml a l iquots of f r ac t i ons fo r a 10 minute react ion time. Table I X A f f i n i t y chromatography of butanol s o l u b i l i z e d a lka l ine phosphatase a c t i v i t y in three gel matrices Gel used A lka l ine phosphatase a c t i v i t y (A 0 D 4 1 Q . m i n )^ Applied to the column Recovered unbound Eluted Concanavalin A Sepharose 3.4 1 . 9 o a AMP-Agarose 3.4 5.6 O b Reactive blue-B-Agaro.se 3.4 5.4 O b a. E lut ion ca r r i ed out using 0.2M a-methyl-D-mannoside b. E lut ion ca r r i ed out using 4mM AMP. 63 I n FRACTION VOLUME (ML) Figure 11. A f f i n i t y chromatography on concanavalin A-Sepharose of butanol extracted a l ka l i ne phosphatase from vegetat ive c e l l s a f t e r treatment with 1% sodium deoxy cho la te . 1 A4 x 1cm column of concanavalin A-Sepharose equ i l i b r a t ed with 5mM Tr i s-C l bu f f e r , pH 7.5 conta in ing 1% sodium deoxycholate was used in th i s experiment. Proteins (x) and a l ka l i ne phosphatase a c t i v i t y (o) were e luted using 0.2M a-methyl-D-mannoside. Protein i s expressed as opt i ca l density at 280nm and a lka l i ne phosphatase a c t i v i t y as the opt i ca l density at 410nm per 0.1ml a l iquots of f r ac t ions fo r a 10 minute incubation per iod . 64 Attempts at p u r i f i c a t i o n by hydrophobic chromatography were a lso unsuccess fu l . The material p rec ip i t a ted from the butanol ext ract using 90% ammonium sulphate was loaded on to an octy l Sepharose column. The e lu t i on was attempted using decreasing concentrat ions of ammonium sulphate and increas ing concentrat ions of T r i ton X-100 and with ethylene g l y c o l . Only 17% of the enzyme a c t i v i t y was recovered by e lu t i on with 1% Tr i ton X-100 and no a c t i v i t y was e luted using other e luants . (Table X). Having es tab l i shed the chromatographic behavior of the butanol-extracted enzyme using a va r ie ty of procedures, attempts were made to pur i f y the butanol extracted a l ka l i ne phosphatase according to the p u r i f i c a t i o n Scheme I. 65 Table X Octyl Sepharose chromatography of butanol extracted a l ka l i ne phosphatase from vegetat ive membranes of D.discoideum A lka i ine phosphatase a c t i v i t y (O.D.min-1) Recovery (percent i n i t i a l a c t i v i t y ) A c t i v i t y appl ied to the column 1.1 100 E lu t ion with 10%(NH 4 ) 2 So 4 0 0 E lu t ion with 25mM Tr i s-C l 0 0 E lu t ion with lOmM Tr i s-C l 0 0 E lu t ion with ImM Tr i s-C l 0 0 E lu t ion with 1% Tr i ton X-100 0.19 17 E lu t ion with 50% ethylene glycol 0 0 66 PURIFICATION SCHEME I Crude vegetat ive membrane Butanol ex t rac t ion Ion exchange chromatography on DEAE - sephacel Treatment with 1% sodium deoxycholate A f f i n i t y chromatography on conA-Sepharose Gel F i l t r a t i o n on Sephacryl S-300 Poly acrylamide gel e lec t rophores is 67 Table XI gives the resu l t s obtained by th i s p u r i f i c a t i o n scheme. It was found that the presence of sodium deoxy cholate fo r prolonged periods in the enzyme suspension, i r r e v e r s i b l y i nh ib i t ed the enzyme. A l s o , the gel f i l t r a t i o n step on Sephacryl S-300 completely inac t i va ted the enzyme (Table XI). A p u r i f i c a t i o n of 220-fold was obtained at the end of the a f f i n i t y chromatography step. In order to assess the progress during the p u r i f i c a t i o n procedure, samples were saved from each stage and electrophoresed on polyacrylamide gels in the presence of e i the r T r i ton X-100 or sodium dodecyl sulphate. It was found that no prote in or enzyme a c t i v i t y from any stage o f the p u r i f i c a t i o n procedure entered the g e l , despi te the fac t that the percentage of acrylamide in the gels was lowered to 5% in one experiment, again suggesting prote in aggregat ion. Treatment of the preparat ions with sodium dodecyl sulphate or T r i ton X-100 p r i o r to e lec t rophores i s d id not improve the r e so lu t i on . Because of the d i f f i c u l t y in disaggregat ion of the a l ka l i ne phosphatase a c t i v i t y in butanol s o l u b i l i z e d ex t r a c t s , a t tent ion was switched to the T r i ton X-100 s o l u b i l i z e d preparat ions. It was found that th i s material could be e lectrophoresed on polyacrylamide gels and sta ined fo r enzyme a c t i v i t y , (see f igure 15). This was only pos s i b l e , however, when 0.05% sodium dodecyl sulphate was present in running and stacking gels and running buf fe r . T r i ton X-100 could not be subst i tu ted fo r SDS, s ince i t produced poor reso lu t ion of the prote in bands. Table XI Purification of butanol solubilized alkaline phosphatase from vegetative membranesof D.discoideum Purification step Total protein (mg) Total activity , (A0.D 4 1 0min" 1) Protein recovery (percent) Activity recovery (percent) Specific activity , , ^0D 4 1 0min"'mg" 1 ) Purification Vegetative membrane 372.8 7.6 100 100 0.02 1 Butanol extraction 87.5 60.9 23.5 801 0.69 34.5 DEAE - Sephacel Chromatography 15.8 22.4 4.2 36.8 1.42 71.0 ConA-Sepharose Chromatography 1.35 5.9 0.36 9.7 4.4 220 Sephacryl S-300 gel f i l trat ion - no recovery - - - -69 b) P u r i f i c a t i o n using Tr i ton X-100 ext rac t ion fo r s o l u b i l i z a t i o n Figure 12 shows the gel f i l t r a t i o n on Sephacryl S-300 of a d ia lyzed T r i ton X-100 ext rac t of vegetat ive membranes. The a l ka l i ne phosphatase a c t i v i t y was included by the column showing that the enzyme was less aggregated than the butanol extracted mate r i a l . The recovery of enzyme a c t i v i t y from the column was 100%. It was a lso observed that during gel f i l t r a t i o n of a co ld- inac t i va ted T r i ton X-100 ext rac t the a l ka l i ne phosphatase was ac t i va ted about 18-fo ld , suggesting that the low molecular weight i n h i b i t o r was removed. Attempts to recover i n h i b i t o r y a c t i v i t y from the column were not success fu l , however. The Tr i ton extract was chromatographed on a concanavalin-A-Sepharose column, which was pre-equl ibrated with 5mM Tr i s-C l buf fer (pH 7.5) conta in ing 1% T r i ton X-100. Only neg l i g i b l e l eve l s of enzyme a c t i v i t y were unbound. The a l ka l i ne phosphatase was e luted by the add i t ion of 0.2M a-methyl-D-mannoside to the column buf fer ( f igure 13). The recovery of enzyme a c t i v i t y was greater than 65%. Aga in , a f f i n i t y chromatography of a co ld- inac t i va ted enzyme caused considerable ac t i va t i on suggesting the removal of the low molecular weight i n h i b i t o r ( s ) during chromatography. The T r i ton extract was also, appl ied to a DEAE-Sephacel column equ i l i b ra ted with 5mM Tr i s-C l buffer (pH 7.5) conta in ing 1% T r i ton X-100, but f a i l e d to bind under these cond i t ions . However, 70 FRACTION VOLUME (ml) Figure 12. Gel f i l t r a t i o n on Sephacryl-S-300 of T r i ton X-100 extracted a l ka l i ne phosphatase from vegetat ive c e l l membranes. 71 FRACTION NUMBER Figure 13. A f f i n i t y chromatography on concanavalin A-Sepharose of T r i ton X-100 extracted a lka l ine phosphatase from vegetat ive c e l l membranes. Protein (x) and a l ka l i ne phosphatase a c t i v i t y (o) were e luted with 0.2M a-methyl-D-mannoside. Protein was determined on 20ul a l iquots of each f r a c t i on by the modif ied Fo l in assay ( 56 ) and i s expressed as opt i ca l densi ty at 650nm.. A lka l ine phosphatase a c t i v i t y i s expressed as op t i ca l densi ty at 410nm produced by lOyl a l i quo ts of f r a c t i o n s f o r 10 minute incubation per iod . Fract ion volumes were 2.5mls. each. 72 lowering the T r i ton X-100 concentrat ion to 0.1%, resu l ted in successful binding of the a l ka l i ne phosphatase to a DEAE-Sephacel column equ l ibra ted with 5mM Tr i s-C l buf fer (pH 7.5) conta in ing 0.1% T r i ton X-10.0. This bound enzyme could then be e luted with a 0 to 1>-M NaCl gradient to give. 70% recovery of enzyme a c t i v i t y ( F ig . 14). In view of th i s requirement fo r low T r i ton X-100 concentrat ions fo r ion-exchange chromatography, attempts were made to perform a f f i n i t y chromatography on conA-Sepharose a lso on 1/10 d i l u t ed T r i t on X-100 ex t rac t s . It was found that the enzyme did not bind to concanaval in -A-Sepharose at T r i ton X-100 concentrat ions below 0.5%. It was a lso found that when e i the r ion exchange or a f f i n i t y chromato-graphy was performed a f t e r gel f i l t r a t i o n of the T r i ton X-100 extract no enzyme a c t i v i t y was recovered. The gel f i l t r a t i o n step was therefore u t i l i z e d at the end of the p u r i f i c a t i o n procedure, but even then i t resu l ted in low recover ies of a c t i v i t y and an unstable enzyme preparat ion . The f i n a l p u r i f i c a t i o n was done according to the P u r i f i c a t i o n Scheme II given below, although fo r some preparat ions the gel f i l t r a t i o n step was replaced by a preparat ive SDS-polyacrylamide step. 73 FRACTION NUMBER Figure 14. Ion exchange chromatography on DEAE-Sephacel of T r i ton X-100 extracted a l ka l i ne phosphatase from vegetat ive c e l l membranes a f t e r a f f i n i t y chromatography. Protein (x) and a l ka l i ne phosphatase a c t i v i t y (o) were e luted with a NaCl gradient of 0 to 1M. Protein concentrat ion and a lka l i ne phosphatase a c t i v i t y are expressed as descr ibed fo r F ig . 13. 74 PURIFICATION SCHEME II Crude vegetat ive membrane Tr i ton X-100 ext rac t ion (1% T r i ton X-100) D i a l y s i s A f f i n i t y chromatography on conA-Sepharose Di lute to 0.1% T r i ton X-100 Ion exchange chromatography on DEAE - Sephacel SDS-pol yacrylami de gel e lec t rophores i s Gel f i l t r a t i o n on Sephacryl-S-300 75 The resu l t s of p u r i f i c a t i o n of a l ka l i ne phosphatase from a vegetat ive membrane preparat ion using th i s scheme i s shown in Table XII. The f i n a l p u r i f i c a t i o n obtained fo r a l ka l i ne phosphatase using the crude membrane as s t a r t i ng material was 620 f o l d . The preparat ion was subjected to SDS-polyacrylamide gel e l ec t rophore r i s at each step of the procedure to assess the progress of p u r i f i c a t i o n and i t was found that the preparat ion e luted from DEAE-Sephacel s t i l l contained several contaminating prote in bands. ( F ig . 15). The a c t i v i t y of th i s preparat ion was found to be very unstable at 4°C or -20°C, l os ing two-thirds of i t s a c t i v i t y in 48 hours at 4 °C and in 14 days at -20°C. However, t h i s p a r t i a l l y p u r i f i e d preparat ion could be stored at -70°C without appreciable loss of a c t i v i t y , f o r more than two months. Attempts to fu r ther pur i f y th i s preparat ion lead to considerable loss of a c t i v i t y . As can be seen in Table XII, 5 '-nuc leot idase , which i s another membrane bound enzyme present in the crude membranes of D.discoideum is copu r i f i ed along with the a l ka l i ne phosphatase. The r a t i o between the two a c t i v i t i e s and the amount of p u r i f i c a t i o n obtained in each step of the p u r i f i c a t i o n process remain s im i l a r throughout the p u r i f i c a t i o n , suggesting that the two a c t i v i t i e s reside in the same p r o t e i n , in two prote ins with very s im i l a r proper t ies or in two prote ins that are f i rm ly attached to each other. Table XII Pu r i f i c a t i on of Tr i ton X-100 s o l u b i l i z e d a l ka l i ne phosphatase and 5-nucleotidase a c t i v i t i e s from vegetative membranes of D.discoideum P u r i f i c a t i o n steps A lka i ine T o t a l 3 a c t i v i t y Phosphatase Spec i f i c ' 3 a c t i v i t y 5 1 -nucleot idase T o t a l C : specific*^ a c t i v i t y a c t i v i t y Vegetative membrane 734.5 1.08 428.4 0.63 Tr i ton X-100 extract ion 884.9 2.56 888.0 2.56 Dialyzed Tr i ton X-100 extract 7902.0 34.25 7161.0 31.10 Concanavalin A-Sepharose chromatography 5875.7 290.10 5644.0 279.50 DEAE-Sephacel chromatography 2569.0 669.70 2308.8 602.90 Sephacryl S-300 gel f i l t r a t i o n 180.0 80.4 N.D. N.D.. a. A lka l ine phosphatase a c t i v i t y expressed as nmol.p-nitrophenol min" b. S p e c i f i c a c t i v i t y expressed as nmol.p-nitrophenol m i n - 1 mg p r o t e i n - 1 c. 5 '-nucleot idase a c t i v i t y expressed as n.mol AMP.min - 1 d. S p e c i f i c a c t i v i t y expressed as n.mol.AMP.min - 1.mg protein-1 N.D.-means not done 77 A B C D E F Figure 15. SDS-polyacrylamide gel e l ec t ro phoresis o f three d i f f e r en t steps in the p u r i f i c a t i o n of the a l ka l i ne phosphatase a c t i v i t y from the vegetat ive c e l l membranes of D.discoideum. Samples of d ia lyzed T r i ton X-100 extracts (lanes A and D), con A - Sepharose eluate (lanes B and E) and the p a r t i a l l y p u r i f i e d enzyme from DEAE -Sephacel (lanes C and F) were subjected to SDS-polyacrylamide gel e lec t rophores is as descr ibed in the methods. Lanes A to C were sta ined fo r a l ka l i ne phosphatase a c t i v i t y (methods) and D to F were sta ined fo r prote in (methods). 78 c) Summary Attempts were made to pur i f y the a l ka l i ne phosphatase a c t i v i t y from vegetat ive c e l l membranes. The butanol extracted enzyme could be p u r i f i e d 220 f o l d by chromatographic methods, but the enzyme was found to be in a h igh ly aggregated state throughout the p u r i f i c a t i o n procedure. T r i ton X-100 s o l u b i l i z e d enzyme was p u r i f i e d 660 f o l d using concanaval in A-Sepharose and DEAE-Sephacel chromatography. SDS -polyacrylamide gel e lec t rophores i s of the p u r i f i e d material revealed several contaminating prote in bands. Attempts on fur ther p u r i f i c a t i o n were unsuccessful due to the i n s t a b i l i t y of the p a r t i a l l y p u r i f i e d enzyme. The 5 '-nucleot idase a c t i v i t y copur i f i ed with the a l ka l i ne phosphatase a c t i v i t y during a l l steps of the p u r i f i c a t i o n procedure. 79 SECTION III PROPERTIES OF ALKALINE PHOSPHATASE AND 5'-NUCLEOTIDASE ACTIVITIES OF THE PARTIALLY PURIFIED PREPARATION a) E f f ec t of f l uo r ide Sodium f l uo r ide was incorporated in the enzyme assay to i n h i b i t ac id phosphatase a c t i v i t y (41). In crude membrane prepara t ions , add i t ion of sodium f l uo r ide i nh ib i t ed about 40% of both a l ka l i ne phosphatase and 5 '-nucleot idase a c t i v i t i e s (Table XII I). In the p a r t i a l l y p u r i f i e d preparat ion a l ka l i ne phosphatase was i nh ib i t ed by f l uo r i de only by 17% and 5 1 -nucleot idase by 11% (Table XIII) again suggesting a c lose s i m i l a r i t y between the two a c t i v i t i e s . b) pH optima In the p a r t i a l l y p u r i f i e d prepara t ion , a l ka l i ne phosphatase was opt imal ly ac t i ve at pH 9.0 and the 5 1 -nucleot idase opt imal ly ac t i ve at pH 8.0 (Figure 16). Since these optima were s l i g h t l y higher than the value reported by Lee et a l . , f o r membrane preparat ions (33), the pH optima in membrane preparat ions were rechecked and found to be iden t i ca l to those found fo r the p a r t i a l l y p u r i f i e d preparat ions, (data not shown). c) Inh ib i t ion of a l ka l i ne phosphatase by inorganic phosphate The p a r t i a l l y p u r i f i e d a l ka l i ne phosphatase was very sens i t i ve to i n h i b i t i o n by inorganic phosphate ( F ig . 17). This i n h i b i t i o n was a mixture of both competit ive and non-competitive 80 Table XIII E f fec t of 30mM sodium f l uo r i de on the a l ka l i ne phosphatase and 5 '-nucleot idase a c t i v i t i e s of vegetat ive c e l l s of D.discoideum. Treatment Alkal ine a d (n.moles i phosphatase : i v i ty_-;. mi n ) 5 1 -nucleot idase a c t i v i t y _-(n.moles, min" . ) Vegetative membrane i P a r t i a l l y p u r i f i e d membrane Vegetative membrane P a r t i a l l y p u r i f i e d membrane No add i t ion + 30mM NaF 3.7 2.2 3.1 2.6 3.2 1.9 2.9 2.58 Note: The data are from one representat ive experiment. S im i l a r resu l t s were obtained in three r e p e t i t i o n s . 81 Figure 16. E f f ec t of pH on the a l ka l i ne phosphatase and 5 '-nucleot idase a c t i v i t i e s in the p a r t i a l l y p u r i f i e d preparat ion . A lka l i ne phosphatase (o) and 5 '-nucleot idase (A ) a c t i v i t i e s were assayed as descr ibed in the methods in T r i s-C l buffers at the ind icated pH. 82 0 20 40 60 80 100 Concert-trotion of Phosphate tmM) Figure 17. Inh ib i t ion of of a l ka l i ne phosphatase a c t i v i t y in vegetat ive c e l l s of D.discoideum by inorganic phosphate. A lka l i ne phosphatase a c t i v i t y in d ia lysed vegetat ive membranes (o) and a p a r t i a l l y p u r i f i e d preparat ion (A ) was assayed in presence of ind icated concentrat ions of KH 2Po 4/Na2HPo» buffer , pH 7.5: . The enzyme a c t i v i t i e s were determined fo r an incubat ion period of 10 minutes and are expressed as percentages of control a c t i v i t i e s of the respect ive preparat ions. 83 i n h i b i t i o n s in that both V n ] a x and were a l te red (F ig . 19A). The i n h i b i t i o n of the a l ka l i ne phosphatase a c t i v i t y of the d ia lysed vegetat ive membranes showed very s im i l a r s e n s i t i v i t y to var ious phosphate concentrat ions (F ig . 17). The phosphate i n h i b i t i o n was not dependent on preincubat ion at 4 ° C , as maximum i n h i b i t i o n was found to occur immediately upon addi t ion of Pi to the assay mixture (Figure 6) . When membranes were preincubated with phosphate and then subsequently incubated at 50°C fo r 30 minutes, the a l ka l i ne phosphatase a c t i v i t y was considerably reduced and the a c t i v i t y could not be recovered by d i a l y s i s . (Table XIV). However, the phosphate i n h i b i t i o n could be p a r t i a l l y reversed by d i a l y s i ng the i nh ib i t ed enzyme (Table XIV). (d) Inh ib i t ion by the putat ive i n h i b i t o r When the p a r t i a l l y p u r i f i e d enzyme was incubated with the putat ive i n h i b i t o r obtained from d ia l ysa tes of the vegetat ive membranes, i t was found that both a l ka l i ne phosphatase and 5 '-nucleot idase a c t i v i t i e s were s i m i l a r l y i nh ib i t ed "within 48 hours. ( F ig . 18). Experiments to t ry to reverse th i s i n h i b i t i o n by d i a l y s i s were not poss ib le due to the i n s t a b i l i t y of these a c t i v i t i e s in the p a r t i a l l y p u r i f i e d preparat ion. 84 Table XIV E f fec t of 50°C treatment and d i a l y s i s on a l ka l i ne phosphatase i nh ib i t ed by inorganic phosphate Sample Treatment A lka l ine phosphatase a c t i v i t y (AQD^iQ/lOmin assay) No addi t ion In presence of lOOmM Pi I Dialysed vegetat ive membrane 24h. at OOC 0.51 0.15 II As above fol lowed by 50OC for 30 min 0.50 0.03 III As fo r II fol lowed by 48h. d i a l y s i s 0.52 0.06 IV As fo r I fol lowed by 48h. dia-lysis 0.51 0.41 Note: The data are from one representat ive experiment. S imi la r resu l t s were obtained in three r e p e t i t i o n s . 85 TIME (Hours) Figure 18. E f f ec t of concentrated d ia l ysa te on the a l ka l i ne phosphatase and 5 1 -nucleot idase a c t i v i t i e s in the p a r t i a l l y p u r i f i e d preparat ion. The p a r t i a l l y p u r i f i e d preparat ion (_0.14m.g_-prote in per ml in 5mM Tr i s-C l pH 7.5, conta in ing 0.1% T r i ton X-100) was mixed with the concentrated d ia l ysa te at a .1:1 r a t i o and incubated at 0 °C . A l iquots (0.05ml) were taken at i n te r va l s and the a c t i v i t i e s of a l ka l i ne phosphatase (o) and 5 1 -nucleot idase (A ) in the mixture were determined and are expressed as percentages of the respect ive control va lues . 86 (e) Substrate s p e c i f i c i t y studies Substrate s p e c i f i c i t y of d ia lysed crude membranes and p a r t i a l l y p u r i f i e d preparat ions was studied at condi t ions used fo r a l ka l i ne phosphatase and 5 1 -nucleot idase assays. Of the 10 substrates included in the study, only four were u t i l i z e d by both preparat ions (Table XV). AMP, ADP and a-naphthyl ac id phosphate were hydrolyzed f as te r at pH 7.5 compared to pH 8.5 by both preparat ions. There was an appreciable decrease in the rate of hydro lys is of ADP and a-naphthyl ac id phosphate r e l a t i v e to pN'PP fo l lowing p u r i f i c a t i o n . ATP was hydrolysed only by the membranes at pH 7.5. The other substrates tested were GMP, GDP, c y c l i c AMP, glucose - 6 - phosphate and ^-glycerophosphate. (f) Determination of Km for AMP and pNPP Lineweaver-Burk p lo ts of the a l ka l i ne phosphatase of the p a r t i a l l y p u r i f i e d preparat ion assayed at pH 8.5 revealed a Km of 1.5 x 10" 4M for pNPP. (Figure 19A). A s im i l a r p lot fo r the _5 5'-nucleotidase a c t i v i t y assayed at pH 7.5 gave a value of 3.4 x 10 M fo r AMP (Figure 19B). Concentration of AMP above 150pM i nh ib i t ed the 5 ' -nucleot idase a c t i v i t y (Figure 19B). In contrast concentrat ions of pNPP 6i5- fo ld greater than the km value produced no i n h i b i t i o n of a l ka l i ne phosphatase. The presence of lOmM inorganic phosphate produced a 5.5 f o l d increase in Km and 3-fold decrease in V„,„„ of a l ka l i ne 87 Table XV Phosphatase-act iv i t ies of vegetat ive membrane and p a r t i a l l y p u r i f i e d preparat ion on various phosphate esters Substrate A c t i v i t y Dialyzed vegetat ive membranes P a r t i a l l y p u r i f i e d prepara tion pH 8 . 5 a pH 7 .5 D pH 8 . 5 a pH 7.5° pNPP 100 100 100 100 5'-AMP 18 180 50 107 ADP 67 350 32 69 a-naphthyl ac id phosphate 170 460 95 111 ATP 0 616 0 0 a. Assay condi t ions were those descr ibed fo r a l ka l i ne phosphatase a c t i v i t y b. Assay condi t ions were those descr ibed fo r 5 1 -nucleot idase a c t i v i t y Note: The data are from one representat ive exper iment . „ S im i l a r r esu l t s were obtained in three r e p e t i t i o n s . 88 • <=-»—M . , , -10 -4 0 4 10 20 40 1/Substrate (miwi) Figure 19A Lineweaver-Burk p lo t fo r the A lka l ine phosphatase a c t i v i t y in the p a r t i a l l y p u r i f i e d preparat ion. The ve l o c i t y of the a l ka l i ne phosphatase a c t i v i t y at various pNPP concentrat ions were determined in the oresence ( ) and absence, (o) of 10 mM KH 2Po 4/Na 2HPo 4 bu f f e r , pH 7.5. . • Note: The data are from one representat ive experiment. 89 Figure 19B. Lineweaver-Burk p lo t fo r the 5 '-nucleot idase a c t i v i t y in the p a r t i a l l y p u r i f i e d preparat ion. Note: The data are from one representat ive experiment. 90 phosphatase ( F ig . 19A). The e f f e c t o f inorganic phosphate on 5 1 -nucleot idase could not be studied as a l l concentrat ions of phosphate i n te r f e red with rad ioact ive adenosine p r e c i p i t a t i o n . g) Summary Several proper t ies of the a l ka l i ne phosphatase and 5'-nucleo-t idase a c t i v i t i e s of the vegetat ive membrane and p a r t i a l l y p u r i f i e d preparat ion were tes ted . Both a c t i v i t i e s were equal ly i nh ib i t ed by sodium f l uo r ide and the i n h i b i t o r from the d i a l y sa tes . The two a c t i v i t i e s exhib i ted d i f f e r en t pH optima; pH 8.0 fo r the 5'-nucleot idase a c t i v i t y and pH 9.0 fo r the a l ka l i ne phosphatase a c t i v i t y . The Km values o f the two a c t i v i t i e s a lso were d i f f e r e n t and the 5'-nucleot idase a c t i v i t y was i nh ib i t ed at high substrate concentrat ions . The a l ka l i ne phosphatase a c t i v i t y in vegetat ive membranes and p a r t i a l l y p u r i f i e d preparat ions were equal ly i nh ib i t ed by inorganic phosphate concentrat ions and th i s i n h i b i t i o n could be p a r t i a l l y reversed by d i a l y s i s . Re l a t i ve l y few phosphorylated substrates were hydrolysed using e i t he r the a l ka l i ne phosphatase or 5 '-nucleot idase assay cond i t i ons . SECTION IV 91 COMPARISON BETWEEN ALKALINE PHOSPHATASE FROM VEGETATIVE AND CULMINATING CELLS a ) Pu r i f i c a t i on of the culminating enzyme Attempts were made to pur i f y the a l ka l i ne phosphatase from culminat ing c e l l s using the procedure worked out fo r the vegetative enzyme. Dialyzed Tr i ton X-100 extracts of crude culminating membranes were prepared and appl ied to a concanavalin A-Sepharose column. No enzyme a c t i v i t y was recovered, e i the r unbound or by e lu t ion with 0.2M or 0.5M a-methyl-D-mannoside. Since the vegetative Tr i ton X-100 ext rac t f a i l e d to bind to a conA-Sepharose in the presence of 0.1% Tr i ton X-100, the concentrat ion of Tr i ton X-100 in the buffer system was lowered to th i s concentrat ion. There was s t i l l no e lu t ion of enzyme a c t i v i t y along with the unbound material or with 0.2M or 0.5M a-methyl-D-mannoside. In a d d i t i o n , culminating enzyme was a lso not e luted from DEAE-Sephacel under the condit ions found useful fo r the vegetat ive enzyme. Rais ing the NaCl concentrat ion in the e lu t ing buf fer to 3M a lso f a i l e d to e lute the enzyme from DEAE-Sephacel. These resu l t s suggested that the culminat ing enzyme d i f f e r s s u f f i c i e n t l y in s t ructure from the vegetat ive enzyme to a l t e r i t s chromatographic proper t ies s i g n i f i c a n t l y . b) Inh ib i t ion by sodium dodecyl sulphate. It was found that while vegetative enzyme a c t i v i t y sta ined in tense ly in SDS-polyacrylamide ge l s , culminating enzyme sta ined only f a i n t l y , suggesting a poss ib le i n h i b i t i o n of the l a t t e r enzyme by SDS (Figure 20). Figure 21 shows that preincubation at 22°C with 0.05% SDS 92 1 2 3 ^ 1 Figure 20. SDS-polyacrylamide gel e l ec t ro phoresis of a lka l ine phosphatase a c t i v i t i e s . Samples of p a r t i a l l y p u r i f i e d preparat ion (lane 1) and d ia lysed Tr i ton X-100 extracts of vegetative (lane 2) and culminat ing (lane 3) c e l l s were electrophoresed in polyacrylamide gels conta in ing 0.05% SDS. The concentrat ions of T r i s buf fer in the running g e l , stacking gel and running buffer were 10 times higher than described in methods. The gel was sta ined fo r a l ka l i ne phosphatase a c t i v i t y as descr ibed under methods. Samples appl ied for lanes 2 and 3 contained approximately equal amounts o f a l ka l i ne phosphatase a c t i v i t y when assayed s p e c t r o p h o t o m e t r y ! l y . 93 18~i .6-£ r—1 1= c O Q • .2-0 0 —.—^  10 T I M E ( H o u r s ) 15 20 Figure 21. E f f ec t of sodium dodecyl sulphate on the a l ka l i ne phosphatase a c t i v i t i e s o f vegetat ive and culminat ing c e l l s . Dialysed T r i ton X-100 extracts of vegetat ive (o) and culminating ( A ) , c e l l membranes were incubated with 0.05% SDS at room temperature ( 2 2 ° C ) . A l iquots (0.1ml each) were removed at the ind ica ted time i n t e r va l s and the a l ka l i ne phosphatase a c t i v i t y was determined. 94 had a marked i nh ib i t o r y e f f e c t on the d ia lyzed Tr i ton X-100 ext rac t of the culminat ing membranes, whereas the vegetat ive c e l l preparat ion was unaf fected. Thus the enzyme from culminating c e l l s i s c l e a r l y more suscept ib le to inac t i va t ion<iy SDS. (c) S t a b i l i t y of vegetat ive and culminat ing enzymes The s t a b i l i t y of a l ka l i ne phosphatase a c t i v i t i e s of d ia l ysed Tr i ton X-100 extracts of vegetat ive and culminating membranes was found to be h ighly dependent on the T r i s-C l concentrat ion and pH of the suspending bu f fe r . In 5mM Tr i s-C l bu f f e r , pH 7.5, the vegetat ive enzyme was r e l a t i v e l y s t ab l e , whereas in 50mM T r i s - C l , pH 7.5, there was considerable loss of a c t i v i t y (Figure 22A). The d i f fe rence in the i nac t i v a t i on of the culminating enzyme at these two T r i s-C l concentrat ions was much less pronounced. (Figure 22A). The vegetat ive enzyme was more stable than the culminat ing enzyme at 5mM T r i s - C l , while at 50mM Tr i s-C l the reverse was t rue . Inact ivat ion of both enzymes was much f as te r as the pH of the preincubat ion was increased from 7.5 to 8.0 (Figure 22 B). This d i f f e r e n t i a l s t a b i l i t y of these enzymes along with the d i f f e rences in s e n s i t i v i t y to SDS suggest that the a l ka l i ne phosphatases obtained from vegetat ive and culminat ing c e l l s may not be the same enzyme. This conclus ion i s substant iated by the fact that the vegetat ive enzyme p u r i f i c a t i o n procedure does not work fo r the culminat ing enzyme. 95 ' o - t — 1 1 I 1 0 30 60 90 120 T I M E ( m i n ) ure 22A S t a b i l i t y of the a l ka l i ne phosphatase a c t i v i t i e s of vegetat ive and culminat ing c e l l s . Dia lyzed T r i ton X-100 extracts of vegetat ive and culminating c e l l membranes were resuspended in 5 or 5.0mM Tr i s-C l buf fer at pH 7.5 and incubated at 50°C. A l iquots (0.1ml each) were removed at ind ica ted in te rva l s and the a l ka l i ne phosphatase a c t i v i t i e s were determined and are expressed as percentages of the 0 hour a c t i v i t y , (o) vegetat ive enzyme at 5mM T r i s - C l . (x) vegetat ive enzyme at 50mM T r i s - C l . (A ) culminat ing enzyme at 5mM T r i s - C l . ( • ) culminating enzyme at 50mM T r i s - C l . 96 T I M E ( m « n ) Figure 22B. S t a b i l i t y of the a l ka l i ne phosphatase a c t i v i t i e s of vegetat ive and culminat ing c e l l membranes at pH 8.0. Deta i l s as fo r Figure 22A, except the change in pH. 97 (d) pH optima The pH optima f o r a l ka l i ne phosphatase and 5 1 -nucleot idase a c t i v i t i e s in the culminat ing c e l l membranes were found to be the same as those found fo r the vegetat ive membranes and the p a r t i a l l y p u r i f i e d preparat ion . A lka l ine phosphatase was opt imal ly act ive at pH 9.0 and 5 '-nucleot idase showed optimal a c t i v i t y at pH 8.0 (Figure 23). (e) Inh ib i t ion by inorganic phosphate It was found that the a l ka l i ne phosphatase a c t i v i t y in the culminat ing membrane was a lso sens i t i ve to i n h i b i t i o n by inorganic phosphate". However, the culminat ing membrane a c t i v i t y showed s l i g h t l y l ess s e n s i t i v i t y than the d ia lysed vegetat ive membrane a c t i v i t y in a l l the phosphate concentrat ions tes ted . (Figure 24) (f) Arrhenius p lo ts Arrhenius p lots fo r the . a l k a l i n e phosphatase a c t i v i t i e s from d ia l yzed vegetat ive and culminat ing membranes over a temperature range of 12°C to 35°C revealed s ing le t r ans i t i on points at approximately 26°C (Figure 25). Thus, the a l te red propert ies of the enzymes from the two d i f f e r e n t stages are not r e f l e c ted by d i f fe rences in the temperature dependence of the a c t i v i t y . 98 g _ J j j , j 1 , 1 7 7.5 8 8.5 9 9.5 p H Figure 23. E f f ec t of pH on the a l ka l i ne phosphatase and the 5 1 -nuc leot idase a c t i v i t i e s in the culminat ing membrane preparat ions. A lka l ine phosphatase (o) and 5 '-nucleot idase ( A ) , a c t i v i t i e s were assayed as descr ibed in T r i s-C l buffers at the ind ica ted pH. 99 0 20 40 60 80 100 Concentration of Phosphate (mM) Figure 24. Inh ib i t ion of a l ka l i ne phosphatase a c t i v i t y in culminat ing c e l l membranes by inorganic phosphate. A lka l i ne phosphatase a c t i v i t y in culminating c e l l membranes ( A ) was assayed in presence of ind icated concentrat ions of KH 2P0 4/Na 2HPo 4 bu f f e r , pH 7.5. Data on the e f f e c t of inorganic phosphate on the enzyme in d ia l yzed vegetat ive membrane (o) are included for comparison. Note: The data are from one representat ive experiment. S imi la r r esu l t s were obtained in three r e p e t i t i o n s . 100 o to o E >• N C 0) O) o 150 100 80 50 150 100 80 50 3 3 3 - 4 3 5 (I/T°K) x 1 0 0 0 Figure 25. Temperature dependence of the a l ka l i ne phosphatase a c t i v i t y . Arrhenius p lots of the a lka l ine phosphatase a c t i v i t i e s in d ia lyzed vegetat ive membranes (o) and culminat ing membranes (A ) were obtained as descr ibed under methods. Enzyme a c t i v i t y i s expressed as n moles of p-nitrophenol produced per 5 minute incubat ion per 0.1ml a l iquo t of samples. 101 (g) Summary Attempts to purify the culminating enzyme employing the same procedure used for the vegetative enzyme were not successful, suggesting possible changes in the protein during development. In addition, the culminating enzyme was found to exhibit different s t a b i l i t y in Tris-Cl and SDS compared with the vegetative enzyme. However, pH optima for the alkaline phosphatase and 5'-nucleotidase a c t i v i t i e s , inhibition of alkaline phosphatase activity by phosphate and the Arrhenius plots for alkaline phosphatase were similar for both vegetative and culminating membranes. 102 DISCUSSION An important observat ion in th i s study was the large ac t i va t i on of a l ka l i ne phosphatase in vegetat ive c e l l membranes of D.discoideum by incubat ion at 50°C (Figs 1 & 2) . This r esu l t was perhaps s u r p r i s i n g , s ince such a marked ac t i va t i on had not been detected by e a r l i e r workers. Gezel ius and Wright ( 18 ) reported that the enzyme to le ra ted incubation at 50°C fo r only f i v e minutes. S imi la r studies conducted by Lee and coworkers ( 33 ) resu l ted in a marginal a c t i va t i on of a l ka l i ne phosphatase when crude membrane preparat ions were incubated fo r 30 minutes at 50°C. Recently, MacLeod and Loomis ( 41 ) while studying the thermal s t a b i l i t y of a l ka l i ne phosphatase in vegetat ive c e l l extracts reported a 50% i nac t i v a t i on of th i s enzyme when incubated fo r 40 minutes at 35°C. However, these workers a l l used d i f f e r e n t methods of c e l l d i s rupt ion and preparat ion of c e l l free ex t rac t s . Moreover, Gezel ius and Wright ( 18) and MacLeod and Loomis ( 41 ) used d i f f e r e n t s t ra ins of D.discoideum making a d i r e c t comparison d i f f i c u l t . The lack of a c t i va t i on observed by Lee et a l . , ( 33 ) might have been due to the fac t that they cooled the i r 50°C treated samples on ice before determining the enzyme a c t i v i t i e s , which may have resu l ted in a loss of a c t i v i t y . A l ka l i ne phosphatases from mammalian c e l l s vary considerably with respect to temperature s t a b i l i t y ( 43 ), and s e n s i t i v i t y to 103 incubat ion at 56°C has been used as a c r i t e r i a fo r t i ssue s p e c i f i c i t y ( 23 )• There has been one report of a 70% increase in the a c t i v i t y of a l ka l i ne phosphatase from calf-duodenum when the preparat ion was incubated at 60°C ( 26 ) and th i s increase was pH dependent. In contrast to the v a r i a b i l i t y in the s t a b i l i t y of a l ka l i ne phosphatase from mammalian c e l l s , the a c t i v i t i e s from several microbia l sources are stable at 60°C. (13,21) The a c t i v i t y in crude c e l l ext rac ts from E_. c o l i has been reported to be stable f o r 30 minutes at 85°C ( 17 ). The very high ac t i v a t i on of a l ka l i ne phosphatase at 50°C that was found in vegetat ive c e l l membranes of D.discoideum in the present study, has not been observed in any other organism. The 50°C ac t i v a t i on of a l ka l i ne phosphatase in vegetat ive c e l l membranes cor re la ted with the d i s soc i a t i on of a low molecular i n h i b i t o r ( s ) from the enzyme, s ince exhaustive d i a l y s i s of these membranes produced a s im i l a r a c t i va t i on (Table I I I ) , and a d ia lysed membrane preparat ion could not be fur ther act ivated by incubation at 50°C (Table II I). Furthermore, i t was poss ib le to reconst i tu te the inac t i va ted enzyme by mixing a d i a l y z e d , ac t iva ted vegetat ive membranes with the i n h i b i t o r ( s ) recovered from the d i a l y s a t e s , and red i ssoc i a te the i n h i b i t o r from the enzyme by fur ther d i a l y s i s (F ig . 5) . The i n h i b i t o r y f ac to r remained t i g h t l y bound to the membrane, a f t e r d i s soc i a t i on from the enzyme at 50°C since repeated washing of the membrane d id not prevent the temperature dependent a c t i v a t i on/ inac t i v a t i on c y c l e . 104 It was important to t ry to obtain highly p u r i f i e d a l ka l i ne phosphatase to study th i s enzyme-inhibitor in te rac t ion in d e t a i l . A l ka l i ne phosphatase in D.discoideum has been shown to be a membrane bound prote in (23,33,52 ). The only s o l u b i l i z a t i o n agents that s a t i s f a c t o r i l y extracted an appreciable amount of a l ka l i ne phosphatase a c t i v i t y in the present study were n-butanol and T r i ton X-100 (Table I). MacLeod and Loomis ( 41 )found that a l ka l i ne phosphatase of e i the r vegetat ive or culminat ion c e l l s was extracted by T r i ton X-100 and Armant and Rutherford ( 3 ) confirmed that a l ka l i ne phosphatase from culminat ing c e l l s was ex t r a c t i b l e by T r i ton X-100. The l a t t e r workers a lso demonstrated that high s a l t was not a good ext rac t ion agent ( 3 ). The a l ka l i ne phosphatase a c t i v i t y extracted by T r i ton X-100 responded s i m i l a r l y to the membrane bound enzyme to incubat ion at 50°C (F ig . 4) or d i a l y s i s (Table III). The ext rac t ion procedure with T r i ton X-100 appeared to s o l u b i l i z e the putat ive i n h i b i t o r along with the enzyme p ro te in . In con t ras t , the butanol s o l u b i l i z e d preparat ion was a lready d ia l ysed to remove a l l t races of butanol and i t was not poss ib le to d i r e c t l y determine i f the i n h i b i t o r was s o l u b i l i z e d by th i s procedure. The butanol s o l u b i l i z e d enzyme was i nh ib i t ed by incubat ion at 50°C (Table II) suggesting a d i f fe rence in the conformation of th i s enzyme compared to the T r i ton X-100 extract or in tac t vegetat ive membrane. When MacLeod and Loomis subjected the i r T r i ton X-100 extracted 105 enzyme to 35 °C , 50% of a l ka l i ne phosphatase a c t i v i t y was l o s t in 40 minutes. ( 41 ) Besides using d i f f e r en t s t ra ins of D.discoideum, a major d i f f e rence between the two studies was that they used c e l l homogenates prepared byi;sonication in the presence of~0.1% T r i ton X-100. In a d d i t i o n , the concentrat ion of T r i ton X-100 that they used ( 41 ) was much lower than that used in the present study and they did not report data on the e f f i c i e n c y of t h e i r s o l u b i l i z a t i o n procedure. Extract ion with n-butanol has been used extens ive ly in pu r i f y ing membrane bound a lka l i ne phosphatase from mammalian c e l l s . ( 1, 19, 45 ) . Butanol s o l u b i l i z e d a l ka l i ne phosphatase from the vegetat ive c e l l s of D.discoideum appeared to be in h igh ly aggregated form and could not be electrophoresed in SDS-polyacrylamide gels even a f t e r p u r i f i c a t i o n by ion exchange and a f f i n i t y chromatography. Hence, T r i t on X-100 s o l u b i l i z e d enzyme was used f o r the f i n a l p u r i f i c a t i o n and considerable p u r i f i c a t i o n of a l ka l i ne phosphatase was achieved (Table XI I ) . The f i n a l preparat ion contained several p ro te ins , but attempts on fu r ther p u r i f i c a t i o n were unsuccessful due to the i n s t a b i l i t y of the p a r t i a l l y p u r i f i e d preparat ion. The only other work on the p u r i f i c a t i o n of a l ka l i ne phosphatase from D.discoideum has been by Armant and Rutherford ( 3 ) who p u r i f i e d to homogeneity the enzyme from culminat ion phase. They a lso found that the s o l u b i l i z e d enzyme tended to aggregate in the absence of detergent. It is apparent that the culminat ion phase 106 p u r i f i e d enzyme obtained by Armant and Rutherford was more stable than the preparat ion obtained in the present study, which probably accounts fo r t he i r greater success in pur i f y ing the prote in (3). It was found in both th i s study and that of Armant and Rutherford that the 5 1 -nucleot idase a c t i v i t y copu r i f i ed with the a l ka l i ne phosphatase a c t i v i t y throughout the procedure (Table XII, 3 ) , suggesting that the two a c t i v i t i e s are due to the same p ro te in . In a d d i t i o n , the two a c t i v i t i e s co-migrated in polyacrylamide gels in the presence of SDS ( 3 ) . There had been several prev ious ly publ ished observat ions supporting the idea that the two a c t i v i t i e s were due to a s ing le enzyme. Both a c t i v i t i e s were found to res ide in plasma membranes of vegetat ive c e l l s and to be regulated co-ordinate ly during development. ( 33 ). In add i t ion both a c t i v i t i e s showed an iden t i ca l spat ia l d i s t r i b u t i o n in the developing culminate; the highest a c t i v i t i e s were l o c a l i z e d in the pres ta lk c e l l region that i s adjacent to the developing spore c e l l s ( 5 , 48 ). Nonetheless, there are several d i f f e rences between the a l ka l i ne phosphatase and 5 1 -nucleot idase a c t i v i t i e s of vegetat ive membranes. The membrane bound 5 '-nucleot idase was not ac t i va ted by incubation at 50°C or by d i a l y s i s ( F i g .8 , Table VI I ) , although both a c t i v i t i e s were equal ly s o l u b i l i z e d by T r i ton X-100 and both a c t i v i t i e s in the extracts were equal ly ac t iva ted by d i a l y s i s (Table XII)'. Thus, l i k e the a l ka l i ne phosphatase, the 107 vegetat ive c e l l 5 1 -nucleot idase a c t i v i t y i s p a r t i a l l y masked and i t s developmental increase may a lso be due to the removal of i n h i b i t o r . However, the membrane bound 5 '-nucleot idase a c t i v i t y i s not as e a s i l y unmasked as the corresponding a l ka l i ne phosphatase a c t i v i t y . Fur ther -more, the a l ka l i ne phosphatase a c t i v i t y i s comparatively stable when membrane preparat ions are incubated at 50 °C , whereas the 5 1 -nucleot idase a c t i v i t y i s markedly i n h i b i t e d . ( F ig . 7 and 8) . Despite these d i f f e rences in unmasking and s t a b i l i t y , the p a r t i a l l y p u r i f i e d a c t i v i t i e s were i nh ib i t ed to s im i l a r extents by the addi t ion of the d ia l ysa te ( F ig . 18). In a d d i t i o n , the pH optima of the p a r t i a l l y p u r i f i e d a c t i v i t i e s from vegetat ive c e l l s were quite d i s t i n c t ; pH 8.0 fo r 5 '-nucleot idase and pH 9.0 fo r a l ka l i ne phosphatase. In con t ras t , however, Gezel ius and Wright ( 18 )found a pH optimum of 9.0 fo r both a l ka l i ne phosphatase and 5 1 -nuc leot idase from both vegetat ive and culminat ing c e l l s and Armant and Rutherford found that the pH optimum for both a c t i v i t i e s in the p u r i f i e d enzyme preparat ion from culminating c e l l s was pH 9.5 (4 ) . The reasons fo r these d i f fe rences are not c l ea r at present. F i n a l l y , the a l ka l i ne phosphatase a c t i v i t y with pNPP as substrate does not exh ib i t substrate i n h i b i t i o n , while the 5 '-nucleot idase a c t i v i t y with AMP as substrate does (F ig . 19), a r e su l t s im i l a r to that observed by Armant and Rutherford fo r the enzyme from culmination c e l l s ( 4 ) . 108 It i s s t i l l not poss ib le to conc lus i ve l y state whether a l ka l i ne phosphatase and 5 '-nucleot idase in D.discoideum are due to the same or d i f f e r e n t p ro te ins . The ava i l ab le data can perhaps be explained i f we assume that the two a c t i v i t i e s are due to a s ing le prote in that i s capable of ex i s t i ng in several conformations, each conformation having a d i s t i n c t substrate s p e c i f i c i t y . Thus the two substrates pNPP and AMP might in te rac t with the enzyme d i f f e r e n t l y ; through d i s t i n c t l y d i f f e r e n t binding s i t e s or through d i f f e r e n t in te rac t ions with the same binding s i t e . It is proposed that the f u l l y ac t ive conformation i s present in culminat ing c e l l s , but a lso i s produced by d i a l y s i s of T r i ton X-100 ext rac ts of vegetat ive c e l l membranes. It contains no i n h i b i t o r and is ac t i ve with both AMP and pNPP. A second conformation i s produced when vegetat ive membranes are d ia lysed or incubated at 50°C. In th i s conformation, there i s pa r t i a l binding of i n h i b i t o r : the hydro lys is of AMP i s i nh ib i t ed while the hydro lys is of pNPP i s not. F i n a l l y a t h i r d conformation that i s r e l a t i v e l y inac t i ve with both AMP and pNPP as substrates due to a more complete binding of i n h i b i t o r i s postulated as the form of the enzyme in f r e sh l y prepared vegetat ive c e l l membranes. One of the ear l y theor ies on the developmental regula t ion of a l ka l i ne phosphatase in D.discoideum was proposed by Solomon and coworkers. They suggested that a novel culminat ion s p e c i f i c enzyme was produced during the l a t e r stages of d i f f e r e n t i a t i o n ( 58 ). They demonstrated th i s new a c t i v i t y as a second band in starch gel e lec t rophores is which appeared only during cu lminat ion. Supporting data came from the i n h i b i t i o n of the 109 accumulation of a l ka l i ne phosphatase a c t i v i t y by i n h i b i t o r s of prote in ( 37 ) and RNA synthesis ( 3 7 , 15) in d i f f e r e n t i a t i n g slime mold c e l l s . It was suggested that the synthesis of the culminat ion s p e c i f i c form of a lka l ine phosphatase was i nh ib i t ed in these s tud ies . Since these ea r l y s t ud i e s , i t has been es tab l i shed that a l ka l i ne phosphatase i s a t i g h t l y membrane bound prote in in D.discoideum ( 2 0 , 2 4 , 3 3 , 5 2 , ) . As Solomon and coworkers ( 58 ) d id not s o l u b i l i z e the enzyme from t h e i r c e l l ext racts before performing starch gel e l e c t ropho res i s , i t is l i k e l y that the addi t iona l band observed in t he i r experiments was an a r t i f a c t produced by pa r t i a l aggregation of the enzyme in culminat ion stage. MacLeod and Loomis ( 41 ) obtained only a s ing le band of a l ka l i ne phosphatase a c t i v i t y during a l l stages of d i f f e r e n t i a t i o n when they electrophoresed Triton X-100 s o l u b i l i z e d c e l l ex t r a c t s . They a lso compared several physical propert ies of enzymes from vegetat ive and culminating c e l l s of the wi ld type and a mutant with a l t e red a l ka l i ne phosphatase a c t i v i t y ( 41 ). As the enzymes from vegetat ive and culminat ing c e l l s were i nd i s t i ngu i shab l e , they concluded that the accumulation of a l ka l i ne phosphatase during culminat ion was due to the increased production of the same enzyme due to increased expression of a s ing le s t ruc tura l gene ( a l p . ) . An a l t e r n a t i v e , novel theory fo r the developmental regula t ion of a l ka l i ne phosphatase comes from the present work. The data ind ica te that the developmental increase of a l ka l i ne phosphatase i s due to the 110 unmasking of a lready ex i s t i ng enzyme by the removal of a low molecular weight i n h i b i t o r . The removal of th i s putat ive i n h i b i t o r was brought about a r t i f i c a l l y in the laboratory by 50°C incubation or d i a l y s i s of vegetat ive c e l l membrane preparat ions. The a c t i v i t y in culminat ing membranes did not apprec iably increase when the two procedures were a p p l i e d , and the enzyme leve l s in ' a c t i v a t ed ' vegetat ive membranes and culminat ing membranes were very s im i l a r (Table IV). The putat ive i n h i b i t o r seems to be f i rm ly membrane bound, since i t was not removed by short per iods of d i a l y s i s or by e lu t ing 50°C act iva ted membranes with bu f f e r . Observations of a s im i l a r nature were made e a r l i e r by Gezel ius and Wright ( 18 ). They found that c e l l f ree extracts from vegetat ive c e l l s o f D.discoideum l o s t 90% of the o r i g i na l a l ka l i ne phosphatase a c t i v i t y when stored at 4°C for e ight days. A sorocarp ext rac t stored s i m i l a r l y l o s t less than 50% of enzyme a c t i v i t y . This i n h i b i t i o n could be reversed in t he i r ext racts by d i l u t i o n of the c e l l f ree extracts 100-fo ld , probably by d i l u t i n g the concentrat ion of the i n h i b i t o r . K i l l i c k and Wright ( 29 ) suggested the ro le o f an endogenous i n h i b i t o r for th is r eve rs ib le i n h i b i t i o n phenomenon. It should be noted that in the present study, however, the i n h i b i t o r could not be removed from the enzyme by d i l u t i n g the extracts (data not shown). I l l Although th i s i s the f i r s t report of the unmasking of a membrane bound enzyme during D.discoideum d i f f e r e n t i a t i o n , the c r yp t i c existence in vegetat ive c e l l s o f another developmental ly regulated enzyme was suggested e a r l i e r by K i l l i c k and Wright ( 2 8 ) . Trehalose - 6 - phosphate synthetase a c t i v i t y was inac t i ve in vegetat ive c e l l ex t r a c t s , but could be unmasked by ammonium sulphate p r e c i p i t a t i o n and incubat ion of the p rec ip i t a t e in presence of UDP-glucose fo r 10 minutes at 35°C. Results reported by Roth and Sussman (. 54 ) a lso suggested the presence of an i n h i b i t o r of Trehalose - 6 -phosphate synthetase in the ear l y stages of d i f f e r e n t a t i o n . They obtained a 56% i n h i b i t i o n of the expected a c t i v i t y of the enzyme, when extracts from 5 hour c e l l s were mixed with those from the preculmination stage. In add i t ion the e x t r a c e l l u l a r c y c l i c AMP s p e c i f i c phosphodiesterase in D. discoideum i s present in two molecular forms showing d i f f e r e n t substrate a f f i n i t i e s . ( 10, 11 ). Kessin et a l . , found that a high molecular weight form with low substrate a f f i n i t y was a complex of enzyme and a prote in i n h i b i t o r , ( 2 7 ) . The enzyme was ac t i va ted 20 to 100 f o l d by i nac t i va t i ng the i n h i b i t o r by treatment of the complex with d i t h i o t h r e i t o l ( 2T)\ They a lso showed that the p u r i f i e d enzyme could be reconst i tu ted to low substrate a f f i n i t y form of the enzyme by add i t ion of p u r i f i e d i n h i b i t o r '(27). -The apparent unmasking of a lka l ine phosphatase during development by the removal of an i n h i b i t o r component i s not necessar i l y incompatible 112 with the data on the i n h i b i t i o n of a l ka l i ne phosphatase accumulation by i n h i b i t o r s of prote in and RNA synthesis- ( 15, 37 ). The removal of the i n h i b i t o r by some as yet unknown phys io log ica l mechanism i s presumably developmentally regulated. The removal of i n h i b i t o r might be cata lyzed by a newly synthesized p ro t e i n , and i n h i b i t i o n of the synthesis of th i s prote in would d i r e c t l y prevent the accumulation of a l ka l i ne phosphatase a c t i v i t y . S i m i l a r l y , observations on a l ka l i ne phosphatase accumulation in c e l l s in which the development had been perturbed in var ious ways ( 25, 44, 51 , 60 ) could be s i m i l a r l y expla ined. The unmasking of a c r yp t i c form of a l ka l i ne phosphatase during development proposed here i s cons is tent with the observat ions of MaCleod and Loomis ( 37 ). They compared several physical propert ies of a l ka l i ne phosphatase from vegetat ive and culminat ing c e l l s of D.discoideum and found them to be ind i s t i ngu i shab le . ( 37 ). Some of the experiments presented here are a lso cons is tent with the concept that the vegetat ive and culmination enzymes are i d e n t i c a l . The culmination phase enzyme is j us t as suscept ib le to the i n h i b i t o r as i t s vegetat ive c e l l counterpart and the pH optimum of the enzyme does not change during development. In a d d i t i o n , Arrhenius p lo ts of a c t i v i t i e s in vegetat ive and culminat ing membranes ind ica te very s im i l a r t r ans i t i on temperatures suggesting a s im i l a r l i p i d i n te rac t ion with the enzymes from the two developmental stages (F ig . 25). F i n a l l y the vegetat ive and culminat ing phase enzymes have iden t i ca l mob i l i t i e s when e lec t ro-phoresed on SDS - polyacrylamide ge l s . (F ig . 20) 113 However, there are de f i n i t e d i f fe rences between the vegetat ive and culminat ing enzymes. It was not poss ib le to pu r i f y the culminat ing enzyme using concanaval in A - Sepharose and DEAE -Sephacel chromatography, techniques that had e f fec ted considerable p u r i f i c a t i o n of the vegetat ive enzyme. There were a lso d i f f e rences in the s e n s i t i v i t y of vegetat ive and culminating enzymes to SDS. ( F ig . 21) , and T r i s-C l buf fe r ( F ig . 22). These resu l t s suggest that post t r ans l a t i ona l modi f i ca t ions of the a l ka l i ne phosphatase may well occur during development and such a change may be important in the unmasking phenomenon. Quiv iger et a l . , found on histochemical s ta in ing of th in sect ions that a l k a l i n e phosphatase a c t i v i t y could only be detected on the con t ra -c t i l e vacuoles dur ing the f i r s t f i v e hours o f d i f f e r e n t i a t i o n . ( 4 8 ) . The a c t i v i t y in con t r a c t i l e vacuoles accounted only fo r 30% of the tota l a c t i v i t y in the c e l l and i t i s poss ib le that the absence of s ta in ing of plasma membrane bound enzyme was due to the i n h i b i t i o n of th i s enzyme during f i x a t i o n procedure. ( 48 ). The con t r a c t i l e vacuoles disappeared a f t e r 5 hours of d i f f e r e n t i a t i o n and i t was impossible to h is tochemica l l y s ta in the enzyme again un t i l the culminat ion stages of development. The enzyme in the culmination stage was l o c a l i s e d in the plasma membrane and some very small v e s i c l e s , and they suggested the p o s s i b i l i t y of t ranspor ta t ion of the enzyme to the plasma membrane through these ves ic les- (48). Another a l t e rna t i ve however would be the complete loss of the con t r a c t i l e vacuole 114 a c t i v i t y and modi f i ca t ion of the plasma membrane enzyme such that i t i s no longer sens i t i ve to f i x a t i o n . In fac t the enzyme from vegetat ive eel 1 s wasmore sens i t i ve to glutaraldehyde than the culminat ion c e l l a c t i v i t y when assayed spectrophotometrycaVTly, in v i t r o . ( 48 ). Moreover, Crean and Rossomando found that the a lka l ine phosphatase from vegetat ive c e l l s bound more t i g h t l y to concanavalin A Sepharose than did enzymes from l a t e r stages of d i f f e r e n t a t i o n , suggesting changes in the carbohydrate components of the g lycoprote in ( ' 1 2 . ) . The resu l t s of these workers ( 12, 48 ) are therefore a lso cons is tent with a modi f i ca t ion of a l ka l i ne phosphatase during d i f f e r e n t i a t i o n . However, s ince the enzymes from vegetat ive and culminating c e l l s migrate i d e n t i c a l l y during e lec t rophores i s in SDS-polyacrylamide gels any modi f i ca t ion must be s l i g h t . The histochemical studies by Quiviger et a l . , do not reveal any a l ka l i ne phosphatase a c t i v i t y between the aggregation and culminat ion phases even though t he i r spectrophotometric determinations on c e l l f ree extracts suggested that approximately 70% of the o r i g ina l a c t i v i t y remained in the c e l l s , ( 4 3 ). S i m i l a r l y , Armant and Rutherford detected 16 to 35% of the maximal a c t i v i t y found in the prestalk-prespore in te r face in the remainder of the culminat ing f r u i t but detected histochemical a c t i v i t y only in the presta lk/prespore i n t e r f a ce . ( 2 ) Hence, i t i s poss ib le that d i f f e r en t regions of . the culminate have d i f f e r e n t molecular forms of the enzyme. 115 The resu l t s of the present study ind ica te that vegetat ive a l ka l i ne phosphatase can be act iva ted approximately 10 f o l d by removal of a putat ive i n h i b i t o r (Table IV). It i s a lso known that the prote in concentrat ion decreases by 50% during d i f f e r e n t i a t i o n . ( 6 1 ) . If i t i s assumed that the a l ka l i ne phosphatase of only 20 to 30% of the populat ion i s ac t i va ted 10 f o l d during d i f f e r e n t i a t i o n by the removal of the putat ive i n h i b i t o r , th i s could account fo r an increase in s p e c i f i c a c t i v i t y fo r a l ka l i ne phosphatase of 5.6 to 7.4 f o l d at culminat ion stage. It should be i n t e res t i ng to f i nd out the fate of the enzyme molecule in the prespore region of the culminating c e l l s . Rutherford and coworkers ( 55 ) using antiserum against p u r i f i e d 5 '-nucleot idase from culminat ing c e l l s have ind icated that the enzyme is present in equal quant i t i es throughout the culminat ing i n d i v i d u a l . Even though de t a i l s of t h i s experiment have not been pub l i shed , i t agrees with the idea that basal quant i t i es of th i s enzyme are present in a l l c e l l s undergoing d i f f e r e n t i a t i o n . It would appear that the enzyme in the prespore region of the culminat ing c e l l s becomes i r r e v e r s i b l y masked during d i f f e r e n t i a t i o n , s ince i t was not poss ib le to apprec iab ly ac t i va te the culminat ing enzyme by treatment at 50°C or by d i a l y s i s (Table IV) . Rutherford et a l . , suggested that the enzyme in the prespore and f u l l y d i f f e r e n t i a t e d s ta lk c e l l s i s i nh ib i t ed by inorganic phosphate and ammonia (55 ). However, while t he i r data on the concentrat ions of ammonia and phosphate in the various c e l l types of the culminate might account fo r the low a c t i v i t y in f u l l y d i f f e r en t i a t ed . 116 stalk c e l l s , i t is d i f f i c u l t to understand how i t could account for the low activity in prespore cells. ( 55 ). Furthermore, the data presented here indicate that the inhibition of the vegetative enzyme by phosphate is freely reversible (Table XIV). The identity of the putative inhibitor or the physiological manner in which i t is removed from the enzyme is not yet known. The production of a developmentally regulated enzyme specific to prestalk cells which removes and degrades the inhibitor in these cells or post translational modification of the enzymes in the prestalk: cells which effects the associations with the inhibitor are just two possible mechanisms worth considering. Further studies on alkaline phosphatase of isolated prestalk and prespore cells would be useful in understanding the changes that occur to the vegetative enzyme during spore and stalk cell differentiation. 117 REFERENCES 1. Ahmed, Z . , and King, E.J. (I960). P u r i f i c a t i o n of p lacental a l ka l i ne phosphatase. Biochim. Biophys. Acta . 40_, 320-328. 2. Armant, D.R., and Rutherford, C L . (1979). 5' -AMP nucleot idase i s l o c a l i s e d in the area of c e l l - c e l l contact of prespore and presta lk regions during culminat ion of P ic tyoste l ium discoideum Mech. Ageing Develop. 10_, 199-217. 3. Armant, D.R., and Rutherford, C L . (1981). Copur i f i c a t i on of a l ka l i ne phosphatase and 5'-AMP s p e c i f i c nucleot idase in Dic tyoste l ium discoideum. J . B i o l . Chem. 256, 12710-12718. 4. Armant, D.R., and Rutherford, C L . (1982). Propert ies of a 5'-AMP s p e c i f i c nucleot idase which accumulates in one c e l l type during development in Dic tyoste l ium discoideum Arch. Biochem. Biophys. 216, 485-494. 5. Armant, D.R., S t e t l e r , D.A., and Rutherford, C L . (1980). Ce l l surface l o c a l i z a t i o n of 5'-AMP nucleot idase in presta lk c e l l s of Dic tyoste l ium discoideum. J . C e l l . S c i . 45_, 119-129. 6. Atryzek, V. (1976). D i ssoc ia t ion of developing slime mold c e l l s does not i n h i b i t the developmentally regulated r i s e in a l ka l i ne phosphatase a c t i v i t y . J . B a c t e r i o l . 1 2 6 , 1005-1008. 7. Bonner, J . T . (1947). Evidence fo r the formation of c e l l aggregates by chemotaxis in the devel cpment of the slime mold Dictyoste l ium discoideum. J . Exp. Zoo l . 106, 1-26. 8. Bonner, J . T . (1967). "The c e l l u l a r slime molds" , 2nd ed. Pr inceton Univ. Press, P r inceton , New Jersey, pp.11. 9. Bonner, J . T . , Chiquoine, A . D . , and Ko lder i e , M.Q. (1955). A histochemical study o f d i f f e r e n t i a t i o n in the c e l l u l a r slime molds. J . Exp. Zoo l . 130, 133-158. 10. Chang, Y.Y. (1968). Cyc l i c 3 ' , 5' - adenosine monophosphate phosphodiesterase produced by the slime mold Dictyoste l ium  discoideum.. Science 161, 57-59. 11. Chassy, B.M. (1972). Cyc l i c nucleot idase phosphodiesterase in Dic tyoste l ium discoideum: Interconversion of two enzyme forms. Science 175_, 1016-1018. 12.. .Crean,; E.V., and Rossomando, E.F. (1977). : Developmental changes in membrane-bound enzymes of Dictyoste l ium discoideum detected by concanaval in A-Sepharose a f f i n i t y chromatography. Biochem. Biophys. Res. Commun. 75, 488-495. 13. Dorn, G.L. (1968). P u r i f i c a t i o n and charac te r i za t ion of Phosphatase I from Asperg i l l us nudulans. J . B i o l . Chem. 243, 3500-3506. 118 14. E l l i n g s o n , J . S . , T e l s e r , A., and Sussman, M. (1971). Regulation of f unc t i ona l l y re la ted enzymes during a l t e rna t i ve developmental programs. Biochim. Biophys. Acta . 244, 388-395. 15. F i r t e l , R.A., Baxter, L., and Lod ish , H.F. (1973). Actinomycin D and the regu la t ion of enzyme biosynthes is during development of Dictyoste l ium discoideum. J . Mol. B i o l . ' 7 9 , 315-327. 16. Fishman, L. (1974). Acrylamide d isc gel e lec t rophores i s of a l ka l i ne phosphatase o f human t i s s u e s , serum and asc i tes f l u i d using T r i ton X-100 in the sample and the gel matr ix. Biochem. Med. 9_, 309-315. 17. Garen, A . , and Lev in tha l , C. (1960) A f ine-s t ruc ture genetic and chemical study o f the a l ka l i ne phosphatase o f E. C o l i . Biochim. Biophys. Acta . 38, 470-483. 18. Geze l ius , K., and Wright, B.E. (1965). A lka l ine phosphatase in Dictyoste l ium discoideum. J . Gen. M i c rob io l . 38, 309-327. 19. Ghosh, N.K., and Fishman, W.H. (1968). P u r i f i c a t i o n and proper t ies o f molecular weight var iants of human placenta l a l ka l i ne phosphatase. Biochem. J . 108, 779-792. 20. G i l k e s , N.R., and Weeks, G. (1977). The p u r i f i c a t i o n and cha rac te r i za t ion of Dictyoste l ium discoideum plasma membranes. Biochim. Biophys. Acta 464, 142-156. 21. Glew, R.H., and Heath, E.C. (1971). Studies on the e x t r a c e l l u l a r a l ka l i ne phosphatase of Micrococcussodonensis. J . B i o l . Chem. 246, 1556-1565. 22. Go ld f i s che r , S., Essner, E., and Nov ikof f , A.B. (1964). The l o c a l i z a t i o n of phosphatase a c t i v i t i e s at the leve l of u l t r a s t r u c tu r e . J . Histochem. Cytochem. 1_2, 72-95. 23. Go lds te in , D. J . , Rogers, C . E . , and Ha r r i s , H. (1980). Expression of a l ka l i ne phosphatase l o c i in mammalian t i s sues . Proc. Na t l . Acad. S c i . USA. 77_. 2857-2860. 24. Green, A . A . , and Newell , P.C. (1974). The i s o l a t i o n and sub-f rac t ionat ion o f plasma membrane from the c e l l u l a r sl ime mold Dictyoste l ium discoideum. Biochem. J . 140, 313-322. 25. Hamilton, I.D., and Ch ia , W.K. (1975). Enzyme a c t i v i t y changes during c y c l i c AMP-induced s ta lk c e l l d i f f e r e n t i a t i o n in p4, a var iant of Dictyoste l ium discoideum. J . Gen. M i c rob io l . 91 295-306. — 119 26. Hofstee, B.H.J. (1955) A lka l ine phosphatase I. Mechanism of act ion of In, Mg, g l y c i n e , versene and hydrogen ions . Arch. Biochem. Biophys. 59, 352-365. 27. Kess in , R.H., Seth, J . O . , Shapiro, R.I., and Franke, J . (1979). Binding of i n h i b i t o r a l t e r s k ine t i c and physical proper t ies of e x t r a c e l l u l a r c y c l i c AMP phosphodiesterase from Pictyostel iurn  discoideum. Proc. Na t l . Acad. S c i . U.S.A. 76, 5450-5454. 28. K i l l i c k , K.A. , and Wright, B.E. (1972). Trehalose synthesis during d i f f e r e n t i a t i o n in Dictyoste l ium discoideum. III. Ir ivitro unmasking of t rehalose 6-phosphate synthetase. J . B i o l . Chem. 247, 2967-2969. 29. K i l l i c k , K.A., and Wright, B.E. (1974). Regulation of enzyme a c t i v i t y during d i f f e r e n t i a t i o n in Dic tyoste l ium discoideum Annu. Rev. M i c r o b i o l . 28, 139-166. 30. Krivanek, J .O . (1956). A lka l ine phosphatase a c t i v i t y in the developing slime mold, Dictyoste l ium discoideum Raper. J . Exp . Zoo l . 133, 459-480. 31. Krivanek, J . O . , and Krivanek, R.C. (1958). The histochemical l o c a l i z a t i o n of ce r ta in biochemical intermediates and enzymes in the developing slime mold, Dictyoste l ium discoideum. Raper J . Exp. Zoo l . 1_37_, 89-115. 32. Laemmli, U.K; (1970). Cleavage of s t ruc tura l prote ins during the assembly of the head of bacteriophage T». Nature (London) 227, 680-685. 33. Lee, A . , Chance, K., Weeks, C . , and Weeks, G. (1975) Studies on the a l ka l i ne phosphatase and 5 1 -nucleot idase of Dictyoste l ium discoideum. Arch. Biochem. Biophys. 171, 407-417. 34. Loomis, W.F. (1972). Role of the surface sheath in the control of morphogenesis in D.discoideum. Nature New B i o l . 240, 6-9-. 35. Loomis, W.F. (1975). D ic tyoste l ium discoideum. A developmental system. Academic Press. New York. pp. 107. 36. Loomis, W.F. (1975). D ic tyoste l ium discoideum. A developmental system. Academic Press , New York. pp.110. 37. Loomis, W.F.; J r . (T969). Developmental regulat ion of a l ka l i ne phosphatase in Dictyoste l ium discoideum. J . B a c t e r i d . 100, 417-422. 120 38. Lowry, O .H . , Rosebrough, N.J . , Fa r r , A . L . , and Randa l l , R.J. (1951). Protein measurement with the Fo l in phenol reagent. J . B i o l . Chem. 193, 265-275. 39. Lumb, J . R . , and D o e l l , R.G. (1970). The biochemical cha rac te r i sa t ion of a l ka l i ne phosphatase from chemical and v i r a l induced thymic lymphomas of C57 BL mice. Cancer. Res. 30, 1391-1396. 40. MacA l i s t e r , T . J . , Coster ton , J .W., Thompson, L, Thompson, J . , and Ingram, J .M. (1972). D i s t r i bu t i on of a l ka l i ne phosphatase within the peripasmic space of gram-negative bac te r i a . J . b a c t e r i o l . I l l , 827-832. 41. MacLeod, C . L . , and Loomis, W.F. (1979). Biochemical and genetic ana lys is o f a mutant with a l te red a l ka l i ne phosphatase a c t i v i t y in D ic tyoste l ium discoideum. Dev. Genet. 1_, 109-121. 42. McComb, R.B., Nowers, G .N . , and Posen, S. (1979). A lka l ine Phosphatase. Plenum Press, New York and London, pp.51. 43. McComb, R.B., Bowers, G .N . , and Posen, S. (1979). A lka l ine Phosphatase. Plenum Press , New York and London, pp.405. 44. McMahon, D., Hoffman, S., Fry , W., and West, C. (1975). The involvement of the plasma membrane in the development of Dic tyoste l ium discoideum, in Pattern formation and gene regulat ion in development. D. McMahon; and C.F. Fox (Eds). W.A. Benjamin. Inc. Palo A l t o . pp. 60-75. 45. Morton, R.K. (1955) Methods of ext rac t ion of enzymes from animal t i s s u e s , in : Methods in Enzymology. (Colowick, S .P. , and Kaplan, N.O., e d s . ) . Academic Press , New York pp.25. 46. Newell , P .C. , Longlands, M., and Sussman, M. (1971) Control of enzyme synthesis by c e l l u l a r i n te rac t ion during development of the c e l l u l a r slime mold Dictyoste l ium discoideum. J . Mol. B i o l . 58, 541-554. 47. Pa r i sh , R.W., and P e l l i , C. (1974). A lka l ine phosphatase of D_. discoideum: Cel l surface loca t ion and co l ch i c i ne 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 Le t t . 48_, 293-296. 48. Qu iv iger , B.; Benichou, J . C , and Ryter, A. (1980) Comparative cytochemical l o c a l i z a t i o n of a l ka l i ne and ac id phosphatases during s tarvat ion and d i f f e r e n t i a t i o n of Di c t y o s t e l i urn di scoi deum. B i o l . C e l l u l a i r e . 37, 241-250. 121 49. Raper, K.B. (1935). Dictyoste l ium discoideum, a new species of sl ime mold from decaying fo res t leaves. J . Agr. Res. 50_, 135-147. 50. Raper, K.B. (1937). Growth and development of Dictyostel iurn discoideum with d i f f e r e n t bac ter ia l assoc ia tes . J . Agr. Res. 55, 289-316. 51. Rickenberg, H.V., T ihon, C , and Guzel Omer. (1977). The e f f e c t of 3' : 5' c y c l i c adenosine monophosphate on enzyme formation in non-aggregated amoebae of D i c t yos t e l i urn di scoideum, in : Developments and d i f f e r e n t i a t i o n in the c e l l u l a r slime moulds. (Cappucc ine l l i , P., and Ashworth, J . , Eds.) E lsev ier/North Hol land, Amsterdam, pp. 173-187. 52. Rossomando, E .F . , and Cutler., L.S. (1975) Loca l i s a t i on of adenylate cyc lase in Dictyoste l ium discoideum. Exp. C e l l . Res. 95, 67-78. 53. Rossomando, E .F . , and Maldonado, B. (1976). Inh ib i t ion of 5'-nucleot idase a c t i v i t y a f t e r growth of Di c t y o s t e l i um di scoideum, Exp. Ce l l Res. 100, 383-388. 54. Roth, R., and Sussman, M. (1966) Trehalose synthesis in the c e l l u l a r slime mold P ictyos te1i um d iscoideum. Biochim. Biophys. Acta . 122, 225-231. 55. Rutherford, C . L . , Tay lo r , R.P., Merkle, R.K., and Frame, L.T. (1982). C e l l u l a r pattern format ion: Di c t yos t e l i um di scoi deum as a system for a biochemical approach. Trends Biochem. S c i . 7, 108-111. 56. Sandermann, H. J r . , and Strominger, J . L . (1972) P u r i f i c a t i o n and proper t ies of Ccc- isopreno id alcohol phosphokinase from Staphylococcus aureus. J . B i o l . Chem. 247, 5123-5131. 57. Sarthy, A . , M i chae l i s , S. , and Beckwith, J . (1981) Delet ion map of the Escher ich ia c o l i s t ruc tura l gene f o r a l ka l i ne phosphatase, PhoA. J . B a c t e r i d . 145, 228-292. 58. Solomon, E.P.; Johnson, E.M., and Gregg, J . H . (1964). Mu l t ip le forms of enzymes in a c e l l u l a r slime mold during morphogenesis. Develop. B io l. 9_, 314-326. 59. S to lbach, L . L . , Krant, M.J., and Fishman, W.H. (1969). Ectopic production of an a l ka l i ne phosphatase in pat ients with cancer. N. Engl . J . Med. 28J_, 757-762. 60. Tuchman,J. , Smart, J . E . , and Lod ish , H.F. (1976). E f fec ts 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 . 51, 77-85. 122 61. Weeks, G. (1973) Agglut inat ion of growing and d i f f e r e n t i a t i n g c e l l s of Dictyostel iurn di scoideum by concanavalin-A. EXD. Ce l l Res. 76, 467-470. 62. White, G . J . , and Sussman, M. (1961) Metabolism of major c e l l const i tuents during slime mold morphogenesis. Biochim. Biophys. Acta 53, 285-293. 63. Wray, W., Bou l ikas , TV, Wray, V . P . , and Hancock, R. (1981). S i l v e r s ta in ing of prote ins in polyacrylamide ge l s . Ana l . Biochem. 118, 197-203. 

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}]}"
                            data-media="{[{embed.selectedMedia}]}"
                            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.831.1-0095886/manifest

Comment

Related Items