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Enzymes of polynucleotide synthesis in two photosynthetic organisms
342
BIOCHIMICA ET BIOPHYSICAACTA
Preliminary Notes PN 6102
Enzymes of polynucleotide synthesis in two photosynthetic organisms The knowledge of enzyme catalyzing the formation of polyribonucleotides is based mainly upon systems which have been isolated from bacteria and animal cells. Little is known, however, about the existence of similar enzymes in the photosynthetic cells of plants. Therefore an attempt has been made to detect and isolate polynucleotide phosphorylase (EC 2.7.7.8 ) and RNA polymerase (EC 2.7.7.6 ) activity in two species of algae representing two different types of cellular organisation : A nacystis nidulans, a blue-green alga which grows exclusively in the fight by bacteria-like cell division, and which also lacks a true cell nucleus; and Chlorella pyrenoidosa, a unicellular green alga capable both of photoautotrophic and heterotrophic growth, and possessing a distinct nucleus within the cell. Anacystis nidulans (Drouet, Kratz and Allen's strain)* was grown bacteria free according to KRATZ AND MYERS1. Chlorella pyrenoidosa was cultivated in a medium which has been described previously 2. Cell-flee extracts were prepared by two methods: either by sonic oscillation of suspensions of cells using a 6o-watt MSE oscillator at 2-5 ° ; or by disrupting the cells with glass beads in a blender under refrigeration. Triethanolamine-HC1 buffer (pH 7.6) and a solution containing 0.05 M Tris-HC1 buffer (pH 7.6 ) o.oi M MgSO 4, I mM EDTA, and I mM mercaptoethanol served as the suspending medium during the preparation of polynucleotide phosphorylase and RNA polymerase, respectively. After centrifugation of the fragmented material at 35 ooo × g crude extracts (containing 25-35 mg of protein per ml) resulted, which served as the starting material for the purification procedures. Polynucleotide phosphorylase activity was determined by measuring the incorporation of E8-z4C]ADP into the acid-insoluble fraction 3. RNA polymerase activity was measured by following the incorporation of E8-14C]ATP into RNA in the presence of unlabelled, GTP, CTP, UTP and DNA 4. Protein was determined by the biuret method; specific enzymic activity is expressed as m/tmoles of substrate incorporated per mg of protein. In order to detect polynucleotide phosphorylase in crude extracts of both organisms in appreciable amounts it was necessary to dilute the samples to a protein content of less than 2.5 mg/ml; the observed inhibition was not caused by inorganic phosphate as extended dialysis failed to remove it. The inhibitory principle disappeared together with the inert proteins during the attempted purification procedure. Solid ammonium sulphate was added to the crude extract and the precipitate, which formed at 30% saturation, was discarded; then the supernatant was adjusted to 800/0 saturation with A culture was kindly supplied by Dr. J. MYERS, Department of Botany and Zoology, University of Texas, Austin, Texas (U.S.A.). *
Biochim. Biophys. A cta, 72. (1963) 342-344
PRELIMINARYNOTES
343
ammonium sulphate. The precipitate which formed was removed by centrifugation and dissolved in triethanolamine-HC1 buffer; the extract was treated with a volume of I °/o protamine sulphate equal to about one quarter of the original extract. After 15 min, the sediment was collected by centrifugation and dialyzed for 6 h against diluted buffer; centrifugation at 35 ooo × g removed the precipitated material. The specific activity of the polynucleotide phosphorylase in the supernatant was greater than that of the crude extract either of Anacystis (52-55 times greater) or of Chlorella (25-30 times greater) (Table I). The supernatants were free from ADP-splitting phosphatases and from myokinase. The incorporation of [8-14CIADP into polyribonucleotides by purified preparations from Anacystis and Chlorella was inhibited by inorganic phosphate; in the presence of GDP, CDP and U D P a smaller rate of incorporation occurred. Net synthesis of polyadenylic acid and of polyribonucleotide copolymers was achieved by incubating ADP or ADP, GDP, CDP and U D P with purified polynucleotide phosphorylase preparations for 1-6 h at 37°; the products were identified by paper chromatography and by their degradation products after alkaline hydrolysis. TABLE I PURIFICATION
OF POLYNUCLEOTIDE PHOSPHORYLASE PHOTOSYNTHETIC ORGANISMS
FROM TWO
The test mixture, o.25o ml, contained: 3/,moles triethanolamine-HC1 buffer (pH 7.6), 1.25/,moles Mg s+, o. I/,mole [8-14C]ADP(specific activity approx. 5" lO6 counts//~mole), o.o30-2.o0 mg protein. After incubation for 3° min at 37° the reaction was stopped with 0.5 ml of lO% perchloric acid, carrier protein was added where necessary, and the mixture was centrifuged for 5 min at 5ooo rev./min. The precipitate was washed 3 times with cold perchloric acid (2 %), once with methanol and with ether; after drying, it was dissolved in o.5 ml of formic acid. Portions were spread on metal planchettes and dried. Radioactivity was measured in a windowless Geiger-Mliller counter. No correction was made for self-absorption.
Fraction
Source of enzyme prepaTations (activity expressed as re#moles of [8-t'C ] A DP incorporated per volume of the #action indicated ("toted") and per mg protein ("specific") A nacystis Total
Crude extract Ammonium sulphate, 3o-80% saturation Protamine eluate after dialysis
Chlorella
Spbciftc
Total
Specific
12 ooo 39 300
4.1 52.0
iooo 2000
0.93 7.5
6 650
214.o
766
26.7
The detection of RNA polymerase in crude extracts of both organisms was impaired by the instability of the enzyme, and by the interfering action of an enzymic activity which catalyzed the incorporation of [8-14CIATP into an acid-insoluble product, probably without any requirement for DNA; addition of the complementary ribonucleoside triphosphates tended to inhibit this reaction. In the presence of DNA, however, an a appreciable incorporation of [8-14C]ATP took place which is considered to be an expression of polymerase activity. Treatment of the crude extracts with bentonit (I00 mg/ml) increased the stability of the polymerase activity, even during the further purification with protamine Biochim. Biophys. Acta, 72 (1963) 342-344
344
PRELIMINARY NOTES
sulphate; the latter was applied as described for polynucleotide phosphorylase. In the case of Anacystis, the eluate obtained from the protamine precipitate showed the incorporation of [8-x4C]ATP only in the presence of GTP, CTP, UTP, and DNA; similar preparations from Chlorella still had a capacity for incorporating [8-14C]ATP as the sole substrate but, in the complete reaction mixture, the specific activity was increased I6-fold over that of the crude extract (Table II). In order to rule out any incorporation of nucleoside diphosphates formed from T A B L E II R N A POLYMHRASE/ACTIVITY IN VARIOUS PREPARATIONS FROM TWO PHOTOSYNTHETIC ORGANISMS The test mixture, 0.25 ° m l , contained: 6/~moles Tris buffer (pH 7.6) ; 1.2/~moles Mgt+; o.12 ~umole E D T A ; o.12 p m o l e m e r c a p t o e t h a n o l ; 1.2 m g b e n t o n i t ; o.1 /*mole [8-aIC]ATP (specific activity approx. I o e counts/pmole) ; o.5 p m o l e of unlabelled GTP, C T P and U T P , each; 1.5 pmoles N a t H P O * (for inhibition of potynucleotide phosphorylase) ; approx. 2 o o p m o l e s t h y m u s D N A ;and o.o4o-2. 5 m g protein. After' incubation for I h at 37 ° t h e samples were deproteinized and p r e p a r e d for c o u n t i n g as described for polynucleotide phosphorylase.
Fraction
Source of emyme preparations (aaitgcly expressed as m¢~ncff.esof [8-t~C.IA TP incorporated per volume of #~ fraaio~,indicag~ ("total"), and per mg protein ("specific") in the complete test raixture. Values in brackets: incorporation with [8-x*C]A TP as sole subavat#, without DNA ) CMoreUa
A nacysBs
Crude. extract after:
Bentonit treatment P r o t a m i n e eluate
Total
S p~dfic
Total
Specific
lO5O (384 o) 117 (nil)
0.42 (I.5) 4.o (nil)
262 (2900) 115 (22o)
1.2 (I3) 18.o (34)
the triphosphates by the action of phosphatases and kinases, inorganic phosphate was added to the test mixtures in sufficient amount to inhibit polynucleotide phosphorylase activity. The results obtained strongly suggest that the functions of polynucleotide phosphorylase and RNA polymerase in photosynthetic cells are rather similar to those in the cells of bacteria and animals. Further details will be given in a later publication. Part of this work was performed at the California Institute of Technology, Division of Biology, Pasadena, Calif. (U.S.A.); it has been supported by the National Institute of Health, U.S. Public Health Service, and by the Deutsche Forschungsgemeinschaft.
Botanisckes Institut, Universitiit T~bingen T~bingen (Germany)
GERHARD RICHTER
x W. A. KRATZ AND J. MYERS, A m . J . B o t a n y , 42 (1955) 282. t N. BISHOP AND H. GA~TRON, B i o c h i m . B i o p h y s . A c t a , 28 (1956) 3.5S M. GRUNBERG-MANAGO, P. J. ORTIZ AND S. OCHOA, Biochira. B i o p h y s . A c t a , 20 (I956) 269. 4 j . j . FURTH, J. HURWITZ AND M. ANDERS, J . Biol. Chem., 237 (I962) 261I.
Received March 18 th, 1963 B i o c h i m . B i o p h y s . Acta, 72 (1963) 342-344
BIOCHIMICA ET BIOPHYSICAACTA
Preliminary Notes PN 6102
Enzymes of polynucleotide synthesis in two photosynthetic organisms The knowledge of enzyme catalyzing the formation of polyribonucleotides is based mainly upon systems which have been isolated from bacteria and animal cells. Little is known, however, about the existence of similar enzymes in the photosynthetic cells of plants. Therefore an attempt has been made to detect and isolate polynucleotide phosphorylase (EC 2.7.7.8 ) and RNA polymerase (EC 2.7.7.6 ) activity in two species of algae representing two different types of cellular organisation : A nacystis nidulans, a blue-green alga which grows exclusively in the fight by bacteria-like cell division, and which also lacks a true cell nucleus; and Chlorella pyrenoidosa, a unicellular green alga capable both of photoautotrophic and heterotrophic growth, and possessing a distinct nucleus within the cell. Anacystis nidulans (Drouet, Kratz and Allen's strain)* was grown bacteria free according to KRATZ AND MYERS1. Chlorella pyrenoidosa was cultivated in a medium which has been described previously 2. Cell-flee extracts were prepared by two methods: either by sonic oscillation of suspensions of cells using a 6o-watt MSE oscillator at 2-5 ° ; or by disrupting the cells with glass beads in a blender under refrigeration. Triethanolamine-HC1 buffer (pH 7.6) and a solution containing 0.05 M Tris-HC1 buffer (pH 7.6 ) o.oi M MgSO 4, I mM EDTA, and I mM mercaptoethanol served as the suspending medium during the preparation of polynucleotide phosphorylase and RNA polymerase, respectively. After centrifugation of the fragmented material at 35 ooo × g crude extracts (containing 25-35 mg of protein per ml) resulted, which served as the starting material for the purification procedures. Polynucleotide phosphorylase activity was determined by measuring the incorporation of E8-z4C]ADP into the acid-insoluble fraction 3. RNA polymerase activity was measured by following the incorporation of E8-14C]ATP into RNA in the presence of unlabelled, GTP, CTP, UTP and DNA 4. Protein was determined by the biuret method; specific enzymic activity is expressed as m/tmoles of substrate incorporated per mg of protein. In order to detect polynucleotide phosphorylase in crude extracts of both organisms in appreciable amounts it was necessary to dilute the samples to a protein content of less than 2.5 mg/ml; the observed inhibition was not caused by inorganic phosphate as extended dialysis failed to remove it. The inhibitory principle disappeared together with the inert proteins during the attempted purification procedure. Solid ammonium sulphate was added to the crude extract and the precipitate, which formed at 30% saturation, was discarded; then the supernatant was adjusted to 800/0 saturation with A culture was kindly supplied by Dr. J. MYERS, Department of Botany and Zoology, University of Texas, Austin, Texas (U.S.A.). *
Biochim. Biophys. A cta, 72. (1963) 342-344
PRELIMINARYNOTES
343
ammonium sulphate. The precipitate which formed was removed by centrifugation and dissolved in triethanolamine-HC1 buffer; the extract was treated with a volume of I °/o protamine sulphate equal to about one quarter of the original extract. After 15 min, the sediment was collected by centrifugation and dialyzed for 6 h against diluted buffer; centrifugation at 35 ooo × g removed the precipitated material. The specific activity of the polynucleotide phosphorylase in the supernatant was greater than that of the crude extract either of Anacystis (52-55 times greater) or of Chlorella (25-30 times greater) (Table I). The supernatants were free from ADP-splitting phosphatases and from myokinase. The incorporation of [8-14CIADP into polyribonucleotides by purified preparations from Anacystis and Chlorella was inhibited by inorganic phosphate; in the presence of GDP, CDP and U D P a smaller rate of incorporation occurred. Net synthesis of polyadenylic acid and of polyribonucleotide copolymers was achieved by incubating ADP or ADP, GDP, CDP and U D P with purified polynucleotide phosphorylase preparations for 1-6 h at 37°; the products were identified by paper chromatography and by their degradation products after alkaline hydrolysis. TABLE I PURIFICATION
OF POLYNUCLEOTIDE PHOSPHORYLASE PHOTOSYNTHETIC ORGANISMS
FROM TWO
The test mixture, o.25o ml, contained: 3/,moles triethanolamine-HC1 buffer (pH 7.6), 1.25/,moles Mg s+, o. I/,mole [8-14C]ADP(specific activity approx. 5" lO6 counts//~mole), o.o30-2.o0 mg protein. After incubation for 3° min at 37° the reaction was stopped with 0.5 ml of lO% perchloric acid, carrier protein was added where necessary, and the mixture was centrifuged for 5 min at 5ooo rev./min. The precipitate was washed 3 times with cold perchloric acid (2 %), once with methanol and with ether; after drying, it was dissolved in o.5 ml of formic acid. Portions were spread on metal planchettes and dried. Radioactivity was measured in a windowless Geiger-Mliller counter. No correction was made for self-absorption.
Fraction
Source of enzyme prepaTations (activity expressed as re#moles of [8-t'C ] A DP incorporated per volume of the #action indicated ("toted") and per mg protein ("specific") A nacystis Total
Crude extract Ammonium sulphate, 3o-80% saturation Protamine eluate after dialysis
Chlorella
Spbciftc
Total
Specific
12 ooo 39 300
4.1 52.0
iooo 2000
0.93 7.5
6 650
214.o
766
26.7
The detection of RNA polymerase in crude extracts of both organisms was impaired by the instability of the enzyme, and by the interfering action of an enzymic activity which catalyzed the incorporation of [8-14CIATP into an acid-insoluble product, probably without any requirement for DNA; addition of the complementary ribonucleoside triphosphates tended to inhibit this reaction. In the presence of DNA, however, an a appreciable incorporation of [8-14C]ATP took place which is considered to be an expression of polymerase activity. Treatment of the crude extracts with bentonit (I00 mg/ml) increased the stability of the polymerase activity, even during the further purification with protamine Biochim. Biophys. Acta, 72 (1963) 342-344
344
PRELIMINARY NOTES
sulphate; the latter was applied as described for polynucleotide phosphorylase. In the case of Anacystis, the eluate obtained from the protamine precipitate showed the incorporation of [8-x4C]ATP only in the presence of GTP, CTP, UTP, and DNA; similar preparations from Chlorella still had a capacity for incorporating [8-14C]ATP as the sole substrate but, in the complete reaction mixture, the specific activity was increased I6-fold over that of the crude extract (Table II). In order to rule out any incorporation of nucleoside diphosphates formed from T A B L E II R N A POLYMHRASE/ACTIVITY IN VARIOUS PREPARATIONS FROM TWO PHOTOSYNTHETIC ORGANISMS The test mixture, 0.25 ° m l , contained: 6/~moles Tris buffer (pH 7.6) ; 1.2/~moles Mgt+; o.12 ~umole E D T A ; o.12 p m o l e m e r c a p t o e t h a n o l ; 1.2 m g b e n t o n i t ; o.1 /*mole [8-aIC]ATP (specific activity approx. I o e counts/pmole) ; o.5 p m o l e of unlabelled GTP, C T P and U T P , each; 1.5 pmoles N a t H P O * (for inhibition of potynucleotide phosphorylase) ; approx. 2 o o p m o l e s t h y m u s D N A ;and o.o4o-2. 5 m g protein. After' incubation for I h at 37 ° t h e samples were deproteinized and p r e p a r e d for c o u n t i n g as described for polynucleotide phosphorylase.
Fraction
Source of emyme preparations (aaitgcly expressed as m¢~ncff.esof [8-t~C.IA TP incorporated per volume of #~ fraaio~,indicag~ ("total"), and per mg protein ("specific") in the complete test raixture. Values in brackets: incorporation with [8-x*C]A TP as sole subavat#, without DNA ) CMoreUa
A nacysBs
Crude. extract after:
Bentonit treatment P r o t a m i n e eluate
Total
S p~dfic
Total
Specific
lO5O (384 o) 117 (nil)
0.42 (I.5) 4.o (nil)
262 (2900) 115 (22o)
1.2 (I3) 18.o (34)
the triphosphates by the action of phosphatases and kinases, inorganic phosphate was added to the test mixtures in sufficient amount to inhibit polynucleotide phosphorylase activity. The results obtained strongly suggest that the functions of polynucleotide phosphorylase and RNA polymerase in photosynthetic cells are rather similar to those in the cells of bacteria and animals. Further details will be given in a later publication. Part of this work was performed at the California Institute of Technology, Division of Biology, Pasadena, Calif. (U.S.A.); it has been supported by the National Institute of Health, U.S. Public Health Service, and by the Deutsche Forschungsgemeinschaft.
Botanisckes Institut, Universitiit T~bingen T~bingen (Germany)
GERHARD RICHTER
x W. A. KRATZ AND J. MYERS, A m . J . B o t a n y , 42 (1955) 282. t N. BISHOP AND H. GA~TRON, B i o c h i m . B i o p h y s . A c t a , 28 (1956) 3.5S M. GRUNBERG-MANAGO, P. J. ORTIZ AND S. OCHOA, Biochira. B i o p h y s . A c t a , 20 (I956) 269. 4 j . j . FURTH, J. HURWITZ AND M. ANDERS, J . Biol. Chem., 237 (I962) 261I.
Received March 18 th, 1963 B i o c h i m . B i o p h y s . Acta, 72 (1963) 342-344