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Microsomal inactivator of deoxycytidylic acid deaminase
228
SHORT COMMUNICATIONS
yet useful tool in a study of the effect of various degrading processes on R N A and in investigations of methods for extracting material of high molecular weight. The authors thank Dr. F. K. SANDERS for his interest and advice in this work, and Mr. F. W. CLOTHIER,Mr. R. HAy and Mr. G. A. WARRINER for technical assistance.
Virus Research Unit, Medical Research Council Laboratories,
Carshalton, Surrey (GreatBritain) 1 A. 2 1~. 3 A. 4 K. 6 H. A. 7 F. s R.
A . T . H . BURNESS A . D . Vlzoso
GIERER, Z. Naturforsch., 13 (I958) 788. y . CHENG, Biochim. Biophys. Acta, 37 (I96o) 238. D. V l z o s o AND A. T. H. BURNESS, Biochem. Biophys. Research Commune., 2 (196o) lO2. MIURA AND K. SUZUKI, Biochim. Biophys. Acta, 22 (1956) 565. S. ROSENKRANZ AND A. BENDICH, J. Am. Chem. Soc., 81 (1959) 902. D. HERSHEY AND E. BUR6I, J. Molecular Biol., 2 (196o) 143. K. SANDERS, Nature, 185 (196o) 802. K. MAIN AND L. J. COLE Arch. Biochem. Biophys., 68 (1957) 186.
Received December I6th, 196o Biochim. Biophys. Acta, 49 (1961) 225-228
Preliminary Notes Microsomal inactivator of deoxycytidylic acid deaminase The enzyme deCMP deaminase, first discovered by E. SCARANO1,2 in developing seaurchin eggs, converts deCMP to deUMP. The enzyme is present also in m a n y m a m m a lian tissues, especially those which are in an active state of proliferation, such as embryonic rat liver*, 3, intestinal mucosa 4, and various tumors 3,s. In contrast to the preparations of deCMP deaminase from sea-urchin eggs which are relatively stable, the attempts to purify this enzyme from animal sources such as embryonic rat liver, monkey liver 6, and other tissues of warm-blooded animals 2 were unsuccessful up to now, due to rapid inactivation. 2.0 1.8
Q = A 280mp_ 1"6 A 2 6 0 r'n~ 1.4
h2 I.O
08 0.6 014
0.2
k/L/z/ k2.0.568 210 lo 60 I I 80 tO0 f = °/o d e C M P c o n v e r t e d to d e U M P Co-C ~ Co
Fig. I. A s s a y of deCMP deaminase.
The circumstances leading to the instability of deCMP deaminase in preparations obtained from animal tumors such as cells of the Novikoff rat tumor ("hepatoma") and Ehrlich ascites mouse tumor were investigated recently in this laboratory. The B,ochim. Biophys. Acta, 49 (I961) 228-23I
PRELIMINARY NOTES
229
assay of deCMP deaminase activity was spectrophotometric, based on empirically found relationship between the ratio of absorbancies at 280 and 260 m/, and the percentage of deCMP conversion (Fig. I). As one m a y see, the relationship is practically linear. The factor responsible for the instability of deCMP deaminase could be traced to the presence of microsomes or the material derived thereof. The supernatants from homogenates (0.25 M sucrose) of Novikoff tumor were considerably more stable in regard to their deCMP deaminase activity when centrifuged at a high speed (30 min at ioo,ooo × g) than those centrifuged at only 3o,ooo x g for 30 min. The latter, when preincubated for 3 ° min at 37 °, lost during this period of time gradually all deCMP deaminase activity, whereas the former preserved 50% of their activity even I h after preincubation. When resuspended subcellular fractions were added in varying amounts to a high-speed supernatant and the mixture preincubated at 37 ° for 15-3 o min, the mixture with microsomes was invariably inactive in deCMP deamination, but purified mitochondria or nuclei washed of microsomes had no effect on the course of inactivation. Frozen (--15 °) microsomes had a similar effect, but heating of the microsomal fraction for io min to 60 ° led to the loss of inactivation. Dialysis against distilled water did not affect the microsomal factor but addition of sodium deoxycholate (0. 5 %) deprived the dialyzed microsomes of their ability to inactivate deCMP deaminase. Microsomes from normal rat liver or kidney could replace tumor microsomes; on the other hand, the effect was the same whether a deCMP deaminase preparation was made from Novikoff tumor, from Ehrlich ascites tumor, or from other sources, so that the microsomal inactivation of deCMP deaminase appears to be a rather general phenomenon. The deCMP deaminase preparations obtained b y extraction with Tris buffer (pH 7.2-7.5) of acetone powder from Ehrlich ascites cells are almost stable for I h at 37 ° but similar extracts from Novikoff tumor often are almost as labile as the homogenates, because the inactivating factor from microsomes is also extracted together with the deaminase. By stirring I g of microsomal acetone powder from normal rat liver with 12 ml Tris buffer, followed b y centrifugation (IO min at 20,000 × g), dialysis of the supernatant against distilled water at p H 8.0 and lyophilization, one obtains approx. 70 mg of water-soluble, thermolabile (5 min at 56°) material which, in a suitable dilution, inactivates progressively, in the linear fashion, the deCMP deaminase from Ehrlich ascites-tumor cells (Fig. 2). The substrate (deCMP) protects the deaminase against the inactivation by the microsomal factor. The course of the reaction and its thermolability do not leave any doubt that the microsomal inactivation of deCMP deaminase is of enzymic nature. While, at the present time, a proteolytic character of this reaction cannot be ruled out, this is made less likely on the basis of our observations which indicate that the activity of other enzymes occurring in the supernatants, e.g. adenosine deaminase, is not affected at all b y preincubation with the microsomal inactivator and that even a longer (I h, 37 °) preincubation of the supernatant with the inactivator does not lead to an increase in polarographically active, protein-bound, sulfhydryl groups. This would occur, in all probability, if a general proteolytic effect emanating from microsomes had taken place. One m a y mention also that the presence of proteolytic enzymes in rat-liver microsomes has not been detected up to date *. I t appears, therefore, that if the effect is proteolytic, it must be limited to certain proteins, such as deCMP deaminase. Biochim. Biophys. Acta, 49 (I961) 228 231
230
PRELIMINARY NOTES
deCMP deaminae, like the microsomal inactivator, is destroyed by heating for 5 min to 56-60 °, but the circumstance that heat denaturation of an enzyme is often delayed or prevented by the presence of an excess substrate, together with the fact Ioo
2.0 ,,~ ~
80 70
oo
60 50
II
40
LO
20 I0 i
5
I=o
o
,
4
15
2o
25
0.3
Preincubation time train)
Fig. 2. I n a c t i v a t i o n of deCMP deaminase b y the microsomal e n z y m e . The experiment consisted of a s e t of t u b e s (1-14) each containing 0. 5 m l of deaminase extract and Tris buffer (pH 7.4) in the a m o u n t s of 0.8 m l (tubes I, 3, 5, 7, 9, i i ) , o.6 m l (tubes 2, 4, 6, 8, io, 12) a n d i.o a n d 0.8 m l in tubes 13 and 14, respectively. D u r i n g 3 o - m i n p r e i n c u b a t i o n of this m i x t u r e at 37 °, the inactivator (0. 5 ml) w a s added to t u b e s i - i 2 - - t o tubes i and 2 at t h e start of preincubation, after 5 min to tubes 3, 4, a f t e r IO m i n to 5, 6, a f t e r 15 m i n to 7, 8, a f t e r 2o m i n to 9, io, a f t e r 25 m i n to i i , 12. N o inactivator w a s added to tubes 13, 14. A t the end of 3 o - m i n preincubation deCMP w a s added (0. 4 m g in the v o l u m e of 0.2 ml) to each even tube in the s e t and all tubes were incubated for another 3 ° m i n . T h e reaction w a s then stopped w i t h 5 m l lO% HC104. After centrifugation the v o l u m e of t h e supernatant w a s adjusted to 25 m l w i t h 5 % HC104. T h e absorbancy at 280 and 26o m/~ was read for each pair of t u b e s . T h e ratio of the differences b e t w e e n absorptions at these w a v e l e n g t h s is: A28O
Qt=
sample
+ deCMP, t --
zt 280 ~*sample, t
+ deCMP, t --
Asample,
A26o
sample
26o t
Q, a c c o r d i n g to Fig. I indicates the % of deCMP converted at time t.
mentioned above that deCMP deaminase is also protected against the microsomal inactivator, was utilized in rendering the deCMP deaminase preparations from Ehrlich ascites-tumor cells stable. Acetone powder was extracted by stirring with icecold Tris buffer (12 vol.) containing 4 mg/ml deCMP. After centrifugation the supernatant was heated to 56o for 5 min. The coagulated inactivator containing protein (approx. 50% of total protein content) was centrifuged off and the supernatant was dialyzed at o ° against redistilled water (pH 8.0 adjusted with diluted ammonia) to get rid of excess deCMP and deUMP formed from it. In contrast to observations of MALEY AND MALEY 3, who reported a complete inactivation during dialysis, the dialyzed, inactivator-free deCMP deaminase preparation does not undergo any loss of activity, and can be further concentrated by ammonium sulfate precipitation as a first step in the purification of this enzyme. This work was supported by a grant in aid of the New York City Research Council No. U lO41.
Department of Pathology, Columbia University and Francis Delafield Hospital, New York, N.Y. (U.S.A.)
SILVIO F I A L A ANNA FIALA
Biochim. Biophys. $cta, 49 (1961) 228-231
PRELIMINARY NOTES
231
b'. SCARANO, Biochim. •iophys. Acta, 29 (1958) 459. 2 E. SCARANO, J. Biol. Chem., 235 (196o) 706. a G. F. MALEY AND F. ~][ALI~Y,J , Biol. Chem., 234 (1959) 29754 D. GUERRITORE, V. ROCCHI AND A. SETZU, Nature, 188 (1961) 146. S. FIALA, A. E. FIALA AND W. GLINSMANN, Naturwissenscha/ten, 47 (196o) 45. 6 E. SCARANO, personal communication. 7 j . BRACHET, Biochemical Cytology, Academic Press, Inc., New York, 1957. 1
Received February I8th, 1961 Biochim. Biophys. Acta, 49 /I96I) 228-231
X-ray induction of synchronized Escherichia coil K12(~) The induction by X- and v-radiation of conventional cultures of KI2(~), a lysogenic strain of Escherichia co!i, has been studied systematically z and shown to be a one-hit, all-or-nothing process suitable for low-level dosimetry. Induction studies on synchronized cultures, the objective of the present work, might be expected to throw light on the question of whether the induction sensitivity ("aptitude") of a single cell of a log-phase culture varies during its growth cycle and, if so, whether this variation can yield useful information concerning the changes in cellular organization which occur during growth and division. We have determined the induction frequency, defined as the quotient of the number of cells induced by the number present, over an age range of o.1-1.5 mean generation time for fixed X-ray dosages of IOOO R, 15oo R and 2000 R. These dosages are well below the inactivation threshold but sufficiently high to raise the induced-cell count well above the free-phage level. Preparation of the synchronized cultures followed quite exactly a quick-filtration procedure 2 which minimizes metabolic disturbance. Growth of the log-phase culture, filtration and incubation of the filtrate were carried out at 3 °0 to improve time resolution in sampling. Taking zero time at the midpoint of the filtration interval, samples were withdrawn at regular intervals. One aliquot of each sample was irradiated and plated immediately with KI2S indicator*. A second aliquot was plated similarly without irradiation for the free-phage assay and a third plated without irradiation or indicator for the total-cell count. All assays were made in triplicate by the agar-layer method with dilutions adjusted to yield lOO-3OO colonies per plate in the total-cell counts. Some care was taken to equalize the sampling-plating intervals in the parallel assays. Supplementary control runs in which aliquots of irradiated cultures were plated with and without indicator established the absence of inactivation effects other than that of induction. The X-ray source was a Machlett AEG-5o-T tube with molybdenum anode operated at 37 kV with 0.03 mm aluminium filtration. The dosage rate was fixed geometrically at IOOO R/rain and dosages adjusted by a timed shutter. No dose-dependent effects of qualitative significance were observed. The results of a single series of measurements, corrected for free-phage background and typical of * K i n d l y supplied, with KI2(~.) b y Dr. J. J. W;EIGLE. The KI2(~.) is LEDERBERGS' W3IIO p r o t o t r o p h m a d e lysogenic to A. KAISER'S wild-type ~. phage.
Biochim. Biophys. Acta, 49 (1961) 231-232
SHORT COMMUNICATIONS
yet useful tool in a study of the effect of various degrading processes on R N A and in investigations of methods for extracting material of high molecular weight. The authors thank Dr. F. K. SANDERS for his interest and advice in this work, and Mr. F. W. CLOTHIER,Mr. R. HAy and Mr. G. A. WARRINER for technical assistance.
Virus Research Unit, Medical Research Council Laboratories,
Carshalton, Surrey (GreatBritain) 1 A. 2 1~. 3 A. 4 K. 6 H. A. 7 F. s R.
A . T . H . BURNESS A . D . Vlzoso
GIERER, Z. Naturforsch., 13 (I958) 788. y . CHENG, Biochim. Biophys. Acta, 37 (I96o) 238. D. V l z o s o AND A. T. H. BURNESS, Biochem. Biophys. Research Commune., 2 (196o) lO2. MIURA AND K. SUZUKI, Biochim. Biophys. Acta, 22 (1956) 565. S. ROSENKRANZ AND A. BENDICH, J. Am. Chem. Soc., 81 (1959) 902. D. HERSHEY AND E. BUR6I, J. Molecular Biol., 2 (196o) 143. K. SANDERS, Nature, 185 (196o) 802. K. MAIN AND L. J. COLE Arch. Biochem. Biophys., 68 (1957) 186.
Received December I6th, 196o Biochim. Biophys. Acta, 49 (1961) 225-228
Preliminary Notes Microsomal inactivator of deoxycytidylic acid deaminase The enzyme deCMP deaminase, first discovered by E. SCARANO1,2 in developing seaurchin eggs, converts deCMP to deUMP. The enzyme is present also in m a n y m a m m a lian tissues, especially those which are in an active state of proliferation, such as embryonic rat liver*, 3, intestinal mucosa 4, and various tumors 3,s. In contrast to the preparations of deCMP deaminase from sea-urchin eggs which are relatively stable, the attempts to purify this enzyme from animal sources such as embryonic rat liver, monkey liver 6, and other tissues of warm-blooded animals 2 were unsuccessful up to now, due to rapid inactivation. 2.0 1.8
Q = A 280mp_ 1"6 A 2 6 0 r'n~ 1.4
h2 I.O
08 0.6 014
0.2
k/L/z/ k2.0.568 210 lo 60 I I 80 tO0 f = °/o d e C M P c o n v e r t e d to d e U M P Co-C ~ Co
Fig. I. A s s a y of deCMP deaminase.
The circumstances leading to the instability of deCMP deaminase in preparations obtained from animal tumors such as cells of the Novikoff rat tumor ("hepatoma") and Ehrlich ascites mouse tumor were investigated recently in this laboratory. The B,ochim. Biophys. Acta, 49 (I961) 228-23I
PRELIMINARY NOTES
229
assay of deCMP deaminase activity was spectrophotometric, based on empirically found relationship between the ratio of absorbancies at 280 and 260 m/, and the percentage of deCMP conversion (Fig. I). As one m a y see, the relationship is practically linear. The factor responsible for the instability of deCMP deaminase could be traced to the presence of microsomes or the material derived thereof. The supernatants from homogenates (0.25 M sucrose) of Novikoff tumor were considerably more stable in regard to their deCMP deaminase activity when centrifuged at a high speed (30 min at ioo,ooo × g) than those centrifuged at only 3o,ooo x g for 30 min. The latter, when preincubated for 3 ° min at 37 °, lost during this period of time gradually all deCMP deaminase activity, whereas the former preserved 50% of their activity even I h after preincubation. When resuspended subcellular fractions were added in varying amounts to a high-speed supernatant and the mixture preincubated at 37 ° for 15-3 o min, the mixture with microsomes was invariably inactive in deCMP deamination, but purified mitochondria or nuclei washed of microsomes had no effect on the course of inactivation. Frozen (--15 °) microsomes had a similar effect, but heating of the microsomal fraction for io min to 60 ° led to the loss of inactivation. Dialysis against distilled water did not affect the microsomal factor but addition of sodium deoxycholate (0. 5 %) deprived the dialyzed microsomes of their ability to inactivate deCMP deaminase. Microsomes from normal rat liver or kidney could replace tumor microsomes; on the other hand, the effect was the same whether a deCMP deaminase preparation was made from Novikoff tumor, from Ehrlich ascites tumor, or from other sources, so that the microsomal inactivation of deCMP deaminase appears to be a rather general phenomenon. The deCMP deaminase preparations obtained b y extraction with Tris buffer (pH 7.2-7.5) of acetone powder from Ehrlich ascites cells are almost stable for I h at 37 ° but similar extracts from Novikoff tumor often are almost as labile as the homogenates, because the inactivating factor from microsomes is also extracted together with the deaminase. By stirring I g of microsomal acetone powder from normal rat liver with 12 ml Tris buffer, followed b y centrifugation (IO min at 20,000 × g), dialysis of the supernatant against distilled water at p H 8.0 and lyophilization, one obtains approx. 70 mg of water-soluble, thermolabile (5 min at 56°) material which, in a suitable dilution, inactivates progressively, in the linear fashion, the deCMP deaminase from Ehrlich ascites-tumor cells (Fig. 2). The substrate (deCMP) protects the deaminase against the inactivation by the microsomal factor. The course of the reaction and its thermolability do not leave any doubt that the microsomal inactivation of deCMP deaminase is of enzymic nature. While, at the present time, a proteolytic character of this reaction cannot be ruled out, this is made less likely on the basis of our observations which indicate that the activity of other enzymes occurring in the supernatants, e.g. adenosine deaminase, is not affected at all b y preincubation with the microsomal inactivator and that even a longer (I h, 37 °) preincubation of the supernatant with the inactivator does not lead to an increase in polarographically active, protein-bound, sulfhydryl groups. This would occur, in all probability, if a general proteolytic effect emanating from microsomes had taken place. One m a y mention also that the presence of proteolytic enzymes in rat-liver microsomes has not been detected up to date *. I t appears, therefore, that if the effect is proteolytic, it must be limited to certain proteins, such as deCMP deaminase. Biochim. Biophys. Acta, 49 (I961) 228 231
230
PRELIMINARY NOTES
deCMP deaminae, like the microsomal inactivator, is destroyed by heating for 5 min to 56-60 °, but the circumstance that heat denaturation of an enzyme is often delayed or prevented by the presence of an excess substrate, together with the fact Ioo
2.0 ,,~ ~
80 70
oo
60 50
II
40
LO
20 I0 i
5
I=o
o
,
4
15
2o
25
0.3
Preincubation time train)
Fig. 2. I n a c t i v a t i o n of deCMP deaminase b y the microsomal e n z y m e . The experiment consisted of a s e t of t u b e s (1-14) each containing 0. 5 m l of deaminase extract and Tris buffer (pH 7.4) in the a m o u n t s of 0.8 m l (tubes I, 3, 5, 7, 9, i i ) , o.6 m l (tubes 2, 4, 6, 8, io, 12) a n d i.o a n d 0.8 m l in tubes 13 and 14, respectively. D u r i n g 3 o - m i n p r e i n c u b a t i o n of this m i x t u r e at 37 °, the inactivator (0. 5 ml) w a s added to t u b e s i - i 2 - - t o tubes i and 2 at t h e start of preincubation, after 5 min to tubes 3, 4, a f t e r IO m i n to 5, 6, a f t e r 15 m i n to 7, 8, a f t e r 2o m i n to 9, io, a f t e r 25 m i n to i i , 12. N o inactivator w a s added to tubes 13, 14. A t the end of 3 o - m i n preincubation deCMP w a s added (0. 4 m g in the v o l u m e of 0.2 ml) to each even tube in the s e t and all tubes were incubated for another 3 ° m i n . T h e reaction w a s then stopped w i t h 5 m l lO% HC104. After centrifugation the v o l u m e of t h e supernatant w a s adjusted to 25 m l w i t h 5 % HC104. T h e absorbancy at 280 and 26o m/~ was read for each pair of t u b e s . T h e ratio of the differences b e t w e e n absorptions at these w a v e l e n g t h s is: A28O
Qt=
sample
+ deCMP, t --
zt 280 ~*sample, t
+ deCMP, t --
Asample,
A26o
sample
26o t
Q, a c c o r d i n g to Fig. I indicates the % of deCMP converted at time t.
mentioned above that deCMP deaminase is also protected against the microsomal inactivator, was utilized in rendering the deCMP deaminase preparations from Ehrlich ascites-tumor cells stable. Acetone powder was extracted by stirring with icecold Tris buffer (12 vol.) containing 4 mg/ml deCMP. After centrifugation the supernatant was heated to 56o for 5 min. The coagulated inactivator containing protein (approx. 50% of total protein content) was centrifuged off and the supernatant was dialyzed at o ° against redistilled water (pH 8.0 adjusted with diluted ammonia) to get rid of excess deCMP and deUMP formed from it. In contrast to observations of MALEY AND MALEY 3, who reported a complete inactivation during dialysis, the dialyzed, inactivator-free deCMP deaminase preparation does not undergo any loss of activity, and can be further concentrated by ammonium sulfate precipitation as a first step in the purification of this enzyme. This work was supported by a grant in aid of the New York City Research Council No. U lO41.
Department of Pathology, Columbia University and Francis Delafield Hospital, New York, N.Y. (U.S.A.)
SILVIO F I A L A ANNA FIALA
Biochim. Biophys. $cta, 49 (1961) 228-231
PRELIMINARY NOTES
231
b'. SCARANO, Biochim. •iophys. Acta, 29 (1958) 459. 2 E. SCARANO, J. Biol. Chem., 235 (196o) 706. a G. F. MALEY AND F. ~][ALI~Y,J , Biol. Chem., 234 (1959) 29754 D. GUERRITORE, V. ROCCHI AND A. SETZU, Nature, 188 (1961) 146. S. FIALA, A. E. FIALA AND W. GLINSMANN, Naturwissenscha/ten, 47 (196o) 45. 6 E. SCARANO, personal communication. 7 j . BRACHET, Biochemical Cytology, Academic Press, Inc., New York, 1957. 1
Received February I8th, 1961 Biochim. Biophys. Acta, 49 /I96I) 228-231
X-ray induction of synchronized Escherichia coil K12(~) The induction by X- and v-radiation of conventional cultures of KI2(~), a lysogenic strain of Escherichia co!i, has been studied systematically z and shown to be a one-hit, all-or-nothing process suitable for low-level dosimetry. Induction studies on synchronized cultures, the objective of the present work, might be expected to throw light on the question of whether the induction sensitivity ("aptitude") of a single cell of a log-phase culture varies during its growth cycle and, if so, whether this variation can yield useful information concerning the changes in cellular organization which occur during growth and division. We have determined the induction frequency, defined as the quotient of the number of cells induced by the number present, over an age range of o.1-1.5 mean generation time for fixed X-ray dosages of IOOO R, 15oo R and 2000 R. These dosages are well below the inactivation threshold but sufficiently high to raise the induced-cell count well above the free-phage level. Preparation of the synchronized cultures followed quite exactly a quick-filtration procedure 2 which minimizes metabolic disturbance. Growth of the log-phase culture, filtration and incubation of the filtrate were carried out at 3 °0 to improve time resolution in sampling. Taking zero time at the midpoint of the filtration interval, samples were withdrawn at regular intervals. One aliquot of each sample was irradiated and plated immediately with KI2S indicator*. A second aliquot was plated similarly without irradiation for the free-phage assay and a third plated without irradiation or indicator for the total-cell count. All assays were made in triplicate by the agar-layer method with dilutions adjusted to yield lOO-3OO colonies per plate in the total-cell counts. Some care was taken to equalize the sampling-plating intervals in the parallel assays. Supplementary control runs in which aliquots of irradiated cultures were plated with and without indicator established the absence of inactivation effects other than that of induction. The X-ray source was a Machlett AEG-5o-T tube with molybdenum anode operated at 37 kV with 0.03 mm aluminium filtration. The dosage rate was fixed geometrically at IOOO R/rain and dosages adjusted by a timed shutter. No dose-dependent effects of qualitative significance were observed. The results of a single series of measurements, corrected for free-phage background and typical of * K i n d l y supplied, with KI2(~.) b y Dr. J. J. W;EIGLE. The KI2(~.) is LEDERBERGS' W3IIO p r o t o t r o p h m a d e lysogenic to A. KAISER'S wild-type ~. phage.
Biochim. Biophys. Acta, 49 (1961) 231-232