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Distribution and metabolism of threonine ethanolamine and serine ethanolamine phosphodiesters in nervous tissues
SHORT COMMUNICATIONS
18 3
C. H. DoY, A. I~IVERA AND P. 1R. SRINIVASAN, Biochem. Biophys. Res. Commun., 4 (196I) 83. 0 C. H. DoY, in preparation. B. D. DAvis AND E. MINGIOLI, J . Bacteriol., 60 (195 o) 17. s C. H. D o v AND F. GIBSON, Biochem. J., 72 (1959) 586. 9 M. I. GIBSON, F. GIBSON, (~. H. DoY AND P. MORGAN, Nature, 195 (1962) 1173. 10 F. GIBSON AND L. M. JACKMAN, Natw,e, 198 (1963) 388. 11 j . R. BECKWITH, A. B. PARDEE, R. AUSTRIAN AND F. JACOB, J. Mol. Biol., 5 (1962) 618. z9 L. GORINI, W. GUNDERSON AND M. BURGER, Cold Spring Harbor Syrup. Quant. Biol. 26 (196I) 173.
Received March 9th, 1964 Biochim. Biophys. Acta, 9o (1964) 18o-183
sc 23o16
Distribution and metabolism of threonine ethanolamine and serine ethanolamine phosphodiesters in nervous tissues L-Threonine ethanolamine phosphate (TEP) has been identified in the tissues of Neoceratodus and Sal~no irideus b y ROSENBERG et al. 1 and in the fishes Esox lucius, Ameiurus nebulosus, Scyliorhynus caniculus and Mustdus mustdus b y PORCELLATI AND CURTI 2, and its chemical and physical properties described ~, 4. L-Serine ethanolamine phosphate (SEP) has been found in some animal species b y ROBERTS AND LOWE ~, ROSENBERG AND ENNOR 6 a n d PORCELLATI AND CURTI z. In this paper new information on the distribution and biochemical properties of T E P in the nervous tissues of fish will be presented, which shows that this compound is actively metabolized in vitro. In addition, some observations on the metabolism of SEP are reported. SEP was isolated in a pure form from the mixed brain and spinal cord of Testudo hermanni and Triton cristatus according to the procedure worked out b y PORCELLATIs, 4 for the isolation of TEP. The compound was precipitated from aqueous methanol as hygroscopic microcrystals. T E P was isolated as the pure compound from the nervous tissues of A. nebulosus, E. lucius and S. caniculus, according to ROSENBERG et al. z, as modified b y PORCELLATI4. Both compounds appeared to be free from a n y contaminating, ninhydrin-reacting material, and were almost completely recovered throughout the whole fractionation procedure. SEP was synthetized according to BEATTY AND MAGRATI~ and T E P as described b y ROSENBERG et al. 1 with only minor modifications 4. The physical properties, infrared spectra, chemical analyses and hydrolysis products of the isolated SEP and T E P were similar to those reported for the synthetic compounds 1, 2, 4, 7. Quantitative estimations of the levels of T E P and SEP in the nervous tissues of some species of fish and of the sea lamprey (Petromyzon marinus), a cyclostom, were then carried out. Samples of the desalted fraction of the first.ion-exchange separation 2-4 were paper-chromatographed, the spots wet-ashed, and the P determined. The results presented in Table I show that SEP is at least I0 or 20 times less concentrated than T E P in the nervous tissues of the Teleostea. I t is virtually absent in those of the two cartilaginous fishes (S. caniculus and M. mustelus), and totally absent in the cyclostom. Some of these findings confirm previous results 2-4. Thus, it appears that in the more primitive classes of fish there are species in which only Abbreviations: TEP, L-threonine ethanolamine phosphate; phosphate.
SEP,-L-serine ethanolamine
Biochim. Biophys. Acta, 90 (1964) 183-186
I84
SHORT COMMUNICATIONS TABLE I THE
DISTRIBUTION OF S E P AND T E P IN THE NERVOUS OF SOME SPECIES OF FISH AND CYCLOSTOM
TISSUES
Values expressed as /,moles of P per g of fresh tissue. Mean levels of 6 estimations each. Abbreviations: A.N., A. nebulosus; E.L., E. lucius; C.C., Cyprinus carpio; S.C., S. caniculua; M.M., M. mustelus; P.M., P. marinus. All the animal species were acclimatized in t h e l a b o r a t o r y for 4-5 days before use. Tissue
Compound
A.N.
E.L.
C.C.
S.C.
M.M.
P.M.
Brain
SEP TEP
0.4 7.4
0.3 4 .1
0.9 6.1
o 6.I
0.06 5.7
o 3 .6
Spinalcord
SEP TEP
0. 4 4.2
o.i 2.2
o 3.6
o 2.6
o 3.i
o --
T E P is found. In terms of systematic fish classification, the Selachii are classified, together with the Acanthodii, Placoderms and Holocephali, as Elasmobranchii. The first three species listed in Table I on the other hand are classified as Actinopterygii, which represents a more advanced stage of evolutionary development than the Elasmobranchii. Thus, it would appear that the distribution of SEP and T E P may be of evolutionary significance. Accordingly, T E P has never been detected in the nervous tissues of reptiles and amphibians, which contain the SEP molecule*:. It has only been found so far in many species of fish and in cyclostom* where the levels are much higher than those of SEP or where SEP is absent. Examination of some more primitive Chordata, as the Tunicata and Cephalochordata, might be of interest therefore in this connection as suggested also by ROSENBERGet al. 1. An enzymic preparation which splits the T E P molecule has been obtained by the procedure reported in Table n from the combined brain and spinal cord of fishes. When the phosphodiester was incubated with the enzymic preparation under the TABLE II THE BREAKDOWN OF T E P IN THE NERVOUS TISSUES OF IRISH 2 g of n e r v o u s tissues were first homogenized in t h e cold w i t h o.2 M 'Iris buffer (pH 7.5 o) (dilution i :6) for IO rain, and t h e n centrifuged at I5OOO × g. The clear s u p e r n a t a n t was passed t h r o u g h a Sephadex Ge25 (fine grade) column, 1.6o cm × 62 cm, and the activity was eluted in t h e cold in 3-ml fractions w i t h o.o2 M NaC1 at a flow rate of 7 ml/h. The combined active fractions were concentrated b y lyophilization, where necessary, and t h e n directly employed. T h e incubation m i x t u r e (3.o ml) in a W a r b u r g flask contained the enzymic p r e p a r a t i o n (4oo m g of fresh originary tissues), 2 5 / , m o l e s T E P , 7 ° p m o l e s Mg 2+, 40o/*moles Tris buffer (pH 7.50). I n c u b a t i o n a t 380 w a s carried o u t u n d e r p u r e Nz. Values expressed as t~moles for the whole incubation m i x t u r e . Compounds in mizture after incubation IncubaNon time _
_ (rain)
TEP
Phosphoryl. ethanolamine
o 3° 60 12o 240
24. 3 17.8 II.9 7.9 1.6
0.2 6.0 11.2 13.o 16.4
Threonine
0.5 5.9 12.I 16.2 23.5
EthanoL*mine
Phosphoryltltreonine
o o 0.3 2.1 9.1
o o o o. 7 1. 4
p~
3.7 4.I 4.4 6.6 11.2
* Owing to the small quantities of material, a rigid isolation of T E P f r o m the s e a - l a m p r e y b r a i n has been precluded.
Biochira. Biophys. Acta, 90 (1964) 183-186
SHORT COMMUNICATIONS
I85
conditions specified in Table II, an apparently stoichiometric formation of threonine and phosphorylethanolamine within the first 30-60 min of incubation was observed. The products were primarily separated from the incubation mixtures by paper chromatography, and then identified and analyzed by ion-exchange procedures 2. A contaminating phosphomonoesterase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1 or EC 3.1.3.2) activity, present in the same enzymic sources, moderately hydrolyzed phosphorylethanolamine, so that after longer periods of incubation, the stoichiometric relationship between phosphorylethanolamine and threonine was no longer observed, and ethanolamine and Pt were produced. At the same time, very little phosphorylthreonine was released b y incubating the T E P molecule with a similar preparation. The same enzymic source was found active also towards synthetic and natural SEP. A phosphodiesterase (orthophosphoric diester phosphohydrolase, EC 3.I.4.1 ) activity towards SEP has been detected in a Similar preparation obtained from the nervous tissues of the reptile Testudo herr~anni, and the formation of stoichiometric amounts of phosphorylethanolamine and serine noticed. ATP
I Phosphotio~lcholine
In preliminary experiments, a fractionation procedure for brain-cell particles 8 would indicate that the enzymic activity towards T E P for both reptiles and fishes is probably confined to the microsomal fraction of a 0.40 M homogenate in sucrose. The results presented in this paper may indicate that the degradative phase of SEP and T E P in the animal species examined could be of some metabolic interest and be linked to some unknown biochemical events. Since the chemical structure of SEP and T E P seems related to that of phosphatidylserine and phosphatidylthreonine respectively, and since SEI~, ~ and probably T E P 1° have low turnover rates, it is possible that the two compounds represent a deposit of phosphorylated moieties further utilized for phosphatidylserine and phosphatidylthreonine biosynthesis. A speculative and provisional scheme which could be postulated similarly for TEP, is shown in the accompanying diagram. It would be interesting to study these relationships, since the reactions leading to the formation of phosphatidylthreonine are completely unknown, and the pathway for phosphatidylserlne biosynthesis is far from clear u. Such experiments are now in progress. This investigation was supported in part by a research grant from the Consiglio Nazionale delle Ricerche, Rome. I am grateful to Professor A. RuFFo, Pavia and Biochim. Biophys. Acta, 90 (I964) I83-186
i86
SHORT COMMUNICATIONS
Professor E. LEONE, Perugia for stimulating discussions. The excellent technical assistance of Mr. I. TANCINI is gratefully acknowledged.
Department o/Biochemistry, Universitieso/ Perugia and Pavia, GIUSEPPE PORCELLATI Perugia and Pavia (Ira/y) t H. ROSENBERG, A. H. ENNOR, D. D. HAGERMAN AND S. SUGAI, Biochem. J., 84 (1962) 536. t G. PORCELLATI AND B. CURTI, Boll. Soc. Ital. Biol. Sper., 38 (1962) 1867, I872. $ G. PORCELLATI, Biochem. J., 89 (I963) 43 P. 4 G. PORCELLATI, Boll. Soc. Ital. Biol. Sper., 39 (1963) 187o. 5 E. ROBERTS AND J . P. LOWE J. Biol. Chem. 21I (1954) I. e H. ROSENEERG AND A. H. ENNOR, J. Biochem. Tokyo, 5° (I96I) 81. I. M. BEATTY AND D. I. MAGRATH, J. Am. Chem. 5oc., 82 (196o) 4983. e R. BALLZS, D. BIESOLD AND K. MAGYAR, J. Neurochem., lO (1963) 685. g P. AYENGAR AND E. ROBERTS, Federalion Proc., 16 (1957) 147. t o G. PORCELLATI, u n p u b l i s h e d results. 11 D. J. HANAHAN AND G. A. THOMPSON, JR., Ann. Rev. Biochem., 32 (1963) 2i 5.
Received January 2oth, 1964 Biochim. Biophys. Acta, 90 (1964) 183-186
SC 2 3 o i o
The effect of streptomycin on the increased rate of metabolism of phosphate, induced by prolonged washing, in sections of etiolated pea stems Evidence has been presented in an earlier paper z to show that cutting I.o-cm sections of etiolated pea stems from intact plants and washing them for 24 h in distilled water or o.o1 M maleate buffer (pH 5.2) resulted in a lo-fold increase in their subsequent ability to absorb and esterify PO~8-. Antibiotics were added to the washing medium to determine whether these apparent increases in the rate of absorption and esterification were due to the build up of bacteria in the sections occurring during the long incubation period. Table I compares the effect of several antibiotics and it is apparent that only streptomycin was able to prevent the appearance of the increased rate of absorption. Even when TABLE I THE EFFECTS OF WASHING I.O-Cm S E C T I O N S O F P E A S T E M S F O R 18 h IN O.OI M M A L E A T E B U F F E R ~ D H 5.2), C O N T A I N I N G V A R I O U S ANTIBIOTICS,
ON THEIR SUBSEQUENT CAPACITY TO ABSORB P O 4 8"- DURING I h FROM I o -5
M~KH~S=P04
No antibiotics were added to the m e d i u m used d u r i n g the u p t a k e period. Tfeafraent
Control 5.2" 1o .5 M s t r e p t o m y c i n + 4.2. lO-~ M penicillin 5.2- lO -5 M s t r e p t o m y c i n 8. 5 • lO -5 M penicillin z. 1o -~ M chloramphenicol
U p ~ e (ml~raoleaper g fresh tt~.per Is) Flesh
Was~.d
1.6
8.z
-----
2. 4 2.2 7.8 7.7
Biochim. Biophys. Acta, 9o (1964) 186-189
18 3
C. H. DoY, A. I~IVERA AND P. 1R. SRINIVASAN, Biochem. Biophys. Res. Commun., 4 (196I) 83. 0 C. H. DoY, in preparation. B. D. DAvis AND E. MINGIOLI, J . Bacteriol., 60 (195 o) 17. s C. H. D o v AND F. GIBSON, Biochem. J., 72 (1959) 586. 9 M. I. GIBSON, F. GIBSON, (~. H. DoY AND P. MORGAN, Nature, 195 (1962) 1173. 10 F. GIBSON AND L. M. JACKMAN, Natw,e, 198 (1963) 388. 11 j . R. BECKWITH, A. B. PARDEE, R. AUSTRIAN AND F. JACOB, J. Mol. Biol., 5 (1962) 618. z9 L. GORINI, W. GUNDERSON AND M. BURGER, Cold Spring Harbor Syrup. Quant. Biol. 26 (196I) 173.
Received March 9th, 1964 Biochim. Biophys. Acta, 9o (1964) 18o-183
sc 23o16
Distribution and metabolism of threonine ethanolamine and serine ethanolamine phosphodiesters in nervous tissues L-Threonine ethanolamine phosphate (TEP) has been identified in the tissues of Neoceratodus and Sal~no irideus b y ROSENBERG et al. 1 and in the fishes Esox lucius, Ameiurus nebulosus, Scyliorhynus caniculus and Mustdus mustdus b y PORCELLATI AND CURTI 2, and its chemical and physical properties described ~, 4. L-Serine ethanolamine phosphate (SEP) has been found in some animal species b y ROBERTS AND LOWE ~, ROSENBERG AND ENNOR 6 a n d PORCELLATI AND CURTI z. In this paper new information on the distribution and biochemical properties of T E P in the nervous tissues of fish will be presented, which shows that this compound is actively metabolized in vitro. In addition, some observations on the metabolism of SEP are reported. SEP was isolated in a pure form from the mixed brain and spinal cord of Testudo hermanni and Triton cristatus according to the procedure worked out b y PORCELLATIs, 4 for the isolation of TEP. The compound was precipitated from aqueous methanol as hygroscopic microcrystals. T E P was isolated as the pure compound from the nervous tissues of A. nebulosus, E. lucius and S. caniculus, according to ROSENBERG et al. z, as modified b y PORCELLATI4. Both compounds appeared to be free from a n y contaminating, ninhydrin-reacting material, and were almost completely recovered throughout the whole fractionation procedure. SEP was synthetized according to BEATTY AND MAGRATI~ and T E P as described b y ROSENBERG et al. 1 with only minor modifications 4. The physical properties, infrared spectra, chemical analyses and hydrolysis products of the isolated SEP and T E P were similar to those reported for the synthetic compounds 1, 2, 4, 7. Quantitative estimations of the levels of T E P and SEP in the nervous tissues of some species of fish and of the sea lamprey (Petromyzon marinus), a cyclostom, were then carried out. Samples of the desalted fraction of the first.ion-exchange separation 2-4 were paper-chromatographed, the spots wet-ashed, and the P determined. The results presented in Table I show that SEP is at least I0 or 20 times less concentrated than T E P in the nervous tissues of the Teleostea. I t is virtually absent in those of the two cartilaginous fishes (S. caniculus and M. mustelus), and totally absent in the cyclostom. Some of these findings confirm previous results 2-4. Thus, it appears that in the more primitive classes of fish there are species in which only Abbreviations: TEP, L-threonine ethanolamine phosphate; phosphate.
SEP,-L-serine ethanolamine
Biochim. Biophys. Acta, 90 (1964) 183-186
I84
SHORT COMMUNICATIONS TABLE I THE
DISTRIBUTION OF S E P AND T E P IN THE NERVOUS OF SOME SPECIES OF FISH AND CYCLOSTOM
TISSUES
Values expressed as /,moles of P per g of fresh tissue. Mean levels of 6 estimations each. Abbreviations: A.N., A. nebulosus; E.L., E. lucius; C.C., Cyprinus carpio; S.C., S. caniculua; M.M., M. mustelus; P.M., P. marinus. All the animal species were acclimatized in t h e l a b o r a t o r y for 4-5 days before use. Tissue
Compound
A.N.
E.L.
C.C.
S.C.
M.M.
P.M.
Brain
SEP TEP
0.4 7.4
0.3 4 .1
0.9 6.1
o 6.I
0.06 5.7
o 3 .6
Spinalcord
SEP TEP
0. 4 4.2
o.i 2.2
o 3.6
o 2.6
o 3.i
o --
T E P is found. In terms of systematic fish classification, the Selachii are classified, together with the Acanthodii, Placoderms and Holocephali, as Elasmobranchii. The first three species listed in Table I on the other hand are classified as Actinopterygii, which represents a more advanced stage of evolutionary development than the Elasmobranchii. Thus, it would appear that the distribution of SEP and T E P may be of evolutionary significance. Accordingly, T E P has never been detected in the nervous tissues of reptiles and amphibians, which contain the SEP molecule*:. It has only been found so far in many species of fish and in cyclostom* where the levels are much higher than those of SEP or where SEP is absent. Examination of some more primitive Chordata, as the Tunicata and Cephalochordata, might be of interest therefore in this connection as suggested also by ROSENBERGet al. 1. An enzymic preparation which splits the T E P molecule has been obtained by the procedure reported in Table n from the combined brain and spinal cord of fishes. When the phosphodiester was incubated with the enzymic preparation under the TABLE II THE BREAKDOWN OF T E P IN THE NERVOUS TISSUES OF IRISH 2 g of n e r v o u s tissues were first homogenized in t h e cold w i t h o.2 M 'Iris buffer (pH 7.5 o) (dilution i :6) for IO rain, and t h e n centrifuged at I5OOO × g. The clear s u p e r n a t a n t was passed t h r o u g h a Sephadex Ge25 (fine grade) column, 1.6o cm × 62 cm, and the activity was eluted in t h e cold in 3-ml fractions w i t h o.o2 M NaC1 at a flow rate of 7 ml/h. The combined active fractions were concentrated b y lyophilization, where necessary, and t h e n directly employed. T h e incubation m i x t u r e (3.o ml) in a W a r b u r g flask contained the enzymic p r e p a r a t i o n (4oo m g of fresh originary tissues), 2 5 / , m o l e s T E P , 7 ° p m o l e s Mg 2+, 40o/*moles Tris buffer (pH 7.50). I n c u b a t i o n a t 380 w a s carried o u t u n d e r p u r e Nz. Values expressed as t~moles for the whole incubation m i x t u r e . Compounds in mizture after incubation IncubaNon time _
_ (rain)
TEP
Phosphoryl. ethanolamine
o 3° 60 12o 240
24. 3 17.8 II.9 7.9 1.6
0.2 6.0 11.2 13.o 16.4
Threonine
0.5 5.9 12.I 16.2 23.5
EthanoL*mine
Phosphoryltltreonine
o o 0.3 2.1 9.1
o o o o. 7 1. 4
p~
3.7 4.I 4.4 6.6 11.2
* Owing to the small quantities of material, a rigid isolation of T E P f r o m the s e a - l a m p r e y b r a i n has been precluded.
Biochira. Biophys. Acta, 90 (1964) 183-186
SHORT COMMUNICATIONS
I85
conditions specified in Table II, an apparently stoichiometric formation of threonine and phosphorylethanolamine within the first 30-60 min of incubation was observed. The products were primarily separated from the incubation mixtures by paper chromatography, and then identified and analyzed by ion-exchange procedures 2. A contaminating phosphomonoesterase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1 or EC 3.1.3.2) activity, present in the same enzymic sources, moderately hydrolyzed phosphorylethanolamine, so that after longer periods of incubation, the stoichiometric relationship between phosphorylethanolamine and threonine was no longer observed, and ethanolamine and Pt were produced. At the same time, very little phosphorylthreonine was released b y incubating the T E P molecule with a similar preparation. The same enzymic source was found active also towards synthetic and natural SEP. A phosphodiesterase (orthophosphoric diester phosphohydrolase, EC 3.I.4.1 ) activity towards SEP has been detected in a Similar preparation obtained from the nervous tissues of the reptile Testudo herr~anni, and the formation of stoichiometric amounts of phosphorylethanolamine and serine noticed. ATP
I Phosphotio~lcholine
In preliminary experiments, a fractionation procedure for brain-cell particles 8 would indicate that the enzymic activity towards T E P for both reptiles and fishes is probably confined to the microsomal fraction of a 0.40 M homogenate in sucrose. The results presented in this paper may indicate that the degradative phase of SEP and T E P in the animal species examined could be of some metabolic interest and be linked to some unknown biochemical events. Since the chemical structure of SEP and T E P seems related to that of phosphatidylserine and phosphatidylthreonine respectively, and since SEI~, ~ and probably T E P 1° have low turnover rates, it is possible that the two compounds represent a deposit of phosphorylated moieties further utilized for phosphatidylserine and phosphatidylthreonine biosynthesis. A speculative and provisional scheme which could be postulated similarly for TEP, is shown in the accompanying diagram. It would be interesting to study these relationships, since the reactions leading to the formation of phosphatidylthreonine are completely unknown, and the pathway for phosphatidylserlne biosynthesis is far from clear u. Such experiments are now in progress. This investigation was supported in part by a research grant from the Consiglio Nazionale delle Ricerche, Rome. I am grateful to Professor A. RuFFo, Pavia and Biochim. Biophys. Acta, 90 (I964) I83-186
i86
SHORT COMMUNICATIONS
Professor E. LEONE, Perugia for stimulating discussions. The excellent technical assistance of Mr. I. TANCINI is gratefully acknowledged.
Department o/Biochemistry, Universitieso/ Perugia and Pavia, GIUSEPPE PORCELLATI Perugia and Pavia (Ira/y) t H. ROSENBERG, A. H. ENNOR, D. D. HAGERMAN AND S. SUGAI, Biochem. J., 84 (1962) 536. t G. PORCELLATI AND B. CURTI, Boll. Soc. Ital. Biol. Sper., 38 (1962) 1867, I872. $ G. PORCELLATI, Biochem. J., 89 (I963) 43 P. 4 G. PORCELLATI, Boll. Soc. Ital. Biol. Sper., 39 (1963) 187o. 5 E. ROBERTS AND J . P. LOWE J. Biol. Chem. 21I (1954) I. e H. ROSENEERG AND A. H. ENNOR, J. Biochem. Tokyo, 5° (I96I) 81. I. M. BEATTY AND D. I. MAGRATH, J. Am. Chem. 5oc., 82 (196o) 4983. e R. BALLZS, D. BIESOLD AND K. MAGYAR, J. Neurochem., lO (1963) 685. g P. AYENGAR AND E. ROBERTS, Federalion Proc., 16 (1957) 147. t o G. PORCELLATI, u n p u b l i s h e d results. 11 D. J. HANAHAN AND G. A. THOMPSON, JR., Ann. Rev. Biochem., 32 (1963) 2i 5.
Received January 2oth, 1964 Biochim. Biophys. Acta, 90 (1964) 183-186
SC 2 3 o i o
The effect of streptomycin on the increased rate of metabolism of phosphate, induced by prolonged washing, in sections of etiolated pea stems Evidence has been presented in an earlier paper z to show that cutting I.o-cm sections of etiolated pea stems from intact plants and washing them for 24 h in distilled water or o.o1 M maleate buffer (pH 5.2) resulted in a lo-fold increase in their subsequent ability to absorb and esterify PO~8-. Antibiotics were added to the washing medium to determine whether these apparent increases in the rate of absorption and esterification were due to the build up of bacteria in the sections occurring during the long incubation period. Table I compares the effect of several antibiotics and it is apparent that only streptomycin was able to prevent the appearance of the increased rate of absorption. Even when TABLE I THE EFFECTS OF WASHING I.O-Cm S E C T I O N S O F P E A S T E M S F O R 18 h IN O.OI M M A L E A T E B U F F E R ~ D H 5.2), C O N T A I N I N G V A R I O U S ANTIBIOTICS,
ON THEIR SUBSEQUENT CAPACITY TO ABSORB P O 4 8"- DURING I h FROM I o -5
M~KH~S=P04
No antibiotics were added to the m e d i u m used d u r i n g the u p t a k e period. Tfeafraent
Control 5.2" 1o .5 M s t r e p t o m y c i n + 4.2. lO-~ M penicillin 5.2- lO -5 M s t r e p t o m y c i n 8. 5 • lO -5 M penicillin z. 1o -~ M chloramphenicol
U p ~ e (ml~raoleaper g fresh tt~.per Is) Flesh
Was~.d
1.6
8.z
-----
2. 4 2.2 7.8 7.7
Biochim. Biophys. Acta, 9o (1964) 186-189