- Email: [email protected]
Observations on lysophosphatide and triglyceride hydrolysis by rat liver preparations
SHORT COMMUNICATIONS
600
I-palmitoyl-3-oleylglycerol. Diglycerides and monoglycerides were separated on a Florisil column I2 and quantitated by glycerol determination after alkaline hydrolysis. Radioactivity activity glycerol,
was measured as above after thin-layer chromatography.
Specific radio-
counts/min per pmole) was found to be 60.0 in intact dipalmityloleyl59.9 in the diglycerides and 55.9 in the monoglycerides.
(103
Financial help from the N.I.H. (Grant AM 04642), Centre National de la Recherche Scientifique (RCP 23) and Commissariat ?I 1’Energie Atomique is gratefully acknowledged.
Institzd de Chimie Biologique, Faculte’ des Sciences, Marseille (France)
I F. H. MATTSON 2 P. SAVARY AND 3 F. H. MATTSON, 4 F. H. MATTSON 5 G. AILHAUD, D.
B. ENTRESSANGLES H. SARI P. DESNUELLE
AND L. W. BECK, J. Biol. Chem., 214 (1955) 115. P. DESNUELLE, Biochim. Biophys. Acta, 21 (1956) 349. J. H. BENEDICT, J. B. MARTIN AND L. W. BECK, J. Nutr., 48 (1952) 335. AND R. A. VOLPENHEIN, J. Lipid Res., 3 (1962) 281. SAMUEL, M. LAZDUNSKI AND P. DESNUELLE, Biochim. Biophys. Acta, 84 (1964)
643. 6 L. HARTMAN, J. Chem. SLC., (1957) 3572. 7 B. DE VRIES, J. Am. Oil Chemists’ Sot., 41 (1964) 403. 8 G. BENZONANA, B. ENTRESSANGLES, G. MARCHIS-MOUREN, L. SARDA AND P. DESNUELLE, in R. M. C. DAWSON AND D. N. RHODES, Metabolism and Physiological Significance of Lipids, Wiley and sons, London 1964, p. 141. 9 B. ENTRESSANGLES, P. SAVARY, M. J. CONSTANTIN AND P. DESNUELLE, Biochim. Bicphys.
Acta, 84 (1964) 140. IO A. E. THOMAS III, J, E. SCHAROUN AND H. RALSTON, J, Am. Oil Chemists’ SLC., 42 (1965) 789. II G. BENZONANA, M. SEMERIVA AND P. DESNUELLE, Bull. Sot. Chim. Biol., in the press. I2 K. K. CARROLL, J. Lipid Res., 2 (1961) 135. Biochim. Biophys. Acta, 125 (1966) 597-600
BBA 53101
Observations
on lysophosphatide
and triglyceride
hydrolysis by rat
liver preparations from diPhospholipase -4 (EC 3.1.1.4), capable of forming lysophosphatides acylglycerophosphatides, is present in a wide range of mammalian tissue+. Several pathways for the metabolism of lysolecithin and lysophosphatidylethanolamine are also known2p7, and the metabolism of lysolecithin in rat liver preparations has been the subject of a recent detailed study8.9. There is evidence that lysophosphatidylserine can be acylated in liver?, but as far as we are aware no other studies have been reported on the metabolism of this lysophosphatide by mammalian tissues. The object of the present work was to obtain data on the rates of hydrolysis of lysophosphatidylserine and other lysophosphatides by phospholipase B (EC 3.1.1.5) of rat liver. In these experiments a lipolytic reaction Biochim. Biophys. Acta, 125 (1966) 600-603
601
SHORT COMMUNICATIONS
was also observed in glycylglycine-buffered systems which was diminished by addition of inorganic phosphate or lysophosphatides; this phenomenon is also described. Rats were killed by decapitation and the livers immediately removed and washed free of obvious blood in 0.9% saline; 10% homogenates were prepared in ,distilled water lo. Lysophosphatides were prepared as previously described7 and stored as solutions in chloroform, in which they were stable at 0’. Aliquots in glass-stoppered test tubes were evaporated to dryness by warming in a stream of N,, and 0.5 ml liver homogenate and 0.5 ml buffer (0.2 M, pH6.5) were added. ‘Tissue-only’ and ‘substrateonly’ controls were also set up and all tubes incubated, with shaking, at 37’, After incubation 4 ml of 2:1 (v/v) chloroform-methanol mixture was added and the tubes were shaken vigorously. After centrifuging, the chloroform layer was isolated quantitatively and evaporated. Aliquots of lipid, in 6 ml chloroform, were treated with 300 mg of silicic acid, to remove phospholipid, and centrifuged; free fatty acids were estimated in the supernatant chloroform solutionX1. Triglycerides in the total lipid extract were separated by thin-layer chromatography on silicic acid and localized with iodine vapour. Triglycerides were eluted from the silicic acid in small columns with diethyl ether, hydrolysed with alcoholic KOH, and the liberated fatty acids extracted and estimated.
TABLE
I
LIBERATION
OF FATTY
ACIDS
BY
RAT
LIVER
HOMOGENATES
IN
PRESENCE
AND
ABSENCE
OF ADDED
LYSOPHOSPHATIDES
Homogenates with and without added lysophosphatide (final concentration 6.25 mM) were incubated for o and 60 min. Values are the means of results from a number of concordant experiments (shown in parentheses), expressed as ymoles fatty acid/g fresh tissue per h. Buger
Phosphate Glycylglycine
Liuer only
7.0(7) 19.1(g)
Change
in j&y
acid liberation in presence oj lysophosphatide
Lysolecithin
Lysophosphatidylethanolamine
+29.7(7) +30.2(g)
+ 1.7(3) -7.1(3)
Lysophosphatidylserinr
Table I shows the mean values obtained for fatty acid production by rat liver homogenates incubated at 37O in phosphate and glycylglycine buffers; also shown is the apparent phospholipase B activity of liver, calculated as the change in fatty acid production resulting from addition of lysophosphatide to the homogenate. Nonenzymic hydrolysis of the three lyso compounds was very small, averaging 0.8% or less of available substrate. In phosphate buffer phospholipase B values with lysolecithin as substrate agree well with previous values3; lysophosphatidylethanolamine and lysophosphatidylserine, on the other hand, are split less actively at about 6% and 24%, respectively, of the rate for lysolecithin. Although phospholipase B activity with lysolecithin appeared to be similar in the two buffers, negative values were obtained for the hydrolysis of the two lysocephalins in glycyglycine buffer. It is, however, evident from Table I that in the absence of added lysophosphatide nearly 3 times as much fatty acid was produced upon incubating liver homogenates in glycylglycine as was found for phosphate-buffered homogenates. A possible interpretation of these Biochim. Biophys.
Acta.
125
(1966) 600-603
602
SHORT COMMUNICATIONS
data in Table
I is that
the extra
fatty
acid production
in tissue-only
controls
in
glycylglycine was reduced by adding lysocephalins to the system, so that calculation of phospholipase B activity against these substrates as described above would lead to low or even negative values. In order to determine the source of the extra free fatty acid liberated in tissueonly controls incubated in glycylglycine buffer, portions of total lipid extracts of homogenates before and after incubation in both buffers were examined by thin-layer chromatography. Only slight breakdown of diacylglycerophosphaticles was observed following incubation for 60 min at 37’ in either buffer. The triglyceride content of the homogenates,
on the other hand, showed 18%
decrease
following
incubation
in
phosphate but 65% decrease after incubation in glycylglycine. The extra triglyceride disappearing in the latter case was virtually completely hydrolysed to free fatty acid, since no significant accumulation of lower glycerides could be detected on the thinlayer chromatograms; furthermore it accounted exactly (101%) for the extra fatty acid produced in tissue-only controls in glycylglycine as compared with phosphate buffer (Table I). In similar experiments buffer
in the incubation
TABLE THE
the presence
medium
of even a small proportion
profoundly
suppressed
of phosphate
the extra hydrolysis
of tri-
II
EFFECT
OF
BUFFER
COMPOSITION
ON
ENDOGENOUS
TRIGLYCERIDE
HYDROLYSIS
IN
RAT
LIVER
HOMOGENATES
Incubation, lipid extraction and estimation were carried out as described in the text. Results are the means of 2 concordant experiments. Final concentration
(M)
Triglyceride Jzydrolysi?
Phosphate
Glycylglycine
0
0.10
23.6
0.001 0.01
0.099 0.09
21.0 II.0
0.05
0.05
8.6
0
8.2
0.10
suppression of extra hydrolysis** 0
ii:
99 IO0 ~~-
,umoles fatty acid liberated/g fresh tissue per h. ** Decrease in triglyceride hydrolysis expressed as percentage of the difference between the values in glycylglycine alone and in phosphate alone. *
glyceride observed in glycylglycine-buffered
homogenates
(15.4
(Table II). Moreover
pmoles)
lysole-
cithin and both lysocephalins, in a final concentration of 6.25 mM, also completely abolished this extra fatty acid production; significant inhibition (38%) was found with 0.625 mM lysolecithin and lysophosphatidylethanolamine but not with lysophosphaticlylserine. Previous work 12 had shown that lysolecithin (2.0 mg/ml) caused partial inhibition of tributyrin hydrolysis by rat brain homogenates. In the glycylglycine-buffered system the presence of lysophosphatide, added as substrate for phospholipase B, would therefore suppress the hydrolysis of endogenous triglyceride to levels similar to those found for phosphate-buffered homogenates in the absence of added lysophosphatide (Table I). It would thus seem more appropriate to substract the value for fatty acid production in tissue-only controls in phosphate from those obtained in glycylglycine in the presence of substrate in order to assess phospholipase B activity in the latter buffer. After adjustment of the Biochim.
Biophys.
Acta,
125 (1966) 600-603
603
SHORT COMMUNICATIONS
in Table I in this way, liver phospholipase B activity with lysolecithin as substrate gave an average value of 42.3 ,umoles/g tissue per h with a range of values above those determined in phosphate buffer. This suggests that phospholipase B activity of rat liver, at least when lysolecithin is used as the substrate, is also enhanced in glycylglycine as compared with phosphate-buffered systems. Similar adjustment of the results for lysocephalin hydrolysis gave positive values of 5.0 and 3.6 ,umoleslg tissue per h for liver phospholipase B activity with lysophosphatidylethanolamine and lysophosphatidylserine as substrates, respectively, The rather wide range of these adjusted values precluded any definite conclusions regarding the enhancement of phospholipase B activity with these substrates in glycylglycine buffer. The question whether the triglyceride hydrolysis observed in these experiments is due to the action of lipoprotein lipase in liver? or of some other type of lipase, and the possible biochemical significance of its inhibition by lysophosphatides are subjects for further study. R. J. IRWIN is grateful to Harvard Medical School and R. J, ALPERN to the University of Rochester School of Medicine for the award of Student Research Fellowships supported respectively by Grants No. 5T5 (6M) 51-04 and zR6 (C-5) from the U.S. Public Health Service. The authors thank Mr. E. PORTMAN for his data
skilled
technical
assistance.
R. J. IRWIN” R. J. ALPERN** G. R. WEBSTER
Department of Chemical Pathology, Guy’s Hosjktat Medical School, London, S.E.r (Great Britain)
I J, GALLAI-HATCHARD AND R. H. S. THOMPSON, Biochim. Biophvs. Acta, g8 (1965) 128. 2 R. M. C. DAWSON, Biochem. J., 64 (1~56) 192. 3 E. A. MARPLES AND R. H. S. THOMPSON, Biochem. J., 74 (1960) 123. 4 6. V. MARINETTI, J. ERBLAND, R. F. WITTER, J. PETZX AND E. STOTZ, B~oc~~rn. Bicphys.
Acta,
30 (vJ58) 223. 5 J. ERBLAND AND G. V. MARINETTI, Federation Proc., 21 (1962) 295. 6 W. E. M. LANDS, J. Biol. Chem., 235 (1~60) 2233. 7 G. R. WEBSTER, Biochim. Biophys. Acta, 98 (rg65) 512. 8 J. F. ERBLAND AND G. V. MARINETTI, Biochim. Biophys. Acta, 106 (1965) 128. 9 J. F. ERBLAND AND G. V. MARINETTI, Biochim. Biophys. Acta, 106 (1gG5) 139. IO G. R. WEBSTER AND A. T. SMITH, Biochem. J.. 90 (1964) 64. II W. G. DUNCOMBE, Biochem. J., 88 (1963) 7. 12 E. A. MARPLES, R. H. S. THOMPSON AND G. R. WEBSTER, J. Neurochem., 4 (1959) 62. 13 E. D. KORN, J. Biol. Chem., 215 (1955) I.
Received May 6th, 1966 * Present address: Harvard Medical School, Boston, Mass. (U.S.A.). ** Present address: Department of Pediatrics, University Hospitals Ohio 44106 (U.S.A.).
of Cleveland,
Cleveland,
Biackim. Biophys. Acta, 125 (1966) 600-603
600
I-palmitoyl-3-oleylglycerol. Diglycerides and monoglycerides were separated on a Florisil column I2 and quantitated by glycerol determination after alkaline hydrolysis. Radioactivity activity glycerol,
was measured as above after thin-layer chromatography.
Specific radio-
counts/min per pmole) was found to be 60.0 in intact dipalmityloleyl59.9 in the diglycerides and 55.9 in the monoglycerides.
(103
Financial help from the N.I.H. (Grant AM 04642), Centre National de la Recherche Scientifique (RCP 23) and Commissariat ?I 1’Energie Atomique is gratefully acknowledged.
Institzd de Chimie Biologique, Faculte’ des Sciences, Marseille (France)
I F. H. MATTSON 2 P. SAVARY AND 3 F. H. MATTSON, 4 F. H. MATTSON 5 G. AILHAUD, D.
B. ENTRESSANGLES H. SARI P. DESNUELLE
AND L. W. BECK, J. Biol. Chem., 214 (1955) 115. P. DESNUELLE, Biochim. Biophys. Acta, 21 (1956) 349. J. H. BENEDICT, J. B. MARTIN AND L. W. BECK, J. Nutr., 48 (1952) 335. AND R. A. VOLPENHEIN, J. Lipid Res., 3 (1962) 281. SAMUEL, M. LAZDUNSKI AND P. DESNUELLE, Biochim. Biophys. Acta, 84 (1964)
643. 6 L. HARTMAN, J. Chem. SLC., (1957) 3572. 7 B. DE VRIES, J. Am. Oil Chemists’ Sot., 41 (1964) 403. 8 G. BENZONANA, B. ENTRESSANGLES, G. MARCHIS-MOUREN, L. SARDA AND P. DESNUELLE, in R. M. C. DAWSON AND D. N. RHODES, Metabolism and Physiological Significance of Lipids, Wiley and sons, London 1964, p. 141. 9 B. ENTRESSANGLES, P. SAVARY, M. J. CONSTANTIN AND P. DESNUELLE, Biochim. Bicphys.
Acta, 84 (1964) 140. IO A. E. THOMAS III, J, E. SCHAROUN AND H. RALSTON, J, Am. Oil Chemists’ SLC., 42 (1965) 789. II G. BENZONANA, M. SEMERIVA AND P. DESNUELLE, Bull. Sot. Chim. Biol., in the press. I2 K. K. CARROLL, J. Lipid Res., 2 (1961) 135. Biochim. Biophys. Acta, 125 (1966) 597-600
BBA 53101
Observations
on lysophosphatide
and triglyceride
hydrolysis by rat
liver preparations from diPhospholipase -4 (EC 3.1.1.4), capable of forming lysophosphatides acylglycerophosphatides, is present in a wide range of mammalian tissue+. Several pathways for the metabolism of lysolecithin and lysophosphatidylethanolamine are also known2p7, and the metabolism of lysolecithin in rat liver preparations has been the subject of a recent detailed study8.9. There is evidence that lysophosphatidylserine can be acylated in liver?, but as far as we are aware no other studies have been reported on the metabolism of this lysophosphatide by mammalian tissues. The object of the present work was to obtain data on the rates of hydrolysis of lysophosphatidylserine and other lysophosphatides by phospholipase B (EC 3.1.1.5) of rat liver. In these experiments a lipolytic reaction Biochim. Biophys. Acta, 125 (1966) 600-603
601
SHORT COMMUNICATIONS
was also observed in glycylglycine-buffered systems which was diminished by addition of inorganic phosphate or lysophosphatides; this phenomenon is also described. Rats were killed by decapitation and the livers immediately removed and washed free of obvious blood in 0.9% saline; 10% homogenates were prepared in ,distilled water lo. Lysophosphatides were prepared as previously described7 and stored as solutions in chloroform, in which they were stable at 0’. Aliquots in glass-stoppered test tubes were evaporated to dryness by warming in a stream of N,, and 0.5 ml liver homogenate and 0.5 ml buffer (0.2 M, pH6.5) were added. ‘Tissue-only’ and ‘substrateonly’ controls were also set up and all tubes incubated, with shaking, at 37’, After incubation 4 ml of 2:1 (v/v) chloroform-methanol mixture was added and the tubes were shaken vigorously. After centrifuging, the chloroform layer was isolated quantitatively and evaporated. Aliquots of lipid, in 6 ml chloroform, were treated with 300 mg of silicic acid, to remove phospholipid, and centrifuged; free fatty acids were estimated in the supernatant chloroform solutionX1. Triglycerides in the total lipid extract were separated by thin-layer chromatography on silicic acid and localized with iodine vapour. Triglycerides were eluted from the silicic acid in small columns with diethyl ether, hydrolysed with alcoholic KOH, and the liberated fatty acids extracted and estimated.
TABLE
I
LIBERATION
OF FATTY
ACIDS
BY
RAT
LIVER
HOMOGENATES
IN
PRESENCE
AND
ABSENCE
OF ADDED
LYSOPHOSPHATIDES
Homogenates with and without added lysophosphatide (final concentration 6.25 mM) were incubated for o and 60 min. Values are the means of results from a number of concordant experiments (shown in parentheses), expressed as ymoles fatty acid/g fresh tissue per h. Buger
Phosphate Glycylglycine
Liuer only
7.0(7) 19.1(g)
Change
in j&y
acid liberation in presence oj lysophosphatide
Lysolecithin
Lysophosphatidylethanolamine
+29.7(7) +30.2(g)
+ 1.7(3) -7.1(3)
Lysophosphatidylserinr
Table I shows the mean values obtained for fatty acid production by rat liver homogenates incubated at 37O in phosphate and glycylglycine buffers; also shown is the apparent phospholipase B activity of liver, calculated as the change in fatty acid production resulting from addition of lysophosphatide to the homogenate. Nonenzymic hydrolysis of the three lyso compounds was very small, averaging 0.8% or less of available substrate. In phosphate buffer phospholipase B values with lysolecithin as substrate agree well with previous values3; lysophosphatidylethanolamine and lysophosphatidylserine, on the other hand, are split less actively at about 6% and 24%, respectively, of the rate for lysolecithin. Although phospholipase B activity with lysolecithin appeared to be similar in the two buffers, negative values were obtained for the hydrolysis of the two lysocephalins in glycyglycine buffer. It is, however, evident from Table I that in the absence of added lysophosphatide nearly 3 times as much fatty acid was produced upon incubating liver homogenates in glycylglycine as was found for phosphate-buffered homogenates. A possible interpretation of these Biochim. Biophys.
Acta.
125
(1966) 600-603
602
SHORT COMMUNICATIONS
data in Table
I is that
the extra
fatty
acid production
in tissue-only
controls
in
glycylglycine was reduced by adding lysocephalins to the system, so that calculation of phospholipase B activity against these substrates as described above would lead to low or even negative values. In order to determine the source of the extra free fatty acid liberated in tissueonly controls incubated in glycylglycine buffer, portions of total lipid extracts of homogenates before and after incubation in both buffers were examined by thin-layer chromatography. Only slight breakdown of diacylglycerophosphaticles was observed following incubation for 60 min at 37’ in either buffer. The triglyceride content of the homogenates,
on the other hand, showed 18%
decrease
following
incubation
in
phosphate but 65% decrease after incubation in glycylglycine. The extra triglyceride disappearing in the latter case was virtually completely hydrolysed to free fatty acid, since no significant accumulation of lower glycerides could be detected on the thinlayer chromatograms; furthermore it accounted exactly (101%) for the extra fatty acid produced in tissue-only controls in glycylglycine as compared with phosphate buffer (Table I). In similar experiments buffer
in the incubation
TABLE THE
the presence
medium
of even a small proportion
profoundly
suppressed
of phosphate
the extra hydrolysis
of tri-
II
EFFECT
OF
BUFFER
COMPOSITION
ON
ENDOGENOUS
TRIGLYCERIDE
HYDROLYSIS
IN
RAT
LIVER
HOMOGENATES
Incubation, lipid extraction and estimation were carried out as described in the text. Results are the means of 2 concordant experiments. Final concentration
(M)
Triglyceride Jzydrolysi?
Phosphate
Glycylglycine
0
0.10
23.6
0.001 0.01
0.099 0.09
21.0 II.0
0.05
0.05
8.6
0
8.2
0.10
suppression of extra hydrolysis** 0
ii:
99 IO0 ~~-
,umoles fatty acid liberated/g fresh tissue per h. ** Decrease in triglyceride hydrolysis expressed as percentage of the difference between the values in glycylglycine alone and in phosphate alone. *
glyceride observed in glycylglycine-buffered
homogenates
(15.4
(Table II). Moreover
pmoles)
lysole-
cithin and both lysocephalins, in a final concentration of 6.25 mM, also completely abolished this extra fatty acid production; significant inhibition (38%) was found with 0.625 mM lysolecithin and lysophosphatidylethanolamine but not with lysophosphaticlylserine. Previous work 12 had shown that lysolecithin (2.0 mg/ml) caused partial inhibition of tributyrin hydrolysis by rat brain homogenates. In the glycylglycine-buffered system the presence of lysophosphatide, added as substrate for phospholipase B, would therefore suppress the hydrolysis of endogenous triglyceride to levels similar to those found for phosphate-buffered homogenates in the absence of added lysophosphatide (Table I). It would thus seem more appropriate to substract the value for fatty acid production in tissue-only controls in phosphate from those obtained in glycylglycine in the presence of substrate in order to assess phospholipase B activity in the latter buffer. After adjustment of the Biochim.
Biophys.
Acta,
125 (1966) 600-603
603
SHORT COMMUNICATIONS
in Table I in this way, liver phospholipase B activity with lysolecithin as substrate gave an average value of 42.3 ,umoles/g tissue per h with a range of values above those determined in phosphate buffer. This suggests that phospholipase B activity of rat liver, at least when lysolecithin is used as the substrate, is also enhanced in glycylglycine as compared with phosphate-buffered systems. Similar adjustment of the results for lysocephalin hydrolysis gave positive values of 5.0 and 3.6 ,umoleslg tissue per h for liver phospholipase B activity with lysophosphatidylethanolamine and lysophosphatidylserine as substrates, respectively, The rather wide range of these adjusted values precluded any definite conclusions regarding the enhancement of phospholipase B activity with these substrates in glycylglycine buffer. The question whether the triglyceride hydrolysis observed in these experiments is due to the action of lipoprotein lipase in liver? or of some other type of lipase, and the possible biochemical significance of its inhibition by lysophosphatides are subjects for further study. R. J. IRWIN is grateful to Harvard Medical School and R. J, ALPERN to the University of Rochester School of Medicine for the award of Student Research Fellowships supported respectively by Grants No. 5T5 (6M) 51-04 and zR6 (C-5) from the U.S. Public Health Service. The authors thank Mr. E. PORTMAN for his data
skilled
technical
assistance.
R. J. IRWIN” R. J. ALPERN** G. R. WEBSTER
Department of Chemical Pathology, Guy’s Hosjktat Medical School, London, S.E.r (Great Britain)
I J, GALLAI-HATCHARD AND R. H. S. THOMPSON, Biochim. Biophvs. Acta, g8 (1965) 128. 2 R. M. C. DAWSON, Biochem. J., 64 (1~56) 192. 3 E. A. MARPLES AND R. H. S. THOMPSON, Biochem. J., 74 (1960) 123. 4 6. V. MARINETTI, J. ERBLAND, R. F. WITTER, J. PETZX AND E. STOTZ, B~oc~~rn. Bicphys.
Acta,
30 (vJ58) 223. 5 J. ERBLAND AND G. V. MARINETTI, Federation Proc., 21 (1962) 295. 6 W. E. M. LANDS, J. Biol. Chem., 235 (1~60) 2233. 7 G. R. WEBSTER, Biochim. Biophys. Acta, 98 (rg65) 512. 8 J. F. ERBLAND AND G. V. MARINETTI, Biochim. Biophys. Acta, 106 (1965) 128. 9 J. F. ERBLAND AND G. V. MARINETTI, Biochim. Biophys. Acta, 106 (1gG5) 139. IO G. R. WEBSTER AND A. T. SMITH, Biochem. J.. 90 (1964) 64. II W. G. DUNCOMBE, Biochem. J., 88 (1963) 7. 12 E. A. MARPLES, R. H. S. THOMPSON AND G. R. WEBSTER, J. Neurochem., 4 (1959) 62. 13 E. D. KORN, J. Biol. Chem., 215 (1955) I.
Received May 6th, 1966 * Present address: Harvard Medical School, Boston, Mass. (U.S.A.). ** Present address: Department of Pediatrics, University Hospitals Ohio 44106 (U.S.A.).
of Cleveland,
Cleveland,
Biackim. Biophys. Acta, 125 (1966) 600-603