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Acrylylcholine: a new naturally occurring pharmacologically active choline ester from Buccinum undatum
Biochemical
Pharmacology,
1958, Vol.
I. pp. 342-346.
Pergamon
Press Ltd.,
Printed
in Great
Britain
ACRYLYLCHOLINE: A NEW NATURALLY OCCURRING PHARMACOLOGICALLY ACTIVE CHOLINE ESTER FROM BUCCINUM UNDATUM V. P. WHUTAKEK Biochemistry
Dep.utment,
Agricultural Research Council, Babraham, Cambridge
Institute
of Animal
Physiology.
Abstract-Extracts of the hypobranchial gland of the prosohranch marine gastropod Buccinunz untlntun7 (common whelk) has been found to have a very high acetylcholinelike activity (equivalent to about 0.3 pmoles acetylcholine g tissue) when assayed on the frog rectus abdominis muscle. So far, high hypohranchial acetylcholine equivalences have been known to occur only in the dye-secreting family of prosohranch gastropods. Muricidae, to which Buccinum does not belong. The active principle has been identified as acrylylcholine and is thus related to the two esters which have hcen isolated previously from Muricidae, urocanylcholine (4(5)-imidazolylacrylylcholine) and 6, p-dimethylacrylylcholine.
gastropods possess, on the under-surface of their mantles and adjacent to their gills, a modified slime gland of unknown function known as the hypobranchial gland. The glands of one particular family of this order of snails, the Muricidae, arc rich in a fluorescent indoxyl derivative which in the presence of crushed glandular material, light and air, is oxidized to the intense purple dye, 5.5’-dibromindigo, the much prized Tyrian Purple of the ancient world. Extracts of the glands are highly toxic to both cold- and warm-blooded animals,’ producing paralysis. The glands also contain a very high content of an acetylcholinc-like substance,” later identified as 4(5)-imidazolyl-), a choline ester with neurourocanylcholine3 (I, R, 7 H, R2 is probably mainly responsible for muscular blocking action, 4. 5 Thus urocanylcholine the toxic properties of the glandular extracts.
PROSOBRANCH
RI
>c
R2
CHCO.OCH,CH,N1
Me,
Any new naturally occurring pharmacologically active ester of cholitle- particularly one possessing neuromuscular blocking action-is of interest not only in connection with the general problem of cholinergic function, but also in relation to possible clinical applications in human and veterinary medicine. Urocanycholine has itself received clinical trial.6 Accordingly a survey was made of a number of marine gastropods and other molluscsi Two other choline esters were discovered. One of these occurs in place of urocanylcholine in the Muricidae Thai.s~floridana (Southern oyster drill) and has been identified as /3,/‘%dimethylacrylylcholine (I,R1 ; R, ~ Me); like urocanylcholine, this has neuromuscular blocking and ganglion-stimulating properties.” 342
Acrylylcholine,
a new active
ester from
Buccinum
undutum
343
The other new ester occurs in Buccinum undatum (the common whelk). The high choline ester content of the hypobranchial glands of this species, which is not a member of the Muricidae, is the first example of this phenomenon in a non dye-secreting snail. This ester has now been identified as the parent substance of the series, acrylylcholine (I,R, = R, = H). The evidence for this is as follows. The active substance was alkali-labile, was destroyed by purified plasma cholinesterase, formed an acid hydroxamate in alkaline solution and yielded choline (identified by paper chromatography) on hydrolysis. The active material is therefore a choline ester. It took up approximately I mole of bromine/mole ester (Table I) suggesting that it is the choline ester of an unsaturated fatty acid. The active material when chromatographed on columns of the ion-exchange resin Amberlite XE-97 buffered to pH 4.3 proved to have a retention volume lower than that of crotonylcholine or its higher homologues.’ TABLE 1. FORMATION OF VOLATILE FATTY ACID FROM, AND UPTAKE OF BROMINE BY HYDROLYSATESOF BUCCINUM ESTER Ester estimated by the ferric hydroxamate method,“ choline fatty acid by steam distillation pH 3 and titration of successive
spectrophotometrically,Y volatile in a Markham still in the presence of excess magnesium sulphate at fractions of distillate’ and bromine uptake by the method of Trappe.12 Figures in brackets are percentage yields. I
Ester (pmoles) 32.8 14.2* 5.0
10.0
Volatile
Choline (pmoles)
,
fatty
Bromine uptake (,:moles)
acid (peq.1
’ 22.5 (6’))
I
_
932%)
12.0* 44.0
~
I.5
i
0.69
0.48 (70)
*Ester hydrolysed by incubating with 10 mg purified human plasma cholinesterase preparation (Kabi A, B., Stockholm) for I5 min at 37” and pH 7.0. This preparation released 120 rmoles acid,!mg per hr from 30 mM acetylcholine in NaHCO, -CO, buffer, pH 7.4. tldentified as acrylic acid by vapour phase chromatography (Fig. 1).
Since retention volumes increase with chain length in both the saturated and unsaturated series,’ this suggested that the new ester was acrylylcholine. On submitting hydrolysates of the ester to steam distillation (Table 1), a volatile fatty acid was recovered in 33-66 per cent yield which was identified (Fig. 1) as acrylic acid by vapour phase chromatography. Synthetic acrylylcholine had the same chromatographic properties (Fig. 2) and relative molar potency on the frog rectus abdominis muscle as the new ester. EXPERIMENTAL Techniques. The extraction of tissue, biological assays, chromatography and chemical analyses were carried out as already described.7 The unit of biological activity (u.) is that amount of extract eliciting a response from the test organ (frog rectus abdominis muscle) equivalent to that from 1 m-pmole acetylcholine.
344
V. P. WHITTAKER
Acrylic acid
Volume
of effluent
nitrogen,
L
FIG. 1. Vapour phase chromatography of acrylic acid and volatile fatty acid from Buccinun~ ester. The graph shows the amount of fatty acid emerging from the column as a function of time or volume of ef7luent nitrogen.
Isolation of ester from Buccinum undatum. A typical preparation was as follows. The hypobranchial’ glands (wt. 413 g) from 62 live Bucchm (obtained from the Marine Biological Laboratory, Plymouth, with the kind co-operation of the Director, Dr. F. S. Russell, F.R.S.) were dissected out and dropped into ice-cold 10 per cent (w/v) trichloroacetic acid (900 ml). The tissue was finely cornminuted in a blender and centrifuged; the precipitate was resuspended in more 10 per cent (w/v) trichloroacetic acid (200 ml) and recentrifuged. The combined supernatants were extracted with ether to pH 4.5, evaporated to a small volume (70 ml) and centrifuged to remove a small amount of insoluble material. This extract contained 120,000 u. of activity, corresponding to a specific activity (‘acetylcholine equivalence’) of the original gland of 290 U./g. The active ester was purified by precipitation as the water insoluble reineckate. Saturated aqueous ammonium reincckate (2.5 vol.) was added to the extract at 0’; the precipitate was collected, dissolved in a small volume of 50 per cent (v/v) aqueous acetone and the reineckate ion replaced by chloride by passing the solution through a column of De-Acidite FF (The Permutit Co., Ltd., London. 16 j 50 mesh) in the chloride form. The effluent wa$ evaporated to dryness giving a crystalline residue containing 78,GOO u. of activity (yield, 65 per cent). Properties of acticc material. Activity was destroyed by heating for IO min at 100’ and pH 1‘0-12 or by incubating with cholinesterase (details in Table I ). C-holine was indentified in the hydrolysate as a compound giving a positive enneaiodide reaction under the conditions described by Appleton, La Du, Levy, Steele and Brady!’ and having an RF value (0.35) identical with that of authentic choline in n-butanolethanol-acetic acid-water (8 : 2 : I : 3). Approximately 1 mole of choline was recovered/mole ester (Table I). On treating solutions of the active material with alkaline hydroxylamine under the conditions given by Hestrin,lO an acylhydroxamic acid was formed which was detected by the formation of a reddish-brown complex with ferric ions. A comparison of the ester content of solutions determined in this way with their biological activity using acetylcholine as a standard enabled their molar potency relative to acetylcholine to be determined. This was 20-40 per cent when the frog rectus was the assay preparation but less than 1 per cent when the guinea pig
Acrylylcholine,
a new active
ester from
Buccinum undrrfctm
345
ileum was used. This difference in the relative molar potencies as determined on these two test organs is also seen with substituted acrylic esters of choline.‘? 8 On submitting hydrolysates of active material to steam distillation at pH 3 in the presence of enough MgSO, to retain HC1,7 a volatile acid was obtained in the distillate. The yield of acid was 33-66 per cent of the ester hydrolysed (Table 1). Acrylic acid was recovered in 63 per cent yield under identical conditions. The acid was concentrated and submitted to vapour phase chromatography’l (Fig. I). It had thes ame retention volume as acrylic acid and could not be separated from the latter when rechromatographed as a mixture. The acid component of the ester was found to combine with bromine in chloroform solutions of the acid were under the conditions given by Trappe. la Chloroform prepared from steam distillates evaporated to dryness under alkaline conditions or from freeze-dried alkaline hydrolysates of the ester after acidification with a few drops of 30 per cent phosphoric acid. After extraction into chloroform, the solution was dried by passing it through a small column of anhydrous Na,SO, and keiselguhr. The amount of bromine taken up (Table 1) corresponded approximately to that required for one double bond in the molecule. Synthesis qf acry/_ylcholirle. Acrylylcholine was synthesized by the method of Fourneau and Page.13 Acrylic acid (3.5 ml) was refluxed with ,%bromoethanol(3*5 ml) in dry benzene (10 ml) in an apparatus with a water trapping device. The presence of an acid catalyst caused the polymerization of acrylic acid. It was therefore omitted, although this resulted in a low (30 per cent) yield of ester. After 1 hr the benzene was evaporated, the residue taken up in ether, freed from unesterified acrylic acid by extracting with 5 per cent (w/v) NaHCO, and washed with water. The ether layer was dried, evaporated and the residue heated with excess anhydrous trimethylamine
FIG. 2. Column chromatography of synthetic acrylylcholine (filled circles) or natural ester (open circles) on columns of the weak acid ion exchange resin Amberlite XE-97 (0.6 cm diameter >: 10 cm height). The resin was buffered to pH 4.35 with 0.1 M-NaH,PO, before use and the esters eluted with 0.1 M-NaH,PO,. The graph shows the distribution of biological activity in successive fractions of eluate.
346
V. P. WHITTAKER
in dry benzene in a sealed tube at 60” for I.5 hr. The glassy deliquescent precipitate of acrylylcholine bromide was washed with dry benzene and dried i/l NXWI. It had a molar potency of 30 per cent relative to acetylcholine on the frog rectus and similar chromatographic properties to the natural ester (Fig. 2). Acrylic acid could be readily separated by vapour phase chromatography from other fatty acids with the exception of propionic acid. The retention volume of the synthetic acrylylcholine and the naturally occurring ester were close to that of propionylcholine which is known to occur in nature.“‘, I5 It is thus difficult to exclude the possibility that some propionylcholine might have been present in the snail extract. However, the presence of the double bond in the molecule and the low relative molar potencies of the ester on the frog rectus and guinea-pig ileum (the corresponding figures for propionylcholine arc 160 per cent and 5 per cent) make this unlikely. AcXno~‘/c,~~~~f7t~~~~--l am grateful to Miss J. M. Gilson for her skilful and painstaking and to Dr. E. F. Annison for advice on vapour phase chromatography.
technical
assistance,
REFERENCES I. R. Dueols,
Ax+.
Zoo/.
Erp.
G&i. (5) 2, 571 ( 1909).
2. D. VINCENT and A. JCLLIEN, C.R. Sec. 3. V. EKSPAMEK and 0.
Biol. Pmis 127. I506 (1938).
B~NATI, Bioclrc,,~~. Z. 324, 66 (1953).
4. V. EKSPAMER and A. GLASSEK, Buir. .I. Pl~unxrc~ol. 12. I76 (I 957). .I. P/WIIKKO/. 13, 103 (1958).
5. M. J. KEYL and V. P. WHITTAKER, &it. 6. S. IX BLASI and U. LEON~. Minwon
nnesrrsiol.
21, I37
( I955
).
J. Ph,~vio/. 139, 334 (1957). S. B. HOLMSTEDT and V. P. W~IIT’I‘AK~R, &it. J. Plmrmaco/. 13. 308 (1958).
7. M. J. KEYL. I. A. MICIIAELSON and
9.
H. D. APPLC rev.
V. P. WHIIIAKIR,
B. N. La Du. B. B. Levy.
J. M. STEFLL.and B. B. BRODIL, J. Biol. c‘llc,~.
803 (1953). IO. S. HESTRIN, J. Bid.
I I.
Chem. 180, 249 (1949).
W. TRAPPE, Biochen~. Z. 296,
I80 (1938).
A. J. P. MARTIN, Bioc/w~)!. J. 50. 679 (1952).
12. A. T. JAMES and 13. E. FOLJR~EAC and
H. J. PA(;E, &r/l. Sot.
cl7ir~. FY. (4) 15. 544 (1914).
14. J. BANISTER, V. P. WHITTAKER and S. WIJCSUNDERA, J. Plr.wio/. 15. J. E. GARDINEK
and V. P.
WHITTAKLII.
Biochem.
J. 58,). 24 (1954
121. 55 (1953).
205,
Pharmacology,
1958, Vol.
I. pp. 342-346.
Pergamon
Press Ltd.,
Printed
in Great
Britain
ACRYLYLCHOLINE: A NEW NATURALLY OCCURRING PHARMACOLOGICALLY ACTIVE CHOLINE ESTER FROM BUCCINUM UNDATUM V. P. WHUTAKEK Biochemistry
Dep.utment,
Agricultural Research Council, Babraham, Cambridge
Institute
of Animal
Physiology.
Abstract-Extracts of the hypobranchial gland of the prosohranch marine gastropod Buccinunz untlntun7 (common whelk) has been found to have a very high acetylcholinelike activity (equivalent to about 0.3 pmoles acetylcholine g tissue) when assayed on the frog rectus abdominis muscle. So far, high hypohranchial acetylcholine equivalences have been known to occur only in the dye-secreting family of prosohranch gastropods. Muricidae, to which Buccinum does not belong. The active principle has been identified as acrylylcholine and is thus related to the two esters which have hcen isolated previously from Muricidae, urocanylcholine (4(5)-imidazolylacrylylcholine) and 6, p-dimethylacrylylcholine.
gastropods possess, on the under-surface of their mantles and adjacent to their gills, a modified slime gland of unknown function known as the hypobranchial gland. The glands of one particular family of this order of snails, the Muricidae, arc rich in a fluorescent indoxyl derivative which in the presence of crushed glandular material, light and air, is oxidized to the intense purple dye, 5.5’-dibromindigo, the much prized Tyrian Purple of the ancient world. Extracts of the glands are highly toxic to both cold- and warm-blooded animals,’ producing paralysis. The glands also contain a very high content of an acetylcholinc-like substance,” later identified as 4(5)-imidazolyl-), a choline ester with neurourocanylcholine3 (I, R, 7 H, R2 is probably mainly responsible for muscular blocking action, 4. 5 Thus urocanylcholine the toxic properties of the glandular extracts.
PROSOBRANCH
RI
>c
R2
CHCO.OCH,CH,N1
Me,
Any new naturally occurring pharmacologically active ester of cholitle- particularly one possessing neuromuscular blocking action-is of interest not only in connection with the general problem of cholinergic function, but also in relation to possible clinical applications in human and veterinary medicine. Urocanycholine has itself received clinical trial.6 Accordingly a survey was made of a number of marine gastropods and other molluscsi Two other choline esters were discovered. One of these occurs in place of urocanylcholine in the Muricidae Thai.s~floridana (Southern oyster drill) and has been identified as /3,/‘%dimethylacrylylcholine (I,R1 ; R, ~ Me); like urocanylcholine, this has neuromuscular blocking and ganglion-stimulating properties.” 342
Acrylylcholine,
a new active
ester from
Buccinum
undutum
343
The other new ester occurs in Buccinum undatum (the common whelk). The high choline ester content of the hypobranchial glands of this species, which is not a member of the Muricidae, is the first example of this phenomenon in a non dye-secreting snail. This ester has now been identified as the parent substance of the series, acrylylcholine (I,R, = R, = H). The evidence for this is as follows. The active substance was alkali-labile, was destroyed by purified plasma cholinesterase, formed an acid hydroxamate in alkaline solution and yielded choline (identified by paper chromatography) on hydrolysis. The active material is therefore a choline ester. It took up approximately I mole of bromine/mole ester (Table I) suggesting that it is the choline ester of an unsaturated fatty acid. The active material when chromatographed on columns of the ion-exchange resin Amberlite XE-97 buffered to pH 4.3 proved to have a retention volume lower than that of crotonylcholine or its higher homologues.’ TABLE 1. FORMATION OF VOLATILE FATTY ACID FROM, AND UPTAKE OF BROMINE BY HYDROLYSATESOF BUCCINUM ESTER Ester estimated by the ferric hydroxamate method,“ choline fatty acid by steam distillation pH 3 and titration of successive
spectrophotometrically,Y volatile in a Markham still in the presence of excess magnesium sulphate at fractions of distillate’ and bromine uptake by the method of Trappe.12 Figures in brackets are percentage yields. I
Ester (pmoles) 32.8 14.2* 5.0
10.0
Volatile
Choline (pmoles)
,
fatty
Bromine uptake (,:moles)
acid (peq.1
’ 22.5 (6’))
I
_
932%)
12.0* 44.0
~
I.5
i
0.69
0.48 (70)
*Ester hydrolysed by incubating with 10 mg purified human plasma cholinesterase preparation (Kabi A, B., Stockholm) for I5 min at 37” and pH 7.0. This preparation released 120 rmoles acid,!mg per hr from 30 mM acetylcholine in NaHCO, -CO, buffer, pH 7.4. tldentified as acrylic acid by vapour phase chromatography (Fig. 1).
Since retention volumes increase with chain length in both the saturated and unsaturated series,’ this suggested that the new ester was acrylylcholine. On submitting hydrolysates of the ester to steam distillation (Table 1), a volatile fatty acid was recovered in 33-66 per cent yield which was identified (Fig. 1) as acrylic acid by vapour phase chromatography. Synthetic acrylylcholine had the same chromatographic properties (Fig. 2) and relative molar potency on the frog rectus abdominis muscle as the new ester. EXPERIMENTAL Techniques. The extraction of tissue, biological assays, chromatography and chemical analyses were carried out as already described.7 The unit of biological activity (u.) is that amount of extract eliciting a response from the test organ (frog rectus abdominis muscle) equivalent to that from 1 m-pmole acetylcholine.
344
V. P. WHITTAKER
Acrylic acid
Volume
of effluent
nitrogen,
L
FIG. 1. Vapour phase chromatography of acrylic acid and volatile fatty acid from Buccinun~ ester. The graph shows the amount of fatty acid emerging from the column as a function of time or volume of ef7luent nitrogen.
Isolation of ester from Buccinum undatum. A typical preparation was as follows. The hypobranchial’ glands (wt. 413 g) from 62 live Bucchm (obtained from the Marine Biological Laboratory, Plymouth, with the kind co-operation of the Director, Dr. F. S. Russell, F.R.S.) were dissected out and dropped into ice-cold 10 per cent (w/v) trichloroacetic acid (900 ml). The tissue was finely cornminuted in a blender and centrifuged; the precipitate was resuspended in more 10 per cent (w/v) trichloroacetic acid (200 ml) and recentrifuged. The combined supernatants were extracted with ether to pH 4.5, evaporated to a small volume (70 ml) and centrifuged to remove a small amount of insoluble material. This extract contained 120,000 u. of activity, corresponding to a specific activity (‘acetylcholine equivalence’) of the original gland of 290 U./g. The active ester was purified by precipitation as the water insoluble reineckate. Saturated aqueous ammonium reincckate (2.5 vol.) was added to the extract at 0’; the precipitate was collected, dissolved in a small volume of 50 per cent (v/v) aqueous acetone and the reineckate ion replaced by chloride by passing the solution through a column of De-Acidite FF (The Permutit Co., Ltd., London. 16 j 50 mesh) in the chloride form. The effluent wa$ evaporated to dryness giving a crystalline residue containing 78,GOO u. of activity (yield, 65 per cent). Properties of acticc material. Activity was destroyed by heating for IO min at 100’ and pH 1‘0-12 or by incubating with cholinesterase (details in Table I ). C-holine was indentified in the hydrolysate as a compound giving a positive enneaiodide reaction under the conditions described by Appleton, La Du, Levy, Steele and Brady!’ and having an RF value (0.35) identical with that of authentic choline in n-butanolethanol-acetic acid-water (8 : 2 : I : 3). Approximately 1 mole of choline was recovered/mole ester (Table I). On treating solutions of the active material with alkaline hydroxylamine under the conditions given by Hestrin,lO an acylhydroxamic acid was formed which was detected by the formation of a reddish-brown complex with ferric ions. A comparison of the ester content of solutions determined in this way with their biological activity using acetylcholine as a standard enabled their molar potency relative to acetylcholine to be determined. This was 20-40 per cent when the frog rectus was the assay preparation but less than 1 per cent when the guinea pig
Acrylylcholine,
a new active
ester from
Buccinum undrrfctm
345
ileum was used. This difference in the relative molar potencies as determined on these two test organs is also seen with substituted acrylic esters of choline.‘? 8 On submitting hydrolysates of active material to steam distillation at pH 3 in the presence of enough MgSO, to retain HC1,7 a volatile acid was obtained in the distillate. The yield of acid was 33-66 per cent of the ester hydrolysed (Table 1). Acrylic acid was recovered in 63 per cent yield under identical conditions. The acid was concentrated and submitted to vapour phase chromatography’l (Fig. I). It had thes ame retention volume as acrylic acid and could not be separated from the latter when rechromatographed as a mixture. The acid component of the ester was found to combine with bromine in chloroform solutions of the acid were under the conditions given by Trappe. la Chloroform prepared from steam distillates evaporated to dryness under alkaline conditions or from freeze-dried alkaline hydrolysates of the ester after acidification with a few drops of 30 per cent phosphoric acid. After extraction into chloroform, the solution was dried by passing it through a small column of anhydrous Na,SO, and keiselguhr. The amount of bromine taken up (Table 1) corresponded approximately to that required for one double bond in the molecule. Synthesis qf acry/_ylcholirle. Acrylylcholine was synthesized by the method of Fourneau and Page.13 Acrylic acid (3.5 ml) was refluxed with ,%bromoethanol(3*5 ml) in dry benzene (10 ml) in an apparatus with a water trapping device. The presence of an acid catalyst caused the polymerization of acrylic acid. It was therefore omitted, although this resulted in a low (30 per cent) yield of ester. After 1 hr the benzene was evaporated, the residue taken up in ether, freed from unesterified acrylic acid by extracting with 5 per cent (w/v) NaHCO, and washed with water. The ether layer was dried, evaporated and the residue heated with excess anhydrous trimethylamine
FIG. 2. Column chromatography of synthetic acrylylcholine (filled circles) or natural ester (open circles) on columns of the weak acid ion exchange resin Amberlite XE-97 (0.6 cm diameter >: 10 cm height). The resin was buffered to pH 4.35 with 0.1 M-NaH,PO, before use and the esters eluted with 0.1 M-NaH,PO,. The graph shows the distribution of biological activity in successive fractions of eluate.
346
V. P. WHITTAKER
in dry benzene in a sealed tube at 60” for I.5 hr. The glassy deliquescent precipitate of acrylylcholine bromide was washed with dry benzene and dried i/l NXWI. It had a molar potency of 30 per cent relative to acetylcholine on the frog rectus and similar chromatographic properties to the natural ester (Fig. 2). Acrylic acid could be readily separated by vapour phase chromatography from other fatty acids with the exception of propionic acid. The retention volume of the synthetic acrylylcholine and the naturally occurring ester were close to that of propionylcholine which is known to occur in nature.“‘, I5 It is thus difficult to exclude the possibility that some propionylcholine might have been present in the snail extract. However, the presence of the double bond in the molecule and the low relative molar potencies of the ester on the frog rectus and guinea-pig ileum (the corresponding figures for propionylcholine arc 160 per cent and 5 per cent) make this unlikely. AcXno~‘/c,~~~~f7t~~~~--l am grateful to Miss J. M. Gilson for her skilful and painstaking and to Dr. E. F. Annison for advice on vapour phase chromatography.
technical
assistance,
REFERENCES I. R. Dueols,
Ax+.
Zoo/.
Erp.
G&i. (5) 2, 571 ( 1909).
2. D. VINCENT and A. JCLLIEN, C.R. Sec. 3. V. EKSPAMEK and 0.
Biol. Pmis 127. I506 (1938).
B~NATI, Bioclrc,,~~. Z. 324, 66 (1953).
4. V. EKSPAMER and A. GLASSEK, Buir. .I. Pl~unxrc~ol. 12. I76 (I 957). .I. P/WIIKKO/. 13, 103 (1958).
5. M. J. KEYL and V. P. WHITTAKER, &it. 6. S. IX BLASI and U. LEON~. Minwon
nnesrrsiol.
21, I37
( I955
).
J. Ph,~vio/. 139, 334 (1957). S. B. HOLMSTEDT and V. P. W~IIT’I‘AK~R, &it. J. Plmrmaco/. 13. 308 (1958).
7. M. J. KEYL. I. A. MICIIAELSON and
9.
H. D. APPLC rev.
V. P. WHIIIAKIR,
B. N. La Du. B. B. Levy.
J. M. STEFLL.and B. B. BRODIL, J. Biol. c‘llc,~.
803 (1953). IO. S. HESTRIN, J. Bid.
I I.
Chem. 180, 249 (1949).
W. TRAPPE, Biochen~. Z. 296,
I80 (1938).
A. J. P. MARTIN, Bioc/w~)!. J. 50. 679 (1952).
12. A. T. JAMES and 13. E. FOLJR~EAC and
H. J. PA(;E, &r/l. Sot.
cl7ir~. FY. (4) 15. 544 (1914).
14. J. BANISTER, V. P. WHITTAKER and S. WIJCSUNDERA, J. Plr.wio/. 15. J. E. GARDINEK
and V. P.
WHITTAKLII.
Biochem.
J. 58,). 24 (1954
121. 55 (1953).
205,