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Purification of glucosamine-6-phosphate N-acetylase from sheep brain
BIOCHIMICA ET BIOPHYSICA ACTA
681
PURIFICATION OF GLUCOSAMINE-6-PHOSPHATE N-ACETYLASE FROM SHEEP BRAIN T. N. P A T T A B I R A M A N
AND B. K. B A C H H A W A T
Neurochemistry Laboratory, Department of Neurology and Neurosurgery, Christian Medical College and Hospital, Vellore (South India) (Received O c t o b e r 25th, 196I)
SUMMARY
The presence of the enzyme glucosamine-6-phosphate acetylase which synthesizes N-acetylglucosamine 6-phosphate from glucosamine 6-phosphate and acetyl-coenzyme A has been established in brain. The enzyme has been purified 75-fold from sheep brain. The enzyme has an optimum pH at 7.4. Free sulfhydryl groups and EDTA activated the enzyme. Km value for glucosamine 6-phosphate is 4.5" lO-4 M and for acetyl-coenzyme A is 2.5" lO-4 M. INTRODUCTION
Amino sugars are widely distributed in animal tissues. In brain these sugars are present primarily as mucopolysaccharides and gangliosidesl, ~. BRANTE and GUHA et al. have suggested that mucopolysaccharides may play an important role in the initial stages of myelination s, 4. According to BOGOCH, neuraminic acid, a main constituent of gangliosides may be the site of attack of viruses which invade the brain s. The biological importance of these macromolecules has lead to the study of the metabolism of amino sugars in recent years in various tissues and micro-organisms. Work in this laboratory has established the enzymic synthesis of GlcNH 2 6-phosphate and UDP-acetylglucosamine in brainS, e. The study of the enzymic acetylation of GlcNH~ 6-phosphate is of primary importance, since the formation of acetylated amino sugar is an obligatory intermediate in the interconversion of amino sugars. In order to establish the complete sequence of enzymic reactions leading to UDP-acetylglucosamine from the common precursor fructose 6-phosphate in brain, the presence of this acetylating enzyme is essential. The presence of this enzyme has been detected in yeast, bacteria and different animal tissuesV, 8. GlcNH z 6-phosphate acetylase has been purified from Neurospora crassa and rabbit liver by ROSEMAN et al. 7. However, ROSEMAN et al. were unable to show the existence of this enzyme in brain. The present paper establishes the presence of GlcNH z. 6-phosphate acetylase in brain. Partial purification and properties of this enzyme are also presented here. MATERIALS AND METHODS
GlcNH 2 6-phosphate was prepared enzymically by phosphorylating GIcNH~ and purified, as described previously 5. ATP and CoA (75 % pure) were obtained from A b b r e v i a t i o n : PCMB, p - c h l o r o m e r c u r i b e n z o a t e .
Biochim. Biophys. Acta, 59 (1962) 6 8 1 - 6 8 9
682
T . N . PATTABIRAMAN, B. K. BACHHAWAT
Sigma Chemical Company. Acetyl-CoA was prepared by chemical acetylation of CoA with acetic anhydride 9. Propionyl-CoA was similarly prepared using propionic anhydride. All other reagents were analytical-grade commercial chemicals. Acetate-activating enzyme system was prepared from Euglena gracilis 1°. The crude extract contained deacetylase and hence the acetate-activating enzyme was to be included in the regular assay during the purification process. The crude preparation contained the powerful deaminase which readily degraded GlcNH, 6-phosphate. To protect the substrate from degradation, it was necessary to include diammonium hydrogen phosphate in the assay system. The effective use of diammonium hydrogen phosphate in the formation of GlcNHAc 6-phosphate in the crude system is shown in Fig. I. It increased the enzymic activity markedly. The optimal concentration of diammonium hydrogen phosphate was found to be o.15 M. Higher concentration inhibited the enzymic reaction.
60
50
E E c
40
E ~- 3f o_
~ 2(1 z 10 _q 0 -
,~
2'0
3'0
.;o
50
(NH4)2 HP04, in ~lmoles Fig. I. Effect of d i a m m o n i u m h y d r o g e n p h o s p h a t e on t h e e n z y m i c f o r m a t i o n of G l c N H A c 6p h o s p h a t e . The a s s a y s y s t e m is t h e s a m e as d e s c r i b e d in t h e t e x t e x c e p t t h a t d i a m m o n i u m h y d r o g e n p h o s p h a t e of v a r y i n g c o n c e n t r a t i o n w a s a dde d.
The regular system for the assay of GlcNH2-6-phosphate acetylase during the purification process contained 0. 4/zmole of GlcNH 2 6-phosphate, 0.02/~mole of CoA, x pmole of ATP, I #mole of cysteine, 0.2/~mote of MgSO 4, 30 ~moles of acetate buffer, pH 6. 5, 3 ° #moles of diammonium hydrogen phosphate, 12o/~g of partially purified acetate-activating enzyme and 0.3-0.8 unit of the brain acetylase in a total volume of 0.2 ml. After i h incubation at 37 ° the reaction was stopped by the addition of o.x ml of 3 ° % trichloroacetic acid. After centrifuging off the precipitated protein, the supernatant fluid was neutralised with 30 % KHCO 3 and GlcNHAc 6-phosphate formed was determined. With the purified enzyme fractions deproteinisation was not found to be necessary. The assay system for the study of the properties of GlcNH 2 6-phosphate acetylase contained 0. 4/~mole of GlcNH~ 6-phosphate, o.15/~mole of acetyl-CoA, 4 °/zmoles of Tris buffer, pH 7-4, and 47.2/~g of the purified enzyme and additions as indicated Biochim. Biophys. Acta, 56) (tq6z) 681-68q
GLUCOSAMINE 6-PHOSPHATEN-AcETYLASE FROM BRAIN
683
in the individual cases, in a total volume of 0.2 ml. After I h incubation at 37 ° the reaction was stopped b y the addition of o.16 M sodium tetraborate and GlcNHAc 6-phosphate formed was determined. GlcNHAc 6-phosphate was assayed b y the method of REISSIG et al. ll with a slight modification TM. Protein was measured spectrophotometrically at 280 and 26o mt~ with a correction for nucleic acid content 13. The concentration of ammonium sulphate is expressed in all cases as g/ml, the saturation at o ° being 0.526 g/ml. The quantitative assay of free sulfhydryl groups was done spectrophotometrically based on the method of BOYER14. Acetyl-CoA was measured as hydroxamate by the method of LIPMANN AND TUTTLE is.
One unit of enzyme is equivalent to IOO mt~moles of GlcNHAc 6-phosphate formed in i h.
Purification of enzyme from sheep brain All operations were conducted between o - 5 °, unless indicated otherwise. Extraction: Sheep brain, obtained immediately after the death of the animal, was chilled in ice, trimmed of white matter and stored at - - 1 8 °. 3oo g of the frozen tissue were homogenised with 6oo ml of o.o3 M Tris buffer, p H 7.4, in a Waring blender for 45 sec. The mixture was magnetically stirred for 15 min and centrifuged at ioooo × g for 2o min in a refrigerated centrifuge. The reddish cloudy supernatant contained 6.8 g of protein in a volume of 49 ° m l . First ammonium sulphate fractionation: The supernatant fluid was brought to 45 % saturation with solid ammonium sulphate b y the slow addition of 131.5 g of the salt and the mixture was magnetically stirred for 2o min. After centrifugation at IOOOO × g for 2o rain the supernatant fluid was collected and brought to 7 ° % saturation b y the addition of 83.8 g of solid ammonium sulphate. The precipitate was collected b y centrifugation and dissolved in 5o ml of o.o3 M Tris buffer, p H 7.4, containing o.ooi M cysteine. The solution was dialysed against 5o times the volume of o.oi M Tris buffer, p H 7.4, for 3 h, with the change of the dialysing solution after an interval of 9 ° min. The reddish clear solution contained 1.77 g of protein in a volume of 75 ml. Gel treatment: To the dialysed solution were added 123.9 ml of calcium phosphate gel (IO mg/ml) bringing the ratio of protein to gel to 1:o.7. The mixture was magnetically stirred for IO min and centrifuged at 4ooo × g for IO min. The gel supernatant contained 1.265 g of protein in a volume of 175 ml. Second ammonium sulphate fractionation: The supernatant fluid was brought to 48 % saturation with solid ammonium sulphate b y the addition of 5o.5 g of the salt and the mixture was magnetically stirred for 2o min. After centrifugation at IO ooo × g for 2o min the supernatant fluid was collected and brought to 7 ° % saturation by the addition of 26. 9 g of solid ammonium sulphate. The precipitate was collected b y centrifugation and dissolved in 12 ml of o.o3 M "Iris buffer, p H 7-4, containing o.ooi M cysteine. The solution was dialysed against 5o times the volume of o.oi M Tris buffer, p H 7.4, for 3 h with the change of the dialysing solution after an interval of 9 ° min. The pale-reddish clear solution contained o.676 g of protein in a volume of 26 ml. Biockim. Biophys. Acta, 59 (1962) 681-689
684
T. N. PATTABIRAMAN, B. K. BACHHAWAT
Heat treatment: The dialysed solution was adjusted to p H 5 with I N acetic acid and heat treated in small fractions of 2 ml each. The solution was immersed in a water bath at 9 ° o, when its temperature reached 65°, it was transferred to a water bath maintained at 65 ° and held at this temperature exactly for 2 rain. I t was cooled rapidly in crushed ice and centrifuged at 2o ooo × g for 2o min. The precipitate was discarded. The supernatant fluid was collected. The colorless clear solution contained 53-4 mg of protein in a volume of 23 ml. RESULTS
The s u m m a r y of the purification process is shown in Table I. A 75-fold purification was obtained by using the above procedure with an overall recovery of 59 % of the original activity. The purified enzyme was found to be free of acetate-activating enzyme and deacetylase. But it contained traces of GlcNH~-6-phosphate deaminase activity. TABLE I PURIFICATION OF GLUCOSAMINE-6-PHOSPHATE N-ACETYLASE Steps Extraction First a m m o n i u m s u l p h a t e fractionation Gel t r e a t m e n t Second a m m o n i u m s u l p h a t e fractionation Heat treatment
S p e ~ activity Total protein (units/ragprotein)
Yield (%)
Volume (~)
Total units
49o
294
6.81 g
o.o43
ioo
75 175
315 280
1.77 g 1.26 g
o.178 o.221
lO7 95.2
26 23
224 174
676 m g 53.4 m g
o.332 3.25
76. 4 59.1
Optimal p H GlcNH,-6-phosphate acetylase from sheep brain has a sharp optimal p H of 7-4 as shown in Fig. 2. This was different from that of N. crassa enzyme v which was found to have a flat optimal range from p H 6-7.1.
Substrate specificity The purified enzyme system exhibited no acetylating activity with GlcNH 2 or GalNH v Acetyl-CoA could be replaced neither by AMP-acetate nor by AMP-acetate and CoA together, This eliminates the possibility of chemical formation of acetyl-CoA in the system. In the presence of acetate-activating enzyme system AMP-acetate and CoA could replace acetyl-CoA. Though propionyl-CoA could transfer the propionyl moiety to the amino group of GlcNH 2 6-phosphate, the reaction was found to be very feeble. With equimolar amounts of propionyl-CoA, the activity was only 15 % compared to that of acetyl-CoA.
Effect of sulfhydryl reagents I t has been reported by ROSEMAN and his associates that the purified enzyme from N. crassa displayed no cofactor requirement 7. Complete inhibition with that enzyme was found at lO -8 M concentration of PCMB. In the case of sheep-brain enzyme PCMB at a concentration of lO -5 M completely inhibited the activity and Biochim. Biophys. Acta, 59 (1962) 68I--689
GLUCOSAMINE 6-PHOSPHATE N-ACETYLASE FROM BRAIN
685
at a concentration of IO-~ M inhibited nearly 50 % of the activity. This inactivation was found to be reversed by cysteine showing that the sheep-brain enzyme requires free sulfhydryl groups for maximal activity and is highly susceptible to sulfhydryl reagents. Cysteine was found to increase the activity of GlcNH2-6-phosphate acetylase from sheep brain (Table n).
55
"5 50
E :L E .c_ 45
i
z
u
3¢
i
L
L
i
5
6
7
8
9
pH Fig. 2. Effect of H + c o n c e n t r a t i o n on t h e e n z y m e reaction. T h e a s s a y s y s t e m is t h e s a m e as described in t h e t e x t e x c e p t t h a t 4 ° # m o l e s of buffer of different p H were included. Q - - O , p h o s p h a t e ; O - - O , Tris buffers. T A B L E II EP'FECT OF CYSTEINE AND SULFHYDRYL REAGENTS T h e e n z y m e (47.2 pg) w a s p r e i n c u b a t e d w i t h p - c h l o r o m e r c u r i b e n z o a t e for io rain a t 25 °. I n t h e control t u b e t h e e n z y m e w a s s i m i l a r l y p r e i n c u b a t e d w i t h o u t t h e a d d i t i o n of s u l f h y d r y l r e a g e n t . A f t e r t h e p r e i n c u b a t i o n t h e s u b s t r a t e s were a d d e d a n d i n c u b a t e d for I h a t 37 °. T h e ~ a e t i o n w a s s t o p p e d b y t h e a d d i t i o n of s o d i u m t e t r a b o r a t e solution. I n t h e e x p e r i m e n t s w i t h cysteine. I p m o l e of c y s t e i n e w a s a d d e d a l o n g w i t h t h e s u b s t r a t e a f t e r t h e p r e i n c u b a t i o n of t h e e n z y m e with p-chloromercuribenzoate. Ezpt. No.
GI~NHAc 6-pb.ospkat~ for,m~ (pt,.ole)
Additio~
None Plus cysteine P l u s i . xo -6 M p - c h l o r o m e r c u r i b e n z o a t e P l u s x. IO-~ M p - c h l o r o m e r c u r i b e n z o a t e and eysteine P l u s I" lO -6 M p - c h l o r o m e r c u r i b e n z o a t e P l u s x- IO-6 M p - c h l o r o m e r c u r i b e n z o a t e and cysteine
0.054 o.o7I o o.o525 o.o 3 o.o54
Effect of E D T A and metal ions N. crassa enzyme was not found to be inhibited by EDTA ~, whereas EDTA was found to activate GlcNH ,-6-phosphate acetylase from sheep brain markedly. To prove that this activation is not even partially due to the inhibition of GlcNH2-6-phosphate deaminase 5, the effect of EDTA was studied at pH 6 where the activity of deaminase Biochim. Biophys. Acta, 59 (i962) 6 8 1 - 6 8 9
686
T. N. PATTABIRAMAN, B. K. BACHHAWAT
was found to be negligible. Under this condition of assay EDTA activated GlcNH,6-phosphate acetylase to the same extent. This enhancing effect of EDTA may be due to the protection of the sulfhydryl groups of the enzyme from heavy metals. Mg 2+ or Mn *+ had a slight but consistent enhancing effect on the enzymic activity. Co s+ had no effect, whereas Ni 2+ inhibited the enzyme (Table III). TABLE EFFECT OF E D T A
III A N D M E T A L IONS
T h e assay system w a s the s a m e as described in the text except that additions were m a d e as indicated. For Expts. 7 and 8, 4 °/*moles of phosphate buffer, p H 6, were used. Exit. No.
I 2 3 4 5 6 7 8
GlcNHA c 6-phosphate formtd (l~mole)
Addition
None Plus x /,mole E D T A Plus o . 1 / , m o l e MgSO 4 Plus o . I / , m o l e MnSO 4 Plus o . I / , m o l e CoCI2 Plus o. I / , m o l e NiCI s None (at p H 6) Plus I / * m o l e E D T A (at p H 6)
0.054 0.096 0.059 o.o6o o.o53 0.039 0.04o o.07o
E f f e a of substrate concentration
Enzymic activity with increasing concentration of GlcNH~ 6-phosphate and acetyl-CoA is presented in Figs. 3 and 4 respectively. The calculated Km for GlcNH 2 6-phosphate is 4-5" lO-4 M and for acetyl-CoA 2.5" lO -4 M. These values were lower compared to the Km values for N. crassa enzyme ~where for both GlcNH 2 6-phosphate and acetyl-CoA the Km was 7.8" lO -4 M. The optimal concentration of acetyl-CoA was found to be 3" IO-S M. Higher concentration of acetyl-CoA inhibited the enzymic activity markedly. A similar observation has been made earlier by ROSEMAN and his associates with the N. crassa enzyme7.
e.
12
120~
10
a
O0 ~. ._c eo~
S/v
E 40 u <
$
v
;
i
3.
.
4
.
5.
.
6
7
8
S {GIc NH2 (5- P concentration) x 10"1M Fig. 3. Effect of G I c N H z 6 - p h o s p h a t e concentration on t h e reaction velocity of the enzyme. The a s s a y s y s t e m w a s t h e s a m e as described in the t e x t except t h a t x / , m o l e of E D T A and GlcNH 2 6 - p h o s p h a t e of v a r y i n g concentration were included. B i o c h i m . B i o p h y s . A c t a , 59 (I962) 681-689
GLUCOSAMINE 6~PHOSPHATE N - A C E T Y L A S E FROM BRAIN
687
Stoichiometry of the reaction Stoichiometry of the reaction is given in Table IV. Equimolar quantities of GlcNHAc 6-phosphate and CoA were formed corresponding to the amount of acetylCoA disappeared.
Stability of the enzyme The enzyme lost 50 % of its activity when stored in the frozen state for two weeks at: tV
"6 E 125
t::
s/v 4
I00 E
3
75 a.t
2
50
I
u
b
;
,o
,;
~
2;
~o ~ 20 4~.... 60~'"
S ( A c CoA concentration) x 10"4H
Fig. 4. Effect of acetyl-CoA c o n c e n t r a t i o n on t h e r e a c t i o n velocity of t h e e n z y m e . T h e a s s a y s y s t e m w a s t h e s a m e as described in t h e t e x t e x c e p t t h a t i / , m o l e of E D T A a n d a c e t y l - C o A of v a r y i n g c o n c e n t r a t i o n were included. T A B L E IV $TOICHIOMETRY OF THE REACTION X /*mole of G l c N H z 6 - p h o s p h a t e , o . 8 / , m o l e of acetyl-CoA, IOO/,moles of Tris, p H 7-4, 0 . 5 / * m o l e of E D T A a n d 136/*g of e n z y m e were i n c u b a t e d for I h a t 37 °. A n a l i q u o t (o.x ml) w a s t a k e n for G l c N H A c 6 - p h o s p h a t e assay, o.5 m l w a s t a k e n for t h e d e t e r m i n a t i o n of free s u l f h y d r y l g r o u p s a p p e a r e d , o.3 m l w a s t a k e n for t h e a s s a y of r e m a i n i n g acetyl-CoA. A b l a n k w a s r u n s i m u l t a n e o u s l y in w h i c h GlcNH~ 6 - p h o s p h a t e w a s a d d e d a f t e r t h e i n c u b a t i o n w a s o v e r a n d t h e d i s a p p e a r a n c e of acetyl-CoA a n d f o r m a t i o n of CoA were d e t e r m i n e d t h e r e also. GlcNHAc 6-pkospkate formed (Umote)
0.44
Acetyl*CoA di.~ppeamd ( Umole)
CoA formed ( Umote)
0.446
0.423
Identification of product as GlcNHAc 6-phosphate IO /zmoles of GlcNH~ 6-phosphate, 5 Izmoles of acetyl-CoA, 2 mmoles of Tris buffer, p H 7.4, IO/~moles of EDTA and 94 °/~g of the purified enzyme in a total volume of 4 ml were incubated for I h at 37 °. The reaction was stopped b y keeping the tubes in a boiling-water b a t h for 2 min. The precipitated protein was centrifuged off. The supernate was passed through Dowex-I in C1- form. N-acetylhexosamine 6-phosphate could be eluted with o.i N HC1 according to the method of ALM et al. 1~. The eluted sugar was again passed through Dowex-5o in H + form and acetylhexosamine phosphate was eluted with water and lyophilized. 3.2 t~moles of N-acetylhexosamine phosphate was formed. Biochim. Biophys. Acta, 59 (1962) 6 8 I ~ 8 9
688
T. N. PATTABIRAMAN, B. K. BACI-IHAWAT
The isolated product was found to enhance the activity of GlcNH2-6-phosphate deaminase to the same extent as an authentic sample of GlcNHAc 6-phosphate when used in equimolar amount 5. The product was further identified b y paper chromatography. 25/zg of the product isolated, was plotted on W h a t m a n 3MM paper and irrigated in the basic water-miscible solvent system of HANES AND ISHERWOODle (propanol-saturated amm o n i a - w a t e r (6:3: I)) and ascending chromatography was performed for 16 h at room temperature. The paper was then dried and sprayed with o.16 M sodium tetraborate solution to ensure complete wetting. The paper was kept in an air oven, maintained at, IOO° for I0 min after which Ehrlich reagent (IO g of p-dimethylaminobenzaidehyde in 9 ° ml of acetic acid and io ml Of io N HC1) was sprayed. A purple spot was formed immediately. The test sample was found to have the same RF value as an authentic sample of GlcNHAc 6-phosphate. The RF value for GlcNHAc 6-phosphate was found to be 0.36. DISCUSSION
Although ROSEMAN a a/. were unable to detect GlcNH~-6-phosphate acetylase in brain *, this work establishes the presence of this enzyme in brain. It is probable that the detection of this enzyme was not possible earlier because of the presence of powerful glucosamine-6-phosphate deaminase in brain and also due to the lower concentration of this enzyme in this tissue. By reversing the deaminase action with the help of diammonium hydrogen phosphate, the presence of this acetylase could be detected. Although ROSEMAN a al. have purified the acetylase from animal tissues so far there had been no studies reported on the properties of this enzyme from animal tissues 7. The present work describes in detail the properties of this acetylase which differs markedly from that of the enzyme from N . crassa ~. Considering the earlier reports from this laboratory on the enzyme related to the amino sugar metabolism, the presence of this acetylase bridges the gap between hexose 6-phosphate and UDP-acetylglucosamine. It m a y be noted that all the enzymes which are known to involve in the biosynthesis of amino sugars are present in brain. On the basis of these facts an outline of the enzymic sequence in the synthesis of amino sugars in brain m a y be given as follows. Fructose 6-phosphate + N H 8 ~
DeamJnase
" Glucosamine 6-phosphate5
Glucosamine 6-phosphate + Acetyl-CoA Acetylase ) N-acetylglueosamine 6-phosphate + CoA N-acetylglucosamine 5-phosphate *
Mutase
* N-acetylglucosamine 1-phosphate
N-acetylglucosamine I-phosphate + U T P +.Pyrophosphoryla~e UDpo acetylglucosamine + pyrophosphate e
All of the above enzymes have been found to be present in brain except mutase. Preliminary investigation in this laboratory indicates that there is an enzyme in brain which participates in the interconversion of N-acetylglucosamine 6-phosphate and N-acetylghicosamine 1-phosphate. I t remains to be determined whether this is a specific mutase for the acetylamino sugar phosphate or a non-specific phosphoglucomutase. Biochim. Biophys. Acta, 59 (1962) 681-689
GLUCOSAMINE 6-PHOSPHATE N-ACETYLASE FROM BRAIN
689
ACKNOWLEDGEMENTS
The keen interest and encouragement of Professor J. CHANDYare deeply appreciated. The first author is a Raptokas Medical Research Fellow. This investigation was supported by a grant from the National Multiple Sclerosis Society (U.S.A.), Grant No. 214-2. REFERENCES 1 G. BRANTE, in D. RICHTER, Metabolism of the Nervous System, P e r g a m o n Press, London, 1957, p. 112. 2 S. BOGOCH, Neurology, Io (196o) 439. 3 G. BRANTE, in F. BRUCKE, Biochemistry of the Central Nervous System, P e r g a m o n Press, London, 1959, p. 291. 4 A. GOHA, B. J. NORTHOVERANY B. K. BACHHHAWAT,J. Sci. Ind. Research (India), I 9 C (196o) 287. 5 T. N. PATTABIRAMAN AND B. K. BACHHAWAT,Biochim. Biophys. Aaa, 54 (1961) 2736 T. N. PATTABIRAMAN AND B. K. BACHHHAWAT,Biochim. Biophys. Acta, 5o (I96I) 129. 7 E. A. DAVIDSON,H. J. BLUMENTHAL AND S. ROSEMAN,J. Biol. Chem., 226 (1957) 125. s D. BROWN, Biochim. Biophys. Acta, 16 (1955) 429. 9 S. OCHOA, in S. P. COLOWICK AND N. O. KAPLAN, Methods in Enzymology, Vol. I, Academic Press, New York, 1955, p. 685. 10 A. ABRAHAM AND g . K. BACHHAWAT,Biochim. Biophys. Acta, in t h e press. xI j . I. REISSIG, J. L. STROMINGER AND L. F. LELOIR, J. Biol. Chem., 217 (1955) 959. i2 T. N. PATTABIRAMAN AND B. K. BACHHAWAT,J. Sci. Ind. Research (India), 2oC (196o) 14. 13 O. WARBURG AND W. CHRISTIAN, Biochem. Z., 31o (1941) 334. 14 E. R. STADTMAN, in S. P. COLOWICK AND N. O. KAPLAN, Methods in Enzymology, Vol. 3, Academic Press, New York, 1957, p. 94 o. 15 F. LIPMANN AND L, C. TUTTLE, J. Biol. Chem., I59 (1945) 21. is C. S. HANES AND F. A. ISHERWOOD, Nature, 164 (1949) 11o 7. 17 R. S. ALM, R. J. P. WILLIAMS AND A. TISELIUS, Acta Chem. Scand., 6 (1952) 826.
Biochim. Biophys. Acta, 59 (1962) 68I--689
681
PURIFICATION OF GLUCOSAMINE-6-PHOSPHATE N-ACETYLASE FROM SHEEP BRAIN T. N. P A T T A B I R A M A N
AND B. K. B A C H H A W A T
Neurochemistry Laboratory, Department of Neurology and Neurosurgery, Christian Medical College and Hospital, Vellore (South India) (Received O c t o b e r 25th, 196I)
SUMMARY
The presence of the enzyme glucosamine-6-phosphate acetylase which synthesizes N-acetylglucosamine 6-phosphate from glucosamine 6-phosphate and acetyl-coenzyme A has been established in brain. The enzyme has been purified 75-fold from sheep brain. The enzyme has an optimum pH at 7.4. Free sulfhydryl groups and EDTA activated the enzyme. Km value for glucosamine 6-phosphate is 4.5" lO-4 M and for acetyl-coenzyme A is 2.5" lO-4 M. INTRODUCTION
Amino sugars are widely distributed in animal tissues. In brain these sugars are present primarily as mucopolysaccharides and gangliosidesl, ~. BRANTE and GUHA et al. have suggested that mucopolysaccharides may play an important role in the initial stages of myelination s, 4. According to BOGOCH, neuraminic acid, a main constituent of gangliosides may be the site of attack of viruses which invade the brain s. The biological importance of these macromolecules has lead to the study of the metabolism of amino sugars in recent years in various tissues and micro-organisms. Work in this laboratory has established the enzymic synthesis of GlcNH 2 6-phosphate and UDP-acetylglucosamine in brainS, e. The study of the enzymic acetylation of GlcNH~ 6-phosphate is of primary importance, since the formation of acetylated amino sugar is an obligatory intermediate in the interconversion of amino sugars. In order to establish the complete sequence of enzymic reactions leading to UDP-acetylglucosamine from the common precursor fructose 6-phosphate in brain, the presence of this acetylating enzyme is essential. The presence of this enzyme has been detected in yeast, bacteria and different animal tissuesV, 8. GlcNH z 6-phosphate acetylase has been purified from Neurospora crassa and rabbit liver by ROSEMAN et al. 7. However, ROSEMAN et al. were unable to show the existence of this enzyme in brain. The present paper establishes the presence of GlcNH z. 6-phosphate acetylase in brain. Partial purification and properties of this enzyme are also presented here. MATERIALS AND METHODS
GlcNH 2 6-phosphate was prepared enzymically by phosphorylating GIcNH~ and purified, as described previously 5. ATP and CoA (75 % pure) were obtained from A b b r e v i a t i o n : PCMB, p - c h l o r o m e r c u r i b e n z o a t e .
Biochim. Biophys. Acta, 59 (1962) 6 8 1 - 6 8 9
682
T . N . PATTABIRAMAN, B. K. BACHHAWAT
Sigma Chemical Company. Acetyl-CoA was prepared by chemical acetylation of CoA with acetic anhydride 9. Propionyl-CoA was similarly prepared using propionic anhydride. All other reagents were analytical-grade commercial chemicals. Acetate-activating enzyme system was prepared from Euglena gracilis 1°. The crude extract contained deacetylase and hence the acetate-activating enzyme was to be included in the regular assay during the purification process. The crude preparation contained the powerful deaminase which readily degraded GlcNH, 6-phosphate. To protect the substrate from degradation, it was necessary to include diammonium hydrogen phosphate in the assay system. The effective use of diammonium hydrogen phosphate in the formation of GlcNHAc 6-phosphate in the crude system is shown in Fig. I. It increased the enzymic activity markedly. The optimal concentration of diammonium hydrogen phosphate was found to be o.15 M. Higher concentration inhibited the enzymic reaction.
60
50
E E c
40
E ~- 3f o_
~ 2(1 z 10 _q 0 -
,~
2'0
3'0
.;o
50
(NH4)2 HP04, in ~lmoles Fig. I. Effect of d i a m m o n i u m h y d r o g e n p h o s p h a t e on t h e e n z y m i c f o r m a t i o n of G l c N H A c 6p h o s p h a t e . The a s s a y s y s t e m is t h e s a m e as d e s c r i b e d in t h e t e x t e x c e p t t h a t d i a m m o n i u m h y d r o g e n p h o s p h a t e of v a r y i n g c o n c e n t r a t i o n w a s a dde d.
The regular system for the assay of GlcNH2-6-phosphate acetylase during the purification process contained 0. 4/zmole of GlcNH 2 6-phosphate, 0.02/~mole of CoA, x pmole of ATP, I #mole of cysteine, 0.2/~mote of MgSO 4, 30 ~moles of acetate buffer, pH 6. 5, 3 ° #moles of diammonium hydrogen phosphate, 12o/~g of partially purified acetate-activating enzyme and 0.3-0.8 unit of the brain acetylase in a total volume of 0.2 ml. After i h incubation at 37 ° the reaction was stopped by the addition of o.x ml of 3 ° % trichloroacetic acid. After centrifuging off the precipitated protein, the supernatant fluid was neutralised with 30 % KHCO 3 and GlcNHAc 6-phosphate formed was determined. With the purified enzyme fractions deproteinisation was not found to be necessary. The assay system for the study of the properties of GlcNH 2 6-phosphate acetylase contained 0. 4/~mole of GlcNH~ 6-phosphate, o.15/~mole of acetyl-CoA, 4 °/zmoles of Tris buffer, pH 7-4, and 47.2/~g of the purified enzyme and additions as indicated Biochim. Biophys. Acta, 56) (tq6z) 681-68q
GLUCOSAMINE 6-PHOSPHATEN-AcETYLASE FROM BRAIN
683
in the individual cases, in a total volume of 0.2 ml. After I h incubation at 37 ° the reaction was stopped b y the addition of o.16 M sodium tetraborate and GlcNHAc 6-phosphate formed was determined. GlcNHAc 6-phosphate was assayed b y the method of REISSIG et al. ll with a slight modification TM. Protein was measured spectrophotometrically at 280 and 26o mt~ with a correction for nucleic acid content 13. The concentration of ammonium sulphate is expressed in all cases as g/ml, the saturation at o ° being 0.526 g/ml. The quantitative assay of free sulfhydryl groups was done spectrophotometrically based on the method of BOYER14. Acetyl-CoA was measured as hydroxamate by the method of LIPMANN AND TUTTLE is.
One unit of enzyme is equivalent to IOO mt~moles of GlcNHAc 6-phosphate formed in i h.
Purification of enzyme from sheep brain All operations were conducted between o - 5 °, unless indicated otherwise. Extraction: Sheep brain, obtained immediately after the death of the animal, was chilled in ice, trimmed of white matter and stored at - - 1 8 °. 3oo g of the frozen tissue were homogenised with 6oo ml of o.o3 M Tris buffer, p H 7.4, in a Waring blender for 45 sec. The mixture was magnetically stirred for 15 min and centrifuged at ioooo × g for 2o min in a refrigerated centrifuge. The reddish cloudy supernatant contained 6.8 g of protein in a volume of 49 ° m l . First ammonium sulphate fractionation: The supernatant fluid was brought to 45 % saturation with solid ammonium sulphate b y the slow addition of 131.5 g of the salt and the mixture was magnetically stirred for 2o min. After centrifugation at IOOOO × g for 2o rain the supernatant fluid was collected and brought to 7 ° % saturation b y the addition of 83.8 g of solid ammonium sulphate. The precipitate was collected b y centrifugation and dissolved in 5o ml of o.o3 M Tris buffer, p H 7.4, containing o.ooi M cysteine. The solution was dialysed against 5o times the volume of o.oi M Tris buffer, p H 7.4, for 3 h, with the change of the dialysing solution after an interval of 9 ° min. The reddish clear solution contained 1.77 g of protein in a volume of 75 ml. Gel treatment: To the dialysed solution were added 123.9 ml of calcium phosphate gel (IO mg/ml) bringing the ratio of protein to gel to 1:o.7. The mixture was magnetically stirred for IO min and centrifuged at 4ooo × g for IO min. The gel supernatant contained 1.265 g of protein in a volume of 175 ml. Second ammonium sulphate fractionation: The supernatant fluid was brought to 48 % saturation with solid ammonium sulphate b y the addition of 5o.5 g of the salt and the mixture was magnetically stirred for 2o min. After centrifugation at IO ooo × g for 2o min the supernatant fluid was collected and brought to 7 ° % saturation by the addition of 26. 9 g of solid ammonium sulphate. The precipitate was collected b y centrifugation and dissolved in 12 ml of o.o3 M "Iris buffer, p H 7-4, containing o.ooi M cysteine. The solution was dialysed against 5o times the volume of o.oi M Tris buffer, p H 7.4, for 3 h with the change of the dialysing solution after an interval of 9 ° min. The pale-reddish clear solution contained o.676 g of protein in a volume of 26 ml. Biockim. Biophys. Acta, 59 (1962) 681-689
684
T. N. PATTABIRAMAN, B. K. BACHHAWAT
Heat treatment: The dialysed solution was adjusted to p H 5 with I N acetic acid and heat treated in small fractions of 2 ml each. The solution was immersed in a water bath at 9 ° o, when its temperature reached 65°, it was transferred to a water bath maintained at 65 ° and held at this temperature exactly for 2 rain. I t was cooled rapidly in crushed ice and centrifuged at 2o ooo × g for 2o min. The precipitate was discarded. The supernatant fluid was collected. The colorless clear solution contained 53-4 mg of protein in a volume of 23 ml. RESULTS
The s u m m a r y of the purification process is shown in Table I. A 75-fold purification was obtained by using the above procedure with an overall recovery of 59 % of the original activity. The purified enzyme was found to be free of acetate-activating enzyme and deacetylase. But it contained traces of GlcNH~-6-phosphate deaminase activity. TABLE I PURIFICATION OF GLUCOSAMINE-6-PHOSPHATE N-ACETYLASE Steps Extraction First a m m o n i u m s u l p h a t e fractionation Gel t r e a t m e n t Second a m m o n i u m s u l p h a t e fractionation Heat treatment
S p e ~ activity Total protein (units/ragprotein)
Yield (%)
Volume (~)
Total units
49o
294
6.81 g
o.o43
ioo
75 175
315 280
1.77 g 1.26 g
o.178 o.221
lO7 95.2
26 23
224 174
676 m g 53.4 m g
o.332 3.25
76. 4 59.1
Optimal p H GlcNH,-6-phosphate acetylase from sheep brain has a sharp optimal p H of 7-4 as shown in Fig. 2. This was different from that of N. crassa enzyme v which was found to have a flat optimal range from p H 6-7.1.
Substrate specificity The purified enzyme system exhibited no acetylating activity with GlcNH 2 or GalNH v Acetyl-CoA could be replaced neither by AMP-acetate nor by AMP-acetate and CoA together, This eliminates the possibility of chemical formation of acetyl-CoA in the system. In the presence of acetate-activating enzyme system AMP-acetate and CoA could replace acetyl-CoA. Though propionyl-CoA could transfer the propionyl moiety to the amino group of GlcNH 2 6-phosphate, the reaction was found to be very feeble. With equimolar amounts of propionyl-CoA, the activity was only 15 % compared to that of acetyl-CoA.
Effect of sulfhydryl reagents I t has been reported by ROSEMAN and his associates that the purified enzyme from N. crassa displayed no cofactor requirement 7. Complete inhibition with that enzyme was found at lO -8 M concentration of PCMB. In the case of sheep-brain enzyme PCMB at a concentration of lO -5 M completely inhibited the activity and Biochim. Biophys. Acta, 59 (1962) 68I--689
GLUCOSAMINE 6-PHOSPHATE N-ACETYLASE FROM BRAIN
685
at a concentration of IO-~ M inhibited nearly 50 % of the activity. This inactivation was found to be reversed by cysteine showing that the sheep-brain enzyme requires free sulfhydryl groups for maximal activity and is highly susceptible to sulfhydryl reagents. Cysteine was found to increase the activity of GlcNH2-6-phosphate acetylase from sheep brain (Table n).
55
"5 50
E :L E .c_ 45
i
z
u
3¢
i
L
L
i
5
6
7
8
9
pH Fig. 2. Effect of H + c o n c e n t r a t i o n on t h e e n z y m e reaction. T h e a s s a y s y s t e m is t h e s a m e as described in t h e t e x t e x c e p t t h a t 4 ° # m o l e s of buffer of different p H were included. Q - - O , p h o s p h a t e ; O - - O , Tris buffers. T A B L E II EP'FECT OF CYSTEINE AND SULFHYDRYL REAGENTS T h e e n z y m e (47.2 pg) w a s p r e i n c u b a t e d w i t h p - c h l o r o m e r c u r i b e n z o a t e for io rain a t 25 °. I n t h e control t u b e t h e e n z y m e w a s s i m i l a r l y p r e i n c u b a t e d w i t h o u t t h e a d d i t i o n of s u l f h y d r y l r e a g e n t . A f t e r t h e p r e i n c u b a t i o n t h e s u b s t r a t e s were a d d e d a n d i n c u b a t e d for I h a t 37 °. T h e ~ a e t i o n w a s s t o p p e d b y t h e a d d i t i o n of s o d i u m t e t r a b o r a t e solution. I n t h e e x p e r i m e n t s w i t h cysteine. I p m o l e of c y s t e i n e w a s a d d e d a l o n g w i t h t h e s u b s t r a t e a f t e r t h e p r e i n c u b a t i o n of t h e e n z y m e with p-chloromercuribenzoate. Ezpt. No.
GI~NHAc 6-pb.ospkat~ for,m~ (pt,.ole)
Additio~
None Plus cysteine P l u s i . xo -6 M p - c h l o r o m e r c u r i b e n z o a t e P l u s x. IO-~ M p - c h l o r o m e r c u r i b e n z o a t e and eysteine P l u s I" lO -6 M p - c h l o r o m e r c u r i b e n z o a t e P l u s x- IO-6 M p - c h l o r o m e r c u r i b e n z o a t e and cysteine
0.054 o.o7I o o.o525 o.o 3 o.o54
Effect of E D T A and metal ions N. crassa enzyme was not found to be inhibited by EDTA ~, whereas EDTA was found to activate GlcNH ,-6-phosphate acetylase from sheep brain markedly. To prove that this activation is not even partially due to the inhibition of GlcNH2-6-phosphate deaminase 5, the effect of EDTA was studied at pH 6 where the activity of deaminase Biochim. Biophys. Acta, 59 (i962) 6 8 1 - 6 8 9
686
T. N. PATTABIRAMAN, B. K. BACHHAWAT
was found to be negligible. Under this condition of assay EDTA activated GlcNH,6-phosphate acetylase to the same extent. This enhancing effect of EDTA may be due to the protection of the sulfhydryl groups of the enzyme from heavy metals. Mg 2+ or Mn *+ had a slight but consistent enhancing effect on the enzymic activity. Co s+ had no effect, whereas Ni 2+ inhibited the enzyme (Table III). TABLE EFFECT OF E D T A
III A N D M E T A L IONS
T h e assay system w a s the s a m e as described in the text except that additions were m a d e as indicated. For Expts. 7 and 8, 4 °/*moles of phosphate buffer, p H 6, were used. Exit. No.
I 2 3 4 5 6 7 8
GlcNHA c 6-phosphate formtd (l~mole)
Addition
None Plus x /,mole E D T A Plus o . 1 / , m o l e MgSO 4 Plus o . I / , m o l e MnSO 4 Plus o . I / , m o l e CoCI2 Plus o. I / , m o l e NiCI s None (at p H 6) Plus I / * m o l e E D T A (at p H 6)
0.054 0.096 0.059 o.o6o o.o53 0.039 0.04o o.07o
E f f e a of substrate concentration
Enzymic activity with increasing concentration of GlcNH~ 6-phosphate and acetyl-CoA is presented in Figs. 3 and 4 respectively. The calculated Km for GlcNH 2 6-phosphate is 4-5" lO-4 M and for acetyl-CoA 2.5" lO -4 M. These values were lower compared to the Km values for N. crassa enzyme ~where for both GlcNH 2 6-phosphate and acetyl-CoA the Km was 7.8" lO -4 M. The optimal concentration of acetyl-CoA was found to be 3" IO-S M. Higher concentration of acetyl-CoA inhibited the enzymic activity markedly. A similar observation has been made earlier by ROSEMAN and his associates with the N. crassa enzyme7.
e.
12
120~
10
a
O0 ~. ._c eo~
S/v
E 40 u <
$
v
;
i
3.
.
4
.
5.
.
6
7
8
S {GIc NH2 (5- P concentration) x 10"1M Fig. 3. Effect of G I c N H z 6 - p h o s p h a t e concentration on t h e reaction velocity of the enzyme. The a s s a y s y s t e m w a s t h e s a m e as described in the t e x t except t h a t x / , m o l e of E D T A and GlcNH 2 6 - p h o s p h a t e of v a r y i n g concentration were included. B i o c h i m . B i o p h y s . A c t a , 59 (I962) 681-689
GLUCOSAMINE 6~PHOSPHATE N - A C E T Y L A S E FROM BRAIN
687
Stoichiometry of the reaction Stoichiometry of the reaction is given in Table IV. Equimolar quantities of GlcNHAc 6-phosphate and CoA were formed corresponding to the amount of acetylCoA disappeared.
Stability of the enzyme The enzyme lost 50 % of its activity when stored in the frozen state for two weeks at: tV
"6 E 125
t::
s/v 4
I00 E
3
75 a.t
2
50
I
u
b
;
,o
,;
~
2;
~o ~ 20 4~.... 60~'"
S ( A c CoA concentration) x 10"4H
Fig. 4. Effect of acetyl-CoA c o n c e n t r a t i o n on t h e r e a c t i o n velocity of t h e e n z y m e . T h e a s s a y s y s t e m w a s t h e s a m e as described in t h e t e x t e x c e p t t h a t i / , m o l e of E D T A a n d a c e t y l - C o A of v a r y i n g c o n c e n t r a t i o n were included. T A B L E IV $TOICHIOMETRY OF THE REACTION X /*mole of G l c N H z 6 - p h o s p h a t e , o . 8 / , m o l e of acetyl-CoA, IOO/,moles of Tris, p H 7-4, 0 . 5 / * m o l e of E D T A a n d 136/*g of e n z y m e were i n c u b a t e d for I h a t 37 °. A n a l i q u o t (o.x ml) w a s t a k e n for G l c N H A c 6 - p h o s p h a t e assay, o.5 m l w a s t a k e n for t h e d e t e r m i n a t i o n of free s u l f h y d r y l g r o u p s a p p e a r e d , o.3 m l w a s t a k e n for t h e a s s a y of r e m a i n i n g acetyl-CoA. A b l a n k w a s r u n s i m u l t a n e o u s l y in w h i c h GlcNH~ 6 - p h o s p h a t e w a s a d d e d a f t e r t h e i n c u b a t i o n w a s o v e r a n d t h e d i s a p p e a r a n c e of acetyl-CoA a n d f o r m a t i o n of CoA were d e t e r m i n e d t h e r e also. GlcNHAc 6-pkospkate formed (Umote)
0.44
Acetyl*CoA di.~ppeamd ( Umole)
CoA formed ( Umote)
0.446
0.423
Identification of product as GlcNHAc 6-phosphate IO /zmoles of GlcNH~ 6-phosphate, 5 Izmoles of acetyl-CoA, 2 mmoles of Tris buffer, p H 7.4, IO/~moles of EDTA and 94 °/~g of the purified enzyme in a total volume of 4 ml were incubated for I h at 37 °. The reaction was stopped b y keeping the tubes in a boiling-water b a t h for 2 min. The precipitated protein was centrifuged off. The supernate was passed through Dowex-I in C1- form. N-acetylhexosamine 6-phosphate could be eluted with o.i N HC1 according to the method of ALM et al. 1~. The eluted sugar was again passed through Dowex-5o in H + form and acetylhexosamine phosphate was eluted with water and lyophilized. 3.2 t~moles of N-acetylhexosamine phosphate was formed. Biochim. Biophys. Acta, 59 (1962) 6 8 I ~ 8 9
688
T. N. PATTABIRAMAN, B. K. BACI-IHAWAT
The isolated product was found to enhance the activity of GlcNH2-6-phosphate deaminase to the same extent as an authentic sample of GlcNHAc 6-phosphate when used in equimolar amount 5. The product was further identified b y paper chromatography. 25/zg of the product isolated, was plotted on W h a t m a n 3MM paper and irrigated in the basic water-miscible solvent system of HANES AND ISHERWOODle (propanol-saturated amm o n i a - w a t e r (6:3: I)) and ascending chromatography was performed for 16 h at room temperature. The paper was then dried and sprayed with o.16 M sodium tetraborate solution to ensure complete wetting. The paper was kept in an air oven, maintained at, IOO° for I0 min after which Ehrlich reagent (IO g of p-dimethylaminobenzaidehyde in 9 ° ml of acetic acid and io ml Of io N HC1) was sprayed. A purple spot was formed immediately. The test sample was found to have the same RF value as an authentic sample of GlcNHAc 6-phosphate. The RF value for GlcNHAc 6-phosphate was found to be 0.36. DISCUSSION
Although ROSEMAN a a/. were unable to detect GlcNH~-6-phosphate acetylase in brain *, this work establishes the presence of this enzyme in brain. It is probable that the detection of this enzyme was not possible earlier because of the presence of powerful glucosamine-6-phosphate deaminase in brain and also due to the lower concentration of this enzyme in this tissue. By reversing the deaminase action with the help of diammonium hydrogen phosphate, the presence of this acetylase could be detected. Although ROSEMAN a al. have purified the acetylase from animal tissues so far there had been no studies reported on the properties of this enzyme from animal tissues 7. The present work describes in detail the properties of this acetylase which differs markedly from that of the enzyme from N . crassa ~. Considering the earlier reports from this laboratory on the enzyme related to the amino sugar metabolism, the presence of this acetylase bridges the gap between hexose 6-phosphate and UDP-acetylglucosamine. It m a y be noted that all the enzymes which are known to involve in the biosynthesis of amino sugars are present in brain. On the basis of these facts an outline of the enzymic sequence in the synthesis of amino sugars in brain m a y be given as follows. Fructose 6-phosphate + N H 8 ~
DeamJnase
" Glucosamine 6-phosphate5
Glucosamine 6-phosphate + Acetyl-CoA Acetylase ) N-acetylglueosamine 6-phosphate + CoA N-acetylglucosamine 5-phosphate *
Mutase
* N-acetylglucosamine 1-phosphate
N-acetylglucosamine I-phosphate + U T P +.Pyrophosphoryla~e UDpo acetylglucosamine + pyrophosphate e
All of the above enzymes have been found to be present in brain except mutase. Preliminary investigation in this laboratory indicates that there is an enzyme in brain which participates in the interconversion of N-acetylglucosamine 6-phosphate and N-acetylghicosamine 1-phosphate. I t remains to be determined whether this is a specific mutase for the acetylamino sugar phosphate or a non-specific phosphoglucomutase. Biochim. Biophys. Acta, 59 (1962) 681-689
GLUCOSAMINE 6-PHOSPHATE N-ACETYLASE FROM BRAIN
689
ACKNOWLEDGEMENTS
The keen interest and encouragement of Professor J. CHANDYare deeply appreciated. The first author is a Raptokas Medical Research Fellow. This investigation was supported by a grant from the National Multiple Sclerosis Society (U.S.A.), Grant No. 214-2. REFERENCES 1 G. BRANTE, in D. RICHTER, Metabolism of the Nervous System, P e r g a m o n Press, London, 1957, p. 112. 2 S. BOGOCH, Neurology, Io (196o) 439. 3 G. BRANTE, in F. BRUCKE, Biochemistry of the Central Nervous System, P e r g a m o n Press, London, 1959, p. 291. 4 A. GOHA, B. J. NORTHOVERANY B. K. BACHHHAWAT,J. Sci. Ind. Research (India), I 9 C (196o) 287. 5 T. N. PATTABIRAMAN AND B. K. BACHHAWAT,Biochim. Biophys. Aaa, 54 (1961) 2736 T. N. PATTABIRAMAN AND B. K. BACHHHAWAT,Biochim. Biophys. Acta, 5o (I96I) 129. 7 E. A. DAVIDSON,H. J. BLUMENTHAL AND S. ROSEMAN,J. Biol. Chem., 226 (1957) 125. s D. BROWN, Biochim. Biophys. Acta, 16 (1955) 429. 9 S. OCHOA, in S. P. COLOWICK AND N. O. KAPLAN, Methods in Enzymology, Vol. I, Academic Press, New York, 1955, p. 685. 10 A. ABRAHAM AND g . K. BACHHAWAT,Biochim. Biophys. Acta, in t h e press. xI j . I. REISSIG, J. L. STROMINGER AND L. F. LELOIR, J. Biol. Chem., 217 (1955) 959. i2 T. N. PATTABIRAMAN AND B. K. BACHHAWAT,J. Sci. Ind. Research (India), 2oC (196o) 14. 13 O. WARBURG AND W. CHRISTIAN, Biochem. Z., 31o (1941) 334. 14 E. R. STADTMAN, in S. P. COLOWICK AND N. O. KAPLAN, Methods in Enzymology, Vol. 3, Academic Press, New York, 1957, p. 94 o. 15 F. LIPMANN AND L, C. TUTTLE, J. Biol. Chem., I59 (1945) 21. is C. S. HANES AND F. A. ISHERWOOD, Nature, 164 (1949) 11o 7. 17 R. S. ALM, R. J. P. WILLIAMS AND A. TISELIUS, Acta Chem. Scand., 6 (1952) 826.
Biochim. Biophys. Acta, 59 (1962) 68I--689