- Email: [email protected]
Preparation and properties of uridinediphosphoglucose-glycogen transferase from rabbit muscle
556
SHORT CO~t~t~NICATIONS
VOL. 33 (1959)
from J.C. had no detectable UDPG-glycogen transferase. The muscle from a case of limit dextrinosis (W.) had the highest phosphorylase content, as well as the highest UDPG-glycogen transferase activity of any tissue examined. These high levels of activity may in part be due to the somewhat dehydrated condition of the tissue. One case of general glycogenosis (P.F.) showed some activity of the transferase in the skeletal muscle but no detectable activity in the heart muscle, although both these tissues had glycogen contents of II %. In another case (C.D.) transferase activity was demonstrated in the heart. Although it has been possible to analyze the tissues from only four cases of general glycogenosis for UDPG-glycogen transferase activity, it appears reasonable to conclude that the activity of this enzyme, since it is not associated with low phosphorylase activity, does not account for the generalized deposition of glycogen in this syndrome. In another paper 6 the preparation and properties of UDPG-glycogen transferase from rabbit skeletal muscle are reported. ROSALIND H A U K *
Department o/ Biological Chemistry, Washington University Medical School, Saint Louis, Mo. (U.S.A.)
BARBARA ILLINGWORTH DAVID H . B R O W N CARL F . CORI
1 G. T. CORI, Mod. Prob. in Ped., 3 (1957) 344. 2 L. F. LELOIR AND C. E. CARDINI, J. Am. Chem. Soc., 79 (1957) 6340. 3 G. T. CORI AND B. ILLINGWORTH, Biochim. Biophys. Acta, 21 (1956) lO 5. 4 j . L. STROMINGER, E. S. MAXWELL, J. AXELROD AND H. M. KALCKAR, J. Biol. Chem., 224 (1957) 79. D. H. BROWN, Proc. Natl. Acad. Sei. U.S., 43 (1957) 783 • 6 R. HAUK AND D. H. BROWN, Biochim. Biophys. Acta, 33 (1959) 556.
Received November 6th, 1958 • N a t i o n a l Science F o u n d a t i o n P r e d o c t o r a l Fellow.
Preparation and properties of uridinediphosphoglucose-glycogen transferase from rabbit muscle In the preceding paper 1, an enzyme has been described in human skeletal and heart muscle which, like the rat-liver enzyme of L E L O I R AND CARDINI i, catalyzes the disappearance of UDPG in the presence of glycogen. In order to provide a basis for the study of this enzyme from human tissues, a similar enzyme has been purified from extracts of rabbit skeletal muscle and its properties have been studied. It may be mentioned that neither phosphorylase a nor b can catalyze any reaction between UDPG and glycogen. When a homogenate of freshly excised rabbit muscle, prepared at high speed in the Virtis homogenizer, was centrifuged at 78,000 × g, most of the transferase activity remained in the sediment. Efforts to render this activity soluble were usually unsuccessful. When the muscle was frozen preliminarily and then homogenized as above, the enzymic activity was largely soluble following centrifugation for I h at 78,000 x g. Accordingly, this latter procedure was followed in all subsequent work. A b b r e v i a t i o n s : U D P , u r i d i n e d i p h o s p h a t e ; U D P G , u r i d i n e d i p h o s p h o g l u c o s e ; Tris, tris(hydroxymethyl)aminomethane; EDTA, ethylenediaminetetraacetate.
VOL. 33 (1959)
SHORT COMMUNICATIONS
557
IOO g of frozen muscle were minced w i t h scissors into 4 vol. cold* o . I M Tris - o . o o i M E D T A , p H 8.o, chilled in ice, a n d t r e a t e d for 3 min a t t o p speed in t h e Virtis homogenizer. The h o m o g e n a t e was centrifuged for 15 m i n at io,ooo × g a n d the s u p e r n a t a n t fluid t h e n centrifuged again for I h at 78,000 × g. To t h e s u p e r n a t a n t fluid, (NH4)2SO 4, s a t u r a t e d a t 5 ° a n d a d j u s t e d w i t h N H 3 to p H 7.1 (measured at 0.25 satn. w i t h a B e c k m a n p H m e t e r set at IO°), was a d d e d to 0.40 satn. The prec i p i t a t e was r e m o v e d b y centfifugation a n d dissolved in cold distilled water. The solution was d i a l y z e d against o . i M T r i s - o . o o I M E D T A , p H 7.2 for 3 h a n d then frozen. A f t e r t h a w i n g t h e solution, a p r e c i p i t a t e was r e m o v e d b y centrifugation. To the s u p e r n a t a n t fluid 0.o3 ml of Ca3(PO4)~ gel (18 m g solids/ml) was a d d e d per m g of t o t a l p r o t e i n as d e t e r m i n e d b y the m e t h o d of ROBINSON AND HOGI)EN 3. The gel was e x t r a c t e d twice using each time a volume of o.Io satd. (NH~)2SO4-o.ooI M E D T A , p H 7.2, which was equal to t h a t of the original solution which h a d been t r e a t e d w i t h t h e gel. The enzyme was e l u t e d from the gel with 0.2 M K p h o s p h a t e - o . o o I M E D T A , p H 7.2. Two elutions were m a d e , using each time a volume of eluent equal to one-half t h e v o l u m e of the original solution from which t h e enzyme h a d been adsorbed. The two eluates were pooled a n d d i a l y z e d for 2 h against o.I M T r i s - o . o o I M E D T A , p H 7.2. The enzyme lost m o s t of its a c t i v i t y when this d i a l y z e d solution was frozen. Accordingly, satd. (NH4)2S04, p H 7.1 (as above), was a d d e d to 0.40 satn,, a n d t h e m i x t u r e which c o n t a i n e d the p r e c i p i t a t e d enzyme was k e p t at 5 ° . I t was stable for a t least 8 weeks. T a b l e I gives the results of such a fractionation. TABLE I PURIFICATION OF U D P G - G L Y C O G E N TRANSFERASE Fraction
78,0OO × g s u p e r n a t a n t
0.40 satd. (NH4)~SO 4 fraction after dialysis and freezing Gel eluate after dialysis 0.4o satd. (NH4)2SO a fraction
Total vol. (ml)
Total protein (rag)
325
357 o
31 31 15
Total activity (units)*
338 24.7 24.7
Speci~c activity (units[rag protein)
494
o.14
273 87.4** 84.5
o.81 3.5*** 3.4
Yield % ioo
55 17-7 17§
* The standard assay system contained : UDPG, 1-3" I o-3 M ; glycogen, 4 mg; MgC12, 3" i o-Z M; cysteine, 7.5" IO-8 M; Tris, 5.7" lO-2 M, and enzyme in a final vol. of o. 4 ml; pH 7.2. Incubation was for 15 rain at 3o°. One unit of activity is the number of #moles of UDPG which disappeared under these conditions. ** When this fraction was assayed in the absence of added glycogen, its total activity was only o.38 unit. *** In other preparations, this fraction has had a specific activity of from 2.4-7.1 units/mg protein and the overall yield has varied from 17-44 %. § The ratio of total phosphorylase activity (a + b) to that of the transferase in this fraction was 57:1. The e n z y m i c a s s a y u s u a l l y used d e p e n d e d u p o n a m e a s u r e m e n t of t h e disa p p e a r a n c e of U D P G via the U D P G d e h y d r o g e n a s e s y s t e m 4. F r e q u e n t l y , t h e formation of U D P also was m e a s u r e d via the p y r u v a t e kinase-lactic d e h y d r o g e n a s e system, b a s e d o n K O R N B E R G ' S o b s e r v a t i o n 5 t h a t U D P is active w i t h p y r u v a t e kinase. CABIB AND LELOIRe h a v e also d e v e l o p e d a U D P a s s a y b a s e d on this fact. Thus, when a r e a c t i o n m i x t u r e containing i n i t i a l l y 0.52/zmole U D P G a n d 4 m g glycogen in 0. 4 m l A l l o p e r a t i o n s w e r e c a r r i e d o u t i n t h e c o l d r o o m a t 5 °.
558
SHORT COMMUNICATIONS
VOL. 3 3
(I959)
Tris, pH 7.2, was incubated with 86/*g enzyme for 15 min at 3 o°, 0.20/*mole UDPG disappeared and 0.20/*mole UDP was formed. Assay of all purified fractions of the enzyme invariably showed perfect stoichiometry between UDPG disappearance and UDP formation. The activity of the most highly purified enzyme was wholly dependent on the addition of glycogen (Table I). That the glycogen served as a primer was shown in experiments in which the structure of the product of enzyme action was studied. In one case, I mg human-liver glycogen and 7/*moles UDPG were incubated with 41o/*g enzyme in a vol. of I.O ml for 69 min under the conditions of the standard assay (Table I). Measurement of UDPG in an aliquot of the reaction mixture showed that 3.08/*moles had disappeared in the whole solution. The glycogen was isolated from the remaining part of the reaction mixture following digestion for I h at IOO° in 4 N NaOH and two precipitations from 50% ethanol. Analysis for total glucose following hydrolysis of the glycogen in HC1 showed that the polysaccharide contained 3.16/,moles more glucose than a control sample which had been incubated with the enzyme in the absence of UDPG. Determination of the outer chain length of the two glycogen samples was done using phosphorylase free of amylo-i,6-glucosidase*. The glycogen which had been incubated with UDPG had 3.02/*moles more glucose units in linkage susceptible to cleavage by phosphorylase than the control sample of glycogen. These findings prove that the UDPG-glycogen transferase adds glucose units in a-I, 4 linkage to the terminal units of preexisting chains in glycogen. It is interesting that in this transferase reaction, no net inversion of glucose configuration occurs. It was found that neither amyloheptaose nor a high molecular weight, soluble, mixed cellodextrin fraction** acted as a primer in this reaction. Although there are similarities between UDPG-glycogen transferase and phosphorylase as shown by the primer requirement and the structure of the polysaccharide chain formed, the enzymes are distinct as shown by their separation in the fractionation procedure used. Thus, the ratio of total phosphorylase activity (a + b) to that of the transferase (calculated as moles of substrate reacting) changed from at least 400 :I in the soluble fraction of a muscle homogenate to 178 :I in the 0.40 satd. (NH4)2SO 4 fraction, and, finally, to 23 :I in the final gel eluate in the case of a preparation not shown in Table I. UDPG-glycogen transferase was inactive when cysteine was omitted from the assay system. The enzyme does not require a bivalent metal ion but is stimulated 2-fold by 6- lO -3 M MgC12. It is not inhibited by a-glucose-l-phosphate (1.5" lO -3 M). The enzyme is inhibited by various nucleotides. Thus, uridine-5'-phosphate competitively inhibits the rate of the reaction by about 50 % when it is present in the usual assay system at a concentration equal to that of the UDPG added. Inosine-5'-phosphate, cytidine-5'-phosphate, and guanosine-5'-phosphate each inhibits to the extent of about 20 % under similar conditions. Adenosine-5'-phosphate also inhibits but to a lesser extent. These inhibitions differentiate the transferase from phosphorylase. On the other hand, phlorizin at 2. lO -3 M inhibits UDPG-glycogen transferase to the extent of 9° %, as it does phosphorylase 7. The specificity of the transferase for UDPG was shown in an experiment in which a sample of uridine]diphosphogalactose containing 3 % UDPG was found to react only * Kindly prepared by Dr. BARBARAILLINGWORTH. ** K i n d l y provided b y Dr. LuIS GLASER.
VOL. 33 (1959)
SHORT COMMUNICATIONS
559
t o t h e e x t e n t of its U D P G c o n t e n t . U r i d i n e d i p h o s p h o - N - a c e t y l g l u c o s a r n i n e y i e l d e d no U D P w h e n i n c u b a t e d w i t h t h e t r a n s f e r a s e a n d g l y c o g e n . T h e a p p a r e n t Michaelis c o n s t a n t of U D P G in t h e t r a n s f e r a s e r e a c t i o n is i . i - l O -3 M . T h i s w a s d e t e r m i n e d in t h e p r e s e n c e of 3" lO -3 M Mg ++ a n d u n d e r c o n d i t i o n s w h e r e t h e r a t e of t h e r e a c t i o n w a s linear. T h e p h y s i o l o g i c a l significance of U D P G - g l y c o g e n t r a n s f e r a s e r e q u i r e s further study. T h i s w o r k has b e e n s u p p o r t e d in p a r t b y a g r a n t ( R G - 4 7 6 I ) f r o m t h e N a t i o n a l I n s t i t u t e s of H e a l t h , U.S. P u b l i c H e a l t h Service.
Department o/Biological Chemistry, Washington University Medical School, Saint Louis, Mo. (U.S.A.)
ROSALIND HAUK* DAVID H . BROWN
1 R. HAUK, B. ILLINGWORTH,D. H. BROWN AND C. F. CORI, Biochim. Biophys. Acta, 33 (1959) 554. 2 L. F. LELOIR AND C. E. CARDINI, J. Am. Chem. Soc., 79 (1957) 6340. 3 H. W. ROBINSON AND C. G. HOGDEN, J. Biol. Chem., 135 (194o) 7o 7. 4 j. L. STROMINGER, E. S. MAXWELL, J. AXELROD AND H. M. KALCKAR, J. Biol. Chem., 224 (1957) 79. 5 A. KORNBERG, in W. D. MCELROY ANn B. GLASS, Phosphorus Metabolism, Vol. I, The Johns Hopkins Press, Baltimore, 1951, p. 392. s E. CABIB AND L. F. LELOIR, J. Biol. Chem., 231 (1958) 259. 7 C. F. CORI, G. T. CORI AND A. A. GREEN, J. Biol. Chem., 151 (1943) 39. R e c e i v e d N o v e m b e r 6th, 1958 * National Science Foundation Predoctoral Fellow.
The calculation of the constant for differential manometers A l t h o u g h n o w a d a y s n o t so w i d e l y u s e d as t h e c o n s t a n t - v o l u m e ( W a r b u r g ) m a n o m e t e r , t h e d i f f e r e n t i a l m a n o m e t e r has a n u m b e r of a d v a n t a g e s , in p a r t i c u l a r t h e i n d e p e n d e n c e of t h e r e a d i n g f r o m c h a n g e s of t h e a t m o s p h e r i c pressure, t h e s m a l l e r effect of c h a n g e s of t e m p e r a t u r e a n d t h e g r e a t e r a c c u r a c y of r e a d i n g t h e m a n o m e t e r . Since, h o w e v e r , t h e r e are c h a n g e s in b o t h t h e v o l u m e a n d t h e p r e s s u r e d u r i n g t h e course of t h e r e a c t i o n i n v o l v i n g an a b s o r p t i o n or an e v o l u t i o n of gas, t h e r e l a t i o n s h i p b e t w e e n t h e m a n o m e t e r r e a d i n g a n d t h e a m o u n t of gas a b s o r b e d or e v o l v e d is r a t h e r c o m p l i c a t e d . DIXON 1 has d e r i v e d t h e e x p r e s s i o n g i v e n in e q u a t i o n (i)*
• = h coso+ ~ ' ~ ( P - p + ~ P ' I -vG 7 "--y~ ~_ v~a --i
~'~.j
Po
~ 273
~
+ ~ " --T-
Po
=hk li)
w h e r e k is t h e m a n o m e t e r c o n s t a n t . T h i s e x p r e s s i o n for t h e m a n o m e t e r c o n s t a n t is n o t i m m e d i a t e l y u s a b l e since it i n c l u d e s A P a n d A P ' , w h i c h c a n n o t be d i r e c t l y r e a d off t h e m a n o m e t e r . M o r e o v e r , • The symbols are the same as those used by Dixon 1. x mm 8 = vol. of gas evolved or absorbed (N.T.P.) ; h mm = manometer reading ; re', va mm 3 = vol. of gas space in left-hand (compensation) and right-hand (reaction) vessels, respectively ; vv ~, vF mm z = v o l . of liquid in left-hand and right-hand vessels respectively; T °, T8 ° = absolute temperature of reaction vessel (bath temperature) and of manometer tube (room temperature) respectively; P, Po, P m m manometer fluid = initial pressure in vessel, standard pressure (760 mm Hg) and vapour pressure of water at T3 ° respectively; LIP', AP mm manometer fluid = increase of pressure in left-hand and righthand vessels, respectively; a m m 3 gas (N.T.P.)/mm a liquid = solubility of gas in reaction solution; 0 ° = angle of manometer scale with vertical; A mm s = area of cross-section of manometer £ube.
SHORT CO~t~t~NICATIONS
VOL. 33 (1959)
from J.C. had no detectable UDPG-glycogen transferase. The muscle from a case of limit dextrinosis (W.) had the highest phosphorylase content, as well as the highest UDPG-glycogen transferase activity of any tissue examined. These high levels of activity may in part be due to the somewhat dehydrated condition of the tissue. One case of general glycogenosis (P.F.) showed some activity of the transferase in the skeletal muscle but no detectable activity in the heart muscle, although both these tissues had glycogen contents of II %. In another case (C.D.) transferase activity was demonstrated in the heart. Although it has been possible to analyze the tissues from only four cases of general glycogenosis for UDPG-glycogen transferase activity, it appears reasonable to conclude that the activity of this enzyme, since it is not associated with low phosphorylase activity, does not account for the generalized deposition of glycogen in this syndrome. In another paper 6 the preparation and properties of UDPG-glycogen transferase from rabbit skeletal muscle are reported. ROSALIND H A U K *
Department o/ Biological Chemistry, Washington University Medical School, Saint Louis, Mo. (U.S.A.)
BARBARA ILLINGWORTH DAVID H . B R O W N CARL F . CORI
1 G. T. CORI, Mod. Prob. in Ped., 3 (1957) 344. 2 L. F. LELOIR AND C. E. CARDINI, J. Am. Chem. Soc., 79 (1957) 6340. 3 G. T. CORI AND B. ILLINGWORTH, Biochim. Biophys. Acta, 21 (1956) lO 5. 4 j . L. STROMINGER, E. S. MAXWELL, J. AXELROD AND H. M. KALCKAR, J. Biol. Chem., 224 (1957) 79. D. H. BROWN, Proc. Natl. Acad. Sei. U.S., 43 (1957) 783 • 6 R. HAUK AND D. H. BROWN, Biochim. Biophys. Acta, 33 (1959) 556.
Received November 6th, 1958 • N a t i o n a l Science F o u n d a t i o n P r e d o c t o r a l Fellow.
Preparation and properties of uridinediphosphoglucose-glycogen transferase from rabbit muscle In the preceding paper 1, an enzyme has been described in human skeletal and heart muscle which, like the rat-liver enzyme of L E L O I R AND CARDINI i, catalyzes the disappearance of UDPG in the presence of glycogen. In order to provide a basis for the study of this enzyme from human tissues, a similar enzyme has been purified from extracts of rabbit skeletal muscle and its properties have been studied. It may be mentioned that neither phosphorylase a nor b can catalyze any reaction between UDPG and glycogen. When a homogenate of freshly excised rabbit muscle, prepared at high speed in the Virtis homogenizer, was centrifuged at 78,000 × g, most of the transferase activity remained in the sediment. Efforts to render this activity soluble were usually unsuccessful. When the muscle was frozen preliminarily and then homogenized as above, the enzymic activity was largely soluble following centrifugation for I h at 78,000 x g. Accordingly, this latter procedure was followed in all subsequent work. A b b r e v i a t i o n s : U D P , u r i d i n e d i p h o s p h a t e ; U D P G , u r i d i n e d i p h o s p h o g l u c o s e ; Tris, tris(hydroxymethyl)aminomethane; EDTA, ethylenediaminetetraacetate.
VOL. 33 (1959)
SHORT COMMUNICATIONS
557
IOO g of frozen muscle were minced w i t h scissors into 4 vol. cold* o . I M Tris - o . o o i M E D T A , p H 8.o, chilled in ice, a n d t r e a t e d for 3 min a t t o p speed in t h e Virtis homogenizer. The h o m o g e n a t e was centrifuged for 15 m i n at io,ooo × g a n d the s u p e r n a t a n t fluid t h e n centrifuged again for I h at 78,000 × g. To t h e s u p e r n a t a n t fluid, (NH4)2SO 4, s a t u r a t e d a t 5 ° a n d a d j u s t e d w i t h N H 3 to p H 7.1 (measured at 0.25 satn. w i t h a B e c k m a n p H m e t e r set at IO°), was a d d e d to 0.40 satn. The prec i p i t a t e was r e m o v e d b y centfifugation a n d dissolved in cold distilled water. The solution was d i a l y z e d against o . i M T r i s - o . o o I M E D T A , p H 7.2 for 3 h a n d then frozen. A f t e r t h a w i n g t h e solution, a p r e c i p i t a t e was r e m o v e d b y centrifugation. To the s u p e r n a t a n t fluid 0.o3 ml of Ca3(PO4)~ gel (18 m g solids/ml) was a d d e d per m g of t o t a l p r o t e i n as d e t e r m i n e d b y the m e t h o d of ROBINSON AND HOGI)EN 3. The gel was e x t r a c t e d twice using each time a volume of o.Io satd. (NH~)2SO4-o.ooI M E D T A , p H 7.2, which was equal to t h a t of the original solution which h a d been t r e a t e d w i t h t h e gel. The enzyme was e l u t e d from the gel with 0.2 M K p h o s p h a t e - o . o o I M E D T A , p H 7.2. Two elutions were m a d e , using each time a volume of eluent equal to one-half t h e v o l u m e of the original solution from which t h e enzyme h a d been adsorbed. The two eluates were pooled a n d d i a l y z e d for 2 h against o.I M T r i s - o . o o I M E D T A , p H 7.2. The enzyme lost m o s t of its a c t i v i t y when this d i a l y z e d solution was frozen. Accordingly, satd. (NH4)2S04, p H 7.1 (as above), was a d d e d to 0.40 satn,, a n d t h e m i x t u r e which c o n t a i n e d the p r e c i p i t a t e d enzyme was k e p t at 5 ° . I t was stable for a t least 8 weeks. T a b l e I gives the results of such a fractionation. TABLE I PURIFICATION OF U D P G - G L Y C O G E N TRANSFERASE Fraction
78,0OO × g s u p e r n a t a n t
0.40 satd. (NH4)~SO 4 fraction after dialysis and freezing Gel eluate after dialysis 0.4o satd. (NH4)2SO a fraction
Total vol. (ml)
Total protein (rag)
325
357 o
31 31 15
Total activity (units)*
338 24.7 24.7
Speci~c activity (units[rag protein)
494
o.14
273 87.4** 84.5
o.81 3.5*** 3.4
Yield % ioo
55 17-7 17§
* The standard assay system contained : UDPG, 1-3" I o-3 M ; glycogen, 4 mg; MgC12, 3" i o-Z M; cysteine, 7.5" IO-8 M; Tris, 5.7" lO-2 M, and enzyme in a final vol. of o. 4 ml; pH 7.2. Incubation was for 15 rain at 3o°. One unit of activity is the number of #moles of UDPG which disappeared under these conditions. ** When this fraction was assayed in the absence of added glycogen, its total activity was only o.38 unit. *** In other preparations, this fraction has had a specific activity of from 2.4-7.1 units/mg protein and the overall yield has varied from 17-44 %. § The ratio of total phosphorylase activity (a + b) to that of the transferase in this fraction was 57:1. The e n z y m i c a s s a y u s u a l l y used d e p e n d e d u p o n a m e a s u r e m e n t of t h e disa p p e a r a n c e of U D P G via the U D P G d e h y d r o g e n a s e s y s t e m 4. F r e q u e n t l y , t h e formation of U D P also was m e a s u r e d via the p y r u v a t e kinase-lactic d e h y d r o g e n a s e system, b a s e d o n K O R N B E R G ' S o b s e r v a t i o n 5 t h a t U D P is active w i t h p y r u v a t e kinase. CABIB AND LELOIRe h a v e also d e v e l o p e d a U D P a s s a y b a s e d on this fact. Thus, when a r e a c t i o n m i x t u r e containing i n i t i a l l y 0.52/zmole U D P G a n d 4 m g glycogen in 0. 4 m l A l l o p e r a t i o n s w e r e c a r r i e d o u t i n t h e c o l d r o o m a t 5 °.
558
SHORT COMMUNICATIONS
VOL. 3 3
(I959)
Tris, pH 7.2, was incubated with 86/*g enzyme for 15 min at 3 o°, 0.20/*mole UDPG disappeared and 0.20/*mole UDP was formed. Assay of all purified fractions of the enzyme invariably showed perfect stoichiometry between UDPG disappearance and UDP formation. The activity of the most highly purified enzyme was wholly dependent on the addition of glycogen (Table I). That the glycogen served as a primer was shown in experiments in which the structure of the product of enzyme action was studied. In one case, I mg human-liver glycogen and 7/*moles UDPG were incubated with 41o/*g enzyme in a vol. of I.O ml for 69 min under the conditions of the standard assay (Table I). Measurement of UDPG in an aliquot of the reaction mixture showed that 3.08/*moles had disappeared in the whole solution. The glycogen was isolated from the remaining part of the reaction mixture following digestion for I h at IOO° in 4 N NaOH and two precipitations from 50% ethanol. Analysis for total glucose following hydrolysis of the glycogen in HC1 showed that the polysaccharide contained 3.16/,moles more glucose than a control sample which had been incubated with the enzyme in the absence of UDPG. Determination of the outer chain length of the two glycogen samples was done using phosphorylase free of amylo-i,6-glucosidase*. The glycogen which had been incubated with UDPG had 3.02/*moles more glucose units in linkage susceptible to cleavage by phosphorylase than the control sample of glycogen. These findings prove that the UDPG-glycogen transferase adds glucose units in a-I, 4 linkage to the terminal units of preexisting chains in glycogen. It is interesting that in this transferase reaction, no net inversion of glucose configuration occurs. It was found that neither amyloheptaose nor a high molecular weight, soluble, mixed cellodextrin fraction** acted as a primer in this reaction. Although there are similarities between UDPG-glycogen transferase and phosphorylase as shown by the primer requirement and the structure of the polysaccharide chain formed, the enzymes are distinct as shown by their separation in the fractionation procedure used. Thus, the ratio of total phosphorylase activity (a + b) to that of the transferase (calculated as moles of substrate reacting) changed from at least 400 :I in the soluble fraction of a muscle homogenate to 178 :I in the 0.40 satd. (NH4)2SO 4 fraction, and, finally, to 23 :I in the final gel eluate in the case of a preparation not shown in Table I. UDPG-glycogen transferase was inactive when cysteine was omitted from the assay system. The enzyme does not require a bivalent metal ion but is stimulated 2-fold by 6- lO -3 M MgC12. It is not inhibited by a-glucose-l-phosphate (1.5" lO -3 M). The enzyme is inhibited by various nucleotides. Thus, uridine-5'-phosphate competitively inhibits the rate of the reaction by about 50 % when it is present in the usual assay system at a concentration equal to that of the UDPG added. Inosine-5'-phosphate, cytidine-5'-phosphate, and guanosine-5'-phosphate each inhibits to the extent of about 20 % under similar conditions. Adenosine-5'-phosphate also inhibits but to a lesser extent. These inhibitions differentiate the transferase from phosphorylase. On the other hand, phlorizin at 2. lO -3 M inhibits UDPG-glycogen transferase to the extent of 9° %, as it does phosphorylase 7. The specificity of the transferase for UDPG was shown in an experiment in which a sample of uridine]diphosphogalactose containing 3 % UDPG was found to react only * Kindly prepared by Dr. BARBARAILLINGWORTH. ** K i n d l y provided b y Dr. LuIS GLASER.
VOL. 33 (1959)
SHORT COMMUNICATIONS
559
t o t h e e x t e n t of its U D P G c o n t e n t . U r i d i n e d i p h o s p h o - N - a c e t y l g l u c o s a r n i n e y i e l d e d no U D P w h e n i n c u b a t e d w i t h t h e t r a n s f e r a s e a n d g l y c o g e n . T h e a p p a r e n t Michaelis c o n s t a n t of U D P G in t h e t r a n s f e r a s e r e a c t i o n is i . i - l O -3 M . T h i s w a s d e t e r m i n e d in t h e p r e s e n c e of 3" lO -3 M Mg ++ a n d u n d e r c o n d i t i o n s w h e r e t h e r a t e of t h e r e a c t i o n w a s linear. T h e p h y s i o l o g i c a l significance of U D P G - g l y c o g e n t r a n s f e r a s e r e q u i r e s further study. T h i s w o r k has b e e n s u p p o r t e d in p a r t b y a g r a n t ( R G - 4 7 6 I ) f r o m t h e N a t i o n a l I n s t i t u t e s of H e a l t h , U.S. P u b l i c H e a l t h Service.
Department o/Biological Chemistry, Washington University Medical School, Saint Louis, Mo. (U.S.A.)
ROSALIND HAUK* DAVID H . BROWN
1 R. HAUK, B. ILLINGWORTH,D. H. BROWN AND C. F. CORI, Biochim. Biophys. Acta, 33 (1959) 554. 2 L. F. LELOIR AND C. E. CARDINI, J. Am. Chem. Soc., 79 (1957) 6340. 3 H. W. ROBINSON AND C. G. HOGDEN, J. Biol. Chem., 135 (194o) 7o 7. 4 j. L. STROMINGER, E. S. MAXWELL, J. AXELROD AND H. M. KALCKAR, J. Biol. Chem., 224 (1957) 79. 5 A. KORNBERG, in W. D. MCELROY ANn B. GLASS, Phosphorus Metabolism, Vol. I, The Johns Hopkins Press, Baltimore, 1951, p. 392. s E. CABIB AND L. F. LELOIR, J. Biol. Chem., 231 (1958) 259. 7 C. F. CORI, G. T. CORI AND A. A. GREEN, J. Biol. Chem., 151 (1943) 39. R e c e i v e d N o v e m b e r 6th, 1958 * National Science Foundation Predoctoral Fellow.
The calculation of the constant for differential manometers A l t h o u g h n o w a d a y s n o t so w i d e l y u s e d as t h e c o n s t a n t - v o l u m e ( W a r b u r g ) m a n o m e t e r , t h e d i f f e r e n t i a l m a n o m e t e r has a n u m b e r of a d v a n t a g e s , in p a r t i c u l a r t h e i n d e p e n d e n c e of t h e r e a d i n g f r o m c h a n g e s of t h e a t m o s p h e r i c pressure, t h e s m a l l e r effect of c h a n g e s of t e m p e r a t u r e a n d t h e g r e a t e r a c c u r a c y of r e a d i n g t h e m a n o m e t e r . Since, h o w e v e r , t h e r e are c h a n g e s in b o t h t h e v o l u m e a n d t h e p r e s s u r e d u r i n g t h e course of t h e r e a c t i o n i n v o l v i n g an a b s o r p t i o n or an e v o l u t i o n of gas, t h e r e l a t i o n s h i p b e t w e e n t h e m a n o m e t e r r e a d i n g a n d t h e a m o u n t of gas a b s o r b e d or e v o l v e d is r a t h e r c o m p l i c a t e d . DIXON 1 has d e r i v e d t h e e x p r e s s i o n g i v e n in e q u a t i o n (i)*
• = h coso+ ~ ' ~ ( P - p + ~ P ' I -vG 7 "--y~ ~_ v~a --i
~'~.j
Po
~ 273
~
+ ~ " --T-
Po
=hk li)
w h e r e k is t h e m a n o m e t e r c o n s t a n t . T h i s e x p r e s s i o n for t h e m a n o m e t e r c o n s t a n t is n o t i m m e d i a t e l y u s a b l e since it i n c l u d e s A P a n d A P ' , w h i c h c a n n o t be d i r e c t l y r e a d off t h e m a n o m e t e r . M o r e o v e r , • The symbols are the same as those used by Dixon 1. x mm 8 = vol. of gas evolved or absorbed (N.T.P.) ; h mm = manometer reading ; re', va mm 3 = vol. of gas space in left-hand (compensation) and right-hand (reaction) vessels, respectively ; vv ~, vF mm z = v o l . of liquid in left-hand and right-hand vessels respectively; T °, T8 ° = absolute temperature of reaction vessel (bath temperature) and of manometer tube (room temperature) respectively; P, Po, P m m manometer fluid = initial pressure in vessel, standard pressure (760 mm Hg) and vapour pressure of water at T3 ° respectively; LIP', AP mm manometer fluid = increase of pressure in left-hand and righthand vessels, respectively; a m m 3 gas (N.T.P.)/mm a liquid = solubility of gas in reaction solution; 0 ° = angle of manometer scale with vertical; A mm s = area of cross-section of manometer £ube.