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Glycogen content and udpglucose-glucan glucosyl-transferase activity of normal human lymphocytes
BIOCHIMICA ET BIOPHYSICA ACTA
£~93
BBA 25722 GLYCOGEN CONTENT AND UDPGLUCOSE-GLUCAN GLUCOSYLT R A N S F E R A S E ACTIVITY OF NORMAL HUMAN LYMPHOCYTES C. J. H E I ) E S K O V , V. E S M A N N AND ~I. I{OSELL P E R E Z
The Departme~# of Medici~e, Marselisborg Hospital, U~Hversity ()f ~4arhas Medical School, .{ re'has (Demnark) a~zd Cdtedra de Bioqzdmica de la Facu/tad de t:arnzacia, UIziversidad de Barcelona, Barcelona (5;pai~) (Received J u n e 24th, 1966)
SUMMARY
I. The glycogen content of lymphocytes isolated from 5 normal subiects was 31 ~- 6 (mean ~ S.E.) mg per lO1° cells or o.9 % on a wet weight basis. 2. UDPglucose-glucan glucosyltransferase (UDPglucose:~-I,4-glucan ~-4-glucosyltransferase, EC 2.4.1.11 ) (transferase or synthetase) activity was found in normal human lymphocytes. The activity of the transferase in freshly prepared homogenates depended on the presence of Glc-6-P and ranged from 1.8o to 3.21 txmoles of glucose incorporated into glycogen per lOs lymphocytes per h. Mg2+ slightly stimulated the enzyme activity. Km for the substrate UDPG (with Glc-6-P present) was 5.o. Io -4 M without Mg2+ and 1.4" lO-4 M with Mg2+ present. The activation constant for Glc-6-P (Ka) ranged from 3.o" IO-~ M to 5.o" IO-3 M in the absence of Mg2+ and from 1. 4, IO-a M to 3.o" IO-a M with Mg2+ added. UDP competed with the substrate UDPG for the enzyme. PI competed at the allosteric site with the activator Glc-6-P. 3. Lymphocytes possess the systems for the interconversion of the D (Glc-6-P dependent) and I (Glc-6-P independent) forms of transferase. It was discovered that the D to I transformation was considerably enhanced by the presence of Mg 2+ in the incubation mixture. The I to D transformation occurred upon addition of Mg-XrP, and it was demonstrated that part of the enzyme was converted to an inactive intermediate form.
INTRODUCTION
The pathway for the synthesis of glycogen in human polymorphonuclear leucocytes has been described recently 1, 2. The presence of glycogen in lymphocytes has been the subject of a number of reports displaying controversial results. Early investigators3, 4 were not able to detect glycogen chemically in lymphocytes from patients with lymphatic leukemia, and histochemically 5 only small amounts of periodic acidSchiff (PAS) positive material were found in a minority of normal lymphocytes. Later, Abbreviation: PAS, periodic acid-Schitt.
l?iochim, t3iophys. Acta, 13o {[966) 393-400
394
C . J . HEDESKOV, V. ESMANN, M. ROSELL t'I'~RE2
however, PAS-positive granules, sensitive to diastase, have been clearly demonstrated in both normal and leukenfic lymphocytes6 10, and chemically demonstrable glycogen has also been reportedlL In addition, the presence of UPDG has been established in chronic lymphatic leukemia cells r'. The present report confirms the presence of chemically demonstrable glycogen in human lymphocytes and presents data on the enzyme systems involved in the synthesis of this polysaccharide. METHODS
Preparation of e~zs,~nefrom (vmpkocvtes The lymphocytes were obtained from healthy blood donors as previously described 1'~. 400 ml of blood was used in each experiment and gave a total yield of 3.o-6.3.1o s cells. The final cell suspensions contained 97-08 % lymphocytes, 2- 3 % polymorphonuclear leukocytes, and 5-6 erythrocytes to each lymphocyte. The isolated lymphocytes were washed twice with isotonic saline and suspended in ice-cold 0.05 M Tris 0.005 M EDTA buffer (pH 7.8) to a final concn, of 8o-15o- IO(~ cells/ml. The cell suspension was sonicated at 4 ° for 2 successive periods of 30 and 15 sec (M.S.E. ultrasonic disintegrator, 2ooo0 kcycles/sec), which conlpletely disrupted the cells as checked by microscopic examination. The suspension thus obtained was used as enzyme source as such or after removal of cellular debris by centrifugation at IOOO ~', g for IO min.
Enzyme assay UDPGlucose:~-I,4-glucan ~-4-glucosyltransferase (transferasc or synthetase) (EC 2.4.1.ii) activity was measured as radioactivity incorporated into glycogen fronl [14Clglucose-labeled UDPG, as previously described 14.The standard assay mixture 2,1~ contained 6" IO a M UDPG (specific activity 8ooo counts/min per/,mole, I °o glycogen, and 0.o 5 M Tris--o.oo5 M EDTA buffer (pH 7.8). When, in addition, the Glc-6-P dependent or D form of the enzyme was assayed, o.oi M Glc-0-P was also present. For the assay of the enzyme, o.o5 ml of the enzyme source was added to o.I nil of the assay mixture and incubated at 3 °° for 5-1o min. The incubation was terminated by adding I ml 6 % trichloroacetic acid containing 1 mg glycogen and 2 mg LiC1. Glycogen was precipitated from the supernatant with ethanol, washed twice and suspended in o.0 mt water. The sample was added to io ml scintillation fluid (4 g 2,5-diphenyloxazole, 2oo mg 1,4-bis-2-(4-methyl-5-phenyloxazolyl)-benzene, 6o g naphthalene, 2o ml ethyleneglycol, IOO ml methanol, and dioxane to iooo inl) and radioactivity was determined by means of a Tri-Carb liquid scintillation spectrometer (Packard). The background was 21 counts/rain.
Glycogen determination Glycogen determinations were made on o.5-I.o-ml aliquots of thrice washed cells suspended in 0. 9 °o NaC1 as described 16. The complete elimination of dextran used in the separation of white cells from erythrocytes was checked by repeating the glycogen determinations after several further washings of the lymphocytes. Glucose was determined by the glucose oxidase method and expressed in terms of glycogen by multiplying by 0.90. Biochim. Biophys..4cta, I3O (1966) 393-400
GLYCOGEN
SYNTHESIS
395
IN L Y M P H O C Y T E S
MATERIAL
The sodium salts of Glc-6-P, U D P and UDPG, Na2-ATP, Tris and glycogen (from rabbit liver) were purchased from Sigma Chemical Co., St. Louis. ~14C]Glucoselabeled U D P G was supplied by New England Nuclear Corp., Boston. Dextran (TDR 2 o 5 - I I - B - I ) was from AB Pharmacia, Uppsala, and glucose oxidase from C. F. Boehringer, Mannheim. RESULTS
Gh, cogen content The average content of glycogen in lymphocytes from 5 normal subjects was 31 :~ 6 (mean :~ S.E.) mg per IO1° lymphocytes.
Transfzrase acEvity Under these experimental conditions the transferase activity detected was, as in normal human polymorphonuclear leucocytes 2, only of the dependent or D form (the enzyme requires addition of Glc-6-P to the test mixtures for activity). With a substrate concentration of 6- IO-3 M U D P G and in the presence of I- IO-z M Glc-6-P the transferase activity in 5 experiments ranged from 1.8o to 3.21 ffmoles per lO8 lymphocytes per h. When MgC12 (5-8.IO -3 M) was added the transferase activity ranged from 2.05 to 3.51 ffmoles per lO8 lymphocytes per h.
Effect of enzyme conce~tratio~ Fig. i shows a perfectly linear relationship between activity and enzyme concentration as expressed by the number of cells from which the actual amount of enzyme has been extracted. In all other assays the amount of enzyme corresponded to 4-7.5' IOG lylnphoeytes.
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F i g . I. R e l a t i o n s h i p b e t w e e n e n z y m e c o n c e n t r a t i o n , a s e x p r e s s e d b y t h e n u m b e r of cells f r o m w h i c h t h e a c t u a l a m o u n t of e n z y m e w a s e x t r a c t e d , a n d r a d i o a c t i v i t y i n c o r p o r a t e d i n t o g l y c o g e n f r o m 1 4 C i g l u c o s e - l a b e l e d U D P G . T h e e n z y m e w a s p r e p a r e d b y s o n i c a t i n g l y m p h o c y t e s s u s p e n d e d in o.o 5 M T r i s - o . o o 5 M E D T A b u f f e r ( p H 7.8). T h e a s s a y m i x t u r e c o n t a i n e d 6- IO a M U D P G , I . Lo 2 M G l c - 6 - P , 1 % g l y c o g e n a n d T r i s - E D T A b u f f e r . T i m e of i n c u b a t i o n w a s l o r a i n a t 3 o . F i g . 2. A c t i v i t y of t r a n s f e r a s e a s a f u n c t i o n of i n c u b a t i o n t i m e . T h e e n z y m e c o n c e n t r a t i o n s p o n d e d t o 4 . 2 - IO 6 l y m p h o c y t e s . O t h e r c o n d i t i o n s w e r e a s in F i g . I.
corre-
13iochim. Biophys..4cta, 13o ( t 9 6 6 ) 3 9 3 4 ° o
39 6
c.j.
HEDESKOV, V. ESMANN, 51. ROSELL PEREZ
Effect of time ineubatio~ i~ the assay Fig. 2 shows t h a t the incorporation of activity is linear with the time of assay for the first 20 inin of incubation.
Effect of substrate concentration o1~ enzyme activity The L i n e w e a v e r - B u r k plot of the enzyme activity versus U D P G concentrations is shown in Fig. 3. Only activity in the presence of Glc-6-P is recorded. In 5 experiments the Km for U D P G was 5.0" IO-4 M (range 2.5-7.0. io -4 M). With Mg 2~ (8. IO-a M) present the Km value decreased to 1. 4. Io -~ M (range 1.3-2.o" IO-~ M).
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Fig. 3. The L i n e w e a v e r - B u r k d i a g r a m of the ITI)PG c o n c e n t r a t i o n d e p e n d e n c e w i t h l y m p h o c y t e t r a n s f e r a s e a c t i v i t y (D form). The a s s a y s y s t e m c o n t a i n e d e n z y m e c o r r e s p o n d i n g to 4.-" I°~ 1y m p h o c y t e s a n d o. 7" l o z M Glc 6-P. O t h e r a d d i t i o n s were as s t a t e d in t h e figure. In t h e e x p e r i m e n t s w i t h U D P 7" l o a M MgC12 was also present. Fig. 4- R e c i p r o c a l p l o t of the GIc-0-P c o n c e n t r a t i o n d e p e n d e n c e w i t h l y m p h o c y t e t r a n s f e r a s e a c t i v i t y (D form). The a s s a y s y s t e m c o n t a i n e d e n z y m e c o r r e s p o n d i n g to 4.-" los l y l n p h o c y t e s . C o n c e n t r a t i o n of U I ) P G was 4' lO a M. O t h e r a d d i t i o n s were as s t a t e d in t h e figure. In t h e exp e r i m e n t s w i t h [ : I ) P a n d Pi 7" to a M MgCI z was also pre s e nt .
I~flue¢aee of the Glc-6-P conce~atratio~ Fig. 4 shows the reciprocal plots of the transferase activity at different concentrations of Glc-6-P. The activation constant Ka (the concentration of C-lc-6-P that gave half maximal activation) obtained at saturating concentrations of U D P G ranged from 3 . o - 5 . o ' I o - a M in the absence of Mg 2+. With Mg 2+ (8.IO -a M) added, I<, ranged from 1.4-3.o. IO-a M.
Effect of Pi and UDP Phosphate ions are inhibitory for the D form of muscle synthetase iv-l'a. U D P has been reported to inhibit the synthetase from rat liver 2°,21 13iochim. Hiophvs..,tcta, 13o (l{#)o) 393 4 oo
GLYCOGEN SYNTHESIS
397
IN LYMPHOCYTES
In human lymphocytes both compounds were found to be inhibitory for the synthetase, but at different levels. U D P (2.3-4.6. io -a M) competed with the substrate U D P G for the enzyme and not with the activator (Figs. 3 and 4), whereas Pt (3.6" lO-.2 M) competed at the allosteric site with the activator Glc-6-P (Fig. 4).
Conversion of D to I form and systems of interconversion The form of the enzyme present in the lymphocyte immediately after isolation was thus dependent on the presence of Glc-6-P in the assay mixture. Incubation of the enzyme source (sonicated lymphocytes suspended in o.o5 M Tris-o.oo5 M EDTA (pH 7.8)) at 3 °° before assay of the enzyme activity, however, revealed a slow but steady conversion of the enzyme from D to I form (Fig. 5), provided that mercaptoethanol (o.o5 M) was added to the incubation mixture. Addition of Mg 2+ (7" I ° - a MI" lO-.2 M) increased the rate of conversion considerably. With Mg .2+ present, practically all the D to I conversion has taken place after 6o rain of preincubation, whereas the I activity in the incubation mixtures without Mg .2+ at the same time is still low and continues to increase slowly. The ratio of activity in the presence of Glc-6-P to that without Gle-6-P changed from 14 at zero time to 1.3 after 6o min of preincubation in the presence of Mg e+, and correspondingly from 14 to 3.2 in the absence of this cation. The increase in I activity in this period corresponded to o.61 ffmole per IOs lymphocytes per h without Mg 2+ and 1.76 /,mole when the cation was present. Fig. 5 also shows the transformation of I to D form of the enzyme obtainable by adding ATP (2. 7. IO-3 M) and Mg 2+ (5.4" IO-a M) to the enzyme source after 60 rain i
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Fig. 5. E f f e c t of t i m e of p r e i n c u b a t i o n a n d M g - A T P a d d i t i o n o n l y m p h o c y t e t r a n s f e r a s e . T h e enzyme was assayed at different time intervals after preincubation with 5' lo a M mercaptoe t h a n o l a n d w i t h or w i t h o u t i . i o 2 M MgC12. A t 6o r a i n M g - A T P (2. 7 .IO - a M - 5 . 4 . I o a M ) w a s a d d e d t o p a r t of t h e i n c u b a t e d e n z y m e . T h e a s s a y s y s t e m c o n t a i n e d e n z y m e c o r r e s p o n d i n g t o 7.3" lo'~ l y m p h o c y t e s , 4" IO a M U D P G , a n d o. 7- i o 2-M G l c - 6 - P w h e n p r e s e n t . O - O, 1 enzyme; O ©, 1 e n z y m e + Mg2+; O - 0 , t o t a l e n z y m e (I + D ) ; • @, t o t a l e n z y m e ([ + D) + Mg'::.
13ioch2m. tCiophys. Acta, r3o (t966) 393 -4 ° 0
398
c . j . HEDESKOV, V. ESMANN, M. ROSELL PI~RE2
of preincubation. The addition did not change the pH from 7.8. In 2o rain the I activity decreased corresponding to o.64 /,mole per lO8 lymphocytes per h in the preparation without Mg2+ and 1.78 /,mole in the one with Mg2+ previously added. As the activity in the presence of Glc-6-P (equal to total I + D activity) in the same period decreased by o.42 and o.29/,mole, respect'vely, the net reversal of I to D form amounts to o.22/,mole per IO8 lymphocytes per h in the first case and 1.49/,mole in the second case, which means that a comparatively larger proportion of the I enzyme wa~ converted to the D enzyme in the experiment with Mg2+. From these experiments i~ vitro it is concluded that normal human lymphocytes possess the systems for the interconversion of the two forms of UDPglucose-glucan glucosyltransferase.
I~flue1~ce of coutamit~ati~g erythrocyles Transferase activity of erythrocyte hemolysates has recently been reported 22. In order to check the percentage of enzymatic activity due to the contaminating red cells, the following experiment was performed. Red cells were sedimented at 37 ~' with dextran and the supernatant plasma was removed. The erythrocytes were further packed by centrifugation at IOOO × g for io min and the supernatant fluid together with the most superficial layer of red cells was discarded. A o.5-ml aliquot was taken from the center of the packed cells, washed twice with cold saline and suspended in buffer to a concn, of 5.5.1o 8 cells/ml. Microscopic examination in a counting chamber did not reveal any white cells among 1. 4. io '~ red cells. The erythrocytes were homogenized and aliquots corresponding to 2.75. io 7 and 5-5" lO7 red cells, which is equivalent to the amount of red cells actually contaminating the lynlphocyte suspensions, were tested for transferase activity. No radioactivity was incorporated into glycogen in the absence of (ilc-(>P. Ill the presence of this compound, with or without Mg('l~, an incorporation o[ 4-8 counts/rain above background was detected, which, if it is assumed to represent transferase activity, corresponds to o.oi 0.o2 /,mole per io a erythrocytes per h, or approx. I °o of the activity of the lymphocytes. The transferase activity found in erythrocytes corresponds to the values reported by COI~NBLATHet ill. 2'2. No gly('()gen could be detected in these pure erythrocyte suspensions. 1)ISCUSSION The presence of glycogen and the enzymes involved in its biosynthetic pathway has been demonstrated in lymphoeytes from normal humans by the experimental work presented here. The glycogen content in lymphocytes was found to be 3 z ~ 6 mg per IOu) cells. In a previous communication ~6, the glycogen content of a mixture of 8o % polymorphonuclear leucocytes and 2o % mononuclear cells was calculated as 148 mg glycogen per lO1° polymorphonuclear leucocytes assuming that all glycogen determined originated from polymorphonuclear leucocytes. In view of the present findings, a recalculation shows the presence of 14o mg glycogen in IOu~ polymorphonuclear leucocytes, when mononuclear cells are credited with 31 mg per IOL° cells. Conversely, the observed contamination of lymphocyte suspensions with 2- 3 % polymorphonuclear leucocytes accounts for 3-4 mg of the measured glycogen content of the lymphocyte suspensions. On a cellular basis the glycogen content of lymphocytes
tli,chim. Hioph),s..lch~. 13° (1960) 393 4 oo
GLYCOGEN SYNTHESIS IN LYMPHOCYTES
39 ()
is thus only one-fifth of t h a t in polymorphonuclear leucocytes. Based on wet weight, however, the glycogen content of lymphocytes (lO l° cells = 3.3 g) is o.9 % and of polymorphonuclear leucocytes (IO1° cells = I o g) 1.4 %. Both values are close to tile glycogen content of muscle and liver from fasting subjects ~a. LRIKINit reported the presence of iz 4 mg glycogen per zo 1° cells in suspensions with more than 9 ° °o normal h u m a n lymphocytes. This value appears to be rather high and would correspond to 3.4 °o of the wet l y m p h o c y t e weight. The activity of the UDPglucose-glucan glucosyltransferase present in these cells is, like that in the polymorphonuclear leucocytes ~, a D form with some characteristics not found in polymorphonuclear leucocytes. Unlike the polymorphonuclear leucocytes, the lymphocytes have the capacity of converting the D form of the enzyme to the I form, which is more active in the sense that it does not require Glc-6-P for activity. The system that carries out this conversion operates i~ vitro more slowly than the similar system found in muscle of rat, rabbit and dog15, ~,'~. On the other hand, the D to I transformation is very much enhanced if a sufficient concentration of Mg "~+ ions is present. This Mg2--activated I) to I conversion, detected here for the first time, has also been demonstrated in peritoneal exudate leucocytes from both normal and alloxan-diabetic rats ~5, whereas the D to I conversion system in rat diaphragmatic muscle is unaffected of Mg 2~ ions (unpublished observations). In normal h u m a n polymorphonuclear leucocytes no I) to I conversion could be d e m o n s t r a t e d - - - e v e n in the presence of Mg 2+ ions "6. The D to I conversion system provides the lymphocytes with a mechanism t h a t would allow them to increase their stores of glycogen without increasing the Glc-6-P pool, the necessary activator of the D form. The switch for the D to I transformation is as yet unknown, but could possibly be produced by some kind of hormone. Thus, insulin seems to have influence on the appearance of such conversion systems in leucocytes of diabetic h u m a n s 26. The system t h a t converts the I to the D form of synthetase is also operational and functions rapidly in a parallel w a y to the system found in muscle preparations ~. The quantitative differences in the transformation of I to D form in the absence or presence of Mg 2 ~ previously added (Fig. 5) could signify t h a t in the absence of this cation the transformation is not carried out completely, the remaining part of the e n z y m e being in an inactive intermediate state 2~, ~. The levels of transferase activity in lymphocytes are on a cellular basis about 2 times lower t h a n those found in polymorphonuclear leucocytes (3.1o-7.o4 /xmoles per IOs cells per h (ref. 2)), but on a weight basis lymphocytes will haw~ an activity approx. 1.5 times t h a t of polymorphonuclear leucocytes. The levels of activity reported here and those previously c o m m u n i c a t e d for polymorphonuclear leucocytes "~ are in agreement with values found b y HUIJING2s and b y MILLER AND VAN ])ER W E N D E 2u, whereas CORNBLATHet a!. 2'~ have reported values 2 - 3 times higher. The fact t h a t Pi competes with the allosteric activator Glc-6-P stresses t h a t the l y m p h o c y t e transferase is a D form of the enzyme. The inhibitory effect of Pl is also found in normal h u m a n polymorphonuclear leucocytes (unpublished observations), whose enzyme form has an ahnost absolute requirement for Glc-6-P. This phosphate effect will provide the cells with one more mechanism for the regulation of their c a r b o h y d r a t e stores b y controlling enzyme activity without modifying enzyme level, e.~.*, bv~ diminishing transferase activity (luring anaerobic conditions. Biochim. I3io/~h>,s..~lcfa, 230 (I9>)} 3~)3 4()O
400
C.J.
HEDESKOV,
V. ESMANN,
M. ROSELI.
I't~RI'~Z
.\CKNOWLEDGEMENTS
We are greatly indebted to Dr. F. KISSMEYER, Chief of the Blood Bank Service, Aarhus K o m m u n e Hospital for supplying us with blood samples and to Prof. I. C. SKOU, Department of Physiology, Aarhus University for access to counting equipment. This investigation was supported by a grant from Statens Atmindelige Videnskabsfond (L 36z65). One of the authors (M.R.P.), collaborator of the Institute J aime Ferran del S.C.I.C. of Spain was supported by U.S. Public Health Service (irant AM ~oo23-o*.
REFERENCES I [. S. |.UGANOVA, z\. D. VLADIMIROVA ANI) |. l ?. SEITS, Ch#Dz. Abstr., 03 (1905) 8822. z M. I{OSELL-PI~REZ AND V. ESMaXN, Acta Chem. Scand., I9 (19~)5) 679. 3 R. \VAGNER, Blood, , (I947) 235. 4 \V. N. VALENTINE, J. H. FOLt~TT£ AND J. S. L.\WRENCI~;, .]. Clil¢. [Iwest., 32 (1953) -'51 5 M. \V,xcHSTEIN, Blood, 4 ( t 9 4 9 ) 54. 0 G. A. ACK~,RMAX, J. Hislochem. Cylochem., 7 (1959) 3187 A. DAHLQUIST, (;. GAHRTON ANI) AA. NORI)I"N, .{cta 5Ied. £'cand., (r902) 31. 8 F. H~CKNER, Acta Haematol., 1() (t95{~) ~. 9 R. V. JONES, G. P. G o F w .x.xi) M. S. R. H u x ' r , J. cli~z. Patho[., 15 (Eq{)_,) 3 o. i o V. E. S,~VaRXSMaX, Chem. Abstr., 03 (1905) 8812. 1 i S. I,. LEIKIN, Proc. Soc. Exptl. Biol. Med., To(3 (190I) 28~). 12 A. l). \~LADIMIROVA AND [. F. SE1TS, Bull. E.rpll. lqiol. ~lled., ~o (1904) 5 o. 13 ('. J. H~z!)E~Kov aNt) V. ESM~.NN, B[~ot, ~'8 (ig()t,) 143. J4 C. \:ILLAR PALaSI AND J. LARNER, Arch. l~ioche*n. Biophys., 94 ( I 9 ( i l ) 430. 15 M. ROSELL PgRF.Z, C. \'II.I.aR P.XLaSl aXl~ J. I.ARNER, Biochemislry, , (~9(i2) 71)3 . 10 V. ESMANN, Scand. J. Clin. Lab. l*lvesl., 13 (I9~)~) 134. 17 M. I(OSELL PfiREZ AND J. LARNIe:R, Biochemistry, 3 (I9(~4) 7518 M. ROSJ~LL P~REZ aND J. LARNER, Biochemistry, 3 (I9(i4) 773. 19 ~I. |{OSELL t)I~REZ AND r . VILLAR I)ALAS1, teCl'. Kspan. Fisiol., 2o ( i 9 0 4 ) 131. 20 D. 1~'. S'I'PSINER AND J. I(IN(;, ./r. t~1701. CJlCltZ., 239 (~904) 1292. 21 D. F. SWEINER, L. YOUN~ AXD J. l,hx~;, Biochemistry, 4 (~905) 4. 22 ~'i. (7ORNBLATH, D. F. STEINER, P. BRYAN \ND J. l'2IX(;, Clin. Chim...tcga, 12 (~965) -'7. 23 J. A. t-[ILDES, S. SHERLOCK AND \7. \VALSHE, CliI2. ,%'Ci., 7 (I949) 287" 24 3,'[. ]{OSELL PI~REZ AND j. EARNER, Igiochemistry, 3 (~904) 81. 25 .'~I. ROSELL PI~REZ, C. J. I-tFDV;SKOV ANI) V. ESM.~,NN, in p r e p a r a t i o n . 20 l\'I. }{OSELL PI~REZ, V. ESMANN AND C. J. HEDESKOV, Proc. 2rid Bleeting, European Assoc. ,S'lu@, Diabetes, ~o a p p e a r in l)iabetologia, ~907. 27 M. t)*OSELI, PI~REZ AND J. EARNER, Biochemistry, ! (I902) 7()q. 28 b'. HUIJING, P*'oc. 6th !nlern. Congr. Biochem., (1904) 23z. 29 \V. L. MH.L~:R AND C. VANDER \ V E N m L Biochim. Biophys. At&t, 77 (1903) 494.
]~;iochim. 13iophys..lcta, 13o (IO()O) 393 4 ° o
£~93
BBA 25722 GLYCOGEN CONTENT AND UDPGLUCOSE-GLUCAN GLUCOSYLT R A N S F E R A S E ACTIVITY OF NORMAL HUMAN LYMPHOCYTES C. J. H E I ) E S K O V , V. E S M A N N AND ~I. I{OSELL P E R E Z
The Departme~# of Medici~e, Marselisborg Hospital, U~Hversity ()f ~4arhas Medical School, .{ re'has (Demnark) a~zd Cdtedra de Bioqzdmica de la Facu/tad de t:arnzacia, UIziversidad de Barcelona, Barcelona (5;pai~) (Received J u n e 24th, 1966)
SUMMARY
I. The glycogen content of lymphocytes isolated from 5 normal subiects was 31 ~- 6 (mean ~ S.E.) mg per lO1° cells or o.9 % on a wet weight basis. 2. UDPglucose-glucan glucosyltransferase (UDPglucose:~-I,4-glucan ~-4-glucosyltransferase, EC 2.4.1.11 ) (transferase or synthetase) activity was found in normal human lymphocytes. The activity of the transferase in freshly prepared homogenates depended on the presence of Glc-6-P and ranged from 1.8o to 3.21 txmoles of glucose incorporated into glycogen per lOs lymphocytes per h. Mg2+ slightly stimulated the enzyme activity. Km for the substrate UDPG (with Glc-6-P present) was 5.o. Io -4 M without Mg2+ and 1.4" lO-4 M with Mg2+ present. The activation constant for Glc-6-P (Ka) ranged from 3.o" IO-~ M to 5.o" IO-3 M in the absence of Mg2+ and from 1. 4, IO-a M to 3.o" IO-a M with Mg2+ added. UDP competed with the substrate UDPG for the enzyme. PI competed at the allosteric site with the activator Glc-6-P. 3. Lymphocytes possess the systems for the interconversion of the D (Glc-6-P dependent) and I (Glc-6-P independent) forms of transferase. It was discovered that the D to I transformation was considerably enhanced by the presence of Mg 2+ in the incubation mixture. The I to D transformation occurred upon addition of Mg-XrP, and it was demonstrated that part of the enzyme was converted to an inactive intermediate form.
INTRODUCTION
The pathway for the synthesis of glycogen in human polymorphonuclear leucocytes has been described recently 1, 2. The presence of glycogen in lymphocytes has been the subject of a number of reports displaying controversial results. Early investigators3, 4 were not able to detect glycogen chemically in lymphocytes from patients with lymphatic leukemia, and histochemically 5 only small amounts of periodic acidSchiff (PAS) positive material were found in a minority of normal lymphocytes. Later, Abbreviation: PAS, periodic acid-Schitt.
l?iochim, t3iophys. Acta, 13o {[966) 393-400
394
C . J . HEDESKOV, V. ESMANN, M. ROSELL t'I'~RE2
however, PAS-positive granules, sensitive to diastase, have been clearly demonstrated in both normal and leukenfic lymphocytes6 10, and chemically demonstrable glycogen has also been reportedlL In addition, the presence of UPDG has been established in chronic lymphatic leukemia cells r'. The present report confirms the presence of chemically demonstrable glycogen in human lymphocytes and presents data on the enzyme systems involved in the synthesis of this polysaccharide. METHODS
Preparation of e~zs,~nefrom (vmpkocvtes The lymphocytes were obtained from healthy blood donors as previously described 1'~. 400 ml of blood was used in each experiment and gave a total yield of 3.o-6.3.1o s cells. The final cell suspensions contained 97-08 % lymphocytes, 2- 3 % polymorphonuclear leukocytes, and 5-6 erythrocytes to each lymphocyte. The isolated lymphocytes were washed twice with isotonic saline and suspended in ice-cold 0.05 M Tris 0.005 M EDTA buffer (pH 7.8) to a final concn, of 8o-15o- IO(~ cells/ml. The cell suspension was sonicated at 4 ° for 2 successive periods of 30 and 15 sec (M.S.E. ultrasonic disintegrator, 2ooo0 kcycles/sec), which conlpletely disrupted the cells as checked by microscopic examination. The suspension thus obtained was used as enzyme source as such or after removal of cellular debris by centrifugation at IOOO ~', g for IO min.
Enzyme assay UDPGlucose:~-I,4-glucan ~-4-glucosyltransferase (transferasc or synthetase) (EC 2.4.1.ii) activity was measured as radioactivity incorporated into glycogen fronl [14Clglucose-labeled UDPG, as previously described 14.The standard assay mixture 2,1~ contained 6" IO a M UDPG (specific activity 8ooo counts/min per/,mole, I °o glycogen, and 0.o 5 M Tris--o.oo5 M EDTA buffer (pH 7.8). When, in addition, the Glc-6-P dependent or D form of the enzyme was assayed, o.oi M Glc-0-P was also present. For the assay of the enzyme, o.o5 ml of the enzyme source was added to o.I nil of the assay mixture and incubated at 3 °° for 5-1o min. The incubation was terminated by adding I ml 6 % trichloroacetic acid containing 1 mg glycogen and 2 mg LiC1. Glycogen was precipitated from the supernatant with ethanol, washed twice and suspended in o.0 mt water. The sample was added to io ml scintillation fluid (4 g 2,5-diphenyloxazole, 2oo mg 1,4-bis-2-(4-methyl-5-phenyloxazolyl)-benzene, 6o g naphthalene, 2o ml ethyleneglycol, IOO ml methanol, and dioxane to iooo inl) and radioactivity was determined by means of a Tri-Carb liquid scintillation spectrometer (Packard). The background was 21 counts/rain.
Glycogen determination Glycogen determinations were made on o.5-I.o-ml aliquots of thrice washed cells suspended in 0. 9 °o NaC1 as described 16. The complete elimination of dextran used in the separation of white cells from erythrocytes was checked by repeating the glycogen determinations after several further washings of the lymphocytes. Glucose was determined by the glucose oxidase method and expressed in terms of glycogen by multiplying by 0.90. Biochim. Biophys..4cta, I3O (1966) 393-400
GLYCOGEN
SYNTHESIS
395
IN L Y M P H O C Y T E S
MATERIAL
The sodium salts of Glc-6-P, U D P and UDPG, Na2-ATP, Tris and glycogen (from rabbit liver) were purchased from Sigma Chemical Co., St. Louis. ~14C]Glucoselabeled U D P G was supplied by New England Nuclear Corp., Boston. Dextran (TDR 2 o 5 - I I - B - I ) was from AB Pharmacia, Uppsala, and glucose oxidase from C. F. Boehringer, Mannheim. RESULTS
Gh, cogen content The average content of glycogen in lymphocytes from 5 normal subjects was 31 :~ 6 (mean :~ S.E.) mg per IO1° lymphocytes.
Transfzrase acEvity Under these experimental conditions the transferase activity detected was, as in normal human polymorphonuclear leucocytes 2, only of the dependent or D form (the enzyme requires addition of Glc-6-P to the test mixtures for activity). With a substrate concentration of 6- IO-3 M U D P G and in the presence of I- IO-z M Glc-6-P the transferase activity in 5 experiments ranged from 1.8o to 3.21 ffmoles per lO8 lymphocytes per h. When MgC12 (5-8.IO -3 M) was added the transferase activity ranged from 2.05 to 3.51 ffmoles per lO8 lymphocytes per h.
Effect of enzyme conce~tratio~ Fig. i shows a perfectly linear relationship between activity and enzyme concentration as expressed by the number of cells from which the actual amount of enzyme has been extracted. In all other assays the amount of enzyme corresponded to 4-7.5' IOG lylnphoeytes.
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F i g . I. R e l a t i o n s h i p b e t w e e n e n z y m e c o n c e n t r a t i o n , a s e x p r e s s e d b y t h e n u m b e r of cells f r o m w h i c h t h e a c t u a l a m o u n t of e n z y m e w a s e x t r a c t e d , a n d r a d i o a c t i v i t y i n c o r p o r a t e d i n t o g l y c o g e n f r o m 1 4 C i g l u c o s e - l a b e l e d U D P G . T h e e n z y m e w a s p r e p a r e d b y s o n i c a t i n g l y m p h o c y t e s s u s p e n d e d in o.o 5 M T r i s - o . o o 5 M E D T A b u f f e r ( p H 7.8). T h e a s s a y m i x t u r e c o n t a i n e d 6- IO a M U D P G , I . Lo 2 M G l c - 6 - P , 1 % g l y c o g e n a n d T r i s - E D T A b u f f e r . T i m e of i n c u b a t i o n w a s l o r a i n a t 3 o . F i g . 2. A c t i v i t y of t r a n s f e r a s e a s a f u n c t i o n of i n c u b a t i o n t i m e . T h e e n z y m e c o n c e n t r a t i o n s p o n d e d t o 4 . 2 - IO 6 l y m p h o c y t e s . O t h e r c o n d i t i o n s w e r e a s in F i g . I.
corre-
13iochim. Biophys..4cta, 13o ( t 9 6 6 ) 3 9 3 4 ° o
39 6
c.j.
HEDESKOV, V. ESMANN, 51. ROSELL PEREZ
Effect of time ineubatio~ i~ the assay Fig. 2 shows t h a t the incorporation of activity is linear with the time of assay for the first 20 inin of incubation.
Effect of substrate concentration o1~ enzyme activity The L i n e w e a v e r - B u r k plot of the enzyme activity versus U D P G concentrations is shown in Fig. 3. Only activity in the presence of Glc-6-P is recorded. In 5 experiments the Km for U D P G was 5.0" IO-4 M (range 2.5-7.0. io -4 M). With Mg 2~ (8. IO-a M) present the Km value decreased to 1. 4. Io -~ M (range 1.3-2.o" IO-~ M).
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Fig. 3. The L i n e w e a v e r - B u r k d i a g r a m of the ITI)PG c o n c e n t r a t i o n d e p e n d e n c e w i t h l y m p h o c y t e t r a n s f e r a s e a c t i v i t y (D form). The a s s a y s y s t e m c o n t a i n e d e n z y m e c o r r e s p o n d i n g to 4.-" I°~ 1y m p h o c y t e s a n d o. 7" l o z M Glc 6-P. O t h e r a d d i t i o n s were as s t a t e d in t h e figure. In t h e e x p e r i m e n t s w i t h U D P 7" l o a M MgC12 was also present. Fig. 4- R e c i p r o c a l p l o t of the GIc-0-P c o n c e n t r a t i o n d e p e n d e n c e w i t h l y m p h o c y t e t r a n s f e r a s e a c t i v i t y (D form). The a s s a y s y s t e m c o n t a i n e d e n z y m e c o r r e s p o n d i n g to 4.-" los l y l n p h o c y t e s . C o n c e n t r a t i o n of U I ) P G was 4' lO a M. O t h e r a d d i t i o n s were as s t a t e d in t h e figure. In t h e exp e r i m e n t s w i t h [ : I ) P a n d Pi 7" to a M MgCI z was also pre s e nt .
I~flue¢aee of the Glc-6-P conce~atratio~ Fig. 4 shows the reciprocal plots of the transferase activity at different concentrations of Glc-6-P. The activation constant Ka (the concentration of C-lc-6-P that gave half maximal activation) obtained at saturating concentrations of U D P G ranged from 3 . o - 5 . o ' I o - a M in the absence of Mg 2+. With Mg 2+ (8.IO -a M) added, I<, ranged from 1.4-3.o. IO-a M.
Effect of Pi and UDP Phosphate ions are inhibitory for the D form of muscle synthetase iv-l'a. U D P has been reported to inhibit the synthetase from rat liver 2°,21 13iochim. Hiophvs..,tcta, 13o (l{#)o) 393 4 oo
GLYCOGEN SYNTHESIS
397
IN LYMPHOCYTES
In human lymphocytes both compounds were found to be inhibitory for the synthetase, but at different levels. U D P (2.3-4.6. io -a M) competed with the substrate U D P G for the enzyme and not with the activator (Figs. 3 and 4), whereas Pt (3.6" lO-.2 M) competed at the allosteric site with the activator Glc-6-P (Fig. 4).
Conversion of D to I form and systems of interconversion The form of the enzyme present in the lymphocyte immediately after isolation was thus dependent on the presence of Glc-6-P in the assay mixture. Incubation of the enzyme source (sonicated lymphocytes suspended in o.o5 M Tris-o.oo5 M EDTA (pH 7.8)) at 3 °° before assay of the enzyme activity, however, revealed a slow but steady conversion of the enzyme from D to I form (Fig. 5), provided that mercaptoethanol (o.o5 M) was added to the incubation mixture. Addition of Mg 2+ (7" I ° - a MI" lO-.2 M) increased the rate of conversion considerably. With Mg .2+ present, practically all the D to I conversion has taken place after 6o rain of preincubation, whereas the I activity in the incubation mixtures without Mg .2+ at the same time is still low and continues to increase slowly. The ratio of activity in the presence of Glc-6-P to that without Gle-6-P changed from 14 at zero time to 1.3 after 6o min of preincubation in the presence of Mg e+, and correspondingly from 14 to 3.2 in the absence of this cation. The increase in I activity in this period corresponded to o.61 ffmole per IOs lymphocytes per h without Mg 2+ and 1.76 /,mole when the cation was present. Fig. 5 also shows the transformation of I to D form of the enzyme obtainable by adding ATP (2. 7. IO-3 M) and Mg 2+ (5.4" IO-a M) to the enzyme source after 60 rain i
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Fig. 5. E f f e c t of t i m e of p r e i n c u b a t i o n a n d M g - A T P a d d i t i o n o n l y m p h o c y t e t r a n s f e r a s e . T h e enzyme was assayed at different time intervals after preincubation with 5' lo a M mercaptoe t h a n o l a n d w i t h or w i t h o u t i . i o 2 M MgC12. A t 6o r a i n M g - A T P (2. 7 .IO - a M - 5 . 4 . I o a M ) w a s a d d e d t o p a r t of t h e i n c u b a t e d e n z y m e . T h e a s s a y s y s t e m c o n t a i n e d e n z y m e c o r r e s p o n d i n g t o 7.3" lo'~ l y m p h o c y t e s , 4" IO a M U D P G , a n d o. 7- i o 2-M G l c - 6 - P w h e n p r e s e n t . O - O, 1 enzyme; O ©, 1 e n z y m e + Mg2+; O - 0 , t o t a l e n z y m e (I + D ) ; • @, t o t a l e n z y m e ([ + D) + Mg'::.
13ioch2m. tCiophys. Acta, r3o (t966) 393 -4 ° 0
398
c . j . HEDESKOV, V. ESMANN, M. ROSELL PI~RE2
of preincubation. The addition did not change the pH from 7.8. In 2o rain the I activity decreased corresponding to o.64 /,mole per lO8 lymphocytes per h in the preparation without Mg2+ and 1.78 /,mole in the one with Mg2+ previously added. As the activity in the presence of Glc-6-P (equal to total I + D activity) in the same period decreased by o.42 and o.29/,mole, respect'vely, the net reversal of I to D form amounts to o.22/,mole per IO8 lymphocytes per h in the first case and 1.49/,mole in the second case, which means that a comparatively larger proportion of the I enzyme wa~ converted to the D enzyme in the experiment with Mg2+. From these experiments i~ vitro it is concluded that normal human lymphocytes possess the systems for the interconversion of the two forms of UDPglucose-glucan glucosyltransferase.
I~flue1~ce of coutamit~ati~g erythrocyles Transferase activity of erythrocyte hemolysates has recently been reported 22. In order to check the percentage of enzymatic activity due to the contaminating red cells, the following experiment was performed. Red cells were sedimented at 37 ~' with dextran and the supernatant plasma was removed. The erythrocytes were further packed by centrifugation at IOOO × g for io min and the supernatant fluid together with the most superficial layer of red cells was discarded. A o.5-ml aliquot was taken from the center of the packed cells, washed twice with cold saline and suspended in buffer to a concn, of 5.5.1o 8 cells/ml. Microscopic examination in a counting chamber did not reveal any white cells among 1. 4. io '~ red cells. The erythrocytes were homogenized and aliquots corresponding to 2.75. io 7 and 5-5" lO7 red cells, which is equivalent to the amount of red cells actually contaminating the lynlphocyte suspensions, were tested for transferase activity. No radioactivity was incorporated into glycogen in the absence of (ilc-(>P. Ill the presence of this compound, with or without Mg('l~, an incorporation o[ 4-8 counts/rain above background was detected, which, if it is assumed to represent transferase activity, corresponds to o.oi 0.o2 /,mole per io a erythrocytes per h, or approx. I °o of the activity of the lymphocytes. The transferase activity found in erythrocytes corresponds to the values reported by COI~NBLATHet ill. 2'2. No gly('()gen could be detected in these pure erythrocyte suspensions. 1)ISCUSSION The presence of glycogen and the enzymes involved in its biosynthetic pathway has been demonstrated in lymphoeytes from normal humans by the experimental work presented here. The glycogen content in lymphocytes was found to be 3 z ~ 6 mg per IOu) cells. In a previous communication ~6, the glycogen content of a mixture of 8o % polymorphonuclear leucocytes and 2o % mononuclear cells was calculated as 148 mg glycogen per lO1° polymorphonuclear leucocytes assuming that all glycogen determined originated from polymorphonuclear leucocytes. In view of the present findings, a recalculation shows the presence of 14o mg glycogen in IOu~ polymorphonuclear leucocytes, when mononuclear cells are credited with 31 mg per IOL° cells. Conversely, the observed contamination of lymphocyte suspensions with 2- 3 % polymorphonuclear leucocytes accounts for 3-4 mg of the measured glycogen content of the lymphocyte suspensions. On a cellular basis the glycogen content of lymphocytes
tli,chim. Hioph),s..lch~. 13° (1960) 393 4 oo
GLYCOGEN SYNTHESIS IN LYMPHOCYTES
39 ()
is thus only one-fifth of t h a t in polymorphonuclear leucocytes. Based on wet weight, however, the glycogen content of lymphocytes (lO l° cells = 3.3 g) is o.9 % and of polymorphonuclear leucocytes (IO1° cells = I o g) 1.4 %. Both values are close to tile glycogen content of muscle and liver from fasting subjects ~a. LRIKINit reported the presence of iz 4 mg glycogen per zo 1° cells in suspensions with more than 9 ° °o normal h u m a n lymphocytes. This value appears to be rather high and would correspond to 3.4 °o of the wet l y m p h o c y t e weight. The activity of the UDPglucose-glucan glucosyltransferase present in these cells is, like that in the polymorphonuclear leucocytes ~, a D form with some characteristics not found in polymorphonuclear leucocytes. Unlike the polymorphonuclear leucocytes, the lymphocytes have the capacity of converting the D form of the enzyme to the I form, which is more active in the sense that it does not require Glc-6-P for activity. The system that carries out this conversion operates i~ vitro more slowly than the similar system found in muscle of rat, rabbit and dog15, ~,'~. On the other hand, the D to I transformation is very much enhanced if a sufficient concentration of Mg "~+ ions is present. This Mg2--activated I) to I conversion, detected here for the first time, has also been demonstrated in peritoneal exudate leucocytes from both normal and alloxan-diabetic rats ~5, whereas the D to I conversion system in rat diaphragmatic muscle is unaffected of Mg 2~ ions (unpublished observations). In normal h u m a n polymorphonuclear leucocytes no I) to I conversion could be d e m o n s t r a t e d - - - e v e n in the presence of Mg 2+ ions "6. The D to I conversion system provides the lymphocytes with a mechanism t h a t would allow them to increase their stores of glycogen without increasing the Glc-6-P pool, the necessary activator of the D form. The switch for the D to I transformation is as yet unknown, but could possibly be produced by some kind of hormone. Thus, insulin seems to have influence on the appearance of such conversion systems in leucocytes of diabetic h u m a n s 26. The system t h a t converts the I to the D form of synthetase is also operational and functions rapidly in a parallel w a y to the system found in muscle preparations ~. The quantitative differences in the transformation of I to D form in the absence or presence of Mg 2 ~ previously added (Fig. 5) could signify t h a t in the absence of this cation the transformation is not carried out completely, the remaining part of the e n z y m e being in an inactive intermediate state 2~, ~. The levels of transferase activity in lymphocytes are on a cellular basis about 2 times lower t h a n those found in polymorphonuclear leucocytes (3.1o-7.o4 /xmoles per IOs cells per h (ref. 2)), but on a weight basis lymphocytes will haw~ an activity approx. 1.5 times t h a t of polymorphonuclear leucocytes. The levels of activity reported here and those previously c o m m u n i c a t e d for polymorphonuclear leucocytes "~ are in agreement with values found b y HUIJING2s and b y MILLER AND VAN ])ER W E N D E 2u, whereas CORNBLATHet a!. 2'~ have reported values 2 - 3 times higher. The fact t h a t Pi competes with the allosteric activator Glc-6-P stresses t h a t the l y m p h o c y t e transferase is a D form of the enzyme. The inhibitory effect of Pl is also found in normal h u m a n polymorphonuclear leucocytes (unpublished observations), whose enzyme form has an ahnost absolute requirement for Glc-6-P. This phosphate effect will provide the cells with one more mechanism for the regulation of their c a r b o h y d r a t e stores b y controlling enzyme activity without modifying enzyme level, e.~.*, bv~ diminishing transferase activity (luring anaerobic conditions. Biochim. I3io/~h>,s..~lcfa, 230 (I9>)} 3~)3 4()O
400
C.J.
HEDESKOV,
V. ESMANN,
M. ROSELI.
I't~RI'~Z
.\CKNOWLEDGEMENTS
We are greatly indebted to Dr. F. KISSMEYER, Chief of the Blood Bank Service, Aarhus K o m m u n e Hospital for supplying us with blood samples and to Prof. I. C. SKOU, Department of Physiology, Aarhus University for access to counting equipment. This investigation was supported by a grant from Statens Atmindelige Videnskabsfond (L 36z65). One of the authors (M.R.P.), collaborator of the Institute J aime Ferran del S.C.I.C. of Spain was supported by U.S. Public Health Service (irant AM ~oo23-o*.
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]~;iochim. 13iophys..lcta, 13o (IO()O) 393 4 ° o