The Phosphorylation and Absorption of Sugars in the Rat

Transcription

1 Vl The Phsphrylatin and Absrptin f Sugars in the Rat 1. HEXOKINASE ACTIVITY IN THE INTESTINAL MUCOSA By M. P. HELE* Bichemical Labratry, University f Cambridge (Received 14 January 1953) and galactse are knwn t be mre rapidly absrbed frm the small intestine f the rat than fructse, mannse and the pentses (e.g. Cri, 1925). The mre rapid absrptin fthe first tw sugars has been attributed t their underging phsphrylatin in the intestinal mucsa during absrptin, a prcess named 'active' r 'selective' absrptin. The less rapidly absrbed sugars have been cnsidered t pass thrugh the mucsa by a prcess f diffusin, knwn as 'passive' r 'nn-selective' absrptin (Verzar & McDugall, 1936). The evidence prduced fr the direct participatin f sugar phsphrylatin in selective absrptin cnsists mainly f studies upn the effect f idacetate pisning (Wilbrandt & Laszt, 1933), r phlrrhizin pisning (Lundsgaard, 1933; Wertheimer, 1933) n sugar absrptin in viv, and upn the accumulatin f phsphrylated sugars in the mucsa during sugar absrptin (Laszt & Sulhnann, 1935; Lundsgaard, 1939; Beck, 1942). Whilst indicating that the phsphrylatin f sugar in the mucsa is prbably a necessary cncmitant f sugar absrptin, this evidence is nt f itself sufficient t demnstrate that phsphrylatin participates directly in sugar absrptin as part f the transfer mechanism. Other investigatrs have shwn that the distinctin' nce drawn between active and passive absrptin may nt be s clear as was nce thught. Esters are knwn t accumulate in the mucsa during the absrptin f fructse (Lundsgaard, 1939; Kjerulf-Jensen, 1942) and ther passively absrbed sugars (Verzar & Sullman, 1937). Xylse, in the cat, can be as rapidly absrbed as glucse (Davidsn & Garry, 194). If the absrptin f sugars frm the small intestine invlves the direct phsphrylatin f these sugars, then it might be expected that the phsphrylatin rates f different sugars in vitr wuld bear the same relatinship t each ther, as d their absrptin rates in viv. The demnstratin f such a relatinship has been attempted here. The results reprted fall int tw grups, the first dealing with the many factrs cncerned in the develpment f an assay fr hexkinase activity in' dispersins f intestinal mucsa, the secnd with a mre detailed * Benn W. Levy Student. study f the hexkinase. The terms 'phsphatase' and 'ATPase' are used t designate certain activities present in such dispersins, nt as identificatin fr specific enzymes. EXPERIMENTAL Piebald Nrwegian rats were used, f bth sexes, and weighing 13-2 g. They were maintained n the fllwing synthetic diet: 4% rice starch, 17% sucrse, 23% light white casein, 15% arachis il, 5% salt mixture, 7.5% dried yeast, 2.5% cd-liver il. A 15g. rat cnsumed abut 15 g. f this mixture (dry weight) daily. The animals were nt fasted befre the experiments. Preparatin f ti8sue suspe8ins. The animal was killed by deeapitatin, and the whle length f the small intestine excised and placed in cld, glass-distilled water. The intestine was pened and gently cleaned with a paintbrush. The intestine was bltted with filter paper, weighed t the nearest *5 g., returned t clean water, and cut int shrt lengths. The pieces were grund fr 3 min. by hand with an equal weight f water in a gla#s hmgenizer kept n ice. The pistn had a smth surface and fitted lsely. The preparatin was squeezed thrugh muslin t remve the pieces f muscle layer. Abut 8-16 ml. were btained, cntaining abut 25 mg. wet tissue/ml. The hexkinase remained active twards glucse fr 2 hr., whilst the ATPase and phsphatase were stable fr at least 2 days. Bacterilgical examinatin shwed that cntaminatin with Escherichia cli and Streptcccus faecali8 was negligible. Histlgical examinatin shwed that after 3 min. treatment in the hmgenizer the clumnar epithelium alne was split frm the mucsa, the cres fthe villi remaining behind, attached t the muscle layer, which appeared t be intact. Very few intact cells were bserved in the suspensin. All f the suspensins used were prepared frm single animals. Assay f hexkinase activity. The final prcedure fr the determinatin f hexkinase activity was as fllws. Tissue suspensins were incubated in test tubes at 3 in the fllwing mixture: -5M vernal-acetate buffer, ph 8., -1 M-MgC12, -6m-NaF, sugars f varying cncentratins (l14--28m) and adensine triphsphate (ATP) f varying cncentratin ( m); the final vlume was 2 ml., cntaining *7 ml. tissue suspensin, equivalent t abut 175 mg. wet tissue; fluride was added last s as t avid the frmatin fa precipitate (Ptter, 1947). Samples f 5 ml. were remved at intervals and treated with Ba(OH)2 and ZnSO4 t remve prtein and phsphrylated sugar befre the estimatin f free sugar by the methd f Nelsn (1944). The rate f the hexkinase reactin was It was fllwed by the rate f disappearance f free sugar. nt fund practicable t fllw the reactin by the dis-

2 858 M. P. HELE I953 appearance f ATP because f the high cncentratin f ATP which had t be used in rder t cunteract the effect f c ATPase. Fr the study f the reactin with galactse, suspensins cl were prepared with the magnesium-ringer slutin described by Meyerhf & Geliazkwa (1947). Bicarbnate was E 8 mitted, as suspensins prepared with bicarbnate-cntaining Ringer were viscus and inactive. Suspensins VI._ prepared with water did nt phsphrylate galactse, but S -4 thse prepared with magnesium-ringer did s, althugh the rate fell ff after the first 2-5 min. All determinatins f the phsphrylatin rate f galactse were made fr this time.lea interval, and mst f the suspensins s examined were als tested with glucse and fructse under the same cnditins. The rates bserved with these tw latter sugars with magnesium-ringer suspensins did nt differ frm the rates fund with water suspensins. A88ay f phsphatase and ATPase activity. A test system similar t that emplyed fr the determinatin f the hexkinase activity was used, withut NaF, and with the apprpriate ester r ATP added instead f sugar and ATP. Fr the phsphatase reactin 5 ml. samples were treated with twice the vlumes f Ba(OH)2 and ZnSO,, 7H2 prescribed by Nelsn (1944) s as t ensure the remval f all the phsphrylated sugar and the free sugars were then determined by Nelsn's methd. Fr the ATPase reactin the samples were deprteinized with 5% trichlracetic acid, and the inrganic and 9 min.-labile P estimated by the methd f Fiske & Subbarw (1925). The rates f these reactins were expressed as 'mg. free sugar (r inrganic phsphate) frmed/ml. tissue suspensin/unit f time'. Preparatin f ATP. ATP was prepared frm rabbit muscle accrding t Bailey (1949). The ATP s btained was cntaminated with a metal, prbably irn, which seriusly interfered with the hexkinase reactin. This metal was remved by stirring the ATP with 8-hydrxyquinline in CHC13 (Waring & Werkman, 1942). The ATP was finally precipitated as the sdium salt, using a mixture f equal vlumes f ether and ethanl, 9 vl. f mixture t 1 vl. f ATP slutin. Sugars. The sugars used, D-glucse, D-galactse, D- fructse, D-mannse, and D-xylse, were Kerft's 'bacterilgical sugars'. Factrs affecting the determinatin f hexkina8e activity in the inte8tinal mucsa Blanks and cntrls. Fur tissue suspensins, examined by the Nelsn prcedure, gave n appreciable reducing value when fresh, r after incubatin with vernal-acetate buffer r with inrganic phsphate, -33M and -16M. These phsphate cncentratins represent the limits f inrganic phsphate appearing in the digest in the presence f ATP during the hexkinase reactin. One fresh suspensin cntained *9 mg. inrganic P/ml. which rse t -12 mg./ml. after 1 min. incubatin. Small amunts f 9 min.-labile P and 3 hr.-hydrlysable P were fund, amunting t -1-2 mg./ml. suspensin. Incubatin f glucse r fructse (-55M) with ten different suspensins in the absence f ATP did nt lead t any disappearance f sugar, althugh in the presence f ATP rapid sugar disappearance was bserved. Incubatin f -28M glucse (fur suspensins) r -28M fructse (tw suspensins) with -65M disdium phsphate gave n disappearance f free sugar, althugh gd sugar dis Time (min.) 5 1 Fig. 1. Sugar disappearance as a measure f the hexkinase reactin. Each pair f curves was btained frm ne animal. The test system emplyed is described in the experimental sectin. The glucse, -, and fructse, -, cncentratins were -55M, and the ATP cncentratin was -33M. appearance was fund with ATP, and the presence f an active phsphatase was indicated by the rapid hydrlysis f added glucse 6-phsphate r fructse 6-phsphate. ATP (-33M) gave a small reducing blank equivalent t abut 1 mg. glucse n each estimatin, and did nt alter n incubatinwith the suspensins in absence f sugar (three suspensins). Buffer slutins. Three suspensins were tested fr hexkinase activity with bth glucse and fructse (-55M) in the fllwing buffers: vernal-acetate buffer (ph 8-,-5M), phsphate buffer (ph 8-,.5M), and bicarbnate buffer (ph 7.5,.2% NaHCO3 with a gas mixture f 95% N2 and 5% CO). The rates apprpriate fr each sugar did nt differ with the buffer used. In glycine buffer (ph 8-5, -5M) the rate with glucse was depressed relatively t the rate with fructse, and a slight increase f inrganic phsphate at the expense f 9 min.-labile P was bserved. In brate buffer (ph 8-, -25M) phsphatase and ATPase activities were depressed by 5 %, and lwer cncentratins f ATP culd be used t give the same rates bserved in vernal-acetate buffer at a higher ATP cncentratin. Added magnesium. The suspensin cntained enugh magnesium t allw sme phsphrylatin t ccur, but the maximf.m rate was nt btained unless added MgCl2 were present in a final cncentratin f 1 M. Time f incubatin. A fall in the reducing pwer f the samples was bserved during the first 1 min., and the disappearance f sugar prceeded at a steady rate during the first 5 min. f the incubatin perid. After the first 1 min. the reducing pwer rse again (Fig. 1) but this culd be prevented by the additin f mre ATP, by the additin f fluride t depress ATPase activity, r by the additin f pyruvate. During the first 1 min. f the reactin the rati between the phsphrylatin rates f glucse and fructse remained the same (Table 1), but after this time the rate f the reactin with fructse fell ff much mre rapidly than that with glucse. This cnstancy f rati during the early part f

3 Vl. 55 PHOSPHORYLATION AND ABSORPTION Table 1. Relative rates f phsphrylatin f fructse and gluc8e (The cncentratins ffructse and glucse were 55m, and that f ATP -33M. Results are expressed as the mean rati x 1 ± S.D. (number f suspensins) f fructse t glucse disappearing (mg. sugar/ml. suspensin) at prgressive intervals.) Time frm start f incubatin (min.) Rati f phsphrylatin fructse/glucse x 1 46±4.2 (19) 48±3.6 (25) 5±J3.7 (49) 42±5 (12) 3±4-6 (12) the incubatin perid was fund with almst every ne f the tissue suspensins examined, nt nly with glucse and fructse, but with mannse and xylse as well. With galactse, hwever, the rate f the reactin fell ff sharply after the first 2-5 min. Stability during incubatin. Samples f suspensins were incubated at 3 fr a given time in the presence r absence f sugar. These samples were then tested fr hexkinase activity in the usual way (Table 2). The inactivating effect f previus incubatin upn hexkinase activity and the stabilizing effect f added sugar during incubatin were fund t be very variable. The rate f the reactin with fructse and galactse fell cnsiderably n incubatin, and neither sugar shwed any stabilizing effect. The rates with glucse, mannse and xylse were unaffected by incubatin in the absence f sugar. ATP had n prtective effect if added during incubatin. Cncentratin f tissue suspensin. The rate f the hexkinase reactin with all five sugars was linear with increasing amunt f suspensin up t a cncentratin f 1 part suspensin in 3 f the digest. The phsphata8e and ATPase reatins. Fig. 2 shws the appearance, ver a perid f time, f free sugar frm glucse 1- and glucse 6-phsphates, frm fructse 6-phsphate and frm fructse 1:6-diphsphate. It als shws the appearance f inrganic phsphate frm ATP. All mnphsphates gave rise t free sugar at the same rate, but the diphsphate did s at nly half the rate. The reactin with ATP ceased when bth the 9 min.-labile P grups were cnverted int inrganic phsphate. The rate f appearance f free sugar increased with increasing cncentratin f mnphsphate esters up t 1M (Table 3). The rate f sugar liberatin with the diphsphate was nly half that bserved with the mnphsphates, and did nt increase with cncentratins abve -7M. The rate f appearance finrganic phsphate frm ATP increased up t an ATP cncentratin f -33M. The inrganic phsphate appearing was equivalent t the 9 min.-labile P disappearing. Bth phsphatase and ATPase activity were raised 3% if MgCl3 (final cncentratin -O1M) was added t the reactin mixture. Fluride (-6M) ablished this effect. Phlrrhizin (.4m) and idacetate (-8M) did nt affect phsphatase r ATPase activity. Pre-incubatin f the suspensin at 3, under cnditins similar t thse used in the study f the hexkinase, had n effect upn either phsphatase r ATPase activity. The rate f bth reactins increased prprtinally t the cncentratin f the suspensin, up t 1 part suspensin in 3 f the digest. "e.s 14- AX )* m q ~ P4 8 6 e s ] b 4.4 C> az t w a2 c CsA CO _ eors O~ C> c t'> > 1 1 _ CO 1 1n r IC I c -H -H -H~ l cc u: 1 CO X P " X 6 aq 1 c- -H j'4h -H c) 1 E- CO? CO (: t CO CO c 1-H -H. C Xg H -H + c x - e -- rq -H -H 1 +' + I., m t m = ~~~~ CD

4 86 M. P. HELE I953 Table 3. The effect f substrate cncentratin upn the rates f the phsphatase and ATPa8e reactin8 (The rates f the phsphatase reactin are expressed as 'mg. free sugar appearing/ml. suspensin/5 min.'. The rates f the ATPase reactin are expressed as 'mg. inrganic phsphate appearing r 9 min.-labile P disappearing/ml. suspensin/ 1 min.'. The results are given as: mean ±s.d. (n. f suspensins).) Substance examined Phsphatase 6-phsphate* Fructse 6-phsphate* 1-phsphate Fructse 1:6-diphsphate Adensine triphsphatase Appearance f inrganic phsphate Disappearance f 9 min.-labile P Phsphrylated sugar (m) *35 *7 *1-28 *48±-21 (8) *78±*4 (8) *99±*6 (8) 96±-5 (8) *49±*3 (8) -84±-4 (8) *99±*5 (8) *9±-3 (8) *49±*4 (3) *8±-11 (3). 1-3±-7 (3) 1-14±-5 (3) *29±-3 (3) *49±-3 (3) -48±- (3) 73± (3) ATP cncentratin (M) * ±-5 (4) *81+-6 (4) *41±*6 (4) -73±-4 (4) * Same suspensin used (3) 1-5±-12 (4) -81±*7 (4) -98±-7 (4) raised t -28M the rate fthe reactin varied with the ATP cncentratin in a manner similar t that bserved with a glucse cncentratin f -55M. Mannse and xylse. At a sugar cncentratin f -55M the rate fthe hexkinase reactin with mannse and xylse was nt appreciably affected by changes in ATP cncentratin (Table 4). At a sugar cncentratin f -22M the rate varied mre markedly with the ATP cncentratin. Gakzte. At a sugar cncentratin f -55M the rate f the phsphrylatin reactin varied with the ATP cncentratin ver the range *13--33M (Table 4). 5 IC Time (min.) Fig. 2. Phsphatase and ATPase activity. One animal was emplyed, using a test system as described in the experimental sectin. The cncentratin f phsphrylated sugars emplyed was 828m, and f ATP -33M. 6-phsphate, -; fructse 6-phsphate, **-; glucse 1-phsphate, x-x; fructse 1:6- diphsphate, E-E); ATP, 3. The effect f ATP cncentratin upn the rate f the hexkinase reactin andfructse. The rate fthe hexkinase-reactin at O55M glucse increased with the ATP cncentratin (.13-.4m), whilst the rate with -55m fructse was unaffected by changes in ATP cncentratin ver the same range (Table 4). At an ATP cncentratin f *13m the rates with bth sugars were the same, whilst at X33M ATP the rate with glucse was nearly twice the rate bserved with fructse. When, hwever, the fructse cncentratin was The effect f sqgar cncentratin upn the rate f the hexkinase reactin andfrutse. The rate fthe reactin with glucse, at a cnstant ATP cncentratin f -33M, shwed a sharp drp with increasing glucse cncentratin, this drp being usually bserved at -16m glucse. With a further increase in the cncentratin the rate rse sharply, until at a cncentratin f -28m glucse the rate was the same as that btained at -55M glucse (Table 5). Out f twenty-five suspensins examined twenty-tw shwed this sharp minlimum. Increasing the ATP cncentratin t -52m led t the flattening f this dip in the glucse cncentratin curve, althugh the rate at *28m r -55m glucse was unaffected. The rate f the hexkinase reactin varied with the glucse cncentratin ver the range ' M, at an ATP cncentratin f -33M(Table 6). In cntrast with the reactin with glucse, the rate f the reactin with fructse at a cnstant ATP cncentratin f -33m increased steadily with the fructse cncentratin t -22M. The rate at the higher fructse cncentratin was nt increased appreciably by raising the ATP cncentratin. Mannse and xylse (Table 5). The rate f the reactin with mannse and xylse increased with sugar cncentratin frm -55 t *22M. The phsphrylatin rate f mannse was a little greater than that f xylse, the rates with bth sugars being less than a third f the rate bserved with glucse at a sugar cncentratin f-55m. At a sugar cncentratin f *28x the rates, especially with mannse, apprached that bserved with glucse at this cncentratin.

5 VI. 55 PHOSPHORYLATION AND ABSORPTION Table 4. The effect f ATP cncentratin upn the rate f the hexkinase reactin (The sugars were tested at the sugar cncentratins indicated. The rates f the reactins are expressed as 'mg. sugar disappearing/ml. suspensin/1 min. (2.5 min. fr galactse)'. The results are given as: mean ±S.D. (number f suspensins), extreme range f values.) Sugar examined in (M) -13 ( 55) (1) Fructse ( 55) (1) * Fructse ( 28) (3) Mannse ( 55) - Mannse ( 22) (4) Xylse ( 55) - Xylse (4) (.22) * Galactse ( 55) -15, 3 (2) -2-64±-3 (1) ±-4 (9) ±-5 (3) ±-7 (4) 9-45 *12±-5 (4) , -6 (2) ATP cncentratin (m) A t4 (9) *8+-6 (7) (9) (7) (3) *46±-8 (3) (4) -24-* (3) *63±*2 (4) *3 (4) *23±-4 (3) * *26+-3 (4) ±+-3 (4) (4) * ,.66 (2) *84, *96 (2) 4 1-9±-6 (7) (7) (3) *38+-6 (4) ±-12 (4) O4 (4) * (4) *84, 1-2 (2) -52 1*79±-6 (3) Table 5. The effect f 8ugar cncentratin upn the rate f the hexkina8se reactin (The sugars were tested at the ATP cncentratin indicated. The rates f the reactins are expressed as: 'mg. sugar disappearing/ml. suspensin/1 min.'. The results are given as: mean ±s.d. (number f suspensins), extreme range f values.) Sugar cncentratin (m) * M ATP (12) -84-1*56-71±-9 (11) (12) ±-6 (1) *54-1*14-97±-1 (1) m ATP (4) -96-1* (4) -6-1*2-94±-8 (4) (4) 1* (4) * Fructse +-33M ATP (8) -42-* (7) *63-*9 1-24±-7 (8) (6) 1* ±-18 (6) Mannse + 33M ATP 3+ 5 (5) (4) ± 7 (5) ±-5 (5) * ?4 (4) * Xylse +-33M ATP -28±-4 (5) -41 ±-6 (4) * * (5) (5) ±-5 (4) -56-*72 Galactse +33M ATP, incubated 2-5 min (6) (6) ±-1 (6) (6) (6) Table 6. The effect f lw glucse cncentratins upn the rate f the hexkina8e reactin (The ATP cncentratin was -33m. The rates f the reactin are expressed as: 'mg. sugar disappearing/ml. suspensin / 5 min.'. The results are given as: mean±s.d. (number f suspensins), extreme range f values.) cncentratin (M) * (5) (5) -54-*72 75± 3 (4) * *8+*4 (5)

6 862 M. P. HELE I953 Table 7. The phsphrykain f 8ugar mixtures (The rates f the reactins are expressed as 'mg. sugar disappearing/ml. suspensin/given unit f time'. The results are given as: mean +s.d. (number f suspensins), extreme range f values.) Mixture used /fructse (1 min.) /mannse (1 min.) /xylse (5 min.) /galactse (2-5 min.) Single sugar, -55m, and ATP, -33M 1-6±-5 (9) ±-5 (4) Fructse -56±-4 (9) Mannse -32±-5 (4) Xylse -96, 1-38 (2) -36, -36 (2) Galactse -64±-6 (3) ±-1 (3) r Mixture f tw sugars, each {55m, and: ATP, -33m (9) ATP, -52m -98±-14 (5) ±-8 (4) -38±-8 (4) , -12 (2) -42, -48 (2) -23±-12 (3) -69±-2 (3) Galact8e. The rate f galactse disappearance varied with increasing galactse cncentratin ver the range m, in a manner similar t that bserved with glucse ver the same range, at the same ATP cncentratin, -33M. The ph8phrylatin f sugar mixtures (Table 7). Mixtures f glucse and f ne ther sugar, each at a cncentratin f -55M, ATP -33M, did nt shw a rate f sugar disappearance equal t the sum f the rates bserved when the sugars were tested separately at the same substrate cncentratins. The rate with the mixture was usually lwer than that bserved with the glucse cntrl, and was in sme cases lwer than the cntrl with the ther sugar. Raising the ATP cncentratin frm -33 t -52M resulted in an increase in the rate f sugar disappearance, althugh a rate equal t the sum f the cntrl rates was never btained. DISCUSSION A pssible surce f errr in the determinatin f hexkinase activity culd lie in the actin f the phsphatase, releasing reducing substance frm phsphrylated derivatives frmed during the hexkinase reactin. The apparent difference bserved between the phsphrylatin rates f tw sugars might thus be due t a difference in the rate f breakdwn f their phsphrylated derivatives. Hwever, glucse and fructse mnphsphates shwed the same dephsphrylatin rate. Mrever, since the rate f the phsphatase reactin is dependent upn the rate ffrmatin f phsphrylated sugar by the hexkinase, it fllws that the rate f the hexkinase reactin must play a part in deciding the dephsphrylatin rate. In the case f glucse, it can be calculated that the bserved hexkinase activity during the first 5 min. f the incubatin perid is at least 7 % f what it shuld be in the absence f cmplicating phsphatase activity. Later n during the incubatin perid the ATP cncentratin falls, the hexkinase reactin slws dwn and the dephsphrylatin reactin becmes mre rapid than the hexkinase reactin, but this can be prevented by increasing the ATP cncentratin, r by depressing the ATPase and phsphatase activity by the use ffluride r brate. It wuld appear that the bserved behaviur f the hexkinase during the first 1 min. f the reactin perid reflects the intrinsic prperties fthe enzyme, and is nt an artifact. The hexkinase f the intestinal mucsa shws tw different patterns in its respnse t changes in sugar cncentratin. With fructse, mannse and xylse the phsphrylatin rate varies with the sugar cncentratin ver the range M. With glucse and galactse the rate falls t a sharp miniimum at sugar cncentratins in the intermediate part f this range, the rate rising again as a cncentratin f -28M is apprached, at which cncentratin the rate bserved is the same as that bserved at -55m. This falling ff in rate can be partially prevented by raising the ATP cncentratin, althugh n effect is bserved upn the rate f the reactin at the tw limits f this sugar cncentratin range. The cause f the sharp drp in the rate f the hexkinase reactin at intermediate sugar cncentratins is nt knwn. Pssibly tw hexkinases might be cncerned, each having different affinities fr glucse, the affinity being determined by the ATP cncentratin. The wrk f Meyerhf & Geliazkwa (1947) shws that the affinity f brain hexkinase fr glucse and fructse may be determined by the ATP cncentratin, but there is n evidence, utside the 'minimum' shwn in the glucse and galactse cncentratin curves, that the hexkinase f the intestinal mucsa behaves in a similar manner. The data presented here with regard t the effect f ATP cncentratin n sugar utilizatin may be interpreted upn the assumptin that

7 Vl. 55 PHOSPHORYLATION AND ABSORPTION the intestinal hexkinase has different affinities fr mucsa. His figures fr Qglucse9 when cnverted different sugars, which are unaffected by changes in int terms f mg. glucse disappearing/ml. suspensin/5 min., yield an average value f 54 mg., the ATP cncentratin. As lng as the sugar cncentratin limits the rate f the reactin, changes in with a range f fr eleven rats. These ATP cncentratin will have a negligible effect figures are a little lwer than thse given in the upn the reactin rate, but as sn as the sugar cncentratin is raised t nn-limiting levels the effect described by Lng is unaffected by changes in present study. The rate f the hexkinase reactin f changes in ATP cncentratin is nticeable. glucse cncentratin ver the range 12- The depressin fphsphrylatin rates bserved 5M, thus differing frm the hexkinase discussed here, but therwise the results f the tw with the intestinal hexkinase when mixtures f sugars are used bradly resembles the findings f investigatins are in agreement. Slein, Cri & Cri (195) upn the phsphrylatin f sugar mixtures by yeast hexkinase. These wrkers shwed that in mixtures f fructse with either glucse r mannse the aldse suppressed the utilizatin fthe ketse, whilst in a glucse-mannse mixture the rates with bth sugars were depressed. With the intestinal hexkinase, all sugar mixtures examined gave a depressin in rate, which suggests that interference takes place. In the case f the glucse-fructse mixture the depressin is nt s great as with sme f the ther mixtures, and it is pssible that in this case the ATP cncentratin becmes the limiting factr. It is als pssible that the additin f high cncentratins f ATP culd alter the affinity f the hexkinase fr the sugars in the mixtures, as suggested by Slein et al. (195). The pssibility exists that tw r mre hexkinases may be present in the intestinal mucsa. The separate existence f a gluckinase and a fructkinase has been demnstrated fr brain (Meyerhf & Geliazkwa, 1947), muscle (Slein et al. 195), and liver (Cri, Ocha, Slein & Cri, 1951). Recently Lng (1952) has published data n glucse phsphrylatin by dispersins f intestinal Bailey, K. (1949). Bichem. J. 45, 479. Beck, L. V. (1942). J. bil. Chem. 143, 43. Cri, C. F. (1925). J. bil. Chem. 66, 691. Cri, G. T., Ocha, S., Slein, M. W. & Cri, C. F. (1951). Bichim. biphys. Acta, 7, 34. Davidsn, J. N. & Garry, R. C. (194). J. Phy8il. 97, 59. Fiske, C. H. & Subbarw, Y. (1925). J. bil. Chem. 66, 375. Kjerulf-Jensen, K. (1942). Acta physil. 8cand. 4, 224. Laszt, L. & Sulimann, H. (1935). Bichem. Z. 278,41. Lng, C. (1952). Bichem. J. 5, 47. Lundsgaard, E. (1933). Bichem. Z. 264, 29, 221. Lundsgaard, E. (1939). Hppe-Seyl. Z. 261, 193. REFERENCES SUMMARY 1. The hexkinase reactin, the dephsphrylatin f hexse phsphates, and adensine triphsphatase activity have been studied in dispersins f intestinal mucsa. 2. Different sugars are phsphrylated at different rates, and the hexkinase shws different patterns in the respnse f the phsphrylatin rates t changes in sugar cncentratin. 3. When the sugar cncentratin is nt the limiting factr the rate with all the sugars examined varies in the same manner with the adensine triphsphate cncentratin. 4. Sugars in mixtures shw a depressin in phsphrylatin rate belw that f single sugar cntrls. 5. The difference in the bserved phsphrylatin rates f glucse and fructse is due t a prperty f the hexkinase, nt t a difference in the rate f liberatin f free sugar frm the phsphrylated sugars frmed, nr t the cnditins f assay. I am very grateful t Dr A. M. Barrett fr histlgical examinatin f specimens f intestinal mucsa. Meyerhf,. & Geliazkwa, N. (1947). Arch. Bichem. 12, 45. Nelsn, N. (1944). J. bil. Chem. 153, 375. Ptter, V. R. (1947). J. bil. Chem. 169, 17. Slein, M. W., Cri, G. T. & Cri, C. F. (195). J. bil. Chem. 186, 763. Verzar, F. & McDugall, E. J. (1936). Absrptin frm the Intestine, 1st ed. Lndn: Lngmans Green and C. Verz4r, F. & Sullmann, H. (1937). Bichem. Z. 289, 323. Waring, W. S. & Werkman, C. H. (1942). Arch. Bichem. 1, 33. Wertheimer, E. (1933). Pflhg. Arch. ges. Physil. 288, 514. Wilbrandt, W. & Laszt, L. (1933). Bichem. Z. 259, 398.