BIOCHIMICA tiT BIOPHYSICA ACTA 2 8 I

T H E D I G E S T I V E F U N C T I O N OF T H E E P I T H E L I U M

OF T H E SMALL I N T E S T I N E

I. AN INTRACELLULAR LOCUS OF DISACCHARIDE AND

SUGAR PHOSPHATE ESTER HYDROLYSIS*

D A V I D M I L L E R * * AND R O B E R T ix2. C R A N E

Department o] Biological Chemistry, Washington University School of Medicine, St. Louis, Mo. (U.S.A.)

(Received F e b r u a r y 2oth, 1061)

S U M M A R Y

The relative distribution between the tissue and the medium of the products ot hydrolysis in vitro of sucrose, maltose and glucose 1-phosphate by hamster small intestine was measured. The concentration relationships found indicate that hydrol- ysis of all three of these compounds takes place within the epithelial cells.

INTRODUCTION

The ways in which the epithelium of the small intestine carries out its various digestive functions have not been clearly defined. With regard to carbohydrates, the small intestine is presented with a mixture of disaccharides and sugar phosphate esters as components of the diet or as the result of the degradation of more complex sub- stances by the action of salivary, gastric and pancreatic secretions. I t has not been certain whether hydrolysis of these compounds to monosaccharides occurs primarily within the lumen of the gut or whether they must first enter the cells of the epithelial lining. A prevalent view is that the hydrolyses are catalysed by enzymes present in the "succus entericus" and that the monosaccharides produced are then absorbed from the luminal contents by the intestinal villi a-6. At one time, hydrolytic enzymes were believed to be secreted by the crypts (or "glands") of Lieberkiihn. With the demonstration that the intervillous crypts are primarily centers of intense mitotic act ivi ty associated with the continuous renewal of the epitheliurrY, ~ and not glandular structures, it now appears that these enzymes are contributed to the intestinal con- tents by the normal shedding of epithelial ceils rather than by specific secretory processes. The concept of digestion as an extra-cellular process has nevertheless persisted because it is the presence of the hydrolytic enzymes in the intestinal content, and not their origin, which has been regarded as relevant.

* A p re l imina ry repor t was m a d e to the 33rd A n n u a l Meet ing of t he Centra l Society for Clinical Research , Chicago, N o v e m b e r 5, 196o (J. Lab. Clin. Med., 56 (196o) 928).

** P re sen t address : D e p a r t m e n t of Hema t o l ogy , Jewish Hospi ta l , St. Louis, Mo. (U.S.A.),

Bioehim. Biophys. Acta, 52 (I961) 281-293

282 D. MILLER, R. K. CRANE

In recent years, evidence has accumulated to show that intestinal hydrolysis of disaccharides, and also of peptides, is an intracellular process; that it is, in fact, an integral function of the intact mucosal epithelium 9-n. In vivo observations have shown that the hydrolytic activities of intestinal fluid are not nearly great enough to account for the rapid disappearance of disaccharides and peptides from the lumen if it is assumed that these substances must be split prior to absorption ~*-14. Sugar phosphate esters have been shown to be rapidly hydrolysed when introduced into the lumen although the luminal contents contain virtually no phosphatase activity ~s. Also, in vitro experiments with sucrose by FRIDHANDLER AND QUASTEL 16 and with dipeptides by NEWEY AND SMYTH 17 indicate that these compounds are hydrolyzed within the epithelial cells. These experiments support the concept that terminal hydrolytic digestion in the small intestine is intracellular. The concept has not gained wide acceptance, however, possibly because of the largely circumstantial nature of the evidence.

I t is the purpose of this report to provide direct evidence that sucrose, maltose and glucose I-phosphate are hydrolysed within the epithelialcells of the small intestine of the go]den hamster. The following communication 18 will present evidence to show that the enzymes, maltase and invertase, are predominantly, if not exclusively, located within the brush border region of the epithelial cells.

MATERIALS AND METHODS

Experimental design

When strips of hamster small intestine are incubated with an actively transported sugar, the sugar accumulates within the tissue to concentrations greatly exceeding those in the medium 19. With compounds such as xylose, which are not actively transported, the final tissue concentration, even after prolonged incubation, is only a fraction of the concentration in the medium and may be assumed to measure the entrance of the sugar by free diffusion into a predominantly extracellular compart- ment of the tissue. However, in both cases, the extent of tissue uptake is dependent upon the concentration of sugar in the medium. In the experiments described below, hamster intestine was incubated with one of the disaccharides, sucrose or maltose, or with glucose 1-phosphate. During incubation, free monosaccharides accumulated in the tissue and in the incubation medium. At various time intervals, the relative concentrations of monosaccharides in the tissue and medium were measured and compared with their relative concentrations found during similar incubations with individual monosaccharides replacing the glucosyl compounds. The experiments were done under conditions suitable for active transport of glucose and also under conditions in which active transport was inhibited by various means. Other experiments were done in which the medium concentrations of monosaccharides derived by hydrolysis of disaccharides or of glucose 1-phosphate were varied over a wide range without otherwise altering the conditions of the experiment. From a comparison of tissue accumulation of free monosaccharide formed by hydrolysis with the uptake of the same free monosaccharide added to the medium under these various experimental conditions, it was possible to decide whether hydrolysis had taken place in the medium, at the epithelial cell surface or inside the cell.

Biochim. Biophys. Acre, 52 (1961) 281--293

AN INTRACELLULAR LOCUS OF INTESTINAL HYDROLASES 283

Incubation technique

The present studies were carried out, in vitro, using small strips or rings of everted hamster intestine as described by CRANE AND MANDELSTAM 19. The accumula- tion of sugar in the tissue is expressed as its millimolar concentration with respect to total tissue water with the same correction for tissue solids (0.8) as that previously used. Tissue concentrations were not routinely corrected for extracellular space as they were in the previous study 19 because the conclusions to be drawn are based on a comparison of tissue and medium concentrations of sugar irrespective of the process by which tissue accumulation occurs and of the location within the tissue of the accu- mulated sugar. The following formula was used to make the calculations:

mmol es (filtrate) × h o m o g e n a t e v o l u m e m m o l e s (tissue) = we igh t of t i s sue × 0.8

The homogenate volume in this formula is equal to the volume of the reagents used to prepare the protein-free filtrate plus the wet weight of the tissue.

The utilization of monosaccharides by the tissue was not routinely measured since utilization could only lower the observed values of monosaccharide uptake and an error in this direction would not affect the conclusions drawn. In several experi- ments with sucrose, net hexose utilization was calculated to be of the order of IOO ~moles/g wet wt. of intestine/h.

Assay procedures

All the sugar determinations were done using protein free filtrates prepared with o.19 M zinc chloride and 0.3 N barium hydroxide according to the method of SOMOGYI ~°.

Glucose was determined enzymically. Generally, glucose oxidase and peroxidase (Worthington Biochemical Corporation) were used and the oxidation of o-dianisidine (Eastman Kodak) was measured spectrophotometrically~k Since these enzyme reagents contained appreciable maltase activity, glucose in the presence of maltose was assayed by the coupled reaction of hexokinase and glucose 6-phosphate dehydro- genase in which the reduction of triphosphopyridine nucleotide is measured spectro- photometrically at 34 ° m~ (see ref. 22). The hexokinase and. triphosphopyridine nucleotide were obtained from Sigma Chemical Company and glucose 6-phosphate dehydrogenase free of maltase activity was obtained from C. F. Boehringer and Sons, Mannheim, Germany.

Sucrose was determined by the ROE method 2~, after first making the samples 0.25 N with respect to sodium hydroxide and heating them at IOO ° for IO min to destroy and avoid interference from fructose 24.

Fructose was usually determined by the RoE method. In the presence of glucose and relatively large amounts of sucrose, fructose was determined as the difference between total reducing sugar measured by the method of SOMOGYI ~s and glucose as determined by a specific enzymic method 21.

Maltose was assayed by measuring the formation of glucose after making the sample 1 N in HC1 and boiling for I h. The hydrolysate was neutralized to phenol red with sodium hydroxide and its glucose concentration compared with that of an untreated sample. Glucose in this case was determined by its reaction with hexokinase and glucose 6-phosphate dehydrogenase as described above.

Biochim. Biophys. Acta, 52 (1961) 281-293

2 8 4 D. MILLER, R. K. CRANE

Malerials

The sugars used were reagent grade and were obtained from Merck (glucose and sucrose) and from Pfanstiehl (fructose and maltose monohydrate). Glucose I-phos- phate was obtained as the dipotassium salt from the Sigma Chemical Company. Fructose was recrystallized from absolute methanol. Maltose was treated with Darco G-5 o charcoal and recrystallized from ethanol. The purity of each sugar was checked by paper chromatography using butanol-pyridine-water (6:4:3) as the solvent 2e and a benzidine reagent to locate the sugars on the chromatograms 27. Enough sample was used to permit the detection of reducing impurities to a level of about o. 5 %. Except for maltose, which contained a trace of a slow-moving component, the sugars used were chromatographically pure to this extent. 1,5-anhydro-D-ESHlglucitol was pre- pared in this laboratory and was assayed by techniques previously described28, 29.

RESULTS

Incubation with monosaccharides

Glucose: The process of active transport by which intestinal strips accumulate glucose against a concentration difference has been shown previously to exhibit Michaelis-Menten kinetics and to have an apparent Km of approx. 2" lO -3 M (see ref. 30). There is also a component of glucose entry which is linearly related to its concentration in the incubation medium 19. In the following experiments short incuba- tion periods were used so that the tissue concentrations achieved by active transport were well below the apparent maximal steady state levels 19 and it is assumed that they are proportional to the initial uptake rates.

The relationship of glucose accumulation in intestinal strips to the glucose con- centration in the medium is shown in Fig. I. At each concentration proportionate volumes of the incubation medium were used to provide approximately the same total amount of substrate in each flask. Thus, the medium concentration even in the lower range did not change appreciably during the experiment. In the lower range of medium glucose concentrations, tissue accumulation was due mainly to an active

15

bJ (J

oZ:,o u~ L,JbJ U3..J

I-

l.- z

bj_l P.LOR=f. ! ~ ~ l0

5

i iI0 c/) s p MEDIUM GLUCOSE CONCKNTRATIOI~I

( M M O L E S / t )

~ ~o ~o ~ MEDIUM GLUCOSE CONCENTRATION

(MMOLE5/ [ )

Fig. I. The u p t a k e of glucose b y in tes t ina l s t r ips i ncuba t ed wi th glucose a t v a r y i n g concen t r a t ions and t he effect of phlorizin, lO -4 M. (a) The ini t ial concen t r a t ion of glucose in t he m e d i u m was in t h e r ange o .5-1o m M and t he i ncuba t ion period was io rain. (b) The ini t ial concen t ra t ion of glucose

was in t he r ange 20-80 m M and t he incuba t ion period was 5 min .

Biochim. Biophys. Acta, 52 (1961) 281-293

AN I N T R A C E L L U L A R LOCUS OF I N T E S T I N A L H Y D R O L A S E S 2 8 5

process which was sensitive to changes in the concentration. The apparent Km of this process, obtained from LINEWEAVER-BuRK 31 plot of the da ta in Fig. I , corrected for non-active entrance, was 3.o" IO -a M, in good agreement with the value previously obtained. In the higher range of glucose concentrations, the rate of tissue accumulation further increased, but in a linear fashion. This increase appears to reflect a non-active process. I t s slope is similar to the slope of entrance in the presence of phlorizin, a compound which inhibits the active transport of glucose a2.

Fructose: Following incubation with fructose, the tissue concentration of this sugar approached a constant value with time which was about 3o % of the medium concentration (Table I), in agreement with previous findings that fructose is not actively transported by the intestine 3~. Glucose was formed from fructose33, 3~ at a rate that was fairly constant with time and over a wide range of tissue and medium fructose concentrations, indicating that fructose was not rate-limiting for this process. When o-dinitrocresol was added, the conversion of fructose to glucose was inhibited and the tissue levels of fructose were correspondingly appreciably increased. In none of the experiments was glucose detectable in the medium.

T A B L E I

THE ENTRY OF FRUCTOSE AND ITS CONVERSION TO GLUCOSE IN INTESTINAL STRIPS

Initial medium fructose Incubation Tissue fructose Tissue glucose period

concentration ( mi~ 0 (raM) (raM) (raM)

IO i o 1.87 i .75 20 2 .29 3 .82

20 i o 3 .85 i .92 20 5 .89 3 .84

4 ° i o 7.02 1 .8o 2o 9-85 3 .93

20* i o 5.71 o 20 7 .85 o

o - D i n i t r o c r e s o l in t h e m e d i u m (lO -4 M ) .

Incubation with disaccharides and glucose z-phosphate

Sucrose : When intestinal strips were incubated in IO m M sucrose, glucose rapidly accumulated within the tissue (Table II). Moreover, tissue uptake, relative to the concentration of glucose in the medium, was much greater than when the tissue was incubated directly with glucose. In an experiment with sucrose, after a Io-min incu- bation, the tissue glucose concentration was 17.1 m M and that in the medium o.17 mM. In incubations with glucose, a comparable tissue uptake required medium concentrations of glucose of the order of 5 to IO m M or at least 25 times the final values observed here. The data in Table I I I show that this relationship holds over a wide range of sucrose concentrations. Since the conversion of fructose to glucose is quanti tat ively small (compare Table I I with Table I), its contribution to the tissue glucose levels has been ignored.

These results are similar to observations of others on the hydrolysis of sucrose and of dipeptides in perfused loops and everted sacs of the intestine of other species 16,17. The findings cannot be explained by tile hydrolysis of substrate in the medium and subsequent absorption by the tissue of the split products.

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286 D. MILLER, R. K. CRANE

T A B L E I I

TH]~ HYDROLYSIS OF SUCROSE BY INTESTINAL STRIPS

T h e i n i t i a l m e d i u m s u c r o s e c o n c e n t r a t i o n w a s i o r a M .

Time Tissue glucose Medium glucose Tissue fructose Medium fructose Medium sucrose Utilization* * (rain) (ram) (mM) plus sucrose* (raM) (raM) (raM) (percent)

IO 17.1 o.17o 2.28 1.12 9.61 - - 7 20 25.5 o.717 2.14 2-39 8.15 - - 4 3 ° 35.1 1.17 2.26 2.66 6.90 3 4 ° 36.0 1.76 2.41 3 .36 5.55 lO 50 36.7 2.22 2.48 4.2o 4.65 7 60 37 .8 3.25 2-46 4-49 3.72 14

* T i s s u e f r u c t o s e w a s n o t m e a s u r e d s e p a r a t e l y in t h i s e x p e r i m e n t . O t h e r d a t a i n d i c a t e t h a t a p p r o x i m a t e l y fou r - f i f th s of t h e c o m b i n e d d e t e r m i n a t i o n c o n s i s t s of f ruc tose .

** P e r c e n t u t i l i z a t i o n r e fe r s t o t h e d i s a p p e a r a n c e of s u c r o s e a n d i t s s p l i t p r o d u c t s f r o m t h e . s y s t e m c a l c u l a t e d as fo l lows :

p e r c e n t u t i l i z a t i o n = I - ( m m ° l e s g l u c ° s e + m m ° l e s f r u c t ° s e + 2 ( m m ° l e s s u c r ° s e ) ) × IOO 2 ( m m o l e s suc ro se i n i t i a l l y p r e s e n t )

Al l q u a n t i t i e s r e f e r t o t h e t o t a l a m o u n t s p r e s e n t i n t h e s y s t e m .

T A B L E I I I

COMPARISON OF THE ACCUMULATION OF TISSUE GLUCOSE IN INTESTINAL STRIPS INCUBATED IN SUCROSE AND IN GLUCOSE

A t t h e v a r i o u s c o n c e n t r a t i o n s t h e v o l u m e of t h e m e d i u m w a s a d j u s t e d t o p r o v i d e t h e s a m e t o t a l a m o u n t of s u b s t r a t e . T h e i n c u b a t i o n t i m e w a s 5 m i n .

Initial mediura Tissue glucose Medium glucose Tissue fructose Tissue sucrose Medium sucrose concentration (raM) (raM) (ram) (ram) (ram) (raM)

Sucrose 5 ° 22.5 1.03 5.75 1.25 48.0 2o 13. 5 o .43o 2.52 o.412 18. 5 IO IO.I o .18o 1.2o o.212 9.1o

5 .0 4 .37 0.045 0.60 o.138 4.85 I.O 0.925 O.OLO o. 16 o .o89 0.925

G lucose I .o 5.09 o. 850 o. 18 - - - -

T A B L E I V

INHIBITION OF GLUCOSE UPTAKE IN INTESTINAL STRIPS BY PHLORIZIN AND O-DINITROCRESOL

T h e i n i t i a l g l u c o s e c o n c e n t r a t i o n i n t h e m e d i u m w a s 5 m M in t h e e x p e r i m e n t w i t h p h l o r i z i n a n d IO m M in t h e e x p e r i m e n t w i t h o -d in i t r oc r e so l . T h e i n c u b a t i o n t i m e w a s i o ra in .

Medium concentration Tissue gluccse Medium glucose (M) CraM) (raM)

P h l o r i z i n o 14.7 4.3 IO - e 1 2 . o 4.5

5" lO-6 lO.6 4.7 lO-5 6.51 4.7

5" lO-5 3 .46 4.9 i o -4 i .92 5.0

2.5" IO -4 0.837 4.8

o - d i n i t r o c r e s o l o 18. 5 9 .38 2o.4 9.45

lO -5 15.1 9.05 15.9 9.15

l ° - 4 3.74 9.15 4.25 9.38

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AN I N T R A C E L L U L A R LOCUS OF I N T E S T I N A L H Y D R O L A S E S 287

T A B L E V

THE EFFECT OF 0-DINITROCRESOL ON THE ACCUMULATION OF GLUCOSJE AND FRUCTOSE FROM SUCROSE

The e x p e r i m e n t a l cond i t ions were as in Tab le [ [ e x c e p t t h a t t h e m e d i u m conta ined o-dini trocresol , I o -~ 3/-,

Time Tissue glucose Medium glucose Tissue fructose Mediura fructose Medium sucrose Utilization (rain) (raM) (raM) and sucr~se (raM) (raM) (percent)

(raM)

i o 2 .88 o . 6 5 o 3 .26 1.17 8 .85 i 2o 3 .52 1 ,26 3 .8o 1 .69 8 .15 2 3 o 3 .6o i , 73 4 .o4 2 .58 7 .6o o 4 ° 3-75 2 ,79 4 .48 4 .41 .5.65 6 5 ° 3 .25 3 .69 4 .38 ,5.15 4 .36 i o 60 2 .8o 4 .07 4 .38 4 .74 3 .93 15

T A B L E V I

TISSUE ACCUMULATION OF GLUCOSE AND FRUCTOSE FROM SUCROSE UNDER ANAEROBIC CONDITIONS

E x p e r i m e n t a l condi t ions as in Table II e x c e p t t h a t the f lasks were gassed 3 ° rain w i t h N 2 - C O 2 (95:5) prior to the addi t ion of the t issue. Tissue strips were prepared and added to the f lasks

under a c o n s t a n t s t r e a m of N ~ - C O = (95 : 5)-

Tirae Tissue glucose Medium glucose Tissue fructose Tissue sucrose Mediura sucrose (m in) (raM) (mM) (raM) (raM) (raM)

5 3 .66 o . 1 4 5 1.48 0 . 3 2 5 9 .65 IO 4 .37 0 .34 ° 1.96 o . 5 o o 9.OO 15 4 .89 O.37 o 1.5 ° O.3OO 8.65 30 4.5 ° 1.4 ° 2 .45 O.666 7 .75

In incubations with glucose the addition to the medium of either o-dinitrocresol or phlorizin, in IO ~ M concentration, was found to prevent accumulation of glucose to tissue concentrations higher than those in the medium (Table IV). In incubations with sucrose, on the other hand, the tissue concentration of glucose derived from sucrose hydrolysis was appreciably greater than its concentration in the medium (Table V) under the same conditions. It seems clear that the concentration difference of glucose in the early time periods of incubation does not depend upon active transport, although a contribution of this process to the ability of the tissue to retain hydrolytically formed glucose is indicated by the lower glucose concentrations in the inhibited tissue. At later periods, the tissue concentration of glucose remained fairly constant while the medium concentration increased steadily due to the progressive hydrolysis of sucrose. Similar results were obtained when anaerobiosis was used to inhibit active transport (Table VI).

Measurements of the relative fructose concentrations in the tissue and medium after incubations with sucrose (Table VII) provide further evidence to show that the products of hydrolysis which accumulate in the tissue are not absorbed from the medium. At short incubation periods the concentration of fructose in the tissue exceeded the concentration in the medium by a large margin. Such a concentration difference cannot occur under any circumstance when fructose is initially plesent only in the medium 3~. In the later time periods the tissue to medium concentration relationsllips which developed were similar to those observed with the glucose moiety in the experiments where active transport was inhibited.

Biochim. Biophys . ,4eta, 52 (1961) 2 8 1 - 2 9 3

288 D. MILLER, R. K. CRANE

T A B L E V I I

THE TISSUE ACCUMULATION OF FRUCTOSE FROM SUCROSE

The e x p e r i m e n t a l condi t ions were the same as in Table I I .

Time Tissue fructose Medium fructose Tissue sucrose (rain) (raM) (raM) (raM)

io 1.o8 o.616 0.27 o.71o 0.26

20 2.16 1.33 o.26 1.92 1.24 - -

3 ° 1.85 1.63 0.29 2.16 1.46 0.25

TABLE VIII

THE TISSUE ACCUMULATION OF GLUCOSE DERIVED FROM MALTOSE

(a) I n i t i a l concen t ra t ion of mal tose, io m M ; (b) I n i t i a l concen t ra t ion of mal tose , 5 raM.

Time Tissue glucose Medium glucose Medium maltose (rain) (ram) (ram) (mMJ

IO 22.3 3.8 7.3 20 34.0 7.0 5.5

a 3 ° 41.8 8.8 3.8 4 ° 39.5 11.2 1.9 5 ° 42 .5 12.3 1.4

5 14.5 0.82o 4.18 b IO 21.9 1.77 3.34

15 29.2 2.26 3.12

Maltose: Following incubation in IO mM maltose, glucose appeared in the medium at a much higher concentration after a given incubation period than when sucrose was the substrate (Table v n I , a). This difference seems to reflect the more rapid hydrolysis of maltose as measured by its disappearance from the medium, and the limited capacity of the tissue to retain the glucose formed. At lower initial concentra- tions of maltose, the results resembled those with sucrose (Table v n I , b).

A direct comparison of the accumulation of glucose in tissue incubated with maltose and with glucose, respectively, is shown in Table IX, a. The tissue levels of glucose following incubation with maltose or with twice its molar concentration of glucose were very similar despite the marked differences in the concentration of glucose in the medium. Moreover, with maltose, as with sucrose, tissue glucose con- centrations were greater than those in the medium even when active transport was inhibited by means of phlorizin (Table IX, b) or o-dinitrocresol (Table IX, c). The relative depression of tissue glucose accumulation by these agents was comparable whether the glucose was absorbed directly from the medium or whether it was derived from the hydrolysis of maltose.

Glucose x-~hosphate: The results obtained in experiments with glucose I-phosphate (Table X) were similar to those found for the disaccharides.

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AN I N T R A C E L L U L A R L O C U S OF I N T E S T I N A L H Y D R O L A S E S 289

T A B L E I X

COMPARISON OF GLUCOSE UPTAKE IN TISSUE INCUBATED IN MALTOSE

AND IN EQUIVALENT CONCENTRATIONS OF GLUCOSE UNDER CONTROL CONDITIONS AND IN THE PRESENCE OF PHLORIZIN AND 0-DINITROCRESOL

T h e v o l u m e o f t h e m e d i u m w a s 20 m l a n d t h e i n c u b a t i o n p e r i o d w a s 5 m i n .

(a) Control Initial concentration

Glucose Maltose (ram) (ram)

Tissue ~lucose Medium glucose (mM) (mM)

2o

4o

8o

IO

20

4 °

11.8 19. 4

11.5 o .75

15 .6 39 .4 16 .o 4o .4 12.9 1.19

14 .9 1.17 19 .6 8 1 . 6

17.8 1.49

(~ Ph~r~m Initial concentration

Glucose Maltose (ram) (ram)

Phlorizin Tissue glucose Medium glucose (M) (ram) (ram)

20 IO -3 3 .15 20 .2 IO IO -3 2 .32 I . I O

4 ° IO -3 6 .73 41-4 20 IO -3 3 .80 1.47

80 lO _3 12.9 81. 5

4 ° lO -3 8 .44 2 .27

20 o 14. 7 19.5 I o o 12 .o i . o 7

(c) o-dinitrocresol Initial concenB'alion

Glucose Maltose (ram) (ram)

o-dinitrocresol Tissue glucose Medium glucose (,.1I) (mM) (mM)

20 lO-4 5-79 19.4 IO lO-4 4 .39 - -

4 ° lO -4 8 .50 40 .3 20 lO -4 6 .16 1 .4o

80 IO 4 13.1 8 o . [

4 ° lO -4 8 .55 1 .89 20 o 12.9 19 .6

IO o 10.2 0. 7

T A B L E X

TISSUE ACCUMULATION OF GLUCOSE FROM GLUCOSE I-PHOSPHATE AND THE EFFECT OF O-DINITROCRESOL

The initial concentration of glucose 1-phosphate was IO ram.

Time o-dinitrocresol Tissue glucose Medium glucose (rain) (M) (ram) (ram)

IO o 16.3 I.O

3 ° o 29 .0 - - 60 o 35 .0 - - i o lO -4 3 . 7 6 1.35 3 ° l o -4 4 .15 2 .86 60 lO -4 3 .14 4 .5 °

B i o c h i m . B i o p k y s . Acta , 52 ( 1 9 6 I ) 2 8 1 - 2 9 3

2 9 0 D. MILLER, R. K. CRANE

Influence of volume changes and of glucose oxidase on the relative tissue and medium concentration of monosaccharide

The data in Table XI were obtained by varying the total volume of the incubation medium in a series of flasks containing similar amounts of tissue and the same initial disaccharide or glucose 1-phosphate concentration. Although the concentration of glucose appearing in the medium was decreased in proportion to the increase in medium volume, the tissue accumulation of glucose remained relatively constant.

In other experiments (Table XlI) the concentration of glucose in the medium

T A B L E X I

T H E E F F E C T OF V A R I A T I O N S IN T H E V O L U M E OF T H E I N C U B A T I O N M E D I U M

ON T H E T I S S U E A C C U M U L A T I O N OF F R E E M O N O S A C C t t A R I D E S F R O M S U C R O S E , M A L T O S E

A N D G L U C O S E I - P H O S P H A T E

Initial concentration of substrate Volume of Tissue glucose Medium glucose Tissue fructose Medium fructose incubation medium (raM) (raM) plus sucrose and incubation period ( ml) (raM) (raM)

Sucrose, IO m M IO 8.45 0.064 IO rain 8.65 0.082

IOO 7-3o o.o12 7.3 ° O.OLO

Maltose, IO m M IO 29.8 6.51 20 min 20 27. 7 3.03

5o 29.0 1.32 ioo 25,1 0.74

Glucose 1-phosphate , 5 m M IO 12.8 0.685 2o rain I 1.4 o.491

5 o 13.1 o.156 11.9 o.159

1.35 0.57 1.5o o.61

1.36 o.o5 1.54 o.o5

T A B L E X l I

T H E E F F E C T OF G L U C O S E O X I D A S E ON T H E M E D I U M C O N C E N T R A T I O N OF

G L U C O S E A N D ON ITS U P T A K E IN I N T E S T I N A L S T R I P S .

Glucose oxidase (Sigma Chemical Company, crude) was added to the m e d i u m to give final concen t ra t ions as shown.

Substrate Glucose oxidase Tissue glucose Medium glucose Incubation time Initial concentration concentration (w/v) (%) (raM) (raM)

(a) 30 rain 1,5-anhydro-D-gluci tol o 9.86* 4.35* 5 m M 0.3 9.92 * 4.65

Glucose o 18.6 1.07 5 m M 0.3 3.08 o.19

Sucrose o 2 4 . 1 2 , 2 I

IO m M 0. 3 26.0 0.39

(b) 20 rain Maltose o 31.6 6.3 ° IO mll.l 34.9 6 . i6

I.O 33.3 0.74 26.3 0.75

(c) 15 rain Glucose I - phos pha t e o 13. 5 o.89o IO m M o. 3 11. 7 O. l l 5

i .o 8.80 o

* Refers to concen t r a t ion of 1,5-anhydro-D-glucitol .

B i o c h i m . B i o p h y s . Ac ta , 52 (1961) 281-293

AN INTRACELLULAR LOCUS OF INTESTINAL HYDROLASES 2 9 I

was lowered by the addition of glucose oxidase. 1,5-Anhydro-D-E3HJglucitol, which is an actively transported but non-utilizable compound '9, was used as a control to show that the glucose oxidase preparation did not affect the permeability of the tissue or its active transport function. The tissue accumulation of glucose following incuba- tion with a disaecharide or glucose 1-phosphate was substantially unaffected by the presence of glucose oxidase.

These experiments show that tissue accumulation of hydrolytically formed monosaccharides is a function of the medium disaccharide or glucose I-phosphate concentration and not of the medium monosaccharide concentration.

The absence of enzymic activity in the medium during the in vitro incubation of intestinal tissue

Following incubation of tissue with sucrose, maltose, and glucose 1-phosphate, measurements were made of the concentration of monosaccharide appearing in the medium at varying times after removal of the tissue. The enzymic activity present in the medium even after tissue had been incubated I h was never more than 5 % of the activity associated with the presence of the tissue. I t is unlikely that progressive inactivation of enzyme released into the medium could account for these results as the activity of mucosal homogenates is stable for at least several hours at room temperature. The small amount of activity which was found in the medium is con- sistent with the shedding of cells and fragments of villi which normally occurs during the incubation.

DISCUSSION

The data presented indicate that in vitro uptake by intestinal tissue of a mono- saccharide formed by hydrolysis of a disaccharide or of glucose 1-phosphate does not depend upon the liberation of the monosaccharide in the incubation medium or at the free surface of the mucosa; hydrolysis occurs at a site from which diffusion into the tissue occurs more rapidly than diffusion into the medium. The only conclusion that it seems possible to draw is that the hydrolases are intracellular enzymes. Were the enzymes external to the cell membrane, one would have expected results such as those that have actually been found in studies of sucrose hydrolysis by yeast ~5. In these studies it was found, following incubation with sucrose, that the concen- tration of invert sugar was higher in the incubation medium than inside the yeast cells, even when glycolysis was inhibited. In contrast to intestinal invertase, the yeast enzyme appears to be located external to the plasma membrane and to act at the cell surface ~6.

Among other studies in keeping with the concept of intracellular disaccharide and sugar phosphate ester hydrolysis which have already been referred to above x2-17, those of ROTHSTEIN et al. ~5 require further comment. These workers observed that the phosphate liberated during the splitting of a sugar phosphate ester failed to equilibrate with the intracellular phosphate of the mueosa and postulated, therefore, a surface location for intestinal phosphatase. Actually, this observation is not in- consistent with the present conclusion that the enzyme is intracellular, as the re- mainder of the discussion will make clear.

Some of the present data suggest that the products of hydrolysis are not

13ioct~im. Biophys. Acta, 52 (I961) 28I -293

292 D. MILLER, R. K. CRANE

uniformly distributed in the tissue but exist in relatively high concentrations in a superficial region. After prolonged incubation with sucrose, the fructose concentration in the medium exceeded that in the tissue. A similar relationship was found with glucose when active transport was inhibited. Since active transport out of the cells into the medium is a highly improbable event, these observations indicate that hydrolysis occurs in a restricted portion of the cell to give a zone of relatively high concentration from which diffusion of the hydrolytic products occurs into the tissue as a whole and into the medium.

Other work has shown that phlorizin inhibits the entrance of glucose into the epithelial cells 37 and that o-dinitrocresol inhibits active transport by limiting energy supplies ~9. It has also been shown that the locus of the active transport process is in or near the mucosal border of the epithelial cells ~. It is thus of particular signi- ficance that phlorizin and o-dinitrocresol have virtually the same effect on tissue accumulation of glucose whether it originates in maltose or in glucose added to the medium (Table IX). These results suggest that disaccharide hydrolysis occurs at a locus which, though it is inside the cell, is external to a diffusion barrier sensitive to phlorizin and external to the active transport process for sugars. The failure of phosphate to penetrate the cells in the experiments of ROTHSTEIN et al. (see above), far from being inconsistent with the present work, would, on the contrary, appear to be added evidence for the presence of a diffusion barrier between the site of hydro- lysis and the major cytoplasmic portion of the cell. The locus of hydrolysis would, thus, appear to be restricted to that portion of the epithelial cell just underlying the limiting membrane of the epithelial surface; namely, the brush border region. Direct evidence that this is, indeed, the location of the hydrolytic enzymes is presented in the following communication is.

ACKNOWLEDGEMENTS

We are indebted to Miss M. L. ROBERTS and to Mr. P. SCHWARTZ for their capable assistance. This investigation was supported by grants No. G-5892 and G-II216 from the National Science Foundation. One of us (D.M.) is a U.S. Public Health Service Postdoctoral Fellow.

R E F E R E N C E S

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Biochim. Biophys. Acta, 52 (1961) 281-293

AN INYI¢.ACEI.I.I'I.AI~ L()CUS ()F IN'[E%TINAI. HYI)ROLASES 2~1.~

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dology, N e w E n g l a n d N u c l e a r Corp. , A t o m l i g h t , No. ~5, D e c e m b e r , ~96o, p. 4" 29 E. HELMREICH AND R. 1K. ('RANE, in F. TURBA, Radi()isolopes i , Pkysioh;gy, Diagnosis at~d

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Biochim. h~iophys..lcla, 52 (1961) 281 203

T H E D I G E S T I V E F U N C T I O N OF T H E

E P I T H E L I U M OF T H E SMALL I N T E S T I N E

II. LOCALIZATION OF DISACCHARIDE HYDROLYSIS IN THE ISOLATED

BRUSH BORDER PORTION OF INTESTINAL E P I T H E L I A L CELLS*

D A V I I ) M I I . I . E R * * A~D R O B E R T K. C R A N E

Deparlme~.¢l o~ Biological Chemistry, Washingto~z Ut~iversity ,qckool o./ 31edicitw. St. Louis, 3Io. (U.S..q.)

( R e c e i v e d F e b r u a r y zo th , 1961i

S U M M A R Y

The epithelial brush border membrane has been isolated as a morphologically distinct entity from homogenates of intestinal mucosa and found to contain virtually all of the invertase and maltase activities of the unfractionated homogenate.

* A pre l iminary report was made to the 33rd Annual Meeting of the Central Society for C l i n i c a l R e s e a r c h , Chicago, N o v e m b e r 5, I96o (.]. Lab. C l i , . 3Ied., 56 (196o) 928).

** P r e s e n t a d d r e s s : D e p a r t m e n t of t t e m a t o l o g y , J e w i s h H o s p i t a l , St. I .ouis, Mo. (U.S.A.) .

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