Distribution of soluble and membranous forms of alkaline phosphatase in the small intestine of the rat

Biochimica et Biophysica Acta, 676 (1981) 257-265

257

Elsevier/North-HollandBiomedicalPress BBA 29663 DISTRIBUTION OF SOLUBLE AND MEMBRANOUS FORMS OF ALKALINE PHOSPHATASE IN THE SMALL INTESTINE OF THE RAT GRAEME P. YOUNG a, STEVENT. YEDLIN and DAVID H. ALPERS b Division o f Gastroenterology, Department o f lnternal Medicine, Washington University School of Medicine, 660 S. Euclid Ave, St. Louis, 1110 63110, (U.S.A.)

(Received October 8th, 1980) (Revised manuscript March 31st, 1981)

Key words: Alkaline phosphatase; Aging; Membrane.bound enzyme; (Rat small intestine)

In the small bowel mucosa of the rot, alkaline phosphatase was found to be present in the 105 000 × g supernatant fraction, in addition to the major brush border membrane-bound form. This soluble enzyme contributed up to 3.6% of total alkaline phosphatase activity in the adult rat. Combined use of inhibitors of serine, thiol and carboxyl proteases during homogenization did not significantly affect the proportion of enzyme activity in the supernatant fraction. In suckling rats a much larger proportion of alkaline phosphatase was soluble, reaching 36.5% in the 14-day-old animal. The soluble and membranous forms were compared in adult and suckling rats by examining their biochemical and immunological characteristics. In the adult rat the specific activity of membranebound alkaline phosphatase showed a progressive fall distal to the duodenum, while maximal specific activity of the supernatant enzyme occurred in the distal ileum. By contrast, in the suckling rat the specific activity of both forms increased from the duodenum to the ileum. Soluble alkaline phosphatases in mature and immature rats were distinct from the membranous forms when examined by polyacrylamide gel electrophoresis in the presence of Triton X-100. Electrophoretic heterogeneity was seen in the soluble adult enzyme, characteristic Rf values occurring for each level of the small bowel (i.e., duodenum, jejunum and ileum). Such heterogeneity was not seen in suckling rats. Antigenically, the soluble and membranous forms were identical to each other in adult and suckling rats. Analysis of mucosal cells isolated sequentially from tip of the villus to crypt showed that both the soluble and membranous isozymes had highest activity towards the tip of the villus. We conclude that the soluble and membranous forms are distinct biochemically and yet are antigenically identical and arise from the same level of the villus. The presence of the soluble form, which is not an artifact of tissue preparation, indicates that alkaline phosphatase is not exclusively an integral membrane protein.

Introduction

In many vertebrate species intestinal alkaline phosphatase, like other hydrolases of the brush border membrane, shows a gradient of activity along the a Present address: University of Melbourne Department of Medicine, Royal Melbourne Hospital, Victoria 3050, Australia. b To whom requests for reprints should be addressed. Abbreviation: SDS, sodium dodecyl sulfate.

small bowel [1]. There may also be heterogeneity of alkaline phosphatase along the intestine, as demonstrated by variations in electrophoretic mobility [2], in immunological characteristics [3] and in substrate specificity [4]. Alkaline phosphatase is an almost universal glycoprotein component of mammalian plasma membranes. The intestinal villous cell, where alkaline phosphatase is concentrated in large amounts in the brush border membrane, is no exception [5]. However, a soluble intestinal alkaline has been observed as the major form in the suckling rat [6].

0304-4165/81/0000-0000/$02.50 © Elsevier/North-HollandBiomedicalPress

258 The presence of a similar form in the adult rat has not been conclusively demonstrated. Evidence in the suckling rat has indicated that the soluble and membranous forms are isozymes originating from the same gene code [7]. The physiological importance of the soluble form is unknown, as is its distribution along the intestine of soluble and membranous alkaline phosphatases in adult and suckling rats. Electrophoretic heterogeneity of the adult soluble enzyme, which is immunologically identical to the membranous enzyme, is described. In the suckling rat the specific activity of both soluble and membranous forms increases from duodenum to ileum. In the mature rat the membranous form shows a reversal in the gradient, whilst the soluble form maintains a similar pattern to that of the suckling animal.

Methods

Animals and tissue preparation. Adult male Wistar rats weighing approx. 250 gm were obtained from National Laboratory Animal Co., O'Fallon, MO. For studies in immature rats, pregnant females were obtained and the pups studied 14 days after delivery. All animals were allowed free access to standard laboratory food, or to suckle the mother. Studies were performed on animals killed at 0900h who had been subjected to normal cycles of daylight/ darkness. The following procedure was followed to prepare supernatant and membranous fractions from intestinal mucosal homogenates. All procedures were carried out at 4°C and were performed rapidly after removal of the gut. Each rat was killed by decapitation. The entire small bowel was removed and carefully trimmed of fat and rinsed with cold isotonic saline. For studies involving separate segments, the duodenum was separated at the ligament of Trietz and the remainder divided into four sections of equal length; proximal and distal jejunum and proximal and distal ileum. Mucosa was collected from adult rat gut by scraping with a glass slide; suckling rat gut was not scraped but was chopped into small pieces (less than 4 mm) with scissors. The mucosa was then suspended in 20-times its volume of 10 mM Tris/50 mM mannitol/0.2 mM MgC12 (pH 7.4)and homogenized in a Potter-Elvehjem homogenizer using seven strokes of the Teflon pestle rotating at full speed. The

homogenate was then centrifuged at 105000Xgav for 60 min and the supernatant fraction carefully drawn off. Pellets were resuspended in 10 mM Tris/2 mM MgC12 (pH 7.4) (hereafter called buffer A). All fractions were stored at -20°C. Assay procedures. All fractions were assayed within 4 days of homogenization. Alkaline phosphatase was assayed at pH 9.2 [8] and acid phosphatase at pH 4.8 [9], with p-nitrophenylphosphate as substrate for both. Samples for disaccharidase assays [10] were diluted at least 1 : 4 in water before assay. Protein was measured [11 ] with bovine serum albumin as standard. Prior to the assay, Triton X-100 (Fisher Scientific Co., NJ. U.S.A.) was added to resuspended membrane pellets to a concentration of 0.1% (v/v). The pellets were then sonicated on ice at 100W for 15 s (Braunsonic 1510, B. Braun, Melsungen, F.R.G.) to ensure an even dispersion of membrane fragments and ready access of substrate to the enzyme. All chemicals, apart from those specifically indicated, were obtained from Sigma Chemical Co., St. Louis, MO, U.S.A. Disc-gel electrophoresis. Polyacrylamide disc-gel electrophoresis [12] was carried out in gels containing 5% total monomer (38 acrylamide: l bisacrylamide; Polysciences, Warrington, PA, U.S.A.). In some experiments the gels, samples and upper tank buffer also contained 0.1% Triton X-100. Samples normally contained 0.05 units of alkaline phosphatase activity. The membranous samples were first solubilized overnight at 4°C in buffer A containing 1% Triton X-100 and then the insoluble material was removed by centrifugation at 45 000 ×g. The supernatant proteins were then precipitated with 70% ethanol (v/v) at -20°C to extract excess detergent and membrane lipids. The membrane proteins were then resolubilized in buffer A containing 0.1% Triton X-100. This precipitation process consistently gave greater than 92% recovery of enzyme activity. In this way we achieved a sharp single band for the membranous alkaline phosphatase; otherwise, multiple blurred bands were frequently seen. On occasion, ethanol precipitation was used to concentrate the soluble form and this did not alter its electrophoretic characteristics. Alkaline phosphatase bands were located histochemically, as previously described, using naphthyl phosphate as substrate [7].

259 In some cases gels were scanned at 555 nm in a modified Beckman DU-2 spectrophotometer. SDS-polyacrylamide gel electrophoresis. This was carried out according to the method of Neville [13] in 9% total monomer gels (110 acrylamide: 1 bisacrylamide) cast in 1 mm thick slabs. Samples containing 0.1 unit of alkaline phosphatase activity were treated with 2.5% SDS at 48°C for 5 min. The following nuclear weight markers were used: /3-gatactosidase (130 000), phosphorylase b (94 000), bovine serum albumin (68000) and ovalbumen (43 000). Enzyme activity was detected as indicated above. Isolation of villous and crypt cells. Cells were isolated as previously described [14]. The following sequential incubation periods were used for adult rats: 10 min (upper third of villus), 10 min (mid villus), 20 min (lower third villus) and 20 rain (crypt). Shorter incubation periods were required for suckling rats because the mucosa was thinner: 5 min, 5 min, 10 min and 15 min, to provide corresponding cell populations. Time periods were verified by histological examination of rings of intestinal tissue obtained at the end of each of the various time periods. These time periods are similar to those described previously [ 15]. Antibody to alkaline phosphatase. Enzyme preparation and characterization has been described MEMIR ANGUS + TRITON

elsewhere [7]. Antiserum was raised in rabbits with 300/.tg of the purified protein with a specific activity of 340 units/mg protein. The antiserum did not crossreact with bone or liver alkaline phosphatase or with acid phosphatase.

Extraction of alkaline phosphatase by liposomes. Synthetic liposomes were prepared from equal amounts of cholesterol, phosphatidylcholine and stearylamine. Lipid solutions in chloroform were evaporated to dryness in the glass receptable of a Potter-Elvehjem homogenizer. Buffer A (1 ml) was added and sonicated as described above. An aliquot of supernatant proteins was added to give a lipid : protein ratio of 10 : 1 and the mixture homogenized. The mixture was then centrifuged at 105 000 Xgav for 60 min and the resultant supernatant fraction subjected to gel electrophoresis in the presence of Triton X-100. The material from three adult rats was studied in this fashion. Results

Electrophoretic forms in small bowel mucosa When the entire small bowel mucosa of adult rats was homogenized as described, 3.6 -+0.2% (~--+ S.E. for seven rats) of alkaline phosphatase activity was present in the supernatant fraction after ultracenS UPS R N A T A N T +TRITON

- TRITON M S S S S S

o

jp

,p

io

o

Jp

Jo

Jp

ID

o

jp

Jo

ip

,o

Fig. 1. Polyacrylamide gel electrophoresis of adult rat intestinal alkaline phosphatase in supernatant and membranous fractions from various levels of the gut. 0.05 unit alkaline phosphatase activity was applied to each gel and electrophoresed and stained as described in Methods. The results shown are all from one rat but are typical of findings in three other studied in the same manner. The various levels of the gut are indicated as follows: duodenum, D; proximal jejunum, JP; distal jejunum, JD; proximal ileum, IP; distal ileum, ID. The group of gels in the center are supernatant fractions electrophoresed in the absence of Triton and those on the right are the same samples in the presence of Triton. Note the hold-up of enzyme activity at the top of the non-Triton gels. Membranous fractions are shown in the group on the left. The S V in the duodenal supernatant gels of this animal was only seen occasionally in other rats. The nlurred opacity (arrowed) is due to interaction of Triton X-100 with bromphenol blue. The dyefront is marked with india ink.

260 MEMB.

SUPERNATANT

D

JP

JD

IP

ID

Fig. 2. Get electrophoresis of suckling rat intestinal alkaline phosphatase from various levels of the gut. Methods of preparation of membranous (MEMB.) and supernatant fractions are as described earlier. Results shown are from one rat and are typical of the findings in three others. Different levels of the gut are indicated in the same manner as in Fig. 1. All samples were electrophoresed in the presence of Triton. trifugation. This percentage did not change significantly when homogenization was performed in isotonic buffer (250 mM mannitol) or by using a Waring blender or Dounce-type homogenizer (data not shown). The electrophoretic characteristics of membranous (i.e., 105 000 Xg pelleted proteins) and supernata.nt alkaline phosphatases are shown in Fig. 1. A single band of activity, designated M, was seen in washed membrane fractions. Six bands of varying intensity were seen in the supernatant fraction; the five faster were designated S I, S II, S III, S IV and S V. S V was seen only intermittently. The slowest corresponded to the M band in the membrane fraction and reflected contamination of the supernatant fraction with brush border alkaline phosphatase, which was not pelleted by ultracentrifugation up to 165 000 ×gay. In the 14-day-old suckling rat, 36.5 + 2.1% (~-+ S.E. for seven rats) of total gut alkaline phosphatase activity was present in the supernatant fraction, a considerable increase above adult proportions. As in the adult rat, the electrophoretic characteristics of supernatant and membranous forms were distinctly different by gel electrophoresis in the presence of Triton X-100 (Fig. 2). The Re of the membranous form was 0.23 and that of the principal

supernatant band was 0.44. On occasion, supernatant fractions from the whole gut homogenates contained a minor band with an Rf of 0.23, corresponding to that of the membranous form. Mucosa from the proximal quarter of the small bowel of four adult rats was homogenized as described in Methods in the combined presence of 10 ~M of each of the following protease inhibitors: leupeptin, pepstatin and phenylmethylsulphonylfluoride. Four control homogenates were prepared without protease inhibitors. Following ultracentrifugation, 1.6 + 0.2% (~-+ S.E.) of alkaline phosphatase activity was found in the supernatant fraction of controls compared with 2.0 + 0.5% in supernatant fractions of homogenates containing the inhibitors ( p > 0 . 0 5 ) . These proportions are typical of the proximal (rather than entire) small bowel in adult rats. The presence of protease inhibitors did not affect the electrophoretic characteristics of supernatant alkaline phosphatase, indicating that in vitro proteolysis was not a factor in producing the soluble forms of alkaline phosphatase.

Enzyme activity gradients along the gut Fig. 3 shows the distribution along the gut of alkaline phosphatase in the supernatant and membranous fractions of adult rat mucosal homogenates centrifuged at 105 000 ×g. The specific activity of the membranous form showed a characteristic fall from duodenum to distal ileum. By contrast, the specific activity of supernatant alkaline phosphatase followed a different pattern, being highest in the distal ileum and lowest in the second half of the jejunum. Only 1% of activity in the duodenum could be found in the supernatant fraction, whereas 40% of distal ileal activity occurred in that fraction. The method of homogenization used released 25-30% of acid phosphatase activity into the supernatant fraction at all levels of the gut, indicating a lack of parallelism between these two phosphatases. Fig. 3 also shows the distribution of maltase and sucrase for comparison. Both dissaccharidases showed characteristic peak levels in the jejunum. The specific activity of supernatant sucrase was low along the entire small bowel, constituting less than 7% of total activity at any level. Specific activity of supernatant maltase rose progressively from duodenum to ileum. The proportion in the supernatant fraction increased from

261

i

i

i

i

0.8 M o f f o s e ~

~o" .~oucrase '

'

t

10.16

/Oz

'

u~

•

~

i'o,~'~o-=

___"~ ~o z

~-~ ~: 81~f I Alkaline ~ 6Ph°spatase~

"~

~o

.

~

~

~

..'-2. 4k

\

]oo~i

o ° -o

.... ~

~

60 ~-'~ ]0.20.15 .., 140

-10.1

:o D

oo,

PJ DJ PI DI GUTSEGMENT

J0

Fig. 3. The distribution of alkaline phosphatase, sucrase and maltase along the small bowel of the adult rat. The various levels of the gut are designated thus: D, duodenum; JP, proximal jejunum; JD, distal jejunum; IP, proximal ileum; ID, distal ileum. Tissue homogenates were prepared and assayed as described in Methods; membranous and supernatant fractions were prepared by centrifugation of homogenates at 105 000 ×gay"

,i

i

i

i

006 - Maltase

i 60

,"~- ~ - o

O.04

~0

_z 0.02

20

o 0.12 Lactase

D

;

0.08

~

0.04

3 1.6 - A l k a l i n e Phosphatase

b0

1.2

///

0.8

40 20

0.4

D

PJ

DJ

PI

DI

GUT SEGMENT Fig. 4. The distribution of alkaline phosphatase, lactase and maltase along the small bowel of the 14-day-old rat. Other details are as in Fig. 3.

duodenum to ileum, in a fashion similar to that of alkaline phosphatase. Fig. 4 shows the distribution along the gut of alkaline phosphatase activity in supernatant and membranous fractions of the suckling rat. The specific activity of alkaline phosphatase increased in both fractions from duodenum to distal ileum, although the increase was steeper for the supernatant enzyme. The proportion of total alkaline phosphatase activity which was present in the supernatant fraction also increased from duodenum to ileum, in a fashion similar to that of the adult. Membranous maltase and lactate showed fairly fiat gradients in specific activity. The increase in specific activity of supernatant lactase, however, was relatively small and, although the proportion increased towards the ileum, the magnitude was much smaller than for either maltase or alkaline phosphatase.

Electrophoretic patterns along the gut (Figs. 1 and 2) In adult and in suckling rats the M band was electrophoretically the same at all levels of the intestine and required the presence of detergent to enter the gel. The slight variation in Rf values of the M band in adult rats between the duodenal and jejunoileal regions (Fig. 1) was not a real difference, since a single, sharp band resulted when they were mixed. However, the supernatant S bands of the adult tended to be localized to characteristic levels of the small bowel (Fig. 1 and Table I). In general, the more distal the site of origin the lower were the Rf values of the S bands. Table I indicates that band S III was the most prominent of the S bands. Fig. 1 also shows that only the S bands of the supernatant fraction would enter 5% polyacrylamide gels in the absence of detergent. The contaminating M band in the supernatant fraction decreased proportionally in more distal segments. Planimetry of scans from four rats of the supernatant gels stained for alkaline phosphatase activity showed the following proportions contributed by the M band: duodenum, 20-35%; proximal jejunum, 18-31%; distal jejunum, 15-30%; proximal ileum, 8-25% and distal ileum, 2-14%. When allowance is made for the activity contributed by the supernatant M band on the specific activity of supernatant alkaline phosphatase, there is little change in the pattern of the gradient shown in Fig. 3.

262 TABLE I CHARACTERISTICS OF THE VARIOUS ELECTROPHORETIC FORMS OF ADULT RAT INTESTINAL ALKALINE PHOSPHATASE Disc gel electrophoresis of detergent-treated fractions was carried out as described in Methods. Gels from four adult rats were prepared and stained for alkaline phosphatase activity as described in the legend of Fig. 1. They were then scanned at 555 nm and the area under each peak measured by planimetry. The proportional contribution of total small bowel activity by each electrophoretic type was calculated in each gut segment based on the assumptions that all forms hydrolysed the substrate equally well and linearly with time during the 5-10 min in which the color was allowed to develop. The calculations were also based on the mean activity present in supernatant and membranous fractions of each segment of bowel of the four rats studied. Total S-band activity as percentage of total activity, 2.7. Band

M

SI

S II

S III

S IV

Rf (mean) Principal location Proportion of total activity (%)

0.13, 0.15 a Duodenum 97.3 b

0.29 Distal ileum 0.2

0.37 Ileum 0.7

0.42 Widespread 1.4

0.54 Duodenum 0.4

a Values for duodenum and jejunoileal regions, respectively. b Includes 0.9% of activity found in the supernatant fraction. Fig. 2 shows the electrophoretic characteristics o f soluble and membranous suckling rat alkaline phosphatases at various levels o f the gut. In contrast to the findings in mature animals, no variation in electrophoretic mobility was seen along the length o f the gut.

Immunological relationship The immunological relationships between membranous and supernatant alkaline phosphatases from three levels o f the gut are shown in Fig. 5. Comparison o f membranous and supernatant forms from various levels o f the gut by Ouchterlony doublediffusion analysis showed that the two forms were antigenically identical, there being a single continuous precipitin line between the two forms in b o t h adult and suckling rats (data not shown). The shapes o f the titration curves for all forms were very similar (Fig. 5), further demonstrating their close, if not identical, antigenic relationship.

Hence, Sephadex G-200 chromatography was carried out to compare further the molecular weights o f the adult forms. Fig. 6 shows that the membranous form 10C

'%'",,,

When the adult forms were examined b y nondenaturing SDS-polyacrylamide gel electrophoresis, two bands were seen for the supernatant and three for the membranous forms. The molecular weight o f the principle band for each form was: soluble, 170000 and membranous, 148000. Since alkaline phosphatase is a glycoprotein, these results are likely to be inaccurate, due to reduced binding in SDS [16].

~l

I

~80

6o

z _o 4o o

2c

0

Molecular weight

I

~ I

'

'

A

~

8

16

32

64

, ~.-- " ~ ~ 128 256 512 1024 2048 4096 1/TITRE

Fig. 5. Titration curves of adult and suckling rat intestinal alkaline phosphatases. The preparation of monospecific antiserum is described in Methods. Alkaline phosphatase was precipitated from the following fractions, which all contained 0.15 units/ml of enzyme activity: Nonidet P40 solubilized adult membranous (o . . . . . . o); adult duodenal supernatant (¢ :); adult jejunal supernatant (m -); adult ileal supernatant (A (o . . . . . . o).

~);

suckling

ileal

supernatant

263 i

>-

> ~U <

O~

O.lOC

i 165 0 0 0

4,

i

i 67000

q,

i

i//i 18000

DISTRIBUTION OF SOLUBLE AND MEMBRANOUS ALKALINE PHOSPHATASE ALONG THE VILLUS-CRYPT AXIS OF THE ADULT RAT Six adult rats were killed and the duodenum and distal 15 cm of the ileum were removed and rinsed with normal saline. Ceils were isolated as described in Methods, rinsed twice in Tris/MgCl~ buffered isotonic saline and homogenized as described in Methods. Membranous and supernatant fractions were collected by ultracentrifugation of homogenates at 105 000 Xgay for 60 min. Results are the average + S.E. of studies of six rats.

0050

~~ 0025 < v

< 0

TABLE II

10 20 FRACTION NUMBER

30 40

Fig. 6. Chromatography of a mixture of adult rat intestinal alkaline phosphatases on Sephadex G-200. A 1 ml sample also containing 0.1% Triton X-100, was applied to a 1 × 50 cm column and 0.53-ml fractions collected by elution with Tris/Mg buffer containing 0.1% Triton X-100. Fractions were assayed for enzyme activity and then analyzed by disc-gel electrophoresis (see Methods). The following molecular weight markers were used: glucose oxidase (I 65 000), bovine serum albumin (67 000), myoglobin (18 000) and are indicated thus (~). Fraction 0 corresponds to V0.

Specific activity of alkaline phosphatase (Ug/mg protein)

Duodenum

villus

Ileum

crypt villus

tip mid base tip mid base

crypt (estimated Mr 172000) behaved as though it were slightly larger than the soluble forms when eluted with 0.1% Triton X-100. When chromatographed in the absence of detergent, the majority of the adult supernatant M band eluted with the void volume (results not shown), thus confirming that this form of alkaline phosphatase is present either in fragments of membrane or as protein aggregates.

Liposome incorporation To examine further the relationship between the M band in the supernatant and membranous fractions, we investigated the affinity o f adult supernatant alkaline phosphatase for synthetic liposomes. If the supernatant M band were due to the presence of small fragments of membrane in the supernatant fraction, then liposomes might extract this band from the supernatant fraction, since the hydrophobic anchor-piece of the enzyme could become associated with the liposome. When supernatant fractions from duodenum, j e j u n u m and ileum were treated in this manner, all M-band activity was consistently incorporated into the liposomes. S-band activity remained in supernatant fractions. This would appear to confirm the supposition that the supernatant

Membranous

Supernatant

2.24 _+0.20 1.39 _+0.35 0.72 + 0.26 0.43 + 0.14 0.053 _+0.003 0.027_+0.010 0.023_+0.003 0.015_+ 0.004

0.075 _+0.011 0.067 +_0.014 0.062 + 0.009 0.049_+0.010 0.033 + 0.003 0.031 _+0.003 0.023_+0.003 0.009_+0.002

TABLE III DISTRIBUTION OF SOLUBLE AND MEMBRANOUS ALKALINE PHOSPHATASES ALONG THE CRYPT-VILLUS AXIS OF THE SUCKLING RAT The duodenum and proximal two-thirds of the ileum of each of six 14-day-old rats were used for cell isolation as described in Methods. Membranous and supernatant fractions were hen prepared as described for adult rats (Table II). Results are the average + &E. of studies of six suckling rats. Specific activity of alkaline phosphatase (U/mg protein)

Duodenum

villus

Ileum

crypt villus

crypt

tip mid base tip mid base

Membranous

Supernatant

0.874_+0.217 0.456_+0.043 0.188_+0.024 0.080 _+0.006 1.026 _+0.075 0.761_+9,953 0.292_+0.024 0.083 _+0.015

0.136_+0.051 0.518_+0.119 0.070 + 0.023 0.029 _+0.006 2.259 _+0.390 0.565_+ 0.383 0.289_+ 0.098 0.042 _+0.003

264 M-band is a membrane-associated enzyme with a hydrophobic anchor-piece.

Villus-crypt gradient of alkaline phosphatase Table II shows that in the adult rat the specific activity of alkaline phosphatase in the membranous fraction decreases from villous tip to crypt, in both the duodenum and the ileum. The same pattern occurs for supernatant alkaline phosphatase, although the rate of fall-off in specific activity towards the crypt is not as great, mid-villus values being almost as high as those in the tip. Similar studies in suckling rats are depicted in Table III. The pattern for the membranous enzyme is the same as in the adult. Whilst the specific activity of the supernatant enzyme is highest at the tip in the ileum, mid-villus cells show the highest levels in the duodenum. Discussion This study demonstrates the presence of a distinctive, soluble form of alkaline phosphatase in the intestinal mucosal cells of adult as well as of suckling rats. The soluble form constituted about 3% of total small bowel activity in the adult rat, whilst over onethird of total activity in the suckling rat was soluble. Hence, this enzyme is not exclusively an integral membrane protein. Despite the difference in physical states of the two forms, they were shown to be antigenically identical by multiple criteria. This supports earlier evidence, based on biochemical analysis of purified proteins [7], that the soluble and membranous enzymes are true isozymes originating from the same gene code. The soluble form is unlikely to be an artifact resulting from in vitro proteolytic solubilization of the membranous enzyme since the combined use of inhibitors of serine, thiol and carboxyl proteases did not prevent the soluble enzyme from appearing in the supernatant fraction of adult rats. In addition, the proportion of enzyme in the supematant fraction was not altered by the use of hypotonic versus isotonic buffers or by different methods of cell disruption. Such findings mean that is is unlikely that the soluble form was a loosely attached peripheral membrane protein. Despite their close antigenit relationship, the gradient of specific activity along the intestine in

adult rats is quite different between soluble and membranous alkaline phosphatases. In agreement with numerous earlier studies, we found the gradient of the membranous alkaline phosphatase to fall steeply from duodenum to distal ileum. As can be seen from Fig. 2, the soluble enzyme followed an almost opposite pattern, with the highest specific activities being seen in the distal half of the jejunum. Another striking difference between soluble and membranous forms was the different electrophoretic properties of the two forms when they were examined by polyacrylamide gel electrophoresis in the presence of Triton X-100. The soluble form always migrated faster than the membranous form and showed electrophoretic heterogeneity along the length of the adult small bowel. The reason for this electrophoretic heterogeneity was not clear. Examination fo the distribution of soluble and membranous forms along the villus-crypt axis showed that the highest specific activities of both forms occurred at the tip of the villus in the adult. This suggests that the same cells on the villis-crypt axis produce both forms. Immunoperoxidase studies in progress support this suggestion (Shields, H., personal communication). It could be that at different levels of the small bowel the soluble form possesses a different carbohydrate composition. Thus its charge: size ratio would change, altering its electrophoretic characteristics (although any change in antigenicity must be minimal). In contrast, similar patterns of distribution of the two forms were observed in suckling rats and these patterns were compared with those of adult rats in an attempt to gain insight into the nature of this phenomenon. There was no electrophoretic heterogeneity of suckling rat soluble alkaline phosphatase. Although the proportion of alkaline phosphate in the supernatant fraction increases in both mature and immature animals from duodenum to ileum, the total proportion which is soluble is considerably higher in the suckling rat. Similar findings were seen for supernatant maltase, whereas relatively low specific activities of lactase and sucrase were found in the supernatant fractions of suckling and adult rats respectively. In adults rats maximal specific activities of the membrane-bound hydrolases had shifted proximally; to duodenum for alkaline phosphatase and to jejunum for disaccharidases. The ileum remained the

265 site of maximal specific activity of the supernatant hydrolases, although the proportion of total activity contributed by the supernatant enzymes fell considerably after maturation. This suggests that the mechanism in the suckling rat which leads to high proportions of supernatant brush border hydrolases in the ileum is still operative in the adult but to a lesser degree. It should be stressed, though, that the mere presence of hydrolases other than alkaline phosphatase in the supernatant fraction does not mean that they are there for the same reason. Rat brush border disaccharidases are released from the cell membrane more readily than is alkaline phosphatase, by a variety of techniques including detergents, proteases and mechanical disruption [17], yet proportionally much more alkaline phosphatase than sucrase is present in the supernatant fraction of adult ileal homogenate. It remains to be shown whether the supernatant forms of these disaccharidases are distinct from their membranous counterparts. This is clearly the case for alkaline phosphatase in view of (a) the electrophoretic differences, (b) the ability of the soluble form to enter polyacrylamide gels in the absence of detergent, (c) the high affinity of the membranous form for liposomes and (d) their different synthetic patterns (Young, G.P., unpublished data). Soluble alkaline phosphatases may be either cytosolic, packaged within very fragile intracellular vesicles or a loosely attached peripheral membrane protein. The present results will have to be compared with those of immunohistological techniques before the question of localization can be answered with certainty. The cause of elevated levels of the soluble form in the intestine of the suckling rat has not yet been answered. The soluble form could accumulate due to a failure of its release from the mucosal cell. Fat-feeding is known to increase serum intestinal alkaline phosphatase levels in adult rats [18] and the relationship of this rise to the soluble tissue enzyme requires investigation.

Acknowledgements The authors thank Ms, Cathy Camp and Ms. Pam

Helms for their assisstance with the manuscript. This work was supported by Internal Research Fellowship F05 TWO 2672 (G.P.Y.) and grants AM 05280, AM 07130 and GM 00371 from the National Institutes of Health, U.S. Public Health Service. G.P.Y. was also supported in part by a Royal Australasian College of Physicians Overseas Fellowship. The helpful advice provided by Drs. Helen Shields and BeUur Seetharam is gratefully acknowledged.

References 1 McComb, R.B., Bowers, G.N. and Posen, S. (1979) Alkaline Phosphatase, p. 83, Plenum Press, New York 2 McComb, R.B., Bowers, G.N. and Posen, S. (1979) Alkaline Phosphatase, p. 448, Plenum Press, New York 3 Moog, F. and Angeletti, P.U. (1962) Biochim. Biophys. Acta 60,440-442 4 Moog, F., Vire, H.R. and Grey, R.D. (1966) Biochim. Biophys. Acta 113,336-349 5 0 n o , K. (1975) Aeta Histochem. 53,117-133 6 Seetharam, B., Yeh, K.-Y. Moog, F. and Alpers, D.H. (1977) Biochim. Biophys. Acta 470,424-436 7 Yedlin, S.T., Young, G.P., Seetharam, B., Goodwin, C.L. and Alpers, D.H.J. Biol. Chem. 256, 5620-5626 8 Forstner, G.G., Sabesin, S. and Isselbaeher, K.J. (1968) Bioehem. J. 106,381-390 9 Henning, R. and Plattner, H. (1974) Biochim. Biophys. Acta 354, 114-120 10 Dahlqvist, A. (1968) Anal. Biochem. 22, 99-107 11 Lowry, O.H., Rosebrough, N.J., Farr, A.O. and Randall, R.J. (1951) J. Biol. Chem. 193,265-275 12 Davis, B.J. (1964) Ann. N.Y. Acad. Sci. 121,404-427 13 Neville, D.M. (1971) J. Biol. Chem. 246, 6328-6334 14 Merchant, J.L. and Heller, R.A. (1977) J. Lipids Res. 18, 722-733 15 Simon, P.M., Kedinger, M., Raul, F., Grenier, J.F. and Haffen, K. (1979) Biochem. J. 178,407-413 16 Pitt-Rivers, R. and Impiobato, F.S.A. (1968) Biochem. J. 109,825-830 17 Eloy, R., Haffen, K. and Grenier, J.F. (1977) in Progress in Gastroenterology, (Glass, G.B.J., ed.) Vol. 3, pp. 454-455 Grune and Stratton, New York 18 Madsen, N.B. and Tuba, J. (1952) J. Biol. Chem. 195, 741-750