independent biosynthesis of soluble and membrane-bound alkaline phosphatases in the suckling rat...

Biochem. J. (1981) 200, 645-654 645Printed in Great Britain

Independent biosynthesis of soluble and membrane-bound alkalinephosphatases in the suckling rat ileum

Graeme P. YOUNG,* Steven T. YEDLIN and David H. ALPERStDivision ofGastroenterology, Department ofInternal Medicine, Washington University School ofMedicine,

660 S. Euclid Avenue, St. Louis, MO 63110, U.S.A.

(Received 20 July 1981/Accepted 31 July 1981)

Enzymically active intestinal alkaline phosphatase exists in both soluble andmembrane-bound forms in the suckling rat. Antiserum prepared against purified solublealkaline phosphatase (anti-AlP) was shown to be monospecific when assessed byOuchterlony double-diffusion analysis and immunoelectrophoresis. The two forms ofalkaline phosphatase were antigenically identical and possessed similar affinities foranti-AlP. To study the biosynthesis of the two forms, 14-day-old rats were injectedintraperitoneally with [ 3Hlleucine. The labelling kinetics of alkaline phosphatase,extracted from supernatant and brush-border membrane fractions with anti-AlP, wasfollowed over 20h. Incorporation of [3Hlleucine into membrane-bound alkalinephosphatase was rapid, reaching a plateau at 6h. The soluble enzyme showed slowerincorporation of label and maximal radioactivity was not reached until 12h afterlabelling, a lag of 6 h behind the membrane-bound enzyme. Soluble alkaline phosphatasecould not have been a precursor of the membrane form, as there was no early peak ofradioactivity in the soluble form. To determine if the soluble enzyme was irreversiblyderived from the membrane enzyme, a newly developed technique of labellingbrush-border membrane proteins in vivo by intraluminal injection of diazotizedf1251liodosulphanilic acid was used. The appearance of 1251 in soluble and membranealkaline phosphatase was then monitored over a 7h period, encompassing the lagbetween maximal leucine labelling of the two forms. The results failed to show either aproportional transfer of radioactivity from membrane to soluble alkaline phosphataseor an absolute increase in radioactivity of the soluble form during degradation ofbrush-border alkaline phosphatase. Therefore there does not appear to be a serialprecursor/product relationship between thesuckling-rat intestinal alkaline phosphatase.

Certain hydrolases of the intestinal brush bordercan occur in the supernatant as well as themembrane fraction after homogenization of mucosa.Hynie & Zbarsky (1973) found that up to 39% ofintestinal alkaline phosphatase was in a soluble formin suckling-rat small bowel. Similarly, Galand &Forstner (1974) found 44% of maltase activity inthe supernatant fraction of suckling rat gut homo-genates. The soluble proportion fell to 7% after

Abbreviation used: anti-AIP, antiserum monospecificfor intestinal alkaline phosphatase.

* Present address: Department of Medicine, Universityof Melbourne, Royal Melbourne Hospital, Melbourne,Vic. 3050, Australia.

t To whom reprint requests should be addressed.

soluble and membrane-bound forms of

weaning, and after treatment of suckling rats withcortisol. Seetharam et al. (1977) confirmed thesefindings and showed that this phenomenon was moremarked in the ileum, that it persisted to a lesserdegree in adult rats, and that the soluble enzymescorresponded to their brush-border but not theirlysosomal counterparts. The cellular location ofthese soluble enzymes was not determined. Forstner& Forstner (1979) have shown that developmentalchanges in soluble maltase are not coincident withthose of lysosomal marker enzymes, indicating thelack of dependence between the soluble enzymephenomenon and the endocytic-lysosomal appar-atus of the villous cell.

The genetic relationship of these two intestinalalkaline phosphatases in uncertain. They may arise

0306-3283/81/120645-10$01.50/1 ©) 1981 The Biochemical SocietyVol. 200

G. P. Young, S. T. Yedlin and D. H. Alpers

from two different gene codes, and the changeswhich occur with maturation could represent achange in gene expression. To clarify their relation-ship, we purified the two forms from the suckling-ratileum (Yedlin et al., 1981). Comparison of thepurified hydrolases revealed similar kinetics formultiple substrates and similar amino acid com-positions, suggesting that they originated from thesame gene code. However, the membrane enzymediffered from the soluble enzyme in a number ofways. First, the membrane enzyme was incorporatedreadily into synthetic liposomes. Second, it pos-sessed a different carbohydrate composition, withmore mannose and less fucose than the solubleenzyme. Third, the principal isoelectric points ofmembrane alkaline phosphatase were more acidicthat those of the soluble form. The soluble enzymewas not an artefact of proteolysis during enzymepurification. It was concluded that the soluble formlacked the hydrophobic anchor-piece necessary formembrane incorporation.

If soluble and membrane-bound alkaline phos-phatase were derived from the same gene code, theymay be biosynthetically related in one of twodifferent ways: synthesis could proceed in parallelfrom a common precursor (e.g. nascent peptide), oralternatively they could have a serial precursor/product relationship. The different carbohydratecompositions of the two forms suggests that thelatter is unlikely (Yedlin et al., 1981), but suchevidence is not conclusive.

Therefore we have further studied the relation-ship between the two forms of intestinal alkalinephosphatase in the suckling rat by using highlyspecific antibody prepared against soluble alkalinephosphatase. The kinetics of incorporation of 1I3H1-leucine in vivo into each form of alkaline phos-phatase have been elucidated. We have also exam-ined the possibility that soluble alkaline phos-phatase is derived from the membrane-bound form,by using a newly developed method of labelling withdiazotized [125lliodosulphanilic acid. We concludethat soluble alkaline phosphatase is neither a pre-cursor nor a product of the membrane enzyme.

Materials and methods

AnimalsPregnant albino Wistar rats were obtained from

National Laboratory Animal Co., O'Fallon, MO,U.S.A. After delivery, all litters were made equal insize, to ensure uniform growth rates.

Preparation ofbrush borders and solublefractionAt the desired age, suckling rats were stunned and

decapitated, and the small bowel was immediatelyremoved and rinsed with cold iso-osmotic saline(0.9% NaCI). The distal half of the small bowel (i.e.

the ileum) was cut into small pieces. This washomogenized at 40C in 2mM-Tris/HCl, pH7.4,containing 50mM-mannitol, with five up-and-downstrokes of a Potter-Elvehjem-type size B tissuegrinder (A. H. Thomas Co., Philadelphia, PA,U.S.A.) with Teflon pestle rotating at 900rev./min.The method of Schmitz et al. (1973) was used toisolate brush-border fragments, which were thenresuspended in 10mM-Tris/HCl, pH7.4, containing0.2 mM-MgCl2 (hereafter called Tris/Mg buffer)before further treatment. The supernatant fractionremaining after isolation of brush borders was thenspun at 105 0OOg for 60min and the resultantsupernatant fraction used as the source of solublealkaline phosphatase.

Enzyme andprotein assaysAlkaline phosphatase was assayed by the'method

of Forstner et al. (1968) at pH9.2 and acidphosphatase was assayed by the method of Henning& Plattner (1974) at pH4.8, with p-nitrophenylphosphate (Sigma Chemical Co., St. Louis, MO,U.S.A.) as substrate in both cases. One unit ofactivity is the amount of enzyme that hydrolyses1,umol of substrate in min at 37°C. Samples'foralkaline phosphatase and acid phosphatase assayswere always pretreated with 0.1% Nonidet P40(BDH Chemicals, Poole, Dorset, U.K.). Protein wasassayed by the method of Lowry et al. (1951), withbovine serum albumin (97-99% pure; Sigma) asstandard.

Polyacrylamide-gel electrophoresisDisc gel electrophoresis was performed by the

method of Davis (1964), with or without 0.1%Triton X-100 (Fisher Scientific Co., Pittsburgh, PA,U.S.A.). Total acrylamide concentration was 5%(acrylamide:bisacrylamide 38:1). On each gel 0.1unit of soluble or solubilized membrane alkalinephosphatase was loaded and electrophoresed at2mA per gel. After electrophoresis, gels were stainedfor alkaline phosphatase activity at room tem-perature with a solution of 2mM-fi-naphthyl acidphosphate, 1 mM-tetrazotized o-dianisidine, and0.4mM-MgCl2 in 7.5 mM-sodium barbital buffer,pH 9.4. The o-dianisidine was added last. Allchemicals were obtained from Sigma.

Detergent solubilization ofmembranefractionsMembrane alkaline phosphatase was solubilized

from either brush borders or total membranepreparations (i.e. the 105 OOOg pellet of intestinalhomogenates). In each case solubilization wasperformed at 40C for 16 h in Tris/Mg buffer. Thefollowing non-ionic detergents were used in variousconcentrations: Nonidet P40, Triton X-100,Emulphogen and Empigen BB (Marchon, White-

1981

646

Intestinal alkaline phosphatases

haven, Cumbria, U.K.). Solubilized material wascollected after centrifugation at 45 0OOg for 15 min.

Ethanol precipitation ofproteinsBoth detergent-solubilized membrane proteins and

soluble tissue proteins were precipitated with 70%(v/v) ethanol precooled to -15°C. Precipitation wasperformed in precooled containers in a freezer roomat -15 0C, for 30min. The precipitated proteins werecollected by centrifugation at 45 0OOg for 15 min.The pellet was redissolved in Tris/Mg buffercontaining 0.1% Nonidet P40 and denatured proteinremoved by repeat centrifugation.

Preparation and characterization ofantiseraAntiserum was prepared against purified soluble

intestinal alkaline phosphatase in rabbits asdescribed in detail by Yedlin et al. (1981). Immuni-zation was performed over a 6-week period by usingFreund's complete adjuvant. Antiserum was adsor-bed with the supernatant fraction from the suckling-rat duodenum before use. Anti-AlP was mono-specific when assessed by Ouchterlony double-diffusion analysis (Fig. la) and immunoelectro-phoresis (Fig. lb). Titration of anti-AlP against thetwo forms of ileal alkaline phosphatase (Fig. 2)revealed a similar titre and affinity for each form.Anti-AlP did not cross-react with rat bone or liveralkaline phosphatase or with rat acid phosphatase.There was no evidence of precipitation of otherbrush-border hydrolases (sucrase, maltase andlactase) during the titration studies.

[3HlLeucine-labelling studiesFor this, 47 14-day-old non-starved suckling rats

from five litters were each' injected intraperitoneallywith 50,uCi of [3H]leucine (specific radioactivity45.7 Ci/mmol; New England Nuclear, Boston, MA,U.S.A.) contained in 0.25ml of 50mM phosphate-buffered iso-osmotic saline, pH7.4. Animals werereturned to their mothers and then killed at thefollowing time points: 10, 20, 30 and 60min, 2, 3, 4,6, 8, 12, 16 and 20h. Ileal homogenates wereprepared in 18ml of buffer as described above. Asample (0.5ml) of homogenate was centrifuged at105OOOg for 60min to allow determination of thetotal amount of enzyme in the soluble and mem-brane fraction of each animal's ileum. The remaining17.5 ml was used to make brush-border andsoluble fractions. After solubilization of brush-border proteins in Nonidet P40, both the solublefraction and detergent-solubilized brush-borderproteins were precipitated by ethanol as describedabove. Precipitated proteins were resolubilized in0.1% Nonidet P40 in Tris/Mg buffer. Alkalinephosphatase was then isolated by immuno-precipitation.

Vol. 200

Fig. 1. (a) Ouchterlony double-diffusion analysis ofanti-AIP against various forms of intestinal alkalinephosphatase, and (b) immunoelectrophoresis of ilealsupernatant fraction by the method of Grabar &

Williams (1953)(a) The central well contains anti-AlP. Commencingfrom the top well and moving clockwise, theperipheral wells contain: ileal supernatant fraction,purified soluble alkaline phosphatase, purified mem-brane alkaline phosphatase, Nonidet-P40-solubilizedileal brush-border proteins, ileal supernatant fractionand Nonidet-P40-solubilized ileal total membranefraction. Immunodiffusion was performed in0.8%-agarose containing 50mM-sodium barbital,pH 8.6, and 0.1% Nonidet P40. (b) Supernatantfraction (105OO0g) from the ileum containing 0.9unit of alkaline phosphatase activity was placed inthe top well. The same amount of protein from aduodenal supernatant fraction was placed in theother well. Electrophoresis was performed towardsthe anode for 2h. A 1:4 dilution of anti-AlP wasthen placed in the trough. Histochemical staining foralkaline phosphatase (see the Materials and methodssection) showed concentration of enzymic activity inthe precipitin line. The support medium was thesame as in Fig. 1(a).

Immunoprecipitation ofalkaline phosphataseAlkaline phosphatase was isolated from brush-

border and soluble fractions by immuno-precipitation from 1 ml samples containing a known

647


100

80\.

60 -

. 40 -

20

0

32 64 128 256

1 /Titre

Fig. 2. Titration curve of anti-AlP againsiof ileal alkaline phosphatase

V, Purified soluble alkaline phosphata

supernatant fractions; O, Nonidet-P44ileal brush-border proteins. The conc(alkaline phosphatase in each case was IFor further details see the Materials a

section.

amount of alkaline phosphatase acirange 5-10 units. To determine thi

antibody required to precipitate at Ienzyme activity, pilot studies were pe

50,ul of sample and 50,ul of dilu

Generally a titre of 1:8 or 1:16 N

Sample/anti-AlP mixtures were then

37°C for 2h and maintained at 4c

Visible immune pellets were collectcfugation at 1200g for 10min, and th

fraction was assayed to ensure that

98% enzyme activity had been precipit

pellets were washed three times in T

containing 0.1% Nonidet P40. Non-sp4

in the immune pellet of radioactiveI

than alkaline phosphatase was determiinon-radioactive concentrated alkaline

to the immune-supernatant fractioniequal to the original enzyme activity.

was repeated with the same amount of

the pellets were washed as described abTo determine the incorporation of3I

phosphatase, washed immune pelletEpended in 50,ul of water and then

scintillation vials containing 0.5ml of

Solubilizer (0.6M; Amersham/SearlHeights, IL, U.S.A.) After solubiliza

temperature for 16h,lOml of a

scintillation fluid was added, each i

contained 6g of 2,5-diphenyloxazole

1,4-bis-(5-phenyloxazol-2-yl)benzene (New EnglandNuclear). Radioactivity was determined in a Pack-ard Tri-Carb liquid-scintillation spectrophotometer(model 3320) until 10000 counts were accumulatedor 50 min had elapsed. Background radioactivitywas 15.7c.p.m. Samples with the lowest radio-activity gave at least twice this number of counts.Counting efficiency determined by the channels-ratiomethod was uniform, in the range 40.5-42.5%. Forsoluble alkaline phosphatase, non-specific trappingof radioactivity was always less than 4.6% of totalradioactivity. When alkaline phosphatase wasimmunoprecipitated from detergent-solubilizedbrush borders, non-specific trapping was fairlyconstant, being no higher than twice background.

512 1024This represented 2-27% of radioactivity in alkaline

512 1024 phosphatase, depending on the time point. Pre-liminary studies showed that if ethanol precipita-

tvarious forms tion of fractions was omitted before immuno-ise;*, ileal precipitation, non-specific trapping could reach 5O-solubilized times background.entration of [3H]Leucine incorporation into soluble or mem-1.4 units/ml. brane alkaline phosphatase was calculated for eachnd methods animal, after correction for non-specific trapping and

background. Incorporation was calculated in twoways: (a) c.p.m. of 3H per unit of enzyme activity;and (b) c.p.m. in the total mass (i.e. c.p.m. per unit ofactivity x total units in each fraction) of ileal soluble

tivity, in the or brush-border-bound alkaline phosphatase. Thee amount of first method fails to account for variations in theleast 98% of total mass of enzyme and for a possible difference in-rformed with catalytic efficiency (units of enzyme activity per molted anti-AlP. of enzyme protein) between the two forms.was required.incubated at Labelling studies with[[25lIiodosulphanilic~C overnight.

ed by centri- [125Ilodosulphanilic acid (>1OOOCi/mmol; Newe supernatant England Nuclear) was converted into the diazoniumgreater than salt according to the instructions provided by New

;ated. Immune England Nuclear and used immediately. The use ofris/Mg buffer this agent in vivo to label brush-border membraneecific trapping proteins of adult rats has been described in detailproteins other elsewhere (Young & Alpers, 1981). Nine 16-day-oldned by adding rats from a single litter were operated on, underphosphatase ether anaesthesia; 65,uCi of diazotized [i25J]-

in an amount iodosulphanilic acid in 0.2 ml of 10 mM-potassium. Precipitation phosphate-buffered iso-osmotic saline, pH7.4, wasanti-AlP and injected via a 27-gauge needle into the distal bowel

)ove. at the jejuno-ileal junction. No obvious peritonealHI into alkaline leak was seen through the small puncture hole. All

were resus- animals recovered rapidly from the procedure aftertransferred to closure of the abdominal wound with acontinuousNCS Tissue silk suture. Animals were returned to their mothers

le, Arlington and killed after 1, 3 or 7 h, three at each time point.tion at room After extensive rinsing of the intestine with phos-toluene-based phate-buffered saline, ileal homogenates were pre-itre of which pared as described above by using eight strokes ofand 75 mg of the homogenizer. Homogenates were then im-

1981

648


mediately treated with 70% ethanol as described andprecipitated for 10min. This extracted over 90% ofnon-protein-bound diazotized [1251]iodosulphanilicacid (assessed by precipitation with 10% trichloro-acetic acid). This made multiple washing of mem-branes unnecessary and achieved separation ofreleased soluble intracellular proteins from free label,thus stopping the reaction. Ethanol-precipitatedhomogenate proteins were redissolved in Tris/Mgbuffer, and soluble alkaline phosphatase wasseparated from membrane-bound alkaline phos-phatase by centrifugation at 105000g for 60min.Supernatant fractions were dialysed overnight at4°C against Tris/Mg buffer. Membrane proteinswere solubilized, precipitated with ethanol andresolubilized in 0.1% Nonidet P40 as described forbrush-border membranes. These treatments resultedin over 95% of diazotized [ '25lliodosulphanilic acidbeing bound to protein, as assessed by trichloro-acetic acid precipitation. Alkaline phosphatase wasimmunoprecipitated from each fraction as describedabove and incorporation of 125I measured in aNuclear-Chicago gamma counter for 10min. Back-ground was 14 c.p.m., counting efficiency was 71%.Results were expressed as c.p.m. in total mass ofsoluble or membrane alkaline phosphatase.

Uptake of 125I into the liver was determined bycounting radioactivity of ml samples of a 10%(w/v) liver homogenate prepared in 50 mM-mannitol/lOmM-Tris/HCI (pH 7.4).

Results

In the 14-day-old rat, 55% of small-bowel alkalinephosphatase activity was found in the ileum. Of thisileal enzyme, 64.9 + 6.7% (mean + S.D.) was found inthe 105 OOOg supernatant fraction. That is, in theileum the pool of soluble enzyme is twice the size ofthat of the membrane-bound enzyme, based on

enzyme activity. In the jejunum, only 10.9 + 4.3%was soluble. Fig. 3 shows the results of poly-acrylamide-gel electrophoresis of ileal supernatantand membrane-bound alkaline phosphatase. Themembrane (i.e. brush-border) form migrated slowly,with a mean RF of 0.23, and would not enter the gelin the absence of non-ionic detergent. The super-natant form entered the gel whether or not detergentwas present, with a mean RF of 0.44 in the presenceof detergent. The slow form was not seen in the ilealsupernatant fraction (105 000g), nor was the fastform seen in washed pellets. Hence centrifugation athigh speed for 1 h was a satisfactory means ofseparating the two.

Acid phosphatase activity was distributed dif-ferently along the intestine from alkaline phos-phatase; 75% of activity was found in the distalhalf of the gut. The amount of activity released intothe supernatant fraction by homogenization was

Vol. 200

Fig. 3. Polyacrylamide-disc-gel electrophoresis of mem-brane (a) and soluble (b and c) fractions of ilealhomogenates, stainedfor alkaline phosphatase activityEnzyme activity was detected in these Tris/glycinegels as described in the Materials and methodssection. Gels and samples for (a) and (b) contained0.1% Triton X-100. No detergent was present in (c).The RF values for alkaline phosphatase are: (a), 0.23(b) 0.044, (c) 0.50. The slightly dense area which isseen behind the dye front in gels (a) and (b) is due tointeraction between Triton X-100 and a componentof Bromophenol Blue.

33.6 + 1.6% (mean + S.E.M.) in the ileum and31.9 ± 3.6% in the jejunum.When untreated ileal supernatant proteins were

fractionated on Sephadex G-200, alkaline phos-phatase activity was eluted with a molecular weightof 97000, in close agreement with the findings ofYedlin et al. (1981) for the purified enzyme.

(a) (b) (c)

649

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.w....NW..1


Immunoprecipitation of these fractions showed

radioactivity to be proportional to the amount ofenzymically active alkaline phosphatase present(Fig. 4). No other peaks of radioactivity were

observed, indicating that anti-AlP did not precipitateenzymically inactive fragments of the alkalinephosphatase molecule or other radioactive proteins.

0.6

c

C)

N

0.4

0.2

;o

C.)

0 >

;:>co

!O la0-

a4

0 4 6 8 10 12 14 16 18Fraction no.

Fig. 4. Immunoprecipitation of alkaline phosphatasefrom ileal supernatant proteins separated on Sephadex

G-200Ileal supernatant fractions from three rats, eachlabelled for 12h with 50,uCi of [3Hlleucine, were

prepared as described in the Materials and methodssection, pooled, and 1 ml was applied to a SephadexG-200 column (50 cm x cm). Fractions (0.75 ml)were eluted with Tris/Mg buffer at a flow rate of6.3 ml/h per cm3. After assay of fractions foralkaline phosphatase, they were pooled in pairs andan equal volume of anti-AlP diluted 1:32 was added(sufficient to precipitate all alkaline phosphataseactivity). Radioactivity of the immunoprecipitatewas determined as described in the Materials andmethods section. 0, Enzyme activity; 0, radio-activity.

This latter observation further confirmed the mono-

specificity of anti-AlP.

Solubilization ofalkaline phosphataseThe effect of solubilization of alkaline phos-

phatase with Nonidet P40 is shown in Table 1.Regardless of whether brush-border or total mem-

brane preparations were used, a high concentrationof detergent was needed to solubilize 95% of theenzyme activity. With Nonidet P40, a 1% con-

centration solubilized 86.5% of alkaline phos-phatase activity from the brush-border preparations.With Triton X-100, Empigen BB or Emulphogen atthe same detergent/protein ratio of 100 :1 (w/w),81.5%, 81.0% and 75.0% solubilization respectivelywas achieved. Detergent had no effect on solublealkaline phosphatase acfivity, but it did give a smallincrease in membrane alkaline phosphatase activity.With Nonidet P40, the mean increase was 9.9%when solubilizing total membranes and 15.2% whensolubilizing brush borders. The percentage increasewas not concentration-dependent between 0.1 and5.0% detergent and presumably resulted from betteraccess of the substrate to the enzyme.Some 99% of alkaline phosphatase activity was

precipitated by 70% ethanol at -15°C. Recoveryafter resolubilization exceeded 95%. Even though itwas necessary to solubilize initially with 5% NonidetP40, the ethanol-precipitated material could beresolubilized was as little as 0.1% Nonidet P40. Thislow detergent concentration simplified enzyme andprotein assays and minimized non-specific trappingof other proteins during immunoprecipitation. Pre-treatment of samples with ethanol did not effectelectrophoretic mobility of alkaline phosphatase,except that sharper bands were obtained (results notshown).

Table 1. Solubilization ofalkaline phosphatasefrom suckling rat ileal mucosa by Nonidet P40The total membrane preparation was collected by centrifugation for 60min at 45 OOOg of pooled ileal homogenatesfrom 15 suckling rats. Homogenates were prepared in 50mM-mannitol/2 mM-Tris/HCl (pH 7.4). Ileal brush borderswere prepared as described in the Materials and methods section from the same homogenate. Solubilization ofthese membrane preparations was performed overnight in Tris/Mg buffer at 40C. Insoluble material was removedby centrifugation for 60min at 45000g, and solubilization of alkaline phosphatase was determined by assayingsupernatant fractions in triplicate. Control values were obtained by remixing pellet and supernatant fractions foreach detergent concentration. The values in parentheses refer to the detergent concentration (v/v).

Alkaline phosphatase activity (units/ml)

Total membranes

Ileal brush borders

Detergent/protein, A

ratio (w/w) Control Supernatant60:1 (5%) 4.0 3.912:1 (1%) 4.1 3.01.2:1 (0.1%) 4.0 1.9

500:1 (5%) 0.60 0.56100:1 (1%) 0.59 0.5110:1(0.1%) 0.59 0.40

Alkaline phosphatasesolubilized (%)

95.473.546.694.986.567.9

1981

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650 owl


[3HlLeucine labellingFig. 5 shows the results of incorporation of

[3Hlleucine in vivo into the total pools of soluble andmembrane alkaline phosphatase. Significant labellingof brush-border alkaline phosphatase occurred asearly as 30 min and reached a plateau at 6 h.

.2

x)

.S

.x

0 1 2 3 4 6 8 12Duration of labelling (h)

Fig. 5. Patterns ofincorporation of [3HIletpools of brush-border or soluble alkaline I

the ileum ofthe 14-day-old raEach rat was injected intraperitoneallyof [3Hileucine and alkaline phospiimmunoprecipitated from soluble andfractions as described in the Materials asection. Each point represents the nporation into the total pool of enzyme ir(E) or membrane (0) fractions. Veindicate S.E.M. Studies continued l,showed that maximal incorporation of rinto the soluble form occurred at 12values for the membrane enzyme for fnot significantly different (unpaired Studnor were those for 12-20h for the solubli

Labelling of soluble alkaline phosphatase proceededmore slowly and reached a plateau at 12 h. The closeagreement in radioactivity incorporated into thetotal pool of ileal soluble and ileal memnbranealkaline phosphatase at 20h (Fig. 5) does not reflectsimilar specific radioactivities. At 20h there were45.4c.p.m./unit of membrane alkaline phosphataseand 23.9 c.p.m./unit of soluble alkaline phos-phatase. Radioactivity in the total pools of soluble ormembrane enzymes was similar, as there was almosttwice as much soluble as membranous enzyme(64.9% was soluble). If the results were plotted asc.p.m./unit of alkaline phosphatase activity ratherthan as shown in Fig. 5, no change in relativepatterns of [3Hlleucine incorporation was seen.

T | Labelling with diazotized [ l2I]iodosulphanilic acidTable 2 shows the results of labelling in vivo of

ileal alkaline phosphatase with diazotized ['2III-iodosulphanilic acid. Radioactivity incorporated intothe membrane alkaline phosphatase pool reacheda maximum at 3h, owing to continuation of thelabelling reaction. The subsequent fall between 3 and

16 20 7h presumably resulted from turnover of themembrane form, as mean total enzyme activity

ucine into total remained constant (Table 3). Over the 7h of study,phosphatase in radioactivity in the soluble pool, expressed as aIt proportion of total radioactivity in both pools, fellwith 50,uCi from 23.9 to 16.6%. A fall in actual radioactivity inhatase was the soluble enzyme pool occurred between 3 and 7 h,membrane during turnover of the membrane enzyme.

nd methods This labelling technique did not label the mem-nean incor- brane form exclusively, as some labelling of the1 the soluble soluble enzyme was observed. However, membrane

yrtical bars alkaline phosphatase was at least 4 times more

radioactivity radioactive than soluble enzyme, there being almost2-20h. The twice as much of the latter. Some non-protein-bound6-20h were diazotized [125lliodosulphanilic acid was able tolent's t test), cross suckling-rat ileal mucosa. For the liver,le form. maximum radioactivity was observed at 1 h (mean

Table 2. Incorporation of radioactivity into alkaline phosphatase after labelling of ileal mucosa in vivo with diazotized[ '25lliodosulphanilic acid

Nine 16-day-old rats from a single litter each received 65,uCi of diazotized [1251]iodosulphanilic acid (ISA) byintraluminal injection into the ileum (see the Materials and methods section). Three animals were killed at each timepoint, and ileal homogenates were separated into soluble and membrane fractions by centrifugation at 105 OOOg for60min. Alkaline phosphatase was extracted from soluble and Nonidet-P40-solubilized membrane fractions withanti-AlP (see the Materials and methods section). Radioactivity is expressed as c.p.m. incorporated into the totalenzyme pool in each fraction, as means + S.E.M. derived from two determinations in each of three animals.

1i-0 x 12511]ISA-labelled alkaline phosphatase (c.p.m.)t~A~A

Membrane3.61 + 0.249.38 + 0.525.34 + 0.13

Supernatant1.13 ± 0.172.31 +0.161.06 + 0.21

[125IIISA bound to supernatantalkaline phosphatase (%)

23.919.716.6

Time(h)137

Vol. 200

651


Table 3. Total alkaline phosphatase activity in soluble and membrane fractions of suckling-rat ileal mucosa labelledwith diazotized [ 25Iliodosulphanilic acid

See Table 2 for details. Results are means + S.D. for three rats at each time-point.Mass of alkaline phosphatase (units/ileum)

Time ,-(h) Membrane1 15.82 + 2.643 16.60+2.467 17.04 + 0.94

Soluble28.6 + 2.027.6 + 3.631.0 + 3.8

Ratiosoluble/membrane

1.81.71.8

496.6 x 103c.p.m. per liver). This represented 3% ofthe total dose. By trichloroacetic acid precipitation,70.8% of radioactivity was protein bound. A similarobservation has been made in adult rats (Young &Alpers, 1981).

Discussion

The results indicate that there are importantbiosynthetic differences between the two immuno-logically identical but structurally different forms ofintestinal alkaline phosphatase found in the ileum ofthe suckling rat. The pool of soluble enzyme is twicethe size of that of membrane-bound enzyme, basedon enzyme activity in supernatant and membranefractions. This is likely to be true for enzyme proteinmass also, as the two forms have similar specificactivities for p-nitrophenyl phosphate and havesimilar molecular weights (Yedlin et al., 1981).When suckling rats were injected with P3Hlleucine,radioactivity appeared more rapidly in the pool ofmembrane enzyme; maximal radioactivity in thesoluble pool was not reached until about 6 h after themaximum in membrane alkaline phosphatase (Fig.5). The absence of an early peak of radioactivity inthe soluble pool means that the soluble enzymicallyactive alkaline phosphatase is most unlikely to be a

precursor of membrane alkaline phosphatase.However, it was possible that soluble alkaline

phosphatase was derived from the brush-borderform without loss of enzyme activity, by action ofluminal proteinases, with or without bile. Theenzymically active soluble protein would sub-sequently have to be taken up intact by thevillous cell. For a number of reasons it seemed thatsolubilization of brush-border alkaline phosphatasein vivo would not account for this soluble form ofthe enzyme. We have shown that the two glyco-proteins have considerably different isoelectricpoints and carbohydrate compositions: in particular,the soluble form possessed more fucose and lessmannose (Yedlin et al., 1981). Such differences insugar composition would require extensivemodification of carbohydrates after membranerelease, involving addition as well as removal of

sugars. In addition, after labelling in vivo with[3Hlleucine, the specific radioactivity of the solubleenzyme (c.p.m./unit) only reached half that achievedin the membrane enzyme. Furthermore, radio-activity in the membrane pool of alkaline phos-phatase did not decline rapidly as it rose in thesoluble pool. For these reasons we decided to studymore directly the possibility that soluble enzyme wasa derived product of the membrane enzyme.

Diazotized [ '25lliodosulphanilic acid has beenused in low concentrations to label plasma-membrane proteins of platelets and erythrocyteswithout affecting cell function (Sears et al., 1971;George et al., 1976). When diazotized F125IIiodo-sulphanilic acid was tested as a label for adult-ratintestinal brush-border proteins in vivo, we foundthat principal proteins were labelled proportionallyto their relative presence in the membrane (Young &Alpers, 1981). In the present study some non-protein-bound label was absorbed, with subsequentlabelling of intracellular soluble proteins, includingalkaline phosphatase. Such labelling must haveoccurred in vivo, because it could not be preventedby immediate extraction of homogenates withethanol to remove label that had not reacted. Thislabelling in vivo of soluble enzyme accounted for lessthan 24% of total alkaline phosphatase labelled andthus did not interfere with the interpretation ofresults. The rationale for labelling brush-borderalkaline phosphatase by this method was thatradioactivity would be transferred to the solubleenzyme pool, if it were derived from the brush-border form. Provided that soluble alkaline phos-phatase was turned over (or degraded) more slowlythan membrane alkaline phosphatase, then a rise inradioactivity would be seen in the soluble enzymepool as it fell in the membrane pool.

The difference in rates of incorporation of[3Hlleucine into soluble and membrane pools ofalkaline phosphatase depends on the ratio ofsynthesis to degradation in each form and does notprovide information about synthesis or degradationindependently. But if soluble enzyme were derivedfrom brush-border enzyme by an irreversibleprocess, relative degradation rates can be deducedfrom pool sizes, because the rate of transfer of

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enzyme to the soluble pool cannot exceed thesynthesis rate of membrane-bound enzyme. Two-way exchange between the pools appears unlikely, asthe soluble enzyme lacks the hydrophobic anchor-piece necessary for incorporation into membranes(Yedlin et al., 1981). Because the soluble enzymepool is twice the size of the membrane pool, thedegradation rate of the soluble enzyme would needto be less than (approximately half) that of mem-brane enzyme to account for the difference in poolsizes, if the two enzyme pools were serially related toeach other. There is also direct evidence that thesoluble enzyme pool is turned over slowly in thesuckling rat. By studying the effect of fat ingestionon intestinal alkaline phosphatase activity in plasmaand small-bowel mucosa, we have shown thatcirculating enzyme arises from the soluble mucosalenzyme (Young et al., 1982). The fractionalclearance rate of the plasma enzyme is 0.13 unit/min,based on the plasma half-life of '25I-labelled enzyme.Mean plasma intestinal alkaline phosphatase insuckling rats is 0.014 unit/ml. It can thus becalculated that soluble enzyme is released into thecirculation at a rate of approx. 0.1 unit/h. This isonly a small fraction of the total soluble pool in theenterocyte, of over 30 units. In fat-fed adult rats, therate of release is 7.5 units/h and the small-bowelsoluble pool is 20-30 units. Thus a much largerfraction of the soluble pool is being released in adultrats.

Despite these arguments suggesting a slow turn-over of the soluble pool, the proportion of total1251I-labelled alkaline phosphatase did not increase inthe soluble pool at the expense of the membranepool, during the 6 h period covering the lag betweenmaximal [3Hlleucine labelling of the two forms. Inparticular, in the 4 h after maximal labelling ofmembrane enzyme, total radioactivity fell in thesoluble pool (Table 2). As a rise in neither theproportion of radioactivity bound to the solubleform nor its specific radioactivity occurred, theevidence is against the soluble enzyme being derivedfrom the brush-border form.

The labelling experiments described here dependupon the fact that there is not a trivial explanationfor the two forms of alkaline phosphatase. They arereadily separable by high-speed centrifugation ofhomogenates, and the soluble enzyme is not derivedfrom the membrane form by proteolysis duringhomogenization (Yedlin et al., 1981). The super-natant form is clearly soluble, as it will enterpolyacrylamide gels in the absence of detergent andas it is eluted from a Sephadex G-200 column at amolecular weight of 97000. Soluble alkaline phos-phatase is not lysosomal because: (a) it is liber-ated completely by homogenization, whereas mostlysosomes remain intact, as determined by acidphosphatase activity; (b) histochemical studies have

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not shown activity in lysosomes (Ono, 1975); and (c)the pH optimum is different from that of lysosomalenzymes. However, it is difficult to be certain of thecellular localization of soluble alkaline phosphatase.It may be cytoplasmic, as it is readily released bygentle homogenization. To support this suggestion,alkaline phosphatase has been observed histo-chemically in the subapical cytoplasm (Ono, 1975)and in the cytosolic fraction of intestinal cellsseparated on continuous density gradients (Batt &Peters, 1978). But it cannot be discounted that it iscontained within fragile intracellular organelles. Inparticular, there is a possibility that soluble alka-line phosphatase is a secretory glycoprotein, as in-creased amounts of alkaline phosphatase are foundin the non-chylomicron fraction of mesenteric lymph,and in the serum, after fat feeding (Barrowman,1979).

It is conceivable that the soluble and membranephosphatases are produced by two different celltypes and would not be expected to be bio-synthetically related to each other (Forstner &Forstner, 1979). This possibility seems unlikely, fortwo reasons. First, both forms are present inmucosal cells isolated from the same level of thevillus-crypt axis (Young et al., 1982). Second, in thesuckling-rat ileum, alkaline phosphatase has beenseen both on the membrane and in the apicalcytoplasm in the same cells (Ono, 1975).As the two forms have very similar amino acid

compositions (Yedlin et al., 1981) and are anti-genically identical (Figs. 1 and 2), it seems mostlikely that they originate from the same gene code.Their ultimate biosynthetic differences may resultfrom an early event in the biosynthesis of thenascent peptide which leads to diverging syntheticpathways. The principal biochemical differenceresides in the absence from soluble alkaline phos-phatase of the hydrophobic anchor-piece (Yedlin etal., 1981). This is known to be C-terminal in pigintestinal alkaline phosphatase (Colbeau & Maroux,1978). Thus a terminal deletion mechanism may beresponsible for formation of the two isoenzymes, i.e.the final C-terminal segment of the nascent peptide tobe synthesized may be removed. This could occurpost-translationally, owing to cleavage by pro-teinases, or it may result from two separate mRNAspecies.

The excellent secretarial assistance of Mrs. C. Campand Mrs. P. Helms is gratefully acknowledged. This workwas supported in part by International Research Fel-lowship F05 TWO 2652 (G.P.Y.) and grants AM 05280,AM 07130 and GM 00371 from the National Institutes ofHealth, U.S. Public Health Service. G.P.Y. was alsosupported in part by a Royal Australasian College ofPhysicians Travelling Fellowship.

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281-292George, J. N., Potterf, R. D., Lewic, P. S. & Sears, D. A.

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Young, G. P., Yedlin, S. T. & Alpers, D. H. (1982)Am. J. Physiol. in the press

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independent biosynthesis of soluble and membrane-bound alkaline phosphatases in the suckling rat...

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