isoform of na+, k+-atpase from rumen epithelium identified and quantified by immunochemical methods

Isoform of Na+, K+-ATPase from rumen epithelium

identi®ed and quanti®ed by immunochemical methods

O . H A N S E N

Department of Physiology, Aarhus University, AÊrhus, Denmark

ABSTRACT

Using biopsies of rumen epithelium papillae a net influx of [86Rb+] was measured corresponding to a

high concentration of Na+, K+-pumps found in [3H]ouabain-binding studies (Kristensen et al. 1995). In

the present study the Na+, K+-ATPase in papillae homogenates is compared with puri®ed (Na+, K+)-

ATPase from different sources, immunochemically characterized with respect to the isoform of the

hydrolytic a subunit and the concentration of pumps substantiated by a novel immunochemical

method. Na+, K+-ATPase puri®ed from bovine kidney was shown to contain one homogeneous high-

af®nity population of [3H]ouabain-binding sites (Kd 1.37 nM). The ouabain-binding capacity was

0.82 nmol (mg protein))1. Site-directed polyclonal antibodies raised to isoform-speci®c sequences of

the three known a-subunit isoforms and monoclonal a1-speci®c antibodies were used for isoform

characterization on western blots of peptides separated by SDS-polyacrylamide gel electrophoresis.

All three isoforms were present in Na+, K+-ATPase prepared from bovine brain. The a isoform of

bovine kidney Na+, K+-ATPase and of rumen epithelium homogenate appeared to be a1 whereas a2

and a3 were undetectable. Using an a1-speci®c antibody and 125I-labelled antimouse IgG the content

of (Na+, K+)-ATPase in rumen epithelium was determined by comparison of the signal from known

amount of bovine kidney Na+, K+-ATPase on western blots. By this method rumen epithelium was

found to contain 2.6 nmol Na+, K+-ATPase (g wet wt))1, i.e. a similarly high or even higher

concentration than previously seen in ouabain-binding studies on biopsies.

Keywords [3H]ouabain binding, a isoforms, bovine kidney, electronic autoradiography, InstantIm-

ager, isoform-speci®c antibodies, Na+, K+-ATPase, Na+/SCFA absorption, rumen epi-

thelium.

Received 1 September 1997, accepted 22 December 1997

A copious secretion of saliva with a high concentration

of Na+ and bicarbonate takes place in ruminants

(Dobson 1963). A great part of this Na+ is reabsorbed

across the rumen epithelium in a tight coupling with

absorption of short-chain fatty acids (SCFA) either di-

rectly or via an amiloride sensitive Na+/H+-exchange

(GaÈbel et al. 1988, 1989). In isolated sheets of rumen

epithelium Ferreira et al. (1966) noticed a Na+- and K+-

dependent short-circuit current that was inhibited by

ouabain at the serosal side of the preparation. The

deeper layers of rumen epithelium and especially stra-

tum basale have been shown to contain (Na+, K+)-

activated ATPase (Schnorr et al. 1969, Currell & Munn

1970, Steven & Marshall 1970). One-®fth of ruminal

papillae O2 consumption appeared to be ouabain sen-

sitive (Kelly et al. 1993).

A high concentration of functional Na+, K+-Pumps

in biopsies of rumen epithelium papillae was shown in

[3H]ouabain binding and [86Rb+]in¯ux studies (Kris-

tensen et al. 1995). Excitable tissues usually have high

concentrations of pumps but ruminal papillae in fact

have values that are two to three times higher than

those reported for skeletal muscle from a variety of

mammals (Hansen & Clausen 1988). A detailed char-

acterization of Na+, K+-ATPase from rumen epitheli-

um thus seems appropriate.

Puri®cation of Na+, K+-ATPase from complex

structures may be misleading (Hansen & Clausen 1988)

and studies on homogenate of rumen epithelium pa-

pillae of Na+, K+-Pump associated activity, e.g. mea-

surement of K+-activated pNPPase hydrolysis,

appeared unfeasible. So, further characterization of the

Na+, K+-ATPase in homogenate of bovine rumen

epithelium papillae was carried out by immunochemical

methods and by comparison with enzyme isolated from

bovine kidney. Na+, K+-ATPase is an ab heterodimer

Correspondence: Otto Hansen, Department of Physiology, Aarhus University, Ole Worms AlleÂ 160, DK-8000 AÊ rhus C, Denmark.

Acta Physiol Scand 1998, 163, 201±208

Ó 1998 Scandinavian Physiological Society 201

and three isoforms (a1, a2 and a3) of the hydrolytic a-

peptide are known (Sweadner 1989). After SDS-elec-

trophoresis the a-isoform from bovine rumen epithe-

lium was characterized in western blots and the high

ouabain-binding capacity per g tissue substantiated by a

novel immunochemical method for quanti®cation of

Na+, K+-ATPase isoforms.

MATERIALS AND METHODS

Tissue and enzyme preparation

Bovine rumen epithelium and kidneys were obtained

from a local abattoir within 30±45 min after slaugh-

tering. For comparison with the experiments carried

out on biopsies, mid-portions of epithelium papillae

from the ventral sac of the rumen were prepared as

described (Kristensen et al. 1995). In short, the whole

length of the ¯at papillae was preserved, whereas the

two edges were cut away. Papillae were homogenized

by an Ultra-Turrax T25 tissue homogenizer in

5 mL g)1 of icecold 250 mM sucrose, 25 mM imidazole

(pH 7.2, 20 °C), 1 mM dithiothreitol and 0.2 mM phe-

nylmethylsulphonyl ¯uoride (PMSF). Heavy cell debris

were separated by slight centrifugation at 700 g for

2 min (Sigma 4±10), and the supernatant frozen and

kept at )80 °C.

Na+, K+-ATPase was prepared from the outer

medulla of the bovine kidney. A crude membrane

fraction was isolated from the homogenate. After mild

SDS-activation (0.37 mg SDS (2 mg protein))1 mL)1)

of this fraction in the presence of ATP, puri®ed Na+,

K+-ATPase was isolated by isopycnic zonal centrifu-

gation according to Jùrgensen (1988) (see also Hansen

et al. 1991). Na+, K+-ATPase from other sources used

in studies on isoform identi®cation was prepared from

pig or mink kidney according to the same procedure.

Na+, K+-ATPase from bovine brain and from grey

matter brain tissue of 200 g rats were isolated as de-

scribed by Klodos et al. (1975).

Isoform-speci®c antibodies to (Na++K+)-ATPase

Hybridomas (splenocytes from BALB/cJ mice fused

with SP 2/0 myeloma cells) producing the monoclonal

antibodies (MA) 3B and F6 to chicken kidney Na+, K+-

ATPase (Fambrough & Bayne 1986, Arystarkhova &

Sweadner 1996) were generous gifts from Dr Douglas

M. Fambrough. Hybridomas producing MAs to Na+,

K+-ATPase were grown in RPMI 1640 + 5% FCS and

the supernatants harvested and screened using an

ELISA (Hansen 1992). Microtitre plates (96-wells

MaxisorbÒ from Tech-Nunc) were coated with Na+,

K+-ATPase, reacted with supernatants diluted with

PBS and then with peroxidase-conjugated antimouse

IgG (Dako, Copenhagen).

Immunoglobulins from supernatants containing

MAs were puri®ed and concentrated by repeated

chromatography on AFFI-TTM thiophilic agarose ac-

cording to the instruction manual by the manufacturer

(Kem-En-Tec, Copenhagen 1992). The ®nal concen-

tration of mouse IgG was estimated by means of an

ELISA kit for quantitative determination of immuno-

globulin from mice (Boehringer Mannheim) and was

found in the range 400±600 lg IgG mL)1.

Monospeci®c polyclonal antibodies (PAs) to iso-

form-speci®c segments of each of the three rat iso-

forms were raised in rabbits and puri®ed essentially as

described by Pressley (1992). The oligopeptides

H-KNPNASEPKHLL-OH (�a1), H-KHEREDSPQ-

SHVL-OH (�a2) and H-KHETEDPNDNRYL-OH

(�a3) were synthesized by Kem-En-Tec, Copenhagen.

Oligopeptides of a purity >95% were coupled to key-

hole limpet haemocyanin (oligopeptide/KLH 1:1 by

mass). Each KLH-coupled oligopeptide was used for

the immunization of two rabbits (Dako, Copenhagen).

Antisera were puri®ed by chromatography on two

consecutive columns of CNBr-activated Sepharose 4B

(Pharmacia Biotech) coupled to KLH (column I) or the

relevant oligopeptide (column II) according to the in-

structions of the company (Pharmacia Biotech 1994).

After disconnection of the system, immunoglobulins

bound to column II were released by acidic glycine

buffer (pH 2.2) and immediately neutralized by addi-

tion of Tris base. The neutralized ef¯uent was con-

centrated on an Amicon ultra®ltration unit equipped

with a YM3 43 mm Amicon ®lter. The protein con-

centration of stock solutions of puri®ed antibody was

0.16±0.48 mg mL)1. The speci®city and cross-reactivity

of the ®nal product were tested with the ELISA using

microplates coated with one of the three synthesized

oligopeptides, Na+, K+-ATPase from pig or mink

kidney (a1) or Na+, K+-ATPase from bovine brain (a2

and a3, mentioned later). Microplates coated with KLH

or solutions of skimmed milk powder were run as

controls of non-speci®c antigen±antibody reactions.

Puri®ed antibodies to a2 and a3 exhibited excellent

speci®city for the relevant antigens, whereas antibody

to a1 cross-reacted weakly with the oligopeptide �a2.

The speci®city of antibodies was also tested on blots

with puri®ed Na+, K+-ATPase isolated from pig and

mink kidney (a1), beef brain (a2 and a3) or rat brain (all

three isoforms), mentioned later.

Gel electrophoresis and immunoblotting

SDS-polyacrylamide gel electrophoresis according to

Laemmli was performed with a mini gel equipment and

a 4±16% gradient polyacrylamide gel. Measures of 3±

15 lg protein of Na+, K+-ATPase or 30±40 lg pro-

tein of rumen epithelium homogenate dissolved in

Rumen epithelium Na+, K+-ATPase � O Hansen Acta Physiol Scand 1998, 163, 201±208

202 Ó 1998 Scandinavian Physiological Society

SDS±mercaptoethanol and glycerol without heating

was applied per lane. The gels were stained with Coo-

massie brilliant blue R-250 or transferred to Immobilon

PVDF membranes in a semidry electroblotter (JKA-

Biotech or Kem-En-Tech, Copenhagen). The blots

were quenched with 0.5% Tween-20 in PBS. In some

cases the blots were stained with 0.1% Amidoschwarz

10B (Merck) in 40% methanol and 10% acetic acid, the

supposed a-bands marked with a pencil and then de-

stained before reaction with antibodies. Blots were in-

cubated with puri®ed MAs diluted 1:667 or with PAs

diluted 1:2500±1:5000. Bound antibodies were detected

with horseradish peroxidase-conjugated antimouse

(Dako-immunoglobulins) or antirabbit IgG (Zymed),

respectively, diluted 1:1000 and substrate prepared

from 3, 3-diaminobenzidine tetrahydrochloride (DAB-

tablets, Kem-En-Tec) plus H2O2.

For quanti®cation of Na+, K+-ATPase in homoge-

nates of rumen epithelium, homogenate and bovine

kidney Na+, K+-ATPase were run in parallel on SDS

gels and electroblotted onto Immobilon PVDF mem-

branes. Blots were incubated with isoform-speci®c

MAs, then with 125I-coupled antimouse IgG (rabbit

antimouse IgG[125I], DuPont NEN) and a-spots ana-

lysed in an electronic autoradiography system (Packard

InstantImager). Negative controls were run by omis-

sion of the primary antibody.

(Na+ + K +)-ATPase activity and ouabain binding

Measurement of (Na+ + K+)-activated ATP hydrolysis

was carried out in a coupled assay (Nùrby 1988) or as

described elsewhere (Hansen 1992). Protein was de-

termined by the Lowry method with BSA from Boeh-

ringer adjusted to 1 mg mL)1 by spectrophotometry at

279 nM as standard. [3H]ouabain was obtained from

Amersham International and puri®ed by chromatogra-

phy on (Na+, K+)-ATPase before use as tracer in

ouabain-binding experiments (Hansen 1976). [3H]oua-

bain binding to (Na+, K+)-ATPase was carried out by

the (Mg2+ + Pi)-facilitated pathway and equilibrium

binding was determined by a ®ltration technique as

described (Hansen 1976, 1984). Equilibrium binding

data obtained after 2 h incubation at different non-

saturating ouabain concentrations were analysed ac-

cording to the one component model: B � a á F/

(F + b) where B is the bound ouabain, F the free

ouabain, a the maximum ouabain binding, and b the

dissociation constant. The speci®c activity of Na+, K+-

ATPase was (1) mink kidney enzyme 33.1, (2) pig

kidney enzyme 27.8, (3) beef kidney enzyme 7.0, (4) rat

brain enzyme 6.9 and (5) bovine brain enzyme

2.76 lmol mg)1 min)1.

RESULTS

(Na+, K+)-ATPase isolated from brain tissue is sup-

posed to contain all three isoforms of the hydrolytic a-

peptide and enzyme from kidney exclusively or pref-

erentially the a1 form. Figure 1 is an illustration of

ouabain binding to Na+, K+-ATPase isolated from the

outer medulla of the bovine kidney. Equilibrium

binding data obtained at different ouabain concentra-

tions after 2 h incubation at 37 °C seem compatible

with one homogeneous population of binding sites. An

apparent dissociation constant as high as 1.37 nM is

noticed. On the other hand, a ouabain-binding capacity

of 0.82 nmol (mg protein))1 would indicate a relatively

low purity of the preparation. A contamination with

unrelated proteins is also seen after SDS-gel electro-

phoresis. In Fig. 2, lanes containing kidney Na+, K+-

ATPase are placed along with lanes containing brain

Figure 1 [3H]ouabain binding to beef

kidney enzyme plotted in a Scatchard type

plot of bound (B) vs. bound over free (B/F)

and as bound vs. log free ouabain (inset).

Binding was determined by membrane

®ltration after incubation of 0.0688 mg

protein mL)1 in a medium containing

Mg2+, Pi and Trisbuffer for 2 h at 37 °C.

The line represents the best ®t of the data to

the one component model.


Acta Physiol Scand 1998, 163, 201±208 O Hansen � Rumen epithelium Na+, K+-ATPase

enzymes or rumen tissue homogenate. In puri®ed en-

zyme from mink kidney only two peptides are visible,

the a-peptide of apparent Mr 95 000 (real size 112 kDa)

and the glycosylated b-peptide (variable size, un-

glycosylated »35 kDa), in other enzyme preparations a

number of other bands also are visible. Brain enzymes,

notably bovine brain enzyme, contain a doublet of

bands at the a-position, a faster moving a1 peptide and

a slower moving (a2 + a3) band (Urayama et al. 1989).

In beef kidney enzyme a heavy band at this position

seems compatible with an a1 form whereas in rumen

homogenate three faint and yet distinct bands are likely

candidates.

Western blots of SDS-gels with enzyme preparations

and homogenate of rumen epithelium papillae placed

exactly as in the coomassie blue stained gel in Fig. 2

were reacted with isoform-speci®c PAs or with MAs.

Antibody binding as disclosed by the peroxidase-cou-

pled anti-IgG reaction is shown in Fig. 3. From the

upper part of the ®gure it is seen that the PAs raised to

rat Na+, K+-ATPase isoforms recognize all three iso-

forms in rat brain enzyme and also a2 and a3 in bovine

brain Na+, K+-ATPase. As predicted (Pressley 1992),

bovine brain and kidney Na+, K+-ATPase are not

recognized by the a1-speci®c PA whereas this is the

case with mink kidney enzyme. None of the PAs hy-

bridize with bovine kidney Na+, K+-ATPase or rumen

epithelium homogenate which indicates the absence of

a2 or a3 in these tissues. The a1-speci®c MA 3B (and

F6, not shown), however, recognizes bovine kidney and

rumen epithelium Na+, K+-ATPase as seen from the

lower part of Fig. 3. Rumen epithelium and bovine

kidney thus seem to contain the a1 isoform of Na+,

K+-ATPase and no other subgroups of this enzyme.

The hydrolytic peptide of rumen epithelium Na+,

K+-ATPase was further identi®ed with the a1-speci®c

MA and 125I-labelled anti-IgG. In Fig. 4 are shown two

individual experiments with blots indicating non-spe-

ci®c reactions after incubation with secondary antibody

(left part) and speci®c plus non-speci®c reaction after

incubation with both antibodies (right part). In lanes

one, two and four increasing concentrations of bovine

kidney enzyme were run and in lane three the homog-

enate of the rumen epithelium. Although badly repro-

duced in the ®gure, a delicate and distinct line

representing a1 was seen in lane three. The speci®c re-

action was identi®ed by subtraction of the non-speci®c

labelling (left part) from the total one (right part). Fig-

ure 5 is identical to Fig. 4 except for the application of

squares for analysis of a spots and their respective blinds

Figure 3 Blots of SDS-gels identical to the one

shown in Fig. 2. In the upper part blots are

shown incubated with a1-speci®c polyclonal

antibody (PA) (a), a2-speci®c PA (b) or

a3-speci®c PA (c) and peroxidase-coupled

antirabbit IgG. In the lower part blots are shown

incubated with a1-speci®c monoclonal antibody

(MA) + peroxidase-coupled antimouse IgG (d)

peroxidase-coupled antimouse IgG only (e) and

peroxidase-coupled antirabbit IgG only (f).

Figure 2 SDS-gel electrophoresis of Na+, K+-ATPase from

rat brain (lane two), mink kidney (lane three), bovine kidney

(lane four), bovine brain (lane ®ve) and homogenate of the rumen

papillae (lane six). MW-standards of 130, 94, 68, 43, 34 and 21.5 kDa

are shown in lanes one and seven.



by means of the electronic autoradiography programme.

A linear relationship between the amount of bovine

kidney enzyme applied to lanes one, two and four and

net counts were obtained. The content of a1 in the

homogenate of the rumen epithelium expressed as bo-

vine kidney enzyme equivalents was found by inter-

polation. As bovine kidney enzyme is well characterized

(Fig. 1) the corresponding number of ouabain-binding

units g)1 rumen epithelium homogenate could be cal-

culated. From Fig. 5 an average content of 2.6 nmol

Na+, K+-ATPase (g wet wt))1 tissue was found.

DISCUSSION

A considerable mucosal to serosal transport of elec-

trolytes and SCFA takes place across the epithelium of

the ruminant forestomach. The surface area of the ru-

men epithelium is substantially increased by a large

number of papillae. A close correlation between Na+

and SCFA absorption is seen across the rumen multi-

layer as well as the small and large intestine monolayer

epithelia (Rechkemmer 1991). The coupling of Na+ and

SCFA transport in rumen epithelium seems non-met-

abolic (GaÈbel et al. 1989) and may be direct or more

likely indirect via a Na+/H+ antiport mechanism with

extrusion of H+ for regulation of intracellular pH and/

or delivery of H+ for non-ionic diffusion of weak

electrolytes such as SCFA. An apical Na+/H+ ex-

changer plays an important role in Na+ reabsorption

and acid secretion in distal ileum and colon enterocytes

(Maher et al. 1997, Wakabayashi et al. 1997). The in-

testinal Na+/H+ antiporter isoform is relatively insen-

sitive to amiloride (Wakabayashi et al. 1997) which may

explain con¯icting results: abolition by amiloride of

SCFA-dependent mucosal to serosal Na+ ¯ux in the

rumen of sheep (GaÈbel et al. 1989) and no signi®cant

effect of amiloride on Na+ ¯ux across the bovine ru-

men (Diernñs et al. 1994).

Irrespective of the mechanism of Na+ transport at

the apical membrane and of any role of the Na+/H+

Figure 4 Autoradiograms of western blots incubated with a1-speci®c monoclonal antibody (MA) and 125I-labelled antimouse IgG. The upper

part and the lower part are the results of separate experiments with different antibody loads. To the left blots are shown incubated with

secondary antibody only, to the right blots successively incubated with primary and secondary antibodies. Lanes one, two and four were loaded

with bovine kidney Na+, K+-ATPase corresponding to 3.44, 6.88 and 13.76 lg protein, respectively, and lane three was loaded with

homogenate of the rumen epithelium papillae corresponding to 31 lg protein.



exchanger in SCFA absorption, an inwardly directed

electrochemical Na+ gradient is a provision for Na+

absorption and for exchange of extracellular Na+ for

intracellular proton. This Na+ gradient is created by

Na+, K+-ATPase preferentially located in stratum ba-

sale of rumen epithelium (see Kristensen et al. 1995).

Biopsies of bovine rumen epithelium papillae were re-

cently shown to have a high concentration of functional

Na+, K+-pumps: a high [3H]ouabain-binding capacity

consistent with the measured K+ or 86Rb+ in¯ux and

Na+ ef¯ux (Kristensen et al. 1995). The rat small in-

testine, has been shown to contain a distinct isoform of

the hydrolytic peptide of (Na+, K+)-ATPase (Zemel-

man et al. 1992, Barada et al. 1994) and recalculation of

data on mucosal scrapes would indicate a high con-

centration of Na+, K+-ATPase in this monolayer epi-

thelium (Barada et al. 1994). In the present paper the

Na+, K+-ATPase of bovine rumen epithelium is further

characterized and the high ouabain-binding capacity

substantiated by immunochemical methods. Even an

approximately quantitative isolation of Na+, K+-AT-

Pase from several tissues seems impossible and a risk of

preferential isolation of a subpopulation of the enzyme

thus also exists (Hansen & Clausen 1988). For these

reasons, characterization as well as quanti®cation of the

enzyme were carried out on the homogenate of the

rumen epithelium except for a gentle separation of

heavy cell debris of this keratinized multilayer epithe-

lium.

Na+, K+-ATPase is an ab heterodimer with known

isoforms of the hydrolytic a peptide as well as the

glycosylated b peptide. No identi®ed enzymatic func-

tion is associated with the b peptide except for the

proper insertion of the ab protomer into the plasma

membrane. The individual a isoforms probably sub-

serve speci®c functions as a remarkable retention of a-

subunit isoform structure during the evolution has been

established (Takeyasu et al. 1990). Isoform-speci®c

regulatory functions seem obvious but the only known

difference in kinetic behaviour is a higher K+-af®nity

and a lower Na+-af®nity of the a3 isoform than those

of a1 and a2 (Munzer et al. 1994).

According to proposals by Takeyasu et al. (1990) and

Pressley (1992) site-directed PAs were raised to an

Figure 5 As with Fig. 4 this illustrates the applied squares for extraction of counts originating from non-speci®c (left part) and speci®c (right

part) labelling of a1 peptide. Rectangles representing background counting are shown above the position of a bands.



isoform-speci®c but not species-speci®c amino acid

sequence of the three known rat a isoforms. With these

antibodies it is shown that Na+, K+-ATPase prepared

from bovine brain at least contains the a2 and a3 iso-

forms whereas both were undetectable in bovine kidney

Na+, K+-ATPase and in bovine rumen epithelium. In

contrast to the broad range of species recognition by

the a2- and a3-speci®c antibodies, the a1-speci®c anti-

body prepared according to the same procedure is

known to recognize the a1 isoform from a limited

number of species (Pressley 1992). In the present study,

Na+, K+-ATPase from mink kidney and from rat brain

are recognized by the a1-speci®c antibody but not by

the bovine brain and kidney Na+, K+-ATPase.

The MAs 3B and F6, however, both recognize an a1

isoform in bovine brain and kidney Na+, K+-ATPase as

well as in bovine rumen epithelium. Both antibodies are

known to be a1 speci®c and to react with a1 from a

variety of species and the epitope seems to be near the

N terminus at the ®rst intracellular loop (Arystarkhova

& Sweadner 1996) in contrast to PA that are directed

towards a domain of the large intracellular loop just

upstream to FITC and nucleotide binding sites. Bovine

kidney Na+, K+-ATPase and rumen epithelium thus

seem to contain the a1 isoform with high Na+ af®nity

but not a2 or a3.

The Na+, K+-ATPase from bovine kidney was also

characterized in [3H]ouabain-binding studies and

equilibrium binding data seem compatible with one

homogeneous population of binding sites, e.g. the

presence of a1b heterodimers only, in contrast to

previous results on Na+, K+-ATPase from bovine

brain (Hansen 1976). The apparent dissociation con-

stant of 1.37 nM is identical with that of the high-

af®nity component previously found in Na+, K+-

ATPase from bovine brain (Hansen 1976). On the

other hand, different isoforms of Na+, K+-ATPase

with the exception of the rodent a1 isoform do not

necessarily vary much with respect to ouabain af®nity.

In most species the a1 isoform has a high ouabain

af®nity, whereas the rat a1 isoform has an extremely

low af®nity (Hansen et al. 1991). As the ouabain-

binding capacity of bovine kidney enzyme seems to

represent one homogeneous population of Na+, K+-

ATPase, i.e. the a1 isoform identi®ed by MA, this

enzyme is an ideal reference in the semiquantitative

immunochemical method further developed.

In this method the secondary antibody is radioac-

tively labelled and the speci®c labelling of the a band is

translated to Na+, K+-ATPase units. Western blots were

obtained after SDS-PAGE of homogenates of the ru-

men epithelium and different amounts of bovine kidney

enzyme, incubated with the a1-speci®c antibody 3B and125I-coupled antibody to IgG. Autoradiography was

then carried out in a special instrument with a device for

electronic data treatment. A linear relationship between

the enzyme applied and the signal was obtained and, by

interpolation, the amount of Na+, K+-ATPase in the

rumen epithelium homogenate was calculated. By this

method it was veri®ed that papillae of the rumen epi-

thelium has a high content of Na+, K+-ATPase. Ex-

pressed as nmol Na+, K+-ATPase (g wet wt))1, roughly

a doubling was found compared to the result of direct

[3H]ouabain binding in biopsies. Although preferentially

localized to stratum basale of the keratinized multilayer

epithelium the content of Na+, K+-ATPase in the ru-

men epithelium expressed per g wet weight is higher

than that found in skeletal muscle and of the same order

as that found in guinea-pig heart (Hansen & Clausen

1988). The immunochemical method may overestimate

the number of Na+, K+-pumps if unrelated peptides

adjacent to the a1 peptide in SDS-gels and western blots

react with the primary antibody 3B. On the other hand,

only faint bands are seen at this position in Coomassie

stained gels (Fig. 2) and apparently only the speci®c one

in peroxidase stained blots (Fig. 3). Another explanation

of the discrepancy could be the inaccessibility of

ouabain to newly synthetized enzyme that had so far not

reached the plasma membrane.

In conclusion, using western blots and isoform-

speci®c antibodies to the hydrolytic peptide of Na+,

K+-ATPase it is shown that the rumen epithelium

contains the a1 isoform. A semiquantitative immuno-

chemical analysis carried out on blots seems compatible

with a similarly high or even higher content of Na+,

K+-ATPase in the rumen epithelium as found in

ouabain binding.

Dr Douglas M. Fambrough, Baltimore MD, is gratefully acknowl-

edged for his kind gift of antibody-producing hybridomas and Dr

Thomas A. Pressley, Lubbock TX, for his introduction to site-

directed antibodies. Thanks are also due to Ms. Edith Bjùrn Mùller

and to Mr Toke Nùrby for excellent technical assistance. The study

was supported by the Danish Biomembrane Research Centre and

Aarhus Universitets Forskningsfond, grant no. F-1996-SUN-1±82.

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isoform of na+, k+-atpase from rumen epithelium identified and quantified by immunochemical methods

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