Biochemical and immunological studies on isolated brush border membranes of human kidney cortex and their membrane surface proteins

179

Clinica C~i~ica Acta, 55 (1974) 179-197 8 Eisevier Scientific Publishing Company, Amsterdam -. Printed in The Netherlands

CCA 6509

BIOCHEMICAL AND IMMUNOLOGICAL STUDIES ON ISOLATED BRUSH BORDER MEMBRANES OF HUMAN KIDNEY CORTEX AND THEIR MEMBRANE SURFACE PROTEINS

J~RGEN E. SC~~R3ERIC~, FRANK W. FALK~NBERG, HILDEGARD M’EjLLER and GERHARD PFLEIDERER

A. WERNER MONGOLS,

Abteilung fiir Nephrologie im Zentrum der Inneren Medizin der Johann Wolfgang Goethe Universitiit, 6000 Frankfurt/Main and Lehrstuhl fiir Bioehemie, Abteilung fiir Chemie der Ruhr-Universitiit Bochum, 4630 Bochum (G.F.R.) (Received March 19,1974)

summary A subfraction rich in brush border (BB) membranes was obtained by differenti~ cent~fugation of plasme membr~es (PM) of human kidney cortex. In addition to alkaline phosphatase, the usual BB marker enzyme, an alanine specific ~~opeptidase (AAP) and a gamma-glut~yltranspeptid~e fg-GTP) were associated with this membrane fraction. The three enzymes are considered specific markers for the brush border region of the proximal tubules. Among the proteins released from the BB membranes by proteolytic treatment these three enzymes were found. The results indicate that AAP and g-GTP are components of the outer membrane surface. Antisera prepared against PM reacted in several immunological techniques with soluble proteins (including the three BB marker enzymes) released from the BB membranes by digestion with papain, as well as with those found in the urine of patients with kidney diseases. AAP from both sources (papain digest and urine) were immunolo~cally indistin~ishable. The molecular weights and some other biochemical parameters of the three enzymes were determined and compared with those reported in the literature. It is suggested that the methods and systems described might be of use for further studies on the structure and function of the membranes of kidney proximal tubules and are of potential value for diagnostic purposes.

sulted

Our previous studies on tubular antigens of the human kidney [1,2] rein the prep~ation of a plasma membrane (PM) fraction. This PM frae-

tion was associated with marker enzymes known to be specific for brush border (BB) membranes. Antisera against this PM fraction revealed a specific luminal fluorescence of the epithelia of proximal tubules in the sandwich staining technique, thus confirming the presence of BB membrane fragments in the PM fraction [ 23 . With the help of the anti-PM sera, we were able to demonstrate that tubular antigens are excreted at an increased rate into the urine of humans with kidney transplants and during acute tubular necrosis [3]. Alteration of the proximal convoluted tubules is known to be caused also by a variety of drugs. In addition, brush border proteins are potential inducers of autologous immune complex nephritis in rats, as was shown by Glassock et al. [4]. Realizing the impor~nce of this tubular structure for kidney function under normal and patholo~c~ conditions, we decided to study in detail the structural and biochemical properties of BB fragments further enriched from PM fractions. In order to obtain more information on the properties of human kidney BB membranes and their proteins, sedimentation characteristics in continuous and discontinuous sucrose density gradients, before and after proteolytic treatment with papain, were studied. Some of the membrane proteins, solubilized by papain digestion, were partially purified and characterized by biochemical and immunological methods. Materials and Methods

Sodium azide, L-canine-2-naphtbyl~ide hydrobromide, L-alanine-pnitranilide, glycylglycin, glutathion, Aminoblack IOB: Merck, Darmstadt; Lleucine-p-nitranilide, aldolase (No. 15372), leucine-aminopeptidase (No. 15006), pyruvate-kinase (No. 15744) and other reagents for enzyme assays (Boehringer Test combinations, substrates and coenzymes): Boehringer, Mannheim; dithiothreitol: Calbiochem, Los Angeles; p-chloromercuribenzoate (PCMB), sodium salt, Fast Blue B Salt, ovalbumin, papain (No. 31600): Serva, Heidelberg; complete Freund’s adjuvant (CFA) : Difco-Laboratories, Detroit; human albumin (RHA 04), agarose (REO 14), anti-rabbit gamma-globulin from goat, fluorescein conjugated gamma-globulin fraction (TKF 04) : Behringwerke AG, M~burg; Sepharose 2B, Sephadex G 200, Blue Dextran 2000: Pharmacia Fine Chemicals, Uppsala, Sweden; malate dehydrogenase (No. 34”401(2)~: Miles Seravac, Konkakee, Ill.; aminopeptidase M: Rijhm and Haas, Darmstadt; leucine aminopeptidase (No. LE 080): Schuchardt, Mtinchen. Preparation of kidney homogenates Human kidneys were obtained by autopsy, perfused with isotonic Ringer solution (containing 1.0 g chloramphenicol per liter) and decapsulated. The cortex was separated from the medulla and homogenized in a Potter-Elvehjem homogenizer with a teflon pestle (Braun-Melsungen) with STED buffer (0.25 M sucrose, 10 mM TV-eth~ol~ine-HCl, 5 mM EDTA-Na, 2 mM dithiothreitol; ratio tissue:buffer 1:7 w/v). Brush border-rich plasma membranes (PM) were isolated by a differenti~ cent~fugation technique, as described previously [ 21.

181

Centrifugation in a discontinuous sucrose density gradient The plasma membranes were centrifuged for 7 h at 91 000 X g, at 0”) in a SW 25.1 rotor (Beckman Spinco ultracentrifuge, model L). The gradient consisted of four layers of 5.0 ml each containing sucrose in TRA buffer (0.1 M tri-ethanolamine-HCl buffer, pH 7.6). The densities chosen were: dl = 1.195, d2 = 1.178, d3 = 1.163, d4 = 1.155 (0”). Isovolumetric fractions were collected after centrifugation. Protein absorbancy was determined simultaneously at 280 nm by a LKBUvicord II. The fractions of each of the four resulting protein bands were combined, homogenized and recentrifuged (25 min, 37 000 X g, 0”) Sorvall RC 2B, SS 34 rotor). The pellets were resuspended in Tris buffer, 0.1 M, pH 7.6 (20” ), and enzyme activities and protein concentrations were determined. Cen trifugation in a linear sucrose density gradient Untreated or papain-treated membranes were centrifuged for 6 h (175 000 X g, 0”) SW 39 L rotor) in a linear sucrose density gradient; density range: 15-45% sucrose in 0.1 M TRA buffer, at 25”. After centrifugation 11 isovolumetric fractions were collected. The enzyme distribution in the gradient was determined according to Beaufay et al. [ 51. Enzyme assays Alkaline phosphatase ( AP), alanine-aminopeptidase ( AAP, substrate alanine-p-nitranilide), alcohol dehydrogenase ( ADH), gamma-glutamyltranspeptidase (g-GTP) , glutamate dehydrogenase (GLDH), glutamate-oxalacetate transaminase (GOT), glucose-6-phosphatase (GP) , glyceraldehyde-phosphate dehydrogenase (GAPDH), lactate dehydrogenase (LDH), malate dehydrogenase (MDH) and pyruvate kinase (PK) were assayed according to Biochemica Informations, Boehringer [ 61. Protein was measured by the method of Lowry et al. [7] using bovine serum albumin as the standard. Papain treatment Membranes were incubated with papain in a ratio of 20:1, membrane protein to papain. Activation of papain was performed as described by Emmelot [8]. After a short incubation (15 min, 37”), the samples were centrifuged (35 min, 40 000 X g, 0”, SW 39 L rotor). The supematant and the resuspended pellet were used for further studies. Gel filtration Sephadex G 200 was used for gel filtration. Elution buffer: Tris-HCl 75 mM, pH 7.6; column size: 2.3 cm X 120 cm. The exclusion volume was determined with Blue Dextran 2000. Fractions were collected in a LKB UltroRat fraction collector at 2”. Protein absorbancy was recorded automatically by a LKB Uvicord II unit at 280 nm. The individual fractions were tested for enzyme activity. Pooled fractions of gel chromatographic separations were concentrated by Amicon Ultrafiltration cells 402 and 10, using Diaflo membranes No. PM 30.

182

An t&era Rabbits were immunized at multiple sites with the particulate or soluble antigens (1.0 mg per animal) emulsified with complete Freund’s adjuvant (CFA). Six weeks after priming the animals were challenged with an equal dose of antigen either intravenously (soluble antigens in saline) or by another injection at multiple sites (particulate antigens in CFA). Serum was prepared from blood collected 10 days after the second injection. Antibodies against human plasma proteins were removed by a polyacrylamide immunosorbent technique [ 91. The gamma-globulin fractions of these absorbed sera, prepared by ammonium sulfate precipitation (35% saturation) were used for immunological studies. Immunoinhibition and immunotitration Samples with known enzymatic activity were incubated for 20 min at 25” with varying volumes of anti-PM sera. After correction for the activity of the corresponding enzymes in the rabbit antiserum, inhibition was expressed in percent of initial activity. By measuring residual activity after incubation with antiserum and centrifugation (15 min, 37 000 X g), conclusions can be drawn concerning the presence of multiple forms of enzymes with similar or identical enzymatic but different immunological properties. If one enzyme species is present and if it is precipitable by antibodies, it will be possible to remove it completely by an excess of antiserum. Electroimmunodiffusion (EID) was performed according to Laurel1 [lo] using the one- and two-dimensional technique. AAP-specific staining of immune-precipitates. Electroimmunodiffusion plates were washed with saline for two days. Enzyme activity was visualized by coupling /3-naphthylamine, the product of splitting of alanine-fl-naphthylamide, with Fast Blue B Salt according to Nachlass et al. [ 111. Urine concentrates Urine of healthy persons and of persons with kidney disorders (verified histopathologically from specimens obtained by transcutaneous biopsy) was collected over 24 h periods. After addition of 0.1% sodium azide the urine samples were stored in the cold for several hours and centrifuged for 20 min (10 000 rev./min, Sorvall, GSA rotor). Concentration of urine samples was performed with Amicon Ultrafiltration Cells TCF-10, 402 and 10. Diaflo membranes No. UM 10, UM 20E, PM 30 and XM 50 were used. The factor by which the urine samples were concentrated is defined as the concentration factor “c.f.” Electron microscopy Electron microscopic examination of PM samples was kindly done by Dr U. Leuschner, Department of Gastroenterology, J.W. Goethe University, Frankfurt/Main according to Reimer [ 121.

183 TABLE

I

SUMMARY

OF CENTRIFUGATION

STEPS

renal cortex

(human)

1 total homogenate

s (P. 3. 2. P. 3.3)

20 min. 17 500 X gmax

1

S

P. 4.1 (P. 4.2.

P. 4.3) STED

PM

$i?f!?~ooo ’gmax I

linear swkrose density range 154 5% 1 6 h. 175000 11 fractions

gradient

1

discontinuous sucrose density (d 1.155.1.163.1.178.1.195)

X gmax

1 4 protein fractions

I h. 91000

gradient

X gmax

bands

4 of absorbance

maxima

1

pellet Pd_l-Pd4

ReSUltIS

Centrifugation studies In the terminal fraction of the differential centrifugation called PM fraction (P 5.1 in Table I), an increase of alkaline phosphatase (AP, EC 3.2.3.1), alanine-aminopeptidase (AAP), and gamma-glutamyltranspeptidase (g-GTP) activities was observed as compared to the total homogenate (Fig. 1). Marker enzymes for endoplasrnic reticulum (GP), mitochondria (GLDH), and cytoplasm (LDH) decreased. AAP and g-GTP activities in the PM were enriched simultaneously, the specific activity of g-GTP reaching a very high level (AAP: 157.2 mU/mg, g-GTP: 29 500 mU/mg). The PM fraction was examined by electron microscopy to obtain visual information on its composition. The picture obtained by this technique revealed a large amount of brush border fragments and microvilli (Fig. 2)

184

q

g

GTP

AP

GP

totalhomqenate p&l

GLDH

fractlo”

LDH

Fig. 1. Relative enrichment of alanine-aminopeptidase (AAP), gamma-glutamyltranspeptidase (g-GTP), alkaline phosphatase (AP), glucose-6-phosphatase (GP), glutamate dehydrogenase (GLDH) and lactate dehydrogenase (LDH) in the human kidney plasma membrane (PM) fraction as compared with the total homogenate.

Fig. 2. Electron

microscopic

photograph

of the PM fraction

of human kidney; magnification

X 20 000.

185

besides unidentified particles and vesicles, At a higher m~ification (not shown), thin axial microfilaments within the “cytoplasmic compartment” of the microvilli were observed. Centrifugation in a discontinuous sucrose density gradient Further enrichment of BB membranes was obtained by discontinuous density gradient centrifugation, especially in the protein band of density d3 = 1.163. In this BB fraction the activities of AP, used as a BB marker enzyme, and AAP were 12.3 and 23.8 times higher than in the total homogenate respectively (Table II and Fig. 3). Continuous ~l~near)sucrose density ~adient centr~fugation In a continuous density gradient, AP and AAP sedimented very closely together. There is a maximum of enzyme activity in the density range d = 1.163. The rank-correlation coefficient AP-AAP over the whole gradient is r = 0.9991, significance related to 0, a < 0.001; regression line y = 1.0346x + 0.068 (Fig. 4). The Gamma-glutamyltranspeptidase has an analogous distribution. maximal g-GTP activity is within the same density range as AP and AAP. The AP-g-GTP correlation over the whole gradient is r = 0.9774, significance related to 0, a < 0.0001; regression line: y = 0.9737x + 0.1266, Biochemical studies Having prepared a’membrane fraction which was further enriched in brush border (BB) membranes, we were interested in studying the biochemical properties of its membrane proteins. Treatment with papain was used for the solubilization of the membrane proteins. Papain treatment of the BB fraction After mild papain digestion of BB followed by centrifugation, AAP is preferentially released into the supernatant. (Abbreviation: BB-Pap-S). BBPap-S contains 82 times more AAP than AP activity, About one-third of the membrane bound g-GTP was solubilized. TABLE

II

SPECIFIC ACTIVITIES OF ALKALINE PHOSPHATASE (AP) AND ALANINE-AMINOPEPTIDASE (AAP) IN THE TOTAL HOMOGENATE, THE PLASMA MEMBRANE FRACTION (PM) AND IN THE FOUR DENSITY ZONES OF THE DISCONTINUOUS SUCROSE DENSITY GRADIENT n = Number of experiments. Mean of specific activity: arithmetic mean f S.D. Fraction

Total homogenate PM fraction Sediment d, = 1.195 Sediment d, = 1.178 Sediment d, = 1.163 Sediment d, = 1.166

n

8 8 5 5 5

5

Mean of specific activity (mu/me

protein)

AP

AAP

20.5 137.6 64.5 146.5 246.3 151.1

1 k f f f f

7.2 61.4 28.5 37.6 12.7 42.1

13.8 167.2 70.0 167.1 329.0 136.7

f f f i f i

4.6 48.7 27.6 42.6 32.8 22.6

186

23 -

?I

-

m

A?

[

4AP

19 -

!:

-

15 -

i

13 -

E

.c

Y ; 11 -

r

a3 02

z-

T;;

P-

TJ _ 7-

r

5 -

3 -

1.J ,. //

1 -

‘, ,,,,

~

4

Sd

I

Sd- 2

Sd

-3

Fig. 3. Relative enrichment of alkaline phosphatass (AP) and alanine-aminopeptidase (AAP) in the zones of ecntrifugation of the PM fraction in a discontinuous suerose gradient. Sd-3, brush border-rich fraction. (Total homogellate, Sd-1 .I

I

2

3

4

5

6

7

*-r.*n--.

Alkal.

Phosphatare

a . . . ..I “.,. . . ..a

Protein

4ntnopepliddse

8

9

10

II

FRACTION

Fig, 4. Disttibution of the alkaline phosphataae {API, &nine-amknopeptidase (AAP) and protein of the brush box&x fraction in ttre continuws ruerose darrait~ gradient f=nle 1545%>.

187

-

.Alkal.

Phosphattse

=----a

Aminopeptidare

l ---=

y-

Glutamyltranrpept.

Fig. 5. Distribution of alkaline phosphatase (AP), transpeptidase (g-GTP) in the continuous sucrose border membranes.

alanine-aminopeptidase density gradient after

(AAP) and gamma-glutamylpapain treatment of the brush

After ultrasonification of the membranes (Branson sonifier, 3 X 1 min, 8 kcycles), however, all three enzymes were equally released into the supernatant. Electron microscopic studies showed heavy destruction and disintegration of the membranes, while after papain digestion, the typical microvillous membrane ultrastructure was preserved (not shown). In continuous sucrose density gradient centrifugation after papain treatment, the membranes with AP and g-GTP activities migrated to the density of d = 1.145. The solubilized AAP, part of the g-GTP and a very small amount of AP remained on top of the gradient (Fig. 5). Nearly all of the AAP activity is released from the membranes; only a very small peak of AAP activity in d = 1.145 represents residual membrane-associated AAP. Gel filtration

By preparative gel filtration on Sephadex G 200, AAP and g-GTP could be purified further from the digest (BB-Pap-S). The material separated into five protein peaks (Fig. 6). The fractions of each of the peaks were pooled and concentrated. Peak II contained the AAP activity and was used for immunization.

188

4

30

50

76

90

I lo

Fig. 6. Gel filtration on Sephadex G 200 of the papain digest of brush border membranes (BB-Pap-S). Elution diagram with the activity peaks of alanine-aminopeptidase (AAP), allmline phosphatase (AP) and gamma-glutamyltranspeptidase (g-GTP).

Molecular weights, calculated from analytical gel filtration on Sephadex G 200 (Fig. 7), are presented in Table III. In addition, the molecular weights of commercially available aminopeptidases were determined on the same column (Table III). The significantly higher molecular weight of the “Aminopeptidase

Fig. 7. Determination of the molecular weights of alanine-aminopeptidase (AAP), allcaline phosphatase (AP) and gamma-glutamyltranspeptidase (g-GTP) solubilfzed from brush border membranes by analytical gel filtration on Sephadex G 200. Reference proteins: pyruvate kinase (PK). alcohol dehydrogenase (ADH), lactate dehydrogenase (LDH). glyceraldehyde-phosphate dehydrogenase (GAPDH), glutamateoxalacetate transaminase (GOT), malate dehydrogenase (MDH), human albumin and ovalbumin.

189 TABLE

III

MOLECULAR

V,.

effluent

WEIGHTS volume;

DETERMINED

V,.

exclusion

BY

ANALYTICAL

Protein AAP AP

G 200

(BB-Pap-S) (BB-Pap-S)

“Leucine

Aminopeptidase”

Boehringer. and

M” Haas,

wt

1.397

240

1.474

200

000 000

1.638

126

000

1.421

230000

1.232

380

from

Mannheim

“Aminopeptidase Riihm

(LAP)

CHROMATOGRAPHY

Mol.

velvo

(BB-Pap-S)

eGTP

SEPHADEX

volume.

from Darmstadt

000

M” of Rohm and Haas will be discussed later in connection with other results. Preliminary experiments showed that the AAP prepared by chromatography on Sephadex G 200 from the papain digest, could be activated by 1.0 mM Co’+ (+31%) and by 0.5 mM Mg2+ (+30%). 0.5 mM Mn” (-15%) and 0.5 mM Zn2’ (-87%) caused inhibition, p-chloromercurybenzoate (PCMB) activated the enzyme in the range 0.125-0.5 mM (+17%) but caused inhibition at a concentration of 1.0 mM (-12%). All experiments were performed under standard conditions (15 min incubation at 25”). The Michaelis-Menten constant of AAP was 9.6 X 10e4 M for alanine-,ynitranilide (0.1 M phosphate buffer, pH 7.5) as substrate. Over a wide range (1.0-0.001 M) PCMB did not influence the g-GTP activity; however, 1 X 10m4 M reduced glutathione caused an inhibition (--32%). The optimum of enzymatic activity of AAP was found in the pH range 7.0, --I 3.0 (Fig. 8).

U ml

2.0 Fig.

8. pH dependence

6.0 of the

7.0 enzymatic

8.0 activity

9.0 of alanine

10.0 aminopeptidase

11

pH (AAP).

190

Immunochemical

characterization of BB-Pap-S proteins

For the biochemical characterization of proteins, a high degree of purity is desirable. Immunological methods can be useful tools to control the purity of protein preparations. For all immunochemical experiments extensively absorbed antisera were used. Efficiency of absorbtion was controlled by showing that no precipitin lines could be produced with normal human plasma in the very sensitive electroimmunodiffusion (EID) technique.

Quantitative gel precipitation using the EID technique Figs 9 and 11, showing the five peaks of the separation of BB-Pap-S on Sephadex G 200, precipitated with an anti-PM serum in the one- and twodimensional electroimmunodiffusion techniques (EID) according to Laurel& give an impression of the heterogeneity of this fraction. In addition to the enzymes (AAP can be stained specifically as indicated by an arrow) a variety of other proteins, so far unidentified, is present in the digest. By using the antiserum obtained after immunization with peak II, the AAP peak (Fig. 6), therefore called “anti-AAP”, the EID pictures show much less heterogeneity of precipitation lines. This means that the antibody population provoked by the injection of the partially purified AAP fraction recognizes only a few of the various proteins in the BB-Pap-S. This is indirect proof of the high purity reached in peak II, but it also shows that there were still a few other proteins in peak II capable of provoking antibodies. Nevertheless these precipitin lines are very faint as compared with the heavy AAPanti-AAP precipitate identified by specific staining, (Figs 10 and 12).

Fig. 9. One-dimensional EID according to Lawell of the 5 elution peaks after Sephadex G 200 chromatography of BB-Pap-S with anti-PM serum in the gel. Fig. 10. One-dimensional EID according to Laurel1 of the 5 elution peaks after Sephadex G 200 chromatography of BB-Pap-S with anti-AAP serum in the gel.

Fig. 11. Two-dimensional EID of BB-Pap-S with anti-PM serum in the gel. Fig. 12. Two-dimensional EID of AAP (peak II) with anti-AAP serum in the gel.

Maximal inhibition of AAP BB-Pap-S by an excess of anti-PM serum was 81%. This result can give no ~fo~ation concerning the homogeneity of the enzyme. The fact that only one pr~ipitin line occurs in the EID pictures that can be stained specifically for AAP activity does not yet prove that only one molecular form of the enzyme is present in the digest. In order to clarify this question, which is of great importance for interpretation and characterization, immunotitration experiments were performed with the anti-PM serum. The results of these experiments (Table IV) clearly show that the three enzymes contained in BB-Pap-S can be completely precipitated by anti-PM sera. Together with the single line stained for AAP activity in the EID picture, this indicates that the AAP is present as a single enzyme in the BB-Pap-S. TABLE

IV

IMMUNOTITRATION SERUM

OF SOLUBILIZED

BRUSH BORDER

0.1 ml BB-Pap-S with specific activities of 15.6.0.79 were used as antigen. Anti-PM serum added 0~1)

0 20 60 180

ENZYMES

(BB-Pap-S) WITH ANTI-PM

and 36.7 U/mg of GAP, AP and g-GTP, respectively,

Residual enzyme activity in the supernatant after centrifugation (%) AAP

AP

g-GTP

100.00 0.22 0.00 0.00

l#O.OO 0.88 0.28 0.00

100.00 2.42 0.55 0.00

192

For the purpose of classification of the AAP solubilized from human kidney BB, commercially available aminopeptidases, all of porcine origin, were tested for their immunological cross-reactivity with anti-human BB-AAP. In addition, rat kidney leucine aminopeptidase (kindly supplied by Dr Kinne, Max Planck Institute for Biophysics, Frankfurt, G.F.R.) and the corresponding antiserum were used. “Aminopeptidase M” (Rohm and Haas) and rat kidney leucine aminopeptidase cross reacted very weakly with the antiserum to human AAP, while “leucine aminopeptidase” (LAP, Boehringer) and “aminopeptidase” (Schuch~dt~ did not show any measurable cross-reacti~ty. With the ~tise~rn directed against rat kidney LAP, no cross-reaction with any of the other antigens could be demonstrated. (Cross-reaction was tested in the very sensitive EID technique with subsequent enzyme specific staining.)

Demonstration of soluble brush border proteins in human pathological urine To show whether anti-PM serum is capable of recognizing soluble human brush border proteins in urine, concentrates of urine from normal individuals and from patients with kidney diseases were examined in the one-dimensional EID. The results of such an experiment are shown in Fig. 13. Normal human plasma (sample 1) and concentrates of normal human urine (sample 5) show no precipitin lines with anti-PM serum in the gel. BB-Pap-S (sample 2) and a concentrate of ~ome~lonephritic urine (sample 3f, however, produce several precipitin lines (stained for protein only).

Fig. 13. One-dimensional EID with anti-PM serum in the gel. The samples contain: 1, normal human plasma; 2, BB-Pap-S; 3, concentrate of acute glomendonephritic urine, concentration factor (c.f.) = 575; 4, mixture of samples 2 and 3 (the same volumes as in samples 2 and 3 are mixed in sample 4): 5, concentrate of normal human urine, c-f. = 3000. Fig. 14. necrosis, 4: urine centrate;

One-dimensional EID of BB-Pap-S and concentrate of the wine of a patient with acute tabular with anti-PM serum in the gel. Samples 1 and 2: PM-Pap-S, 4 and 8 ~1 respectively: samples 3 and concentrate 4 and 8 gl respectively: sample 5: mixture of 4 ~cl BE-Pap-S and 4 ~1 urine consample 6: mixture of 8 J BB-Pap-S and 4 ~1 urine concentrate. Staining for AAP activity.

193

Sample 4 is a mixture of the corresponding volumes of BB-Pap-S with the glomerulonephritic urine concentrate. The results of this experiment show that the anti-PM serum is capable of recognizing soluble BB-proteins in pathological urine. In addition, the “mixing experiment” (sample 4), indicates that at least some of the urine proteins are identical with or so similar to the corresponding BB-Pap-S proteins that immune complexes are formed containing the proteins from both sources. In Fig. 14 the results of another experiment are presented, showing a reaction of immunologic~ identity for the AAP from BB-Pap-S and from a urine concentrate of a case of acute tubular necrosis. In this case, the plates are stained specifically for AAP activity. Samples 5 and 6 are mixtures of the corresponding volumes of samples 1 and 3 and samples 2 and 4, respectively. Samples 1 and 2 contain BB-Pap-S; samples 3 and 4 the urine concentrates. Discussion We have recently reported [2] the preparation of a membrane fraction from human kidney cortex, the plasma membrane (PM) fraction, enriched in brush border (BB) fragments. The BB membrane is a specific~ly differentia~d epitheliar part of the kidney proximal tubules, characterized by high transmembrane transport fluxes. Therefore, the exploration of this luminal membrane structure may be of great interest in the understanding of physiological and pathological events in the human kidney. Histochemic~ly it has been shown that a variety of enzymes are localized in the BB membrane. In particular alkaline phosphat~e (AP) was found to be restricted to this part of the tubulus [13]. Using this enzyme as a marker, we found that an alanine specific aminopeptidase (AAP) is associated with the BB membrane as well [2]. The results of the experiments reported here show that another enzyme, the g~ma-glut~ylt~speptidase (g-GTP) is also bound to the BB structure. The presence of this enzyme in the kidney BB region of another species (rat) was reported by Glossmann et al. [14], The three enzymes concerned, AP, AAP and g_GTP, always sediment together in one fraction in centrifugation experiments using continuous (linear) and discontinuous sucrose density gradients (Figs 3 and 4). Electron microscopic photographs of this fraction revealed a high percentage of BB fragments and microvilli. We have recently obtained further evidence that the three enzymes are bound to a distinct membrane fraction. BB membranes, recognized by electron microscopy, migrate in a distinct peak of electrophoretic mobility when subjected to continuous free flow electrophoresis (Elphor model VAP, Drs Bender and Hobein, Miinchen, G.F.R.). The activities of the three marker enzymes are localized mainly in this ele~trophoreti~ fraction. The fraction d3 of the discontinuous sucrose density gradient (Fig. 3 and Table II), containing membrane particles with the highest specific activity of the three BB marker enzymes, named BB fraction, was used for further experiments. To study the properties of BB proteins a method for their solubilization

194

had to be devised. Of the methods tested the treatment with papain turned out to be most efficient. In addition to other proteins (so far unidentified) the three BB marker enzymes were solubilized from the BB membrane fraction by a short incubation with papain. The sedimentation behavior of the membrane particles after papain treatment differed only slightly from that of the untreated membranes (Fig. 5). This was confirmed by electron microscopy. In contrast to ultrasonication, which leads to complete destruction of the membranes, no major changes in the membrane structure could be detected after papain treatment. Differences could be observed in the degree to which each of the three enzymes is released by papain treatment. While practically all AAP activity and about two-thirds of the g-GTP activity were solubilized, most of the AP activity remained membrane-bound (Fig. 5). From these results, we conclude that the papain treatment leads to a specific release of surface components while not affecting the basic structure of the BB membrane. The two BB marker enzymes AAP and g-GTP might be localized on the membrane surface, thus accessible to papain, while AP, the other BB marker, is integrated deeper in the membrane structure. This is in accordance with results of similar investigations on rabbit and rat kidneys where an aminopeptidase is localized in discrete granula on the outer surface of the BB membrane [ 15,161. Another possible explanation for the lack of release of AP by papain might be that this enzyme is linked to the membrane in a different way, making cleavage by papain impossible. Because in inflammatory and destructive processes in tissues proteolytic enzymes are involved, the release of membrane components into body fluids could be used as an early indication for pathologic events. Especially in the BB region of the kidney, which is very sensitive to changes in its physiological status, the release of surface markers might be highly significant for diagnostic purposes, Therefore the artificial release of surface proteins from a defined kidney membranes structure is a useful model and can be of relevance for future diagnostic investigations on the excretion of kidney proteins in cases of kidney diseases. For immunochemic~ studies we used antisera against whole plasma membranes (PM) as described in our previous paper f2] . These antisera were successfully used for fluorescent staining experiments. They specifically reacted with luminal surface structures of proximal tubules thus indicating that they contained antibodies against BB constituents [ 21. These anti-PM sera now turned out to be a very powerful tool for the study of solubilized membrane structures as well. Proteins released from BB membranes by papain (BB-Pap-S) reacted with anti-PM sera in three different immunochemical techniques: (a) the enzymatic activity of AAP in the BBPap-S was strongly inhibited by anti-PM sera (81%); (b) AAP, AP and g-GTP in BB-Pap-S could be precipitated completely by these antisera (immunotitration experiment, Table IV); (c) solubilized BB proteins in BB-Pap-S formed precipitin lines with anti-PM serum in the EID technique, one of which could be identified by enzyme specific staining as AAP-anti-AAP precipitate (Figs 9 and 11). The conclusion from these experiments is as follows: by immunizing rabbits with a defined particulate fraction, antibodies were produced against

195

components on the surface of these particles. As the solubilizing agent was a protease, the solubilization procedure must have resulted in cleavages of peptide bonds. The fact that the antibodies produced against the membrane bound proteins still reacted very efficiently with the solubilized proteins shows that the proteolytic solubilization can only have caused very minute changes in the three-dimensional structure of the proteins concerned. This is further confirmed by the fact that some of these proteins, those with known enzymatic activities, have not lost their enzymatic properties, although it is not possible to judge if and to what extent changes in their enzymatic properties have occurred. In contrast to our “artificial” solubilization procedure by papain the tissue components found in urine of patients with kidney diseases are “patho-physiological” conditions. These may include solubilized under proteolytic processes too. Using anti-PM sera, we tried to find out if in concentrates of human pathological urine soluble proteins could be detected with anti-PM antisera. Fig. 13 shows the result of such an experiment using the EID technique: antibodies in the anti-PM sera were capable of recognizing soluble proteins in pathological urine. In addition some of these soluble urine proteins showed reactions of immunological identity with those artificially solubilized from BB membranes (Fig. 13, sample 4). In a similar experiment, identifying the AAP-anti-AAP precipitate by an enzyme specific staining procedure, it could be demonstrated that AAP, artificially solubilized from BB membranes, and AAP in the urine of a patient with acute tubular necrosis immunologically behaved like identical proteins when tested with anti-PM sera (Fig. 14) as well as with anti-AAP sera (the latter were produced by immunizing rabbits with peak II of the Sephadex G 200 chromatography of BB-Pap-S (Fig. 6) which contains partially purified AAP). In connection with these results, it is important to discuss the question of the possible origin(s) of proteins excreted in the urine. In many kidney diseases, leakage of plasma proteins through the glomerular membrane into urine occurs (proteinuria) to a much higher extent than under normal physiological conditions. Some enzymes which in very low concentrations are components of normal plasma, can be excreted with the urine too. It has not yet been possible to discriminate definitely between those enzymes and proteins originating from plasma and those from the kidney. However, the much higher concentration of these enzymes in pathological urine as compared to plasma indicates that they are released from the kidney. Greene et al. [ 171 conclude from their studies on protein antigens found in normal urine that a specific concentration mechanism in the kidney might lead to an accumulation of tissue proteins from various sources in normal urine. We doubt whether this process, which possibly occurs in normal intact kidneys, would also be found in pathological conditions. The concentrations of tissue components, especially of the BB marker enzyme AAP, that we could detect with our anti-PM sera in urine of patients with a variety of kidney diseases and in rejection crises after kidney transplantation [3] are much too high to be derived from the plasma alone. Further work on this question of discrimination between urine proteins originating from plasma and those from kidney is under way in our laboratories.

196

Enzyme assays in concentrates of large pools of normal and pathological urine revealed the presence of the three BB marker enzymes AAP, AP and g-GTP (F~kenberg et al., in preparation). In immunotitmtion experiments these enzymes reacted with anti-PM sera, again indicating their immunological identity with the corresponding enzymes in BB-Pap-S. In EID experiments several precipitin lines could be produced with these urine concentrates using anti-PM antisera. However the enzymes AAP, AP and g-GTP obtained from pathological urine displayed a si~ificantly lower molecular weight than the co~esponding enzymes artificially released from BB membranes by papain (Table V). Table V also summarizes the data available from literature for the molecular weights of different preparations of the kidney enzymes concerned. It seems therefore that the molecular weights of enzymes, obtained by solubilization from membranes, considerably depends on the method of solubilization: DOG, SDS, digestion by papain or by other proteolytic enzymes and digestion under “patho-physiologicaI” conditions in urinary tract diseases. Nevertheless all the fragmented enzymes seem to be active enzymatitally and immunologically. This interpretation is further supported by the fact that hog kidney “Aminopeptid~e M” (Rohm and Haas, ~~stadt) shows a weak but distinct cross-reaction with anti-PM serum although its mol. wt was determined as 380 000 (Table III) which differs significantly from that of the corresponding enzymes in BB-Pap-S and urine of human origin. The kinetic data presented in this paper give no further information concerning the classification of the three enzymes. The alanine specificity of the aminopeptidase has been reported in our previous paper [2]. The fact that and rat kidney amino“Aminopeptidase M” of RShm and Haas, Darmstadt, peptidase prepared by Thomas and Kinne [19] weakly cross-reacted with antiAAP serum indicates a certain homology between these aminopeptidases which might be related phylogenetically.

TABLE

V

COMPARISON

OF MOLECULAR

WEIGHTS

OF KIDNEY

ENZYMES

AP obtained by extraction from human kidneys with butanol: 150 000, while extraction with sulted in several AP fractions differing in the proportion of polysaccharides bound to the protein 221. For AP from human urine Butterworth f211 reported a mol. wt of 75 000 which is half kidney enzyme. No reports could be found in the literature concerning the molecular weight of Source

of enzyme

BB-Pap-S Concentrates of pathological urine Rabbit kidney BB aminopeptidase Rat kidney BB aminopeptidase Hog kidney microsomes aminopeptidase

Reference

this paper (Table

Molecular

III,

Fig. 7) Falkenberg et af. (in preparation) Kenny et ai. [151 Thomas et al. [lQl Wachsmuth C201

alcohol reparts 221, that of his g-GTP.

weights

AAP

AP

g-GTP

240 000

200 000

126 000

157 000

110000

136 000 140 000 280 000

86 000

197

Using a suitable membrane fraction (e.g. the BB fraction) for immunization, we hope that it will be possible to trace and quantitate kidney proteins, even those of a defined part of the nephron, in urine concentrates. Further investigations on the enzymes and other tissue proteins in BB-Pap-S and urine are under way and might help us to understand structure and function of BB-membranes under normal physiological and patho-physiological conditions. Acknowledgements This work was supported by Grant No. Mo207/1-2 and Grant No. Fa71/1-3 of the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg. J.E. Scherberich received a fellowship of the Studienstiftung des Deutschen Volkes, Bonn-Bad Godesberg. A.W. Mondorf was supported by a fellowship of the Paul Martini Stiftung, Frankfurt/M. References 1 A.W. Mondorf. R. Kinne. J.E. Scherberich and F. Falkenberg, Verhdl. Dtsch. Ges. Inn. Med., 77 (1971) 702-705 2 A.W. Mondorf. R. Kinne. J.E. Scherberich and F. FaIkenberg, Clin. Chim. Acta, 37 (1972) 25-32 3 A.W. Mondorf, C.B. Carpenter, J.E. Scherberich and J. Merrill, 5th Int. Congr. Nephrol. Oct. 1972, Mexico City 4 R.J. Glassock, T.S. Edgington, J.I. Watson and F. Dixon, J. Exp. Med., 127 (1968) 573-587 5 H. Beaufay, S. BendaIl. P. Bahuin, R. Wattiaux and C. de Duve. Biochem. J., 73 (1959) 628 6 Biochemica Informationen. Boehringer Mannheim. 1970 Edition 7 O.H. Lowry, H.J. Rosebrough, A.L. Farr and R.J. RandaB, J. Biol. Chem., 193 (1951) 265 8 P. Emmelot, A. Visser and E.L. Benedetti, Biochim. Biophys. Acta, 150 (1968) 364 9 S. Carrel, S. Barandum and H. Gerber, in Progr. Immunbiol. Standard, Vol. 4, Karger, Basel. 1970, PP. 95-98 10 C.B. LaureII, AnaIyt. Biochem., 17 (1966) 45 11 M.M. Nachlass, B. Morris. D. Rosenblatt and A.M. Seligman, J. Biophys. Biochem. Cytol., 7 (1960) 261 und Priipamtionsmethoden, 2nd edn, 12 L. Reimer, in Elektronenmikroskopische UntersuchungsSpringer, Bedim, 1967 13 N.O. Jacobsen, F. Jdrgensen and A.C. Thornsen, J. Histochem. Cytochem. 15 (1967) 456 14 H. Glossmann and D.M. NeviBe, Jr, FEBS Lett., 19 (1972) 340 15 A.J. Kenny, S.G. George and S.G.R. Aparicio. Biochem. J., 116 (1969) 18 p 16 H. Pockrandt-Hemstedt. J.E. Schmitz, E. Kinne-Saffran and R. Kinne, Arch. Ges. Physiol.. 333 (1972) 297 17 E.L. Greene, S.P. Halpert and 1.C. PaBavacini, Intern. Arch. Allergy APP~. Immunology, 40 (1971) 861 18 P. Dehm and A. Nordwig. Eur. J. Biochem., 17 (1970) 364 19 L. Thomas and R. Kinne, Biochim. Biophys. Acta. 255 (1972) 144 20 E.D. Wachsmuth. Biochem. Z.. 10 SU, 346 (1967) 446 21 P.J. Butterworth, Biochem. J., 107 (1968) 467 22 A.L. Latner, M. Parson and A.W. Skillen, Enzymologia, 40 (1971) 1