Schoenberger et al.: N-Acetylalanine aminopeptidase activity in cells 375

J. Clin. Chem. Clin. Biochem.Vol. 24, 1986, pp. 375-378© 1986 Walter de Gruyter & Co.

Berlin · New York

N-Acetylalanine Aminopeptidase Activity in Normal and Tumour Cells

By O. L. Schoenberger, H. Schwöbel and W. EbenKrankenhaus Rohrbach, Klinik für Thoraxerkrankungen, Heidelberg

j | (Received November 15, 1985/February 14, 1986)

Summary: The catalytic concentration of N-acetylalanine aminopeptidase was determined in erythrocytes,polymorphonuclear leukocytes, lymphocytes, alveolar macrophages, human lung fibroblasts, human lungcancer cells, human umbilical vein endothelial cells, mice Leydig cells, rat tumour cells, and endothelial cellsfrom hog and bovine lung. The catalytic concentration ranged from%0.5 ±0.1 nU/cell (erythrocytes) to35 nU/cell (rat tumour cell line B Sp 73 ASML). Almost all tumour cells showed higher activity levels. Noactivity was found in human plasma.

N-Acetylalanm-aminopeptidase-Aktivität in normalen Zellen und in TumorzellenZusammenfassung: Die katalytische Konzentration von N-Acetylalanin-aminopeptidase bestimmten wir inErythrocyten, polymorphkemigen Leukocyten, Lymphocyten, Alveolar-Makrophagen, menschlichen Lun-genfibroblasten, menschlichen Lungentumorzellen, menschlichen Nabelschnurendothelzellen, Leydig-Zdlenvon Mäusen, Ratten-Tumorzellen und Endothelzellen aus Schweine- und Rinderlunge. Die katalytischeKonzentration reichte von 0,5 ± 0,1 nU/Zelle (Erythrocyten) bis zu 35 nU/Zelle (Ratten Tumorzell-LinieB Sp 73 ASML). In fast allen Tumorzellen wurde eine erhöhte Aktivität gefunden. Keine Aktivität konnteim menschlichen Plasma nachgewiesen werden.

Introduction We found ̂ w-acetylalamTLZ--mtToanilidz is aN^Acylamino acyl-peptide hydrolases (EC 3.4.19.1) good Substrate for the enzyme from human erythro-have been found in different tissues of a variety of cytes (8). In this publication we show that N-acetylal-vertebrates (l — 10) and in E. coli B (3). Despite small anine aminopeptidase can be detected in many cells.differences in their biocheniical properties, they have The high activity of the enzyme permits a simplemany characteristics in eommpn, for example the quantiative determination.high molecülar weight of about 300000 Palton, theoptimal activity ät afound pH 8, the sensitivity Material and Methode1)against sulphydryl blocking reagents, the lack of any Cellgeffect of cations and the release of acylamino acidsfrpm peptide, The ^bstrates specificity, however, is ̂ ̂ ^^ ̂ ^ ̂ ^ ÜiSlSnot exactly the same for all N-acylamino acyl-peptide frQm bronchial alveolar lavage in cooperation with Dr. Beckerhydrolases In a recently published paper, Jones & at our hospital. Mice Leydig cells were kindly provided by Prof.Manning (10) pointtd ou, te ths pH-optaum can ^ SSSlSJStaSSÄ 5change, depending on the Substrate; for example they \elz Of the Gennan Cancer Research Centre in Heidelberg.obtained for the human erythrocyte enzyme a pH- All other cells were isolated and cultured in our laboratory.Optimum of pH 8 with N-acetyl-alanine-p-nitroani- Erythrocytes and polymorphonuclear leukocytes were isolated, i. . I V * - -KT * * * · from fresh blood of a healthy donor by density centnfugationhde äs Substrate, but with N-acetyl-glutamate^-ni- w,ft a percoll gradient according to Nees A Fink (12).troanilide äs Substrate the pH-optimurn was shifted _to pH 6. 0 Abbreviation: RPMI: Rosewell-Park-Memorial Institute

J. Clin. Chem. Clin. Biochem. / Vol. 24, 1986 / No. 6

376 Schoenberger et al.: N-Acetylalanine aminopeptidase activity in cells

The isolation of human cancer cells will be described in detailelsewhere (13). Briefly, human lung tumour pieces were mincedand then usually digested with l g/l collagenase (Worthington,Cat. No. LS 0004176; catalytic concentration: 180 U/mg). AfterFiltration through a nylon gauze (Schweizerische Seidengazefa-brik. Zürich, Nybolt 81/2 XXX-160) the cells were seeded inculture fiasks (Falcon, Heidelberg, Cat. No. 3024). RPMI-1640(Seromed, Berlin, Cat. No. F-1215) containing 100 ml/1 foetalcalf serum (Seromed, S-0115), 100 · 103 U/l penicillin and 100• l O3 U/l streptomycin (Gibco, Karlsruhe, Cat. No. 043-5140)and 2 mmol/1 glutamine (Flow Laboratories, Meckenheim, Cat.No. 16-30-1-46) was used äs medium for the tumour cells. Amedium change was performed every other day. Usually afterone week the adherent cells were harvested by treatment with1.25 g/l trypsin and 1.25 g/l EDTA (both reagents were fromServa, Heidelberg; Cat. No. 37290 and Cat. No. 11280, respec-tively) solution in phosphate buffered saune. Essentially thesame method was used for human lung fibroblasts.

To obtain endothelial cells, fresh bovine or hog lungs wereperfused with 9 g/l sodium chloride solution. A chymotrypsin/EDTA solution (l g/l oc-Chymotrase®, Hasenclever, Bonn) waspumped through the perfusion tubes into the lung. The flowof the soiution was then stopped, and after 5 min at 37 °C thedetached cells were collected, washed with RPMI-1640 (thistime from Gibco, Cat. No. 048-2400, which has a differentcomposition compared to RPMI-1640 purchased from Sero-med) containing penicillin and streptomycin (each 100 · l O3

U/l), and supplemented with 200 ml/l foetal calf serum. Thecells were cultured in the same medium.

Endothelial cells from human umbilical vein were isolated inthe same way.

The cells were identifled histologically.

For the investigation of whether plasma contains N-acetylala-nine aminopeptidase activity, blood was withdrawn into tubeswith and without beads (NH4-Heparin Monovette and EDTAK Monovette, Sarstedt, Nümbrecht).

Cell lysis

Cells were washed twice with 10 ml 9 g/l sodium chloride solu-tion. After counting the cell number in a haemocytometer andafter checking the viability of the cells with trypan blue, thecell Suspension (usually l O7 cells/lOml) was lysed by shortultrasonic treatment with a Labsonic 1510 (Braun, Melsungen)at 40 W for 30 to 60 s. The membranes were removed bycentrifugation at 25000g and the supernatant was used tomeasure enzymatic activity. In order to be sure that all cellswere disrupted, the pellet was again ultrasonicated. Usually lessthan 5% of the activity emerged, i. e. 95% of the cells werelysed by the first ultrasonic treatment.

Activity assay

The N-acetylalanine aminopeptidase assay was performed in0.1 mol/1 Tris/HCl buffer, pH 8.3 containing 0.01 mol/1 calciumchloride and 0.05 ml/i Triton X-100 at 25 °C. A 200 samplewas added to 795 buffer in a l ml cuvette. The reactionwas started with 5 N-acetyl-L-alanine-/?-nitroanilide (40 g/ldimethylsulphoxide; the Substrate was purchased from Serva,Cat. No. 10160). The release of p-nitroaniline was measured at405 nm with an Uvikon 810 (Kontron, Eching). The activitywas calculated using = 962m2/mol (14).

For the analysis of the haemolysate, the sample volume wasreduced to 100 , because of the strong absorbance of haemo-globin at 405 nm.

Results

N-acetylalanine aminopeptidase activity inhuman blood cells

In a previous paper (8), which deals with the isolationand some characteristics of N-aoetylalanine amino-peptidase from human erythrocytes, we calculatedthat erythrocytes contain 0.6—0.75 nU/cell N-acetyl-alanine aminopeptidase activity, but this calcülatioüwas not based on an exact cell number. The amountof enzyme determined in the present study was 0.5+ 0.1 nU/cell (tab. 1). In addition to the determina-tion of the amoünt of N-acetylalanine aminopepti-dase in erythrocytes, the enzymic activity was alsomeasured in different types of white blood cells(tab. 1). T-lymphocytes showed a slightly higher ac^tivity than erythrocytes and B-lymphocytes, andmonocytes had twice äs much activity äs red bloodcells. In the case of pölyrriorphonuclear leukocytesthe activity was almost four times that in erythro-cytes. The differences were statistically significant(p < 0.05) in a Wilcoxon test (15).

Tab. 1. N-acetylalanine aminopeptidase activity in humanblood cells. Values are means ± S. D. of the numberof cell preparations. Erythrocytes were only isolatedfour times, but 15 dilutions were made from eaeh pre^paration; in this way we obtained 60 samples for thedetermination of N-acetylalanine aminopeptidase ac-tivity.The number of viable cells exceeded l O6 in all prepara-tions.

Catalytic concentration'' ;:(nU/cell)

ErythrocytesT-lymphocytesB-lymphocytesMonocytesPolymorphonuclear leukocytes

0.5 ±0.10.75 ± 0.181.3 ±0.30.98 + 0.171.9 ±0.7

(n = 60)(n= 9)(n= 5)(n= 7)(n= 6)

N-acetylalanine aminopeptidase activity inhuman lung cells

Alveolar macrophages and human lung fibroblasts(tabl. 2) contained about 10 times more activity thanerythrocytes. We found even more activity in humanlung tumour cells (tab. 2). In our laboratory we cul-ture human lung tumour cells routinely. At present,we have two cell lines (HS 24 and HS 57), which havebeen growing for months. Another cell line (HS 34)died after one month, arid three other samples(HS 37, HS 38, and HS 40) lived only a few days. Theamount of N-acetylalanine aminopeptidase in thesecells ranged from 5.6 nU/cell to 29 nU/cell. Thereason for this difference of md/e than one order of

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Schoenberger et al.: N-Acetylalanine aminopeptidase activity in cells 377

magnitude between these basically similar cells is notyet understood, but it could be caused by a differenttumour history and/or by the cell division phase. Aswe collected the cells from the culture flasks we didnot check the relative proportions of S-, G- and M-phase cells (16).

Tab. 2. N-acetylalanine aminopeptidase activity in human lungcells. Values are from one preparation, because the cellswere only once available or cultured. In the latter caseit was impossible to get the same type of cells again.

Alveolar macrophagesFibroblastsTumour HS 24Tumour HS 34Tumour HS 37Tumour HS 38Tumour HS 40Tumour HS 57Tumour HS 105

No. ofviable cells(io6)

5.820148.53.50.65.3

323

Catalyticconcentration(nU/cell)

3.76.7

16105.6

1382.9

16

N-acetylalanine aminopeptidase activity incells from other sources

N-acetylalanine aminopeptidase activity was addi-tionally determined in cells from human umbilicalvein, from rnice and rats, and from hog and bovinelungs (tab. 3). The lowest amounts in these cells werefound in bovine endothelial cells (l nU/cell), and thehighest activity was found the high-metastasizing ratcell line B Sp 73 ASML (35 nU/cell).

Tab. 3. N-acetylalanine aminopeptidase activity in cells fromdifferent sources. for the reasons mentioned in thelegend of table 2, values are from one preparation.

Np. of Catalyticviable cells Concentration(IO6) (nÜ/cell)

Rat B Sp 73 AS cellsRat B Sp 73 ASML cellsMice Leydig cellsHog lung endothelial cellsBovine lung endothelial cellsHuman umbilicalendothelial cells

200301

12100

5.5

9.8351.63.719.8

N-acetylalanine aminopeptidase activity inhuman plasma

In contradiction of Unger & Struck (17) we could notdetect any N-acetylalanine aminopeptidase activityin human plasma. To clarify this point we checkedplasma samples withdrawn from the same donor intodifferent tubes. One tube contained no beads andanother contained some beads for a better Separationbetween plasma and blood cells. N-acetylalanine am-inopeptidase activity was found only in the lattersamples; obviously some erythrocytes were lysed andthe enzyme was released into the plasma.

Discussion

All cells we have checked so far contained N-acetylal-anine aminopeptidase activity. Erythrocytes showedthe lowest activity per cell, but considering the abun-dance of red blood cells, their entire activity isremarkable high^nd could be of physiological impor-tance. Unfortunately, the physiological role of N-acetylalanine aminopeptidase is not known, but thereare some speculations about the function of N-acyl-amino acyl-peptide hydrolases in protein biosynthesis(10, 18) and in intracellular protein turnover (8).

In all human lung cancer cells, in human umbilicalendothelial cells, and in the rat tumour cell linesAS and ASML, the N-acetylalanine aminopeptidaseactivity was quite high. In the case of HS 24 andHS 57 we measured the activity over a period of time(13). The amount of N-acetylalanine aminopeptidasewent up and down, but the activity was always higherthan in all other cells. Whether these high valuesindicate a tumour-specific characteristic or a normalcharacteristic of growing cells, can only be judgedwhen we can successfully culture normal human lungcells. However, the conclusion that the enzyme is anormal characteristic of growing cells is favored bythe high activity in human umbilical endothelial cells.

AcknowledgementThe endothelial cell Isolation method was recommended to usby Dr. Thilo-Körner, Gießen. This work was supported by theTumorzentrum Heidelberg/Mannheim.

References1. Yoshida, A. & Lin,.M. (1972) J. Biol. Chem. 247, 952-957.2. Witheiler, J. & Wilson, D. B. (1972) J. Biol. Chem. 247,

2217-2221.3. Tsunasawa, S., Narita, K. & Ogata, V. (1975) J. Biochem.

77,89-102.

4. Lorentz, K., Petersen, S. & Ritter, U. (1975) Z. Klin. Chem.Klin. Biochem. 13, 45-48.

5. Tsunasawa, S. & Narita, K. (1976) Methods Enzymol. 45,552-561.

6. Gade, W. & Brown, J. L. (1978) J. Biol. Chem. 255012-5018.

J. Clin. Chem, Clin. Biochem. / Vol. 24,1986 / No. 6

378 Schoenberger et al.: N-Acetylalanine aminopeptidase activity in cells

7. Suda, H., Yamamoto, K. & Umezawa, H. (1980) Biochim. 14. Wachsmuth, E. D., Fritze, I. & Pfleiderer, G. (1966) Bio-Biophys. Acta 616, 60-67. chemistry 5, 169-174.

8. Schoenberger, O. L. & Tschesche, H. (1981) Hoppe-Seyler's 15. Ehrengruber, H. (1978) in Klinische Chemie, 4th edn.Z. Physiol. Chem. 352,865-873. (Richterich, R. & Colombo, J. P., eds.) pp. 39-40, S.

9. Marks, N., Lo, E.-S., Stern, F. & Danho, W. (1983) J. Karger, Basel.Neurochem. 41, 201-208. 16. Stein, G. & Bäseraga, R. (1972) Adv. Cancer Res. 5,

10. Jones, W. M. & Manning, J. M. (1985) Biochem. Biophys. 287-330.Res. Commun. 126, 933-940. 17. Unger, T. & Struck, H. (1977) ; t\m. Chim. Acta 78,

11. AuJenbacher, P., Werling, H.-O., Paweletz, N. & Spiess, E. 113-120.(1984) Anticancer Res. 4, 75-82. 18. Narita, K., Tsuchida, I., TsuiDasawa, S. & Ogata, K. (1969)

12. Nees, S. & Fink, W. (1984) Labor Praxis 1314-1317. Biochem. Biophys. Res. 'Commun. 37, 327-332.13. Schoenberger, O. L., Ebert, W. & Drings, P. in preparation.

Dr. Oeyviiid L. SchoenbergerKrankenhaus RohrbachKlinik für ThoraxerkrankungenAmalienstr. 5D-6900 Heidelberg l

J. Clin. Chem. Clin. Biochem. / Vol. 24,1986 / No. 6