Comp. Biochem. Physiol. Vol. 95B, No. 3, pp. 589-595, 1990 0305-0491/90 $3.00 + 0.00 Printed in Great Britain © 1990 Pergamon Press plc

PURIFICATION AND PROPERTIES OF GLUTAMYL AMINOPEPTIDASE FROM CHICKEN EGG-WHITE

SINISA PETROVIC and LJUBINKA VITALE

Department of Organic Chemistry and Biochemistry, "Rudjer Bo~kovir" Institute, 41001 Zagreb, Yugoslavia (Tel: 041 424 987)

(Received 2 August 1989)

Abstract--l. Hydrolytic activities characteristic for different aminopeptidases were detected in the egg-white of unfertilized chicken eggs, and one aminopeptidase was isolated in an electropb.oretically homogeneous form.

2. The isolated aminopeptidase preferentially hydrolyzed bonds of ct-glutamyl residue at the NH2-end of synthetic substrates and peptides.

3. The enzyme is a dimer with an M r of 320,000 and pI of 4.2. Its optimal pH and temperature are 7.6 and 60°C, respectively.

4. EDTA, amastatin, and N-bromosuccinimide are inhibitors, while Ca 2÷ and Mn 2+ are activators of the enzyme. Ca 2÷ also stabilizes the enzyme.

5. According to the observed properties, the isolated chicken egg-white aminopeptidase belongs to the glutamyl aminopeptidases.

INTRODUCTION MATERIALS AND METHODS

Animal eggs as well as plant seeds contain storage proteins which are mobilized during embryo or germ development. Their conversion to active molecules or constituent amino acids requires the action of proteo- lytic enzymes, of both endo- and exopeptidases, some of which themselves undergo previous activation by a proteolytic cleavage.

In the eggs of mammals and several aquatic species different types of endopeptidases have been found and implicated in the fertilization or ovulation pro- cesses (Csernansky et al., 1979; Mumford et al., 1981). Aminopeptidases have been described in hamster, nematode and mullet eggs (Mumford et al., 1981; Tefft and Bone, 1985; Chiou et al., 1988). In the first two cases their activity was only detected, whereas from the mullet roe a broad specificity aminopeptidase was isolated. As far as birds eggs are concerned, there are no data on their aminopeptidases except for the early detection of peptidase activity reported by Lineweaver et al. (1948).

We have analyzed the chicken egg-white, which presents a main compartment for storage proteins, for aminopeptidase activity. We have also isolated an enzyme, which by its specificity corresponds to glutamyl aminopeptidase.

Materials

Fresh unfertilized eggs were obtained from the local chicken farm and the egg-whites were separated manually from yolks and chalazae.

Sephacryl S-200 Superfine, Con A-Sepharose, Sephacryl S-300 Superfine, Mono Q HR 5/5 column, molecular weight markers for polyacrylamide gel electrophoresis (HMW PAGE) and gel filtration, pl markers for isoelectric focusing (IEF), PhastGel gradient media 8-25 and PhastGel IEF media 4.0-6.5, were purchased from Pharmacia Fine Chem- icals, Uppsala, Sweden, Hydroxylapatite (Hypatite C) was from Clarkson Chem. Co., Williamport, PA. Ultrafiltration ceils, Diaflo XM-50 membranes and a Centricon Micro- concentrator System with Diaflo YM-5 membrane were obtained from Amicon, Oosterhout, Netherlands.

Synthetic peptides, Ct-L-Glu-2NA and other aminoacyl- 2NA, Ct-L-Glu-4-nitroanilide (~-L-Glu-pNan), Fast Blue B salt, N-bromosuccinimide (NBS), phenylmethylsulfonyl fluoride (PMSF), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), puromycin. 2HC1, Chelite C and Visking dialysis tubing were products of Serva, Heidelberg. 4-Hydroxy- mercuribenzoic acid (pHMB) was purchased from Cal- biochem, Lucerne and Silica gel 60 F254 0.2 mm thin-layer plates from Merck, Darmstadt.

Bestatin, amastatin and cystatin were the generous gifts of Dr T. Aoyagi, Tokyo, Japan, and Dr J. Kos, Ljubljana, respectively.

Metal salts and other chemicals were of analytical grade, and were supplied by Kemika, Zagreb, and Merck, Darmstadt.

Abbreviations used - -DTNB, 5,5'-dithiobis(2-nitrobenzoic acid); EAP, glutamyl aminopeptidase; FPLC, fast protein liquid chromatography; IEF, isoelectric focus- ing; 2NA, 2-naphthylamide; NBS, N-bromosuccinimide; PAGE, polyacrylamide gel electrophoresis; pHMB, 4- hydroxymercuribenzoic acid; PMSF, phenylmethylsul- fonyl fluoride; pNan, 4-nitroanilide; SDS, sodium dodecyl sulfate.

Enzyme assay and protein content

Aminopeptidase activity was assayed by the colourimetric method according to Nagatsu et aL (1970). The reaction mixture contained 0.1 M Tris-HCl buffer pH 8.0, 5 mM CaC12, 0.136mM ~t-Glu-2NA or another naphthylamide and an appropriate amount of the enzyme. After 15-30 rain at 37°C in a water bath shaker liberated 2-naphthylamine

589

590 SINISA PETROVI6 and LJUBINKA VITALE

was coupled with Fast Blue B salt (0.025%) at pH 4.2 in the presence of 1.6% Tween 20, and the A530 was measured. ~-Glu-pNan was used as a substrate for the determination of kinetic constants. Reactions were carried out at 37°C in 0.02M Tris HC1 buffer, pH 7.6, with 5mM CaCI2, 166 ng/ml of the enzyme and 0.01 1 mM substrate. Liber- ated p-nitroaniline was continuously followed at 405 nm, and the enzyme activity was calculated from the linear part of the curve (Pfleiderer, 1970). K m value was estimated from the Hanes plot (Cornish-Bowden, 1979), and K i values for competing substrates were calculated according to Chu and Orlowski (1985).

One unit of the enzyme activity (U) was defined as the amount of enzyme which hydrolyses 1 pmol of substrate per min at 37c~C.

Enzyme activity on gels was located by incubating the gel in 0.1 M Tris HC1 buffer, pH 8.0, containing 5mM CaCI 2 and 2.6 mM ~-Glu-4-methoxy-2NA. After 15 min at 37°C the gel was shortly rinsed with water and then immersed in a solution of 0.15% Fast Blue B salt in 2.1 M sodium acetate buffer, pH 4.2, with 10% Tween 20.

The hydrolysis of peptides was tested in reaction mixtures containing 1 5 mM substrate, 1 mM CaCI~ and 83 ng of the enzyme in a 0.I ml aqueous solution, pH 7.6. After 60 or 120min, 15/d aliquots were analyzed by thin-layer chromatography on silica gel plates using n-bu- tanol:acetic acid:water (60:15:25) or n-butanol:27% NHaOH (70:30) as solvents. Spots were detected by spray- ing with a solution of 0.2% ninhydrin in ethanol (Brenner et aL, 1969). During chromatographic separations the protein concentration was monitored as an absorbance at 280 nm. In other samples protein content was assayed by the method of Bradford (1976) using bovine serum albumin as a standard.

Relative molecular mass and pl determination

M r was estimated by gel filtration on a Sephacryl S-300 Superfine column (2.5 x 85 cm) which was eluted at a flow rate of 10ml/hr with 0.01M Tris HC1 buffer, pH 8.0, containing 0.5 M NaC1, and by the PhastSystem PAGE on PhastGel gradient 8 25% plates, under denaturing and nondenaturing conditions. For the sodium dodecyl sulfate (SDS)-PAGE the samples were treated with 2.5% SDS and 5% mercaptoethanol for 5min at 100°C (PhastSystem Separation Technique File No. 110 and 120). High molecu- lar weight calibration kits were used as molecular mass markers.

The isoelectric point was determined by the isoelectric focusing of native protein, performed on PhastSystem using PhastGel IEF plates, pH 4.0~5.5 (Separation Technique File No. 100).

Enzyme effectors and stability

Measurements of the inhibitors effects were preceded by 15-30 min preincubation of the enzyme with an inhibitor. The influence of divalent metal ions was assayed using the native enzyme, and the enzyme which was previously treated by 1 mM EDTA and gel filtered through the Sephadex G-25 column in a buffer without metal ions and EDTA. Enzyme activities were expressed as the percentage of activity deter- mined in the absence of metal compounds. For these experiments plastic ware, Milli Q water (18 MQ/cm) and buffer solutions purified by Chelite C, were used.

For the pH stability determinations the enzyme samples (80ng/ml) were incubated at 22°C in 14.3mM Brit- ton-Welford universal buffer of different pH (Long, 1961) and after 210 min, the remaining activity was measured by the previously described assay at pH 7.6. The influence of the pH on the hydrolysis rate was determined in the same universal buffer. Enzyme's heat stability was estimated by determining the loss of its activity after 60 min incubation (80 ng/ml) at different temperatures in 0.01 M Tris-HC1 buffer, pH 7.6, with or without 5 mM CaC12. Heat inactiv- ation at 60°C was followed by the measurement of enzyme activity at different time intervals. Temperature optimum and activation energy were calculated using initial reaction rates obtained at different temperatures.

R E S U L T S

Enzyme purification

Egg-whites were homogenized with an equal vol- ume of 0.25% aqueous NaCI solution, pH 5.0; then the pH was adjusted to 6.0-6.5 with a 5 M sodium formiate buffer, pH 3.0, and the precipitated ovo- mucin was removed by centrifugation (30min, 4080 g). The obtained clear supernatant was assayed for the presence of aminopeptidases using different aminoacyl naphthylamides as substrates. Hydrolytic activities were found towards Met-, Ala-, Leu-, Glu-. Phe- and Arg-2NA, whereas degradation of Ser- and Pro-2NA was not observed.

Q4

~ 3

Q2 i I

0.1

QO 0

b

I~.q , Ooo ~o x / eeeeea°°°Oo o

I00 200 300 Vol. [ ml )

0.6 _~

._c E

0.4 <

._> *5

0.2 o o~

N C U.I

' " 00 40O

Fig. 1. Gel filtration of 1.7 g of active protein fraction on Sephacryl S-200 Superfine column (2.5 x 85 cm) in 0.01 M Tris HC1 buffer, pH 8.0, 0.1 M NaCI. The flow rate was 7ml/hr. Enzyme activity was

determined with ~-GIu-2NA (0 ) and Leu-2NA (O).

Glutamyl aminopeptidase 591

1.5

1.0 E

o

i

I

QOO5M QOO8M

x k°°~° "'"°'~ x 0"00 1

0.4.c_ .~

v ~

i I

1

) al [

N

O008M

" 2 " . . . . ½ OO

Vol. ( I )

Fig. 2. Chromatography of 15 mg of EAP active fractions from Sephacryl S-300 on hydroxylapatite column (2.5 x 10 cm) in sodium phosphate buffer, pH 7.0. Buffer concentration changes are indicated by arrows and by concentration gradient curve. The flow rate was 40 ml/hr. Enzyme activity was determined

with ct-GIu-2NA (O) and Leu-2NA (O).

Proteins in the egg-white homogenate were further fractionated by ammonium sulfate precipitation. The precipitate obtained at salt saturation between 35 and 65% was dissolved in a small amount of 0.01 M Tris-HC1 buffer, pH 8.0, containing 0.04 M NaC1 and dialyzed overnight against the same buffer. The solution was clarified by centrifugation (20min, 12,100g), concentrated by ultrafiltration and loaded on the Sephacryl S-200 column as shown in Fig. 1. A good separation of aminopepti- dases from the main mass of proteins was achieved. In addition, the glutamyl aminopeptidase was par- tially separated from the leucyl aminopeptidase. Fractions containing glutamyl aminopeptidase (EAP) activity were pooled, concentrated and again gel filtered, but now on the Sephacryl S-300 column. Inactive proteins were removed, excluding the en- zyme responsible for Leu-2NA hydrolysis. The separ- ation of the two aminopeptidases was possible through chromatography on hydroxylapatite under the conditions described in Fig. 2. EAP was eluted with a 15-25mM buffer in a very dilute solution. The active fractions were concentrated by ultrafiltra- tion and remaining impurities were eliminated by fast protein liquid chromatography (FPLC) on the Mono Q column. The column was washed with 50 mM Tris-HCl buffer, pH 8.0, containing 1 mM NaC1, and then with NaC1 gradient (1 mM-1 M) in the same buffer. A symmetrical protein peak which appeared at 18 mM NaC1 coincided with the enzyme activity.

Table 1 summarizes a typical purification pro- cedure. A purification factor of approximately 100,000-fold and a 14% recovery indicate that EAP represents less than 0.01% of egg-white proteins. The enzyme preparation was shown to be homogeneous by the criteria of PAGE and IEF.

Molecular properties The Mr determination by gel filtration and by

PAGE in gradient gels gave a value of 320,000 for the isolated EAP. However, zymogram revealed both the main band and a small amount of enzyme having an M r close to 160,000 (Fig. 3). SDS-PAGE of the denatured enzyme gave only one protein band whose

ili~ii!iii~

!i~i;iiii

Fig. 3. Polyacrylamide gradient gel (8-25%) electrophoresis of (I) thyroglobulin, ferritin, catalase, lactate dehydroge- nase and bovine serum albumin, 0.5/~g each; (2) EAP, 0.12 pg, stained by Coomassie brilliant blue R-250; (3) EAP, 0.12 # g, zymogram obtained with ~-Glu-4-methoxy-2NA as

substrate.

Purification step

Table 1. Purification of glutamyl aminopeptidase from chicken egg-white Vol. Prot. conc. Spec. activity Yield (ml) (mg/ml) (mU/mg) (%)

Homogenate* 1000 62 0.20 100 pH adjustment 904 48 0.25 87 35~5% (NH4)2SO4, dialysis, conc. 72 164 0.84 80 Sephacryl S-200, concentration 42.7 6.7 28.1 65 Sephacryl S-300, concentration 116.5 0.066 671.0 42 Hydroxylapatite, concentration 5.7 0.025 14550.0 17 FPLC, concentration 1.1 0.083 19760.0 14 *Obtained from 20 eggs.

5 9 2 SINISA P E T R O V l C a n d L J U B I N K A V I T A L E

100

;e

5O >

60min

o -_.o, 100

~ 50

o

•,• 60 'c

~ o .

o ' - . ,

o ~

\o \o

0 i i i i i i , 1 O i i I i i i

-20 0 20 40 60 0 20 40 60 Temp ( °C ) Time ( rain )

Fig. 4. Thermal stability of EAP. 83 ng/ml EAP in 0.2 M Tris-HC1 buffer, pH 7.6 (O, solid line); the same mixture plus 5 mM CaCI 2 (O, dashed line).

M r was approximately 180,000. Both these observa- tions speak in favor of the dimeric structure of egg-white EAP. The enzyme was firmly bound to Con A-Sepharose, which indicates that its molecule con- tains a glycosidic moiety.

The IEF in polyacrylamide gels established the anionic character of the chicken egg-white EAP with pI of 4.2.

The isolated enzyme was shown to be stable at pH 5-10, during freezing, and also at temperatures up to 45°C. Higher temperatures applied in the course of 1 hr inactivate the enzyme, whereas Ca 2+ ions have a protective effect (Fig. 4).

Catalytic properties The optimal pH for ~-GIu-2NA hydrolysis by

chicken egg-white EAP was 7.6. The hydrolysis rate, measured for 15 min, was increased with a raise of temperature up to 60°C. At higher temperatures inactivation of the enzyme took place. From the Arrhenius plot two values for the activation energy

2.5

2O

o E Z L

1.5

>

03 o

\

\o

\\o \ \ \

\ ] , 0 t i i i i i

3.0 3.1 a2 33 :~ as ! (looo K)-~ T

Fig. 5. Arrhenius plot of temperature dependence of c¢-Glu- 2NA hydrolysis by EAP from chicken egg-white.

can be calculated. The higher one of 76.0 kJ/mol is obtained at temperatures below 36.6°C, whereas at higher temperatures it diminishes to 35.3kJ/mol (Fig. 5).

The isolated EAP hydrolyzed ~-GIu-2NA and ~-Glu-pNan following the Michaelis-Menten kinet- ics. K m and k~ t for the ~-Glu-pNan at pH 7.6 and 37°C are 3.1 x 10-4M (+0.8 x 10 4SD) and 191 sec i respectively. K m for the ~-GIu-2NA was estimated indirectly as K~ in the reaction where this substrate acted as a competitive substrate of ~-Gln- pNan hydrolysis. According to Cornish-Bowden (1979) and Chu and Orlowski (1985), in such cases K i closely corresponds to the Km of the competing substrate. The obtained value is 8.3 × 10-SM (4- 1.6x 10 SSD).

EAP from egg-white has shown high sub- strate specificity. Out of 13 tested aminoacyl naphthylamides, it hydrolyzed only c~-Glu-2NA and at a seven times slower rate ~-Asp-2NA. When different peptides, including chicken egg- white cystatins, were used as substrates, only those having glutamic and aspartic acid at the N-terminal were degraded, regardless of their size (Table 2).

Enzyme effectors The effect of various protease inhibitors on egg-

white EAP is presented in Table 3. The enzyme has a pronounced sensitivity to the chelating agent EDTA, to NBS, a reagent for tryptophan, and to amastatin, the inhibitor of aminopeptidase A. A thiol reagent, DTNB, moderately inhibited EAP, this inhi- bition being about 25% smaller when Ca 2+ was omitted from the preincubation mixture. Serine and cysteine proteinase inhibitors, PMSF and pHMB, as well as bestatin and puromycin, under the same conditions had practically no effect on the activity of the enzyme.

Determination of metal ions influence on the iso- lated EAP revealed a significant activation of the enzyme by 1 mM Mn 2+ and Ca 2+, whereas Ni z+, Zn > and Co 2+ at the same concentration were inhibitory (Fig. 6). An EDTA-treated enzyme could be reactivated to about 90% of its full activity by Ca 2+ and less efficiently by Mn 2+.

Glutamyl aminopeptidase

Table 2. Substrate specificity of EAP from chicken egg-white

593

Hydrolyzed Not hydrolyzed -GIu-2NA -Asp-2NA

Asp-Ala Asp-Leu Glu-Ala-Ala Asp-Arg-Val-Tyr-lle-His-Pro-Phe

7-GIu-2NA, 7-Glu-INA. fl-Asp-2NA, ~t-Asp-Arg-2NA*, AIa-2NA, Leu-2NA, D-Leu-2NA, Phe-2NA, Arg-2NA, Met-2NA, Ser-2NA, GIy-2NA. Pro-2NA, N-GIt-Gly-GIu-Phe-2NA*, 7-Glu-Cys-Gly, Leu-Trp, Leu-Val. Val-Tyr, Phe-Ala, Arg-Phe, Met-Leu, Ser-Met, Gly-Glu, Gly-Leu, His-Leu, Pro-Gly, Cystatin

The hydrolysis of naphthylamides was determined colourimetrically as described in Materials and Methods. The hydrolysis of peptides and asterisk-marked naphthylamides was followed by thin-layer chromatog- raphy after 1 and 2 hr of incubation. All amino acids were of the L configuration.

DISCUSSION

Chicken egg-white was shown to possess hydrolytic activities characteristic of several types of aminopep- tidases. Two corresponding enzymes were discerned and one of them was isolated in the electrophoreti- cally homogeneous form.

Table 3. Effect of inhibitors on the activity of EAP from chicken egg-white

Concentration Activity Inhibitor~" (raM) (%) PMSF* 1.0 96 EDTA 0. I 8

1.0 3 pHMB 0.1 I00 DTNB 0.1 76

1.0 63 NBS+ + 0.001 74

0.01 0 Puromycin* 0.50 100 Bestatin* 0.66 100 Amastatin* 0.003 50 +Enzyme and inhibitor were preincubated for 15 min (or

30rain for those marked with asterisk) at room temperature in 0.1 M Tris-HCl buffer, pH 7.6, con- taining 5 mM CaCI 2.

:~Preincubation was carried out in 0.1 M sodium acetate buffer, pH 5.0, containing 5 mM CaCI z as suggested by Harada et al. (1984).

After the removal of a bulk of egg-white proteins by precipitation and gel filtration, the separation of closely related aminopeptidases was achieved through hydroxylapatite chromatography, Consider- ing the low content of the enzyme in egg-white proteins, the purification procedure is simple and gives a good yield. Among the amino acid naphthyl- amides the enzyme hydrolyzed only ct-Glu-2NA and to a lesser extent ct-Asp-2NA, which means that the enzyme belongs to the glutamyl aminopeptidases. Its Mr of 320,000, obtained both by gel filtration and native protein PAGE, is somewhat higher than the Mr of glutamyl aminopeptidases from other sources, exclusive of the detergent solubilized enzymes from hog kidney and intestine, whose gel filtration behav- ior was changed by the presence of detergent (Danielsen et al., 1980; Benajiba and Maroux, 1980). EAPs seem to be very heterogeneous in size, with Mr spanning from below 100,000 for the enzyme from bacteria (Eriquez and Knight, 1980) and bovine kidney (Chulkova and Orekhovich, 1979) to 300,000 for the enzyme from pig kidney (Tobe et aL, 1980). This heterogeneity might stem from their glyco- protein and polymeric nature. Egg-white EAP has a dimeric structure like that of mammalian membrane- bound enzymes (Danielsen et al., 1980; Benajiba and Maroux, 1980). Its pI is within the acidic pH range,

300

/I I ~D 200 / z/z o

~O

~ 100 n

u.J

-6 -5 -4 -3 -2 log salt conc. ( M )

Fig. 6. The effect of metal ions on the activity of EAP from chicken egg-white. CaCI 2 (O), MnC12 (r-l), CoCI2 (0) , NiCI2 (A, solid line), ZnC12 (A, dashed line). The activities are shown relative to that

determined without added metal ions.

594 SINISA PETROVIC and LJUBINKA V1TALE

which is close to that of human serum and pla- cental EAP (Nagatsu et al., 1970; Yamada et al., 1988). A great similarity between egg-white, porcine kidney and human serum EAP exists in pH optimum and thermal stability which is enhanced by the presence of Ca 2+ ions (Tobe et al., 1980; Lalu et al., 1984). In contrast, egg-white enzyme is stable in the broad pH range, while porcine kidney enzyme loses its activity above pH 8.5 (Danielsen et al., 1980).

An interesting feature of egg-white EAP is that its activation energy decreases above 36°C. Since this is the temperature necessary for chicken embryo devel- opment, facilitated enzyme activity might indicate the enzyme's involvement in this process.

A majority of the so far described EAPs were shown to be metallo peptidases, inhibited by chela- ting agents and activated by Ca 2+ (Glenner and Folk, 1961; Nagatsu et al., 1970; Danielson et al., 1980; Tobe et al., 1980; Lalu et al., 1984; Yamada et al., 1988). The reactivation of EDTA-treated en- zyme was possible with either Ca 2+ or Mn 2+. There is, however, a different Mn 2+ relative efficiency reported for the porcine kidney (Danielsen et al., 1980) and for the egg-white EAP. Cysteine and serine residues do not seem to be essential for the enzyme activity since there was no strong inhibition by pHMB, DTNB and PMSF observed. The activity of EAP from egg-white was completely abolished by 10tiM NBS. This might suggest an essential role of Trp residue, but the inhibition could result from nonspecific reactions of NBS with Cys, Tyr, His and Met residues as well (Spande and Witkop, 1967).

The isolated enzyme has a strict specificity for ~-glutamyl or ~-aspartyl residue at the NH2-end of synthetic substrates and peptides. Hydrolysis of substrates with N-terminal neutral or basic amino acids reported for EAP from other sources (Danielsen et al., 1980; Yamada et al., 1988) was not observed with egg-white enzyme. Its Km for ~-Glu-pNan determined in the presence of Ca 2+ corresponds to the Km value of EAP from rat kidney (Hess, 1965), and Km for ~-Glu-2NA to those of EAP from pig kidney, human serum, and bacteria Neisseria meningitidis and Moraxel la urethralis (Danielsen et al., 1980; Lalu et al., 1984; Eriquez and Knight, 1980). Chicken egg-white EAP is distinct from the soluble aspartyl aminopeptidases found in dog kidney (Cheung and Cushman, 1971) and Salmonella typhimurium (Carter and Miller, 1984) which preferably cleave N-terminal aspartic acid residue from the corresponding substrates. Thus it was shown that unfertilized chicken eggs contain an active aminopeptidase, similar to mammalian mem- brane bound enzymes, which can participate in the degradation of storage proteins. However, owing to its high specificity, its role as a regulatory enzyme with an at present unknown substrate, cannot be excluded.

Acknowledgements--The excellent technical assistance of Mrs Ljerka Dolov~ak is gratefully acknowledged. This work was supported by grants from the Research Councils of SR Croatia and SFR Yugoslavia.

REFERENCES

Benajiba A. and Maroux S. (1980) Purification and charac- terization of an aminopeptidase A from hog intestinal brush-border membrane. Eur. J. Biochem. 107, 381-388.

Bradford M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt. Biochem. 72, 248-254.

Brenner M., Niederwieser A. and Pataki G. (1969) Amino acids and derivatives. In Thin-layer Chromatography (Edited by Stahl E.), pp. 730-785. Springer, Berlin.

Carter T. H. and Miller C. G. (1984) Aspartate-specific peptidases in Salmonella typhimurium: mutants deficient in peptidase E. J. Bacteriol. 159, 453-459.

Cheung H. S. and Cushman D. W. (1971) A soluble aspartate aminopeptidase from dog kidney. Biochim. biophys. Acta 242, 190-193.

Chiou T.-K., Matsui T. and Konosu S. (1988) Purification and properties of an aminopeptidase from mullet, Mugil cephalus, roe. Agric. Biol. Chem. 52, 235-242.

Chu T. G. and Orlowski M. (1985) Soluble metallo-endo- peptidase from rat brain: action on enkephalin-containing peptides and other bioactive peptides. Endocrinology 116, 1418-1425.

Chulkova T. M. and Orekhovich V. N. (1979) Molecular organization of aminopeptidase A from bovine kidney. Biokhimiya 44, 1539-1541.

Cornish-Bowden E. (1979) Fundamentals of Enzyme Kinet- ics, pp. 2627; 82-85. Butterworths, London.

Csernansky J. G., Zimmerman M. and Troll W. (1979) An elastase-like enzyme in the oocytes of Arbaeia punctulata. Devl Biol. 70, 283-286.

Danielsen E. M., Nor6n O., Sj6str6m H., Ingram J. and Kenny A. J. (1980) Proteins of the kidney microvillar membrane. Aspartate aminopeptidase: purification by immunoadsorbent chromatography and properties of the detergent- and proteinase-solubilized forms. Biochem. J. 189, 591~03.

Eriquez L. A. and Knight G. B. (1980) Aminopeptidases highly specific for glutamyl residues from Neisseria meningitidis and Moraxella urethralis. J. clin. Microbiol. 12, 667-671.

Glenner G. G. and Folk J. E. (1961) Glutamyl peptidases in rat and guinea pig kidney slices. Nature, Lond. 192, 338 340.

Harada M., Hiraoka B. Y., Fukasawa K. M. and Fukasawa K. (1984) Chemical modification of dipeptidyl peptidase IV: involvement of an essential tryptophan residue at the substrate binding site. Archs Biochem. Biophys. 234, 622~528.

Hess R. (1965) Arylamidase activity related to angiotensin- ase. Biochim. biophys. Acta 99, 316324.

Lalu K., Lampelo S., Numme!in-Kortelainen M. and Vanha-Perttula T. (1984) Purification and partial charac- terization of aminopeptidase A from the serum of preg- nant and non-pregnant women. Biochim. biophys. Acta 789, 324-333.

Lineweaver H. H., Morris J., Kline L. and Bean R. S. (1948) Enzymes in fresh hen eggs. Arch. Biochem. 16, 443-472.

Long C. (1961) Biochemists' Handbook, pp. 41. E. & F. N. Spon, London.

Mumford R. A., Hartman J. F., Ashe B. M. and Zimmer- man M. (l 981) Proteinase activities of the golden hamster eggs and cells of the cumulus oophorus. Devl Biol. 81, 332-335.

Nagatsu I., Nagatsu T., Yamamoto T., Glenner G. G. and Mehl J. W. (1970) Purification of aminopeptidase A in human serum and degradation of angiotensin II by the purified enzyme. Biochim. biophys. Acta 198, 255-270.

Pfleiderer G. (1970) Particle-bound aminopeptidase from pig kidney. Meth. Enzymol. 19, 514-521.

Glutamyl aminopeptidase 595

Spande T. F. and Witkop B. (1967) Determination of the tryptophan content of proteins with N-bromosuccin- imide. Meth. Enzymol. 11, 498-506.

Tefft P. M. and Bone L. W. (1985) Leucine aminopeptidase in eggs of soybean cyst nematode Heterodera glyeines. J. Nematol. 17, 270-274.

Tobe H., Kojima F., Aoyagi T. and Umezawa H. (1980)

Purification by affinity chromatography using amastatin and properties of aminopeptidase A from pig kidney. Biochim. biophys. Aeta 613, 459-468.

Yamada R., Mizutani S., Kurauchi O., Okano K., Imaizumi H., Narita O. and Tomoda Y. (1988) Purification and characterization of human placental aminopeptidase A. Enzyme 40, 223-230.