Studies on the N-acetyl-β-d-hexosaminidase B from germinating Lupinus luteus L. seeds I. Purification and characterization

110

Biochimica et Biophysica Acta, 1039(1990) 110-118

Elsevier BBAPRO 33657

Studies on the N-acetyl-fl-D-hexosaminidase B from germinating Lupinus luteus L. seeds I. Purification and characterization Istvfin P6csi, Lfiszl6 Kiss and Phi Nfinfisi Institute of Biochemistry, Lajos Kossuth University, Debrecen (Hungary)

(ReceivedAugust 1989) (Revised manuscript received19 February 1990) Key words: HexosaminidaseB; Enzymepurification The N-acetylofl-D-hexosaminidase B of germinating Luplnus luteus L. seeds (McFarlane et al. (1984) Phytochemistry 23, 2431-2433) was partially purified with a six-step purification procedure following extraction. This enzyme consists of one protein chain ( M r 69 000, as determined by SDS-PAGE and 62 500, as obtained by gel filtration on Bio-Gel P-60 Gel) and has a neutral isoelectric point (pl-- 7.05, as determined by chromatofocusing). Moreover, it was found to be very sensitive to low ionic strength, especially in the presence of different gels based on Sephadex. Considering the substrate specificity, the enzyme splits both p-nitropbenyl-2-acetamido-2-deoxy-fl-D-glucosaminide and -galactosaminide substrates, but lacks N,N '-diacetylchitobiase activity. A new mixed-substrate procedure was developed and is presented here to demonstrate that a common active site is responsible for the splitting of both synthetic substrates.

Introduction N-Acetyl-fl-D-hexosaminidase (N-acetyl-fl-D-hexosaminide N-acetylhexosaminohydrolase, EC 3.2.1.52) (hexosaminidase) occurs widely in the vacuolar system of various higher plant cells [1]. In particular, high concentrations of hexosaminidase activity are found in the aleurone grains of germinating seeds [2], suggesting a probable physiological function in the degradation of reserve glycoproteins [3]. Recently, evidence was found which demonstrates that acid glycosidase activities in imbibing cotyledons of several leguminous seeds can be attributed to rehydration of enzyme molecules formed during seed development, rather than to the de novo synthesis [4]. Moreover, no direct relationship could be found between the hexosaminidase activity level and the intensity of the reserve glycoprotein catabolism [4,5], which indicates that hexosaminidase may also take part in the degradation of other cellular glycoproteins. Abbreviations: PNP-GicNAc, p-nitrophenyl-2-acetamido-2-deoxy-flD-glucopyranoside; PNP-GalNAc, p-nitrophenyl-2-acetamido-2-deoxy-fl-D-galactopyranoside; PMSF, phenylmethylsulfonylfluoride; BSA, bovine serum albumin; SDS, sodium dodecyl sulphate; PBE, PolybufferExchanger. Correspondence: L. Kiss, Institute of Biochemistry,Lajos Kossuth University, Egyetemt6r 1, H-4010 Debrecen, Hungary.

On the other hand, some plant hexosaminidases also split chitin and chitin oligomers [6-10]. Thus, some participation in the process of chitin-elicited lignification cannot be excluded [10]. Beyond their physiological significance, plant hexosaminidases are very effective tools in the investigation of the oligosaccharide moieties of, for example, different glycoproteins and glycolipids [11]. In fact, this promising analytical applicability prompted many research groups to isolate more and more effective hexosaminidase preparations from different plant species, and to test their substrate specificity on a wide variety of biological materials [6,8,12-14]. Therefore, it is hardly surprising that while the actions of several fungal and animal hexosaminidases have been very intensively investigated [15-18], only a small amount of information is available on the mechanism of the analogous higher plant enzymes. Considering this problem, we purified and kinetically characterized the N-acetylfl-D-hexosaminidase B [19] of germinating lupin seed. Here, we report on the purification and the substrate specificity of this enzyme. Materials and Methods Chemicals

• DEAE-Sephadex A-25, CM-Sephadex C-50, phenylSepharose CL-4B, Polybuffer Exchanger 94 (PBE 94), Polybuffer 96, Polybuffer 74, Sephadex G-75 (Super-

0167-4838/90/$03.50 © 1990 Elsevier SciencePublishers B.V. (BiomedicalDivision)

111 fine) and Blue Dextran 2000 were purchased from Pharmacia (Uppsala, Sweden). Bio-Gel P-60 Gel (Fine) was from Bio-Rad (Richmond, U.S.A.). M r gel-filtration calibration kit (MS-II) and bovine serum albumin (BSA) were bought from Serva Feinbiochemica (Heidelberg, F.R.G.). p-Nitrophenol was from Fluka (Buchs, Switzerland) and phenylmethylsulfonyl fluoride (PMSF) was from E. Merck (Darmstadt, F.R.G.). p-Nitrophenyl-2-acetamido-2-deoxy-fl-D-glucopyranoside (PNP-GlcNAc) [20], p-nitrophenyl-2-acetamido-2-deoxy-fl-D-glactopyranoside (PNP-GalNAc) [21] and N,N'-diacetylchitobiose [22] were prepared in our own laboratory. Other chemicals, unless stated, were purchased from Reanal (Budapest, Hungary).

Plant material and optimization of the germination time For studying the changes in p-nitrophenyl-fl-D-glucosaminidase (PNP-GlcNAc-ase) and p-nitrophenyl-flD-galactosaminidase (PNP-GalNAc-ase) activities during germination, 5 g of lupin seeds obtained from local dealers (Vet~Smagtermeltet5 6s l~rt6kesit6 V~llalat) were germinated. Germination was carried out in Petri dishes which were placed in the dark at room temperature and irrigated daily with 1 ml of ion-free water up until the day of further processing. Intact cotyledons were removed immediately after imbibition and on the 2nd, 4th, 6th, 10th and 12th day of germination, and were cut into small pieces. One part (about 1 g) of this material was used for the determination of the dry matter content, while another fraction (3 g) was further homogenized in 6 ml of 0.1 M citric acid-sodium citrate buffer (pH 4.5; 0.003 M PMSF) with quartz sand in a mortar. After centrifugation (10 000 x g, 10 min), the crude extract was kept for 3 h at 0 ° C and then recentrifuged (15 rain). The resulting supernatant was used for enzyme activity, protein and dry matter content determinations. For enzyme preparation a larger amount of seed (about 50 g) was germinated for 6 days, essentially under the same conditions.

Enzyme purification, tissue fractionation and partial purification All operations were performed at 4 ° C, unless otherwise indicated, and all buffer solutions contained 0.003 M of NaN 3 as an antimicrobial agent. A crude extract from 50 g of cotyledons was centrifuged (10000 × g, 15 min) and pre-purified with two subsequent ammonium sulphate precipitations (80 and 100% saturations). The precipitated protein was then redissolved in 50 ml of 0.1 M citric acid-sodium citrate buffer (pH 4.5) and was dialyzed against the same buffer overnight. The precipitated reserve proteins [2] were centrifuged off and the supernatant was dialyzed

twice against 3 litres of 0.1 M Tris-HC1 buffer (pH 7.75).

Ion-exchange and hydrophobic interaction chromatography The dialyzate obtained from the previous step was passed through a DEAE-Sephadex column (3 × 45 cm), which had been equilibrated with 0.1 M Tris-HC1 buffer (pH 7.75). The effluent fractions containing unbound hexosaminidase activity were then pooled. In order to complete the separation of the hexosaminidase B from the B1 isoenzyme [19] the enzyme preparation was once more passed through a DEAESephadex A-25 column (3 × 22 cm) under the same conditions. The hexosaminidase B was then dialyzed against 8 litres of 0.05 M NaHEPO4-NaEHPO4 buffer (pH 6.0, 0.15 M NaC1) and was loaded on a CM-Sephadex C-50 column (3 × 25 cm) previously equilibrated in the same phosphate buffer. The column was washed with 200 ml of the same sodium phosphate buffer and was eluted with 1 litre of a linear gradient of 0.15-0.5 M NaC1 in the equilibrating buffer. The flow rate was 75 ml per h and the hexosaminidase was recovered at 0.23 M NaC1. The fractions (about 15 ml each) containing more than 1.0 nkat of PNP-GlcNAc-ase activity were pooled and directly applied to a phenyl-Sepharose CL-4B column (1.5 × 8 cm) that had been equilibrated in 0.05 M NaHEPO4-NaEHPO 4 buffer (pH 6.0; 0.23 M NaC1; flow rate 150 ml/h). After washing the column with 50 ml of the equilibrating buffer the enzyme was eluted with 100 ml of a gradient of 0-80% (v/v) ethylene glycol in the same buffer. The fractions having the most intense enzyme activity (between 45 and 50% of ethylene glycol, calculated from conductivity measurements) were pooled and either used directly for molecular mass determination or dialyzed against 0.05 M sodium phosphate buffer (pH 6.0, 0.15 M NaC1) and utilized in the kinetic experiments. On the other hand, we were unable to dialyze this enzyme preparation against either 0.1 M citric acidsodium citrate buffer (pH 4.5) or 0.1 M Tris-HC1 buffer (pH 7.75) as almost complete inactivation could be observed in both cases.

lsoelectric point determination To determine the isoelectric point of the hexosaminidase isoenzymes, a 20 ml aliquot of the crude extract following exhaustive dialysis against 0.1 M citric acid-sodium citrate buffer (pH 4.5) was further dialyzed against 0.025 M Tris-acetic acid buffer (pH 8.3) and applied to a PBE 94 column (1 × 17 cm) equilibrated with the same buffer. The pH gradient was developed by 250 ml of ll-fold diluted Polybuffer 96/Polybuffer 74 mixture (3:7, v/v, pH 5.0), according to the Phar-

112 macia protocols. Fractions of 2.5 ml were collected at a flow rate of 30 m l / h .

Molecular mass determination by gel filtration (1) A 50 ml sample of the enzyme preparation from CM-Sephadex C-50 chromatography containing 5.1 nkat of PNP-GlcNAc-ase activity was dialyzed against 0.05 M NaH2PO4-Na2HPO 4 buffer (pH 6.0, 0.15 M NaC1) and loaded on a column (1.5 × 8 cm) also filled with CM-Sephadex C-50 gel and equilibrated with the previous phosphate buffer. The bound hexosaminidase was recovered in a vol. of 5 ml by increasing the NaC1 concentration to 0.4 M in one step. 4 ml of this concentrated enzyme sample (protein content was 270/,g, possessing 2.6 nkat of PNP-GlcNAc-ase activity) was applied to an analytical Sephadex G-75 column (2 × 40 cm, flow rate 65 m l / h ) equilibrated and eluted with 0.05 M sodium phosphate buffer (pH 6.0, 0.4 M NaCI). Fractions of 2.2 ml were collected. (2) A 1 ml aliquot of the combined fractions from phenyl-Sepharose CL-4B chromatography containing 35/,g of protein and 0.5 nkat of PNP-GlcNAcase activity was loaded on a Bio-Gel P-60 Gel column (1.5 × 25 cm) which had been equilibrated in 0.05 M sodium phosphate buffer (pH 6.0, 0.23 M NaC1) containing 10% ( v / v ) ethylene glycol. The elution was carried out at a flow rate of 10 m l / h , collecting fractions of 1.5 ml. SDS-polyacrylamide gel electrophoresis (SDS-PA GE) Gel electrophoresis was performed according to Laemmli [23] on 12-14 cm discontinuous slabs and M r determination was carried out by comparison with marker proteins. Protein determinations Protein was determined either by the method of Lowry et al. [24] using bovine serum albumin as a standard or, in particular for dilute solutions, by monitoring the ultraviolet absorption at 280 nm [25]. Enzyme assays and estimation of kinetic parameters All measurements were carried out in 0.1 M citric acid-sodium citrate buffer (pH 4.50) at 37 ° C. The total reaction vol. was 1 ml, 5 min preincubation was used and the reaction was initiated, in all cases, by addition of the enzyme. The reaction was quenched by the addition of 2.0 ml of 0.2 M sodium borate buffer (pH 10.0) and the quantity of the liberated p-nitrophenolate ion was determined spectrophotometrically at 400 nm using calibration curves. In the case of crude extracts, blank solutions consisted of the same components as reaction mixtures, but the enzyme preparation was added after the sodium borate buffer. Substrate blank solution was used in all other cases.

The standard reaction mixtures used for measuring both the PNP-GlcNAc-ase and PNP-GalNAc-ase activities in the subsequent purification steps, as well as in the p I and M r determinations, contained 1.0 mM of the appropriate p-nitrophenylglycoside. In substrate specificity investigations the reaction velocity of the hydrolysis of both PNP-GlcNAc and PNP-GalNAc was determined at seven different substrate concentrations in the range of 0.5-5.0 g m and at three different reaction times, namely at 3, 4 and 5 rain for PNP-GlcNAc and 6, 8 and 10 min for PNP-GalNAc. The liberation of p-nitrophenol was found to be linear in these intervals and the kinetic parameters ( g m , Vmax) were calculated by non-linear least-squares fit of the experimental data to the classic Michaelis-Menten equation [26,27]. To estimate the precision of the curve fit, the average difference (A) and the average relative difference ( A ' ) (Eqn. 1) in the measured and calculated rate values were calculated for each case [27].

f •

(In( 0~,¢, i///)obs,i ))2

A'

= V i=1

-~

(1)

where n represents the number of experimental points. The accuracy of the fitted parameters was characterized by the standard errors (S.E.) [27]. The N, N'-diacetylchitobiase activity was also tested in a reaction mixture of 1 ml containing 10 mM of the disaccharide. The possible changes in the saccharide composition were examined at every 30 min of the incubation time (5 h) on a Kieselgel 60 F 254 thin layer (E. Merck), in a solvent system of 1 - b u t a n o l / a c e t o n e / water ( 5 : 4 : 1 , v/v). The R F values were 0.46 for N, N'-diacetylchitobiose and 0.65 for N-acetyl-D-glucosamine.

Mixed-substrate experiments - theory If a system contains only one single-substrate enzyme which has one active site for the splitting of both substrates S1 and S2 (Scheme I), the total initial velocity of the reaction can be calculated on the basis of Eqn. 2

[281. E + S a ~ ES 1 ---,E + p I + P E + $2 ~ ES2 ---*E + P2 + P

Scheme I VaKz[S1] + V2Kl[S21 v = K 1 K z + K1[$2] + K2[$1]

(2)

where V1, V2 and K1, K 2 a r e the individual maximal velocity values and the Michaelis constants for Sa and S2 substrates, respectively, which were determined from independent experiments.

113 On the basis of Eqn. 2 it is possible to decide from one set of experimental data whether the two substrates are competitive inhibitors [6,10,30-32], however, this procedure gives reasonably reliable results only in the case of homogeneous enzyme preparations [33]. Furthermore, Eqn. 2 can be linearized maintaining either the mole fractions of the substrates [34] or the sum of K1152] + K z [ S 1 ] [29] as constant. Now assuming that Va is greater than V2 and that [Sa] is constant, the first and second derivatives of v with respect to [$2] are zero where [$1] = V 2 K 1 / ( V a Vz); that is, the predicted reaction velocity will be Vz independent of [$2]. This provides a new, simple simulation procedure to demonstrate the presence of a common active site. If two different active sites exist for the substrates S a and Sz, then two additional reaction routes should be taken into consideration (Scheme II) [29]. ES1 + S'2 # ES1S2 ~ ES1 + Pz + P ES2 + $1 # ES2S1 "') ES2 + P1 + P Scheme II

If the two substrates influence both each other's binding and splitting, the appropriate kinetic parameters for the formation and decomposition of ES~S2 and ES2S t complexes will be V3, V4 and K3, g4, respectively. Considering that K I K 3 = K z K 4, and assuming that [Sa] = V z K 1 / ( V 1 - V z ) , the v =f([S2])function will be a hyperbola with a general form of o = (a + b[Sz])/ (c + d[S2] ). The a, b, c and d parameters are summarized in Table I. As can be seen, if the two substrates have no effect on either the binding and splitting of the other (Case 1) or influence only each other's binding (Case 3) the v =f([Sz]) function is always a hyperbola. On the other hand, if they influence each other's decomposition (Cases 2 and 4), v will be equal to Vz independently of [$2] in the extreme case when V3 + V4 = V2.

Generally, Eqn. 2 can be used as a model at any fixed [Sa] or [$2]. In the cases, however, the experimental points should match a hyperbola rather than a straight line. Nevertheless, the analysis of the A and A' values indicates whether the substrates are split at the same active site or not. In mixed substrate experiments both the [$1] and [$2] values were chosen from the ranges of 0.5-5.0 Ka, and 0.5-5.0 K z, respectively. Results Optimization

of the extraction

Physiological studies revealed that the dry matter content of the cotyledons decreased continuously during the entire period of observation (from 42 to 26%), while that in 50 #1 of the extract fell sharply on the first 2 days (from 7 to 2 mg) and thereafter stabilized at about 2 mg. In the same interval the extractable protein content in the same volume decreased up to the 6th day (from 2.7 to 1.1 mg) and then showed mild elevation to 1.8 mg on the 12th day. Regarding the extracted enzyme activities and specific activities, the former quantities showed an increasing tendency over time, while the later changed according to maximum curves, with an absolute maximum at day 6. Considering this we used 6-day-gerrninated seedlings for hexosaminidase preparation. Moreover, it is noteworthy that both the enzyme activities and the specific activities run parallel with each other during the period of observation. Purification

After extraction a six-step purification procedure was used consisting of ammonium sulphate precipitation, acid dialysis, two DEAE-Sephadex chromatographies, CM-Sephadex C-50 chromatography and phenyl-Sepharose CL-4B chromatography (Table II).

TABLE I The parameters, a, b, c and a~ for the v = (a + b[S2]) / (c + diS2] ) type hyberbolas in the case o f I S 1] = V2 K 1 / ( V 1 - V2) in the presence o f two active centres

Case

1

2

3

4

Conditions a

Parameters

v = V2

a

b

c

d

V1=V4, V 2 = V 3 K 1 = K4, K2 = K 3

V2K2

2 V2

K2

1

[s21 = 0

VI"7~V4, V2"-#V3 g I = K4, g z = K 3

1111121(2

½ ( V l + ½ + V4 - V2)

V1K2

vl

( v3 + v4) = v2

K 1 ~ K 4, K2 ~ K 3

V1V2K2K3

V2(V1K 3 + VIK 2 + V2K 2 - V2K3)

VIKzK3

V1K 3 + V2K2 - V 2 K 3

[s:]=o

V 1 * V 4 , V24:V3 K 1 --# K4, K 2 ~ K 3

V1VzK2K3

V2(V1K 3 + V3K z + V4K 2 -

VIK2K 3

V I K 3 + V2K 2 - V 2 K 3

( v~ + v4) = v2

E=v,,v2=v3

a The classification was made as described by Keleti et al. [29].

VzK3)

114 1

The separation of the hexosaminidase B from other isoenzymes [19] was achieved by the application of DEAE-Sephadex A-25 chromatography. Hexosaminidase B was not bound by the column, while the other isoenzyme forms were retained. (Hexosaminidase B1 and A could be recovered at 0.05 and 0.85 M NaC1, respectively.) In the following purification steps the PNP-GlcNAcase and PNP-GalNAc-ase activities co-purified and their ratio remained constant (Table II). This procedure resulted in a partially purified enzyme preparation (Fig. 1) which could be stored for weeks at - 2 0 °C without any loss of activity, and also could be dialyzed against 0.05 M sodium phosphate buffer (pH 6.00, 0.15 M NaC1).

2

3

4

5 M r ( l O -3)

6

7

8 M r ( l O -3)

lsoelectric point and molecular mass determinations

As shown in Fig. 2, three major hexosaminidase peaks could be observed during chromatofocusing of a lupin seed extract. The isoenzymes could be identified in order of decreasing isoelectric points; hexosaminidase B (pI = 7.05), Bt (pI = 6.65) and A (pI = 5.45) [19]. On the other hand, another minor hexosaminidase between hexosaminidase Bt and A (pI = 6.00) was also probably present. Moreover, regarding the first activity peak (hexosaminidase B) a significant zone broadening can be seen, which means that the ionic strength of the eluting buffer mixture was high enough to remove the enzyme from

Fig. 1. SDS-polyacrytamide gel etectrophoresis of hexosaminidase B preparations from germinating lupin seeds obtained in successive purification steps. The applied samples, the protein content of which is indicated in parentheses, were dialyzed against 0.1 M Tris-HC1 buffer (pH 7.75), lyophilized and dissolved in the sample buffer. Lane 1, acidic dialysis (45/~g); lane 2, 1. DEAE-Sephadex A-25 chromatography (52 /~g); lane 3, 2. DEAE-Sepahdex A-25 chromatography (32 ttg); lane 4, CM-Sephadex C-50 chromatography (14 ~g); lanes 5 and 7, phenyl-Sepharose CL-4B chromatography (11 #g); lane 8, Bio-Gel P-60 gel filtration (protein could not be determined); and lane 6, M r determination standards, which were: bovine serum albumin (67000), ovalbumin (45000), chymotrypsinogen A (25000) and myoblobin (17 800).

TABLE II

Purification of the N-acetyl-fl-D-hexosaminidase B from germinating L luteus seeds Purification step

Vol. (ml)

Protein b (mg)

Activity (nkat) (yield (%))

Activity ratio PNP-GlcNAc-ase

Spec. activity (mkat/kg) (purification (fold))

PNP-GlcNAc

PNP-GalNAc

PNP-GalNac-ase

PNP-GlcNAc

PNP-GalNAc

Extraction a

54

2468.8

399.3 (100)

163.4 (100)

2.444

0.16 (1)

0.066 (1)

(NH4)2SO 4 precip. acidic dialysis

96

863.1

310.2 (77.7)

120.2 (73.6)

2.581

0.36 (2.23)

0.14 (2.12)

1. DEAE-Sephadex A-25 chromatography

114

229.9

163.2 (40.9)

86.3 (52.8)

1.891

0.71 (4.41)

0.38 (5.71)

2. DEAE-Sephadex c A-25 chromatography

141

34.0

61.9 (15.5)

32.3 (19.8)

1.916

1.82 (11.3)

0.95 (14.5)

CM-Sephadex C-50 chromatography

213

2.58

22.1 (5.53)

11.6 (7.10)

1.905

8.57 (53.2)

4.50 (68.5)

22

0.40

8.57 (2.15)

4.65 (2.85)

1.843

21.4 (132.9)

11.6 (176.6)

phenyl-Sepharose CL-4B chromatography

50 g of cotyledons from 6-day-old seedlings were extracted. b During extraction and acidic dialysis the protein was determined by the method of Lowry et al. [24]. In all other cases the method of Warburg and Christian [25] was used. c The enzyme preparation from the 1. DEAE-Sephadex A-25 chromatography was further processed in two separated parts. The data from this step represent the sum of two runs. a

115 the PBE 94 gel at a p H of about 8. In a parallel experiment, where a similar sample in 0.1 M Tris-HC1 (pH 7.75)/0.025 M imidazole-HC1 (pH 7.50) buffer mixture (1 : 2, v / v ; p H 7.62; I = 0.05 M) was loaded on the same column, neither hexosaminidase B nor B1 bound to the ion exchanger equilibrated in the same buffer mixture. Furthermore, we could not dialyze such an extract portion against 0.025 M imidazole-HC1 buffer because protein precipitation and enzyme inactivation could be observed. The relative molecular mass of the enzyme was determined by both gel filtration (Fig. 3) and SDS-PAGE (Fig. 1). The gel filtration on a Bio-Gel P-60 column in the presence of 10% ( v / v ) of ethylene glycol gave an electrophoretically homogeneous enzyme preparation (Fig. 1, line 8; Mr 69000) with a yield of 63.5% for PNPGlcNAc-ase and 74.6% for PNP-GalNAc-ase activities. Both activities ran together and reached a common maximum at an Mr values of 62 500. Unfortunately, any attempt to remove the ethylene glycol by dialysis led to almost complete inactivation. As can be seen, the M r values given by SDS-PAGE and Bio-Gel P-60 gel filtration are in accordance, while on a Sephadex G-75 column a significant retardation (apparent M r 15 300) could be observed with a recovery of 22.3% for PNP-GlcNAc-ase and 23.7% for PNPGalNAc-ase activities. Moreover, the application of lower ionic strength resulted in complete inactivation at different p H values (pH 7.75, 6.00 and 4.50). During gel filtrations the effluents had no ab-

5.0

N'~x1

V

5.0

Bio -(Set P-60

togbtr

ex8

~~., , ~

t,..O

Sephadex G-75

IogM~t~.5

2

s 4.0

1.0

'

2.0

V,/Vo

3.0

Fig. 3. Gel filtration and Mr determination of hexosam~dase B. The applied Mr standards were: 1, bovine serum albumin (67000); 2, ovalbumin (45000); 3, chymotrypsinogenA (25000); 4, myoglobin (17800); and 5, cytochrome c (12300).

sorbance at 280 nm and also no protein could be detected by the method of Lowry et al. [24].

Substrate specificity investigations

pH 9

7 1.5 6

=

c Azao

A

008

1.0..~_ ~ ..>_ o

GO6 .

0.5 E

o.o~,

~:

t,~

0.02

0.00 -

0

Yellow lupin hexosaminidase B catalyzed the hydrolysis of both PNP-GlcNAc and PNP-GalNAc, and the dependence of the reaction rate on the substrate concentration obeyed the Michaelis-Menten equation in both cases. On the other hand, in the case of N, N'-diacetylchitobiose, no N-acetyl-D-glucosamine liberation could be observed, even during an incubation time of 5 h. The enzyme bound PNP-GlcNAc less tightly and split it more effectively than PNP-GalNAc. The calculated kinetic parameters: (i) for PNP-GlcNAc were, Km,1 = (9.4 + 0.3)" 10 -4 M, Vm~x,1 = (4.1 + 0.1)" 10 -2 k a t / k g (A = 1.13 • 10 -3 kat/kg, A' = 0.055) and Vmax,1/Km,1 = 4 3 . 6 k a t / k g per M; and (ii) those for PNP-GalNAc; Km, 2 = (5.1 + 0.2)" 10 -4 M, Vmax, 2 = (1.32 + 0.04). 10 -2 k a t / k g (A = 4 . 2 . 1 0 -4 kat/kg, A' = 0.053) and Vm~x,z/Km, 2 = 25.9 k a t / k g per M.

0.0

50 Effluent

100 volume

150

200

(rot)

Fig. 2. Chromatofocusing of the fl-N-acetyl-D-hexosaminidase isoenzymes from yellow lupin seeds, o , pH; O, protein (A2s0); I1, PNPGlcNAe-ase activity; and D, PNP-GalNAc-ase activity. A p H gradient between pH 8 and 5 was applied (P), as described in the text.

Mixed-substrate experiments Mixed-substrate experiments were performed at six different PNP-GlcNAc and PNP-GalNAc concentrations, obtaining 36 data. The representation of the experimental data as a function of the PNP-GlcNAc

116 t,.0

--

3.0

--

pendent variable, also resulted in a series of hyperbolas, which were concave downward below a PNP-GlcNAc concentration of 4 . 4 6 . 1 0 - 4 M and concave upward above this concentration. When [PNP-GlcNAc] was equal to 4.46-10 -4 M, the experimental points correlated well with the v = Vmax.2 line. It is remarkable that the A and A' values for this simulation (A = 1.06.10 -3 k a t / k g , A' = 0.049) were very similar to those gained in parallel substrate specificity investigations.

(~) (~.)

"~

(51

(6)

-- 20

(:3

1.0

Discussion 0.0

'

0.0

'

1.0 [ PNP - GIcNAc ]

2.0

'

( mid )

3.0

Fig. 4. Mixed-substrate experiments with PNP-GIcNAc and PNPGalNAc. The concentration of PNP-GicNAc was changed between 0 - 2 . 5 . 1 0 -3 M. The applied PNP-GalNAc concentrations and the theoretically expected hyperbolas (Eqn. 2) were as follows: ©, no PNP-GalNAc, (1); O, 2.5"10-4 M (2); n, 5.0.10 - 4 M, (3); II, 7.5.10 -4 M (4); o, 1.0.10 - 3 M, (5); and O, 1.5.10 - 3 M, (6). The experimental data gained in the case of 4.46.10 - 4 M of PNP-GlcNAc are not shown.

concentration at constant [PNP-GalNAc] values is shown in Fig. 4. As can be seen, the experimental points show a good correlation, with a series of hyperbolas intersecting each other at the point where [PNPGlcNAc] = Vmax,2Km, 1 / ( V m a x , 1 - Vmax 2) = 4.46 • 1 0 - 4 M and v = Vm~x,2 = 1.32" 10 -2 k a t / k g , as calculated according to Eqn. 2. The reverse plotting of the same set of experimental data (Fig. 5), where the [PNP-GalNAc] was the inde-

4.0

3

.

0

~

(1~

2.0

-

,2,

/

"

~ •

o

"-

~

<31

~

u

1.0

0.0 ~ O.O

1.0

[ PNP

- GaINAc

]

(51 (6) 1/")

2.0 ( mid )

Fig. 5. Mixed-substrate experiments with PNP-GlcNAc and PNPGalNAc. The concentration of PNP-GalNAc was changed between 0-1.5.10 -3 M. The applied PNP-GIcNAc concentrations and the hyperbolas calculated from Eqn. 2 were as follows: C,, no PNPGlcNAc, (6);., 2.5.10 -4 M, (5); D, 4.46-10-4 M, (4); II, 1.0.10 -3 M (3); o, 1.5-10-3 M (2); and O, 2.5-10-3 M (1).

Acid hydrolases of legume cotyledonary cells play a very important role in the mobilization of the reserves of the protein bodies [35,36]. The hexosaminidase content of the germinating seeds together with other acid hydrolases concentrate mainly in the aleurone grains, so that a physiological function in the degradation of the oligosaccharide moieties of the reserve proteins seems to be obvious [2,3]. McFarlane and his co-workers [19] have reported on the presence of three distinct hexosaminidases in yellow lupin seeds. Our investigations support this finding, but the presence of other minor hexosaminidases cannot be excluded (Fig. 2). Physiological studies revealed that in the first 2 days of germination a significant decrease could be observed in both the extractable protein content and the dry matter content of the extract, which indicates rapid rehydration processes in seed tissues. Moreover, both PNP-GlcNAc-ase and PNP-GalNAc-ase activities increased continuously and in parallel during germination, while the specific activities reached their maxima simultaneously on the 6th day. These results suggest that the ratio of the different hexosaminidase isoenzymes probably remained constant during the 12-daylong observation time. Following the further purification of the crude extracts with a six-step procedure (Table II), which resuited in a partially purified hexosaminidase B preparation (Fig. 1), an additional step, gel filtration on Bio-Gel P-60 Gel, gave an electroporetically homogeneous enzyme preparation. However, this enzyme preparation was very sensitive to dialysis and therefore could not be used in further investigations. In parallel, the enzyme was very sensitive to low ionic strength throughout the purification, e.g., crude extract could not be dialyzed against 0.025 M imidazole-HC1 buffer (pH 7.50) and an irreversible adsorption could be observed on a DEAE-Sephadex A-25 column if the concentration of the Tris-HC1 buffer (pH 7.75) was decreased to 0.01 M. On the other hand, hexosaminidase B did not bind either on a PBE 94 column at p H 7.62 if the concentration of the applied

117

buffer mixture was 0.05 M, or on a DEAE-Sephadex A-25 column at pH 7.75 even in 0.04 M Tris-HC1 buffer. Furthermore, the ionic strength of the ll-folddiluted Polybuffer mixture was high enough to initiate the elution of the enzyme from PBE 94 at pH 8.3 (Fig. 2), and hexosaminidase B bound very tightly to phenylSepharose CL-4B column. Finally, it is worth mentioning the considerable retardation and inactivation on a Sephadex G-75 column eluted with 0.05 M sodium phosphate buffer (pH 6.00, 0.4 M NaC1) (Fig. 3). Considering these results we can conclude that only few charges, together with wide hydrophobic regions, must be present on the surface of the enzyme. Nevertheless, this tendency for irreversible adsorption at low ionic strength is not unique among plant hexosaminidases. Mitchell and his co-workers [37] found that N-acetyl-fl-D-glucosaminidase from malted barley inactivated significantly as it was passed through a DEAE-ceUulose column. The same was also reported by Neely and Beevers [5] on the analogous Pisum sativum enzyme. Comparing the M r values determined by SDS-PAGE (M r 69000, Fig. 1) and gel filtration on Bio-Gel P-60 Gel (M r 62 500, Fig. 3) we can establish that they are in good accordance, that is, the enzyme consists of a single protein chain. On the other hand, each M r value is significantly higher than that reported previously by McFarlane et al. [19] (M r 40000, determined by SDSPAGE). However, interestingly, essentially the same M r value (66 000-67 000) was obtained with three independent methods for the A isoenzyme [19]. Substrate-specificity investigations revealed that the enzyme catalyzes the hydrolysis of both PNP-GlcNAc and PNP-GalNAc. In detail, PNP-GlcNAc bound less tightly to the active centre than did PNP-GalNAc, while the Vm~ value and the catalytic efficiency [38] were higher for PNP-GlcNAc. Similar kinetic properties were found for many hexosaminidases isolated from different species, e.g., from the legume Canavalia ensiformis [6], the fungi Tremella fuciformis [39] and Paecilomyces persicinus [40], the mollusc Helocella erceratum [41], the ascidian Halocynthia roretzi [42], the slug Arion rufus [43], the nematoda Turbatrix aceti [17] and the silkworm Bombyx mori [44]. Although previously many papers reported on the ability of several hexosaminidases to liberate N-acetylD-glucosamine monomers from chitin and different chitin oligomers [6-10,17,39,44,45], the lupin hexosaminidase B lacks entirely N, N'-diacetylchitobiase activity, suggesting that a possible protective function against filamentous fungi [10] can be excluded in this case. Mixed-substrate experiments (Figs. 4 and 5) persuasively demonstrated that one catalytic site exists on the enzyme for the splitting of both synthetic substrates. Experimental points were well-matched with a series of hyperbolas calculated from independently determined

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43 Villa.r, E., Cabezas, J.A. and Calvo, P. (1984) Biochimie 66, 291304. 44 Koga, D., Nakashima, M., Matsukura, T., Kimura, S. and Ide, A. (1986) Agric. Biol. Chem. 50, 2357-2368. 45 Cohen, R.J. (1986) Plant Sci. 43, 93-101.