Factors affecting l -glutaminase activity in soils

Soil Bid. Biochem. Vol. 23. No. 9. pp. 8X-8879, 1991 Printed in Greal Britain. All rights -al

FACTORS

Copy@t

AFFECTING

L-GLUTAMINASE IN

0038-0717/91 s3.00 + 0.00 (9 1991PcrgamoaPreu pk

ACTIVITY

SOILS

W. T. FRANKEN~ERGER JR* and M. A. TABATALU~ Department of Agronomy. Iowa State University, Ames, IA 50011. U.S.A. (Accepted 25 February

1991)

Summary-L.-Glutaminase activity in soil protile samples decreased with sample depth. The relationship between L-glutaminasc activity and organic C. using the pooled data (33 samples) from five soil profiles, was highly correlated (r = 0.92**). The soil properties that related to the amounts of t-glutaminasc activities in 25 surface soils included organic C (r = 0.79**) and total N (r = 0.76**). There was no significant relationship between r-glutaminasc activity and pH. percentage of clay or sand. There was, however, a significant correlation between L-glutaminase activity and amidasc (r =0.82**). urcasc (r = 0.78.‘) and L-asparaginax (r = 0.92**) activities in the surface samples studied. Most of the 21 trace elements, I2 herbicides, two fungicides and two insecticides studied inhibited the activity of t&ttaminasc. but the degree of inhibition varied among the soils. When the trace elements were compared by using 5 pmol-’ g-t soil, the average inhibition of L-glutaminasc in three soils showed that Ag(I), Hg(lI). Sn(ll). Cr(IB). Ti(IV) and W(V1)were the most effective inhibitors (av. >25%). The other trace elements studied were lesseffective inhibitors (av. < 25%). When the pesticides were compared by using IO fig of active ingredient g-r soil, the average inhibition of L-glutaminax activity in three soils ranged from 4% with Merpan to 19% with Malaspray.

INTRODUCIION

Knowledge of the rclativc effects of trace clcments and pcsticidcs on L-glutaminase activity is important because this cnzymc is highly sensitive to trace quantities of metal ions and possibly pesticides, and because its substrate, t.-glutamine. is hydrolyzed to a form of nitrogen that is available to plants. We developed a method for assay of L-glutaminase activity in soils and systematically studied the factors affecting the release of NH: from t.-glutamine added to soils (Frankenberger and Tabatabai. 1991~). We have determined the distribution of L-glutaminase activity in soil profile samples and tested the effects of 21 trace elements, 12 herbicides, two fungicides and two insecticides on its activity. The term “trace element” is used here to refer to elements that are, when present in sufficient concentration, toxic to living systems. MATERIALS AND METHODS

A total of 26 Iowa surface soil samples, selected to obtain a wide range in pH (4.6-8.3). organic C (0.43-5.32%). total N (0.041-0.426%) and texture (4-45% clay and l-93% sand) were used. The samples were sieved (2 mm screen) in the field-moist condition, air-dried by spreading the soils in a thin (c 2 cm) layer on clean paper, and left to dry at laboratory temperature (22°C) for 2 h. The samples were stored in tightly-sealed glass containers. *Present address: Department of Soil and Environmental Scicnccs. University of California, Riverside, CA 92521. U.S.A. tAuthor for correspondence.

The profile samples were selected to give a range in organic C and L-glutaminase activity. Of the soil profiles studied, Clarion, Nicollet and Marshall soils were from cornfields, whereas Webster and Marna were from soybean fields, that had been cultivated before samples were taken. Before use, each sample was air-dried and crushed ( < 2 mm). A subsample of each soil was ground (150 pm) for determination of organic C. Organic C, total N and percentage of clay and sand were determined as described by Frankenberger and Tabatabai (198lb). The following soil enzymes were assayed in this investigation: t_-glutaminase (Frankenberger and Tabatabai. 1991~). L-asparaginase (Frankenberger and Tabatabai. 1991a), aliphatic amidase (Frankenberger and Tabatabai, 1980) and urease (Tabatabai and Bremner, 1972). The trace elements used were Fisher certified reagent-grade chemicals (see Table 2). The pesticides used included herbicides, fungicides and insecticides. The active ingredients and trade and common names of the pesticides studied were reported by Frankenberger and Tabatabai (1981 b). In testing the effects of trace elements and pesticides on r-glutaminase activity, a Sg soil sample in a SO-ml volumetric flask was treated with I ml of solution containing either 25pmol of trace element or 50~8 of active ingredient of pesticide. This solution was added dropwise to moisten the whole soil sample. After 30min of equilibration. t.-glutaminase activity was assayed (Frankenberger and Tabatabai, 1991~). The results of t.-glutaminase activity from trace element- or pesticide-treated soils were compared with those obtained with 5 g of soil treated with 1 ml of water. All results reported are averages of duplicate determinations calculated 875

876

W.

T. FLUWSBERCERJR and M. A. TAUTAM

Tabk I. Distribution of L-glutarninase activity and organic C in lckctcd Iowa soil profik sam~ks

Clarion Depth sample (cm)

DC*

GAt

O-15 15-M 3045 45-60 60-75 75-90 90-10s 105-I I5

1.93 I.51 I.11 0.62 0.40 0.30 0.16 0.16

139 58 39 36 40 39 35 29

Nicolkt

Maraa

IL=7

Webster

OC

GA

GC

GA

OC

GA

DC

GA

3.07 1.36 0.93 0.54 0.32 0.18 0.16 ND;

256 162 63 48 33 25 I8 ND

4.19 2.24 I.10 0.76 0.43 0.34 ND ND

340 170 42 20 29

2.19 2.11 1.40 1.04 0.68 0.41 ND ND

110 75

3.14 1.44 0.90 0.61 0.37 0.20 ND ND

316 85 50 I5 I6

N: ND

:: 2 N: ND

N2: ND

lGC, Organic carbon (%). tGA. L-plutaminase activity (pg NH,-N rclcarcd g-’ soil 2 h-l). :NOI determined

on an oven-dry basis. moisture being determined from loss in weight after drying at IOYC for 24h. RESULTS AND DISCUSSION Distribution

of L-glutaminase

activity

in soil profiles

t-Glutaminase activity and organic C content in soil samples decreased considerably with depth (Table I). Enzyme activities in soil profiles usually decrease with sample depth and this decrease is accompanied by a decrease in organic matter content (Khnziev and Burangulova. 1965; Skujins, 1967; Tabatabai, 1977; Frankcnberger and Tabatabai. l98la). Statistical analyses showed that thcrc was relationship between t-glutaminase significant activity and organic C in the following soil profile samples: Clarion (r = 0.82+)+ Nicollct (r = 0.98+‘), Marna (f = 0.99”). Marshall (I = 0.96+*) and Webster (r = 0.98++). The relationship between L-glutaminase activity and organic C of the pooled data (33 samples) of all the soil profiles examined was highly correlated (r = 0.92+*). Statistical analyses of the relationship between organic C and t-glutaminase activities in 25 surface soils tested showed (Fig. 1) that L-glutaminase activity was sig-

600

(

/

nificantly and positively correlated with organic C (t = 0.79.‘). t_-Glutaminase was significantly correlated with total N (r = 0.76**) in the surface soils studied (Fig. 2). This relationship was expected because of the involvement of t-glutaminase in N mineralization of soils. There was no significant correlation between percentage of clay, sand nor pH. The poor relationship between L-glutaminase activity and clay was similar to that reported for t_-asparaginase (Frankcnberger and Tabatabai, 199lb). and is somewhat surprising because most soil enzymes arc thought to be incorporated into a three-dimensional network of clay and humus complexes where they can continue to exhibit activity indcpendcntly from that of microbial populations (Ramirez-Martinet and McLarcn. 1966). There was, howcvcr, a significant relationship between t-glutaminasc activity and amidase (r = 0.82+*), urease (r = O-78*+) and L-asparaginase (r = 0.92++) activities in the 26 surface soils studied (Figs 3-5). The significant correlation between these enzymes is not unusual because similar relationships have been reported for other soil enzymes. For example, Speir (1977) found that soil arylsulfatase activity was significantly correlated with phosphatase and urease activities.

I

700

600 500

t

0

t

--I 4.0

5.0

6.0

ORGANIC c (%)

Fig. 1. Relationship between L-glutaminasc activity and organic C in soils.

TOTAL N (96)

Relationship between L-ghttaminase activity and total N in soils.

877

L-Glutaminase in soil5 Efects of truce elements In studies of the effects of trace elements on L-glutaminase activity in soils, it is important that the pH of the incubation medium be controlled. For the t-glutaminase assay, a buffer was used to maintain the pH @H lo), and deviation in pH values resulting from the addition of trace elements in the presence of THAM buffer did not exceed fO.l pH unit. Therefore, the inhibition of L-glutaminase activity observed in soils in the presence of each of the trace elements studied was not due to changes in pH of the incubation mixture. Also, studies of inhibition of enzymes in soils {or in solution) are normally carried out by comparing the relative inhibition by any compound or ion using mole quantities of inhibitors and assaying the activity of the enzymes under strictly standardized conditions. These requirements were strictly observed. The effects of trace elements on soil t-glutaminase activity in three soils varied considerably (Table 2). When the average inhibition was compared by using 5 pm01 of trace element g-l soil, L-glutaminase activity was inhibited by 5% with Ba(I1) and Co(H) to 81% with Hg(I1). The most effective inhibitors were Ag(1). Hg(II), Sn(iI), Cr(III), Ti(IV) and W(VI), which showed an average inhibition of >25%. The least effective inhibitors (average < 10%) were Ba(II), Co(lI), Fc(Il), Mn(II). Ni(tI), A&III), B(III). Fc(IiI), Se(IV) and Mo(VI). Other tract elcmcnts that markedly inhibited t-glutaminasc activity were Cu(I), Cd(II). Cu(ll). Pb(ll), Zn(ll). As(Il1). B(IiI), V(W) and As(V). Tests with Spmoi of NaCl and &SO, g-i soil indicated that K +, Nat, Cl-, and SOi- associated with the tract clcmcnts studied did not affect t-glutaminase activity in soils, Also, NO; was not inhibitory at this concentration. The results of this study are in good agreement with those rcportcd by Hartman (1971). who found that L-glutamina~ was sensitive to inhibition by

7OC

6OC

5oc

400

Y=1.9X+54 r 3 0.78” N-26

too

t 200

I

300

400

500

UREASE ACTIVIN (pg NH,.N released ~‘soil2h-‘)

4. Relationship between L-glutaminasc activity and urease activity in soils.

fig.

heavy metal ions such as H&II), A&I), Pb(I1) and Cu(I1). but not by other divalent metal ions such as Mg(II). Mn(II) and Ca(I1). We have previously shown that at 5 mM both Ca(ll) and Mg(II) activate soil L-glutaminase activity by an average of 4 and 12%. respcctivcly (Frankcnbcrgcr and Tabatabai, 199lc). The effect of trace elements on soil L-glutaminase activity was comparable to their effects on activities of soii t-asparaginase (Frankcnbcrgcr and Tabatabai. 1991b) and soil amidase (Frankcnbergcr and Tabatabai, 198 I b). Thcsc three amidohydrolases are similar in that Hg(Il) was a very effcctivc inhibitor of their activities, which implies that thiol groups arc involved in their active sites.

800

=

600 -

cx zp % 3% 2;

500-

400”

-a

0

100

200

300

dW

so0

AMIDASE ACTIVITY (pg NH,-N released rlroil 2 k’)

Fig. 3. Relationship between L-glutaminax activity and

amidase activity in soils.

&

300”

i;T in =

200 -

0

20

40

60

80

100

I

L-ASPARAGINASE ACTIVITY (pgNH,-N released g-boil 2 h-l)

Fig. 5. Relationship between t.-glutaminase activity and t.-asparaginase activity in soils.

W. T. FUN ICENBEXGERJR and M. A. TABATABA~

878

Tabk 2. Effects of trace elements on L-nlutatninasc activitv in soils Pcromtagc inhibition of L-glutamina.u activity in soil spcciIied*

Trace clement Element

Oxidation state

Ag

I

63 7

Ba Cd CO cu Fe

II

4 16 3 18 6 82 0 6 8 25 16

cu

Hg M” Ni Pb S” Z”

Hams

Okoboji

AV-XUC

78 14

76 I2

14 7 8S 0 I2 I5 29 I8

3 33 5 II 13 76 0 7 9 36 11

2: 5 I4 9 81 0 8 II 30 17

MWXKiIlC

87 2 2:

a

Al AS B Cr Fe

III

8 I8 I2 39 8

12 27 8 26 II

6 6 II 42 4

9 17 IO 36 8

Se Ti V

IV

8 2s I7

IO 32 23

I6 31 I6

8 29 I9

AS

V

I8

I3

I9

17

MO W

VI

5 16

6 31

6 30

6 26

LSD P < 0.05

0.9

2.3

I.1

‘5 Nmol trace ckmcnt g-’ sotl. For the salts used. see Frankenbcrgcr and Tabutahsi (199th).

Eflects

of pesticides

The cffccts of 12 hcrbicidcs. two fungicides. and two insccticidcs on L-glutaminasc activity in soils arc shown in Table 3. The average inhibition observed. by using 10~8 of active ingredient of pesticide g-’ soil. ranged from 4% with Merpan to 19% with Malaspray. Of all the pesticides tcstcd. the most Table 3. EtTectr of pesticides on L-glutaminare activity in soils Percentage inhlbition of t.-glutrminasc activity in roil specified’ Pesticide

Harps

Muscatinc

Okoboji

Average

Herbicide Aatrax Alanap Amiben Banvcl Bladcr 2.4-D Dinitraminc Eradicanc LaSSO

Paraquat Sutan Treflan

II I3 I6 II I7 I4 I8 I7 I2 IO 16 4

I7 II 9 23 I3 3 I9 I2 I7 I9 IS 7

I3 23 I6 13 I9 2 9 I3 6 15 II 9

I4 16 I4 I6 16 6 IS 14 I2 I5 14 7

8 3

I8 7

II 2

I2 4

6 23

I8 13

3 22

9 19

Fungicide MC”.Xa” Mcrpan Insecticide Dirzinon Malaspray LSD P < 0.05

lIOpg

1.4

1.5

of active ingredient of pesticide g-’ soil.

I.4

ctTcctivc inhibitors (3 IS%) wcrc Alanap, Banvcl, Bladcx. Dinitraminc, Paraquat and Malaspray. The least effective inhibitors (< 10%) wcrc 2.4-D. Trcflan, Merpan and Diazinon. Other pesticides that inhibited L-glutaminase activity included Aatrex, Amibcn, Eradicane. Lasso, Sutan and Menesan. The effects of pesticides on soil L-glutaminasc activity were much lower than their effects on soil and Tabatabai, L-asparaginase (Frankenbergcr 199lb). but somewhat comparable to soil amidasc (Frankenberger and Tabatabai. 1981b). However, the pesticides that were the most effective and lcast effective inhibitors of both L-glutaminase and t.-asparaginase activities were very similar. The degree of inhibition by pesticides was probably not large enough to inhibit these amidohydrolases in soils receiving normal rates of pesticide applications. REFERENCES Frankcnbcrger W. T. Jr and Tabatabai M. A. (1980) Amidase activity in soils. I. Method of assay. Soil Science Society of America Journal 44. 282-287. Frankcnberger W. T. Jr and Tabatabai M. A. (198la) Amidasc activity in soils. 111. Stability and distribution. Soil Science Society of America Journal 45, 333-338. Frankenlxrger W. T. Jr and Tabatabai M. A. (198lb) Amidase activity in soils. IV. Effects of trace elements and pesticides. Soil Science Society of America Journal 45, IIZO-I 124. Frankenbergcr W. T. Jr and Tabatabai M. A. (199la) t_-Asparaginase activity of soils. Biolo,qy and Fertility of Soils 11, 6-12. Frankenberger W. T. Jr and Tabatabai M. A. (199lb) Factors affecting t+-asparaginase activity in soils. Biology and Fertility of Soils 11. l-5.

L-Glutaminasc in soils

Frankcnberger W. T. Jr and Tabatabai M. A. (1991~) L-Glutaminase activity of soils. Soil Biology & Biochemistry u.

869-874.

Hartman S. C. (1971) Glutaminases and y-glutamyltransferases. In The Enzymes (P. D. Boyer. Ed.). Vol. 4, pp. 79-100. Academic Press, New York. Khazicv F. K. and Burangulova M. N. (1965) Activity of enzymes which dephosphorylate organic phosphorus compounds in soil. Priklandnclyo Biokhimiya I Mikrobiologiya 1, 373-379. Ramim-Martinez J. R. and McLaren A. D. (1966) Some factors influencing the determination of phosphatase activity in native soils and in soils sterilized by irradiation. Ewymologia

31. 23-28.

an

Skujins J. J. (1967) Enzymes in soil. In Soif Biochemistry (A. D. McLaren and G. H. Peterson, Eds). Vol. 1. pp. 371-414. Dekkcr. New York. Speir T. M. (1977) Studies on a climosequence of soils in tussock grasslands. II. Urease. phosphatase. and sulphatase activities of topsoils and their relationships with other properties including plant available sulphur. New Zealand Journal of Science 20, 159-166. Tabatabai M. A. (1977) Effects of trace elements on UIWSC activity in soils. Soil Biology & Biochemistry 9, 9-13. Tabatabai M. A. and Bremner J. M. (1972) Assay of urease activity in soils. Soil Biology & Biochemirtry 4, 479-487.