I Zeitschrift fiir Allg. Mikrobiologie 1 14 I 7 I 1974 I 581 -591 1

(Department of Microbiology, M. S. Universit'y of Baroda, Baroda, India)

Nucleotide degrading enzymes in Neurospora crassa')

A. I<. MATTOO and ZARNA M. SHAH

(Eingegangen am 24. 9 .1973)

Considerable amounts of GTP, GMP, adenosine, guanine, and adenine nccnmulated in Neurospom crasaa when a cult'ure grown on low phosphate (0.01%) medium was trans- ferred to a high phosphate (19;) medium. The levels of alkaline phosphatase, nucleotidase, and nucleosidase decreased by 2.4, 5.4, and 3 folds, respectively, in cultures grown on high phosphate medium. Substrate kinetics of these enzymes revealed that: (1) alkaline phos- phatase isolated from the organism grown on low phosphate medium demonstrates non- linear reciprocal plots with two distinct apparent K w values for ,4-glycerophonphate com- pared to one apparent, K m value (associated with 32% decrease in the apparent VIllar value) obtained with tha t grown on high phosphate medium ; (2) nucleotidases and nncleosidases isolated from organisms cultivated on low phosphate and high phosphate media showed the same apparent Km values, 0.25 mM for nucleot'idase and 0.909 miw for nucleosidase. There was, however, >3 times decrease in the catalytic activity of the latter enzymes isolated from organisms grown on high phosphate medium as compared to those grown on low phosphate. Inclusion of inorganic rhosphate in standard assay mixtures of the three enzymes resulted in a considerable inhibition in the catalytic activities of all of them. High levels of phosphate in the medium caused marked repression of three out of six of alkaline phosphatase, two out of three of nucleotidase, and one out of two of nucleosidase isozymic forms detected in the low phosphate grown culture.

Nucleotide degrading enzymes, viz., phosphatase, nucleotidase, and nucleosi- dase have been reported from a number of living cells (BARMAN 1969, HALFORD et al. 1969, KUO and BLUMENTHAL 1961, REESE 1968, REESE and MAGUIRE 1968, SIMPSON and VALLEE 1970, STADTMAN 1961). Studies on these enzymes among fungi have been mostly concentrated on acid and/or alkaline phosphat- ases and have revealed the presence of phosphate repressible and non-repressible enzymes in Neurospora crassa (DAVIS and LEES 1969, 1972, 1973, JACOBS et al. 1971, KADNER and NYC 1969, Kuo andBLUMENTHAL 1961a, 1961b, NYC 1967, NYC et al. 1966) and Aspergillus nidulans (DORN and RIVERA 1966). Comparatively very little is known about the effect of phosphate concentration in tfhe culture media on the levels of nucleotidases and nucleosidases in these organisms. Earlier, MATTOO et al. (1973) reported on the accumulation of aminopurine nucleotides by a carotenogenic (wild) strain of N . crassa and the stimulation of this process by high phosphate levels. We report here on the effects of low and high phosphate levels in the culture medium on the isozymes and properties of alkaline phos- phatase, nucleotidase and nucleosidase in the cell-free extracts of N . crassa.

1) A part of this communication was read a t the 13th Annual Meeting of the Association of Microbiologists of India, held at Ludhiana (India; in 1972. 39 Zeitschrift f . Allg. hlikrobiologie. Bd. 1 4 , H. 7

582 $. K. i l f a ~ ~ o o and ZAXNA 31. SH-4H

X a t e r i a l s a d niethods Cultural conditions and methods for quantitative and qualitative determinations of

aminopurine nucleotides and bases were the same as described earlier (MATTOO e t al. 1973). Cultures grown on the synthetic medium described earlier (MATTOO et aI. 1973). containing 0.Olq.h potassium dihydrogen phosphate (KH,PO,) are referred to as 'low phosphate' grown and those cultivated on

The cell free extracts for enzyme actirities were obtained essentially by the method des- cribed previously (MEHTA rf a / . 1972). Protein was determined by the method of LOWRY e t a l . (1951) using bovine serum albumin as the standard. Enzyme act,irities were measured either immediately after preparation or after the solution was kept, overnight, at, -20 "C and thawed only once. Controls n-ithout substrate or enzyme solution or with boiled enzynie were carried out, correction being made for any change observed in this inconiplet,e test system .

Alkaline phospliatase (orthophosplioric monoester hydrolase, E. C. 3.1.3.1) was measured at 30 ' C by measiiring thc release of inorganic phosphate from sodium B-glycerophosphate. Unless where otherwise indicated, the standard assay mixture contained: tris-HCI buffer (pH 8.0) 100 p o l e s , sodium P-glgcerophosphate 50 pmoles, and an appropriate aliquot of en- zyme solution. The total volume was 3.0 nil. Incubation was carried out a t 30 "C for 30 min to 1 h. The releaeed inorganic phosphate in 59* (w/v) trichloroacetic acid super- natants was measured by the method of FISRE and SCBBAROW (19%). A unit of enzyme activity is defined as that quantity of the enzyme that Xvould catalyse the liberation of 1 nmole of inorganic phosphate per min under the experimental conditions.

The alkaline phosphatase activity at the optimum pH of 8.0, exhibited a linear depen- dence of the extent of reaction on the time of incubation (upto 90 niin) and on the rate of the concentration of enzyme protein (up to 1.5 nig). The enzyme was act'ive over a broad range of incubation temperatures, from 10 'C to 40 'CI but a t higher temperat,ures it showed a sudden fall in enzyme actix-ity? recording more than GOY", decrease a t 50 "C.

Nucleosidase (N-ribosyl purine ribohj-drolase. l3.C'. 3.2.2.1) was estimated by the met.hod of WAXG (1955), free ribose liberated by the enzymic reaction being measured by the me- thod of KELSON (1944). Unless where otherwise indicated, the standard assay mixture of 2.0 ml contained: sodium acetate buffer (pH 6.5) 200 pmoles. GMP 3.0 pmoles, and an appro- priate aliquot of enzyme solution. The mixtures were incubated for 2 hrs. a t 30 "C. The enzyme reaction was stopped with 1 nil of cold 40; perchloric acid and to neutralise the acid 0.5 ml of saturated KHCO, solution was added. The filtrate was assayed for free ribose. A unit of enzyme activity is defined as that quantity of the enzyme that would catalyse the liberation of 1 nniole of free ribose per min under the specified experimental conditions.

The nucleosidase activity, with an optimum pH of 6.5. exhibited a linear dependence of the extent of reaction on the time of incubation (up to 4 hrs. a t 30 "C) and on the rate of the concentration of enzyme protein (0.3- 1 nig).

The activity of a nucleotidase was closely associated with the nncleosidase. The extent of degradation of GRlP by nucleotidase (5'-ribonucleotide phasphohydrolase. E.C. 3.1.3.5) was measured by estimating the inorganic phosphate, released from GMP after an incuba- tion of 2 hrs. at 30 'C, in 50/;, (w/v) trichloroacetic acid supernat,ants of the above mentioned assay mixture (same as for nacleosidase), by the method of FISRE and SZTBBAROW (1925). A iinit of enzyme activity is defined as that quantity of the enzyme that, would catalyse the liberation of 1 nniole nf inorganic phosphate per min under the experimental conditions.

The electrophoretic fractionation of the isozymes of alkaline phosphatase, nucleotidase, and nucleosidase was performed on 7.5'jo polyacrylamide gels as described by DAVIS (1964) using Tris-glycine buffer a t pH 8.3. Thc extract was applied (<0.2 ml) to the top of the gel just before electrophoresis. The current applied was 5 niA (for alkaline phosphatase) and 4 mA (for nucleotidase and nucleosidase) per gel and the electrophoresis was completed a t 5 'C in 75 min. For detection of alkaline phosphatase activitp the gels were incubated a t 25 'C with a' reaction mixture containing 0.9 31 tris-HC'1 buffer pH 8-9 ,O .G mar lead acetate, and 10 mlr sodium 17-glycerophosphate. At the end of incubation (4-6 hrs.) the gels were first washed in distilled water and then in 7 o o acetic acid for 2 niin, and finally immersed in a 1o/b solution of sodium sulphide for 5 niin. The bands of enzyme activity turned black against faint yellow to white background.

For detection of nucleotidase activity, the gels were incubated in a reaction mixture con- taining 0.2 31 citrate-KaOH buffer, pH 6.5. 0.6 niJr lead nitrate, and 2 m~ GMP. At the end of incubation (6 -8 hrs.) the gels were first washed in distilled water and then in 7?/0 acetic

KH,PO, are referred to as 'high phosphat'e' grown.

Nucleotide degrading enzymes in N . crussa 583

acid for 2 min, and finally immersed in a dilute solution of ammonium sulphide for 30 t’o 60 sec. The bands of enzyme activity turned black against faint brown background.

For detection of nucleosidase activity, t,he gels were incubated in a st.aining solut,ion developed by one of us in this laboratory (MATTOO, in preparation). This solution contained GMP 3 mnf, NADPH 0.06 mM, MnCI, 5 nm, DL-isocitric acid 1.4 mM, pig heart isocitrate dehydrogenase (specific activity: 3 - 10 pmoles units/min/mg; t,ype IV; SIGMA), 0.15 pmoles units/ml ; Eremothecium D-ribose reductase (specific activity: 7 pmoles NADP/min/mg; MEHTA et d. 1872) 7 units/ml, phenazine methosulphate 0.3 nig/ml, nitro-blue tetrazolium 0.6 mg/ml, and citrate-NaOH buffer (pH 6.5) 100 mM. Staining was complete in 45 min. The gels were washed in wat,er and then transferred to t,ubes containing 7.50,6 acetsic acid. The enzyme activity bands showed as purple-blue against yellow background.

Results and discussion Higher yields of aminopurines and their nucleotides obtained on transferring

iV. crassa grown on low phosphate medium to high phosphate medium (MATTOO et al. 1973) are accompanied a t 72 hours of the transfer process by increases in the concentrations of GTP, GMP, adenosine, guanine, and adenine by 26, 61, 300, 250, and 50%, respectively, and decreases in t#hose of GDP, ADP, and AMP by 41, 62, and 20%, respectively; no detectable change in the concentrations of guanosine and ATP was obtained (Table 1) . These dynamic changes in the con-

Table 1 Effect of transfer of low phosphate medium grown culture to high phosphate medium on the accumulation of amino- purines and their nucleosides and nucleotides by N . crussd)

Concentration (mg/l)

Before 1 72 hr after transfer I t,ransfer

Adenine 1 1.73 1 2.66 Adenosine 2.53 7.32 AMP 1 4.85 I 3.70 ADP ATP Guanine Guanosine GMP GDP GTP

1.68 2.64 3.12 6.82 1.83 3.44 3.30

0.64 2.30

12.94 6.43 2.93 2.02 4.20

l) Culture was grown in synthetic medium. At 48 hr. cells were collected by centrifugation and transferred to fresh synthetic medium containing l”/b K,HPO,. After 72 hr. of transfer samples were removed and andyzed. Results are an average of 3 separate experiments.

centrations of aminopurines and their nucleotide forms are a reflection of changes/control in the properties and/or formation of enzymes connected with their interconversion, synthesis, and degradation. The levels of alkaline phos- phatase, nucleotidase, and nucleosidase were found to decrease by 2.4, 5.4, and 3 folds, respectively, in high phosphate medium indicating the possibility of repression of all the three enzymes by high phosphate concentrations (Table 2).

The effect of increasing concentrations of /?-glycerophosphate and GMP on the velocity of alkaline phosphatase and nucleosidase are shown in Figs. 1A and 2A. Their kinetic constants are given in Table 3. It is apparent that the

39*

584

Table 2 ~ e i ~ r c s s i o n of S e z t r o s m , o alkaline DhosDhatase. nncleotidase. n n d

Initial AAlkaliiie I

ase2) Snclrot id ase3) , Si~clcosit lasc~) phosphate

concen. in nnioles/niin/mg protein

. ~~ ~ ~~~~

0.01 233.2 42.56 ~ 12.31 1 .oo 95.7 , 7.88 4.13

l ) Xge of the cultures \\-as 7 2 hr. Results are from a repre~ent~n-

?) Substrate (,i-filyceropliosl’liate) conceiitration w e d was 15 ~ B I . 3, Substrate (GMP) concentration used lvas 1.5 m a r .

t i w set of 5 esperirnents parallel in triplicate.

0 02 04 06 r//S)[CSi=mMp-Gp/

Fig. 1 . Effect of sodiuni i?-BlJ’cel.opliospliatt: ($-GI?) coriceritrations on rcaction rates of alkaline phosphatase isolated from S . c r n s s n grown on low phosphate (0) and high phosphate ( 0 ) media (a). I n B, the same data are shown in the form of double reciprocal plots of

velocity and i3-glyccrophosphate concentration

Sucleotide degrading enzymes in N . crnssa 585

A

U 7 2 3 4 V(sl f(sl = ~ M G M P ]

Fig. 2. Effect of GMP concentration on reaction rates of nucleosidase isolated from N . cr(1sccI grown on low phosphate (0) and high phosphate ( 0 ) media (A). In B, the same data, are shown in the fornl of double reciprocal plots of velocity and G M P concen-

tration

substrate saturation curves for alkaline phosphatase from organisms grown on low phosphate and high phosphate media are different and reveal complicated interactions (Fig. 1 A). The enzyme from low phosphate grown culture showed activation a t high substrate concentration whereas the enzyme from high phohpliate grown culture was inhibited a t substrate concentrations greater than 20 mM. The reciprocal plots (Fig. 1 B) of 1/v against l/(p-plycerophos- phate) are non-linear and of the concave-down type with two distinct appa- rent K m values for the substrate, designated a s Knz, (5.26 mM) and Km2 (1.92 mM), for the low phosphate enzyme. The apparent Knz, (5.26 n i ~ ) value agrees well with that reportedearlier (5.9 mM) by KUO and BLUMENTHAL (1961b) in N . C T U S . ~ ~ . MOTZOK (1959) and MOTZOK and BRaNION (1959) in their detailed studies with chick alkaline phosphatases also obtained a LINEWEAVER-BURK plot that angled downwards as i t approached the l / v axis. Such a resolution of the data into two portions permitted the determination of two different K ~ L values, one applicable to dilute and the other applicable to more concrntrnted sub-

586 A. K. MATTQO and ZARNA 81. SiraH

Table 3 Kinetic constants of alkaline phosphatases and nncleosidases from Neurospora crasSa

cultivated on low (0.01:,) and high (19;) phosphate medial)

Enzy nie

~- ~-

Alkaline

phosphat ase

Conditions of assay

( O O ) ~ -

I

tris-HCI, I

33.3 nm 1 0.01

pH 8.0, 30 "C

1 .OO Nucleosidase 100 nix 0.01

1 .OO l sodium acetate,

pH 6.5, 30 "C

.- I

Apparent I Apparent h'7l l VlTlZX

I (nm) ~(nmoIes/niin/mg)

307.69

(2) l.W3) I 210.52

5.26 210.52 0.909 ~ 16.66

0.909 5.20

l ) Synthetic niedium as described earlier (XATTOO ef ( I / . 1973). Only t'he concent'ration of KH,P04 in this medium was varied as indicated. Thc cultures analysed were harvested at 72 hr.

z , Values corresponding to @-glycerophosphate concentration between 10- 36 mni . 3, Values corresponding to B-glyceropliosphate concentration between 1 - 20 mn1.

strate solut'ion. DAVIS and LEES (197.2) also observed the turning of double reci- procal plot's downnrards a t highest substrate concentra'tion test'ed for alkaline phosphatase from LV. crassa. These results suggest) tha t either mult'iple mole- cules of the substrate combine with the enzyme or tha t there are present in t8he cst,ract t,wo (or more) alkaline phosphatases with different kinet'ic paranietms.

In contrast), the high phosphat'e enzyme showed normal MICHAELIS-MENTEN kinetics between 1 .O mivI t o 20 n m substrate concentrat'ions with apparent, Km (5.26 mb1) similar to Kml of the 'low phosphate' enzyme, with inhibition a t high subst,rate concentrations (Fig. 1B and Table 3) . I n acldition, tshe velocit,y of the enzyme decreased (Table 3) which, when considered together with the changes in the kinetic parameters is suggest,ive of select,ive repression of some of the molecular forms of the enzyme in the high phosphate grown culture.

The influence of increasing GXIP concentrat'ions on the velocity of nucleo- sidase (Fig. 2A) indicates normal MICHAELIS-MENTEN kinet'ics between 0.2 mM t o 1 miu substrate concentrations and substrate inhibition a t concent#rations greater than 1 nibf. The douhle reciprocal plot's (Fig. 2B) of velocity and GMP concentration revealed the same apparent' Kwa values (0.909 mM) for GMP irre- spective of the sonrce of the enzyine. However. t.he appa.rent Vnmr value was 3 times lower for thc 'high phosphate' enzyme compa'rcd to the 'low phosphate' enzyme (Table 3). IVhen t,he values obtained with substrate concentrations less than 0.2 miu GhlP were incorporated (not, shown) in the double reciprocal plots (Fig. 2B) thc curves tended tolvarcls nonlinearit'y suggesting differences in the kinetics of the enzymes at low and high substrate concent'rabions. A sharp de- crease in the velocity of the enzyme above 1 mbf GMP (Fig. 2A) leading t o sub- strate inhibit>ion (Fig. 2B) points out to the possible exist'ence of a regulatory mechanism. Inclusion of 10 m~ of inorganic phosphate (a,s sodium salt) in the standard assay mixture of the nucleosidase from the t,mo types of cu1t)ures re- sulte'd in 3 to 4 fold inhibition in t,he enzyme act'ivity a t subsbra'te concentrations between 0.2 ml\.r a n d 1 n n ~ and complete inhibition a t GMP concentlations greater than 2 imi. This data indicate that the products of the enzyme reaction

Nucleotide degrading enzymes in N . crassa 587

may contribute to the control of the enzyme as evidenced in the report,s that inorganic phosphate inhibits AMP nucleosidase from Azotobacter vinelandii (HURWITZ et al. 1957) and that ribose (2.5 mM) in addition to inhibiting GMP nucleosidase from Eremothecium ashbyii in vitro may also lead to repression of t,he enzyme (MEHTA et al. 1972).

A nucleotidase, closely associated with nucleosidase, showed the same apparent K.rn values (0.25 mM) for the enzymes isolated from low phosphate and high phosphate grown cultures, but the apparent Vmax value for the enzyme from the latter was 3 times lower (14 nmoles/min/mg protein) compared to the enzyme from the former (43.5 nmoles/min/mg protein). Addition of 10.5 mM inorganic phosphate in t)he standard assay mixtures resulted in about 5 fold decrease in the catalytic activity of the enzymes from both the types of cultures, a result similar to that of the inhibition of nucleotidases from Lactobacillus pentosus (WANG 1955) and rat liver (SEGAL and BRENNER 1960) by inorganic phosphate.

Alkaline phosphatases from N. crassa (KUO and BLUMENTHAL 1961 a, 1961 b, NYC et al. 1966, HOCHBERG and SARGENT 1973) and A. nidulans (DORN and RIVERA 1966), and AMP nucleosidase from A . vinelandii (YOSHINO et al. 1968) are known to exist in their multiple molecular forms. It may well be that the changed kinetic constants of the 3 nucleotide degrading enzymes studied here (Table 3) in high phosphate grown culture are a reflection due, in part, t o change/repression in their isozymes. To study this, t,he soluble cell-free pre-

Fig. 3. Disc gel electrophoresis of cell-free alkaline phosphatase preparations from N . crassa grown on low (A) and high (B) phosphate media. Each preparation (200 pg) from 72 hr.

harvested cultures was subjected to electrophoresis for 75 niin a t 5 mA/gel in Tris-glycine buffer, p H 8.3

588 A. K. JIATTOO and ZARSA 31. SHAH

parations from cultures g ro~vn on low and high phosphate iiieclia were submitted to polyacrylaniide gel electrophoresis. The results are illustrated in Figs. 3, 4, and 5 .

Development of carving numbers of bands of alkaline phosphatase act'ivity was observed depending on the source of the extract (Fig. 3 A and R). The low phosphate cnzyine preparation showed about 6 bands, out of which 3 to 4 bands (no. 3, 4, 5, 6 in Fig. 3 A ) were very intensely staincd and the ot.lier two (no. 1 and 2) less stained: which are indicatire of the presence of multiplc forms of the enzyme having varying catalytic activitj-. The soluble enzyme prepra t ion from high phosphate medium gron.11 culture. in contrast,, showed only two very intensely stained bands (Fig. 3 B) corresponding to 4t)h and 5th of those in the low phosphate culture (Fig. 3 A). Hornever, when high probein concen- trate of the former preparation was applied on the gels. one additional band appeared (not' shown) which corresponded to the 2nd band of low phosphate enzyme preparation. The presence of 6 isozyines of alkaline phosphstase in our cultures as compared to 5 observed by HOCHBERC: and SARGENT (1973) may be due to the different. cwltivation media employed. \Ve are now undertaking t'o study what' changes occur in isozymes of alkaline phosphatase when cultivated on different culti\-ation media.

Fig. 4. Disc gel electrophoresis of cell-free nncleosidase preparations from K . oassa grown on low (gel 1) and high (gel '7) phosphate niedia. Each preparation (200 pg) from 7 2 hr. liarrested cultures was subjected to electrophoresis for 7 5 min at 4 mA/gel in Tris-glycine

buffer, pH 8.3

Nucleotide degrading enzymes in N . crama 589

YOSHINO et al. (1968) showed the presence of two distinct forms of AMP nucleosidase from A . vinelandii. Neurospora grown on low phosphate medium also exhibited 2 distinct forms of nucleosidase (Fig. 4, no. l ) , one of which was a fast moving band. The soluble enzyme preparation from high phosphate medium grown culture showed only one intensely stained band (Fig. 4. no. 2) which corresponded to the fast moving isozyme in the low phosphate culture. The slow moving component, possibly of high molecular weight. could not be detected in the high phosphate culture.

Cell-free extracts from low phosphate grown culture mere resolved into 3 distinct isozymes of nucleotidase (Fig. 5 , no. l), out of which the fast moving band and the slowest moving band (first from the top) were intensely stained

Fig. 5 . Disc gel electrophoresis of cell-free nucleotidase preparations from N . c m s s u grown on low (gel 1 ) and high (gel 2 and 3) phosphate media. To gel 1 and gel 2 200 $g and to gel 3 400 pg of enzyme protein were applied. Other conditions were the sitme as in Fig. 4

witahin 5 rnin of incmbation in the staining solution. The band of enzyme activi- t y which is second from the top (Fig. 5 , no. 1) staine dafter 4 hr. of incubat'ion. These result's are indicative of the presence of multiple forms of t'he enzyme having varying cahlytic activity. It is interesting to note t#hat, as in nucleosi- dase (Fig. 4), only the fast moving band of nucleotidase act>ivit,y was reta,ined and dectetable in the cultures from high phosphate medium (Fig. 5 , no. 2 and 3). On increasing the concentrat>ion of applied cell-extra'ct from the latt#er no additional band of enzyme activit'y could be detected (Fig. 5 , no. 3).

The above results indicat,e that, selectfive repression of at least three isoenzy- mes of alkaline phosphat,ase, t,wo isozymes of nucleotidase, and one isozyme of nucleosida.se takes place in t'he high phosphate grown culture and that possibly this difference may reflect, on t,he differences observed in the substrat'e kinet'ics

of these enzymes from two cultures. However, further experimentation with each isozyme form is neccssary to confirm our presumptions.

The above data , in addition to revealing interesting substrate kinetics and isozyrnic pattern of the three nucleotide degrading enzymes, also suggest a control niechanism by phosphate ions in the regulation of N . crassa alkaline phosphatase, nucleosidase, and nucleotidase which in turn may in par t reflect in the qualitative and quantitative picture of the accumulated nniinopurines and their nucleotides in the culture filtrates of the organism. The high phos- phate concentration may even have a sparing effect on the nucleotides and nurleosides. thus favouring their accumulation. Further work on these lines is in progress

A c kit owl edge in eiz t s

\\-e thank Professor V. V. NOUI for his interest in this work. We are grateful to our colleague. H. S. CHATTPAR. for extending valuable help in experiments on disc electro- phoresis of alkaline phosphatase isozymes. This investigation was supported in part by UGC grant to post-graduate teachers (L\. I(. 32.)

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Mailing address: Dr. A. K. MATTOO Department of Microbiology, Faculty of Science, M. S. University of Baroda Baroda 390002, India