THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 249, No. 8, Issue of April 25, pp. 2344-2353, 1974

Printed in U.S.A.

Regulation of the Pentose Phosphate Pathway

in the Fungus Aspergillus nidukzns

THE EFFECT OF GROWTH WITH NITRATE*

(Received for publication, August 6, 1973)

OLIVER HANKINSON$ AND DAVID J. COVE

From the Department of Genetics, University of Cambridge, Cambridge CB/, IXH, England

SUMMARY

When grown in medium containing urea and nitrate, wild type Aspergillus nidulans possesses approximately Z-fold higher activities of four enzymes of the pentose phosphate pathway and 3-fold higher activity of glucose phosphate isomerase than when grown in medium containing urea. The activities of one enzyme of the pentose phosphate path- way and of three enzymes of the Embden-Meyerhof-Parnas pathway and of NADP-isocitrate dehydrogenase are either not affected, or only slightly affected, by the presence of nitrate during growth.

The results obtained from mutants of the nitrate reduction pathway show that neither nitrate nor nitrite directly causes the increases in the activities of the four enzymes of the pentose phosphate pathway and glucose phosphate isomerase, that the increases in these activities do not depend upon the metabolism of nitrate and probably do not result from changes in the concentrations of either NADP+ or NADPH. The results suggest that the product of the nirA gene, which is the inducer of the enzymes of the nitrate reduction pathway, is responsible for bringing about the increases in the activities of the enzymes of the pentose phosphate pathway and glucose phosphate isomerase and that the state of activity of the product of the nirA gene for its stimulatory effect on the activities of the four pentose phosphate pathway enzymes and glucose phosphate isomerase is regulated in exactly the same way as is its state of activity for inducing the enzymes of the nitrate reduction pathway.

The nitrate reductase, nitrite reductase, and hydroxylamine reductase of Aspergillus nidulans use NADPH, but not NADH, as coenzyme (3) and NADPH is oxidized to NADP during the conversion of nitrate to ammonium. Some NADPH is also utilized during the assimilation of urea (and ammonium) due to the action of NADP-glutamate dehydrogenase. However, a

* Preliminary reports of this work have appeared (1, 2). $ Present address, Genetics Unit, Children’s Service, Massa-

chusetts General Hospital, Boston, Mass. 02114. Recipient of a Research Studentship from the Medical Research Council.

far greater amount of NADPH is oxidized per atom of nitrogen incorporated into amino acid from nitrate than is oxidized per atom of nitrogen incorporated from urea.

The two dehydrogenases of the pentose phosphate pathway afford a major means whereby fungi reduce NADP+ to NADPH. Osmond and ap Rees (4) showed that when Canida utilis was grown with KNOs as sole nitrogen source it possessed 2.5.fold higher activities of two enzymes of the pentose phosphate path- way than when it was grown with a complex amino acid mixture. Holligan and Jennings (5) have provided evidence that &rate also stimulates glucose oxidation by w-Nay of the pentose phos- phate pathway in the fungus Dendryphiella salina.

The first aim of the present investigation was to determine whether nitrate produced a response in A. nidulans similar to that in C. utilis. To this end the activities of several enzymes of the pentose phosphate pathway and of several other enzymes of carbohydrate metabolism were measured in mycelium of the wild type which had been grown with either 1 y0 glucose + 5 mrvr urea or with 1% glucose + 5 mM urea + 10 mM NaN03. Growth in the latter medium leads to maximal induction of the enzymes of the nitrate reduction pathway (3). It is shown in this paper that growth of A. nidulans in nitrate-containing medium compared with growth in medium containing no nitrate leads to elevated activities of several enzymes of the pentose phosphate pathway. The regulatory mechanism involved in this response to nitrate was then studied. Our approach was to measure the activities of the pentose phosphate pathway enzymes in several mutants of the nitrate reduction pathway of A. nidulans. To assist interpretation of the results obtained, the characteristics, with respect to nitrate metabolism, of the mutants and the wild type are summarized below and in Table I.

Both nitrate and nitrite induce nitrate reductase, nitrite reductase, and hydroxylamine reductase activities in the wild

type (8). Mutations in the niaD locus or in any one of the five cnz loci

can cause loss of nitrate reductase act.ivity (9). The cnl: loci are believed to direct the synthesis of a molybdenum-containing cofactor (9, 10) which binds with the polypeptide specified by the niaD locus to produce an active nitrate reductase molecule (11, 12). Recent evidence indicates that the cnz cofactor has a polypeptide component (13).

&A- mutants lack nitrite reductase and hydroxylamine

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TABLE I

Characteristics of some mutants of nitrate reduction pathway of Aspergillus nidulans

Modified from Cove and Pateman (6) and Cove (7).

Strain

-I- A

‘_ a

Wild-type

niaD- -

cnx Tloci

niid -

nirA- -

nirA’- 1 -

)

N

I bility to use following .s sole nitrogen sources

‘i trate N

I itrite ummnium

Relative specific activities in mycelium grown with the indicated nitrogen sources

Nitrate reductase

5 mM urea 5 r&l urea + 10 mM NaNOs

3 100

0 0

0 0

3 100

3 3

140 140

Inactive product Nitrate reductase Nitrite/ hydroxylomine -nitrate complex reductase

r cnx cofactor

Active product - Necessary for gene expression

L +L L

nir Agene nia Dgene nii A gene

FIG. 1. (Data from Cove (7).) Model for the regulation of the enzymes of the nitrate reduction pathway in A. nidulans.

reductase and it is probable that niiA is the structural gene for an enzyme which possesses both of these activities (8).

&A- mutants are noninducible by nitrate or nitrite for the three enzymes of the nitrat,e reduction pathway (8). The mutation nirAC-1 is allelic to &A- mutations (7) and &AC-l is constitutive for nitrate reductase and semiconstitutive for nitrite-hydroxylamine reductase (14).

It has been found that the majority of niaD- and cnz- mutants are constitutive for nitrite-hydroxylamine reductase while the remainder are inducible for this enzyme (6, 8). Evidence has also been obtained that those niaD- and cnx- mutants that are constitutive for nitrite-hydroxylamine reductase are also con stitutive for the product of the niaD gene (which is altered in the case of the n&D- mutants), whereas those niaD- and cnx- mutants which are inducible for nitrite-hydroxylamine reductase are inducible for the niaD product (9, 11, 12).

A model has been proposed for the regulation of nitrite-hy- droxylamine reductase and the niaD product (6, 7) which is as follows (see also Fig. 1).

The product of the nirA gene (the “nir product”) is necessary

Nitrite reductase and hydroxylamine reductase

5 IUM urea 5 mM urea + IO mN NaNOs

3

majority 100 minority 3

majority 100 minority 3

0

3

30

100

100 100

100 100

0

3

140

for the induction of the n&D product and nitrite-hydroxylamine reductase. In the wild type, the nir product is subject to in- activation by nitrate reductase, so that, in the absence of nitrate, there is a negative feedback restricting the synthesis of the niaD

product and nitrite-hydroxylamine reduct’ase. However, when nitrate binds to nitrate reductase, the latter is altered in such a way that it no longer inactivates the nir product. No judge- ment was made as to at what stage in the synthesis of the en- zymes regulation occurs.

According to the model, the &A- mutants give rise to no product, or to an inactive one, the product of the ,nirA”-1 allele is insensitive to inactivation by nitrate reductase, and the con- stitutive n&D- and constitutive cnx- mutants eit’her produce no nitrate reductase or an altered enzyme which cannot in- activate the nir product, whereas the inducible n&D- and in- ducible cnz- mutants produce an altered nitrate reductase which retains the regulatory properties of t’he wild type enzyme but which is unable to catalyze the reduction of nitrate.

MATERIALS AlUD METHODS

Strains

With the exceptions of b&l niaD-17 nirA--1 and biA-1 cnzG-4 nirA--1, all strains used in this work had been isolated previously. Except for nirAc-1, all mutations of the nitrate reduction pathway were induced either in strain yA-2 WA-3 by ultraviolet irradiation (niaD-3, niaD-6, niall-7, niaD-8, niaI>-14, cnxA-2, cnxA-4, cnxG-2, cnxH-1) (9) or in strain biA-1 by diethylsulfate mutagenesis (cnxG-4, niiA-4, and nirA--1) or ultraviolet irradiation (niaD-17) (8, 15). The origin of strain yA-2 WA-3 (mutations leading to the production of yellow conidia and white conidia, respectively) is given in Iteference 16. biA-1 is a translocation-free strain ob- tained from the Department of Genetics, University of Glasgow, which is auxotrophic for biotin. The strain biA-1 cnxG-2 had been isolated from a cross between yA-2 WA-3 cnxG-2 and biA-1 niaD-17. nirAo:-1 wasinduced in strain yA-2 adE-20 AcrA-1 galA- riboB-2 niiA-9 by N-methyl-N’-nitrosoguanidine mutagenesis (14). biA-1 nirAO-1 was obtained from a cross between yA-2 adE-20 AcrA-1 gaZA-1 riboB-2 niiA-9 nirAa-1 and biA-1 cnxH-4. Strains carrying two mutations of the nitrate reduction pathway were isolated from appropriate crosses. The presence in some double mutants of one of the mutations affecting nitrate metabo- lism could not be established by growth tests. In these cases, the presence of the cryptic mutation was established by demon- strating that the double mutant could not complement in a hetero- karyon with the appropriate parent strain for growth on nitrate.

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Strain biA-1, which is normal with respect to carbon and nitro- gen metabolism, was used as the “wild type” for most of the studies presented in this paper.

Media

The nitrogen-free minimal medium of Cove (17) was used. This was supplemented with 10 pg per liter of biotin or 500 pg per liter of pyridoxine where appropriate. When a carbon source other than 1% glucose was used, this was substituted for the glucose in the minimal medium.

Growth of Mycelium for Enzyme Assays

Conidial suspensions were prepared in sterile distilled water containing 0.1% of the detergent, Tween 80, as described by Cove (17), except that the suspensions were not filtered through muslin. A small volume of the concentrated conidial suspension was added to 200 ml of minimal medium (containing the appropriate vitamin supplements and carbon and nitrogen sources) in a l-liter Erlen- meyer flask to give a final density of approximately 5 X lo6 co- nidia per ml. The flasks were shaken on a reciprocal shaker at 25” and the mycelia harvested as described previously (17). Mycelium was stored for up to 10 days at -70”.

Preparation of Cell-free Extracts

Frozen mycelium (200 to 800 mg) was ground for 2 min in a pestle and mortar with an approximately equal weight of sand and 10 times its weight of buffer. The resulting slurry was centrifuged at 100,000 X 9 for 20 min and the supernatant used as the cell-free extract. The whole process was carried out at O-4”.

Nitrite reductase and 6-phosphogluconate dehvdroaenase were assayed in extracts prepared- w%h 100 mM ‘or&phosphate buffer. DH 7.0. containine 10 mM cvsteine-HCl. Aldolase. nhos- phofr;&okinase, pyruvare kinase,” ribose phosphate isome’rase, and malic enzyme were assayed in extracts prepared with 50 mM Tris-HCl, pH 7.4, and glucose phosphate isomerase, transaldolase, and transketolase in extracts prepared with 50 mM Tris-HCl, pH 7.6. Glucose B-phosphate dehydrogenase and NADP-isocitrate dehydrogenase were assayed in extracts prepared with any one of the three buffers above.

Chemicals

Analytical grade chemicals were used wherever possible. NAD+. NADP+. and NADPH were obtained from the Boehrineer Corpoiation, L&don, and glucose A-phosphate, DL-isocitrate, &d GTP were obtained from Koch-Light Laboratories Ltd, Coln- brook, Buckinghamshire, England. All other substrates, enzymes and coenzymes for the enzyme assays were obtained from the Sigma Chemical Company, St. Louis.

Units of Enzyme Activity

The unit of activity of ribose phosphate isomerase is given in AE520 nm per min at 37”. The activities of all other enzymes are given as nanomoles of substrate (NADPH in the case of nitrite reductase) utilized per minute at 25”. Specific activities are given as units per mg of protein.

Enzyme Assays

A Beckman DB spectrophotometer was used for the assay of ribose nhosuhate isomerase and either a Pve Unicam SP 800 or SP 8006 ult;aviolet recording spectrophoto&eter was used for all other assays. The assay of ribose-phosphate isomerase was carried out at 37”. All other assays were carried out at 25”.

Except for buffers and for FAD, which was sometimes stored as a concentrated solution for up to 2 months at 4”, new solutions of all assay components and new dilutions of the commercial en- zyme preparations were made each day. For some assays, the cell-free extracts were diluted in the extraction buffer. These dilutions were always made immediately before measuring the enzyme activity. All components, including the cell-free ex- tracts, but excluding the assay buffers, were kept at 0”.

Nitrite Reductase (NADPH : Nitrite oxidoreductase, EC 1.6.6.4) -The assay of Pateman et al. (8) was used.

Glucose B-Phosphate Dehydrogenase (D-Glucose-6-phosphate: NADP+ Oxidoreductase, EC 1.1.1.49)-The assay was modified slightly from that of Arst, Jr., et al. (10). The assay mixture

contained 10 pmoles of n-glucose 6-phosphate (disodium salt), 0.6 rmole of NADP+ (disodium salt), 150 pmoles of Tris-HCl, pH 8.0, and 100 ~1 of a 1:8 to 1:2 dilution of cell-free extract in a total volume of 3 ml. In each case it was checked that there was negligible or no activity in the absence of glucose A-phosphate. Gllicose 6-DhosDhate was then added and the initial rate of change of absorbance at 340 nm determined. Activities of up to 75 units were proportional to the amount of extract. Extracts did not lose any glucose B-phosphate dehydrogenase activity over an 8- hour period at 0”.

This assay overestimates the activity of glucose B-phosphate dehydrogenase, since 6-phosphogluconate dehydrogenase, by act- ing on the 6-phosphogluconate produced by glucose 6-phosphate dehydrogenase activity, contributes towards the NADPH pro- duced in the assay mixture.

The optimal concentrations of the substrates, coenzymes, co- factors, and enzymes were determined for each of the following assays, using an extract prepared from the strain biA-1 grown with 1% glucose, 5 mM urea, and 10 mM NaN03 and harvested after 21 hours. Unless otherwise stated, maximum activity was obtained only when all components of the assay mixture were present. No attempt was made to determine the optimum pH values for the assays.

Aldolase (Fructose -i,6-&phosphate D -Glyceraldehyde -3 -phos- phate-Zyase, EC 4.1 .d.lS)-The assay was modified from that of wu and Racker [18]. The assay mixiure contained 30 pmoles of n-fructose l.A-di~hosnhate (sodium salt). 0.6 umole of NADH (disodium salt), i2.5 ;g of mixed crystals’of a-glycerophosphate dehydrogenase and triose phosphate isomerase (i.e. 1250 units of a-glycerophosphate dehydrogenase and 7000 units of triose phos- phate isomerase), 75 pmoles of Tris-HCI, pH 7.4, and 100 ~1 of a 1:20 to 1:6 dilution of cell-free extract in a total volume of 1.5 ml. In each case it was checked that there was no activity in the absence of fructose 1,6-diphosphate. Fructose 1,6-diphosphate was then added and the initial rate of change of absorbance at 340 nm determined. Activities of up to at least 18 units were proportional to the amount of extract. The activity was stable over a B-hour period at 0”.

Glucose Phosphate Isomerase (o-Glucose-6-phosphate Ketol- isomerase, EC 6.5.1.9)-The assay was modified from that of Josephson and Fraenkel (19). The assay mixture contained 6 pmoles of n-fructose 6-phosphate (sodium salt), 0.6 pmole of NADP+, 750 units of glucose 6-phosphate dehydrogenase (Sigma type XV), 75 pmoles of Tris-HCl, pH 7.6, and 100 ~1 of a 1:400 to 1:50 dilution of cell-free extract in a total volume of 1.5 ml. In each case it was checked that there was no activity in the absence of fructose g-phosphate. Fructose B-phosphate was then added and the initial rate of change of absorbance at 340 nm determined. Activities of up to at least 13 units were proportional to the amount of extract. Extracts lost approximately 5% of their glucose phosphate isomerase activities-over a a-ho& period at 0”.

NADP-Isocitrate Dehudroaenase (three-D.-isocitrate: NADP+ Oxidoreductase (Decarboiylacng), EC 1.1. I-.@)-The method was modified from that of Kornberg (20). The assay mixture contained 3 pmoles of DL-isocitric acid (trisodium salt), 0.6 pmole of NADPf, 3 /*moles of M&$.6 H20, 75 rmoles of Tris-HCl, pH 7.4, and 5d to 100 ~1 of ceil-free extract in a total volume of-l.5 ml. The activitv in a control mixture which lacked DL-isocitrate was subtracted from the activity in the assay mixture. The ac- tivity was determined from the initial rate of change of absorbance at 340 nm. Activities of up to 15 units were proportional to the amount of extract. The activity was stable over a 4-hour period at 0”.

There was no activity when NAD+ was substituted for NADP+. The NAD-isocitrate dehydrogenase of A. niger requires AMP (21).

Malic Enzyme (L-Malate:NADP+ Oxidoreductase (Decarboxylat- kg), EC 1 .l .l .&)-The assay was essentially the same as that of Ochoa (22). The assay mixture contained 6 Nmoles of nL-malic acid (solution adjusted td pH 7.4 with KOH), 0.6 pmole of NADP+, 6 umoles of MnSOd.4H20. 75 umoles of Tris-HCl. DH 7.4. and 100 ~1. of cell-free extract in 8: total volume of 1.5 ml: A No activity of this enzyme could be detected on the two occasions of testing.

Phosphofructokinase (ATP:o-Fructose-6-phosphate l-Phospho- transferase, EC 8.7.1 .If)-The method was modified from that of Sols and Salas (23). The assay mixture contained 12 pmoles of D-fructose 6-phosphate, 0.45 rmole of NADH, 0.9 pmole of GTP, 3 pmoles of MgC12.6Hz0, 750 units of aldolase (Sigma grade l),

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12.5 ag of mixed crystals of a-glycerophosphate dehydrogenase and triose phosphate isomerase, 75 pmoles of Tris-HCl, pH 7.4, and 25 to 100 ~1 of cell-free extract in a total volume of 1.5 ml. The control mixture lacked GTP. The activity was determined from the maximum rate of change of absorbance at 340 nm. Ac- tivities of up to 5 units were proportional to the amount of ex- tract. The activity was stable over a 3-hour period at 0”.

With some extracts, the activity varied erratically during the progress of the reaction. This behavior may have been due partly to the presence of mannitol l-phosphate dehydrogenase in these extracts.’ When the assay was carried out against a water-blank, activity was obtained in the absence of GTP. This activity was not reduced in assay mixtures lacking either aldolase or mixed crystals of a-glycerophosphate dehydrogenase and triose phos- phate isomerase and was most probably due to mannitol l-phos- phate dehydrogenase. The phosphofructokinase activity was inhibited by excess GTP. Preliminary experiments had indicated that excess ATP strongly inhibited the enzyme.

B-Phosphogluconate Dehydrogenase (6-Phospho-o-gluconate: NADPC Oxidoreductase (Decarboxylating), EC i .l .I .&)-The assay mixture contained 4 Fmoles of 6-phospho-n-gluconate (tri- sodium salt), 0.6 rmole of NADP+, 10 pmoles of MgC12.6H20, 150 pmoles of Tris-HCl, pH 8.0, and 25 to 100 ~1 of cell-free extract in a total volume of 3 ml. In each case it was checked that there was negligible or no activity in the absence of 6.phosphogluconate. 6-Phosphogluconate was then added and the initial rate of change of absorbance at 340 nm was determined. Activities of up to 70 units were proportional to the amount of extract. MgCb was not required for maximum activity. There was no activity when NAD+ was substituted for NADP+.

Pyruvate Kinase (ATP: Pyruvate Phosphotransferase, EC 2.7.1 .@)-The assay was modified from that of Bucher and Pfleiderer (24). The assay mixture contained 1.5 pmoles of phos- phoenolpyruvate (trisodium salt), 0.6 pmole of NADH, 3 rmoles of ADP (disodium salt), 15 pmoles of MgC12.6H,0, 70,009 units of lactic dehydrogenase (Sigma type II), 75 pmoles of Tris-HCl, pH 7.4, and 100 ~1 of a 1:20 to 1:lO dilution of cell-free extract in a total volume of 1.5 ml. The constant rate of change of absorb- ance at 340 nm which occurred in an assay mixture lacking extract was first determined and this was subtracted from the initial rate of change of absorbance which occurred after the addition of extract. The small amount of activity obtained with an assay mixture lacking extract was proportional to the amount of lactic dehydrogenase added and was probably due to the pyruvate kinase which contaminates the lactic dehydrogenase preparation (Sigma). Activity was not stimulated by 100 mM KCl. Activ- ities of up to at least 22 units were proportional to the amount of extract. Extracts lost approximately 8% of their pyruvate kinase activities over a 3-hour period at 0”.

Ribose Phosphate Isomerase (o-Ribose-5-phosphate Ketol-isom- erase, EC 5.3.1 .B)-The assay was the same as that of Axelrod (25), except that ribose 5-phosphate was used at 2 mg per ml instead of 1 mg per ml. At least two independent determinations of the activity in each extract were carried out. Activities of up to 0.6 unit were proportional to the amount of extract.

Transaldolase (Sedoheptulose-r-phosphate: o-Glyceraldehyde-S- phosphate Dihydroxyacetone Phosphate Transferase, EC 2.2.1.2) -The assay was modified from that of Venkataraman and Racker (26). The assay mixture contained 1.8 pmoles of n-erythrose 4- phosphate (sodium salt), 27 pmoles of n-fructose 6-phosphate, 0.6 pmole of NADH, 12.5 pg of mixed crystals of a-glycerophos- phate dehydrogenase and triose phosphate isomerase, 75 pmoles of Tris-HCl, pH 7.6, and 100 ~1 of a 1:15 to 1:5 dilution of cell-free extract in a total volume of 1.5 ml. The control mixture lacked erythrose 4.phosphate. The activity was determined from the initial rate of change of absorbance at 340 nm. Activities of up to at least 15 units were proportional to the amcunt of extract. The stability of transaldolase activity in extracts kept at 0” was variable.

Activity was not stimulated by 10 mM MgCl,.6H20. Some extracts gave considerable activities with assay mixtures lacking erythrose 4-phosphate (assayed against a water-blank). Manni- to1 l-phosphate dehydrogenase was probably responsible for this activity.

Transketolase (Sedoheptulose - 7 -phosphate: D - Glyceraldehyde -3.

r 0. Hankinson and D. J. Cove, manuscript in preparation.

phosphate Glycolaldehydetransferase, EC 2.2.1 .l.)-The assay was modified from t,hat of Josephson and Fraenkel (19). The assay mixture contained 7.5 pmoles of n-ribose 5-phosphate (di- sodium salt), 0.6 pmole of NADH, 0.45 pmole of thiamine pyro- phosphate chloride, 15 @moles of MgC12.6H20, 1000 units ribose phosphate isomerase (Sigma type I), 12.5 pg of mixed crystals of a-glycerophosphate dehydrogenase and triose phosphate isomer- ase, 75 pmoles of Tris-HCl, pH 7.6, and 100 ~1 of a 1:15 to 1:5 dilution of cell-free extract in a total volume of 1.5 ml. The re- action was started by the addition of ribose 5phosphate. Maxi- mum activity was achieved after about 8 min and thereafter remained constant. The activity was determined from the maxi- mum rate of change of absorbance at 340 nm. Activities of up to 10 units were proportional to the amount of extract. The activity was unstable at 0”.

Excess ribose 5-phosphate and excess thiamine pyrophosphate inhibited activity. These inhibitions may have been caused by impurities in the commercial preparations of these substances.

The assay depends on the presence of ribulose 5-phosphate 3-epimerase in the extract, since the substrates of transketolase are n-ribose 5.phosphate and n-xylulose-5-phosphate, and the latter is formed by the action of ribose 5.phosphate isomerase and ribulose 5-phosphate 3.epimerase on ribose 5-phosphate. Addi- tion of ribulose 5.phosphate 3-epimerase (Sigma type III) to the assay mixture did not stimulate activity, but inhibited it. It is therefore possible that xylulose 5.phosphate, and not ribose 5- phosphate, is an inhibitor of transketolase.

Ribose phosphate isomerase did not increase activity, but maxi- mum activity was achieved faster in its presence than in its ab- sence (in about 8 min as opposed to about 14 min).

MgC12.6HtO was not required for maximum activity.

Protein Determination

The amount of soluble protein in the extracts was determined by the Folin method as modified by Lowry et al. (27).

Polyoxrylamide Gel Electrophoresis

Electrophoresis was performed in the cold using 7% or 10% polyacrylamide gels (pH 8.9) according to the method of Orn- stein (28) and Davis (29). The electrode buffer was Tris-glycine, pH 8.3 (2.88 g of glycine and 0.6 g of Tris in 1000 ml of water).

RESULTS

Enzyme Activities in Wild Type

When the wild type was grown with 1 y. glucose 4 5 mM urea

+ 10 mM NaN03 it possessed approximately 2-fold higher ac-

tivities of glucose &phosphate dehydrogenase, 6-phosphoglu-

conate dehydrogenase, transketolase, and transaldolase, and

3-fold higher activity of glucose phosphate isomerase, than when

it was grown with 1 y. glucose + 5 mM urea (Table II). The

activities of ribose phosphate isomerase, phosphofructokinasc,

aldolase, pyruvate kinase, and NADPisocitrate dehydrogenase

in mycelium grown with urea and nitrate were tither not sig-

nificant,ly greater, or only slightly greater, than those in my-

celium grown with urea.

The activities of the enzymes of the pentose phosphate path-

way in mycelium grown with lyO glucose + 10 mM NaNOp

were no greater than those in mycelium grown with 1% glucose

+ 5 mM urea + 10 mM NaN03. Thus, the presence of urea

in the latter medium did not reduce the effect of nitrate on the

activities of the pentose phosphate pathway enzymes.

When 0.1% glucose was the carbon source, growth with 10

mM NaN03, compared with growth with 5 mM urea, led to in-

creased activities of those same enzymes which were found to

be stimulated by growth with 5 mM urea + 10 mM NaNOr, in

the presence of 170 glucose. The activities of the de-

hydrogenases of the pentose phosphate pathway were also found

to be higher in mycelium grown with nit,rate than in mycclium

grown without nitrate when both 1 To n-fructose and 1 y. ethanol

were used as the sole carbon sources.

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TABLE II Speci$c activities oj enzymes of carbohydrate metabolism in wild type grower with either 5 rnM uuTea or 5 mM urea + 10 m&r lValV03

A few of the results were obtained with strain yA-2 WA-~. The remainder were obtained with strain biA-1. The strains were grown

in minimal medium (supplemented with 10 pg per liter of biotinin the case of biA-1) containing 1% glucose as the initial carbon source and containing the indicated initial nitrogen sources. Mycelium was harvested after 21 hours of growth at 25”. leach of the means is calculated from values obtained from different mycelial samples which were assayed on different occasions.

T Glucose-6-phosphate dehydrogenase 6-phosphogluconate dehydrogenase Ribose-phosphate isomerase Transketolase Transaldolase Glucose-phosphate isomerase Phosphofructokinase Aldolase Pyruvate kinase NADP-isocitrate dehydrogenase

Mean value of the specific activity and 95X' confidence limits in

mycelium grown with:

5 mM urea

671 * 52 352 f 56

1.98* 0.46 232 * 79 417 f 57

2380 f 235 37.6 f 11.1

641 + 59 1132 f 159

93 f 21

5 IIN urea + 10 mM NeN&

1387 * 93 666 f 62

2.28 f 0.44 399 f 86 750 f 69

7582 + 818 37.0 f. 12.8

718 f 88 1415 f 179

106 f 18

-

Mean value of the ratio' and 95%

confidence limits

2.09 f 0.10 26 1.95 + 0.22 12

1.17 f 0.21 6

1.80 f 0.23 9 1.81 f 0.13 6

3.20 f 0.25 9 1.04 f 0.28 8 1.12 f 0.07 7 1.26 f 0.07 7 1.17 f 0.17 8

1 Number of extracts assayed for each growth condition

‘The units of specific activity are defined in the Materials and Methods section. The 95% confidence limits of the mean were calculated as follows: standard error X t

(f) (where 7f) is the value of

Student’s t corresponding to f degrees of freedom).

2 This is the mean value of the following ratio: specific activity in mycelium grown with 5 mM urea + 10 mM NaN03 : specific activity in mycelium grown with 5 mM urea, where the specific activities which were compared wepe determined from mycelial samples which were grown up and assayed on the same occasion as one another.

I I I I ( /+

OURATfOh’ OF GROWTH WITH NoNo ,hours

FIG. 2. Specific activities of glucose A-phosphate dehydrogenase and nitrate reductase as a function of time after the addition of nitrate. biA-1 was grown in minimal medium containing 1% glucose as initial carbon source and 5 mM urea as initial nitrogen source. NaN03 was added to give a final concentration of 10 mM at the indicated times before harvesting. All mycelial sam- ples were harvested after 21 hours of growth. Each point is the mean of the values obtained from two or three mycelial samples which were harvested from different flasks. O--O, glucose 6-phosphate dehydrogenase; w - -H, nitrite reductase.

Fig. 2 shows the time courses of the increases in the specific

activities of glucose 6-phosphate dehydrogenase and nitrite

reductase in the wild type after the addition of nitrate to the

medium.

Test for Presence of Inhibitors or Activators of Glucose 6-Phos- phate Llehydrogenase in Wild Type LCxtracts-The activity of

glucose B-phosphate dehydrogenase in an extract prepared from

b&l grown with urea was completely additive with that in an extract prepared from bik1 grown with urea and nitrate when the extracts were mixed. This result suggests that either the extracts did not contain freely diffusible inhibitors or activators of glucose B-phosphate dehydrogenase or, if th’ey did, that these activators or inhibitors were saturated in the extracts containing

them. Polyacrylamide Gel Uectrophoresis-Samples of mycelium of

biA-1 which had been grown with 1% glucose + 5 mM urea, with 1% glucose + 10 mM NaN03, and with 1 Y0 glucose + 5 mM urea + 10 rnhz NaN03 were used. Extracts were prepared from each of these mycelial samples and also from a mixture containing mycelium grown with 5 mM urea and mycelium grown with 10 mM NaN03. Each sample was extracted with 50 mM Tris-HCl, pH 8.0, and with 50 InM Tris-HCl, pH 8.0, containing 0.5 mM NADP+. (These two extraction media were used because it has been reported that when extracts of Neurospora crassa were prepared with buffer containing 0.5 mM

NAIlI’+ they produced only one band of 6-phosphogluconate dehydrogenase activity, but when they were prepared in the absence of NADP+, they produced three bands of 6-phospho- gluconate dehydrogenase (30) .) Electrophoresis was carried out with 7% gels for all extracts and with 10% gels for some, and the gels were stained for glucose 6-phosphate dehydrogenase activity.

All of the gels produced only one band of glucose g-phosphate dehydrogenase activity.

Activities of Enzymes of Carbohydrate Metabolism in Mutants of Nitrate Reduction Pathway-The results obtained with mu- tants lacking nitrate reductase (the niaD- and cnx- mutants)

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are presented in Table III. Those obtained with a mutant defective in the nitrite reduct,ase structural gene (niiA-4) and with mutants of the regulatory gene of the nitrate reduction pathway (nirA- -1 and nirAC-1) are presented in Table IV and those obtained with various doubly mutant strains are pre- sented in Table V. These results may be summarized as fol- lows.

1. All mutant strains examined were similar to the wild type strains with respect to the activities of those enzymes which in the wild type strains are unaffected by the presence or absence of nitrate (i.e. phosphofructokinase, aldolase, pyruvate kinase, and NADP-isocitrate dehydrogenase (ribose phosphate isomerase was not assayed)).

2. Considering the activities of those enzymes which in wild type strains are higher in the presence of nitrate than in its absence (i.e. glucose B-phosphate dehydrogenase, 6-phospho- gluconate dehydrogenase, transketolase, and glucose phosphate isomerase (transaldolase was not assayed)), we may conclude the following.

a. When they were grown in the absence of nitrate as well as in its presence, those niaD- and cnx- mutants which were con- stitutive for nitrite reductase (i.e. those which had high ac- t,ivities of nitrite reductase even when they are grown without, nitrate) possessed the high activities of these enzymes which the wild type strains possessed only when they were grown in the presence of nitrate.

b. Those niaD- and cnx- mutants which were inducible for nitrite reductase (i.e. those which had high activities of nitrite reductase only when they were grown with nitrate), and also &A-4 behaved similarly to the wild type strains in that they possessed the high activities of these enzymes only when they were grown in the presence of nitrate.

c. The mutant nirA--l, even when grown in the presence of nitrate, had the low activities of these enzymes typical of the wild type grown without nitrate.

3. Extensive data were obtained only for the enzyme glucose 6-phosphate dehydrogenase in the mutant nirAC-I When grown in the absence of nitrate, this mutant possessed activity of glu- cose 6-phosphate dehydrogenase which was significantly higher (at the 5% level of significance) than the activity in the wild type strain grown under the same condition.

4. Considering the activities of glucose B-phosphate dehy- drogenase and nit.rite reductase in the double mutants and in the corresponding single mutants, the following conclusions may be made concerning the effect of the interaction of the mutations in question on t)he regulation of these two enzymes.

a. The niaD-17 cnxG-2 double mutant resembled nid-17

rather than cnxG-2 in that it possessed high activities of glucose 6-phosphate dehydrogenase and nitrite reductase even when it was grown without nitrate. Thus n&D-17 is epist.atic to cnxG-2 with respect to both activities.

b. With respect to glucose 6-phosphate dehydrogenase ac- tivity, niaD-17 niiA-4 resembled niaD-17 rather than niiA-4 and cnxG-4 niiA-4 resembled cnxG-4 rather than niiA-4, since the double mutants possessed high activities of this enzyme even when they were grown without nitrate. Thus, both niaD- 17 and cnxG-4 are epistatic to &A-4 with regard to the activity of glucose 6-phosphate dehydrogenase. The niiA-4 mutation abolishes nitrite reductase activity in both these double mu- tants and, therefore, nothing can be deduced from the results obtained from them concerning the effect of the interaction of the two mutations in question on the regulation of the enzymes of the nitrate reduction pathway.

c. niaD-17 nirA--l resembled nir.A--1 rather than niul)-I7

and cnzG-4 nir.A--1 resembled nirA-1 rather than cnrG-4 in that these double mutants possessed low activities of glucosic 6-phosphate dehydrogenase and nitrite retluctasc even when they were grown in the presence of nitrate. Thus, &X--l is epistatic to both &D-17 and cnxG-4 with respect to the a(:- tivities of both of these enzymes.

5. niaD- and cnx- mutants generally posscssctl somewhat higher activities of glucose G-phosphate dehydrogenasc when

they were grown on urea + nitrate than did the wild type strains. Also, when those &I>- and cnz- mutants which arc constitutive for nitrite reductase were grown on urea, they possessed ac- tivities of glucose 6-phosphate dehydrogcnase which were some- what higher than those in wild type strains grown on urea + nitrate.

Growth of the wild type 9. nidulans in the presence of nitrate compared with growth in its absence led to approximat,cly 2-fold higher activities of four enzymes of the pentose phosphate path- way and 3-fold higher activity of one enzyme, glucose phosphate isomerase, which is common to the pentose phosphate pathway and the Embden-Meyerhof-Parnas pathway. The activities of one enzyme of the pentose phosphate pathway, ribose phosphate isomerase, of NADP-isocitrate dehydrogenase, and of three enzymes of the EmbdenMeyerhof-Parnas pathway were either not affected or only slightly affected by the prcsencc of nitrate in the growth medium.

No investigation was undertaken to ascertain whether the increases in the activities of the pentosc phosphate pathway enzymes result from increases in their rates of synthesis, al- though, in the case of glucose B-phosphate dehydrogcnase, the results indicate that simple inhibition or activation of enzyme activity is not involved.

Carter and Bull (31) showed that an increase in the growth rate of A. niduhns in continuous culture led to an increase in the proportion of glucose metabolized through the osidative pentose phosphate pathway and resulted in a greater elevation in the activity of glucose B-phosphate dehydrogenase than in the activities of hexokinase or aldolase. They also found that, the proportion of glucose metabolized through the oxidativc pentose phosphate pathway increased during growth in batch culture to reach a maximum at the late esponential phase. When 1% glucose is the i&ial carbon source t,he wild type A. niduluns, b&l, grows faster and achieves a greater maximum growth yield with 5 mM urea + 10 mM NaNOs as initial nitrogen source than with 5 mM urea as the initial nitrogen source.* The following results show, however, that the elevations in the ac- tivities of the pentose phosphate pathway enzymes which result from growth with 5 InM urea + 10 mM NaNOJ do not depend upon the stimulation of growth which is caused by the presence of nitrate in the medium. (a) Nitrate caused increases in the activities of these enzymes in certain n&T)- and cnx- mutants although these mutants are unable to metabolize nitrate @I Other niuD- and cnz- mutants possessed the high activities of these enzymes even when they were grown in the absence of nitrate.

The response of A. niduluns to nitrate differs from that of C. utilis. In C. utilis nitrate causes marked increases in the activities of glucose B-phosphate dehydrogenase and trans- ketolase, but only marginal increases in the activities of phos-

2 0. Hankinson, unpublished observations.

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TABL

E III

Spec

iJic

ac

tivitie

s of

en

zym

es

of

carb

ohyd

rate

m

etab

olism

in

nia

D-

and

cnx-

wr

utan

t st

rain

s

The

stra

ins

were

gr

own

with

1%

gl

ucos

e an

d wi

th

eith

er

5 m

M

urea

or

5

rnM

urea

+

10 m

M

NaN0

3 as

ini

tial

nitro

gen

sour

ce.

Myc

elium

wa

s ha

rves

ted

afte

r 21

ho

urs.

I Sp

ecifi

c ac

tivitie

s of

:

zA-2

W

A-3

0 11

4 61

7 10

80

200

592

rA-2

iA

- cn

xA-2

89

93

14

80

1390

71

4 62

6 LA

-2

;A-3

FX

A-4

0 13

9 74

7 16

30

290

684

rA-2

GA

-3

r&7

72

114

1320

14

30

626

616

rA-2

GA

-3

- ;IT

;;D-8

-

1 16

5 47

1 13

00

259

585

zA-2

W

A-3

0 14

1 87

9 15

50

534

901

xA-2

GA

-3

niaD-

8 0

140

560

1480

37

9 87

6 rA

-2

;;A-3

;IT

ao-1

4 67

55

14

10

1500

82

8 88

1 xA

-2

VA-3

-

- a-1

0 17

0 69

1 16

10

420

953

biA-

1 2

176

756

1440

36

7 64

9 =A

-1

niaD

-I7

127

130

1880

19

90

702

775

=A-1

a-

2 0

213

793

2060

33

3 80

4 =A

-1

cnx0

4 16

0 13

1 22

10

1990

85

6 72

4 -

-

Tran

s-

keto

lase

Glu

cose

- Ph

osph

o-

NADP

-

phos

phat

e fru

cto-

Aldo

lase

Py

ruva

te

isocit

rate

isoai

eras

e kin

ase

kinas

e de

hydr

o-

gena

se

!

urea

+

urea

+

urea

+

urea

+

urea

+

urea

+

Irea

~~~0

3 ur

ea

NAN&

ur

ea

&NO3

ur

ea

NaNO

j lJr

ea

NaN0

3 ur

ea

NaNO

s

43

39

99

100

20

18

70

124

30

27

97

Ill

27

36

88

95

262

480

2850

98

50

56

72

722

834

1200

14

20

109

99

480

504

8870

91

50

61

61

625

753

1110

11

60

103

121

332

598

2580

66

90

70

65

648

488

1080

88

0 84

10

0 67

7 73

2 70

40

8210

50

54

55

0 63

7 87

4 92

3 11

0 11

0

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2351

TABLE IV

Specijk activities of enzymes of carbohydrate metabolism in niiA-4, nirA--1, and nirAc:-f muta?lt strains

Procedures were as for Table III.

L Specific acrivitiea of:

- ’ ’ I Strain 1

biA- 1 618 1440 359 717 iTA-1 nirA'-I 904 ,210 555 677 n-1 XA--I 459 324 280 ,*a - -

bikl 6;A-I nirAC-I t?A-I =A--1 RA-I ;;;TA-4 - -

1 169 IWO ISSO 475 786 490 655 2460 6500 41 Y '030 2280 550 876 425 912 2400 8230

2 741 863 449 382 379 398 1850 1890 0 0 lOB0 3290 532 830 400 477 2550 5440

biA-I 640 1080 EA-I - nirA'-I 915 1480

biA- I 714 1210 EA-1 &A=-1 900 2050

biA-1 809 1750 EA-I &AC-l 1360 2270

TABLE V

Specific activities of enzymes of carbohydrate metabolism in various doubly mutant strains

Procedures were as for Table III.

Nitrogen source + 1 urea J”iz&: Iurea kt,:

Strain J

biA- 1 0 162 598 1140 EA-1 cnxG-2 niaD-17 126 123 1740 1750 i;iA-1 GG-4 ;;z;D-17 87 86 1620 1620 CA- 1 XD- 17TiA-4 i 1 1630 1760 nA-1 =A-4 GG-4 GA-4 0 1350 1450 KA-I nlaD-17 =A--1 0 1 501 509 EA- 1 ZG-4 nx--1 6 2 516 486 - - -

phogluconate dehydrogenase and transaldolase (4). The ac- tivity of glucose phosphate isomerase was not measured in this study.

In various organisms, NADPH can be generated by the action of malic enzyme, pyridine nucleotide transhydrogenase, or man- nitol dehydrogenase. It is important to consider whether these activities provide NADPH in A. n&duns.

Malic enzyme activity could not be detected in extracts of A.

nidulans. There have been reports both of the presence (32) and absence (33) of this activity in A. niger extracts. Eagon (34) found only very low activities of pyridine nucleotide transhydrogenase in extracts of the fungi Penicillium chrysogenum and Saccharomyces cerevisiae, while Hochster (35) found no such activity in Aspergillus flavus oryzae. Results to be presented elsewhere* suggest that in A. nidulans growing on glucose very little NADPH is generated by mannitol dehydrogenase.

It is therefore probable that glucose 6-phosphate dehydrogen-

8 0. Hankinson and D. J. Cove, manuscript in preparation.

ase, 6-phosphogluconate dehydrogenase, and NADP-isocitrate dehydrogenase provide the major proportion of NADPH in glu- cose-grown A. nidulans. Furthermore, the results suggest that during growth with nitrate, NADP-isocitrate dehydrogenase contributes less NADPH than do the dehydrogenases of the pentose phosphate pathway.

The end-products of the pentose phosphate pathway, fructose 6-phosphate and glyceraldehyde 3-phosphate, are produced in the ratio of 2 molecules of the former to 1 of the latter. Of the reutilizable carbon, 80% is therefore in the form of fructose 6- phosphate. It is probable that the increase in activity of glucose phosphate isomerase which occurs during growth with nitrate permits extensive recycling of fructose 6-phosphate through the pentose phosphate pathway. It is possible that such recycling also occurs during growth in the absence of nitrate. Although the activity of glucose phosphate isomerase is considerably higher than the activities of the pentose phosphate pathway en- zymes, glucose phosphate isomerase competes with both phos- phofructokinase and mannitol l-phosphate dehydrogenase for fructose 6-phosphate.

It has been reported that no recycling through the pentose phosphate pathway occurs in the yeast Rhoduturula gracilis growing with glucose and NH,NOa (36). However, when A.

nidulans is grown with NH4NOa, it possesses activities of the en- zymes of the nitrate reduction pathway which are considerably lower than those it possesses when it is grown with urea + nitrate (8), and growth with NHdN03 is probably more equivalent to growth with urea than to growth with urea + nitrate with re- spect to both the rate of oxidation of NADPH and the activities of the pentose phosphate pathway enzymes.

Multiple forms of glucose g-phosphate dehydrogenase (37), of 6-phosphogluconate dehydrogenase (37, 38)) transketolase (39)) and transaldolase (40) have been demonstrated in various fungi. It was considered possible that growth of A. nidulans with ni- trate might lead to the appearance of isozymes not present in urea-grown mycelium. However, only one form of glucose 6- phosphate dehydrogenase could be demonstrated in A. nidulans

extracts. Clearly, further efforts to detect isozymes of the pen- tose phosphate pathway enzymes of A. nidulans need to be made.

When the wild type A. nidulans is growing with urea and ni- trate it differs from when it is growing with urea in the following respects. (a) It grows faster (when 1% glucose is the carbon source). (b) Its intracellular concentrations of nitrate and ni- trite are higher. (c) It oxidizes NADPH to NADP+ at a greater rate. (d) It has higher activities of nitrate reductase and nitrite-hydroxylamine reductase. (e) The nitrate reductase is bound with nitrate and the nitrite-hydroxylamine reductase with nitrite. (f) The nir product is in an active state.

The properties of the mutants provide indications as to which of these changes is most directly involved in bringing about the increases in the activities of the enzymes of the pentose phosphate pathway.

As has already been discussed, these increases do not result from an increase in the growth rate which nitrate causes under some conditions.

Since some niaD- and cnx- mutants have high activities of the pentose phosphate pathway enzymes even when they are grown in the absence of nitrate, neither nitrate nor nitrite can be directly responsible for increasing the activities of the pentose phosphate pathway enzymes. The fact that nitrate does not promote an increase in the activities of these enzymes in nirA--1 also indicates that nitrate cannot be directly responsible.

During the reduction of nitrate to ammonium, NADPH is

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oxidized to NADP+. Some niaD- and cnz- mutants are sub- ject to the nitrate-promoted increase in the activities of the pen- tose phosphate pathway enzymes, although they are incapable of reducing nitrat,e. r&A-4 is only capable of reducing nitrate to nitrite but it also remains fully responsive to nitrate. Some niaD- and cnx- mutants have the high activities of the pentose phosphate pathway enzymes when they are grown in the absence of nitrate. These results suggest, but do not provide conclusive evidence (see below), that the increases in the activities of the pentose phosphate pathway enzymes are not caused by a change in the concentration of NADP+ or NADPH.

In the wild type, nitrate causes increases in NADPH-dia- phorase activities with several electron acceptors (cytochrome c, iodophenylnitrophenyltetrazolium, and potassium ferricyanide) (8). Nitrite-hydroxylamine reductase and the product of the niaD gene are responsible for these activities (8). The integrity of the cnx cofactor is not required for the expression of these ac- tivities by the niaD product (8, 9, 11, 12). All of the niaD- and cnx- mutants which have been shown in this paper to have the high activities of the pentose phosphate pathway enzymes even when they are grown without nitrate are constitutive for nitrite reductase (and are probably also constitutive for the n&D product), whereas those that have the high activities of the pentose phosphate pathway enzymes only when they are grown in the presence of nitrate are inducible for nitrite reductase. Therefore, in these mutants, high activities of the pentose phos- phate pathway enzymes should be associated with high activities of the above mentioned diaphorase activities. If nitrite-hy- droxylamine reductase and the n&D product have “diaphorase” activities towards natural electron acceptors (besides nitrate and nitrite) which occur in a soluble form in the cytoplasm, then it is possible that a change in concentration of NADP+ or NADPH produced by these hypothetical activities effects the increases in the pentose phosphate pathway enzyme activities.

The fact that two of the niaD- mutants studied, niaD-3 and n&D-14, have very low niaD product associated activity for NADPH-cytochrome c reductase (9, la), yet have high activities of the pentose phosphate pathway enzymes even when they are grown without nitrate, provides some evidence against this last hypothesis. Stronger evidence against it is provided by results presented elsewhere (41), indicating that those niaD- and cnx- mutants which have high activities of the pentose phosphate pathway enzymes even when they are grown without nitrate, do not, in fact have an enhanced ability to oxidize NADPH to NADP+ when they are grown without nitrate.

In all of the strains studied, except those possessing the niiA-4 mutation, high activities of the pentosc phosphate path- way enzymes are associated with high activities of nitrite-hy- droxylamine reductase. Nitrite-hydroxylamine reductasc might be an inducer of the pentose phosphate pathway enzymes. How- ever, if this is the case, the GA-4 mutation must have led to the loss of the enzymic activit,y but not to the loss of the hypo- thetical regulatory function of the nitrite-hydroxylamine reduc- tase molecule.

A hypothesis in which it is proposed that nitrate reductase is a repressor of the pentose phosphate pathway enzymes and that combination of nitrate reductase with nitrate relieves this re- pression seem consistent with the findings of this paper. (AC-

cording to this hypothesis, the act,ivities of the pentose phosphate pathway enzymes are regulated in the same manner as is the activity of the nir product.) This hypothesis is compatible with the behavior of the wild type, n&4-4, and the niaD- and cnx- mutants. It is not, however, compatible with the results ob-

tained for either &AC-l or &A--l. nirA--1 provides the more

convincing evidence against it. The fact that nirA--1 possesses the low activities of the pentose phosphate pathway enzymes when it is grown in the absence of nitrate could be explained through this hypothesis by the low “escape” synthesis of nitrate reductase which occurs in this mutant (8). However, the hy- pothesis cannot explain the fact that GA--l does not respond to the inductive effect of nitrate on the pentose phosphate path- way enzymes.

The only relatively simple hypothesis that can be proposed which is compatible with all of the findings is as follows. The nir product brings about the increases in the activities of the pentose phosphate pathway enzymes. The state of activity of the nir product for its stimulatory effect on the activities of the pentose phosphate pathway enzymes is regulated in exactly the same way as is its state of activity for inducing the enzymes of the nitrate reduction pathway.

In all situations studied, the higher activities of the pentose phosphate pathway enzymes occur under conditions for which it can be inferred that the nir product is present in a state that is active in inducing the enzymes of the nitrate reduction pathway, and the low activities occur under conditions for which inac- tivity of the nir product can be inferred. +A”-1 might appear to be an exception to this rule, since this mutant, when grown on urea, appears to have act.ivity of glucose B-phosphate dehydro- genase only slightly greater than that of the wild type. G-AC-l is also, however, only semiconstitutive for nitrite-hydroxylamine reductase (16).

Acknowledgments-We should like to thank Dr. H. N. Arst,

Jr., and Dr. D. W. MacDonald for helpful discussions.

1. HANKINSON, O., AND COVE, D. J. (1972) Biochem. J. 127, 18P- 19P

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HANKINSON, O., AND COVE:, I>. J. (1972) Heredity 28, 276 COVF:, 1). J., ARST, H. N., AND SCAZZOCCHIO, C. (1974) in Ad-

vances in Molecular Genetics (FBLCONER, .I., AND COVE, D. J., eds) Vol. 1, Paul Elek (Scientific Books) Ltd., London

OSMOND, C. B., AND AP REM, T. (1969) Lliochim. Biophys. Acta 184, 3542

5. HOLLIGAN, P. M., AND JENNINGS, D. H. (1972) New Phytol. 71, 1119-1133

6. COVE, D. J., AND PATEMAN, J. A. (1969) J. Bacferiol. 97, 1374- 1378

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PATXMI\N. J. A.. COVE. D. J.. REVER. B. M.. AND ROBERTS.

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Mol. Gen. Gellet. 108, 129-145 MACDONALD. D. W. (1969) Ph.D. thesis, Universitv of East

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HOLL, F. B. (1971) Ph.D. thesis, University of Cambridge, U. K.

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Oliver Hankinson and David J. CoveTHE EFFECT OF GROWTH WITH NITRATE

:Aspergillus nidulansRegulation of the Pentose Phosphate Pathway in the Fungus

1974, 249:2344-2353.J. Biol. Chem. 

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