Modulation of nitrate uptake in Anacystis nidulans by the balance between ammonium assimilation and CO2 fixation

ARCHIVES

Vol.

OF BIOCHEMISTRY

256, No. 2, August

AND BIOPHYSICS 1, pp. 5’78-584, 1987

Modulation of Nitrate Uptake in Anacystis nidulans by the Balance between Ammonium Assimilation and CO2 Fixation’ JOSfi M. ROMERO, AND

TOMAS MIGUEL

CORONIL, CATALINA G. GUERRERO

LARA,

Departamento de Bioquimica, Facultad de Biobgib y Consejo Superior Investigackmes Cient$icas, Apart&o 1095, $1080 Se-villa, Spain Received

December

1,1986,

and in revised

form

April

de

7,1987

Gradual inhibition of ammonium assimilation in Anmystis niduluns cells by increasing concentrations of 5hydroxylysine resulted in a progressive enhancement of nitrate uptake. For &hydroxylysine-treated cells, the magnitude of the inhibition of nitrate uptake promoted by added ammonium was dependent on the ammonium assimilation capacity. In cells with a moderate ammonium assimilation activity, acceleration of COz fixation induced by bicarbonate addition antagonized the negative effect of ammonium, allowing full nitrate uptake activity. The results support the contention that nitrate utilization is under the feed-back control exerted by products of its own assimilation via ammonium, the inhibitory effect being potentiated by ammonium addition and alleviated by enhanced CO2 fixation. Results of amino acid analysis in cells exhibiting different capacities to utilize nitrate speak against these compounds as direct effecters of nitrate uptake. 0 1987 Academic Press, Inc. Nitrate utilization by cyanobacteria is promptly and severely inhibited by the presence of ammonium in the outer medium at concentrations as low as 10 PM. This inhibition is reversible, the uptake of nitrate being resumed as soon as the added ammonium is exhausted (l-4). The ammonium effect is prevented by inhibitors of ammonium assimilation, such as L-methionine DL-sulfoximine (MSX)3 (Z-4), azaserine (2-4), and 5-hydroxylysine (HYL) (4, 5), indicating that organic nitrogenous compounds mediate the ammonium effect. The nature of these effecters is still an open question, although amino acids such as

glutamine or glutamine derivatives appear as plausible candidates (3, 4). On the other hand, a tight correlation between the rate of nitrate utilization and that of COz fixation has been evidenced for the cyanobacterium Anacystis niduhns. Interestingly, prevention of ammonium assimilation results in the release of nitrate uptake from its dependence on CO2 fixation (6-9). This and other evidence (see (4)) suggest a common link between the mechanism of the carbon dependence and that of the ammonium inhibition of nitrate uptake, and a model has been proposed for the regulation of nitrate uptake involving the concerted participation of assimilation products of both ammonium and COz (6). In cells with intact ammonium assimilation capacity, an antagonism between ammonium and CO:! in the control of nitrate uptake is not readily apparent, since ammonium overcomes any positive effect of COZ. In this communication we present results concerning the effect of gradually in-

1 Supported by Grant BT85/0045 from Comision Asesora de Investigacibn, Spain. a To whom correspondence and requests for reprints should be addressed. ’ Abbreviations used: chl, chlorophyll a; Cit. citrulline; HYL, 5-hydroxylysine; MSX, L-methionine DL-sulfoximine; Tricine, N-[2-hydroxy-l,l-bis(hydroxymethyl)ethyllglycine.

0003-9861/87 Copyright All righta

$3.00

Q 198’7 by Academic Press. Inc. of reproduction in any form reserved.

578

FEED-BACK

REGULATION

OF

hibiting

ammonium assimilation in intact cells with HYL, a reversible inhibitor of glutamine synthetase (lo), on nitrate utilization in the presence and absence of added ammonium. The effect of CO, availability on the inhibition by ammonium of nitrate uptake has been comparatively studied in normal untreated and HYL-treated cells. The results clearly show that the magnitude of the ammonium-induced inhibition of nitrate uptake depends on the capacity of the cells to assimilate ammonium, and illustrate the antagonism between ammonium and CO2 on the control of nitrate utilization. Indications against a direct involvement of amino acids in the regulation of nitrate uptake have also been obtained. A. nidulans

MATERIALS

AND

METHODS

Organism and culture conditions. A. nidulans 1402I (Synechococcus leopoliensis, GSttingen University, FRG) was grown photoautotrophically at 40°C with uitrate as the sole nitrogen source as described previously (11). Cells were harvested by centrifugation after 24 h growth (15-20 pg chlorophyll a (chl) ml-‘), washed with 25 mM Tricine-NaOH-KOH buffer, pH X.3, and resuspended in the same buffer. Chlorophyll a was estimated after extraction with methanol using the extinction coefficient given in (12). Nitrate and ammonium uptake. The assays were carried out at 40°C with continuous shaking and illumination (160 PE m-* s-i of photosynthetically active radiation (9)) in open conical flasks. The assay medium contained, in a volume of 4 ml: 100 pmol TricineNaOH-KOH buffer, pH 8.3; 1 rmol of either KNOa or NH&l; an amount of cells equivalent to 40 Fg chl; and, when indicated, 40 pmol NaHC03. The reaction was started by simultaneously switching on the light and adding KN03 and/or NH&l and, when required, NaHC03. At regular intervals, 0.5-ml aliquots were withdrawn and, after rapid removal of the cells by filtration (Millipore HA 0.45 pm pore size filter), nitrate or ammonium was determined in the filtrates. Nitrate was determined by optical absorption at 210 nm in acid solution (13), and ammonium was determined by the glutamate dehydrogenase method (14). Treatment with HYL was performed by adding the adequate amount of the compound to the illuminated cell suspensions 15 min before starting the assay. Amino acid analysis. Free amino acids were analyzed by reverse-phase HPLC (15) in the supernatant (12,OOOg, 30 min) of acid cell lysates obtained by adding

NITRATE

UPTAKE

IN

579

Anacystis

aliquots of cell suspensions equivalent to 9 pg chl onto ice-cooled HCl to a final concentration of 0.2 N. Prior to analysis, the samples were neutralized with 0.4 M borate buffer, pH 10, and derivatized with o-phthaldialdehyde/@-mercaptoethanol reagent (a mixture of 0.75 vol of a methanolic solution of o-phthaldialdehyde (20 mM) and /3-mercaptoethanol(57 mM) and 0.25 vol of 0.4 M borate buffer, pH 10) for 2 min. Samples were applied onto a Waters Novapack C-18 column (3.9 X 150 mm, 4 pm particle size) and eluted at 45°C with a gradient of decreasing polarity formed between a mixture of phosphate-acetate buffer (30 mM, pH 6.5): methanol:tetrahydrofuran (96:2:2) and a mixture of methanol:water (65:35), at a flow rate of 1.5 ml mini. The derivatized amino acids were fluorometrically detected (excitation wavelength, 340 nm; emission wavelength, 425 nm). The HPLC equipment was purchased from Waters. Identification of the corresponding peaks was achieved by comparing their retention times with those of standard amino acids, which were also used for calibrating the system. The method does not allow the determination of Pro and Cys. Commercial HYL contained derivatizable amino compounds, which coeluted with Ile, Leu, Lys, Met, Orn, Phe, Ser, Trp, and Val. Therefore these amino acids could not be accurately determined in HYL-treated cells. For calculation of the intracellular levels, the obtained values were corrected for amino acids excreted by the cells to the assay medium, determined in the supernatant of cell suspension aliquots after removing the cells by centrifugation (lO,OOOg, 1.5 min) through a layer of silicone oil (a 21 mixture of Versilube F50 (Aldrich) and silicone 14615-3 from Jansen Chimica). Even after a short assay time (lo-20 min) and using this gentle centrifugation procedure, the relative amount of amino acids found in the outer medium was significant, although it varied for the individual amino acids. Ala, Gly, and Ser were the most abundant. Chemicals. Tricine, ADP, 5-hydroxylysine, standard amino acids, and L-glutamate dehydrogenase were from Sigma Chemical Co. (St. Louis, MO); NADPH was from Boehringer (Mannheim, FRG). Other chemicals were products of Merck (Darmstadt, FRG) and Scharlau (Barcelona, Spain).

RESULTS

Nitrate Uptake by A. nidulans Treated with CHydroxylysine

Cells

Treatment of Anacystis cells with HYL resulted in a concentration-dependent modification of the rate of nitrate uptake (Fig. 1). Concentrations of HYL below 1.5

580

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-.

.-

0

5 [5-hydrorylyslnel

10 (mM)

FIG. 1. Effect of 5-hydroxylysine on nitrate uptake by A. niduhns cells. Cell suspensions (10 ng chl ml-‘) were preincubated for 15 min in the light with HYL at the indicated concentrations. The assay was initiated by addition of KN03 (0.25 mM) and NaHCOa (10 mM) and proceeded for 30 min. 100% corresponds to a rate of 24 rmol nitrate taken up per milligram chl per hour.

were moderately inhibitory, whereas higher HYL concentrations progressively stimulated nitrate uptake to reach, at 10 mM HYL, rates up to 160% of those in untreated cells. The negative effect of HYL at low concentrations on nitrate uptake appears to be due to small amounts of ammonium released from HYL by oxidative deamination (16), and was particularly evident under COz-limiting conditions (data not shown). Treatment of Anacystis cells with concentrations of HYL that stimulated nitrate uptake resulted in excretion by the cells, depending on the HYL concentration used, of part or all of the ammonium derived from reduction of the nitrate taken up (data not shown). Thus, nitrate uptake is enhanced when assimilation of ammonium derived from nitrate reduction is inhibited. It would appear, therefore, that nitrate uptake is under the feed-back control exerted by products of nitrate assimilation via ammonium (2), and that the positive effect of HYL treatment on nitrate uptake is due to the release of the process from such a control.

mM

ET

AL.

Inhibition of Ammonium Uptake by 5Hydroxylysine and Prevention of the Ammonium-Induced Inhibition of Nitrate Uptake As can be expected from a reversible inhibitor of cyanobacterial glutamine synthetase (lo), treatment of Anacystis cells with increasing concentrations of HYL resulted in gradual inhibition of ammonium uptake (Fig. 2A). The ammonium uptake rate was decreased by 50% at HYL concentrations of 1.5 ITIM in the external medium, the inhibition being virtually complete (95%) at 20 mM HYL. This gradual inhibition of ammonium utilization induced by HYL contrasts with the complete suppression of the process that takes place when cells are treated with MSX (3, 17), a powerful inactivator of GS. By treatment with different concentrations of HYL it is, therefore, possible to obtain Anucystis cells exhibiting different rates of ammonium utilization. The in viva inhibition of glutamine synthetase by HYL is apparent from the results in Table I. High levels of glutamine and lower levels of glutamate and other amino acids were present in Anacystis cells utilizing ammonium as the nitrogen source. Treatment of the cells with

[5-hydroxylyr~nel(mM)

[5- hydralylyrlnel

(mt.4)

FIG. 2. Effect of 5-hydroxylysine on ammonium uptake (A) and on nitrate uptake in the presence of added ammonium (B). General conditions were as in Fig. 1, except that NH&l (0.25 mM) was added instead of (A) or in addition to (B) nitrate. 100% in (A) corresponds to a rate of 49 rmol ammonium taken up per milligram chl per hour.

FEED-BACK TABLE

REGULATION

OF

I

INTRACELLULAR LEVELS OF FREE AMINO ACIDS IN UNTREATED AND HYL-TREATED A. nidulans CELLS Intracellular (nmol mg-’

_. Ala Asp Gin Glu Gly Other

Amino

amino

acid

acids”

levels (chl))

Untreated cells

HYL-treated cells

<1 30 5720 370 130 4

60 180
Note. Free amino acids were determined in the supernatant of acid lysates from cells untreated or pretreated with 10 mM HYL, after 15 min in the presence of NH&I (0.25 mM) and NaHC03 (10 mM) in the light. The obtained values have been corrected for amino acids excreted to the outer medium. Data are those of a representative experiment. Variability in the amount of each amino acid was observed in different experiments but the relative proportions of the different amino acids were fairly constant. a Asn, Arg, Cit, His, Thr, and Tyr. In untreated cells, also Ile, Leu, Lys, Met, Orn, Phe, Ser, Trp, and Val.

10 mM HYL, which inhibited ammonium uptake by 85% (Fig. 2A), resulted in a marked accumulation of Glu at the expense of the Gln pool, with no major changes in the levels of other amino acids analyzed, except for increases in Ala, Asp, and Gly. This indicates that the major effect of HYL in Anacystis cells is the inhibition of glutamine synthetase activity. Results in Fig. 2B show the rate of nitrate uptake in the presence of ammonium by Anacystis cell suspensions as a function of the HYL concentration with which the cells had been treated. In the absence of HYL, nitrate uptake was completely inhibited by ammonium. As the HYL concentration increased, nitrate uptake progressively increased, reaching rates similar to those of untreated cell suspensions in the absence of ammonium (about 30 pmol nitrate/mg chl per hour) at HYL concentrations of 5 MM, and higher thereafter. Thus, nitrate uptake was protected against

NITRATE

UPTAKE

IN

Anacystis

581

ammonium inhibition by HYL. Interestingly, the inhibitory effect of HYL on ammonium uptake (Fig. 2A) correlated positively with its prevention of the negative effect of ammonium on nitrate uptake (Fig. 2B). The correlation coefficient found for HYL-treated cells between the rates of ammonium uptake and the rates of nitrate uptake in the presence of ammonium, r = -0.954 (/3 < O.OOl), indicates that the magnitude of the ammonium-promoted inhibition of nitrate uptake depends on the ammonium assimilation capacity. Antagonism between Ammonium and CO, in the Control of Nitrate Uptake

The proposed antagonism between ammonium and COz in the modulation of nitrate uptake (6) is not readily apparent in normal A. nidulans cells with intact ammonium assimilation capacity. As shown in Fig. 3A, the increase in COz fixation promoted by bicarbonate addition resulted in enhanced nitrate uptake in cell suspensions kept in the absence of added ammonium,

6 Cl2

-T g 58 F : L s4 5

FIG. 3. Effect of bicarbonate addition on nitrate uptake by untreated (A) and HYL-treated (B) cells in the absence or presence of added ammonium. Cell suspensions (10 pg chl ml-‘) were preincubated for 15 min in the light under air in the absence (A) or presence of 2 mM HYL (B). At 0 time, 0.25 mM KN03 alone (open symbols) or plus 0.25 mM NH&l (closed symbols) was added. After 16 min (arrow), 10 mM NaHC03 was added to the cell suspensions.

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but had the opposite effect in cells exposed to ammonium. Thus, in normal untreated cells bicarbonate enhances the inhibitory effect of ammonium, as a logical result of the stimulation of ammonium assimilation (about fivefold) induced by bicarbonate (16). HYL-treated cells with restricted capacity for ammonium assimilation have proven, however, useful for verifying the above referred antagonism between ammonium and COz. Figure 3B shows that addition of bicarbonate to cells treated with 2 mM HYL completely released nitrate uptake from the inhibitory effect of ammonium. The resulting rate of nitrate uptake was virtually the same as that in cells kept in the absence of ammonium. It is worth mentioning that 2 mM HYL inhibits nitrate uptake by about 50% when air is the only source of COz; hence the low nitrate uptake activity exhibited by the HYLtreated cells prior to bicarbonate addition. These results indicate that nitrate uptake rates depend on the relative balance between rates of ammonium utilization and of COz fixation. The inhibitory effect of ammonium is overcome by enhancing COz fixation provided that a comparable increase in the ammonium utilization capacity does not take place.

TABLE

ET

AL.

Amino Acid Levels in Cells with &&rent Nitrate Uptake Capacity

The different rates of nitrate uptake observed in experimental situations such as those described above could respond to differences in the levels of some regulatory metabolites. Since amino acids appear to be the most immediate candidates as effectors of nitrate uptake (3, 4), levels of free amino acids have been determined in Anacystis cells showing different capacities to take up nitrate. Results obtained for experimental situations analogous to those shown in Fig. 3 are summarized in Table II. In the absence of ammonium, addition of bicarbonate to Anacystis cells, either untreated or treated with 2 mM HYL, resulted in a marked increase in the rate of nitrate uptake and in higher levels of Glu, Asp, and Ala, whereas the Gln pool remained unaltered (Table II). Other amino acids assayed either increased upon bicarbonate addition following the trend of Glu, Asp, and Ala or remained below the detection limit of the method (data not shown). In general terms, the availability of carbon skeletons seems to be limiting transamination reactions at air levels of COz. Upon acceleration of COz fix-

II

NITRATE UPTAKEAND INTRACELLLILARLEVELSOFFREEAMINOACIDSIN

A.nidulans

Intracellular (nmol mg-’ Condition

Nitrate uptake (gmol mg-’ (chl) h-l)

Gln

Glu

level (chl)) Asp

Ala

Untreated cells Air NaHCOa NH:, air NH:, NaHCOs

9 29 3 1

200 220 1050 560

1000 1620 450 1270

130 540 40 460

140 130 100 230

HYL-treated cells Air NaHC03 NH,+, air NH;, NaHCOa

6 26 0 22

0 0 130 130

1360 2420 980 2120

0 410 0 1200

30 390 20 640

Note. Conditions were as in Fig. 3. Aliquots of cell suspensions for amino acid analysis were withdrawn 10 min after initiating the experiment (air) and 10 min after addition of NaHCOa. The values determined in the supernatant of acid cell lysates have been corrected for amino acids excreted to the outer medium.

FEED-BACK

REGULATION

OF

ation by bicarbonate addition this limitation would disappear, resulting in both an increase in total amino-N and an enhancement of nitrate uptake. In the presence of ammonium, control, untreated cells exhibited higher levels of Gln and lower levels of Glu than in its absence (Table II). Addition of bicarbonate, which enhances ammonium utilization (see above) but not nitrate uptake, promoted an increase in the levels of Glu, Asp, and Ala, while the Gln pool decreased. This pattern could arise from an enhanced availability of amino-N acceptors. HYL-treated cells also had higher levels of Gln in the presence of ammonium. Addition of bicarbonate to these cells enhanced both the rate of nitrate uptake and the intracellular levels of most amino acids with the exception of Gln, which remained virtually unchanged (Table II). In summary, high nitrate uptake rates correspond to high levels of total intracellular free amino acids, a trend followed by most individual amino acids, with the particular exception of Gln. For high nitrate uptake rates, the Gln level was always low, and the converse (i.e., high Gln level and low nitrate uptake rate) was also true in general. Nevertheless, cases of low Gln level and low nitrate uptake rate were also found (Table II). No correlation was evident, therefore, between the size of the Gln pool and the rate of nitrate uptake. The data argue thus against a direct involvement of Gln per se, and of amino acids in general, as negative effecters of nitrate uptake. DISCUSSION

When ammonium

assimilation in A. niby treatment with HYL, nitrate uptake is stimulated, the extent of the stimulation being dependent on the HYL concentration used (Fig. 1). This indicates that in nitrate-utilizing cells the uptake of nitrate is controlled by the assimilation of the ammonium resulting from nitrate reduction. This feed-back control of nitrate uptake is exaggerated by addition of ammonium to the cell suspension, causing complete cessation of nitrate dulans cells is inhibited

NITRATE

UPTAKE

IN

Anac@ti

583

uptake. In HYL-treated cells, the rate of nitrate uptake in the presence of ammonium is inversely related to the rate of ammonium uptake (Fig. 2). This suggests that the control of nitrate uptake is exerted in a gradual fashion, the higher ammonium assimilation capacity the lower nitrate uptake activity. The existence of an antagonism between ammonium and COZ in the control of nitrate uptake becomes evident in HYLtreated cells. When ammonium assimilation is partially limited by HYL treatment, acceleration of CO:! fixation induced by transition from COa-limiting to COz-saturating conditions results in abolishment of the negative effect of ammonium (Fig. 3). These results give strong support to the model proposed for the regulation of nitrate uptake involving the concerted participation of both ammonium assimilation and CO, fixation products (6,8). According to this model, accumulation of some organic nitrogenous compound(s) will inhibit nitrate uptake. The intracellular level of the inhibitor(s) would be determined both by the supply of ammonium from which they are generated and by the provision of some COB-fixation products. The latter would combine with the inhibitory ammonium derivatives, removing them while generating other noninhibitory C,N-intermediates. The gradual inhibition of ammonium assimilation would thus result in a progressively enhanced ability to take up nitrate in the presence of ammonium and, for a given ammonium assimilation capacity, acceleration of COB fixation would favor the removal of accumulated inhibitors of nitrate uptake. This would lead to a loss of the ammonium inhibition. The balance between the relative rates of ammonium assimilation and COz fixation could, therefore, determine the rate of nitrate uptake. The operation of such a regulatory system would allow a fine modulation of nitrate utilization exerted not only by accumulation of some of its own products, but also through the availability of carbon metabolites. Some key issues remain to be solved for a better understanding of this regulatory system, among them, the identification of

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the ammonium derivative(s) acting as negative effector of nitrate uptake. The intracellular levels of these effecters should (a) increase in response to ammonium addition and (b) decrease in response to increased COP fixation. Amino acids, being early products of ammonium assimilation, appear to be good candidates for the role of effecters of nitrate uptake (18). Glutamine is particularly attractive in this regard, since its level is high under ammonium-assimilating conditions and very low when ammonium assimilation is impeded by either MSX (2,3) or HYL (Table I). Also, addition of glutamine to MSX-treated Anacystis cells inhibited nitrate uptake (18). The present results argue against the involvement of amino acids in general and of glutamine in particular as direct negative effecters of nitrate uptake. In general terms, high amino acid levels are found under conditions in which the nitrate uptake rate is high. Only Gln increased after ammonium addition, but remained unaltered after addition of bicarbonate in those situations in which the nitrate uptake rate resulted simultaneously enhanced. This behavior was particularly noticeable in HYL-treated cells, in which nitrate uptake rate was either low or high, depending on the COB availability, whereas the glutamine levels remained practically unchanged. No correlation exists, therefore, between the glutamine pool and the rate of nitrate uptake. The data suggest that glutamine per se is not a direct inhibitor of nitrate uptake. Nevertheless, it may be involved in the feedback control of nitrate uptake. Glutamine could be the precursor of the actual inhibitor. In favor of this possibility are the observations that in Anacystis cells treated with azaserine, nitrate uptake is insensitive both to ammonium addition (3) and to selective inhibition of CO2 fixation (8), despite exhibiting high Gln levels (2,3). This could indicate that transfer of amide-N from Gln is required to promote inhibition of nitrate uptake (3,4). Additionally, glutamine could be involved in a complex control mechanism involving more than one effector. In any case, the possibility of glutamine derivatives other than amino acids, such as carbamyl phos-

ET

AL.

phate, nucleosides, or nucleotides acting as negative effecters must be kept in mind when considering the feedback regulation of nitrate uptake. ACKNOWLEDGMENTS

We are grateful to M. Losada for generous encouragement and to A. Friend and J. Perez de Le6n for skillful secretarial assistance. REFERENCES 1. OHMORI, M., OHMORI, K., AND STROTMANN, H. (1977) Arch. MicrobioL 114,225-229. 2. FLORES, E., GUERRERO, M. G., AND LOSADA, M. (1980) Arch. Micro6ioL 128.137-144. 3. FLORES,E., RAMOS, J. L., HERRERO,A., AND GUERRERO,M. G. (1983) in Photosynthetic Prokaryotes: Ceil Differentiation and Function (Papageorgiou, G. C., and Packer, L., Eds.), pp. 363387, Elsevier, New York. 4. GUERRERO,M. G., AND LARA, C. (1987) in The Cyanobacteria (Van Baalen, C., and Fay, P., Eds.), pp. 163-186, Elsevier, Amsterdam. 5. FLORES, E. (1982) Ph.D. thesis, University of Sevilla. 6. FLORES, E., ROMERO,J. M., GUERRERO,M. G., AND LOSADA, M. (1983) B&him. Bisphys. Actu 725, 529-532.

7. LARA, C., ROMERO, J. M., FLORES, E., GUERRERO, M. G., AND LOSADA, M. (1984) in Advances in Photosynthesis Research (Sybesma, C., Ed.), Vol. II, pp. 715-718, Martinus Nijhoff/Dr. Junk, The Hague. 8. ROMERO, J. M., LARA, C., AND GUERRERO, M. G. (1985) Arch. Biochem Biophys. 237,396-401. 9. LARA, C., AND ROMERO,J. M. (1986) Plant PhysioL 81,686-688.

10. LADHA, J. K., ROWELL, P., AND STEWART, W. D. P. (1978) Biochem 696.

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83,688-

11. HERRERO, A., FLORES, E., AND GUERRERO, M. G. (1981) J. BacterioL 145,175-180. 12. MACKINNEY, G. (1941) J. BioL Chem 140,315-322. 13. CAWSE, P. A. (1967) Analyst 92,311-315. 14. BERGMEYER, H. U. (1974) Methoden der Enzymatischen Analyse, 2nd ed, Verlag Chemie, Weinheim. 15. FLEURY, M. O., AND ASHLEY, D. V. (1983) Anal. Biochem.

133,330-335.

16. ROMERO, J. M. (1986) Ph.D. thesis, University Sevilla.

of

17. STEWART, W. D. P., AND ROWELL, P. (1975) Biochem Biophys. Res. Commun. 65,846-856. 18. ROMERO, J. M., FLORES, E., AND GUERRERO, M. G. (1985) Arch MicrobioL 142, l-5.