In situ behavior of the pyrimidine pathway enzymes in Saccharomyces cerevisiae

ARCHIVES OF BIOCHEMISTRY Vol. 225, No. 2, September,

AND BIOPHYSICS pp. 562-575, 1983

In Situ Behavior of the Pyrimidine Pathway Enzymes in Saccharomyces cerevisiae I. Catalytic

and Regulatory

BERNADETTE Lahrratoire

d %n.zyndogie, Received

of Aspartate

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Centre National October

Properties

de la

27, 1982, and

AND

GUY HERVI?

Recherche S&e&$*, in revised

Transcarbamylase

form

March

91190 Gif-sur- Yvette, France 21, 1983

A permeabilization procedure was adapted to allow the in situ determination of aspartate transcarbamylase activity in Saccharom~ces oerevtiiae. Permeabilization is obtained by treating cell suspensions with small amounts of 10% toluene in absolute ethanol. After washing, the cells can be used directly in the enzyme assays. Kinetic studies of aspartate transcarbamylase (EC 2.1.3.2) in such permeabilized cells showed that apparent Km for substrates and Ki for the feedback inhibitor UTP were only slightly different from those reported using partially purified enzyme. The aspartate saturation curve is hyperbolic both in the presence and absence of UTP. The inhibition by this nucleotide is noncompetitive with respect to aspartate, decreasing both the affinity for this substrate and the maximal velocity of the reaction. The saturation curves for both substrates give parallel double reciprocal plots. The inhibition by the products is linear noncompetitive. Succinate, an aspartate analog, provokes competitive and uncompetitive inhibitions toward aspartate and carbamyl phosphate, respectively. The inhibition by phosphonacetate, a carbamyl phosphate analog, is uncompetitive and noncompetitive toward carbamyl phosphate and aspartate, respectively, but pyrophosphate inhibition is competitive toward carbamyl phosphate and noncompetitive toward aspartate. These results, as well as the effect of the transition state analog Nphosphonacetyl-L-aspartate, all exclude a random mechanism for aspartate transcarbamylase. Most of the data suggest an ordered mechanism except the substrates saturation curves, which are indicative of a ping-pong mechanism. Such a discrepancy might be related to some channeling of carbamyl phosphate between carbamyl phosphate synthetase and aspartate transcarbamylase catalytic sites.

olites. Precise information has been published on the intracellular location and behaviour of the arginine pathway enzymes in Saccharwrrqfce~ cerewzXae (7, 8), New rospora crassa (9), and in the case of Escherichia coli glutamine synthetase (10,ll). In several instances, differences in catalytic or regulatory properties were observed between pure or partially purified enzymes and those tested in their physiological environment (12-18). Pyrimidine pathway enzymes play an important metabolic role, since their cat-

Although most of the steps of the metabolic pathways have been described and in many cases have been extensively investigated in vitro there is more and more concern about the conditions under which enzymes catalyze these steps in the cell. The possible effects of the intracellular environment on the catalytic and regulatory properties of enzymes have been extensively reviewed and discussed (l-6). Increasing information is now available concerning the subcellular localization and the compartmentation of enzymes and metab0003-9861183 Copyright All rights

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0 1983 by Academic of reproduction in

any

Press, Inc. form reserved.

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alytic and regulatory properties contribute to establish the level of the intracellular pools of nucleotides on which DNA replication and cell division depend (19). It has been shown that in S. cerevtiiae the eni zymes catalysing the first two reactions of this pathway (carbamyl-phosphate synthetase and aspartate transcarbamylase), copurify and cosediment during centrifugation analysis, indicating that they are part of a multienzymatic complex (20-23). A possible approach to the determination of the properties exhibited by enzymes in their natural intracellular environment is the use of “permeabilized” cells. Numerous procedures have been proposed to permeabilize different kinds of cells. The methods involve various treatments: osmotic shock (18, 24-27), the influence of organic solvents (12, 13, 28, 29) or other chemicals (17,30-34), and the combination of several physicochemical agents (10, 34-36). In the present work, the S. cerevtiiae cells were permeabilized to the substrates and products of ATCase through the use of diluted toluene and the catalytic and regulatory properties of this enzyme were investigated in situ. MATERIALS

AND

METHODS

Materi& [WlAspartate (specific activity 200 mCi/ was purchased from the Service de Biochimie, CEN, Saclay; carbamyl phosphate, carbamylaspartic acid, succinic acid, and dithiothreitol from Sigma; DLaspartic acid, arginine, uracil from Fluka; ethylene glycol, and orthophosphoric acid from Prolabo; glycerol from Carlo Erba; uridine triphosphate from PL Biochemicals, Inc.; triethyl phosphonoacetate from Aldrich, arsenate (sodium salt) from Merck; N-(phosphonacetyl)-L aspartate (PALA)’ was a generous gift from Dr. G. Stark (Stanford University). The commercial preparation of carbamyl phosphate was recrystallized according to Adair and Jones (37). Phosphonacetic acid was prepared as described by mM)

’ Abbreviations used: ATCase, aspartate transcarbamylase:aspartate carbamoyltransferase (EC 2.1.3.2.); CPSase, carbamyl phosphate synthetase (EC 2.7.2.9); Tris, Tris(hydroxymethy1) aminomethane; PALA, N-phosphonacetyl-L-apartate; bis-Tris, bis(2hydroxyethyl)imino-tris(hydroxymethy1) methane; Caps, cyclohexylaminopropanesulfonic acid.

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Porter et aL (38) and recrystallized according to Balsiger et aL (39). CelLsgrowth The MD 17l.lCmutant strain (a, cpa2, ura3, fur4) used in this study, was isolated by Dr. M. Denis-Duphil and derived from the wild-type FL-100 (40). It was grown in yeast nitrogen base (Difco) medium, supplemented with 2% glucose, 40 mg/liter of both uracil and arginine. Growth was followed at 546 nm using an Eppendorf spectrophotometer. The cells were grown at 28°C on an alternating shaker and harvested in exponential phase (O.D. = 0.4-0.5). After centrifugation the pellet was suspended in 50 mM Trisacetate buffer (pH 7.5), 1 rnM glutamine, 1 mM dithiothreitol, 10% glycerol (v/v), to a concentration of 200 mg cells/ml (wet wt). Preparation of cell-free extracts and aspartate trunscarbamyluse assay. One-milliliter aliquots of the resuspended cells were disrupted in an Eaton press. The resulting material was centrifuged for 30 min at 4°C at 30,OOOg and the supernatant constituted the crude extract. One volume of the same buffer containing 60% ethylene glycol instead of glycerol was then added to the extract in order to preserve the carbamyl phosphate synthetase-aspartate transcarbamylase complex. The aspartate transcarbamylase activity was tested according to Denis-Duphil et aL (23) under the following conditions: the 300-~1 mixture containing 50 mM Tris-acetate (pH 7.5), 10 mM carbamyl phosphate, 50 mM [14C]aspartate (specific radioactivity, 0.05 mCi/mmol) and the permeabilized cells as indicated was incubated for 10 min at 30°C. A 250-~1 sample was then taken and pipetted rapidly into 1.6 ml of 0.2 M acetic acid previously placed on top of a small column of the H+ form of Dowex AG50 (X8) 200 to 400 mesh (0.7 cm X 7 cm). The effluent was collected directly into a glass vial for counting. After 3 rinses of the column with 1 ml of water and addition of 8 ml of Aquasol, the radioactivity of this effluent was measured using an Intertechnique Model SL32 scintillation counter. One unit of ATCase is defined as the amount that catalyzes the formation of 1 pmol of carbamylaspartate in 1 h under the standard conditions. Since this method is based on the separation of Nsubstituted derivatives of aspartate lacking the positive charge, it allows the accurate determination of ATCase in permeabilized cells, which also contain the other enzymes of the pyrimidine pathway. AssayforfeeaTback inhibition by VTP. The feedback inhibition by UTP was tested under the standard conditions described above but in the presence of 2 mM UTP. The percentage of inhibition was calculated as: %inhibition

= 7

X 100

where A0 is the enzymatic activity measured in the absence of UTP and A, is the enzymatic activity measured in its presence. The concentration of UTP so-

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lutions used were controlled by uv absorption and their purity was verified by thin-layer chromatography (41). Assay for uzrimmyl phmphte During the arsenolysis experiments, lOO-~1 samples were taken and their carbamyl phosphate content was determined by incubation with an excess of aspartate (20 mM) and catalytic subunits of E. coli ATCase (26 pg) in a total volume of 300 pl of 50 mM Tris-acetate buffer pH 8. After 1 h of incubation at 37°C the amount of carbamylaspartate formed was measured as described above. RESULTS

A. Pmeabilization of the S. cerevisiae Cells to the Substrates of ATCase under the InfEuence of Toluene Among the various technics devised to permeabilize cells of different organisms, the use of small quantities of toluene proved to be the most convenient in the present case. The optimal conditions for this reaction were determined. The maximal permeabilization is obtained upon addition of 50 ~1 of 10% toluene in absolute ethanol to 1 ml of cell suspension. The ATCase activity measured under these conditions is the same as that of a cell free extract prepared from an identical cell

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suspension. As a control, such a suspension of permeabilized cells was centrifuged and it was verified that the supernatant did not exhibit any significant ATCase activity, showing that no leaking of enzyme from the cells was provoked by this treatment. Two minutes of vigorous agitation using a Vortex mixer ensures maximal permeabilization. Figure la shows that the ATCase reaction is linear over at least 30 min without any lag, indicating that substrates and products diffuse freely in and out of the permeabilized cells. Figure lb shows that the extent of the enzymatic reaction varies linearly with the amount of permeabilized cells, up to at least 36 mg (wet wt) of cells/ 300 /.ll. On the basis of these preliminary assays the permeabilization procedure was standardized as following: to l-ml samples of cells (loo-250 mg wet wt corresponding to 5-12.5 mg of proteins) in 50 mM Tris-acetate buffer (pH 7.5), 1 mM glutamine, 1 mM dithiothreitol, 30% ethylene glycol, and 5% glycerol (storage buffer) cooled at O”C, 50 ~1 of 10% toluene in absolute ethanol were added. The mixture was vigorously agitated on a Vortex mixer at maximum T

k QI 50.9-

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FIG. 1. Estimation of ATCase activity in permeabilized cells. Permeabilization of the cells was performed in the following way: 50 ~1 of 10% toluene in absolute ethanol (v/v) were added to 1 ml of cells suspension (240 mg wet wt). The mixture was immediately vigorously mixed on a Vortex mixer at maximum speed for 2 min. The cells were then spun down, washed twice in 1 ml of 50 mM Tris-acetate, pH 7.5,1 mM glutamine, 1 mM dithiothreitol, 30% ethylene glycol, and 5% glycerol, and resuspended in 1 ml of the same buffer. The ATCase activity was then determined as described under Materials and Methods. (a) Time course of the ATCase reaction using 20-~1 samples of cells suspension. (b) Dependence of the velocity upon cell concentration.

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from pH 6.5 to 10 in the presence of Trisbis-Tris-Caps buffer. The activity increases regularly from pH 6.5 to 9 similar to that of ATCase in cell-free extracts. 2. Saturation curves for substrates. The saturation curve for aspartate in the presence of saturating carbamyl phosphate (10 mM) and the corresponding LineweaverBurk double reciprocal plot indicates an apparent K, for aspartate of 16.6 + 2.6 mM, the standard deviation being calculated from six experiments. The influence of carbamyl phosphate concentration on this saturation curve was investigated. Figure 2 shows the different saturation curves obtained and their double reciprocal plots. The parallel lines given by this representation are indicative of a nonsequential mechanism for the enzymatic reaction. Figure 3 shows the saturation curves for carbamyl phosphate in the presence of

speed for 2 min. The cells were then spun down, washed twice, and resuspended each time using 1 ml of the storage buffer. These cells were immediately used for the enzymatic determinations. At the end of each series of experiments it was verified that no significant enzymatic activity could be detected in the supernatant after centrifugation of the cell suspension. It was ascertained that the concentrations of ethanol and toluene used did not alter the ATCase activity in the crude extracts. When observed by electron microscopy, the permeabilized cells could not be distinguished from untreated cells. B. Characteristics of the Aspartate Transcarbamylase in the Permeabilized Cells 1. pH dependence. The pH dependence of the ATCase activity was determined

0

015 O-05 040 l/[Aspartate] (m M-’ )

FIG. 2. Effect of carbamyl phosphate concentration on the aspartate saturation curve in permeabilized cells. Saturation curves for aspartate were determined as indicated in in the presence of varying carbamyl phosphate: n , 0.333 mM; A, 0.666 mM; A, 1 mM; 0, 10 mM; Cl, 20 mM; 3.5 mg (wet wt) of permeabilised cells were used. Insert: aspartate curves. Main figure: corresponding Lineweaver-Burk double reciprocal plots.

of ATCase Fig. 1 but 3.3 mM; 0, saturation

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FIG. 3. Effect of aspartate concentration on the carbamyl phosphate saturation curve of ATCase in permeabilized cells. Saturation curves for carbamyl phosphate were determined as indicated in Fig. 1, but in the presence of varying aspartate concentrations: 0, 6.6 m!d; A, 12 mM; 0, 20 mM; W, 26.6 mM. 2.5 mg (wet wt) of permeabilized cells were used. Insert: carbamyl phosphate saturation curves. Main figure: corresponding Lineweaver-Burk double reciprocal plots.

varying concentrations of aspartate and the corresponding double reciprocal plots. Again parallel lines are obtained, suggesting a nonsequential mechanism. Extrapolation of intercepts to infinite concentration of aspartate indicate a K, of 1.18 + 0.25 mM for carbamyl phosphate. 3. Products and substrates analogs inhibition studie.s. In order to obtain further information about the possible mechanism of catalysis by ATCase in the permeabilized cells, the pattern of inhibition by the products of the reaction and by several substrates analogs was determined. Figures 4 and 5 show that, under conditions of saturation by carbamyl phosphate and subsaturation by aspartate, both the products carbamylaspartate and phosphate behave as noncompetitive inhibitors towards both carbamyl phosphate and aspartate. In the same figures it can be seen that the corresponding slope and intercept replots are linear. Thus, it appears that both prod-

ucts act as linear noncompetitive inhibitors towards the substrates. In the same manner, the effects of substrates analogs on the saturation curve for the two substrates were investigated. Figure 6 shows that succinate, an analog of aspartate provokes uncompetitive inhibition toward carbamyl phosphate and competitive inhibition toward aspartate. In this case, the slope and intercept replots are also linear. Phosphonacetate was used as an analog of carbamyl phosphate (38). Figure 7 shows that this compound gives a pattern of uncompetitive inhibition toward carbamyl phosphate and noncompetitive inhibition toward aspartate. Again in this case, the slope and intercept replots are linear. When pyrophosphate was used as the carbamyl phosphate analog (38) the same result was obtained for aspartate. However, this compound behaved as a competitive inhibitor for carbamyl phosphate.

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I

a

b

FIG. 4. Product inhibition of ATCase aspartate (a) and carbamyl phosphate (b) but in absence (0) or in the presence of using 4 mg of permeabilized cells (wet intercepts (0) replots.

4. In~Euence of the transition N-phmphonoaeetyl-mzquwtate.

,I I II

activity by carbamylaspartate. The saturation curves for were determined as described under Materials and Methods 22 maa (O), 44 mre mM (A), or 100 mM (m) carbamylaspartate, wt). The inserts show the corresponding slope (X) and

state analog, N-Phos-

phonacetyl+aspartate (PALA) was prepared and used as a transition state analog with chemical groups similar to those involved in the binding of the two substrates, carbamyl phosphate and aspartate (42,43). It has been shown that it binds to E. coli ATCase in competition with carbamyl

phosphate but not aspartate (42) since this enzyme binds the two substrates according to an ordered mechanism in which carbamyl phosphate binds first and then allows the binding of aspartate (42, 44-48). Figure 8 shows that also in S. cerevisiae permeabilized cells, PALA is a competitor of carbamyl phosphate only. This was confirmed by the carbamyl phosphate and as-

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0 0.05 010 l/[Aspartate](mMA)

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FIG. 5. Product inhibition of ATCase activity by phosphate. The saturation curves for aspartate (a) and carbamyl phosphate (b) were determined as described under Materials and Methods but in the absence (0) or in the presence of 100 mM (0) and 300 mM (A) phosphate using respectively 2.8 and 4 mg of permeabilized cells (wet wt). The inserts show the corresponding slope (X) and intercept (0) replots.

partate saturation curves obtained in the presence of PALA, which showed competitive and noncompetitive types of inhibition for carbamyl phosphate and aspartate, respectively. 5. Assay of arsenolysis of a putative carbamyl-enzyme intermediate. Since some of the results reported above suggest a nonsequential ping-pong type of mechanism, it was interesting to investigate the pu-

tative formation of a carbamyl-enzyme intermediate. If such an intermediate exists, one would expect that in the presence of arsenate it would give rise to carbamylarsenate which in turn would decompose to carbamate plus arsenate (49). The overall effect would be an accelerated breakdown of carbamyl phosphate in the presence of enzyme and arsenate. This possibility was investigated in the following way: in a total

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l/LCarbamylphosphateI(mM-l) FIG. 6. Inhibition of ATCase activity by succinate. The saturation curves for aspartate (a) and earbamyl phosphate (b) were determined as described under Materials and Methods but in the absence (0) or in the presence of 5 mM (O), 10 mM (A), and 26 mM (B) succinate, using 7 mg of permeabilized cells (wet wt). The inserts show the corresponding slope (X) and intercept (0) replots.

volume of 1 ml of 0.1 M Tris-acetate buffer, pH 7.5, 40 mg (wet wt) of permeabilized cells were incubated with 10 mM carbamyl phosphate in the presence or absence of 20 mM arsenate. At different time intervals, lOO-~1 samples were taken and assayed for carbamyl phosphate content as described under Materials and Methods. Arsenate did not promote any detectable increase in the carbamyl phosphate breakdown.

6. Feedback inhibition by UTP. It has been reported using semi-purified preparations of the enzyme complex that UTP is a feedback inhibitor of the ATCase reaction (50). However, it was also shown that this complex can be disrupted into a CPSase, which remains sensitive to inhibition by this nucleotide, and an ATCase, which is insensitive to it (23). The sensitivity of the ATCase reaction to UTP was

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4 a

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FIG. 7. Inhibition of AT&se activity by phosphonacetate. The saturation curves for aspartate (a) and carbamyl phosphate (b) were determined as described under Materials and Methods but in the absence (0) or in the presence of 15 mM (0). 30 mM (A), and 50 mrd (m) phosphonacetate, using 4 mg (wet wt) of permeabilized cells. The inserts show the corresponding slope (X) and intercept (Cl) replots.

investigated here in the permeabilized cells. Figure 9 shows the evolution of the feedback inhibition of the ATCase activity

when the concentration of UTP is increased in the presence of 10 mM carbamyl phosphate and four different concentrations of aspartate (from 4 to 50 mM). In each case,

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arbamilphosp&te]

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FIG. 8. Inhibition of ATCase activity by N-phosphonacetyl-L-aspartate (PALA). ATCase activity was measured as described under Materials and Methods in the absence or presence of 0.056 mM PALA. When the aspartate concentration was constant at 50 mM, carbamyl phosphate varied from 0.13 to 10 mM (0). When the carbamyl phosphate concentration was constant at 10 mhr, aspartate varied from 6.5 to 70 mM (0). 4 mg (wet wt) of permeabilized cells were used.

the feedback inhibition reaches a plateau whose level depends on the aspartate concentration. The possible significance of this result will be discussed further. In the same figure the Dixon plot indicates a Ki value of 1.8 mM for UTP. Figure 10 reports the saturation curves for carbamyl phosphate and aspartate in the presence of varying concentrations of UTP. These results show that the presence of the feedback inhibitor does not induce the appearance of a cooperativity in the utilization of the substrates. The saturation curves for aspartate in the presence of increasing amounts of UTP show that the feedback inhibitor decreases both the affinity for aspartate and the maximal velocity of the reaction. Toward carbamyl phosphate UTP behaves as an uncompetitive inhibitor. DISCUSSION

The permeabilization of the yeast cells described here allows the study of the ki-

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netic properties of ATCase inside these cells without leaking of the enzyme. Since the permeabilized cells are indistinguishable from the untreated cells upon electron microscopic examination, the enzyme is tested in an environment which must be very close to the physiological one. This is remarkable in view of the fact that there is evidence that the CPSase-ATCase complex is located in the nucleus (51). No cooperativity for the utilization of the substrates can be detected under the conditions used here in accordance with what has been reported in the case of a partially purified preparation (50). The ATCase from some organisms revealed cooperative binding of the substrates only in the presence of the feedback inhibitor (52). It appears that this is not the case for S. cerevisiae ATCase in situ. The apparent Km for the substrates and the Ki for UTP found under these conditions differ only slightly from the values reported from a partially purified preparation of the complex (variation less than by a factor of 3). The presence of the feedback inhibitor reduces both the affinity for aspartate and the maximal velocity of the reaction. This last effect might be related to the concomitant influence of this nucleotide on the CPSase reaction. The feedback inhibition reaches a plateau, the level of which depends on the aspartate concentration. In the case of E. coli ATCase this phenomenon can be correlated with the allosteric interactions between the catalytic sites for aspartate binding (53). However, in the present case such interactions do not occur even in the presence of the feedback inhibitor and this result must be explained only by a decrease in the affinity for aspartate resulting from UTP binding to sites that are distinct from the catalytic sites. The various kinetic information obtained: parallel double reciprocal plots for both substrates; linear noncompetitive inhibition by products; succinate competitive and uncompetitive inhibition toward aspartate and carbamyl phosphate, respectively; phosphonacetate uncompetitive and noncompetitive inhibition toward carbamy1 phosphate and aspartate, respectively;

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[UTP 1(mM) FIG. 9. Effect of UTP on the ATCase activity. (a) The feedback inhibition of the ATCase activity was measured as described under Materials and Methods in the presence of 10 rnrd carbamyl phosphate and 4 mM (B), 8 mM (A), 20 mM (e), or 50 my (0) asparate, using 2.8 mg (wet wt) of permeabilized cells. (b) Corresponding Dixon plot.

PALA competitive and noncompetitive inhibition toward carbamyl phosphate and aspartate, respectively, all exclude a random mechanism for the reaction catalyzed by ATCase in the permeabilized cells. However, these results do not allow unequivocal discrimination between an ordered and a ping-pong type mechanism (54-57). The product inhibition by carbamylaspartate and the influence of the substrates analogs, pyrophosphate, succinate, and PALA, are indicative of an ordered mechanism but the substrate saturation curves are consistent with an apparent ping-pong mechanism, the effect of succinate and PALA being compatible with both. The negative result of the arsenolysis experiment indicates that a ping-pong mechanism including a carbamyl-enzyme intermediate is not involved. It is important to remark that similar contradictory

results were obtained with a partially purified preparation of the S, cerevisiae enzyme complex (58). Such conflicting results were also obtained in the case of purified ATCase from Phaseolus aweus (59), bovine brain hexokinase (60), and beef kidney 3hydroxyanthranylate oxygenase (61). In these cases it was concluded that these enzymes are acting according to an ordered mechanism in which an essentially irreversible step occurs before the binding of the second substrate. In the present case this complex behavior might be related to the fact that the CPSase-ATCase multienzyme system seems to show some channeling of the carbamyl phosphate from the CPSase catalytic site to that of ATCase (62). In addition, such a process might explain the amazing behavior of phosphonacetate, which appears to be an uncompetitive inhibitor toward carbamyl

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(mM4)

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FIG. 10. Influence of UTP on aspartate and carbamyl phosphate saturation curves. The saturation curves for aspartate (a) and carbamyl phosphate (b) were determined as described under Materials and Methods but in the absence (0) or presence of 0.1 mM (A), 0.2 mm (O), 1 mM (+), and 2 mM (0) UTP, using 6.6 mg (wet wt) of permeabilized cells. Insert: saturation curves; main figure: corresponding double reciprocal plots.

phosphate. These possibilities will be investigated using permeabilized cells of mutants that possessonly one of these two enzymes.

ACKNOWLEDGMENTS The authors are indebted to Dr. George Stark (Stanford University) for generously providing the sample of PALA used in these experiments, to Inda

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Issaly and Maria Nagy for stimulative and to Sathyam Ganessan for reading this manuscript.

discussions, and improving

REFERENCES 1. SOLS, S. A., AND MARCO, R. (1970) in Current Topics in Cellular Regulation (B. L. Horecker and E. R. Stadman, eds.), Vol. 2, pp. 227-273, Academic Press, New York/London. 2. KEMPNER, E. S. (1975) SubcelL. Biochem 4, 213221. 3. MASTERS, C. J. (1977) in Current Topics in Cellular Regulation (B. L. Horecker and E. R. Stadman, eds.), Vol. 12, pp. 75-103, Academic Press, New York/London. 4. WILSON, J. E. (1978) Trends Biochxm. Sci 3,124125. 5. SIES, H. (1980) Twds Biochem Sci 5,182-185. 6. MASTERS, C. J. (1978) Trends Biochem Sci. 3,206208. 7. WIPF, B., AND LEISINGER, TH. (1977) FEMS MicrobioL L&t 2, 239-242. 8. JAUNIAUX, J. C., URRESTARAZU, L. A., AND WIAME, J. M. (1978) J. Bacterid 133,1096-1107. 9. DAVIS, R. H., BOWMAN, B. J., AND WEISS, R. L. (1978) J. Supram Struck. 9, 473-488. 10. MURA, U., AND STADTMAN, E. R. (1981) J. BioL Chem. 256, 13,014-13,021. 11. MURA, U., CHOCK, P. B., AND STADTMAN, E. R. (1981) J. Bid Chem 256, 13,022-13,029. 12. SOLS, S. A., DE LA FUENTE, G., VILLAR-PALASI, C., AND ASENSIO, G. (1958) Bicchim Biophys. Actu 30, 92-101. 13. GACHELIN, G. (1969) Biochem Biuphvs Res Commun. 34,382-387. 14. REEVES, R. E., AND SOLS, A. (1973) Biodwm Bie phys. Rex Commun 50.459-466. 15. WEIT~MAN, P. D. J., AND HEWSON, J. K. (1973) FEBS Lzett 36, 227-231. 16. RYAN, E. D., AND KOHLHAW, G. B. (1974) .I. Bacterid 120, 631-637. 17. BLATT, J. M., AND JACKSON, J. H. (1978) Biochim Biqvhys. Acta 526, 267-275. 18. ARAGON, J. J., FELIU, J. E., FRENKEL, R. A., AND SOLS, S. A. (1980) Proc Nati Ad Sci. USA 77, 6324-6328. 19. BJURSELL, G., AND REICHARD, P. (1973) .J. Bid C&m. 248. 3904-3909. 20. LACROUTE, F. (1968) J. BacterioL 95, 824-832. 21. LUE, P., AND KAPLAN, G. (1969) Biochem. Biophya Res. Commun 34,426-433. 22. LUE, P., AND KAPLAN, G. (1971) Can J. Biochem 49.403-411. 23. DE&-DUPHIL, M., MATHIEN-SHIRE, Y., AND HERVE, G. (1981) J. Bacterid 148.659-669.

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