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An improved method for the production of Neurospora crassa spheroplasts and a new method for measuring transport in fragile cells
ANALYTICAL
An
BIOCHEMISTRY
Improved
crassa
GENE Department
61, 441-447
Method
(1974)
for the
Production
of Neurospora
Spheroplasts and a New Method for Measuring Transport in Fragile Cells1 A. SCARBOROUGH of Biochemistry, Denver,
AND
THOMAS
University of Colorado Colorado 80620
Received January 14, 1974; accepted April
H. SCHULTE School
of Medicine,
15, lQ74
An improved method for t.he production of Neurospora spheroplasta and a procedure for measuring transport by these fragile structures are described. Using these techniques, the activity of the glucose active transport system in spheroplasts has been measured. The results indicate that removal of the Neurospora cell wall does not affect the activity of this transport system. In light of this finding, it is unlikely that soluble periplasmic components play a role in the glucose active transport system of Neurosporn.
Previous reports from this laboratory have described two glucose transport systems in germinated conidia of Neurospora CTCLSSU (1,2). One, a facilitated diffusion system which is present when cells are germinated in the presence of high levels of glucose (Glu I) ; the other an active tranaport system which is repressed by high levels of glucose but is derepressed when cells are germinated in the presence of fructose or low levels of glucose (Glu II). The survival value of this bimodal transport scheme has been discussed (2). With the characterization of the glucose facilitated diffusion system and the glucose active transport system well established, the major goal in this laboratory is an understanding of the molecular mechanisms whereby these transport systems accomplish the facilitated diffusion and the active transport of glucose. As one avenue of approach to these problems we have chosen to investigate the nature of these transport systems in spheroplasts, the membrane-bound structures which result from removal of the rigid Xewospora cell wall. The reasons for studying transport in spheroplasts rather than in whole cells are several. The absence of the cell wall makes it theoretically possible to establish the role of various binding proteins in transport by adding such pro‘This study was supported by Research Grant AM14479 from the United States Public Health Service, National Institutes of Health and Research Grant GRS 256 from the University of Colorado Medical Research Fund. 441 Copyright @ 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.
442
SCARBOROUGH
AND
SCHULTE
teins back to spheroplasts and reconstitut.ing transport activity. In Neurospora, two such binding proteins have already been identified, one for tryptophan (3) and one for arginine (4). In addition, removal of the protective cell wall permits evaluation of the effects of macromolecular membrane perturbing agents, such as lectins, proteases, and phospholipases, on transport activities. Finally the isolation of healthy spheroplasts which are intact with respect to the glucose active transport system is an important prerequisite for the study of this system in isolated plasma membrane vesicles, a technique which has yielded much information regarding the mechanism of transport in bacterial systems (5). This paper describes (1) a method for the rapid preparation of Neurospora spheroplasts, (2) a method for assaying transport by these fragile structures, and (3) some results of the application of these methods to the study of the glucose active transport system in spheroplasts. MATERIALS
AND METHODS
The sources of most materials have been described previously (1,2,10). The /3-glucuronidase (Nelir pomatia) and Trizma base were from Sigma. The Ficoll and Sephadex were from Pharmacia. Sodium Lauryl Sulfate (SDS) was from Fischer Scientific Co. Preparation of /3-glucuroniduse. /?-Glucuronidase powder (200 mg) is dissolved in 2 ml of spheroplasting medium (SM ; 0.1 M succinic acid containing 0.4 M MgSO,, 20 mM KU, and 1 mM mercaptoethanol; adjusted to pH 5.0 with Trizma base) and passed through a small column (1 ml bed vol) of Sephadex G15 under pressure in order to remove a sticky green substance which is present in some of the commercial enzyme preparations. The column is then washed with another 3 ml of SM and the eluate added to the enzyme preparation. Preparation of spheroplasts. This method is a modification of previously described methods (6-9) but requires significantly less time than these procedures. Conidia are germinated in the presence of 50 mM fructose as previously described (1,2) until the optical density increases about fivefold. The cells are then washed and resuspended in SM to a concentration corresponding to 6 mg dry wt of cells/ml. Five milliliters of this cell preparation is combined with 5 ml of the @glucuronidase preparation described above and the mixture is incubated with agitation for 1 hr at 37°C. After chilling, the mixture is centrifuged for 5 min in a swinging bucket clinical centrifuge and the pellet resuspended in a mixture of 10 ml SM and 10 ml water at 4°C. The resulting suspension is dispersed in a glass-teflon tissue homogenizer (clearance approx 0.008 in.) and filtered through glass wool in order to remove the remaining whole cells and cell wall fragments. The spheroplasts in the filtrate are then pelleted in a
GLUCOSE
TRANSPORT
SYSTEM
IN
N. crassa
443
clinical centrifuge and resuspended in SM to a concentration corresponding to approximately 1 mg protein/ml. The yield is 20-40s based upon protein analyses of whole cells and spheroplasts. Protein determination. Protein was estimated by the method of Lowry et al. (11). Cell wall isolation. Cell wall weight was determined by the method of Mahadevan and Tatum (12). The length of the SDS extraction period was increased to 36 hr in these experiments. This method yields a microscopically homogeneous preparation of cell walls from whole cells. Chitin determination. Chitin content was determined essentially by the method of Blumenthal and Roseman (13) using the Elson-Morgan procedure for hexosamine assay as reported by Roseman and Daffner (14). Ficoll centrifugation transport assay. Due to the extreme fragilit,y of the spheroplasts prepared as above, a standard uptake assay based upon Millipore filtration was not feasible since it resulted in nearly complete destruction of the spheroplasts. Thus it was necessary to develop a new type of transport assay. Of a variety of methods tried, by far the most sensitive and reproducible was an assay based on centrifugation of tho spheroplasts through a solution of Ficoll in SM. Incubations are carried out in SM containing 14C-sorbose (10 mM, 10 cpm/nmole) and whole cells (0.2-0.4 mg protein) or spheroplasts (0.05-0.1 mg protein) in a final volume of 0.5 ml. After incubation at 30°C for the appropriate time, 0.4 ml of the test mixture is layered over 4 ml ice cold 10% Ficoll in SM and centrifuged at top speed in a swinging bucket clinical centrifuge for 10 min. After centrifugation, the upper layer containing most of the radioactivity is removed by aspiration and the upper area of the Ficoll solution is then washed three times by layering water over t,he Ficoll and removing it by aspiration. Finally the Ficoll solution is removed by aspiration. When whole cells are assayed by this procedure, the pellet is resuspended in 1.1 ml of water and 1 ml of this suspension is mixed with Patterson-Greene scintillation fluid (15) and counted in a liquid scintillation counter. When transport in spheroplasts or other fragile cells is being measured, the pellet is resuspended in 1.2 ml water and 0.1 ml of this suspension is taken for protein determination while 1 ml is counted as above. Determination of the protein actually present in the pellet corrects for small amounts of cell destruction during the incubation period. This procedure is not necessary when assaying intact cells. A small precipitate forms in the scintillation vials, but this settles out and does not noticeably affect counting efficiency. Attempts to use solutions of sucrose or glycerol in SM as the dense layer were not successful, probably because such solutions contribute greatly to the osmotic pressure of the solution, causing the spheroplasts to shrink as they sediment into
444
SCARBOROUGH
AND
SCHULTE
the dense layer. The advantage of Ficoll (MW approx 400,000) is that a solution of adequate density contributes very little to the osmotic pressure and thus the osmotic environment does not change appreciably as the spheroplasts sediment out of the incubation mixture into the dense layer. RESULTS
AND
DISCUSSION
All experiments reported in this communication were carried out with cells grown on 50 mM fructose or spheroplasts derived from cells grown on 50 mM fructose. The glucose active transport system is largely derepressed under these conditions of growth (1,2). Characterization of spheroplasts. Figure 1 shows photomicrographs of whole cells grown to approximately five times their original optical density and of spheroplasts prepared from such cells. The spheroplasts prepared by the above procedure contain greater than 95% osmotically sensitive structures as estimated by microscopic examination after dilution in water. Cell wall, isolated as described in Methods, constitutes about 16% of the dry weight of whole cells, in agreement with published values (12). Attempts to isolate cell wall from the spheroplast preparation by this procedure yielded no significant cell wall material. Since chitin is known to be a significant constituent of the Neurospora cell wall (12,13,16), determination of the chitin content of whole ceils and spheroplasts provides another index of the amount of cell wall remaining in the spheroplast preparation. In a series of four experiments, the chitin content of spheroplasts was found to be less than 5% that ,of whole cells. Thus, spheroplasts prepared as described above, contain very little cell wall. Transpm-t in spheroplasts. In order to prove that the Ficoll centrifugation method of measuring uptake is comparable in efficiency to the wellestablished Millipore filtration assay (1,2,10), the experiment described in Fig. 2 was carried out. In this experiment, the uptake of sorbose, a nonmetabolizable substrate of the glucose active transport system (2), by whole cells was measured using both the Millipore filtration assay and the Ficoll centrifugation assay. The results indicate that the rate of sorbose uptake as measured by the Ficoll centrifugation assay is comparable to the rate of sorbose uptake as measured by Millipore filtration. (This rate of sorbose uptake, measured in SM at 3O”C, is comparable to the rates measured in more conventional incubation media (1,2,10) .) Thus the Ficoll centrifugation assay is a valid method for measuring transport by whole cells. Figure 3 compares the sorbose transport activity of whole cells, measured by the Millipore filtration assay, and spheroplasts, measured by the
GLUCOSE
FIG. 1. Photomicrographs Microscope magnification,
TRANSPORT
SYSTEM
IN
,y.
CIYXSSQ
445
of whole cells and spheroplasts prepared from such cells. X440.
Ficoll centrifugation assay. In this experiment, germinated conidia were filtered and resuspended in SM. One portion was taken for the spheroplasting procedure while the other was held at 0°C during this period. Both cells and spheroplasts were then assayed for sorbose transport activity. It can be seen that the sorbose transport by spheroplasts is comparable to that of intact cells. The Ficoll centrifugation assay is therefore a valid method for measuring transport in fragile cells such as Neurospora spheroplasts. The difference in the rates of sorbose transport by whole cells in Figs. 2 and 3 reflects day to day variability in the activity of the glucose active transport system in different cultures of cells. Finally, since NeGrospmo spheroplast.s, essentially devoid of cell wall, transport sorbose at, a rate comparable to that of whole cells, there does
446
SCARBOROUGH
AND
SCHULTE
soo-
MINUTES
2. Sorbose uptake by whole cells. Ficoll centrifugation versus Millipore fYtration. The Ficoll centrifugation assay is described in Methods. The Millipore filtration assay was carried out as previously described (l&10) except that incubations were carried out at 30°C in SM and the cell pellets were washed with 1 ml of cold SM. Data points indicate the average of duplicate determinations. (V-V) Ficoll centrifugation assay; (0-O) Millipore filtration assay. FIG.
0
IO
20
30
MINUTES
FIG. 3. Sorbose uptake by whole cells and spheroplasts. Whole cells were assayed by Millipore filtration aa described for Fig. 2. Spheroplasts were assayed by Ficoll centrifugation as described in Methods. Data points indicate the average of duplicate determinations. (V-V 1 Whole cells; (0-O) spheroplasts.
GLUCOSE
TRANSPORT
SYSTEM
IR' S.
crassa
447
not appear to be any involvement of soluble periplasmic components in the glucose active transport system of Newospora, as has been suggested for certain prokaryote transport systems (17j. ACKNOWLEDGMENT The authors wish to acknowledge Owen.
the able technical
assistance of Miss Claudia
REFERENCES 1. SCARBOROUGH, G. A. (1970) J. Viol. Chem. 245, 1694-1698. 2. Ibid., pp. 39853987. 3. WILEY, W. R. (1970) J. Bacterial. 103, 656-662. 4. STUART. W. D., AND DEBUSK, A. G. (1971) Arch. &o&em. Biophys. 144, 512518. 5. KABACK, H. R. (1973) Symp. Sot. Ezp. Biol. 27, 145-174. 6. BACHMANN, B. J., AND BONNER, D. M. (1959) J. Bacterial. 78, 550-561. 7. KINSKY, S. C. (1962) J. Bacterial. 83, 351-358. 8. TREVITHICK, J. R.. AND METZENBERG, R. L. (1964) Biochem. Biophys. Res. Commun. 16, 319-325. 9. HENDRICK, D., .~ND DEBUSK, A. G. (1967) Neurospora Newsbtt. 12, 14. 10. SC~RBOROC~H. G. A. (1971) Biochem. Biophys. Res. Commun. 43, 968-975. 11. LOVCR~, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. C‘hem. 193, 265-275. 12. MAHADEVAN, P. R., AND TATUM, E. I,. (1965) J. Bacterial. 90, 10731081. 13. BLUMENTHAL, H. J., AND &SEMAN, S. (1957) J. Bucteriol. 74, 222-224. 14. ROSEMAN, S., AND DAFFNER, I. (1956) Anal. Chem. 28, 1743-1746. 15. PATTERSON, M. S., AND GREENE, R. C. (1965) Anal. Chem. 37, 854-857. 16. POTGIETER, H. J., AND ALEXANDER, M. (1965) Can. J. Microbial. 11, 122-125. 17. OXENDER. D. L. (1972) Ann. Rev. Biochem. 41, 777-809.
An
BIOCHEMISTRY
Improved
crassa
GENE Department
61, 441-447
Method
(1974)
for the
Production
of Neurospora
Spheroplasts and a New Method for Measuring Transport in Fragile Cells1 A. SCARBOROUGH of Biochemistry, Denver,
AND
THOMAS
University of Colorado Colorado 80620
Received January 14, 1974; accepted April
H. SCHULTE School
of Medicine,
15, lQ74
An improved method for t.he production of Neurospora spheroplasta and a procedure for measuring transport by these fragile structures are described. Using these techniques, the activity of the glucose active transport system in spheroplasts has been measured. The results indicate that removal of the Neurospora cell wall does not affect the activity of this transport system. In light of this finding, it is unlikely that soluble periplasmic components play a role in the glucose active transport system of Neurosporn.
Previous reports from this laboratory have described two glucose transport systems in germinated conidia of Neurospora CTCLSSU (1,2). One, a facilitated diffusion system which is present when cells are germinated in the presence of high levels of glucose (Glu I) ; the other an active tranaport system which is repressed by high levels of glucose but is derepressed when cells are germinated in the presence of fructose or low levels of glucose (Glu II). The survival value of this bimodal transport scheme has been discussed (2). With the characterization of the glucose facilitated diffusion system and the glucose active transport system well established, the major goal in this laboratory is an understanding of the molecular mechanisms whereby these transport systems accomplish the facilitated diffusion and the active transport of glucose. As one avenue of approach to these problems we have chosen to investigate the nature of these transport systems in spheroplasts, the membrane-bound structures which result from removal of the rigid Xewospora cell wall. The reasons for studying transport in spheroplasts rather than in whole cells are several. The absence of the cell wall makes it theoretically possible to establish the role of various binding proteins in transport by adding such pro‘This study was supported by Research Grant AM14479 from the United States Public Health Service, National Institutes of Health and Research Grant GRS 256 from the University of Colorado Medical Research Fund. 441 Copyright @ 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.
442
SCARBOROUGH
AND
SCHULTE
teins back to spheroplasts and reconstitut.ing transport activity. In Neurospora, two such binding proteins have already been identified, one for tryptophan (3) and one for arginine (4). In addition, removal of the protective cell wall permits evaluation of the effects of macromolecular membrane perturbing agents, such as lectins, proteases, and phospholipases, on transport activities. Finally the isolation of healthy spheroplasts which are intact with respect to the glucose active transport system is an important prerequisite for the study of this system in isolated plasma membrane vesicles, a technique which has yielded much information regarding the mechanism of transport in bacterial systems (5). This paper describes (1) a method for the rapid preparation of Neurospora spheroplasts, (2) a method for assaying transport by these fragile structures, and (3) some results of the application of these methods to the study of the glucose active transport system in spheroplasts. MATERIALS
AND METHODS
The sources of most materials have been described previously (1,2,10). The /3-glucuronidase (Nelir pomatia) and Trizma base were from Sigma. The Ficoll and Sephadex were from Pharmacia. Sodium Lauryl Sulfate (SDS) was from Fischer Scientific Co. Preparation of /3-glucuroniduse. /?-Glucuronidase powder (200 mg) is dissolved in 2 ml of spheroplasting medium (SM ; 0.1 M succinic acid containing 0.4 M MgSO,, 20 mM KU, and 1 mM mercaptoethanol; adjusted to pH 5.0 with Trizma base) and passed through a small column (1 ml bed vol) of Sephadex G15 under pressure in order to remove a sticky green substance which is present in some of the commercial enzyme preparations. The column is then washed with another 3 ml of SM and the eluate added to the enzyme preparation. Preparation of spheroplasts. This method is a modification of previously described methods (6-9) but requires significantly less time than these procedures. Conidia are germinated in the presence of 50 mM fructose as previously described (1,2) until the optical density increases about fivefold. The cells are then washed and resuspended in SM to a concentration corresponding to 6 mg dry wt of cells/ml. Five milliliters of this cell preparation is combined with 5 ml of the @glucuronidase preparation described above and the mixture is incubated with agitation for 1 hr at 37°C. After chilling, the mixture is centrifuged for 5 min in a swinging bucket clinical centrifuge and the pellet resuspended in a mixture of 10 ml SM and 10 ml water at 4°C. The resulting suspension is dispersed in a glass-teflon tissue homogenizer (clearance approx 0.008 in.) and filtered through glass wool in order to remove the remaining whole cells and cell wall fragments. The spheroplasts in the filtrate are then pelleted in a
GLUCOSE
TRANSPORT
SYSTEM
IN
N. crassa
443
clinical centrifuge and resuspended in SM to a concentration corresponding to approximately 1 mg protein/ml. The yield is 20-40s based upon protein analyses of whole cells and spheroplasts. Protein determination. Protein was estimated by the method of Lowry et al. (11). Cell wall isolation. Cell wall weight was determined by the method of Mahadevan and Tatum (12). The length of the SDS extraction period was increased to 36 hr in these experiments. This method yields a microscopically homogeneous preparation of cell walls from whole cells. Chitin determination. Chitin content was determined essentially by the method of Blumenthal and Roseman (13) using the Elson-Morgan procedure for hexosamine assay as reported by Roseman and Daffner (14). Ficoll centrifugation transport assay. Due to the extreme fragilit,y of the spheroplasts prepared as above, a standard uptake assay based upon Millipore filtration was not feasible since it resulted in nearly complete destruction of the spheroplasts. Thus it was necessary to develop a new type of transport assay. Of a variety of methods tried, by far the most sensitive and reproducible was an assay based on centrifugation of tho spheroplasts through a solution of Ficoll in SM. Incubations are carried out in SM containing 14C-sorbose (10 mM, 10 cpm/nmole) and whole cells (0.2-0.4 mg protein) or spheroplasts (0.05-0.1 mg protein) in a final volume of 0.5 ml. After incubation at 30°C for the appropriate time, 0.4 ml of the test mixture is layered over 4 ml ice cold 10% Ficoll in SM and centrifuged at top speed in a swinging bucket clinical centrifuge for 10 min. After centrifugation, the upper layer containing most of the radioactivity is removed by aspiration and the upper area of the Ficoll solution is then washed three times by layering water over t,he Ficoll and removing it by aspiration. Finally the Ficoll solution is removed by aspiration. When whole cells are assayed by this procedure, the pellet is resuspended in 1.1 ml of water and 1 ml of this suspension is mixed with Patterson-Greene scintillation fluid (15) and counted in a liquid scintillation counter. When transport in spheroplasts or other fragile cells is being measured, the pellet is resuspended in 1.2 ml water and 0.1 ml of this suspension is taken for protein determination while 1 ml is counted as above. Determination of the protein actually present in the pellet corrects for small amounts of cell destruction during the incubation period. This procedure is not necessary when assaying intact cells. A small precipitate forms in the scintillation vials, but this settles out and does not noticeably affect counting efficiency. Attempts to use solutions of sucrose or glycerol in SM as the dense layer were not successful, probably because such solutions contribute greatly to the osmotic pressure of the solution, causing the spheroplasts to shrink as they sediment into
444
SCARBOROUGH
AND
SCHULTE
the dense layer. The advantage of Ficoll (MW approx 400,000) is that a solution of adequate density contributes very little to the osmotic pressure and thus the osmotic environment does not change appreciably as the spheroplasts sediment out of the incubation mixture into the dense layer. RESULTS
AND
DISCUSSION
All experiments reported in this communication were carried out with cells grown on 50 mM fructose or spheroplasts derived from cells grown on 50 mM fructose. The glucose active transport system is largely derepressed under these conditions of growth (1,2). Characterization of spheroplasts. Figure 1 shows photomicrographs of whole cells grown to approximately five times their original optical density and of spheroplasts prepared from such cells. The spheroplasts prepared by the above procedure contain greater than 95% osmotically sensitive structures as estimated by microscopic examination after dilution in water. Cell wall, isolated as described in Methods, constitutes about 16% of the dry weight of whole cells, in agreement with published values (12). Attempts to isolate cell wall from the spheroplast preparation by this procedure yielded no significant cell wall material. Since chitin is known to be a significant constituent of the Neurospora cell wall (12,13,16), determination of the chitin content of whole ceils and spheroplasts provides another index of the amount of cell wall remaining in the spheroplast preparation. In a series of four experiments, the chitin content of spheroplasts was found to be less than 5% that ,of whole cells. Thus, spheroplasts prepared as described above, contain very little cell wall. Transpm-t in spheroplasts. In order to prove that the Ficoll centrifugation method of measuring uptake is comparable in efficiency to the wellestablished Millipore filtration assay (1,2,10), the experiment described in Fig. 2 was carried out. In this experiment, the uptake of sorbose, a nonmetabolizable substrate of the glucose active transport system (2), by whole cells was measured using both the Millipore filtration assay and the Ficoll centrifugation assay. The results indicate that the rate of sorbose uptake as measured by the Ficoll centrifugation assay is comparable to the rate of sorbose uptake as measured by Millipore filtration. (This rate of sorbose uptake, measured in SM at 3O”C, is comparable to the rates measured in more conventional incubation media (1,2,10) .) Thus the Ficoll centrifugation assay is a valid method for measuring transport by whole cells. Figure 3 compares the sorbose transport activity of whole cells, measured by the Millipore filtration assay, and spheroplasts, measured by the
GLUCOSE
FIG. 1. Photomicrographs Microscope magnification,
TRANSPORT
SYSTEM
IN
,y.
CIYXSSQ
445
of whole cells and spheroplasts prepared from such cells. X440.
Ficoll centrifugation assay. In this experiment, germinated conidia were filtered and resuspended in SM. One portion was taken for the spheroplasting procedure while the other was held at 0°C during this period. Both cells and spheroplasts were then assayed for sorbose transport activity. It can be seen that the sorbose transport by spheroplasts is comparable to that of intact cells. The Ficoll centrifugation assay is therefore a valid method for measuring transport in fragile cells such as Neurospora spheroplasts. The difference in the rates of sorbose transport by whole cells in Figs. 2 and 3 reflects day to day variability in the activity of the glucose active transport system in different cultures of cells. Finally, since NeGrospmo spheroplast.s, essentially devoid of cell wall, transport sorbose at, a rate comparable to that of whole cells, there does
446
SCARBOROUGH
AND
SCHULTE
soo-
MINUTES
2. Sorbose uptake by whole cells. Ficoll centrifugation versus Millipore fYtration. The Ficoll centrifugation assay is described in Methods. The Millipore filtration assay was carried out as previously described (l&10) except that incubations were carried out at 30°C in SM and the cell pellets were washed with 1 ml of cold SM. Data points indicate the average of duplicate determinations. (V-V) Ficoll centrifugation assay; (0-O) Millipore filtration assay. FIG.
0
IO
20
30
MINUTES
FIG. 3. Sorbose uptake by whole cells and spheroplasts. Whole cells were assayed by Millipore filtration aa described for Fig. 2. Spheroplasts were assayed by Ficoll centrifugation as described in Methods. Data points indicate the average of duplicate determinations. (V-V 1 Whole cells; (0-O) spheroplasts.
GLUCOSE
TRANSPORT
SYSTEM
IR' S.
crassa
447
not appear to be any involvement of soluble periplasmic components in the glucose active transport system of Newospora, as has been suggested for certain prokaryote transport systems (17j. ACKNOWLEDGMENT The authors wish to acknowledge Owen.
the able technical
assistance of Miss Claudia
REFERENCES 1. SCARBOROUGH, G. A. (1970) J. Viol. Chem. 245, 1694-1698. 2. Ibid., pp. 39853987. 3. WILEY, W. R. (1970) J. Bacterial. 103, 656-662. 4. STUART. W. D., AND DEBUSK, A. G. (1971) Arch. &o&em. Biophys. 144, 512518. 5. KABACK, H. R. (1973) Symp. Sot. Ezp. Biol. 27, 145-174. 6. BACHMANN, B. J., AND BONNER, D. M. (1959) J. Bacterial. 78, 550-561. 7. KINSKY, S. C. (1962) J. Bacterial. 83, 351-358. 8. TREVITHICK, J. R.. AND METZENBERG, R. L. (1964) Biochem. Biophys. Res. Commun. 16, 319-325. 9. HENDRICK, D., .~ND DEBUSK, A. G. (1967) Neurospora Newsbtt. 12, 14. 10. SC~RBOROC~H. G. A. (1971) Biochem. Biophys. Res. Commun. 43, 968-975. 11. LOVCR~, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. C‘hem. 193, 265-275. 12. MAHADEVAN, P. R., AND TATUM, E. I,. (1965) J. Bacterial. 90, 10731081. 13. BLUMENTHAL, H. J., AND &SEMAN, S. (1957) J. Bucteriol. 74, 222-224. 14. ROSEMAN, S., AND DAFFNER, I. (1956) Anal. Chem. 28, 1743-1746. 15. PATTERSON, M. S., AND GREENE, R. C. (1965) Anal. Chem. 37, 854-857. 16. POTGIETER, H. J., AND ALEXANDER, M. (1965) Can. J. Microbial. 11, 122-125. 17. OXENDER. D. L. (1972) Ann. Rev. Biochem. 41, 777-809.