Purification and properties of a d -fructose 1,6-bisphosphatase from Saccharomyces cerevisiae

ARCHIVES

OF BIOCHEMISTRY

AND

BIOPHYSICS

Vol. 197, No. 1, October 1, pp. 170-177, 1979

Purification

and Properties

of a D-Fructose

Saccharomyces SHIGEHIRO Instituto

FUNAYAMA,‘JESUS

de Enzimologfa

de1 C.S.I.C.,

Facultud

1,6-Bisphosphatase

from

cerevisiae

MOLAN0,2 de Medicimu

de

AND CARLOS

GANCEDO

la Universidad AutBnoma, Madrid-$4,

Spain

Received January 22, 1979; revised April 9, 1979 Fructose 1,6-bisphosphatase (EC 3.1.3.11) from Saccharomyces ceretisiae has been purified to homogeneity. A molecular weight of 115,000 has been obtained by gel filtration. The enzyme appears to be a dimer with identical subunits. The apparent Km for fructose bisphosphatase varies with the MgZ+ concentration of the enzyme, being 1 x 10e6 M at 10 mM Mg2+ and 1 x 10m5M at 2 mM Mg2+. Other phosphorylated compounds are not significantly hydrolyzed by the enzyme. An optimum pH of 8.0 is exhibited by the enzyme. This optimum is not changed by addition of EDTA. AMP inhibits the enzyme with a Ki of 8.0 x 10m5M at 25°C. The inhibition is temperature dependent, the value of Ki increasing with raising temperature. 2-Deoxy-AMP is also inhibitory with a Ki value at 25°C of 1.6 x 10m4M. An ordered uni-bi mechanism has been deduced for the reaction with phosphate leaving the enzyme as the first product and the fructose 6-phosphate as the second one.

The metabolic position of fructose 1,6bisphosphatase (FbPase,3 EC 3.1.3.11) as the terminal irreversible enzyme of the gluconeogenic sequence in microorganisms makes it a likely target for control mechanisms. Indeed controls of synthesis and activity have been described in a variety of organisms (for a review see Ref. (1)). In addition to these classical controls, the FbPase from Sacchromyces cerevisiae is subject to “catabolite inactivation,” i.e., the enzyme is rapidly and irreversibly inactivated when glucose is added to a culture growing on gluconeogenic carbon sources (2). The mechanism of this inactivation has not yet been satisfactorily explained. In order to get a deeper insight in the inactivation process we undertook the purification of the

enzyme from S. cerevisiae. We present some properties of the enzyme in this article. MATERIALS

AND METHODS

Organism and growth conditions. Saccharomyces cerevisiae CJM 13 (originally provided as 8. cerevisiae 1714-24A by Professor D. C. Hawthorne, Washington University) was used along this work. The yeast was grown with aeration at 30°C in a mineral medium (3) containing 0.3% Difco yeast extract and 2% ethanol as carbon source. When the culture attained a cell density of about 2 g wet yeast/liter it was harvested by centrifugation, washed in the cold with distilled water, and stored frozen until used. Reagents. Auxiliary enzymes and biochemicals were from Sigma (St. Louis, MO.) or Boehringer-Mannheim (Federal Republic of Germany), glycerophosphate was from Calbiochem (Switzerland), p-nltrophenylphosphate from Merck (Federal Republic of Germany), and protamine sulfate essentially histone-free from Sigma. 1 Fellow of Coordenacio do Aperfeicoamento de The crystallized trisodium salt from fructose 1,6Pessoal de Nivel Superior-CAPES. On leave of ab- bisphosphate (Boehringer) was used to elute the FbPase sence from the Universidade Federal do Parana, from the phosphocellulose column. Phosphocellulose P 11 Whatman (Great Britain) was Curitiba, Brasil. treated as described by Traniello et al. (4). Sedoheptu2 Present address: Departamento de Laboratorio, Servicio de Bioquhnica, Ciudad Sanitaria La Paz, lose 1,7-bisphosphate was a gift from Dr. 0. Tsolas (Roche Institute for Molecular biology, N. J.). Madrid-34. Assay methods. FbPase was assayed spectrophotos Abbreviationsused: FbPase, Fructose l$-bisphosmetrically or fluorometrically with an enzymatic phate; SDS, sodium dodecyl sulfate; F6P, fructose coupled system as described by Gancedo and Gancedo 6phosphate. 170 00039861/79/110170-08$02.00/0 Copyright All rights

Q 1979 by Academic of reproduction in

Press,

any form

Inc. reserved.

PURIFICATION

OF S. ceretiiae

FRUCTOSE

171

BISPHOSPHATASE

tion in a glass filter. The beads were washed with 90 ml of the same buffer and this liquid was added to the former one. The filtrates were centrifuged at 27,000g for 30 min and the pellet was discarded. To the supernatant of the previous step a freshly prepared 1% solution of protamine sulfate in the extraction buffer was added dropwise until a final concentration of 0.2% was reached. After 30 min stirring it was centrifuged at 27,000g for 10 min and the precipitate discarded. The supernatant was adjusted to pH 7.27.5 with 2 M NaOH and 50 g (wet weight) phosphocellulose was added with constant stirring. The pH was kept constant by addition of 2 M NaOH. The reddish-colored phosphocellulose was removed by filtration with suction through a coarse sintered-glass filter and discarded. The filtrate was adjusted to pH 6.9 with 1 M malonic acid and 50 g (wet weight) phosphocellulose was added and removed as above. The clear filtrate (350 ml) was brought to a final volume of 1 liter with 10 mM malonate buffer pH 6.0. Additional phosphocellulose (about 400 g wet wt) was added until all enzymatic activity had been adsorbed maintaining a constant RESULTS of pH 5.6 by addition of 2 M NaOH. The resin Purification of the Enzyme was collected as above washed on the filter All the following operations were carried with 3.0 liter of 10 MM malonate, pH 6.0, out at a temperature between 0 and 4°C. and suspended in about 500 ml of 10 mM Frozen yeast was homogenized for 5 min malonate buffer. This suspension was poured in a Vibrogen Cell Mill (E. Biihler, Ttibingen, into a glass column (17 cm2 x 34 cm) and further washed with about 3 liters of buffer FRG) with glass beads (0.5 mm diameter) and buffer composed of 20 mM imidazole, until the absorbance in the eluate at 280 nm 20 1?IM Tris, 1 mM EDTA, 1 mM mercaptowas less than 0.02. The enzyme was eluted ethanol, 2 mM phenylmethylsulfonyl fluoride with the same buffer containing 2 lllM FbP, adjusted to pH 8.0 with HCl (30 g yeast/ pH 5.6, at a flow rate of 94 ml/h. Fractions 170 ml glass beads/60 ml buffer). The extract (7.8 ml) were collected and analyzed for was separated from the glass beads by suc- FbPase activity. The active fractions were

(5). The assay mixture had a pH of 7.0 and contained in a final volume of 0.5 ml: 50 mM imidazole, 50 mM KCl, 10 mM MgCl,, 1 IIIM EDTA, 0.25 mM NADP, and 0.5 units of glucose g-phosphate dehydrogenase and glucose B-phosphate isomerase. Hydrolysis of other compounds was followed measuring inorganic phosphate liberation as described by Bernhart and Wreath (6). Hydrolysis of p-nitrophenylphosphate was followed at pH 7.5 measuring the appearance of p-nitrophenol at 410 nm. One unit of enzyme is defined as the amount producing the liberation of 1 pmol of fructose &phosphate or inorganic phosphate per minute under the conditions described. In experiments where the inhibition of the enzyme by fructose g-phosphate was measured the activity was assayed following fructose bisphosphatase disappearance in the conditions described in the legend to Fig. 4. Protein concentration was determined according to Lowry et al. (7) after precipitation of the sample with 5% trichloroacetic acid. Bovine serum albumin was used as standard. Polyacrylamide gel electrophoresis was performed at 5°C in 7.5% acrylamide with 0.06 M Tris-borate buffer, pH 9.0, at 2 mA per gel. Electrophoresis in gels containing 0.1% SDS was performed in 10% acrylamide in 0.1 M Tris-acetate buffer, pH 7.4, at 8 mA per gel and room temperature. Gels were stained with Coomassie brilliant blue R 250 (8) or G 250 (9) and scanned at 550 nm in a Gilford spectrophotometer.

TABLE PURIFICATION

Step Extract Protamine sulfate Phosphocellulose and Diaflo concentration

OF FbPase

I FROM S. cerevisiae

Total activity

Protein

Volume

(units)

(mg)

(ml)

270 310

4300 1500

250 300

67

1.5

3.5

Specific activity (units/mg protein) 0.06 0.20 44.6

Yield (%)

Purification

100 114

1 3

25

740

172

FUNAYAMA

pooled and concentrated about 80 times with a Diaflo PM-30 membrane under 3 kg/cm2 of nitrogen pressure. At this step the enzyme is electrophoretically pure as shown in Fig. 1A. Table I shows a summary of the purification procedure for a typical batch of 90 g yeast. The procedure is easily reproducible and the recoveries ranged from ZO-25% of the original activity. Properties

of the Enzyme

The enzyme has a molecular weight of 115,000 as determined by gel filtration (10) on Sephadex G-200 using markers embracing a range from 99,000 to 237.000 molecular weight.

A

Electrophoresis on polyacrylamide gels containing 0.1% SDS revealed only one kind of subunit with a molecular weight of 56,000 (Fig. 1B). The enzyme therefore behaves as a dimer. Antibodies raised in rabbits against the purified enzyme did not react with FbPase from rat liver or C. utilis. Several bands were seen when the antibody reacted with an extract of derepressed commercial bakers’ yeast (Fig. 2). An analysis of the temperature dependence of the reaction under maximal velocity conditions yielded an Arrhenius plot linear between 10 and 32°C. At this temperature a sham break in the plot was observed (Fig. 3j possibly indicating a change in the

B

I

O.St-

ET AL.

----. 0.5 -

0.4-

5

Oe3-

:: 0.2 2

1

Relative

migration

(cm)

0.1 I

I

0

, 1

I 2

3

4

5

6

7 cm

.+

1I

+

FIG. 1. Gel electrophoresis of purified FbPase from S. cerevisiae. (A) Fifteen micrograms of purified FbPase electrophoresed in polyacrylamide gels without SDS as described under Materials and Methods. (B) Fifteen micrograms of purified FbPase was treated with 2.5% SDS, 2% mercaptoethanol, at 90°C for 10 min and electrophoresed in polyacrylamide gel with 0.1% SDS as described in the text. The inset shows the position of the enzyme subunit in a SDS gel with the corresponding markers:a, RNA polymerase (M, 95,000); b, bovine serum albumin (M, 68,000); c, fructose l,6-bisphosphatase (M, 56,000); d, RNA polymerase (M, 41,000); e, chymotrypsinogen (M, 25,700); and f, cytochrome c (M, 12,000).

PURIFICATION

OF S. cerevisiae FRUCTOSE

BISPHOSPHATASE

173

FIG. 2. Ouchterlony immunodiffusion test with FbPase antibodies. The center well contained FbPase antibodies raised in rabbits. The other wells contained the following: (1) 50 ~1 of extract from ethanolgrown S. ccrevisiae (70 munit/ml FbPase); (2) 100 ~1 of extract from glucose-grown S. cerevisiae (1 munit/ml FbPase); (3) 100 ~1 extract of derepressed commercial bakers’ yeast (40 munit/ml FbPase); (4) 100 ~1 extract of ethanol-grown C. utilis (100 munit/ml FbPase); (5) 100 ~1 of a cytosol fraction from rat liver (200 munit/ml FbPase); (6) 20 ~1 of purified yeast FbPase (4 units/ml). Precipitin bands were stained with Coomassie brillant blue G-250.

conformation of the enzyme (11). An activation energy of 19 kcal/mol was calculated for the first part and one of 5 kcal/mol for the range above 32°C. The Q1,, varied from 4.2 (lo-ZO’C) to 1.3 (32-42°C). 2.5

t

2.0-

\\ “t, *... \ l

log K l

1.5-

\

FIG. 3. Arrhenius plot for the reaction catalyzed by S. ccrevisiae FbPase. The enzyme was assayed as described under Materials and Methods at the temperatures indicated. The reaction mixture was equilibrated at the desired temperature for 10 min. After finishing the reaction the temperature was checked on the cuvette.

The purified preparation showed a PH optimum around 8.0 (Fig. 4). In contrast with the results obtained with C. utilis (1) addition of EDTA to the reaction mixture did not displace the curve (Fig. 4). The same result was obtained when a nonchelating buffer mixture (20 mM N-2-hydroxyethylpiperazine-N ‘-2-ethanesulfonic acid and 20 InM Tris) was used. Substrate Specifiity Fructose 1,6-bisphosphate was the preferred substrate of the enzyme in our experimental conditions. Measuring initial rates corresponding to less than 30% utilization of the initial substrate a value of 1 x 1OP M was obtained as the apparent Michaelis constant. This value varied with the Mg2+ concentration of the assay mixture increasing with decreasing Mg2+ (Fig. 5) although the V remained virtually unchanged. This results suggests a complex fructose 1,6-bisphosphate-Mg as the true substrate for the enzyme. No inhibition by excess substrate was found with a fructose 1,6-bisphosphate concentration up to 1 mM. Table II shows the observed activities toward different esters. As it can be seen other compounds tested even at high concentration were not significant substrates for the enzyme.

174

FUNAYAMA

ET AL. TABLE SUBSTRATE

SPECIFICITY OF THE FbPase FROM s. Ct3%?tiiae”

Compound

I

6

.

1

I

8

IO

PH FIG. 4. Influence of pH on the activity of FbPase. The reaction mixture contained in a final volume of 0.5 ml: 50 mM NJ-bis(2-hydroxyethyl)glycine, 50 mM morpholine ethane sulfonic acid, 50 mM morpholine propane sulfonic acid, 50 mM glycine adjusted with KOH to the indicated pHs, 10 mM MgCl*, 0.25 mM NADP, 0.5 unit of glucose B-phosphate dehydrogenase, 0.5 unit of glucose g-phosphate isomerase, 0.1 mM fructose I$bisphosphate, and an appropriate amount of enzyme. Purified enzyme without EDTA (0). Purified enzyme with I mM EDTA (Xl.

Inhibition

by AMP

and Analogs

As reported for other FbPases, AMP inhibits the enzyme from S. cerevisiae (14). The enzyme presents an increased sensitiv-

II

Fructose I$-bisphosphate Glucose l,6-bisphosphate Sedoheptulose 1,7-bisphosphate Fructose l-phosphate Glucose l-phosphate Phosphoenolpyruvate cr-Glycerophosphate P-Glycerophosphate p-Nitrophenylphosphate

Concentration Cm@

Relative activity (%/o)

0.1 5.0 6.0

100 10

10.0

5.0 2.0 5.0 5.0 2.0

5 5 4 3 2 1 1

(1Hydrolysis of the different compounds was followed as described under Materials and Methods. Fructose l-phosphate was purified according to Sherma and Zweig (12) and assayed as in Ref. (13).

ity to AMP inhibition with decreasing temperature, a result parallel to that found by Taketa and Pogell in rat liver (15). Ki values were 60 PM at lO”C, 80 PM at 25”C, and 160 PM at 35°C. No appreciable cooperativity was found in the plots of inhibition vs concentration of inhibitor at any temperature. Several analogs of AMP were assayed as potential inhibitors. ZDeoxy-AMP showed a similar inhibitory effect (Ki at 25°C 160 PM). IMP essentially free of AMP as shown by a test with adenylate deaminase inhibited the enzyme but the Ki was loo-fold larger than that for AMP. Adenosine, 2’-AMP, and

FIG. 5. Influence of the Mg2+ concentration on the activity of FbPase. The activity was measured fluorometrically as described in the text varying the concentrations of magnesium as indicated.

PURIFICATION

OF S. cerewisiaR FRUCTOSE

175

BISPHOSPHATASE

20 mM F6P

0.012-

0 I/V

10 mM F6P

x Whout

I/

FbP

added

F6P

VM

FIG. 6. Inhibition by fructose 6-phosphate of the FbPase activity. Mixtures containing 50 mM imidasole, 50 mM KCl, 2 mM MgCl%, 1 mM EDTA, pH 7, and the indicated concentrations of fructose 1,6bisphosphate were incubated with purified FbPase at 30°C. At 0, 1, 3, 5, and 8 min aliquots were withdrawn and quickly mixed with 0.1 ml of cold 6 M acetic acid. Eight-tenths milliliter of 0.3 M imidasole was added to raise the pH to 7.1. In this mixtures fructose 1,6-bisphosphate was measured spectrophotometrically with a coupled enzymatic system.

3’-AMP were neither inhibitors relieve the effect of AMP. Mechanism

nor did they

result suggests a binding of fructose 6-phosphate to the free enzyme. The other product of the reaction, orthophosphate, also inhibits the activity toward fructose bisphosphate but the type of inhibition is noncompetitive with respect to the substrate (Fig. ‘7). According to Cleland (16) these results are consistent with the ordered seauential mechanism shown below.

of the Reaction

Hydrolysis of fructose bisphosphate is inhibited by fructose 6-phosphate. This inhibition is competitive with respect to fructose bisphosphate as can be seen in Fig. 6. This

1

FbP E

F6P

pi

I

T

T (E-FbP-E-FGP-P)

E

E-F6P

0.05

*

0.04 -

/

50 mM phosphate

I/V 0.03-

.* 0

x.x/x 25 mM phosphate

without 0.05

I/FbP

added

phosphate

01

PM

FIG. 7. Inhibition by phosphate of the FbPase activity. Activity was measured spectrophotometritally in a mixture that contained in a final volume of 0.5 ml: 50 mM imidasole, 50 mM KCI, 2 mM MgCIZ, pH 7.0, 0.2 mM NADP, 0.5 unit of glucose B-phosphate isomerase, 0.5 unit of glucose B-phosphate dehydrogenase, and the indicated amount of phosphate.

176

FUNAYAMA

ET AL.

of EDTA does not modify the pH profile of the enzyme. FbPase was purified from S. cerevisiae The molecular weight of the S. cerevisiae grown under controlled conditions. Comenzyme is close to that found for the enzyme mercial bakers’ yeast was not an adequate from C. utilis (20). However the enzyme source since after more than 1500-fold purifrom S. cerevisiae dissociates only into two fication of the activity many protein bands subunits in the presence of SDS while the appeared on polyacrylamide gels. Most likely enzyme from C. utilis behaved in these conthe process used to derepress the FbPase activity in commercial bakers’ yeast (2) in- ditions as a tetramer (1). The enzyme is creased the proteolytic activities of the highly specific for fructose 1,6-bisphosphate; organism (17). Caution should therefore be no other substances tested even at high conexerted when using commercial yeast to centrations were significantly hydrolysed. K, for fructose 1,Bbisphosphate is inpurify enzymes for certain purposes since fluenced by the magnesium concentration either it may be difficult to reproduce puriin the assay. A value of 1.0 pM was obtained fication procedures developed by other workers (18) or artifactual enzymatic forms at 10 mM MgCI, while a value of 10 PM was may appear. We also noted that it was found at 2 mM MgC&. This could indicate that the substrate for catalysis is not free of paramount importance to use a highly fructose 1,Bbisphosphate but the correpure preparation of fructose 1,6-bisphosphate sponding magnesium derivative. to achieve a good purification. The results obtained in the inhibition The enzyme purified from S. cerevisiae studies allow us to conclude an ordered seappears to be the native form of the protein quential uni-bi mechanism, with phosphate and not a proteolytic degradation product being released from the enzyme as the first as judged by the following set of facts. Preproduct and fructose 6-phosphate as the cautions have been taken to avoid known second one. This mechanism proceeds withpotential proteolytic processes; the yeast enwas harvested in the exponential phase out the formation of a phosphorylated otherwise phosphate of growth and extracts and purification steps zyme as intermediate would competitively inhibit the enzyme. were carried out in the presence of phenylThis interpretation is consistent with the methanesulfonyl fluoride, an inhibitor of results of Pontremoli et al. (21) who did not yeast proteases B and C (17). Extreme find evidence for the existence of such acidic pH that could favor the action of intermediate with the rabbit liver enzyme. protease A was also avoided. The proposed mechanism differs from that The purified preparation migrates as found for other phosphomonoesterases that a single band on SDS gels while the proteorelease the alcohol component as first lyzed form from rabbit liver migrates as product and phosphate as the last one (22). a doublet in these conditions (4). Moreover The inhibition by analogs of AMP showed as discussed below the calculation of activity that the amino group in position six of the under physiological conditions accounts for purine ring and the phosphate in the 5’ carthe metabolic flux observed in vivo. One fact bon of the ribose are of critical importance that has been puzzling in different FbPase for the inhibiton. The inhibition by AMP preparations of several sources was the does not show appreciable cooperativity, “alkaline” pH needed for activity in the ab- another difference with the enzyme from sence of chelating agents (1). This embarC. utilis (23). AMP has been implicated as rassing question was solved by the groups a regulator in the shift glycolysis-gluconeoof Horecker and Pontremoli (19) who showed genesis owing to the antagonistic effect obthat this behavior was due to partial proteolserved in vitro toward FbPase and phosysis of the native enzyme during the puri- phofructokinase. However in the case of fication. The enzyme from S. cerevisiae has yeast levels of AMP do not vary markedly an optimal pH around 8.0 and shows about in this shift; moreover at the physiological 40% of its optimal activity in the physiologiAMP concentration of 1.0 mM the FbPase cal range of pH of the yeast. The addition would be permanently inhibited about 60%. DISCUSSION

PURIFICATION

OF S. cerevisiae

A calculation of the activity of the FbPase at physiological pH and in the presence of 0.1 mM AMP produces a figure of 30 mU/mg protein, a value in good agreement with the gluconeogenic flux measured by Barwell and Hess (24). Therefore the physiological significance of the AMP inhibition remains far from understood. An interesting aspect of the FbPase from S. cerevisiae is its regulation by inactivation in the shift gluconeogenesisglycolysis (2). No conclusive evidence is yet available regarding the mechanism of this inactivation. The availability of the purified enzyme would allow detailed studies on the inactivation mechanism using immunological procedures. Work toward this goal is in progress in our laboratory.

FRUCTOSE

8. 9. 10. 11. 12.

13. 14.

15. 16.

ACKNOWLEDGMENT 17. The constant encouragement of Dr. Juana M. Gancedo and helpful discussions with her were of invaluable help. 18. REFERENCES 1. PONTREMOLI, S., AND HORECKER, B. L. (1971) in The Enzymes, (Boyer, P. D., ed.), 3rd ed., Vol. 4, pp. 611-646, Academic Press, New York/London. 2. GANCEDO, C. (1971) J. Bacterial. 107, 401-405. 3. B~Eu)s, M., GANCEDO, C., AND GANCEDO, J. M. (197’7) J. Biol. Chem. 252, 6394-6398. 4. TRANIELLO, S., PONTREMOLI, S., TASHIMA, Y., AND HORECKER, B. L. (1971) Arch. Biochem. Biophys. 146, 161- 166. 5. GANCEDO, J. M., AND GANCEDO, C. (1971) Arch. Microbial. 76, 132-138. 6. BERNHART, D. N., AND WREATH, A. R. (1955) Anal. Chem. 27, 440-441. 7. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L.,

19.

20.

21.

22. 23. 24.

BISPHOSPHATASE

177

AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. VESTERBERG, 0. (1971) Biochim. Biophys. Acta 243, 345-348. BLAKESLEY, R. W., ANDBOEZI, J. A. (1977)Anal. Biochem. 82, 580-582. NIMMO, H. G., AND TIPTON, K. F. (1975) Biothem. J. 145, 323-334. TALSKY, G. (1971) Angew Chem. 10, 548-554. SHERMA, J. (ED.), AND ZWEIG, G. (1971) in Paper Chromatography and Electrophoresis, Vol. 2, pp. 485, Academic Press, New YorWLondon. BANDLJRSKI, R. S., AND AXELROD, B. (1952) J. Biol. Chem. 193, 405-412. GANCEDO, C., SALAS, M. L., GINER, A., AND SOLS, A. (1965) Biochem. Biophys. Res. Commun. 20, 15-20. TAKETA, K., AND POGELL, B. M. (1965) J. Biol. Chem. 240, 651-662. CLELAND, W. W. (1970) in The Enzymes (Boyer, P. D., ed.), 3rd ed., Vol. 2, pp. l-65, Academic Press, New York/London. PRINGLE, J. R. (1975) in Methods in Cell Biology (Prescott, D. M., ed.), Vol. 12, pp. 149-184, Academic Press, New York/London. KELLY, P. J., AND CATLEY, B. J. (1976) Anal. Biochem. 72, 353-358. PONTREMOLI, S., MELLONI, E., BALESTRERO, F., FRANZI, A. T., DE FLORA, A., AND HORECKER, B. L. (1973) Proc. Nat. Acad. Sci. USA 70, 303-305. ROSEN, 0. M., COPELAND, P. L., AND ROSEN, S. M. (1966) Proc. Nat. Acad. Ski. USA 56, 1810-1816. PONTREMOLI, S., TRANIELLO, S., LUPPIS, B., AND WOOD, W. A. (1965) J. Biol. Chem. 240, 34593463. Hsu, R. Y., CLELAND, W. W., AND ANDERSON, L. B. (1966) Biochemistry 2, 779-807. COOPERMAN. B. S., AND But, H. (1972) Eur. J. Biochem. 27, 503-512. BARWELL, C. J., ANDHESS, B. (1971)FEBSLett. 19, l-4.