Crystalline fructose 1,6-bisphosphatase from chicken breast muscle

ARCHIVES

OF

BIOCHEMISTRY

Crystalline ANJANAYAKI

Fructose

AND

BIOPHYSICS

I%$,

48-56

(1977)

1,6-Bisphosphatase

E. ANNAMALAI,’

from Chicken

ORESTES

Received

March

TSOLAS,

AND

Breast Muscle B. L. HORECKER

31, 1977

Fructose 1,6-bisphosphatase has been isolated and crystallized in high yield from chicken breast muscle, which is a rich source of this enzyme. The specific activity assayed at pH 7.4 and 25°C in the presence of 0.2 mM MnCl,, 0.1 mM EDTA, and 40 mM ammonium sulfate is 50-60 unitsimg. making this one of the most active fructose bisphosphatases yet described. The K,,, for fructose bisphosphate is 8.3 p.M. AMP (0.4 pM) inhibits the activity at pH 7.4 almost completely. EDTA can be replaced as activator by citrate or histidine, which both give maximum activation at millimolar concentrations. Citrate is as effective as EDTA. The enzyme has a molecular weight of 144,000 and is composed of four subunits having a molecular weight of 36.000. Aminoand carboxyterminal analyses indicate that the subunits are identical.

Fructose 1,6-bisphosphatase (FruP,ase)” (EC 3.1.3.11) occurs in liver and kidney, where it is required for gluconeogenesis. However, high levels of activity of this enzyme have also been found in white skeletal muscle (l-31, and a number of hypotheses (2,4-7) have been proposed for the function of the muscle enzyme. The most attractive of these is that it is involved in the production of heat from the hydrolysis of ATP in a fructose 6-phosphateifructose 1,6-bisphosphate substrate cycle, catalyzed by phosphofructokinase and Fru-P,ase (7). The role of this cycle in heat production in the bumble bee is supported by the elegant experiments of Bloxham et al. (8), and the substrate cycle catalyzed by phosphofructokinase and FruP,ase has also been implicated in heat production in malignant hyperthermia in halothane-sensitive pigs (9, 10). However, its role in normal heat production in mammals has not been demonstrated. Olson and Marquardt (11) have shown that chicken breast muscle is an unusually rich source of Fru-P,ase, comparable to

chicken liver in terms of units of this activity per gram of tissue. They have purified the enzyme from chicken muscle and showed that it differed from the liver enzyme in its immunological and electrophoretie properties. The enzyme from rabbit muscle has also been isolated in homogeneous form (12). However, very little information is available on the structure of muscle Fru-P,ase and its genetic or evolutionary relation to the liver enzyme, and indeed sufficient quantities of the pure enzyme from muscle have not been available to permit such studies. We report here a simple and convenient procedure for the isolation of the enzyme from chicken breast muscle and an examination of some of its properties. MATERIALS

AND

METHODS

Breast muscle from chicken tGollus domesticus 1 was obtained fresh from a local poultry market and stored frozen. Fru-P, tNal salt), NADP, AMP, ATP, and sodium dodecyl sulfate were obtained from Sigma Chemical Co., St. Louis. Missouri. Glucose 6phosphate dehydrogenase, phosphoglucose isomerase. myokinase, pyruvate kinase. and phosphoenolpyruvate were obtained from Boehringer Mannheim Corp., Indianapolis, Indiana. Carboxypeptidase A treated with phenylmethylsulfonyl fluoride and carboxypeptidase B treated with diisopropyl phosphofluoridate were obtained from Worthington Biochemical Corp.. Freehold, New Jersey. Rabbit mus-

’ Present address: Department of Pediatrics, Mayo Memorial Building, University of Minnesota Hospitals, Minneapolis, Minnesota 55455. : Abbreviations used: CM, carboxymethyl; FruP,. n-fructose 1.6-bisphosphate; Fru-P,ase, fructose 1,6-bisphosphatase. 48 Copyright. All rights

,S, 1977 by Academic Press, Inc. of reproduction in any form reserved.

ISSN

0003~9861

CHICKEN cle aldolase gift of Dr.

BREAST

used for Edman degradation C. Y. Lai of this Institute.

MUSCLE was a kind Whatman

phosphocellulose (P-11) was purchased from ReeveAngel Co., Clifton, New Jersey. N-Ethyl morpholine (distilled prior to use), hydrazine (95R+), and phenyl isothiocyanate were obtained from Eastman Kodak, Rochester, New York. N-Allyl-N,N-dimethylamine, distilled pyridine, trifluoroacetic acid, and 3-(2-aminoethyl)indole were obtained from Pierce Chemical Co., Rockford, Illinois. Other chemicals were the best commercial grade available. Fru-P,ase was assayed with the coupled spectrophotometric method described by Pontremoli et al. (13). The standard assay mixture (1 ml) contained 40 mM triethanolamine-40 mM diethanolamine buffer, pH 7.4, 0.1 mM Fru-P,, 0.2 mM NADP-, 0.2 mM MnCl,, 0.1 mM EDTA, 40 mM (NH,),SO,, 2 pg of glucose 6-phosphate dehydrogenase, and 2 pg of phosphoglucose isomerase. Variations in metal, chelator, and salt are as indicated in the text. Since AMP was found to be a strong inhibitor of the muscle enzyme (1, 2), an AMP trapping system (12) was included for assay of the crude extract. One unit of Fru-P,ase activity was defined as the amount of enzyme that catalyzes the hydrolysis of 1 pmol of Fru-P, per minute at 25°C. Protein was routinely determined by Biicher’s turbidimetric method (14). Purified enzyme was also measured by its absorbance; a solution containing 1.0 mg (dry weight) of purified Fru-P2ase per milliliter yielded an absorbance of 0.81 at 280 nm in a light path of 1.0 cm. Disc gel electrophoresis was performed at 4°C with a current of 4 mA per tube according to Davis (15). Standard 7% polyacrylamide gels at pH 8.8 in Tris-glycine buffer or at pH 4.3 in acetic acid buffer were used with a protein sample of about 40 pg per gel. The gels were stained with 0.5% aniline black in 7% acetic acid and destained in 7% acetic acid. Disc gel electrophoresis in the presence of 0.1% sodium dodecyl sulfate was carried out as described by Weber and Osborn (16), except that the staining solution contained 0.1% Coomassie brilliant blue. The protein standards were bovine serum albumin (M, = 67,000), rabbit muscle aldolase (subunit M, = 40,000), a-chymotrypsinogen (M, = 25,600), and bovine pancreatic ribonuclease (M, = 13,700). They were obtained from Boehringer Mannheim Corp., Indianapolis, Indiana. Low-speed sedimentation equilibrium for the molecular weight determinations of the purified enzyme was performed at 6000 rpm. The samples (1.43 mgiml in 0.01 M Tris-HCl buffer, pH 7.4, containing 1 mM EDTA, 0.1 M KCl, and 2 mM P-mercaptoethano11 were centrifuged at 12”C, utilizing a Beckman Model E analytical ultracentrifuge equipped with a monochromator set at 280 nm, a photoelectric scanner, an RTIC temperature control, and an electronic speed control The partial specific volume. 0.744.

FRUCTOSE

49

BISPHOSPHATASE

was calculated

from

the amino

acid composition

ac-

cording to Schachman (17). For performing amino acid analysis, protein (2-3 nmol) was hydrolyzed in 5.7 N HCl in sealed evacuated tubes at 110°C for 24, 48, and 72 h (18). The hydrolyzed samples were analyzed with a JEOL 6AH automatic amino acid analyzer according to the method of Spackman et al. (19). Glutamine in carboxypeptidase digests was identified by using lithium citrate buffers on the amino acid analyzer (20). The tryptophan content of the protein was evaluated by three different methods: (a) by the procedure of Spande and Witkop (21) usingN-bromosuccinimide, (b) fluorometrically after digestion with a mixture of Pronase and chymotrypsin (221, and (c) by amino acid analysis after hydrolysis in 3 N methanesulfonic acid containing 0.2% 3-(2.aminoethyl)indole (23, 24). Carboxymethylation was carried out according to Crestfield et al. (25) using recrystallized iodoacetic acid (26). Fluorescence spectra were recorded with an Aminco-Bowman recording spectrofluorometer. Phosphocellulose was washed according to Peterson and Sober (27). The washed material was suspended in 0.1 M sodium malonate buffer, pH 5.8, containing 1 mM EDTA, with stirring for 5 min, filtered using a Biichner funnel, and resuspended again in the same buffer. The resin was allowed to settle for 2 h and was filtered, and the moist cake was stored at 4°C. Carboxypeptidase digestion was performed on Fru-P,ase dialyzed against 1 liter of 0.1% sodium dodecyl sulfate. The incubation mixture contained 1.09 mg of Fru-PLase, 0.2 M N-ethylmorpholine acetate, pH 8.3, 0.1% sodium dodecyl sulfate, and 0.01 mg of washed carboxypeptidase A. The final volume was 0.8 ml and the temperature was 25°C. Aliquots, 0.2 ml, at given time intervals were pipetted directly into 2.0 ml of the sample dilutor used for the amino acid analyzer, and 0.05 ml of glacial acetic acid was added. The samples were frozen and thawed, the precipitated protein was centrifuged, and the clear supernatant solution was applied on the amino acid analyzer. Hydrazinolysis was performed according to Carlton and Yanofsky (28). Edman degradation was performed according to either Lai (29) or Weiner et al. (30). The amino acid thiazolinone derivatives were converted to free amino acids by hydrolysis in the presence of SnCl, (31) and identified on the amino acid analyzer. RESULTS

Purification of Fru-P,ase Breast Muscle

from

Chicken

1. Extraction. All operations in this and subsequent steps were carried out in the cold. Partially thawed chicken breast mus-

50

ANNAMALAI.

TSOLAS. AND HORECKER

cle (308 g) was homogenized in 1232 ml of 10 mM Tris-HCl buffer, pH 7.0, containing 1 mM EDTA for two 1-min intervals. The homogenate was centrifuged at 20,OOOg for 40 min and the residue was discarded. The supernatant solution, pH 5.8, was adjusted to pH 7.0 with 7.2 ml of 2 N NaOH (extract: 1300 ml, Table I). 2. Heat fractionation. The extract was transferred to a 4-liter beaker and heated to 67°C with constant stirring in an 85°C water bath. The extract was maintained at 67°C for 3 min and then rapidly cooled in an ice bath to 15°C and centrifuged at 2O,OOC& for 20 min. The supernatant solution was filtered through glass wool (heat fraction: 1180 ml). 3. Phosphocellulose adsorption. The heat fraction was diluted with 3 vol of 1 mM EDTA, pH 7.0, and the pH was adjusted to 5.8 with solid malonic acid. To this solution, 118 g of phosphocellulose was added slowly with stirring while the pH was maintained at 5.8 with either solid malonic acid or 1 N NaOH. The resin was allowed to settle and the supernatant solution was assayed for residual enzymatic activity. The suspension was filtered through a coarse sintered glass funnel using a slight vacuum. The resin was washed on the funnel with 1300 ml of 0.1 M sodium malonate, pH 5.8, containing 1 mM EDTA and then with 1500 ml of the same solution at pH 6.8. The resin was then suspended in 0.125 M sodium malonate, pH 6.8, containing 1 mM EDTA, transferred to a column, and washed, and the enzyme was eluted with TABLE PURIFICATION

I

OF CHICKEN

BREAST

Fru-

MUSCLE

P,ase” step

Extract Heat fraction Phosphocellulose eluate Crystals

Volume (ml)

Total units

1300 1180 20 4.5

0 The enzyme was assayed under Materials and Methods. b Protein was determined method (141.

Specific

tivity” (units/mg)

2933 2289 1742

0.25 8.8 62.5

1300

57

ac-

Yield

(%I 100 78 62 44

at pH 7.4 as described by

the

turbidity

the same buffer containing 2 mM Fru-P, (Fig. 1). Fractions with high specific activity (Fractions 22-27) were pooled (phosphocellulose eluate: 20 ml, Table I). 4. Ammonium sulfate precipitation. To 20 ml of phosphocellulose eluate, 5.8 g of ammonium sulfate was added to bring the saturation to 50%. The solution was centrifuged at 10,OOOg for 15 min and the clear supernatant solution was brought to 70% saturation by the slow addition of 2.5 g of solid ammonium sulfate. The suspension was stored at 4°C. 5. Crystallization. The ammonium sulfate fraction was centrifuged at 10,OOOg for 15 min. The precipitate, which contained all of the activity, was suspended in 4.0 ml of 50% saturated ammonium sulfate solution to a protein concentration of 4-5 mgl ml. Cold water was added dropwise with stirring until the suspension was almost clear. To the turbid solution, cold saturated ammonium sulfate solution was added dropwise, over a period of days, until all of the activity was crystallized (Fig.

1.6

1601

8 P

1.2 c 0.8 5 E 0.4 g z 88

96

104

VOLUME

(ml)

112

FIG. 1. Chromatography of chicken breast muscle Fru-P,ase on phosphocellulose. A column (3.5 x 20.5 cm) was packed with t,he resin containing the enzyme and washed with 0.125 M sodium malonate. pH 6.8, containing 1 rnM EDTA until the absorbance of the emuent at 280 nm was 0.02. All of the washings were checked for activity and contained less than 7% of the total units in the heat fraction. Enzymatic activity was eluted from the column with the same buffer, containing 2 mM Fru-P?. Fractions, 4 ml each, were collected at a flow rate of 40 ml/h and assayed for enzymatic activity (O---G) and absorbance at 280 nm ( x-----x 1. Fru-P,ase activity is expressed as micromoles of substrate hydrolyzed per minute per milliliter of effluent.

CHICKEN

FIG.

BREAST

2. Crystals

MUSCLE

of chicken

FRUCTOSE

breast

St ructure and Physical Properties Homogeneity. The pattern of elution & om the column of phosphocellulose sh lowed a single protein peak that cointicled with the activity (Fig. 1) and no in-

muscle

BISPHOSPHATASE

Fru-P!ase

51

(x4.50).

crease in specific activity was observed on crystallization. In polyacrylamide disc gel electrophoresis, a single protein band was obtained at either pH 4.3 or 8.8. Disc gel electrophoresis in the presence of sodium dodecyl sul-

ANNAMALAI,

52

TSOLAS,

AND HORECKER

fate also yielded a single band. Homogeneity with respect to molecular weight was confirmed by low-speed equilibrium sedimentation analysis in the ultracentrifuge (see below). Ultraviolet absorption spectrum. The spectrum of the purified enzyme in 0.1 N HCl and 0.1 N NaOH showed maxima at 270 and 293 nm, respectively. The isosbestic point was at 281 nm, in agreement with the data reported by Beaven and Holiday (32) for proteins containing a mixture of aromatic amino acids. The ratio at A,,,,/ A,,,, in 0.1 N NaOH was 1.40, indicating the absence of significant quantities of tryptophan (see also below). Molecular weight. The molecular weight of the purified native enzyme estimated from sedimentation equilibrium experiments performed as described under Materials and Methods was 143,800. A linear plot of the square of the distance from the center of the rotor, versus the loga-

rithm of the absorbance (not shown), indicated that the enzyme was homogeneous with respect to molecular weight. The subunit molecular weight, estimated by disc gel electrophoresis in the presence of sodium dodecyl sulfate according to Weber and Osborn (16), was 36,000. Amino acid composition. The amino acid composition of chicken muscle FruP,ase differs significantly from those of the rabbit muscle and rabbit liver enzymes (Table II). For the determination of the tryptophan content of chicken muscle FruP,ase, several methods were employed. Titration of the enzyme with N-bromosuccinimide (21) yielded a value of 1.95 mol/ mol. Fluorometric analysis of the protein after proteolytic digestion (22) yielded a value of 0.62 mol/mol. Amino acid analysis after hydrolysis (23, 24) yielded a value for tryptophan of 0.95 mol/mol. Except for the analysis with N-bromosuccinimide (21), the results indicated that the tryptophan

TABLE AMINO

ACID

COMPOSITION

OF CHICKEN

MUSCLE

Fru-P,ase AND

Residue

Chicken Hours

Lysine Histidine Arginine CM-cysteine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan”

II AND

COMPARISON

WITH

THAT

OF RABBIT

MUSCLE

LIVER

Rabbit”.’

muscle”

of hydrolysis”

24

48

72

114 19 42 14 118 82 85 120 60 116 120 95 36 71 134 59 44 -

127 18 41 118 71 71 125 60 125 123 95 42 68 131 56 42 -

125 20 37 131 81 73 125 61 121 116 96 37 68 131 56 44 -

Average

Muscle

122 19 40 14 122 86” 90” 123 60 121 120 95 38 69 132 57 43 -co.3

102 14 47 21 117 68 70 119 62 120 129 102 26 71 134 55 31 0

(I Equivalents per 144,000 g. b A sample of pure enzyme was reduced, carboxymethylated in 8 M urea, 48, and 72 h as described under Materials and Methods. c Data of Abrams et al. (33). ” These values were obtained by extrapolating to zero time. p Determined as described under Materials and Methods.

desalted,

and hydrolyzed

Liver

115 13 40 23 147 71 71 93 60 102 111 104 35 77 111 52 47 CO.5 for 24,

CHICKEN

BREAST

MUSCLE

content of chicken muscle Fru-P,ase was less than 0.3 equiv per subunit. End group analysis. The carboxy-terminal amino acid of chicken muscle FruP,ase was found to be serine. Hydrazinolysis yielded 0.64 mol of this amino acid per mole of subunit and smaller quantities of arginine (0.31 mol) and glycine (0.37 mol). Carboxypeptidase A digestion of the denatured enzyme at 25°C and a protein to enzyme ratio of 109 (w/w) yielded the following quantities of serine (mole per mole subunit): 0 min (0), 5 min (0.90), 15 min (1.55), 60 min (1.99), and 120 min (2.23). At 120 min, in addition to serine, lysine (0.18), histidine (0.19), and glutamine (0.12) were also identified. Digestion with carboxypeptidase A at a ratio of 1:15 (w/w) or with carboxypeptidase B at a ratio of 1:17 (w/w) confirmed the release of serine, lysine, histidine, and glutamine. No serine was released by carboxypeptidase A or B in the absence of denaturant or in the presence of 1 M urea. Edman degradation of the enzyme either by the method of Lai (29) using glass beads as support or by the method of Weiner et al. (30) using sodium dodecyl sulfate as the denaturant gave negative results. Both methods yielded proline and lysine, respectively, from rabbit muscle aldolase and ribonuclease. The results indicate that the amino terminus of chicken muscle Fru-P,ase, like that of the rabbit liver enzyme (34), is blocked. Catalytic

Properties

pH activity profile and effect of chelators. The pH optimum of chicken muscle Fru-P,ase was approximately 8.2 in the presence of Mn2+ or Mg’+. The addition of EDTA or citrate increased the catalytic activity in the neutral pH range and shifted the pH optimum to about 7.4. The highest activity was observed when citrate and L-histidine were both added to the assay mixture, particularly when Mn?+ was the metal cofactor (Fig. 3). Histidine alone was approximately half as effective as the mixture of histidine and citrate (data not shown). The chelators had little or no effect in the alkaline region, particularly when assayed with Mg”+.

FRUCTOSE

53

BISPHOSPHATASE

6

7

8

96

7

8

9

PH

FIG. 3. Effect of chelators on the pH activity profile of chicken muscle Fru-P,ase in the presence of 0.2 rnM MnCl, or 5 mM h%gCl,. The enzyme (0.2-0.4 pg) was assayed as described under Materials and Methods, with no chelators (X-X), lo-” M EDTA (/J-a), lo-:’ M citrate (O-O), or 10-I’ M citrate plus lo-:’ M L-histidine (O---O). The concentrations of chelators used were optimal and similar to those reported for the enzyme from rabbit muscle (35, 36). Fru-P,ase activity is expressed as micromoles of substrate hydrolyzed per minute per milligram of protein.

Effect of substrate concentration. The optimum concentration for Fru-P, in the presence of 0.2 mM MnCl, was 0.1-0.2 mM. Above this concentration, there was some inhibition. The apparent K,,,, estimated from the double-reciprocal plot, was 8.3 x 10-l; M (Fig. 4). Effect of divalent cations. Chicken muscle Fru-P,ase showed an absolute requirement for either Mnl+ or Mg2+. In the absence of EDTA, the apparent K,,,‘s and V’s as determined from double-reciprocal plots were 8.7 x 10-j M and 30.3 units/mg for Mn’+ and 1.1 x lo-” M and 26.3 units/mg for Mgl+. Hill plots of the data showed that the activating effect of Mn”+ was noncooperative with a Hill coefficient of 1.0. Activation by NH,+. The chicken muscle enzyme, like other Fru-P,ases, is activated by NH,+ (12, 36-40). At pH 7.4, in the presence of 5 mM MgCl?, optimum activity was observed at a concentration of NH,+ of 40-50 mM (Fig. 5). Inhibition by AMP. In common with other Fru-P,ases, the chicken muscle enzyme was found to be inhibited by AMP (Fig. 6A). In the presence of 5 mM MgCl, at pH 7.4, the value ofKi for AMP was 42 nM, in agreement with the values previously

54

ANNAMALAI,

TSOLAS,

AND

HORECKER

I

0 FIG. 4. Effect of substrate concentration on FruP,ase activity. The enzyme (0.12 fig) was assayed in the presence of 0.2 mM MnCl,, at pH 7.4, as described under Materials and Methods. The inset shows the plot of the reciprocal of the specific activity (units per milligram) against the reciprocal of Fru-P, concentration. The value for K,,, was estimated to be approximately 8.3 PM. Activity is expressed as indicated in the legend to Fig. 3.

I

I

50 (NH&O4

100 hM)

FIG. 5. The effect of increasing concentration of (NH&SO, on chicken muscle Fru-P,ase at pH 7.4 in the presence of 5 mM MgCl,. The enzyme (0.3 pg1 was assayed as described under Materials and Methods, and the activity is expressed as indicated in the legend to Fig. 3.

reported for the partially purified enzyme (4, 43). A Hill plot of the data showed no evidence of cooperativity with a Hill coefficient of 1.0 (Fig. 6B). Opie and Newsholme (4) have also reported noncooperative inhibition of Fru-P,ase by AMP in chicken breast muscle extracts. DISCUSSION

Chicken breast muscle is not only a very rich source of Fru-P,ase, but the crystalline enzyme isolated from this source has a higher specific activity than has previously been reported for any mammalian Fru-P,ase. The specific activity reported here (57 units/mg) is to be compared with values of 28.5 units/mg for the purified rabbit muscle enzyme (12) and 22.5 units/ mg previously reported for the chicken breast muscle enzyme (11). The procedure described is rapid and reproducible, can be adapted to large-scale purification, and makes possible structural work on the enzyme protein. The molecular weight reported here, 144,000, is consistent with the sedimentation coefficient of 7.05 previously reported by Olson and Marquardt (11). The amino acid composition of the chicken muscle enzyme is significantly different from that of the enzymes isolated from rabbit muscle

Free

AMP

(&MI

log

Free

AMP

1nM)

FIG. 6. The effect of AMP on chicken muscle FruP,ase at pH 7.4, assayed in the presence of 5 mM MgCl,. The enzyme (0.3-1.2 pgl was assayed as described under Materials and Methods, in the presence of increasing concentrations of AMP, and the activity is expressed as indicated in the legend to Fig. 3. The effect of increasing concentration of free AMP, calculated assuming an association constant of 100 M ’ for the Mg-AMP complex (411, is shown in A. The inset shows the reciprocal plot of the percentage inhibition by AMP against the concentration of free AMP. In B, the Hill plot of log V,, - U/V against log free AMP (42) is shown.

and rabbit liver, and there appears to be little homology between the enzymes from these two vertebrate species. Like other Fru-P,ases (44, 451, the chicken muscle enzyme is activated by chelating agents. In the case of the rabbit and rat liver enzymes, this activation has

CHICKEN

BREAST

MUSCLE

been attributed to the removal of an inhibitory ion (461, probably Zn2+ (47). However, the additive effects of histidine and citrate, which together are more effective than EDTA, are difficult to explain in terms of this simple hypothesis. The activating effect of NH,+ was observed with the enzymes isolated from rabbit muscle (12,361 and chicken muscle (reported here) and may prove to be an important physiological regulating mechanism. This effect was first reported for the enzyme from swine liver (48). The present results confirm the extremely high sensitivity of Fru-P,ases from mammalian muscle to inhibition by AMP (2, 4, 36). Previous workers have reported that this inhibition is dependent on the concentration of Mg’+ employed in the assay, but the effect of Mg’+ was attributed to its binding of AMP. We have obtained a value for Ki of 4.2 x lo--* M based on the free AMP concentration. It is probable that the physiological concentration of AMP is much greater than this value (41, suggesting that under normal conditions this enzyme is fully inhibited. This raises interesting questions regarding the physiological function of muscle Fru-P,ases and the regulation of their activity. The availability of large quantities of the pure muscle enzyme should facilitate studies of its catalytic mechanism and regulatory properties.

FRUCTOSE

Biochem.

authors wish to thank Mr. D. Luk of this for the experiments in the ultracentrifuge. REFERENCES

M., VINUELA, E., SALAS, J., AND SOLS, A. (1964) Biochem. Biophys. Res. Commun. 17, 150-155. KREBS, H. A., AND WOODFORD, M. (1965) Biochem.J. 94, 436-445. OPIE, L. H., AND NEWSHOLME, E. A. (1967) Biothem. J. 103, 391-399. OPIE, L. H., AND NEWSHOLME, E. A. (1967) Biothem. J. 104, 353-360. NEWSHOLME, E. A., AND CRABTREE, B. (1970) FEBS Lett. 7, 195-198. NOLTE, J., BRDICZKA, D., AND PETTE, D. (1972) Biochim. Biophys. Acta 284, 497-507. NEWSHOLME, E. A., CRABTREE, B., HIGGINS, S. J., THORNTON, S. D., AND START, C. (1972)

586. 9. CLARK. AND

3. 4. 5.

6. 7.

J. 128. 89-97.

M. G., BLOXHAM, D. P., HOLLAND, P. C., H. A. (1973) Biochem. J. 133, 589-

LARDY.

597. 10. CLARK,

M. G., WILLIAMS, C. H., PFEIFER, W. F., D. P., HOLLAND. P. C., TAYLOR, C. LARDY, H. A. (1973) Nature (London) 245, 99-101. OLSON, J. P., AND MARQUARDT, R. R. (1972) Biochim. Biophys. Acta 268, 453-467. BLACK, W. J., VAN TOL, A., FERNANDO, J.. AND HORECKER, B. L. (1972) Arch. Biochem. Biophys. 1.51, 576-590. PONTREMOLI. S., TRANIELLO, S., LUPPIS, B., AND WOOD, W. A. (1965) J. Biol. Chem. 240, 34593463. Bucher, T. (1947) Biochim. Biophys. Acta 1,292314. DAVIS, B. J. (1964) Ann. N.Y. Acad. Sci. 121, 404-427. WEBER, K., AND OSBORN, M. (1969) J. Biol. Chem. 214, 4406-4412. SCHACHMAN, H. K. (1957) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 4, pp. 32-103, Academic Press, New York. MOORE, S., AND STEIN, W. H. (1963) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 6, pp. 819-831. Academic Press, New York. SPACKMAN, D. H., STEIN, W. H., AND MOORE, S. (1958) Anal. Chem. 30, 1190-1206. BENSON, J. V., JR., GORDON, M. J., AND PATTERSON, J. A. (1967) Anal. B&hem. 18, 228-240. SPANDE, T. F., AND WITKOP, B. (1967) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. 0.. eds.), Vol. 11, pp. 498-506, Academic Press, New York. SASAKI, T., ABRAMS, B., AND HORECKER. B. L. (1975) Anal. Biochem. 65, 396-404. LIU. T.-Y., AND CHANG, Y. H. (1971) J. Bid. Chem. 246, 2842-2848. LIU, T.-Y., as quoted by MOORE, S. (1972) in Chemistry and Biology of Peptides (Meienhofer, J., ed.), pp. 629-653, Ann Arbor Publishers, Michigan. CRESTFIELD, A. M., MOORE, S., AND STEIN. W. H. (1963) J. Biol. Chem. 238, 622-627. TSOLAS, O., AND SUN, S. C. (1975) Arch. Biothem. Biophys. 167, 525-533. PETERSON, E. A., AND SOBER, H. A. (1962) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.). Vol. 5, pp. 3-27, Academic Press, New York. CARLTON, B. C., AND YANOFSKY, C. (1963) J. Bid. Chem. 238, 636-639. BLOXHAM, A., AND

11. 12.

13.

14. 15. 16. 17.

18.

19. 20. 21.

22. 23.

1. SALAS,

2.

55

8. BLOXHAM, D. P., CLARK, M. G., HOLLAND, P. C., AND LARDY, H. A. (1973) Biochem. J. 134, 581-

ACKNOWLEDGMENT The Institute

BISPHOSPHATASE

24.

25. 26. 27.

28.

56

ANNAMALAI,

TSOLAS,

29. LAI, C. Y. (1975)Arch. Biochem. Biophys. 166, 330-338. 30. WEINER, A. M., PLATT, T., AND WEBER, K. (1972) J. Biol. Chem. 247, 3242-3251. 31. MENDEZ, E., AND LAI, C. Y. (1975) Anal. Bio&em. 68, 47-53. 32. BEAVEN, G. H., AND HOLIDAY, E. R. (1952) in Advances in Protein Chemistry (Anson, M. L., Bailey, K., and Edsall, J. T., eds.), Vol. 7, pp. 319-386, Academic Press, New York. 33. ABRAMS, B., SASAKI, T., DATTA, A., MELLONI, E., PONTREMOLI, S., AND HORECKER, B. L. (1975) Arch. Biochem. Biophys. 169, 116-125. 34. EL-DORRY, H. A., CHU, D. K., DZUGAJ, A., TsoLAS, O., PONTREMOLI, S., AND HORECKER, B. L. (1977) Arch. Biochem. Biophys. 178. 200207. 35. DATTA, A. G., ABRAMS, B., SASAKI, T.. VAN DEN BERG, J. W. O., PONTREMOLI, S., AND HoRECKER, B. L. (1974) Arch. Biochem. Biophys. 165, 641-645. 36. VAN TOL, A., BLACK, W. J., AND HORECKER, B. L. (1972) Arch. Biochem. Biophys. 151, 591596. 37. BEHRISCH, H. W. (1971) Biochem. J. 121, 399409. 38. GONZALEZ, A. M., GONZALEZ, F., GOLF, S., AND

AND

HORECKER MARCUS,

F. (1972)

J. Biol.

Chem.

247, 6067-

6070. 39.

J., AND MARCUS, F. (1974)J. Bid. 249, 745-749. NAKASHIMA, K., AND TUBOI, S. (1976) J. Bid. Chem. 251, 4315-4321. PONTREMOLI, S., GRAZI, E., AND ACCORSI, A. (1968) Biochemistry 7, 3628-3633. TAKETA, K., AND POGELL, B. M. (1965) J. Biol. Chem. 240, 651-662. MARQUARDT. R. R., AND OLSON, J. P. (1975) Canad. J. Biochem. 53, 1214-1219. HORECKER, B. L., MELLONI. E., AND PONTREMOLI, S. (1975) in Advances in Enzymology (Meister. A., ed.), Vol. 42, pp. 193-226, WileyInterscience, New York. TEJWANI, G. A., PEDROSA, F. O., PONTREMOLI. S., AND HORECKER, B. L. (1976) Arch. Biothem. Biophys. 17i, 253-264. NIMMO, H. G., AND TIPTON, K. F. (1975) Biothem. J. 14.5, 323-334. TEJWANI, G. A., PEDROSA, F. O., PONTREMOLI, S.. AND HORECKER, B. L. (1975) Proc. Nat. Acad. Sci. USA 73, 2692-2695. HUBERT, E., VILLANUEVA, J., GONZALEZ, A. M., AND MARCUS, F. (1970) Arch. Biochem. Biophys. 138, 590-597. VILLANUEVA,

Chem.

40.

41. 42. 43. 44.

45.

46. 47.

48.