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Fructose diphosphatase from rabbit muscle
ARCHIVES
OF
BIOCHEMISTRY
.lND
Fructose II. Amino
Acid
BIOPHYSICS
Diphosphatase
Composition
of Biological
from
and Activation
J. FERNANDO Institute
370-376 (1969)
129,
Muscle
by Sulfhydryl
Reagents’
S. PONTREMOLI
AND
Chemistry,
Rabbit
University
of Ferrara,
Ferrara,
Italy
AND
B. L. HORECKER Department
of Molecular
Biology,
Received
Albert
September
Einstein
College of Medicine,
27, 1968; accepted
October
Bronx,
New York 10461
4, 1968
The properties of rabbit muscle and rabbit liver fructose 1,6-diphosphatases are compared. It may be concluded that the two enzymes are different proteins with a number of common properties. They differ significantly in primary structure, as indicated by amino acid analysis, although there may be extensive homology. They are similarly activated by treatment with dinitrofluorobenzene or p-mercuribenaoate, or by disulfide exchange with 5,5’-dithio bis(2-nit,robenxoic) acid (DTNB). However, the muscle enzyme is not activated by cystamine, the only known natural activator of liver fructose diphosphatase. The enzyme activated with fluorodinitrobenzene retains normal sensitivity to the allosteric inhibitor AMP; after activation with I>TNB the enzyme is somewhat less sensitive.
We have recently described the purification and general properties of fructose 1,6diphosphatase (FDPase2 EC 3.1.3.11) from rabbit skeletal muscle (1). The enzyme resembled that purified from rabbit liver (2) in its ability to hydrolyze fructose-l, B-P2 or sedoheptulose-1 ,7-P, , and its requirement for Mg++ or &In++. However, it was found to be more sensibive to inhibition by AMP than was the liver enzyme (see also 3,4), and its pH optimum was dependent on the 1 This work was supported by grants from the National Science Foundation (GB 7140), the National Institute of General Medical Sciences, National Institutes of Health (GM 11301, GM 12991), and the Impresa di Enzimologia of the Italian C.N.R. This is Communication No. 143 from the Joan and Lester Avnet Institute of Molecular Biology. For paper No. 1 of this series see (1). 2 The following abbreviations are employed : FDPase, fructose 1,6-diphosphatase; FDP, fructose diphosphate; DTNB, 5,5’-dithio bis(2-nitrobenzoic) acid.
met,hod of purification and t#he assay conditions (1). The results suggested t,hat the liver and muscle enzymes were distinct proteins, but evidence bearing directly on this point was lacking. In the present study we have compared t,he amino acid composition of the muscle and liver enzymes, and the changes in catalytic properties produced by sulfhydryl reagents. The results confirm the nonidentity of the two proteins. In the accompanying paper the enzymes are shown to differ in their immunologic properties and electrophoretic mobility (5).
370
EXPERIMENTAL
PROCEDURES
Materials. Rabbit muscle FDPase was purified by the procedure of Fernando et al. (l), with the following modification: The fractions from the phosphocellulose columns in Procedure 1 were concentrated by vacuum dialysis and then subjected to ammonium sulfate fractionation. FDPase activity was precipitated between 48 and 557, saturation. This preparation had a
Fl:UCTOSl1:
DIPIIOSPHATrlSE
FI:OM
specific activity of 7.0 units/mg at pH i.5 and yielded a single band in polyacrylamide gel electrophoresis at pII 9.3 or 4.5. Pure rabbit liver FI)I’ase was prepared by the method of Pontremoli ct crl. (2); it,s specific activity was 17.5 units/
a Calculated for t,he liver enzyme from equilibrium sedimentation measurements (Sia, C. L., Traniello, S., Pontremoli, F., and Horecker,
371
MUSCLE
Amino Acid”
mg at ~JH 9.2.
Hexosephosphatc isomerase and glucose 6-P dehydrogellase were purchased from BoehringeriXI:~tlllhcim, ~;ermany. TPN, ilMP, cystamine I)-Fructose-l ,&Pi, dihydrochloride, cysteine, cystine, pantotheine, insulill, and oxidized and reduced ghltathione were obtained from the Sigma Chemical Co., St. Louis. Cocnzyme 9 was purchased from Pabst Laboratories, Milwartkee. The sodium salt of p-chloromercllribenzoate was prlrchased from the California Foluldation, Los Angeles, 5,5’-dithio bis(2-llitrobetlzoic) acid (DTNB) from K & K and mercaptoethanol from the Laboratories, Aldrich Chemical Co. 2,&DinitroBuorobenzene was purchased from C. Erba, Milan, Italy, and recrystallized from ethanol at 0”. Fresh solutious in absolllte ethanol were prepared for each experimPut. Ethyl disulfide and 2-hydroxyethyl disulfide were prepared by oxidizing the correspondilig reduced compounds with H&I. Oxidized Coerizyme A was prepared by bubbling oxygen through a solution of the reduced compound in phosphate buffer at pH 7.4. ~lnal~tical procedures. For routine assay of Fl>Pase activity the rate of formation of fructose-G-P was measured spectrophotometrically by following the reduct’ion of TPN in the presence of excess hexosephosphate isomerase and glucose6-P dehydrogenase. The reaction mixture (1 ml) buffer, pH 7.5, 5 mM contained 0.04 M Tris-HCl MgS04, 5 rg each of glucose-6-P dehydrogenase, and hexosephosphate isomerase, 0.1 mM TPN, 0.1 rnM fructose-1,6-Pp and 0.0015-0.003 units of enzyme. One unit of enzyme was defined as the amount which would cause the formation of 1 pmole of fructose-6-P/min under the above conditions. Specific activity is expressed as units/mg of protein. Assays at alkaline pH were carried ollt in 0.04 M glycine buffer, pH 9.2, with 0.5 rnM MnClr. Protein concentrations were calculated from absorbauce at 280 mp. The absorbance of a solution containing 1.0 mg of muscle FDPase (dry weight)) per milliliter in a l.O-cm light path was 0.94 at 280 mr and 0.61 at 260 rnp For liver FDPase the corresponding value at 280 mp was 0.89. Both enzymes were assumed to have a molecular weight of 130,000.3
1:ABBIT
‘Kio. of residues per mole of enzymeb hfuscle
Lysine
H istidine Arginine Aspartic acid Threonine Serilre Glutamic acid Proline Glycine Alanine Valine Isoleucine Leucine Tprosine Phenylalanine Cysteic acid Methionine s~dfone Tryptopha&
89 10 43 103 78 76 118 48 104 111 104 58 119 G3 32 21 21 0
LiverC
117 14 34 128 70 79 81 52 100 106 104 70 94 52 40 20 34 0
(125) (14) (32) (162) (62) (78) (79) (52) (105) (109) (93) (G2) (93) (37) (40) (20) (29)
cl Samples of Fl>Pase were dialyzed against distilled water for 48 hr with several changes and then evaporated to dryness. In some cases the dried enzymes were oxidized with performic acid (9) for 2% hr at O”, diluted x-ith water aud lyophilized. The dry material was dissolved in 5.7 N HCl, divided into three equal portions and hydrolyzed under vacuum for 24, 48, and 72 hr at 110”. Cysteine and methionine residues were determined as cysteic acid and methionine sulfone, respectively. Appropriate corrections were made for the destruction and incomplete hydrolysis of certain amino acids (12). b Calculated for mol wt = 130,000 and rounded off to the nearest whole numbers. c The values in parentheses are those previously reported for the liver enzyme (11). d For details on the determination of tryptophan residues. see text. The reaction of sulfhydryl groups with p-mercuribenzoate was followed spectrophotometrically (6) and standardized against a sample of reduced glutathione of known concentration, analyzed under the same conditions. Measurement of disulfide exchange with DTNB was carried out according to the procedure of Bellman (7). Amino acid analyses were carried out with a B. L., unpublished observations). A similar value has been estimated for the muscle enzyme by sedimentation in sucrose density gradients (1).
372
FERNANDO,
PONTREMOLI,
Beckman model 120B analyzer according to the method of Spackman et al. (8). Cysteine and methionine residues were determined as cysteic acid and methionine sulfone after performic acid oxidation (9). Polyacrylamide gel electrophoreses were performed in 7.57, gel at pH 9.3 and pH 4.5 (10). Cellulose polyacetate electrophoresis was carried out at pH 7.3 using Tris-EDTA-borate buffer (Tris, 0.07 M, EDTA, 3.5 mM, borate, 0.2 M) at 300 V, 5 mA, for 30 min. After the electrophoresis the strips were stained with Ponceau red for 2 min and then destained in 57, acetic acid.
AND
41
HORECKER
/ 10 MOLES
RESULTS
Amino acid composition. Muscle and liver FDPases were found to differ significantly in amino acid composition (Table I). Muscle FDPase was found to contain more glutamic acid, leucine, tyrosine, and arginine, whereas the liver enzyme was richer in aspartic acid, isoleucine, phenylalanine, lysine, and methionine. It is evident from these results that the two proteins differ in primary structure, although there may be considerable homology. The data reported here for the liver enzyme are generally in agreement with our previous findings (ll), although there are significant ORIGIN + M
ORIGIN
1. Cellulose polyacetate electrophoresis of rabbit muscle FDPase (M), liver FDPase (L), and a mixture of the two. Conditions for electrophoresis were as described under Experimental Procedures. Ten micrograms of each enzyme, either alone or in the mixture, was applied at the origin. Specific activities of the muscle and the liver FDPases were 7.2 units/mg (at pH 7.5), and 17.5 units/mg (at pH 9.1), respectively, assayed as described under Experimental Procedures. FIG.
/ 20 FDNB/MOLE
Id
50
ENZYME
FIG. 2. The effect of dinitrophenylation on enzyme activity. The reaction mixture (0.2 ml) contained 0.04 M bicarbonate bueer, pH 9.2, FDPase (0.11 mg, 4.2 X 10m6M) and dinitrofluorobenzene in the quantities indicated. The mixtures were incubated in the dark at 22”. Aliquots were taken after 20 min and activity was measured with 0.5 mM Mn++ and 0.1 mM FDP in 0.04 1u Tris-HCl buffer, pH 7.5, or 0.04 JI glycine buffer at pH 9.3. Control samples incubated under the same conditions but without dinitrofluorobenzene showed no change in activity during this time.
differences in the values for aspnrtic acid, valine, isoleucine, and tyrosine. It is not known whether these differences represent errors in the determinations or are related to the fact that the enzymes were obtained from different strains of animals; these were supplied by a source in Ferrara in the present case, and by Pel-Freez Biologicals, Inc., Rogers, Arkansas, in the previous st,udy. Tryptophan was not detected either by the method of Goodwin and Jlorton (13), or with N-bromosuccinimide in the presence or absence of urea (14, 15). To confirm the absence of tryptophan, both enzymes mere dissolved in 0.1 WIalkali and the spectra compared with that of tyrosine under the same conditions. In each case a single maximum at 294 rnp was obtained; the cont,ent of tyrosine calculated from the height of the peaks was 52 residues for the liver enzyme, and 63 residues for the muscle enzyme, in excellent agreement with the results obtained from the amino acid analyses. Based on the above observations, it was concluded that both enzymes are devoid of tryptophan. Electrophoresis. The two enzymes migrated with different mobilities in electrophoresis on cellulose polyacetate strips at
FRUCTOSE
DIPHOSPHATASE
FROM
pH 7.3 (Fig. 1). In this system, rabbit muscle FDPase migrated towards the anode while the liver enzyme remained at the origin. When a mixture of the two enzymes was analyzed, the two bands were clearly separated. They did not separate in polyacrylamide gel electrophoresis at either pH 9.3 or 4.5. E$ect of Jluorodinitrobenzene on catalytic activity. The addition of small quantities of dinitrofluorobenzene to solutions of muscle FDPase in bicarbonate buffer, pH 9.2, resulted in a marked increase in catalyt,ic activity, particularly when this was tested in the neutral pH range (Fig. 2). Maximum activation was observed when 3-4 equivalents of FDNB per mole of enzyme had been added. With excess fluorodinitrobenzene activity decreased, and in the case of the assay at alkaline pH, fell below that of the native enzyme. The activity of the dinitrophenylated enzyme was dependent on the conditions of the assay. Activation was observed over the entire pH range, but was greatest at pH 7.5, when the enzyme was test’ed with Mn++ as the activating cation (Fig. 3A) ; under these conditions the DKP-enzyme showed the same activity at pH 7.3 as at pH 9.2.
6.0
7.0
9.0
6.0 PH
too
RABBIT
373
MUSCLE
With Mg++ as the cation (Fig. 3B), the native enzyme showed a single pH optimum at about pH 7. In the case of the DNPenzyme an overall decrease in activity was noticed and again the activities between pH 7.0 and 8.0 were nearly equal. By analogy with the liver enzyme it was assumed that dinitrofluorobenzene was reacting with cysteine residues (16). Changes in catalytic activity during titratkm of SH groups with p-mercuribenzoate. Catalytic activity was also enhanced by exposure of the enzyme to low concentrations of pmercuribenzoate (Fig. 4). The titration of 8 SH groups resulted in a 3-fold increase in activity when tested at neutral pH and with Mn++ as the cation (Fig. 4). Less activation was observed with this cation at alkaline pH or with l\Ig++ at either pH. With further addition of p-mercuribenzoate the activity remained high. In the absence of urea, however, only 14 of the 21 SH groups in muscle FDPase could be titrated even with an SOfold excess of p-mercuribenzoate; the reaction of these 14 groups was complete within 20 min. Beyond that time, and with excess of p-mercuribenzoate, measurements could not be contined because of protein precipitation. It was possible to titrate the remaining SH
6.0
7.0
6.0
9.0
10.0
PH
FIG. 3. The effect of pH on the activity of muscle FDPase. (A) Assay with 0.5 mM Mn++. (B) Assay with 5.0 mu Mg ++. The conditions for dinitrophenylation were as described in the legend to Fig. 2 with 0.11 mg of FDPase and 4 equivalents (1.6 X 1W5 M) of fluorodinitrobenzene. After 20 min in the dark, the solution was cooled. Aliquots of the mixture were analyzed as described in Fig. 2. The following buffers (0.04 M) were used: Imidazole at pH 6.5 and 7.0; Tris at pH 7.5 and 8.0; glycine above pH 8.0. The p1-I was measured immediately after each assay.
FERNANDO,
2 6 SULFHYDRYL
PONTREMOLI,
IO 14 16 GROUPS TITRATED
on the FIG. 4. The effect of p-mercuribenzoate catalytic activity of muscle FDPase. Incubation mixtures (1.0 ml) contained 0.04 M Tris buffer, pH 7.5, 0.1 mM EDTA, FDPase (0.37 mg, 2.8 X 10-6 M) and p-mercuribenzoate ranging from 6 X UY6 M to 1 X lO+ M, obtained by successive additions of the reagent. The reaction was followed at 250 rnp (6). When the quantitiy of p-mercuribenzoate bound corresponded to the values indicated in the figure, samples were removed and assayed for activity at pH 7.5 and 9.3, with 5.0 mM Mg++ or 0.5 mM Mn++, respectively. The initial specific activities (lOOyO) were: 6.9 and 4.6 at pH 7.5 with 5.0 mM Mg++ and 0.5 mM Mn”, respectively; 2.3 and 7.2 at pH 9.3 with 5.0 mM Mg* and 0.5 IIIM Mn++, respectively.
groups if the reaction was carried out in the presence of n-propyl alcohol (5 % v/v). Complete loss of activity resulted under these conditions, although the enzyme remained fully active when it was incubated with n-propyl alcohol alone for the same length of time. Activation by the formation of mixed disulJicles. DTNB, an effective activator for the liver enzyme (17), was also found to activate muscle FDPase. When the enzyme was treated with a lo-fold excess of DTNB, and tested in the assay system with Mn++, catalytic activity at either pH 7.5 or pH 9.3 was increased to about 250% of the original value (Fig. 5). When tested at alkaline pH, the activity remained at this elevated level even with a 40-fold excess of the reagent; on the other hand, the assays at neutral pH
AND
HORECKER
reached a maximum with a 4-fold excess of reagent, and then declined to a relatively constant level. In the assay at neutral pH with Mg++ as the cation, little effect of DTNB was observed. The number of SH groups that have exchanged with DTNB was determined spectrophotometrically (7). With a 4-, 6-, and IO-fold excess of DTNB the number of SH groups reacted were 1.5, 1.8, and 4.6, respectively. A number of other disulfides were tested for their ability to activate the enzyme. Of particular interest was the fact that cystamine, which had been found to activate rabbit liver FDPase (17), failed to activate the muscle enzyme. Some loss of activity was observed when excess cystamine was added, similar to that observed with the liver enzyme (17). Ethyl disulfide was the only other disulfide effective in activating the rabbit muscle FDPase. With a IO-fold excess of ethyl disulfide over protein, the activity at neutral pH and with Mn++ as the cofactor increased to 250%. At pH 9.3 with Mn++ only a 50% increase in activity was observed. The’ specific activities of the activated enzyme were approximately equal at pH 7.5 and 9.3. 2-Hydroxyethyl disulfide, which activated the liver enzyme (17), also failed to activate the muscle enzyme. Other disulfides tested and found to be ineffective were oxidized glutathione, insulin, cystine, pantotheine, and oxidized Coenzyme A. Insulin and oxidized glutathione were also negative when tested with catalytic amounts of reduced glutathione or mercaptoethanol. Deactivation of DTNB-treated FDPase by thiols. The activation by DTNB was fully reversed by reduced glutathione (Table II) ; treatment of the activated enzyme with mercaptoethanol or cysteine caused the activity to fall significantly below the original level. Sensitivity of activated muscle FDPase to AMP and excess substrate. Purified muscle FDPase is considerably more sensitive to inhibition by AMP than is the liver enzyme, and this inhibition is also observed with the activated enzyme. In the standard assay (pH 7.5, 5 mM MgSOd), the native enzyme was inhibited 50% by 0.4 X lop6 M AMP;
Fl:UCTOSE
/ 4
DIPHOSPHATASE
I 12
I 20
I 20
DTNB
ADDED,
I 36
FROM
RABBIT
I 44 MOLES/MOLE
MUSCLE
375
I OF ENZYME
5. Activation of muscle FDPase by DTNB. The enzyme was incubated in 0.04 M Tris buffer, pH 7.5, containing 1 rn~ EDTA, FDPase (0.11 mg, 8.5 X 10-’ M) and with varying amounts of DTNB in a final volume of 1.0 ml. Final concentrations of DTNB varied from 3 X 10-e M to 1 X 1V M. After 60 min at 22” aliquots were analyzed as described in the text. The initial specific activities were as described in the legend to Fig. 4. FIG.
TABLE
II
EFFECT OF RISDUCD GLUT.ITHIONE, MERCAPTOETHANOL, AND CYSTPINE: ON THE DTNB-TREATED ENZYME:S Incubationa First
None DTNB DTNB DTNB DTNB
Second
None None GSH Mercaptoethanol Cysteine
Specific activit# pH I.5
pH 9.3
4.5 9.0 5.5 2.5 3.4
7.2 19.4 5.8 4.2 4.6
u FDPase (0.32 mg, 4.9 X 10-O M) was incubated at 22” in a final volume of 0.5 ml containing 0.04 M Tris-HCl bufl’er, pH 7.5, and 5 X 10m5 M DTNB (first incubation). After 60 min, wheu maximum activation was reached, O.l-ml aliquots were removed and incubated for 30 min at 22” in 0.1 M Tris-HCl buffer, pH 7.5, with 1 mM cysteine, 1 mM reduced glutathione, or 10 mM mercaptoethanol (second incubation). b FDPase activity with 0.5 mM Mn++ and 0.1 mM FDP was measured spectrophotometrically in 0.04 M Tris buffer, pH 7.5, or 0.04 M glycine buffer, pH 9.3.
ARIP was required to inhibit the native enzyme by 50 % ; the DNP-enzyme was equally sensitive, but 5- to lo-fold higher concentrations were required for t’he DTNBenzyme. These concentrations (10e4 nr) are comparable to those required t’o inhibit liver FDPase (1s). The activated enzyme was less sensitive to inhibition by high concentrations of substrate. At 2.0 rnM fructose-l ,6-P? , the DTNB-t’reated and the DNP-enzymes were inhibited 15 and 21 c/c, respectively (7.5, 5 mM -\Ig++), compared with 40 % for the untreated enzyme. However, like the untreated enzyme, neither of the activated enzymes was inhibited by this concentration of fructose-l ,%I’2 at alkaline pH in the presence of Ah++. DISCUSSION
The results reported here support the conclusion that muscle and liver FDPases are different proteins, although they possess many properties in common. They differ significantly in amino acid composition, but 3- t,o 5-fold higher concentrations were re- these differences may not greatly affect the three-dimensional structures. Thus they quired to achieve the same degree of inhibishow similar totals for homologous groups of tion with the DNP-enzyme or DTNBactivated enzyme. In the presence of 0.5 m&t amino acids, such as the sum of aspartic acid Mn++ at either pH 7.5 or 9.3, nearly 10-j 11 plus glutamic acid, isoleucine and leucine
376
FERNANDO,
PONTREMOLI,
plus valine, tyrosine plus phenylalanine, and arginine plus lysine (Table I). Neither enzyme contains tryptophan. Both enzymes are activated by treatment with dinitrofluorobenzene, p-mercuribenzoate, and by disulfide exchange reactions. It may be of significance that the muscle enzyme is not activated by disulfide exchange with cystamine, since this compound has not been reported to occur in muscle. This remains the only physiological agent known to activate liver FDPase by modification of sulfhydryl groups. In the accompanying paper (5), immunological evidence is presented for nonidentity of the liver and muscle enzymes. Further studies on the structure of these enzymes mill be required to determine the extent of the differences and similarities. REFERENCES 1. FERNANDO, J., ENSER, M., PONTREMOLI, S., ASD HORIXKER, B. L., ilrch. Biochem. Biophys. 126, 599 (1968). 2. PONTREMOLI, S., TKANIELLO, S., LUPPIS, B., END WOOD, W. A., J. Hiol. Chem. 240, 3459 (1965). 3. KRERS, H. A. AND WOODFORD, M., Hiochem. J. 94, 436 (19G5). 4. OPIE, L. II. AND NEIVSHOLME, E. A., Biochem. J. 104, 353 (1967).
AND
HORECKER
5. ENSE:R, M., SHAPIRO, S., AND HORIXKER, B. L., i2rch. Biochem. Biophys. 128, 554-562 (1968). 6. BOYER, P. D., J. Am. Chem. Sot. 76, 4331 (1954). 7. ELLM~N, G. D., drch. Biochem. Biophys. 82, 70 (1959). 8. SP~ICKMAN, D. H., STEIN, W. H., AND MOORE, S., Anal. Chem. 30, 1190 (1958). 9. HIRS, H. W., J. Biol. Chem. 219, 611 (1956). 10. D;WIS, B. J., Ann. N. Y. Acad. Sci. 212, 404 (1964). 11. PONTRICMOLI, S., LUPPIS, B., TRANIF,LLo, S., I~IPPA, M., ANT) HORECKER, B. L., Arch. Biochem. Biophys. 112, 7 (1965). 12. CRESTFIELD, A. M., MOORE, S., .~ND STEIN, W. H., J. Biol. Chem. 238, 622 (1963). 13. GOODTVIN, T. W. .~ND MORTON, R. A., Biothem. J. 40, 628 (1946). 14. PAT~HORNIK, A., LAID-SON, W. B., AND WITKOP, B., J. Am. Chem. sot. 80, 4747 (1958). 15. FUN~TSV, M., GREEN, N. M., AND WITKOP, B., J. Am. Chem. Sot. 86, 1846 (1964). 16. PONTREMOLI, S., LUPPIS, B., WOOD, W. A., TRANIELLO, S., BND HORIXKER, B. I,., J. Biol. Chem. 240, 3469 (1965). 17. PONTREMOLI, S., TRANIELLO, S., ENSER, PI/I., SI-IAPIRO, S., .\ND HORECKGR, B. L., Proc. ATatl. dead. Sci. 58, 286 (19F7). 18. HOR~CKER, B. L., PONTREMOLI, S., ROSI!ZN, O., AND I~OSEN, S., Federation Proc. 25, 1521 (1966).
OF
BIOCHEMISTRY
.lND
Fructose II. Amino
Acid
BIOPHYSICS
Diphosphatase
Composition
of Biological
from
and Activation
J. FERNANDO Institute
370-376 (1969)
129,
Muscle
by Sulfhydryl
Reagents’
S. PONTREMOLI
AND
Chemistry,
Rabbit
University
of Ferrara,
Ferrara,
Italy
AND
B. L. HORECKER Department
of Molecular
Biology,
Received
Albert
September
Einstein
College of Medicine,
27, 1968; accepted
October
Bronx,
New York 10461
4, 1968
The properties of rabbit muscle and rabbit liver fructose 1,6-diphosphatases are compared. It may be concluded that the two enzymes are different proteins with a number of common properties. They differ significantly in primary structure, as indicated by amino acid analysis, although there may be extensive homology. They are similarly activated by treatment with dinitrofluorobenzene or p-mercuribenaoate, or by disulfide exchange with 5,5’-dithio bis(2-nit,robenxoic) acid (DTNB). However, the muscle enzyme is not activated by cystamine, the only known natural activator of liver fructose diphosphatase. The enzyme activated with fluorodinitrobenzene retains normal sensitivity to the allosteric inhibitor AMP; after activation with I>TNB the enzyme is somewhat less sensitive.
We have recently described the purification and general properties of fructose 1,6diphosphatase (FDPase2 EC 3.1.3.11) from rabbit skeletal muscle (1). The enzyme resembled that purified from rabbit liver (2) in its ability to hydrolyze fructose-l, B-P2 or sedoheptulose-1 ,7-P, , and its requirement for Mg++ or &In++. However, it was found to be more sensibive to inhibition by AMP than was the liver enzyme (see also 3,4), and its pH optimum was dependent on the 1 This work was supported by grants from the National Science Foundation (GB 7140), the National Institute of General Medical Sciences, National Institutes of Health (GM 11301, GM 12991), and the Impresa di Enzimologia of the Italian C.N.R. This is Communication No. 143 from the Joan and Lester Avnet Institute of Molecular Biology. For paper No. 1 of this series see (1). 2 The following abbreviations are employed : FDPase, fructose 1,6-diphosphatase; FDP, fructose diphosphate; DTNB, 5,5’-dithio bis(2-nitrobenzoic) acid.
met,hod of purification and t#he assay conditions (1). The results suggested t,hat the liver and muscle enzymes were distinct proteins, but evidence bearing directly on this point was lacking. In the present study we have compared t,he amino acid composition of the muscle and liver enzymes, and the changes in catalytic properties produced by sulfhydryl reagents. The results confirm the nonidentity of the two proteins. In the accompanying paper the enzymes are shown to differ in their immunologic properties and electrophoretic mobility (5).
370
EXPERIMENTAL
PROCEDURES
Materials. Rabbit muscle FDPase was purified by the procedure of Fernando et al. (l), with the following modification: The fractions from the phosphocellulose columns in Procedure 1 were concentrated by vacuum dialysis and then subjected to ammonium sulfate fractionation. FDPase activity was precipitated between 48 and 557, saturation. This preparation had a
Fl:UCTOSl1:
DIPIIOSPHATrlSE
FI:OM
specific activity of 7.0 units/mg at pH i.5 and yielded a single band in polyacrylamide gel electrophoresis at pII 9.3 or 4.5. Pure rabbit liver FI)I’ase was prepared by the method of Pontremoli ct crl. (2); it,s specific activity was 17.5 units/
a Calculated for t,he liver enzyme from equilibrium sedimentation measurements (Sia, C. L., Traniello, S., Pontremoli, F., and Horecker,
371
MUSCLE
Amino Acid”
mg at ~JH 9.2.
Hexosephosphatc isomerase and glucose 6-P dehydrogellase were purchased from BoehringeriXI:~tlllhcim, ~;ermany. TPN, ilMP, cystamine I)-Fructose-l ,&Pi, dihydrochloride, cysteine, cystine, pantotheine, insulill, and oxidized and reduced ghltathione were obtained from the Sigma Chemical Co., St. Louis. Cocnzyme 9 was purchased from Pabst Laboratories, Milwartkee. The sodium salt of p-chloromercllribenzoate was prlrchased from the California Foluldation, Los Angeles, 5,5’-dithio bis(2-llitrobetlzoic) acid (DTNB) from K & K and mercaptoethanol from the Laboratories, Aldrich Chemical Co. 2,&DinitroBuorobenzene was purchased from C. Erba, Milan, Italy, and recrystallized from ethanol at 0”. Fresh solutious in absolllte ethanol were prepared for each experimPut. Ethyl disulfide and 2-hydroxyethyl disulfide were prepared by oxidizing the correspondilig reduced compounds with H&I. Oxidized Coerizyme A was prepared by bubbling oxygen through a solution of the reduced compound in phosphate buffer at pH 7.4. ~lnal~tical procedures. For routine assay of Fl>Pase activity the rate of formation of fructose-G-P was measured spectrophotometrically by following the reduct’ion of TPN in the presence of excess hexosephosphate isomerase and glucose6-P dehydrogenase. The reaction mixture (1 ml) buffer, pH 7.5, 5 mM contained 0.04 M Tris-HCl MgS04, 5 rg each of glucose-6-P dehydrogenase, and hexosephosphate isomerase, 0.1 mM TPN, 0.1 rnM fructose-1,6-Pp and 0.0015-0.003 units of enzyme. One unit of enzyme was defined as the amount which would cause the formation of 1 pmole of fructose-6-P/min under the above conditions. Specific activity is expressed as units/mg of protein. Assays at alkaline pH were carried ollt in 0.04 M glycine buffer, pH 9.2, with 0.5 rnM MnClr. Protein concentrations were calculated from absorbauce at 280 mp. The absorbance of a solution containing 1.0 mg of muscle FDPase (dry weight)) per milliliter in a l.O-cm light path was 0.94 at 280 mr and 0.61 at 260 rnp For liver FDPase the corresponding value at 280 mp was 0.89. Both enzymes were assumed to have a molecular weight of 130,000.3
1:ABBIT
‘Kio. of residues per mole of enzymeb hfuscle
Lysine
H istidine Arginine Aspartic acid Threonine Serilre Glutamic acid Proline Glycine Alanine Valine Isoleucine Leucine Tprosine Phenylalanine Cysteic acid Methionine s~dfone Tryptopha&
89 10 43 103 78 76 118 48 104 111 104 58 119 G3 32 21 21 0
LiverC
117 14 34 128 70 79 81 52 100 106 104 70 94 52 40 20 34 0
(125) (14) (32) (162) (62) (78) (79) (52) (105) (109) (93) (G2) (93) (37) (40) (20) (29)
cl Samples of Fl>Pase were dialyzed against distilled water for 48 hr with several changes and then evaporated to dryness. In some cases the dried enzymes were oxidized with performic acid (9) for 2% hr at O”, diluted x-ith water aud lyophilized. The dry material was dissolved in 5.7 N HCl, divided into three equal portions and hydrolyzed under vacuum for 24, 48, and 72 hr at 110”. Cysteine and methionine residues were determined as cysteic acid and methionine sulfone, respectively. Appropriate corrections were made for the destruction and incomplete hydrolysis of certain amino acids (12). b Calculated for mol wt = 130,000 and rounded off to the nearest whole numbers. c The values in parentheses are those previously reported for the liver enzyme (11). d For details on the determination of tryptophan residues. see text. The reaction of sulfhydryl groups with p-mercuribenzoate was followed spectrophotometrically (6) and standardized against a sample of reduced glutathione of known concentration, analyzed under the same conditions. Measurement of disulfide exchange with DTNB was carried out according to the procedure of Bellman (7). Amino acid analyses were carried out with a B. L., unpublished observations). A similar value has been estimated for the muscle enzyme by sedimentation in sucrose density gradients (1).
372
FERNANDO,
PONTREMOLI,
Beckman model 120B analyzer according to the method of Spackman et al. (8). Cysteine and methionine residues were determined as cysteic acid and methionine sulfone after performic acid oxidation (9). Polyacrylamide gel electrophoreses were performed in 7.57, gel at pH 9.3 and pH 4.5 (10). Cellulose polyacetate electrophoresis was carried out at pH 7.3 using Tris-EDTA-borate buffer (Tris, 0.07 M, EDTA, 3.5 mM, borate, 0.2 M) at 300 V, 5 mA, for 30 min. After the electrophoresis the strips were stained with Ponceau red for 2 min and then destained in 57, acetic acid.
AND
41
HORECKER
/ 10 MOLES
RESULTS
Amino acid composition. Muscle and liver FDPases were found to differ significantly in amino acid composition (Table I). Muscle FDPase was found to contain more glutamic acid, leucine, tyrosine, and arginine, whereas the liver enzyme was richer in aspartic acid, isoleucine, phenylalanine, lysine, and methionine. It is evident from these results that the two proteins differ in primary structure, although there may be considerable homology. The data reported here for the liver enzyme are generally in agreement with our previous findings (ll), although there are significant ORIGIN + M
ORIGIN
1. Cellulose polyacetate electrophoresis of rabbit muscle FDPase (M), liver FDPase (L), and a mixture of the two. Conditions for electrophoresis were as described under Experimental Procedures. Ten micrograms of each enzyme, either alone or in the mixture, was applied at the origin. Specific activities of the muscle and the liver FDPases were 7.2 units/mg (at pH 7.5), and 17.5 units/mg (at pH 9.1), respectively, assayed as described under Experimental Procedures. FIG.
/ 20 FDNB/MOLE
Id
50
ENZYME
FIG. 2. The effect of dinitrophenylation on enzyme activity. The reaction mixture (0.2 ml) contained 0.04 M bicarbonate bueer, pH 9.2, FDPase (0.11 mg, 4.2 X 10m6M) and dinitrofluorobenzene in the quantities indicated. The mixtures were incubated in the dark at 22”. Aliquots were taken after 20 min and activity was measured with 0.5 mM Mn++ and 0.1 mM FDP in 0.04 1u Tris-HCl buffer, pH 7.5, or 0.04 JI glycine buffer at pH 9.3. Control samples incubated under the same conditions but without dinitrofluorobenzene showed no change in activity during this time.
differences in the values for aspnrtic acid, valine, isoleucine, and tyrosine. It is not known whether these differences represent errors in the determinations or are related to the fact that the enzymes were obtained from different strains of animals; these were supplied by a source in Ferrara in the present case, and by Pel-Freez Biologicals, Inc., Rogers, Arkansas, in the previous st,udy. Tryptophan was not detected either by the method of Goodwin and Jlorton (13), or with N-bromosuccinimide in the presence or absence of urea (14, 15). To confirm the absence of tryptophan, both enzymes mere dissolved in 0.1 WIalkali and the spectra compared with that of tyrosine under the same conditions. In each case a single maximum at 294 rnp was obtained; the cont,ent of tyrosine calculated from the height of the peaks was 52 residues for the liver enzyme, and 63 residues for the muscle enzyme, in excellent agreement with the results obtained from the amino acid analyses. Based on the above observations, it was concluded that both enzymes are devoid of tryptophan. Electrophoresis. The two enzymes migrated with different mobilities in electrophoresis on cellulose polyacetate strips at
FRUCTOSE
DIPHOSPHATASE
FROM
pH 7.3 (Fig. 1). In this system, rabbit muscle FDPase migrated towards the anode while the liver enzyme remained at the origin. When a mixture of the two enzymes was analyzed, the two bands were clearly separated. They did not separate in polyacrylamide gel electrophoresis at either pH 9.3 or 4.5. E$ect of Jluorodinitrobenzene on catalytic activity. The addition of small quantities of dinitrofluorobenzene to solutions of muscle FDPase in bicarbonate buffer, pH 9.2, resulted in a marked increase in catalyt,ic activity, particularly when this was tested in the neutral pH range (Fig. 2). Maximum activation was observed when 3-4 equivalents of FDNB per mole of enzyme had been added. With excess fluorodinitrobenzene activity decreased, and in the case of the assay at alkaline pH, fell below that of the native enzyme. The activity of the dinitrophenylated enzyme was dependent on the conditions of the assay. Activation was observed over the entire pH range, but was greatest at pH 7.5, when the enzyme was test’ed with Mn++ as the activating cation (Fig. 3A) ; under these conditions the DKP-enzyme showed the same activity at pH 7.3 as at pH 9.2.
6.0
7.0
9.0
6.0 PH
too
RABBIT
373
MUSCLE
With Mg++ as the cation (Fig. 3B), the native enzyme showed a single pH optimum at about pH 7. In the case of the DNPenzyme an overall decrease in activity was noticed and again the activities between pH 7.0 and 8.0 were nearly equal. By analogy with the liver enzyme it was assumed that dinitrofluorobenzene was reacting with cysteine residues (16). Changes in catalytic activity during titratkm of SH groups with p-mercuribenzoate. Catalytic activity was also enhanced by exposure of the enzyme to low concentrations of pmercuribenzoate (Fig. 4). The titration of 8 SH groups resulted in a 3-fold increase in activity when tested at neutral pH and with Mn++ as the cation (Fig. 4). Less activation was observed with this cation at alkaline pH or with l\Ig++ at either pH. With further addition of p-mercuribenzoate the activity remained high. In the absence of urea, however, only 14 of the 21 SH groups in muscle FDPase could be titrated even with an SOfold excess of p-mercuribenzoate; the reaction of these 14 groups was complete within 20 min. Beyond that time, and with excess of p-mercuribenzoate, measurements could not be contined because of protein precipitation. It was possible to titrate the remaining SH
6.0
7.0
6.0
9.0
10.0
PH
FIG. 3. The effect of pH on the activity of muscle FDPase. (A) Assay with 0.5 mM Mn++. (B) Assay with 5.0 mu Mg ++. The conditions for dinitrophenylation were as described in the legend to Fig. 2 with 0.11 mg of FDPase and 4 equivalents (1.6 X 1W5 M) of fluorodinitrobenzene. After 20 min in the dark, the solution was cooled. Aliquots of the mixture were analyzed as described in Fig. 2. The following buffers (0.04 M) were used: Imidazole at pH 6.5 and 7.0; Tris at pH 7.5 and 8.0; glycine above pH 8.0. The p1-I was measured immediately after each assay.
FERNANDO,
2 6 SULFHYDRYL
PONTREMOLI,
IO 14 16 GROUPS TITRATED
on the FIG. 4. The effect of p-mercuribenzoate catalytic activity of muscle FDPase. Incubation mixtures (1.0 ml) contained 0.04 M Tris buffer, pH 7.5, 0.1 mM EDTA, FDPase (0.37 mg, 2.8 X 10-6 M) and p-mercuribenzoate ranging from 6 X UY6 M to 1 X lO+ M, obtained by successive additions of the reagent. The reaction was followed at 250 rnp (6). When the quantitiy of p-mercuribenzoate bound corresponded to the values indicated in the figure, samples were removed and assayed for activity at pH 7.5 and 9.3, with 5.0 mM Mg++ or 0.5 mM Mn++, respectively. The initial specific activities (lOOyO) were: 6.9 and 4.6 at pH 7.5 with 5.0 mM Mg++ and 0.5 mM Mn”, respectively; 2.3 and 7.2 at pH 9.3 with 5.0 mM Mg* and 0.5 IIIM Mn++, respectively.
groups if the reaction was carried out in the presence of n-propyl alcohol (5 % v/v). Complete loss of activity resulted under these conditions, although the enzyme remained fully active when it was incubated with n-propyl alcohol alone for the same length of time. Activation by the formation of mixed disulJicles. DTNB, an effective activator for the liver enzyme (17), was also found to activate muscle FDPase. When the enzyme was treated with a lo-fold excess of DTNB, and tested in the assay system with Mn++, catalytic activity at either pH 7.5 or pH 9.3 was increased to about 250% of the original value (Fig. 5). When tested at alkaline pH, the activity remained at this elevated level even with a 40-fold excess of the reagent; on the other hand, the assays at neutral pH
AND
HORECKER
reached a maximum with a 4-fold excess of reagent, and then declined to a relatively constant level. In the assay at neutral pH with Mg++ as the cation, little effect of DTNB was observed. The number of SH groups that have exchanged with DTNB was determined spectrophotometrically (7). With a 4-, 6-, and IO-fold excess of DTNB the number of SH groups reacted were 1.5, 1.8, and 4.6, respectively. A number of other disulfides were tested for their ability to activate the enzyme. Of particular interest was the fact that cystamine, which had been found to activate rabbit liver FDPase (17), failed to activate the muscle enzyme. Some loss of activity was observed when excess cystamine was added, similar to that observed with the liver enzyme (17). Ethyl disulfide was the only other disulfide effective in activating the rabbit muscle FDPase. With a IO-fold excess of ethyl disulfide over protein, the activity at neutral pH and with Mn++ as the cofactor increased to 250%. At pH 9.3 with Mn++ only a 50% increase in activity was observed. The’ specific activities of the activated enzyme were approximately equal at pH 7.5 and 9.3. 2-Hydroxyethyl disulfide, which activated the liver enzyme (17), also failed to activate the muscle enzyme. Other disulfides tested and found to be ineffective were oxidized glutathione, insulin, cystine, pantotheine, and oxidized Coenzyme A. Insulin and oxidized glutathione were also negative when tested with catalytic amounts of reduced glutathione or mercaptoethanol. Deactivation of DTNB-treated FDPase by thiols. The activation by DTNB was fully reversed by reduced glutathione (Table II) ; treatment of the activated enzyme with mercaptoethanol or cysteine caused the activity to fall significantly below the original level. Sensitivity of activated muscle FDPase to AMP and excess substrate. Purified muscle FDPase is considerably more sensitive to inhibition by AMP than is the liver enzyme, and this inhibition is also observed with the activated enzyme. In the standard assay (pH 7.5, 5 mM MgSOd), the native enzyme was inhibited 50% by 0.4 X lop6 M AMP;
Fl:UCTOSE
/ 4
DIPHOSPHATASE
I 12
I 20
I 20
DTNB
ADDED,
I 36
FROM
RABBIT
I 44 MOLES/MOLE
MUSCLE
375
I OF ENZYME
5. Activation of muscle FDPase by DTNB. The enzyme was incubated in 0.04 M Tris buffer, pH 7.5, containing 1 rn~ EDTA, FDPase (0.11 mg, 8.5 X 10-’ M) and with varying amounts of DTNB in a final volume of 1.0 ml. Final concentrations of DTNB varied from 3 X 10-e M to 1 X 1V M. After 60 min at 22” aliquots were analyzed as described in the text. The initial specific activities were as described in the legend to Fig. 4. FIG.
TABLE
II
EFFECT OF RISDUCD GLUT.ITHIONE, MERCAPTOETHANOL, AND CYSTPINE: ON THE DTNB-TREATED ENZYME:S Incubationa First
None DTNB DTNB DTNB DTNB
Second
None None GSH Mercaptoethanol Cysteine
Specific activit# pH I.5
pH 9.3
4.5 9.0 5.5 2.5 3.4
7.2 19.4 5.8 4.2 4.6
u FDPase (0.32 mg, 4.9 X 10-O M) was incubated at 22” in a final volume of 0.5 ml containing 0.04 M Tris-HCl bufl’er, pH 7.5, and 5 X 10m5 M DTNB (first incubation). After 60 min, wheu maximum activation was reached, O.l-ml aliquots were removed and incubated for 30 min at 22” in 0.1 M Tris-HCl buffer, pH 7.5, with 1 mM cysteine, 1 mM reduced glutathione, or 10 mM mercaptoethanol (second incubation). b FDPase activity with 0.5 mM Mn++ and 0.1 mM FDP was measured spectrophotometrically in 0.04 M Tris buffer, pH 7.5, or 0.04 M glycine buffer, pH 9.3.
ARIP was required to inhibit the native enzyme by 50 % ; the DNP-enzyme was equally sensitive, but 5- to lo-fold higher concentrations were required for t’he DTNBenzyme. These concentrations (10e4 nr) are comparable to those required t’o inhibit liver FDPase (1s). The activated enzyme was less sensitive to inhibition by high concentrations of substrate. At 2.0 rnM fructose-l ,6-P? , the DTNB-t’reated and the DNP-enzymes were inhibited 15 and 21 c/c, respectively (7.5, 5 mM -\Ig++), compared with 40 % for the untreated enzyme. However, like the untreated enzyme, neither of the activated enzymes was inhibited by this concentration of fructose-l ,%I’2 at alkaline pH in the presence of Ah++. DISCUSSION
The results reported here support the conclusion that muscle and liver FDPases are different proteins, although they possess many properties in common. They differ significantly in amino acid composition, but 3- t,o 5-fold higher concentrations were re- these differences may not greatly affect the three-dimensional structures. Thus they quired to achieve the same degree of inhibishow similar totals for homologous groups of tion with the DNP-enzyme or DTNBactivated enzyme. In the presence of 0.5 m&t amino acids, such as the sum of aspartic acid Mn++ at either pH 7.5 or 9.3, nearly 10-j 11 plus glutamic acid, isoleucine and leucine
376
FERNANDO,
PONTREMOLI,
plus valine, tyrosine plus phenylalanine, and arginine plus lysine (Table I). Neither enzyme contains tryptophan. Both enzymes are activated by treatment with dinitrofluorobenzene, p-mercuribenzoate, and by disulfide exchange reactions. It may be of significance that the muscle enzyme is not activated by disulfide exchange with cystamine, since this compound has not been reported to occur in muscle. This remains the only physiological agent known to activate liver FDPase by modification of sulfhydryl groups. In the accompanying paper (5), immunological evidence is presented for nonidentity of the liver and muscle enzymes. Further studies on the structure of these enzymes mill be required to determine the extent of the differences and similarities. REFERENCES 1. FERNANDO, J., ENSER, M., PONTREMOLI, S., ASD HORIXKER, B. L., ilrch. Biochem. Biophys. 126, 599 (1968). 2. PONTREMOLI, S., TKANIELLO, S., LUPPIS, B., END WOOD, W. A., J. Hiol. Chem. 240, 3459 (1965). 3. KRERS, H. A. AND WOODFORD, M., Hiochem. J. 94, 436 (19G5). 4. OPIE, L. II. AND NEIVSHOLME, E. A., Biochem. J. 104, 353 (1967).
AND
HORECKER
5. ENSE:R, M., SHAPIRO, S., AND HORIXKER, B. L., i2rch. Biochem. Biophys. 128, 554-562 (1968). 6. BOYER, P. D., J. Am. Chem. Sot. 76, 4331 (1954). 7. ELLM~N, G. D., drch. Biochem. Biophys. 82, 70 (1959). 8. SP~ICKMAN, D. H., STEIN, W. H., AND MOORE, S., Anal. Chem. 30, 1190 (1958). 9. HIRS, H. W., J. Biol. Chem. 219, 611 (1956). 10. D;WIS, B. J., Ann. N. Y. Acad. Sci. 212, 404 (1964). 11. PONTRICMOLI, S., LUPPIS, B., TRANIF,LLo, S., I~IPPA, M., ANT) HORECKER, B. L., Arch. Biochem. Biophys. 112, 7 (1965). 12. CRESTFIELD, A. M., MOORE, S., .~ND STEIN, W. H., J. Biol. Chem. 238, 622 (1963). 13. GOODTVIN, T. W. .~ND MORTON, R. A., Biothem. J. 40, 628 (1946). 14. PAT~HORNIK, A., LAID-SON, W. B., AND WITKOP, B., J. Am. Chem. sot. 80, 4747 (1958). 15. FUN~TSV, M., GREEN, N. M., AND WITKOP, B., J. Am. Chem. Sot. 86, 1846 (1964). 16. PONTREMOLI, S., LUPPIS, B., WOOD, W. A., TRANIELLO, S., BND HORIXKER, B. I,., J. Biol. Chem. 240, 3469 (1965). 17. PONTREMOLI, S., TRANIELLO, S., ENSER, PI/I., SI-IAPIRO, S., .\ND HORECKGR, B. L., Proc. ATatl. dead. Sci. 58, 286 (19F7). 18. HOR~CKER, B. L., PONTREMOLI, S., ROSI!ZN, O., AND I~OSEN, S., Federation Proc. 25, 1521 (1966).