role of emzyme-enzyme imteractions in the … of emzyme-enzyme imteractions in the regulation of...

Role of Emzyme-Enzyme Imteractions in the Regulation of

Gluconeogenesis

PROPERTIES AiND SUBUNIT STRUCTURE 01” FRUCTOSE 1, (~-DI~‘IIOSI’HI~TASI~: E’ROM SWISE I<TDKEY*

(Received for puhlicntion. May 18, 1972)

JOSEPH I\~XDI~INO AND NAXCY &ATOWICH

Flom the Department of Biochemistry, University of Gewlia, Atlrens, Georgia 30~01

Fwm the Department of Chemistry, Clayton FoundatiorL, Biochemical Instifute, I *trioersit!J (!f Texas, hstiw,

Texas 78712

SUMMARY

Fructose 1,6-diphosphatase has been isolated from swine kidney extracts by a procedure which yields large quantities of homogeneous enzyme. The purified preparation has an extinction coefficient (I&&,) of 8.9 in 0.05 M Tris-HCl, pH 8.0 and a specific activity of 25 to 30 pmoles per min per mg. The molecular weight of the native protein, as determined by sedimentation velocity, sedimentation equilibrium analysis, sucrose gradient centrifugation, and gel filtration on Sephadex G-200 is 130,000 + 2,500. Sedi- mentation equilibrium analysis of the enzyme denatured in 4 M guanidine hydrochloride indicated that four polypeptide chains might be present. These results were confirmed by polyacrylamide gel electrophoresis in 0.1% sodium dodecyl sulfate which showed that the native enzyme contained 4 identical subunits with molecular weights of 34,000 i 1,500. Further evidence for the presence of 4 subunits was obtained from a determination of the number of peptides formed after digestion of the enzyme with trypsin. The COOH-terminal amino acid residues of all 4 subunits were identified as alanine by hydrozinolysis and by digestion with carboxypeptidases. The NH,-terminal amino acid was serine. Quantitative Edman degradations on oxidized denatured samples showed that the native enzyme contained 4 serine NHg-terminal residues per mole. All of the determinations gave nearly the same values for the subunit molecular weight of about 34,000, which is in good agreement with a tetrameric structure for the native enzyme with a molecular weight of 130,000. Examination of fructose 1,6-diphosphatase in the electron microscope by the nega- tive staining technique revealed the presence of a flattened ellipsoidal particle with cross-section diameters of 60 and 120 A and a width of about 30 A.

* This investigation was sllpported by United States Public He:\lth Service (irant AM13150 from the Xationitl Institute of Arthritis and Metabolic 1)ise:tses.

The activity of renal frurt’ore L,6-diphos~~hatase (K 3.1 .3.11, n-fructose 1 ,6-diphosphate l~l,llosI)hohS‘tlrolase), which cat,a- lyzes a rate-limiting reaction in gluconcogcnesis in kidney is inhibited by L1sXI1’ and fructose-l, 6-1’2 (1). Studies on the enzyme isolated from rat and rabbit liver have shown that th(I enzyme contains four binding sites for fructose-l, 6-P2 (2) and three to four specific binding sites for AMl’, an allosteric inhibi- tor of the enzyme (3, 4). The swine kidney (5) and rabbit liver enzymes (6) both have molecular weights of 130,000 and thq- probably each contain four subunits.

The catalytic and regulator\- propert& of fructose 1,6-di- phospliatases from various tissues have been found to be ,greatl~- altered by a surprisingly large number of effecters and different reagents which modify proteins. The activity of the liver enzyme was affected by the prcscnce of sulfhytlryl rcagcrrts (7), cystamine (8), cocnzyme h (9), acyl carrier protein (9), EDTA1 (lo), 3-I-‘-glyceratc (lo), AM~1’ (IO), and ATP (10). The activity was also influenced by treatment with l-fiuoro-2,4-d- nitrobenzcne or p-hydrosymercuribenzoate (7).

The sensitivity of the enzyme to inhibition by r\;LIP could also be significantly altered by modification of specific tyrosine residues with dinitrolluorobenzene reaction of t-amino lysine groups with pyridosal 5-phosphate (II), or by treating the cnzymc with cystamine or N-ethglmalcimide (7). The inhibitory effect of X111’ could be abolished by limited hydrolysis of the purified enzyme with papain, and this effect was accompanied by a 3.fold increase in the activity of the enzyme (3). The fructose I, 6-diphosphatase isolated from Cundida utilis could be reversibly dissociated into subunits with one-half the molecular weight of the native enzyme. The dissociatrd enzyme was found to be relatively insensitive to inhibition by ;2J\IP (12). These results suggest that the active catalytic sites which bind fructose-l ,6-I’% and the allostcric sites which bind ,\Ml’ ran be selectively and independrntly modified under appropriate conditions. It is also possible that the two different sites may be located on different subunits in the native enzyme.

The present study was ulltlcrtaken to esamine some of the

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physical and chemical properties of renal fructose 1, B-diphosphatase. The enzyme which was obtained as a homogenous protein had a molecular weight of 130,000 f 2,700 and sedimentation studies indicated the presence of 4 subunits. In addition, the number of peptides formed after digestion with trypsin and the results of electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate supported the conclusion that the enzyme contained 4 identical subunits with molecular weights of about 34,000. The presence of four polypeptide chains was confirmed by quantitative analysis of the NH%-terminal and COOH-terminal amino acids. The results obtained on examination of the purified enzyme in the electron microscope indicated the presence of a flattened ellipsoidal particle with dimensions which correspond to a protein with a molecular weight of about 115,000.

EXPERIMENTAL PROCEl)URE

Purification of Fructose 1, CDiphosphatase jrom Swine Kidney-The enzyme was purified from swine kidney by elution from substituted anionic cellulose columns by procedures developed in previous studies for the preparation of fructose 1 ,6- diphosphatase (1, 5). A purified preparation of the enzyme was obtained directly from crude kidney extracts by absorption and elution from cellulose-P columns (5). Fructose 1, B-diphos- phatases derived from many different tissues show an unusually high affinity for anionic cellulose columns. This rather char- acteristic property of fructose 1,6-diphosphatase has been uti- lized to isolate the enzymes from liver (13), muscle (14), and C. utilis (15). In the present study the final elution from small cellulose-P columns (3 x 4 cm) was carried out with 0.05 M Tris- HCI, pH 8.0-0.001 M AMP-O.001 M fructose-l, 6-1’2 (5). The final purified preparation was homogeneous at this stage and it had a specific activity of 25 to 30 pmoles per min per mg of

protein. Enzyme preparations stored at -20” for more than 1 month gradually began to lose activity. However, all of the activity could be regained by incubating the enzyme at 38” for 15 min in the presence of 30 mM cysteine.

Fructose 1, A-diphosphatase activity was determined by measuring the rate of formation of either fructose-6-l’ or l’i. For routine assays, the activity was determined by following the rate of formation of Pi from fructose-l, 6-1’~ as described previously (5). The reaction mixture was incubated at 38” and contained in 9 ml, 100 mnr Tris-HCl (pH 8.0), 12 m&t XgCls, 5 mM cysteine, 0.1 111~ fructose-1,6-P2, and enzyme. After 5 min the reaction was stopped by the addition of 1 ml of 5 N HzS04- 2.5y0 ammonium molybdate, and the amount of phosphate released was measured (16). The conversion of fructose-l, 6-P2 to fructose-6-l’ was assayed spectrophotometrically by measuring the rate of reduction of TPN in the presence of excess phospho- glucose isomerase and glucose-6-P dehgdrogenase (5).

The enzyme was also assayed at low substrate concentrations with a coupled system which regenerated fructose-l, 6.P2. In the presence of esccss phosphofructokinase (2 units), 0.5 unit of pyruvic kinase, 0.5 unit of lactic dehydrogenase, 0.25 mM ATP, 2 mM MgCln, 100 IIN KCl, 0.2 mM DPNH, and 0.5 mM P-enolpyruvate, the hydrolysis of fructose-l, 6-P2 is accompanied by a stoichiometric oxidation of DPNH (5). Assays were carried out under conditions in which the rate of hydrolysis of the substrate was linear with time and proportional to the concentration of cnzymc (5). One unit of activity in all three assays is defined as that amount of enzyme which will catalyze the convenion of I pmole of fructose-l, 6-Ps to fructo,cc-6-l’ and

Pi per min and specific activity is cxpresscd as units per mg of protein.

Xedimentation Studies-Ultracentrifugation was carried out in a Beckman analytical ultracentrifuge. Sedimentation velocity experiments were performed. at 60,000 rpm at 20” according to Schachman (17), and molecular weight values were calculated from ~20,~ and D20,w values by using the Svedberg equa- tion. High speed sedimentation equilibrium runs were carried out with a 2.8mm liquid column according to the procedure of Yphantis (18) by using a 12.mm double sector cell with sap- phire windows. The time required to reach equilibrium was usually 24 to 30 hours, as determined by measuring the fringe displacement until it was constant. A partial specific volume of 0.735, which was calculated from the amino acid composition of the purified enzyme (5), was used in all calculations. The densities and viscosities of the solvents were obtained from the data of Kawahara and Tanford (19).

Electrophoresis-Disc gel electrophoresis was performed according to the procedure of Davis (20) and Ornstein (21) with a 7.5% polyacrylamide gel. A 3.757, polyacrylamide separat- ing gel was used and some samples were layered on the running gel in a solution containing 0.1 M sucrose and the electrode buffer. Electrophoresis was performed with Tris-glycine buffer at pH 8.3 and with sodium acetate buffer at pH 4.5.

Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate was performed at pH 7.0 with a 57. gel by the method of Shapiro et al. (22). Electrophoresis was carried out 255 hours at 8 ma per tube. In experiments with proteins dissociated by maleylation, electrophoresis was performed with a 7.5% polyacrylamide gel (20) and Trisglycine buffer at pH 9.0. Proteins were treated with maleic anhydride according to the procedure of Sia and Horecker (23). The maleylated samples were dialyzed against 0.05 M Tris-glycine, pH 9.0-0.01 M 2-mercaptoethylamine-0.1 M NaCl before elcctrophoresis.

Hydrolysis with Trypsin and Peptide Xaps-The enzyme was denatured and oxidized with performic acid (24) before it was digested with trypsin. The purified enzyme was dialyzed es- haustively against distilled water and the solution was lyophilized to dryness. About 20 mg of the enzyme was dissolved in a solution containing 9.5 ml of 97% formic acid and 0.5 ml of hy- drogen peroxide. The solution was kept at 3” for 3 hours, and the oxidized enzyme was diluted with water and lyophilized. The protein was dissolved and reprecipitated twice by the addition of acetone and the final pellet was dried under vacuum. About 100 pg of twice-crystallized beef trypsin (Worthington), which was treated with L-l-tosylamido-2-phcnylethyl chloro- methyl ketone (25) to remove any traces of chymotrypsin activity, and IO-mg samples of the oxidized enzyme were dissolved in 5 ml of 0.2 M N-ethyl morpholine acetate, pH 8.5. This reaction mixture was incubated at 37” for 10 hours with agitation. The solution was lyophilized to dryness to remove the volatile buffer, and the soluble peptides were dissolved in water and 2- mg aliquots were applied to Whatman Xo. 3MM paper. The paper was developed with a solvent system containing I-butanol acetic acid-water (4 : 1: 5) by descending chromatography for 12 hours (26). The papers were dried at room temperature and electrophoresis was carried out at pH 3.6 with a solvent containing pyridine-acetic acid-water (1:10:300) at 2000 volts for 75 min. The chromatogram was dried and the peptides were detected with a solution of 0.257, ninhydrin in 95% ethanol, anti in some cases it was then sprayed again with Pauley’s reagent to reveal peptides containing histidine.


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NH&erminal and COOH-terminal Amino Acid Analysis- Preliminary studies with a modification (27) of the dinitrophenyl- ation procedure (28) indicated that serine was the NH&erminal amino acid of swine kidney fructose l,&diphosphatase. In order to confirm this observation the NHn-terminal amino acids of the enzyme were examined by the dansyll-chloride method (29, 30). The procedure was modified slightly to prevent precipitation of fructose 1,6-diphosphatase. The concentration of the reagent was doubled, and 2 volumes of this solution were added to 5 volumes of the solution containing the protein. Two dimensional chromatography on polyamide sheets with, several solvent systems was used to separate the derivatives (29).

The quantitative determination of the amount of each NHz- terminal amino acid was carried out by the method of Richards et al. (31). A modification of the Edman method involving a quantitative reaction between methylisothiocyanate and the enzyme in the presence of added 15N-labeled amino acid, followed by analysis of the methylthiohydantoin derivative in the mass spectrometer was used for the estimation of the NHS-terminal amino acids in this assay (31). The amount of amino acid released from the protein was calculated by measuring the extent of dilution of the added 15N-labeled amino acid.

The COOH-terminal amino acid was determined by hydra- einolysis of the oxidized enzyme (32). The protein was oxidized with performic acid as described previously (24), and the thoroughly dried sample was treated with anhydrous hydrazine at 105” for 8 to 16 hours in a sealed evacuated tube. The unreacted hydrazine was removed by evaporation under reduced pressure. The free amino acids released were then determined with an amino acid analyzer.

The digestion of the oxidized enzyme with carboxypeptidase A and B was carried out at 37”, and samples were removed at various times (33)) diluted, and assayed directly in the amino acid analyzer. The release of amino acids from the COOH-terminal portion of fructose 1,6-diphosphatase was followed for about 4 hours with different concentrations of either carboxypeptidase A or B. Negligible autodigestion was found in control experiments under these conditions.

Methods and Materials-Highly purified urea, guanidine HCl and ammonium sulfate obtained from Mann were used in this study. Urea solutions were deionized by treatment with a mixed bed resin Rexyn 101 (Fisher) before use. Recrystallized sodium dodecyl sulfate was obtained from Gallard-Schlesinger. Enzyme grade tris(hydroxymethyl)aminomethane (General Biochemicals) was used and 8-hydroxyquinoline was purchased from Sigma. Dithiothreitol was obtained from Calbiochem and 2-mercaptoethanol was purchased from Matheson, Coleman and Bell. Sephadex G-200 was obtained from Pharmacia and cellulose-P was purchased from Whatman. Dansyl-chloride was obtained from the Pierce Chemical Co., and thin layer sheets with E-poly- caprolactam bonded to a polyester support were purchased from Cheng-Chin Trading Co., Ltd., No. 75, Sec. 1, Hankow St., Taipei, Taiwan.

Cellulose-P was prepared for use in column chromatography according to the directions furnished by the supplier. Fines were removed by decantation, and the thoroughly equilibrated resin was suspended in 0.05 M Tris-HC1, pH 8.0 and stored at 3”. Trypsin, hemoglobin, n-glyceraldehyde-P dehydrogenase, chy- motrypsimogen, and ovalbumin were obtained from Worthington.

f The abbreviation used is: dansyl, dimethylaminonaphthalene. 5-sulfonyi.

RESULTS

Ultracenkijugation Analysis-The sedimentation velocity pattern of the purified enzyme, after dialysis against 0.05 M Tris- HCl, pH 8.0-0.01 M 2-mercaptoethylamine-0.1 M NaCl, showed the presence of a single peak (Fig. 1). Five determinations with solutions containing from 3 to 10 mg of protein per ml gave SZO,~ values of 7.4 f 0.2. There was very little variation in the sedimentation coefficient at these protein concentrations. A molecular weight of 130,000 f 2,700 was calculated from this data using a partial specific volume of 0.735 cma per g calculated from the amino acid composition and a D20,,,, of 5.44 x lo-’ cm2 set-l (5).

High speed sedimentation equilibrium analysis of the native enzyme, according to the procedure Yphantis (18), gave linear plots of the fringe displacement versus the square of the radial distance, which further indicated that the preparation was homogeneous. A molecular weight of 130,000 f 3,000 was obtained by this method. These values agree well with the molecular weights obtained by sucrose density centrifugation and ex- clusion chromatography on Sephadex G-200 (5).

Molecular Weight of Subunits of Fructose 1,6-Diphosphatase- The enzyme could be readily dissociated into subunits under various conditions such as treatment with sodium dodecyl sulfate and high concentrations of urea or guanidine HCl. Performic acid oxidation and maleylation also caused the native enzyme to break up into subunits.

The enzyme, 10 mg per ml, was dialyzed for 48 hours at 3” against 0.05 M Tris-HCl (pH 8.0), 4 M guanidine HCl, and 0.01 M

dithiothreitol. Sedimentation velocity experiments with enzyme dissociated in 4 M guanidine HCl revealed the presence of a single broad peak. The average sedimentation coefficient of the peak was estimated to be approximately 1.5 S. Since preliminary studies indicated that the enzyme contained 4 subunits, it was of interest to determine the molecular weight of the subunits by the meniscus depletion sedimentation equilibrium method. The dialyzed enzyme in 4 M guanidine HCl was centrifuged at 40,000 rpm at 20” until equilibrium was attained, and the results are summarized in Fig. 2. Plots of the log of the fringe displacement versus the square of the radial distance were linear under these conditions. The molecular weight calculated from the slope of the line in Fig. 2 was found to be 33,000, assuming a value of 0.735 for the partial specific volume of the dissociated enzyme in 4 M guanidine HCl. In order to obtain more definitive informa-

FIG. 1. Sedimentation velocity profile of fmctose 1,6-diphosphatase. The enzyme was dialyzed against 0.05 M .Tris-HCl, pH 8.0 and 0.15 M NaCl. The Drotein concentration was 3.5 me: per ml, and measurementswere made at 60,000 rpm and 20< photographs were taken at a bar angle of 65” at 26 min, and 42 min after attaining maximum speed.


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tion on the subunit structure of fructose 1,6-diphosphatase by this procedure the enzyme was dissociated with maleic anhydride, and the molecular weight of the resulting subunits was determined by sedimentation equilibrium. Studies with maleylated enzyme yielded linear plots of log y versus the square of the radial distance. The molecular weight of the polypeptide chains in this sample was calculated to be about 34,500.

Polyacrylamide Gel Electrophoresis-The analysis of the native enzyme by disc gel electrophoresis was complicated by the fact that the protein aggregated in the absence of high concentrations of salt. When the purified enzyme, which was dialyzed for 6 hours against 0.05 M Tris-HCl, pH 8.0-0.02 M 2-mercaptoethylamine-O.1 N NaCl, was examined by electrophoresis on 7.5 or 4.0$& polyacrylamide gels at pH 8.9, 7.8, or 4.5, essentially all of the protein remained at the origin. Blurred patterns were obtained with enzyme preparations which were precipitated with ammonium sulfate and dissolved in 0.05 M Tris-HCl, pH 8.0 before analysis. However, the major band was always found at the origin, indicating that a significant amount of the enzyme never entered the gel.

The subunit structure of swine kidney fructose 1,6-diphosphatase wa,s examined by polyacrylamide gel electrophoresis in the presence of 0.1% sodium dodecyl sulfate and 0.1 M sodium phosphate, pH 7.0. When the purified enzyme was treated with 2-mercaptoethanol and sodium dodecyl sulfate as described in Fig. 3, a single component was observed. To estimate the size of the polypeptide chains of fructose diphosphatase their mobil- ities were compared with those of standard proteins in the same gel as shown in Fig. 4. The molecular weight was determined from linear plots of the distance of migration versus the logarithm of the molecular weight of the standards. These experiments yielded a molecular weight of 34,000 for the subunits of fructose 1 ,6-diphosphatase in the presence of 0.1 y. sodium dodecyl sulfate. When preliminary incubation for 3 hours in the presence of 1 To 2-mercaptoethanol and 1% sodium dodecyl sulfate was omitted a band corresponding in size to a dimer, 65,000 to 70,000, was also observed in the gels.

It was decided to determine the molecular weight of the subunits formed after dissociating the native enzyme by maleylation. The molecular weights of the polypeptide chains of fruct.ose 1,6- diphosphatase formed after maleylation of the purified enzyme were determined by polyacrylamide gel electrophoresis by using

FIG. 2. Sedimentation equilibrium data on subunits of fructose l,B-diphosphatase in 4 M guanidine HCI. The enzyme (0.2 mg per ml) was dialyzed for 48 hours against a solution containing 4 M guanidine HCl and 0.01 M dithiothreitol. Measurements were made from patterns taken after centrifugation at 40,000 rpm for 30 hours at 20”. The fringe displacement was measured on a mi- crocomparator, and the logarithms were plotted against. the square of the distance from the center of rotation at equilibrium. the same conditions for & and F.

FIG. 3. Polyacrylamide gel electrophoresis of swine kidney fructose 1,6-diphosphatase in 0.1% sodium dodecyl sulfate and 0.01 M sodium phosphate, pH 7.0. A, 20 pg of enzyme treated with 1% sodium dodecyl sulfate and 1’7 X-mercaptoethanol for 2 hours at 37”; B, 30 pg of enzyme and C, 40 rg of enzyme treated as in A ; D, 20 fig of enzyme and E, 40 rg of enzyme dissociated by maleylation as described in the text. Electrophoresis was for 2f/z hours at 8 ma per gel for A, B, and C, and the time was 3 hours under -

proteins maleylated under the same conditions as standards. The molecular weight of the maleylated subunit derived from linear plots obtained with the standard proteins was estimated to be 33,000. The values for the apparent molecular weight of the subunits obtained by both of these procedures are in good agreement, and these results demonstrate that the native enzyme contains four similar polypeptide chains.

Analysis of Peptides Formed by Hydrolysis of Fructose i,6- Diphosphatase with Trypsin-Two-dimensional paper chromatography and electrophoresis of trypsin digests of fructose 1,6- diphosphatase yielded the pattern shown in Fig. 5. On the basis of 48 arginyl and 96 lysyl residues per mole of enzyme (Table I), 144 peptides would be expected if the enzyme consisted of four different polypeptide chains, and 36 peptides would be formed if all 4 subunits were identical. The detection of only 33 to 39 ninhydrin spots upon hydrolysis of several different purified preparations indicates that the enzyme is composed of 4 identical subunits. The variation in the number of peptides in the five preparations examined is due to the presence of some weak ninhydrin spots. Additional evidence in support of this conclusion was obtained by developing the chromatograms with Pauley’s reagent


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7

e 6 b -5

B FRUCTOSE DIPHOSPHATASE

t

(monomer)

C

IO40 DISTANCE (mm)

+- ELECTROPHORESIS

CHROMATOGRAPHY 0 000 0

0 Ooo n Oo"

OG n

0 0” 0

FIG. 4 (left). Determination of the molecular weight of the subunits of fructose 1,6-diphosphatase by electrophoresis on acylamide gels in the presence of 0.1% sodium dodecyl sulfate. The relative distance of migration from the origin to the anode are plotted against the logarithm of the molecular weight, in each case. The standard proteins and their subunit molecular weights were (A) ovalbumin (>3,000), (B) glyceraldehyde-P dehydrogenase (36.000). (0 chvmotrvosinoeen (25.700). and CD) hemoglobin (15;5OOj: -A’, dis”tance”of m&at& bf tge mo&m& and vdimer of fructose 1,6-diphosphatase. Salts were removed from fructose 1,6-diphosphatase preparations by dialysis against 0.1% sodium dodecyl sulfate and 0.01 M sodium phosphate (pH 7.0) to prevent precipitation of the enzyme. All of the-proteins were disiociat.ed by incubation for 2 hours at 37” in 0.01 M sodium phosphate (pH 7.0), 1% sodium dodecyl sulfate, and 1% 2-mercaptoethanol.

to reveal which of the peptides also contained histidine. About three to four of the peptides on the chromatogram also contained histidine. Since the enzyme contains 14 histidyl residues per mole (Table I), these results again indicate that only about 25% of the maximum number of different peptides are being formed on digestion with trypsin. Thus, these results are completely consistent with the number of peptides which would be expected for a protein containing 4 identical subunits.

The amino acid composition of swine kidney fructose 1 , 6-diphosphatase was determined, and it was compared with those of the enzymes isolated from liver, muscle, and chloroplasts (Table

I). The analysis obtained in the present study differs somewhat

from that reported previously (5). The enzyme preparation used in the present study was eluted from cellulose-P columns with a buffer containing 1 pmole each of AMP and fructose- 1, 6-Pz. In the previous study the enzyme was eluted with a NaCl gradient. Both preparations had a specific activity of about 20 to 30 pmoles per min per mg. However, the enzyme eluted with AMP and fructose-l, 6-PZ contained very little or no tryptophan. A protein impurity which contained tryptophan was eluted from the cellulose-P column with NaCl after the elution of fructose 1,6-diphosphatase with AMP and fructose-l, 6- Pz. The histidine, valine, cysteine, and tyrosine content of the two preparations were also different. Precautions were taken to correct for the loss of some of these amino acids during hydrolysis in the experiments described in Table I.

The chemical compositions of the kidney and liver enzymes are very similar, whereas the enzyme from chloroplasts is different from the other three, particularly with respect to the content of cysteine. However, a number of enzymes derived from plant tissues have a higher cysteine content than the corresponding enzymes purified from mammalian tissues, and this may not be a

0.6 t /

WAVELENGTH, mu

FIG. 5 (center). Two-dimensional map of peptides from a tryp- tic digest of swine kidney fructose 1,6-diphosphatase. The peptides showing a positive reaction to ninhvdrin are circled. The conditions for digestion with trypsin and for the separat’ion of t.he peptides are described in the text. The direct-ion of chromatography and electrophoresis are shown by the arrows, and the polar- ity during electrophoresis is shown at the bottom of the chromatogram.

FIG. 6 (right). Ultraviolet absorption spectra of swine kidney fructose 1,6-diphosphatase in 0.05 M Tris-HCl at pH 7.5 and in 0.1 N NaOH. About 0.3 mg per ml of protein was used to obtain the spectrum at pH 7.5, and 0.43 mg per ml was used to obtain the spectrum in alkali.

significant difference. The content of glutamic acid in the enzyme from chloroplasts is twice as high as that in the other three enzymes. This and the 210 residues of half-cystine per mole of enzyme would tend to make this protein somewhat more acidic than the mammalian enzymes. Interestingly, this enzyme was purified from DEAE-cellulose columns, whereas the three mammalian enzymes bind very tightly to cellulose-P and other modified cellulose anion exchange columns, but they pass through DEAE-cellulose columns. The extinction coefficient of a 1% solution of the purified kidney enzyme at 280 nm was 8.9 based on the protein concentration dctcrmincd by dry weight of a dialyzed sample or by the phenol procedure (37). The enzyme contains very little tryptophan. Only very small amounts of this amino acid were detected in hydrolysates when samples of the purified enzyme were hydrolyzed in 6 N HCl in the presence of 4y0 thioglycollic acid (36).

When a 2-mg sample of the purified enzyme was hydrolyzed in the presence of thioglycollic acid, less than 0.005 kmole of tryptophan was released. If each subunit contained only 1 tryptophan residue then at least, 0.062 pmole rould have been formed. Less than 1 mole of tryptophan per mole of enzyme was detected.

The ultraviolet absorption spectra of the purified enzyme at pH 7.5 and in 0.1 N NaOH was examined and the results are shown in Fig. 6. The spectrum at pH 7.5 has an absorption maximum at 278 nm and a minimum at 250 nm. In 0.1 N NaOH only a single peak was observed at 293 nm and the minimum absorption was seen at 275 nm. These results are in agreement with the data obtained by amino acid analysis which indicate that fructose 1 , 6-diphosphatase contains very little tryptophan.

Determination of NH2-Terminal Residues-A micromethod (29), based on a modification of the procedure of Gray (30) was used to analyze for NHs-terminal amino acids in the enzyme.


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TABLE I

Amino acid composition of kidney, liver, muscle, and chloroplast

fructose 1,6-diphosphatase Five-milligram samples of the purified enzyme were dialyzed

and hydrolyzed in evacuated scaled tubes for 24 and 48 hours at a concentration of 1 mg per ml with 6 N HCl at 105”.

Amino acid I-

Lgsine..

Histidine. Arginine. Aspartic acid

Threonine Serine Glutamic acid

Proline. Glycine Alanine

Half-cystine Valine. Methionine.

Isoleucine Leucine. Tyrosine

Phenylalanine Tryptophan’.

Number of residues per mole

Swine kidney’l

Rabbit Rabbit liver” muscles

Spinach 1loroplast”

96 117 89 65 14 14 10 1‘2 48 34 43 30

133 128 103 9s 72” 70 78 28 6Td 79 76 78

100 81 118 216 61 52 48 43

105 100 104 166 111 106 111 64

22e 20 21 210 97d 104 104 67 29e 34 21 14

71d 70 58 57 104 94 119 73

53 52 63 36 38 40 32 25

<1 0 0 0

0 The values are average or extrapolat,ed values calculated from data obtained after 24 and 48 hours of hydrolysis. The number of residues were calculated by using a molecular weight of 130,000 for the enzyme and the values were estimated by taking the

nearest whole number. The recoveries after 24 and 48 hours of hydrolysis were generally greater than 957, and the total number

of residues per mole of enzyme was 1221. b Data obtained from Fernando et al. (34).

c Data obtained from Buchanan et al. (35).

d Extrapolated values to zero time for the hydrolysis of serine and threonine and to infinite time for valine and isoleucine.

e The cysteine and methionine were converted to cysteic acid and methionine sulfoxide by oxidation with performic acid prior

to hydrolysis (24). f Determined on samples hydrolyzed in 6 N HCl in the presence

of 47, thioglycollic acid (36).

Several preparations of purified fructose 1,6-diphosphatase were dialyzed exhaustively against distilled water and lyophilized. The dansyl-amino acid derivatives of the NH%-terminal residues of the enzyme prepared by the modified procedure were separated by thin layer chromatography on polyamide sheets. One NH2- terminal dansyl-amino acid derivative was found, corresponding in position upon chromatography to the dansyl derivative of serine. The only other distinct dansyl derivatives which were detected were 0-dansyl tyrosine, dansyl-NH*, and c-amino-dansyl lysine, which remained near the origin.

These results were confirmed and extended in other experiments in which quantitative determinations were carried out on oxidized samples of the enzyme by a modification of the Edman reaction (31). The enzyme was oxidized with performic acid and it was then reacted with methylisothiocyanate. The only compound detected after isolation of the products was the methylthiohydantoin derivative of serine. The amount of serine released from the enzyme was determined by measuring the extent of dilution of lSN-labeled serine which was added to the

reaction mixture (31). \Vhen a 22.3.mg sample of the enzyme was analyzed by this procedure, 0.7 pmole of serine was found. Based on a molecular weight of 130,000 for kidney fructose 1,6- diphosphatase these values correspond to about 3.9 moles of serine residue per mole of enzyme. The results are consistent with other observations made in the present study, in that they further show that kidney fructose 1 , 6-diphosphatase contains 4 subunits with serine NHz-terminal residues.

Determination of COOH-terminal Residues by IIydrazinolysis and Treatment with Carboxypeptidase B-Four different preparations of purified fructose 1 , 6-diphosphatase, with specific activi- ties of 25.2 units per mg of protein were dialyzed against distilled water and lyophilieed. These preparations were treated with performic acid, and the oxidized samples were reisolated and lyophilized in test tubes over PZOs. The thoroughly dried samples, 15 mg, were dissolved in 0.5 ml of hydrazine. The tubes were sealed under vacuum and kept at 105” for 8 to 16 hours. The unreacted hydrazine was removed by evaporation and the samples were examined with an amino acid analyzer. The only amino acid detected by this procedure was alanine. Approxi- mately 0.21 pmole of alanine was formed from 15-mg samples of the purified enzyme. This value corresponds to about 1.9 moles of alanine per 130,000 g of fructose 1 ,6-diphosphatase. However, only about 507, of the COOH-terminal amino acid is usually recovered by this method (32). Based on this recovery, the release of 1.9 moles of alanine per mole of enzyme is in good agreement with the presence of four COOH-terminal amino acids in the enzyme.

The COOH-terminal region of n-fructose 1,6-diphosphatase was also examined by digestion with carboxypeptidases. The native enzyme was very resistant to digestion with carboxypeptidase. When the enzyme was dissociated by oxidation with performic acid, alanine was released very rapidly by carboxypeptidase A and B. The digestion of 15.mg samples of the oxidized enzyme with carboxypeptidase released about 0.13, 0.21, 0.30, 0.41, and 0.43 pmole of alanine in 2, 4, 6, 8, and 16 hours, re- spectively. During the same time periods, 0.05, 0.08, 0.13, 0.20, and 0.30 pmole of leucine were released. The yield of alanine approached 3.7 residues per mole of enzyme at longer times, based on the release of 0.43 pmole of alanine from 15 mg of enzyme after 16 hours. Collectively these results demonstrate that swine kidney fructose 1 ,6-diphosphatase contains four polypeptide chains and that the COOII~terminal residue of all 4 subunits is alanine.

Shape of Fructose 1,6-Diphosphatase in the Electron iMicro- scope-High magnification electron micrographs of fructose 1,6-diphosphatase negatively stained with methylamine tungstate are shown in Fig. 7. The most striking feature seen on examination of this enzyme in the electron microscope is the large number of images such as Structure B in Fig. 7, Frame 2, which have a high contrast and are thin (30 to 40 A) and long (60 to 90 A). An equally noticeable feature is that perhaps half of the structural components are of low contrast compared to Structure B and they are elliptically shaped. Images typical of this type of projection are marked A in Frames 1 and 2 in Fig. 7. An elliptical structure, with proper orientation, should oc- casionally appear circular. An image with this shape is marked Al in Fig. 7, Frame 1. The high contrast of images of type B and C suggest that t,he thickness of protein along the direction of the electron beam is relatively greater than the thickness in the same direction for images of type A. The minimal observed widths of images designated as B and C in Fig. 7 are about 30 A. If these views are related to images such as A, the thickness of


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these structures is then only about 30A, the dimensions of the structures designated A in Fig. 7 are about 60 X 120 A. These observations suggest that the principal component present in the preparation is an ellipsoidal structure with dimensions of about 30 x 60 x 120 A. Several images of type D and E were also observed, which suggest the presence of an unfolded or a partially dissociated form derived from the more intact images A, B, and C. Previous studies have clearly shown that fructose 1 ,6-diphosphatase does dissociate to subunits with molecular weights of about 60,000 to 70,000 under various conditions (5).

The approximate volume of the flattened prolate spheroid particle with the elliptical cross-section seen in the micrograph was measured by summing the volumes of an oblate spheroid with a semi-major axis of 30 A and a semi-minor axis of 15 A and an ellipsoidal cylinder 60 A long with elliptical cross-section diameters of 30 and 60 A. The sum of these volumes is 1.4 X lo-l9 cn?. If the volume of this particle is multiplied by

Avogodro’s number and divided by the partial specific volume of the enzyme, a molecular weight of 115,000 is obtained. The results obtained on examination of the size of purified enzyme by electron microscopy are consistent with the other physical and chemical measurements reported in this communication.

DISCUSSION

The information presented in this report amply demonstrates that native swine kidney fructose 1 , 6-diphosphatase contains

. . . . . .

FIG. 7. Electron micrographs of swine kidney fructose 1,6- diphosphatase. 1, high concentration of enzyme sprayed in methylamine tungstate, X 300,000. I, same as in 1 but lower concentration of enzyme, X 300,000. A description of the structures in squares is given in the text.

The data obtained in previous studies and that reported here show that the native undissociated enzyme has a molecular weight in the range of 130,000 f 2,700, and these values repre- sent the results of at least three different physical techniques. The chemical data obtained in the present study confirms these results and further shows that the native enzyme contains 4 subunits. High speed sedimentation equilibrium analysis of three different enzyme preparations, and the sedimentation behavior of thcsc preparations on sucrose gradient centrifugation at very low concentrations showed that the enzyme was homogeneous and had a SZO+ value of 7.3 to 7.5. In guanidine HCl the enzyme dissociated into subunits that had minimal molecular weights of about 33,000. Equilibrium sedimentation analysis and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate confirmed the presence of only four polypeptide chains in the native enzyme and further showed that the 4 subunits formed from the native tetramer were identical. Subsequent experiments clearly showed that the enzyme contained four polypeptide chains. Nearly 4 eq of alanine were released when the denatured enzyme was treated with carboxypept.idases, and only alanine was found after hydrazinolysis. Preliminary studies showed that the subunits contained serine as the NHS-terminal amino acid. Quantitative Edman degradations confirmed and extended these results, and showed that the native enzyme contained nearly 4 eq of serine on the NHrterminal ends of the four polypeptide chains.

Analysis of the peptides formed after digestion of fructose 1 , 6-diphosphatase with trypsin provided still more evidence for the presence of 4 identical subunits. On the basis of the number of lysine and arginine residues found by amino acid analysis, about 36 different peptides could be formed on incubation with


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6650

trypsin if the enzyme contained 4 identical subunits. Hydro- lyzed samples of four different enzyme preparations consistently showed from 33 to 39 ninhydrinl)ositive areas after paper chromatography and electrophoresis. Further confirmation for the fact that only half of the theoretical number of peptides were being formed was obtained by spraying the paper chromatograms with l’auly’s reagent. The native enzyme contains 14 histidine residues, and only three to four peptides containing histidine were found after digestion with trypsin. Finally, the shape and size of the structures observed in electron micrographs also indicate that the enzyme may be composed of 4 subunits.

The subunit structure of fructose 1 ,6-diphosphatase is of some importance, since its activity must vary during glycolysis and gluconeogenesis. The data obtained from binding studies indicates that the native enzyme binds 4 eq of fructose 1,6-diphosphate and 4 eq of AMP. Preliminary evidence indicates that both the catalytic and allosteric sites may be present on all four polypeptide chains. The relationship between the ability of various specific effecters to influence the activity of the enzyme and its sensitivity to AMP and bring about specific alterations in the quaternary structure remains to be established.

Acknowledgnzents-ale gratefully acknowledge the participa- tion and excellent assistance of Dr. Thomas Fairwell for perform- ing the quantitative NHz-terminal analysis and Dr. Raymond Rshworth and Dr. James Travis for assistance with the analysis of protein samples. We wish to thank Dr. John Brewer for help with the ultracentrifugation studies. Large quantities of swine kidney were very generously provided by the McEvers Packing Company, Talmo, Georgia.

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Joseph Mendicino, Nancy Kratowich and Robert M. Oliver1,6-DIPHOSPHATASE FROM SWINE KIDNEY

PROPERTIES AND SUBUNIT STRUCTURE OF FRUCTOSE Role of Enzyme-Enzyme Interactions in the Regulation of Gluconeogenesis:

1972, 247:6643-6650.J. Biol. Chem.

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