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Inactivation of phosphofructokinase by dialdehyde-ATP
ARCHIVESOF BIOCHEMISTRYAND BIOPHYSICS Vol. 196, No. 1, August, pp. W-208, 1979
Inactivation of Phosphofructokinase
by Dialdehyde-ATP’
MARTHA R. GREGORY2 AND E. T. KAISER Department
of Chemi.stry,
University of Chicago, Chicago, Illinois 60657
Received January 2, 1979 Rabbit muscle phosphofructokinase (PFK) is rapidly inactivated by a 2’,3’-dialdehyde derivative of adenosine triphosphate (dialdehyde-ATP). When allowed to react with 0.6 mM dialdehyde-ATP in 0.1 M borate buffer (pH 8.6) containing 0.2 mM EDTA and 0.5 mM dithiothreitol, PFK loses essentially all activity (99%) in 30 min. The modified PFK remains inactive following dialysis of the reaction mixture against sodium borate (pH 8.0) containing fructose diphosphate, EDTA, and dithiothreitol. Experiments with [“Cldialdehyde-ATP show that 99% inactivation of PFK corresponds to incorporation of 3 to 4 mol of the ATP analog per PFK protomer. The inactivation of PFK with dialdehyde reagent is not caused by dissociation of the 340,000 M, tetramer to the 170,000 M, dimer, as determined by analytical ultracentrifugation. Adenosine diphosphate or ATP protect PFK from inactivation by dialdehyde-ATP at pH 8.6, but fructose g-phosphate, cyclic 3’,5’-adenosine monophosphate, or fructose diphosphate, which protect PFK from modification by pyridoxal phosphate, provide little protection from inactivation. Amino acid analyses of dialdehyde-inactivated PFK and of a control sample of the enzyme were compared following reaction of each with 2,4-dinitrofluorobenzene. The results show that three or four lysine residues per PFK protomer are modified by dialdehyde-ATP. Additional data indicate that these lysine residues react with dialdehyde-ATP to form dihydroxymorpholine-like adducts rather than Schiff bases.
ate, phosphocreatine, and phosphoenolpyruvate are inhibitors (6-11). Furthermore, kinetic data and chemical modification studies are consistent with the hypothesis that there are separate catalytic and regulatory sites on PFK (12, 20). Although PFK is an important regulatory enzyme in glycolysis, little is known about the composition of the ATP binding sites on the enzyme. Kemp and Krebs (21) reported that rabbit muscle binds 3 mol of ATP per protomer and Lorenson and Mansour (22) reported that sheep heart PFK binds 3.6 mol of ATP per protomer. In the present communication, we report on the modification of rabbit muscle PFK I Supported by National Institutes of Health Research with a 2’,3’-dialdehyde derivative of ATP, Grant GM 19037 (E.T.K.) and National Institutes of a study undertaken with the objective of Health Postdoctoral Fellowship AM 05667 (M.R.G.). identifying basic amino acid residues in the 2 Present address: Department of Pharmacology, catalytic and/or binding sites for ATP. The University of Rochester Medical Center, Rochester, inactivation of PFK with dialdehyde-ATP N. Y. 14642. is discussed with respect to incorporation 3 Abbreviations used: PFK, rabbit muscle phosof 14C-labeled reagent, protection by subphofructokinase; F6P, fructose &phosphate; FDP, strates, and amino acid residues modified. fructose l,&diphosphate; DTT, dithiothreitol; DNFB, A comparison of the modified enzyme and a 2,4-dinitrofluorobenzene.
Phosphofructokinase (PFK)3 catalyzes one of the controlling steps in glycolysis, transfer of the terminal phosphate of adenosine triphosphate (ATP) to the C-l hydroxyl of the pfuranose anomer of fructose 6-phosphate (l-4). The importance of this practically irreversible reaction in the regulation of glycolysis is underscored by the observation that a number of metabolites regulate PFK activity (1, 5). Adenosine monophosphate (AMP), adenosine diphosphate (ADP), fructose 6-phosphate (F6P), and fructose 1,6-diphosphate (FDP) are activators of PFK, while citrate, ATP, 3-phosphoglycer-
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0003-9861/79/090199-10$02.00/O Copyright 6 1979by AcademicPress, Inc. AU rights of reproductionin any form reserved.
GREGORY AND KAISER
control sample of native PFK by analytical ultracentrifugation is also presented. EXPERIMENTAL
PROCEDURE
Materials and methods. NADH (ChromatoPure) was obtained from P-L Biochemicals. All other substrates and effecters for PFK were obtained from Sigma Chemical Company. DEAE-Cellulose (DE-52) used in purification of PFK was obtained from Whatman. [&l’C]ATP, tetrasodium salt (60 mCi/mmol), and Instagel were obtained from New England Nuclear and Packard Instrument Company, Inc., respectively. The charcoal utilized to adsorb dialdehyde-ATP was a product ofTaiyo Kaken Company, Ltd., Tokyo, Japan. Amino acid analyses were carried out with a Beckman Model 121automatic amino acid analyzer. A Beckman Acta MVI spectrophotometer was used for determination of PFK concentration and activity and a Packard Tri-Carb scintillation spectrometer was utilized for counting samples in Instagel. Analytical ultracentrifugation studies were conducted using a Beckman Spinco Model E ultracentrifuge. Buffers used for the modification of PFK with dialdehyde-ATP and for dialysis of the resulting reaction mixtures were 0.1 M Tris-HCl (pH 8.6), 0.1 M sodium borate (pH 8.6), 0.1 M Tris-phosphate (pH 8.0), and 0.1 M sodium borate-O.2 mM FDP (pH 8.0), all containing 0.2 mM EDTA and 0.5 mM dithiothreitol (DTT). These buffers are referred to in the text as Tris-HCl (pH 8.6), borate (pH 8.6), Tris-phosphate (pH 8.0), and borate-FDP (pH 8.0), respectively. Modifications of PFK with DNFB were carried out in 0.1 M borate containing 0.2 mM EDTA only. The errors in determination of dialdehyde incorporation are expressed as standard deviations. PFK was puriPurification of phosphofructokinase. fied according to Kemp and Forest (23) with the following modification. After a 24-h dialysis against 50 mM /3-glycerophosphate, 2 mM ATP, pH 7.2, containing ammonium sulfate at 32% saturation, the supernatant enzyme was dialyzed against 0.1 M Tris-phosphate, 0.2 mM EDTA, 0.2 mM FDP, pH 8.0. The resulting solution was chromatographed on DEAE-cellulose as described by Ling et al. (24). PFK was stored as a suspension in 50 mM P-glycerophosphate, 2 mM EDTA, 0.5 mM DTT, 1 mM FDP, pH 8.0, containing ammonium sulfate at 50% saturation. Purified PFK exhibited an A219/A259 ratio of 1.7. PFK activity was routinely 130-140 U/mg where 1 unit (U) of PFK catalyzes the conversion of 1 pmol of F6P to FDP per minute at pH 8.0, 25°C. Assay of PFK activity. PFK activity was measured spectrophotometrically at pH 8.0, 25”C, in an assay coupled with aldolase, triosephosphate isomerase, and a-glycerophosphate dehydrogenase according to the procedure of Ling et al. (25) as modified by Lad et al. (26). PFK concentration was determined spectrophoto-
metrically using E@” = 1.02 mg-’ ml (27). The AzT9 of the dialdehyde-modified enzyme was determined by subtracting the contribution (at 279 nm) of the incorporated dialdehyde-ATP from the observed absorbance at 279 nm. The A,,, of the incorporated dialdehyde was calculated by utilizing the A2,9/A259 ratios of PFK and dialdehyde-ATP to solve simultaneous equations. The same results were obtained if the A,,, of bound dialdehyde was determined from 14Cincorporation. Preparation of dialdehyde-ATP. Dialdehyde-ATP was prepared as described by Easterbrook-Smith et al. (28) except that the sodium periodate cleavage was carried out at pH 8 instead of pH 7. [14C]DialdehydeATP was prepared by adding 20 &i of [14C]ATP to the cold ATP (0.1 mmol) in the reaction mixture. Reactions were monitored by thin-layer chromatography on polyethyleneimine-cellulose sheets (0.8 M ammonium bicarbonate as solvent) as described by EasterbrookSmith et al. (28). ATP was cochromatographed as a standard and the spots located by uv light. The concentration of dialdehyde-ATP in deionized water solutions was determined spectrophotometrically at 258 nm using l hl = 14,900 cm-’ M-’ (29). Modijication oj’ PFK with dialdehyde-ATP. PFK was dialyzed against borate (pH 8.6). An aliquot of a stock solution of dialdehyde-ATP (usually 3.2 mM in water) was then added to a portion of the dialyzed enzyme. A control experiment was conducted by adding an equivalent amount of borate buffer (pH 8.6) or water to another portion of dialyzed PFK. After a 30to 35-min incubation at room temperature, modified PFK and the control sample were dialyzed against Tris-phosphate (pH 8.0) or borate-FDP (pH 8.0). In some cases, sodium borohydride was added before dialysis; after the 30- to 35-min incubation at room temperature, both solutions were chilled to O’C, and an aliquot of a sodium borohydride solution (0.1 or 0.4 M) was added to each. Both modified PFK and the control sample were assayed following dialysis. An alternative method of removing excess dialdehyde-ATP was also used. Dialdehyde-modified PFK (6 ml) was passed through a charcoal bead column (0.7 x 15 cm) until a constant A279/A259ratio was obtained. The control solution (6 ml) was also passed through a charcoal column of the same length. Charcoal has been used by other workers (27) to adsorb bound ATP from PFK. The control and dialdehyde-modified PFK were assayed at pH 8.0 following charcoal treatment. The concentration dependence of the inactivation was determined by incubating PFK with several concentrations of dialdehyde-ATP at pH 8.6; aliquots were withdrawn for assay during each inactivation. Protection against dialdehyde-mediated inactivation of PFK was determined by incubation of PFK with each effector compound (5.3 mM) for 10 min prior to addition of dialdehyde-ATP (0.2 mM). Aliquots were assayed for PFK activity during the course of each reaction.
DIALDEHYDE-ATP
INACTIVATION
OF PHOSPHOFRUCTOKINASE
201
Zncorporation of [Wldialdehyde-AZ’P. PFK (0.4-4 mg/ml) was modified with [%]dialdehyde-ATP (0.6 mM) in borate (pH 8.6) or Tris-HCl (pH 8.6) as described above. Modified PFK and the control sample were assayed following charcoal treatment or borohydride addition and dialysis. Aliquots of each protein solution were then diluted to 5 ml with deionized water before adding 5 ml of Instagel. A sample containing dialdehyde-ATP alone was included as a standard when counting samples. A molecular weight of 340,000(85,000 per protomer) for PFK was used in calculations of [Wldialdehyde-ATP incorporation (30). Reaction of PFK and dialdehyde-inactivated PFK with dinitrojkorobenzene. PFK (0.5 mg/ml) was
modified with dialdehyde-ATP (0.6 mM) in borate (pH 8.6). After 30 min both the control solution and dialdehyde-modified PFK were dialyzed against 1 liter of 0.1 mM borate (pH 8.0) containing 0.2 mM EDTA (two changes). Then 2 ml of a DNFB solution (0.1 ml DNFB in 10 ml of acetone) was added to 2 ml of each PFK solution at 0°C (control and dialdehyde-inactivated PFK). Both mixtures were brought to room temperature and the reactions were allowed to proceed for ‘70 min. The DNFB was extracted with ethyl ether (four times) before dialyzing both solutions against 0.01 M HCl. The 0.01 M HCl was removed by lyophilization and each sample was hydrolyzed in 6 N HCl, 110°C for 24 h in sealed evacuated tubes. Basic amino acid content of each sample was determined following chromatography on a 17-cm column (PA-35 resin) equilibrated in citrate, pH 5.26 (sodium concentration, 0.35 N). Analytical
ultmcenttifugation. PFK (3-5 mg/ml) was inactivated with dialdehyde-ATP (1.0 mM) in borate buffer (pH 8.6) as described above. After addition of sodium borohydride (1.9 mM) to the control solution and to the reaction mixture containing dialdehyde-ATP, both solutions were dialyzed against borateFDP (pH 8.0). The control sample of PFK and the modified enzyme were then centrifuged simultaneously at 48,000 rpm (An-D rotor), 20°C using double sector cells (12-mm centerpiece, 4” sector), one with a quartz window and one with a + 1” wedge window. Schlieren optics were used to monitor sedimentation velocity and photographs were taken at 4-min intervals (Kodak metallographic plates, Type II) at a diaphragm angle of 50”. Schlieren peak distance to the reference line was measured using a Nikon microcomparator equipped with a Goertner digital readout. Sedimentation constants were determined graphically according to the procedure of Schachman (31).
RESULTS
Inactivation
of PFK in Tris Buffer
PFK (0.5 mg/ml) lost more than 90% of its activity (control activity = 128 U/mg) when
MINUTES
OF
INCUBATION
FIG. 1. Time course of the inactivation of PFK with dialdehyde-ATP in borate buffer (pH 8.6).
incubated with 0.5 mM dialdehyde-ATP in Tris-HCl (pH 8.6). Sodium borohydride (13 mM) was added to the control sample and dialdehyde-inactivated PFK before analysis (Tris-phosphate, pH 8.0) in order to reduce Schiff base linkages which might have been formed. Following dialysis, dialdehyde-modified PFK was 25% as active as the control sample of enzyme (117 U/mg). Inactivation
of PFK in Borate Buffer
Figure 1 summarizes the result of experiments in which PFK (0.5 mg/ml) was incubated with 0.2, 0.4, or 0.6 mM dialdehydeATP in borate buffer (pH 8.6). Incubation of PFK with 0.6 mM dialdehyde-ATP for 30 min at 22°C resulted in essentially complete inactivation (99%). The activity of a control sample of enzyme was 114- 130 U/mg. The modified PFK remained inactive (1% of control activity) following dialysis of the reaction mixture (4 days) against borate-FDP, pH 8.0 (control activity = 114-130 U/mg). In these experiments, sodium borohydride was not added before dialysis so that the effect of borate buffer on the inactivation process could be assessed. The results were the same whether excess reagent was removed by dialysis or by passing the reaction mixture through a charcoal bead column. However, the charcoal beads adsorbed 40-50% of the PFK in addition to the excess
GREGORY AND KAISER
202 TABLE I
PROTECTION FROM DIALDEHYDE-ATP-MEDIATED INACTIVATION OF PFK
Compound added”
PFK activity* (% of control)
None CAMP F6P FDP AMP ATP ADP ATP + F6P
16 26 28 31 33 52 62 67
a The concentration of dialdehyde-ATP was 0.2 mM; each effector had a concentration of 5.3 mM. b PFK was 0.5 mg/ml in Tris-HCl, pH 8.6.
reagent. PFK inactivated in borate buffer (pH 8.6) and then dialyzed for 2 days against Tris-phosphate buffer (pH 8.0) regained 8% activity. Protection against Dialdehyde-Mediated Inactivation of PFK
The effects of substrates and activators on the inactivation of PFK were studied. Table I summarizes the results of experiments in which PFK (0.5 mg/ml in TrisHCl, pH 8.6) was incubated with effector compounds (concentration of each, 5.3 mM) for 10 min prior to addition of dialdehydeATP (0.2 InM). The column on the right lists PFK activity, as a percentage of control activity, 10 min after addition of dialdehydeATP. Adenosine diphosphate or ATP partially prevented inactivation by dialdehydeATP at pH 8.6 but F6P, cyclic 3’,5’-adenosine monophosphate, or FDP provided little protection from inactivation. Incorporation
of [14C]Dialdehyde-ATP
PFK protomer. PFK inactivated in Tris buffer and dialyzed without borohydride treatment incorporated 2.6 mol of 14C-reagent per protomer (79% loss of activity). However, in order to insure removal of “nonspecifically” bound 14C-reagent, 6.6 mM ATP was added to inactivated and control samples after 2 days of dialysis, and dialysis was continued for 2 days more. Incorporation of [14C]dialdehyde-ATP following inactivation of PFK in Tris-HCl (pH 8.6) and dialysis against 0.01 M HCl was 3.3 + 0.1 mol of 14C-reagent per protomer. The PFK activity could not be measured because of the acid treatment. Removal of excess [14C]dialdehyde-ATP upon inactivation of PFK in borate buffer was accomplished by dialysis against borate-FDP (pH 8.0) or by charcoal treatment of the reaction mixtures. Results from experiments in which excess reagent was removed by charcoal treatment showed that 99% inactivation (control activity = 115120 U/mg) of PFK corresponded to incorporation of 3.7 & 0.2 mol of reagent per PFK protomer. Reactions in which dialysis was performed to remove excess reagent were terminated by adding sodium borohydride (0.66 mM) before dialysis. Addition of sodium borohydride resulted in the enzyme regaining some of its activity (a few percent) as measured following dialysis. Dialysis for 7 days in the pH 8.0 borate-FDP buffer was necessary to remove “nonspecifically” bound reagent. Removal of excess reagent was facilitated if the PFK concentration was 4 mg/ml rather than 0.5 mg/ml. A 94% loss of activity (control activity = 130 U/mg) corresponded to incorporation of 3.6 -+ 0.2 mol of reagent per PFK protomer. Hansske et al. (29) reported that the half-life for the decomposition of dialdehyde-AMP at pH 7.0, 4”C, was 17 days. However, the incorporation of [14C]dialdehyde-ATP in PFK was practically the same whether excess reagent was removed by charcoal treatment or by dialysis.
Incorporation of [ 14C]dialdehyde-ATP was measured following incubation of PFK (in Tris or borate buffer) with 0.6 KIM 14Creagent for 30-35 min. PFK inactivated in Determination of the Identity of the Tris buffer was dialyzed (4 days) against Modi$.ed Amino Acid Residues Tris-phosphate (pH 8.0) following addition of 13 mM sodium borohydride. A 75% loss of Lysine was a logical choice for the amino activity corresponded to incorporation of acid residue(s) modified by dialdehyde2.4 f 0.2 mol of [14C]dialdehyde-ATP per ATP (28). Sodium borohydride was added to
DIALDEHYDE-ATP
INACTIVATION
modified PFK before dialysis against Trisphosphate or before amino acid analysis in order to reduce any Schiff base linkages formed. However, dialysis of a borohydridetreated reaction mixture against Trisphosphate (pH 8.0) resulted in partial recovery of activity. Furthermore, amino acid analyses of control and dialdehyde-modified PFK showed no differences regardless of whether or not borohydride treatment was performed. Another possibility for the amino acid residue(s) modified by dialdehyde-ATP was arginine. Riordan et al. (32) found that PFK is rapidly inactivated in borate buffer by 2,3-butanedione. Indeed, chemical modification with 2,3-butanedione, phenylglyoxal, or 1,Zcyclohexanedione has indicated that arginine residues are present in the substrate binding sites of a number of glycolytic enzymes (32-36). Modification of arginine by a-dicarbonyl reagents can be detected by amino acid analysis. Although the two carbony1 groups in dialdehyde-ATP are not a to each other, it seemed possible that arginine could also be modified by this reagent. Accordingly PFK was modified with dialdehyde-ATP (0.6 mM) in borate buffer (pH 8.6); the modified enzyme and a control sample were then dialyzed against 0.01 N HCl prior to lyophilization and 6 N HCl hydrolysis. Amino acid analyses showed no differences in arginine content between dialdehyde modified PFK and the control sample. In order to examine further whether lysine in PFK was modified by dialdehydeATP, an indirect method was used. Since borate buffer appeared to stabilize the dialdehyde-ATP-modified form of PFK, both modified PFK and the control sample were treated with DNFB in borate (pH 8.0) to modify free lysine residues. If lysine residues were modified by dialdehyde-ATP, these residues would be protected from the reaction with DNFB. Reversal of the dialdehyde modification during hydrolysis with 6 N HCl prior to amino acid analysis would result in regeneration of free lysine. Amino acid analysis of a control sample of PFK and of the dialdehyde-modified enzyme (both treated with DNFB) should then show a net difference in lysine content corresponding to the number of lysines (if any) modified by
203
OF PHOSPHOFRUCTOKINASE TABLE
II
LYSINE CONTENT OF PFK REACTED WITH DNFB Treatment Dialdehyde-ATP, DNFB
Lysineiprotomer DNFB
4.2 + 0.1 0.6 + 0.1
dialdehyde-ATP. To achieve good resolution, a 1’7-cm column was used to separate the basic amino acids (rather than the 5-cm column normally used). Reaction of proteins with DNFB can result in modification of thiol, c-amino, a-amino, imidazole, and phenolic groups; on the 17-cm column imdinitrophenyl histidine is resolved from lysine (37). The number of lysines remaining following modification with DNFB was calculated employing the arginine and lysine content (53 and 43 residues, respectively, per protomer of 85,000 M,) of rabbit muscle PFK, as reported by Walker et al. (38) and the ratio of arginine to lysine measured in our chromatograms. The analytical results we obtained for lysine are listed in Table II. As mentioned previously, amino acid analyses of dialdehyde-modified PFK and the control sample showed no difference in arginine content. Analytical
Ultracentrifugation
Modified PFK and the control sample were dialyzed against borate-FDP (pH 8,0), following addition of 1.9 InM sodium borohydride (to reduce excess dialdehyde). Enzymatic assay of dialdehyde-modified PFK before centrifugation revealed 94% inhibition compared to the control (115 U/mg). Figure 2 shows Schlieren patterns of control and inactivated PFK (5 mglml) 23 min after centrifugation was begun. The control sample of enzyme (upper) shows three peaks with sedimentation coefficients of 12, 18, and 25 S. The dialdehyde-inactivated PFK shows two peaks with sedimentation constants of 12 and 18 S. The 25 S species is present only to a small extent, if at all. DISCUSSION
Despite the vast amount of work on PFK, very little is known about the chemistry of the ATP binding sites. Evidence has been
204
GREGORY AND KAISER
presented suggesting that each PFK protomer has three different types of nucleotide binding sites; a catalytic site, a site which binds activators such as AMP, or ADP, and a site which binds ATP as an inhibitor (1% 20, 39-41). PFK has been modified with a number of sulfhydryl reagents, but the resulting inactivation is generally incomplete and varies with the reagent used (42). ATP protects against reaction of the most reactive thiol group with reagents such as 5,5’-dithiobis (Znitrobenzoic acid) and a-bromo-4-hydroxy3-nitroacetophenone, but Kemp (43) and Schwartz et aZ.(41) propose that ATP protects by causing a conformational change which makes the sulfhydryl group inaccessible. In 1973, Bloxham et aZ.(44) reported
that 6-mercapto-9+Dribofuranosylpurine5’-triphosphate completely inactivated rabbit muscle PFK upon incorporation of 6-8 mol of reagent per tetramer (380,000 M,). However, F6P provided greater protection against the inactivation reaction than did ATP. In the present paper, we have reported the results of the modification of rabbit muscle PFK with 2’,3’-dialdehyde-ATP. Incubation of PFK with dialdehyde-ATP in Tris buffer at pH 8.6 resulted in rapid inactivation. However, inactivation of PFK in Tris buffer was incomplete and partially reversible upon dialysis of a borohydridetreated reaction mixture. Later experiments showed that PFK was rapidly and essentially completely inactivated by dial-
FIG. 2. Analytical ultracentrifugation of a control sample of PFK (upper) and of the dialdehydemodified enzyme (lower) (5 mg/ml) at 48,000 rpm, 23 min after reaching speed.
DIALDEHYDE-ATP
INACTIVATION
dehyde-ATP in borate buffer at pH 8.6. Furthermore, activity was not regained upon dialysis of the reaction mixture against borate-FDP buffer, pH 8.0. These results are evidence that borate buffer stabilizes the modification of PFK with dialdehydeATP, since sodium borohydride was not added to modified PFK before dialysis against borate-FDP. The additional activity loss in borate was not caused by the change in buffer (from Tris to borate) since PFK inactivated in borate and then dialyzed against Tris regained 8% activity. Experiments with [14C]dialdehyde-ATP showed that 94-99% inactivation of PFK corresponded to incorporation of 3.6 -t 0.2 mol of reagent per PFK protomer. Excess dialdehyde-ATP was removed by charcoal or by dialysis so that loss of PFK activity could be directly related to incorporation of reagent. The incorporation of modifying agent was practically the same regardless of whether excess reagent was removed by charcoal treatment or dialysis. Kemp and Krebs (21) found that 3 mol of ATP were bound by each rabbit muscle PFK protomer (pH 6.95) and Lorenson and Mansour (22) found that 3.6 mol of ATP were bound by each sheep heart PFK protomer (pH 6.65). However, the inactivation of PFK with dialdehyde-ATP was performed at pH 8.6 where inhibition by ATP does not occur. Determination of the amino acid residues modified by dialdehyde-ATP was accomplished using an indirect method since amino acid analyses of acid hydrolyzates of dialdehyde-ATP inactivated PFK and of control samples showed no differences in lysine or arginine content. In order to determine whether lysine residues were modified by dialdehyde-ATP, the amino acid contents of dialdehyde-inactivated enzyme and control samples were compared following reaction with DNFB. Since hydrolysis with 6 N HCl regenerated the lysines modified by the dialdehyde reagent, a difference of 3.6 lysine residues between the dialdehydeinactivated enzyme and control samples was found. This difference which indicates that three or four lysines are modified per protomer, is in excellent agreement with the results of the incorporation of [14C]
205
OF PHOSPHOFRUCTOKINASE 4-(O,P),OCHz
a.
Ad
Hr”lH I’N’I HO
‘-OSPOCH2
OH
b.
Hr’-tH I’N’I HO AHOH
(CH2)4
I HniCHC02
Ad
L=o 0
’ \
I
FIG. 3. (a) Proposed structure for dialdehyde-ATPlysine derivative. (b) Morpholine derivative isolated from reaction of benzoic acid hydrazide with dialdehyde-AMP (29).
dialdehyde-ATP in the enzyme. The lysine residues modified could react with the dialdehyde-ATP reagent in two possible ways. The first possibility considered was Schiff base formation, as has been observed, for instance, in the modification of pyruvate carboxylase (28) and histone kinase (45) by dialdehyde-ATP. However, this possibility was ruled out for the following reasons. Based on amino acid analyses and activity assays of modified PFK following dialysis in Tris buffer, evidence was obtained that treatment of modified PFK with sodium borohydride did not result in covalently modified lysine. Furthermore, the modified form of PFK obtained by treatment with dialdehyde-ATP is stable in 0.01 N HCl (based on 14C incorporation results), a result inconsistent with the lability expected for a Schiff base linkage. As an alternative, we propose that the product of the reaction of dialdehyde-ATP with lysine in PFK has the dihydroxy morpholine-type structure shown in Fig. 3a. If this structure is correct, the stabilization by borate buffer of the modified form of PFK can be understood since borate could complex the dihydroxy morpholinelike adduct in a manner similar to that observed in arginine modifications with butane dione (46). Hansske et aZ.(29) and Hansske and Cramer (47) have found that carboxylic acid hydrazides react with dialdehyde-AMP to give stable dihydroxy morpholine derivatives (Fig. 3b). Therefore, on the basis of our observations combined with those of Hansske et d(29) it seems reasonable to postulate that the lysine residues of PFK react with dialdehyde-
206
GREGORY
AND KAISER
ATP to form morpholine-like adducts, a result previously not observed upon reaction of lysine residues of protein with aldehyde reagents. The results from the protection experiments show that although neither ADP nor ATP provides full protection against inactivation, these nucleotides do provide the best protection. Since 3-4 mol of the dialdehyde reagent bind irreversibly to PFK, the K, value for the reagent has not been assessed. However, the structure of dialdehyde-ATP and ATP may be similar enough that the dialdehyde species can compete effectively with ATP for ATP binding sites on PFK. Dialdehyde nucleosides exist in several hydrated forms in solution (47) and the structure of the hemiacetal or cis-dihydroxydioxane-type form of dialdehyde-ATP does indeed resemble that of ATP. This form of the reagent may be stabilized by borate buffer. Another possible explanation of the results of the protection experiments is that the lysine residues involved in the modification reaction are near, but not precisely at the ATP binding sites. It is also conceivable that during the inactivation of PFK dialdehyde-ATP binds in a different mode than that in which ATP normally binds. Table I shows that F6P, FDP, or AMP provide very little protection from inactivation by dialdehyde-ATP. This differs from what has been observed in the modification of PFK with pyridoxal phosphate. Uyeda (48) studied the inactivation of rabbit muscle PFK with pyridoxal phosphate and found that F6P provided the best protection from inactivation; AMP or FDP provided some protection against the inactivation process. Setlow and Mansour (40) inactivated sheep heart PFK with pyridoxal phosphate and found that the best protection was afforded by FDP with some protection by ATP. If the same lysine residues are reacting with pyridoxal phosphate and dialdehyde-ATP, the difference in the results of the protection experiments could indicate that the binding sites for the two reagents are distinct, yet close. Analytical ultracentrifugation studies were performed on dialdehyde-ATP inactivated PFK and control samples of the enzyme
to determine whether inactivation was caused by dissociation of the enzyme. Enzymatically active PFK is composed of four identical subunits of molecular weight 85,000 + 10,000, as measured by sedimentation equilibrium and sedimentation velocity (30). However, the tetrameric form of the enzyme can be in rapid equilibrium with higher molecular weight aggregates as well as with inactive dimers, resulting in a multicomponent sedimentation pattern (24, 27, 49). This aggregational behavior is affected by protein concentration, pH, and the ligands present (26, 50). FDP stabilizes the active tetrameric form of PFK (26) and was included in our analytical ultracentrifugation studies because PFK is not stable in borate buffer over long periods of time. The three peaks observed (Fig. 2) upon ultracentrifugation of the control sample had sedimentation coefficients of 12, 18, and 25 S. These results agree with studies reported by Leonard and Walker (30) on the subunit structure of PFK in 0.1 M Tris-phosphate (pH 8.0) containing 0.5 mM FDP. They found that ultracentrifugation of PFK (3-5 mg/ml, 20%) resulted in three Schieren peaks with sedimentation coefficients of 13, 18, and 27 S. The 13 S species corresponded to the tetramer, and the 18 and 27 S species were higher molecular weight aggregates. Centrifugation of dialdehyde-ATP modified PFK resulted in two Schlieren peaks with sedimentation coefficients of 12 and 18 S. However, the 25 S species is largely absent, indicating that dialdehyde-ATP modified PFK is more dissociated than the control enzyme (borate-FDP, pH 8.0). Modification of PFK with dialdehyde-ATP thus affects the aggregational behavior of the enzyme, but the observed inactivation is not caused by an inability of the modified enzyme to form the 340,000 M, tetramer. In conclusion, dialdehyde-ATP rapidly inactivates PFK by a mechanism other than dissociation of the 340,000 M, tetramer. ATP and ADP partially protect PFK from inactivation by dialdehyde-ATP, but F6P, FDP, or AMP, which protect PFK from modification by pyridoxal phosphate, provide little protection from modification by the dialdehyde reagent. This reagent modifies three to four lysine residues per PFK
DIALDEHYDE-ATP
INACTIVATION
protomer. These lysine residues, which we believe are important to the catalytic and/or ATP binding properties of PFK, most probably react with dialdehyde-ATP to form dihydroxymorpholine derivatives rather than Schiff bases. ACKNOWLEDGMENTS We are grateful to Dr. Y. Nakagawa, Division of Renal Medicine, Michael Reese Hospital, for many helpful discussions, to Professor Angelo Scanu, Depsrtments of Medicine and Biochemistry, for allowing us to use the analytical ultracentrifuge, and to Mr. Frank Buckingham for sacrificing the rabbits. REFERENCES 1. STADTMAN, E. R. (1966) A&. Enzymol. 28, 41-154. 2. KOERNER, T. A. W., JR., YOUNATHAN, E. S., ASHOUR, A. L. E., AND VOLL, R. J. (1974) J. Biol. Chem. 249, 5749-5754. 3. FISHBEIN, R., BENKOVIC, P. A., SCHRAY, K. J., SIEWERS, I. H., STJZFFENS,J. J., ANDBENKOVIC, S. J. (19’74) J. Biol. Chem. 249, 6047-6051. 4. SCHRAY, K. J., AND BENKOVIC, S. J. (1978) Accounts Chem. Res. 11, 136-141. 5. LARDY, H. A., AND PARKS, R. E. (1956) in Enzymes: Units of Biological Structure and Function (Gaebler, 0. H., ed.), pp. 584-587, Academic Press, New York. 6. PASSONEAU, J. V., AND LOWRY, 0. H. (1962) Biochem. Biophys. Res. Commun. 7, 10-15. ‘7. PASSONEAU, J. V., AND LOWRY, 0. H. (1963) Biochem. Biophys. Res. Commun. 13,372-379. 8. VIEIUELA, E., SALES, M., AND SOLS, A. (1963) Biochem. Biophys. Res. Commun. 12,140-145. 9. MANSOUR, T. E. (1963) J. Biol. Chem. 238, 2285-2292. 10. MANSOUR, T. E. (1972) Curr. Top. Cell. Regul. 5, l-46. 11. UYEDA, K., ANDRACKER, E. (1965)J. Biol. Chem. 240, 4682-4688. 12. LOWRY, 0. H., AND PASSONEAU, J. V. (1966) J. Biol. Chem. 241, 2268-2279. 13. SALAS, M. L., SALAS, J., AND SOLS, A. (1968) Biochem. Biophys. Res. Commun. 31,461-466. 14. AHLFORS, C. E., AND MANSOUR, T. E. (1969) J. Biol. Chem. 244, 1247-1251. 15. CHAPMAN, A., SANNER, T., AND PIHL, A. (1969) Eur. J. Biochem. 7, 588-593. 16. SETLOW, B., AND MANSOUR, T. E. (197O)J. Bid. Chem. 245, 5524-5533. 17. BRUNSWICK, D. J., AND COOPERMAN, B. S. (1971) Proc. Nat. Acad. Sci. USA 68, 1801-1804. 18. PETTIGREW, D. W., AND FRIEDEN, C. (1978) J. Biol. Chem. 253, 3623-3627.
OF PHOSPHOFRUCTOKINASE
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19. MANSOUR, T. E., AND MARTENSEN, T. M. (1978) J. Biol. Chem. 253, 3628-3634. 20. MANSOUR, T. E., AND COLMAN, R. F. (1978) Biochem. Biophys. Res. Commun. 81, 13701376. 21. KEMP, R. G., AND KREBS, E. G. (1967) Biochemistv 6, 423-434. 22. LORENSON, M. Y., AND MANSOUR, T. E. (1969) J. Biol. Chem. 244, 6420-6431. 23. KEMP, R. G., AND FOREST, P. B. (1968) Biochemistry 7, 2596-2603. 24. LANG, K.-H., MARCUS, F., AND LARDY, H. A. (1965) J. Biol. Chem. 240, 1893-1899. 25. LING, K.-H., PAETKAU, V., MARCUS, F., AND LARDY, H. A. (1966) in Methods in Enzymology (Wood, W. A., ed.), Vol. 9, pp 425-429, Academic Press, New York. 26. LAD, P. M., HILL, P. E., AND HAMMES, G. G. (1973) Biochemistry 12, 4303-4309. 27. PARMEGGIANI, A., LUFT, J. H., LOVE, D. S. AND KREBS, E. G. (1966) J. Biol. Chem. 241, 4625-4637. 28. EASTERBROOK-SMITH, S. B., WALLACE, J. C., AND KEECH, B. D. (1976) Eur. J. Biochem. 62, 125-130. 29. HANSSKE, F., SPRINZL, M., AND CRAMER, F. (1974) Bioorg. Chem. 3, 367-376. 30. LEONARD, K. R., AND WALKER, I. 0. (1972) Eur. J. Biochem. 26, 442-448. 31. 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. 32. RIORDAN, J. F., MCELVANY, K. D., ANDBORDERS, C. L., JR. (1977) Science 195, 884-886. 33. LANGE, L. G., III. RIORDAN, J. F., ANDVALLEE, B. L. (1974) Biochemistry 13, 4361-4370. 34. YANG, P., AND SCHWERT, G. W. (1972) Biochemistry 11, 2218-2224. 35. NAGRADOVA, N. K., AND ASYRANTS, R. A. (1975) Biochim. Biophys. Acta 386, 365-368. 36. ROGERS, K., AND WEBER, B. H. (1977) Arch. Biochem. Biophys. 180, 19-25. 37. HIRS, C. H. W. (ed.) (1976) in Methods in Enzymology, Vol. 11, pp. 548-555, Academic Press, New York. 38. WALKER, I. D., HARRIS, J. I., RUNSWICK, M. J., AND HUDSON, P. (1976) Eur. J. Biochem. 68, 255-269. 39. MATHIAS, M., AND KEMP, R. G. (1972) Biochemistry 11, 578-584. 40. SETLOW, B., ANDMANSOUR, T. E. (1972)Biochim. Biophys. Acta 258, 106-112. 41. SCHWARTZ, K. J., NAKAGAWA, Y., AND KAISER, E. T. (1976) J. Amer. Chem. Sot. 98, 63696378. 42. BLOXHAM, D. B., AND LARDY, H. A. (1972) in The Enzymes (Boyer, P. D., ed.), 3rd ed.,
208
GREGORY
Vol. VIII, pp. 239-278, Academic Press, New York. 43. KEMP, R. G. (1969) Biochemistry 11, 4490-4496. 44. BLOXHAM, D. B., CLARK, M. G., HOLLAND, P. C., AND LARDY, H. A. (1973) Biochemistry 12, 1596-1601. 45. KOCHETKOV, S. N., BULARGINA, T. V., SASHCHENCKO, L. P., AND SEVERIN, E. S. (1977) Eur. J. Biochem. 81, 111-118.
AND KAISER 12, 391546. RIORDAN, J. F. (1973) Biochemistry, 3923. 47. HANSSKE, R., AND CRAMER, F. (1977) Carbohyd. Res. 54, 75-84. 8, 2366-2373. 48. UYEDA, K. (1969) Biochemistv 49. PAETKAU, V., AND LARDY, H. A. (1967) J. Biol. Chem. 242, 2035-2042. 50. AARONSON, P. P., ANDFRIEDEN, C. (1972)J. Biol. Chem. 247, 7502-7509.
Inactivation of Phosphofructokinase
by Dialdehyde-ATP’
MARTHA R. GREGORY2 AND E. T. KAISER Department
of Chemi.stry,
University of Chicago, Chicago, Illinois 60657
Received January 2, 1979 Rabbit muscle phosphofructokinase (PFK) is rapidly inactivated by a 2’,3’-dialdehyde derivative of adenosine triphosphate (dialdehyde-ATP). When allowed to react with 0.6 mM dialdehyde-ATP in 0.1 M borate buffer (pH 8.6) containing 0.2 mM EDTA and 0.5 mM dithiothreitol, PFK loses essentially all activity (99%) in 30 min. The modified PFK remains inactive following dialysis of the reaction mixture against sodium borate (pH 8.0) containing fructose diphosphate, EDTA, and dithiothreitol. Experiments with [“Cldialdehyde-ATP show that 99% inactivation of PFK corresponds to incorporation of 3 to 4 mol of the ATP analog per PFK protomer. The inactivation of PFK with dialdehyde reagent is not caused by dissociation of the 340,000 M, tetramer to the 170,000 M, dimer, as determined by analytical ultracentrifugation. Adenosine diphosphate or ATP protect PFK from inactivation by dialdehyde-ATP at pH 8.6, but fructose g-phosphate, cyclic 3’,5’-adenosine monophosphate, or fructose diphosphate, which protect PFK from modification by pyridoxal phosphate, provide little protection from inactivation. Amino acid analyses of dialdehyde-inactivated PFK and of a control sample of the enzyme were compared following reaction of each with 2,4-dinitrofluorobenzene. The results show that three or four lysine residues per PFK protomer are modified by dialdehyde-ATP. Additional data indicate that these lysine residues react with dialdehyde-ATP to form dihydroxymorpholine-like adducts rather than Schiff bases.
ate, phosphocreatine, and phosphoenolpyruvate are inhibitors (6-11). Furthermore, kinetic data and chemical modification studies are consistent with the hypothesis that there are separate catalytic and regulatory sites on PFK (12, 20). Although PFK is an important regulatory enzyme in glycolysis, little is known about the composition of the ATP binding sites on the enzyme. Kemp and Krebs (21) reported that rabbit muscle binds 3 mol of ATP per protomer and Lorenson and Mansour (22) reported that sheep heart PFK binds 3.6 mol of ATP per protomer. In the present communication, we report on the modification of rabbit muscle PFK I Supported by National Institutes of Health Research with a 2’,3’-dialdehyde derivative of ATP, Grant GM 19037 (E.T.K.) and National Institutes of a study undertaken with the objective of Health Postdoctoral Fellowship AM 05667 (M.R.G.). identifying basic amino acid residues in the 2 Present address: Department of Pharmacology, catalytic and/or binding sites for ATP. The University of Rochester Medical Center, Rochester, inactivation of PFK with dialdehyde-ATP N. Y. 14642. is discussed with respect to incorporation 3 Abbreviations used: PFK, rabbit muscle phosof 14C-labeled reagent, protection by subphofructokinase; F6P, fructose &phosphate; FDP, strates, and amino acid residues modified. fructose l,&diphosphate; DTT, dithiothreitol; DNFB, A comparison of the modified enzyme and a 2,4-dinitrofluorobenzene.
Phosphofructokinase (PFK)3 catalyzes one of the controlling steps in glycolysis, transfer of the terminal phosphate of adenosine triphosphate (ATP) to the C-l hydroxyl of the pfuranose anomer of fructose 6-phosphate (l-4). The importance of this practically irreversible reaction in the regulation of glycolysis is underscored by the observation that a number of metabolites regulate PFK activity (1, 5). Adenosine monophosphate (AMP), adenosine diphosphate (ADP), fructose 6-phosphate (F6P), and fructose 1,6-diphosphate (FDP) are activators of PFK, while citrate, ATP, 3-phosphoglycer-
199
0003-9861/79/090199-10$02.00/O Copyright 6 1979by AcademicPress, Inc. AU rights of reproductionin any form reserved.
GREGORY AND KAISER
control sample of native PFK by analytical ultracentrifugation is also presented. EXPERIMENTAL
PROCEDURE
Materials and methods. NADH (ChromatoPure) was obtained from P-L Biochemicals. All other substrates and effecters for PFK were obtained from Sigma Chemical Company. DEAE-Cellulose (DE-52) used in purification of PFK was obtained from Whatman. [&l’C]ATP, tetrasodium salt (60 mCi/mmol), and Instagel were obtained from New England Nuclear and Packard Instrument Company, Inc., respectively. The charcoal utilized to adsorb dialdehyde-ATP was a product ofTaiyo Kaken Company, Ltd., Tokyo, Japan. Amino acid analyses were carried out with a Beckman Model 121automatic amino acid analyzer. A Beckman Acta MVI spectrophotometer was used for determination of PFK concentration and activity and a Packard Tri-Carb scintillation spectrometer was utilized for counting samples in Instagel. Analytical ultracentrifugation studies were conducted using a Beckman Spinco Model E ultracentrifuge. Buffers used for the modification of PFK with dialdehyde-ATP and for dialysis of the resulting reaction mixtures were 0.1 M Tris-HCl (pH 8.6), 0.1 M sodium borate (pH 8.6), 0.1 M Tris-phosphate (pH 8.0), and 0.1 M sodium borate-O.2 mM FDP (pH 8.0), all containing 0.2 mM EDTA and 0.5 mM dithiothreitol (DTT). These buffers are referred to in the text as Tris-HCl (pH 8.6), borate (pH 8.6), Tris-phosphate (pH 8.0), and borate-FDP (pH 8.0), respectively. Modifications of PFK with DNFB were carried out in 0.1 M borate containing 0.2 mM EDTA only. The errors in determination of dialdehyde incorporation are expressed as standard deviations. PFK was puriPurification of phosphofructokinase. fied according to Kemp and Forest (23) with the following modification. After a 24-h dialysis against 50 mM /3-glycerophosphate, 2 mM ATP, pH 7.2, containing ammonium sulfate at 32% saturation, the supernatant enzyme was dialyzed against 0.1 M Tris-phosphate, 0.2 mM EDTA, 0.2 mM FDP, pH 8.0. The resulting solution was chromatographed on DEAE-cellulose as described by Ling et al. (24). PFK was stored as a suspension in 50 mM P-glycerophosphate, 2 mM EDTA, 0.5 mM DTT, 1 mM FDP, pH 8.0, containing ammonium sulfate at 50% saturation. Purified PFK exhibited an A219/A259 ratio of 1.7. PFK activity was routinely 130-140 U/mg where 1 unit (U) of PFK catalyzes the conversion of 1 pmol of F6P to FDP per minute at pH 8.0, 25°C. Assay of PFK activity. PFK activity was measured spectrophotometrically at pH 8.0, 25”C, in an assay coupled with aldolase, triosephosphate isomerase, and a-glycerophosphate dehydrogenase according to the procedure of Ling et al. (25) as modified by Lad et al. (26). PFK concentration was determined spectrophoto-
metrically using E@” = 1.02 mg-’ ml (27). The AzT9 of the dialdehyde-modified enzyme was determined by subtracting the contribution (at 279 nm) of the incorporated dialdehyde-ATP from the observed absorbance at 279 nm. The A,,, of the incorporated dialdehyde was calculated by utilizing the A2,9/A259 ratios of PFK and dialdehyde-ATP to solve simultaneous equations. The same results were obtained if the A,,, of bound dialdehyde was determined from 14Cincorporation. Preparation of dialdehyde-ATP. Dialdehyde-ATP was prepared as described by Easterbrook-Smith et al. (28) except that the sodium periodate cleavage was carried out at pH 8 instead of pH 7. [14C]DialdehydeATP was prepared by adding 20 &i of [14C]ATP to the cold ATP (0.1 mmol) in the reaction mixture. Reactions were monitored by thin-layer chromatography on polyethyleneimine-cellulose sheets (0.8 M ammonium bicarbonate as solvent) as described by EasterbrookSmith et al. (28). ATP was cochromatographed as a standard and the spots located by uv light. The concentration of dialdehyde-ATP in deionized water solutions was determined spectrophotometrically at 258 nm using l hl = 14,900 cm-’ M-’ (29). Modijication oj’ PFK with dialdehyde-ATP. PFK was dialyzed against borate (pH 8.6). An aliquot of a stock solution of dialdehyde-ATP (usually 3.2 mM in water) was then added to a portion of the dialyzed enzyme. A control experiment was conducted by adding an equivalent amount of borate buffer (pH 8.6) or water to another portion of dialyzed PFK. After a 30to 35-min incubation at room temperature, modified PFK and the control sample were dialyzed against Tris-phosphate (pH 8.0) or borate-FDP (pH 8.0). In some cases, sodium borohydride was added before dialysis; after the 30- to 35-min incubation at room temperature, both solutions were chilled to O’C, and an aliquot of a sodium borohydride solution (0.1 or 0.4 M) was added to each. Both modified PFK and the control sample were assayed following dialysis. An alternative method of removing excess dialdehyde-ATP was also used. Dialdehyde-modified PFK (6 ml) was passed through a charcoal bead column (0.7 x 15 cm) until a constant A279/A259ratio was obtained. The control solution (6 ml) was also passed through a charcoal column of the same length. Charcoal has been used by other workers (27) to adsorb bound ATP from PFK. The control and dialdehyde-modified PFK were assayed at pH 8.0 following charcoal treatment. The concentration dependence of the inactivation was determined by incubating PFK with several concentrations of dialdehyde-ATP at pH 8.6; aliquots were withdrawn for assay during each inactivation. Protection against dialdehyde-mediated inactivation of PFK was determined by incubation of PFK with each effector compound (5.3 mM) for 10 min prior to addition of dialdehyde-ATP (0.2 mM). Aliquots were assayed for PFK activity during the course of each reaction.
DIALDEHYDE-ATP
INACTIVATION
OF PHOSPHOFRUCTOKINASE
201
Zncorporation of [Wldialdehyde-AZ’P. PFK (0.4-4 mg/ml) was modified with [%]dialdehyde-ATP (0.6 mM) in borate (pH 8.6) or Tris-HCl (pH 8.6) as described above. Modified PFK and the control sample were assayed following charcoal treatment or borohydride addition and dialysis. Aliquots of each protein solution were then diluted to 5 ml with deionized water before adding 5 ml of Instagel. A sample containing dialdehyde-ATP alone was included as a standard when counting samples. A molecular weight of 340,000(85,000 per protomer) for PFK was used in calculations of [Wldialdehyde-ATP incorporation (30). Reaction of PFK and dialdehyde-inactivated PFK with dinitrojkorobenzene. PFK (0.5 mg/ml) was
modified with dialdehyde-ATP (0.6 mM) in borate (pH 8.6). After 30 min both the control solution and dialdehyde-modified PFK were dialyzed against 1 liter of 0.1 mM borate (pH 8.0) containing 0.2 mM EDTA (two changes). Then 2 ml of a DNFB solution (0.1 ml DNFB in 10 ml of acetone) was added to 2 ml of each PFK solution at 0°C (control and dialdehyde-inactivated PFK). Both mixtures were brought to room temperature and the reactions were allowed to proceed for ‘70 min. The DNFB was extracted with ethyl ether (four times) before dialyzing both solutions against 0.01 M HCl. The 0.01 M HCl was removed by lyophilization and each sample was hydrolyzed in 6 N HCl, 110°C for 24 h in sealed evacuated tubes. Basic amino acid content of each sample was determined following chromatography on a 17-cm column (PA-35 resin) equilibrated in citrate, pH 5.26 (sodium concentration, 0.35 N). Analytical
ultmcenttifugation. PFK (3-5 mg/ml) was inactivated with dialdehyde-ATP (1.0 mM) in borate buffer (pH 8.6) as described above. After addition of sodium borohydride (1.9 mM) to the control solution and to the reaction mixture containing dialdehyde-ATP, both solutions were dialyzed against borateFDP (pH 8.0). The control sample of PFK and the modified enzyme were then centrifuged simultaneously at 48,000 rpm (An-D rotor), 20°C using double sector cells (12-mm centerpiece, 4” sector), one with a quartz window and one with a + 1” wedge window. Schlieren optics were used to monitor sedimentation velocity and photographs were taken at 4-min intervals (Kodak metallographic plates, Type II) at a diaphragm angle of 50”. Schlieren peak distance to the reference line was measured using a Nikon microcomparator equipped with a Goertner digital readout. Sedimentation constants were determined graphically according to the procedure of Schachman (31).
RESULTS
Inactivation
of PFK in Tris Buffer
PFK (0.5 mg/ml) lost more than 90% of its activity (control activity = 128 U/mg) when
MINUTES
OF
INCUBATION
FIG. 1. Time course of the inactivation of PFK with dialdehyde-ATP in borate buffer (pH 8.6).
incubated with 0.5 mM dialdehyde-ATP in Tris-HCl (pH 8.6). Sodium borohydride (13 mM) was added to the control sample and dialdehyde-inactivated PFK before analysis (Tris-phosphate, pH 8.0) in order to reduce Schiff base linkages which might have been formed. Following dialysis, dialdehyde-modified PFK was 25% as active as the control sample of enzyme (117 U/mg). Inactivation
of PFK in Borate Buffer
Figure 1 summarizes the result of experiments in which PFK (0.5 mg/ml) was incubated with 0.2, 0.4, or 0.6 mM dialdehydeATP in borate buffer (pH 8.6). Incubation of PFK with 0.6 mM dialdehyde-ATP for 30 min at 22°C resulted in essentially complete inactivation (99%). The activity of a control sample of enzyme was 114- 130 U/mg. The modified PFK remained inactive (1% of control activity) following dialysis of the reaction mixture (4 days) against borate-FDP, pH 8.0 (control activity = 114-130 U/mg). In these experiments, sodium borohydride was not added before dialysis so that the effect of borate buffer on the inactivation process could be assessed. The results were the same whether excess reagent was removed by dialysis or by passing the reaction mixture through a charcoal bead column. However, the charcoal beads adsorbed 40-50% of the PFK in addition to the excess
GREGORY AND KAISER
202 TABLE I
PROTECTION FROM DIALDEHYDE-ATP-MEDIATED INACTIVATION OF PFK
Compound added”
PFK activity* (% of control)
None CAMP F6P FDP AMP ATP ADP ATP + F6P
16 26 28 31 33 52 62 67
a The concentration of dialdehyde-ATP was 0.2 mM; each effector had a concentration of 5.3 mM. b PFK was 0.5 mg/ml in Tris-HCl, pH 8.6.
reagent. PFK inactivated in borate buffer (pH 8.6) and then dialyzed for 2 days against Tris-phosphate buffer (pH 8.0) regained 8% activity. Protection against Dialdehyde-Mediated Inactivation of PFK
The effects of substrates and activators on the inactivation of PFK were studied. Table I summarizes the results of experiments in which PFK (0.5 mg/ml in TrisHCl, pH 8.6) was incubated with effector compounds (concentration of each, 5.3 mM) for 10 min prior to addition of dialdehydeATP (0.2 InM). The column on the right lists PFK activity, as a percentage of control activity, 10 min after addition of dialdehydeATP. Adenosine diphosphate or ATP partially prevented inactivation by dialdehydeATP at pH 8.6 but F6P, cyclic 3’,5’-adenosine monophosphate, or FDP provided little protection from inactivation. Incorporation
of [14C]Dialdehyde-ATP
PFK protomer. PFK inactivated in Tris buffer and dialyzed without borohydride treatment incorporated 2.6 mol of 14C-reagent per protomer (79% loss of activity). However, in order to insure removal of “nonspecifically” bound 14C-reagent, 6.6 mM ATP was added to inactivated and control samples after 2 days of dialysis, and dialysis was continued for 2 days more. Incorporation of [14C]dialdehyde-ATP following inactivation of PFK in Tris-HCl (pH 8.6) and dialysis against 0.01 M HCl was 3.3 + 0.1 mol of 14C-reagent per protomer. The PFK activity could not be measured because of the acid treatment. Removal of excess [14C]dialdehyde-ATP upon inactivation of PFK in borate buffer was accomplished by dialysis against borate-FDP (pH 8.0) or by charcoal treatment of the reaction mixtures. Results from experiments in which excess reagent was removed by charcoal treatment showed that 99% inactivation (control activity = 115120 U/mg) of PFK corresponded to incorporation of 3.7 & 0.2 mol of reagent per PFK protomer. Reactions in which dialysis was performed to remove excess reagent were terminated by adding sodium borohydride (0.66 mM) before dialysis. Addition of sodium borohydride resulted in the enzyme regaining some of its activity (a few percent) as measured following dialysis. Dialysis for 7 days in the pH 8.0 borate-FDP buffer was necessary to remove “nonspecifically” bound reagent. Removal of excess reagent was facilitated if the PFK concentration was 4 mg/ml rather than 0.5 mg/ml. A 94% loss of activity (control activity = 130 U/mg) corresponded to incorporation of 3.6 -+ 0.2 mol of reagent per PFK protomer. Hansske et al. (29) reported that the half-life for the decomposition of dialdehyde-AMP at pH 7.0, 4”C, was 17 days. However, the incorporation of [14C]dialdehyde-ATP in PFK was practically the same whether excess reagent was removed by charcoal treatment or by dialysis.
Incorporation of [ 14C]dialdehyde-ATP was measured following incubation of PFK (in Tris or borate buffer) with 0.6 KIM 14Creagent for 30-35 min. PFK inactivated in Determination of the Identity of the Tris buffer was dialyzed (4 days) against Modi$.ed Amino Acid Residues Tris-phosphate (pH 8.0) following addition of 13 mM sodium borohydride. A 75% loss of Lysine was a logical choice for the amino activity corresponded to incorporation of acid residue(s) modified by dialdehyde2.4 f 0.2 mol of [14C]dialdehyde-ATP per ATP (28). Sodium borohydride was added to
DIALDEHYDE-ATP
INACTIVATION
modified PFK before dialysis against Trisphosphate or before amino acid analysis in order to reduce any Schiff base linkages formed. However, dialysis of a borohydridetreated reaction mixture against Trisphosphate (pH 8.0) resulted in partial recovery of activity. Furthermore, amino acid analyses of control and dialdehyde-modified PFK showed no differences regardless of whether or not borohydride treatment was performed. Another possibility for the amino acid residue(s) modified by dialdehyde-ATP was arginine. Riordan et al. (32) found that PFK is rapidly inactivated in borate buffer by 2,3-butanedione. Indeed, chemical modification with 2,3-butanedione, phenylglyoxal, or 1,Zcyclohexanedione has indicated that arginine residues are present in the substrate binding sites of a number of glycolytic enzymes (32-36). Modification of arginine by a-dicarbonyl reagents can be detected by amino acid analysis. Although the two carbony1 groups in dialdehyde-ATP are not a to each other, it seemed possible that arginine could also be modified by this reagent. Accordingly PFK was modified with dialdehyde-ATP (0.6 mM) in borate buffer (pH 8.6); the modified enzyme and a control sample were then dialyzed against 0.01 N HCl prior to lyophilization and 6 N HCl hydrolysis. Amino acid analyses showed no differences in arginine content between dialdehyde modified PFK and the control sample. In order to examine further whether lysine in PFK was modified by dialdehydeATP, an indirect method was used. Since borate buffer appeared to stabilize the dialdehyde-ATP-modified form of PFK, both modified PFK and the control sample were treated with DNFB in borate (pH 8.0) to modify free lysine residues. If lysine residues were modified by dialdehyde-ATP, these residues would be protected from the reaction with DNFB. Reversal of the dialdehyde modification during hydrolysis with 6 N HCl prior to amino acid analysis would result in regeneration of free lysine. Amino acid analysis of a control sample of PFK and of the dialdehyde-modified enzyme (both treated with DNFB) should then show a net difference in lysine content corresponding to the number of lysines (if any) modified by
203
OF PHOSPHOFRUCTOKINASE TABLE
II
LYSINE CONTENT OF PFK REACTED WITH DNFB Treatment Dialdehyde-ATP, DNFB
Lysineiprotomer DNFB
4.2 + 0.1 0.6 + 0.1
dialdehyde-ATP. To achieve good resolution, a 1’7-cm column was used to separate the basic amino acids (rather than the 5-cm column normally used). Reaction of proteins with DNFB can result in modification of thiol, c-amino, a-amino, imidazole, and phenolic groups; on the 17-cm column imdinitrophenyl histidine is resolved from lysine (37). The number of lysines remaining following modification with DNFB was calculated employing the arginine and lysine content (53 and 43 residues, respectively, per protomer of 85,000 M,) of rabbit muscle PFK, as reported by Walker et al. (38) and the ratio of arginine to lysine measured in our chromatograms. The analytical results we obtained for lysine are listed in Table II. As mentioned previously, amino acid analyses of dialdehyde-modified PFK and the control sample showed no difference in arginine content. Analytical
Ultracentrifugation
Modified PFK and the control sample were dialyzed against borate-FDP (pH 8,0), following addition of 1.9 InM sodium borohydride (to reduce excess dialdehyde). Enzymatic assay of dialdehyde-modified PFK before centrifugation revealed 94% inhibition compared to the control (115 U/mg). Figure 2 shows Schlieren patterns of control and inactivated PFK (5 mglml) 23 min after centrifugation was begun. The control sample of enzyme (upper) shows three peaks with sedimentation coefficients of 12, 18, and 25 S. The dialdehyde-inactivated PFK shows two peaks with sedimentation constants of 12 and 18 S. The 25 S species is present only to a small extent, if at all. DISCUSSION
Despite the vast amount of work on PFK, very little is known about the chemistry of the ATP binding sites. Evidence has been
204
GREGORY AND KAISER
presented suggesting that each PFK protomer has three different types of nucleotide binding sites; a catalytic site, a site which binds activators such as AMP, or ADP, and a site which binds ATP as an inhibitor (1% 20, 39-41). PFK has been modified with a number of sulfhydryl reagents, but the resulting inactivation is generally incomplete and varies with the reagent used (42). ATP protects against reaction of the most reactive thiol group with reagents such as 5,5’-dithiobis (Znitrobenzoic acid) and a-bromo-4-hydroxy3-nitroacetophenone, but Kemp (43) and Schwartz et aZ.(41) propose that ATP protects by causing a conformational change which makes the sulfhydryl group inaccessible. In 1973, Bloxham et aZ.(44) reported
that 6-mercapto-9+Dribofuranosylpurine5’-triphosphate completely inactivated rabbit muscle PFK upon incorporation of 6-8 mol of reagent per tetramer (380,000 M,). However, F6P provided greater protection against the inactivation reaction than did ATP. In the present paper, we have reported the results of the modification of rabbit muscle PFK with 2’,3’-dialdehyde-ATP. Incubation of PFK with dialdehyde-ATP in Tris buffer at pH 8.6 resulted in rapid inactivation. However, inactivation of PFK in Tris buffer was incomplete and partially reversible upon dialysis of a borohydridetreated reaction mixture. Later experiments showed that PFK was rapidly and essentially completely inactivated by dial-
FIG. 2. Analytical ultracentrifugation of a control sample of PFK (upper) and of the dialdehydemodified enzyme (lower) (5 mg/ml) at 48,000 rpm, 23 min after reaching speed.
DIALDEHYDE-ATP
INACTIVATION
dehyde-ATP in borate buffer at pH 8.6. Furthermore, activity was not regained upon dialysis of the reaction mixture against borate-FDP buffer, pH 8.0. These results are evidence that borate buffer stabilizes the modification of PFK with dialdehydeATP, since sodium borohydride was not added to modified PFK before dialysis against borate-FDP. The additional activity loss in borate was not caused by the change in buffer (from Tris to borate) since PFK inactivated in borate and then dialyzed against Tris regained 8% activity. Experiments with [14C]dialdehyde-ATP showed that 94-99% inactivation of PFK corresponded to incorporation of 3.6 -t 0.2 mol of reagent per PFK protomer. Excess dialdehyde-ATP was removed by charcoal or by dialysis so that loss of PFK activity could be directly related to incorporation of reagent. The incorporation of modifying agent was practically the same regardless of whether excess reagent was removed by charcoal treatment or dialysis. Kemp and Krebs (21) found that 3 mol of ATP were bound by each rabbit muscle PFK protomer (pH 6.95) and Lorenson and Mansour (22) found that 3.6 mol of ATP were bound by each sheep heart PFK protomer (pH 6.65). However, the inactivation of PFK with dialdehyde-ATP was performed at pH 8.6 where inhibition by ATP does not occur. Determination of the amino acid residues modified by dialdehyde-ATP was accomplished using an indirect method since amino acid analyses of acid hydrolyzates of dialdehyde-ATP inactivated PFK and of control samples showed no differences in lysine or arginine content. In order to determine whether lysine residues were modified by dialdehyde-ATP, the amino acid contents of dialdehyde-inactivated enzyme and control samples were compared following reaction with DNFB. Since hydrolysis with 6 N HCl regenerated the lysines modified by the dialdehyde reagent, a difference of 3.6 lysine residues between the dialdehydeinactivated enzyme and control samples was found. This difference which indicates that three or four lysines are modified per protomer, is in excellent agreement with the results of the incorporation of [14C]
205
OF PHOSPHOFRUCTOKINASE 4-(O,P),OCHz
a.
Ad
Hr”lH I’N’I HO
‘-OSPOCH2
OH
b.
Hr’-tH I’N’I HO AHOH
(CH2)4
I HniCHC02
Ad
L=o 0
’ \
I
FIG. 3. (a) Proposed structure for dialdehyde-ATPlysine derivative. (b) Morpholine derivative isolated from reaction of benzoic acid hydrazide with dialdehyde-AMP (29).
dialdehyde-ATP in the enzyme. The lysine residues modified could react with the dialdehyde-ATP reagent in two possible ways. The first possibility considered was Schiff base formation, as has been observed, for instance, in the modification of pyruvate carboxylase (28) and histone kinase (45) by dialdehyde-ATP. However, this possibility was ruled out for the following reasons. Based on amino acid analyses and activity assays of modified PFK following dialysis in Tris buffer, evidence was obtained that treatment of modified PFK with sodium borohydride did not result in covalently modified lysine. Furthermore, the modified form of PFK obtained by treatment with dialdehyde-ATP is stable in 0.01 N HCl (based on 14C incorporation results), a result inconsistent with the lability expected for a Schiff base linkage. As an alternative, we propose that the product of the reaction of dialdehyde-ATP with lysine in PFK has the dihydroxy morpholine-type structure shown in Fig. 3a. If this structure is correct, the stabilization by borate buffer of the modified form of PFK can be understood since borate could complex the dihydroxy morpholinelike adduct in a manner similar to that observed in arginine modifications with butane dione (46). Hansske et aZ.(29) and Hansske and Cramer (47) have found that carboxylic acid hydrazides react with dialdehyde-AMP to give stable dihydroxy morpholine derivatives (Fig. 3b). Therefore, on the basis of our observations combined with those of Hansske et d(29) it seems reasonable to postulate that the lysine residues of PFK react with dialdehyde-
206
GREGORY
AND KAISER
ATP to form morpholine-like adducts, a result previously not observed upon reaction of lysine residues of protein with aldehyde reagents. The results from the protection experiments show that although neither ADP nor ATP provides full protection against inactivation, these nucleotides do provide the best protection. Since 3-4 mol of the dialdehyde reagent bind irreversibly to PFK, the K, value for the reagent has not been assessed. However, the structure of dialdehyde-ATP and ATP may be similar enough that the dialdehyde species can compete effectively with ATP for ATP binding sites on PFK. Dialdehyde nucleosides exist in several hydrated forms in solution (47) and the structure of the hemiacetal or cis-dihydroxydioxane-type form of dialdehyde-ATP does indeed resemble that of ATP. This form of the reagent may be stabilized by borate buffer. Another possible explanation of the results of the protection experiments is that the lysine residues involved in the modification reaction are near, but not precisely at the ATP binding sites. It is also conceivable that during the inactivation of PFK dialdehyde-ATP binds in a different mode than that in which ATP normally binds. Table I shows that F6P, FDP, or AMP provide very little protection from inactivation by dialdehyde-ATP. This differs from what has been observed in the modification of PFK with pyridoxal phosphate. Uyeda (48) studied the inactivation of rabbit muscle PFK with pyridoxal phosphate and found that F6P provided the best protection from inactivation; AMP or FDP provided some protection against the inactivation process. Setlow and Mansour (40) inactivated sheep heart PFK with pyridoxal phosphate and found that the best protection was afforded by FDP with some protection by ATP. If the same lysine residues are reacting with pyridoxal phosphate and dialdehyde-ATP, the difference in the results of the protection experiments could indicate that the binding sites for the two reagents are distinct, yet close. Analytical ultracentrifugation studies were performed on dialdehyde-ATP inactivated PFK and control samples of the enzyme
to determine whether inactivation was caused by dissociation of the enzyme. Enzymatically active PFK is composed of four identical subunits of molecular weight 85,000 + 10,000, as measured by sedimentation equilibrium and sedimentation velocity (30). However, the tetrameric form of the enzyme can be in rapid equilibrium with higher molecular weight aggregates as well as with inactive dimers, resulting in a multicomponent sedimentation pattern (24, 27, 49). This aggregational behavior is affected by protein concentration, pH, and the ligands present (26, 50). FDP stabilizes the active tetrameric form of PFK (26) and was included in our analytical ultracentrifugation studies because PFK is not stable in borate buffer over long periods of time. The three peaks observed (Fig. 2) upon ultracentrifugation of the control sample had sedimentation coefficients of 12, 18, and 25 S. These results agree with studies reported by Leonard and Walker (30) on the subunit structure of PFK in 0.1 M Tris-phosphate (pH 8.0) containing 0.5 mM FDP. They found that ultracentrifugation of PFK (3-5 mg/ml, 20%) resulted in three Schieren peaks with sedimentation coefficients of 13, 18, and 27 S. The 13 S species corresponded to the tetramer, and the 18 and 27 S species were higher molecular weight aggregates. Centrifugation of dialdehyde-ATP modified PFK resulted in two Schlieren peaks with sedimentation coefficients of 12 and 18 S. However, the 25 S species is largely absent, indicating that dialdehyde-ATP modified PFK is more dissociated than the control enzyme (borate-FDP, pH 8.0). Modification of PFK with dialdehyde-ATP thus affects the aggregational behavior of the enzyme, but the observed inactivation is not caused by an inability of the modified enzyme to form the 340,000 M, tetramer. In conclusion, dialdehyde-ATP rapidly inactivates PFK by a mechanism other than dissociation of the 340,000 M, tetramer. ATP and ADP partially protect PFK from inactivation by dialdehyde-ATP, but F6P, FDP, or AMP, which protect PFK from modification by pyridoxal phosphate, provide little protection from modification by the dialdehyde reagent. This reagent modifies three to four lysine residues per PFK
DIALDEHYDE-ATP
INACTIVATION
protomer. These lysine residues, which we believe are important to the catalytic and/or ATP binding properties of PFK, most probably react with dialdehyde-ATP to form dihydroxymorpholine derivatives rather than Schiff bases. ACKNOWLEDGMENTS We are grateful to Dr. Y. Nakagawa, Division of Renal Medicine, Michael Reese Hospital, for many helpful discussions, to Professor Angelo Scanu, Depsrtments of Medicine and Biochemistry, for allowing us to use the analytical ultracentrifuge, and to Mr. Frank Buckingham for sacrificing the rabbits. REFERENCES 1. STADTMAN, E. R. (1966) A&. Enzymol. 28, 41-154. 2. KOERNER, T. A. W., JR., YOUNATHAN, E. S., ASHOUR, A. L. E., AND VOLL, R. J. (1974) J. Biol. Chem. 249, 5749-5754. 3. FISHBEIN, R., BENKOVIC, P. A., SCHRAY, K. J., SIEWERS, I. H., STJZFFENS,J. J., ANDBENKOVIC, S. J. (19’74) J. Biol. Chem. 249, 6047-6051. 4. SCHRAY, K. J., AND BENKOVIC, S. J. (1978) Accounts Chem. Res. 11, 136-141. 5. LARDY, H. A., AND PARKS, R. E. (1956) in Enzymes: Units of Biological Structure and Function (Gaebler, 0. H., ed.), pp. 584-587, Academic Press, New York. 6. PASSONEAU, J. V., AND LOWRY, 0. H. (1962) Biochem. Biophys. Res. Commun. 7, 10-15. ‘7. PASSONEAU, J. V., AND LOWRY, 0. H. (1963) Biochem. Biophys. Res. Commun. 13,372-379. 8. VIEIUELA, E., SALES, M., AND SOLS, A. (1963) Biochem. Biophys. Res. Commun. 12,140-145. 9. MANSOUR, T. E. (1963) J. Biol. Chem. 238, 2285-2292. 10. MANSOUR, T. E. (1972) Curr. Top. Cell. Regul. 5, l-46. 11. UYEDA, K., ANDRACKER, E. (1965)J. Biol. Chem. 240, 4682-4688. 12. LOWRY, 0. H., AND PASSONEAU, J. V. (1966) J. Biol. Chem. 241, 2268-2279. 13. SALAS, M. L., SALAS, J., AND SOLS, A. (1968) Biochem. Biophys. Res. Commun. 31,461-466. 14. AHLFORS, C. E., AND MANSOUR, T. E. (1969) J. Biol. Chem. 244, 1247-1251. 15. CHAPMAN, A., SANNER, T., AND PIHL, A. (1969) Eur. J. Biochem. 7, 588-593. 16. SETLOW, B., AND MANSOUR, T. E. (197O)J. Bid. Chem. 245, 5524-5533. 17. BRUNSWICK, D. J., AND COOPERMAN, B. S. (1971) Proc. Nat. Acad. Sci. USA 68, 1801-1804. 18. PETTIGREW, D. W., AND FRIEDEN, C. (1978) J. Biol. Chem. 253, 3623-3627.
OF PHOSPHOFRUCTOKINASE
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19. MANSOUR, T. E., AND MARTENSEN, T. M. (1978) J. Biol. Chem. 253, 3628-3634. 20. MANSOUR, T. E., AND COLMAN, R. F. (1978) Biochem. Biophys. Res. Commun. 81, 13701376. 21. KEMP, R. G., AND KREBS, E. G. (1967) Biochemistv 6, 423-434. 22. LORENSON, M. Y., AND MANSOUR, T. E. (1969) J. Biol. Chem. 244, 6420-6431. 23. KEMP, R. G., AND FOREST, P. B. (1968) Biochemistry 7, 2596-2603. 24. LANG, K.-H., MARCUS, F., AND LARDY, H. A. (1965) J. Biol. Chem. 240, 1893-1899. 25. LING, K.-H., PAETKAU, V., MARCUS, F., AND LARDY, H. A. (1966) in Methods in Enzymology (Wood, W. A., ed.), Vol. 9, pp 425-429, Academic Press, New York. 26. LAD, P. M., HILL, P. E., AND HAMMES, G. G. (1973) Biochemistry 12, 4303-4309. 27. PARMEGGIANI, A., LUFT, J. H., LOVE, D. S. AND KREBS, E. G. (1966) J. Biol. Chem. 241, 4625-4637. 28. EASTERBROOK-SMITH, S. B., WALLACE, J. C., AND KEECH, B. D. (1976) Eur. J. Biochem. 62, 125-130. 29. HANSSKE, F., SPRINZL, M., AND CRAMER, F. (1974) Bioorg. Chem. 3, 367-376. 30. LEONARD, K. R., AND WALKER, I. 0. (1972) Eur. J. Biochem. 26, 442-448. 31. 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. 32. RIORDAN, J. F., MCELVANY, K. D., ANDBORDERS, C. L., JR. (1977) Science 195, 884-886. 33. LANGE, L. G., III. RIORDAN, J. F., ANDVALLEE, B. L. (1974) Biochemistry 13, 4361-4370. 34. YANG, P., AND SCHWERT, G. W. (1972) Biochemistry 11, 2218-2224. 35. NAGRADOVA, N. K., AND ASYRANTS, R. A. (1975) Biochim. Biophys. Acta 386, 365-368. 36. ROGERS, K., AND WEBER, B. H. (1977) Arch. Biochem. Biophys. 180, 19-25. 37. HIRS, C. H. W. (ed.) (1976) in Methods in Enzymology, Vol. 11, pp. 548-555, Academic Press, New York. 38. WALKER, I. D., HARRIS, J. I., RUNSWICK, M. J., AND HUDSON, P. (1976) Eur. J. Biochem. 68, 255-269. 39. MATHIAS, M., AND KEMP, R. G. (1972) Biochemistry 11, 578-584. 40. SETLOW, B., ANDMANSOUR, T. E. (1972)Biochim. Biophys. Acta 258, 106-112. 41. SCHWARTZ, K. J., NAKAGAWA, Y., AND KAISER, E. T. (1976) J. Amer. Chem. Sot. 98, 63696378. 42. BLOXHAM, D. B., AND LARDY, H. A. (1972) in The Enzymes (Boyer, P. D., ed.), 3rd ed.,
208
GREGORY
Vol. VIII, pp. 239-278, Academic Press, New York. 43. KEMP, R. G. (1969) Biochemistry 11, 4490-4496. 44. BLOXHAM, D. B., CLARK, M. G., HOLLAND, P. C., AND LARDY, H. A. (1973) Biochemistry 12, 1596-1601. 45. KOCHETKOV, S. N., BULARGINA, T. V., SASHCHENCKO, L. P., AND SEVERIN, E. S. (1977) Eur. J. Biochem. 81, 111-118.
AND KAISER 12, 391546. RIORDAN, J. F. (1973) Biochemistry, 3923. 47. HANSSKE, R., AND CRAMER, F. (1977) Carbohyd. Res. 54, 75-84. 8, 2366-2373. 48. UYEDA, K. (1969) Biochemistv 49. PAETKAU, V., AND LARDY, H. A. (1967) J. Biol. Chem. 242, 2035-2042. 50. AARONSON, P. P., ANDFRIEDEN, C. (1972)J. Biol. Chem. 247, 7502-7509.