phosphofructokinase from ascaris suum

5
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 257,,No. 7, Issue of April 10, pp. 3807-3810. 1982 Printed In U.S.A. Phosphofructokinase from Ascaris suum THE EFFECT OF PHOSPHORYLATION ON ACTIVITY NEAR-PHYSIOLOGICAL CONDITIONS* (Received for publication, September 14, 1981) Hans Werner Hofer, Benja L. Allen, M. R. Kaeini, and Ben G. Harris$ From the Departments of Biochemistry and Biological Sciences, North Texas State University/Texas College of Osteopathic Medicine, Denton, Texas 76203 and the Department of Biology, University of Konstanz, D-7750 Konstanz, Federal Republic of Germany Phosphofructokinase has been purified in the pres- ence (+F) and absence (-F) of sodium fluoride in the buffers. The +F enzyme contained 8.4 mol of phos- phate/tetramer and the -F enzyme contained 3.3 mol of phosphate/tetramer. Both purified forms of the en- zyme exhibited the same specific activity. However, when assayed under “physiological” conditions, the +F enzyme carried out catalysis 2.0 to 2.5 times faster than the -F enzyme. The -F enzyme which contained 3.3 & 0.3 mol of phosphate/tetramer was incubated with [y-”P]ATP and the catalytic subunit of rabbit muscle cyclic AMP-dependent protein kinase. After a period of 1 h, both specific radioactivity and phosphate analyses revealed that 3 mol of phosphate had been incorporated into the phosphofructokinase/mol tetra- mer. Tryptic digestion of the radiolabeled enzyme fol- lowed by peptide fingerprinting and autoradiography revealed twospots containing approximately equal amounts of radioactivity. The [32P]phosphofructoki- nase containing 6.3 & 0.6 mol of phosphate/tetramer was incubated with bacterial alkaline phosphatase for 1 h and radioactivity and phosphate analyses revealed that the enzyme had been dephosphorylated to the level of 2.8 & 0.3 mol ofphosphate/tetramer. When the phos- phorylated and dephosphorylated forms of the enzyme were tested in the assay near-physiological conditions, the phosphorylated enzyme had 2.0 to 2.5 times higher activity than the dephosphorylated form, although both forms exhibited the same maximum velocity. The de- phosphorylated form was preincubated in the physio- logical assay (minus Fru-6-P) with the catalytic subunit of cyclic AMP-dependent protein kinase. When the re- action was started by the addition of Fru-g-P, the rate obtained was 2.5 times faster than that obtained with the control preincubation of dephosphorylated enzyme. Based on these results and other data from this labo- ratory, it is proposed that phosphofructokinase activity in the muscle of the parasite might be regulated in part by phosphorylation by the cyclic AMP-dependent pro- tein kinase. * This work was supported by National Institutes of Health (AI- 12331),the Deutsche Forschungsgemeinschaft (Ho 650/3), and Son- derforschungsbereich 138 “Biologische Grenzflachen and Spezifitat,” the Alexander von Humboldt Stiftung, and North Texas State Uni- versity Organized Research Funds. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact. + Recipient of a Research Career Development Award from NIH (AI-00057). To whom correspondence may be addressed at: Depart- ment of Biological Sciences, North Texas State University, Denton, T X 76203. Several studies have demonstrated that phosphofructoki- nase from muscle can be isolated as a phosphorylated protein (1-7) and some studies have correlated this phosphorylation with an increase in muscle activity (5, 8, 9). However, the function of this phosphorylation in regulating the activity of the enzyme in muscle remains unknown. On the other hand, it has been demonstrated by several workers that phospho- rylation of liver phosphofructokinase results in a form of the enzyme that is inhibited by ATP to a greater extent than the dephosphoform (10-12). Further, phosphorylated liver phos- phofructokinase appears to have lesser affinity for “activating factor” (13-15), fructose 2,6-bisphosphate (16-18). Thus,it appears that phosphorylation of the liver enzyme results in a form that is less active than the nonphosphorylated form (14). In the two preceding papers in this series (19, 20), we have described the purification, physicochemical properties, and the regulatory kinetic properties of phosphofructokinase from the muscle of Ascaris suum. In addition, a “physiological” assay was developed that approximated the intracellular levels of substrates, products, and known effectors of the ascarid phosphofructokinase. In this assay, the activity of the enzyme at physiological levels of substrate was about 1 to 2% of V,,, (20). It was shown further that this level of enzyme activity approximates that found in vivo (20). In the preceding studies (19, 20), the purified phosphofructokinase had at least 2 mol of phosphate bound/mol of subunit of enzyme. The present study describes the purification of a form of the enzyme that has fewer phosphates, exhibits a 2- to 2.5-fold lower activity under physiological conditions and yet has the same V,,, as the phosphorylated enzyme. It is further established that the enzyme can be phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase and dephosphorylated by bacterial alkaline phosphatase. The two forms of the enzyme exhibit similar V,,, activities, but the activity of the dephosphorylated form is 2 to 2.5 times lower under physio- logical assay conditions. EXPERIMENTAL PROCEDURES Methods Purification of Enzymes-The ascarid phosphofructokinase was purified as described in the fwst paper in this series (19). The minus fluoride preparation was carried out by omitting sodium fluoride from all the buffers used in the procedure. The catalytic subunit of the CAMP’-dependent protein kinase was purified by the method of Beavo et al. (21). Phosphate analysis of the phosphofructokinase sample was carried out as described previously (19). Physiological Assay-This assay was carried out as described in the preceding paper (20). The assay contained the following compo- The abbreviations used are: CAMP,cyclic adenosine 3’:5’-mono- phosphate; Fru-6-P, fructose 6-phosphate; Glc-1,6-P2, glucose 1,6- bisphosphate. 3807 by guest on March 20, 2018 http://www.jbc.org/ Downloaded from

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Page 1: Phosphofructokinase from Ascaris suum

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 257,,No. 7, Issue of April 10, pp. 3807-3810. 1982 Printed In U.S.A.

Phosphofructokinase from Ascaris suum THE EFFECT OF PHOSPHORYLATION ON ACTIVITY NEAR-PHYSIOLOGICAL CONDITIONS*

(Received for publication, September 14, 1981)

Hans Werner Hofer, Benja L. Allen, M. R. Kaeini, and Ben G. Harris$ From the Departments of Biochemistry and Biological Sciences, North Texas State University/Texas College of Osteopathic Medicine, Denton, Texas 76203 and the Department of Biology, University of Konstanz, D- 7750 Konstanz, Federal Republic of Germany

Phosphofructokinase has been purified in the pres- ence (+F) and absence (-F) of sodium fluoride in the buffers. The +F enzyme contained 8.4 mol of phos- phate/tetramer and the -F enzyme contained 3.3 mol of phosphate/tetramer. Both purified forms of the en- zyme exhibited the same specific activity. However, when assayed under “physiological” conditions, the +F enzyme carried out catalysis 2.0 to 2.5 times faster than the -F enzyme. The -F enzyme which contained 3.3 & 0.3 mol of phosphate/tetramer was incubated with [y-”P]ATP and the catalytic subunit of rabbit muscle cyclic AMP-dependent protein kinase. After a period of 1 h, both specific radioactivity and phosphate analyses revealed that 3 mol of phosphate had been incorporated into the phosphofructokinase/mol tetra- mer. Tryptic digestion of the radiolabeled enzyme fol- lowed by peptide fingerprinting and autoradiography revealed two spots containing approximately equal amounts of radioactivity. The [32P]phosphofructoki- nase containing 6.3 & 0.6 mol of phosphate/tetramer was incubated with bacterial alkaline phosphatase for 1 h and radioactivity and phosphate analyses revealed that the enzyme had been dephosphorylated to the level of 2.8 & 0.3 mol of phosphate/tetramer. When the phos- phorylated and dephosphorylated forms of the enzyme were tested in the assay near-physiological conditions, the phosphorylated enzyme had 2.0 to 2.5 times higher activity than the dephosphorylated form, although both forms exhibited the same maximum velocity. The de- phosphorylated form was preincubated in the physio- logical assay (minus Fru-6-P) with the catalytic subunit of cyclic AMP-dependent protein kinase. When the re- action was started by the addition of Fru-g-P, the rate obtained was 2.5 times faster than that obtained with the control preincubation of dephosphorylated enzyme. Based on these results and other data from this labo- ratory, it is proposed that phosphofructokinase activity in the muscle of the parasite might be regulated in part by phosphorylation by the cyclic AMP-dependent pro- tein kinase.

* This work was supported by National Institutes of Health (AI- 12331), the Deutsche Forschungsgemeinschaft (Ho 650/3), and Son- derforschungsbereich 138 “Biologische Grenzflachen and Spezifitat,” the Alexander von Humboldt Stiftung, and North Texas State Uni- versity Organized Research Funds. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact. + Recipient of a Research Career Development Award from NIH (AI-00057). To whom correspondence may be addressed at: Depart- ment of Biological Sciences, North Texas State University, Denton, TX 76203.

Several studies have demonstrated that phosphofructoki- nase from muscle can be isolated as a phosphorylated protein (1-7) and some studies have correlated this phosphorylation with an increase in muscle activity (5, 8, 9). However, the function of this phosphorylation in regulating the activity of the enzyme in muscle remains unknown. On the other hand, it has been demonstrated by several workers that phospho- rylation of liver phosphofructokinase results in a form of the enzyme that is inhibited by ATP to a greater extent than the dephosphoform (10-12). Further, phosphorylated liver phos- phofructokinase appears to have lesser affinity for “activating factor” (13-15), fructose 2,6-bisphosphate (16-18). Thus, it appears that phosphorylation of the liver enzyme results in a form that is less active than the nonphosphorylated form (14).

In the two preceding papers in this series (19, 20), we have described the purification, physicochemical properties, and the regulatory kinetic properties of phosphofructokinase from the muscle of Ascaris suum. In addition, a “physiological” assay was developed that approximated the intracellular levels of substrates, products, and known effectors of the ascarid phosphofructokinase. In this assay, the activity of the enzyme at physiological levels of substrate was about 1 to 2% of V,,, (20). It was shown further that this level of enzyme activity approximates that found in vivo (20). In the preceding studies (19, 20), the purified phosphofructokinase had at least 2 mol of phosphate bound/mol of subunit of enzyme. The present study describes the purification of a form of the enzyme that has fewer phosphates, exhibits a 2- to 2.5-fold lower activity under physiological conditions and yet has the same V,,, as the phosphorylated enzyme. It is further established that the enzyme can be phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase and dephosphorylated by bacterial alkaline phosphatase. The two forms of the enzyme exhibit similar V,,, activities, but the activity of the dephosphorylated form is 2 to 2.5 times lower under physio- logical assay conditions.

EXPERIMENTAL PROCEDURES

Methods Purification of Enzymes-The ascarid phosphofructokinase was

purified as described in the fwst paper in this series (19). The minus fluoride preparation was carried out by omitting sodium fluoride from all the buffers used in the procedure. The catalytic subunit of the CAMP’-dependent protein kinase was purified by the method of Beavo et al. (21). Phosphate analysis of the phosphofructokinase sample was carried out as described previously (19).

Physiological Assay-This assay was carried out as described in the preceding paper (20). The assay contained the following compo-

’ The abbreviations used are: CAMP, cyclic adenosine 3’:5’-mono- phosphate; Fru-6-P, fructose 6-phosphate; Glc-1,6-P2, glucose 1,6- bisphosphate.

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3808 Phosphorylation of Ascaris Phosphofructokinase

nents: 50 mM imidazole/HCl, pH 6.8, 5.25 mM potassium phosphate, 8 mM MgC12, 101.3 mM KC], Fru-6-P as indicated, 3 mM ATP, 1 mM ADP, 0.36 mM AMP, 25 p~ Glc-1,6-P2, 0.1 mM dithiothreitol, 0.16 mM NADH, 50 pg of aldolase, 30 p g of glycerol phosphate dehydrogenase, 10 pg of triose phosphate isomerase (coupling enzymes desalted by dialysis overnight against 10 mM triethanolamine/HCl, pH 7.5, 10 mM dithiothreitol), and 3 to 7 pg of phosphofructokinase (dialyzed and diluted as indicated previously, 20). The assays were carried out in a 1-ml volume at 30 “C.

Phosphorylation Studies-The phosphofructokinase and the pro- tein kinase (CAMP-dependent catalytic subunit) were combined (vol- ume of 1 ml ) and dialyzed overnight in 0.1 M triethanolamine/HCl, pH 7.6, and 0.2 M KC1 under an atmosphere of NS. The proteins were then incubated at 30 “C (pH 7.2) and the reaction started by the addition of 0.2 mM [y-”P]ATP and 0.2 mM MgC12. At various time intervals, aliquots of the reaction mixture were removed and spotted on fdter paper that had been soaked in 10% trichloroacetic acid and then dried. After spotting, the fdter papers were washed in five changes of 5% trichloroacetic acid, two changes of 95% ethanol, and then dried and counted. After aliquots had been removed for radio- activity counting, the reaction was stopped by the addition of satu- rated (NH4)2S04 (pH 7.6), the protein precipitated, redissolved in water, and chromatographed on a Sepharose 6B column (90 X 0.9 cm) equilibrated with 0.2 M (NH4)2S04 (pH 7.6).

Dephosphorylation Studies-Phosphofructokinase was dialyzed overnight against 0.1 M Tris-HCI, pH 8.5. Alkaline phosphatase (Esch- erichia coli, Sigma Type 111) was added to the enzyme and incubation was carried out at 30 “C (pH 8.1). Aliquots were removed and precip- itated in trichloroacetic acid, washed, and counted as described above. After incubation, the mixture was concentrated by precipitation with (NH4)2S04 and chromatographed on Sepharose 6B as outlined above.

Tryptic Digestion and Peptide Mapping-The “P-labeled phos- phofructokinase was dialyzed against 0.05 M NH4HCOs, pH 8.2, under an atmosphere of Ns. Trypsin was added to the solution (1 pg of trypsin/lO pg of phosphofructokinase) and incubated for 24 h at 37 “C. After 12 h, an equivalent amount of trypsin was added. The NH4HCO3 was removed in uucuo and the tryptic peptides were redissolved in 100% formic acid. Mapping of peptides was performed on silica gel thin layer plates (HPTLC plates, Merck Darmstadt). Chromatography was performed in the first dimension in 1-butanol/ 2-propanol/pyri&ne/glacial acetic acid/H20 (15:15:25:8:37 by vol- ume). In the second dimension, the peptides were separated by electrophoresis in pyridine/HzO/acetic acid buffer (10% pyridine brought to pH 6.1 with glacial acetic acid) for 2 h (20 mA, 300 V). After drying, the plates were exposed to x-ray film (Kodak No-screen film, Type NS-2T) for a period of time (4 to 12 h) and developed.

Electrophoresis of Inorganic Phosphate-Electrophoresis of tri- chloroacetic acid extracts was carried at 600 V for 30 min on silica gel thin layer plates using the pyridine/H’O/acetic acid buffer (pH 6.1) described above. After drying, the plate was exposed to x-ray film overnight and developed.

Preparation of [y-”’P]ATP-The preparation of the [y3’P]ATP was performed as described by Johnson and Walseth (22).

Materials E . coli alkaline phosphatase (Type 111) was purchased from Sigma.

NADH, adenine nucleotides, Fru-6-P, Glc-1,6-P2, aldolase, glycerol phosphate dehydrogenase, and triose phosphate isomerase were pur- chased from Boehringer Mannheim. The [”’Plphosphate was ob- tained from Amersham-Buchler (Braunschweig, Germany). The re- maining chemicals used were of reagent grade and were obtained from commercial sources.

RESULTS

The phosphofructokinase purified from A. suum contained at least 7 to 8 mol of phosphate/mol of enzyme (19). Therefore, it was of interest to attempt to purify a form of the enzyme that had lower phosphate content and to determine whether there were kinetic differences that could be ascertained be- tween the differentially phosphorylated forms. Since fluoride is considered as a general inhibitor of phosphatases, it was omitted from the buffers and the purification procedure was carried out. Table I gives the results of the purification of phosphofructokinase from two 100-g samples of frozen A. suum in the presence and absence of sodium fluoride. The

procedures were carried out at the same time and required 1 day to complete. In each case, the preparation was shown to be homogenous by sodium dodecyl sulfate gel electrophoresis. The specific activities of the enzymes were essentially the same, but the yield was greater in the absence of fluoride. This was due to a better yield in the phosphocellulose step ( c f . 19). The covalently bound phosphate content of the enzyme pur sed in the absence of fluoride was significantly lower, less than 1 mol/subunit.

The enzymes were dialyzed and diluted (see “Methods”) to the same activity and identical aliquots were tested in the physiological assay at various levels of Fru-6-P. Fig. 1 shows the results of these experiments. Over the concentration range of 50 to 200 ,UM Fru-6-P, the enzyme isolated in the presence of fluoride exhibited 1.8- to 2.5-fold higher activity than the enzyme purified in the absence of fluoride. At every Fru-6-P concentration, an additional assay was carried out in which the inhibitory action of ATP was reversed by the addition of 5 mM AMP (data not shown). Both forms of the enzyme exhibited virtually identical activities under these conditions.

These results suggested that phosphorylation of the enzyme resulted in a form that was less inhibited by ATP and ex- hibited an increased activity under physiological levels of substrates, products, and effectors. It did not, however, rule out other modifications of the enzyme that might be brought about by omission of fluoride from the isolation buffers. For this reason, it was necessary to attempt to phosphorylate and dephosphorylate the enzyme in vitro to demonstrate that the kinetic differences were actually due to phosphate attach- ment. Enzyme which had been isolated in the absence of fluoride and contained 3.3 +. 0.3 mol of phosphate/tetramer was incubated in the presence of the catalytic subunit of CAMP-dependent protein kinase and [y-3zP]ATP and Fig. 2 shows the results of this experiment. During a period of 60

TABLE I Summary ofpurification ofphosphofructokinase from A. suum in

the presence a n d absence of sodium fluoride Experiment Specific Total

activity protein mol enzyme Moles PI/

unitdmg mg B

I (+NaF) 51.2 3.1 51.6 8.4 I1 (-NaF) 50.4 4.5 75.0 3.3

A-

FIG. 1. Fructose 6-phosphate saturation curve in a physio- logical assay of two forms of Ascaris phosphofructokinase isolated in the presence and absence of fluoride. Differentially phosphorylated forms of phosphofructokinase (PFK) (see Table I) were assayed in the physiological assay described under “Methods.” The specific activity of both forms of the enzyme was 32 units/mg at pH 6.8. Ascaris phosphofructokinase (7.2 pg) of each form of the enzyme were added to start the reaction. (a), enzyme isolated in the presence of fluoride; (O), enzyme isolated in the absence of fluoride.

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Phosphorylation of Ascaris Phosphofructokinase 3809

, O L I - I ,

0 2 4 6 8 1 0 6 0 Tame. mtn

FIG. 2. Phosphorylation of Ascaris phosphofructokinase with [y-3’P]ATP and the catalytic subunit of CAMP-dependent protein kinase from rabbit muscle. Two mg (5.2 nmol) of Ascarts phosphofructokinase ( P F K ) (104 units) was incubated with 20 1-18 o f catalytic subunit, 0.2 pmol of [y-’”1’1ATl’ (5.3 X 10‘’ cpm/pmol), 0.25 pmol of MgCI,, 50 pmol of potassium phosphate, pH 7.6, 30 pmol of KF, and 5 pmol of dithiothreitol in a volume of 1 ml. Aliquots were removed and treated as described under “Methods.”

I -,/ 1 -

- 1 o Electrophoresis -

1

I ”

I Start

1

FIG. 3. Autoradiograph of the tryptic peptide fingerprint of 32P-labeled Ascaris phosphofructokinase. Fifty p g of the .“l’- labeled enzyme (Fig. 2) was digested with trypsin, fingerprinted, and exposed to x-ray film, as described under “Methods.” The F denotes solvent front in the chromatography.

min, a total of 3 mol of ‘“P were incorporated/mol of tetramer of phosphofructokinase. Analysis of phosphate content of the product revealed 6.3 +- 0.6 mol of phosphate/tetramer. In order to determine the specificity of phosphorylation by the catalytic subunit, a portion of the labeled protein was sub- jected to tryptic digestion and peptide fingerprinting. Fig. 3 is an autoradiograph of the tryptic fingerprint of the enzyme. There were two peptides separated by the procedure which exhibited approximately equal amounts of radioactivity.

I I

3.04

!\

O l d ~ !~ I 1 ~ - 1 - I 0 10 20 30 40 5 0 60

Time. min.

FIG. 4. Dephosphorylation of Ascaris [:’2P]phosphofructoki- nase by E. coli alkaline phosphatase. The [.’“l’]phosphofructoki- nase (216 p g , 2.9 X 10‘’ cpm) was incubated at 30 “ C with 55 p g of alkaline phosphatase in 50 pmol of Tris-HCI. pH 8.1, and 50 pmol o f (NH,),SO, in a volume of 0 .5 ml. Aliquots were removed and treated as described under “Methods.” PFK, phosphofructokinase.

TAHLE I1 Activity ofphosphoylated and dephosphoylated Ascaris

phosphofructokinase in a physiological assuy

PFK” ___ ~~~~~~~ ~ - - ~ -

” ~

Fnr-6-1’ Activity” Activity’ Activity“ ___ . ”~ ~ . .

P .w Phosphorylated 50 0.33 0.32 3.2

100 0.63 0.64 5.1 Dephosphorylated 50 0.13 0.3 1 3.1

1 00 0.25 0.65 5.2

~~

unlls/mp

___~~. ___- ~. ~ .~

“ The two forms of the phosphofructokinases were from the exper; iments depicted in Figs. 2 and 4.

”The assay consisted of: 50 mM imidazole/HCI, pH 6.8, 5.25 mM potassium phosphate, 8 mM MgCI,, 101.3 mM KCI, 3 mM ATP. 1 mhr

phosphofructokinase (specific activity of both forms was 30 units/mg at pH 6.8). 50 p g of aldolase, 30 p g of glycerol phosphate dehvdrogen- ase, and 10pg of triose phosphate isomerase. The reaction was started by the addition of Fru-6-1’. Final volume, 1 ml; temperature, 30 “C.

‘ Five p g of catalytic suhunit of CAMP-dependent protein kinase was added to the assay 2 min prior to starting the reaction hy the addition of Fru-6-1’.

ADP, 0.36 mM AMI’, 25 pM Gk-l,6-1’~, 0.16 mM KADH, 2 p g of

“ Five mM AMI’ was added to the assay.

Fig. 4 illustrates the effect of incubating E . coli alkaline phosphatase with the labeled phosphofructokinase. Over 90% of the radioactivity was released within 1 h of incubation. Analysis of the phosphate content of the dephosphorylated protein revealed 2.8 +- 0.3 mol of phosphate remaininghetra- mer. In order to establish that the solubilization of the phos- phate was the result of phosphatase activity and not proteo- lytic activity, electrophoresis of the trichloroacetic acid-solu- ble product was conducted. The results of an autoradiograph of this electrophoresis (data not shown) showed that the product co-migrated in electrophoresis with inorganic phos- phate.

Table I1 depicts the activities of the phosphorylated (Fig. 2) and dephosphorylated (Fig. 4) enzymes in the physiological assay at two concentrations of Fru-6-P. In each case, the phosphorylated form of the enzyme exhibited a 2.5-fold higher activity than the dephosphorylated enzyme. Preincubation of the catalytic subunit of the CAMP-dependent protein kinase with the phosphorylated form of the enzyme in the physiolog- ical assay resulted in no change in the rate. However, the rate of the dephosphorylated form increased 2.4- to 2.6-fold after addition of the catalytic subunit. Finally, maximum activities at each concentration, brought about by the addition of 5 mM AMP, were the same for both forms of the enzyme.

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3810 Phosphorylation of Ascaris Phosphofructokinase

DISCUSSION

Two differentially phosphorylated forms of phosphofruc- tokinase have been purified from A. suum muscle. One form contained two phosphates per subunit and one of these phos- phates could be removed presumably by endogenous phos- phatase activity during purification in the absence of fluoride. Hofer and Sgrenson-Ziganke (6) have demonstrated that phosphofructokinase from freeze-clamped, contracting rabbit muscle has about 2 ml of phosphate covalently bound/subunit and the enzyme from resting muscle has 1 mol of phosphate/ subunit. It appears that this single phosphate is not easily removed or exchanged. Tryptic fingerprints (9) of the enzyme labeled in vivo with 32P or in vitro with [y3’P]ATP and the catalytic subunit of CAMP-dependent protein kinase exhibited two labeled peptides, but one contained much more radioac- tivity than the other. With the ascarid enzyme, one phosphate also appeared to be refractory to both endogenous and exog- enous phosphatase activity. A form of the enzyme without phosphate could not be generated.

The high and low phosphate forms of the enzyme isolated from the muscle in the presence and absence of fluoride, respectively, appeared to be similar, if not identical, to the phosphorylated and dephosphorylated forms produced in ui- tro. All forms of the enzymes exhibited similar specific activ- ities in the optimal assay and also the same specific activity in the physiological assay when ATP inhibition was reversed by excess AMP. On the other hand, the forms containing one phosphate per subunit had 2- to 2.5-fold lower activities than did the forms containing two phosphates per subunit. In addition, preincubation of the dephosphorylated form in the physiological assay with protein kinase resulted in a 2.5-fold stimulation of activity. These results suggest that the addition of one phosphate per subunit is sufficient to cause a significant increase in enzymatic activity. This phosphorylation appears to result in a form of the enzyme that is less inhibited by ATP. This is in contrast to the liver phosphofructokinase, which, when phosphorylated, is more inhibited by ATP (10-12).

When the low phosphate enzyme was phosphorylated with the protein kinase, tryptic fingerprints revealed two labeled peptides although less than 1 mol of radioactive phosphate had been incorporated/subunit. It is possible that the two labeled peptides resulted from incomplete tryptic digestion of the native enzyme, although it should be mentioned that tryptic fingerprints of cyanogen bromide-treated enzyme (data not shown) also exhibited two spots of radioactivity. An alternative explanation is that both sites are accessible to the kinase, but the evidence against this is that the alkaline phosphatase removed essentially all of the radioactivity, but phosphate remained on the enzyme. Further characterization of the labeling pattern is necessary to determine the correct alternative.

The muscle of the nematode parasite, A . suum, primarily derives energy from glycolysis and the subsequent malate dismutation in the mitochondria (23, 24). Thus, metabolic pathways in the parasite muscle must depend on these reac- tions for production of ATP. The muscle is specialized for this metabolism since it stores glycogen to a level of 20% of its fresh weight (25, 26). Further, the parasite has no hold-fast organs (hooks, suckers, etc.) and it must constantly “swim” against the distally directed action of the host’s intestine in order to maintain position in the gut. Therefore, it is doubtful that carbohydrate catabolism is ever completely stopped in this muscle (26). On the other hand, the rate of glycolysis might be expected to be elevated when the muscle activity is increased to meet the demands of more rapid intestinal peri- stalsis. Regulation of this increased glycolytic rate must de-

pend on modulation of phosphofructokinase activity (27) by effectors and/or other factors. The results presented here suggest that a portion of the phosphofructokinase activity in Ascaris muscle might be regulated by phosphorylation. This phosphorylation, in turn, could be accomplished in part by the CAMP-dependent protein kinase. Recent work from this laboratory (28) has demonstrated the presence of this kinase in the muscle of the parasite and has also shown that the enzyme is similar to isozyme I of the protein kinase found in mammalian muscle. Furthermore, recent studies on Ascaris muscle segments perfused with the putative hormone sero- tonin (5-hydroxytryptamine) have correlated increased CAMP levels with inactivation of glycogen synthase and activation of glycogen phosphorylase (29). Therefore, it is possible that a coordinate regulation of phosphofructokinase with the glyco- gen-metabolizing enzymes might occur. Further work is being carried out on this aspect at the present time.

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Riquelme, P. T., Hosey, M. M., Marcus, F., and Kernp, R. Y.

Uyeda, K., Miyatake, A,, Luby, L. J., and Richards, E. G. (1978)

Hofer, H. W. (1978) FEBS Proc. Meet. 5b, 413-414 Hofer, H. W., and Sorenson-Ziganke, B. (1979) Biochem. Biophys.

Riquelme, P. T., and Kemp, R. G. (1980) J. Biol. Chem. 255,

Krystek, E., and Hofer, H. W. (1981) Biochem. Biophys. Res.

S~renson-Ziganke, B., and Hofer, H. W. (1979) Biochem. Biophys.

Kagimoto, T., and Uyeda, K. (1979) J. Biol. Chem. 254, 5584-

Castaiio, J . G., Nieto, A,, and Feliu, J. E. (1979) J. Biol. Chem.

Pilkis, S., Schurnpf, J., Pilkis, J., and Claus, T. H. (1979) Biochem.

Furuya, E., and Uyeda, K. (1979) J. Biol. Chem. 255,11656-11659 Furuya, E., and Uyeda, K. (1980) Proc. Natl. Acad. Sci. U.S.A.

Richards, C. S., and Uyeda, K. (1980) Biochem. Biophys. Res. Commun. 97, 1535-1540

Van Schaftingen, E., and Hers, H.-G. (1980) Biochem. Biophys. Res. Commun. 96, 1524-1531

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Page 5: Phosphofructokinase from Ascaris suum

H W Hofer, B L Allen, M R Kaeini and B G Harrisnear-physiological conditions.

Phosphofructokinase from Ascaris suum. The effect of phosphorylation on activity

1982, 257:3807-3810.J. Biol. Chem. 

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