- Email: [email protected]
Yeast phosphofructokinase: Effector-controlled conversion of an ATP-sensitive to an ATP-desensitized form
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
Yeast Phosphofructokinase:
Bioch.elnisches
Znstilut
der
to
Freiburg jar
Gesellschajt
Conversion
an ATP-Desensitized
D. RUPPERT, Cn.iversitdt
(1971)
Effector-Controlled
ATP-Sensitive E.-G. AFTISG,
1&&58i-592
V. HAGMAIER, Breisgau,
.im
and
Strahlenjorschung
Form’ AXD
H. HOLZER
Forschungsgruppe m.b.H.
Received December 11, 1970; accepted January
of an
jiir
Biochenrie
de?-
%I, 1971
The conversion of yeast-PFKs (sensitive phosphofructokinase, inhibited by concentrations of ATP 2 10e4 M) to PFKd (desensitized PFK, with hyperbolic dependency of the reaction rate on the concentration of ATP) is stimulated by ADP. ATP inhibits desensitization. In addition t.o ADP, the following substances are essential to desensitization: Fru6-P, NH,+, Mg*+, and F’. Cyclic 3’,5’-AMP and 5’AMP are not necessary. In the presence of these effecters no “desensitizing protein” is necessary for the conversion of PFKs to PFKd. The protein fraction described in t,he earlier publications as “desensitizing protein” does not affect PFK itself, but, changes the composition of the incubation medium in such a way that the effectorstimulated desensitization of PFR takes place. The biological significance of t.hesc observations is discussed. In 1964 Vifiuels et al. (1) ident,ified t,wo different forms of yeast PFIi (,4TP:wfructose-6-phosphate l-phosphotransferase, EC 2.17.1.11). One form is inhibited by concentra.tions of A4TP higher than 0.1 rnlr and is, therefore, called ATP-sensitive (PFKs). The other form is not inhibited by excess -4TP. This desensitized form has been designat,ed PFKd Q). Vifiuela et al. (1) interpreted t,heir experiments as evidence for a “conversion enzyme,” which, upon incubation with ?tIgATP, IYnF, and cyclic 3’) 5’-4JIP, converts PFK.s to PFKd. Procedures for purification of yeast PFK to a homogeneous st,ate have been described by Lindell and Stellwagen (a), by .Jauch el al. (4), and by At.zpodien and Bode (5). Using t,he highly ,4TP-sensitive preparation of Atzpodien et al. (5) :IY substrate, the “desensitizing protein” 11a.s been purified about SO-fold (6). It has been shown that the two interconvertible forms of PFK are stable (6), and tha.t in addition to the “desensitizing prot.ein”, a hea.&stable fraction from yeast is necessary for desensi-
1 Dedicated to Prof. Dr.Dr.h.c. Feodor T,yncn on the occasion of his Wth birthd:ty.
t,izat,ion (‘2). In analogy 1vit.h t.he regulation of muscle phosphorylase, an enzyme-catalyzed interconversion of PFKs to PFKd via ATP-dependent phosphorylation (1) or some other chemical modification (7, S) of PFI(s has been postulat.ed. In the present article it, is demonst,rated t’hat, what has been designated as “desent.izing prot.ein” does not, in fact, modify PI%, but. alters the composit,ion of the incubation medium in such a way that a negat.ive effectar (ATP) of the transformation of PI;& to PFKd disappears and is replaced by a positive effector (ADP). In addition t,o ADP the following met,:lbolit.es and ions have been found t.o be essent.ial t.o the conversion of PFKs to PFKd: Fru-6-P, 3Ig?+, SH.,+, and F’. M.4TERIALS AN 11 >IETHODS of the etqmes. The ATP-sensitive form of phosphofructokinase (PFKs) was purified from commercial baker’s yeast. (Pleser Hefefsbrik, Darmstadt) according to Btzpodien and Bode (5j. The phosphofructokinase activity was determined by a coupled assay described by Atzpodien and Bode (5) with slight modifications. The assay
587
Purijication
mixture (2 ml) contained 50 mM imidaaole/HCl buffer pH 7.5, 2.5 mu MgCle, 2 mM mercaptoethanol, 0.25 mu Fru-&P, 0.3 mllr NADTI, 0.05 mu or 1.0 mM ATP, and an excess of aldolaae, trioscphosphate isomerase and a-glycerophosphate dehydrogenase. Incubation WBS at 25”. The phosphofructokinase “desensit,izing prot.ein” was prepared as described by Atzpodien et al. (6). The precipitate of the first, ammonium sulfate fractionation st,ep (35-70yc saturaiion) was used in the following experiments. Protein was measured by t.he method of Lowry el al. (9) using boviue serum albumin M standard. Standard inwbation mizlure jar ccmversion of PFKs lo PFKd. The standard incubation mixt.ure for conversion of t,he enzyme forms contained 59 mu pot.assium phosphate buffer ~11 6.8, or 50 rnM imidazole/HCl buffer pH 6.8, 1.0 mM magnesium chloride, 1.0 mM AMP, 0.1 rn.w ATP, 0.07 mM Fru-G-P, and 10 mM sodium fluoride in a total volume of 1.0 ml. Addition t,o this medium of 40-80 ag purified phosphofruct.okinase and 4000 /.lg “desensitizing proicin” comprises the “complete mixture.” The conversion reaction was carried out for 15 min at 20”. Samples were removed from the conversion mixture for enzyme assay. The conversion is arresied by 1Wfold dilution into t,he assay mixture. Xea~uren1en.l of conve,aion by determina&n of 12~l.l’. The dependencies of the activities of t,he two forms of PFK on t.he concentrat.ion of ATP
are shown rai.io
in Fig.
K.&T,. =
1. As in the previous
work
Activity
at 0.05 rnM
Activity
at 1.0 rnM ATP
ATP
was selected as a measure of the extent. of conversion. As can be seen from Fig. 1 an KA’I.I~ valne of 5.0 (A2.0) is typical for form Y and 0.0 (410.4) for form cl. Delerminalion of A ‘I’!‘, AM’, AMP. To derermine the act.ual concentrations of ATP, Al)]‘, and AMP in t,he complete mixture before and after incubation, t,he mixture was ultrafil tered (Amicon Corp., Cambridge, Mass., filter PM 10) and the protein-free filtrate assayed for AI)P and AMP according t,o .4dam (10) and for ATP according t.o Lamprecht. (11). All other ultra!ilt,rations were carried out with the same device. (Jhen&-als. Phosphoenolpyruvate, adenosinetriphosphate, adenosinediphosphate, adenosincmonophosphate, NAI)H, SAl)P, FrnctoseA-phosphate, lactate dehydrogenaae, pyruvate kinase, myokinaac, glucose-(i-phosphate dehydrogenase, aldolase, glycerophosphate dehydrogenase, and t,riosephosphatc isomerase aerc purchased from Boehringer & fioehne, GmbH., Mannheim. All other chemicals used were analyl ical gradcx re:lgelits obtained from commercial sourc’es. RESULTS Experimerlt 1 in Table 1 shows the converRio11 of P121i.s to PFIitl in the complete mix-
“0 x .E
150.
E \ ui x w a
A
----
--+
_-----------
;-
l ’
:
iT2 100 ‘5 -.-
p’s
g
0
I
\
0
ii
d
2 al .-2 % z
,+-+---+---+
\ O\,
50 . \ OYo-
z c % f -t
02
OL
b 0.6 OS
Fro. 1. Dependence of the phosphofructokinase (For assay see Met,hods). 0-0-O PFKs, PFKd as described in legend of Table IV.
(6) the
activit,y X-X-X
[NP]l
on the concentration of ATP. PFKd. PFKs was convert.ed to
CONVERSIOK
OF YISAHT PHOHPIIOFJ~UCTOKINAHI:
ml
that “desensitizing protein” was effect,ivrly excluded from the medium by ultrafilt,ratiou. The upper part, of Table II gives the measured concentrations of the adenosine nucleotides in the conversion medium before and after incubation iu the “complete mixture.” KA.I.~ = activity at 0.0.5 IIIJL ATP/rctivity From column 2 it can be seenthat duriug the at 1.0 III>, .ATP EsperiIncubation mixture _-....._... -.~.. merit no. incubation ATP completely disappears and Before After incubation incubation is recovered to 90% as ADP. A3IP conccntration is uot measurably altered during the 1 Complete mixt,ure 3.5 0.65 incubation. Apparently the convwsiou of 2 Incubation mixture --0.54 ATP to ADP is due to -4TPase activity confrom I’:xpt.. 1 after tained in the “desensitizing protein.” Direct incubation ultramrnsurement of the .4TP:w activity of the filtered, then addi “desensitizing protein” according to I,amCon of PFKs again precllt, cl a/. (12) suffices to account. for the 3 Complete mixture 5.5 3.6 without ATP and observed ATP hydrolysis. The lower part of AMP Table II shons that, while the complete 4.7 4 Incubated mixture mixture minus “desensitizing prot(ain” is inof Rxpt. 3 idtrafileffect.iw in the I’E‘Iis to PJ’Tirl convcrsiou, :I tered, then +ATP, mixt.ure constituted to resemble tlwt found +AMP, +PFKa after incubation is fully effective without, 5 Incubated mixture 0.38 “desensitizing prot&u.” of ICxpt.. 4 + “deEarliw studies had failed to demonstritte a sensitizing prostimulating effect. of ADP on the PFlis to tein” -PFKrl conversion. The results preseutcd in Table III explain this failure. Earliw an ture. In addiCon to pure PlXs and enriched effect of ADP was sought only in the prtw “desensit.i.zing protein,” the mixture con- once of ATP, under which condition little tained ATP, ALIP, I:ru-G-P, Rig?+ and 1:‘. increase in PE’L conversion takes place (line If, after conversion to PlXd, one removes 2 of Table III). In the absenceof ATP, howPFK and “desensitizing protein” by ultraever, the stimulation by ADP (linr 4 of filtration, and t.hen adds fresh PJ’Iis, it, too, Table III) is plainly visible. ATP curtails the is converted to Pl;Iirl in the absenceof “de- XDP-stimulated conversion, presumably besensitizing protein” (see Table I, Expt. 2). cause in its presencethe obligatory effector, This shows1hat. “desensitizing protein” does l’ru-(i-P, is removed by ~~h~~s~~hor~l;~tio~~. not, alter PI:Ji itself, but rather that the medium is somehow altered (perhaps with T.4131,15 1I the participation of PI:R) such that the de.~l,I~:sosrNI~: ~LX’I.EOTII)KS .ANI) I )ESXNSITI~.ITIOX sensitization can take place. ok- PFKs That, the “desensitizing protein” is indeed Standard Standard removed by t.he ultrafiltration t.echniyue is mixture mixture Concentration UIIJI) before after demonstrated in t,he cont.rol experiments $4, incubation incubation and 5. Experiment, 3 shamsthat, in an ot.her- - -.-. wise complete mixture, lacking only AMP ATP 0.1 0.0 and ATP, no conversion of s- to r/-form takes AL)P 0.0 0.00 place. If AMP, ATP, and new PT:I
690
AFTTNG TABLIS
.- ---.
III
b;FFISCTS OF ADP AND I)IGSENSTTIZITTON
ATP
ON THE
E?’ dl,.
not, subsMute for ADP, nor work synergistically wit.h ADP.
OF PFKa J)ISCUSSION
Incubation
i3.4~1~ = activity at 0.05 mu ATP/activity at 1.0 mu ATP
mixture
Before incubation
After incubation
Complete mixture” 6.3 4.8 + ADP” 6.3 3.3 - ATP 3.4 6.3 - ATP, + ADPh 6.3 0.3 6.3 ._._ - - ATP, + ADP,” - AMP -.-..- 0.4 (1Without “desensitizing protein,” + NHd+. * 0.1 mM.
The present, experiments show that, what was earlier described as a “desensitizing protein” does not catalyze a modification of PFK itself, but, :I permissive modification of the medium so that the conversion to the desensitized form of PP’K proceeds on its own. In view of the essent.ial effecters the conversion may be formulated as follows: PP KS
ADP, Frn-6-P, NHf, Mg2+, F’
* PP lid.
Comparative physical measurements of PlqKs and Pl;I
IV
CONVERSION
OF YEAST
;,!I1
PHOSPHOFl~UCTOKINAPI:
Glucose
Fructose
catabolic
products
FIG. 2. Effector-controlled ATP-desensitization of yeast-phosphofnlctokinaze. The two forms of PFK are characterized by Michaelis curves for ATP wit.h (PFKs) and without. (PFKd) substrabe inhibit,ion. @ = stimulation of PFK-desensitization. Fru-6P = fructose-6-phosphate, Fru-1,6-Pz = fructose-1,6-diphosphate.
ACKSOWLEDGMENTS subsequent amidation to glutamine (14, 15). The substrat,e for NHd+ fixation, aMupport,ed by Bundesminist.erium fiir wissenket.oglutarate, is provided by glycolysis in schaftliche Forachung and SonderforschungsbeGrundlagen der Entwicklung.” concert with the citric acid cycle. Thus, it. reich “Molekulare appears also purposeful bhat NHd+ stimuREFERENCES lat,es the conversion to PI%2 and con1. VIRURLA, TX., Sagas, M. I,., S.\L.\S, M., .\SD sequently glycolysis. The specificit,y of the SOLS, A., Biochcm. Biophys. Res. Comnmn. h’H1+ effect supports t,his idea: Ii+, ?rTa+,and 16, 243 (1964). Cs+ are without effect (Aft,ing et al., manu2. GANCEDO, J. M., ATZ~OIHKN, W., AND lfoLzI.:n, script in preparation). H., Pd. Eur. Biochem. Sot. LSK 6, 199 The above discussion relates only to the (1969). effects of various eff ectors on the conversion 3. LINDELL, T. J., AND STELI.\~AGEX, E:., .f. Biol. of the ATP-sensit,ive form of PFK to the Chem. 243, 907 (1968). insensitive form. But, these effect.ors which 4. JAIJCH, R., RIEPEHTINGEH, C., AND LYNEN, F., promote conversion (and a few others) also Hoppe-Seyler’s %. fhysiol. Chetn. 361. 74 (1970). influence the cat.alvt.ic qualities of the W., ;\NI) T~ODIC, H., E.w. .I. Bioenzyme, i.e., the ra$dit?: of I:ru-6-P phos- 5. ATZPODIKL, rhenz. 12, 126 (1970). phorylation. The situ&on is thus more W., GANCKDO, J. M., HAGMAIER, complicated than represented in Fig. 2. One 6. ATZPODIKN, V., AND Ilor,z~a, IT., Eur. J. Biochem. 12, can attempt a comprehensive discussion of 0 (1970). PFK regulation only when more exhaustive 7. VIm?ELA, E., Mamas, M. L., A~VU SOLS, A., investigations of the desensitization of PFK Biochem. Biophys. Hes. Cotntttun. 12, 140 and especially of the propert,ies of PFKs and (1963). PFKd are completed. 8. HOLZER, H., in “Advances in Enzyme RegulaDiscussionsof the catalytic characteristics t,ion” (G. Weber, ed.), Vol. 8, p. 85. Pergamon, New York (1970). of yeast PFKs without, regard to the con9. Lown~, 0. H., ROSEBI~~UGH, S. J., FM~R, version of s-form to d-form have been A. L., :\ND RAND.~LL, R. J., J. Biol. Chetn. undertaken by several earlier investigators 193, 265 (1951). of this enzyme: Jauch et al. (4), &ellwagen 10. ADU, H., in “Methoden der enzymatischen et al. (3, IS), Liebe et al. (17), and Sols Analyse” (H. U. Bergmeyer, ed.), p. 573. et al. (18, 19). Verlag Chemie, Weinheim (19G2).
59”
AFTING El’ AL.
11. LA~PRECHT,W., .~ND TI~~UTSCHOI.D, J., in “Mct.hodeu der enzymatischen Analyse” (II. U. Bergmeyer, ed.), p. 543. Verlag Chemie, Weirtheim (1962). 12. L,ZMPKECHT, W., .\ND ~~~~~~~~~~~~~~~ J., in “Methoden der enzymatischen Analyse” (H. U. Bergmeyer, ed.), p. 2024. Verlag Chemie, Weinheim (1970). 13. HOLZEB,II., Ann. Rev. Biochem. 28, 171 (1959). 14. WITT, I., AND HOLZ~, H., &&em. %. 889, 255 (1984). 15. ITOLZIZR, II., HIEUHOLZEH, G., AND WITT, I., Colloq. Inf. C.X.R.S. No. 124. 407 (1965).
IG. Mtvls,
It. I)., .\su
STKLL~VAGKN,
IC.,J. Hid.
Clhs7u.246, 674 (1970). 17. IJICIIE, ST., IiOPP~:ltSCHI.ii(;K11, (;., hb:Zl~:L,
K., WOLF, J.,
W., NISSL~R,
E., Fed. h’ur. Hiochem.
18. Sors, A.,
AND
K.u..\s,M.
Ellsymology”
AND
IIOIWANS,
Sec. IAl. 8, 20 (1970).
L., in “Methods in
(S. P. Colowick and S. 0.
Kaplan, eds.), Vol. IX, p. 438. Academic Press, New York (1908). 19.
B~LAS,
Aem.
M. L., S.\I,.\S,J., Biophys.
AND
Res. Commun.
SOLS,
A., Bio-
31. 461 (1968).
OF
BIOCHEMISTRY
AND
BIOPHYSICS
Yeast Phosphofructokinase:
Bioch.elnisches
Znstilut
der
to
Freiburg jar
Gesellschajt
Conversion
an ATP-Desensitized
D. RUPPERT, Cn.iversitdt
(1971)
Effector-Controlled
ATP-Sensitive E.-G. AFTISG,
1&&58i-592
V. HAGMAIER, Breisgau,
.im
and
Strahlenjorschung
Form’ AXD
H. HOLZER
Forschungsgruppe m.b.H.
Received December 11, 1970; accepted January
of an
jiir
Biochenrie
de?-
%I, 1971
The conversion of yeast-PFKs (sensitive phosphofructokinase, inhibited by concentrations of ATP 2 10e4 M) to PFKd (desensitized PFK, with hyperbolic dependency of the reaction rate on the concentration of ATP) is stimulated by ADP. ATP inhibits desensitization. In addition t.o ADP, the following substances are essential to desensitization: Fru6-P, NH,+, Mg*+, and F’. Cyclic 3’,5’-AMP and 5’AMP are not necessary. In the presence of these effecters no “desensitizing protein” is necessary for the conversion of PFKs to PFKd. The protein fraction described in t,he earlier publications as “desensitizing protein” does not affect PFK itself, but, changes the composition of the incubation medium in such a way that the effectorstimulated desensitization of PFR takes place. The biological significance of t.hesc observations is discussed. In 1964 Vifiuels et al. (1) ident,ified t,wo different forms of yeast PFIi (,4TP:wfructose-6-phosphate l-phosphotransferase, EC 2.17.1.11). One form is inhibited by concentra.tions of A4TP higher than 0.1 rnlr and is, therefore, called ATP-sensitive (PFKs). The other form is not inhibited by excess -4TP. This desensitized form has been designat,ed PFKd Q). Vifiuela et al. (1) interpreted t,heir experiments as evidence for a “conversion enzyme,” which, upon incubation with ?tIgATP, IYnF, and cyclic 3’) 5’-4JIP, converts PFK.s to PFKd. Procedures for purification of yeast PFK to a homogeneous st,ate have been described by Lindell and Stellwagen (a), by .Jauch el al. (4), and by At.zpodien and Bode (5). Using t,he highly ,4TP-sensitive preparation of Atzpodien et al. (5) :IY substrate, the “desensitizing protein” 11a.s been purified about SO-fold (6). It has been shown that the two interconvertible forms of PFK are stable (6), and tha.t in addition to the “desensitizing prot.ein”, a hea.&stable fraction from yeast is necessary for desensi-
1 Dedicated to Prof. Dr.Dr.h.c. Feodor T,yncn on the occasion of his Wth birthd:ty.
t,izat,ion (‘2). In analogy 1vit.h t.he regulation of muscle phosphorylase, an enzyme-catalyzed interconversion of PFKs to PFKd via ATP-dependent phosphorylation (1) or some other chemical modification (7, S) of PFI(s has been postulat.ed. In the present article it, is demonst,rated t’hat, what has been designated as “desent.izing prot.ein” does not, in fact, modify PI%, but. alters the composit,ion of the incubation medium in such a way that a negat.ive effectar (ATP) of the transformation of PI;& to PFKd disappears and is replaced by a positive effector (ADP). In addition t,o ADP the following met,:lbolit.es and ions have been found t.o be essent.ial t.o the conversion of PFKs to PFKd: Fru-6-P, 3Ig?+, SH.,+, and F’. M.4TERIALS AN 11 >IETHODS of the etqmes. The ATP-sensitive form of phosphofructokinase (PFKs) was purified from commercial baker’s yeast. (Pleser Hefefsbrik, Darmstadt) according to Btzpodien and Bode (5j. The phosphofructokinase activity was determined by a coupled assay described by Atzpodien and Bode (5) with slight modifications. The assay
587
Purijication
mixture (2 ml) contained 50 mM imidaaole/HCl buffer pH 7.5, 2.5 mu MgCle, 2 mM mercaptoethanol, 0.25 mu Fru-&P, 0.3 mllr NADTI, 0.05 mu or 1.0 mM ATP, and an excess of aldolaae, trioscphosphate isomerase and a-glycerophosphate dehydrogenase. Incubation WBS at 25”. The phosphofructokinase “desensit,izing prot.ein” was prepared as described by Atzpodien et al. (6). The precipitate of the first, ammonium sulfate fractionation st,ep (35-70yc saturaiion) was used in the following experiments. Protein was measured by t.he method of Lowry el al. (9) using boviue serum albumin M standard. Standard inwbation mizlure jar ccmversion of PFKs lo PFKd. The standard incubation mixt.ure for conversion of t,he enzyme forms contained 59 mu pot.assium phosphate buffer ~11 6.8, or 50 rnM imidazole/HCl buffer pH 6.8, 1.0 mM magnesium chloride, 1.0 mM AMP, 0.1 rn.w ATP, 0.07 mM Fru-G-P, and 10 mM sodium fluoride in a total volume of 1.0 ml. Addition t,o this medium of 40-80 ag purified phosphofruct.okinase and 4000 /.lg “desensitizing proicin” comprises the “complete mixture.” The conversion reaction was carried out for 15 min at 20”. Samples were removed from the conversion mixture for enzyme assay. The conversion is arresied by 1Wfold dilution into t,he assay mixture. Xea~uren1en.l of conve,aion by determina&n of 12~l.l’. The dependencies of the activities of t,he two forms of PFK on t.he concentrat.ion of ATP
are shown rai.io
in Fig.
K.&T,. =
1. As in the previous
work
Activity
at 0.05 rnM
Activity
at 1.0 rnM ATP
ATP
was selected as a measure of the extent. of conversion. As can be seen from Fig. 1 an KA’I.I~ valne of 5.0 (A2.0) is typical for form Y and 0.0 (410.4) for form cl. Delerminalion of A ‘I’!‘, AM’, AMP. To derermine the act.ual concentrations of ATP, Al)]‘, and AMP in t,he complete mixture before and after incubation, t,he mixture was ultrafil tered (Amicon Corp., Cambridge, Mass., filter PM 10) and the protein-free filtrate assayed for AI)P and AMP according t,o .4dam (10) and for ATP according t.o Lamprecht. (11). All other ultra!ilt,rations were carried out with the same device. (Jhen&-als. Phosphoenolpyruvate, adenosinetriphosphate, adenosinediphosphate, adenosincmonophosphate, NAI)H, SAl)P, FrnctoseA-phosphate, lactate dehydrogenaae, pyruvate kinase, myokinaac, glucose-(i-phosphate dehydrogenase, aldolase, glycerophosphate dehydrogenase, and t,riosephosphatc isomerase aerc purchased from Boehringer & fioehne, GmbH., Mannheim. All other chemicals used were analyl ical gradcx re:lgelits obtained from commercial sourc’es. RESULTS Experimerlt 1 in Table 1 shows the converRio11 of P121i.s to PFIitl in the complete mix-
“0 x .E
150.
E \ ui x w a
A
----
--+
_-----------
;-
l ’
:
iT2 100 ‘5 -.-
p’s
g
0
I
\
0
ii
d
2 al .-2 % z
,+-+---+---+
\ O\,
50 . \ OYo-
z c % f -t
02
OL
b 0.6 OS
Fro. 1. Dependence of the phosphofructokinase (For assay see Met,hods). 0-0-O PFKs, PFKd as described in legend of Table IV.
(6) the
activit,y X-X-X
[NP]l
on the concentration of ATP. PFKd. PFKs was convert.ed to
CONVERSIOK
OF YISAHT PHOHPIIOFJ~UCTOKINAHI:
ml
that “desensitizing protein” was effect,ivrly excluded from the medium by ultrafilt,ratiou. The upper part, of Table II gives the measured concentrations of the adenosine nucleotides in the conversion medium before and after incubation iu the “complete mixture.” KA.I.~ = activity at 0.0.5 IIIJL ATP/rctivity From column 2 it can be seenthat duriug the at 1.0 III>, .ATP EsperiIncubation mixture _-....._... -.~.. merit no. incubation ATP completely disappears and Before After incubation incubation is recovered to 90% as ADP. A3IP conccntration is uot measurably altered during the 1 Complete mixt,ure 3.5 0.65 incubation. Apparently the convwsiou of 2 Incubation mixture --0.54 ATP to ADP is due to -4TPase activity confrom I’:xpt.. 1 after tained in the “desensitizing protein.” Direct incubation ultramrnsurement of the .4TP:w activity of the filtered, then addi “desensitizing protein” according to I,amCon of PFKs again precllt, cl a/. (12) suffices to account. for the 3 Complete mixture 5.5 3.6 without ATP and observed ATP hydrolysis. The lower part of AMP Table II shons that, while the complete 4.7 4 Incubated mixture mixture minus “desensitizing prot(ain” is inof Rxpt. 3 idtrafileffect.iw in the I’E‘Iis to PJ’Tirl convcrsiou, :I tered, then +ATP, mixt.ure constituted to resemble tlwt found +AMP, +PFKa after incubation is fully effective without, 5 Incubated mixture 0.38 “desensitizing prot&u.” of ICxpt.. 4 + “deEarliw studies had failed to demonstritte a sensitizing prostimulating effect. of ADP on the PFlis to tein” -PFKrl conversion. The results preseutcd in Table III explain this failure. Earliw an ture. In addiCon to pure PlXs and enriched effect of ADP was sought only in the prtw “desensit.i.zing protein,” the mixture con- once of ATP, under which condition little tained ATP, ALIP, I:ru-G-P, Rig?+ and 1:‘. increase in PE’L conversion takes place (line If, after conversion to PlXd, one removes 2 of Table III). In the absenceof ATP, howPFK and “desensitizing protein” by ultraever, the stimulation by ADP (linr 4 of filtration, and t.hen adds fresh PJ’Iis, it, too, Table III) is plainly visible. ATP curtails the is converted to Pl;Iirl in the absenceof “de- XDP-stimulated conversion, presumably besensitizing protein” (see Table I, Expt. 2). cause in its presencethe obligatory effector, This shows1hat. “desensitizing protein” does l’ru-(i-P, is removed by ~~h~~s~~hor~l;~tio~~. not, alter PI:Ji itself, but rather that the medium is somehow altered (perhaps with T.4131,15 1I the participation of PI:R) such that the de.~l,I~:sosrNI~: ~LX’I.EOTII)KS .ANI) I )ESXNSITI~.ITIOX sensitization can take place. ok- PFKs That, the “desensitizing protein” is indeed Standard Standard removed by t.he ultrafiltration t.echniyue is mixture mixture Concentration UIIJI) before after demonstrated in t,he cont.rol experiments $4, incubation incubation and 5. Experiment, 3 shamsthat, in an ot.her- - -.-. wise complete mixture, lacking only AMP ATP 0.1 0.0 and ATP, no conversion of s- to r/-form takes AL)P 0.0 0.00 place. If AMP, ATP, and new PT:I
690
AFTTNG TABLIS
.- ---.
III
b;FFISCTS OF ADP AND I)IGSENSTTIZITTON
ATP
ON THE
E?’ dl,.
not, subsMute for ADP, nor work synergistically wit.h ADP.
OF PFKa J)ISCUSSION
Incubation
i3.4~1~ = activity at 0.05 mu ATP/activity at 1.0 mu ATP
mixture
Before incubation
After incubation
Complete mixture” 6.3 4.8 + ADP” 6.3 3.3 - ATP 3.4 6.3 - ATP, + ADPh 6.3 0.3 6.3 ._._ - - ATP, + ADP,” - AMP -.-..- 0.4 (1Without “desensitizing protein,” + NHd+. * 0.1 mM.
The present, experiments show that, what was earlier described as a “desensitizing protein” does not catalyze a modification of PFK itself, but, :I permissive modification of the medium so that the conversion to the desensitized form of PP’K proceeds on its own. In view of the essent.ial effecters the conversion may be formulated as follows: PP KS
ADP, Frn-6-P, NHf, Mg2+, F’
* PP lid.
Comparative physical measurements of PlqKs and Pl;I
IV
CONVERSION
OF YEAST
;,!I1
PHOSPHOFl~UCTOKINAPI:
Glucose
Fructose
catabolic
products
FIG. 2. Effector-controlled ATP-desensitization of yeast-phosphofnlctokinaze. The two forms of PFK are characterized by Michaelis curves for ATP wit.h (PFKs) and without. (PFKd) substrabe inhibit,ion. @ = stimulation of PFK-desensitization. Fru-6P = fructose-6-phosphate, Fru-1,6-Pz = fructose-1,6-diphosphate.
ACKSOWLEDGMENTS subsequent amidation to glutamine (14, 15). The substrat,e for NHd+ fixation, aMupport,ed by Bundesminist.erium fiir wissenket.oglutarate, is provided by glycolysis in schaftliche Forachung and SonderforschungsbeGrundlagen der Entwicklung.” concert with the citric acid cycle. Thus, it. reich “Molekulare appears also purposeful bhat NHd+ stimuREFERENCES lat,es the conversion to PI%2 and con1. VIRURLA, TX., Sagas, M. I,., S.\L.\S, M., .\SD sequently glycolysis. The specificit,y of the SOLS, A., Biochcm. Biophys. Res. Comnmn. h’H1+ effect supports t,his idea: Ii+, ?rTa+,and 16, 243 (1964). Cs+ are without effect (Aft,ing et al., manu2. GANCEDO, J. M., ATZ~OIHKN, W., AND lfoLzI.:n, script in preparation). H., Pd. Eur. Biochem. Sot. LSK 6, 199 The above discussion relates only to the (1969). effects of various eff ectors on the conversion 3. LINDELL, T. J., AND STELI.\~AGEX, E:., .f. Biol. of the ATP-sensit,ive form of PFK to the Chem. 243, 907 (1968). insensitive form. But, these effect.ors which 4. JAIJCH, R., RIEPEHTINGEH, C., AND LYNEN, F., promote conversion (and a few others) also Hoppe-Seyler’s %. fhysiol. Chetn. 361. 74 (1970). influence the cat.alvt.ic qualities of the W., ;\NI) T~ODIC, H., E.w. .I. Bioenzyme, i.e., the ra$dit?: of I:ru-6-P phos- 5. ATZPODIKL, rhenz. 12, 126 (1970). phorylation. The situ&on is thus more W., GANCKDO, J. M., HAGMAIER, complicated than represented in Fig. 2. One 6. ATZPODIKN, V., AND Ilor,z~a, IT., Eur. J. Biochem. 12, can attempt a comprehensive discussion of 0 (1970). PFK regulation only when more exhaustive 7. VIm?ELA, E., Mamas, M. L., A~VU SOLS, A., investigations of the desensitization of PFK Biochem. Biophys. Hes. Cotntttun. 12, 140 and especially of the propert,ies of PFKs and (1963). PFKd are completed. 8. HOLZER, H., in “Advances in Enzyme RegulaDiscussionsof the catalytic characteristics t,ion” (G. Weber, ed.), Vol. 8, p. 85. Pergamon, New York (1970). of yeast PFKs without, regard to the con9. Lown~, 0. H., ROSEBI~~UGH, S. J., FM~R, version of s-form to d-form have been A. L., :\ND RAND.~LL, R. J., J. Biol. Chetn. undertaken by several earlier investigators 193, 265 (1951). of this enzyme: Jauch et al. (4), &ellwagen 10. ADU, H., in “Methoden der enzymatischen et al. (3, IS), Liebe et al. (17), and Sols Analyse” (H. U. Bergmeyer, ed.), p. 573. et al. (18, 19). Verlag Chemie, Weinheim (19G2).
59”
AFTING El’ AL.
11. LA~PRECHT,W., .~ND TI~~UTSCHOI.D, J., in “Mct.hodeu der enzymatischen Analyse” (II. U. Bergmeyer, ed.), p. 543. Verlag Chemie, Weirtheim (1962). 12. L,ZMPKECHT, W., .\ND ~~~~~~~~~~~~~~~ J., in “Methoden der enzymatischen Analyse” (H. U. Bergmeyer, ed.), p. 2024. Verlag Chemie, Weinheim (1970). 13. HOLZEB,II., Ann. Rev. Biochem. 28, 171 (1959). 14. WITT, I., AND HOLZ~, H., &&em. %. 889, 255 (1984). 15. ITOLZIZR, II., HIEUHOLZEH, G., AND WITT, I., Colloq. Inf. C.X.R.S. No. 124. 407 (1965).
IG. Mtvls,
It. I)., .\su
STKLL~VAGKN,
IC.,J. Hid.
Clhs7u.246, 674 (1970). 17. IJICIIE, ST., IiOPP~:ltSCHI.ii(;K11, (;., hb:Zl~:L,
K., WOLF, J.,
W., NISSL~R,
E., Fed. h’ur. Hiochem.
18. Sors, A.,
AND
K.u..\s,M.
Ellsymology”
AND
IIOIWANS,
Sec. IAl. 8, 20 (1970).
L., in “Methods in
(S. P. Colowick and S. 0.
Kaplan, eds.), Vol. IX, p. 438. Academic Press, New York (1908). 19.
B~LAS,
Aem.
M. L., S.\I,.\S,J., Biophys.
AND
Res. Commun.
SOLS,
A., Bio-
31. 461 (1968).