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Metabolism of human erythrocyte glucose-6-phosphate dehydrogenase
ARCHIVES
OF
BIOCHEMISTRY
AND
Metabolism
BIOPHYSICS
of Human Phosphate
VI. Interconversion A. HONSIGNORE,
Institute
147, 493-601 (1971)
Erythrocyte
Glucose-6
Dehydrogenase of Multiple
Molecular
R. CANCEDDA, A. NICOLINI, AND A. DE FLORA of Biochemistry,
University
Forms’ G. DAMIANI
of Genova, Genova, Italy
Received April 29, 1971; accepted September 11, 1971 Human erythrocyte glucose-6-phosphate dehydrogenase exists in two catalytically active forms, i.e., tetramers and dimers, and also monomers which are intrinsically devoid of activity. Several factors are involved in the interconversion of the three species. High pH and ionic strength favor dissociation of the tetramer to dimers, while low values of pH and ionic strength and in addition some divalent cations shift the equilibrium toward the tetramers. NADPH,, formed during the operation of glucose-6-P dehydrogenase, inactivates the enzyme through a specific disaggregation which affects only the dimeric form. Dissociation of the tetramer to inactive monomers can, however, occur through a compulsory mechanism involving t,he previous conversion to dimers which are themselves disaggregated by NADPHe. Therefore, when the enzyme is working in a system where NADPHZ is not immediately reoxidized (i.e., in the absence of glutathione reductase), a sequential tetramer dimer - monomer conversion may occur as a result of the enzymatic activity.
Recent investigations have resolved earlier controversial findings on the molecular size and the subunit structure of human erythrocyte glucose-6-phosphate dehydrogenase (Dglucose-6-phosphate : NADP oxidoreductase, EC 1.1.1.49). These studies led to the following conclusions: (a) the subunits of glucose-6-P dehydrogenase are composed by a single polypeptide chain (l), they are most probably identical (l-4) and catalytically “inactive” (Ogunmola, Cancedda, and Luzzatto, unpublished data). (b) Two discrete polymeric states have been described (2, 5), namely tetramers and dimers, both of which are associated with catalytic activity: the balance between the two active forms is shifted toward the tetramers by low values of pH and ionic strength (2, 5, 6) and also by some divalent cations (7), while dissociation to dimers prevails at alkaline pH and 1 This work was supported by a Grant from the Italian Consiglio Nazionale delle Ricerche.
high salt concentrations (1, 2, 5, 6). (c) At variance with glucose-6-phosphate dehydrogenase from other sources, the erythrocyte enzyme has some tightly bound NADP (S12). This “structural” NADP plays an important role in stabilizing the enzyme in an active conformation (by probably affecting the secondary and tertiary structure) (10, 13, 14) and also in determining the ordered assembly of basic subunits (9, 10, 15, 16). It has been reported that incubation with NADP-glycohydrolase (EC 3.2.2.6) (15) and also washing with both acidic (10) and slightly alkaline (9) ammonium sulfate produce disaggregation and inactivation of erythrocyte glucose-B-phosphate dehydrogenase. The same effects, i.e., dissociation and loss of catalytic activity, have been shown following incubation of the enzyme in presence of its substrate glucose-6-P as well as of the product NADPHz (17, 18). Evidence has also been provided that treatment 493
494
BONSIGNORE
of the monomers obtained by the foregoing procedures with both NADP and SH groups is accompanied by reaggregation and reactivation (9, 10, 15, 17, 18). The present investigation aimed at better elucidating the glucose-6-P (NADPH2)-induced inactivation, which was therefore evaluated under a wide set of conditions displacing the equilibrium between tetramers and dimers. The data reported herein are consistent with the following model, according to which only the dimeric species of glucose6-P dehydrogenase can directly dissociate to monomers, while the tetramers appear to be intrinsically resistant to disaggregation to monomers : Tetramer
2
Dimer
G-6-P(NADPHe)b Monomer
Moreover the two sets of equilibria, i.e. between tetramers and dimers and between dimers and monomers, appear to be closely interdependent on an equilibrium basis. These facts support the view that the catalytic operation of erythrocyte glucose-6-P dehydrogenase in vitro involves a continuous interconversion of multiple molecular forms arising from at least two association-dissociation systems. MATERIALS
AND
METHODS
Glucose-6-P (Na and Ba salts), NADP, glucose-6-phosphate dehydrogenase NADPHs, from yeast (“Zwischenferment”), liver catalase, liver and yeast alcohol dehydrogenase were obtained from Boehringer and Sons, Mannheim, Germany. Tris, glycyl-glycine, bovine serum albumin and NAD (P)-glycohydrolase from Neurospora crassa were purchased from Sigma Chemical Co., St. Louis, MO. EDTA (disodium salt) was purchased from Schuchardt, Munchen, Germany. B-mercaptoethanol was obtained from AG, Buchs, Switzerland, Coomassie Fluka, brilliant Blue from Serva, Heidelberg, Germany and Amido Black from C. Erba, Milan, Italy. All reagents for disc gel electrophoresis were supplied from Canalco, Bethesda, MD. Glucose-g-phosphate dehydrogenase was purified from human red cells as previously described (6). The catalytic activity was assayed as reported elsewhere (7). Protein was determined according to the procedure of Lowry et al. (19). NAD(P)-glycohydrolase was tested for the hydrolysis of NADP according to the method described by Kaplan (23).
ET AL. Centrifugations on 546% linear sucrose gradients were performed in a Spinco ~-65 B preparative ultracentrifuge, using SW 25, or SW 27, or SW 50.1 rotors (1, 15, 17). Gel filtration experiments and calculations of the Stokes radii were carried out as described in a previous paper (1) using Sephadex G-266 in a K-25 column (Pharmacia). Unless otherwise specified, disc gel electrophoreses were routinely run at room temperature (25”) with 7.5yo acrylamide: other conditions were as reported in a previous communication (21) with the exception that 5 X 10-6~ NADP was added to the electrophoretic buffer and to the gels; occasionally, lO+M glucose-6-P was also present in both buffers and gels. For staining of the protein Coomassie brilliant Blue or Amido Black was used (6). For parallel staining of activity the method previously described by us (21) was utilized. RESULTS
Factors Affecting the Tetramer-Dimer Equilibrium Table I summarizes some patterns of the active population of glucose-6-P dehydrogenase, as expressed by physical parameters, under a number of environmental conditions in aqueous solvents. From these experiments it is clear that dissociation of the tetramers to the dimers occurs when the pH and (or) the ionic strength of the buffer are raised, while a reverse effect, namely association, is promoted by low pH and ionic strength as well as by Mg2+ ions. However, tetramerization was observed in presence of other divalent cations (7). All the solvent conditions tested produced a partial loss of catalytic activity (Table I). This inactivation, which is due to either low or alkaline pH, or to the high salt concentrations per se, was found to be markedly variable in extent; moreover it is unrelated to any formation of monomers, as shown by investigations at the electron microscope (Wrigley and De Flora, unpublished data). The foregoing changes of the dimer/tetramer ratio were reproducible at several steps of the purification procedure. Confirmatory evidence of these structural variations depending on solvent parameters was provided with the homogeneous enzyme by means of electrophoresis on polyacrylamide which allows complete separation of the three indi-
INTERCONVERSION TABLE
OF HUMAN
495
G6PD
I
EFFECT OF SOLVENT CONDITIONSON ACTUAL POLYMERICFORMSOF G6PDn
0.02 M phosphate 0.02 M phosphate 0.05 M MgSOh 0.02 M phosphate 0.02 M phosphate 0.05 M MgS04 0.02 M phosphate 0.1 M MgS04 0.02 M phosphate 0.15 M NaCl 0.02 M Tris-HCI 0.01 M Tris-HCl 0.02 M phosphate 1 M NaCl 0.05 M phosphate 0.04 M glycylglycine
+
7.0 7.0
7.39 8.95
51.5
low moderate
+
7.5 7.5
6.81 8.6
-
low moderate
+
7.5
8.2
-
high
+
7.5
6.19
-
moderate
+
8.0 8.5 6.5
6.53 6.29 5.81
43.0 40.7 -
6.5 7.5
8.51 6.62
-
low moderate very high low low
= Centrifugation on linear sucrose gradients and chromatography on Sephadex G-200 were performed as described under Methods. All the buffers contained 0.2% @mercaptoethanol (vol/ vol) and NADP ranging from 2 and 5 PM. GGPD concentrations ranged from 3.0 and 50 pg/ml. b The sedimentation coefficients (a$, w values) of tetramers and dimers are 9.18 S and 5.75 S, respectively (l), while the corresponding Stokes radii are 51.5 d (tetramers) and 40.7 i (dimers).
vidual forms of glucose-6-P dehydrogenase, probably because of the molecular sieving effect displayed by the gel. Dissociation by Glucose-6-P When glucose-6-P dehydrogenase was dialyzed against a large excess of glucose-6-P and then incubated at 37” with this substrate, a decrease of catalytic activity was observed, accounting for about 45% of the original activity. Gel chromatography of the partially inactivated enzyme revealed a single peak of activity (Fig. 1) corresponding to the residual fraction of glucose-6-P dehydrogenase
which
had
escaped
the
inactiva-
tion. The addition of an excess of oxidized NADP to the individual fractions produced a reactivation, the maximum of which was centered behind the peak of the native enzyme. By calibrating the same column with
ml effluent
FIG. 1. Gel chromatography of G6PD following partial inactivation by glucose-6P. Purified GGPD (0.092 mg, corresponding to 16.5 IU) was dialyzed 5 hr at 2” against 1,000volumes of 0.02 M Tris-HCl, pH 8.0, containing 1 X 10-4~EDTA, 1 X 10-6~ NADP, 0.2% &mercaptoethanol (vol/ vol) and 2 X 10-6~ G6P. This treatment led to an inactivation amounting to 45% of the original activity, while no loss of activity occurred by omitting glucose-6-P from the dialysis buffer. 10 mg of N-4 phage (kindly provided by Dr. G. Rialdi) were added to the partially inactivated enzyme (0.9 ml) and the mixture was layered over a K-25 column (Pharmacia) equipped with a flow adaptor and filled with Sephadex G-200 (89 X 2.5 cm, i.d.). Elutionwas accomplished using the same buffer employed in the dialysis. Upward flow was used at a rate of 8 ml/hr and 2.5 ml fractions were collected which were immediately assayed for N-4 phage (by measuring the absorbance at 260 rnp) and for GBPD activity (open circles). Starting from the peak of enzymic activity 0.5 ml samples of each fraction were then made 1.6 X 10-3~ in NADP, incubated 40 min at 25” and again assayed for catalytic activity (filled circles indicate the difference between the second and the first assay of catalytic activity, after appropriately correcting the former values for dilution). The same column was then calibrated with the following standards which were run separately from GBPD : N-4 phage (to measure the void volume), liver catalase, liver and yeast alcohol dehydrogenase, bovine serum albumin and bovine hemoglobin. Elution was as above and assay of the fractions for the standards employed showed that the gel pore parameter, r(22), had nearly identical values within the two chromatographies. Calculation of the Stokes radii was performed as reported above (l), according to the Stokes-Einstein equation (23).
496
BONSIGNORE
ET AL.
+ FIG. 2. Electrophoresis of native, glucose-6-P dissociated and reassociated GGPD. Tracing A: native enzyme. Tracing B: enzyme following dissociation by glucose-6-P. Tracing C: enzyme following reaggregation by NADP and p-mercaptoethanol. 10 pg of purified enzyme were incubated for 60 min at 37” in tightly capped small tubes containing also 0.01 M phosphate buffer, pH 7.0, in 32y0 sucrose (final concentration), 10-S M NADP, 2 X 10-6 M EDTA and 0.040/,. &mercaptoethanol, to a final volume of 0.05 ml (A). Two mixtures, containing also 1 X lO+ M G6P and 0.2% p-mercaptoethanol besides the enzyme and the above components, were incubated in parallel: one of these was directly processed through electrophoresis (B), while the second one, after addition of NADP to a f&al concentration of 2 X lO+ M, was incubated further at 37 for 60 min (C). Electrophoresis of mixtures A, B and C was performed at 25” for 70 min on gels prepared as described previously (21), excepting a 7.5% concentration of acrylamide in all gels and presence of 1 X 10-a M G6P in gel B. The electrophoretic buffer was 0.01 M Tris-HCl, pH 8.4 (final value) containing 1.3 X UYa M EDTA and 1 X WE M NADP (gels A and C) and0.01 M Tris-HCl, pH 7.4, containing 1.3 X lO+ M EDTA and 1 X 1W M G6P (gel B). During the experiment the current was held constant at 5 mA per gel column, which corresponded to 115-150 V. Staining for protein was with Amido Black. Microdensitometric tracings were made at a wavelength of 560 rnp with a Gilford mod 240 spectrophotometer fitted with a mod. 2410 linear transport unit, at a rate of 2 cm/min.
a number of protein standards, the Stokes radii of both active and inactive (i.e., reactivatable) enzyme forms could Obedetermined and found to be 40.7 and 30.7 A, respectively. These values are coincident with those of dimers and monomers, respectively (1). Since the Sephadex gels (24), as well as sucrose (25), have been reported to produce themselves a shift of several association equilibria toward the smaller species, the disso-
ciation of glucose-6-P dehydrogenase by glucose-6-P was further investigated by means of disc gel electrophoresis. Figure 2 shows that with a preparation of native enzyme (tracing A) three electrophoretie bands appear which correspond, as previously demonstrated (6), to tetramers, dimers and monomers, respectively, in order of increased mobility toward the anode. Preincubation of glucose-6-P dehydrogenase with
INTERCONVERSION
L-
30
60 time (minutes]
90
I
FIG. 3. Effect of Mgzf ions on the inactivation by NADPHz. Purified GGPD (0.695 mg) was dialyzed against 1,666 volumes of 5 X lca~ TrisHCI, pH 8.5, containing 1 X 10-Q EDTA, 4 X ~O-@MNADP, 0.1% p-mercaptoethanol. 3.2 pg of the dialyzed enzyme were incubated 15 min at 2.5” with 5 X Wsnn Tris-HCl, pH 8.5, containing EDTA, NADP and @-mercaptoethanol as in the dialysis buffer (filled circles), 2 X 10% MgSOc (half-filled circles) and 3 X 10-2~ MgSOc (open circles). After 15 min incubation at room temperature each of the three mixtures was divided into two parts: both the first half, after receiving NADPHZ to a final concentration of 1.4 X 10-4~~ and the second half (control) were incubated at 37”. At the times indicated aliquots were assayed for catalytic activity and values were corrected for variations of the controls.
the substrate glucose-6-P, under conditions where “autoinactivation” (17,1S) occurs, results in the complete disappearance of both polymeric forms of the enzyme and in the parallel increase of the anodic band, namely of the monomers (tracing B). By reincubating the dissociated enzyme with excess NADP and p-mercaptoethanol, the resulting electrophoretic pattern (C) becomes similar to that of native glucose-6-P dehydrogenase (A), therefore indicating reassembly of the subunits under these conditions. Distinctive Regulatory Properties of Dimers and of Tetramers The marked extent of dimer-dimer association occurring upon addition of divalent cations (7) was utilized to evaluate comparatively the inactivation of tetramers and dimers by NADPHz . The results obtained are represented in Fig. 3. Preincubation of the enzyme with MgSO, , which is known to dedetermine tetramerization, reduces signifi-
OF HUMAN
GGPD
497
cantly the extent of inactivation by NADPHz as compared with a system lacking Mg2+ and containing mainly dimeric glucose6-P dehydrogenase. AU the experiments designed to obtain almost homogeneous dimers or tetramers were affected to some variable extent by the instability of control samples (see the last column of Table I). This unfavorable situation, due to the factors necessary to modify the equilibrium between the two polymeric species, was overcome by following the initial rate of inactivation by NADP-glycohydrolase of the tetramers and the dimers, respectively. This prevented both forms of glucose6-P dehydrogenase from being inactivated by any other environmental parameter than by NADP-glycohydrolase. It has been previously shown, by means of density gradient centrifugation, that the latter enzyme dissociates glucose-6-P dehydrogenase to inactive monomers by primarily hydrolyzing the Ystructural” NADP (15). Table II shows that an almost 100% pure population of tetramers retains full catalytic activity during a 5 min. incubation with excess NADP-glycohydrolase: on the other hand this NADP-cleaving enzyme produces a dramatic inactivation of the same sample of glucose-6-P dehydrogenase where a high ionic strength and a pH of 8.8 have determined a marked dissociation to dimers (73% of active glucose-6-P dehydrogenase, as calculated from the ~~00,~value (2, 7, 12)). The experimental time has been restricted to 5 minutes since longer periods of incubation at 37” resulted in a differential loss of activity of the control samples containing either tetramers or dimers. Relationship Between the Tetramer-Dimer and the Dimer-Monomer Equilibria The apparent discrepancy between the intrinsic resistance of the tetramers to inactivation by dissociating factors (Fig. 3 and Table II), and the complete disappearance of the same molecular form following catalytic operation of glucose-6-P dehydrogenase (Figs. 1 and 2) prompted a careful investigation of the overall tetramerdimer-monomer syst,em in the course of the inactivation by glucose-6-P. To this
498
BONSIGNORE TABLE
II
COMPAR.~TIVE INACTIVATION OF GGPD BY TETRllMERIC GLYCOHYDROLASE NADPh"'YZo; Buffer’
(g-gc
DIMERIC
AND
NADP-
G6PD activity
Kase -__ Units/ml %$ ml
0.02 M phosphate pH 6.0 0.04 M Tris-HCl pH 8.8 + 0.2 M NaCl
S 0’
5’
352
9.15
2.66
2.60
295
6.69
2.58
1.08
a Incubation at 37” in a water bath. GGPD activity was measured within the first 20 see, to minimize cleavage of NADP by NADP-glycohydrolase in the assay mixture. Controls lacking NADP-glycohydrolase and incubated in parallel showed complete stability during 5 min incubation. b Assay of NADP-glycohydrolase was performed according to Kaplan (20) directly on the inactivation mixtures after 5 min incubation. c 0.15 ml of the respective controls were layered over 5-200/, linear sucrose gradients containing the same buffer and centrifuged 11 hr at 4” at 45,000 rpm in a SW 50.1 rotor. 90 see fractions were collected by means of a mod. Perpex LKB peristaltic pump fitted with a 10 ml/hr reduction gear.
purpose, electrophoretic patterns were followed at different time intervals during incubation of the enzyme with its substrate (Fig. 4). Dissociation of glucose-6-P dehydrogenase to monomers is progressive within 45 min, whereas at longer incubation times the equilibrium tends to favor reaggregation of the monomeric units. Fig. 4 shows that during conversion of the enzyme to monomers, the decrease of the middle band (dimers) is accompanied by an almost equivalent reduction of the right band, namely of the tetramers. Such a pattern is consistent with the view that dissociation of the tetramers to the basic monomers is a compulsory pathway involving formation of the dimeric species as intermediate. This hypothesis received further support by experiments of density gradient centrifugation , where the sedimentation of
ET AL.
glucose-6-P dehydrogenase was evaluated in presence and in absence of NADPH2, respectively. With the exception of a low recovery of catalytic activity in the NADPHz-containing sample, which is obviously accounted for by inactivation, no major differences were observed between the migration of activities with and without NADPHz (Fig. 5). The same result was obtained by processing glucose-6-P dehydrogenase through gradient centrifugation, following its partial inactivation by glucose-6-P, namely under the conditions reported in Fig. 4. Both the electrophoretic analysis and the centrifugation experiments showed therefore that conversion of the dimer to monomers involves the secondary dissociation of the tetramer, as it is indicated by a constant dimer/tetramer ratio. DISCUSSION
A number of lines of evidence seem to indicate that of the two active forms of glucose-6-P dehydrogenase, i.e., tetramers and dimers, only the dimeric species can undergo dissociation to monomers. In fact, a) both inactivation and dissociation by NADPH2 are maximal at those pH values (8.5-9.5) where the balance of active species is largely displaced toward the dimers (26) ; b) no dissociation of the tetramers has been found (27) under conditions in which NADPHZ is formed (the actual pH of the experiment was 6.0). The present paper provides additional experimental evidence for the above hypothesis, based on the demonstration that the dimer is highly susceptible to dissociating factors such as NADPH, and NADP-glycohydrolase, while the tetrameric form is intrinsically resistant to both mechanisms (Fig. 3 and Table II). The physiological meaning of the above distinctive features appears however rather doubtful when one considers that the two dissociation equilibria are closely interrelated. We have in fact shown that, if other critical conditions are kept constant (protein concentration, solvent, temperature), the displacement of either equilibrium such as the dimer to monomer conversion,
INTERCONVERSION
OF HUMAN
GGPD
499
r
0’
(no
C6P)
+
10’
(7 ti’ G6P)
90’
(7mE’ GhP)
+ 45’
(7mM G6P)
FIG. 4. Electrophoretic modifications of GGPD during inactivation by NADPH2. Experimental conditions were as described in the legend to Fig. 2, excepting the concentration of G6P in the inactivation mixtures (7 X 1(r3 M instead of 1 X l(r4 M). The rate of scanning was 1 cm/min. Arrows indicate the peaks of the three molecular forms.
does not affect appreciably the other one (Figs. 4 and 5). A consequence of the interdependence of the two dissociation systems should be a depletion of the pool of both active forms
when the enzyme is working in a closed system, i.e., when NADPHZ accumulates as a product of the glucose-6-P dehydrogenase reaction. However, the inactivation by the reduced coenzyme, as well as that
BONSIGNORE
ET AL.
experiments where major parameters were unchanged. The reasons for the incomplete glucose-6-P (NADPHg)-induced inactivation and for its quantitative variations are still unclear. I . A possible explanation could be occurrence of additional enzyme species (28)-which, however, have escaped recognition despite attempts to identify them-involved in an jeven more complicated equilibrium t#han that postulated so far. An alternative hypothesis is that some monomers, arising from dissociation of the dimer, are either intrinsically active or somehow reactivated during the assay of catalytic activity. According to this hypothesis, the effect of NADPHz on a structural basis would be greater than indicated by the loss of activity. This could explain the electrophoretic finding that the dissociating effect of glucose-6-P (NADPH,) is most times complete (Fig. 2) or nearly complete (Fig. 4), at variance with the extent of inactivation, although a further displacement toward dissociation of the dimer-monomer equilibrium during migration on polyacrylfractions amide gel cannot be ruled out. To elucidate FIG. 5. Sucrose gradient sedimentation of acthis point which is closely related to the tive GBPD before and after inactivation by mechanism of inactivation by glucose-6-P NADPH2. Twenty-five micrograms of dialyzed (NADPHI), experiments are in progress to enzyme were incubated at 37” in 0.04 M Tris-HCl, measure the intrinsic molecular activity pH 8.0, containing 1 X IO-% EDTA, 1 X 10e6an of tetramers, dimers and monomers by NADP, 0.2a/, B-mercaptoethanol and 1.4 X 10-Q means of rapid mixing techniques. NADPH2, to a final volume of 1.5 ml. A control The interconversion among structurally lacking NADPHz was incubated in parallel. The and functionally different molecular forms net loss of activity produced by NADPHz within is a mechanism through which the activity 40 min incubation was 0.95 units/ml, starting from an initial value of 3.0 units/ml. After the incubaof several key enzymes can be widely t,ion, 1.0 ml of each of the two mixtures was modulated (18, 29). In most cases the layered together with 5 mg COHb on a 5-2070 balance is dictated by specific superimposed linear sucrose gradient containing the same comenzymes, catalyzing the interconversion, for ponents as the incubation experiments. Centriwhich the term ‘primary regulatory enzymes” fugation was for 40 hr at 5” in a SW 25.1 rotor has been recently proposed (30). Human using a Beckman L-2 centrifuge. Fractions were glucose-6-P dehydrogenase appears to lack collected by means of a Perpex peristaltic pump such a sophisticated regulatory system (LKB) and assayed for catalytic activity (open applying to “dimorphic” or “polymorphic” circles) and for COHb by measuring the absorbenzyme proteins since the interconversion ance at 413 rnN (filled circles). Arrows indicate the among the three discrete species (taking theoretical migration of tetramers and dimers, respectively. place according to the scheme represented in Fig. 6) is more simply ligand-dependent. produced by glucose-6-P, is generally It is, however likely, on the basis of preincomplete and in addition extremely vious experiments (17, IS), that glutathione variable in extent, as shown in several reductase (Reduced-NAD(P) : oxidized-glu-
INTERCONVERSION High pH and
OF HUMAN Glucose 6-PIhmPH,
ionic strength
I ACTIVE)
\--\
IACTIVEI Low pH ond tom strength Mg’*, Mn*’
6. Factors regulating
FIG.
the tetramer-dimer
oxidoreductase, EC 1.6.4.2.) tathione fulfills within the erythrocyte the role of preventing
the inactivation
of glucose-6-P
dehydrogenase by NADPHZ, therefore acting on this protein and being accordingly involved
in the
regulation
of the
pentose
15. 16. 17.
phosphat’e pathway. 18. 19.
ACKNOWLEDGMENTS We are indebted to Professor S. Pontremoli for critical revision of the manuscript. The skillful technical assistance of Mr. F. Giuliano is gratefully acknowledged.
20.
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LORENZONI, FLORA, A.,
21.
Commun. 43, 94
(1971). ROSEMEYER, M. A., Eur. 8, 8 (1969). 3. YOSHIDA, A., J. Biol. Chem. 241, 4966 (1966). 4. YOSHID~, A., Biochem. Genet. 2, 237 (1968). 5. COHEN, P. AND ROSEMEYER, M. A., FEBS 2. COHEN,
P.,
I
DIMER !---
TETRAMER
AND
J. Biochem.
22. 23.
Lett. 1, 147 (1968).
24.
6. BONSIGNORE, A., LORENZONI, I., CANCEDDA, R., NICOLINI, A., DAMIANI, G. AND DE FLORA, A., Ital. J. Biochem. 19, 139 (1970). 7. BONSIGNORE, A., LORENZONI, I., CANCEDDA, M. E. AND DE FLORA, A., R., COSULICH,
Biochem. Biophys. Res. Commun. 43, 159 (1971). 8. KIRKMAN, H. N., J. Biol. Chem. 237, 2364 (1962). 9. KIRKM~N, H. N. AND HENDRICKSON, E. M., J. Biol. Chem. 23’7, 2371 (1962). 10. CHUNG, A, E. AND LANGDON, R. G., .J. Biol. Chem. 238, 2317 (1963). 11. YOSHIDA, A., Biochem. Genet. 1, 81 (1967). 12. BONSIGNORE, A., LORENZONI, I., CANCEDDA, R. .4ND DE FLORA, A., Biochem. Biophys. Res. Com,mun. 39, 142 (1970). 13. MARKS, P. A., SZEINBERG, A. BND BANKS, J., J. Biol. Chem. 236, 10 (1961). 14. LUZZATTO, L. AND ALLAN, N. C., Biochem. Biophys. Res. Commun. 21, 547 (1965).
25. 26.
27.
28.
29. 30.
501
GGPD
MONOMER
-ii
( INACTWE 1 NAOq-SH
and the dimer-monomer
equilibria.
A., DE FLORA, A., MANGIAA., LORENZONI, 1. AND ALEMA, S., Ital. J. Biochem. 16, 117 (1967). RATTAZZI, M. C., Biochem. Biophys. Res. Commun. 31, 16 (1968). BONSIGNORE, A., DE FLORA, A., LORENZONI, I., CANCEDDA, R., SILENGO, L. AND DINA, D., Ital. J. Biochem. 17, 346 (1968). DE FLORA, A., Ital. J. Biochem. 17, 363 (1968). LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L. AND RANDALL, R. J., J. Biol. Chem. 193, 265 (1951). KAPLAN, N. O., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, Eds.), Vol. II, p. 664, Academic Press, New York (1955). DE FLORA, A., LORENZONI, I., MANQIAROTTI, M. A., DINA, D. AND BONSIGNORE, A., Biochem. Biophys. Res. Commun. 31, 501 (1968). ACKERS, G. K., Biochemistry 3, 723 (1964). GOSTING, L. J., in “Advances in protein chemistry,” M. L. Anson, K. Bailey and J. T. Edsall Editors, Vol. 11, p. 449, Academic Press, New York, 522 (1956). DETERMAN, H., “Chromatographie sur gel,” Masson and Cie, Paris, p. 111 (1969). YIELDING, K. L., Biochem. Biophys. Res. Commun. 38. 546 (1970). R., LORENZONI, DE FLORA, A., CANCEDDA, I., NICOLINI, A. AND BONSIGNORE, A., Proceedings 1st International Symposium on “Metabolic Interconversion of Enzymes,” S. Margherita Ligure, p. 35 (1970). YOSHIDA, A. AND HOAGLAND, V. D., JR., Biochem. Biophys. Res. Commun. 40, 1167 (1970). BONSIGNORE, A., LORENZONI, I., CANCEDDA, R., MORELLI, S., GIULIANO, F. AND DE FLORA, A., Ital. J. Biochem. 18, 439 (1969). HOLZER, H., Advances Enzymol. 32, 297 (1969). SOLS, A., Proceedings 1st International Symposium on “Metabolic Interconversion of S. Margherita Ligure, p. 1 Enzymes,” (1970). BONSIONORE, ROTTI, M.
OF
BIOCHEMISTRY
AND
Metabolism
BIOPHYSICS
of Human Phosphate
VI. Interconversion A. HONSIGNORE,
Institute
147, 493-601 (1971)
Erythrocyte
Glucose-6
Dehydrogenase of Multiple
Molecular
R. CANCEDDA, A. NICOLINI, AND A. DE FLORA of Biochemistry,
University
Forms’ G. DAMIANI
of Genova, Genova, Italy
Received April 29, 1971; accepted September 11, 1971 Human erythrocyte glucose-6-phosphate dehydrogenase exists in two catalytically active forms, i.e., tetramers and dimers, and also monomers which are intrinsically devoid of activity. Several factors are involved in the interconversion of the three species. High pH and ionic strength favor dissociation of the tetramer to dimers, while low values of pH and ionic strength and in addition some divalent cations shift the equilibrium toward the tetramers. NADPH,, formed during the operation of glucose-6-P dehydrogenase, inactivates the enzyme through a specific disaggregation which affects only the dimeric form. Dissociation of the tetramer to inactive monomers can, however, occur through a compulsory mechanism involving t,he previous conversion to dimers which are themselves disaggregated by NADPHe. Therefore, when the enzyme is working in a system where NADPHZ is not immediately reoxidized (i.e., in the absence of glutathione reductase), a sequential tetramer dimer - monomer conversion may occur as a result of the enzymatic activity.
Recent investigations have resolved earlier controversial findings on the molecular size and the subunit structure of human erythrocyte glucose-6-phosphate dehydrogenase (Dglucose-6-phosphate : NADP oxidoreductase, EC 1.1.1.49). These studies led to the following conclusions: (a) the subunits of glucose-6-P dehydrogenase are composed by a single polypeptide chain (l), they are most probably identical (l-4) and catalytically “inactive” (Ogunmola, Cancedda, and Luzzatto, unpublished data). (b) Two discrete polymeric states have been described (2, 5), namely tetramers and dimers, both of which are associated with catalytic activity: the balance between the two active forms is shifted toward the tetramers by low values of pH and ionic strength (2, 5, 6) and also by some divalent cations (7), while dissociation to dimers prevails at alkaline pH and 1 This work was supported by a Grant from the Italian Consiglio Nazionale delle Ricerche.
high salt concentrations (1, 2, 5, 6). (c) At variance with glucose-6-phosphate dehydrogenase from other sources, the erythrocyte enzyme has some tightly bound NADP (S12). This “structural” NADP plays an important role in stabilizing the enzyme in an active conformation (by probably affecting the secondary and tertiary structure) (10, 13, 14) and also in determining the ordered assembly of basic subunits (9, 10, 15, 16). It has been reported that incubation with NADP-glycohydrolase (EC 3.2.2.6) (15) and also washing with both acidic (10) and slightly alkaline (9) ammonium sulfate produce disaggregation and inactivation of erythrocyte glucose-B-phosphate dehydrogenase. The same effects, i.e., dissociation and loss of catalytic activity, have been shown following incubation of the enzyme in presence of its substrate glucose-6-P as well as of the product NADPHz (17, 18). Evidence has also been provided that treatment 493
494
BONSIGNORE
of the monomers obtained by the foregoing procedures with both NADP and SH groups is accompanied by reaggregation and reactivation (9, 10, 15, 17, 18). The present investigation aimed at better elucidating the glucose-6-P (NADPH2)-induced inactivation, which was therefore evaluated under a wide set of conditions displacing the equilibrium between tetramers and dimers. The data reported herein are consistent with the following model, according to which only the dimeric species of glucose6-P dehydrogenase can directly dissociate to monomers, while the tetramers appear to be intrinsically resistant to disaggregation to monomers : Tetramer
2
Dimer
G-6-P(NADPHe)b Monomer
Moreover the two sets of equilibria, i.e. between tetramers and dimers and between dimers and monomers, appear to be closely interdependent on an equilibrium basis. These facts support the view that the catalytic operation of erythrocyte glucose-6-P dehydrogenase in vitro involves a continuous interconversion of multiple molecular forms arising from at least two association-dissociation systems. MATERIALS
AND
METHODS
Glucose-6-P (Na and Ba salts), NADP, glucose-6-phosphate dehydrogenase NADPHs, from yeast (“Zwischenferment”), liver catalase, liver and yeast alcohol dehydrogenase were obtained from Boehringer and Sons, Mannheim, Germany. Tris, glycyl-glycine, bovine serum albumin and NAD (P)-glycohydrolase from Neurospora crassa were purchased from Sigma Chemical Co., St. Louis, MO. EDTA (disodium salt) was purchased from Schuchardt, Munchen, Germany. B-mercaptoethanol was obtained from AG, Buchs, Switzerland, Coomassie Fluka, brilliant Blue from Serva, Heidelberg, Germany and Amido Black from C. Erba, Milan, Italy. All reagents for disc gel electrophoresis were supplied from Canalco, Bethesda, MD. Glucose-g-phosphate dehydrogenase was purified from human red cells as previously described (6). The catalytic activity was assayed as reported elsewhere (7). Protein was determined according to the procedure of Lowry et al. (19). NAD(P)-glycohydrolase was tested for the hydrolysis of NADP according to the method described by Kaplan (23).
ET AL. Centrifugations on 546% linear sucrose gradients were performed in a Spinco ~-65 B preparative ultracentrifuge, using SW 25, or SW 27, or SW 50.1 rotors (1, 15, 17). Gel filtration experiments and calculations of the Stokes radii were carried out as described in a previous paper (1) using Sephadex G-266 in a K-25 column (Pharmacia). Unless otherwise specified, disc gel electrophoreses were routinely run at room temperature (25”) with 7.5yo acrylamide: other conditions were as reported in a previous communication (21) with the exception that 5 X 10-6~ NADP was added to the electrophoretic buffer and to the gels; occasionally, lO+M glucose-6-P was also present in both buffers and gels. For staining of the protein Coomassie brilliant Blue or Amido Black was used (6). For parallel staining of activity the method previously described by us (21) was utilized. RESULTS
Factors Affecting the Tetramer-Dimer Equilibrium Table I summarizes some patterns of the active population of glucose-6-P dehydrogenase, as expressed by physical parameters, under a number of environmental conditions in aqueous solvents. From these experiments it is clear that dissociation of the tetramers to the dimers occurs when the pH and (or) the ionic strength of the buffer are raised, while a reverse effect, namely association, is promoted by low pH and ionic strength as well as by Mg2+ ions. However, tetramerization was observed in presence of other divalent cations (7). All the solvent conditions tested produced a partial loss of catalytic activity (Table I). This inactivation, which is due to either low or alkaline pH, or to the high salt concentrations per se, was found to be markedly variable in extent; moreover it is unrelated to any formation of monomers, as shown by investigations at the electron microscope (Wrigley and De Flora, unpublished data). The foregoing changes of the dimer/tetramer ratio were reproducible at several steps of the purification procedure. Confirmatory evidence of these structural variations depending on solvent parameters was provided with the homogeneous enzyme by means of electrophoresis on polyacrylamide which allows complete separation of the three indi-
INTERCONVERSION TABLE
OF HUMAN
495
G6PD
I
EFFECT OF SOLVENT CONDITIONSON ACTUAL POLYMERICFORMSOF G6PDn
0.02 M phosphate 0.02 M phosphate 0.05 M MgSOh 0.02 M phosphate 0.02 M phosphate 0.05 M MgS04 0.02 M phosphate 0.1 M MgS04 0.02 M phosphate 0.15 M NaCl 0.02 M Tris-HCI 0.01 M Tris-HCl 0.02 M phosphate 1 M NaCl 0.05 M phosphate 0.04 M glycylglycine
+
7.0 7.0
7.39 8.95
51.5
low moderate
+
7.5 7.5
6.81 8.6
-
low moderate
+
7.5
8.2
-
high
+
7.5
6.19
-
moderate
+
8.0 8.5 6.5
6.53 6.29 5.81
43.0 40.7 -
6.5 7.5
8.51 6.62
-
low moderate very high low low
= Centrifugation on linear sucrose gradients and chromatography on Sephadex G-200 were performed as described under Methods. All the buffers contained 0.2% @mercaptoethanol (vol/ vol) and NADP ranging from 2 and 5 PM. GGPD concentrations ranged from 3.0 and 50 pg/ml. b The sedimentation coefficients (a$, w values) of tetramers and dimers are 9.18 S and 5.75 S, respectively (l), while the corresponding Stokes radii are 51.5 d (tetramers) and 40.7 i (dimers).
vidual forms of glucose-6-P dehydrogenase, probably because of the molecular sieving effect displayed by the gel. Dissociation by Glucose-6-P When glucose-6-P dehydrogenase was dialyzed against a large excess of glucose-6-P and then incubated at 37” with this substrate, a decrease of catalytic activity was observed, accounting for about 45% of the original activity. Gel chromatography of the partially inactivated enzyme revealed a single peak of activity (Fig. 1) corresponding to the residual fraction of glucose-6-P dehydrogenase
which
had
escaped
the
inactiva-
tion. The addition of an excess of oxidized NADP to the individual fractions produced a reactivation, the maximum of which was centered behind the peak of the native enzyme. By calibrating the same column with
ml effluent
FIG. 1. Gel chromatography of G6PD following partial inactivation by glucose-6P. Purified GGPD (0.092 mg, corresponding to 16.5 IU) was dialyzed 5 hr at 2” against 1,000volumes of 0.02 M Tris-HCl, pH 8.0, containing 1 X 10-4~EDTA, 1 X 10-6~ NADP, 0.2% &mercaptoethanol (vol/ vol) and 2 X 10-6~ G6P. This treatment led to an inactivation amounting to 45% of the original activity, while no loss of activity occurred by omitting glucose-6-P from the dialysis buffer. 10 mg of N-4 phage (kindly provided by Dr. G. Rialdi) were added to the partially inactivated enzyme (0.9 ml) and the mixture was layered over a K-25 column (Pharmacia) equipped with a flow adaptor and filled with Sephadex G-200 (89 X 2.5 cm, i.d.). Elutionwas accomplished using the same buffer employed in the dialysis. Upward flow was used at a rate of 8 ml/hr and 2.5 ml fractions were collected which were immediately assayed for N-4 phage (by measuring the absorbance at 260 rnp) and for GBPD activity (open circles). Starting from the peak of enzymic activity 0.5 ml samples of each fraction were then made 1.6 X 10-3~ in NADP, incubated 40 min at 25” and again assayed for catalytic activity (filled circles indicate the difference between the second and the first assay of catalytic activity, after appropriately correcting the former values for dilution). The same column was then calibrated with the following standards which were run separately from GBPD : N-4 phage (to measure the void volume), liver catalase, liver and yeast alcohol dehydrogenase, bovine serum albumin and bovine hemoglobin. Elution was as above and assay of the fractions for the standards employed showed that the gel pore parameter, r(22), had nearly identical values within the two chromatographies. Calculation of the Stokes radii was performed as reported above (l), according to the Stokes-Einstein equation (23).
496
BONSIGNORE
ET AL.
+ FIG. 2. Electrophoresis of native, glucose-6-P dissociated and reassociated GGPD. Tracing A: native enzyme. Tracing B: enzyme following dissociation by glucose-6-P. Tracing C: enzyme following reaggregation by NADP and p-mercaptoethanol. 10 pg of purified enzyme were incubated for 60 min at 37” in tightly capped small tubes containing also 0.01 M phosphate buffer, pH 7.0, in 32y0 sucrose (final concentration), 10-S M NADP, 2 X 10-6 M EDTA and 0.040/,. &mercaptoethanol, to a final volume of 0.05 ml (A). Two mixtures, containing also 1 X lO+ M G6P and 0.2% p-mercaptoethanol besides the enzyme and the above components, were incubated in parallel: one of these was directly processed through electrophoresis (B), while the second one, after addition of NADP to a f&al concentration of 2 X lO+ M, was incubated further at 37 for 60 min (C). Electrophoresis of mixtures A, B and C was performed at 25” for 70 min on gels prepared as described previously (21), excepting a 7.5% concentration of acrylamide in all gels and presence of 1 X 10-a M G6P in gel B. The electrophoretic buffer was 0.01 M Tris-HCl, pH 8.4 (final value) containing 1.3 X UYa M EDTA and 1 X WE M NADP (gels A and C) and0.01 M Tris-HCl, pH 7.4, containing 1.3 X lO+ M EDTA and 1 X 1W M G6P (gel B). During the experiment the current was held constant at 5 mA per gel column, which corresponded to 115-150 V. Staining for protein was with Amido Black. Microdensitometric tracings were made at a wavelength of 560 rnp with a Gilford mod 240 spectrophotometer fitted with a mod. 2410 linear transport unit, at a rate of 2 cm/min.
a number of protein standards, the Stokes radii of both active and inactive (i.e., reactivatable) enzyme forms could Obedetermined and found to be 40.7 and 30.7 A, respectively. These values are coincident with those of dimers and monomers, respectively (1). Since the Sephadex gels (24), as well as sucrose (25), have been reported to produce themselves a shift of several association equilibria toward the smaller species, the disso-
ciation of glucose-6-P dehydrogenase by glucose-6-P was further investigated by means of disc gel electrophoresis. Figure 2 shows that with a preparation of native enzyme (tracing A) three electrophoretie bands appear which correspond, as previously demonstrated (6), to tetramers, dimers and monomers, respectively, in order of increased mobility toward the anode. Preincubation of glucose-6-P dehydrogenase with
INTERCONVERSION
L-
30
60 time (minutes]
90
I
FIG. 3. Effect of Mgzf ions on the inactivation by NADPHz. Purified GGPD (0.695 mg) was dialyzed against 1,666 volumes of 5 X lca~ TrisHCI, pH 8.5, containing 1 X 10-Q EDTA, 4 X ~O-@MNADP, 0.1% p-mercaptoethanol. 3.2 pg of the dialyzed enzyme were incubated 15 min at 2.5” with 5 X Wsnn Tris-HCl, pH 8.5, containing EDTA, NADP and @-mercaptoethanol as in the dialysis buffer (filled circles), 2 X 10% MgSOc (half-filled circles) and 3 X 10-2~ MgSOc (open circles). After 15 min incubation at room temperature each of the three mixtures was divided into two parts: both the first half, after receiving NADPHZ to a final concentration of 1.4 X 10-4~~ and the second half (control) were incubated at 37”. At the times indicated aliquots were assayed for catalytic activity and values were corrected for variations of the controls.
the substrate glucose-6-P, under conditions where “autoinactivation” (17,1S) occurs, results in the complete disappearance of both polymeric forms of the enzyme and in the parallel increase of the anodic band, namely of the monomers (tracing B). By reincubating the dissociated enzyme with excess NADP and p-mercaptoethanol, the resulting electrophoretic pattern (C) becomes similar to that of native glucose-6-P dehydrogenase (A), therefore indicating reassembly of the subunits under these conditions. Distinctive Regulatory Properties of Dimers and of Tetramers The marked extent of dimer-dimer association occurring upon addition of divalent cations (7) was utilized to evaluate comparatively the inactivation of tetramers and dimers by NADPHz . The results obtained are represented in Fig. 3. Preincubation of the enzyme with MgSO, , which is known to dedetermine tetramerization, reduces signifi-
OF HUMAN
GGPD
497
cantly the extent of inactivation by NADPHz as compared with a system lacking Mg2+ and containing mainly dimeric glucose6-P dehydrogenase. AU the experiments designed to obtain almost homogeneous dimers or tetramers were affected to some variable extent by the instability of control samples (see the last column of Table I). This unfavorable situation, due to the factors necessary to modify the equilibrium between the two polymeric species, was overcome by following the initial rate of inactivation by NADP-glycohydrolase of the tetramers and the dimers, respectively. This prevented both forms of glucose6-P dehydrogenase from being inactivated by any other environmental parameter than by NADP-glycohydrolase. It has been previously shown, by means of density gradient centrifugation, that the latter enzyme dissociates glucose-6-P dehydrogenase to inactive monomers by primarily hydrolyzing the Ystructural” NADP (15). Table II shows that an almost 100% pure population of tetramers retains full catalytic activity during a 5 min. incubation with excess NADP-glycohydrolase: on the other hand this NADP-cleaving enzyme produces a dramatic inactivation of the same sample of glucose-6-P dehydrogenase where a high ionic strength and a pH of 8.8 have determined a marked dissociation to dimers (73% of active glucose-6-P dehydrogenase, as calculated from the ~~00,~value (2, 7, 12)). The experimental time has been restricted to 5 minutes since longer periods of incubation at 37” resulted in a differential loss of activity of the control samples containing either tetramers or dimers. Relationship Between the Tetramer-Dimer and the Dimer-Monomer Equilibria The apparent discrepancy between the intrinsic resistance of the tetramers to inactivation by dissociating factors (Fig. 3 and Table II), and the complete disappearance of the same molecular form following catalytic operation of glucose-6-P dehydrogenase (Figs. 1 and 2) prompted a careful investigation of the overall tetramerdimer-monomer syst,em in the course of the inactivation by glucose-6-P. To this
498
BONSIGNORE TABLE
II
COMPAR.~TIVE INACTIVATION OF GGPD BY TETRllMERIC GLYCOHYDROLASE NADPh"'YZo; Buffer’
(g-gc
DIMERIC
AND
NADP-
G6PD activity
Kase -__ Units/ml %$ ml
0.02 M phosphate pH 6.0 0.04 M Tris-HCl pH 8.8 + 0.2 M NaCl
S 0’
5’
352
9.15
2.66
2.60
295
6.69
2.58
1.08
a Incubation at 37” in a water bath. GGPD activity was measured within the first 20 see, to minimize cleavage of NADP by NADP-glycohydrolase in the assay mixture. Controls lacking NADP-glycohydrolase and incubated in parallel showed complete stability during 5 min incubation. b Assay of NADP-glycohydrolase was performed according to Kaplan (20) directly on the inactivation mixtures after 5 min incubation. c 0.15 ml of the respective controls were layered over 5-200/, linear sucrose gradients containing the same buffer and centrifuged 11 hr at 4” at 45,000 rpm in a SW 50.1 rotor. 90 see fractions were collected by means of a mod. Perpex LKB peristaltic pump fitted with a 10 ml/hr reduction gear.
purpose, electrophoretic patterns were followed at different time intervals during incubation of the enzyme with its substrate (Fig. 4). Dissociation of glucose-6-P dehydrogenase to monomers is progressive within 45 min, whereas at longer incubation times the equilibrium tends to favor reaggregation of the monomeric units. Fig. 4 shows that during conversion of the enzyme to monomers, the decrease of the middle band (dimers) is accompanied by an almost equivalent reduction of the right band, namely of the tetramers. Such a pattern is consistent with the view that dissociation of the tetramers to the basic monomers is a compulsory pathway involving formation of the dimeric species as intermediate. This hypothesis received further support by experiments of density gradient centrifugation , where the sedimentation of
ET AL.
glucose-6-P dehydrogenase was evaluated in presence and in absence of NADPH2, respectively. With the exception of a low recovery of catalytic activity in the NADPHz-containing sample, which is obviously accounted for by inactivation, no major differences were observed between the migration of activities with and without NADPHz (Fig. 5). The same result was obtained by processing glucose-6-P dehydrogenase through gradient centrifugation, following its partial inactivation by glucose-6-P, namely under the conditions reported in Fig. 4. Both the electrophoretic analysis and the centrifugation experiments showed therefore that conversion of the dimer to monomers involves the secondary dissociation of the tetramer, as it is indicated by a constant dimer/tetramer ratio. DISCUSSION
A number of lines of evidence seem to indicate that of the two active forms of glucose-6-P dehydrogenase, i.e., tetramers and dimers, only the dimeric species can undergo dissociation to monomers. In fact, a) both inactivation and dissociation by NADPH2 are maximal at those pH values (8.5-9.5) where the balance of active species is largely displaced toward the dimers (26) ; b) no dissociation of the tetramers has been found (27) under conditions in which NADPHZ is formed (the actual pH of the experiment was 6.0). The present paper provides additional experimental evidence for the above hypothesis, based on the demonstration that the dimer is highly susceptible to dissociating factors such as NADPH, and NADP-glycohydrolase, while the tetrameric form is intrinsically resistant to both mechanisms (Fig. 3 and Table II). The physiological meaning of the above distinctive features appears however rather doubtful when one considers that the two dissociation equilibria are closely interrelated. We have in fact shown that, if other critical conditions are kept constant (protein concentration, solvent, temperature), the displacement of either equilibrium such as the dimer to monomer conversion,
INTERCONVERSION
OF HUMAN
GGPD
499
r
0’
(no
C6P)
+
10’
(7 ti’ G6P)
90’
(7mE’ GhP)
+ 45’
(7mM G6P)
FIG. 4. Electrophoretic modifications of GGPD during inactivation by NADPH2. Experimental conditions were as described in the legend to Fig. 2, excepting the concentration of G6P in the inactivation mixtures (7 X 1(r3 M instead of 1 X l(r4 M). The rate of scanning was 1 cm/min. Arrows indicate the peaks of the three molecular forms.
does not affect appreciably the other one (Figs. 4 and 5). A consequence of the interdependence of the two dissociation systems should be a depletion of the pool of both active forms
when the enzyme is working in a closed system, i.e., when NADPHZ accumulates as a product of the glucose-6-P dehydrogenase reaction. However, the inactivation by the reduced coenzyme, as well as that
BONSIGNORE
ET AL.
experiments where major parameters were unchanged. The reasons for the incomplete glucose-6-P (NADPHg)-induced inactivation and for its quantitative variations are still unclear. I . A possible explanation could be occurrence of additional enzyme species (28)-which, however, have escaped recognition despite attempts to identify them-involved in an jeven more complicated equilibrium t#han that postulated so far. An alternative hypothesis is that some monomers, arising from dissociation of the dimer, are either intrinsically active or somehow reactivated during the assay of catalytic activity. According to this hypothesis, the effect of NADPHz on a structural basis would be greater than indicated by the loss of activity. This could explain the electrophoretic finding that the dissociating effect of glucose-6-P (NADPH,) is most times complete (Fig. 2) or nearly complete (Fig. 4), at variance with the extent of inactivation, although a further displacement toward dissociation of the dimer-monomer equilibrium during migration on polyacrylfractions amide gel cannot be ruled out. To elucidate FIG. 5. Sucrose gradient sedimentation of acthis point which is closely related to the tive GBPD before and after inactivation by mechanism of inactivation by glucose-6-P NADPH2. Twenty-five micrograms of dialyzed (NADPHI), experiments are in progress to enzyme were incubated at 37” in 0.04 M Tris-HCl, measure the intrinsic molecular activity pH 8.0, containing 1 X IO-% EDTA, 1 X 10e6an of tetramers, dimers and monomers by NADP, 0.2a/, B-mercaptoethanol and 1.4 X 10-Q means of rapid mixing techniques. NADPH2, to a final volume of 1.5 ml. A control The interconversion among structurally lacking NADPHz was incubated in parallel. The and functionally different molecular forms net loss of activity produced by NADPHz within is a mechanism through which the activity 40 min incubation was 0.95 units/ml, starting from an initial value of 3.0 units/ml. After the incubaof several key enzymes can be widely t,ion, 1.0 ml of each of the two mixtures was modulated (18, 29). In most cases the layered together with 5 mg COHb on a 5-2070 balance is dictated by specific superimposed linear sucrose gradient containing the same comenzymes, catalyzing the interconversion, for ponents as the incubation experiments. Centriwhich the term ‘primary regulatory enzymes” fugation was for 40 hr at 5” in a SW 25.1 rotor has been recently proposed (30). Human using a Beckman L-2 centrifuge. Fractions were glucose-6-P dehydrogenase appears to lack collected by means of a Perpex peristaltic pump such a sophisticated regulatory system (LKB) and assayed for catalytic activity (open applying to “dimorphic” or “polymorphic” circles) and for COHb by measuring the absorbenzyme proteins since the interconversion ance at 413 rnN (filled circles). Arrows indicate the among the three discrete species (taking theoretical migration of tetramers and dimers, respectively. place according to the scheme represented in Fig. 6) is more simply ligand-dependent. produced by glucose-6-P, is generally It is, however likely, on the basis of preincomplete and in addition extremely vious experiments (17, IS), that glutathione variable in extent, as shown in several reductase (Reduced-NAD(P) : oxidized-glu-
INTERCONVERSION High pH and
OF HUMAN Glucose 6-PIhmPH,
ionic strength
I ACTIVE)
\--\
IACTIVEI Low pH ond tom strength Mg’*, Mn*’
6. Factors regulating
FIG.
the tetramer-dimer
oxidoreductase, EC 1.6.4.2.) tathione fulfills within the erythrocyte the role of preventing
the inactivation
of glucose-6-P
dehydrogenase by NADPHZ, therefore acting on this protein and being accordingly involved
in the
regulation
of the
pentose
15. 16. 17.
phosphat’e pathway. 18. 19.
ACKNOWLEDGMENTS We are indebted to Professor S. Pontremoli for critical revision of the manuscript. The skillful technical assistance of Mr. F. Giuliano is gratefully acknowledged.
20.
REFERENCES 1.
BONSIGNORE, A., CANCEDDA, I., COSULICH, M. E. AND
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Biophys.
Res.
R., DE
LORENZONI, FLORA, A.,
21.
Commun. 43, 94
(1971). ROSEMEYER, M. A., Eur. 8, 8 (1969). 3. YOSHIDA, A., J. Biol. Chem. 241, 4966 (1966). 4. YOSHID~, A., Biochem. Genet. 2, 237 (1968). 5. COHEN, P. AND ROSEMEYER, M. A., FEBS 2. COHEN,
P.,
I
DIMER !---
TETRAMER
AND
J. Biochem.
22. 23.
Lett. 1, 147 (1968).
24.
6. BONSIGNORE, A., LORENZONI, I., CANCEDDA, R., NICOLINI, A., DAMIANI, G. AND DE FLORA, A., Ital. J. Biochem. 19, 139 (1970). 7. BONSIGNORE, A., LORENZONI, I., CANCEDDA, M. E. AND DE FLORA, A., R., COSULICH,
Biochem. Biophys. Res. Commun. 43, 159 (1971). 8. KIRKMAN, H. N., J. Biol. Chem. 237, 2364 (1962). 9. KIRKM~N, H. N. AND HENDRICKSON, E. M., J. Biol. Chem. 23’7, 2371 (1962). 10. CHUNG, A, E. AND LANGDON, R. G., .J. Biol. Chem. 238, 2317 (1963). 11. YOSHIDA, A., Biochem. Genet. 1, 81 (1967). 12. BONSIGNORE, A., LORENZONI, I., CANCEDDA, R. .4ND DE FLORA, A., Biochem. Biophys. Res. Com,mun. 39, 142 (1970). 13. MARKS, P. A., SZEINBERG, A. BND BANKS, J., J. Biol. Chem. 236, 10 (1961). 14. LUZZATTO, L. AND ALLAN, N. C., Biochem. Biophys. Res. Commun. 21, 547 (1965).
25. 26.
27.
28.
29. 30.
501
GGPD
MONOMER
-ii
( INACTWE 1 NAOq-SH
and the dimer-monomer
equilibria.
A., DE FLORA, A., MANGIAA., LORENZONI, 1. AND ALEMA, S., Ital. J. Biochem. 16, 117 (1967). RATTAZZI, M. C., Biochem. Biophys. Res. Commun. 31, 16 (1968). BONSIGNORE, A., DE FLORA, A., LORENZONI, I., CANCEDDA, R., SILENGO, L. AND DINA, D., Ital. J. Biochem. 17, 346 (1968). DE FLORA, A., Ital. J. Biochem. 17, 363 (1968). LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L. AND RANDALL, R. J., J. Biol. Chem. 193, 265 (1951). KAPLAN, N. O., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, Eds.), Vol. II, p. 664, Academic Press, New York (1955). DE FLORA, A., LORENZONI, I., MANQIAROTTI, M. A., DINA, D. AND BONSIGNORE, A., Biochem. Biophys. Res. Commun. 31, 501 (1968). ACKERS, G. K., Biochemistry 3, 723 (1964). GOSTING, L. J., in “Advances in protein chemistry,” M. L. Anson, K. Bailey and J. T. Edsall Editors, Vol. 11, p. 449, Academic Press, New York, 522 (1956). DETERMAN, H., “Chromatographie sur gel,” Masson and Cie, Paris, p. 111 (1969). YIELDING, K. L., Biochem. Biophys. Res. Commun. 38. 546 (1970). R., LORENZONI, DE FLORA, A., CANCEDDA, I., NICOLINI, A. AND BONSIGNORE, A., Proceedings 1st International Symposium on “Metabolic Interconversion of Enzymes,” S. Margherita Ligure, p. 35 (1970). YOSHIDA, A. AND HOAGLAND, V. D., JR., Biochem. Biophys. Res. Commun. 40, 1167 (1970). BONSIGNORE, A., LORENZONI, I., CANCEDDA, R., MORELLI, S., GIULIANO, F. AND DE FLORA, A., Ital. J. Biochem. 18, 439 (1969). HOLZER, H., Advances Enzymol. 32, 297 (1969). SOLS, A., Proceedings 1st International Symposium on “Metabolic Interconversion of S. Margherita Ligure, p. 1 Enzymes,” (1970). BONSIONORE, ROTTI, M.