OF BIOCHEMISTRY AND BIOPHYSICS 185, No. 2, January 30, pp. 419-422, 1978
ARCHIVES
Vol.
Anaerobic LUCIANA
Reaction
AVIGLIANO,*
of Ascorbate GIUSEPPE
BRUNO MONDOVf,* * Istitutz Molecolare
AND
Oxidase
ROTILIO,?
with Ascorbate
SANDRA
ALESSANDRO
URBANELLI,*’
FINAZZI
AGR&
dz Chimaca Bwlogica e Bzochimica Applicata dell’llniuersatci dz Roma e Centro di Biologia de1 C.N.R. Roma; tlstituto di Chimica Biulogica, Uniuersitd di Camerino, Came&o; Slstituto di Chimica Biologica, Uniuersitd di Cagliari, Cagliari, Italy Received
May
16, 1977; revised
August
4, 1977
Ascorbate oxidase is fully reduced by 4 mol of ascorbatc in the absence of air, as monitored by optical and electron paramagnetic resonance spectra. At less than stoichiometric ascorbate concentration there is a slow equilibration between the 605and 330-nm absorption bands: The 605nm chromophore is first reduced, then its color reappears while the 330-nm absorption band decreases. Upon reoxidation with air the process takes place in the opposite direction. Intramolecular rather than intermolecular electron exchange appears to be responsible for this process. The reduced protein 1s about twice as fluorescent as the oxidized protein. The fluorescence quenching in the oxidized protein is related to the 330-nm absorption band rather than to the 605.nm band as previously reported for lactase.
A very interesting problem in dealing with oxidases is to elucidate the electron pathway across the protein from the reducing substrate to oxygen. Many oxidases contain different redox centers which accept electrons most probably in a sequential process. A particular case is that of the so-called “blue oxidases,” which have as the redox component only copper apparently bound in three spectroscopically distinct ways, namely, Type 1, responsible for the large absorption at 610 nm, Type 2, which contributes to the epr’ spectrum, and Type 3, not detectable with epr and associated with a strong absorption band at 330 nm. They show different redox potentials and/or different kinetic properties (l-3). However, the direction of the electron flow inside these proteins is not yet known with certainty. Furthermore, it is still questioned whether the electrons go through the same path in all of the blue oxidases. The latter point is particularly relevant to the larger blue oxidases, i.e., ceruloplasmin and ascorbate oxidase, where the number of copper atoms is I Abbreviation resonance.
used:
epr,
electron
higher than in the laccases, and where even the subdivision in the three types appears to be an oversimplification (3). It seemed therefore worthwhile to study the reaction of green zucchini ascorbate oxidase with ascorbate in anaerobiosis, following the disappearance of the absorption bands related to Type 1 and Type 3 copper. The effect of copper reduction on the intrinsic fluorescence of the protein, which was proposed by Pecht and Goldberg (4) as a useful tool for the study of the reduction of Rhus vernicifera lactase, was also investigated. MATERIALS
AND
METHODS
Reagent grade chemicals were used without further purification. Ascorbate oxidase was purified from green zucchini as previously described (5). An E,M,, = 1 x 10’ at 605 nm was used for concentration measurements (6). The homogeneity of the protein was checked by disc gel electrophoresis at pH 8.9. The enzyme samples used throughout this work ratio of 24 and a mean copper had an E,,IE,,, content of 7-8 Cu atoms/140,000 M,. Anaerobic optical absorption, fluorescence, and epr titrations with ascorbate were performed anaerobically in Thunberg-type cuvettes or in tubes equipped with an airtight rubber cap. Air was substituted with prepurified oxygen-free argon by at least three cycles
paramagnetrc 419
0003-9861/78/1852-0419$02.00/O Copyright 0 1978 by Academic Press, Inc. All rrghts of reproduction in any form reserved
420
AVIGLIANO
of vacuum refilling. Aliquots of oxygen-free ascorbate were then added anaerobically with an airtight syringe through the rubber cap. Optical absorption spectra were recorded with a Beckman DK2A spectrophotometer equipped with a temperature-control unit. Fluorimetric experiments were conducted with a FICA 55L corrected spectrofluorimeter operating at room temperature. The excitation wavelength was 295-300 nm so that relatively concentrated protein samples could be used, which would absorb too much at 280 nm, the fluorescence excitation maximum. Thus, it was possible to follow the optical density change at 605 nm of the same samples used for fluorescence experiments. Low-temperature epr measurements at the Xband were obtained with a Varian V-4502-14 spectrometer. RESULTS
Reduction of Ascorbate Oxidase by Ascorbate The addition of ascorbate to anaerobic samples of ascorbate oxidase leads to the disappearance of the visible optical absorption spectrum and to the decrease of the near-ultraviolet absorption. The difference spectrum between oxidized and reduced samples shows a peak at about 330 nm, as previously described in the other blue oxidases (l-3). Four moles of ascorbate per mole of enzyme are required for full bleaching of the spectrum of the protein. No oxygen leakage occurs in the system, as it remains colorless for hours in the Thunberg cuvette after reduction. At less than the stoichiometric ascorbate concentration, time-dependent variations in the absorption spectrum were observed (see Fig. 1). There was, in fact, a decrease in absorption at 605 nm, immediately after the addition of ascorbate. Then, the blue color partly reappeared with a parallel decrease in absorbancy at 330 nm. The process is slow (half-time, 5-10 min). The rate of the process is temperature independent in the range lo-40°C and is not affected by increasing the viscosity of the medium from 0.89 to 12.39 CP with sucrose. Furthermore, the reaction is also concentration independent. Intramolecular electron transfer seems therefore to be involved in this phenomenon. An analogous process in the opposite direction, i.e., from the 330-nm chromophore to the 605nm
ET
AL
FIG. 1. Optical absorption spectra of ascorbate oxidase reduction by ascorbate. Absorption spectra of 60 pM ascorbate oxidase in phosphate buffer, pH 6, in anaerobiosis (curve 1) and after additions of ascorbate at 100 PM (curve 21, 200 PM (curve 31, and 300 PM (curve 4). Curves 2’ and 3’ were obtained after incubating samples 2 and 3 for 30 and 5 min, respectively, at room temperature.
band, was observed after the addition of less than stoichiometric oxygen to fully reduced ascorbate oxidase samples. Four equivalents of ascorbate are also necessary to fully reduce the epr-detectable copper. Both Type 1 and Type 2 copper disappear gradually as the function of the added ascorbate, with no change in epr parameters. In view of the inhibitory effect of azide on the ascorbate oxidase activity (91, it was of interest to study its influence on anaerobic reduction. It was found that the presence of 0.1 M NaN, had no effect on the reduction by ascorbate. The 410-nm peak due to the N,- to Cu(I1) charge transfer (10) was bleached before full disappearance of the blue color. This seems to indicate either that Type 2 copper is reduced in spite of the formation of its azide complex or that the formation constant of this complex is greatly lowered in the partly reduced protein. Influence of Redox State of the Protein on Its Intrinsic Fluorescence The complete reduction of the protein is accompanied by a twofold increase in the intrinsic fluorescence, without any change in the shape and position of the emission (328 nm) or excitation peak (280 nm) (see Fig. 2). Since the protein contains about 40 tryptophan residues per mole (111, the most reasonable mechanism for the fluo-
REDUCTION
330 Wavelength
390
OF
ASCORBATE
J
ctlrnl
FIG. 2. Intrinsic fluorescence changes in reduced ascorbate oxidase. Intrinsic fluorescence of a 7.5 PM anaerobic ascorbate oxidase solution in 80 mM phosphate buffer, pH 6, in the absence (curve 1) and presence of 6 WM (curve 21, 15 PM (curve 3), 24 PM (curve 4), 85 pM (curve 51, and 400 PM ascorbate (curve 6). Curve 7 was obtained after opening the solution of curve 6 to air.
rescence quenching is a long-range nonradiative energy transfer from the aromatic residues to an unknown acceptor. Pecht and Goldberg (41, who studied a similar effect on Rhus uernicifera lactase, found that the increase in fluorescence parallels the decrease in absorption at 605 nm. They concluded that the blue chromophore is in fact responsible for this fluorescence quenching. However, the overlap between the 605nm absorption band and the tryptophan emission is quite low, and this would require a close proximity of all emitting tryptophans to the blue center(s) in order to get this extent of quenching (12). On the other hand, it is difficult to understand why the disappearance of the 330-nm band, which has a fairly high extinction coefficient (in the range of lo3 M-’ cm-‘) and strongly overlaps the intrinsic fluorescence of the protein, would not affect this fluorescence. In our experiments the increase in fluorescence is related to the disappearance of the 330-nm band and not to that of the 605nm band as determined by checking
421
OXIDASE
the optical density just after the fluorescence measurements. Furthermore, at intermediate reduction stages, a time-dependent fluorescence increase was observed which parallels the decrease of the 330-nm chromophore and the concomitant increase of the 605nm band (see above). Control experiments with lactase showed that the correlation between the blue band disappearance and the fluorescence enhancement is not linear at low and high ascorbatelprotein ratios, while it is almost linear at intermediate values. Here the experimental conditions appear to be more critical: Excess ascorbate must be added for anaerobic bleaching of the visible spectrum, which is complete only after a fairly long period of time using 40 mol of ascorbate per mole of enzyme. Moreover, excess ascorbate may interfere with fluorescence measurements by the inner filter effect since it absorbs in the same region of fluorescence, and this could give a false end point for fluorescence enhancement titrations. Finally, it is not possible to take advantage of a time-dependent equilibration since in this protein it may occur much faster. DISCUSSION
Four equivalents of ascorbate (i.e., eight electrons) are used for full reduction of ascorbate oxidase copper in the absence of oxygen with no formation of spectroscopically distinct intermediates, in the time scale used in the present study. However, this anaerobic reaction is peculiarly time dependent, as an intramolecular electron exchange takes place between the Type 1 and Type 3 chromophores. Though this process is very slow, at variance with fungal lactase (3), and it cannot be related to the steady-state oxidation of the substrate, it may support the hypothesis of a linear array of redox centers which undergo consecutive reduction (3). Type 1 copper, which is responsible for the 605nm band, appears to be nearer to the substrate side, whereas Type 3 copper, presumably responsible for the 330-nm band, would be on the oxygen side. It may be assumed that ascorbate oxidase, like cytochrome oxidase (13, 14) and lactase
422
AVIGLIANt
(151, goes faster after a “pulse,” i.e., after a catalytic cycle. This hypothesis may help to explain the activation of ascorbate oxidase by ascorbate described by Gerwin et al. (16). On the other hand, steady-state experiments show that the enzyme does not follow a ping-pong type of kinetics (A. Finazzi Agrti et al ., in preparation). Nothing can be said concerning Type 2 copper besides the observation that its binding to azide has no effect on its equilibrium reduction by ascorbate, which is at variance with the fluoride effect toward fungal lactase (17). A final remark concerns the intrinsic fluorescence of the protein and its relationship to the absorption bands. It appears that the fluorescence quenching in the oxidized protein is essentially related to the presence of the 330-nm band. Thus, the fluorescence quenching may be used as a probe of the oxidation state of this chromophore in dilute solutions where absorbance measurements can hardly be made. ACKNOWLEDGMENTS The authors wish to thank Mr. Paolo Gerosa for his skillful assistance throughout this work This work was supported in part by a Contributo per la Ricerca Scientifica Cap. IX Art. 13, Bilancio Universitario. REFERENCES 1. MALKIN, R., AND MALMSTR~~M, van. Enzymol. 33, 177-244.
B. G. (1970) Ad-
3 ET
AL
2. MALMSTRBM, B. G., ANDR~ASSON, L. E., AND REINHAMMAR, B. (1975) in The Enzymes (Bayer, P., ed.), 3rd ed., Vol. 12, pp. 507-579. Academic Press, New York. 3. FEE, J. A. (1975) Struct. Bonding 23, l-60. 4. PECHT, I., AND GOLDBERG, M. (1974) Proc. Nat. Acad. Scl. USA 71, 4684-4687. 5. AVIGLIANO, L., GEROSA, P., ROTILIO, G., FINAZZI AGR~I, A., CALABRESE, L., AND MONDOVI, B. (1972) Ital. J. Biochem. 21, 248-255. 6. NAKAMURA, T., MAKINO, N., AND OGURA, Y. (1968) J. Bwchem. (Tokyo) 64, 189-195. 7. ORNSTEIN, L. (1964) Ann. N.Y. Acad. Scz. 121, 312-349. 8. DAVIS, Y. B. (1964) Ann. N.Y. Acad. Sci. 121, 404-427. 9. MONDOVI, B., AVIGLIANO, L., ROTILIO, G., FINAZZI AGR~, A., GEROSA, P., AND GIOVAGNOLI, C. (1975) Mol. Cell. Bsochem. 7, 131-135. 10. MORPURGO, L., ROTILIO, G., FINAZZI AGR~, A., AND MONDOVI, B. (1974) Biochzm. Biophys. Acta 336, 324-328. 11. DAWSON, C. R., STROTHKAMP, K. G., AND KRUL, K. G. (1975) Ann. N.Y. Acad. Sci. 258, 209220. 12. FGRSTER, T. (1948) Ann. Phys. 2, 55-75. 13. ANTONINI, E., BRUNORI, M., COLOSIMO, A., GREENWOOD, C., AND WILSON, M. T. (1977) Proc. Nat. Acad. Sci. USA 74, 3128-3132. 14. ROSBN, S., BRAND&N, R., VKNNGXRD, T., AND MALMSTRGM, B. G. (1977) FEBS Lett. 74, 2530. 15. ANDR%ASSON, L.-E., AND REINHAMMAR, B. (1976) Biochim. Bzophys. Acta 445, 339-355. 16. GERWIN, B., BURSTEIN, S. R., AND WESTLEY, J. (1974) J. Biol. Chem. 249, 2005-2008. 17. MALKIN, R., MALMSTRBM, B. G., AND VANNG.&RD, T. (1969) EUF. J. Biochem. 10, 324-329.
ARCHIVES
Vol.
Anaerobic LUCIANA
Reaction
AVIGLIANO,*
of Ascorbate GIUSEPPE
BRUNO MONDOVf,* * Istitutz Molecolare
AND
Oxidase
ROTILIO,?
with Ascorbate
SANDRA
ALESSANDRO
URBANELLI,*’
FINAZZI
AGR&
dz Chimaca Bwlogica e Bzochimica Applicata dell’llniuersatci dz Roma e Centro di Biologia de1 C.N.R. Roma; tlstituto di Chimica Biulogica, Uniuersitd di Camerino, Came&o; Slstituto di Chimica Biologica, Uniuersitd di Cagliari, Cagliari, Italy Received
May
16, 1977; revised
August
4, 1977
Ascorbate oxidase is fully reduced by 4 mol of ascorbatc in the absence of air, as monitored by optical and electron paramagnetic resonance spectra. At less than stoichiometric ascorbate concentration there is a slow equilibration between the 605and 330-nm absorption bands: The 605nm chromophore is first reduced, then its color reappears while the 330-nm absorption band decreases. Upon reoxidation with air the process takes place in the opposite direction. Intramolecular rather than intermolecular electron exchange appears to be responsible for this process. The reduced protein 1s about twice as fluorescent as the oxidized protein. The fluorescence quenching in the oxidized protein is related to the 330-nm absorption band rather than to the 605.nm band as previously reported for lactase.
A very interesting problem in dealing with oxidases is to elucidate the electron pathway across the protein from the reducing substrate to oxygen. Many oxidases contain different redox centers which accept electrons most probably in a sequential process. A particular case is that of the so-called “blue oxidases,” which have as the redox component only copper apparently bound in three spectroscopically distinct ways, namely, Type 1, responsible for the large absorption at 610 nm, Type 2, which contributes to the epr’ spectrum, and Type 3, not detectable with epr and associated with a strong absorption band at 330 nm. They show different redox potentials and/or different kinetic properties (l-3). However, the direction of the electron flow inside these proteins is not yet known with certainty. Furthermore, it is still questioned whether the electrons go through the same path in all of the blue oxidases. The latter point is particularly relevant to the larger blue oxidases, i.e., ceruloplasmin and ascorbate oxidase, where the number of copper atoms is I Abbreviation resonance.
used:
epr,
electron
higher than in the laccases, and where even the subdivision in the three types appears to be an oversimplification (3). It seemed therefore worthwhile to study the reaction of green zucchini ascorbate oxidase with ascorbate in anaerobiosis, following the disappearance of the absorption bands related to Type 1 and Type 3 copper. The effect of copper reduction on the intrinsic fluorescence of the protein, which was proposed by Pecht and Goldberg (4) as a useful tool for the study of the reduction of Rhus vernicifera lactase, was also investigated. MATERIALS
AND
METHODS
Reagent grade chemicals were used without further purification. Ascorbate oxidase was purified from green zucchini as previously described (5). An E,M,, = 1 x 10’ at 605 nm was used for concentration measurements (6). The homogeneity of the protein was checked by disc gel electrophoresis at pH 8.9. The enzyme samples used throughout this work ratio of 24 and a mean copper had an E,,IE,,, content of 7-8 Cu atoms/140,000 M,. Anaerobic optical absorption, fluorescence, and epr titrations with ascorbate were performed anaerobically in Thunberg-type cuvettes or in tubes equipped with an airtight rubber cap. Air was substituted with prepurified oxygen-free argon by at least three cycles
paramagnetrc 419
0003-9861/78/1852-0419$02.00/O Copyright 0 1978 by Academic Press, Inc. All rrghts of reproduction in any form reserved
420
AVIGLIANO
of vacuum refilling. Aliquots of oxygen-free ascorbate were then added anaerobically with an airtight syringe through the rubber cap. Optical absorption spectra were recorded with a Beckman DK2A spectrophotometer equipped with a temperature-control unit. Fluorimetric experiments were conducted with a FICA 55L corrected spectrofluorimeter operating at room temperature. The excitation wavelength was 295-300 nm so that relatively concentrated protein samples could be used, which would absorb too much at 280 nm, the fluorescence excitation maximum. Thus, it was possible to follow the optical density change at 605 nm of the same samples used for fluorescence experiments. Low-temperature epr measurements at the Xband were obtained with a Varian V-4502-14 spectrometer. RESULTS
Reduction of Ascorbate Oxidase by Ascorbate The addition of ascorbate to anaerobic samples of ascorbate oxidase leads to the disappearance of the visible optical absorption spectrum and to the decrease of the near-ultraviolet absorption. The difference spectrum between oxidized and reduced samples shows a peak at about 330 nm, as previously described in the other blue oxidases (l-3). Four moles of ascorbate per mole of enzyme are required for full bleaching of the spectrum of the protein. No oxygen leakage occurs in the system, as it remains colorless for hours in the Thunberg cuvette after reduction. At less than the stoichiometric ascorbate concentration, time-dependent variations in the absorption spectrum were observed (see Fig. 1). There was, in fact, a decrease in absorption at 605 nm, immediately after the addition of ascorbate. Then, the blue color partly reappeared with a parallel decrease in absorbancy at 330 nm. The process is slow (half-time, 5-10 min). The rate of the process is temperature independent in the range lo-40°C and is not affected by increasing the viscosity of the medium from 0.89 to 12.39 CP with sucrose. Furthermore, the reaction is also concentration independent. Intramolecular electron transfer seems therefore to be involved in this phenomenon. An analogous process in the opposite direction, i.e., from the 330-nm chromophore to the 605nm
ET
AL
FIG. 1. Optical absorption spectra of ascorbate oxidase reduction by ascorbate. Absorption spectra of 60 pM ascorbate oxidase in phosphate buffer, pH 6, in anaerobiosis (curve 1) and after additions of ascorbate at 100 PM (curve 21, 200 PM (curve 31, and 300 PM (curve 4). Curves 2’ and 3’ were obtained after incubating samples 2 and 3 for 30 and 5 min, respectively, at room temperature.
band, was observed after the addition of less than stoichiometric oxygen to fully reduced ascorbate oxidase samples. Four equivalents of ascorbate are also necessary to fully reduce the epr-detectable copper. Both Type 1 and Type 2 copper disappear gradually as the function of the added ascorbate, with no change in epr parameters. In view of the inhibitory effect of azide on the ascorbate oxidase activity (91, it was of interest to study its influence on anaerobic reduction. It was found that the presence of 0.1 M NaN, had no effect on the reduction by ascorbate. The 410-nm peak due to the N,- to Cu(I1) charge transfer (10) was bleached before full disappearance of the blue color. This seems to indicate either that Type 2 copper is reduced in spite of the formation of its azide complex or that the formation constant of this complex is greatly lowered in the partly reduced protein. Influence of Redox State of the Protein on Its Intrinsic Fluorescence The complete reduction of the protein is accompanied by a twofold increase in the intrinsic fluorescence, without any change in the shape and position of the emission (328 nm) or excitation peak (280 nm) (see Fig. 2). Since the protein contains about 40 tryptophan residues per mole (111, the most reasonable mechanism for the fluo-
REDUCTION
330 Wavelength
390
OF
ASCORBATE
J
ctlrnl
FIG. 2. Intrinsic fluorescence changes in reduced ascorbate oxidase. Intrinsic fluorescence of a 7.5 PM anaerobic ascorbate oxidase solution in 80 mM phosphate buffer, pH 6, in the absence (curve 1) and presence of 6 WM (curve 21, 15 PM (curve 3), 24 PM (curve 4), 85 pM (curve 51, and 400 PM ascorbate (curve 6). Curve 7 was obtained after opening the solution of curve 6 to air.
rescence quenching is a long-range nonradiative energy transfer from the aromatic residues to an unknown acceptor. Pecht and Goldberg (41, who studied a similar effect on Rhus uernicifera lactase, found that the increase in fluorescence parallels the decrease in absorption at 605 nm. They concluded that the blue chromophore is in fact responsible for this fluorescence quenching. However, the overlap between the 605nm absorption band and the tryptophan emission is quite low, and this would require a close proximity of all emitting tryptophans to the blue center(s) in order to get this extent of quenching (12). On the other hand, it is difficult to understand why the disappearance of the 330-nm band, which has a fairly high extinction coefficient (in the range of lo3 M-’ cm-‘) and strongly overlaps the intrinsic fluorescence of the protein, would not affect this fluorescence. In our experiments the increase in fluorescence is related to the disappearance of the 330-nm band and not to that of the 605nm band as determined by checking
421
OXIDASE
the optical density just after the fluorescence measurements. Furthermore, at intermediate reduction stages, a time-dependent fluorescence increase was observed which parallels the decrease of the 330-nm chromophore and the concomitant increase of the 605nm band (see above). Control experiments with lactase showed that the correlation between the blue band disappearance and the fluorescence enhancement is not linear at low and high ascorbatelprotein ratios, while it is almost linear at intermediate values. Here the experimental conditions appear to be more critical: Excess ascorbate must be added for anaerobic bleaching of the visible spectrum, which is complete only after a fairly long period of time using 40 mol of ascorbate per mole of enzyme. Moreover, excess ascorbate may interfere with fluorescence measurements by the inner filter effect since it absorbs in the same region of fluorescence, and this could give a false end point for fluorescence enhancement titrations. Finally, it is not possible to take advantage of a time-dependent equilibration since in this protein it may occur much faster. DISCUSSION
Four equivalents of ascorbate (i.e., eight electrons) are used for full reduction of ascorbate oxidase copper in the absence of oxygen with no formation of spectroscopically distinct intermediates, in the time scale used in the present study. However, this anaerobic reaction is peculiarly time dependent, as an intramolecular electron exchange takes place between the Type 1 and Type 3 chromophores. Though this process is very slow, at variance with fungal lactase (3), and it cannot be related to the steady-state oxidation of the substrate, it may support the hypothesis of a linear array of redox centers which undergo consecutive reduction (3). Type 1 copper, which is responsible for the 605nm band, appears to be nearer to the substrate side, whereas Type 3 copper, presumably responsible for the 330-nm band, would be on the oxygen side. It may be assumed that ascorbate oxidase, like cytochrome oxidase (13, 14) and lactase
422
AVIGLIANt
(151, goes faster after a “pulse,” i.e., after a catalytic cycle. This hypothesis may help to explain the activation of ascorbate oxidase by ascorbate described by Gerwin et al. (16). On the other hand, steady-state experiments show that the enzyme does not follow a ping-pong type of kinetics (A. Finazzi Agrti et al ., in preparation). Nothing can be said concerning Type 2 copper besides the observation that its binding to azide has no effect on its equilibrium reduction by ascorbate, which is at variance with the fluoride effect toward fungal lactase (17). A final remark concerns the intrinsic fluorescence of the protein and its relationship to the absorption bands. It appears that the fluorescence quenching in the oxidized protein is essentially related to the presence of the 330-nm band. Thus, the fluorescence quenching may be used as a probe of the oxidation state of this chromophore in dilute solutions where absorbance measurements can hardly be made. ACKNOWLEDGMENTS The authors wish to thank Mr. Paolo Gerosa for his skillful assistance throughout this work This work was supported in part by a Contributo per la Ricerca Scientifica Cap. IX Art. 13, Bilancio Universitario. REFERENCES 1. MALKIN, R., AND MALMSTR~~M, van. Enzymol. 33, 177-244.
B. G. (1970) Ad-
3 ET
AL
2. MALMSTRBM, B. G., ANDR~ASSON, L. E., AND REINHAMMAR, B. (1975) in The Enzymes (Bayer, P., ed.), 3rd ed., Vol. 12, pp. 507-579. Academic Press, New York. 3. FEE, J. A. (1975) Struct. Bonding 23, l-60. 4. PECHT, I., AND GOLDBERG, M. (1974) Proc. Nat. Acad. Scl. USA 71, 4684-4687. 5. AVIGLIANO, L., GEROSA, P., ROTILIO, G., FINAZZI AGR~I, A., CALABRESE, L., AND MONDOVI, B. (1972) Ital. J. Biochem. 21, 248-255. 6. NAKAMURA, T., MAKINO, N., AND OGURA, Y. (1968) J. Bwchem. (Tokyo) 64, 189-195. 7. ORNSTEIN, L. (1964) Ann. N.Y. Acad. Scz. 121, 312-349. 8. DAVIS, Y. B. (1964) Ann. N.Y. Acad. Sci. 121, 404-427. 9. MONDOVI, B., AVIGLIANO, L., ROTILIO, G., FINAZZI AGR~, A., GEROSA, P., AND GIOVAGNOLI, C. (1975) Mol. Cell. Bsochem. 7, 131-135. 10. MORPURGO, L., ROTILIO, G., FINAZZI AGR~, A., AND MONDOVI, B. (1974) Biochzm. Biophys. Acta 336, 324-328. 11. DAWSON, C. R., STROTHKAMP, K. G., AND KRUL, K. G. (1975) Ann. N.Y. Acad. Sci. 258, 209220. 12. FGRSTER, T. (1948) Ann. Phys. 2, 55-75. 13. ANTONINI, E., BRUNORI, M., COLOSIMO, A., GREENWOOD, C., AND WILSON, M. T. (1977) Proc. Nat. Acad. Sci. USA 74, 3128-3132. 14. ROSBN, S., BRAND&N, R., VKNNGXRD, T., AND MALMSTRGM, B. G. (1977) FEBS Lett. 74, 2530. 15. ANDR%ASSON, L.-E., AND REINHAMMAR, B. (1976) Biochim. Bzophys. Acta 445, 339-355. 16. GERWIN, B., BURSTEIN, S. R., AND WESTLEY, J. (1974) J. Biol. Chem. 249, 2005-2008. 17. MALKIN, R., MALMSTRBM, B. G., AND VANNG.&RD, T. (1969) EUF. J. Biochem. 10, 324-329.
