Optical Properties and Denaturation by Guanidinium Chloride and Urea

Biochem. J. (1977) 161, 321-331 321 Printed in Great Britain Optical Properties and Denaturation by Guanidinium Chloride and Urea of the Adenosine Triphosphatase of Micrococcus lysodeikticus A COMPARISON OF FOUR MOLECULAR FORMS OF THE ENZYME By MANUEL NIETO and JUAN A. AYALA Seccion de Bioquimica de Membranas, Centro de Investigaciones Biol6gicas, Veldzquez 144, Madrid-6, Spain (Received 7 July 1976) 1. The fluorescence and circular dichroism offour homogeneous preparations of ATPase (adenosine triphosphatase) fromMicrococcus lysodeikticus differing in molecular structure and enzymic properties were examined at pH 7.5 and 25°C. Emission was maximum at 325 and 335nm and the relative intensities at these wavelengths may be used to characterize the different ATPase preparations. The circular-dichroism spectra exhibited negative extrema at 208 and 220nm, and the relative value of the molar ellipticity at these wavelengths was also different for each molecular form ofthe enzyme. 2. The four preparations undergo two consecutive major unfolding transitions in guanidinium chloride (midpoints at 0.94 and 1.5 M denaturant), with concomitant destruction ofthe quaternary structure of the protein. A comparatively minor alteration in the ATPase structure also occurred in 0.05-0.2M-guanidine and led to complete inactivation ofthe enzyme. The inactivation and the first unfolding transition were reversible by dilution of the denaturant; the transition with midpoint at 1.5M-guanidine was irreversible. 3. Similar results were obtained in urea, except that the successive transitions had midpoints at concentrations of denaturant of 0.4, 2.0 and 4.5M. Low concentrations of urea caused a noticeable activation of the enzyme activity and alterations of the electrophoretic mobility of the ATPase. 4. A model is proposed in which one of the major subunits, a, is first dissociated and unfolded rever- sibly by the denaturants, followed by the irreversible unfolding and dissociation of the other major subunit, P, from subunit a and/or the components of relative mobility 1.0 in dodecyl sulphate/polyacrylamide-gel electrophoresis (p). The ATPase* (bacterial F1, BF1, EC 3.6.1.3) of the 1975). However, we found that those conditions that membranes of Micrococcus lysodeikticus is an led to dissociation of the ATPase molecule into the energy-transducing ATPase similar to those of mito- constituent subunits also led to their irreversible chondria, chloroplasts and other bacteria (Andreu et unfolding and chemical degradation. The observa- al., 1973; Penefsky, 1974; Abrams & Smith, 1974; tion that the enzyme could be inactivated by moderate Pedersen, 1975). It can be solubilized by very mild concentrations of guanidine or urea and the activity treatment in the absence of detergent (Munoz et al., recovered by dilution or dialysis (J. M. Andreu, 1969) and purified to homogeneity (Andreu et al., unpublished work) encouraged us to study the 1973; Andreu & Muiioz, 1975). denaturation by these compounds. In the present The preparation thus obtained was the so-called work we compare the fluorescence and c.d. of the form A of the enzyme. Three additional pure pre- enzyme preparations mentioned above and their parations were obtained from a closely related sub- denaturation by guanidinium chloride and urea. We strain of this micro-organism (forms B; Carreira et show that for all the forms of the enzyme the effect of al., 1976a,b). They differed from form A and from the denaturants takes place in three steps, two of each other in specific activity, stimulation by trypsin which are reversible by dilution. and subunit apparent molecular weight and/or A preliminary account ofpart ofthis work has been composition (Carreira et al., 1976a,b). presented (Nieto & Andreu, 1975). In a previous paper we examined the possibility of achieving reversible denaturation of the ATPase Materials and Methods (preparation A) by variations in the pH (Nieto et al., Chemicals Guanidinium chloride and urea were Ultrapure * Abbreviations: ATPase, adenosine triphosphatase; from Schwarz/Mann (Orangeburg, NY, U.S.A.); c.d., circular dichroism. ATP (disodium salt) was from P-L Biochemicals Vol. 161 L 322 M. NIETO AND J. A. AYALA (Milwaukee, WI, U.S.A.). All other chemicals were below 0.05. Double-glass-distilled water was used in of the best quality commercially available. the preparation of solutions and these, except for the protein, were identical in both the sample and Enzyme preparations reference cuvettes. ATPase preparations A, BAT, BA and B, were Measurements of pH (Radiometer pH M26) and purified as described by Andreu & Munoz (1975) and analytical centrifugation (Spinco model E) by the Carreira et al. (1976a,b). They had the subunit com- high-speed method (Yphantis, 1964) were performed position described by the above authors, and their as described by Nieto et al. (1975). specific acti'vities in pmol/min per mg' of enzyme, measured by the pH-stat method (Nieto et al., 1975), Polyacrylamide-gel electrophoresis were the following: preparation A, 25; preparation Gels (5% acrylamide; 0.6cmxlOcm) were pre- B, (strain PNB, inactive), 2.4; preparation BAT pared and electrophoresis was carried out at pH8.6 (strain PNB, active and stimulated by trypsin), basal in the presence of urea as described by Andreu & activity, 7, stimulated by trypsin (Carreira et al., Munioz (1975). However, in our case the concentra- 1976b) to 14; preparation BA (strain PNB, active, not tion ofurea used in the polymerization ofthe gels and stimulated by trypsin), 23. The assay conditions are the upper buffer chamber was variable from 0 to 8 M. described below. Electrophoresisin sodium dodecylsulphate, pH8.6, was carried out as described by Andreu et al. (1973). Enzyme assay ATPase activity was measured by the pH-stat Denaturation by guanidinium chloride or urea method (Nieto et al., 1975) or colorimetrically To estimate the equilibrium fraction of protein (Vambutas & Racker, 1965; Andreu et al., 1973). remaining native after exposure to a given concentra- Incubations were performed in 30mM-Tris/HCI tion of denaturant, separate solutions (0.5- buffer, pH7.5, at 37°C and had a final ATP/CaCI2 2.5nml) of ATPase in 30mM-Tris/HCI buffer, pH7.5, concentration of 8 mm. The reactions were started by containing guanidinium chloride or urea were adding enzyme (15 or 35pg, depending on the activity maintained at 25°C for 3-5h, and then one or two of of the preparation). The concentration of ATPase the following properties measured: enzymic activity, solutions was estimated by using the value of A m- circular dichroism or fluorescence. The minimal time 6.9 at 276nm as previously determined (Nieto et al., to reach equilibrium had been determined in previous 1975; Carreira et al., 1976a). experiments by recording continuously the time- dependence of the circular dichroism or fluorescence Physical measurements emission at several concentrations ofdenaturant. The U.v. absorption (Cary 16S spectrophotometer) conoentrations of enzyme used in these experiments and circular dichroism (Roussel-Jouan 185 Dicro- were in the range 0.03-0.08mg/mI. In other experi- graph II) were measured at 25 ± 0.1°C and pH7.5 as ments, where reversal of denaturation was tested, the described previously (Nieto etaL, 1975). The values of enzyme concentrations in the solution of denaturant dichroic absorption in a light-path of 1 cm, &A, were were in the range 0.2-0.5mg/ml. After these solutions converted into mean residue molar ellipticity, [0], by had been left at 25°C for 3.5h, they were diluted 5- or means of the relationship, [0]= 3298 AA/c degree 10-fold with 30mM-Tris/HCI buffer, pH7.5, and their cm2 dmol-', where c was the mean residue con- activity, circular dichroism or emission spectra were centration in mol/litre. A value of 109 was used for measured. The concentration of urea and guani- the mean residue weight (Nieto et al., 1975). dinium chloride was estimated from measurements of Fluorescence was measured at 900 to the exciting the refractive index of the solutions (Fasman, 1963; beam (275 nm) with a FICA 55 MK II difference Nozaki, 1972). Two measurements of refractive spectrofluorimeter (Le Mesnil, St.-Denis, France). index were performed, one immediately after setting This apparatus recorded excitation and emission up the denaturation mixture and another after it had spectra, corrected for lamp quantum output and been left for 3-5h at 25°C. Both agreed within 2-3 % detector response respectively. Settings were as in the experiments reported. follows: excitation and emission bandwidths, 7.5nm; measure gain 10; reference voltage, 540V; time- Results constant, 3s; scan speed, lOnm/min. The Rhodamine B cuvette was maintained at 32±0.1°C and the Subunit composition ofthe ATPase preparations sample and reference cuvettes at 25+ 0.1°C by means A brief description of the subunit composition of of jacketed cell-holders through which water was the four ATPase preparations used here should circulated from two independent constant-tempera- facilitate the description of the results and their ture baths (Lauda K2R, Lauda, West Germany). subsequent discussion. Subunits are all designated by Cuvettes with light-paths of 2, 5 and 10mm were Greek letters; subunit a corresponds to subunit x of used as required to keep the absorption ofthe samples Carreira et al. (1976a,b). Preparation A was a3fl3y(p 1977 BACTSIF JAL ATP-ASE DENATURATION 323 (Andreu et al., 1973; Andreu & Munioz, 1975). 7 Preparations B1 and BA were (f'+ C)3335acp; -6 preparation BAT was (a'+ ao)3%i5erEp (Carreira et al., 1976a,b). Preparations of B-type contained two kinds I*e 5. of subunit a, which differed in apparent molecular i.m weight in dodecyl sulphate/polyacrylamide-gel elec- - 4 trophoresis. The difference between forms B1 and BA J3 resided in the relative proportion of these a chains. Preparation BAT was identical with form BA, except 8 that it contained an additional subunit, e, character- I ized as the intrinsic ATPase inhibitor.

Load more