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Slow pH-Dependent Conformational Transitions in Rabbit Skeletal Muscle Phosphofructokinase HECTOR B. RODRIGUEZ* and MICHAEL R. HOLLAWAY Department of Biochemistry, University College London, Gower Street, London WClE 6BT, U.K. The results of some preliminary experiments involving the reaction of the thiol groups of rabbit muscle phosphofructokinase (EC 2.7.1.1 1) with Ellman’s reagent (Ellman, 1955) suggested that a pH-dependent conformation change in the enzyme occurred in the range pH7.0 to 9.0. In this communication we describe an investigation of the rate and extent of this conformational change as monitored by changes in enzyme thiolgroup reactivity. The experimental approach involved subjecting the enzyme to a ‘pHjump’ immediately before reaction with the thiol reagent. Materials and methods Sodium b-glycerophosphate, N-glycylglycine, KCl and EDTA (disodium salt) were analytical-grade reagents from Fisons, Loughborough, Leics., U.K. Ellman’s reagent [5,5’-dithiobis-(2-nitrobenzoic acid)] was purchased from R. N. Emanuel Ltd., Wembley, Middx., U.K., and recrystallized twice from water/ethanol (1 :1, v/v). Rabbit skeletal-muscle phosphofructokinase was prepared by a modification (Rodriguez, 1973) of the method of Kemp & Forest (1968). The specific enzyme activity of these preparations, assayed by a modification of the method of Dyson & Noltman (1965), was in the range 15&220units/mg of protein. The concentration of enzyme solutions were determined spectrophotometrically at 297 nm, by using the value of Ei’,%, = 10.2 given by Parmeggiani et al. (1966). A protomer mol.wt. of 84000 was assumed. Timecourses of the release of 3-carboxylate4nitrothiophenolateanion during the reactionoftheenzyme WithEllman’sreagent werefollowedat412nm byusinga DurrumGibson stopped-flow spectrophotometer (Durrum Instruments Co.,Palo Alto, Calif., U.S.A.) thermostatically maintained at 25°C. The path length of the observation cuvette was 1.7cm. Traces of timecourses, linear in percentage transmission, were recorded on a Tektronix 564 retention oscilloscope, photographed, and absorbanceagainst-time curves computed from measurements from drawings of enlarged projections of the negatives on graph paper.

Results The timecourses of the extent of reaction of enzyme thiol groups with Ellman’s reagent given in Fig. 1 show that the rate of reaction of the different groups depends on the ‘history’ of the enzyme. Preincubation of the enzyme at pH9.0 before reaction at pH8.9 with the thiol reagent leads to enhanced reactivity in about two groups per protomer compared with the enzyme preincubated at pH7.0, ‘jumped’ to pH8.9 and simultaneously mixed with the reagent (Fig. 16). However, preincubation at the higher pH value gave consistently a decrease in the rate of reaction of some more slowly reacting thiols (see Fig. la). It was decided to determine the rate of the pH-dependent conformational transition leading to enhanced reactivity in some thiol groups. A solution of enzyme at pH7 was transferred to a buffer at pH 8.9 and the resulting solution transferred as quickly as possible to one syringe of the stopped-flow apparatus (the other syringe contained a solution of Ellman’s reagent). A time-course of the thiol-group reaction with thereagent was recorded immediately and then at different known times. Progress curves recorded at different times are shown in Fig. 2(a).From these curves it is possible to calculate the number of additional rapidly reacting thiol groups that have appeared at the different * Present address: Facultad de Ciencias, Universidad de 10s Andes, Merida, Venezuela. VOl.

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Time (s) Fig. 1. Time-coursesfor the reaction of phosphofructokinase with Ellman’s reagent at pH8.9 with enzyme preincubated at pH7.0 and 9.0 The time-courses shown are for the reaction of enzyme ( 0 . 7 ~with ~ ) Ellman’s reagent ( 0 . 5 m ~at ) pH 8.9 and 25°C. 0 , Time-coursesfor the reaction of enzyme preincubated at pH7.0 in a buffer comprising 10m-glycerophosphate, 0.1 M-KCland 1mM-EDTA and then mixed with Ellman’s reagent in a buffer comprising 5Om~-glycylglycine, 0.1 M-KCI and 1m~-EDTA,pH9.0. 0 , Reaction where the enzyme was preincubated for l5min in the pH9.0 buffer and the Ellman’s reagent in the pH7.0 buffer. Curves in (a) and (b) are for the same time-course but drawn with different time-scales.

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times (see Fig. 26). The time-course (Fig. 26) shows that the structural change in the enzyme leads to the appearance of 1.5-2 more rapidly reacting groups in a process with a half-lifeof about 4min, correspondingto afirst-orderrateconstant of about 3 x 10-3s-1. Discussion

Kemp & Forest (1968) also reported a pH-dependent conformation change in phosphofructokinase leading to exposure of additional thiol groups with high reactivity towards Ellman’s reagent. However, no report was made of a determination of the time-scale of this change and the contribution this could have made to timecourses followed at different pH values. The present results, demonstrating slow relaxations in 1976

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phosphofructokinase conformation, indicate that timecourses of the reactions of the enzyme thiol groups require an interpretation which takes these slow changes into account. The immediate ‘history’ of the enzyme solution will also be a factor in the observed timecourse. H. B. R. thanks the Facultad de Ciencias and Consejo Desarrollo Cientifico Humanistico, Universidad de 10s Andes, Merida, Venezuela for financial support. M. R. H. thanks The Science Research Council for a grant to purchase the stopped-flow apparatus.

Dyson, J. E. & Noltman, E. A. (1965) Anal. Biochem. 11, 362 Ellman, G. L. (1955) Arch. Biochem. Biophys. 82,70 Kemp. R. G. & Forest, P. B . (1968) Biochemistry 7, 2596-2603 Parmeggiani, A., Luft, J. H., Love, D. S.& Krebs, E.G. (1966)J. Biol. Chem. 241,46254637 Rodriguez, H. B. (1973) Ph.D. Thesis, University of London

An Investigation of the Subunit Structure of Rabbit Skeletal-Muscle Phosphofructokinase IAN A. SIMPSON and MICHAEL R. HOLLAWAY Department of Biochemistry, University College London, Gower Street, London WClE 6BT, U.K.

On the basis of ultracentrifugation experiments in 8M-urea and tryptiopeptide ‘mapping’ studies, Paetkau et al. (1968) concluded that the 93000-mol.wt. protomer of rabbit skeletal-muscle phosphofructokinase comprises four subunits each of mol.wt. about 23000, such that the protomer structure is either AJB or A2BC. Since then, work by Coffee et al. (1972) on the rabbit muscle enzyme and by Brennan et al. (1974) on the sheep heart enzyme indicated that in each case the protomer comprised two similar, if not identical, polypeptide chains, each with a mol.wt. of about 40000. These subunits appeared to be joined in some way that prevented their separation by treatment with guanidine hydrochloride or detergent. The aim of the present study was to investigatethe substructure of the protomer by a method involving specific covalent attachment of the phosphofructokinase molecule to an insoluble matrix and then removing any dissociable components after treatment with urea and detergents. The high reactivity towards Ellman’s reagent [5,5’-dithiobis-(2-nitrobenmic acid)] of the single class-I thiol group per protomer (Kemp, 1969) was exploited to make the specific covalent attachment in the way shown in Scheme 1 (reactions 1 and 2). The rabbit skeletal-muscle phosphofructokinase was prepared by a modification (Rodriguez, 1973) of the method of Kemp & Forest (1968) and gave a specific activity of 120-180units/mg of protein depending on the time of storage. The protomer mol.wt. as measured by the method of Laemmli (1970) was 72000f5000. Thiolated Sepharose 4B (N-acetylhomocysteinelinked via 3,3‘-iminobispropylamineto Sepharose) was prepared by the method of Cuatrecasas (1970) by using Sepharose desulphonated and cross-linked by the methods of Porath et al. (1971). The degree of thiol substitution of the product was 6-18pmol of thiol groupslgm. Steps 1 and 2; attachment of the enzyme to the Sepharose

In a t y p i d experiment 5 d of a solution of enzyme (3mglml) in 0.1M-sodium phosphate buffer, pH7.4, containing 1mM-EDTA was treated with a stoicheiometric amount of Ellman’s reagent to give enzyme derivative I (see Scheme 1). After passage through a column of Sephadex G-25 equilibrated with the pH7.4 buffer, the solution of derivative I (lOml, 1.5mg/ml)was stirred gently with 0.2g of thiolated Sepharose for 1 h at 25°C. The resultant slurry was packed in a chromatography column (0.5cm x 5cm) VOl. 4

Slow pH-dependent conformational transitions in rabbit skeletal muscle phosphofructokinase.

560th MEETING, OXFORD 83 Slow pH-Dependent Conformational Transitions in Rabbit Skeletal Muscle Phosphofructokinase HECTOR B. RODRIGUEZ* and MICHAEL...
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