Norma M. Allewell

A physicochemical analysis of conjugation in Tetrahymena pyriformis

Abstract The process of conjugation in Tetrahymena pyriformis is a useful model system for investigating mechanisms of cellular recognition, adhesion and fusion. As a first step in the biochemical analysis of this process, we have examined the effects of (a) nutrients; (b) metal ions; (c) several pharmacological agents (actinomycin D, cycloheximide, colchicine, theophylline, dithiothreitol and caffeine); and (d) temperature. We find that: 1. 1. While the complete nutrient medium inhibits conjugation, no single compound or group of compounds of the defined medium [1]produces any inhibition. 2. 2. At least trace amounts of Ca2+ are required. 3. 3. All of the pharmacological agents tested, except actinomycin D, inhibit both the preparations for conjugation and pair formation itself, indicating a requirement for both protein synthesis and low intracellular levels of cAMP, as well as the involvement of microtubules. 4. 4. While actinomycin D inhibits the preparations for conjugation, its addition after cells have begun to pair does not block further pairing. This result suggests that a stable RNA which is required for conjugation is produced during the preparations for conjugation. 5. 5. Paired cells may be disrupted for the first 1-1 1 2 i h after pairing by proteose peptone, cycloheximide, theophylline, and dithiothreitol. The cells undergo a transition 1 1 2 h after pairing which renders them resistant to these agents. 6. 6. The period of initiation (the time of starvation required to make cells competent to conjugate, the period of costimulation (the lag time preceding cell pairing after competent cells are mixed), and the rate of cell pairing are all temperature sensitive. Large changes in these parameters occur over narrow temperature ranges, possibly as a result of temperature-induced changes in membrane lipid composition or structure.

A kinetic analysis of the memory of a developmental interaction: Mating interactions in tetrahymena pyriformis☆

Abstract The kinetics of an inductive intercellular interaction and the decay of the memory of that interaction during a developmental process have been analysed. The effects of three inhibitors (actinomycin D, cordycepin and α-amanitin) of RNA synthesis have been examined, in order to explore the role of RNA in both processes. In the protozoan Tetrahymena pyriformis, inductive intercellular interactions between starved cells of complementary mating types result in the ability to form heterotypic cell pairs. These interactions have been shown previously to be blocked by either actinomycin D or rapid agitation [1, 2]. We show here that the effects of both actinomycin D and agitation are restricted to the first phase of the inductive period. This implies that two separable processes take place during the inductive period: (1) a period during which cells are activated by the induction; (2) a maturation period during which preparations for pair formation in activated cells are completed. In contrast to the effects of actinomycin D, neither cordycepin nor α-amanitin blocks the induction of pair-forming ability. Since rapid agitation also independently blocks pair formation, the kinetics of the decay of the memory of the inductive interactions may be analysed by interrupting pairing for various lengths of time by agitation. In the absence of any inhibitors, the decay of the memory is a random, discrete event for any cell, with a half-time of 3.9 ± 1.4 h. While α-amanitin has only a moderate effect on the memory, both actinomycin D and cordycepin dramatically accelerate its decay, reducing the half-time to 35 ± 5 min. A biochemical model which accounts for these observations and which may be applicable to other developmental systems is proposed.

Analysis of self-association of bovine neurophysins by gel chromatography☆

Analytical gel chromatography has been used to examine self-association of bovine neurophysins I and II under several sets of conditions. The data provide no evidence for associated species larger than the dimer. Association constants and Stokes radii of both monomer and dimer are very similar for both proteins in both 0.1 M KOAc, 0.16 M KCl and 0.1 M KPO4, 0.16 M KCl at pH 5.6 and 25 degrees C. The average values derived for the Stokes radii of the monomer and dimer under these conditions are 14.5 +/- 0.7 and 23.0 +/- 0.4 A, respectively. These results confirm the conclusion of Rholam and Nicolas [(1981) Biochemistry 20, 5837-5843] that the monomer and, to a lesser extent, the dimer are highly assymmetric. The Stokes radius of the monomer calculated by Rholam and Nicolas (op cit.) is approximately 30% larger than the value derived here. This discrepancy is probably the result of end-on penetration of the gel by elongated molecules [Y. Nozaki, N. M. Schechter, J. A. Reynolds, and C. Tanford (1976) Biochemistry 15, 3884-3890]. In contrast to Tellam and Winzor [(1980) Arch. Biochem. Biophys. 201, 20-24], it was found that neurophysin II does not exist solely as the dimer in 0.1 M KPO4, pH 5.6, although substitution of 0.1 M KPO4 for 0.1 M KOAc does increase the association constant by a factor of seven. Addition of 1.4 M LiCl at pH 8.1 also increases the association constant sevenfold, as well as increasing the Stokes radius of the monomer approximately 20%. The effects of ionic strength are consistent with the conclusion of Nicolas et al. [(1978) J. Biol. Chem 253, 2633-2639] that formation of the dimer depends upon hydrophobic bonding.

Regulation of neutral protease activity through the life cycle of Tetrahymena pyriformis

Substantial fluctuations in the intracellular specific activity of neutral proteases, as assayed at pH 8 with azocasein as substrate, occur during the life cycle of the protozoan Tetrahymena pyriformis. Specific activity increases during growth in 2% proteose peptone, despite slow secretion into the medium. The most rapid increase occurs during late stationary phase and appears to be a response to one or more low molecular weight (less than 10 000), heat-stable, trypsin-insensitive, polar molecules secreted into the medium. In contrast, intracellular specific activity drops by a factor of 2--5 within the first 2--3 h after transfer to non-nutritive medium. The decrease in activity under these conditions results from an enhanced rate of secretion and the cessation of net synthesis. Its kinetics are unaffected by cycloheximide and concanavalin A.

Use of analytical gel chromatography to analyze tertiary and quaternary structural changes in E. coli aspartate transcarbamylase.

E. coli aspartate transcarbamylase (ATCase) is a large (310 kDa) protein that undergoes major changes in quaternary structure when substrates and regulatory nucleotides bind. We have used analytical gel chromatography to detect quaternary structure changes in both the holoenzyme and its catalytic subunit (c3), to characterize the quaternary structure of single site mutant proteins and to monitor urea-induced dissociation and unfolding of c3. Binding of the bisubstrate analog PALA (N-(phosphonacetyl)-L-aspartate) to ATCase and c3 has been shown to alter s20.w by -3.3% and + 1.4%, respectively [Howlett, G.J. and Schachman, H.K. (1977), Biochemistry 23, 5077-5083]. The corresponding changes in the chromatographic partition coefficient (sigma) are -2.6 +/- 0.3% and 5.5 +/- 1.9% on Sephacryl S400HR and S200, respectively. Partition coefficients of mutant ATCases with single site mutations in the c chain differ from those of the wild-type protein by +/- 0.5% in small zone experiments; for example, mutations Arg 269----Gly and Glu 239----Gln alter the partition coefficient by 0.4% and -0.5%, respectively. The partition coefficient of mutant Glu 50----Gln is identical to the wild type enzyme. In the presence of saturating PALA, partition coefficients of Glu 50----Gln and Arg 269----Gly, but not Glu 239----Gln are identical to those of the wild type. Results for Glu 239----Gln are consistent with measurements of activity, small angle X-ray scattering and sedimentation coefficient that indicate that mutations at this site shift the quaternary structure towards the R state [Ladjimi and Kantrowitz (1988), Biochemistry 27, 276-83; Vachette and Hervé, cited by Kantrowitz and Lipscomb (1988), Science 241, 669-674; Newell and Schachman (1988), FASEB J. 2, A551]. Results for Glu 50----Gln are also consistent with measurements of activity (Ladjimi et al. (1988), Biochemistry 27, 268-276). The changes in tertiary and quaternary structure that result from urea-induced denaturation of c3 result in larger changes in the partition coefficient. Dissociation into folded monomers in 1-1.75 M urea is accompanied by a 4.6% increase in partition coefficient, while denaturation at greater than 5 M urea gives rise to a 43% decrease on S-300 Sephacryl. The bisubstrate analog PALA suppresses dissociation and increases the cooperativity of the unfolding reaction.

Differential scanning calorimetric studies of E. coli aspartate transcarbamylase. III, The denaturational thermodynamics of the holoenzyme with single-site mutations in the catalytic chain

Aspartate transcarbamylase (EC 2.1.3.2) from E. coli is a multimeric enzyme consisting of two catalytic subunits and three regulatory subunits whose activity is regulated by subunit interactions. Differential scanning calorimetric (DSC) scans of the wild-type enzyme consist of two peaks, each comprised of at least two components, corresponding to denaturation of the catalytic and regulatory subunits within the intact holoenzyme (Vickers et al., J. Biol. Chem. 253 (1978) 8493; Edge et al., Biochemistry 27 (1988) 8081). We have examined the effects of nine single-site mutations in the catalytic chains. Three of the mutations (Asp-100-Gly, Glu-86-Gln, and Arg-269-Gly) are at sites at the C1: C2 interface between c chains within the catalytic subunit. These mutations disrupt salt linkages present in both the T and R states of the molecule (Honzatko et al., J. Mol. Biol. 160 (1982) 219; Krause et al., J. Mol. Biol. 193 (1987) 527). The remainder (Lys-164-Ile, Tyr-165-Phe, Glu-239-Gln, Glu-239-Ala, Tyr-240-Phe and Asp-271-Ser) are at the C1: C4 interface between catalytic subunits and are involved in interactions which stabilize either the T or R state. DSC scans of all of the mutants except Asp-100-Gly and Arg-269-Gly consisted of two peaks. At intermediate concentrations, Asp-100-Gly and Arg-269-Gly had only a single peak near the Tm of the regulatory subunit transition in the holoenzyme, although their denaturational profiles were more complex at high and low protein concentrations. The catalytic subunits of Glu-86-Gln, Lys-164-Ile and Asp-271-Ser appear to be significantly destabilized relative to wild-type protein while Tyr-165-Phe and Tyr-240-Phe appear to be stabilized. Values of delta delta G degree cr, the difference between the subunit interaction energy of wild-type and mutant proteins, evaluated as suggested by Brandts et al. (Biochemistry 28 (1989) 8588) range from -3.7 kcal mol-1 for Glu-86-Gln to 2.4 kcal mol-1 for Tyr-165-Phe.

Structural fluctuations in aspartate transcarbamylase. Succinimide quenching and fluorescence depolarization of tryptophan and tyrosine residues.

The effects of binding of various effector ligands on the dynamics of aspartate transcarbamylase (ATCase, c6r6) and on its regulatory (r2) and catalytic (c3) subunits were characterized by examining succinimide quenching of the intrinsic fluorescence, and by measurement of the lifetime-resolved anisotropies. The lifetimes of the tryptophan residues in c3 and c6r6 are about 1.7 ns while those of tyrosine residues in r2 are 2.7 ns. These lifetimes are not significantly altered by the binding of various substrates, substrate analogs and nucleotides. The effects of ligand binding on the accessibility of both tyrosine and tryptophan residues to the quencher are modest in all cases, though the changes are in the same direction as seen using other physicochemical techniques such as hydrogen exchange (M. Lennick and N.M. Allewell, Proc. Natl. Acad. Sci. U.S.A. 78 (1981) 6759). The tryptophan residues in both c3 and c6r6 are immobilized whereas the tyrosine residues of r2 have some motional freedom. Ligands have no effect on the immobilized tryptophan residues in c3 and c6r6, while binding of nucleotides to r2 results in a small decrease in the motional freedom of the tyrosine residues. These results suggest that the protein matrix around the aromatic amino acids in r2, c3 and c6r6 is rather rigid and that local effects of ligands on the dynamics of these residues, and that of the surrounding protein matrix, are minor. They are in general agreement with the results of the crystal structure determination (R.B. Honzatko et al., J. Mol. Biol. 160 (1982) 219).