Analysis of mutations induced by replication of UV-damaged plasmid DNA in HeLa cell extracts.

We have used an SV40-based shuttle vector, pZ189, to investigate the capacity of HeLa cell extracts to reproduce the in vivo process of mutation fixation. We showed previously that when UV-irradiated pZ189 is replicated in these extracts, bypass of UV photoproducts occurs, resulting in base substitution mutations in the supF gene of the vector. Here we report the DNA sequence characterization of a collection of 60 of these UV-induced mutants. Most of the mutations observed are single or tandem double base substitutions at dipyrimidine sites; of these, approximately 90% are G:C-->A:T transitions. Mutations are observed predominantly at a few sites, in particular at positions 155 and 156 in the supF sequence. No dramatic differences in the mutation spectrum were observed when the orientation of the supF gene was reversed with respect to the SV40 origin of replication, suggesting that mutation fixation occurs similarly on both the leading and the lagging strands for DNA replication. Generally, the mutational hot spots observed in vitro are at the same sites as those observed when UV-irradiated pZ189 was passaged in human or monkey cells in culture. Thus, it appears that the replication and mutagenesis of UV-damaged templates in HeLa cell extracts accurately reflects these processes in the intact cell.

Effects of calcium and calmodulin antagonists on calpain II subunit conformations.

Only the 80-kD catalytic subunit of smooth muscle calpain II shows a change in intrinsic fluorescence on binding calcium, but both the 80-kD and 30-kD subunits show fluorescence changes in bound toluidinyl-naphthalenesulphonate as a result of calcium binding. Both subunits also show changes in intrinsic fluorescence in the presence of calmidazolium and felodipine. These studies indicate that both subunits have binding sites for calcium and the calmodulin antagonists, which are probably located in the calmodulin-like domain of each subunit.

Fluorescence spectroscopic analysis of calpain II interactions with calcium and calmodulin antagonists.

1. The intrinsic fluorescence of epoxysuccinyl-inhibited calpain II undergoes a Ca2(+)-dependent decrease which contrasts with the increase observed for calmodulin. 2. Calpain II was inhibited by the calmodulin antagonist toluidinylnaphthalenesulfonate (TNS), and a Ca2(+)-dependent increase in TNS fluorescence intensity was observed for epoxysuccinyl-inhibited calpain II. 3. The calmodulin antagonists calmidazolium CDZ and felodipine both caused decreases in the intrinsic fluorescence of epoxysuccinyl-inhibited calpain II. 4. Increasing concentrations of Ca2+ caused an increase in the fluorescence intensity of the inhibited enzyme in the presence of (CDZ), and a decrease in the presence of felodipine. 5. It is concluded from these studies that Ca2+ and calmodulin antagonists induce conformational changes in calpain II, and that changes occur in regions other than the Ca2(+)-binding domains.

Ca2+, pH and the regulation of cardiac myofilament force and ATPase activity.

When the pH surrounding myofilaments of striated muscle is reduced there is an inhibition of both the actin-myosin reaction as well as the Ca2+-sensitivity of the myofilaments. Although the mechanism for the effect of acidic pH on Ca2+-sensitivity has been controversial, we have evidence for the hypothesis that acidic pH reduces the affinity of troponin C (TNC) for Ca2+. This effect of acidic pH depends not only on a direct effect of protons on Ca2+-binding to TNC, but also upon neighboring thin filament proteins, especially TNI, the inhibitory component of the TN complex. Using fluorescent probes that report Ca2+-binding to the regulatory sites of skeletal and cardiac TNC, we have shown, for example, that acidic pH directly decreases the Ca2+-affinity of TNC, but only by a relatively small amount. However, with TNC in whole TN or in the TNI-TNC complex, there is about a 2-fold enhancement of the effects of acidic pH on Ca2+-binding to TNC. Acidic pH decreases the affinity of skeletal TNI for skeletal TNC, and also influences the micro-environment of a probe positioned at Cys-133 of TNI, a region of interaction with TNC. Other evidence that the effects of acidic pH on Ca2+-TNC activation of myofilaments are influenced by TNI comes from studies with developing hearts. In contrast, to the case with the adult preparations, Ca2+-activation of detergent extracted fibers prepared from dog or rat hearts in the peri-natal period are weakly affected by a drop in pH from 7.0 to 6.5. This difference in the effect of acidic pH appears to be due to a difference in the isoform population of TNI, and not to differences in isotype population or amount of TNC.

Troponin I enhances acidic pH-induced depression of Ca2+ binding to the regulatory sites in skeletal troponin C.

Inhibition of muscle force development by acidic pH is a well known phenomenon, yet the exact mechanism by which a decrease in pH inhibits the Ca2+-activated force in striated myofilaments remains poorly understood. Whether or not the deactivation by acidic pH involves direct competition between Ca2+ and protons for regulatory binding sites on fast skeletal troponin C (TnC) or whether other proteins in thin filament regulation are important remains unclear. We measured the effects of acidic pH on Ca2+-dependent fluorescent changes in TnC labeled with the probe danzylaziridine (Danz), which reports Ca2+ binding to the regulatory (Ca2+-specific) sites. Measurements were also made with TnCDanz complexed with the inhibitory Tn unit, TnI, and in the whole Tn complex. Our results show that a drop in pH from 7.0 to 6.5 is associated with a 1.6-fold increase in the midpoint for the relation between free Ca2+ and Ca2+ binding to the regulatory sites on TnCDanz. However, when TnCDanz was present in its complex with either TnI alone or with TnI-TnT, the increase in midpoint free Ca2+ was increased by 3.5-fold. We tested whether this potentiation in the effect of acidic pH on Ca2+ binding to TnC is due to a pH-induced alteration in the binding of TnI to TnC. A decrease in pH from 7.0 to 6.5 was associated with a halving of the affinity of TnI for TnC. We also probed the effect of acidic pH on TnI. This was done (i) by measuring the intrinsic fluorescence of tryptophan residues in TnI alone and (ii) by measuring fluorescence of TnI (in the Tn complex) labeled at Cys-133 with 5-iodoacetamidofluorescein. A drop in pH from 7.0 to 6.5 was associated with a 15% decrease in intrinsic fluorescence and with a 30% decrease in the fluorescence of the 5-iodoacetamidofluorescein probe. We conclude, therefore, that while protons and Ca2+ may directly affect Ca2+ binding to regulatory sites on fast skeletal TnC, the effect of acidic pH on TnC Ca2+ binding is amplified in the TnI-TnC and Tn complexes by a pH-related effect on the affinity of TnI for TnC.

Calmidazolium, a calmodulin antagonist, stimulates calcium-troponin C and calcium-calmodulin-dependent activation of striated muscle myofilaments.

Although regulatory Ca2+-binding domains of calmodulin (CaM) and troponin C (TnC) are similar, it is interesting that agents that act as CaM antagonists appear to be TnC "agonists" in that they sensitize cardiac myofilaments to activation by Ca2+ (El-Saleh, S., and Solaro, R. J. (1987) Biophys. J. 51, 325 (abstr.). This indicates that the effects of agents that react with Ca2+-binding proteins may depend on protein-protein interactions involved in a particular Ca2+-dependent process. In experiments described here, we have explored this idea by testing effects of calmidazolium (CDZ), a potent calmodulin antagonist on striated muscle myofilaments regulated by cardiac TnC, skeletal TnC, and CaM. CDZ was shown to increase submaximal calcium activation of myofilament force and ATPase activity in both cardiac and skeletal muscle, but the effect was greater in the case of the cardiac preparations. In the presence of 10 microM CDZ, the free Ca2+ giving half-maximal activation was reduced to about 60% of the control value in the case of cardiac myofilaments. Analogous differential effects of CDZ were also seen in studies in which we measured direct effects of CDZ on Ca2+-dependent fluorescence changes of cardiac TnC and skeletal TnC labeled with probes reporting Ca2+ binding to the regulatory sites. Measurements were also done with myofibrillar preparations of psoas muscle in which the native skeletal TnC was removed and exchanged with cardiac TnC and CaM, both of which could substitute for skeletal TnC as a regulatory protein. CDZ was more effective in sensitizing Ca2+-dependent MgATPase activity of skeletal myofibrils containing CaM than in preparations containing the native TnC. However, CDZ was most effective in its Ca2+-sensitizing effect in the case of the preparations containing cardiac TnC. Our results indicate that effects of agents that bind to Ca2+-binding proteins depend not only on the particular variant, but also on the specific environment in which the Ca2+-binding proteins operate.

Alteration of actin-tropomyosin interaction in 2,4-pentanedione-treated rabbit skeletal myofibrils.

In previous work, we (El-Saleh, S., Theiret, R., Johnson, P., and Potter, J. D. (1984) J. Biol. Chem. 259, 11014-11021) presented evidence that Ca2+ activation of skeletal myofilaments depends on a specific actin domain. We showed that rabbit skeletal thin filaments reconstituted with actin modified at Lys-237 activate heavy meromyosin X Mg2+-ATPase activity independently of the Ca2+ ion concentration. The modification, which apparently blocks the inhibitory effects of troponin-tropomyosin (Tn X Tm), on acto-heavy meromyosin X Mg2+-ATPase activity, consisted of conversion of Lys-237 to an enamine by reaction of purified actin with 2,4-pentanedione (PD). In experiments reported here, we have treated myofibrils with PD with the idea of altering actin in its native state within the myofilament lattice. Preparations of native and Tn X Tm free ("desensitized") myofibrils were incubated with PD (100 mol/mol of actin lysine) under rigorous conditions (10 mM 4-morpholinepropanesulfonic acid, pH 7.0, 2.0 nM [ethylenebis(oxyethylenenitrilo)]tetraacetic acid, 0.4 mM dithiothreitol, and 0.15 mM NaN3). Actin isolated from PD X myofibrils contained 0.5 mol of enamine/mol. In the presence of Ca2+, the Mg2+-ATPase activity of PD-treated myofibrils was 110-120% of the maximum Ca2+-stimulated Mg2+-ATPase activity of untreated control myofibrils. In low free Ca2+ (pCa greater than 8), the Mg2+-ATPase activity of the PD-treated myofibrils was not suppressed and remained at 100-106% of the maximum activity of the control myofibrils. Ca2+ sensitivity of the PD-treated myofibrils was restored following treatment with hydroxylamine, which hydrolyzes enamine's products. Preparations of desensitized myofibrils reconstituted with PD-modified or unmodified Tn X Tm demonstrated the same Ca2+-sensitive ATPase activities. On the other hand, preparations reconstituted with unmodified or PD-modified Tn X Tm and PD-modified desensitized myofibrils were insensitive to Ca2+ ion concentration. The Mg2+-ATPase activity of preparations of myosin treated with PD was not activated by modified or unmodified actin. Our results indicate that is is possible to produce an active state(s) of the myofibrils in the absence and presence of Ca2+ by specific alteration of the actin X Tm interaction following modification of myofibrillar actin most likely at Lys-237.

The role of tropomyosin-troponin in the regulation of skeletal muscle contraction.

Steric blocking of actin-myosin interaction by tropomyosin has been a working hypothesis in the study of the regulation of skeletal muscle contraction, yet the simple movement of actin-associated tropomyosin from a myosin-blocking position (relaxation) to a nonblocking position (contraction) cannot adequately account for all of the biophysical and biochemical observations which have been made to date. Ambiguous assignment of tropomyosin positions on actin during contraction, due in part to the limited resolution of reconstruction techniques, may also hint at a real lack of clearcut 'on' and 'off' positioning of tropomyosin and tropomyosin-troponin complex. Recent biochemical evidence suggests processes relatively independent of tropomyosin-troponin may have a governing effect on contraction, involving kinetic constraints on actin-myosin interaction influenced by the binding of ATP and the intermediates of ATP hydrolysis. Based on our current understanding put forth in this review, it is clear that regulatory interactions in muscle contraction do not consist solely of steric effects but involve kinetic factors as well. Where the latter are being defined in systems reconstituted from purified proteins and their fragments, the steric components of regulation are most clearly observed in studies of structurally more intact physiologic systems (e.g. intact or skinned whole muscle fibres). The fine detail of the processes and their interplay remains an intriguing question. Likewise, the precise physical relationship of myosin with actin in the crossbridge cycle continues to elude definition. Refinement of several methodologies (X-ray crystallography, three-dimensional reconstruction, time-resolved X-ray diffraction) will increase the potential for detailing the molecular basis of the regulation of muscle contraction.

Calcium-insensitive binding of heavy meromyosin to regulated actin at physiological ionic strength.

Several conflicting reports have been made regarding the affinity of myosin heads (subfragment 1 and heavy meromyosin (HMM) for regulated actin (actin complexed with tropomyosin and troponin) at low ionic strength (mu = 18-50 mM) and whether or not this interaction is Ca2+ sensitive (Chalovich, J. M., and Eisenberg, E. (1982) J. Biol. Chem. 257, 2432-2437; Chalovich, J. M., and Eisenberg, E. (1984) Biophys. J. 45, 221a; Wagner, P. D., and Stone, D. B. (1983) Biochemistry 22, 1334-1342; and Wagner, P. D. (1984) Biochemistry 23, 5950-5956). Since the low ionic strengths used in the above studies do not represent the physiological ionic strength under which intact muscle exhibits Ca2+-dependent tension development, we investigated the possibility of whether a Ca2+-dependent regulated actin-HMM interaction could be observed at physiological ionic strength (mu = 134 mM, pH 7.4) and in the presence of ATP (at 23-24 degrees C). Direct binding of HMM to varied concentrations of regulated actin (87.7-221 microM free actin) was measured by sedimentation in an air-driven ultracentrifuge. Under the above conditions, we found that the regulated actin activation of HMM-Mg2+-ATPase was about 94% inhibited in the absence of Ca2+ although the association constant (Ka) is only moderately affected in the presence of Ca2+. These results are similar to those obtained by Chalovich and Eisenberg (1982 and 1984) with subfragment 1 and HMM, respectively, at low ionic strength and support their suggestion that in solution tropomyosin-troponin may not act totally by physically blocking the formation of cross-bridges with actin, but instead may act to inhibit a kinetic step in the overall ATPase rate. Whether this holds true in more intact systems (e.g. myosin, thick filaments) remains to be determined. Our results also show a good correlation between levels of ATPase activation and HMM binding by unregulated actin and in regulated actin in the presence of Ca2+.

Modification of Lys-237 on actin by 2,4-pentanedione. Alteration of the interaction of actin with tropomyosin.

It has been possible to specifically label rabbit skeletal muscle actin at Lys-237 with 2,4-pentanedione, producing an enamine. This reaction can be reversed with hydroxylamine. The modification can be carried out with actin in either the G- or F-forms and does not affect polymerization-depolymerization. The modification does affect, however, the interaction of tropomyosin (Tm) with the modified F-actin. In the absence of Ca2+ and Mg2+ (mu = 0.12), Tm failed to bind to the modified F-actin whereas it did bind to unmodified F-actin (1 Tm:7 actins). Tm binding could be restored under these conditions by the addition of either troponin (Tn), Mg2+, or Mg2+ and Ca2+. Under certain conditions, Tm alone has been shown to inhibit actin-activated heavy meromyosin (HMM)-Mg2+-ATPase. This inhibition did not occur with the modified F-actin even though Tm was bound (approximately 1 Tm:7 actins). Even when Tn was added to this system (in the absence of Ca2+), no inhibition of ATPase could be observed. Thus, this modification appears to prevent F-actin X Tm from assuming the "blocking" inhibitory position (conformation). In addition, Tn appears to enhance the activation of heavy meromyosin-Mg2+-ATPase by the modified F-actin X Tm complex whether Ca2+ is present or not. This state may be analogous to the potentiated state (Murray, J. M., Knox, M. K., Trueblood, C. E., and Weber, A. (1982) Biochemistry 27, 906-915) seen with myosin subfragment 1-saturated actin at low ATP levels. Thus, using modified and unmodified F-actin, it is possible to produce three Tm X actin states: off (F-actin X Tm), on (modified F-actin X Tm), and "potentiated" (modified F-actin X Tm X Tn).

Calorimetric studies on monomeric and polymeric actin.

Differential scanning calorimetry of polymeric F-actin at pH 8.0 showed that the polymer had a concentration-independent thermal profile with a single transition temperature of 81 degrees C. In contrast, the thermal profile of G-actin was concentration-dependent, and although it resembled the F-actin profile at lower concentrations, it was found to have a more complex profile at higher protein concentrations.