pH-dependence characteristics of Ca-ATPase activity of heavy meromyosin with modified SH-groups]

Study of pH-dependence of Ca-ATPase activity of heavy meromyosin (HMM) at low and high ionic strength showed essential differences in the modifying effect of two sulfhydryl reagents, p-CMB and silver. Silver ions in conditions studied independently on pH and KCl concentration produce an inhibition of ATP hydrolysis by myosin and HMM, the shape of the pH-dependence curve remaining similar to that of the native enzyme up to 40% of blocking free sulfhydryl groups. At the same degree of binding of sulfhydryl groups with p-CMB at 0,5 M KCl the pH-dependence curve due to activation at neutral pH changes it's shape and becomes similar to that for dissociation of two ionizable groups (at neutral and alkaline regions). In contrast to this, a low or zero concentrations of KCl no activation was observed for the enzyme with 40-50% of SH-Groups modified by p-CMB and Ca-ATPase in this case seemed to be independent of pH. The data obtained suggest that SH-Groups are not included into the active site of myosin, and the activating effect observed for some sulfhydryl reagents, is due to conformational changes and it can be the result of the penetrance of the organic part of the reagent molecule into hydrophobic region of the protein.     

Butanedione monoxime suppresses contraction and ATPase activity of rabbit skeletal muscle

The effects of 2,3-butanedione 2-monoxime (BDM) on mechanical responses of glycerinated fibers and the ATPase activity of heavy meromyosin (HMM) and myofibrils have been studied using rabbit skeletal muscle. The mechanical responses and the ATPase activity were measured in similar conditions (ionic strength 0.06-0.2 M, 0.4-4 mM MgATP, 0-20 mM BDM, 2-20 degrees C and pH 7.0). BDM reversibly reduced the isometric tension, shortening speed, and instantaneous stiffness of the fibers. BDM also inhibited myofibrillar and HMM ATPase activities. The inhibitory effect on the relative ATPase activity of HMM was not influenced by the addition of actin or troponin-tropomyosin-actin. High temperature and low ionic strength weakened BDM's suppression of contraction of the fibers and the ATPase activity of contracting myofibrils, but not of the HMM, acto-HMM and relaxed myofibrillar ATPase activity. The size of the initial phosphate burst at 20 degrees C was independent of the concentration of BDM. These results suggest that the suppression of contraction of muscle fibers is due mainly to direct action of BDM on the myosin molecules.     

Presence of a unit for actin-myosin interaction during the superprecipitation of actomyosin.

The interaction of actin with myosin was studied in the presence of ATP at low ionic strength by means of measurements of the actin-activated ATPase activity of myosin and superprecipitation of actomyosin. At high ATP concentrations the ATPase activities of myosin, heavy meromyosin (HMM) and myosin subfragment 1 (S-1) were activated by actin in the same extent. At low ATP concentrations the myosin ATPase activity was activated about 30-fold by actin, whereas those of HMM and S-1 were stimulated only several-fold. This high actin activation of myosin ATPase was coupled with the occurrence of superprecipitation. The activation of HMM or S-1 ATPase by actin shows a simple hyperbolic dependence on actin concentration, but the myosin ATPase was maximally activated by actin at a 2:1 molar ratio of actin to myosin, and a further increase in the actin concentration had no effect on the activation. These results suggest the presence of a unit for actin-myosin interaction, composed of two actin monomers and one myosin molecule in the filaments.     

Bicarbonate stimulation of mitochondrial ATPase. Effect of physical training.

Bicarbonate stimulation of hepatic mitochondrial ATPase activity decreased in rats subjected to intense physical training and reached minimum values at the end of the third week. The stimulatory effect of bicarbonate on mitochondrial heart ATPase remained unaffected under equal conditions. ATPase stimulation by dinitrophenol and sensitivity to oligomycin, both in mitochondria from rat liver or heart, were not affected by physical training. Results suggest that stimulation by dinitrophenol and bicarbonate might be due to effects on separate sites of the enzyme.     

The amounts of adenosine di- and triphosphates bound to H-meromyosin and the adenosinetriphosphatase activity of the H-meromyosin-F-actin-relaxing protein system in the presence and absence of calcium ions. The physiological functions of the two routes of myosin adenosinetriphosphatase in muscle contraction.

The rates of the ATPase [EC 3.6.1.3] reaction of the H-meromyosin-F-actin-relaxing protein system were measured in 2 mM MgCl2, 50mM KC1, and 10mM Tris-HC1 at pH 7.8 and 20 degrees in the presence and absence of 0.05-0.1 mM Ca2+ ions. The concentrations of H-meromyosin (HMM) and the F-actin-relaxing protein (F-A-PR) complex were 3.4 and 3 mg/ml, respectively, and the ATPase reaction was coupled with 4 mg/ml of pyruvate kinase [EC 2.7.1.40] and 1 or 20 mM phosphoenolpyruvate to regenerate ATP. The amount of ADP bound to HMM during the ATPase reaction was determined by measuring the amount of ADP remaining in the reaction mixture. The amount of ATP bound to HMM was determined by subtracting the amount of bound ADP from the total amount of nucleotides bound to HMM, which was measured by a rapid flow-dialysis method. The following results were obtained. 1. The ATPase activity of the HMM-F-A-RP system increased linearly with increase in the amount of ATP added, and was independent of the presence of 0.05 mM Ca2+, when the amount of ATP added was less than 1 mole/mole of HMM. In the presence of 0.05 mM Ca2+, the ATPase activity reached a maximal level when 1.2-1.5 mole of ATP was added per mole of HMM, and maintained this level even at 3 moles of added ATP/mole of HMM. In the presence of 3mM EGTA, the ATPase activity decreased with increase in the amount of ATP added, from 1.5 to 3 moles of ATP/mole of HMM, and reached the level of the HMM ATPase reaction at 3 moles of added ATP/mole of HMM. Similar results were observed when the concentration of HMM was maintained at 3.4 mg/ml and the concentration of the F-A-RP complex was decreased from 3 to 1 or 0.5 mg/ml.     

pH-dependence of inhibitory action of eosin Y on ATPase activity of smooth muscle actomyosin complex of the uterus

Research of pH-dependence of inhibitory action of eosin Y (2',4',5',7'-tetrabromofluorescin) on ATPase of contractile proteins of smooth muscles of the uterus has shown that the increase of concentration of this inhibitor (from 0.1 to 10 microM) influenced the profile of pH-dependence of ATPase activity of actomyosin: in the presence of 0.1 microM eosin Y the change of optimal value of pH has been observed in more sour side in relation to the control; at the increase of concentration of eosin Y (from 0.5 to 10 microM) the strongly pronounced optimum of pH is absents in general. The ability of eosin Y to inhibit the ATPase activity of contractile complex is dependent on pH of incubation environment. The change of pH from 6.0 to 7.2 results in a 9-fold decrease of magnitude of apparent constant of inhibition Ki (from 6.5 +/- 0.8 microM to 0.74 +/- 0.07 microM). The obtained results indicate that the diminishing of concentration of H+ in an incubation environment favors the increase of affinity ATPase of actomyosin for eosin Y and prove the important role of ionization processes in the system "enzyme-substrate-inhibitor" for realization of inhibitory action of eosin Y.     

Characteristics of affinity modification of myosin ATPase under the action of monoaldehyde derivatives of ADP]

It was shown that the highly purified monoaldehyde derivative of ADP obtained by partial reduction of the dialdehyde derivative of ADP causes strong irreversible inhibition of the Ca-ATPase activity of myosin subfragment I, the inhibiting effect being of the affinity modification type. The addition to the reaction medium of Mg2+ (but not Ca2+) during the subfragment I interaction with the inhibitor fully prevents the inhibiting effect at all substrates used (Ca-, Mg- or K, EDTA-ATPases). Contrariwise, the subfragment I modified in the absence of Mg2+ exhibits the same degree of inhibition for all the three types of the ATPase activity. An unexpected result that was previously unobserved for other affinity modifiers of myosin ATPase is the maintenance of activity in 50% of active centers, when "two-head" forms of the enzyme (the myosin proper and heavy meromyosin, HMM) are modified. Noteworthy that the affinity modification reaction is characterized by the same values of inhibition constants as in the case of myosin subfragment I (Ki = 3.3-3.5 X 10(-4) M; ki = 0.03-0.04 min-1). This finding provides additional evidence in favour of functional asymmetry of myosin heads in the myosin molecule which seems to be due to the screening of the active center of one head by the other one.     

Chicken gizzard heavy meromyosin that retains the two light-chain components, including a phosphorylatable one.

A method was developed to obtain a preparation of chicken gizzard heavy meromyosin (HMM) that retains the two light-chain components of parent myosin: the 20,000-dalton and 17,000-dalton light-chains. The HMM preparation was also shown to retain two characteristics of the ATPase activity of the parent myosin: the characteristic effect of phosphorylation of the 20,000-dalton light-chain component on the ATPase activity, and the characteristic dependence of the ATPase activity on the KCl concentration. 1. Two distinct stages were observed in the Mg-ATPase reaction catalyzed by gizzard HMM and rabbit skeletal actin in the presence of gizzard "native" tropomyosin (NTM) and Ca2+ ions: an early lag phase, in which the reaction rate gradually increased, and a subsequent steady state, in which the reaction proceeded at a high, constant rate. Urea-gel electrophoresis revealed that the 20,000-dalton light-chain component was gradually phosphorylated in the lag phase, and was fully phosphorylated in the steady state. It was also observed that addition of EGTA (to remove Ca2+ ions) at various times in the lag phase caused neither a further increase nor a decrease in the reaction rate, and that addition of EGTA in the steady state caused no change in the reaction rate. These observations imply that the ATPase activity increased as the amount of phosphorylated 20,000-dalton light-chain component increased, and also that Mg-ATPase of acto-phosphorylated HMM was no longer calcium-sensitive. 2. The Mg-ATPase activity of HMM in the presence of gizzard NTM and Ca2+ ions or EGTA was studied as a function of the concentration of rabbit skeletal actin. The maximal activity (Vmax) and the apparent affinity constant of acto-HMM (KA) were thus estimated from the double-reciprocal plot of Eisenberg-Moos: the Vmax and KA values for phosphorylated HMM (in the presence of Ca2+ ions) were 5 S(-1) and 5.5 mg/ml actin, respectively, and the Vmax value for unphosphorylated HMM (in the presence of EGTA) was 0.3 S(-1), assuming that the KA value with unphosphorylated HMM is equal to that with phosphorylated HMM.     

Heavy meromyosin from skipjack tuna, Euthynus pelamis. Preparation and enzymic properties.

A method was developed to obtain heavy meromyosin (HMM) from the tryptic digest of skipjack tuna dorsal myosin. The tuna HMM thus obtained was shown to be homogeneous on gel filtration-gel electrophoresis, and on ultracentrifugation. The sedimentation constant (S20,w) was estimated to be 6.1S for tuna HMM. The ATPase activity of tuna dorsal HMM was found to be very similar to that of rabbit skeletal HMM in many respects: KCl concentration dependence, pH dependence, effect of pCMB, kinetic parameters (Vmax and Ka) in actin activation, and Arrhenius activation energy. The only difference found between tuna HMM and rabbit HMM was in heat denaturation behavior: the ATPase activities of tuna HMM were approximately four times as sensitive to heat inactivation as those of rabbit HMM. Thus, tuna HMM should represent a good experimental material for investigations of the molecular basis of susceptibility to denaturation, and of the characteristics of fish myosins in general. A new type of heat denaturation of myosin was observed. It occurred in a very early stage of heat treatment of either tuna dorsal myosin or rabbit skeletal myosin; however, it did not occur upon heat treatment of HMM of either tuna or rabbit, and it was detectable in terms of the Mg-ATPase activity only when the activity was measured in the presence of untreated actin.     

Study of E. coli penicillin amidase. The pH-dependence of the enzymatic inactivation kinetics]

The pH-dependence of the inactivation rate constant of penicillin amidase at a temperature of 40 degrees C was studied. It was shown that in all cases the enzyme inactivation corresponded to the kinetics of the reaction of the 1st order. The pH-dependence profile was found to be bell-shaped, the effect of transfer from the highest to the lowest values of the inactivation rate constants increasing more than 100 times. On the basis of the data obtained and published earlier it was concluded that the enzyme inactivation proceeded in accordance with the scheme in which out of 3 equilibrium ionic forms of penicillin amidase, i.e. "acid", "neutral" and "alkaline" the neutral form of the active enzyme was most stable. Kinetic analysis of the scheme was carried out and it was shown that the dependence found was in accordance with the theoretical curve in which the pK values of the ionogenic groups controlling the interconvertions between the penicillin amidase forms were equal to 2.4 and 10.1 at a temperature of 40 degrees C. The value of the inactivation rate constant of the "acid" or "alkaline" form was equal to 5.95 min-1, while the "neutral" form of the enzyme was characterized by the inactivation rate constant equal to 5.1.10(-4) min-1. A mechanism for the enzyme inactivation was proposed. According to this mechanism, destruction of the salt bridge in the native structure of penicillin amidase resulted in production of extremely labile forms of the enzyme as compared to the native form.     

Coupling of the enzymic activities of myosin ATPase and creatine kinase and its role in muscular contraction

During muscular contraction the regeneration of ATP, catalysed by creatine kinase (CK), keeps pace with the hydrolysis of ATP by myosin ATPase posing the question of its regulatory mechanism. In the background of F-actin activation of heavy meromyosin (HMM) ATPase activity we have investigated in vitro the role of F-actin in regulating CK's activity in the absence and presence of HMM. For the coupled enzyme system we have also looked into the roles played by the individual reactants. F-actin has been found to appreciably increase CK's activity in the absence of HMM. While HMM alone inhibited CK's activity, there was a several fold increase when F-actin was also present. By a process of elimination we conclude that none of the reactants apart from H+ could be involved in regulating CK's activity in the coupled enzyme system. As no change in the pH of reaction mixture was observed during the reaction, we further conclude that the two enzymic reactions are coupled by proton transfer along F-actin. Implications of the findings for PCr-Cr shuttle and movements of ATP and ADP in sarcomere are discussed.     

Modulation of the actin-activated adenosinetriphosphatase activity of myosin by tropomyosin from vascular and gizzard smooth muscles

Tropomyosins from bovine aorta and pulmonary artery exhibit identical electrophoretic patterns in sodium dodecyl sulfate but differ from tropomyosins of either chicken gizzard or rabbit skeletal muscle. Each of the four tropomyosins binds readily to skeletal muscle F-actin as indicated by their sedimentation with actin and by their ability to maximally stimulate or inhibit actin-activated ATPase activity at a molar ratio of one tropomyosin per seven actin monomers. Smooth and skeletal muscle tropomyosins differ in their effects on activity of skeletal myosin or heavy meromyosin (HMM); the former can enhance activity under conditions in which the latter inhibits. Gizzard and arterial tropomyosins are usually equally effective in stimulating ATPase activity of skeletal acto-HMM, but at high concentrations of Mg2+ gizzard tropomyosin is more effective, a result that cannot be attributed to differences in the binding of the two tropomyosins to F-actin. The effects of tropomyosin also depend on the type of myosin; tropomyosin enhances activity of gizzard myosin under conditions in which it inhibits that of skeletal myosin. Increasing the pH or the Mg2+ concentration can reverse the effect of tropomyosin on actin-stimulated ATPase activity of skeletal HMM from activation to inhibition, but this reversal is not found with gizzard myosin. Activity in the absence of tropomyosin is independent of pH, and the loss of activation with increasing pH is not accompanied by loss of binding of tropomyosin to actin.     

Kinetic study of the pH-dependence of maximal rate of Ca-ATP hydrolysis by myosin]

Curves of V pH-dependence for Ca ATPase of myosin and heavy meromyosin are demonstrated to be well modelled with theoretical curves for the case of proton dissociation at three groups of enzyme-substrate complex with the loss of the activity at some intermediate ionization stage. Variation of pK values for these three groups and the degree of inhibition for intermediate forms of enzyme-substrate complex are found to be sufficient to reproduce main varieties of described in the literature and obtained in this work multiformity of pH-dependence curves of different nucleoside triphosphates hydrolysis by both native and modified enzymes. Calculated pK values and modification data suggest a significant importance of the dissociation of two imidazole groups ("activating" and "inhibitory") and cisteine sulhydryl group for the catalytic activity of myosin. Inhibition of ATPase activity by increasing of KCl concentrations is found to be due first of all to a shift in pK values of "inhibitory" imidazole and sulhydryl groups.     

Trimethylamine N-oxide suppresses the activity of the actomyosin motor

Background

During actomyosin interactions, the transduction of energy from ATP hydrolysis to motility seems to occur with the modulation of hydration. Trimethylamine N-oxide (TMAO) perturbs the surface of proteins by altering hydrogen bonding in a manner opposite to that of urea. Hence, we focus on the effects of TMAO on the motility and ATPase activation of actomyosin complexes.

Methods

Actin and heavy meromyosin (HMM) were prepared from rabbit skeletal muscle. Structural changes in HMM were detected using fluorescence and circular dichroism spectroscopy. The sliding velocity of rhodamine-phalloidin-bound actin filaments on HMM was measured using an in vitro motility assay. ATPase activity was measured using a malachite green method.

Results

Although TMAO, unlike urea, stabilized the HMM structure, both the sliding velocity and ATPase activity of acto-HMM were considerably decreased with increasing TMAO concentrations from 0–1.0 M. Whereas urea-induced decreases in the structural stability of HMM were recovered by TMAO, TMAO further decreased the urea-induced decrease in ATPase activation. Urea and TMAO were found to have counteractive effects on motility at concentrations of 0.6 M and 0.2 M, respectively.

Conclusions

The excessive stabilization of the HMM structure by TMAO may suppress its activities; however, the counteractive effects of urea and TMAO on actomyosin motor activity is distinct from their effects on HMM stability.

General significance

The present results provide insight into not only the water-related properties of proteins, but also the physiological significance of TMAO and urea osmolytes in the muscular proteins of water-stressed animals.     

Subtilisin cleavage of actin inhibits in vitro sliding movement of actin filaments over myosin

Subtilisin cleaved actin was shown to retain several properties of intact actin including the binding of heavy meromyosin (HMM), the dissociation from HMM by ATP, and the activation of HMM ATPase activity. Similar Vmax but different Km values were obtained for acto-HMM ATPase with the cleaved and intact actins. The ATPase activity of HMM stimulated by copolymers of intact and cleaved actin showed a linear dependence on the fraction of intact actin in the copolymer. The most important difference between the intact and cleaved actin was observed in an in vitro motility assay for actin sliding movement over an HMM coated surface. Only 30% of the cleaved actin filaments appeared mobile in this assay and moreover, the velocity of the mobile filaments was approximately 30% that of intact actin filaments. These results suggest that the motility of actin filaments can be uncoupled from the activation of myosin ATPase activity and is dependent on the structural integrity of actin and perhaps, dynamic changes in the actin molecule.     

Effects of urea and guanidine hydrochloride on the sliding movement of actin filaments with ATP hydrolysis by myosin molecules

To evaluate the role of the hydration layer on the protein surface of actomyosin, we compared the effects of urea and guanidine-HCl on the sliding velocities and ATPase activities of the actin-heavy meromyosin (HMM) system. Both chemicals denature proteins, but only urea perturbs the hydration layer. Both the sliding velocity of actin filaments and actin-activated ATPase activity decreased with increasing urea concentrations. The sliding movement was completely inhibited at 1.0 M urea, while actin filaments were bound to HMM molecules fixed on the glass surface. Guanidine-HCl (0-0.05 M) drastically decreased both the sliding velocity and ATPase activation of acto-HMM complexes. Under this condition, actin filaments almost detached from HMM molecules. In contrast, the ATPase activity of HMM without actin filaments was almost independent of urea concentrations <1.0 M and guanidine-HCl concentrations <0.05 M. An increase in urea concentrations up to 2.0 M partly induced changes in the ternary structure of HMM molecules, while the actin filaments were stable in this concentration range. Hydration changes around such actomyosin complexes may alter both the stability of part of the myosin molecules, and the affinity for force transmission between actin filaments and myosin heads.     

Induction of contractile activity by rigor complexes in myofibrils

The relation between ATPase rate and substrate concentration was investigated for myofibrils with varying amounts of added HMM. There was a biphasic, 3 to 5-fold increase in ATPase in the absence of Ca⁺⁺. In the absence of added HMM, the peak activity occurred at ≤ 0.1 mM MgATP. With increasing concentrations of HMM, the position and magnitude of the ATPase peak shifted to larger substrate concentrations and higher rates. The cofactor activity of regulated actin in myofibrils is activated to a similar degree by Ca⁺⁺ as by HMM (rigor links). SDS gel electrophoretic patterns of myofibrils mixed with HMM indicated the soluble HMM binds to myofibrils at 0.1 mM MgATP and is dissociated at higher MgATP concentrations. Thus, in well-regulated myofibrils in the absence of Ca⁺⁺ actin cofactor activity can be activated by rigor complexes.     

Modification of lipoxygenase by hydrogen peroxide and photooxidation.

The kinetic study of fluorescence stopped-flow method suggested that the interaction between lipoxygenase and H2O2 is consistent with a simple irreversible one-step mechanism. The activation energy of the reaction was 7.2 kcal/mol. Participation of an ionizable group with pK about 8.8, possibly a histidine residue, was suggested from the pH-dependence of the rate constant. No further fluorescence quenching of lipoxygenase was observed when the product was added to the lipoxygenase solution before mixing the lipoxygenase and H2O2 solutions. The fluorescence quenching of lipoxygenase by H2O2 was in parallel with the inactivation of the enzyme. Hydroperoxylinoleic acid strongly protects the inactivation of lipoxygenase caused by H2O2. These results are consistent with an interpretation that OH- and/or O- - are produced when the iron of the enzyme is oxidized by H2O2, which in turn will attack some amino acid essential for the enzyme activity. The pH-dependence of the inactivation rate constant of photooxidation of lipoxygenase sensitized by methylene blue indicated that an ionizable group with pK about 8.8 is concerned with the enzymatic activity. In contrast to the inactivation of lipoxygenase by H2O2, the product protected the inactivation of the enzyme by photooxidation only at high concentration.     

Cytoplasmic and plasma membrane adenosine triphosphatase of polymorphonuclear neutrophils: comparison of their enzymatic properties and attempt for a direct determination of myosin ATPase activity using polymorphonuclear neutrophil extract

Enzymatic properties of the ATPase of the plasma membrane and cytoplasmic myosin B from guinea-pig polymorphonuclear neutrophils were compared. In the plasma membrane, Mg2+- and Ca2+-activated ATPases showed the same dependence pattern on KCl concentration and pH, i.e., both ATPases increased with decreasing KCl concentration and with rising pH until pH 9.0. The maximum activation of Mg2+-ATPase was observed at 1 . 10(-3) M Mg2+. On the other hand, EDTA-activated ATPase activity was so low that no clear dependence curve was obtained. In myosin B, Mg2+-ATPase activity was below one-tenth that of the plasma membrane ATPase with the maximum activation at 1 . 10(-2) M Mg2+ and pH 9.0 EDTA- and Ca2+-activated ATPase exhibited almost the same activity and the same KCl-dependence curve, i.e., both ATPases increased and increasing KCl concentration. With regard to pH-dependence, Ca2+-ATPase showed a U-shaped curve with the minimum at pH 7.0, wherease EDTA-activated ATPase indicated a bell-shaped curve with the maximum at pH 9.0. Based on the findings that the EDTA-activated ATPase activity was hardly detected in the plasma membrane but high in myosin B, the distribution of ATPase activity on subcellular fractions was studied and the results obtained that the myosin-ATPase activity could be directly measured using the polymorphonuclear neutrophil extract if the EDTA-activated ATPase activity was used as an enzymatic marker for myosin.     

Dissociation of the effect of caldesmon on the ATPase activity and on the binding of smooth heavy meromyosin to actin by partial digestion of caldesmon

We have proposed earlier that caldesmon inhibits the actin-activated ATPase activity of smooth muscle heavy meromyosin (HMM) by inhibiting the binding of the HMM.ATP complex to the productive site of actin (Hemric, M. E., and Chalovich, J. M. (1988) J. Biol. Chem. 263, 1868-1885). This has been difficult to prove directly because caldesmon also binds to HMM and it is difficult to distinguish the actin-caldesmon-HMM complex from the actin-caldesmon complex in binding studies. We have eliminated the interaction between caldesmon and smooth HMM by digestion of caldesmon with chymotrypsin. This cleaved caldesmon inhibits the actin-activated ATPase rate of smooth HMM and this inhibition is correlated with a decrease in the binding of HMM.ATP to actin. Therefore, caldesmon functions by inhibiting the binding of the myosin-ATP complex to actin regardless of the source of myosin. We have also isolated the myosin-binding region of caldesmon and have performed a partial sequence. Comparison of this sequence with the derived sequence of caldesmon demonstrates, unequivocally, that the myosin-binding region of caldesmon begins at the amino terminus and extends beyond the first Cys residue.