Actin-activated MgATPase of smooth muscle heavy meromyosin is activated by thiophosphorylation of two regulatory light chains, one on each head domain. To understand cooperativity between heads, we examined the kinetics of heavy meromyosin (HMM) with one thiophosphorylated head. Proteolytic gizzard heavy meromyosin regulatory light chains were partially exchanged with recombinant thiophosphorylated His-tagged light chains, and HMM with one thiophosphorylated head was isolated by nickel-affinity chromatography. In vitro motility was observed. By steady-state kinetic analysis, one-head thiophosphorylated heavy meromyosin had a similar K m value for actin but a V max value of ϳ50% of the fully thiophosphorylated molecule. However, single turnover analysis, which is not sensitive to small amounts of active heads, showed that one-head thiophosphorylated heavy meromyosin was 46-120 times more active than unphosphorylated HMM but only 7-19% as active as the fully thiophosphorylated molecule. Discrepancy between the single turnover and steady-state values could be explained by a small fraction of rigor heads. These rigor heads would have a large effect on the steady-state kinetics of one-head thiophosphorylated HMM. In summary, thiophosphorylation of one head leads to a molecule with unique intermediate kinetics suggesting that thiophosphorylation of one head cooperatively alters the kinetics of the partner head and vice versa.The actin-activated ATPase activity and motor properties of smooth muscle and nonmuscle myosins are regulated by phosphorylation of the regulatory light chain (1-3). The unphosphorylated forms of these regulated myosins have low ATPase activity and are unable to move actin filaments, whereas the phosphorylated forms are activated in both respects. Domain requirements for regulation have been elucidated through studies of various proteolytic and expressed subfragments of SMM.1 SMM contains two head domains (S1) attached to a long ␣-helical coiled-coil domain (tail). Single-headed myosin (4) and S1 (5, 6) are active in both unphosphorylated and phosphorylated states. HMM, which lacks the COOH-terminal two-thirds of the tail, is double-headed and well regulated (5, 7), but expressed HMMs with shorter tails failed to form doubleheaded structures and were found to be unregulated (8, 9) as in S1 and single-headed myosin. Therefore, two heads are critical for down-regulation, suggesting that head-head interaction is an important feature of the unphosphorylated state. It is interesting that in scallop HMM (regulated by Ca 2ϩ binding), communication between the nucleotide-and calcium-binding sites requires the presence of two heads (10).The general location of the phosphorylated subunit (RLC) at the junction between the heads and tail (11, 12) suggests that RLC-RLC interactions may be a logical consequence of headhead interactions. We have recently shown that certain residues of the RLC can be cross-linked to the RLC of the other head in smooth HMM but only in the unphosphorylated state (13). These data st...

The calcineurin (CaN) a and b catalytic subunit isoforms are coexpressed within almost all cell types. The enzymatic properties of CaN heterodimers comprised of the regulatory B subunit (CnB) with either the a or b catalytic subunit were compared using in vitro phosphatase assays. CaN containing the a isoform (CnAa) has lower K m and higher V max values than CaN containing the b isoform (CnAb) toward the PO 4 -RII, PO 4 -DARPP-32(20-38) peptides, and p-nitrophenylphosphate (pNPP). CaN heterodimers containing the a or b catalytic subunit isoform displayed identical calmodulin dissociation rates. Similar inhibition curves for each CaN heterodimer were obtained with the CaN autoinhibitory peptide (CaP) and cyclophilin A/cyclosporin A (CyPA/CsA) using each peptide substrate at K m concentrations, except for a five-to ninefold higher IC 50 value measured for CaN containing the b isoform with p-nitrophenylphosphate as substrate. No difference in stimulation of phosphatase activity toward p-nitrophenylphosphate by FKBP12/FK506 was observed. At low concentrations of FKBP12/FK506, CaN containing the a isoform is more sensitive to inhibition than CaN containing the b isoform using the phosphopeptide substrates. Higher concentrations of FKBP12/FK506 are required for maximal inhibition of b CaN using PO 4 -DARPP-32(20-38) as substrate. The functional differences conferred upon CaN by the a or b catalytic subunit isoforms suggest that the a:b and CaN:substrate ratios may determine the levels of CaN phosphatase activity toward specific substrates within tissues and specific cell types. These findings also indicate that the a and b catalytic subunit isoforms give rise to substrate-dependent differences in sensitivity toward FKBP12/FK506.

The effect of ADP and phosphorylation upon the actin binding properties of heavy meromyosin was investigated using three fluorescence methods that monitor the number of heavy meromyosin heads that bind to pyrene-actin: (i) amplitudes of ATP-induced dissociation, (ii) amplitudes of ADP-induced dissociation of the pyrene-actin-heavy meromyosin complex, and (iii) amplitudes of the association of heavy meromyosin with pyrene-actin. Both heads bound to pyrene-actin, irrespective of regulatory light chain phosphorylation or the presence of ADP. This behavior was found for native regulated heavy meromyosin prepared by proteolytic digestion of chicken gizzard myosin with between 5 and 95% heavy chain cleavage at the actin-binding loop, showing that two-head binding is a property of heavy meromyosin with uncleaved heavy chains. These data are in contrast to a previous study using an uncleaved expressed preparation (Berger, C. E., Fagnant, P. M., Heizmann, S., Trybus, K. M., and Geeves, M. A. (2001) J. Biol. Chem. 276, 23240 -23245), which showed that one head of the unphosphorylated heavy meromyosin-ADP complex bound to actin and that the partner head either did not bind or bound weakly. Possible explanations for the differences between the two studies are discussed. We have shown that unphosphorylated heavy meromyosin appears to adopt a special state in the presence of ADP based upon analysis of actin-heavy meromyosin association rate constants. Data were consistent with one head binding rapidly and the second head binding more slowly in the presence of ADP. Both heads bound to actin at the same rate for all other states. Smooth muscle myosin (SMM),1 like other members of the myosin II family, has two heads connected by a coiled-coil tail. SMM and the double-headed subfragment HMM are regulated by phosphorylation of the two regulatory light chains, one on each head (1-3). In contrast, single-headed SMM (4, 5) and the single-headed S1 (6 -8) are not regulated by phosphorylation.Non-muscle HMM IIB is also regulated by phosphorylation, and constructs lacking one motor domain have been shown to be unregulated (9). Therefore, two motor domains are required for regulation.Structural differences between unphosphorylated and phosphorylated HMM have been demonstrated by a number of studies. Reconstruction of images of expressed unphosphorylated HMM in the presence of ATP in two-dimensional crystalline arrays (10, 11) shows an asymmetrical structure with the converter domain of one head bound to the actin-binding site of the other head. No interaction was seen between the motor domains of phosphorylated HMM. This model is supported by data from Berger et al. (12), who demonstrated that only one of the heads of an unphosphorylated expressed smooth muscle HMM-ADP complex binds to actin. Either the binding of the first head prevented the binding of the second head or the second head bound weakly such that no signal was observed for its binding. Both heads bound to actin in the phosphorylated state. These data are inconsistent with t...

Reactions involving proteins frequently involve large changes in volume, which allows the equilibrium position to be perturbed by changes in pressure. Rapid changes in pressure can thus be used to initiate relaxation in pressure; however, this approach is seldom used, because it requires specialized equipment. We have built a microvolume (50 microl) pressure-jump apparatus, powered by a piezoelectric actuator, based on the original design of Clegg and Maxfield [(1976) Rev. Sci. Instrum. 47, 1383-1393]. This equipment can apply pressure changes of +/-20 MPa (maximally) in time periods as short as 80 micros and follow the resulting change in fluorescence signals. The system is relatively simple to use with fast (approx. 1 min) exchange of samples. In the present study, we show that this system can perturb the binding of 2'(3')-O-(N-methylanthraniloyl)-ADP to myosin subfragment-1(S1) from skeletal and smooth muscles. The kinetic data are consistent with previous work, and in addition show that, although 2'(3')-O-(N-methylanthraniloyl)-ADP binds with a similar affinity to both proteins, the increase in molar volume for the skeletal-muscle S1 binding to ADP is half of that for the smooth-muscle protein. This high-volume change for smooth-muscle S1 may be related to the ability of ADP to induce a 23 degrees tilt in the tail of S1 bound to actin.

Dietary procyanidins have emerged as important bioactive components that regulate various metabolic pathways to maintain homeostasis. Grape seed procyanidin extract (GSPE), in particular, has demonstrated regulatory effects on bile acid and lipid metabolism in vivo. While numerous studies in rodent models have shown the potent hypolipidemic action of grape seed extracts, human studies have shown inconsistent results. This review will focus on the molecular mechanisms underlying the hypolipidemic actions of GSPE identified to date, specifically highlighting the effects exerted via nuclear receptors. Such evidence may provide avenues for future research in human subjects with GSPE as a therapeutic treatment for the prevention and amelioration of the metabolic syndrome and cardiovascular disease.