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The effect of [MgADP] on relaxation from isometric tension, initiated by reducing free [Ca2+] through photolysis of the caged photolabile Ca2+ chelator diazo-2, was determined at 20 degrees C in alpha-toxin permeabilized tonic (rabbit femoral artery, Rf) and phasic (rabbit bladder, Rb) smooth muscle. In Rf, the shape of the relaxation curve was clearly biphasic, consisting of a slow "plateau" phase followed by a monotonic exponential decline with rate constant k. The duration of the plateau (d = 44 +/- 4 s, mean +/- SEM, n = 28) was well correlated (R = 0.92) with the total t1/2 of relaxation that was 66 +/- 3 s (n = 28) in the presence of 20 mM creatine phosphate (CP), and was prolonged in the absence of CP (t1/2 = 83 +/- 3 s, n = 7); addition of 100 microM MgADP further slowed relaxation (t1/2 = 132 +/- 7 s, n = 14). In Rb, a plateau was not detectable and t1/2 (= 15 +/- 2 s, n = 6) was not affected by 100 microM MgADP. In Rf the Q10 between 20 degrees C and 30 degrees C was 4.3 +/- 0.4 for d-1 and 2.8 +/- 0.3 for k (n = 8; p = 0.006). The regulatory myosin light chain (MLC20) in Rf was dephosphorylated at 0.07 +/- 0.02 s-1, from 42 +/- 3% before to 20 +/- 2% after photolysis of diazo-2, reaching basal values at a time when force had fallen by only 40%. We conclude that, in the presence of ATP, as during rigor, the affinity of dephosphorylated cross-bridges for MgADP is significantly higher in tonic than in phasic smooth muscle and contributes to the maintenance of force at low levels of phosphorylation. The MgADP dependence of the post-dephosphorylation phase of relaxation is consistent with its being rate-limited by the slow off-rate of ADP from cross-bridges that were dephosphorylated while in force-generating ADP-bound (AM*D) cross-bridge states. The fourfold faster off-rate of ADP from AM*D in the phasic, Rb, compared to tonic, Rf, smooth muscle is a major determinant of the different kinetics of relaxation in the two types of smooth muscle.

The R- and S-enantiomers of inorganic [16O, 17O, 18O]-thiophosphate have been synthesized from the B and A isomers of [alpha-S; alph-18O; alpha beta-17O; beta-17O3]ADP. Each chiral thiophosphate was incorporated into isomer A of ATP beta S with distinct oxygen isotopic labelings, which were characterized by 31P NMR. The 3-phosphoglycerate kinase-catalyzed reaction was shown to proceed by inversion at phosphorus since all other steps in the reaction sequence between inorganic thiophosphate and ATP beta S were of known stereospecificity.

Knowledge of the functional properties of the protein in oriented multilayers, in addition to vesicular dispersions, of membranes such as the isolated sarcoplasmic reticulum (SR), extends the variety of techniques that can be effectively used in studies of the membrane protein's structure or structural changes associated with its function. One technique requiring the use of oriented multilayers to provide more direct time-averaged and time-resolved structural investigations of the SR membrane is x-ray diffraction. Therefore, the kinetics of ATP-induced calcium uptake by isolated SR vesicles in dispersions and hydrated, oriented multilayers were compared. Ca2+ uptake was necessarily initiated by the addition of ATP through flash photolysis of caged ATP, P3-1-(2-nitro)phenylethyl adenosine 5'-triphosphate, with either a frequency-doubled ruby laser or a 200 W Hg arc lamp, and measured with two different detector systems that followed the absorbance changes of the metallochromic indicator arsenazo III, which is sensitive to changes in the extravesicular [Ca2+]. The temperature range investigated was -2 degrees to 26 degrees C. The Ca2+ uptake kinetics of SR membranes in both the vesicular dispersions and oriented multilayers consist of at least two phases, an initial fast phase and a subsequent slow phase. The fast phase, generally believed to be associated with the formation of the phosphorylated enzyme, E approximately P, is kinetically comparable in both SR dispersions and multilayers. The slow phase mathematically follows first-order kinetics with specific rate constants of approximately 0.6 s-1 and approximately 1.2 s-1 for the dispersions at 26 degrees C and multilayers at 21 degrees C, respectively, with the given experimental conditions. The slow phase, generally believed to be associated with the translocation of Ca+2, across the membrane profile, appears to be the same process in SR dispersions and multilayers through their virtually identical rate constants and their identical activation energies of 22 +/-1 kcal mol -1. The stoichiometry of ~2 mol Ca2+/mol ATP hydrolyzed was measured in dispersions for the slow phase of Ca2+ uptake. Photolysis of caged ATP with the lamp and the laser provides comparable results for the Ca2+ uptake kinetics in SR dispersions and multilayers. Laser flash photolysis, however, has the advantages of optimal time resolution and effective synchronization of the ensemble of Ca2+-ATPase molecules in the ATP initiated Ca2+ transport process.