Glutaraldehyde cross-links Lys-492 and Arg-678 at the active site of sarcoplasmic reticulum Ca(2+)-ATPase.

It has been shown previously that glutaraldehyde cross-links the Ca(2+)-ATPase of sarcoplasmic reticulum intramolecularly at the active site, involving residues participating in nucleotide binding and the conformational change that results in Ca2+ release to the vesicle lumen and formation of ADP-insensitive E2-P (Ross, D. C., Davidson, G. A., and McIntosh, D. B. (1991) J. Biol. Chem. 266, 4613-4621). This study shows that 10 nmol of [14C]glutaraldehyde/mg of protein attached irreversibly to the ATPase under conditions optimal for formation of the intramolecular cross-link. Half of this amount (i.e. 1 mol/mol ATPase) was inhibited by nucleotide binding. Thermolysin digestion of derivatized vesicles released two nucleotide-sensitive 14C-labeled species, which were isolated and identified as FSRDR*S AND FSRDR*S FA* FA*VEPS where the missing residues are Lys-492 and Arg-678. The majority of the 14C label was released in the sixth cycle of both Edman degradations, confirming the cross-link position. Lys-492 and Arg-678 are evidently close together in the active site, but their distance apart in the linear sequence suggests that they may arise from separate domains, which together constitute an ATP binding cleft. Residues in both regions, and Lys-492 in particular (McIntosh, D.B., Woolley, D.G., and Berman, M.C. (1992) J. Biol. Chem. 267, 5301-5309), have been derivatized by nucleotide-based affinity probes. Mutations of both of these residues in some of the bacterial P-type ATPases suggest that they do not play an essential catalytic role, and the inability of the cross-linked ATPase to form E2-P and to release Ca2+ to the lumen is probably because an essential tertiary structural movement at the active site is blocked.     

Reaction cycle of solubilized monomeric Ca2+-ATPase of sarcoplasmic reticulum is the same as that of the membrane form

Monomeric Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum dispersed in Triton X-100 is stoichiometrically phosphorylated from Pi in a Ca2+-depleted medium containing dimethyl sulfoxide and catalyzes efficient (80%) phosphoryl transfer to ADP following a jump in water activity in the presence of Ca2+. The Ca2+ concentration dependence of ATP synthesis was sigmoidal (nH = 1.7) and in the millimolar range (K0.5 = 0.3 mM), indicating the involvement of at least two low affinity Ca2+ binding sites. These results, taken together with the properties of the monomer in the forward direction of catalysis, show that the catalytic cycle of the detergent-solubilized monomer is essentially the same as that of the membrane enzyme. The substrate and ion specificity of the catalytic intermediates suggest that the monomer is capable of coupled vectorial transport of Ca2+.     

Interactions between marine bacteria and dissolved-phase and beached hydrocarbons after the Exxon Valdez oil spill.

Turnover times for toluene in Resurrection Bay after the Exxon Valdez grounding were determined to be decades, longer than expected considering that dissolved hydrocarbons were anticipated to drift with the current and stimulate development of additional hydrocarbon-utilizing capacity among the microflora in that downcurrent location. These turnover times were based on the recovery of 14CO2 from added [14C]toluene that was oxidized. The concentrations of toluene there, 0.1 to 0.2 microgram/liter, were similar to prespill values. Oxidation rates appeared to be enhanced upstream near islands in the wake of the wind-blown slick, and even more within the slick itself. Specific affinities of the water column bacteria for toluene were computed with the help of biomass data, as measured by high-resolution flow cytometry. They were a very low 0.3 to 3 liters/g of cells.h-1, indicating limited capacity to utilize this hydrocarbon. Since current-driven mixing rates exceeded those of oxidation, dissolved spill components such as toluene should enter the world-ocean pool of hydrocarbons rather than biooxidize in place. Some of the floating oil slick washed ashore and permeated a coarse gravel beach. A bacterial biomass of 2 to 14 mg/kg appeared in apparent response to the new carbon and energy source. This biomass was computed from that of the organisms and associated naphthalene oxidation activity washed from the gravel compared with the original suspension. These sediment organisms were very small at approximately 0.06 microns 3 in volume, low in DNA at approximately 5.5 g per cell, and unlike the aquatic bacteria obtained by enrichment culture but quite similar to the oligobacteria in the water column.(ABSTRACT TRUNCATED AT 250 WORDS)