Mechanism of thermal uncoupling of Ca2+-ATPase of sarcoplasmic reticulum as revealed by thapsigargin stabilization.

Thermal uncoupling of the Ca2+ pump of skeletal muscle sarcoplasmic reticulum is specifically blocked by binding of Ca2+ to the high affinity sites, having identical characteristics to the Ca2+ transport sites (Berman, M.C., McIntosh, D.B. and Kench, J.E. (1977) J. Biol. Chem. 252, 994-1001). The present study has investigated the nature of the decreased net Ca2+ transport in the uncoupling process. Ca2+ uptake in the presence and absence of oxalate, Ca2+ retention following passive Ca2+ loading and Ca2+-dependent ATPase activity were inactivated at pH 7.0 and 37 degrees C, with rate constants of 0.12, 0.047, 0.053 and 0.001 min-1, respectively. Activation energies were in the range 72-76 kcal/mol, indicating a common irreversible protein conformational transition. A thermodynamic analysis of parallel or consecutive inactivation pathways revealed that loss of Ca2+ transfer and ATPase activity occurred on the same pump unit, making the existence of a predominant uncoupled intermediate unlikely. Decreased passive Ca2+ loading, an index of the number of intact vesicles, correlated with decreased active uptake in the absence of oxalate, indicating increased vesicle permeability. Thapsigargin, at a 1:1 stoichiometry, stabilised the Ca-ATPase against thermal inactivation, while previously inactivated Ca-ATPases appeared not to bind TG. Protection by TG suggests that the origin of inactivation is in the transmembrane and stalk regions of the ATPase. We propose that protein unfolding results in inefficient gating of a small percentage of ATPases with subsequent uncoupling of the entire vesicle.

Rate of growth of Pseudomonas fluorescens in donated blood.

AIMS: To examine how delayed refrigeration of blood affects the growth of Pseudomonas fluorescens, one of the two most important causes of sepsis resulting from transfusion of contaminated blood. METHODS: Two donations of whole blood were each divided into three aliquots and inoculated with 5-10 cfu/ml of a P fluorescens strain from a case of transfusion associated sepsis. From each donation, one aliquot was placed at 4 degrees C, one was held at 20 degrees C for six hours prior to refrigeration and the third was held at 20 degrees C for 24 hours prior to refrigeration. Samples were aseptically withdrawn over 17 days and bacterial counts were determined using a pour plate technique. RESULTS: The rate of growth of P fluorescens in blood at 20 degrees C was increased compared with blood at 4 degrees C. At 24 hours the aliquots held at 20 degrees C for six and 24 hours had, respectively, 174 and 29,000 cfu/ml compared with 15 cfu/ml in aliquots held at 4 degrees C. There was no evidence of increased killing of P fluorescens at the higher temperature. CONCLUSIONS: These results suggest that blood for transfusion should be refrigerated as soon as possible after collection.

Detection of specific and 'constitutive' antibody secreting cells in the gills, head kidney and peripheral blood leucocytes of dab (Limanda limanda).

The relative immunological importance of the gills of fish was investigated in terms of antibody production by enumerating antibody secreting cells (ASC) in the gills, head kidney and blood of dab (Limanda limanda) using the ELISPOT assay. The contribution of 'constitutive' ASC in the gill appeared more substantial than that of elicited specific ASC. The gills were found to contain a mean (+/- SD) of 4227 +/- 1029 'constitutive' ASC/10(6) cells which was fewer than the head kidney which contained a mean (+/- SD) of 15617 +/- 3723 'constitutive' ASC/10(6) cells but more than peripheral blood leucocytes which contained a mean (+/- SD) of 2650 +/- 212 'constitutive' ASC/10(6) cells. The number of specific anti-human gamma globulin (HGG) ASC following parenteral or oral administration of HGG was also determined. Anti-HGG ASC were detected in all three tissues following parenteral immunization, peaking simultaneously, 4 weeks post-immunization. The strongest response was found in the head kidney. After oral immunization, responses were much weaker: again the head kidney was the most active but the gill response was barely detectable. These data were complemented by measurement of specific antibody in the serum by ELISA. Serum antibody titres following immunization were found to correlate closely with the number of specific ASC in the head kidney following parenteral immunization whereas serum antibody titres after oral administration of antigen most closely followed the number of specific ASC in the blood. In the light of these data it is suggested that the primary immunological role of the leucocytes in the gill may be in the earliest stages of defence against infection.

Calcium dependence during single-cycle catalysis of the sarcoplasmic reticulum ATPase.

We have investigated the kinetic and thermodynamic properties of the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum under conditions that result in a single transport cycle. Simultaneous addition of ATP and EGTA to sarcoplasmic reticulum vesicles, preincubated with calcium, resulted in a transient of intermediate species. In the presence of saturating Ca2+ levels, total E-P species reached a maximum of 2.3 nmol/mg at 100 ms, followed by a monoexponential decay with kobs = 3.6 s-1. The data are interpreted in terms of Ca2+ sequestration, either by occlusion as Ca2+ in the phosphorylated enzyme or chelation by EGTA. Maximum Ca2+ uptake was 8.3 nmol/mg with the release of 4.4 nmol/mg Pi. The ratio of Ca2+ uptake to Pi release approached 1.9 over a wide [Ca2+] range. Equilibrium Ca2+ binding, in the absence of ATP, showed a K0.5 of 0.88 microM with a Hill coefficient of 1.9. The Ca2+ concentration dependence of Ca2+ uptake during single-cycle catalysis showed a 10-fold enhanced affinity (K0.5 = 0.06 microM) and was noncooperative (nH = 0.9). Quench with excess EGTA (greater than 2 mM) decreased Ca2+ uptake to 1 nmol/mg, indicating an "off" rate of Ca2+ from high affinity sites that exceeds 100 s-1. The ATP concentration dependence for a single-cycle catalysis showed an apparent K0.5 of 1.1 microM, similar to that for ATP equilibrium binding. It is proposed that enzyme phosphorylation proceeds only following binding of a second calcium ion to externally oriented sites whose intrinsic affinity is in the same range as the calcium dependence of a single-cycle turnover.

Kinetics of thapsigargin-Ca(2+)-ATPase (sarcoplasmic reticulum) interaction reveals a two-step binding mechanism and picomolar inhibition.

Thapsigargin is a high affinity inhibitor of sarco- and endoplasmic reticulum (SERCA) type ATPases. We have used kinetics to determine the dissociation constant of thapsigargin-sarcoplasmic reticulum Ca(2+)-ATPase interaction in the absence and presence of non-ionic detergent. The observed "off" rate constant was measured as 0.0052 s-1 at 26 degrees C by the kinetics of inhibition of ATPase activity following transfer from an inactivated thapsigargin-ATPase complex to native ATPase. Inactive ATPase was produced by cross-linking the active site with glutaraldehyde. The observed dissociation rate constant was increased 7-fold by 0.1% Triton X-100, indicating that perturbation of the transmembrane and stalk region by detergent altered the binding parameters of the inhibitor. In addition, thapsigargin stabilized the ATPase against inactivation caused by detergent in the absence of Ca2+. The observed "on" rate constant of thapsigargin was measured at 26 degrees C as 25 s-1 irrespective of thapsigargin concentration, by the kinetics of thapsigargin- induced change in intrinsic fluorescence. An Arrhenius plot showed a temperature dependence of this rate constant, indicative of a conformational change in the protein with an activation energy of 9.5 kcal/mol for thapsigargin binding. The affinity of the Ca(2+)-ATPase for thapsigargin was calculated to be greater than 2 pM at pH 7.0 and 26 degrees C.

Interaction of valinomycin and monovalent cations with the (Ca2+,Mg2+)-ATPase of skeletal muscle sarcoplasmic reticulum.

The interactions of monovalent cations and of the K+-specific ionophore, valinomycin, with the Ca2+-ATPase of skeletal muscle of sarcoplasmic reticulum have been studied in the absence of cation gradients by their effects on enzyme turnover and on the ATP plus Ca2+-dependent enhanced fluorescence of the ATP analogue, 2',3'-O-(2,4,6-trinitrocyclohexyldienylidine)-adenosine 5'-triphosphate (TNP-ATP) (Watanabe, T., and Inesi, G. (1982) J. Biol. Chem. 257, 11510-11516). Monovalent cations decreased turnover-dependent TNP-ATP fluorescence in the series K+ greater than Rb+ approximately equal to Cs+ greater than Na+ greater than Li+ (K0.5 = 49, 73, 75, 94, and 246 mM, respectively), consistent with the known specificity of the monovalent cation binding site that stimulates turnover and E-P hydrolysis. Valinomycin (200 nmol/mg), in the absence of monovalent cations, decreased ATPase activity by 30% and abolished the stimulatory effects of 150 mM KCl or NaCl on turnover. The ionophore alone enhanced TNP-ATP fluorescence by 20% and altered the specificity and affinity of the site that inhibited TNP-ATP fluorescence to Cs+ greater than Rb+ greater than K+ approximately equal to Na+ greater than Li+ (K0.5 = 79, 111, 134, 136, and 270 mM, respectively), which follows the Hofmeister series for effectiveness of monovalent lyotropic cations. TNP-ATP binding was not affected by either monovalent cations or valinomycin. Inhibition of turnover-dependent TNP-ATP fluorescence appears to be a useful parameter for monitoring monovalent cation binding to the Ca2+-ATPase. It is concluded that the ionophore interacts directly with the Ca2+-ATPase, independent of its K+ conductance effects on the lipid bilayer, and modifies the affinity and specificity of the monovalent cation site, either by direct interaction or by the formation of a valinomycin-monovalent cation-enzyme complex.

Phosphoenzyme conformational states and nucleotide-binding site hydrophobicity following thiol modification of the Ca2+-ATPase of sarcoplasmic reticulum from skeletal muscle.

Enhanced fluorescence of the ATP analogue 2',3'-O-(2,4,6-trinitrocyclohexyldienylidine)adenosine 5'-triphosphate (TNP-ATP), bound to the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum, is closely related to phosphoenzyme levels (Bishop, J. E., Johnson, J. D., and Berman, M. C. (1984) J. Biol. Chem. 259, 15163-15171) and has an emission maximum consistent with decreased polarity of the TNP-ATP-binding site. The phosphoenzyme conformation responsible for increased nucleotide-binding site hydrophobicity has been studied by redistribution of phosphoenzyme intermediates following specific thiol group modification. N-Ethylmaleimide, in the presence of 50 microM Ca2+, 1 mM adenyl-5'-yl imidodiphosphate, pH 7.0, at 25 degrees C for 30 min, selectively modified the SH group essential for phosphoenzyme decomposition, which resulted in decreased ATPase activity, Ca2+ uptake, and a decrease in ATP-induced TNP-ATP fluorescence. Phosphorylated (Ca2+, Mg2+)-ATPase levels from [gamma-32P] ATP remained relatively unaffected (3.1 nmol/mg), but the ADP-insensitive fraction decreased from 56 to 15%. Phosphoenzyme levels from 32Pi were also decreased to the same extent as turnover, with equivalent loss of Pi-induced TNP-ATP fluorescence. The E1 to E2 transition, as monitored by the change in intrinsic tryptophan fluorescence, was unaffected. Modification of thiol groups of unknown function did not modify turnover-induced TNP-ATP fluorescence. It is concluded that the ADP-insensitive phosphoenzyme, E2-P, is responsible for enhanced TNP-ATP fluorescence. This suggests that the conformational transition, 2Ca2+outE1 approximately P----2Ca2+inE2-P, is associated with altered properties of the noncatalytic, or regulatory, nucleotide-binding site.

Effects of nonsolubilizing and solubilizing concentrations of Triton X-100 on Ca2+ binding and Ca2+-ATPase activity of sarcoplasmic reticulum.

The effect of low concentrations of Triton X-100, below that required for solubilization, on the properties of the Ca2+-ATPase of sarcoplasmic reticulum has been investigated. The changes observed have been compared with the changes produced on solubilization of the vesicles at higher concentrations of detergent. In the range 0.02-0.05% (w/v) Triton X-100, concentrations which did not solubilize the vesicles but completely inhibit ATP-mediated Ca2+ accumulation, 8-16 mol of detergent/mol of ATPase was associated with the vesicles. This amount of Triton X-100 altered equilibrium Ca2+ binding and Ca2+ activation of p-nitrophenyl phosphate and of ATP hydrolysis in a manner which lowered the apparent Ca2+ cooperatively (nH = 1 or less), and which increased the K0.5(Ca) value 20-fold. These changes in Ca2+ binding and activation parameters were associated with a 90% lower Ca2+-induced change in fluorescence of fluorescein isothiocyanate modified enzyme. The rates of p-nitrophenyl phosphate and of ATP hydrolysis, at saturating Ca2+ concentrations, were about half that of detergent-free vesicles. The rate constant for phosphoenzyme hydrolysis in the absence of Ca2+, calculated from medium Pi in equilibrium HOH exchange and phosphoenzyme measurements, was lowered from 38 to 11 s-1. The steady-state level of phosphoenzyme formed from Pi in the absence of Ca2+ was slightly increased up to 0.02% Triton X-100 and then decreased about half at 0.05%. The synthesis of ATP in single turnover type experiments was not affected by detergent binding. Pi in equilibrium ATP exchange was inhibited 65%.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanism of inhibition of sarcoplasmic reticulum Ca2(+)-ATPase by active site cross-linking. Impairment of nucleotide binding slows nucleotide-dependent phosphoryl transfer, and loss of active site flexibility stabilizes occluded forms and blocks E2-P formation.

Investigation of the properties of Ca2(+)-ATPase of sarcoplasmic reticulum cross-linked at the active site with glutaraldehyde showed that ATP binding affinity and rate of ATP-dependent phosphorylation and Ca2+ occlusion were decreased 2-3 orders of magnitude compared with the native enzyme. Cross-linkage had little effect on or marginally increased the rate of acetyl phosphate- and p-nitrophenyl phosphate-supported Ca2+ occlusion. Ca2+ binding or Ca2(+)-induced changes in tryptophan fluorescence were unaffected. High levels of phosphoenzyme (up to 4 nmol/mg of protein) were obtained, with 2 mol of Ca2+ occluded/mol of E-P. Dephosphorylation and deocclusion occurred together at a slow rate (k = 0.01 s-1) and were stimulated in a monophasic manner up to 20-fold by ADP. Cross-linking inhibited E2-P formation from Pi in 30% (v/v) dimethyl sulfoxide by more than 95%. Induction of turnover of the native ATPase, under conditions designed to yield high steady state levels of E1 approximately P(2Ca), results in a 3-4-fold increase in reactivity of active site residues to glutaraldehyde. The results show that cross-linkage sterically impairs nucleotide binding, changing ATP and ADP into relatively poor substrates, slowing nucleotide-dependent phosphoryl transfer and Ca2+ occlusion and deocclusion. The forward reaction with smaller substrates is unaffected. Another major effect of the cross-link is to inhibit E2-P formation, causing accumulation of E1 approximately P(2Ca) during enzyme turnover and preventing phosphorylation by Pi in the reverse direction. We suggest that occlusion and deocclusion of cations at the transport site of the native enzyme are linked to a two-step cleft closure movement at the active site and that the crosslink stabilizes occluded forms of the pump because it blocks part of this tertiary structural change. The latter could normally be propagated through linking helices to the distal side of the pump to destabilize the cations and open the transport sites to the lumen.

Modeling the growth of Yersinia enterocolitica in donated blood.

BACKGROUND: Sepsis and death subsequent to the transfusion of blood containing Yersinia enterocolitica is an increasing problem. The organisms probably originate from bacteremia in the donor and can subsequently multiply at low temperature. STUDY DESIGN AND METHODS: Reported here are experiments with a strain of Y. enterocolitica associated with a case of transfusion-associated bacteremia. RESULTS: It was found that the rapid early killing of Y. enterocolitica injected into donated blood does not require viable phagocytes and can be explained by complement-mediated killing. Complement resistance in Y. enterocolitica is known to be plasmid-coded. It is expressed at 37 degrees C, but not at 20 degrees C, and is favored by calcium-deficient culture media. Y. enterocolitica organisms induced to express complement resistance were still killed in donated blood, though the initial rate was slower. Such organisms multiplied in plasma at 37 degrees C, but were killed after 6 hours of incubation at 20 degrees C, presumably because complement resistance genes are switched off at this temperature. CONCLUSION: This experiment is thought to reflect the natural history of Y. enterocolitica contamination of blood, in which complement-resistant organisms in the donor blood encounter lower temperatures after donation. These observations suggest that the practice of plasma depletion may have contributed to the increased incidence of mortality due to Y. enterocolitica contamination of donated blood.

Mechanism of action of the calcium pump of sarcoplasmic reticulum of skeletal muscle.

The Ca2+-adenosine triphosphatase (ATPase) of skeletal muscle sarcoplasmic reticulum is a single protein species that pumps calcium ions at the expense of adenosine triphosphate (ATP). The reaction cycle includes phosphorylated intermediates which change the affinity and orientation of calcium sites. The monomer appears to be fully functional. Cross-linking and fluorescence studies indicate that ATP binds to a domain that approaches the phosphorylation site and becomes occluded during the reaction cycle. Interactions between these and the calcium channel, possibly via an energy transduction domain, ensure efficient coupling of catalytic and transport cycles.