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D B McIntosh

Publications and source records attributed to D B McIntosh.

9 recordsLinked to original sources
PubMed1992

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.

Amino Acid Sequence↗
PubMed1992

2',3'-O-(2,4,6-trinitrophenyl)-8-azido-AMP and -ATP photolabel Lys-492 at the active site of sarcoplasmic reticulum Ca(2+)-ATPase.

2',3'-O-(2,4,6-trinitrophenyl)-8-azido (TNP-8N3)-AMP, -ADP, and -ATP bind tightly to the Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum and become covalently attached on irradiation at alkaline pH, concomitant with inactivation of ATPase activity (Seebregts, C. J., and McIntosh, D. B. (1989) J. Biol. Chem. 264, 2043-2052). The ATPase is derivatized to the extent of 2-3 nmol/mg protein (i.e. approximately 1/2 maximum phosphoenzyme levels) per irradiation period at equimolar concentrations of ATPase and nucleotide. Stability studies of the adduct formed at alkaline pH revealed that the linkage is labile, particularly if the protein is denatured by brief heat (60 degrees C) treatment (t1/2 = 4-8 h at 40 degrees C). Thermolysin digestion of derivatized vesicles resulted in the release of the majority of the TNP chromaphore as an unstable TNP-peptide adduct (t1/2 = 9 h at 25 degrees C) with the sequence FSRDR*SMS, where the missing residue is Lys-492 and is presumably that which is derivatized. The same peptide adduct, and in similar amounts, was isolated from the ATPase derivatized with either TNP-8N3-AMP or -ATP. Several lines of evidence, including the finding that ATP- and not acetyl phosphate- or Pi-dependent phosphorylation is blocked by derivatization, suggest that the lysyl residue is at the catalytic nucleotide binding site, but is not directly involved in phosphoryl transfer. Lys-492 and Phe-487, as well as neighboring Arg-476 and Lys-515 (labeled with fluorescein 5'-isothiocyanate), have all been highly conserved and probably contribute to a subdomain binding the purine and/or proximal phosphoryl groups of ATP.

Adenosine Monophosphate↗
PubMed1991

Crosslinking the active site of sarcoplasmic reticulum Ca(2+)-ATPase completely blocks Ca2+ release to the vesicle lumen.

Intramolecular crosslinking of the active site of the sarcoplasmic reticulum Ca(2+)-ATPase with glutaraldehyde results in substantial inhibition of ATPase activity and stabilization of the ADP-sensitive E1 approximately P(2Ca) intermediate (E, enzyme) with occluded Ca2+ [Ross, D. C., Davidson, G. A. & McIntosh, D. B. (1991) J. Biol. Chem. 266, 4613-4621]. We show here, using conditions of low passive vesicle permeability and absence of ADP, that Ca2+ "deoccludes" more rapidly than it leaks out of the vesicle lumen. Deocclusion is paralleled by dephosphorylation. Therefore, turnover of crosslinked E1 approximately P(Ca) (approximately 5 nmol/min per mg of protein at 25 degrees C) involves Ca2+ release to the vesicle exterior and concomitant phosphoenzyme hydrolysis. Ca2+ release to the lumen, the normal pathway, is apparently blocked completely. In the presence of ADP, Ca2+ is also released to the vesicle exterior, and this release is coupled to the synthesis of ATP. The results suggest that a tertiary structural change at the active site follows phosphorylation and is an absolute requirement for Ca2+ release from the native enzyme to the vesicle lumen.

Animals↗
PubMed1991

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.

Adenosine Triphosphate↗
PubMed1989

2',3'-O-(2,4,6-trinitrophenyl)-8-azido-adenosine mono-, di-, and triphosphates as photoaffinity probes of the Ca2+-ATPase of sarcoplasmic reticulum. Regulatory/superfluorescent nucleotides label the catalytic site with high efficiency.

We have synthesized a new class of ATP photo-affinity analogs, 2',3'-O-(2,4,6-trinitrophenyl)-8-azido (TNP-8N3)-ATP, -ADP, and -AMP, and their radiolabeled derivatives, and characterized their interaction with sarcoplasmic reticulum vesicles. The nucleotides bind with high affinity (Kd = 0.04-0.4 microM) to the catalytic site of the Ca2+-ATPase. TNP-8N3-ATP and TNP-8N3-ADP, at low concentrations (less than 10 microM), accelerate ATPase activity 1.5- and 1.4-fold, respectively, indicating that they bind to a regulatory site. In the same concentration range, they all undergo a large increase in fluorescence ("superfluorescence") during enzyme turnover in the presence of ATP and Ca2+, or on phosphorylation from Pi in a Ca2+-depleted medium. Irradiation at alkaline pH results in specific covalent incorporation of the nucleotide at the catalytic site on the A1 tryptic subfragment. The efficiency of catalytic site labeling is greatest (up to 80% of available sites/irradiation period) in the presence of ATP, Ca2+, and Mg2+, conditions in which the probe binds only to the regulatory and superfluorescent sites. The covalently attached nucleotide exhibits fluorescence enhancement on enzyme turnover in the presence of acetyl phosphate plus Ca2+ or on phosphorylation from Pi in a Ca2+-depleted medium, but not in the presence of ATP plus Ca2+. The results suggest that the catalytic, regulatory, and superfluorescent nucleotide sites are at the same locus and that the binding domain includes portions of the A1 subfragment. The high efficiency with which the site is photolabeled during turnover is ascribed to water exclusion and possibly cleft closure in E2-P.

Adenosine Diphosphate↗
PubMed1979

Occurrence and role of tightly bound adenine nucleotides in sarcoplasmic reticulum of rabbit skeletal muscle.

Freshly isolated sarcoplasmic reticulum vesicles contain 0.05 mol of tightly bound ADP and 0.03 mol of tightly bound ATP per mol of Ca2+, Mg2+-ATPase (ATP phosphohydrolase, EC 3.6.1.3). These values were increased to 0.1-0.2 mol ADP and 0.2-0.3 mol ATP per mol of ATPase after incubation of vesicles in the presence of MgATP and Ca2+ at 25 degrees C and pH 7.0. Half-maximal enrichment of tightly bound nucleotides was obtained with 2.5 mM ATP and 0.32 microM free Ca2+. Uncoupling of calcium transport from ATPase activity by mild acidic conditions or with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid at pH 7.0 decreased the ability of the membranes to be enriched with tightly bound nucleotides and also decreased the content of tightly bound nucleotides of previously enriched membranes. Tightly bound [3H]nucleotides could only be partially displaced by reincubation under enrichment conditions. Tightly bound nucleotides are associated with energized calcium translocation but do not appear to be directly involved in the catalytic cycle.

Adenine Nucleotides↗
PubMed1977

Characteristics of sarcoplasmic reticulum from slowly glycolysing and from rapidly glycolysing pig skeletal muscle post mortem.

The composition and function of fragmented sarcoplasmic reticulum from pig skeletal muscle was examined in the period immediately post mortem. Muscle was defined as being either slowly glycolysing or rapidly glycolysing on the basis of colour, pH and concentrations of glycogen and lactate. The microsomal fraction was separated on a discontinuous gradient of 35, 40 and 45% (w/v) sucrose into heavy and intermediate fractions which sedimented to the interfaces, and a light fraction which remained on the surface of the 35%-sucrose layer. The sarcoplasmic reticulum from rapidly glycolysing muscle had a lower buoyant density than had that from slowly glycolysing muscle. This was reflected in the consistent lack of material in the heavy fraction and a greater proportion in the light fraction. The latter material had significantly lower ratios (w/w) of protein to phospholipid (2.3:1 versus 3.8:1) and of protein to cholesterol (10.4:1 versus 15.6:1). There were no gross differences in phospholipid content or in fatty acid composition of individual phospholipid classes in the membranes from the two types of muscle. Analysis of membrane proteins by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis showed that ATPase (adenosine triphosphatase) was a major component of each fraction and that its contribution to the total protein content of the membrane was greater in rapidly glycolysing muscle, suggesting a loss of non-ATPase proteins. The two fractions of sarcoplasmic reticulum prepared from rapidly glycolysing muscle had approximately one-third the normal activities of Ca(2+) binding and Ca(2+) uptake in the presence of ATP and one-half the passive Ca(2+)-binding capacity in the absence of ATP of the fractions from slowly glycolysing muscle. However, the (Ca(2+)+Mg(2+))-stimulated ATPase activities were similar. Efflux from actively loaded vesicles, after the addition of EDTA, consisted of a rapid and a slow phase. Vesicles from rapidly glycolysing muscle lost 60% of associated Ca(2+) (approx. 0.10mumol of Ca(2+)/mg of protein) during the rapid phase, compared with 30% (approx. 0.17mumol of Ca(2+)/mg of protein) in those from slowly glycolysing muscle. The efflux rate during the slower phase was comparable in both types of vesicles. Analysis of the temperature-dependence of (Ca(2+)+Mg(2+))-stimulated ATPase activity revealed that a high-activation-energy process operating in the temperature range 31-45 degrees C in the intermediate and light fractions from slowly glycolysing muscle was not apparent in vesicles from rapidly glycolysing muscle. Conditions that result in the prolonged activation of glycogenolysis in pig muscle post mortem primarily affect the protein components of the sarcoplasmic-reticular membrane, giving rise to a loss of loosely associated proteins. The function of the membranes observed under these conditions does not appear to be due to enhanced permeability of the membrane to Ca(2+) and may be the result of a defect in the transport of Ca(2+) into the vesicles.

Adenosine Triphosphatases↗
PubMed1977

Proton inactivation of Ca2+ transport by sarcoplasmic reticulum.

The effects of acid on fragmented sarcoplasmic reticulum from rabbit white skeletal muscle have been studied. Brief exposure of sarcoplasmic reticulum membranes to pH values in the range 5.5 to 6.0 at 37 degrees caused rapid inactivation of calcium accumulation measured at 25 degrees in the presence of oxalate (calcium uptake) while (Ca2+, Mg2+)-ATPase (EC 3.6.1.3) activity was enhanced by 75%. ATPase activity, measured at 37 degrees in the absence of oxalate and in the calcium steady state, was unaltered when calcium uptake was inactivated. Calcium efflux from sarcoplasmic reticulum vesicles, previously loaded passibely with 45CaCl2, was only slightly increased when calcium uptake was abolished. At still lower pH values, 5.0 to 5.5, (Ca2+, Mg2+)-ATPase was inactivated while Mg2+ ATPase was more acid-resistant. Acid inactivation of calcium uptake followed simple first order kinetics for at least 80% of the time course. The rate constant, k, increased from 0.043 min-1 to 1.63 min-1 between pH 6.50 and pH 5.35. At pH 4.65, Ea, the energy of activation, was 31 kcal mol-1 between 24 degrees and 43 degrees. Inactivation, once initiated, was irreversible. Aged suspensions of sarcoplasmic reticulum were more sensitive to acid inactivation. Ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid enhanced inactivation, and calcium specifically protected against inactivation with half-maximal effect at 1 to 2 mM. The sulfhydryl reagent, dithiothreitol (1 mM), caused significantly increased rates of inactivation. Calcium binding was studied by dual wavelength spectrophotometry and stopped flow analysis. Acid inactivation distinguished two ATP-induced binding sites, previously described (Entman, M. L., Snow, T. R., Freed, D., and Schwartz, A. (1973) J. Biol. Chem. 248, 7762-7772) as a superficial Mg2+-independent Site A which binds and releases calcium rapidly and a deeper Mg2+-dependent Site B which binds and releases calcium more slowly. Rates of binding to both sites were decreased by acid inactivation. Binding of calcium to Site A increased, however, from 4.6 to 6.4 nmol mg of protein-1 whereas that to Site B decreased from 17.0 to 6.9 nmol mg of protein-1. Passive binding of calcium to sites of medium affinity (K = 7 X 10(4) M-1) was unaffected by acid inactivation of calcium uptake. Temperature dependence of (Ca2+, Mg2+)-ATPase was unchanged in the range 9-34 degrees. Above 34 degrees, the higher activation energy process (Ealpha = 33.7 kcal mol-1) observed in control sarcoplasmic reticulum and thought to arise from a conformational change in the ATPase (Inesi, G., Millman, M., and Eletr, S. (1973) J. Mol. Biol. 81, 483-504) was diminished by acid inactivation (Ealpha = 8.2 kcal mol-1) in a manner suggesting that it is related to active calcium transport. The ATP in equilibrium 32Pi exchange reaction was diminished by acid, but 25% of the activity remained when calcium uptake was completely abolished...

Adenosine Triphosphatases↗