Browsing by Author "Berman, Mervyn C"
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- ItemOpen AccessConformational changes in the (Ca²⁺, Mg²⁺)-ATPase of sarcoplasmic reticulum during energy transduction(1981) Swiel, Denise; Berman, Mervyn CTreatment of SR membranes with mild acid (pH 5.6) (Berman, M.C., McIntosh, D.B. and Kench, J.E. (1977) J. Biol. Chem. 252, 994-1001) or incubation with millimolar concentrations of ethylene glycol bis (β-aminoethyl ether)-N ,N'-tetraacetic acid (EGTA) at neutral pH and 37°C (McIntosh, D. B. and Berman, M. C. (1978) J. Biol. Chem. 253, 5l40-5146) results in a progressive irreversible inhibition of calcium transport while (Ca²⁺, Mg²⁺)-ATPase activity is unimpaired. Possible conformational changes associated with this uncoupling were monitored by following alterations in kinetic mobility of sulphydryl (-SH) groups either by using 5, 5'-dithiobis- (2-nitrobenzoate) (DTNB) and stopped flow analysis or 1-¹⁴C-N-ethylmaleimide (NEM). Kinetic reactivity with DTNB revealed a total of 20 thiol groups/1.5 x 10⁵ g of SR protein (rontaining 1 mole of ATPase protein) in the presence of sodium dodecyl sulphate, which constitute four kinetic classes. In native control vesicles 4.5 thiol groups were unreactive, 0.4 represented the fast reacting class, 0.8 the moderately fast reacting class and 14.4 the slowly reacting class, displaying pseudo-first order rate constants, k, of 159.0-, 22.0- and 6.23 x 10⁻² sec⁻¹, respectively. Inactivation of calcium transport to the extent of 90%, using mild acid conditions, increased the number of fast and moderately fast reacting groups, each by 1.0 - 1.5 sulphydryl groups / mol ATPase. The number of slowly reacting groups decreased by approximately 3 .0 thiol groups/mol ATPase. The kinetics of the reaction with 1-¹⁴C-NEM was essentially similar to that with DTNB. EGTA inactivation of calcium transport, to the extent of 90% and subsequent 1-¹⁴C-NEM modification, resulted in an increase in the number of fast reacting thiol groups by 0.5-1.0 thiol groups/mol ATPase. The total number of reactive thiol groups decreased by 1.0 -2.0 thiol groups/ mol ATPase, probably due to autoxidation of the newly exposed sulphydryl group. Inactivation of transport carried out in the presence of N-ethylmaleimide to prevent autoxidation resulted in an increase of approximately one thiol group/mol ATPase. The rate constant for the increase in reactivity of this group was 1.45 min⁻¹. This thiol group was localized on the ATPase protein of molecular weight approximately 100 000 daltons. Trypsinization of the ATPase produced four fragments of molecular weights 55 000, 45 000, 30 000 and 20 000. More extensive cleavage resulted in a significant decrease in the 55 000 dalton fragment and increased amounts of the 30 000 and 20 000 dalton subfragments. There was increased labelling on all subfragments of EGTA-treated vesicles compared to control, untreated vesicles. However, the greatest relative increase in labelling appeared to be localized on the 55 000 dalton and 20 000 dalton subfragments. Peptide mapping of the purified ATPase revealed 24 ninhydrin-positive peptides. Five of these were labelled in control and EGTAtreated vesicles, four of which showed increased labelling in the latter preparation. Random labelling of the nonoverlapping fragments may be due to the enzyme being "trapped" in a number of intermediate conformations or due to heterogeneity within the ATPase populations. NEM modification of SR membranes did not affect the tryptic cleavage pattern or the mobilities of the tryptic subfragments. It did however, affect the extent of tryptic cleavage resulting in solubilization of NEM-labelled protein into the medium following centrifugation. This protein fraction was identified as consisting largely of the 55 000 dalton molecular weight species on sodium dodecyl sulphate gel electrophoresis. It is concluded that occupancy of high affinity K₀.₅(Ca²⁺)≈10⁻⁶M) calcium binding sites maintain the (Ca²⁺, Mg²⁺)- ATPase in a stable, coupled conformation. Displacement of this calcium induces a conformational change in the protein which results in the loss of the vectorial component of calcium transport.
- ItemOpen AccessThe D-domain of fibrin : structural and functional studies(1987) Purves, Maud; Purvis, Langley R; Berman, Mervyn CThe D-domain of fibrin (ogen) was separated from the parent molecule by plasmin digestion in the presence of calcium and isolated by means of DEAE-anion exchange chromatography followed by gel-filtration in buffer containing 4 M urea. Fluorescent-D-dimer (f-D-dimer) was isolated from a plasmic digest of fibrin clotted in the presence of 2.45 mM dansyl cadaverine, a fluorescent lysine analogue. Fluorescent-D-monomer was a by-product of f-D-dimer purification, the yield being determined by the concentration of dansyl cadaverine. At 2.45 mM f-D-monomer was always present in the digest. The f-D-monomer is probably formed directly and not as a result of degradation of f-D-dimer. The molecule elutes in the fibrinogen-derived-D- monomer position on gel-filtration. A protease was isolated and partially purified from venom of the puffadder (Bitis arietans). Puffadder venom protease is characterized by its ability to cleave D-dimer into symmetrical D-monomers, smaller than plasmin-derived D-monomers from fibrinogen. This characteristic was used to detect the puffadder venom protease activity with fluorescent-D-dimer being used as the substrate. Fractions obtained were assayed for D-dimer cleavage activity and the samples analyzed by means of SDS-PAGE on 4-20% gradient gels under reducing (βME) and non-reducing conditions. The fluorescent bands were located under U.V light and photographed prior to staining with Coomassie Blue. Several methods for the purification of the protease were investigated.
- ItemOpen AccessMembrane reconstitution studies on the irreversibility of inactivation of sarcoplasmic reticulum of rabbit skeletal muscle(1979) Arendse, Michael Peter; Berman, Mervyn CMild acid treatment or incubation in the presence of Ethylene glycol bis (β-aminoethyl ether) - N,N' - tetraacetic acid inactivates calcium transport by sarcoplasmic reticulum membranes but does not inhibit calcium stimulated ATPase activity. This inactivation is apparently irreversible. The purpose of the present study was to determine whether lipid-protein interactions, imposed by the transmembrane nature of the (Ca²⁺, Mg²⁺) - ATPase contributed towards the irreversible nature of the inactivation. This was determined by studying the possibility of reactivating calcium transport in acid-inactivated sarcoplasmic reticulum vesicles by means of membrane reconstitution studies. Calcium transport activity was reconstituted in control and acid-inactivated sarcoplasmic reticulum vesicles by deoxycholate solubilisation and subsequent slow dialysis at room temperature. Reconstituted control sarcoplasmic reticulum had an average specific activity of 0,38 μmol calcium transported /minute /mg of protein. Acid-inactivated sarcoplasmic reticulum vesicles, in which calcium transport had been inactivated to 0.2 μmol Calcium transported/minute/mg of protein (10% of the original transport activity) were studied by reconstitution methods. Following reconstitution, the isolated, reformed vesicles regained up to 1,5-fold transport activity when compared with the original acid-inactivated vesicles, indicating that acid-inactivation was partially reversible. Protein composition of reconstituted control and reconstituted acid-inactivated sarcoplasmic reticulum vesicles was studied by SDS-gel electrophoresis. Both preparations showed that the M55 protein was incorporated into reconstituted vesicles whereas there was a preferential loss of the M45 calcium binding protein (calsequestrin). The removal of deoxycholate into the dialysate was studied by means of (Carboxyl-C¹⁴) -deoxycholate. The kinetics of removal indicate that approximately 0,15 mg DOC remained associated per mg of protein even after exhaustive dialysis. Calcium efflux from reconstituted vesicles was followed by release of calcium into Ethylene glycol bis (β-aminoethyl ether) -N, N' -tetraacetic acid following active uptake in the presence of precipitable phosphate anions. Calcium efflux was slower from reconstituted vesicles than from original sarcoplasmic reticulum. The ability of acid-inactivated sarcoplasmic reticulum to bind Ca²⁺ or adenine nucleotides tightly was investigated. The capacity to bind calcium tightly was decreased from 1.43 nmol Ca²⁺/mg protein in control to 0,96nmol Ca²⁺/mg protein in acid inactivated sarcoplasmic reticulum. Similarly, the capacity to bind adenine nucleotides tightly decreased from 0,20 mol nucleotides/mol ATPase in control vesicles to 0,07 mol nucleotides /mol ATPase in acid inactivated vesicles. Following reconstitution the capacity for tight binding of calcium and adenine nucleotides increased to 2,4 nmol Ca²⁺/mg protein and 0,24 mol nucleotides/mol ATPase respectively indicating that the capacity to bind both calcium and adenine nucleotides tightly is closely related to transport activity but not to calcium dependent ATPase activity. These studies indicate that the protein-lipid interaction restrains the acid-inactivated sarcoplasmic reticulum from returning to its native conformation. Release of these constraints by deoxycholate followed by its removal results in reversal of the conformational change to that of the coupled native sarcoplasmic reticulum membrane.
- ItemOpen AccessProperties of the non-catalytic nucleotide site of the Ca²⁺-ATPase of sarcoplasmic reticulum(1986) Davidson, George Alexander; Berman, Mervyn CProperties of the regulatory nucleotide binding site of the Ca²⁺-ATPase of skeletal muscle sarcoplasmic reticulum have been investigated. Previously, several lines of evidence have indicated the existence of both catalytic and regulatory nucleotide binding sites on the same polypeptide species. The present study concentrates on the interaction of the ATP analogue, 2'-3'-0-(2,4,6-trinitrocyclohexadienylidine) adenosine 5'-triphosphate, (TNP-ATP), with sites on the non-phosphorylated and phosphorylated enzyme. In particular those conformational transitions linking TNP-ATP fluorescence to the phosphoenzyme subspecies have been sought. Previous studies have demonstrated a close relationship between TNP-ATP fluorescence and phosphoenzyme formed from ATP plus Ca²⁺, or from inorganic phosphate (Pi) in the absence of Ca²⁺, in the reverse direction of the cycle. However, the precise relationship of TNP-ATP fluorescence to the energy transducing conformations of the ATPase is controversial. TNP-ATP binding was investigated by spectrophotometric methods and by the synthesis of [ ¹⁴C] TNP-ATP. [ ¹⁴C] TNP-ATP bound to the ATPase site with high affinity ([TNP-ATP] 0. 5 = 0.12 uM), and · a stoichiometry of 5.4 nmol/mg. [ ¹⁴C] ATP binding stoichiometry was 6.1 nmol/mg, demonstrating that TNP-ATP binds to a single family of sites. The nature of the phosphoenzyme intermediate species that results in enhanced TNP-ATP fluorescence was investigated. NEM derivitization, Sr²⁺-transport and Ca²⁺-oxalate uptake have previously been found to alter the distribution or relative levels of phosphoenzyme intermediates. Modification of thiol groups responsible for phosphoenzyme decomposition (SHd), using N-ethylmaleimide (NEM) (0.4 mM) with 50 uM Ca²⁺, 1 mM AMP-PNP at pH 7.0, resulted in a 50% decrease in Ca²⁺-uptake, Ca²⁺-ATPase activity and ADP-insensitive E-P (E₂-P), while total EP (E₁-P + E₂-P = 3.2 nmol/mg), remained unaltered. ATP-dependent TNP-ATP enhanced fluorescence decreased by 50% under these conditions. Ca²⁺-oxalate induced turnover has previously been shown to decrease steady-state E₂-P levels by prevention of Ca²⁺ gradient formation. Oxalate (5 mM) caused a 40% decrease in ATP-induced TNP-ATP fluorescence levels while total EP levels remained relatively unaltered. Previous studies have shown that Sr²⁺-induced turnover favours higher levels of E₂-P by inhibiting the reverse reaction from E₂-P to E₁-P. Strontium-induced turnover increased TNP-ATP fluorescence by 10% as compared to that of Ca²⁺, without affecting steady-state E-P levels, consistent with an E₂-P conformation relationship to enhanced TNP-ATP fluorescence. The binding site for TNP-ATP on the enzyme was investigated by chase studies using millimolar concentrations of nucleotides. ATP and ADP diminished TNP-ATP fluorescence competitively, with apparent Km values of 1.25 and 0.54 mM respectively, consistent with their affinities of binding to the regulatory site. The rates of decrease of fluorescence (25 and 34 sec⁻¹ at 5 ᵒC, respectively), were of the same order of magnitude as the derived "off" rate of TNP-ATP from the site of enhanced fluorescence (33 sec⁻¹), consistent with TNP-ATP being bound to the regulatory site of the enzyme. Enhanced TNP-ATP fluorescence has previously been related to decreased water activity of the probe site. Alteration of water activity by structure- forming (Deuterium oxide) and structure-breaking solutes (KSCN) in relation to fluorescence were explored. Replacement of H₂O by D₂O altered the fluorescence of unbound TNP-ATP. The apparent for TNP-ATP binding to the E₂-P conformation of the regulatory site. The regulatory site appears to be a modified form of the phosphorylated catalytic site. It is proposed that TNP-ATP fluorescence monitors an enzyme conformation related to Ca²⁺ binding to an inward oriented site of low affinity. The mechanism of K⁺ fluorescence quenching appears to be via an acceleration of dephosphorylation, as opposed to a change in affinity of the enzyme for TNP-ATP, as previously suggested. The K⁺ sensitivity of TNP-ATP fluorescence has proved useful in demonstrating a direct interaction of valinomycin with the enzyme through the monovalent cation binding site. Valinomycin appears to bind directly to the enzyme and to selectively accelerate the "off" rate of K⁺ from this site.
- ItemOpen AccessThe role of TNP-Nucleotides, LYS492 and CA²⁺chelators in the skeletal muscle sarcoplasmic reticulum CA²⁺atpase cycle(1998) Wichmann, Janine; McIntosh, David B; Berman, Mervyn CIn the first part of this study, the kinetics of decay of TNP-nucleotide superfluorescence was investigated with a view to understanding the role of nucleotides and Lys492 in later steps in the catalytic cycle of the skeletal muscle Ca²⁺ATPase. It has been found previously, and verified here, that tethering TNP-8N₃-AMP to the Ca²⁺ATPase via Lys492 retarded the Ca²⁺ initiated decay of Pᵢ-induced superfluorescence 10-fold compared with untethered nucleotide. The rapidity of the decay upon addition of EDTA suggested that the E₂ ↔ E₁ → E₁Ca₂ steps were being monitored rather than dephosphorylation per se. Tethered diand triphospho species did not accelerate the decay. While monophasic kinetics was observed with untethered TNP-AMP and TNP-8N₃-AMP, complex kinetics were observed with the di- and triphospho TNP-nucleotides. This was shown to be due to the utilization of TNP-ADP and -ATP, and the azido derivatives, as coupled substrates of the Ca²⁺ATPase in the forward direction of catalysis in the presence of Ca²⁺. The hydrolysis rates of TNP-ADP, TNP-ATP, TNP-8N₃ -ADP, and TNP-8N₃ -ATP were 10, 5, 15 and 10 nomoles/min/mg of protein, respectively, at room temperature and pH 5.5. Ca²⁺ transport was supported by all four nucleotides. This is the first time that a diphosphonucleotide has been shown to support Ca²⁺ transport. A new nonhydrolysable triphospho TNPnucleotide, TNP-AMP-PCP was synthesized and shown to interact with the Ca²⁺ATPase in a similar way, in terms of superfluorescence, as the other TNP-nucleotides. It did not show the complex kinetics on inhibition of the Pcinduced superfluorescence by Ca²⁺, but neither did it accelerate the kinetics. It was concluded that TNP-nucleotides do not accelerate the E₂ ↔ E₁ transition under these conditions, possibly because of the presence of glycerol in the medium. In the second part of the study, it was shown that addition of small amounts of chelators EGTA, EDTA, BAPTA, DTPA, HEDTA and NTA to a Ca²⁺ transport assay in which the free Ca²⁺ concentration is monitored by Fluo-3 causes the Ca²⁺ATPase to pump to apparently lower levels as seen in the [Ca²⁺] lim fluorescence. Addition of chelator retards pump function in the sense that it takes longer for 50 nmols Ca²⁺ to be accumulated. Increased thermodynamic efficiency of the pump and contaminating heavy metal ions were considered as possible mechanisms. To some extend Zn²⁺ and Cd²⁺, but not Fe²⁺ and Cu²⁺, appeared to reverse the partial inhibition. While interpretation of the results is difficult, it is suggested that heavy metal ions interact with luminal loops of the Ca²⁺ATPase and enhance Ca²⁺ release under conditions of high luminal Ca²⁺ concentrations.