Browsing by Subject "Sarcoplasmic reticulum"

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    Membrane reconstitution studies on the irreversibility of inactivation of sarcoplasmic reticulum of rabbit skeletal muscle
    (1979) Arendse, Michael Peter; Berman, Mervyn C
    Mild 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.
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    Photoaffinity labeling the nucleotide sites of the sarcoplasmic reticulum Ca²⁺-ATPase
    (1989) Seebregts, Christopher J; McIntosh, David B
    We have synthesized a new class of photoaffinity analogs, 2',3'-O-(2,4,6-trinitrophenyl)-8-azido-ATP, -ADP and -AMP (TNP- 8N₃ATP, -ADP and -AMP), and their radiolabeled derivatives, and characterized their interaction with the sarcoplasmic reticulum Ca²⁺-ATPase. The TNP-8N₃-nucleotides were synthesized from ATP in three steps involving bromination in the 8-position of the adenine ring followed by displacement with an azido group and then trinitrophenylation of the resulting 8N₃-nucleotide with TNBS. Inclusion of the oxidizing agent, DTNB, in the final reaction was found to be necessary to prevent reduction of the azido group by the released sulfite anion and also elevated the yield of trinitrophenylation to about 80%. Purity was determined spectrophotometrically, as well as by anion exchange TLC and reversed phase HPLC. In the dark, the compounds were found to display most of the features of the parent TNP-nucleotides and interacted with the Ca²⁺-ATPase in a similar way. When activated by illumination, the probes were specifically incorporated into SR vesicles with high efficiency at alkaline pH. The site of labeling was identified as being on the A₁ tryptic fragment.
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    Properties of the non-catalytic nucleotide site of the Ca²⁺-ATPase of sarcoplasmic reticulum
    (1986) Davidson, George Alexander; Berman, Mervyn C
    Properties 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.