Rabbit Skeletal Sarcoplasmic Reticulum Research Articles

Climate Change and Housing Prices: Hedonic Estimates for Ski Resorts in Western North America

Sarcoplasmic reticulum (SR) vesicles prepared from rat ventricle muscle are isolated, and their [3H]ryanodine-binding and calcium transport properties are studied in detail under active loading conditions in the presence of pyrophosphate. Experiments are performed in tandem with rabbit skeletal SR under identical conditions to allow direct comparisons of the mechanisms by which activators and inhibitors influence the calcium release channel. Ca(++)-induced Ca++ release is demonstrated with both preparations and the cardiac channel is about 1.5-fold more sensitive to activation by Ca++, which is in excellent quantitative agreement with the ability of Ca++ to activate [3H]ryanodine-binding sites. The cardiac and skeletal receptors show major quantitative differences with respect to sensitivity to pharmacologic modulators, cations and pH. The inhibitors ruthenium red, Mg++ and neomycin are significantly more potent in inhibiting the skeletal receptor, whereas the activators daunorubicin and caffeine are significantly more potent towards the cardiac receptor. The ATP analog, beta,gamma-methyleneadenosine 59-triphosphate, enhances the binding of [3H]ryanodine to the high-affinity site in skeletal SR by a factor of 4 but has a negligible effect on the cardiac receptor, although at suboptimal Ca++ for the binding of ryanodine, beta,gamma-methyleneadenosine 59-triphosphate activates the cardiac receptor to a greater extent. High levels of salt (1 M NaCl) enhance the rate of [3H]ryanodine association with its binding sites in both preparations, although they selectively reduce the binding-site capacity in skeletal SR due to a failure to maintain a stable equilibrium. Although high- and low-affinity binding of [3H]ryanodine have a similar response to changing pH, the skeletal receptors are significantly more sensitive to pH.(ABSTRACT TRUNCATED AT 250 WORDS)

Aldolase potentiates DIDS activation of the ryanodine receptor in rabbit skeletal sarcoplasmic reticulum

DIDS (4,4'-di-isothiocyanostilbene-2,2'-disulfonate), an anion channel blocker, triggers Ca2+ release from skeletal muscle SR (sarcoplasmic reticulum). The present study characterized the effects of DIDS on rabbit skeletal single Ca2+-release channel/RyR1 (ryanodine receptor type 1) incorporated into a planar lipid bilayer. When junctional SR vesicles were used for channel incorporation (native RyR1), DIDS increased the mean P(o) (open probability) of RyR1 without affecting unitary conductance when Cs+ was used as the charge carrier. Lifetime analysis of single RyR1 activities showed that 10 microM DIDS induced reversible long-lived open events (P(o)=0.451+/-0.038) in the presence of 10 microM Ca2+, due mainly to a new third component for both open and closed time constants. However, when purified RyR1 was examined in the same condition, 10 microM DIDS became considerably less potent (P(o)=0.206+/-0.025), although the caffeine response was similar between native and purified RyR1. Hence we postulated that a DIDS-binding protein, essential for the DIDS sensitivity of RyR1, was lost during RyR1 purification. DIDS-affinity column chromatography of solubilized junctional SR, and MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS analysis of the affinity-column-associated proteins, identified four major DIDS-binding proteins in the SR fraction. Among them, aldolase was the only protein that greatly potentiated DIDS sensitivity. The association between RyR1 and aldolase was further confirmed by co-immunoprecipitation and aldolase-affinity batch-column chromatography. Taken together, we conclude that aldolase is physically associated with RyR1 and could confer a considerable potentiation of the DIDS effect on RyR1.

Calumenin, a multiple EF-hands Ca 2+-binding protein, interacts with ryanodine receptor-1 in rabbit skeletal sarcoplasmic reticulum

Calumenin is a multiple EF-hand Ca 2+-binding protein located in endo/sarcoplasmic reticulum of mammalian tissues. In the present study, we cloned two rabbit calumenin isoforms (rabbit calumenin-1 and -2, GenBank Accession Nos. AY225335 and AY225336, respectively) by RT-PCR. Both isoforms contain a 19 aa N-terminal signal sequence, 6 EF-hand domains, and a C-terminal ER/SR retrieval signal, HDEF. Both calumenin isoforms exist in rabbit cardiac and skeletal muscles, but calumenin-2 is the main isoform in skeletal muscle. Presence of calumenin in rabbit sarcoplasmic reticulum (SR) was identified by Western blot analysis. GST-pull down and co-immunoprecipitation experiments showed that ryanodine receptor 1 (RyR1) interacted with calumenin-2 in millimolar Ca 2+concentration range. Experiments of gradual EF-hand deletions suggest that the second EF-hand domain is essential for calumenin binding to RyR1. Adenovirus-mediated overexpression of calumenin-2 in C2C12 myotubes led to increased caffeine-induced Ca 2+ release, but decreased depolarization-induced Ca 2+ release. Taken together, we propose that calumenin-2 in the SR lumen can directly regulate the RyR1 activity in Ca 2+-dependent manner.

Aldolase potentiates DIDS activation of the ryanodine receptor in rabbit skeletal sarcoplasmic reticulum

Aldolase potentiates DIDS activation of the ryanodine receptor in rabbit skeletal sarcoplasmic reticulum

Oxidative damage to Ca2+‐ATPase sarcoplasmic reticulum by HOCl and protective effect of some antioxidants

Injury of rabbit skeletal sarcoplasmic reticulum (SR) induced by hypochlorous acid (HOCl) was studied. HOCl inhibited Ca2+-ATPase activity in a concentration-dependent manner (IC50=100 micromol/l). The concentration of 13.5 micromol/l HOCl reduced the level of sulfhydryl (SH) groups by 50%, yet it did not influence the enzyme activity. In comparison with SH group oxidation and enzyme activity inhibition, a significantly longer time was necessary for the generation of protein carbonyls in SR injured by HOCl. Protective effects of some antioxidants (stobadine, trolox, EGb 761, Pycnogenol) were studied in SR oxidatively injured by HOCl. Trolox and EGb 761 exerted a protective effect on ATPase activity and on SH groups of SR oxidatively modified by HOCl. Stobadine and Pycnogenol inhibited markedly protein carbonyl formation. Stobadine was the only antioxidant able to scavenge HOCl. In conclusion, the protective effects of antioxidants against decrease of Ca2+-ATPase activity induced by HOCl might be caused by protection of SH groups. The compounds with both antioxidant and Ca2+-ATPase protecting effect offer dual defense against tissue damage occurring, e.g. in aging process.

SERCA structural dynamics induced by ATP and calcium.

We have used time-resolved phosphorescence anisotropy (TPA) to probe rotational dynamics of the rabbit skeletal sarcoplasmic reticulum Ca-ATPase (SERCA), to test the hypothesis, generated from X-ray crystallography, that large-scale structural changes are induced by Ca in this system. Previous TPA studies on SERCA used primarily erythrosin 5'-isothiocyanate (ErITC), which binds to the nucleotide-binding domain and inactivates the enzyme. To investigate rotational dynamics of the active enzyme, we labeled SERCA with erythrosin 5'-iodoacetamide, which binds to the phosphorylation domain and has a minimal effect on the calcium-dependent ATPase activity. In the absence of nucleotide and the presence of calcium, TPA results were similar to those observed previously with ErITC, consistent with the global uniaxial rotation of SERCA monomers and oligomers and small amplitude internal protein dynamics. The removal of Ca had only a slight effect, while the addition of adenosine 5'-triphosphate (ATP) increased the amplitude of internal dynamics and changed the probe's orientation, corresponding to tilting of the phosphorylation domain by at least 20 degrees . Ca partially reversed the ATP effects. A nonhydrolyzable ATP analogue had the same effects as ATP, showing that the observed changes were not dependent on active ion transport. Computational analysis indicates that these ligands affect primarily the internal dynamics of the enzyme, with negligible effects on global dynamics and enzyme association. Melittin, which has been shown to aggregate and inhibit SERCA, eliminated the nucleotide-induced internal dynamics and increased the final anisotropy. We propose that (i) the large Ca-dependent structural changes suggested by SERCA crystallography are more dependent on ATP than on Ca and (ii) aggregation-induced inhibition of SERCA is due to the functional coupling between global and internal protein dynamics.

N-terminal Region of FKBP12 Is Essential for Binding to the Skeletal Ryanodine Receptor

It is known that the two types of FK506-binding proteins FKBP12 and FKBP12.6 are tightly associated with the skeletal (RyR1) and cardiac ryanodine receptors (RyR2), respectively, and their interactions are important for channel functions of the RyR. In the case of cardiac muscle, three amino acid residues (Gln-31, Asn-32, and Phe-59) of FKBP12.6 could be essential for the selective binding to RyR2 (Xin, H. B., Rogers, K., Qi, Y., Kanematsu, T., and Fleischer, S. (1999) J. Biol. Chem. 274, 15315-15319). In this study to identify amino acid residues of FKBP12 that are important for the selective binding to RyR1, we mutated 9 amino acid residues of FKBP12 that differ from the counterparts of FKBP12.6 (Q3E, R18A, E31Q, D32N, M49R, R57A, W59F, H94A, and K105A), and we examined binding properties of these mutants to RyR1 by in vitro binding assay by using glutathione S-transferase-fused proteins of the mutants and Triton X-100-solubilized, FKBP12-depleted rabbit skeletal sarcoplasmic reticulum vesicles. Among the nine mutants tested, only Q3E and R18A lost their selective binding ability to RyR1. Furthermore, co-immunoprecipitation of RyR1 with 33 various mutants for the 9 positions produced by introducing different size, charge, and hydrophobicity revealed that an integration of the hydrogen bonds by the irreplaceable Gln-3 and the hydrophobic interactions by the residues Arg-18 and Met-49 could be a possible mechanism for the binding of FKBP12 to RyR1. Therefore, these results suggest that the N-terminal regions of FKBP12 (Gln-3 and Arg-18) and Met-49 are essential and unique for binding of FKBP12 to RyR1 in skeletal muscle.

Preservation of the native structure and function of Ca2+-ATPase from sarcoplasmic reticulum: solubilization and reconstitution by new short-chain phospholipid detergent 1,2-diheptanoyl-sn-phosphatidylcholine.

The properties of Ca2+-ATPase purified and reconstituted from rabbit skeletal sarcoplasmic reticulum (SR) has been studied in comparison with the preparations obtained by the commonly used detergent poly(oxyethylene)8-lauryl ether (C12E8) and the bile salt detergents cholate and deoxycholate. 1,2-Diheptanoyl-sn-phosphatidylcholine (DHPC) has been shown to be excellent for solubilizing a wide variety of membrane proteins [Kessi, Poiree, Wehrli, Bachofen, Semenza and Hauser (1994) Biochemistry 33, 10825-10836]. The DHPC method consistently gave higher yields of purified Ca2+-ATPase with a greater specific activity than the methods with C12E8, cholate, or deoxycholate. DHPC and C12E8 were superior to cholate and deoxycholate in active enzyme yields and specific activity. DHPC-solubilized Ca2+-ATPase purified on a density gradient retained the E1Ca-E1(*)Ca conformational transition, whereas the enzyme from the C12E8 purification did not retain this transition. The coupling of Ca2+ transported to ATP hydrolysed in the DHPC-purified enzyme was maximal and matched the values obtained with native SR, whereas the coupling was much lower for the C12E8-purified enzyme. The specific activity of Ca2+-ATPase reconstituted into dioleoylphosphatidylcholine vesicles with DHPC was up to 2-fold greater than that achieved with C12E8, and is comparable to that measured in the native SR. Finally, the dissociation of Ca2+-ATPase into monomers by DHPC preserved the ATPase activity, whereas similar dissociation by C12E8 gave only one-sixth the activity of that obtained with DHPC. These studies show that the Ca2+-ATPase solubilized, purified and reconstituted with DHPC is superior to that obtained with C12E8 in significant ways, making it a preparation suitable for detailed studies on the mechanism of ion transport and the role of protein-lipid interactions in the function of membrane proteins.

Comparison of 2,2′-azobis(2-amidinopropane) hydrochloride (AAPH) and 2,2′-azobis(2,4-dimethylvaleronitrile)(AMVN) as free radical initiators: a spin-trapping study

Spin trapping with 5,5-dimethyl-1-pyrroline 1-oxide (DMPO) and its hydrophobic analogue 2,2-dimethyl-4-phenyl-2H-imidazole 1-oxide (DMPIO) was used to identify and to monitor the concentration of participating radical species in oxidation reactions initiated by azo compounds: water-soluble 2,2′-azobis(2-amidinopropane) hydrochloride (AAPH) and lipophilic 2,2′-azobis(2,4-dimethylvaleronitrile)(AMVN). Incubation of AAPH with spin traps in aqueous media produced alkoxyl radical spin adducts with hyperfine splitting constants being aN= 14.62 G, βaH= 15.29 G, γaH= 0.72 G and aN= 13.46 G, βaH= 12.53 G, for DMPO and DMPIO, respectively. In contrast, formation of AMVN-derived peroxyl radicals was detected by both direct EPR and spin trapping in DMSO (dimethylsulfoxide) solutions. In the presence of either rabbit skeletal sarcoplasmic reticulum (SR) membranes (10 g dm–3 of SR protein) or egg phosphatidylcholine liposomes (10 g dm–3 of lipid) preloaded with AMVN no spin adduct formation was observed, for both DMPO and DMPIO spin traps, indicating that AMVN-derived radical species do not escape the lipid environment. Only a small portion of AAPH-derived alkoxyl radicals was trapped by DMPIO in the presence of SR membranes. Spectral characteristics of the DMPIO spin adduct indicate its location at the lipid–water interface. At the same time, there was virtually no effect of SR on the rate of formation and steady-state level of the DMPO-spin adduct formed in the aqueous phase. From these data we suggest that the bulky cytosolic domains of the SR Ca2+-ATPase protect the membrane surface from radicals generated in the bulk (aqueous) solvent. Other evidence also demonstrates different mechanisms for free radical formation by AAPH and AMVN azo-initiators.

Reaction of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole with the (Ca 2+ + Mg 2+) -ATPase protein of sarcoplasmic reticulum at low temperature

Modification of the (Ca 2+ + Mg 2+)-ATPase protein of rabbit skeletal sarcoplasmic reticulum (SR) with 7-chloro-4-nitrobenzo-2-oxa 1, 3-diazole, NBD-Cl, at 4°C for 5 min caused a 63% loss of the Ca 2+-dependent ATPase activity when 1 mol of the adenine analog was incorporated per 10 5 g of protein. At 25°C, above the lipid phase transition, the extent of labeling was 3-fold higher although the Ca 2+-ATPase activity was inhibited to the saine extent. MgATP protected the ATPase activity at 4°C and 25°C but there was little change in the extent of labeling at 4°C suggesting that changes in the fluidity of the lipid moiety made different sites on the ATPase protein accessible to the reagent. At 4°C, addition of sodium deoxycholate enhanced the inactivation (6% ATPase activity remained) but the labeling of the SR-ATPase protein did not increase significantly. Incubation with MgATP prior to solubilization with deoxycholate resulted in the protection of the Ca 2+-ATPaseactivity and only a small decrease in the labeling occurred. At 25°C, a similar pattern was found with deoxycholate but the loss of ATPase activity was less dramatic and the extent of labeling by NBD-Cl was greater than that at 4°C. MgATP induced changes in the conformation of the ATPase protein protecting essential cysteine residues while shifting the reaction of NBD-Cl with the ATPase protein to non-essential sites in the absence or presence of deoxycholate. An analysis of tryptic digests of the NBD-ATPase protein showed that MgATP shifted the labeling from the A 2 subfragment to the A 1 subfragment in the absence of deoxycholate and from the A 1 subfragment to the A 2 subfragment in the presence of deoxycholate. The reagent, NBD-Cl, can distinguish between different temperature dependent conformational states of the ATPase protein.

The interaction of fluorescein isothiocyanate with the ryanodine receptor/Ca2+ release channel of sarcoplasmic reticulum.

Fluorescein 5'-isothiocyanate (FITC) markedly inhibited ryanodine binding to rabbit skeletal muscle junctional sarcoplasmic reticulum. Half-maximal inhibition was obtained with about 20 microM and complete inhibition by 80 microM FITC. Inhibition was enhanced in the presence of high salt and at alkaline pH. The pH dependence of the inactivation of ryanodine binding suggested that FITC bound to a very reactive lysine epsilon-amino group with a pK alpha of about 7.5 or above. Kinetic analysis of the time course of inactivation of ryanodine binding by various concentrations of FITC suggested that the inactivation resulted from the modification of 2 or more amino acid residues. The inhibition of ryanodine binding by FITC was partially prevented by ATP, ADP, adenyl imidodiphosphate, and 3-O-(benzoyl)-benzoyl-ATP (Bz-ATP) but not by AMP. FITC modification of sarcoplasmic reticulum membranes inhibited the photoaffinity labeling by [alpha-32P]Bz-ATP of the 450-kDa protein and the ryanodine receptor with half-maximal inhibition at about 100 microM. The results suggest that the inhibition of ryanodine binding is mainly due to FITC modification of sites which are not involved in ATP binding. The FITC moiety is bound to the 160-, 96-, 76-, and 60-kDa ryanodine receptor tryptic fragments, and the FITC site is apparently on the 21.5-, 18-, and 17-kDa fragments which are formed by the V8 protease. Covalent modification by FITC dramatically affected the activity of single Ca2+ release channels incorporated into planar lipid bilayers. FITC caused a marked increased in channel open probability mainly to a noisy approximately 60% subconductance state. FITC-modified channels were no longer affected by ryanodine but were still abolished by Mg2+ and ruthenium red. We suggest that FITC modifies reactive lysine residues involved in channel activation by transmembrane charge movement in the t-tubular system.

Purification, characterization, and molecular cloning of a 60-kDa phosphoprotein in rabbit skeletal sarcoplasmic reticulum which is an isoform of phosphoglucomutase.

A 60-kDa substrate of calmodulin-dependent protein kinase in rabbit "heavy" skeletal sarcoplasmic reticulum (SR) was characterized by purification and cDNA cloning. Purification was achieved by column chromatography using DEAE-Sephacel, heparin-agarose, and hydroxylapatite in 0.5% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonic acid (CHAPS). Analyses of amino acid sequence and composition indicated that the CHAPS-soluble 60-kDa protein is an isoform of phosphoglucomutase (PGM). cDNAs encoding two isoforms of PGM were isolated from rabbit skeletal muscles. The translated amino acid sequences show that the isoforms, PGM1 and PGM2, differ in the N-terminal 77 amino acids and that PGM2 is identical to the 60-kDa protein in the SR. Northern blot analysis showed that the size of the mRNA encoding PGM2 is 2.4 kilobases. The PGM enzyme activity was markedly inhibited in SR membranes, while perturbation of the membranes with CHAPS or guanidine-HCl recovered the enzyme activity. KCl (0.15-1 M) led to a partial recovery of the enzyme activity suggesting that the charge interaction is not the primary force for PGM-SR interaction. PGM is localized in the heavy fraction of SR, where calsequestrin and Ca2+ release channel are enriched. Our results demonstrate that an isoform of PGM localized in junctional skeletal SR is the 60-kDa substrate of calmodulin-dependent protein kinase.

Inactivation of calcium uptake by EGTA is due to an irreversible thermotropic conformational change in the calcium binding domain of the Ca(2+)-ATPase.

Calcium uptake by rabbit skeletal sarcoplasmic reticulum (SR) is inhibited with an effective inactivation temperature (TI) of 37 degrees C in EGTA with no effect on ATPase activity. Since the Ca-ATPase denatures at a much higher temperature (49 degrees C) in EGTA, this suggests that a small or localized conformational change of the Ca-ATPase at 37 degrees C results in inability to accumulate calcium by the SR. Using a fluorescent analogue of dicyclohexylcarbodiimide, N-cyclohexyl-N'-[4-(dimethylamino)-alpha-naphthyl]-carbodiimide (NCD-4), the region of the calcium binding sites of the SR Ca-ATPase was labeled. Steady-state and frequency-resolved fluorescence measurements were subsequently performed on the NCD-4-labeled Ca-ATPase. Site-specific information pertaining to the hydrophobicity and segmental flexibility of the region of the calcium binding sites was derived from the steady-state fluorescence intensity, lifetime, and rotational rate of the covalently bound NCD-4 label as a function of temperature (0-50 degrees C). A reversible transition at approximately 15 degrees C and an irreversible transition at approximately 35 degrees C were deduced from the measured fluorescence parameters. The low-temperature transition agrees with the previously observed break in the Arrhenius plot of ATPase activity of the native Ca-ATPase at 15-20 degrees C. The high-temperature transition conforms well with the conformational transition, resulting in uncoupling of Ca translocation from ATP hydrolysis as predicted from the irreversible inactivation of Ca uptake at 31-37 degrees C in 1 mM EGTA.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of melittin on molecular dynamics and Ca-ATPase activity in sarcoplasmic reticulum membranes: electron paramagnetic resonance.

We have performed electron paramagnetic resonance (EPR) experiments on nitroxide spin labels incorporated into rabbit skeletal sarcoplasmic reticulum (SR), in order to investigate the physical and functional interactions between melittin, a small basic membrane-binding peptide, and the Ca-ATPase of SR. Melittin binding to SR substantially inhibits Ca(2+)-dependent ATPase activity at 25 degrees C, with half-maximal inhibition at 9 mol of melittin bound per mole of Ca-ATPase. Saturation transfer EPR (ST-EPR) of maleimide spin-labeled Ca-ATPase showed that melittin decreases the submillisecond rotational mobility of the enzyme, with a 4-fold increase in the effective rotational correlation time (tau r) at a melittin/Ca-ATPase mole ratio of 10:1. This decreased rotational motion is consistent with melittin-induced aggregation of the Ca-ATPase. Conventional EPR was used to measure the submicrosecond rotational dynamics of spin-labeled stearic acid probes incorporated into SR. Melittin binding to SR at a melittin/Ca-ATPase mole ratio of 10:1 decreases lipid hydrocarbon chain mobility (fluidity) 25% near the surface of the membrane, but only 5% near the center of the bilayer. This gradient effect of melittin on SR fluidity suggests that melittin interacts primarily with the membrane surface. For all of these melittin effects (on enzymatic activity, protein mobility, and fluidity), increasing the ionic strength lessened the effect of melittin but did not alleviate it entirely. This is consistent with a melittin-SR interaction characterized by both hydrophobic and electrostatic forces. Since the effect of melittin on lipid fluidity alone is too small to account for the large inhibition of Ca-ATPase rotational mobility and enzymatic activity, we propose that melittin inhibits the ATPase primarily through its capacity to aggregate the enzyme, consistent with previous observations of decreased Ca-ATPase activity under conditions that decrease protein rotational mobility.

Effect of the detergent C12E8 on the binding of monoclonal antibodies to the (Ca2+-Mg2+)-ATPase of rabbit skeletal sarcoplasmic reticulum

Effect of the detergent C12E8 on the binding of monoclonal antibodies to the (Ca2+-Mg2+)-ATPase of rabbit skeletal sarcoplasmic reticulum

Rotational dynamics of the calcium ATPase in sarcoplasmic reticulum studied by time-resolved phosphorescence anisotropy

We have investigated the microsecond rotational motions of the Ca-ATPase in rabbit skeletal sarcoplasmic reticulum (SR), by measuring the time-resolved phosphorescence anisotropy of erythrosin 5-isothiocyanate (ERITC) covalently and specifically attached to the enzyme. Over a wide range of solvent conditions and temperatures, the phosphorescence anisotropy decay was best fit by a sum of three exponentials plus a constant term. At 4 degrees C, the rotational correlation times were phi 1 = 13 +/- 3 microseconds, phi 2 = 77 +/- 11 microseconds, and phi 3 = 314 +/- 23 microseconds. Increasing the solution viscosity with glycerol caused very little effect on the correlation times, while decreasing the lipid viscosity with diethyl ether decreased the correlation times substantially, indicating that the decay corresponds to rotation of the protein within the membrane, not to vesicle tumbling. The normalized residual anisotropy (A infinity) is insensitive to viscosity and temperature changes, supporting the model of uniaxial rotation of the protein about the membrane normal. The value of A infinity (0.20 +/- .02) indicates that each of the three decay components can be analyzed as a separate rotational species, with the preexponential factor Ai equal to 1.25X the mole fraction. An empirically accurate measurement of the membrane lipid viscosity was obtained, permitting a theoretical analysis of the correlation times in terms of the sizes of the rotating species. At 4 degrees C, the dominant correlation time (phi 3) is too large for a Ca-ATPase monomer, strongly suggesting that the enzyme is primarily aggregated (oligomeric).(ABSTRACT TRUNCATED AT 250 WORDS)

Fluorescence properties of the Ca 2+Mg 2+-ATPase protein of sarcoplasmic reticulum labeled with 7-chloro-4-nitrobenzo-2-oxa-1,3- diazole

The fluorescence intensity of the Ca 2+,Mg 2+-ATPase protein of rabbit skeletal sarcoplasmic reticulum that incorporated about 2 mol of 7-chloro-4-nitrobenzo-2-oxa-1, 3-diazole (NBD-Cl) was enhanced at high MgATP concentrations with or without 50 μM calcium. The observed enhancement indicates that the fluorophore, NBD-Cl, can detect conformational changes in the ATPase protein.

Modification of the (Ca2 + Mg2)-ATPase protein of sarcoplasmic reticulum with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole

The (Ca2+ + Mg2+)-ATPase (ATP phosphohydrolase (Ca2+-transporting), EC 3.6.1.38) protein of rabbit skeletal sarcoplasmic reticulum (SR) rapidly incorporated 2 mol of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) per 10(5) g of protein with little change in the Ca2+-dependent ATPase activity. When 2 additional mol of the reagent were bound the Ca2+-ATPase, activity was inhibited. The same pattern was found for modified intact SR and the Ca2+ uptake ability was inhibited. MgATP, CaATP and MgADP protected the Ca2+-ATPase activity concurrent with a decrease of about 1 mol of the NBD group per 10(5) g protein, but the Ca2+ uptake ability was not protected. Calcium alone had no effect on the modification. The modified ATPase protein or SR formed non-serial oligomers or aggregates, but the ATPase protein remained the predominant species present. In the presence of MgATP, oligomer formation was reduced partially but the major changes in the Ca2+-ATPase activity were due to the modification of the ATPase monomer. Thiolysis of the NBD-ATPase protein with dithiothreitol did not restore the Ca2+-ATPase activity, although more than 1 mol of the NBD group was removed from cysteine residues. Cysteine residues were modified in the NBD-ATPase protein or SR when the enzyme activity was inhibited. Trypsin digestion of NBD-SR or its ATPase protein released the A, B, A1, and A2 fragments. The A fragment and its subfragment A2 contained most of the label. Substrate MgATP protection studies showed that the A1 and A2 fragments were involved in maintaining the Ca2+-ATPase activity. Reagent-induced conformational changes of these fragments rather than direct active site group labeling accounted for the loss of ATPase activity.

The interaction of [formula omitted] with phospholipid bilayers

Long chain acylamides of ethanolamine were previously found to increase in the infarcted canine myocardium. Subsequent in vitro experiments established a number of interesting biological and physiological properties of these compounds including alteration of rabbit skeletal sarcoplasmic reticulum function and inhibition of permeability dependent calcium release from heart mitochondria. These results suggested an interaction between the N- acylethanolamines and biological membranes. In the present work we show that the most potent species in previous studies, N- oleylethanolamine , forms stable complexes with phospholipid vesicles, lowers species in previous studies, N- oleylethanolamine , forms stable complexes with phospholipid vesicles, lowers diphenylhexatriene polarization ratios in dimyristoylphosphatidylcholine and dipalmitoylphosphatidylcholine uni- and multilamellar bilayer vesicles, and also produces a concentration dependent decrease in the phase transitions of these lipid structures. In addition studies with parinaric acids also suggested that N- oleylethanolamine partitions preferentially into more fluid areas of the bilayer. The results are discussed in terms of possible effects on biological membranes.

Transient-state kinetics of phosphoenzyme transformation in the rabbit skeletal sarcoplasmic reticulum calcium-dependent adenosine triphosphatase reaction. Two distinct modes of ADP and K+ regulation.

We have observed two modes each of ADP and K+ regulation of phosphoenzyme (EP) intermediates formed in the early phase of skeletal sarcoplasmic reticulum hydrolysis of ATP at 20 degrees C, using, for the first time, a five-syringe quench flow apparatus for transient-state kinetic measurements. The total acid-stable EP formed for 20.5 and 116 ms in the K+ medium appears to be composed of either two monomers in rapid equilibrium, E1P in equilibrium E'1P, or a dimer of the two subunits, PE1E'1P. The ADP-sensitive E1P may form an acid-labile ADP X E1P (or ATP X E1) complex rapidly, giving ATP as a consequence of acid quenching. The ADP may also induce decomposition of the ADP-reactive E'1P. Monomeric and dimeric mechanisms are introduced to account for the hyperbolic relation between the rate constant of the ADP-induced E'1P decomposition and [ADP], consistent with the fact that the E'1P may also give ATP in the presence of ADP. As to the K+ effects, the K+, which is bound to the unphosphorylated enzyme and possibly becomes occluded during EP formation, may either facilitate the one-to-one E1P in equilibrium E'1P equilibrium or maintain the dimeric functional unit. The subsequent forward transformation of the E'1P to the ADP-insensitive K+-sensitive E'2P, possibly the rate-determining step for the catalytic cycle, is found to be K+ independent. The major effect of the K+ in the medium is its catalytic cleavage of the E'2P, which is detected as the missing EP under these conditions. When K+ is not involved in the EP formation, the forward sequential transformation E1P----E'1P----E'2P----E2P or PE1E'1P----PE'2E2P is apparent in the time range from 20.5 to 116 ms after EP formation, and the E'2P may accumulate in the K+ devoid medium and be detected as the major component of the total acid-stable EP. The Mg2+-sensitive E2P represents the EP missing in the medium containing no ADP and K+.