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S Fleischer

Publications and source records attributed to S Fleischer.

At least 109 records · Page 6Linked to original sources
PubMed1989

Synthesis of spin-labeled 2-azido-ATP: evidence for distinct nucleotide-binding sites in calcium pump protein from sarcoplasmic reticulum.

A spin-labeled and photoreactive derivative of ATP was synthesized with the spin label attached to the 2'- or 3'-position of the ribose moiety and an azido group to C2 of the adenine ring (SL-2N3-ATP). Irradiation of this compound at 350 nm generates a nitrene, which then reacts with nucleophiles in its vicinty. SL-2N3-ATP, in the presence of Ca2+, was hydrolyzed by the calcium pump protein (Ca2+-ATPase) of fast twitch skeletal muscle sarcoplasmic reticulum. The SL-2N3-ATP-enzyme complex in the absence of Ca2+ exhibited strongly immobilized ESR spectra. ESR spectra obtained after covalent incorporation of SL-2N3-ATP into Ca2+-ATPase and removal of freely tumbling SL-2N3-ATP exhibited motionally constrained species indicative of distinct and possibly adjacent ATP-binding sites. By contrast, with SL-ATP devoid of the azido group or with the corresponding 'non-cleavable' beta, gamma-methylene triphosphate analogue (SL-AMP-PCP), two distinct sites were not as well resolved in the ESR spectra due to spectral overlap with the signal from the freely tumbling fraction even with the enhanced spectral resolution provided by perdeuteration of the spin label. Thus, SL-2N3-ATP may have general application for ESR studies of ATP-dependent proteins under conditions in which non-covalent interactions are too weak for motionally restricted species to be resolved.

Adenosine Triphosphate↗
PubMed1989

Coenzyme binding by 3-hydroxybutyrate dehydrogenase, a lipid-requiring enzyme: lecithin acts as an allosteric modulator to enhance the affinity for coenzyme.

The role of phospholipid in the binding of coenzyme, NAD(H), to 3-hydroxybutyrate dehydrogenase, a lipid-requiring membrane enzyme, has been studied with the ultrafiltration binding method, which we optimized to quantitate weak ligand binding (KD in the range 10-100 microM). 3-Hydroxybutyrate dehydrogenase has a specific requirement of phosphatidylcholine (PC) for optimal function and is a tetramer quantitated both for the apodehydrogenase, which is devoid of phospholipid, and for the enzyme reconstituted into phospholipid vesicles in either the presence or absence of PC. We find that (i) the stoichiometry for NADH and NAD binding is 0.5 mol/mol of enzyme monomer (2 mol/mol of tetramer); (ii) the dissociation constant for NADH binding is essentially the same for the enzyme reconstituted into the mixture of mitochondrial phospholipids (MPL) (KD = 15 +/- 3 microM) or into dioleoyl-PC (KD = 12 +/- 3 microM); (iii) the binding of NAD+ to the enzyme-MPL complex is more than an order of magnitude weaker than NADH binding (KD approximately 200 microM versus 15 microM) but can be enhanced by formation of a ternary complex with either 2-methylmalonate (apparent KD = 1.1 +/- 0.2 microM) or sulfite to form the NAD-SO3- adduct (KD = 0.5 +/- 0.1 microM); (iv) the binding stoichiometry for NADH is the same (0.5 mol/mol) for binary (NADH alone) and ternary complexes (NADH plus monomethyl malonate); (v) binding of NAD+ and NADH together totals 0.5 mol of NAD(H)/mol of enzyme monomer, i.e., two nucleotide binding sites per enzyme tetramer; and (vi) the binding of nucleotide to the enzyme reconstituted with phospholipid devoid of PC is weak, being detected only for the NAD+ plus 2-methylmalonate ternary complex (apparent KD approximately 50 microM or approximately 50-fold weaker binding than that for the same complex in the presence of PC). The binding of NADH by equilibrium dialysis or of spin-labeled analogues of NAD+ by EPR spectroscopy gave complementary results, indicating that the ultrafiltration studies approximated equilibrium conditions. In addition to specific binding of NAD(H) to 3-hydroxybutyrate dehydrogenase, we find significant binding of NAD(H) to phospholipid vesicles. An important new finding is that the nucleotide binding site is present in 3-hydroxybutyrate dehydrogenase in the absence of activating phospholipid since (a) NAD+, as the ternary complex with 2-methylmalonate, binds to the enzyme reconstituted with phospholipid devoid of PC and (b) the apodehydrogenase, devoid of phospholipid, binds NADH or NAD-SO3- weakly (half-maximal binding at approximately 75 microM NAD-SO3- and somewhat weaker binding for NADH).(ABSTRACT TRUNCATED AT 400 WORDS)

Allosteric Regulation↗
PubMed1989

Cooperativity in lipid activation of 3-hydroxybutyrate dehydrogenase: role of lecithin as an essential allosteric activator.

3-Hydroxybutyrate dehydrogenase (BDH) is a lecithin-requiring mitochondrial enzyme which catalyzes the interconversion of 3-hydroxybutyrate and acetoacetate with NAD(H) as coenzyme. The purified enzyme devoid of lipid (i.e., the apodehydrogenase or apoBDH) can be reactivated with soluble lecithin or by insertion into phospholipid vesicles containing lecithin. Two different models have been proposed to explain the sigmoidal lipid activation curves. For both models, activation of BDH is assumed to require the binding of two lecithin molecules per functional unit. Activation of soluble enzyme (dimeric form) by short-chain (soluble) lecithin is consistent with a model in which lecithin binding is noncooperative, whereas activation of the membrane-bound enzyme (tetrameric form) indicates cooperativity between the lecithin binding sites. A new comprehensive model is presented in which lecithin is considered to be an essential allosteric activator that shifts the equilibrium between conformational states of the enzyme. Resonance energy transfer data, reflecting NADH binding to membrane-bound and soluble apoBDH, are consistent with such a lecithin-induced conformational change. Apparent dissociation constants for binding of NADH to BDH are approximately 10 microM and approximately 37 microM for BDH activated by bilayer and soluble lecithin, respectively. The maximal fluorescence resonance energy transfer (delta F max) increases with higher mole fraction of lecithin in the bilayer. The largest changes occur between mole fractions 0 and 0.13, thereby correlating with enzymic function. Essentially no binding of NADH is observed in the absence of lecithin.(ABSTRACT TRUNCATED AT 250 WORDS)

Allosteric Regulation↗
PubMed1989

Three-dimensional architecture of the calcium channel/foot structure of sarcoplasmic reticulum.

The calcium channel responsible for the release of Ca2+ from the sarcoplasmic reticulum of skeletal muscle during excitation-contraction coupling has recently been identified and purified. The isolated calcium channel has been identified morphologically with the 'foot' structures which are associated with the junctional face membrane of the terminal cisternae of sarcoplasmic reticulum. In situ, the foot structure extends across the gap of the triad junction from the terminal cisternae of the reticulum to the transverse tubule. We describe here the three-dimensional architecture (3.7 nm resolution) of the calcium channel/foot structure from fast-twitch rabbit skeletal muscle, which we determined from electron micrographs of isolated, non-crystalline structures that had been tilted in the electron microscope. The reconstruction reveals two different faces and an internal structure in which stain accumulates at several interconnected locations, which could empty into the junctional gap of the triad junction. The detailed architecture of the channel complex is relevant to understanding both the physical path followed by calcium ions during excitation-contraction coupling and the association of the terminal cisternae and the transverse tubules in the triad junction.

Animals↗
PubMed1989

Positive cooperativity of ryanodine binding to the calcium release channel of sarcoplasmic reticulum from heart and skeletal muscle.

Ryanodine is a specific ligand for the calcium release channel which mediates calcium release in excitation-contraction coupling in muscle. In this study, ryanodine binding in sarcoplasmic reticulum from heart muscle and skeletal muscle is further compared and correlated with function. The new findings include the following: (1) Two types of binding, high affinity (KD1 approximately 5-10 nM) and low affinity (KD2 approximately 3 microM), can now be discerned for the skeletal muscle receptor. KD1 is approximately the same as and KD2 of similar magnitude to that previously reported for heart. (2) The dissociation rates for the high-affinity binding have been directly measured for both heart and skeletal muscle (t1/2 approximately 30-40 min). These rates are more rapid than previously reported (t1/2 approximately 14 h). (3) KD1's obtained from the ratio of the dissociation and association rate constants agree with the dissociation constant measured by equilibrium binding Scatchard analysis. (4) Ryanodine binding to the low-affinity site can be correlated with a decrease in the dissociation rate constant (k-1) of the high-affinity site, and thereby in the apparent dissociation constant (KD1). The inhibition constant (KI) for inhibiting the high-affinity off rate obtained from a double-reciprocal plot of the change in off rate vs [ryanodine] is practically the same in heart (0.66 microM) and skeletal muscle (0.64 microM) and in the range of the KD2. The binding of cold ryanodine to the low-affinity site appears to lock the bound [3H]ryanodine onto the high-affinity site rather than to exchange with it. Thus, in this sense, the ryanodine receptor exhibits "positive cooperativity".(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaloids↗
PubMed1989

Comparison of the calcium release channel of cardiac and skeletal muscle sarcoplasmic reticulum by target inactivation analysis.

The calcium release channel of sarcoplasmic reticulum which triggers muscle contraction in excitation-contraction coupling has recently been isolated. The channel has been found to be morphologically identical with the feet structures of the junctional face membrane of terminal cisternae and consists of an oligomer of a unique high molecular weight polypeptide. In this study, we compare the target size of the calcium release channel from heart and skeletal muscle using target inactivation analysis. The target molecular weights of the calcium release channel estimated by measuring ryanodine binding after irradiation are similar for heart (139,000) and skeletal muscle (143,000) and are smaller than the monomeric unit (estimated to be about 360,000). The target size, estimated by measuring polypeptide remaining after irradiation, was essentially the same for heart and skeletal muscle, 1,061,000 and 1,070,000, respectively, indicating an oligomeric association of protomers. Thus, the calcium release channel of both cardiac and skeletal muscle reacts uniquely with regard to target inactivation analysis in that (1) the size by ryanodine binding is smaller than the monomeric unit and (2) a single hit leads to destruction of more than one polypeptide, by measuring polypeptide remaining. Our target inactivation analysis studies indicate that heart and skeletal muscle receptors are structurally very similar.

Alkaloids↗
PubMed1989

Modulation of the calcium release channel of sarcoplasmic reticulum by adriamycin and other drugs.

The calcium release channel of sarcoplasmic reticulum mediates Ca2+ release which triggers muscle contraction in excitation-contraction coupling. The channels have been identified morphologically with the feet structures, which are involved in junctional association of terminal cisternae of sarcoplasmic reticulum with the transverse tubules to form the triad junction. In this study, we further characterize the action of drugs on the calcium release channel from sarcoplasmic reticulum fused into planar bilayers. Adriamycin is an effective cancer chemotherapeutic drug, which is limited by its cardiotoxicity. The drug, when added to the myoplasmic side (cis side), activates channel opening at microM concentrations in a dose dependent manner. Adriamycin together with ATP (mM) gives optimal activation, with an open probability (Po) of approximately 1.0. Ruthenium red added to the cis side, equivalent to the cytoplasmic (myoplasmic) domain, completely blocks channel opening. Qualitatively similar results are obtained with adriamycinol, the major metabolite of adriamycin. The inhibition by adriamycin is not reversed by reperfusion to wash out the drug. Silver ions are also found to activate the channel. The conductance of the channel activated by adriamycin, adriamycinol or Ag+ is approximately 100 ps, similar to that previously reported for activation of the channel with Ca2+ and ATP. Ruthenium red has previously been observed to block channel activation from the cytoplasmic side.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1989

Molecular cloning and characterization of the ryanodine receptor/junctional channel complex cDNA from skeletal muscle sarcoplasmic reticulum.

Major progress has been made in elucidating the calcium release mechanism involved in excitation-contraction coupling. The ryanodine receptor of sarcoplasmic reticulum has been isolated and found to be morphologically identical to the foot structure, which is involved in the junctional association of terminal cisternae with the transverse tubule. The foot structure also contains the calcium release channel itself. For this reason, we refer to the foot structure as the junctional channel complex (JCC). The JCC consists of an oligomer of a single high molecular weight protein. Although progress has been made in characterizing important aspects of the structure and function of the JCC, further understanding of the JCC protein subunit awaits the molecular cloning of the JCC. We report on the isolation of cDNA clones encoding portions of the JCC from rabbit fast-twitch skeletal muscle and its tissue distribution and expression. The large size and lack of solubility of the JCC protein posed particular challenges to cloning this molecule. Among these was the necessity to develop techniques for partially digesting the JCC protein subunit with endoproteases in the presence of detergent. With this approach we obtained partial amino acid sequences from regions of the JCC and designed oligonucleotide primers and probes to synthesize and screen cDNA libraries. The rabbit skeletal muscle JCC mRNA encodes an approximately 16-kilobase mRNA present in skeletal, heart, and aortic smooth muscle, as determined by RNA blot analysis with a 700-base-pair cDNA probe. Whereas the JCC mRNA appears to be relatively abundant in adult rabbit fast-twitch skeletal muscle, it is much less abundant in heart and smooth muscle. The JCC mRNA in BC3H1 (a myoblast cell line) is reversibly regulated by growth factors in a manner similar to muscle-specific contractile protein genes.

Amino Acid Sequence↗
PubMed1988

Characterization of junctional and longitudinal sarcoplasmic reticulum from heart muscle.

Longitudinal tubules and junctional sarcoplasmic reticulum (SR) were prepared from heart muscle microsomes by Ca2+-phosphate loading followed by sucrose density gradient centrifugation. The longitudinal SR had a high Ca2+ loading rate (0.93 +/- 0.08 mumol.mg-1.min) which was unchanged by addition of ruthenium red. Junctional SR had a low Ca2+ loading rate (0.16 +/- 0.02 mumol.mg-1.min) which was enhanced about 5-fold by ruthenium red. Junctional SR had feet structures observed by electron microscopy and a high molecular weight protein with Mr of 340,000, whereas longitudinal SR was essentially devoid of both. Thus, these subfractions have similar characteristics to longitudinal and junctional terminal cisternae of SR from fast twitch skeletal muscle. Ryanodine binding was localized to junctional cardiac SR as determined by [3H]ryanodine binding. Scatchard analysis of the binding data showed two types of binding (high affinity, Kd approximately 7.9 nM; low affinity, Kd approximately 1 microM), contrasting with skeletal junctional terminal cisternae where only one site with Kd of approximately 50 nM was observed. The ruthenium red enhancement of Ca2+ loading rate in junctional cardiac SR was blocked by pretreatment with low concentrations of ryanodine as reported for junctional terminal cisternae of skeletal muscle SR. The Ca2+ loading rate of junctional cardiac SR was enhanced by preincubation with high concentrations of ryanodine. The apparent inhibition constant (Ki approximately 7 nM) and stimulation constant (Km approximately 1.1 microM) for ryanodine on junctional SR corresponded to the Kd for high affinity binding (Kd approximately 7.9 nM) and low affinity binding (Kd approximately 1.1 microM), respectively. These results suggest that high affinity ryanodine binding locks the Ca2+ release channels in the open state and that low affinity binding closes the Ca2+ release channels of the junctional cardiac SR. The characteristics of the Ca2+ release channels of junctional cardiac SR appear to be similar to that of skeletal muscle SR, but the Ca2+ release channels of cardiac SR are more sensitive to ryanodine.

Animals↗
PubMed1988

Identification and purification of a transverse tubule coupling protein which binds to the ryanodine receptor of terminal cisternae at the triad junction in skeletal muscle.

In fast twitch skeletal muscle, the signal for excitation-contraction coupling is transferred from transverse tubule across the triad junction; calcium is thereby released from the terminal cisternae of sarcoplasmic reticulum triggering muscle contraction. Recently, the feet structures of terminal cisternae, which bridge the gap at the triad junction, have been identified as the ryanodine receptor and in turn with the calcium release channels of sarcoplasmic reticulum. The latter consists of an oligomer of a single high molecular weight polypeptide (Mr 360,000). This study attempts to identify the component in the transverse tubule which ligands with the foot structure to form the triad junction. The purified ryanodine receptor, derivatized with sulfosuccinimidyl-2-(p-azidosalicylimido)-1,3'-dithiopropionate (SASD), a thiol-cleavable, 125I-iodinatable, and photoactive probe, was shown to selectively cross-link to a protein with Mr of 71,000 in isolated transverse tubules. This coupling protein was purified from transverse tubule by solubilization with the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) and then purified by sequential column chromatography. In the absence of sulfhydryl agents, the purified polypeptide has an Mr of 61,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A complementary approach using SASD was employed to confirm association of the coupling protein with the ryanodine receptor of terminal cisternae. We conclude that the transverse tubule coupling protein together with the ryanodine receptor (foot structure) is involved in the liganding between transverse tubule and terminal cisternae of sacroplasmic reticulum.

Amino Acids↗
PubMed1988

Reconstitution of purified cardiac muscle calcium release channel (ryanodine receptor) in planar bilayers.

The purified ryanodine receptor of heart sarcoplasmic reticulum (SR) has been reconstituted into planar phospholipid bilayers and found to form Ca2+-specific channels. The channels are strongly activated by Ca2+ (10 nM) in the presence of ATP (1 mM) and ryanodine, and inactivated by Mg2+ (3 mM) or ruthenium red (30 microM). These characteristics are diagnostic of calcium release from heart SR. The cardiac ryanodine receptor, which has previously been identified as the foot structure, is now identified as the calcium release channel. A similar identity of the calcium release channel has recently been reported for skeletal muscle. The characteristics of the calcium release channel from skeletal muscle and heart are similar in that they: 1) consist of an oligomer of a single high molecular weight polypeptide (Mr 360,000 for skeletal muscle and 340,000 for heart); 2) exist morphologically as the foot structure; 3) are activated (ATP, Ca2+, ryanodine) and inhibited (ruthenium red and Mg2+) by a number of the same ligands. Important differences include: 1) Ca2+ activation at lower concentration of Ca2+ for the heart; 2) more dramatic stabilization by ryanodine of the open state for the skeletal muscle channel; and 3) different relative permeabilities (PCa/PK).

Adenosine Triphosphate↗
PubMed1988

Clonal analysis of human T cell activation by the Mycoplasma arthritidis mitogen (MAS).

Mycoplasma arthritidis produces an as yet undefined soluble molecule (MAS) that has a potent mitogenic effect on T cells of several species. We have used cloned human cytotoxic and proliferative T lymphocytes to dissect the molecular mechanism of T cell activation by this mitogen. Reactivity to MAS is clonally expressed among T cell receptor (TcR) alpha/beta chain-expressing T cell clones of CD4+ or CD8+ phenotype, as well as CD4-8- TcR alpha/beta chain-negative T lymphocyte clones expressing the CD3-associated TcR gamma chain. MAS is able to induce cytotoxicity and/or proliferation in these T cell clones. For triggering of these T cells, regardless of their phenotype of specificity, the presence of autologous, allogeneic or xenogeneic major histocompatibility complex (MHC) class II molecules on accessory cells or target cells is necessary. However, T cells do not immunologically recognize MAS on class II molecules, since a direct action of MAS on the T cells themselves can be demonstrated. Triggering of T cells by MAS can be blocked by monoclonal antibodies against CD2, CD3 and the TcR alpha/beta chain dimer. We discuss as a possible explanation that MAS is a functionally bivalent molecule cross-linking TcR and MHC class II molecules. Thus, the mechanism of T cell activation by MAS has striking similarities to the mechanisms by which Staphylococcal enterotoxins activate T cells. It is intriguing that a similar mitogenic principle has been developed by two evolutionary distinct pathogenic microorganisms.

Antigen-Antibody Reactions↗
PubMed1988

Comparison of 3-hydroxybutyrate dehydrogenase from bovine heart and rat liver mitochondria.

3-Hydroxybutyrate dehydrogenase is a lipid-requiring enzyme with an absolute requirement of phosphatidylcholine for enzymatic activity. Purification of the enzyme to homogeneity from bovine heart mitochondria was described more than a decade ago [H. G. Bock and S. Fleischer (1975) J. Biol. Chem. 250, 5774-5781]. We have modified the purification procedure so that it is faster, the yield has been improved, and the specific activity is greater by approximately 50%. The updated procedure has also been applied to isolate the enzyme from rat liver mitochondria. Characteristics of the enzyme from bovine heart and rat liver mitochondria have been compared and found to be similar with respect to: (1) purification characteristics; (2) amino acid composition; (3) pH optimum for enzymatic activity; (4) kinetic characteristics; (5) molecular weight as determined by sedimentation equilibrium in guanidine hydrochloride; (6) peptide maps; (7) immunological cross-reactivity. These studies show that 3-hydroxybutyrate dehydrogenase from bovine heart and rat liver mitochondria, though similar, are not identical.

Amino Acids↗