Biomedical subjects

S Fleischer

Publications and source records attributed to S Fleischer.

At least 163 records · Page 9Linked to original sources

PubMed1985

Localization of Ca2+ release channels with ryanodine in junctional terminal cisternae of sarcoplasmic reticulum of fast skeletal muscle.

The mechanism of Ca2+ release from sarcoplasmic reticulum, which triggers contraction in skeletal muscle, remains the key unresolved problem in excitation-contraction coupling. Recently, we have described the isolation of purified fractions referable to terminal and longitudinal cisternae of sarcoplasmic reticulum. Junctional terminal cisternae are distinct in that they have a low net energized Ca2+ loading, which can be enhanced 5-fold or more by addition of ruthenium red. The loading rate, normalized for calcium pump protein content, then approaches that of longitudinal cisternae of sarcoplasmic reticulum. We now find that the ruthenium red-enhanced Ca2+ loading rate can be blocked by the previous addition of ryanodine. The inhibition constant is in the nanomolar range (20-180 nM). Ryanodine and ruthenium red have no effect on the Ca2+ loading rate of longitudinal cisternae. Direct binding studies with [3H]ryanodine localized the receptors to the terminal cisternae and not to longitudinal cisternae. Scatchard analysis of the binding data gives a dissociation constant for ryanodine in the range of the drug action on the terminal cisternae (approximately 100 nM range) with approximately 4 to 20 pmol bound per mg of protein. Ryanodine is known to be toxic in animals, leading to irreversible muscle contractures. These studies provide evidence on the mode of action of ryanodine and its localization to the terminal cisternae. The low concentration at which the drug is effective appears to account for its toxicity. Ryanodine locks the Ca2+ release channels in the "open state," so that Ca2+ is not reaccumulated and the muscle fiber cannot relax.

Alkaloids↗

PubMed1984

Subcellular distribution of bile acids, bile salts, and taurocholate binding sites in rat liver.

We have quantitated bile acids and their conjugates in rat liver using high-pressure liquid chromatography. Over 95% of the hepatic bile acid pool in rat liver homogenates is present as taurocholate and tauromuricholate. Although over 60% of the bile acid pool is recovered in the supernatant, evidence is presented suggesting that taurocholate redistributes among the subcellular fractions during their isolation. Taurocholate (TC) binding to purified subcellular fractions from rat liver was determined by using equilibrium dialysis in a TC concentration range from 0.1 to 100 microM. This is well below the critical micellar concentration of taurocholate (3 mM). All of the fractions investigated exhibited low-affinity binding with dissociation constants from 80 to 240 microM as did membrane lipid vesicles. Therefore, low-affinity binding appears referable to taurocholate nonspecifically partitioning into the lipid bilayer. High-affinity binding is present in plasma membranes, Golgi, and cell supernatant. The high-affinity binding sites in Golgi have a mean dissociation constant (A1) of 1.0 microM and bind 0.15 nmol of TC/mg of protein. Similarly, the high-affinity binding sites of plasma membrane have an A1 of 1.3 microM and bind 0.15 nmol of TC/mg of protein. For cell supernatant, the A1 was 4.8 microM, and 0.35 nmol of TC was bound per mg of protein. Mitochondria, smooth and rough microsomes, and Golgi liposomes showed no detectable amounts of high-affinity binding. These results are compatible with a role for the Golgi complex, cytoplasmic component(s), and plasma membranes in transhepatic bile acid transport.

Animals↗

PubMed1984

Complex formation between nucleotides and D-beta-hydroxybutyrate dehydrogenase studied by fluorescence and EPR spectroscopy.

D-beta-Hydroxybutyrate dehydrogenase (D-3-hydroxybutyrate:NAD+ oxidoreductase, EC 1.1.1.30) is a lipid-requiring enzyme which specifically requires phosphosphatidylcholine for enzymic activity. The phosphatidylcholine modifies the binding and orientation of the coenzyme, NAD(H), with respect to the enzyme. In the present study, two derivatives of NAD, spin-labeled either at N-6 or C-8 of the adenine ring, were found to be active as coenzyme. The binding affinity of NADH to the enzyme was opitimized by increasing the salt concentration and increasing the pH from 6 to 8, with the pK at 6.8. Monomethylmalonate, a substrate analogue, was found to enhance NADH binding (Kd is reduced from 4 to 1 microM). Sulfite strongly enhances the binding of NAD+ via the enzyme-catalyzed formation of an adduct of sulfite with the nucleotide; the Kd for binding of NAD-sulfite is in the micromolar range, whereas NAD+ binding is more than a magnitude weaker. The binding of spin-labeled NAD(H) was further characterized by EPR spectroscopy. Increased sensitivity and resolution were obtained with the use of NAD(H) analogues perdeuterated in the spin-label moiety. For these analogues bound to D-beta-hydroxybutyrate dehydrogenase in phospholipid vesicles, EPR studies showed the spin-label moiety to be constrained and revealed two distinct components. Increasing the viscosity of the medium by addition of glycerol affected the EPR spectral characteristics of only the component with the smaller resolved averaged hyperfine splitting. The stage is now set to study motional characteristics of the enzyme, using these spin-labeled probes which mimic the coenzyme.

Animals↗

PubMed1984

The synthesis of 15N- and deuterium-substituted, spin-labeled analogues of NAD+ and their use in EPR studies of dehydrogenases.

Two spin-labeled analogues of NAD+ were synthesized with an 15N and perdeuterated nitroxide radical, 4-amino-2,2,6,6-[2H17, 15N]tetramethylpiperidone-1-oxyl, which was attached to either the C-6 or C-8 position of the purine ring. The EPR spectra of these derivatives exhibit an approx. 6-fold increase in sensitivity compared with the corresponding 14N, protonated analogues due to a decrease in both the number of nuclear manifolds (from three to two) and the linewidth. The enhanced spectral resolution obtained with (2H17, 15N)spin-labeled-NAD+ analogues has facilitated simulation of the EPR lineshape of the nucleotide bound to lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27). The spin-label moiety exhibits highly constrained motion indicative of a single environment. The motion of the spin label does not reflect the overall motion of the enzyme; rather, it is characteristic of some limited mobility relative to the lactate dehydrogenase. By contrast, the spin label on the membrane-bound enzyme, D-beta-hydroxybutyrate dehydrogenase (D-beta-hydroxybutyrate:NAD+ oxidoreductase, EC 1.1.1.30), is completely immobilized and exhibits two distinct spectral components for spin-labeled NAD+, which appear to differ in the polarity of the environment of the nitroxide.

Electron Spin Resonance Spectroscopy↗

PubMed1984

Half-site reactivity of an essential thiol group of D-beta-hydroxybutyrate dehydrogenase.

D-beta-Hydroxybutyrate dehydrogenase is a lipid-requiring enzyme, which is a tetramer both in the mitochondrial inner membrane and as the purified enzyme reconstituted with phospholipid. For the active enzyme-phospholipid complex in the absence of ligands, we previously found that reaction with N-ethylmaleimide (at 5 mol/mol of enzyme subunit) resulted in progressive loss of enzymic activity with an inactivation stoichiometry of 1 equiv of sulfhydryl derivatized per mole of enzyme and a maximum derivatization of 2 equiv [Latruffe, N., Brenner, S. C., & Fleischer, S. (1980) Biochemistry 19, 5285-5290]. We now find, in the presence of nucleotide or substrate, that the rate of inactivation is significantly reduced, which indicates that these ligands afford protection of the essential sulfhydryl. Further, in the presence of ligands, the inactivation stoichiometry is 0.5, consistent with half-of-the-site reactivity of the essential sulfhydryl. Thus, at a low ratio of N-ethylmaleimide to enzyme, nucleotide or substrate affords essentially complete protection of the nonessential sulfhydryl from derivatization. The binding characteristics of NADH to both the native and N-ethylmaleimide-derivatized enzyme have been compared by fluorescence spectroscopy. Quenching of intrinsic tryptophan fluorescence of the protein shows that the enzyme, derivatized with N-ethylmaleimide either in the absence or in the presence of NAD+, binds NADH but with a reduced Kd (approximately 50 microM as compared with approximately 20 microM for native enzyme). However, a critical change has occurred in that resonance energy transfer from protein to bound NADH, observed in the native enzyme, is abolished in the N-ethylmaleimide-derivatized enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗

PubMed1984

Reversible modification of D-beta-hydroxybutyrate dehydrogenase by diamide.

D-beta-Hydroxybutyrate dehydrogenase is a lipid-requiring enzyme with a specific requirement of lecithin for function. The purified enzyme devoid of lipid (apodehydrogenase) is inactive but can be reactivated by forming a complex with phospholipid containing lecithin. We find that, of the six half cysteines present in D-beta-hydroxybutyrate dehydrogenase, only two are in the reduced form and available for modification with N-ethylmaleimide, even after denaturation in sodium dodecyl sulfate. Diamide treatment of either the inactive apodehydrogenase or the active enzyme-phospholipid complex resulted in complete loss of enzymic activity, the apodehydrogenase being assayed after addition of phospholipid. The inactivation by diamide can be reversed by the addition of dithiothreitol with full recovery of activity. Derivatization using N-[14C]ethylmaleimide showed that diamide modified only one sulfhydryl per enzyme monomer. The other sulfhydryl appears not to be essential for function since full activity can be restored after this sulfhydryl had been covalently derivatized with N-ethylmaleimide. Protein cross-linking was not observed after diamide modification of D-beta-hydroxybutyrate dehydrogenase, indicating that a disulfide bridge was not formed between enzyme subunits. The diamide-modified enzyme retains the ability to bind coenzyme, NAD(H), as detected by quenching of the intrinsic fluorescence of the protein. However, resonance energy transfer from protein to bound NADH and enhancement of NADH fluorescence were not observed, indicating that diamide modification of the protein alters the nucleotide binding site.(ABSTRACT TRUNCATED AT 250 WORDS)

Azo Compounds↗

PubMed1984

Two distinct mechanisms for taurocholate uptake in subcellular fractions from rat liver.

As part of the enterohepatic circulation, hepatocytes take up bile acids from the intestines via the hepatic portal blood using a sodium-dependent carrier mechanism and resecrete the bile acids into the bile. In order to assess whether intracellular organelles are involved in the transcellular secretion of bile acids, we measured directly the ability of purified subcellular fractions of rat liver to take up taurocholate using a Millipore filtration assay. Two distinct uptake mechanisms can be discerned, one localized in the plasma membranes and the other in the Golgi and smooth microsomal fractions. Plasma membranes prepared by the method of Fleischer and Kervina (Fleischer, S., and Kervina, M. (1974) Methods Enzymol. 31, 6) take up taurocholate in a saturable manner with an apparent Vmax of 2.4 nmol min-1 mg protein-1 and a Km of 190 microM at 37 degrees C. After preincubation of the membranes with K+ ions, a sodium gradient (100 mM outside) stimulates the uptake rate by 90% with the observed Km unchanged. The stimulation is inhibited by phalloidin but not by bromosulfophthalein. Bile canalicular plasma membranes made according to Kramer et al. (Kramer, W., Bickel, U., Buscher, H. P., Gerok, W., and Kurz, G. (1982) Eur. J. Biochem. 129, 13-24) do not take up taurocholate. The transport by Golgi vesicles and smooth microsomes differs from that in the plasma membrane fraction in that it is not stimulated by a sodium gradient, has a Vmax of 12 nmol min-1 mg protein-1 and a Km of 440 microM at 37 degrees C, and is inhibited by bromosulfophthalein but not by phalloidin. Taurocholate uptake into smooth microsomes is abolished by filipin, an antibiotic that complexes with cholesterol to disrupt the membrane. This suggests that taurocholate uptake occurs into a nonendoplasmic reticulum subfraction since endoplasmic reticulum membranes contain negligible amounts of cholesterol. Little uptake was observed using rough microsomes or mitochondria. A model of transhepatic transport compatible with our observations is that taurocholate uptake into the cytoplasm occurs via the plasma membranes on the sinusoidal side of the hepatocyte; taurocholate is then taken up into smooth vesicles and the Golgi complex and is secreted into the bile by exocytosis as the vesicles fuse with the canalicular plasma membranes.

Animals↗

PubMed1984

The effect of phenothiazines on Ca2+ fluxes in skeletal muscle sarcoplasmic reticulum.

The effect of phenothiazines (trifluoperazine, chlorpromazine, methochlorpromazine, and imipramine) on Ca2+ fluxes in light and heavy sarcoplasmic reticulum (SR) isolated from rabbit fast-twitch skeletal muscle was investigated. These drugs inhibited Ca2+ loading and (Ca2+,Mg2+)-ATPase activity, but had no effect on unidirectional Ca2+ efflux from vesicles loaded either actively or passively with Ca2+. Chlorpromazine, which is membrane permeable, and its quaternary analog, methochlorpromazine, which is membrane impermeable, gave identical results. It is concluded that (a) the enhancement of net Ca2+ release by phenothiazines is due to inhibition of Ca2+ influx mediated by the Ca2+ pump rather than to the opening of a Ca2+ channel; and (b) phenothiazines act at the outer (myoplasmic) face of the SR membrane.

Animals↗

PubMed1984

Interaction of liver microsomal cytochrome P-450 and NADPH-cytochrome P-450 reductase in the presence and absence of lipid.

Phospholipid has been reported to be necessary for optimal catalytic activity of a number of mammalian cytochrome P-450 (P-450) systems. We also confirm that a number of individual phospholipids and mixtures, used as soluble monomers or phospholipid vesicles, show activation of 7-ethoxycoumarin O-deethylase activity by an enzyme system composed of rat liver microsomal P-450PB-B and NADPH-P-450 reductase. However, by preincubating a mixture of P-450 and NADPH-P-450 reductase at high concentrations, optimal activity can be obtained in the absence of phospholipid. The catalytic activity of the complex formed is concentration dependent in the absence of lipid or in the presence of soluble lipid. The activity in phospholipid vesicles is optimal and concentration independent. The apparent Km for NADPH-P-450 reductase in P-450-dependent oxidation systems is lowered severalfold in the presence of phospholipid. The apparent Km for the P-450 substrate, 7-ethoxycoumarin, and the temperature dependence of 7-ethoxycoumarin O-deethylase activity were unaffected by the addition of phospholipid to a preformed complex of P-450PB-B and NADPH-P-450 reductase. The effect of lipid on a number of other P-450 isozymes was also examined and in no case did lipid enhance the catalytic activity of the preformed complex. These results lead to the conclusion that the major effect of phospholipids in P-450-based enzyme systems is the facilitation of an active P-450:NADPH-P-450 reductase complex. This is the first report that maximum P-450 supported monooxygenase activity can be obtained in the absence of phospholipid.

7-Alkoxycoumarin O-Dealkylase↗

PubMed1984

Calcium-induced calcium release from purified cardiac sarcoplasmic reticulum vesicles. General characteristics.

Isolated canine cardiac sarcoplasmic reticulum exhibits Ca2+-induced Ca2+ release from both actively and passively loaded vesicles. The rate and extent of Ca2+ release depend on the extravesicular ionized Ca2+ concentration ( [Ca2+]o) at the onset of release. Maximal release following ATP-dependent, phosphate-facilitated Ca2+ loading (up to 360 nmol of Ca2+/mg of protein/min at 37 degrees C) occurs at 1.5-2 microM [Ca2+]o, with reduced release at both lower and higher Ca2+ concentrations (half-maximal Ca2+ release at approximately 0.8 and 5.5 microM [Ca2+]o). Only a portion of the accumulated Ca2+ is released and the release is followed by reuptake of Ca2+. A similar Ca2+ dependence is obtained in the absence of ATP and Pi by measuring unidirectional Ca2+ efflux from passively loaded vesicles (maximal Ca2+ efflux at 1 microM [Ca2+]o; half-maximal Ca2+-dependent efflux at approximately 0.15 and 13 microM [Ca2+]o). Although the Ca2+ release rates observed in this study are several orders of magnitude lower than the rate of Ca2+ release which occurs in muscle cells in vivo, this Ca2+ release phenomenon may be related to the Ca2+-induced Ca2+ release which has been described for skinned cardiac cells ( Fabiato , A. (1983) Am. J. Physiol. 245, C1-C14). Ca2+ release occurs in the presence of an ATP-regenerating system and is not accompanied by a reduction in ATP hydrolysis. Also, since unidirectional Ca2+ efflux (as high as 860 nmol of Ca2+/mg of protein/min at 37 degrees C) exceeds net Ca2+ release under similar conditions, Ca2+ influx proceeds during the period of net Ca2+ release. Therefore, Ca2+ release does not involve reversal or cessation of inward Ca2+ pumping. Other data indicate that Ca2+ release is not mediated through the Ca2+ pump protein, but occurs through a separate Ca2+-dependent efflux pathway, possibly a channel.

Adenosine Triphosphate↗

PubMed1984

Inhibition of calcium-induced calcium release from purified cardiac sarcoplasmic reticulum vesicles.

A variety of reagents (local anesthetics, phenothiazines, ruthenium red, ryanodine, dicyclohexylcarbodiimide, R 24571) inhibit Ca2+-induced Ca2+ release from purified canine cardiac sarcoplasmic reticulum (SR). Most of these compounds also increase the rate of net Ca2+ uptake by cardiac SR while moderately inhibiting Ca2+-dependent ATP hydrolysis, and together these two effects produce increased coupling ratios for ATP-dependent Ca2+ transport (Ca2+/ATP approximately equal to 2) compared to controls (Ca2+/ATP approximately equal to 1). We conclude that Ca2+ efflux normally occurs during net Ca2+ uptake by isolated cardiac SR vesicles and that this phenomenon is responsible for the low coupling ratios generally observed for cardiac SR preparations. Blockers of sarcolemmal Ca2+ channels (nitrendipine, diltiazem, methoxyverapamil, dantrolene), at concentrations much greater than those effective for sarcolemmal Ca2+ fluxes, do not affect either Ca2+ uptake or Ca2+ release by cardiac SR. Furthermore, the effects of local anesthetics and phenothiazines on Ca2+ release from cardiac SR are different from those previously reported for skeletal muscle SR. These results indicate that the Ca2+ release "channels" in cardiac SR are distinct from Ca2+ channels present in cardiac sarcolemma or in skeletal muscle SR. The common hydrophobic nature but structural dissimilarity of various inhibitors of Ca2+ release from cardiac SR suggest that in some cases a hydrophobic interaction with the membrane might be involved in blocking Ca2+ efflux. On the other hand, half-maximal inhibition by 80 nM ruthenium red is suggestive of a specific ionic interaction with some component of the Ca2+ efflux pathway.

Adenosine Triphosphate↗

PubMed1984

Protein-mediated exchange of synthetic phosphatidylcholines into synaptosomal membranes.

A phosphatidylcholine (PC) exchange protein from bovine liver was used to exchange endogenous synaptosomal membrane PC's with PC's of defined fatty-acid composition from phospholipid vesicles. Up to 50% of the total synaptosomal PC could be exchanged during a 3 h incubation with PC's which were in the liquid-crystalline state at the temperature of incubation (dimyristoyl-, dioleoyl- and dielaidoyl-PC). The biphasic kinetics of the exchange of 14C-labeled 1-palmitoyl-2-oleoyl-PC into isolated synaptic plasma membrane vesicles indicated that the half-time for transbilayer equilibrium of PC in these membranes was about 10 h. Hence, the observed 50% exchange of total synaptosomal PC probably represented nearly complete exchange of PC in the outer face of the synaptosomal plasma membrane. This extensive exchange was accomplished without apparent loss of synaptosomal function, including membrane potential and high-affinity uptake of choline and gamma-aminobutyric acid. PC's in the gel state (dipalmitoyl- and distearoyl-PC) could not be exchanged extensively into the synaptosomal membranes. However, from within gel-state distearoyl-PC liposomes, a trace amount of fluid 1-palmitoyl-2-oleoyl-PC (Tm less than 10 degrees C) could be preferentially exchanged into the synaptosomes at 32 degrees C with little transfer of the saturated PC.

Androgen-Binding Protein↗

PubMed1984

Target size of calcium pump protein from skeletal muscle sarcoplasmic reticulum.

The oligomeric size of calcium pump protein (CPP) in fast skeletal muscle sarcoplasmic reticulum membrane was determined using target theory analysis of radiation inactivation data. There was a parallel decrease of Ca2+-ATPase and calcium pumping activities with increasing radiation dose. The loss of staining intensity of the CPP band, observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, also correlated directly with the loss of activity. The target size molecular weight of the CPP in the normal sarcoplasmic reticulum membrane ranged between 210,000 and 250,000, which is consistent with a dimeric structure. Essentially the same size is obtained for the non-phosphorylated CPP or for the phosphoenzyme form generated from either ATP (E1 state) or inorganic phosphate (E2 state). Hence, the oligomeric state of the pump does not appear to change during the catalytic cycle. Similar results were obtained with reconstituted sarcoplasmic reticulum membrane vesicles with different lipid to protein ratios. We conclude that the CPP is a dimer in both native and reconstituted sarcoplasmic reticulum membranes. The target size of the calcium-binding protein (calsequestrin) was found to be 50,000 daltons, approximating a monomer.

Animals↗

PubMed1984

Spontaneous calcium release from sarcoplasmic reticulum. Assessment of other ionic influences.

A form of spontaneous Ca2+ release from purified light sarcoplasmic reticulum has been recently described (Palade, P., Mitchell, R. D., and Fleischer, S. (1983) J. Biol. Chem. 258, 8098-8107). It is characterized by rapid Ca2+ efflux (1-10 mumol/min X mg protein) which begins only after a delay following preloading and depletion of extravesicular Ca2+. In the present study, the influences of a number of ionic factors modulating the spontaneous calcium release phenomenon are described. The divalent metal ions Mn2+ and Sr2+ and higher external [Mg2+] inhibit release. Mn2+ appears to inhibit from outside, whereas Sr2+ must be taken up to inhibit. Decreasing the phosphate concentration during preloading eliminates spontaneous release, but it can be partially restored with salts of other nonprecipitating anions. No such release was obtained with other Ca2+-precipitating anions, i.e. pyrophosphate, oxalate, and fluoride. The release characteristics are strongly dependent on the monovalent cation present. Spontaneous Ca2+ release is optimal at pH 6.8-7.0 and decreases sharply at higher and lower pH. Ca2+ release can be enhanced by addition, during the lag period, of concentrated salt solutions consisting of more permeant cations than anions, which may indicate that an inside positive membrane potential enhances the rate of calcium release. Spontaneous Ca2+ release can take place in the presence of sucrose, and Ca2+ efflux is not accompanied by a corresponding efflux of preloaded sucrose, demonstrating that the vesicles remain sealed during spontaneous release. The spontaneous Ca2+ release process is distinct from several other forms of Ca2+ release from sarcoplasmic reticulum.

Adenosine Triphosphate↗

PubMed1984

Calcium release from sarcoplasmic reticulum of normal and dystrophic mice.

Contraction of skeletal muscle is triggered by release of calcium from the sarcoplasmic reticulum. In this study, highly purified normal and dystrophic mouse sarcoplasmic reticulum vesicles were compared with respect to calcium release characteristics. Sarcoplasmic reticulum vesicles were actively loaded with calcium in the presence of an ATP-regenerating system. Calcium fluxes were followed by dual wavelength spectrophotometry using the metallochromic indicators antipyrylazo III and arsenazo III, and by isotopic techniques. Calcium release from sarcoplasmic reticulum vesicle was elicited by (a) changing the free calcium concentration of the assay medium (calcium-induced calcium release); (b) addition of a permeant anion to the assay medium, following calcium loading in the presence of a relatively impermeant anion (depolarization-induced calcium release); (c) addition of the lipophilic anion tetraphenylboron (TPB-) to the assay medium and (d) using specific experimental conditions, i.e. high phosphate levels and low magnesium (spontaneous calcium release). Drugs known to influence Ca2+ release were shown to differentially affect the various types of calcium release. Caffeine (10 mM) was found to enhance calcium-induced calcium release from isolated sarcoplasmic reticulum. Ruthenium red (20 microM) inhibited both calcium-induced calcium release and tetraphenylboron-induced calcium release, and partially inhibited spontaneous calcium release and depolarization-induced calcium release. Local anesthetics inhibited spontaneous calcium release in a time-dependent manner, and inhibited calcium-induced calcium release instantaneously, but did not inhibit depolarization-induced calcium release. Use of pharmacological agents indicates that several types of calcium release operate in vitro. No significant differences were found between normal and dystrophic sarcoplasmic reticulum in calcium release kinetics or drug sensitivities.

Animals↗

PubMed1984

Decavanadate is responsible for vanadate-induced two-dimensional crystals in sarcoplasmic reticulum.

Two-dimensional protein crystals of the calcium pump protein of sarcoplasmic reticulum (SR) from fast skeletal muscle were induced using Na3VO3 as first described by Dux and Martonosi. These crystals exhibit repeat rows approximately 11 nm apart which contain discrete units with approximately 7 nm repeats. Four different methods of sample preparation for electron microscopy, i.e., negative staining, freeze-drying, freeze-fracturing, and thin-sectioning electron microscopy, each give complimentary repeat units. The SR-membrane crystals exhibit surface structure by the freeze-drying technique and row-like structures on the normally smooth outer face of normal SR. The formation of the membrane crystals is dependent on the pH and concentration of the vanadate. Only conditions favoring the presence of decavanadate yield crystals. At low concentrations and neutral pH, decavanadate is unstable and with time converts to smaller oligomers and the monomer. The presence of membrane crystals was correlated with the life span of the decavanadate. Membrane crystals were obtained in the SR membrane from fast twitch muscle from light and heavy SR, referable to longitudinal and terminal cisternae as well as from reconstituted SR. Canine cardiac SR did not crystallize under these conditions.

Animals↗

PubMed1984

Ionic requirements for taurocholate transport in rat liver plasma membrane vesicles.

As part of the enterohepatic circulation, taurocholate is taken up by hepatocytes by a Na+-gradient-dependent, carrier-mediated process. The dependence of taurocholate uptake on the presence of a Na+ gradient, outside greater than inside, has been studied in isolated rat liver plasma membranes. The uptake is specific for sodium, and a cotransport stoichiometry of 2 Na+ per taurocholate taken up was found. The presence of K+ ions inside the vesicles was also found to be essential for maximum Na+-stimulated uptake of taurocholate, although a K+ gradient is not required. Mg2+ was almost as effective as K+ in this regard. The symport of Na+ and taurocholate during uptake was shown to be electrogenic, so that K+ may act as an exchange counterion preventing the accumulation of positive charge within the vesicles.

Animals↗

PubMed1984

Phospholipid asymmetry in the isolated sarcoplasmic reticulum membrane.

The total phospholipid content and distribution of phospholipid species between the outer and inner monolayers of the isolated sarcoplasmic reticulum membrane was measured by phospholipase A2 activities and neutron diffraction. Phospholipase measurements showed that specific phospholipid species were asymmetric in their distribution between the outer and inner monolayers of the sarcoplasmic reticulum lipid bilayer; phosphatidylcholine (PC) was distributed 48/52 +/- 2% between the outer and inner monolayer of the sarcoplasmic reticulum bilayer, 69% of the phosphatidyl-ethanolamine (PE) resided mainly in the outer monolayer of the bilayer, 85% of the phosphatidylserine (PS) and 88% of the phosphatidylinositol (PI) were localized predominantly in the inner monolayer. The total phospholipid distribution determined by these measurements was 48/52 +/- 2% for the outer/inner monolayer of the sarcoplasmic reticulum lipid bilayer. Sarcoplasmic reticulum phospholipids were biosynthetically deuterated and exchanged into isolated vesicles with both a specific lecithin and a general exchange protein. Neutron diffraction measurements directly provided lipid distribution profiles for both PC and the total lipid content in the intact sarcoplasmic reticulum membrane. The outer/inner monolayer distribution for PC was 47/53 +/- 1%, in agreement with phospholipase measurements, while that for the total lipid was 46/54 +/- 1%, similar to the phospholipase measurements. These neutron diffraction results regarding the sarcoplasmic reticulum membrane bilayer were used in model calculations for decomposing the electron-density profile structure (10 A resolution) of isolated sarcoplasmic reticulum previously determined by X-ray diffraction into structures for the separate membrane components. These structure studies showed that the protein profile structure within the membrane lipid bilayer was asymmetric, complementary to the asymmetric lipid structure. Thus, the total phospholipid asymmetry obtained by two independent methods was small but consistent with a complementary asymmetric protein structure, and may be related to the highly vectorial functional properties of the calcium pump ATPase protein in the sarcoplasmic reticulum membrane.

Animals↗