Absence of a characteristic cell wall lipopolysaccharide in the phototrophic bacterium Chloroflexus aurantiacus.

Two strains of the gliding phototrophic bacterium Chloroflexus aurantiacus were investigated for the presence of lipopolysaccharide (LPS). With both strains, all fractions of hot phenol-water extracts and the extracted cell residues from whole cells or cell homogenates were found to be free from characteristic LPS constituents, such as 3-hydroxy fatty acids, 2-keto-3-deoxyoctonate, heptoses, or O-chain sugars. Phenolchloroform-petroleum ether extracts were also free from precipitable LPS. A lipid A fraction could not be obtained, and there was no hint for glucosamine as a possible lipid A backbone amino sugar. Absence of LPS was confirmed by sodium deoxycholate gel electrophoresis.     

Measuring electric impedance of organs--methodologic principles]

Ischemia causes changes in organ tissue (e.g. during operation or transplantation) which may finally lead to irreversible injury, so that the organ can no longer be resuscitated. To the extent that these changes affect the electrical properties of the tissue they are manifested in the impedance spectrum. As an example, the course of impedance of a HTK-protected porcine liver is presented in the frequency range of 0.1 Hz to 10 MHz, which includes two dispersion--alpha- and beta-dispersion. Using a suitable electrical equivalent circuit analogue to the structure of the liver, the behavior of the alpha- and beta-dispersion is explained on the basis of gap junction closure and narrowing of the extracellular space due to cell swelling.     

Calmodulin activation and inhibition of skeletal muscle Ca2+ release channel (ryanodine receptor).

The calmodulin-binding properties of the rabbit skeletal muscle Ca2+ release channel (ryanodine receptor) and the channel's regulation by calmodulin were determined at < or = 0.1 microM and micromolar to millimolar Ca2+ concentrations. [125I]Calmodulin and [3H]ryanodine binding to sarcoplasmic reticulum (SR) vesicles and purified Ca2+ release channel preparations indicated that the large (2200 kDa) Ca2+ release channel complex binds with high affinity (KD = 5-25 nM) 16 calmodulins at < or = 0.1 microM Ca2+ and 4 calmodulins at 100 microM Ca2+. Calmodulin-binding affinity to the channel showed a broad maximum at pH 6.8 and was highest at 0.15 M KCl at both < or = 0.1 MicroM and 100 microM Ca2+. Under condition closely related to those during muscle contraction and relaxation, the half-times of calmodulin dissociation and binding were 50 +/- 20 s and 30 +/- 10 min, respectively. SR vesicle-45Ca2+ flux, single-channel, and [3H]ryanodine bind measurements showed that, at < or = 0.2 microM Ca2+, calmodulin activated the Ca2+ release channel severalfold. Ar micromolar to millimolar Ca2+ concentrations, calmodulin inhibited the Ca(2+)-activated channel severalfold. Hill coefficients of approximately 1.3 suggested no or only weak cooperative activation and inhibition of Ca2+ release channel activity by calmodulin. These results suggest a role for calmodulin in modulating SR Ca2+ release in skeletal muscle at both resting and elevated Ca2+ concentrations.     

Isolation of sarcoplasmic reticulum by zonal centrifugation and purification of Ca2+-pump and Ca2+-binding proteins

A procedure for the isolation of highly purified sarcoplasmic reticulum vesicles from rabbit skeletal muscle has been described using sucrose gradient centrifugation in zonal rotors. The yield of our purest fraction was 300 mg of sarcoplasmic reticulum protein using 1 kg muscle. The sarcoplasmic reticulum vesicles were relatively simple in composition. The Ca²⁺-pump protein accounted for most (approx. two-thirds) of the sarcoplasmic reticulum protein. Two other protein components, a Ca²⁺-binding protein and a M₅₅ protein (approx. 55 000 daltons) each accounted for about 5–10% of the protein. Enrichment in the level of phosphoenzyme by the Ca²⁺-pump protein was regarded as an important index of the purification of sarcoplasmic reticulum vesicles. The sarcoplasmic reticulum vesicles were capable of forming 6.4 nmoles of ³²P-labelled phosphoenzyme per mg protein and had a high capacity of energized Ca²⁺ uptake. The Ca²⁺-dependent formation of phosphoenzyme has been used to estimate the sarcoplasmic reticulum protein content in rabbit skeletal muscle and found to be about 2.5% of the total muscle protein.The Ca²⁺-pump and Ca²⁺-binding proteins were isolated with a purity of 90% or more by treating the purified sarcoplasmic reticulum vesicles with bile acids in the presence of salt. The solubilized Ca²⁺-pump protein reaggregated during dialysis together with phospholipid to form membranous vesicles which were capable of forming approx. 9 nmoles ³²P-labelled phosphoenzyme per mg protein. The Ca²⁺-binding protein was water soluble and contained a high percentage of acidic amino acids (35% of total residues).Ca²⁺ binding by sarcoplasmic reticulum vesicles and by the Ca²⁺-pump and Ca²⁺-binding proteins was studied by equilibrium dialysis. Sarcoplasmic reticulum vesicles and Ca²⁺-pump protein contained nonspecific high-affinity Ca²⁺ binding sites with a capacity of 90–100 and 55–70 nmoles Ca²⁺ per mg protein, respectively. Both of them specifically bound 10–15 nmoles Ca²⁺ per mg protein. The binding constants for nonspecific and specific Ca²⁺ binding by both preparations were approx. 1 μM^?1. The Ca²⁺-binding protein nonspecifically bound 900–1000 nmoles Ca²⁺ per mg protein with a binding constant of about 0.25 μM^?1.     

Ca 2+ uptake in reconstituted sarcoplasmic reticulum vesicles

The reconstitution of functional sarcoplasmic reticulum vesicles capable of Ca²⁺ transport has been achieved. Sarcoplasmic reticulum vesicles are first solubilized with deoxycholate and then reassembled into membranous vesicles by removal of the detergent using dialysis. The Ca²⁺ pump protein can, by itself, be reconstituted to form membranous vesicles capable of energized Ca²⁺ binding and uptake. The lipid content of the reconstituted vesicles is about the same as that of the original sarcoplasmic reticulum vesicles. The reconstituted vesicles have an elevated ATPase activity. Ca²⁺ binding and uptake in the presence of ATP are restored to about 25% and 50%, respectively.     

Sodium-calcium ion exchange in skeletal muscle sarcolemmal vesicles

Summary The Ca²⁺ permeability of rabbit skeletal muscle sarcolemmal vesicles was investigated by means of radioisotope flux measurements. A membrane vesicle fraction highly enriched in sarcolemma, as revealed by enzymatic markers, was obtained from the 22–27% region of sucrose gradients after isopycnic centrifugation. The ability of sarcolemmal vesicles to exchange Na⁺ for Ca²⁺ was investigated by measuring Ca²⁺ influx into and efflux from sarcolemmal vesicles in the presence and absence of a Na⁺ gradient. It was found that Ca²⁺ movements were enhanced in the direction of the higher Na⁺ concentration. When intra- and extravesicular Na⁺ concentrations were high, Na⁺–Na⁺ exchange predominated and Na⁺–Ca²⁺ exchange was low or absent. The presence of the Ca²⁺ ionophore A23187 in the dilution medium resulted in the rapid release of Ca²⁺ and the elimination of the Na⁺-enhanced efflux of Ca²⁺, suggesting that internal rather than bound external Ca²⁺ was exchanged with Na⁺. La³⁺ abolished Na⁺–Ca²⁺ exchange and decreased overall membrane permeability. Na⁺–Ca²⁺ exchange was not due to sarcoplasmic reticulum or mitochondrial contaminants. This investigation suggests that skeletal muscle, like cardiac muscle and neurons, is capable of a transmembranous Na⁺–Ca²⁺ exchange.     

Evidence for a K+, Na+ permeable channel in sarcoplasmic reticulum

Summary Potassium and sodium cation permeabilities of skeletal sarcoplasmic reticulum vesicles were characterized by means of³H-choline,²²Na⁺ and⁸⁶Rb⁺ isotope efflux and membrane potential measurements. Membrane potentials were generated by diluting K gluconate filled sarcoplasmic reticulum vesicles and liposomes into Tris or Na gluconate media, in the presence or absence of valinomycin, and were measured using the voltage-sensitive membrane probe 3,3-dipentyl-2,2-oxacarbocyanine. About 2/3 of the sarcoplasmic reticulum vesicles, designated Type I, were found to be permeable to Rb⁺, K⁺ and Na⁺. The remaining 1/3, Type II vesicles, were essentially impermeable to these ions. The two types of vesicles were impermeable to larger cations such as choline or Tris. Both were present in about the same ratio in fractions derived from different parts of the reticulum structure. Studies with cations of different size and shape suggested that in Type I vesicles permeation was restricted to molecules fitting through a pore with a cross-section of 4–5 Å by 6 Å or more. When vesicles were sonicated, vesicles permeable to K⁺ decreased more than those impermeable to K⁺. These data suggest the existence of K⁺, Na⁺ permeable channels which are probably randomly dispersed in the intact reticulum structure at an estimated density of 50 pores/m². The function of the channel may be to allow rapid K⁺ movement to counter Ca²⁺ fluxes during muscle contraction and relaxation.     

The muscle ryanodine receptor and its intrinsic Ca2+ channel activity

In skeletal and cardiac muscle, contraction is initiated by the rapid release of Ca²⁺ ions from the intracellular membrane system, sarcoplasmic reticulum. Rapid-mixing vesicle ion flux and planar lipid bilayer-single-channel measurements have shown that Ca²⁺ release is mediated by a high-conductance, ligand-gated Ca²⁺ channel. Using the Ca²⁺ release-specific probe ryanodine, a 30 S protein complex composed of four polypeptides ofM _r 400,000 has been isolated. Reconstitution of the purified skeletal and cardiac muscle 30 S complexes into planar lipid bilayers induced single Ca²⁺ channel currents with conductance and gating kinetics similar to those of native Ca²⁺ release channels. Electron microscopy revealed structural similarity with the protein bridges (feet) that span the transverse-tubule-sarcoplasmic reticulum junction. These results suggest that striated muscle contains an intracellular Ca²⁺ release channel that is identical with the ryanodine receptor and the transverse-tubule-sarcoplasmic reticulum spanning feet structures.     

Multiple conductance states of the purified calcium release channel complex from skeletal sarcoplasmic reticulum.

The CHAPS-solubilized and purified 30S ryanodine receptor protein complex from skeletal sarcoplasmic reticulum (SR) was incorporated into planar lipid bilayers. The resulting electrical activity displayed similar responses to agents such as Ca2+, ATP, ryanodine, or caffeine as the native Ca2+ release channel, confirming the identification of the 30S complex as the Ca2+ release channel. The purified channel was permeable to monovalent ions such as Na+, with the permeability ratio PCa/PNa approximately 5, and was highly selective for cations over anions. The purified channel also showed at least four distinct conductance levels for both Na+ and Ca2+ conducting ions, with the major subconducting level in NaCl buffers possessing half the conductance value of the main conductance state. These levels may be produced by intrinsic subconductances present within the channel oligomer. Several of these conductances may be cooperatively coupled to produce the characteristic 100 +/- 10 pS unitary Ca2+ conductance of the native channel.