Non-invasive Providencia alcalifaciens strains fail to attach to HEp-2 cells.

We investigated the adherence properties of six P. alcalifaciens strains with previously characterized differential invasive capabilities in HEp-2 cells. Highly invasive strains were found to attach to HEp-2 cell monolayers within 2 h post-infection and in large numbers on the eukaryotic cell surfaces within 3 h post-infection. In contrast, weakly or non-invasive P. alcalifaciens strains were non-adherent to HEp-2 cells even at 3 h post-infection. Highly invasive isolates were found to weakly bind F-actin using the fluorescent actin staining assay although these strains were negative for Escherichia coli attachment and effacing gene (eaeA) of enteropathogenic E. coli (EPEC). These results suggest that the strain variation in the ability of P. alcalifaciens to invade HEp-2 cells previously noted by several investigators may be linked to expression of key adhesin(s) on the cell surface of invasive isolates.

Morphological, immunological and biochemical characterization of purified transverse tubule membranes isolated from rabbit skeletal muscle.

A microsomal fraction consisting of membranes of transverse tubule origin has been purified by a modification of the calcium-loading procedure initially described by Rosemblatt et al. (J Biol Chem 256:8140-8, 1981). Enzymatic analysis of this fraction shows an enrichment of the vesicles in the Mg++ ATPase (basal) activity characteristic of the T-tubules and an absent or very low Ca++-dependent-ATPase activity. Stereological analysis of freeze fracture replica of the membranes in the purified fraction indicates that they have a very low density of particles in their P faces and lack the structural manifestation of the caveolae typical of the sarcolemma. Immunological analysis performed with monoclonal antibodies prepared against purified T-tubule and sarcoplasmic reticulum membranes define some T-tubule specific antigens and confirm the morphological and biochemical data regarding the origin and purity of the T-tubule preparation.

Oligovanadate binding to sarcoplasmic reticulum ATPase. Evidence for substrate analogue behavior.

Solutions of vanadate were controlled through concentration and pH adjustment to give specific compositions of mono- and oligovanadates. By monitoring the EPR spectrum of iodoacetamide spin-labeled ATPase, it is shown that decavanadate and the oligovanadate species present at neutral pH exhibit behavior typical of a substrate analogue. This is seen in terms of Ca2+ binding site affinity (microM), outward Ca2+ site orientation, and conformational effects on the enzyme normally associated with enzyme activation. In contrast, monovanadates exhibit behavior identical to that observed with Pi, with one exception: the vanadoenzyme is stable to Ca2+ in the concentration range of high affinity binding at the vanadate concentrations used here (200 microM). It is further demonstrated that Ca2+ binding in the 100 microM range directly induces enzyme devanadation of the monovanadate enzyme complex through Ca2+ binding to internal sites. Extensive array formation of dimeric ATPase units is found only with decavanadate in the absence of Ca2+, and then stoichiometric amounts are sufficient. Electron micrographs of dimeric arrays show evidence of increased penetration into the lipid bilayer, including freeze-fracture replicas which show evidence of corresponding "pits" in the inner leaflet of the bilayer. In turn, EPR spectra provide a means of following vanadate binding to the ATPase per se, as well as monitoring Ca2+-induced changes in the vanadoenzyme conformation, as only binding to specific sites on the enzyme affect the EPR spectrum.

Calcium-ryanodine receptor complex. Solubilization and partial characterization from skeletal muscle junctional sarcoplasmic reticulum vesicles.

The Ca2+-ryanodine receptor complex is solubilized in functional form on treating sarcoplasmic reticulum (SR) vesicles from rabbit fast skeletal muscle with 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS) (1 mg/mg protein) and 1 M NaCl at pH 7.1 by shaking for 30 min at 5 degrees C. The heavy membrane preparations obtained from pyrophosphate homogenates frequently exhibit junctional feet and appear to be derived primarily from the terminal cisternae of the SR. The characteristics of [3H]ryanodine binding are similar for the soluble receptor and the heavy SR vesicles with respect to dependence on Ca2+, pharmacological specificity for inhibition by six ryanoids and ruthenium red, and lack of sensitivity to voltage-dependent Ca2+-channel blockers, inositol 1,4,5-trisphosphate, or doxorubicin. In contrast, the cation sensitivity is decreased on receptor solubilization. The soluble receptor is modulated by cyclic nucleotides and rapidly denatured at 50 degrees C. Saturation experiments reveal a single class of receptors (Kd = 9.6 nM), whereas kinetic measurements yield a calculated association constant of 5.5 X 10(6) min-1 M-1 and a dissociation constant of 5.7 X 10(-4) min-1, suggesting that the [3H]ryanodine receptor complex ages with time to a state which is recalcitrant to dissociation. Sepharose chromatography shows that the receptor complex consists primarily of two protein fractions, one of apparent Mr 150,000-300,000 and a second, the [3H]ryanodine binding component, of approximately Mr 1.2 X 10(6). Preliminary analysis of the soluble receptor preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals subunits of Mr greater than 200,000 and major bands of calsequestrin and Ca2+-transport ATPase. These findings indicate that [3H]ryanodine binds to the Ca2+-induced open state of the channel involved in the release of contractile Ca2+.

High-affinity and low-affinity vanadate binding to sarcoplasmic reticulum Ca2+-ATPase labeled with fluorescein isothiocyanate.

Conditions were found that allowed both the fluorescence detection of vanadate binding to the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum and the vanadate-induced formation of two-dimensional arrays of the enzyme. The fluorescence intensity of fluorescein isothiocyanate-labeled Ca2+-ATPase increased with high-affinity vanadate binding (Ka = 10(6) M-1) as reported by Pick and Karlish (Pick, U. and Karlish, S.D. (1982) J. Biol. Chem. 257, 6120-6126). The Ca2+ and Mg2+ dependencies for high-affinity vanadate binding were similar but not identical to those for orthophosphate. In addition, it was found that there is low-affinity (Ka = 380 M-1) vanadate binding, which causes a 25% decrease in fluorescence. The Ca2+ and Mg2+ dependencies of the low-affinity vanadate binding were different from those of orthophosphate or high-affinity vanadate binding. The covalent attachment of fluorescein isothiocyanate (FITC) in the ATP site of the Ca2+-ATPase did not affect the formation of two-dimensional arrays, as detected by negatively stained electron micrographs. Vanadate concentrations high enough to saturate the low-affinity binding caused two-dimensional arrays as reported by Dux and Martonosi (Dux, L. and Martonosi, A. (1983) J. Biol. Chem. 258, 2599-2603). In addition, freeze-fracture replicas of quick-frozen specimens showed rows of indentations in the inner leaflet of the bilayer that corresponds to the arrays seen on the outer leaflet. This appearance of indentations suggests that low-affinity vanadate binding causes a transmembrane movement of the Ca2+-ATPase. By contrast, high-affinity vanadate binding was shown to cause neither array formation nor the appearance of indentations.

Electron microscopic evidence for the transmembrane displacement of calcium ATPase.

Incubation of the Ca2+-ATPase in vanadate solutions leads to the formation of two-dimensional arrays in the sarcoplasmic reticulum membrane. Electron micrographic freeze fracture replicas show depressions on the inner leaflet for the first time. This indicates that the ATPase has moved perpendicular to the plane of the membrane. Our results also suggest that aggregation of the Ca2+-ATPase into the two-dimensional arrays occurs before they move into the membrane. These phenomena were observed as soon as 15 minutes after vanadate was added. The effects of vanadate appear to be completely reversible. When SR was incubated in the vanadate solutions and was then diluted into a buffer containing Ca2+ and ATP, the ATPase activity was normal for up to several hours of incubation and only somewhat reduced after 3 days.

The effect of trifluoroperazine on the sarcoplasmic reticulum membrane.

The inhibitory effect of trifluoroperazine (25-200 microM) on the sarcoplasmic reticulum calcium pump was studied in sarcoplasmic reticulum vesicles isolated from skeletal muscle. It was found that the lowest effective concentrations of trifluoroperazine (10 microM) displaces the Ca2+ dependence of sarcoplasmic reticulum ATPase to higher Ca2+ concentrations. Higher trifluoroperazine concentrations (100 microM) inhibit the enzyme even at saturating Ca2+. If trifluoroperazine is added to vesicles filled with calcium in the presence of ATP, inhibition of the catalytic cycle is accompanied by rapid release of accumulated calcium. ATPase inhibition and calcium release are produced by identical concentrations of trifluoroperazine and, most likely, by the same enzyme perturbation. These effects are related to partition of trifluoroperazine ino the sarcoplasmic reticulum membrane, and consequent alteration of the enzyme assembly within the membrane structure, and of the bilayer surface properties. The effect of trifluoroperazine was also studied on dissociated ('chemically skinned') cardiac cells undergoing phasic contractile activity which is totally dependent on calcium uptake and release by sarcoplasmic reticulum, and is not influenced by inhibitors of slow calcium channels. It was found that trifluoroperazine interferes with calcium transport by sarcoplasmic reticulum in situ, as well as with the role of sarcoplasmic reticulum in contractile activation.

III. Three-dimensional electron microscopy of mammalian cardiac sarcoplasmic reticulum at 80 kV.

The Golgi black reaction method was combined with stereoscopic techniques to obtain three-dimensional views of cardiac sarcoplasmic reticulum (SR) using a conventional electron microscope operating at 80 kV. We have previously described the SR in avian and mammalian skeletal muscles with similar techniques. It was necessary to modify these earlier techniques for cardiac muscle. Two regions of mammalian heart were explored: trabecular and papillary muscles. These muscles presented striking differences with regard to relative volume of mitochondria and myofibrils, but both muscles presented similar dispositions of the inner tubules of SR. The SR near myofibrils appeared heterogeneous and consisted of fenestrated collar, bulbous extensions at the Z line (corbular SR), and flat extended regions (cisternal SR). The SR near mitochondria, however, always formed a simple rete with occasional cisternal SR. Specific "staining" of the inner tubules of cardiac SR by the Golgi method offers new views of cardiac fibers that suggest a more extensively developed SR than previously acknowledged.

The sarcoplasmic reticulum of an ultrafast lobster muscle: first evidence of a tubular configuration.

The sarcoplasmic reticulum (SR) of the remarkably fast remotor muscle of lobster second antenna was investigated with regard to ultrastructure, calcium uptake and protein composition. The SR of this unique muscle dominates the volume of the cell. We were able to preserve the configuration of the SR in its native state simply by processing the muscle for electron microscopy at 0-4 degree C. For the first time the SR is seen as larger (0.1 micrometer diam.) tubules that crowd the space between myofibrils, Previous observations of this organelle have been reported that showed a rather unsatisfying discontinuous vesicular configuration of the SR. This report indicates that these structures were fixation artifacts. The uptake of calcium measured in a microsomal subfraction in the presence of ATP and oxalate does not differ significantly from similar experiments with vertebrate SR. However, gel electrophoresis of lobster SR demonstrate a single intrinsic Ca-ATPase protein of 100,000 mol, wt. with neither of the typical lower molecular weight proteins found in vertebrate SR.

Stereological analysis of freeze-fracture subfractions from skeletal muscle. I. Relative intrinsic protein. II. Relative lipid content and protein-to-lipid ratio.

Standard microsomal subfractions from biological tissues are not homogeneous but mixtures of membranes derived from the various cellular organelles. In the case of skeletal muscle, freeze-fracture replicas show both smooth concave faces and concave faces densely populated with 90-A particles. Stereological sampling techniques have been applied to such replicas and the relative surface area of sarcoplasmic reticulum (SR) membrane calculated. Expressions are derived that estimate the relative fraction of SR intrinsic protein and lipid as a function of the relative surface area. Although most of the protein in our subfraction is SR protein, a significant amount of lipid is non-SR lipid. The effect of this on measurements of the protein-to-lipid ratio is discussed.

Microsomal T system: a stereological analysis of purified microsomes derived from normal and dystrophic skeletal muscle.

Heterogeneous populations of microsomes obtained from normal and dystrophic chicken pectoralis muscle were separated into two subfractions by an iterative loading technique. The buoyant density of the sarcoplasmic reticulum (SR) microsomes was increased after loading them with calcium oxalate. Several incubations in the transport medium were necessary to load all of the SR. The fraction that did not form a pellet contained microsomes which displayed freeze-fracture faces that had a low density of particles. A stereological analysis was used on membrane fracture faces of intact muscle to generate reference particle density distributions, which were compared with the distributions measured on the microsomal fracture faces. The concave microsomal fracture faces of purified microsomes which did not load calcium oxalate had particle distributions nearly identical to the distributions of intact P-face T tubules. The morphological data suggest that this subfraction is microsomal T system. Biochemical measurements show negligible amounts of specific Na+, K+-ATPase activity, suggesting that there was little contamination from the surface membrane in this subfraction. Furthermore, an active Ca2+-ATPase is demonstrated in both normal and dystrophic T-tubular membranes.