Fatty acid effects on calcium influx and efflux in sarcoplasmic reticulum vesicles from rabbit skeletal muscle

Low concentrations of fatty acids inhibited initial Ca uptake by sarcoplasmic reticulum vesicles, the extent of inhibition varying with chain length and unsaturation in a series of C₁₄–C₂₀ fatty acids. Oleic acid was a more potent inhibitor of initial Ca uptake than stearic acid at 25°C, whereas at 5°C there was less difference between the inhibitory effects of low concentrations of these fatty acids. When the fatty acids were added later, during the phase of spontaneous Ca release that follows Ca uptake in reactions carried out at 25°C, 1–4 μM oleic and stearic acids caused Ca content to increase. This effect was due to marked inhibition of Ca efflux and slight stimulation of Ca influx. At concentrations of >4 μM, both fatty acids inhibited the Ca influx that occurs during spontaneous Ca release; in the case of oleic acid, this inhibition resembled that of initial Ca uptake at 5°C. The different effects of fatty acids at various times during Ca uptake reactions may be explained in part if alterations in the physical state of the membranes occur during the transition from the phase of initial Ca uptake to that of spontaneous Ca release.     

The Ca2+ permeability of sarcoplasmic reticulum vesicles II. Ca2+ efflux in the energized state of the calcium pump

Ca²⁺ efflux from sarcoplasmic reticulum vesicles was studied by measurements of net Ca²⁺ uptake, ⁴⁵Ca²⁺ flux and hydrolysis of energy-rich phosphate. The maximal Ca²⁺ uptake capacity (150–200 nmol/mg protein at pH 6.7, 10 mM MgCl₂ and μ=0.26) was independent of the nature and concentration of the energy-donating substrate (ATP or carbamyl phosphate) and of temperature (15–35°C), suggesting coupling between influx and efflux of Ca²⁺. In the presence of high concentrations of ATP, this efflux of Ca²⁺ was much higher than the passive Ca²⁺ permeation, measured after ATP or Ca²⁺ depletion of the reaction medium. Ca²⁺ efflux was imperceptible at vesicle filling levels below 35–40 nmol Ca²⁺/mg protein, and uncorrelated to the inhibition of the Ca²⁺-ATPase by high intravesicular Ca²⁺ concentrations. Analysis of the data indicated that Ca²⁺ efflux under our conditions probably is associated with one of the Ca²⁺-ATPase partial reactions occurring after dephosphorylation, rather than with a reversal of the Ca²⁺ translocation step in the phosphorylated state of the enzyme. Furthermore, passive Ca²⁺ permeation may be concurrently reduced during the enzymatically active state. It is proposed that both Ca²⁺ efflux and passive Ca²⁺ permeation (Ca²⁺ outflow) proceed via the same channels which are closed (occluded) during part of the Ca²⁺-ATPase reaction cycle.     

Lysophospholipid-mediated alterations in the calcium transport systems of skeletal and cardiac muscle sarcoplasmic reticulum

Summary The effects of various lysophospholipids on the calcium transport activity of sarcoplasmic reticulum (SR) from rabbit skeletal and canine cardiac muscles were examined. The lipids decreased calcium transport activity in both membrane types; the effectiveness being in the order lysoPC > lsyoPS, lysoPG > lysoPE. The maximum inhibition induced by lysoPC, lysoPG and lysoPS was greater than 85% of the normal Ca²⁺-transport rate. In cardiac SR lysoPE had a maximal inhibition of about 50%. Half maximal inhibition of calcium transport by lysoPC was achieved at 110 nmoles lysoPC/mg SR. At this concentration of lysoPC, the (Ca²⁺ + Mg²⁺)-ATPase and Ca²⁺-uptake activities were inhibited to the same extent (about 60%) in skeletal sarcoplasmic reticulum, while in cardiac sarcoplasmic reticulum, there was less than 20% inhibition of the Ca²⁺ + Mg²⁺-ATPase activity. Studies with EGTA-induced passive calcium efflux showed that up to 200 nmoles lysoPC/mg SR did not alter calcium permeability significantly in cardiac sarcoplasmic reticulum. In skeletal muscle membranes the lysophospholipid mediated decrease in calcium uptake correlated well with the increase in passive calcium efflux due to lysophosphatidylcholine. The difference in the lysophospholipid-induced effects on the sarcoplasmic reticulum from the two muscle types probably reflects variations in protein and other membrane components related to the respective calcium transport systems.     

Synthesis of the calcium transport ATPase of sarcoplasmic reticulum and other muscle proteins during development of muscle cells in vivo and in vitro

The effect of medium Ca²⁺ concentration upon the concentration and the rate of synthesis of muscle proteins was investigated in chicken pectoralis muscle cultures.There is an easily identifiable class of muscle protein which includes the Ca²⁺-ATPase of sarcoplasmic reticulum, myosin, troponin C, ATP : creatine phosphotransferase, muscle specific actin, tropomyosin 1 and 2, and muscle hemagglutinin, which show a large increase in concentration during normal development. The increased synthesis of these proteins was inhibited, without inhibition of cell proliferation, in culture media of relatively low Ca²⁺ concentration, 0.05–0.3 mM, where fusion was prevented. Similar medium Ca²⁺ concentration was required for the expression of all these proteins, suggesting their coordinate regulation. The proteins are denoted as ‘calcium-modulated proteins’. The increased Ca²⁺ transport activity of sarcoplasmic reticulum in cultured chicken pectoralis muscle cells during development at 1.8 mM medium calcium concentration represents de novo synthesis of the Ca²⁺ transport ATPase, as shown by immunoprecipitation, active site labeling and direct identification of the Ca²⁺ transport ATPase on two-dimensional gel electropherograms of whole muscle homogenates.The concentration and the turnover rate of the majority of the muscle proteins is not affected significantly by medium Ca²⁺ concentration between 0.06 and 1.8 mM.It is proposed that increase in cytoplasmic free Ca²⁺ concentration during fusion plays a central role in the regulation of the synthesis of calcium-modulated proteins.     

Inhibition of sarcoplasmic reticulum calcium pump by cytosolic protein(s) endogenous to heart and slow skeletal muscle but not fast skeletal muscle

Cytosol from rabbit heart and slow and fast skeletal muscles was fractionated using (NH₄)₂SO₄ to yield three cytosolic protein fractions, viz., CPF-I (protein precipitated at 30% saturation), CPF-II (protein precipitated between 30 and 60% saturation), and cytosol supernatant (protein soluble at 60% saturation). The protein fractions were dialysed and tested for their effects on ATP-dependent, oxalate-supported Ca²⁺ uptake by sarcoplasmic reticulum from heart and slow and fast skeletal muscles. CPF-I from heart and slow muscle, but not from fast muscle, caused marked inhibition (up to 95%) of Ca²⁺ uptake by sarcoplasmic reticulum from heart and from slow and fast muscles. Neither unfractionated cytosol nor CPF-II or cytosol supernatant from any of the muscles altered the Ca²⁺ uptake activity of sarcoplasmic reticulum. Studies on the characteristics of inhibition of sarcoplasmic reticulum Ca²⁺ uptake by CPF-I (from heart and slow muscle) revealed the following: (a) Inhibition was concentration- and temperature-dependent (50% inhibition with approx. 80 to 100 μg CPF-I; seen only at temperatures above 20°C). (b) The inhibitor reduced the velocity of Ca²⁺ uptake without appreciably influencing the apparent affinity of the transport system for Ca²⁺. (c) Inhibition was uncompetitive with respect to ATP. (d) Sarcoplasmic reticulum washed following exposure to CPF-I showed reduced rates of Ca²⁺ uptake, indicating that inhibition results from an interaction of the inhibitor with the sarcoplasmic reticulum membrane. (e) Concomitant with the inhibition of Ca²⁺ uptake, CPF-I also inhibited the Ca²⁺-ATPase activity of sarcoplasmic reticulum. (f) Heat-treatment of CPF-I led to loss of inhibitor activity, whereas exposure to trypsin appeared to enhance its inhibitory effect. (g) Addition of CPF-I to Ca²⁺-preloaded sarcoplasmic reticulum vesicles did not promote Ca²⁺ release from the vesicles. These results demonstrate the presence of a soluble protein inhibitor of sarcoplasmic reticulum Ca²⁺ pump in heart and slow skeletal muscle but not in fast skeletal muscle. The characteristics of the inhibitor and its apparently selective distribution suggest a potentially important role for it in the in vivo regulation of sarcoplasmic reticulum Ca²⁺ pump, and therefore in determining the duration of Ca²⁺ signal in slow-contracting muscle fibers.     

The ADP- and Mg2+-reactive calcium complex of the phosphoenzyme in skeletal sarcoplasmic reticulum Ca2+-ATPase

The effects of ATP on Ca²⁺ binding in the absence of added Mg²⁺ to the purified sarcoplasmic reticulum Ca²⁺-ATPase were studied at pH 7.0 and 0°C. ATP increased the number of Ca²⁺-binding sites of the enzyme from 2 to 3 mol per mol of phosphorylatable enzyme. The association constant for the ATP-induced Ca²⁺ binding was 4·10⁵ M^?1, which was not significantly different from that obtained in the absence of ATP. AdoP[CH₂]PP has little effect on the Ca²⁺-binding process. The amount of phosphoenzyme formed was equivalent to the level of ATP-induced Ca²⁺ binding. ADP decreased the level of ATP-induced Ca²⁺ binding and phosphoenzyme by the same amount. These results suggest that ATP-induced Ca²⁺ binding exists in the form of an ADP-reactive phosphoenzyme·Ca complex. In addition, the Ca²⁺ bound to the enzyme in the presence of ATP was released on the addition of 1 mM MgCl₂; after the release of Ca²⁺, the phosphoenzyme decayed. These observations suggest that Mg²⁺, added after the ATP-induced Ca²⁺-binding process, may replace the Ca²⁺ on the phosphoenzyme and initiate phosphoenzyme decomposition.     

Three-dimensional electron microscopy of the sarcoplasmic reticulum and T-system in embryonic chick skeletal muscle cells in vitro

Summary The formation of the sarcoplasmic reticulum (SR) and the transverse tubular system (T-system) in embryonic chick skeletal muscle cells in vitro was studied by either the critical point drying-physical rupturing or physical rupturing-freeze drying together with rotary shadowing. In these cells, two membranous systems were observed. One was composed of flattened sacs which were either isolated or were connected to each other with slender processes to form mostly longitudinally oriented strands. Initially, these sacs had small granules at their surface and were found mainly under the sarcolemma. Later, they became smooth at their surface, extending throughout the cytoplasm to form irregular and dense networks. At later phases, the networks tended to be disposed at right angle to nascent myofibrils, exhibiting a characteristic honeycomb appearance. From the similarities in thin section images, they were identified as developing SR.The other membranous system were tubules with many enlargements. They were frequently associated with coated vesicles which appeared to take part in the formation, elongation, and anastomosing of developing tubules. These tubules could be impregnated with a tannic acid-glutaraldehyde-potassium ferrocyanide complex and, thus, were identified as T-tubules.Abbreviations CPD critical-point drying - ES exoplasmic surface of the sarcolemma - FD freeze-drying - PR physical rupture - PS protoplasmic surface of the sarcolemma - SR sarcoplasmic reticulum - TAGPF tannic acid-glutaraldehyde-potassium ferrocyanide - T-system transverse tubular system     

Cholesterol distribution and structural differentiation in the sarcoplasmic reticulum of rat cardiac muscle cells

Summary The polyene compound, filipin, was used as a probe to localize cholesterol in the membranes of the rat cardiac muscle cell, with particular reference to the sarcoplasmic reticulum (SR). Filipin binds specifically to cholesterol (and related 3--hydroxysterols) in membranes, producing distinct deformations which can be viewed by freeze-fracture and used as markers for the presence of cholesterol-rich regions in the membrane plane. In freeze-fracture replicas of filipin-treated rat myocardium, the muscle cells revealed abundant deformations in their plasma membranes, no deformations in mitochondrial membranes, and an intermediate response in the SR. These results are in agreement with the levels of cholesterol reported in isolated fractions of the different membrane types, and confirm the specificity of filipin action. Within the SR, the filipin-induced deformations were not randomly distributed but occurred more commonly in free SR at or near the Z-region of the sarcomere than in other parts of the free SR or the junctional SR. This finding is interpreted as evidence for a non-homogeneous distribution of cholesterol in cardiac muscle cell SR. The possible significance of cholesterol in relation to structural differentiation and function of the SR is discussed.     

Daily changes in nitrate influx,efflux and metabolism in maize and pearl millet

Maize (Zea mays L.) and pearl millet (Pennisetum americanum (L.) Leeke) seedlings were exposed to [¹⁵N]nitrate for 1-h periods at eight times during a 24-h period (16–8 h light-dark for maize; 14–10 h for millet). Influx of [¹⁵N]nitrate as well as its reduction and translocation were determined during each period. The efflux of previously absorbed [¹⁴N]nitrate to the uptake solution was also estimated. No marked diurnal changes in [¹⁴N]nitrate efflux or [¹⁵N]nitrate influx were evident in maize. In contrast, [¹⁴N]nitrate efflux from millet increased and eventually exceeded [¹⁵N]nitrate influx during the late dark and early light periods, resulting in net nitrate efflux from the roots. The dissimilarity of their diurnal patterns indicates that influx and efflux are independently regulated. In both species, [¹⁵N]nitrate reduction and ¹⁵N translocation to shoots were curtailed more by darkness than was [¹⁵N]nitrate influx. In the light, maize reduced 15% and millet 24% of the incoming [¹⁵N]nitrate. In darkness, reduction dropped to 11 and 17%, respectively. Since the accumulation of reduced-¹⁵N in shoots declined abruptly in darkness, whereas that in roots was little affected, it is suggested that in darkness [¹⁵N]nitrate reduction occurred primarily in roots. The decrease in nitrate uptake and reduction in darkness was not related to efflux, which remained constant in maize and did not respond immediately to darkness in pearl millet.Paper No. 6722 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh     

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 vesicles 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 μM) 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.     

Characterization of sarcolemma and sarcoplasmic reticulum isolated from skeletal muscle of the freeze tolerant wood frog, Rana sylvatica: the beta(2)-adrenergic receptor and calcium transport systems in control, frozen and thawed states

In freeze tolerant wood frog Rana sylvatica, the freeze-induced liberation of glucose plays a critical role in survival in response to sub-zero temperature exposure. We have shown that the glycaemic response is linked to selective changes in the expression of hepatic adrenergic receptors through which catecholamines act to produce their hepatic glycogenolytic effects. The purpose of the present study was to determine if skeletal muscle, another catecholamine-sensitive tissue with glycogenolytic potential, displayed similar or different changes. In order to achieve these objectives, skeletal muscle derived from Rana sylvatica was studied in control, frozen and thawed states. In isolated sarcolemmal fractions, freezing effected an 88% decrease in beta(2)-adrenergic receptor expression but was without effect on the calcium pump; while thawing resulted in a recovery of the beta(2)-adrenergic receptor to 60% of control levels and a 2.4-fold increase in calcium transport. In isolated sarcoplasmic reticular fractions, freezing effected a 52% decrease in calcium binding and a 92% decrease in oxalate-stimulated calcium uptake; while thawing elicited partial normalization to control levels to 70% with respect to calcium binding and to 47% with respect to calcium uptake. Freezing and thawing were associated with increases and decreases, receptively, in blood glucose levels but were without effect on skeletal muscle glycogen content. Thus these muscle changes in Rana sylvatica in freezing and thawing are not linked to glycogen breakdown, are different from those previously seen in liver, and may provide a role in recovery of muscle function during thawing by protecting glycogen stores for contraction and maximizing extracellular calcium for excitation-contraction coupling in the frozen state. The involvement of thyroid hormone in triggering these muscle changes is discussed.     

The inside pH determines rates of electron and proton transfer in vesicle-reconstituted cytochrome c oxidase

Cytochrome c oxidase is the terminal enzyme in the respiratory chains of mitochondria and many bacteria where it translocates protons across a membrane thereby maintaining an electrochemical proton gradient. Results from earlier studies on detergent-solubilized cytochrome c oxidase have shown that individual reaction steps associated with proton pumping display pH-dependent kinetics. Here, we investigated the effect of pH on the kinetics of these reaction steps with membrane-reconstituted cytochrome c oxidase such that the pH was adjusted to different values on the inside and outside of the membrane. The results show that the pH on the inside of the membrane fully determines the kinetics of internal electron transfers that are linked to proton pumping. Thus, even though proton release is rate limiting for these reaction steps (Salomonsson et al., Proc. Natl. Acad. Sci. USA, 2005, 102, 17624), the transition kinetics is insensitive to the outside pH (in the range 6-9.5).     

Thymidine transport in cultured mammalian cells. Kinetic analysis,temperature dependence and specificity of the transport system

The transport of thymidine has been characterized kinetically and thermodynamically in Novikoff rat hepatoma cells grown in culture and, less extensively, in mouse L cells, Chinese hamster ovary cells, P388 murine leukemia cells and HeLa cells. That the characterizations pertained to the transport system per se was ensured, (i) by employing recently developed methods for rapid sampling of cell/substrate mixtures in order to follow isotope movements within a few seconds after initial exposure of cells to substrate; (ii) by utilizing cells rendered, by genetic or chemical means, incapable of metabolizing thymidine; and, (iii) by demonstrating conformity of the transport data to an integrated rate equation derived for a simple, carrier-mediated system. The results indicate that thymidine is transported into mammalian cells by a functionally symmetrical, non-concentrative system for which the carrier : substrate dissociation constant ranges from about 100 μM in Chinese hamster ovary cells, to 230 μM in Novikoff hepatoma cells. In all cell lines investigated, the velocity of transport was sufficient to nearly completely equilibrate low concentrations of thymidine across the membrane within 15 s. Temperature dependence of transport velocity and substrate : carrier dissociation were continuous ($\text{E}{}_{\mathrm{<mtext>A</mtext>}}\text{= 18.3}\text{kcal/mol}\text{, ΔH}{}^0\text{′ = 9.3}\text{kcal/mol}$, respectively), and showed no evidence of abrupt transitions. Several natural and artificial nucleosides and nucleic acid based inhibited influx of radiolabeled thymidine, apparently by competing with thymidine for the transport carrier.     

Regulatory and structural EF-hand motifs of neuronal calcium sensor-1: Mg 2+ modulates Ca 2+ binding, Ca 2+ -induced conformational changes, and equilibrium unfolding transitions

Neuronal calcium sensor-1 (NCS-1) is a major modulator of Ca²⁺ signaling with a known role in neurotransmitter release. NCS-1 has one cryptic (EF1) and three functional (EF2, EF3, and EF4) EF-hand motifs. However, it is not known which are the regulatory (Ca²⁺-specific) and structural (Ca²⁺- or Mg²⁺-binding) EF-hand motifs. To understand the specialized functions of NCS-1, identification of the ionic discrimination of the EF-hand sites is important. In this work, we determined the specificity of Ca²⁺ binding using NMR and EF-hand mutants. Ca²⁺ titration, as monitored by [¹⁵N,¹H] heteronuclear single quantum coherence, suggests that Ca²⁺ binds to the EF2 and EF3 almost simultaneously, followed by EF4. Our NMR data suggest that Mg²⁺ binds to EF2 and EF3, thereby classifying them as structural sites, whereas EF4 is a Ca²⁺-specific or regulatory site. This was further corroborated using an EF2/EF3-disabled mutant, which binds only Ca²⁺ and not Mg²⁺. Ca²⁺ binding induces conformational rearrangements in the protein by reversing Mg²⁺-induced changes in Trp fluorescence and surface hydrophobicity. In a larger physiological perspective, exchanging or replacing Mg²⁺ with Ca²⁺ reduces the Ca²⁺-binding affinity of NCS-1 from 90 nM to 440 nM, which would be advantageous to the molecule by facilitating reversibility to the Ca²⁺-free state. Although the equilibrium unfolding transitions of apo-NCS-1 and Mg²⁺-bound NCS-1 are similar, the early unfolding transitions of Ca²⁺-bound NCS-1 are partially influenced in the presence of Mg²⁺. This study demonstrates the importance of Mg²⁺ as a modulator of calcium homeostasis and active-state behavior of NCS-1.     

The unfolded protein response to endoplasmic reticulum stress in cultured astrocytes and rat brain during experimental diabetes

Oxidative-nitrosative stress and inflammatory responses are associated with endoplasmic reticulum (ER) stress in diabetic retinopathy, raising the possibility that disturbances in ER protein processing may contribute to CNS dysfunction in diabetics. Upregulation of the unfolded protein response (UPR) is a homeostatic response to accumulation of abnormal proteins in the ER, and the present study tested the hypothesis that the UPR is upregulated in two models for diabetes, cultured astrocytes grown in 25 mmol/L glucose for up to 4 weeks and brain of streptozotocin (STZ)-treated rats with diabetes for 1–7 months. Markers associated with translational blockade (phospho-eIF2α and apoptosis (CHOP), inflammatory response (inducible nitric oxide synthase, iNOS), and nitrosative stress (nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase, GAPDH) were not detected in either model. Nrf2 was present in nuclei of low- and high-glucose cultures, consistent with oxidative stress. Astrocytic ATF4 expression was not altered by culture glucose concentration, whereas phospho-IRE and ATF6 levels were higher in low- compared with high-glucose cultures. The glucose-regulated chaperones, GRP78 and GRP94, were also expressed at higher levels in low- than high-glucose cultures, probably due to recurrent glucose depletion between feeding cycles. In STZ-rat cerebral cortex, ATF4 level was transiently reduced at 4 months, and p-IRE levels were transiently elevated at 3 months. However, GRP78 and GRP94 expression was not upregulated, and iNOS, amyloid-β, and nuclear accumulation of GAPDH were not evident in STZ-diabetic brain. High-glucose cultured astrocytes and STZ-diabetic brain are relatively resistant to diabetes-induced ER stress, in sharp contrast with cultured retinal Müller cells and diabetic rodent retina.     

Flagellar hook structures of Caulobacter and Salmonella and their relationship to filament structure

Native flagellar hooks from a polarly flagellated bacterium, Caulobacter crescentus, and polyhooks from a peritrichously flagellated bacterium, Salmonella typhimurium. have been studied by densitometry of electron micrographs of negatively stained specimens, followed by computerized Fourier analysis and three-dimensional reconstruction. The two structures are remarkably similar. In both cases, the subunits are arranged along a right-handed basic helix of 2.3 nm pitch with successive subunits separated by an azimuthal angle of 64 to 65 °, and there is a pronounced system of continuous 6-start grooves and ridges on the surface of the structures. The subunit of Salmonella (M_r 42,000, versus 70,000 for Caulobacter) is somewhat thinner and yields a smaller overall hook diameter. The “bent finger” subunit shape and orientation in both cases suggests that the hook could bend readily by a sliding motion in the 11-start direction at inner radii, with the 6-start groove preventing collision at outer radii. The basic helical pitch of the Salmonella hook structure, and the number of subunits per basic helical turn (5.56) makes it highly compatible with the Salmonella flagellar filament (2.6 nm pitch. 5.51 subunits per turn); so also does the elongated shape and tilt angle of the hook and flagellin subunits in the respective structures. The two structures may therefore conjoin directly in the intact flagellum, although participation of a minor protein is not ruled out by the data.     

Crystal structure of the C-terminal domain of Mu phage central spike and functions of bound calcium ion

Bacteriophage Mu, which has a contractile tail, is one of the most famous genus of Myoviridae. It has a wide host range and is thought to contribute to horizontal gene transfer. The Myoviridae infection process is initiated by adhesion to the host surface. The phage then penetrates the host cell membrane using its tail to inject its genetic material into the host. In this penetration process, Myoviridae phages are proposed to puncture the membrane of the host cell using a central spike located beneath its baseplate. The central spike of the Mu phage is thought to be composed of gene 45 product (gp45), which has a significant sequence homology with the central spike of P2 phage (gpV). We determined the crystal structure of shortened Mu gp45Δ1-91 (Arg92–Gln197) at 1.5 Å resolution and showed that Mu gp45 is a needlelike structure that punctures the membrane. The apex of Mu gp45 and that of P2 gpV contained iron, chloride, and calcium ions. Although the C-terminal domain of Mu gp45 was sufficient for binding to the E. coli membrane, a mutant D188A, in which the Asp amino acid residue that coordinates the calcium ion was replaced by Ala, did not exhibit a propensity to bind to the membrane. Therefore, we concluded that calcium ion played an important role in interaction with the host cell membrane.