The localization of calcium by X-ray microanalysis in myopathic muscle fibers

An electron histochemical study was undertaken to localize calcium with ammonium oxalate precipitation technique in soleus muscle of rat in normal cases and in myopathy induced experimentally by a prolonged treatment of 2,4-dichlorophenoxyacetate (2,4-D). The calcium content of precipitates was detected by energy-dispersive X-ray microanalysis. In normal cases, the electron dense precipitates containing calcium were mainly found in the vesicles of sarcoplasmic reticulum, whereas in 2,4-D induced myopathy the deposits were shifted near the Z line into the myofibrils. Calcium, because the uptake into sarcoplasmic vesicles was inhibited by 2,4-D, could attach to other binding sites, such as to the troponin-C.A long-lasting binding of calcium might lead to a prolonged activation of the actin-myosin system.     

Enhanced Ca2+-induced calcium release by isolated sarcoplasmic reticulum vesicles from malignant hyperthermia susceptible pig muscle

To further define the possible involvement of sarcoplasmic reticulum calcium accumulation and release in the skeletal muscle disorder malignant hyperthermia (MH), we have examined various properties of sarcoplasmic reticulum fractions isolated from normal and MH-susceptible pig muscle. A sarcoplasmic reticulum preparation enriched in vesicles derived from the terminal cisternae, was further fractionated on discontinuous sucrose density gradients (Meissner, G. (1984) J. Biol. Chem. 259, 2365-2374). The resultant MH-susceptible and normal sarcoplasmic reticulum fractions, designated F0-F4, did not differ in yield, cholesterol and phospholipid content, or nitrendipine binding capacity. Calcium accumulation (0.27 mumol Ca/mg per min at 22 degrees C), Ca2+-ATPase activity (0.98 mumol Pi/mg per min at 22 degrees C), and calsequestrin content were also similar for MH-susceptible and normal sarcoplasmic reticulum fraction F3. To examine sarcoplasmic reticulum calcium release, fraction F3 vesicles were passively loaded with 45Ca (approx. 40 nmol Ca/mg), and rapidly diluted into a medium of defined Ca2+ concentration. Upon dilution into 1 microM Ca2+, the extent of Ca2+-dependent calcium release measured after 5 s was significantly greater for MH-susceptible than for normal sarcoplasmic reticulum, 65.9 +/- 2.8% vs. 47.7 +/- 3.9% of the loaded calcium, respectively. The C1/2 for Ca2+ stimulation of this calcium release (5 s value) from MH-susceptible sarcoplasmic reticulum also appeared to be shifted towards a higher Ca2+-sensitivity when compared to normal sarcoplasmic reticulum. Dantrolene had no effect on calcium release from fraction F3, however, halothane (0.1-0.5 mM) increased the extent of calcium release (5 s) similarly in both MH-susceptible and normal sarcoplasmic reticulum. Furthermore, Mg2+ was less effective at inhibiting, while ATP and caffeine were more effective in stimulating, this Ca2+-dependent release of calcium from MH-susceptible, when compared to normal sarcoplasmic reticulum. Our results demonstrate that while sarcoplasmic reticulum calcium-accumulation appears unaffected in MH, aspect(s) of the sarcoplasmic reticulum Ca2+-induced calcium release mechanism are altered. Although the role of the Ca2+-induced calcium release mechanism of sarcoplasmic reticulum in situ is not yet clear, our results suggest that an abnormality in the regulation of sarcoplasmic reticulum calcium release may play an important role in the MH syndrome.     

A morphological and histological comparison of the initiation and development of pecan (Carya illinoinensis) somatic embryogenic cultures induced with naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid

Summary Somatic embryos produced in vitro may exhibit structural abnormalities that affect their subsequent germination and conversion into plants. To assess the influence of auxin type on embryo initiation and development, a morphological and histological comparison was made of pecan (Carya illinoinensis) somatic embryogenic cultures induced on media with naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid (2,4-D), using light and scanning electron microscopy. Both auxins promoted enhanced cell division, particularly in subepidermal cell layers. However, notable differences were observed in mitotic activity, location of embryogenic cell proliferation, epidermal continuity, callus growth, and embryo morphology. Cultures induced on naphthaleneacetic acid had embryogenic regions composed of homogeneous, isodiametric, meristematic cells. Embryos derived from these cultures generally had a normal morphology, were single, and had a discrete apical meristem. In contrast, tissues induced on media with 2,4-D had more intense and heterogeneous regions of cell division. Proliferating cell regions were composed of meristematic cells interspersed with callus and involved more extensive regions of the mesophyll. Marked callus proliferation caused epidermal rupture in some areas. Embryos induced on medium with 2,4-D had a higher incidence of abnormalities that included fasciated, fan-shaped, and tubular embryos. Defined apical meristems were often lacking or partially obliterated due to callus proliferation. The heterogeneous, often intensive proliferation of cells in cultures induced with 2,4-D may interfere with normal patterns of embryo development.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - NAA naphthaleneacetic acid - SEM scanning electron microscopy     

Effect of thymol on calcium handling in mammalian ventricular myocardium

Concentration-dependent effects of thymol on calcium handling were studied in canine and guinea pig cardiac preparations (Langendorff-perfused guinea pig hearts, canine ventricular trabeculae, canine sarcoplasmic reticular vesicles and single ryanodine receptors). Thymol induced a concentration-dependent negative inotropic action in both canine and guinea pig preparations (EC(50) = 297 +/- 12 microM in dog). However, low concentrations of thymol reduced intracellular calcium transients in guinea pig hearts without decreasing contractility. At higher concentrations both calcium transients and contractions were suppressed. In canine sarcoplasmic reticular vesicles thymol induced rapid release of calcium (V(max) = 0.47 +/- 0.04 nmol s(-1), EC(50) = 258 +/- 21 microM, Hill coefficient = 3.0 +/- 0.54), and decreased the activity of the calcium pump (EC(50) = 253 +/- 4.7 microM, Hill coefficient = 1.62 +/- 0.05). Due to the less sharp concentration-dependence of the ATPase inhibition, this effect was significant from 50 microM, whereas the thymol-induced calcium release only from 100 microM. In single ryanodine receptors incorporated into artificial lipid bilayer thymol induced long lasting openings, having mean open times increased with 3 orders of magnitude, however, the specific conductance of the channel remained unaltered. This effect of thymol was not voltage-dependent and failed to prevent the binding of ryanodine. In conclusion, the negative inotropic action of thymol can be explained by reduction in calcium content of the sarcoplasmic reticulum due to the combination of the thymol-induced calcium release and inhibition of the calcium pump. The calcium-sensitizer effect, observed at lower thymol concentrations, indicates that thymol is likely to interact with the contractile machinery also.     

Regulatory mechanism of contraction in the proboscis retractor muscle of a sipunculid worm,Phascolosoma scolops

Summary Regulatory mechanism of contraction in the proboscis retractor muscle of Phascolosoma scolops was studied by physiological measurements and cytochemical electron microscopy. The magnitude of K⁺-contracture was dependent on external Ca²⁺ concentration and the contracture disappeared in Ca²⁺-free solution. The K⁺-contracture was suppressed by application of procaine and Mn²⁺. Caffeine induced contracture even when external Ca²⁺ was absent. Ultrastructural observations of the retractor muscle cells showed the presence of a large number of vesicles (subsarcolemmal vesicles), corresponding to the sarcoplasmic reticulum in vertebrate skeletal muscle, underneath the plasma membrane. For the cytochemical electron microscopy, the muscle fibers were fixed with 1% OsO₄ solution containing 2% K-pyroantimonate. In the relaxed fibers, pyroantimonate precipitates were localized along the inner surface of plasma membrane and in the subsarcolemmal vesicles. In the contracting fibers, the precipitates were uniformly distributed in the myoplasm. The X-ray microanalysis revealed that the precipitates contained Ca. These results suggest that the contractile system is activated by the influx of extracellular Ca²⁺ as well as by the release of Ca²⁺ from the intracellular structures such as the inner surface of the plasma membrane and subsarcolemmal vesicles.     

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.     

Further characterization of light and heavy sarcoplasmic reticulum vesicles. Identification of the ‘sarcoplasmic reticulum feet’ associated with heavy sarcoplasmic reticulum vesicles

Light and heavy sarcoplasmic reticulum vesicles were isolated from rabbit leg muscle using a combination of differential centrifugation and isophycnic zonal ultracentrifugation. Light sarcoplasmic reticulum vesicles obtained from the 30–32.5% and heavy sarcoplasmic reticulum vesicles obtained from the 38.5–42% sucrose regions of the linear sucrose gradient were determined to be free of surface and mitochondrial membrane contamination by marker enzyme analysis and electron microscopy. Thin sections of the light vesicles revealed empty vesicles of various sizes and shapes. Freeze-fracture replicas of the light vesicles showed an asymmetric distribution of intramembranous particles with the same orientation and distribution as the longitudinal sarcoplasmic reticulum in vivo. Heavy vesicles appeared as rounded vesicles of uniform size filled with electron dense material, similar to that seen in the terminal cisternae of the sarcoplasmic reticulum. The cytoplasmic surface of the membrane was decorated by membrane projections, closely resembling the ‘feet’ which join the sarcoplasmic reticulum to the transverse tubules in the intact muscle fiber. Freeze-fracture replicas of the heavy vesicles revealed an asymmetric distribution of particles which in some areas of the vesicle's surface are larger and less densely aggregated than those of the light vesicles. In the best quality replicas, some regions of the luminal leaflet were not smooth but showed evidence of pits. These structural details are characteristic of the area of sarcoplasmic reticulum membrane which is covered by the ‘feet’ in the intact muscle.Heavy vesicles contained greater than six times the calcium content of light vesicles, 54 vs. 9 nmol Ca²⁺/μl of water space. After KCl washing both contained less than 4 nmol Ca²⁺/μl of water space. Although they transported at the same rate and the same total amount of calcium, the rate of passive Ca²⁺ efflux from the heavy vesicles was double that of light vesicles. The higher rate of calcium efflux from the heavy vesicles was inhibited by dantrolene, an inhibitor of Ca²⁺ release. High resolution sodium dodecyl sulfate gel electrophoresis showed that the light vesicles contained predominantly Ca²⁺-ATPase along with several approx. 55 000-dalton proteins and a 5000-dalton proteolipid, while the heavy vesicles contained Ca²⁺-ATPase and calsequestrin along with several approx. 55 000-dalton proteins, extrinsic 34 000- and 38 000-dalton proteins, intrinsic 30 000- and 33 000-dalton proteins and two proteolipids of 5000 and 9000 daltons. KCl washing of the heavy vesicles removed both the approx. 34 000- and 38 000-dalton proteins, and the ‘sarcoplasmic reticulum feet’ were no longer seen on the heavy vesicles. The KCl supernatant was enriched in the 34 000- and 38 000-dalton proteins, indicating that these proteins are possible components of the sarcoplasmic reticulum feet. The biochemical and morphological data strongly support the view that the light vesicles are derived from the longitudinal sarcoplasmic reticulum and that the heavy vesicles are derived from the terminal cisternae containing junctional sarcoplasmic reticulum membrane with the intact ‘sarcoplasmic reticulum feet’.     

Programmed cell death: Cytochemical and X-ray microanalytical characterization of calcium compartments in neuromuscular junctions during the normal breakdown of the intersegmental muscles in the giant silkmothAntheraea polyphemus

Summary Calcium stores were cytochemically demonstrated using a combined oxalate—pyroantimonate method in the neuromuscular junctions of the degenerating intersegmental muscles in the giant silkmothAntheraea polyphemus. The elemental composition of punctate precipitates of the reaction product was determined by electron probe X-ray microanalysis of unstained thin sections by energy-dispersive spectrometry and wavelength-dispersive spectrometry. The wavelength-dispersive spectra collected over terminal axons demonstrate a significant calcium signal and a trace of antimony.During the rapid lytic phase of spontaneous muscle degeneration, the calcium punctate deposits were detected in presynaptic terminals in the following sites: the synaptic vesicles and the mitochondria. Calcium precipitates were also found in the dense bodies and the mitochondria encountered in the glial convolutions. No calcium deposit was seen in the synaptic clefts and intercellular spaces of the subsynaptic reticulum of type I and type II. A comparison of calcium to antimony ratios between the terminal axons and the sarcoplasmic lysosomes revealed highly significant differences (P<0.001). Such a variability of the calcium to antimony ratio may be related to different conditions of precipitation or antimony diffusion in the different cell compartments. It was concluded that such synaptic terminals do not appear damaged in spite of the muscle degeneration and presumably continue to perform vital functions while the muscles are no longer contractile 20 h after adult ecdysis.     

Localization of the high affinity calcium binding protein and an intrinsic glycoprotein in sarcoplasmic reticulum membranes

Several proteins in sarcoplasmic reticulum preparations move in a band with a mobility, in sodium dodecyl sulfate-polyacrylamide gels (0.1 M phosphate buffer, pH 7.0), corresponding to a molecular mass of about 55,000 daltons. Only one of these proteins is the high affinity calcium binding protein. An intrinsic glycoprotein is also present in this band, and it is this glycoprotein which is found in vesicles reconstituted after dissolution of sarcoplasmic reticulum in deoxycholate. Both of these proteins are found in rather constant ratios with the ATPase in light, intermediate, and heavy sarcoplasmic reticulum vesicles. Transverse tubular vesicles can be isolated from the heavy sarcoplasmic reticulum vesicles after disruption of the membrane in a French pressure cell (Lau, Y.H., Caswell, A.H., and Brunschwig, J.P. (1977) J. Biol. Chem. 252, 5565-5574). These vesicles are enriched in their content of the high affinity calcium binding and depleted of the intrinsic glycoprotein. Cycloheptaamylose . fluorescamine complex (CFC) labels the intrinsic glycoprotein heavily indicating that it is at least partially exposed on the cytoplasmic surface of sarcoplasmic reticulum membranes. Since the carbohydrate component of the protein must lie in luminal spaces, it is inferred that the intrinsic glycoprotein is a transmembrane protein. The high affinity calcium binding protein is not labeled by CFC indicating that it is not exposed on the cytoplasmic surface of sarcotubular vesicles. The protein is also not affected by proteolytic digestion of sarcoplasmic reticulum vesicles and can be isolated intact from trypsin-digested vesicles. It is not removed from sarcoplasmic-reticulum vesicles by washing with buffers containing Chelex 100 or ethylene glycol bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA). These data show that the high affinity calcium binding protein is localized in the interior of the sarcotubular system and suggest that it might be common to both sarcoplasmic reticulum and transverse tubular membranes.     

Enzymatic activity of dystrophic chicken sarcoplasmic reticulum

We have isolated sarcoplasmic reticulum from normal and dystrophic chicken muscle, using an improved isolation procedure. Dystrophic sarcoplasmic reticulum has a reduced level of calcium-sensitive ATPase activity, phosphoenzyme formation, and steady-state calcium transport. Anion-stimulated calcium transport by dystrophic sarcoplasmic reticulum is also reduced when measured under the proper conditions, and dystrophic sarcoplasmic reticulum shows no alteration in calcium efflux rate. Active calcium phosphate loading of the normal and dystrophic sarcoplasmic reticulum preparations indicates that a reduced percentage jof the dystrophic vesicles are capable of active calcium transport. The loaded dystrophic sarcoplasmic reticulum vesicles exhibit the same relative reductions in enzymatic activity as the starting sarcoplasmic reticulum preparations. However, the enzyme activities of normal and dystrophic sarcoplasmic reticulum are similar in the presence of detergent and exogenous phospholipid. On the basis of these results, we suggest that the lipid microenvironment of the dystrophic enzyme is altered.     

Abnormal ryanodine receptor channels in malignant hyperthermia.

Previous studies have demonstrated a defect associated with the calcium release mechanism of sarcoplasmic reticulum (SR) from individuals susceptible to malignant hyperthermia (MH). To examine whether SR calcium release channels were indeed altered in MH, SR vesicles were purified from normal and MH susceptible (MHS) porcine muscle. The Ca2+ dependence of calcium efflux rates from 45Ca2(+)-filled SR vesicles was then compared with the Ca2+ dependence of single-channel recordings of SR vesicles incorporated into planar lipid bilayers. The rate constants of 45Ca2+ efflux from MHS SR were two to threefold larger than from normal SR over a wide range of myoplasmic Ca2+. Normal and MHS single channels were progressively activated in a similar fashion by cis Ca2+ from pCa 7 to 4. However, below pCa 4, normal channels were inactivated by cis Ca2+, whereas MHS channels remained open for significantly longer times. The altered Ca2+ dependence of channel inactivation in MHS SR was also evident when Ca2+ was increased on the trans side while cis Ca2+ was held constant. We propose that a defect in a low-affinity Ca2+ binding site is responsible for the altered gating of MHS SR channels. Such a defect could logically result from a mutation in the gene encoding the calcium release channel, providing a testable hypothesis for the molecular basis of this inherited disorder.     

Physiological and cytochemical studies on activator calcium in contraction by smooth muscle of a sea cucumber,Isostichopus badionotus

Summary The physiological properties of mechanical responses and the intracellular localization and translocation of calcium as a pyroantimonate precipitate were studied in the longitudinal retractor muscle (LRM) of a Bermuda sea cucumber. Acetylcholine (ACh)-induced contraction was reduced by lowering the external Ca concentration, and suppressed completely by prolonged soaking in Ca-free solution. The magnitude of ACh-induced contraction was decreased by Mn and La ions. Furthermore, procaine reduced the ACh-induced contraction. The complete removal of Ca and Mg ions from the external medium induced a socalled Ca · Mg-removal contraction. Electron microscopically, numerous subsarcolemmal vesicles were observed in the LRM fibers. In the resting fibers, pyroantimonate precipitates were localized in the subsarcolemmal vesicles and along the inner surface of plasma membrane. While, in the fiber fixed during mechanical activity, the pyroantimonate precipitates were decreased remarkably in the subsarcolemmal vesicles and at the plasma membrane, and diffusely distributed in the myoplasm. Electronprobe X-ray microanalysis showed that the precipitate contains Ca in a significant amount. These results indicate that the contraction of the LRM fibers is caused not only by Ca-influx but also by Ca-release from the intracellular storage sites, such as the subsarcolemmal vesicles and the inner surface of plasma membrane.     

Cortical alveoli of Paramecium: a vast submembranous calcium storage compartment

The plasma membrane of Paramecium is underlain by a continuous layer of membrane vesicles known as cortical alveoli, whose function was unknown but whose organization had suggested some resemblance with muscle sarcoplasmic reticulum. The occurrence of antimonate precipitates within the alveoli first indicated to us that they may indeed correspond to a vast calcium storage site. To analyze the possible involvement of this compartment in calcium sequestration more directly, we have developed a new fractionation method, involving a Percoll gradient, that allows rapid purification of the surface layer (cortex) of Paramecium in good yield and purity and in which the alveoli retain their in vivo topological orientation. This fraction pumped calcium very actively in a closed membrane compartment, with strict dependence on ATP and Mg2+. The pumping activity was affected by anti-calmodulin drugs but no Triton-soluble calmodulin binding protein could be identified, using gel overlay procedures. The high affinity of the pump for calcium (Km = 0.5 microM) suggests that it plays an important role in the normal physiological environment of the cytosol. This may be related to at least three calcium-regulated processes that take place in the immediate vicinity of alveoli: trichocyst exocytosis, ciliary beating and cytoskeletal elements dynamics during division.     

The mechanism of increase in the ATPase activity of sarcoplasmic reticulum vesicles treated with n-alcohols.

Treatment of sarcoplasmic reticulum vesicles with aqueous n-alcohols caused inhibition of calcium uptake and enhancement of ATPase activity. With increasing alcohol concentration, the ATPase activity reached a maximum (in the case of n-butanol, at about 350 mM) and then decreased. The effect of n-butanol was extensively studied. The purified ATPase enzyme and leaky vesicles treated with Triton X-100 or phospholipase A showed high ATPase activity in the absence of n-butanol. With increasing n-butanol concentration, their atpase activities began to decrease above about 250 mM n-butanol, without any enhancement. In the presence of ATP, the turnover rate of calcium after calcium accumulation had reached a steady level was the same as that at the initial uptake. n-Butanol did not affect these rates. Kinetic analyses of these experiments were carried out. The mechanisms of calcium transport and of increase of ATPase activity in the presence of alcohol were interpreted as follows. After calcium accumulation had reached a steady level, fast influx and efflux continued; the influx was coupled with phosphorylated enzyme (E-P) formation and most of the efflux was coupled with rephosphorylation of ATP from ADP and E-P. The observed ATPase activity is the difference between these two reactions. If alcohol molecules make the vesicles leaky, calcium ions will flow out without ATP synthesis and the apparent ATPase activity will increase. The effect of alcohols on sarcoplasmic reticulum vesicles was separated into two actions. The enhancement of ATPase activity was attributed to a leakage of calcium ions from the vesicles, while the decrease of ATPase activity at higher concentrations of alcohols was attributed to denaturation of the ATPase enzyme itself. The two effects were interpreted in terms of equilibrium binding of alcohol molecules to two different sites of the vesicles; leakage and denaturation sites. Similar analysis was carried out for various n-alcohols from methanol to n-heptanol. The apparent free energies of binding of the methylene groups of n-alcohols were evaluated to be -863 cal/mol for the leakage site, and -732 cal/mol for the denaturation site.     

ATP- and growth substance-dependent cell-free enlargement of plasma membrane vesicles from soybean

Growth hormone-responsive and nucleoside triphosphate-dependent enlargement of inside-out vesicles of plasma membranes from soybeans prepared by aqueous two-phase partition and everted by freezing and thawing has been achieved in a cell-free system. In the presence of 100 microM ATP in 40 mM HEPES buffer, pH 7, enlargement of isolated plasma membrane vesicles was accelerated by the synthetic plant growth factor, 2,4-dichlorophenoxyacetic acid (2,4-D), compared to ATP alone, 2,4-D alone or no additions. After 20 min with 1 microM 2,4-D, vesicles increased in diameter, 20% on average. Although vesicle diameters in the presence or absence of 2,4-D overlapped, the means were clearly separated. The 20% increases in diameter corresponded to a doubling of vesicle volume. Both 100 microM ATP and 1 microM 2,4-D were necessary to stimulate the cell-free vesicle enlargement. In the presence of 1 microM 2,4-D, enlargement observed with 100 microM ATP was greater than with either 10 microM ATP or 500 microM ATP alone. In the presence of 100 microM ATP, vesicle enlargement was proportional to the logarithm of 2,4-D concentration. With the growth-inactive 2,4-D analog, 2,3-D, no vesicle enlargement was observed either alone or in the presence of 100 microM ATP. Right side-out vesicles did not enlarge in response to either ATP, 2,4-D or the two in combination suggesting that the responsible ATP site was on the inside of the cell.     

The formation of intravesicular calcium phosphate deposits in microsomes of smooth muscle

Summary The calcium uptake in the microsomial fraction isolated from the smooth muscle of the antrum of the pig stomach is stimulated by phosphate. The microsomial vesicles which are loaded with calcium phosphate can be purified by differential centrifugation. A purification of 36 times in terms of calcium content was reached. Electron microscopy of the freshly prepared material revealed calcium phosphate deposits in the form of needles of crystalline calcium phosphate. This structure differs from that of the deposits which appear in the fragmented sarcoplasmic reticulum of skeletal muscle. Their morphology is that of non-crystalline calcium phosphate. However, on standing these deposits convert slowly into crystalline calcium phosphate. This difference reflects different kinetics of crystallization of the precipitates in the two preparations. After negative staining of the calcium phosphate loaded microsomes of skeletal and of smooth muscle, only few deposits are preserved because a release of calcium occurs as a consequence of the action of the stain and also of the dilution and warming up of the suspension. Smooth muscle microsomes partially purified by loading with calcium phosphate were studied by freeze etching and rotary replication. Membrane fragments displaying subunit intramembrane particles similar to those observed in sarcoplasmic reticulum of skeletal muscle could be identified. However, in the smooth muscle microsomes the intramembrane particles were much less densely packed. Part of these particles could correspond to calcium transport sites.     

Lack of effects of calcium X calmodulin-dependent phosphorylation on Ca2+ release from cardiac sarcoplasmic reticulum

Canine cardiac sarcoplasmic reticulum is phosphorylated by an endogenous calcium X calmodulin-dependent protein kinase and phosphorylation occurs mainly on a 27 kDa proteolipid, called phospholamban. To determine whether this phosphorylation has any effect on Ca2+ release, sarcoplasmic reticulum vesicles were phosphorylated by the calcium X calmodulin-dependent protein kinase, while non-phosphorylated vesicles were preincubated under identical conditions but in the absence of ATP to avoid phosphorylation. Both non-phosphorylated and phosphorylated vesicles were centrifuged to remove calmodulin, and subsequently used for Ca2+ release studies. Calcium loading was carried out either by the active calcium pump or by incubation with high (5 mM) calcium for longer periods. Phosphorylation of sarcoplasmic reticulum by calcium X calmodulin-dependent protein kinase had no appreciable effect on the initial rates of Ca2+ released from cardiac sarcoplasmic reticulum vesicles loaded under passive conditions and on the apparent 45Ca2+-40Ca2+ exchange from cardiac sarcoplasmic reticulum vesicles loaded under active conditions. Thus, it appears that calcium X calmodulin-dependent protein kinase mediated phosphorylation of cardiac sarcoplasmic reticulum is not involved in the regulation of Ca2+ release and 45Ca2+-40Ca2+ exchange.     

Lack of effects of calcium · calmodulin-dependent phosphorylation on Ca2+ release from cardiac sarcoplasmic reticulum

Canine cardiac sarcoplasmic reticulum is phosphorylated by an endogenous calcium · calmodulin-dependent protein kinase and phosphorylation occurs mainly on a 27 kDa proteolipid, called phospholamban. To determine whether this phosphorylation has any effect on Ca²⁺ release, sarcoplasmic reticulum vesicles were phosphorylated by the calcium · calmodulin-dependent protein kinase, while non-phosphorylated vesicles were preincubated under identical conditions but in the absence of ATP to avoid phosphorylation. Both non-phosphorylated and phosphorylated vesicles were centrifuged to remove calmodulin, and subsequently used for Ca²⁺ release studies. Calcium loading was carried out either by the active calcium pump or by incubation with high (5 mM) calcium for longer periods. Phosphorylation of sarcoplasmic reticulum by calcium · calmodulin-dependent protein kinase had no appreciable effect on the initial rates of Ca²⁺ released from cardiac sarcoplasmic reticulum vesicles loaded under passive conditions and on the apparent ⁴⁵Ca²⁺ − ⁴⁰Ca²⁺ exchange from cardiac sarcoplasmic reticulum vesicles loaded under active conditions. Thus, it appears that calcium · calmodulin-dependent protein kinase mediated phosphorylation of cardiac sarcoplasmic reticulum is not involved in the regulation of Ca²⁺ release and ⁴⁵Ca²⁺ − ⁴⁰Ca²⁺ exchange.     

2,4-D诱导固氮螺菌和慢生型大豆根瘤菌在小麦上的结瘤作用及其效果

分别在水培和砂培条件下进行了2,4-D诱导固氮螺菌和慢生型大豆根瘤菌在小麦根上的结瘤试验,结果表明2,4-D能诱发它们在小麦根系上形成“类根瘤”,扫描电镜结果证明只有个别细菌进入小麦根瘤细胞内,在细胞间隙有较多的细菌。用乙炔还原法仅检测到接种大豆根瘤菌的小麦根瘤有微量的固氮酶活性,但在盆栽植株的生长方面,看不到2,4-D,2,4-D+固氮螺菌或2,4-D+根瘤菌对小麦生长的促进作用。     

Formation of magnesium-phosphoenzyme and magnesium-calcium-phosphoenzyme in the phosphorylation of adenosine triphosphatase by orthophosphate in sarcoplasmic reticulum. Models of a reaction sequence

The aim of the present study was to test simple reaction sequences which describe calcium-independent plus calcium-dependent phosphorylation of sarcoplasmic reticulum transport. ATPase by orthophosphate including the function of magnesium in phosphoenzyme formation. The reaction schemes considered were based on the reaction sequence for calcium-independent phosphorylation proposed previously; namely that the transport enzyme (E) forms a ternary complex (Mg . E . Pi), by random binding of free magnesium and free orthophosphate, which is in equilibrium with the magnesium-phosphoenzyme (Mg . E-P). Phosphorylation, performed at pH 7.0 20 degrees C and a constant free orthophosphate concentration using sarcoplasmic reticulum vesicles either unloaded or loaded passively with calcium in the presence of 5 mM or 40 mM CaCl2, resulted in a gradual decrease in the apparent magnesium half-saturation constant and an increase in maximum phosphoprotein formation with increasing calcium loads. When phosphorylation of sarcoplasmic reticulum vesicles preloaded in the presence of 5 mM CaCl2 was performed at a constant free magnesium concentration, a decrease in the apparent orthophosphate half-saturation constant and an increase in maximum phosphoprotein formation was observed as compared with vesicles from which calcium inside has been removed by ionophore X-537A plus EGTA treatment; however, both parameters remained unchanged by increasing free magnesium from 20 mM to 30 mM. When phosphorylation of sarcoplasmic reticulum vesicles passively loaded with calcium in the presence of 40 mM CaCl2, at which the saturation of the low-affinity calcium binding sites of the ATPase is presumably near maximum, was performed at increasing concentrations of free orthophosphate, there was a parallel shift of phosphoprotein formation as a function of free magnesium and vice versa, with no change in the maximum phosphoenzyme formation. Comparison of the experimental data with the pattern of phosphoprotein formation predicted from model equations for various theoretical possible reaction sequences suggests that phosphoenzyme formation from orthophosphate possesses the following features. Firstly, calcium present at the inside of the sarcoplasmic reticulum membrane binds to the free enzyme and in sequential order to E . Mg . Pi or Mg . E-P or to both, but neither to E. Mg nor to E . Pi. Secondly, calcium-independent and calcium-dependent phosphoproteins are magnesium-phosphoenzymes. Calcium-dependent phosphoenzyme is a magnesium-calcium-enzyme phosphate complex with 1 magnesium, 2 calciums and 1 orthophosphate (the last covalently) bound to the enzyme [Mg . E-P . (Cai)2], and not a 'calcium-phosphoprotein' without bound magnesium.