Phosphorylation of troponin I by protein kinase C: mechanism of inhibition by calmodulin and troponin C

The total phospholipid content of excised rat muscle, liver, brain and kidney and of human muscle biopsies was estimated by natural abundance 13C-NMR after complete solubilization of the tissue membranes with excess halothane. An external dioxane capillary, calibrated against pure palmitic acid and phospholipid vesicles with known phosphate concentration, was inserted into the tissues, and the repeating methylene carbon peak area in the spectra of the halothane-treated tissues was integrated versus the dioxane reference peak area. The amount of tissue used for NMR analysis was quantitated by dry weight determination after 13C spectroscopy was completed. The phospholipid content estimated by the indirect NMR method was in good agreement with that measured by direct phosphate analysis and with literature data. For human muscle biopsies, the NMR method can also estimate the fraction of the total phospholipids which are mobile without treating the muscles with halothane. In this respect human muscles could be separated into three different groups: normal and nonspecific muscle diseases, myotonia and myopathy, Duchenne dystrophy; with increasing fraction of the mobile phospholipids in this order.     

Characterization of the broad resonance in 31P NMR spectra of excised rat brain

31P NMR spectra of excised rat brain showed a broad resonance between-12 and -13 ppm. Subcellular fractions of brain, rich in membranes, exhibited the broad resonance and it was also present in isolated myelin, the major membrane component of brain. However, it was absent in brain cytosol (161,100 X g supernatant). Raising the temperature of the brain above 50 degrees C caused a gradual downfield chemical shift of the broad resonance, to about -1 ppm at 90 degrees C. An even larger downfield shift was produced by halothane or deoxycholate with concomitant narrowing of the line width of this resonance. Vesicles prepared from the phospholipids of excised brain or isolated myelin showed the broad resonance, and halothane produced the same downfield shift and peak sharpening in brain phospholipid vesicles as that in the intact brain. The chemical shift anisotropy was estimated to be 45 ppm for both myelin and the brain, as characteristic for biological membranes. The T1 and T2 relaxation times of the perpendicular 31P chemical shift tensor component of the broad resonance were 0.66 sec and 1.6 msec, respectively, in the same range as those for other biological membranes. Halothane-treatment of the brain increased both the T1 and T2 times considerably, as expected from the disruption of the phospholipid bilayer in a membrane. These data indicate that the broad resonance in the 31P NMR spectrum of excised rat brain originates exclusively from the phosphate head group of membrane bound phospholipids. Similar broad resonances were found in autopsied human brain and porcine spinal cord and to a lesser extent in excised rat liver and kidney.     

Effect of denervation on the phospholipid content in subcellular fractions of tonic and tetanic muscles.

Four weeks after denervation, various changes were observed in the phospholipid composition of the sarcolemmal and sarcoplasmic fractions of skeletal muscles with different functions. Neurotomy also affected the innervated contralateral muscles and produced opposite changes in the phospholipid content of subcellular fractions. The increase in the amount of phospholipids in the sarcolemmal fractions of the denervated muscles was only apparent. The difference between the denervated and contralateral muscles was also due to the decrease of phospholipids in the contralateral muscles. These changes were more pronounced in the tetanic (fast-twitch) than in the tonic (slow-twitch) muscles. In the sarcoplasmic fraction of the denervated tetanic muscle an increase, while in that of the tonic one a slight decrease of phospholipids appeared. In contrast, the phospholipid content in the sarcoplasmic fractions of contralateral muscles did not decrease, while it increased slightly in the tonic muscle. The amount of plasmalogens (fatty aldehyde: lipid phosphorus ratio) decreased only in the subcellular fractions of the denervated muscles while there was no change in those of the contralateral muscles.     

Tissue and subcellular distribution of 3H-dioxane in the rat and apparent lack of microsome-catalyzed covalent binding in the target tissue

Using ³H-dioxane, the distribution of dioxane among a number of tissues and various subcellular fractions of rat liver was studied. At various times after i.p. injection, dioxane was found to distribute more or less uniformly among various tissues (liver, kidney, spleen, lung, colon and skeletal muscle), consistent with its polar/nonpolar nature. Studies of the nature of dioxane binding, however, revealed that the extent of “covalent” binding (as measured by incorporation into lipid-free, acid-insoluble tissue residues) was significantly higher in the liver (the main carcinogenesis target tissue), spleen and colon than that in other tissues. Investigations of the subcellular distribution in liver indicated that most of the radioactivity was in the cytosol, followed by the microsomal, mitochondrial and nuclear fractions. The binding of dioxane to the macromolecules in the cytosol was mainly noncovalent. The percent covalent binding was highest in the nuclear fraction, followed by mitochondrial and microsomal fractions and the whole homogenate. Pretreatment of rats with inducers of microsomal mixed-function oxidases had no significant effect on the covalent binding of dioxane to the various subcellular fractions of the liver. There was no microsome-catalyzed $\text{in}$$\text{vitro}$ binding of ³H- or ¹⁴C-dioxane to DNA under conditions which brought about substantial binding of ³H-benzo[a]pyrene.     

The effect of phosphate deficiency on membrane phospholipid composition of bean (Phaseolus vulgaris L.) roots

Plasma membranes were isolated from roots of bean (Phaseolus vulgaris L.) plants cultured on phosphate sufficient or phosphate deficient medium. The phospholipid composition of plasma membranes was analyzed and compared with that of the microsomal fraction. Phosphate deficiency had no influence on lipid/protein ratio in microsomal as well as plasma membrane fraction. In phosphate deficient roots phospholipid content was lower in the plasma membrane, but did not change in the microsomal fraction. Phosphatidylcholine and phosphatidylethanolamine were two major phospholipids in plasmalemma and microsomal membranes (80 % of the total). After two weeks of phosphate starvation a considerable decrease (about 50 %) in phosphatidylcholine and phosphatidylethanolamine in microsomal membranes was observed. The decline in two major phospholipids was accompanied by an increase in phosphatidic acid and lysophosphatidylcholine content. The effect of alterations in plasma membrane phospholipids on membrane function e.g. nitrate uptake is discussed.     

Effect of endurance training on the phospholipid content of skeletal muscles in the rat

Only few data are available on the effect of training on phospholipid metabolism in skeletal muscles. The aim of the present study was to examine the effect of 6 weeks of endurance training on the content of particular phospholipid fractions and on the incorporation of blood-borne [14C]-palmitic acid into the phospholipids in different skeletal muscles (white and red sections of the gastrocnemius, the soleus and the diaphragm) of the rat. Lipids were extracted from the muscles and separated using thin-layer chromatography into the following fractions: sphingomyelin, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, cardiolipin and neutral lipids (this fraction being composed mostly of triacylglycerols). It was found that training did not affect the content of any phospholipid fraction in soleus muscle. It increased the content of sphingomyelin in white gastrocnemius muscle, cardiolipin and phosphatidylethanolamine in red gastrocnemius muscle and phosphatidylinositol in white gastrocnemius muscle and diaphragm. The total phospholipid content in red gastrocnemius muscle of the trained group was higher than in the control group. Training reduced the specific activity of sphingomyelin and cardiolipin in all muscles, phosphatidylcholine in soleus, red, and white gastrocnemius muscles, phosphatidylserine in all muscles, phosphatidylinositol in all except the soleus muscle, and phosphatidylethanolamine in hindleg muscles, but not in the diaphragm compared to the corresponding values in the sedentary group. It was concluded that endurance training affects skeletal muscle phospholipid content and the rate of incorporation of the blood-borne [14C]palmitic acid into the phospholipid moieties.     

Intervesicular phospholipid exchange. An NMR study.

Phospholipid exchange between phosphatidylinositol and phosphatidylcholine vesicles has been studied by NMR spectroscopy with use of hydrophilic paramagnetic lanthanide probes (Pr³⁺ and Eu³⁺ ions). The dependence of the lanthanide induced shifts in the ¹H and ³¹P NMR spectra on the phospholipid composition of the vesicles could be used for its quantitative evaluation. The method has been proved to be applicable for studying phospholipid exchange stimulated by soluble proteins (postmicrosomal supernatant fraction) from rat liver. Furthermore it has been shown that the phospholipid molecules newly introduced by protein-stimulated exchange are predominantly incorporated into the outer monolayer of the vesicular bilayer membrane. This makes it possible to produce liposomes with asymmetric distribution of the phospholipids across the bilayer.     

2H and31P nuclear magnetic resonance studies of membranes containing bovine rhodopsin

Summary Purified, delipidated rhodopsin is recombined with phospholipid using octyl-glucoside (OG) and preformed vesicles. Normal egg phosphatidylcholine, phosphatidylcholine in which the N-methyl groups are fully deuterated, and dioleoyl phosphatidylcholine labeled with deuterium at carbons 9 and 10 were used.³¹P nuclear magnetic resonance (NMR) and²H NMR measurements were obtained of the pure phospholipids and of the recombined membranes containing rhodopsin.³¹P NMR of the recombined membrane (containing the deuterated phospholipid) showed two overlapping resonances. One resembled a normal phospholipid bilayer, and the other was much broader, representing a motionally restricted phospholipid headgroup environment. The population of phospholipids in the motionally restricted environment can be modulated by conditions in the media.²H NMR spectra of the same recombined membranes showed only one component. These experimental results agree with a theoretical analysis that predicts an insensitivity of²H NMR to lipids bound to membrane proteins. A model containing at least three different phospholipid environments in the presence of the membrane protein rhodopsin is described.Deceased.     

Hormone (3-indoleacetic acid)-phospholipid interaction: 1H, 13C and 31P nuclear magnetic resonance studies

The interaction between the plant hormone, 3-indoleacetic acid (IAA), and some phospholipids in CDCL₃ has been studied by ¹H, ¹³C and ³¹P nuclear magnetic resonance (NMR) spectroscopy. Upon interaction with IAA, significant changes occurred in resonance positions of the phospholipid head group nuclei. Alteration of the fatty acid composition influenced the effects of IAA on these nuclei. These effects were observed in the ethanolamine and phosphate groups of the phosphatidylethanolamines, and in the choline, phosphate and glycerol groups of the phosphatidylcholines. Changes in resonance positions of the phospholipid head group nuclei were used for the determination of dissociation constants (K_d). In all cases, K_d values were approx. 10^?2 molal for 1 : 1 interaction. The NMR results suggest an interaction orientation in which the aromatic ring system of IAA interacts with the quaternary nitrogen function of the head group, and the phosphate group becomes hydrogen-bonded to the NH or carboxyl proton of 1AA.     

Characterization of a Phospholipid Adduct Formed in Sprague Dawley Rats by Chloroform Metabolism: NMR Studies

The formation of a covalent adduct to a single phospholipid by the oxidative chloroform metabolite, phosgene, is demonstrated in liver mitochondria of phenobarbital-pretreated Sprague Dawley (SD) rats treated with CHCl₃. The densitometric analysis of the phosphorus stained extracted phospholipids showed that the formation of this adduct in liver mitochondria is accompanied by a decrease of phosphatidylethanolamine and cardiolipin. The characterization of this adduct was performed with a multinuclear NMR approach by comparison with the decreased phospholipids. Treatment of rats with [¹³C]chloroform resulted in an intense ¹³C NMR peak from either an esteric or amidic carbonyl. Very strong similarities in fatty acid composition were found between phosphatidylethanolamine and the phosgene-modified PL, using ¹³C and ¹H NMR spectroscopy. A multiplet at 3.91 ppm coupled to a signal at 3.41 ppm was shown by two-dimensional ¹H NMR in the adduct spectrum. This cross peak was interpreted as arising from the shifted resonances of the two PE head group methylene groups, due to the binding with phosgene. ³¹P spectrum of the adduct was identical to that of phosphatidylethanolamine. We concluded that the chloroform adduct is a modified phosphatidylethanolamine, with the phosgene-derived carbonyl bound to the amine of the head group. © 1997 John Wiley & Sons, Inc. J Biochem Toxicol 12: 93–102, 1998     

Effect of triiodothyronine on phospholipid metabolism in skeletal muscles of the rat.

The aim of the present study was to examine the effect of treatment with triiodothyronine (T3) on certain aspects of phospholipid metabolism in skeletal muscles. Rats were injected with triiodothyronine (T3) daily (10 microg x 100 g(-1) b.w., s.c.) for six days. Saline-treated rats served as controls. 24 h after the last dose of T3, 14C palmitic acid suspended in the serum of a donor rat, was administered intravenously. Thirty min later, samples of the soleus, white and red section of the gastrocnemius and blood from the abdominal aorta were taken. The muscle phospholipids were extracted and separated into different fractions by means of thin layer chromatography. The following fractions were obtained: shingomeylin, phosphatidylcholine phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine and cardiolipin. The phospholipids were quantified and their radioactivity was measured. The plasma free fatty acid concentration and radioactivity was also determined. Treatment with T3 reduced the content of phosphatidylinositol and phosphatidylserine in each muscle type, whereas the concentration of other phospholipids remained stable. T3 increased markedly incorporation of the blood-borne fatty acids into each phospholipid fraction in the muscles. It is concluded that an excess of T3 influences the metabolism of phospholipids in skeletal muscles.     

Microbial Assimilation of Hydrocarbons: Identification of Phospholipids

The distribution of phospholipids derived from Micrococcus cerificans was determined under a variety of nutritive conditions. Cells were grown with hexadecane, heptadecane, or acetate serving as the sole carbon source. Total lipid was isolated by chloroform-methanol extraction, and the phospholipid fraction was isolated by silicic acid column chromatography. The phospholipids were characterized by silicic acid chromatography, by thin-layer chromatography, and by identification of water-soluble products resulting from acid hydrolysis of purified phospholipids. Major phospholipids characterized were phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin. Minor phospholipids were phosphatidylglycerol phosphate and phosphatidylserine. Trace amounts of methylated derivatives of phosphatidylethanolamine were determined by incorporation of ¹⁴C from ¹⁴C-methylmethionine. These experiments demonstrated the presence of phosphatidyl-N-methylethanolamine, phosphatidyl-N,N′-dimethylethanolamine, and phosphatidylcholine in trace quantities. Pulse labeling with ¹⁴C-serine demonstrated the direct incorporation of serine into phosphatidylserine followed by decarboxylation to phosphatidylethanolamine.     

Generation second messengers by prostanoids in the iris-sphincter and ciliary muscles of cows, cats and humans

We have examined the generation of second messengers after stimulation of feline, bovine, human iris-sphincter and ciliary muscles by selected prostaglandins (PGs). The tissues, labeled or unlabeled with ³H-myo-inositol, were stimulated by a range of concentrations of 16, 16-dimethyl PGE₂, 11-deoxy PGE₁, 17-phenyl trinor PGE₂ and PGF_2α. In both tissues of all three species, 16, 16-dimethyl PGE₂ and 11-deoxy PGE₁ stimulated the formation of cyclic AMP. Butaprost, an EP₂ receptor agonist, which was tested only in feline ciliary muscle, generated cyclic AMP. In the feline iris-sphincter and in bovine and feline ciliary muscles, 17-phenyl trinor PGE₂, an EP₁ receptor agonist, significantly increased inositol phosphate turnover. The FP receptor agonist, PGF_2α stimulated inositol phosphate turnover in the bovine, feline, and human iris-sphincter muscles and in human ciliary muscles. Feline and bovine ciliary muscles did not respond to PGF_2α.These results suggest that EP₁ receptors are present in feline iris-sphincter muscle and in bovine and feline ciliary muscles. The EP₂ receptors exist in both tissue. These results also suggest the presence FP receptors in bovine, feline, and human iris-sphincter and in human ciliary muscles. Bovine and feline ciliary muscles do not appear to express FP receptors.     

31P-NMR study of brain phospholipid structures in vivo.

31P-NMR has been used extensively for the study of cytosolic small molecule phosphates in vivo and phospholipid structures in vitro. We present in this paper a series of studies of the brain by 31P-NMR, both in vivo and in extracts, showing the information that can be derived about phospholipids. 31P-NMR spectra of mouse brain at 73 mHz are characterised by almost a complete absence of the large phosphodiester peak in comparison to equivalent spectra at 32 mHz. Proton decoupled spectra in vivo, and spectra of extracts, show that the phosphodiester peak observed in 32 mHz spectra in vivo is mainly due to phospholipid bilayers. Homogenates of quaking and control mouse brains, and of bovine grey matter, show another narrower phosphodiester peak possibly from small phospholipid vesicles. This peak is increased in intensity in the affected mice. These experiments demonstrate the presence of three major components contributing to the phosphodiester resonance: bilayer phospholipids, more mobile phospholipids, and the freely soluble cytosolic molecules glycerophosphocholine and glycerophosphoethanolamine. These NMR methods for non-invasive investigation of phospholipid structures in the brain might be extended to studies of patients with membrane involved diseases such as multiple sclerosis.     

31P NMR and freeze fracture studies of sarcoplasmic reticulum membranes from normal and malignant hyperthermic pigs: effect of halothane and dantrolene.

The effects of halothane and dantrolene on sarcoplasmic reticulum membranes isolated from normal and malignant hyperthermia pig muscle have been investigated using 31P NMR and freeze fracture electron microscopy. The dynamical and structural changes are estimated from the second moment, as calculated from 31P NMR spectra. For both membranes, addition of halothane induces a similar decrease in the spectral second moment. At high concentration of halothane, freeze fracture replicas show small unilamellar vesicles or mixed micelles, uniformly sprayed in the case of malignant hyperthermia membranes but mainly aggregated for the normal ones. The effect of halothane on both membranes is partially inhibited by adding dantrolene. These results suggest that (i) the malignant hyperthermia syndrome is not directly related to the polar heads of phospholipids and (ii) dantrolene counteracts unspecifically the disturbing effect of halothane at the lipid level.     

Identification of capillaries in sections from skeletal muscle by use of lectins and monoclonal antibodies reacting with histo-blood group ABH antigens

This study was performed to evaluate the application of different lectins and monoclonal antibodies against ABH antigens to detect and characterize carbohydrate structures in capillaries of skeletal muscle from humans and laboratory animals. Blood group specific lectins (Griffonia simplicifolia, Griffonia simplicifolia isolectin B4,Lotus tetragonlobus, Ulex europaeus, andDolichos biflorus) and monoclonal antibodies reacting with histo-blood group carbohydrate antigens belonging to type 1 (Le^a) and type 2 (H, A and Le^y) chains were used as histological markers for capillaries in sections from skeletal muscle. The material consisted of 20 human masseter muscle biopsies from individuals with known blood types: (eight blood group O, nine blood group A, two blood group B, and one blood group AB) and masseter muscles specimens from different laboratory animals (mouse, rat, rabbit, cat, dog, pig, cow, and macaca monkey). Unfixed sections and an avidin alkaline phosphatase method were used to visualize the specific reaction.Ulex lectin stained capillaries in all human biopsies either strongly or moderately. Strong muscle capillary reaction was observed in biopsies from O, B and AB individuals while capillaries from A individuals were only moderately stained.Griffonia simplicifolia marked capillaries in A, B, and AB individuals andGriffonia simplicifolia isolectin B4 stained capillaries in muscle biopsies from B and AB donors.Dolichos biflorus was a weak marker of muscle capillaries from A individuals. Only capillaries from O individuals were stained with the antibody against H type 2. Capillary reaction was not observed with the other antibodies used.Girffonia simplicifolia was an excellent marker for capillaries in mouse muscle whileGriffonia simplicifolia isolectin B4 is recommended for rat muscles. Periodic acid treatment and subsequentLotus tetragonolobus staining is suitable to visualize capillaries in mouse, rat and pig muscle. Using a sensitive histochemical technique for staining with lectins and monoclonal antibodies reacting with blood group related antigens the microvascular density in human skeletal muscle may be estimated. Further, the carbohydrate compounds in the muscle capillaries reflect the individual blood type. A selection of lectins is suitable for demonstration of capillaries in animal skeletal muscle.     

Lipophorin from Rhodnius prolixus: Purification and partial characterization

The lipophorin of adult females of Rhodnius prolixus was radioactively labelled with ³²P exclusively in the phospholipid moiety and purified on a KBr ultracentrifugation gradient. The density of purified [³²P]phospholipid labelled lipophorin on the fifth day after a blood meal was 1.1211 ± 0.0017 g/ml. By weight it contained 51.7% protein, 0.7% sugar and 47.6% lipid. The protein moiety was composed of three apoproteins of 226 ± 11, 86 ± 2 and 16 ± 1 kDa. Mannose and N-acetylglucosamine were the only sugars detected. Among the lipids, 66.3% were neutral lipids and 33.8% were phospholipids. Analysis by thin-layer chromatography showed that in the total phospholipids fraction ³²P was distributed as follows: phosphatidylethanolamine (54.4%), phosphatidylcholine (44.7%), cardiolipin (2.1%), phosphatidylserine (0.7%), phosphatidylinositol (0.4%), sphingomyelin (0.3%) and phosphatidic acid (0.2%). The total phosphate content was 0.53 ± 0.03 nmol/μg of protein.     

Older adults with sarcopenia have distinct skeletal muscle phosphodiester,phosphocreatine, and phospholipid profiles

The loss of skeletal muscle mass and function with age (sarcopenia) is a critical healthcare challenge for older adults. 31‐phosphorus magnetic resonance spectroscopy (³¹P‐MRS) is a powerful tool used to evaluate phosphorus metabolite levels in muscle. Here, we sought to determine which phosphorus metabolites were linked with reduced muscle mass and function in older adults. This investigation was conducted across two separate studies. Resting phosphorus metabolites in skeletal muscle were examined by ³¹P‐MRS. In the first study, fifty‐five older adults with obesity were enrolled and we found that resting phosphocreatine (PCr) was positively associated with muscle volume and knee extensor peak power, while a phosphodiester peak (PDE2) was negatively related to these variables. In the second study, we examined well‐phenotyped older adults that were classified as nonsarcopenic or sarcopenic based on sex‐specific criteria described by the European Working Group on Sarcopenia in Older People. PCr content was lower in muscle from older adults with sarcopenia compared to controls, while PDE2 was elevated. Percutaneous biopsy specimens of the vastus lateralis were obtained for metabolomic and lipidomic analyses. Lower PCr was related to higher muscle creatine. PDE2 was associated with glycerol‐phosphoethanolamine levels, a putative marker of phospholipid membrane damage. Lipidomic analyses revealed that the major phospholipids, (phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol) were elevated in sarcopenic muscle and were inversely related to muscle volume and peak power. These data suggest phosphorus metabolites and phospholipids are associated with the loss of skeletal muscle mass and function in older adults.     

Effect of Water Quality on Relationships Between Cationic Species and Leaf Lipids at Two Different Development Stages in Cotton

Lipid and phospholipid content of cotton leaves were measured in plants irrigated with fresh water and brackish water. The correlation between K⁺ and Na⁺ in leaf tissue and the changes in total lipids and phospholipids was determined at the vegetative and fruiting stages. Data demonstrated that total lipids and phospholipids can be associated with changes in the K/Na ratio and with the two developmental stages of the cotton plant. Plants irrigated with brackish water had more total phosphate incorporated into the phospholipids in the fruiting stage.     

Principles of multiparametric optimization for phospholipidomics by 31P NMR spectroscopy

Phospholipids have long been known to be the principal constituents of the bilayer matrix of cell membranes. While the main function of cell membranes is to provide physical separation between intracellular and extracellular compartments, further biological and biochemical functions for phospholipids have been identified more recently, notably in cell signaling, cell recognition and cell–cell interaction, but also in cell growth, electrical insulation of neurons and many other processes. Therefore, accurate and efficient determination of tissue phospholipid composition is essential for our understanding of biological tissue function. ³¹P NMR spectroscopy is a quantitative and fast method for analyzing phospholipid extracts from biological samples without prior separation. However, the number of phospholipid classes and subclasses that can be quantified separately and reliably in ³¹P NMR spectra of tissue extracts is critically dependent on a variety of experimental conditions. Until recently, little attention has been paid to the optimization of phospholipid ³¹P NMR spectra. This review surveys the basic physicochemical properties that determine the quality of phospholipid spectra, and describes an optimization strategy based on this assessment. Notably, the following experimental parameters need to be controlled for systematic optimization: (1) extract concentration, (2) concentration of chelating agent, (3) pH value of the aqueous component of the solvent system, and (4) temperature of the NMR measurement. We conclude that a multiparametric optimization approach is crucial to obtaining highly predictable and reproducible ³¹P NMR spectra of phospholipids.