Interaction of spin-labelled phosphatidyl choline with Ca-dependent ATPase from sarcoplasmic reticulum

The interaction between 3-acyl-2-(6-doxylpalmitoyl) phosphatidyl choline used as a hydrophobic spin probe and Ca2+-dependent ATPase from sarcoplasmic reticulum membranes of rabbit and carp white skeletal muscles was studied. The spin label incorporation into ATPase preparations was performed at initial steps of ATPase isolation by incubating reticulum membranes with the spin probe in the presence of cholic acid. A comparison of the molecular mobility of the probe incorporated into ATPase preparations and into liposomes formed from ATPase phospholipids demonstrated that the presence of the protein in the membrane produces the same effect on the probe mobility as does the decrease of temperature by 10-15 degrees C. The molecular mobility of the probe in the ATPase preparation is increased during protein thermal denaturation. The breaks on the Arrhenius plots for the probe molecular mobility are revealed at the same temperatures (25 degrees for rabbit reticulum and 16 degrees for carp reticulum) as those for the ATPase activity.     

Low levels of taurine introgression in the current Brazilian Nelore and Gir indicine cattle populations

Background

Nelore and Gir are the two most important indicine cattle breeds for production of beef and milk in Brazil. Historical records state that these breeds were introduced in Brazil from the Indian subcontinent, crossed to local taurine cattle in order to quickly increase the population size, and then backcrossed to the original breeds to recover indicine adaptive and productive traits. Previous investigations based on sparse DNA markers detected taurine admixture in these breeds. High-density genome-wide analyses can provide high-resolution information on the genetic composition of current Nelore and Gir populations, estimate more precisely the levels and nature of taurine introgression, and shed light on their history and the strategies that were used to expand these breeds.

Results

We used the high-density Illumina BovineHD BeadChip with more than 777 K single nucleotide polymorphisms (SNPs) that were reduced to 697 115 after quality control filtering to investigate the structure of Nelore and Gir populations and seven other worldwide populations for comparison. Multidimensional scaling and model-based ancestry estimation clearly separated the indicine, European taurine and African taurine ancestries. The average level of taurine introgression in the autosomal genome of Nelore and Gir breeds was less than 1% but was 9% for the Brahman breed. Analyses based on the mitochondrial SNPs present in the Illumina BovineHD BeadChip did not clearly differentiate taurine and indicine haplotype groupings.

Conclusions

The low level of taurine ancestry observed for both Nelore and Gir breeds confirms the historical records of crossbreeding and supports a strong directional selection against taurine haplotypes via backcrossing. Random sampling in production herds across the country and subsequent genotyping would be useful for a more complete view of the admixture levels in the commercial Nelore and Gir populations.

Electronic supplementary material

The online version of this article (doi:10.1186/s12711-015-0109-5) contains supplementary material, which is available to authorized users.     

2,2,4-Trimethylpentane induces Ca2+ release from the sarcoplasmic reticulum terminal cisterns.

Using quin2, the effects of aliphatic hydrocarbons on the system of Ca(2+)-induced Ca2+ release in isolated membranes of rabbit skeletal muscle terminal cisterns have been studied. The hydrocarbons were inserted into the membranes by means of hydrocarbon-containing liposomes. 2,2,4-Trimethylpentane (isooctane) caused a rapid release of 70-75% of Ca2+ taken up by the terminal cistern vesicles during the Ca(2+)-pump operation. This effect was inhibited by the caffeine-induced Ca2+ release blockers--Mg2+, ruthenium red and tetracaine. The same was observed with a decrease in the concentration of ATP that is known to activate the terminal cistern Ca2+ channels. The effect of 2,2,4-trimethylpentane on the longitudinal cistern fractions practically devoid of Ca(2+)-channels was insignificant. Heptane, hexane and octane caused a slow release of 5-10% of the accumulated Ca2+ from the terminal cistern vesicles; no such effect was induced by decane.     

The blocking effect of aliphatic hydrocarbons on Ca2+ leakage channels formed by aggregates of Ca2+-ATPase of the sarcoplasmic reticulum

The effects of aliphatic hydrocarbons within the liposomes on the Ca2+ transport function of isolated sarcoplasmic reticulum (SR) membranes of rabbit skeletal muscle, vesiculate preparation of Ca2+ dependent ATPase and proteoliposomes reconstituted from Ca2+-ATPase and egg phosphatidylcholine, were studied. It was shown that liposomes prepared from dipalmitoyl phosphatidylcholine containing aliphatic hydrocarbons increase 2 to 3 times Ca2+ accumulation by Ca2+-dependent ATPase from rabbit skeletal muscle SR. Ca2+ transport by SR vesicles increases in the presence of hydrocarbons by 15--20%. The activating effect of hydrocarbons on Ca2+ transport by proteoliposomes depends on the lipid/protein ratio. The proteoliposomes with a high lipid/protein ratio are practically insensitive to the effects of hydrocarbons. It was suggested that activation of Ca2+ transport by hydrocarbons is due to blocking of Ca2+ leakage channels formed during the aggregation of Ca2+-ATPase molecules. Treatment of membranes by formaldehyde results in the oligomerization of Ca2+-ATPase and decreases 2--4-fold the ATP-dependent accumulation of Ca2+. Subsequent addition of decane restores Ca2+ transport practically completely.     

Vitamin E protection of membranes of the sarcoplasmic reticulum against the damaging effect of free fatty acids

Feeding rats the diet enriched with vitamin E or addition of alpha-tocopherol to the suspension of sarcoplasmic reticular membranes of rat and rabbit skeletal muscles protects Ca2+-dependent ATPase against thermal inactivation aggravated by the action of free fatty acids.     

Release of Ca2+ ions from the sarcoplasmic reticulum of skeletal muscle induced by heparin. Relation between the Ca2+ release caused by Ca2+ ions and caffeine

Using a Ca2+-selective electrode and Quin 2 and chlortetracycline fluorescence, a Ca2+ release from terminal cysterns of skeletal muscle sarcoplasmic reticulum under effects of heparin, caffeine and Ca2+ has been studied. It was shown that Ca2+ release induced by heparin is insensitive to the blockers of Mg2+-dependent system of Ca2+-induced Ca2+ release, i.e., Mg2+, tetracaine and dimethylsulfoxide. Preliminary release of Ca2+ in the presence of caffeine, which activates Mg2+-dependent Ca2+ release, does not prevent the heparin-induced Ca2+ release. At the same time, after Ca2+ release caused by Ca2+ in a Mg2+-independent system, heparin cannot cause additional efflux of Ca2+. It has been shown that the heparin-induced release of Ca2+ diminishes with a decrease in a decrease in Ca2+ concentration. This effect is less pronounced in the presence of Na+ than with K+. The data obtained suggest that sarcoplasmic reticulum terminal cysterns contain two systems of Ca2+-induced release of Ca2+, i.e., a Mg2+-dependent, caffeine-sensitive and a Mg2+-independent heparin-sensitive ones. The mechanism of activation of both systems by caffeine and heparin consists, in all probability, in their increased affinity for Ca2+.     

Temperature-dependent changes in the profile of the sarcoplasmic reticulum membrane hydrophobic zones]

The dependence of the state of the hydrophobic zone of rabbit sarcoplasmic reticulum (SR) membranes on temperature of the membrane fragment suspension before rapid freezing was studied by the freeze fracturing technique. It was shown that within the temperature range of--15-- +37 degrees C the amount of intramembrane particles and their distribution in the membrane plane and between their convex and concave surfaces do not practically depend on the temperature of the SR membrane suspension. This is indicative of the lack of correlation between the physical state of the phospholipid matrix (gel -- liquid crystal) before freezing and the nature of the profile of the membrane hydrophobic zone revealed after fracturing. The disturbances in the protein -- lipid interactions in the membrane under the effects of mersalyl or aqueous solutions of diethyl ester followed by complete inactivation of Ca2+-dependent ATPase lead to a decrease in the amount of intramembrane particles, which is especially well-pronounced at 37 degrees and -15 degrees C.     

The role of a GTP-binding protein in coupling of a muscarinic cholinergic receptor and Na,K-ATPase in myocardial sarcolemma]

The effects of the cholinergic agonist carbachol (Cch) and guanine nucleotides on the Na,K-ATPase and K-dependent p-nitrophenylphosphatase (K-p-NPPase) activities in rabbit and dog myocardial sarcolemma vesicles in the presence of the pore-forming antibiotic alamethicin (20 micrograms/ml), was studied. Cch (0.01-100 microM) inhibited the both enzymatic activities by 40-45% (IC50 = 0.3-0.5 microM) only after addition of GTP (50 microM) or its analogs: GTP gamma S (0.1-1.0 microM) and Gpp(NH)p (10 microM). The muscarinic acetylcholine receptor (mAchR) antagonist atropine (10 microM) blocked the effect of Cch. GTP gamma S alone produced a concentration-dependent decrease in the both Na,K-ATPase and K-p-NPPase activities by 40-45% (IC50 = 1-2 microM) with a lag period of about 3 minutes; this lag disappeared in the presence of the agonist. The GDP analog GDP beta S (0.01-100 microM) neither affected these activities nor promoted the inhibiting effect of Cch. Pretreatment of sarcolemmal vesicles with 20 micrograms/ml of pertussis toxin in the presence of 100 microM NAD abolished the inhibiting effect of Cch on the Na,K-ATPase and phosphatase activities. Under these conditions pertussis toxin catalyzed the ADP-ribosylation of alpha-subunits of the inhibitory GTP-binding protein (G1) which were identified immunochemically as alpha i2, alpha i3 and, possibly, alpha i1. The data obtained testify to the involvement of G1 in the mAchR-mediated inhibition of myocardial sarcolemmal Na,K-ATPase as well as in the signal transduction from the receptor to the enzyme.     

Concentration of components of the adenylate system in heavy and light fractions of the sarcoplasmic reticulum of skeletal muscles and sensitivity of these fractions to the effects of imidazole-containing compounds and caffeine]

Fragments of sarcoplasmic reticulum from rabbit sceletal muscles sedimented within the range from 2000 g to 8000 g (heavy fraction) and 8000 g to 40000 g (light fraction) and washed with 0.6 M KCl, were practically free of adenylatecyclase activity. Phosphodiesterase cAMP was not found in the light fraction, while its activity in the heavy fraction was 500 pmol of cAMP/min per mg of protein. Both fractions contain bound cAMP (1-2 pmol/mg of protein) and specific sites of cAMP binding, the binding constant being approximately 10(6)M-1. The number of binding sites is 60 pmol/mg of protein for the heavy and 30 pmol/mg of protein for the light fractions. The level of phosphodiesterase activity in the heavy fraction correlates with its sensitivity to imidazole, anserine and caffeine. Imidazole and anserine increase in 1.5-1.8 times the value of Ca2+/ATP in the heavy fraction and produce no effect on Ca2+ transport by the light fraction. Caffeine decreases almost twice the Ca2+/ATP value in the heavy fraction and has practically no effect on Ca2+ absorption by enzymes of the light reticulum fraction. Imidazole and anserine activate membrane-bound phosphodiesterase, while caffeine inhibits it. It is suggested that structural rearrangements of membrane-bound phosphodiesterase under the effect of caffeine, imidazole and anserine are responsible for changes in the efficiency of Ca2+ transport by fragments of the heavy reticulum fractions.