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.     

ON THE POSSIBILITY OF LYSOPHOSPHATIDE FORMATION DURING WALLERIAN DEGENERATION

—During an extensive decomposition of phospholipids, at the end of the second week of Wallerian degeneration, the decomposition of glycerophosphatides were studied in detail. In a degenerative process lasting for 2 weeks about one-third of the choline phosphatides, two-thirds of the ethanolamine phosphatides, one-third of the serine phosphatides and one-quarter of the inositol phosphatides, were destroyed. The amount of lysophosphatidylcholine decreased proportionally to the destruction of choline phosphatide. On the other hand, the amount of lysophosphatides formed from‘kephalin'-containing fatty aldehyde, during the marked destruction of these phospholipids, remained constant or increased to a small extent and its percentage distribution increased 2 or 3 times compared with other phospholipids. Ethanolamine phosphatides having a high fatty aldehyde content can be regarded as mainly responsible for the relative accumulation of lysophosphatides.     

Experimental investigations on hypokinesis of skeletal muscles with different functions, III. Changes in protein fractions of subcellular components

Changes occurring in the protein fractions of rabbits' immobilized skeletal muscles with different functions were studied. Disuse of the muscles resulted in a gradual reduction in the contractile proteins. The specific proteins of the tonic muscle (m. soleus) were degraded to a greater extent than those of the tetanic (white) muscles (m. gastrocnemius). Parallel with the decrease in the structural proteins the sarcoplasmic protein exhibited a relative increase. The tonic muscles underwent greater damage than the tetanic muscles, indicating that the dedifferentiation was more marked in the tonic muscle. The results are explained by the biological importance of the function and activity of the cell: disuse leads to changes in the physiological and biochemical characteristics of the muscle, and to dedifferentiation of the cells.     

Temperature adaptation of biological membranes. Compensation of the molar activity of cytochrome c oxidase in the mitochondrial energy-transducing membrane during thermal acclimation of the carp (cyprinus carpio L.)

The phospholipid composition, fatty acid pattern and cholesterol content are studied in mitochondria of red lateral muscle of carp acclimated to high and low environmental temperatures.The results of the experiments are: mitochondria from cold-acclimated carp contain higher proportions of ethanolamine phosphatides than mitochondria from warm-acclimated fish, the opposite is true for the choline phosphatides. Thus, at constant pH, the membrane phospholipids are slightly more negatively charged at low acclimation temperature. The total plasmalogen content is reduced in the cold; this reduction is caused by a decrease in the proportion of the choline plasmalogens. The ethanolamine phosphoglycerides contain approx. 20% of the alk-1-enyl acyl type, irrespective of the acclimation temperature. There is no temperature-dependent difference in the low proportion of cholesterol.The fatty acids of total mitochondrial phospholipids are characterized by large amounts of the n-3 and n-6 families. The ratio of unsaturated to saturated fatty acids and the unsaturation index are remarkably higher than those reported for comparable mammalian phospholipids. Cold acclimation of carp does not significantly increase the unsaturation of total phospholipids. A fatty acid analysis of the main isolated phospholipids, however, shows that cold acclimation considerably increases unsaturation of the neutral phosphatidylcholine, whereas it dramatically decreases unsaturation of the negatively charged cardiolipin. It is suggested that the observed fatty acid substitution in phosphatidylcholine indicates a temperature-induced fluidity adaptation within the mitochondrial lipid bilayer, whereas the inverse acclimation pattern of cardiolipin provides a suitable lipid to accommodate the temperature-dependent modifications in the dynamic surface shape of integral membrane proteins.     

A Sensitive Radial Diffusion Method for Detection of Esterolytic Activities of Proteases

The influence of under-nutrition (sub-maintenance feeding) and ad libitum feeding on the deposition of proteins in different subcellular sarcoplasmic fractions of red (tonic) and white (phasic) muscles of growing broilers was investigated. The relative concentration of overall sarcoplasmic proteins was lesser in red than in white muscles from ad libitum fed broilers. The content of mitochondrial proteins was slightly more and that of lysosomal and microsomal proteins and of true soluble proteins was lesser in red than in white muscles. Besides, the relative amount of some specific molecular weight proteins in each subcellular fractions differed by more than 50% between red and white muscle.

There was also conspicuous differences in the influence of under-nutrition on the proteins in red and white muscles. Some reduction in mitochondrial, lysosomal, microsomal, and soluble protein content occurred only in white muscle, whereas little change was found in subcellular fractions in red muscle from underfed broilers. The relative amount of some proteins in each subcellular fraction of both muscles remained unaffected, and that of others either decreased or increased more than 20 to 50% due to nutritional stress.     

Lipids from nerve tissues of the horseshoe crab, Limulus polyphemus.

1. The predominant lipids of nerve cords, ganglion and brain from horseshoe crabs were cholesterol (11% of lipid) and phospholipid (81% of lipid). 2. Major phospholipids were phosphatidyl ethanolamine and phosphatidyl choline with lesser amounts of phosphatidyl serine and phosphatidyl inositol and sphingomyelin. 3. The phospholipid fraction was characterized by a high content of plasmalogen, i.e. alk-1-enyl acyl phosphatides, so that 42% of the ethanolamine phosphatides were the plasmalogen, phosphatidal ethanolamine. 4. Phosphatidyl choline and phosphatidyl ethanolamine were high in polyunsaturation with 20:4 and 20:5 major fatty acids. Sphingomyelin had predominantly long chain saturated fatty acids. 5. Cerebrosides and gangliosides, which are associated with vertebrate nerve tissues, were absent from nerves of horseshoe crabs.     

THE INCORPORATION OF [1-14C]LINOLENATE INTO LIPIDS OF DEVELOPING RAT BRAIN DURING ESSENTIAL FATTY ACID DEPRIVATION

Abstract— Twenty-one-day old essential fatty acid (EFA) deprived rats incorporated about twice the radioactivity from [1-¹⁴C]linolenate into brain lipid fractions as did controls. At 5 min after injection, 2/3 of the radioactivity was associated with the less polar lipid fraction of both control and EFA deprived animals. By 30 min after injection, 70% of the radioactivity was in the phospholipid fraction. This value increased to 90% at later time points.
The specific activity of brain phospholipids from EFA deprived rats was always greater than that of controls. This held true for the individual phosphatide fractions also. In general, phosphatidylcholine (PC) was labeled most rapidly. With increasing time intervals, radioactivity was transferred to phospha-tidylethanolamine (PE) and phosphatidylserine + phosphatidylinositol (PS + PI).
The transfer of fatty acid radioactivity into phospholipid and the distribution of radioactivity among individual phosphatides did not appear to be affected by the dietary state. However, the total amount of radioactivity incorporated was related to the amount initially retained by brain after injection. Our data suggest that one or more components of the less polar lipid fraction may act as a 'trap' or reservoir for fatty acids which are required for phospholipid synthesis.     

Ratio and composition of the plasmalogen and diacylated forms of phospholipids in subcellular fractions of the avian brain

Studies have been made on the specific content of plasmalogen and diacylated forms of phosphatidylethanolamine and phosphatidylcholine in subcellular fractions (myelin, nuclei, microsomes, mitochondria, synaptosomes) from the brain of pigeons, as well as in the myelin fraction from the brain of the crow Corvus cornix and the hawk Accipiter gentelis. Fatty acid composition and fatty aldehyde composition of these two main phospholipids of the brain were studied in the subcellular fractions obtained. It was shown that plasmalogen forms of phospholipids are localized in birds mainly in the myelin fraction which exhibits the highest plasmalogen concentration as compared to the same fraction of all the vertebrates investigated. With respect to fatty acid and fatty aldehyde composition, as well as to the degree of their unsaturation, myelin plasmalogens from birds are similar to those from other cold-blooded and warm-blooded animals. This fact indicates that high relative content of plasmalogens together with their high unsaturation account for normal functional activity of myelin membranes in all vertebrates.     

Distribution of newly formed fatty acids among glycerolipids of isolated perfused rat liver.

Livers of chow fed rats were perfused 1-3 h with buffer, glucose, albumin, and red blood cells, made up in 100 percent D(2)O. Glycerolipids were isolated and the deuterated fatty acids determined by gas chromatography with mass spectrometry on Silar 5 CP. Percentage of replacement by deuterated acids ranged from 1 to 14, of which palmitate was 87 percent. Differences were found in total lipid class and in subcellular distribution of the newly synthesized acids. Microsomes had 37 percent more deuterated acids than the total or floating fat. At 3 h the highest replacement was found in diacylglycerols (17 percent) and free fatty acids (11 percent). Of the palmitate in hepatic choline and ethanolamine phosphatides, 6.9 percent and 4.7 percent, respectively, contained dueterium. The serine and inositol phosphatides had a higher proportion of deuterated palmitate (7.7 percent) than other phosphatides. The data support the hypothesis that palmitate is incorporated into glycerolipids largely via de novo synthesis while stearate enters them by deacylation-acyl transfer replacement.     

Temperature adaptation of biological membranes. The effects of acclimation temperature on the unsaturation of the main neutral and charged phospholipids in mitochondrial membranes of the carp (cyprinus carpio L.)

The phospholipid composition, fatty acid pattern and cholesterol content are studied in mitochondria of red lateral muscle of carp acclimated to high and low environmental temperatures.The results of the experiments are: mitochondria from cold-acclimated carp contain higher proportions of ethanolamine phosphatides than mitochondria from warm-acclimated fish, the opposite is true for the choline phosphatides. Thus, at constant pH, the membrane phospholipids are slightly more negatively charged at low acclimation temperature. The total plasmalogen content is reduced in the cold; this reduction is caused by a decrease in the proportion of the choline plasmalogens. The ethanolamine phosphoglycerides contain approx. 20% of the alk-1-enyl acyl type, irrespective of the acclimation temperature. There is no temperature-dependent difference in the low proportion of cholesterol.The fatty acids of total mitochondrial phospholipids are characterized by large amounts of the $\text{n-3}\text{and}\text{n-6}$ families. The ratio of unsaturated to saturated fatty acids and the unsaturation index are remarkably higher than those reported for comparable mammalian phospholipids. Cold acclimation of carp does not significantly increase the unsaturation of total phospholipids. A fatty acid analysis of the main isolated phospholipids, however, shows that cold acclimation considerably increases unsaturation of the neutral phosphatidylcholine, whereas it dramatically decreases unsaturation of the negatively charged cardiolipin. It is suggested that the observed fatty acid substitution in phosphatidylcholine indicates a temperature-induced fluidity adaptation within the mitochondrial lipid bilayer, whereas the inverse acclimation pattern of cardiolipin provides a suitable lipid to accommodate the temperature-dependent modifications in the dynamic surface shape of integral membrane proteins.     

A chemical and histochemical study of the lipides of the pyloric cecum of the starfish,Asterias forbesi

The lipides of the diverticula of Asterias forbesi have been studied by histochemical and biochemical means. Correlations between results obtained by histochemical examination of sections, and chemical analysis of isolated lipide have been made, particularly with respect to phosphatides, steroids, and aldehyde lipides. The results of the histochemical study were in good agreement with the chemical data as to the nature of the phosphatide fraction, the presence of acetone-soluble aldehyde lipides, and the composition of the free droplet fat. Homogenized diverticula were differentially centrifuged in order to establish the distribution of types of lipides in the various cellular components. In addition, data have been presented which demonstrate a direct correlation between the titer of alpha-glycerol ethers and that of acetone-soluble lipide acetals in the unsaponifiable fraction.     

A CHEMICAL AND HISTOCHEMICAL STUDY OF THE LIPIDES OF THE PYLORIC CECUM OF THE STARFISH, ASTERIAS FORBESI

The lipides of the diverticula of Asterias forbesi have been studied by histochemical and biochemical means. Correlations between results obtained by histochemical examination of sections, and chemical analysis of isolated lipide have been made, particularly with respect to phosphatides, steroids, and aldehyde lipides. The results of the histochemical study were in good agreement with the chemical data as to the nature of the phosphatide fraction, the presence of acetone-soluble aldehyde lipides, and the composition of the free droplet fat. Homogenized diverticula were differentially centrifuged in order to establish the distribution of types of lipides in the various cellular components. In addition, data have been presented which demonstrate a direct correlation between the titer of α-glycerol ethers and that of acetone-soluble lipide acetals in the unsaponifiable fraction.     

Active Ca transport of sacroplasmic reticulum during experimental uremia. Changes in kinetics and lipid composition.

The Ca-transport system of sarcoplasmic vesicles of rabbits is altered by experimental uremia. 1. The influx rate constant of the experimental membranes decrease with a resulting decrease of the calcium influx rate. 2. The experimental membranes transport a smaller amount of Ca2+ per mol of ATP split than the controls, i.e. their transport ratio is discussed. 3. The calcium permeability of the experimental membranes increases with a resulting decreased concentrating ability. 4. The phosphatide content but not the cholesterol content of the experimental membranes decreases with a consequent increase of the cholesterol/phosphatide ratio. 5. The fatty acid pattern of total phosphatides of the experimental membranes changes. A relative decrease of palmitic acid and oleic acid occurs and a relative increase of stearic, arachidonic and higher unsaturated fatty acids. 6. The altered lipid composition of the membranes does not change the temperature dependence of the kinetics.     

The Effects of Acetylcholine on the Turnover of Phosphatidic Acid and Phosphoinositide in Sympathetic Ganglia, and in Various Parts of the Central Nervous System in Vitro

The effect of acetylcholine on the incorporation of P³² into the individual phosphatides in slices of various structures of the nervous system has been studied. There was a marked stimulation of P³² incorporation into phosphoinositide and phosphatidic acid, but not into phosphatidyl choline and phosphatidyl ethanolamine, in the cat stellate and celiac ganglia in vitro. Acetylcholine stimulated P³² incorporation into certain phosphatides, primarily phosphoinositide and phosphatidic acid, in several structures of the cat and guinea pig brain; there was little or no effect of acetylcholine on phosphatide turnover in the inferior corpora quadrigsemina and cerebellar cortex. The suggestion is made that the phospholipid effect can best be explained as being concerned with the active transport of sodium ions out of the cell across the postsynaptic membrane of cholinergic neurons in response to acetylcholine.     

Radiotracer evidence implicating phosphoryl and phosphatidyl bases as intermediates in betaine synthesis by water-stressed barley leaves

In barley, glycine betaine is a metabolic end product accumulated by wilted leaves; betaine accumulation involves acceleration of de novo synthesis from serine, via ethanolamine, N-methylethanolamines, choline, and betaine aldehyde (Hanson, Scott 1980 Plant Physiol 66: 342-348). Because in animals and microorganisms the N-methylation of ethanolamine involves phosphatide intermediates, and because in barley, wilting markedly increases the rate of methylation of ethanolamine to choline, the labeling of phosphatides was followed after supplying [¹⁴C]ethanolamine to attached leaf blades of turgid and wilted barley plants. The kinetics of labeling of phosphatidylcholine and betaine showed that phosphatidylcholine became labeled 2.5-fold faster in wilted than in turgid leaves, and that after short incubations, phosphatidylcholine was always more heavily labeled than betaine. In pulse-chase experiments with wilted leaves, label from [¹⁴C]ethanolamine continued to accumulate in betaine as it was being lost from phosphatidylcholine. When [¹⁴C]monomethylethanolamine was supplied to wilted leaves, phosphatidylcholine was initially more heavily labeled than betaine. These results are qualitatively consistent with a precursor-to-product relationship between phosphatidylcholine and betaine.     

DECOMPOSITION OF PHOSPHOLIPIDS DURING WALLERIAN DEGENERATION J. DOMONKOS

—Of the lipid metabolic changes in Wallerian degeneration, the decomposition of phosphatides was studied in the early stages, 2–14 days after the nerve had been cut. It was found that the decomposition of phosphatides begins only 6 days after section. The amounts of lipid-P and fatty acid ester decrease in parallel. However, a greatcr decrease in the amount of fatty aldehyde was observed than of the lipid-P and fatty acid ester; this indicates that the vinyl ether bonds of glycerophosphatides are more sensitive to the degenerative process than the fatty acid ester or phosphorus. The fatty acid ester: lipid-P ratio, calculated from the results, does not change; however, the fatty aldehyde: lipid-P ratio shows a marked decrease during this same period. The values of the ratios indicate that in the 2nd week following nerve section, decomposition of glycerophosphatides containing fatty aldehyde (i.e. plasmalogens), in contrast to glycerophosphatides of diacyl form, may be only partial. Thus, in the 2nd week of Wallerian degeneration the formation of lysophosphatides may be presumed to come from the plasmalogens.     

The subcellular fractionation of the bovine caudate nucleus

Two synaptosomal fractions could be obtained from bovine caudate nucleus on sucrose density gradients one of which had a much greater capacity for high affinity choline uptake than the other but comparable amounts of CAT and choline kinase activity. Specific binding of QNB was widely distributed among all the subcellular fractions except the mitochondrial fraction and in quantitative terms by far the greatest amount was in the microsomal fraction. Only the microsomal fraction contained measurable amounts of glycerophosphocholine phosphodiesterase.This paper is dedicated to Dr. Derek Richter on his seventy-fifth birthday.     

Distribution of newly formed palmitate and stearate among molecular species of choline and ethanolamine phosphatides.

The choline and ethanolamine phosphatides derived from isolated rat livers during perfusion with 75 percent deuterated water (Kuksis, A., Myher, J.J., Marai L., Yeung, S.K.F., Steiman, I. & Mookerjea, S. (1975) Can. J. Biochem. 53, 509-518) were resolved into molecular species by argentation thin-layer chromatography. The time course of percentage replacement of the newly synthesized fatty acids in each molecular species was determined by gas chromatography with mass spectrometry. The results confirmed the earlier postulated differential utilization of palmitic and stearic acids in glycerolipid biosynthesis as well as supported the hypothesis of a precursor-product relationship between the oligoenoic and tetranoic species of both phosphatides. Calculations of half-lives gave values of 14-19 h for palmitoyl oligoenes, 40-50 h for palmitoyl tetraenes, and 22-28 h for palmitoyl hexaenes of both choline and ethanolamine phosphatides. The corresponding stearoyl species had half-lives which ranged from 89 to 200 h. Evidence was obtained for a metabolic heterogeneity among subsets of molecular species recognized on the basis of combinations of new and old glycerol and fatty acids in the same glycerolipid molecule.     

Interrelationships among the ethanolamine phosphatides in myelinating rat brain

Abstract— —The ethanolamine phosphatide fraction was isolated from rat brain at 17, 19, and 22 days of age. Analysis by gas-liquid chromatography of the liberated fatty aldehydes and alkyl glyceryl ethers demonstrated a chain length composition quite distinct from that of the fatty acids in the comparable 1(3)-position of the diacyl phosphatides. [1-¹⁴C]-Acetate was administered intraperitoneally to 17-day-old rats. With the exception of the polyunsaturated fatty acids, isotope was readily incorporated into the individual side chains of the 1- and 2-positions of the glycerol moiety. Time studies revealed no readily discernible precursor-product relationships among the linkages in question. Therefore, although the long chain precursors for the alkenyl and alkyl ethers may be related by biosynthetic interconversion, the isotope data are suggestive of independent pathways of biosynthesis for the alkenyl ether, alkyl ether, and ester linkages.     

Changes in fatty acid composition of phospholipids and triglycerides of Musca domestica resulting from choline deficiency

The fatty acid composition of the phospholipids and triglycerides extracted from housefly larvae reared on diets containing no added fatty acids but containing differing concentrations of choline has been determined. Reducing the choline content of the diet resulted in a graded reduction of the percentage of phosphatidylcholine present in the phospholipids of the larvae. This was accompanied by changes in the fatty acid composition, choline deficiency causing an increased utilization of 16-C rather than 18-C acids by the phospholipids. Changes in the fatty acid composition of the triglyceride fraction were also observed but these were associated with insects containing very low levels of phosphatidylcholine. Examination of the fatty acids in the different classes of phospholipids showed that the major change resulting from choline deficiency was in the fatty acids of the phosphatidylethanolamine fraction—the phospholipid which increased as the phosphatidylcholine decreased.Although the fatty acid composition of the different classes of phospholipids was not completely fixed, some preferential utilization of certain fatty acids by certain classes was observed, in both larval and adult insects. The fatty acid composition of the phospholipids extracted from larval gut, muscle, fat body, cuticle, trachea, nervous tissue, and haemolymph was determined. Changes resulting from choline deficiency similar to those seen in the whole larva were observed in all tissues except the nervous tissue. The effect of rearing larvae at temperatures between 24 and 35°C resulted in only minor changes in the fatty acid composition of both phospholipid and triglyceride fractions but the difference due to choline deficiency was observed at all temperatures. The possibility that the observed changes in the fatty acids of the phospholipids are compensatory to the changes in the proportion of the choline to the ethanolamine phospholipids is discussed.