Phospholipid biosynthesis in sarcoplasmic reticulum membrane during development.

Biosynthesis of phosphatidic acid, phosphatidylcholine and phosphatidylethanolamine in the sarcoplasmic reticulum membrane has been investigated. The results show that sarcoplasmic reticulum, in addition to its main function, i.e. transport and accumulation of Ca2+, is able to synthetize phospholipids by the same pathways as endoplasmic reticulum of other tissues. The changes of activity of enzymes involved in phospholipid biosynthesis during muscle development have been analysed. The extent of sn-glycero-3-phosphate and lysophosphatidylcholine acylation by acyl-CoA or free fatty acids in the presence of ATP and CoA is the same at every stage of development. The specific activity of glycerolphosphate acyltransferase(s) increases progressively during development up to about the 10th day of postnatal life and then decreases to the adult level. Linoleate esterifies sn-glycero-3-phosphate to a higher extent than palmitate, especially during postnatal period. The main product of sn-glycero-3-phosphate acylation is phosphatidic acid. The specific activity of lysolecithin acyltransferase increases from the embryonic period to a maximum between the 4th and the 9th day of postnatal life followed by a decrease to the adult value. the low embryonic value to a maximum at about the 3rd day of postnatal life, followed by a decrease to the adult value. The activity of cholinephosphotransferase decreases from a high value observed during the earliest embryonic period studied until the 3rd day before birth, and then begins to increase again from about the 5th day of postnatal life. The activity of ethanolaminephosphotransferase decreases continuously with age. The main product of phosphatidylethanolamine methylation is phosphatidylmonomethylethanolamine. The specific activity of phosphatidylethanolamine methyltransferase increases from

Phospholipase C and diacylglycerol lipase activities associated with plasma membranes of chromaffin cells isolated from bovine adrenal medulla.

The plasma membranes of bovine adrenal chromaffin cells were isolated and the activities of enzymes involved in arachidonic acid liberation were investigated. Only a minute activity of phospholipase A2 (phosphatide 2-acylhydrolase, EC 3.1.1.4) could be detected using externally added phosphatidylcholine (PC) and phosphatidylethanolamine (PE) as substrate. When membranes were treated with exogenous phospholipase C (orthophosphoric acid diester phosphohydrolase, EC 3.1.4.1) there was a liberation of free fatty acids from the sn-2 position of PC. The enzyme responsible for this effect could be demonstrated to be a diacylglycerol lipase (glycerol ester hydrolase, EC 3.1.1.3) localized in the plasma membrane. Using phosphatidylinositol (PI) as a substrate, it was found that an endogenous phospholipase C exists which co-purifies with the membrane preparation. The produced diacylglycerol is subsequently hydrolyzed by diacylglycerol lipase liberating arachidonic acid. The two enzymes, phospholipase C and diacylglycerol lipase were characterized. Phospholipase C was found to be calcium dependent and PI specific, showing an activity of 60 pmol/micrograms protein per h (1.2 mM Ca2+), whereas the diacylglycerol lipase was calcium independent hydrolyzing diacylglycerol at a rate of 7.2 pmol/micrograms protein per h. The lipase but not the phospholipase C was inhibited 50% by 1.7 mM para-bromophenacylbromide.

Thyroid hormones control lipid composition and membrane fluidity of skeletal muscle sarcolemma.

Sarcolemma membrane lipid phase of skeletal muscles of hyperthyroid animals was compared to that of control (euthyroid) ones. Hyperthyroidism caused 15% decrease in cholesterol and 70% increase in the phospholipid content of the membrane. This was accompanied by the alterations in proportions between individual phospholipid classes, and was followed by changes in the composition of phospholipid fatty acids. The calculated fatty acid unsaturation index was higher for membrane lipid phase of hyperthyroid animals than of euthyroid ones. Thyroxine-induced alterations in the lipid composition of sarcolemma caused changes in the membrane fluidity and the activity of calmodulin-stimulated (Ca(2+)-Mg(2+)-ATPase. Measurements of the steady-state fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene indicated that the lipid phase transition of membrane vesicles occurred at 25.9 degrees C and at 28.9 degrees C for preparations isolated from hyperthyroid and euthyroid rabbits, respectively. Arrhenius plot break-point temperature for CaM-stimulated (Ca(2+)-Mg(2+)-ATPase activity was lower in membrane preparations isolated from hyperthyroid (26.9 degrees C) than from euthyroid ones (30.0 degrees C). Thus, the increase of the membrane fluidity presumably caused that the enzyme was characterized by the lower activation energy value. This phenomenon may be viewed as a supplementary mechanism for activation of the enzyme by thyroid hormones to previously reported elevation of the amount of (Ca(2+)-Mg(2+)-ATPase protein exerted by hyperthyroidism (Famulski et al. (1988) Eur. J. Biochem., 171, 363-368; Famulski and Wrzosek (1988) in The Ion Pumps-Structure, Function and Regulation (Stein, W.D., ed.), pp. 355-360, Alan R. Liss, New York).

The response of some organic microsensor coatings to a variety of vapors.

Six organic compounds were spray-coated onto surface acoustic wave devices which were then exposed to vapors of acetone, diethyl ether, dichloromethane, chlorobenzene, benzene, and acetonitrile. Changes in the resonant frequency of the device or in the resistance of the coating were collected by computer-controlled data acquisition. Different patterns of response to the six vapors were observed for each of the coatings.

Properties and topology of enzymes methylating phosphatidylethanolamine to phosphatidylcholine in sarcoplasmic reticulum.

1. The synthesis of phosphatidylcholine (PC) by stepwise methylation of phosphatidylethanolamine (PE) is carried out by two enzymes in sarcoplasmic reticulum (SR) membrane of rabbit fast-twitch skeletal muscles. 2. Two methyltransferases (Met I and Met II) have a different pH optimum and affinity for methyl donor--S-adenosyl-L-methionine (SAM). 3. Met I is an integral SR membrane protein which active site faces the cytoplasmic surface of the membrane. 4. Met II is a peripheral, loosely bound protein, localized mainly on the extracytoplasmic (luminal) part of the SR membrane.

Changes in the structure, composition and function of sarcoplasmic-reticulum membrane during development.

The structure, chemical composition and function of the microsomal fraction, isolated by differential centrifugation and purified on sucrose gradients, from muscle of fetal, newborn and young rabbits were characterized and compared with those of sarcoplasmic reticulum vesicles from adult muscle. Negative staining shows that the microsomal vesicles isolated from muscles of embryos and newborn animals are smooth, in contrast to vesicles obtained from adult muscle which contain 4-nm particles on their surface. The particles appear first in the microsomal vesicles from muscles of 5--8-day-old rabbits. Their number increases with the age of the animals. Ca2+-pump protein, with molecular weight about 100000, accounts for 10% of the total protein content in sarcoplasmic reticulum membrane, isolated at the earliest stages of development analysed. Its amount increases continuously with the rabbit's age to the adult value of about 70% of total sarcoplasmic reticulum protein. The low amount of 100000-dalton protein and lack of 4-nm surface particles in sarcoplasmic reticulum vesicles obtained from fetal and newborn rabbits are strictly correlated with the low activity of Ca2+-dependent ATPase and the ability to take up Ca2+. These activities rise in parallel with the age of the rabbits. On the other hand, Mg2+-dependent ATPase activity is very high at the early stages of development and declines continuously to a low value in sarcoplasmic reticulum from adult muscle. The sarcoplasmic reticulum membrane from fetal and newborn rabbits contains a higher amount of lipids as compared with the membrane present in the muscle of adult animals. The ratio of both phospholipid to protein and neutral lipid to protein decreases with the age of the rabbits. The composition of sarcoplasmic reticulum phospholipids also changes during development.

The effect of thyroxine on the calmodulin-dependent (Ca2+-Mg2+)ATPase activity and protein phosphorylation in rabbit fast skeletal muscle sarcolemma.

Enzymatic properties and the protein pattern of sarcolemma fractions isolated from three groups of rabbits: euthyroid, hyperthyroid and hypothyroid, were studied. The amount of phosphorylated intermediate formed by the calmodulin-dependent (Ca2+-Mg2+)ATPase and the activity of this enzyme as well as that of (Na+-K+)ATPase were the highest in membranes isolated at the hyperthyroid state. On the other hand, sarcolemma obtained from the hypothyroid animals exhibited a decreased activity of (Na+-K+)ATPase, while the activity of calmodulin-dependent (Ca2+-Mg2+)ATPase was the same as in the preparations obtained from euthyroid animals. Thyroid hormones also changed the protein pattern of muscle sarcolemma. Membranes isolated from hyperthyroid animals lacked peptides of apparent molecular masses of 41 kDa and 53 kDa, while a peptide of the apparent molecular mass of 63 kDa was enriched in the preparation from hypothyroid animals. Thyroid hormones affected endogenous cAMP-dependent protein phosphorylation. The sarcolemma fraction obtained from hyperthyroid animals exhibited a decreased phosphorylation of peptides of apparent molecular masses of 30 kDa and 47 kDa, while the cAMP-independent phosphorylation of several other peptides was augmented. Moreover, sarcolemma preparations isolated from hyperthyroid animals showed higher activity of cAMP-independent protein kinase(s) and lower activity of cAMP-dependent protein kinase when compared to the euthyroid preparations. It is proposed that thyroxine increases the content of calmodulin-dependent (Ca2+-Mg2+)ATPase protein and affects the activity of cAMP-independent and cAMP-dependent protein kinases bound to sarcolemma.