Phospholipid metabolism in roots and microsomes of sunflower seedlings: Inhibition of choline phosphotransferase activity by boron

The action of boron on phospholipid composition and synthesis in roots and microsomes from sunflower seedlings has been studied. The fatty acid composition and relative amounts of individual molecular species of phospholipids in roots and microsomes were very similar. In both the content of phospholipids was decreased and the relative levels of their component fatty acids changed by treatment with 50 ppm of boron. This concentration of boron in the culture medium was found to inhibit the in vivo [1-^14C] acetate incorporation into root lipids and that of [Me-¹⁴C] choline into phosphatidylcholine of root microsomes. Cytidine-5-diphospho (CDP)-[Me-¹⁴C] choline incorporation into phosphatidylcholine of isolated microsomes was also inhibited by 50 ppm of boron when present in the growth medium of seedlings. These results indicate that the decrease in phosphatidylcholine labelling from [¹⁴C] choline observed when root microsomes were treated with boron would be caused by a decrease in CDP-choline phosphotransferase activity.     

Changes in both acyl-CoA:cholesterol acyltransferase activity and microsomal lipid composition in rat liver induced by distal-small-bowel resection.

The acyl-CoA:cholesterol acyltransferase (ACAT) activity and lipid composition of hepatic microsomal membrane were investigated 6 weeks after both 50 and 75% distal-small-bowel resection (SBR). A significant decrease in hepatic cholesteryl ester levels was observed after SBR, with a significant increase in the cholesteryl ester content of the livers of 75% SBR compared with the 50% SBR. Hepatic total acylglycerols, free cholesterol and phospholipid levels were not modified after the surgical operation. Microsomal free cholesterol was increased after both 50 and 75% SBR. However, a decrease in both microsomal ACAT activity and cholesteryl ester levels were found in microsomes (microsomal fractions) of resected rats, both changes being higher after 75 than after 50% resection. The total phospholipid content of the microsomes did not change after the surgical operation. The microsomal phospholipid fatty acid composition indicated higher changes after 75 than after 50% SBR. These results demonstrated that, in resected animals: (1) the activity of the enzyme responsible for catalysing cholesterol esterification (ACAT) is decreased, and (2) hepatic microsomal free cholesterol does not appear to influence the activity of ACAT.     

Transfer of tyrosinase to melanosomes in Harding-Passey mouse melanoma

The transfer of tyrosinase from microsomes into melanosomes, without passing through the cytosol in the Harding-Passey mouse melanoma cell, was confirmed by experiments carried out using a combination of radioisotope tracer techniques and immunoprecipitation. 3H-Labeled amino acid incorporation into tyrosinase present in the microsome, melanosome, and soluble fractions confirmed the precursor-product relationship of the enzyme in the microsome fraction and in the melanosome fraction. However, two forms of the enzyme, Ts1- and Ts2-tyrosinase, separated from the soluble fraction by polyacrylamide gel electrophoresis, were shown to play no role in the transfer since little or no incorporation of radioactivity into tyrosinase in this fraction was found. It is suggested that most tyrosinase observed in the soluble fraction does not leak from the melanosomes or the microsomes during homogenization, but comes from necrotic tumor cells. It appears that melanosomal and microsomal tyrosinase might be released from the membrane of necrotic cells modified by various degradation enzymes, considering the data on the recovery of tyrosinase from the soluble fraction, where one-third of total enzyme activity in the postnuclear fraction could not be increased, even when the postnuclear fraction of the tumor was further homogenized radically.     

A comparative study of the composition of the microsomal membranes of the liver, brain and skeletal muscles in vertebrates]

Phospholipid and cholesterol amounts, intrinsic protein/lipid ratios in liver, brain and skeletal muscle microsomal membranes of 14 species of vertebrate animals have been studied. No significant differences between phospholipid amounts in tissues as well as vertebrate classes have been discovered. The highest cholesterol amount has been found in brain microsomes, the smallest one in sarcoplasmic reticulum membranes. In reptile brain and muscle microsomes a higher amount of cholesterol compared to that in species of other vertebrate classes has been found. In brain membranes intrinsic protein and lipid amounts are approximately equal, while in liver and muscle microsomes a protein component predominates. Phospholipid/protein ratio is larger in brain membranes than in liver and muscle ones. Cholesterol/protein ratio reaches the highest values in microsomal membranes of reptile tissues. Brain membranes of vertebrate animals are characterized by a greater stability of protein-lipid composition than liver and muscle ones.     

A phospholipid requirement for dolichol pyrophosphate N-acetylglucosamine synthesis in phospholipase A2-treated rat lung microsomes

The role of phospholipids in the activity of UDP-Glc-NAc:dolichol phosphate GlcNAc-1-phosphate transferase of rat lung microsomes has been investigated. Treatment of microsomes with phospholipase A2 in the presence of delipidated bovine serum albumin resulted in a time-dependent loss of 65 to 75% of the enzyme activity and approximately 30% of the phospholipids. Addition of phosphatidylglycerol to the enzyme assay system containing phospholipase A2-treated microsomes restored activity to that obtained with native microsomes and phosphatidylglycerol. Addition of phosphatidylinositol, phosphatidylcholine, or cardiolipin resulted in only partial restoration of activity, whereas phosphatidylserine and phosphatidylethanolamine were without effect. Triton X-100 was not by itself capable of restoring activity, but was required for the phospholipid effect. Measurements of the phospholipase A2 hydrolysis products released from the microsomes during digestion, and other control experiments of adding fatty acids and lysophospholipids to the enzyme assay system, indicated that the loss of UDP-GlcNAc:dolichol phosphate GlcNAc-1-phosphate transferase activity was not due to product inhibition.     

Phospholipid-transfer proteins in human liver and primary liver carcinoma

Investigations have been carried out on phospholipid-transfer activity of the cytosol and the phospholipid composition of subcellular membranes from human liver and primary liver carcinoma. In both human liver and primary liver carcinoma cytosolic fractions, the transfer activity for phosphatidylcholine (PC), phosphatidylethanolamine (PE) and sphingomyelin has been observed for the first time. The transfer rate of PC and PE in normal human liver was almost equal, whereas sphingomyelin-transfer activity was much slower. In carcinoma cells, the transfer activity for PE and PC was significantly enhanced, while sphingomyelin transfer remained unchanged. Comparative investigations with HepG2 cultured cells have revealed a high PE-transfer activity in this cell line. Parallel with the phospholipid-transfer activity modifications in neoplasic cells, changes in the phospholipid composition of microsomes and mitochondria have been observed. The content of PC and PE in hepatocarcinoma cells was decreased in microsomes, while in the mitochondria it was increased. The possible role of the phospholipid-transfer proteins in the maintenance of membrane composition and structure is discussed.     

Phospholipid requirement of the vanadate-sensitive ATPase from maize roots evaluated by two methods

The activation of the vanadate-sensitive ATPase from maize (Zea mays L.) root microsomes by phospholipids was assessed by two different methods. First, the vanadate-sensitive ATPase was partially purified and substantially delipidated by treating microsomes with 0.6% deoxycholate (DOC) at a protein concentration of 1 milligram per milliliter. Vanadate-sensitive ATP hydrolysis by the DOC-extracted microsomes was stimulated up to 100% by the addition of asolectin. Of the individual phospholipids tested, phosphatidylserine and phosphatidylglycerol stimulated activity as much as asolectin, whereas phosphatidylcholine did not. Second, phospholipid dependence of the ATPase was also assessed by reconstituting the enzyme into proteoliposomes of differing phospholipid composition. In these experiments, the rate of proton transport and ATP hydrolysis was only slightly affected by phospholipid composition. DOC-extracted microsomes reconstituted with dioleoylphosphatidylcholine had rates of proton transport similar to those found with microsomes reconstituted with asolectin. The difference between the two types of assays is discussed in terms of factors contributing to the interaction between proteins and lipids.     

Comparison of phospholipid effects on insulin-sensitive low Km cyclic AMP phosphodiesterase in adipocyte plasma membranes and microsomes

Both adipocyte plasma membranes and microsomes possess insulin-sensitive low Km cyclic AMP phosphodiesterase activity. The activity of the enzyme from both sources was susceptible to activation by several anionic phospholipids. Activators of the plasma membrane enzyme were lysophosphatidylglycerol greater than lysophosphatidylcholine greater than lysophosphatidylserine greater than phosphatidylserine greater than phosphatidylglycerol. These same phospholipids activated the microsomal enzyme but the extent of activation by each phospholipid was reversed. Neutral phospholipids and other anionic phospholipids were without effect. The phospholipids had no effect on high Km cAMP phosphodiesterase in either membrane. The results suggest that the phospholipid headgroup was an important determinant for enzyme activation by phospholipid. The increased susceptibility of the plasma membrane enzyme to lysophospholipid may be attributed to a difference in the plasma membrane enzyme compared to the microsomal membrane enzyme or to differences in plasma membrane and microsomal membrane phospholipid composition and their ability to regulate low Km cAMP phosphodiesterase activity.     

Use of protein-mediated lipid exchange in the study of membrane-bound enzymes. The lipid dependence of glucose-6-phosphatase.

The ability of liver lipid-exchange proteins to introduce foreign phospholipids into microsomes was used in a study of the lipid dependence of glucose-6-phosphatase. Supplementation of intact rat liver and hepatoma microsomes with exogeneous aminophospholipids prevents the decline of glucose-6-phosphatase activity during incubation, whereas the introduction of exogeneous phosphatidylcholine has no protective effect. On the contrary with deoxycholate-disrupted hepatoma microsomes, introduction of additional phosphatidylcholine causes activation while phosphatidylethanolamine has only little effect. The results are explained by assuming that the transport unit and the catalytic moiety of the glucose-6-phosphatase system have different lipid requirements, the activity of the former protein depending mainly on phosphatidylethanolamine and phosphatidylserine and that of the catalytic protein depending on phosphatidylcholine. In deoxycholate-disrupted liver microsomes (in which both the glucose-6-phosphatase activity and the phosphatidylcholine content are much higher than in hepatoma microsomes) incubation with phosphatidylcholine and lipid-exchange proteins alters neither the phospholipid composition nor the enzyme activity. THis suggests that the diminished activity of glucose-6-phosphatase in hepatomas may be partly due to a low level of phosphatidylcholine.     

Effect of distal small bowel resection on ACAT activity and microsomal lipid composition in rat small intestine

1. The acyl-CoA:cholesterol acyltransferase (ACAT) activity and lipid composition of intestinal microsomal membrane were investigated 6 weeks after both 50 and 75% distal small bowel resection (DSBR). 2. No changes in both microsomal ACAT activity and cholesteryl ester levels were found, while microsomal non-esterified cholesterol content was increased after the surgical operation. 3. The total phospholipid content of the microsomes did not change as a result of DSBR. 4. The microsomal phospholipid fatty acid composition showed a significant increase in saturated fatty acids together with no changes in both total monounsaturated and total polyunsaturated fatty acids after resection. 5. An increase in the levels of linoleic acid accompanied by a decrease in arachidonic acid was found in remnant intestine of resected rats.     

Membrane regulation of liver and lung microsomes under low oxygen tension

A highly monitorized animal model has been developed for the study of the influence of low oxygen tension on lipid composition, microviscosity and regulation of enzyme activities involved in the phospholipid synthesis of hepatic and pulmonary microsomes. Microviscosity decreased in liver microsomes whereas no difference was shown in that of microsomal membrane core of hypoxemic lung. Nevertheless, phospholipid and cholesterol content of both liver and lung membranes changed significantly. Microsomal membranes of hypoxemic liver increased the unsaturation degree of fatty acids, whereas hypoxemic lung membranes become more saturated, mainly due to the increase of palmitic acid. The adaptive response of lung was confirmed by the high increase of the deacylation-reacylation mechanism.     

Studies on the reactions between human tyrosinase, superoxide anion, hydrogen peroxide and thiols.

Human tyrosinase (5.5 mg) has been purified from a single human melanotic melanoma metastasis (50.5 g). In the presence of dioxygen, L-tyrosine proved to be a very poor substrate for this enzyme with barely detectable activity compared to L-dopa. However, saturating superoxide anion (i.e., greater than 5 x 10(-3) M) enhanced the oxidation rate of L-tyrosine to dopachrome 40-fold. Hydrogen peroxide was shown to be a competitive inhibitor of tyrosinase when L-tyrosine was the substrate. This reversible inhibition is based on a slow pseudocatalase activity for tyrosinase. Monothiols and dithiols inhibit tyrosinase by different mechanisms. Reduced human thioredoxin and 2,3-dithiopropanol are allosteric inhibitors of tyrosinase yielding bis-cysteinate complexes with one of the copper atoms in the enzyme active site. Bis-cysteinate tyrosinase activity is down-regulated to 30% of native enzyme activity in the L-dopa assay; suggesting a true regulatory role for dithiols. Monothiols such as reduced glutathione and beta-mercaptoethanol are much less reactive with tyrosinase although 10(-3) M monothiol totally inhibits enzyme activity. Reduced thioredoxin inhibits tyrosinase 23-fold more than reduced glutathione under the same experimental conditions.     

Effect of prolactin on the phospholipid composition and cholesterol level in liver microsomes of the carp during acclimation to different temperatures

The phospholipid composition, content of cholesterol and its esters in the carp (Cyprinus carpio L.) liver microsomes depend on the environmental temperature. The free cholesterol amount and cholesterol/phospholipids ratio in microsomes decrease after the lowering of temperature from 20 to 5 degrees C. The temperature elevation to 30 degrees C results in an increase of the cholesterol ester content. The relative proportions of phosphatidyl choline, phosphatidyl ethanolamine, sphingomyelin, phosphatidyl inositol, phosphatidyl serine, phosphatidic acid increase with a significant decrease of the unidentified phospholipids amount at 30 degrees C. Prolactin affects the cholesterol content and phospholipid composition of liver microsomes. The hormone has a more pronounced effect at subextremal temperatures (5 and 30 degrees C). The actions of prolactin and temperature on the cholesterol content are similar. The hormone influence on the membrane phospholipid composition is opposite to the effect of the temperature acclimation. The possible role of prolactin in the temperature adaptation of the membrane lipids metabolism in poikilotherms is discussed.     

Effects of tyrosinase activity on the cytotoxicity of 3,4-dihydroxybenzylamine and buthionine sulfoximine in human melanoma cells

The rationale for melanoma specific dihydroxybenzene containing antitumor agents is based in part upon the ability of the enzyme tyrosinase to oxidize these pro drugs to toxic intermediates. In situ tyrosinase activity was demonstrated to be affected by both cell density and time from plating in pigmented melanoma cells. Phenylthiourea, which completely inhibited tyrosinase activity with minimal cytotoxicity was found to block the growth inhibitory activity of the antitumor dopamine analog 3,4-dihydroxybenzylamine (3,4-DHBA) (NSC 263475). The antioxidant dithioerythritol was also found to inhibit tyrosinase activity and to block the growth inhibitory effects of 3,4-DHBA in pigmented melanoma cell lines. Buthionine sulfoximine (BSO) was shown to be cytotoxic to melanoma cells and its growth inhibitory effects appears to correlate with tyrosinase levels. Furthermore, BSO was shown to potentiate the growth inhibitory effects of 3,4-DHBA on marginally pigmented human melanoma cell lines.     

Effect of dietary fat saturation on acylcoenzyme A:cholesterol acyltransferase activity of rat liver microsomes

The saturation of the fat contained in the diet has been observed to affect the acylcoenzyme A:cholesterol acyltransferase (ACAT) activity of rat liver microsomes. ACAT activity in microsomes (Mp) prepared from livers of rats fed a polyunsaturated fat-enriched diet containing 14% sunflower seed oil was 70-90% higher than in microsomes (Ms) prepared from livers of rats fed a saturated fat-enriched diet containing 14% coconut oil. This difference was observed within 20 days after the diets were begun, the earliest time tested, and persisted throughout the 70-day experimental period. The difference was noted at all [1-14C]palmitoyl CoA concentrations tested, 2.5-33 micronM, and at temperatures between 18 and 40 degrees C. Arrhenius plots revealed a single transition in enzyme activity, occurring at 29 degrees C in both microsomal preparations. Likewise, the activation energy above this transition was the same in Mp and Ms, 12.5 KCal/mol. Addition of albumin to the incubation medium increased the ACAT activity of both microsome preparations, but the difference between Mp and Ms persisted. Mp was enriched in polyenoic fatty acids, primarily 18:2 and 20:4, while Ms was enriched in monoenoic acids. Although the 20:4 increase in Mp occurred in all phosphoglycerides, it was especially pronounced in the serine and inositol phosphoglyceride fraction. There were no differences in the phospholipid or cholesterol content, phospholipid head group composition, or protein composition of the two microsomal preparations. The possibility is discussed that the changes in ACAT activity result from the differences in fatty acid composition of the microsomes. Other microsomal enzymes exhibited varying responses to these dietary fatty acid modifications. Palmitoyl CoA hydrolase and NADPH cytochrome c reductase activities were unchanged. UDP glucuronyl transferase activity was 50% higher in Mp, but glucose-6-phosphatase and NADH cytochrome b5 reductase activities were 25% higher in Ms. Therefore, dietary fat modifications do not produce a uniform effect on the activity of microsomal enzymes.     

BIOGENESIS OF ENDOPLASMIC RETICULUM MEMBRANES : II. Synthesis of Constitutive Microsomal Enzymes in Developing Rat Hepatocyte

The constitutive enzymes of microsomal membranes were investigated during a period of rapid ER development (from 3 days before to 8 days after birth) in rat hepatocytes. The activities studied (electron transport enzymes and phosphatases) appear at different times and increase at different rates. The increase in the enzyme activities tested was inhibited by Actinomycin D and puromycin. G-6-Pase and NADPH-cytochrome c reductase activities appeared first in the rough microsomes, and subsequently in smooth microsomes, eventually reaching a uniform concentration as in adult liver. The evidence suggests that the enzymes are synthesized in the rough part, then transferred to the smooth part, of the ER. Changes in the fat supplement of the maternal diet brought about changes in the fatty acid composition of microsomal phospholipids but did not influence the enzymic pattern of the suckling. Microsomes from 8-day-old and adult rats lose 95% of PLP and 80% of NADH-cytochrome c reductase activity after acetone-H₂O (10:1) extraction. However, one-half the original activity could be regained by adding back phospholipid micelles prepared from purified phospholipid, or from lipid extracts of heart mitochondria, or of liver microsomes of 8-day or adult rats, thus demonstrating an activation of the enzyme by nonspecific phospholipid. The results suggest that during development the enzymic pattern is not influenced by the fatty acid or phospholipid composition of ER membranes.     

Comparison of phospholipid effects on insulin-sensitive low Km cyclic AMP phosphodiesterase in adipocyte plasma membranes and microsomes

Both adipocyte plasma membranes and microsomes possess insulin-sensitive low K_m cyclic AMP phosphodiesterase activity. The activity of the enzyme from both sources was susceptible to activation by several anionic phospholipids. Activators of the plasma membrane enzyme were lysophosphatidylglycerol > lysophosphatidylcholine > lysophosphatidylserine > phosphatidylserine > phosphatidylglycerol. These same phospholipids activated the microsomal enzyme but the extent of activation by each phospholipid was reversed. Neutral phospholipids and other anionic phospholipids were without effect. The phospholipids had no effect on high K_m cAMP phosphodiesterase in either membrane. The results suggest that the phospholipid headgroup was an important determinant for enzyme activation by phospholipid. The increased susceptibility of the plasma membrane enzyme to lysophospholipid may be attributed to a difference in the plasma membrane enzyme compared to the microsomal membrane enzyme or to differences in plasma membrane and microsomal membrane phospholipid composition and their ability to regulate low K_m cAMP phosphodiesterase activity.     

The modulation of choline phosphoglyceride metabolism in human colon cancer

Colorectal cancer has a high incidence of morbidity and mortality in the North American population. Elevated levels of plasmalogens have been reported in some neoplastic tissues including colon tumors, but the mechanism for this increase has not been defined. Since changes in plasmalogen level are usually associated with changes in the other phospholipid subclasses, a general increase in all phospholipid subclasses may also be found in colonic neoplasms. In this study, the levels of the major phospholipids, including their plasmalogen and diacylphospholipid subclasses, were found to be elevated in human malignant colonic tissues. Since phosphatidylcholine is the most prominent type of phospholipid found in both malignant and control tissues, the mechanism for its accumulation during malignancy was investigated. Decreases in phospholipase C and D activities were observed in tumor samples, but an enhancement of the CTP: phosphocholine cytidylyltransferase activity was also detected. Immunoblotting analysis revealed that the elevated cytidylyltransferase activity was caused by a three-fold increase in the level of enzyme protein during tumor development. Based on these enzyme studies, we conclude that the high level of phosphatidylcholine in colon tumors resulted from a decrease in its turnover and an increase in its expression.     

Differential response of chick liver and brain membranes to short ethanol treatment

The effect of 60 hr ethanol ingestion on lipid composition of liver and brain membranes from 2-day-old chicks was investigated. Analysis of hepatic membrane cholesterol shows that ethanol induced a slight increase in microsomes exclusively due to free cholesterol while mitochondria was not affected. In brain, both fractions showed a clear increase in their cholesterol content, while a high decrease was observed in myelin. Free cholesterol was also the main responsible for the changes found in brain. The ethanol-treated animals showed an alteration in their phospholipid composition exclusively in brain microsomes and myelin. Despite all these changes, the values of cholesterol/phospholipid molar ratio in both liver and brain membranes remained unaltered after short ethanol treatment. Our results indicate that neonatal chick brain membranes appears to be especially sensitive to the presence of ethanol.     

Lipid peroxidation and physical properties of smooth and rough hepatoma microsomes

Smooth and rough endoplasmic reticulum of two Morris hepatomas, the slow growing 9618A and the fast growing 3924A, have been isolated, and their biochemical composition, supramolecular organization, and response to the action of peroxidative agents have been studied. Cytochrome P450 content and lipid availability are the limiting factors of their peroxidizability. The hemoprotein content is reduced about 80% in hepatoma 9618A and is virtually absent in hepatoma 3924A. The peroxidizability decreases with increasing growth rate of the tumor. The protein, phospholipid, and cholesterol content, the fatty acid composition as well as the double bond index, and the saturated and unsaturated fatty acid content are reported. Differences have been found between normal liver and tumors and between the fractions within a given tumoral tissue. The molecular order, as determined by fluorescence anisotrophy decay of DPH, increases in total microsomes and in the smooth fraction going from liver 9618A to 3924A, whereas for the rough fraction it is the same in liver and hepatoma 9618A; in 3924A it increases of about 30%. Fluidity decreases in total microsomes going from liver to 3924A, to 9618A. In both the purified fractions it decreases with increasing deviation of the tumor.