A study of the Influence of mevalonic acid and its metabolites on the morphology of swiss 3T3 cells.

We used two model systems to investigate the effect of compactin, a competitive inhibitor of beta-hydroxy beta-methylglutarylcoenzyme A reductase, on the shape of Swiss 3T3 cells. We maintained cells in a quiescent state in medium deficient in platelet-derived growth factor (PDGF), or we added PDGF to quiescent cells to initiate traverse through a single cell cycle. In both systems, the cells responded to compactin by acquiring a characteristic rounded shape. Cell rounding seemed to depend on an induced deficiency of mevalonic acid (MVA) since the response could be prevented or reversed by adding MVA to the culture medium. Compactin-induced rounding appeared in PDGF-stimulated cells concomitantly with a compactin-mediated inhibition of DNA synthesis, and both effects had similar sensitivities to exogenous compactin and MVA. However, cell rounding seemed to be unrelated to other, previously observed effects of MVA deficiency. Compactin did not influence the total content of cell cholesterol, and little cholesterol was formed when we added radioactive MVA to round cells to effect shape change reversal. Measurement of the dolichol-dependent glycosylation of cell protein revealed no evidence of dolichol deficiency. In addition, reversal of cell rounding by MVA was not prevented by concentrations of tunicamycin that effectively blocked the incorporation of radioactive mannose into cell protein or by concentrations of cycloheximide that blocked protein synthesis. Taken together, our results suggest a new role for MVA or its products in the maintenance of cell shape.

Identification of geranylgeranyl-modified proteins in HeLa cells.

Previous studies have shown that animal cells contain isoprenoid-modified proteins and that one of these proteins, lamin B, contains a thioether-linked farnesyl group that is attached to cysteine. In the present study, a novel isoprenoid-modification was identified by labeling HeLa cells with [3H]mevalonic acid and analyzing proteolytic digests of the total cell protein. Radioactive fragments were purified from these digests and treated with Raney nickel. The released, labeled material was analyzed by gas-liquid chromatography (GC) and mass spectrometry (MS). This approach revealed that an all-trans geranylgeranyl group was a major isoprenoid modification.

Prenyl proteins in eukaryotic cells: a new type of membrane anchor.

Recent studies have indicated that eukaryotic cells contain proteins that are post-translationally modified by long-chain, thioether-linked prenyl groups. These proteins include yeast mating factors, ras proteins and nuclear lamins. The modification occurs on a cysteine residue near the C terminus and appears to initiate a set of additional protein modification reactions that promote attachment of the proteins to specific membranes.

Plasma lipoproteins in familial lecithin: cholesterol acyltransferase deficiency: lipid composition and reactivity in vitro.

Plasma lipoproteins from patients with familial lecithin:cholesterol acyltransferase (LCAT) deficiency have been fractioned by preparative ultra-centrifugation and gel filtration and their lipid content and reactivity studied. All of the lipoproteins are abnormal with respect to lipid concentration or relative lipid content. The low density lipoproteins (LDL) and high density lipoproteins (HDL) appear to react normally with partially purified LCAT from normal plasma. Also, the lipids of the very low density lipoproteins (VLDL) and LDL, like those of the corresponding lipoproteins of normal plasma, are indirectly altered by the action of LCAT on normal HDL. Thus, during incubation in vitro VLDL cholesteryl ester is increased and VLDL triglyceride is decreased, as described by others for VLDL from hyperlipemic plasma, and both the unesterified cholesterol and lecithin of the VLDL and LDL are decreased. The patients' VLDL and LDL are abnormal, however, in that they lose unesterified cholesterol and lecithin to normal HDL in the absence of LCAT. Also, the patients' HDL lose these lipids to erythrocyte membranes in the absence of the enzyme. Our results provide further evidence that the abnormal cholesterol and phospholipid composition of the patients' lipoproteins is caused by the LCAT deficiency. They support the postulate that an excess of unesterified cholesterol and lecithin develops as VLDL are converted to LDL and HDL and suggest that in the absence of LCAT this excess lipid distributes among plasma lipoproteins and plasma membranes. They further suggest that LCAT normally reduces this excess lipid through a combination of direct and indirect effects.

Plasma lipoproteins in familial lecithin: cholesterol acyltransferase deficiency: physical and chemical studies of low and high density lipoproteins.

LOW DENSITY LIPOPROTEINS (LDL) AND HIGH DENSITY LIPOPROTEINS (HDL) FROM THE PLASMA OF PATIENTS WITH FAMILIAL LECITHIN: cholesterol acyltransferase (LCAT) deficiency have been characterized by gel filtration, analytical ultracentrifugation, and gel electrophoresis, and their relative content of lipid and protein has been determined. The LDL of d 1.019-1.063 g/ml show marked heterogeneity. A subfraction of the LDL emerges from columns of 2% agarose gel with the void volume, has corrected flotation rates (S(f) degrees ) in the range of 20-400, and contains 4-10 times as much unesterified cholesterol, phosphatidylcholine, and triglyceride per mg protein as normal LDL. A major subfraction of the LDL emerges from the gel in the same general position as normal LDL, but exhibits somewhat higher flotation rates and contains 1.5-3 times as much unesterified cholesterol and phosphatidylcholine and 13 times as much triglyceride per mg protein. The HDL, shown to be heterogeneous in earlier studies, are mainly comprised of molecules which have flotation rates of F(1.20) 3-20, migrate in the alpha(1)-alpha(2) region on electrophoresis, and contain about 12 times as much unesterified cholesterol and 5 times as much phosphatidylcholine per mg protein as normal HDL. Smaller molecules are also detected, which have flotation rates of F(1.20) 0-3, migrate in the prealbumin region on electrophoresis, and contain only slightly more unesterified cholesterol and phosphatidylcholine per mg protein than normal HDL.

Plasma lipoproteins in familial lecithin: cholesterol acyltransferase deficiency: structure of low and high density lipoproteins as revealed by elctron microscopy.

The low density lipoproteins (LDL) of d 1.019-1.063 g/ml of patients with familial lecithin: cholesterol acyltransferase (LCAT) deficiency show marked heterogeneity when viewed with the electron microscope. At least two types of particles are present, one large and the other small. The large particles predominate in a LDL subfraction of large molecular weight isolated by gel filtration on 2% agarose gel. They appear to be flattened structures with diameters mainly in the range of 900-1200 A. The small particles predominate in a LDL subfraction of smaller molecular weight isolated by filtration on the same type of gel. They are 210-250 A in diameter and are similar to normal LDL in size and shape. The high density lipoproteins (HDL) also are heterogeneous. The majority of particles are disc-shaped structures 150-200 A in diameter. The discs are mainly present in stacks which have a periodicity of 50-55 A and a variable length. Each disc appears to be made up of a rosette of smaller globular units 50 A in diameter. The appearance of these large molecular weight HDL contrasts with that of normal HDL, which are 70-100 A in diameter and aggregate in monolayers that show hexagonal packing of particles. A small percentage of the patients' HDL consists of structures 45-60 A in diameter. These predominate in a smaller molecular weight HDL subfraction isolated by gel filtration on Sephadex G200. The particles are present in monolayer aggregates but never form stacked structures similar to those seen in the large molecular weight HDL subfraction.

Plasma lipoproteins in familial lecithin: cholesterol acyltransferase deficiency. Further studies of very low and low density lipoprotein abnormalities.

Plasma lipoproteins of d<1.006 g/ml, d 1.006-1.019 g/ml, and d 1.019-1.063 g/ml from patients with familial lecithin:cholesterol acyltransferase deficiency yielded abnormal subfractions upon being separately filtered through 2% agarose gel. A subfraction that emerged with the void volume and contained unusually large amounts of unesterified cholesterol and phosphatidylcholine was present in each lipoprotein group, and in each group this subfraction was less prominent in the nonlipemic plasma of one patient than in the lipemic plasma of other patients. A subfraction containing smaller lipoproteins also was present in each lipoprotein group. These lipoproteins were of the same size as normal lipoproteins of the corresponding density, but contained abnormally small amounts of cholesteryl ester. The lipoproteins of 1.019-1.063 g/ml contained abnormal components of intermediate molecular weight as well as large and small abnormal components similar to those described previously. The intermediate components were more prominent in the nonlipemic plasma but were easily recognized in the hyperlipemic plasma as a peak of S(f) 20-30 in the analytical ultracentrifuge. Also they could be recognized, upon electron microscopy of the lipoproteins of d 1.019-1.063 g/ml, as particles 340-1000 A in diameter. The data suggest that related large, abnormal particles pervade the patients' very low and low density lipoproteins, and that the large particles are affected by, but are not dependent on, the lipemia that frequently accompanies the disease. The smaller very low and low density lipoproteins appear to be counterparts of lipoproteins present in normal plasma. Their abnormal composition is compatible with the possibility that lecithin:cholesterol acyltransferase normally decreases the triglyceride and phosphatidylcholine and increases the cholesteryl ester of very low density and low density plasma lipoproteins in vivo.

sn-1,2-Diacylglycerols and phorbol diesters stimulate thromboxane synthesis by de novo synthesis of prostaglandin H synthase in human promyelocytic leukemia cells.

We studied the regulation of thromboxane (TX) synthesis in promyelocytic leukemia cells during macrophage differentiation. Cells treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) showed rates of TXB2 synthesis from exogenous arachidonic acid that exceeded that of control cells by a factor of up to 81. Cells treated with sn-1,2-dioctanoylglycerol (diC8) showed similarly high TXB2 synthesis rates when diC8 was added concomitantly with a subthreshold concentration of TPA or when given in multiple doses. These activities depended on de novo synthesis of prostaglandin H (PGH) synthase because: microsomal PGH synthase activity showed large increases in Vmax values, and mass measurements of PGH synthase revealed the presence of PGH synthase in differentiating cells whereas the enzyme was undetectable in control cells. These results indicate that macrophage differentiation is associated with stimulation of TXB2 synthesis that requires both activation of protein kinase C and de novo synthesis of PGH synthase.

Increased fluid pinocytosis, induced by action of platelet-derived growth factor on quiescent arterial smooth muscle cells, does not require increased cholesterol biosynthesis.

In the current report we provide evidence that the increased rate of cholesterol biosynthesis mediated by platelet-derived growth factor in the cell cycle of monkey (Macaca nemestrina) arterial smooth muscle cells can be separated from the increased rate of fluid pinocytosis using inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

Cholesteryl ester hydrolytic acitivity of rat liver plasma membrane.

Cholesteryl ester hydrolyzing activity of rat liver plasma membranes was studied using acetone-dispersed [4-14-C] cholesteryl oleate as substrate. In contrast to whole liver homogenates which displayed ample activity at both acid (4.5) and neutral (6.2-7.4) pH, purified plasma membrane fractions contained little activity at neutral pH as compared to acid pH. Moreover, rate-zonal sucrose density-gradient centrifugation patterns of plasma membrane rich fractions suggested a specific association with plasma membrane only in the case of the acid activity. These findings suggest that in vivo hepatic cell surface membranes contain little or no cholesteryl ester hydrolytic activity at extracellular pH. They support the possibility that plasma lipoprotein cholesteryl esters enter hepatic parenchymal cells prior to hydrolysis.

Studies of the interaction between apolipoproteins A and C and triacylglycerol-rich particles.

We studied the interaction of apolipoproteins A-I, C, and HDL2 with phospholipid-stabilized, triacylglycerol-rich particles to learn more about the molecular mechanisms that underlie the metabolism of chylomicrons. Apolipoproteins A-I, C-I, C-III1 and C-III2 all bound to and destabilized triacylglycerol-rich particles, apparently by removing phospholipids from the particle surface. None of the apolipoprotein C tested, at any concentration, was, however, able to equal the disruptive effect of apolipoprotein A-I. The destabilizing effects of apolipoproteins A-I and C were not additive. Apolipoprotein C-III1 seemed to lessen the disruptive effect of apolipoprotein A-I by binding competitively to triacylglycerol-rich particles. Unexpectedly, previous binding of apolipoprotein A-I to triacylglycerol-rich particles nearly tripled the ability of these particles to bind apolipoprotein C. Destabilization of triacylglycerol-rich particles by apolipoprotein A-I was prevented by HDL2. The protective effect of HDL2 seemed to depend partly on transfer of unesterified cholesterol from HDL2, since the amounts of unesterified cholesterol and apolipoprotein A-I bound to the particles surface showed a strong negative correlation.

In vitro effects of lecithin:cholesterol acyltransferase on apolipoprotein distribution in familial lecithin:cholesterol acyltransferase deficiency.

Action of LCAT on the plasma of patients afflicted with familial LCAT deficiency shifts the distribution of C apolipoproteins from lipoproteins of d less than 1.019 g/ml to lipoproteins of d greater than 1.109 g/ml, and causes an opposite shift in the distribution of apolipoprotein E. The altered distribution of apolipoprotein E appears to depend primarily on enzyme-related effects on HDL. Loss of apolipoprotein E from HDL occurs as cholesteryl esters are formed and transfer to other lipoproteins; disc-shaped HDL, rich in apolipoprotein E, are converted into spherical particles; and the population of HDL as a whole is converted first into particles the size of HDL2 and HDL3 and then into intermediate-sized particles. Transfer of apolipoprotein E to artificially prepared triglyceride-rich particles occurs at a nearly linear rate that is slow than the rates of formation and transfer of cholesteryl esters or the rate of formation of "HDL2" and "HDL3." Transfer of apolipoprotein E is faster, however, when the patients' disc-shaped HDL are incubated with triglyceride-rich particles in the presence of normal plasma lipoproteins of d greater than 1.063 g/ml. Since the disc-shaped HDL, rich in apolipoprotein E, resemble particles reported to be released from perfused rat livers, they may be nascent lipoproteins of hepatic origin. If so, it appears that action of LCAT on these lipoproteins may be one of the factors that regulates the content of apolipoprotein E in VLDL.

Plasma lipoproteins in familial lecithin: cholesterol acyltransferase deficiency: effects of dietary manipulation.

To study the metabolism of the abnormal plasma lipoproteins in familial lecithin:cholesterol acyltransferase deficiency we performed five dietary experiments designed to perturb their distribution and composition. Four patients with the disease were given successive diets that differed in triglyceride, carbohydrate, or cholestrol content, and after each dietary period the lipoproteins were analyzed by combinations of preparative and analytical ultracentrifugation, gel filtration, chromatography, and disc gel electrophorsis. Lowering the intake of long chain, dietary triglyceride descreased the concentrations of the large very low density lipoproteins, the large and intermediate low density lipoproteins, and the small high density lipoproteins by as much ad 79 %, but either increased or did not change the concentrations of the small very low and low density lipoproteins. Re-adding long chain triglycerdine to the diet generally reversed these effects, but increasing the dietary cholesterol without lowering the dietary triglyceride only decreased the concentration of plasma cholesteryl ester. We conclude that the concentrations of the large very low and low sensity lipoproteins, the intermediate-sized low density lipoproteins, and the small high density lipoproteins are related to the absorption and subsequent transport of long chain dietary fatty acids. Since these lipoproteins are rich in unesterified cholesterol and lecithin, two polar lipids that form a substantial part of the surfaces of chylomicrons, components of chylomicron surfaces may accumulate in the patient's plasma following enzymic removal of chylomicron triglyceride and contribute to several of the abnormal lipoproteins.