Guido V. Marinetti

Tightly (covalently) bound fatty acids in cell membrane proteins

Abstract We find tightly (covalently) bound fatty acids in membrane proteins from human red cells and polymorphonuclear cells and several tissues of the rat. These fatty acids are not removed by exhaustive extraction with organic solvents, by phospholipase A 2 treatment, or by sodium dodecyl sulfate (SDS). The fatty acids are released by refluxing with hot methanolic-HCl. They account for 0.2–6.8% of the total fatty acids. 50–60% of the tightly bound fatty acids are removed by mild alkaline hydrolysis. [ 3 H]Palmitic acid is incorporated into lipids and proteins of the red cell membrane. In red cell ghosts incorporation is stimulated by ATP and CoA but maximal incorporation is achieved with [ 14 C]palmityl-CoA. The labeling of lipids and proteins by [ 14 C]palmityl-CoA is greater in ghosts than in cells, however basal incorporation of [ 3 H]palmitic acid into lipids and proteins is greater in cells than in ghosts. [ 3 H]Palmitic acid is also incorporated into proteins of polymorphonuclear cells. The incorporation in polymorphonuclear cell proteins is inhibited by cycloheximide but not by actinomycin D. Labeled stearic, linoleic, linoleic, and palmitic acid are incorporated into red cell membrane proteins. These incorporated fatty acids are not removed by exhaustive solvent extraction, by prolonged dialysis or by SDS. On SDS-polyacrylamide gel electrophoresis several protein bands are labeled by all the fatty acids although each fatty acid gives a different pattern of labeling. In all cases, the major protein band which is labeled stains very weakly with Coomassie blue and runs between band 4.2 and 5. Other proteins which have appreciable label are bands 1, 2 and 3. We postulate that fatty acids are covalently attached to certain proteins as a post-translational event and act to direct, insert, and anchor the proteins in cell membranes. A similar postulate has been enunciated by Schmidt and Schlessinger (Cell 17, 813–819 (1979)).

alpha 2-Adrenergic receptors in human polymorphonuclear leukocyte membranes

Human polymorphonuclear cell membranes contain alpha 2-adrenergic receptors which are measured by binding of the alpha 2-adrenergic antagonist [3H]yohimbine. The alpha 1-adrenergic antagonist [3H]prazosin showed no specific binding. High and low affinity sites were detected which had Kd values of 2.38 +/- 0.4 and 139 +/- 12 nM, respectively, and which bound maximally 4.82 +/- 0.9 and 81 +/- 9 fmoles of [3H]yohimbine/mg membrane protein. The high and low affinity sites were also detected by competition studies with phentolamine, epinephrine and norepinephrine and by dissociation kinetics of bound [3H]yohimbine. [3H]Yohimbine binding was stereospecifically inhibited by (-)- and (+)-epinephrine and norepinephrine. [3H]Yohimbine binding to intact cells showed about 500 high affinity sites per cell (Kd 0.5 nM) and approximately 4000 lower affinity sites per cell (Kd 3-4 nM). Yohimbine enhanced the (-)-norepinephrine stimulation of cAMP production in intact cells.

Composition and metabolism of phospholipids of human leukocytes

Human mononuclear (MN) and polymorphonuclear (PMN) leukocytes were analyzed for their phospholipid, triglyceride, cholesterol and fatty acid content. The phospholipid/cholesterol ratio was 1.24 for both cells. MN cells contain more phosphatidylcholine (PC), but less phosphatidylserine (PS), Phosphatidylethanolamine (PE) and sphingomyelin (SPH) than PMN cells when expressed as percent of total phospholipid. When expressed on the basis of lipid content per cell, MN cells contain less PS, PE and SPH but more triglyceride than PMN cells. PMN cells incorporate palmitic, stearic, linoleic and linolenic acids into their phospholipids, triglycerides or cholesterol esters. The incorporation into triglycerides was highest for all fatty acids. Of the phospholipids, the incorporation was highest into PC. Labeled fatty acids also were found in proteins which had been delipidized by exhaustive extraction with organic solvents. These represent tightly or covalently bound fatty acids. The incorporation [3H] palmitic acid into this protein fraction is stimulated by insulin.

Lipid analysis of cells and chromatophores of Rhodopseudomonas sphaeroides

Abstract The phospholipids and fatty acid analysis of four strains of Rhodopseudomonas sphaeroides and of chromatophores from two strains show some differences and also show the presence of an unusual polar neutral lipid which is ninhydrin positive and which on acid hydrolysis yields ornithine and an unidentified amino compound. This lipid is called aminolipid-X and has a fatty acid composition very different from the phospholipids. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) contain a very small amount of plasmalogen forms as determined by combined mild alkaline hydrolysis, acetic acid hydrolysis and phospholipase A2 hydrolysis. The reaction of intact cells and chromatophores with trinitrobenzenesulfonate (TNBS), fluorodinitrobenzene (FDNB) and isethionylacetimidate (IA) show that 78% of the total PE in chromatophores is localized on the outer membrane surface. In intact cells about 15–35% of the total PE is localized on the outer surface of the plasma membrane.

The interaction of γ-globulin with lipids☆

Abstract γ-globulin interacts with cardiolipin (CL), phosphatidic acid (PA) and phosphatidylserine (PS) to yield an insoluble complex. The bound PA and PS can be nearly all removed by extraction with organic solvents, but about 50 per cent of the bound CL is not extracted. Amino acid analysis shows that lysine, arginine, histidine, tyrosine, methionine and cysteine are involved in the binding of CL to γ-globulin. The binding of CL to γ-glubulin exhibits a sharp p H profile in phosphate buffers with a maximum between 5.7 to 6.1. The interaction is influenced by ionic strength and shows a broad peak at 0.06 M NaCl. Freshly prepared cardiolipin (in water) shows maximal activity. The activity decreases with aging of the aqueous cardiolipin dispersion. Lecithin and cholesterol alone do not form an insoluble complex with γ-globulin and have either no influence or slightly inhibit the Cl-γ-globulin reaction. However, when lecithin and cholesterol are mixed they interact with γ-globulin to yield an insoluble complex. Moreover, the lecithin—cholesterol mixture greatly enhances the Cl—γ-globulin reaction. Sphingomyelin can not substitute for lecithin. The bound lecithin and cholesterol in the γ-globulin precipitate are completely extracted by organic solvents. A variety of γ-globulins were studied and all react with CL and with lecithin—cholesterol or lecithin—cholesterol mixtures but to varying degrees. The reaction of γ-globulin with CL is markedly inhibited by p-hydroxymercuribenzoate and iodoacetate. Bence—Jones protein reacts very weakly with CL but quite strongly with a mixture of CL—lecithin—cholesterol.

Characterization of an 11,000-dalton beta-bungarotoxin: binding and enzyme activity on rat brain synaptosomal membranes.

The binding and phospholipase A2 activity of an 11,000-dalton beta-bungarotoxin, isolated from Bungarus multicincutus venom, have been characterized using rat brain subcellular fractions as substrates. 125I-labeled beta-bungarotoxin binds rapidly (k = 0.14 min-1 and 0.11 min-1), saturably (Vmax = 130.1 +/- 5.0 fmoles/mg and 128.2 +/- 7.1) fmoles/mg), and with high affinity (apparent Kd = 0.8 +/- 0.1 nM and 0.7 +/- 0.1 nM) to rat brain mitochondria and synaptosomal membranes, respectively, but not to myelin. The binding to synaptosomal membranes is inhibited by divalent cations and by pretreatment with trypsin. The binding results suggest that the toxin binds to specific protein receptor sites on presynpatic membranes. The 11,000-dalton toxin rapidly hydrolyzes synaptosomal membrane phospholipids to lysophosphatides and manifests relative substrate specificity in the order phosphatidyl ethanolamine greater than phosphatidyl choline greater than phosphatidyl serine. These results indicate that the 11,000-dalton beta-bungarotoxin is a phospholipase A2 and can use presynaptic membrane phospholipids as substrates. The binding, phospholipase activity and other biological properties of the 11,000-dalton toxin are contrasted with those of the beta-bungarotoxin found in highest concentration in the venom (the 22,000-dalton beta-bungarotoxin), and the two toxins are shown to have qualitatively similar properties. Finally the results are shown to support the hypothesis that beta-bungarotoxins act in a two-step fashion to inhibit transmitter release: first, by binding to a protein receptor site on the presynatic membrane associated with Ca2+ entry, and second, by perturbing through enzymatic hydrolyses the phospholipid matrix of the membrane and thereby causing an increase in passive Ca2+ permeability.

Dietary Management of Elevated Blood Lipids

High levels of plasma cholesterol, in particular LDL-cholesterol, are associated with premature atherosclerosis and heart disease. Therefore, it is important to know how drug therapy and dietary management can be used to lower blood cholesterol levels. The effect of dietary management, particularly of dietary lipids, will be considered first, since for most people this approach to treatment of of hyperlipidemia is tried before drug therapy.

Light-enhanced cross-linking of rhodopsin in rod outer segment membranes as detected by chemical probes

Bovine rod outer segment membranes were treated with cross-linking reagents before and after light exposure. Bleached membranes showed enhanced cross-linking with difluorodinitrobenzene or methyl acetimidate compared to dark-adapted membranes. The light-induced enhancement of cross-linking may be due to increased association of rhodopsin monomers in the light and/or due to increased reactivity of amino and sulfhydryl groups of bleached rhodopsin. In some instances, the band ascribed to the rhodopsin monomer in gel electrophoresis appears as a partially resolved doublet. Treatment of bleached rod outer segment membranes with methyl acetimidate improved the resolution of the doublet into two closely migrating bands.

The feasibility of dating rock paintings from Brandberg, Namibia, with 14C

The composition of six rock paintings from the Brandberg in Namibia has been examined by highly sensitive chemical separation in combination with chromatographic identification and by surface excitation probes. This study concludes that the paintings examined contain no organic material suitable for carbon dating. These tests differ from previous analyses (van der Merwe et al., 1987) in that they attempt to isolate specific organic compounds. Our results suggest that the technique used to produce paintings at Brandberg was not like those described in accounts of rock and body painting which include the use of fats to bond inorganic pigments (Rudner, 1982, 1983).

Synthesis of 3-O-acyl esters of serine and threonine

Abstract The 3- O -acyl derivatives of serine and threonine have been prepared by reacting oleoyl chloride and palmitoyl chloride with N - t -butoxycarbonyl (N-T-BOC) serine and N - t -BOC threonine. The t -BOC group was removed by treatment with 4 N HCl in dioxane. The products were identified by proton magnetic resonance spectroscopy, infrared spectroscopy, elemental analysis and chromatographic properties. The O -acyl serines and O -acyl threonines were converted to their methyl esters by treatment with boron trifluoride in methanol and were converted to their dinitrophyl derivatives by treatment with dinitrofluorobenzen (DNFB). The yield of the dinitrophenyl derivatives was very high but the yield of methyl esters was low due mainly to methanolysis and loss of the fatty acyl group. The O -acyl serines and O -acyld threonines prepared will provide standards for researchers who are interested in identifying fatty acids esterified to serine and threonine hydroxyl groups in membrane proteins.