Long-chain fatty acids bound to alpha-fetoprotein and to serum albumin from fetal and adult pig

1. Pig alpha-fetoprotein (AFP) and albumin were isolated from fetal serum by DEAE-Sephadex ion exchange chromatography combined with Cibacron Blue-Sepharose and trypsin-Sepharose adsorptions. 2. AFP, fetal albumin and adult albumin carried 2.6, 2.4, and 1.9 moles of fatty acids per mole of protein, respectively. 3. Most of fatty acids bound to AFP were polyunsaturated: mainly arachidonic (20:4, n-6) and docosahexaenoic (22:6, n-3) acids, which accounted respectively for 21.7 and 18.8% of the total fatty acids. 4. By contrast, the fatty acids found in the albumins (fetal and adult) were preferentially saturated and monounsaturated. 5. Arachidonic acid was a minor component in both albumins, and no docosahexaenoic acid was detected.     

Fatty acids bound to alpha-fetoprotein and albumin during rat development

The time-course levels and composition of the fatty acids bound to rat alpha-fetoprotein (AFP) and albumin from several sources, were determined throughout development, and related to the intake of lipids from milk and the compositional changes in brain and liver fatty acids. The major fatty acids bound to AFP were acids bound to AFP were polyunsaturated and mainly docosahexaenoic acid (22:6(n-3], either from fetal serum (23.1%) or whole fetuses (21.6%), whereas palmitic (34.1%) and oleic (29.9%) acids were the main acids bound to albumin from the same sources. Amniotic fluid AFP contained less fatty acids (0.8 mol/mol protein) than that of fetal serum (1.4 mol/mol protein), and especially noticeable was a reduced amount of 22:6 (9.6%). Both AFP-concanavalin A microforms showed identical fatty acid composition. Levels of 22:6 bound to AFP decreased quickly after birth until a minimum at 8-10 days, increasing moderately thereafter. This minimum is coincident in time with a maximal accumulation of this fatty acid by brain and a loss of 22:6 by liver. Except for colostrum, levels of 22:6 in milk lipids were low and fairly constant, but always greater than those of its precursor, linolenic acid (18:3 (n-3]. These results support a specialized role of AFP in the plasma transport and tissue delivery of polyunsaturated fatty acids, and mainly docosahexaenoic acid.     

Detection of squalene in alpha-fetoprotein and fetal serum albumin from bovine

Alpha-fetoprotein and fetal serum albumin have been simultaneously purified from fetal bovine serum by mild procedures utilizing ammonium sulfate, hydrophobic interaction, immobilized metal (nickel) affinity chromatography, and isoelectric focusing. The lipidic extract from each protein was analyzed by gas chromatography and the peak appearing just after the arachidonic acid was identified as squalene by gas chromatography-mass spectrometry. This isoprenoid was not detected formerly in these proteins from human, rat, bovine, and pig. Until recently, in the analysis of the fatty acid composition of the alpha-fetoprotein and serum albumin from mammals, a peak has been assigned in the last part of the chromatographic profile, after arachidonic acid, to docosahexaenoic acid. In the present work, it was found that the peak corresponds to squalene instead of docosahexaenoic acid. Furthermore, we conclude that bovine alpha-fetoprotein and fetal serum albumin carry squalene, but not docosahexaenoic acid. These results agree with others obtained analyzing the same proteins from chick embryo.     

Electrophoretic isolation and partial characterization of a major secretory glycoprotein from the submandibular glands of the mouse

After either cholinergic or adrenergic stimulation of the submandibular glands of the mouse, a major protein of the incubation medium could be isolated by electrophoresis, designated the AM2 protein. About 5 per cent of the secreted proteins and 2.4 per cent of the secreted protein-bound sialic acid was recovered as the purified AM2 protein. The AM2 protein appeared to be electrophoretically pure in 7.5% polyacrylamide gel both at pH 8.9 and at pH 4.3. In sodium dodecyl sulfate-electrophoresis the molecular weight was estimated to be about 80 000 for the major component and about 40 000 for the minor component. By isoelectric focusing the isoelectric point has been determined to be 4.7. The amino acid analysis indicated Glx, Asx, Leu and Ala as the major amino acids, comprising 15.0, 10.6, 9.2 and 9.1 per cent of the amino acid residues, respectively. The ratio of the acidic amino acids and their amides (Glx plus Asx) to the basic amino acids (Lys plus Arg) was 2.2. The sugar analysis showed that the AM2 glycoprotein consists of 17.3 per cent of carbohydrate, with as major carbohydrate component glucosamine. The molar ratio of the sugars was Man : Gal : Glc : GlcNH2 : sialic acid = 2.3 : 1.0 : 4.7 : 9.8 : 2.9. Galactosamine could be detected as a trace component and fucose was not detectable.     

Calcium ionophore A 23187 induces arachidonic acid release from phosphatidylcholine in cultured human endothelial cells

Cultured endothelial cells from human umbilical vein were incubated with (³H)arachidonic acid for 24 hours. The label was incorporated into phospholipids (79.3 %), neutral lipids (15.6 %) and non-esterified fatty acids (4.7 %). Upon challenge with the calcium ionophore A 23187, 5.3 % of the total radioactivity were found in supernatant and corresponded to 6-keto-prostaglandin F_1α (1.6 %) and free arachidonic acid (3.7 %). This release was accompanied by a concomitant and selective decrease of phosphatidylcholine. It is concluded that the entry of calcium promoted by A 23187 activates a phospholipase A₂ regulating the availability of arachidonic acid to the prostacyclin synthetase.     

Characterization and quantitation of fatty acids covalently bound to erythrocyte membrane proteins: anion transporter contains 1 mol of fatty acid thiol ester

Human erythrocyte membranes which had been thoroughly extracted with organic solvents contained 20 nmol of fatty acids/mg dry wt. The major fatty acids were palmitic and stearic with their monoethenoic derivatives as minor constituents. No other fatty acids were detected. When solvent-extracted membranes were digested with Pronase about 90% of the original content of fatty acids was retained in the insoluble residue. Fatty acids were linked to membrane proteins through alkali-labile bonds of which 30% were of a thiol ester and the remainder of an O-ester type. This conclusion is based on differential liberation of fatty acids by hydroxylamine at pH 7.0 and pH 11.0. Two extracts of membranes enriched in peripheral proteins (bands 1, 2, 5 and 2.1, 4.1, 4.2, 6) were prepared and extracted with organic solvents but each contained about six times less fatty acids than the parent solvent-extracted membranes. Glycophorin A contains little if any covalently bound fatty acids. Anion transporter (band 3) contains about 1 mol of thiol ester of fatty acid. This accounts for about half of the thiol ester-linked fatty acids in the parent solvent-extracted membranes. Most of the O-ester-linked fatty acids are linked to an undisclosed membrane protein.     

Studies on human alpha-fetoprotein. Isolation and characterization of monomeric and polymeric forms and amino-terminal sequence analysis.

Human alpha-fetoprotein has been isolated from the serum and ascitic fluid of a patient with hepatoma by a combination of immunoadsorbent column chromatography and Sephadex G-150 gel filtration. Human alpha-fetoprotein is a sialylated glycoprotein with an estimated molecular weight of 67 500, composed of a single-chain polypeptide of approximately 580 amino acid residues and 3.6% carbohydrate. It is a negatively charged protein with an acid isoelectric point (pH 4.57). In addition to the monomeric form of alpha-fetoprotein, we have identified human alpha-fetoprotein polymers, including dimeric and trimeric forms, which dissociate to the monomer only upon exposure to disulfide-reducing reagents, implying that their formation is dependent upon intermolecular disulfide bonds. These polymers are found in human alpha-fetoprotein isolated by isoelectric focusing in both the major (pI 4.57) and minor (pI 5.2) alpha-fetoprotein fractions. The first 17 residues of the NH2-terminal amino acid sequence of the hepatoma-derived human alpha-fetoprotein have been identified. Fetal alpha-fetoprotein is indistinguishable from hepatoma alpha-fetoprotein by several criteria, including immunoelectrophoresis, acryalmide gel electrophoresis, and proclivity for dimerization.     

Human alpha-fetoprotein-fatty acid interaction

Human alpha-fetoprotein (AFP) is able to bind arachidonic acid with high affinity Ka = 10(7)M-1 and a limited number of binding sites NS = 3. Thirty different fatty acids were tested by competition using labelled arachidonic acid as tracer. This experiment demonstrated a great specificity for the fatty acid-AFP interaction. Only polyunsaturated long chain fatty acids were able to bind to AFP with high affinity. The metabolic products of arachidonic acid i.e. prostaglandins presented no affinity for the AFP molecule.     

Isolation and characterization of alpha-fetoprotein from the mouse hepatoma BW7756.

Purification of alpha-fetoprotein from mouse hepatoma BW7756 extracts was performed using ammonium sulfate precipitations, gel filtration, ion-exchange chromatography and isoelectric focusing. These procedures produced a 5.6% yield of alpha-fetoprotein with 96% purity. Polyacrylamide slab gel electrophoresis, extended agarose electrophoresis and immunoelectrophoresis demonstrated that mouse hepatoma alpha-fetoprotein migrated at pH 8.6 as a rapid alpha1, or postalbumin globulin. Crossed antibody electrophoresis of the agarose zone containing alpha-fetoprotein failed to demonstrate microheterogeneity. Molecular weight analysis of the mouse hepatoma alpha-fetoprotein on a calibrated Sephadex G-200 column yielded a value of 72 000-73 000 for the native protein. Sodium dodecyl sulfate gel electrophoresis subsequently demonstrated a single polypeptide chain with a molecular weight of 72 000. Amino acid analysis showed the alpha-fetoprotein to be an acidic protein dominated by hydrophobic residues. The total carbohydrate content was 5.5%, and 3 mol of sialic acid were detected per mol of alpha-fetoprotein. Although neutral sugars were the principal class present, galactosamine was the most abundant single sugar detected.     

Fatty acids bound to vitamin D-binding protein (DBP) from human and bovine sera

Human and bovine vitamin D-binding protein (DBP) have been isolated from serum by a method that does not involve denaturing steps. This method includes Cibacron Blue-Sepharose chromatography, gel filtration, DEAE-Sephadex chromatography and albumin immunoadsorption. Analysis of fatty acids bound to the isolated human and bovine DBP showed molar ratios of fatty acid to protein of 0.4 and 1.3 respectively meanwhile human and bovine albumin have bound 1.8 and 1.5 moles per mol respectively. Most of fatty acids bound to human and bovine DBP are monounsaturated and saturated, mainly oleic and palmitic acids, which together account for 50% of the total of fatty acids in both species. By contrast, polyunsaturated fatty acids represented a minor component, less than 5%.     

High affinity of alpha-foetoprotein for arachidonate and other fatty acids.

Fatty acid analysis of purified bovine alpha-foetoprotein showed it to contain 2.7 mol of fatty acid/mol of alpha-foetoprotein. Purified alpha-foetoprotein focused at isoelectric point 4.8. Removal of bound ligands from alpha-foetoprotein by charcoal treatment changed its isoelectric point to 5.2. This change could be reversed by addition of exogenous fatty acids to the defatted alpha-foetoprotein. Albumin isolated from the same foetal calf serum source as alpha-foetoprotein contained 1.4 mol of fatty acid/mol of protein. alpha-Foetoprotein and albumin contained comparable amounts of fatty acids with 14 to 18 carbon atoms, but alpha-foetoprotein contained 16 times as much of the long-chain polyunsaturated fatty acids as albumin. alpha-Foetoprotein was found to have slightly higher affinity for palmitate and linoleate and severalfold higher affinity for arachidonate than albumin. These findings suggest that alpha-foetoprotein may play a role in the foetal metabolism of the long-chain polyunsaturated fatty acids.     

Studies on the structure of sphingomyelinase. Amino acid composition and heterogeneity on isoelectric focusing.

Sphingomyelinase, purified to apparent homogeneity from human placenta, is an acidic protein, as judged from its amino acid composition and by isoelectric focusing of the carboxymethylated protein. The amino acid composition is characterized by an approximately equal content of hydrophobic and polar amino acid residues. The reduced-alkylated polypeptides were separated into two groups. Most of the polypeptides were heterogeneous with pI values of 4.4-5.0, but an additional more minor component was observed at pI 5.4. Liquid isoelectric focusing resolved the purified enzyme into a single major component (pI 4.7-4.8), a minor component (pI 5.0-5.4) and a plateau region of activity (pI 6-7). On thin-layer isoelectric focusing, the protein profile obtained from each of these regions was the same. In addition, the substrate specificity, Km values and effect of inhibitory substances were identical. We conclude that sphingomyelinase is an acidic, microheterogeneous protein that likely exists as a holopolymer of a single major polypeptide chain. the heterogeneity of the intact protein on isoelectric focusing appears to reflect this microheterogeneity, which is influenced by a tendency to associate with itself and with detergents such as Triton X-100.     

Lipid and cell metabolic changes associated with essential fatty acid enrichment of articular chondrocytes

Observations of impaired chondrocyte metabolism in essential fatty acid (EFA) deficiency as well as EFA protection against development of osteoarthrosis in inbred mice suggest the existence of a relationship between EFA, chondrocyte metabolism, and cartilage degeneration. To explore this relationship further, the fatty acid content of lipids in normal fetal bovine chondrocytes was manipulated by in vitro exposure to media supplemented with 100 microM arachidonic acid (20:4) or oleic acid (18:1). Chondrocytes rapidly and differentially incorporated both fatty acids into their lipid pools. The predominant acceptor was triacylglycerols. A 980% enrichment of arachidonic acid was associated with increased concentrations of fatty acids, increased 35SO4 and [3H]proline incorporation into matrix macromolecules (170% and 54-103%, respectively), and a 24-fold elevation in chondrocyte prostaglandin synthesis. No metabolic effects elicited observed in cells enriched by 377% with 18:1 oleic acid. The metabolic effects elicited by 20:4 arachidonic acid were abolished by pretreatment of cells with indomethacin, suggesting that the cellular responses to essential fatty acid loading may be associated with induced increases in prostaglandin synthesis. The data indicate that excessive in vitro accumulation of arachidonic acid is associated with an increase in synthetic activity that is causally related to increased prostaglandin synthesis and elevated levels of cellular fatty acids.     

The fatty acid composition of placenta in intrauterine growth retardation

To examine the impact of intrauterine growth retardation (IUGR) on essential fatty acids in human placenta, fatty acid composition in total acylglycerol and in the major phosphoglycerides phosphatidylcholine (PC) and phosphatidylethanolamine (PE), of 15 placentas from small for gestational age (SGA) births was compared with that of 7 control placentas. The acylglycerol fatty acid content was similar between the two groups, but the proportion of fatty acids of the linoleic acid series, including arachidonic acid, was significantly lower in SGA placentas. When the fatty acid composition in PC was studied, the reduction in fatty acids of the linoleic acid series was even more striking, and fatty acids of the linolenic acid series was also significantly less in the SGA group. These fatty acid changes in placenta membrane phospholipids can affect the transport of important nutrients to the fetal compartment. The decreased level of arachidonic acid and docosahexanoic acid might also lead to a disturbed formation of fetal thromboxane and prostacyclin. However, cord plasma PC fatty acid patterns were nearly identical in the two groups suggesting that in IUGR, the essential fatty acids will be transported to the fetus at the expense of the placenta.     

Kinetic properties of arachidonoyl-coenzyme A synthetase in rat brain microsomes

Free arachidonic acid is released rapidly in the brain at the onset of ischemia and during convulsions. The transient nature of this phenomenon indicates the existence of an active reacylation system for this fatty acid, likely an arachidonoyl-CoA synthetase-arachidonoyl transferase. The first of these enzymatic activities in brain microsomes was studied and it was found that [1-14C]arachidonic acid is rapidly activated and shows an absolute requirement for ATP and CoA. MgCl2 enhances this activity 10-fold. The optimum pH is 8.5, and the apparent Km values for the radiolabeled substrate, ATP, CoA, and MgCl2 are 36, 154, 8, and 182 microM, respectively. The apparent Vmax is 32.4 nmol/min/mg protein for arachidonic acid. The presence of Triton X-100 (0.1%) in the assay medium caused a significant reduction in apparent Km (9.4 microM) and Vmax (25.7 nmol/min/mg protein) values. The enzymatic activity is thermolabile with a T1/2 of less than 1 min at 45 degrees C and a maximal activity at 40 degrees C. The breaking point or transition temperature is 25 degrees C in an Arrhenius plot. The activation energies were 95 kJ/mol from 0 to 25 degrees C and 30 kJ/mol from 25 to 40 degrees C. Fatty acid competition studies showed inhibition by unlabeled docosahexaenoic and arachidonic acids with a Ki of 31 and 37 microM, respectively, in the absence and 18 and 7.7 microM in the presence of Triton X-100. Palmitic acid and oleic acid slightly inhibited the reaction whereas linoleic acid inhibited it to a moderate extent. It is concluded that this very active enzyme can activate arachidonic acid as well as docosahexaenoic acid in brain microsomes. In addition, this reaction may be involved in regulating the pool size of these free fatty acids in brain by rapid removal through activation, thus limiting eicosanoid formation. Moreover, the rapid formation of polyenoic acyl-coenzyme A may participate in the retention of essential fatty acids in the central nervous system.     

Alpha-fetoprotein-mediated uptake of fatty acids by human T lymphocytes.

The binding to resting and activated T lymphocytes of two radiolabelled fatty acids (oleic and arachidonic) was studied in the presence or in the absence of alpha-fetoprotein (AFP) as carrier protein. Fatty acid binding by resting and activated T lymphocytes was determined at 4 degrees C as a function of the concentration of fatty acid and AFP. Under the conditions employed, the following observations were made: (1) in the presence of AFP, fatty acids (oleic and arachidonic acid) are bound to cells by a two-component pathway; one is a saturable process, evidenced when the fatty acid to AFP (FA/AFP) molar ratio was fixed at 1 and the concentration of the fatty acid and the protein varied from 0.1 to 3.2 microM, and the second is a nonsaturable function of FA/AFP molar ratio and was linearly related to the unbound fatty acid concentration in the medium over the entire range studied; (2) in the absence of AFP, the nonsaturable process appears to be the only component of fatty acid binding; 3) at all tested concentrations of free (unbound) fatty acid in the medium, net fatty acid binding by either resting or activated T cells was considerably greater in the presence than in the absence of AFP; (4) in the presence of AFP, fatty acid binding was much higher in activated T cells than in resting T cells, whereas in the absence of AFP, nonsignificant differences were observed between activated and resting T cells; and (5) the time course of fatty acid and AFP binding at 4 degrees C revealed that, at equilibrium, the number of fatty acid molecules bound to the cell was much greater than that of AFP suggesting an accelerated dissociation of the fatty acid upon interaction of the AFP-fatty acid complex with putative cell receptors. It is concluded to the existence of an AFP/AFP-receptor pathway that facilitates the binding of fatty acids to T lymphocytes, particularly upon their blast transformation. This pathway may fulfill the increased requirement for fatty acids characteristic of proliferating cells and may serve to regulate the endocytosis of fatty acids with modulatory effects on lymphocyte function and to protect cells from their cytotoxic potential when internalized in excess.     

Endogenous fatty acids are covalently and noncovalently bound to interphotoreceptor retinoid-binding protein in the monkey retina

Interphotoreceptor retinoid-binding protein (IRBP) purified from monkey interphotoreceptor matrix contains relatively high concentrations of endogenous fatty acids, 6.51 mol/mol of protein. Sixty-five percent of the total fatty acid bound to IRBP was found to be noncovalently attached, with the remainder covalently bound. The fatty acids are not residual components of phospholipids or neutral lipids, as judged by microchemical methods. The major fatty acids bound to IRBP are: palmitic (35%), stearic (21%), palmitoleic (7%), oleic (29%), linoleic (6%) and docosahexaenoic acids (2%). These fatty acids account for about 90% of the total fatty acid bound to interphotoreceptor matrix proteins extracted with organic solvents. Thus, IRBP may function as an intercellular fatty acid carrier and may depend on the covalently bound fatty acids for anchoring in the outer leaflet of cell membranes.     

Alpha-fetoprotein favours accumulation of estrone but not arachidonic acid into the fetal and new-born rat brain

Tritriated estrone or arachidonic acid, two high affinity ligands for rat alpha-fetoprotein (AFP), were injected into adult, pregnant or newborn Sprague Dawley rats in order to evaluate their possible transfer into the brain. This study shows that the developing brain accumulates the estrogen but not the fatty acid, suggesting that the uptake of AFP by the developing brain is a mechanism for transporting estrogens, but not fatty acids.     

Marked acceleration of exogenous fatty acid incorporation into cellular triglycerides by fetuin.

Fetuin belongs to a group of fetal glycoproteins whose specific function is not known. In this study we investigated the effect of bovine fetuin on exogenous fatty acid incorporation into lipid classes by fetal rabbit aortic smooth muscle cells (SMC) and human fetal skin fibroblasts. When compared with albumin, the addition of fetuin to the culture medium caused a dramatic increase in labeled fatty acid incorporation (nanomoles/mg of protein) by SMC into triglycerides (albumin (control) 2.8 +/- 0.3 + fetuin 178.3 +/- 13.7). This effect was noted at a wide range of fetuin concentrations (0.2-5%) at oleate:fetuin molar ratios of 3.3-0.13, respectively. Similar effects were noted using human fetal skin fibroblasts with both labeled oleic and arachidonic acids (0.1 mM) as substrates (arachidonic acid incorporation into triglycerides, albumin (control) 76.9 +/- 16.2 + fetuin 684.6 +/- 64.1). Stimulation of fatty acid incorporation into di- and monoglycerides was also noted. Although the amount of unbound fatty acid in the presence of fetuin was greater than with albumin, experiments done under conditions that create identical unbound oleate levels (by varying fatty acid concentration) still showed increased fatty acid incorporation into triglycerides by SMC when exposed to fetuin. This marked effect of fetuin on triglyceride accumulation in cells was confirmed by lipid analysis, strong positive staining with oil red O, and transmission of electron microscopy. Furthermore, the potential physiological role of fetuin in terms of fatty acid and transport was attested by (a) the presence of significant amounts of free fatty acids associated with fetuin; and (b) by the stimulatory effect of fetuin, even when added to culture media containing other fatty acid carriers. These results show that (a) fetuin is far more efficient than albumin in incorporating fatty acids into cells; and (b) this might represent a novel function for fetuin during development.     

Production of lysophospholipids and free fatty acids by a sarcolemmal fraction from canine myocardium.

The potential for injury of myocardial sarcolemma by endogenous lipases was studied. The sarcolemmal fraction was incubated for 30 min under conditions found optimal for hydrolysis of exogenous phosphatidylethanolamine (5 mM calcium, pH 7.0, 37°C). Incubation of the sarcolemmal fraction increased significantly the level of total free fatty acids (14.1 to 31.1 nmoles/mg protein, P < 0.001); in addition, production of arachidonic acid was increased significantly (P < 0.01). Lysophosphatidylcholine was increased significantly (P < 0.01) but the content of lysophosphatidylethanolamine was unchanged. A large proportion of the above free fatty acids (10.2 nmoles/mg protein) was derived from the hydrolysis of triacylglycerols. These data demonstrate that the sarcolemmal fraction preferentially hydrolyses endogenous membrane phosphatidylcholine at neutral pH in the presence of calcium with the formation of lysophosphatidylcholine and free fatty acids including arachidonic acid.