Characterization, primary structure, and evolution of lamprey plasma albumin.

The most abundant protein found in blood plasma from the sea lamprey (Petromyzon marinus) has the hallmarks of a plasma albumin: namely, high abundance, solubility in distilled water, a small number of tryptophans, and a high content of cysteines and charged residues. As in other vertebrate albumins, not all the cysteines are disulfide bonded. An unusual feature of this protein is its molecular weight of 175,000, roughly 2.5 times the size of other vertebrate albumins. Its amino acid sequence, deduced from a series of overlapping cDNA clones, can be aligned with other members of the gene family including plasma albumin, alpha-fetoprotein, and vitamin-D binding protein, confirming that it is indeed an oversized albumin. An unusual feature of the sequence is a 28-amino acid stretch consisting of a serine-threonine repeat with the general motif (STTT). Lamprey albumin contains a 23-amino acid putative signal peptide and a 6-residue putative propeptide, which, when cleaved, yield a mature protein of 1,394 amino acids with a calculated molecular weight of 157,000. The sequence also includes nine potential N-linked glycosylation sites (Asn-X-Ser/Thr), consistent with observation that lamprey albumin is a glycoprotein. If all the potential glycosylation sites were occupied by clusters of 2,000 molecular weight each, the total molecular weight would be 175,000. Like other members of the gene family, lamprey albumin is composed of a series of 190-amino acid repeats, there being seven such domains all together. Quantitative amino acid sequence comparisons of lamprey albumin with the other members of the gene family indicate that it diverged from an ancestral albumin prior to the gene duplications leading to this diverse group. This notion is confirmed by the pattern of amino acid insertions and deletions observed in a consideration of all domains that compose this family. Furthermore, it suggests that the invention of albumin antedates the vertebrate radiation.     

The Antarctic toothfish (Dissostichus mawsoni) lacks plasma albumin and utilises high density lipoprotein as its major palmitate binding protein

Plasma from the Antarctic toothfish, Dissostichus mawsoni, a member of the advanced teleost Nototheniidae family, was analysed. Agarose gel electrophoresis showed a major diffuse anionic protein that bound [14C]palmitic acid but not 63Ni2+, and two more cationic proteins that bound 63Ni2+ but not palmitate. Oil Red O staining following cellulose acetate electrophoresis indicated that the palmitate binding protein was a lipoprotein. Two-dimensional electrophoresis showed that this palmitate binding band was composed of three proteins with M(r) of 11, 30, and 42 kDa, without any trace of material at approximately 65 kDa, the mass of albumin. N-terminal sequencing of the palmitate binding band gave a major sequence of DAAQPSQELR-, indicating a high degree of homology to apolipoprotein A-I (apo-AI), the major apolipoprotein of high density lipoprotein (HDL). N-terminal sequencing of the major nickel binding band produced a sequence with no homology to albumin. When ultracentrifugation was used to isolate the lipoproteins from Antarctic toothfish plasma, the palmitate binding protein was found solely in the lipoprotein fraction. In competitive binding experiments, added human albumin did not prevent palmitate binding to toothfish HDL. In conclusion, there is no evidence for albumin in Antarctic toothfish plasma and HDL assumes the role of fatty acid transport.     

A stereochemical investigation of phosphoryl transfer catalysed by phosphoglucomutase by the use of alpha-D-glucose 1-[(S)-16O,17O,18O]phosphate.

Fluorescence spectra of protoporphyrin bound to its most affinitive site on human serum albumin, bound to human haemopexin and dissolved in human plasma reveal that, when present in plasma, at least 90% of this porphyrin is bound to albumin. Human serum albumin binds protoporphyrin with an affinity KA = 3 X 10(9)M-1 in phosphate-buffered saline. The affinity of haemopexin for protoporphyrin is 4 times smaller. From these data it is concluded that less than 1% of plasma protoporphyrin is bound to haemopexin. Implications of the data for protoporphyrin transport and clearance are discussed.     

A physical-chemical model for cellular uptake of fatty acids: prediction of intracellular pool sizes

If the uptake of fatty acids by liver is a physical, not a biological, process, then the size and location of the intrahepatic pool of fatty acids can be predicted from uptake rates and thermodynamic data. The purpose of the experiments in this paper was to test the accuracy of this idea. Rat livers were perfused with palmitate bound to albumin, and the total amounts of palmitate removed from the perfusate were measured at 3-s intervals. The intrahepatic pools of palmitate calculated from these data were 13.8 and 23.0 nmol/g of liver at ratios of palmitate/albumin (mol/mol) (afferent side) of 2/1 and 4/1, respectively, in the steady state. The intrahepatic pools of palmitate calculated from the distributions of palmitate between membranes, H2O, albumin, and fatty acid binding protein and the measured first-order rate constants for acyl-CoA ligases in mitochondria and microsomes were 12.1 and 34.6 nmol/g for perfusate ratios of palmitate/albumin of 2/1 and 4/1, in the steady state. Intrahepatic pools of palmitate measured after establishment of a steady-state rate of uptake were 15.0 and 31.8 nmol/g for these ratios of palmitate/albumin of 2/1 and 4/1.     

Bilirubin and haeme binding to human serum albumin studied by spectroscopy methods

Cobinding of bilirubin and of haeme to human serum albumin was investigated by means of difference absorption spectroscopy and fluorescence spectroscopy. Two specific sites for bilirubin and two for haeme binding occur on the albumin molecule. The primary binding site for bilirubin (Ka = 2.5 microM-1) is different from the primary heame binding site (Ka = 50 microM-1; Beaven et al., Eur J. Biochem. 41, 539-546, 1974), the former, however, might be identical with the secondary center for haeme binding. Similarly, the primary haeme binding center might be identical with the secondary bilirubin binding site.     

Palmitate uptake by neonatal rat myocytes and hepatocytes. Role of extracellular protein.

The role of extracellular binding proteins in the rate of [3H]palmitate uptake by neonatal cardiac myocytes and hepatocytes was investigated using a model-independent approach. Binding proteins used in this study included alpha1-acid glycoprotein [isoelectric point (pI) approximately 2.7], conalbumin (pI approximately 6.4), lysozyme (pI approximately 11.0), albumin (pI approximately 4.9), and albumin which had been modified to yield proteins with pI values of 3.5, 4.7, 7.5 and 8.6. All uptake studies were conducted at similar unbound ligand fractions. There was a linear relationship between the rate of neonatal hepatocyte [3H]palmitate clearance and protein pI (r2 = 0.98). In contrast, there was an overall poor relationship between neonatal cardiac myocyte [3H]palmitate-clearance rate and protein pI (r2 = 0.48). However, the relationship improved when the data on [3H]palmitate-clearance were analyzed using only the modified albumins. The study indicates that an ionic interaction between extracellular proteins and the hepatocyte surface enhances the overall uptake of [3H]palmitate. This interaction may be limited to albumin for neonatal cardiac myocytes.     

Levetiracetam protects against kainic acid-induced toxicity

We investigated the Levetiracetam (LVT) ability to protect the brain against kainic acid (KA) induced neurotoxicity. Brain injury was induced by intraperitoneal administration of KA (10 mg/kg). Sham brain injury rats were used as controls. Animals were randomized to receive either LVT (50 mg/kg) or its vehicle (1 ml/kg) 30 min. before KA administration. Animals were sacrificed 6 hours after KA injection to measure brain malonildialdehyde (MDA), glutathione levels (GSH) and the mRNA for interleukin-1beta (IL-1beta) in the cortex and in the diencephalon. Behavioral changes were also monitored. Intraperitoneal administration of LVT decreased significantly MDA in the cortex (KA + vehicle = 0.25 +/- 0.03 nmol/mg protein; KA + LVT = 0.13 +/- 0.01 nmol/mg protein; P < 0.005), and in the diencephalons (KA + vehicle = 1,01 +/- 0.2 nmol/mg protein; KA + LVT = 0,33 +/- 0,08 nmol/mg protein; P < 0.005), prevented the brain loss of GSH in both cortex (KA + vehicle = 5 +/- 1 micromol/g protein; KA + LVT = 15 +/- 2 micromol/g protein; P < 0.005) and diencephalons (KA + vehicle = 9 +/- 0.8 micromol/g protein; KA + LVT = 13 +/- 0.3 micromol/g protein; P < 0.05), reduced brain IL-1beta mRNA and markedly controlled seizures. Histological analysis showed a reduction of cell damage in LVT treated samples. The present data indicate that LVT displays neuro-protective effects against KA induced brain toxicity and suggest that these effects are mediated, at least in part, by inhibition of lipid peroxidation.     

Effects of aliphatic fatty acids on the binding of Phenol Red to human serum albumin.

Binding of Phenol Red to human serum albumin at pH 7.0 was studied by ultrafiltration (n1 = 1, K1 = 3.9 X 1-(4) M-1, n2 = 5, K2 = 9.6 X 10(2) M-1). The presence of 1 mol of octanoate or decanoate per mol of albumin caused a decrease in dye binding (dye/protein molar ratio 1:1), which, in contrast with additional fatty acid, was very pronounced: 1-8 mol of palmitate or stearate resulted in a small, and apparently linear, displacement of Phenol Red. The displacement effect of 1-5 mol of oleate, linoleate or linolenate per mol of albumin was comparable with that of the equimolar concentrations of palmitate or stearate. A higher molar ratios the unsaturated acids caused a drastic decrease in dye binding. The different Phenol Red-displacement effects of low molar ratios of medium-chain and long-chain fatty acids indicate that these acids have different high-affinity binding sites. In accordance with this proposal, low concentrations of stearate had only a small effect on the Phenol Red-displacement effect of octanoate. Phenol Red-binding curves in the presence of 1 mol of octanoate, 8 mol of stearate and 6 or 7 mol of linolenate per mol of albumin respectively indicated that the dye and the fatty acids do not complete for a common primary binding site. In contrast, a secondary Phenol Red-binding site could be identical with the primary octanoate-binding site. Furthermore, the primary Phenol Red-binding site could be the same as a secondary linolenate-binding site. Assignment of the different primary binding sites for Phenol Red and for medium-chain and long-chain fatty acids to a model of the secondary structure of albumin is attempted.     

Palmitate-binding, serum albumin-like proteins in salmonids

There has been considerable controversy over the existence of serum albumin in fish. One of the physiological functions of albumin is to bind free fatty acids. This characteristic was used to screen the plasma of seven species of salmonids. Each species contains a protein fraction that (i) binds palmitate, (ii) has a molecular mass similar to that of human serum albumin, and (iii) is one of the most rapidly migrating proteins when salmonid plasma is subjected to anodal polyacrylamide gel electrophoresis. We conclude therefore, that salmonids have serum albumins that are homologous to the serum albumin of higher vertebrates.     

A theoretical analysis of the binding of palmitate by human serum albumin

The distribution of palmitate between the form bound by human serum albumin and the free form in plasma was calculated by use of 12 stepwise equilibrium constants and a computer program. Computations were carried out for molar ratios of palmitate to serum albumin of 0.5, 1,2,3, and 4. At most 0.0003% of the palmitate would be in the unbound form, and the remainder distributed among different complexes with albumin. At low molar ratios, the complexes with 1 to 2 moles of palmitate/albumin would predominate while at the highest ratio the complexes of 3 and 4 moles of palmitate/albumin would be most abundant. In the delivery of palmitate to tissues the relative contribution of the different complexes would change, as the molar ratio of fatty acid to albumin changed.     

Fragments of bovine serum albumin produced by limited proteolysis. Conformation and ligand binding.

Twelve fragments of bovine serum albumin, isolated following limited tryptic or peptic hydrolysis, have been studied to define secondary structure and locate ligand-binding sites. Based on circular dichroism, the conformational pattern of albumin (68% alpha helix and 18% beta structure) is substantially retained by individual fragments, indicating that secondary configuration is locally determined and is not destroyed during the cleavage process nor during fragment purification. The strong bilirubin-binding site of bovine serum albumin is present in 3 of the 12 fragments. Residues 186-238 are common to the three fragments and absent from those fragments which do not bind bilirubin; consequently the strong bilirubin-binding site is suggested to involve this region. By similar reasoning, the presence of palmitate-binding sites in some fragments and not in others indicates that the three strongest sites for the binding of palmitate are located in the carboxyl-terminal two-thirds of the molecule. The first site (KA approximately 2 X 10(7) M-1) is suggested as residues 377-503; the second site (KA approximately 8 X 10(6) M-1), residues 239-306; the third site (KA approximately 2 X 10(6) M-1), residues 307-377. Bromocresol Green, a reagent used in the assay of ablumin, was bound by fragments rougly in proportion to their size but showed particular affinity for the region of the strong bilirubin-binding site. The fluorescent probe, 8-anilino-1-naphthalensulfonate, was in general bound by large fragments, supporting the concept that this ligand is held principally in clefts between domains of the macromolecule.     

Fatty acid enhancement of human serum albumin binding properties. A spin label study.

The introduction of a new spin-labeled anionic ligand, 1-gamma-aminobutyrate-5-N-(1-oxyl-2,2,6,6-tetramethyl-4-aminopiperidinyl)-2,4-dinitrobenzene, is reported. Under the experimental conditions, the first molar equivalent of this ligand is 93% bound to human serum albumin. With the addition of palmitate, the free spin label concentration decreases greatly, by almost 80%, in the presence of a fatty acid:albumin ratio of 3:1 to 4:1. The spectral characteristics of the bound spin label are also affected. The changes seen in the intensity of and the splitting between the high and low field extrema are indicative of perturbations of the protein molecule. It is seen then that the binding of each molar equivalent of fatty acid effects the conformation state of albumin and allosterically affects albumin binding properties. Computer spectral subtractions, furthermore, suggest that the binding of the first molar equivalent of palmitate specifically increases the affinity of the first two 1-gamma-amino-butyrate-5-N-(1-oxyl-2,2,6,6-tetramethyl-4-aminopiperidinyl)-2,4-dinitrobenzene binding sites. The present results indicate that fluctuations in serum free fatty acid levels within the physiological range may have a major modulatory effect on the free serum levels of certain drugs and/or physiological substances that bind to albumin.     

Modeling of palmitate transport in the heart

Summary Transport of palmitate from the albumin-palmitate complex in the plasma to inside mitochondria where it undergoes -oxidation is a multistep process. Albumin's large size prevents permeation via interendothelial clefts. Palmitate dissociation from albumin in solution is too slow to provide an adequate supply of the unbound palmitate. The discovery that the dissociation occurs upon albumin binding to an endothelial surface receptor resolves the conundrum. Palmitate transport across the luminal surface membrane may be either carrier-mediated or passive. Fatty-acid binding protein inside endothelial and cardiac muscle cells facilitates diffusion through cytosol while maintaining the unbound palmitate concentration at a very low level. Within the interstitium, albumin is again the palmitate carrier. Still controversial is whether or not there is a saturable sarcolemmal transporter or simply passive exchange. Inside the myocyte palmitate is again bound to the fatty acid binding protein which buffers the free palmitate concentration, facilitates diffusion, and may facilitate further intracellular reactions.     

The effect of ligand presaturation on the interaction of serum albumins with an immobilized Cibacron Blue 3G-A studied by affinity gel electrophoresis.

The interaction of the immobilized triazine dye Cibacron Blue 3G-A with rat, rabbit, sheep, goat, bovine and human serum albumins was studied by affinity gel electrophoresis. Dissociation constants were estimated in each instance and showed human serum albumin to have a significantly higher affinity for the dye than did albumin from any other species. Pretreatment of the defatted proteins with bilirubin (3 mol of bilirubin/mol of protein) did not increase the dissociation constants of the serum albumins, whereas pretreatment with palmitate (7 mol of palmitate/mol of protein) increased the dissociation constant in all cases: 3-fold for human serum albumin, 15-fold for other serum albumins. Increasing the bilirubin/albumin ratio (to 7:1) did not affect the dissociation constant of the albumins studied. Decreasing the palmitate/albumin ratio decreased the dissociation constant for human serum albumin, but did not affect those of bovine and rat albumins. Altering the chain length of the presaturating fatty acid dramatically changed the dissociation constant of both human and bovine serum albumins. Butyrate, hexanoate, octanoate and decanoate did not significantly influence the dissociation constants of bovine and human serum albumins for Cibacron Blue, whereas laurate, myristate and palmitate greatly increased the dissociation constant. These data are discussed in relationship to the behaviour of albumins during dye--agarose column chromatography. In Addendum the effect of nucleotide presaturation on the interaction between Bacillus stearothermophilus 6-phosphogluconate dehydrogenase and the immobilized triazine dyes Cibacron Blue 3G-A and Procion Red HE-3B was examined, and the implications for dye--ligand chromatography are discussed.     

Photoaffinity labelling of the cardiac calcium channel. (-)-[3H]azidopine labels a 165 kDa polypeptide, and evidence against a [3H]-1,4-dihydropyridine-isothiocyanate being a calcium-channel-specific affinity ligand.

The arylazide 1,4-dihydropyridine (-)-[3H]azidopine binds to a saturable population of sites in guinea-pig heart membranes with a dissociation constant (KD) of 30 +/- 7 pM and a density (Bmax.) of 670 +/- 97 fmol/mg of protein. This high-affinity binding site is assumed to reside on voltage-operated calcium channels because reversible binding is blocked stereoselectively by 1,4-dihydropyridine channel blockers and by the enantiomers of Bay K 8644. A low-affinity (KD 25 +/- 7 nM) high-capacity (Bmax. 21.6 +/- 9 pmol/mg of protein) site does not bind (-)- or (+)-Bay K 8644, but is blocked by high concentrations (greater than 500 nM) of dihydro-2,6-dimethyl-4-(2-isothiocyanatophenyl)-3,5-pyridinedicarboxy lic acid dimethyl ester (1,4-DHP-isothiocyanate) or, e.g., (+/-)-nicardipine. (-)-[3H]Azidopine was photoincorporated covalently into bands of 165 +/- 8, 39 +/- 2 and 35 +/- 3 kDa, as determined by SDS/polyacrylamide-gel electrophoresis. Labelling of the 165 kDa band is protected stereoselectively by 1,4-dihydropyridine enantiomers at low (nM) concentrations and by (-)- and (+)-Bay K 8644, whereas the lower-Mr bands are not. Thus, only the 165 kDa band is the calcium-channel-linked 1,4-dihydropyridine receptor. Photolabelling of the 39 or 35 kDa bands was only blocked by 10 microM-1,4-DHP-isothiocyanate or 50 microM-(+/-)-nicardipine but not by 10 microM-(-)-Bay K 8644. [3H]-1,4-DHP-isothiocyanate binds to guinea-pig heart membranes with a KD of 0.35 nM and dissociates with a k-1 of 0.2 min-1 at 30 degrees C. [3H]-1,4 DHP-isothiocyanate irreversibly labels bands of 39 and 35 kDa which are protected by greater than 10 microM-(+/-)-nicardipine or unlabelled ligand but not by 10 microM-(-)-Bay K 8644. Thus, [3H]-1,4-DHP-isothiocyanate is not an affinity probe for the calcium channel.     

Effect of fatty acid-binding proteins on intermembrane fatty acid transport studies on different types and mutant proteins.

Liposomes of different charge fixed to nitrocellulose filters were used to study the transfer of fatty acids to rat heart or liver mitochondria in the presence of fatty acid-binding protein (FABP) or albumin. [14C]Palmitate oxidation was used as a parameter. Different FABP types and heart FABP mutants were tested. The charge of the liposomes did not influence the solubilization and mitochondrial oxidation of palmitate without FABP and the amount of solubilized palmitate in the presence of FABP. Mitochondria did not show a preference for oxidation of FABP-bound palmitate over their tissue-specific FABP type. All FABP types increased palmitate oxidation by heart and liver mitochondria with neutral, positive and negative liposomes by 2.5-fold, 3.2-fold and twofold, respectively. Ileal lipid-binding protein and H-FABP mutants that do not bind fatty acid had no effect. Other H-FABP mutants had different effects, dependent on the site of mutation. The effect of albumin was similar to, but not dependent on, liposome charge. The ionic strength had only a slight effect. In conclusion, the transfer of palmitate from liposomal membranes to mitochondria was increased by all FABP types to a similar extent. The membrane charge had a large effect in contrast to the origin of the mitochondria.     

Protein binding of palmitate measured by transmembrane diffusion through polyethylene

Thin polyethylene membranes permit ready diffusion of protonated long-chain fatty acids but are impermeable to protein and ions. This circumstance recommends polyethylene for measuring the free fraction of fatty acids in the presence of a binding protein and for estimating the ionization constant with which to compute the equilibrium constant for the binding of fatty acid anions. As an example of this approach we report the binding of tracer palmitate to bovine albumin and bovine beta-lactoglobulin. We find a binding constant for the high-affinity site on albumin that is close to that calculated by others from heptane:H2O partition ratios. Our procedure is simpler, however, and free of the theoretical objection that heptane may alter the binding characteristics of the protein. Our estimate of the pKa for palmitic acid is 4.9, a finding that conforms to the widely predicted but heretofore unconfirmed expectation that long-chain fatty acids should have a pKa of about 4.8. Unidirectional flux measurements exclude direct exchange of palmitate between albumin and polyethylene.     

Mitochondria from the lamprey (Lampetra fluviatilis). Oxidative phosphorylation and related processes.

1. High efficiency of oxidative phosphorylation and a good respiratory control in liver, heart and somatic muscle mitochondria of the lamprey (Lampetra fluviatilis) were observed when the particles were isolated in a complex sucrose medium containing EDTA, heparin and nicotinamide. The coupling properties of these mitochondria were further improved by including serum albumin in the incubation medium. 2. The content of total adenine nucleotides in lamprey mitochondria was between 4 and 6 nmoles/mg protein. The translocation of these nucleotides across mitochondrial membrane was stimulated by serum albumin. 3. Lamprey mitochondrial phospholipids contain a large proportion (64-72%) of polyunsaturated fatty acids. 4. Electron micrographs of mitochondria from lamprey liver, heart and somatic muscle are presented.     

Determination of the distribution of fatty acids and diethylstilbestrol between serum albumin and alpha-fetoprotein by concanavalin A affinity chromatography

The distribution of fatty acids and diethylstilbestrol between serum albumin and alpha-fetoprotein was measured in vitro by a new method based on the separation of the two proteins by virtue of the binding specificity of concanavalin A for the carbohydrate moiety of alpha-fetoprotein. Human and bovine proteins were investigated. It was found that palmitate and oleate were distributed almost equally between albumin and alpha-fetoprotein, while docosahexaenoate and diethylstilbestrol bound preferentially to alpha-fetoprotein even at an albumin: alpha-fetoprotein ratio of 10:1. The results confirm the binding specificity of alpha-fetoprotein for polyunsaturated fatty acids and also show that alpha-fetoprotein binds diethylstilbestrol much more strongly than albumin does. This suggests that alpha-fetoprotein may play a role in the fetal uptake of diethylstilbestrol.