The effect of commercial bovine serum albumin and other proteins on the activity of bovine milk galactosyltransferase

The widespread use of bovine serum albumin preparations for the stabilization of purified glycosyltransferases has prompted us to study the effects of different preparations of albumins on the galactosyltransferase activity of bovine milk. For comparison, several other proteins were tested as well. The albumins caused a large stimulation of transferase activity (400-700%) which varied depending on the source of the albumin and the treatment to which it had been subjected. Several other unrelated proteins were tested for their effects on transferase activity. Some proteins stimulated, while others had little effect. Lysozyme stimulated the activity by 178% and poly-L-lysine had little effect. Other proteins stimulated to variable extents. The stimulations obtained with albumin and myelin basic protein were noteworthy. The stimulation was considerably less marked when the enzyme was incorporated into lipid vesicles. These results emphasize the need for caution when adding proteins such as bovine serum albumin to purified enzymes for the purpose of stabilizing the activity of the enzyme.     

The Use of Human,Bovine, and Camel Milk Albumins in Anticancer Complexes with Oleic Acid

Oleic acid (OA) is a monounsaturated fatty acid that upon binding to milk proteins, such as α-lactalbumin and lactoferrin, forms potent complexes, which exert selective anti-tumor activity against malignant cells but are nontoxic for healthy normal cells. We showed that the interaction of OA with albumins isolated from human, bovine, and camel milk results in the formation of complexes with high antitumor activity against Caco-2, HepG-2, PC-3, and MCF-7 tumor cells. The antitumor effect of the complexes is mostly due to the action of oleic acid, similar to the case of OA complexes with other proteins. Viability of tumor cells is inhibited by the albumin-OA complexes in a dose dependent manner, as evaluated by the MTT assay. Strong induction of apoptosis in tumor cells after their treatment with the complexes was monitored by flow cytometry, cell cycle analysis, nuclear staining, and DNA fragmentation methods. The complex of camel albumin with OA displayed the most pronounced anti-tumor effects in comparison with the complexes of OA with human and bovine albumins. Therefore, these results suggest that albumins have the potential to be used as efficient and low cost means of tumor treatment.     

Activation of delta5-3-ketosteroid isomerase of bovine adrenal microsomes by serum albumins.

The Δ⁵-3-ketosteroid isomerase (EC 5.3.3.1) of bovine adrenal microsomes is activated as much as 10- to 20-fold by micromolar concentrations of bovine serum albumin. Comparable activations are observed with the serum albumins of 10 other mammalian species, but are not seen with ovalbumin or conalbumin. Evidence that the activation is attributable to the serum albumins, rather than to a small, firmly-bound ligand, is based on: (1) Failure to remove the stimulatory activity from the albumin by chloroform extraction, dialysis, or gel filtration; (2) Destruction of the activity by heating or by trypsin digestion; (3) Precipitation of the stimulatory activity of bovine serum albumin by specific antibody. Bovine serum albumin induces small decreases in the Michaelis constant for Δ⁵-androstene-3,17-dione, but most of the activational effect reflects an increase in the maximum velocity. Low concentrations of Triton X-100, which are without effect on the isomerase activity, prevent the activation by bovine serum albumin.     

The effects of serum albumin and phospholipid on sterol exretion in tissue culture cells

Serum albumin was an effective as whole serum or α-globulins in facilitatting sterol release from strain L mouse fibroblasts. Commercial bovine serum albumin preparations, however, had markedly different absolute effects in this regard. These differences were attributable to the variation in phospholipid content of these products. All but one of these albumins enhanced sterol release when supplemented with phospholipid. The exception was fatty acid-poor albumin which contained an adequate amount of phospholipid. Among the phospholipids examine, lecithin proved to be most effective, while phosphatidylethanolamine had little potentiating influence. As the unsaturation of the test lecithins increased, enhancement of sterol release decreased. The potentiating effect of the phospholipid was in turn dependent on the protein used, since the phenomenon was not observed with non-serum proteins like ovalbumin or with non-transport serum proteins such as γ-globulins. The results of these studies raise the possibility that serum albumin together with phospholipid can play an important role in sterol release in tissue cultured cells.     

Stimulatory and Inhibitory Actions of Proteins and Amino Acids On Copper-Catalysed Free Radical Generation in the Bulk Phase

The effect of a variety of proteins and amino acids was investigated on oxygen free radical activity as assessed by copper/hydrogen peroxide induced benzoate hydroxylation as well as copper-catalysed ascor-bate autoxidation. Serum albumins from a variety of species (human, bovine and dog) had both inhibitory and stimulatory effects depending on the molar copper to protein ratio; low ratios were inhibitory and high stimulatory. Some other proteins tested (lysozyme, soybean trypsin inhibitor and conalbumin) also had dual (inhibitory and stimulatory) effects, as did both histidine and polyhistidine, but all effects occurred at different molar ratios presumably dependent on the relative affinities for the copper ions. In contrast, metallolhioncin and cacruloplasmin, proteins specialised to bind copper in vivo had no stimulatory effects. In this paper we show that in addition to their fairly well documented inhibitory effects, under certain conditions some proteins also stimulate radical reactions. The possible role of this phenomenon in vivo is discussed.     

The effect of soluble rat liver proteins on the activity of microsomal stearoyl-CoA and linoleoyl-CoA desaturase.

1. The influence of bovine serum albumin and soluble rat liver proteins on the activity of rat liver microsomal delta9 and delta6 desaturases has been studied. 2. In the absence of bovine serum albumin, the delta9 desaturase which converts stearoyl-CoA into oleoyl-CoA, shows a non-linear correlation between enzyme activity and protein concentration. 3. Optimum concentrations of bovine serum albumin have three main effects on the enzyme activity: (i) establishes a linear relationship between enzyme activity and protein concentration, (ii) stimulates the enzyme activity 2--3-fold and (iii) raises the optimum substrate concentration from 10 to 100 muM. 4. A highly purified soluble liver protein of molecular weight 24 000 also stimulated the enzyme activity and brought about a linear relationship between enzyme activity and protein concentration. 5. It was concluded that the non-linear kinetics were due to limiting amounts of substrate binding protein in the microsomal preparations. 6. The delta6 desaturase which converts linoleoyl-CoA into gamma-linolenoyl-CoA was also stimulated by bovine serum albumin and soluble liver proteins. 7. The significance of the fatty acid-binding proteins is discussed.     

Extraction of an erythrotropin-like factor from bovine serum albumin (cohn fraction V)

Summary Different batches of commercially available bovine serum albumin (Cohn fraction V) were tested in a serum-free medium for their ability to stimulate thymidine incorporation in erythroid cells of fetal bovine liver. All preparations stimulated thymidine incorporation. Crystallized, charcoal-treated, or fatty acid-free albumin had substantially lower thymidine incorporation-stimulating activities than the crude preparations. The albumin preparations also had a synergistic effect with respect to erythropoietin on erythroid cells from rat liver, a typical property of erythrotropins. One gram of one of the batches of Cohn fraction V was fractionated by reversed-phase high performance liquid chromatography (HPLC). The fraction with thymidine incorporation-stimulating activity had a similar elution position as erythrotropin isolated from fetal bovine serum. Further purification using reversed-phase HPLC in the presence of trifluoroacetic acid and heptafluorobutyric acid and gel permeation HPLC resulted, in the isolation of a factor that is very similar to fetal bovine serum erythrotropin. It has practically the same specific activity as the purified fetal peptide in the rat liver bioassay. These results suggest that many of the beneficial effects of the albumin preparations added as supplement of serum-free tissue culture media may be due to the presence of erythrotropin-like factors. The work was supported by grants MT-6072 and ME-9031 from the Medical Research Council of Canada. The author is a Chercheur-Boursier of the Fonds de la Recherche en Santé du Quebec.     

Various proteins modulate the kinase activity of the insulin receptor

Previous studies of the substrate specificity of the purified insulin receptor tyrosine kinase using synthetic random polymers have demonstrated that the receptor kinase phosphorylates poly (Glu, Tyr) 4:1 but not poly (Glu, Tyr) 1:1. In the present study, insulin treatment of Chinese hamster ovary cells overexpressing the human insulin receptor was found to stimulate the ability of their membrane extracts to phosphorylate poly (Glu, Tyr) 1:1. It was concluded that this activity was due to the receptor itself because: 1) it was precipitated with a monoclonal antibody to the receptor; 2) the addition of various membrane extracts to purified insulin receptor preparations stimulated the ability of these preparations to phosphorylate poly (Glu, Tyr) 1:1; and 3) certain purified proteins, including bovine serum albumin and casein, were also capable of stimulating the purified receptor to phosphorylate poly (Glu, Tyr) 1:1. The effect of albumin was dose-dependent; 0.5 and 10 mg/ml bovine serum albumin stimulated the phosphorylation of poly (Glu, Tyr) 1:1 by 2- and 230-fold, respectively. In contrast, albumin had no effect on the phosphorylation of poly (Glu, Tyr) 4:1. These results indicate that the activity of the insulin receptor kinase on certain substrates can be modulated by the presence of other proteins.     

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.     

Removal by bovine serum albumin of fatty acids from membrane vesicles and its effect on proline transport activity in Escherichia coli.

Bovine serum albumin appreciably stimulated the initial rate and the steady-state level of proline uptake in membrane vesicles, while it had no effect on the oxidase activity for ascorbate-phenazine methosulfate, on which the transport activity depends. Bovine serum albumin showed the strongest stimulatory effect on the transport system among the proteins tested. Three other proteins did not show any effect, while beta-lactoglobulin showed a weaker but appreciable effect on the transport activity. The incubation of membrane vesicles with bovine serum albumin resulted in extensive removal of fatty acids, while none of the other membrane components, including proteins and phospholipids, was removed by this treatment. Fatty acids inhibited the proline transport activity, while the inhibited activity was appreciably restored by incubation with the albumin. An experiment with radioactive fatty acids showed that exogenously-added fatty acids bound firmly to the membrane vesicles and were removed by subsequent incubation with the albumin. The incubation of membrane vesicles for several hours resulted in an increase of fatty acids with a concomitant loss of the transport activity. Subsequent incubation with the albumin resulted in the removal of fatty acids and the partial restoration of the transport activity. Based on these results, it is concluded that bovine serum albumin specifically removed fatty acids from membrane vesicles, resulting in activation of the proline transport system.     

The role of free amino groups of peptides and proteins in the Folin-Lowry and biuret methods

On an equal weight basis polymyxin B and EM 49 which do not contain tyrosine or tryptophan yielded the same colour intensity as proteins in the Folin-Lowry and biuret methods. But, in the absence of reagent C (alkaline copper reagent) polymyxin B and EM 49 yielded no colour in the Folin-Lowry method. Mono-, di- and tri-formyl polymyxins B formed identical amounts of coloured complexes as polymyxin B in the two methods. However, the tetra- and penta-formyl polymyxins B yielded only one-fifth and one-sixth, respectively, of the expected colour in the Folin-Lowry method. Similarly, 40% and 30%, respectively, of the anticipated amount of colour is formed in the biuret method. Formylated and methylated lysozyme and bovine serum albumins form only 70–75% of the expected colour in the Folin-Lowry method. Since formation of colour by reduction of Folin reagent, in the Folin-Lowry method, is at least partly due to complexes of copper, it was inferred that polymyxin B as well as its mono-, di- and tri-formyl derivatives on the one hand and the tetra- and penta-formyl derivatives on the other differ in their ability to complex Cu(II) The former group of compounds was indeed found to complex as many as three Cu(II) ions whereas the tetra- and penta-formyl polymyxins B complexed only one equivalent, under conditions of excess Cu(II). Under conditions of low Cu(II), polymyxin B and all its derivatives complexed only one Cu(II). In proteins, sites other than amino groups which complex Cu(II) probably play a major role in the reduction of the Folin reagent, since methylated lysozyme and bovine serum albumin yield 70–75% of the colour formed by the unmodified proteins in the Folin-Lowry reaction.     

Comparison of the antioxidant activity of albumin from various animal species

We measured the antioxidant activity of human, rat, bovine, rabbit, and guinea pig albumins against the superoxide, hydroxyl, and 1,1-diphenyl-2-picryl-hydrazyl (DPPH) radicals. The albumins of different animal species did not differ in antioxidant activity against superoxide. Human and rat albumins exhibited antioxidant activity against hydroxyl radicals, but bovine, rabbit, and guinea pig albumins showed weaker antioxidant activity than human and rat albumins. Human, rat, rabbit, and guinea pig albumins, but not bovine albumin, exhibited strong antioxidant activity against DPPH radicals. Human and rat albumins with strong antioxidant activity against hydroxyl radicals contained methionine-123 in domain 1, but bovine, rabbit, and guinea pig albumins did not. Rat, rabbit, and guinea pig albumins with strong antioxidant activity against DPPH radicals had methionine-264 in domain 2. Human albumin did not have methionine-264, but methionine-298 and methionine-329 in domain 2. Bovine albumin, with the weakest antioxidant activity against DPPH radicals, contained no methionine residues in domain 2. These results suggest that methionine residues in domain 1 or 2 influence the antioxidant activity of albumin.     

Effect of Bovine Serum Albumin Incubation on Lettuce Chloroplasts Digested by Trypsin

1. Bovine serum albumin stimulates the DCIP photoreduction activity of lettuce chloroplasts which has been treated with trypsin. When these chloroplast preparations were washed by tricine buffer such "reversible action" can still be obtained. It is possible that bovine serum albumin may be incorporated into trypsin destroyed site of the membrane. 2. Trypsin-induced CCCP inhibitory effect on DCIP photoreduction activity is reversed by bovine serum albumin. 3. Bovine serum albumin partially reverses the trypsin-induced unstacking of lettuce chloroplast membranes. 4. After trypsin digestion, there are absorbance decreases around 500–640 nm. Bovine serum albumin has no effect on these absorbance decreases. It is concluded that the membrane-bound proteins responsible for different functions of chloroplast are heterogeneous. The results also show that there are gate and channel near the position of PSⅡ on chloroplast membrane.     

Stimulatory and Inhibitory Actions of Proteins and Amino Acids On Copper-Catalysed Free Radical Generation in the Bulk Phase

The effect of a variety of proteins and amino acids was investigated on oxygen free radical activity as assessed by copper/hydrogen peroxide induced benzoate hydroxylation as well as copper-catalysed ascor-bate autoxidation. Serum albumins from a variety of species (human, bovine and dog) had both inhibitory and stimulatory effects depending on the molar copper to protein ratio; low ratios were inhibitory and high stimulatory. Some other proteins tested (lysozyme, soybean trypsin inhibitor and conalbumin) also had dual (inhibitory and stimulatory) effects, as did both histidine and polyhistidine, but all effects occurred at different molar ratios presumably dependent on the relative affinities for the copper ions. In contrast, metallolhioncin and cacruloplasmin, proteins specialised to bind copper in vivo had no stimulatory effects. In this paper we show that in addition to their fairly well documented inhibitory effects, under certain conditions some proteins also stimulate radical reactions. The possible role of this phenomenon in vivo is discussed.     

Influence of fatty acids and bovine serum albumin on the growth of human hepatoma and immortalized human kidney epithelial cells

Summary The protective influence of bovine serum albumin against growth inhibition caused by fatty acids was studied in human hepatoma (HepG2) and immortalized human kidney epithelial (IHKE) cells. In general, growth inhibition by unsaturated fatty acids (0.15 mmol/liter) increased with increasing number of double bonds. For HepG2 cells crude albumin (1g/100 ml) did not greatly modify growth inhibition by arachidonic, eicosapentaenoic, and docosahexaenoic acid. With oleic, linoleic, and linolenic acids, crude and defatted albumin stimulated cell growth. In contrast, for IHKE cells both albumins counteracted growth inhibition by unsaturated fatty acids to approximately the same extent. When HepG2 cells were cultured in the presence of saturated fatty acids (0.3 mmol/liter), C2, C6, and C8 had no or little inhibitory effect. C10 and C12 inhibited cell growth appreciably, whereas C14, and especially C16, had poor inhibitory effects. Crude albumin counteracted growth inhibition by all these fatty acids. In contrast, defatted albumin had little or no effect (except against C10 and C12), and even increased the growth inhibition by C14 and C16. With unsaturated fatty acids there seemed to be an inverse relationship between cell growth and the concentration of thiobarbituric acid reactive substances (TBARS) in media. Vitamin E abolished growth inhibition (and the increase in TBARS concentration) by unsaturated fatty acids. The complex interaction between fatty acids and albumins calls for great caution when interpreting data on growth effects.     

Molecular characteristics of methylglyoxal-modified bovine and human serum albumins. Comparison with glucose-derived advanced glycation endproduct-modified serum albumins

The amino acid modification, gel filtration chromatographic, and electrophoretic characteristics of bovine and human serum albumins irreversibly modified by methylglyoxal (MG-SA) and by glucose-derived advanced glycation endproducts (AGE-SA) were investigated. Methylglyoxal selectively modified arginine residues at low concentration (1 mM); at high methylglyoxal concentration (100 mM), the extent of arginine modification increased and lysine residues were also modified. Both arginine and lysine residues were modified in AGE-SA. Analytical gel filtration HPLC of serum albumin derivatives suggested that the proportion of dimers and oligomers increased with modification in both low and highly modified MG-SA and AGE-SA derivatives relative to unmodified serum albumins. In SDS-PAGE analysis, dimers and oligomers of low-modified MG-SA were dissociated into monomers, but not in highly modified MG-SA. MG-SA had increased anodic electrophoretic mobility under nondenaturing conditions atpH 8.6, indicating an increased net negative charge, which increased with extent of modification; highly modified MG-SA and AGE-SA had similar high electrophoretic mobilities. MG-SA derivatives were fluorescent: the fluorescence was characteristic of the arginine-derived imidazoloneN -(5-methyl-4-imidazolon-2-yl)ornithine, but other fluorophores were also present. AGE-SA had similar fluorescence, attributed, in part, to glucose-derived imidazolones. AGE formed from glucose-modified proteins and AGE-like compounds formed from methylglyoxal-modified proteins may both be signals for recognition and degradation of senescent macromolecules.Abbreviations AGE advanced glycation endproduct - BSA bovine serum albumin - HSA human serum albumin - MG-SA methylglyoxal-modified serum albumin - MG-BSA methylglyoxal-modified bovine serum albumin - MG-HSA methylglyoxal-modified human serum albumin - AGE-SA AGE-modified serum albumin - AGE-BSA AGE-modified bovine serum albumin - AGE-HSA AGE-modified human serum albumin - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - HPLC high-performance liquid chromatography - FFI 2-(2-furoyl)-4(5)-(2-furanyl)-1H-imidazole     

Post-feeding induction of trypsin in the midgut of Aedes aegypti L. (Diptera: Culicidae) is separable into two cellular phases

The induction of trypsin activity in the midgut of the mosquito, Aedes aegypti, was studied following meals of chicken blood, and several protein and peptide diets. Various concentrations of bovine serum albumin (BSA) in 0.15 M NaCl stimulated trypsin activity, in a similar fashion to the initial increase observed after a normal blood meal. Trypsin synthesis was also initiated when Ae. aegypti were fed on glutaraldehyde cross-linked BSA and on BSA fragments prepared by both pepsin and cyanogen bromide cleavage. Non-soluble proteins, in the form of glutaraldehyde-fixed erythrocyte ghosts, induced a delayed and reduced trypsin response, whilst small peptides from neutralized liver digests did not induce trypsin activity until 8–10 h after feeding. Metabolic inhibitors had varying effects on the post-feeding activity of trypsin stimulated by BSA feeding. Cycloheximide, a peptidyl transferase inhibitor prevented expression of all activity in vivo, whereas α-amanitin (RNA-polymerase inhibitor) did not affect trypsin activity in the first 10 h after feeding. At 20 μg/ml concentration in the diet, actinomycin D (RNA synthesis inhibitor) caused temporary superinduction followed by inhibition of trypsin activity, but at lower concentrations, the later phase of trypsin activity was inhibited. The results suggest that post-feeding induction of trypsin activity in Ae. aegypti is a two-phase process regulated at the midgut cellular level. The first phase of trypsin synthesis is stimulated by soluble proteins of variable molecular weights, and only involves translation of messenger RNA already available within the midgut cells. The second phase is stimulated by small peptides and requires complete synthesis of new mRNA from DNA.     

Haemolytic and bactericidal activity of serum from the ring dove (Streptopelia risoria) after treatment with exogenous prolactin

The purpose of this study was to elucidate the effect of exogenous prolactin on the haemolytic and bactericidal capacity of serum obtained from ring doves (Streptopelia risoria) previously injected with either bovine serum albumin or saline solution. Haemolytic activity was measured in CH-50 units (which represents the capacity of serum complement to lyse 50% of sheep red blood cells in the presence of specific antibody) and the bactericidal activity was estimated from the number of colony-forming units ofStaphylococcus aureus which survived after 24 h of incubation in the presence of serum. The results indicated that: (1) bovine serum albumin stimulated both haemolytic and bactericidal activity, the highest values occurring 24 h and 4 days after administration, respectively. (2) Prolactin induced an increase in the haemolytic activity of complement. (3) The administration of bovine serum albumin to animals previously treated with prolactin produced a greater stimulation than either bovine serum albumin or prolactin alone.Abbreviations BSA bovine serum albumin - CFT complement fixation test - CFU colony-forming units - CH-50 units, the reciprocal of the complement dilution with 50% lysis of the hemolysin-treated erythrocytes - IU international units - PBS phosphate-buffered saline solution - SS saline solution     

The effect of bovine and human serum albumins on the mechanical properties of human erythrocyte membranes

Crystalline bovine serum albumin increased the mechanical resistance of fresh human erythrocytes to lysis by hydrodynamic shear forces. A saturation effect suggests that the bovine alubmin molecules are adsorbed on to a finite number of “attachment sites” on the erythrocyte surface, possibly by displacing human proteins already occupying these sites. A heterogeneous fraction of human serum albumins does not exhibit the same marked protection effect, nor displace adsorbed bovine albumin molecules from the erythrocyte surface. The precise nature and extent of the interaction between any given concentration of either human or bovine serum albumin and the intact erythrocyte membrane depends upon the chronological age of the cell concerned.     

Inactivation by detergents of the proline transport system in membrane vesicles from Escherichia coli and its reactivation by bovine serum albumin

The proline transport system of membrane vesicles from Escherichia coli was inactivated by a low concentration of detergents such as deoxycholate, dodecyl sulfate and Triton X-100. The addition of a large amount of bovine serum albumin to membrane vesicles which had been treated with one of these detergents resulted in the restoration of the proline transport activity. The restoration of the transport activity by bovine serum albumin was most remarkable with the deoxycholate-inactivated membrane vesicle. 80% inactivation of the transport system with 0.005% deoxycholate was completely overcome by the addition of albumin. The degree of restoration was dependent on the concentration of albumin. Although albumin stimulated the proline transport activity itself, the stimulatory effect could not account for the restoration transport activity. The binding of deoxy[¹⁴C]cholate to the membrane vesicle was roughly proportional to the amount of detergent added. Deoxycholate once bound to the membrane vesicle was removed almost completely by the incubation with albumin. It is concluded that the removal of detergent from the membrane vesicle by bovine serum albumin results in the restoration of the proline transportactivity.