Purification and characterization of mouse α-lactalbumin from lactating mammary glands

The whey protein, α-lactalbumin, was purified from lactating mammary glands of mice at high yields. It exists as two major charge forms (pI values of 6.2 and 5.8) with similar molecular weights (approx. 14 00). Antibodies prepared against these peptides precipitate newly synthesized and secreted α-lactalbumin from organ cultures of mid-pregnancy mammary glands. The antibody is specific for mouse α-lactalbumin as it does not react with mouse casein, mouse serum or purified bovine α-lactalbumin or galactosyl transferase. In addition, it blocks enzymatic activity of α-lactalbumin in mouse milk but has no effect on guinea pig or human milk. A very sensitive radioimmunoassay has been developed with this antibody which can detect α-lactalbumin levels as low as 0.25 ng.     

Interaction of the components of the lactose synthetase system

The modifier action of α-lactalbumin upon galactosyl transferase is pH-dependent. When either N-acetyl glucosamine or glucose is the acceptor, the pH profile of activity is altered in the presence of α-lactalbumin. The effect of α-lactalbumin upon the kinetic constants at a series of pH's has been interpreted in terms of a molecular mechanism of modifier activity.Fluorescence polarization studies indicated that a definite molecular complex between α-lactalbumin and galactosyl transferase is formed in the presence of substrate. Estimates of the equilibrium constants have been made.     

Biosynthesis of keratan sulfate: purification and properties of a galactosyltransferase from bovine cornea.

A soluble galactosyltransferase was purified 22,000-fold from bovine cornea. The enzyme catalyzes the transfer of galactose from UDP-galactose to N-acetyl-d-glucosamine, α- and β-glucosaminides, bovine cornea and nasal septum agalactokeratan, and to glycoproteins containing terminal nonreducing N-acetylglucosaminyl units. When N-acetyl-d-glucosamine served as acceptor, the product formed by the cornea transferase contained galactose glycosidically linked to carbon atom 4 of N-acetyl-d-glucosamine; the same glycosidic linkage was found in [¹⁴C]keratan preparations isolated from reaction mixtures where keratan containing terminal nonreducing N-acetylglucosaminyl units served as acceptor. The cornea enzyme exhibited a markedly lower K_m with keratan than with N-acetyl-d-glucosamine. The physical and kinetic properties of the cornea galactosyltransferase and of the milk A-protein (A-protein + α-lactalbumin = lactose synthase), including modulations of acceptor specificity by α-lactalbumin, were compared. The results of these studies strongly suggest that the two glycosyltransferases are similar, if not identical. Efforts to demonstrate the presence of other soluble galactosyltransferases in cornea were unsuccessful; no change in the ratios of products formed with several acceptors was observed at any stage of purification. It is suggested that in bovine tissues a single galactosyltransferase participates in the synthesis of both high and low molecular weight galactosides including the assembly of the repeating disaccharide [O-β-galactopyranosyl-(1 → 4)-N-acetylglucosamine] of cornea keratan sulfate.     

Microchip electrophoresis for monitoring covalent attachment of poly(ethylene glycol) to proteins

A microchip electrophoretic method was applied to monitor and characterize the covalent attachment of poly(ethylene glycol) (PEGylation) of two proteins, α-lactalbumin and bovine serum albumin, using several poly(ethylene glycol) (PEG) derivatives with molecular weights from 1 to 20 kDa. This method effectively separated multi-PEGylated proteins in a size-based manner and allowed monitoring of the PEGylation pattern with the advantages of high speed, minimal sample consumption, and high reproducibility. Microchip electrophoresis would be a very useful tool for protein PEGylation studies such as reaction monitoring, purity checks, and characterization of PEGylated protein products.     

Incorporation of methionine by a soluble enzyme system from Escherichia coli

A soluble fraction from Escherichia coli B was found to incorporate methionine into 95°C CCl₃COOH-insoluble fraction. The incorporation required methionyl-tRNA synthetase, methionine tRNA, ATP, Mg²⁺ and bovine milk casein. The casein could be replaced by arginylated bovine serum albumin and arginylated bovine α-lactalbumin. A mixture of 19 amino acids other than methionine and GTP had no effect on the incorporation. KCl was rather inhibitory. Puromycin, RNase A and trypsin inhibited the incorporation, while DNase I did not. The soluble fraction also incorporated the methionyl moiety of methionyl-tRNA. This incorporation was not affected by the addition of free methionine.     

The amino acid sequence of goat alpha-lactalbumin

The amino acid sequence of goat α-lactalbumin has been established from the structures of peptides isolated from trypsin and thermolysin digests of the reduced aminoethylated protein and from a chymotrypsin digest of the reduced carboxamidomethylated protein. The amino-terminal sequence was confirmed by automatic sequencer analysis. Of the previously sequenced species variants of α-lactalbumin, the bovine protein is most similar to the goat, differing in only 12 amino acid substitutions. One difference between these proteins corresponds to a substitution found in the bovine A genetic variant (Arg¹⁰ → Gln). The relevance of the structure to the evolutionary relationships in the α-lactalbumin-lysozyme family of proteins is discussed.     

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.     

Binding of naphthalene dyes to the N and A conformers of bovine α-lactalbumin

Contrary to earlier findings, monomeric native α-lactalbumin does bind naphthalene dyes such as ANS and TNS with marked enhancement of their fluorescence. Nanosecond decay measurements indicate there to be two dye binding sites per protein molecule with lifetimes of ca. 2 and 15 ns for ANS and 5 and 11 ns for TNS. The fluorescence titrations curves of α-lactalbumin with ANS and TNS reflect this site multiplicity, i.e., it was not possible to analyze such curves with a single K_diss. The apparent dissociation constants for binding of ANS and TNS to native bovine α-lactalbumin, as determined by an ultracentrifugal technique, ca. 950 and 900 μm, respectively, indicate that such binding is considerably weaker than previously supposed. The A conformer (metal ion-free form) of α-lactalbumin binds ANS and TNS more tightly than the N (native) form of the protein with marked fluorescence enhancement. The A conformer has two dye binding sites with lifetimes for ANS and TNS comparable with those seen with native protein.     

Conformational changes of α-lactalbumin and its fragment,Phe31-Ile59, induced by sodium dodecyl sulfate

Conformational changes of bovine α-lactalbumin in sodium dodecyl sulfate (SDS) solution were studied with the circular dichroism (CD) method using a dilute phosphate buffer ofpH 7.0 and ionic strength 0.014. The proportions of α-helix and β-structure in α-lactalbumin were 34% and 12%, respectively, in the absence of SDS. In the SDS solution, the helicity increased to 44%, while the β-structure disappeared. In order to verify the structural change from β-structure to α-helix, the moiety, assuming the β-structure in the α-lactalbumin, was isolated by a chymotryptic digestion. The structure of this α-lactalbumin fragment, Phe³¹-Ile⁵⁹, was almost disordered. However, the fragment adopted a considerable amount of α-helical structure in the SDS solution. On the other hand, the tertiary structure of α-lactalbumin, detected by changes of CD in the near-ultraviolet region, began to be disrupted before the secondary structural change in the surfactant solution. Dodecyl sulfate ions of 80 mol were cooperatively bound to α-lactalbumin. Although the removal of the bound dodecyl sulfate ions was tried by the dialysis against the phosphate buffer for 5 days, 4 mol dodecyl sulfates remained per mole of the protein. The remaining amount agreed with the number of stoichiometric binding site, determined by the Scatchard plot, indicating that the stoichiometric binding was so tight.     

The membrane bound retinol dehydrogenase from bovine rod outer segments

Retinol dehydrogenase from bovine rod outer segments was solubilized in detergent and partially purified 25-fold through a combination of hydroxyapatite and retinyl-Sepharose chromatography. Alltrans retinol solubilized in protein solutions of bovine serum albumin or β-lactalbumin was a better substrate for the enzyme than retinol solubilized in detergents or suspended in buffer. Retinol dehydrogenase was sensitive to the carbonyl reagent pyridoxal-5′-phosphate but was not inhibited by retinal followed by reduction with NaBH₄. The solubilized enzyme requires phospholipids to maintain enzymatic activity, as was evidenced by the inactivating effect of phospholipase A₂ on the partially purified enzyme.     

Arginyl-tRNA-protein transferase in eucaryotic protists

Soluble extracts of $\text{Saccharomyces cerevisiae}$ and $\text{Blastocladiella emersonii}$ were found to catalyze the specific transfer of arginine from a mixture of [¹⁴C] aminoacyl-tRNAs into protein. Arginine transfer was stimulated by bovine serum albumin. Glu-Ala, Asp-Ala and cystinyl-$\text{bis}$-Ala inhibited incorporation into protein, whereas dipeptides with other NH₂-terminal residues linked to alanine did not. These results indicate the presence of an enzyme in eucaryotic protists with the same donor and acceptor specificity as mammalian arginyl-tRNA-protein transferase.     

Immunological reactivities of rat and human α-lactalbumin

• 1.1. Antisera to rat α-lactalbumin immediately displayed precipitation bands to human, bovine, pig and goat α-lactalbumin, whereas antisera to human α-lactalbumin displayed similar cross reactivities only at a later time.
• 2.2. Cross reactivity of the antisera was determined by radioimmune assays to rat and human α-lactalbumin.

     

β-Lactoglobulin Suppresses Melanogenesis in Cultured Human Melanocytes

The effects of whey proteins from bovine milk on melanogenesis in cultured human melanocytes were examined. Among the major protein components of milk whey including β-lactoglobulin (BLG), α-lactalbumin, serum albumin, and IgG, only BLG exhibited the depigmenting effect at a concentration of 1 mg/ml. Also, BLG suppressed the activity of tyrosinase in these cells. Retinol, to which BLG is known to bind, slightly increased the pigmentation of the cells at concentrations in the range of 1–100 nM, and retinoic acid, a metabolite of retinol, exhibited a strong pigmentation-promoting effect within the same concentration range. Treatment of the cells with 1 mg/ml BLG completely abrogated the pigmentation induced by these A vitamins. These results demonstrate a novel biological activity of BLG and suggest that this activity is dependent on its ability to bind retinol.     

The Effect Of Commercial Bovine Serum Albumin And Other Proteins On The Activity Of Bovine Milk Galactosyltransferase

Abstract

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 Vittle 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.     

The primary structure of wheat germ tRNAArg--the substrate for arginyl-tRNAArg:protein transferase

Besides its major role in protein synthesis, wheat germ arginyl-tRNAArg can serve as an amino acid donor in an enzymatic reaction to bovine serum albumin catalysed by the enzyme arginyl-tRNAArg: protein transferase. The nucleotide sequence of the tRNAArg involved in this reaction was determined to be: pG-A-C-U-C-C-G-U-m1G-m2G-C-C-C-A-A-D-Gm-G-A-X-A-A-G-G-C-m2(2) G-C-U-G-G-U-Cm-U-I-C-G-m2A-A-A-C-C-A-G-A-G-A-D-U-m5C-U-G-G-G-T-psi -C-G-m1 A-U-C-C-C-C-A-G-C-G-G-A-G-U-C-G-C-C-AOH. We suggest that the decapentanucleotide 5'-G-U-Pu-m2G-C-N-C-A-A-D-Gm-G-A-X-A-3', localized in the D-region, interacts specifically with the protein transferase.     

Investigation on the effect of fluorescence quenching of bovine serum albumin by cefoxitin sodium using fluorescence spectroscopy and synchronous fluorescence spectroscopy

The reaction mechanism of cefoxitin sodium with bovine serum albumin was investigated using fluorescence spectroscopy and synchronous fluorescence spectroscopy at different temperatures. The results showed that the change of binding constant of the synchronous fluorescence method with increasing temperature could be used to estimate the types of quenching mechanisms of drugs with protein and was consistent with one of fluorescence quenching method. In addition, the number of binding sites, type of interaction force, cooperativity between drug and protein and energy‐transfer parameters of cefoxitin sodium and bovine serum albumin obtained from two methods using the same equation were consistent. Electrostatic force played a major role in the conjugation reaction between bovine serum albumin and cefoxitin sodium, and the type of quenching was static quenching. The primary binding site for cefoxitin sodium was sub‐hydrophobic domain IIA, and the number of binding sites was 1. The value of Hill's coefficients (n_H) was approximately equal to 1, which suggested no cooperativity in the bovine serum albumin–cefoxitin sodium system. The donor‐to‐acceptor distance r < 7 nm indicated that static fluorescence quenching of bovine serum albumin by cefoxitin sodium was also a non‐radiation energy‐transfer process. The results indicated that synchronous fluorescence spectrometry could be used to study the reaction mechanism between drug and protein, and was a useful supplement to the conventional method. Copyright © 2015 John Wiley & Sons, Ltd.     

Effect of albumin on the in vitro conjugation of bilirubin by rat liver microsomes

These studies were carried out to determine whether bovine serum albumin (BSA), which is usually included in the incubation mixture for the in vitro determination of bilirubin-UDP-glucuronyl transferase (GT) activity, affects GT activity. Using bilirubin as substrate, addition of BSA to the enzyme reaction mixture at concentrations varying from 2 to 30 mg/ml resulted in a dose-related inhibition of "native" GT activity of rat liver microsomes. When detergent-activated enzyme was employed, increasing concentrations of BSA also required higher concentrations of deoxycholate, digitonin, or Triton X-100 to produce maximal bilirubin conjugation. Low BSA concentrations (2 mg/ml) prevented enzyme activation by both detergents and UDP-N-acetyl glucosamine. When BSA was omitted and bilirubin dissolved in dimethyl sulfoxide, UDP-N-acetyl glucosamine failed to enhance GT activity, and activation by detergents was only 15-25% of that observed in the presence of optimal concentrations of BSA. When rat albumin was substituted for BSA, a similar dose-related inhibition of in vitro bilirubin conjugation by untreated microsomes was observed, although at any given albumin concentration, GT activity was lower with rat than with bovine albumin. Additionally, both detergents and UDP-N-acetyl glucosamine produced similar GT activation regardless of the rat albumin concentration. Finally, these effects of BSA and rat albumin could not be reproduced when beta-lactoglobulin was employed and/or when p-nitrophenol was the acceptor substrate of GT. These findings indicate that albumin, in particular BSA, profoundly and selectively influences the in vitro activity of microsomal GT toward bilirubin as the acceptor substrate.     

Is thermally denatured protein unfolded? The example of α-lactalbumin

The degree of unfolding of various forms of human and bovine α-lactalbumin was characterized by the chromatographically determined distribution coefficient. Molecular size increases as follows: native protein ≈ apo form < acid form ⪡ thermally unfolded form ≈ guanidine hydrochloride-unfolded protein.     

Plausible Novel Ribose Metabolism Catalyzed by Enzymes of the Methionine Salvage Pathway in Bacillus subtilis

Although several studies have shown that milk protein components have a wide range of biological activities, the potential role of these proteins in the gastrointestinal mucosal defense system is less well elucidated. In this study, we investigated the effect of the major proteins in cow’s milk on gastric mucosal injury by using two acute ulcer models in Wistar rats. Gastric mucosal injury was induced by either intragastric 60% ethanol-HCl or water-immersion restraint stress (23°C, 7 h). Each test milk protein was orally administered 30 min before the induction of gastric injury. Among the major milk proteins, α-lactalbumin (α-LA) is demonstrated to have a marked protective effect against ethanol-induced gastric injury, with the same potency as that of the typical antiulcer agent, Selbex. Whey protein isolate (WPI), which contained 25% α-LA, also protected against gastric injury, while casein showed no effect. Comparative studies on the protective effect of the four major components of WPI, β-lactoglobulin, α-LA, bovine serum albumin and γ-globulins (immunoglobulins), on the basis of their contents in WPI revealed that α-LA was responsible for the protective effect of WPI, being about 4-fold more effective than WPI itself. α-LA showed dose-dependent protection against gastric injury induced by stress as well as ethanol. Pretreatment with indomethacin (10 mg/kg body weight, s.c.), which is a potent inhibitor of endogenous prostaglandin synthesis, resulted in a significant reduction in the protective effect of α-LA. These results indicate that α-LA has marked antiulcer activity as an active component of cow’s milk protein, and suggest that α-LA intake may serve to protect against gastric mucosal injury, in part through endogenous prostaglandin synthesis.     

Effect of pH and Glucose on the Stability of α-lactalbumin

The objective of this study is to understand the influence of pH and effect of cosolvent (glucose) on the stabilization of bovine α-lactalbumin by using ultrasonic techniques. Values of density, ultrasonic velocity and viscosity were measured for bovine α-lactalbumin (5 mg/ml) dissolved in phosphate buffer (pH 2, 5, 7, 9 and 12) solutions mixed with and without the cosolvent at 30 °C. These measurements were used to calculate few thermo-acoustical parameters such as adiabatic compressibility, intermolecular free length, acoustic impedance, relaxation time, relative association constant, the partial apparent specific volume and the partial apparent specific adiabatic compressibility for the said systems. The obtained results revealed a strong comparison between the effects of acidic and alkaline pH values on protein denaturation, i.e., the acidic pH are instantaneous and are of less magnitude whereas alkaline pH are slower but sharper. Further the present study supports the fact that the presence of glucose stabilizes α-lactalbumin against denaturation due to pH variation, which may be due to the strengthening of non-covalent interactions and the steric exclusion effect.