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At least 19 recordsLinked to original sources
PubMed1984

Evolution of alpha-lactalbumins. The complete amino acid sequence of the alpha-lactalbumin from a marsupial (Macropus rufogriseus) and corrections to regions of sequence in bovine and goat alpha-lactalbumins.

alpha-Lactalbumin was purified from a whey protein fraction of the milk of the red-necked wallaby (Macropus rufogriseus). The complete amino acid sequence was determined from the results of automatic sequenator analyses of the intact protein, the three cyanogen bromide fragments, and of peptides generated from the larger, COOH-terminal CNBr fragment by digestion with trypsin or staphylococcal protease. This is the first sequence to be determined of an alpha-lactalbumin from a marsupial and differs from known eutherian alpha-lactalbumins in size and locations of deletions in alignments with the homologous type c lysozymes, as well as in having amino acid substitutions at 8 sites that are invariant in known eutherian proteins. Some corrections are also reported for two regions of sequence in both bovine and goat alpha-lactalbumins. The new and previously published information on alpha-lactalbumin sequences is analyzed in relation to the evolutionary history of the alpha-lactalbumin line as well as the relationship of structure to function in these proteins.

Amino Acid Sequence↗
PubMed2003

Alpha-lactalbumin unfolding is not sufficient to cause apoptosis, but is required for the conversion to HAMLET (human alpha-lactalbumin made lethal to tumor cells).

HAMLET (human alpha-lactalbumin made lethal to tumor cells) is a complex of human alpha-lactalbumin and oleic acid (C18:1:9 cis) that kills tumor cells by an apoptosis-like mechanism. Previous studies have shown that a conformational change is required to form HAMLET from alpha-lactalbumin, and that a partially unfolded conformation is maintained in the HAMLET complex. This study examined if unfolding of alpha-lactalbumin is sufficient to induce cell death. We used the bovine alpha-lactalbumin Ca(2+) site mutant D87A, which is unable to bind Ca(2+), and thus remains partially unfolded regardless of solvent conditions. The D87A mutant protein was found to be inactive in the apoptosis assay, but could readily be converted to a HAMLET-like complex in the presence of oleic acid. BAMLET (bovine alpha-lactalbumin made lethal to tumor cells) and D87A-BAMLET complexes were both able to kill tumor cells. This activity was independent of the Ca(2+)site, as HAMLET maintained a high affinity for Ca(2+) but D87A-BAMLET was active with no Ca(2+) bound. We conclude that partial unfolding of alpha-lactalbumin is necessary but not sufficient to trigger cell death, and that the activity of HAMLET is defined both by the protein and the lipid cofactor. Furthermore, a functional Ca(2+)-binding site is not required for conversion of alpha-lactalbumin to the active complex or to cause cell death. This suggests that the lipid cofactor stabilizes the altered fold without interfering with the Ca(2+)site.

Amino Acids↗
PubMed2005

Effects of the stabilization of the molten globule state on the folding mechanism of alpha-lactalbumin: a study of a chimera of bovine and human alpha-lactalbumin.

The N-terminal half of the alpha-domain (residues 1 to 34) is more important for the stability of the acid-induced molten globule state of alpha-lactalbumin than the C-terminal half (residues 86 to 123). The refolding and unfolding kinetics of a chimera, in which the amino acid sequence of residues 1 to 34 was from human alpha-lactalbumin and the remainder of the sequence from bovine alpha-lactalbumin, were studied by stopped-flow tryptophan fluorescence spectroscopy. The chimeric protein refolded and unfolded substantially faster than bovine alpha-lactalbumin. The stability of the molten globule state formed by the chimera was greater than that of bovine alpha-lactalbumin, and the hydrophobic surface area buried inside of the molecule in the molten globule state was increased by the substitution of residues 1 to 34. Peptide fragments corresponding to the A- and B-helix of the chimera showed higher helix propensity than those of the bovine protein, indicating the contribution of local interactions to the high stability of the molten globule state of the chimera. Moreover, the substitution of residues 1-34 decreased the free energy level of the transition state and increased hydrophobic surface area buried inside of the molecule in the transition state. Our results indicate that local interactions as well as hydrophobic interactions formed in the molten globule state are important in guiding the subsequent structural formation of alpha-lactalbumin.

Amino Acid Sequence↗
PubMed1977

Plasma free amino acid levels in human subjects after meals containing lactalbumin, heated lactalbumin, or no protein.

Plasma free amino acids were measured in six adult human subjects at four consecutive 1/2 hr intervals following meals containing either 50 g lactalbumin, 50 g heated lactalbumin, or no protein. After the lactalbumin meal, all essential and several nonessential amino acids increased in the plasma. After the heated lactalbumin meal, these increases were either much less or did not appear. Following the no protein meal all measured plasma amino acids fell. The absence of a response to the heated lactalbumin meal was considered to be related to a drop in digestibility of heated lactalbumin as measured in rats.

Amino Acids↗
PubMed1975

A fluorimetric study of the interactions of insolubilized human alpha-lactalbumin with galactosyl transferase (A-protein) and with anti-alpha-lactalbumin antibodies.

Intrinsic as well as extrinsic fluorescence of an immobilized protein was used for the study of the interactions between alpha-lactalbumin-Sepharose and protein ligands. The fluorescence peak of the human alpha-lactalbumin-agarose conjugate was shifted to the blue and quenched in the presence of the galactosyl transferase (A-protein), indicating the probable formation of a complex between both proteins. The natural fluorescence of human alpha-lactalbumin bound to Sepharose was specifically quenched in presence of antihuman alpha-lactalbumin antibodies. This change in fluorescence appears to be due to binding of the antibodies to the immobilized antigen. Furthermore, the extrinsic fluorescence of a bound dye such as 2-p-toluidinylnaphthalene-6-sulfonate was used to confirm the existence of binding between antibodies and alpha-lactalbumin-agarose, and to obtain values for the association constant. A value of 5.6-10(+6) M(-1) for the binding constant was reported, which compares favorably with other data obtained by equilibrium dialysis.

Animals↗
PubMed1982

Amino acid sequence of rat alpha-lactalbumin: a unique alpha-lactalbumin.

The amino acid sequence of rat alpha-lactalbumin has been determined. Unlike other alpha-lactalbumins which contain 122 or 123 amino acids, rat alpha-lactalbumin is unique in that it contains 140 amino acids. The extra amino acids are a 17 amino acid extension at the carboxyl terminus. The amino acid sequence of this extension is Gly124-Ala-Pro-Ala-Leu-Val-Val130-Pro-Ala-Leu-Asp-Gly135-Glu-Thr-Pro-Val-Pro140 . The extension is proline rich, which may contribute to the anomalous structural properties of rat alpha-lactalbumin. The amino acid sequence from residues 1 to 123 is similar to that of other alpha-lactalbumins. One possible explanation for the 17 amino acid extension is a mutation at the termination codon.

Amino Acid Sequence↗
PubMed1980

Bovine galactosyltransferase: interaction with alpha-lactalbumin and the role of alpha-lactalbumin in lactose synthase.

Bovine alpha-lactalbumin has been dansylated to give an enzymatically fully active, highly fluorescent derivative. This derivative is uniquely labeled on the N-terminal glutamic acid residue of alpha-lactalbumin. This fluorescent derivative of alpha-lactalbumin has been covalently cross-linked to galacto-syltransferase by using dimethyl pimelimidate. Resonance energy transfer measurements using cobalt bound to the transferase as the acceptor of energy transfer from the dansyl group on the alpha-lactalbumin indicate that the dansyl group is 32 A from the cobalt of the transferase. A model of the active site of the transferase and its interaction with alpha-lactalbumin is proposed on the basis of these and previous studies.

Animals↗
PubMed1978

The interference of endogenous antibodies to bovine lactalbumin in the radioimmunoassay of human lactalbumin in serum.

Using a radioimmunoassay for the milk protein lactalbumin in human serum, we have found that falsely high results can be caused by cross-reacting human antibodies to bovine lactalbumin. A reliable method of circumventing this problem is described. When such interference had been eliminated, circulating lactalbumin was not detected in normal men or post-menopausal women, but could still be found in a proportion of non-pregnant, non-lactating pre-menopausal women.

Adolescent↗
PubMed1984

Conformational changes induced by zinc and terbium binding to native bovine alpha-lactalbumin and calcium-free alpha-lactalbumin.

Terbium at submillimolar concentrations appears to bind to the calcium site of apo bovine alpha-lactalbumin and stabilizes the N conformation (fluorescence criterion; Kronman, M. J., Sinha, S., and Brew, K. (1981) J. Biol. Chem. 256, 8582-8586). At millimolar concentrations however, it binds additionally to a low affinity site of both apo- and calcium-liganded protein, inducing a time-dependent conformational change to an "expanded A-like state." The pH dependence of the transformation implicates the alpha-amino group of glutamic acid 1 of the protein in the binding process. The Zn2+ concentration dependence of the fluorescence of the calcium-free protein indicates there to be two binding sites for this metal ion in agreement with the binding studies with Zn2+ (Bratcher, S.C., and Kronman, M. J. (1984) J. Biol. Chem. 259, 10875-10886). Binding of Zn2+ at submillimolar concentrations stabilizes the A conformation of the protein in contrast to what was observed with Tb3+ at comparable metal ion concentrations. Millimolar concentrations of Zn2+ induce a time-dependent conformational change in both calcium-free and calcium-liganded alpha-lactalbumin to produce an "expanded A-like state" comparable to that seen with terbium at similar concentrations. In contrast to the "expanded A state" induced by high concentrations of zinc or terbium, a "collapsed A state" results from binding of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer species, EDTA, and ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (Kronman, M. J., and Bratcher, S. C. (1983) J. Biol. Chem. 258, 5707-5709), lysine methyl ester, arginine methyl ester, and histidine and by increasing ionic strength of the medium. Since the Zn2+ form of the protein (A conformation) promotes lactose synthetase activity (Kronman et al. cited above), the conformational flexibility of alpha-lactalbumin is likely to be of importance in the formation of the catalytically active complex with galactosyltransferase.

Animals↗
PubMed1990

Comparison of the X-ray structure of baboon alpha-lactalbumin and the tertiary predicted computer models of human alpha-lactalbumin.

The previously predicted structures of human alpha-lactalbumin by homology with hen egg white lysozyme by an automatic method, after the alignment stage, are compared to the X-ray determined structure of baboon alpha-lactalbumin. The root mean square by rotation method (RMSR) deviations for 122 C-alpha atoms between the two models and the X-ray structure are 2.0 A and 2.3 A. The RMSR deviations for all atoms, except for differences in human and baboon sequences, are 2.8 A and 3.1 A. If the flexible C-terminus (residues 112-122) are removed then these RMSR deviations are reduced to 2.4 A and 2.3 A respectively. These results are consistent with the fact that the RMSR deviation between the human and baboon X-ray structures increases from residue 112 onwards and is conformationally flexible.

Amino Acid Sequence↗
PubMed2001

A comparative study of peptide models of the alpha-domain of alpha-lactalbumin, lysozyme, and alpha-lactalbumin/lysozyme chimeras allows the elucidation of critical factors that contribute to the ability to form stable partially folded states.

alpha-Lactalbumin (alpha LA) forms a well-populated equilibrium molten globule state, while the homologous protein hen lysozyme does not. alpha LA is a two-domain protein and the alpha-domain is more structured in the molten globule state than is the beta-domain. Peptide models derived from the alpha-subdomain that contain the A, B, D, and 3(10) helices of alpha LA are capable of forming a molten globule state in the absence of the remainder of the protein. Here we report comparative studies of a peptide model derived from the same region of hen lysozyme and a set of chimeric alpha-lactalbumin--lysozyme constructs. Circular dichroism, dynamic light scattering, sedimentation equilibrium, and fluorescence experiments indicate that the lysozyme construct does not fold. Chimeric constructs were prepared to probe the origins of the difference in the ability of the two isolated subdomains to fold. The first consists of the A and B helices of alpha LA cross-linked to the D and C-terminal 3(10) helices of lysozyme. This construct is highly helical, while a second construct that contains the A and B helices of lysozyme cross-linked to the D and 3(10) helices of alpha LA does not fold. Furthermore, the disulfide cross-linked homodimer of the alpha LA AB peptide is helical, while the homodimer of the lysozyme AB peptide is unstructured. Thus, the AB helix region of alpha LA appears to have an intrinsic ability to form structure as long as some relatively nonspecific interactions can be made with other regions of the protein. Our studies show that the A and B helices plays a key role in the ability of the respective alpha-subdomains to fold.

Amino Acid Sequence↗
PubMed1995

The utilization of lupin (Lupinus angustifolius) and faba bean globulins by rats is poorer than of soybean globulins or lactalbumin but the nutritional value of lupin seed meal is lower only than that of lactalbumin.

The effects of dietary sweet lupin (Lupinus angustifolius, Unicrop) seed meal or its insoluble fiber (nonstarch polysaccharides + lignin) on performance, digestibility and nitrogen utilization in growing rats were studied in four experiments. Globulin proteins isolated from lupin, faba bean (Vicia faba L. minor) or soybean (Glycine max) were also incorporated into purified diets as replacements for lactalbumin (control) and the nutritional effects were evaluated. Isocaloric, legume-based diets supplemented with amino acids were used. Final weight gain, gain:feed ratios, nitrogen retention and net protein utilization of the animals fed whole lupin meal-based diets for 10 d were inferior to those of controls. In contrast, adding lupin insoluble fiber to a control diet produced no adverse effects. Ileal starch and apparent nitrogen digestibilities, and fecal digestibility of starch in lupin-fed rats were higher than those of controls, but fecal true nitrogen digestibility was lower. Replacement of lactalbumin with globulin proteins from lupin or faba bean depressed food intake and protein utilization, but only performance was affected by consumption of soybean globulins. Rats consuming lupin or faba bean globulins excreted significantly more nitrogen, particularly as urea through the urine. This did not occur in rats fed soybean globulins. Urea concentration in plasma was higher in rats fed diets containing lupin meal or legume globulins. The concentrations of urea, arginine and ornithine in plasma increased significantly compared with control values after 3 to 9 h of a lupin diet. After 9 h, plasma lysine was also decreased. We concluded that the main reasons for the low nutritional value of sweet lupin seed meal are likely to be related to the chemical structure of the globulin proteins and their adverse effects on growth and nitrogen metabolism, and not to any known antinutritional factor or poor digestibility.

Amino Acids↗
PubMed1977

Heterogeneity in alpha-lactalbumins. I. Human alpha-lactalbumin.

alpha-Lactalbumin from human milk shows an heterogeneous behaviour when subjected to ion exchange chromatography with DEAE-Sephadex. Two components have been separated, showing identical patterns in the following studies: amino acid compositions, fluorescence and circular dichroism spectra, transition temperature of denaturation, antigenicity, lactose synthase specifying activity and hydrodynamic properties. After rechromatography of either peak, these two components appeared to be in equilibrium. This equilibrium varies with the temperature and the pH of chromatography. Moreover, an increase of n-alcohol concentration in the eluting buffer also induces an increase of the second protein peak eluting at higher ionic strength. These two peaks seem to be the result of some conformational change induced upon the binding of the protein to the solid anionic matrix.

Amino Acids↗
PubMed2005

Characterization of tryptic hydrolysis of alpha-lactalbumin/saponin mixture and structural change of alpha-lactalbumin interacting with soybean saponin.

Bovine milk alpha-lactalbumin (alpha-La) was mixed with soybean saponin, and the resulting mixture was hydrolyzed by trypsin. Saponin increased the tryptic-hydrolysis level of alpha-La only at relatively high phosphate buffer concentrations (> or = 0.05 M). T(1) experiments with acetylated soybean saponin demonstrated that there were some interactions between alpha-La and saponin not only at high concentrations of phosphate buffers but even at low concentrations as well. Circular dichroism spectra of alpha-La showed that the tertiary structure of alpha-La was changed through interactions with saponin only at high buffer concentrations. Furthermore, by analyzing the tryptic peptides from an alpha-La/saponin mixture, hydrolyzing rates at all or some of K5, R10, and K16 of alpha-La were accelerated by saponin interactions. The increase in the tryptic hydrolysis of alpha-La by saponin addition was considered due to modification of the tertiary structure of alpha-La by saponin.

Animals↗
PubMed1995

Lactation is disrupted by alpha-lactalbumin deficiency and can be restored by human alpha-lactalbumin gene replacement in mice.

Mice carrying either a deletion of the murine alpha-lactalbumin (alpha-lac) gene (null allele) or its replacement by the human alpha-lac gene (humanized allele) have been generated by gene targeting. Homozygous null females are alpha-lac-deficient, produce reduced amounts of thickened milk containing little or no lactose, and cannot sustain their offspring. This provides definitive evidence that alpha-lac is required for lactose synthesis and that lactose is important for milk production. Females homozygous for the humanized allele lactate normally, indicating that human alpha-lac can replace murine alpha-lac. Mouse and human alpha-lac expression was compared in mice heterozygous for the humanized allele. The human gene expressed approximately 15-fold greater mRNA and approximately 14-fold greater protein than the mouse, indicating that the major determinants of human alpha-lac expression are close to, or within, the human gene and that the mouse locus does not exert a negative influence on alpha-lac expression. Variations in alpha-lac expression levels in nondeficient mice did not affect milk lactose concentration, but the volume of milk increased slightly in mice homozygous for the humanized allele. These variations demonstrated that alpha-lac expression in mice is gene dosage dependent.

Alleles↗
PubMed2000

The structure of denatured alpha-lactalbumin elucidated by the technique of disulfide scrambling: fractionation of conformational isomers of alpha-lactalbumin.

The structure of denatured alpha-lactalbumin (alpha-LA) has been characterized using the method of disulfide scrambling. Under denaturing conditions (urea, guanidine hydrochloride, guanidine thiocyanate, organic solvent or elevated temperature) and in the presence of thiol initiator, alpha-LA denatures by shuffling its four native disulfide bonds and converts to a mixture of fully oxidized scrambled structures. Analysis by reversed-phase HPLC reveals that the denatured alpha-LA comprises a minimum of 45 fractions of scrambled isomers. Among them, six well populated isomers have been isolated and structurally characterized. Their relative concentrations, which represent the fingerprinting of the denatured alpha-LA, vary substantially under different denaturing conditions. These results permit independent plotting of the denaturation and unfolding curves of alpha-LA. Most importantly, unique isomers of partially unfolded alpha-LA were shown to populate at mild and selected denaturing conditions. Organic solvent disrupts preferentially the hydrophobic alpha-helical domain, generating a predominant isomer containing two native disulfide bonds at the beta-sheet domain and two scrambled disulfide bonds at the alpha-helical region. Thermal denaturation selectively unfolds the beta-sheet domain of alpha-LA, producing a prevalent isomer that exhibits structural characteristics of the molten globule state of alpha-LA.

Acetonitriles↗