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At least 19 recordsLinked to original sources
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↗
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↗
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↗
PubMed1976

The kinetic mechansim of bovine milk galactosyltransferase. The role of alpha-lactalbumin.

Initial rate parameters obtained with bovine galactosyltransferase at saturating Mn2+ concentrations, and a variety of acceptors including N-acetylglucosamine, glucose, ovalbumin, and di-N-acetylglucosamine are inconsistent with an ordered addition of UDP-galactose and acceptor substrates to the enzyme-Mn2+ complex. Inhibition patterns with N-acetylglucosamine or UDP-glucose as inhibitors of the galactosylation of ovalbumin indicated that either UDP-galactose or N-acetylglucosamine can bind to an enzyme-Mn2+ complex by a random equilibrium mechanism. Initial rate studies also indicate that alpha-lactalbumin may bind to either an enzyme-Mn2+-acceptor complex or an enzyme-Mn2+-UDP-galactose complex, suggesting that lactose synthesis also proceeds by a random equilibrium addition of substrates and alpha-lactalbumin. From the initial rate data assuming the random equilibrium mechanism, the dissociation constants for UDP-galactose, acceptor substrates, and alpha-lactalbumin from the appropriate complexes have been calculated. These values are in good agreement with those obtained independently by nonkinetic methods, providing additional support for the proposed random equilibrium mechanism. From similar studies with a cross-linked complex of alpha-lactalbumin and transferase, dissociation constants for UDP-galactose and acceptor substrates from the enzyme-Mn2+-alpha-lactalbumin complex were calculated. Comparison of each of the dissociation constants in the substrate addition phase shows that the binding of acceptor substrates and alpha-lactalbumin to enzyme-Mn2+ complexes is highly synergistic; the affinity of alpha-lactalbumin for the enzyme-Mn2+ acceptor complex is about 2 orders of magnitude greater than for the enzyme-Mn2+ complex. Similarly, the affinity of the acceptor for the enzyme-Mn2+-alpha-lactalbumin complex is about 2 orders of magnitude greater than the enzyme-Mn2+ complex. Synergism is also observed between alpha-lactalbumin and UDP-galactose binding but the synergism is much less than that observed with acceptor substrates and alpha-lactalbumin. Thus, the large decrease in the Michaelis constant for glucose in the presence of alpha-lactalbumin, which is observed for lactose synthesis by the galactosyltranferase, is primarily the result of the high degree of synergism in the binding of alpha-lactalbumin and glucose to enzyme-Mn2+ complexes. This synergism also accounts for the activation of N-acetyllactosamine synthesis by alpha-lactalbumin at low concentrations (less than 2 mM) of N-acetylglucosamine. An abortive enzyme-Mn2+-UDP-acceptor complex in the product release phase of the reaction appears to account for the inhibition of either lactose, or N-acetyllactosamine synthesis at a high concentration of either N-acetylglucosamine or glucose. This abortive complex is further stabilized by alpha-lactalbumin, thus the resulting substrate inhibition is observed at much lower acceptor concentrations in the presence of alpha-lactalbumin.

Acetylglucosamine↗
PubMed1978

Prolactin stimulation of alpha-lactalbumin in normal primate mammary gland.

An in vitro system has been developed to examine the effects of PRL on the normal primate mammary gland. alpha-Lactalbumin a milk protein, was found in breast tissue samples from 17 of 19 primates representing several Macaca and Papio species; concentrations ranged from 10-768 ng/mg protein. That none of the animals was pregnant or lactating and half were nulliparous indicates that milk protein production takes place under normal circumstances, even in breast tissue of nonlactating animals. Studies of the effect of PRL on alpha-lactalbumin production in these tissues in organ culture revealed that PRL maintained existing or stimulated new production of alpha-lactalbumin for periods of up to 9 days. Measurement of alpha-lactalbumin in medium bathing mammary tissue from three animals revealed that mean alpha-lactalbumin production during days 7-9 when PRL was added (100 and 1000 ng/ml) was 11 and 59 times greater, respectively, than control. Simultaneous measurement of tissue concentrations of alpha-lactalbumin revealed that those tissues maintained with PRL (1000 ng/ml) had a mean concentration of alpha-lactalbumin that was 61 times that of controls without PRL. PRL consistently maintained or increased alpha-lactalbumin production in tissues from all 22 primates tested. Even in those premenarchal animals in whose mammary tissue alpha-lactalbumin was undetectable initially, PRL stimulated alpha-lactalbumin production in a dose-related fashion. In contrast, when PRL was absent from medium, alpha-lactalbumin concentrations decreased at 9 days to less than 20% of the initial 3-day value in all cases. These studies provide evidence that mammary tissue from normal nonlactating, nonpregnant primates produces milk proteins and that when tissues are exposed to PRL in culture, production of alpha-lactalbumin is stimulated.

Animals↗
PubMed1975

Fluorimetric study of conformational changes of various alpha-lactalbumins on agarose carriers.

1. Various insoluble alpha-lactalbumins (bovine, bovine glyco-alpha-lactalbumin, human and human nitrated) have been prepared by coupling these proteins on to an agarose gel with use of cyanogen bromide. 2. Some intrinsic fluorescence properties, such as fluorescence maximum and pH dependence, were considered in order to study conformational changes of the alpha-lactalbumins covalently bound to an insoluble matrix. Examination of the pH-fluorescence profiles as well as the position of the maximum in the emission spectrum indicates that the Sepharose matrix does not appreciably modify the conformation of human and bovine glyco-alpha-lactalbumins. Some changes in the fluorescence spectrum (peak shifting towards longer wavelength) was observed for bovine alpha-lactalbumin and appeared to be due to alteration of the environment of the tryptophan side-chains in the protein upon coupling to the agarose gel. The emission spectrum of the insolubilized human nitrated alpha-lactalbumin indicates that the polypeptide chain of this protein gained some native conformation when covalently bound to the carrier. 3. The extrinsic fluorescence of a bound dye, such as 2-p-toluidinylnaphthalene-6-sulfonate, was used to study and to compare the hydrophobic sites on the surface of insoluble alpha-lactalbumins with the same proteins in solution. Considering the fluorescence properties of the protein with dye complexes it was found that both states of alpha-lactalbumins (insoluble and free in solution) bind the dye with similar association constants. However, the positions of the maxima in the emission spectra are all somewhat shifted towards longer wavelengths, suggesting that the dye binding site is located in a more polar environment when the proteins are bound to agarose. The human nitrated alpha-lactalbumin retains about equal possibility of binding this fluorescent dye. 4. As shown for bovine alpha-lactalbumin in solution, the binding of 2-p-toluidinylnaphthalene-6-sulfonate towards the various insoluble alpha-lactalbumins was not appreciably modified by the presence of various small compounds such as sugars and UDP, which are effectors in the lactose synthetase function.

Animals↗
PubMed1977

Studies on human alpha-lactalbumin: radioimmunoassay measurements in normal human breast and breast cancer.

A sensitive and specific radioimmunoassay for human alpha-lactalbumin, a milk protein, has been developed in order to examine the effect of prolactin on the human breast in normal and diseased states. Samples of milk from nursing mothers and from men and women with galactorrhea were found to contain milligram concentrations of this protein. In serum, 8 of 25 normal men and 18 of 44 normal women had detectable concentrations of alpha-lactalbumin. Significantly higher levels of alpha-lactalbumin were found in 17 of 19 women during pregnancy who were not actively lactating. All nursing mothers were found to have distinctly elevated serum alpha-lactalbumin concentrations. In a group of 17 female patients with phenothiazine induced prolactin elevations (mean 29.4 ng/ml), the mean serum alpha-lactalbumin of 17.3 ng/ml was significantly higher than in normal female volunteers. Patients with gynecomastia were not noted to have elevated alpha-lactalbumin. In vitro, homogenates of normal breast and carcinoma tissue from the same individuals revealed that in 9 of 17 patients alpha-lactalbumin was present in higher concentrations in normal than in cancerous tissue. Overall, alpha-lactalbumin was found in 48.5% of homogenates and 41% of organ cultures of normal breast tissue from cancer patients. In contrast, it was present in only 19% of homogenates and 21% of cultures of carcinoma tissue, indicating that the cancer tissue may lose its ability to produce alpha-lactalbumin. Differences in biologic behavior were found in some tumors. In 2 cases homogenates of breast cancer tissue had much higher concentrations of alpha-lactalbumin than the normal tissue, and in 3 of 33 tumors studied in organ culture prolactin increased alpha-lactalbumin output.

Adolescent↗
PubMed1977

alpha-Lactalbumin levels in human mammary tumors, sera, and mammary cell culture lines.

Antiserum to purified human alpha-lactalbumin was produced in rabbits and used to develop a radioimmunoassay capable of detecting 0.1 ng of alpha-lactalbumin per ml of sample. Human breast diseases were analyzed for alpha-lactalbumin levels. A high percentage of breast carcinomas contained varying levels of alpha-lactalbumin. Lymph node metastases from primary carcinomas that synthesized alpha-lactalbumin also contained it. Analysis of serum from breast cancer patients indicated that approximately 25 percent had measurable levels of alpha-lactalbumin before surgery, but no alpha-lactalbumin was found in postsurgery sera. alpha-Lactalbumin was not detected in the urine of early lactational women, although it was present in the sera. Human cell culture lines derived from pleural effusions of mammary carcinomas contained little, if any, alpha-lactalbumin. Other human cell lines derived from mammary carcinomas and grown as solid tumors in athymic mice did not contain measurable levels of alpha-lactalbumin.

Breast Neoplasms↗
PubMed1977

Relationships between radioimmunoassays of alpha lactalbumin and prolactin in bovine skim milk.

A radioimmunoassay developed for alpha-lactalbumin was sensitive between 5 and 140 ng alpha lactalbumin. Addition of increasing volumes of milk to assay tubes progressively decreased binding of iodine-125-labeled alpha-lactalbumin to the antisera in a manner which paralleled the binding curve generated by increasing concentrations of standard alpha-lactalbumin. The addition of 10, 20, 30, or 40 ng of alpha-lactalbumin to diluted milk samples gave 90, 100, 105, and 102% recoveries. Alpha-lactalbumin antisera did not crossreact with 1000 ng of bovine casein, blood serum albumin, beta-lactoglobulin, prolactin, or growth hormone. Milk from each of approximately 100 Holstein Friesian cows at different stages of lactation was sampled monthly for 12 mo. Concentrations of alpha-lactalbumin (1.63 mg/ml) and prolactin (24.9 ng/ml) in samples of skim milk collected in the 1st mo of lactation were greater than those in the remaining months of lactation. Monthly concentrations of alpha-lactalbumin and prolactin in skim milk did not change significantly during seasons of the year. The correlation between concentrations of prolactin and alpha-lactalbumin pooled within subclasses of month of lactation within month of year was .08 for 1125 pairs.

Animals↗
PubMed1975

On the interaction of alpha-lactalbumin and galactosyltransferase during lactose synthesis.

The regulatory effect of alpha-lactalbumin in the lactose synthase system has been ascribed to its reversible association with a complex of galactosyltransferase with Mn2+ and UDP-galactose, prior to the binding of monosaccharides; the resulting complex has a higher affinity for various monosaccharides. Two steps in the postulated catalytic cycle have been investigated; UDP-galactose binding to enzyme-Mn2+ by equilibrium dialysis and alpha-lactalbumin binding to enzyme-Mn2+-UDP-galactose by sedimentation velocity and kinetics. There is a single binding site for UDP-galactose on the enzyme-Mn2+ complex, and the dissociation constant for UDP-galactose from enzyme-Mn2+-UDP-galactose was found to be 72 muM at 37 degrees. The formation of a complex between galactosyltransferase and alpha-lactalbumin in the presence of Mn2+ and UDP-galactose was observed as an increase in sedimentation coefficient of enzyme activity So20,w from 3.25 +/- 0.03 in the absence of alpha-lactalbumin to 4.22 +/- 0.03 at saturating concentrations of alpha-lactalbumin, a value closely similar to that of a cross-linked 1:1 complex of the proteins under the same conditions (4.35 +/- 0.03). No interaction was observed in the absence of substrates or with UDP-galactose and EDTA. From the ultracentrifuge data and steady state kinetics, dissociation constants for alpha-lactalbumin from the enzyme-Mn2+-UDP-galactose-alpha-lactalbumin complex were determined at several temperatures and salt concentrations. These showed good internal agreement. The free energy change delta G degrees for the association of the two proteins is calculated, and the results are discussed in relation to the nature of the interaction.

Animals↗
PubMed1976

A comparison of the interactions of galactosyltransferase with a glycoprotein substrate (Ovalbumin) and with alpha-lactalbumin.

Sedimentation velocity ultracentrifugation and other procedures have been used to investigate macromolecular interactions of bovine colostrum galactosyltransferase with a glycoprotein substrate (ovalbumin) and with the lactose synthase regulatory protein, alpha-lactalbumin. The determination of equilibrium binding constants for these interactions and the effects of ligands and combinations of ligands on the equilibria have clarified several aspects of the mechanism of galactosyltransferase and its regulation by alpha-lactalbumin. 1. The attachment of Mn2+ at the tight binding site on galactosyltransferase (site I, Kd 2.3 muM) is an essential prerequisite for interactions with ovalbumin and with alpha-lactalbumin. 2. The attachment of Mn2+ or Ca2+ at the weaker metal binding site (site II, Kd 1 to 2 mM) does not significantly affect the interaction of galactosyltransferase with either protein. This is consistent with the hypothesis derived from kinetic studies that site II is functionally connected with the binding of UDP-derivatives. 3. While the binding of ovalbumin to galactosyltransferase in the presence of Mn2+ alone can be observed by ultracentrifugation, this interaction is too weak to cause binding of galactosyltransferase to ovalbumin-Sepharose. Binding to ovalbumin-Sepharose could only be detected by affinity chromatography in the presence of both Mn2+ (10 mM) and UDP (0.3 MM). Sedimentation studies showed that the association of galactosyltransferase with ovalbumin is pressure-dependent, and that the presence of UDP in the complex increases the equilibrium association constant by a factor of 46. The enzyme Mn2+-UDP-ovalbumin complex has unusual hydrodynamic properties. 4. The presence of saturating concentrations of UDP-galactose potentiates the binding of alpha-lactalbumin at high concentrations of Mn2+, as shown by a 25-fold increase in the association constant. Competitive inhibition by alpha-lactalbumin, with respect to ovalbumin that is observed by steady state kinetics, is attributed to the mutally exclusive binding of the proteins with an enzyme complex containing Mn2+ and UDP-galactose. 5. Monsaccharides (N-acetylglucosamine and glucose) strongly enhance the binding of alpha-lactalbumin to enzyme complexes containing Mn2+, in the presence or absence of UDP-glucose. The binding of alpha-lactalbumin and monosaccharide to form enzyme complexes containing both is random, and evaluation of the four associated equilibrium constants shows that the binding is strongly synergistic. 6. Although the significance of some of the many equilibria studied cannot be ascertained, it appears than an element of randomness may be present in reactions catalyzed by galactosyltransferase...

Animals↗
PubMed1975

The low-temperature luminescence properties of bovine alpha-lactalbumin.

The luminescence of bovine alpha-lactalbumin at 77 K has been studied and compared with that of lysozyme. Alpha-Lactalbumin has several unusual properties, including a fluorescence spectrum showing vibrational fine structure, an abnormal phosphorescence spectrum, a high fluorescence: phosphorescence ratio and an abnormal phosphorescence decay. These properties are largely due to the proximity of tryptophan residues to disulphide bonds. Reduction of all these bonds causes considerable changes in alpha-lactalbumin luminescence, as does denaturation in acid solution. Reduction of a single labile disulphide bond has little effect, and the properties of alpha-lactalbumin III, a variant lacking one disulphide bond and one trypotophan residue, are similar to those of the normal protein. Several differences between alpha-lactalbumin and lysozyme are reported. The results support the suggestion that the two tryptophan residues found in the active site cleft of alpha-lactalbumin may be largely responsible for its luminescence.

Animals↗
PubMed1975

The interaction of N-acetylglucosamine and an affinity-label analogue with alpha-lactalbumin and lactose synthetase.

We have attempted to detect binding of N-acetylglucosamine (NAG) to alpha-lactalbumin, the B protein of lactose synthetase, under conditions in which binding of NAG to lysozyme, a protein to which alpha-lactalbumin has a significant sequence homology, is observed. Using 1H nuclear magnetic resonance spectroscopy, uv difference spectroscopy, competition of NAG with N-methylnicotinamide chloride, and fluorescence spectroscopy, no binding was detected. The synthesis of a NAG analogue, N-diazoacetyl-glucosamine (diazoNAG), was carried out, and the molecule was demonstrated to be an active galactose acceptor in the lactose synthetase reaction. Use of this molecule in photochemical labeling experiments resulted in a large amount of nonspecific labeling of alpha-lactalbumin, lactose synthetase A protein, ribonuclease, and lysozyme, but competition experiments in the presence of an excess of NAG revealed some specific labeling in the case of A protein and lysozyme, but not with alpha-lactalbumin or a ribonuclease control. Thus, it is highly questionable that a NAG binding site is retained in alpha-lactalbumin; furthermore, it appears that the galacyosyl acceptor makes significant contacts with the A protein rather than alpha-lactalbumin in the lactose synthetase complex.

Acetylglucosamine↗
PubMed1978

Purification and characterization of rat alpha-lactalbumins: apparent genetic variants.

Rat alpha-lactalbumin, from the milk of Fischer 344 (CDF) rats, was isolated and purified by a combination of gel filtration and diethylaminoethyl-cellulose ion exchange chromatography. Three electrophoretically distinct proteins had alpha-lactalbumin activity. Staining for carbohydrate indicated that at least two of the three forms were glycoproteins. The low molecular weight protein fraction from the wheys of two additional strains of laboratory rat were compared to ascertain whether the composition of this fraction was common in the divergent strains. Outbred Wistar and Long-Evans dams yielded wheys containing up to six forms of alpha-lactalbumin. Either one or both of two groups of three alpha-lactalbumins were in a given milk sample. The two groups of three alpha-lactalbumins appear to represent two genetic variants upon which is imposed a polymorphic character. All forms of alpha-lactalbumin, within and between strains, were immunologically identical.

Animals↗
PubMed1977

On the reactivities of the tryptophan residues of human alpha-lactalbumin to 2-hydroxy-5-nitrobenzyl bromide.

The reaction of human alpha-lactalbumin with the tryptophan reagent 2-hydroxy-5-nitrobenzyl bromide has been studied. This protein has 3 tryptophan residues (Trp-60, Trp-104 and Trp-118) all of which are accessible to the reagent at pH 2.7 or 7. Trp-60 of human alpha-lactalbumin is much more reactive than Trp-60 of bovine alpha-lactalbumin (Barman, T. E. (1972) Biochim. Biophys. Acta 257, 297-313). As with bovine alpha-lactalbumin, at pH 2.7, 2-hydroxy-5-nitrobenzyl bromide is specific for tryptophan but at pH 7 His-32 also reacts. When treated with the tryptophan reagent, both alpha-lactalbumins lose their specifier protein activities in the lactose synthase (UDPgalactose:D-glucose 4-beta-galactosyltransferase, EC 2.4.1.22) reaction.

2-Hydroxy-5-nitrobenzyl Bromide↗
PubMed1978

Antibodies to lactalbumin interfere with its radioimmunoassay in human plasma.

Two radioimmunoassays for human lactalbumin have been established using a rabbit antiserum. One assay uses a second antibody to separate bound from free label; the other uses polyethylene glycol to precipitate gamma globulin non-specifically. We have confirmed that about half the normal human population have a substance in their blood which inhibits the binding of lactalbumin to the rabbit antibody. Comparison of the two assays has demonstrated that this material is not lactalbumin but a naturally occurring antibody. We have shown that it is in the IgG fraction of human plasma and is probably a cross-reacting antibody to bovine lactalbumin. None out of fifteen males and fourteen out of fifty eight non-pregnant, non-lactating females had low levels of lactalbumin in the their blood (0.6--2.0 ng/ml). Our assay could not detect a statistically significant difference between normal women and those with either benign breast disease or metastatic mammary carcinoma.

Animals↗