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In spite of the fact that beta-lactoglobulin (beta-lg) was first discovered in bovine milk more than fifty years ago, and that it represents the main whey protein component in all the milks in which it has been found, its biological role and genetic evolution still remain rather uncertain. From comparative studies of the primary and tertiary structures of beta-lg and of other proteins of a similar size, the existence of a new superfamily of proteins with the function of transporter of hydrophobic molecules has been conjectured. The elucidation of the structure of beta-lg either from different species or from different genetic variants of the same species should give useful information on the evolution and function of this protein family. With this aim in mind we have now undertaken the isolation and characterization of a recently discovered, new genetic variant of bovine beta-lg. A two-step purification procedure involving preparative HPLC gel filtration and preparative IEF-IPG has been successfully carried out; it affords a good recovery of the new beta-lg in highly purified form.

A corticosteroid-binding protein was detected in the whey of human colostrum and milk which resembles serum corticosteroid-binding globulin in certain respects: the equilibrium association constants for cortisol and progesterone binding and the apparent molecular size, as determined by Sephadex G-200 chromatography, were similar, and cortisol andd progesterone competed strongly for binding to the same site in each instance. Dexamethasone-binding activity could not be detected. The concentration of corticosteroid-binding protein in the colostrum obtained before parturition is about 0.1 muM; the concentration declines rapidly after parturition to about 0.01 muM. A corticosteroid-binding protein was found, also, in the whey of mature rat milk at levels of about 0.3 muM. This protein resembles rat serum corticosteroid-binding globulin: the equilibrium association constants for cortisol, corticosterone, and progesterone binding, and the apparent molecular size, as determined by Sephadex G-200 chromatography, were similar; the elution behavior of the respective proteins on anion exchange chromatography with DEAE-Sephadex A-50 was similar, also. Identity of the corticosteroid-binding proteins in whey with corticosteroid-binding globulin in serum is not presumed, however. Rat and human whey exhibited very little testosterone- or 17 beta-estradiol-binding activity. It is suggested the corticosteroid-binding proteins may play a significant physiological role in regulating the concentration of the bound and unbound forms of progesterone and cortisol in the fluids bathing the epithelial cells lining the mammary ducts and acini.

Fat globule membrane material, isolated by churning cream that had been washed four times, was extracted sequentially with .6 M potassium chloride and centrifuged to yield pellet and supernatant fractions. Compositional data indicated that lipid components were removed preferentially into the supernatant fractions. Electropherograms of the pellet and supernatant fraction in 10% and 5% sodium dodecyl sulfate-containing polyacrylamide gels revealed that the protein species originally present in the membrane associated into higher molecular weight species concomitant with the removal of lipid moieties. Thus, a contribution of the lipid moiety to membrane integrity is suggested. Approximately 17 protein-stained zones, ranging in molecular weight from 13,500 to 208,000 daltons, were observed in gels of the membrane material.

A new protein with a particular thermoprecipitability was isolated from bovine milk and tentatively termed milk pyroglobulin. The protein which was soluble at a relatively cold temperature was precipitated by raising the temperature to a certain degree depending on the concentration of the protein. The precipitate disappeared on recooling. This protein had the electrophoretic mobility of gamma globulin but did not carry either antigenic specificities of immunoglobulins or of free secretory component. The molecular weight was estimated to be approximately 60,000 in thin-layer gel filtration on Sephadex G-200 superfine gel, but the protein appeared to be convertible to molecules with a lower molecular weight of approximately 20,000 in the presence of bovine serum albumin. The presence of the albumin inhibited the thermoprecipitation as did alpha-lactalbumin but not IgG immunoglobulin from bovine colostrum. In SDS-polyacrylamide gel electrophoresis, the protein was separated into two components having a molecular weight of 19,000 and 10,000, respectively. Both components were thermoprecipitable and carried identical antigenic determinants.

The hypothesis that a zinc-binding ligand (ZBL) recently discovered in human milk but absent from cow's milk might be related to zinc nutrition in the neonate was investigated. The zinc-binding characteristics of rat milk were examined to determine if the rat was a suitable model. By gel filtration, rat milk was found to contain a ZBL with characteristics similar to those of the ZBL found in human milk. A similar ZBL was identified in the intestinal mucosa of rats 16 days of age and older but was absent in rats from birth to 16 days. These results support the hypothesis that the ZBL of maternal milk may enhance zinc transport in the neonatal period before the development of intestinal mechanisms for zinc absorption.

Bovine free secretory component was purified from whey by salt precipitation, gel filtration, DEAE-cellulose and phosphocellulose chromatography, and immunoadsorption. It was obtained in immunologically pure form and in 56% yield. The Stokes radius of pure free secretory component was found to be 4.3 nm by gel filtration, and an (see article) of 4.1 S was determined by the ultracentrifuge. The molecular weight was 79,000 by sodium dodecyl sulfate gel electrophoresis and by sedimentation dquilibrium in the ultracentrifuge, using a v of 0.73 determined by ultracentrifugation in D2O and H2O. A minimal axial ratio of approximately 5 was calculated. Amino acid analysis of bovine free secretory component showed remarkable similarity to that of human, dog, and rabbit but carbohydrate analysis showed significant differences. In contrast to the human, bovine free secretory compoennt has 2 methionine residues/mol. The NH2-terminal sequence was found to be Lys-Ser-Pro-Ile-PPHE-Gly-Pro-Glu-Glu-Val-Asp-Ser-Val. This sequence is identical with that the human and dog. However, the poor immunological cross-reactivity between the dog, human, and bovine proteins suggests that significant structural differences will be found in other regions of the molecule.

A major whey protein which appears in milk from the tammar wallaby (Macropus eugenii) only during the second half of lactation (late lactation protein-A, LLP-A) was purified to apparent homogeneity by ion-exchange chromatography and gel filtration. An Mr of 21,600 +/- 2000 was calculated from its amino acid composition. A computer-based comparison of the sequence of the first 69 amino acid residues with the Atlas of Protein Sequence data base showed no significant homology with known proteins. Antiserum to LLP-A was prepared in rabbits, and single radial immunodiffusion was used to measure the amounts of LLP-A in milk during the first 40 weeks of lactation. LLP-A was first detected at 26 weeks; thereafter its concentration increased abruptly, to reach a maximum of 26 g/l at approx. 36 weeks of lactation. Explants prepared from mammary gland biopsies at 20 and 35 weeks of lactation were exposed to [3H]amino acids for 8 h; immunoprecipitation of tissue extracts showed that, whereas the rate of casein synthesis was the same at both stages of lactation, LLP-A was synthesized only by the 35-week mammary gland.

Lipid globule membranes were isolated from human and bovine milk and from the milk of sheep, goat, pig, rat and guinea pig, and their polypeptide compositions were analyzed. The major polypeptides with molecular weights similar to that of bovine butyrophilin were separated by gel electrophoresis, isolated and characterized with respect to isoelectric point, molecular weight, immunological cross-reactivity and peptide composition after proteolytic cleavage. We show that in all species examined these proteins are similar to bovine butyrophilin in (i) their relative insolubility in buffers of low and high ionic strength and in non-denaturing detergents, (ii) the occurrence of several isoelectric variants, and (iii) patterns of peptides obtained by protease digestion. It is concluded that closely related proteins are major constituents of the cytoplasmic coat structures associated with milk lipid globule membranes of many species, and we propose the name butyrophilins for this group of proteins. Bovine and human butyrophilins are glycosylated with relatively large amounts of glucosamine, mannose, glucose and galactose but little fucose, sialic acids or galactosamine. Most if not all of the sugar residues are associated with an acetone-soluble peptide fragment of Mr 12000-16000 focusing at about pH 4.0. We suggest that this fragment contains a membrane-spanning peptide sequence and is involved in the attachment of the cytoplasmic coat to the membrane of the milk lipid globule.

A growth factor, mammary-derived growth factor 1 (MDGF1), has been purified to apparent homogeneity from human milk. The factor is a pepsin-sensitive, reducing agent-insensitive protein with a molecular mass of 62 kDa and a pI of 4.8. An apparently identical factor has been isolated from human mammary tumors, suggesting that MDGF1 might be made by and act as an autocrine growth factor for mammary cells. High affinity receptors for MDGF1 have been detected on mouse mammary cells, normal rat kidney cells, and A431 epidermoid cells (KD = 2 X 10(-10) M). MDGF1 at picomolar levels stimulates the growth of mammary cells and greatly amplifies their production of collagen, apparently via elevating collagen mRNA levels, an effect that is demonstrated for normal rat kidney cells. The responsiveness of mammary cells to MDGF1 is attenuated when the cells are grown on a basement membrane collagen substratum, a component of the extracellular matrix upon which these cells normally rest in vivo. MDGF1 thus may regulate the production of new basement membrane as mammary epithelium invades the stroma during proliferation.

Attempts at isolating individual human milk proteins showed that cross interactions made it difficult to obtain of homogeneous components. A new method was devised, based on complete precipitation of milk proteins with saturated ammonium sulphate and progressive solubilization of the precipitate on a column of Sephadex G10 with a linear gradient of ammonium sulphate (from saturation to water). Three fractions were obtained. The first contained lactoferrin, serum albumin, lysozyme and traces of alpha-lactalbumin. Lysozyme could be obtained free from contaminants by chromatography on Ultrogel AcA 54. Lactoferrin and serum albumin coeluting as a single peak, were separated by a further chromatography on DEAE-cellulose. From the other two fractions recovered on Sephadex G10, it should be possible to prepare immunoglobulins, alpha-lactalbumin and the bulk of caseins. The homogeneity of the preparations of lysozyme, lactoferrin and serum albumin was assessed by SDS polyacrylamide gel electrophoresis, acrylamide agarose electrophoresis and immunoelectrophoresis.

A 39 kilodalton glycoprotein has been isolated from bovine mammary secretions by heparin-agarose affinity chromatography and gel filtration. It is a minor whey protein in mammary secretions collected during the nonlactating period, but is clearly detectable by affinity chromatographic and immunoblotting techniques. It is not detectable by these techniques in milk or colostrum. This protein is not immunologically related to milk proteins, serum proteins or cytoskeletal proteins. The N-terminal amino acid sequence (36 amino acids) is not similar to other known proteins. Isolating this novel 39 kilodalton protein provides a specific marker for mammary function during the nonlactating period.

The salt soluble proteins from the fat globule membrane of cow's milk were resolved into three fractions by Sephadex column chromatography in sodium dodecyl sulfate. One of the fractions, termed glycoprotein B, was purified by rechromatography to essentially one band on sodium dodecyl sulfate gel electrophoresis. It was found to contain 14% carbohydrate including sialic acid, mannose, galactose, glucose, glucosamine and galactosamine. The amino acid composition of glycoprotein B was determined; it has amino terminal serine and carboxyl terminal leucine. The molecular weight of this glycoprotein as estimated by sodium dodecyl sulfate gel electrophoresis is 49 500.

Proteose-peptone component 8F (or '8-fast') has been prepared from bovine milk. Sedimentation equilibrium analysis, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate and gel filtration in urea-containing buffers all gave molecular weight values between 3300 and 3900. The N-terminal sequence was found to be Arg-Glu- by dansylation and Edman degradation. Hydrazinolysis released lysine from the C-terminus. A mixture of carboxypeptidases A and B showed that the C-terminal sequence was -Thr-(Arg,Ile,Asn)-Lys. The phosphate content was 3.8 mol/mol and was completely released by a short alkaline hydrolysis indicating linkage to serine. This and all other aspects of the composition were entirely consistent with the identification of this proteosepeptone as residues 1--28 of the beta-casein molecule. This identity was confirmed by a peptide mapping procedure. Thus proteose-peptone component 8F represents the N-terminal fragment when the gamma1-caseins are formed by proteolysis of beta-casein.

The possibility of isolating lactic whey proteins (LWP) in the form of insoluble complexes (IC) with apple pectin was studied. The effect of pH, ionic strength (mu, NaCl), temperature (T degree C), pectin weight fraction (X3%) and the total concentration of macromolecular components in the system (Ws) on isolation has been considered. The process has been characterized by LWP yield in the composition IC--P, percentage and the extent of protein concentration in the concentrated phase (IC)--F. The mixing of lactic whey with a pectin solution usually yielded an IC (10% less than or equal to X3 less than or equal to 90%). The dependence of P on X3 is of an extreme nature with a maximum of 85% at X3 = 60%. The following conditions correspond to the maximum LWP yield (90%) in the complex composition: pH 3.4, mu = 0.01, T = 5-20 degrees C, X3 = 60%, Ws = 0.5%. At compositions of the system corresponding to the maximum P value (X3 = 60%) practically all the LWP fractions are present in the concentrated phase. If X3 much greater than 60% or X3 much less than 60% alpha-lactalbumin is practically absent in the concentrated phase. Usually, minimum F values (2.5-4.0) correspond to maximum protein yield at X3 = 60%. At X3 greater than 70% and X3 less than 50% F values may be considerably higher (20 times and more). A decrease in the pectin methylation degree from 56.7% down to 15.4% does not affect F. The maximum protein yield (94%) occurs when low methylated pectin is used. The character of the dependence of F on X3 is explained according to similar processes of complex gel formation and the processes of gel formation in polymer solutions.

The milk-fat-globule membrane (MFGM) was isolated from guinea-pig milk and the membrane-associated proteins and glycoproteins characterized by electrophoretic techniques. Major components of the membrane included PAS-I, a sialoglycoprotein of Mr greater than or equal to 200000, the redox enzyme xanthine oxidase and the glycoprotein, butyrophilin. Membrane preparations also contained two other glycoproteins, GP-80 and GP-55, of Mr 80000 and 55000, respectively. Comparison of guinea-pig xanthine oxidase and butyrophilin with proteins from bovine MFGM by peptide mapping procedures, showed that the two proteins in both species were similar, but not identical. GP-55 may also be related to glycoproteins of Mr 45000 and 48000 in the bovine membrane. The integral and peripheral components of guinea-pig MFGM were identified by treating membrane preparations with sodium carbonate solutions at high pH and by partitioning the membrane proteins in solutions of Triton X-114. By these criteria xanthine oxidase and GP-55 appeared to be peripheral components and GP-80 an integral protein of the membrane. PAS-I and butyrophilin displayed hydrophilic properties in Triton X-114 solutions, but could not be removed from membrane preparations with sodium carbonate. Possible reasons for these ambiguous data are discussed. The observed similarity between several of the proteins of guinea-pig and bovine MFGM implies that these proteins may have specific functions related to milk secretion in mammary tissue, e.g. in the budding of milk-fat globules or the exocytosis of milk protein and lactose at the apical surface.

A method is described which allows the simultaneous separation of all polymorphic protein fractions in cow's milk in one single run. The separation could be achieved by isoelectric focusing in ultrathin-layer polyacrylamide gels. The method is especially suitable for screening purposes because it combines low costs, high resolution and short separation time.

1. Milk proteins of the Galapagos fur seal (Arctocephalus galapagoensis) were separated adequately into whey and casein fractions using bovine milk analysis methods. 2. In samples from days 5-30 of lactation 40% of the total proteins were whey and 60% caseins; in mid-lactation, day 150, 25% were whey and 75% casein proteins. 3. Electrophoretic and isoelectric focusing patterns of fur seal whey protein differed widely from bovine patterns, whereas those of caseins were similar. 4. Polymorphisms of fur seal whey and casein proteins were noted and did not seem related to different stages of lactation. 5. C-16 and C-18 fatty acids contributed about 70% of fatty acids; 63% of the total acids in milk fat were unsaturated.

A camel milk whey protein has been isolated by reverse-phase high performance liquid chromatography. The protein is, like caseins, rich in proline (25% of the whole protein). The N-terminal amino acid sequence shows that the protein is homologous with a C-terminal region of beta-caseins analyzed from other species. The protein is concluded to be a fragment of beta-casein, derived from a non-tryptic type of cleavage of the parent molecule, and increasing the multiplicity of known casein products.

Fractionation of bovine whey concentrate was performed by gel filtration on Sephadex G-75 both on a laboratory scale and on a large scale. By a recycling procedure and improved separation was obtained and the whey proteins were resolved into four fractions in the weight ratio 3:12:1:4. The fractions were analysed by polyacrylamide gel (PAG) electrophoresis and the apparent molecular weights were determined by thin layer gel chromatography (TLG) and by sodium dodecylsulfate (SDS) gel electrophoresis.

A glycoprotein that reacted to the antisoluble glycoprotein of bovine milk fat globule membrane was purified from the proteose-peptone of whey and designated lactophorin. Lactophorin was separated into seven components. Lactophorin and the seven components were rich in aspartic acid, threonine, serine, glutamic acid, leucine, and lysine. The content of threonine, glycine, isoleucine, lysine, and arginine varied in each component. The ratio of fucose, mannose, galactose, N-acetylgalactosamine, N-acetylglucosamine, and sialic acid of lactophorin, which contains about 18% saccharide, were 1, 6.6, 10.3, 5.5, 9.7, and 11.6, respectively, while the respective ratio of the seven components were 1, 5 to 6, 7 to 9, 3 to 4, 6 to 8, and 4 to 12. Sialic acid content varied in each component. Protein-carbohydrate linkage was N- and o-glucoside linkage. Lactophorin consisted of seven polypeptides (I to VII) with apparent molecular weights 17,000 to 67,000. Bands I, II, VI, and VII were glycoprotein. Bands VI and VII were major and had antigenicity to anti-soluble glycoprotein, while bands I to V were minor polypeptides. Component 1 consisted of only one polypeptide (VII), whereas the components 2 to 7 contained two major (VI, VII, or both) and several minor polypeptides. The sedimentation pattern of each component was a single and almost symmetrical peak. Sedimentation coefficient was 3.79 to 5.64 S and also varied in lactophorin. The results indicate that lactophorin has multiple forms.

During the last twenty years heat coagulation has become a more important technical procedure for the isolation of milk proteins because of its very efficient utilization of raw material. Experiments are reported to get approximation equations for the mass transfer in the production of milk protein coprecipitates from skim milk and mixtures of skim milk with rennet and acid whey by means of statistical planning and interpreting of experiments. Good exactness was reached. The coagula consist of casein fractions, whey protein fractions, calcium, and phosphates. The products are thermically rather stable, sensorically indifferent and contain water insoluble protein. They are suitable to improve the fat: protein ration of sausages, but also for the protein enrichment of other foods and for dietetic purposes.

Bovine whey proteins and caseins were separated by hydrophobic interaction chromatography with the new Pharmacia fast protein liquid chromatography column, phenyl-Superose. Total casein was separated using a decreasing gradient of 0.8 to 0.05 M sodium phosphate and a constant 3.75 M urea concentration at pH 6.0. The order of elution of caseins was beta less than gamma, alpha s2 less than kappa less than alpha s1, and beta-casein was always eluted first. Whey proteins were separated with a decreasing salt gradient of 1.5 to 0 M ammonium sulphate in 0.05 M sodium phosphate at pH 7.0. The order of elution was beta-lactoglobulin less than bovine serum albumin less than immunoglobulin less than alpha-lactalbumin. The elution order of proteins from the column did not correlate with the calculated average hydrophobicities but the method was considered to be a measure of the "effective" hydrophobicity of proteins and therefore of more use for attempting to relate hydrophobicity to functional properties of proteins. The method shows significant advantages over conventional techniques allowing rapid optimization of elution conditions and reducing run times from 24 h or more to less than 2 h.

Chitosan, a cationic carbohydrate polymer manufactured from chitin in shrimp and crab wastes, coagulated suspended solids in cheese whey as effectively or more so than ten commercial synthetic polymers. Concentrations of suspended solids after settling were reduced over 90% by coagulation at pH 6.0 with a ratio of chitosan to suspended solids of 2.15% (1:46.5). The yield of coagulated solids was approximately 2,270 mg/liter. The proximate composition of the coagulated solids after freeze drying was 73% protein, 6% lactose, 10% ash, and 7% moisture. Without the aid of a coagulating agent, setting for 1 or 3 h reduced suspended solids by only 34% and 49%, respectively. Rat feeding studies showed nn significant differences in the protein efficiency ratio of casein, coagulated whey solids containing chitosan, and whey solids containing no polymer. These results provide evidence of utility for coagulated whey solids as a protein supplement.

A fast protein liquid chromatography system was used to fractionate the major proteins of sweet and acid wheys. Fifty to 500 microliter of whey were fractionated with a stepwise ionic strength gradient using water (buffer A) and increasing concentrations of .7 M sodium acetate (buffer B). Six well-resolved peaks were obtained: 1) amino acids (tentative identification), 2) low molecular weight peptides (tentative identification), 3) highly enriched alpha-lactalbumin, 4) highly enriched serum albumin 5) electrophoretically pure beta-lactoglobulin B, and electrophoretically pure beta-lactoglobulin A. A poor baseline or unresolved peaks resulted when .02 M bis-tris or .02 M histidine was used for buffer A and .7 M sodium acetate in .02 M bis-tris or histidine was used for buffer B. When sodium chloride was used in place of sodium acetate, beta-lactoglobulins A and B were poorly resolved. The column was cleaned after each run by injecting 2 ml of the following reagents: glacial acetic acid, 2 N sodium chloride, 2 N sodium hydroxide, 2 N sodium chloride, 2% detergent, and 100% acetonitrile. Time required to run each sample including column cleanup was 40 min.

Crude, delipidated milk and the acid:ethanol extracts of primary human breast tumors contain several activities that biologically resemble transforming growth factors (TGFs) in that they promote the anchorage-independent growth of normal rat kidney and Mm5mt/c1 mouse mammary tumor cells in soft agar. Three major TGF species with isoelectric points (pl) of about 4.0, 6.0-6.5, and 7.0 have been detected in both tumors and milk. The pl 4.0 species from milk has been purified about 10,000-fold by isoelectric focusing and high-performance liquid chromatography. This species, designated milk-derived growth factor II (MDGFII), coelutes from gel filtration columns with an authentic human epidermal growth factor standard when using a low ionic strength eluting buffer. However, on the same column, MDGFII is completely resolved from human epidermal growth factor with high ionic strength eluting buffers. Nevertheless, MDGFII purified by the latter technique still competes with 125I-epidermal growth factor for receptor binding to A431 cell membranes. Additionally the TGF activity of MDGFII present in the pl 4.0 fraction of milk is markedly inhibited by anti-epidermal growth factor receptor antibody preparations. Consequently MDGFII appears to be an alpha-TGF. MDGFII is a pepsin-sensitive, disulfide reducing agent-sensitive, heat-stable protein that may be physiologically important for the mammary gland or the neonate.

Identification of human milk proteins and formulation of a two-dimensional map is a first step in a project which intends to survey human milk proteins by two-dimensional electrophoresis. Thirty-four proteins have been identified using the Iso-Dalt method of separation and Western blot with immunoprobes. Identification confirms that milk is species-specific, and, therefore, breast feeding confers a decided advantage for the infant. Using antisera for identification has revealed relationships between molecules which have not been previously noted. The antibody recognizes a common epitope between the IgA alpha chain and lactoferrin, and between the IgD d chain and beta casein. Milk protein concentrations vary longitudinally, diurnally, and individually. Identification of the proteins contributes meaning to the varying patterns. Knowledge of human milk proteins will help to elucidate human nutrition and health needs.

A human milk protein was isolated to apparent homogeneity and shown to be immunologically related to gp55, the major envelope glycoprotein of murine mammary tumor virus. Through the development of ultrasensitive protocols, which have wide applicability, immunological relatedness was corroborated by the demonstration of homologous protein sequences between the human and viral proteins.

Binding of an added radionuclide to milk protein and casein components of cow's milk fractionated by the combination of Sephadex G-150 gel filtration and diethylaminoethyl-cellulose chromatography was determined. Iron-59 labeled ferric chloride was added directly to raw whole milk at a concentration of .02 and 10 ppm isotope and carrier, respectively, and held overnight at 4 C. Five milliliters of the skin milk were chromatographed on a Sephadex G-150 column and fractionated into casein, whey protein, and nonprotein materials. The casein fraction was chromatographed on a diethylaminoethyl-cellulose column and separated into its components, alphas-, beta-, and kappa-caseins. Casein bound about 85% of the added iron in skim milk; of this amount, 72, 21, and 4% were associated with the alphas-, beta, and kappa-caseins.

Gradient-polyacrylamide-gel electrophoresis of human milk serum separated three high-Mr glycoprotein bands. The properties of the components corresponding to the three bands (tentatively termed 'Components C, B and A' in their order of migration) were compared by staining with four monoclonal antibodies and lectins. Components B and C both reacted with the four antibodies, but Component A did not. Components B and C were stained with peanut (Arachis hypogaea) agglutinin (PNA) and wheat (Triticum)-germ agglutinin (WGA), Component A being stained with soya-bean (Glycine max) agglutinin as well as PNA and WGA. These results suggest that Components B and C were related molecules, whereas Component A was markedly different from them. The reactivities of Components B and C were the same as those of PAS-0, a high-Mr periodate/Schiff (PAS)-positive glycoprotein previously isolated from human milk fat-globule membrane (MFGM). Component C, whose electrophoretic mobility was the same as PAS-0, could have been a soluble form of PAS-0. A high-Mr glycoprotein having the same properties as Component A was also observed in MFGM. The amino acid composition of the isolated Component A was significantly different from that of Component C and PAS-0, high threonine and serine contents being characteristic of Component A. The carbohydrate content of Component A was 65-80%, and was much higher than that of Component C and PAS-0. Fucose, galactose, N-acetylglucosamine, N-acetylgalactosamine and sialic acid were each detected as constituent sugars of Component A. Component A represents, therefore, a new high-Mr glycoprotein species in human milk serum and MFGM. Since these glycoproteins were high-Mr mucin-like glycoproteins, the names 'HM glycoprotein-A' and 'HM glycoprotein-C' were proposed for Component A and Component C (PAS-O) respectively.

A large acidic glycoprotein, PAS-I, was purified from the fat-globule membrane of guinea-pig milk. Threonine and serine accounted for over 30 mol% of the amino acids, and galactose, N-acetylgalactosamine, N-acetylglucosamine, mannose and sialic acid were the principal sugars detected. On a molar basis, sialic acid accounted for over 60% of the total sugar. Removal of sialic acid by treatment with neuraminidase revealed the presence of binding sites for peanut (Arachis hypogaea) agglutinin, a lectin specific for the sugar sequence beta-D-Gal-(beta 1----3)-D-GalNac (the T antigen). The distribution of PAS-I-related epitopes, defined by five monoclonal antibodies, was determined in the mammary gland and in other guinea-pig tissues. PAS-I was maximally expressed on the apical surfaces of secretory cells in lactating mammary tissue and was either absent, or present in much lower amounts, in the glands of virgin or pregnant animals. PAS-I epitopes were not detected in liver, heart, spleen, pancreas, ovary, uterus, lung or intestine, either by immunofluorescence microscopy or by immunoblotting techniques. Several of the PAS-I-specific antibodies bound to mucins of high Mr in human fat-globule membrane, and similarities and differences between PAS-I and the human mucins are discussed. PAS-I and epitopes of this glycoprotein will be useful as indicators of differentiation in mammary cells and of markers of the apical surface of these cells during lactation.

Children with or without cow's milk protein intolerance were investigated for serum antibodies to native cow's milk proteins, processed cow's milk proteins and native- and pepsin-digested beta-lactoglobulin. Antibodies of IgG and IgA isotypes were determined with ELISA and those of IgE isotype with RAST. Children who exhibited slowly appearing untoward reactions to cow's milk feeding had significantly higher titres of IgG antibodies against both native and digested beta-lactoglobulin than children with an immediate type of reaction or no intolerance. The IgG antibodies to pepsin-digested beta-lactoglobulin were efficiently inhibited by native beta-lactoglobulin even at low concentrations, which suggests that there were no antibodies specific for the degraded proteins. The children with slow reactions to cow's milk also tended to have higher antibody levels of the IgG and IgA isotypes to both native and processed cow's milk. Antibodies to this mix of antigens discriminated less well, however, than the antibodies to isolated beta-lactoglobulin between the groups of children with slowly appearing reactions, with immediate reactions and the controls. Seven out of nine children with an immediate type of reaction to cow's milk protein had IgE antibodies against cow's milk protein determined with the RAST, while this type of antibodies could not be demonstrated in any child in the two other groups. Using processed or digested protein as antigen did not increase the sensitivity of the antibody determinations.

Milk proteins in acid whey were separated into five fractions according to molecular size by gel filtration chromatography. The second peak, P2, contained proteins between approximately 250,000 and 100,000 daltons. Proteins in P2 were concentrated. After separation into albumins and globulins, each protein group was isolated by DEAE chromatography and hydrophobic interaction chromatography, Isolated albumin fractions were a yellow-colored protein of 89,000 daltons, an unidentified protein of 73,000 daltons, a beta-lactoglobulin of 18,300 daltons, and a red-colored protein of 87,000 daltons. Two types of globulin fractions were isolated: 1) a globulin fraction that coagulated in saturated sodium sulfate but did not coagulate when dialyzed against deionized water included a brown-colored protein of 150,000 daltons, and 2) a bovine serum albumin of 67,000 daltons with unidentified 170,000 and 30,000 daltons bands. A true globulin fraction contained a 77,000 dalton unidentified protein with several faint bands. The red-colored protein was identified as lactoferrin and the brown-colored protein as xanthine oxidase (EC 1.2.3.2.). A yellow-colored protein was concluded to be the denatured protein of contaminated lactoperoxidase (EC 1.11.1.7).

Using a fermentative process of whey through Kluyveromyces fragilys, Jörgensen, the Author prepared two edible products: Biomass I (yeast) and Biomass II (yeast plus protein of whey). Biomass I offered 53% of protein, and the yield was 22,3 g/1 whey. Biomass II, 62% of protein and yield of 27,7 g/1 whey. The test of food efficiency for Biomass II was similar to that presented by casein; the protein eficiency ratio at the level of 5% was the same, both for Biomass I and II. More research is needed specially to determine the economical convenience of the process.

Caseins were separated from whey proteins by acid precipitation of skimmed rabbit milk. Whole casein was resolved by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis into three major bands with apparent relative molecular masses (Mr of 31 000, 29 000 and 25 000. On agarose/urea-gel electrophoresis whole casein gave three bands with electrophoretic mobilities alpha, beta and gamma. The three components were purified by DEAE-cellulose chromatography under denaturing and reducing conditions. Each was shown to have a different amino acid, hexose and phosphorus content, as well as non-identical peptide fragments after proteinase digestion. The 31 000 Da (dalton) protein, of alpha-electrophoretic mobility, had a high phosphorus content (4.38%, w/w); the 29 000 Da peptide, of gamma-mobility, had the highest hexose content (2.2%, w/w), contained 0.8 cysteine residue per 100 amino acid residues and was susceptible to chymosin digestion corresponding thus to kappa-casein; the 25 000 Da protein migrated to the beta-position. The rabbit casein complex is composed of at least three caseins, two of which (alpha- and kappa-caseins) are analogous to the caseins from ruminants. Although caseins are poor immunogens, specific antibodies were raised against total and purified polypeptides. The antiserum directed against whole casein recognized each polypeptide, each casein corresponding to a distinct precipitation line. The antisera directed against each casein polypeptide reacted exclusively with the corresponding casein and no antiserum cross-reaction occurred between the three polypeptides. From whey, several proteins were isolated, characterized and used as antigens to raise specific antibodies. An iron-binding protein with an apparent Mr of 80 000 was shown to be immunologically and structurally identical with serum transferrin.

The antigenic relationship between the milk fat globule membrane (MFGM) and the hydrophobic fraction of proteose-peptone (HFPP) was demonstrated, using a mono-specific anti-HFPP antibody.

A new peptide of 20,000 daltons was found in human milk as a constituent of the casein micelle. Enzymic digestion with plasmin or trypsin revealed that the peptide was identical with a degradation product of human beta-casein. The amino acid composition of the degradation product and the previously reported sequence in the N-terminal region of human beta-casein suggested that the peptide was a fragment of beta-casein lacking the C-terminal region. The thermal sensitivity of this peptide was higher than that of beta-casein, but the peptide lost the property of calcium-dependent precipitation, which intact beta-casein possesses.

The immunoregulatory properties of several proteins, isolated from human milk, were investigated. Secretory component and galactothermin exhibited immunoregulatory activities in the in vivo and in vitro assays, generating helper cells, changing the ARFC levels and the resistance of thymocytes to hydrocortisone (HC). In addition, the immunoregulatory action of the P protein and its four fractions was studied. The bulk of the activity was found in the fraction II and III. The results suggest that the postneonatal development of the mammalian immune system is under the surveillance of various immunoregulatory proteins contained in maternal secretory fluids.

A group of four electrophoretically distinct but related proteins has been isolated from rat milk whey. These have been classified as P.1 to P.4 in order of their decreasing electrophoretic mobility in an alkaline buffer system. All four members of this group of proteins are immunologically identical apparently constitute a single protein which is differentially phosphorylated in the ratio of 3:2:1:0. Sodium dodecyl sulfate gel electrophoresis yielded constant estimates of molecular weight of approximately 20,700. Enzymatic dephosphorylation of purified P.1 generated the other three members of the group, demonstrating the commonality of the peptide component. This group of proteins in total makes a significant contribution of the total whey proteins being approximately 5 mg/ml in whole rat milk in the approximate proportion of 1:1.5:2:1.5. The P.1, P.2, and P.3 were isolated in sufficient quantities to permit further characterization. The partial amino acid analyses of each of the three forms were similar. They had commonextinction coefficients, E1(1)% cm A280, of 4.9 and A280/A290 ratio of about 1.36.

A milk fraction containing whey proteins of 10-30 kDa was injected into one mammary gland of lactating goats via the teat canal. This fraction produced a temporary dose-dependent reduction in milk yield in the treated gland; the milk yield of the other gland, which received an equal volume of carrier solution, was not affected. Injection of a second fraction, containing whey proteins of greater than 30 kDa, affected milk secretion only at high doses, and this effect was not wholly specific to the treated gland. The 10-30 kDa fraction and the greater than 30 kDa fraction produced similar transient changes in the concentrations of several ions and lactose in milk of the treated gland, but not in that of the untreated gland. These data indicate that a milk constituent present in the 10-30 kDa whey inhibits milk secretion in a temporary and reversible manner. The results are discussed in relation to regulation of milk secretion through local feedback inhibition.

Three new proteins have now been isolated from goat milk obtained after colchicine is infused into the mammary gland. Two of the proteins are proline-rich, and a third is a very acidic phosphoprotein. One of the proline-rich proteins is related compositionally to a sheep colostrum proline-rich protein, which has been shown to have a regulatory effect on the immune response (Janusz, M., Stavoscik, K., Zimecki, M., Wieczorek, Z., and Lisowski, J. (1981) Biochem. J. 199, 9-15). Other aspects of colchicine-treated milks are described.