Rae Greenberg

Identification of the milk fat globule membrane proteins. I. Isolation and partial characterization of glycoprotein B

The salt soluble proteins from the fat globule membrane of cows 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.

αs1-Casein A (Bos Taurus): A probable sequential deletion of eight amino acid residues and its effect on physical properties

Abstract 1. 1. The major casein of cows milk (αs1− occurs in four known polymorphic forms, A,B, C and D. 2. 2. αs1− Casein A is devoid of eight amino acid residues (apparently in sequence) which are present in the more common variants, B and C. 3. 3. αs1− A is soluble in calcium chloride solutions above 0·10 M at 1°C while the B and C variants are insoluble. 4. 4. The increased solubility of the A variant appears to have arisen as a result of either less extensive hydrophobic bonding and/or a conformational change of the protein brought about by the deletion of the nonpolar amino acids alanine, valine, two phenylalanines and three leucines and one polar amino acid, arginine.

Plasmin cleaves human β-casein

Abstract Plasmin cleaves isolated human β-casein to form specific fragments in a manner similar to the generation of γ 1 -, γ 2 -, and γ 3 -caseins from the bovine homologue. Identification of a protein previously isolated from human milk as a specific plasmin cleaved portion of β-casein indicates that endogenous plasmin is active in whole milk. These findings suggest that protease activity should be considered in casein quantitation or isolation of components from human milk.

End-group analysis of αs1-caseins A, B, and C

Abstract Three genetically determined variants of αs1-casein have been prepared, and the purity of each has been verified by starch-gel-urea electrophoresis. The amino-terminal end group was determined qualitatively by two independent chemical methods. Arginine was the only amino-terminal amino acid revealed by each method for all three variants. The results of the action of carboxypeptidase A can be interpreted most simply to infer a leucyl-tryptophan carboxyl-terminal sequence for all three proteins. The kinetics of amino acid release by carboxypeptidase was similar for the three αs1-caseins. A molecular weight of ~31,000 was determined from the carboxypeptidase data, assuming a single polypeptide chain. The results indicate that all amino acid differences among the variants must occur within the polypeptide chain, if the latter assumption is valid.

Analysis of proteolytic digests of genetic variants of αs1-casein

Abstract The genetic variants, A, B, and C, of αs1-casein from cows milk were digested under controlled conditions with trypsin, chymotrypsin, and pepsin. The resulting peptides were examined by a mapping procedure and the patterns were compared within each set of digests. The B and C variants are known to differ in composition by a single amino acid substitution—a glutamic acid residue in B being replaced by a glycine in C. Probable difference peptides containing this substitution were observed in each set of proteolytic digestions. The results of specific staining for arginine, histidine, methionine, tyrosine, and tryptophan residues suggest that the presumed difference peptides are related and indicate the presence of several of these amino acids in the vicinity of the amino acid substitution. The αs1-casein A variant, which differs from the other two proteins quite substantially in amino acid composition, revealed peptide patterns almost identical to those of B and C, with the main difference being the absence of one or two major spots in A. The hypothesis is proposed that αs1-casein A, which represents an unusual type of genetic variant, is devoid of a portion of the molecule, with the remaining sequence similar to the B protein.

The carboxyl terminal amino acids of β-lactoglobulins A and B

Abstract The release of amino acids from the carboxyl terminal end of β-lactoglobulins A and B has been studied with the use of carboxypeptidases A and B. The action of carboxypeptidase A catalyzes the hydrolysis of two moles of isoleucine and about one mole of histidine per mole of native protein at pH 8.5. The S-sulfo derivative releases two moles of isoleucine and two moles of histidine under the same conditions. When the native proteins were hydrolyzed with a combination of carboxypeptidases A and B, a wide spectrum of amino acids was found with leucine appearing as the amino acid following histidine. It appears that the two phenotypes, β-lactoglobulin A and β-lactoglobulin B, do not differ in the sequence of amino acids in the carboxyl-terminal portion of the molecules. In addition, they do not seem to differ in the manner they are hydrolyzed by the carboxypeptidase preparations.

Amino acid composition of β-caseins from the milks of Bos indicus and Bos taurus cows: A comparative study

Abstract 1. 1. β-Caseins B ( Bos taurus ), B Z and D ( Bos indicus ) were examined for amino acid composition. 2. 2. B Z differs from B in content of several neutral amino acids and possibly glutamic acid, as well as in tryptic “fingerprints”. 3. 3. β-D differs from B Z notably in content of arginine (+1), lysine (+1) and histidine (−1), but also in one additional residue each of threonine, serine and alanine. 4. 4. β-D (like β-C, Bos taurus ) lacks 1–2 residues of phosphorus as compared with B. 5. 5. It is likely that β-C ( Bos taurus ), itself, occurs as polymorphs.

Isolation and properties of goat β2-microglobulin

Abstract 1. 1. Goat β 2 - microglobulin was isolated and purified from colostrum. 2. 2. Comparisons of the amino acid composition and amino-terminal sequence the goat protein with the bovine and human homologues, indicates a high degree of similarity. Both goat and bovine β 2 - microglobulins differ slightly in composition from the human molecule, most notably in threonine and proline values. 3. 3. For the first 32 residues, bovine and goat differ only at two positions, one of which is a valyl/isoleucyl substitution consistent with the amino acid compositions. The equivalent goat/human sequence comparison shows seven differences. 4. 4. Immunological studies, using the ELISA method, also confirm the close relatedness of goat and bovine β 2 - microglobulin and their more distant relatedness to the human homologue.

Spectrophotometric estimation of protein concentration in the presence of tryptophan modified by 2-hydroxy-5-nitrobenzyl bromide.

A spectrophotometric method makes it possible to determine the concentration of a protein after covalent modification of tryptophan residues by 2-hydroxy-5-nitrobenzyl bromide. Molar absorption coefficients for the 2-hydroxy-5-nitrobenzyl chromophore, reported here in the pH range from 4.0 to 10.9, can be used to correct the protein absorbance values at 280 nm, which then provides the basis for calculating protein concentration in the usual way. The method was tested with alpha-lactalbumin, beta-lactoglobulin, pepsin, and soybean trypsin inhibitor; spectrophotometrically estimated concentrations of these proteins agreed closely with values obtained by amino acid analysis.

Effects of reductive alkylation on peptide retention times.

Reductive alkylation of primary amino groups is used to introduce nuclear magnetic resonance or radioactive probes into proteins. Because of electrostatic and conformational effects, reductive alkylation is not always complete. We describe herein a rapid high-performance liquid chromatographic method for separating and quantitating mixtures of native and reductively alkylated peptides. Small synthetic peptides were chosen to illustrate the effects of methylation and isopropylation of primary amino groups on chromatographic retention times. Mixtures of unmodified and reductively methylated or isopropylated peptides (Gly-Leu-Tyr, Gly-Gly-Lys-Arg, Arg-Lys-Asp-Val-Tyr and Pro-Gly-Lys-Ala-Arg) could be separated. Chromatography was on a 5-micron, 25 cm x 0.4 I.D., C18 reversed-phase column with 0.1% trifluoroacetic acid in a 10 to 80% linear gradient of acetonitrile in water, a system appropriate for protein digests. The relative concentrations of native, and singly and doubly alkylated peptide were determined as well as the effective retention coefficients for dimethyl and isopropyl groups. The method shows promise for the peptide mapping of partially alkylated proteins.