Marvin P. Thompson

Calcium-induced associations of the caseins: A thermodynamic linkage approach to precipitation and resolubilization

Calcium-induced changes in protein solubility play a role in a variety of important biological processes including the deposition of bone and dentin and the secretion of milk. The phenomena of salt-induced (calcium) precipitation of proteins (salting-out), and the resolubilization of these proteins at higher salt concentrations (salting-in) have been studied and quantitated using an approach based on the concepts of Wymans thermodynamic linkage. Salting-out has been described by a salt-binding constant, k1, the number of moles of salt bound per mole of protein, n, and S1, the fraction soluble at saturation of n; salting-in has been described by corresponding constants k2, m, and S2. Analysis of salt-induced solubility profiles was performed using nonlinear regression analysis. Results of calcium-induced solubility profiles of two genetic variants of alpha s1-casein (alpha s1-A), (alpha s1-B), and beta-casein C (beta-C) at 37 degrees C, where hydrophobic interactions are maximized, showed no salting-in behavior and for salting-out, yielded k1 values of 157, 186, and 156 liters.mol-1 and n values of 8, 8, and 4, respectively. The values of k1 can be correlated with the apparent association constant for calcium binding to casein, while the values of n can be correlated with the number of calcium binding sites of the respective caseins. At 1 degree C, where hydrophobic interactions are minimized, nominally only hydrophilic and electrostatic interactions can be linked to the salt-induced solubility profiles; here beta-C is totally soluble at all calcium concentrations and alpha s1-B and alpha s1-A were now found to have salting-in parameters, k2 and m, of 2.5 liters.mol-1 and 4, and 11 liters.mol-1 and 8, respectively. alpha s1-A is more readily salted-in and studies on the variation of S1 with added KCl for this protein at 1 degree C indicated that salting-in is also mainly electrostatic in nature and may result from competition between K+ and Ca2+ for binding sites rather than from solute-solvent interactions as previously proposed. Comparison of k1 and k2 values between the two genetic variants, coupled with the known sequence differences (the A variant is a linear deletion of 13 amino acids) suggest the existence of a hydrophobically stabilized ion pair in alpha s1-B which is deleted in alpha s1-A; it is speculated that such bonds may play a role in other calcium-induced changes in protein solubility.

Identification of human milk α-lactalbumin as a cell growth inhibitor

SummaryA growth inhibitory protein, mammary inhibitory activity (MIA), was purified to apparent homogeneity from human milk. At concentrations of 5 to 10 ng/ml, the factor inhibited the growth of mammary epithelial cells by 30–80% and also inhibited the growth of normal rat kidney cells. Whereas the cell division of normal human mammary epithelium in primary culture was inhibited by MIA, cell division by fibroblasts from the same tissues was unresponsive. Inhibition was dose and time dependent and readily reversed when MIA was removed. MIA also inhibited growth in culture for three cell lines. The growth inhibitory protein migrated as a 14 kDa protein under reducing conditions on polyacrylamide gels in the presence of sodium dodecyl sulfate. The apparent isoelectric point was pI 5.0. The amino acid composition of MIA resembled that of α-lactalbumin, and sequence analysis of the N-terminal region comprising residues 1–24 and an isolated peptide were identical with the N-terminal and residues 66–81 of human α-lactalbumin. In addition, MIA was active in the lactose synthase system. The results strongly suggest that MIA and α-lactalbumin are identical proteins. Consistent with these results, α-lactalbumin preparations from several mammalian species, including human, goat, cow and camel, were all found to be growth inhibitory for cultured mammary epithelial cells. The inhibitory activity associated with human α-lactalbumin was destroyed by digestion with pepsin or chymotrypsin, by carboxymethylation of cysteine, or by cleavage of methionine 90 following cyanogen bromide treatment. The results raise the possibility that during lactation α-lactalbumin, a product of mammary cell differentiation, could be a physiologically relevant feed-back inhibitor of mammary cell growth and perhaps of other cell types as well.

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.

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 calcium-dependent electrophoretic shift of α-lactalbumin, the modifier protein of galactosyl transferase

alpha-Lactalbumin, the modifier protein of galactosyl transferase in the synthesis of lactose by the mammary gland, has been shown to undergo a Ca2+-dependent electrophoretic shift. Such shifts, characteristic of most calcium modulated proteins, are related to gross conformational changes upon binding calcium when detected in the presence of detergent (SDS-PAGE). However, we detected the calcium shift for alpha-lactalbumin using non-denaturing PAGE (ND-PAGE) where electrical charge changes are observed upon binding calcium. In order for a shift to be observed between the apo and calcium bound protein, calcium ion binding to proteins must have minimal dissociation constants (Kdiss) of 10(-7) M; alpha-lactalbumin is reported to bind calcium at Kdiss = 10(-10) to 10(-12) M. The electrophoretic shift identifies alpha-lactalbumin in complex milk whey patterns of many species of mammals.

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.

Influence of preparative method on the lipid profiles of bovine fat globule membrane

Abstract The lipid profiles of bovine fat globule membrane preparations were obtained using a procedure that separates neutral and polar lipids on a silica cartridge, analyzes components of these two lipid groups by high performance liquid chromatography, and identifies fatty acids of total lipid as well as neutral and polar lipids separately by gas chromatography. Fat globule membrane preparation involved either churning the cream, followed by ammonium sulfate precipitation of membrane, or a combined isolation-extraction with dimethyl sulfoxide. In preparations that did not include a delipidation step, the carryover of neutral lipids was significant. In contrast, preparations that were delipidated after ammonium sulfate precipitation or as part of the isolation-extraction had much lower levels of neutral lipids. For the latter samples, values for neutral and polar lipids were comparable to those reported previously. The relative ease with which fat globule membrane is obtained using the isolation-extraction procedure makes it the preferred method for subsequent isolation and purification of fat globule membrane proteins.

Regulation of Terpenoid Biosynthesis in Tapped Latex

The tapped latex of Euphorbia lathyris can convert acetate to several structurally different triterpenols (TOH) and their fatty acid esters (TE) (1). Simple low speed centrifugation of the latex affords a pellet that utilizes mevalonic acid as a triterpenoid precursor (2). Biosynthetic activity in the latex pellet is absolutely dependent upon the presence of an osmoticum (0.4 M sorbitol), indicating that biosynthesis occurs in an osmotically sensitive organelle. Calcium constitutes about 7.5% of dry latex weight, and the addition of Ca2+ to an EGTA-treated latex pellet stimulates triterpene biosynthesis. This study was undertaken to investigate whether the calcium binding protein, calmodulin, mediates Ca2+ effects upon triterpenoid biosynthesis.