Antony G. Mackinlay

Effect of casein micelle composition and casein dephosphorylation on coagulation and syneresis

The effect of varying the casein composition of artificial micelle milk on rennet coagulation time and syneresis was examined in order to determine whether either of these processes is dependent on the concentration of particular casein components. It was found that the levels of κ-and β-caseins had a significant effect on coagulation, whereas syneresis was only affected by the level of β-casein. Partial dephosphorylation of preformed micelles or the incorporation of dephosphorylated or partly dephosphorylated β-casein into artificial micelle milk was found to have an adverse effect on both coagulation and syneresis. It was concluded that the phosphate groups of casein, particularly those of β-casein, are directly involved in the micelle-micelle interactions which occur during coagulation and syneresis.

NMR studies of the phosphoserine regions of bovine αs1- and β-casein: Assignment of 31P resonances to specific phosphoserines and cation binding studied by measurement of enhancement of 1H relaxation rate

(1) High-resolution 31P-NMR was used to study the environment of the phosphoserine residues of the phosphoproteins, alpha s1-casein B, beta-casein A2 and beta-casein C. For reference purposes 31P-NMR spectra of phosvitin and ovalbumin were also collected. (2) 31P resonances were assigned to specific phosphoserine residues as a result of comparisons of the high-resolution 31P-NMR spectra for alpha s1- and beta-caseins and for peptide fragments of these proteins obtained by cyanogen bromide and trypsin cleavage. (3) Measurements of the enhancement of the relaxation rate for water protons (1H) on addition of Mn2+ to alpha s1-casein B and to a fragment alpha s1-CN3, obtained by cyanogen bromide cleavage, gave approximate pK values for the binding groups and suggest the possibility of a conformational change induced by varying the concentration of divalent cation.

Effect of heat induced interaction between β-lactoglobulin and κ-casein on syneresis

The effect of preheating skim milk and artificial micelle milk on curd syneresis was studied. The inhibition of syneresis caused by heat was dependent on the presence of β-lactoglobulin (β-lg) and to a lesser extent α-lactalbumin. The degree of inhibition increased with increasing amounts of added β-lg and preheating temperature. This agrees with the hypothesis that the detrimental effect of preheating on syneresis is due to complex formation between β-lg and κ-casein. This complex appeared to be mediated via thiol–disulphide exchange and its formation appeared to interfere with the micelle–micelle interactions responsible for syneresis.

Complete nucleotide sequence of the bovine β‐lactoglobulin gene

Abstract In this paper we report the complete bovine β‐lactoglobulin gene sequence, consisting of seven exons within a total of 4724 base pairs. The inferred amino acid sequence agrees completely with that determined directly for the ? variant. The bovine β‐lactoglobulin gene sequence shows 89% homology to the corresponding ovine sequence, with complete conservation of gene organization and splice sites, though a significant proportion of CG dinucleotides have been lost since the species diverged. Comparison with the previously reported cDNA sequence reveals a number of nucleotide substitutions in addition to those which distinguish the A and ? variants. Three of these are silent mutations, however it appears that the occurrence in the cDNA sequence of a Val residue at position 105 may represent a new variant provisionally designated β‐lactoglobulin K, derived from β‐lactoglobulin A.

Protein kinase activity from lactating bovine mammary gland

Abstract 1. 1.Protein kinase activity (ATP:protein phosphotransferase, EC 2.7.1.37) has been detected in crude extracts from lactating bovine mammary gland. 2. 2.This activity has been partially purified and shown to consist of several components, separable by gel filtration. 3. 3.The properties of the two main fractions of enzyme activity have been compared and shown to be similar, however, they may be distinguished by their sensitivity to adenosine 3′,5′-monophosphate. 4. 4.Analysis of the patterns of phosphorylation of the components of whole casein appears to offer a useful method for distinguishing protein kinases with different substrate specificities.

The binding of β-casein to hydroxyapatite: the effect of phosphate content and location

The elution behaviour of beta-casein from columns of hydroxyapatite has been studied and the effect examined of the enzymic addition of an extra phosphate residue. When the additional phosphate is located near pre-existing phosphate residues stronger binding to hydroxyapatite is observed. When the additional phosphate is remote from the pre-existing phosphates no increase in strength of binding is observed. It is suggested that the clustering of phosphate residues which characterizes alphas- and beta-caseins can be rationalized on the basis that it facilitates co-operative interactions between these phosphates and the Ca phosphate of the casein micelle and/or basic residues within casein polypeptide chains.

Histone and casein kinases of lactating bovine mammary gland.

Abstract 1. 1. It is shown that adenosine 3′,5′-monophosphate (cyclic AMP) causes dissociation of the previously described histone-preferring protein kinase I obtained from lactating bovine mammary gland. 2. 2. A method is reported for the preparation from lactating bovine mammary gland of a crude protein kinase fraction for which casein is a better phosphate acceptor than histone. Unlike protein kinase I, this fraction is not stimulated by cyclic AMP. 3. 3. Ion-exchange chromatography of this fraction separates it into three components. For one of these, kinase C, α s -casein which has been dephosphorylated proves a better acceptor than untreated α s -casein. 4. 4. A phosphate-rich peptide obtained from α s -casein by CNBr cleavage becomes an excellent phosphate acceptor in the kinase C reaction following partial dephosphorylation. 5. 5. Analysis of tryptic and acid hydrolysates of phosphorylated α s -casein and the derived peptide indicates that kinase C is able to phosphorylate serine residues which are normally phosphorylated in vivo.