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PubMed1989

[The Bos taurus casein genes. Isolation and characterization of the kappa-casein gene].

A region spanning 25 kb of genomic DNA containing the kappa-casein gene, has been isolated from two genomic libraries in EMBL3 and EMBL4 phage vectors. Five phage clones containing kappa-casein gene have been found. Gene organisation has been determined using restriction mapping and a partial sequencing the 5' and 3' flanking regions. The kappa-casein gene includes 5 exons, the first of them coding for 64 nucleotides from the 5' untranslated mRNA zone. The gene is 12.5 kb long, which is almost 16 times longer than the corresponding mRNA. The first intron spans 2.5 kb, the second is the largest one and spans 5.5 kb. The 5' flanking region sequence has been analysed; it contains a TATA box from -30 to -25 bp, somewhat different from the canonic sequence, and a CAAT box at -80 bp.

Animals↗
PubMed2002

Identification of syntaxin-1A sites of phosphorylation by casein kinase I and casein kinase II.

Casein kinases I (CKI) are serine/threonine protein kinases widely expressed in a range of eukaryotes including yeast, mammals and plants. They have been shown to play a role in diverse physiological events including membrane trafficking. CKI alpha is associated with synaptic vesicles and phosphorylates some synaptic vesicle associated proteins including SV2. In this report, we show that syntaxin-1A is phosphorylated in vitro by CKI on Thr21. Casein kinase II (CKII) has been shown previously to phosphorylate syntaxin-1A in vitro and we have identified Ser14 as the CKII phosphorylation site, which is known to be phosphorylated in vivo. As syntaxin-1A plays a key role in the regulation of neurotransmitter release by forming part of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex, we propose that CKI may play a role in synaptic vesicle exocytosis.

Amino Acid Sequence↗
PubMed1994

Phosphorylation of synthetic fragments of inhibitor-2 of protein phosphatase-1 by casein kinase-1 and -2. Evidence that phosphorylated residues are not strictly required for efficient targeting by casein kinase-1.

The major phosphorylation site for both casein kinase-2 (CK2) and casein kinase-1 (CK1) in protein phosphatase-1 (PP-1) inhibitor-2 (I-2) is Ser86. Minor phosphorylation sites affected by either CK2 or CK1 are Ser120/Ser121 and Ser174, respectively. A synthetic peptide of 25 amino acids encompassing residues 67-93 of I-2 is phosphorylated by either CK2 or CK1 at its seryl residue corresponding to Ser86 with higher Vmax and Km values similar to those of the intact protein (9 vs 7.2 microM and 14.2 vs 5.3 microM with CK2 and CK1, respectively). No detectable phosphorylation of this peptide which also includes the glycogen synthase kinase-3 (GSK-3) site (Thr72), could be observed with either GSK-3 or p34cdc2 kinase whether or not its seryl residue equivalent to Ser86 had been previously phosphorylated by CK2. Shorter derivatives of I-2(67-93), encompassing residues 72-93 and 78-93, are also readily phosphorylated by both CK1 and CK2, with phosphorylation efficiencies similar to those of the parent peptide. A synthetic heptadecapeptide reproducing the phosphoacceptor site around Ser120/Ser121 is phosphorylated by CK2, but not to any detectable extent by CK1, with a Km value fivefold higher than that of the corresponding pentadecapeptide including Ser86 (78-93). A synthetic pentadecapeptide (166-180) reproducing the phosphoacceptor site around Ser174 is phosphorylated by CK1 less efficiently than the pentadecapeptide including its main phosphorylation site (78-93) (Km 280 microM vs 33 microM). This peptide is readily phosphorylated by CK2 as well, although it lacks the canonical consensus sequence for CK2 and its Ser174 is almost unaffected by CK2 in intact I-2. These data provide the clear-cut demonstration that the consensus sequence with N-terminal prephosphorylated residue(s), SerP/ThrP-Xaa-Xaa-Ser/Thr, [Flotow, H., Graves, P. R., Wang, A., Fiol, C. J., Roeske, R. W. & Roach, P. J. (1990) J. Biol. Chem. 265, 14264-14269; Meggio, F., Perich, J. W., Reynolds, E. C. & Pinna, L. A. (1991) FEBS Lett. 283, 303-306] is not always required to achieve efficient and high-affinity phosphorylation by CK1. They also show that the specificity determinants for I-2 phosphorylation by either CK2 or CK1, but not by GSK3, are entirely grounded on local structural features of the phosphoacceptor site, being only marginally affected by the overall structure of I-2.

Amino Acid Sequence↗
PubMed1996

Semi-preparative isolation of phosphopeptides derived from bovine casein and dephosphorylation of casein phosphopeptides.

Semi-preparative isolation of casein phosphopeptides (CPP) from a tryptic hydrolysate of bovine casein was performed applying a three-step procedure consisting of solid phase extraction, reversed phase HPLC and ion exchange chromatography. Dephosphorylation of CPP was achieved using immobilized alkaline phosphatase. The purified phosphopeptides and their dephosphorylated forms obtained by these methods are suitable for comparative studies on biological activities, especially mineral binding and immunmodulation.

Animals↗
PubMed2006

Reconstituted micelle formation using reduced, carboxymethylated bovine kappa-casein and human beta-casein.

In milk, kappa-casein, a mixture of disulfide-bonded polymers, stabilizes and regulates the size of the unique colloidal complex of protein, Ca2+ and inorganic phosphate (Pi) termed the casein (CN) micelle. However, reduced, carboxymethylated bovine kappa-CN (RCM-kappa) forms fibrils at 37 degrees C and its micelle-forming ability is in question. Here, the doubly- and quadruply-phosphorylated human beta-CN forms and 1:1 (wt:wt) mixtures were combined with RCM-kappa at different beta/kappa weight ratios. Turbidity (OD(400 nm)) and a lack of precipitation up to 37 degrees C were used as an index of micelle formation. Studies were with 0, 5 and 10 mM Ca2+ and 4 and 8 mM Pi. The RCM-kappa does form concentration-dependent micelles. Also, beta-CN phosphorylation level influences micelle formation. Complexes were low-temperature reversible and RCM-kappa fibrils were seen. There appears to be equilibrium between fibrillar and soluble forms since the solution still stabilized after fibril removal. The RCM-kappa stabilized better than native bovine kappa-CN.

Amyloid↗
PubMed1983

Peptide substrates for chymosin (rennin): conformational studies of kappa-casein and some kappa-casein-related oligopeptides by circular dichroism and secondary structure prediction.

Circular dichroism spectra of a series of synthetic, kappa-casein-related oligopeptide substrates for chymosin in water and in surfactant solution were determined. The results show that there is a good correlation between the beta-structure forming potential of these peptides as found by using structure-predictive methods and the conformation in dilute sodium dodecyl sulfate solutions. The results support earlier suggestions concerning enzyme-substrate interaction which were made on the basis of X-ray analysis of acid proteinases. A predicted secondary structure of the whole kappa-casein molecule obtained by using a combination of three methods is also presented.

Animals↗
PubMed1983

Phosphorylation of acetyl-coenzyme A carboxylase by casein kinase I and casein kinase II.

Two cAMP-independent acetyl-CoA carboxylase (ACC) protein kinases have been partially purified from rat liver cytosol and microsomal extracts. The first kinase, present in greatest activity in microsomal extracts, appears to be identical to casein kinase I by characteristic molecular size on gel filtration (Mr 40,000) and sodium dodecyl sulfate-gel electrophoresis (Mr 34,000), autophosphorylation of this single subunit, inability to efficiently utilize GTP, and resistance to inhibition by heparin and 2,3-diphosphoglycerate. The second kinase, predominant in cytosol, appears to be identical to casein kinase II by characteristic molecular size on gel filtration (Mr 150,000), an autophosphorylated subunit of Mr 25,000, a Km for GTP nearly equal to that of ATP, inhibition by heparin and 2,3 DPG, and relative substrate specificity. Despite the incorporation of up to 2 mol 32P/mol carboxylase subunit (kinase I) and 0.6 mol/subunit (kinase II), phosphorylation by either kinase causes no change in carboxylase activity. The site(s) phosphorylated by each kinase and by the cAMP-dependent protein kinase on carboxylase appear to be clustered on a Mr 16,000 cyanogen bromide peptide that is readily released on incubation with trypsin. The potential roles of these kinases in the regulation of ACC remain to be clarified.

Acetyl-CoA Carboxylase↗
PubMed1979

Hydration of casein micelles: kinetics and isotherms of water sorption of micellar casein isolated from fresh and heat-treated milk.

Water vapour sorption isotherms of casein micelles prepared from raw milk and various heat-treated milks were determined. The equilibrium water contents of the heated preparations were markedly lower than that of the raw-milk casein over the whole range of vapour pressures studied. An analysis of the sorption isotherms in the relative vapour pressure range 0.1--0.45, according to the Brunauer, Emmett & Teller (1938) equation, showed that there were significant differences between preparations in the computed monolayer contents. Differences in the rates of water sorption were also observed between the different preparations. As judged from the amount of absorbed water, the influence of the heating methods could be ranked in the order: HTST (92 degrees C) approximately UHT (direct) less than UHT (indirect) less than HTST (72 degrees C).

Animals↗
PubMed1979

Covalent bonds formed in proteins during milk sterilization: studies on caseins and casein peptides.

Formation of covalent bonds at milk sterilization temperatures was studied using caseins and casein peptides. At 120 degrees C lysinoalanyl residues produced even at pH 7.0 were derived from intra-molecular interactions between phosphoserine and lysine; the conditions of formation were determined. It was also found that the formation fo isopeptidic cross-links was significant with conditions more severe than those used for milk sterilization.

Amino Acids↗
PubMed1994

Disulphide arrangement in bovine caseins: localization of intrachain disulphide bridges in monomers of kappa- and alpha s2-casein from bovine milk.

Naturally occurring monomeric kappa-casein and alpha s2-casein in bovine milk were purified by ion-exchange chromatography in order to localize potential intrachain disulphide bridges. Enzymic cleavage of the proteins followed by mass spectrometry and amino acid sequence analysis of cystine-containing peptides revealed the presence of an intrachain disulphide bond in both proteins.

Amino Acid Sequence↗
PubMed2004

Casein proteolysis in human milk: tracing the pattern of casein breakdown and the formation of potential bioactive peptides.

The protein and peptide fraction of human milk samples collected from mothers of pre- and full-term infants in the first week after parturition was analysed by use of liquid chromatography-mass spectrometry and tandem mass spectrometry. By characterising the peptide sequence, we defined the pathway of casein hydrolysis which leads to the formation of small peptides through intermediate oligopeptides. It was found that the action of a plasmin-like enzyme acting on specific lysine residues is the primary step in casein degradation. This is followed by endopeptidases and/or exopeptidases mediated cleavage of the oligopeptides which, in turn, produces a multiplicity of short peptides differing by one or more amino acid residues. In this process, a series of potentially bioactive peptides (opioid, phosphopeptides) and their precursors are produced.

Amino Acid Sequence↗
PubMed1998

Regulation of casein kinase I epsilon and casein kinase I delta by an in vivo futile phosphorylation cycle.

Casein kinase I delta (CKIdelta) and casein kinase I epsilon (CKIepsilon) have been implicated in the response to DNA damage, but the understanding of how these kinases are regulated remains incomplete. In vitro, these kinases rapidly autophosphorylate, predominantly on their carboxyl-terminal extensions, and this autophosphorylation markedly inhibits kinase activity (Cegielska, A., Gietzen, K. F., Rivers, A., and Virshup, D. M. (1998) J. Biol. Chem. 273, 1357-1364). However, we now report that while these kinases are able to autophosphorylate in vivo, they are actively maintained in the dephosphorylated, active state by cellular protein phosphatases. Treatment of cells with the cell-permeable serine/threonine phosphatase inhibitors okadaic acid or calyculin A leads to rapid increases in kinase intramolecular autophosphorylation. Since CKI autophosphorylation decreases kinase activity, this dynamic autophosphorylation/dephosphorylation cycle provides a mechanism for kinase regulation in vivo.

3T3 Cells↗
PubMed1987

The effects of intraruminal infusions of urea, casein, glucose syrup and a mixture of casein and glucose syrup on nitrogen digestion in the rumen of cattle receiving grass-silage diets.

In an incomplete 5 X 5 Latin square experiment, four cattle were given grass silage in two meals per d to satisfy 1.15 maintenance energy requirements. In addition, water or casein (21 g nitrogen and 0.17 kg organic matter (OM)/d) or urea (U; 28 g N/d) or a glucose syrup (G; 0.87 kg OM/d) or casein and glucose syrup (CG; 17 g N and 0.93 kg OM/d) were infused intraruminally at a constant rate. A 24 h collection of duodenal digesta was made using chromic oxide for flow estimation and 35S as a marker of microbial N entering the small intestine. Samples of rumen fluid were also taken for estimation of rumen pH, and concentrations of ammonia-N and volatile fatty acids. The intraruminal infusions had no significant effects on rumen pH, concentrations of volatile fatty acids or their molar proportions. Infusion of either C or U significantly (P less than 0.05) increased rumen NH3-N concentrations whereas infusions of either G or CG lowered rumen NH3-N concentrations. Infusions of C or U had no significant effect on the quantities of OM, acid-detergent fibre (ADF) or N constituents which entered the small intestine. Infusions of G or CG increased the quantities of OM (G P less than 0.05, CG P less than 0.01), ADF (CG P less than 0.05), non-NH3-N (G P less than 0.05, CG P less than 0.01), amino acid N (G P less than 0.05, CG P less than 0.01) and microbial N (G P less than 0.05, CG P less than 0.01) which entered the small intestine. The efficiency of rumen microbial N synthesis was unchanged by the infusion of C, U or G (P greater than 0.05) but increased significantly (P less than 0.05) when CG were infused.

Animal Nutritional Physiological Phenomena↗
PubMed1992

Effect of casein, casein phosphopeptides and calcium intake on ileal 45Ca disappearance and temporal systolic blood pressure in spontaneously hypertensive rats.

Paracellular 45Ca absorption and temporal systolic blood pressure (SBP) measurements were recorded in spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats fed on casein (C) and soya-bean-protein isolate (S) diets, containing 20 (H), 5 (H) and 0.5 (L) g Ca/kg. Similar measurements were also taken in SHR rats only fed on C-M and S-M diets supplemented with 30 g caseinophosphopeptides (CPP)/kg. Absorption of 45Ca from the ileal loop was equivalent in both SHR and WKY animals and largely affected by the level of dietary Ca. In addition, animals fed on C diets exhibited significantly (P < 0.05) greater ileal absorption of 45Ca compared with S-fed animals. This result was attributed to the presence of CPP and a greater (P < 0.05) proportion of soluble 45Ca in the contents of the ileum. Animals fed on S diets supplemented with CPP confirmed this finding. The SBP of SHR rats was higher (P < 0.01) than WKY controls after 9-10 weeks of age. The temporal pattern of observed hypertension was independent of dietary influence in the SHR. The severity of hypertension in SHR rats was affected only by dietary Ca deficiency, and not by Ca supplementation or CPP enhancement of Ca bioavailability. These findings suggest that tryptic digestion products of casein in milk can enhance Ca bioavailability by increasing Ca solubility; however, this action had no effect in reducing hypertension in SHR.

Animals↗
PubMed1985

Production and characterization of monoclonal antibodies to human casein. A monoclonal antibody that cross-reacts with casein and alpha-lactalbumin.

Four hybridomas have been produced that secrete monoclonal antibodies to human beta-casein, a protein synthesized by fully differentiated breast epithelial cells. The antibodies have been characterized on immunoblots and have been shown to react with methanol: acetone-fixed sections of human lactating mammary gland. Immunoblots show that three of these antibodies react with casein whereas one, F20.10, also recognizes an epitope present on human alpha-lactalbumin. Computer analysis of the amino acid sequences of these two milk proteins reveals very little sequence homology, leading to the conclusion that the three-dimensional shape, rather than the primary sequence, is important in this cross-reactivity.

Amino Acid Sequence↗
PubMed1990

Ribofuranosyl-benzimidazole derivatives as inhibitors of casein kinase-2 and casein kinase-1.

5,6-Dichloro-1-(beta-D-ribofuranosyl)benzimidazole (DiCl-RB) is a powerful inhibitor of casein kinase-2 (CK-2) [Zandomeni, R. et al. (1986) J. Biol. Chem. 261, 3414-3420]. Here a series of 17 analogues of DiCl-RB has been employed for studying the specificity and the mode of action of this family of CK-2 inhibitors. The two halogen substituents on the benzene ring are shown to play a prominent role in inhibition, the 5,6-dibromo derivative (DiBr-RB) being fivefold more effective than DiCl-RB (Ki = 2 microM, with GTP as substrate), whereas the difluoro derivative (DiF-RB) is nearly as ineffective as unsubstituted 1-(beta-D-ribofuranosyl)benzimidazole. On the other hand, although some modifications of the ribose group significantly decrease the inhibitory efficiency, the sugar moiety is not strictly required, since dichlorobenzimidazole itself (DiCl-Bz) is an inhibitor almost as good as DiCl-RB. Inhibition of CK-2 by DiCl-RB and by its analogues, DiCl-Bz included, is of the competitive type with respect to the nucleotide substrate, the Ki values being lower with GTP than with ATP. The Ki values of the most potent inhibitor, DiBr-RB, with ATP and GTP, are 6 microM and 2 microM, respectively, denoting an affinity for the enzyme higher than that of the physiological substrates, ATP and GTP. DiBr-RB has been assayed for its inhibitory capacity toward several protein kinase other than CK-2. Protein kinase-C, cAMP-dependent protein kinase, the Ser/Thr protein kinase expressed by Pseudorabies virus, and four different tyrosine protein kinases from spleen, proved insensitive to DiBr-RB concentrations capable of almost entirely suppressing the activity of rat liver and maize seedling CK-2. Casein kinase-1 however is nearly as sensitive as CK-2 to DiBr-RB. Inhibition of CK-1 is also of the competitive type with respect to ATP (Ki = 14 microM). Although the inhibitory spectrum of CK-1 by the various analogues is reminiscent of that observed with CK-2, a remarkable difference is revealed by 5'-phosphorylation of ribose which increases the Ki with CK-2 while decreasing that with CK-1.

Animals↗
PubMed1992

Purification and characterization of maize seedling casein kinase IIB, a monomeric enzyme immunologically related to the alpha subunit of animal casein kinase-2.

Casein kinase IIB (CKIIB), a protein kinase related to animal casein kinase-2 (CK2), has been purified to homogeneity. It appears to be a monomeric enzyme, composed by an individual 39 kDa subunit, homologous to the alpha/alpha' subunits of animal CK2 and devoid of the autophosphorylatable 25-kDa alpha subunit of animal CK2, which display an heterotetrameric alpha 2 beta 2/alpha alpha' beta 2 structure. Such a conclusion is supported by the following lines of evidence: (1) CKIIB displays an apparent 39,000 Mr by gel filtration on Ultrogel AcA 34 and it gives rise to a single prominent protein band of similar Mr (38,000) upon SDS/PAGE; (2) upon incubation of the enzyme with [32P]ATP, no radiolabeled bands are detectable which might be attributable to either canonical or atypical beta subunits; (3) the 39-kDa band immunoreacts with antisera that recognize the alpha subunit of rat and chicken CK2; (4) conversely, no component immunologically related with the beta subunit could be detected in CKIIB by Western-blot analyses with antisera that recognize animal beta subunits; (5) the recombinant beta subunit of human CK2 is readily phosphorylated by CKIIB, the reaction being prevented, rather than stimulated, by polylysine, a behaviour typical of animal CK2 autophosphorylation. While the responsiveness of CKIIB to either heparin inhibition or polylysine stimulation are reminiscent of those of animal CK2, its peptide substrate specificity is significantly different and its thermolability is increased. Altogether these data would indicate that maize seedling CKIIB represents a naturally occurring monomeric form of CK2 devoid of non-catalytic subunits. Its properties, compared to those of animal CK2, suggest that the beta subunits of animal CK2 may be responsible for structural modifications conferring an altered specificity and an increased stability to the catalytic subunit.

Adenosine Triphosphate↗
PubMed1995

Preparation of casein phosphopeptides from casein micelles by ultrafiltration.

Casein phosphopeptides (CPPs), which inhibit the precipitation of calcium phosphate in the intestines, were prepared as CPP-calcium phosphate complexes from casein micelles by the ultrafiltration method. The prepared CPPs hardly contained any other peptides, so there was no need to treat with active carbon to remove the bitter peptides. The sum of alpha s1-CN-5P(f59-79) and beta-CN-4P(f1-25) comprised 60% of the CPP composition by an analysis of the elution pattern from a Q-Sepharose FF column. The content of alpha s1-CN-5P(f59-79), which has high retention ability for calcium phosphate, was the highest in the CPPs.

Animals↗