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PubMed1988

[Group separation of caseins from cow milk on hydroxyapatite].

Ion exchange-chromatography method with the use of hydroxy apatite is considered one of the methods used for the casein fractionation particularly those containing phosphate in its composition. Simplicity of hydroxyapatite-preparation and its easily use in casein fractionation in batches are advantages of this method. Sephadex G-25 or Agarose is used to easement the sample passage through the column. By using of hydroxy-apatite in batches, alpha s-casein can easily separated with the use of buffer solution 0.006 M phosphate at pH 6.6 and the elution is carried out by buffer solution containing 0.25 M KH2PO4, 0.2 M KCl, 4.5 M urea and 0.002 M di-thiotreitol. Before the elution of alpha s-casein, beta- and kappa-casein are separated using buffer solutions containing 0.005 M and 0.1 M KH2PO4 in addition to the previously mentioned components.

Animals↗

PubMed1996

Quantitative comparison of casein and rapeseed proteolysis by pancreatin.

The problem of quantitative comparison of kinetic curves was solved for casein and rapeseed pancreatin hydrolysis in a membrane reactor, which ensured the measurement of proteolysis kinetics for the products with a molecular weight of less than 1000. Coordinates were derived which provided good linearization of kinetic curves and the determination of relative rate constants irrespective of reagent concentrations, E0/S0 ratio and time intervals of kinetic measurements. When the relative rate constants of the release of the individual amino acid residues in the low-weight proteolysis products were compared, trypsin-dependent constants (for Lys and Arg residues) were found to be two times less for rapeseed than for casein, and chymotrypsin-dependent constants (for Tyr and Phe residues) were approximately 1.3 times higher for rapeseed than for casein. Statistical analysis demonstrated that the distribution of constants was narrower for rapeseed than for casein. Differences between target (Arg, Lys, Tyr and Phe) and non-target constants of release in the form of peptides and free amino acids, or in the form of free amino acids only, were attributed on the differences in the peptide bond masking for casein and rapeseed proteins. Computer simulation of proteolysis kinetics was performed by PROTEOLYSIS program package to confirm the dependence of rate constant distribution on the state of masking.

Amino Acids↗

PubMed1985

Phosphorylation of the insulin receptor by casein kinase I.

Insulin receptor was examined as a substrate for the multipotential protein kinase casein kinase I. Casein kinase I phosphorylated partially purified insulin receptor from human placenta as shown by immunoprecipitation of the complex with antiserum to the insulin receptor. Analysis of the phosphorylated complex by polyacrylamide gel electrophoresis under nonreducing conditions showed a major phosphorylated band at the position of the alpha 2 beta 2 complex. When the phosphorylated receptor was analyzed on polyacrylamide gels under reducing conditions, two phosphorylated bands, Mr 95,000 and Mr 135,000, were observed which corresponded to the alpha and beta subunits. The majority of the phosphate was associated with the beta subunit with minor phosphorylation of the alpha subunit. Phosphoamino acid analysis revealed that casein kinase I phosphorylated only seryl residues. The autophosphorylated alpha 2 beta 2 receptor purified by affinity chromatography on immobilized O-phosphotyrosyl binding antibody was also a substrate for casein kinase I. Reduction of the phosphorylated alpha 2 beta 2 receptor indicated that casein kinase I incorporated phosphate into seryl residues only in the beta subunit.

Binding Sites↗

PubMed1992

Expression and regulation of casein kinase 2 during heat shock in Verticillium dahliae.

A homogeneous preparation of casein kinase 2 has been isolated from the phytopathogenic fungus Verticillium dahliae (the parasite of cotton). The enzyme consists of three subunits with molecular masses of 53, 41, and 38 kDa. Highly specific immune serum against casein kinase 2 has been obtained. By means of immunoblotting, enzyme-linked immunosorbent assay (ELISA), and immunochemical isolation on protein A-Sepharose, it is shown that the amount of casein kinase 2 increases under heat shock conditions (at least in part due to the synthesis de novo), while the synthesis of the majority of other proteins falls. The activity of casein kinase 2 is supressed during heat shock and so does not correlate with its content. The results give an evidence for the two-step model of casein kinase 2 regulation during heat shock.

Blotting, Western↗

PubMed2006

Determination of the phosphorylation level and deamidation susceptibility of equine beta-casein.

beta-Casein was isolated from Haflinger mare's milk by RP-HPLC, and displayed microheterogeneity by urea-electrophoresis and 2-DE probably due to a variable degree of phosphorylation. To investigate the degree of phosphorylation, the primary structure of equine beta-casein was determined by tryptic hydrolysis and MS of peptides released and by MS of the protein treated by alkaline phosphatase. The molecular mass found for the apo-form of Haflinger mare's beta-casein (25 514 +/- 3 Da) was close to the theoretical mass of the reported sequence (GenBank AAG43954) modified by insertion of a region (residues 27-34) encoded by an exon sometimes out-spliced (25 511.40 Da). Hence, the beta-casein isolated from Haflinger mare's milk corresponded to a variant of 226 amino acid residues. The latter was composed by highly multi-phosphorylated isoforms with three to seven phosphate groups, and pIs, determined by 2-DE, ranging from 4.74 to 5.30. Moreover, the equine beta-casein was able to deamidate spontaneously, at the level of Asn in the potential deamidation motif (135)Asn-Gly(136). Approximately 80% of the protein was deamidated after 96 h of incubation under physiological conditions.

Alkaline Phosphatase↗

PubMed1995

Intracellular routing and release of caseins and growth hormone produced into milk from transgenic mice.

Growth hormone (GH) secretion, in mammary tissue from transgenic mice, containing a chimeric gene composed of the regulatory region of whey acidic protein gene and the structural region of GH gene, was compared to casein secretion. GH was expressed in milk and for a small percentage (1:1000) in blood as revealed by SDS-polyacrylamide gel electrophoresis and radioimmunoassay. As attested by immunofluorescence and immunogold electron microscopy, caseins and GH followed the same secretory pathway. However, contrary to caseins, which are essentially in micellar form, GH was detected in a nonaggregated form in secretory vesicles and in the lumen of the acini. Newly synthesized caseins and GH were carried simultaneously, mainly to the lumen of the acini, but also to the base of the cell. Secretion of newly synthesized proteins was increased by prolactin (PRL). As shown by immunoblotting, the proportion of GH versus other proteins, secreted in the presence of PRL was not modified, suggesting that GH secretion is subjected to the same hormonal regulation by PRL as other milk proteins. These results show that, in lactating mammary epithelial cells from transgenic mice, a recombinant GH and the caseins are carried simultaneously to the lumen and suggest that secretion of both proteins is increased by PRL during the same time course. Transport of these newly synthesized proteins occurs also to the base of the cell.

Animals↗

PubMed1999

Construction of phosphorylatable chimeric monoclonal antibody CC49 with a casein kinase I recognition site.

Phosphorylation sites for casein kinase I were introduced into chimeric monoclonal antibody CC49 (MAb-chCC49) by inserting a synthetic fragment (CK1) encoding two casein kinase I phosphorylation sites into an expression vector. The phosphorylation sites were created by incorporating the predicted consensus sequences for phosphorylation by the casein kinase I at the carboxyl terminus of the heavy-chain constant region of the MAb-chCC49. The resultant modified MAb-chCC49 (MAb-chCC49CK1) was expressed and purified. The MAb-chCC49CK1 protein can be phosphorylated by the casein kinase I with [gamma-32P]ATP to high radiospecific activity. The 32P-labeled MAb-chCC49CK1 protein binds to cells expressing TAG-72 antigens. The introduction of phosphorylation sites into MAb provides new reagents for the diagnosis and treatment of cancer. This demonstrates that, as was described for the cAMP-dependent protein kinase site, the casein kinase I recognition site can also be used to introduce phosphorylation sites into proteins.

Amino Acid Sequence↗

PubMed1991

Interaction between casein and vitamin A during food processing.

A particular relation exists between casein micelle and vitamin A. In vitro, large amounts of retinol are fixed to acid casein and its different fractions by hydrophobic bindings. The binding on the hydrophobic amino-acid residues (Trp, Phe) is greatly facilitated by a configurational change in the molecule exposed to physico-chemical parameters: alcalinity and heat treatments. Another amount of retinol is even more strongly fixed by a binding which can only be broken by saponification. Casein plays an important role in stabilizing retinol which does not degrade over time or during heat treatments. In vivo, when the retinol availability is measured by the vitamin A content in the liver, acid casein somewhat increases the retinol efficiency and, even more so, the retinal efficiency. Nevertheless, this favourable action is only observed under particular conditions: balanced diet, casein from milk, etc. For the moment, the animal data cannot be directly linked with the properties of binding appearing in vitro.

Animals↗

PubMed1991

Comparison of bovine beta-casein hydrolysis by PI and PIII-type proteinases from Lactococcus lactis subsp. cremoris [corrected].

The action of the cell-wall-associated proteinases from Lactococcus lactis subsp. cremoris strains H2 and SK112 on bovine beta-casein was compared. The proteinase from the H2 strain was characterised as a PI-type proteinase since it did not hydrolyse alpha s1-casein and the initial trifluoroacetic acid-soluble products of beta-casein hydrolysis were identical to those previously identified as hydrolysis products of PI-type lactococcal proteinase action. The time-course of product formation by the proteinase from the H2 strain indicated that the bonds Tyr193-Gln194 and Gln182-Arg183 were the first to be hydrolysed. Cleavage of the bonds Gln175-Lys176, Ser168-Lys169, Ser166-Gln167 and Leu163-Ser164 was also very rapid. Four of the five bonds in beta-casein most susceptible to hydrolysis by the PIII-type proteinase from strain SK112 were different from those cleaved by the PI-type proteinase, initial hydrolysis being at the sites Tyr193-Gln194, Leu192-Tyr193, Asp43-Glu44, Gln46-Asp47 and Phe52-Ala53. Early hydrolysis at the three sites in the N-terminal region of beta-casein, leading to cleavage of the N-terminal phosphopeptide and rapid precipitation of the residual fragment, represents a marked contrast to the action of PI-type proteinases where cleavage at sites in the N-terminal region occurs only very slowly.

Amino Acid Sequence↗

PubMed1995

Casein kinase 2 inactivation by Mg2+, Mn2+ and Co2+ ions.

Mg2+ as well as Mn2+, and Co2+, which may substitute Mg2+ in the mental ion requirement of casein kinase 2 (Gatica et al., FEBS Lett: 315:173-173, 1993), have been repeatedly reported to display an optimal concentration at which activity of casein kinase 2 is maximal. As far as we know this intriguing property has always been observed with casein as substrate. This phosphoprotein is not the natural substrate of the enzyme, and it is well known that it binds divalent metal ions, which provoke the aggregation and precipitation of the protein. Since an optimal concentration of metal ion might have a regulatory role, we have examined if it is a consequence of the particular properties of casein, or it is an inherent property of the enzyme, extensive to other substrates. We have used the type II regulatory subunit of protein kinase A which is a physiological substrate of the enzyme, and the peptide RRREEETEEE as a specific substrate. No optimal concentration of Mg2+ is observed when these two substrates are used. The results explain, however, why that optimum is observed with casein. Although low concentration of Mn2+, and Co2+ render about 25% of the maximal activity found with Mg2+, they inactivate the enzyme almost fully at concentrations at which Mg2+ yield the maximal activity.

Amino Acid Sequence↗

PubMed1993

Casein kinase II in signal transduction and cell cycle regulation.

Casein kinase II is a protein serine/threonine kinase that is ubiquitously distributed in eukaryotes. Molecular cloning studies and protein sequence analysis of purified proteins have demonstrated the existence of two related, but distinct, isoenzymic forms of its catalytic subunit in mammals and birds. At present, the precise role of the individual casein kinase II isoforms in biological responses is poorly understood. However, a great deal of evidence indicates that casein kinase II is an important component of signalling pathways that control the growth and division of cells. In particular, casein kinase II is known to phosphorylate, and in several cases, regulate the activity of a variety of regulatory nuclear proteins including nuclear oncoproteins, transcription factors, and enzymes involved in other aspects of DNA metabolism. In this review, we will summarize evidence relating to the involvement of casein kinase II in signal transduction events that are relevant to cell proliferation.

Amino Acid Sequence↗

PubMed1994

Proteolysis of bovine alpha s2-casein by chymosin.

Proteolysis of bovine alpha s2-casein by chymosin (E. C. 3.4.23.4) in solution in 100 mM Na phosphate buffer, pH 6.5, at 30 degrees C was studied by reversed-phase (RP)-HPLC and urea-polyacrylamide gel electrophoresis (PAGE). Chymosin hydrolyzed alpha s2-casein in solution to eight peptides detectable by urea-PAGE. Peptides soluble in acetate buffer, pH 4.6, were isolated by RP-HPLC on a C18 column using an acetonitrile/water gradient and identified from their N-terminal amino acid sequence. The chymosin cleavage sites were at the bonds Phe88-Tyr89, Tyr95-Leu96, Gln97-Tyr98, Tyr98-Leu99, Phe163-Leu164, Phe174-Ala175 and Tyr179-Leu180. Chymosin cleavage sites were restricted to the hydrophobic regions of the molecule. The bond-type in alpha s2-casein cleaved by chymosin was in agreement with that found to be susceptible to chymosin in other caseins. The primary site of chymosin action on alpha s2-casein appeared to be at Phe88-Tyr89.

Amino Acid Sequence↗

PubMed1981

Effects of casein and fibrin on calcium absorption and calcium homeostasis in the rat.

To test the hypothesis that individual proteins have specific effects on calcium absorption, two proteins, casein and fibrin, were pair-fed in diets to rats for 3 weeks. Each protein was fed at normal (1.2%) and low (0.02%) calcium intakes. Diets were matched in other nutrients. Calcium absorption, measured in vitro as serosal-to-mucosal concentration ratio of 45Ca developed by duodenal sacs, was the same for both protein groups when calcium intake was normal and was increased by both low-calcium diets. Comparing low-calcium diets, absorption was greater in fibrin-fed than casein-fed groups. Balance studies showed that casein- and fibrin-fed rats taking normal calcium diets excreted the same amounts of calcium and phosphorus in urine and feces. Fibrin-fed rats taking low calcium excreted twice as much calcium as casein-fed rats and had decreased serum calcium. The balance data suggest that, compared to casein, fibrin prevents absorption or promotes excretion of calcium when calcium intake is low, and the response to calcium depletion is enhanced calcium absorption. It is concluded that individual proteins can alter calcium transport through effects on overall calcium homeostasis.

Animals↗

PubMed1985

Kappa casein, lactalbumin and GCDFP 70 localization in human breast carcinomas: an immunohistochemical study using the avidin-biotin-peroxidase complex method.

A retrospective study of 67 human breast carcinomas of various types and grades was conducted using the avidin-biotin-peroxidase complex (ABC) to localize casein, lactalbumin, and GCDFP 70 on paraffin sections. Estrogen and progesteron receptors also were evaluated. This study demonstrated the following: (1) Casein positive cells were present in all cases with a variable distribution and degree of staining, whereas lactalbumin and GCDFP 70 were seen in only 40 and 43% of the cases, respectively. (2) No significant relationship was observed between casein, lactalbumin, GCDFP 70 and the histologic types of tumors or the extent of stromal elastosis, with the exception GCDFP 70, which was observed more often in well-differentiated ductal carcinomas. (3) No significance was established in the relationship between antigens and steroid receptor content, with the exception of casein; strong casein immunostaining was significantly related to high progestin receptor levels. (4) Lactalbumin and GCDFP 70 were significantly associated with each other, but independently so of the histologic grades and types, the extent of stromal elastosis, and the steroid receptor content of the tumor cells.

Adenocarcinoma, Mucinous↗

PubMed1999

Type I (insulin-dependent) diabetes mellitus and cow milk: casein variant consumption.

Previously published Type I (insulin-dependent) diabetes mellitus incidence in 0 to 14-year-old children from 10 countries or areas was compared with the national annual cow milk protein consumption. Countries which were selected for study had appropriate milk protein polymorphism studies, herd breed composition information and low dairy imports from other countries. Total protein consumption did not correlate with diabetes incidence (r = +0.402), but consumption of the beta-casein A1 variant did (r = +0.726). Even more pronounced was the relation between beta-casein (A1+B) consumption and diabetes (r = +0.982). These latter two cow caseins yield a bioactive peptide beta-casomorphin-7 after in vitro digestion with intestinal enzymes whereas the common A2 variant or the corresponding human or goat caseins do not. beta-casomorphin-7 has opioid properties including immunosuppression, which could account for the specificity of the relation between the consumption of some but not all beta-casein variants and diabetes incidence.

Adolescent↗

PubMed2005

Speciation analysis of calcium, iron, and zinc in casein phosphopeptide fractions from toddler milk-based formula by anion exchange and reversed-phase high-performance liquid chromatography-mass spectrometry/flame atomic-absorption spectroscopy.

Casein phosphopeptides (CPP) are phosphorylated casein-derived peptides that can be released by in-vitro or in-vivo enzymatic hydrolysis of alpha(s1)-casein, alpha(s2)-casein, and beta-casein (CN). Many of these peptides contain a highly polar acidic sequence of three phosphoseryl groups followed by two glutamic acid residues. These domains are binding sites for minerals such as calcium, iron, and zinc and play an important role in mineral bioavailability. The aim of this study was speciation analysis of calcium, iron, and zinc in CPP fractions from the soluble fraction of a toddler milk-based formula. Methods for CPP separation by anion-exchange high-performance liquid chromatography (AE-HPLC) were combined with CPP identification by reversed-phase high performance liquid chromatography-electrospray ionization mass spectrometry and determination of the calcium, iron, zinc, and phosphorus content of the fractions obtained by AE-HPLC. Calcium and phosphorus were detected in all the analyzed AE-HPLC fractions. Calcium and zinc could be bound to CPP derived from alpha(s1)-CN and alpha(s2)-CN in fraction 3. Iron could be bound to CPP in fraction 4 in which beta-CN(15-34)4P was present with the cluster sequence S(P)S(P)S(P)EE. The results obtained prove the different distribution of calcium, iron, and zinc in heterogeneous CPP fractions.

Amino Acid Sequence↗

PubMed1981

Cyclic nucleotide independent casein kinase (G type) in bovine adrenal cortex: purification and properties of two molecular forms.

Two soluble cyclic nucleotide independent protein kinase (ATP: protein-phosphotransferase, EC 2.7.1.37) activities have been purified from bovine adrenal cortex cytosol. Both purified enzymes exhibit the best affinity for acidic substrates such as casein and can use GTP as well as ATP as phosphoryl donor. They can thus be classified as casein kinase of the G type as previously proposed (Cochet C. et al., (1980) Endocrinology 106, 750-757). Whereas the two moieties could be separated using their different affinities toward a phosphocellulose resin, both purified enzymes appeared indistinguishable on the basis of several molecular and catalytic properties. Both G type casein kinase moieties have an identical sedimentation behavior (5.5 S in the presence of 0.5 M NaCl), yield similar patterns upon electrophoresis under denaturing conditions with three major protein components (42 000, 38 000 and 27 000), and show an ability to undergo self-phosphorylation mostly on the 27 000 component. Both enzymes have the same protein and nucleotide (ATP and GTP) substrate specificity, show similar increases in activity in the presence of polyamines and Mg2+ (optimum at 50 mM) and similar inhibition by NaCl above 0.2 M. The only difference between the two forms of casein kinase (i.e., affinity for phosphocellulose) could not be explained by a different degree of self-phosphorylation or by a limited proteolytic process during handling and purification. These results suggest that the two active moieties may represent isoenzymatic forms of the G type casein kinase activity in bovine adrenal cortex cytosol.

Adenosine Triphosphate↗

PubMed1983

Regulation of hepatic receptor-dependent degradation of LDL by mevinolin in rabbits with hypercholesterolemia induced by a wheat starch-casein diet.

Rabbits fed a wheat starch-casein diet develop a marked hypercholesterolemia and have a slower rate of removal of rabbit 125I-labeled low density lipoproteins (LDL) from plasma. Treating rabbits with mevinolin, a highly potent competitive inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase, at a daily dose of 20 mg per animal prevents the increase in plasma and LDL cholesterol. The mevinolin effect is mediated through an increased rate of removal of rabbit 125I-labeled LDL from plasma. To study the role of mevinolin on the regulation of the hepatic LDL receptor in rabbits, the binding of 125I-labeled LDL and 125I-labeled beta-VLDL (beta-migrating very-low-density lipoproteins) to liver membranes prepared from rabbits fed the wheat starch-casein diet with or without mevinolin was investigated. Liver membranes from wheat starch-casein-fed rabbits have no demonstrable EDTA-sensitive binding activity of 125I-labeled LDL and low (37 ng/mg protein) binding activity of 125I-labeled beta-VLDL. Treatment of the wheat starch-casein fed rabbits with mevinolin results in high levels of specific EDTA-sensitive binding of 125I-labeled LDL (28.7 ng/mg protein) and 125I-labeled beta-VLDL (120 ng/mg protein). To assess the functional role of the hepatic LDL receptor in response to mevinolin, the catabolism of 125I-labeled LDL by perfused rabbit livers was studied. Perfused livers from mevinolin-treated rabbits show a 3.3-fold increase in the rate of receptor-dependent catabolism of 125I-labeled LDL (4.6% X h-1) when compared with that of livers from rabbits not treated with mevinolin (1.4% X h-1). Thus, these studies demonstrate that mevinolin prevents the increase of plasma LDL cholesterol level in rabbits fed a wheat starch-casein diet by regulating the levels of hepatic LDL-binding sites and the rate of receptor-dependent catabolism of LDL by the liver.

Animals↗