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PubMed2005

Effect of ingestion of cow's milk protein hydrolysate formulas on alpha-casein-specific immunoglobulin E and G1 antibody responses in naïve and sensitized mice.

OBJECTIVES: Cow's milk protein hydrolysate formulas are widely used for genetically predisposed atopic infants. Whether hydrolysate formulas can induce oral tolerance to alpha-casein was studied for the first time in naive and sensitized mice. METHODS: Using immunoblotting, residual antigenicity to alpha-casein was examined for in animals fed hydrolysate formulas. Naïve mice fed hydrolysate formulas for 1 to 4 weeks were later sensitized with alpha-casein. Another group of mice sensitized first with alpha-casein were then fed hydrolysate formulas continually for 12 weeks. RESULTS: Oral tolerance measured by immunoglobulin (Ig)E and IgG1 antibody responses to alpha-casein was induced in naïve mice fed NAN for 1 week or NAN-HA for 4 weeks. IgE responses to alpha-casein were suppressed in mice fed NAN-HA for 1 week or Neoangelac FL for 4 weeks. In contrast, mice fed Alfare, Pepti-Junior, or Pregestimil for 1 to 4 weeks did not develop tolerance to alpha-casein. Antibody responses to alpha-casein were not significantly suppressed in sensitized mice fed NAN or hydrolysate formulas for 12 weeks. CONCLUSIONS: Primary IgE responses to alpha-casein are readily suppressed in naïve mice first fed cow's milk formula or partially hydrolyzed formula for 1 week. Conversely, ongoing IgE, IgG1, and IgG antibody responses to alpha-casein are poorly suppressed in previously sensitized mice even after prolonged feeding of cow's milk formula or hydrolysate formulas.

Animals↗
PubMed1975

[Purification of kappa-caseins from sheep. Analysis of the glycan and peptide components (author's transl)].

Starting from whole individual ovine casein prepared according to the method of Shahani, K. M. & Sommer, H. H. [J. Dairy Sci. 34, 1003-1009 (1951)], kappa-casein was isolated and purified by successive steps of chromatography on columns of dextran gel and hydroxyapatite. On filtration through Sephadex G-150 in a buffer containing urea, the bulk of the kappa-casein behaved as aggregates appearing in the void volume. Dissociation of these aggregates by reductive cleavage of disulfide bonds with 2-mercaptoethanol, followed by a second filtration step on Sephadex G-150 in the presence of both urea and 2-mercaptoethanol, resulted in retardation of the kappa-casein, with separation from a contaminant representing 10-12% of the material applied. Further purification was achieved by chromatography on hydroxyapatite which eliminated the alpha-s- and beta-caseins. The purified kappa-casein had a molecular weight of about 20000, an absorption coefficient (see journal for formula) at 280 nm of 10.85 and a sialic acid and phosphorous content of 0.3% (w/w) each. The sugar fraction liberated on acid hydrolysis of the caseinomacropeptide showed the presence of N-acetylgalactosamine, galactose and neuraminic acid in equimolar ratio. Neuraminic acid existed mainly as the N-glycolyl derivative. The polypeptide chain of the ovine kappa-casein was composed of about 170 amino-acids residues. Compared to bovine kappa-caseins, the most notable difference was the presence of one additional cysteinyl and four additional aspartyl residues. Starch-gel and polyacrylamide-gel electrophoresis clearly revealed the heterogeneity of ovine kappa-casein. Chromatographic fractionation of whole kappa-casein on DEAE-cellulose also led to the separation of several fractions, the main characteristics of which are presented. Analysis of these fractions indicated that only those components which were firmly bound to DEAE-cellulose were glycosylated.

Amino Acids↗
PubMed1990

Characterization in mammalian brain of a DARPP-32 serine kinase identical to casein kinase II.

DARPP-32, a dopamine- and cyclic AMP-regulated phosphoprotein of Mr 32,000, is phosphorylated in vitro by casein kinase II at a site which is also phosphorylated in intact cells. In the present study, we show that a protein kinase activity, present in caudate-putamen cytosol, phosphorylates DARPP-32 on a seryl residue located on the same thermolytic peptide that is phosphorylated by purified casein kinase II. This DARPP-32 serine kinase was indistinguishable from casein kinase II on the basis of a number of biochemical criteria. Excitotoxic lesions of the caudate-putamen and immunocytochemistry revealed the presence of casein kinase II in the medium-sized striatonigral neurons which are known to contain DARPP-32. Casein kinase II activity was high in all rat brain regions studied, and casein kinase II-like immunoreactivity was detected in most brain neurons, although some neuronal populations (e.g., cortical pyramidal cells and large striatal neurons) were stained more intensely than others. In rat caudate-putamen, 45% of the total casein kinase II activity was in the cytosol and 20% in the synaptosomal fraction. In mouse cerebral cortex and caudate-putamen, casein kinase II activity was high at embryonic day 16, and remained elevated during development. In addition to DARPP-32, several major substrates for casein kinase II were observed specifically in brain, but not in liver extracts. The high activity of casein kinase II in brain from the embryonic period to adult age and the existence of a number of specific substrates suggest that this enzyme may play an important role in both developing and mature brain, possibly in modulating the responsiveness of target proteins to various extracellular signals.

Animals↗
PubMed1998

Specificity of the human IgE response to the different purified caseins in allergy to cow's milk proteins.

BACKGROUND: Cow's milk is one of the most frequent food allergens. Whole casein appears to be highly allergenic. It corresponds to an association of four different proteins, alpha(s1)-, alpha(s2)-, beta- and kappa-caseins in approximate proportions of 40, 10, 40, and 10%, respectively. METHODS: These different components were thus purified and used as immobilized antigens in an original enzyme immunoassay to measure specific serum IgE response in a population of 58 children (median age 11 months) allergic to cow's milk who were sensitive to whole casein. RESULTS: A great variability was observed in the affinity and specificity of specific IgE responses in milk-allergic patients' sera. 85% of the patients presented IgE against each of the four caseins. Statistically higher amounts of specific IgE were found to be directed against the most abundant fractions (alpha[s1]- and beta-casein). Co- and/or cross-sensitization to the different caseins were seen in most of the patients sensitive to whole casein. CONCLUSION: These results suggest that both distinct and common epitopes may occur on these different caseins. The major site of phosphorylation which is the most conserved domain in three caseins could be involved in the IgE response to casein and in immunocross-reactivity between these proteins.

Animals↗
PubMed1997

Distribution of casein-like proteins in various organs of rat.

Casein-like proteins were detected in various organs of rat by use of a specific antiserum raised against rat milk caseins. The antiserum specifically recognized alpha 1-, alpha 2-, beta-, and gamma-caseins in rat milk by Western blot analysis, whereas no immunoreactive band was observed in sera of rat and fetal bovine and in bovine caseins. Immunohistochemical studies of this antiserum on formalin-fixed mammary glands showed that immunoreactive caseins were localized to the apical portion of the cytoplasm in lactating mammary epithelial cells and in the luminal secretion, which indicates a directional secretion of caseins to the lumen by the mammary epithelial cells. With this antiserum, immunoreactive substances were detected in various organs, including the pancreatic ducts and islets of Langerhans, the secretory ducts of salivary glands, zona fasciculata cells and ganglion cells of adrenal gland, distal tubules and convoluted collecting tubules of kidney, epithelial cells of bronchioles and large pneumocytes of the lung, hair follicles, sebaceous glands, and the prickle cell layer of skin, uterine glands and epithelium of the endometrium, hepatic bile ducts, and brain. In Western blot analysis, major immunoreactive substances in the above organ extracts showed a similarity in molecular weight to alpha 2-casein of rat milk. Skin was the only tissue that expressed both alpha 2- and beta-caseins. There were no other immunoreactive bands with similarity to beta- and gamma-caseins in the other organ extracts, but higher molecular weight immunoreactive bands (> 100 kD) were detected in some organ extracts, such as salivary gland, kidney, liver, lung, and uterus. These findings suggest that the alpha 2-casein-like substance is localized not only in the mammary gland but also in a variety of organs and may play an important role as a functional molecule in those organs.

Animals↗
PubMed1986

Identification of human milk kappa-casein on polyacrylamide gels by differential staining with Ethyl-Stains-all and chymosin sensitivity.

Ethyl-Stains-all (ESA), a cationic carbocyanine dye that stains phosphorylated, sialylated, and unmodified proteins differentially, was used to stain a human casein fraction enriched for its kappa-casein-like characteristics. The staining properties and chymosin sensitivity of this fraction were compared with those of human milk and bovine casein proteins. Phosphorylated human and bovine beta caseins stained blue with ESA. The sialic acid-containing bovine kappa-casein stained blue-green. The human kappa-like fraction was enriched for a protein that stained blue-green with ESA. Both bovine kappa-casein and the human blue-green-staining protein were susceptible to chymosin digestion at lower concentrations of chymosin than that required for digestion of beta-caseins. In each case, following chymosin digestion, a green-staining peptide of lower molecular weight replaced the original protein and para-kappa-casein was formed. Identification of human kappa-casein on SDS-polyacrylamide gels was based on its differential staining with ESA and chymosin sensitivity with respect to beta-casein.

Animals↗
PubMed2006

Progesterone receptor repression of prolactin/signal transducer and activator of transcription 5-mediated transcription of the beta-casein gene in mammary epithelial cells.

Prolactin (PRL) and glucocorticoids act synergistically to stimulate transcription of the beta-casein milk protein gene. Signal transducer and activator of transcription 5 (Stat5) mediates PRL-dependent trans-activation, and glucocorticoid potentiation occurs through cross talk between glucocorticoid receptor (GR) and Stat5 at the beta-casein promoter. In the mouse, progesterone withdrawal leads to terminal differentiation and secretory activation of the mammary gland at parturition, indicating progesterone's role in repressing milk protein gene expression during pregnancy. To investigate the mechanism of the inhibitory action of progesterone, experiments were performed with cell culture systems reconstituted to express progesterone receptor (PR), the PRL receptor/Stat5 signaling pathway, and GR, enabling evaluation of PR, GR, and Stat5 interactions at the beta-casein promoter. With COS-1, normal murine mammary gland, HC-11, and primary mammary epithelial cells, progestin-PR directly repressed the PRL receptor/Stat5a signaling pathway's mediation of PRL-induced beta-casein transcription. Progestin-PR also inhibited glucocorticoid-GR enhancement of PRL induced trans-activation of beta-casein. Inhibition depended on a functional PR DNA binding domain and specific PR-DNA interactions at the beta-casein promoter. Chromatin immunoprecipitation assays in HC-11 cells revealed recruitment of PR and Stat5a to the beta-casein promoter by progestin or PRL, respectively. Recruitment was disrupted by cotreatment with progestin and PRL, suggesting a mutual interference between activated PR and Stat5a. Without PRL, progestin-PR also recruited Stat5a to the beta-casein promoter, suggesting that recruitment of an unactivated form of Stat5a may contribute to inhibition of beta-casein by progesterone. These results define a negative cross talk between PR and Stat5a/GR that may contribute to the physiological role of progesterone to repress lactogenic hormone induction of the beta-casein gene in the mammary gland during pregnancy.

Animals↗
PubMed1999

Nutrient-specific preferences by lambs conditioned with intraruminal infusions of starch, casein, and water.

We hypothesized that lambs discriminate between postingestive effects of energy and protein and associate those effects with a food's flavor to modify food choices. Based on this hypothesis, we predicted that 1) lambs would acquire a preference for a poorly nutritious food (grape pomace) eaten during intraruminal infusions of energy (starch) or protein (casein) and that 2) shortly after an intraruminal infusion of energy or protein (preload), lambs would decrease their preferences for foods previously conditioned with starch or casein, respectively. Thirty lambs were allotted to three groups and conditioned as follows. On d 1, lambs in each group received grape pomace containing a different flavor and water was infused into their rumens as they ate the pomace. On d 2, the flavors were switched so each group received a new flavor and a suspension of starch (10% of the DE required per day) replaced the water infusion. On d 3, the flavors were switched again, and a suspension of casein (2.7 to 5.4% of the CP required per day) replaced the starch infusion. Conditioning was repeated during four consecutive trials. Lambs in Trial 1 had a basal diet of alfalfa pellets (e.g., free access from 1200 to 1700) and 400 g of rolled barley. Lambs in Trials 2, 3, and 4 received a restricted amount of alfalfa pellets (990 g/d) as their basal diet. After conditioning, all animals received an infusion of water, and, 30 min later, they were offered a choice of the three flavors previously paired with water, starch, or casein. On the ensuing days, the choice was repeated, but starch, casein, and barley replaced the water preload. The nutrient density of the infused preloads was increased during consecutive trials. Lambs preferred the flavors paired with starch > water > casein during Trial 1 (P < .05) and the flavors paired with starch > casein > water during Trials 2 (P < .05), 3 (P < .001), and 4 (P < .001). Preloads of casein decreased preferences for flavors previously paired with casein (P < .10 [Trial 2]; P < .001 [Trial 3], and increased preferences for flavors paired with starch (P < .05 [Trial 2]; P < .001 [Trial 3]). Preloads of energy (barley) had the opposite effect (P < .05 [Trial 3]). These results indicate that lambs discriminated between the postingestive effects of starch and casein and associated the effects with specific external cues (i.e., added flavors) to regulate macronutrient ingestion.

Animal Nutritional Physiological Phenomena↗
PubMed2005

Contributions of terminal peptides to the associative behavior of alphas1-casein.

The N- and C-terminal segments of bovine alphas1-casein-B (f1-23 and f136-196) were characterized under conditions that promoted or inhibited self-association to determine the relative contributions of each fragment to the interaction of alphas1-casein with itself or with other caseins. In earlier studies of f1-23, nuclear magnetic resonance (NMR) data and circular dichroism (CD) spectra showed that its conformation was thermostable between 10 degrees and 25 degrees C. In contrast, NMR studies of f136-196 indicated temperature sensitivity between 10 and 60 degrees C, as did near-UV and far-UV CD data, suggesting a molten globule-like structure at higher temperatures. To compare the effects of temperature on conformational attributes of alphas1-casein and its terminal peptides, additional CD studies were conducted over a broader temperature range (10 to 70 degrees C). The far-UV CD spectra indicated little temperature sensitivity for alphas1-casein, and the N-terminal peptide remained thermostable. During molecular dynamics simulations, the N-terminal peptide conformation did not change significantly, but the conformation of the C-terminal peptide (f136-196) was dramatically altered. These changes are correlated with the thermal instability observed by both CD and NMR in f136-196. Analytical ultracentrifugation studies of the self-association reactions of genetic variants A, B, and C of alphas1-casein showed that at 37 degrees C the associative state is primarily dimeric; the amounts of higher order polymers significantly decreased when temperature was increased from 20 to 37 degrees C. In all 3 genetic variants, the C-terminal portion of the whole molecule showed thermal instability with respect to aggregation to higher polymers, confirming the predictions of CD data and molecular dynamics simulations. The temperature dependency of these conformational changes suggests a possible function for alphas1-casein in facilitating casein-casein interactions in casein micelle formation.

Caseins↗
PubMed2006

Focusing on the goat casein complex.

The analysis of casein polymorphisms in goat species is rather difficult, because of a large number of mutations at each locus, and the tight linkage involving the 4 casein genes. Three goat breeds from Northern Italy, Orobica, Verzasca, and Frisa, were analyzed at the casein complex by milk isoelectrofocusing and analyses at the DNA level to identify the majority of all known polymorphisms. The casein gene structure of the 3 local breeds at alpha(S1)-casein (CSN1S1), beta-casein (CSN2), alpha(S2)-casein (CSN1S2), and kappa-casein (CSN3) was compared with that of Camosciata, a more widely distributed breed. A new allele was identified and characterized at CSN2 gene, which seemed to be specific to the Frisa breed. It was named CSN2*E, and was characterized by a transversion TCT --> TAT responsible for the amino acid exchange Ser(166) --> Tyr(166) in the mature protein. The casein haplotype structure is highly different among breeds. A total of 26 haplotypes showed a frequency higher than 0.01 in at least 1 of the 4 breeds considered, with 12, 3, 5, and 19 haplotypes in Frisa, Orobica, Verzasca, and Camosciata breeds, respectively. Only 13 haplotypes occurred at a frequency higher than 0.05 in at least 1 breed. With the molecular knowledge of each locus, the ancestral haplotype coding for CSN1S1*B, CSN2*A, CSN1S2*A, and CSN3*B protein variants can be postulated. A protein evolutionary model considering the whole casein haplotype is proposed.

Alleles↗
PubMed1984

Heritability of milk casein and genetic and phenotypic correlations with production traits.

Individual milk samples were obtained monthly from November 1979 to November 1981 from approximately 2,800 Holstein cows in 63 herds enrolled in the Quebec Dairy Herd Analysis Service. These milk samples were analyzed for fat, total protein, casein, and serum protein. After editing, the data comprised 2,813 lactations representing 109 sires. By approximate maximum likelihood procedures, the model included herd (absorbed), year-month, and parity fixed effects, and sire and error random effects. Lactation casein yield increased with advancing parity to parity five and then decreased slightly. Lactation casein percent and number (percent casein in protein) declined until parities four and three, respectively, and essentially remained constant thereafter. Lactation casein yield was high for lactations initiated during winter months as opposed to summer months. No seasonal trend was apparent for casein percent or number. Heritabilities for lactation casein yield, percent, and number were .11, .26, and .08. Genetic and phenotypic correlations of casein yield with milk, fat, and protein yields were large and positive. Genetic correlation of casein percent with milk yield was negative (-.76) but positive (.96) with protein percent. Genetic correlations involving casein number tended to be small.

Animals↗
PubMed1992

The binding ability of bovine milk caseins to mutagenic heterocyclic amines.

The binding ability of bovine milk caseins with mutagenic heterocyclic amines was investigated. Binding was determined with 2 mg of casein and 20 micrograms of heterocyclic amine in .40 ml of pH 7.4, 50 mM phosphate buffer, at 37 degrees C, in a shaker for 10 min. The unbound heterocyclic amine in protein-free ultrafiltrate was analyzed by HPLC. The binding ability of whole casein, alpha s-casein, beta-casein, and kappa-casein, respectively, was 90.08, 83.06, 90.92, and 96.70% with 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole; 85.48, 51.54, 63.62, and 82.71% with 3-amino-1-methyl-5H-pyrido[4,3-b]indole; and 87.03, 59.77, 97.04, and 88.30% with 2-amino-6-methyldipyrido[1,2-a:3',2'-d]-imidazole. Higher binding of 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole and 3-amino-1-methyl-5H-pyrido[4,3-b]indole to alpha s-casein, beta-casein, and kappa-casein was observed at pH above 7.4, and the binding was inhibited at pH below 6.5. The maximum binding of 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole to these caseins was at pH 6.6 and 7.4. The binding was inhibited at alkaline pH above 8.5 and acidic pH below 6.5.

Animals↗
PubMed1991

Phosphorylation of neuromodulin (GAP-43) by casein kinase II. Identification of phosphorylation sites and regulation by calmodulin.

Neuromodulin (P-57, GAP-43, B-50, F-1) is a neurospecific calmodulin-binding protein believed to play a role in regulation of neurite outgrowth and neuroplasticity. Neuromodulin is phosphorylated by protein kinase C, and this phosphorylation prevents calmodulin from binding to neuromodulin (Alexander, K. A., Cimler, B. M., Meier, K. E. & Storm, D. R. (1987) J. Biol. Chem. 262, 6108-6113). The only other protein kinase known to phosphorylate neuromodulin is casein kinase II (Pisano, M. R., Hegazy, M. G., Reimann, E. M. & Dokas, L. A. (1988) Biochem. Biophys. Res. Commun. 155, 1207-1212). Phosphoamino acid analyses revealed that casein kinase II modified serine and threonine residues in both native bovine and recombinant mouse neuromodulin. Two serines located in the C-terminal end of neuromodulin, Ser-192 and Ser-193, were identified as the major casein kinase II phosphorylation sites. Thr-88, Thr-89, or Thr-95 were identified as minor casein kinase II phosphorylation sites. Phosphorylation by casein kinase II did not affect the ability of neuromodulin to bind to calmodulin-Sepharose. However, calmodulin did inhibit the phosphorylation of neuromodulin by casein kinase II with a Ki of 1-2 microM. Calmodulin inhibition of casein kinase II phosphorylation was due to calmodulin binding to neuromodulin rather than to the protein kinase. These data suggest that the minimal secondary and tertiary structure exhibited by neuromodulin may be sufficient to juxtapose its calmodulin-binding domain, located at the N-terminal end, with the neuromodulin casein kinase II phosphorylation sites at the C-terminal end of the protein. We propose that calmodulin regulates casein kinase II phosphorylation of neuromodulin by binding to neuromodulin and sterically hindering the interaction of casein kinase II with its phosphorylation sites on neuromodulin.

Amino Acid Sequence↗
PubMed1991

Role of acidic residues as substrate determinants for casein kinase I.

Sites phosphorylated by casein kinase I have been characterized by the presence of acidic amino acids NH2-terminal to the modified residue. Recently, phosphoserine was shown to be a particularly effective determinant for casein kinase I action when present in the motif -S(P)-X-X-S- (Flotow, H., Graves, P. R., Wang, A., Fiol, C. J., Roeske, R. W., and Roach, P. J. (1990) J. Biol. Chem. 265, 14264-14269). Nonetheless, nonphosphorylated substrates for casein kinase I are well documented. In this study, we examined the efficacy of Asp and Glu residues as determinants of casein kinase I action using synthetic peptide substrates. Peptides with runs of Asp residues in the motif Dn-X-X-S- were substrates for casein kinase I. Peptides with n = 3 or 4 were the most effective substrates, much better than n = 2. The peptide with n = 1, a single Asp residue, was a very poor substrate. A block of 4 Glu residues was a little less effective as a substrate determinant than 4 Asp residues in an otherwise identical peptide. The most effective substrate, with the motif -D-D-D-D-X-X-S-, was specific for casein kinase I and was not detectably phosphorylated by cyclic AMP-dependent protein kinase, casein kinase II, glycogen synthase kinase 3, or phosphorylase kinase and thus will be useful for the specific assay of casein kinase I. This peptide was nonetheless significantly worse as a substrate than peptides in which casein kinase I action was determined by phosphoserine in the -3 position. Still, the fact that Asp or Glu residues can specify a casein kinase I substrate suggests that acidic character has a role in substrate selection by this protein kinase.

Amino Acid Sequence↗
PubMed1990

Modulation of casein kinase II activity by the polar head group of an insulin-sensitive glycosyl-phosphatidylinositol.

A phospho-oligosaccharide, whose production is stimulated by insulin, modulated the activity of partially purified casein kinase II. Whereas at 2 microM the phospho-oligosaccharide stimulated casein kinase II 1.3-fold, higher concentrations of this molecule were inhibitory. 50% inhibition of the enzyme was obtained at 15 microM phospho-oligosaccharide. This biphasic effect of the phospho-oligosaccharide on casein kinase II activity was observed using as substrate both casein or the specific peptide for casein kinase II, Arg-Arg-Arg-Glu-Glu-Glu-Thr-Glu-Glu-Glu. The effect of the phospho-oligosaccharide on casein kinase II was still observed after gel filtration. Deamination of the phospho-oligosaccharide with nitrous acid abolished both the activation and the inhibition of casein kinase II. The glycophospholipid precursor of the phospho-oligosaccharide did not affect casein kinase II activity. Moreover, modulation of casein kinase II activity was not observed with other compounds structurally related to the phospho-oligosaccharide, when used in the micro-molar range. In conclusion, the present results indicate that the phospho-oligosaccharide that mimics and might mediate some of the actions of insulin modulates casein kinase II activity in vitro.

Amino Acid Sequence↗
PubMed1985

The absence from the oocyte secretory apparatus of a protein kinase capable of phosphorylating sequestered caseins.

The lactating guinea-pig mammary gland synthesizes and secretes four major milk proteins, i.e., three caseins and alpha-lactalbumin. Of these, the caseins are highly phosphorylated, a post-translational event which in the mammary gland involves a specific casein kinase, which is an integral membrane protein probably of Golgi origin. The microinjection of milk protein mRNA into Xenopus oocytes in the presence of [35S]methionine leads to the synthesis, sequestration, and secretion of proteins which coelectrophorese with alpha-lactalbumin and with partially processed caseins. That the secreted caseins were not phosphorylated was shown by the use of 32P. Either the oocytes were injected with mammary gland mRNA followed by incubation with [32P]phosphate containing media or the mRNA was co-injected with [gamma-32P]ATP and the oocytes were then incubated. In neither case were 32P-labeled caseins secreted. Golgi-rich fractions, identified by the marker enzyme galactosyltransferase, were isolated from the postnuclear supernatant of both oocytes and lactating mammary gland by sucrose density gradient fractionation. In contrast to the mammary gland fractions those derived from the oocytes contained no detectable casein kinase activity. Homogenates of oocytes do effect the phosphorylation of casein but the enzyme activity appears to be present in the soluble fraction and is not membrane bound. It is concluded that the Xenopus oocyte lacks the specific kinase that in the mammary gland phosphorylates sequestered caseins and that the phosphorylation of the caseins is not a prerequisite for their secretion by the oocyte.

Adenosine Triphosphate↗
PubMed1985

Methylation and expression of rat kappa-casein gene in normal and neoplastic rat mammary gland.

The kappa-casein mRNA was evaluated in the rat mammary gland during functional differentiation and neoplastic growth. Using a dot-blot assay, the mRNA was barely detectable in the virgin gland; it steadily increased from the onset of gestation and leveled off during lactation. In rat mammary tumors, either primary (7,12-dimethylbenz(a)anthracene-induced) or transplanted (MTW9), the level of kappa-casein mRNA was about 2.5-fold lower than in the lactating gland, but an extensive variation among individual tumors was observed. There was no detectable kappa-casein mRNA in rat liver. In the mammary gland of virgin, 10-day pregnant, and nonlactating females, the DNA sequences within and/or around the kappa-casein gene were found to be hypermethylated at the HpaII-MspI sites as compared to 10-day lactating females. In the two tumors studied, the kappa-casein gene was partially methylated at the same sites. Prolactin treatment induced kappa-casein gene expression in the virgin rat mammary gland but did not result in a change of the methylation status at the HpaII-MspI sites. Under similar conditions of prolactin treatment, however, the methylation of the Sau96I sites was reduced, and an inverse correlation between the onset of kappa-casein gene methylation and kappa-casein gene expression was evident in both the virgin gland and the tumors. Thus, the expression of kappa-casein was found to be inversely correlated with the extent of methylation of the kappa-casein gene, except in the case of the prolactin-stimulated virgin gland.

Animals↗
PubMed1994

In vitro phosphorylation of caveolin-rich membrane domains: identification of an associated serine kinase activity as a casein kinase II-like enzyme.

Caveolae are flask-shaped micro-invaginations associated with the plasma membrane of a wide variety of cell types. Caveolin, an integral membrane component of caveolae, was first identified as the major phosphoprotein whose phosphorylation was elevated in v-Src transformed cells. As both v-Src transformation and elevated caveolin phosphorylation were dependent on membrane attachment of v-Src, it has been suggested that caveolin is a critical target in v-Src transformation. Although an increase in tyrosine phosphorylation of caveolin was evident, the increase in caveolin phosphorylation was predominantly on serine residues. In accordance with these in vivo observations, isolated caveolin-rich membrane domains undergo phosphorylation in vitro predominantly on serine and contain an unidentified serine kinase activity. Here, we have identified this serine kinase activity as a casein kinase II-like enzyme, since the phosphorylation of caveolin-rich membrane domains is stimulated and inhibited by known effectors of casein kinase II (poly-L-lysine, endogenous polyamines, and a casein kinase II inhibitor peptide), but is unaffected by modulators of other known kinases. In support of these observations, caveolin contains a consensus sequence for casein kinase II phosphorylation in its cytoplasmic N-terminal domain (Ser-88). A peptide containing this sequence inhibits the in vitro phosphorylation of caveolin-rich membrane domains, while many other peptides derived from the N-terminal domain of caveolin do not affect phosphorylation. Caveolin-rich membrane domains were also a substrate for exogenously added purified casein kinase II, but not casein kinase I. Finally, immunoblotting of these domains with an antibody directed against the alpha and alpha' subunits of casein kinase II reveals two bands with apparent molecular weights consistent with the known molecular weights of the alpha and alpha' subunits of casein kinase II. As casein kinase II appears to play a role in mitogenic signalling events and casein kinase II activators (endogenous polyamines) are required for v-Src transformation, our results may have implications for understanding the mechanism of v-Src oncogenesis.

Amino Acid Sequence↗