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PubMed2001

Analysis of casein using two-dimensional electrophoresis, western blot, and computer imaging.

Casein from milk of 20 mothers, 8 weeks postpartum, was analyzed using 2-dimensional electrophoresis. The casein micelle under 2-D denaturing conditions gives approximately 15 major spots. Beta-Casein, kappa-casein, para-kappa-casein, and casein peptides can be tentatively identified by molecular size, isoelectric point, and spot shape characteristics. All spots are not present in all samples. Two spots thought to represent different phosphorylated forms of beta-casein were selected for area and density measurements by means of the NIH-Image program. The larger area at approximately 26 kDa, isoelectric point 5.3, consisted of 1 to 3 closely linked spots, perhaps reflecting phenotype. The smaller spot 24kDa, isoelectric point 5.4, was always a single unit. Visual scanning of gels suggests a wide range of casein concentration. Computer comparison will allow a statistical analysis.

Blotting, Western↗
PubMed1989

Effects of dietary casein and soy protein on metabolism of radiolabelled low density apolipoprotein B in rabbits.

Rabbits fed semipurified diets containing casein have elevated plasma cholesterol levels compared to those fed soy protein. As part of continuing studies on the mechanism of casein-induced hypercholesterolemia, two groups of six rabbits were fed these diets for 14 to 16 weeks. Animals fed the casein diet were found to have significantly higher plasma concentrations of protein, cholesterol, triacylglycerol, phospholipid and apolipoprotein B (apo B) associated with low density lipoprotein (LDL) than those fed the soy protein diet. Kinetic studies showed that the fractional catabolic rate of LDL-apo B was significantly lower in animals fed casein than in those fed soy protein regardless of whether the tracer LDL was obtained from donors fed casein or soy protein. The production rate of LDL-apo B was higher in casein-fed animals but this was not statistically significant. These results show that the efficiency of removal of LDL is significantly reduced in animals fed casein compared to those fed soy protein, and that the source of LDL did not affect the efficiency of its subsequent removal. The accumulation of LDL in casein-fed animals is consistent with down-regulation of the LDL receptor.

Animals↗
PubMed1997

Genomic organization and chromosomal localization of the human casein gene family.

Five yeast artificial chromosome (YAC) clones containing the human casein gene family were isolated and characterized to study the control mechanisms for the expression of these genes. Partial restriction analysis in conjunction with the chromosomal fragmentation method and fluorescence in situ hybridization (FISH) analysis were performed to construct a detailed physical map of the casein gene family and to determine the chromosomal localization of these genes. The isolated YAC clones 748F3, 750D11, 882G11, 886B3 and 960D2 were 1.2 Mb, 860 kb, 800 kb, 1.5 Mb and 1.5 Mb in size, respectively. The clones 748F3, 882G11, 886B3 and 960D2 contained the entire casein gene family, while the kappa-casein gene was absent in 750D11. The human alphaS1-, beta- and kappa-casein genes were found to be closely linked and arranged in the order alphaS1-beta-kappa. The distance between alphaS1 and beta, and between alphaS1 and kappa was approximately 10 and 300 kb, respectively. The beta-casein gene was oriented in the opposite direction to the alphaS1- and kappa-casein genes. The casein gene family was localized to chromosome 4q21.1 by FISH analysis.

Caseins↗
PubMed1987

Water interactions with varying molecular states of bovine casein: 2H NMR relaxation studies.

The caseins occur in milk as spherical colloidal complexes of protein and salts with an average diameter of 1200 A, the casein micelles. Removal of Ca2+ is thought to result in their dissociation into smaller protein complexes stabilized by hydrophobic interactions and called submicelles. Whether these submicelles actually occur within the micelles as discrete particles interconnected by calcium phosphate salt bridges has been the subject of much controversy. A variety of physical measurements have shown that casein micelles contain an inordinately high amount of trapped water (2 to 7 g H2O/g protein). With this in mind it was of interest to determine if NMR relaxation measurements could detect the presence of this trapped water within the micelles, and to evaluate whether it is a continuum with picosecond correlation times or is associated in part with discrete submicellar structures with nanosecond motions. For this purpose the variations in 2H NMR longitudinal and transverse relaxation rates of water with protein concentration were determined for bovine casein at various temperatures, under both submicellar and micellar conditions. D2O was used instead of H2O to eliminate cross-relaxation effects. From the protein concentration dependence of the relaxation rates, the second virial coefficient of the protein was obtained by nonlinear regression analysis. Using either an isotropic tumbling or an intermediate asymmetry model, degrees of hydration, v, and correlation times, tau c, were calculated for the caseins; from the latter parameter the Stokes radius, r, was obtained. Next, estimates of molecular weights were obtained from r and the partial specific volume. Values were in the range of those published from other methodologies for the submicelles. Temperature dependences of the hydration and Stokes radius of the casein submicelles were consistent with the hypothesis that hydrophobic interactions represent the predominant forces responsible for the aggregation leading to a submicellar structure. The same temperature dependence of r and v was found for casein under micellar conditions; here, the absolute values of both the Stokes radii and hydrations were significantly greater than those obtained under submicellar conditions, even though tau c values corresponding to the great size of the entire micelle would result in relaxation rates too fast to be observed by these NMR measurements. The existence of a substantial amount of trapped water within the casein micelle is, therefore, corroborated, and the concept that this water is in part associated with submicelles of nanosecond motion is supported by the results of this study.

Caseins↗
PubMed1991

Purification and characterization of polyamine-stimulated protein kinase (casein kinase II) from bovine spermatozoa.

Casein kinase II from bovine epididymal spermatozoa was purified to apparent homogeneity by repeated chromatography with phosphocellulose and gel filtration with sephacryl S-200. The purified enzyme exhibited a molecular mass of 130 kDa by gel filtration and displayed three polypeptide bands with molecular masses of 26, 33, and 36 kDa by SDS-polyacrylamide gel electrophoresis. Antibodies raised against calf thymus casein kinase II cross reacted with the three sperm polypeptides. Incubation of the holoenzyme with either [gamma-32P]ATP or [gamma-32P]GTP resulted in the phosphorylation of the 26-kDa subunit. The enzymatic activity with casein as substrate was strongly inhibited by nanomolar heparin and greatly stimulated by micromolar spermine. With casein as substrate, the specific activity of the pure enzyme (0.5 mumol/min/mg protein) was comparable to that of casein kinase II from other sources. Endogenous substrates of the kinase were demonstrated by incubating sperm cytosolic extracts with [gamma-32P]GTP, under conditions that limit the expression of other protein kinases, and analyzing the products by SDS-PAGE and autoradiography. Similar results were obtained when sperm extracts, suitably diluted to minimize endogenous casein kinase II, were incubated with [gamma-32P]GTP and aliquots of pure sperm casein kinase II. Low concentrations (50 microM) spermine strongly enhanced the phosphorylation of 92- and 106-kDa cytosolic proteins. Our results clearly show that casein kinase II is present in spermatozoa and that it shares many of the properties of the enzyme from other sources. Further, they indicate that the enzyme plays a role in mediating the phosphorylation state of sperm proteins.

Animals↗
PubMed1976

Removal of phosphate groups from casein with potato acid phosphatase.

Potato acid phosphatase (EC 3.1.3.2) was used to remove the eight phosphate groups from alphas1-casein. Unlike most acid phosphatases, which are active at pH 6.0 or below, potato acid phosphatase can catalyze the dephosphorylation of alphas1-casein at pH 7.0. Although phosphate inhibition is considerable (K1=0.42 mM phosphate), the phosphate ions produced by the dephosphorylation of casein can be removed by dialysis, allowing the reaction to go to completion. The dephosphorylated alphas1-casein is homogeneous on gel electrophoresis with a slower mobility than native alphas1-casein and has an amino acid composition which is identical to native alphas1-casein. Thus the removal of phosphate groups from casein does not alter its primary structure. Potato acid phosphatase also removed the phosphate groups from other phosphoproteins, such as beta-casein, riboflavin binding protein, pepsinogen, ovalbumin, and phosvitin.

Acid Phosphatase↗
PubMed1989

Turnover of 125I-VLDL and 131I-LDL apolipoprotein B in rabbits fed diets containing casein or soy protein.

Rabbits fed low-fat, cholesterol-free, semi-purified diets containing casein developed a marked hypercholesterolemia compared to rabbits fed a similar diet containing soy protein (plasma cholesterol 281 +/- 31 vs. 86 +/- 9 mg/dl; P less than 0.05). Turnover studies (three per dietary group) were carried out in which homologous 125I-labeled VLDL and 131I-labeled LDL were injected simultaneously into casein- (n = 8) or soy protein- (n = 9) fed rabbits. ApoB-specific activities were determined in VLDL, IDL and LDL isolated from the pooled plasma of two or three rabbits per dietary group. The production rate of VLDL apoB (1.20 +/- 0.3 vs. 1.09 +/- 0.1 mg/h per kg) was similar for the two dietary groups. The fractional catabolic rate of VLDL apoB was lower for the casein group (0.15 +/- 0.03 vs. 0.23 +/- 0.01.h-1; 0.05 less than P less than 0.10). Although the pool size of VLDL apoB was higher in the casein group (8 +/- 2 vs. 5 +/- 0.3 mg/kg), this value did not reach statistical significance. For LDL apoB, the increased pool size in casein-fed rabbits (30 +/- 5 vs. 5 +/- 1 mg/kg; P less than 0.01) was associated with a decreased fractional catabolic rate (0.03 +/- 0.005 vs. 0.08 +/- 0.008.h-1; P less than 0.01) and a 2-fold increase in the production rate of LDL apoB (1 +/- 0.3 vs. 0.4 +/- 0.06 mg/kg per h; 0.05 less than P less than 0.10) compared to rabbits fed soy protein. Analysis of precursor-product relationships between the various lipoprotein fractions showed that casein-fed rabbits synthesized a higher proportion of LDL apoB (95% +/- 2 vs. 67% +/- 2; P less than 0.001) independent of VLDL catabolism. These results support the concept that the hypercholesterolemia in casein-fed rabbits is associated with impaired LDL removal consistent with a down-regulation of LDL receptors. These changes do not occur when the casein is replaced by soy protein.

Animals↗
PubMed1988

Increase in plasma ammonia and amino acids when rats are fed a 44% casein diet.

Rats were trained to eat a 6% casein basal diet during a 3-hour period per day. They were then fed either the same 6% casein diet or a 44% casein diet for 3 hours. No food intake depression was observed in the rats eating 44% casein diet during the 3-hour period. Plasma ammonia and amino acids and brain amino acids were measured at 0, 4, 12 and 24 hours after presentation of the 6% or 44% casein diets. Plasma ammonia rose to 134 (p less than 0.01) and 110 micromolar (p less than 0.05) in the 44% casein fed rats at 4 and 12 hours, respectively, as compared to 67 and 53 micromolar, respectively, for the 6% casein fed rats. All plasma amino acid concentrations except methionine and glutamate were elevated (p less than 0.05) at 4 hours. In the brain, threonine, glutamine and tyrosine concentrations were elevated (p less than 0.05) at 4 hours after diet presentation. At 24 hours, valine, isoleucine, leucine, phenylalanine, and methionine concentrations were also elevated (p less than 0.05). Because intake of the 44% casein diet decreases the second day of its presentation, as noted in an earlier experiment, the increases in plasma ammonia and its possible entry into the brain as reflected by increased brain glutamine together with changes in amino acid concentrations should be considered collectively among possible metabolic signals affecting intake of high protein diets.

Amino Acids↗
PubMed1986

Orally induced tolerance to casein in mice on normal mouse chow.

We examined the ability of bovine casein to induce oral tolerance in BDF1 mice raised on a normal mouse chow diet. Giving 20 mg casein 1 X or 4 X by intragastric tube or for 28 days at 1 mg/ml in drinking water failed to have a significant effect (P greater than 0.2) on the subsequent immune response to parenteral casein, suggesting that casein was not an effective oral tolerogen. However, we discovered that most normal mouse chow contains casein. When BDF1 mice were raised on a casein-free diet, and treated as above, they showed marked suppression of subsequent responses to parenteral casein (P less than 0.01). Our results indicated that mice on the normal diet were presuppressed by the dietary exposure. Examination of several other normal mouse chows revealed that they all contained casein as a protein source. A possible effect of prior exposure must be considered in all experimental animal studies involving immune responses to antigens which may be present in the environment.

Administration, Oral↗
PubMed1988

Arachidonic acid metabolism and casein secretion in lactating rabbit mammary epithelial cells: effects of inhibitors of prostaglandins and leukotrienes synthesis.

The metabolism of arachidonic acid (AA) in fragments of lactating rabbit mammary glands in vitro was studied by considering the distribution of 13-[14C]AA in the cells, and the effects of inhibitors of cyclooxygenase and lipoxygenase pathway on the basal and prolactin (PRL)-stimulated casein secretion. 13-[14C]AA was incorporated in all classes of lipids and PRL increased transiently the percentage of free fatty acid after 1 and 5 min. Ten microM ETYA (5,8,11,14-Eicosatetraynoic acid), a tetrayne analogue of AA inhibited prostaglandins F2 alpha (PGF2 alpha) production but not leukotrienes B4 and C4 (LTB4 and LTC4) production and increased basal casein secretion. 10(-4) M DCHA (Docosahexaenoic acid) a competitive inhibitor of prostaglandin-synthetase inhibited PGF2 alpha production but did not affect basal nor PRL-stimulated casein secretion. Fourteen microM indomethacin inhibited PGF2 alpha and LTC4 production and PRL-stimulated casein secretion. Ten microM NdgA (nordihydroguaiaretic acid) an inhibitor of lipoxygenase pathway, inhibited LTB4 and LTC4 production, increased basal level of casein secretion and inhibited PRL-stimulated casein secretion. Hundred microM caffeic acid, an inhibitor of glutathione-S-transferase (GST), a class of enzymes implied in the transformation of LTA4 into LTC4, had the same effect that NDGA on basal and PRL-stimulated casein secretion. These findings show that inhibitors of AA metabolites alter casein secretion.

5,8,11,14-Eicosatetraynoic Acid↗
PubMed1979

Prolactin-mediated transcriptional and post-transcriptional control of casein gene expression.

The mechanism by which prolactin, a peptide hormone, regulates casein gene expression has been studied in mammary gland organ culture. After prolactin addition, a 2-4 fold increase in the rate of casein mRNA transcription was observed within 1 hr and maintained for at least 24 hr. This increased rate of transcription is not sufficient to account for the mass accumulation of casein mRNA. The half-life of casein mRNA is also increased 17-25 fold in the presence of prolactin. This change in casein mRNA half-life, coupled with a 2-4 fold increase in the rate of transcription, can account for the normal accumulation of casein mRNA observed after prolactin addition. This hormone-induced change in casein mRNA half-life appeared to be selective, since prolactin was found to exert only a slight effect (1-4 fold) on the half-life of poly(A) RNA determined under identical pulse-chase conditions. The hormonal regulation of casein gene expression thus does not app-ar to be an "all or none" process occurring only at the transcriptional or post-transcriptional levels, but rather may involve a coordinated response at several levels to permit the efficient expression of specialized differentiated functions.

Animals↗
PubMed1996

Effect of dietary casein levels on activation of promutagens in the spiral Salmonella mutagenicity assay. I. Studies with noninduced rat liver S9.

Xenobiotic metabolism can be influenced by various nutritional factors, including protein. In the present study, we have examined the effect of dietary protein (casein) levels on the ability of rat liver S9 to activate the promutagens aflatoxin B1 (AFB), 2-aminoanthracene (2AN) and benzo[a]pyrene (BAP) in strain TA98 using the spiral Salmonella mutagenicity assay. S9s were derived from individual male F344 rats fed for 6 weeks on semisynthetic diets containing 8%, 12% or 22% methionine-supplemented casein as the sole source of protein (diets were made isocaloric by adjusting the corn starch content). Rats were housed in large, raised-bed cages by groups of three per diet. S9 activation mixtures were prepared at 5 mg of S9 protein/ml of S9 mix. Slopes from the linear portions of the mutagenicity dose-response curves were analyzed by ANOVA comparisons. Assays used to elucidate the phase I activities of microsomal preparations were cytochrome P450 content, cytochrome-c reductase activity, flavin-containing monooxygenase activity, 7-ethoxyresorufin O-deethylation (EROD) activity, N-demethylation of benzphetamine and para-nitrophenol O-deethylation. Phase II activities in cytosolic preparations were assayed by estimation of glutathione (GSH) content and glutathione S-transferase activity through metabolism of 1-chloro-2,4-dinitrobenzene (CDNB). Increased levels of dietary casein increased liver wet weights and decreased the ability of the S9 to activate 2AN. Dietary casein levels did not influence the S9-mediated activation of BAP; and consistent but nonsignificant increases in activation of AFB were produced by S9 from animals fed the 22% casein diet. The phase I and phase II activities measured here were not altered significantly by dietary casein levels; thus, other, more specific enzymatic activities may account for the mutagenesis data. These results illustrate the complex interaction between dietary levels of casein and promutagen activation mechanisms, which prevents drawing broad generalizations regarding the influence of dietary casein levels on the capacity of hepatic S9s to activate promutagens.

Aflatoxin B1↗
PubMed1983

Phosphorylation of K-casein by glycogen synthase kinase-3 from rabbit skeletal muscle.

Glycogen synthase kinase-3 (ATP:protein phosphotransferase, EC 2.7.1.37) phosphorylated K-casein 20-fold more rapidly than beta-casein, while alpha S1-casein was not a substrate. This distinguished it from casein kinase-I and casein kinase-II, which phosphorylate the beta-casein variant preferentially. Glycogen synthase kinase-3 phosphorylated a serine residue(s) in the C-terminal cyanogen bromide fragment on K-casein. In contrast, cyclic AMP-dependent protein kinase phosphorylated the N-terminal fragment, and phosphorylase kinase the N-terminal and intermediate cyanogen bromide fragments. The results emphasize the potential value of casein phosphorylation as a means of classifying protein kinases.

Animals↗
PubMed1994

The plasminogen activation system in bovine milk: differential localization of tissue-type plasminogen activator and urokinase in milk fractions is caused by binding to casein and urokinase receptor.

We have analyzed the occurrence of components of the plasminogen activation system in bovine milk. Zymographic analyses showed that tissue-type plasminogen activator (t-PA) occurred in association with casein micelles, partially as a complex with type-1 plasminogen activator inhibitor (PAI-1), whereas urokinase-type plasminogen activator (u-PA) was confined to milk leukocytes. Whey contained a component with a plasminogen dependent proteolytic activity which was shown to be plasma prekallikrein (PPK). The u-PA in the milk leukocytes was shown to be bound to urokinase receptor (u-PAR). A purification to near-homogeneity of the bovine u-PAR was undertaken. Investigating the novel t-PA binding to casein micelles by ligand blotting and Sepharose immobilized casein, multimeric forms of kappa-casein and dimeric alpha s2-casein were identified as t-PA binding components. The kappa-casein gene and the fibrinogen gene are believed to have evolved from a common ancestor. Thus, the recent finding that casein enhances t-PA catalyzed plasminogen activation (Marcus, G., Hitt, S., Harvey, S.R. and Tritsch, G.L. (1993) Fibrinolysis 7, 229-236), and the observed t-PA/casein binding suggests that the casein micelle, which also contains plasminogen, may serve as a matrix for t-PA-catalyzed plasminogen activation in milk.

Animals↗
PubMed1992

A distal region enhances the prolactin induced promoter activity of the rabbit alpha s1-casein gene.

Casein gene expression is induced in the rabbit mammary gland by prolactin (PRL). alpha s1-casein is the major casein secreted into milk. In order to define the position of the DNA sequences involved in the control of rabbit alpha s1-casein gene regulation by PRL, chimeric genes were constructed between upstream regions of the rabbit alpha s1-casein gene and the chloramphenicol acetyl transferase (CAT) reporter gene. A series of 5'-deleted fusion genes was obtained by nuclease digestion of the alpha s1-casein gene upstream region. These gene constructs were transfected into rabbit primary mammary cells, or cotransfected in CHO cells with the plasmid coding for the rabbit mammary receptor (PRL-R). A regulatory region has been located between nt -3768 and -3155. This region enhances the prolactin induced promoter activity of the alpha s1-casein gene. It might possess or cooperate with prolactin responsive elements located further downstream in the alpha s1-casein gene.

Animals↗
PubMed1980

Composition and size distribution of bovine casein micelles.

Chromatography of glutaraldehyde-fixed skim-milk on controlled-pore glass (CPG-10, 300 nm) gave three micellar fractions whose averaged diameters, measured by electron microscopy, decreased progressively with increasing elution volume. Casein micelles with diameters up to 680 nm were detected. The casein composition of the same fractions from unfixed skim-milk was determined. As the fraction elution volume increased, kappa-casein varied from 7.7 to 11.4% of total casein, giving alpha s/kappa ratios of 6.1, 4.7 and 3.3. A plot of kappa-casein content versus micelle surface-to-volume ratio for skim-milk and the column fractions approximated to a straight line. Re-calculation of the published results from two other studies also gave linear relationships between kappa-casein content and surface area for artificial micelles. The three regression lines thus obtained had small intercepts. It was concluded that the data indicated the same fundamental structure for casein micelles, with a predominant surface location for kappa-casein, whether the micelles are natural or artificial and whether they are aggregated by Ca2+ alone or by Ca2+ together with calcium phosphate-citrate complex.

Animals↗
PubMed1991

Cortisol induces rapid accumulation of whey acid protein mRNA but not of asl and b-casein mRNA in rabbit mammary explants.

Mammary explants from rabbit were cultured in the presence of various combinations of insulin, cortisol and prolactin. The concentration of whey acidic protein (WAP) asl-casein and b-casein mRNA was measured using specific cDNA probes. Medium alone was added to the explants for one day. Prolactin with and without cortisol was then added to the medium. Prolactin alone induced rapidly asl-and b-casein gene but not WAP gene. When cortisol was added with prolactin, the asl- and b-casein genes were induced at the same rate as in the absence of the steroid. In contrast, the WAP mRNA was then rapidly accumulated. This induction process was not altered by cycloheximide for two hours and it was blocked at a later stage. In a second experiment, insulin and prolactin were first added for 24 hours in the culture medium. Cortisol was then added and the concentration of the three mRNA was measured. Cortisol did not significantly modify the level of asl- and b-casein mRNA. On the contrary, the WAP mRNA was rapidly accumulated. These data indicate that the well-established amplificatory effect of glucocorticoids on casein gene expression is a slow process whereas their effect on the WAP gene is rapid. This suggests that glucocorticoids induce casein gene expression through an indirect cellular mechanism not involving a glucocorticoid receptor element in casein gene promoters and that WAP gene is more classically stimulated through the direct binding of the steroid receptor to a glucocorticoid receptor element located in its promoter.

Actins↗
PubMed2003

Association behavior of beta-casein.

The association behavior of beta-casein, a protein with a distinct amphipathic character, was studied. beta-Casein exhibits markedly temperature-dependent association behavior; at low temperatures (<10-15 degrees C), monomers predominate, but as the temperature is increased, monomers associate, via hydrophobic bonding, into micelles. beta-Casein micelles have a hydrodynamic radius of approximately 12 nm, a radius of gyration of approximately 8.3 nm, and an interaction radius of approximately 15 nm. These data are fully consistent with a previous fluffy particle. The association behavior of beta-casein is also strongly affected by concentration and solvent quality. At low concentrations beta-casein exhibits a critical micelle concentration (CMC) of approximately 0.05%, w/v, at 40 degrees C. In the presence of 6 M urea the temperature dependence of beta-casein's association behavior is eliminated, leaving monomers predominantly. Temperature-dependent transformations in micelle morphology can be explained by changes in solvent quality, i.e., the temperature-protein hydrophobicity and temperature-voluminosity profiles of beta-casein. The results obtained are consistent with the shell model as developed by Kegeles, in which a distribution of micelle sizes is formed. They contrast with the traditional description of the micellization of beta-casein by a two-state model or by the closed-association model, i.e., monomers if micelles.

Caseins↗