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PubMed1994

Reconstitution of heterologous and chimeric casein kinase II with recombinant subunits from human and Drosophila: identification of species-specific differences in the beta subunit.

Casein kinase II is composed of two catalytic (alpha) and two regulatory (beta) subunits, the amino acid sequences of the alpha and beta subunits are highly conserved between species. To examine whether heterologous casein kinase II could be formed, recombinant alpha and beta subunits from human and Drosophila were reconstituted from inclusion bodies. Casein kinase II containing either human alpha and Drosophila beta or Drosophila alpha and human beta subunits exhibited enzymatic properties similar to those of the homologous holoenzymes with regard to specific activity, salt optima, and autophosphorylation. However, renaturation and reconstitution of casein kinase II was dependent on the type of beta subunits and the redox conditions, with the Drosophila beta subunits requiring more reduced conditions. Chimeric beta subunits prepared from human and Drosophila cDNA revealed that the N-terminal region was responsible for the requirement for the reduced redox state during renaturation. The N-terminal region also affected solubility and electrophoretic mobility of the beta subunit.

Amino Acid Sequence↗
PubMed1996

An efficient expression of human growth hormone (hGH) in the milk of transgenic mice using rat beta-casein/hGH fusion genes.

In order to produce human growth hormone (hGH) in the milk of transgenic mice, two expression vectors for hGH differing in their 3' flanking sequences were constructed by placing the genomic sequences of hGH gene under the control of the rat beta-casein gene promotor. The 3' flanking sequences of the expression constructs were derived from either the hGH gene (pBCN1GH) or the rat beta-casein gene (pBCN2GH). Transgenic lines bearing pBCN1GH expressed hGH more efficiently than those bearing pBCN2GH in the milk (19-5500 micrograms/mL vs 0.7-2 micrograms/mL). In particular, one of the BCN1GH lines expressed hGH as much as 5500 +/- 620 micrograms/mL. Northern blot analysis showed that the transgene expression was specifically confined to the mammary gland and developmentally regulated like the endogenous mouse beta-casein gene in the mammary gland. However, a low level of nonmammary expression was also detected with more sensitive assay methods. In conclusion, the rat beta-casein/hGH fusion gene could direct an efficient production of hGH in a highly tissue-and stage-specific manner in the transgenic mice and the 3' flanking sequences of hGH gene had an important role for the efficient expression.

Animals↗
PubMed2000

Influence of heat treatment of casein in presence of reducing sugars on Zn solubility and Zn uptake by Caco-2 cells after in vitro digestion.

The effect of the heat treatment of casein in presence of reducing sugars on some aspects of Zn availability was investigated. Samples were prepared by mixing casein with glucose-fructose, and were used unprocessed (C) or heated (HC). Changes in Zn speciation after the in vitro digestion of the samples, both as part of a diet and in isolation, were studied. The uptake of soluble Zn from the digested samples was investigated in Caco-2 cells. After in vitro digestion, the percentage of precipitated Zn was significantly higher with the HC sample, both when digested alone and as a part of the diet. In assays with Caco-2 cells, a significant decrease in Zn uptake was observed when the uptake buffer contained the sample C digest, by comparison with the control buffer, without casein digest. When the digested heated mixture was added, Zn uptake by the cells was significantly lower than in either of the two other cases. It may be concluded that the heat treatment of casein in the presence of glucose-fructose has a negative effect on Zn availability because, after in vitro digestion, Zn insolubilization was enhanced and Zn uptake by the enterocyte was impaired, compared with the unheated mixture. In addition, the usefulness of Caco-2 cells in this kind of research has been shown.

Caco-2 Cells↗
PubMed2004

An equilibrium thermodynamic model of the sequestration of calcium phosphate by casein phosphopeptides.

Sequestration of calcium phosphate by caseins occurs in the Golgi region of mammary secretory cells during lactation, where it helps to prevent calcification of the gland and to deliver high concentrations of calcium and phosphate to the neonate in the form of milk. Calcium phosphate nanoclusters are formed when a core of amorphous calcium phosphate is sequestered within a shell of casein or casein phosphopeptides. The nanoclusters can form spontaneously from a supersaturated solution or by dispersion of a precipitate of calcium phosphate, demonstrating that they are thermodynamically stable complexes. The average size and chemical composition of the complexes are largely independent of the solution conditions (pH, temperature, peptide concentration, salt composition and rate of reaction) under which they form. Larger, metastable, colloidal particles can form if there is not enough of the phosphopeptide to sequester all the calcium phosphate, or, transiently, if the salt and peptide solutions are mixed together without sufficient care. A thermodynamic model of the sequestration process is presented which makes use of an invariant ion activity product observed in nanocluster-containing solutions. In any given solution that has thermodynamic stability, the extent of the sequestration reaction can be calculated from the empirical formula of the nanoclusters using the criterion that the solution should have the equilibrium value of the invariant ion activity product. Other members of the paralogous group of secretory calcium-binding phosphoproteins to which caseins belong may also be able to sequester calcium phosphate in biological fluids such as saliva and in the extracellular matrix of mineralizing tissues.

Animals↗
PubMed2004

An equilibrium thermodynamic model of the sequestration of calcium phosphate by casein micelles and its application to the calculation of the partition of salts in milk.

An equilibrium thermodynamic model of the interaction of calcium, phosphate and casein in milk is described in which the micellar calcium phosphate is assumed to be in the form of calcium phosphate nanoclusters. A generalized empirical formula for the nanocluster is used to define the molar ratios of small ions (Ca, Mg, P(i) and citrate) to a casein phosphorylated sequence (phosphate centre, PC). From this model, a method of calculating the partition of milk salts into diffusible and non-diffusible fractions is obtained. No arbitrary assumptions are made, no fitting of adjustable parameters is done and the PCs in the caseins are defined by inspection of their primary structures. In addition to the salt partition, the mole fractions of the individual caseins not complexed to the calcium phosphate through one or more of their PCs are computed and a generic stability rule for milks is derived. The use of the model is illustrated by calculations of the partition of salts in a standard milk and by comparison with experimental data on the partition of salts in the milk of individual cows. The generic stability rule is applied to the individual milks to determine whether the micellar calcium phosphate is thermodynamically stable. According to the calculations, compositions that might lead to pathological calcification in the lumen of the mammary gland were seldom found in primiparous healthy cows in early or mid lactation but occurred more often in multiparous animals, in late lactation and during mastitic infection.

Animals↗
PubMed2002

Intraluminal casein model of necrotizing enterocolitis for assessment of mucosal destruction, bacterial translocation, and the effects of allopurinol and N-acetylcysteine.

An intraluminal casein model (ICM) of necrotizing enterocolitis (NEC) is able to produce small-bowel changes reminiscent of human NEC in neonatal animals. We studied bacterial translocation (BT) in NEC induced by using the ICM in neonatal piglets. We also studied whether allopurinol (AL) and N-acetylcysteine (NAC) have an effect on BT and mucosal changes in the ICM of NEC. Twenty-eight neonatal piglets were randomized into four groups. NEC was induced in 21 by injecting casein-d-gluconate into a loop of terminal ileum: group Cas (n = 7) had no premedication, in group Cas/AL (n = 7) intravenous (i.v.) Al (100 mg/kg), and in group Cas/NAC (n = 7) i.v. NAC (200 mg/kg) was given. Group Sham (n = 7) had the ileum injected with 0.9% saline with no premedication. Immediately after the injection a mesenteric lymph node (MLN) adjacent to the loop was harvested for quantitative aerobic bacterial culture; 4 h after the injection another MLN and samples of spleen, liver, kidney, and lung were harvested and cultured. Comparison of the incidence of samples with positive bacterial cultures and the number of colony-forming units (CFU) in samples was made between groups. The severity of NEC in the ileum was graded from 0 to 3 according to macroscopic and histologic findings. NEC changes in the bowel were most severe in Cas piglets, less severe in Cas/NAC piglets ( P < 0.5), and sham piglets had the least severe changes ( P < 0.05). piglets with NEC changes in the ileum had a higher incidence of BT into the MLN than piglets without NEC changes ( P < 0.05), but the difference in CFU was not significant ( P > 0.05). In Cas and Cas/NAC piglets a high incidence of BT into the MLN was noted as early at -5 min after casein injection. The incidence of BT into the MLN was significantly higher in Cas and Cas/NAC piglets than in Sham piglets ( P < 0.05), the difference in CFU being not significant ( P > 0.05). BT in Cas/Al piglets was not significantly different from that of Cas piglets ( P > 0.05), but less than in Cas/NAC piglets ( P < 0.05). Four hours after casein injection into the ileum there was significant BT into the MLN. Premedication with NAC was associated with less severe NEC changes, but neither NAC nor AL significantly affected BT.

Acetylcysteine↗
PubMed1984

Interaction of polyamines and magnesium with casein kinase II.

In reticulocytes, polyamines appear to be physiologically relevant activators of casein kinase II [Hathaway, G. M. and Traugh, J. A. (1984). J. Biol. Chem. 259, 7011-7015]. The mechanism by which polyamines and Mg2+ interact to activate casein kinase II has been investigated. These studies were conducted by holding ionic strength constant at 0.10 M. At low Mg2+ (2.5 mM), activation by spermine resulted in a 33% decrease in the apparent Km for casein. Under these conditions, a 2.3-fold increase in the maximum velocity of the reaction was observed, and half-maximal stimulation was obtained with 275 microM spermine. At a kinetically optimal Mg2+ concentration of 12.5 mM, the effects of spermine on Km and Vmax were reduced, and the concentration of spermine required to give 50% of maximal stimulation was increased to 750 microM. Kinetic data obtained at the two Mg2+ concentrations indicated that Mg2+ and spermine competed for the same form of the enzyme. Double-reciprocal plots of velocity versus Mg2+ concentration showed downward curvature at Mg2+ concentrations higher than 1 mM, and these results were interpreted as evidence for two binding sites on the enzyme with an apparent Km of 0.5 and 2.5 mM. Experiments carried out with ATP-Mg2+ in the absence of excess MgCl2 gave results consistent with an absolute requirement of the enzyme for the metal ion which could not be replaced by spermine. These results are consistent with the formation of an enzyme-activator complex. A model is proposed where spermine activates casein kinase II at one site on the enzyme at which MgCl2 can also bind, while a second, high-affinity site exists exclusively for the metal ion.

Adenosine Triphosphate↗
PubMed1988

Polyamines stimulate the activity of glycogen synthase (casein) kinase-1 from bovine kidney and different rat tissues.

Previous studies have established that casein kinase-2 (CK-2) is stimulated by polyamines. In this study it is shown that glycogen synthase (casein) kinase-1 (CK-1) can be activated similarly. Using casein as the substrate, bovine kidney CK-1 was stimulated 7-, 2-, and 0.5-fold by spermine, spermidine, and putrescine, respectively. Half-maximal activation of CK-1 by these polyamines was observed at 0.25, 0.70, and 0.50 mM, respectively. CK-1 was optimally activated by spermine at low ionic strength and low Mg2+ concentrations (1-3 mM). Using phosvitin as the substrate, CK-1 was stimulated at low concentrations (0-0.8 mM) and inhibited at higher concentrations of spermine. By contrast CK-2 was inhibited at all concentrations of spermine when phosvitin was used as substrate. Using calcineurin (not a substrate for CK-2) as a substrate, CK-1 from bovine kidney or from three rat tissues (liver, kidney, and testis) was stimulated greater than 2-fold by spermine. It is further shown that heparin inhibits CK-1 and this inhibition can be reversed by spermine. The Vmax of CK-1 for both casein and ATP is increased by spermine while the Km remains unchanged by the polyamine. These studies indicate that CK-1, like CK-2, is a heparin-inhibited and polyamine-activated protein kinase. The results also suggest that CK-1 may be activated by spermine in vivo.

Animals↗
PubMed1988

Calcium-induced associations of the caseins: a thermodynamic linkage approach to precipitation and resolubilization.

Calcium-induced changes in protein solubility play a role in a variety of important biological processes including the deposition of bone and dentin and the secretion of milk. The phenomena of salt-induced (calcium) precipitation of proteins (salting-out), and the resolubilization of these proteins at higher salt concentrations (salting-in) have been studied and quantitated using an approach based on the concepts of Wyman's thermodynamic linkage. Salting-out has been described by a salt-binding constant, k1, the number of moles of salt bound per mole of protein, n, and S1, the fraction soluble at saturation of n; salting-in has been described by corresponding constants k2, m, and S2. Analysis of salt-induced solubility profiles was performed using nonlinear regression analysis. Results of calcium-induced solubility profiles of two genetic variants of alpha s1-casein (alpha s1-A), (alpha s1-B), and beta-casein C (beta-C) at 37 degrees C, where hydrophobic interactions are maximized, showed no salting-in behavior and for salting-out, yielded k1 values of 157, 186, and 156 liters.mol-1 and n values of 8, 8, and 4, respectively. The values of k1 can be correlated with the apparent association constant for calcium binding to casein, while the values of n can be correlated with the number of calcium binding sites of the respective caseins. At 1 degree C, where hydrophobic interactions are minimized, nominally only hydrophilic and electrostatic interactions can be linked to the salt-induced solubility profiles; here beta-C is totally soluble at all calcium concentrations and alpha s1-B and alpha s1-A were now found to have salting-in parameters, k2 and m, of 2.5 liters.mol-1 and 4, and 11 liters.mol-1 and 8, respectively. alpha s1-A is more readily salted-in and studies on the variation of S1 with added KCl for this protein at 1 degree C indicated that salting-in is also mainly electrostatic in nature and may result from competition between K+ and Ca2+ for binding sites rather than from solute-solvent interactions as previously proposed. Comparison of k1 and k2 values between the two genetic variants, coupled with the known sequence differences (the A variant is a linear deletion of 13 amino acids) suggest the existence of a hydrophobically stabilized ion pair in alpha s1-B which is deleted in alpha s1-A; it is speculated that such bonds may play a role in other calcium-induced changes in protein solubility.

Calcium↗
PubMed1991

Casein kinase I phosphorylates the 25-kDa mRNA cap-binding protein.

The 25-kDa mRNA cap-binding protein (eIF-4E) exists in both phosphorylated and dephosphorylated forms in eukaryotic cells. Phosphorylated eIF-4E appears to be preferentially associated with 48 S initiation complexes and with the 220-kDa subunit of eIF-4F. In addition, dephosphorylation of eIF-4E has been observed during heat shock and mitosis which are accompanied by decreased protein synthesis. However, the control of eIF-4E phosphorylation and its regulatory role remain poorly understood. Using eIF-4E as a substrate we have identified and purified from rabbit reticulocytes a protein kinase that phosphorylates eIF-4E in vitro. This enzyme phosphorylated eIF-4E on both serine and threonine residues with an apparent Km of 3.7 microM. The molecular mass of the enzyme and specificity for substrates other than eIF-4E suggested that this enzyme was a species of casein kinase I. This was confirmed by comparing the phosphopeptide map of the purified reticulocyte enzyme with that of rabbit skeletal muscle casein kinase I and by comparing phosphopeptide maps of eIF-4E phosphorylated in vitro by each enzyme. We conclude that casein kinase I phosphorylates eIF-4E in vitro and suggest that eIF-4E may be phosphorylated by casein kinase I in intact cells under some physiologic conditions.

Animals↗
PubMed1979

Glycosylation of kappa-casein. I. Localization and characterization of sialyltransferase in bovine mammary gland.

A sialyltransferase (CMP-N-acetylneuraminate:D-galactosyl-glycoprotein N-acetylneuraminyltransferase, EC 2.4.99.1) which attaches N-acetylneuraminic acid to the terminal end of the carbohydrate chain of kappa-casein was found to be concentrated in Golgi apparatus-enriched fractions of bovine mammary gland. Maximum sialyltransferase activity was obtained at pH 5.5 and 37 degrees C in the presence of 1 mM dithiothreitol and Triton X-100. A Km of 0.19 mg asialo-kappa-casein/ml (0.01 mM) was obtained for the sialyltransferase. Native kappa-casein also served as acceptor for N-acetylneuraminic acid transferase of Golgi apparatus-enriched fractions although at a slower rate than did asialo-kappa-casein. The sialyltransferase has a divalent cation requirement for maximum activity which was best satisfied by the presence of 10 mM Mn2+.

Animals↗
PubMed1975

Infrared spectroscopy of the water vapor sorption process of caseins.

Infrared spectra of as-, beta- and micellar casein were studied at relative water vapor pressures (p/po) ranging from 0 to 0.98. The samples were prepared as self-supporting films by evaporating concentrated aqueous suspensions of the caseins under study. An infrared cell and a vacuum apparatus were constructed which allowed exposure of the casein films either to vacuum or to sorbate vapor. Following the increase in intensities of the OH and O2H absorption bands during hydration, a sigmoid-shaped curve was observed, similar to the type II isotherm usually obtained by gravimetric sorption measurements. The pronounced frequency and intensity changes in the amide I, II and III bands in the p/po range from 0 to about 0.10 lead to the conclusion that water molecules are already attached to the peptide repeat unit at very low humidities. Based on calculations of the amount of polar groups per casein molecule it was shown that much less than one water molecule per polar group is needed to cause these significant spectral changes.

Animals↗
PubMed1978

The involvement of one of the three histidine residues of cow kappa-casein in the chymosin-initiated milk clotting process.

Cow kappa-casein has been modified by photo-oxidation in the presence of rose bengal and by the chemical reagents diethyl pyrocarbonate, 2-hydroxy-5-nitro-benzyl bromide and iodoacetic acid. Photo-oxidation resulted in the destruction of histidine and tryptophan residues and all of the histidines could be ethoxy-formylated by treatment with diethyl pyrocarbonate. Both procedures caused a loss in the susceptibility of the Phe-Met linkage of kappa-casein to chymosin hydrolysis. Treatment of kappa-casein with 2-hydroxy-5-nitrobenzyl bromide and iodoacetic acid caused the loss of tryptophan and methionine residues respectively but, in both cases, the susceptibility of the modified protein to chymosin hydrolysis remained unaffected. Of the amino acids examined it is concluded that only the histidine residues of cow kappa-casein are important for the hydrolytic action of chymosin and, furthermore, the treatment with diethyl pyrocarbonate suggests that only one of the three histidines plays an essential role.

Amino Acid Sequence↗
PubMed1980

14C-Methylated beta-casein as a substrate for plasmin, and its application to the study of milk protein transformations.

A method was investigated for the sensitive radiochemical assay of milk protein transformations. beta-Casein was reductively methylated with NaBH4 and H14CHO giving a product of spec. act. 4.42 microCi/mg in which a maximum of 12% of the lysine residues were labelled. Methylation did not alter the electrophoretic or chromatographic properties of the protein, or its pattern of proteolysis by plasmin. The substrate susceptibility of 14C-methylated beta-casein towards plasmin was determined by measuring radioactivity transfer to the proteolytic fragments gamma 2- and gamma 3-casein. Compared with the native protein, no decrease in substrate susceptibility was detected. The presence in milk of the plasmin-like enzyme, milk proteinase, was demonstrated and its activity at 4 degrees C was quantified by examination of the fragmentation pattern of added 14C-methylated beta-casein. It was concluded that 14C-methylated protein substrates, prepared by reductive methylation, are well suited to the study of hydrolytic enzymes and that they can provide valuable information on milk protein transformations. In particular, no interference was encountered with the rate of cleavage by plasmin when the level of methylation was kept to a minimum.

Animals↗
PubMed1984

Plasmin cleaves human beta-casein.

Plasmin cleaves isolated human beta-casein to form specific fragments in a manner similar to the generation of gamma 1-, gamma 2-, and gamma 3-caseins from the bovine homologue. Identification of a protein previously isolated from human milk as a specific plasmin cleaved portion of beta-casein indicates that endogenous plasmin is active in whole milk. These findings suggest that protease activity should be considered in casein quantitation or isolation of components from human milk.

Amino Acid Sequence↗
PubMed1984

Effects of estrogen-depletion on rat casein gene expression.

Mammary tissue from rats that had been ovariectomized and adrenalectomized 4 weeks previously was compared to that from intact rats in terms of epithelial content and hormone-responsiveness in vitro. The endocrinectomy resulted in about a 30% enlargement of the gland, but led to a loss of only about 12% of the epithelium. This estrogen-depleted epithelium was able to acquire full responsiveness in vitro to insulin in terms of the accumulation of alpha-aminoisobutyric acid, and induction of glucose-6-phosphate and gluconate-6-phosphate dehydrogenases. It was also fully responsive to cortisol in relation to the induction of NADH-cytochrome C reductase, and to prolactin in terms of total RNA synthesis. However, estrogen-depletion led to an 82% loss in the ability of a unit amount of the epithelium to synthesize casein in response to these 3 hormones, and to a similar loss in relation to the accumulation of 25K casein mRNA. Estrogen administration in vivo could prevent and reverse the casein lesion. The disparity between constitutive and casein hormone-responsiveness in the absence of estrogen is discussed in relation to cell commitment.

Adrenalectomy↗
PubMed1986

Phosphorylation of isolated plasma membranes of AH-66 hepatoma ascites cells by casein kinase 1.

The isolated plasma membranes of AH-66 hepatoma cells were phosphorylated by casein kinase 1 purified from the cytosol fraction of AH-66 cells. Casein kinase 2 purified from the same source had little effect on the phosphorylation of the plasma membranes. Two-dimensional gel electrophoresis and autoradiography showed that casein kinase 1 enhanced the phosphorylation of approx. 10 plasma membrane proteins that are phosphorylated only faintly in the isolated plasma membranes by endogenous protein kinase. Among these phosphoproteins, tubulin was identified as judged from their molecular weights and isoelectric points. These results suggest that one of the physiological functions of casein kinase 1 is phosphorylation of plasma membrane and plasma membrane-associated proteins.

Amino Acids↗
PubMed1987

Cloning and sequence analysis of bovine beta-casein cDNA.

A bovine beta-casein cDNA clone was isolated from a cDNA library prepared from mammary gland mRNA. Sequence analysis revealed 25 nucleotides (nt) of the 5' noncoding region, 672 nt of the complete sequence coding and a 3' region of approximately 500 nt. When the nucleotide sequence of bovine beta-casein cDNA is compared to rat beta-casein cDNA (5), a high degree of homology is observed in the first 100 nt corresponding to the signal peptide of the pre-beta-caseins.

Animals↗