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PubMed1992

Proteolytic activity of proteinases on macropeptide isolated from kappa-casein.

Proteolytic activities of chymosin, bovine pepsin, Mucor miehei rennet, Cryphonectria parasitica (formerly Endothia parasitica) rennet, trypsin, and chymotrypsin on kappa-casein macropeptide were measured. Macropeptide solutions (10 mg/ml of .05 M, pH 6.6 phosphate buffer) were incubated with the enzymes at 37 degrees C for various times, and their reactions were stopped by adding .025 ml of pepstatin (1 mg/ml of methanol). Peptides released from kappa-casein macropeptide were then fractionated using reverse-phase HPLC. At the pH of milk (pH 6.6), kappa-casein macropeptide was resistant to enzymic action by chymosin, bovine pepsin, and M. miehei and C. parasitica rennets. Bovine pepsin hydrolyzed kappa-casein macropeptide at pH 3. kappa-Casein macropeptide was readily hydrolyzed at pH 6.6 by trypsin and chymotrypsin. Possible physiological functions of the kappa-casein macropeptide are discussed in light of these findings.

Carbohydrate Sequence↗
PubMed1993

Review and update of casein chemistry.

Of all food proteins, bovine milk proteins are probably the most well characterized chemically, physically, and genetically. The primary structures are known for most genetic variants of alpha s1-, alpha s2-, beta-, and kappa-caseins, beta-lactoglobulin, and alpha-lactalbumin. Secondary and tertiary structures of the whey proteins have been determined, and secondary structures of the caseins have been predicted from spectral studies. The caseins, although less ordered in structure and more flexible than the typical globular whey proteins, have significant amounts of secondary and, probably, tertiary structure. The amphipathic structure of the caseins is especially noteworthy; thus, these proteins most likely are divided into polar and hydrophobic domains. The presence of anionic phosphoseryl residue clusters in the calcium-sensitive casein polar domains is particularly significant because of their interaction with calcium ions, or calcium salts, or both, and the formation of micelles. Flexibility of casein structures is reflected by their susceptibilities to limited proteolysis, which dramatically changes functionality.

Amino Acid Sequence↗
PubMed1994

Antihypertensive effect of the peptides derived from casein by an extracellular proteinase from Lactobacillus helveticus CP790.

Peptides derived from alpha s1- and beta-caseins by the Lactobacillus helveticus CP790 proteinase were investigated for their inhibitory activities against angiotensin I-converting enzyme. The antihypertensive effect of casein hydrolysates in strain SHR spontaneously hypertensive rats was also investigated. Both alpha s1- and beta-casein hydrolysates inhibited this enzyme. Some of these peptides showed enzyme inhibitory activity, and one of them from beta-casein inhibited the enzyme greatly; the concentration of an angiotensin I-converting enzyme inhibitor needed to inhibit 50% of the enzyme activity was 4 microM. The hydrolysate of casein demonstrated antihypertensive activity in spontaneously hypertensive rats at an orally administered dosage of 15 mg/kg of body weight. MILK fermented with L. helveticus CP790, containing about .3% peptides, also showed antihypertensive activity in SHR rats with 5 ml/kg of body weight (15 mg of peptide/kg); however, the milk fermented with L. helveticus CP791, a variant defective for proteinase activity, did not show this activity. Results suggested that the peptides liberated from casein by the proteinase in the culture medium showed antihypertensive effect in SHR rats.

Amino Acid Sequence↗
PubMed1994

A cell culture model to identify biologically active peptides generated by bacterial hydrolysis of casein.

Consumption of fermented dairy foods has been linked to reduced incidence of colon cancer in population groups. Recently, biologically active compounds have been isolated from these products. Bacterial proteinases, produced by dairy starter cultures, generate a variety of peptides from casein. Some of these casein-derived peptides are likely to alter intestinal cell kinetics. Effects on colon cell kinetics because of the presence of casein-derived peptides may be a mechanism through which fermented dairy foods reduce the risk of colon cancer. We have used two intestinal cell lines (IEC-6 cells, derived from normal rat intestine, and Caco-2 cells, derived from human colon adenocarcinoma) to identify casein peptides that affect intestinal cell kinetics. Cell culture media containing casein were inoculated with three commercial starter cultures and incubated for 4, 8, or 24 h. The bacteria-conditioned media were then filter-sterilized and incubated with the intestinal cells for 6 or 24 h. Rates of [3H]thymidine incorporation and cell cycle kinetics determined by flow cytometry were affected by the culture-modified media in both cell lines. The IEC-6 cells tended to reduce, and Caco-2 cells to increase, rates of cell division after exposure to the media. Intestinal cell response varied among the starter cultures. The results support the use of intestinal cell cultures to identify casein peptides generated by dairy starter cultures, which affect intestinal cell kinetics.

Adenocarcinoma↗
PubMed1995

Preparation of casein using carbon dioxide.

The effects of pressure, temperature, residence time, and mass of skim milk on some characteristics of casein, prepared by precipitation with high pressure CO2, were examined in a batch reactor. For a 500-g milk sample, precipitation occurred at pressures > 2760 kPa and temperatures > 32 degrees C. Residence time was not significant and was held at 5 min. Yields were maximum at 2750 to 5520 kPa and at 38 to 49 degrees C for a 500-g milk sample. The resulting whey had a pH of 6.0. The casein product had an acceptable appearance and had greater solids, ash, and Ca contents than commercial acid caseins. Particle size distribution studies showed that the mean particle size was sensitive to precipitation pressure and temperature and was similar to that of acid caseins produced under laboratory conditions. The HPLC studies of the casein and whey fractions showed that precipitation by CO2 did not result in fractionation of casein or whey proteins to their component proteins.

Animals↗
PubMed1998

Changes in the secondary structure of bovine casein by Fourier transform infrared spectroscopy: effects of calcium and temperature.

Bovine casein submicelles and reformed micelles, produced by addition of Ca2+, were examined by Fourier transform infrared spectroscopy at 15 and 37 degrees C in aqueous salt solutions of K+ and Na+. Previous measurements of caseins, made in D2O and in the solid form, can now be made in a more realistic environment of H2O. When analyzed in detail, data obtained by Fourier transform infrared spectroscopy have the potential to show subtle changes in secondary structural elements that are associated with changes in protein environment. Electrostatic binding of Ca2+ to casein resulted in a redistribution of the components of the infrared spectra. Addition of Ca2+ in salt solutions of K+ and Na+ led to apparent decreases in large loop or helical structures at 37 degrees C with concomitant increases in the percentage of structures having greater bond energy, such as turns and extended helical structures. At 15 degrees C, Na+ and K+ have differential effects on the Ca(2+)-casein complexes. All of these observations are in accordance with the important role of serine phosphate side chains as sites for Ca2+ binding in caseins and the swelling of the casein structure upon incorporation into reformed micelles at 37 degrees C. This new open, hydrated structure is buttressed by a change in backbone as evidenced by a shift in absorbance to higher wave numbers (greater bond energies) as colloidal micelles are reformed.

Animals↗
PubMed1987

Measurement by radioimmunoassay of casein content in rabbit mammary gland during pregnancy and after prolactin stimulation in organ culture.

A specific homologous radioimmunoassay was developed to measure rabbit beta-casein in rabbit mammary gland with a sensitivity of 0.5 ng/ml protein. It was used to measure casein concentration during pregnancy and in organ culture of mammary gland explants. Casein was detectable in virgin mammary glands, showed a small increase during the first half of pregnancy, increased more than 20-fold between Days 21 and 27, and diminished somewhat on the first days of lactation. After 24 hr of culture, mammary gland explants had no detectable casein, but the addition of increasing concentrations of prolactin to a culture medium which contained insulin (5 micrograms/ml) and cortisol (0.5 microgram/ml) induced a regular increase in the casein content of the tissue. Casein started to increase when 10 ng/ml of prolactin was present and maximal values were achieved for 100 ng/ml of the hormone.

Animals↗
PubMed1999

Alterations in C3 activation and binding caused by phosphorylation by a casein kinase released from activated human platelets.

A casein kinase released from activated human platelets phosphorylates a number of plasma proteins extracellularly, and that activation of platelets in systemic lupus erythematosus patients parallels an increase in the phosphate content of plasma proteins, including C3. The present study was undertaken to characterize this platelet protein kinase and to further elucidate the effect(s) on C3 function of phosphorylation by platelet casein kinase. The phosphate content of human plasma C3 was increased from 0.15 to 0.60 mol phosphate/mol of C3 after platelet activation in whole blood or platelet-rich plasma. The platelet casein kinase was distinct from other casein kinases in terms of its dependence on cations, inhibition by specific protein kinase inhibitors, and immunological reactivity. C3 that had been phosphorylated with platelet casein kinase was tested for its susceptibility to cleavage by trypsin or the classical and alternative pathway convertases and its binding to EAC and IgG. Phosphorylation did not affect the cleavage of C3 into C3a and C3b, but the binding of fragments from phosphorylated C3 to EAC14oxy2 cells and to IgG in purified systems and in serum was increased by 1.6-4.5 times over that of unphosphorylated C3. A covariation was seen between the enhanced binding of C3 fragments to IgG after phosphorylation and an increased ratio of glycerol/glycine binding, from 2.0 for unphosphorylated C3 to 4.9 for phosphorylated C3. The present study suggests that an overall effect of phosphorylation of C3 by platelet casein kinase is to enhance the opsonization of immune complexes.

Animals↗
PubMed2001

Effect of casein diet on gonadotropin releasing hormone antagonist induced changes in adrenal gonadal functions in male rats.

Adult male rats received daily injections (sc) of gonadotropin releasing hormone antagonist (0.2 mg/kg(-1) x day(-1)) for 21 days when they were sacrificed on day 22, adrenal weight, adrenal A5-3beta (delta 5-3beta) hydroxysteroid dehydrogenase (Delta5-3beta-HSD) activity and serum level of corticosterone were increased significantly while testicular 17beta (17beta) hydroxysteroid dehydrogenase (17beta-HSD) activity and serum level of testosterone and spermatogenesis were decreased in the rats fed on 5% casein diet. GnRH antagonist treated rats fed on 20% casein diet, resulted significant decrease in adrenal weight, serum corticosterone and adrenal A5-3beta-HSD activity while testicular 17beta-HSD activity serum testosterone levels and the weights of sex organs were increased with respect to anti GnRH treated rats fed on 5% casein diet. But the GnRH antagonist treated rats fed on 20% casein diet showed decreased spermatogenesis quantitatively and sperm count appeared similar to anti GnRH treated rats fed on 5% casein diet. These results indicate that high casein diet protects adrenocortical activity and stimulates testosterone synthesis without effecting spermatogenic arrest in GnRH antagonist treated rats. It may be concluded that GnRH antagonist in presence of high milk protein diet may be considered to be a suitable antihormone in the development of an ideal male contraceptive.

Adrenal Glands↗
PubMed2002

[Murine beta-casein gene sequences direct a human tissue plasminogen activator variant expressed in the milk of transgenic mice].

To investigate the ability of our cloned murine beta-casein locus to direct the exogenous gene expression in the milk of transgenic mice, the human t-PA variant mammary gland expression vector under the control of murine beta-casein gene regulatory elements was constructed, in which the human t-PA variant signal-pro peptide sequence was replaced with murine beta-casein signal peptide sequence and the human t-PA variant mature peptide cDNA was inserted into the second exon of beta-casein gene. The fusion gene was microinjected in the fertilized mice eggs. A total of 285 embryos were microinjected and transferred into 13 surrogate mother mice. Twelve positive transgenic mice were identified through PCR and Southern blot analysis among 42 new born mice. Human t-PA variant was expressed in the milk of 7 transgenic mice, the highest expression level attained to 3.6593 micrograms/ml. The results demonstrated that the murine beta-casein gene regulatory elements can direct the human t-PA variant gene successfully express in the milk of transgenic mice. It lays great foundation for the research on the beta-casein knock-in mice mammary gland bioreactor model construction.

Animals↗
PubMed1992

Influence of casein and soy flour proteins on aminoacid content in the liver of experimental animals.

We have observed a significantly increased content of fats and decreased content of proteins in the liver of experimental rats fed a diet supplemented with 25% casein proteins in comparison with the application of de-fatted soy flour. Casein proteins have a higher content of methionine in relation to cystine than baked soy flour. But the soy diet in contrast to the casein diet has a high content of free aminoacids which are not present in casein at all: aspartic acid, asparagine, alpha-aminoadipic acid, methionine, norleucine, lysine, phenylalanine, beta-alanine, ethanolamine, histidine, proline, gamma-aminobutyric acid, taurine. Differences in free valine, alanine, arginine, glycine, ornithine and cysteic acid are also significant. The content of free aminoacids in the liver of experimental animals fed a soy diet is high in the content of cystine, cystathionine, ornithine, beta-aminoisobutyric acid, beta-alanine, gamma-aminobutyric acid, leucine. We have also found accumulation of methionine, glycine, alpha-aminobutyric acid, taurine and citrulline in free aminoacids from the liver of animals fed a casein diet. Citrulline and glycine in free aminoacids from the liver of animals fed a soy protein supplement were not recorded. Our investigations have shown that the application of a soy diet enriched with cystine acts protectively on methionine and that methionine is preferentially utilized for protein synthesis. The catabolic pathway of methionine prevails in animals on a casein diet.

Alanine↗
PubMed1992

A pregnancy-specific mammary nuclear factor involved in the repression of the mouse beta-casein gene transcription by progesterone.

The sequence-specific binding pattern of mammary nuclear proteins to the 545-base pair (bp) 5'-flanking region of the mouse beta-casein gene was compared between pregnant and lactating mice by a gel mobility shift assay. By using appropriate probes, two complexes were detected only during pregnancy, whereas an additional four complexes were detected during both pregnancy and lactation. The two pregnancy-specific complexes showed identical electrophoretic mobility and were cross-competed with two unlabeled DNA fragments used to generate the probes. Methylation interference experiments indicated that the two binding regions involved guanosine residues at nucleotides -350 and -8, and the sequences around each guanosine residue have a common palindromic sequence, 5'-TGAT/ATCA-3'. This binding factor is termed pregnancy-specific mammary nuclear factor. In gene transfection experiments, expression of the chimeric beta-casein-CAT gene linked to the 545 bp of mouse beta-casein gene promoter region was maximally induced when transfected mammary epithelial cells were cultured with the lactogenic hormones prolactin, hydrocortisone, and insulin, whereas it was very low when insulin alone was added. The addition of progesterone together with the lactogenic hormones inhibited the hormonal induction of the chimeric gene, whereas co-transfection with an oligonucleotide containing the pregnancy-specific mammary nuclear factor binding site substantially overcome the progesterone-mediated repression of transcription. Furthermore, mutation of the pregnancy-specific mammary nuclear factor binding sites of the chimeric beta-casein-CAT gene resulted in attenuation of the inhibitory effect of progesterone without blocking the stimulatory effect of the lactogenic hormones. These results indicate the presence of a pregnancy-specific mammary nuclear factor(s) that bind to two separate sites of the beta-casein gene promoter and suggest that it may serve as a repressor that mediates the inhibitory action of progesterone on beta-casein gene transcription.

Animals↗
PubMed2004

[The ht-PAm cDNA knock-in the goat beta-casein gene locus].

The production of recombinant protein is one of the major successes of biotechnology, animal cells are required to synthesize proteins with the appropriate post-translational modifications. Transgenic animal mammary gland bioreactor are being used for this purpose. Gene targeting is a more powerful method to produce mammary gland bioreactor, and nuclear transfer from cultured somatic cells provides an wonderful means of cell-mediated transgensis. Here we describe efficient and reproducible gene targeting in goat fetal fibroblasts to place the human tissue plasminogen activator mutant (ht-PAm) cDNA at the beta-casein locus, and would produce the transgenic goat by nuclear transfer. To construct the gene targeting vector pGBC4tPA, the milk goat beta-casein genomic DNA sequence for homologous arms had been cloned firstly. The left arm was 6.3 kb fragment including goat beta-casein gene 5' flanking sequence, and the right arm was 2.4 kb fragement including beta-casein gene from exon 8 to exon 9. The ht-PAm cDNA was subcloned in the goat beta-casein gene exon 2, and the endogenous start condon was replaced by that of ht-PAm. The bacterial neomycin (neo) gene as positive selection marker gene, was placed in the beta-casein gene intron 7, the thymidine kinase (tk) as the negative selection marker gene, was just outside the right arm. The validity of the positive-negative selection vector (PNS), was tested, and targeting homologous recombination (HR) were elevated to 5-fold with the negative selection marker using the drug GANC. The DNA fragment in which two LoxP sequence was delected effectively using Cre recombinase in vitro. Goat fetal fibroblasts were thawed and cultured to subconfluence before transfection, about 10(7) fibroblasts were electoporated at 240V, 600 microF in 0.8 mL PBS buffer containing linear pGBC4tPA. transfected cells were cultured in collagen-coated 96-wellplate for 24h without selection, then added the drug G418 (600 microg/mL) and GANC (2 micromol/L). After 12 days of selection, well separated G418r/GANCr clones were isolated and expanded in 24-wellplate. 244 clones were selected, and only 90 clones could grow and be tested by PCR screening for targeting. The primary result demonstrated that 31 targeting cell clones with homologous recombination events were obtained, and 2 cell clones was verified by DNA sequence analysis on the homologous recombination region.

Animals↗
PubMed1991

Casein kinase I is regulated by phosphatidylinositol 4,5-bisphosphate in native membranes.

Casein kinase I activity is present in cells as a cytosolic and a membrane-bound enzyme. Previously, the erythroid membrane-bound casein kinase I was shown to associate with purified integral membrane proteins; this association and protein kinase activity was regulated by phosphatidylinositol 4,5-bisphosphate (PIP2) (Bazenet, C.E., Brockman, J.L., Lewis, D., Chan, C., and Anderson, R.A. (1990) J. Biol. Chem. 265, 7369-7376). Here we show that both the membrane-bound and the cytosolic casein kinase interact with native membranes and that this interaction is regulated by the membrane content of PIP2. On native membranes, casein kinase I activity is potently inhibited by small increases (10-20%) in the membrane content of either exogenously added or intrinsic PIP2. However, the majority of the intrinsic content of PIP2 in isolated membranes does not inhibit casein kinase, suggesting that this PIP2 is not accessible. Regulation of the casein kinases on membranes is sensitive to detergents and to chymotrypsin treatment of membranes.

Animals↗
PubMed1991

Characterization of the catalytic subunit of casein kinase II expressed in Escherichia coli and regulation of activity.

The catalytic (alpha) subunit of casein kinase II from Drosophila, cloned and expressed in Escherichia coli (Saxena, A., Padmanabha, R., and Glover, C. V. C., (1987) Mol. Cell. Biol. 7, 3409-3417), has been purified and characterized, and the properties have been compared to those of the holoenzyme. The catalytic subunit exhibits protein kinase activity with casein as substrate and is autophosphorylated. The specific activity of the purified subunit is 6% of the activity of the holoenzyme from reticulocytes or from Drosophila. The alpha subunit is a monomer, eluting at Mr = 40,000 upon gel filtration in high salt, but as part of an aggregate in low salt. The alpha subunit has been purified to apparent homogeneity by sequential chromatography on DEAE-cellulose, Mono S, and Mono Q. A single band, Mr = 37,000, is detected by silver staining following polyacrylamide gel electrophoresis. The isolated alpha subunit displays apparent Km values for beta casein, ATP, and GTP similar to those of the holoenzyme. The activity of the alpha subunit is inhibited by heparin with an I50 of 0.1-0.3 micrograms/ml, a value similar to that observed for the holoenzyme; autophosphorylation is also inhibited by heparin. Polylysine has no stimulatory effect on the activity of the catalytic subunit, as measured with casein and by autophosphorylation, but stimulates both activities with the holoenzyme. When physiological substrates for casein kinase II are examined, glycogen synthase and eukaryotic initiation factor 3 (eIF-3) (p120) are phosphorylated by the alpha subunit at a rate equivalent to that of the holoenzyme, while phosphorylation of eIF-3 (p67) is reduced 9-fold and eIF-2 beta is not modified. From these data, it can be concluded that the alpha subunit of casein kinase II is sufficient for catalysis, is autophosphorylated, and can be directly inhibited by heparin, whereas the beta subunit mediates the effects of basic stimulatory compounds and is involved in recognition and/or binding to specific physiological substrates.

Animals↗
PubMed1991

Phosphorylation of maize RAB-17 protein by casein kinase 2.

The maize gene RAB-17, which is responsive to abscisic acid, encodes a basic glycine-rich protein containing, in the middle part of its sequence, a cluster of 8 serine residues followed by a putative casein kinase 2-type substrate consensus sequence. This protein was found to be highly phosphorylated in vivo. Here, we show that RAB-17 protein is a real substrate for casein kinase 2. RAB-17 protein is phosphorylated in vitro by casein kinase 2 isolated from rat liver cytosol and from maize embryos. A maximum of 4 mol of phosphate were incorporated per mol of RAB-17 protein following incubation with casein kinase 2. Phosphopeptide mapping experiments show that the peptide phosphorylated by casein kinase 2 in vitro is identical to that derived from the protein phosphorylated in vivo. Purification by high performance liquid chromatography and partial sequencing of the phosphopeptide indicate that it corresponds to the region of the protein (residues 56-89) containing the cluster of serine residues. Our results indicate that RAB-17 is phosphorylated by casein kinase 2 or a kinase with a similar specificity and that phosphorylation takes place in the serine cluster region of the protein both in vitro and in vivo.

Amino Acid Sequence↗
PubMed1990

[Inhibition of casein kinase type 2 activity by formation of complexes with mRNA in vivo].

It is known that casein kinase 2 possesses, besides the protein kinase, an RNA-binding activity. Using ligand blotting it has been demonstrated that the both activities are localized on the alpha- and alpha'-subunits of the enzyme. Casein kinase 2 is suppressed in vitro by polyuridylic acid. A part of the intracellular pool of casein kinase 2 is found in the informosomes. The informosomes and free proteins were separated by centrifugation in a sucrose density gradient, and each fraction was incubated with casein and [gamma-32P]ATP. The informosome-bound protein kinase is completely inhibited, while the free protein kinases heavily phosphorylate casein. It is concluded that the activity of casein kinase 2 can be regulated by the reversible formation of complexes with RNA.

Animals↗
PubMed1990

The 204-kDa smooth muscle myosin heavy chain is phosphorylated in intact cells by casein kinase II on a serine near the carboxyl terminus.

The heavy chain of smooth muscle myosin was found to be phosphorylated following immunoprecipitation from cultured bovine aortic smooth muscle cells. Of a variety of serine/threonine kinases assayed, only casein kinase II and calcium/calmodulin-dependent protein kinase II phosphorylated the smooth muscle myosin heavy chain to a significant extent in vitro. Two-dimensional maps of tryptic peptides derived from heavy chains phosphorylated in cultured cells revealed one major and one minor phosphopeptide. Identical tryptic peptide maps were obtained from heavy chains phosphorylated in vitro with casein kinase II but not with calcium/calmodulin-dependent protein kinase II. Of note, the 204-kDa smooth muscle myosin heavy chain but not the 200-kDa heavy chain isoform was phosphorylated by casein kinase II. Partial sequence of the tryptic phosphopeptides generated following phosphorylation by casein kinase II yielded Val-Ile-Glu-Asn-Ala-Asp-Gly-Ser*-Glu-Glu-Glu-Val. The Ser* represents the Ser(PO4) which is in an acidic environment, as is typical for casein kinase II phosphorylation sites. By comparison with the deduced amino acid sequence for rabbit uterine smooth muscle myosin (Nagai, R., Kuro-o, M., Babij, P., and Periasamy, M. (1989) J. Biol. Chem. 264, 9734-9737), we have localized the phosphorylated serine residue to the non-helical tail of the 204-kDa isoform of the smooth muscle myosin heavy chain. The ability of the 204-kDa isoform, but not the 200-kDa isoform, to serve as a substrate for casein kinase II suggests that these two isoforms can be regulated differentially.

Amino Acid Sequence↗