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PubMed1997

The alpha4beta1 integrin can mediate leukocyte adhesion to casein and denatured protein substrates.

An understanding of the binding specificity of leukocyte integrins is important to determine the range of ligands that interact with these receptors during inflammatory processes. In this study we show that the alpha4beta1 integrin can interact with casein and denatured albumin and promote leukocyte adhesion through these interactions. This was demonstrated with the use of blocking antibodies directed to alpha4beta1 and peptide adhesion competitors containing the alpha4beta1 binding tripeptide, Leu-Asp-Val (LDV). Consistent with this data, the adhesion is completely divalent cation-dependent and is stimulated by known activators of leukocyte integrin function, namely phorbol ester and the beta1 integrin activating antibody, 8A2. It is interesting to note that neither bovine alpha-casein or human albumin contain an LDV site (present in the CS-1 site of alternatively spliced fibronectin) or an IDS site (present in VCAM-1) yet they promote adhesion through this integrin. Furthermore, alpha4beta1 directly binds to Sepharose columns containing casein, casein fragments, or denatured albumin but does not bind columns containing native albumin. These data suggest that the binding specificity for the alpha4beta1 integrin is considerably broader than previously realized. This work has implications for how subsets of leukocytes may interact with damaged proteins during tissue injury and inflammation.

Amino Acid Sequence↗

PubMedDate not supplied

Casein-hydrolyzing activity of sIgA antibodies from human milk.

During pregnancy and immediately after delivery (i.e. at the beginning of lactation), the female organism is frequently characterized by an immune status similar to that of patients with autoimmune diseases. In addition, lactation is associated with an appearance of catalytically active antibodies or abzymes (Abzs) with DNAse, RNase, ATPase, amylolitic, protein kinase and lipid kinase activities in breast milk. However, until now there were no examples of human milk Abzs with a proteolytic activity. We present the first evidence that electrophoretically and immunologically homogeneous human milk sIgAs possess a beta-casein-hydrolyzing activity different from known proteases. Abzs specifically hydrolyze both human and bovine beta-caseins but not many other proteins tested. Using different methods including in situ analysis of proteolytic activity in a gel after SDS-PAGE it was shown that the observed proteolytic activity is an intrinsic property of human milk polyclonal sIgAs. Specific inhibitors of acidic and thiol proteases demonstrated a weak effect on proteolytic activity of Abzs, while a specific inhibitor of serine proteases (AEBSF) significantly inhibited the proteolytic activity of the abzymes. The K(M) value for human casein as a substrate was estimated (7.3 microM). Our findings suggest that the immune system of clinically healthy mothers can generate IgAs with a beta-casein-specific serine protease-like activity.

Antibodies, Catalytic↗

PubMed1994

Synthesis of lactoferrin and casein by explants of bovine mammary tissue.

Synthesis of lactoferrin and casein by the bovine mammary gland was determined in an experimental model where lactation was maintained in one mammary half, while involution was induced in the contralateral half. Culture of explants with prolactin had no consistent effect on synthesis of casein or lactoferrin in tissue from either mammary half. Endotoxin and tumor necrosis factor-alpha generally decreased synthesis of casein and lactoferrin, suggesting that these inflammatory mediators are not directly responsible for increasing lactoferrin synthesis during mammary inflammation or involution. Synthesis of lactoferrin was increased and casein decreased in the involuting mammary half vs. the lactating half. These results suggest that local factors in the mammary gland play a role in the regulation of lactoferrin synthesis during involution.

Animals↗

PubMed1996

K-casein gene phylogeny of higher ruminants (Pecora, Artiodactyla).

To assess phylogenetic relationships among the higher ruminants (infraorder Pecora, order Artiodactyla), we analyzed K-casein DNA sequences, including 434 nucleotides of the fourth exon. The higher ruminant families Bovidae, Cervidae, Giraffidae, and Antilocapridae each have monophyletic K-casein sequences. Maximum parsimony and distance analyses identify Giraffidae as a sister group to either Cervidae or a Bovidae-Cervidae clade and Antilocapridae as a sister group to a Bovidae-Cervidae-Giraffidae clade. At a higher level these four families occur as a monophyletic clade relative to Tragulidae and Suidae. Within Cervidae, the subfamily Odocoileinae is monophyletic and Cervinae and Muntiacinae occur as independent lineages within a separate clade. Within Bovidae, the subfamilies Bovinae and Caprinae are monophyletic. Genera within Cervinae (Cervus, Elaphurus) and Bovinae (Bison, Bos) are paraphyletic. There is intraspecific allelic variation in Cervus elaphus, Odocoileus hemionus, and Bison bison. The rate of K-casein fourth exon DNA sequence evolution is estimated to be about 0.004 nucleotide substitutions per million years. The K-casein phylogeny is discussed relative to other molecular and morphological data.

Animals↗

PubMed1998

Expression and characterization of the recombinant catalytic subunit of casein kinase II from the yeast Yarrowia lipolytica in Escherichia coli.

The alpha catalytic subunit of casein kinase II from the yeast Yarrowia lipolytica has been cloned and overexpressed using the pT7-7 expression vector in Escherichia coli. Casein kinase activity is found in the bacterial extracts. The catalytic subunit is partially expressed in a soluble and active form, which is purified to electrophoretic homogeneity. Most of the enzyme was found in inclusion bodies. In this form, the enzyme, which is almost pure, exhibits a low specific activity. We have focused our efforts on methods to activate the protein from the inclusion bodies. We have studied the renaturation of urea-denaturated CKII catalytic subunit. We have tried different renaturation buffers and found that renaturation by dilution was more efficient than renaturation by dialysis. Treatment of the enzyme found in the inclusion bodies with different nondetergent sulfobetaines (NDSB) led to a time-dependent activation. NDSB195 is a V-type activator of the recombinant catalytic subunit of casein kinase II. The NDSB195-activated enzyme remained active at the room temperature for weeks. Kinetic properties of the recombinant casein kinase II subunit are similar to those of the purified holoenzyme (low Km for ATP and inhibition by heparin). Kinetic study indicates that the beta subunit could interact with the alpha subunit at the level of the catalytic site to enhance activity and to modify the kinetic behavior of the enzyme.

Base Sequence↗

PubMed1976

Casein-induced murine amyloidosis: amyloid and immunoglobulin production and proliferative capacity of splenocytes.

We have examined some aspects of lymphocyte and macrophage function in experimental murine amyloidosis. Casein-induced murine amyloidosis is a good model for studying secondary human amyloidosis while myeloma-associated murine amyloidosis is a poor model for human myeloma-associated amyloidosis. The amyloid of casein-induced and myeloma-associated murine amyloidosis cross-reacted immunologically. Neither form of amyloidosis was associated with L chain fragments or excess L chain production. Cellular immunologic reactivity of casein-induced amyloidotic mice, as assessed by lymphocyte transformation with mitogens, was abnormal using spleen lymphocytes but completely normal when thymus and peripheral blood lymphocytes were examined. The depressed activity could be attributed to splenic amyloid deposits. Intracellular amyloid was detected in the spleens of casein-injected mice prior to extracellular amyloid deposits. Amyloid containing cells could also be cultured from the spleen in a much higher proportion than that found in vivo. These cells may represent a subpopulation committed to amyloid synthesis.

Amyloid↗

PubMed1986

Effect of severely alkali-treated casein on gastrointestinal transit and selected intestinal enzyme activities.

Alkaline treatment of proteins leads to chemical changes which alter the proteins' digestibility. Severely alkali-treated casein (0.2N NaOH, 80 degrees C, 1 hour) in the diet reduces food intake and growth of young but not of adult Sprague Dawley rats. Gastrointestinal transit time is not reduced significantly in either young or adult rats. Food intake and growth rate are improved by amino acid supplementation. In this case, protein content and total leucine aminopeptidase activity are increased in the distal part of the small intestine whereas gamma-glutamyl-transpeptidase and maltase activities are increased in both the proximal and distal parts. Alkaline phosphatase activity remains unchanged. These intestinal adaptations differ from those observed in rats receiving a diet containing untreated casein and graded levels of a synthetic trypsin inhibitor. In the latter, protein digestibility remains high, gamma-glutamyltranspeptidase and also maltase activities are increased in the proximal and medial parts of the small intestine only. Intestinal adaptation in rats receiving alkali-treated casein does not result from a deficiency of pancreatic proteases activity. Ileal accumulation of undigested peptides from insufficient hydrolysis of alkali-treated casein may account for these mucosal adaptations.

Alkaline Phosphatase↗

PubMed1991

Specificity of a cell-envelope-located proteinase (PIII-type) from Lactococcus lactis subsp. cremoris AM1 in its action on bovine beta-casein.

The action of the cell-envelope proteinase (PIII-type) from Lactococcus lactis ssp. cremoris AM1 on bovine beta-casein was studied. The results were compared with those obtained earlier with (PI-type) proteinases from the cell envelope of other L. lactis strains. From a 4-h digest (pH 6.2; 15 degrees C) of beta-casein made with the PIII-type proteinase, 24 peptides were isolated and purified by selective precipitation followed by semi-preparative reversed-phase HPLC. Altogether, these peptides accounted for the preferential splitting of 16 peptide bonds in beta-casein by the PIII-type proteinase. In nine cases the primary cleavage site (P1-P'1) was a Glx-X or X-Glx peptide bond. In ten cases at least one large hydrophobic residue (Met, Leu, Tyr, Phe) formed part of the cleavable bond. The P2-P3 and/or P'2-P'3 regions of the substrate consisted of hydrophobic and/or negatively charged side chains or of side chains potentially involved in hydrogen bonds. Nine of the peptide bonds split were reported previously to be also susceptible to cleavage by PI-type proteinases, although the kinetics may be different. The PIII-type proteinase shows a broader specificity in its initial cleavage of beta-casein than does the PI-type.

Amino Acid Sequence↗

PubMed1993

Insulin receptor serine kinase activation by casein kinase 2 and a membrane tyrosine kinase.

The insulin receptor (IR) tyrosine kinase can apparently directly phosphorylate and activate one or more serine kinases. The identities of such serine kinases and their modes of activation are still unclear. We have described a serine kinase (here designated insulin receptor serine (IRS) kinase) from rat liver membranes that co-purifies with IR on wheat germ agglutinin-agarose. The kinase was activated after phosphorylation of the membrane glycoproteins by casein kinase-1, casein kinase-2, or casein kinase-3 (Biochem Biophys Res Commun 171: 75-83,1990). In this study, IRS kinase was further characterized. The presence of vanadate or phosphotyrosine in reaction mixtures was required for activation to be observed. Phosphoserine and phosphothreonine are only about 25% as effective as phosphotyrosine, whereas sodium fluoride and molybdate were ineffective in supporting activation. Vanadate and phosphotyrosine support IRS kinase activation by apparently inhibiting phosphotyrosine protein phosphatases present among the membrane glycoproteins. IR beta-subunit, myelin basic protein, and microtubule-associated protein-2 are good substrates for IRS kinase. The kinase prefers Mn2+ (Ka = 1.3 mM) as a metal cofactor. Mg2+ (Ka = 3.3 mM) is only 30% as effective as Mn2+. The kinase activity is stimulated by basic polypeptides, with greater than 30-fold activation achieved with polylysine and protamine. Our results suggest that both serine/threonine and tyrosine phosphorylation are required for activation of IRS kinase. Serine phosphorylation is catalyzed by one of the casein kinases, whereas tyrosine phosphorylation is catalyzed by a membrane tyrosine kinase, possibly IR tyrosine kinase.

Animals↗

PubMed1992

Solubility and reactivity of caseins and beta-lactoglobulin in protic solvents.

The study of the solubility of unstructured proteins (alpha s1-, beta-, and kappa-casein) and well-structured globulin (beta-lactoglobulin) in low water binary solvent systems demonstrated the crucial importance of solvent polarity and neutralization of protein polar functions on the final outcome of solubility experiments. The solubilities up to 38, 56, and 96% in CHCl3/CH3OH (1/1, v/v) acidified with HCl and up to 5, 10, and 25% in CHCl3/CH3OH (1/1, v/v) in the presence of triethylamine (TEA) were obtained for kappa-, alpha s1-, and beta-casein, respectively. The importance of protein charge neutralization was apparent when the solubilization was performed in basified CHCl3/CH3OH media, giving the optimal results when the studied proteins were brought before to their isoionic point. The maximum solubility of beta-casein at its pI in 30-70% methanol in CHCl3 was reaching 50-60% with triethylamine (TEA) added. beta-lactoglobulin could be solubilized up to 70% in CHCl3/CH3OH (7/3, v/v) acidified with HCl and up to 40% in CHCl3/CH3OH (3/7, v/v) in the presence of TEA. The observed yield of reductive alkylation of beta-lactoglobulin was much higher (98%) when performed in studied solvent system than in aqueous conditions (75%). Apparently, steric hindrance of the well-folded beta-barrel (in aqueous conditions) structure masks the portion of epsilon-NH2 groups. In the case of unstructured aqueous media beta-casein, 90% alkylation yields were obtained in organic and aqueous conditions.

Alkylation↗

PubMed1982

Association of aflatoxin M1 with casein.

Equilibrium dialysis was used to determine whether aflatoxin M1 (AFM1) binds to casein. Simulated milk ultrafiltrate (SMUF) containing 10 or 20 ng/ml of AFM1 was dialyzed against SMUF containing casein micelles. After 24 h at 7 degrees C, the casein suspension contained 2.5- or 2.9-fold, respectively, more toxin than that found in SMUF. An average of 17.9 or 55.3 micrograms of AFM1, respectively, per gram of casein was bound. In a separate experiment, milk naturally contaminated with AFM1 was treated with a proteolytic enzyme. An average of 30.7% more toxin was found in treated than in untreated milk. This result also suggests binding of AFM1 by milk protein.

Aflatoxin B1↗

PubMed1987

Influence of phytate on in vitro digestibility of casein under physiological conditions.

The phytic acid content of four different varieties of beans under different processing conditions was estimated. It was highest in red kidney (1.86-2.13%) slightly lower in pigeon (1.86-2.03%), white (1.80-1.96%) and black eyed beans (1.15-1.64%). There was no significant change in phytic acid content of beans after soaking at 25 degrees C for 22 hours. However, both soaking and cooking revealed 26-37% loss of phytic acid in all four varieties of beans. The rate of in vitro casein digestibility with and without phytic acid at concentrations found in legumes was determined at pH 8 and 37 degrees C using multienzyme technique. Addition of 5 mg Na-phytate reduced the casein digestibility up to 20% compared to the control. However, only 25% reduction of casein digestibility was observed in the presence of 25 mg of Na-phytate. Higher concentration of Na-phytate had no significant effect on the rate of casein digestibility. Data strongly suggest the formation of protein phytate complex at alkaline pH of small intestine.

Caseins↗

PubMed1981

[Immunological estimation of bovine casein in sheep cheese products (author's transl)].

Casein fraction of cow's milk and sheep's milk are very similar in their immunological behavior. Therefore, in the presence of sheep's milk, precipitation reaction with commercially obtained serum are not specific. Results from comparative immunoelectrophoresis according to Grabar and Williams also are equivocal, for heat and other denaturing factors caused shifts in the pattern of just the small group of differing elecrophoretic bonds present among many identical bonds. Qualitative identification of large amounts of cow's milk added to sheep's milk or of bovine casein to sheep cheese or similar products are possible in principle by two dimensional immunoelectrophoresis or so called crossed immunoelectrophoresis as described by Clarke and Freeman, but this estimation takes a lot of time and is not suitable for analysis of a series of samples. Sufficient sensitivity for certain and semiquantitative statements about the content of cows' milk casein in products from sheep's milk are obtained by the technique of radial double diffusion by Ouchterlony in Agarose gels only by the saturation of commercial anti-bovine-casein-serum by means of a suitable protein-standardized sheep's milk extract and its subsequent work-up and lyophilisation to yield a stable serum.

Animals↗

PubMed1991

Heat-induced changes in casein-derived phosphopeptides.

Phosphopeptides derived from casein may function as carriers for calcium and trace elements. In regard to such specific nutritive effects, the heat-induced changes in tryptic phosphopeptides liberated from bovine sodium caseinate as a model system were investigated. Both microwave and oven heating resulted in a marked loss of peptide-bound phosphorous (dephosphorylation) and a decrease of casein-phosphopeptides in the soluble part of the tryptic hydrolysate. It is concluded that hydrolysis of phosphoseryl to seryl residues was the prevailing degradation step to soluble proteolytic products, whereas lysinoalanyl-casein is claimed to be present almost exclusively in the pH 4.6-insoluble part of the tryptic digest.

Animals↗

PubMed1992

Detection of alpha S1-casein in vomit from bottle-fed babies by enzyme-linked immunosorbent assay.

This study describes a sensitive enzyme-linked immunosorbent assay (ELISA) using rabbit anti-bovine alpha S1-casein antibody for the detection of commercial milk and milk-containing vomit. The antibody does not react with other human body fluids such as breast milk. The stability of alpha S1-casein antigenic activity was examined after storage at different temperatures and enzyme digestion. There was no decrease after storage for one year at room temperature but 40% of the activity was lost after 6 months at 37 degrees C. Enzyme digestion (6 hours, 37 degrees C) resulted in 65 approximately 70% loss of activity but the antibody reacted with the peptide fragments of alpha S1-casein. Vomit samples from 3 normal infants were tested by ELISA, and alpha S1-casein could be detected in 1 cm2 stain.

Animals↗

PubMed1978

Structural relatedness of kappa-casein and fibrinogen gamma-chain.

kappa-Caseins, involved in the milk clotting process, and human fibrinogen gamma-chain, involved in the blood clotting process, show structural similarities. Several long kappa-casein sections, together corresponding to 80% of the whole protein molecule, have their counterparts in the gamma-chain of fibrinogen, in that 31--42% of the amino acid residues occupy identical positions. The section of kappa-casein which contains the chymosin-sensitive bond has a counterpart not only in the gamma but also in the Bbeta-chain of fibrinogen. Furthermore, the secondary structures of the kappa-caseins and of the gamma-chain predicted according to the method of Chou and Fasman present several common features.

Amino Acid Sequence↗

PubMed1996

Particle sizes of purified kappa-casein: metal effect and correspondence with predicted three-dimensional molecular models.

kappa-Casein as purified from bovine milk exhibits a rather unique disulfide bonding pattern as revealed by SDS-PAGE. The disulfide-bonded caseins present range from dimer to octamer and above and preparations contain about 10% monomer. All of these heterogenous polymers, however, self-associated into nearly spherical uniform particles with an average radius of 8.9 nm as revealed by negatively stained transmission electron micrographs. Evidence is presented that multivalent cations play a role in the stabilization of these spherical particles. Treatment with EDTA causes disruption of the kappa-casein particles and leads to a broder size distribution as judged by electron microscopy and dynamic light scattering. The size and shape of the particles are in accord with earlier proposed 3D models for kappa-casein that actually predicted participation of divalent cations in the structure.

Amino Acids↗

PubMed1994

Comparison of the three-dimensional molecular models of bovine submicellar caseins with small-angle X-ray scattering. Influence of protein hydration.

To test the applicability of two energy-minimized, three-dimensional structures of the bovine casein submicelle, theoretical small-angle X-ray scattering curves in the presence and absence of water were compared to experimental data. The published method simulates molecular dynamics of proteins in solution by employing adjustable Debye-Waller temperature factors (B factors) for the protein, for the solvent, and for protein-bound water. The programs were first tested upon bovine pancreatic trypsin inhibitor beginning with its known X-ray crystal structure. To approximate the degree of protein hydration previously determined by NMR relaxation experiments (0.01 g water/g protein), 120 water molecules were docked into the large void of the kappa-casein portion of the structure for both the symmetric and asymmetric casein submicelle models. To approximate hydrodynamic hydration (0.244 g water/g protein), 2703 water molecules were added to each of the above structures using the "droplet" algorithm in the Sybyl molecular modeling package. All structures were then energy-minimized and their solvation energies calculated. Theoretical small-angle X-ray scattering curves were calculated for all unhydrated and hydrated structures and compared with experimentally determined scattering profiles for submicellar casein. Best results were achieved with the 120-bound-water structure for both the symmetric and asymmetric submicelle models. Comparison of results for the protein submicelle models with those for the theoretical and literature values of bovine pancreatic trypsin inhibitor demonstrates the applicability of the methodology.

Animals↗