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PubMed1985

Kinetic study of the action of bovine chymosin and pepsin A on bovine kappa-casein.

A study was carried out to determine the Michaelian parameters relative to the action of chymosin and pepsin A on bond Phe105-Met106 of bovine kappa0-casein (carbohydrate-free fraction in micellar state). The reaction was performed in citrate buffer, pH 6.2, at 30 degrees C. The reaction mixture was analysed by reverse phase HPLC. Dosages of peptide 106-169 (caseino macropeptide) at different reaction times from recordings of its absorbance at 220 nm gave the initial rates of reaction at each substrate concentration. From these values the following parameters were determined: kcat = 68.5 s-1, Km = 0.048 mM, kcat/Km = 1,413 mM-1 s-1 for chymosin, and kcat = 45 s-1, Km = 0.018 mM, kcat/Km = 2,439 mM-1 s-1 for pepsin A. For chymosin they are similar to those obtained previously in dimethyl glutarate buffer, pH 6.6, at 30 degrees C, using fragment 98-111 of kappa-casein as substrate. It can thus be concluded that neither the micellar state nor the presence of the whole peptide chain of kappa-casein (our conditions) significantly affect the action of chymosin on fragment 98-111, which seems to contain all information that makes bond 105-106 highly sensitive to chymosin. For pepsin A, only the information contained in fragment 103-108 appears to be required.

Amino Acids↗

PubMed1989

Casein kinase II activity and polyamine-stimulated protein phosphorylation of cytosolic and plasma membrane proteins in bovine sperm.

A highly purified preparation of sperm cytosolic protein kinase was obtained by repeated chromatography with phosphocellulose. The preferred substrate of the enzyme was casein and the activity was not stimulated by added Ca2+, calmodulin, or cAMP. With casein as substrate, both ATP and GTP served as phosphate donors and the activity was inhibited by low micromolar heparin and stimulated by low millimolar spermine and spermidine. These properties are characteristic of casein kinase II from other cells. Endogenous protein substrates of the enzyme in sperm cytosolic fractions and in plasma membranes were demonstrated by incubating the preparations with [gamma-32P]GTP, under conditions unfavorable to other protein kinases, and analyzing the products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Spermine greatly enhanced the phosphorylation of three (55, 92, and 106 kDa) proteins in both cytosolic and plasma membrane preparations. Our results indicate that polyamines play a role in modulating the phosphorylation state of proteins in sperm and may further regulate sperm function through this mechanism.

Animals↗

PubMed1990

Interactions of triply phosphorylated human beta-casein: monomer characterization and hydrodynamic studies of self-association.

The triply phosphorylated form of human beta-casein comprises about 15% of that fraction and is thus a significant component about midway between the two extremes of zero and five phosphoryls. Its partial specific volume, v, of 0.74 +/- 0.01 and absorbancy, E1% 1 cm, 280 nm, of 6.2 +/- 0.2 are almost identical to the other human beta-caseins. Equilibrium dialysis gave an average of 3.1 +/- 0.4 major Ca2+ binding sites at 37 degrees C with Kdiss = 8.6 x 10(-4) M. Sedimentation and viscosity at low temperatures or in 3.3 M urea suggested a prolate ellipsoidal monomer with 1.4 g H2O/g protein, 10 nm in length and 1.4 nm in width. The concentrated charge of the phosphoryls may be near one end of the ellipsoid, allowing the molecules to align with the flow in the viscometer at low concentration but, due to intermolecular electrostatic interactions, not when concentration is high. This would provide a reason for the heretofore unexplained curvature in the plots of reduced viscosity, eta red, vs beta-casein protein concentration. Self-association increased with temperature. At 37 degrees C in low salt buffer, s20,W was 16 S, which increased to about 33 S as ionic strength, I, was increased to 0.2 and above. At the same time, eta red in low salt buffer decreased from about 22 ml/g at 4 degrees C to a constant value of about 5 ml/g above 23 degrees C. A similar value for eta red at 37 degrees C, which was almost independent of protein concentration, was obtained at I greater than 0.25, giving an extrapolated intrinsic viscosity value of [eta] = 4.0 ml/g. Using this value and assuming a spherical aggregate, calculations suggest a radius of 9 nm with about 48 monomers and 0.86 g H2O/g protein.

Calcium↗

PubMed1990

Characteristics of the interaction of calcium with casein submicelles as determined by analytical affinity chromatography.

Interaction of calcium with casein submicelles was investigated in CaCl2 and calcium phosphate buffers and with synthetic milk salt solutions using the technique of analytical affinity chromatography. Micelles that had been prepared by size exclusion chromatography with glycerolpropyl controlled-pore glass from fresh raw skim milk that had never been cooled, were dialyzed at room temperature against calcium-free imidazole buffer, pH 6.7. Resulting submicelles were covalently immobilized on succinamidopropyl controlled-pore glass (300-nm pore size). Using 45Ca to monitor the elution retardation, the affinity of free Ca2+ and calcium salt species was determined at temperatures of 20 to 40 degrees C and pH 6.0 to 7.5. Increasing the pH in this range or increasing the temperature strengthened the binding of calcium to submicelles, similar to previous observations with individual caseins. However, the enthalpy change obtained from the temperature dependence was considerably greater than that reported for alpha s1- and beta-caseins. Furthermore, the elution profiles for 45Ca in milk salt solutions were decidedly different from those in CaCl2 or calcium phosphate buffers and the affinities were also greater. For example, at pH 6.7 and 30 degrees C the average dissociation constant for the submicelle-calcium complex is 0.074 mM for CaCl2 and calcium phosphate buffers, vs 0.016 mM for the milk salt solution. The asymmetric frontal boundaries and higher average affinities observed with milk salts may be due to binding of calcium salts with greater affinity in addition to the binding of free Ca2+ in these solutions.

Animals↗

PubMed1991

Tertiary and quaternary structural differences between two genetic variants of bovine casein by small-angle X-ray scattering.

The casein complexes of bovine milk consist of four major protein fractions, alpha s1, alpha s2, beta, and kappa. Colloidal particles of casein (termed micelles) contain inorganic calcium and phosphate; they are very roughly spherical with an average radius of 650 A. Removal of Ca2+ leads to the formation of smaller protein aggregates (submicelles) with an average radius of 94 A. Two genetic variants, A and B, of the predominant fraction, alpha s1-casein, result in milks with markedly different physical properties, such as solubility and heat stability. To investigate the molecular basis for these differences, small-angle X-ray scattering was performed on the respective colloidal micelles and submicelles. Scattering curves for submicelles of both variants showed multiple Gaussian character; data for the B variant were previously interpreted in terms of two concentric regions of different electron density, i.e., a "compact" core and a relatively "loose" shell. For the submicelle of A, there was a third Gaussian, reflecting a negative contribution due to interparticle interference. Molecular parameters for submicelles of both A and B are in agreement with hydrodynamic data in the literature. Data for the micelles, for which scattering yields cross-sectional information, were fitted by a sum of three Gaussians for both variants; for these, the corresponding two lower radii of gyration represent the two concentric regions of the submicelles, while the third reflects the average packing of submicelles within the micellar cross section. Most of the molecular parameters obtained showed small but consistent differences between A and B, but for submicelles within the micelle several differences were particularly notable: A has a greater molecular weight for the "compact" region of the constituent submicelle (82,000 vs 60,000) and a much greater submicellar packing number (6:1 vs 3:1). Reasons for these and other differences are to be sought in sequence differences and in differences in calcium-binding sites and charge distribution.

Animals↗

PubMed1975

A comparison of the interfacial interactions of the apoprotein from high density lipoprotein and beta-casein with phospholipids.

The conformations adopted by beta-casein and the total apoprotein from serum high density lipoprotein when spread at the air-water interface are compared; the monolayer data are consistent with the apoprotein being alpha-helical and the beta-casein being disordered with segments distributed in loops and trains. The penetration of these hydrophobic proteins into phosphatidylcholine monolayers in different physical states was investigated. More protein can penetrate into monolayers when they are in the liquid-expanded state; for penetration at constant total surface area the lateral compressibility of the lipid is an important factor. The charge and conformation of the polar group of the phospholipid does not have a major influence on the interaction. The mixed films of lipid and protein have a mosaic structure; probably the beta-casein is in a compressed state whereas the apoprotein is extended as alpha-helices in the plane of the interface. The chain-length depedences of the interaction of the apoprotein with phosphatidylcholine monolayers and bilayers are different; when the apoprotein binds to bilayers of shorter-chain phosphatidylcholines it alters the shape of the lipid-water interface whereas with monolayers the interface remains planar throughout.

Apoproteins↗

PubMed1981

A study with model substrates of the structure of the sites phosphorylated by rat liver casein kinase TS.

Two new sites phosphorylated by rat liver cyclic AMP-independent casein kinase TS have been identified in denatured pepsin and soybean antiprotease C-II, exhibiting the sequences: Cys-Ser-Ser(P)-Ile-Asp-Ser and His-Ser3(P)-Asp-Asp-Glu, respectively. Their phosphorylation efficiency has been compared to that of previously identified sites and the effects of chemical modifications in the vicinity of the phosphorylatable residue have been studied. The results obtained support the following conclusions: 1. All sites affected by casein kinase TS conform to the sequence: Ser/Thr-X-Glu/Asp which is also believed to be required by the mammary gland casein kinase. Threonine appears to be less suitable for phosphorylation then serine. The presence of some additional residues on the C-terminal side also appears to be required. 2. X can be either an additional acidic residue or a natural one, but not a basic residue. The contiguity of an acidic cluster to the C-terminal side of the target greatly improves the phosphorylation efficiency. 3. The residues N-terminal to the target one do not seem to be relevant for determining the site recognition by the protein kinase. 4. The predicted secondary structure constantly occurring at the phosphorylation sites is the beta-turn: apparently the bend must include both the target residue and the acidic determinant at the n + 2 position.

Amino Acid Sequence↗

PubMed1984

Inhibition of lipid peroxidation by casein. Evidence of molecular encapsulation of 1,4-pentadiene fatty acids.

The capability of cyclodextrins to form molecular inclusion complexes with linoleate appeared in a lipoxygenase-linoleate model reaction as inhibition of oxygenation. The inhibited rates were established instantaneously upon addition of the complexant and maintained until linoleate was exhausted. Total cessation of the reaction was not obtained with cyclodextrins. All these features were reproduced also in casein-inhibited reaction mixtures. Both casein and cyclodextrins protected linoleate also against autoxidation although they did not change free radical generation by xanthine oxidase or Fe2+ reactions. Since neither of the inhibitors affected the enzyme directly, casein may also act by forming linoleate complexes which via a standing equilibrium reduce the oxidizable monomer fatty acids and cause substrate-limited reaction rates. Comparisons at acidic and alkaline pH, in the presence of increasing amounts of the complexants, detergent and hydroperoxides supported this view.

Caseins↗

PubMed1981

Effect of chymosin action on the hydrodynamic diameter of casein micelles.

Quasi-elastic light scattering shows an initial decrease of about 5 nm in the hydrodynamic radius of casein micelles after adding chymosin, assuming the decrease to be equal for all micelles. This is consistent with the hypothesis that casein micelles have a hairy outer layer that is partly made up of the caseino-macropeptide part of kappa casein.

Caseins↗

PubMed1984

Phosphorylation of rat heart ornithine decarboxylase by type-2 casein kinase.

Highly purified preparations of rat heart ornithine decarboxylase are readily phosphorylated by rat liver type-2 casein kinase-TS at the same 54 KDa protein band which is also radiolabeled by 3H-DFMO. The reaction, which is stimulated by polylysine leads to the incorporation of up to 0.8 mol P/mol ornithine decarboxylase at seryl residue(s) included in a single 8.6 KDa CNBr fragment. Partially purified preparations of ornithine decarboxylase contain a type-2 casein kinase which promotes the phosphorylation of ornithine decarboxylase at the same CNBr fragment affected by rat liver casein kinase-TS.

Animals↗

PubMed1989

Ionic strength-dependent interaction of rat liver casein kinase I with plasma membrane.

Casein kinase I binding to rat liver plasma membrane was rapidly released from membrane by increasing the ionic strength above physiological level. The released activities at 250-300 mM NaCl were 3-4-fold higher than those obtained under lower ionic strength below 100 mM NaCl. This reaction occurred nonenzymatically and was reversible. By lowering the ionic strength from 250 mM to 50 mM NaCl by dilution at least 50% of the released enzyme was rebound to plasma membrane. By gel filtration analysis, most of the released enzyme activity under higher NaCl concentration was recovered around the molecular mass of 35,000 which corresponded to that of casein kinase I. This enzyme showed the tendency to aggregate under lower ionic strength (50 mM NaCl), but existed as monomer under higher ionic strength (250 mM NaCl). These results suggest that the release of casein kinase I from plasma membrane and the rebinding to membrane induced by the alteration of ionic strength seem to be an important regulatory mechanism in determining the subcellular distribution of this enzyme.

Animals↗

PubMed1991

The phosphotransferase activity of casein kinase II is required for its physiological function in vivo.

We have previously shown that the inviability associated with disruption of both catalytic subunits of casein kinase II in Saccharomyces cerevisiae can be rescued by plasmids expressing the catalytic subunit of the Drosophila enzyme (Padmanabha et al., 1990, Mol. Cell. Biol. 10, 4089). Here we describe the construction of mutant forms of the Drosophila catalytic subunit in which residues known to be crucial for catalytic activity in other protein kinases have been altered by site-directed mutagenesis. Mutation of either Lys66 or Asp173, which correspond to Lys72 and Asp184 of cAMP-dependent protein kinase, respectively, yields a casein kinase II catalytic subunit which fails to rescue a yeast strain lacking both endogenous catalytic subunit genes. The data indicate that the phosphotransferase activity of casein kinase II is required for its physiological function in vivo.

Animals↗

PubMed1991

Casein kinase I in bovine sperm: purification and characterization.

A highly purified preparation of sperm casein kinase I was obtained by sequential chromatography with phosphocellulose, gel filtration on sephacryl S-300, Affi-gel blue and DEAE-Cellulose. The chromatographic behavior and properties of the enzyme suggest that the sperm enzyme is similar to casein kinase I from other tissues. Antibodies against calf thymus casein kinase I cross-react with the sperm enzyme. A special feature of the sperm enzyme is that the activity is stimulated by spermine.

Animals↗

PubMed1987

Stimulation of 5-lipoxygenase activity in polymorphonuclear leukocytes of rats by caseinate treatment.

In the present study, the lipoxygenase activity in polymorphonuclear (PMN) leukocytes of rats was characterized, and the change in lipoxygenase activity in peritoneal and peripheral PMN leukocytes challenged by caseinate was investigated. Peritoneal PMN leukocytes produced 5-HETE and LTB4 as the major 5-lipoxygenase metabolites from arachidonic acid. The 5-lipoxygenase activity was calcium dependent. Caseinate treatment in rats significantly stimulated the 5-lipoxygenase activity in peripheral and peritoneal PMN leukocytes. Although it also stimulated the cyclooxygenase metabolic pathway, the effect was not as evident as that on the 5-lipoxygenase pathway. Since LTB4 is a potent chemotactic factor for PMN leukocyte migration, the present results might explain the mechanism on the PMN leukocyte infiltration caused by caseinate treatment.

Animals↗

PubMed1976

[The primary structure of bovine alpha s2 caseins].

The bovine alphas2-, alphas3-, alphas4- and alphas6-caseins [1] were isolated. The 4 proteins had the same amino-acid composition and C-terminal sequence, but different phosphorus contents. From a mixture of these proteins (designated as 'alphas2-complex') and from alphas3-casein a single and identical N-terminal sequence was obtained by Edman degradation. It seems therefore that the 4 proteins have the same peptide chain and only differ in their phosphorus content. For this reason we propose to modify the nomenclature of Annan and Mason [1] and to use in future the single term alphas2 to designate the caseins which have been previously called alphas2,alphas3,alphas4 and alphas6 by these authors. The study of the primary structure of the peptide chain, which has confirmed these results, was undertaken on the S-carboxymethylated alphas2-complex. From a cyanogen bromide digest and from a tryptic hydrolyzate of the alphas2-complex, 5 and 25 peptides were obtained respectively, both sets of peptides accounting for the whole peptide chain. Examination of the tryptic peptides containing methionine combined with the N- and C-terminal sequences of the alphas2-complex and some CNBr peptides, gave the order of the CNBr peptides, H.CN4--CN2--CN5--CN1--CN3.OH, which contain 4, 22, 115, 49 and 17 residues respectively. A partial sequence accounting for half of the peptide chain of the alphas2-complex is given. This peptide chain is likely composed of 207 amino-acid residues.

Amino Acid Sequence↗

PubMed1983

Effect of sodium butyrate on the stimulation of casein gene expression by prolactin.

Sodium butyrate, but not isobutyrate, inhibits prolactin action on the induction of casein synthesis and casein mRNA accumulation in rabbit mammary explants. Sodium butyrate specifically prevents the generation of the prolactin relay which can be released from isolated membranes incubated with prolactin and which stimulates directly casein gene transcription when added to isolated mammary nuclei. This indicates that sodium butyrate exerts its inhibitory action essentially at the membrane level.

Animals↗

PubMed1986

Chemical characterization and opioid activity of an exorphin isolated from in vivo digests of casein.

The in vivo formation of an opioid peptide (exorphin) derived from beta-casein has been proved for the first time. It was isolated from duodenal chyme of minipigs after feeding with the milk protein casein. The exorphin has been identified as a beta-casein fragment by end-group determinations and qualitative amino acid analysis of the purified peptide. This peptide, named beta-casomorphin-11, displayed substantial opioid activity in an opiate receptor-binding assay.

Amino Acids↗

PubMed1987

C-terminal labelling of beta-casein.

This paper is the first to report specific labelling of a native protein at its C-terminal end by carboxypeptidase Y-catalyzed transpeptidation between beta-casein and tritiated Phe amide. A tryptic digest of the radiolabelled protein was resolved by reversed-phase HPLC and a single labelled peptide was isolated therefrom. Sequence determination and FAB mass spectrometry showed that the last 2 residues (Val-209, Ile-208) of beta-casein had been deleted and Ile 207 substituted by Phe, deamidation presumably occurring after transpeptidation. Identical results were obtained by transpeptidating the isolated C-terminal tryptic heptapeptide (203-209) of native beta-casein.

Carboxypeptidases↗