Amino acids content and electrophoretic profile of camel milk casein from different camel breeds in Saudi Arabia

This study aimed to evaluate amino acids content and the electrophoretic profile of camel milk casein from different camel breeds. Milk from three different camel breeds (Majaheim, Wadah and Safrah) as well as cow milk were used in this study.Results showed that ash and moisture contents were significantly higher in camel milk casein of all breeds compared to that of cow milk. On the other hand, casein protein of cow milk was significantly higher compared to that of all camel milk breeds. Molecular weights of casein patterns of camel milk breeds were higher compared to that of cow milk.Essential (Phe, Lys and His) and non-essential amino acids content was significantly higher in cow milk casein compared to the casein of all camel milk breeds. However, there was no significant difference for the other essential amino acids between cow casein and the casein of Safrah breed and their quantities in cow and Safrah casein were significantly higher compared to the other two breeds. Non-essential amino acids except Arg and the essential amino acids (Met, Ile, Lue and Phe) were also significantly higher in cow milk α-casein compared to α-casein from all camel breeds. Moreover, essential amino acids (Val, Phe and His) and the non-essential amino acids (Gly and Ser) content was significantly higher in cow milk β-casein compared to the β-casein of all camel milk breeds and the opposite was true for Lys, Thr, Met and Ile. However, Met, Ile, Phe and His were significantly higher for β-casein of Majaheim compared to the other two milk breeds. The non-essential amino acids (Gly, Tyr, Ala and Asp) and the essential amino acids (Thr, Val and Ile) were significantly higher in cow milk κ-casein compared to that for all camel milk breeds. There was no significant difference among all camel milk breeds in their κ-casein content of most essential amino acids.Relative migration of casein bands of camel milk casein was not identical. The relative migration of α_s-, β- and κ-casein of camel casein was slower than those of cow casein. The molecular weights of α_s-, β- and κ-casein of camel caseins were 27.6, 23.8 and 22.4 KDa, respectively. More studies are needed to elucidate the structure of camel milk.     

The structure of caseins from cow milk]

The paper presents a review of up-to-date data on the primary structure of alphas1-, beta- and kappa-casein of cow milk, gives amino acid substitutes in the genetic forms, arrangement of bitter peptides resulting from enzymic degradation and discusses the possible mechanism of phosphorylation. With respect to the primary structure alimentary specificity of caseins is discussed.     

Chemical and immunochemical characterization of caseins and the major whey proteins of rabbit milk.

Caseins were separated from whey proteins by acid precipitation of skimmed rabbit milk. Whole casein was resolved by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis into three major bands with apparent relative molecular masses (Mr of 31 000, 29 000 and 25 000. On agarose/urea-gel electrophoresis whole casein gave three bands with electrophoretic mobilities alpha, beta and gamma. The three components were purified by DEAE-cellulose chromatography under denaturing and reducing conditions. Each was shown to have a different amino acid, hexose and phosphorus content, as well as non-identical peptide fragments after proteinase digestion. The 31 000 Da (dalton) protein, of alpha-electrophoretic mobility, had a high phosphorus content (4.38%, w/w); the 29 000 Da peptide, of gamma-mobility, had the highest hexose content (2.2%, w/w), contained 0.8 cysteine residue per 100 amino acid residues and was susceptible to chymosin digestion corresponding thus to kappa-casein; the 25 000 Da protein migrated to the beta-position. The rabbit casein complex is composed of at least three caseins, two of which (alpha- and kappa-caseins) are analogous to the caseins from ruminants. Although caseins are poor immunogens, specific antibodies were raised against total and purified polypeptides. The antiserum directed against whole casein recognized each polypeptide, each casein corresponding to a distinct precipitation line. The antisera directed against each casein polypeptide reacted exclusively with the corresponding casein and no antiserum cross-reaction occurred between the three polypeptides. From whey, several proteins were isolated, characterized and used as antigens to raise specific antibodies. An iron-binding protein with an apparent Mr of 80 000 was shown to be immunologically and structurally identical with serum transferrin.     

Separation and partial characterization of guinea-pig caseins.

1. Guinea-pig caseins A, B and C were purified free of each other by a combination of ion-exchange chromatography and gel filtration. 2. Determination of the amino acid composition showed all three caseins to contain a high proportion of proline and glutamic acid, but no cysteine. This apart, the amino acid composition of the three caseins was markedly different, though calculated divergence values suggest that some homology may exist between caseins A and B. Molecular-weight estimates based on amino acid composition were in good agreement with those based on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 3. N-Terminal analysis showed lysine, methionine and lysine to be the N-terminal residues of caseins A, B and C respectively. 4. Two-dimensional separation of tryptic digests revealed a distinctive pattern for each casein. 5. All caseins were shown to be phosphoproteins. The casein C preparation also contained significant amounts of sialic acid, neutral and amino sugars. 6. The results suggest that each casein represents a separate gene product, and that the low-molecular-weight proteins are not the result of a post-translational cleavage of the largest. All were distinctly different from the whey protein alpha-lactalbumin.     

Disulphide bonds in casein micelle from milk

Mammary epithelial cells synthesised and secreted caseins, the major milk proteins in most mammals, as large aggregates called micelles into the alveolar lumen they surround. We investigated the implication of the highly conserved cysteine(s) of kappa-casein in disulphide bond formation in casein micelles from several species. Dimers were found in all milks studied, confirming previous observation in ruminants. More importantly, the study of interchain disulphide bridges in mouse and rat casein micelles revealed that any casein possessing a cysteine is engaged in disulphide bond interchange; these species express four or five cysteine-containing caseins, respectively. We found that the main rodent caseins form both homo- and heterodimers. Additionally, disulphide bond formation among milk proteins was specific since the interaction of the caseins with cysteine-containing whey proteins was not observed in native casein micelles.     

Free Amino Acids and Phosphorylethanolamine in Milk Whey of Cow

Free amino acids in the milk of cow were investigated in comparison with those in the plasma. The concentrations of most free amino acids in the milk except for a few amino acid were lower than those in the plasma. It appears that the percentage of each amino acid in the milk against the corresponding amino acid in the plasma is the reflexion of casein synthesis in the mammary gland. Nutritional alteration influenced on the level of some amino acids in the milk. Free phosphorylserine, glycerylphosphorylethanolamine, and phosphorylethanolamine were observed in the milk. Phosphorylethanolamine was present in significantly high concentration in one animal as control, whereas was almost absent in another animal as experimental.     

Substrate specificity of the cell envelope-located proteinase of Lactococcus lactis subsp. lactis NCDO 763.

1. The specificity of the cell envelope-located proteinase of Lactococcus lactis subsp. lactis NCDO 763 towards caseins has been submitted to a statistical study. Positive and negative relations have been evidenced between several amino acids and positions P6 to P'2 of the cleaved bonds. 2. Fragment 1-23 of alpha s1 and oxidized B chain of insulin are well cleaved by the proteinase while CMP (fragment 106-169 of kappa-casein) is a poor substrate. 3. Comparison with other cell envelope-located proteinase has been done. The enzyme of the strain 763 hydrolyses alpha s1-casein and fragment 1-23 of alpha s1-casein as the enzyme of the strain Sk11 and beta-casein as the enzyme of the strain Wg2. 4. The specificity of these proteinases and the comparison of their amino acid sequences let us postulate a more complex substrate binding area for these lactococcal proteinases than for the subtilisin.     

Identification of human milk kappa-casein on polyacrylamide gels by differential staining with Ethyl-Stains-all and chymosin sensitivity

Ethyl-Stains-all (ESA), a cationic carbocyanine dye that stains phosphorylated, sialylated, and unmodified proteins differentially, was used to stain a human casein fraction enriched for its kappa-casein-like characteristics. The staining properties and chymosin sensitivity of this fraction were compared with those of human milk and bovine casein proteins. Phosphorylated human and bovine beta caseins stained blue with ESA. The sialic acid-containing bovine kappa-casein stained blue-green. The human kappa-like fraction was enriched for a protein that stained blue-green with ESA. Both bovine kappa-casein and the human blue-green-staining protein were susceptible to chymosin digestion at lower concentrations of chymosin than that required for digestion of beta-caseins. In each case, following chymosin digestion, a green-staining peptide of lower molecular weight replaced the original protein and para-kappa-casein was formed. Identification of human kappa-casein on SDS-polyacrylamide gels was based on its differential staining with ESA and chymosin sensitivity with respect to beta-casein.     

Cloned transgenic cattle produce milk with higher levels of beta-casein and kappa-casein

To enhance milk composition and milk processing efficiency by increasing the casein concentration in milk, we have introduced additional copies of the genes encoding bovine beta- and kappa-casein (CSN2 and CSN3, respectively) into female bovine fibroblasts. Nuclear transfer with four independent donor cell lines resulted in the production of 11 transgenic calves. The analysis of hormonally induced milk showed substantial expression and secretion of the transgene-derived caseins into milk. Nine cows, representing two high-expressing lines, produced milk with an 8-20% increase in beta-casein, a twofold increase in kappa-casein levels, and a markedly altered kappa-casein to total casein ratio. These results show that it is feasible to substantially alter a major component of milk in high producing dairy cows by a transgenic approach and thus to improve the functional properties of dairy milk.     

Isolation and some effects of functional, low-phenylalanine kappa-casein expressed in the milk of transgenic rabbits

Patients suffering certain metabolic diseases (e.g. phenylketonuria) need a low-phenylalanine diet throughout their lives. Transgenic rabbits were created to express low-phenylalanine kappa-casein in their milk. The aim was to demonstrate for the first time the feasibility of producing a modified milk protein in addition to normal milk proteins. A gene construct containing the coding region of the rabbit kappa-casein gene was modified by site-specific oligonucleotide directed mutagenesis. Four of the five phenylalanine amino acids present in the mature protein were mutated and the gene construct was used to create two transgenic rabbit lines. The transgenic rabbits produced the recombinant kappa-casein at a high level in their milk causing a reduction in the average size of the casein micelles. The low-phenylalanine kappa-casein was digestible with chymosin and it was separated from its native counterpart and from the other milk proteins by a one-step HPLC method on a reversed-phase column. In the future, low-phenylalanine casein produced in transgenic animals could be used as dietary replacements to meet the special requirements of certain consumer groups.     

Nucleotide sequence of cloned cDNA coding for mouse epsilon casein

We isolated cDNA clones, which correspond to the mRNA coding for the smallest of the seven mouse caseins. From the nucleotide sequence of the cDNA we deduced the amino acid sequence of the protein, which we named epsilon casein. Mouse epsilon casein and cow alpha s2 casein show amino acid sequence homologies in the N-terminal region of the mature protein. The signal peptide of mouse epsilon casein shows in length and sequence remarkable homology to the signal peptides of the calcium-precipitable caseins of other species. In accordance with this group of caseins mouse epsilon casein contains in the sequence -Ser-Ser-Glu-Glu- a site for potential multiple phosphorylation.     

Expression of kappa-casein in normal and neoplastic rat mammary gland is under the control of prolactin

An 820-nucleotide-long cDNA clone for the kappa-casein (the casein micelle-stabilizing protein) from rat mammary gland was isolated, and its nucleotide sequence was determined. The deduced amino acid sequence from the nucleotide sequence revealed a signal peptide, 21 amino acids long, and a mature protein of 157 amino acids. The signal peptide of rat kappa-casein was highly homologous to that of the precursor to ovine kappa-casein. However, little homology was apparent when the mature kappa-casein protein sequences from ovine or bovine sources were compared with rat kappa-casein. The kappa-casein mRNA content of the mammary tissue was found to increase during its functional differentiation. Prolactin appears to modulate the production of kappa-casein mRNA. Mammary glands of virgin females had no detectable kappa-casein mRNA; however, a marked induction of kappa-casein mRNA was obtained by intravenous infusion of prolactin. Mammary carcinomas did not follow the same pattern. 7,12-Dimethylbenz[a]anthracene-induced mammary carcinomas had normally low levels of kappa-casein mRNA, but intravenous prolactin infusion increased the levels by 2-fold. The MTW9 mammary carcinoma that grows only in the presence of high levels of mammotropic hormones had kappa-casein mRNA content equivalent to that in 10-day lactating rat mammary gland. Continuous venous infusion of prolactin to MTW9 mammary carcinoma did not modify the kappa-casein mRNA levels. Nitrosomethylurea-induced mammary carcinomas had no detectable kappa-casein mRNA, and intravenous prolactin infusion was unable to induce it.     

草地藏系绵羊乳游离氨基酸质量分数及蛋白遗传多态性研究

用反相高效液相色谱法检测草地藏系绵羊乳游离氨基酸质量分数,用聚丙烯酰胺凝胶电泳法研究乳蛋白组分及其遗传多态性。结果表明:草地藏系绵羊乳中检测到17种游离氨基酸,其中质量分数最高的为Arg;与金堂黑山羊乳比较17种游离氨基酸中,甲硫氨酸的质量分数极显著高于金堂黑山羊(p<0.01),而天冬氨酸、甘氨酸、赖氨酸、谷氨酸、苏氨酸、丙氨酸和缬氨酸的质量分数均显著低于金堂黑山羊(p<0.05),其余9种游离氨基酸质量分数未发现明显差异,但两种羊乳中的必需氨基酸总量基本相同,差异不明显(p>0.05)。草地藏系绵羊乳蛋白组分主要包括α-La、β-Lg、CN、IgG等,CN的相对质量分数约50%~52%;研究还发现4种分子量类型的乳上皮粘蛋白MUC1,分子量分别为214kD、209kD、207kD、205kD;CN、β-Lg均未检测到多态性,说明草地藏系绵羊乳蛋白遗传多态性较为贫乏。     

Involvement of enzyme-substrate charge interactions in the caseinolytic specificity of lactococcal cell envelope-associated proteinases.

Three series of oligopeptides were synthesized to investigate the proposal that a major factor in determining the differences in specificity of the lactococcal cell surface-associated proteinases against caseins is the interactions between charged amino acids in the substrate and in the enzyme. The sequences of the oligopeptides were based on two regions of kappa-casein (residues 98 to 111 and 153 to 169) which show markedly different susceptibilities to PI- and PIII-type lactococcal proteinases. In each series, one oligopeptide had an identical sequence to that of the kappa-casein region, while in the others, one or more charged residues were substituted by an amino acid of opposite charge, i.e., His<-->Glu. Generally, substitution of His by Glu in the oligopeptides corresponding to residues 98 to 111 of kappa-casein resulted in reduced cleavage of susceptible bonds by the PI-type proteinase and increased cleavage of susceptible bonds by the PIII-type proteinase. In the case of the oligopeptide corresponding to residues 153 to 169 of kappa-casein, one major cleavage site was evident, and the bond was hydrolyzed by both types of proteinase (even though this sequence in kappa-casein itself is extremely resistant to the PI-type enzyme). Substitution of Glu by His in this oligopeptide, even in the P7 position, resulted in increased cleavage of the bond by the PI-type proteinase and reduced cleavage by the PIII-type proteinase. C-terminal truncation of this oligopeptide resulted in a 100-fold decrease in the rate of hydrolysis of the susceptible bond and a change in the pattern of cleavage.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amyloid fibril formation by bovine milk kappa-casein and its inhibition by the molecular chaperones alphaS- and beta-casein

Caseins are a unique and diverse group of proteins present in bovine milk. While their function is presumed to be primarily nutritional, caseins have a remarkable ability to stabilize proteins, i.e., to inhibit protein aggregation and precipitation, that is comparable to molecular chaperones of the small heat-shock protein (sHsp) family. Additionally, sHsps have been shown to inhibit the formation of amyloid fibrils. This study investigated (i) the fibril-forming propensities of casein proteins and their mixture, sodium caseinate, and (ii) the ability of caseins to prevent in vitro fibril formation by kappa-casein. Transmission electron microscopy (TEM) and X-ray fiber diffraction data demonstrated that kappa-casein readily forms amyloid fibrils at 37 degrees C particularly following reduction of its disulfide bonds. The time-dependent increase in thioflavin T fluorescence observed for reduced and nonreduced kappa-casein at 37 degrees C was suppressed by stoichiometric amounts of alphaS- and beta-casein and by the hydrophobic dye 8-anilino-1-naphthalene sulfonate; the inhibition of kappa-casein fibril formation under these conditions was verified by TEM. Our findings suggest that alphaS- and beta-casein are potent inhibitors of kappa-casein fibril formation and may prevent large-scale fibril formation in vivo. Casein proteins may therefore play a preventative role in the development of corpora amylacea, a disorder associated with the accumulation of amyloid deposits in mammary tissue.     

Isolation and properties of human kappa-casein

Human kappa-casein was isolated from human whole casein by gel filtration with Sephadex G-200 and hydroxylapatite chromatography in the presence of sodium dodecyl sulfate (SDS). The kappa-casein was calcium-insensitive and did stabilize human beta-casein and bovine alpha s1-casein against precipitation by calcium ions. Formation of micelles from human beta- and kappa-caseins, and calcium ions was confirmed by electron microscopic observation. On SDS-polyacrylamide gel electrophoresis (SDS-PAGE), a single band was obtained. The formation of para-kappa-caseins by chymosin was confirmed by SDS-PAGE. Two para-kappa-caseins with apparent molecular weights of 13,000 and 11,000 appeared. The molecular weight of intact human kappa-casein was estimated to be approximately 33,000. The human kappa-casein contained about 40% carbohydrate (15% galactose, 3% fucose, 15% hexosamines, and 5% sialic acid) and 0.10% (1 mol/mol) phosphorus. Its amino acid composition was similar to that of bovine kappa-casein except for serine, glutamic acid, and lysine contents.     

Uptake of free plasma amino acids by the lactating cow''s udder and amino acid composition of udder lymph

1. Total α-amino N and the amounts of 24 ninhydrin-positive substances were determined in several samples of plasma and lymph from the cow's udder. The arteriovenous differences of these substances across the mammary glands were measured in several experiments performed on lactating cows and in one experiment on a `dry' cow. Udder lymph obtained from live lactating cows by a lymph fistula and taken after killing lactating cows was analysed. 2. The concentrations of the individual free amino acids in udder lymph obtained from the live cow were similar to those found in cow's plasma. The concentrations of many amino acids in udder lymph taken immediately after death were two- to four-fold higher than those of the corresponding amino acids in udder lymph obtained from the live cow. 3. Most amino acids of the blood showed a considerable decrease in concentration by passage across the lactating mammary gland. Ornithine, a non-casein amino acid, showed arteriovenous differences of up to 60% of the arterial plasma concentration. No substantial amino acid uptake by the udder could be demonstrated in the experiment on the non-lactating cow. 4. The arteriovenous differences obtained for arginine, glutamine, isoleucine, leucine, lysine, valine, threonine and histidine were probably large enough to provide all the respective amino acid residues in milk protein. 5. The uptake of aspartic acid, asparagine, glutamic acid, serine and proline by the lactating cow's udder was not sufficient to account for all these respective amino acid residues found in milk protein.     

Similarities and specificities of fungal keratinolytic proteases: comparison of keratinases of Paecilomyces marquandii and Doratomyces microsporus to some known proteases

Based on previous screening for keratinolytic nonpathogenic fungi, Paecilomyces marquandii and Doratomyces microsporus were selected for production of potent keratinases. The enzymes were purified and their main biochemical characteristics were determined (molecular masses, optimal temperature and pH for keratinolytic activity, N-terminal amino acid sequences). Studies of substrate specificity revealed that skin constituents, such as the stratum corneum, and appendages such as nail but not hair, feather, and wool were efficiently hydrolyzed by the P. marquandii keratinase and about 40% less by the D. microsporus keratinase. Hydrolysis of keratin could be increased by the presence of reducing agents. The catalytic properties of the keratinases were studied and compared to those of some known commercial proteases. The profile of the oxidized insulin B-chain digestion revealed that both keratinases, like proteinase K but not subtilisin, trypsin, or elastase, possess broad cleavage specificity with a preference for aromatic and nonpolar amino acid residues at the P-1 position. Kinetic studies were performed on a synthetic substrate, succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. The keratinase of P. marquandii exhibited the lowest Km among microbial keratinases reported in the literature, and its catalytic efficiency was high in comparison to that of D. microsporus keratinase and proteinase K. All three keratinolytic enzymes, the keratinases of P. marquandii and D. microsporus as well as proteinase K, were significantly more active on keratin than subtilisin, trypsin, elastase, chymotrypsin, or collagenase.     

Inhibition of subtilisin BPN' by reaction site P1 mutants of Streptomyces subtilisin inhibitor

It has been shown that the P1 site (the center of the reactive site) of protease inhibitors corresponds to the specificity of the cognate protease, and consequently specificity of Streptomyces subtilisin inhibitor (SSI) can be altered by substitution of a single amino acid at the P1 site. In this paper, to investigate whether similar correlation between inhibitory activity of mutated SSI and substrate preference of protease is observed for subtilisin BPN', which has broad substrate specificity, a complete set of mutants of SSI at the reaction site P1 (position 73) was constructed by cassette and site-directed mutagenesis and their inhibitory activities toward subtilisin BPN' were measured. Mutated SSIs which have a polar (Ser, Thr, Gln, Asn), basic (Lys, Arg), or aromatic amino acid (Tyr, Phe, Trp, His), or Ala or Leu, at the P1 site showed almost the same strong inhibitory activity toward subtilisin as the wild type (Met) SSI. However, the inhibitory activity of SSI variants with an acidic (Glu, Asp), or a beta-branched aliphatic amino acid (Val, Ile), or Gly or Pro, at P1 was decreased. The values of the inhibitor constant (Ki) of mutated SSIs toward subtilisin BPN' were consistent with the substrate preference of subtilisin BPN'. A linear correlation was observed between log(1/Ki) of mutated SSIs and log(1/Km) of synthetic substrates. These results demonstrate that the inhibitory activities of P1 site mutants of SSI are linearly related to the substrate preference of subtilisin BPN', and indicate that the binding mode of the inhibitors with the protease may be similar to that of substrates, as in the case of trypsin and chymotrypsin.(ABSTRACT TRUNCATED AT 250 WORDS)     

A highly active and oxidation-resistant subtilisin-like enzyme produced by a combination of site-directed mutagenesis and chemical modification

The subtilisins are known to be susceptible to chemical oxidation due to the conversion of Met222 into the corresponding sulfoxide. A number of derivatives with resistance towards oxidation have previously been prepared by replacement of this group with the other 19 amino acid residues. Unfortunately, the activities of these enzymes were of the order of 1-10% of that obtained with the wild-type enzyme. In contrast, the oxidation-labile cysteine mutant exhibited much higher activity, suggesting that this is associated with the presence of a sulphur atom in the amino acid at position 222. It is shown here that it is possible to maintain a sulphur atom in the amino acid at position 222 without the enzyme becoming labile towards oxidation. A subtilisin from Bacillus lentus, subtilisin 309, in which Met222 was replaced with a cysteinyl residue by site-directed mutagenesis was modified with thioalkylating reagents. Treatment of such enzyme derivatives with H2O2 revealed that their stabilities towards oxidation had increased significantly compared to both wild-type and unmodified [Cys222]subtilisin. One of the chemically modified enzyme derivatives, [Me-S-Cys222]subtilisin, exhibited a kcat/Km value of 56% of that obtained with the wild-type enzyme when assayed against the substrate Suc-Ala-Ala-Pro-Phe-NH-Ph-NO2 (Suc, succinyl) and it exhibited 89% activity when tested in an assay with dimethyl casein as a substrate. The corresponding values obtained for unmodified [Cys222]subtilisin were lower, i.e. 39% for the dimethyl casein activity and 46% for the kcat/Km for the hydrolysis of Suc-Ala-Ala-Pro-Phe-NH-Ph-NO2. This demonstrates the feasibility of replacing the oxidation-labile methionyl residue group in a subtilisin enzyme with a group stable towards oxidation without substantially reducing the activity.