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1.
The content of alpha-lactalbumin and three species of caseins, 42K, 29K, and 25K, have been measured along with the levels and activities of their mRNAs in the rat mammary gland. Changes in these values were followed during gestation and lactation. An increment of 3- to 4-fold over the virgin level was observed for both alpha-lactalbumin and 42K casein during the 1st day of gestation. From this point on, the level of 42K remained unchanged during the 1st week of gestation and increased thereafter. After the increment of the 1st day, the alpha-lactalbumin content decreased rapidly during the 2nd day of gestation, continued to decrease more slowly until the 12th day, and then started to increase thereafter. During the 2nd and 3rd week of gestation. the amounts of alpha-lactalbumin within the gland increased continuously but not uniformly and caseins accumulated rapidly with a tendency to plateau around the 13th to 16th day of gestation. The relative proportions remained, respectively, 42K greater than 29K greater than 25K greater than alpha-lactalbumin until parturition. At the onset of lactation, both alpha-lactalbumin and casein content increased sharply, the relative proportion for caseins changed to 42K greater than 25K greater than 29K greater than alpha-lactalbumin and remained so throughout the lactation period. alpha-Lactalbumin and casein mRNA activity, as judged by the wheat germ translational system, remained unchanged during the 1st week of gestation, then showed a steady but not uniform increase from the 7th day of gestation until parturition. These activities increased sequentially during lactation, alpha-lactalbumin reaching a plateau by the 1st week, caseins between the 1st and 2nd week, and other mRNAs by the end of the 2nd week of lactation. By the 21st day of lactation, the activity of all mRNA had declined. The levels of alpha-lactalbumin mRNA and 16 S doublet casein mRNA sequences measured with the cDNA probes increased by about 8-fold for alpha-lactalbumin mRNA and 6-fold for casein mRNA during the 1st week of gestation. These levels declined slightly early in the 2nd week and then continued to increase until parturition with a shoulder in the levels around the 13th to 16th day. During lactation, these levels increased until the 8th to 12th day and from then on declined. The content of alpha-lactalbumin and caseins, as well as the measurement of sequences and activities of their mRNAs, showed that in the rat mammary gland these differentiated functions are already expressed at the onset of gestation. Both concentration and activity of mRNA are out of phase with protein levels during the 1st week of gestation but they remain in phase thereafter.  相似文献   

2.
Two species of alpha-lactalbumin, alpha-lactalbumin1 and alpha-lactalbumin2, were separated from rat milk and purified to homogeneity by gel filtration, followed by the DEAE-cellulose chromatography. alpha-Lactalbumin1 is a bigger molecule in contrast to other known alpha-lactalbumins, and has a molecular weight of 21,500 as determined by sedimentation equilibrium and sodium dodecyl sulfate-polyacrylamide gel analysis. alpha-Lactalbumin2 has a molecular weight of 16,000 measured by sedimentation analysis. alpha-Lactalbumin2, however, exhibits abnormally high molecular weight of 22,500 on sodium dodecyl sulfate polyacrylamide gels. Both alpha-lactalbumins are active in lactose synthase assay and are glycoproteins containing 7 to 9% carbohydrate. Antiserum raised against alpha-lactalbumin1 cannot discriminate between the two species in a radioimmunoassay.  相似文献   

3.
Progesterone prevents lactation during pregnancy. This anti-lactogenic effect includes suppression of the advent of alpha-lactalbumin activity, an effect which prevents the formation of lactose. Alpha lactalbumin activity can be induced to some extent in pregnant rat mammary explants by insulin and hydrocortisone alone, and to a greater extent with prolactin in addition, or with EGF in addition. Physiological levels of progesterone markedly inhibit the induction in the presence of prolactin plus insulin and hydrocortisone, only weakly inhibit in the presence of insulin and hydrocortisone alone, and have no inhibitory effect in the presence of EGF plus insulin and hydrocortisone. Prolactin permits some inhibition in the presence of EGF. The results suggest that progesterone does not subvert the essential insulin or glucocorticoid signals. It also appears that transduction of the prolactin signal is required in order that progesterone effectively block induction of alpha-lactalbumin activity.  相似文献   

4.
Cortisol was previously shown to exert different, concentration-dependent, effects on the accumulation of casein and alpha-lactalbumin in mammary glands from mid-pregnant mice cultured in the presence of insulin and prolactin [Ono & Oka (1980) Cell 19, 473-480]. The present study demonstrated that the addition of 30nM-cortisol to the medium containing insulin and prolactin resulted in a marked enhancement of the rate of synthesis of both alpha-lactalbumin and casein in cultured tissue. The addition of 3 microM-cortisol in combination with insulin and prolactin caused a marked decrease in the rate of alpha-lactalbumin synthesis, but increased casein synthesis substantially. Similar changes were also observed in the amount of translatable mRNA for alpha-lactalbumin and casein in mammary explants cultured with insulin, prolactin and the two concentrations of cortisol. The study of the turnover of the milk proteins in cultured explants showed that virtually all of the casein synthesized remained intact in tissue explants cultured with 3 microM cortisol, whereas about 45% of casein disappeared in 40h from explants cultured with 30nM-cortisol. In contrast, the two concentrations of cortisol did not differentially affect the disappearance of alpha-lactalbumin, which was about 55% in 40h. These results indicate that the concentration-dependent differential actions of cortisol on the accumulation of alpha-lactalbumin and casein are exerted through its effects on the rate of synthesis and turnover of the two proteins as well as on the accumulation of their mRNA species.  相似文献   

5.
A mild and rapid method is described for isolating various milk proteins from bovine rennet whey. beta-Lactoglobulin from bovine rennet whey was easily adsorbed on and desorbed from a weak anion exchanger, diethylaminoethyl-Toyopearl. However, alpha-lactalbumin could not be adsorbed onto the resin. alpha-Lactalbumin and beta-lactoglobulin from rennet whey could also be adsorbed and separated using a strong anion exchanger, quaternary aminoethyl-Toyopearl. The rennet whey was passed through a strong cation exchanger, sulphopropyl-Toyopearl, to separate lactoperoxidase and lactoferrin. alpha-Lactalbumin and beta-lactoglobulin were adsorbed onto quaternary aminoethyl-Toyopearl. alpha-Lactalbumin was eluted using a linear (0-0.15 M) concentration gradient of NaCl in 0.05 M Tris-HCl buffer (pH 8.5). Subsequently, beta-lactoglobulin B and beta-lactoglobulin A were eluted from the column with 0.05 M Tris-HCl (pH 6.8), using a linear (0.1-0.25 M) concentration gradient of NaCl. The yields were 1260 mg alpha-lactalbumin, 1290 mg beta-lactoglobulin B and 2280 mg beta-lactoglobulin A from 1 l rennet whey.  相似文献   

6.
1. Both alpha-lactalbumin and beta-lactoglobulin-like proteins were detected in the whey fraction of feline milk by immunoblotting with rabbit antisera to alpha-lactalbumin and beta-lactoglobulin, respectively. 2. alpha-Lactalbumin was found to occur in both glycosylated and unglycosylated forms in approximately equal concentrations. No polymorphism of feline alpha-lactalbumin was found. 3. Feline beta-lactoglobulin-like proteins produced complex electrophoretic patterns that appear to be determined by three distinct loci. Between two and five genetic variants are expressed by each locus. 4. Lysozyme was detected at levels of approximately 1 mg/ml in skim milk. 5. The identifications of the proteins as alpha-lactalbumin, beta-lactoglobulin and lysozyme were confirmed by determination of N-terminal amino acid sequences.  相似文献   

7.
Ovariectomy on day 19 of pregnancy augmented galactosyl transferase activity 24 h after surgery preceding by 6 h the significant alpha-lactalbumin accumulation. Progesterone, injected immediately after ovariectomy showed a clear inhibitory effect on both galactosyl transferase and alpha-lactalbumin concentration, measured 30 h after ovariectomy. However, once the synthesis of lactose has been induced, progesterone is no longer inhibitory. Oestrogen induced a significant increase in lactose synthetase activity but no effect was obtained on galactosyl transferase activity. Progesterone, in a time and dose dependent relationship, was capable of preventing the effect of estrogen on lactogenesis. The lactogenic action of oestrogen in ovariectomized pregnant rats might be due to a direct effect at the mammary gland level facilitating the action of prolactin or through an indirect effect mediated via an increase on prolactin release.  相似文献   

8.
9.
alpha-Lactalbumin, a modifier protein that changes the substrate specificity of galactosyltransferase, to promote the synthesis of lactose, is found in the mammary glands of lactating mammals and in milk. Molecules similar to mammary gland alpha-lactalbumin but distinct in their modifier activity have been found in rat epididymal fluid. We report here, using a rat mammary gland alpha-lactalbumin cDNA clone as a hybridization probe, RNA sequences homologous to alpha-lactalbumin mRNA were detected in total RNA from the rat epididymis. This finding suggests that alpha-lactalbumin or similar molecules, in addition to regulating lactose synthesis in the mammary gland, may have other important functions in mammalian reproduction.  相似文献   

10.
1. Lysozyme and alpha-lactalbumin from the milk of the common ringtail possum have been purified and partially sequenced. 2. The lysozyme had similar enzymic activity to the c-type lysozyme of the domestic hen and 43% homology over the N-terminal 49 residues. 3. alpha-Lactalbumin was present in the milk in two biologically active forms; the more acidic form had 66% sequence homology with the N-terminal 35 residues of red-necked wallaby, 54% with human and 43% with bovine alpha-lactalbumin. 4. SDS polyacrylamide-gel electrophoresis of milk samples showed that alpha-lactalbumin was present in the milk throughout lactation but that lysozyme first appeared only in mid-lactation. The implications of this functional adaptation are discussed.  相似文献   

11.
Clusterin is an extracellular mammalian chaperone protein which inhibits stress-induced precipitation of many different proteins. The conformational state(s) of proteins that interact with clusterin and the stage(s) along the folding and off-folding (precipitation-bound) pathways where this interaction occurs were previously unknown. We investigated this by examining the interactions of clusterin with different structural forms of alpha-lactalbumin, gamma-crystallin and lysozyme. When assessed by ELISA and native gel electrophoresis, clusterin did not bind to various stable, intermediately folded states of alpha-lactalbumin nor to the native form of this protein, but did bind to and inhibit the slow precipitation of reduced alpha-lactalbumin. Reduction-induced changes in the conformation of alpha-lactalbumin, in the absence and presence of clusterin, were monitored by real-time (1)H NMR spectroscopy. In the absence of clusterin, an intermediately folded form of alpha-lactalbumin, with some secondary structure but lacking tertiary structure, aggregated and precipitated. In the presence of clusterin, this form of alpha-lactalbumin was stabilised in a non-aggregated state, possibly via transient interactions with clusterin prior to complexation. Additional experiments demonstrated that clusterin potently inhibited the slow precipitation, but did not inhibit the rapid precipitation, of lysozyme and gamma-crystallin induced by different stresses. These results suggest that clusterin interacts with and stabilises slowly aggregating proteins but is unable to stabilise rapidly aggregating proteins. Collectively, our results suggest that during its chaperone action, clusterin preferentially recognises partly folded protein intermediates that are slowly aggregating whilst venturing along their irreversible off-folding pathway towards a precipitated protein.  相似文献   

12.
HAMLET (human alpha-lactalbumin made lethal to tumor cells) is a tumoricidal complex of apo alpha-lactalbumin and oleic acid, formed in casein after low pH treatment of human milk. This study examined if HAMLET-like complexes are present in casein from different species and if isolated alpha-lactalbumin from those species can form such complexes with oleic acid. Casein from human, bovine, equine, and porcine milk was separated by ion exchange chromatography and active complexes were only found in human casein. This was not explained by alpha-lactalbumin sequence variation, as purified bovine, equine, porcine, and caprine alpha-lactalbumins formed complexes with oleic acid with biological activity similar to HAMLET. We conclude that structural variation of alpha-lactalbumins does not preclude the formation of HAMLET-like complexes and that natural HAMLET formation in casein was unique to human milk, which also showed the highest oleic acid content.  相似文献   

13.
This study describes an alpha-lactalbumin folding variant from human milk with bactericidal activity against antibiotic-resistant and -susceptible strains of Streptococcus pneumoniae. The active complex precipitated with the casein fraction at pH 4.6 and was purified from casein by a combination of anion exchange and gel chromatography. Unlike other casein components, the active complex was retained on the ion-exchange matrix and eluted only with high salt. The eluted fraction showed N-terminal and mass spectrometric identity with human milk alpha-lactalbumin, but native alpha-lactalbumin had no bactericidal effect. Spectroscopic analysis demonstrated that the active form of the molecule was in a different folding state, with secondary structure identical to alpha-lactalbumin from human milk whey, but fluctuating tertiary structure. Native alpha-lactalbumin could be converted to the active bactericidal form by ion-exchange chromatography in the presence of a cofactor from human milk casein, characterized as a C18:1 fatty acid. Analysis of the antibacterial spectrum showed selectivity for streptococci; Gram-negative and other Gram-positive bacteria were resistant. The folding variant of alpha-lactalbumin is a new example of naturally occurring molecules with antimicrobial activity.  相似文献   

14.
O Mulner  F Megret  J E Alouf  R Ozon 《FEBS letters》1985,181(2):397-402
Progesterone triggers the first meiotic cell division of Xenopus oocyte and inhibits cAMP synthesis. The effect of pertussis toxin purified from Bordetella pertussis was tested on the maturation of Xenopus oocyte. The toxin did not inhibit progesterone-induced resumption of meiosis or the hormone-induced drop in cAMP level. This indicates that progesterone action is not mediated by the Ni subunit of the oocyte adenylate cyclase. Furthermore, pertussis toxin caused a reduction in the time course of maturation correlated with the precocious appearance of an alkali stable 47 kDa phosphoprotein, a marker of the maturation promoting factor (MPF) activity. Pertussis toxin effects mimicked those of 2-glycerophosphate suggesting that both agents act on the steady-state level of phosphorylation implicated in MPF activity.  相似文献   

15.
Effects of Soybean Saponin on Protease Hydrolyses of beta-Lactoglobulin and alpha-Lactalbumin The effects of soybean saponin on tryptic and chymotryptic hydrolyses of whey proteins were evaluated. beta-lactoglobulin and alpha-lactalbumin became more sensitive to both trypsin and chymotrypsin by interacting with saponin in contrast to serum albumin. Soybean saponin was shown to have different effects on various proteins according to their nature.  相似文献   

16.
1. Individual mRNA species encoding guinea-pigs caseins A, B and C, and alpha-lactalbumin, were purified by hydridization to recombinant milk-protein plasmid DNA immobilized on diazobenzyloxymethyl-paper or diazobenzyloxymethyl-cellulose. Addition of the purified mRNA species to a reticulocyte-lysate cell-free system, in the presence or absence of a dog pancreas microsomal membrane fraction, established a precursor-product relationship between the primary translation products and those sequestered within microsomal vesicles, as determined by polyacrylamide-gel analysis in one and two dimensions. 2. Three sequestered variants of sequestered casein A were identified, but only single forms of sequestered casein B and alpha-lactalbumin. Sequestered variants of casein C proved to be unexpectedly basic, and did not focus on the pH gradient utilized. 3. Comparative analysis of milk proteins synthesized in the reticulocyte-lysate and wheat-germ cell-free systems by two-dimensional gel electrophoresis demonstrated both quantitative and qualitative differences. In particular, marked but variable heterogeneity was apparent within the primary translation products of casein A and casein B. Pre-casein C did not focus. Limited N-terminal processing of the primary translation products was also evident. These observations are discussed in relation to (i) unscheduled post-translational modifications by cell-free protein-synthesizing systems and (ii) multiplicity of signal sequences. 4. Overall we demonstrate that complex precursor-product relationships between primary translation products and their sequestered variants, programmed in vitro by a mixed mRNA population, may be readily analysed by using individual mRNA sequences purified by hybridization to immobilized cloned complementary-DNA sequences.  相似文献   

17.
Hydrocortisone acetate injected into pseudopregnant rabbits induced casein synthesis and a parallel accumulation of casein mRNA. These effects were not accompanied by any enrichment of total RNA in the mammary cell. Hydrocortisone acetate did not favour the attachment of polysomes to endoplasmic reticulum. Casein mRNA concentration was enhanced in free and membrane-bound polysomes. After long treatments, the concentration of casein mRNA reached a plateau in membrane bound polysomes whereas it continued to be accumulated in free polysomes, suggesting that a substantial part of casein synthesis is then carried out by free polysomes. Progesterone injected with high doses of prolactin was unable to prevent the stimulatory action of prolactin on the synthesis of casein, the accumulation of casein mRNA and mammary gland growth, as judged by DNA content. By contrast, the increase in the total RNA content of mammary gland was still significantly reduced by progesterone. In addition, progesterone inhibited almost completely the formation of membrane-bound polysomes and the anchorage of casein mRNA to endoplasmic reticulum. From these data, it was concluded that the formation of the endoplasmic reticulum is not a prerequisite for the initiation of casein synthesis. Glucocorticoids do not play a major role in the formation of the endoplasmic reticulum and the Golai apparatus and in the binding of casein synthesizing polysomes to membranes. Progesteronne is capable of inhibiting preferentially and gradually the stimulation of cellular functions requiring the most potent prolactin stimulation.  相似文献   

18.
alpha-Lactalbumin, the modifier protein of galactosyl transferase in the synthesis of lactose by the mammary gland, has been shown to undergo a Ca2+-dependent electrophoretic shift. Such shifts, characteristic of most calcium modulated proteins, are related to gross conformational changes upon binding calcium when detected in the presence of detergent (SDS-PAGE). However, we detected the calcium shift for alpha-lactalbumin using non-denaturing PAGE (ND-PAGE) where electrical charge changes are observed upon binding calcium. In order for a shift to be observed between the apo and calcium bound protein, calcium ion binding to proteins must have minimal dissociation constants (Kdiss) of 10(-7) M; alpha-lactalbumin is reported to bind calcium at Kdiss = 10(-10) to 10(-12) M. The electrophoretic shift identifies alpha-lactalbumin in complex milk whey patterns of many species of mammals.  相似文献   

19.
Proteolytic digestion of alpha-lactalbumin by pepsin, trypsin and chymotrypsin yielded three polypeptide fragments with bactericidal properties. Two fragments were obtained from the tryptic digestion. One was a pentapeptide with the sequence EQLTK (residues 1-5) and the other, GYGGVSLPEWVCTTF ALCSEK (residues (17-31)S-S(109-114)), was composed of two polypeptide chains held together by a disulfide bridge. Fragmentation of alpha-lactalbumin by chymotrypsin yielded CKDDQNPH ISCDKF (residues (61-68)S-S(75-80)), also a polypeptide composed of two polypeptide chains held together by a disulfide bridge. The three polypeptides were synthesized and found to exert antimicrobial activities. The polypeptides were mostly active against Gram-positive bacteria. Gram-negative bacteria were only poorly susceptible to the bactericidal action of the polypeptides. GYGGVSLPEWVCTTF ALCSEK was most, EQLTK least bactericidal. Replacement of leucine (23) with isoleucine, having a similar chemical structure but higher hydrophobicity, in the sequence GYGGVSLPEWVCTTF ALCSEK significantly reduced the bactericidal capacity of the polypeptide. Digestion of alpha-lactalbumin by pepsin yielded several polypeptide fragments without antibacterial activity. alpha-Lactalbumin in contrast to its polypeptide fragments was not bactericidal against all the bacterial strains tested. Our results suggest a possible antimicrobial function of alpha-lactalbumin after its partial digestion by endopeptidases.  相似文献   

20.
alpha-Lactalbumin was purified to apparent homogeneity from mouse milk by combined use of gel filtration, chromatography on DEAE-cellulose and hydroxyapatite, and concanavalin A-Sepharose affinity chromatography. Mouse alpha-lactalbumin exists in several species with different charges and in two molecular-size forms. The smaller form, which constituted over 90% of total alpha-lactalbumin, included two major and two minor species, each of which showed different electrophoretic mobility on polyacrylamide-gel electrophoresis, but gave the same single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis in two different buffer systems and over the range 10-15% acrylamide concentrations. The molecular weight was estimated as 14 100. The two major species of the smaller form had the same amino acid composition and contained no significant amount of carbohydrate. The larger form of alpha-lactalbumin, consisting of two species with different charges, was present in a small amount (less than 10%) in the milk and was isolated by its ability to interact with concanavalin A-Sepharose. Each of the two species also gave the same single band of apparent mol.w.t 18 500 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. However, this value may be anomalous, since this larger form appears to be glycosylated, and glycoproteins can behave anomalously on sodium dodecyl sulphate/polyacrylamide gels by binding less sodium dodecyl sulphate. All species of mouse alpha-lactalbumin from milk were active in the lactose synthase reaction and showed identical immunological properties, as determined by the mono-specific antibody prepared against the small major species. The presence of both the larger and the smaller forms, each in a percentage concentration similar to that found in milk, was also demonstrated in alpha-lactalbumin induced by hormones in organ cultureof pregnant-mouse mammary gland.  相似文献   

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