scholarly journals Guinea-pig milk-protein synthesis. Isolation and characterization of messenger ribonucleic acids from lactating mammary gland and identification of caseins and pre-α-lactalbumin as translation products in heterologous cell-free systems

1976 ◽  
Vol 160 (1) ◽  
pp. 57-74 ◽  
Author(s):  
R K Craig ◽  
P A Brown ◽  
O S Harrison ◽  
D McIlreavy ◽  
P N Campbell
Keyword(s):  
Molecular Weight ◽  
Protein Synthesis ◽  
Mammary Gland ◽  
Guinea Pig ◽  
Wheat Germ ◽  
Milk Proteins ◽  
N Terminus ◽  
Lactating Mammary Gland ◽  
Heterologous Cell ◽  
Alpha Lactalbumin

1. The major milk proteins synthesized by the lactating mammary gland of the guinea pig were identified and designated as caseins A, B and C and alpha-lactalbumin, with estimated mol.wts. of 28000, 25500, 20500 and 14500 respectively. 2. Antisera to the total casein fraction and to alpha-lactalbumin were prepared from rabbits. The milk proteins were also iodinated with either 131I or 125I. 3. A poly(A)-rich RNA fraction was isolated from lactating guinea-pig mammary glands. Isolation was by affinity chromatography on oligo(dT)-cellulose. 4. Examination of this RNA fraction by electrophoresis on polyacrylamide gels containing formamide indicated three major species 1, 2 and 3, with estimated wol.wts. of 5.4 × 10(5) and 3.3 × 10(5), and the apparent absence of rRNA species. 5. The poly(A)-rich RNA stimulated protein synthesis in heterologous cell-free systems based on wheat germ, Krebs II ascites-tumour cells, and the latter supplemented with an initiation factor-3 fraction from rabbit reticulocyte ribosomes. 6. Between 80 and 90% of the protein synthesis directed by the mRNA was for milk proteins. 7. Analysis of the proteins immunoprecipitated by the alpha-lactalbumin antiserum showed in the wheat-germ system that the product was a protein with a molecular weight greater than that of alpha-lactalbumin, whereas in the ascites-tumour-cell systems both this protein and alpha-lactalbumin were found. When the larger protein was treated with CNBr and the resulting peptides were examined, it was shown that the extra peptide was at the N-terminus. This and other evidence is adduced for the initial translation product of alpha-lactalbumin being a precursor with an addition of about ten amino acids at the N-terminus. 8. Similar analysis of the casein immlnospecific proteins produced under the direction of mRNA indicated that the products had a molecular weight that was apparently a littel smaller than that of the caseins synthesized in vivo. This was not consistent with higher-molecular weight casein precursors. 9. Possible explanations for the results obtained are discussed, especially in terms of the physiological significance of the pre-alpha-lactalbumin as a secretory protein.

Utilization of dipeptides by the caprine mammary gland for milk protein synthesis

1994 ◽  
Vol 267 (1) ◽  
pp. R1-R6 ◽  
Author(s):  
F. R. Backwell ◽  
B. J. Bequette ◽  
D. Wilson ◽  
A. G. Calder ◽  
J. A. Metcalf ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Mammary Gland ◽  
Blood Cell ◽  
Red Blood Cell ◽  
Milk Protein ◽  
Common Mechanism ◽  
Dairy Goats ◽  
Peptide Hydrolysis ◽  
Lactating Mammary Gland

Specific use by the mammary gland in vivo of amino acids (AA) of peptide origin has been demonstrated in lactating dairy goats using a dual-labeled tracer technique involving close-arterial (external pudic artery, EPA) infusion of 13C-labeled dipeptides. The extent of utilization does not appear to differ for glycyl-L-[1-13C]phenylalanine and glycyl-L-[1-13C]leucine, perhaps indicative of a common mechanism by which AA are incorporated from peptide into milk protein. [1-13C]phenyl-alanine of peptide origin appears to be concentrated within the red blood cell, suggesting a role for the erythrocyte in peptide metabolism in vivo. In conclusion, it appears that the lactating mammary gland of goats has the ability to utilize AA of peptide origin for milk protein synthesis, and while the mechanism by which [1-13C]AA are incorporated into milk protein is not clear, it may involve peptide hydrolysis by either mammary cell surface or red blood cell hydrolases followed by uptake of liberated AA by the mammary gland.

scholarly journals Relative distribution of post-nuclear poly(A)-containing RNA abundance groups within the nuclear and post-nuclear polyadenylated and non-polyadenylated RNA populations of the lactating guinea-pig mammary gland

1980 ◽  
Vol 192 (2) ◽  
pp. 489-498 ◽  
Author(s):  
Ian C. Bathurst ◽  
Roger K. Craig ◽  
David G. Herries ◽  
Peter N. Campbell
Keyword(s):  
Mammary Gland ◽  
Guinea Pig ◽  
Milk Protein ◽  
Regulation Of Gene Expression ◽  
Milk Proteins ◽  
Relative Distribution ◽  
Free System ◽  
Low Salt ◽  
Nuclear Rna ◽  
System 2

1. RNA isolated from the post-nuclear supernatant of the lactating guinea-pig mammary gland was fractionated with oligo(dT)–cellulose into three populations; those that bound at ‘low salt’ [long poly(A) tracts, 78–32 nucleotides]; those that bound at ‘high salt’ [shorter poly(A) tracts, 48–21 nucleotides]; and those that did not bind [no poly(A) or short poly(A) tracts, <20 nucleotides]. Nuclear RNA was fractionated into two populations, those that bound in ‘low salt’ and those that did not bind. All the post-nuclear RNA fractions directed the synthesis of milk proteins in a Krebs II ascites cell-free system. 2. 3H-labelled DNA complementary to the post-nuclear poly-(A)-containing RNA population (low-salt fraction) was fractionated into abundant (milk-protein mRNA), moderately abundant and scarce sequences. This complementary DNA was then used to investigate the distribution of the mRNA sequences in the different RNA populations. This showed that all sequences were present in polyadenylated and non-polyadenylated fractions, but that major quantitative differences were apparent. The abundant milk-protein mRNA sequences predominated in the ‘low-salt’ post-nuclear poly(A)-containing RNA fraction, whereas the moderately abundant sequences predominated in the non-polyadenylated post-nuclear RNA fraction. In total cellular RNA, those sequences deemed initially to be moderately abundant within the ‘low-salt’ poly(A)-containing RNA population were present at a concentration very similar to those of the abundant milk-protein mRNA (approx. 6×105 copies of each sequence/cell). Similarly, analysis of the nuclear RNA populations showed that the ‘abundant’ and so-called ‘moderately abundant’ sequences were present in essentially identical concentrations (2×103 copies of each sequence/cell). The majority of these (90–95%) were non-polyadenylated. 3. The results are discussed in terms of the post-transcriptional mechanisms involved in the regulation of gene expression in the lactating guinea-pig mammary gland.

scholarly journals The protein-synthesizing activity of ribosomes isolated from the mammary gland of lactating and pregnant guinea pigs

1971 ◽  
Vol 123 (5) ◽  
pp. 865-874 ◽  
Author(s):  
E. Fairhurst ◽  
Diana McIlreavy ◽  
P. N. Campbell
Keyword(s):  
Protein Synthesis ◽  
Mammary Gland ◽  
Guinea Pig ◽  
Guinea Pigs ◽  
Cyanogen Bromide ◽  
Mammary Glands

1. Polyribosome preparations were made from the deoxycholate-treated post-nuclear fractions obtained by the disruption of mammary glands from lactating and pregnant guinea pigs. 2. A high proportion of large polyribosomes was obtained from the glands of lactating animals whereas mainly small polyribosomes were obtained from the glands of pregnant animals. The isolated preparations incorporated [14C]phenylalanine into protein. The polyribosomes from the glands of pregnant animals were less active than those from the glands of lactating animals but the activity of the former was stimulated more by poly(U) than was the latter. 3. The ribosomes from mammary gland could be dissociated into subunits after incubation, under conditions necessary for protein synthesis, in the presence of puromycin. The subunits could be recombined to give a preparation that actively polymerized [14C]phenylalanine in the presence of poly(U). The subunits from guinea-pig mammary gland could be combined with subunits from liver of either guinea pig or rat. Hybrid ribosomes were also formed from subunits derived from glands of pregnant and lactating animals. The hybrids were as active as were the ribosomes formed by reassociation of subunits from the same tissue, suggesting that in this respect the ribosomes from pregnant animals were not defective. 4. Polyribosomes from mammary glands of lactating animals when incubated with cell sap from the same source were tested for their ability to synthesize α-lactalbumin. The polyribosomes were incubated in the presence of [3H]leucine and α-lactalbumin was isolated from the supernatant. The protein was finally treated with cyanogen bromide and the C-terminal and N-terminal fragments were separated and their radioactivity was determined. Both fragments were radioactive consistent with the synthesis of α-lactalbumin. 5. The results are discussed in relation to protein synthesis in the mammary gland after parturition.

scholarly journals Differential expression of α-lactalbumin and casein genes during the onset of lactation in the guinea-pig mammary gland

1981 ◽  
Vol 194 (3) ◽  
pp. 999-1006 ◽  
Author(s):  
L J Burditt ◽  
D Parker ◽  
R K Craig ◽  
T Getova ◽  
P N Campbell
Keyword(s):  
Mammary Gland ◽  
Guinea Pig ◽  
Post Partum ◽  
Mammary Tissue ◽  
Milk Protein Gene ◽  
Milk Protein Gene Expression ◽  
Gene Transcripts ◽  
Casein Genes ◽  
In Pregnancy ◽  
Alpha Lactalbumin

1. The expression of alpha-lactalbumin and casein genes was examined in guinea-pig mammary tissue taken from animals both pre- and post-partum. 2. Analysis of total RNA by RNA excess hybridization with sequence-specific complementary DNA probes demonstrated that alpha-lactalbumin mRNA was present late in pregnancy, and that maximum concentrations were present at parturition. Casein gene transcripts were absent late in pregnancy (62 days), but by parturition were present at concentrations identical to those found at all time points examined throughout lactation. 3. Studies using mammary explants in organ culture showed that tissue from pregnant animals, or animals at parturition, synthesized and secreted only alpha-lactalbumin. After parturition, at the onset of casein synthesis, differential rates of secretion of alpha-lactalbumin and the caseins were observed. 4. The results are discussed in terms of the multiple intracellular mechanisms involved in the regulation of milk protein gene expression in the guinea-pig mammary gland.

scholarly journals The differential actions of cortisol on the synthesis and turnover of alpha-lactalbumin and casein and on accumulation of their mRNA in mouse mammary gland in organ culture

1983 ◽  
Vol 212 (2) ◽  
pp. 507-515 ◽  
Author(s):  
Y Nagamatsu ◽  
T Oka
Keyword(s):  
Mammary Gland ◽  
Marked Decrease ◽  
Milk Proteins ◽  
Mammary Glands ◽  
Mouse Mammary Gland ◽  
Marked Enhancement ◽  
Pregnant Mice ◽  
Translatable Mrna ◽  
Casein Synthesis ◽  
Alpha Lactalbumin

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.

scholarly journals Utilization of l-alanine and l-glutamine by lactating mammary gland of the rat. A role for l-alanine as a lipogenic precursor

1981 ◽  
Vol 196 (3) ◽  
pp. 757-762 ◽  
Author(s):  
J R Viña ◽  
D H Williamson
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Mammary Gland ◽  
Pyruvate Dehydrogenase ◽  
Blood Concentration ◽  
Mammary Glands ◽  
Cafeteria Diet ◽  
Arterial Blood ◽  
Lactating Mammary Gland

1. Lactation is associated with an increase in the arterial blood concentration of L-alanine and L-glutamate, but a decrease in that of L-glutamine compared with the corresponding values for virgin rats. 2. Virgin rats fed a ‘cafeteria diet’ that induces hyperphagia have increased arterial concentrations of L-alanine, L-glutamate and L-glutamine. During lactation L-alanine and L-glutamate concentrations are even higher. 3. The removal of L-alanine is decreased in hepatocytes from lactating rats fed either a chow or cafeteria diet. 4. Measurements of arteriovenous differences across lactating mammary glands indicate that appreciable amounts of L-glutamine and L-alanine are extracted by the gland. 5. A high proportion of the L-alanine metabolized by isolated acini from fed lactating rats is converted into lipid. 6. Metabolism of L-alanine in acini from starved lactating rats is limited by the activity of pyruvate dehydrogenase. 7. It is concluded that L-alanine and certain other amino acids taken up by the gland in excess of the requirements for protein synthesis can be converted into lipid.

scholarly journals Autocrine regulation of milk secretion by a protein in milk

1995 ◽  
Vol 305 (1) ◽  
pp. 51-58 ◽  
Author(s):  
C J Wilde ◽  
C V P Addey ◽  
L M Boddy ◽  
M Peaker
Keyword(s):  
Mammary Gland ◽  
Whey Protein ◽  
Mammary Epithelial Cells ◽  
Feedback Inhibition ◽  
Primary Cultures ◽  
Milk Proteins ◽  
Mammary Epithelial ◽  
Milk Secretion ◽  
Lactating Goats ◽  
Lactating Mammary Gland

Frequency or completeness of milk removal from the lactating mammary gland regulates the rate of milk secretion by a mechanism which is local, chemical and inhibitory in nature. Screening of goat's milk proteins in rabbit mammary explant cultures identified a single whey protein of M(r) 7600 able to inhibit synthesis of milk constituents. The active whey protein, which we term FIL (Feedback inhibitor of Lactation), also decreased milk secretion temporarily when introduced into a mammary gland of lactating goats. FIL was synthesized by primary cultures of goat mammary epithelial cells, and was secreted vectorially together with other milk proteins. N-terminal amino acid sequencing indicated that it is a hitherto unknown protein. The evidence indicates that local regulation of milk secretion by milk removal is through autocrine feedback inhibition by this milk protein.

Effects of increased supply of essential amino acids on the metabolism of the isolated perfused guinea-pig mammary gland

1979 ◽  
Vol 46 (1) ◽  
pp. 59-67 ◽  
Author(s):  
Andrew R. Peters ◽  
Stephen Alexandrov ◽  
T. Ben Mepham
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Mammary Gland ◽  
Amino Acid ◽  
Guinea Pig ◽  
Amino Acid Uptake ◽  
Essential Amino Acids ◽  
Acid Uptake ◽  
Isoleucine Leucine ◽  
Group 1

SUMMARYThe effects of high rates of infusion of essential amino acids on amino acid uptake by the isolated perfused guinea-pig mammary gland were studied. Infusion of methionine, tyrosine, phenylalanine, histidine and tryptophan (designated group 1) resulted in significant increases in the uptakes of tyrosine, phenylalanine and histidine. Methionine, tryptophan and other essential amino acids were not significantly affected. Infusion of threonine, valine, isoleucine, leucine, lysine and arginine (designated group 2) resulted in significant increases in uptake of all these amino acids. Group 1 amino acid uptake was not significantly affected. Infusion of all the essential amino acids (i.e. groups 1 and 2 together) resulted in significant increases in all their uptakes. Using as index ‘the predicted rate of protein synthesis’, infusion of group 1 and 2 together led to an apparent 27% increase in protein synthesis. The above results are discussed in relation to the control of milk protein synthesis by limiting essential amino acids.

Protein synthesis and degradation in the mammary gland of lactating goats

1988 ◽  
Vol 55 (2) ◽  
pp. 143-154 ◽  
Author(s):  
V. Hutton Oddy ◽  
Derek B. Lindsay ◽  
Ivan R. Fleet
Keyword(s):  
Protein Synthesis ◽  
Mammary Gland ◽  
Milk Proteins ◽  
Mammary Tissue ◽  
Lactating Goats ◽  
Significant Difference ◽  
The Difference ◽  
Image Position ◽  
Protein Synthesis And Degradation ◽  
Synthesis And Degradation

SummaryLactating goats were given a close arterial infusion of [1-14C]leucine and [4,5-3H]4-methyl-2-oxopentanoic acid into one half of the mammary gland at 2–3 weeks and 34–39 weeks after kidding. Rates of protein synthesis, degradation and net output were determined from measurements of arteriovenous difference and blood flow using a model of leucine metabolism previously developed for muscle (Oddy & Lindsay, 1986). Protein leucine output in milk (Y μmol/min) correlated well with the difference between synthesis and degradation (X μmol/min) derived from the model:There was substantial synthesis and degradation of protein within the mammary gland. Although only an approximate value could be obtained for the partitioning of protein synthesis and degradation between tissue and milk proteins, there was evidence of appreciable turnover of both. There was no significant difference between mammary leucine and protein metabolism in early and late lactation other than that imparted by a greater mass of mammary tissue in early lactation, although there was a tendency for greater oxidation of leucine in late lactation.

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