Lactating Rabbit Mammary Gland Research Articles

Prolactin induces release of a factor from membranes capable of stimulating beta-casein gene transcription in isolated mammary cell nuclei.

Crude microsomes from lactating rabbit mammary gland were incubated with prolactin. The incubation mixture was centrifuged and the supernatant was incubated with isolated mammary cell nuclei from lactating rabbits treated for 4 days by bromocryptin to antagonize prolactin and to deinduce casein gene transcription. Nuclei were incubated with HgCTP, and the newly synthesized mercurated RNA was isolated on SH-Sepharose columns. The content of beta-casein mRNA sequences in the fraction eluted with 2-mercaptoethanol was estimated with a [(3)H]cDNA probe obtained from partially purified beta-casein mRNA. The supernatant markedly stimulated beta-casein gene transcription but not 28S rRNA transcription. The same effect was obtained with other lactogenic hormones such as human growth hormone and ovine placental lactogen but was not observed with bovine growth hormone, insulin, parathyroid hormone, luteotropic hormone, or epidermal growth factor. Prolactin and human growth hormone were totally inactive when added directly to nuclei. The factor stimulating beta-casein gene transcription was also generated by membranes containing prolactin receptors such as those from liver, ovary, adrenals, and brain but not by membranes from heart, lung, and muscle, which do not bind prolactin. The factor stimulated beta-casein transcription when added to mammary nuclei from pseudopregnant or bromocryptin-treated lactating rabbits, in which the transcription rate is submaximal, but was ineffective on mammary nuclei prepared from untreated fully lactating rabbits. The factor was unable to induce beta-casein gene transcription in nuclei isolated from rabbit liver and reticulocytes. The factor did not stimulate the transcription of globin genes in nuclei isolated from reticulocytes or the transcription of mammary "housekeeping" genes evaluated by a cDNA probe prepared from total mRNA isolated from an unstimulated mammary gland. The transcription of beta-casein genes was abolished by adding alpha-amanitin to the medium in the presence or in the absence of the factor, indicating that the generation of mercurated beta-casein mRNA sequences depended upon the transcriptional activity of RNA polymerase II. The addition of the factor to the incubation mixture did not enhance total and alpha-amanitin-sensitive RNA synthesis. These data suggest that the binding of prolactin to its receptor in vitro induces the formation of a second messager, which specifically stimulates the transcription of prolactin-sensitive genes.

Isolation and partial characterization of secreted hamster pituitary prolactin.

PRL secreted by hamster anterior pituitaries was purified by gel filtration on Sephadex G-100 and ion exchange chromatography on DEAE-cellulose. Hamster PRL eluted from Sephadex G-100 with an elution volume to void volume ratio of 1.85 and at a salt concentration of 0.13 M NaCl on DEAE-cellulose. Electrophoretic homogeneity of the hormone was demonstrated in several gel systems, and electrophoresis in the presence of sodium dodecyl sulfate indicated a molecular weight of 22,000 +/- 2,000. Hamster PRL did not cross-react with antiserum to mouse PRL. Hamster PRL displayed lactogenic activity in an in vitro mouse mammary gland assay. Hamster PRL displaced [125I]iodo-PRL from lactating rabbit mammary gland receptors. Thus, with respect to its exclusion properties on SEphadex G-100, its molecular weight, and its biological activity, hamster PRL closely resembles other mammalian PRLs.

Mammary blood flow and cardiac output in the conscious lactating rabbit

1. Cardiac output and regional blood flow were measured in conscious lactating and non-lactating rabbits. 2. At 14 days post-partum the cardiac output in lactating rabbits was 30% higher than that measured in virgin animals. 3. The mammary gland was the only organ to show an increase in relative weight (% body weight) during lactation and also the only organ to receive an increased proportion of the cardiac output per g tissue weight during lactation. 4. These results are discussed in the context of net substrate uptake by the lactating rabbit mammary gland.

Medium-chain fatty acid synthesis in lactating-rabbit mammary gland. Intracellular concentration and specificity of medium-chain acyl thioester hydrolase.

The concentration of medium-chain acyl thioester hydrolase and of fatty acid synthetase was determined by rocket immunoelectrophoresis in nine different particle-free supernatant fractions from lactating-rabbit mammary gland. The molar ratio of the hydrolase to fatty acid synthetase was 1.99 +/- 0.66 (mean +/- S.D.). A rate-limiting concentration of malonyl-CoA was required to ensure the predominant synthesis of medium-chain fatty acids when 2 mol of the hydrolase was added per mol of fatty acid synthetase. The interaction of the hydrolase with fatty acid synthetase was concentration-dependent, though an optimum concentration of hydrolase to synthetase could not be obtained. The lactating-rabbit mammary gland hydrolase altered the pattern of fatty acids synthesized by fatty acid synthetases prepared from cow, goat, sheep and rabbit lactating mammary glands, rabbit liver and cow adipose tissue.

Dephosphorylation and activation of Acetyl-CoA-carboxylase from lactating rabbit mammary gland

Dephosphorylation and activation of Acetyl-CoA-carboxylase from lactating rabbit mammary gland

The Effect of Cycloheximide on Protein Glycosylation in Lactating Rabbit Mammary Gland

The Effect of Cycloheximide on Protein Glycosylation in Lactating Rabbit Mammary Gland

Lipid-linked oligosaccharides containing glucose in lactating rabbit mammary gland.

1. Microsomal fractions of lactating rabbit mammary gland incubated with UDP-glucose formed lipid-linked mono- and oligo-saccharides. The lipid-linked monosaccharide had chromatographic properties similar to those of dolichol phosphate mannose and yielded glucose on acid hydrolysis. 2. Incubation of the microsomal fraction with GDP-[U14C]-mannose yielded an oligosaccharide lipid of approximately seven monosaccharide units. Further incubation with UDP-glucose increased the size of the oligosaccharide by approximately two units. 3. Explants of lactating rabbit mammary gland incorporated [U-14C]glucose into both lipid-linked mono- and oligo-saccharides. The oligosaccharide lipid was of approx. 11 monosaccharide units. 4. Considerable redistribution of radioactive label occurred in the explant system, and radioactively labelled glucosamine and mannose, as well as glucose, were detected on acid hydrolysis of the oligosaccharide lipid. 5. Glucose was also detected in the acid hydrolysate of explant proteins. Radioactive glucosamine, galactosamine, galactose and mannose were also found in this fraction.

Purification and physicochemical properties of fatty acid synthetase and acetyl-CoA carboxylase from lactating rabbit mammary gland.

Fatty acid synthetase was purified 13-fold from lactating rabbit mammary glands by a procedure which involved chromatography on DEAE-cellulose, ammonium sulphate precipitation and gel filtration on Sepharose 4B. The preparation was completed within two days and over 100 mg of enzyme was isolated from 100--150 g of mammary tissue, which represented a yield of over 40%. The preparation was homogeneous by the criteria of polyacrylamide gel electrophoresis and ultracentrifugal analysis. The sedimentation constant, S20,w was 13.3 S, the absorption coefficient, A280nm1%, measured refractometrically was 10.0 +/- 0.1, and the amino acid composition was determined. The subunit molecular weight determined by gel electrophoresis in the presence of sodium dodecyl sulphate was 252,000 +/- 6,000, and the molecular weight of the native enzyme measured by sedimentation equilibrium was 515,000. These experiments indicate that at the concentrations which exist in mammary tissue (2--4 mg/ml) fatty acid synthetase is a dimer. The purified enzyme did however show a tendency to dissociate to a monomeric 9-9S species on storage for several days or following exposure to a low ionic strength buffer at pH 8.3. There was only a small quantity of alkali labile phosphate (0.2 molecules per subunit) bound covalently to the purified enzyme. Acetyl-CoA carboxylase was purified 300-fold in a 50% yield within 24 h by ammonium sulphate and polyethylene glycol precipitations [Hardie, D.G. and Cohen, P. (1978) FEBS Lett. 91, 1--7]. The preparation was in a state approaching homogeneity as judged by polyacrylamide gel electrophoresis, gel filtration on Sepharose 4B and ultracentrifugal analysis. The sedimentation constant, S20,w, was 50.5 S, the absorption index, A280nm1%, was 14.5 +/- 0.7, and the amino acid composition was determined. The subunit molecular weight of acetyl-CoA carboxylase determined by gel electrophoresis in the presence of sodium dodecyl sulphate was identical to that of fatty acid synthetase (252,000) as shown by electrophoresis of a mixture of the two proteins. The preparations also contained two minor components of molecular weight 235,000 and 225,000, which appear to be derived from the major species of mol. wt 252,000. A large emount of phosphate (3.2 molecules per subunit) was found to be bound covalently to the purified enzyme. The properties of fatty acid synthetase and acetyl-CoA carboxylase are compared to those obtained by other workers.

Oxytocin binding sites in lactating rabbit mammary gland

Material which specifically binds oxytocin was prepared from a crude preparation of lactating rabbit mammary gland by purification on a sucrose density gradient. On examination of activities of enzyme markers and the molar ratio of cholesterol to phospholipid, this material was considered to be a highly purified plasma membrane fraction. For the determination of specificity and time course of oxytocin binding, a Scatchard plot analysis was carried out for the crude and purified fractions. Dissociation constant (Kd) and binding capacity values were found to be as follows: crude, Kd equals 1.83 X 10(-9) M, capacity equals 670 fmol/mg protein; purified, Kd equals 2.8 X 10(-9) M, capacity equals 1700 fmol/mg protein. Treatment of the purified material with different detergents resulted in loss of all [3H]oxytocin binding capacity. However, preincubation of this material with [3H]oxytocin prior to detergent treatment resulted in solubilization of a receptor-hormone complex. This complex remained in the supernatant even after centrifugation at 210 000 X g for 30 min. Using oxytocin analogs, we have shown this solubilized complex to be oxytocin specific.

Uptake of substrates for milk-fat synthesis by lactating-rabbit mammary gland.

1. A cannulation technique is described for measuring arteriovenous differences across the lactating-rabbit mammary gland. 2. Analysis of milk obtained before and after surgery shows no effect of cannulation on milk constituents. 3. Results of blood analysis show significant net changes in the concentrations of glucose, acetate, 3-hydroxybutrate, triacylglycerols and non-esterified fatty acids across the mammary gland. 4. The molar proportions of individual fatty acids in both the triacylglycerol and non-esterified fatty acid fractions did not alter between the arterial and venous samples. 5. The extraction rates are compared with those obtained from other species.

The regulation of fatty acid biosynthesis: Simple procedure for the purification of acetyl CoA carboxylase from lactating rabbit mammary gland, and its phosphorylation by endogenous cyclic AMP-dependent and -independent protein kinase activities

A number of key regulatory enzymes in eukaryotic cells are now known to be controlled by phosphorylation-dephosphorylation mechanisms, and there is increasing evidence that these interconversion reactions underlie the short term regulation of metabolism by external physiological stimuli (reviewed [I] ). The rate at which cytoplasmic acetyl CoA is converted to long chain fatty acids is stimulated by insulin in fat cells [2] and inhibited by glucagon in hepatocytes [3] . Acetyl CoA carboxylase has been implicated as the enzyme at which these hormonal effects are exerted, but the mechanisms are not understood in molecular terms. Acetyl CoA carboxylase is completely dependent on the allosteric activator citrate for activity, and it is inhibited by very low concentrations of palmityl CoA [4] . There is also preliminary evidence which indicates that acetyl CoA carboxylase may be regulated by a phosphorylation-dephosphorylation mechanism. Relatively crude preparations from rat liver [S] or bovine mammary gland [6] undergo a time-dependent inactivation if they are incubated with magnesium ions and [Y-~~P] ATP [7-91 and 32P-radioactivity is incorporated into a protein(s) which is precipitated by antiserum raised to a proteolytic fragment of rat liver acetyl CoA carboxylase [7-91 . Furthermore the inactivation can be reversed by incubation with a protein phosphatase preparation partially purified from hen oviduct [9] . If isolated fat cells are incubated with 32P, a protein with a subunit molecular weight corresponding to acetyl CoA carboxylase becomes labelled, and this protein can be precipitated using a monospecific antibody raised to acetyl CoA carboxylase from rat mammary gland [lo] . This experiment shows that the enzyme is phosphorylated in intact cells. We describe here an extremely simple method for the purification of large amounts of undergraded acetyl CoA carboxylase and demonstrate that this preparation can be phosphorylated by two distinct protein kinase activities that are present as trace endogenous contaminants in the preparation.

The formation of lipid-linked sugars by cell-free preparations of lactating rabbit mammary gland.

1. A lactating rabbit mammary-gland microsomal system catalysed the incorporation of mannose from GDP-[U-14C]mannose into three endogenous acceptors, (i) polyprenyl phosphate mannose, (ii) lipid-linked oligosaccharide and (iii) protein. 2. Synthesis of polyprenyl phosphate mannose was stimulated by addition of dolichol phosphate to the incubation medium and was reversed by addition of GDP. The product had properties identical with those of authentic dolichol phosphate mannose. 3. The oligosaccharides derived from acid hydrolysis of the lipid-linked oligosaccharide fraction were of six, eight and nine to ten monosaccharide units, the octasaccharide being the major species formed. The oligosaccharide appeared to be attached to the lipid via a pyrophosphate bridge, since strong alkaline hydrolysis liberated an oligosaccharide phosphate. 4. Polyprenyl phosphate mannose served as a mannose donor to lipid-linked oligosaccharides and protein. When added as exogenous substrate it gave rise to a lipid-linked oligosaccharide of about six units. 5. Incorporation of radioactivity in protein was low, but polyacrylamide-gel electrophoresis of the protein fractions indicated that polypeptides of mol.wts. 115000, 75000 and 33000 were labelled.

Early effects of prolactin on lactating rabbit mammary gland. Ultrastructural changes and stimulation of casein secretion.

Effects of prolactin on the secretion of milk proteins have been investigated by incubating mammary tissue fragments from lactating rabbits. Within 15 min of adding the hormone to the incubation medium, cell morphology is modified: the relative volume occupied by the Golgi region is greatly increased. When prolactin is added immediately after a pulse labelling of proteins (3 min with 3H-L-leucine), the amount of labelled caseins secreted during one hour is significantly increased. This increase proceeds neither from an acceleration of intracellular transit of caseins (as shown by electron microscopic autoradiography) nor by an enhancement of amino acid uptake (as measured by incorporation of non-metabolizable amino acids) nor by an increase of overall protein synthesis, during the first hour. The action of prolactin on the morphology of such subcellular organelles as the Golgi apparatus and its influence on casein secretion are discussed.

TThe effect of tunicamycin on glycoprotein biosynthesis in lactating-rabbit mammary gland [proceedings

TThe effect of tunicamycin on glycoprotein biosynthesis in lactating-rabbit mammary gland [proceedings

Immunological properties of medium-chain acyl- thioester hydrolase and fatty acid synthetase from lactating-rabbit mammary gland

The cytosol from lactating-rabbit mammary gland contains a medium-chain acyl-thioester hydrolase. This hydrolase terminates chain lengthening of the fatty acids synthesised by fatty acid synthetase so as to release C 8:0 and C 10:0 fatty acids which are characteristic of rabbit milk. The medium-chain hydrolase and the fatty acid synthetase present in this cytosol have been shown to be immunologically distinct. When fatty acid synthetase was purified from this cytosol it showed unexpected immunological reactivity towards antiserum raised to the medium-chain hydrolase. The precipitate formed was not due to fatty acid synthetase, but to medium-chain hydrolase contaminating the synthetase. However, the proportion of this medium-chain hydrolase which was recovered with the purified synthetase was too small to be detected by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and was too small to elicit an antibody response in sheep. Immunological techniques have shown that the medium-chain hydrolase appears in rabbit mammary gland between days 17 and 22 of pregnancy. This coincides with the onset of milk-fat synthesis. The medium-chain hydrolase could not be detected in the cytosol from lactating-rabbit liver.

Purification and some properties of a medium-chain acyl-thioester hydrolase from lactating-rabbit mammary gland which terminates chain elongation in fatty acid synthesis

Purification and some properties of a medium-chain acyl-thioester hydrolase from lactating-rabbit mammary gland which terminates chain elongation in fatty acid synthesis

The interaction of fatty acid synthetase with cytoplasmic protein in the control of the chain-length of fatty acids synthesised by the lactating rabbit mammary gland

1. 1. Fatty acid synthetase from rabbit mammary gland can be separated from the protein which modifies the chain-length at which fatty acids are released from the enzyme complex in the soluble fraction. This can be achieved by ultracentrifugation, precipitation with specific antibody or ammonium sulphate. 2. 2. The chain-length modifying protein in the supernatant fraction from rabbit mammary gland was less active towards cow mammary gland fatty acid synthetase than rabbit mammary gland fatty acid synthetase in the synthesis of medium-chain fatty acids. The fatty acid synthetases from these two tissues are also immunologically non-identical. 3. 3. It is proposed that there is a loose but specific interaction of rabbit mammary gland fatty acid synthetase with the chain-length modifying protein in regulating product chain length which is dependent on the concentration of interacting proteins. 4. 4. The chain-lengthening effect of added malonyl-CoA decreases with increasing concentration of interacting proteins, but differences in the fatty acid chain-length with malonyl-CoA synthesised in situ by acetyl-CoA carboxylase and with added malonyl-CoA indicate that the product chain-length is sensitive to the availability of malonyl-CoA for enlongation in all but the most tightly coupled situations.

Purification and some properties of a medium-chain acyl-thioester hydrolase from lactating-rabbit mammary gland which terminates chain elongation in fatty acid synthesis

1. An acyl-thioester hydrolase was isolated from the cytosol of lactating-rabbit mammary gland. The purified enzyme terminates fatty acid synthesis at medium-chain (C8:0-C12:0) acids when it is incubated with fatty acid synthetase and rate-limiting concentrations of malonyl-CoA. These acids are characteristic products of the lactating gland. 2. The mol.wt. of the enzyme is 29000+/-500 (mean+/-S.D. of three independent preparations), as estimated by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. 3. The enzyme also hydrolyses acyl-CoA esters of chain lengths C10:0-C16:0 when these are used as model substrates. The greatest activity was towards dodecanoyl-CoA, and the three preparations had specific activities of 305, 1130 and 2010 nmol of dodecanoyl-CoA hydrolysed/min per mg of protein when 56muM substrate was used. 4. The way in which this enzyme controls the synthesis of medium-chain fatty acids by fatty acid synthetase is briefly discussed.

Isoenzymes of an acyltransferase from rabbit mammary gland: Solubilization of the micelle-specific species with Triton X-100

The micelle-specific palmityl-CoA: monopalmityl-sn-glycerol 3-phosphate palmityltransferase isoenzyme from lactating rabbit mammary gland microsomes is selectively solubilized in Triton X-100 but not Tween 80. Both detergents inactivate the monomer-specific isoenzyme. The possibility that, in vivo , physiological surfactants regulate the relative activities of these two isoenzymes is discussed.

Purification and some properties of acetyl-coenzyme A carboxylase from rabbit mammary gland.

1. Acetyl-Coa carboxylase from lactating-rabbit mammary gland was purified to homogeneity by the criterion of polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. 2. Use of phosphate buffer throughout the purification gave low recovery of enzyme. Consequently, Tris buffers were used in the extraction and in selected stages of the purification procedure. 3. The purified enzyme had a specific activity of 5.15 +/- 0.3 mumol of bicarbonate incorporated/min per mg of protein (mean +/- S.E.M. of five preparations). This represents a purification of 257 +/- 16-fold and a yield of 4.3 +/- 0.13%. 4. The kinetic parameters of the purified enzyme were similar to those reported for the enzyme from other tissue sources. 5. The enzyme was assayed by a spectrophotometric assay and by a [14C]bicarbonate-fixation assay. Short incubation were used in the radio-chemical assay to avoid substantial loss of [14C]bicarbonate.