Pore properties of the Golgi membrane from lactating-rat mammary gland. Effects of pH and temperature and reconstitution into phospholipid vesicles.
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ABSTRACT:
SUBMITTER: White MD
PROVIDER: S-EPMC1153209 | biostudies-other | 1984 Jan
REPOSITORIES: biostudies-other
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Project description:The ability of rat mammary-gland Golgi membranes to produce monosaccharide-specific pores in phospholipid vesicles was investigated. The apparent ability of Triton X-100 extracts of Golgi membranes to form such pores was re-evaluated, since we have now found that an apparent pore is produced by the detergent alone. We therefore incorporated intact Golgi membranes (1 mg of protein) into egg-yolk phospholipid vesicles by direct sonication in the absence of any detergent. These vesicles retained about 0.6% of the total sucrose, but demonstrated selective permeability towards glucose compared with sucrose, with 19.8% of the glucose being lost during gel filtration on Sepharose 4B. This phenomenon seemed to be enhanced by the presence of acidic phospholipids and lysophosphatidylcholine, but was inhibited by inclusion of cholesterol in the vesicles. The best mixture of phospholipids comprised 6.5 mg of egg-yolk phospholipid, 1 mg of phosphatidylserine and 0.05 mg of lysophosphatidylcholine, where 32.9% of the glucose was lost. By using this optimum phospholipid mixture the pores were shown to be permeable to both glucose and mannitol, whereas sucrose and lactose were retained by the vesicles. Chaps (3- [(3-cholamidopropyl)dimethylammonio] propane-1-sulphonate)-solubilized membranes produced similar permeability in vesicles produced by dialysis of a solution of the phospholipids mixed with the membrane extract. This technique resulted in a greater loss of glucose, 33% loss requiring about 0.25 mg of protein. The pore-forming ability of both intact Golgi membranes and Chaps extracts was sensitive to boiling and proteolysis, indicating that a membrane protein was likely to be involved in pore formation.
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Project description:The Golgi-membrane vesicles present in particulate preparations of lactating rat mammary gland were biosynthetically loaded with [14C]lactose. This lactose was effectively retained by particles sedimented after exposure to 0.25 M-disaccharide, but was partly lost after exposure to 0.25 M-glucose or other solutes of similar size. Loss of lactose was time-, concentration- and temperature-dependent and varied with the solute structure. This behaviour is ascribed to the presence of protein in the Golgi membrane, forming a specific carrier or channel that serves to supply glucose for lactose synthesis.
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Project description:1. The ability of phlorrhizin to inhibit the galactosylation of glucose was re-examined with Golgi membrane vesicles purified from rat mammary gland, and extended to the galactosylation of several glucose analogues and N-acylglucosamines. 2. The inhibition is ascribed, contrary to previous conclusions, to a general annealing of leaky membranes comprising a minority of the vesicles. 3. Three thiol reagents were able to inhibit the galactosylation of N-acylglucosamines with less, or no, inhibition of galactosylation of glucose. This demonstrates the existence of a Golgi membrane carrier that distinguishes between glucose and N-acylglucosamines.
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Project description:1. Golgi membrane vesicles, isolated from lactating-rat mammary gland and greatly enriched in galactosyltransferase (EC 2.4.1.22), contained over 40 separate bands of protein, including some periodic acid)(Schiff-staining material and free thiol groups, when analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 2. The membrane lipids were enriched in phosphatidylcholine, phosphatidylethanolamine and unesterified cholesterol. 3. Membrane fluidity, as monitored by the fluorescence polarization of 1,6-diphenylhexa-1,3,5-triene, increased linearly over 5-37 degrees C. 4. The vesicle membranes were impermeable to lactose over a wide pH range, but admitted electrolytes of molecular weight below about 300. 5. These properties are discussed with respect to other cellular membranes and the secretion of milk products.
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Project description:1. Purified Golgi-membrane vesicles of lactating-rat mammary gland were penetrated by glucose. 3-O-methylglucose, mannose, fructose, sorbitol and mannitol, but not by lactose or sucrose. 2. The kinetics of mannitol uptake and release were followed at 2-6 degrees C with the aid of fine filters (0.45 micrometers pore size) to separate the vesicles from the medium. 3. Mannitol efflux exhibited apparent first-order kinetics with k approximately 1 min-1. Neither saturability, nor inhibition by excess sorbitol or glucose, could be observed. 4. Mannitol efflux at 18 degrees C was about seven times faster than at 1 degrees C, and rates at higher temperatures were too fast to be measured. The rate of glucose efflux at 2-6 degrees C exceeded that of mannitol severalfold. 5. These findings imply a channel or carrier of definite, but limited, specificity straddling the Golgi membrane and able to supply glucose for lactose synthesis.
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Project description:BackgroundThe mammary gland is a dynamic organ that undergoes important physiological changes during reproductive cycles. Until now, data regarding the characterisation of miRNA in the mammary gland have been scarce and mainly focused on their abnormal expression in breast cancer. Our goal was to characterise the microRNA (miRNA) involved in mechanisms regulating the mammary function, with particular focus on the lactation stage.Methodology/principal findingsUsing high-throughput sequencing technology, the exhaustive repertoires of miRNA expressed (miRNome) in mouse and bovine mammary glands during established lactation were identified, characterized and compared. Furthermore, in order to obtain more information on miRNA loading in the RNA-induced silencing complex (RISC), the miRNome was compared with that obtained from RNA associated with the AGO2 protein (AGO2-miRNome) in mouse lactating mammary gland. This study enabled the identification of 164 and 167 miRNA in mouse and bovine, respectively. Among the 30 miRNA most highly expressed in each species, 24 were common to both species and six of them were preferentially highly expressed in lactating than non-lactating mammary gland. The potential functional roles of these 24 miRNA were deduced using DIANA-miRPath software, based on miRNA/mRNA interactions. Moreover, seven putative novel miRNA were identified. Using DAVID analysis, it was concluded that the predicted targets of two of these putative novel miRNA are involved in mammary gland morphogenesis.Conclusion/significanceOur study provides an overview of the characteristics of lactating mouse and bovine mammary gland miRNA expression profiles. Moreover, species-conserved miRNA involved in this fundamental biological function were identified. These miRNomes will now be used as references for further studies during which the impact of animal breeding on the miRNA expression will be analysed.
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Project description:RNA-Seq enables the generation of extensive transcriptome information providing the capability to characterize transcripts (including alternative isoforms and polymorphism), to quantify expression and to identify differential regulation in a single experiment. Our aim in this study was to take advantage of using RNA-Seq high-throughput technology to provide a comprehensive transcriptome profiling of the sheep lactating mammary gland. Eight ewes of two dairy sheep breeds with differences in milk production traits were used in this experiment (four Churra and four Assaf ewes). Milk samples from these animals were collected on days 10, 50, 120 and 150 after lambing to cover the various physiological stages of the mammary gland across the complete lactation. RNA samples were extracted from milk somatic cells. The RNA-Seq dataset was generated using an Illumina HiSeq 2000 sequencer. The information reported here will be useful to understand the biology of lactation in sheep, providing also an opportunity to characterize their different patterns on milk production aptitude.
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