Most of the lipid present in lactating mammary gland cytosol was associated with a high molecular weight aggregate isolated from cytosol by gel exclusion chromatography or by density gradient centrifugation. The major polypeptide constituent of this lipoprotein aggregate was the monomer of fatty acid synthase (FAS). The major milk lipid globule proteins, butyrophilin (8u) and xanthine oxidase (XO), as well as the small GTP-binding protein ARF, also were present. This lipoprotein complex was abundant in cytosol from lactating but not from involuting mammary glands. HPTLC analysis of lipids extracted from the low density FAS (LDFAS) complex demonstrated the presence of the five major milk phospholipids as well as triacylglycerols, cholesterol, unesterified fatty acids, and diacylglycerols. ³²P-labeled phospholipids present in cytosol could be transferred to microlipid droplets (MLD) and endoplasmic reticulum (ER), in vitro, and could be precipitated along with FAS, and other polypeptide constituents of the LDFAS complex. Complexed FAS could be separated from noncorrlplexed FAS by density gradient centrifugation, native PAGE, and gel exclusion chromatography. A large amount of phospholipid consistently was retained with the complexed form of FAS. These results suggest that FAS migrates to a low density fraction by virtue of its association to other proteins and lipids.
FAS was found to be associated with ER, intracellular lipid droplets, and the milk lipid globule membrane (MLGM). A similar complex to LDFAS was isolated from ER from liver and mammary gland homogenates following incubation in buffer containing ATP. Polypeptide constituents of this complex had similar electrophoretic patterns to LDFAS, but behaved differently from LDFAS constituents when fractionated with the detergent TX-114. While most of the polypeptides in LDFAS partitioned equally into the detergent and aqueous phases, a constituent with an approximate molecular weight of 70 kDa was enriched in the detergent phase. For the ER-derived FAS complex, most of the polypeptides remained in the aqueous phase but the detergent phase also was enriched with a polypeptide similar in size to the LDFAS detergent enriched constituent. Western blot analysis failed to detect Bu in the ER-derived complex. However, protein disulfide isomerase (PDI) was detected in this complex as well as a polypeptide with approximate molecular weight 50 kDa that cross-reacted with PDI antibody. Extraction of lipids from this ER-derived complex demonstrated the presence of large quantities of unesterified fatty acids, with relatively low amounts of complex lipids.
In studies using ¹²⁵I labeled LDFAS, labeled polypeptides were shown to associate with ER and intracellular lipid droplets and their dissociation was stimulated by ATP. Immunocytochemistry using antibody to rat liver FAS revealed distribution of FAS at localized regions of the cytoplasmic surface of rough endoplasmic reticulum and on surfaces of intracellular lipid droplets. Electron micrographs of the LDFAS complex showed a homogeneous morphology of granular, symmetrical particles ranging in size from 40 nm to 170 nm in diameter. These particles resembled low density lipoprotein (LDL) in morphology.
From the available data, the following model was proposed for a possible involvement of FAS in lipid droplet secretion in the mammary gland. During active lipogenesis, FAS is targeted to ER membrane by association with a signal or targeting peptide(s) in the cytosol. The signal peptide then binds to selected regions of ER where signal receptors reside. Binding of FAS may initiate synthesis and accumulation of triacylglycerol between ER membrane bilayers. Upon the achievement of a "critical mass", the lipid core may be released into the cytoplasm in an ATP-dependent manner, surrounded by the membrane components that provided the hydrophobic pocket for lipid accumulation. Butyrophilin and the 70 kDa detergent-extractable constituent released from the ER and present in LDFAS are possible sources of such a function. Polypeptides from the cytosolic leaflet of the ER, and proteins peripherally associated with the leaflet then would comprise the polypeptide constituents of the lipid particle. / Ph. D.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/37700 |
| Date | 04 May 2006 |
| Creators | Keon, Brigitte H. |
| Contributors | Biochemistry and Nutrition, Keenan, Thomas W., Bevan, David R., Potts, Malcolm, Akers, Robert Michael, Kennelly, Peter J. |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Language | English |
| Detected Language | English |
| Type | Dissertation, Text |
| Format | xiv, 131 leaves, BTD, application/pdf, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
| Relation | OCLC# 28732772, LD5655.V856_1993.K466.pdf |
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