Vesicles fractionation

Sub-cellular fractionation of five strains reveal the same numbers of bands. The distribution of PG activity in sub-cellular organelles was broadly similar in these five strains. PG activity was detected in low-density vesicles, vacuoles and ER fractions in samples harvested during the early exponential phase of growth. However, PG levels were always lower (at least 1.5 fold) than those found in wild type. Cells of the mutants harvested during stationary phase of growth showed that 84% of total intracellular PG activity was located in the vesicle fraction. No intracellular PG activity was found in stationary phase wild type cells. 

Wuthier, R. E., Gore, S. T. Partition of inorganic ions and phospholipids in isolated cell, membrane and matrix vesicle fractions Evidence for Ca-Pj-acidic phospholipid complexes. Calc. Tiss. Res. 24, 163 (1977)... 

The —50 kDa band (48-53 kDa) is identified as dysbindin-1 A in our WESTERNS, because it runs close to the molecular mass of our histidine-tagged recombinant mouse dysbindin-1 A. Its identity is confirmed by the fact that it is recognized by antibodies we have recently generated to amino acid sequences in the CTR of human dysbindin-lA, but not found in dysbindin-lB, -2, or -3. The —50 kDa band is the most consistently observed dysbindin-1 band across tissues. We find it in all tissues examined to date the adrenal gland, heart, kidney, liver, lung, spleen, skeletal muscle, testes, spinal cord, cerebellum, striatum, hippocampus, and cerebral cortex (e.g., Figure 2.2-12a and c). In mouse and human synaptosomes, the —50 kDa isoform is heavily concentrated in the PSD fraction with a much lesser amount in the presynaptic membrane and no detectable amount in the synaptic vesicle fraction (Talbot et al., in preparation). 

The —37 kDa band (36-38 kDa) is close in molecular mass to dysbindin-lB, which cannot be a degradation product of dysbindin-1 A because of its unique C-terminus (see Section 2.2.2.2.1). We cannot detect this band in the mouse brain with any of the dysbindin-1 antibodies we have tested, including one (UPenn 331) which has a high affinity for the —37 kDa band in human tissue. (UPenn 331 also recognizes the 50 kDa band in humans and mice, albeit with much less affinity, and hence can recognize both dysbindin-1 A and - IB, but not dysbindin-lC or other dysbindin paralogs as expected from the fact that it was raised against an aa sequence in the NTR of human dysbindin-lA and -IB absent in dysbindin-lC, -2 or -3.) In synaptosomes of the human brain, the —37 kDa isoform is heavily concentrated in synaptic vesicle fractions with much lesser amounts in the PSD fractions and very little in presynaptic membrane fractions (Talbot et al., submitted). 

Protein expression of dysbindin-1 is also ubiquitous in the body and is detectable in cell bodies of virtually all neuronal populations. Levels of somatal protein are variable, however, with the highest levels found in areas listed above where gene expression is highest. High levels of dysbindin-1 protein expression are also seen in certain synaptic fields. Where these have been examined with immunoEM, dysbindin-1 has been found mainly along microtubules of dendrites and axons, in PSDs of dendritic spines, and around synaptic vesicles. Tissue fractionation of whole brain tissue reveals that dysbindin-1 A is most highly concentrated in PSD fractions, dysbindin -IB in synaptic vesicle fractions, and dysbindin-1C in both PSD and synaptic vesicle fractions. 

Synaptobrevin, SNAP-25, and syntaxin are all highly abundant membrane proteins in the mammalian CNS. Thus, a sufficiently enriched membrane preparation can be obtained by crude subfractionation techniques. While this approach is convenient, particularly for laboratories with no expertise in molecular techniques, it has a number of shortcomings due to the inherent property of the proteins to form toxin-resistant complexes (Hayashi ef.al., 1994). A procedure based on a crude tissue extract is given below. Better results can be achieved when using more highly purified subcellular fractions, e.g., synaptic vesicle fractions, for the assay of synaptobrevin-cleaving toxins. 

Further studies in our laboratory revealed that chronic treatment with morphine in both rats and mice, on the other hand, produced opposite effects to acute treatment, with significant increases in synaptosomal Ca2+ levels being observed (50, 51, 52). The increases were reported to be localized in the synaptic vesicle fraction (51,52) and synaptic plasma membrane fraction (SPM) (53). Naloxone treatment blocked the increase in Ca2+ levels, while naloxone-precipitated withdrawal resulted in a return to control Ca2+ levels within 15 minutes after injection (50). Both -endorphin and methionine-enkephalin were also noted to cause Ca2+ depletion of synaptic vesicles and SPM after acute treatment ( 3). 

Burton, R. M. and Gibbons, J. M. (1964) Lipid composition of a rat brain synaptic-vesicle fraction. Biochim. Biophys. Acta. (Amst.J, 84,220-223. 

Partial Characterization of Chromatin-Binding Properties of Sea Urchin Egg MVl, MV2c , and MV2/3 Vesicle Fractions "... 

Figure 7.5 2D 1H-31P -HMQC-T0CSY of membrane vesicle fraction of nuclear envelope of Lytechinus pictus as explained in text. The projection of the 2D peaks onto the left side of the figure gives a NMR spectrum. Along the top, the projection gives the NMR spectrum. [Reproduced from Larijani et ai. (2000) Lipids 35(11) 1289-1297]...

Kontro P, Marnela K M, and Oja S S (1980) Free amino acids m the synaptosome and synaptic vesicle fractions of different bovine brain areas Bram Res 184, 129-141... 

This approach can also be used to identify novel or unexpected proteins that contribute to cytoskeletal function on membranes. We have successfully isolated and identified individual protein bands from the SDS-PAGE gels and identified them using mass spectrometry (Chen et al., 2004). This has been particularly feasible with the vesicle fractions and liposome binding assays where the background from resident Golgi proteins is greatly reduced or absent (Fig. 3). 

A 10 min period of electrical stimulation in the presence of Pi led to increased labelling of synaptosomal phosphatidate (Bleasdale Hawthorne, 1975) but no consistent, increase in phosphatidylinositol specific radioactivity. The labelling of ATP was unaffected. When sub-synaptosomal membrane fractions were prepared, the increased phosphatidate labelling was seen to be associated with the synaptic vesicle fraction, as in the acetylcholine experiments. Further work (Hawthorne Bleasdale, 1975) showed that electrical stimulation only increased the labelling of phosphatidate in the vesicle fraction when the medium contained calcium ions. This and the time course of labelling (changes seen 2 min after the onset of stimulation) suggest that the metabolism of phosphatidate is closely associated with the process of transmitter release. 

Preparation of sub-synaptosomal fractions showed that after labelling vivo, phosphatidate of Fraction E which contains marker enzymes for endoplasmic reticulum and plasma membrane (but not in higher concentration than some other fractions) had the highest specific radioactivity (Table 1). The most active phosphatidylinositol was in the synaptic vesicle fraction. Electrical stimulation provoked loss of these particular pools of phospholipid, while there was little effect on either phospholipid in the remaining fractions, as can be seen from the Table. 

Breakdown and resynthesis of phosphatidylinositol and phosphatidic acid are closely associated with the events following depolarization of isolated synaptosomes. The most important of these events is transmitter release and the weight of the present evidence favours the view that exocytosis is the mechanism of release. This is supported by our finding that major phospholipid effects are seen in the synaptic vesicle fraction. Work with pre-labelled synaptosomes showed that stimulation led to loss of phosphatidylinositol from this fraction. The enzyme converting it to diacylglyce rol is most likely to be involved. Entry of calcium... 

In our previous paper we proposed that the endoglucanase may be synthesized in light microsomal vesicles and then transferred to a heavy vesicle fraction in an inactive state. This issue has been examined in experiments presented in this paper. The fractions were prepared from mycelia at two different time intervals of growth, at 18 h when synthesis was high and secretion was low and at 24 h when the reverse was true. The... 

Amine Half life in vas deferens (min) % recovered in storage-vesicle fraction % released by electrical stimulation... 

The activity of the E.R. marker enzyme, CDP-choline DAG transferase for the top 15 fractions of a gradient is shown in figure 3. CDP-choline DAG transferase activity like LPC-AT (figure 2) copurifies with the enzymes of TAG synthesis in the dense fraction (9-13),but is almost undetectable in the light vesicle fraction (3-5). Marker enzymes for the plastids, cytosol, mitochondria and chlorophyll were also assayed (data not shown), however these organelles were found not to be associated with TAG synthesis. 

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