Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum.

Fujiki, Yukio; Hubbard, Ann L.; Fowler, S; Lazarow, Paul B.

doi:10.1083/jcb.93.1.97

Cited by 1,718 publications

(1,305 citation statements)

References 30 publications

Supporting

Mentioning

1,264

Contrasting

Unclassified

Order By: Relevance

“…These authors reported a 67-fold increase in the specific activity of DHAP-AT as compared to a 42-fold increase in our procedure. However, the contamination, as calculated by the method of Fujiki et al [35], with microsomes and mitochondria amounted to 9 and 88, respectively, and were somewhat higher than the 4.5 and 6.6% found in our adapted method.…”

Section: Discussioncontrasting

confidence: 66%

Rat liver dihydroxyacetone-phosphate acyltransferase: Enzyme characteristics and localization studies

Hardeman

Bosch

1988

Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metaboli

View full text Add to dashboard Cite

Peroxisomes were isolated from rat liver by pelleting a light mitochondrial (L) fraction over a 30% (w/v) Metrizamide layer. Peroxisomes were recovered as a loose pellet from the bottom of the tube and the purity of the peroxisomal fraction was calculated to be about !H%. The characteristics of dihydroxyacetone-phosphate acyltransferase (DHAP-AT) in the light mitochondrial fraction and the purified peroxisomal fraction were compared. The behaviour of the enzyme in the two fractions was very similar, except for the effect of sodium fluoride, which stimulated the activity in the L fraction 5-lo-fold and in the peroxisomal fraction only l&fold. This difference could be explained by the action of fluoride-sensitive acid phosphatases present in the L fraction that dephosphorylate palmitoyl-coenzyme A, a substrate for DHAP-AT. The localizations of DHAP-AT and alkyldihydroxyacetone-phosphate synthase in the rat liver peroxisomal membrane were studied. It is shown that in intact peroxisomes, DHAP-AT and alkyl-DHAP synthase are resistant to proteolytic inactivation by trypsin, as is fatty acid b-oxidation activity, which served as a marker for the intactness of the peroxisomal membrane. Catalase was found not to be a suitable marker to assess peroxisome intactness in view of its relative insensitivity to trypsin. In l-lauroyllysophosphatidylcholine-permeabilized peroxisomes, DHAP-AT, alkyl-DHAP synthase and B-oxidation activities were rapidly inactivated by trypsin. It is concluded that in rat liver peroxisomes, at least the active sites of the integral membrane proteins DHAP-AT and alkyl-DHAP synthase are localized exclusively at the inner surface of the peroxisomal membrane.

show abstract

Section: Discussioncontrasting

confidence: 66%

Rat liver dihydroxyacetone-phosphate acyltransferase: Enzyme characteristics and localization studies

Hardeman

Bosch

1988

Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metaboli

View full text Add to dashboard Cite

show abstract

“…The pellet and the supernatant fractions were assayed for the presence of DAD1 by immunoblotting analysis using the antibody to DAD1. When the same volume of pellets was treated with either high salt or sodium carbonate (which removes the peripheral membrane proteins (Fujiki et al 1982)), DAD1 proteins remained in the pellet (P) and not in the supernatant (S) (Fig. 1B, lanes 3-6).…”

Section: Dad1 Is An Integral Membrane Proteinmentioning

confidence: 99%

The highly conserved DAD1 protein involved in apoptosis is required for N‐linked glycosylation

Makishima¹,

Nakashima²,

Nagata-Kuno³

et al. 1997

Genes to Cells

View full text Add to dashboard Cite

show abstract

“…In order to verify if Hex behave as a peripheral membrane protein associated with the lipid bilayer by ionic and/or hydrogen interaction involving transmembrane proteins or phospholipids resident in LM, lysosomal membraneenriched fraction P3 was treated with 1 M NaCl or with sodium carbonate at the extreme pH value of 11.5 [33]. In the first condition Hex remained associated to LM as well as the lysosomal transmembrane protein LAMP-2, while the extreme pH conditions produced the complete solubilisation of both Hex subunits but not of the integral lysosomal membrane LAMP-2 ( Figure 4).…”

Section: Discussionmentioning

confidence: 99%

“…Lysosomal membrane-enriched fraction P3 was resuspended in 0.1 M Na 2 CO 3 pH 11.5 [33] or in 10 mM Na/P containing 1M NaCl pH 6.0. As control, fraction P3 was resuspended in 10 mM Na/P pH 6.0.…”

Section: Nacl and Carbonate Extractionsmentioning

confidence: 99%

“…Using the procedure of Fujiki [33], aliquots of the lysosomal membrane-enriched fraction P3 were incubated with 0.1 M Na 2 CO 3 pH 11.5, with 1 M NaCl in 10 mM Na/P pH 6.0, and with 10 mM Na/P pH 6.0, for 30 min. After the incubation, the mixtures were subjected to ultracentrifugation and the supernatant (S) and pellet (P) fractions obtained were analyzed by immunoblotting.…”

Section: Peripheral Membrane Protein Extraction and Triton X-114 Phasmentioning

confidence: 99%

See 1 more Smart Citation

Identification and characterization of mature β-hexosaminidases associated with human placenta lysosomal membrane

et al. 2008

View full text Add to dashboard Cite

Hex (β-hexosaminidase) is a soluble glycohydrolase involved in glycoconjugate degradation in lysosomes, however its localization has also been described in the cytosol and PM (plasma membrane). We previously demonstrated that Hex associated with human fibroblast PM as the mature form, which is functionally active towards GM2 ganglioside. In the present study, Hex was analysed in a lysosomal membrane-enriched fraction obtained by purification from highly purified human placenta lysosomes. These results demonstrate the presence of mature Hex associated with the lysosomal membrane and displaying, as observed for the PM-associated form, an acidic optimum pH. When subjected to sodium carbonate extraction, the enzyme behaved as a peripheral membrane protein, whereas Triton X-114 phase separation confirmed its partially hydrophilic nature, characteristics which are shared with the PM-associated form of Hex. Moreover, two-dimensional electrophoresis indicated a slight difference in the pI of β-subunits in the membrane and the soluble forms of the lysosomal Hex. These results reveal a new aspect of Hex biology and suggest that a fully processed membrane-associated form of Hex is translocated from the lysosomal membrane to the PM by an as yet unknown mechanism. We present a testable hypothesis that, at the cell surface, Hex changes the composition of glycoconjugates that are known to be involved in intercellular communication and signalling.

show abstract

Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum.

Cited by 1,718 publications

References 30 publications

Rat liver dihydroxyacetone-phosphate acyltransferase: Enzyme characteristics and localization studies

Rat liver dihydroxyacetone-phosphate acyltransferase: Enzyme characteristics and localization studies

The highly conserved DAD1 protein involved in apoptosis is required for N‐linked glycosylation

Identification and characterization of mature β-hexosaminidases associated with human placenta lysosomal membrane

Contact Info

Product

Resources

About