Production and characterization of antibodies against the cross-reacting determinant of glycosyl-phosphatidylinositol-anchored acetylcholinesterase

Jäger, Karin; Meyer, Pascale; Stieger, S.; Brodbeck, Urs

doi:10.1016/0167-4838(90)90272-h

Cited by 12 publications

(1 citation statement)

References 30 publications

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“…This phospholipase D is also capable of cleaving the acylated anchor of human erythrocyte acetylcholinesterase (Roberts et al, 1988). Several anti-(cross-reacting determinant) (CRD) antisera are now available which recognize the G-PI anchors of unrelated proteins following endogenous or exogenous digestion by phospholipase C (Zamze et Jager et al, 1990;Hooper et al, 1991). Alternatively, if a suitable cell line is available, the protein of interest can be biosynthetically labelled with components ofthe anchor structure (e.g.…”

Section: Introductionmentioning

confidence: 99%

Glycosyl-phosphatidylinositol-anchored membrane proteins can be distinguished from transmembrane polypeptide-anchored proteins by differential solubilization and temperature-induced phase separation in Triton X-114

Hooper

Bashir

1991

Biochemical Journal

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Treatment of kidney microvillar membranes with the non-ionic detergent Triton X-114 at 0 degrees C, followed by low-speed centrifugation, generated a detergent-insoluble pellet and a detergent-soluble supernatant. The supernatant was further fractionated by phase separation at 30 degrees C into a detergent-rich phase and a detergent-depleted or aqueous phase. Those ectoenzymes with a covalently attached glycosyl-phosphatidylinositol (G-PI) membrane anchor were recovered predominantly (greater than 73%) in the detergent-insoluble pellet. In contrast, those ectoenzymes anchored by a single membrane-spanning polypeptide were recovered predominantly (greater than 62%) in the detergent-rich phase. Removal of the hydrophobic membrane-anchoring domain from either class of ectoenzyme resulted in the proteins being recovered predominantly (greater than 70%) in the aqueous phase. This technique was also applied to other membrane types, including pig and human erythrocyte ghosts, where, in both cases, the G-PI-anchored acetylcholinesterase partitioned predominantly (greater than 69%) into the detergent-insoluble pellet. When the microvillar membranes were subjected only to differential solubilization with Triton X-114 at 0 degrees C, the G-PI-anchored ectoenzymes were recovered predominantly (greater than 63%) in the detergent-insoluble pellet, whereas the transmembrane-polypeptide-anchored ectoenzymes were recovered predominantly (greater than 95%) in the detergent-solubilized supernatant. Thus differential solubilization and temperature-induced phase separation in Triton X-114 distinguished between G-PI-anchored membrane proteins, transmembrane-polypeptide-anchored proteins and soluble, hydrophilic proteins. This technique may be more useful and reliable than susceptibility to release by phospholipases as a means of identifying a G-PI anchor on an unpurified membrane protein.

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Section: Introductionmentioning

confidence: 99%

Glycosyl-phosphatidylinositol-anchored membrane proteins can be distinguished from transmembrane polypeptide-anchored proteins by differential solubilization and temperature-induced phase separation in Triton X-114

Hooper

Bashir

1991

Biochemical Journal

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Characterization of an antibody to the cross-reacting determinant of the glycosyl-phosphatidylinositol anchor of human membrane dipeptidase

Broomfield

Hooper

1993

Biochimica et Biophysica Acta (BBA) - Biomembranes

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The cation‐independent mannose‐6‐phosphate receptor binds to soluble GPI‐linked proteins via mannose‐6‐phosphate

Green¹,

Ferguson²,

Robinson³

et al. 1995

FEBS Letters

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The cation-independent mannose-6-phosphatelinsulinlike growth factor II receptor has been observed to bind to soluble forms of glycosyl-phosphatidylinositol-linked molecules, one of mammalian origin (rat Thy-1) and two of protozoan origins. Of the two phosphate groups found on the soluble forms of the protozoan glycosyl-phosphatidylinositol-linked molecules: (i) the internal mannose-6-phosphate diester (which forms a part of the ethanolamine bridge) and (ii) the inositol-l,2 cyclic phosphate group (which arises after cleavage of the membrane associated form with phosphatidylinositol-specific phospholipase C), only the former appears to be recognized by the mannose-6-phosphate/ insulin-like growth factor II receptor, as mild acid hydrolysis which destroys the latter has been observed not to affect the receptor binding site.

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Production and characterization of antibodies against the cross-reacting determinant of glycosyl-phosphatidylinositol-anchored acetylcholinesterase

Cited by 12 publications

References 30 publications

Glycosyl-phosphatidylinositol-anchored membrane proteins can be distinguished from transmembrane polypeptide-anchored proteins by differential solubilization and temperature-induced phase separation in Triton X-114

Glycosyl-phosphatidylinositol-anchored membrane proteins can be distinguished from transmembrane polypeptide-anchored proteins by differential solubilization and temperature-induced phase separation in Triton X-114

Characterization of an antibody to the cross-reacting determinant of the glycosyl-phosphatidylinositol anchor of human membrane dipeptidase

The cation‐independent mannose‐6‐phosphate receptor binds to soluble GPI‐linked proteins via mannose‐6‐phosphate

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