Document Detail

Transacylase-mediated alkylacyl-GPC synthesis and its hydrolysis by phospholipase D occur in separate cell compartments in the human neutrophil.
MedLine Citation:
PMID:  8836876     Owner:  NLM     Status:  MEDLINE    
Subcellular localizations of CoA-independent transacylase and phospholipase D enzymes have been investigated in human neutrophils performing a two-step gradient system to separate plasma membranes from internal membranes and from the bulk of granules. The internal membranes were constituted by endoplasmic reticulum and by a subpopulation of specific and tertiary granules. The enzymes activities were assayed in vitro on gradient fractions using exogenous substrates. Following cell prelabelling with [3H]alkyllyso-GPC, we also analyzed the in situ localization of labelled products involving the action of both enzymes. The CoA-independent transacylase activity, together with the CoA-dependent transacylase and acyltransferase activities were only located in the internal membranes. Following 15 min cell labelling, part of the [3H]alkylacyl-GPC was recovered in plasma membranes indicating a rapid redistribution of the acylated compound. Very high contents in arachidonate containing [3H]alkylacyl-GPC were recovered both in plasma membranes and internal membranes. Phospholipase D activity being assayed in the presence of cytosol, GTP gamma S and gradient fractions, only the plasma membrane fractions from resting or stimulated cells allowed the enzyme to be active. The [3H]alkylacyl-GP and [3H]alkylacyl-GPethanol, phospholipase D breakdown products from [3H]alkylacyl-GPC, obtained after neutrophil prelabelling and activation by phorbol myristate acetate, were exclusively present in the plasma membranes. In contrast, the secondary generated [3H]alkylacylglycerols were equally distributed between plasma and internal membranes. No labelled product was recovered on azurophil granules. These data demonstrate that internal membranes are the site of action of the CoA-independent transacylase and plasma membranes are the site of action of the phospholipase D. This topographical separation between CoA-independent transacylase which generated substrate and phospholipase D which degraded it, suggested that subcellular localisation and traffic of substrates within the cell can be important to regulate the enzymes.
G Ribbes; A Cane; V Planat; M Breton; H Chap; G Béréziat; M Record; O Colard
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Journal of cellular biochemistry     Volume:  62     ISSN:  0730-2312     ISO Abbreviation:  J. Cell. Biochem.     Publication Date:  1996 Jul 
Date Detail:
Created Date:  1996-12-11     Completed Date:  1996-12-11     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  8205768     Medline TA:  J Cell Biochem     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  56-68     Citation Subset:  IM    
INSERM Unité 326, Hôpital Purpan, Toulouse, France.
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MeSH Terms
Amidohydrolases / metabolism*
Cell Compartmentation
Cell Membrane / metabolism
Cytoplasmic Granules / metabolism
Intracellular Membranes / metabolism
Neutrophils / metabolism*
Phosphatidylcholines / biosynthesis*
Phospholipase D / metabolism*
Subcellular Fractions / metabolism
Reg. No./Substance:
0/Phosphatidylcholines; EC D; EC 3.5.-/Amidohydrolases; EC

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