Biomedical subjects

J Edgeworth

Publications and source records attributed to J Edgeworth.

4 recordsLinked to original sources

PubMed1991

Identification of p8,14 as a highly abundant heterodimeric calcium binding protein complex of myeloid cells.

In this report we describe the biochemical characterization of neutrophil and monocyte p8 and p14. Together the two proteins comprise approximately 45% of cytosolic protein in neutrophils and approximately 40-fold less in monocytes. They fractionated together in several chromatographic procedures and were found to exist as a noncovalently associated complex with a stoichiometry of 1:1, named p8,14. Cross-linking experiments showed p8,14 to form heterodimers under conditions simulating the cytosol. An apparent molecular mass of 35,000 daltons was obtained for the p8,14 complex in molecular sizing experiments which suggests the presence of modifications or distinctive structural features. Two major forms of p14 can be identified by two-dimensional gel electrophoresis, both of which form heterodimers with p8. The lower molecular weight variant of p14 lacks Cys-3 (Met-Thr-Cys-Lys-Met...) suggesting that differing translational start sites account for these two forms of p14. A protocol has been devised for the rapid purification of milligram quantities of p8 and p14 from neutrophil cytosol using fast-protein liquid chromatography.

Blotting, Western↗

PubMed1989

Ionomycin-regulated phosphorylation of the myeloid calcium-binding protein p14.

Two associated calcium-binding proteins (CaBPs) have recently been identified specifically in cells of myeloid origin. These proteins have relative molecular masses (Mr) of 8,000 and 14,000 and are variously referred to as the cystic fibrosis antigen, the L1 light chain, MRP-8 or p8, and the L1 heavy chain, MRP14 or p14, respectively. The expression of p8 and p14 seems to be confined to a specific stage of myeloid cell differentiation, because both proteins are expressed in circulating neutrophils and monocytes but not in normal tissue macrophages. In chronic inflammatory conditions, however, such as rheumatoid arthritis, macrophages in affected tissues express both p8 and p14. These proteins are members of a family of CaBPs of low Mr, which include S-100 alpha and beta proteins, calcyclin (2A9), intestinal CaBP and p11. All the proteins have an Mr of approximately 10,000 with the exception of p14 which has a longer C-terminal sequence after the second calcium-binding domain. Little is known about their function, although by analogy with calmodulin they could be molecules involved in intracellular signalling that are activated by an increase in the intracellular Ca2+ concentration ([Ca2+]). Here we report that p14 is phosphorylated in both monocytes and neutrophils. The level of p14 phosphorylation can be increased by elevating the [Ca2+]i using the ionophore ionomycin, but is not affected by activation of protein kinase C using phorbol 12,13-dibutyrate. The phosphorylated residue is threonine at position 113, which is the penultimate amino acid in p14 and contained in the longer 'tail' sequence. Part of this sequence is identical to the neutrophil immobilizing factors NIF-1 and NIF-2, indicating that the phosphorylation event could have a role in the generation of NIF activity in the p14 protein.

Amino Acid Sequence↗

PubMed1989

Sequence identity.

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Amino Acid Sequence↗

PubMed1989

Monoclonal antibody 5.5 reacts with p8,14, a myeloid molecule associated with some vascular endothelium.

The movement of mononuclear phagocytes and neutrophils from the circulation into tissues is a process which is not completely understood. Monoclonal antibody 5.5 is specific for an 8/14-kDa molecule known variously as the CF antigen, L1 molecule or MRP8 and 14. We show that this molecule, which will be named p8,14 in this study, is expressed in all circulating monocytes and neutrophils as an intracellular product (as well as some types of epithelium). Tissue staining patterns suggest that when monocytes and neutrophils adhere to vascular endothelium, they release this molecule onto the associated endothelium. This process occurs with single monocytes and when monocytes form part of an inflammatory infiltrate. Monoclonal antibody 5.5 does not react with cultured endothelial cells even when stimulated with phorbol ester, tumor necrosis factor, interferon-gamma or interleukin 1 alpha providing further evidence that myeloid cells are the source of the p8,14 in this interactive process. Monocytes which have moved further into such tissues and tissue macrophages in general are monoclonal antibody 5.5 negative, suggesting that the ability to synthesize this molecule may be lost when monocytes leave the circulation and enter tissues. These results indicate that p8,14 plays a role in the interaction between myeloid cells and the vascular endothelium to which they adhere prior to leaving the circulation.

Antibodies, Monoclonal↗