The membrane proteins of the vacuolar system I. Analysis of a novel method of intralysosomal iodination - PubMed (original) (raw)

The membrane proteins of the vacuolar system I. Analysis of a novel method of intralysosomal iodination

W A Muller et al. J Cell Biol. 1980 Jul.

Abstract

A method has been developed to deliver an idoinating system into the confines of the phagolysosome, allowing us to study the nature of the phagolysosomal membrane. Lactoperoxidase (LPO) is covalently coupled to carboxylated latex spheres (LPO-latex) in a stable, enzymatically active form. The addition of LPO-latex to cultured macrophages leads to their rapid attachment, ingestion, and enclosure in a plasma membrane-derived phagocytic vacuole. These organelles rapidly fuse with preexisting lysosomes and are converted to phagolysosomes (PL) that demonstrates both acid phosphatase and lactoperoxidase activities. The exposure of LPO-latex containing cells to 125I- and an extracellular peroxide-generating system, glucose oxidase-glucose, at 4 degrees C leads to incorporation of label into TCA-precipitable material. The incorporated cel-associated label was present as monoiodotyrosine, and negligible amounts were found in lipids. Cell viability remained > 99%. Autoradiography at both the light and EM level revealed that > 97% of the cells were labeled, and quantitative analysis demonstrated the localization of grains to LPO-latex containing PL. PL were separated on sucrose gradients, and their radiolabel was confined almost exclusively to the membrane rather than soluble contents. SDS-polyacrylamide gel electrophoretic analysis of the peptides iodinated from within PL demonstrated at least 24 species with molecular weights ranging from 12,000 to 250,000. A very similar group of proteins was identified on the plasma membrane (PM) after surface iodination, and on latex phagosomes derived from iodinated PM. No novel proteins were detected in PL, either immediately after phagosome-lysosome fusion or after 1 h of intracytoplasmic residence. We conclude that the membrane proteins accessible to LPO-catalyzed iodination on the luminal surface of the PL and on the external face of the PM are similar, if not identical.

PubMed Disclaimer

Cited by

Intralysosomal accumulation of polyanions. I. Fusion of pinocytic and phagocytic vacuoles with secondary lysosomes.
Kielian MC, Steinman RM, Cohn ZA. Kielian MC, et al. J Cell Biol. 1982 Jun;93(3):866-74. doi: 10.1083/jcb.93.3.866. J Cell Biol. 1982. PMID: 6181074 Free PMC article.
Electron microscopic studies of the endocytotic process of cationized ferritin in cultured human retinal pigment epithelial cells.
Akeo K, Tanaka Y, Fujiwara T. Akeo K, et al. In Vitro Cell Dev Biol. 1988 Jul;24(7):705-10. doi: 10.1007/BF02623609. In Vitro Cell Dev Biol. 1988. PMID: 3397370
Production of the superoxide adduct of myeloperoxidase (compound III) by stimulated human neutrophils and its reactivity with hydrogen peroxide and chloride.
Winterbourn CC, Garcia RC, Segal AW. Winterbourn CC, et al. Biochem J. 1985 Jun 15;228(3):583-92. doi: 10.1042/bj2280583. Biochem J. 1985. PMID: 2992450 Free PMC article.
Iodination by stimulated human neutrophils. Studies on its stoichiometry, subcellular localization and relevance to microbial killing.
Segal AW, Garcia RC, Harper AM, Banga JP. Segal AW, et al. Biochem J. 1983 Jan 15;210(1):215-25. doi: 10.1042/bj2100215. Biochem J. 1983. PMID: 6303312 Free PMC article.

References

1. J Histochem Cytochem. 1966 Apr;14(4):291-302 - PubMed
1. J Biol Chem. 1969 May 10;244(9):2459-64 - PubMed
1. J Cell Biol. 1969 Oct;43(1):90-104 - PubMed
1. J Cell Biol. 1971 Sep;50(3):859-86 - PubMed
1. J Biol Chem. 1971 Oct 25;246(20):6328-34 - PubMed

The membrane proteins of the vacuolar system I. Analysis of a novel method of intralysosomal iodination - PubMed (original) (raw)