Externally disposed plasma membrane proteins. II. Metabolic fate of iodinated polypeptides of mouse L cells

Ann Louise Hubbard, Z. A. Cohn

Research output: Contribution to journalArticle

Abstract

The fate of the L cell plasma membrane proteins labeled by enzymatic iodination was studied. The disappearance of label from growing cells exhibits a biphasic behavior, with 5-20% lost rapidly (t1/2 2 hr) and 80-90% lost relatively slowly (t1/2 25-33 hr). The loss is temperature dependent and serum independent, and is accompanied by the appearance of 51% [125I]monoiodotyrosine (MIT) in the medium by 47 hr. A variable amount (1-14%) of acid insoluble label can be recovered in the medium over 47 hr. Sodium dodecyl sulfate (SDS) polyacrylamide gel labeling patterns from cells cultured up to 48 hr after iodination reveal no change in the relative distribution of radioactivity, indicating similar rates of degradation for most of the labeled membrane proteins. The fate of the labeled membrane proteins was studied at various times after phagocytosis of nondigestible polystyrene particles. Iodinated L cells phagocytose sufficient 1.1 μm latex beads in 60 min to interiorize 15-30% of the total cell surface area. Electron microscope autoradiography confirmed that labeled membrane is internalized during phagocytosis. The latex containing phagocytic vacuoles are isolated by flotation in a discontinuous sucrose gradient. 15-30% of the total incorporated label and a comparable percentage of alkaline phosphodiesterase I activity (PDase, a plasma membrane enzyme marker) are recovered in the phagocytic vacuole fraction. Lysosomal enzyme activities are found in the latex vacuole fraction, indicating formation of phagolysosomes. SDS gel analyses reveal that all of the radioactive proteins initially present on the intact cell's surface are interiorized to the same relative extent. Incorporated label and PDase activity disappear much more rapidly from the phagolysosomes than from the whole cell. In the phagolysosomal compartment, >70% of the TCA precipitable labeled proteins and all of the PDase activity are lost rapidly (t1/2 = 1-2 hr) but 30% of the labeled proteins in this compartment are degraded with a 17-20 hr half life. The slowly degraded label is due to specific long lived polypeptides, of 85,000 and 8,000-15,000 daltons, which remain in the phagolysosomal membrane up to 40 hr after phagocytosis.

Original languageEnglish (US)
Pages (from-to)461-479
Number of pages19
JournalJournal of Cell Biology
Volume64
Issue number2
StatePublished - 1975
Externally publishedYes

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Blood Proteins
Membrane Proteins
Vacuoles
Phagocytosis
Cell Membrane
Phagosomes
Peptides
Halogenation
Latex
Sodium Dodecyl Sulfate
Monoiodotyrosine
Phosphodiesterase I
Cytophagocytosis
Proteins
Membranes
Polystyrenes
Enzymes
Autoradiography
Microspheres
Radioactivity

ASJC Scopus subject areas

  • Cell Biology

Cite this

Externally disposed plasma membrane proteins. II. Metabolic fate of iodinated polypeptides of mouse L cells. / Hubbard, Ann Louise; Cohn, Z. A.

In: Journal of Cell Biology, Vol. 64, No. 2, 1975, p. 461-479.

Research output: Contribution to journalArticle

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abstract = "The fate of the L cell plasma membrane proteins labeled by enzymatic iodination was studied. The disappearance of label from growing cells exhibits a biphasic behavior, with 5-20{\%} lost rapidly (t1/2 2 hr) and 80-90{\%} lost relatively slowly (t1/2 25-33 hr). The loss is temperature dependent and serum independent, and is accompanied by the appearance of 51{\%} [125I]monoiodotyrosine (MIT) in the medium by 47 hr. A variable amount (1-14{\%}) of acid insoluble label can be recovered in the medium over 47 hr. Sodium dodecyl sulfate (SDS) polyacrylamide gel labeling patterns from cells cultured up to 48 hr after iodination reveal no change in the relative distribution of radioactivity, indicating similar rates of degradation for most of the labeled membrane proteins. The fate of the labeled membrane proteins was studied at various times after phagocytosis of nondigestible polystyrene particles. Iodinated L cells phagocytose sufficient 1.1 μm latex beads in 60 min to interiorize 15-30{\%} of the total cell surface area. Electron microscope autoradiography confirmed that labeled membrane is internalized during phagocytosis. The latex containing phagocytic vacuoles are isolated by flotation in a discontinuous sucrose gradient. 15-30{\%} of the total incorporated label and a comparable percentage of alkaline phosphodiesterase I activity (PDase, a plasma membrane enzyme marker) are recovered in the phagocytic vacuole fraction. Lysosomal enzyme activities are found in the latex vacuole fraction, indicating formation of phagolysosomes. SDS gel analyses reveal that all of the radioactive proteins initially present on the intact cell's surface are interiorized to the same relative extent. Incorporated label and PDase activity disappear much more rapidly from the phagolysosomes than from the whole cell. In the phagolysosomal compartment, >70{\%} of the TCA precipitable labeled proteins and all of the PDase activity are lost rapidly (t1/2 = 1-2 hr) but 30{\%} of the labeled proteins in this compartment are degraded with a 17-20 hr half life. The slowly degraded label is due to specific long lived polypeptides, of 85,000 and 8,000-15,000 daltons, which remain in the phagolysosomal membrane up to 40 hr after phagocytosis.",
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