Purification and characterization of a cytosolic 65-kilodalton phosphoprotein in human leukocytes whose phosphorylation is augmented by stimulation with interleukin 1

Journal Article · Tue May 17 00:00:00 EDT 1988 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00410a037· OSTI ID:6994159

Matsushima, K; Shiroo, M; Kung, H; Copeland, T D

The authors have recently shown that glucocorticoids dramatically increase the number of interleukin 1 (IL 1) receptors on human peripheral blood mononuclear cells (PBMC) and that IL 1 selectively induces the phosphorylation of a cytosolic 65-kilodalton (kDa) protein (pp 65) in glucocorticoid-pretreated PBMC. They describe here the purification and biochemical characteristics of pp 65. /sup 32/P-Labeled pp 65 was purified to homogeneity from the cytosol fraction of IL 1 stimulated (/sup 32/P)orthophosphate-labeled PBMC by sequential chromatography on Sephacryl S-200, high-performance liquid chromatography (HPLC) anion exchange, and hydroxyapatite HPLC. The purified pp 65 was homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The unphosphorylated 65-kDa protein (p 65) was also purified to homogeneity in a similar way. About 40 ..mu..g of purified 65-kDa protein was recovered from 5 x 10/sup 8/ PBMC. Analysis of the amino-terminal sequence of the purified pp 65 revealed the amino terminus of pp 65 to be blocked. Amino acid sequence analysis of a cyanogen bromide cleaved peptide showed pp 65 to be a unique protein whose protein sequence has not yet been reported. Studies of the distribution of p(p) 65 based on Western blotting using specific polyclonal rabbit antibody to p(p) 65 showed that p(p) 65 exists in a variety of cells such as neutrophils, monocytes, B lymphocytes, and myeloid cells. It could not be detected in the T cell leukemia cell line (MOLT), melanoma cells, and fibroblasts. Although the role of the pp 65 has not yet been established, the protein sequence should provide useful information for the cloning of cDNA encoded for pp 65.

• Research Organization: Frederick Cancer Research Facility, MD (USA)
• OSTI ID: 6994159
• Journal Information: Biochemistry; (United States), Vol. 27:10
• Country of Publication: United States
• Language: English

Related Subjects

62 RADIOLOGY AND NUCLEAR MEDICINE
59 BASIC BIOLOGICAL SCIENCES
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PROTEINS
AUTORADIOGRAPHY
PHOSPHORYLATION
PURIFICATION
AMINO ACID SEQUENCE
ELECTROPHORESIS
GAS CHROMATOGRAPHY
GLUCOCORTICOIDS
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BIOLOGICAL MATERIALS
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BODY FLUIDS
CHEMICAL REACTIONS
CHEMISTRY
CHROMATOGRAPHY
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DAYS LIVING RADIOISOTOPES
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HYDROXY COMPOUNDS
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KETONES
LIGHT NUCLEI
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NUCLEI
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STEROIDS
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