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Title: Detection of phosphorylated forms of moloney murine leukemia virus major capsid protein p30 by immunoprecipitation and two-dimensional gel electrophoresis

Abstract

The authors detected phosphorylation of the major Moloney murine leukemia virus (M-MuLV) capsid polypeptide, p30, by using /sup 32/P/sub i/-labeled virions. This was observed both on two-dimensional polyacrylamide gels directly or on one-dimensional gels of viral lysates that had been immunoprecipitated with monospecific goat anti-p30 serum. The phosphorylation event had been difficult to detect because pp12 the major virion phosphoprotein incorporates almost all of the /sup 32/P label added to infected cells. When immunoprecipitates from M-MuLV lysates labeled with /sup 32/P/sub i/ were compared with those labeled with (/sup 35/S)methionine, it was calculated that the degree of phosphorylation at the p30 domain of Pr65/sup gag/ was only 0.22 to 0.54% relative to phosphorylation at the p12 domain. Two-dimensional gel electrophoresis of the /sup 32/P-labeled p30 immunoprecipitates showed that there were three phosphorylated p30 forms with isoelectric points (pIs) of 5.7, 5.8, and 6.0. These forms were generally more acidic than the (/sup 35/S) methionine-labeled p30 forms, which had pIs of 6.0, 6.1, 6.3 (the major constituent with > 80% of the label), and 6.6. The predominant phosphoamino acid of the major phosphorylated p30 form (pI 5.8) was phosphoserine. Further, tryptic peptide analysis of this p30 form showed that only onemore » peptide was predominantly phosphorylated. Based on a comparison of specific labeling of p30 tryptic peptides with (/sup 14/C)sesrine, (/sup 35/S)methionine, and /sup 32/P/sub i/, we tentatively assigned the phosphorylation site to a 2.4-kilodalton NH/sub 2/-terminal peptide containing triple tandem serines spanning the region from amino acids 4 to 24.« less

Authors:
;
Publication Date:
Research Org.:
Louisiana State Univ. Medical Center, New Orleans
OSTI Identifier:
5299272
Resource Type:
Journal Article
Resource Relation:
Journal Name: J. Virol.; (United States); Journal Volume: 62:1
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; LABELLED COMPOUNDS; TWO-DIMENSIONAL ELECTROPHORESIS; LEUKEMIA VIRUSES; IMMUNOLOGY; PROTEINS; CARBON 14 COMPOUNDS; METHIONINE; MOLECULAR WEIGHT; PHOSPHORUS 32; PHOSPHORYLATION; PRECIPITATION; SERINE; SULFUR 35; AMINO ACIDS; BETA DECAY RADIOISOTOPES; BETA-MINUS DECAY RADIOISOTOPES; CARBOXYLIC ACIDS; CHEMICAL REACTIONS; DAYS LIVING RADIOISOTOPES; DRUGS; ELECTROPHORESIS; EVEN-ODD NUCLEI; HYDROXY ACIDS; ISOTOPES; LIGHT NUCLEI; LIPOTROPIC FACTORS; MICROORGANISMS; NUCLEI; ODD-ODD NUCLEI; ONCOGENIC VIRUSES; ORGANIC ACIDS; ORGANIC COMPOUNDS; ORGANIC SULFUR COMPOUNDS; PARASITES; PHOSPHORUS ISOTOPES; RADIOISOTOPES; SEPARATION PROCESSES; SULFUR ISOTOPES; VIRUSES; 550701* - Microbiology- Tracer Techniques

Citation Formats

Ikuta, K., and Luftig, R.B.. Detection of phosphorylated forms of moloney murine leukemia virus major capsid protein p30 by immunoprecipitation and two-dimensional gel electrophoresis. United States: N. p., 1988. Web.
Ikuta, K., & Luftig, R.B.. Detection of phosphorylated forms of moloney murine leukemia virus major capsid protein p30 by immunoprecipitation and two-dimensional gel electrophoresis. United States.
Ikuta, K., and Luftig, R.B.. Fri . "Detection of phosphorylated forms of moloney murine leukemia virus major capsid protein p30 by immunoprecipitation and two-dimensional gel electrophoresis". United States.
@article{osti_5299272,
title = {Detection of phosphorylated forms of moloney murine leukemia virus major capsid protein p30 by immunoprecipitation and two-dimensional gel electrophoresis},
author = {Ikuta, K. and Luftig, R.B.},
abstractNote = {The authors detected phosphorylation of the major Moloney murine leukemia virus (M-MuLV) capsid polypeptide, p30, by using /sup 32/P/sub i/-labeled virions. This was observed both on two-dimensional polyacrylamide gels directly or on one-dimensional gels of viral lysates that had been immunoprecipitated with monospecific goat anti-p30 serum. The phosphorylation event had been difficult to detect because pp12 the major virion phosphoprotein incorporates almost all of the /sup 32/P label added to infected cells. When immunoprecipitates from M-MuLV lysates labeled with /sup 32/P/sub i/ were compared with those labeled with (/sup 35/S)methionine, it was calculated that the degree of phosphorylation at the p30 domain of Pr65/sup gag/ was only 0.22 to 0.54% relative to phosphorylation at the p12 domain. Two-dimensional gel electrophoresis of the /sup 32/P-labeled p30 immunoprecipitates showed that there were three phosphorylated p30 forms with isoelectric points (pIs) of 5.7, 5.8, and 6.0. These forms were generally more acidic than the (/sup 35/S) methionine-labeled p30 forms, which had pIs of 6.0, 6.1, 6.3 (the major constituent with > 80% of the label), and 6.6. The predominant phosphoamino acid of the major phosphorylated p30 form (pI 5.8) was phosphoserine. Further, tryptic peptide analysis of this p30 form showed that only one peptide was predominantly phosphorylated. Based on a comparison of specific labeling of p30 tryptic peptides with (/sup 14/C)sesrine, (/sup 35/S)methionine, and /sup 32/P/sub i/, we tentatively assigned the phosphorylation site to a 2.4-kilodalton NH/sub 2/-terminal peptide containing triple tandem serines spanning the region from amino acids 4 to 24.},
doi = {},
journal = {J. Virol.; (United States)},
number = ,
volume = 62:1,
place = {United States},
year = {Fri Jan 01 00:00:00 EST 1988},
month = {Fri Jan 01 00:00:00 EST 1988}
}