Detection of phosphorylated forms of moloney murine leukemia virus major capsid protein p30 by immunoprecipitation and two-dimensional gel electrophoresis
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.
- Research Organization:
- Louisiana State Univ. Medical Center, New Orleans
- OSTI ID:
- 5299272
- Journal Information:
- J. Virol.; (United States), Journal Name: J. Virol.; (United States) Vol. 62:1; ISSN JOVIA
- Country of Publication:
- United States
- Language:
- English
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59 BASIC BIOLOGICAL SCIENCES
AMINO ACIDS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CARBON 14 COMPOUNDS
CARBOXYLIC ACIDS
CHEMICAL REACTIONS
DAYS LIVING RADIOISOTOPES
DRUGS
ELECTROPHORESIS
EVEN-ODD NUCLEI
HYDROXY ACIDS
IMMUNOLOGY
ISOTOPES
LABELLED COMPOUNDS
LEUKEMIA VIRUSES
LIGHT NUCLEI
LIPOTROPIC FACTORS
METHIONINE
MICROORGANISMS
MOLECULAR WEIGHT
NUCLEI
ODD-ODD NUCLEI
ONCOGENIC VIRUSES
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PARASITES
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PHOSPHORYLATION
PRECIPITATION
PROTEINS
RADIOISOTOPES
SEPARATION PROCESSES
SERINE
SULFUR 35
SULFUR ISOTOPES
TWO-DIMENSIONAL ELECTROPHORESIS
VIRUSES