Hydrophobicity and subunit interactions of rod outer segment proteins investigated using Triton X-114 phase partitioning.
Triton X-114 phase partitioning, a procedure used for purifying integral membrane proteins, was used to study protein components of the mammalian visual transduction cascade. An integral membrane protein, rhodopsin, and two isoprenylated protein complexes, cyclic GMP phosphodiesterase and Gt beta gamma, partitioned into the detergent-rich phase. Arrestin, a soluble protein, accumulated in the aqueous phase. Gt alpha distributed about equally between phases whether GDP (Gt alpha.GDP) or GTP (Gt alpha.GTP) was bound. Gt beta gamma increased recovery of Gt alpha.GDP but not Gt alpha.GTP in the detergent phase. Trypsin-treated Gt alpha, which lacks the fatty acylated amino-terminal 2-kDa region, accumulated to a greater extent in the aqueous phase than did intact Gt alpha. Trypsinized cGMP phosphodiesterase, which lacks the isoprenyl group, partitioned into the aqueous phase. A carboxyl-terminal truncated mutant (Val-331 stop) of Gt alpha accumulated more in the aqueous phase then did recombinant full-length Gt alpha, supporting the role of the carboxyl terminus in increasing its hydrophobicity. N-Myristoylated recombinant Go alpha was more hydrophobic than recombinant Go alpha without myristate. ADP-ribosylation of Gt alpha catalyzed by NAD:arginine ADP-ribosyltransferase, but not by pertussis toxin, increased hydrophilicity. Triton X-114 phase partitioning can thus semiquantify the hydrophobic nature of proteins and protein domains. It may aid in evaluating changes associated with post-translational protein modification and protein-protein interactions in a defined system.
Justice
J M
JM
Pulmonary-Critical Care Medicine Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892, USA.
Murtagh
J J
JJ
Moss
J
J
Vaughan
M
M
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000737:chemistry*; D000246:Adenosine Diphosphate Ribose / Q000737:chemistry; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D017931:DNA Primers; D019204:GTP-Binding Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D019814:Myristic Acid; D009227:Myristic Acids / Q000737:chemistry; D010449:Peptide Mapping; D011994:Recombinant Proteins / Q000737:chemistry; D012243:Rhodopsin / Q000737:chemistry*; D012374:Rod Cell Outer Segment / Q000737:chemistry*; D014357:Trypsin / Q000737:chemistry
D017931:DNA Primers; D009227:Myristic Acids; D011994:Recombinant Proteins; D019814:Myristic Acid; D000246:Adenosine Diphosphate Ribose; D012243:Rhodopsin; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D014357:Trypsin; D019204:GTP-Binding Proteins