The molecular heterogeneity of the smg-21/Krev-1/rap1 proteins, a GTP-binding protein having the same effector domain as ras p21s, in bovine aortic smooth muscle membranes.
smg p21 is a member of the ras p21/ras p21-like small GTP-binding protein (G protein) superfamily, having the same putative effector domain as ras p21s. In the preceding report, we showed that smg p21 was a major G protein in bovine aortic smooth muscle membranes. Recently, two different smg p21 cDNA clones, designated smg-21A and -B, were isolated from a bovine brain cDNA library. In the present studies, we resolved the bovine aortic smg p21 fraction into two distinct G protein fractions on hydroxyapatite column chromatography and purified them separately to near homogeneity (22K G1 and -2). Both 22K G1 and -2 were specifically recognized by an anti-smg p21 polyclonal antibody. 22K G1 and -2 were identified as smg p21B and -A, respectively, by peptide map and amino acid sequence analyses. Purified smg p21A and -B showed GDP/GTP-binding and GTPase activities similar to each other. The GTPase activities of smg p21A and -B were equally stimulated by smg p21 GTPase activating protein 1 and -2. Moreover, both smg p21A and -B were phosphorylated by cyclic AMP-dependent protein kinase with a stoichiometry of one phosphate/molecule of protein. These results indicate that smg p21A and -B coexist in bovine aortic smooth muscle membranes and suggest that smg p21A and -B may serve as intermediates for cyclic AMP actions.
Kawata
M
M
Department of Internal Medicine (1st Division), Kobe University School of Medicine, Japan.
Kawahara
Y
Y
Sunako
M
M
Araki
S
S
Koide
M
M
Tsuda
T
T
Fukuzaki
H
H
Takai
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D001011:Aorta / Q000502:physiology*; D002417:Cattle; D002845:Chromatography; D002850:Chromatography, Gel; D017886:Durapatite; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006882:Hydroxyapatites; D015151:Immunoblotting; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009131:Muscle, Smooth, Vascular / Q000502:physiology*; D010449:Peptide Mapping; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D020668:rap GTP-Binding Proteins
D006882:Hydroxyapatites; D011518:Proto-Oncogene Proteins; D017886:Durapatite; D019204:GTP-Binding Proteins; D016283:Proto-Oncogene Proteins p21(ras); D020668:rap GTP-Binding Proteins