Actin Amino-Acid Sequences. Comparison of Actins from Calf Thymus, Bovine Brain, and SV40-Transformed Mouse 3T3 Cells with Rabbit Skeletal Muscle Actin

Abstract

Actin was purified from calf thymus, bovine brain and SV40-transformed mouse 3T3 cells grown in tissue culture. Isoelectric focusing analysis showed the presence of the two actin polypeptides p and y typical for non-muscle actins in all three actins. Tryptic and thermolytic peptides accounting for the complete amino-acid sequence of the cytoplasmic actins were separated and isolated by preparative fingerprint techniques. All peptides were characterized by amino-acid analysis and compared with the corresponding peptides from rabbit skeletal muscle actin. Peptides which differed in amino-acid composition from the corresponding skeletal muscle actin peptides were subjected to sequence analysis in order to localize the amino-acid replacement. The results obtained show that all three mammalian cytoplasmic actins studied contain the same amino-acid exchanges indicating that mammalian cytoplasmic actins are very similar if not identical in amino-acid sequence.The presence of two different isoelectric species p and 1' in cytoplasmic actins from higher vertebrates is accounted for by the isolation of two very similar but not identical amino-terminal peptides in all three actin preparations. The nature of the amino-acid replacements in these two peptides not only accounts for the different isoelectric forms but also shows that p and y cytoplasmic actins are the products of two different structural genes expressed in the same cell. The total number of aminoacid replacements so far detected in the comparison of these cytoplasmic actins and skeletal muscle actin is 25 for the p chain and 24 for the y chain. With the exception of the amino-terminal three or four residues, which are responsible for the isoelectric differences, the replacements do not involve charged amino acids. The exchanges are not randomly distributed. No replacements were detected in regions 18 -75 and 299 -356 while the regions between residues 2 -17 and 259 -298 show a high number of replacements.In addition documentation for a few minor revisions of the amino acid sequence of rabbit skeletal muscle actin is provided

The chromosomal arrangement of two linked actin genes in the sea urchin S. purpuratus

“…On the restriction map (Fig. 2) (18,19). Above Gln 121 is the consensus sequence of the exon-intron boundary (20).…”

The amino acid sequence of actin from chicken skeletal muscle actin and chicken gizzard smooth muscle actin

“…The peptide comprising the 3 N-terminal residues of chicken gizzard actin and the peptide comprising the 4 N-terminal residues of chicken skeletal muscle actin, which form a cluster of very acidic residues [ 1, 3,4] migrate from the paper under the electrophoretic conditions used for optimal separation of the other peptides. These residues can however be studied easily as a part of the N-terminal tryptic peptide covering the 17 (gizzard) or 18 (skeletal muscle) N-terminal residues [I].…”

“…The mixture of soluble tryptic peptides was further cleaved with the glutamoyl-specific protease ofStaphyZocuccus aweus and the insoluble tryptic 'core' peptides were further hydrolyzed with thermolysin. The resulting two sets of fully soluble and relatively small peptides (average size 6.5 residues) were resolved in individual peptides by a preparative two-d~ension~ fmgerp~nt system as in [3]. Peptides were detected with a dilute fluorescamine stain [9] and aliquots hydrolyzed.…”

“…These residues can however be studied easily as a part of the N-terminal tryptic peptide covering the 17 (gizzard) or 18 (skeletal muscle) N-terminal residues [I]. Table 1 shows the amino acid compositions of those peptides from chicken gizzard actin which differ in composition from the corresponding rabbit skeletal muscle actin peptides [2,3]. The positioning of the amino acid substitutions were made as follows:…”

“…These actins are: two cytoplasmic actins typical of non-muscle tissue; two smooth muscle actins and two striated muscle actins, i.e., cardiac muscle actin and skeletal muscle actin [ 11. Complete amino acid sequences are currently available only for rabbit skeletal muscle actin [2] and for the two mammalian cytoplasmic actins [3]. Sequence data on smooth muscle actins however are still incomplete [ 1,4].…”