Biochemical evidence for the presence of an actin protein in Tetrahymena pyriformis.

Abstract

A protein from an ATP extract prepared from an acetone powder of Tetrahymena pyriformis GL was identified as actin. The protein migrated slightly behind muscle actin on sodium dodecyl sulphate (SDS)/10% polyacrylamide gels (SDS/PAGE) with an apparent molecular weight of 47 500 (47.5 X 10(3) Mr). Partial proteolysis of this band with Staphylococcus aureus V-8 protease followed by electrophoresis revealed a pattern of peptides in which at least four peptides were similar to those observed after digestion of rabbit skeletal muscle actin. The 47.5 X 10(3) Mr protein appeared particularly susceptible to endogenous proteolytic cleavage, which was inhibited by leupeptin. An ATP extract prepared with leupeptin was applied to a DNase I-affinity column and a distinct peak was eluted with 3 M-guanidine . HCl; the DNase I-binding protein appeared as a distinct band on SDS/PAGE with an apparent molecular weight of 47.5 X 10(3) Mr. In the absence of leupeptin, the DNase I-binding protein appeared as a broad 34 X 10(3) Mr band on gels. Both the ATP extract and the DNase I-binding protein showed reactivity with commercially available antiserum raised against native chicken skeletal muscle actin as determined by an enzyme-linked immunosorbance assay (ELISA). Immuno-blotting studies and affinity purification of this antiserum showed that the recognition was not specific to the 47.5 X 10(3) Mr protein. However, using affinity-purified anti-actin antibodies raised against denatured actin from chick smooth muscle, recognition of the 47.5 X 10(3) Mr protein and a 34 X 10(3) Mr protein was shown. In negatively stained preparations from an ATP extract after two cycles of polymerization and depolymerization there were filaments, 8-12 nm diameter, which did not decorate with subfragment S-1 of myosin, but which resembled intermediate filaments. Analysis of these filaments on SDS/PAGE indicated an intensely stained 54 X 10(3) Mr band. It is suggested that, in vitro, Tetrahymena intermediate filaments assemble under conditions expected to assemble actin filaments. Thus, in Tetrahymena there is a protein that resembles actin in its extractability, molecular weight, peptide pattern after partial proteolysis, DNase I-binding capacity and reactivity with anti-actin antibodies. However, this protein did not assemble into actin filaments in crude extracts.