Abstract
A partially purified preparation of human erythrocyte protein 4.9, consisting of 48-, 52-, and 55-kilodalton polypeptides, is capable of bundling rabbit muscle actin in vitro (Siegel, D. L., and Branton, D. (1985) J. Cell Biol. 100, 775-785). Purification schemes, peptide mapping, antibody cross-reactivity, and chemical cross-linking techniques show that the 48- and 52-kDa polypeptides are sequence-related phosphorylated components, whereas the 55-kDa polypeptide is not. Purified protein 4.9 (dematin), consisting of 48- and 52-kDa polypeptides, effectively bundles actin in vitro; under similar conditions, the isolated 55-kDa polypeptide does not bundle actin. In fact, when added back to purified dematin, fractions containing the 55-kDa polypeptide can completely abolish dematin's actin-bundling activity. The basis for this inhibitory activity is an endogenous protein kinase that phosporylates both the 48- and 52-kDa isoforms of dematin, thus abolishing dematin's actin-bundling activity (Husain-Chishti, A., Levin, A., and Branton, D. (1988) Nature 334, 718-721). Although the endogenous kinase often co-purifies with the 55-kDa polypeptide, it can be separated from the 55-kDa polypeptide and has the characteristics of a catalytic subunit of a cyclic AMP-dependent protein kinase.
Highlights
These two forms were not separated from each other in a variety of different chromatographic conditions, yielded similar butnot identical peptides and phosphopeptides when subjected to enzymatic or chemical cleavage (Fig. 3, Miniprint), andcross-reacted with antibodies affinity-purified against each form (Fig. 4, Miniprint). Both isoformsparticipated in trimer formation(Fig.5), sedimented with actin bundles, and, when separated by preparative gel electrophoresis, either polypeptide alone could be renatured into trimers that had actin bundling activity. These results show that both the 48- and 52-kDa polypeptides are able to form homotrimers, we have not been able to determine if the native trimers are homotrimers or contain a mixture of the 48- and 52-kDa polypeptides
While the 48- and 52-kDa isoforms of dematin we have studied share many similarities, the decreased mobility of the 52-kDa polypeptide in the presence of reducing agent (Fig. 5, lune 2, Miniprint) suggests thatit may contain intramolecular sulfhydryl bridges not found in the 48-kDa form whose mobility remained the same in the presence or absence of DTT
The presence of more than one isoform of dematin in circulating erythrocytes could arise from post-translational modification, alternative mRNA splicing,or independentgene products
Summary
Dematinthe 55-kDa polypeptide does not bundle actin or contribute containing fractions (100mM NaCl), as assayed by SDS-PAGE, were to the bundling activity of the 48- and 52-kDa components. One- and Two-dimensionalPeptide Mapping-Coomassie-stained fied dematin were added to tritium-labeled rabbit muscle F-actin in polypeptides were excised from 10% gels and resolved on an 18% the bundling assay mixture. Tritium-labeled G-actin was separately added/mol of actin in thaessay mixture; when impure protein polymerized by the addition of50 mM KC1 and 2 mMMgC12 and containing the 55-kDa polypeptide was added
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