S. J. Singer

Bifunctional imidoesters as cross-linking reagents

Abstract Bifunctional small molecule reagents have been important in the study of a number of problems in protein chemistry (cf. Wold, 1961 a, b; Zahn and Meienhofer, 1958 ; Singer, 1959 ). For most purposes it is desirable that the chemical modifications produced by the bifunctional reagent cause as little alteration as possible in the conformation of the protein molecule. The exhaustive amidination of ɛ-NH 3 + groups with monofunctional imidoesters ( Hunter and Ludwig, 1962 ) preserves the positive charges near these sites, and was shown to produce little or no significant changes in the conformational properties or biological activities of proteins ( Wofsy and Singer, 1963 ). It was therefore anticipated that bifunctional imidoesters would be highly useful cross-linking agents, in that they would not affect the charge distribution of the proteins which they modified. Preliminary investigations along these lines are reported in this paper.

A disulfide-bridge bifunctional imidoester as a reversible cross-linking reagent.

Abstract A disulfide-bridged bifunctional imidoester, dimethyl 3, 3′ dithio-bispropionimidate (DTP) has been prepared and investigated as a reagent to introduce covalent cross-links in proteins that can subsequently be broken by mild reduction. Such reversible cross-links were shown to be introduced by DTP in the soluble subunit proteins aldolase and Concanavalin A. DTP was also used to modify human intact erythrocytes. Such modification rendered the erythrocytes resistant to hypotonic lysis; subsequent treatment with mercaptoethanol lysed the cells. After DTP-modification of the cells, the hemoglobin contained in them could still be reversibly oxygenated and deoxygenated.

The two components of spectrin, filamin, and the heavy chain of smooth muscle myosin show no detectable homologies to one another by two-dimensional mapping of iodinated tryptic peptides.

Abstract The possible structural relationships among four high molecular weight mechanochemical proteins has been examined using two-dimensional mapping of the tryptic peptide fragments prepared from 125I-labeled proteins (Elder et al., J. Biol. Chem. 252 :6510–6515 (1977)). Erythrocyte spectrin bands 1 and 2 protein, the heavy chain of smooth muscle (uterine) myosin, a filamin from human and rabbit were studied. The maps of the four proteins within each species differed considerably from each other, with no apparent homologies evident among them, whereas maps of the same individual protein between the two species showed a high degree of homology.

Affinity labeling of antibodies to the p-azophenyltrimethylammonium hapten and a structural relationship among antibody active sites of different specificities

Abstract In studies from this laboratory (Wofsy, et al. , 1962; Metzger, et al. , 1963; Metzger, et al. , 1964; Singer, et al. , 1965) the method of affinity labeling of the active sites of protein molecules was proposed and investigated experimentally. In this method, a labeling agent is utilized which 1) combines specifically and reversibly with the particular active site; and 2) reacts with one or more amino acid residues in the site to form irreversible covalent linkages. Two anti-hapten antibody (Ab) systems, anti-p-azobenzenearsonate (anti-R) and anti-2,4 dinitrophenyllysyl (anti-DNP), have previously been investigated. To complement these studies with Ab to the negatively charged R and the neutral DNP haptenic determinants, we have now investigated Ab to the positively charged p-azophenyltrimethylammonium (TMA) determinant. The labeling reagent used with the anti-TMA Ab was p-(trimethylammonium) benzenediazonium difluoborate (TDF). As in our previous studies, the diazonium group was the reactive functional group of the labeling reagent, and the formation of covalent and irreversible azo-linkages between the reagent and the Ab was followed spectrophotometrically. The results obtained with all three systems are so closely similar as to suggest some significant generalizations about the composition and structure of Ab active sites.

Muscle G-actin is an inhibitor of ATP-induced erythrocyte ghost shape changes and endocytosis

Summary Muscle G-actin included in erythrocyte ghosts is an effective inhibitor, with an apparent K m ∼ 20 μg/ml, of ATP-induced shape changes and endocytosis. Pancreatic DNAase I, which forms a tightly bound complex with G-actin, prevents the actin inhibition. G-actin has no effect on the degree of phosphorylation of spectrin component 2 by ATP but it binds effectively to the ghost membrane under the conditions where it is an inhibitor of the shape changes. DNAase I and pyrophosphate significantly reduce such binding. These results are consistent with the existence of a competition between the added G-actin and the endogenous erythrocyte actin during ATP-induced shape changes and endocytosis, and therefore suggest an important function for the endogenous actin in these processes.