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Biomedical subjects

R F Doolittle

Publications and source records attributed to R F Doolittle.

At least 145 records · Page 8Linked to original sources
PubMed1980

Antibodies specific for the carboxy- and amino-terminal regions of simian virus 40 large tumor antigen.

Antibodies specific for the amino- and carboxy-terminal portions of simian virus 40 large tumor (T) antigen were obtained by immunization of rabbits with synthetic peptides corresponding to these regions. The amino-terminal synthetic peptide has the sequence Ac-Met-Asp-Lys-Val-Leu-Asn-Arg-(Tyr). The tyrosine residue was introduced in order to couple the peptide to bovine serum albumin with bis-diazotized benzidine. The carboxy-terminal peptide has the sequence Lys-Pro-Pro-Thr-Pro-Pro-Pro-Glu-Pro-Glu-Thr. It was coupled to bovine serum albumin with glutaraldehyde. The antisera against both peptides reacted with large T antigen. The specificity of the immune reaction was demonstrated by inhibition experiments using excess synthetic peptides. Furthermore, fragments of T antigen encoded by the nondefective adenovirus 2-simian virus 40 hybrid viruses Ad2+ND2 and Ad2+ND4, which contain the carboxy terminus and lack the amino terminus of large T antigen, were precipitated only with antiserum to the carboxy-terminal peptide. Small T antigen was not precipitated with either serum, suggesting that the amino terminus of small T antigen has a conformation different from that of large T antigen or that it is sterically hindered by a host protein. The procedures used here are of general importance for identification and characterization of gene product.

Adenoviruses, Human↗
PubMed1979

Amino acid sequence studies on the alpha chain of human fibrinogen. Complete sequence of the largest cyanogen bromide fragment.

The largest fragment produced by complete cyanogen bromide digestion of the alpha chain of human fibrinogen contains 236 residues and has a calculated molecular weight of 23,949. The complete amino acid sequence of the fragment was determined by the isolation of peptides generated by plasmin, trypsin (including digestion of citraconylated material), staphylococcal protease, and chymotrypsin. In addition, some key subfragmentation was achieved by selective chemical cleavage at tryptophan residues. The fragment has an unusual amino acid composition, more than half of its residues being glycine, serine, threonine, and proline. There are very few nonpolar residues, although 7 of the alpha-chain's 10 tryptophans occur in this fragment. The fragment contains 2 cysteine residues located 30 residues apart which are connected by an intrachain disulfide bond in the native molecule. The tryptophans occur with a definite periodicity that highlights a series of 13-residue homology repeats. The fragment also contains the two principal alpha-chain cross-linking sites.

Amino Acid Sequence↗
PubMed1979

Amino acid sequence studies on the alpha chain of human fibrinogen. Exact location of cross-linking acceptor sites.

Human fibrinogen was clotted under conditions that promote latent factor XIII activity and in the presence of a radioactive substitute cross-linking donor ([14C]glycine ethyl ester). The labeled fibrin was reduced and alkylated in the presence of 6 M guanidinium chloride. After dialysis and freeze-drying, the preparation was separated into its constituent polypeptide subunits by chromatography on (carboxymethyl)cellulose in the presence of 8 M urea. Under the incorporation conditions used, the radioactivity was limited to gamma chains (one donor molecule/chain) and alpha chains (two donor molecules/chain). The labeled alpha chains were digested with cyanogen bromide and fractionated on Sephadex G-50. All the radioactivity was found in a fragment previously designated H alpha CNI, the largest of the cyanogen bromide fragments in the alpha chain. The fragment was further fragmented by digestion with plasmin, trypsin, chymotrypsin, and/or staphylococcal protease. The incorporated radioactivity was found to reside in equal amounts at two different sites located 38 residues apart. These were determined to be positions 88 and 126 in H alpha CNI, which correspond to glutamine-328 and glutamine-366 in the alpha chain.

Amino Acid Sequence↗
PubMed1979

Amino acid sequence studies on the alpha chain of human fibrinogen. Overlapping sequences providing the complete sequence.

The complete amino acid sequence of the alpha chain of human fibrinogen has been determined. It contains 610 amino acid residues and has a calculated molecular weight of 66,124. The chain has 10 methionines, and fragmentation with cyanogen bromide yields 11 peptides [Doolittle, R.F., Cassman, K.G., Cottrell, B.A., Friezner, S.J., Hucko, J.T., & Takagi, T. (1977) Biochemistry 16, 1703]. The arrangement of the 11 fragments was determined by the isolation of peptide overlaps from plasmic and staphylococcal protease digests of fibrinogen and/or alpha chains. In addition, certain of the cyanogen bromide fragments, preliminary reports of whose sequences have appeared previously, have been reexamined in order to resolve several discrepancies. The alpha chain is homologous with the beta and gamma chains of fibrinogen, although a large repetitive segment of unusual composition is absent from the latter two chains. The existence of this unusual segment divides the sequence of the alpha chain into three zones of about 200 residues each that are readily distinguishable on the basis of amino acid composition alone.

Amino Acid Sequence↗
PubMed1979

The amino acid sequence of the alpha-chain of human fibrinogen.

The amino acid sequence of the human fibrinogen alpha-chain reveals a structure that can be divided into three zones of unique amino acid composition. The middle of these contains the two primary alpha-chain cross-linking acceptor sites and consists of a remarkable series of internal duplications.

Amino Acid Sequence↗
PubMed1979

Amino acid sequence of the beta chain of human fibrinogen.

The beta chain of human fibrinogen contains 461 amino acid residues, 15 of which are methionines. The calculated molecular weight, independent of a single carbohydrate cluster, is 52 230. In this regard, we have isolated and characterized all 16 cyanogen bromide fragments. In one case (CNI), we have concentrated on a disputed portion of a previously reported fragment. The arrangement of the cyanogen bromide peptides was deduced by the use of overlap fragments obtained from the tryptic digestion of modified and unmodified beta-chains and from digestions with staphylococcal protease, as well as by considerations involving the plasmic digestion products of fibrinogen. In one case two adjacent fragments were aligned by homology with the corresponding segments of the gamma chain. The homology of the beta chain with the gamma chain is especially strong over the course of the carboxy-terminal two-thirds of the sequence. Neither of these chains appears to be homologous with the alpha chain in these regions. With a few minor exceptions, the sequence reported in this article is in agreement with data reported by other groups in Stockholm and Munich.

Amino Acid Sequence↗
PubMed1978

Amino acid sequence of the beta chain of human fibrinogen: homology with the gamma chain.

The beta chain of human fibrinogen is composed of 452 +/- 5 amino acid residues, 14 of which are methionines. Consistent with these findings we have isolated and characterized 15 fragments after cyanogen bromide digestion of carboxymethylated beta chains. The arrangement of several of these peptides was deduced on the basis of overlapping peptides isolated from the fragments D and E produced by the plasmic digestion of fibrinogen and/or from a tryptic digest of citraconylated beta chains. Most of the other cyanogen bromide fragments can be aligned by homology with the alpha and/or gamma chains from human fibrinogen, although the positioning of a few of the smallest peptides is still ambiguous. The homology of the beta chain with the gamma chain is especially strong in certain regions of the domain that includes fragment D.

Amino Acid Sequence↗
PubMed1978

Synthetic peptide derivatives that bind to fibrinogen and prevent the polymerization of fibrin monomers.

A series of small peptides corresponding to the amino termini of the fibrin alpha- and beta-chains has been synthesized. The peptides glycyl-L-prolyl-L-arginyl-L-proline and glycyl-L-prolyl-L-arginylsarcosine are potent inhibitors of fibrin polymerization. Moreover, these peptides have a natural stability stemming from their inherent resistance to proteolysis because of the involvement of amino acids in each of their peptide bonds. The peptide glycyl-L-prolyl-L-arginyl-L-proline binds to fibrinogen and to fragment D, in both cases with an association constant of approximately 5 x 10(4); it does not bind to fragment E. The number of binding sites is two for fibrinogen and one for fragment D. The tripeptide glycyl-L-prolyl-L-arginine binds less tightly and is less than half as effective in preventing polymerization. The peptide glycyl-L-histidyl-L-arginyl-L-proline, which corresponds exactly to the amino terminus of the fibrin beta-chain, does not inhibit the aggregation of fibrin monomers under the conditions used. It does bind weakly to fibrinogen, however, suggesting the involvement of sites other than those binding the alpha-chain analogues. Various other peptides were found not to inhibit polymerization; these included glycine-L-proline, L-prolyl-L-arginine and glycyl-L-prolyl-L-seryl-L-proline. The last-named corresponds to the serine/arginine amino acid replacement previously reported for a defective human fibrinogen.

Amino Acid Sequence↗
PubMed1977

Amino acid sequence studies on the alpha chain of human fibrinogen. Characterization of 11 cyanogen bromide fragments.

The alpha chain of human fibrinogen consists of 600 +/- 25 amino acid residues, 10-11 of which are methionines. In this regard, we have identified and characterized 11 cyanogen bromide peptide fragments of 2, 3, 26, 28, 28, 37, 51, 56, 60 +/- 5, 64 +/- 5, and 260 +/- 20 residues, respectively. The sequences of five of these and a portion of a sixth have been reported previously. We now report the complete amino acid sequences of another of these fragments (56 residues), partial sequences for four others, and a preliminary characterization of the largest fragment. In a companion study (Doolittle, R. F., Cassman, K. G., Cottrell, B. A., Friezner, S. J., and Takagi, T. (1977), Biochemistry 16 (following paper in this issue)), we have obtained key overlap sequences from plasmic digests of fibrinogen which allow all but one of these cyanogen bromide peptides to be arranged in order. The sequences of some of these newly reported fragments have revealed an internal homology in the alpha chain, as well as structural similarities to the corresponding portions of the beta and gamma chains.

Amino Acid Sequence↗
PubMed1977

Amino acid sequence studies on the alpha chain of human fibrinogen. Covalent structure of the alpha-chain portion of fragment D.

The alpha-chain portion of fragment D has been purified from an exhaustive plasmic digest of human fibrinogen. The major polypeptide species has 91 amino acid residues, although a small amount of a 97-residue chain representing an earlier digestion stage remains. The amino acid sequence of the first 44 residues was determined by stepwise degradation with an automatic solid-phase sequencer. Another large stretch of sequence was revealed by the finding that the alpha chain of fragment D overlaps the cyanogen bromide fragments alphaCNIVA and alphaCNIII (Doolittle, R. F. Cassman, K. G., Cottrell, B. A., Friezner, S. J. Hucko, J. T., and Takagi, T. (1977), Biochemistry 16 (preceding paper in this issue)). The automatic sequencer results were confirmed and extended by the isolation and characterization of 18 of 19 expected tryptic peptides from the fragment D alpha chain. As a result, almost the entire sequence has been obtained. The overlap with key cyanogen bromide fragments has also allowed us to propose an order for the first 198 residues of the fibrinogen alpha chain. A striking homology with the gamma chain and beta chain is apparent which has interesting structural implications.

Amino Acid Sequence↗
PubMed1977

Amino acid sequence studies on the alpha chain of human fibrinogen. Isolation and characterization of two linked alpha-chained cyanogen bromide fragments from fully cross-linked fibrin.

Fully cross-linked human fibrin was digested with cyanogen bromide and the resulting fragments were characterized and compared with the fragments produced upon cyanogen bromide treatment of fibrinogen alpha chains. The largest molecular-weight fraction isolated by gel filtration on Sephadex G-150 was reduced and alkylated, and upon rechromatography on Sephadex G-150 it eluted at the same place as the original material. This large molecular weight fraction was subjected to amino acid analysis and the amino-terminal sequences of its constituent chains were determined by both dimethylaminonaphthyl sulfonation (Dns) and thioacetylation procedures. The identified sequences corresponded to two cyanogen bromide fragments previously found in alpha chains isolated from fibrinogen, one of which has a molecular weight of about 30 000 and the other 6000. The latter is thought to be the carboxy-terminal penultimate cyanogen bromide fragment of the alpha chain.

Amino Acid Sequence↗