John E. Mole

High-sensitivity sequence determination of proteins quantitatively recovered from sodium dodecyl sulfate gels using an improved electrodialysis procedure

Abstract An improved electrodialysis procedure has been developed to recover quantitatively proteins from sodium dodecyl sulfate (SDS)-polyacrylamide gels. The efficiency of the method has been demonstrated successfully on H-2K k , β 2 -microglobulin, complement factor D, and viral structural protein p27. The results indicate that yields exceeding 93% are obtainable, and that extended amino acid sequences of the eluted proteins in microgram quantities can be obtained in the presence of SDS without intrinsic and/or extrinsic labeling with radioisotopes.

An improved procedure for high-sensitivity microsequencing: Use of aminoethyl aminopropyl glass beads in the beckman sequencer and the Ultrasphere ODS column for PTH amino acid identification

An improved procedure for high-sensitivity microsequencing is described. The method employs (i) aminoethyl aminopropyl glass beads for in situ purification of quadrol by absorbing α-amino group reactive impurities commonly found even in highly purified quadrol and (ii) an Altex 5-μm Ultrasphere ODS column and highly purified methanol which is available commercially to identify phenylthiohydantoin derivatives of amino acids by high-pressure liquid chromatography. The efficiency of the method has been demonstrated by the results of sequence analyses of peptides obtained by acid and cyanogen bromide digestions of viral proteins. A significant increase in the initial and repetitive yields has been consistently observed by this procedure.

A simple modification on the vacuum system of the Beckman automated sequencer to improve the efficiency of edman degradation

Abstract A simple modification of the high-vacuum system of the Beckman automated sequencer is described. The air cylinder controlling the high vacuum to the reaction cup has been replaced with an electrically operated stainless-steel solenoid valve. The system maintains better high vacuum without the necessity for frequent oil changes. The efficiency has been demonstrated by the results of extended amino acid sequence analysis of various unknown proteins and peptides.

Use of fluorescamine as an effective blocking reagent to reduce the background in protein sequence analyses by the beckman automated sequencer

As an effective aid to further develop the strategies for extended amino acid sequence determinations, the use of fluorescamine (fluram) as a novel blocking reagent to chemically reduce the newly generated amino termini responsible for the progressively increasing background is described. The method involves interruption of the run when proline has been determined to be the amino terminus, deletion of PITC delivery, in situ treatment of the sample with fluram within the spinning cup extraction of the reaction products using an ethyl acetate: benzene mixture, HFBA delivery, and second extraction with 1-chlorobutane. The normal program is then allowed to continue until the next proline is reached and the entire extended sequence information on nanomole amounts of proteins. The efficiency of the method has been demonstrated by direct comparison of the results of sequence analysis of amyloid P-component with (63 residues) and without (46 residues) fluram treatment.

Computer-Assisted high-pressure liquid chromatography of radio-labelled phenylthiohydantoin amino acids

Abstract A computer-controlled high-pressure liquid chromatographic (HPLC) system is described to identify vitro phenyl [35S]isothiocyanate-labelled phenylthiohydantoin (PTH) amino acids from a solid-phase sequencer. Each radio-labelled amino acid from the sequencer is added to a PTH amino acid standard and the mixture separated by HPLC using a computer, programmed to detect a slope change in the absorbance. Individual fractions corresponding to the PTH amino acids are collected and counted. The sensitivity of the system is demonstrated on 700 pmoles of lysozyme.

The H-2Kk antigen: isolation using monoclonal immunoadsorbent chromatography and sequence analysis without radioisotopes.

Monoclonal immunoadsorbent chromatography has been used to isolate milligram quantities of detergent-solubilized H-2Kk antigen. Using the procedure described in this paper 10(12) cells may be processed yielding 10 mg of homogenous H-2Kk which represents 70% of the allotypic serological activity present in the original homogenate. NH2-Terminal sequence data of the first 30 residues of the H-2Kk heavy chain are presented. The cell line selected as the source of antigen and the criteria of purity of the antigen have been found to be critical as proteins of molecular weight 42,000 and 12,000 were copurified with H-2Kk from the BW5147 cell line. The additional components were observed in gradient gel electrophoresis or two-dimensional electrophoresis, but not in conventional Laemmli gel electrophoresis.

Purification and characterization of the gag gene products of avian-type C retroviruses by high-pressure liquid chromatography

Abstract A rapid and efficient method for separating the translational products of the gag gene of avian-type C retroviruses by employing a high-resolution gel permeation column, high-pressure liquid chromatography and volatile solvents has been described. The viral structural components p27, p19, and p15 have been purified in milligram quantities to near homogeneity and in high yields. The purity of the products of separation has been confirmed by amino acid analysis and amino and carboxy terminal amino acid sequence determination, and their molecular integrity was established by immunological methods.

Sequence analysis of human J chain. Amino terminal location of a disulfide bond linking the immunoglobulin heavy chain

Abstract Carboxyamidomethylated J chain was shown to be an excellent substrate for the enzyme, pyrollidone carboxylyl peptidase, which specifically removed the cyclized amino terminal glutamyl residue. J chain lacking the “blocked” PCA group was subjected to automated Edman degradation and the amino terminal amino acid sequence determined as: PCA-Glu-Asp-Glu-Arg-Ile-Val-Leu-Val-Asp-Asn-Lys-CMCys-Lys-CMCys-Ala-Arg. Previous studies by others have identified a disulfide bridge between the heavy chain of immunoglobulins and a tripeptide identical in composition with the sequence at positions 15–17 in the J chain. These two sets of data locate the linkage of immunoglobulin heavy chain with Cys 15 of the J chain.

The amino acid sequence of the C- terminal cyanogen bromide peptide of human J chain

Abstract The carboxyl-terminal peptide obtained from human J chain treated with cyanogen bromide (CNBr) 1 has been isolated and the amino acid sequence determined as Val - Glx - Thr - Ala - Leu - Thr - Pro - Asx - Ala - CMCys - Tyr - Pro - Asx. Comparisons with the primary structures of human lambda, kappa, alpha, or mu chains failed to disclose analogous regions with these immunoglobulins.

The Molecular Structure and Interrelationships of Murine T Lymphoid Cell-Surface Antigens

The current understanding of the immune system is that of a complex, self-regulating network of cell interactions involving four components: bone-marrowderived (B) lymphocytes, thymus-derived (T) lymphocytes, macrophages, and the stimulus for the immune reaction, the antigen (Jerne, 1974). It has become increasingly apparent that T lymphocytes play a major role in the quantitative control of antibody responses and possibly, through clonal selection, in qualitative control. The functions of help, suppression, and cell-mediated cytotoxicity have been shown to be the properties of distinct subclasses of T cells (Cantor and Weissman, 1976; Feldmann et al, 1976). Although these functions are regulated by the immune response (Ir) region of the mouse major histocompatibility complex, the functional subclasses of T cells can be correlated with the differential expression of certain of their surface markers, in particularly Ly (Cantor and Boyse, 1976) and Qa (Stanton et al, 1978). In addition, studies on cytotoxic T-cell reponses have resulted in descriptions of systems involving H-2 restriction (Shearer, 1974; Bevan, 1975; Doherty et al.,1976), suggesting that there is a requirement for the specific presentation of antigen with the H-2 products. The antigen may act as a modifying agent of the major histocompatibility antigens or as a cofactor in dual recognition. These observations implicate the H-2 products of the major histocompatibility region in immunosurveillance and tolerance, in addition to their role as alloantigens.