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E Appella

Publications and source records attributed to E Appella.

At least 37 records · Page 2Linked to original sources
PubMed1978

Hepatic microsomal epoxide hydrase. Involvement of a histidine at the active site suggests a nucleophilic mechanism.

The effects of a wide variety of chemical modification reagents on the activity of purified rat liver microsomal epoxide hydrase have been investigated. Alkylating agents, such as the phenacyl bromides and benzyl bromide are potent inhibitors of epoxide hydrase. 2-Bromo-4'-nitroacetophenone (p-nitrophenacyl bromide) specifically and irreversibly inactivates epoxide hydrase. Pseudo-first order kinetics of inhibition is observed at higher inhibitor/enzyme ratios. The rate of inactivation is controlled by a group on the enzyme with an apparent pKa of 7.6. Inactivation of the enzyme with 14C-labeled 2-bromo-4'-nitroacetophenone leads to the incorporation of approximately 1 mol of radioactive inhibitor/mol of protein. Epoxide hydrase can be protected against this inactivation by the substrate phenanthrene-9,10-oxide. These results are consistent with the interpretation that 2-bromo-4'-nitroacetophenone acts as an active site-directed inhibitor. The site of alkylation by 2-bromo-4'-nitroacetophenone is a histidine residue of epoxide hydrase. The N-alkylated histidine derivative has been identified as 1-(p-nitrophenacyl)-4-histidine. A possible mechanism for the enzymatic hydration catalyzed by epoxide hydrase is discussed which involves a histidine residue of the enzyme serving as a general base catalyst for the nucleophilic addition of water.

Amino Acids↗
PubMed1978

Structural characterization of H-2 antigens purified from mouse liver.

Papain solubilized H-2a histocompatibility antigens (H-2Kk plus H-2Dd) have been purified by a large-scale procedure that can routinely provide 2-3 mg of heavy chain from 1 kg of mouse liver. The heavy chains were homogeneous by sodium dodecyl sulfate electrophoresis. Disc gel electrophoresis resolved two protein bands that were identified as H-2Dd and H-2Kd by immune complex formation and autoradiography. Comparative amino acid composition and NH2-terminal sequence analyses of unfractionated H-2a, H-2Kk, and HLA suggested close structural relationships. However, the following observations suggest that papain cleaves these membrane bound antigens at different positions with respect to the COOH terminus: the molecular weight of the peptide portion of papain solubilized HLA is smaller than that of H-2 (30,000 versus 33,700); the COOH-terminal sequences are different; and, finally, papain-solubilized H-2 contains a free cysteine residue in addition to the two disulfide bridges that are present in both H-2 and HLA.

Amino Acid Sequence↗
PubMed1978

Immunochemical evidence of a tumor-specific surface antigen obtained by detergent solubilization of the membranes of a chemically induced sarcoma, meth-A.

We reported previously the partial purification of detergent-solubilized specific tumor rejection antigen of a chemically induced sarcoma, Meth-A. During the course of the study, rabbit antiserum against a partially purified specific tumor rejection antigen preparation was raised and rendered specific by in vivo absorption. In this report we show that an antigenic molecule defined by in vivo-absorbed rabbit antiserum, which we tentatively refer to as tumor-specific surface antigen, was solublized by detergent Nonidet P40, and extensive attempts at purification were carried out by a sequence of procedures including gel filtration, isotachophoresis, and polyacrylamide gel electrophoresis. The most highly purified tumor-specific surface antigen retained some specific tumor rejection antigen activity, suggesting an association of the two activities. Both antigens were shown to have a molecular weight of about 60,000 and an electrophoretic mobility of alpha-globulin.

Animals↗
PubMed1977

Solubilized TSTA and the major viral structural proteins, gp70 and p30, in the immune response to murine leukemias induced by Friend and Rauscher virus.

Antigens present in gp70 and p30 purified from Rauscher virus, were tested for immunogenicity in various assays measuring the anti-tumor immune response against lymphocytic leukemias of Friend (FBL-3) or Rauscher (RBL-5) virus origin. p30 had no effect on in vitro cytotoxicity against tumor cell targets mediated by either an anti RBL-5 serum or lymphocytes from animals immunized with FBL-3 cells. gp70 had had no effect on serum-mediated cytotoxicity but used at high concentrations it inhibited cell-mediated cytotoxicity. When used to immunize mice directly against subsequent challenge with the RBL-5 and FBL-3 leukemias, p30 had no discernible effect, while gp70 afforded partial protection against RBL-5 but only at high concentrations. Cell-free preparations of tumor membranes containing negligible amounts of gp70 were antigenically superior to gp70 in both the in vitro and in vivo assays. It is concluded that antigens on these purified proteins that are also expressed on tumor cells are not major targets of the anti-tumor immune response in this system.

Animals↗
PubMed1977

Amino acid sequence of the first 217 residues of a mouse heavy chain (MOPC 47A) with a domain deletion.

Cyanogen bromide cleavage of the deleted heavy (alpha) chain of mouse IgA 47A yielded five peptides, CNBr 1-5, consisting of 34, 50, 28, 70, and 160 residues. A 217-residue NH2-terminal sequence, which comprises the variable region and the first domain of the constant region (CH1), and a 19-residue COOH-terminal sequence of the chain were obtained from the sequences of CNBr 1-4 and the NH2-terminal and COOH-terminal sequences of CNBr 5. The tryptic peptides of the remainder of the chain have been partially characterized. Comparison of these data with the sequences of other, nondeleted, chains reveals that the 47A chain terminates exactly at the end of the CH2 domain and that there are no deletions up to that point. It is concluded that the deletion in the chain is a single, large deletion consisting of the entire CH3 domain. The 47A chain also differs from other, nondeleted, mouse alpha chains derived from BALB/c strains in that it contains a labile cysteine at position 135, which is believed to participate in the light-heavy chain bond, and contains galactosamine in the hinge region. This may mean that the 47A alpha chain, in addition to being deleted, represents a mouse IgA subclass analogous to human IgA1.

Amino Acid Sequence↗
PubMed1977

Quantitative in vivo studies of soluble simian virus 40 tumor-specific transplantation antigens of the mouse.

Quantitative studies have been performed on the immunogenicity of a membrane-bound antigen of a simian virus 40 (SV40) -induced sarcoma in syngeneic BALB/c mice and of subcellular fractions derived from this tumor. The objectives of the investigation were: a) to develop a quantitative in vivo assay of the tumor-specific transplantation antigen (TSTA) and b) to compare the distribution of histocompatibility antigens, H-2, with that of the SV40 TSTA during several fractionation steps. The immunogenicity of the TSTA-containing fractions was assessed from dose-response curves relating tumor size and the amount of protein used for immunization. After digestion of the tumor cell membranes with a limited amount of papain, H-2 as well as TSTA were present in a soluble form. A single immunization with only 2 microng of the solubilized TSTA reduced the tumor size by 70% compared to that in nonimmunized control animals. The results of several fractionation steps suggest that H-2 and the TSTA are not tightly associated in the solubilized immunogenic material.

Animals↗
PubMed1977

Subcellular distribution of the tumor-specific transplantation antigen of simian virus 40-transformed cells.

TU-5, a simian virus 40 (SV40)-transformed cell line of BALB/c origin, expressed the SV40-specific T-antigen and a transplantation antigen (TSTA). Nuclei and plasma membranes were prepared from these cells and shown on the basis of the distribution of T-antigen and histocompatibility (H-2) antigens to be relatively free of cross contamination. Most of the TSTA, estimated by tumor rejection, was associated with the nuclear fraction.

Animals↗
PubMed1977

Biologic and biochemical properties of detergent-solubilized tumor-specific transplantation antigen from a simian virus 40-induced neoplasm: brief communication.

The detergent Nonidet P40 was used to solubilize tumor-specific transplantation antigens (TSTA) from crude membranes obtained from dissociated cells of simian virus 40-induced sarcoma of BALB/c mice. A good recovery of specific tumor rejection activity was observed. One fraction, fraction V, was obtained following polyacrylamide-agarose filtration of the solubilized material, and this fraction contained most of the activity. An increased specific activity followed gel filtration. Preliminary data from lectin column chromatography of the active fraction V indicated a separation of TSTA activity from H-2 activity.

Animals↗
PubMed1977

Biological and biochemical properties of Nonidet P40-solubilized and partially purified tumor-specific antigens of the transplantation type from plasma membranes of a methylcholanthrene-induced sarcoma.

Tumor-specific transplantation antigen (TSTA) was solubilized from cell membranes of sarcoma Meth-A with non-ionic detergent Nonidet P40. Soluble TSTA was partially characterized by chromatographic separation and electrophoresis. The antigen responsible for tumor rejection activity had a molecular weight of approximately 70,000 daltons in the presence of detergent and an electrophoretic mobility of alpha-globulin. TSTA was well separated from mouse histocompatibility antigen H-2 by a sequence of procedures, including gel filtration, lectin affinity chromatography, column electrophoresis, and rechromatography on agarose, showed only three major bands on polyacrylamide gel electrophoresis. TSTA was specific for sarcoma Meth-A.

Animals↗