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J L Strominger

Publications and source records attributed to J L Strominger.

At least 469 records · Page 26Linked to original sources
PubMed1978

A membrane enzyme from Staphylococcus aureus which catalyzes transpeptidase, carboxypeptidase, and penicillinase activities.

Staphylococcus aureus H membranes were found to contain four major binding components: Mr = 115,000; Mr = 100,000 doublet; and Mr = 46,000. The low molecular weight protein bound penicillin reversibly and was purified by prebinding membranes with penicillin prior to affinity chromatography. The purified protein catalyzed transpeptidase and carboxypeptidase reactions using di[14C]acetyl-L-lysyl-D-alanyl-D-alanine as the substrate and glycine and hydroxylamine as the acceptors. In addition, the enzyme catalyzed a penicillinase reaction. Kinetic analysis of these reactions revealed similar Vmax values suggesting that, if there is a single active site, the rate-determining steps (i.e. deacetylation) are similar. Rapid denaturation of the enzyme.substrate complex resulted in the detection of covalent penicilloyl- and diacetyl-L-lysyl-D-alanyl.enzyme complexes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Acyltransferases↗
PubMed1978

Utilization of a depsipeptide substrate for trapping acyl-enzyme intermediates of penicillin-sensitive D-alanine carboxypeptidases.

The penicillin-sensitive D-alanine carboxypeptidases of Bacillus subtilis, Escherichia coli, and Staphylococcus aureus catalyzed the hydrolysis of the D-lactic acid residue from the depsipeptide diacetyl-L-lysyl-D-alanyl-D-lactic acid. The ester substrate was hydrolyzed faster than the peptide analogue, diacetyl-L-lysyl-D-alanyl-D-alanine, by the B. subtilis (15-fold) and E. coli (4-fold) carboxypeptidases, presumably because acylation (k(2)), which is the rate-limiting step of the peptidase reaction, occurred more rapidly during cleavage of the ester bond than during cleavage of the amide bond. No rate acceleration was observed with the S. aureus carboxypeptidase for which deacylation (k(3)) is already the rate-determining step with the peptide substrate. The efficiency of utilization of the depsipeptide (V(max)/K(m)) was greatly enhanced (19- to 147-fold) for all three enzymes. After incubation of the B. subtilis carboxypeptidase and [(14)C]diacetyl-L-lysyl-D-alanyl-D-lactic acid at pH 5.0 and lowering of the pH to 3.0, a radioactive acyl-enzyme intermediate containing 0.43 mol of substrate per mol of enzyme was isolated by Sephadex G-50 chromatography. After acetone precipitation, the acyl group of the denatured acyl-enzyme complex appeared to be bound to the protein by an ester bond. Acyl enzymes were also detected for the S. aureus and E. coli carboxypeptidases after sodium dodecyl sulfate/polyacrylamide gel electrophoresis and fluorography of enzyme incubated with [(14)C]depsipeptide and precipitated with acetone.

Acylation↗
PubMed1978

Translocation of a hydrocarbon fluorescent probe between Epstein-Barr virus and lymphoid cells: an assay for early events in viral infection.

Translocation of the hydrocarbon fluorescent probe diphenylhexatriene (DPH) between membranes was studied by fluorescence polarization (P) analysis. First, using a model system, the high P value (0.324) of DPH-labeled cholesterol/phosphatidylcholine liposomes and the low P value (0.157) of DPH-labeled phosphatidylcholine liposomes allowed detection of DPH translocation between interacting liposomes. This was monitored by the change in P in either direction. Early events during cell-virus interactions were similarly studied by monitoring DPH translocation. The P value of DPH-labeled Epstein-Barr Virus (EBV) was significantly higher (0.350-0.392) than the P value of DPH-labeled lymphoid cells (0.238-0.289). Hence, DPH translocation could be detected by changes in P following incubation of DPH-labeled EBV and nonlabeled cells. A marked decrease in P was observed after incubation of DPH-labeled EBV with either nonlabeled lymphoblastoid Raji cells or fresh human B lymphocytes. However, only a slight decrease in P was obtained when DPH-labeled EBV was incubated with either nonlabeled fresh human T lymphocytes or fresh T or B rabbit lymphocytes. Moreover, incubation of fresh human B lymphocytes with the purified C3 component of complement (a putative inhibitor for the EBV receptor) prior to the addition of DPH-labeled EBV abolished the observed decrease in the P value. Most of these experiments were carried out with both the P3HR-1 and the B95-8 strains of EBV. DPH translocation, as determined by fluorescence polarization analysis, is, therefore, measuring some early event during interaction of this enveloped virus and mammalian cells. The potential applicability of this technique to other viruses is illustrated by an experiment with Semliki Forest virus.

Cells, Cultured↗
PubMed1978

Induction of secondary cytotoxic T lymphocytes by purified HLA-A and HLA-B antigens reconstituted into phospholipid vesicles.

Mouse cytotoxic T lymphocytes were induced by culturing primed spleen cells with cells, membranes, or detergent-solubilized and dialyzed membranes from the human lymphoblastoid cell line JY. Cytotoxic T cells could also be induced by coculturing primed spleen cells with phospholipid vesicles containing purified HLA-A and -B antigens derived from JY cells. The induction of killer cells by all subcellular fractions demonstrated an optimal response as a function of the amount of material added and, in the case of HLA-containing liposomes, this optimum was dependent upon the density of the HLA molecules in the liposomes. In addition, the maximal level of cytotoxicity elicited was also dependent upon this density. Finally, the cytotoxic cells demonstrated specificity as judged by a lower level of lysis on an HLA-unrelated lymphoblastoid cell line than on the appropriate target, JY. These results suggest that major histocompatibility antigens alone are sufficient for the induction of a secondary cytotoxic T lymphocyte response. It is proposed that this system may be useful in defining possible sites on the HLA molecule recognized by cytolytic T lymphocytes.

Animals↗
PubMed1978

Phosphorylation in vivo and in vitro of human histocompatibility antigens (HLA-A and HLA-B) in the carboxy-terminal intracellular domain.

HLA-A and -B antigens are phosphorylated in transformed lymphoblastoid cells and peripheral blood lymphocytes, both incubated with 32Pi. The phosphate group is attached to HLA-A and -B heavy chain (p44) as identified by immunoprecipitation with anti-beta2-microglobulin IgG, sodium dodecyl sulfate/polyacrylamide gel electrophoresis, isoelectric focusing, and susceptibility to limited proteolysis by papain and trypsin. The site(s) of phosphorylation is identified as a serine residue(s) located in the hydrophilic carboxy terminus of the p44 chain. HLA antigens are also phosphorylated in isolated membranes from transformed lymphoblastoid cells that are incubated with [gamma32P]ATP. The phosphorylation of the carboxy terminus of HLA-A and -B antigens in vivo is good evidence that this portion of the molecule is intracellular. Furthermore, this modification suggests a general way in which interactions between membrane proteins and cytoskeletal elements may be regulated.

Amino Acid Sequence↗
PubMed1978

Reconstitution of purified detergent-soluble HLA-A and HLA-B antigens into phospholipid vesicles.

Purified detergent-soluble human histocmpatibility antigens (HLA-A and HLA-B) were reconstituted into phospholipid vesicles by mixing the protein and lipid together in the presence of either octylglucoside (octyl-beta-D-glucopyranoside) or deoxycholate and removing the detergent by dialysis. The resulting preparation consisted of lipid vesicles containing all or most of the added protein. The protein in the vesicles was antigenically active, as demonstrated by specific binding to anti-beta2-microglobulin IgG-Sepharose beads and by specific inhibition of alloantibody and complement-mediated cytotoxicity. Protein incorporated into vesicles at a protein/phospholipid ratio of 1:10 showed an asymmetric distribution of the HLA-A and HLA-B molecules, with virtually all of the antigens oriented facing the external medium. Cleavage experiments with proteases showed that the molecule was attached to the vesicle membrane via the COOH terminus, consistent with its proposed structure in intact cellular plasma membranes. Electron micrographs of the vesicles showed 50-60 A knobs on the outer surface similar to structures observed for other membrane proteins. HLA-A and HLA-B could also be incoporated into vesicles together with Semliki Forest virus membrane proteins. The resulting preparations should be useful in defining the molecular interactions involving HLA-A and HLA-B antigens in the immune response.

Cholesterol↗
PubMed1978

Human (HLA-A and HLA-B) and murine (H-2K and H-2D) histocompatibility antigens are cell surface receptors for Semliki Forest virus.

The proteins coded for by the HLA-A and HLA-B loci in man and the H-2K and H-2D loci in mice were identified as cell surface receptors for Semliki Forest virus. This conclusion is based on the following observations: (i) Water-soluble octamers of viral coat proteins inhibit the complement-dependent cytotoxicity of antibodies directed against H-2K and H-2D antigens in mouse cells. (ii) Isolated detergent-soluble HLA-A and HLA-B antigens reconstituted in lipid vesicles inhibit the binding of viral proteins to human cells (as do the water-soluble antigens to a lesser extent). (iii) Reconstituted HLA-A and HLA-B vesicles interact in solution with Semliki Forest virus (or with vesicles containing viral spike proteins), as demonstrated by coprecipitation with antisera. (iv) Complexes between viral spoke proteins and HLA-A and HLA-B antigens or H-2K and H-2D antigens can be isolated from the cell surface by utilizing affinity chromatography or immunoprecipitation.

Animals↗
PubMed1978

Possible detection of HLA-DR alloantigenic specificities in man with unabsorbed rabbit antisera.

Thirty-six rabbits were immunized with precipitin lines formed between detergent solubilized membrane fractions from HLA-D-homozygous cells and a rabbit antiserum to human B cells (anti-p 23/30). Thirty-one of the rabbits produced B-cell specific cytotoxic antibodies and twelve of the antibodies were also precipitating in gel diffusion. In cytotoxicity tests six of the antisera showed clear preferential reactivity with the immunizing HLA-D (DR) antigen. Thus immunization of rabbits might prove valuable for the production of HLA-DR typing reagents.

Animals↗
PubMed1978

Mapping of the mecillinam-resistant, round morphological mutants of Escherichia coli.

Genes responsible for round morphology in mecillinam-resistant, round morphological mutants of Escherichia coli have been mapped. Three mutants, called rodX, mapped at around 14 min, and two, called rodY, mapped at around 70 min by P1 transduction. These are either the same or very close to the loci reported, respectively, for the rodA (H. Matsuzawa, K. Hayakawa, T. Sato, and K. Imahori, J. Bacteriol. 115:436-442, 1973) and envB genes (B. Westling-Häggström and S. Normark, J. Bacteriol. 123:75-82, 1975). This suggests that mecillinam can be used very efficiently to select for found morphological mutants of rodA and envB after nitrosoguanidine treatment.

Amdinocillin↗
PubMed1978

Morphology of an Escherichia coli mutant with a temperature-dependent round cell shape.

Mutants of Escherichia coli capable of growing in the presence of 10 microgram of mecillinam per ml were selected after intensive mutagenesis. Of these mutants, 1.4% formed normal, rod-shaped cells at 30 degrees C but grew as spherical cells at 42 degrees C. The phenotype of one of these rod(Ts) mutants was 88% cotransducible with lip (14.3 min), and all lip+ rod(Ts) transductants of a lip recipient had the following characteristics: (i) growth was relatively sensitive to mecillinam at 30 degrees C but relatively resistant to mecillinam at 42 degrees C; (ii) penicillin-binding protein 2 was present in membranes of cells grown at 30 degrees C in reduced amounts and was undetectable in the membranes of cells grown at 42 degrees C. The mecillinam resistance, penicillin-binding protein 2 defect, and rod phenotypes all cotransduced with lip with high frequency. Thus the mutation [rodA(Ts)] is most likely in the gene for penicillin-binding protein 2 and causes the organism to grow as a sphere at 42 degrees C, although it grows with normal rodlike morphology at 30 degrees C. At 42 degrees C, cells of this strain were round with many wrinkles on their surfaces, as revealed by scanning electron microscopy. In these round cells, chromosomes were dispersed or distributed peripherally, in contrast to normal rod-shaped cells which had centrally located, more condensed chromosomes. The round cells divided asymmetrically on solid agar, and it seemed that the plane of each successive division was perpendicular to the preceding one. On temperature shift-down in liquid medium many cells with abnormal morphology appeared before normal rod-shaped cells developed. Few abnormal cells were seen when cells were placed on solid medium during temperature shift-down. These pleiotropic effects are presumably caused by one or more mutations in the rodA gene.

Amdinocillin↗
PubMed1978

Suppression of in vitro Epstein-Barr virus infection: a new role for adult human T-lymphocytes.

In vitro EBV infection of lymphocytes from different sources indicated that B-cells of adult peripheral or fetal cord blood transform with equal efficiency whether assayed by DNA synthesis induction or immortalization. Unfractionated adult lymphocytes transformed much less efficiently than those from fetal cord blood. This effect was due to the suppression of B-cell proliferation by adult T-lymphocytes.

Adult↗
PubMed1977

Hydroxylaminolysis of penicillin binding componenets is enzymatically catalyzed.

The hydroxylaminolysis of the penicilloyl moiety from [14C]penicillin G binding component (PBC) complexes of the Bacillus subtilis D-alanine carboxypeptidase and of the mixture of PBC's of Staphylococcus aureus was inhibited by denaturation of the complexes by heat (55 degrees), detergent (1% sodium dodecyl sulfate), or trichloroacetic acid. The kinetics of inhibition by denaturation were comparable to those of the inhibition of [14C]penicillin G binding to the PBC's and of carboxypeptidase activity of the B. subtilis enzyme under identical denaturing conditions. These data establish that the hydroxylaminolysis is an enzymatically catalyzed process suggesting that penicillin G is bound to an enzymatically active site. Treatment of the denatured [14C]penicillin G-carboxypeptidase complex with sodium borohydride or at pH 12 resulted in the release of the penicilloyl moiety. These results are consistent with a carboxylic ester bond for the penicilloyl-PBC instead of a thiolester linkage as was initially presumed.

Alanine↗
PubMed1977

Purification of HLA-linked B lymphocyte alloantigens in immunologically active form by preparative sodium dodecyl sulfate-gel electrophoresis and studies on their subunit association.

The HLA-linked B cell alloantigen (p29,34) is composed of two subunits of 29,000 (p29) and 34,000 (p34) molecular weight. The partially purified HLA-linked B cell alloantigen was purified by a final step of preparative sodium dodecyl sulfate-gel electrophoresis. An antiserum was prepared against p29,34 which specifically lysed B lymphocytes. In sodium dodecyl sulfate at 21 degrees, p29 and p34 remained noncovalently associated and retained immunologic activity; subunit dissociation at higher temperatures correlated with loss of immunologic activity. Although the pI values of p29 and p34 are 6.1 and 5.2, respectively, the subunits co-electrofocus under nondenaturing conditions. In addition, cross-linking studies showed the B cell antigen has a (p29)1(p34)1 subunit structure.

Antigen-Antibody Reactions↗
PubMed1977

Suppression of in vitro Epstein-Barr virus infection. A new role for adult human T lymphocytes.

Studies have been performed on in vitro infection by Epstein-Barr virus (EBV) of subpopulations of human lymphocytes. B cells of adult peripheral or fetal cord blood transform with equal efficiency, whether assayed by DNA synthesis induction or by outgrowth of transformed lymphocytes. In contrast, unfractionated adult lymphocytes transform much less efficiently than those from fetal cord. Reconstitution experiments of different cell preparations indicated that this difference was due to a suppression of B-cell proliferation by adult Ig-negative lymphocytes which fetal Ig-negative lymphocytes were unable to perform. Separation of Ig-negative lymphocytes into various subpopulations revealed that the suppression was performed by T cells. Macrophages and null cells play little or no role in suppression. The relevance of this phenomenon to infection and recovery from EBV infection during and after infectious mononucleosis is discussed.

Adult↗
PubMed1977

Purification and structural characterisation of human HLA-linked B-cell antigens.

The human B cell-specific alloantigen which is closely linked genetically to HLA contains two non-covalently associated, sialogycoprotein subunits of molecular weight (MW) 29,000 (p29), and 34,000 (p34). Although p29 and p34 have different amino-terminal sequences, their tyrosine peptide maps indicate considerable similarity in other portions of their polypeptide chains. Thus the genes for their proteins may have evolved by duplication of a common ancestral gene. Another lymphocyte cell surface protein of MW 16,000 (p16) has also been characterised. Both p16 and p44 (the heavy chain of HLA-A,B antigens) have been compared with p29 and p34.

Amino Acid Sequence↗
PubMed1977

Detergent solubilization, purification, and separation of specificities of HLA antigens from a cultured human lymphoblastoid line, RPMI 4265.

HLA antigens have been purified to homogeneity after detergent solubilization from RPMI 4265, a human lymphoblastoid line. The inhibition of cytotoxicity assay for HLA antigen was modified, using preincubation with bovine serum albumin of antigen samples containing detergent to prevent lysis of target cells by detergent. Solubilization was tested with many types of detergents. A polyethyleneglycol oleyl ether nonionic detergent mixture, Brij 99:Brij 97 (2:1) was selected for solubilization, since it selectively solubilized HLA antigens, had a low absorbance at 280 nm and was uncharded. HLA antigens were then purified by Lens culinaris lectin affinity chromatography and Bio-Gel A-5m filtration. The antigen specifity HLA-A2 was separated from specificities HLA-B7,12 by isoelectric focusing. Purified HLA antigens contained a subunit of Mr=44,000 with NH2-terminal glycine, and a subunit of Mr=12,000, beta2-microglobulin, with NH2-terminal isoleucine.

Amino Acids↗