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J L Strominger

Publications and source records attributed to J L Strominger.

At least 433 records · Page 24Linked to original sources
PubMed1980

Sequence of active site peptides from the penicillin-sensitive D-alanine carboxypeptidase of Bacillus subtilis. Mechanism of penicillin action and sequence homology to beta-lactamases.

It has been proposed that penicillin and other beta-lactam antibiotics are substrate analogs which inactivate certain essential enzymes of bacterial cell wall biosynthesis by acylating a catalytic site amino acid residue (Tipper, D.J., and Strominger, J.L. (1965) Proc. Natl. Acad. Sci. U.S.A. 54, 1133-1141). A key prediction of this hypothesis, that the penicilloyl moiety and an acyl moiety derived from substrate both bind to the same active site residue, has been examined. D-Alanine carboxypeptidase, a penicillin-sensitive membrane enzyme, was purified from Bacillus subtilis and labeled covalently at the antibiotic binding site with [14C]penicillin G or with the cephalosporin [14C]cefoxitin. Alternatively, an acyl moiety derived from the depsipeptide substrate [14C]diacetyl L-Lys-D-Ala-D-lactate was trapped at the catalytic site in near-stoichiometric amounts by rapid denaturation of an acyl-enzyme intermediate. Radiolabeled peptides were purified from a pepsin digest of each of the 14C-labeled D-alanine carboxypeptidases and their amino acid sequences determined. Antibiotic- and substrate-labeled peptic peptides had the same sequence: Tyr-Ser-Lys-Asn-Ala-Asp-Lys-Arg-Leu-Pro-Ile-Ala-Ser-Met. Acyl moieties derived from antibiotic and from substrate were shown to be bound covalently in ester linkage to the identical amino acid residue, a serine at the penultimate position of the peptic peptide. These studies establish that beta-lactam antibiotics are indeed active site-directed acylating agents. Additional amino acid sequence data were obtained by isolating and sequencing [14C]penicilloyl peptides after digestion of [14C]penicilloyl D-alanine carboxypeptidase with either trypsin or cyanogen bromide and by NH2-terminal sequencing of the uncleaved protein. The sequence of the NH2-terminal 64 amino acids was thus determined and the active site serine then identified as residue 36. A computer search for homologous proteins indicated significant sequence homology between the active site of D-alanine carboxypeptidase and the NH2-terminal portion of beta-lactamases. Maximum homology was obtained when the active site serine of D-alanine carboxypeptidase was aligned correctly with a serine likely to be involved in beta-lactamase catalysis. These findings provide strong evidence that penicillin-sensitive D-alanine carboxypeptidases and penicillin-inactivating beta-lactamases are related evolutionarily.

Amino Acid Sequence↗
PubMed1980

The mechanism of action of penicillin. Penicillin acylates the active site of Bacillus stearothermophilus D-alanine carboxypeptidase.

Penicillin kills susceptible bacteria by specifically inhibiting the transpeptidase that catalyzes the final step in cell wall biosynthesis, the cross-linking of peptidoglycan. It was hypothesized (Tipper, D., and Strominger, J. (1965) Proc. Natl. Acad. Sci. U.S.A. 54, 1133-1141) that 1) penicillin is a structural analog of the acyl-D-alanyl-D-alanine terminus of the pentapeptide side chains of nascent peptidoglycan, and that 2) penicillin, by virtue of its highly reactive beta-lactam structure, irreversibly acylates the active site of the cell wall transpeptidase. Although the cell wall transpeptidase has proven elusive, a closely related penicillin-sensitive cell wall enzyme, D-alanine carboxypeptidase, has been purified from membranes of Bacillus stearothermophilus by penicillin affinity chromatography. By amino acid sequence analysis of 14C-labeled cyanogen bromide peptides generated and purified from this carboxypeptidase covalently labeled with either [14C]penicillin G or the substrate, [14C]diacetyl-L-lysyl-D-alanyl-D-lactate, it was shown that the penicillin and substrate were both bound as esters to a serine at residue 36. Therefore, the second hypothesis stated above was proven to be correct for D-alanine carboxypeptidase. Several new methods were developed in the course of this work, including 1) a rapid penicillin-binding assay, 2) use of hydroxylamine to protect peptides against carbamylation during ion exchange chromatography in concentrated urea solutions, and 3) gel filtration chromatography in 70% formic acid, a universal solvent for peptides.

Amino Acid Sequence↗
PubMed1980

Enzymatic activities in cultured human lymphocytes that dephosphorylate dolichyl pyrophosphate and dolichyl phosphate.

Enzymatic activities which dephosphorylate dolichyl phosphate (Dol-P) and dolichyl pyrophosphate (Dol-P-P) have been observed in membranes from cultured human lymphocytes. Neither activity requires divalent metals. Dol-P phosphatase is inhibited by inorganic phosphate but not by other phosphate-containing compounds. Dol-P-P phosphatase is inhibited by bacitracin but not by phosphate-containing compounds including the methylene analogue of pyrophosphate. These reactions are similar to those previously found in the cycle of bacterial wall peptidoglycan biosynthesis. A chemical synthesis of [32P]Dol-P and [32P]Dol-P-P is reported.

Bacitracin↗
PubMed1980

Demonstration of structural polymorphism among HLA-DR light chains by two-dimensional gel electrophoresis.

Human HLA-DR antigens were immunoprecipitated from Nonidet P-40 extracts of [35S]methionine-labeled B lymphoblastoid cell lines and compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing (IEF). Two-dimensional (2-D) gel analyses, combining SDS-PAGE in the first dimension and IEF in the second dimension, revealed that the heavy (alpha) and light (beta) chains of each DRw specificity displays microheterogeneity of charge. However, the pattern of the heavy chain did not vary among different DRw specificities. In contrast, the light chains of different DRw types varied both in apparent size and charge distribution. Removal of sialic acids with neuraminidase or inhibition of glycosylation with tunicamycin reduced the microheterogeneity of both DR subunits. However, the heavy and light chains each still focused as two major bands, suggesting that other post-translational modifications contribute to the microheterogeneity or that there are two nonallelic DR-like molecules. After treatment with either neuraminidase or tunicamycin, the DR light chains, but not the heavy chains, were still structurally polymorphic. The DR light chains of serologically cross-reactive specificities displayed similar 2-D gel patterns suggesting that the structural polymorphism of the DR light chains is the basis for the serologically detected polymorphism of the HLA-DR antigens. Two additional polypeptides were observed in immunoprecipitates of DR antigens. These proteins, designated M1 and M2, both had a basic isoelectric point and were invariant among different cell lines. The protein M1 may be intracellular because it can not be immunoprecipitated from the cell surface.

Amino Acid Sequence↗
PubMed1980

Virus-immune cytotoxic T cells recognize structural differences between serologically indistinguishable HLA-A2 molecules.

The self-specificity of human influenza virus-immune cytotoxic T cells has been analyzed in order to identify the relationship between the self-determinants which they recognize and the serologically defined HLA-A and -B antigenic determinants. Virus-immune T cells were generated in vitro by culture of normal adult peripheral blood lymphocytes with A/HK influenza virus. Virus-immune effectors from HLA-A2 positive donors were tested on panels of virus-infected target cells from donors who were either HLA-mismatched or matched only for the HLA-A2 specificity. Virus-immune T cells from 11/11 A2-positive donors lysed all A2-matched virus-infected target cells (and no HLA-mismatched targets), except that each of these effector cell populations consistently failed to lyse the virus-infected target cells from one A2-positive donor (designated M7). Although the A2 antigen of donor M7 could also be distinguished from the A2 antigen of other donors by alloimmune cytotoxic T cells, no differences in the A2 antigen of donor M7 could be defined by extensive serological analyses. Results of isoelectric focusing of A2 molecules from three individuals plus M7 demonstrated that the M7 A2 heavy-polypeptide chain is structurally distinct. These results indicate that: 1) there is a strong but incomplete association between a self antigen recognized by virus-immune T cells and the serologically defined HLA-A2 specificity; and 2) there may be at least two structurally and functionally distinct epitopes on the same A2 molecule: one is the serologically defined HLA-A2 antigenic determinant; the other is the self determinant recognized by T cells on HLA-A2 molecules.

Cell Line↗
PubMed1980

Molecular cloning of a human histocompatibility antigen cDNA fragment.

A clone (pHLA-1) containing HLA-specific cDNA was constructed by reverse transcription of partially purified HLA mRNA from the human lymphoblastoid cell line LKT. The identity of pHLA-1 was established by its ability to hybridize to HLA heavy chain mRNA and by nucleotide sequence analysis. The pHLA-1 cDNA insert (approximately 525 base pairs) corresponds to the COOH-terminal 46 amino acids of an HLA-A, -B, or -C antigen (15 residues from the hydrophobic region and the remainder from the COOH-termial hydrophilic region), together with a portion of the 3' untranslated region of the mRNA.

Base Sequence↗
PubMed1980

High molecular weight antigens present on human T cells.

A series of eight high molecular weight (140,000-220,000) glycoproteins on human peripheral T cells were recognized by radioimmunoprecipitation with a rabbit antiserum. The pattern of antigens present on each of eight human T cell lines studied was unique, and no line displayed the range of antigens present on peripheral T cells. The pattern of bands on peripheral T cells changed after allogeneic or lectin stimulation. Adsorption/elution experiments with antiserum showed that some of these proteins were antigenically related, and at least three different groups of proteins were present. Two of these groups could be partially distinguished by their ability to bind to ricin or lentil lectin and by their reactivity with two additional rabbit antisera. On some cell lines, it was found that proteins bound by lentil lectin but not ricin were precursors of higher molecular weight material recognized by ricin. Taken together, the data suggest that these proteins may be the products of a multigenic or multiallelic system, probably equivalent to the murine Ly 5 antigens.

Antigens, Surface↗
PubMed1980

Characterization of a monoclonal antibody that defines an immunoregulatory T cell subset for immunoglobulin synthesis in humans.

This study characterizes a monoclonal antibody (3A1), and partially characterizes the cell surface antigen and the functional peripheral blood T cell subset that it defines. The 3A1 antigen is present on the surface of several human T cell lines (HSB-2, CEM, MOLT-4, and others) in various amounts but is absent from the T cell line YT4E and all human B cell lines tested. Immunoprecipitation of an HSB-2 extract with 3A1 yielded one specific band with a molecular weight of approximately 40,000 in the presence of reducing agent. With directly fluoresceinated 3A1 antibody, fluorescence-activated cell sorter analysis showed that 85% of peripheral blood E-rosette-positive T cells were positive for the 3A1 antigen. After E-rosette-positive cells had been separated into 3A1+ and 3A1- cell suspensions, the 3A1+ cells helped autologous peripheral blood B cell suspensions toward pokeweed mitogen-driven proliferation and intracytoplasmic Ig production, whereas 3A1-T cells did not. Further, addition of 3A1- cells from some but not all normal subjects to cocultures of 3A1+ cells and B cells actively suppressed intracytoplasmic Ig production. However, the 3A1+T cell subset could be activated by concanavalin A to maximally suppress B cell Ig synthesis in vitro. Thus, the 3A1 antibody defines a major functional subject of peripheral blood T cells and should provide a useful marker for the study of human T cell function.

Antibody Formation↗
PubMed1980

Cloning and mapping of BamHi endonuclease fragments of DNA from the transforming B95-8 strain of Epstein-Barr virus.

DNA from the B95-8 strain of Epstein-Barr virus was cleaved into 29 different fragments by BamHI endonuclease (EC 3.1.23.6). All of the fragments except the terminal fragments have been inserted into the pBR322 cloning vector and replicated in Escherichia coli. The location of each cloned DNA fragment in the viral genome has been determined, providing a more detailed physical map of the genome than has been available previously.

Cell Transformation, Viral↗
PubMed1980

Development and characterization of allospecific long-term human cytolytic T-cell lines.

Two long-term human cytolytic T lymphocyte (CTL) lines (VE and JR), whose cytolytic activity was dependent upon both irradiated JY cells (the stimulating alloantigen) and T-cell growth factor, were established. These lines were monitored in culture for 6-8 months. Both lines were specific for HLA-A or B antigens or both and the JR line was allospecific for HLA-B7. These CTL lines killed specific target cells at an effector-to-target ratio of 0.4 (VE) or 0.08 (JR). All of the cells, which grow in suspension, rosetted with sheep erythrocytes and reacted with an antiserum specific for human T cells. The CTL line VE was used to raise rabbit antisera that immunoprecipitated two specific polypeptides (78,000 and 33,000 daltons) from labeled membranes of these CTL lines.

Animals↗
PubMed1980

An evaluation of the significance of amino acid sequence homologies in human histocompatibility antigens (HLA-A and HLA-B) with immunoglobulins and other proteins, using relatively short sequences.

A computer search was carried out for homologies between HLA-A and HLA-B antigen sequences and the sequences of constant and variable regions of immunoglobulins and of all other sequenced proteins. Searches were made both with relatively short peptide sequences from the HLA antigens and with those longer peptide sequences which were available in 1978. Significant homology of HLA antigen sequences to immunoglobulin constant region sequences was found in two cases: (1) a short decapeptide sequence which includes the fourth cysteine residue of HLA-B7 and (2) an 89-amino-acid residue (Ac-2) C-terminal fragment of the papainsolubilized HLA-B7 molecule. The difficulty of establishing statistically significant sequence homology with relatively short peptide sequences is emphasized by computer-based comparisons of the decapeptide sequence with randomly generated peptide sequences. It is concluded that statistically significant homology with short sequences can be assured only when extraordinarily high degrees of homology are present and additional constraints are included in the matches, for example, matches at relatively rare amino acid residues such as Cys, His and Trp. The homology of the 89-amino-acid residue sequence to constant region sequences of immunoglobulins is as great as or greater than that of beta 2-microglobulin. These findings and the unique domain structure involving a disulphide loop of comparable size strongly favour a common evolutionary origin for this region of HLA-A and -B, beta 2-microglobulin and immunoglobulin constant regions.

Amino Acid Sequence↗
PubMed1980

Human T lymphocyte surface antigens: partial purification and characterization utilizing a high-titer heteroantiserum.

A high-titer human T lymphocyte-specific antiserum (A99) has been prepared by immunization of rabbits with a preparation partially purified from membranes of the T cell line, CEM. The specificities recognized by this antiserum included those recognized by TH1 and HTL antisera. Several experiments indicated that these antisera recognized several different proteins. A partial purification of material recognized by A99 serum was obtained by 1) isolation of plasma membranes, 2) detergent solubilization and chromatography on DEAE-cellulose, and 3) affinity chromatography on a wheat germ lectin-Sepharose 4B column. These three steps yielded material about 100-fold purified, and an estimate was made in several ways that on overall purification of 3500-fold would be required to achieve homogeneity. Immunoprecipitation of 125I-labeled 100-fold purified material showed that the major bands recognized by A99 serum had m.w. of 52,000, 96,000 (seen only after reduction), 120,000, 152,000, and 195,000. These data are compared to other published reports of human T lymphocyte-specific proteins.

Animals↗
PubMed1979

Complete amino acid sequence of a papain-solubilized human histocompatibility antigen HLA-B7. 1. Isolation and amino acid composition of fragments and of tryptic and chymotryptic peptides.

As a part of the overall strategy for determining the complete covalent structure of the papain-solubilized portion of the heavy chain of the human histocompatibility antigen HLA-B7, the protein was dissected into various fragments by a combination of partial acid hydrolysis and cyanogen bromide cleavage. After purification by chromatographic procedures, these fragments have been used as a source for tryptic and chymotryptic peptides. Thirty-three major tryptic and twenty-two major chymotryptic peptides were purified in nanomole amounts and their amino acid compositions determined. These peptides account for the whole extent of the polypeptide chain with the exception of the amino-terminal CNBr pentapeptide. They provide the basis for the formal alignment of the acid cleavage and cyanogen bromide fragments of the molecule as well as the source material for the elucidation of the primary structure of the HLA-B7 heavy chain.

Amino Acid Sequence↗
PubMed1979

Complete amino acid sequence of a papain-solubilized human histocompatibility antigen, HLA-B7. 2. Sequence determination and search for homologies.

The complete amino acid sequence of the papain-solubilized heavy chain of a human histocompatibility antigen (HLA-B7) has been elucidated. It consists of a polypeptide of 271 residues (31 333 daltons). A single glycan moiety is attached to an asparagine residue at position 86 by an N-glycosidic bond. Two intrachain disulfide bonds, arranged linearly, involve half-cystine residues at positions 101 and 164 and at positions 203 and 259. They form two loops of 62 and 55 residues, respectively, separated by 38 residues. Computer analysis of the sequence suggests the existence of internal homology between the amino-terminal portion (residues 1--90) and the region of the first disulfide loop (residues 91--180). There is a significant homology between the second disulfide loop region of the chain (residues 182-271) and immunoglobulin (Ig) constant domains and beta 2-microglobulin [Orr, H. T., Lancet, D., Robb, R. J., López de Castro, J. A., & Strominger, J. L. (1979A) Nature (London) (in press)]. However, no such homology to Ig is apparent in the amino-terminal or in the first disulfide loop regions.

Amino Acid Sequence↗
PubMed1979

Cephalosporin-sensitive penicillin-binding proteins of Staphylococcus aureus and Bacillus subtilis active in the conversion of [14C]penicillin G to [14C]phenylacetylglycine.

Breakdown of the covalent complex formed between [14C]penicillin G and higher molecular weight, cephalosporin-sensitive penicillin-binding proteins was studied using mixtures of the purified proteins isolated from membranes of Staphylococcus aureus and Bacillus subtilis. These penicillin-binding proteins were found to release the bound 14C label in a first order process characterized by half-lives of 10 to 300 min at 37 degrees C. Denaturation of the penicilloyl.penicillin-binding proctein complex prevented this release, indicating that the process is enzyme-catalyzed. [14C]Phenylacetylglycine was identified as the major labeled fragmentation product, indicating that these cephalosporin-sensitive penicillin-binding proteins, for which no in vitro transpeptidase or carboxypeptidase activity has been found, catalyze the same fragmentation of the bound penicilloyl moiety previously described for several penicillin-sensitive D-alanine carboxypeptidases.

Bacillus cereus↗