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J L Strominger

Publications and source records attributed to J L Strominger.

At least 451 records · Page 25Linked to original sources
PubMed1979

The heavy chain of human histocompatibility antigen HLA-B7 contains an immunoglobulin-like region.

The 88-residue fragment (ac-2) containing the second disulphide loop from HLA-B7 heavy chain is shown to have statistically significant homology with Ig constant domains, including both matches at invariant positions and conservative substitutions of structurally important residues. Thus, both the light chain (beta 2m) and a segment of the heavy chain of HLA antigens may be structurally and evolutionarily related to immunoglobulins.

Amino Acid Sequence↗
PubMed1979

Studies of the high molecular weight penicillin-binding proteins of Bacillus subtilis.

Seven or eight penicillin-binding proteins (PBPs) were detected in Bacillus subtilis membranes. By introducing covalent affinity chromatography employing cephalosporins as ligands, milligram amounts of three high molecular weight PBPs (PBP 1 ab, Mr = 120,000; PBP 2b, Mr = 94,000; and PBP 4, Mr = 78,000) were obtained without any contamination of the major PBP 5, the D-alanine carboxypeptidase. Small amounts of pure PBP 2b could be isolated by manipulation of the affinity chromatography conditions. Structural and physical properties of these proteins as well as the generation of one major penicilloyl peptide from each PBP by digestion with pepsin suggest that each PBP is the product of a separate gene. No enzymatic activity could be found in mixtures of these high molecular weight PBPs employing substrates used for the transpeptidase and D-alanine carboxypeptidase assays in particulate membrane fractions.

Bacillus subtilis↗
PubMed1979

Cleavage of a COOH-terminal hydrophobic region from D-alanine carboxypeptidase, a penicillin-sensitive bacterial membrane enzyme. Characterization of active, water-soluble fragments.

D-Alanine carboxypeptidase (CPase), a detergent-soluble penicillin-sensitive membrane enzyme of Bacillus stearothermophilus, Mr = 46,500, was digested with either trypsin or alpha-chymotrypsin to yield water-soluble fragments, designated T-CPase and Chy-CPase, respectively, each of Mr = approximately 45,000. These fragments were generated and purified in milligram quantities by digestion of CPase covalently immobilized on a penicillin affinity column. They retained full enzymatic activity, became significantly more resistant to thermal inactivation, and lost micellar detergent binding upon proteolysis. Each was derived from CPase by loss of a COOH-terminal hydrophobic peptide. CPase was reconstituted into bacterial lipid vesicles in an enzymatically active form. Penicillin-binding sites were equally distributed on both sides of the lipid bilayer, suggesting a random orientation of the CPase molecules. Neither T-CPase nor Chy-CPase reconstituted into lipid vesicles when treated in an identical manner. CPase was slowly cleaved from the surface of these vesicles by either trypsin or alpha-chymotrypsin, yielding T-CPase and Chy-CPase, respectively. These results demonstrate that CPase is comprised of a water-soluble catalytic domain and a COOH-terminal hydrophobic region which mediates the anchoring of this enzyme to the bacterial membrane.

Carboxypeptidases↗
PubMed1979

Assembly and maturation of HLA-A and HLA-B antigens in vivo.

HLA-A and HLA-B antigens are integral membrane glycoproteins which consist of a glycosylated heavy chain embedded in the membrane in noncovalent association with beta 2-microglobulin, a water-soluble polypeptide. The assembly and maturation of these antigens has been studied in vivo in the human B lymphoblastoid cell line T5-1 (HLA-A1, -A2, -B8, -B27). Two antigenically distinct populations of HLA-A and -B heavy chains can be detected by antisera which recognize determinants sensitive to the conformation of the heavy chain. One heavy chain population is associated with beta 2-microglobulin, whereas the other population is not. These populations can be further distinguished by their oligosaccharide structure and their localization within the cell. Pulse-chase experiments demonstrate a precursor-product relationship between these heavy chain populations and suggest the following pathway for the assembly and maturation of HLA-A and -B antigens. The completed heavy chains initially carry high mannose oligosaccharides and are largely or wholly associated with beta 2-microglobulin. During the next 10-15 min, association with beta 2-microglobulin occurs and the heavy chain conformation is altered. Beginning at about 30 min after synthesis, the oligosaccharides are converted from the high mannose form to the complex form, and between 60 and 80 min after synthesis, the mature antigens appear at the cell surface. These observations are discussed in relation to in vivo and in vitro studies on the biosynthesis of a variety of secreted proteins and membrane proteins.

Cell Line↗
PubMed1979

Dissociation and exchange of the beta 2-microglobulin subunit of HLA-A and HLA-B antigens.

Human histocompatibility antigens HLA-A, HLA-B, and HLA-C are a complex of two noncovalently associated subunits: a heavy chain glycoprotein (alpha) carrying the genetic polymorphism and an invariant light chain, beta 2-microglobulin (beta 2m). Upon incubation of papain-solubilized HLA with radiolabeled urinary beta 2m, the latter is incorporated into HLA, where it substitutes for the preexisting beta 2m that has dissociated from the complex. The association-dissociation equilibrium that governs this beta 2m exchange reaction was investigated and found to be characterized by a long lifetime of the complex (half-life of 80 min at 37 degrees C) and a relatively low Kd (4 nM). The beta 2m exchange was used as the basis of a radioimmunoassay for HLA antigens with radiolabeled beta 2m as a unique label for all HLA specificities. In a similar fashion, radiolabeled beta 2m can be incorporated into HLA at the cell surface. Although the process is slower and less extensive than in solution, it can be used as a means to tag cells with specific probes for HLA antigens.

Beta-Globulins↗
PubMed1979

Both chains of HLA-DR bind to the membrane with a penultimate hydrophobic region and the heavy chain is phosphorylated at its hydrophilic carboxyl terminus.

The HLA-DR antigen, a complex of two glycoproteins of 29,000 and 34,000 daltons, can be isolated from the membranes of human B-lymphoblastoid cell lines. Extensive proteolysis releases only 5-10% of the antigen, whereas detergent solubilizes all of it. Detergent solubilization after papain proteolysis of membranes produces antigen with chains cleaved near the carboxyl termini. Comparison of these three preparations demonstrates that each chain contains a carboxyl-terminal hydrophilic region that is sensitive to proteolytic degradation and a penultimate hydrophobic region, responsible for membrane binding, that is more resistant to papain. This two-step cleavage of each chain is also observed during the proteolysis of detergent-solubilized HLA-DR antigen. Both chains of HLA-DR in the membrane can be labeled with the lipophilic photoactivatable carbene reagent adamantane diazirine. This label is released from both chains during the second cleavage. The heavy chain can be reduced and alkylated under mild conditions, and this label is also lost during the second cleavage. The heavy chain is phosphorylated in vivo, and this label is lost upon the first cleavage. This observation suggests that the carboxyl terminus of the heavy chain is intracellular. Cumulatively, these data suggest that both chains of HLA-DR antigens are comprised of large extracellular NH2-terminal regions, small penultimate intramembranous regions, and small carboxyl-terminal intracellular regions.

Amino Acid Sequence↗
PubMed1979

Cell-free translation of the mRNAs for the heavy and light chains of HLA-A and HLA-B antigens.

Cell-free translation of poly(A)-containing RNA from the JY lymphoblastoid cell line followed by immunoprecipitation has indicated the presence of mRNAs for both the heavy and light chains of HLA-A and HLA-B antigens in these preparations. Both chains are synthesized with an NH2-terminal extension, approximately 20 residues in length for the light chain, and 20 or 24 residues for the heavy chains. The precursors can be processed by dog pancreatic microsomes to products similar to those obtained in vivo. Immunoprecipitation of the cell-free products has been employed as an assay for partial purification of the mRNAs. Investigation of the Daudi cell line, which cannot synthesize the small subunit, beta 2-microglobin, has indicated that the heavy chains of HLA-A and HLA-B antigens are synthesized intracellularly in vivo and can also be translated from their cognate RNAs in vitro. The implications of these findings for biosynthesis of membrane proteins in general and multimeric membrane proteins in particular, as well as the role of beta 2-microglobulin in expression of HLA-A and HLA-B antigens, are discussed.

Amino Acid Sequence↗
PubMed1979

Mechanism of penicillin action: penicillin and substrate bind covalently to the same active site serine in two bacterial D-alanine carboxypeptidases.

It has been hypothesized that penicillin acts as a structural analog of the acyl-D-alanyl-D-alanine terminus of nascent bacterial cell wall and that it consequently binds to and acylates the active site of the enzyme(s) that crosslinks the cell wall to form an inactive penicilloyl enzyme [Tipper, D.J. & Strominger, J.L. (1965) Proc. Natl. Acad. Sci. USA 64, 1133-1138]. This study directly proves that penicillin acylates the active site of two penicillin-sensitive enzymes, D-alanine carboxypeptidases from Bacillus stearothermophilus and Bacillus subtilis. Active site peptides were generated by chemical or enzymatic cleavage of these carboxypeptidases after covalently labeling with [14C]penicillin G or after trapping an acyl-enzyme intermediate derived from the depsipeptide substrate. [14C]diacetyl-L-lysyl-D-alanyl-D-lactate. The amino acid sequences of the penicillin- and substrate-labeled peptides were identical. Both penicillin and substrate were covalently bound via an ester linkage to the same active site residue, a serine at position 36 of the B. stearothermophilus carboxypeptidase and the corresponding serine in the B. subtilis carboxypeptidase. The two D-alanine carboxypeptidases showed significant homology around the active site. Moreover, homology between these two enzymes and four beta-lactamases of known sequence suggests that these two groups of enzymes are evolutionally related.

Amino Acid Sequence↗
PubMed1979

Heavy chain of HLA-A and HLA-B antigens is conformationally labile: a possible role for beta 2-microglobulin.

The three-dimensional organization of HLA antigens has been investigated by spectroscopic and immunochemical techniques. Measurement of the circular dichroism shows that in papain-solubilized HLA the heavy chain as well as the previously studied light chain (beta 2-microglobulin) consists predominantly of beta-pleated sheet structures. When heavy chain is separated from the light chain under denaturing conditions and is allowed to renature, about 50% of the beta structure is lost, concomitantly with most of the alloantigenic activity. Analysis of the two acid-cleaved fragments of HLA-B7 heavy chain shows that beta structure is preferentially lost from the COOH-terminal region of the heavy chain. Exposure to denaturants per se does not inevitably result in irreversible loss of antigenic activity. However, recovery of antigenic properties does seem to depend on reassociation of the two chains. The results reported here provide further evidence for (i) the similarity of HLA antigens and immunoglobulins at the three-dimensional level and (ii) two distinct and physiologically important conformations of the HLA heavy chain, depending upon whether it is associated with the light chain.

Beta-Globulins↗
PubMed1979

Comparison of amino acid sequences of two human histocompatibility antigens, HLA-A2 and HLA-B7: location of putative alloantigenic sites.

The complete amino acid sequence for papain-solubilized HLA-B7 heavy chain is compared with the partial sequences of HLA-A2 and H-2Kb heavy chains. Although these molecules are highly conserved (i.e., 80% homology in comparing HLA-B7 with HLA-A2; 72% and 74% homology in comparing H-2Kb with HLA-A2 and HLA-B7, respectively), two stretches of greater variability are observed. These clusters of variability are discussed in terms of their possible involvement in the alloantigenic determinant(s) characteristic of these highly polymorphic membrane antigens.

Amino Acid Sequence↗
PubMed1979

Mutational evidence for identity of penicillin-binding protein 5 in Escherichia coli with the major D-alanine carboxypeptidase IA activity.

The defect in D-alanine carboxypeptidase IA activity in the dacA11191 mutant of Escherichia coli was correlated with a defect in the release of penicillin G from penicillin-binding protein 5. The results suggest that penicillin-binding protein 5 catalyzes the major D-alanine carboxypeptidase IA activity of the wild type and that the mutation results in a defect in the deacylation step catalyzed by this enzyme.

Bacterial Proteins↗
PubMed1978

Sequence of the COOH-terminal hydrophilic region of histocompatibility antigens HLA-A2 and HLA-B7.

Detergent-solubilized HLA antigens were isolated from a human lymphoblastoid cell using an anti-beta2-microglobulin immunoaffinity column. The HLA-A and HLA-B locus products were separated by thin layer isoelectric focusing. Cleavage of the p44 chain of HLA-A2 and -B7 antigens with cyanogen bromide led to the isolation of a 31-amino-acid fragment from each. The fragments were sequenced and shown to be from the COOH-terminal end of the intact chains using carboxypeptidase Y. The fragment from the HLA-B7 chain, 55% of whose amino acids were polar, contained the 2 cysteine residues not found in the papain-derived molecule. The tentative sequence of the fragment from the HLA-A2 chain was similar to that of the HLA-B7 fragment but appeared not to contain any cysteine residues. The hydrophilic COOH-terminal region of HLA antigens, which directly follows the hydrophobic, membrane-binding segment, began with a cluster of basic amino acids. This arrangement of amino acids resembles that found at the COOH terminus of the red blood cell membrane protein, glycophorin.

Amino Acid Sequence↗
PubMed1978

Partial purification and properties of the Epstein-Barr virus-associated nuclear antigen.

The Epstein-Barr virus (EBV)-associated nuclear antigen (EBNA) was purified 85-fold from a nuclear pellet derived from an EBV-transformed B lyphoblastoid cell line by a five-step procedure consisting of preparation of extract, heating at 80 degrees C in phosphate buffer, ammonium sulfate precipitation, preparative ultracentrifugation, and affinity chromatography on double-stranded DNA-cellulose. The purified complement fixing antigen specifically blocked the anticomplement immunofluorescence assay for EBNA. Several properties indicate a close association of EBNA with chromatin, viz. 1) precipitation of antigenic activity by phosphate buffer and subsequent thermal fractionation; 2) partial sensitivity of antigenic activity to DNase (but not to RNase) and restoration of activity by addition of calf thymus DNA; and 3) specific binding of EBNA to double-stranded DNA-cellulose. Other properties of EBNA, including its unusual heat stability, are described.

Antibodies, Antinuclear↗
PubMed1978

Effects of sulfhydryl reagents on the binding and release of penicillin G by D-alanine carboxypeptidase IA of Escherichia coli.

Purified D-alanine carboxypeptidase IA of Escherichia coli is inhibited by penicillin G and binds penicillin G reversibly. The binding of penicillin to the enzyme is relatively insensitive to sulfhydryl reagents, while release of penicillin from the enzyme is severely inhibited by these reagents. The inhibition of release parallels the inhibition of carboxypeptidase activity by the sulfhydryl reagents. In the presence of the sulfhydryl reagent p-chloromercuribenzoate, an acyl-enzyme intermediate, produced by the reaction of carboxypeptidase IA with diacetyl-L-lysyl-D-alanyl-D-alanine, accumulates and can be isolated. These results indicate that binding of penicillin to carboxypeptidase IA occurs by an acylation step of the carboxypeptidase reaction, while penicillin release occurs by a deacylation step of the reaction. Only the latter is inhibited by sulfhydryl reagents.

Acylation↗