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hXBP-1 is a transcription factor of the leucine zipper (b-zip) family important in the expression of the class II major histocompatibility complex gene, DRA. Studies with mouse-human hybrids have mapped hXBP-1 hybridizing fragments to human chromosomes 5 and 22 and the frequent detection of two mRNA transcripts suggested that hXBP-1 may be a member of a small gene family. To analyze the structure and regulation of hXBP-1 further, cosmid clones from both chromosomes were isolated. Mapping and sequence analyses reveal that chromosome 22 contains the functional hXBP-1 gene while chromosome 5 contains a processed pseudogene. hXBP-1 promoter analysis has revealed that cis-active elements within the 5'-untranslated region of hXBP-1 are essential for full promoter activity. One such element, hX2, is identical to the hXBP-1 target sequence in the DRA promoter. Mutagenesis of the hX2 site substantially decreases promoter activity. This element interacts with four distinct protein complexes in mature B cells and cross-competition experiments show that two of these complexes (complex 1 and complex 4) also interact with the hXBP-1 target sequence (X2) from the DRA promoter. The similarities of the hXBP-1 promoter and of the DRA promoter (the gene that the hXBP-1 protein regulates) are further emphasized by the fact that a Y box element is located 3' of both hX2 and X2.

The ability of bacitracin to inhibit the growth of Halobacterium salinarium suggested that glycosylation of the major envelope component, a high molecular weight glycoprotein, might occur via a pathway involving lipid intermediates. This report demonstrates that the cells have enzymatic activities for formation of lipid-linked sugar compounds having the expected properties of such intermediates. Whole cell homogenate catalyzed the transfer of sugar from UDP-glucose, GDP-mannose, and UDP-N-acetyglucosamine to endogenous lipid acceptors. Two lipid products were formed from UDP-glucose, two from GDP-mannose, and one from UDP-N-acetylglucosamine. Characterization of the partially purified lipids by ion exchange chromatography, thin layer chromatography, and mild acid and base hydrolysis showed the major product in each case to have the properties expected for polyisoprenyl phosphoglucose, polyisoprenyl phosphomannose, and polyisoprenyl pyrophospho-N-acetylglucosamine. Estimates of chain length by thin layer chromatography indicate that the lipid has 11 to 12 isoprene identity as a C55-60-polyisoprenyl pyrophospho-N-acetylglucosamine. The N-acetylglucosamine transferase, present in cell envelope preparations, was partially characterized. The enzyme was found to be extremely halophilic, specifically requiring a high concentration of KCl. Optimum activity was obtained at 4 m KCl and partial substitution of K+ by Na+ resulted in a decrease in activity.

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.

Penicillin-binding proteins (PBPs) 5 and 6 in the cytoplasmic membranes of Escherichia coli K12, which had previously been co-purified as a penicillin-sensitive D-alanine carboxypeptidase IA (Tamura, T., Imae, Y., and Strominger, J. L. (1976) J. Biol. Chem. 251, 414-423), were each purified to protein homogeneity. Purification involved selective solubilization of PBPs 1a, 5, and 6 from membranes by Triton X-100 at low ionic strength, covalent penicillin affinity chromatography, and CM-cellulose column chromatography. Purified PBP 5 and PBP 6 each catalyzed a D-alanine carboxypeptidase I activity using various natural and synthetic substrates including linear uncross-linked peptidoglycan. PBP 5 showed 3- to 4-fold higher specific activities toward these substrates than PBP 6. Both PBPs also catalyzed a model transpeptidase activity using glycine as a transpeptidation acceptor, and showed similar pH profiles and MgCl2 sensitivities for their D-alanine carboxypeptidase I activities. Both PBPs bound a stoichiometric amount of [14C]penicillin G at saturation. Peptide mapping by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after partial proteolysis by proteases and cyanogen bromide demonstrated that these PBPs are distinct polypeptides.

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.

A monoclonal antibody (B-5G10) was produced which specifically recognizes the Mr 150,000/130,000 VLA-4 complex on the surface of human cells. Cross-linking studies indicated that the Mr 150,000 alpha 4 subunit of VLA-4 is in noncovalent 1:1 association with the Mr 130,000 VLA beta subunit. In the absence of cross-linking, the VLA-4 alpha 4 beta subunit complex was easily dissociated, especially in Nonidet P-40 detergent, or at elevated pH (above 8.0). Studies of dissociated subunits showed that B-5G10 recognizes an epitope on the Mr 150,000 alpha 4 subunit of VLA-4, whereas the beta subunit is immunologically identical to the Mr 130,000 beta subunit common to all VLA heterodimers. VLA-4 is widely distributed on hematopoietic cells, including thymocytes, peripheral blood lymphocytes, monocytes, activated T cells, T and B lymphoblastoid cell lines, and myeloid cell lines. However, VLA-4 is only weakly expressed on most adherent cell lines tested. Immunoprecipitates of VLA-4 often contain additional proteins of Mr 80,000 and Mr 70,000. These are probably derived from the Mr 150,000 alpha 4 subunit because: 1) they are both recognized by anti-alpha 4 sera, but not anti-beta sera; 2) the sum of their sizes is equal to the size of alpha 4; 3) they are selectively coexpressed with alpha 4 and not other VLA alpha subunits; 4) the Mr 80,000 protein has an identical NH2-terminal sequence to alpha 4; 5) like alpha 4, the Mr 70,000 and 80,000 peptides can variably associate with the VLA beta subunit; and 6) trypsin appears to cleave the Mr 150,000 alpha 4 subunit into products of Mr 70,000 and 80,000.

Three homogeneous preparations of D-alanine carboxypeptidases I have been obtained from Escherichia coli strain H2143, termed enzymes IA, IB, and IC. Enzyme IA purified from the membrane after extraction with Triton X-100 appeared on sodium dodecyl sulfate gel electrophoresis to be a polypeptide doublet whose monomer molecular weights were about 32,000 and 34,000. In addition to D-alanine carboxypeptidase activity, it catalyzed a transpeptidase reaction with several substrates, bound [14C]penicillin G, had a weak penicillinase activity, but was devoid of endopeptidase activity. Enzyme IB obtained from the membrane after LiCl extraction and enzyme IC obtained from the supernatant solution were either identical or extremely similar. They were composed of a single polypeptide whose monomer molecular weight was about 41,000. In addition to carboxypeptidase activity, they catalyzed an endopeptidase reaction, had weak penicillinase activity, and had very poor transpeptidase activity, but did not bind [14C]penicillin G. Some data relating to the mechanism of catalysis by these enzymes are described. Their possible physiological role is discussed.

The site of phosphorylation of the HLA-B7 antigen in vivo is serine 335, which is located in the intracellular region. Pseudomonas fragi protease was used in limited proteolysis experiments of HLA antigens to identify the position of the phosphoserine residue. The intracellular region is composed of 30 amino acids at the carboxyl terminus of the heavy chain; up to nine serine residues are located in this region. Four of the serines are found at the distal end, and are encoded by exon 7 in both human and murine class I MHC antigens. Alignment of the protein and DNA sequences of the intracellular regions of human and murine class I antigens demonstrates conservation of the serine positions located within this exon. Realignment of exon 7, by introducing a gap in the murine sequences, increases homology across species, and reveals two conserved serines at 332 and 335 within the conserved sequence Ser-Asp/Glu-X-Ser(P)-Leu. The preservation of this sequence and the site of phosphorylation suggests that this modification of the intracellular region of histocompatibility antigens is functionally significant.

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.

The high molecular weight penicillin-binding proteins (PBP(s) ) Bacillus subtilis PBPs 1, 2, and 4 and Bacillus stearothermophilus PBPs 1-4 were shown to catalyze peptidoglycan synthesis from the undecaprenol-containing lipid intermediate substrate in two assay systems. In a filter paper assay system, high levels of substrate polymerization occurred when reaction mixtures were incubated on Whatman 3MM filter paper. The pH optimum for peptidoglycan synthesis was 7.5 for B. subtilis PBPs 1, 2, and 4 and 8.5 for B. stearothermophilus PBPs 1-4. Polymerization was Mg2+-independent and was unaffected by sulfhydryl reagents. Reconstitution with membrane lipids or addition of detergent (optimal concentration, 0.1%) was necessary for synthesis to occur. Bacitracin, penicillin, and cephalothin did not affect polymerization while vancomycin, ristocetin, moenomycin, and macarbomycin were strong inhibitors. In a test tube assay system, optimal synthesis occurred either in the presence of 10% ethylene glycol, 10% glycerol, and 8% methanol or in the presence of 10% N-acetylglucosamine. The products of lysozyme digestion of the synthesized peptidoglycan were analyzed by gel filtration and paper chromatography. B. stearothermophilus PBPs 1-4 synthesized a peptidoglycan product that was 5-7% cross-linked. No evidence for cross-linking was apparent in the peptidoglycan product of B. subtilis PBPs 1, 2, and 4.

Penicillin-binding proteins 5 and 6 have been purified to homogeneity from the dacA mutant strain of Escherichia coli (JE 11191). Protein 6 from the mutant strain appears to be identical to that from the wild type, but protein 5 is a mutant protein which has no D-alanine carboxypeptidase activity. Moreover, the mutant protein 5 binds, but does not release, [14C]penicillin G. Correspondingly, an acyl-enzyme intermediate derived from a synthetic substrate is accumulated by the mutant protein. A comparison of the acylation site for the synthetic substrate and for penicillin G by limited proteolysis and some other properties of the mutant protein are described.

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.

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.

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.

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.

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.

The localization of the active site of penicillin-binding protein 5 from the dacA mutant of Escherichia coli strain TMRL 1222 has been determined. The protein was purified to homogeneity and labeled with [14C] penicillin G. The labeled protein was digested with trypsin, and the active site tryptic peptide was purified by a combination of gel filtration and high-pressure liquid chromatography. Sequencing of the purified [14C]penicilloyl peptide yielded the sequence Arg-Asp-Pro-Ala-Ser-Leu-Thr-Lys, which corresponds to residues 40-47 of the gene sequence (Broome-Smith, J., Edelman, A., and Spratt, B. G. (1983) in The Target of Penicillin (Hakenbeck, R., Holtje, J.-V., and Labischinski, H., eds) pp. 403-408, Walter de Gruyter, Berlin). The catalytic amino acid residue that forms a covalent bond with penicillin was identified by treating the purified [14C]penicilloyl peptide with a mixture of proteases and then separating the radioactive products using high-pressure liquid chromatography. Analysis of the radioactive peaks by amino acid analysis confirmed that it is the serine residue that reacts with the beta-lactam ring of penicillin.

Soluble, truncated mutant and wild-type forms of penicillin-binding protein 5 (sPBP 5) from Escherichia coli were produced in large amounts by placing the dacA gene that encodes PBP 5 under the control of the trp-lac fusion promoter. The 3' end of the dacA gene used in this study contains a stop codon that results in the deletion of 15 amino acids from the carboxyl terminus and the production of a soluble protein. Using oligonucleotide-directed mutagenesis, the role of cysteine 115 in the mechanism of sPBP 5 was investigated. Alkylation of cysteine 115 with sulfhydryl reagents has previously been shown to inhibit severely the D-alanine carboxypeptidase activity of PBP 5. Alkylation also inhibits the hydrolysis of bound penicillin G, with only a slight effect on its binding. Cysteine 115 in sPBP 5 was changed to either a serine (sPBP 5C-S) or an alanine (sPBP 5C-A) residue. The wild-type and mutant sPBPs were purified in milligram amounts from induced cultures by ampicillin affinity chromatography. The mutant PBPs showed only a 2-fold increase in the half-life of the penicilloyl-PBP complex, and had a binding affinity for penicillin G identical to wild-type PBP 5. The Km for the release of D-alanine from the peptide L-Ala-D-gamma-Glu-L-Lys-D-Ala-D-Ala was 5.0, 3.5, and 7.8 mM for PBP 5, PBP 5C-S, and PBP 5C-A, respectively, while the values for Vmax were 2.5, 3.3, and 5.1 mumol/min/mg. From these data it was concluded that the cysteine residue does not directly participate in the enzymatic mechanism.

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.

The kinetics of hydrolysis and transpeptidation of the synthetic substrate diacetyl-L-lysyl-D-alanyl-D-alanine and of the natural substrate UDP-acetylmuramyl pentapeptide and related compounds catalyzed by the D-alanine carboxypeptidases of Bacillus subtilis and Bacillus stearothermophilus in the presence of the nucleophiles hydroxylamine or glycine have been examined. These kinetic data suggest that an acyl-enzyme intermediate is formed in the first step of the reaction and that the transpeptidation is the consequence of the partitioning of this intermediate between water and the nucleophile in the second step.

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.

D-Alanine carboxypeptidase is a penicillin-sensitive intrinsic membrane enzyme which is composed of a hydrophilic NH2-terminal catalytic domain (Mr congruent to 45,000 to 47,000) and a COOH-terminal membranous segment (approximately 20 to 30 amino acids in length) (Waxman, D. J., and Strominger, J. L. (1979) J. Biol. Chem. 254, 4863-4875; Waxman, D. J., and Strominger, J. L. (1981) J. Biol. Chem. 256, 2059-2066). The primary structures of the COOH-terminal 30 amino acids of two D-alanine carboxypeptidase purified from bacterial membranes were determined (residues numbered from the COOH terminus): Bacillus stearothermophilus: (formula see text) Water-soluble fragments of the B. stearothermophilus D-alanine carboxypeptidase were shown to be formed by cleavage after Phe27 or after Leu25 as indicated by carboxypeptidase A and B analysis and by the release of the four COOH-terminal chymotryptic peptides (Val26-Leu25, Ser24-Phe16, Val15-Trp12, and Thr11-Leu1) upon formation of water-soluble chymotrypsin D-alanine carboxypeptidase. This indicates that the membranous fragment is largely contained within the COOH-terminal 24 residues. Thus, this bacterial membrane protein probably does not contain the significant cytoplasmic domain characteristic of transmembrane proteins such as glycophorin. The absence of an uninterrupted stretch of 20 to 25 uncharged residues suggests that the membrane anchoring of D-alanine carboxypeptidase may differ from that of simple transmembrane proteins. Possible structures for the membranous segment of D-alanine carboxypeptidase are discussed.

The heavy chain of the HLA-A2 antigen is phosphorylated by cyclic AMP-dependent protein kinase at two serine residues of the intracellular region. Limited proteolysis was performed on purified [32P]HLA-A2 antigens in order to define the sites of phosphorylation. Both of the phosphorylated serine residues are located in the carboxyl terminus of the heavy chain; one is encoded by exon 5, while the other is encoded by exon 6. The phosphoserine encoded by exon 5 is part of the conserved sequence Arg-Arg-Lys-Ser-Ser. This protein sequence contains the proper arrangement of amino acids for recognition and phosphorylation by the catalytic subunit of cyclic AMP-dependent protein kinase. In the murine class I antigens (H-2), exon 5 encodes a similar sequence of basic residues followed by one intervening residue and a threonine rather than a serine residue in the last amino acid position. A composite figure is presented that compares the carboxyl-terminal sequences of human and murine class I antigens and illustrates the known sites of phosphorylation recognized by various kinases. Each site of phosphorylation in the carboxyl terminus is contained within a conserved protein sequence encoded by one of the three exons. A separation and preservation of unique sites of phosphorylation could explain why there is segmentation in the genomic arrangement of class I molecules.

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.

Physicochemical techniques were used to investigate the activation of C55-isoprenoid alcohol phosphokinase by synthetic lecithins. Complexes of the enzyme with phospholipids were prepared using a method employing sodium dodecyl sulfate as a protein-solubilizing agent. Circular dichorism and the intrinsic fluorescence of the kinase were used as optical probes of protein conformation with these complexes. No evidence for a major lipid-dependent conformational change in the protein was observed when these complexes were studied under conditions where the lipid mesomorphic transitions occurred. EPR studies of mixtures of synthetic lecithins and the C55-isoprenoid alcohol indicated a correlation between kinase activity and the rotational diffusion rate within the hydrophobic phase. It is concluded that the lipid physical state probably does not affect the enzyme activity by altering the protein conformation but more likely does so by affecting the motion of the molecular participants in the reaction.

The activation of kinase by neutral detergents has been examined. Of those detergents tested, only those with short chain and unsaturated chain hydrophobes were successful activators at 25 degrees. In addition to these structural requirements, a dependence upon hydrophilic-lipophilic balance number was observed when mixing hydrophobic and hydrophilic detergents. Most pairs tested showed an optimal hydrophilic-lipophilic balance number for kinase activation in the hydrophobic range of 6 to 9. Thus, there appears to be both a structural and a relative hydrophobic requirement for activation, and hydrophilic-lipophilic balance number may be a measure of the physical properties of the lipid required for activation.

Human class II histocompatibility antigens were purified from the Epstein-Barr virus-transformed human B lymphoblastoid cell line LG-2 by immunoaffinity chromatography. This is the first time all three subsets have been prepared as nonradioactive materials on a milligram scale. The yields of DR, DQ, and DP from 10 g of cells were approximately 12, 2, and 0.2 mg, respectively. Cross-contamination of the subsets was found to be less than 2% when assayed by measuring the binding of antigen-specific monoclonal antibodies to antigen immobilized on fixed erythrocytes. The three purified subsets were extensively characterized. They contained no detectable invariant chain. The three proteins were distinguished by their migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing. The denatured antigens were susceptible to partial removal of carbohydrate by endoglycosidase H and apparently complete removal of carbohydrate by endoglycosidase F. The isolated, denatured chains differed in their affinities for radiolabeled lectins, suggesting differences in carbohydrate structures. A water-soluble form of each antigen was prepared by a controlled papain digestion of the native antigen. Both native and denatured antigens were analyzed for their reactivities with a panel of class II antigen-specific monoclonal antibodies, allowing a precise definition of the specificities of the antibodies.

The glycoprotein which accounts for approximately 50% of the protein and all of the nonlipid carbohydrate of the cell envelope of Halobacterium salinarium (Mescher, M. F., Strominger, J. L., and Watson S. W. (1974) J. Bacteriol. 120, 945-954) has been purified and partially characterized. The glycoprotein has an apparent molecular weight of 200,000, is extremely acidic, and has a carbohydrate content of approximately 10 to 12%. The carbohydrate included neutral hexoses, amino sugar, and uronic acid. Information regarding the number, composition, and mode of attachment of the carbohydrate chains was obtained by isolation and examination of the glycopeptides derived from degradation of cell envelope protein with trypsin and pronase. Trypsin digestion resulted in two glycopeptides. One of these was large (approximately 55,000 daltons) and had most of the neutral hexose linked to it. The carbohydrate moieties consisted of di- and trisaccharides of glucosylgalactose and (uronic acid, glucose)-galactose attached via O-glycosidic linkages between galactose and threonine. The other tryptic glycopeptide had a relatively large heterosaccharide attached to it via an alkaline-stable linkage. The heterosaccharide contained 1 glucose, 8 to 9 galactose, 1 mannose, and 10 to 11 glucosamine residues, and approximately 6 residues of an unidentified amino augar. The alkaline stability of the linkage and the amino acid composition of glycopeptides resulting from Pronase digestion of the tryptic glycopeptide showed that the heterosaccharide was attached to an asparagine residue, presumably via an N-glycosylamine bond to the amide group. The intact glycoprotein has a single N-linked heterosaccharide, 22 to 24 O-linked disaccharides, and 12 to 14 O-linked trisaccharides per molecule. N- and O-glycosidic linkages are the most common carbohydrate-protein linkages in mammalian glycoproteins but, to our knowledge, this is the first report of either type of linkage in a prokaryotic cell envelope protein.

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.

The structure of monomeric and disulfide-bonded dimeric forms of HLA antigens has been studied. Detergent-soluble HLA antigen heavy chains contain one or two easily reduced sulfhydryl groups not found in papain-solubilized HLA antigens, as demonstrated by amino acid analysis (Springer, T. A., and Strominger, J.L. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 2481-2485, and Terhorst, C., Parham, P., Mann, D.L., and Strominger, J.L. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 910-914) and by labeling with iodo[3H]acetate. Dimer formation occurred during purification, since it was prevented by pretreatment of membranes containing HLA antigen with iodoacetamide. Cross-linking studies showed that the non-disulfide-bonded form of HLA antigens contains one subunit each of the Mr = 44,000 heavy chain and the Mr = 12,000 light chain (beta2-microglobulin).

The very late antigen complexes VLA-1 and VLA-2 which appear on long-term activated human T cells have been characterized with respect to 1) subunit arrangement, 2) location of monoclonal antibody (MAb) binding sites, 3) carbohydrate content, and 4) protein homology. Cross-linking experiments showed that the VLA-1 complex is a heterodimer composed of an Mr 210,000 subunit (alpha 1) in acid-labile association with an Mr 130,000 subunit (beta). The VLA-2 complex is a heterodimer with an Mr 165,000 subunit (alpha 2) in base-labile association with the Mr 130,000 beta subunit. The subunits of VLA-1 (alpha 1 beta) and VLA-2 (alpha 2 beta) each appear to be arranged with 1:1 stoichiometry. The MAb A-1A5 has been shown to bind to an epitope on the common beta subunit, consistent with its recognition of both the VLA-1 and VLA-2 heterodimers. On the other hand, MAb TS2/7 bound to an epitope of the alpha 1 subunit, thus explaining the specific recognition of the VLA-1 heterodimer by TS2/7. Digestion of the alpha 1, alpha 2, and beta subunits with neuraminidase and with endoglycosidase F revealed that each subunit contains substantial sialic acid and N-linked carbohydrate. By one-dimensional peptide mapping, the alpha 1, alpha 2, and beta subunits were shown to be highly nonhomologous with respect to each other, although each subunit from different T cell sources appeared highly homologous if not identical.

HL-A antigens having specificities HL-A2, HL-A7, HL-A12 have been solubilized by papain treatment of membrane preparations from the cultured human lymphoblastoid cell line RPMI 4265 and purified about 80-fold by chromatography on carboxymethylcellulose, Sephadex G-150, and diethylaminoethylcellulose columns. Separation of HL-A2 from a mixture of HL-A7 and HL-A12 was achieved on the final DEAE-cellulose column. The yield was about 1 mg of protein of each antigen preparation per 100 g of packed, frozen cells. On sodium dodecyl sulfate gel electrophoresis both preparations showed two polypeptide bands. The smaller subunit of 12,000 daltons is common to all HL-A preparations and has been shown to be identical with beta2-microglobulin. The larger subunit is a glycopeptide and in the HL-A7, 12 preparation was resolved into a duplex of 34,000 and 37,000 daltons. The HL-A2 preparation showed a single band at 34,000 daltons. On isoelectric focusing under nondenaturing conditions, the preparation showed multiple bands, all of which contained both subunits and retained antigenic activity. On isoelectric focusing in the presence of 6 M urea a single band for beta2-microglobulin and multiple bands for the larger subunit were seen. This charge heterogeneity of the larger subunit has been shown to be due to variable amounts of sialic acid. When HL-A antigen preparations were subjected to Sephadex G-100 chromatography in the presence of 3 M KCl, no separation of the two subunits was observed.