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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.

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 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.

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.

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.

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.

Lysine-requiring mutants of Bacillus sphaericus 9602 were isolated and classified into three groups by their mutation site in the pathway of lysine biosynthesis. The Group I mutant lacks meso-alpha, epsilon-diaminopimelic acid (meso-Dap) decarboxylase activity, but Group II and III mutants have a normal level of Dap decarboxylase activity. A Group II mutant makes dipicolinic acid from an intermediate in the lysine pathway, but Group III mutants do not. In the absence of meso-Dap in the culture, muramic lactam content in the spore cortex of Group II and III mutants is very low, compared to the wild type content. Addition of meso-Dap to the culture causes an increase of muramic lactam content. Since meso-Dap is detectable only in the spore cortex of B. sphaericus 9602, almost all of the muramic lactam in the spore is probably also located in the cortex. Group I mutants grown in the presence of L-lysine sporulate normally. Group II and III mutants produce oval and nonrefractile spores under the same conditions but the addition of meso-Dap to the culture results in the production of round and refractile spores. Thus, the presence of cortex in the spore is essential to give the round and refractile spores in B. sphaericus. The presence of cortex is also required for the accumulation of dipicolinic acid in the sporulating cells. Furthermore, 1-octanol resistance of the spore depends only on the presence of cortex but both cortex and dipicolinic acid are required for heat resistance of the spore.

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.

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.

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.

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.

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.