Purification of the peptidoglycan transglycosylase of Bacillus megaterium

The peptidoglycan transglycosylase of Bacillus megaterium has been purified approximately 500-fold from a crude membrane fraction. This protein is likely to be the one previously called PG-II and was assayed by its ability to reconstitute with a crude phospho-N-acetyl-muramyl-pentapeptide translocase preparation and partially purified N-acetylglucosaminyl transferase to give peptidoglycan synthesis from nucleotide precursors. The protein was identified as the peptidoglycan transglycosylase by its ability to synthesize lysozyme-sensitive peptidoglycan from undecaprenylpyrophosphoryl-disaccharide-pentapeptide. The enzyme is inhibited by vancomycin but not by bacitracin, penicillin G, or tunicamycin. The enzyme has no detectable transpeptidase activity, but it does bind penicillin.     

Membrane intermediates in the peptidoglycan metabolism of Escherichia coli: possible roles of PBP 1b and PBP 3.

The two membrane precursors (pentapeptide lipids I and II) of peptidoglycan are present in Escherichia coli at cell copy numbers no higher than 700 and 2,000 respectively. Conditions were determined for an optimal accumulation of pentapeptide lipid II from UDP-MurNAc-pentapeptide in a cell-free system and for its isolation and purification. When UDP-MurNAc-tripeptide was used in the accumulation reaction, tripeptide lipid II was formed, and it was isolated and purified. Both lipids II were compared as substrates in the in vitro polymerization by transglycosylation assayed with PBP 1b or PBP 3. With PBP 1b, tripeptide lipid II was used as efficiently as pentapeptide lipid II. It should be stressed that the in vitro PBP 1b activity accounts for at best to 2 to 3% of the in vivo synthesis. With PBP 3, no polymerization was observed with either substrate. Furthermore, tripeptide lipid II was detected in D-cycloserine-treated cells, and its possible in vivo use in peptidoglycan formation is discussed. In particular, it is speculated that the transglycosylase activity of PBP 1b could be coupled with the transpeptidase activity of PBP 3, using mainly tripeptide lipid II as precursor.     

Amidase activity involved in peptidoglycan biosynthesis in membranes of Micrococcus luteus (sodonensis).

Membrane suspensions prepared from Micrococcus luteus (sodonensis) in both the exponential and stationary phases of growth contained a transglycosidase activity capable of synthesizing linear peptidoglycan. Exponential-phase membranes also contained an N-acetylmuramyl-L-alanine amidase activity which degraded the peptidoglycan as it was formed. The product of this amidase was purified and found to be free pentapeptide. The amidase was specific for peptidoglycan and could not attack lower-molecular-weight substrates even though the susceptible bond was present. Crude cell wall preparations isolated from exponential-phase cells also contained high levels of amidase. This cell wall-bound amidase would preferentially degrade in vitro-synthesized peptidoglycan over its own cell wall. Amidase activity could be solubilized from both cell walls and membranes by Triton X-100 treatment, butanol extraction, or LiCl extraction. Both membrane- and cell wall-derived amidases, solubilized by LiCl extraction, appeared to be of high molecular weight (greater than 150,000). Once solubilized, these wall- and membrane-derived amidases could attack the cross-bridged peptidoglycan of purified native cell walls, whereas bound amidases could not.     

Phosphorylation of a low molecular weight polypeptide in beef heart Na plus, K plus-ATPase preparations.

Sodium dodecyl sulfate-polyacrylamide gel profiles of a NaI-treated beef heart Na⁺,K⁺-ATPase preparation revealed the presence of two protein kinase substrates of low molecular weight, whereas a more purified citrate beef heart Na⁺,K⁺-ATPase preparation contained one low molecular weight polypeptide substrate. This enzyme preparation was phosphorylated in the presence of protein kinase, and phosphorylation was inhibited by protein kinase inhibitor. The phosphorylated product was identified as a phosphoester. Half maximal stimulation of protein kinase-catalyzed phosphorylation occurred at approximately 9 × 10^?8m cyclic AMP. The low molecular weight (11,700) protein kinase substrate present in the heart preparations was eluted from polyacrylamide slab gels. The polypeptide fraction was reelectrophoresed and the polypeptide was removed from the gels, hydrolyzed, and analyzed for amino acid content. This polypeptide was different from other low molecular weight protein kinase substrates including troponin components, myosin light chains, and histones and is most likely of plasma membrane origin.     

Biochemical characterisation of the chlamydial MurF ligase, and possible sequence of the chlamydial peptidoglycan pentapeptide stem

Chlamydiaceae are obligate intracellular bacteria that do not synthesise detectable peptidoglycan although they possess an almost complete arsenal of genes encoding peptidoglycan biosynthetic activities. In this paper, the murF gene from Chlamydia trachomatis was shown to be capable of complementing a conditional Escherichia coli mutant impaired in UDP-MurNAc-tripeptide:D-Ala-D-Ala ligase activity. Recombinant MurF from C. trachomatis was overproduced and purified from E. coli. It exhibited ATP-dependent UDP-MurNAc-X-γ-D-Glu-meso-A(2)pm:D-Ala-D-Ala ligase activity in vitro. No significant difference of kinetic parameters was seen when X was L-Ala, L-Ser or Gly. The L-Lys-containing UDP-MurNAc-tripeptide was a poorer substrate as compared to the meso-A(2)pm-containing one. Based on the respective substrate specificities of the chlamydial MurC, MurE, MurF and Ddl enzymes, a sequence L-Ala/L-Ser/Gly-γ-D-Glu-meso-A(2)pm-D-Ala-D-Ala is expected for the chlamydial pentapeptide stem, with Gly at position 1 being less likely.     

Purification and partial characterization of a stimulatory factor for lamb thymus RNA polymerase II.

A heat-stable protein (HSF) that stimulates the activity of lamb thymus RNA polymerase II has been purified 2500-fold and partially characterized. This factor stimulates the activity of RNA polymerase II up to 13 times and retains complete activity when heated at 90 degrees C for 5 min. Stimulation is observed only in the presence of RNA polymerase II and requires native DNA as template. The stimulatory factor has a sedimentation coefficient of 2.7 S, a diffusion coefficient of 9.55 x 10(-7) cm2/s, and an isoelectric point of 8.0. Calculated from the sedimentation and diffusion data, the factor has a molecular weight of about 24,000. Electrophoresis of the purified factor on polyacrylamide gels in the presence of sodium dodecyl sulfate results in a single band corresponding to a molecular weight of 25,000. The number-average length of the RNA synthesized by RNA polymerase II is increased in the presence of the factor. Sedimentation velocity and exclusion chromatography experiments suggest that the stimulatory factor interacts with RNA polymerase II. These results suggest that the factor stimulates RNA synthesis through a direct interaction with RNA polymerase II. The stoichiometry of the HSF-RNA polymerase binding appears to be about 1:1. HSF is located in the nucleus, as determined by cell fractionation studies.     

Modification of the vitamin K-dependent carboxylase assay

Methods are presented that describe alternative protocols for the isolation of rat liver microsomes containing the vitamin K-dependent carboxylase and the procedure in which the solubilized enzyme is assayed. The method for determining the rate of 14CO2 incorporation into low molecular weight, acid soluble substrates by the rat liver microsomal vitamin K-dependent carboxylase has been modified in order to optimize safety, accuracy and simplicity. For these studies the rat liver microsomes containing the vitamin K-dependent carboxylase were isolated by CaCl2 precipitation. These Triton X-100 solubilized microsomes were found to be equivalent to the microsomes obtained by high speed ultracentrifugation with regard to protein concentration, pentapeptide carboxylase activity, carboxylase activity, preprothrombin concentration and total carboxylatable endogenous protein substrate. This modified assay procedure requires fewer steps and pipetting transfers and is quantitatively equivalent to previously employed protocols. The described technique can be adapted for any assay where 14CO2 or H14CO3- is incorporated into non-volatile products. This newly developed assay procedure was employed to assess conditions necessary for optimal vitamin K-dependent carboxylation of the less expensive substrate, N-t-Boc-L-glutamic acid alpha-benzyl ester. The optimal conditions for the carboxylation of N-t-Boc-L-glutamic acid alpha-benzyl ester by the carboxylase were found to be 10 mM N-t-Boc-L-glutamic acid alpha-benzyl ester, 10 mM MgCl2 at 15-18 degrees C. The rate of N-t-Boc-L-glutamic acid alpha-benzyl ester carboxylation under these optimized conditions was found to be higher (1.5-fold) than the rate of carboxylation of 1 mM Phe-Leu-Glu-Glu-Ile in the presence of the cation activator, MgCl2.     

Recruitment of penicillin-binding protein PBP2 to the division site of Staphylococcus aureus is dependent on its transpeptidation substrates

Staphylococcus aureus penicillin-binding protein PBP2 is an enzyme involved in the last stages of peptidoglycan assembly and is an important player in the mechanism of methicillin resistance of this pathogen. PBP2 localized to the division site but its recruitment to the forming division septum was prevented after acylation by oxacillin. The presence of the antibiotic did not affect FtsZ ring maintenance nor the localization of externalized peptidoglycan precursors. Delocalization of PBP2 was also observed when its pentapeptide substrate was eliminated by addition of d-cycloserine or blocked by addition of vancomycin. Taken together these observations suggest that PBP2 is recruited to the division site by binding to its substrate, which is localized at that place. In methicillin-resistant S. aureus, addition of oxacillin does not result in delocalization of PBP2 indicating that acylated PBP2 can be maintained in place by functional PBP2A, the central element of this resistance mechanism.     

Characterization of an alkaline protease associated with a granulosis virus of Plodia interpunctella.

An alkaline protease was found to be associated with the granulosis virus of the Indian meal moth. Plodia interpunctella. The protease was located within the protein matrix of the occluded virus and hydrolyzed the major constituent of this matrix, a 28,000-dalton protein (granulin), to a mixture of polypeptides ranging in molecular weight from 10,000 to 27,000. A rapid, sensitive assay for the protease was developed using radioactively labeled granulosis virus as substrate. With this assay, the proteolytic activity could be detected by measuring the release of acid-soluble peptides from the labeled virus. The protease had a pH optimum of 10.5 and a temperature optimum of 40 degrees C and was inhibited by diisopropyl phosphorofluoridate, phenylmethylsulfonyl fluoride, and L-(1-tosylamido-2-phenyl) ethyl chloromethyl ketone. Purification of the protease from matrix protein was achieved by anion-exchange and gel permeation chromatography. The molecular weight of the isolated protease, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, was approximately 14,000.     

Deformations in the cytoplasmic membrane of Escherichia coli direct the synthesis of peptidoglycan. The hernia model.

To explain the growth of the Gram-negative envelope and in particular how it could be strengthened where it is weakest, we propose in the hernia model that local weakening of the peptidoglycan sacculus allows turgor pressure to cause the envelope to bulge outwards in a hernia; the consequent local alteration in the radius of curvature of the cytoplasmic membrane causes local alterations in phospholipid structure and composition that determine both the synthesis and hydrolysis of peptidoglycan. This proposal is supported by evidence that phospholipid composition determines the activity of phospho-N-acetylmuramic acid pentapeptide translocase, UDP-N-acetylglucosamine:N-acetylmuramic acid-(pentapeptide)-P-P-bactoprenyl-N-acetylglucosamine transferase, bactoprenyl phosphate phosphokinase, and N-acetylmuramyl-L-alanine amidase. We also propose that the shape of Escherichia coli is maintained by contractile proteins acting at the hernia. Given the universal importance of membranes, these proposals have implications for the determination of shape in eukaryotic cells.     

Purification and partial characterization of megamodulin, a heat-stable protein factor from E. coli and its stimulatory effect on RNA polymerase holoenzyme

Megamodulin, a heat-stable protein from Escherichia coli was isolated and purified near homogeneity as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis. It had a molecular weight of 71,000 and pl between 3.5 and 4.0. This factor stimulated E. coli RNA polymerase 71-fold in the presence of a synthetic template such as poly (rA).p(dT). When TATAAA sequence was used as template, the RNA polymerase activity was increased 68 times by this factor. The possible mechanism by which this protein factor may regulate the RNA polymerase activity has been described.     

Evidence for participation of a multiprotein complex in yeast DNA replication in vitro

Fractions containing a high molecular weight form (Mr approximately equal to 2 X 10(6] of the activity that replicates in vitro both the 2-micron yeast DNA plasmid and the chromosomal autonomously replicating sequence ars 1 can be prepared from cells of the budding yeast Saccharomyces. Protein complexes from the fractions associate in vitro with the replication origins of these DNA elements, as determined by electron microscopy. In the present study, the high molecular weight replicative fraction has been characterized in further detail. The DNA synthetic activity in the high molecular weight fraction was bound to the DNA and could be isolated with it. This binding of the replicating activity to the DNA was greatly reduced in the absence of the 2-micron origins of replication. Association of the protein complexes with DNA depended on the amount of replicating activity added, was sensitive to 0.2 M KCl, and exhibited a requirement for rATP and deoxyribonucleoside triphosphates. It was not blocked, however, by the DNA polymerase inhibitor aphidicolin or by the RNA polymerase inhibitor alpha-amanitin. The lack of inhibition by aphidicolin suggests that the deoxyribonucleoside triphosphates may function as cofactors in the binding of protein complexes to DNA or as substrates for a polymerizing activity such as a primase. Binding of the protein complexes as well as actual DNA replication were heat sensitive in the high molecular weight fraction prepared from the temperature-sensitive mutant of the cell division cycle cdc 8. This suggests that the cdc 8 gene product is present in a replicative protein complex and strengthens the conclusion that the presence of the protein complexes on the DNA is associated with replication. Using independent enzyme assays, several other possible replication proteins (including DNA polymerase I, DNA ligase, DNA primase, and DNA topoisomerase II) have been identified directly in the high molecular weight replicative fraction. All of these results provide support for the idea that a protein complex (or replisome ) is involved in the replication of both the extrachromosomal 2-micron DNA and chromosomal DNA in yeast.     

Biochemical characterization of aspartyl phosphate phosphatase interaction with a phosphorylated response regulator and its inhibition by a pentapeptide

The RapA and RapB proteins are aspartyl phosphate phosphatases that specifically dephosphorylate the Spo0F approximately P intermediate response regulator of the phosphorelay signal transduction system for sporulation initiation in Bacillus subtilis. The approximately 48-kDa His-tag derivative proteins were purified by metal affinity chromatography, and their molecular and biochemical characteristics were studied. RapA and RapB were found to be dimers in solution. Enzymatic activity was strongly dependent upon maintaining reducing conditions during purification and storage. RapA phosphatase activity on Spo0F approximately P is inhibited in vivo by a pentapeptide generated from the phrA gene. Native gel assays demonstrated that the RapA dimer forms a stable complex with two molecules of Spo0F approximately P or with its PhrA pentapeptide inhibitor. The pentapeptide was shown to displace Spo0F approximately P from a preformed complex with RapA. The structural organization of Rap phosphatases in tetratricopeptide repeats provides insights on the mechanisms of RapA interaction with its substrate and its inhibitor.     

Kinetics of carboxylation of endogenous and exogenous substrates by the vitamin K-dependent carboxylase

The vitamin K-dependent carboxylation of the exogenous pentapeptide, Phe-Leu-Glu-Glu-Ile, and endogenous liver microsomal protein was studied in solubilized rat liver microsomes. The MnCl2 stimulation of the vitamin K-dependent pentapeptide carboxylation rate, which is conducted at subsaturating concentrations of pentapeptide, is due to the cation's ability to lower the Km of the substrate. Although there are clear kinetic differences observed between the carboxylation rates for the pentapeptide and the endogenous protein substrates, several lines of evidence suggest that the same carboxylase system is responsible for both. These points of evidence are (i) the initial velocity of endogenous protein carboxylation is lowered in the presence of 3 mM pentapeptide; (ii) the presence of endogenous microsomal protein substrate causes an initial lag in pentapeptide carboxylation; and (iii) this initial lag phase is not observed when the total endogenous substrate pool is carboxylated by a preincubation reaction prior to the addition of pentapeptide.     

Evidence that the N-terminal part of the S-layer protein from Bacillus stearothermophilus PV72/p2 recognizes a secondary cell wall polymer.

The S-layer of Bacillus stearothermophilus PV72/p2 shows oblique lattice symmetry and is composed of identical protein subunits with a molecular weight of 97,000. The isolated S-layer subunits could bind and recrystallize into the oblique lattice on native peptidoglycan-containing sacculi which consist of peptidoglycan of the A1gamma chemotype and a secondary cell wall polymer with an estimated molecular weight of 24,000. The secondary cell wall polymer could be completely extracted from peptidoglycan-containing sacculi with 48% HF, indicating the presence of phosphodiester linkages between the polymer chains and the peptidoglycan backbone. The cell wall polymer was composed mainly of GlcNAc and ManNAc in a molar ratio of 4:1, constituted about 20% of the peptidoglycan-containing sacculus dry weight, and was also detected in the fraction of the S-layer self-assembly products. Extraction experiments and recrystallization of the whole S-layer protein and proteolytic cleavage fragments confirmed that the secondary cell wall polymer is responsible for anchoring the S-layer subunits by the N-terminal part to the peptidoglycan-containing sacculi. In addition to this binding function, the cell wall polymer was found to influence the in vitro self-assembly of the guanidinium hydrochloride-extracted S-layer protein. Chemical modification studies further showed that the secondary cell wall polymer does not contribute significant free amino or carboxylate groups to the peptidoglycan-containing sacculi.     

ATP x Mg-dependent protein phosphatase from rabbit skeletal muscle. I. Purification of the enzyme and its regulation by the interaction with an activating protein factor

An ATP x Mg-dependent protein phosphatase (FC) was purified to near homogeneity from rabbit muscle. The enzyme was completely devoid of any spontaneous activity but could be activated by a protein activator (FA) in the presence of ATP and Mg ions. The inactive phosphatase migrated as a single protein band on sodium dodecyl sulfate-gel electrophoresis, and in discontinuous gel electrophoresis, where the potential phosphatase activity was located in the main protein band. The molecular weight determined by sodium dodecyl sulfate electrophoresis or by sucrose density centrifugation was found to be 70,000. FC migrated on gel filtration as a 140,000 molecular weight species. The activation by FA was not paralleled by an incorporation of [32P]-phosphate into the ATP x Mg-dependent phosphatase, and from the kinetics of activation a protein-protein interaction with ATP x Mg as a necessary factor, can be inferred as the mechanism of activation. After activation by FA and ATP X Mg, the purified enzyme had a specific activity of 10,000 units/mg of protein, and a Km for rabbit muscle phosphorylase a of approximately 1.0 mg/ml. The activated enzyme did not release [32P]phosphate from 32[-labeled rabbit muscle synthase b, prepared from glucagon-treated dogs. It did, however, remove all the 32P label from phosphorylase b kinase, autophosphorylated to the level of 2.0 mol/mol of 1.3 X 10(6) molecular weight.     

Human deoxyuridine triphosphate nucleotidohydrolase. Purification and characterization of the deoxyuridine triphosphate nucleotidohydrolase from acute lymphocytic leukemia.

A new fast assay procedure for increasing deoxyuridine triphosphate nucleotidohydrolase activity was developed. With this assay procedure, this enzyme derived from blast cells of patients with acute lymphocytic leukemia was purified at least 1218-fold. The molecular weight was estimated by gel filtration to be 43,000. The enzyme exhibited optimal activity over a pH range of 7 to 8 and the activation energy was estimated to be 6.5 kcal/mol at pH 7.5. While the enzyme had activity in the absence of added divalent cations, the activity could be inhibited by EDTA but not by phenanthroline. The inhibition caused by EDTA could be reversed by Mg2+ or Zn2+. The enzyme had maximal activity in the presence of Mg2+ (40 muM) and Mg2+ (4 mM) stabilized the enzyme at 37 degrees C. Cupric ion (0.5 mM) inhibited (50%) enzyme activity in the presence or absence of Mg2+. The substrate for the enzyme was dUTP and the apparent Km was 1 muM. No other deoxyribonucleoside or ribonucleoside triphosphate served as a substrate for the enzyme.     

Balance between two transpeptidation mechanisms determines the expression of beta-lactam resistance in Enterococcus faecium

The d,d-transpeptidase activity of high molecular weight penicillin-binding proteins (PBPs) is essential to maintain cell wall integrity as it catalyzes the final cross-linking step of bacterial peptidoglycan synthesis. We investigated a novel beta-lactam resistance mechanism involving by-pass of the essential PBPs by l,d-transpeptidation in Enterococcus faecium. Determination of the peptidoglycan structure by reverse phase high performance liquid chromatography coupled to mass spectrometry revealed that stepwise selection for ampicillin resistance led to the gradual replacement of the usual cross-links generated by the PBPs (d-Ala(4) --> d-Asx-Lys(3)) by cross-links resulting from l,d-transpeptidation (l-Lys(3) --> d-Asx-Lys(3)). This was associated with no modification of the level of production of the PBPs or of their affinity for beta-lactams, indicating that altered PBP activity was not required for ampicillin resistance. A beta-lactam-insensitive l,d-transpeptidase was detected in membrane preparations of the parental susceptible strain. Acquisition of resistance was not because of variation of this activity. Instead, selection led to production of a beta-lactam-insensitive d,d-carboxypeptidase that cleaved the C-terminal d-Ala residue of pentapeptide stems in vitro and caused massive accumulation of cytoplasmic precursors containing a tetrapeptide stem in vivo. The parallel dramatic increase in the proportion of l-Lys(3) --> d-Asx-Lys(3) cross-links showed that the enzyme was activating the resistance pathway by generating the substrate for the l,d-transpeptidase.     

Identification of a new protein involved in the regulation of the anticoagulant activity of activated protein C. Protein S-binding protein

The apparent molecular weight of functional protein S in citrated plasma was observed to be between 115,000 and 130,000 as measured by sedimentation equilibrium in the air-driven ultracentrifuge. The molecular weight of the functional protein decreased to approximately 62,000 when copper ions were added to the plasma. This suggested the presence of a protein S-binding protein in plasma, which was confirmed by gel filtration experiments. Frontal analysis of plasma indicated that functional protein S could exist in as many as three forms. Addition of copper ions to plasma reduced the number of forms to one. In order to isolate the binding protein, plasma was fractionated first on a column of immobilized iminodiacetic acid that had been equilibrated with copper ions. The proteins that eluted in a 0.6 M NaCl wash were passed over a column of protein S immobilized on agarose beads. A protein, eluted in the 0.6 M NaCl wash, was observed to bind to protein S in gel filtration experiments. When added to plasma depleted of both protein S and the binding protein, the binding protein was observed to enhance the anticoagulant activity of activated protein C only in the presence of protein S. Protein S-binding protein was also observed to enhance the rate of factor Va inactivation by activated protein C and protein S.